 stranded DNA, which is in the form of a covalently closed  circle (and so designated ccc ds DNA). Additional genes  that usually just confer upon the cell a survival advantage  under certain circumstances may also be contained upon  plasmids; these are usually similar in structure to chro-  mosomes but much smaller and replicate independently  (Chapters 3 and 13). The total complement of genes pos-  sessed by a cell, i.e. those in the chromosome, plasmid(s)  and any received from other sources, e.g. bacteriophages  (bacterial viruses), is referred to as the genome of the cell.   Typically bacterial chromosomes are 1 mm or more in  length and contain about 1000-3000 genes. As many bac-  terial cells are approximately 1 pm long, it is clear that the  chromosome has to be tightly coiled in order to fit in the  available volume. Although all the genes are contained on  a single chromosome (rather than being distributed over  two or more), it is possible for a cell to contain several  copies of that chromosome at any one time. Usually there  are multiple copies during periods of rapid cell division,  but some species seem to have many copies all the time.  The mechanisms by which bacterial genes may be trans-  ferred from one organism to another are described in  Chapter 3.   Plasmids usually resemble chromosomes except that  they are approximately 0. 1—1.0% of the size of a bacterial  chromosome, and there are a few that are linear rather  than circular. Plasmid genes are not essential for the  normal functioning of the cell but may code for a prop-  erty that affords a survival advantage in certain environ-  mental conditions; bacteria possessing the plasmid in  question would therefore be selected when such condi-  tions exist. Properties which can be coded by plasmids  include the ability to utilize unusual sugars or food  sources, toxin production, production of pili that facili-  tate the attachment of a cell to a substrate (e.g. intestinal  epithelium) and antibiotic resistance. A cell may contain  multiple copies of any one plasmid and may contain two  or more different plasmids. However, some plasmid com-  binations cannot coexist inside the same cell and are said  to be incompatible; this phenomenon enables plasmids  to be classified into incompatibility groups.   Plasmids replicate independently of the chromosome  within the cell, so that both daughter cells contain a copy  of the plasmid after binary fission. Plasmids may also be  passed from one cell to another by various means  (Chapter 3). Some plasmids exhibit a marked degree of  host specificity and may only be transmitted between  different strains of the same species, although others, par-  ticularly those commonly found in Gram-negative intes-  tinal bacteria, may cross between different species within    a genus or between different genera. Conjugative (self-  transmissible) plasmids code for genes that facilitate  their own transmission from one cell to another by the  production of pili. These sex pili initially establish contact  between the two cells and then retract, drawing the  donor and recipient cells together until membrane fusion  occurs.   Eukaryotic microorganisms (yeasts, moulds, algae and  protozoa) possess a nucleus that normally contains one  or more pairs of linear chromosomes, in which the ds  DNA is complexed with protein. The cells may divide  asexually and the nucleus undergoes mitosis — a sequence  of events by which the nucleus and the chromosomes  within it are replicated to give copies identical to the  originals. Most eukaryotes also have the potential for  sexual reproduction during which the nucleus undergoes  meiosis, i.e. a more specialized form of nuclear and chro-  mosome division creating new gene combinations, so the  offspring differ from the parents. Despite this potential,  there are some eukaryotic cells, particularly fungi, in  which a sexual stage in the life cycle has never been  observed. Many eukaryotic microorganisms possess plas-  mids, and some fungal plasmids are based on RNA  instead of DNA.   Microorganisms may adapt rapidly to new environments  and devise strategies to avoid or negate stressful or poten-  tially harmful circumstances. Their ability to survive  adverse conditions may result from the organism using  genes it already possesses, or by the acquisition of new  genetic information. The term ‘genotype’ describes the  genetic composition of an organism, i.e. it refers to the  genes that the organism possesses, regardless of whether  they are expressed or not. It is not uncommon for a  microbial cell to possess a particular gene but not to  express it, i.e. not to manufacture the protein or enzyme  that is the product of that gene, unless or until the  product is actually required; this is simply a mechanism  to avoid wasting energy. For example, many bacteria  possess the genes that code for (3-lactamases; these  enzymes hydrolyse and inactivate (3-lactam antibiotics  (e.g. penicillins). In many organisms (3-lactamases are  only produced in response to the presence of the antibi-  otic. This form of non-genetic adaptation is termed phe-  notypic adaptation, and there are many situations in  which bacteria adopt a phenotypic change to counter  environmental stress. But microorganisms may also use    an alternative strategy of genetic adaptation, by which  they acquire new genes either by mutation or by conjuga-  tion (Chapter 3); subsequently, a process of selection  ensures that the mutant organisms that are better  suited to the new environment become numerically  dominant.   In bacteria, mutation is an important mechanism by  which resistance to antibiotics and other antimicrobial  chemicals is achieved, although the receipt of entirely  new genes directly from other bacteria is also clinically  very important. Spontaneous mutation rates (rates not  influenced by mutagenic chemicals or ionizing radiation)  vary substantially depending on the gene and the organ-  ism in question, but rates of 10 5 — 10 7 are typical. These  values mean that, on average, a mutant arises once in  every 100000 to every 10 million cell divisions. Although  these figures might suggest that mutation is a relatively  rare event, the speed with which microorganisms can  multiply means, for example, that mutants exhibiting  increased antibiotic resistance can arise quite quickly  during the course of therapy.   Table 2.4 indicates the ways in which the different types  of microorganism are considered relevant in pharmacy.  The importance of viruses derives exclusively from their  pathogenic potential, and because of their lack of intrin-  sic metabolism they are not susceptible to antibiotics.  Partly for these reasons, viral infections are among the  most dangerous and difficult to cure, and of all the cat-  egories of microorganism, only viruses appear in (the  most serious) Hazard Category 4 as classified by the  Advisory Committee on Dangerous Pathogens. Because  they are not free-living, viruses are incapable of growing  on manufactured medicines or raw materials, so they do  not cause product spoilage, and they have no synthetic  capabilities that can be exploited in medicines manufac-  ture. Viruses are relatively easy to destroy by heat, radia-  tion or toxic chemicals, so they do not represent a  problem from this perspective. In this, they contrast with  prions; although some authorities would question the  categorization of these infectious agents as microorgan-  isms, they are included here because of their undoubted  ability to cause, as yet incurable, fatal disease, and their  extreme resistance to lethal agents. Pharmacists and  healthcare personnel in general should be aware of the  ability of prions to easily withstand sterilizing conditions   that would be satisfactory for the destruction of all other  categories of infectious agent.   There are examples of bacteria that are important in  each of the different ways indicated by the column head-  ings of Table 2.4. Many of the medically and pharmaceu-  tically important bacteria are pathogens, and some of  these pathogens are of long-standing notoriety as a result  of their ability to resist the activity of antibiotics and  biocides (disinfectants, antiseptics and preservatives). In  addition to these long-established resistant organisms,  other bacteria have given more recent cause for concern  including meticillin-resistant Staphylococcus aureus  (MRSA), vancomycin-resistant enterococci (VSE) and  multiply resistant Mycobacterium tuberculosis (Chapter  13). While penicillin and cephalosporin antibiotics are  produced by fungal species, the majority of the other  categories of clinically important antibiotics are pro-  duced by species of bacteria, notably streptomycetes. In  addition, a variety of bacteria are exploited commercially  in the manufacture of other medicines including steroids,  enzymes and carbohydrates. The ability of bacteria to  grow on diverse substrates ensures that their potential as    agents of spoilage in manufactured medicines and raw  materials is well recognized, and the ability of many  species to survive drying means that they survive well in  dust and so become important as contaminants of manu-  factured medicines. The ability to survive not only in dry  conditions but in other adverse environments (heat,  radiation, toxic chemicals) is well exemplified by bacterial  spores, and their pre-eminence at or near the top of the  Teague table’ of resistance to lethal agents has resulted in  spores acting as the indicator organisms that have to be  eliminated in most sterilization processes (Chapter 21).   Like bacteria, fungi are able to form spores that survive  drying, so they too arise commonly as contaminants of  manufactured medicines. However, the degree of resist-  ance presented by the spores is usually less than that  exhibited by bacteria, and fungi do not represent a steri-  lization problem. Fungi do not generally create a signifi-  cant infection hazard either; relatively few fungal species  are considered major pathogens for animals that possess  a fully functional immune system. There are, however,  several fungi which, while representing little threat to  immunocompetent individuals, are nevertheless capable    of initiating an infection in persons with impaired  immune function; the term ‘opportunist pathogens’ is  used to describe microorganisms (of all types) possessing  this characteristic. In this context it is worth noting that  the immunocomopromised represent an increasingly  large group of patients, and this is not just because of  HIV/AIDS. Several other conditions or drug treatments  impair immune function, e.g. congenital immunodefi-  ciency, cancer (particularly leukaemia), radiotherapy and  chemotherapy, the use of systemic corticosteroids and  immunosuppressive drugs (often following tissue or  organ transplants), severe burns and malnutrition.   Protozoa are of significance largely owing to the path-  ogenic potential of a few species. Because protozoa do not  possess cell walls they do not survive drying well (unless  in the form of cysts), so they are not a problem in the  manufacturing environment — and even the encysted  forms do not display resistance to sterilizing processes to  match that of bacterial spores. It should be noted that  protozoal infections are not currently a major problem  to human health in temperate climates, although they are  more troublesome in veterinary medicine and in the  tropics. There are concerns that the geographical ranges  of protozoal infections such as malaria may extend sub-  stantially if current fears about global warming translate  into reality.   In addition to their uses in the manufacture of medicines,  microorganisms are employed in a variety of tests and  assays, particularly those to measure the activity of anti-  microbial chemicals. Useful organisms, therefore, need to  be correctly preserved in order to ensure that their desir-  able properties are not changed during storage or, worse,  the culture dies completely and is irreplaceable. Many  bacteria and fungi can conveniently be stored for a few  days, or possibly weeks, in the form of liquid cultures in  tubes, or as colonies on Petri dishes. Organisms that  readily form spores — Bacillus and Clostridium species of    bacteria and most fungi — can be stored for months or  even years in this way provided that the culture medium  does not evaporate to dryness, but non-sporing organ-  isms vary substantially in their survival capacities. Gram-  positive bacteria generally tend to survive better than  Gram-negative ones: species like Pseudomonas aerugi-  nosa , for example, may die in a few weeks, even at refrig-  eration temperatures, if maintained as colonies on  unsealed Petri dishes. Even if a culture that is to be pre-  served does not die completely when stored in the refrig-  erator, there is a risk that the cells that do survive are not  typical of the population as a whole; they may, for  example, be mutants that have increased resistance to  adverse conditions in general, and so fail to give the  expected results when used in tests on antibiotic activity.  The dual aims of a culture preservation procedure there-  fore are to maintain the viability of the highest possible  percentage of cells and to minimize the risk of selecting  atypical mutants.   The most common procedures for long-term storage  are by freezing at — 80 °C (or lower) in refrigerators, by  storage in liquid nitrogen at — 196°C in special vessels, or  by freeze-drying (also called lyophilization). In each case,  cryoprotectant chemicals — compounds like glycerol or  dimethylsulphoxide — are incorporated at a concentra-  tion of about 10% v/v in the liquid culture of the organ-  ism in order to minimize both the formation of damaging  ice crystals and osmotic stresses that can accelerate cell  death during freezing and thawing.   Reference cultures, those with well-defined biosyn-  thetic capabilities or resistance properties, can be obtained  in a freeze-dried form from internationally accessible  culture collections like the American Type Culture  Collection (cultures having the designation ATCC before  a reference number) or the UK National Collection of  Industrial and Marine Bacteria (NICMB). Increasingly,  pharmacopoeias and regulatory agencies are requiring  tests that employ microorganisms to be conducted with  cultures or test suspensions of cells that are no more than  five subcultures from the reference material obtained  from the designated culture collection.   The smallest free-living microorganisms are the prokary-  otes, comprising bacteria and archaea (see Chapter 2).  Prokaryote is a term used to define cells that lack a true  nuclear membrane; they contrast with eukaryotic cells  (e.g. plants, animals and fungi) that possess a nuclear   8 Detection, identification and characterization of organisms of  pharmaceutical and medical significance 38    membrane and internal compartmentalization. Indeed, a  major feature of eukaryotic cells, absent from prokaryotic  cells, is the presence in the cytoplasm of membrane-  enclosed organelles. These and other criteria differentiat-  ing eukaryotes and prokaryotes are shown in Table 2.1.   Bacteria and archaea share many traits and it was not  until the early 1980s that differences first became evident  from analyses of gene sequences. One major difference is    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Hodges, Sean P. Gorman,  Brendan F. Gilmore.    the composition of cell walls. A more striking contrast is  in the structure of the lipids that make up their cytoplas-  mic membranes. Differences also exist in their respective  patterns of metabolism: most archaea are anaerobes, and  are often found inhabiting extreme environments. It is  possible that their unusual membrane structure gives  archaeal cells greater stability under extreme conditions.  Of notable interest is the observation that no disease-  causing archaea have yet been identified; the vast  majority of prokaryotes of medical and pharmaceutical  significance are bacteria.   Bacteria represent a large and diverse group of micro-  organisms that can exist as single cells or as cell clusters.  Moreover, they are generally able to carry out their life  processes of growth, energy generation and reproduction  independently of other cells. In these respects they are  very different from the cells of animals and plants, which  are unable to live alone in nature and can exist only as  part of a multicellular organism. They are capable of  growing in a range of different environments and can not  only cause contamination and spoilage of many pharma-  ceutical products but also a range of different diseases.  For this reason, only bacteria are referred to throughout  the remainder of this chapter.   Bacterial diversity can be seen in terms of variation in cell  size and shape (morphology), adaptation to environmen-  tal extremes, survival strategies and metabolic capabili-  ties. Such diversity allows bacteria to grow in a multiplicity  of environments ranging from hot sulphur springs (65 °C)  to deep freezers (— 20°C), from high (pH 1) to low (pH  13) acidity and high (0.7m) to low osmolarity (water).  In addition, they can grow in both nutritionally rich  (compost) and nutritionally poor (distilled water) situa-  tions. Hence, although each organism is uniquely suited  to its own particular environmental niche and rarely  grows out of it, the presence of bacteria maybe considered  ubiquitous. Indeed, there is no natural environment that  is free from bacteria. This ubiquity is often demonstrated  by terms used to describe organisms that grow and/or  survive in particular environments. An example of such  descriptive terminology is shown in Table 3.1.   Bacteria are the smallest free-living organisms, their size  being measured in micrometres (microns). Because of    this small size a microscope affording a considerable  degree of magnification (X400-1000) is necessary to  observe them. Bacteria vary in size from a cell as small as  0. 1-0.2 pm in diameter to those that are >5 pm in diam-  eter. Bacteria this large, such as Thiomargarita namibien-  sis, are extremely rare: the majority of bacteria are 1-5 pm  long and 1-2 pm in diameter. By comparison, eukaryotic  cells may be 2 pm to >200 pm in diameter. The small size  of bacteria has a number of implications with regard to  their biological properties, most notably increased and  more efficient transport rates. This advantage allows bac-  teria far more rapid growth rates than eukaryotic cells.   While the classification of bacteria is immensely  complex, nowadays relying very much on 16S ribosomal  DNA sequencing data, a more simplistic approach is to  divide them into major groups on purely morphological  grounds. The majority of bacteria are unicellular and  possess simple shapes, e.g. round (cocci), cylindrical (rod,  also called bacillus, spelt with a lower case initial letter to  distinguish from Bacillus , the genus) or ovoid. Some rods  are curved (vibrios), while longer rigid curved organisms  with multiple spirals are known as spirochaetes. Rarer  morphological forms include the actinomycetes which  are rigid bacteria resembling fungi that may grow as  lengthy branched filaments; the mycoplasmas which  lack a conventional peptidoglycan (murein) cell wall and  are highly pleomorphic organisms of indefinite shape;  and some miscellaneous bacteria comprising stalked,  sheathed, budded and slime-producing forms often asso-  ciated with aquatic and soil environments.   Often bacteria remain together in specific arrange-  ments after cell division. These arrangements are usually  characteristic of different organisms and can be used as    part of a preliminary identification. Examples of such  cellular arrangements include chains of rods or cocci,  paired cells (diplococci), tetrads and clusters.   Compared with eukaryotic cells, bacteria possess a fairly  simple base cell structure, comprising cell wall, cytoplas-  mic membrane, nucleoid, ribosomes and occasionally  inclusion granules (Figure 3.1). Nevertheless it is impor-  tant for several reasons to have a good knowledge of these  structures and their functions. First, the study of bacteria  provides an excellent route for probing the nature of  biological processes, many of which are shared by multi-  cellular organisms. Secondly, at an applied level, normal  bacterial processes can be customized to benefit society  on a mass scale. Here, an obvious example is the large-  scale industrial production (fermentation) of antibiotics.  Thirdly, from a pharmaceutical and healthcare perspec-  tive, it is important to be able to know how to kill bacte-  rial contaminants and disease-causing organisms. To  treat infections antimicrobial agents are used to inhibit  the growth of bacteria, a process known as antimicrobial  chemotherapy. The essence of antimicrobial chemother-  apy is selective toxicity (Chapters 11, 12, 14 and 15),  which is achieved by exploiting differences between the  structure and metabolism of bacteria and host cells.  Selective toxicity is, therefore, most efficient when a  similar target does not exist in the host. Examples of such  targets will be noted in the following sections.   The bacterial cell wall is an extremely important struc-  ture, being essential for the maintenance of the shape and  integrity of the bacterial cell. It is also chemically unlike  any structure present in eukaryotic cells and is therefore  an obvious target for antibiotics that can attack and kill  bacteria without harm to the host (Chapter 12).   The primary function of the cell wall is to provide a  strong, rigid structural component that can withstand the  osmotic pressures caused by high chemical concentra-  tions of inorganic ions in the cell. Most bacterial cell walls  have in common a unique structural component called  peptidoglycan (also called murein or glycopeptide);  exceptions include the mycoplasmas, extreme halophiles  and the archaea. Peptidoglycan is a large macromolecule  containing glycan (polysaccharide) chains that are cross-  linked by short peptide bridges. The glycan chain acts as  a backbone to peptidoglycan, and is composed of alter-  nating residues of N-acetyl muramic acid (NAM) and  N - acetyl glucosamine (NAG). To each molecule of NAM   Figure 3.1 Diagram of a bacterial cell. Features represented  above the dotted line are only found in some bacteria, whereas  those below the line are common to all bacteria.    is attached a tetrapeptide consisting of the amino acids  L-alanine, D-alanine, D-glutamic acid and either lysine or  diaminopimelic acid (DAP). This glycan tetrapeptide  repeat unit is cross-linked to adjacent glycan chains,  either through a direct peptide linkage or a peptide inter-  bridge (Figure 3.2). The types and numbers of cross-  linking amino acids vary from organism to organism.  Other unusual features of the cell wall that provide  potential antimicrobial targets are DAP and the presence  of two amino acids that have the D-configuration.   Bacteria can be divided into two large groups, Gram-  positive and Gram-negative, on the basis of a differential  staining technique called the Gram stain. Essentially, the  Gram stain consists of treating a film of bacteria dried on  a microscope slide with a solution of crystal violet, fol-  lowed by a solution of iodine; these are then washed with  an alcohol solution. In Gram-negative organisms the cells  lose the crystal violet-iodine complex and are rendered  colourless, whereas Gram-positive cells retain the dye.  Regardless, both cell types are counter-stained with a dif-  ferent coloured dye, e.g. carbolfuchsin, which is red.    Hence, under the light microscope Gram-negative cells  appear red while Gram-positive cells are purple. These  marked differences in response reflect differences in cell  wall structure. The Gram-positive cell wall consists prima-  rily of a single type of molecule whereas the Gram-negative  cell wall is a multilayered structure and quite complex.   The cell walls of Gram-positive bacteria are quite thick  (20-80 nm) and consist of between 60% and 80% pepti-  doglycan, which is extensively cross-linked in three  dimensions to form a thick polymeric mesh (Figure 3.3).  Gram-positive walls frequently contain acidic polysac-  charides called teichoic acids; these are either ribitol  phosphate or glycerol phosphate molecules that are con-  nected by phosphodiester bridges. Because they are  negatively charged, teichoic acids are partially responsible  for the negative charge of the cell surface as a whole.    Their function may be to effect passage of metal cations  through the cell wall. In some Gram-positive bacteria  glycerol-teichoic acids are bound to membrane lipids  and are termed lipoteichoic acids. During an infection,  lipoteichoic acid molecules released by killed bacteria  trigger an inflammatory response. Cell wall proteins, if  present, are generally found on the outer surface of the  peptidoglycan.   The wall, or more correctly, envelope of Gram-negative  cells is a far more complicated structure (Figure 3.4).  Although it contains less peptidoglycan (10-20% of  wall), a second membrane structure is found outside  the peptidoglycan layer. This outer membrane is  asymmetrical, composed of proteins, lipoproteins, phos-  pholipids and a component unique to Gram-negative  bacteria, lipopolysaccharide (LPS). Essentially, the outer    membrane is attached to the peptidoglycan by a lipopro-  tein, one end of which is covalently attached to peptidog-  lycan and the other end is embedded in the outer  membrane. The outer membrane is not a phospholipid  bilayer although it does contain phospholipids in the  inner leaf, and its outer layer is composed of LPS, a  polysaccharide-lipid molecule. Proteins are also found in  the outer membrane, some of which form trimers that  traverse the whole membrane and in so doing form  water- filled channels or porins through which small mol-  ecules can pass. Other proteins are found at either the  inner or outer face of the membrane.   The LPS (Figure 3.5) is an important molecule because  it determines the antigenicity of the Gram-negative cell  and it is extremely toxic to animal cells. The molecule  consists of three regions, namely lipid A, core polysac-  charide and O-specihc polysaccharide. The lipid A  portion is composed of a disaccharide of glucosamine  phosphate bound to fatty acids and forms the outer  leaflet of the membrane. It is the lipid A component that  is responsible for the toxic and pyrogenic properties of  Gram-negative bacteria. Lipid A is linked to the core  polysaccharide by the unique molecule ketodeoxyocto-  nate (KDO), and at the other end of the core is the  O-polysaccharide (O-antigen), which usually contains  six-carbon sugars as well as one or more unusual deoxy  sugars such as abequose.   Although the outer membrane is relatively permeable  to small molecules, it is not permeable to enzymes or  large molecules. Indeed, one of the major functions of  the outer membrane may be to keep certain enzymes that  are present outside the cytoplasmic membrane from dif-  fusing away from the cell. Moreover, the outer membrane  is not readily penetrated by hydrophobic compounds and  is, therefore, resistant to dissolution by detergents.   The region between the outer surface of the cytoplas-  mic membrane and the inner surface of the outer mem-  brane is called the periplasm. This occupies a distance of  about 12-15 nm, is gel-like in consistency and, in addi-  tion to the peptidoglycan, contains sugars and an abun-  dance of proteins including hydrolytic enzymes and  transport proteins. Table 3.2 summarizes the major dif-   Biochemically, the cytoplasmic membrane is a fragile,  phospholipid bilayer with proteins distributed randomly  throughout. These are involved in the various transport  and enzyme functions associated with the membrane.  A major difference in chemical composition between  prokaryotic and eukaryotic cells is that eukaryotes have  sterols in their membranes (e.g. cholesterol) whereas  prokaryotes do not. The cytoplasmic membrane serves  many functions, including transport of nutrients, energy  generation and electron transport; it is the location for  regulatory proteins and biosynthetic proteins, and it acts  as a semipermeable selectivity barrier between the cyto-  plasm and the cell environment.   Invaginations of the cytoplasmic membrane are referred  to as mesosomes. Those that form near the septum of Gram-  positive cells serve as organs of attachment for the bacte-  rial chromosome.   The cytoplasm consists of approximately 80% water and  contains enzymes that generate ATP directly by oxidizing  glucose and other carbon sources. The cytoplasm also    contains some of the enzymes involved in the synthesis  of peptidoglycan subunits. Ribosomes, the DNA genome  (nucleoid) and inclusion granules are also found in the  cytoplasm.   The bacterial chromosome exists as a singular, covalently  closed circular molecule of double-stranded DNA  comprising approximately 4600 kilobase pairs. It is com-  plexed with small amounts of proteins and RNA, but  unlike eukaryotic DNA, is not associated with histones.  The DNA, if linearized, would be about 1 mm in length.  In order to package this amount of material the cell  requires that the DNA is supercoiled into a number of  domains (c.50) and that the domains are associated  with each other and stabilized by specific proteins into  an aggregated mass or nucleoid. The enzymes, topoi-  somerases, that control topological changes in DNA  architecture are different from their eukaryotic counter-  parts (which act on linear chromosomes) and therefore  provide a unique biochemical target for antibiotic  action.   Plasmids are relatively small, circular pieces of double-  stranded extrachromosomal DNA. They are capable of  autonomous replication and encode for many auxiliary  functions that are not usually necessary for bacterial  growth. One such function of great significance is that  of antibiotic resistance (Chapter 13). Plasmids may  also transfer readily from one organism to another,  and between species, thereby increasing the spread of  resistance.   The cytoplasm is densely packed with ribosomes. Unlike  eukaryotic cells these are not associated with a membra-  nous structure; the endoplasmic reticulum is not a com-  ponent of prokaryotic cells. Bacterial ribosomes are 70S  in size, made up of two subunits of 30S and 50S. This is  smaller than eukaryotic ribosomes, which are 80S in size  (40S and 60S subunits). Differences will therefore exist in  the size and geometry of RNA binding sites.   Bacteria occasionally contain inclusion granules within  their cytoplasm. These consist of storage material com-  posed of carbon, nitrogen, sulphur or phosphorus and  are formed when these materials are replete in the envi-  ronment to act as repositories of these nutrients when   The surface of the bacterial cell is the portion of the  organism that interacts with the external environment  most directly. As a consequence, many bacteria deploy  components on their surfaces in a variety of ways that  allow them to withstand and survive fluctuations in the  growth environment. The following sections describe a  few of these components that are commonly found,  although not universally, that allow bacteria to move,  sense their environment, attach to surfaces and provide  protection from harsh conditions.   Bacterial motility is commonly provided by flagella, long  (c. 12 pm) helical-shaped structures that project from the  surface of the cell. The filament of the flagellum is built  up from multiple copies of the protein flagellin. Where  the filament enters the surface of the bacterium, there is  a hook in the flagellum, which is attached to the cell  surface by a series of complex proteins called the flagellar  motor. This rotates the flagellum, causing the bacterium  to move through the environment. The numbers and  distribution of flagella vary with bacterial species. Some  have a single, polar flagellum, whereas others are flagel-  late over their entire surface (peritrichous); intermediate  forms also exist.   Fimbriae are structurally similar to flagella, but are not  involved in motility. Although they are straighter, more  numerous and considerably thinner and shorter (3|im)  than flagella, they do consist of protein and project from  the cell surface. There is strong evidence to suggest that  fimbriae act primarily as adhesins, allowing organisms to  attach to surfaces, including animal tissues in the case of  some pathogenic bacteria, and to initiate biofilm forma-  tion. Fimbriae are also responsible for haemagglutination  and cell clumping in bacteria. Among the best character-  ized fimbriae are the type I fimbriae of enteric (intestinal)  bacteria.   Pili are morphologically and chemically similar to fim-  briae, but they are present in much smaller numbers  (<10) and are usually longer. They are involved in the  genetic exchange process of conjugation (section 6.3).   Many bacteria secrete extracellular polysaccharides (EPS)  that are associated with the exterior of the bacterial cell.  The EPS is composed primarily of c.2% carbohydrate and  98% water, and provides a gummy exterior to the cell.  Morphologically, two extreme forms exist: capsules,  which form a tight, fairly rigid layer closely associated  with the cell, and slimes, which are loosely associated with  the cell. Both forms function similarly, to offer protection  against desiccation, to provide a protective barrier against  the penetration of biocides, disinfectants and positively  charged antibiotics, to protect against engulfment by  phagocytes and protozoa and to act as a cement binding  cells to each other and to the substratum in biofilms (see  below). One such polymer that performs all these func-  tions is alginate, produced by Pseudomonas aeruginosa-,  dextran, produced by Leuconostoc mesenteroides, is  another. Both polymers may be harvested and used vari-  ously as pharmaceutical aids, surgical dressings and drug  delivery systems, although the preferred source of algi-  nate is seaweed rather than bacteria.   S-layers are the most common cell wall type amongst the  archaea. These consist of a two-dimensional paracrystal-  line array of proteins or glycoproteins which show various  ordered symmetries when viewed under the electron  microscope. In many species of bacteria, S-layers are  present on their outer surfaces in addition to other cell  wall components such as polysaccharides. In such  arrangements the S-layer is always the outermost layer. In  addition to increasing the structural robustness of the  cell, S-layers can act to a certain extent as an external  permeability barrier.   Any surface, whether it is animate or inanimate, is of  considerable importance as a microbial habitat owing to  the adsorption of nutrients. A nutrient-rich microenvi-  ronment is thus produced in a nutrient-poor macroenvi-  ronment whenever a surface-liquid interface exists.  Consequently, microbial numbers and activity are usually  much greater on a surface than in suspension. Hence, in  many natural, medical and industrial settings bacteria  attach to surfaces and form multilayered communities  called biofilms. These commonly contain more than one  species of bacteria, which exist cooperatively together as  a functional, dynamic consortium. Moreover, biofilms    commonly possess unique properties that are distinct to  unattached cells. Biofilm formation usually begins with  pioneer cells attaching to a surface, either through the use  of specific adhesins such as fimbriae, or non-specifically  by EPS. Once established, these cells grow and divide to  produce microcolonies, which with time, eventually coa-  lesce to produce a biofilm. A key characteristic of biofilms  is the enveloping of the attached cells in a matrix of EPS  and other macromolecules. This helps to cement cells to  the surface and to each other, and protects the bacteria  from hazardous materials such as antibiotics and bio-  cides, from desiccation and from engulfment by macro-  phages and phagocytes in much the same way as the  capsules and slime layers mentioned above. In addition,  strands of EPS hold the bacterial cells at a distance from  one another, enabling small water channels to form in the  biofilm. These channels act as a primitive circulatory  system carrying trapped nutrients and oxygen to the  enclosed cells and take waste products away.   Biofilms have a number of significant implications in  medicine and industry. In the human body the resident  cells within the biofilm are not exposed to attack by the  immune system and in some instances can exacerbate the  inflammatory response. An example of this is shown by  the growth of Ps. aeruginosa as an alginate-enclosed  biofilm in the lungs of cystic fibrosis patients. Bacterial  biofilms are also profoundly less susceptible to antimi-  crobial agents than their free-living, planktonic counter-  parts. As a consequence, bacterial biofilms that form on  contaminated medical implants and prosthetic devices,  manufacturing surfaces or fluid conduit systems are vir-  tually impossible to eliminate with antibiotics or bio-  cides. In these situations antimicrobial resistance occurs  as a population or community response. Biofilms are  considered in more detail in Chapter 8.   In a few bacterial genera, notably Bacillus and Clostridium,  a unique process takes place in which the vegetative cell  undergoes a profound biochemical change to give rise to  a specialized structure called an endospore or spore  (Figure 3.6). This process of sporulation is not part of a  reproductive cycle, but the spore is a highly resistant cell  that enables the producing organism to survive in adverse  environmental conditions such as lack of moisture or  essential nutrients, or exposure to toxic chemicals, radia-  tion or high temperatures. Because of their extreme  resistance to radiation, ethylene oxide and heat, all steri-    lization processes for pharmaceutical products have been  designed to destroy the bacterial spore (Chapter 21).  Removal of the environmental stress may lead to germi-  nation of the spore back to the vegetative cell form.   Endospores are differentiated cells that possess a grossly  different structure to that of the parent vegetative cell in  which they are formed. The structure of the spore is  much more complex than that of the vegetative cell in  that it has many layers surrounding a central core (Figure  3.7). The outermost layer is the exosporium composed of  protein; within this are the spore coats, which are also  proteinaceous but with a high cysteine content, the cortex  that consists of loosely cross-linked peptidoglycan and  the central core that contains the genome. Characteristic  of the spore is the presence of dipicolinic acid and high  levels of calcium ions which complex together. The core  is also partially dehydrated, containing only 10-30% of  the water content of the vegetative cells. Dehydration has  been shown to increase resistance to both heat and chem-  icals. In addition, the pH of the core is about 1 unit lower  than the cytoplasm of the vegetative cell and contains  high levels of core-specific proteins that bind tightly to  the DNA and protect it from potential damage. These  core-specific proteins also function as an energy source  for the outgrowth or germination of a new vegetative cell  from the endospore.   During endospore formation the vegetative cell under-  goes a complex series of biochemical events in cellular  differentiation, and many genetically directed changes in  the cell that underpin the conversion occur in a series of   distinct stages. Sporulation requires that the synthesis of  some proteins involved in vegetative cell function cease  and that specific spore proteins are made. This is accom-  plished by activation of a variety of spore-specific genes  such as spo and ssp. The proteins coded by these genes  catalyse a series of events leading ultimately to the pro-  duction of a dry, metabolically inert but extremely resist-  ant endospore. The whole process can take only a matter  of hours to complete under optimal conditions.   Although endospores can lie dormant for decades, they  can revert back to a vegetative cell very rapidly. Activation  of the process may occur through removal of the stress  inducer that initiated sporulation. During germination  loss of resistance properties occurs along with a loss of  calcium dipicolinate and cortex components, and degra-  dation of the core-specific proteins. Outgrowth occurs,  involving water uptake and synthesis of new RNA, pro-  teins and DNA until eventually, after a matter of minutes,  the vegetative cell emerges from the fractured spore coat  and begins to divide again.   Although bacteria are associated with disease, only a few  species are disease-producing or pathogenic for healthy  individuals (Chapter 7). Of greater concern are those  organisms that, if presented with the correct set of condi-  tions, can cause disease, i.e. opportunist pathogens.  Examples include Staphylococcus epidermidis , a beneficial  organism when present on the skin (its normal habitat)  yet potentially fatal if attached to a synthetic heart valve,    and Ps. aeruginosa, a non-pathogenic environmental  organism but again potentially lethal in immunocompro-  mised patients.   The pathogens cause host damage in a number of  ways. In most cases pathogens produce a variety of mol-  ecules or factors that promote pathogenesis, among  which are the toxins: products of bacteria that produce  immediate host cell damage. Toxins have been classified  as either endotoxin, i.e. cell wall-related, or exotoxin,  products released extracellularly as the organism grows.   Endotoxin is the lipid A component of LPS (see section  2.2.1). It possesses multiple biological properties includ-  ing the ability to induce fever, initiate the complement  and blood cascades, activate B lymphocytes and stimulate  production of tumour necrosis factor. Endotoxin is gen-  erally released from lysed or damaged cells. Care must be  taken to eliminate or exclude such heat-resistant material  from parenteral products and their delivery systems  through a process known as depyrogenation (Chapters  21 and 22).   Most exotoxins fall into one of three categories on the  basis of their structure and activities. These are the A-B  toxins, the cytolytic toxins and the superantigen toxins.  The A-B toxins consist of a B subunit that binds to a host  cell receptor and is also covalently bound to the A subunit  that mediates the enzymic activity responsible for toxic-  ity. Most exotoxins (e.g. diphtheria toxin, cholera toxin)  are of the A-B category. The cytolytic toxins such as  haemolysins and phospholipases do not have separable A  and B portions but work by enzymatically attacking cell  constituents, causing lysis. The superantigens also lack an  A-B type structure and act by stimulating large numbers  of immune response cells to release cytokines, resulting  in a massive inflammatory reaction. An example of this  type of reaction is Staphylococcus aureus - mediated toxic  shock syndrome.   The majority of bacterial cells multiply in number by a  process of binary fission. That is, each individual will  increase in size until it is large enough to divide into two  identical daughter cells. At the point of separation  each daughter cell must be capable of growth and repro-  duction. While each daughter cell will automatically  contain those materials that are dispersed throughout the  mother cell (mRNA, rRNA, ribosomes, enzymes, cyto-    chromes, etc.), each must also carry at least one copy of  the chromosome. The bacterial chromosome is circular  and attached to the cytoplasmic membrane where it is  able to uncoil during DNA replication. The process of  DNA replication proceeds at a fixed rate dependent on  temperature, therefore the time taken to copy an entire  chromosome depends on the number of base pairs  within it and the growth temperature. For Escherichia coli  growing at 37 °C, replication of the chromosome will  take approximately 45 minutes. These copies of the chro-  mosome must then segregate to opposite sides of the  cell before cell division can proceed. Division occurs in  different ways for Gram-positive and Gram-negative  bacteria. Gram-negative cells do not have a rigid cell wall  and divide by a process of constriction followed by  membrane fusion. Gram-positive cells, on the other  hand, having a rigid cell wall, must develop a cross-wall  (see Figure 2.1a) that divides the cell into two equal  halves. Constriction and cross-wall formation takes  approximately 15 minutes to complete. DNA replication,  chromosome segregation (C-phase) and cell division (D-  phase) occur sequentially in slow-growing cells with gen-  eration times of greater than 1 hour and are the final  events of the bacterial cell cycle. Cells are able to replicate  faster than once every hour by initiating several rounds  of DNA replication at a time. Thus partially replicated  chromosomes become segregated into the newly formed  daughter cells. In this fashion it is possible for some  organisms growing under their optimal conditions to  divide every 15-20 minutes. Rod-shaped organisms  maintain their diameter during the cell cycle and increase  their mass and volume by a process of elongation. When  the length of the cell has approximately doubled then the  division/constriction occurs centrally. Coccal forms  increase in size by radial expansion, with the division  plane going towards the geometric centre. In some genera  the successive division planes are always parallel. Under  such circumstances the cells appear to form chains (i.e.  streptococci). In staphylococci successive division planes  are randomized, giving dividing clusters of cells the  appearance of a bunch of grapes. Certain genera, e.g.  Sarcina, rotate successive division planes by 90 ° to form  tetrads and cubical octets. The appearance of dividing  cells under the microscope can therefore be a useful  initial guide to identification.   When placed in favourable conditions populations of  bacteria can increase at remarkable rates, given that each  division gives rise to two identical daughter cells, then    each has the potential to divide again. Thus cell numbers  will increase exponentially as a function of time. For a  microorganism growing with a generation time of 20  minutes, one cell will have divided three times within an  hour to give a total of eight cells. After 20 hours of con-  tinued division at this rate then the accumulated mass of  bacterial cells would be approximately 70kg (the weight  of an average man). Ten hours later the mass would be  equivalent to the combined body weight of the entire  population of the UK. Clearly this does not happen in  nature; rather, the supply of nutrients becomes exhausted  and the organisms grow considerably more slowly, if  at all.   The time interval between one cell division and the  next is called the generation time. When considering a  growing culture containing thousands of cells, a mean  generation time is usually calculated. As one cell doubles  to become two cells, which then multiply to become four  cells and so on, the number of bacteria n in any genera-  tion can be expressed as:    n= 1x2 = 2'  n=lx2x2 = 2 2  n=lx2x2x2 = 2 3  n = 1 x 2 X = 2 X    For an initial population of N 0 cells, as distinct from  one cell, at the xth generation the cell population will be:   where N is the final cell number, N 0 the initial cell number  and x the number of generations. To express this equation  in terms of x, then:   x = (log N - log N 0 )/ log 2 = (logN — logN 0 )/0.30 1  = 3.3(logN-logN 0 )   The actual generation time is calculated by dividing x  into t where t represents the hours or minutes of expo-  nential growth.   If microorganisms are immobilized on a solid surface  from which they can derive nutrients and remain  moist, cell division will cause the daughter cells to form  a localized colony. In spite of the small size of the indi-  vidual organisms, colonies are easily visible to the  naked eye. Indeed, microbial growth can often be seen on    the tonsils of an infected individual or as colonies on  discarded or badly stored foods. In the laboratory solidi-  fied growth media are deployed to separate different  types of bacteria and also as an aid to enumerating  viable cell numbers. These media comprise a nutrient  soup (broth) that has been solidified by the addition of  agar (see Chapter 2). Agar melts and dissolves in  boiling water but will not resolidify until the temperature  is below 45 °C. Agar media are used in the laboratory  either poured as a thin layer into a covered dish (Petri  dish or plate) or contained within a small, capped  bottle (slant). If suspensions of different species of bac-  teria are spread on to the surface of a nutrient agar plate  then each individual cell will produce a single visible  colony. These may be counted to obtain an estimate of  the original number of cells. Different species will  produce colonies of slightly different appearance, ena-  bling judgements to be made as to the population diver-  sity. The colour, size, shape and texture of colonies of  different species of bacteria vary considerably and  form a useful diagnostic aid to identification. Transfer of  single colonies from the plate to a slant enables pure  cultures of each organism to be maintained, cultured and  identified.   When growing on a solid surface the size of the resultant  colony is governed by the local availability of nutrients.  These must diffuse through the colony. Eventually growth  ceases when the rate of consumption of nutrients exceeds  the rate of supply. When grown in liquids the bacteria,  being of colloidal dimensions and sometimes highly  motile, are dispersed evenly through the fluid. Nutrients  are therefore equally available to all cells. When consider-  ing growth of bacterial populations in liquids it is neces-  sary to consider whether the environment is closed or  open with respect to the acquisition of fresh nutrient.  Closed systems are typified by batch culture in closed  glass flasks. In these waste products of metabolism are  retained and all the available nutrients are present at the  beginning of growth. Open systems, on the other hand,  have a continual supply of fresh nutrients and removal of  waste products.   Figure 3.8 shows the pattern of population growth  obtained when a small sample of bacteria is placed within  a suitable liquid growth medium held in a glass vessel.  As the increase in cell numbers is exponential (1, 2, 4, 8,  16, etc.) then during active growth a logarithmic plot of    cell number against time gives a straight line (B). This  period is often referred to as the logarithmic growth phase,  during which the generation or doubling time may be  calculated from the slope of the line. However, the  exponential phase is preceded by a lag period (A), during  which time the inoculum adapts its physiology to that  required for growth on the available nutrients. As growth  proceeds nutrients are consumed and waste materials  accumulate. This has the effect of reducing the rate of  growth ( late logarithmic phase) towards an eventual  halt (stationary phase, C). Starvation during the station-  ary phase will eventually lead to the death of some of  the cells and adaptation to a dormant state in others  (decline phase, D). Patterns of growth such as this  occur within inadequately preserved pharmaceutical  products, in water storage tanks and in industrial  fermentations.   Except under circumstances of feast-famine, growth of  bacteria in association with humans and in our environ-  ment is subject to a gradual but continuous provision of  nutrients and a dilution of waste products. Under such  circumstances the rate of growth of bacteria is governed  by the rate of supply of nutrients and the population  size. Accordingly, bacteria in our gastrointestinal tracts  receive a more or less continuous supply of food and  excess bacteria are voided with the faeces (indeed,  bacteria make up >90% of the dry mass of faeces). In  many situations the bacteria become immobilized, as a  biofilm, upon a surface and extract nutrients from the  bulk fluid phase.   Figure 3.8 Typical bacterial growth curve in closed batch  liquid culture: (A) lag or adaptive phase; (B) logarithmic or  exponential phase; (C) stationary phase; (D) decline phase.   For many years it was thought that bacteria, dividing by  binary fission, had no opportunity for the exchange of  genetic material and could only adapt and evolve through  mutation of genes. This is not only untrue but masks  the profound ability of bacteria to exchange and share  DNA across diverse genera. This is of particular signifi-  cance because it enables bacterial populations to adapt  rapidly to changes in their environment, whether this is  related to the appearance of a novel food or to the deploy-  ment of antibacterial chemicals and antibiotics. Three  major processes of genetic exchange can be identified in  bacteria — transformation, transduction and conjugation.  Further details of these processes are given in Chapter 13  dealing with the development and spread of antibiotic  resistance.   In 1928 Griffith noticed that a culture of Streptococcus  pneumoniae that had mutated to become deficient in  capsule production could be restored to its normal cap-  sulate form by incubation with a cell-free filtrate taken  from a culture of the normal strain. While this discovery  preceded the discovery of DNA as the genetic library and  was only poorly understood at the time, it demonstrated  the ability of certain types of bacteria to absorb small  pieces of naked DNA from the environment that may  recombine into the recipient chromosome. The process  has become known as transformation and is likely to  occur naturally in situations such as septic abscesses and  in biofilms where high cell densities are associated with  death and lysis of significant portions of the population.  Transformation is also exploited in molecular biology as  a means of transferring genes between different types of  bacteria.   Viruses are discussed more fully elsewhere (Chapter 5);  however, there is a group of viruses, called bacteriophages,  which have bacterial cells as their hosts. These bacteri-  ophages inject viral DNA into the host cell. This viral  DNA is then replicated and transcribed at the expense of  the host and assembled into new viral particles. Under  normal circumstances the host cell becomes lysed in  order to release the viral progeny, but in exceptional cir-  cumstances, rather than enter a replication cycle the viral  DNA becomes incorporated, by recombination, into the  chromosome of the bacterium. This is known as a tem-  perate phage. The viral DNA thus forms part of the bacte-  rial chromosome and will be copied to all daughter cells.    Temperate phage will become active once again at a low  frequency and phasing between temperate and lytic  forms ensures the long-term survival of the virus.  Occasionally during this transition back to the lytic form  the excision of the viral DNA from the bacterial chromo-  some is inaccurate. The resultant virus may then either  be defective, if viral DNA has been lost, or it may carry  additional DNA of bacterial origin. Subsequent temper-  ate infections caused by the latter virions will result in  this bacterial DNA having moved between cells: a process  of gene movement known as transduction. As the host  range of some bacteriophages is broad then such proc-  esses can move DNA between diverse species.   Conjugation is thought to have evolved through trans-  duction, and relates to the generation of defective viral  DNA. This can be transcribed to produce singular viral  elements, which cannot assemble or lyse the host cell.  Such DNA strands are known as plasmids. They are cir-  cular and can either be integrated into the main chromo-  some, in which case they are replicated along with the  chromosome and passed to daughter cells, or they are  separate from it and can replicate independently. The  simplest form of plasmid is the F-factor (fertility factor);  this can be transcribed at the cell membrane to generate  an F-pilus within the cell envelope and cells containing  an F-factor are designated F + . The F-pilus is a hollow  appendage that is capable of transferring DNA from one  cell to another, through a process that is very similar to  the injection of viral DNA into a cell during infection. In  its simplest form an unassociated F-factor will simply  transfer a copy to a recipient cell, and such a transfer  process is known as conjugation. Integration with, and  dissociation of, the F-factor with the chromosome occurs  randomly. When it is in the integrated form, designated  Flfr (high frequency of recombination), then not only  can a copy of the plasmid DNA be transferred across  the F-pilus but so also can a partial or complete copy of  the donor chromosome. Subsequent recombination  events incorporate the new DNA into the recipient  chromosome.   Just as the excision of temperate viral DNA from the  host chromosome could be inaccurate, and lead to addi-  tions and deletions from the sequence, so too can the  F-factor gather chromosomal DNA as the host cells  change from Flfr to F + . In such instances the plasmid that  is formed will transfer not only itself but also this  additional DNA into recipient cells. This is particularly  significant because the unassociated plasmid can repli-    cate autonomously from the chromosome to achieve a  high copy number. It can also be transferred simultane-  ously to many recipient bacteria. If the transported  DNA encoded a mechanism of antibiotic resistance  (Chapter 13) it would not be difficult to imagine how  whole populations could rapidly acquire the resistance  characteristics.   The rate of growth of a microbial population depends on  the nature and availability of water and nutrients, tem-  perature, pH, the partial pressure of oxygen and solute  concentrations. In many laboratory experiments the  microorganisms are provided with an excess of complex  organic nutrients and are maintained at optimal pH and  temperature. This enables growth to be very rapid and  the results visualized within a relatively short time period.  Such idealized conditions rarely exist in nature, where  microorganisms not only compete with one another for  nutrients but also grow under suboptimal conditions.  Particular groups of organisms are adapted to survive  under particular conditions; thus Gram-negative bacteria  tend to be aquatic whereas Gram-positive bacteria tend  to prefer more arid conditions such as the skin. The next  two sections of this chapter will consider separately the  physicochemical factors that affect growth and survival  of bacteria, and the availability and nature of the available  nutrients.   Earlier in this chapter various classes of bacteria (ther-  mophile, mesophile, etc.) were described according to the  range of temperatures under which they could grow. The  majority of bacteria that have medical or pharmaceutical  significance are mesophiles and have optimal growth  temperatures between ambient and body temperature  (37 °C). Individual species of bacteria also have a range  of temperatures under which they can actively grow and  multiply (permissive temperatures). For every organism  there is a minimum temperature below which no growth  occurs, an optimum temperature at which growth is most  rapid and a maximum temperature above which growth  is not possible (Figure 3.9). As temperatures rise, chemi-  cal and enzymic reactions within the cell proceed more   rapidly, and growth becomes faster until an optimal rate  is achieved. Beyond this temperature certain proteins  may become irreversibly damaged through thermal lysis,  resulting in a rapid loss of cell viability.   The optimum temperature for growth is much nearer  the maximum value than the minimum, and the range of  the permissive temperatures can be quite narrow (3-4 °C)  for obligate pathogens yet broad ( 10-20 °C) for environ-  mental isolates, reflecting the range of temperatures that  they are likely to encounter in their specialized niches. If  the temperature exceeds the permissive range then pro-  vided that lethal temperatures are not achieved (c.60°C  for most Gram-negative mesophiles) the organisms will  survive but not grow. Temperatures of 105 °C and above  are rapidly lethal and can be deployed to sterilize materi-  als and products. Generally bacteria are able to survive  temperatures beneath the permissive range provided that  they are gradually acclimatized to them.   As for temperature, each individual microorganism has  an optimal pH for growth and a range about that  optimum where growth can occur albeit at a slower pace.  Unlike the response to temperature, pH effects on growth  are bell-shaped (Figure 3.10), and extremes of pH can be  lethal. Generally those microorganisms that have medical  or pharmaceutical significance have pH growth optima  of between 7.4 and 7.6 but may grow suboptimally at pH  values of 5-8.5. Thus growth of lactobacilli within the  vaginal vault reduces the pH to approximately 5.5 and  prevents the growth of many opportunist pathogens.  Accordingly, the pH of a pharmaceutical preparation may  dictate the range of microorganisms that could poten-  tially cause its spoilage.   Water is essential for the growth of all known forms of  life. Gram-negative bacteria are particularly adapted to  an existence in, and are able to extract trace nutrients  from, the most dilute environments. This adaptation has  its limitations because the Gram-negative cell envelope  cannot withstand the high internal osmotic pressures  associated with rapid rehydration after desiccation and  the organisms are unable to grow in the presence of high  concentrations of solute. The availability of water is  reflected in the water activity of a material or liquid.  Water activity (A*) is defined as the vapour pressure of  water in the space above the material relative to the  vapour pressure above pure water at the same tempera-  ture and pressure. Pure water by definition has an A„ of  1.00. Pharmaceutical creams might have A w values  of 0.8-0.98, whereas strawberry jam might have an A w of  c.0.7. Generally Gram-negative bacteria cannot grow if  the A„ is below 0.97, whereas Gram-positive bacteria can  grow in materials with A w of 0.8-0.98 and can survive  rehydration after periods of desiccation, hence their  dominance in the soil. Yeasts and moulds can grow at low  A w values, hence their appearance on moist bathroom  walls and on the surface of jam. The water activity of a  pharmaceutical product can markedly affect its vulnera-  bility to spoilage contaminants (see Chapter 17).   For many aerobic microorganisms oxygen acts as the ter-  minal electron acceptor in respiration and is essential for  growth. Alternative terminal electron acceptors are  organic molecules whose reduction leads to the genera-  tion of organic acids such as lactic acid. They can some-  times be utilized under conditions of low oxygen or where    carbon substrate is in excess (fermentation), and highly  specialized groups of microorganisms can utilize inor-  ganic materials such as iron as electron acceptors (e.g.  iron-sulphur bacteria). Different groups of organisms  therefore vary in their dependenceon oxygen. Paradoxically,  there are many bacteria for which oxygen is highly toxic  ( obligate anaerobes), so the presence or absence of oxygen  within a nutrient environment can profoundly affect both  the rate and nature of the microbial growth obtained.  Strongly oxygen-dependent bacteria will tend to grow as  a thin pellicle on the surfaces of liquid media where oxygen  is most available. Special media and anaerobic chambers  are required to grow obligate anaerobes within the labora-  tory, yet such organisms persist and actively grow within  the general environment. This is because the close proxim-  ity of strongly aerobic cells and anaerobes will create an  anoxic microenvironment in which the anaerobe can  flourish. This is particularly the case for the mouth and  gastrointestinal tract where obligate anaerobes such as  Bacteroides and Fusobacter can be found in association  with strongly aerobic streptococci.   The inability of oxygen to diffuse adequately into a  liquid culture is often the factor that causes an onset of  stationary phase, so culture density is limited by oxygen  demand. The cell density at stationary phase can often be  increased, therefore, by shaking the flask or providing  baffles. Diffusion of oxygen may also be a factor limiting  the size of bacterial colonies formed on an agar surface.   Bacteria vary considerably in their requirements for  nutrients and in their ability to synthesize for themselves  various vitamins and growth factors. Clearly the major  elemental requirements for growth will match closely the  elemental composition of the bacteria themselves. In this  fashion there is a need for the provision of carbon, nitro-  gen, water, phosphorus, potassium and sulphur with a  minor requirement for trace elements such as magne-  sium, calcium, iron, etc. The most independent classes of  bacteria are able to derive much of their nutrition from  simple inorganic forms of these elements. These organ-  isms are called chemolithotrophs and can even utilize  atmospheric carbon dioxide and nitrogen as sources of  carbon and nitrogen. Indeed, such bacteria are, in addi-  tion to the green plants and algae, a major source of  organic molecules and so they are more beneficial than  problematic to humans. The majority of bacteria require  a fixed carbon source, usually in the form of a sugar, but  this may also be obtained from complex organic mole-  cules such as benzene, paraffin waxes and proteins.    Nitrogen can generally be obtained from ammonium  ions but is also available by deamination of amino acids,  which can thus provide both carbon and nitrogen sources  simultaneously. Many classes of bacteria are auxotrophic  and can grow on simple sugars together with ammonium  ions, a source of potassium and trace elements. Such  bacteria can synthesize for themselves all the amino acids  and ancillary factors required for growth and division.  These bacteria, e.g. pseudomonads and Achromobacter  species, are generally free-living environmental strains  but they can sometimes cause infections in immunocom-  promised people. In the laboratory they can be grown in  simple salts media with few, if any, complex supplements.  The rate of growth of such organisms depends not only  on temperature and pH but also on the nature of the  carbon and nitrogen sources. Thus, a faster rate of growth  is often obtained when glucose or succinate is the carbon  source rather than lactose or glycerol, and when amino  acids are provided as sources of nitrogen rather than  ammonium salts. If faced with a choice of carbon and  nitrogen sources then the bacteria will adapt their physi-  ology to the preferred substrate and only when this is  depleted will they turn their attention to the less preferred  substrate. Growth in liquid cultures with dual provision  of substrate such as this is often characterized by a second  lag phase during the logarithmic growth period while  this adaptation takes place. This is called diauxic growth  (Figure 3.11).   As the association between bacteria and higher life  forms becomes closer then more and more preformed  biosynthetic building blocks become available without  the need to synthesize them from their basic elements.  Thus a pathogenic organism growing in soft tissues   Figure 3.11 Diauxic growth on a mixture of glucose and  lactose.    will have available to it glucose and metal ions from the  blood and a whole plethora of amino acids, bases, vita-  mins, etc. from lysed tissue cells. While most bacteria will  utilize these when they are available, a number of bacteria  that have become specialized pathogens have lost their  ability to synthesize many of these chemicals and so  cannot grow in situations where the chemicals are not  provided in the medium. Consequently many pathogens  require complex growth media if they are to be cultured  in vitro.   All that has been discussed earlier about the physico-  chemical and nutritional constraints on bacterial growth  has been based on laboratory studies. By definition, the  only bacteria that we can describe in this way are those  that can be cultured artificially. It cannot be overstated  that a majority of bacterial species and genera cannot be  cultured in the laboratory. In the past the presence of  such non-culturable bacteria has been attributed to  moribund cells. With the advent of modern molecular  tools, however, it has now been realized that these organ-  isms are viable. By amplifying their DNA and sequence  mapping, the genetic relationship of such bacteria to  the culturable ones can be demonstrated and whole  new families of hitherto unrecognized bacteria are being  identified. It is possible that in the future many disease  states currently thought to have no microbiological  involvement could be identified as being of bacterial  origin. A recent example of this has been the association  of Helicobacter pylori with gastric ulcers and gastric  cancers.   8 Detection, identification and  characterization of organisms of  pharmaceutical and medical significance   There are many situations in which microorganisms  must not only be detected and enumerated but where  they must also be identified either to make a specific  diagnosis of infection or to ensure the absence of speci-  fied bacteria from certain types of product. In such cir-  cumstances various cultural approaches are available that  deploy enrichment and selection media. Once a microor-  ganism has been isolated in pure culture, usually from a  single colony grown on an agar plate, then further char-  acterization may be made by the application of micros-  copy together with some relatively simple biochemical  tests. Over the last 20 years the biochemical characteriza-  tion of individual organisms has become simplified by  the introduction of rapid identification systems. In recent    years molecular approaches have enabled identification  of organisms without the need to culture them.   Conventional approaches to microbiological examina-  tion of specimens require that they be cultured to assess  the total numbers of specific groups of microorganisms  or to determine the presence or absence of particular  named species. The majority of samples taken for exami-  nation contain mixtures of different species, so simple  plating onto an agar surface may fail to detect an organ-  ism that is present at <2% of the total viable population.  Various enrichment culture techniques may therefore be  deployed to detect trace numbers of particular patho-  gens, prior to confirmatory identification.   The simplest way in which to enumerate the microorgan-  isms that contaminate an object or liquid sample is to  dilute that sample to varying degrees and inoculate the  surface of a predried nutrient agar with known volumes  of those dilutions (see Chapter 2). Individual viable bac-  teria that are able to grow on the nutrients provided and  under the conditions of incubation will produce visible  colonies that can be counted and the numbers related  back to the original sample. Such counting procedures  are often lengthy and tedious; the number of colonies  formed might not relate to the viable number of cells, as  clumps of cells will only produce a single colony and they  will only detect a particular subset of the viable bacteria  present in the sample that can grow under the chosen  conditions. Accordingly a variety of different media and  cultural conditions are deployed to enumerate different  categories of organism.   A number of techniques are currently being developed  in order speed up the enumeration process, although  some of these rapid enumeration techniques indirectly  measure the most probable number of viable cells.   Enumeration media will only ever culture a subset of cells  towards which the medium and incubation conditions  are directed. Thus, simple salts media with relatively  simple sugars as carbon sources and trace levels of amino  acids are often used to enumerate bacteria associated  with water (e.g. R2A medium). Such plates may be incu-  bated under aerobic or anaerobic conditions at a range  of temperatures. Different temperatures will select for  different subsets of cells, therefore any description of a  viable bacterial count must specify the incubation condi-    tions. In medical microbiology temperatures akin to the  human body are often deployed because only those bac-  teria able to grow at such temperatures are likely to cause  infection. However, psychrophilic Gram-negative bacte-  ria (growing in water at 10 °C) can be a major source of  bacterial pyrogen, so a variety of incubation tempera-  tures are often used in monitoring pharmaceutical waters  and products. Highly nutritious media, e.g. blood agar,  are also used as enumeration media. This is particularly  the case when looking for microorganisms such as  staphylococci that are usually found in association  with animals and humans. Such agar plates may be  deliberately exposed to air (settle plates) and the number  of colonies formed related to the bacteria content of a  room. In the pharmaceutical industry microbiological  monitoring will generally report the total aerobic  count and, less commonly, the total anaerobic counts  obtained on a moderately rich medium such as tryptone  soya agar. Sometimes inhibitors of bacterial growth (e.g.  Rose Bengal) can be added to a medium in order to select  for moulds.   The detection and quantification of components of bac-  terial cells is considerably faster than those approaches  requiring the growth of colonies, and estimates of total  viable cell number can thereby be obtained within  minutes rather than hours and days.   Some of the rapid methods that have been used for  bacteria and other microorganisms, e.g. bioluminescence,  epifluorescence and impedance techniques, have been  described in Chapter 2, but there are other rapid methods  that have found more limited application; these will be  considered here. In the examination of pharmaceutical  waters and aqueous pharmaceutical products electronic  particle counters, e.g. Coulter counters, can be used to  determine bacterial concentration, although these instru-  ments do not discriminate between living and dead cells.  Similar counters are available that are able to analyse  particles found in air. Other rapid techniques aim to  detect microbial growth rather than to visualize indi-  vidual cells and colonies. As bacteria grow in liquid  culture they not only alter the conductivity of the culture  (see Chapter 2), they also generate small quantities of  heat. The time taken to detect this heat can be directly  related to the numbers of viable cells present by means  of microcalorimeters. Once again this is a considerable  improvement over conventional culture, but unlike par-  ticle counting and bioluminescence can only detect those  organisms that are able to grow in the chosen medium.    None of the rapid techniques are able to isolate indi-  vidual organisms. They do not therefore aid in the char-  acterization or identification of the contaminants.   Enrichment cultures are intended to increase the domi-  nance of a numerically minor component of a mixed  culture such that it can be readily detected on an agar  plate. Enrichment media are always liquid and are  intended to provide conditions that are favourable for the  growth of the desired organism and unfavourable for  the growth of other likely isolates. This can be achieved  either through manipulation of the pH and tonicity of  the medium or by the inclusion of chemicals that inhibit  the growth of unwanted species. Thus, MacConkey  broth contains bile salts that will inhibit the growth of  non-enteric bacteria and may be used to enrich for  Enterobacteriaceae. Several serial passages through  enrichment broths may be made, and after enrichment it  is not possible to relate the numbers of organisms  detected back to that in the original sample.   Selective media are solidified enrichment broths, so again  they are intended to suppress the growth of particular  groups of bacteria and to allow the growth of others. The  methods of creating this situation are the same as for  enrichment broths. Thus mannitol salts agar will favour  the growth of micrococci and staphylococci, and cetrim-  ide agar will favour the growth of pseudomonads. The  use of selective media is an adjunct to characterizing the  nature of contaminants. Counts of colonies obtained on  selective solid media are often documented as presump-  tive counts, so for example, colonies formed on a  MacConkey agar (containing bile salts) might be cited as  a presumptive coliform count.   Identification media contain nutrients and reagents that  indicate, usually through some form of colour formation,  the presence of particular organisms. This enables them  to be easily detected against a background of other  species. In this fashion inclusion of lactose sugar and  a pH indicator into MacConkey agar facilitates the  identification of colonies of bacteria that can ferment  lactose. Fermentation leads to a reduction in pH within  these colonies and can be detected by an acid shift in the  pH indicator, usually to red. Lactose-fermenting colif-  orms ( Escherichia spp., Klebsiella spp.) can therefore be  easily distinguished from non-fermentative coliforms    ( Salmonella spp., Shigella spp.). Similarly, the inclusion of  egg-yolk lecithin into an agar gives it a cloudy appearance  that clears around colonies of organisms that produce  lecithinase (a virulence factor in staphylococci). While  there are numerous types of selective and diagnostic  media available, they can only be used as a guide to iden-  tification, but microscopy and biochemical or genetic  characterization are much more definitive.   Observation of stained and wet preparations of clinical  specimens (blood, pus, sputum) and isolated pure cul-  tures of bacteria from the manufacturing environment  provides rapid and essential information to guide further  identification. The application of simple stains such as  the Gram stain can divide the various genera of bacteria  into two convenient broad groups. The size and shapes  of individual cells and their arrangement into clusters,  chains and tetrads will also guide identification, as will  specific stains for the presence of endospores, capsules,  flagella and inclusion bodies. Examination of wet prepa-  rations can give an indication as to the motility status of  the isolate, and these procedures all represent an impor-  tant first stage in the identification process.   The differing ability of bacteria to ferment sugars, glyco-  sides and polyhydric alcohols is widely used to differenti-  ate the Enterobacteriaceae and in diagnostic bacteriology  generally. Fermentation can be indicated by pH changes  in the medium with or without gas production visualized  by the collection of bubbles in inverted tubes. More spe-  cialized media examine the ability of certain strains to  oxidize or reduce particular substrates. There are many  hundreds of individual biochemical tests available that  each separately seek the presence of a particular enzyme  or physiological activity. Taxonomic studies have led to  the recognition that certain of these tests in combination  characterize particular species of bacteria. Various  manuals such as Bergey’s Manual (Holt, 1994) and Cowan  and Steel’s Manual for the Identification of Medically  Important Bacteria (Barrow & Feltham, 2004) provide a  logical and sequential framework for the conduct of such  tests. Identification of particular species and genera by  such processes is time-consuming, expensive and may  require numerous media and reagents.   This process has become simplified in recent years by  the development of rapid identification methods and    kits. The latter often use multiwell microtitration plates  that can be inoculated in a single operation either with  an inoculated wire or with a suspension of a pure culture.  Each individual well contains the medium and reagents  for the conduct of a single biochemical test. Identification  kits vary in their complexity and also in the precision of  the identification made. Simple kits may perform only  8-15 tests, more complex ones are capable of performing  96 simultaneous biochemical evaluations. Scoring of each  test and entry into a computer database then allows the  pattern of test results to be compared with a large panel  of organisms and a probability of identity calculated. As  different sets of tests will be required for different classes  of bacteria, guidance as to the initial choice of kit is given  on the basis of the Gram stain reaction, and the results  of oxidase and catalase tests performed directly on iso-  lated colonies. In large diagnostic laboratories and in  quality assurance laboratories automated systems are  deployed that can inoculate, incubate and analyse hun-  dreds of individual samples at a time.   The need to identify microorganisms rapidly has led to  the development of a number of molecular identification  and characterization tools. These have not yet become  routinely adopted in the analytical or diagnostic labora-  tory but probably will be in the future. One such tech-  nique (denaturing gradient gel electrophoresis; DGGE)  isolates and amplifies 16S ribosomal DNA and, following  sequencing of the bases, compares this with known  sequences held in a reference library. This approach  enables phylogenetic relationships to be derived even for  those bacteria that have not previously been identified.  Other systems examine the patterns of key constituents  of the cells such as fatty acids and assign identities based  on similarity matches to known reference cultures.   Molecular approaches can be of especially useful when  attempting to detect a particular species. Thus, gene  probes carrying fluorescent dyes can be used in hybridi-  zation procedures with the collected clinical material.  Examination under the fluorescent microscope will show  the targeted organism as fluorescent against a back-  ground of non-fluorescent organisms.   Microorganisms of medical and pharmaceutical rele-  vance can be broadly classified into those organisms that    are harmful or problematic, and those that can be used production, are based on microorganisms, and selected   to our advantage. Some microorganisms, depending on species can be used to test disinfectant efficacy and to   cause some of the most important diseases of humans respectively, list examples of some of the more pharma-   and animals and they can also be found as major con- ceutically relevant beneficial and problematic microor-   Used to validate and monitor dry heat  and ethylene oxide sterilization processes   General environmental contaminant  Quintessential opportunist pathogen  High resistance to antibiotics and biocides  Biofilm-former   Barrow, G. 8c Feltham, R.K.A. (eds) (2004) Cowan and Steel’s  Manual for the Identification of Medical Bacteria , 3rd edn.  Cambridge University Press, Cambridge.   Costerton, J.W., Lewandowski, Z., deBeer, D., Caldwell, D.,  Korber, D. 8c James, G. (1994) Biofilms, the customised  microniche. / Bacteriol , 176 , 2137-2142.   Gould, G.W. (1985) Modification of resistance and dormancy.  In: Fundamental and Applied Aspects of Bacterial Spores (eds  G.J. Dring, D.J. Ellar 8c G.W. Gould), pp. 371-382. Academic  Press, London.   Holt, J.G. (ed.) (1994) Bergey’s Manual of Determinative  Bacteriology , 9th edn. Williams 8c Wilkins, Baltimore, MD.    Madigan, M.T., Martinko, J.M. 8c Parker, J. (2000) Brock Biology  of Microorganisms , 9th edn. Prentice-Hall, New Jersey.   Roitt, I.M. (2006) Essential Immunology , 11th edn. Blackwell  Scientific, Oxford.   Bauman, R.W. (2009). Microbiology with Diseases by Body  System , 2nd edn. Pearson Benjamin Cummings, San Francisco,  CA.   Sutherland, I.W. (1985) Biosynthesis and composition of Gram-  negative bacterial extracellular and wall polysaccharides.  Anna Rev Microbiol, 10 , 243-270.   Yeast, such as brewers’ yeast, and moulds, such as  Penicillium chrysogenum which produces the antibiotic  penicillin, are classified as fungi. Yeast cells tend to grow  as single cells which reproduce asexually in a process  known as budding, although a minority of species (e.g.  Schizo saccharomyces pombe) reproduce by fission. Many  yeast species are capable of sexual reproduction and the  formation of spores. In contrast, moulds grow as masses  of overlapping and interlinking hyphal filaments and  reproduce by producing masses of spores in a variety of  structures. This division between yeast and moulds based  on growth morphology is not clear-cut since some yeast  can produce hyphae under specific conditions (e.g.  Candida albicans) while many normally filamentous  fungi possess a yeast-like phase at some point in their life  cycle. Fungi are eukaryotic organisms, i.e. their cells  possess a nuclear membrane, consequently there are  many similarities between the biochemistry of fungal  cells and vertebrate (human) cells. Fungi are widely dis-  tributed in nature, occurring as part of the normal flora    on the body of warm-blooded animals, as decomposers  of organic matter and as animal and plant pathogens.  Medically, fungi are an extremely important group of  microbes, being responsible for a number of potentially  fatal diseases in humans (Table 4.1), but a significant  number of fungi are of great benefit to humanity in terms  of the production of alcoholic beverages, bread, enzymes,  antibiotics and recombinant proteins (Table 4.2). Fungi  have also been utilized for a range of molecular biological  applications.   Fungal taxonomy is extremely difficult and there is  much discussion as to the number and inter-relatedness  of fungal classes. Using one classification system, the  kingdom Fungi can be subdivided into six classes. The  class Oomycetes contains the mildews and water moulds,  the class Ascomycetes contains the mildews, some moulds  and most yeast species (including Saccharomyces cerevi-  siae) i, the class Basidiomycetes contains the mushrooms  and bracket fungi, the class Teliomycetes contains the rust  fungi (plant pathogens), the class Ustomycetes contains  the smuts (plant pathogens) and the class Deuteromycetes  contains species such as Aspergillus, Fusarium and  Penicillium (see Figure 4.1). In contrast, phylogenetic    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.    analysis has revealed that there are four distinct phyla  within the fungal kingdom; these are the Chytridiomycota,  Zygomycota, Ascomycota and Basidiomycota.   The typical yeast cell is oval in shape and is surrounded  by a rigid cell wall which contains a number of structural  polysaccharides and may account for up to 25% of the  dry weight of the cell wall (see Figure 4.2). Glucan  accounts for 50-60%, mannan for 15-23% and chitin for  1-9% of the dry weight of the wall, respectively, with  protein and lipids also present in smaller amounts. The  thickness of the cell wall may vary during the life of the  cell but the average thickness in the yeast C. albicans  varies from 100 to 300 nm. Glucan, the main structural  component of the fungal cell wall, is a branched polymer  of glucose which exists in three forms in the cell: (3-1,6-  glucan, (3-1,3-glucan and P-l,3,-(3-l,6-complexed with  chitin. Mannan is a polymer of the sugar mannose and is  found in the outer layers of the cell wall. The third prin-  cipal structural component, chitin, is concentrated in bud  scars that are areas of the cell from which a bud has  detached. Proteins and lipids are also present in the cell    wall and under some conditions may represent up to 30%  of the cell wall contents. Mannoproteins form a fibrillar  layer that radiates from an internal skeletal layer that is  formed by the polysaccharide component of the cell wall.  The innermost layer is rich in glucan and chitin which  provides rigidity to the wall and is important in regulat-  ing cell division.   Enzymatic or mechanical removal of the cell wall  leaves an osmotically fragile protoplast which will burst  if not maintained in an osmotically stabilized environ-  ment. Incubation of protoplasts in an osmotically  stabilized agar growth medium will allow the resynthesis  of the wall and the resumption of normal cellular func-  tions. The ability to generate fungal protoplasts opens  the possibility of fusing these under defined condi-  tions to generate strains with novel biotechnological  applications.   The periplasmic space is a thin region that lies directly  below the cell wall. It contains secreted proteins that do  not penetrate the cell wall and is the location for a  number of enzymes required for processing nutrients  prior to entry into the cell. The cell membrane or plas-  malemma is located directly below the periplasmic space  and is a phopholipid bilayer which contains phospholip-  ids, lipids, protein and sterols. The plasmalemma is    approximately lOnm thick and in addition to being com-  posed of phospholipids also contains globular proteins.  The dominant sterol in fungal cell membranes is ergos-  terol which is the target of the antifungal agent ampho-  tericin B. Sterols are important components of the  plasmalemma and represent regions of rigidity in the  fluidity provided by the phospholipid bilayer.    Most of the cell’s genome is concentrated in the  nucleus which is surrounded by a nuclear membrane  which contains pores to allow communication with the  rest of the cell (see Figure 4.2). The nucleus is a discrete  organelle and, in addition to being the repository of the  DNA, also contains proteins in the form of histones. Yeast  chromosomes vary in size from 0.2 to 6 Mb and the  number per yeast is also variable with S. cerevisiae having  as many as 16 while the fission yeast Sch. pombe has as  few as 3. In addition to the genetic material in the nucleus  the yeast cell often has extrachromosomal information in  the form of plasmids. For example, the 2 pm plasmid is  present in S. cerevisiae, although its function is unclear,  and there are killer plasmids in the yeast Kluyveromyces  lactis which encode a toxin.   Actively respiring fungal cells possess a distinct mito-  chondrion which has been described as the ‘powerhouse’  of the cell (Figure 4.2). The enzymes of the tricarboxylic  acid cycle (Krebs’ cycle) are located in the matrix of the  mitochondrion while electron transport and oxidative  phosphorylation occur in the mitochondrial inner mem-  brane. The outer membrane contains enzymes involved  in lipid biosynthesis. The mitochondrion is a semi-  independent organelle as it possesses its own DNA and is  capable of producing its own proteins on its own ribos-  omes which are referred to as mitoribosomes.   The fungal cell contains a vast number of ribosomes  which are usually present in the form of polysomes —  lines of ribosomes strung together by a strand of mRNA.  Ribosomes are the site of protein biosynthesis. The  system which mediates the export of proteins from the  cell involves a number of membranous compartments  including the Golgi apparatus, the endoplasmic reticu-  lum and the plasmalemma. In addition, the vacuole is  employed as a ‘storage space’ where nutrients, hydrolytic  enzymes or metabolic intermediates are retained until  required.   Fungi represent a significant group of pathogens capable  of causing a range of diseases in humans under the right  set of conditions. Although the majority of fungi appear  to be harmless to humans it is worth bearing in mind that  a normally non-pathogenic fungus can cause a clinically  relevant problem if the immune system is suppressed as  a result of therapy (e.g. for receipt of organ transplant)  or disease (e.g. cancer). In a case of profound immuno-   compromise a wide range of fungi can present as capable  of inducing disease.   The most common fungal pathogens of humans can  be divided into three broad classes: yeasts, moulds and  dermatophytes. The yeast C. albicans is the most fre-  quently encountered human fungal pathogen, being  responsible for a wide range of superficial and systemic  infections. The superficial infections include oropharyn-  geal and genital conditions, the former occurs predomi-  nantly in HIV-positive individuals, geriatric patients and  premature infants and may arise when a weakened or  immature immune system is present. Genital candidosis  is very common and approximately 75% of women are  affected by vulvovaginal candidosis (WC) during their  life with a further 5-12% suffering from recurring bouts  of infection over a prolonged period of time.   The mould Aspergillus fumigatus is the dominant  fungal pulmonary pathogen of humans and generally  presents as a problem in those with pre-existing  lung disease or damage. In addition to pulmonary infec-  tion other sites may be affected including the brain,  kidneys and sinuses depending upon the level of immu-  nocompromise of the individual. Groups particularly  susceptible to colonization by Aspergillus species include  those with cavities due to tuberculosis, patients  affected with asthma or cystic fibrosis and those with  profound immunosuppression due to leukaemia (neu-  tropenia). Aspergillosis presents as a serious problem  in patients immunosuppressed in advance of organ  transplantation.   Dermatophyte is the term applied to a range of fungi  capable of colonizing the skin, nails or hair. The principal  dermatophytic fungi are Trichophyton, Microsporum and    Epidermophyton species. The most commonly encoun-  tered dermatophytic infections are athlete’s foot (infec-  tion of the foot) and ringworm (fungal infection of the  scalp or skin).   The choice and dose of an antifungal will depend upon  the nature of the condition, whether there are any under-  lying diseases, the health of the patient and whether anti-  fungal resistance has been identified as compromising  therapy. Part of the difficulty in designing effective anti-  fungal agents lies in the fact that fungi are eukaryotic  organisms so agents that will kill fungi may also have a  deleterious effect on human tissue. The ideal antifungal  drug should target a pathway or process specific to the  fungal cell, so reducing the possibility of damaging tissue  and inducing unwanted side effects.   Polyene antifungals are characterized by having a large  macrolide ring of carbon atoms closed by the formation  of an internal ester or lactone (Figure 4.3). In addition,  polyenes have a large number of hydroxyl groups distrib-  uted along the macrolide ring on alternate carbon  atoms. This combination of highly polar and non-polar  regions within the molecule renders the polyenes  amphiphatic, i.e. having hydrophobic and hydrophilic  regions in the one molecule, which assists solubility in  lipid membranes.   The principal polyenes are amphotericin B and nysta-  tin. Amphotericin B is produced by the bacterium    Figure 4.3 Structures of polyene  (amphotericin B) and azole  (itraconazole, fluconazole, miconazole  and ketoconazole) antifungal agents.    Streptomyces nodosus and its activity is due to the ability  to bind ergosterol, which is the dominant sterol in fungal  cell membranes, and consequently increases membrane  permeability by the formation of pores (Figure 4.4).  The action of amphotericin B seems to rely on the forma-  tion of pores through which intracellular contents  can escape from the cell. Amphotericin B can lead to  renal damage during prolonged antifungal therapy.  Amphotericin B is active against a broad range of fungal  pathogens and is considered the ‘gold standard’ against    which the activity of other antifungal agents is measured.  Because of its renal toxicity amphotericin B tends to be  reserved for severe cases of systemic fungal disease but  recent formulations in which the drug is encapsulated  within liposomes have been shown to have reduced  toxicity.   Nystatin was discovered in 1950 and exhibits the same  mode of action as amphotericin B but tends to have lower  solubility, which has restricted its use to the treatment of  topical infections. Although nystatin was effective for the    Figure 4.4 Modes of action of polyene  (amphotericin B) and azole antifungal  agents.    treatment of conditions such as oral and vaginal candi-  dosis, its use has been overtaken by the introduction of  azole antifungal drugs.   The first generation of azole antifungals revolutionized  the treatment of mucosal and invasive fungal infections,  and azoles are still the most widely used group of anti-  fungal agents. The azole derivatives are classified as imi-  dazoles or triazoles on the basis of whether they have two  or three nitrogen atoms in the five-membered azole ring  (Figure 4.3). The azoles in current clinical use are clot-  rimazole, miconazole, econazole and ketoconazole; newer    drugs such as itraconazole, fluconazole and voriconazole  have important applications in the treatment of systemic  infections. Azoles function by interfering with ergosterol  biosynthesis by binding to the cytochrome P-450 medi-  ated enzyme known as 14-a-demethylase (P-450 DM ). This  blocks the formation of ergosterol by preventing the  methylation of lanosterol (a precursor of ergosterol)  (Figure 4.4). This result in a reduction in the amount of  ergosterol in the fungal cell membrane which leads to  membrane instability, growth inhibition and cell death.  An additional consequence of the block in ergosterol bio-  synthesis is the build-up of toxic intermediates which can  prove fatal to the fungal cell.    Azoles exhibit a broad spectrum of activity in vitro,  being capable of inhibiting the growth of most Candida,  Cryptococcus and Aspergillus species, and dermatophytes.  Miconazole was the first azole used to treat systemic  fungal infections but demonstrated a number of toxic  side effects. Ketoconazole produced high serum concen-  trations upon oral administration but had poor activity  against aspergillosis. In addition, ketoconazole was  associated with a range of side effects which limited its  applicability. Newer triazoles such as fluconazole and  itraconazole have increased the options for dealing with  fungal infections. Fluconazole was introduced for clinical  use in 1990, is water soluble and shows good penetration  and deposition into the pulmonary tissues; it also reaches  high levels in the cerebrospinal fluid and the peritoneal  fluids. Fluconazole has proved highly effective in the  treatment of infections caused by C. albicans but shows  limited activity against Aspergillus. Itraconazole became  available for clinical use in the late 1980s and was the first  azole with proven efficacy against Aspergillus. Itraconazole  is effective in treating severe Aspergillus infections and  exhibits both fungicidal and fungistatic effects. Upon  ingestion itraconazole undergoes extensive hepatic  metabolism which yields up to 30 metabolites, a number  of which retain antifungal activity. Itraconazole is cur-    rently available as an intravenous formulation and is  widely used for the treatment of severe Aspergillus infec-  tion in this form. Fluconazole and itraconazole demon-  strate significantly reduced side effects compared to  ketoconazole. Novel azole drugs with increased ability to  inhibit the fungal 14-a demethylase are also becoming  available. These agents, which include voriconazole,  posaconazole and ravuconazole, have a wider spectrum  of activity than fluconazole and it has been suggested  that some of them show fungicidal effects to some species  (e.g. Aspergillus spp.). Voriconazole is one of the newest  second-generation triazole antifungal drugs and it shows  good activity against pulmonary aspergillosis and cere-  bral aspergillosis.   The echinocandins are a relatively new group of antifun-  gal drug and are semisynthetic lipopeptides comprising  a cyclic hexapeptide core connected to a lateral fatty acid  chain. Three compounds of this group are currently in  use: caspofungin, micafungin and anidulafungin (Figure  4.5). Unlike conventional antifungal therapy that targets  ergosterol or its synthesis, the echinocandins target the  synthesis of (3-1,3-glucan, the major polymer of the  fungal cell wall. The cell wall is essential to the fungus as    it provides physical protection, maintains osmotic stabil-  ity, regulates cell shape, acts as a scaffold for proteins,  mediates cell-cell communication and is the site of a  number of enzymatic reactions. Inhibition of (3-1,3-  glucan synthesis disrupts the structure of the growing cell  wall, resulting in osmotic instability and ballooning out  of the intracellular contents as a result of high osmotic  pressure, and ultimately ends in cell lysis.   Caspofungin has demonstrated in vitro antifungal  activity against various filamentous fungi and yeasts. It  has activity against different Aspergillus species including  A. fumigatus, A. flavus, A. niger and A. terreus but is con-  sidered to be more fungistatic than fungicidal. Conversely,  caspofungin is particularly fungicidal against a range of  Candida species including species that are resistant (e.g.  C. krusei) or isolates that are less susceptible (e.g. C. dub-  liniensis, C. glabrata) to azoles, or resistant to amphoter-  icin B.   The fungal cell wall represents an attractive target and  the echinocandins have proven to offer a safer alternative  to conventional antifungal therapies, (i.e. polyenes and  azoles). Echinocandins display an unique mode of action  which results in defects in cell wall morphology and  osmotic instability. As the cell wall is an essential compo-  nent for stability and ultimately virulence, the targeting  of the wall by echinocandins results in the efficient  destruction of the fungal cell. To avoid future problems  with resistance, researches need to clarify the precise inter-  actions of the echinocandins with the target enzyme, and  fully examine the cell’s complex response to this agent.   Flucytosine is a synthetic fluorinated pyrimidine which  has been used as an oral antifungal agent and demon-  strates good activity against a range of yeast species and  moderate levels of activity against Aspergillus species.  Two modes of action have been proposed for flucytosine.  One involves the disruption of protein synthesis by the  inhibition of DNA synthesis while the other possible  mode of action is the depletion in the amino acid pools  within the cell as a result of inhibition of protein synthe-  sis. In general yeast cells increase in size when exposed to  levels of flucytosine lower than the minimum inhibitory  concentration (MIC) and display alterations in their  surface morphology, both of which can be interpreted as  a result of an imbalance in the control of cellular growth.  Many fungi are inherently resistant to flucytosine or  develop resistance after a relatively short exposure and  resistance has been attributed to alteration in the enzyme  (cytosine deaminase) required to process flucytosine    once inside the cell or to an elevation in the amount of  pyrimidine synthesis. The problem of resistance has  limited the use of flucytosine so that now it is generally  used in combination with an antifungal agent (e.g.  amphotericin B) where it can potentiate the effect of the  second agent.   The yeast C. albicans is an opportunistic fungal pathogen  which can be present as a normal part of the body’s  microflora. C. albicans is also responsible for a range of  systemic infections and many of these can start as super-  ficial infections. Infection of the gastrointestinal tract is  seen in diabetics, cancer patients and people with AIDS;  the oesophagus is a common site of infection, rendering  swallowing difficult. The urinary tract can also be the site  of candidosis which may be due to renal infection, other  underlying disease(s) or cystitis. The presence of an ind-  welling urinary catheter may also predispose to Candida  infection.   A range of factors are capable of predisposing the indi-  vidual to superficial or systemic candidal infections.  Factors which impair the host’s immune system such as  the presence of underlying disease (AIDS, cancer, diabe-  tes), the use of immunosuppressive therapy during organ  transplantation and broad-spectrum antibiotic therapy  can leave the individual susceptible to candidosis. Other  factors that may predispose to Candida infection include  the presence of indwelling catheters and skin damage as  a result of burns or other trauma.   C. albicans displays a variety of virulence factors which  aid colonization and persistence in the body. One of the  most important of these factors is the ability to adhere to  host tissue using a variety of mechanisms (Figure 4.6A).  The importance of adherence may be illustrated by the  ability of C. albicans to adhere to various mucosal sur-  faces and to withstand forces that may lead to its removal  from the body, such as the bathing/washing action of  body fluids. A hierarchy exists among Candida species  indicating that the more common etiological agents of  candidosis (C. albicans and Candida tropicalis) are more  adherent to host tissue in vitro than relatively non-  pathogenic species such as C. krusei and Candida  guilliermondii.   C. albicans can exist in two morphologically distinct  forms: budding blastospores or hyphae (Figure 4.6B).   Figure 4.6 (A) Cells of C. albicans (arrows) adhering to a human buccal epithelial cell. (B) Germ tubes of C. albicans. Y, yeast cells;  H, hyphal cells.    Figure 4.7 Growth morphologies of  Candida albicans: A, budding  morphology; B, hyphal formation; C,  germ-tube formation leading to hyphal  formation; D, pseudohyphal formation.    The yeast can switch between each form and is usually  encountered in tissue samples in both morphological  forms (Figure 4.7). The hyphae are capable of thigmotro-  pism (contact sensing) which may aid in finding the line  of least resistance between and through layers of cells in  tissue. C. albicans produces a range of extracellular  enzymes which facilitate adherence and/or tissue pene-  tration. Phospholipases A, B, C and lysophospholipase  may function to damage host cell membranes and facili-  tate invasion. C. albicans produces a range of acid protei-  nases which have been shown to aid adherence and  invasion but which also play an important role in the  degradation of the immunoglobulins IgG and IgA. There  are now known to be 10 members of the secreted aspartic    proteinase (SAP ) family and these have a low pH optimum  which may assist in the colonization of the vagina.  Haemolysin production by C. albicans has also been  documented and seems to be important in allowing the  yeast access iron released from ruptured red blood cells.  An important immune evasion tactic of C. albicans is the  ability to bind to platelets via fibrinogen binding ligands  which results in the fungal cell being surrounded by a  cluster of platelets.   C. albicans is capable of giving rise to a variety of  interconvertible phenotypes which can be considered as  providing an extra dimension to the existing virulence  factors associated with this yeast. A number of switching  systems have been identified and phenotypic switching    may have evolved to compensate for the lack of variation  achieved in other organisms that utilize sexual reproduc-  tion. Phenotypic switching allows the yeast to exploit  microniches in the body and alters a variety of factors  (e.g. antifungal drug resistance, adherence, extracellular  enzyme production) in addition to the actual phenotype  and so may be considered as the ‘dominant’ or ‘control-  ling’ virulence factor.   In terms of tissue colonization and invasion, adher-  ence is the initial step in the process. Once the yeast has  adhered, enzymes (phospholipase and proteinase) can  facilitate adherence by damaging or degrading cell mem-  branes and extracellular proteins. Hyphae may be pro-  duced and penetrate layers of cells using thigmotropism  to find the line of least resistance. The passage through  cells is undoubtedly aided by the production of extracel-  lular enzymes. Once endothelial cells are reached enzymes  may assist in the degradation of tissue and allow the yeast  enter the host’s blood stream where phenotypic switching  or coating with platelets may be used to evade the  immune system. While in the bloodstream the haemo-  lysin may function to burst blood cells and release iron  which is essential for growth. Escape from the blood-  stream involves adherence to the walls of capillaries and  passage across the wall.   Aspergillus fumigatus is a saprophytic fungus which is  widely distributed in nature where it is frequently  encountered growing on decaying vegetation and damp  surfaces (Figure 4.8). A. fumigatus can present as an  opportunistic pathogen of humans and is the commonest  etiological agent of pulmonary aspergillosis, being  responsible for 80-90% of cases. Although the incidence  of disease due to Aspergillus species is less than that  due to Candida, aspergillosis results in greater mortality  rates.   A. fumigatus produces a number of extracellular  enzymes which facilitate growth in the lung and dissemi-  nation through the body. Phospholipase production has  been shown in clinical isolates of this fungus with optimal  production occurring at 37 °C. This enzyme plays a  critical role in tissue degradation and may facilitate exit  of the fungus from the lung into the bloodstream. Fungal  proteases are responsible for tissue degradation and  neutralization of the immune system and probably  evolved to allow the fungus to degrade animal and  plant material. Elastin constitutes almost 30% of lung  tissue and many A. fumigatus isolates display elastinolytic  activity while isolates incapable of elastinase produc-    tion display reduced virulence in mice. A. fumigatus  produces two elastinases: a serine protease and a metal-  loproteinase. Apart from their direct role in tissue  degradation, A. fumigatus proteases (in particular the  serine protease) may also function as allergens which  may be important in the induction and persistence of  allergic aspergillosis. Local inflammation due to the pres-  ence of proteases results in airway damage and in vitro  studies have shown that proteases are capable of inducing  epithelial cell detachment from basement membranes.  Such desquamation may partly explain the extent of  damage seen in the lungs of asthmatic and cystic fibrosis  patients affected with allergic aspergillosis. In addition  proteases induce the release of the proinflammatory IL-6  and IL-8 cytokines in cell lines derived from airway  epithelial cells, which may induce mucosal inflammatory  response and subsequent damage to the surrounding  tissue.   The production and secretion of toxins into surround-  ing tissues by the proliferating fungus is regarded as  an important virulence attribute and may facilitate  fungal growth in the lung. Gliotoxin is the main  toxin produced by A. fumigatus, others include helvolic  acid, fumigatin and fumagillin. It is believed that toxins  play a significant role in facilitating the colonization  of the lung by A. fumigatus since they can act to  retard elements of the local immunity. Significantly,  Aspergillus species that do not produce toxins to the same  extent as A. fumigatus isolates are rarely seen in clinical  cases.   Histoplasma capsulatum is a dimorphic fungus which  is the cause of histoplasmosis, the most prevalent fungal    pulmonary infection in the USA. Histoplasmosis is  common amongst AIDS patients but is also found among  infants and elderly people living in endemic areas  who may be susceptible to infection due to impaired  immune function. The mortality rate among infants  exposed to a large dose of H. capsulatum may be 40-50%.  H. capsulatum grows in the mycelial form in soil while  in tissue it is encountered as round, budding cells. Its  natural habitat is soil that has been enriched with the  droppings of bats or birds, and disturbance of such soil  by natural (e.g. wind) or human (e.g. agriculture) activi-  ties releases large numbers of airborne spores that  upon inhalation can establish pulmonary infection in  individuals or in populations distant from the original  source of spores.   Upon inhalation of H. capsulatum pulmonary macro-  phages engulf the yeast cells and provide a protected  environment for the yeast to multiply and disseminate  from the lungs to other tissues. The ability to survive  within the hostile environment provided by the macro-  phage is the key to the success of H. capsulatum as a  pathogen. Normal individuals who inhale a large number  of H. capsulatum spores can develop a non-specific flu-  like illness associated with fever, chills, headache and  chest pains after a 3 week incubation period that resolves  without treatment. Chronic pulmonary histoplasmosis is  seen in men in the 45-55 year age group who may have  been exposed to low levels of spores over a long period  of time. The condition is progressive, leading to a loss of  lung function and death if untreated.   Crypotococcus neoformans is an encapsulated yeast that is  most frequently associated with infection in immuno-  compromised patients, particularly those with AIDS,  where meningitis is the most common clinical manifesta-  tion. C. neoformans is a facultative intracellular pathogen  that is capable of surviving and replicating within mac-  rophages and withstanding the lytic activity within these  cells. In order to survive within macrophages C. neofor-  mans appears to accumulate polysaccharides within cyto-  plasmic vesicle. As part of its survival strategy C.  neoformans also produces melanin, which has the ability  to bind and protect against microbicidal peptides. In  addition, melaninization may allow the cell withstand the  effects of harmful hydroxyl radicals and so survive in  hostile environments . The capsule of C. neoformans is a  virulence factor in that it protects the cell from the  immune response, and capsular material (glucuronoxy-    lomannans) induces the shedding of host cell adherence  molecules required for the migration of host inflamma-  tory cells to the site of infection, thus explaining the  reduced inflammatory response to this yeast.   In immunocompromised patients pulmonary infec-  tion can lead to disseminated forms of the disease where  the eyes, skin and bones become infected. Cryptococcal  meningitis is particularly associated with AIDS patients  where it is a major cause of death. While cryptococcosis  may be controlled by antifungal therapy, in AIDS patients  there is a danger of relapse unless antifungal therapy is  constantly maintained.   The dermatophytes are a group of keratinophilic fungi  which can metabolize keratin — the principal protein in  skin, nails and hair (Figure 4.9). Tinea capitis is defined  as the infection of the hair and scalp with a dermato-  phyte, usually Microsporum canis or Trichophyton viol-  aceum. In Europe and North Africa T. violaceum may  be responsible for approximately 60% of cases of tinea  capitis. This condition is characterized by a mild scaling  and loss of hair and in some cases it may be contagious.  Tinea corporis is characterized by infection of the skin of  the trunk, leg and arms with a dermatophyte and is  usually caused by Trichophyton spp., Microsporum sp. or  Epidermophyton floccosum. Lesions are itchy, dry and  show scaling. Typically lesions retain a circular morphol-  ogy and the condition is often referred to as ‘ringworm’.  Tinea cruris is seen where the skin of the groin is infected  with a dermatophytic fungus, usually Epidermophyton  floccosum or Trichophyton rubrum. This condition is  highly contagious via fomites (towels, sheets, etc.) and up  to 25% of patients show recurrence following antifungal  therapy. Tinea pedis presents as infection of the feet with  a dermatophyte; it is very common and easily contracted.  The principal fungi responsible for this condition are T.  rubrum and E. floccosum and the sites of infection may  include the webs of the toes, the sides of the feet or the  soles of the feet. Tinea manuum is a fungal infection of  the hands and can be caused by a range of fungi most  notably Trichophyton mentagrophytes. Infection is often  seen in association with eczema and can result from  transmission of fungi from another infected body site.  Tinea unguium is defined as infection of the fingernails  or toenails and is often described as onychomycosis,  which includes infections due to a range of other fungi,  bacteria and nail damage associated with certain disease  states (e.g. psoriasis).   Figure 4.9 Dermatophytic infections. Hyphae of Microsporum canis (A) and a dermatophyte infection of the skin (B).   In recent years a number of fungal species previously  regarded as non-pathogenic have emerged as causes of  disease in certain groups of patients (e.g. HIV-positive  patients, cancer patients and transplant patients). While  a number of these fungal species may have been causing  disease previously, their emergence as significant patho-  gens is a cause of great concern. Undoubtedly the ability  to keep immunocompromised patients alive for long  periods is providing a set of novel niches for fungi, as is  the introduction of novel drugs and therapies. This  section focuses on a number of emerging fungal patho-  gens and demonstrate how the availability of novel niches  has facilitated the appearance of this group of fungal  pathogens.   The yeast S. cerevisiae is widely distributed in nature. It  has been used for millennia in the production of bread  and alcoholic beverages, and can be consumed directly as  a dietary supplement. Traditionally it is better known as  brewer’s or baker’s yeast. Because of the ease with which  it can be genetically and biochemically manipulated it is  probably the most studied and best-characterized organ-  ism on the planet. Recently a considerable number of    reports have implicated this yeast as a cause of disease in  immunocompromised patients but also in those with no  apparent predisposing condition. As a result, S. cerevisiae  is no longer regarded as a GRAS (‘generally regarded as  safe’) organism but is now classified as a Biosafety Level  1 pathogen, indicating that it can cause superficial or  mild systemic infections in certain instances. This change  of status has serious implications for industries using this  yeast and indicates that, in cases of impairment of the  immune system, what was once regarded as a harmless  yeast is capable of causing life-threatening illness.   S. cerevisiae has been implicated in a number of super-  ficial and systemic cases of disease and often in patients  who have no apparent predisposing illness. In the major-  ity of cases it has been found in association with other  microbes, but in a significant number of instances it  was identified as the sole pathogen. It has been identified  as a cause of vulvovaginitis in women, being respon-  sible for possibly up to 5% of cases, and is responsible for  pneumonia and widespread dissemination in AIDS  patients. Other conditions attributed to this yeast include  septicaemia, postoperative peritonitis and the induction  of fever and coughs in transplant patients. In one instance  a bone marrow transplant (BMT) patient taking a  self-prescribed course of brewer’s yeast tablets developed  a fever, cough and chest pains. S. cerevisiae was isolated  from the lung and identified as the source of the    infection. In relation to BMT patients the contamination  of food with S. cerevisiae is now regarded as a potential  source of infection.   Isolates of S. cerevisiae that have been associated with  disease display a number of attributes that facilitate their  persistence and dissemination in the host. Clinical iso-  lates display the ability to grow at 42 °C, which is signifi-  cantly greater than the upper temperature range for  brewing yeast. This ability is regarded as important since  febrile patients can attain this temperature and it is  advantageous for a pathogenic microorganism to survive  at this elevated temperature. Pathogenic isolates are  capable of producing a number of extracellular enzymes  such as acid proteinases and phospholipases which play  a role in modulating the immune system’s response to  infection and allow the degradation of cell membranes,  respectively. Isolates also demonstrate the ability to grow  in a pseudohyphal form which may assist in the penetra-  tion of tissue. Brewing strains of S. cerevisiae are known  to flocculate at the end of the fermentation and this phe-  nomenon is also seen in pathogenic isolates where it  plays a role in obstructing capillaries particularly in the  brain with concomitant damage to surrounding tissue.  Pathogenic isolates adhere to epithelial tissue via a pro-  teinaceous adhesin which is critical to the survival of the  yeast in areas where it could be washed away by the action  of swallowing as in the mouth. Potentially the most  important attribute of virulent isolates is their ability to  alter their phenotype. In clinical isolates of S. cerevisiae  this ability contributes to the yeast’s persistence in the  body where in complement factor 5 mice isolates were  capable of persisting in the brain for up to 7 days without  being cleared.   Conventional therapy for superficial or systemic S. cer-  evisiae infection relies on the use of azoles or polyenes.  Interestingly, clinical isolates demonstrate a high level of  resistance to fluconazole, which has been regarded as the  first choice for the treatment of C. albicans- induced  superficial candidosis in HIV-positive individuals. While  the mechanism that confers resistance to fluconazole in  S. cerevisiae is still poorly characterized it is thought to  be mediated via a multidrug efflux pump, which would  remove the drug from the cell before it can act.   The rather cumbersome term ‘non-albicans Candida  species’ covers a range of Candida species that have  emerged as significant human pathogens in recent years.  The principal species are Candida dubliniensis, Candida  krusei and Candida glabrata, although it must be empha-    sized that other Candida species may be problematic in  specific situations. All the emerging Candida species  share a number of common characteristics i.e. they were  either unknown or regarded as inconsequential patho-  gens until recently and their emergence as significant  causes of disease has occurred by exploiting a series of  novel niches produced either as a result of therapy or  disease.   Candida dubliniensis was first identified in 1995 in  samples taken from HIV-positive patients suffering from  oropharyngeal candidosis. Upon streaking samples on  CHROMagar plates C. albicans appeared green but the  newly discovered C. dubliniensis produced colonies dis-  playing a different shade of green. The yeast is similar to  C. albicans in many ways but displays different carbohy-  drate assimilation and DNA restriction patterns. This  yeast has been identified in HIV-positive and -negative  populations from many parts of the world and is now the  dominant cause of oral candidosis in the former group.  The discovery of C. dubliniensis demonstrates that the  provision of a novel niche allows previously unrecog-  nized pathogens to emerge and out-compete the per-  ceived dominant pathogen.   C. krusei was regarded as a harmless, transient com-  mensal, being commonly found in the environment (on  fruit) and on the human body, but now it is regarded as  a significant cause of disease in HIV-postive patients, dia-  betics and cancer patients (both solid tumour and leu-  kaemia where it is capable of colonizing the hastrointestinal,  respiratory and urinary tracts). In terms of virulence  attributes C. krusei demonstrates a reduced ability to  adhere to epithelial cells compared to C. albicans although  it does display a high cell surface hydrophobicity which  allows it stick to and colonize catheters and implants. Its  main virulence attribute is the high level of inherent  resistance to fluconazole. Upon the introduction of this  drug in 1990 to treat oropharyngeal candidosis in AIDS  patients and systemic candidosis in transplant patients  there was a reduction in the number of cases of disease  caused by C. albicans. However the elimination of this  yeast may have facilitated the emergence of C. krusei  as the dominant fungal pathogen in certain classes of  patient.   C. glabrata has emerged in recent years as a serious  cause of disease in neutropenic cancer patients and has  been responsible for mortality rates of 5-38% in some  surveys. It appears to be a particular problem in the late  stages of haematological malignancies, where mortality  rates of 70-100% have been described. The incidence of  C. glabrata infection has increased in recent years; it is    now the fourth most commonly isolated Candida species  and this increase may be attributable, in part at least, to  an alteration in local epidemiology. Since C. glabrata is  common amongst leukaemia patients and bone marrow  recipients, a range of other risk factors such as prolonged  hospitalization, use of cytotoxic drugs and catheteriza-  tion may also play a role in its prevalence. C. glabrata is  recognized as being a yeast of relatively low virulence but  its emergence as a serious pathogen has been attributed  to it being partially resistant to fluconazole. In addition,  other factors that may have contributed to its appearance  as a serious pathogen have been identified as azole proph-  ylaxis which eliminates C. albicans but not C. glabrata or  C. krusei and local factors such as the use of broad-  spectrum antibiotics or vascular catheters or the presence  of neutropenia as part of the disease state.   Until recently Penicillium marneffei was regarded as a  very rare and inconsequential cause of disease in humans.  Now, however, it is the most frequent cause of fungal  disease in AIDS patients who reside in, or have visited,  South-East Asia. The principal areas of infection are  Thailand and southern China although cases have been  reported from Malaysia, Taiwan, Japan and Hong Kong.  While the nature of the infection in humans is well char-  acterized, the natural habitat or reservoir of this fungus  is still unknown although it may be soil or decaying veg-  etation. P. marneffei is an asexual, dimorphic fungus  growing as a mycelium at 37 °C in tissue and as single cells  at 28 °C. It reproduces by a process known as fission. In  the yeast form of growth it can be difficult to distinguish  microscopically from Histoplasma capsulatum and  Cryptococcus neoformans, so monoclonal antibody based  assays specific for the detection of mannoproteins associ-  ated with P. marneffei have been developed. PCR  (polymerase chain reaction) fingerprinting is also used to  identify P.marneffei and to distinguish between infecting  strains.   Infection in humans follows inhalation of fungal  material, and in AIDS patients dissemination throughout  the body can result. Pulmonary involvement is often seen  particularly in AIDS patients but the conditions most  associated with infection are fever, weight loss, anaemia,  skin lesions, and liver and spleen inflammation. The con-  dition is fatal if untreated with antifungal drugs. While  the condition responds well to therapy if it is initiated at  an early stage, relapse is common in AIDS patients and  continuing antifungal therapy may be required. Because  of the immunocompromised nature of AIDS patients    other infections such as tuberculosis and pneumonia are  often seen along with P. marneffei infection.   Perhaps one of the most important discoveries regarding  the beneficial use of fungi for humans was the identifica-  tion in 1929 by Sir Alexander Fleming that an isolate of  Penicillium notatum produced a substance capable of  killing Gram-positive bacteria. This compound was sub-  sequently identified as penicillin and was the first member  of the (3-lactam class of antibiotics to be discovered.  These compounds function by inhibiting peptidoglycan  synthesis in bacteria and their use has reduced the impor-  tance of the Gram-positive bacteria as a cause of disease.  Subsequent to the identification of penicillin production  by P. notatum, a screen revealed that Penicillium chrysoge-  num was a superior producer. Following a series of muta-  genic and selection procedures the strain used in  conventional fermentations is capable of producing peni-  cillin at a rate of 7000 mg/L compared to the 3mg/L of  Fleming’s P. notatum isolate. A typical penicillin fermen-  tation yields three types of penicillin, namely F, G and V.  The latter can be used directly, however G is modified by  the action of penicillin acylase to give a variety of semi-  synthetic penicillins which show resistance to the action  of bacterial penicillinases which are implicated in confer-  ring antibacterial drug resistance.   The majority of antibiotics obtained from fungi are  produced by fermentation and most are secondary  metabolites, production of which occurs in the stationary  phase and is linked to sporulation. Catabolite repression  can inhibit antibiotic production and one way to avoid  this is to use low levels of glucose in the fermentation  medium or to obtain a mutant which is not catabolite  repressed. The chemical content of the medium must be  monitored since high levels of nitrogen or phosphate  (P0 4 ) retard antibiotic production. One problem that  seriously affects the productivity of antibiotic fermenta-  tions is feedback inhibition, where the antibiotic builds  to high intracellular levels and retards production or kills  the cell. One means of reversing this is to introduce low  levels of the antifungal agent amphotericin B, which  increases membrane permeability, leading to a decrease  in intracellular antibiotic levels and a concomitant  increase in production.   Antibiotic production can be maximized by optimiz-  ing production as a result of random mutagenesis and  selection. Another approach has been to fuse or mate    high-producing strains with good secretors. Rational  selection is a process where a chelating agent is intro-  duced into the fermentation to complex all the metal ions  present and consequently has a beneficial effect on anti-  biotic production. More recently genetic manipulation  has been employed to express the genes for antibiotic  production in another species which has the possibility  of producing hybrid antibiotics with novel targets.   Calderone, R.A. & Fonzi, W.A. (2001) Virulence factors of  Candida albicans. Trends Microbiol , 9, 327-335.   Cotter, G. & Kavanagh, K. (2000) Adherence mechanisms of  Candida albicans. Br J Biomed Sci , 57, 241-249.    Deresinski, S.C. & Stevens, D.A. (2003) Caspofungin. CID Rev  Anti- Infect Agents, 36, 1445-1457.   Daly, R & Kavanagh, K. (2001) Pulmonary aspergillosis: clinical  presentation, diagnosis and therapy. Br J Biomed Sci, 58,  197-205.   Haynes, K. (200 1 ) Virulence in Candida species. Trends Microbiol,  9, 591-596.   Kavanagh, K. (ed.) (2005) Fungi: Biology and Applications. John  Wiley & Sons, New York.   Kavanagh, K. (ed.) (2007) New Insights in Medical Mycology.  Springer, New York.   Richardson, M. & Johnson, E. (2000) The Pocket Guide to Fungal  Infection. Blackwell Science, Oxford.   Walker, G. (1998) Yeast Physiology and Biotechnology. John Wiley  & Sons, Chichester.   Viruses were first discovered at the end of the 19th  century, although the symptoms they cause were identi-  fied much earlier. One of the earliest pieces of evidence  is the symptoms of poliomyelitis in an Egyptian priest  depicted on a hieroglyph. Virus discovery came about  when the cause of an infectious disease (rabies), could  not be explained by the presence of bacteria. Unlike bac-  teria, the ‘infectious materials’ were not retained by filtra-  tion and thus viruses were then referred to as ‘filterable  agents’ or ‘filterable viruses’. Until the advent of electron  microscopy in the 1940s, only the chemical nature of  viruses (i.e. proteins and nucleic acid) could be identified    and their infectivity was mainly studied in animal models.  The observation of a virus by electron microscopy and  the development of cell tissue culture started the golden  era of virology. Since then a large number of viruses have  been isolated, their structure identified and their replica-  tion understood, leading to the design of potent antiviral  drugs and effective vaccines. Progress in virology over the  last 50 years have been considerable, leading to the eradi-  cation of smallpox following a worldwide vaccination  programme, and the likely future eradication of poliovi-  rus. For the first time in human history an infectious  disease has been vanquished. Despite such an achieve-  ment, much progress is still required to combat other  viruses. For example, despite large financial investment  and high profile studies, a cure or vaccine for HIV is still    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    not available. The number of commercially available  antivirals is also still limited, when compared to the  number of antibiotics. Within the last few years, diseases  caused by viruses in human and animals have reminded  us that viral infections can easily spread and cause epi-  demics and pandemics. Recent examples are ‘swine flu’  and ‘avian flu’, both caused by influenza viruses, and also  an outbreak of severe acute respiratory syndrome (SARS),  the virus originating from Asia and spreading worldwide,  and outbreaks of foot and mouth virus affecting cattle,  sheep and pigs that had important economic conse-  quences in the UK and mainland Europe.   Viruses are extremely diverse in size and in structure. The  smallest virus is approximately 28 nm in size (poliovirus),  while the largest is 750 nm (mimivirus). In simplistic  terms, a virus consists of viral nucleic acid within a  protein core, the capsid (also referred to as the coat), pos-  sibly surrounded by a lipidic envelope (Table 5.1). In  reality, there are many differences between viruses in  terms of nucleic acid, capsid structure, number of coats  and envelope composition. Such differences account for  the high diversity of viruses and the differences in their  properties, notably their resistance to antiviral drugs and  viricidal agents. Viral classification (Figure 5.1) is based  on the physical and chemical properties of viruses, their  structure and morphology (Figure 5.2).   The viral genome is composed of either DNA or RNA. It  can be double stranded (ds) or single-stranded (ss), linear  (e.g. poliovirus) or circular (e.g. hepatitis B virus), con-  taining several segments (e.g. influenza — eight segments  of minus-sense ss RNA) or one molecule (e.g. poliovi-  rus). Six groups can be distinguished depending on their  nucleic acid content (Table 5.2). Viruses that contain  plus-sense ss RNA (e.g. poliovirus) can have their genome  translated directly by the host ribosome. The nature of  the viral nucleic acid is important for the effectiveness of  antiviral treatments (see below). For example, retroviri-  dae such as HIV require a specific virus-encoded enzyme,  a reverse transcriptase, to convert their ss RNA into ss  DNA, to be able to replicate within the host cell. This  enzyme is a primary target site of many antiviral drugs.   On some occasions, the viral genome has been shown  to be infectious, i.e. to cause an infection. Such an obser-  vation is important when one considers viricidal agents    that damage the viral capsid or envelope but not the  viral nucleic acid. Furthermore, in laboratory conditions,  the phenomenon of multiplicity reactivation has been  observed with poliovirus whereby random damage to  viral capsid and nucleic acid following treatment with  hypochlorite, a biocide intensively used for surface disin-  fection, resulted in complementary reconstruction of an  infectious particle by hybridization of the gene pool of  the inactivated virus. This again underlines the necessity  of rendering the viral nucleic acid non-infectious follow-  ing a viricidal treatment.   The function of the capsid is to protect the viral nucleic  acid from detrimental chemical and physical conditions  (e.g. disinfection). The capsid is composed of a number  of subunits named capsomeres genetically encoded by the  viral genome. The nature and association of the cap-  someres are fundamental for the virus, as they give the  shape of the capsid, but also provide the virus with resist-  ance to chemical and physical agents. The assembly of the  capsomeres results in two different architectural styles —  icosahedral and helical symmetries (Figure 5.3); in mam-  malian viruses, a more complex structure can be found,  where several proteinaceous structures envelope the viral  genome core (e.g. poxviruses, rhabdoviruses). Bacterial  viruses (bacteriophages) also show a complex structure  consisting of a capsid head, a tail and tail fibres (see  section 8.1). The nature of the capsomeres in certain  viruses allows for the self-assembly of the capsid within  the host cell. The capsomeres are held together by non-  covalent intermolecular forces. Such assembly also allows  the release of the viral genome following dissociation of  the non-covalently bonded subunits. These subunits offer  considerable economy of genetic information within the  viral genome since only a small number of different sub-  units contribute to the formation of the capsid. Viruses  with an icosahedral capsid usually have capsomeres in the  form of pentons and hexons. The number of these subu-  nits varies considerably between viruses: for example,  adenovirus is constructed from 240 hexons and 12  pentons, whereas the poliovirus is composed of 20 hexons  and 12 pentons, forming a much smaller structure.  Viruses with a helical capsid (e.g. influenza and mumps  viruses) have their subunits symmetrically packed in a  helical array, appearing like coils of wound rope under  electron microscopy. Although the core of such a virus is  hollow, the viral nucleic acid is embedded into ridges on  the inside of each subunit and does not fill the hollow  core. Such a close association between viral nucleic acid    Variola is the smallpox virus, it produces a  systemic infection with a characteristic  vesicular rash affecting the face, arms and  legs, and has a high mortality rate   Vaccinia has been derived from the  cowpox virus and is used to immunize  against smallpox   Commonly cause upper respiratory tract  infections; tend to produce latent  infections in tonsils and adenoids; will  produce tumours on injection into  hamsters, rats or mice   HSV1 infects oral membranes in children;  >80% are infected by adolescence.  Following the primary infection the  individual retains the HSV 1 DNA in the  trigeminal nerve ganglion for life and has  a 50% chance of developing ‘cold sores’  HSV2 is responsible for recurrent genital  herpes   Causes chickenpox in children; virus  remains dormant in any dorsal root  ganglion of the CNS; release of immune  control in elderly people stimulates  reactivation resulting in shingles   CMV is generally acquired in childhood as  a subclinical infection. About 50% of  adults carry the virus in a dormantstate in  white blood cells. The virus can cause  severe disease (pneumonia, hepatitis,  encephalitis) in immunocompromised  patients. Primary infections during  pregnancy can induce serious congenital  abnormalities in the fetus   Infections occur by salivary exchange. In  young children they are commonly  asymptomatic but the virus persists in a  latent form in lymphocytes. Infection  delayed until adolescence often results in  glandular fever. In tropical Africa, a severe  EBV infection early in life predisposes the  child to malignant facial tumours  (Burkitt’s lymphoma)    Spherical enveloped  particle 42 nm in  diameter enclosing  an inner icosahedral  27 nm nucleocapsid    In areas such as South-East Asia and  Africa, most children are infected by  prenatal transmission. In the Western  world the virus is spread through contact  with contaminated blood or by sexual  intercourse. There is strong evidence that  chronic infections with HBV can progress  to liver cancer   Multiply only in epithelial cells of skin and  mucous membranes causing warts. There  is evidence that some types are associated  with cervical carcinoma    Enveloped particles,  100 nm in diameter  with a helically  symmetric capsid;  haemagglutinin and  neuraminidase spikes  project from the  envelope    These viruses are capable of extensive  antigenic variation, producing new types  against which the human population does  not have effective immunity. These new  antigenic types can cause pandemics of  influenza. In natural infections the virus  only multiplies in the cells lining the  upper respiratory tract. The constitutional  symptoms of influenza are probably  brought about by absorption of toxic  breakdown products from the dying cells  on the respiratory epithelium   Infection in children produces  characteristic swelling of parotid and  submaxillary salivary glands. The disease  can have neurological complications, e.g.  meningitis, especially in adults   Very common childhood fever, immunity  is lifelong and second attacks are very rare    The virus has a very wide host range,  infecting all mammals so far tested; dogs,  cats and cattle are particularly susceptible.  The incubation period of rabies is  extremely varied, ranging from 6 days up  to 1 year. The virus remains localized at  the wound site of entry for a while before  passing along nerve fibres to central  nervous system, where it invariably  produces a fatal encephalitis    An inner core is  surrounded by two  concentric  icosahedral shells  producing particles  70 nm in diameter    Spherical particles  70 nm in diameter; a  tightly adherent  envelope surrounds  an icosahedral capsid    A very common cause of gastroenteritis in  infants. It is spread through poor water  supplies and when standards of general  hygiene are low. In developing countries it  is responsible for about a million deaths  each year   One of a group of enteroviruses common  in the gut of humans. The primary site of  multiplication is the lymphoid tissue of  the alimentary tract. Only rarely do they  cause systemic infections or serious  neurological conditions like encephalitis  or poliomyelitis   The common cold viruses; there are over  100 antigenically distinct types, hence the  difficulty in preparing effective vaccines.  The virus is shed copiously in watery nasal  secretions   Causes German measles in children. An  infection contracted in the early stages of  pregnancy can induce severe multiple  congenital abnormalities, e.g. deafness,  blindness, heart disease and mental  retardation    Spherical particles  40 nm in diameter  with an inner core  surrounded by an  adherent lipid  envelope   Spherical particles  40 nm in diameter  consisting of an  inner core  surrounded by an  adherent lipid  envelope    The virus is spread to humans by  mosquito bites; the liver is the main target;  necrosis of hepatocytes leads to jaundice  and fever    The virus is spread through blood  transfusions and blood products. Induces  a hepatitis which is usually milder than  that caused by HBV   Long filamentous  rods composed of a  lipid envelope  surrounding a helical  nucleocapsidlOOO nm  long, 80 nm in  diameter   The virus is widespread amongst  populations of monkeys. It can be spread  to humans by contact with body fluids  from the primates. The resulting  haemorrhagic fever has a 90% case fatality  rate   Spherical enveloped  virus lOOnm in  diameter; icosahedral  cores contain two  copies of linear RNA  molecules and  reverse transcriptase   HTLV is spread inside infected  lymphocytes in blood, semen or breast  milk. Most infections remain  asymptomatic but after an incubation  period of 10-40 years in about 2% of  cases, adult T-cell leukaemia can result   Differs from other  retroviruses in that  the core is cone-  shaped rather than  icosahedral   HIV is transmitted from person to person  via blood or genital secretions. The  principal target for the virus is the CD4+  T-lymphocyte cells. Depletion of these  cells induces immunodeficiency   An RNA- containing  virus that can only  replicate in cells  co-infected with  HBV. The spherical  coat of HDV is  composed of HBV  capsid protein   The presence of the satellite HDV  exacerbates the pathogenic effects of HBV  producing severe hepatitis   Figure 5.2 The morphology of a variety of virus particles. The large circle indicates the relative size of a Staphylococcus cell.    and capsid proteins can explain the damage caused to the  nucleic acid following disaggregation of the capsid after  chemical or physical treatments.   The viral capsid can be surrounded by a lipidic envelope,  which originates from the host cell. The envelope is added  during the replication process or following excision of the  viral progeny from the host cells. The envelope can come  from the host cell nuclear membrane (e.g. herpes simplex  virus) or the cytoplasmic membrane (e.g. influenza  virus). One characteristic of the viral envelope is that host  proteins are excluded, but proteins encoded by the viral  genome are present. These viral proteins play an impor-  tant serological role. Enveloped viruses are generally con-    sidered to be the most susceptible to chemical and  physical conditions and do not survive well on their own  outside the host cell (e.g. on surfaces), although they can  persist longer in organic soil (e.g. blood, exudates, faeces).  Lipids in viruses are generally phospholipids from the  host envelope, although glycolipids, neutral fats, fatty  acids, fatty aldehydes and cholesterol can be found.   In addition to these structures, glycoproteins can be  found usually protruding from the viral capsid or embed-  ded in the envelope. These virus-encoded structures are  important for viral infectivity as they recognize the host  cell receptor site conveying viral specificity. In bacteri-  ophages, these structures can take the shape of tail fibres.    Table 5.2 Examples of types of nucleic acid in human and bacterial viruses   ss RNA with mRNA identical in  base sequence to virion RNA   ss RNA with mRNA complementary  in base sequence to virion RNA    * Hepadnaviridae genome contains partially ds DNA and partially ss DNA.   An icosahedral virus  particle composed of  252 capsomeres  240 being hexons and  12 being pentons   A helical virus partially  disrupted to show the  helical coil of viral  nucleic acid embedded  in the capsomeres   Viruses need to interact with a host cell as they cannot  reproduce on their own. They have no metabolism and  cannot synthesize their own proteins, lipids or nucleic  acids. Thus viruses can be considered as true intracellular  parasites that grow within living cells and use their energy  and synthetic machinery to produce viral components.  The production and excision of viruses from the host cell  will result in cell death, although this might not be imme-  diate. Following the replication of one virus within the  host cell, hundreds of new viruses (virus progeny or  virions ) can be released and infect adjacent cells (within  a tissue). The propagation from one infected cell to new  cells, and the subsequent destruction of tissue or cells,  provides signs of the viral disease.   On the basis of host specificity, three major viral  groups can be distinguished: (1) viruses of bacteria and  blue-green algae, (2) plant viruses and (3) animal (includ-  ing insect) viruses. Viruses are usually very specific and  rarely cross species barriers, although there are some  exceptions, such as rabies and influenza that can cause  diseases in both animals and humans. Viruses can also be  asymptomatic in certain hosts where they do not cause  an infection; the host becomes a reservoir and can trans-    mit the virus to a susceptible recipient (e.g. transmission  of yellow fever to humans by mosquitoes).   Viruses can interact with the host cell in five different  ways: (1) multiplication of the virus and destruction of  the host cell upon release of the viral progeny, (2) multi-  plication of the virus and release of the virions without  the immediate destruction of the host cell, (3) survival  of the virus in a latent stage without noticeable changes  to the infected cell, (4) survival of the infected cell in a  dramatically altered or transformed state (e.g. transfor-  mation of a normal cell to one having the properties of  a cancerous cell) and (5) incorporation of the viral  nucleic acid in the host cell genome without noticeable  changes to the infected cell. The interaction with the host  cell will vary between viruses, but will generally follow  one of these five routes.   There is a great diversity in viral infections and viral  diseases. Many viral infections are asymptomatic or  ‘silent’ whereby the virus replicates within the host but  does not produce symptoms of a disease. Other common  infections produce some mild symptoms, such as a low-  grade fever and a ‘runny nose’. This is the case of the  common cold, caused by rhinoviruses, from which  patients make a full recovery within a few days. At the  other end of the spectrum, some viruses kill their host  very quickly following infection, as in the case of haemor-  rhagic viruses such as the Ebola virus. On other occasions  a range of symptoms can be observed in different hosts.  This has been the case recently with ‘swine flu’, which  produced a range of symptoms, from a slight fever to full  influenza including a high fever, vomiting, and dizziness.  Other problematic viruses might not cause immediate  symptoms, but following the systematic destruction of  host cells will lead to an incurable disease; e.g. HIV and  oncogenic (tumour) viruses.   HIV is an enveloped virus with a cone-shaped nucleocap-  sid containing two copies of a positive-sense ss RNA mol-  ecule and the enzyme reverse transcriptase. The viral  genome encodes for the following genes: env, envelope  proteins; gag, capsid proteins; pol, enzymes involved in  viral multiplication; and tat, enzymes regulating host  metabolism. Seventy glycoprotein spikes (gpl20) project  from the envelope and interact specifically with the CD4  protein receptor on the T-lymphocyte (Chapter 9). The  HIV core penetrates the cell cytoplasm following mem-  brane fusion and is uncoated releasing the two RNA mol-  ecules and the reverse transcriptase into the cytoplasm.  The RNA is copied by reverse transcriptase into ss DNA,    which is then duplicated to form a ds DNA copy of the  original viral RNA genome. This DNA moves into the  host cell nucleus where it is integrated as a provirus into  a host cell chromosome.   The provirus can lie dormant in the cell or can be  expressed, producing viral mRNA and proteins and  resuming the multiplication cycle producing virions. The  viral mRNA is polycistronic, producing polyproteins that  need to be cleaved by a specific virus-encoded protease.  Following the assembly of viral proteins and viral RNA,  the virions bud off the infected cells.   At present there is no prospect of any drugs that can  be developed to eliminate proviruses from infected cells.  Current therapy has evolved around maintaining a high  count of T4 helper lymphocytes by regulating the viral  load produced by infected cells (up to 10 10 viral particles  produced continuously per day). Indeed, the ultimate  decrease in T4 helper cells (below 200/ml of blood)  seriously compromises the host immune system and  allows infection by a range of opportunist pathogens  including fungi, protozoa, bacteria and other viruses.  Currently, antiviral treatments (highly active antiretrovi-  ral therapy — HAART) combining a protease inhibitor  and two reverse transcriptase inhibitors, reduce the  number of HIV particles and slow the progression of the  disease by restoring and maintaining the number of T4  helper lymphocytes. However, this triple therapy does not  eliminate the virus completely. Patients who stop using  the drugs experience a rapid rebound in levels of the virus  in the blood and progression of the disease. The inability  to eliminate the virus completely calls for lifelong therapy  that is prohibitively expensive for countries with very  limited health budgets.   There is a suggestion that 20% of human cancers  might have a viral origin. Virus infected cells change  dramatically, acquiring the characteristics of tumour  cells exhibiting uncontrolled growth. For example, the  Epstein-Barr virus (EBV) has been associated with the  formation of lymphomas and nasopharyngeal carcino-  mas, the hepatitis B and C viruses with hepatocellular  carcinoma, human papilloma viruses with cervical cancer,  human T-cell lymphotrophic virus type 1 with adult  T-cell leukaemia/lymphoma syndrome and HIV with  Kaposi’s sarcoma.   There are no identified single mechanisms by which  viruses induce tumours. The acquisition of viral genes  by the host must be followed by other events such  as environmental or dietary exposures to chemical    carcinogens for cancer to occur. For example, there might  be an association between EBV and malaria to trigger  Burkitt’s lymphoma in young children in Africa. EBV and  consumption of smoked fish might trigger nasopharyn-  geal carcinoma in adults in China. The development of  liver cancer following infection with hepatitis B virus  (E1VB) might be triggered by high alcohol consumption,  smoking and exposure to fungal toxin (aflatoxins), events  that damage the liver.   It is known that the viral genome of oncogenic viruses  can integrate the host DNA. Indeed, viral DNA has been  recovered from infected cells. Following integration of  the provirus, the regulation of cell growth and division  can be affected. There is no means of eradicating provi-  ruses. However, progress in the prevention of papilloma-  virus has been made by designing an effective vaccine  combined to a successful vaccination programme that is  responsible for an important decrease in cervical cancer  cases.   The objective of the replication cycle is to ensure the mul-  tiplication of the virus with the formation of identical viral  progeny. Viruses differ in their replication cycle and the  time to produce and release new virions. The multiplica-  tion cycle of human viruses is generally slow, from 4 to  more than 40 hours. Bacterial viruses are generally faster  and can take as little as 20 minutes to replicate within the  bacterial host. The replication cycle can be divided into six  distinct phases (Figure 5.4) that are common to all viruses,  although detail within each phase varies greatly between  viruses. Understanding the viral multiplication process is  crucial for the development of new antiviral drugs.   Viral attachment to the cell surface can be divided into  three phases: (1) an initial contact mainly dependent    Figure 5.4 Diagrammatic representation of the production and release of viral particles from an infected cell.    on brownian motion, (2) a reversible phase during  which electrostatic repulsion is reduced and (3) irrevers-  ible changes in virus-receptor-host-receptor configura-  tion that initiates viral penetration through the cell  membrane.   All viruses possess receptors on their surface, usually in  the form of glycoproteins embedded in the viral envelope  or protruding as spike from the viral capsid. These struc-  tures recognize and bind receptors on the host cell and  provide the virus with its high specificity although differ-  ent viruses might share the same receptor. The virus-cell  recognition event is similar to any protein-protein inter-  action in that it occurs through a stereospecific network  of hydrogen bonds and lipophilic associations. For  example, the haemagglutinin receptor of influenza virus  binds the terminal glycoside residues of gangliosides (cell  surface glycolipids) of the target cell leading directly to  the virus particle adhering to the cell. Similarly, the inter-  action between the HIV receptor (i.e. gpl20) and the  T-lymphocyte receptor (i.e. CD4) has been intensively  studied.   Following the irreversible attachment of the virus to the  host cell, penetration of the virus through the cell mem-  brane is initiated following two energy-dependent mech-  anisms, endocytosis or fusion. A third mechanism has  been identified in some bacteriophages that can inject  their nucleic acid inside the bacterium (see section 8.1).  During endocytosis, the association between virus recep-  tor and host receptor triggers a number of mechanisms  that draw the cell membrane to engulf the virus particle  forming a cytosolic vacuole. This process is widespread  among non-enveloped viruses, but is also used with some  enveloped viruses such as influenza (orthomyxoviruses).  Certain enveloped viruses (e.g. herpes simplex virus,  HIV) can penetrate following fusion of their envelope  with the host cell membrane, liberating the viral capsid  within the cell cytoplasm.   Following penetration of the virus in a vacuole or directly  into the cell cytoplasm, the viral nucleic acid then needs  to be released from the capsid/coat(s) to initiate viral  replication. This is the uncoating process. For viruses that  penetrate by endocytoses, the acidification of the cytosolic  vacuoles following endosome fusion induces a conforma-  tional change in the capsid and the release of viral nucleo-  capsid (some helper proteins are associated with the viral  nucleic acid) into the cytoplasm. For certain viruses, such    as reovirus, only a partial uncoating is necessary for the  expression of the viral genome. The release of the nucleo-  capsid from vacuoles can occur in the cytoplasm, close to  the nucleus or within the cell nucleus.   4.4 Replication of viral nucleic acids and  translation of the genome   This stage of viral replication ensures that (1) the host  cell synthesis machinery is taken over by the virus, and  (2) the viral genome is replicated. The structure, size and  nature of the viral genome are extremely diverse and thus  this stage of the viral multiplication cycle reflects this  diversity. Three main mechanisms are, however, common  to all viruses: the transcription of viral genes into viral  mRNA, the translation of the viral genome into proteins,  and the replication of the viral genome. Early transcrip-  tion and translation usually occurring immediately after  the release of the nucleocapsid in the cytoplasm is also  common, and ensures the production of early proteins  such as viral polymerases, and the hijacking of the cell  synthesis machinery. In addition, some viruses can  encode for genes the products of which regulate the host  synthetic processes according to the needs of the virus  (e.g. tat gene in HIV).   The replication of the viral genome depends on the  type of nucleic acid carried by the virus. The positive  strand RNA in viruses such as the poliovirus can be used  directly as mRNA following the acquisition of a terminal  sequence from the host cell. Negative strand RNA (e.g. in  influenza virus) is transcribed into a positive RNA com-  plementary in base sequence to the parent RNA using an  RNA-dependent RNA polymerase carried by the virus. In  ds DNA viruses (e.g. adenoviruses), the nucleic acid  passes into the nucleus where it is usually transcribed by  a host DNA-dependent RNA polymerase. In some viruses  (e.g. poxvirus), this enzyme is contained within the virus  and released during uncoating, allowing the viral genome  to be replicated in the cell cytoplasm. In retroviruses (e.g.  HIV), a single-stranded proviral DNA is produced from  the viral ss RNA by a viral enzyme called reverse tran-  scriptase. This unique enzyme acts both as an RNA- and  DNA-directed DNA polymerase, and has associated  RNAase activity. The proviral DNA can be transported to  the cell nucleus where it can be integrated within the cell  host genome by a viral integrase.   One important difference between the host cell and  the virus is in the nature of their mRNA. Host cell mRNA  encodes directly for functional proteins, whereas viral  mRNA is polycistronic, which means several distinct pro-  teins are encoded within a single piece of mRNA. This    implies that the virus needs to use a virus-specific pro-  tease to cut at the correct place the polyprotein produced  by translation to restore the functionality of viral  proteins.   Late protein synthesis during the replication cycle con-  cerns the production of structural components (e.g. cap-  someres) of the new virions.   Towards the end of the multiplication process, large  amounts of viral materials accumulate within the host.  Viral capsid starts to form from individual structural pro-  teins. In certain viruses (e.g. poliovirus) the capsid self-  assembles. The replicated viral genome and some viral  proteins become packaged within the capsid. Most non-  enveloped viruses accumulate within the cytoplasm or  nucleus and are only released when the cell lyses.  Packaging of viral components can occur within the cyto-  plasm or in the cell nucleus. For example, with influenza  virus, the capsomeres are transported to the cell nucleus  where they combined with the viral RNA and assemble  into helical capsids. The envelope of enveloped viruses  originates from the host. With the influenza virus, viral  proteins such as neuraminidase and haemagglutinin  migrate to the cell membrane, displacing cell protein. The  assembled nucleocapsids pass out from the nucleus to the  cytoplasm and as they impinge on the altered cytoplasmic  membrane they cause it to bulge and bud off completed  enveloped particles from the cell. In the herpesvirus, the  envelope originates from the nucleus membrane. The  nucleocapsid assembles into the nucleus and it acquires  its envelope as it passes through the inner nuclear mem-  brane. The complete virus is then incorporated into a  vesicle which migrates to the cell surface.   The maturation of viruses and their assembly is not  well understood at present. The presence of chaperone  proteins may play an important role in the interac-  tion between the viral nucleic acid and the structural  proteins.   At the end of the multiplication process, the mature  virions are released from the host cell. This can occur in  a number of different ways. For most non-enveloped  viruses, the virus progeny accumulates within the host  cell cytoplasm and is released following cell lysis. Some  viruses (e.g. bacteriophages) produce a lytic enzyme  (peptide) or proteases to lyse the host enabling the  release of infectious particles, although the host often    self-disintegrates as it cannot maintain normal house-  keeping functions during a viral infection. Enveloped  viruses are usually released by a budding process over a  period of hours. Ultimately the host cell will die following  damage to its metabolism and housekeeping functions  during viral replication.   The study and identification of viruses (for diagnosis)  depends on our ability to propagate them. In the early  days of virology, viruses were propagated in a host. To  some extent, this is still the case today with the use of the  chick embryo (see below). However, progress in cell  culture has revolutionized the cultivation of many viruses,  enabling a better understanding of their replication prop-  erties, more rapid diagnosis and the easier production of  vaccine (Table 5.3).   Cells to support the growth of human viruses are usually  derived from humans or other primates, or from rodents.  Cell cultures may be divided into three types according  to their history: (1) primary, (2) secondary and (3) con-  tinuous cell culture. Primary and secondary cells are  usually diploid cell lines. Primary cell lines are derived  directly from an intact tissue such as human embryo  kidney or monkey kidney. Secondary cell cultures are  derived from primary cultures, usually those arising from  embryonic tissue. These cells are more homogenous,  better characterized, but might not be as susceptible to  viral infection as primary cell lines. In addition a limited  number of subcultures can be performed with these cells,  generally up to a maximum of about 50 before the cells  degenerate. Continuous cell lines are usually derived  from malignant tissue (e.g. HeLa cells derived from a  cervical carcinoma) and have the capacity to multiply  indefinitely in vitro.   In principle, cell culture for propagating viruses relies  on the growth of cells in a semiconfluent monolayer  attached to a surface (e.g. the bottom of a flask). To sub-  culture, the cells are separated from the monolayer or  relevant tissue usually with trypsin to form a suspension  of single cells. These suspended cells are then used to seed  a new flask. Following growth at 37 °C, cells will multiply,  attach to the surface and form a new monolayer within a  few days. The media used to grow the cells consists of  basic nutrients and salts (the composition of the medium  varies depending on the type of cell) supplemented with   Human diploid cell line, continuous  cell line or primary, secondary or  tertiary monkey kidney cells    serum (usually bovine albumin) to provide growth  factors; antibiotics and antifungals are included to  prevent bacterial and fungal contamination in such a rich  growth medium.   The established cell monolayer will support viral rep-  lication from which viruses can be harvested. Many types  of viruses, upon inoculation of a cell culture, will pro-  duced a characteristic morphological change in the  infected cells. This is called a cytopathic effect and usually  indicates cell death. The cytopathic effect can take the  form of cell shrinkage or ballooning, or the detachment  of cells from the surface of the flask; in some instances  cellular effects will be detected using various staining  solutions. Cytopathic effects usually spread to adjacent  cells and will result in the formation of a plaque that can  easily be identified following staining. These plaques are  used for the enumeration of viruses assuming one plaque  results from infection by one virus.    To confirm the identity of a viral pathogen (e.g. herpes  simplex virus, cytomegalovirus, influenza), viruses are  grown in cell culture and following the appearance of a  cytopathic effect, the identity of the virus is confirmed  using an appropriate viral antisera labelled with a fluo-  rescent dye.   Mammalian cells used for vaccine production are  obtained from an approved cell bank. All such cells  need to be checked for infectious agents and tumorigenic-  ity (in the case of live vaccines). Such cells are also  characterized biochemically (isoenzyme analysis), immu-  nologically (histocompatibility antigens) and by cytogenic  markers and are shown to be free from contaminating cells  (nucleic acid fingerprint analysis). Depending on their  origin, cells will be examined for the absence of specific  infectious agents, for the absence of bacterial, fungal and  mycoplasma contaminants, and for retroviruses — using  product-enhanced reverse transcriptase (PERT) assay,    transmission electron microscopy and, if necessary, infec-  tivity assays. The same controls apply to insect cells.   Fertile chicken eggs, 9-11 days old, are used as a conven-  ient cell system to grow a number of human pathogenic  viruses. Their usefulness derives from the many types of  different tissues found in the eggs, tissues that will  support the growth of different viruses (Table 5.3). The  use of chicken eggs is expensive and the use of cell culture  is preferred wherever possible. The use of chicken eggs  for the production of vaccine is necessarily subject to  many controls. The eggs have to be free from specific  pathogens and originate from healthy flocks. The process-  ing of the fertilized eggs must be conducted under aseptic  conditions in an area where no other infectious agents or  cells are handled at the same time.   Some animals (e.g. rodents, primates) have to be used to  culture certain viruses in order to study antiviral and  vaccine effectiveness, and also as a source of cell lines for  cell cultures. The use of animals follows strict ethical  guidelines and is extremely expensive. A number of con-  trols ensuring that the animals are free from diseases  must be observed. Where animals are used to grow  viruses or to test an antiviral or vaccine, growth of the  virus is indicated by signs of disease or death.   A number of antivirals are in use in the UK ( British  National Formulary, 2009) for a range of viral infections,  including HIV, herpesvirus infections, viral hepatitis,  influenza and respiratory syncytial virus. Antiviral treat-  ments are particularly important for persons at high risk,  notably immunocompromised patients. Most antivirals  are prodrugs that need to be activated within the cell,  usually by a kinase, and other cellular enzymes.   Antivirals can act at different stages of the viral replica-  tion cycle, with the most effective treatments targeting  unique viral enzymes, such as proteases, polymerases and  the reverse transcriptase (Table 5.4 and Chapter 11). A  number of targets are being investigated to prevent viral  attachment to the host cell: competition for CD4 recep-  tors using a pentapeptide identical in sequence to the  terminal amino acids of HIV gpl20; inhibition of herpes  simplex virus (HSV) ribonucleotide reductase; competi-    tion for the cell receptor using a hexapeptide fusion  sequence at the N-terminus of the influenza haemagglu-  tinin viral receptor. Proteases are particularly important  for the uncoating process preventing the release of viral  nucleocapsid and for the cleavage of viral polypeptide  gene products (e.g. indinavir sulphate). The replication  of viral DNA is also a well-exploited target with the use  of nucleoside analogues (e.g. idoxiuridine is incorporated  into viral and cellular DNA instead of thymidine), non-  nucleoside analogues (e.g. nevirapine and foscarnet) and  oligonucleotides (Table 5.5). These nucleic acid oligom-  ers with base sequence complementary to conserved  regions of proviral DNA have been successful use in the  prevention of viral mRNA function. The inhibition of  HIV reverse transcriptase has led to the synthesis of many  successful antivirals (Table 5.4). The release of the mature  virions after the multiplication process can also be  blocked. This is the case of neuraminidase inhibitors (e.g.  zanamivir and ozaltamivir) preventing the shedding of  virions.   Unfortunately, antiviral chemotherapy is associated  with a number of problems. Many viral diseases only  become apparent after extensive viral multiplication and  tissue damage have occurred, delaying treatments. Many  antivirals are toxic (e.g. nucleoside analogues) since viral  replication often depends on the use of host cell enzymes.  There is also scope for improving the pharmacokinetic  properties of antivirals, providing a better penetration  and retarding drug degradation. The use of prodrugs has  improved drug adsorption. Finally, antiviral mono-  therapy often leads to the development of virus resist-  ance. Emerging HIV resistance has been well documented  and current treatments are based on a triple therapy.   There is no cure for HIV infections as yet. The role of  antivirals is to slow or halt disease progression. Since their  discovery and use, these drugs (Table 5.4), called antiret-  rovirals, have considerably prolonged the life expectancy  of patients, although not without some important side  effects. Antiretroviral treatments aim to reduce HIV  plasma levels for as much and as long as possible. Several  antiretroviral drugs are usually given together to avoid  emerging viral resistance. Initiation of HIV treatment  (HAART) is therefore complex and involves two nucle-  oside reverse transcriptase inhibitors and two non-  nucleoside reverse transcriptase inhibitors. Alternative  regimens are possible following treatment failure and  deterioration of a patient’s condition. The use of antiret-  rovirals for prophylaxis after exposure is also possible,   Hepatitis C, RSV, herpes  virus, adenovirus, poxvirus,  Lassa fever virus, influenza,  measles, mumps, HIV   Triggers the formation of double  stranded duplex, inhibiting viral  RNA replication   Production of synthetic decoys  corresponding to a specific nucleic  acid sequence which binds virally  encoded regulatory proteins    where a patient has been exposed to HIV-contaminated  materials (e.g. needle injury). Such use follows guidelines  available locally (e.g. hospital) or nationally (e.g.  Department of Health, British Association for Sexual  Health and HIV).   The immune reconstitution syndrome and the lipod-  ystrophy syndrome have been associated with antiretro-  viral treatments. The latter includes fat redistribution,  insulin resistance, hyperglycaemia and dyslipidaemia. In  addition, these antivirals can be damaging to liver func-  tion and have been associated with osteonecrosis follow-  ing long-term combination treatments. A number of side  effects are commonly associated with the use of antiret-  rovirals: gastrointestinal disturbance, anorexia, pancrea-  titis, liver damage, dyspnoea, cough, headache, insomnia,  dizziness, fatigue, blood disorders, myalgia, arthralgia,  rash, urticaria and fever. Protease inhibitors are metabo-  lized by cytochrome P-450 and therefore have a signifi-  cant potential for drug interactions. Non-nucleoside  reverse transcriptase inhibitors have been shown to inter-  act with a number of drugs metabolized in the liver. They  have been associated with a number of side effects such  as rash, psychiatric and central nervous system distur-  bances, and even fatal hepatitis.   Herpesviridae is a family of viruses that include the  herpes simplex virus, chickenpox (varicella), shingles  (herpes zoster) and cytomegalovirus. Mild herpes simplex  virus infections in healthy individuals are treated with a  topical antiviral drug (e.g. treatment of cold sores).  However, for primary herpetic gingivostomatitis a change  of diet and analgesics are recommended. For severe infec-  tions (e.g. neonatal herpes infection, infection in immu-  nocompromised patients) a systemic antiviral drug is  used (Table 5.4). Antiviral treatments for chickenpox are  recommended in patients at risk and in neonates to  reduce risks of severe diseases. In healthy adults, treat-  ment taken with 24 hours of the appearance of a rash may  decrease the duration and severity of symptoms. Systemic  antivirals are used to decrease the severity and duration  of shingles when taken within 72 hours of the onset of  rash. Antivirals for herpes are also associated with a  number of side effects which vary depending on the drug,  but may include nausea, vomiting, stomach pain, head-  ache, fatigue, rash, and increase in serum and urine uric  acid. Antivirals for the treatment of cytomegalovirus are  usually given to immunocompromised patients and they  tend to be more toxic with notable nephrotoxicity (e.g.    cidofovir) and a number of documented side effects (e.g.  ganciclovir, foscarnet)   Hepatitis B and C are major causes of viral chronic hepa-  titis. The initial treatment for acute hepatitis B is  with interferons (peginterferon alfa-2a) which may  reduce the risk of chronic infection. However, the use of  interferon is limited by a poor response rate in patients  and frequent relapse. A number of antivirals are licensed  for the treatment of chronic hepatitis B (Table 5.4). The  choice of antivirals depends upon the initial response to  peginterferon-alfa, emerging viral resistance, and co-  infection with HIV. For the treatment of chronic hepatitis  C a combination of ribavarin and peginterferon-alfa is  recommended, although the choice and duration of  treatment depends upon the viral genotypes and viral  load. These antivirals are also associated with a number  of side effects including nausea, vomiting, abdominal  pain and diarrhoea.   Two antivirals are recommended for the treatment of  influenza according to the National Institute of Health  and Clinical Excellence (NICE) guidelines (Table 5.4).  Oseltamivir was extensively used for the prevention and  control of the swine flu outbreak in the UK in 2009.  Following an intensive use, at least two major limitations  in the usefulness of the drug have been identified. First,  the drug needs to be taken within a few hours of the onset  of symptoms, which proved very difficult with a number  of symptoms from mild ‘cold-like’ to severe ‘flu-like’  symptoms reported. Second, the side effects, especially in  young children and adolescents, have been very severe,  prompting many parents to stop the medication, decreas-  ing the willingness to give the antivirals to children who  have been possibly exposed to the virus.   Respiratory syncytial virus (RSV) is responsible for severe  bronchiolitis notably in infants. A monoclonal antibody  (palivizumab) or an antiviral drug (ribavirin) is indicated  for the treatment of RSV (Table 5.4). The antiviral is  associated with a number of severe side effects.   Vaccination is undoubtedly the most successful measure  against microbial infections, and particularly viral infec-  tions. Remarkably, human protection against smallpox    was achieved by Jenner in 1796 with the inoculation of  cowpox, well before the ‘germ theory’ of disease was pos-  tulated. A worldwide vaccination program initiated in  1966 led to the eradication of smallpox in 1980. The  poliovirus is almost completely eradicated following an  intensive worldwide vaccination programme by the  World Health Organization.   Vaccines are preparations containing antigens that  elicit a specific and active immunity against an infecting  agent; they can induce the innate and the adaptive (cel-  lular, humoral) parts of the immune system (Chapter 9).  There are currently a number of viral vaccines available  against a diverse range of human viruses (Table 5.6). The  success of vaccination relies on the prevention of a disease  from occurring. Vaccination is particularly indicated to  protect persons at risk (e.g. hepatitis B, varicella zoster  vaccines for healthcare workers), prior to travelling (hep-  atitis A virus, Japanese encephalitis, yellow fever, tick-  borne encephalitis virus) or to prevent cancer (human  papillomavirus vaccine preventing cervival cancer). A  vaccine can also be given to protect from a viral outbreak  (e.g. measles, mumps and rubella vaccine — MMR) or  following exposure to rabies.    Viral vaccines are prepared using different methods,  the most common being the use of live attenuated  viruses, inactivated viruses or the use of viral compo-  nents. Viruses or their components can be prepared  from animals, fertilized hen’s eggs, in suitable cell cultures  (see section 5) or in suitable tissues, or by culture of  genetically engineered cells. MMR vaccine and live (oral)  poliomyelitis vaccines are based on the use of live  attenuated viruses, which are not as virulent as the  original virus. The attenuated viruses will cause a  strong immune response without causing the disease.  Hepatitis A virus and influenza vaccines rely on chemi-  cally inactivated (formaldehyde, propiolactone) virus  particles or components (surface antigens). Hepatitis  B virus vaccine is a recombinant vaccine where the  viral DNA encoding for a virus surface antigen (HBsAg)  is expressed in yeast ( Saccharomyces cerevisiae) or mam-  malian cells (Chinese hamster ovary cells or other  suitable cell lines). The surface antigen is then purified.  Human papillomavirus (HPV) vaccine contains  virus-like particles and recombinant capsid protein  expressed in yeast or using a baculovirus as an expression  system.   Virus-like particle composed of the major capsid  protein (LI) of HPV type 6, 11, 16 and 18    HBsAg, hepatitis B virus surface antigen; HPV, human papillomavirus; MMR, measles, mumps and rubella.    It should be noted that most viral vaccines contain one  or more adjuvants, e.g. aluminium salts (antigens are  absorbed to the aluminium salts), monophosphoryl lipid  A, to increase or modulate the host immune response to  the antigens.   Immunoglobulin may play a role in the protection of  patients with a compromised immunity against viral  infections. Human normal immunoglobulin (HNIG) is  prepared from a pool of donated human plasma that has  been checked to be non-reactive for hepatitis B surface  antigen, hepatitis C virus and HIV (types 1 and 2) but  contains immunoglobulin G (IgG) and antibodies against  viruses that are prevalent in the general population  including hepatitis A, measles, mumps, rubella and vari-  cella. Intramuscular normal immunoglobulin is thus  used to protect against hepatitis A virus in immunocom-  promised patients visiting areas where the disease is  highly endemic and to protect against or attenuate  measles infection in immunocompromised patients. It  can also be used for pregnant women against rubella  virus, where the risk of termination of pregnancy is  unacceptable.   Disease-specific immunoglobulins are prepared from  a pool of plasma obtained from specific human donors  who have high-levels of the specific antibody required.  Disease-specific hepatitis B immunoglobulin is used fol-  lowing accidental inoculation by a risk material (e.g.  needlestick injury) or for infants born from mothers  infected with the virus. Disease-specific rabies immu-  noglobulin is available following the bite of an animal  suspected of carrying the disease or originating from an  area where the disease is endemic. Disease-specific vari-  cella zoster immunoglobulin is indicated for individuals  who are at a high risk such as neonates whose mothers  develop chickenpox, or for those exposed to the virus  while requiring intensive care or prolonged special care,  and for immunocompromised individuals.   Viruses are generally transmitted via surfaces and there-  fore the use of viricidal disinfectants on hard surfaces and  viricidal antiseptics on skin is important. In addition,  viruses are often associated with organic materials, such  as secretions from the host, blood, faeces etc., which  enable them to persist on surfaces for longer periods of  time (weeks, months, and rarely years) and to survive  better viricidal challenges.   In general, viruses are not particularly resistant to  chemical or physical agents, although some exceptions    exist. In terms of susceptibility to viricidal agents, small  non-enveloped viruses (e.g. poliovirus) are more resist-  ant than large enveloped viruses (e.g. HIV, influenza), the  lipid-rich envelope being damaged easily by chemical and  physical agents. The susceptibility of large non-enveloped  viruses varies, but it is generally considered to be between  that of the small naked and large enveloped viruses,  however some rotaviruses are proving particularly diffi-  cult to destroy.   The viricidal activity of biocides (antiseptics, disinfect-  ants) varies and not all biocides show a strong viricidal  activity against non-enveloped viruses (e.g. cationic bio-  cides, phenolics and alcohols). In addition, biocidal  activity depends upon a number of factors, such as con-  centration, contact time, presence of soiling, and formu-  lation (Chapters 18 and 19). Soiling is particularly an  issue with water- and food-borne viruses. Indeed, even  enveloped viruses can survive for many days on a soiled  surface even when exposed to biocides. The interactions  between biocides and viruses have been poorly studied.  In general, viruses present only a few target sites to bio-  cides, mainly the envelope (when present), glycoproteins,  the capsid and viral nucleic acid (Chapter 20). Some bio-  cides (e.g. cationic) are likely to interact with the envelope  and glycoproteins, inhibiting viral infectivity, without  altering the viral capsid and genome. The main target site  is most probably the capsid which has been shown to be  severely damaged in the presence of highly reactive bio-  cides such as aldehydes (e.g. glutaraldehyde) and oxidiz-  ing agents (e.g. peracetic acid, hydrogen peroxide). Less  reactive biocides, such as quaternary ammonium com-  pounds (QACs) and biguanides (e.g. chlorhexidine) have  been shown to damage viral capsid to a lesser extent,  explaining the limited activity of these agents against  non-enveloped viruses. The viral genome is the infec-  tious part of the virus and is the ideal target for biocides.  The destruction/alteration of the viral nucleic acid  would ensure complete viral inactivation. However, only  a limited number of reactive biocides, mainly oxidiz-  ing agents (e.g. hydrogen peroxide, chlorine dioxide)  have been shown to penetrate within the capsid and  damage viral nucleic acid, or to damage viral nucleic acid  released from a damaged capsid. The nature of the asso-  ciation of the viral nucleic acid with the capsid also plays  a role in the susceptibility of the virus to chemical and  physical agents. Viruses with a helical capsid structure  might be more susceptible since the destruction/alteration  of capsid is more likely to cause damage to the viral  nucleic acid which is closely associated to this type of  structure.    Physical agents such as heat and irradiation are viricidal  and play an important role in the control of viral con-  taminants in pharmaceutical products. Most viruses are  susceptible to exposure to temperatures above 60 °C for  30 minutes. Such susceptibility is used for the inactivation  of viral contaminants, such as HIV, in blood products.  However, other viruses such as the hepatitis B virus are  less susceptible and appropriate assurance of the absence  of such a virus is needed. Viruses survive well at low  temperatures and they can be routinely stored at — 40°C  to — 70 °C. In addition to thermal processes, UV irradiation  and ionizing radiation (y-ray and accelerated electrons)  are viricidal mainly following the destruction of the viral  nucleic acid. Ionizing irradiation and thermal processes  are used for terminal sterilization processes applied to  medical and pharmaceutical products (Chapter 21).   The presence of certain viruses needs to be controlled in  pharmaceutical products derived from human and  animal origin. This includes blood products such as  human plasma for fractionation intended for the manu-  facture of human antithrombin III, human coagulation  factor VII, VIII, IX, XI, dried prothrombin complex, dried  fibrinogen, normal immunoglobulin, human a r  proteinase inhibitor, and human von Willebrand factor,  for which tests for the presence of antibodies against  HIV-1 and HIV-2, hepatitis B surface antigen (HBsAg),  and antibodies against hepatitis C virus are required. For  the urine-derived urofollitropin (obtained from post-  menopausal women), tests for hepatitis virus antigens  and HIV antigen are needed.   The risk of a pharmaceutical product being conta-  minated by viruses depends mainly on the origin of  the product component (e.g. species, organ, tissue), the  history of the donor, the amount of material used,  the manufacturing process and its capacity to remove/  destroy any contaminants. In addition, the infectivity and  pathogenicity of possible viral contaminants must be  taken into consideration, notably when considering the  route of administration of the medicinal product (i.e.  transdermal delivery would carry less risk than an injec-  tion). A risk assessment is generally carried out for these  products containing a component from human or animal  origin, which takes into consideration factors affecting  the potential level of infectious particles and those related  to the use of the product.   Thus stringent controls are applied to the raw materi-  als, in process samples and to the final product. In addi-    tion, one or more validated procedures to remove or  destroy viruses can be applied. The type of inactivation  measures used must be validated against a range of rep-  resentative viruses (i.e. enveloped, non-enveloped, DNA,  and RNA viruses) with different degrees of resistance to  that type of treatment. Furthermore, early inactivation  limits the extent of contamination. For the preparation  of vaccines, the inactivation process must ensure that it  does not affect the antigenicity while killing the virus and  other potential contaminants such as mycoplasmas; for  example for the preparation of influenza vaccine, the  inactivation process chosen must cause minimum altera-  tion of the haemagglutinin and neuraminidase antigens.   Certain viruses or virus components are now used as  vectors for the delivery of genes to targeted cells. A  number of viruses are being used in gene transfer medici-  nal products (GTMP) and these include adenoviruses  (AAV), poxviruses, retroviruses, lentiviruses, adeno-  associated viruses and herpesviruses. Viral vectors for  human use are freeze-dried or liquid preparations of  recombinant viruses, genetically modified to transfer  genetic material to human somatic cells in vivo (i.e.  injected directly into the patient’s body) or ex vivo (i.e.  transferred into host cells before administration).   There are different approaches for the design and con-  struction of viral vectors. The chosen approach depends  upon the type of virus used. The procedure aims to mini-  mize the risk of generating replicating viruses or to elimi-  nate helper viruses when used during production. In  addition, a number of stringent controls are performed  ensuring the complete genetic and phenotypic charac-  terization of the viral vector is carried out. These include  the complete sequence of the genome of the viral vector,  verification of genomic integrity of the vector, determi-  nation of the concentration of the infectious vector,  residual host-cell protein and DNA, and residual reagents  including antibiotics.   For retroviridae-derived vectors, genetic modification  aims to ensure that the recombinant retroviruses are ren-  dered replication-incompetent. Adeno-associated virus  vectors (rAAV) are deficient adenovirus in which certain  genes (i.e. cap and rep) necessary for viral replication have  been replaced. A helper virus is thus needed during pro-  duction of the rAAV and needs to be eliminated from the  final GTMP. As with other viral vectors, the sequence  integrity of the viral genes and expression cassette as well    as genetic stability need to be controlled, and the absence  of wild-type virus (e.g. AAV) verified.   Bacteriophages (phages) are viruses that infect only bac-  teria. They were first described at the end of the 19th  century. They are typically 20-200 nm in size and are  highly diverse in their structure and host range and it is  likely that all bacterial species can be infected by a phage.  Phages are extremely specific in their host range and  some will only infect a specific bacterial strain. Such a  high specificity is used for bacterial typing as discussed  below. The most studied phages are complex ones (e.g.  T-phage) sometimes referred to as ‘tadpole-shaped’ con-  sisting of a head (often icosahedral) that contains the  viral genome and a tail which function is to recognize the  host receptor, attach and subsequently serve as a nucleic  acid injection device (Figure 5.5). Indeed, one of the  main differences between such phages and common  mammalian viruses is that these phages inject their viral  genome inside the host cell.   Phages have proved to be very useful genetic tools over  the years, since they are easy to propagate to high con-  centration and easy to study. Because of their similarity  to mammalian viruses, it is not surprising that phages  have been used to elucidate the viral multiplication cycle  and their study has led to many discoveries such as  mRNA, the understanding of the genetic code and the  control of genes, contributing to important advances in  molecular biology.    From the study of phage replication cycles, two sce-  narios have emerged, one resulting in the lysis of the  bacterial host — the lytic cycle — and the other resulting in  the viral nucleic acid being integrated into the host  genome — the lysogenic cycle (Figure 5.6). Infection with  a lytic phage, also called virulent phage, results in the  replication of the phage within the susceptible bacteria  and the release of infectious phage progeny from the host  cell following cell lysis. Such a lytic property is used to  enumerate phages. Phages inoculated on to a lawn of a  susceptible host bacterium form clear ‘holes’ (plaques)  which result from phage infection and lysis of a bacterial  host and the release of phage progeny, subsequently  infecting, replicating in, and lysing adjacent cells, ulti-  mately forming these plaques which are easily identifiable  with the naked eye (Figure 5.7). Since each of these  plaques is assumed to result front the infection from a  single phage, the number of plaques counted is used to  represent the number of phages.   In the lysogenic cycle, the viral nucleic acid which has  integrated the host genome is called prophage, and the  host cell that contains the viral genome lysogenic.  Following infection with lysogenic phages, both a lytic  and lysogenic responses are observed. The integration of  the prophage ensures that the viral genome is passed on  to the daughter cells following bacterial cell replication.  Lysogeny is an extremely common phenomenon and  through evolution most bacteria will host several  prophages. Indeed, sequencing of the whole bacterial  genome often indicates the presence of prophages (or  their remnants), that have become disabled with time. On  occasions, a prophage dormant in its host can be reacti-  vated and resume a lytic cycle. Upon excision from the   The prophage is passed on to the daughter cells on cell division.  These descendent lysogenic cells are immune to infection by the  phage carried as prophage    Figure 5.6 Scheme to illustrate the lytic and lysogenic responses of bacteriophages.   Figure 5.7 Plaques formed by phage on a plate seeded with  Bacillus subtilis.    host genome, the prophage can take adjacent bacterial  genes that become incorporated in the virion and trans-  mitted to a new susceptible host cell. Genes carried on  the prophage can then be expressed in the new host. This  process is called transduction and is responsible for gene  transfer between bacteria. Sometimes, genetic factors  transferred by transduction encode for antibiotic resist-  ant determinants and/or virulence factors such as toxins.  The induction of prophages to a lytic cycle can be artifi-  cially triggered by exposure to chemical and physical  agents such as mitomycin C and ultraviolet light. If the  use of lytic phages might be appropriate to kill bacteria,  the use of lysogenic phages is best confined to the genetic  engineering of bacteria.   The discovery of the lytic property of phages quickly  resulted in their use as a potential bactericidal agent at  the beginning of the 20th century, to combat bacteria  responsible for dysentery outbreaks, first against Vibrio    cholera (work of Hankin) and then Shigella shiga (work  of d’Herelle). Phages targeting bacteria causing a number  of diseases such as anthrax, scarlet fever, cholera and  diphtheria were quickly isolated, but with the exception  of ‘cholera’ phages these did not result in useful treat-  ments. The introduction of antibiotics in the early 1940s  resulted in the end of phage therapy in the West, although  it continues in the former Soviet Union, where it was used  to treat a variety of bacterial infections in both the First  and Second World Wars and is still used nowadays in  clinical practice (Chapter 27).   With the threat of bacterial resistance to antibiotics,  there has been a renewed interest in the use of phages to  control bacterial infection and product contamination  and this has led to the licensing of phage products for a  number of applications; the use of phages to combat  Listeria monocytogenes in ready-to-eat meat and poultry  products was authorized by the US Food and Drug  Administration (FDA) in 2006 and in cheeses by the EU  commission in 2009. In the UK, a phage-based product  to combat Pseudomonas aeruginosa in ear infection is  now in phase III clinical trials. Phage preparations are  also successfully employed in aquaculture, notably against  Lactococcus garvieae, the cause of a serious fish disease.   There are a number of ways to prepare a phage product.  ‘Natural’ phages from the environment can be selected  on the basis of their activity and incorporated into a  product on the basis that they are lytic and do not contain  any detrimental genes encoding for example antibiotic  resistance or bacterial virulence factors. Often several  phages (cocktail) attacking the same species or strain are  added to the product to minimize the risk of emerging  bacterial resistance. Non-replicating phages have been  used with some degree of success, but in this case the lytic  effect is short lived. Genetically modified phages have also  been used, whereby detrimental genes can be removed  and phage virulence genes added. Another advantage of  using genetically modified phages is commercial where  patents might be easier to file. Finally, lysogenic phages  can be used following treatment to remove their lys-  ogenic property.   Phage-based products can be developed using phage  components, mainly phage lytic enzymes which are  employed during phage penetration and during virion  excision from the host cell. Phage lytic enzymes (e.g. pep-  tides) can be harvested and use on their own to lyse/kill  bacteria (Chapter 27).   The use of phages for surface disinfection and antisep-  sis or for the treatment of a bacterial infection (phage  therapy) is at an early stage and further work is needed    to develop appropriate phage-based products, notably  the effect of the different routes of administration on  phage viability and effectiveness. Animal studies have  shown that the route of administration is crucial for  phage efficacy. Phage therapy is unlikely to supplant anti-  biotic therapy in the future; however, it is highly probable  that commercially available phage products will increase  as the advantages of phage therapy outweigh its disad-  vantages (Table 5.7 and Chapter 28).   The high specificity of phage against their host leads to  other applications. Phage typing is a method that differ-  entiates distinct strains of the same bacterial species on  the basis of their susceptibility to phages. This has been  particularly relevant with bacteria such as Salmonella  enterica serovar Typhi and to some extent Staphylococcus  aureus that can be ‘typed’ during an outbreak, and thus  provides some important epidemiological information.  Molecular techniques based on genetic differences are  now preferred for S. aureus.   A further application for phage is in developing rapid  diagnostics, notably against slow-growing bacteria such  as Mycobacterium tuberculosis. One specific kit relies on  the protection of TB phages from an externally applied  viricide when inside the targeted host, M. tuberculosis if  present; if the bacterium is not present in the sputum or  other biological fluid sample then the phages will be  unprotected and destroyed. Phages that are protected and  able to propagate are then harvested and inoculated to a  lawn of rapidly growing susceptible mycobacteria. The  presence of plaques will indicate that the phages were able  to replicate first within M. tuberculosis, i.e. a positive  sample.   The causative agents of the neurodegenerative diseases  bovine spongiform encephalopathy (BSE), scrapie in  sheep and Creutzfeldt-Jakob disease (CJD) in humans  used to be referred to as slow viruses. Flowever, it is now  clear that they are caused by a distinct class of infectious  agents termed prions that have unique and disturbing  properties. They can be recovered from the brains of  infected individuals as rod-like structures which are oli-  gomers of a 30kDa glycoprotein. They are devoid of  nucleic acid and are extremely resistant to heating and  ultraviolet irradiation. They also fail to produce an  immune response in the host. Just how such proteins can   Bacterial resistance can emerge — the use of a cocktail  containing several distinct phages is often proposed   Phage therapy could result in toxic shock from  bacterial lysis; i.e. release of bacterial components  such as lipopolysaccharide wall material  Potentially immunogenic   Phages can be developed against a wide range of bacterial hosts  Believed that every bacterium will possess at least one phage  due to natural selection   The genomes of selected phages need to be sequenced  to ensure they do not contain unwanted genes   Can be easily manipulated; e.g. incorporated into capsules, etc.  for ease of delivery   Quality assurance controls are complex and will need  to address biological variation, phage yield, and  endotoxin limits   Development and production costs may be less than a new  antibiotic   replicate and be infectious has only recently been under-  stood. It seems that a glycoprotein (designated PrPc)  with the same amino acid sequence as the prion (PrPsc)  but with a different tertiary structure, is present in the  membranes of normal neurons of the host. The evidence  suggests that the prion form of the protein combines  with the normal form and alters its configuration to  that of the prion. The newly formed prion can then in  turn modify the folding of other PrPc molecules. In  this way the prion protein is capable of autocatalytic  replication. As the prions slowly accumulate in the brain,  the neurons progressively vacuolate. Holes eventually  appear in the grey matter and the brain takes on a sponge-  like appearance. The clinical symptoms take a long time  to develop, up to 20 years in humans, but the disease has  an inevitable progression to paralysis, dementia and  death.   It is now clear that the large-scale outbreak of BSE that  began in the UK during the 1980s resulted from feeding  cattle with supplements prepared from sheep and cattle  offal. The recognition of this fact led to changes in animal  feed policies and eventually to the imposition of a ban on  the human consumption of bovine brain, spinal cord and    lymphoid tissues that were considered to be potentially  infectious. Unfortunately people had been consuming  potentially contaminated meat for a number of years.  Concerns that the agent had already been disseminated  to humans in the food chain were realized in 1996 with  the advent of a novel human disease that was called  variant or vCJD. This condition was unusual as it attacked  young adults with an average age of 30 rather than the  60-year-olds who typically succumb to classical sporadic  CJD. Studies on the experimental transmission of prions  to mice provided evidence that vCJD represents infection  by the BSE agent. The pathology in the mouse brain  induced by the vCJD agent and the incubation time of  the disease are different from that of classical CJD and  very similar to that of BSE. Gel electrophoresis of the  polypeptides from the brains of infected mice revealed  that the different transmissible spongiform encephalitis  agents have characteristic molecular signatures. These  signatures are based on the lengths of protease-resistant  fragments and the glycosylation patterns on the prion  molecules. The patterns from vCJD agent were very dif-  ferent patterns from those of the classical CJD but  remarkably similar to those formed by BSE.    Since 1996 there has been a slow but gradual increase  in the numbers of confirmed cases of vCJD. By November  2010 the number of deaths in the UK from vCJD had  reached 170. As the average incubation time for vCJD  is not yet known, it is difficult to estimate how many  more cases will develop. The measures taken to protect  the public will hopefully have prevented any further  human infections, but sadly no effective treatment is  available for those who have already contracted the  disease.   Balfour, H.H. (1999) Antiviral drugs. N Engl J Med, 340,  1255-1268.   British Pharmacopoeia (2011). The Stationery Office, London.  (The most recent volume should be consulted.)    British National Formulary 58. (2009) British Medical Associa-  tion and Pharmaceutical Press, London. (New editions of the  BNF appear at regular intervals and it is available online at  http://bnf.org/bnf/index.htm.)   Hanlon, G.W. (2007) Bacteriophages: an appraisal of their role  in the treatment of bacterial infections. Int J Antimicrob  Agents, 30, 118—128.   Maillard, J-Y. (2001) Virus susceptibility to biocides: an under-  standing. Rev Med Microbiol, 12, 63-74.   Morrison, L. (2001) The global epidemiology of HIV/AIDS. Br  Med Bull, 58, 7-18.   Oxford, J.S., Coates, A.R.M., Sia, D.Y., et al. (1989) Potential  target sites for antiviral inhibitors of human immunodefi-  ciency virus (HIV). J Antimicrob Chemother, 23, S9— S27.   Soto, C. (2006) Prions: The New Biology of Proteins. CRC Press,  London.   Parasitism is a specific type of interaction between two  organisms that has many features in common with other  infectious processes, but host-parasite interactions often  operate over a longer timescale than those seen with  other pathogens. This extended process results in signifi-  cant host-parasite interaction at the cellular and organ-  ismal level. It is known, for example, that some parasites  alter the behaviour of the host, while others, such as  Giardia lamblia, induce biochemical change in the host  cells at the site of infection (the duodenal epithelium).    Most parasites have a life cycle that often involves several  hosts; this means that survival and transmission between  different hosts requires the parasite to exhibit more than  one physiologically distinct form.   Parasites inhabit a wide range of habitats within their  hosts. Some parasites will inhabit only one site through-  out their life cycle, but many move to various sites within  the body. Such movement may require the formation of  motile cellular forms, and it will produce a significant  change in the physiology and morphology of the parasite  as a result of environmental change. Parasites moving  from the gut to other tissues, for example, will encounter    Hugo and Russell's Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.    higher levels of oxygen, changes in pH and significant  exposure to the host immune response. When life cycles  involve more than one host organism these changes are  greater. The reasons why parasites move to various sites  in with the host is driven by evasion of host immune  attack and to aid transmission.   Parasitic protozoa, like their free-living counterparts, are  single-celled eukaryotic organisms that utilize flagella,  cilia or amoeboid movement for motility. The complexity  of some parasite life cycles means that some species may  exhibit, at different times, more than one form of motil-  ity. All pathogenic protozoa are heterotrophs, using car-  bohydrates or amino acids as their major source of  carbon and energy. Some parasitic protozoa utilize  oxygen to generate energy through oxidative phosphor-  ylation, but many protozoan parasites lack functional or  ‘typical’ mitochondria, or have mitochondria that do not  function like those in mammalian cells. As a result of this  adaptation many parasites exhibit a fermentative metab-  olism that functions even in the presence of oxygen. The  reason for the utilization of less efficient fermentative  pathways is not clear, but it is presumably due in part to  the fact that such parasites survive in environments  where oxygen is only present occasionally or at low levels.  For some parasites oxygen is toxic, and they appear to  utilize it possibly in an effort to remove it and thus main-  tain an anaerobic metabolism.   The metabolism of parasites is highly adapted, with many  possessing unique organelles such as kienetoplasts and  hydrogenosomes. Many synthetic pathways that are found  in other eukaryotes are absent because many metabolic  intermediates or precursors such as lipids, amino acids and  nucleotides are actively scavenged from their environment.  This minimizes energy expenditure, which is finely balanced  in parasites and means that the membrane of parasitic pro-  tozoa is rich in transporters. Secretion of haemolysins,  cytolysins, proteolytic enzymes, toxins, antigenic and immu-  nomodulatory molecules that reduce host immune response  also occurs in pathogenic protozoa.   Survival of parasites is partly due to their high rate of  reproduction, which may be either sexual or asexual;  some organisms such as Plasmodium exhibit both forms  of reproduction in their life cycle. Simple fission is char-  acteristic of many amoeba, but some species also undergo  nuclear division in the cystic state (cysts are forms  required for survival outside the host) with each nucleus  giving rise to new trophozoites (the growing, motile and  pathogenic form).   This section considers the life cycles, disease and pathol-  ogy of some blood and tissue parasites; this is not an  exhaustive list but covers some of the most important  species. These diseases are commonly associated with  travel to tropical and subtropical countries, but diseases  such as leishmaniasis are frequently seen in southern  Spain and France. It should also be noted that climate  change is altering the geographical distribution of many  parasitic diseases.   Malaria has been a major disease of humankind for thou-  sands of years. Despite the availability of drugs for treat-  ment, malaria is still one of the most important infectious  diseases of humans, with approximately 200-500 million  new cases and 1-2.5 million deaths each year. Protozoa  of the genus Plasmodium cause malaria and four species  are responsible for the disease in humans: P. falciparum,  P. vivax, P. ovale and P. malariae. P. falciparum and P.  vivax account for the vast majority of cases, although P.  falciparum causes the most severe disease. Other species  of plasmodia infect reptiles, birds and other mammals.  Malaria is spread to humans by the bite of female mos-  quitoes of the genus Anopheles but transmission by inoc-  ulation of infected blood and through congenital routes  is also seen. These mosquitoes feed at night and their  breeding sites are primarily in rural areas.   The most common symptom of malaria is fever, although  chills, headache, myalgia and nausea are frequently seen  and other symptoms such as vomiting, diarrhoea, abdomi-  nal pain and cough occasionally appear. In all types of  malaria, the periodic febrile response (fever) is caused by  rupture of mature schizonts (one of the cell forms arising  as part of the life cycle). In P. vivax and P. ovale malaria  fever occurs every 24-48 hours, whereas in P. malariae,  maturation occurs every 72 hours. In falciparum malaria  fever may occur every 48 hours, but is usually irregular,  showing no distinct periodicity. Apart from anaemia, most  physical findings in malaria are often non-specific and  offer little aid in diagnosis, although enlargement of some  organs may be seen after prolonged infection. If the diag-  nosis of malaria is missed or delayed, especially with P.  falciparum infection, potentially fatal complicated malaria  may develop. The most frequent and serious complications  of malaria are cerebral malaria and severe anaemia.   Plasmodia have a complex life cycle (Figure 6.1) involving  a number of life cycle stages and two hosts. The human  infective stage comprises the sporozoites (c. 1-7 pm),  which are produced by sexual reproduction in the midgut  of the mosquito (vector) and migrate to its salivary gland.  When an infected Anopheles mosquito bites a human,  sporozoites are injected into the bloodstream and are  thought to enter liver parenchymal cells within 30  minutes of inoculation. In these cells the parasite differ-  entiates into a spherical, multinucleate schizont which  may contain 2000-40000 uninucleate merozoites. This  process of growth and development is termed exoeryth-  rocytic schizogony. This exoerythrocytic phase usually  takes between 5 and 21 days, depending on the species of  Plasmodium; however, in P. vivax and P. ovale the matura-  tion of schizonts may be delayed for up to 1-2 years.  These ‘quiescent’ parasites are called hypnozoites. Clinical  illness is caused by the erythrocytic stage of the parasite  life cycle; no disease is associated with sporozoites, the  developing liver stage of the parasite, the merozoites  released from the liver, or gametocytes.   The common symptoms of malaria are due to the  rupture of erythrocytes when erythrocytic schizonts  mature (Figure 6.2a). This release of parasite material  triggers a host immune response, which in turn induces  the formation of inflammatory cytokines, reactive  oxygen intermediates and other cellular products. These  proinflammatory molecules play a prominent role in  pathogenesis, and are probably responsible for the  fever, chills, sweats, weakness and other systemic symp-  toms associated with malaria. In P. falciparum malaria,  infected erythrocytes adhere to the endothelium of capil-  laries and postcapillary venules, leading to obstruction of  the microcirculation and localized anoxia. The pathogen-  esis of anaemia appears to involve haemolysis or  phagocytosis of parasitized erythrocytes and ineffective  erythropoiesis.   The family Trypanosomatidae consists of two genera,  Trypanosoma and Leishmania. These are important path-  ogens of humans and domestic animals and the diseases  they cause constitute serious medical and economic  problems. Because these protozoans have a requirement  for haematin obtained from blood, they are called haemo-  flagellates. The life cycles of both genera involve insect  and vertebrate hosts and have up to eight life cycle stages,  which differ in the placement and origin of the flagellum.  Trypanosomatids have a unique organelle called the kine-    toplast. This appears to be a special part of the mitochon-  drion and is rich in DNA. Two types of DNA have been  found in the kinetoplast: maxicircles that encode many  mitochondrial enzymes, and minicircles, which serve a  function in the process of RNA editing. Replication of  trypanosomatids occurs by single or multiple fission,  involving first the kinetoplast, then the nucleus, and  finally the cytoplasm. There are four major diseases asso-  ciated with this group: Chagas disease is caused by  Trypanosoma cruzi; sleeping sickness (African trypano-  somiasis) is associated with T. brucei; cutaneous and  mucocutaneous leishmaniasis are caused by a range of  species including Leishmania tropica, L. major, L. mexi-  cana, L. amazonensis and L. braziliensis; and visceral leish-  maniasis, which is also known as kala-azar, is typically  caused by L. donovani.   Chagas disease begins as a localized infection that is fol-  lowed by parasitaemia and colonization of internal  organs and tissues. Infection may first be evidenced by  a small tumour (chagoma) on the skin. Symptoms of  the disease include fever, oedema and myocarditis  (infection of the heart muscle) with or without heart  enlargement, and meningoencephalitis in children. The  acute disease is frequently subclinical and patients may  become asymptomatic carriers; this chronic phase may  result, after 10-20 years, in cardiopathy. Chagas disease  is transmitted by several genera of triatomine bugs  ( Triatoma , Rhodnius and Panstrongylus) and in nature  the disease exists among wild mammals and their associ-  ated triatomines. Human trypanosomiasis is seen in  almost all countries of the Americas, including the south-  ern USA, but the main foci are in poor rural areas of Latin  America.   T. cruzi exhibits two cell types in vertebrate hosts, a  blood form termed a trypomastigote, and in the tissues  (mainly heart, skeletal and smooth muscle, and reticu-  loendothelial cells) the parasite occurs as an amastigote  (Figure 6.3). Trypomastigotes ingested when the insect  takes a blood meal from an infected host transform into  epimastigotes in the intestine. Active reproduction occurs  and in 8-10 days metacyclic trypomastigote forms appear  which are flushed out of the gut with the faeces of the  insect. These organisms are able to penetrate the verte-  brate host only through the mucosa or abrasions of the  skin; hence, transmission does not necessarily occur at  every blood meal. Within the vertebrate the trypomastig-  otes transform into amastigotes, which, after a period of  intracellular multiplication at the portal of entry, are    Figure 6.1 The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito  inoculates sporozoites into the human host (1). Sporozoites infect liver cells (2) and mature into schizonts (3), which rupture and  release merozoites (4). (Of note, in P. vivax and P. ovale a dormant stage (hypnozoites) can persist in the liver and cause relapses by  invading the bloodstream weeks, or even years later.) After this initial replication in the liver (exo-erythrocytic schizogony, A), the  parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony, B). Merozoites infect red blood cells (5). The  ring stage trophozoites mature into schizonts, which rupture releasing merozoites (6). Some parasites differentiate into sexual  erythrocytic stages (gametocytes) (7). Blood-stage parasites are responsible for the clinical manifestations of the disease. The  gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood  meal (8). The parasites multiplication in the mosquito is known as the sporogonic cycle (C). While in the mosquito’s stomach, the  microgametes penetrate the macrogametes, generating zygotes (9). The zygotes in turn become motile and elongated (ookinetes)  (10), which invade the midgut wall of the mosquito where they develop into oocysts (11). The oocysts grow, rupture and release  sporozoites (12), which make their way to the mosquito’s salivary glands. Inoculation of the sporozoites into a new human host  perpetuates the malaria life cycle (1).    Figure 6.2 (a) Mature schizonts of Plasmodium vivax. P. vivax schizonts are large, have 12—24 merozoites, and may fill the red  blood cell (RBC). RBCs are enlarged 1.5-2 times and may be distorted. Under optimal conditions Schiiffner’s dots may be seen, (b)  Leishmania tropica amastigotes within an intact macrophage, (c) Toxoplasma gondii trophozoites in the bronchial secretions from  an HIV-infected patient, (d) Trophozoites of Giardia intestinalis. Each cell has two nuclei and is 10— 20 mm in length, (e)  Trophozoites of Entamoeba histolytica with ingested erythrocytes, which appear as dark inclusions, (f) Oocysts of Cryptosporidium  parvum (upper left) and cysts of Giardia intestinalis (lower right) labelled with immunofluorescent antibodies, (g) Trophozoites of  Trichomonas vaginalis.    released into the blood as trypanosomes; these invade  other cells or tissues, becoming amastigotes again.   The pathology of the infection is associated with  inflammatory reactions in infected tissues. These can lead  to destruction of the infected tissue and if it involves    heart tissue this can cause acute myocarditis. Parasite  enzymes may also cause cell and tissue damage. In the  absence of parasites, an autoimmune pathological process  seems to be mediated by T lymphocytes (CD4+) (see  Chapter 9) and by the production of certain cytokines;   Triatomine bug takes a blood meal  Q (passes metacyclic trypomastigotes  v in faeces; trypomastigotes enter bite  wound or mucosal membranes such  as the conjunctiva)    0 Metacyclic trypomastigotes  penetrate various cells at bite  wound site. Inside cells they  transform into amastigotes   0 Amastigotes multiply  by binary fission in cells  of infected tissues    a Intracellular amastigotes  v transform into trypomastigotes,  then burst out of the cell and  enter the bloodstream    Figure 6.3 An infected triatomine insect vector (or ‘kissing’ bug) takes a blood meal and releases trypomastigotes in its faeces near  the site of the bite wound. Trypomastigotes enter the host through the wound or through intact mucosal membranes, such as the  conjunctiva (1). Common triatomine vector species for trypanosomiasis belong to the genera Triatoma , Rhodinius and  Panstrongylus. Inside the host, the trypomastigotes invade cells, where they differentiate into intracellular amastigotes (2). The  amastigotes multiply by binary fission (3) and differentiate into trypomastigotes, and then are released into the circulation as  bloodstream trypomastigotes (4). Trypomastigotes infect cells from a variety of tissues and transform into intracellular amastigotes  in new infection sites. Clinical manifestations can result from this infective cycle. The bloodstream trypomastigotes do not replicate  (different from the African trypanosomes). Replication resumes only when the parasites enter another cell or are ingested by  another vector. The ‘kissing’ bug becomes infected by feeding on human or animal blood that contains circulating parasites (5).   The ingested trypomastigotes transform into epimastigotes in the vector’s midgut (6). The parasites multiply and differentiate in  the midgut (7) and differentiate into infective metacyclic trypomastigotes in the hindgut (8). Trypanosoma cruzi can also be  transmitted through blood transfusions, organ transplantation, transplacentally, and in laboratory accidents.    these induce a polyclonal activation of B-lymphocytes  and the secretion of large quantities of autoantibodies.   Sleeping sickness (African trypanosomiasis) is caused by  Trypanosoma brucei, of which there are two morphologi-  cally indistinguishable subspecies: T. brucei rhodesiense  and T. brucei gambiense. After infection the parasite    undergoes a period of local multiplication then enters  the general circulation via the lymphatics. Recurrent  fever, headache, lymphadenopathy and splenomegaly  may occur. Later, signs of meningoencephalitis appear,  followed by somnolence (sleeping sickness), coma and  death.   T. brucei, unlike T. cruzi, multiplies in the blood or  cerebrospinal fluid. Trypanosomes ingested by a feeding    fly must reach the salivary glands within a few days, where  they reproduce actively as epimastigotes attached to the  microvilli of the salivary gland where they transform into  metacyclic trypomastigotes, which are found free in the  lumen. Around 15-35 days after infection the fly becomes  infective through its bite.   The pathology of the infection is due to inflammatory  changes associated with an induced autoimmune demy-  elination of nerve cells. Interestingly, the immunosup-  pressive action of components of the parasite’s membrane  is probably responsible for frequent secondary infections  such as pneumonia. Liberation of common surface anti-  gens (the mechanism involved in immune evasion) in  every trypanolytic crisis (episode of trypanosome lysis)  leads to antibody and cell-mediated hypersensitivity  reactions. It is believed that some cytotoxic and patho-  logical processes are the result of biochemical and  immune mechanisms.   Leishmaniasis is the term used for diseases caused by  species of the genus Leishmania that are transmitted by  the bite of infected sand flies. The lesions of cutaneous  and mucocutaneous leishmaniasis are localized to the  skin and mucous membranes. Visceral leishmaniasis is a  much more severe disease, which involves the entire retic-  uloendothelial system, and is discussed in section 2.2.4 of  this chapter. Cutaneous leishmaniasis appears 2-3 weeks  after the bite of an infected sand fly as a small cutaneous  papule; this slowly develops and often becomes ulcerated  and develops secondary infections. Secondary or diffuse  lesions may develop. The disease is usually chronic but  may occasionally be self-limiting. Leishmaniasis from a  primary skin lesion may involve the oral and nasopha-  ryngeal mucosa. Leishmania species that infect humans  are all morphologically similar and only exhibit one  form, the intracellular amastigotes (3-6 pm long and  1.5-3 pm in diameter). Promastigotes (Figure 6.2b) are  found in the sand fly.   In mammalian hosts amastigotes are phagocytosed by  macrophages, but resist digestion and divide actively in  the phagolysosome (Figure 6.4). The female sand fly  ingests parasites in the blood meal from an infected  person or animal and these pass into the stomach where  they transform into promastigotes, and multiply actively.  The parasites attach to the walls of the oesophagus,  midgut and hindgut of the fly, and some eventually reach  the proboscis and are inoculated into a new host.   The obvious symptoms of this infection are caused  by the uptake of parasites by local macrophages. Host    response to infection produces tubercle-like structures  designed to limit the spread of infected cells. Some lesions  may resolve spontaneously after a few months but other  types of lesion may become chronic, sometimes with lym-  phatic and bloodstream dissemination. In infections due  to L. braziliensis there is a highly destructive spread of  infected macrophages to the oral or nasal mucosa. In L.  mexicana, L. amazonensis and L. aethiopica infections the  disease becomes more disseminated. The immunological  response of the host plays an important factor in deter-  mining the precise pathology of the disease and this is  apparent from the more severe type of infection seen in  individuals with HIV. In Europe and Africa several rodents  may act as reservoirs of the disease, but in countries such  as India, transmission can occur in a human-sand fly-  human cycle without rodent intervention. In rural semi-  arid zones of Latin America, both wild and domestic dogs  enter the epidemiological chain and the vector is a  common sand fly, Lutzomyia longipalpis, abundant in and  around houses. The disease is more common in children  in both Latin America and the Mediterranean area.   Like cutaneous leishmaniasis, visceral leishmaniasis  begins with the formation of a nodule at the site of inocu-  lation but this lesion rarely ulcerates and usually disap-  pears in a few weeks. However, symptoms and signs of  systemic disease such as undulating fever, malaise, diar-  rhoea, organ enlargement and anaemia subsequently  develop. In more serious cases of visceral leishmaniasis  the parasites, which can resist the internal body tempera-  ture, invade internal organs (liver, spleen, bone marrow  and lymph nodes), where they occupy the reticuloen-  dothelial cells. The pathogenic mechanisms of the disease  are not fully understood, but enlargement occurs in those  organs that exhibit marked cellular alteration such as  hyperplasia. Parasitized macrophages replace tissue in the  bone marrow. Patients with advanced disease are prone  to superinfection with other organisms which are pre-  dominantly bacterial.   The term coccidia describes a group of protozoa that con-  tains the genus Cryptosporidium (see intestinal parasites)  as well as a number of important veterinary parasites.  Toxoplasma gondii is an intestinal coccidian but the major  pathology of infection is associated with other tissues and  organs. T. gondii infects members of the cat family as  definitive hosts and has a wide range of intermediate  hosts. Infection is common in many warm-blooded   Figure 6.4 Leishmaniasis is transmitted by the bite of female phlebotomine sandflies. The sandflies inject the infective stage,  promastigotes, during blood meals (1). Promastigotes that reach the puncture wound are phagocytosed by macrophages (2) and  transform into amastigotes (3). Amastigotes multiply in infected cells and affect different tissues, depending in part on the  Leishmania species (4). This originates the clinical manifestations of leishmaniasis. Sandflies become infected during blood meals  on an infected host when they ingest macrophages infected with amastigotes (5, 6). In the sandfly’s midgut, the parasites  differentiate into promastigotes (7), which multiply and migrate to the proboscis (8).    animals, including humans. In most cases infection is  asymptomatic, but devastating disease can occur congeni-  tally in children as a result of infection during pregnancy.  T. gondii infection in humans is a worldwide problem,  although the rates of human infection vary from country  to country. The reasons for these variations include envi-  ronmental factors, cultural habits and the presence of  domestic and native animal species. The frequency of  postnatal toxoplasmosis acquired by eating raw meat  and by ingesting food contaminated by oocysts from cat  faeces (oocyst formation is greatest in the domestic cat)  is not well established but is thought to be significant.  Widespread natural infection is possible because infected  animals may excrete millions of resistant oocysts, which  can survive in the environment for prolonged periods    (months-years). Mature oocysts are approximately 12 pm  in diameter and contain eight infective sporozoites.   T. gondii infection in most animals including humans  is asymptomatic. Severe disease in humans is observed  only in congenitally infected children and in immuno-  suppressed individuals. The most common symptom  associated with postnatal infection in humans is lym-  phadenitis which may be accompanied by fever, malaise,  fatigue, muscle pains, sore throat and headache (flu-like  symptoms). Typically infection resolves spontaneously in  weeks or months, but in immunosuppressed individuals,  a fatal encephalitis may occur producing symptoms such  as headache, disorientation, drowsiness, hemiparesis,  reflex changes and convulsions. Prenatal T. gondii infec-  tions often target the brain and retina and can cause a    wide spectrum of clinical disease. Mild disease may  consist of impaired vision, whereas severely diseased chil-  dren may exhibit a ‘classic tetrad’ of signs: retinochoroidi-  tis, hydrocephalus, convulsions and intracerebral  calcifications. Hydrocephalus is the least common but  most dramatic lesion of congenital toxoplasmosis.   The life cycle of T. gondii was only fully described in  the early 1970s when felines including domestic cats were  identified as the definitive host and various warm-  blooded animals were identified as intermediate hosts. T.  gondii is transmitted by three mechanisms: congenitally,  through the consumption of uncooked infected meat and  via faecal matter contamination. Figure 6.5 shows the life  cycle of T. gondii. Cats acquire Toxoplasma by ingesting  any of three infectious stages of the organism: the rapidly  multiplying forms, tachyzoites, the dormant bradyzoites  (cysts) in infected tissue and the oocysts shed in faeces.  The probability of infection and the time between infec-  tion and the shedding of oocysts varies with the stage of  T. gondii ingested. Fewer than 50% of cats shed oocysts  after ingesting tachyzoites or oocysts, whereas nearly all  cats shed oocysts after ingesting bradyzoites. When a cat  ingests tissue cysts, the cyst wall is dissolved by intestinal  and gut proteolytic enzymes, which causes the release of  bradyzoites. These enter the epithelial cells of the small  intestine and initiate the formation of numerous asexual  generations before the sexual cycle begins. At the same  time that some bradyzoites invade the surface epithelia,  other bradyzoites penetrate the lamina propria and begin  to multiply as tachyzoites (trophozoites) (Figure 6.2c).  Within a few hours, tachyzoites may disseminate to other  tissues through the lymph and blood. Tachyzoites can  enter almost any type of host cell and multiply until the  cell becomes packed with parasites. The host cell then  lyses and releases more tachyzoites to enter new host cells.  The host usually controls this phase of infection, and as  a result the parasite enters the ‘resting’ stage in which  bradyzoites are isolated in tissue cysts. Tissue cysts are  formed most commonly in the brain, liver and muscles.  These cysts usually cause no host reaction and may  remain dormant for the life of the host. In intermediate  hosts, such as humans, the extraintestinal cycle of T.  gondii is similar to the cycle in cats except that there is no  sexual stage.   Most cases of toxoplasmosis in humans are probably  acquired by the ingestion of either tissue cysts in infected  meat or oocysts in food contaminated with cat faeces.  Bradyzoites from the tissue cysts or sporozoites released  from oocysts invade intestinal epithelia and multiply. T.  gondii may spread both locally to mesenteric lymph    nodes and to distant organs by invading the lymphatic  and blood systems. Focal areas of necrosis (caused by  localized cell lysis) may develop in many organs. The  extent of the disease is usually determined by the extent  of injury to infected organs, especially to vital and vulner-  able organs such as the eye, heart and adrenals.   Opportunist toxoplasmosis in immunosuppressed  patients usually represents reactivation of chronic infec-  tion. The predominant lesion of toxoplasmosis —  encephalitis in these patients — is necrosis, which often  results in multiple abscesses, some as large as a tennis ball.   Gut protozoan parasites include Entamoeba histolytica,  Giardia lamblia. Dientamoeba fragilis. Balantidium sp.,  Isospora sp. and Cryptosporidium parvum. All these  organisms are transmitted by the faecal-oral route and  most of them are cosmopolitan in their distribution. A  good example of this is Giardia, which is found in nearly  all countries of the world. In many developed countries,  including the UK and USA, it is one of the most com-  monly identified waterborne infectious organisms.  Cryptosporidium, like Toxoplasma, has a complex life  cycle utilizing both sexual and asexual reproduction. In  contrast, Giardia and Entamoeba have simple life cycles  utilizing only asexual reproduction. These latter organ-  isms are members of a small group of eukaryotes that do  not have mitochondria. It had long been assumed that  they never had mitochondria, but recent studies showing  the presence of mitochondrial-like enzymes and struc-  tural proteins suggest that it is more likely that this  organelle was lost as a result of metabolic/physiological  adaptation.   Giardia duodenalis (syn lamblia and intestinalis) is the  causative agent of giardiasis, a severe diarrhoeal disease.  The incidence of Giardia infection worldwide ranges  from 1.5% to 20% but is probably significantly higher in  countries where standards of hygiene are poor. The most  common route of spread is via the faecal-oral route,  although spread can also occur through ingestion of con-  taminated water and these modes of transmission are  particularly prevalent in institutions, nurseries and  daycare centres. Recent outbreaks and epidemics in the  UK, USA and eastern Europe have been caused by drink-  ing contaminated water from community water supplies    Both oocysts and tissue cysts transform  into tachyzoites shortly after ingestion.  Tachyzoites localize in neural and muscle  tissue and develop into tissue cyst  bradyzoites. If a pregnant woman becomes  infected, tachyzoites can infect the fetus  via the bloodstream   2) Direct identification of the parasite from peripheral  blood, amniotic fluid, or in tissue sections    Figure 6.5 Members of the cat family (Felidae) are the only known definitive hosts for the sexual stages of Toxoplasma gondii and  thus are the main reservoirs of infection. Cats become infected with T. gondii by carnivorism (1). After tissue cysts or oocysts are  ingested by the cat, viable organisms are released and invade epithelial cells of the small intestine where they undergo an asexual  cycle followed by a sexual cycle and then form oocysts, which are then excreted. The unsporulated oocyst takes 1—5 days after  excretion to sporulate (become infective). Although cats shed oocysts for only 1—2 weeks, large numbers may be shed. Oocysts can  survive in the environment for several months and are remarkably resistant to disinfectants, freezing and drying, but are killed by  heating to 70 °C for 10 minutes. Human infection may be acquired in several ways: (A) ingestion of undercooked infected meat  containing Toxoplasma cysts (2); (B) ingestion of the oocyst from faecally contaminated hands or food (3); (C) organ  transplantation or blood transfusion; (D) transplacental transmission; (E) accidental inoculation of tachyzoites. The parasites form  tissue cysts, most commonly in skeletal muscle, myocardium and brain; these cysts may remain throughout the life of the host.    or directly from rivers and streams. Many animals  harbour Giardia species that are indistinguishable from  the human infective types. There is now clear evidence  from genotyping studies (see section 6.1.2) that the G.  duodenalis species is made up of a number of genetically  distinct groups which may represent species. This has  raised the question of the existence of animal reservoirs    of Giardia. Recent findings of Giardia - infected animals  in watersheds from which humans acquired giardiasis,  and the successful interspecies transfer of these organ-  isms, suggests that human giardiasis can be acquired by  zoonotic transfer, however it is not clear if the major  route of transfer is from animal to human or from human  to animal. More recently, it has been recognized that    Giardia infection may be transmitted by sexual activity,  particularly among homosexual men.   This organism exhibits only two life cycle forms:  the vegetative binucleate trophozoite ( 10-20 |im long x 2  -3 pm wide) (Figure 6.2d) and the transmissible  quadranucleate cyst (10-12 pm long X 1-3 pm wide).  Trophozoites have four pairs of flagella and an adhesive  disc, which is thought to help adhesion to the intestinal  epithelium. Division in trophozoites is by longitudinal  fission.   It was long believed that Giardia was a non-pathogenic  commensal. However, we now know that Giardia can  produce disease ranging from a self-limiting diarrhoea to  a severe chronic syndrome. Immune-competent indi-  viduals with giardiasis may exhibit some or all of the  following signs and symptoms: diarrhoea or loose, foul-  smelling stools; steatorrhoea (fatty diarrhoea); malaise;  abdominal cramps; excessive flatulence; fatigue and  weight loss. Infected individuals with an immune defi-  ciency or protein-calorie malnutrition may develop a  more severe disease and will exhibit symptoms such as  interference with the absorption of fat and fat-soluble  vitamins, retarded growth, weight loss, or a coeliac  disease-like syndrome.   Giardia infection is initiated by ingestion of viable  cysts (Figure 6.6), the infective dose of which can be as  low as one cyst, although infection initiated by 10-100  viable cysts is more likely. As the cysts pass through the  stomach the low pH and elevated C0 2 induce excystation  (cyst-trophozoite transformation). From each cyst two  complete trophozoites emerge and these rapidly undergo  division then attach to the duodenal and jejunal epithe-  lium. Once attached, they will undergo division, and 4-7  days later they will detach and begin to round up and  form cysts (encystment). This process is thought to be  induced in response to bile. The first cysts are found in  faeces after 7-10 days.   The underlying pathology of giardiasis is not fully  understood. The trophozoites do not invade the mucosa,  and although their presence may have some physical  effects on the surface it is more likely that some of the  pathology is caused by inflammation of the mucosal cells  of the small intestine causing an increased turnover rate  of intestinal mucosal epithelium. The immature replace-  ment cells have less functional surface area and less diges-  tive and absorptive ability. This would account for the  microscopic changes seen in infected epithelia. It has  been suggested that other mechanisms may exist, e.g.  toxin production, but to date no such molecule has been  observed.   Entamoeba histolytica is the causative agent of amoebic  dysentery, another infection transmitted via the faecal-  oral route. The severity of this and related pathologies  caused by this organism can vary from diarrhoea associ-  ated with the intestinal infection to extraintestinal amoe-  biasis producing hepatic and often lung infection. The  prevalence of amoebiasis in developing countries reflects  the lack of adequate sanitary systems. It had long been  known that most infections associated with E. histolytica  are asymptomatic or exhibit minimal symptomology, but  in the late 1980s a separate, but morphologically and  biochemically similar species, E. dispar, was identified.  This organism exhibits limited pathogenicity and in  many cases produces no symptoms but is commonly  misidentified as E. histolytica. This species is the most  likely cause of ‘asymptomatic Entamoeba infection’.   E. histolytica has a relatively simple life cycle and, like  Giardia, exhibits only two morphological forms: the tro-  phozoite and cyst stages. Trophozoites (Figure 6.2e) vary  in size from 10 to 60 pm and are actively motile. The cyst  is spherical, 10-20 pm in diameter, with a thin transpar-  ent wall. Fully mature cysts contain four nuclei.   Symptoms of amoebic dysentery are associated with  mucosal invasion and ulceration. Mucosal erosion  causes diarrhoea, the severity of which increases with the  level of invasion and colonization. Symptoms can also  be affected by the site of the infection. Peritonitis as a  result of perforation has been reported in connection  with severe amoebic infection. Extraintestinal amoebiasis  is usually associated with liver infection, causing  abscesses and/or enlargement. The abscess appears as a  slowly enlarging liver mass and will cause noticeable  pain. Jaundice may also occur due to blockage of the  bile. Pleural, pulmonary, and pericardial infection  results from metastatic spread from the liver, but can  also manifest in other parts of the viscera or give rise  to a brain abscess. However, these complications are  uncommon.   The life cycle of E. histolytica (Figure 6.7) is simple, but  the ability of trophozoites to infect sites other than the  intestine make it more complex than that of Giardia.  Infection is initiated by ingestion of mature cysts, and  again, excystation occurs during transit through the gut.  After this, trophozoites rapidly divide by simple fission to  produce four amoebic cells which undergo a second divi-  sion; thus each cyst yields eight trophozoites. Survival  outside the host depends on the resistant cyst form.   The pathology of the disease is only partially under-  stood. The process of tissue invasion has been well studied    Figure 6.6 Cysts are resistant forms and are responsible for transmission of giardiasis. Both cysts and trophozoites can be found in  the faeces (diagnostic stages) (1). The cysts are hardy, and can survive several months in cold water. Infection occurs by the  ingestion of cysts in contaminated water, food or by the faecal— oral route (hands or fomites) (2). In the small intestine, excystation  releases trophozoites (each cyst produces two trophozoites) (3). Trophozoites multiply by longitudinal binary fission, remaining in  the lumen of the proximal small bowel where they can be free or attached to the mucosa by a ventral sucking disc (4). Encystation  occurs as the parasites transit toward the colon. The cyst is the stage found most commonly in non-diarrhoeal faeces (5). Because  the cysts are infectious when passed in the stool or shortly afterwards, person-to-person transmission is possible. Although animals  are infected with Giardia , their importance as a reservoir is unclear.    and involves binding and killing of the host cells by spe-  cific adhesin molecules and the action of a pore-forming  protein, amoebapore. The initial superficial ulcer may  deepen into the submucosa and become chronic. Spread  may occur by direct extension, by undermining of the  surrounding mucosa until it sloughs, or by penetration  that can lead to perforation. If the trophozoites gain  access to the vascular or lymphatic circulation, metastases    may occur first to the liver and then by direct extension  or further metastasis to other organs, including the brain.   Cryptosporidium parvum is a ubiquitous coccidian para-  site that causes cryptosporidiosis in humans; however,  other species are known to cause infection in immuno-  compromised patients and in total this genus comprises   Figure 6.7 Cysts are passed in faeces (1). Infection by Entamoeba histolytica occurs by ingestion of mature cysts (2) in faecally  contaminated food, water or hands. Excystation (3) occurs in the small intestine and trophozoites (4) are released, which migrate  to the large intestine. The trophozoites multiply by binary fission and produce cysts (5), which are passed in the faeces (1). Because  of the protection conferred by their walls, the cysts can survive days to weeks in the external environment and are responsible for  transmission. (Trophozoites can also be passed in diarrhoeal stools, but are rapidly destroyed once outside the body, and if ingested  would not survive exposure to the gastric environment.) In many cases, the trophozoites remain confined to the intestinal lumen  (A, non-invasive infection) of individuals who are asymptomatic carriers, passing cysts in their stool. In some patients the  trophozoites invade the intestinal mucosa (B, intestinal disease), or through the bloodstream, extraintestinal sites such as the liver,  brain and lungs (C, extraintestinal disease), with resultant pathological manifestations. It has been established that the invasive and  non-invasive forms represent two separate species, respectively, E. histolytica and E. dispar. However, not all persons infected with  E. histolytica will have invasive disease. These two species are morphologically indistinguishable. Transmission can also occur  through faecal exposure during sexual contact (in which case not only cysts, but also trophozoites could prove infective).    19-20 distinct species. Recently there has been a re-  naming of the major species in this group to reflect the  host specificity of the particular species or genotype. The  life cycle of the parasite is complex but is completed in a  single host. Infection follows the ingestion of oocysts    associated with contaminated water or food. According to  the World Health Organization (WHO) the health signifi-  cance of C. parvum is high due to the persistence of the  organism in the environment. Cattle represent the most  important reservoir of C. parvum but other mammals,    domestic and wild, can be infected and act as carriers of  the disease, even if asymptomatic. It is now known that a  number of genetically distinct subspecies exist that can be  divided into two groups. Group I organisms infect  humans only (soon to be reclassified as C. hominis); group  II organisms infect a wider range of hosts. There have  been several large outbreaks of this infection in the UK  and USA in which water was identified as the initial  vehicle for transmission. Members of this genus are intra-  cellular parasites infecting the intestinal mucosal epithe-  lium. The two major life cycle forms are the oval oocyst  and the sporozoite.   C. parvum infections are often asymptomatic, but  symptoms such as profuse watery diarrhoea, stomach  cramps, nausea, vomiting and fever are typical. The  symptoms can last from several days to a few weeks in  immunocompetent individuals, but in immunocompro-  mised patients infection can become chronic, lasting  months or even years. The mean infective dose for  immunocompetent people is dependent on the strain of  C. parvum but it is considered to be approximately 100  cells, and infants are more vulnerable to infection.  Diarrhoea is a major cause of childhood mortality and  morbidity as well as malnutrition in developing coun-  tries. Cryptosporidium is the third most common cause  of infective diarrhoea in children in such countries, and  consequently it plays a role in the incidence of childhood  malnutrition.   Cryptosporidium infection has a higher nutritional  impact in boys than girls, becausee of the need for micro-  nutrients in boys to build up larger muscle mass.  However, breast-feeding does offer some protection  against infection. In immunocompromised individuals  Cryptosporidium infection causes a severe gastroenteritis,  and often the parasites infect other epithelial tissues  causing pneumonia; the mortality rate due to C. parvum  in AIDS patients is between 50% and 70%. Infection  occurs when oocysts (Figure 6.2f) excyst following envi-  ronmental stimuli (typical intestinal conditions) and  parasitize the epithelial cells which line the intestine wall  (Figure 6.8). After several further stages of the cycle, two  forms of oocyst are produced; soft-walled oocysts reiniti-  ate infection of neighbouring enterocytes while hard-  walled cysts are expelled in the faeces.   Little is known about the mechanism by which these  organisms cause disease. They are known to invade cells  but this process is atypical in that the parasites form a  vacuole just below the epithelial cell membrane. During  infection a variety of changes are seen such as partial  villous atrophy, crypt lengthening and inflammation;  these responses are probably due in part to cell damage    that occurs during the growth of the intracellular forms.  It has also been proposed that parasite enzymes and/or  immune-mediated mechanisms may also be involved. It  should be remembered, however, that cryptosporidiosis  is resolved by the immune system in healthy patients  normally within 3 weeks.   Trichomonas vaginalis is a common sexually transmitted  parasite. Infection rates vary from 10% to 50%, with the  highest reported rates found in the USA. Infections are  usually asymptomatic or mild although symptomatic  infection is most common in women. Trichomonads  are all anaerobes and contain hydrogenosomes. This  organelle is found in very few other anaerobic eukaryotes  and is often termed the ‘anerobic mitochondrion’. A  number of functions have been assigned to it, and it has  been shown to function in the generation of ATP. This  organism does not exhibit a life cycle as only the motile  (flagellate/amoeboid) trophozoite (Figure 6.2g) has been  seen and division is by binary fission. Trichomonads have  a pear-shaped body 7-15 (im long, a single nucleus, three  to five forward-directed flagella, and a single posterior  flagellum that forms the outer border of an undulating  membrane.   Trichomoniasis in women is frequently chronic and is  characterized by vaginal discharge and dysuria. The  inflammation of the vagina is usually diffuse and is char-  acterized by reddening of the vaginal wall and migration  of polymorphonuclear leucocytes into the vaginal lumen  (these form part of the vaginal discharge).   Because there is no resistant cyst, transmission from  host to host must be direct. The inflammatory response in  trichomoniasis is the major pathology associated with this  organism; however, the mechanisms of induction are not  known. It is likely that mechanical irritation resulting  from contact between the parasite and vaginal epithelium  is a major cause of this response but the organism pro-  duces high concentrations of acidic end-products and  polyamines, both of which would also irritate local tissues.   The free-living opportunist amoebas are an often-  forgotten group of protozoans. The two major groups,  Naegleria and Acanthamoeba , infect humans and both  can cause fatal encephalitis. Both types of infections are  rare, with less than 200 cases of Naegleria fowleri infec-  tion recorded worldwide and approximately 100-200    Figure 6.8 Life cycle of Cryptosporidium. Sporulated oocysts, containing four sporozoites, are excreted by the infected host through  faeces and possibly other routes such as respiratory secretions (1). Transmission of Cryptosporidium parvum occurs mainly through  contact with contaminated water (e.g. drinking or recreational water). Occasionally food sources, such as chicken salad, may serve  as vehicles for transmission. Many outbreaks in the USA have occurred in water parks, community swimming pools and daycare  centres. Zoonotic transmission of C. parvum occurs through exposure to infected animals or exposure to water contaminated by  faeces of infected animals (2). Following ingestion (and possibly inhalation) by a suitable host (3), excystation (a) occurs. The  sporozoites are released and parasitize epithelial cells (b, c) of the gastrointestinal tract or other tissues such as the respiratory tract.  In these cells, the parasites undergo asexual multiplication (schizogony or merogony) (d, e, f) and then sexual multiplication  (gametogony) producing microgamonts (male, g) and macrogamonts (female, h). Upon fertilization of the macrogamonts by the  microgametes (i), oocysts , k) develop that sporulate in the infected host. Two different types of oocysts are produced, the  thick- walled oocyst, which is commonly excreted from the host (j) and the thin- walled oocyst (k), which is primarily involved in  autoinfection. Oocysts are infective upon excretion, thus permitting direct and immediate faecal— oral transmission. (From Juranek,  D.D. Cryptosporidiosis. In: Hunter’s Tropical Medicine, 8th edn, ed. G.T. Strickland.)    cases of Acanthamoeba ulcerative keratitis per year. This  disease is commonly associated with contact lens use and  it is thought that infection is caused by a combination of  corneal trauma and dirty contact lenses. Both types of  amoeba produce resistant cysts and Naegleria also exhib-  its a flagellate form. Both Acanthamoeba and Naegleria  are free-living inhabitants of fresh water and soil, but  Naegleria fowleri (the human pathogen) reproduces  faster in warm waters up to 46 °C. Treatment of water by  chlorination or ozonolysis does not entirely eliminate  cysts and both amoebae have been isolated from air-  conditioning units.   Naegleria fowleri is the causative agent of primary  amoebic meningoencephalitis, a rapidly fatal disease that  usually affects children and young adults. In all cases,  contact with amoebae occurs as a result of swimming in  infected fresh water. The organisms enter the brain via  the olfactory tract after amoebae are inhaled or splashed  into the olfactory epithelium. The incubation period  ranges from 2 to 15 days and depends both on the size of  the inoculum and the virulence of the strain. The disease  appears with the sudden onset of severe frontal headache,  fever, nausea, vomiting and stiff neck. Symptoms develop  rapidly to lethargy, confusion and coma and in all cases  to date the patient died within 48-72 hours.   Acanthamoeba castellanii, A. culbertsoni and other  pathogenic Acanthamoeba species can cause opportunist  lung and skin infections in immunocompromised indi-  viduals. Where amoebae spread from such lesions to the  brain, they can cause a slowly progressive and usually  fatal encephalitis. In addition, Acanthamoeba can cause  an ulcerating keratitis in healthy individuals, usually in  association with improperly sterilized contact lenses. The  presence of cysts and trophozoites in alveoli or in multi-    ple nodules or ulcerations of the skin characterizes acan-  thamoebic pneumonitis and dermatitis. Spread of  amoebae to the brain produces an encephalitis, charac-  terized by neurological changes, drowsiness, personality  changes and seizures in the early stages of infection,  which progress to altered mental status, lethargy and  cerebellar ataxia. The end point of infection is usually  coma followed by death of the patient. Acanthamoeba  keratitis is characterized by painful corneal ulcerations  that fail to respond to the usual anti-infective treatments.  The infected and damaged corneal tissue may show a  characteristic annular infiltrate and congested conjuncti-  vae. If not successfully treated, the disease progresses to  corneal perforation and loss of the eye or to a vascular-  ized scar over thinned cornea, with impaired vision.   Mechanisms to control parasitic protozoa are similar to  those utilized for other infectious agents; they can be  divided into non-specific mechanism(s) and specific  mechanism(s) involving the immune system. The best-  studied non-specific mechanisms include those that  affect the entry of parasites into the red blood cell. The  sickle cell haemoglobin trait and lack of the Duffy factor  on the erythrocyte surface make the red cell more resist-  ant to invasion by Plasmodium. These traits are com-  monly found in populations from malaria-endemic  regions. A second example of a non-specific factor is the  presence of trypanolytic factors in the serum of humans  which confer resistance to T. brucei, although non-specific  factors can play a key role in resistance, usually they work  in conjunction with the host’s immune system.   Unlike most other types of infection, protozoan diseases  are often chronic, lasting for months to years. When asso-  ciated with a strong host immune response, this type of  long-term infection is apt to result in a high incidence  of immunopathology. Until recently the importance of  host immune response in controlling many parasite  infections was not fully appreciated, but the impact of  HIV infection on many parasitic diseases has highlighted  this relationship.   Different parasites elicit different humoral and/or  cellular immune responses (Chapter 9). In malaria  and trypanosome infections, antibody appears to play a  major role in immunity, although it would seem that  for many organisms both humoral and cellular immunity  are required for killing of parasites. Cellular immunity  is believed to be the most important mechanism in  the killing of Leishmania and Toxoplasma. Cytokines  are involved in the control of both the immune response  and also the pathology of many parasitic diseases. Helper  (h) and cytotoxic (c) T cells play major roles in the  induction/control of the response. The various subsets  of these produce different profiles of cytokines. For  example, the Thl subset produces gamma interferon  (IFN-y) and interleukin-2 (IL-2) and is involved in cell-  mediated immunity. In contrast, the Th2 subset produces  IL-4 and IL-6, and is responsible for antibody-mediated  immunity. The induction of the correct T-cell response  is key to recovery. The Thl subset and increased IFN-y  are important for the control of Leishmania, T. cruzi and  Toxoplasma infections, whereas the Th2 response is more  important in parasitic infections in which antibody is a  major factor. It is important to recognize that the  cytokines produced by one T-cell subset can up- or  down-regulate the response of other T-cell subsets; IL-4  will down-regulate Thl cells for example. The cytokines  produced by T and other cell types do not act directly on  the parasites but induce changes in the metabolism of  glucose, fatty acid and protein in other host cells.  Cytokines can also stimulate cell division and, therefore,  clonal expansion of T- and B-cell subsets. This can lead  to increased antibody production and/or cytotoxic T-cell  numbers. The list of cytokines and their functions is  growing rapidly, and it would appear that these chemical  messages influence all phases of the immune response.  They are also clearly involved in the multitude of physi-  ological responses (fever, decreased food intake, etc.)  observed in an animal’s response to a pathogen, and in  the pathology that results.   The protozoa can elicit humoral responses in which  antigen-antibody complexes are formed and these can  trigger coagulation and complement systems. Immune  complexes have been found circulating in serum and  deposited in the kidneys where they may contribute to  conditions such as glomerulonephritis. In other tissues  these complexes can also induce localized hypersen-  sitivities. It is thought that this type of immediate  hypersensitivity is responsible for various clinical  syndromes including blood hyperviscosity, oedema and  hypotension.   Another important form of antibody-mediated  pathology is autoimmunity. Autoantibodies to a number  of different host antigens (e.g. red blood cells, laminin,  collagen and DNA) have been demonstrated. These  autoantibodies may play a role in the pathology of para-  sitic diseases by exerting a direct cytotoxic effect on the  host cells, e.g. autoantibodies that coat red blood cells  produce haemolytic anaemia; they may also cause damage  through a build-up of antigen-antibody complexes.   Many parasites can elicit the symptoms of disease  through the action of their surface molecules such as the  pore-forming proteins of E. histolytica that induce  contact-dependent cell lysis, and trypanosome glycopro-  teins that can fix and activate complement resulting in  the production of biologically active and toxic comple-  ment fragments. A range of parasite-derived enzymes  such as proteases and phospholipases can cause cell  destruction, inflammatory responses and gross tissue  pathology.   Parasites exhibit a number of mechanisms that allow  them to evade host immune response. Two such mecha-  nisms are displayed by trypanosomes, which are able to  exhibit both antigenic masking and antigenic variation.  Masking means the parasite becomes coated with host  components and therefore is not recognized as foreign.  In addition parasites can undergo antigenic variation  which results in surface antigens being changed during  the course of an infection. The ultimate goal of these is  that the hosts immune response is evaded. Parasites can  also suppress the host’s immune response either to the  parasite specifically or to foreign antigens in general.  This, however, can cause a number of problems, as  general immune suppression may make the individual  more susceptible to secondary infection.   The detection of parasites in the host and environment  (including foods) is vital for proper treatment and also  for understanding mechanisms of transmission. In addi-  tion the use of molecular typing is of major benefit in  studying epidemiology and for some of the zoonotic  potential of parasites.   It is not always easy or possible to culture parasites, so the  detection of these organisms in samples requires the use  of methods such as microscopy and DNA amplification.  The most commonly used method involves microscopy  and this can be applied to clinical as well as environmental  samples. For some organisms this approach remains the  ‘gold standard’ for detection and identification. The  advantages of microscopy are speed, cost and availability.  In addition, fluorescent-labelled antibodies raised to  species-specific antigens can be utilized to help identify  organisms to species or subspecies level, and other stains  can be used to help determine the viability of cells.  Examples of such stains include fluorescein diacetate  which is cleaved by esterases in viable cells, releasing fluo-  rescein (gives a green fluorescence) and propidium iodide  which is excluded from viable cells but taken up by cells  with damaged membranes (gives red fluorescence).  However, there are a number of obvious limitations  including the requirement for well-trained staff to  perform the microscopy, limits of sensitivity of the method  and, for some parasites, difficulty in differentiating species  based on morphology. In addition, the parasite may not  be present in easily available samples. Thus a variety of  other approaches are used to help in detection.   For the major protozoan parasites a number of  immunology-based methods exist to detect the presence  of organisms in clinical samples. These include the use of  agglutination, complement fixation and enzyme-linked  immunosorbent assay (ELISA). The most commonly  used method is the ELISA and this can be used to detect  the presence of antigens in samples (direct assay) or  antigen-specific antibodies in patients’ serum (indirect).  The ELISA method has a number of advantages in that  it can be automated and has good specificity and sensitiv-  ity. Although a number of ELISA-based detection kits are  available their cost can be a limitation to use especially in  those developing countries where parasitic infections are    endemic. The use of indirect ELISA methods for deter-  mining the infection status of the patient can also be  difficult because previous exposure to the parasite can  cause problems and the immune status of the patients  can also impact on antibody production.   Nucleic acids, DNA or RNA, are found in all types of  microorganism and the sequences of these molecules can  be used to help in the identification of species and indi-  viduals. The robust nature of DNA has allowed research-  ers to use these sequence in a variety applications  including forensics, archaeology (sequences from animal  tissues over 100 years old have routinely been deter-  mined) and epidemiology. The advent of highly sensitive  DNA amplification technologies such as the polymerase  chain reaction (PCR) has allowed the development of  molecular tools for the identification and detection of  parasites. Initial work on these techniques focused on  their ability to help define species and many of these  studies utilized the ribosomal DNA genes and were  very successful. Using these genes it was possible to dis-  criminate many genera and species, but the genes lacked  the level of sequence variation required for separation  below the level of species (important for some patho-  genic organisms and for epidemiological studies). In  addition ribosomal DNA sequencing was also not  always appropriate for identifying species that are highly  diverse. Other DNA markers that have been used  included genes that encode for metabolic enzymes and  structural proteins. For example Cryptosporidium can be  speciated by using a combination of target genes includ-  ing the Cryptosporidium oocyst wall protein (COWP),  heat shock protein (hsp70), dihydrofolate reductase  (DHFR) and 18S ribosomal DNA. Complete genomes  are now available for the major protozoan parasites  and this has helped in the development of improved  methods for species identification. These genomes can be  accessed at http://eupathdb.org/eupathdb/ and this  website links to many others resources including the  National Centre for Biotechnology Information (http://  www.ncbi.nlm.nih.gov/). Other DNA-based techniques  are now available such as the multiplex PCR (amplifica-  tion of more than one gene at a time) and quantitative  real-time PCR (qPCR), and these have improved  specificity and sensitivity in detection and increased the  usefulness of DNA technology for the detection of para-  sites in environmental as well as clinical samples. It is also  possible to assess the viability of protozoan parasites    using reverse transcription PCR (RT-PCR). This is due to  the fact that mRNAs degrade quickly once parasites are  killed. However, it should be noted that increasing evi-  dence exists to show that mRNA can survive in some cells  for up to 24 hours   A recent method, DNA microarray, is a fast developing  technology. This is a ‘chip’ based system that measures the  binding of target DNA sequences (parasite) from samples  (fluorescently labelled using PCR) to complementary  sequences bound to the chip surface. The binding of  parasite sequence DNA and the amount that is bound can  be detected using fluorescence (Figure 6.9). In theory this  technique allows the detection of multiple parasite  species at the same time in one sample.   Alternative methods are being developed for the detec-  tion of parasites; these include mass spectrometry (MS)  and biosensors. MS can be used by identifying known  surface characteristics of appropriate life cycle forms.  This type of MS uses low-energy electrons to generate a  fingerprint of the cells. Other mass spectrometry methods  can generate fingerprints based on parasite-derived pep-  tides and metabolites. These can be detected easily with  a high level of sensitivity and can be used in complex  mixtures that would be present in clinical and environ-  mental samples. Over the past 10 years the production of  kits for near-patient testing has driven the development  of detection technology and this has generated interest in    the use of biosensors. These sensors detect the presence  of ‘marker molecules’ that are specific to a particular  pathogen and utilize mechanical, electrochemical or pie-  zoelectric methods to generate a signal that can be  detected. Such sensors can be combined onto ‘chips’ to  detect a range of pathogens.   Diagnosis of parasitic infection is dependent on the dem-  onstration of the parasite in appropriate samples. The  type of samples can vary from blood where preparation  can be minimal (e.g. sample of smears for microscopy)  to faeces or intestinal aspirates. Faecal samples require  more processing: for example, fresh or preserved stools  can be concentrated to increase the yield of the parasites  by sedimentation using the formol-ether or formol-ethyl  acetate techniques or by faecal parasite flotation method  using copper sulphate. These concentrates can be stained  for microscopy. For the extraction of DNA from samples  a number of methods exist; however, there are various  resin-based kits that will separate DNA from complex  biological samples.   Many parasites have life cycle forms that can survive in  the environment. These act to initiate infections in sus-  ceptible hosts; for some parasites this only requires inges-   Binding of the fluorescent labelled  amplified target DNA to immobilized  complementary DNA strands  Binding is detected by monitoring  fluorescence    tion of no more than 10 cells or cysts, thus the ability to  detect them in the environment is vital. This section  focuses on water and foods as they are often major routes  of transmission for parasites such as Giardia and  Cryptosporidium which are included in the monitoring  standards for potable waters (drinking-waters) by regula-  tory bodies in both the European Union and the USA. In  addition, food and pharmaceutical companies that utilize  water in their processes are required to test for a number  of pathogens, including waterborne parasites. Foods can  also be a source of infection and methods for the detec-  tion of pathogens in foods are as important as those for  water samples.   As outlined in section 6.1, a number of methods can  be used to detect and to identify parasites but the per-  formance of these techniques will often be impaired  when used on real samples. This is due to a number of  factors that include:   • high levels of protein, lipids and carbohydrates in  samples (foods).   Often these factors combine to make detection diffi-  cult. For example, if levels of a parasite in water are low  (<1/100 ml) several litres must be collected and analysed.  This is possible, but would require the sample volume to  be reduced for analysis. This can be achieved either by  filtration or centrifugation. If the sample contains  sediment, both of these approaches are difficult; however,  methods such as tangential flow filtration (fluid  passed parallel to the filter) can be used. Waters can  also contain organic acids that can inhibit the PCR  process so these must be removed from the sample  before analysis. DNA-based technology may also fail to  detect the presence of a parasite if the DNA present in  the sample is degraded, e.g. by other organisms. In  foods, the process can be even more complex and  separation of parasites from the foodstuff is often the  factor that impacts most on the limits (sensitivity) of the  method.   It is now clear that the best approach for the successful  control of parasites requires the integration of a number  of methods which draw upon our increasing understand-    ing of the parasites’ life cycle, epidemiology and host  response to infection.   The origins of chemotherapy are closely linked to the  development of antiparasitic agents, but there has been  slow progress in the development of new and novel anti-  protozoal agents over the past 30 years. Recently, with  the support of the WHO and government-sponsored  research, new antiparasitic drugs are slowly coming into  the market. Interestingly there are still a number of pro-  tozoan parasite infections such as cryptosporidiosis for  which there is no effective treatment.   For many of the commonly used antiprotozoal drugs the  modes of action and mechanisms of selective toxicity  are well understood, although for some the precise  mechanism remains unclear. The most common antipro-  tozoal drugs and their modes of action are shown in  Table 6.1.   Considering the drugs in relation to modes of action,  dapsone and the sulphonamides block the biosynthesis  of tetrahydrofolate by inhibiting dihydropteroate syn-  thetase, while the 2,4-diamino-pyrimidines (proguanil  and pyrimethamine) block the same pathway but at a  later step catalysed by dihydrofolate reductase.   The drugs that interfere with nucleic acid synthesis  include those that bind to the DNA and intercalate with  it such as chloroquine, mefloquine and quinine, and also  pentamidine, which is unable to intercalate but probably  interacts ionically. Other compounds such as benznida-  zole and metronidazole may alkylate DNA through acti-  vation of nitro groups via a one-electron reduction step.  Several of these compounds, however, including chloro-  quine, mefloquine, quinine and metronidazole, have  more than one potential mode of action. Chloroquine,  for example, inhibits the enzyme haem polymerase,  which functions to detoxify the cytotoxic molecule haem  that is generated during the degradation of haemoglobin.  Metronidazole is reduced in the parasite cell and forms a  number of cytotoxic intermediates, which can cause  damage not only to DNA but also to membranes and  proteins.   Tetracycline targets protein synthesis in Plasmodium  via a similar mechanism to that seen in bacteria: inhibi-  tion of chain elongation and peptide bond formation.  Eflornithine interferes with the metabolism of the amino  acid ornithine in T. brucei gambiense by acting as a suicide  substrate for the enzyme ornithine decarboxylase.    Albendazole has recently been shown to have signifi-  cant antigiardial activity, although its mode of action is  unclear. In Leishmania, amphotericin B binds to ergos-  terol in the membrane making it leaky to ions and small  molecules (e.g. amino acids), while the anti-protozoal  drugs atovaquone and primaquine bind to the cyto-  chrome focj complex and inhibit electron flow. The anti-  trypanosomal drug melarsaprol is most likely to act by  blocking glycolytic kinases, especially the cytoplasmic  pyruvate kinase, although it may also disrupt the reduc-  tion of trypanothione.   As with bacteria, drug resistance in some parasites such  as Plasmodium is a major problem and tends to appear    where chemotherapy has been used extensively. This  problem is exacerbated by the fact there are so few drugs  available for the control of some parasites, which utilize  the same five basic resistance mechanisms that are dis-  played by bacteria: ( 1 ) metabolic inactivation of the drug;  (2) use of efflux pumps; (3) use of alternative metabolic  pathways; (4) alteration of the target; (5) elevation of the  amount of target enzyme.   The early success in malaria control can be attributed  to the use of professional spray teams who treated the  inside of huts with DDT, without any direct involvement  of the infected population. However, the problem with  pesticides like DDT is that they lack specificity, and as    application is not always well directed there is often  destruction of a wide range of insects, which may have  undesirable side effects. Further problems include the  accumulation of pesticide residues in the food chain and  pesticide resistance in the target organism. Window  screens and bed nets do prevent mosquito bites, however,  and there has been a lot of interest in using bed nets  impregnated with insecticide. Environmental control was  a major strategy used before the development of modern  insecticides. A good example of this is mosquito control  through the removal of breeding sites by drainage, land  reclamation projects, removal of vegetation overhanging  water, speeding up water flow in canals, and periodic  drainage and drying out of canals. Life cycle forms that  enter the water system, such as cysts and oocysts of  Giardia and Cryptosporidium, can present a major public  health problem. These forms are often resistant to  common disinfection methods and require physical  removal from waters. Cysts and oocysts can be destroyed  by use of proper sewage treatments such as anaerobic  digestion, but these systems require regular maintenance  in order to remain effective.   Biological control is an active but developing area.  Genetic control of insect vectors, particularly the use of  irradiated sterile males, has been widely publicized, and  the release of chemically sterile males has been attempted  to control anopheline mosquitoes. Other similar methods  include the release of closely related species within  the environment in order to produce sterile hybrids.  Genetically modified mosquitoes are currently being  developed that are resistant to Plasmodium infection, and  larvivorous fish have also been employed for mosquito  control; other organisms considered for the same purpose  include bacteria, fungi, nematodes and predatory  insects. One of the best-studied agents is the bacterium  Bacillus thuringiensis; the spore or the isolated toxin from  this species can be used as a very effective and specific  insecticide.   Where exposure to infection is likely to occur, killing the  parasite as it enters the host is a sensible approach to  control. There are two options available, chemoprophy-  laxis or vaccination. Unfortunately long-term chemo-  therapy can have adverse side effects, and in the absence  of symptoms members of the at-risk population may fail  to take the treatment. Vaccination would seem to be the  ideal method of parasite control, as lifelong resistance    may result from just a single treatment. Despite a huge  amount of effort, the only successful parasite vaccines are  those for the control of veterinary parasites. However,  there has been significant success with the development  of recombinant vaccines for the control of malaria. The  recent development of DNA vaccines may be of use in  the control of parasites. In this method the DNA encod-  ing an important parasite protein is injected into host  cells and the foreign ‘vaccinating’ protein is synthesized  in or on the surface of the cell. This intracellular foreign  protein enters the cell’s major histocompatibility complex  (MHC) class 1 pathway resulting in a cell-mediated  immune response. In contrast, a protein that is extracel-  lular enters the MHC class 2 pathway, which results pri-  marily in an antibody or humoral response.   Despite advances in technology, few commercially  available parasite vaccines exist and there are none for use  in humans. Several vaccines are being evaluated in clini-  cal trials and most of these are against malaria.  Information about these can be found at the Malaria  Vaccine Initiative website (www.malariavaccine.org). In  addition there are vaccines being evaluated against  other parasites, for example a Leishmatiia vaccine co-  administered with BCG (bacillus of Calmette and Guerin)  is under trial. Recently, DNA and viral vector-based vac-  cines have been tested in clinical trials including one uti-  lizing an adenovirus human serotype 35-based vector  encoding the malarial circumsporozoite protein.   The figures for this chapter originate in the copyright-  free DPDx website maintained by the United States  Centre for Disease Control Division of Parasitic Diseases;  this source is gratefully acknowledged.   Gilles, H.M. (2000) Protozoal Diseases: a Comprehensive Guide.  Hodder Arnold, London.   Wiser, M. (2010) Protozoa and Human Disease. Garland Science,  New York.   Rune Stensvold, Marianne Lebbad, Jaco J. Verweij, The impact  of genetic diversity in protozoa on molecular diagnostics.  Trends in Parasitology, Volume 27, Issue 2, February 2011,  Pages 53-58.   Nicole Mideo, Parasite adaptations to within-host competition,  Trends in Parasitology, Volume 25, Issue 6, June 2009, Pages  261-268.   Microorganisms are ubiquitous, and most of them are  free-living and derive their nutrition from inert organic  and inorganic materials. The association of humans with  such microorganisms is generally harmonious, as the  majority of those encountered are benign and, indeed, are  often vital to commerce, health and a balanced ecosystem.  The ability of bacteria and fungi to establish infections of  plants, animals and humans varies considerably. Some    are rarely, if ever, isolated from infected tissues, while  opportunist pathogens (e.g. Pseudomonas aeruginosa or  Staphylococcus epidermidis) can establish themselves only  in compromised individuals. Only a few species of bac-  teria may be regarded as obligate pathogens, for which  animals or plants are the only reservoirs for their exist-  ence (e.g. Neisseria gonorrhoeae, Mycobacterium tubercu-  losis and Treponema pallidum). Viruses (Chapter 5), on  the other hand, must parasitize host cells in order to  replicate and are therefore inevitably associated with  disease. Even among the viruses and obligate bacterial    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    pathogens the degree of virulence varies, in that some  (particularly the bacteria) are able to coexist with the host  without causing overt disease (e.g. Staph, aureus), while  others will always cause some detriment to the host (e.g.  rabies virus). Organisms such as these invariably produce  their effects, directly or indirectly, by actively growing on  or in the host tissues.   Other groups of microorganisms may cause disease  through ingestion of substances (toxins) produced during  microbial growth on foods (e.g. Clostridium botulinum,  botulism; Bacillus cereus, vomiting). In this case, the  organisms themselves do not have to proliferate in the  host for the effects of the toxin to be manifested.   Animals and plants constantly interact with bacteria  present within their environment. For an infection to  develop, such microorganisms must remain associated  with host tissues and increase their numbers more  rapidly than they can be either eliminated or killed. This  balance relates to the ability of the bacterium to mobilize  nutrients and multiply in the face of innate defences  and a developing immune response by the now compro-  mised host.   The greater the number of bacterial cells associated  with the initial challenge to the host, the greater will be  the chance of disease. If the pathogen does not arrive at  its ‘portal of entry’ to the body or directly at its target  tissues in sufficient number, then an infection will not  ensue. The minimum number of viable microorganisms  that is required to cause infection and thereby disease is  called the minimum infective number (MIN). The MIN  varies markedly between the various pathogens and is  also affected by the general health and immune status of  the individual host. The course of an infection can be  considered as a sequence of separate events that includes  initial contact with the pathogen, its consolidation and  spread between and within organs and its eventual elimi-  nation (Figure 7.1). Growth and consolidation of the  microorganisms at the portal of entry commonly involves  the formation of a microcolony (biofilm, see Chapters 3  and 8). Biofilms and microcolonies are collections of  microorganisms that are attached to surfaces and envel-  oped within exopolymers (biofilm matrix) composed of  polysaccharides, glycoproteins and/or proteins. Growth  within the matrix not only protects the pathogens against  opsonization and phagocytosis by the host but also mod-  ulates their microenvironment and reduces the effective-  ness of many antibiotics. The localized high cell densities  present within the biofilm communities also initiate pro-  duction, by the colonizing organism, of extracellular  virulence factors such as toxins, proteases and siderophores    (low molecular weight ligands responsible for the solubi-  lization and transport of iron(III) in microbial cells).  These are associated with a phenomenon termed quorum  sensing (see section 4.2) and help the pathogen to combat  the host’s innate defences and also promote the acquisi-  tion of nutrients.   Viruses are incapable of growing extracellularly and  must therefore rapidly gain entry to cells (normally epi-  thelial) at their initial site of entry. Once internalized in  the non-immune host, they are to a large extent protected  against the non-specific host defences. Following these  initial consolidation events, the organisms may expand  into surrounding tissues and/or disperse, via the blood,  plasma, lymph or nerves, to distant tissues in order to  establish secondary sites of infection or to consolidate  further. In some instances microorganisms are able to  colonize the host indefinitely and remain viable for many  years (e.g. the herpesvirus varicella zoster which causes  chickenpox, and herpes simplex virus 1 responsible for  cold sores), but more generally they succumb to the  heightened defences of the host, and in order to survive  must either infect other individuals or survive in the  general environment.   Even though some parts of the human body are free from  microorganisms ( axenic state), the body harbours mil-  lions of mutualistic and commensal symbionts. Each of  us unwittingly carries a population of bacterial cells on  our skin and in our mouth and digestive tract that out-  numbers cells carrying our own genome by approxi-  mately ten to one. Mutualistic relationships occur when  both organisms (microbial and host cell) benefit from the  interactions, whereas commensalism is a state in which  one member of the relationship benefits without signifi-  cantly affecting the other. Microorganisms that colonize  body surfaces (internal and external) without normally  causing disease constitute the microbiome. The microbi-  ome comprises a number of distinct microbiotas which  are characteristic of the region of the body colonized.   The microbiome is present throughout life and may  comprise predominantly bacteria, with some fungi  and protozoa, the majority of which are commensal. The  microbiome begins to develop when the amniotic mem-  brane surrounding the unborn child ruptures, allow-  ing contact with vaginal, faecal and skin-associated  microorganisms from the mother during childbirth.  Microorganisms enter the infant’s mouth and nose  during passage through the birth canal, colonization of  the upper respiratory tract occurs with the first breath of  air, and the beginning of the colon microbiota occurs  during feeding. The development of the resident micro-  biota is therefore initiated during the first few months of  life. By comparison, transient microbiotas remain in the  body for only a few hours, days or months. They are  found in the same locations as the resident flora, but  cannot persist because of their inability to compete with  the microbiome, to resist elimination by the body’s  defence mechanisms or to tolerate the chemical and  physical changes encountered.   Changes in relative abundance of normal microbiota,  for whatever reason (e.g. major changes to diet, antibiotic  treatment, hormonal changes, chemo- or radiotherapy),  may allow one or more members of the microbiome to  become opportunistic pathogens. For example, reduc-  tions in numbers of protective lactobacilli within the  vagina brought about through antibiotic use can allow  Candida albicans, a minority member of the normal  microbiota, to grow more prolifically, resulting in an  opportunistic vaginal yeast infection such as vaginal can-  didiasis. Following appropriate treatment, the normal  flora is typically re-established in adults. In some areas of    the body, such as the gastrointestinal tract, it is claimed  that the recolonization by desired species can be encour-  aged by administration of probiotics. These are live cul-  tures of intestinal bacteria that are marketed as conferring  a health benefit and preventing digestive problems.  Prebiotics, (normally fibre-like carbohydrates) that rep-  resent preferred or selective growth substrates for probi-  otic bacteria already present within the digestive tract are  also of demonstrable benefit.   The part of the body that is most widely exposed to  microorganisms is the skin. Intact skin is usually imper-  vious to microorganisms, and its surface is of acid pEI  and contains relatively few nutrients that are favourable  for microbial growth. The vast majority of organisms  falling on to the skin surface will die, while the survivors  must compete with the commensal microflora for  nutrients in order to grow. These commensals, which  include coryneform bacteria, staphylococci and yeasts,  derive nutrients from compounds like urea, hormones  (e.g. testosterone) and fatty acids found in the apocrine  and epocrine secretions. Such organisms are highly  adapted to growth in this environment and will normally  prevent the establishment of chance contaminants of the  skin. Infections of the skin itself, such as ringworm  ( Trichophyton mentagrophytes ) and warts (human papil-  lomavirus, HPV), rarely, if ever, involve penetration of  the epidermis. Infection can, however, occur through the  skin following trauma such as burns, cuts and abrasions  and, in some instances, through insect or animal bites or  the injection of contaminated medicines. In recent years  extensive use of intravascular and extravascular medical  devices and implants has led to an increase in the occur-  rence of hospital-acquired infection. Commonly, these  infections involve the growth of skin commensals such as  Staph, epidermidis when associated with devices that pen-  etrate the skin barrier. The organism grows as an adhesive  biofilm on the surfaces of the device, and infection arises  either from contamination of the device during its  implantation or by growth along it of the organism from  the skin. In such instances the biofilm sheds bacterial cells  to the body and may give rise to a bacteraemia (the pres-  ence of bacteria in the blood).   The weak spots, or Achilles heels, of the body occur  where the skin ends and mucous epithelial tissues begin  (mouth, anus, eyes, ears, nose and urinogenital tract).    These mucous membranes present a much more favour-  able environment for microbial growth than the skin, in  that they are warm, moist and rich in nutrients. Such  membranes, nevertheless, possess certain characteristics  that allow them to resist infection. Most of them, for  example, possess their own highly adapted microbiotas  (see section 2) which reduce the chances of infection by  any invading organisms through a process termed coloni-  zation resistance. The resident flora varies greatly between  different sites of the body, but is usually common to  particular host species. Each site can be additionally pro-  tected by physicochemical barriers such as extreme acid  pH in the stomach and bile salts in the large bowel, the  presence of freely circulating non-specific antibodies  and/or opsonins, and/or by macrophages and phagocytes  (see Chapter 9). All infections start from contact between  these tissues and the potential pathogen. Contact may be  direct, from an infected individual to a healthy one; or  indirect, and may involve inanimate vectors such as soil,  food, drink, air and airborne particles. These may directly  contact the body or be ingested or inhaled or enter  wounds via infected bed linen and clothing. Indirect  contact may also involve animal vector intermediates  (carriers).   Air contains a large amount of suspended organic matter  and, in enclosed occupied spaces, may hold up to 1000  microorganisms/m 3 . Almost all of these airborne organ-  isms are non-pathogenic bacteria and fungi, of which the  average person inhales approximately 10000/day. The  respiratory tract is protected against this assault by a  mucociliary blanket that envelops the upper respiratory  tract and nasal cavity. Both present a tortuous path down  which microbial particles travel and impact on these sur-  faces to become trapped within an enveloping blanket of  mucus. Beating cilia move the mucus coating to the back  of the throat where it, together with adherent particles, is  swallowed. The alveolar regions of the lower respiratory  tract have additional protection in the form of alveolar  macrophages. To be successful, a pathogen must avoid  being trapped in the mucus and swallowed; if deposited  in the alveolar sacs it must avoid phagocytosis by macro-  phages, or if phagocytosed it must resist subsequent  digestion by them. The possession of surface adhesins,  specific for epithelial receptors, aids attachment of the  invading microorganism and avoidance of removal by the  mucociliary blanket. Other strategies include the export  of ciliostatic toxins (i.e. Corynebacterium diphtheriae)  that paralyse the cilial bed. As the primary defence of the    respiratory tract is the mucociliary blanket, it is easy to  envisage how infection with respiratory viruses (i.e. influ-  enza virus, which kills respiratory epithelia) or the  chronic inhalation of tobacco smoke, which increases  mucin production and decreases the proportion of cili-  ated epithelial cells, increases the susceptibility of indi-  viduals to infection.   The intestinal tract must contend with whatever it is  given in terms of food and drink. The extreme acidity and  presence of digestive enzymes in the stomach will kill  many of the bacteria challenging it, and the gastrointes-  tinal tract carries its own commensal flora of yeast and  lactobacilli that afford protection by, for example, com-  peting with potential pathogens like Helicobacter pylori.  Bile salts are mixed with the semidigested solids exiting  from the stomach into the small intestine. These salts not  only neutralize the stomach acids but also represent bio-  logical detergents or surfactants that are able to solubilize  the outer membrane of many Gram-negative bacteria.  Consequently, the small intestine is normally colonized  by lower numbers of bacteria than the colon. The lower  gut on the other hand is highly populated by commensal  microorganisms (10 12 g _1 gut tissue) that are often associ-  ated with the intestinal wall, either embedded in layers of  protective mucus or attached directly to the epithelial  cells or attached to particulate food residues. The patho-  genicity of incoming bacteria and viruses depends on  their ability to survive passage through the stomach and  duodenum and their capacity for attachment to, or pen-  etration of, the gut wall, despite competition from the  commensal flora and the presence of secretory antibodies  (Chapter 9).   In healthy individuals, the bladder, ureters and urethra  are sterile, and sterile urine constantly flushes the urinary  tract. Organisms invading the urinary tract must avoid  being detached from the epithelial surfaces and washed  out during urination. Because the male urethra is long  (c.20cm), bacteria must be introduced directly into the  bladder, possibly through catheterization, in order to ini-  tiate an infection. The female urethra is much shorter  (c. 5 cm) and is more readily traversed by microorganisms  that are normally resident within the vaginal vault.  Bladder infections are therefore much more common  in the female. Spread of the infection from the bladder to  the kidneys can easily occur through reflux of urine  into the ureter. As for the implantation of devices across    the skin barrier (above), long-term catheterization of the  bladder will promote the occurrence of bacteriuria (the  presence of bacteria in the urine) with all of the associ-  ated complications.   Lactic acid in the vagina gives it an acidic pH (5.0);  this, together with other products of metabolism, inhibits  colonization by most bacteria, except some lactobacilli  that constitute the commensal flora. Other types of bac-  teria are unable to establish themselves in the vagina  unless they have become extremely specialized. These  species of microorganism tend to be associated with  sexually transmitted infections.   The conjunctiva is usually free of microorganisms and  protected by the continuous flow of secretions from lach-  rymal and other glands, and by frequent mechanical  cleansing of its surface by the eyelid periphery during  blinking. Lachrymal fluids contain a number of inhibitory  compounds, together with lysozyme, that can enzymically  degrade the peptidoglycan of Gram-positive bacteria  such as staphylococci. Damage to the conjunctiva, caused  through mechanical abrasion or reductions in tear flow,  will increase microbial adhesion and allow colonization  by opportunist pathogens. The likelihood of infection is  thus promoted by the use of soft and hard contact lenses,  physical damage, exposure to chemicals, or damage and  infection of the eyelid border (blepharitis).   To be successful, a pathogen must be able to survive at its  initial portal of entry, frequently in competition with the  commensal flora and generally while subject to the atten-  tion of macrophages and wandering white blood cells.  Such survival invariably requires the organism to attach  itself firmly to the epithelial surface, eventually forming  a biofilm. The initial attachment must be highly specific  in order to displace the commensal microflora, and sub-  sequently governs the course of an infection. Attachment  can be mediated through provision, on the bacterial  surface, of adhesive substances such as mucopeptide and  mucopolysaccharide slime layers, fimbriae (Chapter 3),  pili (Chapter 3) and agglutinins (Chapter 9). These are  often highly specific in their binding characteristics, dif-  ferentiating, for example, between the tips and bases of  villi in the large bowel and the epithelial cells of the upper,  mid and lower gut. Secretory antibodies, which are  directed against such adhesions, block the initial attach-   The outcome of the encounter between the tissues and  potential pathogens is governed by the ability of the  microorganism to multiply at a faster rate than it is  removed from those tissues. Factors that influence this  are the organism’s rate of growth, the initial number  arriving at the site and their ability to resist the efforts of  the host tissues at removing/starving/killing them. The  outcome of an encounter between a microorganism and  a host can therefore be described as a balance between  the accumulation of the pathogen and its elimination by  the host.   The definition of virulence (i.e. the degree of patho-  genicity caused by a microorganism) for pathogenic  microorganisms must include the MIN. This will vary  between individuals, but will invariably be lower in com-  promised hosts such as those who are catheterized, dia-  betics, smokers and cystic fibrosis patients, and those  suffering trauma such as malnutrition, chronic infection  or physical damage. A number of the individual factors  that contribute towards virulence are discussed below.   Because the initial inoculum of a pathogen is usually too  small to cause immediate damage to the host, it must  acquire sufficient nutrients to allow it to multiply and  increase in number. Not all nutrients, vitamins or growth  factors are soluble and present in adequate quantities to  allow pathogens to multiply. Moreover, trace elements  may also be in short supply and can influence establish-  ment of the pathogen. One such example is that of iron  (Fe 3+ ) which is essential for microbial growth and func-  tion. Normally this is complexed with host iron-binding  proteins such as lactoferrin or transferrin, resulting in  insufficient iron being made available to the pathogen. In  order to survive and multiply, pathogens need to be able  to compete with the host (and normal microbiota) for  iron. Some bacteria can do this through production of  iron-chelating compounds called siderophores which have  a greater affinity for iron than the host’s iron-binding  proteins, while others secrete hydrolytic enzymes that  release iron from the host. Some organisms, such as Ps.  aeruginosa, possess three or more different mechanisms  of iron capture, giving them great versatility.   Cell attachment, and subsequent biofilm formation, is a  means by which pathogens can remain in a favourable  environment (i.e. one where there are plenty of nutrients)    without getting washed away. As a consequence, bacterial  cell numbers and activities can become quite high.  Biofilms can form on any surface (e.g. soft tissue, bone,  medical implants) and may contain only one or two  species (e.g. Staph, aureus mediated osteomyelitis) or  more commonly several species of bacteria (e.g. dental  plaque). Hence, biofilms may be considered as a func-  tional microbial community. Within a biofilm intracel-  lular signalling molecules (e.g. N-acyl homoserine  lactones) are produced that when sufficient (threshold)  concentrations are reached, up-regulate biofilm-specific  genes. This process is known as quorum sensing, and is  responsible for the formation and maintenance of the  biofilm.   Once formed, biofilms can be extremely difficult, if not  impossible, to remove. Their size and morphology can  help protect the underlying cells, resisting physical forces  or removal, phagocytosis and penetration of toxic mole-  cules such as antibiotics. In addition, biofilms allow cells  to live in close proximity to each other, thereby facilitating  intercellular communication and genetic exchange.  Finally, due to their profound resistance, biofilms provide  foci of infection which often can only be removed by  surgery.   Most bacterial infections confine themselves to the  surface of epithelial tissue (e.g. Bordetella pertussis,  Corynebacterium diphtheriae. Vibrio cholerae). This is, to  a large extent, a reflection of their inability to combat that  host’s deeper defences. Survival at these sites is largely due  to firm attachment to the epithelial cells. Such organisms  manifest disease through the production and release of  toxins (see below).   Other groups of organisms regularly establish systemic  infections after traversing the epithelial surfaces (e.g.  Brucella abortus. Salmonella enterica serovar Typhi,  Streptococcus pyogenes). This property is associated with  their abilities either to gain entry into susceptible cells  and thereby enjoy protection from the body’s defences,  or to be phagocytosed by macrophages or polymorphs  and yet resist their lethal action and multiply within  them. Other organisms are able to multiply and grow  freely in the body’s extracellular fluids. Microorganisms  have evolved a number of different strategies that allow  them to suppress the host’s normal defences and thereby  survive in the tissues.   Growth of microorganisms releases cellular products into  their surrounding medium, many of which cause non-    specific inflammation associated with dilation of blood  vessels. This increases capillary flow and access of phago-  cytes to the infected site. Increased lymphatic flow from  the inflamed tissues carries the organisms to lymph  nodes where further antimicrobial and immune forces  come into play (Chapter 9). Many of the substances that  are released by microorganisms in this fashion are chem-  otactic towards polymorphs that tend, therefore, to  become concentrated at the site of infection: this is in  addition to the inflammation and white blood cell accu-  mulation that is associated with antibody binding and  complement fixation (Chapter 9). Many organisms have  adapted mechanisms that allow them to overcome these  initial defences. Thus, virulent strains of Staph, aureus  produce a mucopeptide (peptidoglycan), which sup-  presses early inflammatory oedema, and related factors  that suppress the chemotaxis of polymorphs.   Resistance to phagocytosis is sometimes associated with  specific components of the cell wall and/or with the pres-  ence of capsules surrounding the cell wall. Classic exam-  ples of these are the M-proteins of the streptococci, the  polysaccharide capsules of the pneumococci and the  alginate-like slime associated with Ps. aeruginosa infec-  tions of the cystic-fibrotic lung. The acidic polysaccharide  K-antigens of Escherichia coli and S. enterica serovar  Typhi behave similarly, in that (1) they can mediate  attachment to the intestinal epithelial cells, and (2) they  render phagocytosis more difficult. Generally, possession  of an extracellular capsule and/or slime will reduce the  likelihood of phagocytosis.   Microorganisms are more readily phagocytosed when  coated with antibody (opsonized). This is due to the  presence on the white blood cells of receptors for the  Fc fragment of IgM and IgG (discussed in Chapter 9).  Avoidance of opsonization will clearly enhance the  chances of survival of a particular pathogen. A substance  called protein A is released from actively growing strains  of Staph, aureus, which acts by non-specific binding to  IgG at the Fc region (see also Chapter 9), at sites both  close to, and remote from, the bacterial surface. This  blocks the Fc region of bound antibody masking it from  phagocytes. Protein A-IgG complexes remote from the  infection site will also bind complement, thereby deplet-  ing it from the plasma and negating its actions near to  the infection site.   Death of microorganisms following phagocytosis can be  avoided if the microorganisms are not exposed to the    killing and digestion processes within the phagocyte. This  is possible if fusion of the lysosomes with phagocytic  vacuoles can be prevented. Such a strategy is employed  by virulent Mycobacterium tuberculosis, although the  precise mechanism is unknown. Other bacteria seem able  to grow within the vacuoles despite lysosomal fusion  (. Listeria monocytogenes, S. enterica serovar Typhi). This  can be attributed to cell wall components that prevent  access of the lysosomal substances to the bacterial  membranes (e.g. Brucella abortus, mycobacteria) or to the  production of extracellular catalase which neutralizes  the hydrogen peroxide liberated in the vacuole (e.g.  staphylococci).   If microorganisms are able to survive and grow within  phagocytes, they will escape many of the other body  defences such as the lymph nodes, and be distributed  around the body. As the lifespan of phagocytes is  relatively short, such bacteria will eventually be delivered  to the liver and gastrointestinal tract where they are  ‘recycled’.   An alternative strategy is for the microorganism to kill  the phagocyte. This can be achieved by the production of  leucocidins (e.g. staphylococci, streptococci) which  promote the discharge of lysosomal substances into the  cytoplasm of the phagocyte rather than into the vacuole,  thus directing the phagocyte’s lethal activity towards  itself.   Once established, the course of a bacterial infection can  proceed in a number of ways. These can be related to the  relative ability of the organism to penetrate and invade  surrounding tissues and organs. The vast majority of  pathogens, being unable to combat the defences of the  deeper tissues, consolidate further on the epithelial  surface. Others, including most viruses, penetrate the  epithelial layers, but no further, and can be regarded as  partially invasive. A small group of pathogens are fully  invasive. These permeate the subepithelial tissues and  are circulated around the body to initiate secondary  sites of infection remote from the initial portal of entry  (Figure 7.1).   Other groups of organisms may cause disease through  ingestion by the victim of substances produced during  microbial growth on foods. Such diseases maybe regarded  as intoxications rather than as infections and are consid-  ered later (section 6.1.1). Treatment in these cases is    usually an alleviation of the harmful effects of the toxin  rather than elimination of the pathogen from the body.   Bordetella pertussis (the aetiological agent of whooping  cough) is probably the best described of these pathogens.  This organism is inhaled and rapidly localizes on the  mucociliary blanket of the lower respiratory tract. This  localization is very selective and is thought to involve  agglutinins on the organism’s surface. Toxins produced  by the organism inhibit ciliary movement of the epithelial  surface and thereby prevent removal of the bacterial cells  to the gut. A high molecular weight exotoxin is also pro-  duced during the growth of the organism which, being  of limited diffusibility, pervades the subepithelial tissues  to produce inflammation and necrosis. Corynebacterium  diphtheriae (the causal organism of diphtheria) behaves  similarly, attaching itself to the epithelial cells of the res-  piratory tract. This organism produces a diffusible toxin  of low molecular weight, which enters the blood circula-  tion and brings about a generalized toxaemia.   In the gut, many pathogens adhere to the gut wall and  produce their effect via toxins that pervade the surround-  ing gut wall or enter the systemic circulation. V. cholerae  and some enteropathic E. coli strains localize on the gut  wall and produce toxins that increase vascular permeabil-  ity. The end result is a hypersecretion of isotonic fluids  into the gut lumen, acute diarrhoea and, as a conse-  quence, dehydration that may be fatal in young or elderly  people. In all these instances, binding to epithelial cells is  not essential but increases permeation of the toxin and  prolongs the presence of the pathogen.   Some bacteria, and most viruses, are able to attach to the  mucosal epithelia and then penetrate rapidly into the  epithelial cells. These organisms multiply within the pro-  tective environment of the host cell, eventually killing  it and inducing disease through erosion and ulceration  of the mucosal epithelium. Typically, members of the  genera Shigella and Salmonella utilize such mechanisms  in infections of the gastrointestinal tract. These bacteria  attach to the epithelial cells of the large and small intes-  tines, respectively, and, following their entry into these  cells by induced pinocytosis, multiply rapidly and pene-  trate laterally into adjacent epithelial cells. The mecha-  nisms for such attachment and movement are unknown  but involve a transition from a non-motile to motile phe-  notype. Some species of salmonellae produce, in addi-  tion, exotoxins that induce diarrhoea (section 6.1.4).  There are innumerable serotypes of Salmonella, which    are primarily parasites of animals but are important to  humans in that they colonize farm animals such as pigs  and poultry and ultimately infect foods derived from  them. Salmonella food poisoning (salmonellosis), there-  fore, is commonly associated with inadequately cooked  meats, eggs and also with cold meat products that have  been incorrectly stored following contact with the  uncooked product. Dependent upon the severity of the  lesions induced in the gut wall by enteric pathogens, red  blood cells and phagocytes pass into the gut lumen, along  with plasma, and cause the classic ‘bloody flux’ of bacil-  lary dysentery. Similar erosive lesions are produced by  some enteropathic strains of E. coli.   Viral infections such as influenza and the ‘common  cold’ (in reality 300-400 different strains of rhinovirus)  infect epithelial cells of the respiratory tract and nasophar-  ynx, respectively. Release of the virus, after lysis of the  host cells, is to the void rather than to subepithelial  tissues. The residual uninfected epithelial cells are rapidly  infected, resulting in general degeneration of the tracts.  Such damage not only predisposes the respiratory tract  to infection with opportunist pathogens such as N. men-  ingitidis and Haemophilus influenzae but also causes the  associated fever.   Invasive pathogens either aggressively invade the tissues  surrounding the primary site of infection (active spread)  or are passively transported around the body in the  blood, lymph, cerebrospinal, axonal or pleural fluids  (passive spread). Some, especially aggressive organisms,  move both passively and actively, setting up multiple,  expansive secondary sites of infection in various organs.   Active spread of microorganisms through normal sub-  epithelial tissues is difficult in that the gel-like nature of  the intercellular materials physically inhibits bacterial  movement. Induced death and lysis of the tissue cells  produces, in addition, a highly viscous fluid, partly due  to undenatured DNA. Physical damage, such as wounds,  is rapidly sealed with fibrin clots, thereby reducing the  effective routes for spread of opportunist pathogens.  Organisms such as Strep, pyogenes, Cl. perfringens and, to  some extent, the staphylococci, are able to establish them-  selves in tissues by virtue of their ability to produce a wide  range of extracellular enzyme toxins. These are associated  with killing of tissue cells, degradation of intracellular  materials and mobilization of nutrients. A selection of  such toxins will be considered briefly.    • Haemolysins are produced by most of the pathogenic  staphylococci and streptococci. They have a lytic effect on  red blood cells, releasing iron-containing nutrients.   • Fibrinolysins are produced by both staphylococci  (staphylokinase) and streptococci (streptokinase). These  toxins indirectly activate plasminogen and so dissolve  fibrin clots that the host forms around wounds and  lesions to seal them. The production of fibrinolysins  therefore increases the likelihood of the infection  spreading. Streptokinase may be employed clinically in  conjunction with streptodornase (Chapter 26) in the  treatment of thrombosis.   • Collagenases and hyaluronidases are produced by most  of the aggressive invaders of tissues. These are able to  dissolve collagen fibres and the hyaluronic acids that  function as intercellular cements; this causes the tissues  to break up and produce oedematous lesions.   • Phospholipases are produced by organisms such as Cl.  perfringens (a-toxin). These toxins kill tissue cells by  hydrolysing the phospholipids that are present in cell  membranes.   • Amylases, peptidases and deoxyribonucleases mobilize  many nutrients that are released from lysed cells.  They also decrease the viscosity of fluids present at  the lesion by depolymerization of their biopolymer  substrates.   Organisms possessing the above toxins, particularly  those also possessing leucocidins, are likely to cause  expanding oedematous lesions at the primary site of  infection. In the case of Cl. perfringens, a soil microorgan-  ism that has become adapted to a saprophytic mode of  life, infection arises from an accidental contamination of  deep wounds when a process similar to that seen during  the decomposition of a carcass ensues (gangrene). This  organism is most likely to spread through tissues when  blood circulation, and therefore oxygen tension, in the  affected areas is minimal.   Abscesses formed by streptococci and staphylococci  can be deep-seated in soft tissues or associated with  infected wounds or skin lesions; they become localized  through the deposition of fibrin capsules around the  infection site. Fibrin deposition is partly a response of the  host tissues, but is also partly a function of enzyme toxins  such as coagulase. Phagocytic white blood cells can  migrate into these abscesses in large numbers to produce  significant quantities of pus. Such pus, often carrying the  infective pathogen, might be digested by other phago-  cytes in the late stages of the infection or discharged to  the exterior or to the capillary and lymphatic network. In  the latter case, blocked capillaries might serve as sites for    secondary lesions. Toxins liberated from the microorgan-  isms during their growth in such abscesses can freely  diffuse to the rest of the body to set up a generalized  toxaemia.   S. enterica serovar Typhi, S. enterica serovar Paratyphi  and S. enterica serovar Typhimurium are serotypes of  Salmonella (section 4.2) that are not only able to pene-  trate into intestinal epithelial cells and produce exotoxins,  but are also able to penetrate beyond into subepithelial  tissues. These organisms therefore produce a characteris-  tic systemic disease (typhoid and enteric fever), in addi-  tion to the usual symptoms of salmonellosis. Following  recovery from such infection the organism is commonly  found associated with the gallbladder. In this state, the  recovered person will excrete the organism and become  a reservoir for the infection of others.   When invading microorganisms have crossed the epithe-  lial barriers they will almost certainly be taken up  with lymph in the lymphatic ducts and be delivered to  filtration and immune systems at the local lymph  nodes. Sometimes this serves to spread infections further  around the body. Eventually, spread may occur from  local to regional lymph nodes and thence to the blood-  stream. Direct entry to the bloodstream from the primary  portal of entry is rare and will only occur when the  organism damages the blood vessels or if it is injected  directly into them. This might be the case following an  insect bite or surgery. Bacteraemia such as this will often  lead to secondary infections remote from the original  portal of entry.   Damage caused to the host organism through infection  can be direct and related to the destructive presence of  microorganisms (or to their production of toxins) in  particular target organs; or it can be indirect and related  to interactions of the antigenic components of the  pathogen with the host’s immune system. Effects can  therefore be closely related to, or remote from, the  infected organ.   Symptoms of the infection can in some instances be  highly specific, relating to a single, precise pharmacologi-  cal response to a particular toxin; or they might be non-  specific and relate to the usual response of the body to  particular types of trauma. Damage induced by infection  will therefore be considered in these categories.   For the host, the consequences of infection depend to  a large extent upon the tissue or organ involved. Soft  tissue infections of skeletal muscle are likely to be less  damaging than, for instance, infections of the heart  muscle and central nervous system. Infections associated  with the epithelial cells that make up small blood vessels  can block or rupture them to produce anoxia or necrosis  in the tissues that they supply. Cell and tissue damage  is generally the result of a direct local action by the  microorganisms, usually concerning action at the cyto-  plasmic membranes. The target cells are usually phago-  cytes and are generally killed (e.g. by Brucella, Listeria,  Mycobacterium). Interference with membrane function  through the action of enzymes such as phospholipase  causes the affected cells to leak. When lysosomal mem-  branes are affected, the lysosomal enzymes disperse into  the cells and tissues causing them, in turn, to autolyse.  This is mediated through the vast battery of enzyme  toxins available to these organisms (section 4). If these  toxins are produced in sufficient concentration they may  enter the circulatory systems to produce a generalized  toxaemia. During their growth, other pathogens liberate  toxins that possess very precise, singular pharmacological  actions. Diseases mediated in this manner include diph-  theria, tetanus and scarlet fever.   In diphtheria, the organism C. diphtheriae confines  itself to epithelial surfaces of the nose and throat and  produces a powerful toxin which affects an elongation  factor involved in eukaryotic protein biosynthesis. The  heart and peripheral nerves are particularly affected,  resulting in myocarditis (inflammation of the myocar-  dium) and neuritis (inflammation of a nerve). Little  damage is produced at the infection site.   Tetanus occurs when Cl. tetani, ubiquitous in the soil  and the faeces of herbivores, contaminates wounds, espe-  cially deep puncture-type lesions. These might be the  result of a minor trauma such as a splinter, or a major  one such as a motor vehicle accident. At these sites, tissue  necrosis ,and possibly also microbial growth, reduce the  oxygen tension to allow this anaerobe to multiply. Its  growth is accompanied by the production of a highly  potent toxin that passes up peripheral nerves and diffuses  locally within the central nervous system. The toxin has  a strychnine-like action and affects normal function at  the synapses. As the motor nerves of the brainstem are  the shortest, the cranial nerves are the first affected, with  twitches of the eyes and spasms of the jaw (lockjaw).    A related organism, Cl. botulinum, produces a similar  toxin that may contaminate food if the organism has  grown in it and if conditions are favourable for anaerobic  growth. Meat pastes and pates are likely sources. This  toxin interferes with acetylcholine release at cholinergic  synapses and also acts at neuromuscular junctions. Death  from this toxin eventually results from respiratory failure.   Many other organisms are capable of producing intox-  ication following their growth on foods. Most common  among these are the staphylococci and strains of B. cereus.  Some strains of Staph, aureus produce an enterotoxin  which acts on the vomiting centres of the brain. Nausea  and vomiting therefore follow ingestion of contaminated  foods and the delay between eating and vomiting varies  between 1 and 6 hours depending on the amount of toxin  ingested. B. cereus also produces an emetic toxin but its  actions are delayed and vomiting can follow up to 20  hours after ingestion. The latter organism is often associ-  ated with rice products and will propagate when the rice  is cooked (spore activation) and subsequently reheated  after a period of storage.   Scarlet fever is produced following infection with  certain strains of Strep, pyogenes. These organisms  produce a potent toxin that causes an erythrogenic skin  rash that then accompanies the more usual effects of a  streptococcal infection.   If the infective agent damages an organ and affects its  functioning, this can manifest itself as a series of second-  ary disease features that reflect the loss of that function  to the host. Thus, diabetes may result from an infection  of the islets of Langerhans, paralysis or coma from infec-  tions of the central nervous system, and kidney malfunc-  tion from loss of tissue fluids and its associated  hyperglycaemia. In this respect virus infections almost  inevitably result in the death and lysis of the host cells.  This will result in some loss of function by the target  organ. Similarly, exotoxins and endotoxins can also be  implicated in non-specific symptoms, even when they  have well-defined pharmacological actions. Thus, a  number of intestinal pathogens (e.g. V. cholerae, E. coli)  produce potent exotoxins that affect vascular permeabil-  ity. These generally act through adenylate cyclase, raising  the intracellular levels of cyclic AMP (adenosine mono-  phosphate). As a result of this the cells lose water and  electrolytes to the surrounding medium, the gut lumen.  A common consequence of these related, yet distinct,  toxins is acute diarrhoea and haemoconcentration.  Kidney malfunction might well follow and in severe cases    lead to death. Symptomologically there is little difference  between these conditions and the food poisoning induced  by ingestion of staphylococcal enterotoxin (above).   Inflammatory materials are released not only from  necrotic cells but also directly from the infective agent.  Endotoxins are derived from constituents of the bacterial  cell rather than being deliberately exported cellular prod-  ucts. Thus, during the growth and autolysis of Gram-  negative bacteria components of their cell envelopes,  such as lipopolysaccharide (see Chapter 3) are shed to the  environment. Endotoxins tend to be less toxic than exo-  toxins and have much less precise pharmacological  actions. Indeed, it is not always clear to what extent these  can be related to actions by the host or by the pathogen.  Reactions include local inflammation, elevations in body  temperature, aching joints, and head and kidney pain.  Inflammation causes swelling, pain and reddening of the  tissues, and sometimes loss of function of the organs  affected. These reactions may sometimes be the major  sign and symptom of the disease.   While various toxic effects have been attributed to  these endotoxins, their role in the establishment of  the infection, if any, remains unclear. The most notable  effect of these materials is their ability to induce a high  body temperature (pyrogenicity) (Chapters 3 and 22).  The pyrogenic effect of lipopolysaccharide relates to  the action of the lipid A component directly upon the  hypothalamus and also to its direct action on macro-  phages and phagocytes. Elevation of body temperature  follows within 1-2 hours. In infections such as meningitis  the administration of antibiotics may cause such a release  of pyrogen that the resultant inflammation and fever is  fatal. In such instances antibiotics are co- administered  with steroids to counter this effect. The pyrogenic effects  of lipid A are unaffected by moist heat treatment (auto-  clave). Growth of Gram-negative organisms such as  Pseudomonas aeruginosa in stored water destined for use  in terminally sterilized products will cause the final  product to be pyrogenic. Processes for the destruction of  pyrogen associated with glassware, and tests for the  absence of pyrogen in water and product, therefore form  an important part of parenteral drug manufacture  (Chapter 22).   Many microorganisms minimize the effects of the  host’s defence system against them by mimicking the  antigenic structure of the host tissue. The eventual  immunological response of the host to infection then  leads to its autoimmune self-destruction. Thus, infec-    tions with Mycoplasma pneumoniae can lead to produc-  tion of antibody against normal group O erythrocytes,  with concomitant haemolytic anaemia.   If antigen released from the infective agent is soluble,  antigen-antibody complexes are produced. When anti-  body is present at a concentration equal to or greater than  the antigen, such as in the case of an immune host, these  complexes precipitate and are removed by macrophages  present in the lymph nodes. When antigen is present in  excess, the complexes, being small, continue to circulate  in the blood and are eventually filtered off by the kidneys,  becoming lodged in kidney glomeruli and in the joints.  Localized inflammatory responses in the kidneys are  sometimes then initiated by the complement system  (Chapter 9). Eventually the filtering function of the  kidneys becomes impaired, producing symptoms of  chronic glomerulonephritis.   The primary requirement for recovery is that multiplica-  tion of the infective agent is brought under control, so  that it ceases to spread around the body and that the  damaging consequences of its presence are arrested and  repaired. Such control is brought about by the combined  functions of the phagocytic, immune and complement  systems. A successful pathogen will not seriously debili-  tate its host; rather, the continued existence of the host  must be ensured in order to maximize the dissemination  of the pathogen within the host population. From the  microorganism’s perspective the ideal situation is where  it can persist permanently within the host and be con-  stantly released to the environment. Although this is the  case for a number of virus infections ( chickenpox, herpes)  and for some bacterial ones, it is not common. Generally,  recovery from infection is accompanied by complete  destruction of the organism and restoration of a sterile  tissue. Alternatively, the organism might return to a com-  mensal relationship with the host on the epithelial and  skin surface.   Where the infective agent is an obligate pathogen, a  means must exist for it to infect other individuals before  its eradication from the host organism. The route of exit  is commonly related to the original portal of entry  (Figure 7.1). Thus, pathogens of the intestinal tract are  liberated in the faeces and might easily contaminate food  and drinking water. Infective agents of the respiratory  tract might be exhaled during coughing, sneezing or    talking, survive in the associated water droplets, and  infect nearby individuals through inhalation. Infective  agents transmitted by insect and animal vectors may be  spread through those same vectors, the insects/ animals  having been themselves infected by the diseased host. For  some ‘fragile’ organisms (e.g. N. gonorrhoeae, T. palli-  dum), direct contact transmission is the only means of  spread between individual hosts. In these cases, intimate  contact between epithelial membranes, such as occurs  during sexual contact, is required for transfer to occur.  For opportunist pathogens, such as those associated  with wound infections, transfer is less important because  the pathogenic role is minor. Rather, the natural habitat  of the organism serves as a constant reservoir for  infection.   Spread of a microbial disease through a population of  individuals can be considered as vertical (transferred  from one generation to another) or horizontal (transfer  occurring within genetically unrelated groups). The latter  can be divided into common source outbreaks, relating to  infection of a number of susceptible individuals from a  single reservoir of the infective agent (i.e. infected foods),  or propagated source outbreaks, where each individual  provides a new source for the infection of others.   Common source outbreaks are characterized by a  sharp onset of reported cases over the course of a single  incubation period (Figure 7.2) and relate to a common  experience of the infected individuals (e.g. a contami-  nated food product). The number of cases will persist  until the source of the infection is removed. If the source  remains (i.e. a reservoir of insect vectors) then the disease  becomes endemic to the exposed population, with a con-  stant rate of infection. Propagated source outbreaks, on  the other hand, are brought about by person-to-person  spread, and show a gradual increase in reported cases  over a number of incubation periods and eventually  decline when most of the susceptible individuals in the  population have been affected (Figure 7.2). Factors that  contribute to propagated outbreaks of infectious disease  are the infectivity of the agent (I), the population density  (P) and the numbers of susceptible individuals in it (F).  The likelihood of an epidemic occurring is given by the  product of these three factors (i.e. FIP). An increase in  any one of them might initiate an outbreak of the disease  in epidemic proportions. Thus, reported cases of particu-  lar diseases show periodicity, with outbreaks of epidemic    Figure 7.2 Comparison of timescale for common source  outbreak and propagated source outbreak infections.   proportion occurring only when FIP exceeds certain  critical threshold values, related to the infectivity of the  agent. Outbreaks of measles and chickenpox therefore  tend to occur annually in the late summer among chil-  dren attending school for the first time. This has the effect  of concentrating all susceptible individuals in one, often  confined, space at the same time. The proportion of sus-  ceptible individuals can be reduced through rigorous vac-    cination programmes (Chapter 9). Provided that the  susceptible population does not exceed the threshold FIP  value, then herd immunity against epidemic spread of the  disease will be maintained.   Certain types of infectious agent (e.g. influenza virus)  are able to combat herd immunity such as this through  undergoing major antigenic changes. These render the  majority of the population susceptible, and their occur-  rence is often accompanied by spread of the disease across  the entire globe (pandemics).   Bauman, R.W. (2009). Microbiology with Diseases by Body  System, 2nd edn. Pearson Benjamin Cummings, San Francisco,  CA.   Roitt, I. M. (2006) Essential Immunology, 11th edn. Blackwell  Scientific, Oxford.   Smith, H. (1990) Pathogenicity and the microbe in vivo. J Gen  Microbiol, 136, 377-393.   Wilson, M., McNab, R. & Henderson, B. (2002) Bacterial Disease  Mechanisms. Cambridge University Press, Cambridge.   Howard Ceri , 1 Sean P. Gorman 2 and Brendan F. Gilmore 2   Traditionally, microbiologists have grown bacteria as sus-  pension cultures in rich media, in order to optimize cell  yield. This planktonic mode of growth also became part  of the standard assay on which all existing antimicrobials  were selected and developed, and continues to be the  basis for the selection of antimicrobials for specific  patient treatment. We now recognize that in most envi-  ronments, including our bodies, bacteria typically exist as  adherent microcolonies termed biofilms, which afford  bacteria a number of growth advantages including an  inherent lack of susceptibility to antimicrobials. This  antimicrobial tolerance differs from classical genetic  resistance in that this reduced susceptibility disappears  when the biofilm is returned to planktonic growth.  Biofilm tolerance is multifactorial, which includes the  spatial and structural parameters of the biofilm as well as    the increased phenotypic diversity within the biofilm  population. Biofilms are believed to be associated with  approximately 60% of human infections including  chronic, recurrent and device-related infections, there-  fore treatment of biofilm infections has become an  important focus in modern medicine. As planktonic  susceptibility testing, via the minimal inhibitory concen-  tration (MIC) test, provides little guidance in the selec-  tion of antimicrobials to treat biofilms, a change in  paradigm is required to determine appropriate treatment  of biofilms and for the discovery of next-generation  antimicrobials.   Biofilms are microcolonies of one or more species of  bacteria or fungi typically growing adherent to a biotic    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    or abiotic surface. Biofilms form to allow bacteria to  maintain themselves in a niche of their choosing rather  than being washed away by the shear force of running  water in the natural environment or the movement of  body fluids and mucins in the body. Biofilms provide a  more energy-efficient means of growth, capturing nutri-  ents as they flow past and easily expelling waste. They also  provide a more secure environment for sustainability,  making it difficult for phagocytes, found both in nature  and as part of the immune system, to eradicate the  biofilm. Also, as a biofilm, bacteria and fungi are less  susceptible to antimicrobials, allowing them to be more  tolerant than their planktonic brethren to antibiotics  found in nature and those used clinically. A cartoon of  the biofilm life cycle of bacteria is shown in Figure 8.1.  In the centre of the figure bacteria exist in a mature  biofilm that may be formed from many species, as in a  consortium formed on the face of a rock in a stream or  those found in the mouth, as part of our dental plaque.  Chemical signals regulate the interactions between  members of the biofilm just as hormones regulate the  cells of our body. For example, under specific stress con-  ditions appropriate signalling may lead to an increase in  phenotypic diversity within the biofilm to accommodate    the stress or these signals may cause bacteria to revert to  their more motile planktonic phenotype and leave the  biofilm to establish new microcolonies that will give rise  to a mature biofilm.   Bacteria exist often as multispecies biofilms in nature,  which may allow pathogens to survive in nature away  from their natural host where they may serve as a nidus  for reinfection. Examples of this are enteric organisms  that form biofilms in drinking-water pipes or in wells.  Following an original contamination event, biofilms  allow for sustainability of these populations, which then  serve to shed further organisms into potable water sup-  plies, even after the apparent clearance of the original  contamination event. Contamination of our groundwater  sources pose concerns for the future of our potable water  supplies; this threat is accentuated by the stability that  biofilms of these contaminants bring into the equation.   Food-borne outbreaks of infections are often associated  with biofilms formed on the hard surfaces of food-    Figure 8.1 The main stages in microbial surface attachment and biofilm formation (From Harrison, J.J. et al. (2005) Biofilms. Am  Sci, 93, 508-515, with permission from American Scientist.)    processing plants, such as tables, knives and processing  equipment. The inherent resistance of biofilms to many  biocides used in the cleaning process allows these bacteria  to multiply and contaminate food products.   Nosocomial infections within healthcare facilities often  result from pathogens surviving in the environment as  biofilms. Biofilms may facilitate carriage by patients, hos-  pital staff, or visitors, as for example in colonization of  nasal passages by meticillin-resistant Staphylococcus  aureus (MRSA). Biofilms may also become associated  with hard surfaces, drains or waterpipes within the facil-  ity. Infections may also be associated with instruments or  devices used in the hospital, such as the contamination  of endoscopes by biofilms, where they are responsible for  the passage of infectious organisms from one patient to  another. Whether biofilms form on animate or inanimate  surfaces, their recalcitrant nature makes it difficult to  remove them from the environment.   Medical devices have become an essential aspect of  patient care, with tens of millions of implantable or ind-  welling medical devices (such as catheters, endotracheal  tubes, artificial joint protheses and so on) used each year  in patients worldwide. However, despite the evolution of  medical devices and the biomaterials from which they are  manufactured, their use in vivo is significantly compro-  mised by their seemingly ubiquitous propensity to  succumb to microbial colonization and biofilm forma-  tion, otherwise known as medical device-associated infec-  tion. Immediately after implantation, the device surface  becomes modified by the adsorption of host- derived pro-  teins, extracellular matrix proteins, coagulation products,  etc., depending on the site. This ‘conditioning film’  renders the surface of the device favourable for microbial  adhesion and is often followed by rapid primary attach-  ment of microorganisms to the material surface and  biofilm formation.   The microorganisms responsible for causing medical  device-associated infections may be either from exoge-  nous (carers, visitors, healthcare environment) or endog-  enous sources (via the migration of microorganisms  from normally colonized body sites). Although site-  dependent, the main causative organisms of medical  device-associated nosocomial infections are frequently  normal skin biota including Staph, aureus and coagulase-  negative staphylococci, predominantly Staph, epider-  midis, which is the most common causative organism of    infections related to intravascular catheters and other  implanted medical devices. A number of other key  microorganisms have been shown to be significant causa-  tive organisms of medical device-related nosocomial  infections, including Pseudomonas aeruginosa (ventilator-  associated pneumonia, VAP), enterococci, Escherichia  coli (urinary tract infection (UTI), septicaemia) and  Proteus species such as Proteus mirabilis (UTI, device  encrustation).   At least half of all cases of healthcare-associated infec-  tions are estimated to be due to biofilm-mediated,  medical device-associated infections, with medical device  use now regarded as the greatest external predictor of  healthcare-associated infections. The development of  medical device-associated infections generally necessi-  tates the complete removal and replacement of the device,  with the level of clinical intervention depending on  the nature and site of the device implantation. Systemic  antibiotics (often a combination therapy of two or  more antimicrobial agents) represent the conventional  approach to the treatment of device-associated infec-  tions; however, given the high degree of tolerance to anti-  microbial challenge that is a feature of biofilm populations,  eradication proves extremely difficult and infection  relapses frequently occur. This has led to the development  of a range of anti-infective and antimicrobial biomateri-  als for use in device manufacture, though the long-term  efficacy of these devices in the reduction of medical  device-associated infection is an area of considerable  debate.   Healthcare-associated infections typically occur at  four main body sites (urinary tract, respiratory tract, sur-  gical sites and bloodstream infections), three of which  (UTI, pneumonia, bloodstream infections) are com-  monly associated with the use of indwelling devices.  Indeed, around 95% of nosocomial UTIs reported are  linked to the use of urological devices (mainly urinary  catheters), and more than 85% of nosocomial respiratory  infections (mainly VAPs) are device -related. Central  venous catheters pose the greatest risk of mortality due  to catheter-related bloodstream infections, with inci-  dences in the USA ranging from 100000 to 500000 cases  annually, resulting in more than 25 000 deaths per year.  Although these represent the most common device-  associated infections, it is worth noting that all types of  implantable medical device are susceptible to infection:  for example, peritoneal catheter infections in peritoneal  dialysis, orthopaedic implant infections, and biofilm for-  mation on prosthetic heart valves. In addition to patient  morbidity and mortality, device-associated infections    impose significant financial burdens on healthcare pro-  viders, related primarily to increased hospitalization time  and associated care costs. Despite this, the use of and  dependence on implantable, indwelling medical devices  increases annually, correlated to an increasing ageing  population in industrialized nations.   With the discovery of antibiotics the world changed.  Acute infectious diseases, the leading cause of morbidity  and mortality in humans, became treatable, resulting in  an increase in life expectancy and quality of life. While  infections continue to be a leading contributor to mortal-  ity, they are often associated with pre-existing conditions  that compromise the patient. However, what became  known as the ‘antibiotic era’ is now being compromised  itself by the emergence of more and more antibiotic-  resistant strains of bacteria that have caused modern  medicine to question if we are not now entering the  ‘postantibiotic era’.   What is often ignored in these discussions of  antimicrobial resistance and our reduced ability to treat  infection is the fact that even in the halcyon times of  antimicrobial therapy chronic or recurrent infections  were poorly resolved with antibiotics. In fact, the designa-  tion of these infections as chronic was presumably  derived due to their lack of responsiveness to antimicro-  bial therapy allowing them to become chronic or to recur  in the face of therapy. Diseases such as recurrent ear  infections in children, recurrent UTIs in women, and  medical device-associated infections were and still remain  a challenge to antimicrobial therapy, even when the iso-  lates of these infections have been shown in vitro to be  susceptible to antibiotics used in their treatment.   It is now recognized that these chronic infections  involve bacteria associated within biofilms. In fact the US  Food and Drug Administration (FDA) and Centers for  Disease Control (CDC) both state that more than 60%  of North American infections involve biofilms. We also  now recognize that the confounding issue in the treat-  ment of chronic infections is the inherent tolerance of  biofilms to antibiotics predicted to have efficacy against  the organism on the basis of planktonic susceptibility  testing. In fact, Ceri and colleagues demonstrated that for  an antibiotic to be effective in biofilms can require a  concentration over 1000 times that needed to treat the  same planktonic population (Ceri et al. 1999); concentra-  tions that cannot be achieved or used safely in patient    treatment. This altered tolerance to drugs is an adapta-  tion of the biofilm, as bacteria derived from the biofilm  show the same susceptibility profile as planktonics when  the organisms are returned to the planktonic growth  phase.   This reduced susceptibility to antibiotics differs from  antibiotic resistance in a number of fundamental ways.  First, this tolerance is only demonstrated when the isolate  is in the biofilm mode of growth and is lost when the  culture is returned to planktonic growth, hence it is not  a permanent genetic change. Secondly, tolerance implies  that the biofilm is not killed by the antimicrobial but it  may not necessarily be able to grow in the presence of the  drug, whereas in resistance the organism can grow in the  presence of the antimicrobial. The tolerance of biofilm  populations to antimicrobials would imply that in many  instances of chronic infections microbes are in fact  exposed to sublethal concentrations of drug, which may  be an important implication in the development of clas-  sical antimicrobial resistance.   It is now believed that the tolerance to antimicrobials  displayed by biofilms is a multifactorial process involving,  to some degree or another, a number of different mecha-  nisms contributing to the survival of the population, if  not the individual cell. A model for this multifactorial  tolerance is shown in Figure 8.2. Contributing to the  tolerance of biofilms are factors ranging from the struc-  tural components of the biofilm, the physiological poten-  tial of cells spread throughout the biofilm and the  expression of the genetic phenotypes of disparate popu-  lations of cells, all derived from the original clonal  population(s) that made up the biofilm.   The hypothesis that the extracellular matrix acts as the  gatekeeper for the penetration of antimicrobials into the  biofilm, as shown in point 3 in Figure 8.2, has engendered  many studies and a great deal of controversy. When bio-  films were first visualized using both transmission and  scanning electron microscopy, the dehydrated matrix  seen in these original micrographs led to the belief that  biofilms were very flat and dense structures where the  compact and highly charged matrix around the biofilm  would prevent penetration of antibiotics into the biofilm;  hence this diffusion barrier would render them resistant  to antimicrobial treatment. Stabilization of the matrix   Figure 8.2 Factors contributing to antimicrobial tolerance of biofilms. ( 1 ) Low 0 2 and decreased nutrient levels near the centre of  the microcolony result in slow microbial bacterial growth and hence tolerance to antibiotic drugs which are more effective against  fast-growing cells. (2) Intercellular signals can alter biofilm physiology, resulting in expression of molecular efflux pumps which  expel antibiotics from the cells allowing the community to grow even in the presence of antimicrobial drug. (3) Negatively charged  biofilm matrix may bind positively charged antimicrobials retarding their penetration into the biofilms. (4) Persister cells, a  specialized population of cells which do not grow but do not die in the presence of antibiotic. Persister cells can, upon removal of  the antimicrobial challenge, give rise to a normal bacterial colony. (5) Genetic and physiological diversity within the population act  as an ‘insurance policy’, improving the chances that some cells survive any challenge. (From Harrison, J.J. et al. (2005) Biofilms. Am  Sci, 93, 508—515, with permission from American Scientist.)    and cross-sections through the biofilm revealed a very  different picture of the biofilm, where cells were seen to  exist within a very hydrated matrix containing channels  to allow for nutrient transfer into the biofilm and the  diffusion of waste out. The matrix is now believed to be  composed of bacterially derived carbohydrate, the com-  position of which is dependent upon the bacterial species,  nutrient availability and the growth conditions of the  biofilm. Recently it has been established that DNA is an  important component of the matrix, which may be spe-  cifically transported into the matrix. The role of DNA in  the matrix is only now being deciphered. It has been  shown to play a role in the conformation of the carbohy-  drate and hypothesized to serve as a gene pool for the    diversity seen within the biofilm. The highly anionic  charge of this matrix could be hypothesized to still play  an important role in preventing charged antibiotics from  effectively entering the biofilm and thereby still act as a  primary inhibitor of antibiotic killing, as was originally  proposed. Several studies of antibiotic penetration into  biofilms demonstrated that the charge of the antibiotic  could affect its penetration. For example, fluoroquinolo-  nes (ciprofloxacin, ofloxacin) that are not highly charged  easily penetrate the matrix, while the penetration of  charged aminoglycosides (tobramycin, gentamicin) is  delayed. These studies have not, however, resolved the  issue of the importance of the matrix in the resistance  of biofilms. The rapid entry of fluoroquinolones, for    example, may be only into water channels of the biofilm  and not into areas where cells of the biofilm are found,  while the delay in entry of aminoglycosides may affect the  rate of entry but may not affect final concentration sig-  nificantly enough to alter susceptibility of the biofilm.  Further, penetration of antimicrobials alone may not be  as key an issue as the physiological state of the cells, which  is also affected by the structure and organization of the  biofilm. The diffusion into the biofilm of multiple factors,  not limited to just the antibiotics themselves, may impact  the biofilm’s physiological status, thereby affecting the  efficacy of antibiotics against the biofilm.   The physiological state of the biofilm is also affected by  its organizational structure, as diffusion of oxygen, nutri-  ents and waste will ultimately affect all properties associ-  ated with growth and sustainability of the biofilm (shown  in part 1 of Figure 8.2). Sophisticated experiments based  on microelectrode probing of the biofilm and confirmed  by dye distribution confocal microscopy assays have  established the presence of oxygen and pH gradients  within the biofilm. Gradients of nutrients and end prod-  ucts are also implicated in defining the different growth  properties throughout the biofilm, which, as described  above, has been linked to antibiotic susceptibility. The  hypothesis predicts that antibiotics dependent on cell  growth for activity will be less effective against biofilms  because of the variability of growth within the biofilm.  However biofilms, prove to be as recalcitrant to antibiot-  ics that are not dependent upon cell growth as to those  that do. Further, live dead staining of biofilms does not  always correlate with cell death occurring most rapidly at  the outer edges of the biofilm, where nutrient and oxygen  levels are at their highest and hence where growth should  be most rapid. In mixed species biofilms, where each  component of the population may exist within its own  niche for optimal growth, this model becomes even more  complex to understand. The complexity of these interac-  tions will make dissecting the process of antimicrobial  tolerance more challenging. One can, however, argue that  targeting one member of the mixed population within  the biofilm may alter the susceptibility patterns of other  species that are dependent upon the symbiotic interac-  tions within the biofilm.   Another focus of study in biofilm resistance related to  spatial orientation and physiology has been to look at  how biofilms deal with oxidative stress. The mechanism  of killing of many antimicrobials, including antibiotics,  biocides and metals, is often associated with redox reac-    tions involving various cellular components. These reac-  tions may oxidize sensitive cellular thiol (RSH) groups or  result in the production of reactive oxygen species (ROS),  such as superoxides, hydroxy radicals, or hydrogen per-  oxide. The regulation of antioxidant pathways within the  cell, such as glutathione (GSH) and thioredoxin pathways  that manage thiol-disulfide homeostasis, and the oxyRS,  soxRS and marR regulons that render cells resistant to  ROS, play an important role in the susceptibility of bio-  films to antimicrobials. The difference in oxygen tension  in the biofilm and of the cells’ response to oxygen stress  may prove vital in survival of biofilms to naturally occur-  ring antibiotics or those used in patient treatment. The  role of the redox potential of E. coli to metals has dem-  onstrated a very complex picture of these interactions  and has shown a difference in mechanisms between  planktonic and biofilm populations.   Our perspective of the microbial lifestyle has changed  from one where bacteria exist mainly as solitary inde-  pendent planktonic populations to one where bacteria  form adherent communal populations of bacteria organ-  ized into microcolonies called biofilms. This shift in life-  style suggests the presence of specific signalling between  cells to allow them to organize these complex structures.  Many different genes have been identified that can alter  biofilm formation or antimicrobial susceptibility, but two  global signalling pathways have come to the forefront as  biofilm regulators in many different species of bacteria  (see point 2 in Figure 8.2). Although models of biofilm  formation have been proposed that do not require cell  signal molecules, the importance of the following mole-  cules in biofilm formation and antimicrobial resistance  is well established and has even led to attempts to  develop signal antagonists for treatment of biofilm  disease or to create greater efficacy of existing antimicro-  bials by returning biofilms to a planktonic-like level of  susceptibility.   Quorum sensing (QS) has been recognized as a key regu-  latory process associated with biofilm formation and  antibiotic susceptibility. Well studied in Vibrio fischeri, QS  involves an enzyme Luxl that produces a small signalling  molecule, or autoinducer, that diffuses out of the cell.  Upon reaching a threshold concentration the autoin-  ducer will diffuse back into the cell, where cellular tran-  scription is altered when the autoinducer binds the    transcription regulator LuxR and initiates QS-specific  gene expression. In Gram-negative organisms the autoin-  ducer is typically an acyl-homoserine lactone (AHL), but  in some organisms multiple QS systems exist. For  example, in Ps. aeruginosa signalling involves interactions  of two distinct AHL compounds, produced by the Luxl  homologues Lasl and Rhll respectively, that interact with  their cognate receptors LasR and RhlR. Yet a third signal  system, PQS, is also active in Ps. aeruginosa. In Gram-  positive bacteria QS is typically carried out by autoinduc-  ing peptides. As QS is an integral step in biofilm formation  and antibiotic tolerance, it has become a target for new  therapeutics. Inhibitors of the QS signal pathway, assayed  for their ability to either block biofilm formation or the  expression of QS-dependent genes, may provide new  approaches to treatment of biofilm disease.   The universal use of nucleotides as signalling molecules  is well recognized in biology. The importance of cyclic  diguanylate (c-di-GMP) as a switch to move bacteria  from a motile planktonic lifestyle to that of an adherent  biofilm is now just being systematically explored. As with  other nucleotide regulation systems, two components are  involved in the regulatory pathway. The first is responsi-  ble for the synthesis of the signal, in this case a diguan-  ylate cyclase (DGC) defined by proteins expressing  GGDEF domains. The second component, a phosphodi-  esterase (PDE), degrades the active signal and is associ-  ated with two distinct domains, EAL and HD-GYP. The  high level of redundancy of these domains makes under-  standing the mechanisms by which c-di-GMP regulates  biofilm formation and virulence a complex issue, where  patterns of temporal and spatial separation remain to be  resolved.   The final mechanism by which biofilms become less sus-  ceptible to antimicrobials is sketched in parts 4 and 5 of  Figure 8.2. Biofilms are able to give rise to unique sub-  populations; in some cases these may be part of the  normal diverse metabolic activity found within the nutri-  ent and oxygen gradients of the biofilm, an example  being persister cell populations, or they may be subpopu-  lations that are derived in response to stress but which  are not classically resistant populations according to defi-  nitions discussed previously. Being part of a population,  as opposed to being a single cell, allows for the adaption  of subpopulations within the biofilm through pheno-  typic expression of unique gene sets, which can ensure    the survival of the population as a whole, but often at the  expense of individuals within the biofilm. This mecha-  nism can be considered either altruism of a subset of the  population or simply adaptation to the gradients of  growth conditions present in the biofilms discussed in  section 2 that result in the expression of different pheno-  types. There have been many proposed models for  phenotypic plasticity of a bacterial population but we  will focus on two prominent hypotheses demonstrated  in Figure 8.2. These are persister cell populations and  the genetic diversity associated with the insurance  hypothesis.   Clonal populations of cells, grown either as a planktonic  or biofilm culture, give rise to persistent subpopulations  of cells that resist killing by high concentrations of anti-  microbials. Persistence is not the same as resistance, as  persister populations possess no resistance mechanisms  carried on transposable elements. They survive but do  not grow in the presence of the selective agent, and when  regrown the persister population recapitulates the killing  curve of the original population when challenged again  with the same antimicrobial. These persister populations  typically represent about 0.1% of a logarithmic plank-  tonic culture and up to about 10% of the initial popula-  tion in a biofilm and they may therefore account for the  higher level of antimicrobial tolerance seen in biofilms.  Although persister cells were first reported in the 1940s  the molecular mechanisms responsible for their proper-  ties are still a subject of debate. Persister cells are not  produced in response to a challenge but pre-exist in the  population and can be selected from any homogenous  population of cells for being a slow growing, physiologi-  cal distinct subpopulations of small cells capable of toler-  ance to environmental stress. The mechanism by which  persistence is manifested remains a focus of many studies.  Persistence in E. coli has been mapped to the high persist-  ence operon {hip), containing the hipA gene that encodes  a toxin and the hipB gene that encodes an antitoxin that  functions as a DNA-binding protein that both binds  HipA and also autoregulates the expression of the hip  operon itself. Homologues of the hip operon are found  across many bacterial genera, suggesting this is a common  mechanism of resistance. Interestingly, in E. coli and  other species redundant toxin-antitoxin genes have been  identified, suggesting the possible specialization of sets of  genes to deal with specific stress factors. Recently a toxin-  antitoxin pair, yafQ-dinJ has been shown to act on biofilm  but not planktonic populations to protect them against    very specific antimicrobials, which may provide a possi-  ble rationale for the redundancy seen in these systems.   Biofilms are made up of cells that have adapted to a wide  range of physiological states associated with the nutri-  tional gradients within the biofilm, and hence even if the  biofilm is initiated from a clonal population it will display  heterogeneity at both the phenotypic and genotypic level.  This diversity is enhanced when the biofilm is placed  under antimicrobial stress. Persister cell populations, dis-  cussed above, represent only one adaptive state that con-  tributes to the increased tolerance of biofilms to  antimicrobials. The combination of diverse populations  found in the biofilm that contribute to tolerance has been  referred to as the insurance hypothesis, where diversity is  increased in an attempt to ensure a population will survive  the stress of antimicrobial challenge or increased metal  or toxin concentrations in nature. This is an area of  immense interest and a field of study that is just in its  infancy. At this point numerous variants, separated on  morphological criteria from challenged populations, have  been identified, but only now with the advent of new-  generation sequencing can these populations be screened  for possible mutations that lead to their tolerant pheno-  type. This is an area worth keeping an eye on in the future.   As already stated, chronic, recurrent and device-related  infections are now recognized to be associated with biofilm  formation. Biofilms, as discussed above, possess multiple  mechanisms that render them less susceptible to antibiotic  treatment than the same isolate in the planktonic mode of  growth. The antibiotics we have today were all selected for  efficacy against planktonic cultures and all our diagnostics  are based on planktonic assays. Finally, we now recognize  that MIC values provide us with little relevant information  on how to treat biofilm infections. Clearly, anew paradigm  for treatment of biofilms is needed.   As part of the development of the next generation of  antimicrobials, techniques must be developed and set in  place to determine efficacy of these ‘new’ antimicrobials  against cells grown in biofilms. Antimicrobial assays  described in Chapter 18 include those targeted at identi-    fying antimicrobials effective against biofilms. It is tempt-  ing to speculate that, since screening of all combinatorial  libraries of antibiotics was based on efficacy against  planktonic targets, potential new antibiotics with efficacy  against chronic disease may have been overlooked. The  potential of identifying ‘next-generation’ antibiotics in  existing libraries by appropriate biofilm screens is a dis-  tinct possibility. The ability to now produce multiple  reproducible biofilms of defined target size provides the  potential for high-throughput screens for antibiotic effi-  cacy against biofilms.   Although all existing antimicrobials were selected for  their efficacy against planktonic cultures and this efficacy  has not always translated to activity against biofilms, it  may still be possible to make better use of existing anti-  microbials to treat biofilm disease. A marked trend in  treating chronic infections of many kinds is the use of  antibiotic combinations. The possible existence of syner-  gies between existing antimicrobials represents one area  of progress in treating biofilm infections. Rational  approaches to the application of combinations of antimi-  crobials, based on the differences of their targets, have not  provided clear use predictions. Most combinations are  still empirically derived from past experience. The  bioFILM PA assay used to select combinations of antibi-  otics for the treatment of Ps. aeruginosa lung infections  in cystic fibrosis patients is one of the first diagnostics to  receive regulatory approval (in Canada) and has had a  measure of success in the treatment of seriously ill  patients. This test is now in clinical trials as a standard  diagnostic in cystic fibrosis. Based on the Calgary Biofilm  Device (see Chapter 18), which is the only high-  throughput device for selecting antimicrobials with effi-  cacy against biofilms, this represents one of the first  attempts to address the differences in susceptibility of  organisms in the biofilm growth mode.   It is clear that biofilms represent an important challenge  in the treatment of infectious diseases. Biofilm are  involved in more than 60% of infections, typically associ-  ated with chronic, recurring and device-related infec-  tions. Furthermore, we now know that that existing  antimicrobials have been selected for efficacy against  planktonic organisms and are often much less effective  against biofilms. What is clearly needed is a paradigm  shift for the selection and development of antimicrobials,  which will ensure efficacy against biofilms.   There is a great deal of discussion about the development  of future antibiotics and whether this is the correct  direction to follow. It is noteworthy that every antibiotic  on the market has been derived or modified from natural  products produced by microbial life forms. It is also  interesting to note that these life forms continue to syn-  thesize antibiotics. If they had lost efficacy in nature one  would expect that these organisms would have evolved  to stop wasting energy on their synthesis, suggesting that  in nature antibiotics continue to have efficacy. The ques-  tion is then, what disconnect is there between how anti-  biotics function in nature and how they are used in  medicine?   For one thing, in the production of antibiotics an  active compound is purified from the fermentation  liquor and tested as an antibiotic against a planktonic  target population of bacteria. This approach assumes that  the planktonic population is a relevant target for antibi-  otic selection. It also assumes that other components of  the liquor are not important components in the activity  of the antibiotic. It is very possible that these discarded  components may define activity against the biofilms  found in nature. It is more difficult, if not impossible, to  bring complex mixtures of active ingredients through  regulatory process; however, this may be the active form  of antibiotics in nature that are able to function against  biofilms.   It should now be obvious that to find antibiotics capable  of treating biofilm infections we require a biofilm assay  of antibiotic efficacy. The lack of such an assay and the  inability to deal with biofilm infections has in part slowed  the adoption of the importance of biofilms in modern  medicine. High-throughput screening of antibiotics  against biofilms must be achieved for this field to move  forward. More diagnostics like the bioFILM PA assay  must be developed, and our understanding of the com-  position of natural antibiotic fermentations must be  better defined for us to understand the synergies that  might exist between existing antibiotics and to formulate  the composition of ingredients of future antibiotics yet  to be defined.    otic tolerance of Pseudomonas aeruginosa in biofilms.  Antimicrob Agents Chemother, 48 , 2659-2664.   Ceri, H., Olsen, M.E., Stremick, C., Read, R.R. & Buret, A. (1999)  The Calgary Biofilm Device: new technology for rapid deter-  mination of antibiotic susceptibilities of bacterial biofilms. J.  Clin. Microbiol, 37(6), 1771-1776.   DeBeer, D., Stoodley, P., Roe, F. & Lewandowski, Z. (1994) Effects  of biofilm structure on oxygen distribution and mass trans-  port. Biotechnol Bioeng, 43 , 1131-1138.   Harrison, J.J., Turner, R.J., Marques, L.L.R. & Ceri, H. (2005)  Biofilms. Am Sci, 93, 508-515.   Harrison, J.J., Ceri, H. Sc Turner, R.J. (2007) Multiple metal  resistance and tolerance in microbial biofilms. Nat Rev.  Microbiol, 5, 928-938.   Harrison, J.J., Wade, W.D., Akierman, S. et al. (2009) The chro-  mosomal toxin gene yafQ is a determinant of multidrug toler-  ance for Escherichia coli growing in a biofilm. Antimicrob  Agents Chemother, 53 , 2253—2258.   Huang, C., Xu, K.D., McFeters, G.A. 8c Stewart, P.S. (1998)  Spatial patterns of alkaline phosphatase expression within  bacterial colonies and biofilms in response to phosphate star-  vation. Appl Environ Microbiol, 64 , 1526—1531.   Hunter, R.C. & Beveridge, T.J. (2005) Application of a pHsensi-  tive fluoroprobe (C-SNARF-4) for pH microenvironment  analysis in Pseudomonas aeruginosa biofilms. Appl Environ  Microbiol, 71 , 2501-2510.   Jonas, K, Melefors, O. & Romling, U. (2009) Regulation of c-di-  GMP in biofilms. Future Microbiol, 4, 341-358.   Lawrence, J.R., Korber, D.R., Hoyle, B.D., Costerton, J.W. 8;  Caldwell, D.E. (1991) Optical sectioning of microbial bio-  films. J Bacteriol, 173 , 6558—6567.   Lewis, K. (2007) Persister cells, dormancy and infectious disease.  Nat Rev Microbiol, 5, 48—56.   McFeters, G.A. 8c Stewart, P.S. (2000) Biofilm resistance to anti-  microbial agents. Microbiology, 146 , 547-549.   Nadell, C.D., Xavier, J.O. 8c Foster, K.R. (2008) The sociobiology  of biofilms. FEMS Microbiol Rev, 33, 206-224.   Pesavento, C. a8c Hengge, R. (2009) Bacterial nucleotide-based  second messengers. Curr Opin Microbiol, 12 , 170-176.   Schertzer, J.W., Boulette, M.L. 8c Whiteley, M. (2009) More than  a signal, non-signaling properties of quorum sensing mole-  cules. Trends Microbiol, 17 , 189-195.   Stewart, RS. (2002) Mechanisms of antibiotic resistance in bac-  terial biofilms. Int J Med Microbiol, 292, 107—113.   Stewart, P.S. 8c Costerton, J.W. (2001) The antibiotic resistance  of bacteria in biofilms. Lancet, 358, 135-138.   Tamayo, R., Pratt, J.T., and Camilli, A. (2007) Roles of cyclic  diguanylate in the regulation of bacterial pathogenesis. Annu  Rev Microbiol, 61 , 131-148.   Walters III, M.C., Roe, F., Bugnicourt, A., Franklin, M.J. 8c  Stewart, P.S. (2003) Contributions of antibiotic penetration,  oxygen limitation and low metabolic activity to tolerance in  Pseudomonas aeruginosa biofilms to ciprofloxacin and  tobramycin. Antimicrob Agents Chemother, 47 , 317—323.    Werner, E., Roe, F., Bugnicourt, A. et al. (2004) Stratified growth  in Pseudomonas aeruginosa biofilms. Appl Environ Microbiol ,  70, 6188-6196.   Williams, R & Camara, M. (2009) Quorum sensing and envi-  ronmental adaptation in Pseudomonas aeruginosa: a tale of  regulatory networks and multifunctional signal molecules.  Curr Opin Microbiol, 12, 182-191.   Xu, K.D., Franklin, M.J. & Stewart, P.S. (2003) Contributions of  antibiotic penetration, oxygen limitation, and low metabolic    activity to tolerance of Pseudomonas aeruginosa biofilms to  ciprofloxacin and tobramycin. Antimicrob Agents Chemother,  47,317-323.   Xu, K.D., Stewart, P.S., Xia, F., Huang, C. & McFeters, G.A. (1998)  Spatial physiological heterogeneity in Pseudomonas aerugi-  nosa biofilm is determined by oxygen availability. Appl  Environ. Microbiol, 64, 4035-4039.   Progress in immunological science has been driven by the  need to understand and exploit the generation of immune  states exemplified now by the use of modern vaccines.  From almost the first recorded observations, it was rec-  ognized that persons who had contracted and recovered  from certain infectious diseases were not susceptible (i.e.  were immune) to the effects of the same disease when  re-exposed to the infection. Thucydides, over 2500 years    ago, described in detail an epidemic in Athens (which  could have been typhus or plague) and noted that suffer-  ers were ‘touched by the pitying care of those who had  recovered because they were themselves free of apprehen-  sion, for no one was ever attacked a second time or with  a fatal result’.   Since that time many attempts have been made to  induce this immune state. In ancient times the process of  variolation (the inoculation of live organisms of smallpox  obtained from the diseased pustules of patients who were  recovering from the disease) was practised extensively in  India and China. The success rate was very variable and  often depended on the skill of the variolator. In the late    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    18th century Edward Jenner, an English country doctor,  observed the similarity between the pustules of smallpox  and those of cowpox, a disease that affected the udders  of cows. He also observed that milkmaids who had con-  tracted cowpox by the handling of diseased udders were  immune to smallpox. Jenner deliberately inoculated a  young boy with cowpox, and after the boy’s recovery,  inoculated him again with the contents of a pustule taken  from a patient suffering from smallpox; the boy did not  succumb to infection from this first, or any subsequent  challenges, with the smallpox virus. Even though the  mechanisms by which this protected against smallpox  were not understood, Jenner’s work had shown proof of  principle that the harmless stimulation of our adaptive  immune system (see section 1.2) was capable of generat-  ing an immune state against a specific disease, and thereby  provided the basis for the process we now understand as  vaccination. The cowpox virus is otherwise known as the  vaccinia virus and the term vaccine was introduced by  Pasteur to commemorate Jenner’s work.   In 1801 Jenner prophesied the eradication of small-  pox by the practice of vaccination. In 1967 smallpox in-  fected 10 million people worldwide. The World Health  Organization (WHO) initiated a programme of confine-  ment and vaccination with the aim of eradicating  the disease. In Somalia in 1977 the last case of naturally  acquired smallpox occurred, and in 1979 WHO an-  nounced the total eradication of smallpox, thus fulfilling  Jenner’s prophecy. Many vaccine products are now  available designed to provide protection against a range  of infectious diseases. Their value has been proven in  national vaccination programmes leading to dramatic  reductions in morbidity and mortality of such diseases  as diphtheria, pertussis, mumps, measles, rubella, and  hepatitis A and B.   Further progress in the understanding of the complex  nature and functioning of the immune system has been  gained through the recognition that many varied forms  of pathology, beyond that of infectious disease per se,  have an underlying immunological basis, including such  diseases as asthma, diabetes, rheumatoid arthritis and  many forms of cancer. A basic knowledge of how the  immune system functions is essential for health profes-  sionals involved in understanding the nature of disease  and rationalizing therapeutic strategies. This chapter  aims to provide a sound overview of the structure and  functioning of the immune system and impart the reader  with knowledge which will serve as a platform for the  study of more complex specialized texts if and when  required.   The primary function of the immune system is to defend  against and eliminate ‘foreign’ material, and to minimize  any damage that may be caused as a result of the presence  of such material. The term ‘foreign’ includes not only  potentially pathogenic microorganisms but also cells rec-  ognized as ‘non-self’ and therefore foreign such as the  human body’s own virally infected or otherwise trans-  formed (e.g. cancerous) host cells. Foreign material  would also include allogeneic (within species) or xenoge-  neic (between species) transplant tissue and therapeutic  proteins administered as medicines if they arose from a  different species or were of human origin but had under-  gone inappropriate post-translational modifications  during manufacture or contained impurities. It is also  possible that small organic-based drugs may form  adducts with endogenous proteins leading to the genera-  tion of an immunogen. A good example of such adduct  formation is that between the serum protein albumin and  the glucuronide metabolite of some non-steroidal anti-  inflammatory drugs. This adduct is proposed as the basis  of some hypersensitivity reactions.   For clarity there are a number of terms that should be  defined at this point. An organism which has the ability  to cause disease is termed a pathogen. The term virulence  is used to indicate the degree of pathogenicity of a given  strain of microorganism. Reduction in the virulence of a  pathogen is termed attenuation; this can eventually result  in an organism losing its virulence completely and it is  then said to be avirulent.   An antigen is a component of the ‘foreign’ material that  gives rise to the primary interaction with the body’s  immune system. If the antigen elicits an immune response  it may then be termed an immunogen. Within a given  antigen, e.g. a protein, there will be antigenic determinants  or epitopes, which actually represent the antigen recogni-  tion sites for our adaptive immune system (see below). For  example, within a protein antigen, an epitope for an  antibody response will consist of 5-20 amino acids that  form either part of a linear chain or a cluster of  amino acids brought together conformationally by the  folding of the protein. Antibodies (otherwise known as  immunoglobulins — Ig) are produced and secreted into  biological fluids by our adaptive immune system, are  widely used in in vitro diagnostics and have been inves-  tigated in therapeutics as a means of targeting drugs to  specific sites in the body. A monoclonal antibody is an  antibody nominally recognizing only a single antigen  (e.g. a single protein) and within which only a single    common epitope (e.g. clusters comprising a common  single specific amino acid sequence or pattern) is recog-  nized. In contrast, a polyclonal antibody is an antibody  nominally recognizing only a single antigen but within  which a number of different epitopes (e.g. clusters com-  prising different amino acid sequences or patterns) are  recognized.   The immune system is broadly considered to exhibit  two forms of response:   • The innate immune response, which is non-specific,  displays no time lag in responsiveness, and is not intrinsically  affected by prior contact with infectious agent.   • The adaptive immune response, which displays a time  lag in response, involves highly specific recognition of  antigen and affords the generation of immunological  memory. An example of immunological memory is that  provided by the generation of specific lymphocyte  memory cell populations following vaccination with  an antigen (e.g. diphtheria toxoid). These memory  cells reside over a long term in our lymphoid tissue and  permit a more rapid and pronounced protective  immunological response on future exposure to the same  antigen. The adaptive immune system is further  subdivided into:   ° Humoral immunity, within which the effector cells  are B-lymphocytes and where antigen recognition occurs  through interactions with antibodies.   ° Cell-mediated immunity, within which the effector  cells are T-lymphocytes and where antigen recognition  occurs through interactions of peptide antigen (presented  on the surface of other cell types) with T-cell receptors  ( TCR ) on the plasma membrane of T-lymphocytes. In  cell-mediated immunity the peptide antigen must be  presented to T-lymphocytes by other cell types in  association with a class of plasma membrane molecules  termed major histocompatibility complex (MHC) proteins.   A schematic overview of the cells involved in both the  innate and adaptive components of the immune response  is shown in Figure 9.1. Most of the cells involved in the  immune system arise from progenitor cell populations  within the bone marrow. The differentiation of these  progenitor cells is under the control of a variety of growth  factors, e.g. granulocyte- or macrophage-colony stimu-  lating factors (G-CSF and M-CSF, respectively) released  by monocyte and macrophage cells as well as by fibrob-  lasts and activated endothelial cells. These growth  factors promote the growth and maturation of monocyte  and granulocyte populations within the bone marrow   The principal cells of the innate immune system  include the following:   • Mononuclear phagocytic cells, which are short-lived  (<8 hours) monocytes in the blood circulation that  migrate into tissues and undergo further differentiation  to give rise to the long-lived and key effector cell — the  macrophage.   • The granulocyte cell populations which include the  neutrophil, basophil and eosinophil.   • The mast cell which is a tissue-resident cell that is  triggered by tissue damage or infection to release  numerous initiating factors leading to an inflammatory  response. Such initiating factors include histamine,  leukotrienes B4, C4 and D4, proinflammatory cytokines  (signal proteins released by leucocytes — white blood  cells), such as tumour necrosis factor-a (TNF-a), and  chemotactic substances such as interleukin-8 (IL-8). The  sudden degranulation of the contents of mast cells is also  responsible for the acute anaphylactic reactions to bee  stings, penicillins, nuts, etc.   • The natural killer (NK) cell which has a phenotype  similar to that of lymphocytes but lacks their specific  recognition receptors. The NK cell exploits non-specific  recognition to elicit cytotoxic actions against host cells  infected with virus and those host cells that have acquired  tumour cell characteristics.   The lymphocyte populations also arise from bone  marrow progenitor cells. The B-lymphocytes mature or  differentiate in the bone marrow before leaving to circu-  late in the blood and lymph, while T-lymphocytes  undergo maturation in the thymus. Antibodies mediat-  ing the effector functions of the humoral immune  system are produced and secreted from a differentiated  B-lymphocyte cell population termed plasma cells.   Innate defence against the passage of potentially patho-  genic microorganisms across epidermal and mucosal  barriers involves a range of non-specific mechanisms.  Commensal microorganisms living on mucosal surfaces  and on membranes such as skin and conjunctiva  constitute one such mechanism (see Chapter 8). These  commensals are, under normal circumstances, non-  pathogenic, and help prevent colonization by pathogenic   Figure 9.1 An overview of the cells involved in the immune response: both innate and adaptive components. The cells arise from a  pluripotent progenitor cell within the bone marrow, with their growth and differentiation controlled by numerous growth factors.  The T-lymphocytes differentiate in the thymus gland.    strains. There are also a number of physical and chemical  barriers against microbial entry, including the flow of  fluid secretions from tear ducts, the urogenital tract and  the skin. Many of these secretions possess bacteristatic or  bactericidal activity due to their low pH or the presence  of hydrolytic enzymes such as lysozyme (a peptidoglycan  hydrolase). Similarly, the mucus barrier covering mucosal  surfaces such as the epithelium of the lung serves as a false  binding platform for microorganisms, preventing them  from interacting with the underlying host cells. In the  normal state the hydrated mucus barrier is efficiently  cleared under the driving force of beating cilia. The  serious lung infections seen in cystic fibrosis arise because  patients are unable to clear the bacteria-laden dehydrated  mucus effectively.   The function of the innate defence system against micro-  organisms that have penetrated into interstitial tissues  and the vascular compartment relies largely on the proc-  esses of phagocytosis (see section 2.2.3) and of activation  of the alternative complement pathway (see section  2.2.4). However, the functions of the innate system when  exposed to microbial infection are also critical in the  recruitment and activation of cells of the adaptive  immune response (see later).   The main cells mediating phagocytosis are the mono-  nuclear phagocytic cells and granulocyte cell popula-  tions; of the latter, neutrophils are particularly important.  For such cells to function, they must possess receptors to    sense signals from their environment. In executing their  effector functions they need to secrete a range of mole-  cules that will recruit or activate other immune cells to a  site of infection.   Before consideration of the process of phagocytosis, an  overview of the mononuclear phagocytic cell and granu-  locyte cell populations is useful.   The mononuclear phagocytic cells include monocytes  and macrophages. Monocytes make up approximately  5% of the circulating blood leucocyte population and are  short-lived cells (circulating in blood for <8 hours), but  migrate into tissue to give rise to tissue macrophages. The  macrophages constitute a long-lived, widely distributed  heterogeneous population of cell types which bear differ-  ent names within different tissues, such as the migrating  Kupffer cell within the liver or the fixed mesangial cell  within the kidney glomerulus.   The mononuclear phagocytic cells secrete a wide range  of molecules too numerous to list in full here. However,  these secretions include:   • Molecules which can break down or permeabilize  microbial membranes and thereby mediate extracellular  killing of microorganisms, e.g. enzymes (lysozyme or  cathepsin G), bactericidal reactive oxygen species and  cationic proteins.   • Cytokines which can provide innate protective antiviral  (e.g. interferon (IFN)-a or -(3) and antitumour (e.g. TNF-a)  activity against other host cells. A group of cytokines termed  chemokines can also serve to chemoattract other leucocytes  into an area of ongoing infection or inflammation, for  example IL-8 which attracts neutrophils. Yet another group  of cytokines has proinflammatory actions (e.g. IL-1 and  TNF-a) which, among other outcomes, leads to activation  of endothelial and leucocyte cells promoting increased  leucocyte extravasation into tissues and, in the case of IL-1,  activation of T-lymphocyte populations.   • Bioactive lipids (e.g. thromboxanes, prostaglandins and  leukotrienes), which further promote the inflammatory  response through actions to increase capillary vasodilation  and permeability.   The mononuclear phagocytic cells also possess numer-  ous receptors that interact with their environment. These  cells possess, among others:   • Receptors for chemotaxis toward microorganisms, e.g.  receptors for secreted bacterial peptides such as  formylmethionyl peptide.   • Receptors for complement proteins that serve as  leucocyte activators (e.g. C3a and C5a; see section 2.2.4)  or complement proteins that serve to coat (opsonize)    microorganisms (e.g. C3b). An opsonized microbial  surface more readily adheres to a phagocyte membrane,  with the opsonin triggering enhanced activity of the  phagocyte itself.   • Receptors for promoting adherence, such as lectin  receptors interacting with carbohydrate moieties on the  surface of the microorganism, or receptors for Fc domains  (non-antigen-recognition domains) of antibodies which  opsonize microorganisms (e.g. the receptor for the Fc  domain of IgG is Fcg), or integrin receptors for cell-cell  adhesion (e.g. promoting interaction between a  macrophage and T-lymphocyte).   • Receptors for cytokines including those involved in  macrophage activation (e.g.IFN.-y) or limiting macrophage  mobility (e.g. macrophage inhibitory factor, MIF) and hence  increasing cell retention at a site of infection.   The granulocyte cell populations include the neutrophils,  basophils and eosinophils. The short-lived (2-3 days)  neutrophil is the most abundant granulocyte (compris-  ing >90% of all circulating blood granulocytes) and is the  most important in terms of phagocytosis; indeed, this is  the main function of the neutrophil. The receptors and  secretions of the neutrophil are similar to those of the  macrophage, although notably the neutrophil does not  present antigen via MHC class II proteins (see later). The  neutrophil is recruited to sites of tissue infection or  inflammation by a neutrophil-specific chemotactic factor  (IL-8) and is also chemoattracted and activated by some  of the same factors described for mononuclear phago-  cytic cells, including complement protein C3a, bacterial  formylmethionyl peptides and leukotrienes. Like macro-  phages, neutrophils undergo a respiratory burst and are  very effective generators of reactive oxygen species.   Eosinophils are poor phagocytic cells and have a spe-  cialized role in the extracellular killing of parasites such  as helminths, which cannot be physically phagocytosed.  Basophils are non-phagocytic cells.   Macrophages and neutrophils in particular demonstrate  a high capacity for the physical engulfment of particles  such as microorganisms or microbial fragments from  their immediate extracellular environment. This process  (Figure 9.2) is made up of a number of steps:   • Chemotaxis of the phagocyte toward the microorganism  through signals arising from the microorganism itself  (e.g. formylmethionyl peptide), signals arising from  complement proteins (e.g. C3a and C5a) generated as  part of the activation of the alternative complement    Figure 9.2 Schematic of phagocytosis  showing: (step A) adherence of the  microorganism to the surface of the  phagocyte; (step B) membrane  activation of the phagocyte; (step C)  enclosure of phagocytosed material  within the phagosome and subsequently  the phagolysosome. The mediators of  the phagolysosome degradation of the  microorganism are shown in the  enlarged phagolysosome insert.    pathway (see section 2.2.4), or signals due to release of  inflammatory factors (e.g. leukotrienes) secreted by other  leucocyte cells situated at the site of an infection.   • Adherence of the microorganism to the surface of the  phagocyte (step A in Figure 9.2), involving adhesion  through lectin receptors present on the surface of the  phagocyte which interact with carbohydrate moieties on  the surface of the microorganism; adhesion through  complement C3b receptors present on the surface of the  phagocyte interacting with C3b molecules that have  opsonized the surface of the microorganism; and  adhesion through Fc receptors which interact with the Fc  domain of antibodies that have opsonized the surface of  a microorganism.   • Membrane activation of the phagocyte actin-myosin  contractile network to extend pseudopodia around the  attached microorganism (step B in Figure 9.2). Membrane  activation will also lead to the generation of a ‘respiratory  burst’ by the phagocyte which involves an increase in the  activity of the phagocyte membrane NADPH oxidase  which converts molecular oxygen into bactericidal  reactive oxygen species such as superoxide anion ( - CT),  hydrogen peroxide (H 2 0 2 ), and in particular hydroxyl  radicals ( - OH) and halogenated oxygen metabolites  (HOC1 ).    • The enclosure of phagocytosed material, initially  within a membranous vesicle termed a phagosome. FFere,  cationic proteins such as defensins and reactive oxygen  species begin microbial membrane degradation. This is  followed within minutes by fusion of the phagosome  with a lysosome to form a phagolysosome, whose contents  are at an acidic pFI of about 5 which is optimal for the  continued active breakdown of microbial structural  components (step C in Figure 9.2).   The alternative complement pathway fulfils a critical role  in innate immune defence. The complement system com-  prises at least 20 different serum proteins; many are  known by the letter C and a number, e.g. C3. Many of the  complement proteins are zymogens, i.e. proenzymes  requiring proteolytic cleavage to be enzymically active  themselves; some are regulatory in function. The cleavage  products of complement proteins are distinguished from  their precursor by the suffix ‘a’ or ‘b’, e.g. C3a and C3b,  with the suffix ‘b’ generally denoting the larger fragment  that stays associated with a microbial membrane, and  the suffix ‘a’ generally denoting the smaller fragment  that diffuses away. The activation of the complement  pathway occurs in a cascade sequence, with amplification    occurring at each stage, such that each individual enzyme  molecule activated at one stage generates multiple acti-  vated molecules at the next. In the ‘resting’ state, in the  absence of infection, the complement proteins are inac-  tive or have only a low level of spontaneous activation.  The cascade is tightly regulated by both soluble and  membrane-bound associated proteins. The regulation of  the complement pathway prevents inappropriate activa-  tion of the cascade (i.e. when no infection is present) and  also minimizes damage to host cells during an appropri-  ate complement response to a microbial infection.  Complement activation is normally localized to the  site(s) of infection.   There are three main biological functions of the alter-  native complement pathway.   • Opsonization of microbial membranes. This involves the  covalent binding of complement proteins to the surface  of microbial membranes. This opsonization or coating  by complement proteins promotes adherence of the  opsonized microbial component(s) to the cell membranes  of phagocytic cells. The complement protein C3b is a  potent opsonin.   • Activation of leucocytes. This involves complement  proteins acting on leucocytes, either at the site of infection  or at some distance away, with the result of raising the  level of functioning of the leucocytes in immune defence.  For example, C3a is a potent leucocyte chemoattractant  and also an activator of the respiratory burst.   • Lysis of the target cell membrane. This involves a  collection of complement proteins associating on the  surface of a microbial membrane to form a membrane  attack complex (MAC), which leads to the formation of  membrane pores and, ultimately, microbial cell lysis.   Figure 9.3 shows a highly schematized view of the  activation cascade for the alternative complement  pathway on a microbial membrane surface. The activa-  tion steps in the alternative pathway are also shown in  Figure 9.7, which contrasts with the activation  steps in the classical complement pathway involving  antibody.   The pivotal protein in the alternative pathway is C3  (195 kDa). Under normal circumstances (in the absence  of infection) C3 is cleaved very slowly through reaction  with water or trace amounts of proteolytic enzyme to give  C3b and C3a. The C3b formed is susceptible to nucle-  ophilic attack by water and is rapidly inactivated to give  iC3b. The C3a is not generated in sufficient amounts to  lead to leucocyte activation and is rapidly inactivated.  This normal low-level cleavage of the C3 molecule is  termed ‘C3 tickover’ and it provides low levels of starting    material, i.e. C3b, which will be required for full activa-  tion of the alternative complement pathway in the case  of a microbial infection.   In the presence of a microbial membrane the C3b  formed by C3 tickover will be susceptible to nucleophilic  attack by hydroxyl or amine groups on the membrane  surface, leading to the covalent attachment of C3b to the  membrane (step A in Figure 9.3). Once C3b has attached  to the membrane, factor B can bind to form a molecule  termed C3bB. This complex is stabilized by a soluble  protein called properdin. Factor D then enzymically  cleaves the bound factor B to generate a molecule termed  C3bBb which is the C3 convertase of the alternative  pathway (Figure 9.3 inset).   This newly generated stable C3 convertase enzymically  cleaves C3 to generate further C3b and C3a molecules,  leading to leucocyte activation (by C3a) and greater dep-  osition of C3b on the microbial membrane and hence  further generation of C3 convertase molecules. In effect  the microbial membrane has activated a positive feedback  loop with cleavage of C3 to generate high amounts of  C3b and C3a molecules.   The deposited C3b not only leads to the formation of  the C3 convertase but also coats the microbial membrane  as an opsonin and so promotes binding to phagocyte cell  membranes. Some of the deposited C3b associates with  the newly formed C3 convertase to generate a complex  termed C3bBb3b, which is the C5 convertase of the alter-  native pathway (step B in Figure 9.3). This C5 convertase  binds the complement protein C5 and cleaves it into C5a  (a leucocyte activator) and C5b (an opsonin). The C5b  remains associated with the membrane and acts as a plat-  form for the sequential binding of complement proteins  C6, C7, C8 (step C in Figure 9.3). The a-chain of the C8  molecule penetrates into the microbial membrane and  mediates conformational changes in the incoming C9  molecules such that C9 becomes amphipathic (simulta-  neously containing hydrophilic and hydrophobic groups).  In this form it is capable of insertion through the micro-  bial membrane where it mediates a polymerization  process that gives the MAC. The MAC generates trans-  membrane channels within the microbial membrane  with the osmotic pressure of the cell leading to an influx  of water and eventual microbial cell lysis.   Differences between host cell membranes and micro-  bial cell membranes mean that the cascade is only acti-  vated in the presence of microorganisms, so C3 tickover  cannot give rise to full activation of the alternative  pathway in the absence of microbial membrane. Stable  deposition of a functional C3 convertase only occurs on   Figure 9.3 A highly schematized overview of the activation cascade for the alternative complement pathway on a microbial  membrane surface. In the presence of a microbial membrane the C3b formed by C3 tickover deposits on the microbial membrane  (step A). C3a diffuses away, leading to leucocyte activation. The deposited C3b leads to the generation of a stabilized C3 convertase  (step B) which, through a positive feedback loop, leads to the amplified cleavage of more C3. Some C3b associates with the C3  convertase to generate a C5 convertase (step C) which will eventually lead to the generation of an MAC.    the microbial cell surface. The differences that exist  include, for example:   • the high sialic acid content of host cell membranes that  promotes the dissociation of any C3 convertase formed  on host surfaces   • the presence of specific host cell membrane proteins  that also serve a key regulatory function.   Decay activating factor (DAP) or complement receptor  type 1 (CR1) are host cell membrane proteins that serve  to competitively block the binding of factor B with C3b  and hence inhibit formation of a C3 convertase; they  also promote disassembly of any C3 convertase formed.    Membrane cofactor protein (MCP) and CR1 are further  host cell membrane proteins that promote the displace-  ment of factor B from its binding with C3b. Host cell  membranes also possess a protein, CD59, which prevents  the unfolding of C9 — a required step for membrane  insertion to form an effective MAC.   The humoral immune response is mediated through  antibody-antigen interactions. B-lymphocytes in their  naive state, unstimulated by antigen, possess antibody  molecules on their cell membrane, which serve a surveil-    lance function to recognize any invading antigen. A  B-lymphocyte that has bound antigen is capable of dif-  ferentiating into a plasma cell which, under the influence  of signals from helper T-cells, produces a fuller repertoire  of antibody molecules (i.e. a fuller range of antibody  classes), which are then secreted from the plasma cell into  the extracellular environment to bring about a range of  humoral effector functions.   As briefly discussed in section 1.2, a B-cell antigen is a  substance or molecule specifically interacting with an  antibody, and which may lead to the further production  of antibody and an immunological response. Typically  B-lymphocyte antigens are proteins within which the  epitopes each consist of clusters of 5-20 amino acid resi-  dues. B-lymphocyte epitopes arise most commonly from  the three-dimensional folding of proteins (i.e. conforma-  tional epitopes), although they may also consist of a  sequential linear sequence of amino acids within the  polypeptide chain (linear epitopes). As a general rule,  there is a gradient of increasing immunogenicity with  increasing molecular weight of protein. Further, the  higher the structural complexity of the protein or  polypeptide antigen, the higher the level of immuno-  genicity it is likely to exhibit. Thus, a polypeptide com-  prising a single amino acid such as polylysine will be  expected to be a weaker immunogen than a protein of  equivalent molecular weight made up of a diverse range  of amino acids.   Polysaccharides tend not to be good immunogens  for B-lymphocytes. When a polysaccharide serves as the  sole immunogen, the humoral response obtained is  termed ‘T-cell-independent’ because the polysaccharide  does not elicit helper T-lymphocyte cooperation (see  section 4). The consequences of a T-cell-independent  humoral response include the lack of production of  memory B-cell populations and the lack of synthesis by  the plasma cell of the full range of antibody subclasses,  i.e. T-cell-independent humoral responses mainly involve  the production of IgM antibody. For improved immuno-  genicity carbohydrate antigens are conjugated to  proteins which allow a more effective ‘T-cell dependent’  humoral response, i.e. one that affords the generation  of memory B-cell populations and of the synthesis of  the full range of antibody subclasses. This strategy is  used in a number of current vaccine products, e.g.  meningococcal group C conjugate vaccine contains the  capsular polysaccharide antigen of Neisseria meningitidis  group C conjugated to Corynebacterium diphtheriae    protein. Pure nucleic acid and lipid serve as very poor  antigens.   Figure 9.4 shows an antibody monomer with a four-  polypeptide subunit structure, where the subunits are  linked through disulphide bonding. The basic monomer  structure can be considered the same for all the different  classes of antibody (see below) even though some may  form higher-order structures, e.g. IgM is a pentamer  made up of five antibody monomer units.   The subunits of the antibody monomer comprise two  identical ‘heavy’ polypeptide chains and two identical  ‘light’ polypeptide chains, with each of these containing  a ‘constant’ region and a ‘variable’ region. The light chain  variable regions (V L ) and the heavy chain variable regions  (V H ) are the parts of the antibody molecule involved in  antigen recognition. Specifically, antibodies produced by  different B-lymphocytes or plasma cells will have variable  regions possessing different amino acid sequences leading  to differences in antibody variable region surface confor-  mation. At the extreme tips of the variable regions are  hypervariable domains that serve the specific antigen rec-  ognition function discriminating between, for example,  diphtheria toxin and tetanus toxin. The structural differ-  ences in the variable and hypervariable domains enable  different antibodies to recognize different structural  epitopes; this meets the needs of the immune system to  combat a large and diverse range of antigens.   A horizontal line of symmetry can be drawn through  the antibody structure in Figure 9.4, bisecting the mole-  cule into two equivalent halves each containing a single  heavy chain and a single light chain and clearly showing  the antibody monomer to possess bivalency in its ability  to interact with antigen, i.e. each antibody monomer can  bind two epitopes, although the epitopes bound by a  single antibody must be identical. The antigen recogni-  tion domain of an antibody monomer is termed the Fab  domain. The structure of the constant region of the heavy  chain (C H ) does not influence the antigen recognition  function of the molecule but defines the different classes  of antibody that are produced and hence the effector  functions arising from antigen-antibody interaction; this  heavy chain constant region is termed the Fc domain.   An analogy that may assist visualization of the func-  tion of an antibody molecule is one that views it as a  hand (Fab domain) attached to the arm (Fc domain)  (Figure 9.4). The palm of the hand (variable region) can  take up different shapes to allow the fingertips (hyper-  variable regions) to gain a very precise interaction with    Figure 9.4 An antibody monomer  consisting of a four-polypeptide subunit  structure, where the subunits are linked  through disulphide bonding. The Fab  fragment is concerned with antigen  recognition, while the Fc region  determines the various effector  functions of antibodies. A horizontal  line of symmetry can be drawn through  the antibody structure bisecting the  molecule into a single heavy chain and a  single light chain and clearly showing  the bivalency in antigen recognition.    an object (antigen). At the wrist (hinge region) the hand  is highly flexible relative to the arm (Fc domain) to allow  the hand and fingertips (Fab domain) maximum flexibil-  ity to orientate an interaction with objects (antigen). The  structure of the arm (Fc domain) does not influence  interaction with an object (antigen). Once the object  (antigen) has interacted with the fingertips (hypervaria-  ble regions) of the hand then the arm (Fc domain) can  mediate a variety of effector functions.   A B-lymphocyte and plasma cell can produce different  classes of antibody depending on the stage of immune  activation and on the intercellular signals that the  B-lymphocyte and plasma cell receive from other effector  cells within the immune system. As stated above, the class  of antibody is determined by the structure of the Fc  domain and the different classes of antibodies possess  different effector functions. The basic classes of antibod-  ies are: IgM (heavy chain constant region defined as p);  IgA (heavy chain constant region defined as a); IgD  (heavy chain constant region defined as 8); IgG (heavy  chain constant region defined as y) and IgE (heavy chain  constant region defined as e). The different classes of  antibody can be remembered using the acronym MADGE.  In addition to the heavy chain constant region classes,  there are two light chain constant region classes, K and X;  however, these do not mediate different antibody effector  functions.   Each B-lymphocyte and the plasma cell that derives  from it is capable of producing all the different antibody  classes. However, all the antibody classes produced by a  single B-lymphocyte and its derived plasma cell will rec-  ognize only a single epitope, i.e. only a single specific set  of chemical features within a sequence or pattern of  amino acid residues. In other words, all antibodies pro-    duced by a single B-lymphocyte, and its derived plasma  cell, possess the same Fab domain recognizing the same  antigenic determinant but clearly may possess different  Fc domains capable of mediating different effector func-  tions. Thus the same epitope can stimulate various dif-  ferent forms mediated via the IgM, IgA, IgD, IgG, IgE  classes of humoral immune attack.   Within the antibody pool it is estimated that there are  approximately 10 9 different epitope recognition specifici-  ties, sufficient to cover the range of pathogens likely to be  encountered in life. This enormous diversity in antigen  recognition is due to the amino acid sequence diversity  in the variable and hypervariable domains of the anti-  body molecule. However, this large diversity cannot  result from the presence of an equivalent number of  separate protein-coding genes; the human genome  project has estimated there to be only approximately  30 000 protein-coding genes. Rather, the clonal diversity  in antigen recognition is due in the main to a process  termed gene rearrangement , which occurs in each  B-lymphocyte during maturation in the bone marrow.  For example, the DNA coding for a single heavy chain  molecule will result from the splicing together of genes  from four separate regions termed a variable region gene  (V), a diversity region gene (D), a joining region gene (J)  and a constant region gene (C). There are approximately  100 V genes, 25 D genes and 50 J genes. Gene rearrange-  ment will allow combinatorial freedom for any V, D and  J genes to splice together, providing a large number of  VDJ combined gene product permutations and hence  diversity in antigen recognition. Inaccurate splicing  together of the regional genes at the V-D and D-J junc-  tions further increases diversity, as does the process of  random nucleotide insertion. The C genes dictate the    different classes of antibody and not the antigen recogni-  tion specificity. An additional process which occurs in a  B-lymphocyte memory cell population while it resides  within the lymphoid tissue is that of somatic mutation,  in which only very slight changes in antibody Fab  domains occur through single base mutations. Sometimes  these mutations prove advantageous by increasing the  affinity of an antibody to the same original epitope.  Under these circumstances the antibody clone with the  highest binding affinity to the original target epitope will  proliferate and dominate. The light chain gene also has  V, J and C regions and the V and J genes undergo a similar  rearrangement to that described for the heavy chain, and  hence further add to diversity. The heavy chain and light  chain polypeptides are joined together via disulphide  bond formation following protein synthesis of the indi-  vidual heavy and light chains. In summary, all antibodies  produced by a single B-lymphocyte and its derived  plasma cell are ‘programmed’ to recognize only a single  antigen recognition feature determined by the recombi-  nation pattern of the V, D and J genes (heavy chain) and  the V and J genes (light chain). The class of antibody is  determined by further excisions within the DNA to allow  the same VDJ gene combination to lie next to a different  C gene, which codes for the structure of the antibody  constant region and therefore determines antibody class.  The five C gene classes are m, a, d, g and e, although  various subclasses also exist. Antibody class switching is  not a random process but one that is regulated by helper  T-lymphocyte cytokine secretions.   Within the body there may exist at any one time only a  handful of naive B-lymphocytes capable of recognizing  the same epitope. The meeting of an antigen and a naive  B-lymphocyte capable of recognizing an epitope within  the antigen occurs through the delivery of antigen to  lymphoid tissues of the spleen, lymph nodes and local  lymphoid tissue within mucosal surfaces (mucosal asso-  ciated lymphoid tissue, MALT) and skin (SALT). This  lymphoid tissue is rich in lymphocytes. Further, a pro-  portion of B-lymphocytes will always be recirculating  from the lymphoid tissue through the lymph and blood  circulations and so able to encounter circulating antigen.   Antigen will be specifically recognized by IgM mole-  cules present on the surface of the naive B-lymphocyte.  Following this antigen-driven selection of a specific  B-lymphocyte clone, the clone will undergo repeated cell  divisions. Some of the daughter cells will differentiate  into short-lived (2-3 days) plasma cells able to secrete    antibody of different classes to combat the initial primary  antigen exposure. Other clonal daughter cells will become  long-lived B-lymphocyte memory cells populating the  lymphoid tissue and spreading around the body through  the lymph and blood circulations. These cells will provide  ‘immunological memory’ able to generate a more rapid  and pronounced secondary response on subsequent  exposure to the original antigen (Figure 9.5).   This process of clonal selection and expansion to form  memory cell populations is the basis of vaccination. The  initial introduction of antigen gives rise to a primary  response (Figure 9.6) in which there is a significant latent  period before increased serum antibody levels are  observed; the main antibody response is IgM production,  although some IgG is also synthesized and secreted. On  re-exposure to the same antigen a secondary response is  elicited. The features of the secondary response include:   • a reduced latent period between antigen challenge and  increases in serum antibody (e.g. latent period of 5-7  days for the secondary response vs 7-20 days for the  primary response)   • an antibody response dominated by IgG which is more  pronounced with higher serum levels achieved.   In the absence of helper T-lymphocyte involvement  (T-cell-independent humoral responses, e.g. where  antigen is carbohydrate alone) B-lymphocyte memory  cell populations are not produced, and antibody class  switching is restricted. Hence, under these circumstances,  the primary and secondary antibody responses to antigen   Figure 9.5 Clonal proliferation of B-lymphocytes. Following  antigen- driven selection of a specific B-lymphocyte clone, it  will undergo repeated cell divisions to give effector cell  populations and memory cell populations.    Figure 9.6 Primary and secondary responses to antigen. A  primary response of the humoral system involves a significant  latent period before elevated serum antibody levels are seen;  the major serum antibody generated is IgM. Memory cell  populations provide the basis of the secondary response,  which displays a significant reduction in the latency period to  achieve elevated serum antibody. The antibody serum levels  are greater than in the primary response and involve mainly  IgG.    challenge are essentially indistinguishable and exhibit  a prolonged latent period, relatively low levels of  serum antibody produced, and IgM as the main serum  antibody.   The humoral immune response is mediated by the initial  antibody-antigen interaction, but with the different anti-  body classes offering a range of effector functions. The  effector functions of antibodies include those described  below.   Antibody on the surface of naive or memory B-  lymphocytes serves to recognize and bind specific antigen;  IgM serves this main cognitive function. It exists as a  pentamer of five monomer units with an antigen valency  of 10 and is extremely efficient at binding antigen. IgD  appears to function mainly on the surface of B-lymphocytes  and may also contribute to cognition in some way.   Secreted antibody, in particular IgG, IgA and IgM, can  bind antigen and sterically hinder the interaction of  toxins, viruses, bacteria, etc. with host cell surfaces. In the  circulatory and interstitial fluids IgG (which exists as a  monomer with an antigen valency of 2) is the main anti-  body that fulfils this role in the secondary response, while  IgM is the main antibody produced in the primary  response. IgA has specific roles in mucosal immunity.   Secreted antibody, in particular IgG, opsonizes antigenic  material and in doing so promotes association (e.g.  through Fcg receptors) of the antigenic material with  phagocyte membranes. Occupancy of the Fc receptor by  the antibody also serves to activate a phagocyte’s killing  mechanisms.   Mucosal immunity involves the interaction of antibody  with antigen at mucosal surfaces such as those of the  gastrointestinal tract, lung or urogenital tract. The major  antibody of the mucosal lining fluid is IgA, which exists  as a dimer of two monomer units (antigen valency of 4).  IgA is actively secreted across mucosal epithelium into  the lining fluid; it will neutralize antigen and may also  serve as an opsonin. IgA is also present in secretions such  as tears, saliva, etc. but it has a limited role in systemic  immunity.   Through specific binding to antigen on the surface of  membranes perceived as ‘foreign’, e.g. microbial cells or  host cells virally infected or otherwise transformed, anti-  body can direct (through its Fc domains) the close asso-  ciation of ‘killing’ cells, such as neutrophils, eosinophils,  NK cells and even cytotoxic T-lymphocytes, with the  ‘foreign’ membrane. This close association depends on  the antibody’s Fc domain binding to the respective Fc  receptor present on the surface membrane of the ‘killing’  cell. Such close proximity to the ‘foreign’ cell enables the  efficient and targeted release of cytotoxic molecules into  the extracellular environment. IgG is the main antibody  of systemic body fluids and is an important mediator of  ADCC, although IgE and IgA may undertake this role in  certain circumstances, e.g. against certain parasites IgE  directs ADCC mediated by eosinophils.   Mast cells express high-affinity receptors (Fee) that bind  the Fc domain of IgE antibodies. In the absence of  antigen these receptors are occupied by the IgE monomer  (antigen valency of 2) secreted previously from plasma  cells. In this circumstance the IgE molecules are serving  a cognitive function which, on appropriate antigen  binding, results in aggregation of the membrane-bound  IgE and causes immediate mast cell degranulation and  release of inflammatory mediators. Mast cells possess in  their membranes IgE monomers able to recognize  different antigenic epitopes. This contrasts with each  single B-lymphocyte, which possesses IgM antibody on  its surface membrane that performs a cognitive function  but is capable of recognizing only a single epitope  specificity.   The neonate lacks the ability to mount a full immuno-  logical response; accordingly, maternal IgG is transported  across the placenta late in pregnancy and is also  absorbed across the gastrointestinal tract from breast  milk. Maternal IgA secreted into breast milk will also  provide mucosal protection for the neonate.   A complement cascade similar to that of the alternative  pathway can be activated through specific antibody-    antigen interactions. The antibodies that activate the clas-  sical complement pathway are IgM and IgG.   Key steps in the activation of the classical pathway are  shown in Figure 9.7, where this pathway is also compared  to the alternative pathway. In the classical pathway the  initiating step is the specific binding of IgG or IgM to  antigen. Once this occurs, a complement protein termed  Cl (which comprises a single Clq subunit, two Clr subu-  nits and two Cls subunits) binds to adjacent Fc domains  in the antibody-antigen complex. This binding of Cl  activates the catalytic activity of the Clr subunits, and in  turn the Cls subunits. The activated Cls subunits cleave  C4 into C4b and C4a; the latter can diffuse away and serve  as a leucocyte activator. The C4b covalently associates  with the antibody-antigen complex on the surface of a  microbial membrane and can serve as an opsonin. A  further complement protein, C2, binds to this membrane  complex to give C4b2. The Cls subunit then enzymically  cleaves the bound C2a to generate on the membrane a  new complex termed C4b2b, which is the C3 convertase  of the classical pathway. (In some texts the C2a is referred  to as the larger subunit remaining with the membrane  while C2b is the smaller subunit that diffuses away.)   This C3 convertase molecule is distinct from that  within the alternative pathway, but it is from this point  onwards that parallels can be drawn between the two  cascades.   The host proteins that serve key regulatory functions  within the alternative pathway (DAF, CR1 factor I, CD59)    also serve similar functions within the classical pathway.  However, in contrast to the alternative pathway the acti-  vation step in the classical pathway requires specific  antibody-antigen interactions. In this context the Cl  protein can only become catalytically active when it is  bound to at least two adjacent Fc domains. In the case of  the IgG and IgM molecules the Fc domains will only align  adjacent to each other when the corresponding Fab  domains bind antigen. Further, when Cl is free in the  circulation it is bound to a protein termed Cl inhibitor  (Cl-INH) which prevents any possible activation of Cl  in the absence of antibody. Once Cl binds to adjacent Fc  domains within an antibody-antigen complex Cl-INH is  displaced.   The functions of the classical complement pathway are  similar to those described for the alternative pathway, i.e.  opsonization, leucocyte activation and membrane lysis of  target cells. The classical pathway can additionally lead to  complement protein deposition on insoluble antibody-  antigen immune complexes circulating within blood, and  in doing so promote the clearance of such potentially  harmful complexes by Kupffer cells of the liver. The pres-  ence of two complement pathways provides for rapid  (alternative) and specific (classical) activation of a key  defence mechanism, and offers greater protection against  the development of microbial resistance mechanisms.   Cell-mediated adaptive immune responses are mediated  by T-lymphocytes which arise from bone marrow pro-  genitor cells and undergo maturation in the thymus  before release into the systemic blood and lymph  circulations (Figure 9.1). A number of parallels can be  drawn between the B-lymphocyte-mediated immune  response and the T-lymphocyte-mediated response. First,  membrane-bound antibodies serve the cognitive function  for B-lymphocytes, while the cognitive function for  T-lymphocytes is served by T-lymphocyte receptor (TCR)  present on the cell’s plasma membrane surface. Second,  in response to antigen T-lymphocytes, like B-lymphocytes,  will undergo clonal proliferation and form a population  clone of memory T-cells specific for a single epitope.  Third, each single B-lymphocyte and plasma cell that  derives from it is capable of producing antibodies that  will only recognize a single epitope. In the same manner  each single T-cell is programmed to make TCR of only a  single specificity able to recognize only a single specific    set of chemical features within a T-cell epitope. This is  achieved for the TCR in a similar manner to that for  antibodies in that different gene segments termed V-D-J  are brought together by the process of gene rearrange-  ment into single RNA products. The recombined RNA  will code for the polypeptide chains that make up the  TCR. When all the possible recombinations are consid-  ered, the number of different TCR molecules that an  individual can make is in excess of 10 9 , a number similar  to the primary antigen recognition repertoire of T-cells.   There are two general classes of T-lymphocytes: helper  T-lymphocytes and cytotoxic T-lymphocytes. The latter  function to kill host cells that have undergone a transfor-  mation such as a viral infection or cancer and they rec-  ognize specific antigens on the surface of host cells  that have arisen as a result of such cell transformation.  Through this specific antigen recognition, the cytotoxic  T-cell becomes closely apposed to the target host cell and  is activated to synthesize and release cytotoxic secretory  products (e.g. pore-forming molecules such as perforins)  leading to lysis of the affected host cell. In contrast, the  helper T-cell can be viewed as the coordinator of the  adaptive immune system, providing appropriate activa-  tion signals, in the form of secreted cytokines, to promote  the functioning of both the cytotoxic T-cell populations  and that of the antibody-producing B-lymphocyte and  plasma cell populations. The actions of the helper T-cell  populations also promote the function of the innate  immune system, for example IFN-y released by helper  T-cells increases the phagocytic activity of macrophages.  The helper T-cells are further divided into T H 1 or T H 2  subpopulations depending on the nature of cytokines  they secrete and, as a consequence, the arms of the  immune system they predominantly influence. The T H 1  helper T-cells mainly regulate cell-mediated immunity  while T h 2 helper T-cells regulate humoral immunity.   Epitopes for T-lymphocytes comprise exclusively linear  peptide sequences. T-lymphocytes are unable to respond  to carbohydrate, lipid or nucleic acid material and they  only respond to peptide antigen when it is presented to  the T-lymphocyte by surface proteins on the plasma  membrane of host cells. These surface proteins are termed  MHC proteins and can be subdivided into two main  classes:   • MHC class I proteins are expressed on the surface of all  nucleated host cell membranes and present peptide  antigen to cytotoxic T-lymphocytes.    • MHC class II proteins are expressed only on a more  specialized group of cells termed antigen-presenting  cells (APCs), and present peptide antigen to helper  T-lymphocytes.   Such a distinct cellular distribution of MHC proteins  and restriction in presentation to discrete T-lymphocyte  subpopulations may be remembered by considering the  different T-lymphocyte functions. That is, all cells of  the body have the potential to become infected with virus  and undergo a cancerous change, and hence all cells  must have the capacity to be destroyed by the actions of  cytotoxic T-cells. As such, all cells of the body must  possess MHC I molecules to afford antigen presentation  to cytotoxic T-cells. In contrast, as a coordinator cell of  the immune system, the helper T-cell must be able to  respond to its environment in order to give appropriate  signals or ‘help’ to other immune cells. Specialized APCs  with the capacity to phagocytose interstitial proteina-  ceous material therefore undertake the function of ‘envi-  ronmental sampling’. MHC class II proteins expressed on  the surface of APCs will present peptide antigen to helper  T-lymphocytes.   APCs include the macrophage tissue cell population,  specialized APCs such as dendritic cells within the lym-  phatic system or Langerhans cells within the skin.  B-lymphocytes also serve the function of an APC because  they interact with protein antigen through high-affinity  surface IgM molecules. Subsequently, they internalize the  protein antigen for processing to generate peptides that  will be presented by MHC II molecules expressed on the  B-lymphocyte cell surface. Another cell type that can  serve the function of an APC is the endothelial cell, which  can be induced to express MHC II molecules by the  action of the cytokine IFN-y. It should not be overlooked  that the APC can itself become infected with virus and    undergo cancerous transformation, and therefore the  APC, in addition to MHC II molecules, will also express  the full complement of MHC I molecules on its surface.   This process of MHC presentation of peptide and  interaction with T-lymphocyte receptor is shown in  Figure 9.8. A foreign peptide presented by a MHC mol-  ecule will be recognized by a TCR expressed on the  surface of an appropriate T-lymphocyte. Once a particu-  lar TCR recognizes a peptide sequence as foreign, intrac-  ellular signals to activate the T-cell are sent via the CD3  complex present within the T-cell membrane. The recog-  nition of peptide as foreign will lead to an immune  response. Beyond antigen presentation, the interaction  between MHC molecule and T-lymphocyte also serves to  identify that the T-lymphocyte and host cell membrane  arise from the same embryonic tissue. Tremendous inter-  individual differences, or more specifically polymor-  phisms, exist in the MHC proteins within a population.  The T-lymphocyte undertakes this MHC surveillance  through the possession of accessory molecules. Cytotoxic  T-lymphocytes possess CD8+ molecules which interact  with MHC I (Figure 9.8a), while helper T-lymphocytes  possess CD4 + molecules which interact with MHC II  (Figure 9.8b); hence the use of the terms CD8 + lym-  phocytes to refer to cytotoxic T-cells and CD4 + lym-  phocytes to refer to helper T-cells.   4.2 Processing of proteins to allow  peptide presentation by MHC molecules   Peptide epitope presented by MHC I is derived from  the processing of proteins (e.g. a viral protein) synthe-  sized within the actual cell that eventually will present  the peptide to cytotoxic T-lymphocytes. The MHC I  molecule is composed of two polypeptide chains, an a-  chain which has al, a2 and a3 domains, and a second    Figure 9.8 The process of MHC  presentation of peptide and interaction  with T-lymphocyte receptor (TCR).  Cytotoxic T-lymphocytes (CD8 + ) interact  with MHC I (a), while helper  T-lymphocytes (CD4 + ) interact with  MHC II (b).    polypeptide termed (3 2 -microglobulin. The al and (32  domains form a peptide-binding cleft which can accom-  modate peptides up to 11 amino acids in length. Figure  9.9a shows the processing of a protein into peptide frag-  ments for presentation by MHC I. The synthesized  protein (indicated by an asterisk) is present in the cyto-  plasm of the cell and is degraded by a subcellular organelle  termed a proteasome. The derived peptide fragments are  actively transported, via a TAP peptide transporter, into  the lumen of the endoplasmic reticulum (ER) where they  fit within the binding clefts of MHC I molecules. From  the ER the MHC I with bound peptide is transported to  the trans-Golgi network (TGN), from which it is trans-  ported via endosomes to the plasma membrane where  the MHC I molecule with bound peptide is accessible to  surveillance by cytotoxic CD8 + T-lymphocytes.    Peptide epitopes presented by MHC II are derived  from proteins present within the extracellular fluid and  are presented to helper T-lymphocytes by APCs. The  MHC II molecule is composed of two polypeptide chains,  an a-chain which has al and a2 domains, and a (3-chain  which has (3 1 and (32 domains. The al and (31 domains  form a peptide-binding cleft that can accommodate pep-  tides up to 20 amino acids in length. Figure 9.9b shows  the processing of a protein into peptide fragments for  presentation by MHC II. In this case the protein (indi-  cated by an asterisk) is internalized from the extracellular  fluid by the APC and restricted to an endosomal com-  partment without access to the APC’s cytoplasm. The  endosome delivers the protein to a lysosome compart-  ment which degrades the protein into peptide fragments,  after which the peptide fragments are returned to an    Figure 9.9 Schematic of the processing of a protein into peptide fragments for presentation by MHC. (a) The synthesized protein   is present in the cytoplasm of the cell and is degraded into peptide by the proteasome. The derived peptide fragments are  presented by MHC I to cytotoxic CD8 + T-lymphocytes. (b) Protein  is endocytosed by an APC and processed into peptide  within lysosomes. The derived peptide fragments are presented by MHC II to helper CD4 + T-lymphocytes.    endosome compartment. In the lumen of the ER the  MHC II molecule becomes associated with another  protein termed an invariant chain which blocks access of  peptides to the binding cleft of the MHC II molecule; the  MHC II-invariant chain complex is transferred to the  TGN and then to an endosomal compartment. At this  point the endosomes that contain the processed peptide  and the MHC II molecules merge, and the invariant  chain disintegrates, allowing peptides access to the MHC  II-binding cleft. The MHC II molecules with bound  peptide are transported to the plasma membrane where  they are accessible to surveillance by helper CD4 +  T-lymphocytes.   Following maturation in the thymus, mature but naive  CD4 + helper T-cells access the systemic blood and lym-  phatic circulations. In this naive state they have yet to be  stimulated by antigen. The antigen-driven activation  signals involve initially TCR interactions with MHC-  presented peptide and subsequent CD3 activation. This  is followed by the interaction of a variety of costimula-  tory molecules on the surface of the APC (e.g. CD80,  CD86) with surface receptors (e.g. CD28) on the helper  T-cell. Once the helper T-cell is activated it can proliferate  in an autocrine or paracrine fashion driven by secreted  IL-2. These proliferating helper T-cells will then differen-  tiate depending on their cytokine environment; for  example, IFN-y and IL-12 drive the differentiation to a  T h 1 subpopulation of cells while IL-4 drives the differen-  tiation to a T h 2 subpopulation of cells.   Apart from IL-2 the main cytokines produced by T H 1  cells are IFNy and TNF(3, and the main cell partner for  T h 1 cells are the APCs. The T H 1 cells classically promote  cell-mediated immune responses maximizing the effec-  tiveness of APCs and the proliferation of cytotoxic CD8 +  T-cells. Apart from IL-2 the main cytokines produced by  T h 2 cells are IL-4, 5,6,10 and 13, while the main cell  partner for T H 2 cells is the B-cell. The T H 2 cells classically  promote the humoral immune responses stimulating  B-cells to proliferate to undergo Ig class switching and  increase Ig production and secretion.   The above model of helper T-cell subpopulations and  how various cytokines can serve to promote the differen-  tiation pathway to either T H 1 or T H 2 phenotype is recog-  nized to be an oversimplification. However, the basic  model serves to emphasize that distinct populations of   T-regulatory cells (T regs ) are a subset of T lymphocytes  that serve an immune suppressor function leading to  peripheral tolerance to self- or foreign antigens. T regs  are characterized by a CD4 + /CD8~ phenotype, but among  a number of other identity markers that these cells  display the expression of the Foxp3 transcription factor  is the main distinguishing feature. T regs are distinct  from those effector T-cells which are induced to switch  to secrete immunosuppressive cytokines as a typical  immune response progresses with time from an immu-  nostimulatory to immunoinhibitory character. The T regs  represent approximately 10% of all CD4 + T-cells and  can acquire the immunosuppressive phenotype in the  thymus or via induction in the periphery. In the thymus  this subset of CD4 + T-cells are positively induced by  interactions with MHC molecules and the recognition of  agonist peptide. This contrasts to the situation for effec-  tor T-cells where TCR recognition in the thymus of  MHC-presented peptide normally triggers the positive  deletion of the affected T-cell. In particular TGF(3 appears  particularly important for the expression of the T regs phe-  notype while IL-2 as the key T-cell mitogen is also  important.   Tregs essentially serve to suppress immune responses of  effector T-cells, effector B-cells and APCs leading to  peripheral immune tolerance. Direct cell-cell contact as  well as cytokine signalling are mechanisms important in  mediating their actions. These cells play an important  role in self-limiting immune responses. T regs display a  number of disease associations, with decreased numbers  of T regs or reduced function in a range of autoimmune  diseases. T regs also appear to fulfil a role in the active  immune evasion of tumours, with the experimental  depletion of T regs improving natural antitumour immu-  nity and effectiveness of active immunotherapy.   The vast majority of T-cells possess a TCR comprised of  two polypeptide chains, a single a-chain and a single 13-  chain. y5 T-cells possess a TCR made of a single y-chain  and a single 8-chain. The antigenic molecules or ligands  that activate y8 T-cells remain essentially unknown,  although they appear not to require antigen processing  or MHC presentation. These cells have characteristics  of both innate and adaptive immune cells possessing a  TCR, but also undergoing early activation capable of    phagocytosis and rapid production of cytokines that  regulate inflammation and pathogen removal.   This section is intended to provide a brief overview of  some clinical issues that exemplify the basic aspects of  immune system functioning discussed previously.   Transplantation is the process of transferring cells, tissues  or organs — termed a graft — from one location to another.  An autologous graft is a transplant between two sites  within the same individual, e.g. skin graft from the thigh  to the hand. An allogeneic graft is a transplant between  two genetically different individuals of the same species,  e.g. kidney transplant from a donor to a recipient indi-  vidual. A xenogenic graft is a transplant across different  species, e.g. pig to human.   • Hyperacute rejection occurs within minutes to hours  following revascularization of a graft. The cause is due to  the presence of preformed circulating antibody (IgG)  that reacts with the blood cell antigens (the ABO system),  or MHC I molecules or other poorly defined antigens.  This should now be a rare event clinically as recipients  are tested (cross-matched) before transplantation for the  presence of antibodies reactive with cells of the donor.   • Acute rejection occurs within weeks to months following  transplantation and involves humoral (antibody) and  cell-mediated induced cytotoxicity. Damage may be  reversed with early diagnosis and more aggressive  immunosuppressive therapy.   • Chronic rejection occurs many months or even years  following transplantation. The pathology is characterized  by fibrosis and may require differential diagnosis to  distinguish between a chronic rejection event and the  recurrence of the original disease that necessitated  transplantation in the first place.   The major alloantigens (i.e. antigens responsible for  rejection of allogeneic grafts) are the MHC proteins.  Although there are two distinct classes of MHC protein  (described in section 4.1), the MHC molecules actually  have a number of subclasses which vary further in the  general nature of peptides that they will accept within  their binding clefts. The MHC I molecules are composed  of three subclasses, MHC IA, MHC IB and MHC IC, on    each nucleated cell of the body; all three subclasses are  simultaneously expressed. The MHC II molecules are  also made up of three subclasses, MHC II DR, MHC II  DP and MHC II DQ, and again on each APC all three  subclasses of MHC II molecule are simultaneously  expressed. APCs, like other cells in the body, will also  express MHC I molecules on their surface in addition to  MHC II.   As indicated previously, the major cause of allogeneic  tissue transplantation rejection is the polymorphic  nature of the MHC phenotype between individuals.  Polymorphism in MHC arises within the population  because the genes for each of the MHC subclasses can  exist in multiple different forms or alleles. For example,  in humans there are at least 52 different forms of the  MHC IB gene and at least 24 different forms of the MHC  IA gene. It follows that individuals in a population can  possess any one of the 52 different forms of MHC IB gene  and any one of the 24 different forms of MHC IA gene,  so the number of different combinations for the six  classes of MHC proteins is many millions. The situation  is further complicated by the fact that each individual  inherits and coexpresses a set of MHC I and II genes from  each parent. This means that on each nucleated cell of the  body there will be coexpressed paternally derived and  maternally derived versions of the MHC IA, MHC IB and  MHC IC molecules. The same principle will apply for  coexpression on APCs of paternal and maternal MHC II  protein subclasses.   This tremendous polymorphism is important in  immune defence because it allows the broadest possible  scope of peptide antigen presentation, and thus the best  chance of survival of a population as a whole, but it also  confers the very high probability of MHC mismatch  during allogeneic transplantation. As a result of the mode  of MHC inheritance, the highest probability of a MHC  tissue match between individuals who are not genetically  identical twins will be that obtained between siblings,  where there is a 1 in 4 chance of a sibling possessing an  exact match for all the MHC I and MHC II subclasses.  The MHC proteins are also termed human leucocyte  antigens (HLA), and HLA tissue typing is undertaken  routinely before transplantation to gain improved  matches between donor and recipient. In kidney trans-  plantation it has been found that matching the MHC IA,  IB and IIDR genes in particular appears to improve  short- and long-term graft survival.   The main target for the modern immunosuppressants  such as ciclosporin and tacrolimus is inhibition of  cytokine gene transcription in a highly selective manner    in the helper T-lymphocyte populations. The conse-  quence of this is to inhibit helper T-cell autoactivation  and helper T-cell coactivation of cytotoxic T-lymphocytes  and of B-lymphocytes, and thus considerably ‘damp  down’ cell-mediated and humoral immune responses to  the graft.   Hypersensitivity can be defined as an exaggerated  response of the immune system leading to host tissue  damage. However, some of the immune responses  described in the hypersensitivity classification below are,  in some circumstances, appropriate responses to invading  antigen. For example, a component in what is an appro-  priate immune response to tissue transplant rejection can  be defined as a type II hypersensitivity reaction.   The highly influential Cell and Coombs classification  scheme defines four categories of hypersensitivity:   • Type I — immediate hypersensitivity. This is also called  anaphylactic or acute hypersensitivity. It involves IgE  antibody and is mediated via degranulation of mast cells  leading to release of preformed factors which promote an  influx of immune cells to the site of mast cell activation  and initiation of a rapid inflammatory reaction. In the  extreme case the inflammatory response extends beyond  the localized site of initiation and affects systemic tissues  leading to life-threatening anaphylactic reactions such  as those documented to penicillin, to peanut antigen,  or to bee-sting antigen. Examples of localized type I  hypersensitivity include hay fever. The term ‘allergy’ has  become synonymous with type I hypersensitivity.   • Type II hypersensitivity — antibody-mediated cytotoxicity.  This is caused by antibodies that are directed against cell  surface antigens. IgG and IgM are the key antibodies  involved that direct cytotoxic events against the cell  surface with which they interact. The cytotoxic events  include activation of the classical complement pathway  leading to the formation of a MAC, and the attraction and  activation of killing cells such as NK cells or phagocytes  which can bind to the antigen-antibody complex via  receptors for antibody Fc domains or complement C3b.  Type II hypersensitivity disorders include blood  transfusion reactions arising from mismatch of the blood  ABO antigens between donor and recipient, or haemolytic  disease of the newborn. Autoimmune disorders such as  myasthenia gravis, Goodpasture’s syndrome and  autoimmune haemolytic anaemias are initiated by  autoantibodies reacting against ‘self’ tissue.   • Type III hypersensitivity — complex-mediated. This  involves the formation of large antigen-antibody    complexes that circulate in the blood, are usually coated  by complement proteins and are removed by phagocytosis.  If this process is compromised for any reason then the  antigen-antibody complexes will be deposited in tissue  capillary beds, with kidney deposition being clinically the  most important site. This deposition of high molecular  weight antigen-antibody complexes in the glomerular  capillaries of the kidney can lead to a condition termed  glomerulonephritis which involves disruption of the  glomerular basement membrane, destruction of glomeruli  and ultimately renal failure which may necessitate organ  transplantation. Systemic lupus erythematosus is a  condition where autoantibodies are directed against the  host’s DNA and RNA with subsequent complement-  coated immune complexes deposited throughout systemic  tissues such as in the kidney, skin, joints and brain.   • Type IV hypersensitivity — cell-mediated. This results  from inappropriate accumulation of macrophages at a  localized site, and may or may not involve the presence  of antigen. Under conditions of ongoing localized  infection or inflammation, macrophages release proteases,  which destroy infected or otherwise damaged tissue.  However, with the inappropriate recruitment and/or  activation of excessive numbers of macrophages,  continuing damage to normal tissue may result, leading  to chronic inflammation. The recruitment and activation  of macrophages in type IV hypersensitivity is augmented  by the activity of helper T-lymphocytes (specifically the  T h 1 subpopulation). Examples of type IV hypersensitivity  include granuloma formation and contact dermatitis.  Granulomas are initiated and maintained by the  recruitment of macrophages into the site of a persistent  source of antigen or toxic material. A granuloma is a  fibrotic core of tissue composed of tissue cells and  macrophages surrounded by lymphocytes and then  further surrounded by layers of calcified collagenous  material. Sarcoidosis is a granulomatous disease of  unknown cause but characterized by granuloma nodule  formation in the lung and skin, among other sites.   The immune system is a complex body system whose  various functions display a high level of inter-regulation.  As such, any attempt to describe the functioning of the  immune system within a single chapter will inevitably  represent an oversimplification. However, the authors  consider this chapter to be a comprehensive, but never-  theless basic, overview of the immune system that will    serve as a sound foundation for further reading on the  clinical immunological basis of disease or for the consul-  tation of more specialized texts on immunological  function.   The discussion in this chapter is structured by delin-  eating the immune system into innate and adaptive  responses. The innate system, responding immediately  but non-specifically to antigen, is complementary to the  adaptive immune system which reacts in a highly specific  manner to antigen but which displays a delay in its  response. It should not be forgotten, however, that the  functioning of the two systems are intimately related,  showing dependency on each other for the optimal main-  tenance of health.   Abbas, A.K., Lichtman, A.H. & Pober, J.S. (2009) Cellular and  Molecular Immunology, 6th edn. W.B. Saunders, Philadelphia.  (Strong on experimental observations that form the basis for  the science of immunology at the molecular, cellular, and  whole-organism levels, and the resulting conclusions.)    Bonneville, M., O’Brien, R.L. & Born, W.K. (2010) y5 T-cell  effector functions: a blend of innate programming and  acquired plasticity. Nat Rev. Immunol , 10 , 467-478. (Review  on y5 T-cells.)   Clancy, J. & Morgan, J. (2001) Basic Concepts in Immunology:  A Student’s Survival Guide. McGraw-Hill, New York.  (Foundation text.)   Delves, P.J., Martin, S., Burton, D., & Roitt, R. (2006). Roitt’s  Essential Immunology, 11th edn. Blackwell Science, Oxford  (Classic introductory text).   Janeway, C., Travers, P., Capra, J.D., Walport, M.J. & Shlomichik,  M. (2004) Immunobiology: The Immune System in Health  and Disease, 6th edn. Garland Press, New York. (Medical and  basic immunology with emphasis on concepts.)   O’Garra, A. & Arai, N. (2000). The molecular basis of T-helper  1 and T-helper 2 cell differentiation. Trends Cell Biol. 10 ,  542-550. (Overview of cytokine regulation of helper T-cell  differentiation into T H 1 and T H 2 subpopulations, and the  effector functions of the subpopulations.)   Parkin, J. & Cohen, B. (2001) An overview of the immune  system. Lancet, 357, 1777—1789.   Playfair, J.H.L. (2000) Immunology at a Glance. Blackwell  Science, Oxford. (Pictorial based primer for immunological  novices.)   Rabson, A., Roitt, I. & Delves, P.J. (2004) Really Essential Medical  Immunology, 2nd edn. Blackwell Science, Oxford. (Contains  essential immunological information for medical students  and other health professionals.)   People rarely suffer from the same infectious disease  twice. Reinfections normally occur, primarily (1) when  the infectious agent exhibits antigenic plasticity such as  with the common cold and influenza; (2) if the patient is  immunocompromised, due for example to immunosup-  pressive therapy or immunological disorders; or (3) when  a significant amount of time has passed after the first  infection. Alternatively, the patient may have failed to  eliminate the primary infection which remained latent    and emerged later in a modified of similar form as for  example with herpes simplex (oral and genital herpes),  herpes zoster, (chickenpox) and HIV/AIDS.   Immunity against reinfection was recognized long  before the discovery of the causal agents of infectious  disease. Consequently, efforts were made towards devel-  oping treatment strategies that could generate immunity  to infection without the individual suffering the infec-  tion. An early development was the attempted prevention  of smallpox (variola major) through the dermal inocula-  tion of healthy individuals with material taken from  active smallpox lesions. Such treatments often produced    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    single localized lesions and commonly, but not always,  protected the recipient from contracting full-blown  smallpox. The process became known as variolation and,  unknown to its practitioners, protected against the  disease by changing the route of infection of the causal  organism from respiratory transmission to cutaneous.  Unfortunately, occasional cases of smallpox resulted from  such practices and variolated individuals could also  (rarely) infect others, resulting in infection. Further  developments recognized that immunity developed  towards one pathogen may be associated with cross-  immunity towards related infectious agents. Cowpox is a  disease of cattle that can be transmitted to humans. The  symptoms are similar to those of smallpox, but consider-  ably less severe. Following the observation that individu-  als exposed to cowpox were conferred protection against  smallpox, Edward Jenner substituted material taken from  active cowpox (vaccinia) into the variolation procedures.  This conferred much of the protection against smallpox  that had become associated with variolation but without  the associated risks. This discovery, made over two cen-  turies ago, became known as vaccination and heralded a  new era in disease control. The term vaccination was  originally used to refer to prophylactic measures that use  living microorganisms or their products to induce immu-  nity, but the term is now used to refer to all immunization  procedures.   Vaccination is used to protect individuals against infec-  tion and also to protect communities against epidemic  disease. Such public health measures have met with spec-  tacular success and in instances where there is no reservoir  of the pathogen other than in infected individuals and  survival of the pathogen outside the host is therefore  limited, vaccination has the potential to eradicate the  disease permanently. This has already been achieved for  smallpox where the coordinated deployment of an effec-  tive vaccine over many decades led to the eradication of  this disease. The global eradication of smallpox was  endorsed by the World Health Assembly on 8 May 1980.  Another candidate disease for global eradiation by vacci-  nation is poliomyelitis, where effective vaccination pro-  grammes have reduced the annual incidence to fewer than  2000 cases. The virus persists, however, in India, Pakistan,  Afghanistan and Nigeria. The Global Polio Eradication  Initiative, spearheaded by the World Health Organization  (WHO), Rotary International, the U.S. Centers for Disease  Control (CDC) and the United Nations Children’s Fund  (UNICEF) is now actively working towards the eradica-  tion of this virus. It has been calculated that eradication is  a more cost-effective option than containing the disease,    as well as reducing morbidity associated with the residual  cases. The effectiveness of poliomyelitis vaccines is clearly  indicated by data shown in Figure 10.1.   Infectious diseases may either be spread from a common  reservoir (common source) of the infectious agent that is  distinct from the diseased individuals, or through a pop-  ulation by serial transfer from diseased to healthy, suscep-  tible individuals (propagated source; Figure 10.2).   In common source infections, potential reservoirs of  infection include infected drinking-water, contaminated  water vapour from a cooling tower, or contaminated  food. In the simplest of cases the source of the infection  is transient (i.e. food sourced to a single retail outlet, or  to an isolated event such as a dinner party). In such  instances, the onset of new cases will be phased over a  timescale approximately equivalent to one incubation  period, and the decline in new cases closely follows the  elimination of the source (Figure 10.2). This leads to an  acute outbreak of infection, limited to those linked  with the source. Such incidents are epitomized by the  1996 outbreak of Escherichia coli 0157 infections in  Lanarkshire, Scotland, that resulted in 5 deaths and left  280 people ill. Similarly, in Clemenstone, South Wales, an  outbreak resulted in 157 cases of E. coli including the  death of a 5-year-old boy in 2005. Both of these outbreaks  were linked to a single retail outlet.   If the source of the infection persists beyond the onset,  then the incidence of new cases may be maintained at a  level that is commensurate with the infectivity of the  pathogen and the frequency of exposure of individuals.  For those infectious diseases that are transmitted to  humans via insect vectors that may act as reservoirs of  infection, onset and decline phases of epidemics are  rarely observed, other than as reflections of the seasonal  variation in the prevalence of the insect. Diseases such as  these are generally controlled by public health measures  and environmental control of the insect vector with vac-  cination and immunization being deployed to protect  individuals (e.g. yellow fever vaccination).   Propagated outbreaks of infection relate to the direct  transmission of an infective agent from a diseased    Figure 10.1 Reported incidence of  paralytic poliomyelitis in England and  Wales during the 1950s and 1960s. After  the introduction of vaccination  programmes the incidence of this disease  dropped from an endemic incidence  of around 5000 cases per year to fewer  than 10.    Figure 10.2 Incidence pattern for common-source  outbreaks of infection where the source persists (■)  and where it is short-lived (•).    individual to a healthy, susceptible one. Mechanisms of  such transmission have been described in Chapter 7 and  include inhalation of infective aerosols such as with  measles, mumps and diphtheria; direct physical contact  such as may occur with syphilis, herpesvirus and  human papillomavirus; and, where sanitation standards  are poor, through the introduction of infected faecal  material into drinking-water (i.e. cholera and typhoid  fever) or onto food (e.g. Salmonella and Campylobacter).  The ease of transmission, and hence the rate of onset  of an epidemic, relates to the susceptibility status and  general state of health of the individuals concerned, the  virulence properties of the organism, the route of  transmission, the duration of the infective period associ-  ated with the disease, behavioural patterns, age of the  population group and the population density (i.e. urban  versus rural).   Each infectious individual will be capable of transmit-  ting the disease to the susceptible individuals that they  encounter during their infectious period. The number of  persons to which a single infectious individual might  transmit the disease and hence the rate of occurrence of  the infection within the population will depend upon the  population densities of susceptible and infective indi-  viduals, the degree and nature of their social interaction  and the duration and timing of the infective period. If    infectivity precedes the manifestation of disease, then  spread of the infection may be greater than if these were  concurrent. As each infected individual will in turn,  become a source of infection, this leads to a near expo-  nential increase in the incidence of disease. Figure 10.3  shows the incidence of disease within a population group.  This group is perfectly mixed and all individuals are sus-  ceptible to the infection. The model infection has an  incubation period of 1 day and an infective period of 2  days commencing at the onset of symptoms with recov-  ery occurring 1 day later. For the sake of this illustration  it has been assumed that each infective individual will  infect two others per day until the entire population  group has contracted the disease (solid lines). In reality,  the rate of transmission will decrease as the epidemic  progresses, because recovered individuals may become  immune to further infection, reducing the population  density of susceptible individuals, and thereby the likeli-  hood of onward transmission. Epidemics therefore often  cease before all members of the community have been  infected (Figure 10.3, dotted line). If the proportion of  immune individuals within a population group can be  maintained above this threshold level then the likelihood  of an epidemic arising from a single isolated infection  incident is small (this is referred to as herd immunity).  The threshold level itself is a function of the infectivity   Figure 10.3 Propagated outbreaks of  infection showing the incidence of new  cases (•), diseased individuals (■) and  recovered immune individuals (A). The  dotted line indicates the incidence  pattern for an incompletely mixed  population group.    of the agent and the population density. Outbreaks of  measles and chickenpox therefore tend to occur annually  in the late summer among children attending school for  the first time. This has the effect of concentrating all  susceptible individuals in one, space and thereby reduc-  ing the proportion of immune subjects to a value below  the threshold for propagated transmission. An effective  vaccination programme that maintains the proportion of  individuals who are immune to a given infectious disease  above the critical threshold level for herd immunity. Such  a programme will not prevent isolated cases of infection  but will prevent these from becoming epidemic.   There is the potential to develop a protective vaccine/  immunization programme for all infectious diseases,  although some pathogens are considerably more chal-  lenging candidates than others. Whether or not such vac-  cines are developed and deployed is related to the severity  and economic impact of the disease on the community  as well as the effects upon the individual. Various factors  governing the likelihood of an immunization programme  being adopted are discussed below, while the principles  of immunity, and of the production and quality control  of immunological products are discussed in Chapters 9  and 24 respectively.   The severity of the disease in terms of its morbidity and  mortality, the probability of permanent injury to its sur-  vivors and the likelihood of infection must be sufficient  to warrant the costly development of a vaccine and its  subsequent use. Thus, although influenza vaccines are  constantly reviewed and stocks maintained, the control  of influenza epidemics through vaccination is not recom-  mended. Rather, those groups of individuals, such as  elderly people, who are at special risk from the infection,  are protected.   Vaccines to be included within national immunization  and vaccination programmes should be chosen to reflect  the infection risks within that country. Additional immu-  nizations, appropriate for persons travelling abroad, are  intended to protect the at-risk individual, but also to  prevent importation of the disease into an unprotected  home community.   Vaccination and immunization programmes seldom  confer 100% protection against the target disease. More  commonly the degree of protection is 60-95%. In such  instances, although individuals receiving treatment have  a high probability of becoming immune, virtually all  members of a community must be treated in order to  reduce the actual proportion of susceptible individuals to  below the threshold for epidemic spread of the disease.  Antidiphtheria and antitetanus prophylaxes, which utilize  toxoids, are among the most efficient immunization  programmes, whereas the performance of BCG is highly  variable.   No medical or therapeutic procedure comes without  some risk to the patient, but all possible steps are taken  to ensure safety, quality and efficacy of vaccines and  immunological products (see Chapter 24). The risks  associated with immunization procedures are constantly  reviewed and balanced against the risks associated with  contracting the disease. In this respect, the incidence of  paralytic poliomyelitis in the USA and UK in the late  1990s was low, with the majority of cases being related to  vaccine use (vaccine-associated paralytic polio or VAPP).  As the worldwide elimination of poliomyelitis approaches,  there is debate as to the value of the live (Sabin) vaccine  outside endemic areas, and the inactivated polio vaccine  (IPV) is now the vaccine of choice in the UK for prophy-  laxis against paralytic polio.   Public confidence in the safety of vaccines and immuni-  zation procedures is essential if compliance is to match  the needs of the community. The correlation between  actual risk and perception of risk is not always reliable,  however. In this respect, public concern and anxiety in  the mid 1970s over the perceived safety of pertussis  vaccine led to a reduction in coverage of the target group  from about 80% to 30%. Major epidemics of whooping  cough, with over 100000 notified cases, followed in the  late 1970s and early 1980s. By 1992, public confidence  had returned and coverage had increased to 92%, with a  considerable associated decrease in disease incidence.  Similarly, links have been claimed between the incidence  of autism in children and the change in the UK from  single measles and German measles vaccines to the com-  bined measles, mumps and rubella (MMR) vaccine.  Such claims have been proved to be unfounded beyond    reasonable doubt but have nevertheless decreased the  uptake of the MMR vaccine and thereby increased the  likelihood and magnitude of measles epidemics.   Cheap, effective vaccines are an essential component of  the global battle against infectious disease. It was esti-  mated that the 1996 costs of the USA childhood vaccina-  tion programme, directed against polio, diphtheria,  pertussis, tetanus, measles and tuberculosis, was $1 for  the vaccines and $14 for the programme costs. The  newer vaccines, particularly those that have been geneti-  cally engineered, are considerably more expensive,  putting the costs beyond the budgets of many developing  countries.   The ideal of any vaccine is to provide lifelong protection  of the individual against disease. Immunological memory  (Chapter 9) depends on the survival of cloned popula-  tions of B- and T-lymphocytes (memory cells). Although  these lymphocytes can persist in the body for many  decades, the duration of protection varies from one indi-  vidual to another and depends on the vaccine; commonly  ranging between 10 and 20 years. Thus, if the immune  system is not boosted, either by natural exposure to the  organism or by reimmunization, protective immunity  gained in childhood may be lost by the age of 30. Those  vaccines that provide only poor protection against disease  may have proportionately reduced timespans of effective-  ness. Equally, vaccines may be less effective and have a  shorter duration when administered to neonates. Yellow  fever vaccination, which is highly effective, must there-  fore be repeated at 10-year intervals, while the typhoid  vaccine is only effective for up to 3 years. Whether or not  immunization in childhood is boosted at adolescence or  in adulthood depends on the relative risks associated with  the infection as a function of age and the longevity of  immunity conferred by the vaccine.   The theoretical background that underlies immunity to  infection has been discussed in detail in Chapter 9.  Immunity to infection may be passively acquired through  the receipt of preformed, protective antibodies or it may  be actively acquired through an immune response follow-  ing deliberate or accidental exposure to microorganisms  or their component parts. Active, acquired immunity    might involve either or both of the humoral and cell-  mediated responses.   Humoral antibodies of the IgG class are able to cross the  placenta from mother to fetus. These antibodies will  provide passive protection of the newborn against those  diseases which involve humoral immunity and to which  the mother is immune. In this manner, most newborn  infants in the UK will have passive protection against  tetanus, but not against tuberculosis. Protection against  the latter relies to a large extent on cell-mediated immu-  nity. Secreted (IgA) antibodies are also passed to the gut  of newborn, together with the first deliveries of breast  milk (colostrum). Such antibodies provide some passive  protection against infections of the gastrointestinal tract.  Maternally acquired antibodies will react with antigens  associated with an infection but also with antigens  introduced to the body as part of an immunization pro-  gramme. Premature immunization, i.e. before degradation  of the maternal antibodies, may reduce the potency of an  administered vaccine. This aspect of the timing of a  course of vaccinations is discussed later.   Administration of preformed antibodies taken from  animals, pooled human serum, or human cell lines is  often used to treat existing infections (e.g. tetanus, diph-  theria) or condition (e.g. venomous snake bite). Pooled  serum may also be administered prophylactically, within  a slow-release vehicle, for individuals travelling to parts  of the world where diseases such as hepatitis A are  endemic. Such administrations confer no long-term  immunity and may interfere with concurrent vaccination  procedures.   Active immunity (Chapter 9) relates to exposure of the  immune system to antigenic materials and the subse-  quent response. Such exposure might be related to an  infection or to the multiplication of an attenuated  vaccine strain, or it might be associated with the direct  introduction of non-viable antigenic material into the  body e.g. a non-living or inactivated vaccine. The route  of exposure to antigen will influence the nature of the  subsequent immune response. Thus, injection of antigen  will lead primarily to humoral (IgG, IgM) production,  while exposure of epithelial tissues (gut, respiratory tract)  will lead to the production of secretory antibodies  (IgA, IgE) and to the stimulation of humoral antibody  production.    The magnitude and specificity of an immune response  depends upon the duration of the exposure to antigen  and on its time-concentration profile. During a naturally  occurring infection (or the administration of a live, atten-  uated vaccine), the levels of antigen in the host may be  low at the onset and localized to the portal of entry to the  host. As the amounts of antigen are small, they will react  only with a small, highly defined subgroup of small lym-  phocytes. These may undergo transformation to produce  various antibody classes specific to the antigen and  undergo clonal expansion. These immune responses and  the progress of the infection may progress simultane-  ously. With time, microorganisms will produce greater  amounts of antigenic materials that will, in turn, react  with an increasing number of cloned lymphocytes to  produce yet more antibodies. Eventually the antibody  levels may be sufficient to eliminate the infecting organ-  ism from the host. Antibody levels will then decline, with  the net result of this encounter being the clonal expan-  sion of particular small lymphocytes relating to a highly  specific ‘immunological memory’ of the encounter.   This situation should be contrasted with the injection  of a killed or non-living vaccine where the amount of  antigen introduced is relatively high when compared with  the levels present during the initial stages of an infection.  In a non-immune animal, the antigens may react not only  with those lymphocytes that are capable of producing  antibody of high specificity but also with those of a lower  specificity. Antibody of both high and lower specificity  may react with and remove the residual antigen. The  immune response will cease after this initial (primary)  challenge. On a subsequent (secondary) challenge (during  a course of vaccinations), the antigen will react with  residual preformed antibody relating to the first chal-  lenge, together with a more specific subgroup of the  original cloned lymphocytes. As the number of chal-  lenges is increased, the proportion of stimulated lym-  phocytes that are specific to the antigen rises. After a  sufficient number of consecutive challenges the magni-  tude and specificity of the immune response matches that  which would occur during a natural infection with an  organism bearing the antigen. This pattern of exposure  brings with it certain problems. Firstly, as the introduced  immunogen will react preferentially with preformed  antibody rather than lymphocytes then sufficient time  must elapse between exposures to allow the natural loss  of antibody to occur. Secondly, immunity to infection  will only be complete after the final challenge with immu-  nogen. Thirdly, low specificity antibody produced during  the early exposures to antigen might be capable of cross-    reaction with host tissues to produce an adverse response  to the vaccine.   Vaccines may comprise living, attenuated microorgan-  isms, killed microorganisms, or purified bacterial and  viral components (component vaccines). Recent innova-  tions include the development of DNA vaccines that  encode for the transcription of antigen when introduced  directly into host tissues or vaccines that might be deliv-  ered nasally by non-pathogenic bacteria (e.g. Lactococcus  lactis) or otherwise by viral vectors (e.g. adenovirus).  Some aspects of these vaccine classes are discussed below.   Live, infective microorganisms, attenuated with respect  to their pathogenicity but retaining their ability to infect,  can be used to confer protective immunity. Two major  advantages stem from the use of live vaccines. First, the  immunization mimics the course of a natural infection  such that only a single exposure is required to render an  individual immune. Secondly, the exposure may be medi-  ated through the natural route of infection (e.g. oral),  thereby stimulating an immune response that is appro-  priate to a particular disease (e.g. secretory antibody as a  primary defence against poliomyelitis virus in the gut  with oral polio vaccine (OPV); see below). Disadvantages  associated with the use of live vaccines are also apparent.  Live, attenuated vaccines, administered through the  natural route of infection, will replicate in the patient and  could be transmitted to others. If attenuation is reduced  during the replication process, infections might result  (see OPV below). A second, major disadvantage, of live  vaccines is that the course of their action, and possible  side effects, might be affected by the infection and immu-  nological status of the patient.   Since these vaccines are unable to evoke a natural infec-  tion profile with respect to the release of antigen, they  must be administered on a number of occasions.  Immunity may not reach optimal levels until the course  of immunization is complete and, with the exception of  toxin-dominated diseases such as diphtheria and tetanus  where the immunogen is a toxoid, are unlikely to match  the performance of a live vaccine. The specificity of the  immune response generated in the patient may initially  be low. This is particularly the case when the vaccine is    composed of a relatively crude cocktail of killed cells,  where the immune response is directed only partially  towards antigenic components of the pathogen. This  increases the possibility of adverse reactions in the  patient. Release profiles of these immunogens can be  improved through their formulation with adjuvants  (Chapters 9 and 24), and the immunogenicity of certain  purified bacterial components such as polysaccharides  can be improved by their conjugation to a carrier.   A development associated with research into gene therapy  has been the use of DNA encoding specific virulence  factors of defined pathogens to evoke an immune  response. The DNA is introduced directly into tissue  cells by means of a transdermal ‘gene gun’ and is tran-  scribed by the recipient cells. Accordingly, the host  responds to the antigenic material produced as though it  were an infection. The course of release of the antigen  reflects that of a natural infection and, therefore, a highly  specific response is invoked. Eventually the introduced  DNA is lost from the recipient cells and antigen release  ceases. To date, few experimental trials have demon-  strated convincing protection in humans, but this  remains a promising approach. Protective immunity has  been reported in a trial of a human vaccine for bird  flu and a West Nile virus vaccine for horses has been  approved.   Poliovirus, a picornavirus, has three immunologically  distinct serotypes (I, II and III) and there may be three  phases of the disease. The first is an acute infection of  lymphoid tissues associated with the gastrointestinal tract  (Peyer’s patches), during which time the virus can be  found in the throat and in faeces. The second phase is  characterized by an invasion of the bloodstream, and in  the third phase the virus migrates from the bloodstream  into the meninges. Infections range in severity from  asymptomatic (the majority of cases) to paralytic polio-  myelitis which may cause permanent neurological  damage and muscle paralysis. Paralytic poliomyelitis is a  major illness, but occurs in only 0.1-2% of cases. It is  characterized by the destruction of large nerve cells in the  anterior horn of the brain, resulting in varying degrees of  paralysis, and unvaccinated adults are at greater risk of    paralytic infection than children. The infection is trans-  mitted by the faecal-oral route.   Polio is the only disease, at present, for which both  live and killed vaccines compete. Since the introduction  of the killed virus (Salk) in 1956 and the live, attenuated  virus (Sabin) in 1962 there has been a remarkable  decline in the incidence of poliomyelitis (Figure 10.1).  The inactivated polio (Salk) vaccine (IPV) contains  formalin-killed poliovirus of all three serotypes. On  injection, the vaccine stimulates the production of  antibodies of the IgM and IgG class that neutralize the  virus in the second stage of infection. A course of three  injections at monthly intervals produces long-lasting  immunity to all three poliovirus types. The live, oral  polio (Sabin) vaccine (OPV) is now less commonly  used. Advantages over the IPV vaccine include lower costs  and easier administration. OPV contains attenuated  poliovirus of each of the three types and is administered,  as a liquid, onto the tongue. The vaccine strains infect  the gastrointestinal mucosa and oropharynx, promoting  the common immune response, and involving both  humoral and secretory antibodies. IgA, secreted within  the gut epithelium, provides local resistance to the first  stages of poliomyelitis infection. OPV therefore provides  protection at an earlier stage of the infection than does  IPV. Infection of epithelial cells with one strain of entero-  virus, however, may inhibit simultaneous infection by  related strains. At least three administrations of OPV are  therefore given, with each dose conferring immunity to  one of the vaccine serotypes. These doses must be sepa-  rated by a period of at least 1 month in order to allow  the previous infection to lapse. Booster vaccinations are  also provided to cover the eventuality that some other  enterovirus infection, present at the time of vaccination,  had reduced the response to the vaccine strains. Faecal  excretion of vaccine virus will occur and may last for up  to 6 weeks after administration. Such released virus  may spread to close contacts and infect or (re)immunize  them. Vaccine-associated poliomyelitis may occur  through reversion of the attenuated strains to the virulent  wild type, particularly with types II and III and has been  estimated to occur once per 4 million doses. Since the  introduction of OPV, notifications of paralytic poliomy-  elitis in the UK have markedly dropped. However, from  1985 to 1995, 19 of the 28 notified cases of paralytic  poliomyelitis were associated with reverted vaccine  strains (14 recipients, 5 contacts). As the risk of natural  infections with poliomyelitis within developed countries  has now diminished markedly, the live vaccine strains  present the greatest risk. Consequently, OPV has now    been replaced with IPV as the polio vaccine of choice in  the UK and USA.   Measles, mumps and rubella (German measles) are infec-  tious diseases with respiratory routes of transmission and  infection and each is caused by distinct members of the  paramyxovirus group. Each virus only has one serotype.  Although the primary multiplication sites of these viruses  are within the respiratory tract, the diseases are associated  with viral multiplication elsewhere in the host.   Measles is a severe, acute, highly contagious infection  that frequently occurs in epidemic form. After multiplica-  tion within the respiratory tract, the virus is tran-  sported throughout the body, particularly to the skin  where a characteristic maculopapular rash develops.  Complications of the disease can occur, including measles  encephalitis, which can cause permanent neurological  injury and death, and subacute, sclerosing panencephali-  tis (SSPE), which is a rare form of progressive encepha-  litis associated with persistence of the measles virus that  primarily affects children and young adults. It is incura-  ble, although early treatment can slow progression or  improve the remission rate.   A live, attenuated vaccine strain of measles was intro-  duced in the USA in 1962 and to the UK in 1968. A single  injection produces high-level immunity in over 95% of  recipients. Moreover, as the vaccine induces immunity  more rapidly than the natural infection, it may be used  to control the impact of measles outbreaks. The measles  virus cannot survive outside an infected host. Widespread  use of the vaccine therefore has the potential, as with  smallpox, of eliminating the disease worldwide. Mass  immunization has markedly reduced the incidence of  measles, although a 15-fold increase in the incidence was  noted in the USA between 1989 and 1991 because of poor  compliance with the vaccine.   Mumps virus infects the parotid glands to cause swelling  and a general viraemia. Complications include pancrea-  titis, meningitis and orchitis, the last occasionally leading  to male sterility. Infections can also cause permanent uni-  lateral deafness. In the absence of vaccination, infection  occurs in more than 90% of individuals by age 15 years.  A live, attenuated mumps vaccine has been available since  1967 and has been part of the childhood vaccination    programme in the UK since 1988 when it was included  as part of the MMR triple vaccine (see below).   Rubella is a mild, often subclinical infection that is  common among children aged between 4 and 9 years.  Infection during the first trimester of pregnancy brings  with it a major risk of abortion or congenital deformity  in the fetus (congenital rubella syndrome, CRS). Rubella  immunization was introduced to the UK in 1970 for pre-  pubertal females and non-immune women intending to  start families. The vaccine utilizes a live, cold-adapted  strain of the virus. The major disadvantage of the vaccine  is that, as with the wild type, the fetus can be infected.  While there have been no reports of CRS associated with  use of the vaccine, the possible risk makes it imperative  that women do not become pregnant within 1 month of  vaccination. Prepubertal females were immunized to  extend the period of immunity through the childbearing  years. Until 1988 boys were not routinely protected  against rubella. Their susceptibility to the virus was  thought to maintain the natural prevalence of the disease  in the community and thereby reinforce the vaccine-  induced immunity in vaccinated, adult females. This  proved not to be the case and in fact cases of CRS could  be related to incidence of the disease in younger children  within the family. Rubella vaccine is now given to both  sexes at the age of about 13 months as part of the MMR  programme (below).   MMR vaccine was introduced to the UK in 1988 for  young children of both sexes, replacing the single measles  vaccine. It consists of a single dose of a lyophilized  preparation of live attenuated strains of the measles,  mumps and rubella viruses. The MMR vaccine had previ-  ously been deployed in the USA and Scandinavia for a  significant number of years without any indication of  increased adverse reaction or of decreased seroconversion  over separate administration of the component parts.  Immunization results in seroconversion to all three  viruses in >95% of recipients. For maximum effect,  MMR vaccine is recommended for children of both sexes  aged 12-15 months but can also be given to non-immune  adults. From October 1996 a second dose of MMR was  recommended for children aged approximately 4 years in  order to prevent the reaccumulation of sufficient suscep-  tible children to sustain future epidemics.   In 1998 a research paper attempted to associate an  increase in autism to the introduction of the triple    vaccine. This led to a decreased public confidence in the  vaccine. Detailed examination of the data and also the  results of several clinical studies have indicated that there  is no association between use of the triple vaccine and  autism. This is backed up by over 20 years of successful  deployment of the vaccine outside of the UK. Currently,  much effort is being made to restore confidence in the  vaccine in order to avoid the lack of compliance leading  to the occurrence of measles epidemics.   Tuberculosis (TB) is a major cause of death and morbid-  ity worldwide, particularly where poverty, malnutrition  and poor housing prevail. Human infection is acquired  by inhalation of Mycobacterium tuberculosis or M. bovis.  Tuberculosis is primarily a disease of the lungs, causing  chronic infection of the lower respiratory tract, but may  spread to other sites or proceed to a generalized infection  (miliary tuberculosis). Active disease can result either  from a primary infection or from a subsequent reactiva-  tion of a quiescent infection. Following inhalation, the  mycobacteria are taken up by alveolar macrophages  where they survive and multiply. Circulating macro-  phages and lymphocytes, attracted to the site, carry the  organism to local lymph nodes where a cell-mediated  immune response is triggered. The host, unable to elimi-  nate the pathogen, contains the bacteria within small  granulomas or tubercles. If high numbers of mycobacte-  ria are present then the cellular responses can result in  tissue necrosis. The tubercles contain viable pathogens  that may persist for the remaining life of the host.  Reactivation of the healed primary lesions is thought to  account for over two-thirds of all newly reported cases of  the disease.   The incidence of TB in the UK declined 10-fold  between 1948 and 1992, to just over 5000 new cases being  notified each year. Those most at-risk include pubescent  children, health service staff and individuals intending to  stay for more than 1 month in countries where TB is  endemic. However, since 1994 there has been a gradual  increase in the number of notified cases in England and  Wales to approximately 7000 new cases per year which  can be associated with age group (20-45-year-olds more  susceptible), sex (males slightly more susceptible), geo-  graphical region and ethnicity. The rise in notifications  can be attributed to a number of different factors, includ-  ing increased immigration from countries where TB is  more prevalent, crowded conditions (e.g. prisons, hostels,  wards, etc.), a greater number of susceptible individuals  now in society (e.g. substance abusers, immunocompro-    mised individuals) and limited training for GPs in recog-  nizing the symptoms of TB. Fortunately this latter issue  has now been addressed, along with the re-emergence of  TB research programmes.   A live vaccine is required to elicit protection against  TB and both antibody and cell-mediated immunity are  required for protective immunity. Vaccination with BCG  (bacille Calmette-Guerin), derived from an attenuated  M. bovis strain, is commonly used in countries where TB  is endemic. The vaccine was introduced in the UK in  1953. Efficacy in the UK has been shown to be over 70%,  with protection lasting at least 15 years. In other coun-  tries, where the general state of health and well-being of  the population is less than in the developed world, the  efficacy of the vaccine has been shown to be markedly  lower than this.   Because of the risks of adverse reaction to the vaccine  by individuals who have already been exposed to the  disease, a sensitivity test must be carried out before  immunization with BCG. A Mantoux skin test assesses an  individual’s sensitivity to a purified protein derivative  (PPD) prepared from heat-treated antigens (tuberculin)  extracted from M. tuberculosis. A positive test implies past  infection or past, successful immunization. Those with  strongly positive tests may have active disease and should  be referred to a chest clinic. However, many people with  active TB, especially disseminated TB, seroconvert from  a positive to a negative skin test. Results of the skin test  must therefore be interpreted with care.   Much debate surrounds the use of BCG vaccine, a  matter of some importance, considering that TB kills  around 3 million people annually and that drug-resistant  strains have emerged. Although the vaccine has demon-  strated some efficacy in preventing childhood TB, it has  little prophylactic effect against postprimary TB in those  already infected. One solution is to bring forward the  BCG immunization to include neonates. Immunization  at 2-4 weeks of age will ensure that immunization pre-  cedes infection, and also negates the requirement for a  skin test. Passive, acquired maternal antibody to TB is  unlikely to interfere with the effectiveness of the immu-  nization as immunity relates primarily to a cell-mediated  response. Alternative strategies involve improvement of  the vaccine, possibly through the introduction into the  BCG strain of genes that encode protective antigens of  M tuberculosis.   The current UK policy (introduced in September  2005) is to reserve the BCG vaccine for those considered  to be at highest risk based on location or familial links  to TB endemic areas. As such, this targeted approach will    seek to immunize all infants in areas where the incidence  of TB is 40 or more per 100000; infants with at least  one parent or grandparent who was born in a country  with a TB incidence of 40 or more per 100000; previously  unvaccinated new immigrants from high-prevalence  countries for TB; those who have lived in the same  household or had prolonged, close contact with someone  with TB; or at-risk workers (doctors, nurses, social  workers, etc.).   Immunization against these three unrelated diseases is  considered together because the vaccines are non-living  and are often co-administered as a triple vaccine as part  of the childhood vaccination programme.   This is an acute, non-invasive infectious disease associ-  ated with the upper respiratory tract (Chapter 7). The  incubation period is 2-5 days although the disease  remains communicable for up to 4 weeks. A low molecu-  lar weight toxin is produced which affects the myocar-  dium, nervous and adrenal tissues. Death results in 3-5%  of infected children. Diphtheria immunization stimulates  the production of an antitoxin which protects against the  disease but not against infection/colonization of the res-  piratory tract. The immunogen is a toxoid, prepared by  formaldehyde treatment of the purified toxin (Chapter  24) and administered while adsorbed to an adjuvant,  usually aluminium phosphate or aluminium hydroxide.  The primary course of diphtheria prophylaxis consists of  three doses starting at 2 months of age and separated by  an interval of at least 1 month. The immune status of  adults may be determined by administration of Schick  test toxin, which is essentially a diluted form of the  vaccine.   Tetanus results from the production of a toxin by germi-  nating spores and vegetative cells of Clostridium tetani  that can potentially infect deep wounds. The organism,  which may be introduced into the wound, grows anaero-  bically at such sites. The toxin is adsorbed into nerve cells  and has a profound effect on nerve synapses resulting in  spastic paralysis in affected individuals (Chapter 7).  Mortality rates are highest in individuals over 60 years of  age and the unvaccinated. Tetanus immunization employs  a toxoid and protects by stimulating the production of  antitoxin. This antitoxin will neutralize the toxin as the    organisms release it and before it can be adsorbed into  nerves. As the toxin is produced only slowly after infec-  tion, the vaccine, which acts rapidly, may be used prophy-  lactically in non-immunized individuals who have  recently suffered a high-risk injury. The toxoid, as with  diphtheria toxoid, is formed by reaction with formalde-  hyde and is adsorbed onto an inorganic adjuvant. The  primary course of tetanus vaccination consists of three  doses starting at 2 months of age and is separated by an  interval of at least 1 month.   Whooping cough is caused by the non-invasive respira-  tory pathogen Bordetella pertussis (Chapter 7). This  disease may be complicated by bronchopneumonia, or by  repeated post-tussis vomiting leading to weight loss and  to cerebral hypoxia associated with a risk of brain damage.  Until the mid 1970s the mortality from whooping cough  was about 1 per 1000 notified cases, with a higher rate for  infants under 1 year of age. A full course of vaccine now  consists of highly purified selected components of the  Bordetella pertussis organism (i.e. acellular pertussis)  which are treated with formaldehyde or glutaraldehyde  and absorbed on to aluminium phosphate or aluminium  hydroxide adjuvants. This vaccine gives considerably  lower incidence of local and systemic reactions in com-  parison to the whole-cell pertussis vaccine that preceded  it and gives comparable protection (>80%). The primary  course of pertussis prophylaxis consists of three doses  starting at 2 months of age and separated by an interval  of at least 1 month.   The primary course of DTaP protection consists of three  doses of a combined vaccine, each dose separated by at  least 1 month and commencing not earlier than 2 months  of age. In such combinations, the pertussis component of  the vaccine acts as an additional adjuvant for the toxoid  elements. Tetanus and diphtheria vaccines are also avail-  able in a combined tetanus/diphtheria/inactivated polio  vaccine (Td/IPV) vaccine.   The primary course of pertussis vaccination is  considered sufficient to confer lifelong protection, espe-  cially as the mortality associated with disease declines  markedly after infancy. The risks associated with tetanus  and diphtheria infection however persist throughout life.  The Td/IPV vaccination is therefore repeated before  school entry, at 4-5 years of age, and once again at  puberty.   Meningococcus (Neisseria meningitidis) is a bacterium  that exclusively colonizes and/or infects humans; there is  no animal reservoir. It is present as part of the normal  microbiota of the pharynx in approximately 10% of indi-  viduals but can rarely spread through the bloodstream  and to the brain through poorly understood mechanisms,  causing meningitis and septicaemia. These are life-  threatening, systemic infections; overall mortality from  meningococcal disease is approximately 10%, assuming  symptoms are recognized and treatment is commenced  without delay, but rises considerably if there are delays.  Diagnosis is therefore considered a medical emergency.  At least 12 subtypes or serogroups of meningococcus  have been identified, but groups B and C account for the  majority of cases in Europe and the Americas. In the UK,  group B accounts for approximately two-thirds of  reported cases with group C accounting for the remain-  ing third. Neisseria meningitidis group A does not nor-  mally cause disease in the UK but is an endemic cause of  meningitis in other parts of the world, particularly sub-  Saharan Africa in an area between Senegal (West Africa)  to Ethiopia (East) that has been termed the ‘meningitis  belt’. It is believed that reasons for the hyperendemic inci-  dence of group A meningococcal meningitis in this part  of the world include high incidence of upper respiratory  tract infections during the dry (dusty) season, combined  with overcrowding, migration and pilgrimages.   Whilst group C meningitis is most common in the  under 1 -year-old group in the UK, mortality is highest in  adolescents. Although meningitis accounts for the major-  ity of invasive meningococcal disease, in 15-20% of cases  septicaemia predominates and is associated with signifi-  cantly higher mortality. There is currently no vaccine  available for group B meningococcus but vaccines are  available for groups A and C. As with the Hib vaccine (see  below), the preparations are intended to invoke protec-  tive immunity towards the polysaccharide component of  the bacterium. Early vaccines, composed of purified  polysaccharide, worked in adults but had poor efficacy in  infants — the most at-risk group. The new MenC conju-  gate vaccine comprises capsular polysaccharide compo-  nents conjugated to a carrier protein (usually diphtheria  or tetanus toxoid). This vaccine is effective in the very  young and is therefore suitable for protecting infants. The  vaccine is normally administered along with DTaP and  Hib at 3, 4 and 12 months and a single dose is sufficient    to immunize individuals over 12 months of age; it has  also been used to provide prophylaxis for teenagers, ado-  lescents and young adults. Group A vaccine is available  for those travelling to areas of the world where the infec-  tion is epidemic.   Haemophilus influenzae can cause infections ranging  from bronchitis and otitis media to life-threatening, inva-  sive disease (meningitis and bacteraemia). Invasive infec-  tions, which are most common in young children, are  normally caused by encapsulated strains of the bacterium  that can be serologically differentiated into six typeable  capsular serotypes (a-f). Before the introduction of vac-  cination, H. influenzae type b (Hib) was the most preva-  lent of these in invasive disease. Non-invasive haemophilus  disease is most often caused by non-encapsulated strains  that are not amenable to typing based on capsular serol-  ogy. Although the most common form of invasive Hib  disease is meningitis, accounting for 60% of cases, Hib  can also cause other infections, including pneumonia and  pericarditis.   The fatality rate for treated Hib meningitis infections  is approximately 5% and complications include deafness  and intellectual impairment (in c. 10% of cases). Hib  often forms part of the normal microbiota of the  nasopharynx in healthy individuals and the frequency of  carriage before the Hib vaccine was introduced was  approximately 4 in every 100 for preschool children.  Carriage of this bacterium is now rare because of the  effectiveness of the Hib vaccination. Hib meningitis is  rare in children under 3 months and peaks in its inci-  dence at around 10-11 months of age; infection is  uncommon after 4 years of age. Before the introduction  of Hib vaccination the incidence of the disease in the UK  was estimated at 34 per 100000.   The vaccine utilizes purified preparations of the  polysaccharide capsule of the major serotypes of the bac-  terium associated with disease. Polysaccharides are poorly  immunogenic and must be conjugated onto a protein  carrier (diphtheria or tetanus toxoids) to enhance their  efficacy. The Hib vaccine is given as part of a combined  product of H. influenzae type b, diphtheria, tetanus, acel-  lular pertussis and inactivated polio vaccine (DTaP/IPV/  Hib), or as the Hib/MenC conjugate vaccine.   Streptococcus pneumoniae (pneumococcus) is an encap-  sulated Gram-positive coccus. As with meningococcus    and Hib, the capsule is an important virulence factor for  this bacterium (non-capsulated strains are normally avir-  ulent). Many different capsular types have been charac-  terized but approximately 66% of the serious infections  in adults and 80% of invasive pneumococcal infections  in children are caused by about 10 capsular types.  Pneumococci are often part of the normal microbiota of  the nasal cavity and associated tissues but commensal  strains are often avirulent. Conversely, invasive infections  (i.e. meningitis and septicaemia) are often caused by  strains not considered to be normal commensals. S. pneu-  moniae is a versatile pathogen which can cause sinusitis  or otitis media (infections of the sinuses or middle ear).  The bacterium may also cause deep lung infections  (pneumonia), which accounts for its species name, and is  also capable of causing systemic infections including  bacteraemic pneumonia, bacteraemia and meningitis.  The incidence of infection by pneumococci is highest in  the winter, and transmission by aerosols or direct contact  with respiratory secretions is believed to require either  frequent or prolonged close contact. Two distinct vac-  cines have been developed to provide protection against  this bacterium: the pneumococcal polysaccharide   vaccine (PPV) which contains purified capsular polysac-  charide from 23 capsular types of bacterium and the  pneumococcal conjugate vaccine (PCV) which comprises  capsular polysaccharides from 7 common capsular types.  Importantly, PCV is conjugated to protein in a manner  similar to the Haemophilus influenzae type b (Hib) and  MenC vaccines. While PPV is an effective vaccine in  adults, the effectiveness of pneumococcal prophylaxis is  considerably improved in children by protein conjuga-  tion; PCV is immunogenic in children and the childhood  vaccination schedule recommends that doses be given at  2 and 4 months of age with a booster at 13 months. The  PPV vaccine is recommended for adults over 65 years and  at-risk groups aged 2 years or over. It will provide addi-  tional prophylaxis to individuals who have already  received the PCV vaccine because it protects from addi-  tional serotypes.   Human papillomaviruses (HPV) are a group of viruses  that infect squamous epithelia, including the skin and  mucosal surfaces of the upper respiratory and anogenital  tracts. Approximately 100 types of HPV have been identi-  fied, of which around 40 infect the genital tract. The  majority of genital infections are asymptomatic but in  some cases may be associated with genital warts and (the    reason for the development of the HPV vaccine), cervical  cancer. The time between infection and development of  cancers may range from 12 months to over 10 years. It is  important to note that HPV is associated with genital and  anal cancers in both men and women and also with  cancers of the mouth and throat. Importantly, not all  types of HPV are carcinogenic; the risk of cancer devel-  opment associated with different strains is variable and  HPV viruses that cause warts (a common sexually trans-  mitted infection) may be low risk for carcinogenicity and  in some cases strains are not considered carcinogenic.  Genital HPV infections are transmitted primarily through  sexual intercourse with infected individuals and the use  of condoms reduces the risk of sexual transmission. HPV  may also be transmitted vertically from mother to child.  Persistent HPV infection with HPV 16 and HPV 18  (high-risk strains) is associated with the majority of cer-  vical cancers. There are however, strains other than HPV  16 and HPV 18 that are carcinogenic.   The HPV vaccine comprises recombinant subunits  expressed in yeast or in cells of insect origin such that  the vaccine contains non-infectious, virus-like particles.  The current UK vaccine, Cervarix, affords protection  against HPV 16 and HPV 18 and is very effective at pre-  venting precancerous lesions associated with these virus  strains for at least 6 years, probably longer. Since the main  aim of the HPV vaccine programme is to reduce the  incidence of cervical cancer, the vaccine is currently  administered to females (not males) between the age of  12 and 13 with a catch-up programme for older females  up to the age of 18.   The timing of the various components of the childhood  vaccination programme is subject to continual review. In  the 1960s, the primary course of DTP vaccination con-  sisted of three doses given at 3, 6 and 12 months of age,  together with OPV. This separation gave adequate time  for the levels of induced antibody to decline between  successive doses of the vaccines. Current recommenda-  tions (Table 10.1) accelerate the vaccination programme  with no reductions in its efficacy. Thus, the MMR vac-  cination has replaced separate measles and rubella proph-  ylaxis and BCG vaccination may now be given at birth,  but only for infants living in areas of the UK where the  annual incidence of TB is 40 per 100000 or greater and  those with familial or other links to high-risk countries.    The BCG vaccine (for TB prophylaxis) is now administered only to children considered to be at increased risk of developing severe  disease and/or of exposure to TB infection.    The table is intended as a general guide to the vaccines used. Policies can and do change. For definitive information, see the  UK Department of Flealth website: (http://www.dh.gov.Uk/prod_consum_dh/groups/dh_digitalassets/@dh/@en/documents/  digitalasset/dh_120973.pdf) Accessed 5 November 2010.    DTaP vaccination occurs at 2, 3 and 4 months to coincide  with administration of Hib and IPV. It is imperative that  as many individuals as possible benefit from the vaccina-  tion programme. Fewer visits to the doctor’s surgery  translate into improved patient compliance and less like-  lihood of epidemic spread of the diseases in question. The  current recommendations minimize the number of sepa-  rate visits to the clinic while attempting to maximize the  protection generated.   While not recommended for routine administration, vac-  cines additional to those represented in the childhood    programme are available for individuals in special risk  categories. These categories relate to occupational risks  or risks associated with travel abroad. Such immuniza-  tion protocols include those directed against cholera,  typhoid, meningitis (group A), anthrax, hepatitis A and  B, influenza, Japanese encephalitis, rabies, tick-borne  encephalitis and yellow fever.   The authors acknowledge the contributions of Peter  Gilbert (1951-2008) who was a coauthor on previous  versions of this chapter. Much of the information in  this chapter was originally obtained from Salisbury et al    (2006 and earlier versions); an authoritative source of  information about the composition and schedules for  current UK vaccines and vaccination policies.    Mims, C.A., Nash, A. & Stephen, J. (2001) Mims’ Pathogenesis of  Infectious Disease , 5th edn. Academic Press, London.  Salisbury, D.M., Ramsay, M. & Noakes K. (2006) Immunization  Against Infectious Disease. The Stationery Office, London (fre-  quently updated).   Salyers, A. A. & Whitt, D.D. (1994) Bacterial Pathogenesis: A  Molecular Approach. ASM Press, Washington.   10 Newer antibiotics for MRSA and other Gram-positive cocci  infections 188   An antibiotic was originally defined in the 1940s as a  substance produced by one microorganism which, in low  concentrations, inhibited the growth of other microor-  ganisms. This definition necessarily meant that an anti-  biotic was a naturally occurring substance, a microbial  metabolite, so any antimicrobial agent that was manufac-    tured by chemical synthesis fell outside the definition.  The use of the term has changed over the years, both  because an increasing number of synthetic analogues or  derivatives of ‘true’ antibiotics have come on to the  market, and because several agents that bear little or no  resemblance to natural microbial metabolites have also  been developed which, for all practical purposes, mimic  traditional antibiotics in their potency, low toxicity and,  crucially, systemic action. Consequently, drugs like tri-  methoprim, metronidazole, imidazole derivatives and    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    fluoroquinolones are commonly referred to as antibiot-  ics, and the term ‘antibiotic’ will be used in this chapter  to include these and other agents with systemic antimi-  crobial activity.   By this broader definition, sulphonamides, introduced  into therapy in the 1930s, were the first significant  antibiotics and predated penicillin by about 10 years.  Although Fleming received the credit for discovering  penicillin in 1929, much of the development work  was undertaken at Oxford University over the next 10  years, and the onset of the Second World War was  the impetus for the American pharmaceutical industry  to convert the discovery into a medicine that could be  manufactured on a large scale. Benzylpenicillin was the  original antibiotic in this class, but in the late 1940s  and early 1950s it was joined both by other penicillins  and by several other classes of antibiotic that are still in  widespread use today, such as tetracyclines, macrolides  and aminoglycosides. All of these were ‘true’ antibiotics  in the traditional sense, i.e. they were extracted from  large-volume cultures of Streptomyces bacteria or fungi  (in the case of penicillins), but the 1960s saw the advent  of semisynthetic antibiotics — penicillins particularly — in  which the naturally occurring substance was extracted  from the microbial culture, purified and then structurally  modified by conventional chemical means. Since then, an  increasing number of antibiotics have been totally  synthesized.   Although bacterial resistance was a problem that was  recognized from the start of the antibiotic era, the  international pharmaceutical industry developed new  antibiotics steadily throughout the period 1950-1970,  so that new drugs regularly became available to replace  those to which resistance developed; this ability to  keep ahead of the problem lead to a degree of compla-  cency and a belief that the industry would maintain  its supremacy indefinitely. The naivety of this assumption  became steadily apparent during the remaining years  of the century as antibiotic resistance became a major  problem and hospital ‘superbugs’ like MRSA (meticillin-  resistant Staphylococcus aureus) and ‘C. diff ’ ( Clostridium  difficile) rose to prominence, whilst at the same time  the industry diverted research resources away from  antibiotics.   Most of the antibiotics developed during the period  1970-2000 were structural modifications of existing  ones, and it was not until the new millennium that  genuinely new antibiotics like linezolid and quinupristin/  dalfopristin (Synercid) came into use. Unfortunately,  though, despite the need for new drugs to treat the    superbugs mentioned above and others that have more  recently come into prominence, such as multiply  resistant tuberculosis (MRTB) and vancomycin-resistant  enterococci (VRE), the short duration of therapy and the  likelihood of eventual resistance and falling sales mean  that the commercial incentive to develop new antibiotics  is still much lower than that for drugs treating chronic  diseases like diabetes or hypertension, or ‘lifestyle’ prob-  lems like obesity. Consequently, the supply of new anti-  biotics is likely to remain limited, and the use and  distribution of those that are developed will be carefully  managed to avoid their indiscriminate use which predis-  poses to resistance development (see Chapter 15 on anti-  microbial stewardship). The effects of short durations of  therapy (typically 5-10 days for many infections), anti-  microbial stewardship policies limiting their prescribing,  relatively short patent life, and the likelihood of some  degree of resistance eventually developing anyway, all  combine to make modern antibiotics very expensive  drugs. This trend of escalating cost is starkly illustrated  by comparing the UK price differential of more than  700-fold between trimethoprim (from the 1960s) and  linezolid (marketed in 2000) (based on a single day’s  treatment using current British National Formulary  prices).   Despite the fact that few new classes of antibiotics have  been developed in recent years, there are, nevertheless,  a large number of different antibiotics in current use.  Some of these are only available in particular countries;  and even within a single country the list of available  antibiotics changes year by year as new products replace  old ones that are withdrawn for commercial reasons or  as a result of toxicity concerns. Currently in the UK  there are approximately 70 antibacterial antibiotics, 20  antifungals and 40 antiviral agents on the market, and  numbers are similar in other countries, so it is not the  intention in this chapter to describe, or necessarily even  to mention, all of these drugs. Instead, the approach will  be to consider the important constituents of the major  antibiotic classes and refer the reader to more detailed  sources for information about some of the less frequently  used agents.   The various classes of antibacterial antibiotics will be  considered in their order of importance in terms of usage,  which will be taken to mean the frequency with which  they are prescribed. Surprisingly perhaps, this does not  necessarily coincide with their rank order in terms of the  amount manufactured by the pharmaceutical industry    UK antibiotic use in defined daily doses per 1000 inhabi-  tants per day based on ambulatory outpatients. Based  on data from the European Surveillance of Antimicro-  bial Consumption project: http://www.esac.ua.ac.be/esac_  service/applet/eidb.html    each year, because a significant fraction of the annual  production is used in animal feed supplements. According  to one estimate, the amount used in this way in the USA  has been as much as eight times that used to treat human  infections.   Table 11.1 shows the relative frequency of prescribing  of the major antibiotic classes as determined by recent  data from an ongoing Europe-wide survey of hospital  outpatient prescriptions. The relative numbers would  be likely to change somewhat if the survey were to be  conducted on inpatient prescriptions or on those issued  by community medical practitioners, but some general  trends are clear. P-Lactam antibiotics are, by far, the most  frequently prescribed class, and of these, prescriptions  for penicillins substantially outnumber those for  cephalosporins. Trimethoprim features strongly, and as  an individual drug would rank very highly even  in comparison to penicillins given that the value of  5.854 represents total prescriptions for nearly 10 different  penicillins (see section 2 below). Antibiotics that are  available primarily as parenteral products, e.g. aminogly-    cosides and glycopeptides, are much less likely to be pre-  scribed for outpatients than for inpatients because of  the problems of administration, so they appear low in  the rank order on this particular survey. It is worth  emphasizing that this is part of a European survey, and  the popularity of particular antibiotics and, consequently,  the rank order of the table, is likely to vary from one  country to another.   For 20 years after their introduction, the penicillins were  the only category of P-lactam antibiotics. They were  joined, in the mid 1960s, by the cephalosporins, and  towards the end of the 20th century by carbapenems and  monobactams. These antibiotics all have the same mech-  anism of antibacterial action and all possess the P-lactam  ring as an integral part of their structure, but they differ  widely in other characteristics.   The penicillins (general structure shown in Figure 1 1.1 A)  may be considered as being of the following types:   • Naturally occurring. For example, those produced by  fermentation of moulds such as Penicillium notatum  and P. chrysogenum. The most important examples  are benzylpenicillin (penicillin G) and phenoxy-  methylpenicillin (penicillin V).   • Semisynthetic. In 1959, scientists at Beecham Research  Laboratories succeeded in isolating the penicillin ‘nucleus’,  6-aminopenicillanic acid (6-APA; Figure 11.1 A: R  represents H). During the commercial production of  benzylpenicillin, phenylacetic (phenylethanoic) acid  (C 6 H 5 .CH 2 .COOH) is added to the medium in which the  Penicillium mould is growing. This substance is a  precursor of the side chain (R; see Figure 11.2) in  benzylpenicillin. Growth of the organism in the absence  of phenylacetic acid led to the isolation of 6-APA; this has  a different R f value from benzylpenicillin, which allowed  it to be detected chromatographically.   A second method of producing 6-APA came with the  discovery that certain microorganisms produce enzymes,  penicillin amylases (amylases), which catalyse the removal  of the side chain from benzylpenicillin (Figure 11. IB).  Acylation of 6-APA with appropriate substances results  in new penicillins being produced which differ only in  the nature of the side chain (Table 11.2; Figure 11.2).  Some of these penicillins have considerable activity  against Gram-negative as well as Gram-positive bacteria,   Figure 11.1 (A) General structure of  penicillins. (B) Removal of the side  chain from benzylpenicillin. (C) Site of  action of (i-lactamases.   Figure 11.2 Examples of the side chain R in various penicillins (the numbers 1—9 correspond to those in Table 11.2). Number 8  (temocillin) has a methoxy (-OCH3) group at position 6a. Number 9 (pivmecillinam) is a 6-(i-amidinopenicillanic acid.    and are thus broad-spectrum antibiotics. Pharmacokinetic  properties may also be altered.   The sodium and potassium salts are very soluble in  water, but they are hydrolysed in solution at a temperature-  dependent rate to the corresponding penicilloic acid  (Figure 11. 3 A) which is not antibacterial. Penicilloic acid  is produced at alkaline pH or (via penicillenic acid; Figure  11. 3B) at neutral pH, but at acid pH a molecular rear-  rangement occurs, giving penillic acid (Figure 11. 3C).  Susceptibility to hydrolysis means that penicillins cannot  be formulated as aqueous products, so oral syrups and  mixtures must be manufactured as dry granules for resus-  pension in water, and injections freeze dried in vials or    ampoules. Typically, aqueous solutions of penicillins lose  10% or more of their activity in 24 hours at room tem-  perature. Instability in acid medium logically precludes  oral administration, since the antibiotic may be destroyed  in the stomach; for example at pH 1.3 and 35 °C ben-  zylpenicillin has a half-life of less than 5 minutes and is  therefore not administered orally, whereas ampicillin,  with a half-life of 600 minutes, is obviously suitable for  oral use. Benzylpenicillin is also rapidly excreted, but this  can be overcome by the use of sparingly soluble salts (ben-  zathine, benethamine and procaine) which slowly release  penicillin into the circulation over a period of time, thus  giving a continuous high concentration in the blood.   +, applicable; — , inapplicable; NR, not relevant: pivmecillinam has no effect on Gram-positive bacteria; ±, variable result depending on species and strain.  a Except Ps. aeruginosa. All penicillins show some degree of activity against Gram-negative cocci.   2 Most penicillins are active against Gram-positive bacteria, although in the case of Staph, aureus this may depend upon the resistance of the antibiotic to P-lactamase.   3 The table is not intended to list every penicillin available worldwide; for encyclopaedic data, see the books edited by Bryskier (2005) and Finch et al. (2003).    Antibiotics and synthetic antimicrobial agents: their properties and uses 1 73    Figure 11.3 Degradation products of benzylpenicillin in solution: (A) penicilloic acid; (B) penicillenic acid; (C) penillic acid.    Many bacteria produce enzymes, (3-lactamases (for-  merly called penicillinase; see Chapter 13), which may  inactivate a penicillin by opening the (3-lactam ring, as in  Figure 11. 1C. The only clinically significant (3-lactamase  produced by a Gram-positive species is that of Staph,  aureus , but most, if not all, Gram-negative bacteria have  the potential to produce the enzymes, albeit in such small  amounts in some cases that they are of no clinical impor-  tance. The Gram-negative (3-lactamases exhibit small  interspecies, and even interstrain, differences in chemical  structure which can have profound effects on their ability  to hydrolyse the various (3-lactam antibiotics. Some  penicillins (Table 11.2) are considerably more enzyme-  resistant than others, and consequently may be extremely  valuable in the treatment of infections caused by (3-  lactamase-producing bacteria.   The older penicillins, benzylpenicillin and phe-  noxymethylpenicillin, are primarily active against Gram-  positive bacteria; they are also (3-lactamase sensitive.  These shortcomings were overcome to varying degrees by  the semisynthetic penicillins developed in the 1960s.  Ampicillin remained (3-lactamase sensitive, but its pos-  session of an amino group on the benzyl side chain gave  the molecule a much broader spectrum of activity than  its parent, benzylpenicillin. The oral absorption of ampi-  cillin was found to be adequate rather than good; a figure  of less than 50% is commonly quoted. This was signifi-  cantly improved by the inclusion of a p-hydroxyl group  on the benzene ring of the ampicillin side chain, thus  creating amoxicillin, which has largely superseded the  older antibiotic. Both ampicillin and amoxicillin are  effective against many Gram-negative bacteria including  Haemophilus influenzae, Escherichia coli, Salmonella,    Shigella and Proteus species, though not Pseudomonas  aeruginosa. This last organism has represented a problem  in antibiotic therapy for many years, and carbenicillin,  also developed in the early 1960s, was the first penicillin  showing antipseudomonal activity, although it has now  been largely replaced by ticarcillin. Piperacillin, an acyl  derivative of ampicillin, also possesses activity against  Pseudomonas, and, like ticarcillin, is moderately suscepti-  ble to (3-lactamases, so both antibiotics are normally used  as combination products with (3-lactamase inhibitors (see  section 2.3).The property of resistance to Gram-negative  (3-lactamases, strongly exhibited by temocillin, is con-  ferred by the possession of a 6(X-methoxy group, although  this causes the molecule to exhibit an antibacterial spec-  trum confined almost exclusively to Gram-negative  species. A similar spectrum arises with pivmecillinam, an  amidinopenicillin, but in this case the enzyme resistance  is much weaker.   Penicillins possess a carboxylic acid group on C3  which can be esterified to create lipophilic prodrugs with  enhanced absorption from the gastrointestinal tract, after  which tissue esterases hydrolyse the ester to release the  active antibiotic. This strategy has been particularly suc-  cessful in remedying the poor oral absorption of ampicil-  lin and resulted in the development of bacampicillin,  pivampicillin and talampicillin; the first two of these, in  particular, are widely available elsewhere, but not cur-  rently in the UK.   The problem of sensitivity to staphylococcal 13-  lactamase was overcome by the development of meticillin  in which the bulky substituent groups of the side chain  largely prevented enzyme binding. Meticillin, which was  only available as an injection, has largely been replaced    with other (3-lactamase-stable penicillins, particularly the  orally active flucloxacillin, although there are several  related drugs whose availability varies from country to  country. The great majority of Staph, aureus strains  remained sensitive to meticillin for about 15 years after  its introduction in I960, but the emergence of MRSA  gathered momentum from the mid 1970s; the incidence  in the USA was 2.4% in 1975 but rose to 35% by 1996.   Penicillins generally are of low toxicity, with allergic  reactions as the only serious problem; these arise more  commonly with benzylpenicillin and ampicillin than the  rest. All penicillins, but particularly those administered  orally, can cause diarrhoea, and, rarely, pseudomembra-  nous colitis; this is more of a problem with ampicillin  because a higher proportion of an oral dose remains in  the colon to disturb the natural flora. Penicillins are  excreted primarily in the urine, in which they achieve  much higher levels than in the blood, and accumulation  of sodium and potassium may arise with high-dose injec-  tions in patients with poor kidney function.   There are quite a number of similarities between cepha-  losporins and penicillins, including their early history. In  both cases, the academic research was conducted at  Oxford University and the American pharmaceutical  companies converted that early work into a marketed  product. The discovery of the first cephalosporin was  made in 1948 in Sardinia, the research was conducted at  the William Dunn School of Pathology during the 1950s  and the first antibiotics in the class, cephalothin and  cephaloridine, became available in the mid 1960s. Since  then many cephalosporins have been synthesized, and  more than 60 have been marketed in various countries  over the last 40 years, although not all of them are still  available. As with the penicillins, the policy in this chapter  is to consider the general properties of this class of anti-  biotics and to provide more detail about selected impor-  tant cephalosporins, rather than provide a comprehensive  listing.   Cephalosporins consist of a six-membered dihydrothi-  azine ring fused to a (3-lactam ring (top structure in  Figure 11.4). The position of the double bond in  A 3 -cephalosporins is important, since A 2 -cephalosporins  (double bond between 2 and 3) are not antibacterial,  irrespective of the composition of the side chains. The  similarity to the basic penicillin structure is immediately  apparent, but the crucial difference is that there is much  greater scope for structural modification of the cepha-  losporins because of the presence of two side chains (on    carbons 3 and 7) in contrast to the single side chain of  penicillins. Again, the fundamental properties of acid sta-  bility (and hence oral availability), antimicrobial spec-  trum, resistance to (3-lactamases and pharmacokinetics  can all be substantially varied by side-chain modifica-  tions. Several of the cephalosporins act as good inducers  of (3-lactamases.   The many cephalosporins have been classified into  ‘generations’, although the usefulness of such a classifica-  tion has been questioned. There is general acceptance of  four generations, but ceftobiprole has been claimed as the  first member of the fifth. The assignment of cepha-  losporins into the first four generations has broadly fol-  lowed the time course of their introduction, but there has  been some overlap, so certain drugs that have been clas-  sified into one particular generation were marketed in  some countries after the earliest ones of the next. The  situation is further complicated by the fact that there is  not universal agreement on the generation to which some  cephalosporins belong; cefaclor, for example, is consid-  ered a first-generation antibiotic in Japan, but is regarded  as second-generation in most other countries.   The general trends have been for an increase in activity  towards Gram-negative species (usually with a corre-  sponding loss of antistaphylococcal action), and increased  resistance to (3-lactamase as the development of cepha-  losporins has progressed through the generations. First-  generation drugs are those that have moderate  antimicrobial activity and resistance to staphylococcal,  but not Gram-negative, (3-lactamases. These were origi-  nally used primarily as alternative antibiotics for the  treatment of staphylococcal infections, and are rarely  first-choice therapy. Like the oral penicillins, the oral  cephalosporins may disturb the gut flora and give rise to  diarrhoea. The possession of good resistance to both sta-  phylococcal and Gram-negative (3-lactamases is the prin-  cipal characteristic distinguishing the second- from the  first-generation antibiotics, although improved potency,  particularly towards H. influenzae and enterobacteria, is  also a feature. Yet higher activity towards Gram-negative  bacteria is displayed by third-generation drugs, to an  extent that some of them have little or no value in the  treatment of staphylococcal infections. The parenterally  administered third-generation cephalosporins, e.g. cefo-  taxime and ceftazidime, are sometimes used in combina-  tion with gentamicin or other aminoglycosides with  the intention of achieving synergy. The usefulness of the  third-generation drugs has diminished somewhat since  their introduction as a consequence of the spread  of strains capable of producing extended-spectrum   H 2 N S N.OCH 3 CH(CH 3 ) 2 )    Figure 11.4 General structure of cephalosporins and examples of side chains R 1 and R 2 (R 3 is H in all examples in this figure).    P-lactamases (see Chapter 13) and it was this deficiency  that the fourth-generation cephalosporins were intended  to remedy. Cefpirome and cefepime exhibit extremely  good enzyme resistance, but otherwise have much the  same antibacterial spectrum as ceftazidime and other  third-generation molecules.   The activity of cephalosporins (and other (3-lactams)  against Gram-positive bacteria depends on antibiotic  affinity for penicillin-sensitive enzymes (PSEs) also  known as penicillin binding proteins (PBPs). Resistance  results from altered PBPs or, more commonly, from  (3-lactamases. Activity against Gram-negative bacteria  depends on penetration of (3-lactams through the outer  membrane, resistance to (3-lactamases found in the  periplasmic space and binding to PBPs. (For further  information on mechanisms of action and bacterial  resistance, see Chapters 12 and 13). Modification of the  cephalosporin nucleus (Figure 11.4) at 7a (i.e. R 3 ) by    addition of a methoxy group increases (3-lactamase sta-  bility but decreases activity against Gram-positive bacte-  ria because of reduced affinity for PBPs; molecules  possessing a 7a-methoxy group, e.g. cefoxitin, are termed  cephamycins.   Side chains containing a 2-aminothiazolyl group at R 1 ,  e.g. cefotaxime, ceftriaxone and ceftazidime, yield  cephalosporins with enhanced affinity for PBPs of Gram-  negative bacteria and streptococci. An iminome-  thoxy group (-C=N. 0 CFl 3 ) in, for example, cefuroxime,  provides (3-lactamase stability against common  plasmid-mediated (3-lactamases. A propylcarboxy group  ((CFl 3 ) 2 -C-COOH) as in ceftazidime increases 13-  lactamase resistance and also provides activity against Ps.  aeruginosa, while at the same time reducing (3-lactamase  induction capabilities. In cephalosporins susceptible to  (3-lactamases, opening of the (3-lactam ring occurs with  concomitant loss of the substituent at R 2 (except in  cefalexin, where R 2 represents H; see Figure 11.4). This is  followed by fragmentation of the molecule.    The nature of the R 2 substituent influences both the  pharmacokinetic properties of the molecule and its  ability to enter bacterial cells — particularly to cross the  outer membrane of Gram-negative bacteria via porins.  For good oral absorption: (1) the R 2 substituent must be  small, non-polar and stable; a methyl group is considered  desirable but might decrease antibacterial activity; and  (2) the 7-acyl group (R 1 ) must be based on phenylglycine  and the amino group must remain unsubstituted.  Esterification of the carboxylic acid group at C4 can, as  with the penicillins, result in enhanced oral absorption  provided that the ester is rapidly hydrolysed by tissue  esterases; this is exemplified in both cefuroxime axetil and  cefpodoxime proxetil. The possession of a quaternary  nitrogen on the side chain at position 3 has two benefits:  it reduces the affinity of the cephalosporin for Gram-  negative (3-lactamases, in other words, makes it more  resistant to enzyme attack, and it makes the molecule  zwitterionic which increases the rate at which it can pass  through the porin channels into the Gram-negative cell.   Side chains of the various cephalosporins, including  those most recently developed, are presented in Figure  1 1.4 and a summary of the properties of these antibiotics  in Table 11.3.   The strategy of protecting vulnerable penicillins against  enzyme-mediated hydrolysis by combining them with a  (3-lactamase inhibitor resulted in the first such combina-  tion product, co-amoxiclav, in 1981. Table 11.1 shows  that penicillins with (3-lactamase inhibitors represent  approximately 6.5% of the antibiotics prescribed to UK  outpatients in the European hospital survey, and co-  amoxiclav, which consists of amoxicillin plus clavulanic  acid, is by far the most important combination available.  Clavulanic acid has also been used to protect ticarcillin  from (3-lactamase attack, and two penicillanic acid sul-  phones, sulbactam and tazobactam, have been used to  protect ampicillin and piperacillin respectively. In each  case the protecting molecule is itself a (3-lactam antibi-  otic, but one possessing little antimicrobial activity in  its own right. The benefit afforded is an extension of  the antimicrobial spectrum of the antibiotic receiving  protection; this is achieved by negating the effects of  (3-lactamases produced by staphylococci and some Gram-  negative species, which would otherwise be resistant. In  the case of co-amoxiclav for example, the combination  exhibits activity not only against many strains of Staph,  aureus, but also against strains of E. coli, H. influenzae and  Klebsiella species, against which amoxicillin alone would    be ineffective. This means that co-amoxiclav should,  in theory, be reserved for infections known, or likely,  to be due to amoxicillin-resistant (3-lactamase-producing  strains, but unfortunately it is not always used in this  prudent manner.   Clavulanic acid was isolated from Streptomyces cla-  vuligerus and belongs to a class of (3-lactams termed  clavems, which differ from penicillins in two respects:  namely the replacement of sulphur in the penicillin thia-  zolidine ring (Figure 11.1) with oxygen in the clavam  oxazolidine ring (Figure 11. 5A), and the absence of the  side chain at position 6. Clavulanic acid also affords some  protection against enzymes that are primarily active  against cephalosporins rather than penicillins, but this  protection is quite modest compared with its activity  against ‘penicillinases’.   Sulbactam and tazobactam (Figure 11.5 B and C) can  be regarded as (3-lactam molecules that resemble penicil-  lins except that the sulphur atom of the thiazolidine  ring is converted to a sulphone, and again, there is no  side chain at position 6. Sulbactam is effective against a  similar range of (3-lactamases to clavulanic acid, although  it is not quite as potent. In both cases, the range of  enzymes does not normally include those manufactured  by Ps. aeruginosa and other problem Gram-negative  organisms. Sulbactam has been combined with both  ampicillin and cefoperazone, but in both products the  two ingredients were separate entities; this poses the  potential problem that their pharmacokinetics might not  perfectly match, so that the two agents might not appear  at the infection site in the optimal concentration ratio at  the same time. In the case of sulbactam and ampicillin  this problem was partly overcome by covalently linking  the two molecules to create sultamicillin which is well  absorbed following oral administration and then hydro-  lysed to liberate equimolar proportions of the individual  components.   The spread of organisms that had developed various  forms of resistance to the early penicillins and cepha-  losporins lead to a search for other (3-lactam antibiotics  that avoided these resistance problems. Many naturally  occurring and synthetic compounds were examined but  only a few were developed to become marketed products,  and the most useful of these were the carbapenems. This  group may be considered as penicillin or cephalosporin  derivatives in which the sulphur atom has been replaced  with a carbon. The nomenclature is confusing because  some reference sources used terms like carbapenems,    +, active; I, injection; O, oral; R, resistant; V, varies between strains, or weak activity.  “Depending on dose.  b As axetil ester.    Figure 11.5 |3-Lactamase inhibitors.  (A) clavulanic acid; (B) sulbactam;  (C) tazobactam.    Figure 11.6 Carbapenems and aztreonam: (A), imipenem; (B), meropenem; (C), ertapenem; (D), aztreonam.    olivanic acids and thienamycins as if they were synony-  mous. In fact, carbapenems is the generic term for the  group which includes olivanic acids (of which there are  no products in therapeutic use) and the thienamycins.  The earliest thienamycins were discovered in the 1970s  but proved difficult to develop because of their poor sta-    bility. The N-formimidoyl derivative of thienamycin,  which was named imipenem (Figure 11. 6A), proved to  combine the desirable properties of in vitro stability, a  broad spectrum of antimicrobial activity and good resist-  ance to almost all of the then-known (3-lactamases. Its  only shortcoming was poor in vivo stability because it was    vulnerable to hydrolysis by mammalian renal dipepti-  dase, but this was solved by the development of a renal  dipeptidase inhibitor, cilastatin, with which imipenem  was marketed. Meropenem, marketed more recently, is  more stable than imipenem to dipeptidase and may thus  be administered without cilastatin; its chemical structure  is depicted in Figure 11. 6B. Ertapenem (Figure 11. 6C)  has properties similar to those of meropenem but affords  the additional advantage of once daily dosing.   Examination of the structure-activity relationships  of the early (3-lactam antibiotics led to an expectation  that molecules possessing only the |3-lactam ring without  a second ring fused to it would have no antimicrobial  activity. This proved not to be so when such naturally  occurring antibiotics, termed monobactams, were dis-  covered, and found not only to possess activity, but to  exhibit good resistance to (3-lactamases. The naturally  occurring monobactams were not developed for clinical  use, but an analogue, aztreonam, produced totally by  conventional chemical synthesis, was marketed in 1986.  It is highly active against most Gram-negative bacteria  and stable to most types of (3-lactamases, although its  resistance to staphylococcal (3-lactamases is irrelevant  because it is inactive against all strains of Staph, aureus as  well as other Gram-positive species and anaerobes. Like  the carbapenems, it is formulated as an intravenous injec-  tion and, as such, it tends to be limited to use in hospitals  for the treatment of serious Gram-negative infections,  including those due to Ps. aeruginosa. As with the carbap-  enems and third-generation cephalosporins, it exhibits  synergy with aminoglycosides like gentamicin and  tobramycin, and these combinations are employed for the  treatment of Pseudomonas lung infections in cystic  fibrosis.   Although penicillins are among the safest antimicrobial  agents available, allergic reactions, particularly skin aller-  gies, may occur in 1-10% of patients receiving them, and  the much more serious, and potentially fatal, anaphylac-  tic reactions occur in 0.005-0.05% of cases. Patients sen-  sitive to one penicillin will be sensitive to all others  because it is the basic penicillin structure that is respon-  sible for the hypersensitivity, but benzylpenicillin and  ampicillin are the ones most likely to cause anaphylaxis  and skin allergy respectively. Although hypersensitivity  reactions can occur after administration via any route,  the more severe reactions are most likely to arise after  intravenous injection. About 10% of patients allergic to  penicillins will also be sensitive to cephalosporins.   The tetracyclines are a group of broad-spectrum antibi-  otics that are declining in use as a result of increasing  bacterial resistance. Despite that, they remain important  antibiotics for several dangerous, but relatively rare,  infections due to chlamydia (e.g. trachoma), rickettsia  (e.g. typhus and Q-fever) and spirochaetes (e.g. Lyme  disease) as well as those caused by ‘typical’ bacteria (e.g.  brucellosis and bubonic plague). They also represent  useful alternatives to macrolides (see section 4 of this  chapter) and to (3-lactams (particularly in cases of allergy)  for the treatment of more common infections, including  those of the respiratory tract.   The tetracyclines (Figure 11.7) were first developed  during the 1940s and 1950s and several that are still in  use date from that time, e.g. tetracycline itself, oxytetra-  cycline and chlortetracycline. Doxycycline and minocy-  cline are more potent semisynthetic analogues discovered  in 1966 and 1972 respectively, after which there were no  significant developments until the introduction, in 2005,  of tigecycline (a glycylglycine derivative), which is gener-  ally more potent again than other tetracyclines and main-  tains activity against some organisms that have become  resistant to earlier members of the group.   Although the tetracyclines can exhibit bactericidal  activity by inhibiting ribosome function at concentra-  tions that might be used in the laboratory, they are bac-  teristatic at concentrations that can safely be achieved in  the body. They are active against Gram-positive bacteria,  although many strains of Staph, aureus have become  resistant to all but tigecycline which, as a consequence, is  of value in the treatment of MRSA infections. Many  Gram-negative species are also sensitive to tetracyclines,  and although the proportion of strains responding to  treatment has significantly diminished in recent years  there are substantial geographical variations in resistance  patterns; Ps. aeruginosa and Proteus species are normally  resistant. In addition to the infections mentioned above,  tetracyclines are prescribed for the treatment of acne,  various genital infections, the eradication of Helicobacter  pylori in gastric and peptic ulcer disease (as part of a  multidrug regimen) and, in the case of doxycycline  particularly, for the prophylaxis of drug-resistant Plasmo-  dium falciparum malaria.   Resistance to the tetracyclines (see also Chapter 13)  develops relatively slowly, but there is cross-resistance, i.e.  an organism resistant to one member is usually resistant  to all other members of this group, but there are excep-   Figure 11.7 Tetracycline antibiotics: (1) oxytetracycline; (2) tetracycline; (3) demeclocycline; (4) lymecycline; (5) doxycycline; (6)  minocycline; (7) tigecycline.    tions: tigecycline (mentioned above), and tetracycline-  resistant Staph, aureus strains may still be sensitive to  minocycline. Superinfection (‘overgrowth’) with natu-  rally tetracycline-resistant organisms, for example  Candida albicans and other yeasts, affecting the mouth,  upper respiratory tract or gastrointestinal tract, may  occur as a result of the suppression of tetracycline-  susceptible microorganisms.   Tetracyclines are absorbed from the gastrointestinal  tract and oral products are the only form in which they  are currently available in the UK, although ophthalmic,  topical and injectable products have been used in the past  and some are still available in other countries. Absorption  of tetracyclines is inhibited by food, antacids, milk or  products containing di- or trivalent cations, so tetracy-  cline, oxytetracycline and chlortetracycline have to be  administered four times a day in order to maintain ade-  quate tissue concentrations. Such impaired absorption is  much less evident in the more recently developed drugs,    and partly as a consequence of both this improvement  and of extended half-lives, lymecycline, minocycline and  tigecycline are administered twice daily, and doxycycline  just once. The newer tetracyclines are also more lipophilic  than the early ones, so doxycycline, and to an even greater  extent minocycline, exhibit good tissue distribution and  achieve concentrations in the biliary tract, liver, kidneys  and other organs which may substantially exceed those  in the blood.   Dose-dependent nausea and vomiting are the most  common side effects, and diarrhoea may arise as a con-  sequence of alterations in the bacterial flora of the colon.  The ability to chelate with calcium results in tetracyclines  being deposited in bones and teeth, and precludes their  administration to children younger than 12 years or to  women in late pregnancy. Their use in patients with poor  kidney function is also contraindicated because most of  the tetracyclines accumulate in this situation; again, dox-  ycycline and minocycline are exceptions.   The macrolide antibiotics are large molecules comprising  12-16-membered lactone rings linked through glycosidic  bonds with amino sugars. Erythromycin was the first  member of the group to be discovered in 1952 and it is  still an important antibiotic today. It was quickly followed  2 years later by spiramycin and oleandomycin but,  although still available in certain countries, these last two  are now little used. Erythromycin suffers from several  disadvantages: its antimicrobial spectrum is largely  restricted to Gram-positive species, it has poor acid sta-  bility so its absorption is erratic, it commonly exhibits  gastrointestinal side effects and bacteria acquire resist-  ance to it relatively easily. These shortcomings prompted  the search for new macrolides, and several semisynthetic  derivatives were forthcoming: roxithromycin was mar-  keted in 1987, clarithromycin and azithromycin in 1991  and the most recent, telithromycin, in 200 1 . Erythromycin  (Figure 11.8) and roxithromycin are chemically similar in  possessing a 14-membered ring structure. A distinction  is sometimes drawn between them and both azithromy-  cin which, strictly speaking, is an azalide (a 15-membered  ring containing an additional nitrogen atom) and tel-  ithromycin which is a ketolide (a 14-membered ring with  an additional keto group). The term macrolide, however,  is commonly used to describe all five antibiotics, and that  terminology will be used here.   The macrolides are active against most Gram-positive  bacteria, Neisseria and H. influenzae but, with the excep-    tion of azithromycin, not against the Enterobacteriaceae.  Because their antibacterial spectrum is similar to those of  the early penicillins, the macrolides were, and still are,  considered alternatives for patients with penicillin allergy.  They are commonly used for respiratory, skin and soft  tissue infections, but one of the factors that stimulated  the development of the more recent macrolides was their  activity against emerging pathogens like species of  Legionella, Campylobacter, Helicobacter and Chlamydia,  as well as some mycoplasmas and rickettsias and the  Mycobacterium avium complex to which AIDS/HIV  patients are susceptible. The semisynthetic macrolides do  not afford a significant advantage over erythromycin in  terms of their activity against staphylococci, streptococci  and enterococci, but they represent an advantage in  several other respects: they are generally more active  against the other organisms mentioned above; they  exhibit better stability and pharmacokinetics, thus per-  mitting less frequent dosage and better tissue penetra-  tion; they generally have fewer side effects; and,  particularly in the case of telithromycin and other  ketolides, they may be active against some strains that  have acquired resistance to erythromycin, and they are,  themselves, less vulnerable to resistance development.   The macrolides all act by inhibiting protein synthesis  in bacteria and they are regarded as bacteristatic drugs,  although bactericidal activity may be achieved at high  concentrations. The antimicrobial activity of erythromy-  cin is pH-dependent, increasing with pH up to about 8.5,  and the same effect occurs to varying degrees with other  members of the group. The macrolides are extremely  bitter and their tablets are often coated, both to disguise  the taste and to protect the antibiotic from stomach acid.  Erythromycin exhibits particularly poor acid stability  and erratic oral absorption, and a variety of esters have  been used to minimize these problems which, although  present, are much less evident in the semisynthetic  molecules.   All the macrolides are orally active and they are con-  centrated intracellularly, particularly into neutrophils by  which they are transported to infection sites. The longer  elimination half-lives of the newer drugs permit less fre-  quent dosing than that required for erythromycin. The  group as a whole are regarded as relatively safe antibiotics  which do not exhibit severe adverse reactions, although  gastrointestinal disturbances (nausea, vomiting, abdomi-  nal pain and, infrequently, diarrhoea) are relatively  common with erythromycin and much reduced or absent  in the others. These and other characteristics are sum-  marized in Table 1 1.4.   Especially poor acid stability, erratic oral absorption and  gastrointestinal side effects   Very good acid stability and oral absorption. Tissue concentrations  very much higher than those in the plasma   Slightly less active than erythromycin, but long half-life and good  tissue concentration   More active against Gram-negative bacteria, including  Enterobacteriaceae, than erythromycin; very high concentration in  neutrophils   Active against bacteria resistant to penicillins and other macrolides,  with recommendations that its use be restricted for that purpose   Sulphonamides were discovered by Domagk in 1935. It  had been shown that a red azo dye, prontosil, had a cura-  tive effect on mice infected with (3-haemolytic strepto-  cocci; it was subsequently found that in vivo, prontosil  was converted into sulphanilamide (Figure 11.9A). The    basis of the antimicrobial activity of the sulphonamides  is their structural similarity to p-aminobenzoic acid  (PABA) which is an integral part of the B vitamin, folic  acid. In sensitive bacteria, sulphonamides compete  with PABA with the result that folic acid synthesis is  reduced. Because the vitamin is essential for the manu-  facture of nucleic acids (and other important biochemi-  cals), this leads to a reduction in, or cessation of, bacterial  growth.    Chemical modifications of sulphanilamide (see Figure  1 1.9A) gave compounds with higher antibacterial activity  or special properties like prolonged activity. The drugs  were extensively used from the 1930s to the 1970s, after  which their popularity declined due to resistance devel-  opment and the introduction of safer and more effective  antibiotics. A few sulphonamides are still used in topical  therapies(notably silver sulphadiazine for burns) and in  veterinary medicine, while sulphadiazine itself remains  available for the prevention of rheumatic fever. Dapsone  (Figure 1 1.9B) is a sulphonamide derivative that has been  used extensively in the past for the treatment of leprosy,  but again, although still available and currently employed  as part of a multidrug treatment for leprosy, its use has  declined as a result of resistance development and the  introduction of better antibiotics.   In the body, folic acid must be reduced to dihydrofolic  acid and then tetrahydrofolic acid in order to become  active, and it was discovered that a group of synthetic  drugs called diaminopyrimidines could inhibit the  enzymes responsible for this reduction. These dihydro-  folate reductase inhibitors, of which trimethoprim  (11.9C) is the most important, were found to act syner-  gistically with sulphonamides because they blocked suc-  cessive steps in the synthesis of reduced folic acid.  Consequently trimethoprim was introduced in 1969 as a  combination product with sulphamethoxazole (co-  trimoxazole) for the treatment of urinary tract and, less  commonly, respiratory infections. Unfortunately, the  advantages of using the combination product were not as  great as anticipated partly because pharmacokinetic dif-  ferences between the two drugs resulted in relative con-  centrations in the body which were far from optimal for  synergy. This, together with increasing evidence that the  antibacterial activity of co-trimoxazole was due largely to  the trimethoprim component with the more toxic sul-  phamethoxazole contributing little, lead to the use of  trimethoprim alone from the mid 1970s. Co-trimoxazole  is currently recommended only for treatment of pneu-  mocystis pneumonia, toxoplasmosis and nocardiasis.   Trimethoprim remains one of the least expensive  orally active agents available for the treatment of urinary  tract infections, for which it is still widely prescribed,  although it, too, is suffering from increasing resistance  development and the trend is towards its replacement  with fluoroquinolones (see section 6). Other trimetho-  prim analogues, notably tetroxoprim, have been intro-  duced as antibacterial agents, but have not demonstrated  significant advantages over trimethoprim itself. The  other important diaminopyrimidine possessing antimi-    crobial activity is pyrimethamine. This is used in combi-  nation with sulphadoxine or other drugs for the treatment  (but not any longer for the prophylaxis) of malaria. It is  used on its own for the treatment of toxoplasmosis and  pneumocystis pneumonia.   The quinolones and the cephalosporins have much in  common in terms of their history and development: both  classes of antibiotic were first introduced in the early  1960s and the first members of each class, nalidixic acid  and cephalothin respectively, had limited applications.  However, both groups were extensively developed over  the next 50 years with four generations of antibiotics  being produced in both classes, each an improvement on  its predecessor, so that the quinolones and the cepha-  losporins are currently amongst the most widely used  and valuable antibiotics available.   Nalidixic acid (Figure 11.10B) was developed as a  treatment for urinary tract infections, and it, together  with other first-generation quinolones produced in the  1960s and 1970s (e.g. cinoxacin, acrosoxacin and pipe-  midic acid), had an antibacterial spectrum that was  largely restricted to E. coli and other Enterobacteriaceae.  As with the cephalosporins, there is no universal accept-  ance of the definitions of each generation of the antibiot-  ics, so particular quinolones have been assigned to  different generations in various textbooks. There is agree-  ment, however, that the distinguishing feature of the first  generation is the absence of a fluorine atom at position  6, so they are simply designated ‘quinolones’, whereas the  second, third and fourth generations are termed fluoro-  quinolones. The first-generation drugs are now little  used, but those that followed in the 1980s, e.g. cipro-  floxacin, norfloxacin, ofloxacin and levofloxacin (the  L-isomer of ofloxacin) (Figure 11.10C-E), are still very  much in use today and owe their popularity both to their  greater activity against E. coli and other Gram-negative  urinary pathogens and their wider antibacterial spectrum  which includes Gram-positive cocci and Ps. aeruginosa.  The third-generation quinolones, e.g. moxifloxacin  (Figure 11.10F), possess the attributes of their predeces-  sors but exhibit greater activity against Strep, pneumo-  niae. The fourth generation, e.g. trovafloxacin (not  available in Europe), are also active against anaerobes.   All the quinolones are bactericidal, and act by inhibit-  ing the bacterial enzymes responsible for coiling of DNA  (see Chapter 12); they are all orally active, and several are   Figure 11.10 (A) Fluoroquinolone; (B)  nalidixic acid; (C) ciprofloxacin; (D)  norfloxacin; (E) levofloxacin and  ofloxacin; (F) moxifloxacin.    available as intravenous injections. The first-generation  drugs were only used for the treatment of urinary infec-  tions, but the much broader spectra of those that fol-  lowed permitted their use for respiratory, soft tissue, bone  and joint, gastrointestinal and sexually transmitted infec-  tions as well. Quinolones have been shown to cause rare  cases of tendon damage, including rupture, and for this  reason are not generally prescribed for children, but their  most common minor side effect is gastrointestinal distur-  bances, which are reported in about 5% of patients. They  are relatively susceptible to resistance development, and  Gram-positive species tend to mutate to resistance at  higher frequencies than Gram-negatives. Some degree of  cross-resistance usually occurs, so that bacteria that  become resistant to one quinolone are often less suscep-  tible to others. They are now the most widely prescribed  antibiotics for adults in the USA, and the growing resist-  ance problem has been attributed in part to their indis-  criminate use in human medicine and to cross-resistance  between the drugs used for human infections and the  several quinolones licensed exclusively for veterinary  medicine which have been overused, particularly in  Europe, for prophylaxis of bacterial infections in poultry  flocks and other intensively reared animals.    The characteristics of some of the important quinolo-  nes are shown in Table 11.5.   The aminoglycosides are a large group of broad-spectrum  antibiotics possessing significant activity against many  Gram-negative bacteria and a more limited range of  Gram-positive organisms. The antibiotics are all  bactericidal and administered by injection because they  are poorly absorbed from the gastrointestinal tract.  They are cationic, water-soluble drugs that interfere  with protein synthesis in bacteria by binding to the  30S ribosome subunit (which is not possessed by  mammals).   The earliest aminoglycoside was streptomycin, discov-  ered in 1944, and at least 10 more were discovered over  the next 30 years although not all were marketed.  However, their susceptibility to resistance development  by inactivating enzymes and other mechanisms (see  Chapter 13), and their potential to cause damage to the  kidneys and the eighth cranial nerve (to the ear) limited  the use of the naturally occurring aminoglycosides and   Has good activity against Gram-negative species but  only moderate activity against Gram-positives. Used  primarily for UTIs, respiratory (but not pneumococcal  pneumonia), GI and bone and joint infections   Primarily for UTIs and lower respiratory infections, as  well as gonorrhoea and genital infections   As for ciprofloxacin, and as a second-line treatment  for community- acquired pneumonia    lead to the development of several semisynthetic members  of the group.   Streptomycin was the first effective antibiotic for the  treatment of tuberculosis and it is still used as a second-  line drug for that purpose, although it now has few  other applications. Neomycin was also discovered in the  1940s, but its high toxicity curtailed its use as a systemic  drug and it is now largely restricted to ophthalmic and  topical products, although in this limited field its use is  widespread, and it is often encountered with other anti-  biotics or with steroids in antibiotic/anti-inflammatory  creams. Oral preparations of neomycin are employed,  again often in combination with other antibiotics, to  reduce the bacterial population of the colon prior to  surgery.   Three of the most important aminoglycosides cur-  rently available are gentamicin, tobramycin and ami-  kacin. The first two of these are naturally occurring drugs  discovered in the 1960s, whilst amikacin is a semisyn-  thetic derivative of kanamycin, which it has superseded.  Gentamicin, the drug of choice in the UK, is used alone  or in combination with (3-lactam antibiotics (with which  it exhibits synergy) both for ‘blind’ therapy of infections  prior to identification of the infecting organism, and for  the treatment of bacterial endocarditis and serious Gram-  negative infections; in common with other members of  the group, it has no activity against anaerobes. Amikacin    has similar applications, but is more stable to inactivation  by bacterial enzymes, though rather less potent, than gen-  tamicin. All three antibiotics possess useful activity  against Ps. aeruginosa and are particularly valuable, again  with (3-lactams, for the eradication or suppression of this  organism in the lungs of cystic fibrosis patients; tobramy-  cin is slightly more active than gentamicin against Ps.  aeruginosa and for this reason it is more frequently used  for this purpose than any other.   Aminoglycosides have in the past often been adminis-  tered twice or three times per day, but the more recent  trend has been towards a single, higher, daily dose.  Although not suitable in all situations, once-daily dosing  is undoubtedly more convenient and considered to be at  least as safe and efficacious. Regardless of the dosing  schedule however, monitoring of serum levels during  treatment to avoid potential toxic high concentrations or  ineffective low ones is still an integral part of aminogly-  coside therapy.   The only two important glycopeptide antibiotics cur-  rently available are vancomycin and teicoplanin. Like  many of the other antibiotics in current use, vancomycin  is a relatively old drug, having been introduced in 1958,    but its activity against MRSA resulted in it becoming  progressively more valuable as MRSA became more prev-  alent. It has a complex tricyclic glycopeptide structure  and its large molecular size means that it cannot pene-  trate through the outer membrane of most Gram-  negative bacteria, so its use is effectively restricted to the  treatment of infections by aerobic or anaerobic Gram-  positive species. In addition to Staph, aureus, it is active  against Staph, epidermidis, streptococci. Cl. difficile and  Ent. faecalis, although resistant enterococci are posing an  increasing clinical problem.   Vancomycin is bactericidal to most susceptible bacte-  ria at concentrations near its minimum inhibitory con-  centration (MIC) and is an inhibitor of bacterial cell wall  peptidoglycan synthesis, although at a site different from  that of (3-lactam antibiotics. Employed as the hydrochlo-  ride and administered by dilute intravenous injection,  vancomycin is indicated in potentially life-threatening  infections that cannot be treated with other, less toxic,  antibiotics. Oral vancomycin, which is not absorbed from  the gastrointestinal tract, is the drug of choice in the  treatment of antibiotic-induced pseudomembranous  colitis associated with the administration of antibiotics  such as clindamycin and lincomycin (section 11).  Although not chemically related to the aminoglycosides,  vancomycin suffers the same toxicity problems and has  the potential to damage both the kidney and the ears, and  it too is normally subject to blood-level monitoring  during therapy.   Because of its potential toxicity, its poor penetration  of bile and cerebrospinal fluid, the requirement for twice-  daily dosing, and pain on intramuscular administration  that effectively limits vancomycin to the intravenous  route, there was scope for the introduction of a second  glycopeptide antibiotic in which these deficiencies were  eliminated or reduced; accordingly, teicoplanin was mar-  keted in Europe in the early 1990s. Teicoplanin has the  same mode of action and antimicrobial spectrum as van-  comycin, as well as a similar chemical structure, but, cru-  cially, teicoplanin possesses more fatty acid side chains  which (1) make the molecule more acidic, thereby per-  mitting the formulation of a sodium salt that can be given  both by intravenous and intramuscular injection, and (2)  make teicoplanin more lipophilic, which affords better  tissue penetration and a longer half-life; as a consequence,  teicoplanin is normally administered once daily rather  than twice. Other advantages over vancomycin are a  slightly higher potency against most target organisms and  a better toxicity profile, thereby eliminating the need for  routine blood monitoring.   The antibiotics used for the treatment of tuberculosis  belong to a variety of chemical classes, but it is convenient  to consider them together in the same section for two  reasons: first, most of them are used exclusively for tuber-  culosis, and second, therapy extends over several months,  during which time resistance development is a significant  possibility. This risk is minimized by the use of two or  three antibiotics in combination, so it is appropriate to  consider them together because that represents their  normal pattern of use.   Streptomycin (Section 7), introduced in the late 1940s,  was the first effective treatment for tuberculosis, but its  use in isolation was short lived because of the ease with  which the bacteria became resistant to a single antibiotic.  Isoniazid, which became available about 5 years later, was  used first in combination with streptomycin then with  rifampicin after the latter was introduced in 1967. The  combination of isoniazid and rifampicin has been  the mainstay of tuberculosis therapy from that time  until the present day, although other drugs like pyrizina-  mide and ethambutol have been added to the combina-  tion, and multidrug-resistant tuberculosis has become an  increasing problem in recent years; this is, by definition,  simultaneous resistance to isoniazid and rifampicin.   Tuberculosis may be caused by any one of three  Mycobacterium species, but all three characteristically  grow slowly in the body and may persist for long  periods in a near dormant state. Because the course of  therapy is normally 4-6 months, antibiotics that are  orally effective are much preferred; this is because unsu-  pervised patients are more likely to take oral medicines,  and in many countries where patients may have difficulty  travelling to clinics the daily administration of injectable  antibiotics is not feasible. The ideal antitubercular drug  should also have the potential to kill rather than merely  inhibit the growth of the infecting organism. Because  mycobacteria can survive, and even reproduce, within  macrophages, relying on the immune system to eradicate  an infection following the use of a bacteristatic drug is  unlikely to be an effective strategy. Rifampicin kills  dormant bacteria, while pyrizinamide is active against  bacteria that are slowly reproducing in acidic environ-  ments; isoniazid is most useful against more rapidly  growing cells.   The current approach is to treat tuberculosis in  two phases: an initial phase of 2 months using  isoniazid, rifampicin and pyrizinamide (with or without    ethambutol), and a 4 month continuation phase with  isoniazid and rifampicin. If, however, the infecting organ-  ism is resistant to any of the above, second-line drugs may  be used and the duration of treatment possibly extended.  The status of streptomycin is equivocal; it is now rarely  used in the UK and is not a first-choice treatment recom-  mendation of the European Respiratory Society either,  but it is more commonly used in front-line therapy in the  USA. Second-line drugs available for infections caused by  resistant organisms, or when first-line drugs cause unac-  ceptable side effects, include amikacin, capreomycin,  cycloserine, newer macrolides (e.g. azithromycin and  clarithromycin) and moxifloxacin. Drug regimens are  often indicated using a shorthand notation with single  letters to indicate the drugs employed, initial numbers  indicating months of therapy and following numbers  (which may be in parentheses or subscripts) indicating  days per week. For example, 2RHZ(E)/4HR(3) or  2RHZ(E)/4HR 3 would mean 2 months of rifampicin, iso-  niazid and pyrizinamide (with the possible addition of  ethambutol) followed by 4 months of isoniazid and  rifampicin three times per week. Several of the antituber-  cular drugs have specific contraindications or require  monitoring during use; some of these requirements and  cautions are shown in Table 11.6.   Rifampicin is the only one of the common antituber-  cular drugs that is used in the treatment of non-  mycobacterial infections. It is active against Gram-positive  bacteria and some Gram-negative species, but not    Enterobacteriaceae or pseudomonads. Rifampicin pos-  sesses significant bactericidal activity at very low concen-  trations against staphylococci. Unfortunately, resistant  mutants may arise very rapidly, both in vitro and in vivo,  so it has been recommended that rifampicin should be  combined with another antibiotic, e.g. vancomycin, in  the treatment of staphylococcal infections. Rifabutin, a  semisynthetic rifamycin, may be used in the prophylaxis  of M. avium complex infections in immunocompro-  mised patients and in the treatment, with other drugs, of  pulmonary tuberculosis and non-tuberculous mycobac-  terial infections.   It is not only MRSA that has become a major problem  organism in recent years; VRE and penicillin-resistant  Strep, pneumoniae (pneumococci) have also become  much more prevalent, prompting fears of serious treat-  ment problems due to the small number of antibiotics  that remained effective. The emergence of Staph, aureus  strains which showed intermediate sensitivity to vanco-  mycin or to glycopeptides in general (designated VISA or  GISA respectively) caused particular concern, and accel-  erated the introduction of three new antibiotics intended  primarily for the treatment of Gram-positive cocci  infections.   Visual acuity check required before therapy and plasma  drug monitoring required during use   Hearing and balance function and serum potassium  checks recommended before and during treatment   Avoided in patients with a history of epilepsy,  neurological and psychiatric problems    1, first-choice therapy; 2, second-choice therapy; IM, intramuscular; IV, intravenous; O, oral.    The first of these, approved in 1999, is a fixed-ratio  combination of the two streptogramin antibiotics, dalfo-  pristin and quinupristin (30 : 70). This bactericidal intra-  venous product is recommended for the treatment of  serious infections with vancomycin-resistant Ent. faecium  and multiresistant strains of staphylococci and pneumo-  cocci. The second, linezolid (2001), is an orally active,  synthetic antimicrobial possessing an essentially bacteri-  static action against Gram-positive organisms, with no  useful activity against Gram-negatives. It is used for the  treatment of MRSA, VRE and pneumococcal infections,  and although isolated early reports of resistance did arise,  the development of resistance during treatment has been  rare. Daptomycin (2003) is a lipopeptide with a novel  mode of bactericidal action destabilizing the bacterial cell  membrane, which means that cross-resistance with exist-  ing drugs is unlikely to arise. It is administered intrave-  nously and used for skin and soft tissue infections by  Gram-positive organisms and for Staph, aureus endocar-    ditis; it is particularly useful for infections by VISA and  GISA strains of this species.   Clindamycin (Figure 11.11 A) is another antibiotic  possessing significant bacteristatic activity towards  Gram-positive cocci (including MRSA, but not Ent. fae-  calis), although rather less activity is shown towards  Gram-negative cocci and none at all against enterobacte-  ria. Clindamycin is not related structurally to the mac-  rolides (section 4) but has a similar mechanism of action,  so cross -resistance may occur between them. The strep-  togramins also have a similar mechanism of action, but  it is claimed that the quinupristin-dalfopristin combina-  tion does not demonstrate cross-resistance to clindamy-  cin or the macrolide antibiotics.   Figure 11.11 Miscellaneous antibiotics: (A) clindamycin; (B) fusidic acid; (C) mupirocin; (D) colistin; (E) chloramphenicol; (F)  metronidazole; (G) nitrofurantoin.    Clindamycin is currently recommended for the oral  treatment of staphylococcal bone and joint infections,  acne, peritonitis, infections by anaerobic bacteria and fal-  ciparum malaria. Together with oral cephalosporins, it is  one of the antibiotics most firmly associated with pseu-  domembranous colitis caused by Cl. difficile , and this  reputation limits its use.   Fusidic acid (Figure 11.1 IB) is a steroid-like, bactericidal  antibiotic used primarily for its activity against staphylo-  cocci. It does possess activity against other Gram-positive  species, although streptococci are relatively resistant and  Gram-negative bacilli completely so. It is active against  penicillin-resistant strains of Staph, aureus, including  MRSA, and may be administered in combination with  erythromycin or clindamycin for severe staphylococcal  infections. Fusidic acid is available as a paediatric oral  suspension, cream and ointment, and, in the form of its  water-soluble sodium salt, as tablets or an intravenous  injection. Resistance arises with relative ease in vitro, but  despite this, resistance is relatively uncommon in clinical  isolates.   Mupirocin (Figure 11.1 1C) is an antibiotic active against  staphylococci, streptococci and a limited range of Gram-  negative species. Its use is largely confined to topical  treatment of Staph, aureus infections, particularly the  eradication of MRSA from the nose; for this purpose it  is claimed normally to be more effective than chlorhexi-  dine or fusidic acid.   Colistin (Figure 1 1.1 ID) is the only member of the poly-  myxin group of peptide antibiotics that is still in use. It  is active against many types of Gram-negative bacteria,  but not against cocci, Serratia marcescens and Proteus  spp.; it is inactive against Gram-positive organisms. Its  use is largely restricted to the treatment of Ps. aeruginosa  lung infections in cystic fibrosis sufferers, in which case  it is nebulized or given by intravenous injection, and in  the treatment of infections, particularly in burns, caused  by Acinetobacter species.   Chloramphenicol (Figure 11.1 IE) is a true antibiotic but  is manufactured totally by chemical synthesis. It has a  broad spectrum of activity including some rickettsias and  larger viruses, but aplastic anaemia, which is dose-related,    may result from treatment in a proportion of patients  and this has largely restricted its use as a systemic antibi-  otic to life-threatening infections with H. influenzae. It is,  however, still used significantly in the treatment of oph-  thalmic infections and in veterinary medicine.   The nitroimidazoles are a group of synthetic antimicrobi-  als that are unusual in possessing activity against a wide  range of organisms including bacteria, protozoa, and  some helminths. They are similar in terms of their struc-  ture, mode of action, uses and toxicity and the principal  factor distinguishing them is their pharmacokinetics.  Metronidazole (Figure 11.1 IF) is by far the most com-  monly used, and is the only one considered in this section,  although nimorazole (no longer available in the UK) and  tinidazole are alternative drugs that may afford the  advantage of less frequent dosing. They may all be  regarded as prodrugs in the sense that they become active  only after reduction of the nitro group in low redox envi-  ronments, so they are able to kill cells growing anaerobi-  cally by damaging their DNA. This damage could, in  theory, occur in any type of cell regardless of its taxo-  nomic status, and the fact that sufficiently reducing con-  ditions do not arise in mammalian cells is the reason for  the drugs’ lack of toxicity for humans.   Metronidazole was introduced in 1960 for the treat-  ment of vaginitis caused by the protozoan Trichmonas  vaginalis, and its wider application for the treatment of  bacterial anaerobic infections was recognized later.  Currently metronidazole is used alone, or quite com-  monly in combination with other antibiotics, for the  treatment of a wide variety of bacterial infections shown  in Table 11.7. For the treatment of amoebiasis, giardiasis  or trichomonal vaginitis, metronidazole is often used  alone. Metronidazole is available in more dosage forms  (oral, topical, injectable or suppositories) than most  other antibiotics, and by the oral route is often given two  or three times daily. Toxicity and side effects are relatively  uncommon, although it does exhibit a disulphiram-like  reaction with alcohol in some patients.   Nitrofurantoin (Figure 11.1 1G) is the one remaining  member of the nitrofuran group of drugs that is still in  common use. Like metronidazole, nitrofurantoin requires  its nitro group to be reduced in order to exhibit antimi-  crobial activity, and it too exhibits bactericidal activity by  damaging DNA. It has a wide spectrum of activity which    Table 11.7 Some common bacterial infections for which metronidazole may be used    includes Gram-positive cocci and many Gram-negative  enteric bacteria, but after oral administration the blood  levels achieved are very low and a significant fraction of  the dose is rapidly excreted in the urine, so its use is  restricted to the treatment of cystitis. The size of the drug  crystals used in tablet manufacture has an effect on the  dissolution of the drug, and it is claimed that the macro-  crystalline form affords steadier release. Its antimicrobial  activity is substantially greater in acid urine, which unfor-  tunately conflicts with the common symptomatic treat-  ment of cystitis by alkalizing the urine with potassium  citrate or similar compounds. Nitrofurantoin is unusual  in being one of the few antimicrobial drugs to which  resistance has not significantly increased since its intro-  duction, and for this reason there is a degree of renewed  interest in it.   There has been a significant increase in the number of  both systemically and topically acting antifungal agents  in recent years; this has been prompted in part by the  increase in patients with impaired immunity who are  particularly vulnerable to such infections. For much of  the second half of the 20th century nystatin, amphoter-  icin and griseofulvin were the principal antifungal anti-  biotics available, and these were supplemented with a  range of synthetic imidazoles which were used primarily  for superficial rather than systemic fungal infections. The  introduction of the triazole antifungals in the 1980s was  a major advance and, more recently, the echinocandins    have further increased the range of drugs available for  severe infections.   Lack of toxicity is, as always, of paramount impor-  tance, but the differences between bacterial and fungal  cells in both structure and biosynthetic processes mean  that the low-toxicity antibacterial antibiotics are usually  inactive against fungi. This limitation is further compli-  cated by the fact that both fungal and human cells are  eukaryotic in structure, which means that there are few  differences that can be exploited in order to achieve selec-  tive toxicity towards fungi whilst leaving the human cells  unharmed. Fungal infections are normally less virulent  in nature than are bacterial or viral ones but may, never-  theless, pose major treatment problems in individuals  with a depressed immune system, particularly in the case  of systemic infection.   The azoles may be considered as two subgroups: the older  imidazole drugs, the majority of which were introduced  as topical products or pessaries for the treatment of super-  ficial infections by dermatophytes (skin pathogens),  Pityriasis species (causing flaky skin and dandruff) and C.  albicans , and the more recently developed, more versatile  and, in some cases, much more expensive, triazoles.   The imidazoles are a large and diverse group of com-  pounds with activity against bacteria and protozoa (met-  ronidazole and tinidazole), helminths (mebendazole) and  fungi (clotrimazole, miconazole, ketoconazole, econazole,  sulconazole and tioconazole). Table 11.8 lists infections   Oral systemic treatment of dermatophytes or Candida resistant to other drugs  or patients intolerant to them    NS, nail solution; P, pessary; Pdr, powder; S, spray; Sham, shampoo; Soln, solution or gel for external or oral use; T, topical cream  or ointment; Tab, tablets; VC, vaginal cream.    for which the common antifungal drugs are employed;  other imidazoles are available in various countries.   As Table 11.8 indicates, the imidazoles are available in  a wide variety of dosage forms, but most of them have  the same uses. They exhibit modest activity against some  Gram-positive bacteria, but they are all essentially fungi-  static drugs which might exert fungicidal action during  prolonged exposure to high concentrations. Miconazole  may be given orally for the treatment of intestinal fungal  infections, and in this situation may show some absorp-  tion into the systemic circulation, although this may be  more of a problem from the perspective of potential drug  interactions than a benefit. Ketoconazole, however,  achieves greater absorption from the gastrointestinal  tract, but it has been largely superseded by newer and less  toxic drugs so its use tends to be restricted to situations  where resistance or patient intolerance to those drugs is  a problem.   Fluconazole (Figure 1 1. 12A) and itraconazole were intro-  duced in the 1980s and posaconazole and voriconazole  much more recently. The two earlier drugs are more  widely used, whilst the recent ones tend to be reserved for  severe, possibly life-threatening infections, in which other  antibiotics have failed or are inappropriate. All four of the  triazole drugs are orally active and all but posaconazole  are available in injection form.   Fluconazole is better absorbed from the gastrointesti-  nal tract than itraconazole and, in addition to the treat-  ment of dermatophytes, pityriasis and Candida infections,    it is valuable in cases of cryptococcosis which, although  uncommon, is very dangerous in immunocompromised  patients. Itraconazole is used for similar infections to flu-  conazole but, in addition, it is more commonly selected  as an alternative to amphotericin (see below) in cases of  systemic Aspergillus infection (again, more common in  the immunocompromised) and other rare systemic  mycoses. It is more frequently associated with liver toxic-  ity than fluconazole.   Polyene antibiotics are characterized by possessing a large  ring containing a lactone group and a hydrophobic  region consisting of a sequence of four to seven conju-  gated double bonds. The only important polyenes are  amphotericin B and nystatin.   Amphotericin B (Figure 11.12C) is active against most  fungal pathogens and is used for systemic mycoses as a  potentially more toxic, but possibly more effective, alter-  native to itraconazole. It is poorly absorbed from the  gastrointestinal tract and is thus usually administered by  intravenous injection under strict medical supervision.  Lipid-based and liposomal formulations of amphotericin  are available which exhibit lower toxicity than conven-  tional aqueous formulations; they may therefore be given  in higher doses. Nystatin is administered orally in the  treatment of C. albicans infections in the intestine or the  mouth (often referred to as thrush), and by cream or  pessary for skin or vaginal infections by that organism. It  is rarely used for the treatment of other infections and is  too toxic to be given by injection.   Figure 11.12 Antifungal agents: (A) fluconazole; (B) flucyctosine; (C) amphotericin; (D) terbinafine; (E) tolnaftate.   The echinocandins are a new class of semisynthetic  lipopeptide antibiotics that are fungicidal towards  Aspergillus spp., Candida spp. and Pneumocystis jirovecii  (previously known as P. carinii). Caspofungin, the  first member of the class, is given as an intravenous infu-  sion recommended for invasive aspergillosis or candidia-  sis that is unresponsive to treatment with amphotericin  or itraconazole. Anidulafungin and micafungin are  similarly injectable products licensed for the treatment  of invasive candidiasis, and they, like caspofungin,  tend to be used as reserve drugs, both to minimize the  risk of resistance development and due to cost  considerations.   Flucytosine (5-fluorocytosine, Figure 11.12B) is a narrow-  spectrum antifungal agent with greatest activity against  yeasts such as Candida, Cryptococcus and Torulopsis. It is    normally used in combination with fluconazole or as a  synergistic combination with amphotericin which  permits amphotericin dose reduction and a lower risk of  toxicity. Terbinafine (Figure 11.12D), a member of the  allylamine class of antimycotics, is orally active, fungi-  cidal, effective against a broad range of dermatophytes  and yeasts and is the drug of choice for fungal nail infec-  tions. Griseofulvin too is employed for the treatment of  dermatophyte infections of hair, skin and nails, but  usually only when topical therapy has failed, and with the  exception of Trichophyton infections in children, it is no  longer regarded as a drug of choice. It is orally active,  although the particle size of the powder used to manu-  facture the tablets has a significant effect on bioavailabil-  ity. Tolnaftate (Figure 1 1 . 12E) is a synthetic thiocarbamate  which is used topically in the treatment or prophylaxis of  tinea (commonly referred to as ringworm, although not  due to worms at all) and amorolfine is used as a cream  or nail lacquer for the same purpose.   Most of the antibacterial and antifungal agents described  earlier in this chapter have little or no activity against  viruses because they target structures or enzyme systems  that are only found in bacterial and fungal cells. In con-  trast to other microorganisms, viruses do not possess the  enzymes necessary for their own replication. After entry  into the host cell, the virus uses the enzymes already  present, or induces the formation of new ones, in order  to synthesize the individual components of the virus  particle which are then assembled and released from the  host cell. Because viruses literally ‘take over’ the machin-  ery of an infected human cell, there are very few unique  features of viral replication that can be exploited for the  purposes of achieving selective toxicity, i.e. creating anti-  viral agents that inhibit or kill the virus without harming  the human host.   Prior to the identification of the HIV virus in 1983  there was a very limited range of effective synthetic anti-  viral agents; the fourth edition of this book, published in  1987, described only nine. The HIV/AIDS pandemic pro-    vided a major stimulus for fundamental research into the  structure and reproduction of viruses in general and ret-  roviruses in particular, and this, together with (1) better  understanding of the role played by some viruses in the  development of specific cancers, (2) more sophisticated  diagnostic methods, and (3) elucidation of the genomes  of several viruses, has led to a wealth of new antiviral  drugs; Table 11.9 lists 21, and a further 20 that are used  largely or exclusively for the treatment of HIV are shown  in Table 11.10. Although these new drugs may be catego-  rized on the basis of their chemical structure (Figure  11.14) or mode of action, most of them are licensed for  use against a limited number of viruses, and often just a  single one, so the most convenient and useful way of  considering antivirals is on the basis of the infections they  are intended to treat.   There is a large and progressively increasing variety of  antiretroviral agents available to treat HIV, and their  use requires specialist knowledge. A detailed account of  the characteristics of each individual drug is beyond  the scope of this chapter, but it is possible to gain a    Table 11.9 Antiviral drugs used in the treatment of selected viral infections    Nucleoside reverse transcriptase inhibitors (nucleoside analogues) Abacavir, didanosine, emtricitabine, lamivudine, stavudine,   Non-nucleoside reverse transcriptase inhibitors  Miscellaneous agents with unique mechanisms of action    good understanding of the principles of HIV chemo-  therapy by considering the life cycle of the virus (Figure  11.13) in relation to the modes of action of the drugs in  current use.   The virus particle initially binds to a CD4 protein  receptor and one of two coreceptors on the surface of a  T-lymphocyte. Maraviroc, an antagonist of the CCR5  coreceptor, is licensed in the UK for the treatment of  patients exclusively infected with CCR5 -tropic HIV. The  bound virus then fuses with the host cell membrane and  the viral RNA is released into the cell. This step is targeted  by enfuvirtide, a fusion inhibitor, that is used for manag-  ing infection that has failed to respond to a regimen of  other antiretroviral drugs. The single-stranded viral RNA  is used as a template from which a complementary DNA  strand is manufactured by viral reverse transcriptase;  there are several nucleoside analogue inhibitors of this  enzyme (Table 11.10), together with a smaller number of  non-nucleoside inhibitors (e.g. efavirenz, etravirine, and  nevirapine). The DNA is duplicated, and in its double-    stranded form it enters the cell nucleus where an HIV  enzyme integrates it into the host DNA to create what is  termed a provirus. The integrase enzyme can be inhibited  by raltegravir, a drug which again is largely reserved  for the treatment of HIV infection resistant to multiple  antiretrovirals.   The provirus may remain latent (dormant) within  the cell nucleus for a period of time varying from 2 weeks  to 20 years; it is then activated by regulatory proteins  termed transcription initiators which cause the viral  DNA to be transcribed into viral mRNA and several  long protein molecules. It is the latent provirus that rep-  resents the major hurdle to complete eradication of  HIV, and recent research interest has focused on the tran-  scription initiators as alternative potential targets for  antiretroviral drug action. The long proteins only become  functional after being split into smaller molecules by  viral protease enzymes and there are now many protease  inhibitor drugs available for the treatment of HIV  (Table 11.10).   Figure 11.14 Structures of nucleosides and selected antiviral agents: (A) thymidine; (B) guanosine; (C) ribavirin; (D) zidovudine;  (E) aciclovir; (F) ganciclovir; (G) foscarnet; (H) tenofovir; (I) saquinavir; (J) efavirenz.    HIV infection cannot be cured, but strict adherence to  a regimen of antiretroviral drugs can substantially extend  survival. However, there are several problems that may  arise during therapy, of which one of the most significant  is the risk of the virus becoming resistant. This risk may  be minimized by using combinations of three or more  drugs with different mechanisms of action in regimens  that have become known as highly active antiretroviral  therapy (HAART). Treatment is normally initiated with  two nucleoside reverse transcriptase inhibitors and a  non-nucleoside reverse transcriptase inhibitor; the regi-  mens currently recommended in the UK contain either    tenofovir, emtricitabine and efavirenz, or abacavir, lami-  vudine, and efavirenz. Retinovir, used in low concentra-  tions at which it has no intrinsic antiviral activity, has  been shown to increase the duration of effective blood  concentrations of almost all the other protease inhibitors  listed in Table 11.10, and these synergistic combinations  have given rise to the term boosted protease inhibitor, a  combination product of retinovir and lopinavir is com-  mercially available. Regimens containing two nucleoside  reverse transcriptase inhibitors and a boosted protease  inhibitor are reserved for patients with resistance to first-  line treatment. Synergy is often observed between antiret-    roviral drugs, both between agents having the same, and  different, modes of action.   Other problems of HIV therapy are drug toxicity and  patient adherence to their prescribed medication, and  these two are often linked. The variety and severity of the  side effects, particularly those relating to redistribution  of body fat, make it more difficult for patients to achieve  the adherence and persistence required for effective treat-  ment. All the common drugs used in therapy are orally  active because any drug that required lifelong daily injec-  tions would so predispose to non-adherence as to preju-  dice its commercial viability. Several of the antiretroviral  drugs listed in Table 11.10, particularly those with unique  mechanisms of action, are used in such restricted circum-  stances that they are extremely expensive.   There are eight herpesviruses capable of causing human  infection, but of these the most important are:   • the two herpes simplex viruses, HSV-1 and HSV-2,  which, respectively, cause cold sores on the face and lips,  and genital herpes   • the cytomegalovirus (CMV) which may cause retinitis  (inflammation of the retina) and, infrequently, similar  symptoms to infectious mononucleosis.   The first four of the drugs listed in Table 11.9 for the  treatment of herpes infections are the most important.  Inosine pranobex is an orally active immunomodulator  the effectiveness of which has not been proven, and  idoxuridine, a pyrimidine analogue used as a solution  in dimethylsulphoxide for the treatment of cutaneous  herpes, has largely been superseded by aciclovir.   Many antiviral drugs are nucleoside analogues, and  aciclovir together with its prodrug valaciclovir, and  penicyclovir and its prodrug famciclovir, are important  examples of this group. Aciclovir and peniciclovir are  structurally very similar and they act in the same way  both to inhibit viral DNA polymerase and to cause pre-  mature termination of DNA synthesis. Both drugs are  only effective when they have been phosphorylated in the  cell, and selective toxicity arises because phosphorylation  is achieved much more efficiently by virus-encoded thy-  midine kinase than by the corresponding mammalian  enzyme, so human cell DNA synthesis is little affected.  All four drugs are used primarily to treat herpes simplex    and varicella zoster infections; CMV is normally resistant  to them and Epstein-Barr virus shows intermediate sen-  sitivity. Aciclovir is available as an intravenous injection,  tablets and a cream, but the last two of these need to be  administered five times daily in order to maintain effec-  tive levels. Its prodrug valaciclovir has a much longer  half-life and the dose frequency is correspondingly  reduced to 2-3 times a day. Peniciclovir has little oral  activity and is used topically, primarily for cold sores, but  it is applied every 2 hours during waking hours. The  orally active famciclovir is taken between one and three  times daily for the treatment of genital herpes and  shingles.   Because CMV does not produce thymidine kinase it is  not normally susceptible to aciclovir and peniciclovir, so  a different group of drugs is used to treat it. Ganciclovir  has a similar structure to aciclovir but it is phosphor-  ylated more effectively in infected cells than healthy  ones, albeit by a host cell enzyme. It is injected intrave-  nously for the treatment of life-threatening or sight-  threatening CMV infections in immunocompromised  patients only, or for the prevention of CMV infection  following organ transplants. Valganciclovir is an expen-  sive, orally active, valine ester prodrug of ganciclovir  which is used in similar circumstances. Both drugs are  toxic, and the latter carries a specific warning in the  UK British National Formulary about potential tera-  togenic and carcinogenic activity. Cidofovir is a cytosine  analogue that is active against most herpesviruses, and  is used by injection in AIDS patients to treat CMV  retinitis that is unresponsive to other drugs. Foscarnet,  too, is given for CMV retinitis when ganciclovir cannot  be used; it has a relatively simple phosphonoformic acid  structure capable of chelating metal ions, which is  thought to be the basis of its inhibitory action on  polymerase enzymes.   Hepatitis, inflammation of the liver, can be caused by  various drugs and toxins, but hepatitis due to viral infec-  tion is more common. The viruses most frequently  responsible are the hepatitis viruses A-E (which are not  all related), but about 5% of cases of viral hepatitis are  due to other viruses, e.g. herpesvirus, CMV, Epstein-Barr  virus, etc. Hepatitis virus A (formerly known as infectious  hepatitis) is a self-limiting, rarely fatal, food-borne infec-  tion that does not result in permanent liver damage and  is not normally treated with antiviral drugs. Hepatitis E  is similarly self-limiting and relatively uncommon,  and hepatitis D can only arise as a co-infection with the    hepatitis B virus, so it is hepatitis viruses B and C (HBV  and HCV respectively) that are the most problematic and  which require antiviral therapy.   It has been estimated that approximately one third  of the world’s population are infected with HBV and  just over one tenth of that number with HCV. The two  infections have several features in common, although  the viruses are not of the same family. In both cases  the disease may be acute or chronic and in the latter  case there may be progression to liver damage and  a higher risk of contracting liver cancer. Acute HBV cases  do not normally receive antiviral drugs, and that  was formerly the case for HCV also, but recent evidence  suggests that early treatment of HCV has a higher  success rate and shorter treatment time than that  required for chronic disease, so this practice is now more  common.   HBV is unusual in that it is not a retrovirus like HIV  but it does use reverse transcriptase in its replication  process. For this reason two of the drugs used to treat  HIV infection, lamivudine and tenofovir, are also effec-  tive for HBV; in addition, adefovirdipivoxil, entecavir and  telbivudine are also used, typically for 6 months or more  for all five drugs. In patients with decompensated liver  disease (liver cirrhosis with fluid accumulation in the  abdomen) lamivudine or adefovir are recommended.  Adefovir is effective in lamivudine-resistant HBV infec-  tion, but telbivudine should not be used because cross-  resistance may arise. Entecavir is effective in patients not  previously treated with nucleoside analogues, but resist-  ance can occur in patients who have received lamivudine.  Again, all of these antivirals are available as relatively  expensive, oral products.   There are several genotypes of HCV which exhibit  different drug sensitivities, so the genotype should be  determined before drug selection. Interferon- a is used  together with ribovirin for the treatment of chronic  infection by both HCV and HBV. Interferons are low  molecular weight proteins produced by virus-infected  cells that themselves induce the formation of a second  protein inhibiting the transcription of viral rnRNA.  Interferon-a needs to be injected on a daily basis or at  least three times weekly, but in its polyethylene glycol-  derivatized form (peginterferon-a2a) it is much longer  acting and requires less frequent dosing. The combina-  tion of ribavirin and interferon-a is less effective against  HCV than the combination of peginterferon-a2a and  ribavirin. Peginterferon-a2a alone should be used if riba-  virin is contraindicated or not tolerated, but ribavirin  alone is ineffective.   There are three related influenza viruses, A, B and C, of  which C is relatively rare and causes only mild infections.  All three reproduce in the same way and possess the  enzyme neuraminidase which is responsible for liberat-  ing the newly formed virus particles from the host cell.  There are two neuraminidase inhibitors available, osel-  tamivir which is formulated as an ethyl ester prodrug for  oral administration, and zanamivir which is administered  by inhalation. Neither drug has activity against other  viruses.   Both oseltamivir and zanamivir are most effective if  taken within 48 hours of the onset of symptoms, when  they may reduce the duration of the symptoms by about  1—1.5 days; they may also reduce the risk of complications  in elderly patients or those with chronic diseases. They  are useful too for postexposure prophylaxis of influenza,  but again need to be administered within a few hours of  exposure. Mutant strains of influenza viruses have devel-  oped resistance to oseltamivir but currently these are  uncommon, and no such resistance has so far been  observed with zanamivir.   Amantadine is a drug that inhibits an ion-channel  protein in influenza A but not influenza B virus (which  does not possess the target molecule). Again, it is effective  if given soon after the onset of symptoms, but resistance  has now become widespread so the drug, although still  available, is no longer recommended in the UK.   Respiratory syncytial virus (RSV) is a commonly  occurring virus related to those causing measles and  mumps. It infects most infants by the age of 2 years, but  unfortunately there is no long-lasting immunity follow-  ing infection and no vaccine available. Ribavirin (Section  13.3) is one of the few antiviral agents used in the treat-  ment of RSV, but its value is very much in doubt.  Palivizumab is a monoclonal antibody used for prevent-  ing serious lower respiratory tract disease caused by RSV  in children at high risk of the disease.   British National Formulary.: British Medical Association & The  Pharmaceutical Press, London. (The chapter on drugs used in  the treatment of infections is a particularly useful section.  New editions of the BNF appear at regular intervals, and it is  available online at http://bnf.org/bnf/.)    Bryskier, M.D. (ed.) (2005) Antimicrobial Agents: Antibacterials  and Antifungals. ASM Press, Washington, DC.   Cunha, B.A. (2009) Antibiotics Essentials, 8th edn. Jones 8c  Bartlett, Sudbury, MA.   Finch, R.G., Greenwood, D., Norrby S.R. 8c Whitley R. (2003)  Antibiotic and Chemotherapy, 8th edn. Churchill Livingstone,  London.    Finch, R.G., Greenwood, D., Davey P. 8c Wilcox M. (2007)  Antimicrobial Chemotherapy, 8th edn. Oxford University Press  Oxford.   Walsh, C. (2003) Antibiotics: Actions, Origins, Resistance .: ASM  Press, Washington, DC.    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Hodges, Sean P. Gorman,  Brendan F. Gilmore.   The antibiotics and synthetic anti-infective agents  described in Chapter 11 are used to treat infections  caused by bacteria, fungi and protozoa. Most exert a  highly selective toxic action on their target microbial cells  but have little or no toxicity towards mammalian cells.  They can therefore be administered at concentrations  sufficient to kill or inhibit the growth of infecting organ-  isms without damaging mammalian cells. By compari-  son, the disinfectants, antiseptics and preservatives  described in Chapter 17 are too toxic for systemic treat-  ment of infections. Figure 12.1 illustrates the five broad  target areas of the major groups of antibiotics and syn-  thetic agents used to treat microbial infections. Note that  most of the agents are selective antibacterials; relatively  few agents are available for treatment of fungal or proto-  zoal infections. Study of the mechanism of action reveals  the basis for the selective toxicity.   Peptidoglycan is a vital component of the cell wall of  virtually all bacteria. About 50% of the weight of the wall  of Gram-positive bacteria is peptidoglycan; smaller  amounts occur in mycobacterial walls (30%) and Gram-  negative bacterial cell walls (10-20%). It is a macromol-    ecule composed of sugar (glycan) chains cross-linked by  short peptide chains (Figure 12.2). The glycan chains  contain alternating units of N-acetylmuramic acid and  N-acetylglucosamine. Each N-acetylmuramic acid con-  tains a short peptide substituent made up of four amino  acids (the stem peptides). A key feature of peptidoglycan  is the occurrence of the D-isomers of some amino  acids in the stem peptides (particularly D-alanine and  D-glutamic acid) and unusual amino acids such as meso-  diaminopimelic acid which are not found in proteins. In  some organisms (e.g. Escherichia coli) cross-linking of the  stem peptides involves a direct peptide bond between the  fourth amino acid of the stem peptide on one chain and  the third amino acid in the stem peptide on an adjacent  chain. In other organisms (e.g. Staphylococcus aureus) the  linkage is made by a short peptide bridge (e.g. five gly-  cines) between the stem peptides. The precise composi-  tion of peptidoglycan varies between different organisms  but the overall structure is the same.   In all cases the peptidoglycan plays a vital role: it is  responsible for maintaining the shape and mechanical  strength of the bacterial cell. If it is damaged in any way,  and particularly if its synthesis is inhibited, then the  shape of the cells becomes distorted, they swell and will  eventually burst (lyse) as a result of the high internal  osmotic pressure. Mammalian cells do not possess a cell  wall and contain no other macromolecules resembling  peptidoglycan. Consequently, antibiotics which interfere  with the synthesis and assembly of peptidoglycan show  excellent selective toxicity.    Figure 12.1 Schematic diagram of a  typical bacterial cell showing the sites of  action of the major classes of antibiotics  and antimicrobial agents used to treat  infections. Agents listed without brackets  are used to treat bacterial infections.  Agents used against mycobacterial,  fungal or protozoal infections are  indicated by the use of ,  and )}  brackets, respectively.    Figure 12.2 Pathway for the biosynthesis of peptidoglycan in bacterial cells showing the sites of action of cycloserine, glycopeptide  and p-lactam antibiotics.   D-Cycloserine interferes with the early stage of synthesis  of peptidoglycan involving the assembly of the dipeptide  D-alanyl-D-alanine. This occurs inside the cytoplasm and  involves a racemase enzyme which converts L-alanine to  D-alanine and a ligase which couples two D-alanines  together (Figure 12.2). Both of these enzymes are inhib-  ited by D-cycloserine, which bears some structural simi-  larities to D-alanine. The antibiotic binds to the pyridoxal  phosphate cofactor of the enzymes, effectively preventing  them from forming D-alanyl-D-alanine. Subsequent  stages of peptidoglycan synthesis, involving coupling of  the dipeptide to three other amino acids forming the  stem peptide on UDP-N-acetylmuramic acid, are blocked.  Note that initially the peptide contains five amino acids,  terminating in D-alanyl-D-alanine. The terminal D-alanine  is removed on insertion into the cell wall during the final  step in which the cross-links are formed between stem  peptides on adjacent glycan strands.   The peptidoglycan macromolecule is assembled in the  cell wall by the sequential action of two enzymes (trans-  glycosylases and transpeptidases) which are located on  the outer face of the cytoplasmic membrane. To reach the  assembly site (i.e. a region of cell wall growth) the precur-  sors, which are assembled in the cytoplasm, must cross  the cell membrane. They do this linked to a lipid, undeca-  prenylphosphate, which acts as a carrier molecule, cycling  between the inner and outer faces of the membrane. The  biochemical details of this process are outlined in Figure  12.2. Antibiotics interfering with this stage of peptidog-  lycan synthesis have been identified, e.g. bacitracin, but  they have not found major applications in the treatment  of infections.   The glycopeptides vancomycin and teicoplanin act  at the stage where the peptidoglycan precursors are  inserted into the cell wall by the transglycosylase enzyme  on the outer face of the cell membrane. This enzyme  assembles linear glycan chains that are not initially  cross-linked to the existing peptidoglycan in the cell wall.  The linear glycan chains are assembled by the transglyco-  sylase by transfer of the growing glycan chain to the  disaccharide peptidoglycan precursor on the lipid carrier  as it crosses the cell membrane. Glycopeptides block  this process by binding, not to the enzyme itself, but to  the disaccharide peptidoglycan precursor, specifically to  the D-alanyl-D-alanine portion on the stem peptide. The  presence of the bulky glycopeptides tightly bound to each  D-alanyl-D-alanine residue prevents the transglycosylase    from carrying out the transfer reaction. Binding involves  formation of a network of five hydrogen bonds between  amino acid residues on the glycopeptide antibiotics and  D-alanyl-D-alanine. Resistance to this unusual mecha-  nism of enzyme inhibition can result from alteration in  the D-alanyl-D-alanine substrate to D-alanyl-D-lactate,  which occurs in glycopeptide (e.g. vancomycin) -resistant  enterococci (VRE). Vancomycin does not penetrate the  cell membrane of bacteria and is thought to bind to the  disaccharide-pentapeptides on the outer face of the cyto-  plasmic membrane. It has been suggested that two vanco-  mycin molecules form a back-to-back dimer which  bridges between pentapeptides on separate glycan chains,  thus preventing further peptidoglycan assembly.  Teicoplanin also binds tightly to the D-alanyl-D-alanine  region of the peptidoglycan precursor. Flowever, as a  lipoglycopeptide it may act slightly differently from van-  comycin, by locating itself in the outer face of the cyto-  plasmic membrane and binding the pentapeptide as the  precursors are transferred through the membrane.  Glycopeptides must cross the cell wall to reach the outer  face of the cell membrane where transglycosylation takes  place. They are too large to penetrate the outer membrane  of most Gram-negative bacteria and are consequently  used for treatment of serious Gram-positive infections.   The final stage of peptidoglycan assembly is the cross-  linking of the linear glycan strands assembled by transg-  lycosylation to the existing peptidoglycan in the cell wall.  This reaction is catalysed by transpeptidase enzymes,  which are also located on the outer face of the cell mem-  brane. They first remove the terminal D-alanine residue  from each stem peptide on the newly synthesized glycan  chain. The energy released from breaking the peptide  bond between the two alanines is used in the formation  of a new peptide bond between the remaining D-alanine  on the stem peptide and a free amino group present on  the third amino acid of the stem peptides in the existing  cross-linked peptidoglycan. In many organisms, includ-  ing E. coli, this acceptor amino group is supplied by the  amino acid diaminopimelic acid. In other organisms, e.g.  Staph, aureus, the acceptor amino group is supplied by  the amino acid L-lysine. Although there is considerable  variation in the composition of the peptide cross-link  among different species of bacteria, the essential  transpeptidation mechanism is the same. Therefore, vir-  tually all bacteria can be inhibited by interference with  this group of enzymes.    The P-lactam antibiotics inhibit transpeptidases by  acting as alternative substrates. They mimic the D-alanyl-  D-alanine residues and react covalently with the trans-  peptidases (Figure 12.3). The P-lactam bond (common  to all members of the P-lactam antibiotics) is broken but  the remaining portion of the antibiotic is not released  immediately. The half-life for the transpeptidase-  antibiotic complex is of the order of 10 minutes; during  this time the enzyme cannot participate in further rounds    of peptidoglycan assembly by reaction with its true sub-  strate. The vital cross-linking of the peptidoglycan is  therefore blocked while other aspects of cell growth con-  tinue. The cells become deformed in shape and eventually  burst through the combined action of a weakened cell  wall, high internal osmotic pressure and the uncontrolled  activity of autolytic enzymes in the cell wall. Penicillins,  cephalosporins, carbapenems and monobactams all  inhibit peptidoglycan cross-linking through interaction   Figure 12.3 The action of transpeptidase (TPase) with its natural substrate (upper) and penicillin G (lower). The -OH group on a  serine residue at the active site of the TPase attacks the peptide bond between the terminal D-alanyl-D-alanine residues of the stem  peptide in the non-cross-linked glycan chain. The terminal D-alanine is released and a new peptide cross-link is formed with  existing peptidoglycan in the cell wall. Penicillin G blocks this process by forming a stable penicilloyl-TPase complex which cannot  release the penicillin and therefore cannot form the peptide cross-link in the peptidoglycan.    of the common (3-lactam ring with the transpeptidase  enzymes. However, there is considerable variation in the  morphological effects of different (3-lactams owing to  the existence of several types of transpeptidase. The  transpeptidase enzymes are usually referred to as  penicillin-binding proteins (PBPs) because they can be  separated and studied after reaction with 14 C-labelled  penicillin. This step is necessary because there are very  few copies of each enzyme present in a cell. They are  usually separated according to their size by electrophore-  sis and are numbered PBP1, PBP2, etc., starting from the  highest molecular weight species. In Gram-negative bac-  teria most of the high molecular weight transpeptidases  also possess transglycosylase activity, i.e. they have a dual  function in the final stages of peptidoglycan synthesis  with the transglycosylase and transpeptidase activities  located in separate regions of the protein structures.  Furthermore, the different transpeptidases have special-  ized functions in the cell; all cross-link peptidoglycan but  some are involved with maintenance of cell integrity,  some regulate cell shape and others produce new cross  wall between elongating cells, securing chromosome seg-  regation prior to cell division. The varying sensitivity of  the PBPs towards different (3-lactams helps to explain  the range of morphological effects observed in treated  bacteria. For example, penicillin G (benzylpenicillin),  ampicillin and cephaloridine are particularly effective  in causing rapid lysis of Gram-negative bacteria such  as E. coli. These antibiotics act primarily uponPBPlB, the  major transpeptidase of the organism. Other (3-lactams  have little activity against this PBP, e.g. mecillinam binds  preferentially to PBP2 and it produces a pronounced  change in the cells from a rod shape to an oval form.  Many of the cephalosporins, e.g. cephalexin, cefotaxime  and ceftazidime, bind to PBP3 resulting in the formation  of elongated, filamentous cells. The lower molecular  weight PBPs, 4, 5 and 6, do not possess transpeptidase  activity. These are carboxypeptidases, which remove the  terminal D-alanine from the pentapeptides on the linear  glycans in the cell wall but do not catalyse the cross-  linkage. Their role in the cells is to regulate the degree  of cross-linking by denying the D-alanyl-D-alanine sub-  strate to the transpeptidases but they are not essential for  cell growth. Up to 90% of the amount of antibiotic react-  ing with the cells may be consumed in inhibiting the  carboxypeptidases, with no lethal consequences to the  cells.   Gram-positive bacteria also have multiple transpepti-  dases, but fewer than Gram-negatives. Shape changes    are less evident than with Gram-negative rod-shaped  organisms. Cell death follows lysis of the cells mediated  by the action of endogenous autolytic enzymes  (autolysins) present in the cell wall which are activated  following (3-lactam action. Autolytic enzymes able to  hydrolyse peptidoglycan are present in most bacterial  walls; they are needed to re-shape the wall during  growth and to aid cell separation during division. Their  activity is regulated by binding to wall components such  as the wall and membrane teichoic acids. When peptidog-  lycan assembly is disrupted through (3-lactam action,  some of the teichoic acids are released from the cells,  which are then susceptible to attack by their own  autolysins.   Expression of (3-lactamase enzymes is the most impor-  tant mechanism through which organisms become resist-  ant to (3-lactams. Over 300 different (3-lactamase enzymes  have been described and they can be classified either by  amino acid sequence or by their biochemical properties.  The majority of the enzymes have a serine residue at their  active site and bear structural and mechanistic similari-  ties to the carboxypeptidases from which they are thought  to have evolved. Unlike the transpeptidases and carbox-  ypeptidases, the (3-lactamases hydrolyse (3-lactam antibi-  otics very efficiently, releasing fragments of the antibiotics  rapidly instead of remaining bound to the ring-opened  forms for several minutes. A number of successful inhibi-  tors, including clavulanic acid, sulbactam and tazobactam  have been developed for use in combination with suscep-  tible (3-lactams (amoxicillin, ampicillin and piperacillin,  respectively), protecting them from inactivation by the  (3-lactamases. The inhibitors are hydrolysed by the 13-  lactamases in the same manner as susceptible (3-lactam  antibiotics, the (3-lactam ring being broken by attack by  a serine residue in the active site of the enzyme. Instead  of undergoing rapid release from the active site serine,  the inhibitors remain bound and undergo one of several  different fates. It is thought that the hydrolysed inhibitors  can interact with a second enzyme residue in the active  site of the (3-lactamase, forming a covalently cross-linked,  irreversibly inhibited complex. Other categories of 13-  lactamase enzymes have zinc atoms at their active sites  and hydrolyse the (3-lactam ring by a different mecha-  nism to the serine-based enzymes. These metallo-(3-  lactamases are not inhibited by clavulanic acid, sulbactam  and tazobactam.   The cell walls of mycobacteria contain an arabinoga-  lactan polysaccharide in addition to the peptidoglycan,  plus a variety of high molecular weight lipids, including  the mycolic acids, glycolipids, phospholipids and waxes.  The lipid-rich nature of the mycobacterial wall is respon-  sible for the characteristic acid-fastness on staining  and serves as a penetration barrier to many antibiotics.  Isoniazid and ethambutol have long been known as spe-  cific antimycobacterial agents, exerting no activity  towards other bacteria, but their mechanisms of action  have only recently been established.   Isoniazid interferes with mycolic acid synthesis by inhib-  iting an enoyl reductase (InhA) which forms part of the  fatty acid synthase system in mycobacteria. Mycolic acids  are produced by a diversion of the normal fatty acid  synthetic pathway in which short-chain (16-carbon)  and long-chain (24-carbon) fatty acids are produced by  addition of 7 or 11 malonate extension units from  malonyl coenzyme A to acetyl coenzyme A. InhA inserts  a double bond into the extending fatty acid chain at the  24-carbon stage. The long-chain fatty acids are further  extended and condensed to produce the 60-90-carbon  P-hydroxymycolic acids which are important compo-  nents of the mycobacterial cell wall. Isoniazid is converted  inside the mycobacteria to a free radical species by a cata-  lase peroxidase enzyme, KatG. The active free radicals  then attack and inhibit the enoyl reductase, InhA, by  covalent attachment to the active site.   Ethambutol blocks assembly of the arabinogalactan  polysaccharide by inhibition of an arabinotransferase  enzyme. Cells treated with ethambutol accumulate the  isoprenoid intermediate decaprenylarabinose, which  supplies arabinose units for assembly in the arabinoga-  lactan polymer.   These members of the echinocandin group of antifungal  agents have been developed for treatment of serious  fungal infections. They interfere with the synthesis of the  (3-1,3-D-glucan polymer in the fungal cell wall. Without  the glucan polymer, the integrity of the fungal cell wall is  compromised, yeast cells lose their rigidity and become  like protoplasts; the effect is especially pronounced in  Candida and Aspergillus species.   Figure 12.4 outlines the process of protein synthesis  involving the ribosome, mRNA, a series of amino-acyl  transfer RNA (tRNA) molecules (at least one for each  amino acid) and accessory protein factors involved in  initiation, elongation and termination. As the process is  essentially the same in prokaryotic (bacterial) and  eukaryotic cells (i.e. higher organisms and mammalian  cells) it is surprising that there are so many selective  agents which act in this area (see Figure 12.1).   Bacterial ribosomes are smaller than their mammalian  counterparts. They consist of one 30S and one 50S  subunit (the S suffix denotes the size, which is derived  from the rate of sedimentation in an ultracentrifuge).  The 30S subunit comprises a single strand of 16S rRNA  and over 20 different proteins that are bound to it. The  larger 50S subunit contains two single strands of rRNA  (23S and 5S) together with over 30 different proteins. The  subunits pack together to form an intact 70S ribosome.  The equivalent subunits for mammalian ribosomes are  40S and 60S, making an 80S ribosome. Some agents  exploit subtle differences in structure between the bacte-  rial and mammalian ribosomes. The macrolides, azalides  and chloramphenicol act on the 50S subunits in bacteria  but not the 60S subunits of mammalian cells. By contrast,  the tetracyclines derive their selective action through  active uptake and concentration within microbial cells  but only limited penetration of mammalian cells.   Most of the information on the mechanisms of action of  aminoglycoside-aminocyclitol (AGAC) antibiotics comes  from studies with streptomycin. One effect of AGACs is  to interfere with the initiation and assembly of the bacte-  rial ribosome (Figure 12.4). During assembly of the ini-  tiation complex, N-formylmethionyl-tRNA (fmet-tRNA)  binds initially to the ribosome binding site on the  untranslated 5' end of the mRNA together with the 30S  ribosomal subunit. Three protein initiation factors (des-  ignated IF- 1, 2 and 3) and a molecule of guanosine tri-  phosphate (GTP) are involved in positioning the  fmet-tRNA on the AUG start codon of mRNA. IF-1 and  IF-3 are then released from the complex, GTP is hydro-  lysed to guanosine diphosphate (GDP) and released with  IF-2 as the 50S subunit joins the 30S subunit and mRNA    Figure 12.4 Outline of the process of protein synthesis (translation of messenger RNA) in bacterial cells. The four stages of  synthesis are shown: initiation, elongation, translocation and termination with the sites of action of antibiotics. AUG is the start  codon on messenger RNA (mRNA) specifying the first amino acid in bacterial proteins, N-formylmethionine. UAG, UAA and  UGA are termination codons specifying no amino acid. 30S and 50S are the subunits of the ribosome. Other protein factors  involved in protein synthesis are initiation factors (IF- 1, 2, 3), elongation factors (EF-Tu and EF-G) and release factors (RF-1, 2, 3).    to form a functional ribosome. The fmet-tRNA occupies  the peptidyl site (P site) leaving a vacant acceptor site (A  site) to receive the next aminoacyl-tRNA specified by the  next codon on the mRNA. Streptomycin binds tightly to  one of the protein components of the 30S subunit.  Binding of the antibiotic to the protein, which is the  receptor for IF-3, prevents initiation and assembly of the  ribosome.   Streptomycin binding to the 30S subunit also distorts  the shape of the A site on the ribosome and interferes  with the positioning of the aminoacyl-tRNA molecules  during peptide chain elongation. Streptomycin therefore  exerts two effects: inhibition of protein synthesis by freez-  ing the initiation complex, and misreading of the codons  through distortion of the 30S subunit. Simple blockage  of protein synthesis would be bacteriostatic rather than  bactericidal. As streptomycin and the other AGACs exert  a potent lethal action, it seems that the formation of toxic,  non-functional proteins through misreading of the  codons on mRNA is a more likely mechanism of action.  This can be demonstrated with cell-free translation  systems in which isolated bacterial ribosomes are sup-  plied with artificial mRNA template such as poly(U) or  poly(C) and all the other factors, including aminoacyl-  tRNAs needed for protein synthesis.   In the absence of an AGAC the ribosomes will produce  artificial polypeptides, polyphenylalanine (as specified by  the codon UUU) or polyproline (as specified by the  codon CCC). However, when streptomycin is added, the  ribosomes produce a mixture of polythreonine (codon  ACU) and polyserine (codon UCU). The misreading of  the codons does not appear to be random: U is read as A  or C, and C is read as A or U. If such misreading occurs  in whole cells, the accumulation of non-functional or  toxic proteins would eventually prove fatal to the cells.  There is some evidence that the bacterial cell membrane  is damaged when the cells attempt to excrete the faulty  proteins.   The effectiveness of the AGACs is enhanced by their  active uptake by bacteria, which proceeds in three phases.  First, a rapid uptake occurs within a few seconds of  contact, which represents binding of the positively  charged AGAC molecules to the negatively charged  surface of the bacteria. This phase is referred to as the  energy-independent phase (EIP) of uptake. In the case of  Gram-negative bacteria the AGACs damage the outer  membrane causing release of some lipopolysaccharide,  phospholipid and proteins but this is not directly lethal  to the cells. Second, there follows an energy-dependent  phase of uptake (EDP I) lasting about 10 minutes, in    which the AGAC is actively transported across the cyto-  plasmic membrane. A second energy-dependent phase  (EDP II) which leads to further intracellular accumula-  tion follows after some AGAC has bound to the ribos-  omes in the cytoplasm. Although the precise details of  uptake by EDP I and EDP II are not clear, both require  organisms to be growing aerobically. Anaerobes do not  take up AGACs by EDP I or EDP II and are consequently  resistant to their action.   This group of antibiotics is actively transported into bac-  terial cells, possibly as the magnesium complex, achieving  a 50-fold concentration inside the cells. Mammalian cells  do not actively take up the tetracyclines (small amounts  enter by diffusion alone) and it is this difference in uptake  that determines the selective toxicity. Resistance to the  tetracyclines occurs through failure of the active uptake  system or the action of active efflux pumps, which remove  the drug from the cells before it can interfere with ribos-  ome function. Other resistance mechanisms involve  ribosomal protection and modification. Protein synthesis  by both bacterial and mammalian ribosomes is inhibited  by the tetracyclines in cell-free systems. The action is on  the smaller subunit. Binding of just one molecule of tet-  racycline to the bacterial 30S subunit occurs at a site  involving the 3' end of the 16S rRNA, a number of associ-  ated ribosomal proteins and magnesium ions. The effect  is to block the binding of aminoacyl-tRNA to the A site  of the ribosome and halt protein synthesis. Tetracyclines  are bacteriostatic rather than bactericidal, consequently  they should not be used in combination with (3-lactams,  which require cells to be growing and dividing to exert  their lethal action.   Of the four possible optical isomers of chloramphenicol,  only the d -threo form is active. This antibiotic selectively  inhibits protein synthesis in bacterial ribosomes by  binding to the 50S subunit in the region of the A site  involving the 23S rRNA. The normal binding of the  aminocyl-tRNA in the A site is affected by chlorampheni-  col in such a way that the peptidyl transferase cannot  form a new peptide bond with the growing peptide  chain on the tRNA in the P site. Studies with aminocyl-  tRNA fragments containing truncated tRNA chains  suggest that the shape of the region of tRNA closest to  the amino acid is distorted by chloramphenicol. The  altered orientation of this region of the aminoacyl-tRNA  in the A site is sufficient to prevent peptide bond forma-    tion. Chloramphenicol has a broad spectrum of activity,  which covers Gram-positive and Gram-negative bacteria,  mycoplasmas, rickettsia and chlamydia. It has the valua-  ble property of penetrating into mammalian cells and is  therefore the drug of choice for treatment of intracellular  pathogens, including Salmonella enterica serovar Typhi,  the causative organism of typhoid. Although it does not  inhibit 80S ribosomes, the 70S ribosomes of mammalian  mitochondria are sensitive and therefore some inhibition  occurs in rapidly growing mammalian cells with high  mitochondrial activity.   Erythromycin is a member of the macrolide group of  antibiotics; it selectively inhibits protein synthesis in a  broad range of bacteria by binding to the 50S subunit.  The site at which it binds is close to that of chlorampheni-  col and involves the 23S rRNA. Resistance to chloram-  phenicol and erythromycin can occur by methylation of  different bases within the same region of the 23S rRNA.  The sites are therefore not identical, but binding of one  antibiotic prevents binding of the other. Unlike chloram-  phenicol, erythromycin blocks translocation. This is the  process by which the ribosome moves along the mRNA  by one codon after the peptidyl transferase reaction has  joined the peptide chain to the aminoacyl-tRNA in the A  site. The peptidyl-tRNA is moved (translocated) to the P  site, vacating the A site for the next aminocyl-tRNA.  Energy is derived by hydrolysis of GTP to GDP by an  associated protein elongation factor, EF-G. By blocking  the translocation process, erythromycin causes release of  incomplete polypeptides from the ribosome. It is assumed  that the azalides, such as azithromycin, have a similar  action to the macrolides. The azalides have improved  intracellular penetration over the macrolides and are  resistant to the metabolic conversion which reduces the  serum half-life of erythromycin.   This agent binds selectively to a region of the 50S ribos-  omal subunit close to that of chloramphenicol and eryth-  romycin. It blocks elongation of the peptide chain by  inhibition of peptidyl transferase.   The two unrelated streptogramins, quinupristin and dal-  fopristin, have been used in combination (in a 30:70  ratio) to treat infections caused by staphylococci and  enterococci, particularly methicillin-resistant Staph.    aureus (MRSA) and VRE. Their action is synergistic,  and is generally bactericidal compared with either  agent used alone or compared with antibiotics in the  macrolide group. The main target is the bacterial 50S  ribosome, with the formulation acting to inhibit protein  synthesis. The agents bind sequentially to the 50S subunit;  dalfopristin alters the shape of the subunit so that more  quinupristin can bind. Dalfopristin blocks an early  step in protein synthesis by forming a bond with the  ribosome, preventing elongation of the peptide chain by  the peptidyl transferase. Quinupristin blocks a later  step by preventing the extension of peptide chains and  causing incomplete chains to be released. The overall  effect is to block elongation. Use of streptogramins is  limited by vasculitis, causing pain on intravenous  administration.   Oxazolidinones such as linezolid act at the early stage of  protein synthesis, preventing the formation of the initia-  tion complex between the 30S subunit, mRNA and  fmet-tRNA.   The target of mupirocin is one of a group of enzymes  which couple amino acids to their respective tRNAs for  delivery to the ribosome and incorporation into protein.  The particular enzyme inhibited by mupirocin is involved  in producing isoleucyl-tRNA. The basis for the inhibition  is a structural similarity between one end of the mupi-  rocin molecule and isoleucine. Protein synthesis is halted  when the ribosome encounters the isoleucine codon  through depletion of the pool of isoleucyl-tRNA.   This steroidal antibiotic does not act on the ribosome  itself, but on one of the associated elongation factors,  EF-G. This factor supplies energy for translocation by  hydrolysis of GTP and GDP. Another elongation factor,  EF-Tu, promotes binding of aminoacyl-tRNA molecules  to the A site through binding and hydrolysis of GTP. Both  EF-G and EF-Tu have overlapping binding sites on the  ribosome. Fusidic acid binds the EF-G : GDP complex to  the ribosome after one round of translocation has taken  place. This prevents further incorporation of aminoacyl-  tRNA by blocking the binding of EF-Tu: GTP. Fusidic  acid owes its selective antimicrobial action to active  uptake by bacteria and exclusion from mammalian cells.  The equivalent elongation factor in mammalian cells,  EF-2, is susceptible to fusidic acid in cell-free systems.   These agents bind to the 23S rRNA component of the 50S  bacterial ribosome and block peptide bond formation by  interfering with the binding of the peptidyl transferase  region with the aminoacyl-tRNA substrates in the A and  P sites on the ribosome. This mechanism is different to  that of other peptidyl transferase inhibitors (chloram-  phenicol and clindamycin) so cross-resistance to these  agents does not occur.   4.1 Basis for the selective inhibition of  chromosome replication and function   As with protein synthesis, the mechanisms of chromo-  some replication and function are essentially the same in  prokaryotes and eukaryotes. There are, however, impor-  tant differences in the detailed functioning and proper-  ties of the enzymes involved and these differences are  exploited by a number of agents as the basis of selective  inhibition. The microbial chromosome is large in com-  parison with the cell that contains it (approximately 500  times the length of E. coli). It is therefore wound into a  compact, supercoiled form inside the cell. During replica-  tion the circular double helix must be unwound to allow  the DNA polymerase enzymes to synthesize new comple-  mentary strands. The shape of the chromosome is  manipulated by the cell by the formation of regions of  supercoiling. Positive supercoiling (coiling in the same  sense as the turns of the double helix) makes the chromo-  some more compact. Negative supercoiling (generated by  twisting the chromosome in the opposite sense to the  helix) produces localized strand separation which is  required both for replication and transcription. In a bac-  terium such as E. coli four different topoisomerase  enzymes are responsible for maintaining the shape of  DNA during cell division. They act by cutting one or both  of the DNA strands; they remove and generate supercoil-  ing, then reseal the strands. Their activity is essential for  the microbial cell to relieve the complex tangling of the  chromosome (both knotting and chain link formation)  which results from progression of the replication fork  around the circular chromosome. Type I topoisomerases  cut one strand of DNA and pass the other strand through  the gap before resealing. Type II enzymes cut both strands  and pass another double helical section of the DNA  through the gap before resealing. In E. coli topoisomer-  ases I and III are both type I enzymes while topoisomer-  ases II and IV are type II enzymes. Topoisomerase II (also    known as DNA gyrase) and topoisomerase IV are essen-  tial enzymes which are inhibited by the fluoroquinolone  group of antimicrobials. Topoisomerase II is responsible  for introducing negative supercoils into DNA and for  relieving torsional stress, which accumulates ahead of  sites of transcription and replication. Topoiosomerase  IV provides a potent decatenating (unlinking) activity  that removes links and knots generated behind the repli-  cation fork.   The basic sequence of events for microbial chromo-  some replication is described below.   Purines, pyrimidines and their nucleosides and nucleo-  side triphosphates are synthesized in the cytoplasm. At  this stage the antifolate drugs (sulphonamides and dihy-  drofolate reductase inhibitors) act by interfering with the  synthesis and recycling of the cofactor dihydrofolic acid  (DHF). Thymidylic acid (2-deoxythymidine monophos-  phate, dTMP) is an essential nucleotide precursor of  DNA synthesis. It is produced by the enzyme thymidylate  synthetase by transfer of a methyl group from tetrahydro-  folic acid (THF) to the uracil base on uridylic acid  (2-deoxyuridine monophosphate, dUMP) (Figure 12.5).  THF is converted to DHF in this process and must be  reverted to THF by the enzyme dihydrofolate reductase  (DHFR) before the cycle can be repeated. By inhibiting  DHFR, the antifolates effectively block the production of  dTMP and hence DNA synthesis.   The antifungal agent 5-fluorocytosine also interferes  with these early stages of DNA synthesis. Through con-  version to the nucleoside triphosphate it subsequently  blocks thymidylic acid production through inhibition of  the enzyme thymidylate synthetase (Figure 12.6).   The antiviral nucleosides aciclovir and ganciclovir  are also converted to their respective nucleoside  triphosphates in the cytoplasm of infected cells. They  proceed to inhibit viral DNA replication either by inhibi-  tion of the DNA polymerase or by incorporation into  DNA with subsequent termination of chain extension.  Finally, the anti-HIV drug AZT acts in an analogous  manner, being converted to the corresponding triphos-  phate and inhibiting viral RNA synthesis by the HIV  reverse transcriptase.   As described in section 4.1, the DNA double helix must  unwind to allow access of the polymerase enzymes to  produce two new strands of DNA. This is facilitated by  topoisomerase II (DNA gyrase) which is the target of the    Figure 12.5 Conversion of uridylic acid to thymidylic acid by the enzyme thymidylate synthetase, a vital early stage in the synthesis  of DNA.    Figure 12.6 Conversion of the antifungal agent 5-fluorocytosine (5-FC) to 5-fluorouracil by a deaminase enzyme inside fungal  cells and subsequent inhibition of fungal DNA synthesis through inhibition of thymidylate synthetase.    fluoroquinolones. Some agents interfere with the unwind-  ing of the chromosome by physical obstruction. These  include the acridine dyes, of which the topical antiseptic  proflavine is the most familiar, and the antimalarial acri-  dine mepacrine. They prevent strand separation by inser-  tion (intercalation) between base pairs from each strand,  but exhibit very poor selective toxicity.   The unwound DNA strands are kept unfolded during  replication by binding a protein called Albert’s protein. A  series of enzymes produce new strands of DNA using  each of the separated strands as templates. One strand is  produced continuously. The other is produced in a series  of short strands called Okazaki fragments that are joined    by a DNA ligase. The entire process is carefully regulated,  with proofreading stages to check that each nucleotide is  correctly incorporated as specified by the template  sequence. There are no therapeutic agents yet known  which interfere directly with the DNA polymerases.   The process of transcription, the copying of a single  strand of mRNA sequence using one strand of the chro-  mosome as a template, is carried out by RNA polymerase.  This is a complex of four proteins (2 a, 1 (3 and 1 (3'  subunits) which make up the core enzyme. Another small  protein, the o factor, joins the core enzyme, which binds  to the promoter region of the DNA preceding the gene  that is to be transcribed. The correct positioning and    orientation of the polymerase is obtained by recognition  of specific marker sites on the DNA at positions —10 and  —35 nucleotide bases before the initiation site for tran-  scription. The O factor is responsible for recognition of  the initiation signal for transcription and the core enzyme  possesses the activity to join the nucleotides in the  sequence specified by the gene. Mammalian genes possess  an analogous RNA polymerase but there are sufficient  differences in structure to permit selective inhibition of  the microbial enzyme by the semisynthetic rifamycin  antibiotics rifampicin and rifabutin.   The fluoroquinolones selectively inhibit topoisomerases  II and IV, which are not found in mammalian cells. The  enzymes, both tetramers comprising two A and two B  subunits, are capable of catalysing a variety of changes in  DNA topology. The topoisomerases bind to the chromo-  some at points where two separate double-stranded  regions cross. This can be at a supercoiled region, a  knotted or a linked (catenane) region. The A subunits  (gyrA for topoisomerase II and parC for topoisomerase  IV) cut both DNA strands on one chain with a 4 base  pair stagger; the other chain is passed through the break  which is then resealed. The B subunits (gyrB for topoi-  somerase II and parE for topoisomerase IV) derive energy  for the reaction by hydrolysis of ATP. The precise details  of the interaction are not clear but it appears that the  fluoroquinolones do not simply eliminate enzyme  function, they actively poison the cells by trapping the  topoisomerases as drug-enzyme-DNA complexes in  which double-stranded DNA breaks are held together by  the enzyme protein alone. The enzymes are unable to  reseal the DNA, with the result that the chromosome in  treated cells becomes fragmented. The number of frag-  ments (approximately 100 per cell) is comparable to the  number of supercoils in the chromosome. The action of  the fluoroquinolones probably triggers secondary  responses in the cells which are responsible for death.  One notable morphological effect of fluoroquinolone  treatment of Gram-negative rod-shaped organisms is the  formation of filaments. In Gram-positive cocci topoi-  somerase IV may be the more important target for fluo-  roquinolone action.   These agents also cause DNA strand breakage, but by a  direct chemical action rather than by inhibition of a  topoisomerase. Metronidazole is active only against  anaerobic organisms. The nitro group of metronidazole    is converted to a nitronate radical by the low redox  potential within cells. The pyruvate:ferredoxin oxidore-  ductase (POR) is a major metabolic pathway in anaerobic  bacteria and protozoa used for generation of ATP.  This system converts metronidazole to its active  form which then attacks the DNA, producing strand  breakage. Another nitroimidazole, tinidazole, and the  nitrofuran nitrofurantoin are thought to act in a similar  manner.   Rifampicin acts on the (3 subunit of RNA polymerase.  Binding of just one molecule of rifampicin inhibits the  initiation stage of transcription in which the first nucle-  otide is incorporated in the RNA chain. Once started,  transcription itself is not inhibited. It has been suggested  that the structure of rifampicin resembles that of two  adenosine nucleotides in RNA; this may form the basis  of the binding of the antibiotic to the (3 subunit. One  problem is the rapid development of resistance in organ-  isms due to alterations in the amino acids comprising one  particular region of the (3 subunit. These changes do not  affect the activity of the polymerase but render it insensi-  tive to rifampicin. The action of rifampicin is specific for  the microbial RNA polymerase, the mammalian version  being unaffected. Rifabutin, which has enhanced activity  against Mycobacterium avium complex, is thought to act  in the same way as rifampicin.   This antifungal agent inhibits DNA synthesis at  the early stages involving production of the nucleotide  thymidylic acid (TMP). 5-Fluorocytosine (5-FC) is  converted by a deaminase inside fungi to 5-fluorouracil,  then to the corresponding nucleoside triphosphate,  5-fluorodeoxyuridine monophosphate (5-F-dUMP)  which then acts as an inhibitor of thymidylate synthetase  (Figure 12.6). This enzyme normally produces thymidylic  acid (TMP) from deoxyuridine monophosphate (dUMP)  by addition of a methyl group (supplied by a folate cofac-  tor, section 4.1.1) to the 5 position of the uracil ring. As  this position is blocked by the fluoro group in 5-FC, the  phosphate acts as an inhibitor of the enzyme. 5-FC can  be considered as a prodrug; it has the value of being taken  up by fungi as the nucleoside, whereas the active triphos-  phate produced inside the cells would not be taken up  because of its negative charge. Although 5-FC is an  important antifungal agent in the treatment of life-  threatening infections, resistance can occur due to active  efflux of the drug from the cells before it can inhibit DNA  synthesis.   Folic acid is an important cofactor in all living cells. In  the reduced form, tetrahydrofolate (THF), it functions  as a carrier of single-carbon fragments, which are used  in the synthesis of adenine, guanine, thymine and methio-  nine (Figure 12.7). One important folate-dependent  enzyme is thymidylate synthetase, which produces TMP  by transfer of the methyl group from THF to UMP. In  this and other folate-dependent reactions THF is con-  verted to dihydrofolic acid (DHF), which must be reduced  back to THF before it can participate again as a carbon  fragment carrier. The enzyme responsible for the reduc-  tion of DHF to THF is dihydrofolate reductase (DHFR)  which uses the nucleotide NADPH 2 as a cofactor. Bacteria,  protozoa and mammalian cells all possess DHFR but  there are sufficient differences in the enzyme structure for  inhibitors such as trimethoprim and pyrimethamine to  inhibit the bacterial and protozoal enzymes selectively  without damaging the mammalian form. In the case of  protozoa such as the Plasmodium species responsible for  malaria, the DHFR is a double enzyme which also con-  tains the thymidylate synthetase activity.   There is another fundamental difference between  folate utilization in microbial and mammalian cells    (Figure 12.7). Bacteria and protozoa are unable to take  up exogenous folate and must synthesize it themselves.  This is carried out in a series of reactions involving first  the synthesis of dihydropteroic acid from one molecule  each of pteridine and p-aminobenzoic acid (PABA).  Glutamic acid is then added to form DHF, which is  reduced by DHFR to THF. Mammalian cells do not make  their own DHF, instead they take it up from dietary nutri-  ents and convert it to THF using DHFR.   Sulphonamides (e.g. sulphamethoxazole and dapsone)  are structural analogues of PABA (Figure 12.8). They  competitively inhibit the incorporation of PABA into  dihydropteroic acid and there is some evidence for their  incorporation into false folate analogues, which inhibit  subsequent metabolism. The presence of excess PABA  will reverse the inhibitory action of sulphonamides, as  will thymine, adenine, guanine and methionine. However  these nutrients are not normally available at the site of  infections for which the sulphonamides are used.   Trimethoprim is a selective inhibitor of bacterial DHFR.  The bacterial enzyme is several thousand times more sen-  sitive than the mammalian enzyme. Pyrimethamine, like-  wise, is a selective inhibitor of plasmodial DHFR. Both    Figure 12.7 Pathways of folate metabolism and  use in microbial cells (upper) and mammalian  cells (lower). Bacterial and protozoal cells must  synthesize dihydrofolic acid (DHF) from  pteridine, p-aminobenzoic acid (PABA) and  glutamate. DHF is converted to tetrahydrofolic  acid (THF) by the enzyme dihydrofolate  reductase (DHFR). THF supplies single carbon  units for various pathways including DNA, RNA  and methionine synthesis. Mammalian cells do  not make DHF, it is supplied from the diet,  conversion to THF occurs via a DHFR enzyme as  in microbial cells.    Mammalian cells cannot make  DHF or THF, DHF is supplied in diet    Figure 12.8 Structural relationships  between dihydrofolate reductase  inhibitors (trimethoprim and  pyrimethamine), sulphonamides (e.g.  sulphamethoxazole, sulphadiazine and    are structural analogues of the dihydropteroic acid  portion of the DHF substrate (Figure 12.8). Crystal struc-  tures of the bacterial, plasmodial and mammalian  DFIFRs, each containing either bound substrate or the  inhibitors, have been determined by X-ray diffraction  studies. These show how inhibitors fit tightly into the  active site normally occupied by the DHF substrate,  forming a pattern of strong hydrogen bonds with amino  acid residues and water molecules lining the site. Another  DHFR is proguanil, a guanidine-containing prodrug  which is metabolized in the liver to cycloguanil, an  active selective inhibitor of plasmodial DHFR.  Methotrexate is a potent DHFR inhibitor that has an  analogous structure to the whole DHF molecule, includ-  ing the glutamate residue. It has no selectivity towards  microbial DHFR and therefore cannot be used to treat  infections; however, it is widely used as an anticancer  agent. A derivative of methotrexate that is used for  treatment of Pneumocystis jirovecii infections in AIDS  patients is trimetrexate. Although it is very toxic to mam-  malian cells, simultaneous administration of leucovorin  (formyl-THF or folinic acid) as an alternative source  of folate which cannot be taken up by the organism pro-  tects host tissues. DHFR inhibitors can be used in com-  bination with a sulphonamide to achieve a double  interference with folate metabolism. Suitable combina-  tions with matching pharmacokinetic properties are    sulphamethoxazole with trimethoprim (the antibacterial  co-trimoxazole) and dapsone with pyrimethamine (the  antimalarial Maloprim).   The integrity of the cytoplasmic membrane is vital for  the normal functioning of all cells. Bacterial membranes  do not contain sterols and in this respect differ from  membranes of fungi and mammalian cells. Fungal mem-  branes contain predominantly ergosterol as the sterol  component whereas mammalian cells contain choles-  terol. Gram-negative bacteria contain an additional  outer-membrane structure that provides a protective  penetration barrier to potentially harmful substances,  including many antibiotics. The outer membrane has an  unusual asymmetric structure in which phospholipids  occupy the inner face and the lipopolysaccharide (LPS)  occupies the outer face. The outer membrane is attached  to the peptidoglycan by proteins and lipoproteins. The  stability of all membranes is maintained by a combina-  tion of non-covalent interactions between the constitu-  ents involving ionic, hydrophobic and hydrogen bonding.  The balance of these interactions can be disturbed by the    intrusion of molecules (membrane-active agents) which  destroy the integrity of the membrane, thereby causing  leakage of cytoplasmic contents or impairment of meta-  bolic functions associated with the membrane. Most  membrane-active agents that function in this way, e.g. the  alcohols, quaternary ammonium compounds and bis-  biguanides (considered in Chapters 17 and 18) have very  poor selectivity. They cannot be used systemically because  of their damaging effects upon mammalian cells; instead  they are used as skin antiseptics, disinfectants and pre-  servatives. A few agents can be used therapeutically: the  polymyxins (colistin), which act principally upon the  outer membrane of Gram-negative bacteria, and the anti-  fungal polyenes, which act upon fungal membranes.  Other antifungal agents, the imidazoles, triazoles and ter-  binafine act by blocking the synthesis of ergosterol, the  major sterol present in fungal membranes (see also  Chapter 4).   Polymyxin E (colistin) is used in the treatment of serious  Gram-negative bacterial infections, particularly those  caused by Pseudomonas aeruginosa. It binds tightly to the  lipid A component of LPS in the outer membrane of  Gram-negative bacteria. The outer leaflet of the mem-  brane structure is distorted, segments of which are  released and the permeability barrier is destroyed. The  polymyxin molecules can then penetrate to the cytoplas-  mic membrane where they bind to phospholipids, disrupt  membrane integrity, and cause irreversible leakage of  cytoplasmic components. Their detergent-like properties  are a key feature of this membrane-damaging action,  which is similar to that of quaternary ammonium com-  pounds. With increasing resistance to the major groups  of antibiotics, some multiresistant organisms (e.g.  Acinetobacter species) remain sensitive only to membrane-  active agents such as colistin. However, some Gram-  negative bacteria produce LPS that does not bind  polymyxins (e.g. Bacteroides species and Burkholderia  cenocepacia) while resistance can occur in some normally  sensitive organisms such as E. coli and Pseudomonas aeru-  ginosa through modification of their LPS structure (e.g.  by addition of aminoarabinose or aminoethanol sub-  stituents to the lipid A regions of their LPS).   This negatively charged bactericidal cyclic lipopetide  binds to the surface of the Gram-positive bacterial cell  membrane. The binding is dependent on calcium ions.  The acyl tail portion of the compound inserts itself into    the cytoplasmic membrane and drug molecules aggregate  together forming channels. The leakage of potassium  ions from the cells results in inhibition of macromolecu-  lar synthesis and cell death.   Amphotericin B and nystatin are the most commonly  used members of this group of antifungal agents. They  derive their action from their strong affinity towards  sterols, particularly ergosterol. The hydrophobic polyene  region binds to the hydrophobic sterol ring system within  fungal membranes. In so doing, the hydroxylated portion  of the polyene is pulled into the membrane interior,  destabilizing the structure and causing leakage of cyto-  plasmic constituents. It is possible that polyene molecules  associate together in the membrane to form aqueous  channels. The pattern of leakage is progressive, with small  metal ions such as K + leaking first, followed by larger  amino acids and nucleotides. The internal pH of the cells  falls as K + ions are released, macromolecules are degraded  and the cells are killed. The selective antifungal activity  of the polyenes is poor, depending on the higher affinity  for ergosterol than cholesterol. Kidney damage is a major  problem when polyenes are used systemically to treat  severe fungal infections. The problem can be reduced, but  not eliminated by administration of amphotericin as a  lipid complex or liposome.   The azole antifungal drugs act by inhibiting the synthesis  of the sterol components of the fungal membrane (see  also Chapter 4). They are inhibitors of one step in the  complex pathway of ergosterol synthesis involving the  removal of a methyl group from lanosterol (Figure 12.9).  The 14-oc-demethylase enzyme responsible is dependent  on cytochrome P-450. The imidazoles and triazoles cause  rapid defects in fungal membrane integrity due to  reduced levels of ergosterol, with loss of cytoplasmic con-  stituents leading to similar effects to the polyenes. The  azoles are not entirely specific for fungal ergosterol syn-  thesis and have some action on mammalian sterol metab-  olism; for example, they reduce testosterone synthesis.   This synthetic antifungal agent inhibits the enzyme  squalene epoxidase at an early stage in fungal sterol bio-  synthesis. Acting as a structural analogue of squalene,  terbinafine causes the accumulation of this unsaturated  hydrocarbon, and a decrease in ergosterol in the fungal  cell membrane (Figure 12.9).   Figure 12.9 Pathway for synthesis of the essential fungal sterol ergosterol and the sites of inhibition by the antifungal agents  terbinafine, imidazoles and triazoles.   Dutton, C.J., Haxwell, M.A., McArthur, H.A.I. & Wax, R.G.  (2002) Peptide Antibiotics: Discovery, Modes of Action and  Applications, Marcel Dekker, New York.   Finch, R.G., Greenwood, D., Norrby, S.R., & Whitley, R.J. (2003)  Antibiotic and Chemotherapy: Anti- Infective Agents and their  Use in Chemotherapy, 8th edn. Churchill Livingstone,  Edinburgh.   Franklin, T.J. & Snow, G.A. (2005) Biochemistry and Molecular  Biology of Antimicrobial Drug Action, 6th edn. Springer, New  York.    Greenwood, D., Finch, R., Davey, P. & Wilcox, M. (2007)  Antimicrobial Chemotherapy, 5th edn. Oxford University  Press, Oxford.   Greenwood, D., Slack, R.C.B., Pleutherer, J.F. & Barer, M.R.  (2007) Medical Microbiology: A Guide to Microbial Infections:  Pathogenesis, Immunity, Laboratory Diagnosis and Control,  17th edn. Churchill Livingstone/Elsevier, London.   Walsh, C. (2003) Antibiotics: Actions, Origins, Resistance. ASM  Press, Washington, DC.   It is both a cliche and a truism to state that antibiotic  resistance has been around for as long as antibiotics  have been used to treat infection. Indeed, the origin of  antibiotic resistance extends much further back in evolu-  tionary terms and reflects the attack and counter-attack  of complex microbial flora in order to establish ecological  niches and survive. It is true to say that early treatment  failures with antibiotics did not represent a significant  clinical problem because other classes of agents, with  different cellular targets, were available. It is the emer-  gence of multiple resistance, i.e. resistance to several types    of antibiotic agent, that is causing major problems in the  clinic today. Several factors drove this situation in the  1970s and 1980s, including the introduction of extended-  spectrum agents and advances in medical techniques,  such as organ transplantation and cancer chemotherapy.  The net result has been a huge selective pressure in  favour of multiply resistant species. Coupled with this,  there has been a sharp decline in the introduction of  agents acting on new cellular targets over the last 30 years  compared with the 20-year period following the Second  World War. There are a number of resistant organisms  causing concern at present. Notable Gram-positive  organisms include meticillin-resistant Staphylococcus  aureus (MRSA) and coagulase-negative staphylococci,    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    glycopeptide-intermediate sensitivity Staphylococcus  aureus (GISA), vancomycin-resistant Enterococcus  (VRE) species and penicillin-resistant Streptococcus pneu-  moniae. Concerns among the Gram-negative organisms  include multidrug-resistant Pseudomonas aeruginosa,  Stenotrophomonas maltophilia and Acinetobacter bau-  mannii and members of the Enterobacteriaceae with  extended-spectrum (3-lactamases. Multidrug resistance in  the acid-fast bacilli Mycobacterium tuberculosis and M.  avium complex pose major health threats worldwide.   Some bacteria are said to have innate resistance against  antibiotics and this typically reflects variations in the  structure of their cell envelope. These will be identified  in subsequent sections on resistance mechanisms.  Resistance or reduced susceptibility may also be pheno-  typic, resulting from adaptation to growth within a spe-  cific environment. A characteristic of such phenotypic  resistance is reversion to antibiotic susceptibility upon  subculture in conventional laboratory media and failure  to isolate genotypic resistant mutants (section 17). The  origins of antibiotic resistance genes are unclear;  however, studies using clinical isolates collected before  the introduction of antibiotics demonstrated susceptibil-  ity, although conjugative plasmids (section 16.1) were  present. Resistance can be achieved by horizontal acquisi-  tion of resistance genes, mobilized via insertion sequences,  transposons and conjugative plasmids, by recombination  of foreign DNA into the chromosome, or by mutations  in different chromosomal loci. Given that it is only  60 years since the introduction of antibiotics, mutation  of common ancestral genes could not be the only resist-  ance mechanism. Many resistance genes will have derived  from the diverse gene pool present in environmental  microorganisms, most likely produced as protective  mechanisms by antibiotic-producing organisms. Genetic  exchange is likely to arise in soil and the general environ-  ment as well as in the gut of humans and animals. Rapid  mutation can occur and there is clearly a heavy selective  pressure resulting from the overuse of antibiotics in  medical practice. Agricultural and veterinary use of  antibiotics also makes an important and unhelpful con-  tribution. The mutation process is not a static event  and a complex network of factors influences the rate and  type of mutants that can be selected under antibiotic  selective pressure. Antibiotic concentration, physiological  conditions such as nutrient availability and stress    can each regulate mutation rates. The structure of a  gene is relevant to mutability. Size is not the main factor,  as not every mutation in a gene that encodes an antibiotic  target leads to resistance. Resistance only occurs by  mutations which are both permissive (i.e. not lethal or  leading to an unacceptable reduction in ‘fitness’ or  ability to cause infection) and able to produce a resistance  phenotype. The probability that such a mutation  arises will be proportional to the number of target  sites within the gene. In Escherichia coli, mutations in  the gyrA gene, encoding the GyrA subunit of topoi-  somerase II and leading to fluoroquinolone resistance  (section 8) have been identified in at least seven locations,  whereas mutational changes in only three positions in the  parC gene, encoding a subunit of topoisomerase IV,  have been observed. As a consequence, the prediction  that the mutation rate would be higher in gyrA than  parC is correct. Such observations and predictions  cannot be extrapolated to other organisms. Indeed, the  opposite is true for fluoroquinolone resistance in Strep,  pneumoniae.   Resistance to antimicrobial agents typically occurs by one  or more of the following mechanisms:   In this chapter, resistance will be examined by agent,  but attention will be drawn to mechanisms which can  permit resistance to multiple, chemically different agents.  This is most commonly associated with efflux and this  will be described in section 16.4.   (3-Lactam antibiotics act by inhibiting the carboxy/  transpeptidase or penicillin-binding proteins (PBPs)  involved in the late stages of peptidoglycan biosynthesis.  Although introduced nearly 60 years ago, (3-lactam anti-  biotics still represent the most widely used class of agents  in the clinic today. Resistance to many (3-lactam agents is  common and is most often caused by (3-lactamases or by  mutation in the PBPs resulting in reduced affinity.  Reduced uptake and efflux are also seen, but they are less  significant.   A number of different (3-lactamases have been described,  but all share the feature of catalysing the ring-opening of  the (3-lactam moiety (Figure 13.1). Thus, the structural  homology with the terminal D-Ala-D-Ala of maturing  peptidoglycan, shared by all (3-lactam antibiotics, is lost.   (3-Lactamases may be chromosomal or plasmid-borne,  inducible or constitutive, and for this reason their termi-  nology can be confusing. A number of classification  systems have been proposed, including classes A-D based  on peptide sequence. Classes A, C and D have a serine at  the active site, whereas class B enzymes have four zinc  atoms at their active site and these are also called metallo-  (3-lactamases. Class A enzymes are highly active against  benzylpenicillin; class B (3-lactamases are effective against  penicillins and cephalosporins. Class C enzymes are  usually inducible, but mutation can lead to overexpres-    sion. Class D consists of the OXA-type enzymes, which  can hydrolyse oxacillin. Increasing resistance to (3-lactam  agents, mainly by (3-lactamase, prompted the discovery  and introduction of agents with greater (3-lactam stability  such as cephalosporins, carbapenems and monobactams.  Resistance first appeared in organisms such as Enterobacter  cloacae and Pseudomonas aeruginosa, due to mutations  causing overproduction of the class C chromosomal  AmpC (3-lactamase. Subsequently, in the late 1980s,  resistance occurred in organisms such as Klebsiella pneu-  moniae and E. coli that lack an inducible AmpC enzyme.  Resistance was found to be mediated by plasmids encod-  ing extended-spectrum (3-lactamases (ESBLs). These  arose from mutational development of more limited-  spectrum (3-lactamases such as TEM and SHV that either  increased the size of the active-site pocket or altered its  binding characteristics to allow the larger cephalosporins  to enter and be broken down. TEM derivatives predomi-  nate, possibly favoured by the use of ceftazidime and  other slowly penetrating cephalosporins. These muta-  tions also increase the binding of clavulanic acid and so  these ESBLs remain susceptible to inhibition by this and  other (3-lactamase inhibitors such as sulbactam and tazo-  bactam, which are generally ineffective against class C  (3-lactamases.   Continuing use of the third-generation cephalosporins  and the introduction of (3-lactamase inhibitor combina-  tions (clavulanate with amoxycillin or ticarcillin, sul-  bactam with ampicillin, and tazobactam with piperacillin;  see section 4.2) resulted in the appearance of plasmids  encoding class C (3-lactamases. After several unconfirmed  reports, the first proof that a class C (3-lactamase had  been captured on a plasmid came in 1990 when transmis-  sible resistance to a-methoxy and oxyimino-(3-lactams  was shown to be mediated by an enzyme whose gene  was 90% identical to the ampC gene of Ent. cloacae. They  have subsequently been found worldwide. Strains with  plasmid-mediated AmpC enzymes are typically resistant  to aminopenicillins (ampicillin or amoxycillin), carbox-  ypenicillins (carbenicillin or ticarcillin) and ureidopeni-  cillins (piperacillin). The enzymes also provide resistance  to the oxyimino cephalosporins (ceftazidime, cefotaxime,  ceftriaxone) and the 7-oc-methoxy group (cefoxitin,  cefmetazole and moxalactam) as well as the monobactam  aztreonam.   In December 2009, the first report of a carbapene-  mase (3-lactamase, referred to as New Delhi metallo-(3  -lactamase (NDM-1), was recorded. It was discovered in  a carbapenem-resistant K. pneumoniae strain isolated  in Sweden from a Swedish national who acquired the    infection in India. The enzyme is one of the class of B  metallo-(3-lactamases and is conferred by the gene Wandm-  This is considered a serious threat to the carbapenem  family of antibiotics.   In addition to introducing agents with increased stability  to p-lactamase inhibition, P-lactamase inhibitors includ-  ing clavulanic acid, sulbactam and tazobactam have been  developed (Figure 13.2). Clavulanic acid is produced by  a streptomyces and is a suicide inhibitor of P-lactamases  from a number of Gram-negative and Gram-positive  organisms. These P-lactamase inhibitors do not have any  significant antimicrobial activity against bacterial  transpeptidases, but their combination with a P-lactam  antibiotic (see above) has extended the clinical usefulness  of the latter.   Altered PBPs are responsible for reduced sensitivity to  P-lactam agents by Strep, pneumoniae (PBPla, PBP2b  and PBP2x) and Haemophilus influenzae (PBP3a and  PBP3b), but by far the most clinically significant example    is MRSA. By the early 1950s, the acquisition and spread  of plasmid-encoded P-lactamases had blunted the effec-  tiveness of penicillin for treating Staph, aureus infections  such as boils, carbuncles, pneumonia, endocarditis and  osteomyelitis. The P-lactamase-stable agent meticillin  was introduced in 1959, but by 1960, meticillin-resistant  strains were identified. This was the result of Staph,  aureus acquiring the mecA gene, which encodes an altered  PBP gene, PBP2a. The mecA gene is chromosomal and  expression is either constitutive or inducible, but not by  meticillin. PBP2a has low affinity for most P-lactam  antibiotics.   Vancomycin and teicoplanin are the two glycopeptides  used clinically. They bind the terminal D-alanyl-D-alanine  side chains of peptidoglycan and prevent cross-linking in  a number of Gram-positive organisms. They are not  active against Gram-negative organisms because of the  presence of the outer membrane. Vancomycin use  increased dramatically in response to the increasing inci-  dence of MRSA and resistance was first reported in the  enterococci in 1988. VRE now account for more than  20% of all enterococcal infections. Resistance is greatest  amongst Ent. faecium strains, but significant numbers of  the more clinically significant Ent. faecalis are also resist-  ant. Five types of resistance, VanA-VanE, have now been  reported. Phenotypic VanA resistance is the most  common and confers high-level resistance to vancomycin  and teicoplanin. VanA resistance is mediated by a seven-  gene cluster on the transposable genetic element Tnl546  (Figure 13.3).   Resistance to vancomycin is via a sensor histidine  kinase (VanS) and a response regulator (VanR). VanFl  encodes a D-lactate dehydrogenase/a-keto acid reductase  and generates D-lactate, which is the substrate for VanA,  a D-Ala-D-Lac ligase. The result is cell wall precursors  terminating in D-Ala-D-Lac to which vancomycin binds  with very low affinity. This change in affinity is mediated  by one hydrogen bond. The complex formed between  vancomycin and D-Ala-D-Ala is stabilized by five hydro-    gen bonds, whereas only four hydrogen bonds can form  between vancomycin and D-Ala-D-Lac and the complex  is unstable (Figure 13.4). Further, VanX encodes a D-Ala-  D-Ala dipeptidase which can modify endogenous d-  Ala-D-Ala precursors. Recent genetic analysis has  identified close homology between this cluster and  genes present in the vancomycin-producing organism  Amycolatopsis orientalis, suggesting that selective pressure  has forced genes originally present to protect antibiotic-  producing organisms to jump to other species. VanB  resistance is also acquired and the peptidoglycan precur-  sor is again D-Ala-D-Lac, but isolates often remain sus-  ceptible to teicoplanin. VanC resistance is intrinsic and  chromosomally encoded in some enterococcal species  such as Ent. gallinarum and the peptidoglycan precursor  is D-Ala-D-Ser. Less is known of VanD and VanE resist-  ance, but both are acquired. VanD uses D-Ala-D-Lac and  VanE uses D-Ala-D-Ser.   There is major concern that high-level, VanA-type resist-  ance could transfer to staphylococci, particularly MRSA.  Experimental transfer of the enterococcal VanA system to  Staph, aureus on the skin of mice has been reported, but  other mechanisms resulting in intermediate-level resist-  ance occur in clinical isolates. In the 1960s and 1970s  MRSA was not feared because several other treatment  options existed, including use of tetracyclines, macrolides  and aminoglycosides. But multiple resistance was accu-  mulating and by the 1980s empirical therapy of staphy-  lococcal infections, particularly nosocomial sepsis, was  changed to the glycopeptide antibiotic vancomycin.  MRSA levels were rising and the early 1990s saw a major  increase in vancomycin use. The inevitable consequence  of the selective pressure was the isolation in 1997 of the  first Staph, aureus strain with reduced susceptibility to  vancomycin and teicoplanin (vancomycin MIC = 8pg/  ml). At the beginning of the 21st century, MRSA is  responsible for up to 25% of nosocomial infections in the  USA and reports of community-acquired MRSA infec-  tions are increasing. While reports of ‘superbugs’ resistant  to all known antibiotics abound, it is important to dis-  tinguish between reduced susceptibility and resistance,  recognizing that there are conflicting definitions of resist-  ance and resistance breakpoints. Strains with MIC values  <4pg/ml are considered sensitive, 8-16pg/ml intermedi-  ate and >32pg/ml resistant. Thus the acronyms VISA  (vancomycin-intermediate Staph, aureus) and GISA  (glycopeptide-insensitive Staph, aureus) are used to    denote strains with vancomycin or teicoplanin MICs of  8pg/ml, whereas VRSA (vancomyin-resistant Staph,  aureus) is reserved for strains with MIC values >32 pg/ml.  The mechanism of glycopeptide resistance is poorly  understood, but strains show longer doubling times and  decreased susceptibility to lysostaphin. Increased quanti-  ties of PBP2 and PBP2' and cell wall precursors are pre-  sumed to trap vancomycin, while amidation of glutamine  residues in cell wall muropeptides reduces the cross-  linking and consequently the number of vancomycin  target molecules.   The aminoglycosides are hydrophilic sugars possessing a  number of amino and hydroxy substituents. The amine  groups are protonated at biological pH and it is the poly-  cationic nature of the molecules that affords them  their affinity for nucleic acids, particularly the acceptor  (A) site of 16S ribosomal RNA. Aminoglycoside binding  to the A site interferes with the accurate recognition of  cognate tRNA by rRNA during translation and may also  perturb translocation of the tRNA from the A site to the  peptidyl-tRNA site (P site). While high-level resistance in  aminoglycoside-producing microorganisms is by meth-  ylation of the rRNA, this is not the mechanism of resist-  ance in previously susceptible strains. The most common  mechanism for clinical aminoglycoside resistance is their  structural modification by enzymes expressed in resistant  organisms, which compromises their ability to interact  with rRNA. There are three classes of these enzymes:  aminoglycoside phosphatases (APHs), aminoglycoside  nucleotidyltransferases (ANTs) and aminoglycoside  acetyltransferases (AACs). Within each class, there are  enzymes with differing specificities around the sugars.  There are four ANTs (ANT(6), ANT(4'), ANT(3") and  ANT(2")), seven APHs (APH(3'), APH(2"), APH(3"),  APH(6), APH(9), APH(4) and APH(7")) and four AACs  (AAC(2'), AAC(6'), AAC(l) and AAC(3)). There is also a  bifunctional enzyme, AAC(6 , )-AAC(2"). Aminogly-  cosides are typically susceptible to attack by multiple  enzymes (Figure 13.5). Attempts to circumvent these  modifying enzymes have centred on structural modifica-  tions. Examples include tobramycin which lacks the  3'-hydroxyl group and is thus not a substrate for APH(3'),  and amikacin which has an acylated N- 1 group and is not  a substrate for several modifying enzymes. Other strate-  gies are exemplified by experimental compounds such   Figure 13.4 Mechanism of high-level vancomycin resistance. Hydrogen bonds are denoted by dotted lines. The key hydrogen bond  present in the stable complex with D-Ala-D-Ala, but missing in the unstable complex with D-Ala-D-Lac, is shown in bold type.   Figure 13.5 Structure of kanamycin B showing system of ring  numbering and sites of action of some aminoglycoside-  modifying enzymes. AAC, aminoglycoside acetyltransferase;  ANT, aminoglycoside nucleotidyltransferase; APH,  aminoglycoside phosphotransferase.    as 3'-oxo-kanamycin. This molecule is a substrate for  APH (3'), but the phosphorylation product is unstable  and regenerates the original antibiotic.   Chlortetracycline and oxytetracycline were discovered  in the late 1940s and studies of representative popula-  tions before their widespread use suggests that emergence  of resistance is a relatively modern event. More than  60% of Shigella flexneri isolates are resistant to tetracy-  cline; resistant isolates of Salmonella enterica serovar  Typhimurium are becoming more common and among  Gram-positive species, approximately 90% of MRSA  strains and 60% of multiply resistant Strep, pneumoniae  are now tetracycline-resistant. The major mechanisms of  resistance are efflux and ribosomal protection. One  exception is the tet(X) gene that encodes an enzyme  which modifies and inactivates the tetracycline molecule,  although this does not appear to be clinically significant.  The Tet efflux proteins belong to the major facilitator  superfamily (MFS). These proteins exchange a proton for  a tetracycline-cation (usually Mg 2+ ) complex, reducing  the intracellular drug concentration and protecting the  target ribosomes in the cell. In Gram-negative bacteria,  the efflux determinants comprise divergently oriented  efflux and repressor proteins that share overlapping pro-  moter and operator regions. In the absence of a  tetracycline-Mg 2+ complex, the repressor protein binds  and blocks transcription of both genes. Drug binding  alters the conformation of the repressor so that it can no  longer bind the DNA operator region and block tran-    scription. This method of regulation probably applies to  all of the Gram-negative efflux systems including fef(A),  fef(C), fef(D), fef(E), tef(G) and tef(H).   No repressor proteins have been identified in the  Gram-positive fef(K) or fef(L) genes and regulation of  plasmid-borne tetracycline resistance appears to be by  translational attenuation, involving stem-loop mRNA  structures and tetracycline-induced unmasking of the  ribosome binding site permitting translation of the efflux  protein. Regulation of chromosomal fef(L) expression  involves tetracycline-promoted stalling of the ribosomes  during translation of early codons of the leader peptide,  which allows reinitiation of translation at the ribosome  binding site for the structural gene. Ribosomal protection  is mediated by cytoplasmic proteins that inhibit tetracy-  cline and also confer resistance to doxycycline and  minocycline. These proteins share homology with the  elongation factors EF-Tu and EF-G, and expression of  Tet(M) and Tet(O) proteins appears to be regulated. A  400-bp region upstream from the coding region for  tet( O) is needed for full expression, but the mechanism(s)  has not been characterized. The widespread emergence  of efflux- and ribosome protection-based resistance to  first- and second-generation tetracyclines has prompted  the development of the 9-glycinyltetracyclines (9-  glycylcyclines). 9-Amino-acylamido derivatives of mino-  cycline have similar activity to earlier compounds;  however, when the acyl group is modified to include  an N,N-dialkylamine or 9-f-butyl-glycylamido moiety  (Figure 13.6), antimicrobial activity is retained and the  compounds are active against strains containing tet  genes responsible for resistance by efflux and ribosomal  protection.   Fluoroquinolones bind and inhibit two bacterial topoi-  somerase enzymes: DNA gyrase (topoisomerase II) which  is required for DNA supercoiling, and topoisomerase IV  which is required for strand separation during cell divi-  sion. DNA gyrase tends to be the major target in Gram-  negative bacteria, whereas both topoisomerases are  inhibited in Gram-positive bacteria. Each topoisomerase  is termed a heterotetramer, being composed of two copies  of two different subunits designated A and B. The A and  B subunits of DNA gyrases are encoded by gyrA and gyrB,  respectively, whilst topoisomerase IV is encoded by parC  and parE ( grlA and grlB in Staph, aureus). Mutations in    Figure 13.6 Structural modifications at the 9 position of the  tetracycline antibiotic minocycline conferring increased  stability against resistance mechanisms.    gyrA, particularly involving substitution of a hydroxyl  group with a bulky hydrophobic group, induce confor-  mational changes such that the fluoroquinolone can no  longer bind. Mutations have also been detected in the B  subunit, but these are probably less important. Alterations  involving Ser80 and Glu84 of Staph, aureus grlA and  Ser79 and Asp83 of Strep, pneumoniae parC have led to  quinolone resistance. Like GyrB, mutations in ParE  leading to resistance are not common. While changes in  GyrA and ParC give resistance to the older fluoroqui-  nolones, MIC values do not always rise above clinically  defined breakpoints for newer agents such as gemi-  floxacin and moxifloxacin.   Topoisomerases are located in the cytoplasm and thus  fluoroquinolones must cross the cell envelope to reach  their target. Changes in outer-membrane permeability  have been associated with resistance in Gram-negative  bacteria, but permeability does not appear to be an issue  with Gram-positive species. Efflux, however, does make a  contribution to resistance, mainly low level, in both  Gram-positive and Gram-negative bacteria. The NorA-  mediated efflux system in Staph, aureus was characterized    in 1990. It is expressed weakly in wild-type strains and  resistance is thought to occur via mutations leading to  increased expression of nor A. NorA is a member of the  MFS and homologues are also present in Streptococcus  pneumoniae and Bacillus sp. There is a tendency for it to  be more effective for hydrophilic fluoroquinolones, but  there is no strict correlation. Fluoroquinolones are now  being used for treating M. avium and multidrug-resistant  M. tuberculosis and efflux-mediated resistance has been  identified. A number of efflux pumps have been identi-  fied among Gram-negative bacteria, including AcrA in E.  coli, which is regulated in part by the multiple-antibiotic  resistance (Mar) operon (section 16.3).   Although chemically distinct, members of the macrolide,  lincosamide and streptogramin (MLS) group of antibiot-  ics all inhibit bacterial protein synthesis by binding to a  target site on the ribosome. Gram-negative bacteria are  intrinsically resistant due to the permeability barrier of  the outer membrane, and three resistance mechanisms  have been described in Gram-positive bacteria. Target  modification, involving adenine methylation of domain  V of the 23S ribosomal RNA, is the most common mech-  anism. The adenine-N 6 -methyltransferase, encoded by  the erm gene, results in resistance to erythromycin and  other macrolides (including the azalides), as well as the  lincosamides and group B streptogramins. Streptogramin  A-type antibiotics are unaffected and streptogramin A/B  combinations remain effective. Expression of the erm  gene may be constitutive or inducible. When expression  is inducible, resistance is seen only against 14- and  15-membered macrolides; lincosamide and strepto-  gramin antibiotics remain active. Telithromycin (Figure  13.7), the first of a new class of ketolide agents in the MLS  family, does not induce MLS resistance and also retains  activity against domain V-modified ribosomes and inhi-  bition of protein synthesis through strong interaction  with domain II. The second resistance mechanism is  efflux. Expression of the mef gene confers resistance to  macrolides only, whereas msr expression results in resist-  ance to macrolides and streptogramins. Efflux-mediated  resistance of Staph, aureus to streptogramin A antibiotics  is also conferred by vga and vgaB gene products. A third  resistance mechanism, involving ribosomal mutation, has  been reported in a small number of clinical isolates of  Strep, pneumoniae.    Figure 13.7 Structure of telithromycin, a ketolide macrolide  antibiotic that retains activity against 23S domain V modified  ribosomal RNA.   Chloramphenicol inhibits protein synthesis by binding  the 50S ribosomal subunit and preventing the peptidyl-  transferase step. Decreased outer-membrane permeabil-  ity and active efflux have been identified in Gram-negative  bacteria; however, the major resistance mechanism is  drug inactivation by chloramphenicol acetyltransferase.  This occurs in both Gram-positive and Gram-negative  species, but the cat genes, typically found on plasmids,  share little homology.    negative bacteria and as such linezolid has been approved  for treating various Gram-positive infections, including  MRSA. Resistance is appearing, although rare at present.  Mutation in the central loop of domain V of the compo-  nent 23S rRNA subunit appears to be the main mecha-  nism, including a G2576T mutation in three isolates of  linezolid-resistant MRSA.   Trimethoprim competitively inhibits dihydrofolate  reductase (DHFR) and resistance can be caused by over-  production of host DHFR, mutation in the structural  gene for DHFR and acquisition of the dfr gene encoding  a resistant form. There are at least 15 DHFR enzyme types  based on sequence homology and acquisition of dfr genes  encoding alternative DHFR of type I, II or V is the most  common mechanism of trimethoprim resistance among  the Enterobacteriaceae.   Nasal carriage of MRSA strains has been identified as an  important target for infection control protocols aimed at  reducing spread and acquisition. Mupirocin (pseudo-  monic acid A) is an effective topical antimicrobial used  in MRSA eradication. It is an analogue of isoleucine that  competitively binds isoleucyl-tRNA synthetase (IRS) and  inhibits protein synthesis. Low-level resistance (MIC  4-256 pg/ml) is usually due to mutation of the host IRS,  whereas high-level resistance (MIC >512pg/ml) is due to  acquisition of a distinct IRS that is less sensitive to inhibi-  tion. The mupA gene, typically carried on transferable  plasmids, is found in Staph, aureus and coagulase-  negative staphylococci, and encodes an IRS with only  30% homology to the mupirocin-sensitive form.   Linezolid is the first of a new class of oxazolidinone anti-  microbials with a novel target in protein synthesis.  Linezolid does not interfere with translation initiation at  the stage of mRNA binding or formation of 30S preinitia-  tion complexes; rather, it involves binding the 50S rRNA.  Its affinity for 50S rRNA from Gram-positive bacteria is  twice that for the corresponding molecule in Gram-   Many peptide antibiotics have been described and can be  broadly classified as non-ribosomally synthesized pep-  tides; they include the polymyxins, bacitracins and  gramicidins as well as the glycopeptides (section 5)  and the ribosomally synthesized peptides such as the  antimicrobial peptides of the innate immune system.  Polymyxins and other cationic antimicrobial peptides    have a self-promoted uptake across the cell envelope and  perturb the cytoplasmic membrane barrier. Addition of  a 4-amino-4-deoxy-L-arabinose (L-Ara4N) moiety to the  phosphate groups on the lipid A component of Gram-  negative lipopolysaccharide has been implicated in resist-  ance to polymyxin. Details of the pathway for L-Ara4N  biosynthesis from UDP glucuronic acid, encoded by the  pmr operon, are emerging.   The nature of mycobacterial infections, particularly  tuberculosis, means that chemotherapy differs from  other infections. Organisms tend to grow slowly (long  generation time) in a near dormant state with little meta-  bolic activity. Hence, a number of the conventional anti-  microbial targets are not suitable. Isoniazid is bactericidal,  reducing the count of aerobically growing organisms.  Pyrazinamide is active only at low pH, making it well  suited to killing organisms within necrotic foci early in  infection, but less useful later on when these foci have  reduced in number. Rifampicin targets slow-growing  organisms. Resistance mechanisms have now been  described and multiple resistance poses a serious threat  to health. Current treatment regimens result in a high  cure rate and the combination of agents makes it highly  unlikely that there will be a spontaneous resistant isolate  to all the components. Problems most commonly occur  in patients who receive inadequate therapy, which pro-  vides a serious selection advantage. Resistance can occur  to single agents and subsequently to multiple agents.  Resistance to rifampicin arises from mutation in the (3  subunit of RNA polymerase encoded by rpoB and resist-  ant isolates show decreased growth rates. Modification of  the catalase gene katG results in resistance to isoniazid,  mainly by reduced or absent catalase activity. Catalase  activity is absolutely required to convert isoniazid to the  active hydrazine derivative. Interestingly, animal model  studies suggest that M. tuberculosis strains in which the  katG gene is inactivated are attenuated compared with  wild-type strains. Low-level rifampicin resistance can be  obtained by point mutations in inhA leading to its over-  expression. Pyrazinamide is a prodrug requiring pyrazi-  namidase to produce the active pyrazinoic acid. Most  cases of resistance are due to mutations in the pyrazi-  namidase gene ( pncA ), but gene inactivation by the inser-  tion sequence IS6110 has been reported. Streptomycin  resistance can arise through mutations in rrs and rpsL    which affect streptomycin binding. However, these  account for only half of the resistant isolates, so further  resistance mechanisms await definition. Ethambutol  resistance has been noted in M. tuberculosis and other  species such as M. smegmatis. Ethambutol inhibits the  polymerization of arabinan in the arabinogalactan  and lipoarabinomannan of the mycobacterial cell wall  and one of its likely targets is the family of arabinosyl-  transferases encoded by the emb locus. Missense muta-  tions in the embB gene in this locus confer resistance to  ethambutol.   Several issues of multiple drug resistance have already  been raised in this chapter. Notable examples are MRSA,  which can harbour both small cryptic plasmids and  larger plasmids encoding resistance to antiseptics, disin-  fectants, trimethoprim, penicillin, gentamicin, tobramy-  cin and kanamycin, and multidrug-resistant M.  tuberculosis. Of equal concern are instances where isolates  can become resistant to multiple, chemically distinct  agents in a single biological event. One of the earliest  examples was in Japan in 1959. Previously sensitive E. coli  became resistant to multiple antibiotics through acquisi-  tion of a conjugative plasmid (R-factor) from resistant  Salmonella and Shigella isolates. A number of R-factors  have now been characterized including RP4, encoding  resistance to ampicillin, kanamycin, tetracycline and neo-  mycin, found in Ps. aeruginosa and other Gram-negative  bacteria; Rl, encoding resistance to ampicillin, kanamy-  cin, sulphonamides, chloramphenicol and streptomycin,  found in Gram-negative bacteria and pSH6, encoding  resistance to gentamicin, trimethoprim and kanamycin,  found in Staph, aureus.   Many Gram-negative resistance genes are located in gene  cassettes. One or more of these cassettes can be integrated  into a specific position on the chromosome termed an  integron. More than 60 cassettes have been identified,  each comprising only a promotor- less single gene (usually  antibiotic resistance) and a 59-base element forming a  specific recombination site. This recombination site  confers mobility because it is recognized by specific  recombinases encoded by integrons that catalyse integra-  tion of the cassette into a specific site within the integron.  Thus, integrons are genetic elements that recognize and    capture multiple mobile gene cassettes. As the gene typi-  cally lacks a promoter, expression is dependent on correct  orientation into the integron to supply the upstream pro-  moter. Four classes of integron have been identified,  although only one member of class 3 has been described  and class 4 integrons are limited to Vibrio cholerae.  Analysis of the resistant Shigella strains isolated in Japan  has shown that some of the conjugative plasmids included  an integron with one or two integrated cassettes.   The multiple-antibiotic resistance {mar) locus was first  described in E. coli by Stuart Levy and colleagues at  Tufts University and has since been recognized in other  enteric bacteria. The locus consists of two divergently  transcribed units, marC and marRAB. Little is known of  marC and marB; however, marR encodes a repressor of  the operon, and marA encodes a transcriptional activator  affecting expression of more than 60 genes. Increased  expression of the MarRAB operon resulting from muta-  tions in marO or marR, or from inactivation of MarR  following exposure to inducing agents such as salicylate,  leads to the Mar phenotype. This phenotype is character-  ized by resistance to structurally unrelated antibiotics,  organic solvents, oxidative stress and chemical disinfect-  ants. A number of effector mechanisms have been identi-  fied, including increased expression of the acrAB-tolC  multidrug efflux system (section 16.4) and the soxRS  regulon.   Whereas some efflux pumps excrete only one drug or  class of drugs, a multidrug efflux pump can excrete a wide  range of compounds where there is often little or no  chemical similarity between the substrates. One common  characteristic may be agents with a significant hydropho-  bic domain. For this reason, hydrophilic compounds  such as the aminoglycoside antibiotics are not exported  by these systems. A distinction needs to be drawn between  those efflux systems, typically in Gram-positive bacteria,  that pump their substrate across the cytoplasmic mem-  brane, such as the QacA and Smr pumps which both  export quaternary ammonium compounds and basic  dyes, and those which efflux across the cytoplasmic and  outer membranes of Gram-negative bacteria. There are  some examples of single membrane systems in Gram-  negative bacteria, such as the EmrE protein in E. coli,  but they are not of great clinical significance. The major-  ity of Gram-negative pumps span both membranes and    Figure 13.8 Schematic diagram of the MexAB-OprM efflux  pump from Pseudomonas aeruginosa. CM, cytoplasmic  membrane; OM, outer membrane. (Kindly supplied by  K. Poole.)    include the AcrAB-TolC system in E. coli and the MexAB-  OprM system in Ps. aeruginosa. Genomic analyses are  revealing numerous homologues. Using the MexAB-  OprM system as the prototypic example (Figure 13.8),  MexA is the linker protein and MexB is in the cytoplasmic  membrane. MexB is a resistance-nodulation-division  (RND) family member and is predicted to be a proton  antiporter with 12 membrane-spanning a-helices. OprM  shows homology with outer-membrane channels of  systems thought to export such diverse molecules as  nodulation signals and alkaline proteases. Mutations in  regulatory genes such as nalB cause overexpression of  MexAB-OprM and consequently multidrug resistance.  MexB is a proton antiporter and efflux by this and other  members of the RND family is energized by the proton  motive force. This contrasts with mammalian multi-  drug efflux pumps (MDR) that are powered by ATP  hydrolysis.   The resistance mechanisms described in this chapter  typically lead to an increase in MIC value, although it  should be remembered that this does not always equate  to clinical failure. If the MIC value remains below the  breakpoint value, which can itself be difficult to deter-  mine, then the antibiotic will remain effective in the    clinic. But such arguments assume that MIC values,  which are typically determined when the isolate is  growing in complex, sensitivity-test broth, equate with  the sensitivity of the organism when growing in the  many subtly different environments encountered during  infection in vivo. Unfortunately, the antibiotic literature  contains numerous examples of treatment failures  despite apparent sensitivity in the test tube and this resist-  ance is referred to as being phenotypic. In other words,  resistance is a consequence of the adaptation of the  organism to grow and survive within the in vivo environ-  ment, and subculture into conventional laboratory  growth medium rarely shows the existence of resistant  mutants. There are several key factors at play here, par-  ticularly slow/no growth, nutrient depletion and mode of  growth. There are numerous papers showing a tendency  for nutrient depletion, i.e. restricted or non-availability  of an important nutrient, and slow/no growth to be asso-  ciated with reduced susceptibility to antibiotics and  biocides.   There is now increasing concern over the role played  by microbial biofilms in infection (see Chapter 8). These  include the well-known examples of medical device-  related infections, such as those associated with artificial  joints, prosthetic heart valves and catheters. Many chronic  infections, not related to medical devices, are also due to  bacteria either not growing and relatively dormant or  growing slowly as biomasses or adherent biofilms on  mucosal surfaces. A bacterial biofilm is typically defined  as a population of cells growing as a consortium on a  surface and enclosed in a complex exopolymer matrix.  Commonly in the wider environment but less so in infec-  tions, the population is mixed and also of heterogeneous  physiologies. Growth as a biofilm almost always leads to  a large increase in resistance to antimicrobial agents,  including antibiotics, biocides and preservatives, com-  pared with cultures grown in suspension (planktonic) in  conventional liquid media, but there is no generally  agreed mechanism to account for this resistance. Although  there is general acceptance that there are numerous  planktonic phenotypes, many papers refer to ‘the biofilm  phenotype’, implicitly assuming (wrongly) that there is  only one. Those same parameters known to influence  planktonic physiology and antibiotic susceptibility,  including growth rate and/or specific nutrient limitation,  also apply to biofilm physiology and antibiotic suscepti-  bility. The general resistance of biofilms is clearly pheno-  typic. The well-characterized resistance mechanisms  described above — lack of antibiotic penetration, inacti-  vation, efflux and repair — make contributions in some    circumstances. However, compelling evidence that they  are uniquely responsible for biofilm resistance is  lacking. Reduced growth rate probably has an involve-  ment, particularly in that it is associated with responses  to stress. During stress responses, key structures are pro-  tected and cellular processes close down to a state of  dormancy, and it has been proposed that exceptional veg-  etative cell dormancy is the basic explanation of biofilm  resistance.   It has been said that in the early years of the 21st century  we are in an interim between the first antibiotic era,  exemplified by the (3-lactams, macrolides, tetracyclines,  aminoglycosides and fluoroquinolones, and a second era  of new agents directed against targets waiting to be  revealed by genomics and proteomics research. In this  interim, the goals must be to reduce antibiotic usage and  encourage the return of a susceptible human commensal  flora. Finally, it must be remembered that antibiotics are  not a human invention. Microorganisms have used them  in attack and counter-attack against each other for bil-  lions of years. Our efforts over the last 60 years seem  trivial in comparison.   Alekshun, M.N. 8t Levy, S.B. (1999) The mar regulon: multiple  resistance to antibiotics and other toxic chemicals. Trends  Microbiol , 7, 410-413.   Chopra, I. & Roberts, M. (2001) Tetracycline antibiotics:  mode of action, applications, molecular biology, and epide-  miology of bacterial resistance. Microbiol Mol Biol Rev , 65 ,  232-260.   Fluit, A.C., Visser, M.R. & Schmitz, F.J. (2001) Molecular detec-  tion of antimicrobial resistance. Clin Microbiol Rev , 14 ,  836-871.   Gillespie, S.H. (2002) Evolution of drug resistance in  Mycobacterium tuberculosis: clinical and molecular perspec-  tive. Antimicrob Agents Chemother, 46 , 267—274.   Kotra, L.R, Haddad, J. & Mobashery, S. (2000) Amino-glycosides:  perspectives on mechanism of action and resistance and strat-  egies to counter resistance. Antimicrob Agents Chemother, 44 ,  3249-3256.   Martinez, J.L. & Baquero, F. (2000) Mutation frequencies and  antibiotic resistance. Antimicrob Agents Chemother, 44 ,  1771-1777.   Murray, B.E. (2000) Vancomycin-resistant enterococcal infec-  tions. N Engl J Med, 342 , 710-721.    Nikaido, H. (1996) Multidrug efflux pumps of gram-negative  bacteria. / Bacteriol 178, 5853-5859.   Philippon, A., Arlet, G. & Jacoby, G.A. (2002) Plasmid-  determined AmpC-type (3-lactamases. Antimicrob Agents  Chemother, 46, 1—11.   Piddock, L.J.V. (2006) Multidrug resistance efflux pumps — not  just for resistance. Nature Rev Microbiol 4, 629-636.    Recchia, G.D. & Hall, R.M. (1997) Origins of the mobile gene  cassettes found in integrons. Trends Microbiol , 5, 389-394.   Yong, D., Toleman, M.A., Giske, C.G. et al. (2009). Characteri-  sation of a new metallo-beta-lactamase gene, bla (NDM-1),  and a novel erythromycin esterase gene carried on a unique  genetic structure in Klebsiella pneumoniae sequence type 14  from India. Antimicriob Agents Chemother , 53, 5046-5054.   1 St Mary’s Hospital, Imperial College Healthcare NHS Trust, London, UK   2 Nottingham University Hospitals NHS Trust, City Hospital Campus, Nottingham, UK   The worldwide use of antimicrobial drugs continues  to rise; in 2005 these agents accounted for an expenditure  of approximately £26 billion. In the UK, prescribing  in general practice accounts for approximately 90% of  all antibiotics and largely involves oral and topical  agents. Hospital use accounts for the remaining 10% of  antibiotic prescribing, with a much heavier use of inject-  able agents. Although this chapter is concerned with  the clinical use of antimicrobial drugs, it should be  remembered that these agents are also extensively used    in veterinary practice and, to a diminishing extent, in  animal husbandry as growth promoters. In humans the  therapeutic use of anti-infectives has revolutionized  the management of most bacterial infections, many  parasitic and fungal diseases and, with the availability of  aciclovir and a growing number of antiretroviral  agents (see Chapters 5 and 11), selected herpesvirus  infections and HIV infection, respectively. Although  originally used for the treatment of established bacterial  infections, antibiotics have proved useful in the preven-  tion of infection in various high-risk circumstances;  this applies especially to patients undergoing various  surgical procedures where perioperative antibiotics    Hugo and Russell's Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   The advantages of effective antimicrobial chemother-  apy are self-evident, but this has led to a significant  problem in ensuring that they are always appropriately  used. Prescribers face a dilemma: initial antimicrobial  therapy must be effective against all likely infective organ-  isms for the individual presentation, but excessive use of  broad-spectrum agents contributes to the development  and selection of drug-resistant organisms. Hence, anti-  infectives are the only class of drug where inappropriate  use in one patient can jeopardize the efficacy of treatment  in other individuals.   Examples of inappropriate antimicrobial use include  prescribing in situations where antibiotics are either inef-  fective, such as viral infections, or where the selected  agent, its dose, route of administration or duration of use  are inappropriate. Of particular concern is the unneces-  sarily prolonged use of antibiotics for surgical prophy-  laxis. Apart from encouraging superinfection by  drug-resistant organisms, prolonged use is wasteful of  health resources and unnecessarily increases the risk of  adverse drug reactions. Thus, it is essential that the clini-  cal use of these agents be based on a clear understanding  of the principles that have evolved to ensure safe, yet  effective, prescribing.   Further information about the properties of antimi-  crobial agents described in this chapter can be found in  Chapter 11.   Drug selection should be based on knowledge of its  activity against infecting microorganisms. Selected  organisms may be predictably susceptible to a particular  agent, and laboratory testing is therefore rarely per-  formed. For example, Streptococcus pyogenes is uniformly  sensitive to penicillin. In contrast, the susceptibility of  many Gram-negative enteric bacteria is less predictable  and laboratory guidance is essential for safe prescribing.  The susceptibility of common bacterial pathogens  and widely prescribed antibiotics is summarized in  Table 14.1. It can be seen that, although certain bacteria  are susceptible in vitro to a particular agent, use of  that drug may be inappropriate, either on pharmacologi-  cal grounds or because other less toxic agents are  preferred.   In vitro susceptibility testing does not always predict  clinical outcome. Host factors play an important part in  determining outcome and this applies particularly to cir-  culating and tissue phagocytic activity. Infections can  progress rapidly in patients suffering from either an abso-  lute or functional deficiency of phagocytic cells. This  applies particularly to those suffering from various hae-  matological malignancies, such as the acute leukaemias,  where phagocyte function is impaired both by the disease  and also by the use of potent cytotoxic drugs which  destroy healthy, as well as malignant, white cells. Under  these circumstances it is essential to select agents that are  bactericidal, as bacteriostatic drugs, such as the tetracy-  clines or sulphonamides, rely on host phagocytic activity  to clear bacteria. Widely used bactericidal agents include  the aminoglycosides, broad-spectrum penicillins, the  cephalosporins and quinolones (see Chapter 11).   In some infections the pathogenic organisms are  located intracellularly within phagocytic cells and there-  fore remain relatively protected from drugs that penetrate  cells poorly, such as the penicillins and cephalosporins.  In contrast, erythromycin, rifampicin and the fluoroqui-  nolones readily penetrate phagocytic cells. Fegionnaires’  disease is an example of an intracellular infection and is  treated with erythromycin with or without rifampicin.   Clinical efficacy is also dependent on achieving satisfac-  tory drug concentrations at the site of the infection; this  is influenced by the standard pharmacological factors of  absorption, distribution, metabolism and excretion. If an  oral agent is selected, gastrointestinal absorption should  be satisfactory. However, it may be impaired by factors  such as the presence of food, drug interactions (including  chelation), or impaired gastrointestinal function either as  a result of surgical resection or malabsorptive states.  Although effective, oral absorption may be inappropriate  in patients who are vomiting or have undergone recent  surgery; under these circumstances a parenteral agent  will be required and has the advantage of providing  rapidly effective drug concentrations.   Antibiotic selection also varies according to the ana-  tomical site of infection. Lipid solubility is of importance  in relation to drug distribution. For example, the  aminoglycosides are poorly lipid-soluble and although  achieving therapeutic concentrations within the extracel-  lular fluid compartment, penetrate the cerebrospinal  fluid (CSF) poorly. Likewise the presence of inflamma-  tion may affect drug penetration into the tissues. In the    +, Sensitive; R, resistant; ±, some strains resistant; , not appropriate therapy; *, rare strains resistant.    presence of meningeal inflammation, (3-lactam agents  achieve satisfactory concentrations within the CSF, but as  the inflammatory response subsides drug concentrations  fall. Hence it is essential to maintain sufficient dosaging  throughout the treatment of bacterial meningitis. Other  agents such as chloramphenicol are little affected by the  presence or absence of meningeal inflammation.   Therapeutic drug concentrations within the bile duct  and gallbladder are dependent on biliary excretion. In the  presence of biliary disease, such as gallstones or chronic  inflammation, the drug concentration may fail to reach  therapeutic levels. In contrast, drugs that are excreted  primarily via the liver or kidneys may require reduced  dosaging in the presence of impaired renal or hepatic  function. The malfunction of excretory organs may not  only risk toxicity from drug accumulation, but will also  reduce urinary concentration of drugs excreted primarily  by glomerular filtration. This applies to the aminoglyco-  sides and the urinary antiseptics nalidixic acid and nitro-  furantoin, where therapeutic failure of urinary tract  infections may complicate severe renal failure.   Drug resistance may be a natural or an acquired charac-  teristic of a microorganism. This may result from  impaired cell wall or cell envelope penetration, enzymatic  inactivation, altered binding sites or active extrusion  from the cell as a result of efflux mechanisms (Chapter  13). Acquired drug resistance may result from mutation,  adaptation or gene transfer. Spontaneous mutations  occur at low frequency, as in the case of Mycobacterium  tuberculosis where a minority population of organisms is  resistant to isoniazid. In this situation the use of isoniazid  alone will eventually result in overgrowth by this sub-  population of resistant organisms.   Genetic resistance may be chromosomal or transfera-  ble on transposons or plasmids. Plasmid-mediated  resistance has been increasingly recognized among Gram-  negative enteric pathogens. By the process of conjugation  (Chapter 13), resistance plasmids may be transferred  between bacteria of the same and different species and  also different genera. Such resistance can code for multi-  ple antibiotic resistance. For example, the penicillins,  cephalosporins, chloramphenicol and the aminoglyco-  sides are all subject to enzymatic inactivation, which may  be plasmid-mediated. Knowledge of the local epidemiol-  ogy of resistant pathogens within a hospital, and espe-  cially within high-dependency areas such as intensive  care and haemodialysis units, is invaluable in guiding  appropriate drug selection.   In recent years multidrug resistance has increased among  certain pathogens. These include Staphylococcus aureus,  enterococci and M. tuberculosis. Staph, aureus resistant to  meticillin is known as meticillin-resistant Staph, aureus  (MRS A). These strains are resistant to many antibiotics  and have been responsible for major epidemics world-  wide, usually in hospitals where they affect patients in  high-dependency units such as intensive care units, burns  units and cardiothoracic units. MRSA have the ability to  colonize staff and patients and to spread readily among  them. Several epidemic strains are currently circulating  in the UK. The glycopeptides vancomycin or teicoplanin  and the oxazolidinone linezolid are the currently recom-  mended agents for treating patients infected with these  organisms. Newer agents such as daptomycin and tigecy-  cline are also active against MRSA and are increasingly  used in the hospital setting; meanwhile, some strains  retain sensitivity to older agents such as tetracyclines,  rifampicin and clindamycin, and combinations of these  agents are sometimes used, but only following full analy-  sis of microbiological sensitivities.   Another serious resistance problem is that of drug-  resistant enterococci. These include Enterococcus faecalis  and, in particular, E. faecium. Resistance to the glycopep-  tides has again been a problem among patients in high-  dependency units. Four different phenotypes are  recognized (VanA, VanB, VanC and VanD). The VanA  phenotype is resistant to both glycopeptides, while the  others are sensitive to teicoplanin but demonstrate high  (VanB) or intermediate (VanC) resistance to vancomycin;  VanD resistance has only recently been described and  remains uncommon. Those fully resistant to the glyco-  peptides are increasing in frequency and causing great  concern as they are essentially resistant to almost all  antibiotics.   Extended-spectrum (3-lactamase (ESBL) producing  Gram-negative organisms are an increasing problem in  hospitals, and occasionally seen as a cause of urinary tract  infection in primary care; ESBLs can hydrolyse most  cephalosporins and penicillins, limiting therapeutic  options to carbapenems or aminoglycosides. ESBLs can  be chromosomally mediated (e.g. Pseudomonas spp.,  Citrobacter spp.) or plasmid-mediated (e.g. Klebsiella  spp.), the latter often being implicated in hospital out-  breaks. Furthermore, carbapenem resistance, via metallo-  (3-lactamase, has been seen in outbreak strains of  Pseudomonas aeruginosa and Acinetobacter baumannii; in  such situations, potentially toxic agents such as colistin  are often the only effective choice.    Tuberculosis is on the increase after decades in which  the incidence had been steadily falling. Drug-resistant  strains have emerged largely among inadequately treated  or non-compliant patients. These include the homeless,  alcoholic, intravenous drug misusing, HIV-positive  and immigrant populations. Resistance patterns vary  but increasingly include rifampicin and isoniazid.  Furthermore, outbreaks of multidrug-resistant tubercu-  losis have been increasingly reported from a number of  hospital centres in the USA and more recently Europe,  including the UK. These infections have occasionally  spread to healthcare workers and are giving rise to con-  siderable concern.   The underlying mechanisms of resistance are consid-  ered in Chapter 13.   Antibiotics are generally used alone, but may on occasion  be prescribed in combination. Combining two antibiotics  may result in synergism, indifference or antagonism. In  the case of synergism, microbial inhibition is achieved at  concentrations below that for each agent alone and may  prove advantageous in treating relatively insusceptible  infections such as enterococcal endocarditis, where a  combination of penicillin and gentamicin is synergisti-  cally active. Another advantage of synergistic combina-  tions is that it may enable the use of toxic agents where  dose reductions are possible. For example, meningitis  caused by the fungus Cryptococcus neoformans responds  to an abbreviated course of amphotericin B when it is  combined with 5-flucytosine, thereby reducing the risk  of toxicity from amphotericin B.   Combined drug use is occasionally recommended to  prevent resistance emerging during treatment. For  example, treatment may fail when fusidic acid is used  alone to treat Staph, aureus infections, because resistant  strains develop rapidly; this is prevented by combining  fusidic acid with flucloxacillin. Likewise, tuberculosis is  initially treated with a minimum of three agents, such as  rifampicin, isoniazid and pyrazinamide; again drug  resistance is prevented, which may result if either agent  is used alone.   The most common reason for using combined therapy  is in the treatment of confirmed or suspected mixed  infections where a single agent alone will fail to cover all  pathogenic organisms. This is the case in serious abdomi-  nal sepsis where mixed aerobic and anaerobic infections  are common and the use of metronidazole in combina-  tion with either an aminoglycoside or a broad-spectrum  cephalosporin is essential. Finally, drugs are used in com-    bination in patients who are seriously ill and about whom  uncertainty exists concerning the microbiological nature  of their infection. This initial ‘blind therapy’ frequently  includes a broad-spectrum penicillin or cephalosporin in  combination with an aminoglycoside. The regimen  should be modified in the light of subsequent microbio-  logical information.   Regrettably, all chemotherapeutic agents have the poten-  tial to produce adverse reactions with varying degrees of  frequency and severity, and these include hypersensitivity  reactions and toxic effects. These maybe dose-related and  predictable in a patient with a history of hypersensitivity  or a previous toxic reaction to a drug or its chemical  analogues. However, many adverse events are idiosyn-  cratic and therefore unpredictable.   Hypersensitivity reactions range in severity from fatal  anaphylaxis, in which there is widespread tissue oedema,  airway obstruction and cardiovascular collapse, to minor  and reversible hypersensitivity reactions such as skin  eruptions and drug fever. Such reactions are more likely  in those with a history of hypersensitivity to the drug,  and are more frequent in patients with previous allergic  diseases such as childhood eczema or asthma. It is impor-  tant to question patients closely concerning hypersensi-  tivity reactions before prescribing, as it precludes the use  of all compounds within a class, such as the sulphona-  mides or tetracyclines, while cephalosporins and carbap-  enems should be used only with caution in patients who  are allergic to penicillin, because these agents are struc-  turally related. They should be avoided entirely in those  who have had a previous severe hypersensitivity reaction  to penicillin.   Drug toxicity is often dose-related and may affect a  variety of organs or tissues. For example, the aminogly-  cosides are both nephrotoxic and ototoxic to varying  degrees; therefore, dosaging should be individualized and  the serum assayed, especially where renal function is  abnormal, to avoid toxic effects and non-therapeutic  drug concentrations. An example of dose-related toxicity  is chloramphenicol-induced bone marrow suppression.  Chloramphenicol interferes with the normal maturation  of bone marrow stem cells and high concentrations may  result in a steady fall in circulating red and white cells  and also platelets. This effect is generally reversible with  dose reduction or drug withdrawal. This dose-related  toxic reaction of chloramphenicol should be contrasted  with idiosyncratic bone marrow toxicity which is unre-  lated to dose and occurs at a much lower frequency of    approximately 1 : 40 000 and is frequently irreversible,  ending fatally. Toxic effects may also be genetically  determined. For example, peripheral neuropathy may  occur in those who are slow acetylators of isoniazid,  while haemolysis occurs in those deficient in the red cell  enzyme glucose-6-phosphate dehydrogenase, when  treated with sulphonamides, primaquine, quinolones or  nitrofurantoin.   Anti-infective drugs not only affect the invading organ-  ism undergoing treatment but also have an impact on the  normal bacterial flora, especially of the skin and mucous  membranes. This may result in microbial overgrowth of  resistant organisms with subsequent superinfection. One  example is the common occurrence of oral or vaginal  candidiasis in patients treated with broad-spectrum  agents such as ampicillin or tetracycline. A more serious  example is the development of pseudomembranous  colitis from the overgrowth of toxin-producing strains of  Clostridium difficile present in the bowel flora following  the use of clindamycin or broad-spectrum antibiotics,  though any antimicrobial can precipitate this condition.  C. difficile-associated diarrhoea is managed by drug with-  drawal and oral vancomycin, or oral/intravenous metro-  nidazole. Intravenous immunoglogbulin is occasionally  used in severe cases, and rarely, colectomy (excision of  part or whole of the colon) maybe necessary. Once estab-  lished, C.difficile infection is transmissible, particularly in  the hospital setting; isolation of symptomatic patients  and strict observation of hygiene practices (e.g. hand  washing) are therefore key in preventing outbreaks.   An increasingly important use of antimicrobial agents is  that of infection prevention, especially in relationship to  surgery. Infection remains one of the most important  complications of many surgical procedures, and the rec-  ognition that perioperative antibiotics are effective and  safe in preventing this complication has proved a major  advance in surgery. The principles that underlie the che-  moprophylactic use of antibacterials relate to the predict-  ability of infection for a particular surgical procedure, in  terms of its occurrence, microbial aetiology and suscep-  tibility to antibiotics. Therapeutic drug concentrations  present at the operative site at the time of surgery rapidly  reduce the number of potentially infectious organisms  and prevent wound sepsis. If prophylaxis is delayed to the  postoperative period, then efficacy is markedly impaired.  It is important that chemoprophylaxis be limited to the    perioperative period, the first dose being administered  approximately 1 hour before surgery for injectable agents;  for many procedures and operative sites, a single dose is  now considered sufficient. Prolonging chemoprophylaxis  beyond this period is not cost-effective and increases the  risk of adverse drug reactions and superinfection. One of  the best examples of the efficacy of surgical prophylaxis  is in the area of large-bowel surgery. Before the wide-  spread use of chemoprophylaxis, postoperative infection  rates for colectomy were often 30% or higher; these have  now been reduced to around 5%.   Chemoprophylaxis has been extended to other surgical  procedures where the risk of infection may be low but  its occurrence has serious consequences. This is especially  true for the implantation of prosthetic joints or heart  valves. These are major surgical procedures and although  infection may be infrequent its consequences are  serious and on balance the use of chemoprophylaxis is  cost-effective.   Examples of chemoprophylaxis in the non-surgical  arena include the prevention of pneumococcal infection  with penicillin V in asplenia or patients with sickle-cell  disease, and the prevention of secondary cases of menin-  gococcal meningitis with rifampicin or ciprofloxacin  among household contacts of an index case.   The choice of antimicrobial chemotherapy is initially  dependent on the clinical diagnosis. In some circum-  stances the clinical diagnosis implies a microbiological  diagnosis which may dictate specific therapy. For example,  typhoid fever is caused by Salmonella enterica serovar  Typhi, which is generally sensitive to co-trimoxazole,  ceftriaxone and ciprofloxacin. However, for many infec-  tions, establishing a clinical diagnosis implies a range of  possible microbiological causes and requires laboratory  confirmation from samples collected, preferably before  antibiotic therapy is begun. Laboratory isolation and sus-  ceptibility testing of the causative agent establish the  diagnosis with certainty and make drug selection more  rational. However, in many circumstances, especially in  general practice, microbiological documentation of an  infection is not possible. Hence knowledge of the usual  microbiological cause of a particular infection and its  susceptibility to antimicrobial agents is essential for effec-  tive drug prescribing. The following section explores a  selection of the problems associated with antimicrobial  drug prescribing for a range of clinical conditions.   Infections of the respiratory tract are among the com-  monest of infections, and account for much consultation  in general practice and a high percentage of acute hospi-  tal admissions. They are divided into infections of the  upper respiratory tract, involving the ears, throat, nasal  sinuses and the trachea, and the lower respiratory tract  (LRT), where they affect the airways, lungs and pleura.   Acute pharyngitis presents a diagnostic and therapeutic  dilemma. The majority of sore throats are caused by a  variety of viruses; fewer than 20% are bacterial and hence  potentially responsive to antibiotic therapy. However,  antibiotics are widely prescribed and this reflects the  difficulty in discriminating streptococcal from non-  streptococcal infections clinically in the absence of  microbiological documentation. Nonetheless, Strep, pyo-  genes is the most important bacterial pathogen and this  responds to oral penicillin. However, up to 10 days’ treat-  ment is required for its eradication from the throat. This  requirement causes problems with compliance as symp-  tomatic improvement generally occurs within 2-3 days.   Although viral infections are important causes of both  otitis media and sinusitis, they are generally self-limiting.  Bacterial infections may complicate viral illnesses, and  are also primary causes of ear and sinus infections.  Streptococcus pneumoniae and Haemophilus influenzae  are the commonest bacterial pathogens. Amoxicillin is  widely prescribed for these infections as it is microbio-  logically active, penetrates the middle ear and sinuses, is  well tolerated and has proved effective.   Infections of the LRT include pneumonia, lung abscess,  bronchitis, bronchiectasis and infective complications of  cystic fibrosis. Each presents a specific diagnostic and  therapeutic challenge, which reflects the variety of patho-  gens involved and the frequent difficulties in establishing  an accurate microbial diagnosis. The laboratory diagnosis  of LRT infections is largely dependent upon culturing  sputum. Unfortunately this may be contaminated with  the normal bacterial flora of the upper respiratory tract  during expectoration. In hospitalized patients, the empir-  ical use of antibiotics before admission substantially  diminishes the value of sputum culture and may result in  overgrowth by non-pathogenic microbes, thus causing  difficulty with the interpretation of sputum culture  results. Alternative diagnostic samples include needle  aspiration of sputum directly from the trachea or of fluid    within the pleural cavity. Blood may also be cultured and  serum examined for antibody responses or microbial  antigens. In the community, few patients will have their  LRT infection diagnosed microbiologically and the choice  of antibiotic is based on clinical diagnosis.   The range of pathogens causing acute pneumonia  includes viruses, bacteria and, in the immunocompro-  mised host, parasites and fungi. Table 14.2 summarizes  these pathogens and indicates drugs appropriate for their  treatment. Clinical assessment includes details of the evo-  lution of the infection, any evidence of a recent viral  infection, the age of the patient and risk factors such as  corticosteroid therapy or pre-existing lung disease. The  extent of the pneumonia, as assessed clinically or by  X-ray, is also important.   Streptococcus pneumoniae remains the commonest  cause of pneumonia and still responds well to penicillin  despite a global increase in isolates showing reduced sus-  ceptibility to this agent. So-called ‘respiratory quinolones’  such as levofloxacin and moxifloxacin, which exhibit  increased activity against Gram positive organisms com-  pared to ciprofloxacin, are an alternative. A number of  atypical infections may cause pneumonia and include  Mycoplasma pneumoniae, Legionella pneumophila, psit-  tacosis and occasionally Q fever. With psittacosis there  may be a history of contact with parrots or budgerigars;  while legionnaires’ disease has often been acquired during  hotel holidays in the Mediterranean area. The atypical  pneumonias, unlike pneumococcal pneumonia, do not  respond to penicillin. Legionnaires’ disease is treated  with erythromycin and, in the presence of severe pneu-  monia, rifampicin is added to the regimen. Mycoplasma  infections are best treated with either erythromycin or  tetracycline, while the latter drug is indicated for both  psittacosis and Q fever.   Destruction of lung tissue may lead to abscess formation  and is a feature of aerobic Gram-negative bacillary and  Staph, aureus infections. In addition, aspiration of  oropharyngeal secretion can lead to chronic low-grade  sepsis with abscess formation and the expectoration of  foul-smelling sputum that characterizes anaerobic sepsis.  The latter condition responds to high-dose penicillin,  which is active against most of the normal oropharyngeal  flora, while metronidazole may be appropriate for  strictly anaerobic infections. In the case of aerobic  Gram-negative bacillary sepsis, aminoglycosides, with or    Table 14.2 Microorganisms responsible for pneumonia and the therapeutic agent of choice    without a broad-spectrum cephalosporin, are the  agents of choice. Acute staphylococcal pneumonia is an  extremely serious infection and requires treatment with  high-dose flucloxacillin alone or in combination with  fusidic acid.   Cystic fibrosis is a multisystem congenital abnormality  that often affects the lungs and results in recurrent  infections, initially with Staph, aureus, subsequently with  H. influenzae and eventually leads on to recurrent  Pseudomonas aeruginosa infection. The last organism is  associated with copious quantities of purulent sputum  that are extremely difficult to expectorate. Ps. aeruginosa  is a co-factor in the progressive lung damage that is even-  tually fatal in these patients. Repeated courses of antibiot-  ics are prescribed and although they have improved the  quality and longevity of life, infections caused by Ps. aeru-  ginosa are difficult to treat and require repeated hospitali-  zation and administration of parenteral antibiotics such  as an aminoglycoside, either alone or in combination    with an antipseudomonal penicillin or cephalosporin.  The dose of aminoglycosides tolerated by these patients  is often higher than in normal individuals and is associ-  ated with larger volumes of distribution for these and  other agents. Some benefit may also be obtained from  inhaled aerosolized antibiotics. Unfortunately drug  resistance may emerge and makes drug selection more  dependent upon laboratory guidance.   Urinary tract infection is a common problem in both  community and hospital practice. Although occurring  throughout life, infections are more common in pre-  school girls and women during their childbearing years,  although in the elderly the sex distribution is similar.  Infection is predisposed by factors that impair urine flow.  These include congenital abnormalities, reflux of urine  from the bladder into the ureters, kidney stones and  tumours and, in males, enlargement of the prostate gland.  Bladder catheterization is an important cause of urinary  tract infection in hospitalized patients.   In those with structural or drainage problems the risk  exists of ascending infection to involve the kidney and  occasionally the bloodstream. Although structural abnor-  malities may be absent in women of childbearing years,  infection can become recurrent, symptomatic and  extremely distressing. Of greater concern is the occur-  rence of infection in the pre-school child, as normal  maturation of the kidney may be impaired and may result  in progressive damage which presents as renal failure in  later life.   From a therapeutic point of view, it is essential to  confirm the presence of bacteriuria (a condition in which  there are bacteria in the urine), as symptoms alone are  not a reliable method of documenting infection. This  applies particularly to bladder infection, where the symp-  toms of burning micturition (dysuria) and frequency can  be associated with a variety of non-bacteriuric condi-  tions. Patients with symptomatic bacteriuria should  always be treated. However, the necessity to treat asymp-  tomatic bacteriuric patients varies with age and the pres-  ence or absence of underlying urinary tract abnormalities.  In the preschool child it is essential to treat all urinary  tract infections and maintain the urine in a sterile state  so that normal kidney maturation can proceed. Likewise  in pregnancy there is a risk of infection ascending from  the bladder to involve the kidney. This is a serious com-  plication and may result in premature labour. Other indi-  cations for treating asymptomatic bacteriuria include the  presence of underlying renal abnormalities such as stones,  which may be associated with repeated infections caused  by Proteus spp.   The antimicrobial treatment of urinary tract infection  presents a number of interesting challenges. Drugs must  be selected for their ability to achieve high urinary con-  centrations and, if the kidney is involved, adequate tissue  concentrations. Safety in childhood or pregnancy is  important as repeated or prolonged medication may be  necessary. The choice of agent will be dictated by the  microbial aetiology and susceptibility findings, because  the latter can vary widely among Gram-negative enteric  bacilli, especially in patients who are hospitalized. Table  14.3 shows the distribution of bacteria causing urinary  tract infection in the community and in hospitalized  patients. The greater tendency towards infections caused  by Klebsiella spp. and Ps. aeruginosa should be noted as  antibiotic sensitivity is more variable for these pathogens.  Drug resistance has increased substantially in recent years  and has reduced the value of formerly widely prescribed  agents such as the sulphonamides and ampicillin.   Uncomplicated community-acquired urinary tract  infection presents few problems with management.  Drugs such as trimethoprim, ciprofloxacin and ampici-  llin are widely used. Cure rates are close to 100% for  ciprofloxacin, about 80% for trimethoprim and about  50% for ampicillin — to which resistance has been steadily  increasing. Treatment for 3 days is generally satisfactory  and is usually accompanied by prompt control of symp-  toms. Single-dose therapy with amoxicillin 3 g has also  been shown to be effective in selected individuals.  Alternative agents include nitrofurantoin, nalidixic acid  and norfloxacin, although these are not as well tolerated.  Oral cephalosporins and co-amoxiclav are also used.    Reprinted from Journal of Infection, 46(2):94- 1 00, Farrell, D.J et al, Urinary tract infection — distribution of  pathogenic bacteria in the community and hospitalized patients. Copyright (2003), with permission from  Elsevier    It is important to demonstrate the cure of bacteriuria  with a repeat urine sample collected 4-6 weeks after treat-  ment, or sooner should symptoms fail to subside.  Recurrent urinary tract infection is an indication for  further investigation of the urinary tract to detect under-  lying pathology that may be surgically correctable. Under  these circumstances it also is important to maintain the  urine in a sterile state. This can be achieved with repeated  courses of antibiotics, guided by laboratory sensitivity  data. Alternatively, long-term chemoprophylaxis for  periods of 6 months to control infection by either preven-  tion or suppression is widely used. Trimethoprim is the  most commonly prescribed chemoprophylactic agent  and is given as a single nightly dose. This achieves high  urinary concentrations throughout the night and gener-  ally ensures a sterile urine. Nitrofurantoin is an alterna-  tive agent.   Infection of the kidney demands the use of agents  that achieve adequate tissue as well as urinary concentra-  tions. As bacteraemia (a condition in which there are  bacteria circulating in the blood) may complicate infec-  tion of the kidney, it is generally recommended that  antibiotics be administered parenterally. Although ampi-  cillin was formerly widely used, drug resistance is now  common and agents such as cefotaxime, co-amoxiclav or  ciprofloxacin are often preferred, because the aminogly-  cosides, although highly effective and preferentially con-  centrated within the renal cortex, carry the risk of  nephrotoxicity.   Infections of the prostate tend to be persistent, recur-  rent and difficult to treat. This is in part due to the more  acid environment of the prostate gland, which inhibits  drug penetration by many of the antibiotics used to treat  urinary tract infection. Agents that are basic in nature,  such as erythromycin, achieve therapeutic concentrations  within the gland but unfortunately are not active against  the pathogens responsible for bacterial prostatitis.  Trimethoprim, and quinolones, however, are useful  agents as they are preferentially concentrated within the  prostate and active against many of the causative patho-  gens. It is important that treatment be prolonged for  several weeks, as relapse is common.   The gut is vulnerable to infection by viruses, bacteria,  parasites and occasionally fungi. Virus infections are the  most prevalent but are not susceptible to chemothera-  peutic intervention. Bacterial infections are more readily  recognized and raise questions concerning the role of    antibiotic management. Parasitic infections of the gut are  beyond the scope of this chapter.   Bacteria cause disease of the gut as a result of either  mucosal invasion or toxin production or a combination  of the two mechanisms, as summarized in Table 14.4.  Treatment is largely directed at replacing and maintain-  ing an adequate intake of fluid and electrolytes. Antibiotics  are generally not recommended for infective gastroen-  teritis, but deserve consideration where they have been  demonstrated to abbreviate the acute disease or to prevent  complications including prolonged gastrointestinal  excretion of the pathogen where this poses a public health  hazard.   It should be emphasized that most gut infections are  self-limiting. However, attacks can be severe and may  result in hospitalization. Antibiotics are used to treat  severe Campylobacter and Shigella infections; erythromy-  cin and ciprofloxacin, respectively, are the preferred  agents. Such treatment abbreviates the disease and elimi-  nates gut excretion in Shigella infection. However, in  severe Campylobacter infection the data are currently  equivocal, although the clinical impression favours the  use of erythromycin for severe infections. The role of  antibiotics for Campylobacter and Shigella infections  should be contrasted with gastrointestinal salmonellosis,  for which antibiotics are contraindicated as they do not  abbreviate symptoms, are associated with more pro-  longed gut excretion and introduce the risk of adverse  drug reactions. However, in severe salmonellosis, espe-  cially at extremes of age, systemic toxaemia and blood-  stream infection can occur and under these circumstances  treatment with either ciprofloxacin or trimethoprim is  appropriate.   Typhoid and paratyphoid fevers (known as enteric  fevers), although acquired by ingestion of salmonellae,  Sal. enterica serovar Typhi and Sal. enterica serovar  Paratyphi, respectively, are largely systemic infections  and antibiotic therapy is mandatory; ciprofloxacin is now  the drug of choice although trimethoprim or chloram-  phenicol are satisfactory alternatives. Prolonged gut  excretion of Sal. enterica serovar Typhi is a well-known  complication of typhoid fever and is a major public  health hazard in developing countries. Treatment with  ciprofloxacin or high-dose ampicillin can eliminate the  gall bladder excretion which is the major site of persistent  infection in carriers. However, the presence of gallstones  reduces the chance of cure.   Cholera is a serious infection causing epidemics through-  out Asia. Although a toxin-mediated disease, largely  controlled with replacement of fluid and electrolyte losses,    tetracycline has proved effective in eliminating the causa-  tive vibrio from the bowel, thereby abbreviating the  course of the illness and reducing the total fluid and  electrolyte losses.   Traveller’s diarrhoea may be caused by one of many  gastrointestinal pathogens (Table 14.4). However, entero-  toxigenic Escherichia coli is the most common pathogen.  While it is generally short-lived, traveller’s diarrhoea can  seriously mar a brief period abroad, be it for holiday or  business purposes. Although not universally accepted, the  use of short-course trimethoprim or quinolone such as  ciprofloxacin can abbreviate an attack in patients with  severe disease.   Infections of the skin and soft tissue commonly follow  traumatic injury to the epithelium but occasionally may  be blood-borne. Interruption of the integrity of the skin  allows ingress of microorganisms to produce superficial,  localized infections which on occasion may become more  deep-seated and spread rapidly through tissues. Skin  trauma complicates surgical incisions and accidents,  including burns. Similarly, prolonged immobilization  can result in pressure damage to skin from impaired    blood flow. It is most commonly seen in patients who are  unconscious.   Microbes responsible for skin infection often arise  from the normal skin flora, which includes Staph, aureus.  In addition Strep, pyogenes, Ps. aeruginosa and anaerobic  bacteria are other recognized pathogens. Viruses also  affect the skin and mucosal surfaces, either as a result of  generalized infection or localized disease as in the case of  herpes simplex. The latter is amenable to antiviral therapy  in selected patients, although for the majority of patients,  virus infections of the skin are self-limiting.   Strep, pyogenes is responsible for a range of skin infec-  tions: impetigo is a superficial infection of the epidermis  which is common in childhood and is highly contagious;  cellulitis is a more deep-seated infection which spreads  rapidly through the tissues to involve the lymphatics and  occasionally the bloodstream; erysipelas is a rapidly  spreading cellulitis commonly involving the face, which  characteristically has a raised leading edge due to lym-  phatic involvement. Necrotizing fasciitis is a more serious,  rapidly progressive infection of the skin and subcutane-  ous structures including the fascia and musculature.  Despite early diagnosis and high-dose intravenous anti-  biotics, this condition is often life-threatening and may  require extensive surgical debridement of devitalized    tissue and even limb amputation to ensure survival. A  fatal outcome is usually the result of profound toxaemia  and bloodstream spread. Penicillin is the drug of choice  for all these infections, usually in combination with  other agents such as an aminoglycoside and metronida-  zole in the case of necrotizing fasciitis; in severe instances  parenteral administration is appropriate. The use of  topical agents, such as tetracycline, to treat impetigo may  fail as drug resistance is now recognized.   Staph, aureus is responsible for a variety of skin infec-  tions which require therapeutic approaches different  from those of streptococcal infections. Staphylococcal  cellulitis is indistinguishable clinically from streptococcal  cellulitis and responds to flucloxacillin, but generally  fails to respond to penicillin owing to penicillinase ((3-  lactamase) production. In hospital-acquired infection,  and occasionally in community practice, MRSA must be  considered as a possibility, particularly where the patient  is known to be colonized. Staph, aureus is an important  cause of superficial, localized skin sepsis which varies  from small pustules to boils and occasionally to a more  deeply invasive, suppurative skin abscess known as a car-  buncle. Antibiotics are generally not indicated for these  conditions. Pustules and boils settle with antiseptic  soaps or creams and often discharge spontaneously,  whereas carbuncles frequently require surgical drainage.  Staph, aureus may also cause postoperative wound  infections, sometimes associated with retained suture  material, and settles once the stitch is removed. Antibiotics  are only appropriate in this situation if there is  extensive accompanying soft tissue invasion. Rarely,  strains of Staph, aureus may express a toxin complex  known as Panton- Valentine Leukocidin (PVL); these  strains can cause severe sepsis and an often fatal necrotiz-  ing pneumonia in young, otherwise fit, patients. The  treatment for such infections usually aims to minimize  toxin production using protein synthesis inhibitors such  as clindamycin plus rifampicin, in combination with  linezolid.   Anaerobic bacteria are characteristically associated  with foul-smelling wounds. They are found in association  with surgical incisions following intra-abdominal proce-  dures and pressure sores, which are usually located over  the buttocks and hips where they become infected with  faecal flora. These infections are frequently mixed and  include Gram-negative enteric bacilli, which may mask  the presence of underlying anaerobic bacteria. The prin-  ciples of treating anaerobic soft tissue infection again  emphasize the need for removal of all foreign and devital-  ized material. Antibiotics such as metronidazole or clin-   The treatment of infected burn wounds presents a  number of peculiar facets. Burns are initially sterile,  especially when they involve all layers of the skin.  However, they rapidly become colonized with bacteria  whose growth is supported by the protein-rich exudate.  Staphylococci, Strep, pyogenes and, particularly, Ps. aeru-  ginosa frequently colonize burns and may jeopardize sur-  vival of skin grafts and occasionally, and more seriously,  result in bloodstream invasion. Treatment of invasive Ps.  aeruginosa infections requires combined therapy with an  aminoglycoside, such as gentamicin or tobramycin, and  an antipseudomonal agent, such as ceftazidime or pipera-  cillin. This produces high therapeutic concentrations  which generally act in a synergistic manner. The use of  aminoglycosides in patients with serious burns requires  careful monitoring of serum concentrations to ensure  that they are therapeutic yet non-toxic, as renal function  is often impaired in the days immediately following a  serious burn.   The brain, its surrounding covering of meninges and  the spinal cord are subject to infection, which is generally  blood-borne but may also complicate neurosurgery,  penetrating injuries or direct spread from infection in  the middle ear or nasal sinuses. Viral meningitis is the  most common infection but is generally self-limiting.  Occasionally destructive forms of encephalitis occur;  an example is herpes simplex encephalitis. Bacterial  infections include meningitis and brain abscesses and  carry a high risk of mortality, while in those who  recover, residual neurological damage or impairment of  intellectual function may follow. This occurs despite the  availability of antibiotics active against the responsible  bacterial pathogens. Fungal infections of the brain,  although rare, are increasing in frequency, particularly  among immunocompromised patients who either have  underlying malignant conditions or are on potent cyto-  toxic drugs.   The treatment of bacterial infections of the central  nervous system highlights a number of important thera-  peutic considerations. Bacterial meningitis is caused by a  variety of bacteria although their incidence varies with  age. In the neonate, E. coli and group B streptococci  account for the majority of infections, while in the pre-  school child H. influenzae was the commonest pathogen  before the introduction of a highly effective vaccine.  Neisseria meningitidis has a peak incidence between 5 and    15 years of age, while pneumococcal meningitis is pre-  dominantly a disease of adults.   Ceftriaxone is the drug of choice for the treatment of  group B streptococcal, meningococcal and pneumococcal  infections but, as discussed earlier, CSF concentrations of  penicillin are significantly influenced by the intensity of  the inflammatory response. To achieve therapeutic con-  centrations within the CSF, high dosages are required,  and in the case of pneumococcal meningitis should be  continued for 10-14 days. Resistance among Strep, pneu-  moniae to penicillin has increased worldwide; in travel-  lers returning from endemic areas, vancomycin may be  indicated. Alternative agents include meropenem.   Resistance of H. influenzae to ampicillin has increased  in the past two decades and varies geographically. Thus,  it can no longer be prescribed with confidence as initial  therapy, and cefotaxime or ceftriaxone are now the pre-  ferred alternatives. Flowever, once laboratory evidence for  (3-lactamase activity is excluded, ampicillin can be safely  substituted.   E. coli meningitis carries a mortality of greater than  40% and reflects both the virulence of this organism and  the pharmacokinetic problems of achieving adequate  CSF antibiotic levels. The broad-spectrum cephalosporins  such as cefotaxime, ceftriaxone or ceftazidime have been  shown to achieve satisfactory therapeutic levels and are  the agents of choice to treat Gram-negative bacillary  meningitis. Treatment again must be prolonged for  periods ranging from 2 to 4 weeks.   Brain abscess presents a different therapeutic chal-  lenge. An abscess is locally destructive to the brain  and causes further damage by increasing intracranial  pressure. The infecting organisms are varied but those  arising from middle ear or nasal sinus infection are  often polymicrobial and include anaerobic bacteria,  microaerophilic species and Gram-negative enteric  bacilli. Less commonly, a pure Staph, aureus abscess may  complicate blood-borne spread. Brain abscess is a neuro-  surgical emergency and requires drainage. Flowever, anti-    biotics are an important adjunct to treatment. The  polymicrobial nature of many infections demands  prompt and careful laboratory examination to determine  optimum therapy. Drugs are selected not only on their  ability to penetrate the blood-brain barrier and enter  the CSF but also on their ability to penetrate the brain  substance. Metronidazole has proved a valuable alterna-  tive agent in such infections, although it is not active  against microaerophilic streptococci, which must be  treated with high-dose benzylpenicillin. The two are  often used in combination. Chloramphenicol is an alter-  native agent.   Fungal infections are divided into superficial or deep-  seated infections. Superficial infections affect the skin,  nails or mucosal surfaces of the mouth or genital tract.  In contrast, deep-seated fungal diseases may target the  lung or disseminate via the bloodstream to organs such  as the brain, spleen, liver or skeletal system.   The fungal infections of the skin and nails include  tinea pedis (athlete’s foot), tinea capitis and tinea cor-  poris (ringworm), Candida intertrigo (usually groin  and submammary regions) and pityriasis ( Malassezia ). A  variety of topical and systemic antifungal agents are avail-  able. The imidazole class of drugs includes clotrimazole  and miconazole, which are highly effective topically.  Systemic antifungals used to treat superficial fungal  infections include griseofulvin and terbinafine, which is  an allylamine. Both agents are ineffective in the treatment  of deep-seated fungal infections that may be caused  by yeasts ( Cryptococcus neoformans), yeast-like fungi  ( Candida spp.) or the filamentous fungi ( Aspergillus spp).  These produce a variety of syndromes for which different  antifungal agents are indicated (Table 14.5). The polyenes  include amphotericin B, which after many years  remains the agent of choice for the treatment of a wide  variety of life-threatening fungal diseases which often  complicate cancer chemotherapy, organ transplantation    and immunodeficiency diseases, such as AIDS.  Nephrotoxicity is common but can be avoided by careful  dosaging or the use of liposomal formulations. The  second major class of systemic antifungals is the triazoles,  which include fluconazole and newer, broader-spectrum  agents such as itraconazole, voriconazole and posacona-  zole. These are extremely well tolerated but may interact  with a number of drugs and drug classes such as the  sulphonylureas, antihistamines and lipid-lowering agents  among others. The echinocandins (caspofungin, anidu-  lafungin) are the newest class of antifungal agents, and  are increasingly used to treat invasive fungal infections;  they have a fungicidal action against Candida spp., and  are fungistatic against many other organisms, including  Aspergillus spp.   A wide variety of medical devices are increasingly used  in clinical practice. These range from vasculature and  urinary catheters, prosthetic joints and heart valves,  shunts and stents for improving the flow of CSF, blood  or bile according to their site of use, to intracardiac  patches and vascular pumps. Unfortunately infection is  the most frequent complication of their use and may  result in the need to replace or remove the device,  sometimes with potentially life-threatening and fatal  consequences.   Infections are often caused by organisms arising from  the normal skin flora, which gain access at the time of  insertion of the device. Staph, epidermidis is among the  most frequent of isolates. Following attachment to the  surface of the device, the organisms undergo multiplica-  tion with the formation of extracellular polysaccharide  material (glycocalyx) which contains slowly replicating  cells to form a biofilm. Microorganisms within a biofilm  are less vulnerable to attack by host defences (phagocytes,  complement and antibodies) and are relatively insuscep-  tible to antibiotic therapy despite the variable ability of  drugs to penetrate the biofilm.   Management approaches have therefore emphasized  the need for prevention through the addition of good  sterile technique at the time of insertion. Manufacturers  have also responded by using materials and creating  surface characteristics of implanted materials inclement  to microbial attachment. Likewise the use of prophylactic  antibiotics at the time of insertion of deep-seated devices  such as joint and heart valve prostheses has further  reduced the risk of infection. Once a medical device  becomes infected, management is difficult. Treatment  with agents such as flucloxacillin, vancomycin and most    recently linezolid is often unsuccessful and the only  course of action is to remove the device.   The plethora of available antimicrobial agents presents  both an increasing problem of selection to the prescriber  and difficulties for the diagnostic laboratory as to which  agents should be tested for susceptibility. Differences in  antimicrobial activity among related compounds are  often of minor importance but can occasionally be of  greater significance and may be a source of confusion to  the non-specialist. This applies particularly to large  classes of drugs, such as the penicillins and cepha-  losporins, where there has been an explosion in the avail-  ability of new agents in recent years. Guidance, in the  form of an antibiotic policy, has a major role to play in  providing the prescriber with a range of agents appropri-  ate to his/her needs and should be supported by labora-  tory evidence of susceptibility to these agents.   In recent years, increased awareness of the cost of  medical care has led to a major review of various aspects  of health costs. The pharmacy budget has often attracted  attention as, unlike many other hospital expenses, it is  readily identifiable in terms of cost and prescriber. Thus,  an antibiotic policy is also seen as a means whereby the  economic burden of drug prescribing can be reduced or  contained. There can be little argument with the recom-  mendation that the cheaper of two compounds should  be selected where agents are similar in terms of efficacy  and adverse reactions. Likewise, generic substitution is  also desirable provided that there is bio-equivalence. It  has become increasingly impractical for pharmacists to  stock all the formulations of every antibiotic currently  available, and here again an antibiotic policy can produce  significant savings by limiting the amount of stock held.  A policy based on a restricted number of agents also  enables price reduction on purchasing costs through  competitive tendering. The above activities have had a  major influence on containing or reducing drug costs,  although these savings have often been lost as new and  often expensive preparations become available, particu-  larly in the field of biological and anticancer therapy.   Another increasingly important argument in favour of  an antibiotic policy is the occurrence of drug-resistant  bacteria within an institution. The presence of sick  patients and the opportunities for the spread of micro-  organisms can produce outbreaks of hospital infection.    The excessive use of selected agents has been associated  with the emergence of drug-resistant bacteria which have  often caused serious problems within high-dependency  areas, such as intensive care units or burns units where  antibiotic use is often high. One oft-quoted example is  the occurrence of a multiple antibiotic-resistant K. aero-  genes within a neurosurgical intensive care unit in which  the organism became resistant to all currently available  antibiotics and was associated with the widespread use of  ampicillin. By prohibiting the use of all antibiotics, and  in particular ampicillin, the resistant organism rapidly  disappeared and the problem was resolved.   Currently the most important hospital-acquired path-  ogen is methicillin-resistant Staph, aureus, which is  responsible for a range of serious infections such as pneu-  monia, postoperative wound infection and skin infec-  tions which may in turn be complicated by bloodstream  spread. The use of vancomycin and teicoplanin has esca-  lated as a consequence, and in turn has been linked to the  emergence of vancomycin-resistant enterococci. Likewise,  the increased prevalence of ESBL-producing Gram-  negative pathogens in intensive care settings often leads  to increased usage of carbapenems, and a corresponding  rise in carbapenem-resistant Klebsiella and Pseudomonas  species on these units.   In formulating an antibiotic policy, it is important that  the susceptibility of microorganisms be monitored and  reviewed at regular intervals. This applies not only to the  hospital as a whole, but to specific high-dependency units  in particular. Likewise general practitioner samples  should also be monitored. This will provide accurate  information on drug susceptibility to guide the prescriber  as to the most effective agent.   There are a number of different approaches to the organi-  zation of an antibiotic policy. These range from a deliber-  ate absence of any restriction on prescribing to a strict  policy whereby all anti-infective agents must have expert  approval before they are administered. Restrictive policies  vary according to whether they are mainly laboratory-  controlled, by employing restrictive reporting, or whether  they are mainly pharmacy-controlled, by restrictive dis-  pensing. In many institutions it is common practice to  combine the two approaches.   The advocates of a free prescribing policy argue that strict  antibiotic policies are both impractical and limit clinical  freedom to prescribe. It is also argued that the greater the    number of agents in use the less likely it is that drug  resistance will emerge to any one agent or class of agents.  However, few would support such an approach, which is  generally an argument for mayhem.   Another approach that is widely practised in the UK is  that of restricted reporting. The laboratory, largely for  practical reasons, tests only a limited range of agents  against bacterial isolates. The agents may be selected pri-  marily by microbiological staff or following consultation  with their clinical colleagues. The antibiotics tested will  vary according to the site of infection, as drugs used to  treat urinary tract infections often differ from those used  to treat systemic disease.   There are specific problems regarding the testing of  certain agents such as the cephalosporins, where the  many different preparations have varying activity against  bacteria. The practice of testing a single agent to represent  first-generation, second-generation or third-generation  compounds is questionable, and with the new com-  pounds susceptibility should be tested specifically to that  agent. By selecting a limited range of compounds for use,  sensitivity testing becomes a practical consideration  and allows the clinician to use such agents with greater  confidence.   As mentioned above, the most draconian of all antibiotic  policies is the absolute restriction of drug dispensing  pending expert approval. The expert opinion may be pro-  vided by either a microbiologist or infectious disease spe-  cialist. Such a system can only be effective in large  institutions where staff are available 24 hours a day. This  approach is often cumbersome, generates hostility and  does not necessarily create the best educational forum for  learning effective antibiotic prescribing.   A more widely used approach is to divide agents  into those approved for unrestricted use and those for  restricted use. Agents on the unrestricted list are appro-  priate for the majority of common clinical situations. The  restricted list may include agents where microbiological  sensitivity information is essential, such as for vancomy-  cin and certain aminoglycosides. In addition, agents that  are used infrequently but for specific indications, such as  parenteral amphotericin B, are also restricted in use.  Other compounds that may be expensive and used for  specific indications, such as broad-spectrum (3-lactams in  the treatment of Ps. aeruginosa infections, may also be  justifiably included on the restricted list. Items omitted    from the restricted or unrestricted list are generally not  stocked, although they can be obtained at short notice as  necessary.   Such a policy should have a mechanism whereby desir-  able new agents are added as they become available and  is most appropriately decided at a therapeutics commit-  tee. Policing such a policy is best effected as a joint  arrangement between senior pharmacists and microbiol-  ogists. This combined approach of both restricted report-  ing and restricted prescribing is extremely effective and  provides a powerful educational tool for medical staff  and students faced with learning the complexities of  modern antibiotic prescribing.   In an attempt to ensure antimicrobials are prescribed  appropriately in hospitals, antimicrobial stewardship  teams have emerged to advise and educate staff while  monitoring compliance with prescribing policies as well  as ensuring good standards of patient management.  Typically these teams comprise, at their core, a consultant  in infectious diseases and/or clinical microbiology, and a  senior pharmacist specializing in infectious diseases, and  may also include infection control practitioners. The  team takes a lead in reviewing the therapy of individual  patients and setting treatment plans, and, often as part of  a wider team, will coordinate the writing and review of  antibiotic treatment policies. Other responsibilities may    include the education and training of clinical staff, the  audit of how well prescribers are adhering to the carefully  written policies, and the provision of feedback to pre-  scribers. Evidence suggests that this multidisciplinary  approach, aligned with targeted and timely feedback, can  improve adherence to prescribing policy, reduce drug  expenditure, and improve patient outcomes. Antibiotic  stewardship is discussed further in Chapter 15.   Cohen, J. & Powderley, D. (2003) Infectious Diseases, 2nd edn.  Mosby, Philadelphia.   Farrell, D.J., Morrissey, I., De Rubeis, D., Robbins, M.,  Felmingham, D. (2003) A UK multicentre study of the anti-  microbial susceptibility of bacterial pathogens causing urinary  tract infection. / Infect, 46, 94-100.   Finch, R.G. (2001) Antimicrobial therapy: principles of use.  Medicine, 29, 35-40.   Finch, R.G., Greenwood, D., Norrby, R. & Whitley, R. (2002)  Antibiotic and Chemotherapy, 8th edn. Churchill Livingstone,  Edinburgh.   Mandell, G.L., Bennett, J.E., & Dolin, R. (2004) Principles and  Practice of Infectious Diseases, 6th edn. Churchill Livingstone,  Philadelphia, PA.   In testimony to a US government committee in June  2010, the Infectious Diseases Society of America (IDSA)  stated that   Most commonly used antibiotics cost only a few dollars for  a typical course of treatment . . . (and) a single course of  antibiotics has the potential to protect and preserve many  quality years of life for many people. No other type of  medicine can claim such an achievement at such a price.  These statements were made as part of the argument pre-  sented by the IDSA to promote both antibiotic research and  appropriate use (‘stewardship’) of antibiotics.   The wider point being made was that antibiotics  have, since their discovery in the 1940s, revolutionized  medicine, and many of the procedures that are taken  for granted now — transplantation, cancer treatment, the  care of premature babies and several forms of surgery —  would be impossible without them. Yet, unfortunately,  largely because they have been taken for granted, the  antibiotics we already possess are becoming less effective  as a result of bacterial resistance, and the prospects for  producing new antibiotics currently look bleak. It was    recognized right from the start of the antibiotic era that  bacteria had the potential to develop resistance to anti-  microbial drugs, but it was quite some time before the  perception of antibiotic resistance changed from one in  which it was regarded as unusual to one where it was  expected; in other words, a recognition that long-term  efficacy was the exception, and resistance was the rule.   As the 20th century drew to a close the emergence of  the antibiotic-resistant pathogens described in Chapter  13 brought with it both the spectre of untreatable infec-  tions where the organisms responsible were resistant to  all available agents and a growing sense of urgency to take  steps to preserve the usefulness of the antibiotics we cur-  rently have. The situation was exacerbated by a reduction  in the number of new antibiotics coming into clinical use.  Figure 15.1 shows that this number has continued to  decline steadily for the last 20 years — a trend that would  be difficult to reverse in the short term simply because  several of the major international pharmaceutical com-  panies are moving out of antimicrobials as a research and  development area. Unfortunately, antibiotics have, in a  sense, become victims of their own success: the more  effective an antimicrobial is, the shorter the likely dura-  tion of treatment, so the lower the pay-back to the  company that developed it. Many courses of antibiotic    Hugo and Russell's Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   Figure 15.1 Number of new antibacterial  agents licensed for use by the FDA, 1983-  2007. Spellberg (2007). Reproduced  with permission of the University of  Chicago Press.    treatment last for a week or less, so the sales accruing  from them are far inferior to those from drugs treating  chronic conditions like diabetes and hypertension. This  fact, together with ( 1 ) increasing pressure to use antibiot-  ics sparingly anyway, (2) the expectation that the drug  will ultimately become less effective due to resistance and  (3) difficulties in establishing clinical trials for antibiotics  that satisfy the US Food and Drugs Administration  (FDA) criteria, all combined to create a climate in which  antibiotics became an unattractive commercial proposi-  tion (except in the case of HIV/AIDS therapies that have  to be taken throughout the patient’s life).   Certainly the problem of growing antibiotic resistance  had been recognized for some time before the end of  the 20th century and policies designed to improve the  quality of antibiotic prescribing and restrict resistance in  hospitals became progressively more common in Europe  and North America from the 1970s onwards. However,  the increasing frequency in the new millennium of  infections due to the so-called ‘ESKAPE’ pathogens  (. Enterococcus faecium. Staphylococcus aureus, Klebsiella  species, Acinetobacter baumannii, Pseudomonas aerugi-  nosa and extended spectrum (3-lactamase-producing  strains of Escherichia coli and Enterobacter species)  together with the drying up of the pipeline of new anti-  biotics from the pharmaceutical industry has further  increased the tempo of measures to preserve what is  increasingly being seen as a precious, and perhaps irre-  placeable, resource that society has a duty to pass on to    future generations rather than squander. These measures  include the ‘10 x 20’ initiative to establish an interna-  tional research effort to develop 10 new antibiotics by  the year 2020, and the STAAR (Strategies to Address  Antimicrobial Resistance) Act, which is, at the time of  writing, being considered by the US Congress.   The cost of antibiotic resistance should, of course, be  measured primarily in terms of the suffering that results  from the failure of antibiotics to cure infections against  which they were formerly effective. The antibiotic-  resistant pathogens responsible for these infections can  arise both in the home or the hospital environment, but  it is in the latter that they are, by far, more common and  problematic. Hospital-acquired infections account for a  substantial number of deaths each year and the treatment  of such infections is time-consuming, difficult and costly.  In its testimony to Congress as part of the consultation  process accompanying the STAAR Act, the IDSA stated  that antibiotic-resistant infections acquired within hospi-  tals were responsible for 90000 deaths each year in the  USA and cost the healthcare system between $21 and $34  billion annually. There are less obvious consequences of  resistance too: when the first-line drugs cease to be effec-  tive it is sometimes necessary to revert to alternatives that  are more toxic. Acinetobacter infections are a good  example of this situation because the organism is natu-  rally multidrug resistant and the incidence of isolates  resistant to all first-line antibiotics has risen in the USA  from 5% to 40% in 10 years, so now colistin, a drug that    became virtually obsolete in the 1960s because of the  significant risk of kidney damage is, for many patients,  the most likely antibiotic choice.   There is widespread agreement that greater use of anti-  biotics predisposes to the development of resistance. The  strength of the link between use and resistance varies  from one antibiotic to another, but for many antibiotics  the connection is irrefutable. However, the situation is far  from simple: there is substantial evidence, for example,  that heavy use of one antibiotic may be a risk factor for  the acquisition of infections by organisms resistant to  other, unrelated antibiotics — heavy cephalosporin use  has been shown to increase the risk of vancomycin-  resistant enterococci, and fluoroquinolone use has been  associated with the prevalence of meticillin-resistant  Staph, aureus (MRSA). The selective pressure created by  the use of one antibiotic will often select for resistance in  others because plasmids within the bacterial cell may  carry resistance genes for multiple antibiotics from dif-  ferent chemical groups. If, for example, an organism pos-  sessed a plasmid with genes for both rifampicin and  gentamicin resistance, constant exposure to gentamicin  would represent a selective pressure that afforded an  advantage to that organism so, not only would the inci-  dence of isolates with gentamicin resistance be expected  to rise, but so too would the incidence of rifampicin  resistant isolates.   Arguments for curtailing antibiotic use in order to  restrict resistance development have been supported by  audits and surveys of antibiotic prescribing and costs. It  has been estimated that up to 50% of antibiotic prescrib-  ing is either inappropriate (wrong drug, duration, dose,  etc.) or unnecessary (not required at all). Antibiotic  consumption varies widely throughout Europe: a 2005  survey showed that France, for example, used three  times as much antibiotics per head of population as the  Netherlands, but this difference was not justified by  higher infection rates or better cure rates, so logic sug-  gests that part of the antibiotic use in France was  unnecessary. Inappropriate prescribing and consump-  tion, together with the fact that antibiotics can represent  up to 30% of a hospital pharmacy budget, have provided  further impetus for measures designed to achieve more  prudent prescribing. Such thinking is not new, however;  it was the rationale for the introduction of antibiotic  policies in the 1970s and 1980s which, in addition to  setting out the general standards for safe and appropriate  prescribing, advised on the selection of antibiotics for  specific infections, for special situations like surgical  prophylaxis and for the treatment of specific groups of    patients, e.g. the newborn, those with poor kidney func-  tion and the immunocompromised. What has changed  since then is the recognition of the need for a much  broader approach to the problem — in other words, the  need for a comprehensive antimicrobial stewardship  strategy that incorporates, but also extends, the policies  formulated in the last century.   There is not a universally accepted definition of, or  agreement upon, what constitutes an antimicrobial  stewardship programme. For most people the term  applies particularly, or even exclusively, to the manner in  which antibiotics are used and distributed in hospitals.  However, some see it in a much broader sense as a range  of initiatives which, together, impact upon antibiotic  resistance but are not necessarily even confined to  antibiotic-related practices in the hospital or the home.  The great majority of the annual global production of  antibiotics is not used in the treatment of human or  animal infection anyway. Most of the antibiotic output of  the international pharmaceutical industry is used as a  food additive to increase weight gain in cattle, pigs and  poultry — some estimates put this proportion as high as  80% — and yet more is used in plant production, but this  fraction is ill-defined. Although antibiotics that are  used to treat human infections have been banned as  growth promoters in Europe for many years it is still a  common practice in many countries, and even the legiti-  mate veterinary use of antibiotics is considerable: the  total volume of antibiotics used in the UK for agricultural  purposes in 2007 was 387 tonnes. Curtailment of the use  of antibiotics for growth promotion — which many see as  inappropriate and a likely contributor to resistance  development — together with better-targeted and -pro-  moted use of vaccines (that would reduce the need for  antibiotics), better diagnostic agents which would more  rapidly and accurately identify the infecting organism  and so inform the selection of the best antibiotic, better  epidemiological data and computer analysis to provide  early warnings of resistance trends, and changes in other  medical practices like the early removal of catheters  and cannulas which are, themselves, a means by which  pathogens can enter the body, might all be seen as part  of a stewardship programme. But from the perspective of  controlling the incidence and spread of antibiotic-  resistant organisms within a hospital it cannot be over-  emphasized that a comprehensive infection control  programme is of paramount importance (see Chapter  16) and the best results are achieved when data from  antibiotic stewardship and infection control can be linked  and analysed together.    It is generally accepted that the principal goals of a  stewardship programme are to:   • Reduce resistance levels, or at least slow the rate of  resistance development   Following the international financial crisis that began  in 2007 it is likely, at least in many European countries,  that the last of these goals will receive particular atten-  tion, so it is worth emphasizing that stewardship pro-  grammes can be self-financing. Although there is an  initial start-up cost, there is substantial evidence that this  is rapidly recovered by cost savings resulting from reduc-  tions in antimicrobial use that some reports have esti-  mated to vary from 22 to 36%.   There are no nationally or internationally accepted  guidelines on the structure of a stewardship programme,  so they vary from country to country and even from  one hospital to another within a geographical region.  Indeed, even the titles vary, so terms like ‘good antimi-  crobial practice programmes’, ‘antibiotic management  programmes’ and ‘antibiotic control programmes’  have all been used. There is, however, a measure of agree-  ment on the personnel comprising an antimicrobial stew-  ardship team. They are multidisciplinary and usually  consist of:   In the USA the IDSA and the Society for Heathcare  Epidemiology of America (SHEA) set forth stewardship  guidelines in 2007 that have been widely adopted both in  the USA and elsewhere; but in Europe the situation is less  uniform. Here, an expert group reported in 2000 that  little was known about which stewardship measures were  employed in Europe and which were optimal. As a con-  sequence, the European Commission Concerted Action  Antibiotic Resistance Prevention and Control (ARPAC)  survey of stewardship practices in 263 hospitals through  32 European countries was undertaken, and it reported  in 2009. Table 15.1 lists both the components of the US    guidelines and the six key indicators of stewardship that  were the subject of the ARPAC survey.   Before considering in more detail the elements of a  stewardship programme, it is worth explaining the  meaning of some of the terms used in Table 15.1. In this  context a prospective audit means a review of the future  delivery of healthcare to ensure that best practice is being  carried out, so a prospective audit with intervention and  feedback is a process in which the use of antibiotics is  monitored and suggestions made for improvement,  where necessary, while the course of treatment is still in  progress. The feedback element is the provision of infor-  mation to the prescriber about the drug in question, e.g.  local resistance patterns and dose information based  upon pharmacokinetic data.   Most hospitals have a drugs and therapeutics commit-  tee (DTC; sometimes also known as a pharmacy and  therapeutics committee) whose function is to evaluate  drugs for inclusion in a hospital formulary on the basis  of their efficacy, toxicity and cost. The intention is to  avoid the unnecessary stocking of multiple drugs from  the same class which have similar or identical properties;  few hospitals stock every available aminoglycoside or  third-generation cephalosporin, for example. An antibi-  otics committee would be a subgroup of the DTC with  responsibility for selecting the drugs for inclusion in the  antibiotics formulary. The strategy of formulary restric-  tion and preauthorization could limit the availability of  certain antibiotics simply by excluding them from the  formulary (although they could usually be obtained in  exceptional circumstances if required). The preauthori-  zation element would simply be a requirement to seek the  recommendation (or even written approval) of senior  clinical staff when prescribing antibiotics in a restricted  category — usually those drugs whose effectiveness needs  to be preserved because of their value in treating infec-  tions for which few drugs are effective, e.g. linezolid or  daptomycin, or antibiotics which may predispose to  other problems, e.g. clindamycin because of its link to  Clostridium difficile outbreaks.   The ARPAC stewardship indicator that the hospital  should have a strategic management goal of improving  prescribing has important implications for the provision  of resources and granting the authority to implement  decisions. Establishing a stewardship programme costs  money: staff time must be available for (1) the prepara-  tion of the antibiotics policy document and formulary,  (2) intervention and feedback, and (3) monitoring of  antibiotic use and resistance patterns. It is easier to gain  approval for the necessary budget allocation if there is a    Core strategies and elements of a stewardship  programme as recommended by the ISDA and SHEA    Two core strategies (not necessarily mutually exclusive):  Prospective audit with intervention and feedback  Formulary restriction and preauthorization   Guidelines and clinical pathways  Antimicrobial order forms  De-escalation of therapy  Parenteral to oral conversion  Dose optimization  Antimicrobial cycling  Combination therapy   A written antibiotic formulary  A formulary that included a restricted antibiotic list  A drugs and therapeutic committee  A strategic management goal of improving prescribing    ISDA, Infectious Diseases Society of America; SHEA, Society for Healthcare Epidemiology of America.    publically stated management goal of improving pre-  scribing. Where preauthorization for selected antibiotics  is a part of the programme, the greater the authority  bestowed on the person(s) making the recommenda-  tions, the greater the effectiveness of the policy has been  shown to be.   How effective a particular action is will be determined by  the parameter used to measure it. In the case of steward-  ship programmes almost all of the individual compo-  nents of the two IDSA/SHEA strategies (Table 15.1) can  be shown to reduce resistance development, adverse  effects or costs, but there are few actions that result in  favourable changes in all three criteria.   Both of the core strategies of prospective audit and  formulary restriction are active measures and, as such, are  more effective than passive ones. Prospective audit/  intervention has been shown to reduce inappropriate use  of antibiotics, achieve cost savings and, in some cases,  restricted the isolation of particular antibiotic-resistant  organisms. Formulary restriction/preauthorization has  similarly been shown to reduce antibiotic consumption,  and this may be immediate and significant, but its long-  term impact on restricting resistance development is not    proven. Education of patients to dissuade them from  pressurizing prescribers for antibiotics — particularly for  colds and flu or other viral infections — is now well estab-  lished, but education of prescribers (conference presenta-  tions, teaching sessions, e-mail alerts and bulletins) is a  passive approach which, by itself, has been shown to have  only a marginal and short-term impact. Clinical path-  ways (also known as critical care pathways or care maps)  are intended to reduce the variability both in the quality  of care and in patient outcomes by the adoption of  defined, standardized and sequenced procedures for  patients with specific conditions — in this context, infec-  tions. The use of a clinical pathway that includes a  specified antibiotic regimen in the treatment of  community-acquired pneumonia, for example, has been  shown to be capable of reducing the duration of hospital  stay and duration of antibiotic therapy, and there is  strong evidence that practice guidelines and clinical path-  ways incorporating local resistance patterns can generally  improve antimicrobial utilization.   Four other elements of the IDSA/SHEA that have  been shown to afford clear benefits are the use of antimi-  crobial order forms, de-escalation of therapy, parenteral  to oral conversion and optimized dosing. Order forms  are particularly useful when antibiotics are prescribed  for prophylactic purposes to reduce the incidence of  infection following surgery. In this situation there is a  tendency to continue the course of treatment for an    unnecessarily long period after the operation, and  studies have shown that order forms with a default stop  date have diminished drug consumption with no adverse  effect. When hospital treatment for an infection begins,  it is often the case that the organism responsible has  not been identified, so initial treatment is empirical or  ‘blind’ and, in order to maximize the probability of inhib-  iting the pathogen, it may involve the use of either a  broad-spectrum antibiotic, or of two or more different  drugs. Once the organism has been identified it is good  practice to replace broad-spectrum antibiotics (or redun-  dant components of a combination) with a drug having  more specific activity, because the continued unnecessary  use of broad-spectrum therapy contributes to selection  of resistant pathogens. Converting from intravenous to  oral therapy affords a benefit primarily in terms of cost  reduction but may also permit earlier removal of intra-  venous lines which facilitate the establishment of infec-  tions by skin pathogens like Staph, epidermidis. Oral  antibiotics are usually cheaper than intravenous ones,  quite simply because the use of the latter is largely  restricted to hospitals anyway, so their manufacturers  have to recover the development costs from lower lifetime  sales. Optimized dosing is, as the name implies, modify-  ing the antibiotic dose to suit an individual patient’s cir-  cumstances. Factors that may influence the dose, and  hence the effectiveness of the therapy, include the patient’s  physical characteristics (weight, age, immune status, renal  function), the site of infection and the pharmacokinetics  of the drug which determine its access to, and concentra-  tion at, that infection site.   Two procedures that have been advocated as means  of restricting resistance development are cycling of anti-  biotics and routinely using them in combination, par-  ticularly for infections with a long time-course of  treatment. However, there is little evidence to support the  use of routine cycling — the planned replacement of one  specific antibiotic, or category of antibiotics, with another  at predetermined intervals — as a means of controlling  resistance. True cycling involves the return into use of  the original antibiotic after a specified time, and although  the first switch may lead to a reduced incidence of resist-  ance to the first drug, some studies have shown that when  it is subsequently reintroduced the original resistance  level is quickly restored. Using antibiotics in combination  as a means of restricting resistance development is well  established and of undoubted benefit in the treatment of  both tuberculosis, for which the duration of therapy is  typically 6 months, and in HIV/AIDS which requires life-  long treatment. During such long time periods the    number of pathogen replication cycles is so large, and in  these two examples the mutation frequency is so high,  that the probability of a resistant mutant arising and  being selected as the predominant strain at the site of  infection is significant, so using two or more agents with  different modes of action is both logical and effective.  However, the same logic has been applied to other, rela-  tively short-term, infections without clear evidence of  benefit.   To get the most out of a newly implemented steward-  ship programme (or a change to an existing one) it  is necessary to have the means by which to measure  its effect. It is, therefore, necessary to plan in advance  what parameters will be measured and what will be the  baseline data against which the changes will be judged.  Some of the parameters might include, but are not  restricted to:   • Antibiotic consumption and costs, both in total and by  specific drug class   • Costs associated with prescribing potentially toxic  antibiotics, e.g. gentamicin and vancomycin blood level  monitoring   One of the problems that has dogged reviewers trying  to assess the extent of the benefits of stewardship pro-  grammes is that in many of the cases reported in the  medical literature multiple changes to an established pro-  gramme have been introduced together, or they have  overlapped in time so that evaluating the contribution of  each change has been difficult. It is, therefore, worthwhile  deciding in advance when a new policy or practice will  be implemented and when its effect will be assessed.  The input of information technology specialists and hos-  pital epidemiologists to a stewardship management team  becomes important since they, together, can decide how  the data will be recorded and analysed to best effect.   The more sophisticated antibiotic control and infor-  mation systems do not simply record data on antibiotic  consumption, cost and resistance, but are capable of  relating infection control data to antibiotic use and would  be expected to draw attention to situations where a  change in use of a particular antibiotic was associated    with increasing isolation of a particular pathogen. Such  an association does not, of course, mean that one caused  the other, but it does raise staff awareness of that poten-  tial. Computer-assisted surveillance of hospital-acquired  infection (HAI) has been shown, in some cases, to be  more effective than manual monitoring and reporting; as  long ago as 1986 one study reported that 90% of  antibiotic-resistant HAIs were detected by computer  compared to 76% manually. There is also the potential to  improve antibiotic prescribing by minimizing the risk of  adverse effects when information systems provide  patient-specific warnings on allergies, immune and renal  functions and the potential for interaction with the  patient’s other drugs.   It is not surprising, perhaps, that there are marked  differences in antibiotic resistance patterns from one  country to another. This is illustrated in Figure 15.2,  which shows that penicillin resistance in Streptococcus    pneumoniae can vary from less than 5% in some European  countries to more than 50% in others. Smaller, but nev-  ertheless significant, variations may also arise between  different regions of a single country; Figure 15.3 shows  the corresponding Strep, pneumoniae data for the UK  from the national Heath Protection Agency. However,  data on local resistance patterns are of paramount impor-  tance and well-structured monitoring programmes  should be capable of identifying unforeseen consequences  of changes in antibiotic use such as that arising when a  preapproval policy for cephalosporins was introduced in  a New York hospital in an attempt to control cepha-  losporin resistance in Klebsiella species. The policy did  achieve a 7 1 % reduction in ceft azidime-resistant Klebsiella  isolated in intensive care units, but monitoring revealed  a concomitant rise in imipenem use and a 69% increase  in imipenem-resistant Ps. aeruginosa that was attributed  largely to the preapproval policy.   Figure 15.2 Incidence of penicillin-resistant Strep, pneumoniae in Europe, 2008. (From the European Antimicrobial Resistance  Surveillance System; reproduced with permission.)    Figure 15.3 Incidence of Strep,  pneumoniae in different regions of the  UK, 2007. (From the UK Health  Protection Agency; reproduced with  permission).   5 The impact on resistance of antibiotic  availability to the public   The ease of availability of antibiotics to the general public  varies throughout the world. In Europe the Council of  the European Union recommended in 2001 that member  states should support the prudent use of antibiotics by  restricting systemic antibacterial agents to prescription-  only availability. In many other countries, though, anti-  biotics are supplied without a prescription — either  because the practice is not illegal, or because the law is  not enforced. Even in the USA this is so: in its testimony  to Congress referred to in the first sentence of this chapter  the IDSA posed the question Antibiotics’ true value: “pre-  cious resource” or a “giveaway marketing tool”?’ They  were referring to the practice ‘where grocery stores and  pharmacies give prescribed antibiotics away for free as a  marketing ploy to lure customers into their stores’. Self-  medication with antibiotics (obtained via the internet or  simply by patients using left-over antibiotics prescribed  at an earlier date for unrelated infections) is another  practice that contributes to uncontrolled and often inap-  propriate antibiotic use and so contributes to the resist-  ance problem.   In the UK there has been a slow movement of topical  anti-infective agents from prescription-only medicine  (POM) to pharmacy-only (P) status; thus, clotrimazole,  chloramphenicol and aciclovir creams, and others,  have all undergone this reclassification, but in late 2008  azithromycin became the first systemic antibiotic availa-  ble in pharmacies without prescription, albeit for the    defined purpose of treating chlamydial infection. This  change in policy by the UK Medicines and Elealthcare  products Regulatory Agency (MHRA) received a mixed  reception. It was welcomed by some as a step towards the  desirable objectives of increasing patient choice and  encouraging patients to take responsibility for their own  healthcare, but executive officers of the British Society of  Antimicrobial Chemotherapy strongly criticized the  change as a retrograde step that is likely to lead to  increased use without professional advice and, as a con-  sequence, increased resistance. The situation was exacer-  bated by proposals for similar reclassification of other  antibiotics, e.g. trimethoprim and nitrofurantoin, both  for the treatment of cystitis, but in March 2010 the UK  government stepped in to halt MEIRA reconsideration of  the trimethoprim application.   Apart from the likely increase in use leading to  increased resistance, one of the strongest arguments  against the availability of antibiotics without prescription  is that it would even remove the means by which the  consumption of a particular antibiotic could be moni-  tored and correlated with any resistance trends —  prescriptions can be counted, but sales are not! The  ability to operate such surveillance systems is an integral  component of stewardship programmes, so any change  in the legal status of systemic antibiotics to make them  available without prescription would not only flout the  EU recommendation but undermine the government’s  stated support for prudent antibiotic use. It would seem  that the decision to halt MEIRA consideration of the pro-  posal to reclassify trimethoprim means that the UK gov-  ernment has heeded the warnings that can be summarized    in the following quotation from the Chief Medical  Officer’s report for 2008:   The potency of one of the key weapons in the medical  armoury is being eroded. The harm caused by each unnec-  essary prescription is not visible at the time, and so society  fails to take action that is necessary to stop the problem  worsening. Correcting this situation will require a para-  digm shift in thinking. The effectiveness of antibiotics  should be seen as a common and collective public good.  Every antibiotic expected by a patient, every unnecessary  prescription written by a doctor, every uncompleted course  of antibiotics, and every inappropriate or unnecessary use  in animals or agriculture is potentially signing a death  warrant for a future patient.   Bruce, J., MacKenzie, E, Cookson, B., et al. (2009) Antibiotic  stewardship and consumption: findings from a pan-European  hospital study. / Antimicrob Chemother, 64, 853—860.   Chief Medical Officer’s Report (2008) Antimicrobial Resistance:  Up Against the Ropes, http://www.dh.gov.uk/prod_consum_   Dellit, T.H., Owens, R.C., McGowan, J.E. Jr, etal (2007) Infectious  Diseases Society of America and the Society for Healthcare  Epidemiology of America Guidelines for Developing an  Institutional Program to Enhance Antimicrobial Stewardship.  Clin Infect Dis , 44, 159-177.   Dryden, M.S., Cooke, J. & Davey, P. (2009) Antibiotic stewardship —  more education and regulation not more availability? /  Antimicrob Chemother, 64, 885—888.   Infectious Diseases Society of America (2010) Antibiotic resist-  ance: promoting critically needed antibiotic research and  development and appropriate use (‘stewardship’) of these pre-  cious drugs. Testimony presented by Brad Spellberg before the  House Committee on Energy and Commerce Subcommittee  on Heath, June 9, 2010. http://www.idsociety.org/WorkArea/  DownloadAsset.aspx?id= 16656 (accessed 17 July 2010).   Infectious Diseases Society of America (2010) The 10 x 20 ini-  tiative: pursuing a global commitment to develop 10 new  antibacterial drugs by 2020. Clin Infect Dis, 50, 159-177.   MacDougall, C. & Polk, R.E. (2005) Antimicrobial stewardship  programmes in health care systems. Clin Microbiol Rev,  18(40), 638-656.   The critical and defining element of all infections and  infectious diseases is their capacity to spread. The fact  that infection can spread from person to person (patient  to patient) by direct contact, or by the hands and clothes  of healthcare workers, or by contamination of equipment  or the environment, means that there is a public health  and infection control aspect to all infections. For every    patient who acquires an infection there is a need for  specific treatment for that patient, but we also need to  address the two infection control issues — from where (or  from whom) has the infection come, and how can we  minimize the risk of it spreading to others. This is the  subject of infection prevention and control.   Among the broad range of infections, those that  develop as complications of other illnesses or of the treat-  ment of those illnesses, possibly by cross-infection but  also by infection from the patient’s own bacterial flora,    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    are a major concern to health services throughout the  world. These are variously known as hospital acquired  infections, hospital cross-infections, or more generally  now, healthcare-associated infections (HCAIs) to reflect  the fact that they occur in patients throughout health and  social care settings, not just those in hospital. An increas-  ingly elderly population with a range of chronic medical  conditions and the tremendous success of modern  medical and surgical practice in increasing life expect-  ancy and treating previously fatal conditions means that  health services have to deal with many patients who are  highly vulnerable to infection. Invasive clinical proce-  dures are an essential part of most medical care, as are  treatment requirements causing significant immunosup-  pression, both of which are key risks for the patient devel-  oping an HCAI.   One of the main responsibilities of all clinicians is that  they should, as a priority, not harm their patients through  the investigations and treatment they give. Clearly, with  modern medical and surgical practice, all risks cannot be  eliminated, but the risks must be minimized, in particular  the risk of these vulnerable patients acquiring an infec-  tion as a direct result of their healthcare. Hence there is  a specific focus in all health services on the prevention  and control of HCAI.   HCAIs encompass a considerable variety of infections of  different body sites and systems caused by a wide range  of bacteria and some viruses and fungi. The types of  infection reflect all the body sites and systems that can be  the subject of medical intervention.   Any wound, accidental or surgical, breaches one of the  key barriers to infections — the skin. With accidental  wounds, contamination with dirt, soil and environmental  bacteria may be inevitable, but with ‘deliberate’ surgical  wounds, every effort must be made to minimize the risk  of postoperative wound/surgical site infection (SSI; the  term reflects the importance of infection at any part of  the surgical site, not only the obvious external wound).  These SSIs may come from the patient’s own normal  body flora (e.g. from the intestinal bacteria after abdomi-  nal surgery), from nose or skin carriage of wound patho-  gens such as Staphylococcus aureus by the patient, or by  cross-infection from other patients or staff as a result of    a breakdown of aseptic procedures and proper clinical  care. Protocols for clinical care of surgical wounds aim to  minimize the risk of the cross-infection, and antibiotic  prophylaxis combined with careful surgical practice aims  to minimize the risk from the patient’s own endogenous  bacteria.   Non-surgical soft tissues sites are also prime sources  of HCAI, particularly peripheral ulcers (vascular,  diabetic, etc.) and pressure sores (decubitus ulcers)  where the initial vascular insufficiency and tissue break-  down provides an ideal environment for bacterial  infection.   Potentially the most severe types of HCAI in terms of  outcome are bloodstream infections (bacteraemias). The  blood and the cardiovascular system should be sterile  and the presence of bacteria in the blood is an alarm  signal for a patient’s healthcare. Many bacteraemias are  part of infectious diseases not linked to healthcare risks  (bacterial meningitis, community acquired pneumonia,  acute pylonephritis) but others are important complica-  tions of healthcare. Almost all hospital treatment in  modern medical practice requires invasive procedures  with the insertion of various synthetic tubes and pros-  thetic devices. The insertion of indwelling intravascular  catheters and cannulae penetrates the protective layer of  the skin and provides a portal of entry for bacteria.  Central venous catheters are used increasingly for a range  of clinical investigations and treatments and carry a sig-  nificant risk of infection which starts on the artificial  surface of the catheter and then seeds the bloodstream  more generally, with the risk of clinical sepsis and infec-  tion of cardiovascular structures such as the heart valves  (endocarditis) or other metastatic infection sites. Short  peripheral intravenous cannulae carry less individual risk  of infection, but so many are used in modern clinical  practice that they are in fact the source of more HCAI  bacteraemias than central catheters. Other local sites of  HCAI (wounds, ulcers, urinary tract, respiratory tract)  can also lead to bacteraemia.   The commonest form of HCAI (see below) are urinary  tract infections, mostly as a result of indwelling urinary  catheters which inevitably become contaminated and  colonized with bacteria, then leading to infection of the  bladder and the lower urinary tract, then the ureters and  potentially the kidneys (pylonephritis). This can be an  important cause of bacteraemia.   Respiratory infections are some of the most common  types of infection in the general community and these  can also affect hospital patients, but some clinical condi-  tions and treatments predispose patients to healthcare-  associated respiratory tract infections. During the  postoperative period surgical patients are particularly  vulnerable to pneumonia; they will have undergone  endotracheal intubation for their anaesthesia and post-  operative discomfort and inactivity may lead to inade-  quate ventilation of their lungs and inability (or  disinclination) to cough, resulting in postoperative pneu-  monia. This risk is greatly magnified in patients needing  intensive care and undergoing prolonged intubation and  artificial ventilation. Ventilator-associated pneumonia  (VAP) is one of the major challenges to successful inten-  sive care. As well as the local effects of VAP on the respira-  tory tract, it is also a significant cause of bacteraemia.   Infectious diarrhoea is a well-recognized community  syndrome. Some of the issues that result in diarrhoea and  vomiting in the population at large, such as mass catering  leading to food poisoning with Salmonella and  Campylobacter spp. also apply in hospitals and other  healthcare and social care settings but other gastrointes-  tinal infections cause particular problems in relation to  healthcare settings. Norovirus vomiting and diarrhoea  spreads so readily in closed communities that it causes  numerous and frequent outbreaks in all health and social  care settings and is the commonest cause of ward closures  in the NHS in England to control the spread of this virus.  One bacterial cause of potentially severe, indeed fatal,  diarrhoeal disease that is much more specifically an  HCAI is Clostridium difficile infection (CDI). This infec-  tion generally affects only those whose normal gut bacte-  rial flora has been disturbed by treatment with  broad-spectrum antibiotics (or some other infection  proven or suspected) which enables the Cl. difficile spores  to germinate and then the vegetative bacteria to produce  their damaging toxins.   Although some infectious agents causing HCAI are also  involved in community acquired infectious diseases  (norovirus, Salmonella spp., Staph, aureus), many HCAIs  are caused by organisms particularly associated with  healthcare. These bacteria are generally characterized by    two properties: they tend to be resistant to many antibiot-  ics and have been selected by pressure of antibiotic usage  in healthcare, and they are also generally opportunist  pathogens with less capacity to produce illness in other-  wise fit and healthy people but able to cause severe and  life-threatening infection in those who are vulnerable and  most susceptible because of their underlying diseases  and/or their treatment. Some, in particular, can be associ-  ated with artificial implants and prostheses.   Staph, aureus is inextricably linked with human health  and disease. Its natural habitat is the human skin particu-  larly the anterior nares and the warm, moist skin folds of  the perineum (and groin) and axilla and at any one time  about one third of people are colonized with Staph,  aureus, alternatively known as ‘carriers’. The nose is the  principle carriage site in most (probably all but not  always detectable) carriers with the skin sites also colo-  nized in a smaller number. Staph, aureus has always been  the most common cause of wound infections (accidental  and surgical) and a leading cause of HCAIs. When peni-  cillin was first used clinically in the early 1940s, almost  all Staph, aureus strains were susceptible to it and it  was widely used. By the late 1950s, 95% were resistant  to penicillin due to natural selection of penicillinase  ((3-lactamase)-producing strains. Meticillin (formerly  known as methicillin), and the later oxacillin, cloxacillin  and flucloxacillin, was developed to resist breakdown by  (3-lactamase and restore treatment options. Within a year  of introduction, the first meticillin-resistant Staph, aureus  (MRSA) strain was described. MRSA were interesting  rarities in the 1960s and through the 1970s. In the 1980s,  some MRSA strains (now called epidemic strains; EMRSA  with sequential numbers) caused isolated and restricted  outbreaks of HCAI which were mostly contained by a  ‘search and destroy’ approach (i.e. isolate and treat the  patient; screen all contacts among patients and staff for  carriage; give decolonization treatment to any found to  be positive). However, in the early 1990s in the UK, two  EMRSA strains, 15 and 16, emerged with greater capacity  to spread in healthcare settings and to cause a higher  proportion of severe disease. Their spread through the  healthcare system was not controlled and these strains  became a major HCAI problem and the subject of media  publicity, pressure group campaigns, and political  concern in the early years of the 21st century. These  strains were difficult to treat and caused significant mor-  bidity and mortality. However, it must be remembered    that MRSA is not a disease; it is a group of Staph, aureus  strains that cause a wide range of HCAIs — wound and  soft tissue infections, VAP, catheter-associated UTI, as  well as the bacteraemias that have been a major focus for  surveillance and preventive measures as being the most  severe end of the spectrum of MRSA disease.   Everyone carries Staph, epidermidis, and sometimes other  coagulase-negative staphylococci (CNS), on their skin as  part of the normal flora. These CNS are often antibiotic  resistant (including being meticillin resistant) but have  much less pathogenic potential than Staph, aureus for  healthy people. However, they are one of the most  common causes of HCAI associated with indwelling arti-  ficial devices and prosthesis. Despite the emphasis on  MRSA bacteraemia, S. epidermidis is the most common  cause of intravenous line-associated bacteraemia, but  with more low-grade or ‘grumbling’ clinical presentations  and fewer severe and fatal infections than MRSA. It is also  the commonest cause of late-onset deep infections of  implanted prostheses such as hip and knee joints and  heart valves. In this respect they cause significant morbid-  ity and treatment often requires removal and replace-  ment of the original implant.   Several distinct groups of Gram-negative bacilli have the  capacity to cause opportunist infections in vulnerable  patients, particularly those with various forms of immu-  nosuppression. All share a general characteristic of being  able to survive in moist environmental conditions and  being resistant to a range of antibiotics so that their sur-  vival and spread is selected by the widespread use of  antibiotics in hospitals and other healthcare settings.  This group of opportunist pathogens has long included  organisms such as Pseudomonas aeruginosa (or other  Pseudomonas species), Acinetobacter spp. and the resistant  enterobacterial relatives of Escherichia coli — Enterobacter,  Serratia and Klebsiella spp. All have caused serious infec-  tions in immunocompromised patients, e.g. patients  undergoing chemotherapy for a malignant disease in  whom Gram-negative bacteraemias have a high inci-  dence, often leading to clinical sepsis and death. Ps. aeru-  ginosa causes similar infections but also has a long  association with pulmonary infection in cystic fibrosis  patients.   Two further challenges from Gram-negative bacteria  are causing concern with the emergence of new antibiotic    resistance markers. The (3-lactam antibiotics (penicillins  and cephalosporins) have been mainstays of treatment of  E. coli and related Gram-negative bacteria for many years.  Strains of E. coli have now emerged that have acquired  genes for the production of extended spectrum 13-  lactamases that break down all (3-lactam agents. These  ESBL-producing E. coli have become widely established  in patients in hospitals and in other health and social care  settings. Gut colonization is linked to urinary tract infec-  tion, especially when the patients are catheterized, and  this can then result in bacteraemias that are very difficult  to treat. Carbapenen antibiotics (e.g. imipenem) are impor-  tant for treatment of these resistant Gram-negative HCAIs,  so it is of special concern that some carbapenemase-  producing Klebsiella spp. and other Gram-negative  species are now being found in some UK and other  European hospitals.   Enterococci (Enterococcus faecalis and Ent. faecium ) have  been recognized causes of HCAI in immunocompro-  mised patients, e.g. post-transplant (including bone  marrow recipients) and cancer chemotherapy. These  organisms are intrinsically resistant to many antibiotics  and the mainstays of treatment where the glycopeptides  vancomycin and teicoplanin. There was particular  concern in the early 2000s when glycopeptide-resistant  strains of Ent. faecium caused small but severe outbreaks  in transplant and chemotherapy units. A suggested link  to glycopeptide use in agriculture has not been proven,  and although such strains still cause some serious infec-  tions, they have not become a widespread problem in  other patient groups.   Cl. difficile was discovered to be the major cause of  antibiotic-associated diarrhoea and colitis simultane-  ously by groups in the UK and the USA in 1978. Although  the subject of much interest and research amongst anaer-  obe microbiologists and a few infectious diseases physi-  cians, it did not reach popular notoriety until the middle  of the first decade of the 21st century. Cl. difficile is an  anaerobic spore-forming organism found in the gut of  many species of animal, including humans. With conven-  tional bacteriological methods, about 3% of healthy  people are found to carry Cl. difficile in their faeces.  However, the figure is much greater in hospitalized  patients and in residential nursing and care home resi-  dents. Patients with Cl. difficile diarrhoea excrete large  numbers of spores into their surrounding environment    where they can survive for weeks, months and even years.  People with a normal, healthy gut flora can swallow Cl.  difficile spores without ill effect, but in patients whose  normal intestinal flora is disturbed, especially and most  commonly by use of broad-spectrum antibiotics, the  normal inhibition of Cl. difficile germination and growth  is removed and Cl. difficile grows rapidly. The vegetative  C. difficile cells attach to the gut mucosa and produce two  toxins (A and B) that cause the disease. Toxin A is specifi-  cally called an enterotoxin whereas toxin B is a cytotoxin,  but they show considerable structural and functional  overlap and toxin B in particular can produce severe  disease on its own when a strain that is A-negative/B-positive  is the cause of the infection. The toxins cause diarrhoea,  ranging from mild to profuse and debilitating, and  colonic ulceration (which appears as erupting volcanoes  on microscopy and biopsy specimens) that can develop  into pseudomembranous colitis with, at worst, toxic  megacolon and colonic perforation.   The directly attributable mortality in several well-  documented outbreaks has been around 10% during the  initial stages of infection, but in the cohorts of elderly  patients who have had CDI, the overall mortality over a  period of 2-3 months can be as high as 40%.   The precipitating factor of antibiotic use and the range  of severity of CDI have been recognized since the late  1970s and the infection was well documented in the  1980s in surgical patients from over- use of clindamycin  and the cephalosporin antibiotics for surgical prophylaxis  (and in immunocompromised groups), but it was not  regarded as a priority. In an outbreak amongst elderly  patients in hospital in the winter of 1991 in Manchester,  there were more than 170 cases with 17 deaths. The  patients were on open ‘Nightingale’ wards and most had  received antibiotics for chest infections. For the first time,  strain typing (by pyrolysis mass spectrometry at that  stage) proved that the outbreak was caused by a single  strain spread by cross-infection in the hospital setting. A  subsequent Department of Health and Public Health  Laboratory Service review and report provided guidance  and recommendations that remained relevant a decade  later but were not generally applied and acted upon with  the rigour that was needed. The number of cases reported  rose steadily during the 1990s and reached epidemic pro-  portions by the early 2000s when the general increase was  compounded by the emergence (in North America, the  UK and Europe) of a new strain designated ribotype 027  with increased virulence and specific resistance to fluoro-  quinolone antibiotics which were being used increasingly  in community and hospital practice. The serious impact    of this disease meant that health services in the UK and  elsewhere had to take specific and vigorous actions to  bring down the unacceptably high incidence of CDI  which was compromising the delivery of healthcare. Not  only were patients suffering from a serious infection that  compromised their underlying medical problems, but the  management of these patients placed great pressure on  hospital services overall and were a serious drain on  health service resources.   Norovirus infections cause very significant disruption to  hospital and social care services. This virus infection  causes an explosive vomiting and diarrhoea with rapid  onset and little preliminary warning that the patient is  about to be ill. The vomitus and diarrhoea are heavily  laden with the virus and the aerosols created by both are  key to its rapid spread among contacts. The virus can  spread directly by aerosol to the mucous membranes  (mouth, nose, pharynx) of people in the same area. It will  also contaminate items throughout the environment that  can then themselves be a source of infection when those  items of furniture, fittings, equipment, etc. are handled.  The infection has a very high attack rate among those  exposed and the postinfection immunity is generally very  short-lived, so previous infection provides little protec-  tion. Both patients or residents and staff are usually  affected in these outbreaks. Control of norovirus infec-  tion requires prompt and vigorous infection control  precautions.   Fortunately, although unpleasant and debilitating  when it occurs, norovirus infection is usually short-lived  and recovery is quick in most patients. The acute vomit-  ing and diarrhoea symptoms usually last for only 36-48  hours and most people then have only a few days of  convalescence before feeling well again. Staff are usually  able to resume work about 72 hours after the vomiting  and diarrhoea has ceased, by which time they are no  longer infectious. However, in health and social care set-  tings where patients and residents are elderly and often  frail, with severe underlying conditions, norovirus can be  a more severe disease with significant associated morbid-  ity and some mortality.   An assessment of the number of cases of HCAI and the  risk of infection for different patient groups is based on    a series of different measurements. Point prevalence  studies have been undertaken in many countries to give  a snapshot of the number, and the percentage, of patients  with any type of HCAI in a particular healthcare setting  on a specific day. Prevalence studies in various developed  countries over the last decade have shown overall preva-  lence rates between 5% and 10% (Table 16.1). A preva-  lence study in the four UK countries and the Republic of  Ireland in 2006 showed an overall prevalence of 7.6%,  ranging from 4.9% in the Republic of Ireland to 8.2% in  England. The low rates in the Republic of Ireland prob-  ably reflected a rather younger patient population overall  and the higher number in England was linked almost  entirely to much higher numbers of cases of CDI (2006  was the height of the epidemic of CDI in hospitals in  England). The commonest types of infection were urinary  tract infection, followed by skin and soft tissue infections  and wound infections, respiratory infections, and gas-  trointestinal infections (Table 16.2). Bloodstream infec-  tions accounted for only 7% of ElCAIs but represent the  most severe end of the spectrum of disease. MRSA infec-  tions of all types accounted for 16% of the ElCAIs  and CDI was 17% of the total in hospitals in England but  only 5% in Wales, Northern Ireland and the Republic of  Ireland.   Prevalence studies provide valuable comparisons  between hospitals and countries and show the general  contribution of the various types of HCAI. However, they  do not represent the actual number of cases of different  HCAIs over time, i.e. the incidence of infection in differ-  ent hospitals, wards or patient groups. For example, a  point prevalence of 8.2% does not mean that 8.2% of the  patients admitted to the hospitals in England developed  an HCAI because the infected patients tend to be more  seriously ill and have longer stays in hospital both from  their underlying illness and as a result of their HCAI;  therefore, the incidence is always less than the point prev-  alence. Incidence is measured by ongoing and continued  surveillance of specific infections in which all cases of the  infection are recorded and related to the number of  patients at risk. The HCAIs that are most commonly  subject to surveillance programmes are bacteraemias  (bloodstream infections) caused by specific healthcare-  associated pathogens such as MRSA or ESBL-producing  E. coli (because they represent the most severe types of  HCAI), SSIs (one of the key indicators of the quality of  a surgical service or an individual surgeon), and CDI.  Surveillance data are necessary for monitoring infection  prevention and control activities at national and regional  level, which can provide useful comparisons between    Table 16.2 Comparative prevalence of different types of  healthcare-associated infections (HCAIs)    hospitals for patients who may choose where they wish  to have their treatment. However, surveillance with timely  feedback and data is particularly important at local  level, within individual hospitals to ensure delivery of a  high quality of care. Most surveillance programmes have  depended on voluntary reporting of the infections to  regional or national programmes but in several countries  some high-profile surveillance programmes have been  made mandatory to ensure that infection prevention    and control is made a high priority for health service  managers and clinicians alike. Mandatory surveillance of  MRSA bacteraemia was introduced in England in 2001,  followed CDI, glycopeptide-resistant enterococcal bacter-  aemia and SSIs in orthopaedic surgery in 2004. Similar  approaches have been applied in other UK countries, the  Republic of Ireland, European countries and the USA  (mostly at an individual state level).   A further development of basic surveillance (i.e.  reporting numbers of cases) is the application of enhanced  surveillance in which information about patient demo-  graphics, risk factors, and outcomes is included. This can  then be linked to root cause analysis of individual cases  to provide valuable information about target areas for  preventive measures.   Mandatory surveillance of MRSA bacteraemia in England  showed a steady rise in cases to a peak of 7700 in 2004.  Public and political concern about MRSA resulted in the  government setting a target for a 50% reduction in MRSA  bacteraemia over the 3 years 2005-2008. The surveillance  data enabled active performance management through  all levels of the (national) health service, provided pres-  sures for improved infection prevention and control  actions, and showed show the target was being achieved.  Information from enhanced surveillance within this pro-  gramme showed the importance of patients’ ages and  underlying conditions as particular risk factors, and most  significantly showed the importance of intravenous cath-  eters and cannulae, renal dialysis catheters, urinary cath-  eters and chronic wounds and ulcers as sites of MRSA  infection leading to bacteraemia.   A similar approach was taken with CDI when manda-  tory surveillance showed that there were 55 000 cases in  patients over 65 years of age in England in 2006; as these  represent about 75% of cases, this means that there were  around 70 000 cases overall. Guidance on prevention and  control measures (based on the 1994 guidance) and a  target to reduce the number of cases by 30% by 2011  focused activities in NHS hospitals and the 30% reduc-  tion was achieved within the first year (2008-2009).   SSI surveillance in orthopaedic surgery has been a  national requirement in all UK countries for several  years. This is a type of surgery with relatively low rates of  infection (<5%) but surveillance has focused attention on  infective complications, shown differences between sur-  gical procedures, and also shown a steady improvement  in rates in all the countries. However, the very short  length of stay now usual for patients after these opera-    tions means that some infections only manifest them-  selves when the patient is at home. This has shown the  need for including some system of postdischarge surveil-  lance for surgical site infection to give a realistic picture  of these HCAIs. This approach is being pioneered in the  UK and the Republic of Ireland in relation to infections  following caesarean section births where there is a clear  opportunity for professional input to postdischarge sur-  veillance because the mothers are seen at home by a  midwife or health visitor.   The surveillance programmes in various countries have  shown the scale of the challenge set by HCAI in modern  health and social care services. Why has this situation  developed? There is a strong argument that during the  last quarter of the 20th century, infections (including  HCAI) had not been regarded as an important part of  modern medical practice. There had been a view from the  late 1960s onwards that infectious diseases had been con-  quered and that antibiotics and vaccines provided the  answer. The importance of infection prevention and  control measures was not given its former position in  clinical training of doctors and nurses. Modern medicine  was making tremendous progress in the treatment of  malignant diseases, cardiovascular disease, transplanta-  tion, and chemotherapy, and in the management of  chronic diseases. Life expectancy increased markedly and  the proportion of very elderly people in the population,  with their inevitable healthcare needs, rose rapidly. This  created a vulnerable patient population at high risk of  infection, but these infections were regarded as incidental  nuisances rather than the major risk to a patients’ health  and potential mortality which they are. Infections were  regarded as the province of the infection specialists  (medical microbiologists, infection control nurses) who  had plenty of interesting work to do but a generally low  profile. The situation changed with the advent of the 21st  century and the recognition of the human and financial  costs of HCAIs in a modern healthcare service.   The responsibility for infection prevention and control is  shared across all who have a role in the delivery of health  and social care, from the most senior managers to the  most junior members of staff at ward or unit level — often  described as the ‘board to ward’ approach. Clinicians    (doctors, nurses and other professional colleagues) have  a personal and professional responsibility for the care of  their patients. Their responsibility for patient safety  includes minimizing the risk of infection by implement-  ing best clinical practice protocols, antibiotic steward-  ship, etc., and delivering the highest standards of clinical  care and treatment for those who develop an infection,  to treat those infections and minimize the risk of trans-  mission to others.   Health and social care service managers and oversee-  ing boards have an overarching responsibility for provid-  ing the corporate environment in which infection  prevention and control has a high priority and all staff  know what is expected of them. They need surveillance  data and performance audits, coupled with inclusion of  infection prevention and control in the individual per-  formance review and personal appraisal of all their staff.  In this way, infection prevention and control becomes  embedded in the culture of the healthcare organization.   Governments, departments of health and health  service national managers are the third party in this part-  nership. They are responsible for making HCAI preven-  tion and control a top priority throughout health and  social care services and holding local managers and  boards to account for delivery of a high quality and  patient care — and low rates of HCAI. They use perform-  ance management arrangements to require high stand-  ards from healthcare providers and can set targets for  HCAI reductions (or ceilings for those with good  records). They decide which HCAIs should be subject to  mandatory surveillance. Governments also have the ulti-  mate authority in terms of legislation that may be used  to support HCAI prevention and control. The UK gov-  ernment was the first to use this type of legislation in  relation to healthcare in England. The Health Act 2006  implemented a statutory code of practice for the preven-  tion and control of HCAI.   • assess the risks of acquiring HCAI and take actions to  reduce such risks   • provide and maintain a clean and appropriate  environment for healthcare   • provide information when patients move from one  healthcare body to another   • adhere to policies and protocols applicable to infection  prevention and control.   • prevention of occupational exposure to blood-borne  viruses including prevention of sharps injuries   • management of occupational exposure to blood-borne  viruses and postexposure prophylaxis   • reporting of HCAI to the Health Protection Agency as  directed by the Department of Health   • policies for the control of infections with specific alert  organisms.   The code of practice also required NHS bodies to  ensure (as far as reasonably practicable), that healthcare  workers are free of and/or protected from exposure to  communicable diseases during the course of their work,  and the staff are suitably educated in the prevention and  control of HCAI. The latter duty within the code of prac-  tice reinforced an essential element for all approaches to  prevention and control of HCAI — a requirement for  education and training of all healthcare workers on the  principles of infection prevention and control and the  part they need to play.   The combination of all these elements represents the  fact that there is personal and corporate responsibility for  HCAI prevention and control at all levels in any health  and social care system.   There is no single ‘silver bullet’ to solve the challenge of  HCAI. Infection prevention and control requires a com-  bination of actions and activities, each of which provides  an essential component to the whole.   The commitment of senior management to making  infection prevention and control a high priority sets a    culture of a health of social care organization. Management  should monitor surveillance and audit data at all levels of  the organization (‘from board to ward’) and ensure that  all the staff play their part. This helps general a culture of  pride in delivery of a quality service.   Up-to-date surveillance data on the incidence of key  infections should be collected, analysed and returned to  the clinical units with minimum delay. In this way the  data are seen to be ‘real’ by the staff responsible for the  care of the patients. It is now mandatory in several coun-  tries to collect surveillance data on MRSA infections  (particularly bacteraemias), CDI, and some type of surgi-  cal site infections. Although national data collection is  focused on whole hospitals or hospital groups, effective  action within hospital depends upon the data being  assessed and acted upon in individual wards or other  clinical units. The essential nature of surveillance data is  encapsulated in the dictum ‘you have to measure it to  manage it’.   There are two main reasons why patients are at risk of  developing an HCAI — their underlying medical condi-  tion making them vulnerable to infections and the  treatment and clinical interventions they are subjected  to. These interventions often include invasive procedures  that bypass the normal defences of the skin, urinary  and respiratory tract. It is, therefore, essential that  clinical staff exercise all due care and attention when  performing these procedures. To this end, an approach  to clinical practice has been developed variously known  as ‘care bundles’ (in the USA in particular) and ‘high-  impact interventions’ (HII; the UK approach). These  bundles are care protocols setting out in a simple bullet-  point format the five or six essential elements needed  to minimize the infection risk associated with the indi-  vidual aseptic procedures. The aim is for each element  to be performed correctly on every occasion and the  care bundle/HII incorporates a simple audit tool for  self- or peer-assessment on a frequent and regular basis  of the performance of the clinical staff doing those  procedures. The procedures that had been the main  focus of the bundles comprise the following invasive  interventions:   For the HCAI programme in England these were set  out in packages called ‘Saving Lives’ for secondary care  and ‘Essential Steps’ for primary care use. The packages  were complemented by a care bundle for CDI and  guidance presented in a similar way based upon key  elements for antibiotic prescribing, isolation and  cohorting of infected patients and the collection of blood  cultures which form the basis of the diagnosis of a  bacteraemia.   One of the most basic and ancient approaches to prevent-  ing the spread of infection (or ‘contagion’ in previous  centuries) is isolation of the affected patient. Segregation  of infected patients from those vulnerable to infection  (including various forms of quarantine) is an essential  element of all infection prevention and control practice.  It is particular important in HCAIs where the infection  often occurs in vulnerable or debilitated patients and in  a setting where transmission of infection between patients  can occur readily. In many hospitals, the number of single  rooms available for patient isolation is limited. The best  use of the available rooms should be made for those with  infections, but where the capacity of single rooms is  exceeded by the number of cases of infection, it may then  be necessary and appropriate for patients with the same  infection to be nursed together in a cohort ward physi-  cally separated from other ward areas and with dedicated  staff who do not move between the cohort ward and  other clinical areas (i.e. cohorting should apply to nursing  staff as well as patients).   One of the main routes of transmission of HCAI patho-  gens such as MRSA is via the hands of healthcare workers  as they move between patients. For many years staff were  recommended to wash their hands between patient con-  tacts and especially before and after performing clinical  procedures. However, very frequent hand washing is time  consuming, can damage the skin if done as often as  should be required, and can also be quite impracticable  when there is inadequate provision of wash-hand  basins for staff. The answer to this problem has been the  introduction of alcohol hand rubs that can be used  repeatedly and quickly at every point of patient contact.  Alcohol is highly effective against vegetative bacteria    Table 16.3 High-impact intervention no. 1: central venous catheter care bundle   Consider antimicrobial impregnated catheter if duration 1-3 weeks and risk of  CR-BSI high   Preferably use 2% chlorhexidine gluconate in 70% isopropyl alcohol and allow  to dry   If patient has a sensitivity use a single patient use povidone-iodine application   Gloves are single-use items and should be removed and discarded immediately  after the care activity   Eye and face protection is indicated if there is a risk of splashing with blood or  body fluids   Decontaminate hands before and after each patient contact  Use correct hand hygiene procedure   Gown, gloves and drapes as indicated should be used for the insertion of  invasive devices   Use a sterile, transparent, semipermeable dressing to allow observation of  insertion site   Sharps container should be available at point of use and should not be overfilled;  do not disassemble needle and syringe; do not pass sharps from hand to hand   Decontaminate hands before and after each patient contact  Use correct hand hygiene procedure   Use aseptic technique and swab ports or hub with 2% chlorhexidine gluconate in  70% isopropyl alcohol prior to accessing the line for administering fluids or  injections   Following administration of blood, blood products — immediately  Following total parenteral nutrition — after 24 h (72 h if no lipid)  With other fluid sets — after 72 h   Source: DH (2007) Saving Lives: Reducing Infection, Delivering Clean and Safe Care. Department of Health, London.    causing HCAI such as MRSA and the Gram-negative  bacteria. The hand rubs should be made available at  every patient bedside and in every clinical area, and at  the entrance to cubicles and single rooms. Personal dis-  pensers can also be carried attached to a belt or the  clothing of healthcare staff to use as they move  between patients. In many hospitals, alcohol hand rub  dispensers have also been placed at the hospital  entrance and/or at every ward entrance to emphasize to  patients and visitors, as well as staff, the absolute impor-  tance of hand hygiene. However, the crucial times and  places for hand hygiene relate to direct clinical contact  with patients. The hand hygiene campaign based on  alcohol hand rubs has been promoted internationally  by the World Health Organization (WHO) and in the  UK by the National Patient Safety Agency through its  ‘cleanyourhands’ campaign. The WHO campaign high-  lights the five opportunities (and requirements) for hand  hygiene:   The need for effective and appropriate hand hygiene  has also been included in all the care bundles/HIIs (see  above).   However, in one area of infection prevention and  control, hand hygiene with alcohol hand rubs does not  replace the absolute requirement for hand washing: this  is in relation to diarrhoeal infections caused by norovirus  or Cl. difficile. Alcohol is not effective against norovirus  or against the spores of Cl. difficile, so for these common  diarrhoeal infections, hand washing is essential before  and after each patient contact or contact with the envi-  ronment around infective patients.   The audit of hand hygiene (alcohol hand rub and  hand washing) is an important part of monitoring  compliance with clinical protocols for infection preven-  tion and control and should include direct observation  of all grades of healthcare staff and also measurement  of the volume of alcohol hand rub and liquid soaps  used as a general proxy measurement of hand hygiene  practice.   Whenever there are problems or outbreaks of HCAI,  there is popular outcry over dirty hospitals, giving the  impression that environmental cleanliness equates to    prevention of infection. Whereas there is no question  that hospitals and other healthcare premises should be  clean and that a clean environment promotes good  healthcare practice, there is less of a direct correlation  between general cleanliness and rates of HCAI. However,  there is good evidence that bacteria and viruses from  infected patients contaminate the general environment  around those patients from where they can be picked up,  e.g. on the hands, by other vulnerable patients or by  healthcare workers and transmitted to other patients. The  evidence is particularly clear with C. difficile spores,  which can be found on all environmental services in  rooms where there are patients with CDI. They are par-  ticularly prevalent around toilets, or on commodes, or  near bed pan washers.   General hospital cleaning is based on a detergent and  water cleaning regimen, but when there are patients with  known HCAIs, it is advisable to supplement detergent  cleaning with use of environmental disinfectants. This is  particularly the case in outbreaks of norovirus or CDI.  The most effective disinfectants for these viruses and for  the spores of Cl. difficile are those based on chlorine-  releasing agents (see Chapter 19). These should be used  routinely in areas where there are cases of norovirus or  CDI.   Good antibiotic stewardship and the prudent use of these  valuable drugs is an important part of the prevention and  control of HCAI. Most bacterial HCAIs are caused by  antibiotic-resistant organisms that flourish under the  selective pressure of antibiotic use, and because of the fact  that many of the bacteria are resistant to several different  types of antibiotic, even the use of individual antibiotics  can select for bacteria resistant to a wide range of agents.  Furthermore, many of the resistance genes are carried on  transferable genetic elements that can transfer among  bacterial populations, particularly in a selective environ-  ment such as a hospital. There are many examples of links  between use of particular antibiotics and cases of HCAI  caused by resistant organisms. There are also more  general links between use of agents such as the fluoroqui-  nolone antibiotics and the rising incidence of MRSA  colonization and infection. With CDI, the link is even  more direct with the use of broad-spectrum antibiotics  being a key precipitating factor for this disease. Outbreaks  of CDI have been linked to widespread use of cepha-  losporins and, more recently, fluoroquinolone antibiotics  when the Cl. difficile strains such as ribotype 027 have  been specifically resistant to these agents.    All healthcare organizations should have antibiotic  prescribing protocols to promote and audit good stew-  ardship. This is a requirement of the statutory Code of  Practice in England. The guidance in the ‘Saving Lives’  package recommends that antibiotic stewardship pro-  gramme should have the following elements:   • an antimicrobial formulary and guidelines for  antimicrobial treatment and prophylaxis   • intravenous therapy should only be used for severe  infections or where oral antimicrobials are not appropriate   • intravenous antimicrobials should only be used for 2  days before review and switch to an oral agent where  possible and appropriate   • all antimicrobial prescriptions should include a stop  date — generally a maximum of 5-7 days without  represcription   • a single dose at induction of anaesthesia for most  operations where antimicrobial prophylaxis is indicated   The implementation of the range of infection prevention  and control practices in any health or social care setting  can only be successful if there is a comprehensive  approach to staff education and training through  which all can learn that everyone has a role to play in  preventing HCAI. Basic training in infection prevention  and control is mandatory for all staff in many hospitals  and healthcare settings in the UK, Europe and North  America, usually with regular (generally annual) required  updates and specialist training for particular professional  groups. Completion of this training is generally a require-  ment for successful appraisal and performance review for  all staff.   Whereas surveillance of key HCAIs is necessary for moni-  toring the changing pattern and the incidence of infec-  tions, audit of implementation of clinical protocols is  equally necessary for maintaining a high level of infection  prevention and control practice. There should be regular    audits of hand hygiene compliance, adherence to antibi-  otic prescribing guidelines and the implementation of the  clinical protocols in the care bundles/HIIs. The results of  these audits should be reviewed in a timely manner at all  levels of management in the health and social care organ-  izations so that those implementing the protocols have  ownership of the procedures and their effective applica-  tion. Performance management at all levels depends  upon a combination of the data from surveillance of the  infections and audit of the clinical practice.   As a result of the implementation of the measures out-  lined above, the rates of particular HCAIs have fallen  significantly in several countries. The application of the  central venous catheter care bundle by intensive care  units in the USA has resulted in a steep reduction in cases  of catheter-related bacteraemia and, in some cases, long  periods with no such infections. In England, the package  of measures aimed at MRSA bacteraemias resulted in a  65% decrease in cases reported from the 2004 target base-  line to 2009. These improvements have enabled the pro-  motion of a zero tolerance approach to HCAI. This does  not mean that there will be no infections (this is micro-  biologically and clinically implausible) but does mean  that we can apply a zero tolerance approach to avoidable  infections and to poor clinical practice such as inade-  quate compliance with hand hygiene requirements and  imprudent antibiotic prescribing. The aim is to do eve-  rything right every time.   The general principles of infection management apply  to HCAIs as they do to all types of infection, but it is  particularly important to have reliable application of the  principles where there is the risk of spread amongst vul-  nerable patients. Clinical staff need to have a high index  of suspicion that a patient may be developing an infection  and initiative appropriate confirmative diagnostic tests  quickly. There should be prompt isolation of a patient  suspected of being infected and specific treatment insti-  tuted along with infection prevention and control meas-  ures to prevent further spread. As well as these universal  principals, there are some specific prevention and control  measures aimed at particular infections.   The reductions in MRSA bacteraemia achieved in UK  hospitals and elsewhere as part of the targeted approach    has been mostly the result of emphasis on hand hygiene,  improved aseptic practices and the care bundles/HII  approach for invasive procedures, particularly intrave-  nous central catheter and peripheral cannula insertion  and care. Implementation of these measures has been  backed by the commitment of managers to reduce infec-  tion rates. These general improvements in infection pre-  vention and control procedures would also be expected  to help prevent other courses of bacteraemias linked to  intravenous catheters and cannulae.   The further measure that is specific to the prevention  of MRSA infections overall (wound and soft tissues infec-  tions, VAP, etc., as well as bacteraemias) is screening of  patients before admission to hospital (when admission is  planned or ‘elective’) or on admission in respect of emer-  gency admissions. The principles behind such screening  are that colonization of the nose and/or skin sites gener-  ally precedes clinical infection and that a colonized  patient (otherwise referred to as a ‘carrier’) is at risk of  developing an MRSA infection themselves and also a  potential source of transmission to others. For such  screening a swab is taken from the anterior nares, and  also from the other skin carriage sites of perineum  and axilla if considered appropriate, as well as from any  surface lesions such as a chronic ulcer. Laboratory exami-  nation of the swab can be based upon convention selec-  tive culture for MRSA (which has a minimum turnaround  time of 24-48 hours) or more expensive molecular  methods based on PCR (polymerase chain reaction)  methodology when a rapid result is considered to give  significant benefit. Patients found to be colonized with  MRSA are generally then given a ‘decolonization’ or ‘sup-  pressive’ treatment regimen of nasal mupirocin cream  and an antiseptic skin wash and shampoo for 5 days. This  is very effective in reducing the bioburden in MRSA colo-  nization in the short term, thus reducing the risk of  infection for the individual patient and the risk of trans-  mission to other. However, colonization may recur over  a period of several months in 40% or more individuals.  Nevertheless, the suppression of colonization will have  covered the period of particular vulnerability and the  time when they would be more likely to be a source of  transmission. Screening and decolonization were part of  the ‘search and destroy’ approach to MRSA infection  developed in the UK in thel980s. It was also adopted  elsewhere and has continued to be very effective in  helping maintain low levels of MRSA infection and  low levels of colonization in countries such as the  Netherlands, parts of Scandinavia, and Western Australia.  This approach was not maintained in most parts of the    UK during the 1990s but has been reintroduced as part of  the MRSA control measures in various ways in the UK  countries in more recent years. Screening of all patients  admitted to NHS hospitals has been introduced in  England, whereas other countries have adopted wide-  spread but more restricted, risk-based approaches to  selecting patients for screening. The risk factors are gen-  erally age (>65 or 70 years), previous MRSA carriage,  previous hospital admission, residence in a nursing home  or residential care home, and the presence of a chronic  disease. The most appropriate approach will become  evident as these different regimens are applied in differ-  ent healthcare settings.   Screening for Staph, aureus more generally, not specifi-  cally MRSA, has been used as part of outbreak control  measures over many years but has not been adopted on  a routine basis. However, modern approaches to MRSA  screen could also enable screening for any Staph, aureus  strain in particular vulnerable groups.   The emergence of CDI has been a complication of  modern medical care compounded by inadequate atten-  tion to antibiotic stewardship and infection prevention  and control measures. Elderly patients are the most vul-  nerable to this infection (75% of cases are in people >65  years old) but severe disease can occur in younger patients  also. The major precipitating factor is the use of broad-  spectrum antibiotics. In the UK, guidance produced in  1994 was reviewed and an updated document was pub-  lished in 2009. This recommended the application of a  mnemonic protocol (SIGHT) for managing suspected  and then proven cases:   • Suspect that a case of diarrhoea maybe infective when  there is not a clear alternative cause for the diarrhoea   • Gloves and aprons must be worn for all contact with  the patient and their environment   • Hand washing with soap and water before and after  each contact with the patient and their environment   • Test the stool for Cl. difficile toxins by sending a  specimen immediately.   CDI itself then needs to be treated as a major diagnosis  in its own right (not just a minor complication of the  underlying disease). Sufficient isolation capacity is  required for single room accommodation of cases, but  in outbreak situations it may be necessary to cohort  patients in designated CDI isolation wards. Cleanliness  and use of sporicidal disinfectants (currently only   There should also be a major focus on antibiotic stew-  ardship. The guidance recommends that hospitals should  establish antimicrobial management teams comprising  antimicrobial pharmacists, consultant microbiologist, or  infectious diseases specialist, and other clinicians as  appropriate. The team should develop restricted guide-  lines to promote the use of narrow-spectrum agents  and avoid, where reasonably possible, clindamycin and  the second- and third-generation cephalosporins (espe-  cially in elderly patients), while minimizing the use of  fluoroquinolones, carbapenems and prolonged courses  of aninopenicillins.   It is difficult to prevent norovirus introduction because  of the rapid onset of illness, but it should be stressed to  all staff, patients and visitors that people should stay away  from health and social care settings if they or their fami-  lies are suffering from this type of vomiting and diar-  rhoea. When cases occur in health or residential care  settings, prompt action is essential. Patients should be  isolated at the first signs of the infection (which is often  dramatic onset of the vomiting and diarrhoea). Patients  or residents and staff who were in the same area should  be quarantined to reduce the risk of wider spread as more  cases occur in those exposed. Individual cases often  trigger outbreaks and as soon as this is recognized to be  happening, the ward or residential unit should be closed  to further admissions until the outbreak has ended  amongst those (patients/residents and staff) already  exposed. Because of the extensive environmental con-  tamination, cleaning and disinfection of the affected  areas is an important part of control. Areas contaminated  with vomitus and faeces, which is common in these infec-  tions, should be promptly cleaned and disinfected with  chlorine-based disinfectant. When the outbreak is over,  patients or residents need to be moved elsewhere and a  thorough deep clean of the affected area (ward, unit, etc.)  including disinfection with a chlorine-releasing disinfect-  ant should be done before any patients or residents are  readmitted.   Throughout this chapter, it has been stressed that infec-  tion prevention and control are the responsibility of all    staff (clinical, managerial and support staff). However, it  is also an area that requires the leadership and expertise  of clinicians specifically trained in infection prevention  and control. All healthcare and social care organizations  should have access to such expertise and should have an  infection prevention and control team and committee to  deliver the expert service and support for the staff of all  the clinical and social care units. The infection control  team in hospitals should generally comprise nursing,  medical and pharmaceutical professionals with specific  training and expertise in infection prevention and control  and in antibiotic prescribing. The nurse should be a  trained infection prevention and control practitioner and  the medical input is generally provided by a consultant  medical microbiologist or infectious diseases physician  whose training and experience has included the specific  area of HCAI prevention and control. The pharmacist  member of the team should have specific experience and  expertise in antimicrobial prescribing. The team has an  important role in outbreak investigation and manage-  ment but, most importantly, provides the guidance and  support for the delivery and effective infection preven-  tion and control throughout the hospital or health and  social care organization.   DH (2007) Saving Lives: Reducing Infection, Delivering Clean and  Safe Care. Department of Health, London.   DH (2006) Essential Steps to Safe, Clean Care. Department of  Health, London.   DH (2009) Health and Social Care Act 2008 — Code of Practice for  the Prevention and Control of Healthcare Associated Infections.  Department of Health, London.   DH and Health Protection Agency (2009) Clostridium Difficile  Infection: How to Deal with the Problem. Department of  Health, London.   National Patient Safety Agency (2004) Ready, Steady, Go! The  Full Guide to Implementing the cleanyourhands Campaign in  your Trust. National Patient Safety Agency, London.   National Audit Office (2009) Reducing Healthcare Associated  Infections in Hospitals in England. 12 June 2009.   Smyth, E.T.M. et al. on behalf of the Hospital Infection Society  Prevalence Survey Steering Group (2008) Four country  healthcare associated infection prevalence survey 2006: over-  view of the results. / Hosp Infect, 69, 230-248.   Pharmaceutical products used in the prevention, treat-  ment and diagnosis of disease contain a wide variety of  ingredients, often in quite complex physicochemical    states. Such products must not only meet current good  pharmaceutical manufacturing practice (GPMP) require-  ments for quality, safety and efficacy, but also must be  stable and sufficiently attractive to be acceptable to  patients. Products made in the pharmaceutical industry  today must meet high microbiological specifications; i.e.    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    if not sterile, they are expected to have no more than a  minimal microbial population at the time of product  release.   Nevertheless, from time to time a few rogue products  with an unacceptable level and type of contamination  will occasionally escape the quality assurance net. The  consequences of such contamination may be serious and  far-reaching on several accounts, particularly if contami-  nants have had the opportunity to multiply to high levels.  First, the product may be spoiled, rendering it unfit for  use through chemical and physicochemical deterioration  of the formulation. Spoilage and subsequent wastage of  individual batches usually result in major financial prob-  lems for the manufacturer through direct loss of faulty  product. Secondly, the threat of litigation and the  unwanted, damaging publicity of recalls may have serious  economic implications for the manufacturer. Thirdly,  inadvertent use of contaminated products may present a  potential health hazard to patients, perhaps resulting in  outbreaks of medicament-related infections, and ironi-  cally therefore contributing to the spread of disease. Most  commonly, heavy contamination of product with oppor-  tunist pathogens, such as Pseudomonas spp., has resulted  in the spread of nosocomial (hospital-acquired) infec-  tions in compromised patients; less frequently, low levels  of contamination with pathogenic organisms, such as  Salmonella , have attracted considerable attention, as have  products contaminated with toxic microbial metabolites,  such as mycotoxins in herbal medicines. The conse-  quences of microbial contamination in pharmaceutical  products are discussed in more detail below.   Microorganisms form a major part of the natural recy-  cling processes for biological matter in the environment.  As such, they possess a wide variety of degradative capa-  bilities, which they are able to exert under relatively mild  physicochemical conditions. Mixed natural communities  are often far more effective cooperative biodeteriogens  than the individual species alone, and sequences of  attack of complex substrates occur where initial attack by  one group of microorganisms renders them susceptible  to further deterioration by secondary, and subsequent,  microorganisms. Under suitable environmental selection  pressures, novel degradative pathways may emerge with  the capability to attack newly introduced synthetic chem-    icals (xenobiotics). However, the rates of degradation of  materials released into the environment can vary greatly,  from half-lives of hours (phenol) to months (‘hard’  detergents) to years (halogenated pesticides).   The overall rate of deterioration of a chemical depends  on its molecular structure; the physicochemical proper-  ties of a particular environment; the type and quantity of  microbes present; and whether the metabolites produced  can serve as sources of usable energy and precursors for  the biosynthesis of cellular components, and hence the  creation of more microorganisms.   Pharmaceutical formulations may be considered as  specialized microenvironments and their susceptibility  to microbial attack can be assessed using conventional  ecological criteria. Some naturally occurring ingredients  are particularly sensitive to attack, and a number of syn-  thetic components, such as modern surfactants, have  been deliberately constructed to be readily degraded  after disposal into the environment. Crude vegetable and  animal drug extracts often contain a wide assortment of  microbial nutrients besides the therapeutic agents. This,  combined with frequently conducive and unstable physi-  cochemical characteristics, leaves many formulations  with a high potential for microbial attack unless steps are  taken to minimize it.   • Therapeutic agents. Through spoilage, active drug  constituents may be metabolized to less potent or  chemically inactive forms. Under laboratory conditions,  it has been shown that a variety of microorganisms  can metabolize a wide assortment of drugs, resulting  in loss of activity. Materials as diverse as alkaloids  (morphine, strychnine, atropine), analgesics (aspirin,  paracetamol), thalidomide (still used in the treatment of  some forms of cancer), barbiturates, steroid esters and  mandelic acid can be metabolized and serve as substrates  for growth. Indeed, the use of microorganisms to carry  out subtle transformations on steroid molecules forms  the basis of the commercial production of potent  therapeutic steroidal agents (see Chapter 26). In practice,  reports of drug destruction in medicines are less frequent.  There have, however, been some notable exceptions: the  metabolism of atropine in eye drops by contaminating  fungi; inactivation of penicillin injections by (3-lactamase-  producing bacteria (see Chapters 11 and 13); steroid  metabolism in damp tablets and creams by fungi;  microbial hydrolysis of aspirin in suspension by esterase-  producing bacteria; and chloramphenicol deactivation   • Surface-active agents. Anionic surfactants, such as the  alkali metal and amine soaps of fatty acids, are generally  stable because of the slightly alkaline pH of the  formulations, although readily degraded once diluted  into sewage. Alkyl and alkylbenzene sulphonates and  sulphate esters are metabolized by co-oxidation of their  terminal methyl groups followed by sequential (3-  oxidation of the alkyl chains and fission of the aromatic  rings. The presence of chain branching involves additional  a-oxidative processes. Generally, ease of degradation  decreases with increasing chain length and complexity of  branching of the alkyl chain.   • Non-ionic surfactants, such as alkylpolyoxyethylene  alcohol emulsifiers, are readily metabolized by a wide  variety of microorganisms. Increasing chain lengths  and branching again decrease ease of attack. Alkylphenol  polyoxyethylene alcohols are similarly attacked, but are  significantly more resistant. Lipolytic cleavage of the  fatty acids from sorbitan esters, polysorbates and sucrose  esters is often followed by degradation of the cyclic  nuclei, producing numerous small molecules readily  utilizable for microbial growth. Ampholytic surfactants,  based on phosphatides, betaines and alkylamino-  substituted amino acids, are an increasingly important  group of surfactants and are generally reported to be  reasonably biodegradable. The cationic surfactants used  as antiseptics and preservatives in pharmaceutical  applications are usually only slowly degraded at high  dilution in sewage. Pseudomonads have been found  growing readily in quaternary ammonium antiseptic  solutions, largely at the expense of other ingredients such  as buffering materials, although some metabolism of the  surfactant has also been observed.   • Organic polymers. Many of the thickening and  suspending agents used in pharmaceutical formulations  are subject to microbial depolymerization by specific  classes of extracellular enzymes, yielding nutritive  fragments and monomers. Examples of such enzymes,  with their substrates in parentheses, are: amylases  (starches), pectinases (pectins), cellulases (carboxy-  methylcelluloses, but not alkylcelluloses), uronidases  (polyuronides such as in tragacanth and acacia),  dextranases (dextrans) and proteases (proteins). Agar (a  complex polysaccharide) is an example of a relatively  inert polymer and, as such, is used as a support for  solidifying microbiological culture media. The lower  molecular weight polyethylene glycols are readily  degraded by sequential oxidation of the hydrocarbon    chain, but the larger congeners are rather more  recalcitrant. Synthetic packaging polymers such as nylon,  polystyrene and polyester are extremely resistant to  attack, although cellophane (modified cellulose) is  susceptible under some humid conditions.   • Humectants. Low molecular weight materials such as  glycerol and sorbitol are included in some products to  reduce water loss and may be readily metabolized unless  present in high concentrations (see section 2.3.3).   • Fats and oils. These hydrophobic materials are  usually attacked extensively when dispersed in aqueous  formulations such as oil-in-water emulsions, aided by the  high solubility of oxygen in many oils. Fungal attack has  been reported in condensed moisture films on the surface  of oils in bulk, or where water droplets have contaminated  the bulk oil phase. Lipolytic rupture of triglycerides  liberates glycerol and fatty acids, the latter often then  undergoing (3-oxidation of the alkyl chains and the  production of odiferous ketones. Although the microbial  metabolism of pharmaceutical hydrocarbon oils is rarely  reported, this is a problem in engineering and fuel  technology when water droplets have accumulated in oil  storage tanks and subsequent fungal colonization has  catalysed serious corrosion.   • Sweetening, flavouring and colouring agents. Many of  the sugars and other sweetening agents used in pharmacy  are ready substrates for microbial growth. However, some  are used in very high concentrations to reduce water  activity in aqueous products and inhibit microbial attack  (see section 2.3.3). At one time, a variety of colouring  agents (such as tartrazine and amaranth) and flavouring  agents (such as peppermint water) were kept as stock  solutions for extemporaneous dispensing purposes, but  they frequently supported the growth of Pseudomonas  spp., including Ps. aeruginosa. Such stock solutions  should now be preserved, or freshly made as required by  dilution of alcoholic solutions which are much less  susceptible to microbial attack.   • Preservatives and disinfectants. Many preservatives and  disinfectants can be metabolized by a wide variety of  Gram-negative bacteria, although most commonly  at concentrations below their effective ‘use’ levels.  Growth of pseudomonads in stock solutions of  quaternary ammonium antiseptics and chlorhexidine  has resulted in infection of patients. Pseudomonas spp.  have metabolized 4-hydroxybenzoate (parabens) ester  preservatives contained in eye-drops and caused serious  eye infections, and have also metabolized the preservatives  in oral suspensions and solutions. In selecting suitable  preservatives for formulation, a detailed knowledge of    the properties of such agents, their susceptibility to  contamination and limitations clearly provides invaluable  information.   Microbial contaminants usually need to attack formula-  tion ingredients and create substrates necessary for  biosynthesis and energy production before they can rep-  licate to levels where obvious spoilage becomes apparent.  Thus, for example, 10 6 microbes will have an overall  degradative effect around 10 6 times faster than one  cell. However, growth and attack may well be localized  in surface moisture films or very unevenly distributed  within the bulk of viscous formulations such as  creams. Early indications of spoilage are often organolep-  tic, with the release of unpleasant smelling and tasting  metabolites such as ‘sour’ fatty acids, ‘fishy’ amines,  ‘bad eggs’, bitter, ‘earthy’ or sickly tastes and smells.  Products may become unappealingly discoloured by  microbial pigments of various shades. Thickening and  suspending agents such as tragacanth, acacia or car-  boxymethylcellulose can be depolymerized, resulting in  loss of viscosity and sedimentation of suspended ingre-  dients. Alternatively, microbial polymerization of sugars  and surfactant molecules can produce slimy, viscous  masses in syrups, shampoos and creams, and fungal  growth in creams has produced ‘gritty’ textures. Changes  in product pH can occur depending on whether acidic  or basic metabolites are released, and become so  modified as to permit secondary attack by microbes pre-  viously inhibited by the initial product pH. Gaseous  metabolites may be seen as trapped bubbles within  viscous formulations.   When a complex formulation such as an oil-in-water  emulsion is attacked, a gross and progressive spoilage  sequence may be observed. Metabolism of surfactants  will reduce stability and accelerate ‘creaming’ of the oil  globules. Lipolytic release of fatty acids from oils will  lower pH and encourage coalescence of oil globules and  ‘cracking’ of the emulsion. Fatty acids and their ketonic  oxidation products will provide a sour taste and unpleas-  ant smell, while bubbles of gaseous metabolites may be  visible, trapped in the product, and pigments may discol-  our it (see Figure 17.1).   By understanding the influence of environmental param-  eters on microorganisms, it may be possible to manipu-    late formulations to create conditions which are as  unfavourable as possible for growth and spoilage, within  the limitations of patient acceptability and therapeutic  efficacy. Furthermore, the overall characteristics of a par-  ticular formulation will indicate its susceptibility to  attack by various classes of microorganisms.   Successful formulation of products against microbial  attack involves an element of prediction. An understand-  ing of where and how the product is to be used and the  challenges it must face during its life will enable the for-  mulator to build in as much protection as possible against  microbial attack. When failures inevitably occur from  time to time, knowledge of the microbial ecology and  careful identification of contaminants can be most useful  in tracking down the defective steps in the design or  production process.   Low levels of contaminants may not cause appreciable  spoilage, particularly if they are unable to replicate in a   Figure 17.1 Section (xl.5) through an inadequately preserved  olive oil, oil-in-water, emulsion in an advanced state of  microbial spoilage showing: A, discoloured, oil-depleted,  aqueous phase; B, oil globule-rich creamed layer; C, coalesced  oil layer from ‘cracked’ emulsion; D, fungal mycelial growth on  surface. Also present are a foul taste and evil smell.    product; however, an unexpected surge in the contami-  nant bioburden may present an unacceptable challenge  to the designed formulation. This could arise if, for  example, raw materials were unusually contaminated;  there was a lapse in the plant-cleaning protocol; a biofilm  detached itself from within supplying pipework; or the  product had been grossly misused during administration.  Inoculum size alone is not always a reliable indicator of  likely spoilage potential. Low levels of aggressive pseu-  domonads in a weakly preserved solution may pose a  greater risk than tablets containing fairly high numbers  of fungal and bacterial spores.   When an aggressive microorganism contaminates a  medicine, there may be an appreciable lag period before  significant spoilage begins, the duration of which  decreases disproportionately with increasing contami-  nant loading. As there is usually a considerable delay  between manufacture and administration of factory-  made medicines, growth and attack could ensue during  this period unless additional steps are taken to prevent it.  On the other hand, for extemporaneously dispensed for-  mulations some control can be provided by specifying  short shelf-lives, for example 2 weeks.   The isolation of a particular microorganism from a  markedly spoiled product does not necessarily mean that  it was the initiator of the attack. It could be a secondary  opportunist contaminant which had overgrown the  primary spoilage organism once the physicochemical  properties had been favourably modified by the primary  spoiler.   The simple nutritional requirements and metabolic  adaptability of many common spoilage microorganisms  enable them to utilize many formulation components as  substrates for biosynthesis and growth. The use of crude  vegetable or animal products in a formulation provides  an additionally nutritious environment. Even demineral-  ized water prepared by good ion-exchange methods will  normally contain sufficient nutrients to allow significant  growth of many waterborne Gram-negative bacteria such  as Pseudomonas spp. When such contaminants fail to  survive, it is unlikely to be the result of nutrient limitation  in the product but due to other, non-supportive, physico-  chemical or toxic properties.   Acute pathogens require specific growth factors nor-  mally associated with the tissues they infect but which are  often absent in pharmaceutical formulations. They are  thus unlikely to multiply in them, although they may  remain viable and infective for an appreciable time in   Microorganisms require readily accessible water in appre-  ciable quantities for growth to occur. By measuring a  product’s water activity, A m it is possible to obtain an  estimate of the proportion of uncomplexed water that is  available in the formulation to support microbial growth,  using the formula Av = vapour pressure of formulation/  vapour pressure of water under similar conditions.   The greater the solute concentration, the lower is the  water activity. With the exception of halophilic bacteria,  most microorganisms grow best in dilute solutions (high  A,) and, as solute concentration rises (lowering A*),  growth rates decline until a minimal growth-inhibitory  Ao is reached. Limiting Ay, values are of the order of 0.95  for Gram-negative rods; 0.9 for staphylococci, micrococci  and lactobacilli; and 0.88 for most yeasts. Syrup-  fermenting osmotolerant yeasts have spoiled products  with A w levels as low as 0.73, while some filamentous  fungi such as Aspergillus glaucus can grow at 0.61.   The A« of aqueous formulations can be lowered to  increase resistance to microbial attack by the addition of  high concentrations of sugars or polyethylene glycols.  However, even Syrup BP (67% sucrose; A* = 0.86) has  occasionally failed to inhibit osmotolerant yeasts and  additional preservation may be necessary. With a con-  tinuing trend towards the elimination of sucrose from  medicines, alternative solutes which are not thought to  encourage dental caries such as sorbitol and fructose have  been investigated. A w can also be reduced by drying,  although the dry, often hygroscopic medicines (tablets,  capsules, powders, vitreous ‘glasses’) will require suitable  packaging to prevent resorption of water and consequent  microbial growth (Figure 17.2).   Tablet film coatings are now available which greatly  reduce water vapour uptake during storage while allow-  ing ready dissolution in bulk water. These might contrib-  ute to increased microbial stability during storage in  particularly humid climates, although suitable foil strip  packing may be more effective, albeit more expensive.   Condensed water films can accumulate on the surface  of otherwise ‘dry’ products such as tablets or bulk oils  following storage in damp atmospheres with fluctuating  temperatures, resulting in sufficiently high localized A„ to  initiate fungal growth. Condensation similarly formed on  the surface of viscous products such as syrups and creams,  or exuded by syneresis from hydrogels, may well permit  surface yeast and fungal spoilage.   Figure 17.2 Fungal growth on a tablet which has become  damp (raised A w ) during storage under humid conditions.  Note the sparseness of mycelium, and conidiophores. The  contaminant is thought to be a Penicillium sp.   The ability of microbes to grow in an environment is  influenced by their oxidation-reduction balance (redox  potential), as they will require compatible terminal elec-  tron acceptors to permit their respiratory pathways to  function. The redox potential even in fairly viscous emul-  sions may be quite high because of the appreciable solu-  bility of oxygen in most fats and oils.   Spoilage of pharmaceuticals could occur potentially over  the range of about — 20 °C to 60 °C, although it is much  less likely at the extremes. The particular storage tem-  perature may selectively determine the types of microor-  ganisms involved in spoilage. A deep freeze at — 20 °C or  lower is used for long-term storage of some pharmaceuti-  cal raw materials and short-term storage of dispensed  total parenteral nutrition (TPN) feeds prepared in hos-  pitals. Reconstituted syrups and multidose eye drop  packs are sometimes dispensed with the instruction to  ‘store in a cool place’ such as a domestic fridge (2-8 °C),  partly to reduce the risk of growth of contaminants inad-  vertently introduced during use. Conversely, Water for  Injections (EP) should be held at 80 °C or above after  distillation and before packing and sterilization to prevent   Extremes of pEl prevent microbial attack. Around neu-  trality bacterial spoilage is more likely, with reports of  pseudomonads and related Gram-negative bacteria  growing in antacid mixtures, flavoured mouthwashes and  distilled or demineralized water. Above pH 8 (e.g. with  soap-based emulsions) spoilage is rare. In products with  low pH levels (e.g. fruit-juice-flavoured syrups with a pH  3-4), mould or yeast attack is more likely. Yeasts can  metabolize organic acids and raise the pH to levels where  secondary bacterial growth can occur. Although the use  of low pH adjustment to preserve foodstuffs is well estab-  lished (e.g. pickling, coleslaw, yoghurt), it is not practica-  ble to make deliberate use of this for medicines.   Packaging can have a major influence on microbial stabil-  ity of some formulations in controlling the entry of con-  taminants during both storage and use. Considerable  thought has gone into the design of containers to prevent  the ingress of contaminants into medicines for parenteral  administration, because of the high risks of infection by  this route. Self-sealing rubber wads must be used to  prevent microbial entry into multidose injection contain-  ers (Chapter 22) following withdrawals with a hypoder-  mic needle. Wide-mouthed cream jars have now been  replaced by narrow nozzles and flexible screw-capped  tubes, thereby removing the likelihood of operator-  introduced contamination during use of the product.  Similarly, hand creams, previously supplied in glass jars,  are now packed in closed, disposable dispensers. Where  medicines rely on their low A w to prevent spoilage, pack-  aging such as strip foils must be of water-vapour-proof  materials with fully efficient seals. Cardboard outer pack-  aging and labels themselves can become substrates for  microbial attack under humid conditions, and preserva-  tives are often included to reduce the risk of damage.   The survival of microorganisms in particular environ-  ments is sometimes influenced by the presence of rela-  tively inert materials. Thus, microbes can be more  resistant to heat or desiccation in the presence of poly-  mers such as starch, acacia or gelatin. Adsorption on to  naturally occurring particulate material may aid estab-  lishment and survival in some environments. There is a    belief, but limited hard evidence, that the presence of  suspended particles such as kaolin, magnesium trisilicate  or aluminium hydroxide gel may influence contaminant  longevity in those products containing them, and that the  presence of some surfactants, suspending agents and pro-  teins can increase the resistance of microorganisms to  preservatives, over and above their direct inactivating  effect on the preservative itself.   Nowadays, it is well recognized that the inadvertent use  of a contaminated pharmaceutical product may also  present a potential health hazard to the patient. Although  isolated outbreaks of medicament-related infections  had been reported since the early part of the 20th century,  it was only in the 1960s and 1970s that the significance  of this contamination to the patient was more fully  understood.   Inevitably, the infrequent isolation of true pathogens,  such as Salmonella spp. and the reporting of associated  infections following the use of products contaminated  with these organisms (tablets with pancreatin and thyroid  extract), attracted considerable attention. More often, the  isolation of common saprophytic and non-fastidious  opportunist contaminants with limited pathogenicity to  healthy individuals has presented a significant challenge  to compromised patients.   Gram-negative contaminants, particularly Pseudomo-  nas spp., which have simple nutritional requirements and  can multiply to significant levels in aqueous products,  have been held responsible for numerous outbreaks of  infection. For example, while the intact cornea is quite  resistant to infection, it offers little resistance to pseu-  domonads and related bacteria when scratched, or  damaged by irritant chemicals; loss of sight has frequently  occurred following the use of poorly designed ophthal-  mic solutions which had become contaminated by Ps.  aeruginosa and even supported its active growth.  Pseudomonads contaminating ‘antiseptic’ solutions have  infected the skin of badly burnt patients, resulting in the  failure of skin grafts and subsequent death from Gram-  negative septicaemia. Infections of eczematous skin and  respiratory infections in neonates have been traced to  ointments and creams contaminated with Gram-negative  bacteria. Oral mixtures and antacid suspensions can  support the growth of Gram-negative bacteria and  serious consequences have resulted following their inad-  vertent administration to patients who were immuno-    compromised as a result of antineoplastic chemotherapy.  Growth of Gram-negative bacteria in bladder washout  solutions has been held responsible for painful infections.  In more recent times, Pseudomonas contamination of  TPN fluids during their aseptic compounding in the hos-  pital pharmacy caused the death of several children in the  same hospital.   Fatal viral infections resulting from the use of con-  taminated human tissue or fluids as components of med-  icines are well recorded. Examples of this include HIV  infection of haemophiliacs by contaminated and inade-  quately treated factor VIII products made from pooled  human blood, and Creutzfeldt-Jakob disease (CJD) from  injections of human growth hormone derived from  human pituitary glands, some of which were infected.   Pharmaceutical products of widely differing forms are  known to be susceptible to contamination with a variety  of microorganisms, ranging from true pathogens to a  motley collection of opportunist pathogens (see Table  17.1). Disinfectants, antiseptics, powders, tablets and  other products providing an inhospitable environment to  invading contaminants are known to be at risk, as well as  products with more nutritious components, such as  creams and lotions with carbohydrates, amino acids, vita-  mins and often appreciable quantities of water.   The outcome of using a contaminated product may  vary from patient to patient, depending on the type and  degree of contamination and how the product is to be  used. Undoubtedly, the most serious effects have been  seen with contaminated injected products where general-  ized bacteraemic shock and in some cases death of  patients have been reported. More likely, a wound or sore  in broken skin may become locally infected or colonized  by the contaminant; this may in turn result in extended  hospital bed occupancy, with ensuing economic conse-  quences. It must be stressed, however, that the majority  of cases of medicament-related infections are probably  not recognized or reported as such. Recognition of these  infections presents its own problems. It is a fortunate  hospital physician who can, at an early stage, recognize  contamination shown as a cluster of infections of rapid  onset, such as that following the use of a contaminated  intravenous fluid in a hospital ward. The chances of a  general practitioner recognizing a medicament-related  infection of insidious onset, perhaps spread over several  months, in a diverse group of patients in the community,  are much more remote. Once recognized, of course, there  is a moral obligation to withdraw the offending product;  subsequent investigations of the incident therefore  become retrospective.   Gram-negative bacteria contain lipopolysaccharides  (endotoxins) in their outer cell membranes (Chapter 22);  these can remain in an active condition in products even  after cell death and some can survive moist heat steriliza-  tion. Although inactive by the oral route, endotoxins can  induce a number of physiological effects if they enter the  bloodstream via contaminated infusion fluids, even in  nanogram quantities, or via diffusion across membranes    from contaminated haemodialysis solutions. Such effects  may include fever, activation of the cytokine system,  endothelial cell damage, all leading to septic and often  fatal febrile shock.   The acute bacterial toxins associated with food poison-  ing episodes are not commonly reported in pharmaceuti-  cal products, although aflatoxin-producing aspergilli  have been detected in some vegetable and herbal ingre-  dients. However, many of the metabolites of microbial    deterioration have quite unpleasant tastes and smell even  at low levels, and would deter most patients from using  such a medicine.   Regardless of whether manufacture takes place in indus-  try or on a smaller scale in the hospital pharmacy, the  microbiological quality of the finished product will be  determined by the formulation components used, the  environment in which they are manufactured and  the manufacturing process itself. As discussed in Chapter  23, quality must be built into the product at all stages of  the process and not simply inspected at the end of  manufacture:   • Raw materials, particularly water and those of natural  origin, must be of a high microbiological standard.   • All processing equipment should be subject to planned  preventive maintenance and should be properly cleaned  after use to prevent cross-contamination between batches.   • Cleaning equipment should be appropriate for the task  in hand and should be thoroughly cleaned and properly  maintained.   • Manufacture should take place in suitable premises,  supplied with filtered air, for which the environmental  requirements vary according to the type of product being  made.   • Staff involved in manufacture should not only have  good health but also a sound knowledge of the importance  of personal and production hygiene.   • The end-product requires suitable packaging which  will protect it from contamination during its shelf-life  and is itself free from contamination.   Manufacture in hospital premises raises certain addi-  tional problems with regard to contamination control.   Mains water in hospitals is frequently stored in large roof  tanks, some of which may be relatively inaccessible and  poorly maintained. Water for pharmaceutical manufac-  ture requires some further treatment, usually by distilla-  tion, reverse osmosis or deionization or a combination of  these, depending on the intended use of water. Such proc-  esses need careful monitoring, as does the microbiologi-  cal quality of the water after treatment. Storage of water  requires particular care, as some Gram-negative oppor-    tunist pathogens can survive on traces of organic matter  present in treated water and will readily multiply to high  numbers at room temperature. Water should therefore be  stored at a temperature in excess of 80 °C and circulated  in the distribution system at a flow rate of 1-2 m/s to  prevent the build-up of bacterial biofilms in the piping.   The microbial flora of the hospital pharmacy environ-  ment is a reflection of the general hospital environment  and the activities undertaken there. Free-living oppor-  tunist pathogens, such as Ps. aeruginosa, can normally be  found in wet sites, such as drains, sinks and taps. Cleaning  equipment, such as mops, buckets, cloths and scrubbing  machines, may be responsible for distributing these  organisms around the pharmacy; if stored wet they  provide a convenient niche for microbial growth, result-  ing in heavy contamination of equipment. Contamination  levels in the production environment may, however, be  minimized by observing good manufacturing practices  (CMP), by installing heating traps in sink U-bends, thus  destroying one of the main reservoirs of contaminants,  and by proper maintenance and storage of equipment,  including cleaning equipment. Additionally, cleaning of  production units by contractors should be carried out to  a pharmaceutical specification.   Sacking, cardboard, card liners, corks and paper are  unsuitable for packaging pharmaceuticals, as they are  heavily contaminated, for example with bacterial or  fungal spores. These have now been replaced by non-  biodegradable plastic materials. In the past, packaging in  hospitals was frequently reused for economic reasons.  Large numbers of containers may be returned to the  pharmacy, bringing with them microbial contaminants  introduced during use in the wards. Particular problems  have been encountered with disinfectant solutions where  residues of old stock have been ‘topped up’ with fresh  supplies, resulting in the issue of contaminated solutions  to wards. Reusable containers must therefore be thor-  oughly washed and dried, and never refilled directly.   Another common practice in hospitals is the repackag-  ing of products purchased in bulk into smaller contain-  ers. Increased handling of the product inevitably increases  the risk of contamination, as shown by one survey when  hospital-repacked items were found to be contaminated  twice as often as those in the original pack (Public Health  Laboratory Service Report, 1971).   Pharmaceutical manufacturers may justly argue that their  responsibility ends with the supply of a well-preserved  product of high microbiological standard in a suitable  pack and that the subsequent use, or indeed abuse, of the  product is of little concern to them. Although much less  is known about how products become contaminated  during use, their continued use in a contaminated state  is clearly undesirable, particularly in hospitals where it  could result in the spread of cross-infection. All rnulti-  dose products are vulnerable to contamination during  use. Regardless of whether products are used in hospital  or in the community environment, the sources of con-  tamination are the same, but opportunities for observing  it are greater in the former. Although the risk of contami-  nation during product use has been much reduced in  recent years, primarily through improvements in packag-  ing and changes in nursing practices, it is nevertheless  salutary to reflect upon past reported case histories.   During normal usage, patients may contaminate their  medicine with their own microbial flora; subsequent use  of such products may or may not result in self-infection  (Figure 17.3).   Topical products are considered to be most at risk, as  the product will probably be applied by hand, thus intro-  ducing contaminants from the resident skin flora of sta-  phylococci, Micrococcus spp. and diphtheroids but also  perhaps transient contaminants, such as Pseudomonas  or coliforms, which would normally be removed with  effective hand-washing. Opportunities for contamina-  tion may be reduced by using disposable applicators for  topical products or by giving oral products by disposable  spoon.   In hospitals, multidose products, once contaminated,  may serve as a vehicle of cross-contamination or cross-  infection between patients. Zinc-based products packed  in large stockpots and used in the treatment and preven-    tion of bedsores in long-stay and geriatric patients were  reportedly contaminated during use with Ps. aeruginosa  and Staphylococcus aureus. If unpreserved, these products  permit multiplication of contaminants, especially if water  is present either as part of the formulation, for example  in oil/water (o/w) emulsions, or as a film in w/o emul-  sions which have undergone local cracking, or as a con-  densed film from atmospheric water. Appreciable  numbers of contaminants may then be transferred to  other patients when the product is reused. Clearly the  economics and convenience of using stockpots need to  be balanced against the risk of spreading cross-infection  between patients and the inevitable increase in length  of the patients’ stay in hospital. The use of stockpots  in hospitals has noticeably declined over the past two  decades or so.   A further potential source of contamination in hospi-  tals is the nursing staff responsible for medicament  administration. During the course of their work, nurses’  hands become contaminated with opportunist pathogens  which are not part of the normal skin flora but which are  easily removed by thorough hand-washing and drying. In  busy wards, hand-washing between attending to patients  may be overlooked and contaminants may subsequently  be transferred to medicaments during administration.  Hand lotions and creams used to prevent chapping of  nurses’ hands may similarly become contaminated, espe-  cially when packaged in multidose containers and left at  the side of the hand-basin, frequently without lids. Hand  lotions and creams should be well preserved and, ideally,  packaged in disposable dispensers. Other effective control  methods include the supply of products in individual  patient’s packs and the use of non-touch techniques for  medicament administration. The importance of thor-  ough hand-washing in the control of hospital cross-  infection cannot be overemphasized. In recent years  hospitals have successfully raised the level of awareness  on this topic among staff and the general public through  widespread publicity and the provision of easily accessi-  ble hand disinfection stations on the wards.   Small numbers of airborne contaminants may settle in  products left open to the atmosphere. Some of these will  die during storage, with the rest probably remaining at a  static level of about 10 2 -10 3 colony forming units (CFU)  per gram or per millilitre. Larger numbers of waterborne  contaminants maybe accidentally introduced into topical  products by wet hands or by a ‘splash-back mechanism’  if left at the side of a basin. Such contaminants generally    have simple nutritional requirements and, following  multiplication, levels of contamination may often exceed  10 6 CFU/g. In the past this problem has been encountered  particularly when the product was stored in warm hos-  pital wards or in hot steamy bathroom cupboards at  home. Products used in hospitals as soap substitutes for  bathing patients are particularly at risk and soon not only  become contaminated with opportunist pathogens such  as Pseudomonas spp., but also provide conditions condu-  cive to their multiplication. The problem is compounded  by stocks kept in multidose pots for use by several patients  in the same ward over an extended period of time.   The indigenous microbial population is quite different  in the home and in hospitals. Pathogenic organisms are  found much more frequently in the latter and conse-  quently are isolated more often from medicines used in  hospital. Usually, there are fewer opportunities for con-  tamination in the home, as patients are generally issued  with individual supplies in small quantities.   Patients and nursing staff may use a range of applicators  (pads, sponges, brushes and spatulas) during medica-  ment administration, particularly for topical products.  If reused, these easily become contaminated and may  be responsible for perpetuating contamination between  fresh stocks of product, as has indeed been shown in  studies of cosmetic products. Disposable applicators or  swabs should therefore always be used.   In hospitals today a wide variety of complex equip-  ment is used in the course of patient treatment.  Humidifiers, incubators, ventilators, resuscitators and  other apparatus require proper maintenance and decon-  tamination after use. Chemical disinfectants used for this  purpose have in the past, through misuse, become con-  taminated with opportunist pathogens, such as Ps. aeru-  ginosa , and ironically have contributed to, rather than  reduced, the spread of cross-infection in hospital patients.  Disinfectants should only be used for their intended  purpose and directions for use must be followed at all  times.   Most reports of medicament-borne contamination in the  literature tend to be anecdotal in nature, referring to a  specific product and isolated incident. Little information  is available on the overall risk of products becoming con-  taminated and causing patient infections when subse-    quently used. Such information is considered invaluable  not only because it may indicate the effectiveness of exist-  ing practices and standards, but also because the value of  potential improvements in patient quality can be bal-  anced against the inevitable cost of such processes.   Investigations carried out by the Swedish National Board  of Health in 1965 revealed some startling findings on the  overall microbiological quality of non-sterile products  immediately after manufacture. A wide range of products  was routinely found to be contaminated with Bacillus  subtilis, Staph, albus, yeasts and moulds, and in addition  large numbers of coliforms were found in a variety of  tablets. Furthermore, two nationwide outbreaks of infec-  tion in Sweden were subsequently traced to the inadvert-  ent use of contaminated products. Two hundred patients  were involved in an outbreak of salmonellosis, caused  by thyroid tablets contaminated with Salmonella bareilly  and Sal. muenchen (now known as Salmonella enterica  subsp. enterica serovar Bareilly and Sal. enterica serovar  Muenchen respectively); and eight patients had severe eye  infections following the use of a hydrocortisone eye oint-  ment contaminated with Ps. aeruginosa. The results of  this investigation had a profound effect on the manufac-  ture of all medicines; not only were they then used as a  yardstick to compare the microbiological quality of non-  sterile products made in other countries, but also as a  baseline upon which international standards could be  founded.   Under the UK Medicines Act 1968, pharmaceutical  products made in industry were expected to conform to  microbiological and chemical quality specifications. The  majority of products have since been shown to conform  to a high standard, although spot checks have occasion-  ally revealed medicines of unacceptable quality and so  necessitated product recall. By contrast, pharmaceutical  products made in hospitals were much less rigorously  controlled, as shown by several surveys in the 1970s in  which significant numbers of preparations were found to  be contaminated with Ps. aeruginosa. In 1974, however,  hospital manufacture also came under the terms of  the Medicines Act and, as a consequence, considerable  improvements were subsequently seen not only in the  conditions and standard of manufacture, but also in the  chemical and microbiological quality of finished prod-  ucts. Hospital manufacturing operations were later  rationalized. Economic constraints caused a critical eval-  uation of the true cost of these activities. Competitive  purchasing from industry in many cases produced    cheaper alternatives, and small-scale manufacturing was  largely discouraged. Where licensed products were avail-  able, NHS policy dictated that these were to be purchased  from a commercial source and not made locally.   Removal of Crown immunity from the NHS in 1991  meant that manufacturing operations in hospitals were  then subject to the full licensing provisions of the  Medicines Act 1968, i.e. hospital pharmacies intending to  manufacture were required to obtain a manufacturing  licence and to comply fully with the EC Guide to Good  Pharmaceutical Manufacturing Practice (Anon, 1992,  revised in 1997, 2002 and 2007). Among other require-  ments, this included the provision of appropriate envi-  ronmental manufacturing conditions and associated  environmental monitoring. Subsequently, the Medicines  Control Agency (MCA) issued guidance in 1992 on  certain manufacturing exemptions, by virtue of the  product batch size or frequency of manufacture. The  need for extemporaneous dispensing of ‘one-off’ special  formulae continued in hospital pharmacies, although this  work was largely transferred from the dispensing bench  to dedicated preparative facilities with appropriate envi-  ronmental control. Today hospital manufacturing is con-  centrated on the supply of bespoke products from a  regional centre or small-scale specialist manufacture of  those items currently unobtainable from industry.  Repacking of commercial products into more convenient  pack sizes is, however, still common practice.   Higher rates of contamination are invariably seen in  products after opening and use and, among these, medi-  cines used in hospitals are more likely to be contaminated  than those used in the general community. The Public  Health Laboratory Service Report of 1971 expressed  concern at the overall incidence of contamination in  non-sterile products used on hospital wards (327 of 1220  samples) and the proportion of samples found to be  heavily contaminated (18%> 10 4 CFU/g or CFU/ml).  Notably, the presence of Ps. aeruginosa in 2.7% of samples  (mainly oral alkaline mixtures) was considered to be  highly undesirable.   By contrast, medicines used in the home are not only  less often contaminated but also contain lower levels of  contaminants and fewer pathogenic organisms. Generally,  there are fewer opportunities for contamination here  because individual patients use smaller quantities.  Medicines in the home may, however, be hoarded and  used for extended periods of time. Additionally, storage  conditions may be unsuitable and expiry dates ignored;    thus problems other than those of microbial contamina-  tion may be seen in the home.   Although impossible to quantify, the use of contaminated  medicines has undoubtedly contributed to the spread of  cross-infection in hospitals; undeniably, such nosocomial  (hospital-acquired) infections have also extended the  length of stay in hospital with concomitant costs. A  patient’s response to the microbial challenge of a con-  taminated medicine may be diverse and unpredictable,  perhaps with serious consequences. Clinical reactions  may not be evident in one patient, yet in another may be  indisputable, illustrating one problem in the recognition  of medicament-borne infections. Clinical reactions may  range from inconvenient local infections of wounds or  broken skin, caused possibly from contact with a con-  taminated cream, to gastrointestinal infections from the  ingestion of contaminated oral products, to serious wide-  spread infections such as a bacteraemia or septicaemia,  possibly resulting in death, as caused by the administra-  tion of contaminated infusion fluids. Undoubtedly, the  most serious outbreaks of infection have been seen in the  past where contaminated products have been injected  directly into the bloodstream of patients whose immu-  nity is already compromised by their underlying disease  or therapy.   The outcome of any episode is determined by a com-  bination of several factors, among which the type and  degree of microbial contamination, the route of admin-  istration and the patient’s resistance are of particular  importance.   Microorganisms that contaminate medicines and cause  disease in patients may be classified as true pathogens or  opportunist pathogens. Pathogenic organisms like  Clostridium tetani and Salmonella spp. rarely occur in  products, but when present cause serious problems.  Wound infections and several cases of neonatal death  have resulted from use of talcum powder containing Cl.  tetani. Outbreaks of salmonellosis have followed the  inadvertent ingestion of contaminated thyroid and pan-  creatic powders. On the other hand, opportunist patho-  gens like Ps. aeruginosa, Klebsiella, Serratia and other  free-living organisms are more frequently isolated from    medicinal products and, as their name suggests, may be  pathogenic if given the opportunity. The main concern  with these organisms is that their simple nutritional  requirements enable them to survive in a wide range  of pharmaceuticals, and thus they tend to be present in  high numbers, perhaps in excess of 10 6 -10 7 CFU/g or  CFU/ml. The product itself, however, may show no visible  sign of contamination. Opportunist pathogens can  survive in disinfectants and antiseptic solutions that are  normally used in the control of hospital cross-infection,  but which, when contaminated, may even perpetuate the  spread of infection. Compromised hospital patients, i.e.  elderly, burned, traumatized or immunosuppressed  patients, are considered to be particularly at risk from  infection with these organisms, whereas healthy patients  in the general community have given little cause for  concern.   The critical dose of microorganisms that will initiate  an infection is largely unknown and varies not only  between species but also within a species. Animal and  human volunteer studies have indicated that the infecting  dose may be reduced significantly in the presence of  trauma or foreign bodies or if accompanied by a drug  having a local vasoconstrictive action.   As stated previously, contaminated products injected  directly into the bloodstream or instilled into the eye  cause the most serious problems. Intrathecal and epi-  dural injections are potentially hazardous procedures. In  practice, epidural injections are frequently given through  a bacterial filter. Injectable and ophthalmic solutions are  often simple solutions and provide Gram-negative  opportunist pathogens with sufficient nutrients to mul-  tiply during storage; if contaminated, a bioburden of  10 6 CFU as well as the production of endotoxins should  be expected. TPN fluids, formulated for individual  patients’ nutritional requirements, can also provide more  than adequate nutritional support for invading contami-  nants. Ps. aeruginosa , the notorious contaminant of eye  drops, has caused serious ophthalmic infections, includ-  ing the loss of sight in some cases. The problem is com-  pounded when the eye is damaged through the improper  use of contact lenses or scratched by fingernails or cos-  metic applicators.   The fate of contaminants ingested orally in medicines  may be determined by several factors, as is seen with  contaminated food. The acidity of the stomach may  provide a successful barrier, depending on whether the  medicine is taken on an empty or full stomach and also    on the gastric emptying time. Contaminants in topical  products may cause little harm when deposited on intact  skin. Not only does the skin itself provide an excellent  mechanical barrier, but few contaminants normally  survive in competition with its resident microbial flora.  Skin damaged during surgery or trauma or in patients  with burns or pressure sores may, however, be rapidly  colonized and subsequently infected by opportunist  pathogens. Patients treated with topical steroids are also  prone to local infections, particularly if contaminated  steroid drugs are inadvertently used.   A patient’s resistance is crucial in determining the  outcome of a medicament-borne infection. Hospital  patients are more exposed and susceptible to infection  than those treated in the general community. Neonates,  elderly people, diabetics and patients traumatized by  surgery or accident may have impaired defence mecha-  nisms. People suffering from leukaemia and those treated  with immunosuppressants are most vulnerable to infec-  tion; there is an undeniable case for providing all medi-  cines in a sterile form for these patients.   An antimicrobial ‘preservative’ may be included in a for-  mulation to minimize the risk of spoilage and preferably  to kill low levels of contaminants introduced during  storage or repeated use of a multidose container. However,  where there is a low risk of contamination, as with tablets,  capsules and dry powders, the inclusion of a preservative  may be unnecessary. Preservatives should never be added  to mask poor manufacturing processes.   The properties of an ideal preservative are well recog-  nized: a broad spectrum of activity and a rapid rate of  kill; selectivity in reacting with the contaminants and not  the formulation ingredients; non-irritant and non-toxic  to the patient; and stable and effective throughout the life  of the product.   Unfortunately, the most active antimicrobial agents  are often non-selective in action, interacting significantly  with formulation ingredients as well as with patients  and microorganisms. Having excluded the more toxic,  irritant and reactive agents, those remaining generally  have only modest antimicrobial efficacy, and no preserva-  tives are now considered sufficiently non-toxic for use in    highly sensitive areas, e.g. for injection into central  nervous system tissues or for use within the eye. A  number of microbiologically effective preservatives used  in cosmetics have caused a significant number of cases  of contact dermatitis, and are thus precluded from use  in pharmaceutical creams. Although a rapid rate of  kill may be preferable, this may only be possible for rela-  tively simple aqueous solutions such as eye drops or  injections. For physicochemically complex systems  such as emulsions and creams, inhibition of growth and  a slow rate of killing may be all that can be realistically  achieved.   In order to maximize preservative efficacy, it is essen-  tial to have an appreciation of those parameters that  influence antimicrobial activity.   Changes in the efficacy of preservatives vary exponen-  tially with changes in concentration. The effect of changes  in concentration (concentration exponent, 77, Chapter  18) varies with the type of agent. For example, halving  the concentration of phenol (77 = 6) gives a 64-fold (2 s )  reduction in killing activity, whereas a similar dilution for  chlorhexidine (77 = 2) reduces the activity by only four-  fold (2 2 ). Changes in preservative activity are also seen  with changes in product temperature, according to the  temperature coefficient, Q 10 . Thus, a reduction in tem-  perature from 30 °C to 20 °C could result in a significantly  reduced rate of kill for Escherichia coli , fivefold in the case  of phenol (Q 10 = 5) and 45-fold in the case of ethanol  (Qio = 45). If both temperature and concentration vary  concurrently, the situation is more complex; however, it  has been suggested that if a 0.1% chlorocresol (77 = 6,  Q 10 = 5) solution completely killed a suspension of E. coli  at 30 °C in 10 minutes, it would require around 90  minutes to achieve a similar effect if stored at 20 °C and  if slight overheating during production had resulted in a  10% loss in the chlorocresol concentration (other factors  remaining constant).   Preservative molecules are used up as they inactivate  microorganisms and as they interact non-specifically  with significant quantities of contaminant ‘dirt’ intro-  duced during use. This will result in a progressive and  exponential decline in the efficiency of the remaining  preservative. Preservative ‘capacity’ is a term used to  describe the cumulative level of contamination that a  preserved formulation can tolerate before becoming so  depleted as to become ineffective. This will vary with  preservative type and complexity of formulation.   Most preservatives interact in solution to some extent  with many of the commonly used formulation ingredi-  ents via a number of weak bonding attractions as well  as with any contaminants present. Unstable equilibria  may form in which only a small proportion of total  preservative present is ‘available’ to inactivate the rela-  tively small microbial mass; the resulting rate of kill  may be far lower than might be anticipated from the  performance of simple aqueous solutions. Flowever,  ‘unavailable’ preservative may still contribute to the  general irritancy of the product. It is commonly believed  that where the solute concentrations are very high,  and A w is appreciably reduced, the efficiency of preserva-  tives is often significantly reduced and they may be  virtually inactive at very low A„. The practice of including  preservatives in very low A* products such as tablets  and capsules is ill advised, as it only offers minimal  protection for the dry tablets; should they become  damp, they would be spoiled for other, non-microbial,  reasons.   In the weakly acidic preservatives, activity resides prima-  rily in the unionized molecules and they only have sig-  nificant efficacy at pFl values where ionization is low.  Thus, benzoic and sorbic acids (pff a =4.2 and 4.75,  respectively) have limited preservative usefulness above  pFl 5, while the 4(p)-hydroxybenzoate (parabens) esters  with their non-ionizable ester group and poorly ionizable  hydroxyl substituent (pK a c.8.5) have a moderate protec-  tive effect even at neutral pFl levels. The activity of qua-  ternary ammonium preservatives and chlorhexidine  probably resides with their cations; they are effective in  products of neutral pH. Formulation pH can also directly  influence the sensitivity of microorganisms to preserva-  tives (see Chapter 18).   In a multiphase formulation, such as an oil-in-water  emulsion, preservative molecules will distribute them-  selves in an unstable equilibrium between the bulk  aqueous phase and (1) the oil phase by partition, (2) the  surfactant micelles by solubilization, (3) polymeric  suspending agents and other solutes by competitive dis-  placement of water of solvation, (4) particulate and  container surfaces by adsorption and (5) any microor-  ganisms present. Generally, the overall preservative  efficiency can be related to the small proportion of pre-    servative molecules remaining unbound in the bulk  aqueous phase, although as this becomes depleted some  slow re-equilibration between the components can be  anticipated. The loss of neutral molecules into oil and  micellar phases may be favoured over ionized species,  although considerable variation in distribution is found  between different systems.   In view of these major potential reductions in pre-  servative efficacy, considerable effort has been directed to  devise equations in which one might substitute variously  derived system parameters (such as partition coefficients,  surfactant and polymer binding constants and oikwater  ratios) to obtain estimates of residual preservative levels  in aqueous phases. Although some modestly successful  predictions have been obtained for very simple labora-  tory systems, they have proved of limited practical value,  as data for many of the required parameters are unavail-  able for technical grade ingredients or for the more  complex commercial systems.   Preservative availability may be appreciably reduced by  interaction with packaging materials. Phenolics, for  example, will permeate the rubber wads and teats of  multidose injection or eye drop containers and also inter-  act with flexible nylon tubes for creams. Quaternary  ammonium preservative levels in formulations have been  significantly reduced by adsorption on to the surfaces of  plastic and glass containers. Volatile preservatives such as  chloroform are so readily lost by the routine opening and  closing of containers that their usefulness is somewhat  restricted to preservation of medicines in sealed, imper-  vious containers during storage, with short in-use lives  once opened.   8 Quality assurance and the control of  microbial risk in medicines   Manufacturers of medicinal products must comply with  the requirements of their marketing authorization  (product licence) and ensure that their products are fit  for their intended use in terms of safety, quality and effi-  cacy. A quality management system (QMS) must there-  fore be in place so that senior management can ensure  that the required quality objectives are met through a  comprehensively designed and properly implemented  system of quality assurance (QA), encompassing both  GPMP and quality control (QC).    QA encompasses, in turn, a scheme of management  which embraces all the procedures necessary to provide  a high probability that a medicine will conform consist-  ently to a specified description of quality. It includes for-  mulation design and development (R&D), GPMP, as well  as QC and postmarketing surveillance. As many micro-  organisms may be hazardous to patients or cause spoilage  of formulations under suitable conditions, it is necessary  to perform a risk assessment of contamination for each  product. At each stage of its anticipated life from raw  materials to administration, a risk assessment should be  made and strategies should be developed and calculated  to reduce the overall risk(s) to acceptably low levels. Such  risk assessments are complicated by uncertainties about  the exact infective and spoilage hazards likely for many  contaminants, and by difficulties in measuring their  precise performance in complex systems. As the conse-  quences of product failure and patient harm will inevita-  bly be severe, it is usual for manufacturing companies to  make worst-case presumptions and design strategies to  cover them fully; lesser problems are also then encom-  passed. As it must be assumed that all microorganisms  may be potentially hazardous for those routes of admin-  istration where the likelihood of infection from contami-  nants is high, then medicines to be given via these routes  must be supplied in a sterile form, as is the case with  injectable products. It must also be presumed that those  administering medicines may not necessarily be highly  skilled or motivated in contamination control techniques;  additional safeguards to control risks may be included in  these situations. This may include detailed information  on administration as well as training, in addition to pro-  viding a high quality formulation.   The risk of microbial infection and spoilage arising from  microbial contamination during manufacture, storage  and use could be eliminated by presenting all medicines  in sterile, impervious, single-dosage units. However,  the high cost of this strategy restricts its use to situations  where there is a high risk of consequent infection  from any contaminants. Where the risk is assessed as  much lower, less efficient but less expensive strategies are  adopted. The high risk of infection by contaminants  in parenteral medicines, combined with concerns about  the systemic toxicity of preservatives, almost always  demands sterile single-dosage units. With eye drops for  domestic use the risks are perceived to be lower, and  sterile multidose products with preservatives to combat    the anticipated in-use contamination are accepted; sterile  single-dose units are more common in hospitals where  there is an increased risk of infection. Oral and topical  routes of administration are generally perceived to  present relatively low risks of infection and the emphasis  is more on the control of microbial content during man-  ufacture and subsequent protection of the formulation  from chemical and physicochemical spoilage.   As part of the design process, it is necessary to include  features in the formulation and delivery system that  provide as much suitable protection as possible against  microbial contamination and spoilage. Owing to poten-  tial toxicity and irritancy problems, antimicrobial pre-  servatives should only be considered where there is clear  evidence of positive benefit. Manipulation of physico-  chemical parameters, such as A m the elimination of par-  ticularly susceptible ingredients (e.g. natural ingredients  such as tragacanth powder, used as a thickening agent),  the selection of a preservative or the choice of container  may individually and collectively contribute significantly  to overall medicine stability. For ‘dry’ dosage forms where  their very low provides protection against microbial  attack, the moisture vapour properties of packaging  materials require careful examination.   Preservatives are intended to offer further protection  against environmental microbial contaminants. However,  as they are relatively non-specific in their reactivity (see  section 7), it is difficult to calculate with any certainty  what proportion of preservative added to all but the sim-  plest medicine will be available for inactivating such con-  tamination. Laboratory tests have been devised to  challenge the product with an artificial bioburden. Such  tests should form part of formulation development and  stability trials to ensure that suitable activity is likely to  remain throughout the life of the product. They are not  normally used in routine manufacturing QC.   Some ‘preservative challenge tests’ (preservative effi-  cacy tests) add relatively large inocula of various labora-  tory cultures to aliquots of the product and determine  their rate of inactivation by viable counting methods  (single challenge tests), while others reinoculate repeat-  edly at set intervals, monitoring the efficiency of inactiva-  tion until the system fails (multiple challenge test). This  latter technique may give a better estimate of the pre-  servative capacity of the system than the single challenge  approach, but is both time-consuming and expensive.  Problems arise when deciding whether the observed per-  formance in such tests gives reliable predictions of real  in-use efficacy. Although test organisms should bear  some similarity in type and spoilage potential to those    met in use, it is known that repeated cultivation on con-  ventional microbiological media (nutrient agar, etc.) fre-  quently results in reduced virulence of strains. Attempts  to maintain spoilage activity by inclusion of formulation  ingredients in culture media give varied results. Some  manufacturers have been able to maintain active spoilage  strains by cultivation in unpreserved, or diluted aliquots,  of formulations.   The British Pharmacopoeia and the European Pharma-  copoeia describe a single challenge preservative test that  routinely uses four test organisms (two bacteria, a yeast  and a mould), none of which has any significant history  of spoilage potential and which are cultivated on conven-  tional media. However, extension of the basic test is rec-  ommended in some situations, such as the inclusion of  an osmotolerant yeast if it is thought such in-use spoilage  might be a problem. Despite its accepted limitations and  the cautious indications given as to what the tests might  suggest about a formulation, the test does provide some  basic, but useful indicators of likely in-use stability. UK  product licence applications for preserved medicines  must demonstrate that the formulation at least meets the  preservative efficacy criteria of the British Pharmacopoeia  or a similar test.   The concept of the D-value as used in sterilization  technology (Chapter 21) has been applied to the inter-  pretation of challenge testing. Expression of the rate of  microbial inactivation in a preserved system in terms  of a D-value enables estimation of the nominal time to  achieve a prescribed proportionate level of kill. Problems  arise, however, when trying to predict the behaviour of  very low levels of survivors, and the method has its critics  as well as its advocates.   GPMP is concerned with the manufacture of medicines,  and includes control of ingredients, plant construction,  process validation, production and cleaning (see also  Chapter 23). Current GPMP (cGPMP) requirements are  found in the Medicines and Healthcare Products  Regulatory Agency (MHRA) Rules and Guidance for  Pharmaceutical Manufacturers and Distributors, known  as the Orange Guide (Anon 2007), and its 20 annexes.  QC is that part of GPMP dealing with specification, doc-  umentation and assessing conformance to specification.   With traditional QC, a high reliance has been placed  on testing samples of finished products to determine the  overall quality of a batch. This practice can, however,  result in considerable financial loss if non-compliance is    detected only at this late stage, leaving the expensive  options of discarding or reworking the batch. Additionally,  some microbiological test methods have poor precision  and/or accuracy. Validation can be complex or impossi-  ble, and interpretation of results can prove difficult. For  example, although a sterility assurance level of less than  one failure in 10 6 items submitted to a terminal steriliza-  tion process is considered acceptable, conventional ‘tests  for sterility’ for finished products (such as that in the  European Pharmacopoeia) could not possibly be relied  upon to find one damaged but viable microbe within  the 10 6 items, regardless of allowing for its cultivation  with any precision (Chapter 21). Moreover, end-product  testing will not prevent and may not even detect the  isolated rogue processing failure.   It is now generally accepted that a high assurance  of overall product quality can only come from a  detailed specification, control and monitoring of all the  stages that contribute to the manufacturing process.  More realistic decisions about conformance to specifica-  tion can then be made using information from all  relevant parameters (parametric release method), not  just from the results of selective testing of finished  products. Thus, a more realistic estimate of the microbial  quality of a batch of tablets would be achieved from  a knowledge of specific parameters (such as the microbial  bioburden of the starting materials, temperature  records from granule drying ovens, the moisture level of  the dried granules, compaction data, validation records  for the foil strip sealing machine and microbial levels  in the finished tablets), than from the contaminant  content of the finished tablets alone. Similarly, paramet-  ric release is now accepted as an operational alternative  to routine sterility testing for batch release of some fin-  ished sterile products. Through parametric release the  manufacturer can provide assurance that the product is  of the stipulated quality, based on the evidence of suc-  cessful validation of the manufacturing process and  review of the documentation on process monitoring  carried out during manufacturing. Authorization for  parametric release is given, refused or withdrawn by  pharmaceutical assessors, together with GMP inspectors;  the requirements are detailed in Annex 17 of the 2007  Orange Guide.   It may be necessary to exclude certain undesirable con-  taminants from starting materials, such as pseudomon-  ads from bulk aluminium hydroxide gel, or to include  some form of pretreatment to reduce their bioburdens by  irradiation, such as for ispaghula husk, herbal materials  and spices. For biotechnology-derived drugs produced in    human or animal tissue culture, considerable efforts are  made to exclude cell lines contaminated with latent host  viruses. Official guidelines to limit the risk of prion con-  tamination in medicines require bovine-derived ingredi-  ents to be obtained from sources where bovine spongiform  encephalopathy (BSE) is not endemic.   By considering the manufacturing plant and its envi-  rons from an ecological and physiological viewpoint of  microorganisms, it is possible not only to identify areas  where contaminants may accumulate and even thrive to  create hazards for subsequent production batches, but  also to manipulate design and operating conditions in  order to discourage such colonization. The facility to  clean and dry equipment thoroughly is a very useful  deterrent to growth. Design considerations should  include the elimination of obscure nooks and crannies  (where biofilms may readily become established) and the  ability to clean thoroughly in all areas. Some larger items  of equipment now have cleaning-in-place (CIP) and  sterilization-in-place (SIP) systems installed to improve  decontamination capabilities.   It may be necessary to include intermediate steps  within processing to reduce the bioburden and improve  the efficiency of lethal sterilization cycles, or to prevent  swamping of the preservative in a non-sterile medicine  after manufacture. Some of the newer and fragile  biotechnology-derived products may include chromato-  graphic and/or ultrafiltration processing stages to ensure  adequate reductions of viral contamination levels rather  than conventional sterilization cycles.   In a validation exercise, it must be demonstrated  that each stage of the system is capable of providing the  degree of intended efficiency within the limits of varia-  tion for which it was designed. Microbial spoilage aspects  of process validation might include examination of the  cleaning system for its ability to remove deliberately  introduced contamination. Chromatographic removal of  viral contaminants would be validated by determining  the log reduction achievable against a known titre of  added viral particles.   While there is general agreement on the need to control  total microbial levels in non-sterile medicines and to  exclude certain species that have previously proved  troublesome, the precision and accuracy of current  methods for counting (or even detecting) some microbes  in complex products are poor. Pathogens, present in low  numbers, and often damaged by processing, can be very  difficult to isolate. Products showing active spoilage    can yield surprisingly low viable counts on testing.  Although present in high numbers, a particular organism  may be neither pathogenic nor the primary spoilage  agent, but may be relatively inert, e.g. ungerminated  spores or a secondary contaminant which has outgrown  the initiating spoiler. Unevenly distributed growth in  viscous formulations will present serious sampling prob-  lems. The type of culture medium (even different batches  of the same medium) and conditions of recovery and  incubation may significantly influence any viable counts  obtained from products.   An unresolved problem concerns the timing of sam-  pling. Low levels of pseudomonads shortly after manu-  facture may not constitute a spoilage hazard if their  growth is checked. However, if unchecked, high levels  may well initiate spoilage.   The European Pharmacopoeia has introduced both  quantitative and qualitative microbial standards for non-  sterile medicines, which may become enforceable in some  member states. It prescribes varying maximum total  microbial levels and exclusion of particular species  according to the routes of administration. The British  Pharmacopoeia has now included these tests, but suggests  that they should be used to assist in validating GPMP  processing procedures and not as conformance standards  for routine end-product testing. Thus, for a medicine to  be administered orally, the total viable count (TVC)  should not be more than 10 3 aerobic bacteria or 10 2 fungi  per gram or millilitre of product, and there should be an  absence of Escherichia coli. Higher levels may be permis-  sible if the product contains raw materials of natural  origin, as in the case of herbal products where the TVC  should not exceed 10 5 aerobic bacteria, 10 4 fungi and 10 3  Enterobacteria and Gram-negatives, with the absence of  E.colil gram or millilitre and Salmonella/ 10 gram or  millilitres.   Most manufacturers perform periodic tests on their  products for total microbial counts and the presence of  known problem microorganisms; generally these are  used for in-house confirmation of the continuing effi-  ciency of their cGPMP systems, rather than as conven-  tional end-product conformance tests. Fluctuation in  values, or the appearance of specific and unusual species,  can warn of defects in procedures and impending  problems.   In order to reduce the costs of testing and shorten  quarantine periods, there is considerable interest in  automated alternatives to conventional test methods for  the detection and determination of microorganisms.    Although not in widespread use at present, promising  methods include electrical impedance, use of fluorescent  dyes and epifluorescence, and the use of ‘vital’ stains.  Considerable advances in the sensitivity of methods for  estimating microbial ATP using luciferase now allow the  estimation of extremely low bioburdens. The recent  development of highly sensitive laser scanning devices for  detecting bacteria variously labelled with selective fluo-  rescent probes enables the apparent detection even of  single cells.   Endotoxin (pyrogen) levels in parenteral and similar  products must be extremely low in order to prevent  serious endotoxic shock on administration (Chapter 22).  Formerly, this was checked by injecting rabbits and noting  any febrile response. Most determinations are now per-  formed using the Limulus test in which an amoebocyte  lysate from the horseshoe crab ( Limulus polyphemus)  reacts specifically with microbial lipopolysaccharides to  give a gel and opacity even at very high dilutions. A variant  of the test using a chromogenic substrate gives a coloured  end point that can be detected spectroscopically. Tissue  culture tests are under development where the ability of  endotoxins to induce cytokine release is measured directly.   Sophisticated and very sensitive methods have been  developed in the food industry for detecting many other  microbial toxins. For example, aflatoxin detection in  herbal materials, seedstuffs and their oils is performed by  solvent extraction, adsorption onto columns containing  antibodies selective for the toxin, and detection by expo-  sure to ultraviolet light.   Although it would be unusual to test for signs of active  physicochemical or chemical spoilage of products as part  of routine product QC procedures, this may occasionally  be necessary in order to examine an incident of antici-  pated product failure, or during formulation develop-  ment. Many of the volatile and unpleasant-tasting  metabolites generated during active spoilage are readily  apparent. Their characterization by high performance  liquid chromatography or gas chromatography can be  used to distinguish microbial spoilage from other, non-  biological deterioration. Spoilage often results in physico-  chemical changes which can be monitored by conventional  methods. Thus, emulsion spoilage may be followed by  monitoring changes in creaming rates, pH changes, par-  ticle sedimentation and viscosity.   Despite extensive development and a rigorous adherence  to procedures, it is impossible to guarantee that a medi-    cine will never fail under the harsh abuses of real-life  conditions. A proper quality assurance system must  include procedures for monitoring in-use performance  and for responding to customer complaints. These must  be meticulously followed up in great detail in order to  decide whether carefully constructed and implemented  schemes for product safety require modification to  prevent the incident recurring.   Prevention is undoubtedly better than cure in minimiz-  ing the risk of medicament-borne infections. In manu-  facture the principles of GMP must be observed, and  control measures must be built in at all stages. Thus,  initial stability tests should show that the proposed for-  mulation can withstand an appropriate microbial chal-  lenge; raw materials from an authorized supplier should  comply with in-house microbial specifications; environ-  mental conditions appropriate to the production process  should be subject to regular microbiological monitoring;  and finally, end-product analysis should indicate that the  product is microbiologically suitable for its intended use  and conforms to accepted in-house and international  standards.   Based on present knowledge, contaminants, by virtue  of their type or number, should not present a potential  health hazard to patients when used.   Contamination during use is less easily controlled.  Successful measures in the hospital pharmacy have  included the packaging of products as individual  units, thereby discouraging the use of multidose contain-  ers. Unit packaging (one dose per patient) has clear  advantages, but economic constraints have prevented  this desirable procedure from being realized. Ultimately,  the most fruitful approach is through the training  and education of patients and hospital staff, so that  medicines are used only for their intended purpose. The  task of implementing this approach inevitably rests  with the clinical and community pharmacists of the  future.   With thanks to Edgar Beveridge who contributed a  chapter on spoilage and preservation in earlier editions  of this book.   Alexander, R.G., Wilson, D.A. & Davidson, A.G. (1997) Medicines  Control Agency investigation of the microbial quality of  herbal products. Pharm J, 259, 259—261.   Anon (1992), (1997), (2002) The Rules Governing Medicinal  Products in the European Community, Vol IV. Office for Official  Publications of the EC, Brussels.   Anon (1994). Two children die after receiving infected TPN  solutions. Pharm J, 252, 596.   Anon (2007) Rides and Guidance for Pharmaceutical Manufacturers  and Distributors. Pharmaceutical Press, London.   Attwood, D. & Florence, A.T. (1983) Surfactant Systems, Their  Chemistry, Pharmacy and Biology. Chapman & Hall, London.   Baines, A. (2000) Endotoxin testing. In: Handbook of  Microbiological Control: Pharmaceuticals and Medical Devices  (eds R.M. Baird, N.A. Hodges & S.P. Denyer), pp. 144-167.  Taylor & Francis, London.   Baird, R.M. (1981) Drugs and cosmetics. In: Microbial Biodeterioration  (ed. A.H. Rose), pp. 387M26. Academic Press, London.   Baird, R.M. (1985) Microbial contamination of pharmaceutical  products made in a hospital pharmacy. Pharm J, 234, 54-55.   Baird, R.M. (1985) Microbial contamination of non-sterile  pharmaceutical products made in hospitals in the North East  Regional Health Authority. J Clin Hosp Pharm, 10, 95—100.   Baird, R.M. (2004) Sterility assurance: concepts, methods and  problems. In: Principles and Practice of Disinfection,  Preservation and Sterilization (eds A. Fraise, P. Lambert & J-Y.  Maillard), 4th edn, pp. 526—539. Blackwell Scientific, Oxford.   Baird, R.M. & Shooter R.A. (1976) Pseudomonas aeruginosa  infections associated with the use of contaminated medica-  ments. Br Med J, ii, 349-350.   Baird, R.M., Brown, W.R.L. & Shooter, R.A. (1976) Pseudomonas  aeruginosa in hospital pharmacies. Br Med f, i, 511—512.   Baird, R.M., Elhag, K.M. & Shaw, E.J. (1976) Pseudomonas tho-  masii in a hospital distilled water supply. J Med Microbiol, 9,  493-495.   Baird, R.M., Parks, A. & Awad, Z.A. (1977) Control of  Pseudomonas aeruginosa in pharmacy environments and  medicaments. Pharm J, 119, 164—165.   Baird, R.M., Crowden, C.A., O’Farrell, S.M. & Shooter R.A.  (1979) Microbial contamination of pharmaceutical products  in the home. J Hyg, 83, 277—283.   Baird, R.M. & Bloomfield, S.F.L. (1996) Microbial Quality  Assurance of Cosmetics, Toiletries and Non-sterile  Pharmaceuticals. Taylor 8t Francis, London.   Baird, R.M., Hodges, N.A. & Denyer, S.P. (2000). Handbook of  Microbiological Control: Pharmaceuticals and Medical Devices.  Taylor & Francis, London.   Bassett, D.C.J. (1971) Causes and prevention of sepsis due to  Gram-negative bacteria: common sources of outbreaks. Proc  R Sac Med, 64, 980-986.    Brannan, D.K. (1995) Cosmetic preservation. / Soc Cosmet  Chem , 46, 199-220.   Denyer SP. & Baird RM. (2007). Guide to Microbiological Control  in Pharmaceuticals and Medical Devices. 2nd edn. CRC Press,  Boca Raton, FL.   Fraise, A., Lambert, P & Maillard, J-Y. (2004) Principles and  Practice of Disinfection, Preservation and Sterilization , 4th edn.  Blackwell Science, Oxford.   Gould, G.W. (1989) Mechanisms of Action of Food Preservation  Procedures. Elsevier Science Publishers, Barking.   Hills, S. (1946) The isolation of Cl. tetani from infected talc. N  Z Med /, 45, 419-423.   Hugo, W.B. (1995) A brief history of heat, chemical and radia-  tion preservation and disinfectants. Int Biodet Biodegrad , 36,  197-217.   Railings, L.O., Ringertz, O, Silverstolpe, L. & Ernerfeldt, F. (1966)  Microbiological contamination of medicinal preparations.  1965 Report to the Swedish National Board of Health. Acta  Pharm Suecica, 3, 219-228.   Maurer, I.M. (1985) Hospital Hygiene , 3rd edn. Edward Arnold,  London.    Meers, P.D., Calder, M.W., Mazhar, M.M. & Lawrie, G.M. (1973)  Intravenous infusion of contaminated dextrose solution: the  Devonport incident. Lancet , ii, 1189-1192.   Morse, L.J., Williams, H.I., Grenn, F.P., Eldridge, E.F. & Rotta,  J.R. (1967) Septicaemia due to Klebsiella pneumoniae originat-  ing from a handcream dispenser. N Engl J Med , 277 ',  472-473.   Myers, G.E. & Pasutto, F.M. (1973) Microbial contamination of  cosmetics and toiletries. Can J Pharm Sci, 8, 19-23.   Noble, W.C. & Savin, J.A. (1966) Steroid cream contaminated  with Pseudomonas aeruginosa. Lancet , i, 347-349.   Parker, M.T. (1972) The clinical significance of the presence of  microorganisms in pharmaceutical and cosmetic prepara-  tions. / Soc Cosmet Chem , 23, 415-426.   Public Health Laboratory Service Working Party Report (1971)  Microbial contamination of medicines administered to hos-  pital patients. Pharm /, 207, 96-99.   Smart, R. & Spooner D.F. (1972) Microbiological spoilage in  pharmaceuticals and cosmetics. / Soc Cosmet Chem, 23,  721-737.   Stebbing, L. (1993) Quality Assurance: The Route to Efficiency  and Competitiveness , 2nd edn. Ellis Horwood, Chichester.   Brendan F. Gilmore 1 , Howard Ceri 2 and Sean P. Gorman 1   Laboratory evaluation of antimicrobial agents remains a  cornerstone of clinical microbiology and antimicrobial/  biocide discovery and development. The development of  robust and reproducible assays for determining microbial  susceptibility to antimicrobial agents is of fundamental  importance in the appropriate selection of therapeutic  agents and biocides for use in infection control, disinfec-  tion, preservation and antifouling applications. Such    laboratory assays form the basis for high-throughput  screening of compounds or biological extracts in the dis-  covery, isolation and development of new antimicrobial  drugs and biocides. Such assays facilitate identification of  antimicrobial agents from various sources and in lead  antimicrobial compound optimization. In the control  of human and animal infection, laboratory evaluation of  candidate agents yields crucial information which can  inform choice of antimicrobial agent(s) where the causa-  tive organism is known or suspected. As the number of  microorganisms exhibiting resistance to conventional    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    antimicrobial agents increases, laboratory evaluation of  antimicrobial susceptibility is increasingly important for  the selection of appropriate therapeutic antimicrobials.  Evaluation of the potential antimicrobial action and  nature of the inhibitory or lethal effects of established  and novel therapeutic agents and biocides are important  considerations in the success of therapeutic interventions  and infection/contamination control procedures.   Significant concerns that the extensive use of biocidal  agents may be linked to the development of antimicrobial  resistance exist. Recent concerns regarding significant  global public health issues such as the increasing threat  of bioterrorism, the prevalence of healthcare associated  infections, severe acute respiratory syndrome (SARS),  avian influenza (H5N1) and the 2009 World Health  Organization declaration of the swine flu (H1N1) pan-  demic (the first pandemic of the 21st century) have seen  global demand for biocides increase dramatically. In  addition, the emergence of new infectious agents (e.g.  prions) and the increasing transmission rates of signifi-  cant blood-borne viruses (e.g. HIV, hepatitis B and C)  which may readily contaminate medical instruments or  the environment has focused attention on the need for  effective and proven disinfecting and sterilizing agents.   Finally, increasing appreciation of the role played by  microbial biofilms in human and animal infectious dis-  eases and their ubiquitous distribution in natural ecosys-  tems has led to the development of novel approaches for  the laboratory evaluation of antimicrobial susceptibility  of microorganisms growing as surface-adhered sessile  populations. These studies have demonstrated that  microorganisms in the biofilm mode of growth are phe-  notypically different from their planktonic counterparts  and frequently exhibit significant tolerance to antimicro-  bial challenge (see Chapter 8). This has implications for  the environmental control of microorganisms and in the  selection of appropriate concentrations of antibiotic or  biocide necessary to eradicate them. As such, biofilms  may constitute a reservoir of infectious microorganisms  which may remain following antimicrobial treatment,  even if antimicrobial selection is based on standard labo-  ratory evaluations of antimicrobials which are based on  planktonic cultures of microorganisms. Tests for evaluat-  ing candidate antimicrobial agents to be used in human  and animal medicine as well as environmental biocides  remain significant laboratory considerations.   Key terms such as disinfection, preservation, antisepsis  and sterilization are defined in Chapters 19 and 21. A    number of other important terms used to describe the  antimicrobial activity of agents are also commonly  used. A biocide may be defined as a chemical or physical  agent which kills viable organisms, both pathogenic and  nonpathogenic. This broad definition clearly includes  microoganisms, but is not restricted to them. The term  microbicide is therefore also used to refer specifically to  an agent which kills microorganisms ( germicide may also  be used in this context, but generally refers to pathogenic  microorganisms). The terms biocidal, bactericidal, fungi-  cidal and viricidal therefore describe an agent with killing  activity against a specific class or classes of organism indi-  cated by the prefix, whereas the terms bacteriostatic and  fungistatic refer to agents which inhibit the growth of  bacteria or fungi (Figure 18.1), but do not necessarily kill  them. It should be noted, however, that some microor-  ganisms that appear non-viable and non-cultivable fol-  lowing antimicrobial challenge may be revived by  appropriate methods, and that organisms incapable of  multiplication may retain some enzymatic activity.   In the laboratory evaluation of antibacterial agents, the  terms minimum inhibitory concentration (MIC) and  minimum bactericidal concentration (MBC) are most  commonly used. Recently published British Society for  Chemotherapy (BSAC) guidelines for the determination  of minimum inhibitory concentrations (see Further  Reading) define the MIC as the lowest concentration of   Figure 18.1 Effect on the subsequent growth pattern of  inhibitory (static, A) or cidal (□) agents added at time X (the  normal growth pattern is indicated by the • line).    antimicrobial which will inhibit the visible growth of a  microorganism after overnight cultivation and the MBC  as the lowest concentration of antimicrobial that will  prevent the growth of a microorganism after subculture  onto antibiotic-free media. Generally, MIC and MBC  values are recorded in milligrams per litre or per millilitre  (mg/L or mg/ml). With most cidal antimicrobials, the  MIC and MBC are frequently near or equal in value,  although with essentially static agents (e.g. tetracycline),  the lowest concentration required to kill the microorgan-  ism (i.e. the MBC) is invariably many times the MIC and  often clinically unachievable without damage to the  human host. As with microbicides, cidal terms can be  applied to studies involving not just bacteria but other  microbes, e.g. when referring to cidal antifungal agents  the term minimum fungicidal concentration (MFC) is  used. Recently, thanks to developments in the design of  high-throughput laboratory screens for biofilm suscepti-  bility, the minimum biofilm eradication concentration  (MBEC) can be accurately determined for organisms  grown as single or mixed species biofilms. The MBEC is  the minimum concentration of an antimicrobial agent  required to kill a microbial biofilm. For conventional  antibiotics and biocides the MBEC value may be 1000-  fold higher than the MBC value for the same planktonic  microorganisms. Further studies have shown that often  no correlation exists between the MIC and the MBEC,  indicating the potential limitations of therapeutic antibi-  otic selection based on determined MIC values.   The term tolerance implies the ability of some bacterial  strains to survive (without using or expressing resistance  mechanisms), but not grow, at levels of antimicrobial  agent that should normally be cidal. This applies particu-  larly to systems employing the cell-wall-active (3-lactams  and glycopeptides, and to Gram-positive bacteria such as  streptococci. Normally, MIC and MBC levels in such tests  should be similar (i.e. within one or two doubling dilu-  tions); if the MIC/MBC ratio is 32 or greater, the term  tolerance is used. Tolerance may in some way be related  to the Eagle phenomenon (paradoxical effect), where  increasing concentrations of antimicrobial result in  reduced killing rather than the increase in cidal activity  expected (see Figure 18.2). Tolerance to elevated antimi-  crobial challenge concentrations is also a characteristic  of microbial biofilm populations. Finally, the term resist-  ance has several definitions within the literature, however,  it generally refers to the ability of a microorganism to  withstand the effects of a harmful chemical agent, with  the organism neither killed nor inhibited at concentra-  tions to which the majority of strains of that organism   Figure 18.2 Survival of Enterococcus faecalis exposed to a  fluoroquinolone for 4 hours at 37 °C. Three initial bacterial  concentrations were studied, 10 7 Cfu/ml (□); 10 6 CFU/ml (A)  and 10 5 CFU/ml (O). This clearly demonstrates a paradoxical  effect (increasing antimicrobial concentrations past a critical  level reveal decreased killing), and the effects of increased  inoculum densities on subsequent killing. (Courtesy of Dr Z.  Hashmi.)   are susceptible. Resistance mechanisms generally involve  modification of the normal target of the antimicrobial  agent either by mutation, enzymatic changes, target  substitution, antibiotic destruction or alteration, antibi-  otic efflux mechanisms and restricted permeability to  antibiotics.   The activity of antimicrobial agents on a given organism  or population of organisms will depend on a number of  factors which must be reflected in the tests used to define  their efficacy. For example, the activity of a given antimi-  crobial agent will be affected by nature of the agent,  the nature of the challenge organism, the mode of growth  of the challenge organism, concentration of agent, size of  the challenge population and duration of exposure    of that population to the active agent. Furthermore,  environmental/physical conditions (temperature, pH,  presence of extraneous organic matter) are also impor-  tant considerations in modelling the activity of biocidal  agents. Laboratory tests for the evaluation of biocidal  activity must be carefully designed to take into account  these factors which may significantly influence the rate of  kill within the microbial challenge population.   The work of Kronig and Paul in the late 1890s, dem-  onstrated that the rate of chemical disinfection was  related to both concentration of the chemical agent and  the temperature of the system, and that bacteria exposed  to a cidal agent do not die simultaneously but in an  orderly sequence. This led to various attempts at applying  the kinetics of pure chemical reactions (the mechanistic  hypothesis of disinfection) to microbe/disinfectant inter-  actions. However, since the inactivation kinetics depend  on a large number of defined and undefined variables,  such models are often too complicated for routine use.  Despite this, the Chick-Watson model (equation 1),  based on first-order reaction kinetics, remains the basic  rate law for the examination of disinfection kinetics:   where N is the number of surviving microbes after time  t and k 0 is the disinfection rate constant. The Chick-  Watson model may be further refined to account for  biocide concentration (equation 2):    where Ay is the concentration-independent rate constant,  B is the biocide concentration and n is the dilution coef-  ficient. The Chick-Watson model predicts that the  number of survivors falls exponentially at a rate governed  by the rate constant and the concentration of disinfect-  ant. A general assumption is that the concentration of  biocide remains constant throughout the experiment;  however, there are a number of situations when this  appears not to be the case (sequestering) and may result  in observed departures from linear reaction kinetics. The  factors influencing the antimicrobial activity of disinfect-  ant agents are discussed below.   The susceptibility of microorganisms to chemical disin-  fectants and biocides exhibits tremendous variation    Figure 18.3 Survival of stationary phase broth cultures of  Legionella pneumophila and amoebae-grown L. pneumophila  after exposure to 32mg/L benzisothiazolone (Proxel) at 35 °C  in Ringer’s solution. (Adapted from Barker et al. (1992), Appl  Environ Microbiol, 58, 2420—2425, with permission.)   across various classes and species. Bacterial endospores  and the mycobacteria (e.g. Mycobacterium tuberculosis )  possess the most innate resistance, while many vegetative  bacteria and some viruses appear highly susceptible  (see Chapter 19). In addition, microorganisms adhering  to surfaces as biofilms or present within other cells  (e.g. legionellae within amoebae), may reveal a marked  increase in resistance to disinfectants and biocides (Figure  18.3). Therefore, when evaluating new disinfectants, a  suitable range of microorganisms and environmental  conditions must be included in tests. The European sus-  pension test (EN 12054) for hospital-related studies and  the European/British Standard suspension test (EN 1276)  for studies relating to food, industrial, institutional and  domestic areas, both include Pseudomonas aeruginosa,  Escherichia coli. Staphylococcus aureus and Enterococcus  hirae as the challenge organisms to be used in the test.  For specific applications, additional strains may be  chosen from Salmonella enterica serovar Typhimurium,  Lactobacillus brevis and Enterobacter cloacae.   Many disinfectants require adsorption to the microbial  cell surface prior to killing, therefore dense cell popula-  tions or sessile populations may sequester all the available    disinfectant before all cells are affected, thus shielding a  proportion of the population from the toxic effects of the  chemical agent. Therefore, from a practical point of view,  the larger the number of microorganisms present, the  longer it takes a disinfectant to complete killing of all cells.  For instance, using identical test conditions, it has been  shown that 10 spores of the anthrax bacillus ( Bacillus  anthracis) were destroyed in 30 minutes, while it took 3  hours to kill 100 000 (10 5 ) spores. The implications of  predisinfection washing and cleaning of objects (which  removes most of the microorganisms) becomes obvious.  However, when evaluating disinfectants in the laboratory,  it must be remembered that unlike sterilization, kill curves  with disinfectants may not be linear and the rate of killing  may decrease at lower cell numbers (Figure 18.3). Hence  a 3 -log killing may be more rapidly achieved with 10 s than  10 4 cells. Johnston et al. (2000) demonstrated that even  small variations in the initial inoculum size (Staph,  aureus) had a dramatic effect on log reductions over time,  using a constant concentration of sodium dodecyl sul-  phate (SDS). The authors argue that the presence of  microbes quenches the action of the biocide (self-  quenching), since cell and membrane components of  lysed bacteria (e.g. emulsifiers such as triacylglycerols and  phosphatidyl ethanolamine), are similar in action to  emulsifiers (such as Tween and lecithin) used in standard  biocide quenching/neutralizing agents employed in dis-  infectant tests. However, this may not hold true across all  biocides where similar inoculum size dependency of dis-  infection is observed (see Russell et al., 1997). Initial  bioburden/cell numbers must, therefore, be standardized  and accurately quantified in disinfectant efficacy (suspen-  sion) tests and agreement reached on the degree of lulling  required over a stipulated time interval (see Table 18.1).    Table 18.1 Methods of recording viable cells remaining  after exposure of an initial population of 1 000000 ( 10 s )  CFU to a cidal agent   The effects of concentration or dilution of the active  ingredient on the activity of a disinfectant are of major  importance. With the exception of iodophors, the more  concentrated a disinfectant, the greater its efficacy, and  the shorter the time of exposure required to destroy the  population of microorganisms, i.e. there is an exponen-  tial relationship between potency and concentration.  Therefore, a graph plotting the logi 0 of the death time (i.e.  the time required to kill a standard inoculum) against the  log 10 of the concentration is typically a straight line, the  slope of which is the concentration exponent (lj).  Expressed as an equation:    Thus r] can be obtained from experimental data  either graphically or by substitution in equation (3) (see  Table 18.2).   It is important to note that dilution does not affect the  cidal attributes of all disinfectants in a similar manner.  Lor example, mercuric chloride with a concentration  exponent of 1 will be reduced by the power of 1 on dilu-  tion, and a threefold dilution means the disinfectant  activity will be reduced by the value 3 1 , i.e. to a third of  its original activity. Phenol, however, has a concentration  exponent of 6, so a threefold dilution in this case will  mean a decrease in activity of 3 6 or 729 times less active  than the original concentration. Thus, the likely dilution  experienced by the disinfectant agent in use must be  given due consideration when selecting an appropriate  biocidal agent for a given application.   Known and proven influences include temperature, pH  and mineral content of water (‘hardness’).   As with most chemical/biochemical reactions, the cidal  activity of most disinfectants increases with increase in  temperature, since temperature is a measure of the kinetic  energy within a reaction system. Increasing the kinetic  energy of a reaction system increases the rate of reaction  by increasing the number of collisions between reactants  per unit time. This process is observed up to an optimum    temperature, beyond which reaction rates fall again, due  to thermal denaturation of some component(s) of the  reaction. As the temperature is increased in arithmetical  progression the rate (velocity) of disinfection increases in  geometrical progression. Results may be expressed quan-  titatively by means of a temperature coefficient, either the  temperature coefficient per degree rise in temperature  (0), or the coefficient per 10°C rise (the Q 10 value) (Hugo  & Russell, 1998). As shown by Koch, working with phenol  and anthrax ( B . anthracis) spores over 120 years ago,  raising the temperature of phenol from 20 °C to 30 °C  increased the killing activity by a factor of 4 (the Qm  value).   where t, is the extinction time at T, °C, and t 2 the extinc-  tion at T 2 °C (i.e. T, + 1°C).    Qio values may be calculated easily by determining the  extinction time at two temperatures differing by exactly  10°C. Then:   It is also possible to plot the rate of kill against the  temperature.   While the value for Q 10 of chemical and enzyme-  catalysed reactions lies in a narrow range (between 2 and  3), values for disinfectants vary widely, e.g. 4 for phenol,  45 for ethanol, and almost 300 for ethylene glycol monoe-  thyl ether. Clearly, relating chemical reaction kinetics to  disinfection processes is potentially dangerous. Most  laboratory tests involving disinfectant-like chemicals are  now standardized to 20 °C, i.e. around ambient room  temperatures.   Effects of pH on antimicrobial activity can be complex.  As well as directly influencing the survival and rate of  growth of the microorganism under test, changes in pH  may affect the potency of the agent and its ability to  interact with cell surface sites. In a many cases (where the  biocidal agent is an acid or a base), the ionization state  (or degree of ionization) will depend on the pH. As is the  case with some antimicrobials (e.g. phenols, acetic acid,  benzoic acid), the non-ionized molecule is the active state  (capable of crossing the cell membrane/partitioning) and  alkaline pHs which favour the formation of ions of such  compounds will decrease the activity. For these biocidal  agents a knowledge of the molecule’s pK a is important in  predicting the pH range over which activity can be  observed, since in situations where the pH of the system  equals the p K„ of the biocide molecule, ionized and  unionized species are in equilibrium. Others, such as glu-  taraldehyde and quaternary ammonium compounds  (QACs), reveal increased cidal activity as the pH rises and  are best used under alkaline conditions, possibly due to  enhanced interaction with amino groups on microbial  biomolecules. The pH also influences the properties of  the bacterial cell surface, by increasing the proportion  of anionic groups and hence its interaction with cidal  molecules. Since the activity of many disinfectants  requires adherence to cell surfaces; increasing the external  pH renders cell surfaces more negatively charged and  enhances the binding of cationic compounds such as  chlorhexidine and QACs.   The presence of divalent cations (e.g. Mg 2+ , Ca 2+ ), for  example in hard water, has been shown to exert an antag-  onistic effect on certain biocides while having an additive  effect on the cidal activity of others. Metal ions such as  Mg 2+ and Ca 2+ may interact with the disinfectant itself to  form insoluble precipitates and also interact with the  microbial cell surface and block disinfectant adsorption  sites necessary for activity. Biguanides, such as chlorhexi-  dine, are inactivated by hard water. Hard water should  always be employed for laboratory disinfectant and  antiseptic evaluations to reflect this, with recommended  formulae employing various concentrations of MgCl 2  and CaCl 2 solutions, available from the World Health  Organization and the British Standard (BS EN 1276). On  the other hand, cationic compounds may disrupt the  outer membrane of Gram-negative bacteria and facilitate  their own entry.   The presence of extraneous organic material such as  blood, serum, pus, faeces or soil is known to affect the  cidal activity of many antimicrobial agents. Therefore,  it is necessary to determine the likely interaction between  organic matter and the disinfectant by including this  parameter in laboratory evaluations of their activity. In  order to simulate ‘clean’ conditions (i.e. conditions of  minimal organic contaminant), disinfectants are tested  in hard water containing 0.3 g/L bovine albumin, with  the albumin being used to mimic ‘dirty’ conditions.  This standardized method replaces earlier approaches,  some of which employed dried human faeces or yeast to  mimic the effects of blood, pus or faeces on disinfectant  activity. Disinfectants whose activities are particularly  attenuated in the presence of organic contaminant  include the halogen disinfectants (e.g. sodium hypochlo-  rite) where the disinfectant reacts with the organic  matter to form inactive complexes, biguanides, phenolic  compounds and QACs. The aldehydes (formaldehyde  and glutaraldehyde) are largely unaffected by the pres-  ence of extraneous organic contaminants. Organic  material may also interfere with cidal activity by  adherence to the microbial cell surface and blockade of  adsorption sites necessary for disinfectant activity.  For practical purposes and to mirror potential in-  use situations, disinfectants should be evaluated under  both clean and dirty conditions. Alternatives to albumin  have also been suggested, for example sheep blood or  mucin.   Phenol coefficient tests were developed in the early  20th century when typhoid fever was a significant  public health problem and phenolics were used to disin-  fect contaminated utensils and other inanimate objects.  Details of such tests can be found in earlier editions of  this book. However, as non-phenolic disinfectants  became more widely available, tests that more closely  paralleled the conditions under which disinfectants were  being used (e.g. blood spills) and which included a more  diverse range of microbial types (e.g. viruses, bacteria,  fungi, protozoa) were developed. Evaluation of a disin-  fectant’s efficacy was based on its ability to kill microbes,  i.e. its cidal activity, under environmental conditions  mimicking as closely as possible real life situations. As an  essential component of each test was a final viability  assay, removal or neutralization of any residual disinfect-  ant (to prevent ‘carryover’ toxicity) became a significant  consideration.   The development of methods to evaluate disinfectant  activity in diverse environmental conditions and to deter-  mine suitable in-use concentrations/dilutions to be used  led to the development by Kelsey, Sykes and Maurer of  the so-called capacity-use dilution test which measured  the ability of a disinfectant at appropriate concentrations  to kill successive additions of a bacterial culture. Results  were reported simply as pass or fail and not a numerical  coefficient. Tests employed disinfectants diluted in hard  water (clean conditions) and in hard water containing  organic material (yeast suspension to simulate dirty con-  ditions), with the final recovery broth containing 3%  Tween 80 as a neutralizer. Such tests are applicable for use  with a wide variety of disinfectants (see Kelsey & Maurer,  1974). Capacity tests mimic the practical situations of  housekeeping and instrument disinfection, where sur-  faces are contaminated, exposed to disinfectant, recon-  taminated and so forth. The British Standard (BS  6907:1987) method for estimation of disinfectants used  in dirty conditions in hospitals by a modification of the  original Kelsey-Sykes test is the most widely employed  capacity test in the UK and Europe. In the USA, effective-  ness test data for submission must be obtained by  methods accepted by the Association of Official Analytical  Chemists, known collectively as Disinfectant Effectiveness  Tests (DETs).   However, the best information concerning the fate of  microbes exposed to a disinfectant is obtainable by    counting the number of viable cells remaining after expo-  sure of a standard suspension of cells to the disinfectant  at known concentration for a given time interval —  suspension tests. Viable counting is a facile technique used  in many branches of pure and applied microbiology.  Assessment of the number of viable microbes remaining  (survivors) after exposure allows the killing or cidal activ-  ity of the disinfectant to be expressed in a variety of ways,  e.g. percentage kill (e.g. 99.999%), as a log 10 reduction in  numbers (e.g. 5-log killing), or by log 10 survival expressed  as a percentage. Examples of such outcomes are shown in  Table 18.1.   Unfortunately, standardization of the methodology to  be employed in these efficacy tests has proven difficult, if  not impossible, to obtain, as has consensus on what level  of killing represents a satisfactory and/or acceptable  result. It must be stressed, however, that unlike tests  involving chemotherapeutic agents where the major aim  is to establish antimicrobial concentrations that inhibit  growth (i.e. MICs), disinfectant tests require determina-  tions of appropriate cidal levels. Levels of killing required  over a given time interval tend to vary depending on the  regulatory authority concerned. While a 5 -log killing of  bacteria (starting with 10 6 CFU/ml) has been suggested  for suspension tests, some authorities require a 6-log  killing in simulated use tests. With viruses, a 4-log killing  tends to be an acceptable result, while with prions it has  been recommended that a titre loss of 10 4 prions should  be regarded as an indication of appropriate disinfection  provided that there has been adequate prior cleaning.  With simulated use tests, cleaning followed by appropri-  ate disinfection should result in a prion titre loss of at  least 10 7 .   Various regulatory authorities in Europe (e.g. European  Standard or Norm, EN; British Standards, BS; Germany,  DGHM; France, AFNOR) and North America (e.g. Food  and Drug Administration, FDA; Environmental  Protection Authority, EPA; Association of Official  Analytical Chemists, AOAC) have been associated with  attempts to produce some form of harmonization of dis-  infectant tests. Perhaps the most readily accessible and  recent guide to the methodology of possible bactericidal,  tuberculocidal, fungicidal and viricidal disinfectant effi-  cacy tests, is that of Kampf and colleagues (2002). This  publication summarizes and provides references to  various EN procedures (e.g. prEN 12054).   While varying to some degree in their methodology, most  of the proposed procedures tend to employ a standard  suspension of the microorganism in hard water contain-  ing albumin (dirty conditions) and appropriate dilutions  of the disinfectant — so-called suspension tests. Tests are  carried out at a set temperature (usually around room  temperature or 20 °C), and at a selected time interval  samples are removed and viable counts are performed  following neutralization of any disinfectant remaining in  the sample. Neutralization or inactivation of residual dis-  infectant can be carried out by dilution, or by addition  of specific agents (see Table 18.3). Using viable counts, it  is possible to calculate the concentration of disinfectant  required to kill 99.999% (5-log kill) of the original sus-  pension. Thus 10 survivors from an original population  of 10 6 cells represents a 99.999% or 5-log kill. As bacteria  may initially decline in numbers in diluents devoid of  additional disinfectant, results from tests incorporating  disinfectant-treated cells can be compared with results  from simultaneous tests involving a non-disinfectant-  containing system (untreated cells). The bactericidal  effect B e can then be expressed as:   where N c and N D represent the final number of CFU/ml  remaining in the control and disinfectant series,  respectively.   Unfortunately, viable count procedures are based on  the assumption that one colony develops from one viable  cell or one CFU. Such techniques are, therefore, not ideal  for disinfectants (e.g. QACs such as cetrimide) that  promote clumping in bacterial suspensions, although the  latter problem may be overcome by adding non-ionic  surface active agents to the diluting fluid.   Apart from suspension tests, in-use testing of used  medical devices, and simulated use tests involving instru-  ments or surfaces deliberately contaminated with an  organic load and the appropriate test microorganism have  been incorporated into disinfectant testing protocols. An  example is the in-use test first reported by Maurer in 1972.  It is used to determine whether the disinfectant in jars,  buckets or other containers in which potentially contami-  nated material (e.g. lavatory brushes, mops) has been  placed contain living microorganisms, and in what  numbers. A small volume of fluid is withdrawn from the   Lubrol W and egg lecithin or  Tween 80 and lecithin  (Letheen)   ‘Other appropriate enzymes can be considered, e.g. inacti-  vating or modifying enzymes for chloramphenicol and  aminoglycosides, respectively.    in-use container, neutralized in a large volume of a suit-  able diluent, and viable counts are performed on the  resulting suspension. Two plates are involved in viable  count investigations, one of which is incubated for 3 days  at 32 °C (rather than 37 °C, as bacteria damaged by disin-  fectants recover more rapidly at lowered temperatures),  and the other for 7 days at room temperature. Growth of  one or two colonies per plate can be ignored (a disinfect-  ant is not usually a sterilant), but 10 or more colonies  would suggest poor and unsatisfactory cidal action.   Simulated use tests involve deliberate contamination  of instruments, inanimate surfaces, or even skin surfaces,  with a microbial suspension. This may either be under  clean conditions or may utilize a diluent containing  organic material (e.g. albumin) to simulate dirty condi-  tions. After being left to dry, the contaminated surface is  exposed to the test disinfectant for an appropriate time  interval. The microbes are then removed (e.g. by rubbing  with a sterile swab), resuspended in suitable neutralizing  medium, and assessed for viability as for suspension tests.  New products are often compared with a known compa-  rator compound (e.g. 1 minute application of 60% v/v  2-propanol for hand disinfection products — see EN 1500)  to show increased efficacy of the novel product.   Mycobacteria are hydrophobic in nature and, as a result,  exhibit an increased tendency to clump or aggregate in  aqueous media. It may be difficult, therefore, to prepare  homogeneous suspensions devoid of undue cell clump-  ing (which may contribute to their resistance to chemical  disinfection). As Mycobacterium tuberculosis is very slow  growing, more rapidly growing species such as M. terrae,  M. bovis or M. smegatis can be substituted in tests (as  representative of M. tuberculosis). Recent global public  health concerns regarding the increasing incidences of  tuberculosis (including co-infections with HIV) in devel-  oping, middle-tier and industrialized nations brings into  sharp focus the necessity for representative evaluations of  agents with potential tuberculocidal activity. This is par-  ticularly true given the high proportion of cases classified  as multidrug resistant tuberculosis (MDR-TB). Apart  from vegetative bacterial cells, bacterial or fungal spores  can also be used as the inoculum in tests. In such cases,  incubation of plates for the final viability determination  should be continued for several days to allow for germi-  nation and growth.   Compared with suspended (planktonic) cells, bacteria  on surfaces as biofilms are invariably phenotypically    more tolerant to antimicrobial agents. With biofilms, sus-  pension tests can be modified to involve biofilms pro-  duced on small pieces of an appropriate glass, metal or  polymeric substrate, or on the bottom of microtitre tray  wells. After being immersed in, or exposed to, the disin-  fectant solution for the appropriate time interval, the cells  from the biofilm are removed, e.g. by sonication, and  resuspended in a suitable neutralizing medium. Viable  counts are then performed on the resulting planktonic  cells. Reduction in biomass following antimicrobial chal-  lenge can be monitored using a standard crystal violet  staining technique, however, viable counting permits  evaluation of rate of kill. The Calgary Biofilm Device,  discussed in section 9.1, permits the high-throughput  screening of antimicrobial agents against biofilms  grown on 96 polycarbonate pegs in a 96-well microtitre  plate. Some important environmental bacteria survive  in nature as intracellular parasites of other microbes,  e.g. Legionella pneumophila within the protozoan  Acanthamoeba polyphaga. Biocide activity is significantly  reduced against intracelluar legionellae (see Figure 18.3).  Disinfectant tests involving such bacteria should there-  fore be conducted both on planktonic bacteria and on  suspensions involving amoebae-containing bacteria.  With the latter, the final bacterial viable counts are per-  formed after suitable lysis of the protozoan host. The  legionella/protozoa situation may also be further compli-  cated by the fact that the microbes often occur as  biofilms.   Suspension-type efficacy tests can also be performed on  other microbes, e.g. fungi, viruses, using similar tech-  niques to that described above for bacteria, although sig-  nificant differences obviously occur in parts of the tests.   In order for disinfectants to claim fungicidal activity, or  for the discovery of novel fungicidal activities, a range of  standard tests have been devised. Perhaps the main  problem with fungi concerns the question of which mor-  phological form of fungi to use as the inoculum.  Unicellular yeasts can be treated as for bacteria, but  whether to use spores (which may be more resistant than  the vegetative mycelium) or pieces of hyphae with the  filamentous moulds, has yet to be fully resolved. Spore  suspensions (in saline containing the wetting agent  Tween 80) obtained from 7-day-old cultures are presently  recommended. The species to be used may be a known  environmental strain and likely contaminant, such as    Aspergillus niger, or a pathogen, such as Trichophyton  mentagrophyes, other strains such as Penicillium variabile  are also employed. Clearly the final selection of organism  will vary depending on the perceived use for the disin-  fectant under test. In general, spore suspensions of at least  10 6 CFU/ml have been recommended. Viable counts are  typically performed on a suitable media (e.g. malt extract  agar, sabouraud dextrose agar) with incubation at 20 °C  for 48 hours or longer. EN 1275:1997 regulations for  fungicidal activity require a minimum reduction in via-  bility by a factor of 10 4 within 60 minutes; test fungi were  Candida albicans and A. niger. Further procedures may  be obtained by reference to EN 1650:1998 (quantitative  suspension test for evaluation of fungicidal activity of  chemical disinfectants and antiseptics used in food,  industrial, domestic and institutional areas) and AOAC  Fungicidal activity of disinfectants (955.17).   The evaluation of disinfectants for viricidal activity is a  complicated process requiring specialized training and  facilities; viruses are obligate intracellular parasites and  are therefore incapable of independent growth and rep-  lication in artificial culture media. They require some  other system employing living host cells. Suggested test  viruses include rotavirus, adenovirus, poliovirus, herpes  simplex viruses, HIV, pox viruses and papovavirus,  although extension of this list to include additional  blood-borne viruses such as hepatitis B and C, and sig-  nificant animal pathogens (e.g. foot and mouth disease  virus) could be argued, given the potential impact on  public health or the economy of a nation.   Briefly, the virus is grown in an appropriate cell line  that is then mixed with water containing an organic load  and the disinfectant under test. After the appropriate  time, residual viral infectivity is determined using a tissue  culture/plaque assay or other system (e.g. animal host,  molecular assay for some specific viral component). Such  procedures are costly and time-consuming, and must be  appropriately controlled to exclude factors such as disin-  fectant killing of the cell system or test animal. A reduc-  tion of infectivity by a factor of 10 4 has been regarded as  evidence of acceptable viricidal activity (prEN 14476).  For viruses that cannot be grown in the laboratory (e.g.  hepatitis B), naturally infected cells/tissues must be used.  Further test procedures are detailed in British Standard  BS EN 13610 (quantitative suspension test for the evalu-  ation of viricidal activity against bacteriophages of chem-  ical disinfectants used in food and industrial areas). The  use of bacteriophage as model viruses in this procedure    most likely reflects their ease of growth and survivor enu-  meration via standard plaque assay on host bacterial  lawns grown on solid media.   Prions are a unique class of acellular, proteinaceous infec-  tious agent, devoid of an agent-specific nucleic acid  (DNA or RNA). Infection is associated with the abnormal  isoform of a host cellular protein called prion protein  (PrP c ). Prions exhibit unusually high resistance to con-  ventional chemical and physical decontamination  methods, presenting a unique challenge in infection  control. Although numerous published studies on prion  inactivation by disinfectants are available in the literature,  inconsistencies in methodology make direct comparison  difficult. For example, strain differences of prion (with  respect to sensitivity to thermal and chemical inactiva-  tion), prion concentration in tissue homogenate, expo-  sure conditions and determination of log reductions  from incubation period assays instead of end-point titra-  tions. Furthermore, since most studies of prion inactiva-  tion have been conducted with tissue homogenates, the  protective effect of the tissue components may offer some  protective role and contribute to resistance to disinfec-  tion approaches. Despite this, a consistent picture of  effective and ineffective agents has emerged and is sum-  marized in Table 18.4. Although most disinfectants are  inadequate for the elimination of prion infectivity, agents  such as sodium hydroxide, a phenolic formulation, gua-  nidine thiocyanate and chlorine have all been shown to  be effective.   Solid disinfectants usually consist of a disinfectant sub-  stance diluted by an inert powder. Phenolic substances  adsorbed onto kieselguhr (diatomite) form the basis of  many disinfectant powders; another widely used solid  disinfectant is sodium dichloroisocyanurate. Other disin-  fectant or antiseptic powders used in medicine include  acriflavine and compounds with antifungal activity such  as zinc undecenoate or salicylic acid mixed with talc.  These disinfectants may be evaluated by applying them  to suitable test organisms growing on a solid agar  medium. Discs may be cut from the agar and subcultured  for enumeration of survivors. Inhibitory activity is evalu-  ated by dusting the powders onto the surface of seeded  agar plates, using the inert diluents as a control. The  extent of growth is then observed following incubation.   The decontamination and disinfection of air is an impor-  tant consideration for both infection and contamination  control. A large number of important infectious diseases  are spread via microbial contamination of the air. This  cross-infection can occur in a variety of situations (hos-  pitals and care facilities, airplanes, public and institutional  buildings), while stringent control of air quality with  respect to airborne contaminants and particulates is criti-  cal for contamination control in many aseptic procedures.  With the increasing public concern regarding the per-  ceived heightened threat of bioterrorism, effective air  disinfection procedures have been reviewed as a potential  counter-measure. The microorganisms themselves may  be contained in aerosols, or may occur as airborne parti-  cles liberated from some environmental source, e.g. agita-  tion of spore-laden bed linen, decaying vegetation, etc.  Disinfection of air can be carried out by increased ventila-  tion, filtration of air through high-efficiency particulate  air (FIEPA) filters, chemical aerosol/vapour/fumigation  or by ultraviolet germicidal irradiation (UVGI). Although  UVGI disinfectant approaches have demonstrated efficacy  against a range of airborne pathogens and contaminating  organisms, it is often more practical to use some form of  chemical vapour or aerosol to kill them. The use of for-  maldehyde vapour is the most commonly employed agent  for fumigation procedures (not strictly air disinfection),  although vaporized hydrogen peroxide may be used as an  alternative agent. Due to the potential for formation of  carcinogenic bis(chloromethyl) ether when used with  hydrochloric acid and chlorine containing disinfectants,  formaldehyde should not be used with hypochlorites.   The work of Robert Koch in the late 1880s demon-  strated that the numbers of viable bacteria present in air  can be assessed by simply exposing plates of solid nutri-  ent media to the air. Indeed, this same process is still  exploited in environmental monitoring in the form of  settle plates. Any bacteria that fall on to the plates after a  suitable exposure time can then be detected following  an appropriate period of incubation. These gravitational  methods are obviously applicable to many microor-  ganisms, but are unsuitable for viruses. However, more  meaningful data can be obtained if force rather than  gravity is used to collect airborne particles. A stream of  air can be directed on to the surface of a nutrient  agar plate (impaction; slit sampler) or bubbled through  an appropriate buffer or culture medium (liquid impin-  gement). Various commercial impactor samplers are   Effective (>3 log 10 reduction within 1 hour of temperatures  20 °C — 55 °C)   Copper, 0.5 mmol/L and H 2 0 2 , 100 mmol/L   Processes may be listed in both columns (ineffective/effective), due to different testing parameters or testing methods. All experi-  ments conducted without prior cleaning. Adapted from Rutala & Weber, 2010    available. Filtration sampling, where the air is passed  through a porous membrane, which is then cultured, can  also be used. For experimental evaluation of potential air  disinfectants, bacterial or fungal airborne ‘suspensions’  can be created in a closed chamber, and then exposed to  the disinfectant, which may be in the form of radiation,  chemical vapour or aerosol. The airborne microbial pop-  ulation is then sampled at regular intervals using an  appropriate forced-air apparatus such as the slit sampler.  With viruses, the air can be bubbled through a suitable  liquid medium, which is then subjected to some appro-  priate virological assay system. In all cases, problems arise  in producing a suitable airborne microbial ‘suspension’  and in neutralizing residual disinfectant, which may  remain in the air.   Preservatives are widely employed in the cosmetic and  pharmaceutical industries as well as in a variety of other    manufacturing industries. The addition of preservatives  to pharmaceutical formulations to prevent microbial  growth and subsequent spoilage, to retard product dete-  rioration and to restrain growth of contaminating micro-  organisms is commonplace for non-sterile pharmaceutical  formulations as well as low-volume aseptically prepared  formulations intended for multiple use from one con-  tainer. Indeed, adequate preservation (and validation of  effectiveness) is a legal requirement for certain formula-  tions. Effective preservation prevents microbial and, as a  consequence, related chemical, physical and aesthetic  spoilage that could otherwise render the formulation  unacceptable for patient use, therapeutically ineffective  or harmful to the patient (due to presence of toxic metab-  olites, microbial toxins). The factors which influence  the activity of the cidal agent employed as a preservative  are largely those which affect disinfectant activity  (described in section 2.4), however, when considering the  activity of the cidal agent the interactions with formula-  tion components (adsorption to suspended particles,  oil-water partitioning, etc.) should be considered as   While the inhibitory or cidal activity of the chemical  to be used as the preservative can be evaluated using an  appropriate in vitro test system (see sections 3.2.1  and 8.1.2), its continued activity when combined with the  other ingredients in the final manufactured product  must be established. Problems clearly exist with some  products, where partitioning into various phases may  result in the absence of preservative in one of the phases,  e.g. oil-in-water emulsions where the preservative may  partition only into the oily phase, allowing any contami-  nant microorganisms to flourish in the aqueous phase. In  addition, one or more of the components may inactivate  the preservative. Consequently, suitably designed simu-  lated use challenge tests involving the final product are,  therefore, required in addition to direct potency testing  of the pure preservative. In the challenge test, the final  preserved product is deliberately inoculated with a suit-  able environmental microorganism which may be fungal  (e.g. C. albicans or A. niger) or bacterial (e.g. Staph, aureus,  E. coli, Ps. aeruginosa). For oral preparations with a high  sucrose content, the osmophilic yeast Zygosaccharomyces  rouxii is a recommended challenge organism. The subse-  quent survival (inhibition), death or growth of the inocu-  lum is then assessed using viable count techniques.  Different performance criteria are laid down for inject-  able and ophthalmic preparations, topical preparations  and oral liquid preparations in the British Pharmacopoeia  (Appendix XVI C) and the European Pharmacopoeia,  which should be consulted for full details of the experi-  mental procedures to be used. In some instances, the  range and/or spectrum of preservation can be extended  by using more than one preservative at a time. Thus a  combination of parabens (p-hydroxybenzoic acid) with  varying water solubilities may protect both the aqueous  and oil phases of an emulsion, while a combination of  Germall 115 and parabens results in a preservative system  with both antibacterial (Germall 115) and antifungal  (parabens) activity.   In most of the tests mentioned above, results are not  available until visible microbial growth occurs, at least in  the controls. This usually takes 24 hours or more. The  potential benefits of rapid antimicrobial susceptibility  screening procedures are obvious, particularly in aggres-  sive infections or rapidly progressing nosocomial infec-    tions of immunocompromised patients where appropriate  antimicrobial selection is critical. To date only a few  ‘rapid’ methods for detecting microbial viability or  growth are presently employed in assessing the efficacy of  antimicrobials. These include epifluorescent and biolu-  minescence techniques. The former relies on the fact that  when exposed to the vital stain acridine orange and  viewed under UV light, viable cells fluoresce green or  greenish yellow, while dead cells appear orange. Live/dead  staining of sessile bacterial populations has the potential  to yield important data with respect to antimicrobial sus-  ceptibility, but requires skilled personnel and specialized  microscopy equipment.   With tests involving liquid systems the early growth of  viable cells can be assessed by some light-scattering proc-  esses, blood culture techniques have classically used the  production of C0 2 as an indicator of bacterial metabo-  lism and growth. In addition, the availability of molecular  techniques, such as quantitative PCR, may be useful in  demonstrating the presence or growth of microorgan-  isms that are slow or difficult to culture under usual labo-  ratory conditions, e.g. viruses. This may obviate the need  to neutralize residual disinfectant with some assays.   Recently, rapid colorimetric assays for antimicrobial  susceptibility have been developed including the com-  mercially available Vitek and Vitek2 systems (Biomerieux)  and colourimetric tests based on the extracellular reduc-  tion of tetrazolium salts 2-(2-methoxy-4-nitrophenyl)-  3- (4-nitrophenyl) - 5- (2, 4-disulfophenyl) -2H- tetrazolium  monosodium (WST-8) and 2,3-bis[2-methyloxy-4-nitro  -5-sulfophenyl] -2IT-tetrazolium-5-carboxanilide (XTT).  These latter studies have demonstrated the potential for  the tetrazolium salts WST-8 and XTT to be used in the  rapid, accurate and facile screening of antimicrobial sus-  ceptibility and MIC determination in a range of bacteria,  including staphylococci, extended (3-lactamase producing  clinical isolates ( E . coli, Ent. faecalis) and Ps. aeruginosa  (see Tunney et al, 2004). Using this method, MIC values  in agreement with those obtained using standard methods  were obtained after 5 hours.   Unlike tests for the evaluation of disinfectants, where  determination of cidal activity is of paramount impor-  tance, tests involving potential chemotherapeutic agents  (antibiotics) invariably have determination of MIC as  their main focus. Tests for the bacteriostatic activity of    antimicrobial agents are valuable tools in predicting  antimicrobial sensitivity/tolerance in individual patient  samples and for detection and monitoring of resistant  bacteria. However, correlation between MIC and  therapeutic outcome are frequently difficult to predict,  especially in chronic biofilm-mediated infections. The  determination of MIC values must be conducted under  standardized conditions, since deviation from standard  test conditions can result in considerable variation in  data.   The historical gradient plates, ditch-plate and cup-plate  techniques (see Hugo & Russell, 1998) have been replaced  by more quantitative techniques such as disc diffusion  (Figure 18.4), broth and agar dilution, and E-tests  (Figure 18.5). All employ chemically defined media (e.g.  Mueller-Hinton or Iso-Sensitest) at a pH of 1 .2-1 A,  and in the case of solid media, agar plates of defined  thickness. Regularly updated guidelines have been pro-  vided by the National Committee for Clinical Laboratory  Standards (NCCLS) and are widely used in many coun-  tries, although the British Society for Antimicrobial  Chemotherapy has produced its own guidelines and  testing procedures (Andrews, 2009).   These are really modifications of the earlier cup or ditch-  plate procedures where filter-paper discs impregnated  with the antimicrobial replace the antimicrobial-filled  cups or wells. For disc tests, standard suspensions (e.g.  0.5 McFarland standard) of log-phase growth cells are  prepared and inoculated on to the surface of appropriate  agar plates to form a lawn. Commercially available filter-  paper discs containing known concentrations of antimi-  crobial agent (it is possible to prepare your own discs for  use with novel drugs) are then placed on the dried lawn  and the plates are incubated aerobically at 35 °C for 18  hours. The density of bacteria inoculated on to the plate  should produce just confluent growth after incubation.  Any zone of inhibition occurring around the disc is then  measured, and after comparison with known standards,  the bacterium under test is identified as susceptible or  resistant to that particular antibiotic. For novel agents,  these sensitivity parameters are only available after exten-  sive clinical investigations are correlated with laboratory-  generated data. Disc tests are basically qualitative;  however, the diameter of the zone of inhibition may be  correlated to MIC determination through a linear regres-  sion analysis (Figure 18.6).   Figure 18.4 Disc test with inhibition zones around two (1, 2)  of five discs. The zone around disc 1 is clear and easy to  measure, whereas that around disc 2 is indistinct. Although  none of the antimicrobials in discs 3, 4 or 5 appear to inhibit  the bacterium, synergy (as evidenced by inhibition of growth  between the discs) is evident with the antimicrobials in discs 3  and 5. Slight antagonism of the drug in disc 1 by that in disc 3  is evident.   Figure 18.5 E-test on an isolate of Candida albicans.  Inhibition zone edges are distinct and the MICs for  itraconazole (IT) and fluconazole (FL) (0.064 mg/L and  1.5mg/L, respectively) are easily decipherable.    Figure 18.6 A scattergram and  regression line analysis correlating zone  diameters and MICs. The breakpoints of  susceptible (MIC < 2.0mg/L, zone  diameter > 21 mm) and resistant  (MIC > 8.0mg/L, zone < 15 mm) are  shown by the dotted lines. For a  complete correlation between MICs and  zone diameter, all susceptible,  intermediate and resistant isolates should  fall in boxes A, B and C, respectively.  Errors (correlations outside these boxes)  occur. (Courtesy of Dr Z. Hashmi.)    Although subtle variations of the disc test are used in  some countries, the basic principles behind the tests  remain similar and are based on the original work of  Bauer and colleagues (Kirby-Bauer method). Some tech-  niques employ a control bacterial isolate on each plate so  that comparisons between zone sizes around the test and  control bacterium can be ascertained (i.e. a disc potency  control). Provided that discs are maintained and handled  as recommended by the manufacturer, the value of such  controls becomes debatable and probably unnecessary.  Control strains of bacteria are available which should  have inhibition zones of a given diameter with stipulated  antimicrobial discs. Use of such controls endorses the  suitability of the methods (e.g. medium, inoculum  density, incubation conditions) employed. For slow-  growing microorganisms, the incubation period can be  extended. Problems arise with disc tests where the inocu-  lum density is inappropriate (e.g. too low, resulting in an  indistinct edge to the inhibition zone following incuba-    tion), or where the edge is obscured by the sporadic  growth of cells within the inhibition zone, i.e. the initial  inoculum although pure contains cells expressing  varying levels of susceptibility — so-called heterogeneity.  As the distance from the disc increases, there is a loga-  rithmic reduction in the antimicrobial concentration;  the result is that small differences in zone diameter with  antimicrobials (e.g. vancomycin) which diffuse poorly  through solid media may represent significantly different  MICs. Possible synergistic or antagonistic combinations  of antimicrobials can often be detected using disc tests  (Figure 18.4).   These usually employ liquid media but can be modified  to involve solid media. Doubling dilutions, usually in the  range 0.008-256 mg/L of the antimicrobial under test, are  prepared in a suitable broth medium, and a volume of  log-phase cells is added to each dilution to result in a final    cell density of around 5 x 10 5 CFU/ml. After incubation  at 35°C for 18 hours, the concentration of antimicrobial  contained in the first clear tube is read as the MIC.  Needless to say, dilution tests require a number of con-  trols, e.g. sterility control, growth control, and the simul-  taneous testing of a bacterial strain with known MIC to  show that the dilution series is correct. Endpoints with  dilution tests are usually sharp and easily defined,  although ‘skipped’ wells (inhibition in a well with growth  either side) and ‘trailing’ (a gradual reduction in growth  over a series of wells) may be encountered. The latter is  especially evident with antifungal tests (see below).  Nowadays, the dilution test for established antimicrobials  has been simplified by the commercial availability of 96-  well microtitre plates which have appropriate antimicro-  bial dilutions frozen or lyophilized onto wells in the plate.  The appropriate antibacterial suspension (in 200-400 pi  volumes) is simply added to each well, the plate is incu-  bated as before, and the MIC is read.   Dilution tests can also be carried out using a series of  agar plates containing known antimicrobial concentra-  tions. Appropriate bacterial suspensions are inoculated  on to each plate and the presence or absence of growth  is recorded after suitable incubation. Most clinical labo-  ratories now employ agar dilution breakpoint testing  methods. These are essentially truncated agar dilution  MIC tests employing only a small range of antimicrobial  concentrations around the critical susceptible/resistant  cut-off levels. Many automated identification and sensi-  tivity testing machines now use a liquid (broth) variant  of the agar breakpoint procedure. Similar breakpoint  antimicrobial concentrations are used with the presence  or absence of growth being recorded by some automated  procedure (e.g. light-scattering, colour change) after a  suitable incubation period.   Perhaps the most convenient and presently accepted  method of determining bacterial MICs, however, is the E  (Epsilometer)-test. The concept and execution of the  E-test is similar to the disc diffusion test except that a  linear gradient of lyophilized antimicrobial in twofold  dilutions on nylon carrier strips on one side are used  instead of the filter-paper impregnated antimicrobial  discs. On the other side of the nylon strip are a series of  lines and figures denoting MIC values (Figure 18.5). The  nylon strips are placed antimicrobial side down on the  freshly prepared bacterial lawn and, after incubation, the  MIC is determined by noting where the ellipsoid (pear-  shaped) inhibition zone crosses the strip (Figure 18.5).    For most microorganisms, there appears to be excellent  correlation between dilution and E-test MIC results. As  with standard disc diffusion tests, resistant strains may be  isolated from within the zone of inhibition.   With some of the emerging antimicrobial-resistant  bacterial pathogens, e.g. vancomycin-resistant entero-  cocci (VRE), meticillin-resistant Staph, aureus (MRSA),  vancomycin-intermediate Staph, aureus (VISA), the  standard methodology described above may fail to detect  the resistant phenotype. This is due to a variety of factors  including heterogeneous expression of resistance (e.g.  MRSA, VISA), poor agar diffusion of the antimicrobial  (e.g. vancomycin) and slow growth of resistant cells (e.g.  VISA). Disc tests are unsuitable for VRE, which should  have MICs determined by E-test or dilution techniques.  With MRSA, a heavier inoculum should be used in tests  and 2-4% additional salt (NaCl) included in the medium  with incubation for a full 48 hours. Reducing the incuba-  tion temperature to 30 °C may also facilitate detection of  the true MIC value. Although 100% of MRSA cells may  contain resistance genes, the phenotype may only be  evident in a small percentage of cells under the usual  conditions employed in sensitivity tests. Expression is  enhanced at lower temperatures and at higher salt con-  centrations. With VISA, MIC determinations require  incubation for a full 24 hours or more because of the  slower growth rate of resistant cells.   MBC testing is required for the evaluation of novel  antimicrobials. The MBC is the lowest concentration (in  mg/L) of antimicrobial that results in 99.9% or more  killing of the bacterium under test. The 99.9% cut-off is  an arbitrary in vitro value with 95% confidence limits  that has uncertain clinical relevance. MBCs are deter-  mined by spreading 0.1ml (100 pi) volumes of all clear  (no growth) tubes from a dilution MIC test onto separate  agar plates (residual antimicrobial in the 0.1 ml sample is  ‘diluted’ out over the plate). After incubation at 35 °C  overnight (or longer for slow-growing bacteria), the  numbers of colonies growing on each plate are recorded.  The first concentration of drug that produces <50 colo-  nies after subculture is considered the MBC. This is based  on the fact that with MICs, the initial bacterial inoculum  should result in about 5 x 10 5 CFU/ml. Inhibition, but  not killing of this inoculum, should therefore result in the  growth of 50000 bacteria from the 0.1ml sample. A  99.9% (3-log) kill would result in no more than 50 colo-    nies on the subculture plate. With most modern antibac-  terial drugs, the concentration that inhibits growth is very  close to the concentration that produces death, e.g. within  one or two dilutions. In general, only MICs are deter-  mined for such drugs.   As fungi have become more prominent human patho-  gens, techniques for investigating the susceptibility of  isolates to the growing number of antifungal agents have  been developed. These have been largely based on the  established bacterial techniques (disc, dilution, E-test)  mentioned above, with the proviso that the medium used  is different (e.g. use of RPMI 1640 plus 2% dextrose) and  that the inoculum density (yeast cells or spores) used is  reduced (c. 10 4 CFU/ml). With yeast disc and E-tests, a  lawn producing just separated/distinct colonies is prefer-  able to confluent growth (see Figure 18.5). Addition of  methylene blue (0.5 mg/ml) to media may improve the  clarity of inhibition zone edges. Problems of ‘tailing’ or  ‘trailing’ in dilution tests, and indistinct inhibition zone  edges are often seen in tests involving azoles and yeasts  and appear in some way related to the type of buffer  employed in the growth medium. However, their pres-  ence has prompted studies into evaluating the use of  other techniques as an indicator of significant fungistasis —  e.g. 50% reduction in growth (rather than complete inhi-  bition) as the end point, use of a dye (e.g. Alamar blue)  colour change to indicate growth, and sterol (ergosterol)  quantitation. Most of these are presently outside the  scope of most routine laboratories.   As with MBC estimations, MFC evaluation is an exten-  sion of the MIC test. At the completion of the MIC test  (e.g. 72 hours for filamentous fungi), 20ml are subcul-  tured on to a suitable growth medium from each optically  clear microtitre tray well and the growth control well.  These plates are then incubated at 35 °C until growth is  evident on the growth control subculture (24—48 hours).  The MFC is the lowest drug concentration showing no  growth or fewer than three colonies per plate to obtain  approximately 99-99.5% killing activity.   The potential interaction between two antimicrobials can  be demonstrated using a variety of laboratory proce-  dures, e.g. ‘chequerboard’ MIC assays where the microor-  ganism is exposed to varying dilutions of each drug alone  and in combination, disc diffusion tests (see Figure 18.4)    and kinetic kill curve assays. With the former, results can  be plotted in the form of a figure called an isobologram  (see Figure 18.7).   In the case of kill curves, the microorganism is inoculated  into tubes containing a single concentration of each anti-  microbial alone, the same concentrations of each antimi-  crobial in combination, and no antimicrobial — i.e. four  tubes. All tubes are then incubated and viable counts are  performed at regular intervals on each system. With results  plotted on semilogarithmic paper, synergy is defined as a  greater than 100-fold increase in killing of the combina-  tion compared with either drug alone. Antagonism is  defined as at least a 100-fold decrease in killing of the  combination when compared with the most active agent  alone, while an additive or autonomous combined effect  results in a less than 10-fold change from that seen with  the most active single drug. Both chemotherapeutic agents  and disinfectants are amenable to kill curve assays.   Figure 18.7 Diagrammatic representation of MIC values  obtained with two synergistic antimicrobials, penicillin and  gentamicin. The resulting graph or isobologram (A) is  obtained by linking MIC values for each drug alone and in  various dilution combinations. The MIC values for penicillin  and gentamicin alone are 1 .0 mg/L and 32 mg/L, respectively.  The slope of the isobologram for purely additive or  antagonistic combinations is shown by B and C, respectively.   As discussed in Chapter 8 and earlier in this chapter,  biofilms present an additional problem to antimicro-  bial testing as the biofilm may be resistant to more than  1000 times the MIC concentration of antimicrobial.  Antimicrobial testing of biofilms under standardized  conditions has really only become available since 1999  and the development of the MBEC Assay (Calgary  Biofilm Device). Previous techniques for biofilm suscep-  tibility testing suffered from a lack of replicates, as in the  case of flow cell technology, or the need for continuous  pumping of fluids and bacteria that presented a leakage  and contamination risk that was not tolerable in a diag-  nostic laboratory. Forming biofilms directly in 96-well  plates provided replicate numbers, but in this assay  system the initial inoculum could not be calculated and  the efficacy of treatment was based not on viable cell  counts but on a dye absorbance assay that could be meas-  uring a change in extracellular matrix rather than a  change in viable bacterial cell number. The MBEC assay  placed pegs protruding from the lid of the plate into each  well of a 96-well plate. Shear force created by gyration of  the plate initiated bacterial adhesion to the peg and  biofilm formation, the density of which could be deter-  mined by sonication of the biofilm back into a suspen-  sion culture and enumeration of viable cell number by  standard plate counts. The peg-borne biofilms could then  be used as a biofilm inoculation in a standard 96-well  MIC assay, only in this case the susceptibility of a biofilm  rather than a planktonic population would be deter-  mined. Following antimicrobial exposure bacteria would  again be sonicated from the pegs and counted to deter-  mine the biofilm MIC (BMIC), biofilm bactericidal con-  centration (BMBC) and biofilm eradication concentration  (MBEC) in a highly standardized and reproducible assay  based on existing MIC technology. Mycobacteria and  fungi can be assayed using a similar format allowing the  biofilm susceptibility of these organisms to be tested.   The reduced susceptibility of biofilms to antimicrobials  often results in the effective in vitro drug concentration  to far exceed a safe or achievable dose. Combinations of  drugs or drugs and other cofactors are proving to be more  effective against biofilms than single drug therapies.  Synergies in biofilm testing have been defined on formu-  las based on the American Society for Microbiology  standards. The calculation of synergy as defined in    Harrison et al. (2008) where the mathematical definition  of synergy was based on the sum of the fraction bacteri-  cidal concentration (FBC) value or FBC index for each  combination of antimicrobial agents. Therefore in a  two-component assay of agents A+B synergy would be  defined as follows:   Andrews, J.M. (2001) Determination of minimum inhibitory  concentrations . J Antimicrob Chemother , 48(Suppl SI), 5-16.   Andrews, J.M. (2009) BSAC standardized disc susceptibility  testing method (version 8). / Antimicrob Chemother , 64,  454-489.   Barker, J., Brown, M.R.W., Collier, P.J., Farrell I., 8c Gilbert P.  (1992). Relationship between Legionella pneumophila and  Acanthamoeba polyphaga : physiological status and suscepti-  bility to chemical inactivation. Appl Environ Microbiol , 58,  2420-2425.   Buttner, M.P., Willeke K. 8c Grinshpun S.A. (2002) Sampling  and analysis of airborne microorganisms. In: Manual of  Environmental Microbiology (editor-in-chief C.J. Hurst), 2nd  edn, pp. 814-826. ASM Press, Washington, DC.   Espinel-Ingroff, A., Chaturvedi, V., Fothergill, A. 8c Rinaldi, M.G.  (2002) Optimal testing conditions for determining MICs and  minimum fungicidal concentrations of new and established  antifungal agents for uncommon molds: NCCLS collabora-  tive study. / Clin Microbiol , 40, 3776-3781.   Harrison, J.J., Turner, R.J., Joo, D.A., et al (2008) Copper and  quaternary ammonium cations exert synergistic bactericidal  and antibiofilm activity against Pseudomonas aeruginosa.  Antimicrob Agents Chemother, 52(8), 2870-2881.   Hugo, W.B. 8c Russell, A.D. (1998) Evaluation of non- antibiotic  antimicrobial agents. In: Pharmaceutical Microbiology (eds  W.B. Hugo 8c A.D. Russell), 6th edn, pp. 229—255. Blackwell  Science, Oxford.   Johnston, M.D., Simons, E.-A. 8c Lambert, R.J.W. (2000) One  explanation for the variability of the bacterial suspension test.  } Appl Microbiol, 88, 237-242.   Kampf, G., Rudolf, M., Labadie, J-C. 8c Barrett, S. P. (2002)  Spectrum of antimicrobial activity and user acceptability of  the hand disinfectant agent Sterillium ® Gel. J Hosp Infect, 52,  141-147.    Kelsey, J.C. 8c Maurer, I.M. (1974) An improved Kelsey- Sykes test  for disinfectants. Pharm /, 30, 528-530.   Koneman, E.W., Allen, S.D., Janda, W.M., Schreckenberger, P.C.  8c Winn, W.C. (1997) Antimicrobial susceptibility testing. In:  Color Atlas and Textbook of Diagnostic Microbiology, 5th edn,  pp. 785-856. Lippincott- Raven, Philadelphia, PA.   National Committee for Clinical Laboratory Standards (1997)  Reference method for broth dilution antifungal susceptibility  testing of yeasts; approved standard. NCCLS document M27-A,  Wayne, PA.   National Committee for Clinical Laboratory Standards (2000)  Methods for dilution antimicrobial susceptibility tests for bacte-  ria that grow aerobically; approved standard, 5th edn. NCCLS  document M7-A5, Wayne, PA.   National Committee for Clinical Laboratory Standards (2000)  Performance standards for antimicrobial disk susceptibility tests;  approved standard, 7th edn. NCCLS document M2-A7,  Wayne, PA.   National Committee for Clinical Laboratory Standards (2002)  Reference method for broth dilution antifungal susceptibility  testing of filamentous fungi; approved standard. NCCLS docu-  ment M3 8 -A, Wayne, PA.    Russell, A.D., Purr, J.R. 8c Maillard, J-Y. (1997) Microbial suscep-  tibility and resistance to biocides. ASM News, 63, 481-487.   Rutala, W.A. (1999) Selection and use of disinfectants in health-  care. In: Hospital Epidemiology and Infection Control (ed. C.G.  Mayhall), 2nd edn, pp. 1161-1187. Lippincott Williams 8c  Wilkins, Philadelphia, PA.   Rutala, W.A. 8c Weber, D.J. (2001) Creutzfeldt-Jakob disease:  recommendations for disinfection and sterilization. Clin  Infect Dis, 32, 1348-1356.   Tunney, M.M., Ramage, G., Field, T.R., Moriarty, T.P. 8c Storrey,  D.G. (2004) Rapid colorimetric assay for antimicrobial sus-  ceptibility testing of Pseudomonas aeruginosa. Antimicrob  Agents Chemother , 48, 1879-1881.   Walsh, S.E., Maillard, J-Y., Russell, A.D., Catrenich, C.E.,  Charbonneau, D.L. 8c Bartolo, R.G. (2003) Development of  bacterial resistance to several biocides and effects on antibi-  otic susceptibility. / Hosp Infect, 55, 98-107.   Widmer, A.P. 8c Frei, R. (1999) Decontamination, disinfection,  and sterilization. In: Manual of Clinical Microbiology (eds P.R.  Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover 8c R.H. Yolken),  7th edn, pp. 138-164. ASM Press, Washington, DC.    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Hodges, Sean P. Gorman,  Brendan F. Gilmore.   Disinfectants, antiseptics and preservatives are chemical  agents that have the ability to destroy or inhibit the  growth of microorganisms. They play a major role in  medical and health care but also have widespread use  in the management of livestock, the environment,  paints and coatings, plastics, pharmaceutical, food and  beverage manufacture, textiles, the catering industry and  consumer products. The term biocide is increasingly used  to describe this group of chemical agents and this  term is used widely throughout European Union (EU)  directives.   Regulation of biocides continues to develop within the  EU and many other countries wherein guidance is defined  for both manufacturers and users. Specific guidance doc-  uments agreed between the EU Commission services and  the competent authorities of the Member States include,  importantly, the Biocidal Products Directive 98/8/EC  and the related directives: Medicinal Products for Human  Use Directive 2001/83/EC and the Veterinary Medicinal  Products Directive 2001/82/EC. Additionally, ‘agricul-  tural pesticides’, are regulated by the Plant Protection  Products Directive 91/414/EC.   The term ‘biocidal product’ (98/8/EC) encompasses  active substances and preparations containing one or  more active substances, put up in the form in which they  are supplied to the user, intended to destroy, deter, render  harmless, prevent the action of, or otherwise exert a con-  trolling effect on any harmful organism by chemical or  biological means.   The European Commission has proposed a new  Regulation of the European Parliament and of the  Council concerning the placing on the market and use of  biocidal products. The proposed European Regulation  will, over a period of time, revise and replace the current  regulatory framework for the marketing and use of bio-  cidal products contained in the Biocidal Products  Directive (BPD) 98/8/EC (as amended). The BPD is cur-  rently transposed into domestic UK law by the Biocidal  Products Regulations (BPR) 2001 ([SI 2001/880] as  amended). The UK Government Health and Safety  Executive (HSE) engages in negotiations with all 27  Member States of the EU, the European Commission and  the European Parliament in the development of a new  directly- acting EU law. The new draft regulation which is  scheduled to come into effect on 1 January 2013 proposes    several important changes to the current approach to  dealing with biocides, including:   • Extending the scope of the regime to cover treated  articles and materials containing biocides   • Adopting a Community authorization scheme for  certain types of products   Disinfection is the process of removing microorganisms,  including pathogens, from the surfaces of inanimate  objects. The British Standards Institution (BSI) further  defines disinfection as not necessarily killing all microor-  ganisms but reducing them to a level acceptable for a  defined purpose, for example, a level that is harmful  neither to health nor to the quality of perishable goods.  Chemical disinfectants are capable of different levels  of action (Table 19.1). The term high-level disinfection  indicates destruction of all microorganisms but not  necessarily bacterial spores; intermediate-level disinfection  indicates destruction of all vegetative bacteria including  Mycobacterium tuberculosis but may exclude some  resistant viruses and fungi and implies little or no  sporicidal activity; low-level disinfection can destroy  most vegetative bacteria, fungi and viruses, but this  will not include spores and some of the more resistant  microorganisms. Some high-level disinfectants have  good sporicidal activity and are described as liquid  chemical sterilants or chemosterilants to indicate that they  can effect a complete kill of all microorganisms, as in  sterilization. In defining each of these disinfection 1  evels the activity and outcome is determined by  correct use of the disinfectant product in regard to con-  centration, time of contact and prevailing environmental  conditions as described in subsequent sections of this  chapter.   Antisepsis is defined as destruction or inhibition of  microorganisms on living tissues having the effect of lim-  iting or preventing the harmful results of infection. It is  not a synonym for disinfection (BSI). The chemicals used  are applied to skin and mucous membranes, so as well as  having adequate antimicrobial activity they must not be  toxic or irritating for these tissues. Antiseptics are mostly    used to reduce the microbial population on the skin  before surgery or on the hands to help prevent spread of  infection by this route. Antiseptics are sometimes formu-  lated as products containing significantly lower concen-  trations of agents used for disinfection.   Preservatives are included in pharmaceutical and many  other types of formulations, both to prevent microbial  spoilage of the product and to minimize the risk to the  consumer of acquiring an infection when the preparation  is administered. Preservatives must be able to limit pro-  liferation of microorganisms that may be introduced  unavoidably into non-sterile products such as oral and  topical medications during their manufacture and use. In  sterile products, where multiuse preparations remain  available, preservatives should kill any microbial con-  taminants introduced inadvertently during use. It is  essential that a preservative is not toxic in relation to the  intended route of administration of the preserved prepa-  ration. Preservatives tend to be employed at very low  concentrations and consequently levels of antimicrobial  action also tend to be of a lower order than for disinfect-  ants or antiseptics. This is illustrated by the European  Pharmacopoeia requirements for preservative efficacy  where a degree of bactericidal activity is necessary,  although this should be obtained within a few hours or  over several days of microbial challenge depending on the  type of product to be preserved. Other terms are consid-  ered in Chapter 18.   The international antimicrobial chemical market, partic-  ularly in disinfectants, is expected to grow significantly    over the coming years, on the basis of concerns  about bacterial and other pathogenic threats and the  increasing emphasis on hygiene in the home and work-  place. The turnover of the US antimicrobial chemical  industry is estimated to be in the region of $2 billion per  annum. Key disinfectant products in use contain alde-  hydes, iodophors, nitrogen compounds (quaternary  ammonium compounds and amine compounds), orga-  nometallics, organosulphurs, chloroisocyanurates and  phenolics. There are around 250 chemicals that have been  identified as active components of microbicidal products  in the EU.   The aim of this chapter is to introduce the range of  chemicals in common use and to indicate their activities  and applications.   Choice of the most appropriate antimicrobial compound  for a particular purpose depends on many factors and the  key parameters are described.   The process of killing or inhibiting the growth of micro-  organisms using an antimicrobial agent is basically that  of a chemical reaction and the rate and extent of this  reaction will be influenced by concentration of agent,  temperature, pH and formulation. The influence of these  factors on activity is considered in Chapter 18, and is  referred to in discussing the individual agents in section  3. Tissue toxicity influences whether a chemical can be    used as an antiseptic or preservative, and this limits the  range of agents for these applications or necessitates the  use of lower concentrations of the agent. This is discussed  further in section 2.5.   The types of microorganism present and the levels of  microbial contamination (the bioburden) both have a  significant effect on the outcome of treatment. If  the bioburden is high, long exposure times and/or  higher concentrations of antimicrobial may be required.  Microorganisms vary in their sensitivity to the action of  chemical agents. Some organisms merit attention  either because of their resistance to disinfection (for  further discussion see Chapter 20) or because of their  significance in cross-infection or nosocomial (hospital-  acquired) infections. Of particular concern is the  significant increase in resistance to disinfectants resulting  from microbial growth in biofilm form rather than free  suspension (see Chapter 8). Microbial biofilms form  readily on available surfaces, posing a serious problem for  hospital infection control committees in advising suitable  disinfectants for use in such situations.   The efficacy of an antimicrobial agent must be inves-  tigated by appropriate capacity, challenge and in-use tests  to ensure that a standard is obtained which is appropriate  to the intended use (Chapter 18). In practice, it is not  usually possible to know which organisms are present  on the articles being treated. Thus, it is necessary to  categorize agents according to their antimicrobial  activity and for the user to be aware of the level of anti-  microbial action required in a particular situation (see  Table 19.1).   At in-use concentrations, chemicals used for disinfection  should be capable of killing bacteria and other organisms  expected in that environment within a defined contact  period. This includes ‘problem’ organisms such as  methicillin-resistant Staphylococcus aureus (MRSA),  vancomycin-resistant enterococci (VRE) and species of  Listeria, Campylobacter and Legionella. Antiseptics  and preservatives are also expected to have a broad  spectrum of antimicrobial activity but at their in-use  concentrations, after exerting an initial biocidal (killing)  effect, their main function may be biostatic (inhibitory).  Gram-negative bacilli, which are a major causes of  nosocomial infections, are often more resistant than    Gram-positive species. Pseudomonas aeruginosa, an  opportunistic pathogen (see also Chapter 7), has gained  a reputation as the most resistant of the Gram-negative  organisms. However, problems mainly arise when a  number of additional factors such as heavily soiled arti-  cles or diluted or degraded disinfectant solutions are  employed.   M. tuberculosis (the tubercle bacillus) and other myco-  bacteria are resistant to many bactericides. Resistance is  either ( 1 ) intrinsic, mainly due to reduced cellular perme-  ability or (2) acquired, due to mutation or the acquisition  of plasmids (Chapter 13). Tuberculosis remains an  important public health hazard, and indeed the annual  number of tuberculosis cases is rising in many countries.  The greatest risk of acquiring infection is from the  undiagnosed patient. Equipment used for respiratory  investigations can become contaminated with mycobac-  teria if the patient is a carrier of this organism. It is  important to be able to disinfect the equipment to a safe  level to prevent transmission of infection to other patients  (Table 19.2).   Bacterial spores can exhibit significant resistance to  even the most active chemical disinfectant treatment.  The majority of disinfectants have no useful sporicidal  action in a pharmaceutical context, which relates to  disinfection of materials, instruments and environ-  ments that are likely to be contaminated by the spore-  forming genera Bacillus and Clostridium. However,  certain aldehydes, halogens and peroxygen compounds  display very good activity under controlled conditions  and are sometimes used as an alternative to physical  methods for sterilization of heat-sensitive equipment.  In these circumstances, correct usage of the agent is of  paramount importance, as safety margins are lower  in comparison with physical methods of sterilization  (Chapter 21).   Clostridium difficile is a particularly problematic con-  taminant in hospital environments, resulting in high  levels of morbidity and mortality. In addition to stringent  hand-washing, meticulous environmental disinfection  procedures must be in place, e.g. using solutions of 5.25-  6.15% sodium hypochlorite for routine disinfection.  When high-level disinfection of Cl. difficile is required,  2% glutaraldehyde, 0.55% o-phthalaldehyde and 0.35%  peracetic acid are effective.    The antibacterial activity of some disinfectants and  antiseptics is summarized in Table 19.2.   The vegetative fungal form is often as sensitive as vegeta-  tive bacteria to chemical antimicrobial agents. Fungal  spores (conidia and chlamydospores; see Chapter 4) may  be more resistant, but this resistance is of much lesser  magnitude than that exhibited by bacterial spores. The  ability to rapidly destroy pathogenic fungi such as the  important nosocomial pathogen Candida albicans, fila-  mentous fungi such as Trichophyton mentagrophytes, and  spores of common spoilage moulds such as Aspergillus  niger is put to advantage in many applications of use.  Many disinfectants have good activity against these fungi    (Table 19.3). In addition, ethanol (70%) is rapid and reli-  able against Candida species.   Susceptibility of viruses to antimicrobial agents can  depend on whether the viruses possess a lipid envelope.  Non-lipid viruses are frequently more resistant to disin-  fectants and it is also likely that such viruses cannot be  readily categorized with respect to their sensitivities to  antimicrobial agents. These viruses are responsible for  many nosocomial infections, e.g. rotaviruses, picornavi-  ruses and adenoviruses (see Chapter 5) and it may be  necessary to select an antiseptic or disinfectant to suit  specific circumstances. Certain viruses, such as Ebola and  Marburg, which cause haemorrhagic fevers, are highly    infectious and their safe destruction by disinfectants is of  paramount importance. Hepatitis A is an enterovirus  considered to be one of the most resistant viruses to  disinfection.   There is much concern for the safety of personnel  handling articles contaminated with pathogenic viruses  such as hepatitis B virus (HBV) and HIV. Disinfectants  must be able to treat rapidly and reliably accidental  spills of blood, body fluids or secretions from HIV-  infected patients. Such spills may contain levels of HIV  as high as 10 4 infectious units/ml. Fortunately, HIV is  inactivated by most chemicals at in-use concentrations.  However, the recommendation is to use high-level  disinfectants (see Table 19.2) for decontamination of  HIV- or HBV-infected reusable medical equipment. For  patient care areas, cleaning and disinfection with  intermediate-level disinfectants is satisfactory. Flooding  with a liquid germicide is required only when large spills  of cultured or concentrated infectious agents have to be  dealt with.   The World Health Organization (WHO) and epidemi-  ologists in many countries track outbreaks of influenza,  especially in relation to potential epidemic and pandemic  situations arising. The H1N1 outbreak in 2009 generated  considerable concern. As an influenza A virus, however,  it is susceptible to a large number of disinfectant products  when they are used on hard, non-porous surfaces that  may be contaminated. Although no research has been  conducted on the susceptibility of 2009 H1N1 influenza  virus to chlorine and other disinfectants in swimming  pools and spas, studies have demonstrated that free chlo-    rine levels of l-3mg/L (1-3 ppm) are adequate to disin-  fect avian H5N1 influenza virus.   Acanthamoeba spp. can cause acanthamoeba keratitis  with associated corneal scarring and loss of vision in  wearers of soft contact lenses. The cysts of this protozoan  present a particular problem in respect of lens disinfec-  tion. The chlorine-generating systems in use are generally  inadequate. Polyaminopropyl biguanide with or without  chlorhexidine (0.003%) and polyhexamethylene bigua-  nide (0.0005%) both show ability as an acanthamoebi-  cide in combating 10 3 levels of cysts. Hydrogen peroxide  -based disinfection is considered completely reliable and  consistent in producing an acanthamoebicidal effect.   Prions are generally considered to be the infectious agents  most resistant to chemical disinfectants and sterilization  processes; strictly speaking, however, they are not micro-  organisms because they have no cellular structure nor  do they contain nucleic acids. As small proteinaceous  infectious particles they are a unique class of infectious  agent causing spongiform encephalopathies such as  bovine spongiform encephalopathy (BSE) in cattle and  Creutzfeldt-Jakob disease (CJD) in humans. There is  considerable concern about the transmission of these  agents from infected animals or patients. Risk of infectiv-  ity is highest in brain, spinal cord and eye tissues. Prions  are considered resistant to most disinfectant procedures.  For heat-resistant medical instruments that come into    contact with high infectivity tissues or high-risk contacts,  immersion in sodium hydroxide (1M) or sodium  hypochlorite (20 000 ppm available chlorine) for 1 hour  is advised in WHO guidelines and this must be followed  by further treatment including autoclaving, cleaning and  routine sterilization. Recently, a formulation of 0.2%  sodium docecyl sulphate, 0.3% NaOH in 20% n-propanol,  has achieved potent decontamination of steel carriers  contaminated with PrPTSE, the biochemical marker for  prion infectivity, from 263K scrapie hamsters (5.5log 10  units reduction) or patients with sporadic or variant  Creutzfeldt-Jacob disease. No low-temperature steriliza-  tion technology is effective.   The intended application of the antimicrobial agent,  whether for preservation, antisepsis or disinfection, will  influence its selection and also affect its performance. For  example, in medicinal preparations the ingredients in  the formulation may antagonize preservative activity. The  risk to the patient will depend on whether the antimicro-  bial is in close contact with a break in the skin or mucous  membranes or is introduced into a sterile area of the  body.   In disinfection of instruments, the chemicals used  must not adversely affect the instruments, e.g. cause cor-  rosion of metals, affect clarity or integrity of lenses, or  change the texture of synthetic polymers. Many materials  such as fabrics, rubber and plastics are capable of adsorb-  ing certain disinfectants, e.g. quaternary ammonium  compounds (QACs) are adsorbed by fabrics, while phe-  nolics are adsorbed by rubber, the consequence of this  being a reduction in the concentration of active com-  pound. A disinfectant can only exert its effect if it is in  contact with the item being treated. Therefore, access to  all parts of an instrument or piece of equipment is essen-  tial. For small items, total immersion in the disinfectant  must also be ensured.   Organic matter can have a drastic effect on antimicrobial  capacity either by adsorption or chemical inactivation,  thus reducing the concentration of active agent in solu-  tion or by acting as a barrier to the penetration of the  disinfectant. Blood, body fluids, pus, milk, food residues  or colloidal proteins, even when present in small amounts,  all reduce the effectiveness of antimicrobial agents to  varying degrees, and some are seriously affected. In their  normal habitats, microorganisms have a tendency to  adhere to surfaces and are thus less accessible to the    chemical agent. Some organisms are specific to certain  environments and their destruction will be of paramount  importance in the selection of a suitable agent, e.g.  Legionella in cooling towers and non-potable water  supply systems, Listeria in the dairy and food industry  and HBV in blood-contaminated articles.   Dried organic deposits may inhibit penetration of the  chemical agent. Where possible, objects to be disinfected  should be thoroughly cleaned. The presence of ions in  water can also affect activity of antimicrobial agents, thus  water for testing biocidal activity can be made artificially  ‘hard’ by addition of ions.   These factors can have very significant effects on activ-  ity and are summarized in Table 19.4.   In choosing an antimicrobial agent for a particular appli-  cation some consideration must be given to its toxicity.  Increasing concern for health and safety is reflected in the  Control of Substances Hazardous to Health (COSHH)  Regulations that specify the precautions required in han-  dling toxic or potentially toxic agents. In respect of dis-  infectants these regulations affect, particularly, the use of  phenolics, formaldehyde and glutaraldehyde. Toxic vola-  tile substances, in general, should be kept in covered con-  tainers to reduce the level of exposure to irritant vapour  and they should be used with an extractor facility. Limits  governing the exposure of individuals to such substances  are now listed, e.g. 0.7mg/m 3 (0.2 ppm) glutaraldehyde  for both short- and long-term exposure. Many disinfect-  ants including the aldehydes, glutaraldehyde less so than  formaldehyde, may affect the eyes, skin (causing contact  dermatitis) and induce respiratory distress. Face protec-  tion and impermeable nitrile rubber gloves should be  worn when using these agents. Table 19.4 lists the toxicity  of many of the disinfectants in use and other concerns of  toxicity are described below for individual agents.   The COSHH Regulations specify certain disinfectants  that contain active substances not supported under the  BPD that had to be phased out by 2006. Specified disin-  fection procedures applied to laboratories in relation to  spills and routine use state that certain phenolic agents  (including 2,4,6-trichlorophenol and xylenol) can no  longer be employed in disinfectant products.   Because of the historically high number of occupa-  tional asthma cases caused by glutaraldehyde (an  alkylating agent) products in chemical disinfection of  endoscopes, an HSE report (2007) sought alternatives to  this agent. The report recommended the preferential  use of an oxidizing agent such as a chlorine-based or   OES: 1 ppm/3 mg/m 3 , 10 min;  0.5ppm/1.5mg/m 3 , 8h   Use in well-ventilated area.  Gloves, goggles and apron worn  for preparation   Dichloroisocyanurate likely to  produce Cl gas when used to  disinfect acidic urines   OES: 0. 1 ppm/ 1 mg/m 3 , 10 min only  Protect skin and eyes    MEL, maximum exposure limit; OES: occupational exposure standard; QAC, quaternary ammonium compound.   MELs are the time-weighted average upper limits of a substance permitted in the breathing zone of a person. OES levels are the maximum concentrations of a substance  in air to which individuals may be exposed during their working life and at which present knowledge indicates there will be no ill effects. Often similar to 8 h MEL levels.  (MELs have now been replaced by workplace exposure limits, WELs.)    HSE, Health and Safety Executive; COSHH, Control of  Substances Hazardous to Health.    peroxygen-based product rather than a product contain-  ing an alkylating agent. However, it was recognized that  consideration must be given to incompatibility of disin-  fectants with endoscope construction materials in some  cases (Table 19.5).   In all situations where the atmosphere of a workplace  is likely to be contaminated by disinfectant, sampling  and analysis of the atmosphere may need to be carried  out on a periodic basis with a frequency determined by  conditions.   The following section presents, in alphabetical order by  chemical grouping, the agents most often employed for  disinfection, antisepsis and preservation. This informa-  tion is summarized in Table 19.6.   Antimicrobial activity, within a pharmaceutical context,  is generally found only in the organic acids. These are  weak acids and will, therefore, dissociate incompletely to  give the three entities HA, H + and A~ in solution. As the  undissociated form, HA, is the active antimicrobial agent,  the ionization constant, K a , is important and the p K a of  the acid must be considered, especially in formulation  of the agent.   This is an organic acid, C 6 H 5 COOH, which is included,  alone or in combination with other preservatives, in  many pharmaceuticals. Although the compound is  often used as the sodium salt, the non-ionized acid is the  active substance. A limitation on its use is imposed by the  pH of the final product as the p K a of benzoic acid is   4.2 at which pH 50% of the acid is ionized. It is advisable  to limit use of the acid to preservation of pharmaceu-  ticals with a maximum final pH of 5.0 and if possible  less than 4.0. Concentrations of 0.05-0.1% are suitable  for oral preparations. A disadvantage of the compound  is the development of resistance by some organisms, in  some cases involving metabolism of the acid resulting  in complete loss of activity. Benzoic acid also has some  use in combination with other agents, salicylic acid  for example, in the treatment of superficial fungal  infections.   This compound is a widely used preservative as the acid  or its potassium salt. The pK a is 4.8 and, as with benzoic  acid, activity decreases with increasing pH and ioniza-  tion. It is most effective at pH 4 or below. Pharmaceutical  products such as gums, mucilages and syrups are usefully  preserved with this agent.   Sulphur dioxide has extensive use as a preservative in  the food and beverage industries. In a pharmaceu-  tical context, sodium sulphite and metabisulphite or  bisulphite have a dual role, acting as preservatives and  antioxidants.   A series of alkyl esters (Figure 19.1) of p-hydroxybenzoic  acid was originally prepared to overcome the marked  pH dependence on activity of the acids. These parabens,  the methyl, ethyl, propyl and butyl esters, are less readily  ionized, having pA a values in the range 8-8.5, and exhibit  good preservative activity even at pH levels of 7-8,  although optimum activity is again displayed in acidic  solutions. This broader pH range allows extensive and  successful use of the parabens as pharmaceutical pre-  servatives. They are active against a wide range of fungi  but are less so against bacteria, especially the pseudomon-  ads which may utilize them as a carbon source. They are  frequently used as preservatives of emulsions, creams  and lotions where two phases exist. Combinations of    Table 19.6 Examples of the main antimicrobial groups as antiseptics, disinfectants and preservatives   a Also used in combination with other agents, e.g. chlorhexidine, iodine.  b Several forms available having x% chlorhexidine and 10x% cetrimide.  QAC, quaternary ammonium compound.   Figure 19.1 p-Hydroxybenzoates (R is methyl, ethyl, propyl,  butyl, or benzyl).   esters are most successful for this type of product in that  the more water-soluble methyl ester (0.25%) protects  the aqueous phase, whereas the propyl or butyl esters  (0.02%) give protection to the oil phase. Such combina-  tions are also considered to extend the range of activity.  As inactivation of parabens occurs with non-ionic sur-  factants due care should be taken in formulation with  both materials.    Figure 19.2 Structural formulae of alcohols used in preserving  and disinfection: (A) 2-phenylethanol; (B) 2-phenoxyethanol,  (C) chlorbutol (trichloro-tert-butanol); (D) bronopol  (2-bromo-2-nitropropan-l,3-diol).   The aliphatic alcohols, notably ethanol and isopropanol,  are used for disinfection and antisepsis. They are bacte-  ricidal against vegetative forms, including Mycobacterium  species, but are not sporicidal. Overall cidal activity drops  sharply below 50% concentration. Alcohols have poor  penetration of organic matter and their use is, therefore,  restricted to clean conditions. They possess properties  such as a cleansing action and volatility, are able to  achieve a rapid and large reduction in skin flora and have  been widely used for skin preparation before injection  or other surgical procedures. The risk of transmission of  infection due to poor hand hygiene has been attributed  to lack of compliance with hand-washing procedures.  An alcohol hand-rub offers a rapid, easy-to-use alterna-  tive that is more acceptable to personnel and is frequently  recommended for routine use. However, the contact  time of an alcohol-soaked swab with the skin prior to  venepuncture is so brief that it is thought to be of doubt-  ful value.   Ethanol (CH 3 CH 2 OH) is widely used as a disinfectant  and antiseptic. The presence of water is essential for  activity, hence 100% ethanol is relatively ineffective.  Concentrations between 60% and 95% are bactericidal  and a 70% solution is usually employed for the disinfec-  tion of skin, clean instruments or surfaces. At higher con-  centrations, e.g. 90%, ethanol is also active against fungi  and most lipid-containing viruses, including HIV, though  less so against non-lipid-containing viruses. Ethanol is  also a popular choice in pharmaceutical preparations and  cosmetic products as a solvent and preservative, but it is  not recommended for cleaning class II recirculating  safety cabinets; ethanol vapours are flammable and the    lower explosive limit (LEL) is easily attained. Mixtures  with other disinfectants, e.g. with formaldehyde (100 g/L),  are more effective than alcohol alone.   Isopropyl alcohol (isopropanol, CH 3 .CHOH.CH 3 ) has  slightly greater bactericidal activity than ethanol but is  also about twice as toxic. It is less active against viruses,  particularly non-enveloped viruses, and should be con-  sidered a limited-spectrum viricide. Used at concentra-  tions of 60-70%, it is an acceptable alternative to ethanol  for preoperative skin treatment and is also employed as  a preservative for cosmetics.   The aralkyl alcohols and more highly substituted aliphatic  alcohols (Figure 19.2) are used mostly as preservatives.  These include:   • Benzyl alcohol (C 6 H 5 CH 2 OH). This has antibacterial  and weak local anaesthetic properties and is used as an  antimicrobial preservative at a concentration of 2%,  although its use in cosmetics is restricted.   • Chlorbutol (chlorobutanol; trichlorobutanol; trichloro-  t-butanol) is typically used at a concentration of 0.5%  and is employed as a preservative in injections and eye  drops. It is unstable, decomposition occurring at acid pH  during autoclaving, while alkaline solutions are unstable  at room temperature.   • Phenylethanol (phenylethyl alcohol; 2-phenylethanol),  having a typical in-use concentration of 0.25-0.5%, is  reported to have greater activity against Gram-negative  organisms and is usually employed in conjunction with  another agent.   • Phenoxyethanol (2-phenoxyethanol). Typical in-use  concentration: 1%. It is more active against Ps. aeruginosa    than against other bacteria and is usually combined with  other preservatives such as the hydroxybenzoates to  broaden the spectrum of antimicrobial activity.   • Bronopol (2-bromo-2-nitropropan-l,3-diol). Typical  in-use concentration: 0.01-0.1%. It has a broad spectrum  of antibacterial activity, including Pseudomonas species.  The main limitation on the use of bronopol is that when  exposed to light at alkaline pH, especially if accompanied  by an increase in temperature, solutions decompose,  turning yellow or brown. A number of decomposition  products including formaldehyde are produced. In  addition, nitrite ions may be produced and react with  any secondary and tertiary amines present forming  nitrosamines, which are potentially carcinogenic.   A number of aldehydes possess broad-spectrum antimi-  crobial properties, including sporicidal activity. These  highly effective biocides can be employed in appropriate  conditions as chemosterilants.   Glutaraldehyde (CHO(CH 2 ) 3 CHO) has a broad spec-  trum of antimicrobial activity and rapid rate of kill, most  vegetative bacteria being killed within a minute of expo-  sure, although bacterial spores may require 3 hours or  more. The kill rate depends on the intrinsic resistance of  spores, which may vary widely. It has the further advan-  tage of not being affected significantly by organic matter.  The glutaraldehyde molecule possesses two aldehyde  groupings which are highly reactive and their presence is  an important component of biocidal activity. The mono-  meric molecule is in equilibrium with polymeric forms,  and the physical conditions of temperature and pH have  a significant effect on this equilibrium. At a pH of 8,  biocidal activity is greatest but stability is poor due to  polymerization. In contrast, acid solutions are stable but  considerably less active, although as temperature is  increased, there is a breakdown in the polymeric forms  which exist in acid solutions and a concomitant increase  in free, active dialdehyde, resulting in better activity. In  practice, glutaraldehyde is generally supplied as an acidic  2% or greater aqueous solution, which is stable on pro-  longed storage. This is then ‘activated’ before use by  addition of a suitable alkalizing agent to bring the pH of  the solution to its optimum for activity. The activated  solution will have a limited shelf life, of the order of 2  weeks, although more stable formulations are available.  Glutaraldehyde is employed mainly for the cold liquid  chemical sterilization of medical and surgical materials    that cannot be sterilized by other methods. Endoscopes,  including for example arthroscopes, laparascopes, cysto-  scopes and bronchoscopes, may be decontaminated by  glutaraldehyde treatment (see section 2.5 concerning tox-  icity issues). Times employed in practice for high-level  disinfection are often considerably less than the many  hours recommended by manufacturers to achieve sterili-  zation. The contact time for sterilization can be as long  as 10 hours. Times for general disinfection generally  range from 20-90 minutes at 20 °C depending on formu-  lation and concentration.   Orrfio-phthalaldehyde (OPA) is a relatively recent  addition to the aldehyde group of high-level disinfect-  ants. This agent has demonstrated excellent mycobacte-  ricidal activity with complete kill of M. tuberculosis  within 12 minutes at room temperature. OPA has several  other advantages over glutaraldehyde. It requires no acti-  vation, is considerably less irritant to the eyes or nasal  passages and has excellent stability over the pH range 3-9.  It can be used for disinfection of endoscopes (Table 19.5).   Formaldehyde (HCHO) can be used in either the liquid  or the gaseous state for disinfection purposes. In the  vapour phase it has been used for decontamination of  isolators, safety cabinets and rooms. The combination of  formaldehyde vapour with low-temperature steam  (LTSF) has been employed for the sterilization of heat-  sensitive items (Chapter 21). Formaldehyde vapour is  highly toxic and potentially carcinogenic if inhaled, thus  its use must be carefully controlled. It is not very active  at temperatures below 20 °C and requires a relative  humidity of at least 70%. The agent is not supplied as a  gas but either as a solid polymer, paraformaldehyde, or a  liquid, formalin, which is a 34-38% aqueous solution.  The gas is liberated by heating or mixing the solid or  liquid with potassium permanganate and water. Formalin,  diluted 1:10 to give 4% formaldehyde, may be used for  disinfecting surfaces. In general, however, solutions of  either aqueous or alcoholic formaldehyde are too irritant  for routine application to skin, while poor penetration  and a tendency to polymerize on surfaces limit its use as  a disinfectant for pharmaceutical purposes.   Various formaldehyde condensates have been developed  to reduce the irritancy associated with formaldehyde  while maintaining activity, and these are described as   Noxythiolin (N-hydroxy N-methylthiourea) is sup-  plied as a dry powder and on aqueous reconstitution  slowly releases formaldehyde and N-methylthiourea.  The compound has extensive antibacterial and antifungal  properties and has been used both topically and in  accessible body cavities as an irrigation solution and  in the treatment of peritonitis. Polynoxylin (poly-  [methylenedi(hydroxymethyl)urea]) is a similar com-  pound available in gel and lozenge formulations.   Taurolidine (bis- ( 1 , 1 -dioxoperhydro- 1 ,2,4-thiadiazinyl-4)  methane) is a condensate of two molecules of the amino  acid taurine and three molecules of formaldehyde. It is  more stable than noxythiolin in solution and has similar  uses.   Chlorhexidine is an antimicrobial agent first synthesized  in 1954. The chlorhexidine molecule, a bisbiguanide, is  symmetrical with a hexamethylene chain linking two  biguanide groups, each with a pnra-chlorophenyl radical  (Figure 19.3).   Chlorhexidine base is not readily soluble in water;  therefore its freely soluble salts, acetate, gluconate and  hydrochloride, are used in formulation. Chlorhexidine  exhibits the greatest antibacterial activity at pH 7-8  where it exists exclusively as a dication. The cationic  nature of the compound results in activity being reduced  by anionic compounds, including soap, due to the forma-  tion of insoluble salts. Anions to be wary of include bicar-  bonate, borate, carbonate, chloride, citrate and phosphate,  with avoidance of hard water if possible. Deionized or  distilled water should preferably be used for dilution pur-  poses. Reduction in activity will also occur in the pres-  ence of blood, pus and other organic matter. Chlorhexidine    has widespread use, in particular as an antiseptic. It has  significant antibacterial activity, although Gram-negative  bacteria are less sensitive than Gram-positive organisms.  A concentration of 0.0005% prevents growth of, for  example, Staph, aureus, whereas 0.002% prevents growth  of Ps. aeruginosa. Reports of pseudomonad contamina-  tion of aqueous chlorhexidine solutions have prompted  the inclusion of small amounts of ethanol or isopropanol.  Chlorhexidine is ineffective at ambient temperatures  against bacterial spores and M. tuberculosis. Limited anti-  fungal activity has been demonstrated, which unfortu-  nately restricts its use as a general preservative. Skin  sensitivity has occasionally been reported although, in  general, chlorhexidine is well tolerated and non-toxic  when applied to skin or mucous membranes and is an  important preoperative antiseptic.   The antimicrobial activity of the bisbiguanide chlorhexi-  dine exceeds that of monomeric biguanides. This stimu-  lated the development of polymeric biguanides containing  repeating biguanide groups linked by hexamethylene  chains. One such compound is a commercially available  heterodisperse mixture of polyhexamethylene biguanides  (PHMB, polyhexanide) having the general formula  shown in Figure 19.4.   Within the structure, n varies with a mean value of 5.5.  The compound has a broad spectrum of activity against  Gram-positive and Gram-negative bacteria and has low  toxicity. PHMB is employed as an antimicrobial agent in  various ophthalmic products.   Chlorine and iodine have been used extensively since  their introduction as disinfecting agents in the early 19th  century. Preparations containing these halogens, such  as Dakin’s solution and tincture of iodine, were early  inclusions in many pharmacopoeias and national formu-    laries. More recent formulations of these elements have  improved activity, stability and ease of use.   A large number of antimicrobially active chlorine  compounds are commercially available, one of the most  important being liquid chlorine. This is supplied as an  amber liquid made by compressing and cooling gaseous  chlorine. The terms liquid and gaseous chlorine refer to  elemental chlorine, whereas the word ‘chlorine’ itself is  normally used to signify a mixture of OCU, Cl 2 , HOC1  and other active chlorine compounds in aqueous solu-  tion. The potency of chlorine disinfectants is usually  expressed in terms of parts per million (ppm) or percent-  age of available chlorine (avCl).   Hypochlorites (bleach) are the oldest and remain the  most useful of the chlorine disinfectants, being readily  available, inexpensive and compatible with most anionic  and cationic surface-active agents. They exhibit a rapid  kill against a wide spectrum of microorganisms, includ-  ing fungi and viruses. High levels of available chlorine  will enable eradication of mycobacteria and bacterial  spores. Their disadvantages are that they are corrosive,  suffer inactivation by organic matter and can become  unstable. Hypochlorites are available as powders or  liquids, most frequently as the sodium or potassium salts  of hypochlorous acid (HOC1). Sodium hypochlorite  exists in solution as follows:   Undissociated hypochlorous acid is a strong oxidizing  agent and its potent antimicrobial activity is dependent  on pH as shown:   At low pH the existence of HOC1 is favoured over  OC1 (hypochlorite ion). The relative microbicidal effec-  tiveness of these forms is of the order of 100 : 1. By lower-  ing the pH of hypochlorite solutions the antimicrobial  activity increases to an optimum at about pH 5. However,    this is concurrent with a decrease in stability of the solu-  tions. This problem may be alleviated by addition of  NaOH (see equation 1) in order to maintain a high pH  during storage for stability. The absence of buffer allows  the pH to be lowered sufficiently for activity on dilution  to use-strength. It is preferable to prepare use-dilutions  of hypochlorite on a daily basis.   Undiluted bleach stored at room temperature in a  closed container has a shelf life of about 6 months.  Storage of stock or working solutions of bleach in open  containers causes release of chlorine gas, especially at  elevated temperatures, and this considerably weakens the  antimicrobial activity of the solution. Working solutions  should be prepared on a daily basis.   A number of organic chlorine, or chloramine, com-  pounds are now available for disinfection and antisepsis.  These are the N-chloro (=N-C1) derivatives of, for  example, sulphonamides giving compounds such as  chloramine-T and dichloramine-T, and halazone (Figure  19.5), which may be used for the disinfection of contami-  nated drinking-water.   A second group of compounds, formed by N-chloro  derivatization of heterocyclic compounds containing a  nitrogen in the ring, includes the sodium and potassium  salts of dichloroisocyanuric acid (e.g. NaDCC). These are  available in granule or tablet form and, in contrast to  hypochlorite, are very stable on storage if protected from  moisture. In water they will give a known chlorine con-  centration. The antimicrobial activity of the compounds  is similar to that of the hypochlorites when acidic condi-  tions of use are maintained. It is, however, important to  note that where inadequate ventilation exists, care must  be taken not to apply the compound to acidic fluids or  large spills of urine in view of the toxic effects of chlorine  production. The HSE has set the occupational exposure  standard (OES) short-term exposure limit at 1 ppm (see  also section 2.5).   Chloroform (trichloromethane, CHC1 3 ) has a narrow  spectrum of activity. It has been used extensively as a    preservative of pharmaceuticals since the 19th century,  although more recently it has had limitations placed on  its use. Marked reductions in concentration may occur  through volatilization from products, resulting in the  possibility of microbial growth.   Iodine has a wide spectrum of antimicrobial activity.  Gram-negative and Gram-positive organisms, bacterial  spores (on extended exposure), mycobacteria, fungi and  viruses are all susceptible. The active agent is the elemen-  tal iodine molecule, I 2 . As elemental iodine is only slightly  soluble in water, iodide ions are required for aqueous  solutions such as Aqueous Iodine Solution, BP 1988  (Lugol’s Solution) containing 5% iodine in 10% potas-  sium iodide solution. Iodine (2.5%) may also be dis-  solved in ethanol (90%) and potassium iodide (2.5%)  solution to give Weak Iodine Solution, BP 1988 (Iodine  Tincture).   The antimicrobial activity of iodine is less dependent  than chlorine on temperature and pH, although alkaline  pH should be avoided. Iodine is also less susceptible to  inactivation by organic matter. Disadvantages in the use  of iodine in skin antisepsis are staining of skin and fabrics  coupled with possible sensitizing of skin and mucous  membranes.   In the 1950s iodophors (iodo meaning iodine and phor  meaning carrier) were developed, to eliminate the disad-  vantages of iodine while retaining its antimicrobial activ-  ity. These allowed slow release of iodine on demand from  the complex formed. Essentially, four generic compounds  may be used as the carrier molecule or complexing agent.  These give polyoxymer iodophors (i.e. with propylene or  ethyene oxide polymers), cationic (quaternary ammo-  nium) surfactant iodophors, non-ionic (ethoxylated)  surfactant iodophors and polyvinylpyrrolidone iodo-  phors (PVP-I or povidone-iodine). The non-ionic or  cationic surface-active agents act as solubilizers and car-  riers, combining detergency with antimicrobial activity.  The former type of surfactant, especially, produces a  stable, efficient formulation, the activity of which is  further enhanced by the addition of phosphoric or citric  acid to give a pH below 5 on use-dilution. The iodine is  present in the form of micellar aggregates which disperse  on dilution, especially below the critical micelle concen-  tration (cmc) of the surfactant, to liberate free iodine.   When iodine and povidone are combined, a chemical  reaction takes place forming a complex between the two.    Some of the iodine becomes organically linked to povi-  done, although the major portion of the complexed  iodine is in the form of tri-iodide. Dilution of this iodo-  phor results in a weakening of the iodine linkage to the  carrier polymer with concomitant increases in elemental  iodine in solution and antimicrobial activity.   The amount of free iodine the solution can generate is  termed the ‘available iodine’. This acts as a reservoir for  active iodine, releasing it when required and therefore  largely avoiding the harmful side effects of high iodine  concentration. Consequently, when used for antisepsis,  iodophors should be allowed to remain on the skin for 2  minutes to obtain full advantage of the sustained-release  iodine.   Cadexamer-I 2 is an iodophor similar to povidone-  iodine. It is a 2-hydroxymethylene cross-linked (1-4) a-  D-glucan carboxymethyl ether containing iodine. The  compound is used especially for its absorbent and  antiseptic properties in the management of leg ulcers  and pressure sores where it is applied in the form of  microbeads containing 0.9% iodine.   Mercury and silver have antibacterial properties and  preparations of these metals were among the earliest used  antiseptics; however, they have been largely replaced by  less toxic compounds. Silver has enjoyed a renaissance  recently as an antimicrobial frequently incorporated in  urethral catheters for the prevention of device-related  infection. Various forms of silver are employed such as  nanoparticulate silver, silver halides, silver oxide and  combinations such as silver-palladium. A hard surface  disinfectant formulation based on silver dihydrogen  citrate is shown to be effective against a wide range of  bacteria, fungi and viruses using as little as 30 ppm silver.   The organomercurial derivatives thiomersal and phe-  nylmercuric nitrate or acetate (PMN or PMA) (Figure  19.6) have been primarily employed as preservatives. Use    of both compounds has declined considerably as a result  of concerns about mercury toxicity and risk of hypersen-  sitivity or local irritation. They are absorbed from solu-  tion by rubber closures and plastic containers to a  significant extent.   Hydrogen peroxide and peracetic acid are high-level dis-  infectants because of their production of the highly reac-  tive hydroxyl radical. They have the added advantage that  their decomposition products are non-toxic and biode-  gradable. The germicidal properties of hydrogen peroxide  (H 2 0 2 ) have been known for more than a century, but  use of low concentrations of unstable solutions did little  for its reputation. However, stabilized solutions are now  available and because of its unusual properties and anti-  microbial activity, hydrogen peroxide has a valuable role  for specific applications. Its activity against the protozoan  Acanthamoeba, which can cause keratitis in contact lens  wearers, has made it popular for disinfection of soft  contact lenses. Concentrations of 3-6% are effective for  general disinfection purposes. At high concentrations (up  to 35%) and increased temperature, hydrogen peroxide  is sporicidal. Use has been made of this in vapour-phase  hydrogen peroxide decontamination of equipment and  enclosed spaces (Chapter 21).   Peracetic acid (CH 3 COOOH) is the peroxide of acetic  acid and is a more potent biocide than hydrogen perox-  ide, with excellent rapid biocidal activity against bacteria,  including mycobacteria, fungi, viruses and spores. It can  be used in both the liquid and vapour phases and is active  in the presence of organic matter. It is finding increasing  use at concentrations of 0.2-0.35% as a chemosterilant  of medical equipment such as flexible endoscopes. Its  disadvantages are that it is corrosive to some metals. It is  also highly irritant and must be used in an enclosed  system. The combination of hydrogen peroxide and per-  acetic acid is synergistic and is marketed as a cold sterilant  for dialysis machines.   Phenols (Figure 19.7) are widely used as disinfectants and  preservatives. They have good antimicrobial activity and  are rapidly bactericidal but generally are not sporicidal.  Their activity is markedly diminished by dilution and is  also reduced by organic matter. They are more active at  acid pH. Major disadvantages include their caustic effect  on skin and tissues and their systemic toxicity. The more  highly substituted phenols are less toxic and can be used    as preservatives and antiseptics; however, they are also  less active than the simple phenolics, especially against  Gram-negative organisms. To improve their poor aqueous  solubility, phenolic disinfectants are often formulated  with soaps, synthetic detergents, and/or solvents.   Phenol (Figure 19. 7A) no longer plays any significant role  as an antibacterial agent. It is largely of historical interest,  as it was used by Lister in the 1860s as a surgical antiseptic  and has been a standard for comparison with other dis-  infectants in tests such as the Rideal-Walker test.   Phenols obtained by distillation of coal or petroleum can  be separated by fractional distillation according to their  boiling point range into phenols, cresols, xylenols and  high boiling point tar acids. As the boiling point increases  bactericidal activity increases and tissue toxicity decreases,  but there is increased inactivation by organic matter and  decreased water solubility.   Clear soluble fluids are produced from cresols or xyle-  nols. The preparation known as Lysol (Cresol and Soap  Solution BP 1968) is a soap-solubilized formulation of  cresol (Figure 19. 7B) that has been widely used as a  general-purpose disinfectant but has largely been super-  seded by less irritant phenolics. A higher boiling point  fraction consisting of xylenols and ethylphenols (Figure  19. 7C and D) produces a more active, less corrosive  product that retains activity in the presence of organic  matter. A variety of proprietary products for general dis-  infection purposes are available.   Black fluids and white fluids are prepared by solubiliz-  ing the high boiling point tar acids. Black fluids are  homogeneous solutions that form an emulsion on dilu-  tion with water, whereas white fluids are finely dispersed  stable emulsions. Both types of fluid have good bacteri-  cidal activity. Preparations are very irritant and corrosive  to skin; however, they are relatively inexpensive and are  useful for household and general disinfection purposes.   Many derivatives of phenol are now made by a synthetic  process. A combination of alkyl or aryl substitution and  halogenation of phenolic compounds has produced  useful derivatives. Two of the best known chlorinated  derivatives are p-chloro-m-cresol (chlorocresol, Figure  19.7E) which was frequently employed as a preservative  at a concentration of 0.1%, and p-chloro-m-xylenol    (chloroxylenol, Figure 19. 7F) which is sometimes used  for skin disinfection. Chloroxylenol is sparingly soluble  in water and must be solubilized, for example, in a suit-  able soap solution in conjunction with terpineol or pine  oil. Its antimicrobial capacity is weak and is reduced by  the presence of organic matter. Other phenol derivatives  of note are: 2-benzyl-4-chlorophenol (Figure 19. 7G),  2-phenylphenol (Figure 1 9.7H) and p-tert-amylphenol  (Figure 19.71).   Bisphenols are composed of two phenolic groups con-  nected by various linkages. Triclosan (Figure 1 9.7J) is the  most widely used. It has been incorporated into medi-  cated soaps, lotions and solutions and is also included in  household products such as plastics and fabrics. There is  some concern about bacterial resistance developing to  triclosan.   Surface-active agents or surfactants are classified as  anionic, cationic, non-ionic or ampholytic according to  the ionization of the hydrophilic group in the molecule.    A hydrophobic, water-repellent group is also present.  Within the various classes a range of detergent and dis-  infectant activity is found. The anionic and non-ionic  surface-active agents, for example, have strong detergent  properties but exhibit little or no antimicrobial activity.  They can, however, render certain bacterial species more  sensitive to some antimicrobial agents, possibly by alter-  ing the permeability of the outer envelope. Ampholytic  or amphoteric agents can ionize to give anionic, cationic  and zwitterionic (positively and negatively charged ions  in the same molecule) activity. Consequently, they display  both the detergent properties of the anionic surface-  active agents and the antimicrobial activity of the cationic  agents. They are used quite extensively in Europe for  presurgical hand-scrubbing, medical instrument disin-  fection and floor disinfection in hospitals.   Of the four classes of surface-active agents the cationic  compounds play the most important role in an antimi-  crobial context.   The cationic agents used for their antimicrobial activity  all fall within the group known as the quaternary ammo-    nium compounds (QACs, quats or onium ions). These  are organically substituted ammonium compounds  (Figure 19. 8A) where the R substituents are alkyl or het-  erocyclic radicals to give compounds such as benzalko-  nium chloride (Figurel9.8B), cetyltrimethylammonium  bromide (cetrimide) (Figure 19. 8C) and cetylpyridinium  chloride (Figure 19. 8D). Inspection of the structures of  these compounds (Figure 19. 8B and C) indicates that a  chain length in the range C 8 -C 18 in at least one of the R  substituents is a requirement for good antimicrobial  activity. In the pyridinium compounds (Figure 19. 8D),  three of the four covalent links may be satisfied by the  nitrogen in a pyridine ring. Several ‘generational’ changes  have arisen in the development of QACs. Compounds  such as alkyldimethylbenzyl ammonium chloride, alkyld-  imethylethylbenzyl ammonium chloride and didecyld-  imethylammonium chloride have roles in disinfection  where HIV and HBV are present. Polymeric quaternary  ammonium salts such as polyquaternium 1 are finding  increasing use as preservatives.   The QACs are most effective against microorganisms  at neutral or slightly alkaline pH and become virtually  inactive below pH 3.5. Not surprisingly, anionic agents  greatly reduce the activity of these compounds.  Incompatibilities have also been recorded with non-ionic  agents, possibly due to the formation of micelles. The  presence of organic matter such as serum, faeces and milk  will also seriously affect activity.   QACs exhibit greatest activity against Gram-positive  bacteria, with a lethal effect observed using concentrations  as low as 0.0005%. Gram-negative bacteria are more  resistant, requiring a level of 0.0033%, or higher still if Ps.  aeruginosa is present. A limited antifungal activity is  exhibited and they have no useful sporicidal activity. This  relatively narrow spectrum of activity limits the usefulness    of the compounds, but as they are generally well tolerated  and non-toxic when applied to skin and mucous mem-  branes they have considerable use in treatment of wounds  and abrasions. Benzalkonium chloride and cetrimide are  employed extensively in surgery, urology and gynaecology  as aqueous and alcoholic solutions and as creams. In many  instances they are used in conjunction with a biguanide  disinfectant such as chlorhexidine. The detergent proper-  ties of the QACs also provide a useful activity, especially  in hospitals, for general environmental cleaning.   The full range of chemicals that can be shown to have  antimicrobial properties is beyond the scope of this  chapter. The agents included in this section have limited  use or are of historic interest.   The activity of diamidines is reduced by acid pH and in  the presence of blood and serum. Propamidine and  dibromopropamidine, as the isethionate salts, have been  employed as antimicrobial agents in eye drops (0.1%) for  amoebic infection and for topical treatment of minor  infections.   Crystal violet (Gentian violet), brilliant green and mala-  chite green are triphenylmethane dyes used to stain bac-  teria for microscopic examination. They have a static  activity but are no longer applied topically for the treat-  ment of infections because of carcinogenicity.   The acridine dyes acriflavine and aminacrine have  been employed for skin disinfection and treatment of  infected wounds or burns but are slow acting and mainly  bacteriostatic.    Figure 19.8 Quaternary ammonium compounds  (QACs): (A) general structure of QACs; (B)  benzalkonium chloride ( n = 8—18); (C) cetrimide  (n = 12—14 or 16); (D) cetylpyridinium chloride.   The quinoline derivatives of pharmaceutical interest are  little used now. The compound most frequently used is  dequalinium chloride, a bisquaternary ammonium deriv-  ative of 4-aminoquinaldinium which was formulated as  a lozenge for the treatment of oropharyngeal infections.   There is no ideal disinfectant, antiseptic or preservative.  All chemical agents have their limitations in terms of  either their antimicrobial activity, resistance to organic  matter, stability, incompatibility, irritancy, toxicity or cor-  rosivity. To overcome the limitations of an individual  agent, formulations consisting of combinations of agents  are available. For example, ethanol and isopropanol have  been combined with chlorhexidine, QACs, sodium  hypochlorite and iodine to produce more active prepara-  tions. The combination of chlorhexidine and cetrimide  is also considered to improve activity. QACs and phenols  have been combined with glutaraldehyde and formalde-  hyde so that the same effect can be achieved with lower,  less irritant concentrations of the aldehydes. Some com-  binations are considered to be synergistic, e.g. hydrogen  peroxide and peroxygen compounds. Care must be taken  in deciding on disinfectant combinations, as the concen-  tration exponents associated with each component of a  disinfectant combination will have a considerable effect  on the degree of activity (Chapter 18).   Research into the resistance of microbial biofilms pro-  vides potential for improving elimination of this prob-  lematical microbial mode of growth. Bacteria often use a  communication system, quorum sensing (QS), to regu-  late virulence factor production and the formation of  biofilms. Increased understanding of how chemicals can  block QS could help provide effective prevention and  elimination of biofilm-related infection. The incorpora-  tion of antimicrobial agents into materials that form  working and contact surfaces or those of medical devices  and implants has been positive but much further devel-  opmental research is required. Such ‘bioactive’ surfaces  can be formed, for example, by incorporation of silver  salts and alloys, biguanides and triclosan, and have the  ability to reduce infection arising from microbial adher-  ence and biofilm formation.   Other means are available to potentiate the activity of  disinfectants. Ultrasonic energy in combination with  suitable disinfectants such as aldehydes and biguanides  has been demonstrated to be useful in practice and ultra-  violet radiation increases the activity of hydrogen perox-    ide. Superoxidized water provides an extremely active  disinfectant with a mixture of oxidizing species produced  from the electrolysis of saline. The main products are  hypochlorous acid (144mg/L) and free chlorine radicals.  The antimicrobial activity is rapid against a wide range  of microorganisms in the absence of organic matter.   The aim of a disinfection policy is to control the use of  chemicals for disinfection and antisepsis and give guide-  lines on their use. The preceding descriptions within this  chapter of the activities, advantages and disadvantages of  the many disinfectants available allow considerable scope  for choice and inclusion of agents in a policy to be applied  to such areas as industrial plant, walls, ceilings, floors, air,  cleaning equipment and laundries and to the extensive  range of equipment in contact with hospital patients.   The control of microorganisms is of prime importance  in hospital and industrial environments. Where pharma-  ceutical products (either sterile or non-sterile) are manu-  factured, contamination of the product may lead to its  deterioration and to infection in the user. In hospital  there is the additional consideration of patient care,  therefore protection from nosocomial (hospital-acquired)  infection and prevention of cross-infection must also be  covered. Hospitals will have a disinfection policy and  the degree of adherence to, and implementation of, the  policy content will require stringent monitoring. A spe-  cialized infection control committee or similar, compris-  ing a number of specialized personnel such as the  pharmacist, the consultant medical microbiologist and  senior nurse responsible for infection control, should for-  mulate a suitable policy (Chapter 16). This core team may  usefully be expanded to include, for example, a physician,  a surgeon, nurse teachers and nurses from several clinical  areas, the sterile services manager and the domestic  superintendent; purchasing. This expanded committee  will meet regularly to help with the implementation of  the policy and reassess its efficiency. Tables 19.2-19.4  indicate the susceptibility of various microorganisms to  the range of agents available and Table 19.6 presents  examples of the range of formulations available. Although  scope exists for choice of disinfectant in many of the areas  covered by a policy, in certain instances specific recom-  mendations are made as to the type, concentration and  usage of disinfectant.   Categories of risk (to patients) may be assigned to  equipment coming into contact with a patient, dictating   High level disinfection  with aldehyde or  peroxygen; contact  times up to 30 min    the level of decontamination required and degree of  concern (Table 19.7). High-risk (critical) items have close  contact with broken skin or mucous membranes or are  those introduced into a sterile area of the body and  should, themselves, be sterile; they include instruments,  gloves, catheters, syringes and needles. Liquid chemical  disinfectants should only be used if heat or other methods  of sterilization are unsuitable. Intermediate-risk (semic-  ritical) items are in close contact with skin or mucous  membranes and disinfection will normally be applied.  Endoscopes, respiratory and anaesthetic equipment,  wash bowls, bed-pans and similar items are included in  this category. Low-risk (non-critical) items or areas  include those detailed earlier such as walls and floors,  which are not in close contact with the patient. Cleaning  is obviously important with disinfection being required,  for example, in the event of contaminated spillage.   Alvarado, C.J. & Reichelderfer, M. (2000) APIC guideline for  infection prevention and control in flexible endoscopy. Am }  Infect Control, 28, 138-155.   Eggers, H.J. (1990) Experiments on antiviral activity of hand  disinfectants. Some theoretical and practical considerations.  Zentralhl Bakteriol , 273, 36—51.    EC (2009) European Commission proposals for new biocide  regulations. http://eurlex.europa.eu/LexUriServ/LexUriServ  .do?uri=CELEX:DKEY=496597:EN:NOT   HSE (2005) Workplace Exposure Limits: Containing the list of  workplace exposure limits for use with the Control of Substances  Hazardous to Health Regulations 2002 (as amended). Health  and Safety Executive, Sheffield.   HSE (2007) Updates to 2003 Supplement to Occupa-  tional Exposure Limits EH40/2002. Health and Safety  Executive, Sheffield, http://news.hse.gov.uk/2008/01/14/  workplace-exposure-limits-table/   HSE (2009) Control of substances hazardous to health  (COSHH). COSHH Essentials information. http://www.coshh-  essentials.org.uk/   Niven, K. (2006) An Evaluation of Chemical Disinfecting Agents  Used in Endoscopy Suites in the NHS. RR445. Health and Safety  Executive, Sheffield.   Scott, E.M., Gorman, S.P. & McGrath, S.J. (1986) An assessment  of the fungicidal activity of antimicrobial agents used for  hard-surface and skin disinfection. / Clin Hosp Pharm, 11,  199-205.   Traore, O., Springthorpe, V.S. & Sattar, S.A. (2002) Testing chem-  ical germicides against Candida species using quantitative  carrier and fingerpad methods. / Hosp Infect , 50, 66-75.   van Bueren, J., Salman, H. & Cookson, B.D. (1995) The Efficacy  of Thirteen Chemical Disinfectants Against Human Immuno-  deficiency Virus (HIV). Evaluation Report. Medical Devices  Agency, London.   The group of agents which comprises antiseptics,  disinfectants, chemical sterilants and preservatives  (often collectively called biocides) have frequently  been classified as non-specific protoplasmic poisons.  Such a broad generalization is, however, far from the true  position.   It is often convenient to consider the modes of action  of biocides in terms of their targets within the bacterial  cell, in particular the region of the cell in which their    activity is deemed to predominate. Thus agents have been  described variously as cell wall, membrane and cytoplasm-  active. This characterization, whilst having the benefits of  simplicity, does not necessarily describe their mechanism  of action; this is best classified by effects on functional  structures and cellular processes. The range and complex-  ity of the reactions involved will become apparent from  this account and Table 20.1, and it is worth emphasizing  here that many of these substances exhibit concentration-  dependant dual or even multiple effects. More detailed  treatments of the subject will be found in the references  at the end of this chapter.    Hugo and Russell's Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   Crosses, indicating activity, which appear in several rows for a given compound, demonstrate the multiple actions for the compound concerned. This activity is nearly  always concentration-dependent, and the number of crosses indicates the order of concentration at which the effect is elicited, i.e. +, elicited at low concentrations, +-H-,  elicited at high concentrations;   QACs, quaternary ammonium compounds; PHMB, polyhexamethylene biguanides; TCS, tetrachlorosalicylanilide; TCC, trichlorocarbanilide.   For a chemical to exhibit antimicrobial activity it usually  has to undergo a sequence of events that begins with  adsorption on to the microbial cell surface. This initial  uptake is a physicochemical phenomenon which can be  generally characterized into one of several uptake iso-  therms (Figure 20.1); it bears a relationship to the con-  centration exponent (Chapter 19) which describes the  influence of concentration on activity (Table 20.2). In the  many cases where the chemical has an intracellular site  of action, adsorption must be followed by passage  through porin channels in Gram-negative cells (Chapter  3), diffusion across, or into, the lipid-rich cytoplasmic  membrane, and finally, interaction with proteins,  enzymes, nucleic acids or other targets within the cyto-  plasm. These processes are markedly influenced by the  physicochemical characteristics of the biocide, e.g. ioni-  zation constant and lipid solubility, so the wide diversity  of structures exhibited by biocide molecules (Chapter 19)  complicates the prediction of antimicrobial potency and  explanation of their mechanisms of action. Despite this,  it is important to recognize that there is a basis upon  which the mode of action might be deduced, because  there are certain molecular features of biocides that are  associated with activity against particular cellular targets.   Antimicrobial activity is often strongly influenced by the  affinity of the biocide for structural or molecular com-  ponents of the cell, and this, in turn, may depend upon  the attraction of dissimilar charges or on hydrophobic  interactions. Antimicrobial drugs whose active species is  positively charged, e.g. quaternary ammonium com-  pounds (QACs) and chlorohexidine, display an affinity  for the negative charges of sugar residues on the microbial  cell surface or phosphate groups on the membrane(s);  adsorption of these biocides, and thus their antimicrobial  activity, is increased as the pH rises and the cell surface  becomes more electronegative. Antimicrobial chemicals  possessing a long alkyl chain, on the other hand, may  integrate into the hydrophobic region of phospholipid  molecules within the membrane and so cause membrane  disruption and fatal permeability changes. Further exam-  ples of structure-activity relationships are afforded by  aldehydes, particularly glutaraldehyde, which is an elec-  trophile that is able to react with molecules possess-  ing thiol (SH) or amino groups, e.g. proteins. This  reaction, too, increases with pH, so aldehydes are more  active in alkaline conditions. Biocides containing heavy  metal ions, e.g. silver or mercury, also damage or inacti-  vate enzymes and structural proteins by virtue of   Figure 20.1 Typical uptake isotherms associated with the initial biocide-bacterium interaction, (a) S-shape, cooperative sorption  occurs as applied biocide concentration increases; (b) L-shape, Langmuir uptake with biocide molecules orientated at a fixed  number of binding sites; (c) H-shape, special form of the L-shape isotherm indicative of high affinity uptake; (d) C-shape,  constant partition of biocide from solution until bacterial surface is fully saturated; (e) Z-shape, enhanced uptake following  breakdown in cell structure at a critical applied biocide concentration.   Strong chemical or ionic bonding with target site, frequently  promoting cytoplasm leakage or membrane disruption;  uptake limited by binding sites available. Typical examples  include QACs and chlorhexidine   Initial biocide uptake enhanced by cooperative adsorption,  often with specific orientation of adsorbed molecules. A  mixed group of biocides typically exercising both chemical  and physical effects on cellular components; examples include  some phenols and lipophilic acids   Weakly physical interaction, often with partition into  lipophilic components of the cell envelope; a plateau in  uptake can be reached when the envelope is fully saturated  although new sorption sites may be generated with some  biocides at a critical applied concentration (Z-shape). Typical  examples include phenols and 2-phenoxyethanol which are  active as membrane disruptors and proton conductors    interactions with thiol groups. A number of phenols and  bisphenols incorporate a hydroxyl group that is capable  of generating a labile proton, i.e. they are weak acids. A  weakly acidic nature combined with significant lipid solu-  bility are properties associated with uncoupling agents,  i.e. those molecules that can disrupt the proton-motive  force that is responsible for oxidative phosphorylation in  the cell. It is thought that these molecules dissolve in the  lipid bilayer of the membrane and act as proton conduc-  tors by virtue of their ionizability (section 4.4.2). This  property, possessed by biocides such as phenoxythanol  and fentichlor, results in the failure of many important  energy-requiring processes in the cell, including the con-  centration and retention of sugars and amino acids.   In any consideration of mechanism of action, due regard  should be given to the initial health of the organism,  duration of contact with the biocide, and the concentra-  tion of biocide employed. Antibacterial effects may  progress from early, sublethal events to multiple lesions  of bactericidal consequence. Figure 20.2 identifies events  in order of severity, but should not be interpreted as  defining the normal progression of cell injury. As dis-  closed in the following sections, the biocide interaction    may induce particular lesions over others; this will most  certainly be in a concentration-dependant manner.   Biocides with oxidizing (electron-withdrawing) ability  are widely used as disinfectants and chemical sterilants,  and include the halogens (chlorine, hypochlorites,  bromine, iodophors) and peroxygens (hydrogen perox-  ide, peracetic acid and chlorine dioxide). They can exert  specific effects on essential microbial macromolecules    causing, variously: strand breakage and adduct formation  on DNA and RNA with disruption of replication, tran-  scription and and translocation processes; degradation  of, particularly, unsaturated fatty acids leading to loss of  membrane fluidity and subsequent reduced functionality  of membrane-bound proteins; and specific modifications  to amino acid residues, most notably disulphide bonds,  leading to changes in protein primary structure and con-  formation with consequent disruption of structural  enzymic functions. An accumulation of these effects can  be particularly devastating to the microbial cell.   The aldehydes formaldehyde, glutaraldehyde and  orthophthalaldehyde, and the sterilant alkylating agents  ethylene oxide and propylene oxide, are both highly reac-  tive chemical classes. The alkylating agents exhibit par-  ticularly strong reactions with guanine residues causing  cross-linking between DNA strands, inhibiting DNA  unwinding and RNA translation. The amino, carboxyl,  sulphydryl and hydroxyl groups of structural or enzymic  proteins are also susceptible to alkylation, causing cross-  links between adjacent amino acid chains and also with  other amino acid-containing structures such as peptidog-  lycan. The aldehydes are generally more specific with  greatest effect against the amino groups of surface-  exposed lysine or hydroxylysine residues of proteins,  again causing extensive cross-linking.   In all instances, progressive cross-linking leads to mac-  romolecule malfunction causing inhibition or arrest of  essential cell functions. It is safe to say that there is no  single fatal reaction but that death results from the accu-  mulated effect of many reactions in a manner similar to  oxidizing agents (section 4.1).   The cross-linking reactions identified in section 4.2 give  rise to macromolecule denaturation which can be recog-  nized under electron microscopy as intracellular coagula-  tion. Coagulative effects are not unique to aldehydes and  alkylating agents, however, and high concentrations of  disinfectants such as chlorhexidine, phenol, ethanol and  mercuric salts will also coagulate the cytoplasm. This  most likely arises from the precipitation of protein caused  by a variety of interactions including ionic and hydro-  phobic bonding and the disruption of hydrogen bonds.   The integrity and functions of the bacterial cell are  dependant upon critical macromolecular structural    arrangements including within the cell wall and cytoplas-  mic membrane (Chapter 3). A number of biocides can  have a profound effect on these organelles.   This structure is the traditional target for a group of  antibiotics which includes the penicillins (Chapter 11),  but a little-noticed report which appeared in 1948 showed  that low concentrations of disinfectant substances  caused cell wall lysis such that a normally turbid suspen-  sion of bacteria became clear. It is thought that these low  concentrations of disinfectant cause enzymes whose  normal role is to synthesize the cell wall to reverse their  role in some way and effect its disruption or lysis. In the  original report, these low concentrations of disinfectants  (formalin, 0.12%; phenol, 0.32%; mercuric chloride,  0.0008%; sodium hypochlorite, 0.005% and merthiolate,  0.0004%) caused lysis of Escherichia coli, streptococci,  and staphylococci.   Divalent cations, in addition to their role as enzyme  cofactors, also stabilize cell wall, membrane and ribos-  omal structures. In particular, magnesium serves to link  the lipopolysaccharide (LPS) of Gram-negative bacteria  to the outer membrane. Chelators, particularly ethylen-  ediamine tetraacetic acid (EDTA), have been used to  disrupt this link and cause the release of LPS into the  medium. The loss of outer membrane integrity and sub-  sequent permeabilization has been exploited in the  potentiation of biocides, including combinations of  EDTA with chloroxylenol, cetrimide, phenylethanol and  the parahydroxy benzoic acid esters (Chapter 19).   The bacterial cytoplasmic membrane consists of an  impermeable, negatively-charged, fluid phospholipid  bilayer incorporating an organized array of membrane-  associated proteins. Through the membrane-bound elec-  tron transport chain aerobically, or the membrane-bound  adenosine triphosphatase (ATPase) anaerobically, the  bacterium succeeds in maintaining a transmembrane  gradient of electrical potential and pH such that the inte-  rior of the cell is negative and alkaline. This proftm motive  force, as it is called, drives a number of energy-requiring  functions which include the synthesis of ATP, the cou-  pling of oxidative processes to phosphorylation, a meta-  bolic sequence called oxidative phosphorylation, and the  transport and concentration in the cell of metabolites  such as sugars and amino acids. This, put briefly, is the  basis of the chemiosmotic theory linking metabolism to  energy-requiring processes.    Certain chemical substances have been known for  many years to uncouple oxidation from phosphorylation  and to inhibit active transport, and for this reason they  are named uncoupling agents. They are believed to act  by partitioning into the membrane and rendering it per-  meable to protons, hence short-circuiting the potential  gradient or protonmotive force. Some examples of anti-  bacterial agents which owe at least a part of their activity  to this ability are tetrachlorosalicylanilide (TCS), tricar-  banilide, trichlorocarbanilide (TCC), pentachlorophenol,  di-(5-chloro-2-hydroxyphenyl) sulphide (fentichlor),  2-phenoxyethanol, and lipophilic acids and esters.   The membrane, as well as providing a dynamic link  between metabolim and transport, serves to maintain the  pool of metabolites within the cytoplasm. A general  increase in membrane permeability brought about by the  association and likely insertion of biocide molecules into  the lipid bilayer was recognized early as being one effect  of many disinfectant substances.   Treatment of bacterial cells with appropriate concen-  trations of such substances as cetrimide and other QACs,  chlorhexidine, polyhexamethylene biguanides, phenol  and hexylresorcinol causes a leakage of a group of char-  acteristic chemical species. The potassium ion, being a  small entity, is the first substance to appear when the  cytoplasmic membrane is damaged. Amino acids, purines,  pyrimidines and pentoses are examples of other sub-  stances which will leak from treated cells. If the action of  the drug is not prolonged or exerted only in low concen-  tration, the damage may be reversible and leakage may  only induce bacteriostasis. There is however, evidence that  a depletion of intracellular potassium caused by mem-  brane damage can lead to the activation of latent ribonu-  cleases and the consequent breakdown of RNA. Several  biocides, including cetrimide and some phenols, are  known to cause the release of nucleotides and nucleosides  following an autolytic process. This is irreversible and has  been proposed as an autocidal (suicide) process, commit-  ting the injured cell to death (Denyer & Stewart, 1998).   Surface-associated proteins within the membrane  fulfil a number of important roles including wall  biosynthesis, nutrient transport and respiration. Usually  enzymes, these macromolecules are often topologically  organized and uniquely exposed to disruption by  biocidal agents. Thus, hexachlorophane inhibits the elec-  tron transport chain in bacteria, chlorhexidine has  been shown to inhibit ATPase, and thiol-containing  membrane dehydrogenases are highly susceptible to  mercury-containing antibacterials, silver, 2-bromo-2-  nitropropan-l,3-diol (bronopol) and isothiazolinones.   Mention has already been made of the use of permeabiliz-  ing chelators to enhance the penetration of biocides to  their target (section 4.4.1). Much effort has also been  expended in the search for synergistic combinations of  biocides which, when added together, will greatly amplify  the bactericidal effect. While theoretically possible, and  potentially predictable from mechanism of action studies,  in practice this effect is elusive; combinations of phenylm-  ercuric acetate with benzalkonium chloride, lipophilic  weak acids with fatty alcohols, and chlorocresol with phe-  nylethanol have been reported. The most likely route to  enhancing activity lies with optimizing biocide formula-  tions to ensure maximum availability of the active  moiety — particularly important in situations of pH-  dependency or poor water solubility. In the area of phar-  maceutical and cosmetic preservation, constructive use of  formulation ingredients, each with some intrinsic anti-  microbial activity, has successfully built on the activity of  the original preservative agent to create a cumulative bac-  tericidal effect, an approach called ‘hurdle technology’.   Bacterial resistance to biocides has been reported since  the 1950s, notably with QACs, biguanides and phenolics.  Overall there has been more documented evidence of  bacterial resistance to antiseptics than to disinfectants  (Table 20.3). It is worth mentioning that some bacteria  surviving in biocidal formulations have been associated  with outbreaks and pseudo-outbreaks of infection (Weber  et al . , 2007) . Bacterial resistance to all known preservatives  has also been reported (Chapman et al, 1998). Recently,  much interest has focused on bacteria surviving high-  level disinfection, which is usually employed for the dis-  infection of medical devices. Thus bacteria surviving  exposure to the in-use (high) concentration of highly  reactive biocides (e.g. glutaraldehyde, chlorine dioxide,  hydrogen peroxide) have been isolated and studied. In  2009, a large outbreak of atypical mycobacteria in at least  38 hospitals in Brazil was reported. These mycobacteria  were traced to endoscope contamination and were resist-  ant to 2% w/v glutaraldehyde but also to the clinical  concentration of front-line antibiotics against mycobac-  teria (Duarte et al., 2009). This was the first time that  biocide resistance was linked to antibiotic resistance,  nosocomial infection and a large infection outbreak.   Burkholderia cepacia *, Serratia marcescens *, Achromobacter xylosoxydans * ,  Pseudomonas aeruginosa *   Pseudomonas spp., Burkholderia cepacia , Flavobacterium meningosepticum , Serratia  marcescens , Rastonia pickettii *, Achromobacter xylosoxidans   Pseudomonas spp.*, Achromobacter spp., Enterobacter aerogenes. Pseudomonas  kingii , Burkholderia cepacia *, Serratia marcescens, Mycobacterium chelonae,  Mycobacterium abscessus    * Bacteria associated with pseudo-outbreak or outbreak.  Adapted from Weber et al. (2007).    Over the last 10 years, much progress has been made  in understanding the mechanisms conferring resistance  to biocides in bacteria. Interestingly, some mechanisms  that were thought to occur only with antibiotics have now  been described with biocides.   A number of mechanisms conferring some level of resist-  ance to biocide exposure have been documented.  Traditionally mechanisms of resistance have been divided  into intrinsic and acquired resistance. Intrinsic (or innate )  resistance is a natural property of the bacteria and pro-  vides some explanation as to why some bacteria are less  susceptible than others. Intrinsic mechanisms often  involve a structural difference, for example, a difference  in the permeability of the bacterial membrane to bio-    cides, but also the expression of chromosomal genes such  as those encoding for an efflux pump or a degradative  enzyme. Acquired resistance refers to the acquisition of a  new property by the bacteria through mutation and  genetic transfer; such a property can be the mutation of  a target site or the transfer of a gene encoding for an  efflux pump or a degradative enzyme. It should be noted  that when gene transfer occurs, often several genes  present on the same conjugative plasmid or transposon  can be transferred to a recipient cell at the same time. In  this case the term co-resistance is often used to denote the  simultaneous acquisition of a number of genes confer-  ring resistance to a number of antimicrobials, both bio-  cides and antibiotics.   Bacterial resistance to a biocide arises often from the  presence of several mechanisms that work together to    decrease the detrimental concentration of the biocide to  a level that is no longer harmful for the bacterium (Table  20.4). The expression of only one mechanism confers  low-level resistance often measured as an increase in  minimum inhibitory concentration (MIC), but rarely  high-level resistance, which can be measured as an  increase in minimum bactericidal concentration. Finally,  a distinction can be made between mechanisms expressed  by a single bacterium and the mechanisms of resistance  that arise from a community of bacteria such as in bacte-  rial biofilms.   The decrease in biocide penetration arising from changes  in cell permeability is well established and has been    described with different bacterial genera, notably with  Gram-negative bacteria and mycobacteria. It is also the  case with bacterial endospores, which are discussed later  in this chapter. In Gram-negative bacteria, the outer  membrane, and notably the composition of LPS, offers  some protection to the cell, by reducing biocide penetra-  tion. The role of LPS has been exemplified by researchers  with the use of permeabilizing agents and notably ion  chelators such as EDTA. EDTA contributes to the removal  (loss) of LPS from the outer membrane by scavenging  dications involved in the stabilization of LPS in the mem-  brane (section 4.4.1). By losing LPS, the outer membrane  becomes more permeable and biocides can penetrate  better resulting in enhanced activity. A change in the  structure of the outer membrane following a change in   Outer membrane: lipopolysaccharide; cation  content (intrinsic/acquired/ environmental)  Porins: reduction in number, size (intrinsic/  acquired/ environmental)   Increase in pyruvate synthesis and fatty acid  production via an altered metabolic pathway  ( intrinsic/acquired)    protein, fatty acid or phospholipid composition has been  associated with a decrease in the efficacy of cationic bio-  cides. In particular, a decrease in the number of porin  proteins as a result of biocide exposure has been associ-  ated with high-level resistance to QACs in pseudomon-  ads. Recently, a change in surface charge in Pseudomonas  aeruginosa has been associated with a decrease in suscep-  tibility to QACs.   In mycobacteria, the lipid-rich outer cell wall (respon-  sible for the waxy appearance of the colonies), and par-  ticularly the presence of a mycoylacylarabinogalactan  layer and the composition of the arabinogalactan/  arabinomannan within the cell wall, account for a reduc-  tion in biocide penetration; increasing the permeability  of the mycobacterial outer cell wall, for example with  ethambutol, enhances the activity of biocides and  antibiotics.   Efflux pumps are cross-membrane proteins which pump  out various substrates including biocides and antibiotics.  A large number of efflux pumps in bacteria have been  identified and have been divided into five main classes  depending on their structure and activity: the small  multidrug resistance (SMR) family; the major facilitator  superfamily (MFS); the ATP-binding cassette (ABC)  family; the resistance-nodulation-division (RND) family;  and the multidrug and toxic compound extrusion  (MATE) family (Figure 20.3).   The role of efflux pumps is to remove harmful sub-  stances from the bacterial cytoplasm, including biocides  (e.g. QACs and phenolics), to levels that are not damag-  ing for the cell. The quantity of antimicrobial pumped  out depends upon the number of pumps present, their  expression and efficacy. Some studies have shown that  high-level resistance can be achieved by efflux, for  instance against the bisphenol triclosan, but usually, the  effect is an increase in MIC.   Enzymatic degradation plays a role in reducing the  harmful concentration of a biocide and has been observed  with aldehydes (e.g. aldehyde dehydrogenase), oxidizing  agents (e.g. catalases, superoxide dismutase, hydroxyper-  oxidases), phenolics and parabens. In the case of metallic  salts such as silver, the ionic form is reduced to the inac-  tive metal. The role of enzymatic inactivation has not  been widely studied, but it is unlikely that a bacterial  enzyme will contribute to high-level resistance to a  biocide.   To date, resistance conferred by the modification of a  biocide target site has only been observed with triclosan.  At a low concentration this bisphenol interacts specifi-  cally with a bacterial enoyl-acyl reductase carrier protein,  which is involved in the synthesis of fatty acid. Triclosan  has been shown to interact with a number of structurally  related enzymes in many bacterial genera. A modification  of the enzyme confers a low-level resistance to triclosan,  although some studies claim that a high-level resistance  has been observed. This is unlikely, since at a high con-  centration triclosan interacts with multiple target sites to  bring about a bactericidal effect.   A change in metabolic pathway that confers resistance to  a biocide is a relatively new concept that was thought to  occur only with sulphonamides. However, bacterial adap-  tation to triclosan, as measured by an extended lag phase  of growth followed by a normal exponential phase,  has been observed in several bacterial genera. The  recent use of a microarray in Salmonella enterica serovar  Typhimurium enabled the identification of a ‘triclosan  resistance network’ including an alternative pathway to  the production of pyruvate and fatty acid. In Staphylococcus  aureus showing reduced sensitivity to triclosan, a change  in lipid composition of the cell membrane was associated  with altered expression of various genes involved in fatty  acid metabolism. Low-level QAC resistance in Serratia  marcescens may arise from a change in synthetic or meta-  bolic pathways.   The induction of antimicrobial resistance in bacteria fol-  lowing biocide exposure is a relatively recent concern  which is particularly pertinent to the increasing number  of commercially available products containing a low con-  centration of a biocide. This low, often subinhibitory,  concentration can induce the expression of resistance  mechanisms that can confer bacterial survival to biocide  and/or antibiotic exposure. Low-level resistance as meas-  ured by an increase in MIC has often been observed. Prior  to the use of genomics, proteomics and metabolomics,  induction of resistance in bacteria was observed with  an increase in lag phase of growth and a decrease in  growth rate, an overexpression of efflux pumps and the  production of guanosine 5'-diphosphate 3'-diphosphate  (ppGpp). An increase in DNA repair was also associated  with an increase in bacterial survival following biocide  exposure.   Figure 20.3 Examples of efflux pumps in bacteria. Note the  wide range of substrates used by these pumps. (From Piddock,  L.J.V. (2006) Multidrug resistance efflux pumps — not just for  resistance. Nat Rev Microbiol , 4, 629-636. Reprinted by  permission from Macmillan Publishers Ltd, Nature Reviews  Microbiology.)    More recently, a change in expression of regulons com-  manding a number of responses, such as a stress type of  response (and repair mechanism), increased efflux,  change in membrane composition, and a change in  metabolic and synthetic pathways, has been recorded fol-  lowing exposure to a low concentration of biocides.  Such a global response is of concern as it also confers a  decreased susceptibility to antibiotics, and recent evi-  dence suggests it might also lead to overexpression of  virulence determinants.   It can be noted that the concentration of a biocide  that promotes mutation in bacteria might be low. Indeed,  a number of investigations have observed that the  mutation rate increases in the presence of an active  efflux system. The effect of bacterial mutation in the  development of resistance has not been widely investi-  gated, with the exception of triclosan. It is also possible  that biocides that interact with the bacterial genome  (e.g. dyes, oxidizing agents) might produce a higher  mutation rate.   Surprisingly, the dissemination of biocide resistance  mechanisms between bacteria has been little studied.  The acquisition of new genetic determinants, notably  by the process of conjugation, is of concern as it is  often dependent on the presence of large transferable  plasmids, and transposons which encode for many  genes including bacterial resistance to antibiotics and  virulence factors. When several resistance genes are  transferred at the same time, the term coresistance is  used. For example, the resistance to the QAC benzalko-  nium chloride in Staphylococcus aureus has been associ-  ated with the presence of plasmids containing qac, bla  and tet resistant genes (encoding for efflux pumps and a  (3-lactamase). A number of mechanisms of resistance  such as efflux and degradative enzymes have been docu-  mented to be transferred between bacteria. The extent of  such dissemination is difficult to measure, although it is  thought to readily occur in bacterial communities such  as a biofilm.   Equally important to the horizontal transfer of resist-  ance is the maintenance of resistant determinants (plas-  mids) following the continuous presence of biocides.  Although this issue has not been widely studied, it is of  interest with the use of biocidal products which are docu-  mented to leave a residual concentration of biocide on  surfaces, antimicrobial surfaces, and the continuous pres-  ence of biocides in certain applications, such as drinking-  water chlorination.   The formation of a spore is a mechanism of bacterial  survival when growth conditions are detrimental for  the vegetative form. Such adverse conditions include lack  of food but also the presence of biocides and other det-  rimental physical and chemical conditions. The spore  structure is unique and confers upon the spore high-  level resistance to biocides. Hence only a few biocides,  mainly highly reactive ones such as aldehydes, oxidizing  agents and chlorine-releasing agents, are sporicidal, while  others such as biguanides, QACs and phenolics are  sporostatic despite their bactericidal effect on the vegeta-  tive bacteria. It should also be noted that sporicides take  time to kill spores, usually a minimum of 5 minutes  contact, and at a high concentration; aldehydes such as  glutaraldehyde and formaldehyde need a much longer  contact time and, in the case of orthophthalaldehyde, a  raised temperature.   Sporulation, a process in which a bacterial spore develops  from a vegetative cell (Chapter 3), involves seven  stages (I-VII); of these, stages IV-VII (cortex and coat  development) are the most important in relation to  the development of biocide resistance. Resistance to bio-  cidal agents develops during sporulation and may be an  early, intermediate or late/very late event. For example,  resistance to chlorhexidine occurs at an intermediate  stage, at about the same time as heat resistance, whereas  decreasing susceptibility to glutaraldehyde is a very late  event.   Bacillus spore coatless mutants and chemically-  induced coatless spores have shown the role of the spore  coats in limiting access of biocides to the spore core. The  cortex also acts as a barrier to some extent.   During germination and/or outgrowth, metabolism  and biosynthetic processes increase and cells regain their  sensitivity to antibacterial agents. Some inhibitors act at  the germination stage (e.g. phenolics, parabens), whereas  others such as chlorhexidine and the QACs do not affect  germination but inhibit outgrowth. Glutaraldehyde, at  low concentrations, is an effective inhibitor of both  stages.   The spore structure (Chapter 3) and the interaction  between a biocide and the spore have been particularly  well documented in the genus Bacillus. The spore core  (protoplast, sometimes refered to as the germ cell) is  enclosed within a cell wall which is surrounded by the    cortex and several spore coats. Sometimes an exosporium  may surround the spore.   The spore core is the target site of sporicides since it is  the location of RNA, DNA, dipicolinic acid (DPA) and  most of the calcium, potassium, manganese and phos-  phorus. Also present are substantial amounts of low  molecular weight basic proteins, the small acid-soluble  spore proteins (SASPs) which are rapidly degraded  during germination. SASPs, comprising about 10-20% of  the protein in the dormant spore, exist in two forms (ot/(3  and y) and are essential for expression of spore resistance  to ultraviolet radiation and also appear to be involved in  resistance to some biocides e.g. hydrogen peroxide.  Spores (or/(3~) deficient in cx/(3-type SASPs are much  more peroxide-sensitive than are wild-type (normal)  spores. It has been proposed that in wild-type spores  DNA is saturated with oc/p-type SASPs and is thus pro-  tected from free radical damage.   Bacteria are generally associated with surfaces in a  complex community called biofilms (see also Chapter 8).  Following attachment to a surface, a bacterium will go    through a series of metabolic and phenotypic changes,  leading to the formation of microcolonies embedded  within a matrix of secreted exopolysaccharides. Bacteria  in biofilms have been shown to be less susceptible to  antibiotics and biocides than planktonic bacteria.  There are several biocide resistance mechanisms, all  contributing to a ‘biofilm-associated phenotype’: reduc-  tion in biocide penetration, reduced bacterial metabo-  lism, quiescence, enzymatic inactivation and efflux  (Table 20.5).   Biocides have been observed to change the composi-  tion of a complex biofilm, composed of different bacterial  genera or/and species. For example, polyhexamethylene  biguanides (PHMB), chlorhexidine and Bardac (a QAC)  have been shown to select for pseudmonads to the detri-  ment of Gram-positive bacteria. The bisphenol triclosan  was shown to reduce the genera diversity of a complex  waste drain biofilm and to decrease the overall suscepti-  bility of the remaining population.   The indiscriminate use of biocides in an increasing  number of applications and, notably, the use of   Dormant cells (which regrow rapidly in the presence of exudates  released from lysed community)    sub-optimal low concentrations has fuelled the debate on  emerging bacterial cross-resistance to antibiotics used for  human and animal medicine. This is based on in vitro  evidence that some mechanisms conferring a decrease in  biocide susceptibility can also lead to resistance to thera-  peutic concentrations of antibiotics. Some of the most  common mechanisms involved include expression and  over-expression of efflux pumps and changes in cell per-  meability and metabolism. However, there is no useful  rule of thumb to predict cross-resistance between biocide  and antibiotic resistance in bacteria. In addition, emerg-  ing cross-resistance following biocide exposure in situ has  not been widely reported. The most significant study to  date was reported in 2009 and concerned an outbreak in  38 hospitals of an isolate of Mycobacterium massiliense  resistant to 2% glutaraldehyde and to antimycobacterial  therapeutic antibiotics.   It is generally accepted that viruses can be divided into  two groups according to their susceptibility to biocides.  Lipophilic viruses that possess a viral envelope derived  from their host (e.g. HIV, herpes simplex virus, influenza  virus) are the most susceptible to biocides. The hydrophilic  viruses comprise all the non-enveloped viruses and differ  tremendously in size and structure. Among these, the  small non-enveloped viruses such as the picornaviruses  (e.g. poliovirus, hepatitis A virus, foot-and-mouth disease  virus) are often considered to be the least susceptible  to biocide exposure, although some larger viruses such  as adenoviruses and rotaviruses can also be quite resilient.  Overall, the viricidal activity of biocides has been little  studied and often conflicting information can be found  in the peer-reviewed literature. Discrepancies in reported  viricidal activity often can be traced to the difference in  efficacy test methodology used, and notably the lack of  an appropriate neutralizer to quench the activity of the  biocides (see Chapter 18). In general terms if the  membrane-active biocides such as biguanides, phenolics,  QACs and alcohols have a good efficacy against enveloped  viruses, their activity against non-enveloped viruses is  limited. A recent study, however, indicated that the limi-  tation in activity of the biguanide PHMB was caused by  the formation of viral aggregates.   One of the biggest challenges for biocide activity  against viruses is that viruses on surfaces are often associ-  ated with soiling and fomites. Such an association allows  viral survival (notably for enveloped viruses) on surfaces    for long periods of time, as fomites/soiling appear  to protect viruses from desiccation. In addition, these  organic materials can protect viruses from detrimental  chemical agents such as biocides. Viricidal tests do not  always consider viruses embedded in an organic load.   In terms of mechanisms of action, the goal of a viricide  should be the destruction of the viral nucleic acid. In  reality, very few biocides have been shown to affect the  viral genome; cationic biocides and alcohols damage  the viral envelope releasing an intact viral capsid and  genome, although they have been shown to affect the  capsid in some instances, but not the viral genome.  Biocides that have been shown to interact and break open  the capsid (e.g. chlorine-releasing agents) might not  have a damaging effect on the viral nucleic acid. To date,  only a few biocides (i.e. mainly oxidizing agents) have  been observed to damage the viral nucleic acid within  the capsid.   The main mechanism of viral resistance to biocides is  the formation of viral aggregates before or during biocide  exposure. These clumps protect some viruses from the  damaging effect of biocides that fail to penetrate deep  within these clumps. Chlorine-releasing agents (e.g.  hypochlorite) and PHMB have been shown to produce  viral aggregates, limiting their viricidal efficacy. To some  extent a change in capsid configuration has been shown  to alter the susceptibility of viruses to a lower concentra-  tion of biocides (e.g. glutaraldehyde). Finally, multiplicity  reactivation, a process that has been observed only in  vitro, concerns the reassembly of intact viruses that have  been structurally damaged by a biocide intervention, but  where the genome remains intact. This process, together  with the suggestion that the viral genome of certain  viruses (e.g. hepatitis B virus) can remain infectious,  emphasizes the importance for viricides to destroy the  viral nucleic acid.   The activity of biocides has been described in a number  of amoebae, notably in Giardia spp. and Cryptospori-  dium spp., which are major waterborne pathogens, and  Acanthamoeba spp., a pathogen mainly associated with  contact lenses and contact lens solutions. Trophozoites  (the actively-growing form) of Acanthamoeba spp.  have been shown to be susceptible to low concentrations  of chlorhexidine, PHMB, QACs, oxidizing agents  (hydrogen peroxide, chlorine dioxide, peracetic acid,  ozone), chlorine-releasing agents and isothiazolinones.    Glutaraldehyde might possess only a poor trophocidal  activity. In general higher concentrations and much  longer contact times are needed to achieve a cysticidal  activity (e.g. hydrogen peroxide 3% for 4 hours) and the  concentration of biocide (e.g. PHMB, QAC) used for the  disinfection of contact lenses might be ineffective against  Acanthamoeba cysts. Other biocides such as iodine and  bromine and the isothiazolinones have been shown to  have no activity against Acanthamoeba cysts. It should be  noted that differences in inactivation using the same bio-  cides and parameters have been observed against cysts of  different species.   Since most biocides have a poor cysticidal efficacy at a  low concentration, the combination of biocides or the  formulation excipients can become important. For  example, a combination of chlorhexidine and thiomersal  and/or EDTA has been shown to be cycticidal within 24  hours. QAC cysticidal activity can be improved when  combined with tributyltin neodecanoate. A combination  of hydrogen peroxide (3%) with catalase and potassium  iodide (50jaM) was shown to enhance significantly cysti-  cidal activity against A. polyphaga.   The mechanisms of action of biocides against tropho-  zoites are similar to those observed on bacterial struc-  tures. For example, cationic biocides have been shown to  damage the cytoplasmic membrane and to induce pentose  leakage in Acanthamoeba spp.   Amoebal trophozoites undergo encystation when  exposed to detrimental conditions, which include biocide  (e.g. diamidines, chlorhexidine) exposure. Cysts are a  dormant form which enable survival for many years in  the environment. They are a dehydrated structure with a  double wall composed of cellulose and relatively small  numbers of proteins. The outer ectocyst wall is composed  mainly of protein and lipid containing materials and the  inner endocyst wall contains cellulose.   Encystation is a relatively rapid process that can be  divided into three principal stages: induction, during  which cellular components are degraded; immature cysts,  during which the first cell wall is synthesized; and mature  cysts, during which the second cell wall is formed.   The composition and morphological aspects of the  cyst wall vary between species and depend upon the com-  position of the media used during encystation.   The double cyst wall represents a permeability barrier  for biocides (e.g. chlorhexidine, PHMB, diamidines). In  addition, the metabolically-dormant nature of the cyst  might affect the cysticidal activity of biocides. It is thus  not surprising that cysts represent a challenge for disin-  fection (Table 20.6). This is now of particular concern    following a report that protozoal cysts can protect intra-  cellular bacterial pathogens from disinfection.   Fungi and their spores are a major potential source of  contamination in pharmaceutical product preparation  and aseptic processing as they are ubiquitous in the envi-  ronment. The activity of biocides against fungi has not  been widely documented (Table 20.6). It is often assumed  that the interactions of biocides with fungal and yeast  cells can be extrapolated from what is known of the inter-  actions of these agents against bacteria. However, the  fungal cell wall is fundamentally different from that of  the bacteria and little is known about its capacity to  impede the penetration of biocides. The interactions of  QACs and biguanides with the fungal cell have been  studied to some extent. Available information tentatively  links cell wall glucan, wall thickness and consequent rela-  tive porosity to the sensitivity of Saccharomyces cerevisiae  to chlorhexidine. Moulds tend to be less susceptible to  biocides than yeasts, although more evidence is needed  since only a limited number of fungal genera have been  investigated.   Fungi have been shown to possess additional mecha-  nisms of resistance to biocide intervention. The expres-  sion of degradative enzymes notably against metallic salts  (e.g. copper, mercury) has been documented. In S. cerevi-  siae the production of hydrogen sulphide combining  with heavy metal results in insoluble sulphides which are  better tolerated by the microorganism. The expression of  formaldehyde dehydrogenase to decrease the effect of  formaldehyde has also been reported in Penicillium  species. The presence of efflux pumps has now been  widely reported in fungi, although their role in biocide  resistance has been little investigated.   Prions are the cause of transmissible spongiform enceph-  alopathies, a group of fatal neurological diseases such as  scrapie, Creutzfeld-Jakob disease (CJD), new variant  Creutzfeld-Jakob disease (vCJD), bovine spongiform  encephalopathy (BSE), kuru and Gerstman-Straussler-  Sckeinker syndrome (GSS). It is now widely accepted that  prions are an abnormal, protease-resistant form of a  normal harmless host protein (PrP). The prion protein  undergoes a conformational change from four a-helices   Bacillus used as biological indicators for  sterilization processes due to their high  intrinsic resistance   Environmental mycobacteria, M. chelonae and  M. massiliense might show capacity to  develop resistance to repeated microbicide  exposure, and might become a challenge for  high-level disinfection   Might harbour pathogenic bacteria and thus  aid their survival when exposed to  microbicides   There is very little information on  microbicide activity against fungal spores   Pseudomonas spp. and Burkholderia spp. are  particularly challenging for preservative  systems; pathogenic E. coli (e.g. 0157)  associated with surface contamination often  embedded within organic matter   Yeasts usually considered more susceptible  than moulds. Overall very little information  available on yeast susceptibility to  microbicides   Less complex cell walls than Gram-negative  bacteria, generally allowing easier biocide  access to target sites    a Listed in order of resistance to biocides from high to low.    to an infectious (3-sheet form. These conformational  changes cause degeneration of nervous tissue which,  under the microscope, exhibits a sponge-like appearance.  Prions are found in association with a wide range of  tissues and are of particular significance in a pharmaceu-  tical context because of the need to decontaminate surgi-  cal or other hospital equipment that has been in contact  with such diseased tissue. Prions are very stable in the  environment, may form aggregates, and are highly  resistant to conventional disinfection and sterilization  methodologies.   Prions are considered highly resistant to various  types of biocidal products (formulations) (Table 20.6);  this includes strong acids (e.g. 8M hydrochloric acid  for 1 hour), alkylating agents (e.g. glutaraldehyde, (3-  propiolactone), iodine and iodophors, phenolics, alco-  hols, oxidizing agents in their liquid form (e.g. hydrogen  peroxide, peracetic acid) and proteolytic enzymes. Mild  detergents were also reported inactive although sodium  dodecyl sulphate (SDS) has shown some activity.   Alkali (e.g. lMNaOH for 1 hour) is usually effective  against prions and as such has been widely used in the  laboratory, industrial and clinical environments. However,  alkali efficacy might depend on the prion’s nature (host),  and residual prion infectivity following treatment has  been documented. More aggressive treatments combin-  ing alkali and gravity-displacement autoclaving at 121 °C  for 30 minutes have been used. The use of sodium  hypochlorite containing 20 000 ppm of available chlorine  for 1 hour has been recommended for use in practice.  Sodium dichloroisocyanurate (NaDCC) might not be  equally effective against some prion proteins, as some  infectivity following treatment has been reported.  Hydrogen peroxide in a gaseous form has been shown to  be active against prions, although reported efficacy to  date depends much on the type of vapourized hydrogen  peroxide generator used.   Formulations play an important role in prion decon-  tamination. Complex formulations containing liquid  hydrogen peroxide and copper have been shown to be  active against prions. The combination of alkali, chelating  agents, surfactants and various buffers have been particu-  larly effective as they combined prion removal from sur-  faces and prion inactivation. A combination of proteinase  K, pronase and SDS has been shown to degrade PrP res  material from highly concentrated vCJD-infected brain  preparations. Slight changes in formulations might bring  a loss of efficacy against prions, however, and new for-  mulations need to be carefully assessed for antiprion  activity.   Biocides are valuable compounds used in a wide range of  applications. They exercise a variety of mechanisms of  action, often in multiplicity, which can lead to their suc-  cessful and widespread application in preservation and  disinfection. It should also be noted that biocides are used  in complex formulations where excipients might affect  microbicidal efficacy. There is a renewed interest in  biocide use with the rising impact of antibiotic resistance  in bacteria and the perceived decline in hygiene standards  in healthcare facilities. The number of products com-  mercially available containing a biocide is increasing,  which is a concern when the concentration of a biocide  is sub-optimal. Bacterial resistance to biocides has now  been widely described, with novel mechanisms, such as  alterations in a metabolic pathway, still emerging. The use  of genomic, proteomic, transcriptomic and metabolomic  tools has helped secure a better understanding of the  mechanisms involved, but also, and importantly, a better  understanding of the induction of resistance as a result  of biocide exposure. Resistant bacteria can be readily iso-  lated from clinical settings. The efficacy of a biocidal  product can be hampered by a number of external factors  such as level of soiling, type of surface, temperature and  contact time, not forgetting the nature of the target  microorganism. The lack of understanding of these  factors combined with inappropriate in-use concentra-  tions of a biocide in certain applications (e.g. antimicro-  bial surfaces, impregnated textiles) might be conducive  not only to the selection of emerging microbial resistance  to biocides but also to antibiotics. The concentration of  a microbicide is paramount for its lethal activity.   Chapman, J.S., Diehl, M.A. & Fearnside, K.B. (1998) Preservative  tolerance and resistance. Int J Cosmetics , 20, 31-39.   Denyer, S.P. & Hugo, W.B. (1991) Mechanisms of Action of  Chemical Biocides: their Study and Exploitation. Society for  Applied Bacteriology Technical Series No. 27. Blackwell  Scientific, Oxford.   Denyer, S.P. & Stewart, G.S.A.B (1998) Mechanisms of action of  disinfectants. Int Biodet Biodegrad, 41, 261-268  Duarte, R.S., Lourenco, M.C.S., Fonseca, L.D. et al. (2009)  Epidemic of postsurgical infections caused by Mycobacterium  massiliense. J Clin Microbiol, 47, 2149—2155.   Fraise, A., Lambert, P. & Maillard, J-Y. (2004) Russell,  Hugo & Ayliffe’s Principles and Practice of Disinfection,   Maillard, J-Y. (2007) Bacterial resistance to biocides in the  healthcare environment: shall we be concerned? / Hosp Infect,  65 (suppl 2), 60-72.   Maillard, J-Y. (2011) Innate resistance to sporicides and poten-  tial failure to decontaminate. J Hosp Infect, 77, 21-24.   Maillard, J-Y. & Denyer, S.P. (2009) Emerging bacterial resistance  following biocide exposure: should we be concerned? Chem  Oggi, 27, 26-28.   McDonnell, G.E. (2007) Antisepsis, Disinfection and Sterilization:  Types, Action and Resistance. ASM Press, Washington, DC.    Piddock, L.J.V. (2006) Multidrug resistance efflux pumps — not  just for resistance. Nat Rev Microbiol, 4, 629-636.   Webber, M.A., Coldham, N.G., Woodward, M.J. & Piddock,  L.J.V. (2008) Proteomic analysis of triclosan resistance in  Salmonella enterica serovar Typhimurium. / Antimicrob  Chemother, 62, 92-97.   Weber, D.J., Rutala, W.A. & Sickbert- Bennett, E.E. (2007)  Outbreaks associated with contaminated antiseptics and dis-  infectants. Antimicrob Agents Chemother, 51, 4217-4224.   Stephen P. Denyer 1 , Norman Hodges 2 and Catherine Talbot 1   Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Hodges, Sean P. Gorman,  Brendan F. Gilmore.   Sterilization is an essential stage in the processing of any  product destined for parenteral administration, or for  contact with broken skin, mucosal surfaces, or internal  organs, where the threat of infection exists. In addition,  the sterilization of microbiological materials, soiled  dressings and other contaminated items is necessary to  minimize the health hazard associated with these  articles.   Sterilization processes involve the application of a bio-  cidal agent or physical microbial removal process to a  product or preparation with the object of killing or  removing all microorganisms. These processes may  involve elevated temperature, reactive gas, irradiation or  filtration through a microorganism-proof filter. The  success of the process depends on a suitable choice  of treatment conditions, e.g. temperature and duration  of exposure. It must be remembered, however, that with  all articles to be sterilized there is a potential risk  of product damage, which for a pharmaceutical prepara-  tion may result in reduced therapeutic efficacy, stability  or patient acceptability. Thus, there is a need to achieve  a balance between the maximum acceptable risk of  failing to achieve sterility and the maximum level of  product damage that is acceptable. This is best deter-  mined from a knowledge of the properties of the steriliz-  ing agent, the properties of the product to be sterilized  and the nature of the likely contaminants. A suitable  sterilization process may then be selected to ensure  maximum microbial kill/removal with minimum product  deterioration.   The general pattern of resistance of microorganisms to  biocidal sterilization processes is independent of the type  of agent employed (heat, radiation or gas), with vegeta-  tive forms of bacteria and fungi, along with the larger  viruses, showing a greater sensitivity to sterilization proc-  esses than small viruses and bacterial or fungal spores.  The choice of suitable reference organisms for testing the  efficiency of sterilization processes (see section 12.3) is  therefore made from the most durable bacterial spores;  these are usually represented by Bacillus stearother-  mophilus for moist heat, certain strains of B. subtilis for  dry heat and gaseous sterilization, and B. pumilus for  ionizing radiation.    Ideally, when considering the level of treatment neces-  sary to achieve sterility a knowledge of the type and total  number of microorganisms present in a product, together  with their likely response to the proposed treatment, is  necessary. Without this information, however, it is usually  assumed that organisms within the load are no more  resistant than the reference spores or than specific resist-  ant product isolates. In the latter case, it must be remem-  bered that resistance may be altered or lost entirely by  repeated laboratory subculture and the resistance charac-  teristics of the maintained strain must be regularly  checked.   A sterilization process may thus be developed without  a full microbiological background to the product, instead  being based on the ability to deal with a ‘worst case’ con-  dition. This is indeed the situation for official sterilization  methods, which must be capable of general application,  and modern pharmacopoeial recommendations are  derived from a careful analysis of experimental data on  bacterial spore survival following treatments with heat,  ionizing radiation or gas.   However, the infectious agents responsible for  spongiform encephalopathies (prions, see Chapters 5  and 20) such as bovine spongiform encephalopathy  (BSE) and Creutzfeldt-Jakob disease (CJD) exhibit  exceptional degrees of resistance to many lethal agents.  Recent work has even cast doubt on the adequacy of the  process of 18 minute exposure to steam at 134-138 °C  which has been recommended for the destruction of  prions (and which far exceeds the lethal treatment  required to achieve adequate destruction of bacterial  spores).   When exposed to a killing process, populations of micro-  organisms generally lose their viability in an exponential  fashion, independent of the initial number of organisms.  This can be represented graphically with a ‘survivor  curve’ drawn from a plot of the logarithm of the fraction  of survivors against the exposure time or dose (Figure  21.1). Of the typical curves obtained, all have a linear  portion which may be continuous (plot A), or may be  modified by an initial shoulder (B) or by a reduced rate  of kill at low survivor levels (C). Furthermore, a short  activation phase, representing an initial increase in viable  count, may be seen during the heat treatment of certain  bacterial spores. Survivor curves have been employed  principally in the examination of heat sterilization  methods, but can equally well be applied to any biocidal  process.   Figure 21.1 Typical survivor curves for bacterial spores  exposed to moist heat or gamma radiation.   The resistance of an organism to a sterilizing agent can  be described by means of the D-value. For heat and radia-  tion treatments, respectively, this is defined as the time  taken at a fixed temperature or the radiation dose required  to achieve a 90% reduction in viable cells (i.e. a 1 log cycle  reduction in survivors; Figure 21.2A). The calculation of  the D-value assumes a linear type A survivor curve  (Figure 21.1), and must be corrected to allow for any  deviation from linearity with type B or C curves. Some  typical D-values for resistant bacterial spores are given in  Table 21.1.   For heat treatment, a D-value only refers to the resistance  of a microorganism at a particular temperature. In  order to assess the influence of temperature changes on  thermal resistance, a relationship between temperature  and log D-value can be developed, leading to the expres-  sion of a Z-value, which represents the increase in tem-  perature needed to reduce the D-value of an organism by  90% (i.e. 1 log cycle reduction; Figure 21. 2B). For bacte-  rial spores used as biological indicators for moist heat  (B. stearothermophilus) and dry heat (B. subtilis) steriliza-  tion processes, mean Z- values are given as 10 °C and  22 °C, respectively. The Z-value is not truly independent  of temperature but may be considered essentially con-  stant over the temperature ranges used in heat steriliza-  tion processes.   The term ‘sterile’, in a microbiological context, means no  surviving organisms whatsoever. Thus, there are no  degrees of sterility; an item is either sterile or it is not,  and so there are no levels of contamination which  may be considered negligible or insignificant and there-  fore acceptable. From the survivor curves presented, it  can be seen that the elimination of viable microorgan-  isms from a product is a time-dependent process, and  will be influenced by the rate and duration of biocidal  action and the initial microbial contamination level. It is  also evident from Figure 21.2A that true sterility, repre-  sented by zero survivors, can only be achieved after an  infinite exposure period or radiation dose. Clearly, then,  it is illogical to claim, or expect, that a sterilization  procedure will guarantee sterility. Thus, the likelihood of  a product being produced free of microorganisms is  best expressed in terms of the probability of an organism  surviving the treatment process, a possibility not enter-  tained in the absolute term ‘sterile’. From this approach  has arisen the concept of sterility assurance or a microbial  safety index which gives a numerical value to the prob-  ability of a single surviving organism remaining to  contaminate a processed product. For pharmaceutical  products, the most frequently applied standard is that  the probability, poststerilization, of a non-sterile unit is  no more than 1 in 1 million units processed (i.e. <1(T 6 ).  The sterilization protocol necessary to achieve this  with any given organism of known D-value can be estab-  lished from the inactivation factor (IF) which may be  defined as:    Table 21.1 Inactivation factors (IF) for selected sterilization protocols and their corresponding biological indicator (BI)  organisms    where t is the contact time (for a heat or gaseous steriliza-  tion process) or dose (for ionizing radiation) and D is the  D-value appropriate to the process employed.   Thus, for an initial burden of 10 2 spores an inactiva-  tion factor of 10 8 will be needed to give the required  sterility assurance of 1(T 6 (Figure 21.3). The sterilization  process will therefore need to produce sufficient lethality  to achieve an 8 log cycle reduction in viable organisms;  this will require exposure of the product to eight times  the D-value of the reference organism (8D). In practice,  it is generally assumed that the contaminant will have the  same resistance as the relevant biological indicator spores  unless full microbiological data are available to indicate  otherwise. The inactivation factors associated with  certain sterilization protocols and their biological indica-  tor organisms are given in Table 21.1.   A sterilization process should always be considered a  compromise between achieving good antimicrobial    activity and maintaining product stability. It must, there-  fore, be validated against a suitable test organism and its  efficacy continually monitored during use. Even so, a  limit will exist as to the type and size of microbial chal-  lenge that can be handled by the process without signifi-  cant loss of sterility assurance. Thus, sterilization must  not be seen as a ‘catch-all’ or as an alternative to Good  Manufacturing Practice but must be considered as only  the final stage in a programme of microbiological control.  The European Pharmacopoeia recognizes five methods for  the sterilization of pharmaceutical products: (1) steam  sterilization (heating in an autoclave); (2) dry heat; (3)  ionizing radiation; (4) gaseous sterilization; and (5) fil-  tration. In addition, other approaches involving steam  and formaldehyde and UV light have evolved for use in  certain situations. For each method, the possible permu-  tations of exposure conditions are numerous, but experi-  ence and product stability requirements have generally  served to limit this choice. Nevertheless, it should be  remembered that even the recommended methods  and regimens do not necessarily demonstrate equivalent  biocidal potential (see Table 21.1), but simply offer   Figure 21.3 Sterility assurance. At Y, there is (literally) 10 -1  bacterium in one bottle, i.e. in 10 loads of single containers,  there would be one chance in 10 that one load would be  positive. Likewise, at Z, there is (literally) 1 0 fl bacterium in  one bottle, i.e. in 1 million ( 10 s ) loads of single containers,  there is one chance in 1 million that one load would be  positive.    alternative strategies for application to a wide variety of  product types. Thus, each should be validated in their  application to demonstrate that the minimum required  level of sterility assurance can be achieved (sections 2.3  and 9).   In the following sections, factors governing the suc-  cessful use of these sterilizing methods will be covered  and their application to pharmaceutical and medical  products considered. Methods for monitoring the effi-  cacy of these processes are discussed in section 12.   Heat is the most reliable and widely used means of steri-  lization, affording its antimicrobial activity through  destruction of enzymes and other essential cell constitu-  ents. These lethal events proceed most rapidly in a fully  hydrated state, thus requiring a lower heat input (tem-    perature and time) under conditions of high humidity  where denaturation and hydrolysis reactions predomi-  nate, rather than in the dry state where oxidative changes  take place. This method of sterilization is limited to ther-  mostable products, but can be applied to both moisture-  sensitive and moisture-resistant items for which dry  (160-180°C) and moist (121-134 °C) heat sterilization  procedures are respectively used. Where thermal degra-  dation of a product might possibly occur, it can usually  ~ be minimized by selecting the higher temperature range,   In any heat sterilization process, the articles to be treated  must first be raised to sterilization temperature and this  involves a heating-up stage. In the traditional approach,  timing for the process (the holding time) then begins. It  has been recognized, however, that during both the  heating-up and cooling-down stages of a sterilization  cycle (Figure 21.4), the product is held at an elevated  temperature and these stages may thus contribute to the  overall biocidal potential of the process.   A method has been devised to convert all the  temperature-time combinations occurring during the  heating, sterilizing and cooling stages of a moist heat  (steam) sterilization cycle to the equivalent time at 121 °C.  This involves following the temperature profile of a load,  integrating the heat input (as a measure of lethality), and  converting it to the equivalent time at the standard tem-  perature of 121 °C. Using this approach, the overall  lethality of any process can be deduced and is defined as  the F- value; this expresses heat treatment at any tempera-  ture as equal to that of a certain number of minutes at  1 2 1 °C. In other words, if a moist heat sterilization process  has an T-value of x, then it has the same lethal effect on  a given organism as heating at 121 °C for x minutes, irre-  spective of the actual temperature employed or of any  fluctuations in the heating process due to heating and  cooling stages. The F-value of a process will vary accord-  ing to the moist heat resistance of the reference organism;  when the reference spore is that of B. stearothermophilus  with a Z-value of 10 °C, then the T-value is known as the  T 0 -value.   A relationship between F- and D-values, leading to an  assessment of the probable number of survivors in a load  following heat treatment, can be established from the  following equation:   where D is the D-value at 121 °C, and N 0 and N represent,  respectively, the initial and final number of viable cells  per unit volume.   The F-concept has evolved from the food industry and  principally relates to the sterilization of articles by moist  heat. Because it permits calculation of the extent to which  the heating and cooling phases contribute to the overall  killing effect of the autoclaving cycle, the F-concept  enables a sterilization process to be individually devel-  oped for a particular product. This means that adequate  sterility assurance can be achieved in autoclaving cycles  in which the traditional pharmacopoeial recommenda-  tion of 15 minutes at 121 °C is not achieved. The holding  time may be reduced below 15 minutes if there is a sub-  stantial killing effect during the heating and cooling  phases, and an adequate cycle can be achieved even if the  ‘target’ temperature of 121 °C is not reached. Thus, F-  values offer both a means by which alternative sterilizing  cycles can be compared in terms of their microbial  killing efficiency, and a mechanism by which overprocess-  ing of marginally thermolabile products can be reduced  without compromising sterility assurance. The European  Pharmacopoeia emphasizes that when a steam steriliza-  tion cycle is designed on the basis of F„ data, it may be  necessary to perform continuous and rigorous microbio-  logical monitoring of the bioburden (section 10) during  routine manufacturing in order consistently to achieve an  acceptable sterility assurance level.   F 0 values may be calculated either from the area under  the curve of a plot of autoclave temperature against time  constructed using special chart paper on which the tem-  perature scale is modified to take into account the pro-  gressively greater lethality of higher temperatures, or by  use of the equation:    where At is the time interval between temperature meas-  urements, T is the product temperature at time t, and Z  is (assumed to be) 10 °C.   Thus, if temperatures were being recorded from a ther-  mocouple at 1.00 minute intervals then At = 1.00, and a  temperature of, for example, 115°C maintained for 1  minute would give an F 0 value of 1 minute x io (115-121>/10 ,  which is equal to 0.25 minutes. In practice, such calcula-  tions could easily be performed on the data from  several thermocouples within an autoclave using suitable  software, and, in a manufacturing situation, these  would be part of the batch records. Application of the  F-value concept has been largely restricted to steam steri-  lization processes, although there is a less frequently  employed, but direct parallel in dry heat sterilization (see  section 4.3).   Moist heat has been recognized as an efficient biocidal  agent from the early days of bacteriology, when it was  principally developed for the sterilization of culture  media. It now finds widespread application in the process-  ing of many thermostable products and devices. In the  pharmaceutical and medical sphere it is used in the steri-  lization of dressings, sheets, surgical and diagnostic  equipment, containers and closures, and aqueous injec-  tions, ophthalmic preparations and irrigation fluids, in  addition to the processing of soiled and contaminated  items (Chapter 22).   Sterilization by moist heat usually involves the use of  steam at temperatures in the range 121-134 °C, and while  alternative strategies are available for the processing of  products unstable at these high temperatures, they rarely  offer the same degree of sterility assurance and should be  avoided if at all possible. The elevated temperatures gen-  erally associated with moist heat sterilization methods   can only be achieved by the generation of steam under  pressure.   By far the most commonly employed standard  temperature/time cycles for bottled fluids and porous  loads (e.g. surgical dressings) are 121 °C for 15 minutes  and 134°C for 3 minutes, respectively. Not only do high-  temperature-short time cycles often result in lower frac-  tional degradation, they also afford the advantage of  achieving higher levels of sterility assurance due to greater  inactivation factors (Table 21.2). Before the publication  of the 1988 British Pharmacopoeia the 115°C for 30  minute cycle was considered an acceptable alternative to  121 °C for 15 minutes, but it is no longer considered suf-  ficient to give the desired sterility assurance levels for  products which may contain significant concentrations  of thermophilic spores.   To act as an efficient sterilizing agent, steam should be  able to provide moisture and heat efficiently to the article  to be sterilized. This is most effectively done using satu-  rated steam, which is steam in thermal equilibrium with  the water from which it is derived, i.e. steam on the phase  boundary (Figure 21.5). Under these circumstances,  contact with a cooler surface causes condensation and  contraction, drawing in fresh steam and leading to the  immediate release of the latent heat, which represents  approximately 80% of the total heat energy. In this way,  heat and moisture are imparted rapidly to articles being  sterilized and dry porous loads are quickly penetrated by  the steam.   Steam for sterilization can either be generated within  the sterilizer, as with portable bench or ‘instrument and  utensil’ sterilizers, in which case it is constantly in contact   with water and is known as ‘wet’ steam, or can be supplied  under pressure (350-400 kPa) from a separate boiler as  ‘dry’ saturated steam with no entrained water droplets.  The killing potential of ‘wet’ steam is the same as that of  ‘dry’ saturated steam at the same temperature, but it is  more likely to soak a porous load, creating physical dif-  ficulties for further steam penetration. Thus, major  industrial and hospital sterilizers are usually supplied  with ‘dry’ saturated steam and attention is paid to the  removal of entrained water droplets within the supply  line to prevent introduction of a water ‘fog’ into the  sterilizer.    If the temperature of ‘dry’ saturated steam is increased,  then, in the absence of entrained moisture, the relative  humidity or degree of saturation is reduced and the  steam becomes superheated (Figure 21.5). During steri-  lization this can arise in a number of ways, for example  by overheating the steam-jacket (see section 4.2.2), by  using too dry a steam supply, by excessive pressure reduc-  tion during passage of steam from the boiler to the steri-  lizer chamber, and by evolution of heat of hydration  when steaming overdried cotton fabrics. Superheated  steam behaves in the same manner as hot air as condensa-  tion and release of latent heat will not occur unless the  steam is cooled to the phase boundary temperature.  Thus, it proves to be an inefficient sterilizing agent and,  although a small degree of transient superheating can be  tolerated, a maximum acceptable level of 5 °C superheat  is set, i.e. the temperature of the steam is never greater  than 5 °C above the phase boundary temperature at that  pressure.   The relationship between temperature and pressure  holds true only in the presence of pure steam; adultera-  tion with air contributes to a partial pressure but not to  the temperature of the steam. Thus, in the presence of air  the temperature achieved will reflect the contribution  made by the steam and will be lower than that normally  attributed to the total pressure recorded. Addition of  further steam will raise the temperature but residual air  surrounding articles may delay heat penetration or, if a  large amount of air is present, it may collect at the bottom  of the sterilizer, completely altering the temperature  profile of the sterilizer chamber. It is for these reasons that  efficient air removal is a major aim in the design and  operation of a boiler-fed steam sterilizer.   Steam sterilizers, or autoclaves as they are also known, are  stainless steel vessels designed to withstand the steam  pressures employed in sterilization. They can be: ‘porta-  ble’ sterilizers, which generally have internal electric  heaters to produce steam and are used for small pilot or  laboratory-scale sterilization and for the treatment of  instruments and utensils; or large-scale sterilizers for  routine hospital or industrial use, operating on ‘dry’ satu-  rated steam from a separate boiler (Figure 21.6). Because  of their widespread use within pharmacy this latter type  will be considered in greatest detail.   There are two main types of large sterilizers, those  designed for use with porous loads (i.e. dressings) and  generally operated at a minimum temperature of 134 °C,  and those designed as bottled fluid sterilizers employing    a minimum temperature of 121 °C. The stages of opera-  tion are common to both and can be summarized as air  removal and steam admission, heating-up and exposure,  and drying or cooling. Many modifications of design  exist and in this section only general features will be  considered. Fuller treatments of sterilizer design and  operation can be found in the relevant Department of  Health technical memorandums (DH 1995, DH 1997).   Steam sterilizers are constructed with either cylindrical  or rectangular chambers, with preferred capacities  ranging from 400 to 800 L. They can be sealed by either  a single door or by doors at both ends (to allow through-  passage of processed materials; see Chapter 23). During  sterilization the doors are held closed by a locking mecha-  nism which prevents opening when the chamber is under  pressure and until the chamber has cooled to a preset  temperature, typically 80 °C.   In the larger sterilizers the chamber maybe surrounded  by a steam jacket which can be used to heat the autoclave  chamber and promote a more uniform temperature  throughout the load. The same jacket can also be filled  with water at the end of the cycle to facilitate cooling and  thus reduce the overall cycle time. The chamber floor  slopes towards a discharge channel through which air and  condensate can be removed. Temperature is monitored  within the opening of the discharge channel and by ther-  mocouples in dummy packages; jacket and chamber  pressures are followed using pressure gauges. In hospitals  and industry, it is common practice to operate sterilizers  on an automatic cycle, each stage of operation being con-  trolled by a timer responding to temperature- or pressure-  sensing devices.   1 Air removal and steam admission. Air can be removed  from steam sterilizers either by downward displacement  with steam, evacuation or a combination of the two. In  the downward displacement sterilizer, the heavier cool air  is forced out of the discharge channel by incoming hot  steam. This has the benefit of warming the load during  air removal, which aids the heating-up process. It finds  widest application in the sterilization of bottled fluids  where bottle breakage may occur under the combined  stresses of evacuation and high temperature. For more  air-retentive loads (i.e. dressings), however, this tech-  nique of air removal is unsatisfactory and mechanical  evacuation of the air is essential before admission of the  steam. This can either be to an extremely high level (e.g.  2.5 kPa) or can involve a period of pulsed evacuation and   Figure 21.6 Main constructional features of a large-scale steam sterilizer (autoclave).    steam admission, the latter approach improving air  extraction from dressings packs. After evacuation, steam  penetration into the load is very rapid and heating-up is  almost instantaneous. It is axiomatic that packaging and  loading of articles within a sterilizer be so organized as  to facilitate air removal.   During the sterilization process, small pockets of  entrained air may still be released, especially from pack-  ages, and this air must be removed. This is achieved in a  porous load autoclave with a near-to-steam thermostatic  valve incorporated in the discharge channel. The valve  operates on the principle of an expandable bellows con-  taining a volatile liquid which vaporizes at the tempera-  ture of saturated steam thereby closing the valve, and  condenses on the passage of a cooler air-steam mixture,  thus reopening the valve and discharging the air.  Condensate generated during the sterilization process  can also be removed by this device. Small quantities of  air will not, however, lower the temperature sufficiently  to operate the valve and so a continual slight flow of    steam is maintained through a bypass around the device  in order to flush away residual air.   It is common practice to package sterile fluids, especially  intravenous fluids, in flexible plastic containers. During  sterilization these can develop a considerable internal  pressure in the airspace above the fluid and it is therefore  necessary to maintain a proportion of air within the steri-  lizing chamber to produce sufficient overpressure to  prevent these containers from bursting (air ballasting). In  sterilizers modified or designed to process this type of  product, air removal is therefore unnecessary but special  attention must be paid to the prevention of air ‘layering’  within the chamber. This is overcome by the inclusion of  a fan or through a continuous spray of hot water within  the chamber to mix the air and steam. Air ballasting can  also be employed to prevent bottle breakage.   2 Heating-up and exposure. When the sterilizer reaches  its operating temperature and pressure the sterilization  stage begins. The duration of exposure may include a  heating-up time in addition to the holding time and this   3 Drying or cooling. Dressings packs and other porous  loads may become dampened during the sterilization  process and must be dried before removal from the  chamber. This is achieved by steam exhaust and applica-  tion of a vacuum, often assisted by heat from the steam-  filled jacket if fitted. After drying, atmospheric pressure  within the chamber is restored by admission of sterile  filtered air.   For bottled fluids the final stage of the sterilization  process is cooling, and this needs to be achieved as rapidly  as possible to minimize thermal degradation of the  product and to reduce processing time. In modem  sterilizers, this is achieved by circulating water in the  jacket that surrounds the chamber or by spray-cooling  with retained condensate delivered to the surface of  the load by nozzles fitted into the roof of the sterilizer  chamber. This is often accompanied by the introduction  of filtered, compressed air to minimize container  breakage due to high internal pressures (air ballasting).  Containers must not be removed from the sterilizer until  the internal pressure has dropped to a safe level, usually  indicated by a temperature of less than 80 °C. Occasionally,  spray-cooling water maybe a source of bacterial contami-  nation and its microbiological quality must be carefully  monitored.   The lethal effects of dry heat on microorganisms are due  largely to oxidative processes, which are less effective than  the hydrolytic damage which results from exposure to  steam. Thus, dry heat sterilization usually employs higher  temperatures in the range 160-180 °C and requires expo-  sure times of up to 2 hours depending on the temperature  employed.   Again, bacterial spores are much more resistant than  vegetative cells and their recorded resistance varies mark-  edly depending on their degree of dryness. In many early  studies on dry heat resistance of spores their water  content was not adequately controlled, so conflicting data  arose regarding the exposure conditions necessary to  achieve effective sterilization. This was partly responsible  for variations in recommended exposure temperatures  and times in different pharmacopoeias.   Dry heat application is generally restricted to glassware  and metal surgical instruments (where its good penetra-  bility and non-corrosive nature are of benefit), non-  aqueous thermostable liquids and thermostable powders  (see Chapter 22). In practice, the range of materials that    are actually subjected to dry heat sterilization is quite  limited, and consists largely of items used in hospitals.  The major industrial application is in the sterilization of  glass bottles which are to be filled aseptically, and here  the attraction of the process is that it not only achieves  an adequate sterility assurance level, but that it may also  destroy bacterial endotoxins (products of Gram-negative  bacteria, also known as pyrogens, that cause fever when  injected into the body). These are difficult to eliminate  by other means. For the purposes of depyrogenation of  glass, temperatures of approximately 250 °C are used.   The F-value concept that was developed for steam  sterilization processes has an equivalent in dry heat steri-  lization although its application has been limited. The F H  designation describes the lethality of a dry heat process  in terms of the equivalent number of minutes exposure  at 170°C, and in this case a Z - value of 20 °C has been  found empirically to be appropriate for calculation pur-  poses; this contrasts with the value of 10 °C which is  typically employed to describe moist heat resistance.   Dry heat sterilization is usually carried out in a hot-air  oven which comprises an insulated polished stainless  steel chamber, with a usual capacity of up to 250 L, sur-  rounded by an outer case containing electric heaters  located in positions to prevent cool spots developing  inside the chamber. A fan is fitted to the rear of the oven  to provide circulating air, thus ensuring more rapid equi-  libration of temperature. Shelves within the chamber are  perforated to allow good airflow. Thermocouples can be  used to monitor the temperature of both the oven air and  articles contained within. A fixed temperature sensor  connected to a chart or digital recorder provides a per-  manent record of the sterilization cycle. Appropriate  door-locking controls should be incorporated to prevent  interruption of a sterilization cycle once begun.   Recent sterilizer developments have led to the use of  dry heat sterilizing tunnels where heat transfer is achieved  by infrared irradiation or by forced convection in filtered  laminar airflow tunnels. Items to be sterilized are placed  on a conveyer belt and pass through a high-temperature  zone (250-300+ °C) over a period of several minutes.   Articles to be sterilized must be wrapped or enclosed  in containers of sufficient strength and integrity to  provide good poststerilization protection against con-  tamination. Suitable materials are paper, cardboard tubes  or aluminium containers. Container shape and design    must be such that heat penetration is encouraged in order  to shorten the heating-up stage; this can be achieved by  using narrow containers with dull, non-reflecting sur-  faces. In a hot-air oven, heat is delivered to articles prin-  cipally by radiation and convection; thus, they must be  carefully arranged within the chamber to avoid obscuring  centrally placed articles from wall radiation or impending  air flow. The temperature variation within the chamber  should not exceed ±5°C of the recorded temperature.  Heating-up times, which may be as long as 4 hours for  articles with poor heat-conducting properties, can be  reduced by preheating the oven before loading. Following  sterilization, the chamber temperature is usually allowed  to fall to around 40 °C before removal of sterilized arti-  cles; this can be accelerated by the use of forced cooling  with filtered air.   The chemically reactive gases ethylene oxide [(CH 2 ) 2 0]  and formaldehyde [(methanal, H.CHO)] possess broad-  spectrum biocidal activity, and have found application in  the sterilization of reusable surgical instruments, certain  medical, diagnostic and electrical equipment, and the  surface sterilization of powders. Sterilization processes  using ethylene oxide sterilization are far more commonly  used on an international basis than those employing  formaldehyde.   Ethylene oxide treatment can also be considered as an  alternative to radiation sterilization in the commercial  production of disposable medical devices (Chapter 22).  These techniques do not, however, offer the same degree  of sterility assurance as heat methods and are generally  reserved for temperature-sensitive items.   The mechanism of antimicrobial action of the two  gases is assumed to be through alkylation of sulphydryl,  amino, hydroxyl and carboxyl groups on proteins and  imino groups of nucleic acids. At the concentrations  employed in sterilization protocols, type A survivor  curves (section 2.1, Figure 21.1) are produced, the  lethality of these gases increasing in a non-uniform  manner with increasing concentration, exposure tem-  perature and humidity. For this reason, sterilization pro-  tocols have generally been established by an empirical  approach using a standard product load containing suit-  able biological indicator test strips (section 12.3).  Concentration ranges (given as weight of gas per unit  chamber volume) are usually of the order of 800-  1200 mg/L for ethylene oxide and 15-100 mg/L for for-    maldehyde, with operating temperatures in the region of  45-63 °C and 70-75 °C, respectively. Even at the higher  concentrations and temperatures, the sterilization proc-  esses are lengthy and therefore unsuitable for the resteri-  lization of high-turnover articles. Further delays occur  because of the need to remove toxic residues of the gases  before release of the items for use. In addition, because  recovery of survivors in sterility tests is more protracted  with gaseous sterilization methods than with other proc-  esses, an extended quarantine period may also be required.   As alkylating agents, both gases are potentially muta-  genic and carcinogenic (as is the ethylene chlorohydrin  that results from ethylene oxide reaction with chlorine);  they also produce symptoms of acute toxicity including  irritation of the skin, conjunctiva and nasal mucosa.  Consequently, strict control of their atmospheric concen-  trations is necessary and safe working protocols are  required to protect personnel. Table 21.3 summarizes  the comparative advantages afforded by ethylene oxide  and low-temperature steam and formaldehyde (LTSF)  processes.   Relatively slow to form solid  polymers (with the potential  to block pipes, etc.)   With long exposure times it  is possible to sterilize at  ambient temperatures    Less hazardous because  formaldehyde is not  flammable and is more  readily detected by smell   The gas is obtained readily  from aqueous solution  (formalin) which is a  more convenient source  than gas in cylinders   Ethylene oxide gas is highly explosive in mixtures of  more than 3.6% v/v in air; in order to reduce this explo-  sion hazard it is usually supplied for sterilization pur-  poses as a 10% mix with carbon dioxide, or as an  8.6% mixture with HFC 124 (2-chloro-l,l,l,2 tetrafluor-  oethane), which has replaced fluorinated hydrocarbons  (freons). Alternatively, pure ethylene oxide gas can be  used below atmospheric pressure in sterilizer chambers  from which all air has been removed.   The efficacy of ethylene oxide treatment depends on  achieving a suitable concentration in each article and this  is assisted greatly by the good penetrating powers of the  gas, which diffuses readily into many packaging materials  including rubber, plastics, fabric and paper. This is not  without its drawbacks, however, as the level of ethylene  oxide in a sterilizer will decrease due to absorption during  the process and the treated articles must undergo a des-  orption stage to remove toxic residues. Desorption can be  allowed to occur naturally on open shelves, in which case  complete desorption may take many days, e.g. for materi-  als like PVC, or it may be encouraged by special forced-  aeration cabinets where flowing, heated air assists gas  removal, reducing desorption times to between 2 and 24  hours.   Organisms are more resistant to ethylene oxide treat-  ment in a dried state, as are those protected from the gas  by inclusion in crystalline or dried organic deposits.  Thus, a further condition to be satisfied in ethylene  oxide sterilization is attainment of a minimum level of  moisture in the immediate product environment. This  requires a sterilizer humidity of 30-70% and frequently  a preconditioning of the load at relative humidities of  more than 50%.   An ethylene oxide sterilizer consists of a leak-proof and  explosion-proof steel chamber, normally of 100-300 L  capacity, which can be surrounded by a hot-water jacket  to provide a uniform chamber temperature. Successful  operation of the sterilizer requires removal of air  from the chamber by evacuation, humidification and  conditioning of the load by passage of subatmospheric-  pressure steam followed by a further evacuation period  and the admission of preheated vaporized ethylene oxide  from external pressurized canisters or single-charge car-  tridges. Forced gas circulation is often employed to mini-  mize variations in conditions throughout the sterilizer  chamber. Packaging materials must be air-, steam- and  gas-permeable to permit suitable conditions for steriliza-    tion to be achieved within individual articles in the load.  Absorption of ethylene oxide by the load is compensated  for by the introduction of excess gas at the beginning or  by the addition of more gas as the pressure drops during  the sterilization process. The same may also be true for  moisture absorption, which is compensated for by sup-  plementary addition of water to maintain appropriate  relative humidity.   After treatment, the gases are evacuated either directly  to the outside atmosphere or through a special exhaust  system. Filtered, sterile air is then admitted either for a  repeat of the vacuum/air cycle or for air purging until the  chamber is opened. In this way, safe removal of the eth-  ylene oxide is achieved, reducing the toxic hazard to the  operator. Sterilized articles are removed directly from the  chamber and arranged for desorption. The operation of  an ethylene oxide sterilizer should be monitored and con-  trolled automatically. A typical operating cycle for pure  ethylene oxide gas is shown in Figure 21.7.   Formaldehyde gas for use in sterilization is produced by  heating formalin (37% w/v aqueous solution of formal-  dehyde) to a temperature of 70-75 °C with steam, leading  to the process known as LTSF. Formaldehyde has a similar  toxicity to ethylene oxide and although absorption to  materials appears to be lower, similar desorption routines  are recommended. A major disadvantage of formalde-  hyde is low penetrating power, and this limits the packag-  ing materials that can be employed to principally paper  and cotton fabric.   An LTSF sterilizer is designed to operate with  subatmospheric-pressure steam. Air is removed by evacu-  ation and steam is admitted to the chamber to allow  heating of the load and to assist in air removal. The steri-  lization period starts with the release of formaldehyde by  vaporization from formalin (in a vaporizer with a steam-  jacket) and continues through either a simple holding  stage or through a series of pulsed evacuations and  steam and formaldehyde admission cycles. The chamber  temperature is maintained by a thermostatically control-  led water jacket, and steam and condensate are removed  via a drain channel and an evacuated condenser. At the  end of the treatment period formaldehyde vapour is  expelled by steam flushing and the load is dried by alter-  nating stages of evacuation and admission of sterile, fil-  tered air. A typical pulsed cycle of operation is shown in  Figure 21.8.   Hydrogen peroxide (H 2 0 2 ), 30-35%w/v, and peracetic  acid (CH3CO3H), 3.5%w/v, are used as highly effective  oxidizing agents to kill microorganisms (Chapters 19 and  20). The liquids are heated to vaporize them and are held  in a sealed chamber where all surfaces which come into  contact with the vapour will be sterilized. These methods  are widely used for the sterilization of equipment used  for aseptic preparation and manufacture such as isola-  tors, with the vaporized hydrogen peroxide (VHP) often  used for the routine decontamination of cleanrooms.   Several types of radiation find a sterilizing application in  the manufacture of pharmaceutical and medical prod-  ucts, principal among which are accelerated electrons  (particulate radiation), gamma rays and UV light (both  electromagnetic radiations). The major target for these  radiations is believed to be microbial DNA, with damage  occurring as a consequence of ionization and free-radical  production (gamma rays and electrons) or excitation  (UV light). This latter process is less damaging and less  lethal than ionization, and so UV irradiation is not as  efficient a sterilization method as electron or gamma irra-  diation. As mentioned earlier (section 2), vegetative bac-  teria generally prove to be the most sensitive to irradiation  (with notable exceptions, e.g. Deinococcus ( Micrococcus )  radiodurans), followed by moulds and yeasts, with bacte-  rial spores and viruses as the most resistant (except in  the case of UV light, where mould spores prove to be  most resistant). The extent of DNA damage required to  produce cell death can vary and this, together with the  ability to carry out effective repair, probably determines  the resistance of the organism to radiation. With ionizing  radiations (gamma ray and accelerated electrons), micro-  bial resistance decreases with the presence of moisture or  dissolved oxygen (as a result of increased free-radical pro-  duction) and also with elevated temperatures.   Radiation sterilization with high-energy gamma rays  or accelerated electrons has proved to be a useful method  for the industrial sterilization of heat-sensitive products.  However, undesirable changes can occur in irradiated  preparations, especially those in aqueous solution where  radiolysis of water contributes to the damaging processes.  In addition, certain glass or plastic (e.g. polypropylene,  PTFE) materials used for packaging or for medical  devices can also suffer damage. Thus, radiation steriliza-  tion is generally applied to articles in the dried state; these    include surgical instruments, sutures, prostheses, unit-  dose ointments, plastic syringes and dry pharmaceutical  products (Chapter 22). With these radiations, destruction  of a microbial population follows the classic survivor  curves (see Figure 21.1) and a D-value, given as a radia-  tion dose, can be established for standard bacterial spores  (e.g. B. pumilus ) permitting a suitable sterilizing dose to  be calculated. In the UK it is usual to apply a dose of  25kGy (2.5Mrad) for pharmaceutical and medical prod-  ucts, although lower doses are employed in the USA and  Canada.   UV light, with its much lower energy, causes less  damage to microbial DNA. This, coupled with its poor  penetrability of normal packaging materials, renders UV  light unsuitable for sterilization of pharmaceutical dosage  forms. It does find applications, however, in the sterilization  of air, for the surface sterilization of aseptic work areas, and  for the treatment of manufacturing-grade water.   Gamma rays for sterilization are usually derived from a  cobalt-60 ( 60 Co) source (caesium-137 may also be used),  with a half-life of 5.25 years, which on disintegration  emits radiation at two energy levels of 1.33 and 1.17MeV.  The isotope is held as pellets packed in metal rods, each  rod carefully arranged within the source and containing  up to 20kCi (740 x 10 12 Bq) of activity; these rods are  replaced or rearranged as the activity of the source either  drops or becomes unevenly distributed. A typical 60 Co  installation may contain up to lMCi (3.7 x 10 16 Bq) of  activity. For safety reasons, this source is housed within a  reinforced concrete building with walls some 2 m thick,  and it is raised from a sunken water-filled tank only when  required for use. Control devices operate to ensure that  the source is raised only when the chamber is locked and  that it is immediately lowered if a malfunction occurs.  Articles being sterilized are passed through the irradia-  tion chamber on a conveyor belt or monorail system and  move around the raised source, the rate of passage regu-  lating the dose absorbed (Figure 21.9).   Radiation monitors are continually employed to  detect any radiation leakage during operation or source  storage, and to confirm a return to satisfactory back-  ground levels within the sterilization chamber following  operation. The dose delivered is dependent upon source  strength and exposure period, with dwell times typically  up to 20 hours. The difference in radiation susceptibilities  of microbial cells and humans may be gauged from the    fact that a lethal human dose would be delivered by an  exposure of seconds or minutes.   Two types of electron accelerator machine exist, the elec-  trostatic accelerator and the microwave linear accelerator,  producing electrons with maximum energies of 5MeV  and 10 MeV, respectively. Although higher energies would  achieve better penetration into the product, there is a risk  of induced radiation and so they are not used. In the  first, a high-energy electron beam is generated by accel-  erating electrons from a hot filament down an evacuated  tube under high potential difference, while in the  second, additional energy is imparted to this beam in a  pulsed manner by a synchronized travelling microwave.  Articles for treatment are generally limited to small  packs and are arranged on a horizontal conveyor belt,  usually for irradiation from one side but sometimes from  both. The sterilizing dose is delivered more rapidly in an  electron accelerator than in a “Co plant, with exposure  times for sterilization usually amounting to only a few  seconds or minutes. Varying extents of shielding, depend-  ing upon the size of the accelerator, are necessary to  protect operators from X-rays generated by the bremsst-  rahlung effect.   The optimum wavelength for UV sterilization is around  260 nm. A suitable source for UV light in this region is a  mercury lamp giving peak emission levels at 254 nm.  These sources are generally wall- or ceiling-mounted for  air disinfection, or fixed to vessels for water treatment.  Operators present in an irradiated room should wear  appropriate protective clothing and eye shields.   The process of filtration is unique among sterilization  techniques in that it removes, rather than destroys,  microorganisms. Further, it is capable of preventing the  passage of both viable and nonviable particles and can  thus be used for both the clarification and sterilization of  liquids and gases. The principal application of sterilizing-  grade filters is the treatment of heat-sensitive injections  and ophthalmic solutions, biological products and air  and other gases for supply to aseptic areas (see Chapters  22 and 23).   Filters may also be required in industrial applications  where they become part of venting systems on ferment-    ers, centrifuges, autoclaves and freeze-driers. Certain  types of filter (membrane filters) also have an important  role in sterility testing, where they can be employed to  trap and concentrate contaminating organisms from  solutions under test. These filters are then placed on a  solid nutrient medium or in a liquid medium and  incubated to encourage colony growth or turbidity  (section 13.1).   The major mechanisms of filtration are sieving,  adsorption and trapping within the matrix of the filter  material. Of these, only sieving can be regarded as abso-  lute as it ensures the exclusion of all particles above a  defined size. It is generally accepted that synthetic mem-  brane filters, derived from cellulose esters or other poly-  meric materials, approximate most closely to sieve filters;  while fibrous pads, sintered glass and sintered ceramic  products can be regarded as depth filters relying princi-  pally on mechanisms of adsorption and entrapment.  Some of the characteristics of filter media are summa-  rized in Table 21.4. The potential hazard of microbial  multiplication within a depth filter and subsequent  contamination of the filtrate (microbial grow-through)  should be recognized.   In order to compare favourably with other methods of  sterilization, the microorganism removal efficiency of  filters employed in the processing of liquids must be high.  For this reason, membrane filters of 0.2-0.22 pm nominal  pore diameter are chiefly used, while sintered filters are  used only in restricted circumstances, i.e. for the process-  ing of corrosive liquids, viscous fluids or organic solvents.  It may be tempting to assume that the pore size is the  major determinant of filtration efficiency and two filters  of 0.2 pm pore diameter from different manufacturers  will behave similarly. This is not so, because, in addition  to the sieving effect, trapping within the filter matrix,  adsorption and charge effects all contribute significantly  towards the removal of particles. Consequently, the depth  of the membrane, its charge and the tortuosity of the  channels are all factors which can make the performance  of one filter far superior to that of another. The major  criterion by which filters should be compared, therefore,  is their titre reduction values, i.e. the ratio of the number  of organisms challenging a filter under defined conditions  to the number penetrating it. In all cases, the filter medium  employed must be sterilizable, ideally by steam treatment;  in the case of membrane filters this may be for once-only  use, or, in the case of larger industrial filters, a small,  fixed number of resterilizations; sintered filters may be    resterilized many times. Filtration sterilization is an  aseptic process and careful monitoring of filter integrity is  necessary as well as final product sterility testing (section  13).   Membrane filters, in the form of discs, can be assem-  bled into pressure-operated filter holders for syringe  mounting and in-line use or vacuum filtration tower  devices. Filtration under pressure is generally considered  most suitable, as filling at high flow rates directly into the  final containers is possible without problems of foaming,  solvent evaporation or air leaks. To increase the filtration  area, and hence process volumes, several filter discs can  be used in parallel in multiple-plate filtration systems or,  alternatively, membrane filters can be fabricated into  plain or pleated cylinders and installed in cartridges.  Membrane filters are often used in combination with a  coarse-grade fibreglass depth prefilter to improve their  dirt-handling capacity.   The principal application for filtration sterilization of  gases is in the provision of sterile air to aseptic manufac-  turing suites, hospital isolation units and some operating  theatres. Filters employed generally consist of pleated  sheets of glass microfibres separated and supported by an  aluminium framework; these are employed in ducts, wall  or ceiling panels, overhead canopies, or laminar airflow  cabinets (Chapter 23). These high-efficiency particulate  air (FFEPA) filters can remove up to 99.997% of particles  more than 0.3 pm in diameter and thus are acting as  depth filters. In practice, their microorganism removal  efficiency is rather better as the majority of bacteria are  found associated with dust particles and only the larger  fungal spores are found in the free state. Air is forced  through HEPA filters by blower fans, and prefilters are  used to remove larger particles to extend the lifetime of  the FfEPA filter. The operational efficiency and integrity  of a FFEPA filter can be monitored by pressure differential  and airflow rate measurements, and dioctylphthalate  smoke particle penetration tests.   Other applications of filters include sterilization of  venting or displacement air in tissue and microbiological  culture (carbon filters and hydrophobic membrane  filters); decontamination of air in mechanical ventilators  (glass fibre filters); treatment of exhausted air from  microbiological safety cabinets (FFEPA filters); and the  clarification and sterilization of medical gases (glass wool  depth filters and hydrophobic membrane filters).   Heat is the means of terminal sterilization that is pre-  ferred by the regulatory authorities because of its relative  simplicity and the high sterility assurance that it affords.  However, a significant number of traditional pharmaceu-  tical products and many recently developed biotechnol-  ogy products are damaged by heat, as are many  polymer-based medical devices and surgical implants; for  such products alternative sterilization processes must be  adopted. Whilst radiation is a viable option for many dry  materials, radiation-induced damage is common in  aqueous drug solutions, and gaseous methods are also  inappropriate for liquids. Aseptic manufacture from indi-  vidually sterilized ingredients is a suitable solution to the  problem of making sterile thermolabile products, but it  affords a lower degree of sterility assurance than steam  sterilization and is both time-consuming and expensive.  For these reasons alternative sterilization strategies have    been developed in recent years. Two processes that have  progressed to the stage of commercial exploitation are  those employing high-intensity light and low- temperature  plasma. It must be stressed, however, that although the  need to develop alternative strategies for the terminal  sterilization of protein- or nucleic acid-containing bio-  technology products is one of the stimuli for the investi-  gation of new methods in general, these particular  processes are unsuitable for such products.   UV light has long been known to have the potential to  kill all types of microorganisms, but its penetrating  power is so poor that it has found practical application  only in the decontamination of air (e.g. in laminar-flow  workstations and operating theatres), shallow layers of  water and surfaces. UV light does not penetrate metal at  all, nor glass to any useful degree, but it will penetrate  those polymers that do not contain unsaturated bonds or  aromatic groups (e.g. polyethylene and polypropylene,  but not polystyrene, polycarbonate or polyvinyl chlo-  ride). High-intensity light sterilization is based on the  generation of short flashes of broad-wavelength light  from a xenon lamp that has an intensity almost 100000  times that of the sun; approximately 25% of the flash is  UV light. The procedure has been applied to the steriliza-  tion of water and studied as a means of terminal steriliza-  tion for injectables in UV-transmitting plastic ampoules  in a blow-fill-seal operation. Although pulsed light is  unlikely to be useful for coloured solutions or those that  contain solutes with a high UV absorbance, it is likely that  the procedure will be readily applicable not only to water  but to some simple solutions of organic molecules, e.g.  dextrose-saline injection.   Plasma is a gas or vapour that has been subjected to an  electrical or magnetic field which causes a substantial  proportion of the molecules to become ionized. It is thus  composed of a cloud of neutral species, ions and elec-  trons in which the numbers of positive and negatively  charged particles are equal. Plasmas may be generated  from many substances but those from chlorine, glutaral-  dehyde and hydrogen peroxide have been shown to  possess the greatest antimicrobial activity.   Low-temperature plasma is a method of sterilization  that is applicable to most of the items and materials  for which ethylene oxide is used, i.e. principally  medical devices rather than drugs; it cannot be used to  sterilize liquids, powders and certain fabrics. Commercial    plasma sterilizers, which have been available since the  early 1990s, typically consist of a sterilization chamber  of about 75 L; this is evacuated, then filled with hydrogen  peroxide vapour which is subsequently converted to a  plasma by application of an electric field. An alternative  commercial plasma sterilizer utilizes alternating cycles  of peracetic acid vapour and a plasma containing oxygen,  hydrogen and an inert carrier gas. The cycle times  are typically from 60 to 90 minutes and the operating  temperatures are less than 50 °C. Major benefits of  plasma sterilization include elimination of the require-  ment to remove toxic gases at the end of the cycle (in  contrast to ethylene oxide and LTSF processes); there  is also no requirement for the treated device to be  aired to remove residual gas, and there is no significant  corrosion or reduction in sharpness of exposed surgical  instruments.   To be labelled ‘sterile’, a product must be free of viable  microorganisms. To achieve this, the product, or its  ingredients, must undergo a sterilization process of suf-  ficient microbiocidal capacity to ensure a minimum level  of sterility assurance. It is essential that the required con-  ditions for sterilization be achieved and maintained  through every operation of the sterilizer. Some examples  of typical conditions employed in sterilization are shown  in Table 21.5.   Historically, the quality control of sterile products  consisted largely, or in some cases, even exclusively, of a  sterility test, to which the product was subjected at the  end of the manufacturing process. However, a growing  awareness of the limitations of sterility tests in terms of  their ability to detect low concentrations of microorgan-  isms has resulted in a shift in emphasis from a crucial  dependence on end-testing to a situation in which the  conferment of the status ‘sterile’ results from the attain-  ment of satisfactory quality standards throughout the  whole manufacturing process. In other words, the quality  is ‘assured’ by a combination of process monitoring and  performance criteria; these maybe considered under four  headings:    Table 21.5 Examples of typical conditions employed in the  sterilization of pharmaceutical and medical products   160°C for 120 min  170°C for 60 min  180°C for 30 min    In well-understood and well-characterized steriliza-  tion processes (e.g. heat and irradiation), where physical  measurements may be accurately made, sterility can be  assured by ensuring that the manufacturing process as    a whole conforms to the established protocols for the  first three of the above headings. In this case the process  has satisfied the required parameters thereby permitting  parametric release (i.e. release based on process data)  of the product without recourse to a sterility test (see  Chapter 22).   The term bioburden is used to describe the concentration  of microorganisms in a material; this may be either a  total number of organisms per millilitre or per gram,  regardless of type, or a breakdown into such categories  as aerobic bacteria or yeasts and moulds. Bioburden  determinations are normally undertaken by the supplier  of the raw material, whose responsibility it is to ensure  that the material supplied conforms to the agreed speci-  fication, but they may also be checked by the recipient.  The maximum permitted concentrations of contami-  nants may be those specified in various pharmacopoeias  or the levels established by the manufacturer during  product development.   The level of sterility assurance that is achieved in a  terminally sterilized product is dependent on the design  of the sterilization process itself and on the bioburden  immediately prior to sterilization (Chapters 22 and 23).  However, the adoption of high standards for the quality  of the raw materials is not, in itself, a strategy that will  ensure that the product has an acceptably low bioburden  immediately prior to sterilization. It is necessary also to  ensure that the opportunities for microbial contamina-  tion during manufacture are restricted (see below), and  that those organisms that are present initially do not  normally find themselves in conditions conducive to  growth. It is for these reasons that manufacturing  processes are designed to utilize adverse temperatures,  extreme pH values and organic solvent exposures in  order to prevent an increase in the microbial load. For  example, water is the most common, and potentially the  most significant, source of contamination in the manu-  factured product, and maintenance of water at elevated  temperatures is commonly employed as a means of limit-  ing the growth of organisms such as Pseudomonas  spp., which can proliferate during storage, even in dis-  tilled or deionized water. Precautions such as these  ensure that chemically synthesized raw materials have  bioburdens that are generally much lower than those  found in ‘natural’ products of animal, vegetable or  mineral origin.   The levels of microbial contamination in the manufac-  turing areas (Chapter 23) are monitored on a regular  basis to confirm that the numbers do not exceed specified  limits. The concentrations of bacteria and of yeasts/  moulds in the atmosphere may be determined either by  use of ‘settle plates’ (Petri dishes of suitable media  exposed for fixed periods, on which the colonies are  counted after incubation) or by use of air samplers  which cause a known volume of air to be passed over an  agar surface. Similarly, the contamination on surfaces,  including manufacturing equipment, may be measured  using swabs or contact plates (also known as Rodac —  replicate organism detection and counting — plates)  which are specially designed Petri dishes slightly over-  filled with agar, which, when set, projects very slightly  above the plastic wall of the dish. This permits the  plate to be inverted on to or against any solid surface,  thereby allowing transfer of organisms from the surface  on to the agar.   Less commonly, environmental monitoring can  extend also to the operators in the manufacturing  area whose clothing, e.g. gloves or face masks, may be  sampled in order to estimate the levels and types of  organisms that may arise as product contaminants from  those sources.   There are several definitions of ‘validation’ but, in simple  terms, the word means demonstrating that a process will  consistently produce the results that it is intended to.  Thus, with respect to sterile products, validation would  be necessary for each of the individual aspects of the  manufacturing process, e.g. environmental monitoring,  raw materials quality assessment, the sterilization process  itself and the sterility testing procedure. Of these, it is the  sterilization process that is likely to be subject to the most  detailed and complex validation procedures, and these  will be used to exemplify the factors to be considered. A  typical validation procedure for a steam sterilization  process is likely to incorporate most, or all, of the follow-  ing features:   • Calibration and testing of all the physical instruments  used to monitor the process, e.g. thermocouples, pressure  gauges and timers    • Production of evidence that the steam is of the desired  quality (e.g. that the chamber temperature is that expected  for pure steam at the measured pressure)   • Conduct of leak tests and steam penetration tests  using both an empty chamber and a chamber filled with  the product to be sterilized in the intended load  conformation   • Use of biological indicators either alone or in  combination with bioburden organisms to demonstrate  that the sterilization cycle is capable of producing an  acceptable level of sterility assurance under ‘worst case’  conditions   • Production of data to demonstrate repeatability of the  above (typically for three runs)   of adequate sterility assurance in steam sterilization  depending upon the thermostability and knowledge of  the presterilization bioburden. Where the product is  known to be stable, an overkill approach may be adopted  in which biological indicators (section 12.3) containing  10 6 test organisms are inactivated in half the proposed  exposure time (thus achieving a 12-log reduction and  a sterility assurance level of 1(T 6 in the full exposure  period). For a marginally thermostable product the cycle  could be validated on the basis of measurements of the  worst case bioburden level and the heat resistance of the  known bioburden organisms; such an approach would  necessitate rigorous control of the bioburden during  routine manufacturing. In the UK, biological indicators  are used primarily in validation rather than routine mon-  itoring of heat sterilization processes, although their use  in routine manufacturing may be required in other coun-  tries. Chemical indicators of sterilization (section 12.2)  are more convenient to use than biological indicators, but  as they provide no direct measure of the efficacy of the  process in terms of microbial killing they are considered  to be less useful. In certain instances these are no longer  routinely used. Physical measurements of temperature,  pressure, time, relative humidity, etc. are of such funda-  mental importance to the assurance of sterility that  records of these parameters are retained for each batch of  sterilized product.   In heat sterilization processes, a temperature record is  made of each sterilization cycle with both dry and moist  heat (i.e. autoclave) sterilizers; this chart/digital record    forms part of the batch documentation and is compared  against a master temperature record (MTR). It is recom-  mended that the temperature be taken at the coolest part  of the loaded sterilizer. Further information on heat dis-  tribution and penetration within a sterilizer can be  gained by the use of thermocouples placed at selected  sites in the chamber or inserted directly into test packs or  bottles. For gaseous sterilization procedures, elevated  temperatures are monitored for each sterilization cycle by  temperature probes, and routine leak tests are performed  to ensure gas-tight seals. Pressure and humidity measure-  ments are recorded. Gas concentration is measured inde-  pendently of pressure rise, often by reference to weight of  gas used. In radiation sterilization, a plastic (often  Perspex) dosimeter which gradually darkens in propor-  tion to the radiation absorbed gives an accurate measure  of the radiation dose and is considered to be the best  technique currently available for following the radiosteri-  lization process.   Sterilizing filters are subject to a bubble point pressure  test, which is a technique employed for determining  the pore size of filters, and may also be used to check the  integrity of certain types of filter device (membrane  and sintered glass; section 7) immediately after use. The  principle of the test is that the wetted filter, in its assem-  bled unit, is subjected to an increasing air or nitrogen  gas pressure differential. The pressure difference recorded  when the first bubble of gas breaks away from the  filter is related to the maximum pore size. When the  gas pressure is further increased slowly, there is a  general eruption of bubbles over the entire surface. The  pressure difference here is related to the mean pore size.  A pressure differential below the expected value would  signify a damaged or faulty filter. A modification to  this test for membrane filters involves measuring the dif-  fusion of gas through a wetted filter at pressures below  the bubble point pressure (diffusion rate test); a faster  diffusion rate than expected would again indicate a  loss of filter integrity. In addition, a filter is considered  ineffective when an unusually rapid rate of filtration  occurs.   Efficiency testing of HEPA filters used for the supply  of sterile air to aseptic workplaces (Chapter 23) is nor-  mally achieved by the generation upstream of dioctyl-  phthalate (DOP) or sodium chloride particles of known  dimension followed by detection in downstream filtered  air. Retention efficiency is recorded as the percentage  of particles removed under defined test conditions.  Microbiological tests are not normally done.   Chemical monitoring of a sterilization process is based  on the ability of heat, steam, sterilant gases and ionizing  radiation to alter the chemical and/or physical character-  istics of a variety of chemical substances. Ideally, this  change should take place only when satisfactory condi-  tions for sterilization prevail, thus confirming that the  sterilization cycle has been successfully completed. In  practice, however, the ideal indicator response is not  always achieved and so a necessary distinction is made  between (1) those chemical indicators which integrate  several sterilization parameters (i.e. temperature, time  and saturated steam) and closely approach the ideal; and  (2) those which measure only one parameter and conse-  quently can only be used to distinguish processed from  unprocessed articles. Thus, indicators which rely on the  melting of a chemical substance show that the tempera-  ture has been attained but not necessarily maintained.   Chemical indicators generally undergo melting or  colour changes, the relationship of this change to the  sterilization process being influenced by the design of the  test device (Table 21.6). It must be remembered, however,  that the changes recorded do not necessarily correspond  to microbiological sterility and consequently the devices  should never be employed as sole indicators in a steriliza-  tion process. Nevertheless, when included in strategically  placed containers or packages, chemical indicators are  valuable monitors of the conditions prevailing at the  coolest or most inaccessible parts of a sterilizer.   Biological indicators (Bis) for use in thermal, chemical or  radiation sterilization processes consist of standardized  bacterial spore preparations which are usually in the form  either of suspensions in water or culture medium or of  spores dried on paper, aluminium or plastic carriers. As  with chemical indicators, they are usually placed in  dummy packs located at strategic sites in the sterilizer.  Alternatively, for gaseous sterilization these may also be  placed within a tubular helix (Line-Pickerill) device.  After the sterilization process, the aqueous suspensions  or spores on carriers are aseptically transferred to an  appropriate nutrient medium, which is then incubated  and periodically examined for signs of growth. Spores of  B. stearothermophilus in sealed ampoules of culture  medium are used for steam sterilization monitoring, and  these may be incubated directly at 55 °C; this eliminates  the need for an aseptic transfer. Aseptic transfers are also  avoided by the use of self-contained units where the spore    Table 21.6 Examples of chemical indicators that have been used for monitoring sterilization processes   Sealed tubes partly filled with a solution which changes colour at  elevated temperatures; rate of colour change is proportional to  temperature, e.g. Browne’s tubes   Usually a temperature-sensitive white wax concealing a black marked  or printed (paper) surface; at a predetermined temperature the wax  rapidly melts, exposing the background mark(s)   Usually an organic chemical in a printing ink base impregnated into  a carrier material. A combination of moisture and heat produces a  darkening of the ink, e.g. autoclave tape. Devices of this sort can be  used within dressings packs to confirm adequate removal of air and  penetration of saturated steam (Bowie-Dick test)   Consists of a blue dye in a waxy pellet, the melting-point of which is  depressed in the presence of saturated steam. At autoclaving  temperatures, and in the continued presence of steam, the pellet  melts and travels along a paper wick forming a blue band the length  of which is dependent upon both exposure time and temperature   Indicator paper impregnated with a reactive chemical which  undergoes a distinct colour change on reaction with EO in the  presence of heat and moisture. With some devices rate of colour  development varies with temperature and EO concentration   Gas concentration,  temperature, time (selected  devices); NB a minimum  relative humidity (rh) is  required for device to function   Based on the same ‘migration along wick’ principle as Thermalog S.  Optimum response in a cycle of 600mg/L EO, temperature 54 °C, rh  40-80%. Lower EO levels and/or temperature will slow response time   Indicator paper impregnated with a formaldehyde-, steam- and  temperature-sensitive reactive chemical which changes colour during  the sterilization process   Plastic devices impregnated with radiosensitive chemicals which  undergo colour changes at relatively low radiation doses   Acidified ferric ammonium sulphate or ceric sulphate solutions  respond to irradiation by dose-related changes in their optical density   >2.5 min at 54 °C, 60% relative  humidity and 600 mg/L EO    strip and nutrient medium are present in the same device  ready for mixing after use.   The bacterial species to be used in a BI must be selected  carefully, as it must be non-pathogenic and should  possess above-average resistance to the particular sterili-  zation process. Resistance is adjudged from the spore  destruction curve obtained upon exposure to the sterili-  zation process; recommended BI spores and their decimal  reduction times (D-values; section 2.2.1) are shown in  Table 21.7. Great care must be taken in the preparation  and storage of Bis to ensure a standardized response to  sterilization processes. Indeed, while certainly offering  the most direct method of monitoring sterilization proc-  esses, it should be realized that Bis may be less reliable  monitors than physical methods and they are not recom-  mended for routine use, except in the case of gaseous  sterilization.   One of the long-standing criticisms of Bis is that the  incubation period required in order to confirm a satisfac-  tory sterilization process imposes an undesirable delay on  the release of the product. This problem has been over-  come, with respect to steam sterilization at least, by the  use of a detection system in which a spore enzyme, a-  glucosidase (reflective of spore viability), converts a non-  fluorescent substrate into a fluorescent product in as little  as 1 hour.   Filtration sterilization requires a different approach  from biological monitoring, the test effectively measuring  the ability of a filter to produce a sterile filtrate from a  culture of a suitable organism. For this purpose, Serratia  marcescens, a small Gram-negative rod-shaped bacterium  (minimum dimension 0.5 pm), has been used for filters    of 0.45 pm pore size, and a more rigorous test involv-  ing Brevundimonas diminuta (formerly Pseudomonas  diminuta) having a minimum dimension of 0.3 pm is  applied to filters of 0.22 pm pore size. The latter filters are  defined as those capable of completely removing Brev.  diminuta from suspension. In this test, using this organ-  ism, a realistic inoculum level must be adopted, as the  probability of bacteria appearing in the filtrate rises as the  number of Brev. diminuta cells in the test challenge  increases; a standardized inoculum size of 10 7 cells cm~ 2  is normally employed. The extent of the passage of this  organism through membrane filters is enhanced by  increasing the filtration pressure. Thus, successful sterile  filtration depends markedly on the challenge conditions.  Such tests are used as part of the filter manufacturer’s  characterization and quality assurance process, and a  user’s initial validation procedure. They are not employed  as a test of filter performance in use.   A sterility test is essentially a test which assesses whether  a sterilized pharmaceutical or medical product is free  from contaminating microorganisms by incubation of  either the whole or a part of that product with a nutrient  medium. It thus becomes a destructive test and is of  questionable suitability for testing large, expensive or  delicate products or equipment. Furthermore, by its very  nature such a test is a statistical process in which part of  a batch is sampled and the chance of the batch being  passed for use then depends on the sample passing the    sterility test. Random sampling should be applied to  products that have been processed and filled aseptically.  With products sterilized in their final containers, samples  should be taken from the potentially coolest or least  sterilant-accessible part of the load.   A further limitation is that which is inherent in a pro-  cedure intended to demonstrate a negative. A sterility test  is intended to demonstrate that no viable organisms are  present, but failure to detect them could simply be a  consequence of the use of unsuitable media or inappro-  priate cultural conditions. To be certain that no organisms  are present it would be necessary to use a universal culture  medium suitable for the growth of any possible contami-  nant and to incubate the sample under an infinite variety  of conditions. Clearly, no such medium or combination  of media are available and, in practice, only media capable  of supporting non-fastidious bacteria, yeasts and moulds  are employed. Furthermore, in pharmacopoeial tests, no  attempt is made to detect viruses, which on a size basis,  are the organisms most likely to pass through a sterilizing  filter. Nevertheless, the sterility test does have an impor-  tant application in monitoring the microbiological  quality of filter-sterilized, aseptically filled products and  does offer a final check on terminally sterilized articles.  In the UK, test procedures laid down by the European  Pharmacopoeia must be followed; this provides details of  the sample sizes to be adopted in particular cases. The  principles of these tests are discussed below.   • The direct inoculation method involves introducing  test samples directly into nutrient media. The European  Pharmacopoeia recommends two media: ( 1 ) fluid mercap-  toacetate medium (also known as fluid thioglycollate  medium), which contains glucose and sodium mercap-  toacetate (sodium thioglycollate) and is particularly  suitable for the cultivation of anaerobic organisms  (incubation temperature 30-35 °C); and (2) soyabean  casein digest medium (also known as tryptone soya  broth), which will support the growth of both aerobic  bacteria (incubation temperature 30-35 °C) and fungi  (incubation temperature 20-25 °C). Other media may be  used provided that they can be shown to be suitable  alternatives. Limits are placed upon the ratio of the weight  or volume of added sample relative to the volume of  culture medium so as to avoid reducing the nutrient  properties of the medium or creating unfavourably high  osmotic pressures within it.    • Membrane filtration is the technique recommended by  most pharmacopoeias and, consequently, the method by  which the great majority of products are examined. It  involves filtration of fluids through a sterile membrane  filter (pore size <0.45 pm), any microorganism present  being retained on the surface of the filter. After washing  in situ, the filter is divided aseptically and portions are  transferred to suitable culture media which are then  incubated at the appropriate temperature for the required  period of time. Water-soluble solids can be dissolved in a  suitable diluent and processed in this way and oil-soluble  products may be dissolved in a suitable solvent, e.g.  isopropyl myristate.   • A sensitive method for detecting low levels of  contamination in intravenous infusion fluids involves the  addition of a concentrated culture medium to the fluid in  its original container, such that the resultant mixture is  equivalent to single strength culture medium. In this way,  sampling of the entire volume is achieved.   With the techniques discussed above, the media  employed should previously have been assessed for nutri-  tive (growth-supporting) properties and a lack of toxicity  using specified organisms. It must be remembered that  any survivors of a sterilization process may be damaged  and thus must be given the best possible conditions for  growth.   As a precaution against accidental contamination,  product testing must be carried out under conditions of  strict asepsis using, for example, a laminar airflow cabinet  to provide a suitable environment.   The European Pharmacopoeia indicates that it is neces-  sary to conduct control tests that confirm the adequacy  of the facilities by sampling of air and surfaces and car-  rying out tests using samples ‘known’ to be sterile (nega-  tive controls). In reality, this means samples that have  been subjected to a very reliable sterilization process, e.g.  radiation, or samples that have been subjected to a steri-  lization procedure more than once. In order to minimize  the risk of introducing contaminants from the surround-  ings or from the operator during the test itself, isolators  are often employed which physically separate the opera-  tor from the materials under test.   Where an antimicrobial agent comprises the product or  forms part of the product, for example as a preservative,  its activity must be nullified in some way during sterility  testing so that an inhibitory action in preventing the  growth of any contaminating microorganisms is over-  come. This is achieved by the following methods.   An appropriate inactivating (neutralizing) agent (Table  21.8) is incorporated into the culture media. The inacti-  vating agent must be non-toxic to microorganisms, as  must any product resulting from an interaction of the  inactivator and the antimicrobial agent.   Although Table 21.7 lists only benzylpenicillin and  ampicillin as being inactivated by (3-lactamase (from B.  cereus), other (3-lactams may also be hydrolysed by 13-  lactamases. Other antibiotic-inactivating enzymes are  also known (Chapter 13) and have been considered as  possible inactivating agents, e.g. chloramphenicol acetyl-  transferase (inactivates chloramphenicol) and enzymes  that modify aminoglycoside antibiotics.   The antimicrobial agent is diluted in the culture medium  to a level at which it ceases to have any activity, for  example phenols, cresols and alcohols (see Chapter 18).  This method applies to substances with a high dilution  coefficient, r/.   This method has traditionally been used to overcome the  activity of antibiotics for which there are no inactivating  agents, although it could be extended to cover other  products if necessary, e.g. those containing preservatives  for which no specific or effective inactivators are availa-    ble. Basically, a solution of the product is filtered through  a hydrophobic-edged membrane filter that will retain any  contaminating microorganisms. The membrane is  washed in situ to remove any traces of antibiotic adhering  to the membrane and is then transferred to appropriate  culture media.   It is essential to show that microorganisms will actually  grow under the conditions of the test. For this reason  positive controls have to be carried out; in these, the  ability of small numbers of suitable microorganisms to  grow in media in the presence of the sample is assessed.  The microorganism used for positive control tests with a  product containing or comprising an antimicrobial agent  must, if at all possible, be sensitive to that agent, so that  growth of the organism indicates a satisfactory inactiva-  tion, dilution or removal of the agent. The European  Pharmacopoeia suggests the use of designated strains of  Staphylococcus aureus. Bacillus subtilis and Pseudomonas  aeruginosa as appropriate aerobic organisms, Clostridium  sporogenes as an anaerobe and Candida albicans or  Aspergillus niger as fungi.   In practice, a positive control (medium with added  test sample) and a negative control (medium without  it) are inoculated simultaneously, and the rate and  extent of growth arising in each should be similar.  However, the negative control without the test sample,  is, in effect, exactly the same as the growth promotion  control that is also described in the test procedure, so,  for the organisms concerned, it is not necessary to do  both.   All the controls may be conducted either before, or in  parallel with, the test itself, providing that the same  batches of media are used for both. If the controls are  carried out in parallel with the tests and one of the con-  trols gives an unexpected result, the test for sterility may  be declared invalid, and, when the problem is resolved,  the test may be repeated.   Specific details of the sterility testing of parenteral prod-  ucts, ophthalmic and other non-injectable preparations,  and surgical sutures will be found in the European  Pharmacopoeia. These procedures cannot conveniently  be applied to items like surgical dressings and medical  devices because they are too big. In such cases the most  convenient approach is to immerse the whole object in  culture medium in a sterile flexible bag, but care must be    taken to ensure that the liquid penetrates to all parts and  surfaces of the material.   A sterility test attempts to infer the state (sterile or non-  sterile) of a batch from the results of an examination of  part of a batch, and is thus a statistical operation. Suppose  that p represents the proportion of infected containers in  a batch and q the proportion of non-infected containers,  then, p + q = \ ox q = l - p.   Suppose also that a sample of two items is taken from  a large batch containing 10% contaminated containers.  The probability of a single item taken at random being  contaminated is p = 0.1 (10% = 0.1), whereas the prob-  ability of such an item being non-contaminated is given  by q = 1 — p = 0.9. The probability of both items being  contaminated is p 2 = 0.01, and of both items being  non-contaminated, q 2 = (1 — p) 1 = 0.81. The probability  of obtaining one contaminated item and one non-  contaminated item is 1 — (0.01 + 0.81) = 0.18 = 2 pq.   In a sterility test involving a sample size of n contain-  ers, the probability p of obtaining n consecutive ‘steriles’  is given by q" = (1 — p)". Values for various levels of p (i.e.  proportion of infected containers in a batch) with a con-  stant sample size are given in Table 21.9, which shows  that the test cannot detect low levels of contamination.  Similarly, if different sample sizes are employed (also  based on ( 1 — p)") it can be shown that as the sample size  increases, the probability of the batch being passed as  sterile decreases.   It can be seen from the above that a sterility test can  only show that a proportion of the products in a batch is  sterile. Thus, the correct conclusion to be drawn from a  satisfactory test result is that the batch has passed the  sterility test not that the batch is sterile.   Under certain circumstances a sterility test may be  repeated, but the only justification for repeating the test  is unequivocal evidence that the first test was invalid; a  retest cannot be viewed as a second opportunity for the  batch to pass when it has failed the first time. Circumstances  that may justify a retest would include, for example,  failure of the air filtration system in the testing facility  which might have permitted airborne contaminants to  enter the product or media during testing, non-sterility  of the media used for testing, or evidence that contami-  nation arose during testing from the operating personnel  or a source other than the sample under test.   The techniques discussed in this chapter comprise an  attempt to achieve, as far as possible, the continuous  monitoring of a particular sterilization process. The ste-  rility test on its own provides no guarantee as to the steril-  ity of a batch; however, it is an additional check and, as  it will detect gross failure, continued compliance with the  test does give confidence as to the efficacy of a steriliza-  tion or aseptic process. Failure to carry out a sterility test,  despite the major criticism of its inability to detect other  than gross contamination, may have important legal and  moral consequences.   Baird, R.M., Hodges, N.A. & Denyer, S.P. (2000) Handbook of  Microbiological Quality Control: Pharmaceuticals and Medical  Devices. Taylor & Francis, London.   = q 20 ; 20 is the sample size required by the European    British Pharmacopoeia (2011) The Stationery Office, London.  (The most recent edition should be consulted.).   Denyer, S.P. & Baird, R.M. (2007) Guide to Microbiological  Control in Pharmaceuticals and Medical Devices. CRC Press,  London.   DH (1995) Sterilisers. Health Technical Memorandum. HTM  2010. Department of Health, London.   DH (1997) Clean steam for sterilization. Health Technical  Memorandum HTM 2031. Department of Health, London.   European Pharmacopoeia , 6th edn (2009) Council of Europe,  Strasbourg. (This pharmacopoeia consists of volumes and  supplements. The most recent should be consulted.)    Fraise, A.P., Lambert, P.A. & Mailland, J.-Y. (2004) Principles and  Practice of Disinfection, Preservation and Sterilization, 5th edn.  Blackwell Scientific, Oxford.   Medicines and Healthcare Products Regulatory Agency (2007)  Rules and Guidance for Pharmaceutical Manufacturers and  Distributors. Pharmaceutical Press, London.   Parenteral Society (2001) Microbiology special issue. Eur J  Parenteral Sci, 6(4).    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Hodges, Sean P. Gorman,  Brendan F. Gilmore.   Parenteral drug delivery systems and many medicinal  products, such as dressings and sutures, must be sterile  in order to avoid the possibilities of microbial degrada-  tion or infection occurring as a result of their use. Sterility  is also important for any material or instrument likely to  contact broken skin or internal organs. Although patho-  genic bacteria, fungi or viruses pose the most obvious  danger to a patient, it should be also realized that micro-  organisms usually regarded as non-pathogenic and which  inadvertently gain access to body cavities in sufficient  numbers may cause a severe, possibly fatal infection.  Consequently, injections, ophthalmic preparations, irri-  gation fluids, dialysis solutions, sutures and ligatures,  implants, and certain surgical dressings, as well as instru-  ments necessary for their use or administration, must be  presented in a sterile condition.   Although there is always a chance of an idiosyncratic  reaction between a medicine and a patient caused by  sensitivity, allergic reaction or unwanted side effects, for  sterile products there is the added requirement that they  must be free of viable microorganisms. This consequently  means the product should be manufactured in a manner  that reduces to the lowest likelihood the risk of microbial  contamination. Thus a sterile product should not contain  viable bacteria, yeasts or fungi, nor other microorganisms  such as rickettsiae, mycoplasmas, protozoa or viruses.  The absence of prion particles is also desirable but diffi-  cult to demonstrate (see Chapter 2). Sterilization proc-  esses concentrate on the destruction or removal of  microorganisms. Each process is designed to remove the  most problematic microorganism (i.e. the smallest bac-  teria in filtration or the most heat-resistant bacterial  spores in heat sterilization processes) on the basis that,  once a sterilization process has been chosen, elimination  of the most problematic species will have led to the elimi-  nation of all less resistant microorganisms.   The principles behind the sterilization processes are  described in Chapter 21. The choice of method is deter-  mined largely by the ability of the formulation and con-  tainer to withstand the physical stresses applied during  the sterilization process. All products intended for steri-  lization should be manufactured under clean conditions  and therefore will be of low microbial content (biobur-  den) prior to sterilization. Under these conditions, the  sterilization process will not be overtaxed and will gener-  ally be within the safety limits needed to provide the  required level of sterility assurance (Chapter 21). The next    section emphasizes parenteral products, but the practices  described apply to many other types of sterile product.   The most obviously recognized sterile pharmaceutical  preparations are injections. These vary from very small-  volume antigenic products to large-volume, total  parenteral nutrition products. Other sterile products  include ophthalmic preparations, creams and dusting  powders. This section describes their formulation and  packaging and the constraints imposed by sterilization on  stability, formulation and packaging of some of the more  common sterile products.   Injections may be aqueous solutions, oily solutions  (because of poor aqueous solubility or the necessity for  a prolongation of drug activity), aqueous suspensions or  oily suspensions. They may be aseptically produced or  terminally sterilized in their final containers (Chapter  23). Those drugs that are unstable in solution may be  presented as a freeze-dried (lyophilized) powder. The  choice of final packaging should not determine the  method of sterilization.   An injection must be manufactured under conditions  that result in a product containing the minimum possible  levels of particles and pyrogenic substances (section 4.4).  Its formulation and packaging must maintain physical  and chemical stability throughout the production proc-  esses, the intended shelf life and during administration.  To achieve this, excipients such as buffers and antioxi-  dants may be required to ensure chemical stability, and  solubilizers, such as propylene glycol or polysorbates,  may be necessary for drugs with poor aqueous solubility  to maintain the drug in solution. Table 22.1 lists some  chemical constituents of common injections and oph-  thalmic preparations.   Many injections are formulated as aqueous solutions,  with Water for Injections (see Chapter 23) as the vehicle.  Their formulation depends on several factors including  the aqueous solubility of the active ingredient, the dose,  its thermal stability, the route of administration, and  whether the product is to be offered as a multiple-dose  product (i.e. with doses removed on different occasions)  or as a single-dose form (as the term suggests, only one  dose per container). Most injections are prepared in    Table 22.1 Some examples of excipients used in formulations and reasons for their inclusion    single-dose form but this is mandatory for certain routes,  e.g. spinal injections where the intrathecal route is used,  and large-volume intravenous infusions. Multiple-  dose injections may require the inclusion of a suitable  preservative to prevent contamination following the  removal of each dose. Injections used for several routes,  including the intrathecal and intracardiac routes, must  not contain a preservative because of potential long-  term damage to the patient. A review of the preservatives  used in parenteral products has been given by Meyer &  Shi (2009).   Some types of injections must be isotonic with blood  serum. This applies particularly to large-volume intrave-  nous infusions if at all possible; hypotonic solutions may  cause lysis of red blood corpuscles and thus must not be  used for this purpose. Conversely, hypertonic solutions  can be employed; these induce shrinkage, but not lysis, of  red cells, which recover their shape later. Intraspinal  injections must also be isotonic to reduce pain at the site  of injection; so should intramuscular and subcutaneous    injections. Adjustment to isotonicity can be determined  from either the depression of freezing point or from  sodium chloride equivalents. The depression of the freez-  ing point depends on the number of dissolved particles  (molecules or ions) present in a solution. The equation:   where W is the percentage (w/v) of adjusting substance,  a is the freezing point of unadjusted solution and b is the  depression of the freezing point of water induced by 1%  w/v of adjusting substance, allows the determination of  how much adjusting substance is required to produce  isotonicity with blood plasma.   Alternatively the sodium chloride equivalent, which is  produced by dividing the value for the depression of freez-  ing point produced by a solution of the substance by the  corresponding value of a solution of sodium chloride of  the same strength, may be used. Fuller details of each  method maybe found in the Pharmaceutical Codex ( 1994) .   Intravenous infusions consist of large-volume injections  or drips (500 ml or more) that are infused at various rates  (50-500 ml/h) into the venous system. They are generally  sterilized in an autoclave. Examples include isotonic solu-  tions of sodium chloride or glucose that are used to  maintain fluid and electrolyte balance, for replacement of  extracellular body fluids (e.g. after surgery or prolonged  periods of fluid loss), as a supplementary energy source  (1L of 5% w/v glucose yields 714kJ of energy) or as a  vehicle for drugs. Other important examples are blood  products, which are collected and processed in sterile  containers, and plasma substitutes, e.g. dextrans and  degraded gelatin. Dextrans are glucose polymers in which  the glucose monomers are joined by l-6-0t links; they are  produced by certain bacteria of the genus Leuconostoc,  e.g. Leuconostoc mesenteroides.   A common hospital practice is to add drugs to infusions  immediately before administration. Regularly used  additives include potassium chloride, lidocaine (ligno-  caine), heparin, certain vitamins and antibiotics.  Potentially this can be a hazardous practice. For instance,  the drug may precipitate in the infusion fluid because of  the pH (e.g. amphotericin) or the presence of calcium  salts (e.g. thiopentone); the drug may degrade rapidly  (e.g. ampicillin in 5% w/v glucose); multiple additions  may lead to precipitation of one or both of the drugs or  to accelerated degradation; and finally, drug loss may  occur because of sorption by the container. For instance,  insulin is adsorbed by glass or by polyvinyl chloride  (PVC); glyceryl trinitrite and diazepam are absorbed by  PVC. Apart from these problems, if the addition is not  carried out under strict aseptic conditions the fluid can  become contaminated with microorganisms during the  procedure. Thus any addition should be made in a  laminar-flow workstation or isolator, and the fluid  should, ideally, be administered within 24 hours of  preparation.   Another approach to the problem of providing an  intravenous drug additive service is to add the drug to a  small volume (50-100 ml) infusion in a collapsible plastic  container and store the preparation at —20 °C in a freezer.  The infusion can be removed when required and thawed  rapidly in a microwave oven. Many antibiotics are stable  for several months when stored in minibags at —20 °C and  are unaffected by the thawing process. Other antibiotics,  e.g. ampicillin, degrade even when frozen.   Total parenteral nutrition (TPN) is the use of mixtures  of amino acids, vitamins, electrolytes, trace elements and  an energy source (glucose and fat) in the long-term  feeding of patients who are unconscious or unable to take  food. All or most of the ingredients to feed a patient for  1 day are combined aseptically in one large (3 L capacity)  collapsible plastic bag, the contents of which are infused  over a 12-24 hour period. Transfer of amino acid, glucose  and electrolyte infusions, and the addition of vitamins  and trace elements must be carried out with great care  under aseptic conditions to avoid microbial contamina-  tion. These solutions often provide good growth condi-  tions for bacteria and moulds. Fats are administered  as oil-in-water emulsions comprising small droplets of a  suitable vegetable oil (e.g. soyabean) emulsified with  egg lecithin and sterilized by autoclaving. In many cases,  the fat emulsion is added to the 3L bag. Thus TPN  fluids are complex mixtures and a multitude of potential  interactions, both chemical and physicochemical, may  occur between their individual components resulting in  decomposition, creaming, precipitation or even the for-  mation of toxic by-products. Trace elements, calcium,  vitamins and lipids are particularly prone to affecting the  stability.   Although many vitamins may be administered as a  single dose at various time intervals, many of the patient’s  requirements will be found in what is basically an emul-  sion formulation, prepared aseptically and thus with no  terminal sterilization. The product usually contains both  essential and non-essential amino acids rather than fully  formed protein, and energy is provided at a ratio of 0.6-  1.1 MJ per gram of protein nitrogen. A mixture of carbo-  hydrate (glucose) and fat (as an emulsion) provides the  energy and electrolytes, trace elements and vitamins are  included as required. Thus the TPN fluid is prepared to  suit the individual patient’s needs. The fact that the  product contains so many ingredients makes TPN  fluids extremely difficult to prepare, and once vitamins  are added, their chemical instability reduces the shelf  life. During preparation, TPN fluids are compounded  from individual solutions or emulsions. Generally, the  bulk of the final volume is derived from glucose solu-  tions, amino acid solutions and fat emulsions; small-  volume solutions are added to these before filling. During  compounding, electrolytes are added to the amino acid  solutions and phosphate salts to the dextrose (glucose)  solutions, which are then mixed and the lipid emulsions  added. This order of mixing is adopted because the pH    of glucose solutions decreases due to degradation during  their sterilization and addition of emulsions to this low  pH solution might cause emulsion instability. The mixing  of the amino acids with the glucose solutions provides a  vehicle with some degree of buffering capacity. Calcium  might precipitate as the phosphate if its salts were to be  added directly to the phospholipid emulsion. Vitamins  are added to the lipid emulsion or to the bag immediately  before use.   A number of other difficulties may be encountered.  Polyunsaturated acids are subject to hydrolysis. Any  residual air might cause oxidation of labile vitamins, e.g.  vitamin C. Lipids (the fat emulsions and fat-soluble vita-  mins formulated as an emulsion) may extract plasticizer  from a plastic container, especially if the bag is based on  PVC. Any electrolytes may compromise emulsion stabil-  ity by altering the electrochemistry around the dispersed  oil droplets, thus allowing the droplets to move closer to  each other (due to a disruption of the Stern layer) and  coalesce; a less noticeable problem would be changes in  the globule size. Additionally, the plastic bag might  absorb the oil-soluble vitamins and care has to be taken  in the selection of the container to avoid moisture  loss. As a final example of the complex nature of TPN  fluids, amino acids may undergo the Maillard reaction  with glucose, resulting in discoloration. An account of  the clinical aspects of TPN can be found in Harper &  Lamerton (2009).   This category includes single-dose injections, usually of  1-2 ml but as high as 50 ml, dispensed in borosilicate  glass ampoules, plastic (polyethylene or polypropylene)  ampoules or, rarely, multiple-dose glass vials of 5-25 ml  capacity stoppered with a rubber closure through which  a hypodermic needle can be inserted, e.g. insulins, vac-  cines. The closure is designed to reseal after withdrawal  of the needle. It is unwise to include too many doses in a  multiple-dose container because of the risk of microbial  contamination during repeated use. Preservatives must  be added to injections in multiple-dose containers to  prevent contamination during withdrawal of successive  doses. However, preservatives may not be used in  injections in which the total volume to be injected at one  time exceeds 15 ml. This may occur if the solubility of a  drug is such that a therapeutic dose can only be  achieved in this volume of solvent. There is also an abso-  lute prohibition on the inclusion of preservatives in  intra-arterial, intracardiac, intrathecal or subarachnoid,   Certain small-volume injections are available where the  drug is dissolved in a viscous oil because it is insoluble  in water and therefore a non-aqueous solvent is used.  In addition, drugs in non-aqueous solvents provide a  depot effect, e.g. for hormones. The intramuscular  route of injection must be used. The vehicle may be a  metabolizable fixed oil such as arachis oil or sesame  oil (but not a mineral oil) or an ester, such as ethyl  oleate, which is also capable of being metabolized. The  latter is less viscous and therefore easier to administer, but  the depot effect is of shorter duration. The drug is nor-  mally dissolved in the oil, filtered under pressure and  distributed into ampoules. After sealing, the ampoules  are sterilized by dry heat. A preservative is probably inef-  fective in such a medium and therefore offers very little  protection against contamination in a multiple-dose oily  injection.   In brief, freeze-drying (lyophilization) consists of prepar-  ing the drug solution (with buffers and cryoprotectants),  filtering through a bacteria-proof filter, dispensing into  containers, removing water in a freeze-drier, then capping  and closing the containers. Many biotechnology products  are freeze-dried.   Freeze-drying is an aseptic process whereby water is  removed from a frozen product mainly by sublimation,  i.e. by the conversion of ice directly into the vapour state  without the intermediary of liquid water. It is a batch  process, of relatively long duration, and is used frequently  for drugs of poor stability. Drugs are reconstituted into  solution immediately prior to injection. The process con-  sists of three stages:   • primary drying, whereby energy is provided to the  system and a vacuum applied to expedite the removal of  moisture at subambient temperatures   • secondary drying, whereby the product is heated to  remove the last traces (2%) of water.   A number of characteristics of the formulation control  the behaviour of the product during the lyophilization  cycle. These include the glass transition temperature and  the collapse temperature. The maintenance of sterility  and retention of the appropriate sterility assurance level    (SAL) is implicit in the freeze-drier design (Pikal, 2007).  Although membrane-filtered sterile solutions may be  used to fill containers to be placed into the freeze-drier,  other measures to maintain sterility are also employed.  These include using steam sterilization of the drier;  gaseous sterilization has not been widely adopted. The  temperature of shelves is regulated using a circulating  fluid such as dimethylsiloxane oil. Electronics and com-  puterization have led to the accumulation of better data  for validation. Stoppering systems allow the successful  sealing of the containers and gas entering the drier may  be filtered to effect sterilization.   The packaging and closures must prevent loss of vehicle,  excipient or drug during sterilization and storage.  Additionally, ingress of microorganisms must be pre-  vented. The packaging must not contribute any signifi-  cant amounts of extractable chemicals to the contents,  e.g. vulcanizing agents from rubbers or plasticizers from  PVC infusion containers.   Single-dose injections are usually packed in glass  ampoules containing 1, 2 or 5 ml of product. To ensure  removal of the correct dose volume by syringe and needle,  it is necessary to add an appropriate overage to the  ampoule. Thus a 1 ml ampoule will actually contain 1 . 1 ml  of product and a 2 ml ampoule should contain 2.15 ml of  product.   Many injectables are sealed with a rubber closure held  on by an aluminium screw-cap or crimp-on ring. The  rubber should be non-fragmenting, not release soluble  extractives, and be sufficiently soft and pliable to seal  around the needle inserted immediately prior to use.  Although filled bottles are sterilized by autoclaving, it is  still possible for the infusions in glass bottles to become  contaminated with microorganisms through the seal  before use. For instance, during the final part of the auto-  clave cycle, bottles may be spray-cooled with water to  hasten the cooling process. However, if there is a poor fit  between bottle lip and rubber plug (a skirted inset type  is used) it is possible for the spray-cooling water to spread  by capillary movement between bottle thread and screw-  cap and even to enter the bottle contents. Failure may also  result from any imperfection of the bottle or plug.  Microorganisms may gain access to the product within  the containers during storage if hairline cracks (due to  bad handling or rough treatment) are present which    permit fluid seepage. Finally, contamination may occur  during use (1) if poor aseptic techniques are applied  when setting up the infusion, (2) via an ineffective air  inlet (which allows replacement of the infused fluid with  air in glass bottles), or (3) when changing the giving set  or bottle.   Three types of glass are suitable for use in the manu-  facture of containers for injectable preparations. These  are a neutral borosilicate glass, a sulphated soda glass and  a soft, moderately hydrolytic resistant glass. The glasses  are classified by their hydrolytic resistance. The choice for  a container depends on the properties of the solution  they are used to package. The advantages of glasses as  container materials include their chemical resistance, the  fact that they do not absorb or leach organic materials,  their impermeability to water vapour and other gases,  their transparency, their ability to form rigid strong stable  containers which resist puncture, their ability to hold a  vacuum and their overall stability to moist heat or dry  heat sterilization. However, glass containers may break  and crack during the sterilization process, they are  attacked by alkaline solutions (and so may be a problem  with, for example, sodium citrate bladder irrigation),  they are heavy and require venting during administration  of their contents.   Closures are made of a polymer and their formulation  include curing agents, activators, antioxidants, plasticiz-  ers, fillers and pigments. They have to be selected with  the drug product in mind to avoid chemical incompati-  bility and possible reaction with the ingredients in the  product formulation. Sorption of the preservative from  multiple-dose formulations has frequently been a  problem and closures may therefore require saturation  with the ingredients in the product prior to packaging.   Closures should be flexible, to conform to the shape of  the vial; resilient, so as to reseal after each needle punc-  ture; tough, so that low fragmentation levels occur when  punctured; non-thermoplastic, so that the heat steriliza-  tion process is tolerated; and chemically compatible with  the drug formulation. Early closures were sulphur-based,  and easily cured with accelerators to speed up the curing  rate. Unfortunately, a high degree of water-extractable  by-products could be taken up by the product that they  were intended to protect. Consequently they have been  replaced by modern polymer formulations with low  extract curatives. Bromobutyl and chlorobutyl rubbers  show superior performance although special polymers,    e.g. nitrile rubbers, are used for mineral oil products.  Problems of incompatibility may be overcome by film  bonding a fluorocarbon barrier film to the surfaces of the  closures.   Most infusions are now packed in plastic containers. The  plastic material should be pliable, thermoresistant, trans-  parent and non-toxic. The plastics may contain antioxi-  dants, stabilizers, lubricants, plasticizers, fillers and  colorants. Suitable materials are PVC (which may present  a problem with moisture loss) and polyethylene. The  former is transparent and very pliable, allowing the pack  to collapse as the contents are withdrawn (consequently  no air inlet is required). These packs are also amenable  to the inclusion of ports into the bag, allowing greater  safety during use. Such ports may be protected by sterile  overseals.   Two problems arise: (1) the possibility of toxic extrac-  tives, e.g. diethyl phthalate, from the plastic entering the  fluid if poor quality PVC is used, and (2) moisture per-  meability leading to loss of water if the packs are not  protected by a water-impermeable outer wrap. Bags of  high-quality polyethylene are readily moulded (although  separate ports cannot be included), translucent and free  from potential toxic extractives. Again, these packs nor-  mally collapse readily during infusion. An important  advantage of all plastic packs is that the containers are  hermetically sealed prior to autoclaving and therefore  spray-cooling water cannot enter the pack unless there is  seal failure, an easily detected occurrence. However, auto-  claving of plastic bags is more complex than that of  bottled fluids because a steam/air mixture is necessary to  prevent bursting of the bags when heated (air ballasting);  adequate mixing of the steam and air is therefore required  to prevent layering of gases inside the chamber.   Blow-fill technology is an aseptic process whereby the  container is formed from thermoplastic granules, filled  with sterile solution and sealed, all within one automatic  operation. The bulk solution should have a low biobur-  den and is delivered to the machine through a filling  system that has been previously sanitized and steam steri-  lized in situ. Concern has been expressed that the machine  itself may generate particles. The plastic granules are  composed usually of polyethylene, polypropylene or one  of their copolymers and are heat extruded at about 200 °C  into a tube. The two halves of a mould close around this  tube and seal the base. The required quantity of sterile    fluid is filled into the container, which is then sealed.  Products packed in this way include intravenous solu-  tions, and small-volume parenteral, ophthalmic and neb-  ulizer solutions. The technique offers lower costs than  conventional packaging.   Small-volume injections may also be packaged in car-  tridges or directly into disposable syringes. The latter are  immediately available for use but have a high cost of  production and their fixed content may lead to waste of  material that remains uninjected after single use.  Cartridges are lower cost and may be fitted into injection  pens; many insulin products are produced in this manner  because of their low waste, ease of use and not requiring  the patient to draw a dose volume into a separate syringe.  Cartridges have a plunger stopper at one end of a cylin-  drical glass body containing the product for injection,  and the other end is sealed with a rubber-lined crimp cap.  Processing steps include preparing the bulk sterile solu-  tion for injection, washing and siliconizing the plunger  stoppers, caps and glass cartridges, inserting the plunger  stopper, filling and closing. The product is then sterilized,  but care has to be taken that the internal pressures that  develop during the autoclave cycle do not force the car-  tridge plunger out of the cartridge. The industry is devel-  oping a range of devices designed to breach the skin’s  defences to allow transdermal delivery (Arora et al.,  2008). These include microneedle arrays and needle-free  injections.   Because of the possible clinical consequences (such as  granuloma of the lung) of injecting solid particles into  the bloodstream, the number of particles present in injec-  tions and other solutions used in body cavities must be  restricted. The British Pharmacopoeia (2010) states that  injectable preparations which are solutions ‘when exam-  ined under suitable conditions of visibility are clear and  practically free from particles’. It also sets limits for sub-  visible particles in injections based on the principle of  light blockage. Not more than 100 particles/ml greater  than 5 pm and not more than 50 particles/ml greater than  10 pm should be generally obtained. The British  Pharmacopoeia (2010) describes a microscopic method  for determination of the particulate contamination of  injections and intravenous infusions. The counting    methods should estimate extraneous particles, but not  bubbles, that are unintentionally present in the solutions.  If the method provides a means for identifying and  detecting the particles, insight may be gained into their  possible origin. Filtration and observation using light  microscopy have clear advantages, including simplicity  and allowing the operator to visualize the particles.   All parenterally injected solutions should be checked  for particulate contamination, but the above procedure  is clearly impractical as a bulk screening exercise. Those  products contaminated with particulate matter should be  rejected. In practice, all products may be tested individu-  ally by a human observer against split white/black screens  and/or under polarized light for obvious particulate con-  tamination, and again there is a method described in  pharmacopoeias based on the split-screen technique.  Nowadays optical control equipment can take over this  arduous and boring task.   The integrity of sealing of ampoules should be assessed  on an individual basis. Two techniques are available that  depend on dye ingress under vacuum or electronic  means. With dye intrusion, the ampoules are submerged  in a dye solution and under an applied vacuum. Any  container that has cracks in its structure or is not sealed  will admit the dye when the vacuum is reduced. On  washing, badly sealed ampoules will be coloured. This  technique underestimates the problem of bad sealing. In  the alternative technique, high-frequency spark testing,  the presence of a leak causes a change in a high-frequency  electrical signal placed across the ampoule. The method  is limited to aqueous products with a high conductivity.  It is a very sensitive technique and detects weak seals not  detected by the dye test. In reality, both tests should be  used in parallel.   There are many other types of solution in a sterile form,  for use particularly in hospitals.   This is sterile water, not necessarily of injectable water  standards, which is used widely during surgical proce-  dures for wound irrigation, moistening of tissues, washing  of surgeons’ gloves and instruments during use and, when  warmed, as a haemostat. Isotonic saline may also be used.  Topical water (as it is often called) is prepared in 500 ml  and 1 1 polyethylene or polypropylene containers with a  wide neck and tear-off cap to allow for ease of pouring.   These are used for rinsing of the urinary tract to aid tissue  integrity and cleanliness during or after surgery. Either  water or glycine solution is used, the latter eliminating  the risk of intravascular haemolysis when electrosurgical  instruments are used. These are sterile solutions pro-  duced in collapsible or semirigid plastic containers of up  to 3 L capacity.   Peritoneal dialysis solutions are admitted into the  peritoneal cavity as a means of removing accumulated  waste or toxic products following renal failure or poison-  ing. They contain electrolytes and glucose (1.4-7% w/v)  to provide a solution equivalent to potassium-free  extracellular fluid; lactate or acetate is added as a source  of bicarbonate ions. Slightly hypertonic solutions are  usually employed to avoid increasing the water content  of the intravascular compartment. A more hypertonic  solution containing a higher glucose concentration is  used to achieve a more rapid removal of water. In  fact, the peritoneal cavity behaves as if it were separated  from the body organs by a semipermeable membrane.  Warm peritoneal solution (up to 5L) is perfused into  the cavity for 30-90 minutes and then drained out  completely. This procedure can then be repeated as  often as required. As the procedure requires larger  volumes, these fluids are commonly packed in 2.5 L  containers. It is not uncommon to add drugs (for  instance potassium chloride or heparin) to the fluid prior  to use.   Haemodialysis is the process of circulating a patient’s  blood through a machine via tubing composed of a  semipermeable material such that waste products  permeate into the dialysing fluid and the blood then  returns to the patient. Haemodialysis solutions need not  be sterile but must be free from heavy bacterial  contamination.   In cases of severe asthmatic attacks, bronchodilators and  steroids for direct delivery to the lungs may be needed in  large doses. This is achieved by direct inhalation via a  nebulizer device; this converts a liquid into a mist or fine  spray. The drug is diluted in small volumes of Water for  Injections before loading into the reservoir of the  machine. This vehicle must be sterile and preservative-  free and is therefore prepared as a terminally sterilized  unit dose in polyethylene nebules.   Medication intended for instillation on to the surface of  the eye is formulated in aqueous solution as eye drops or  lotion or in an oily base as an ointment. Because of the  possibility of eye infection occurring, particularly after  abrasion or damage to the corneal surface, all ophthalmic  preparations must be sterile. As there is a very poor blood  supply to the anterior chamber, defence against microbial  invasion is minimal; furthermore, it appears to provide a  particularly good environment for growth of bacteria. As  well as being sterile, eye products should also be relatively  free from particles that might cause damage to the cornea.  However, unlike aqueous injections the recommended  vehicle is purified water because the presence of pyrogens  is not clinically significant.   Another type of sterile ophthalmic product is the  contact lens solution. However, unlike the other types this  is not used for medication purposes but merely as wetting,  cleaning and soaking conditions for contact lenses.   Some typical excipients for eye drops are given in  Table 22.1. Eye drops are presented for use in (1) sterile  single-dose plastic sachets (often termed Minims) con-  taining 0. 3-0.5 ml of liquid, (2) multiple-dose amber  fluted eye dropper bottles including the rubber teat  as part of the closed container or supplied separately, or  (3) plastic bottles with integral dropper. A breakable  seal indicates that the dropper or cap has not been  removed prior to initial use. Although a standard design  of bottle is used in hospitals, many proprietary products  are manufactured in plastic bottles designed to improve  safety and care of use. The maximum volume in each  container is limited to 10 ml. Because of the likelihood of  microbial contamination of eye dropper bottles during  use (arising from repeated opening or contact of the  dropper with infected eye tissue or the hands of the  patient), it is essential to protect the product with a  preservative (Matthews & Skinner, 2006). Eye drops for  surgical theatre use should be supplied in single-dose  containers.   Examples of preservatives are phenylmercuric nitrate  or acetate (0.002% w/v), chlorhexidine acetate (0.01%  w/v), thiomersal (0.01% w/v) and benzalkonium chloride  (0.01% w/v). Chlorocresol is too toxic to the corneal epi-  thelium, but 8-hydroxyquinoline and thiomersal may be  used in specific instances. The principal consideration in  relation to antimicrobial properties is the activity of the    bactericide against Pseudomonas aeruginosa, a major  source of serious nosocomial eye infections. There is  some concern over the toxicity of mercurials, and their  use is becoming less common. Although benzalkonium  chloride is probably the most active of the recommended  preservatives, it cannot always be used because of its  incompatibility with many compounds commonly used  to treat eye diseases, nor should it be used to preserve eye  drops containing anaesthetics. As benzalkonium chloride  reacts with natural rubbers, silicone or butyl rubber  teats should be substituted and products should not be  stored for more than 3 months after manufacture because  silicone rubber is permeable to water vapour. As with  all rubber components, the rubber teat should be  pre-equilibrated with the preservative before use.  Thermostable eye drops and lotions are sterilized at  121 °C for 15 minutes. For thermolabile drugs, filtration  sterilization followed by aseptic filling into sterile con-  tainers is necessary. Eye drops in plastic bottles are pre-  pared aseptically.   In order to lessen the risk of eye drops becoming  heavily contaminated, either by repeated inoculation or  by the growth of resistant organisms in the solution,  use is restricted to 1 month after the container is first  opened. This is usually reduced to 7 days for hospital  ward use on one eye of a single patient. The period is  shorter in the hospital environment because of the greater  danger of contamination by potential pathogens, particu-  larly pseudomonads.   Eye lotions are isotonic solutions used for washing or  bathing the eyes. They are sterilized by autoclaving in  relatively large-volume containers (100 ml or greater) of  coloured fluted glass with a rubber closure and screw-cap,  or packed in plastic containers with a screw-cap or tear-  off seal. They may contain a preservative if intended for  intermittent domiciliary use for up to 7 days. If intended  for first aid or similar purposes, however, no bactericide  is included and any remaining solution is discarded after  24 hours.   Eye ointments are prepared in a semisolid base — e.g.  Simple Eye Ointment BP, which consists of yellow soft  paraffin (8 parts), liquid paraffin (1 part) and wool fat (1  part). The base is filtered when molten to remove parti-  cles and sterilized at 160°C for 2 hours. The drug is  incorporated prior to sterilization if heat-stable, or added  aseptically to the sterile base. Finally the product is    aseptically packed in clear sterile aluminium or plastic  tubes. As the product contains virtually no water,  the danger of bacteria proliferating in the ointment is  negligible.   Most contact lenses are worn for optical reasons as an  alternative to spectacles. Contact lenses are of two types:  hard lenses, which are hydrophobic, and soft lenses,  which may be either hydrophilic or hydrophobic. The  surfaces of lenses must be wetted before use and wetting  solutions are used for this purpose. Hard, and more espe-  cially, soft lenses become heavily contaminated with  protein material during use and therefore must be cleaned  before disinfection. Contact lenses are potential sources  of eye infection and, consequently, microorganisms  should be removed before the lens is again inserted into  the eye. Lenses must also be clean and easily wettable by  lachrymal secretions. Contact lens solutions are thus  sterile solutions of the various types described below.  Apart from achieving their stated functions, either singly  or in combination, all solutions must be non-irritating or  must protect against microbial contamination during use  and storage.   These are used to hydrate the surfaces of hard lenses after  disinfection. As they must also cope with chance con-  tamination, they must contain a preservative as well as a  wetting agent. They may be isotonic with lachrymal  secretions and be formulated to a pH of about 7.2 for  compatibility with normal tears.   These are responsible for the removal of ocular debris  and protein deposits, and contain a cleaning agent that  consists of a surfactant and/or an enzyme product. As  they must also cope with chance contamination, they  contain a preservative, are isotonic and have a pH of  about 7.2.   These are solutions for disinfection of lenses but  also maintain the lenses in a hydrated state. The antimi-  crobial agents used for disinfecting hard lenses are those  used in eye drops (benzalkonium, chlorhexidine, phe-  nylmercuric acetate or nitrate, thiomersal and chlorbu-  tol). Ethylenediamine tetraacetic acid (EDTA) is usually  present as a synergist. Benzalkonium chloride and    chlorbutol are strongly bound to hydrophilic soft contact  lenses and therefore cannot be used in storage solutions  for these; chlorhexidine and thiomersal are usually  employed. It must be added that the concentrations of all  preservatives used in contact lens solutions are lower than  those employed in eye drops, to minimize irritancy.  Hydrogen peroxide is becoming commonly used but  must be inactivated before the lenses are inserted onto  the eyes. Finally, heat may be utilized as an alternative  method to disinfect soft contact lenses, especially the  hydrophilic types. Lenses are boiled in isotonic saline.   Dressings and surgical materials are used widely in medi-  cine, both as a means of protecting and providing comfort  for wounds and for many associated activities such as  cleaning and swabbing. They may or may not be used on  areas of broken skin. If there is a potential danger of  infection arising from the use of a dressing then it must  be sterile. For instance, sterile dressings must be used on  all open wounds, both surgical and traumatic, on burns,  and during and after catheterization at a site of injection.  It is also important to appreciate that sterile dressings  must be packaged in such a way that they can be applied  to the wound aseptically.   Dressings are described in the British Pharmacopoeia  (2010). Methods for their sterilization include autoclav-  ing, dry heat, ethylene oxide and ionizing radiation. Any  other effective method may be used. The choice is gov-  erned principally by the stability of the dressing constitu-  ents to the stress applied and the nature of their  components. Most celluloses and synthetic fibres with-  stand autoclaving, but there are exceptions. For instance,  boric acid tenderizes cellulose fibres during autoclaving,  and dressings containing waxes cannot be sterilized by  moist heat. Certain constituents are also adversely affected  on exposure to large doses of gamma radiation. Examples  of dressings that are required to be sterile are listed in  Table 22.2, together with other dressings and materials  that may be sterilized when required.   A very important aspect of the production of dressings  is packaging. The packaging material must allow correct  sterilization conditions (e.g. permeation of moisture or  ethylene oxide), retain the dressing in a sterile condition  and allow for its removal without contamination prior to  use. All dressings intended for aseptic handling and appli-  cation must be double wrapped. For steam sterilization  they may be individually wrapped in fabric, paper or  nylon and sterilized in metal drums, cardboard boxes or  bleached Kraft paper. The choice of method also deter-    mines the design of the autoclave cycle. Providing that  adequate steam penetration is assured, dressings may be  sterilized in downward displacement autoclaves which  rely on displacement of air by steam. However, high pre-  vacuum autoclaves in which virtually all the air is removed  before the admission of steam are much more commonly  employed. This method ensures rapid heating up of  dressings, reduces the time needed to achieve sterilization  (e.g. 134 °C for 4 minutes) and shortens the overall steri-  lization cycle.   A recent development is the use of spray-on dressings.  A convenient type is an acrylic polymer dissolved in  ethyl acetate and packed as an aerosol. This should be  self-sterilizing. The film after application is able to main-  tain the sterility of a clean wound for up to 2 weeks.  However, they can only be used on clean, relatively dry  wounds.   Implants are small, sterile cylinders of drug, inserted  beneath the skin or into muscle tissue to provide  slow absorption and prolonged action therapy. This is  principally based on the fact that such drugs, invariably  hormones, are almost insoluble in water and yet the  implant provides a rate of dissolution sufficient for a  therapeutic effect. Implants are manufactured from the  pure drug made into tablet form by compression or  fusion. No other ingredient can be included because  this may be insoluble or toxic, or, most importantly,  may influence the rate of drug release. Copolymers such  as polylactic acid/polyglycolic acid may be used as the  implant matrix to provide a controlled rate of drug  delivery.   Compression of sterile drugs must be conducted  under aseptic conditions using sterile machine parts and    materials. After manufacture, the outer surface of the  implant is sterilized by immersion in 0.002% w/v phe-  nylmercuric nitrate at 75 °C for 12 hours. After the surface  has been dried, each implant is placed aseptically into  a sterile glass vial with a cotton wool plug at both ends.  This prevents damage and reduces the risk of glass  specules, formed when the vial is opened, adhering to the  implant. This compression process is not ideal and fusion  processes may be used provided that the drug is heat-  stable. The pure drug is melted at 5-10 °C above its  melting temperature and poured into moulds. The inte-  rior of the implant will be automatically sterilized by this  process if the melting temperature is high enough. It is  also possible to dry heat sterilize the implant after pack-  aging provided that the melting temperature is above  160°C. Clearly, it is easier to manufacture sterile implants  by fusion as the process does not require presterilized  ingredients or aseptic processing. The implant hardness  is also very convenient.   The reduction of blood loss during or after surgical pro-  cedures where suturing or ligature is either impractical  or impossible can often be accomplished by the use of  sterile, absorbable haemostats. These consist of a soft  pad of solid material packed around and over the wound  that can be left in situ and absorbed by body tissues  over a period of time, usually up to 6 weeks. The  principal mechanism of action of these is their ability to  encourage platelet fracture because of their fibrous or  rough surfaces, and to act as a matrix for complete blood  clotting. Four products commonly used are oxidized cel-  lulose, absorbable gelatin sponge, human fibrin foam and  calcium alginate.   This consists of cellulose material that has been partially  oxidized. White gauze is the most common form,  although lint is also used. It can be absorbed by the body  in 2-7 weeks, depending on the size. Its action is based  principally on a mechanical effect and it is used in the  dry state. As it inactivates thrombin, its activity cannot be  enhanced by thrombin incorporation.   This insoluble foam is produced by whisking warm  gelatin solution to form a uniform foam, which is then  dried. It can be cut into suitable shapes, packed in metal  or paper containers and sterilized by dry heat (150°C for  1 hour). Moist heat destroys the physical properties of the    material. Immediately before use, it can be moistened  with normal saline containing thrombin. It behaves as a  mechanical haemostat, providing the framework on  which blood clotting can occur.   This is a dry sponge of human fibrin prepared by  clotting a foam of human fibrinogen solution with  human thrombin. It is then freeze-dried, cut into shapes  and sterilized by dry heat at 130 °C for 3 hours. Before  use it is saturated with thrombin solution. Blood coagula-  tion occurs in contact with the thrombin in the interstices  of the foam.   This is composed of the sodium and calcium salts of  alginic acid formed into a powder of fibrous material and  sterilized by autoclaving. It aids clotting by forming a  sodium-calcium alginate complex in contact with tissue  fluids, acting principally as a mechanical haemostat. It is  relatively slowly absorbed and some residues may occa-  sionally remain in the tissues.   The use of strands of material to tie off blood or other  vessels (ligature) and to stitch wounds (suture) is an  essential part of surgery. Both absorbable and non-  absorbable materials are available for this purpose.   This consists of absorbable strands of collagen derived  from mammalian tissue, particularly (despite its name)  the intestines of sheep. Because of its source, it is particu-  larly prone to bacterial contamination, and even anaero-  bic spores may be found in such material. Sterilization is  therefore a particularly difficult process. As collagen is  converted to gelatin when exposed to moist heat, auto-  claving cannot be used. The official method is to pack the  ‘plain’ catgut strands (up to 350cm in length) on a metal  spindle in a glass or other suitable container with a tubing  fluid, the purpose of which is to maintain both flexibility  and tensile strength after sterilization. Probably the most  suitable method is to expose the material to gamma radi-  ation. There is minimal loss of tensile strength and the  container can be overwrapped before sterilization to  provide a sterile container surface for opening aseptically.  The alternative method involves placing the coiled suture  immersed in a tubing fluid (commonly 95% ethyl alcohol  with or without 0.002% w/v phenylmercuric nitrate) and  storing for sufficient time to ensure sterilization. The    outer surface of the vial must be sterilized before opening  to avoid contamination of the suture when removed.  Therefore the vial is immersed in 1% w/v formaldehyde  in ethanol before use. It cannot be heated. A non-official  method of sterilization is to immerse the catgut in a non-  aqueous solvent (naphthalene or toluene) and heat at  160 °C for 2 hours. The catgut becomes hard and brittle  during the process, and is aseptically transferred to an  aqueous tubing fluid to restore its flexibility and tensile  strength.   Catgut is packed in single threads up to 350 cm in  length of various thicknesses related to tensile strength in  single-use glass or plastic containers that cannot be  resealed after use. Any remaining material should be dis-  carded. Hardened catgut is prepared by treating strands  with certain agents to prolong resistance to digestion. If  hardened with chromium compounds, the material is  known as chromicized catgut.   Sutures and ligatures are also made from many materials  not absorbed by the body tissues. These consist of  uniform strands of metal or organic material that will  not cause any tissue reactions and are capable of  being sterilized. Depending on the physical stability of  each material, they are preferably sterilized by autoclav-  ing or gamma radiation. They are packed in single-dose  sachets, either dry or surrounded by a preserving  fluid with or without a bactericide. The different materi-  als are described in the British Pharmacopoeia (2010);  they include linen (adversely affected by gamma rays),  nylon (either monofilament or plaited), silk and  polypropylene.   The method chosen for sterilization of instruments (see  Table 22.3) depends on the nature of the components  and the design of the item. The wide range of instru-  ments that may be required in a sterile condition  includes syringes (glass or plastic disposable), needles,  giving sets, metal surgical instruments (e.g. scalpels, scis-  sors, forceps), rubber gloves, catheters, etc. Relatively  complicated equipment such as pressure transducers,  pacemakers, kidney dialysis equipment, incubators and  aerosol machine parts may also be sterilized. Artificial  joints could also be included in the vast range of items  required in a sterile condition in modern medical prac-  tice. The choice of method depends largely on the physi-  cal stability of the items and the appropriate technique in  particular situations. For instance, incubators necessitate    a chemical method of sterilization. On the other hand,  even delicate instruments like pressure transducers are  now available that can withstand autoclaving.   Sterilization processes are discussed in detail in Chapter  21. However, it is axiomatic that whatever method is  chosen, the process should not cause damage to the  product. By reference mostly to moist heat sterilization  processes (the reader should remember that there are  parallel approaches to other methods of sterilization) this  section illustrates the factors that must be considered in  the design of a sterilization process.   The simplest method of sterilization, for an aqueous  product, is to expose it to the standard moist heat steri-  lization conditions, i.e. holding the product at 121 °C for  15 minutes, a process termed overkill. These conditions  are quite severe and therefore milder conditions might be  considered, i.e. a lower holding temperature than 121 °C,  or a shorter holding period than 15 minutes for a product  prone to degradation. The minimum holding period for  moist heat sterilization might be considered to be 8  minutes at 121 °C. However, in reality a slightly shorter  holding period may be satisfactory if the lethality of the  whole autoclave cycle (including heat-up and cooling  phases) is calculated using P 0 values (Chapter 21) and  shown to afford the requisite SAL. F 0 values of 8 minutes  or more are normally considered satisfactory. Use of  lower temperatures and times gives an autoclave process  partly based on the initial bioburden and partly on the  known stability of the product.   Where it is not possible to sterilize a product in its final  container by terminal heat sterilization at 121 °C for 15  minutes, decisions have to be made to use an alternative  method. The options include filtration in combination  with aseptic processing, but readers should note that  aseptic processing by itself is not a method of steriliza-  tion, rather of preventing contamination of the product  whilst it is manufactured from individually sterilized  components.   The European Agency for the Evaluation of Medicinal  Products in 2000 produced an Annex for Guidance on  Development Pharmaceuticals (CPMP/QWP/ 155/96)  showing decision trees for the selection of sterilization  methods. The tree for the sterilization choices for aqueous  products is shown in Figure 22.1. The initial premise is   Autoclave not recommended; difficulty with  steam penetration unless plungers and barrels  sterilized separately   Cutting edges should be protected from  mechanical damage during the process   If autoclave used, care should be taken with  drying at end of process. Little oxidative  degradation when high-vacuum autoclave used   Chemicals not recommended; may be  microbiologically ineffective, may present  hazard to patient safety by compromising the  safety devices on the machine    Moist heat  ( low- temperature  steam, or hot  water at 80°C) C   b Ethylene oxide sterilization is a difficult process to control and the UK Department of Health discourages its use in hospitals.  c Low- temperature steam with formaldehyde is of value in the sterilization/disinfection of some heat- sensitive materials.  d Chemical agents, e.g. glutaraldehyde, hypochlorite.    Figure 22.1 Decision tree for  sterilization choices for aqueous  products. (From EMA (2000), CPMP/  QWP/054/98.)   that if the products may be sterilized at 121 °C for 15  minutes, that process should be used. The next alternative  is that if the product is stable when an F 0 of 8 minutes or  more can be used, then the reduced moist heat process  should be undertaken. If heat processes are unsuitable  (an F 0 < 8 minutes will not achieve the necessary SAL),  then filtration through a microbial filter should be chosen  as the process to render the product sterile. If that process  cannot be utilized, then presterilizing of stable compo-  nents and aseptic compounding and filling must be con-  sidered. The described methods generally show decreasing  levels of sterility assurance on moving down the tree. It  is therefore imperative to remember that the highest level  of sterility assurance is achieved in conjunction with the  lowest presterilization bioburden. The use of inappropri-  ate heat-labile packaging material cannot by itself be the  reason for the use of aseptic processing, and any manu-  facturer should use the best sterilization method achiev-  able for a given formulation before selecting the packaging    material. The manufacture of biotechnology products,  which are typically heat-labile peptides, proteins or  nucleic acids, will provide a challenge as their overall  stability dictates their positioning near the bottom of the  decision tree. They may require sterilization by submi-  cron (<0.1|im) filtration and filling and finishing using  aseptic processes. The overall SAL for terminally steri-  lized products should be less than 1(T 6 and for aseptically  produced products less than 1CT 3 .   Figure 22.2 gives the decision tree for sterilization  choices for non-aqueous liquid, semisolid or dry powder  products. Intermediary decisions, based on radiation and  not found with aqueous products, may be seen in the tree.   Certain issues of product instability may be resolved by  formulation or careful selection of vehicle. Aminophylline  injection, for example, is a solution of the drug in Water  for Injections free from carbon dioxide, as the presence   Figure 22.2 Decision tree for  sterilization choices for non-aqueous  liquid, semisolid or dry powder products.  (From EMA (2000), CPMP/  QWP/054/98.)    of this gas causes precipitation of the active ingredient.  Similarly, promethazine injection is a solution of the  active ingredient in Water for Injections free from dis-  solved air, as the presence of oxygen would cause  promethazine oxidation. Removal of these gases can be    accomplished by prior boiling; additionally, the product  may be packed under an atmosphere of nitrogen to elimi-  nate oxygen from the headspace in the ampoule.   Formulations may be further stabilized by the inclu-  sion of inactive ingredients with specific functions.    Although the British Pharmacopoeia (2010) describes  chloramphenicol eye drops as a sterile solution of chlo-  ramphenicol in purified water, normally the system is  buffered for stability with a boric acid/sodium borate  buffer (see Table 22.1). Sodium metabisulphite may be  found in many products as an antioxidant to prevent  degradation of the active, examples being promethazine  injection and adrenaline injection. The presence of anti-  microbial preservatives may be found in multiple-dose  products, to prevent microbial growth following con-  tamination during use. Many of these formulation  considerations relate to stability of the product during  storage, but an understanding of thermostability is  required for the selection of the appropriate sterilization  process.   The choice of sterilization method depends on the  thermostability of the active ingredient. Moist heat steri-  lization can only be applied to drugs that are heat-stable  in aqueous solution and are not subject to hydrolysis.  Where aqueous solutions are so unstable that chemical  stabilization is impossible, consideration should be given  to sterilization of the drug itself by dry heat processes  (160°C for 2 hours or its equivalent at higher tempera-  tures) in its final container and dissolution immediately  before use by the addition of sterile Water for Injections  BP. For drugs which are both thermolabile and unstable  in aqueous solution, a sterile solution of the drug may be  freeze-dried in its final container and is again reconsti-  tuted as above just before use. Examples include many  antibiotics and Hyaluronidase Injection BP.   It is not the aim of this section to review the entirety of  quality control of sterile products or of the chemical  assays and requirements of drugs and excipients prior to  formulation. Only those techniques with importance  either to microbiology or the confirmation of sterility of  the final product are introduced here.   It should be obvious from previous sections that a suc-  cessful sterilization process is dependent on a product  having a low presterilization bioburden. This will also be  true of the individual ingredients, which must have low  levels of microbial contamination, or else there is a  danger that the contaminants will find their way into the  final product or be a source of pyrogens (see section 4.4).    Sterilization should normally be considered as the  removal of the bioburden, but the high heat resistance of  bacterial endotoxins means that successful steam sterili-  zation does not necessarily guarantee that the product  will pass a pharmacopoeial endotoxin test; dead bacteria  are likely to remain pyrogenic.   Underestimating the level of microbial contamination  prior to the terminal sterilization process will lead to a  miscalculation of the sterilization dose requirements to  achieve the desired SAL. The bioburden must be main-  tained within certain limits to justify the chosen steriliza-  tion process. When a higher number of organisms or  more resistant microorganisms are encountered during  manufacture of batches than was determined during the  initial validation, those batches must be assumed not to  be sterile. The bioburden is an estimate of the total viable  count of microorganisms present before sterilization, and  a knowledge of the resistance characteristics of these  organisms is often an integral part of the sterility assur-  ance calculation. To build some degree of safety into the  sterilization process the sterilization conditions should be  set to destroy all the bioburden by assuming that all the  contaminating microorganisms are the most resistant of  the species identified in that bioburden. Sterility assur-  ance, as implied in the schemes shown in Figures 22.1 and  22.2, can only be achieved with a low bioburden and with  fully validated, correctly functioning sterilizers.   The broad basis of the test for sterility is that it examines  samples of the final product for the presence of microor-  ganisms. Theoretically, the test for sterility should be  applied to all products that are designated as sterile.  However, the test does not examine all samples in a batch,  and its results can only be considered valid if all items in  a batch are treated similarly ( British Pharmacopoeia,  2010). Clearly, for products which are terminally steri-  lized this might seem a reasonable assumption but only  if there is uniform heat distribution in an autoclave or  hot air oven or uniform delivery of a radiation dose. With  aseptically produced products there are dangers because  not all items in a batch may have been treated similarly.  A successful test only shows that no microbial contami-  nation was found in the samples examined under the  test conditions. Extension of the result to a whole batch  requires the assurance that every unit in the batch was  manufactured in such a manner that it would also have  passed the test with a high degree of probability. This  highlights the weakness of the test for sterility and why  the controls of sterilization processes are very important    and probably of greater assurance in confirming the ste-  rility of a batch. The test, however, remains one of few  analytical methods that examine a product for sterility;  the practical aspects of sterility tests are considered in  Chapter 21 and the limitations of sterility testing have  been discussed by Moldenhaur & Sutton (2004).   As there are significant limitations with the test for steril-  ity, many authorities place considerable reliance on the  validation and reliable performance of sterilizers and  their sterilization cycles. Parametric release takes this reli-  ance a step further by allowing batches of terminally  sterilized products to be released without being subjected  to the test for sterility. The sterilization cycle will be vali-  dated to have a SAL of 1(T 6 or less as the minimum safety  factor. Validation studies would include heat distribution,  heat penetration, bioburden, container closure and cycle  lethality studies. For a product to be subject to parametric  release, presterilization bioburden testing on each batch  would be completed, and the comparative resistance of  isolated spore-formers checked. Each cycle would include  the use of chemical or biological indicators. It is hoped  that these actions will provide a significantly higher level  of assurance of sterility than provided by the test for  sterility. This requires confirmation that each part of the  manufacturing process has been satisfactorily completed,  the initial presterilization bioburden is within agreed  limits, that the controls for the sterilizing cycle were sat-  isfactory and that the correct time cycles were achieved.  In practice parametric release should only be used when  experience has been gained on a reliably controlled and  adequately validated process and where a relationship has  been proved between end-product testing and in-process  monitoring.   Clearly, reproducibility, regular monitoring and docu-  mentation are required. However, parametric release  would imply abandoning the sterility test, an option that  many manufacturers have not yet adopted, possibly  because of the fear of litigation based on the premise that  any sterile product would, if tested, have passed the test  for sterility.   The discovery that aqueous solutions may lead to an  increase in body temperature when injected into a patient  dates back to the 19th century. The agents responsible for  this fever were termed ‘pyrogens’. In theory a pyrogen is  any substance that, when injected into a mammal, elicits  a rise in body temperature, and substances produced by    some Gram-positive bacteria, mycobacteria, fungi and  also viruses conform to this definition. The most common  pyrogens, however, and those of major significance to the  pharmaceutical industry, are produced by Gram-negative  bacteria and are known as endotoxins; they are lipopoly-  saccharides (LPS) found in the cell envelope (Chapter 3).  The presence of pyrogens in aqueous solutions was first  demonstrated by injection into rabbits whose body tem-  perature was recorded. More sensitive methods have since  been developed, mostly based on the discovery that a  fraction of the horseshoe crab blood reacts with LPS as a  clotting agent.   Two pharmacopoeial limit tests exist. That for pyro-  gens uses rabbits to assess pharmacological activity and  therefore the presence of pyrogens of all kinds. The test  for bacterial endotoxins uses lysed amoebocytes (blood  cells) of the horseshoe crab and is therefore termed the  Limulus amoebocyte lysate (LAL) test. This may be  extended to many drug and device products and clearly  will be developed in the future to assess the presence of  endotoxins in biotechnology products.   The most characteristic effect following injection of  pyrogens into humans is a rise of body temperature, but  it is only one of a number of dose-dependent diverse  effects. Pyrogens elevate the circulating levels of inflam-  matory cytokines, which may be followed by fever, blood  coagulation, hypotension, lymphopenia, neutrophilia,  elevated levels of plasma cortisol and acute-phase pro-  teins. Low doses of pyrogens induce asymptomatic  inflammatory reactions. Moderate doses induce fever  and changes in plasma composition. Injection of high  pyrogenic doses results in shock, characterized by  cardiovascular dysfunction, vasodilation, vasoconstric-  tion, endothelium dysfunction and multiple organ dys-  function or failure and death.   The release of LPS from bacteria takes place after death  and lysis. Many Gram-negative bacteria, e.g. Escherichia  coli and Proteus, Pseudomonas, Enterobacter and Klebsiella  species produce pyrogenic LPS which is composed of two  main parts: a hydrophilic polysaccharide chain with anti-  genic regions, and a hydrophobic lipid group termed  lipid A which is responsible for many of the biological  activities. The molecular size of the polysaccharide chain  is very variable, and consequently the molecular weight  of the LPS may vary from a few thousand to several  million daltons. LPS is unusually thermostable and insen-    sitive to pH changes. Molecules are able to withstand  120 °C for over 3 hours. Extremes of pH are required for  rapid destruction of the LPS.   The sources of pyrogens in parenteral products include  water used at the end stages of the purification and crys-  tallization of the drug or excipients; water used during  processing; packaging components; and the chemicals,  raw materials or equipment used in the preparation of  the product. The presence of endotoxins on devices may  be attributed to water in the manufacturing process, the  washing of components such as filter media to be used  for the manufacture of filters, or the washing/rinsing of  tubing or other plastic devices prior to their sterilization.  Additionally, if the drug is biologically produced, incom-  plete removal of the microorganisms during purification  can result in high endotoxin levels.   Pyrogens have traditionally been assessed using rabbits  which are stored in carefully controlled conditions and  whose temperature is monitored before the administra-  tion of the test product. The British Pharmacopoeia  (2010) describes a test initially based on three rabbits;  the number is progressively increased if the results fall  between the two values (Table 22.4). Samples of the  product under test are injected into the marginal ear vein  at a dose no greater than 10 ml/kg. The animals are moni-  tored for the 3 hour period immediately after injection,  at 30 minute intervals. The test assumes that the  maximum rise in temperature will be detected in this 3  hour period immediately after injection. Table 22.4  describes the criteria for pass or fail as the number of  rabbits used increases to the maximum of 12.   A number of limitations of the rabbit pyrogen test are  recognized. Repeated use of animals leads to endotoxin  tolerance. There is low reactivity to the endotoxin pro-  duced by certain species, e.g. Legionella. There is also  variability in control results when identical standardized  endotoxin preparations are used, which is probably  related to interlaboratory factors and variations due to  seasons, rabbit species and other biological sources. Care  must be taken in testing radiopharmaceuticals, and  certain drugs may themselves elicit a rise in temperature  on administration. The test is therefore inadequate for  radiopharmaceuticals, cancer chemotherapeutic agents,  hypnotics and narcotics, vitamins, steroids and some  antibiotics. The presence of pyrogens may be hidden by  the pharmacological activity of the product’s compo-    nents. Finally the rabbit test is insufficiently sensitive to  detect endotoxin in intrathecal products where only low  levels of pyrogens are acceptable.   EMA (2009) is to encourage the replacement of the  rabbit test with the monocyte activation test (Hoffmann,  2005) for plasma-derived medicinal products. Human  monocytes from cultured cell lines mimic the human  fever reaction in vitro by producing cytokines. Cytokine  release can be determined, usually using enzyme-linked  immunoassay (ELISA).   The LAL test is considerably more sensitive than the  pyrogen test. As mentioned above, although the Legionella  endotoxin is not very pyrogenic to rabbits it is easily  detected by the LAL test. It has been estimated that there  is a 1000-fold difference in sensitivity between the two  tests, but the LAL test only detects endotoxins of Gram-  negative bacteria and not all pyrogens. However, the LAL  test may be used for radiopharmaceuticals.   LAL test reagent comes from the American horseshoe  crab Limulus polyphemus. The endotoxin-induced  coagulation of its blood is based on an enzyme-mediated  interaction of LAL with endotoxins. The reagents are  obtained from the blood of freshly captured horseshoe  crabs whose amoebocytes are concentrated, washed and  lysed with endotoxin-free water. The LAL is separated  from the remaining cellular debris and its activity opti-  mized using metallic cations, pH adjustment and addi-  tives and then freeze-dried. Certain preparations  interfere with the interaction between LAL and endo-  toxin. Chemical inhibitors may cause chelation of the  divalent cations necessary for the reaction, protein  denaturation or inappropriate pH changes. Physical  inhibition may result from adsorption of endotoxin or  be caused by viscosity of the product. Even the type    of glassware may affect the test. Siliconized glassware  or plastic can inhibit gel-clot formation, or prevent accu-  rate spectrophotometric readings of the reaction  end-point.   The samples of products are incubated with LAL at  37 °C. If endotoxins are present a solid gel forms, indicat-  ing the presence of endotoxins. Th e British Pharmacopoeia  (2010) describes six separate methodologies for the test  for endotoxin. These are (A) gel-clot limit test; (B) gel-  clot: semi quantitative; (C) turbidimetric kinetic method;  (D) chromogenic kinetic method; (E) chromogenic end-  point method; and (F) turbidimetric end-point method.  There are checks for interfering factors. Any validated  method may be used, but the gel-clot method is the  referee test in the case of dispute. Coloured products  cannot be tested by turbidimetric and chromogenic  methods, as precipitate formation may be mistaken for a  positive response.   Kinetic LAL methods are claimed to increase the effi-  ciency of large-scale testing, probably important when  validation of depyrogenation cycles or preparation of  components for aseptic processing are required. For all  procedures, test validation must be conducted to rule  out interference, which may be either inhibition or  enhancement. Depyrogenated glassware must be used  throughout.   The gel-clot method is most commonly used. The test  is conducted by adding the LAL reagent to an equal  volume of test solution, agitating and storing at 37 °C  for 1 hour when the end-point is determined by inversion  of the tubes. If a solid clot remains intact, the product  is considered to contain endotoxins. Chromogenic  methods utilize colorimetry but do not depend on the  clottable protein. A synthetic substrate is used that con-  tains an amino acid sequence similar to that of coagulo-  gen, the clottable protein. The activated proclotting  enzyme cleaves a p-nitroanilide chromophore from the  synthetic substrate and the colour produced is propor-  tional to the amount of endotoxin. The turbidimetric  LAL method is based on the fact that an increase in endo-  toxin concentration will cause a proportional increase in  turbidity caused by the precipitation of the clottable  protein, coagulogen. The optical density is read spectro-  photometrically either at a fixed time or constantly for  kinetic assays as turbidity develops. The kinetic methods  depend on the relationship between the logarithm of the  response and the logarithm of the endotoxin concentra-  tion. The end-point methods relate endotoxin levels to  the quantity of chromophore released or the amount of  precipitation.   The limits for endotoxin are based on the dose of the  product. Put simply, the endotoxin limit, EL, which rep-  resents the maximum amount of endotoxin that is  allowed in a specific dose, is inversely related to the dose  of the drug; it may be assessed from the following equa-  tion ( United States Pharmacopeia, 2010):   where K is the threshold human pyrogenic dose of endo-  toxin per kg body weight and M is the maximum human  dose of the product in kg body weight that would be  administered in a single 1 hour period. M recognizes that  the pharmacological effects of endotoxin are dose-  dependent. The endotoxin limit is the level at which a  product is adjudged pyrogenic or non-pyrogenic. Gel-  clot reagent sensitivities are generally in the range 0.015-  0.5EU/ml. As examples of endotoxin limits, the United  States Pharmacopeia (2010) states limits of no more than  0.5EU/ml for Dextrose Infusion, no more than 5EU/mg  promethazine in Promethazine Injection USP, no more  than lOEU/mg of mitomycin in Mitomycin for Injection  USP and no more than 24EU/mg warfarin sodium in  Warfarin Sodium for Injection. The British Pharmacopoeia  (2010) has a limit of 0.25IU/ml in Glucose Intravenous  Infusion; this value is similar for many BP intravenous  infusions. As another example, insulin should contain no  more than lOIU/mg of endotoxin. The endotoxin limit  for drugs gaining access to the cerebrospinal fluid is  reduced to 0.2EU/kg because the intrathecal route is the  most toxic route for endotoxins.   Pyrogens and endotoxins are difficult to remove from  products once present and it is easier to keep components  relatively endotoxin-free rather than to remove them  from the final product. Rinsing or dilution is one way of  eliminating pyrogenic activity provided that the rinsing  fluid is apyrogenic. Closures and vials should be washed  with pyrogen-free water before sterilization. Pyrogens in  vials or glass components may be destroyed by dry heat  sterilization at high temperatures. A recommended con-  dition for depyrogenation of glassware and equipment is  heating at 250 °C for 45 minutes. Pyrogens are also  destroyed at 650 °C in 1 minute or at 180°C in 4 hours.  The British Pharmacopoeia (2010) states that dry heat  at temperatures above 220 °C may be used for the depy-  rogenation of glassware. Sterilizing tunnels are designed    not only to sterilize at 250-300 °C but also to remove  pyrogens (Chapter 21). These processes equate to incin-  eration, although removal by washing, also termed dilu-  tion, may be used. Filtration, irradiation or ethylene  oxide treatment have limited value in reducing pyrogen  or endotoxin loads.   The removal of pyrogens from Water for Injections  (Chapter 24) may be effected by distillation or reverse  osmosis. Distillation is the most reliable method for  removing endotoxin. Care has to be taken to avoid splash-  ing in the still as pyrogens have been carried over in  droplets. Another source of endotoxins is the Water for  Injection system. Generally, circulating hot water at tem-  peratures above 75 °C provides an environment that is  not conducive to microbial growth and thus the forma-  tion of endotoxin. Circulating water at approximately  60 °C causes some concern as some Gram-negative  organisms, e.g. Legionella pneumophila, will survive and  grow at 57 °C. The water-producing systems may be sani-  tized by circulating water at 75-80 °C.   Pyrogen-free water can be produced using an ultrafil-  tration membrane with a nominal molecular weight  limit that is low enough to ensure the removal of  endotoxins under all conditions. Hollow fibre polysul-  phone membranes can be sanitized with sodium hydrox-  ide, which efficiently destroys pyrogens. A nominal  molecular weight limit of 5000 Da should efficiently  remove endotoxins. However, many endotoxin-producing  microorganisms multiply in ambient temperature Water  for Injection systems, especially reverse osmosis (RO)  systems, in which the filters are not absolute and may  be used in series in order to manufacture pyrogen-  free water.   The contribution of Professor Mike Allwood, who wrote  the chapter on sterile products in previous editions of this  book, is gratefully acknowledged. The European Agency  for the Evaluation of Medicinal Products (now the  European Medicines Agency) is acknowledged for per-  mission to use Figures 22.1 and 22.2.    British Pharmacopoeia Commission (2010) British Pharma-  copoeia. The Stationery Office, London.   Cooper, J.F. (2001) The bacterial endotoxins test: past, present  and future. Ear J Parenteral Sci, 6, 89—93.   EMA (2000) Annex to Note for Guidance on Development  Pharmaceutics (CPMP/QWP/155/96): Decision Trees for  Selection of Sterilisation Methods. CPMP/QWP/054/98. http://  www.ema.europa.eu/pdfs/human/qwp/005498en.pdf.  European Medicines Agency, London.   EMA (2009) Guideline on the replacement of rabbit pyrogen  testing by an alternative test for plasma derived medicinal prod-  ucts. EMEA Doc. Ref. EMEA/CHMP/BWP/452081/2007  European Medicines Agency London.   Guerret, J. & Murano, R.A. (2002) The unique challenges of  manufacturing parenteral nutrition products. Eur J Parenteral  Sci, 7, 127-130.   Harper, L. & Lamerton, L. (2009) Parenteral nutrition and dialy-  sis. In: Pharmaceutical Practice, 4th edn (eds A.J. Wingfield,  J.A. Rees & I. Smith.), Chapter 41, pp. 467-481. Churchill  Livingstone, Edinburgh.   Hoffmann, S., Peterbauer, A., Schindler, S. et al. (2005)  International validation of novel pyrogen tests based on  human monocytoid cells, / Immunol Methods, 298, 161-173.   Matthews, B.R. 8c Skinner, F.S. (2006) Aspects of the preservative  requirements for multiple dose eye care products. Eur J  Parenter Pharm Sci, 11, 23-28.   Meyer, B.K. & Shi, L. (2009) Antimicrobial preservatives use in  parenteral products: an overview. Eur J Parenter Pharm Sci,  14, 115-117.   Moldenhaur, J. & Sutton, S.V.W. (2004) Towards an improved  sterility test. / Pharm Sci Technol , 58, 284—286.   Pikal,M. (2007) Freeze drying. In '.Encyclopedia of Pharmaceutical  Technology, 3rd edn (ed. J. Swarbrick), pp. 1807—1833. Informa  Healthcare, New York.   Williams, K.L. (2007) Endotoxin relevance and control over-  view. In: Endotoxins: Pyrogens, LAL Testing and Depyrogen-  ation, 3rd edn (ed. K.L. Williams), Chapter 2, pp. 27—45.  Interpharma Healthcare, New York.    Arora, A., Prausnitz, M.R. & Mitragotri, S. (2008) Micro-scale  devices for transdermal drug delivery Int J Pharm, 364,  227-236.   Robert W. Jones, Shaqil Chaudary, Touraj Ehtezazi and  James L. Ford   Regulatory authorities require that pharmaceutical prod-  ucts be manufactured according to the principles of good  manufacturing practice (GMP) (also referred to as  current good manufacturing practice, cGMP). Such  authorities include the European Union (EU), the UK  Medicines and Health Care Products Regulatory Agency  (MHRA) and the US Food and Drug Administration    (FDA). Products manufactured in the UK for the US  market must satisfy the FDA. GMP guidelines were first  given statutory authority in the USA and published in the  UK in 1971 (see Immel, 2000 and Sharp, 2009 for a more  detailed history).   Compliance must not, however, be seen as a regulatory  burden. Failure in GMP can have massive consequences  for the well-being of the patient and the finances of the  manufacturer. For the manufacturer it can lead to litiga-  tion, losses associated with recall of product or loss of    Hugo and Russell's Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.    licence with attendant bad publicity. Furthermore, an  incident in 2004 of failure in GMP for vaccine manufac-  ture at a UK company led to a major shortage of flu  vaccine in the USA.   Some products, such as injections, must be sterile (see  Chapter 22), while others, such as oral drugs, need not  be sterile but must be free from pathogens that can be  contracted via the oral route (British Pharmacopoeia  Commission, 2010, Appendix XVI D). More space in  the literature is dedicated to quality of sterile products  than that of non-sterile products, but this reflects the  additional quality assurance requirements compared to  those for non-sterile products (Sharp, 2000; Butson &  Hawitt, 2008).   The manufacture of sterile products is carried out in  both industry and hospitals. In the latter, batches tend to  be much smaller, sometimes only one item, and the prod-  ucts are stored for a much shorter time, usually less than  24 hours (Beaney, 2005).   This chapter summarizes measures for the control  during manufacture of one important feature of product  quality: the level of microbial contamination. The chapter  is designed to complement and be read in conjunction  with Chapters 7, 17, 19, 21 and 22. It is not intended as a  manual, but as an explanation of the principles involved,  and more detailed information can be found in Section  8 (References and further reading).   Several terms used in industrial and hospital production  must be defined to enable the reader to follow this  chapter. These definitions are given in sections 2. 1-2.6.  More detailed definitions are to be found in The Rules  and Guidance for Pharmaceutical Manufacturers and  Distributors (2007). The inter-relationship between  quality assurance (QA), GMP, quality control (QC) and  in-process control is shown in Figure 23.1.   The UK Orange Guide ( The Rules and Guidance for  Pharmaceutical Manufacturers and Distributors, 2007)  emphasizes the fundamental point:   Quality assurance is particularly important, and this type  of manufacture must strictly follow carefully established  and validated methods of preparation and procedure. Sole  reliance for sterility or other quality aspects must not be  placed on any terminal sterilization process or finished  product test.   The difficulty in demonstrating quality is that the tests  carried out are designed to show the absence of quality.   Figure 23.1 The inter-relationship between quality assurance,  good manufacturing practice, quality control and in-process  control.    For example, the test for sterility (Chapter 21) involves  taking samples and testing for microorganisms. If 20  samples are tested, 3.4% of the batch needs to be con-  taminated to have a 50% chance of detecting that con-  tamination. That level of contamination represents gross  failure of GMP and problems would normally be detected  by environmental monitoring. Indeed, it is not unknown  for a batch to pass a sterility test but be rejected due to  problems detected during environmental monitoring  (section 5.2). Therefore it is important that a product be  manufactured in a suitable environment by a procedure  that minimizes the possibility of contamination occur-  ring. At the end of this process the tests can be performed  as an additional measure of quality.   There are many definitions of quality (see Sharp, 2000).  For the purpose of pharmaceutical products the term  quality is usually taken to mean fitness for purpose. Not  only must the product have the desired therapeutic prop-  erties, it must also be safe for administration by the route  intended. Sharp (2000, 2001) discussed several meanings  of quality but summarizes it as follows:   in a nutshell, it is fit to be given to a patient in the confi-  dence that it will have the desired effects and not damage  him or her, in any way, through faults in manufacture.   Manufacture is the complete cycle of production of a  medical product. This cycle includes the acquisition of all    raw materials, their processing into a final product, and  subsequent packaging and distribution.   Quality assurance is a wide ranging concept covering all  matters which individually or collectively influence the  quality of a product. It is the total sum of the procedures  needed to ensure the fitness of a pharmaceutical product  for its intended use. QA incorporates GMP plus other  factors.   GMP is that part of QA which is aimed at ensuring the  product is consistently manufactured to a quality appro-  priate for its intended use and to meet the requirements  of the regulatory authorities. GMP requires that: (1) the  manufacturing process is fully defined before it begins;  and (2) the necessary facilities are provided. In practice,  this means that:   • the poor chance of the patient detecting that anything  is wrong   • the dangers to patients of even only a small number  of defective or wrongly labelled items in a batch (and  it is very difficult to detect a small number of defectives).   QC is that part of GMP concerned with sampling,  specifications and testing, as well as the organization,  documentation and release procedures which ensure  that the necessary and relevant tests are carried out, and  that materials are not released for use, nor products  released for sale or supply, until their quality has been  judged satisfactory. For sterile products QC includes  testing for sterility and pyrogens (see Chapters 21 and  22). The Rules and Guidance for Pharmaceutical  Manufacturers and Distributors (2007) states that QC is  not confined to laboratory operations, but must be  involved in all decisions which may affect the quality of    the product. The independence of QC from production  is considered fundamental to the satisfactory operation  of QC.   This comprises any test on a product, the environment  or the equipment that is used during the manufacturing  process. An example of this is testing that an autoclave is  functioning correctly (Gardner & Peel, 1998).   A documented programme that provides a high degree  of assurance that a specific process, method or system  will consistently produce a result meeting predetermined  acceptance criteria.   A pharmaceutical product may become contaminated by  a number of means and at several points during manu-  facture. There are several ways in which this risk can be  minimized. Any such measures require an understanding  of the risks involved (Chapter 17).   GMP is informed by past mistakes and case studies have  been valuable (Friedman, 2004). However a proactive  approach is required. Nowadays a manufacturer is  expected to demonstrate to the regulatory authorities  that an extensive risk assessment has been carried out.  Risk analysis must comply with ICH 9Q (EMEA, 2006)  and is underpinned by a sound understanding of the  process and of the microbial ecology of the environment  and ingredients.   Several methods are employed (see EMEA, 2006;  Kirupakar, 2007), including hazard analysis critical  control points (HACCP), failure mode and effects analy-  sis (FMEA), fault tree analysis (FTA), risk ranking and  filtering (RRF) and hazard operability analysis (HAZOP).  Only HACCP and FMEA are discussed here.   HACCP has been widely used in the food industry and  is becoming more commonly used in the pharmaceu-  tical industry (McCullogh, 2007; Sharp, 2000; WHO,  2003; Whyte, 2010). The original HACCP had seven  steps:    1 Conduct a hazard analysis and identify preventive  measures for each step of the process.   5 Establish the corrective action to be taken when moni-  toring indicates that the critical control points are not in  a state of control.   6 Establish a system to verify that the EIACCP procedure  is working effectively.   However, HACCP has been modified so that it can be  applied quantitatively not only to microbiology but also  to pyrogens and particles (Tidswell, 2004; Whyte, 2010).   FMEA was first used in the engineering industry  (Stamatis, 2003). It involves breaking the process down  into many discrete steps. For each step scales are set for  severity, occurrence and detection. The scores are multi-  plied and compared to an informed score at which risk  becomes unacceptable.   Microorganisms may be transferred to a product from  working surfaces, fixtures and equipment. Pooled stag-  nant water is a frequent source of contamination. Thus  it is essential that all working areas are kept clean, dry and  tidy. Any cracks where microorganisms may accumulate  must be eliminated. All walls, floors and ceilings should  be easy to clean. This entails impervious and washable  surfaces, free from open joints or ledges. Coving should  be used at junctions between walls and floors or ceilings.  All services such as pipes, light fittings and ventilation  points should be sited so that inaccessible recesses are  avoided. A rigorous disinfection policy must be in place  (Chapter 19; Pharmig 2006). All equipment must be easy  to dismantle and clean and should be inspected for clean-  liness before use.   Fall-out of dust- and droplet-borne microorganisms  from the atmosphere is an obvious route for contamina-  tion. ‘Clean’ air (section 4.1.4) is therefore a prerequisite  during manufacturing processes and the spread of dust  during manufacture or packaging must be avoided.  Microorganisms may thrive in certain liquid preparations  and creams and ointments (Chapter 22). The manufac-  ture of such products should, as far as possible, be in a  closed system; this serves a dual purpose as it also pre-  vents evaporative loss.    Personnel are another source of potential contamina-  tion. High standards of personal hygiene are essential.  Operatives should be free from communicable disease  and open lesions on exposed body surfaces. To ensure  high standards of personal cleanliness, adequate hand-  washing and hand-disinfecting facilities and protective  garments, including headgear and gloves, must be pro-  vided. Staff should be trained in the principles of GMP  and in the practice (and theory) of the tasks assigned.  Staff employed in the manufacture of sterile products  should also receive training in basic microbiology.   Raw materials account for a high proportion of the  microorganisms introduced during the manufacture of  pharmaceuticals, and the selection of materials of good  microbiological quality aids in the control of contamina-  tion levels in both products and the environment. It is,  however, common to have to accept raw materials which  have some non-pathogenic microorganisms present and  this must be considered during risk assessment. Whatever  the means of prevention of growth or survival by chemi-  cal or in-process treatment, it should be regarded as criti-  cal and controlled accordingly (Sharp 2000).   Untreated raw materials that are derived from a natural  source usually support an extensive and varied micro-  flora. Products from animal sources such as gelatin,  desiccated thyroid, pancreas and cochineal may be con-  taminated with animal-borne pathogens. For this reason  some statutory bodies such as the British Pharmacopoeia  require freedom of such materials from Escherichia coli  and Salmonella spp. at a stated level before they can be  used in the preparation of pharmaceutical products. The  microflora of materials of plant origin such as gum acacia  and tragacanth, agar, powdered rhubarb and starches  may arise from those indigenous to plants and may  include bacteria such as Erwinia spp.. Pseudomonas spp.,  Lactobacillus spp., Bacillus spp., streptococci, moulds  such as Cladosporium spp., Alternaria spp. and Fusarium  spp. and non-mycelated yeasts, or those introduced  during cultivation. For example, the use of untreated  sewage as a fertilizer may result in animal-borne patho-  gens such as Salmonella spp. being present. Some refining  processes modify the microflora of raw materials; for  example, drying may concentrate the level of spore-  forming bacteria and some solubilizing processes may  introduce waterborne bacteria such as Escherichia coli.  Synthetic raw materials are usually free from all but inci-  dental microbial contamination.   The storage condition of raw materials, particularly  hygroscopic substances, is important, and as a minimum    water activity (Av) of 0.70 is required for osmophilic  yeasts, 0.80 for most spoilage moulds and 0.91 for most  spoilage bacteria, precautions should be taken to ensure  that dry materials are held below these levels (Chapter  17). Some packaging used for raw materials, such as  unlined paper sacks, may absorb moisture and may itself  be subject to microbial deterioration and so contaminate  the contents; for this reason polythene-lined sacks are  preferable. Some liquid or semisolid raw materials  contain preservatives, but others such as syrups depend  upon osmotic pressure to prevent the growth of  osmophiles, which are often present. With this type of  material it is important that they are held at a constant  temperature, as any variation may result in evaporation  of some of the water content followed by condensation  and dilution of the surface layers to give an A„ value  which may permit the growth of osmophiles and spoil  the syrup.   The use of natural products with a high non-  pathogenic microbial count is possible if a sterilization  stage is included either before or during the manufactur-  ing process. Such sterilization procedures (see also  Chapter 21) may include heat treatment, filtration, irra-  diation, recrystallization from a bactericidal solvent such  as an alcohol, or for dry products, where compatible,  ethylene oxide gas. If the raw material is only a minor  constituent and the final product is adequately preserved  either by low A*> chemically or by virtue of its pH, sugar  or alcohol content, an in-process sterilization stage may  not be necessary. If, however, the product is intended  for parenteral or ophthalmic use a sterilization stage is  essential.    The handling of contaminated raw materials as  described previously may increase the airborne contami-  nation level, and if there is a central dispensing area  precautions may be necessary to prevent airborne cross-  contamination, as well as that from contaminated meas-  uring and weighing equipment. This presents a risk for  all materials but in particular those stored in the liquid  state where contamination may result in the bulk being  spoiled.   Many grades of water are used in pharmaceutical manu-  facturing (Table 23.1). Water for manufacturing may be  potable mains water, water purified by ion exchange,  reverse osmosis or distillation, or water for injection pur-  poses (EMEA, 2002).   Most types of water are derived from municipal sup-  plies. Such water is treated, sometimes by filtration, and  always by chemicals, usually chlorine, to render it free  from coliforms. This water is, however, not sterile. Its  microbial and chemical content varies from region to  region and the microbial count can increase on storage.   Water used for parenteral products, known as Water  for Injections or Water For Injection (WFI), must  be virtually apyrogenic (Chapter 22). The British  Pharmacopoeia (British Pharmacopoeia Commission,  2010) and the US Pharmacopeia (2009a) specify an endo-  toxin level of no more than 0.25 IU/ml for WFI. In Europe  such water is usually produced in a still specially designed  to prevent pyrogens from being mechanically carried  over into the distillate. In other countries reverse osmosis  may also be used (US Pharmacopoeia, 2009a), but in    Europe reverse osmosis is not approved (EMEA, 2002).  WFI can be used immediately for the preparation of  injections, provided it is sterilized within 4 hours of water  collection. Alternatively, the water can be kept for longer  periods at a temperature above 65 °C (typically 80 °C) to  prevent bacterial growth with consequent pyrogen pro-  duction. Ultraviolet radiation may be useful for treating  WFI in order to reduce the bacterial count, but this must  not be regarded as a sterilization process (Chapter 21). A  more detailed account of water for pharmaceutical use  may be found in EMEA (2002) and US Pharmacopeia  (2009b).   The manufacturing process must be fully defined and  capable of providing, with the facilities available, a  product that is microbiologically acceptable and con-  forms to specifications. The process must be fully vali-  dated before starting to ensure that it is suitable for  routine production operations. Processes and procedures  must also be subject to frequent reappraisal and should  be re-evaluated when any significant changes are made in  the equipment or materials used.   The lower the microbiological count of the starting mate-  rials, the more readily the quality of the product can be  controlled. Microbiological standards should be set for all  raw materials as well as microbial limits for in-process  samples and the final product. Microbiological quality  assurance also covers the validation of cleaning and dis-  infectant solutions and the monitoring of the production  environment by microbial counts. This monitoring  should be carried out while normal production opera-  tions are in progress. In addition, sterile manufacture  requires extra safeguards. Operators must be adequately  trained and their aseptic technique monitored both by  observation and microbiological testing. Air filter and  sterilizer efficiency must also be evaluated (Chapter 21),  whilst sterility testing (Chapter 21) and, where necessary  testing for pyrogens (Chapter 22), are the final tests on  the finished product.   Documentation is a vital part of quality assurance.  Details of starting materials, packaging materials, and  intermediate, bulk and finished products should be  recorded so that the history of each batch may be traced.  Distribution records must be kept. This information is of  paramount importance in the event that a defective batch  has to be recalled.   Packaging serves a number of functions; it keeps the con-  tents in, it should keep contaminants out and is labelled to  permit identification of its contents. The product is con-  tained within primary packaging. In industry these packages  are then placed inside secondary packaging for storage and  transport. This secondary packaging may take the form of  cartons, boxes, trays or shrink wrapping.   Consideration must be given to both the fabric of  the packaging and its cleaning, and to the actual process  of packaging. Where terminal sterilization is carried out,  the packaging must be suitable for the process. Packaging  of aseptically processed products into a sterile container  (section 3.7) must be carried out in a grade A environ-  ment (Table 23.2).   Packaging material has a dual role and acts both to  contain the product and to prevent the entry of micro-  organisms or moisture which may result in spoilage, and  it is therefore important that the source of contamination  is not the packaging itself. The microflora of a packaging  material is dependent upon both its composition and  storage conditions. This, and a consideration of the type  of pharmaceutical product to be packed, determines  whether a sterilization treatment is required.   Glass containers are sterile on leaving the furnace, but  are often stored in dusty conditions and packed for  transport in cardboard boxes. As a result they may  contain mould spores of Cladosporium spp., Penicillium  spp., Aspergillus spp. and bacteria such as Bacillus spp.  and Micrococcus spp. which originate from the cardboard,  although it can be treated to remove these contaminants.  It is commonplace either to air-blow or wash glass  containers to remove any glass spicules or dust which  may be present, and it is often advantageous to include   Maximum permitted number of particles/  m 3 equal to or above    a disinfection stage if the product is a liquid or semisolid  preparation. Plastic bottles that are either blow- or  injection-moulded have a very low microbial count and  may not require disinfection. They may, however, become  contaminated with mould spores if they are transported  in a non-sanitary packaging material such as unlined  cardboard. Packaging materials that have a smooth,  impervious surface, free from crevices or interstices, e.g.  cellulose acetate, polyethylene, polypropylene, polyvinyl  chloride (PVC), and metal foils and laminates, all have a  low surface microbial count.   Closure liners of pulpboard or cork, unless specially  treated with a preservative, foil or wax coating, are often a  source of mould contamination for liquid or semisolid  products. A closure with a plastic flowed-in liner is less  prone to introduce or support microbial growth than one  stuck in with an adhesive, particularly if the latter is based  on a natural product such as casein. Closures can be steri-  lized by either formaldehyde or ethylene oxide gas if required.   In the case of injectables and ophthalmic preparations  which are manufactured aseptically but do not receive a  sterilization treatment in their final container the packag-  ing has to be sterilized (Figure 23.2b). Dry heat at 170°C  is often used for vials and ampoules. Containers and clo-  sures may also be sterilized by moist heat, chemicals and  irradiation, but consideration of the destruction or  removal of bacterial pyrogens may be necessary (see  Chapter 22). Regardless of the type of sterilization, the  process must be validated and critical control points or  other risk assessment parameters (section 3.1) must be  established.   Methods of sterilization are discussed in Chapter 21 and  the various types of sterile product are described in    Chapter 22. For production purposes an important dis-  tinction exists between sterile products which have been  terminally sterilized (Figure 23.2a) and those which have  not. Terminal sterilization involves the product being  sealed in its container and then sterilized, usually by heat,  but ionizing radiation or, less commonly, ethylene oxide  may be employed. Such a product must be manufactured  in a clean area (sections 4.1. 1-4.1. 8). A product which  cannot be terminally sterilized is prepared aseptically  (Figure 23.2b) from previously sterilized materials or by  sterile filtration; in either case, aseptic filling is a post-  sterilization step. Strict aseptic conditions are required  throughout (section 4.1).   Vaccines, consisting of dead microorganisms, micro-  bial extracts or inactivated viruses (see Chapter 24) may  be filled in the same premises as other sterile medicinal  products, so the completeness of killing or removal of  live organisms must be validated before processing.  Separate premises are needed for the filling of live or  attenuated vaccines and for the preparation of other  products derived from live organisms. Non-sterile prod-  ucts and sterile products must not be processed in the  same area.   Sterile production should be carried out in a purpose-  built unit separated from other manufacturing areas and  thoroughfares. The unit should be designed to encourage  separation of each stage of production but should ensure  a safe and organized workflow. A plan of such a facility  is shown in Figure 23.3. Sterilized products held in quar-  antine pending sterility test results (Chapter 21; Sharp,  1997) must be kept separate from those awaiting  sterilization.    Figure 23.3 Example of a diagrammatic  representation of the layout and workflow  of a sterile products manufacturing unit. 1,  the changing area in this example is built  on the black (A)-grey (B)-white (C)  principle; passage into the clean area is  through A and B (see section 3.1.6) whereas  entry to the aseptic area is first through A  and B followed by C (see section 3.2.2). 2,  Dividing step-over sill. 3, For details of  aseptic area requirements, see text; a  laminar airflow work station would be  included in this area. 4i-4iv, These areas are  clean areas. In filling rooms for terminally  sterilized products, care should be exercised  to protect containers from airborne  contamination. The final rinse point (i.e.  where the containers are finally washed)  should be sited as near as possible to the  filling point. 5, Articles which are to be  transferred directly to the aseptic area from  elsewhere must be sterilized by passage  through a double-ended sterilizer. Solutions  manufactured in the clean area may be  brought into the aseptic area through a  sterilizing- grade membrane filter. 6,  Double-doored hatchway through which  presterilized articles may be passed into the  aseptic area (see section 3.2.3). Note:  inspection, holding and final packaging  areas have been omitted. Direction of   Particulate, as well as microbial, contamination must be  prevented. To this end all surfaces must be smooth and  impervious in order to: (1) prevent accumulation of dust  or other particulate matter; and (2) permit easily repeated  cleaning and disinfection. Smooth rounded coving  should be used where the wall meets the floor and the  ceiling.   Suitable flooring may be provided by welded sheets of  PVC; cracks and open joints which might harbour dirt  and microorganisms must be avoided. The preferred sur-  faces for walls are plastic, epoxy-coated plaster, plastic  fibreglass or glass-reinforced polyester. Often the final    finish for the floor, wall and ceiling is achieved using  continuous welded PVC sheeting. False ceilings should be  adequately sealed to prevent contamination from the  space above. Use should be made of well-sealed glass  panels, especially in dividing walls, to ensure good visibil-  ity and allow satisfactory supervision. Doors and windows  should be flush with the walls. Windows should not be  openable.   Internal fittings such as cupboards, drawers and shelves  should be kept to a minimum. They must be sited where  they do not interfere with the laminar flow of the filtered  air supply. Stainless steel or laminated plastic are the  preferred materials for such fittings. Stainless steel    trolleys may be used to transport equipment and materi-  als within the clean and aseptic areas but must remain  confined to their respective units. Equipment must be  designed so that it may be easily cleaned and sterilized or  disinfected.   Clean and aseptic areas must be adequately illuminated;  lights are best housed in translucent panels set in a false  ceiling. Electrical switches and sockets must be flush with  the wall or fitted outside. When required, gases should be  pumped in from outside the unit. Pipes and ducts, if they  must be brought into the clean area, must be sealed  through the walls. Additionally, in order to prevent dust  accumulation, pipes and ducts must be boxed in or  readily cleanable. Alternatively, they may be sited above  false ceilings.   Sinks should be of stainless steel with no overflow, and  water must be of at least potable quality. Wherever pos-  sible, drains should be avoided. If installed they must be  fitted with effective, readily cleanable traps and with air  breaks to prevent backflow. Any floor channels should be  open, shallow and cleanable and connected to drains  outside the area; they should be monitored microbiologi-  cally. Sinks and drains should be excluded from aseptic  areas except where radiopharmaceuticals are being proc-  essed when sinks are a requirement.   Areas for sterile manufacture are classified according to  the required characteristics of the environment. Each  operation requires an appropriate level of microbial and  particulate cleanliness; four grades (Table 23.2) are speci-  fied in The Rules and Guidance for Pharmaceutical  Manufacturers and Distributors (2007). Environmental  quality is substantially influenced by the air supplied to  the manufacturing environment. The grades of air  required for specific manufacturing activities are listed in  Table 23.3.   Filtered air (Chapter 22) is used to achieve the neces-  sary standards; this should be maintained at positive  pressure throughout a clean or aseptic area, with the  highest pressure in the most critical rooms (aseptic or  clean filling rooms) and a progressive reduction through  the preparation and changing rooms (Figure 23.4); a  minimum pressure differential of lOkPa is normally  required between each class of room. A minimum of  20 changes of air per hour is usual in clean and aseptic  rooms. The air inlet points should be situated in or  near the ceiling, with the final filters placed as close as    Table 23.3 Operations carried out in the various grades  of air   Wash and dry hands and forearms.  Put on sterile hood and mask.  Rewash and dry hands, forearms    possible to the point of input to the room. Equipment or  furnishings must be sited so as not to interfere with  laminar flow.   The greatest risk of contamination of a product comes  from its immediate environment. Additional protection  is needed both in the filling area of the cleanroom and in  the aseptic suite. This can be provided by a workstation    supplied with a unidirectional flow of filtered sterile air.  This is known as a laminar flow cabinet. Displacement of  air may be vertical or horizontal with a typical homoge-  neous air flow of 0.45 m/s at the working position.  Consequently airborne contamination is not added to the  work space, and any generated by manipulation is swept  away by the laminar air currents. A fuller description of  high efficiency particulate air (HEPA) filters in laminar  flow cabinets is given by Gardner and Peel (1998).   The efficacy of the filters through which the air is  passed should be monitored at predetermined intervals.  Air quality may be monitored for bacteria and fungi by  slit sampler or settle plate. Particles are measured using a  discrete airborne particle counter. The latest edition of  The Rules and Guidance for Pharmaceutical Manufacturers  and Distributors (2007) states that particles must be  monitored continuously in a grade A area and recom-  mends it for grade B areas. It should be noted that grade  A air is not the purest that can be obtained; four even  cleaner grades are used in the electronics industry  (IS014644-1).   Clothing worn in a clean area must be of non-shedding  fibres; polyester is a suitable fabric. Airborne contamina-  tion, both microbial and particulate, is reduced when  trouser suits, close-fitting at the neck, wrists and ankles,  are worn. Clean suits should be provided once a day, but  fresh headwear, overshoes and powder-free gloves are  necessary for each working session. Special laundering  facilities are desirable. Additional requirements for  aseptic rooms are discussed in section 5.1.   Entry to a clean or aseptic area should be through a  changing room fitted with interlocking doors; this design  acts as an airlock to prevent influx of air from the outside.  This route is for personnel only, not for the transfer of  materials and equipment. Staff entering the changing  room should already be clad in the standard factory  or hospital protective clothing. For entry into a clean  area, passage through the changing room should be from  a ‘black’ to a ‘grey’ area, via a dividing step-over sill  (Figure 23.4). Movement through these areas and finally  into the cleanroom is permitted only when observing a  strict protocol, whereby outer garments are removed in  the ‘black’ area and cleanroom trouser suits donned in  the ‘grey’ area. Only after hand-washing in a sink fitted  with elbow- or foot-operated taps may the operator enter  the cleanroom.    The changing procedure for entry to an aseptic area is  described in section 5.1.2.   A strict, validated disinfection policy is necessary if  microbial contamination is to be kept to a minimum.  Cleaning agents used include alkaline detergents and  ionic and non-ionic surfactants. A wide range of chemi-  cal disinfectants is available (Chapter 19). Clear, soluble  phenolics are commonly used for interior services and  fittings. Disinfectants for working surfaces are alcohols  (70% ethanol or isopropanol) or, less commonly,  chlorine-based agents such as hypochlorites. Skin may be  disinfected with cationic detergents such as cetrimide or  chlorhexidine, usually formulated with 70% alcohol to  avoid the need for rinsing. Gloved hands may be disin-  fected with these detergents or 70% alcohol. The former  have the advantage of offering residual activity. Rotation  of different disinfectants reduces the risk of the emer-  gence of resistant strains, but such rotation should be  validated. In-use dilutions must not be used unless steri-  lized. Disinfectants and detergents for use in grade A/B  areas must be sterile prior to use and formulated with  water for injections. Modern sprays are fitted with devices  to prevent air being sucked back, extending the life of the  disinfectant. As mentioned in section 4.1.2, smooth pol-  ished surfaces are more readily cleaned. Floors and hori-  zontal surfaces should be cleaned and disinfected daily,  walls and ceilings as often as required, but the interval  should not exceed 1 month. Regular microbiological  monitoring should be carried out to determine the effi-  cacy of disinfection procedures. Records must be kept  and immediate remedial action taken should normal  levels for that area be exceeded.   The number of persons involved in sterile manufacture  should be kept to a minimum to avoid the inevitable  turbulence and shedding of particles and organisms asso-  ciated with the operatives. All operations should be  undertaken in a controlled and methodical manner as  excessive activity may increase turbulence and particle  shedding.   Containers made from fibrous materials such as paper,  cardboard and sacking are generally heavily contami-  nated (especially with moulds and bacterial spores)  and should not be taken into clean areas. Ingredients  which must be brought into clean areas must first be  transferred to suitable metal or plastic containers.  Containers and closures for terminally sterilized products    must be thoroughly cleaned before use and should  undergo a final washing and rinsing process in apyro-  genic distilled water (which has been passed through a  bacteria-proof membrane filter) immediately prior to  filling. Containers and closures for use in aseptic manu-  facture must, in addition, be sterilized after washing and  rinsing in preparation for aseptic filling (Figure 23.2).   Additional requirements for aseptic areas, over and above  those discussed in sections 4. 1.1-4. 1.8, are discussed  below.   Requirements in addition to those in section 4.1.5 are  needed for aseptic areas. The operative is a potential  source of microorganisms and it is imperative that steps  are taken to prevent this contamination. The operative  must wear sterile protective headwear totally enclosing  hair and beard, spectacles, powder-free rubber or plastic  gloves (often two pairs are worn), a non-fibre-shedding  facemask (to prevent the release of droplets) and foot-  wear. A suitable garment is a one- or two-piece trouser  suit. Fresh sterile clothing should be provided each time  a person enters an aseptic area.   Entry to an aseptic suite is usually through a ‘black-grey-  white’ changing procedure (Figure 23.4), where white rep-  resents the highest level of cleanliness. Movement from  ‘black’ to ‘white’ is via two changing rooms, the ‘grey’ area  also serving as an entry to the cleanroom (Figure 23.4 and  section 4.1.6). There are several types of entry system in  use. More details may be found in Whyte (2010).   Any articles entering the aseptic area should ideally be  sterilized, but may be disinfected. In order to achieve this,  articles should be transferred via a double-ended steri-  lizer or hatch ( i.e . with a door at each end). If they are  not to be discharged directly to the aseptic area, they  should be (1) double-wrapped before sterilization; (2)  transferred immediately after sterilizing into a clean envi-  ronment until required; (3) transferred from this clean  environment via a double-doored hatch (where the outer  wrapping is removed) to the aseptic area (where the inner    wrapper is removed at the workbench). Hatches and  sterilizers must be designed so that only one door may be  opened at any one time. Solutions manufactured in the  cleanroom may be brought into the aseptic area through  a sterile 0.22 pm membrane filter.   Workbenches, including laminar flow units, and  equipment, should be disinfected immediately before and  after each work session. Equipment must be of the sim-  plest design possible for the operation being performed.   Aseptic manipulations must be carried out in the  grade A air of a laminar flow cabinet or isolator. Speed,  accuracy and economy of movement are essential fea-  tures of good aseptic technique. It is therefore essential  that workers are well trained and motivated and familiar  with the task in hand. Observation and microbiological  monitoring of the operator and of the environment are  very important. Under no circumstances must living  microorganisms, including those used for vaccine prepa-  ration (Chapter 24) and for biological monitoring be  introduced into the aseptic area.   Monitoring of the environment is essential during manu-  facturing. It ensures that environmental requirements are  being met and also helps spot trends.   Air is monitored for particles (section 4.1.4) and  microorganisms. Microorganisms are usually sought  using settle plates or active samplers, such as the slit-to-  agar sampler. Settle plates rely on organisms falling from  the atmosphere and settling onto an exposed agar plate.  After a specified time (usually 4 hours) the plate will be  covered and incubated. A slit-to-agar sampler draws in a  specified volume of air, forcing organisms onto the  surface of an agar plate. This latter method is able to give  a viable count per volume, but organisms may be damaged  and hence rendered non-viable by the capture process.  Limits of viable counts for different grades of air are  shown in Table 23.4. One of the limitations of traditional  microbial detection is the time taken to culture bacteria  and fungi. There is a great deal of interest in developing  rapid (Denyer, 2007) or instantaneous (Jiang, 2009)  methods of microbial detection.   The nature of contamination can be informative. For  example, the presence of Staphylococcus spp. suggests  human-borne contamination. The adequacy of changing  facilities and gowning would then be checked. In con-  trast, Bacillus spores would suggest environmental con-  tamination and the entry of equipment into the  cleanroom would be scrutinized.    Glove prints are taken by applying four fingers and a  thumb to an agar plate. This ensures that disinfection of  gloves is adequate. Surfaces may be monitored by swap-  ping or by using contact plates. The latter have the advan-  tage of providing a quantitative measure of surface  contamination, but there is a risk of leaving agar deposits  on the surface (Butson & Hawitt, 2008).   The greatest source of contamination in the cleanroom  comes from the operating staff (Whyte & Hejab, 2007).  Movement of staff can increase particle shedding and  disrupting laminar airflow. It is not surprising, therefore,  that modern practices seek to minimize or even eliminate  humans from the aseptic production area. This can be  achieved by the use of automation, of isolators and of  restricted access barriers (RABs).   All aseptic packaging should be carried out in a grade  A environment with a grade B background (Table 23.2).  Advances in technology now permit the production of  self-contained workstations, or isolators, which incorpo-  rate many of the design principles of cleanrooms and  laminar flow cabinets.   The isolator both protects the product from contamina-  tion by the operator and the operator from any  hazardous materials. Direct interaction between the  operator and the product is minimized by providing  a grade A laminar flow of air with a positive pressure,  the internal space being accessed by means of a glove/  sleeve system (Figure 23.5). A grade D background  (Table 23.2) is considered adequate for such operations.  A fuller account of isolators is given by Midcalf et al.  (2004).   Restricted access barrier systems (RABS) provide a level  of control intermediate between an isolator and a clean-  room (Agalloco & Akers, 2006). They allow for easier  intervention than an isolator but require a grade B  background.   Blow-fill-seal units are purpose-built pieces of equip-  ment which carry out these three steps in a continuous  process within a controlled environment. Containers are  formed from thermoplastic granules and blown to form  containers which are then filled and heat-sealed. These  units are fitted with a grade A air shower and operated in  a grade C environment for aseptic manufacture and a  grade D background for products which are to be termi-  nally sterilized.   Between 1971 and 1983 the essential features of GMP  were covered in the UK by three editions of the Guide to  Good Pharmaceutical Manufacturing Practice , frequently  referred to as the ‘Orange Guide.’ This guide was prepared  by the UK Medicines Inspectorate in consultation with  industrial, hospital, professional and other interested  parties. The principles of this national guide were subse-  quently assimilated into the EC Guide to Good  Manufacturing Practice for Medicinal Products in 1989  and are now published as Rules and Guidance for  Pharmaceutical Manufacturers and Distributors (2007) by  the MHRA. The FDA has published FDA Requirements  for cGMP Compliance (2007). Two important publica-  tions from the Pharmaceutical Press are Quality in the  Manufacture of Medicines and Other Health Care Products  (Sharp, 2000) and Quality Assurance of Aseptic Preparation  Services (Beaney, 2005), which discusses manufacturing  in hospitals.   Compliance with GMP is one of the major factors  considered by the licensing authority when examining an  application for a licence to manufacture under the  Medicines Act (1968). Similar codes exist in the USA and  other countries.   GMP is not just a process; it is a way of thinking. All staff  should be well trained and motivated and be working to  a common goal: the production of a pharmaceutical  product of a quality that is safe for the patient. The pro-  cedures should not be seen as a chore or burden to make  work more difficult, but essential steps in the production  of a safe, satisfactory product. Self-inspection and exter-  nal audit of procedures are important processes in main-  taining standards of cleanliness. Even after manufacture  and distribution it is vital that the products are used  properly, especially multiuse containers that are subject  to potential in-use contamination.   The manufacture of non-sterile products requires that  certain standards of cleanliness, personal hygiene, pro-  duction methods and storage must be met. Many such  products are for oral and topical use and one might  wonder why such stringent parameters need be in place.  However, there have been controlled hospital studies and  case reports associating these products with nosocomial    (hospital-acquired) infection (Chapter 16). Furthermore,  methods of controlling pathogens also control spoilage  organisms (Chapter 17), which could cause the industry  considerable expense. Spoilage organisms can alter the  aesthetic qualities (such as smell, taste and appearance),  physical properties (pH, viscosity) and efficacy of the  product, in addition to producing toxins.   Greater stringency is required for terminally sterilized  products. Such environmental and process controls  might seem overzealous, but it is better to minimize risk  at all stages rather than to rely on final product testing  (section 2.5). The lower the bioburden, the easier it is to  achieve the required sterility assurance level with the ter-  minal sterilization process (Chapter 21). It is also impor-  tant to exclude pyrogens and particulate matter which  would not be removed by the sterilization process.   Where products are processed aseptically, even higher  standards of cleanliness are necessary. The importance of  the knowledge and commitment of the operatives cannot  be overemphasized, both in hospital and industry. Most  reported incidents of defective products have been traced  to human rather than technological error.   Agalloco, A.P. & Akers, A.J. (2006) RABS and Advanced Aseptic  Processing PharmTech, May 1 http://pharmtech.ftndpharma.  com/pharmtech/article/articleDetail.jsp?id=322984 (accessed 3  November 2009).   Beaney, A.M. (2005) Quality Assurance of Aseptic Preparation  Services , 4th edn. Pharmaceutical Press, London.   British Pharmacopoeia Commission (2010) British Pharmaco-  poeia. The Stationery Office, London.   Butson, P. & Hawitt, K. (2008) Microbiological Control for  Non-Sterile Pharmaceuticals, Pharmig Monograph No. 2,  Pharmaceutical Quality Group Monograph No. 12. Pharmig,  Stanstead Abbotts/Chartered Quality Institute, London.   Denyer, S.P. (2007) Monitoring microbiological quality: applica-  tion of rapid microbiological methods to pharmaceuticals. In:  Guide to Microbiological Quality Control in Pharmaceuticals  and Medical Devices, 2nd edn. (eds S.P. Denyer & R.M. Baird),  pp. 183-196. CRC Press, Boca Raton, FL.   EMEA (2002) Notes on guidance on quality of water for pharma-  ceutical use. EMEA document EMEA/CVMP/1 15/01 http://  www. emea.europa.eu/pdfs/human/qwp/01 5801en.pdf  (accessed 3 November 2009).   EMEA (2006) ICH Q9 Quality risk management. EMEA Doc. Ref.  EXT/24235/2006. http://www.emea.europa.eu/Inspections/docs/  ICHQ9Step4QRM.pdf (accessed 3 November 2009).   FDA (2007) FDA Requirements for cGMP Compliance. Editor  Cantor Verlag, Aulendorf.    Friedman, R.L. (2004) Routes of contamination: aseptic process-  ing case studies. Eur J Parenter Pharm Sci , 10, 3-7.   Gardner, J.F. 8c Peel, M.M. (1998) Sterilization, Disinfection and  Infection Control. Churchill Livingstone, London.   Immel, B.K. (2000) A brief history of the GMPs: the power of  storytelling. BioPharm , 13, 26-36. http://immelresources.com/  HistoryofGMPs.pdf (accessed 3 November 2009).   Jiang, J.P. (2009) How instantaneous microbial detection can be  used by pharmaceutical manufacturers. Eur J Parenter Pharm  Sci , 14, 103-109.   Kirupakar, B.R. (2007) Quality risk management for pharma-  ceutical industry. Pharm Rev , 5(1). http:/ / www.pharmainfo.net/  reviews/quality-risk-management-pharmaceutical-industry  (accessed 3 November 2009).   Midcalf, B., Phillips, M., Neiger, J.S. 8c Coles, T.J. (2004)  Pharmaceutical Isolators — A Guide to their Application, Design  and Control. Pharmaceutical Press, London.   McCullogh, K.Z. (2007) Bacterial endotoxin testing. In:  Environmental Monitoring for Cleanrooms and Controlled  Environments (ed. A. M. Dixon), pp. 129-179. Informa  Healthcare, New York.   Pharmig (2006) A Guide to Disinfectants and their use in the  Pharmaceutical Industry. Pharmig, Stanstead Abbotts.   Rules and Guidance for Pharmaceutical Manufacturers and  Distributors (2007) Pharmaceutical Press, London.   Sharp, J. (1997) Quality Rules in Storage, Materials Handling and  Distribution. John Sharp, Woodley.   Sharp, J. (2000) Quality in the Manufacture of Medicines  and Other Health Care Products. Pharmaceutical Press,  London.   Sharp, J. (2001) Quality Rules: A Short Guide to Drug Products  GMP. CRC Press, Boca Raton, FL.    Sharp, J. (2009) GMP — origins to today: some personal reflec-  tions. Eur J Parenter Pharm Sci, 14, 24-30.   Stamatis, D.H. (2003) Failure Mode and Effect Analysis: FMEA  from Theory to Execution. ASQ Quality Press, Milwaukee.   Tidswell, E.C. (2004) Risk profiling pharmaceutical manufactur-  ing processes. Eur J Parenter Pharm Sci, 9, 49-55.   Tidswell, E.C. 8c McGarvey, B. (2007) Quantitative risk model-  ling assists parenteral batch disposition. Eur J Parenter Pharm  Sci, 12, 51-57.   US Pharmacopeia (2009a) <123 1> Water for injection USP32-  NF27 (pp. 3870—3871). United States Pharmacopeial  Convention, Rockville. MD.   US Pharmacopeia (2009b) <123 1> Water for pharmaceutical  purposes USP32-NF27 (pp. 741-752). United States  Pharmacopeial Convention Rockville. MD.   WHO (2003) Application of hazard analysis and critical control  point (HACCP) methodology to pharmaceuticals. In: WHO  Expert Committee on Specifications for Pharmaceutical  Preparations, 37th report. (WHO Technical Report Series No.  908), Annex 7. World Health Organization, Geneva.   Whyte, W. (2010) Cleanroom Technology: Fundamentals of  Design, Testing and Operation. John Wiley 8c Sons, Chichester.   Whyte, W. 8c Hejab, M. (2007) Particle and microbial air-borne  dispersion from people. Eur / Parenter Pharm Sci, 12, 39-46.   There are two relevant computer-aided learning (CAL) packages  produced by COACS (www.coacs.com, accessed 1 November  2009): Good Manufacturing Practice and Pharmaceutical  Microbiology Assessment.   A range of GMP training videos is available from MVI/Micron  Training, Portsmouth (http://www.mvitraining.com/, accessed  1 November 2009).   Immunological products comprise a group of pharma-  ceutical preparations of varied composition but with a  common pharmacological purpose: the modification of  the immune status of the recipient, either to provide  immunity to infectious disease, or in the case of in vivo  diagnostics, to provoke an indication of immune status  usually signifying previous exposure to the sensitizing    agent. The immunological products that are currently  available are of the following types: vaccines; in vivo diag-  nostics; immune sera; human immunoglobulins; mono-  clonal antibodies; and antibody-targeted therapeutics  and diagnostics. For the purpose of this chapter, cell  biology-derived immunomodifiers with a non-specific  action, e.g. cytokines or chemokines, are not included.   Vaccines are by far the most important immunological  products. They have enabled the control or eradication  of numerous infectious diseases affecting humans and    Hugo and Russell's Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.    their domesticated animals. For example, the systematic  application of smallpox vaccine, deployed under the aegis  of the World Health Organization (WHO), achieved the  eradication of one of the most devastating infections.  Similarly, the universal application of poliomyelitis  vaccine has brought poliomyelitis to the verge of eradica-  tion. Diphtheria, tetanus, pertussis (whooping cough),  measles and rubella vaccines have been applied world-  wide through national or UNICEF-sponsored healthcare  programmes and have virtually eliminated these diseases  in those countries in which there have been the resources  and the will to deploy them effectively. Vaccines that  provide protection against many other infections are  available for use in appropriate circumstances. Some,  such as hepatitis B and conjugate vaccines against  Haemophilus influenzae b (Hib), meningococci and  pneumococci, have had a huge impact on morbidity and  mortality wherever they have been applied.   The range of disorders that may be prevented or  treated by vaccines has enlarged considerably beyond  infectious diseases. Vaccines are currently undergoing  evaluation for several other purposes including therapy  of cancer; prevention of allergies; desensitization of  allergic patients; fertility control; and treatment of  addictions.   In vivo diagnostics such as tuberculins, mallein, histo-  plasmin, coccidioidin and brucellin, are used to demon-  strate an immune response, and hence previous exposure,  to specific pathogens as an aid to diagnosis. Allergen skin  test diagnostics are used to indicate sensitization to mate-  rials of biological origin that may be present in the envi-  ronment or in specific products. Others, such as the  Schick test (diphtheria) toxin are used to detect the pres-  ence of protective immunity. Because of their clinical and  pharmaceutical limitations, the trend has been to phase  out these preparations, and tuberculins (as purified  protein derivative, PPD) are now by far the most impor-  tant of this group.   Immune sera, which were once very widely used in the  prophylaxis and treatment of many infections, have more  limited use today. Vaccines and antibiotics have super-  seded some and lack of proven therapeutic benefit has  caused others to be relegated to immunological history.  However, some still play an important role in the man-  agement of specific conditions. Thus, diphtheria and  botulinum antitoxins prepared in horses remain the only  specific treatments for diphtheria and botulism respec-  tively. Equine tetanus antitoxin is still used as an effective  prophylactic in some parts of the world, although largely  replaced by human tetanus immunoglobulin in devel-    oped countries. Similarly, antivenins prepared in horses,  sheep, goats or other animals against the venoms of  snakes, spiders, scorpions and marine invertebrates still  provide the only effective treatment for venomous bites  and stings and are important therapeutic agents in some  parts of the world.   Human immunoglobulins have important but limited  uses, for example in the prophylaxis of hepatitis A, hepa-  titis B, tetanus and varicella zoster. Additional specific  immunoglobulins against diphtheria and botulism toxins  are under development and vaccinia immunoglobulin  may be reintroduced. Monoclonal antibodies to bacterial  endotoxin, to cytokines involved in the pathogenesis of  septic shock and to specific infectious agents have been  developed and evaluated clinically but have yet to enter  into general use. Monoclonal antibodies against specific  cell receptors have undergone a rapid development and  are employed successfully in cancer therapy and are  under development for treatment of autoimmune disease.  Immune sera and human immunoglobulins depend for  their protective effects on their content of antibodies  derived, in the case of immune sera, from immunized  animals and, in the case of immunoglobulins, from  humans who have been immunized or who have high  antibody titres as a consequence of prior infection. The  form of immunity conferred is known as passive immu-  nity and is achieved immediately but is limited in its  duration to the time that protective levels of antibodies  remain in the circulation (see also Chapter 10).   Vaccines achieve their protective effects by stimulating  the immune system of the recipient to produce T-cells  and/or antibodies that impede the attachment of infec-  tious agents, promote their destruction or neutralize their  toxins. This form of protection, known as active immu-  nity, develops in the course of days following infection  and in the case of many vaccines develops adequately  only after two or three doses of vaccine have been given  at intervals of days or weeks. Once established, this  immunity can last for years but it may need to be rein-  forced by booster doses of vaccine given at relatively long  intervals. The immunogenicity of some vaccines can be  improved by formulating them with adjuvants. These are  a heterogeneous group of substances which enhance the  immune response. Aluminium hydroxide gel (hydrated  aluminium oxide) and aluminium phosphate are the only  ones currently in general use in human vaccines. A much  wider range of substances including oily emulsions,  saponin, immune- stimulating complexes (ISCOMS),  monophosphoryl lipid A, CpG motif contained in oligo-  deoxynucleotide (CpG-ODN) and others are used in    veterinary vaccines, and some are under investigation for  use in human vaccines.   Different types of infectious agent require preferential  mobilization of different arms of the immune response.  For example, toxigenic bacterial infections require the  production of toxin neutralizing antibodies whereas  intracellular bacterial infections such as tuberculosis  require cell-mediated responses involving mixed  T-lymphocytes and activated macrophages, whereas  many viral infections will require neutralizing antibody  and cytotoxic T-cell responses for effective protection.  Achieving the appropriate response can be difficult and  in the past has had to be approached empirically. This is  why most successful viral vaccines have been based on  live attenuated strains, which simulate natural infection.  Non-living vaccines have been effective against many  bacterial infections but markedly less so against those  requiring sustained cell-mediated responses. The devel-  opment of more selective vaccine adjuvants and delivery  systems promises to put the future process of vaccine  design on a more rational basis.   A property common to vaccines, immune sera and  human immunoglobulins is their high specificity of  action. Usually each provides immunity to only one  infection, although in some cases cross-protection can  occur, e.g. BCG protects against both tuberculosis and  leprosy. Where it is necessary to protect against more than  one type of agent, monospecific preparations can be  combined. For example, botulism antitoxin usually  covers types A, B and E; meningococcal polysaccharide  vaccine may cover groups A, C, W125 and Y; and pneu-  mococcal polysaccharide vaccine usually covers 23 sero-  types. Heterologous preparations may also be combined  as in measles/mumps/rubella and diphtheria/tetanus/  pertussis vaccines. With the increasing number of vac-  cines for infants and young children, the trend is to  produce more complex combinations such as diphtheria  /tetanus/pertussis/hepatitis B/inactivated polio/Hib vaccine,  to minimize the number of injections. The possible addi-  tive or interactive effects of the various components on  the immune system have raised concerns about the safety  of such combinations. While some evidence of reduced  responses to certain components has been obtained, there  is little to support suggestions of serious adverse effects  from current combinations.   In addition to the three main types of immunological  products that are widely available, more specialized prep-  arations include: synthetic peptide immune response  modifiers such as those used to block T-cell responses in    multiple sclerosis; monoclonal antibodies for cancer  therapy or diagnosis; and hybrid toxins containing a bac-  terial or plant toxin subunit attached to an antibody or  human cell receptor-binding protein, and also intended  mainly for cancer therapy. These have rather limited  applications and for the most part, are designed to sup-  press or exploit the specificity of immune responses  rather than to stimulate them.   Principles of immunity are discussed in Chapter 9, and  Chapter 10 describes a vaccination and immunization  programme.   The vaccines currently used for the prevention of infec-  tious diseases of humans are all derived, directly or indi-  rectly, from pathogenic microorganisms. The basis of  vaccine manufacture thus consists of procedures which  produce from infectious agents, their components or  their products, immunogenic preparations that are  devoid of pathogenic properties but which, nonetheless,  can still induce a protective response in their recipients.  The methods that are used in vaccine manufacture are  constrained by technical limitations, cost, problems of  delivery to the recipient/patient, by regulatory issues and,  most of all, by the biological properties of the pathogens  from which vaccines are derived. Those vaccines cur-  rently in use in conventional immunization programmes  are of several readily distinguishable types.   These are preparations of live bacteria, viruses or other  agents which, when administered by an appropriate  route, cause subclinical or mild infections. In the course  of such an infection the components of the microorgan-  isms in the vaccine evoke an immune response which  provides protection against the more serious natural  disease. Live vaccines have a long history, dating from the  development of smallpox vaccine. Initially, material from  mild cases of smallpox was used for inoculation. This  process of ‘variolation’ was hazardous and could produce  fatalities and secondary smallpox cases. A much safer  alternative was introduced in 1796 by the Gloucestershire  physician Edward Jenner, following observations made  by Benjamin Jesty, a local farmer, that an attack of the  mild condition known as cowpox (probably a rodent    pox) protected milkmaids from smallpox during epidem-  ics of this dreaded disease. For many years the cowpox  vaccine was propagated by serial transfer from person to  person and at some point evolved into a distinctive virus,  vaccinia, with some features of both cowpox and small-  pox viruses but quite probably derived from a now extinct  poxvirus. Vaccinia was eventually used to eradicate small-  pox. Its significance was that it could stimulate a high  degree of immunity to smallpox while producing only a  localized infection in the recipient.   The natural occurrence of cross-protective organisms  of low pathogenicity seems to be a rare event and attenu-  ated strains have usually had to be selected by laboratory  manipulation. Thus the bacille Calmette-Guerin (BCG)  strain of Mycobacterium bovis used to protect against  human tuberculosis caused by the related species M.  tuberculosis, was produced by many sequential subcul-  tures on ox bile medium. This process resulted in deletion  of many genes present in virulent M. bovis, including  some essential for pathogenicity. Similarly, treatment of  a virulent strain of Salmonella enterica serovar Typhi with  nitrosoguanidine, which produced multiple mutations,  gave rise to the live attenuated typhoid vaccine strain  Ty21A. More recently developed attenuated strains of S.  enterica serovar Typhi and Vibrio cholerae have been  selected by directed mutagenesis processes which can  produce defined mutations in specific genes.   Perhaps surprisingly, nearly all of the most successful  attenuated viral vaccine strains in current use were pro-  duced by empirical methods long before the genetic basis  of pathogenesis by the specific pathogen was understood.  Thus, attenuated strains of polio virus for use as a live,  oral vaccine (Sabin) were selected by growth of viruses  isolated from human cases under cultural conditions that  did not permit replication of neuropathogenic virus.  Comparable procedures were used to select the attenu-  ated virus strains that are currently used in live measles,  mumps, rubella and yellow fever vaccines. A more recent  approach has been to use genetic reassortants to produce  live rotavirus vaccines.   Now, attenuated strains of pathogens can be selected  by deliberate selective modification of genes responsible  for encoding factors determining pathogenesis, such as  toxins or immunomodulators, or metabolites essential  for in vivo growth. Live vaccine strains can also be geneti-  cally modified by incorporating genes that encode pro-  tective antigens of other infectious agents. Several of  these are under evaluation at present e.g. for vaccination  against malaria and tuberculosis.   Killed vaccines are suspensions of bacteria, viruses or  other pathogenic agents that have been killed by heat or  by disinfectants such as phenol, ethanol or formaldehyde.  Killed microorganisms obviously cannot replicate and  cause an infection and so it is necessary for each dose of  a killed vaccine to contain sufficient antigenic material to  stimulate a protective immune response. Killed vaccines  therefore usually have to be relatively concentrated sus-  pensions. Even so, such preparations are often rather  poorly protective, possibly because of partial destruction  of protective antigens during the killing process or inad-  equate expression of these during in vitro culture. At the  same time, because they contain all components of the  microorganism they can be somewhat toxic. It is thus  often necessary to divide the total amount of vaccine that  is needed to induce protection into several doses that are  given at intervals of a few days or weeks. Such a course  of vaccination takes advantage of the enhanced ‘second-  ary’ response that occurs when a vaccine is administered  to an individual person whose immune system has been  sensitized (‘primed’) by a previous dose of the same  vaccine. The best-known killed vaccines are whooping  cough (pertussis), typhoid, cholera, plague, inactivated  polio vaccine (Salk type) and rabies vaccine. The trend  now is for these rather crude preparations to be phased  out and replaced by better-defined subunit vaccines con-  taining only relevant protective antigens, e.g. acellular  pertussis and typhoid Vi polysaccharide vaccines.   Toxoid vaccines are preparations derived from the toxins  that are secreted by certain species of bacteria. In the  manufacture of such vaccines, the toxin is separated from  the bacteria and treated chemically to eliminate toxicity  without eliminating immunogenicity, a process termed  ‘toxoiding’.   A variety of reagents have been used for toxoiding,  but by far the most widely employed and generally suc-  cessful has been formaldehyde. Under carefully control-  led conditions this reacts preferentially with the amino  groups of proteins although many other functional  groups potentially may be affected. Ideally the toxoided  protein will be rendered non-toxic but retain its  immunogenicity.   The treated toxins are sometimes referred to as formol  toxoids. Toxoid vaccines are very effective in the preven-  tion of those diseases such as diphtheria, tetanus, botu-  lism and clostridial infections of farm animals, in which    the infecting bacteria produce disease through the toxic  effects of secreted proteins which enzymically modify  essential cellular components. Many of the clostridial  toxins are lytic enzymes with very specific substrates  such as neural proteins. Detoxification is also required for  the pertussis toxin component of acellular pertussis  vaccines.   Anthrax adsorbed vaccine is not toxoided but relies  on the use of cultural conditions that favour production  of the protective antigen (binding and internalization  factor) rather than the lethal factor (protease) and  oedema factor (adenyl cyclase) components of the toxin.  Selective adsorption to aluminium hydroxide or phos-  phate also slows release of residual toxin.   Rather than use whole cells, which may contain undesir-  able and potentially reactogenic components such as  lipopolysaccharide endotoxins, a more precise strategy is  to prepare vaccines from purified protective components.  These are of two main types, proteins and capsular  polysaccharides. Often more than one component may  be needed to ensure protection against the full range of  prevalent serotypes. The potential advantage of such vac-  cines is that they evoke an immune response only to the  component, or components, in the vaccine and thus  induce a response that is more specific and effective. At  the same time, the amount of unnecessary material in  the vaccine is reduced and with it the likelihood of  adverse reaction. Vaccines that have been based on one  or more capsular polysaccharides include Hib vaccine;  the Neisseria meningitidis ACWY vaccines; the 23-valent  pneumococcal polysaccharide vaccine; and the typhoid  Vi vaccine. These have the disadvantage that they are  T-cell-independent antigens and thus do not evoke  immunological memory or effective protective responses  in the very young. This problem can be overcome by  chemically coupling the polysaccharides to T-cell-  dependent protein carriers.   The pertussis vaccine is another example where, tradi-  tionally, whole bacterial cells have been used, but recent  developments have led to an acellular pertussis vaccine  that may contain detoxified toxin, either alone or com-  bined with several other bacterial antigens.   The performance of certain types of antigen that give  weak or inappropriate immune responses can often be  improved by chemically conjugating them to more  immunogenic carriers. Among others, polysaccharide-    protein, peptide-protein, protein-protein, lipid-protein  and alkaloid-protein conjugate vaccines may be prepared  in this way. These have a wide range of applications,  including prevention of infection, tumour therapy, fertil-  ity control and treatment of addictions. This approach  has been very successful against infections caused by  bacteria that produce polysaccharide capsules. The latter  are T-independent antigens and induce weak responses  without immunological memory. They are particularly  ineffective in the very young.   Three viral subunit vaccines are widely available, two  influenza vaccines and a hepatitis B vaccine. The influ-  enza vaccines are prepared by treating intact influenza  virus particles from embryonated hens’ eggs infected with  influenza virus with a surface-active agent such as a noni-  onic detergent. This disrupts the virus particles, releasing  the virus subunits. The two types that are required in the  vaccine, haemagglutinin and neuraminidase, can be  recovered and concentrated by centrifugation methods.  The hepatitis B vaccine was, at one time, prepared from  hepatitis B surface antigen (HbsAg) obtained from the  blood of carriers of hepatitis B virus. This very con-  strained source of antigen has been replaced by produc-  tion in yeast or mammalian cells that have been genetically  engineered to express HbsAg during fermentation. The  human papillomavirus (HPV) vaccines for prevention of  genital warts and cervical cancer also contain recom-  binant viral proteins.   For further information see Ada (1994), Ellis (1999),  Mizrahi (1990), Pastoret etal. (1997), Perlmann & Wigzell  (1999), Plotkin, Orenstein & Offit (2008) and Powell &  Newman (1995).   The starting point for the production of all microbial  vaccines is the isolation of the appropriate infectious  agent. Such isolates have usually been derived from  human infections and in some cases have yielded strains  suitable for vaccine production very readily; in other  instances a great deal of manipulation and selection in  the laboratory have been needed before a suitable strain  has been obtained. For example, bacterial strains may  need to be selected for high toxin yield or production of  abundant capsular polysaccharide; viral strains may need  to be selected for stable attenuation or good growth in  cell cultures.   Once a suitable strain is available, the practice is to  grow, often from a single viable unit, a substantial volume    of culture which is distributed in small amounts in a large  number of ampoules and then stored at — 70 °C or below,  or freeze-dried. This is the original seed lot. From this  seed lot, one or more ampoules are used to generate the  working seed from which a limited number of batches of  vaccine are generated. These are first examined exhaus-  tively in laboratory and animal tests and then, if found to  be satisfactory, tested for safety and efficacy in clinical  trials. Satisfactory results in the clinical trials validate the  seed lot as the material from which batches of vaccine for  routine use can subsequently be produced.   It is important that the full history of the seed is  known, including the nature of the culture media used to  propagate the strain since isolation. If at all possible,  media prepared from animal products should be avoided.  If this is not practicable, media components must be  from sources certified free of transmissible spongiform  encephalopathy (TSE) agents.   2.3 Production of the bacteria and the  cellular components of bacterial vaccines   The bacteria and cellular components needed for the  manufacture of most bacterial vaccines are prepared in  laboratory media by well-established fermentation  methods. The end-product of the fermentation, the  harvest, is processed to provide a concentrated and puri-  fied bulk lot of vaccine component that may be conven-  iently stored for long periods or even sold to other  manufacturers prior to further processing. It is important  that the materials, equipment, facilities and working  practices are of a standard acceptable for the manufacture  of pharmaceutical products. The requirements for this  are defined as Good Manufacturing Practice (GMP).  Guidelines on the basic requirements have been pub-  lished by WHO (1999a, 1999b).   The production of a bacterial vaccine batch begins with  the recovery of the bacterial seed contained in an ampoule  of the seed lot stored at — 70 °C or below, or freeze-dried.  The resuscitated bacteria are first cultivated through  one or more passages in preproduction media. Then,  when the bacteria have multiplied sufficiently, they are  used to inoculate a batch of production medium. Again,  all media used must be from sources certified free of  TSEs. Wherever possible, medium components of animal  origin, especially human and ruminant, should be  avoided.   The production medium is usually contained in a large  fermenter, the contents of which are continuously stirred.    Usually the pH and the oxidation/reduction potential of  the medium are monitored and adjusted throughout the  growth period to provide conditions that will ensure the  greatest bacterial yield. In the case of rapidly growing  bacteria the maximum yield is obtained after about a day  but in the case of bacteria that grow slowly, e.g. M. bovis  BCG, the maximum yield may not be reached before 2  weeks. At the end of the growth period the contents of  the fermenter, which are known as the harvest, are ready  for the next stage in the production of the vaccine   The harvest is a very complex mixture of bacterial cells,  metabolic products and exhausted medium. In the case  of a live attenuated vaccine it should be innocuous, and  all that is necessary is for the bacteria to be separated and  resuspended under aseptic conditions in an appropriate  diluent, possibly for freeze-drying. In a vaccine made  from a virulent strain of pathogen the harvest may be  intensely dangerous and great care is necessary in the  subsequent processing. Adequate containment will be  required and for class 3 pathogens such as S. enterica  serovar Typhi or Yersinia pestis or bulk production of  bacterial toxins, dedicated facilities that will provide  complete protection for the operators and the environ-  ment are essential.   • Killing. This is the process by which heat and  disinfectants are used to render the live bacteria in the  culture non-viable and harmless. Heat and/or formalin  or thiomersal are used to kill the cells of Bordetella  pertussis used to make whole-cell pertussis vaccines,  whereas phenol was used to kill the V. cholerae and the S.  enterica serovar Typhi cells used in the now obsolete  whole-cell cholera and typhoid vaccines.   • Separation. The process by which the bacterial cells are  separated from the culture fluid and soluble products.  Centrifugation using either a batch or continuous flow  process, or ultrafiltration, is commonly used. Precipitation  of the cells by reducing the pH has been used as an  alternative. In the case of vaccines prepared from cells,  the supernatant fluid is discarded and the cells are  resuspended in a saline diluent; where vaccines are made  from a constituent of the supernatant fluid, the cells are  discarded.   • Fractionation. This is the process by which components  are extracted from bacterial cells or from the medium  in which the bacteria are grown and obtained in a purified  form. The polysaccharide antigens of N. meningitidis  are usually separated from the bacterial cells by treatment  with hexadecyltrimethylammonium bromide followed by    extraction with calcium chloride and selective precipitation  with ethanol. Those of Streptococcus pneumoniae are usually  extracted with sodium deoxycholate, deproteinized and  then fractionally precipitated with ethanol. The purity of an  extracted material may be improved by resolubilization in a  suitable solvent and reprecipitation. These procedures are  often supplemented with filtration through membranes or  ultrafilters with specific molecular size cut-off points. After  purification, a component may be freeze-dried, stored  indefinitely at low temperature and, as required, incorporated  into a vaccine in precisely weighed amount at the blending  stage.   • Detoxification. The process by which bacterial toxins  are converted to harmless toxoids. Formaldehyde is  used to detoxify the toxins of Corynebacterium diphtheriae,  Clostridium botulinum and Cl. tetani. The detoxification  may be performed either on the whole culture in the  fermenter or on the purified toxin after fractionation.  Traditionally the former approach has been adopted,  as it is much safer for the operator. However, the  latter gives a purer product. The pertussis toxin used in  acellular vaccines may be detoxified with formalde-  hyde, glutaraldehyde, or both, hydrogen peroxide or  tetranitromethane. In the case of genetically detoxified  pertussis toxin, a treatment with a low concentration of  formaldehyde is still performed to stabilize the protein.   • Further processing. This may include physical or  chemical treatments to modify the product. For  example polysaccharides may be further fractionated to  produce material of a narrow molecular size specification.  They may then be activated and conjugated to carrier  proteins to produce glycoconjugate vaccines. Further  purification may be required to eliminate unwanted  reactants and by-products. These processes must be done  under conditions that minimize extraneous microbial  contamination. If sterility is not achievable then strict  bioburden limits are imposed.   • Adsorption. This describes the adsorption of the  components of a vaccine on to a mineral adjuvant or  carrier (aluminium hydroxide or aluminium phosphate;  rarely calcium phosphate). Their effect is to increase the  immunogenicity and decrease the toxicity, local and  systemic, of a vaccine. Diphtheria vaccine, tetanus  vaccine, diphtheria/tetanus vaccine and diphtheria/  tetanus/pertussis (whole-cell or acellular) vaccine, are  generally prepared as adsorbed vaccines.   • Conjugation. The linking of a vaccine component that  induces an inadequate immune response with a vaccine  component that induces a good immune response. For  example, the immunogenicity for infants of the capsular    polysaccharide of H. influenzae type b is greatly enhanced  by the conjugation of the polysaccharide with diphtheria  or tetanus toxoid, or with the outer-membrane protein  of N. meningitidis. More recently, in attempts to improve  efficacy, protein carriers that themselves induce a  protective immune response against the pathogen have  been conjugated to the capsular polysaccharide e.g.  Panton-Valentine leucocidin conjugated to Staph, aureus  capsular polysaccharide.   2.5 Production of the viruses and the  components of viral vaccines   Viruses replicate only in living cells, so the first viral  vaccines were necessarily made in animals: smallpox  vaccine in the dermis of calves and sheep, and rabies  vaccines in the spinal cords of rabbits and brains of  mice. Such methods are no longer used in advanced  vaccine production; the only intact animal hosts that are  still used are embryonated hens’ eggs. Almost all the virus  that is needed for viral vaccine production is obtained  from cell cultures infected with virus of the appropriate  strain.   Embryonated hens’ eggs are still the most convenient  hosts for growth of the viruses that are needed for influ-  enza and yellow fever vaccines. Influenza viruses accumu-  late in high titre in the allantoic fluid of the eggs, and  yellow fever virus accumulates in the nervous system of  the embryos. A few bacterial vaccines, e.g. against rickett-  sial and chlamydial agents, are also prepared in embryo-  nated eggs. It is important to use eggs from disease-free  flocks and emphasis is placed on screening the latter for  various avian viruses. The allantoic fluid or embryos  must be harvested under conditions that minimize extra-  neous microbial contamination.   Where cell cultures are used for virus production, they  must be of known origin, obtained from validated sources  and shown to be free of extraneous agents. The media  used in their production should not contain components  of human or animal origin, unless the latter are from  TSE-free sources.   The processing of the virus-containing material from  infected embryonated eggs may take one of several forms.  In the case of influenza vaccines the allantoic fluid is  centrifuged to provide a concentrated and partially puri-  fied suspension of virus. This concentrate is treated with  organic solvent or detergent to split the virus into its    components when split virion or surface antigen  vaccines are prepared. The chick embryos used in the  production of yellow fever vaccine are homogenized  in sterile water to provide a virus-containing pulp.  Centrifugation then precipitates most of the embryonic  debris and leaves much of the yellow fever virus in an  aqueous suspension. Further purification can then be  performed as required.   Cell cultures provide infected fluids that contain little  debris and can generally be satisfactorily clarified by  filtration. Because most viral vaccines made from cell  cultures consist of live attenuated virus, there is no inac-  tivation stage in their manufacture. There are, however,  two important exceptions: inactivated poliomyelitis virus  vaccine is inactivated with dilute formaldehyde or (3-  propiolactone and rabies vaccine is inactivated with (3-  propiolactone. The preparation of these inactivated  vaccines also involves a concentration stage — by adsorp-  tion and elution of the virus in the case of poliomyelitis  vaccine and by ultrafiltration in the case of rabies vaccine.  When processing is complete the bulk materials may be  stored until needed for blending into final vaccine.  Because of the lability of many viruses, however, it is  necessary to store most purified materials at tempera-  tures of-70°C.   Blending is the process in which the various components  of a vaccine are mixed to form a final bulk. It is under-  taken in a large, closed vessel fitted with a stirrer and  ports for the addition of constituents and withdrawal of  the final blend. When bacterial vaccines are blended, the  active constituents usually need to be greatly diluted and  the vessel is first charged with the diluents, usually con-  taining a preservative. Thiomersal has been widely used  in the past but is now being phased out and replaced by  phenoxyethanol or alternatives. A single-component final  bulk is made by adding bacterial suspension, bacterial  component or concentrated toxoid in such quantity that  it is at the required concentration in the final product. A  multiple-component final bulk of a combined vaccine is  made by adding each required component in sequence.  When viral vaccines are blended, the need to maintain  adequate antigenicity or infectivity may preclude dilu-  tion, and tissue culture fluids, or concentrates made  from them, are often used undiluted or, in the case of  multicomponent vaccines, merely diluted one with  another. After thorough mixing, a final bulk may be  divided into a number of moderate-sized volumes to  facilitate handling.   As vaccine is required to meet orders, bulk vaccine is  distributed into single-dose ampoules or into multidose  vials as necessary. Vaccines that are filled as liquids are  sealed and capped in their containers, whereas vaccines  that are provided as dried preparations are freeze-dried  before sealing.   The single-component bacterial vaccines are listed in  Table 24.1. For each vaccine, notes are provided of the  basic material from which the vaccine is made, the salient  production processes and tests for potency and for safety.  The multicomponent vaccines that are made by blending  together two or more of the single-component vaccines  are required to meet the potency and safety requirements  for each of the single components that they contain. The  best-known of the combined bacterial vaccines is the  adsorbed diphtheria, tetanus and pertussis vaccine  (DTPer/Vac/Ads) that is used to immunize infants, and  the adsorbed diphtheria and tetanus vaccine (DT/Vac/  Ads) that is used to reinforce the immunity of school  entrants. The trend is to produce increasingly complex  combinations, and hepta- and octavalent preparations  are now available.   The single-component viral vaccines are listed in  Table 24.2, with notes similar to those provided with the  bacterial vaccines. The only combined viral vaccine that  is widely used is the measles, mumps and rubella vaccine  (MMR Vac). In a sense however, both the inactivated  (Salk) poliovaccine (Pol/Vac (inactivated)) and the live  (Sabin) poliovaccine (Pol/Vac (oral) are combined vac-  cines in that they are both mixtures of virus of each of  the three serotypes of poliovirus. Influenza vaccines, too,  are combined vaccines in that they usually contain com-  ponents from several virus strains, usually from two  strains of influenza A and one strain of influenza B.   The quality control of vaccines is intended to provide  assurances of both the probable efficacy and the safety of  every batch of every product. It is achieved in three ways:  (1) in-process control; (2) final product control; and (3)  requirements that for each product the starting materials,  intermediates, final product and processing methods are  consistent.   The results of all quality control tests must be recorded  in detail and authorized by a qualified person as, in those  countries in which the manufacture of vaccines is regu-  lated by law, they are part of the evidence on which  control authorities judge the acceptability or otherwise of  each batch of each preparation.    Table 24.1 Bacterial vaccines used for the prevention of infectious disease in humans   3-1-3 quantal assay in guinea-  pigs using challenge with B.  anthracis   Cultures of live BCG  cells in liquid or on  solid media   3 + 3 quantal assay in guinea-  pigs using intradermal challenge  with diphtheria toxin or  serological assay for antitoxin   Inoculation of guinea-pigs to  exclude untoxoided toxin  Toxin assay in Vero cell cultures   Cultures of H.  influenzae type b  Protein carrier  ( tetanus/diphtheria  toxoid, CRM 197)   Cultures of N.  meningitidis of  serotypes A, C,Wi 35 , Y  Carrier protein ;  tetanus/diphtheria  toxoid, CRM 197   Cultures of selected  serotypes of Strep,  pneumonia  Carrier protein;  tetanus toxoid, CRM  197, H. influenzae  OMP   3 + 3 quantal assay in mice using  subcutaneous challenge with  tetanus toxin or measurement of  serological response   Cultures of Bord.  pertussis grown in  liquid or on solid  media   Estimation of bacteria to limit  content to 20 X 10 9 per human  dose   2 Extraction, detoxification and  blending of cell components (pertussis  toxin, filamentous haemagglutinin,  pertactin, fimbriae)   Immunogenicity assay (product  specific) or modified quantal  assay using intracerebral  challenge with live Bord. pertussis    a Vaccines used to provide additional protection when circumstances indicate a need.  b Vaccines used in conventional immunization schedules.   Diphtheria and pertussis vaccines are seldom used as single-component vaccines but as components of diphtheria/tetanus vaccines and diphtheria/ tetanus/pertussis vac-  cines. Combined diphtheria/ tetanus/pertussis/Hib and diphtheria/ tetanus/pertussis/Hep B vaccines with or without inactivated polio vaccine are available.   Bacterial vaccines of restricted availability include anthrax, botulism, cholera, plague, Q fever, typhus and tularaemia vaccines.    Table 24.2 Viral vaccines used for the prevention of infectious diseases in humans   Allantoic fluid from embryonated  hens’ eggs infected with influenza  viruses A and B   Allantoic fluid from embryonated  hens’ eggs infected with influenza  viruses A and B   Human diploid cell cultures infected  with each of the three serotypes of  poliovirus   Inoculation of cell cultures and  monkey or transgenic mouse  spinal cords to exclude live virus   Cell cultures infected with  attenuated poliovirus of each of the  three serotypes   Aqueous homogenate of chick  embryos infected with attenuated  yellow fever virus 17D    a Vaccines used to provide additional protection when circumstances indicate a need.   b Vaccines used in conventional immunization programmes. Measles, mumps and rubella vaccines are generally administered in the form of a combined measles/mumps/  rubella vaccine (MMR vaccine).   Viral vaccines of restricted application include Congo Crimean haemorrhagic fever vaccine, dengue fever vaccine, Japanese encephalitis B vaccine, rabies vaccine, smallpox  vaccine, tick-borne encephalitis vaccine and Venezuelan equine encephalitis vaccine.   In-process quality control is the control exercised over  starting materials and intermediates. Its importance  stems from the opportunities that it provides for the  examination of a product at the stages in its manufacture  at which testing is most likely to provide the most mean-  ingful information. The WHO recommendations and  national authorities stipulate many in-process controls  but manufacturers often perform tests in excess of those  stipulated, especially sterility tests (Chapter 21) as, by so  doing, they obtain assurance that production is proceed-  ing normally and that the final product is likely to be  satisfactory. Numerous examples of in-process control  exist for various types of vaccine but three demonstrate  the principle.   The quality control of both diphtheria and tetanus  vaccines requires that the products are tested for the pres-  ence of free toxin, i.e. for specific toxicity due to inade-  quate detoxification with formaldehyde, at the final  product stage. By this stage, however, the toxoid concen-  trates used in the preparation of the vaccines have been  much diluted and, as the volume of vaccine that can be  inoculated into the test animals (guinea-pigs) is limited,  the tests are relatively insensitive. In-process control,  however, provides for tests on the undiluted concentrates  and thus increases the sensitivity of the method at least  100-fold.   An example from virus vaccine manufacture is the  titration, prior to inactivation, of the infectivity of the  pools of live poliovirus used to make inactivated polio-  myelitis vaccine. Adequate infectivity of the virus from  the tissue cultures is an indicator of the adequate virus  content of the starting material and, as infectivity is  destroyed in the inactivation process, there is no possibil-  ity of performing such an assay after formaldehyde  treatment.   A more general example from virus vaccine produc-  tion is the rigorous examination of tissue cultures to  exclude contamination with infectious agents from the  source animal or, in the cases of human diploid cells or  cells from continuous cell lines, to detect cells with  abnormal characteristics. Monkey kidney cell cultures are  tested for simian herpes B virus, simian virus 40, myco-  plasma and tubercle bacilli. Cultures of human diploid  cells and continuous line cells are subjected to detailed  karyological examination (examination of chromosomes  by microscopy) to ensure that the cells have not under-  gone any changes likely to impair the quality of a vaccine  or lead to adverse effects.   Vaccines containing killed microorganisms or their prod-  ucts are generally tested for potency in assays in which  the amount of the vaccine that is required to protect  animals from a defined challenge dose of the appropriate  pathogen, or its product, is compared with the amount  of a standard vaccine that is required to provide the same  protection. The usual format of the test is the 3 + 3 dose  quantal assay that is used to estimate the potency of  whole-cell pertussis vaccine ( British Pharmacopoeia,  2010). Three logarithmic serial doses of the test vaccine  and 3 of the standard vaccine are made and each is used  to inoculate a group of 16 mice. In the case of both the  test vaccine and the standard, the middle dose is chosen  on the basis of experience, so that it is sufficient to induce  a protective response in about 50% of the animals to  which it is given. Each lower dose may then be expected  to protect less than 50% of the mice to which it is given  and each higher dose to protect more than 50% of the  animals. Fourteen days later all of the mice are inoculated  (‘challenged’) with a suitable virulent Bordetella pertussis  strain and, after a further 14 days, the number of mice  surviving in each of the 6 groups is counted. The number  of survivors in each group is used to calculate the potency  of the test vaccine relative to the potency of the standard  vaccine by the statistical method of probit analysis  (Finney, 1971). The potency of the test vaccine may be  expressed as a percentage of the potency of the standard  vaccine. However, as the standard vaccine will have an  assigned potency in international units (IU), it is more  usual to express the potency of the test vaccine in similar  units. Tests similar to that used to estimate the potency  of pertussis vaccine are prescribed for the potency deter-  minations of diphtheria vaccine and tetanus vaccines. In  these cases the respective bacterial toxins are used as the  challenge material ( British Pharmacopoeia, 2010). Tests  that do not involve challenge but involve titration of the  antitoxin response in vitro, e.g. by ELISA (enzyme-linked  immunosorbent assay), are now being adopted.   Vaccines containing live microorganisms are generally  tested for potency by determining their content of viable  particles. In the case of the most widely used live bacterial  vaccine, BCG vaccine, dilutions of vaccine are prepared  in a medium which inhibits clumping of cells, and fixed  volumes are dropped on to solid media capable of sup-  porting mycobacterial growth. After a fortnight the colo-  nies generated by the drops are counted and the live    count of the undiluted vaccine is calculated. The potency  of live viral vaccines is estimated in much the same way  except that a substrate of living cells is used. Dilutions of  vaccine are inoculated on to tissue culture monolayers in  Petri dishes or in plastic trays, and the infective particle  count of the vaccine is calculated from the infectivity of  the dilutions as indicated by plaque formation, cyto-  pathic effect, haemadsorption or other effect and the  dilution factor involved.   Because many vaccines are derived from basic materials  of intense pathogenicity — the lethal dose of tetanus toxin  for a mouse is estimated to be 3 x lCT 2 ng — safety testing  is of paramount importance. Effective testing provides a  guarantee of the safety of each batch of every product and  most vaccines in the final container must pass one or  more safety tests as prescribed in a pharmacopoeial mon-  ograph. This generality does not absolve a manufacturer  from the need to perform in-process tests as required, but  it is relaxed for those preparations that have a final for-  mulation that makes safety tests on the final product  either impractical or meaningless.   Bacterial vaccines are regulated by relatively simple  safety tests. Those vaccines composed of killed bacteria  or bacterial products must be shown to be completely  free from the living microorganisms used in the produc-  tion process. Inoculation of appropriate bacteriological  media with the final product provides an assurance that  all organisms have been killed. Those vaccines prepared  from toxins, for example, diphtheria and tetanus toxoids,  require in addition, a test system capable of revealing  inadequately detoxified toxins; this can be done by inocu-  lation of guinea-pigs, which are exquisitely sensitive to  both diphtheria and tetanus toxins. A test for sensitiza-  tion of mice to the lethal effects of histamine is used to  detect active pertussis toxin in pertussis vaccines. An  improved non-lethal method is also available. The trend  is to replace in vivo assays by cell culture methods where  possible but these do not always emulate in vivo effects.  Inoculation of guinea-pigs is also used to exclude the  presence of abnormally virulent organisms in BCG  vaccine. Molecular genetic methods, such as nucleic acid  amplification to probe for genes specific to virulent  strains, are now available but not yet in routine use for  vaccine testing.   Viral vaccines can present problems of safety testing  far more complex than those experienced with most bac-  terial vaccines. With killed viral vaccines the potential    hazards are those due to incomplete virus inactivation  and the consequent presence of residual live virus in the  preparation. The tests used to detect such live virus  consist of the inoculation of susceptible tissue cultures  and of susceptible animals. The cultures are examined for  cytopathic effects, and the animals for symptoms of  disease and histological evidence of infection at autopsy.  This test is of particular importance in inactivated polio-  myelitis vaccines, the vaccine being injected intraspinally  into monkeys or mice transgenic for the poliovirus recep-  tor. At autopsy, sections of brain and spinal cord are  examined microscopically for the histological lesions  indicative of proliferating poliovirus.   With attenuated viral vaccines the potential hazards  are those associated with reversion of the virus during  production to a degree of virulence capable of causing  disease in recipients. To a large extent this possibility is  controlled by very careful selection of a stable seed  but, especially with live attenuated poliomyelitis vaccine,  it is usual to compare the neurovirulence of the vaccine  with that of a vaccine known to be safe in field use. The  technique involves the intraspinal inoculation of monkeys  with both the reference vaccine and the test vaccine fol-  lowed by comparison of the neurological lesions and  symptoms, if any, that are caused. If the vaccine  causes abnormalities in excess of those caused by the  reference it fails the test. A modification of this test which  uses transgenic mice instead of monkeys is now available.  An in vitro method (MAPREC test) which relies on  detecting RNA sequences specific to virulent virus has  also been developed. A widespread problem with safety  testing of live viral vaccines is that the host specificity of  many viruses limits the availability of suitable animal  models.   In addition to the tests designed to estimate the potency  and to exclude the hazards peculiar to each vaccine there  are a number of tests of more general application. These  relatively simple tests are as follows.   • Sterility. In general, vaccines are required to be sterile.  The exceptions to this requirement are smallpox vaccine  made from the dermis of animals and bacterial vaccines  such as BCG, Ty21A and tularaemia vaccine, which  consist of living but attenuated strains. These have a  bioburden limit which defines the number of permissible  microorganisms but excludes pathogens. WHO recommen-  dations and pharmacopoeial monographs stipulate, for  vaccine batches of different size, the numbers of    containers that must be tested and found to be sterile.  The preferred method of sterility testing is membrane  filtration, as this technique permits the testing of large  volumes without dilution of the test media. The test  system must be capable of detecting aerobic and anaerobic  bacteria and fungi (see Chapter 21).   • Freedom from abnormal or general toxicity. The purpose  of this simple test is to exclude the presence in a final  container of a highly toxic contaminant. Five mice of  17-22 g and two guinea-pigs of 250-350 g are inoculated  with one human dose or 1.0 ml, whichever is less, of the  test preparation. All must survive for 7 days without signs  of illness. Current pharmacopoeial monographs usually  do not require this test if another in vivo test has been  performed on the product.   • Pyrogenicity or endotoxin content. The pyrogenicity of  a specified dose of product when administered to rabbits  can be assayed by a standard pharmacopoeial method but  the trend is to replace this with an in vitro assay for  endotoxin (Chapter 22). The capacity of the product to  induce gelation of Limulus polyphemus amoebocyte lysate  is determined against a reference endotoxin preparation  and the result is expressed as IU of endotoxin. For  pyrogens other than endotoxin, a monocyte stimulation  test is available.   • Presence of aluminium and calcium. The quantity  of aluminium in vaccines containing aluminium  hydroxide or aluminium phosphate as an adjuvant is  limited to 1.25 mg per dose and it is usually estimated  compleximetrically. The quantity of calcium is limited to  1.3 mg per dose and is usually estimated by atomic  absorption spectrometry.   • Free formaldehyde. Inactivation of bacterial toxins with  formaldehyde may lead to the presence of small amounts  of free formaldehyde in the final product. The  concentration, as estimated by colour development with  acetylacetone, must not exceed 0.02%.   • Phenol concentration. When phenol is used to preserve  a vaccine its concentration must not exceed 0.25% w/v  or, in the case of some vaccines, 0.5% w/v. Phenol is  usually estimated by the colour reaction with amino-  phenazone and hexacyanoferrate.   • pFl. The potentiometric determination of pFI is made  by measuring the potential difference between two  appropriate electrodes immersed in the solution to be  examined: one of these electrodes is sensitive to hydrogen  ions and the other is the reference electrode. The pH  apparatus is calibrated with the buffer solution of  potassium hydrogen phthalate and one other buffer  solution of different pH. The pH in the test sample   • Osmolality. Osmolality is a practical means of giving  an overall measure of the contribution of the various  solutes present in a solution to the osmotic pressure of  the solution. Osmolality is determined by measurement  of the depression of freezing point of the test sample  using appropriate apparatus. The osmolality of the  test sample should comply with the limits approved  for the particular products, for example minimum  240 mOsmol/kg.   The most widely used of these are the tuberculins  employed to detect sensitization by mycobacterial pro-  teins and hence the possible presence of infection. These  are prepared by growing approved strains of M. tubercu-  losis (or M. bovis or M. avium in preparations intended  for veterinary use) in a protein-free medium for several  weeks. The culture is then steamed for a prolonged period  to kill surviving bacteria and to facilitate release of tuber-  culoproteins from the cells. The culture supernatant is  recovered by centrifugation and further concentrated by  evaporation and sterile filtered to make a product known  as Old Tuberculin. The crude material may then be stand-  ardized against a reference preparation by titration in the  skin of guinea-pigs sensitized to M. tuberculosis. In prac-  tice, further purification is usually performed by precipi-  tation with trichloracetic acid or other protein precipitant  to produce purified protein derivative, which is standard-  ized by in vivo assay. Concentrated preparations contain-  ing 1 00 000 IU/ml are used to formulate working strengths  such as 1000, 100 or 10 IU/ml. These have to be diluted  in a medium containing a Tween surfactant to reduce  adsorption to glass. The concentrated material can be  used for intradermal testing by a multiprong device such  as the Heaf or Tine method.   Apart from standardization of potency, which also serves  as an identity test, the material must be checked for steril-  ity and for the absence of viable mycobacteria. Because  of their slow growth the latter may not be detected by  conventional sterility tests and it is usual to perform  check tests by guinea pig inoculation, or by prolonged  culture on Lowenstein-Jensen medium. The product is  also checked for absence of reactogenicity in unsensitized    guinea-pigs and if required by the regulatory authority,  for abnormal toxicity.   Analogous intradermal test reagents such as mallein,  histoplasmin and coccidioidin, are produced by similar  methods. Their use has declined, however, as they, like the  tuberculin test, detect previous exposure and sensitiza-  tion to the antigens of the agent but not necessarily active  infection.   Immune sera are preparations derived from the blood of  animals, usually horses, but mules, donkeys, sheep or  goats are also used. The animals must be in good health,  free of infections and obtained from sources free of trans-  missible spongiform encephalopathies, and kept under  veterinary supervision. To prepare an immune serum,  horses or other animals are injected with a sequence of  spaced doses of an antigen until a trial blood sample  shows that the injections have induced a high titre of  antibody to the injected antigen. An adjuvant may be  used if required. A large volume of blood is then removed  by venepuncture and collected into a vessel containing  sufficient citrate solution to prevent clotting. The blood  cells are allowed to settle and the supernatant plasma is  drawn off. Alternatively, the blood can be mechanically  defibrinated. The crude plasma can be sterilized by filtra-  tion and dispensed for use, but it is preferable to fraction-  ate it to separate the immune globulin. This is done by  fractional precipitation of the plasma by the addition of  ammonium sulphate. The globulin fraction is recovered  and treated with pepsin to yield a refined immune  product containing the Fab fragment. This refined globu-  lin contains no more than a trace of the albumin and  other proteins that were present in the plasma. It is less  antigenic, has a longer half-life in the circulation and is  less likely to provoke anaphylaxis or serum sickness than  whole serum or crude globulin (Harms, 1948). The anti-  body content of the refined product is determined by  specific assay, the product is diluted to the required con-  centration and transferred into ampoules. Two or more  monovalent immune sera may be blended together to  provide a multivalent immune serum.   The quality of immune sera is controlled by potency tests  and by conventional tests for safety and sterility. The  potency tests have a common design in that, in the case    of all immune sera, the potency is estimated by compar-  ing the amount of the product that is required to neutral-  ize an effect of a homologous toxin with the amount of  a standard preparation that is required to achieve the  same effect. Serial dilutions of the immune serum and of  a standard preparation are made and to each is added a  constant amount of the homologous antigen. Each  mixture is then inoculated into a group of animals,  usually guinea-pigs or mice, and the dilutions of the  immune serum and of the standard, which neutralize the  effects of toxin, are noted. As the potencies of the stand-  ard preparations are expressed in IU, the potencies of the  immune sera are determined in corresponding units per  millilitre ( British Pharmacopoeia , 2010).The quality of  globulin fractions is usually monitored by gel electro-  phoresis to detect contaminating proteins and uncleaved  immunoglobulin, andby size-exclusion high-performance  liquid chromatography to detect aggregates and small  fragments. The immune sera are also tested for contami-  nating viruses by inoculation on to cell cultures capable  of detecting a wide range of viruses relevant to the par-  ticular product.   Table 24.3 lists the immune sera for which there is  currently a demand, or a potential need, and indicates  their required potencies and the salient features of the  potency assay methods.   Human immunoglobulins are preparations of the immu-  noglobulins, principally immunoglobulin G (IgG) sub-  classes, that are present in human blood. They are derived  from the plasma of donated blood and from plasma  obtained by plasmapheresis. Normal immunoglobulin,  that is immunoglobulin that has relatively low titres of  antibodies representative of those present in the popula-  tion at large, is prepared from pools of plasma obtained  from not fewer than 1000 individuals. Specific immu-  noglobulins, that is immunoglobulins with a high titre of  a particular antibody, are usually prepared from smaller  pools of plasma obtained from individuals who have suf-  fered recent infections or who have undergone recent  immunization and who thus have a high titre of a par-  ticular antibody. Each contribution of plasma to a pool  is tested for the presence of hepatitis B surface antigen  (HBsAg), for antibodies to HIV 1 and 2 and for antibod-  ies to hepatitis C virus in order to identify, and to exclude  from a pool, any plasma capable of transmitting infection    Table 24.3 Immune sera used in the prevention or treatment of infections in humans   Neutralization of the lethal effects of botulinum toxins A, B and E  in mice. Similar assays can also be used to titrate the activity of  monoclonal antibodies which can include activity against types A— G   500 IU/ml type A  500 IU/ml type B  50 IU/ml type E   Neutralization of the erythrogenic effect of diphtheria toxin in the  skin of guinea-pigs   The activity of the sera can also be titrated by ELISA or by  neutralization of toxin activity in Vero cell cultures   Neutralization of the paralytic effect of tetanus toxin in mice  The antitoxin activity can also be titrated by ELISA    In each of the assays of potency the amount of the immune serum and the amount of a corresponding standard antitoxin that  are required to neutralize the effects of a defined dose of the corresponding toxin are determined. The two determined amounts  and the assigned unitage of the standard antitoxin are then used to calculate the potency of the immune serum in international  units (IU).    from donor to recipient. Donors who have been resident  for 6 months or more in areas endemic for transmissible  spongiform bovine encephalopathy (BSE), which cur-  rently includes the UK, are also excluded. The solvent-  detergent process, one of the methods used to inactivate  enveloped viruses, uses treatment with a combination of  tributyl phosphate and octoxinol 10; these reagents are  subsequently removed by oil extraction or by solid phase  extraction ( British Pharmacopoeia, 2010).   The immunoglobulins are obtained from the plasma  pools by fractionation methods that are based on ethanol  precipitation in the cold with rigorous control of protein  concentration, pH and ionic strength (Cohn et al., 1946).  The variation of Kistler & Nitschmann (1962) is widely  used. Some of the fractionation steps may contribute to  the safety of immunoglobulins by inactivating or remov-  ing contaminating viruses that have not been detected by  tests on the blood donations. The immunoglobulins may  be presented either as a freeze-dried or a liquid prepara-  tion at a concentration that is at least 10 times that in the  initial pooled plasma. Glycine may be added as a stabi-  lizer. Multidose preparations contain an antimicrobial  preservative but single-dose preparations do not ( British  Pharmacopoeia, 2010).   The quality control of immunoglobulins includes potency  tests and conventional tests for safety and sterility. The    potency tests consist of toxin or virus neutralization tests  that parallel those used for the potency assay of immune  sera, except that for in-process control of some immu-  noglobulins wider use is made of in vitro assays. In  addition to the safety and sterility tests, total protein is  determined by nitrogen estimations, the protein compo-  sition by sodium dodecyl sulphate (SDS)-polyacrylamide  gel electrophoresis and molecular size by high perform-  ance liquid chromatography. The presence of immu-  noglobulins derived from species other than humans  is excluded by precipitin tests. Table 24.4 lists six  human immunoglobulins and their requisite potencies  and indicates the methods by which the potencies are  determined.   Monoclonal antibodies are immunoglobulins or a frag-  ment of an immunoglobulin, with defined specificity,  produced by a single clone of cells. They can be obtained  from immortalized B-lymphocytes that are cloned and  expanded as continuous cell lines (murine and human  monoclonal antibodies) or from rDNA-engineered  mammalian or bacterial cell lines (engineered mono-  clonal antibodies). Production of monoclonal antibodies  is based on a seed lot system using a master cell bank and  a working cell bank derived from the cloned cells. Two  approaches are currently in use: single harvest (produc-   Neutralization tests in cell cultures or in  animals, or in vitro ELISA estimations   Measurable amounts of one  bacterial antibody and of one  viral antibody for which there  are international standards    In each of the assays of potency the amount of the immunoglobulin and the amount of a corresponding standard preparation  that are required to neutralize the infectivity or other biological activity of a defined amount of virus or to neutralize a defined  amount of a bacterial toxin are determined. The two determined amounts and the assigned unitage of the standard preparation  are then used to calculate the potency of the immunoglobulins in international units (IU). ELISA, enzyme-linked immunosorbent  assay.    tion at finite passage level) and multiple harvest  (continuous-culture production). In the first method, the  cells are cultivated up to a defined maximum number of  passages or population doublings (in accordance with the  stability of the cell line). In the second method, cells are  continuously cultivated for a defined period (in accord-  ance with the stability of the system and production con-  sistency) . In this case, monitoring is necessary throughout  the life of the culture; the required frequency and type of  monitoring will depend on the nature of the production  system. Bulk harvests can be made by pooling individual  harvests before purification. The purification process to  remove unwanted host cell derived proteins, nucleic acids  and carbohydrates is also designed to remove and/or  inactivate non-enveloped and enveloped viruses and  other impurities.   The quality of therapeutic monoclonal antibodies is con-  trolled by rigorous characterization of the products by  chemical and biological methods. The final product  should be tested for bioburden and bacterial endotoxins,  purity, integrity and potency by suitable analytical    methods, comparing with a reference preparation. The  identity test can be done by suitable methods comparing  the product with the reference preparation. Molecular  size distribution can be determined by size-exclusion  chromatography. Depending on the nature of the mono-  clonal antibody, its microheterogeneity and isoforms, a  number of different tests can be used to demonstrate  molecular identity and structural integrity. These tests  may include peptide mapping, isoelectric focusing, ion-  exchange chromatography, hydrophobic interaction  chromatography, oligosaccharide mapping, monosaccha-  ride content and mass spectrometry. The purity can be  examined by SDS-polyacrylamide gel electrophoresis  under reducing and non-reducing conditions or capillary  electrophoresis. For monoclonal antibodies with specific  antimicrobial activity, an appropriate assay is performed  to determine the level of this. Monoclonal antibodies  have been developed for postexposure prophylaxis of  anthrax (anti-Protective Antigen), botulism (anti-toxins  A-G) and plague (anti-V antigen). Monoclonal antibod-  ies directed against the core region of Gram-negative  bacterial endotoxins and intended for treatment of sepsis,  have met with little success.   Ada, G.L. (1994) Strategies in Vaccine Design. RG Landes,  Austin, TX.   British National Formulary. British Medical Association and  Pharmaceutical Press, London. (This publication contains a  useful section on immunological products. New editions  appear twice yearly. Also available from http://bnf.org/bnf/  index.htm)   Cohn, E.J., Strong, L.E., Hughes, W.L., Hulford, D.J., Ashworth,  J.N., Melin, M. & Taylor, H.I. (1946) Preparation and proper-  ties of serum proteins IV. / Am Chem Soc, 68, 459-475.   Ellis, R.W. (1999) Combination Vaccines. Development, Clinical  Research and Approval. Humana Press, Totowa, NJ.   Harms, A.J. (1948) The purification of antitoxic plasmas by  enzyme treatment and heat denaturation. Biochem /, 42,  340-347.   Kistler, P. & Nitschmann, H.S. (1962) Large scale production of  human plasma fractions. Vox Sang , 7, 414-424.    Mizrahi, A. (1990) Bacterial vaccines. Advances in Biotechno-  logical Processes 15. Wiley- Liss, New York.   Pastoret, P., Blancou, J., Vannier, P. & Verschueren, C. (1997)  Veterinary Vaccinology. Elsevier, Amsterdam.   Perlmann, P. & Wigzell, H. (1999) Vaccines. Handbook of  Experimental Pharmacology , Vol. 133. Springer, Berlin.   Plotkin, S.A. & Fantini, B. (eds) (1996) Vaccinia, vaccination,  vaccinology — Jenner, Pasteur and their successors. Elsevier,  Paris.   Plotkin, S.A., Orenstein, W.A. & Offit, P.A. (eds) (2008) Vaccines,  5th edn. Saunders Elsevier, Phildelphia, PA.   Powell, M.F. & Newman, M.J. (1995) Vaccine Design. The Subunit  and Adjuvant Approach. Plenum Press, New York.   WHO (1999a) A WHO Guide to Good Manufacturing Practice  (GMP) Requirements. Part 1. Standard Operating Procedures  and Master Formulae. WHO/VSQ/97.01. World Health  Organization, Geneva.   WHO (1999b) A WHO Guide to Good Manufacturing Practice  (GMP) Requirements. Part 2. Validation. WHO/VSQ/97.02.  World Health Organization, Geneva.   The rapid developments in biotechnology and the appli-  cations of genetic engineering to practical human prob-  lems have allowed the advancement of pharmaceutical  biotechnology at a staggering pace. Furthermore, the  release of the human genome sequence has also been key  for the identification of human genetic diseases and the  design of revolutionary approaches for their treatment.   Genetic engineering involves altering DNA molecules  outside an organism, making the resultant DNA mole-    cules function in living cells. Many of these cells have  been genetically engineered to produce substances that  are medically useful to humans. Pharmaceutical biotech-  nology involves the use of living organisms such as  microorganisms to create new pharmaceutical products,  or safer and more effective versions of conventionally  produced pharmaceuticals, more cost-effectively.   Since the manufacture of the first recombinant phar-  maceutical, insulin, there has been a burst in the  generation of new recombinant drugs, some of which will  be covered later on in this chapter. Furthermore, the use  of recombinant DNA technology has spread further  allowing the development not only of subunit vaccines,    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    such as the one used in the prevention of hepatitis B,  but also of attenuated vaccines, vector vaccines and  DNA vaccines. One of pharmaceutical biotechnology’s  great potentials lies in gene therapy, which consists in  the modification of the genetic material of living cells  to prevent, control or cure disease. It encompasses   repairing or replacing defective genes and, for example,  making tumours more susceptible to other kinds of  treatment.   This chapter aims to describe some essential genetic  manipulation techniques and to illustrate, with some  key examples, their use for the generation of recombinant  pharmaceutical drugs. Applications of recombinant  DNA techniques in the diagnosis of diseases will also be  covered.   To understand how recombinant pharmaceuticals are  manufactured we first need to review some of the essen-  tial DNA manipulation techniques used to generate these  products. We will start by looking at ways to cut and join  fragments of DNA and then examine step by step how    these techniques can be exploited to clone and express  genes from eukaryotic and prokaryotic cells.   DNA isolated from any type of cell can be fragmented  using restriction endonucleases. These are enzymes pro-  duced by microorganisms which cut foreign DNA and  can restrict the proliferation of infecting viruses. Some of  these enzymes cut at specific points known as restriction  sites which are palindromic sequences (complementary  sequences with identical nucleotide sequences when read  in the 5' to 3' direction) of various lengths. For example,  £coRI ( Escherichia coli restriction enzyme I) has specifi-  city for the sequence GAATTC and hydrolyses the G-A  phosphodiester bond. Enzymes which recognize and cut  at restriction sites of 4-8 base pairs (bp) are particularly  useful, as the probability of a site appearing in a random  DNA fragment is inversely proportional to its length  There are two different types of DNA ends that can be  generated using restriction enzymes: cohesive or sticky  and blunt ends (Table 25.1). DNA fragments obtained by  restriction enzyme digestion can be covalently joined  together using the enzyme DNA ligase. There is a limita-  tion, however, with regards to the type of ends this    enzyme is able to bond together. Only blunt ends  generated by some restriction enzymes (e.g. Dral and  EcoRV) or compatible sticky ends generated by either the  same restriction enzymes (e.g. EcoRI) or by enzymes  that generate complementary overhanging ends (e.g.  SauiAl and BatnHI) will be bonded by the DNA ligase  (Table 25.1).   Genes present in DNA fragments that have been excised  with restriction endonucleases can be maintained (repli-  cated) and expressed by inserting them (cloning) into  vectors after ligation. These cloning vectors are of differ-  ent types and are relatively small DNA molecules that  have the ability to self-replicate in a host cell. The main  type of vectors used for gene cloning are plasmids,  cosmids and bacteriophages, which are normally used  according to the size of the DNA fragments that need to  be cloned.   To allow the cloning of small DNA fragments, generally  up to 10 kb, plasmids are the main vectors of choice.   Plasmids are circular extrachromosomal DNA molecules  that replicate independently within cells using their own  origin of replication. There are a number of features gen-  erally found in plasmids used as cloning vectors:   • Antibiotic resistance markers. These are genes coding for  proteins that confer resistance to specific antibiotics.  These markers therefore allow the selection of hosts  carrying the plasmids.   • Multiple cloning sites (MCS). These are stretches of  DNA designed to contain a number of unique different  restriction sites, providing a choice of possible restriction  enzymes to be used for the cloning of DNA fragments.   • Origin of replication. Required for plasmid replication  in a specific host. These DNA sequences also determine  the number of copies at which the plasmid will replicate,  from just one to several hundreds per cell. Plasmids  having more than one host-specific origin of replication  are known as shuttle vectors.   • Insertional inactivation markers. These facilitate the  selection of recombinant from non-recombinant  plasmids. The most commonly used is the lacZ gene  coding for (3-galactosidase. This enzyme cleaves 5-bromo-  4-chloro-3-indolyl-p-D-galactopyranoside (X-Gal), an  artificial substrate that mimics galactose, which results in    the generation of an insoluble blue product. Insertion of  a recombinant DNA fragment in lacZ will result in the  disruption of (5-galactosidase production and the inability  to cleave X-Gal. Hence colonies from bacteria carrying an  intact lacZ gene, i.e. from non-recombinant plasmids,  appear blue on agar plates containing X-Gal. In contrast,  those with a successful insertion in lacZ, i.e. harbouring  recombinant plasmids, appear white.   Figure 25.1 shows all these features in a simplified  diagram of the cloning vector pUC18.   An ideal plasmid for cloning should be small (2-10kb)  and conjugation-defective, i.e. non-self-mobilizable from  cell to cell, and produce a selectable phenotype in host  cells. It should also contain a large MCS and replicate at  a high copy number (> 10 copies per cell).   Sometimes there is a need to clone large fragments of  DNA, for example for the isolation of complete gene   Figure 25.1 Simplified diagram of the plasmid pUC18. lacZ  represents the insertional inactivation marker coding for  (3-galactosidase activity. A multiple cloning site (MCS) is  present within the lacZ gene, allowing the cloning of DNA  fragments. ‘Ori’ represents the origin of replication which, in  this case, is functional in Escherichia coli , and Amp r represents  an ampicillin resistance marker.    clusters. An example would be the cloning of large  eukaryotic genes or genes required for the synthesis of a  certain molecule. Sometimes the synthesis of a com-  pound requires more than 10 different genes and these  are frequently organized in operons cotranscribed in a  single mRNA molecule. To enable the cloning of large  genes or full-length operons, vectors such as bacteri-  ophages, cosmids or BACs need to be used.   The most popular has been the E. coli X (lambda) bacte-  riophage, which is composed of a tubular protein tail and  a protein head packed with approximately 50 kb of  double-stranded (ds) DNA (Figure 25.2). After injection  of the viral DNA into E. coli, bacteriophage X can multi-  ply and enter a lytic cycle leading to the lysis of the host  cell and the subsequent release of a large number of    phage particles. Alternatively, injection of the DNA can  lead to a lysogenic cycle in which the phage DNA is inte-  grated into the E. coli chromosome where it is maintained  until the environmental conditions change and is then  excised, entering a lytic cycle. Out of the 50 kb that make  the X bacteriophage less than half are essential for its  propagation and therefore around 20 kb can be replaced  by recombinant DNA, hence their name X replacement  vectors. For this reason these are very useful vectors for  the generation of genomic libraries. DNA in X bacteri-  ophages contain at each end small single-stranded (ss)  complementary DNA fragments called X cohesive ends  (A cos ends). Recombinant phages can be assembled in a  test tube into phage-like particles by enzymes which rec-  ognize and process the Xcos ends, provided that they are  35-45 kb apart. These enzymes as well as the head and  tails required for the assembly process are commercially   Figure 25.2 Cloning of DNA into X replacement vectors. The DNA from the X bacteriophage can be purified from the phage  particles and digested with restriction enzymes to remove an internal fragment of around 15 kb which is not required for the life  cycle of the bacteriophage. This fragment can be replaced with other fragments of DNA such as those coming from digested  bacterial chromosomal DNA. As the X bacteriophage can only pack DNA fragments flanked by Xcos ends which are 35-45 kb apart,  any recombinant fragments larger or smaller than that will not be successfully packed.    available as part of in vitro packaging kits. The in vitro  packaging results in the formation of recombinant phages  that can be transduced to E. coli cells. Transduced E. coli  will be identified by the formation of lysis plaques in agar  plates seeded with a mixture of E. coli and recombinant  A bacteriophages.   Cosmids are plasmids maintained in E. coli that have been  engineered to carry A ,cos sequences. This allows their  packaging in vitro into A phage particles to be transduced  into E. coli cells for replication. Once inside the bacterial  host the cosmid DNA is circularized by the joining of the  A cos ends and thereafter it replicates as a normal plasmid.  This implies that the cloned DNA will be available as E.  coli colonies and not as plaques like with the A bacteri-  ophages. As less DNA is required for plasmid than for  bacteriophage replication, cosmids can carry up to 40 kb  of cloned DNA still enabling the packaging into A phage  particles.   Eukaryotic and more particularly mammalian genomic  DNA rich in multiple repeated elements can be subject  to deletions and rearrangements when cloned in standard  multicopy cosmids and replicated in bacteria. This incon-  venient can be reduced by using special cosmids in which  the multicopy origin of replication has been replaced by  the single-copy origin of replication from the E. coli F'  factor. These cloning vectors are known as fosmids.   Although cosmids and fosmids enable the cloning of rela-  tively large DNA fragments, the amount of DNA that can  be packed into a A bacteriophage head is limited to  around 50 kb. To clone even larger DNA fragments  cloning vectors derived from the single-copy E. coli F'  factor, similar to the fosmids, have been designed. In this  case the A cos ends are absent or not used for packaging  and instead the recombinant plasmids are introduced  directly into the E. coli cells by transformation. This  allows the cloning of DNA fragments of several hundreds  of kilobases resulting in what are then called bacterial  artificial chromosomes (BACs).   For the expression and maintenance of recombinant  genes the recombinant vectors harbouring them need to  be introduced into suitable hosts. The four main methods  used to achieve this are transformation, electroporation,  conjugation and transduction.    • Transformation is the direct incorporation of DNA into  host cells. Bacteria such as E. coli can uptake recombinant  plasmid DNA when treated with ice-cold CaCl 2 until they  reach a ‘competent’ state in which they are ready to take  up DNA. These cells are then mixed with the recombinant  plasmid and exposed briefly to a heat shock of 42 °C  which causes them to take up the DNA.   • Electroporation is, however, the most efficient way of  introducing DNA not only in bacteria but also in  eukaryotic cells. This technique is based on the induction  of free DNA uptake by the cells after subjecting them to  a strong electric field.   • In some cases conjugation can be used as a natural  transmission of plasmid DNA from a donor cell to a  recipient cell by direct contact through cell-cell junctions.  Only plasmid cloning vectors containing conjugative  elements can be transferred by conjugation. This  procedure requires direct contact between the donor and  the recipient cell. Conjugation is not as frequently used  as electroporation as most plasmid vectors used for the  cloning of recombinant DNA lack conjugative functions,  preventing these plasmids from being passed to other  cells inadvertently.   • Finally, in transduction (prokaryotes) and transfection  (eukaryotes) the transfer of recombinant non-viral  DNA to a cell is achieved by a virus. This is the method  of choice for the introduction of recombinant A  bacteriophages, cosmids and fosmids into E. coli cells.   Before we study how genomic libraries are made we first  need to understand the differences between the genetic  organization in eukaryotic and prokaryotic cells. Bacterial  genes are uninterrupted sequences of nucleotides encod-  ing the genetic information required for the synthesis of  a protein. These genes can sometimes be cotranscribed  with adjacent genes of related function into the same  mRNA molecule. This set of cotranscribed genes is called  an operon (Figure 25.3). The mRNA in bacteria does not  generally need to be processed before translation, and  transcription and translation occur simultaneously.   In contrast, genes from eukaryotic cells contain non-  coding sequences called introns and coding sequences  called exons. The former are removed after transcription  by a process called ‘splicing’ that occurs in the nucleus of  the cell. In addition the mRNA is subjected to further  processing involving the addition of a methylated guanine  (M 7 Gppp) called CAP on its 5' end, required for transla-  tion, and a polyadenine (poly(A))tail on its 3' end (Figure  25.3). Mature mRNA is then exported from the nucleus    Figure 25.3 Genetic organization in bacteria (prokaryotes) and eukaryotes. In prokaryotes genes can sometimes be grouped in  operons and hence transcribed together in a single molecule of mRNA. In these organisms the whole process of transcription and  translation takes place simultaneously in the cytoplasm. In contrast, in eukaryotes, genes are organized in single transcriptional  units incorporating introns. Upon transcription in the nucleus, eukaryotic mRNA is firsdy modified by the addition of a CAP and  a poly(A) tail and then by the splicing out of the introns. The mature mRNA is then exported into the cytoplasm where it is  translated into proteins. P, promoter; T.T., transcriptional terminator; RBS, ribosome binding site.    into the cytoplasm, where it is translated into proteins.  Eukaryotic genes appear to be transcribed individually, as  operons have not been described in eukaryotes.   To enable the cloning and isolation of a specific gene(s)  from a cell several steps are required. The first consists of  choosing the source of genetic material which, in  prokaryotic cells, is normally the chromosomal DNA. In  contrast, in eukaryotic cells this is more often the mature  mRNA, as it is not interrupted by introns and conse-  quently codes for complete, active proteins. The second  step consists of the preparation of the purified DNA or  RNA for cloning. This step is more straightforward when  using prokaryotic DNA (section 2.4.1) than eukaryotic  RNA (section 2.4.2). The result will be the construction  of a collection of cloned DNA fragments propagated in  bacteria that is called the genomic library. This library  should ideally contain representatives of every sequence  in the chromosome of a prokaryotic cell and every    expressed gene in the case of a eukaryotic cell. The final  step consists of the screening of the recombinant clones  to identify the required gene(s).   The construction of a prokaryotic gene library can  be achieved by a technique called ‘shotgun cloning’  (Figure 25.4). This involves the purification and partial  digestion of the genomic (chromosomal) DNA from a  prokaryotic organism with restriction endonucleases to  produce a random mixture of fragments of different  sizes. Chromosomal DNA can also be mechanically  sheared and in this case the extremities must be repaired  and made blunt with DNA polymerase in the presence of  deoxynucleotides (dNTPs). These fragments are then  fractionated into different sizes and ligated into a  cloning vector appropriately digested. The recombinant  vectors are then transformed, in the case of plasmids, or    Figure 25.4 Construction of prokaryotic  genomic libraries by shotgun cloning. The  shotgun approach for the construction of  genomic libraries involves the purification  and digestion of chromosomal DNA from  the prokaryotic organisms, followed by the  cloning into a digested suitable vector using  DNA ligase. The recombinant vector is  then introduced into the host cell using the  appropriate method and then the colonies  or plaques are screened for the presence of  the recombinant gene of interest.   Full restriction digest with  enzymes that generate  ends compatible with  those of the digested  chromosomal DNA    transfected, in the case of bacteriophages and cosmids,  into the host cell of choice. The resulting genomic  library can then be screened for the presence of the  recombinant gene of interest by a number of methods  (see section 2.5).   The shotgun approach cannot be applied for the con-  struction of eukaryotic gene libraries because of the pres-  ence of introns in the DNA, which prevents the direct  cloning of functional genes from digested chromosomal  DNA. Instead, mature mRNA from the cytoplasm of cells  expressing the desired gene is used as the source of genetic  material. For example, to make a genomic library con-  taining the insulin gene, RNA from pancreatic cells  expressing this gene have to be isolated. Remember that  cells show distinct differentiation in different tissues, and    express only a small percentage of the whole genome  according to their role in the tissue of which they form  part. Consequently, it is not possible to purify RNA  coding for insulin from, for example, cells of the pituitary  gland. Therefore, the cells expressing the gene of interest  have to be isolated first, and then their mRNA purified.  As mentioned earlier, virtually every eukaryotic mRNA  has a poly(A) tail on its 3' end. This provides a convenient  way to isolate mature mRNA from total cellular RNA,  most of which (98%) is ribosomal RNA (rRNA) and  transfer RNA (tRNA). The total RNA purified from a cell  can be passed through an affinity column packed with  cellulose linked to deoxythymidine oligonucleotides  (oligo(dT)). As the total RNA passes through the column,  only the mRNA molecules which have poly(A) tails will  bind to the oligo(dT) while the rest will flow through the  column to be discarded. The purified mRNA then has to    be converted into ds cDNA (complementary DNA) to  enable its cloning into a suitable vector.   There are generally two main strategies used for the syn-  thesis of cDNA from mRNA: replacement synthesis and  primer adaptor synthesis. For both strategies the first  strand cDNA synthesis is based on the priming of the  mRNA with an oligo-dT which anneals to the poly(A) tail  of the mRNA molecule and, consequently, with the action  of the enzyme reverse transcriptase and in the presence  of dNTPs, the synthesis of the first cDNA strand takes  place. This results in the formation of a mRNA/cDNA  heteroduplex hybrid. The second stage is different for the  two strategies mentioned. The most commonly used is  the replacement synthesis, which is based on the use of  ribonuclease H (RNaseH), an enzyme that cleaves the  RNA moiety of RNA/DNA hybrids and has 5' to 3' and  3' to 5' direction exonuclease activities. This results in  partial digestion of the RNA in both directions. The  resulting RNA fragments can serve as primers for DNA  synthesis using DNA polymerase I. This enzyme, with its  5' to 3' direction exonuclease and polymerase activities  will fill the nicks and effectively remove the RNA primers.  The cDNA fragments synthesized will be joined using  DNA ligase. This method causes the loss of some nucle-  otides at the 5' end of the mRNA, including the CAP  region.   The primer adaptor method for the synthesis of the  second strand of cDNA starts with the removal of the  RNA strand from the mRNA/cDNA hybrid, by treatment  with alkali. This is followed by the addition of a poly(C)  tail to the 3' end of the DNA strand using an enzyme  called terminal transferase. This enables the hybridiza-  tion of a complementary poly(G) primer that will be  the starting point for the synthesis of the second cDNA  strand by the DNA polymerase (Figure 25.5). This  method, in contrast to the replacement synthesis, gener-  ates cDNA molecules with a complete 5' CAP region.  However, it requires more steps and the terminal trans-  ferase step is difficult to control.   Finally, the cloning of the resulting cDNA is aided by  the addition of a poly(C) tail at the 3' ends of the cDNA  fragments using terminal transferase and the ligation of  these to a linearized vector containing a complementary  poly(G) tail also generated by the terminal transferase.   There are a number of points to take into consideration  when choosing a strategy to generate a genomic library.    The larger the insert size, the lower the number of clones  required to have full representation of an entire genome.  To reduce the number of recombinant clones to be  screened before the gene of interest is identified, cosmids,  X bacteriophages or BACs should be used as they can take  larger DNA inserts. Furthermore, if we want to isolate a  large DNA fragment containing, for example, all the  genes required for the biosynthesis of an antibiotic,  plasmid libraries might not be the right choice. In con-  trast, if we are trying to isolate a single gene and the  extent of the screening is not a problem, plasmid libraries  are ideal as they reduce the subcloning steps required to  single out the gene of interest. Table 25.2 shows a com-  parison between the insert sizes taken by different vectors,  their hosts and the genomic libraries for which they can  be suitable.   Once the genomic library has been generated it is neces-  sary to screen for the gene of interest within thousands  of recombinant clones. The choice of screening method  will very much depend on the availability of reagents and  the information on the target gene to be isolated.   This technique is used when some of the DNA sequence  for the gene screened for is known, or when a fragment  of this gene is available from a previous cloning.  Alternatively, a DNA fragment from a closely related gene  can be used as a probe for the isolation of the gene of  interest. The hybridization technique requires plating the  library on a set of agar plates to generate a replica, on  nitrocellulose or nylon membranes, of the plaques or  colonies, each containing a different recombinant DNA  fragment. This process transfers a portion of each plaque  or colony to the membranes and is done in such a way  that the pattern of plaques/colonies on the original plate  is maintained on the filters. The membranes are then  hybridized with a radiolabelled DNA probe containing  part of the sequence to be isolated from the library. The  probe will only bind/hybridize to the recombinant clones  containing that sequence. After this process, the mem-  branes are exposed to X-ray film (autoradiography). The  presence of dark spots on the films represents the loca-  tion of colonies containing the target gene. By orienting  the film with the original agar plate, the colony/plaque  carrying the complementary sequence can be identified  and the desired clone isolated. An alternative to the use  of radio-isotopes for the probes resides in the use of  nucleotides labelled with a molecule such as digoxigenin    Figure 25.5 Synthesis and cloning of  cDNA. Cloning of eukaryotic genes  involves the isolation of mRNA from the  cytoplasm of the cells expressing the  gene of interest. To allow the cloning of  the mRNA the synthesis of ds cDNA is  first required. This involves the synthesis  of the first cDNA strand by reverse  transcriptase using an oligo(dT) primer.  To generate the second strand of cDNA  there are two main methods. The  replacement synthesis method involves  the generation of nicks in the mRNA  strand by the RNaseH followed by the  synthesis of the complementary strand,  using the RNA fragments generated by  the RNaseH as primers, by the DNA  polymerase. The DNA fragments  generated are then joined together by the  DNA ligase. In contrast, the primer  adaptor method requires the degradation  of the mRNA strand by alkali followed  by the addition of a poly(C) tail at the 3'  end of the cDNA strand by the terminal  transferase. For the synthesis of the  second strand of cDNA, addition of an  oligo(dG) primer and DNA polymerase  are required. The ds cDNA generated by  either method can be cloned into any  vector upon addition of poly(C) sticky  ends by the terminal transferase,  provided that the vector has  complementary poly(G) ends created by  this enzyme.    (DIG). In that case detection of the hybridized probes is  performed with anti-DIG antibodies conjugated to an  enzyme such as alkaline phosphatase, which reacts with  a substrate to produce chemiluminescence.   This technique is used when we need to isolate a gene  coding for a protein for which there are antibodies avail-  able. The success of this technique relies on the expres-    sion of the gene of interest, as it requires the synthesis of  the target protein from the target recombinant gene. The  screening steps are similar to those used for the hybridi-  zation screening, with the difference that the membranes  containing portions of plaques or colonies have to be  incubated with the antibodies that will recognize the  target protein. This antibody, called the primary anti-  body, will bind tightly to those colonies/plaques contain-  ing the recombinant gene of interest, provided that the    Table 25.2 Comparison of vectors used for the construction of genomic libraries    protein encoded by this gene has been synthesized. The  position of the bound antibody is revealed by incubating  the membranes with a labelled antibody (secondary anti-  body) that recognizes the primary antibody. There are  different types of labels for antibodies, all of which can  easily be detected.   This type of screening is limited to proteins that have a  specific activity that can easily be identified within a large  population of recombinant clones. Needless to say, to  detect a protein activity the gene coding for this protein  must be expressed and an active protein must be pro-  duced. Understanding of this technique can be helped by  illustrating this screening with an example. Suppose we  want to isolate a gene coding for a bacterial haemolytic  toxin from a genomic library. As we know that this toxin  lyses red blood cells, we could plate the library on plates  containing agar mixed with blood. Those colonies/  plaques expressing the haemolytic toxin could easily be  identified by the presence of a haemolytic halo around  them resulting from the action of the toxin on the red  blood cells.   The primary objective of pharmaceutical companies  involved in the production of recombinant drugs is the  maximal expression of recombinant genes to generate  large quantities of these drugs. Unfortunately, the cloning  of a gene into a vector does not ensure that it will be  properly expressed. To improve expression of a cloned  gene the different stages that lead to the synthesis of the  protein therefore have to be optimized. This is achieved  by the use of so-called expression vectors (Figure 25.6).    Some expression vectors have been designed to produce  large quantities of protein in specific cell hosts. For  example, the bacmids are shuttle expression vectors  derived from a baculovirus ds DNA circular genome and  are used to transfect insect cells in order to produce large  quantities of a recombinant protein in fermenters.   To optimize transcription it must be ensured that the  recombinant gene is placed after a promoter (Figure 25.6)  that will be recognized by the RNA polymerase of the  host cell where the gene is going to be expressed. Two  types of promoters can be used: (1) constitutive promot-  ers, which are expressed all the time and (2) inducible  promoters, where expression is turned off during culture  growth and turned on, for example, upon the addition of  an inducing molecule to the culture, usually shortly  before harvesting, when high numbers of bacteria are  present in the culture. Inducible promoters are very  useful when expressing foreign genes coding for proteins  toxic to the bacterial hosts as their premature expression  could lead to growth impairments and consequently low  yields of recombinant protein.   Furthermore, to ensure that transcription finishes after  the 3' end of the recombinant gene, a transcriptional ter-  minator (Figure 25.6) must be placed just downstream of  this gene.   A key feature that determines whether a gene is going to  be efficiently translated by a certain host is the nucleotide  sequence of the ribosome binding site (RBS), located  upstream of the gene (Figure 25.6), which needs to be  efficiently recognized by the ribosomes of this host. In  addition, the distance between the RBS and the transla-   Figure 25.6 Expression vectors and the generation of fusion  proteins. Expression vectors have optimized all the signals  required for transcription (inducible promoter and  transcriptional terminator) and for translation (ribosome  binding site). Some of them carry the gene for (3-galactosidase  with a multiple cloning site that allows the insertion of small  genes for the generation of fusion proteins. RBS, ribosome  binding site.   tion start codon needs to be optimal to enable the right  interactions between the mRNA and the ribosomes and  start the protein synthesis. There are commercially  available vectors carrying sequences for RBSs and transla-  tion start codons which are optimally recognized by the  ribosomes of the host cells, ensuring that any recom-   Small proteins are frequently susceptible to proteolytic  degradation when expressed in a foreign host. This deg-  radation can be avoided by expressing them fused to a  larger protein. This is normally achieved by cloning  the small gene downstream of a gene coding for a  protein such as (3-galactosidase. To obtain the fusion  protein (Figure 25.6) it is essential to ensure that the  reading frame is conserved and that no translation stop  codons are present between the (3-galactosidase and  the target gene, enabling the ribosomes to read  through. Interestingly, affinity columns that will bind  the fused polypeptides are available, which facilitates the  purification of the recombinant protein by affinity  chromatography.   Although high levels of protein production may be  achieved by optimizing transcription and translation of  a gene, the obtained protein may still need to undergo  post-translational modifications before it is active. Some  of these modifications include correct disulphide bond  formation, proteolytic cleavage of a precursor, glycosyla-  tion and additions to amino acids such as phosphoryla-  tion, acetylation, sulphation, acylation, etc. Unfortunately,  the practical E. coli host, in which most recombinant  proteins are produced, does not possess the same type of  cellular machinery required for these modifications.  Recently, Campylobacter jejuni has been found to possess  an eukaryotic-like system for protein glycosylation and  efforts are being made to genetically engineer E. coli  strains to perform the adequate glycosylation of recom-  binant proteins as mammalian cells. Hence, it is essential  to select a suitable host for the expression of the target  gene that can carry out the required post-translational  modifications that will enable the synthesis of large  amounts of a biologically authentic product. Table 25.3  shows a comparison of a selection of hosts currently used  for the expression of recombinant proteins.   The polymerase chain reaction (PCR) is an extremely  simple and powerful technique that was perfected by  Kary Mullis in the mid-1980s and has revolutionized  many studies in molecular biology, currently having  applications ranging from forensic studies to the develop-  ment of new recombinant drugs. This technique allows  the generation of large amounts of copies of a specified   Table 25.3 Comparison of different hosts used for the expression of recombinant genes   Easy to grow in large-scale volumes  Transcriptional and translational control well known  Successfully used in the manufacture of insulin,  interferon and human somatotropin   Difficult to achieve export of some  proteins into growth medium  Degradation of small proteins by proteases  Unable to undertake most post-  translational modifications, e.g.  glycosylation   Many proteins can be exported into growth medium  Easy to grow in large-scale volumes   Lack of high-level expression vectors  Unable to carry out most post-  translational modifications, e.g.  glycosylation   Wide range of high-level expression systems available  Recombinant proteins do not form insoluble  aggregates in the cytoplasm   Gene expression can be difficult to control  Sometimes fails to achieve accurate  post-translational modification of  recombinant proteins   Precise post-transcriptional modifications  Easy to generate large amount of recombinant  protein, e.g. one goat can generate 1 kg of  recombinant protein in milk per year  Relatively inexpensive    DNA sequence from a single DNA molecule without the  need for cloning.   The PCR exploits certain characteristics of DNA rep-  lication, as it uses single-stranded DNA (ss DNA) as a  template for the synthesis of complementary new strands  in a 5' to 3' direction. The ss DNA templates can be gener-    ated by heating ds DNA above 90 °C. DNA polymerase  synthesizes ds DNA by extending the complementary  strand of a template. Hence the DNA polymerase can be  directed to synthesize a specific region of DNA by using  a synthetic, complementary oligonucleotide primer that  will anneal to the template when the temperature is    lowered. The PCR reaction uses a special DNA polymer-  ase ( Taq DNA polymerase from the thermophilic bacte-  rium Thermus aquaticus) that can withstand temperatures  above 100 °C and has an optimal activity at 72 °C, which  has the advantage of reducing non-specific primer  annealing that may occur at lower temperatures.   In PCR both strands of a target DNA serve simultane-  ously as templates upon the addition of a pair of primers,  one for each strand of DNA. A typical PCR amplification  is shown in Figure 25.7. Every PCR cycle is normally  repeated up to 30 times. The net result of a PCR is  that, at the end of n cycles, it will generate 2" ds DNA  copies of a single DNA fragment located between the two  primers.   There are some obvious advantages of using PCR. The  main one is specificity, as it allows, using the appropriate  primers, the amplification of specific DNA fragments  from a population of different cells. It is also a very  rapid technique, as it only takes a few hours to amplify  a fragment of DNA compared with days using conven-  tional cloning methods. An important feature of PCR is  its versatility, as it allows the incorporation of mismatches  on the 5' end of the primers provided that the 3' end  has perfect complementary with the targeted strand.  This can be exploited to add restriction sites to enable  subsequent cloning of the amplified DNA, or introducing  specific mutations into genes. Furthermore, the equip-  ment used for PCR is relatively inexpensive and allows  the analysis of a large number of samples at one  time. Finally, PCR does not require purified template  DNA and can amplify genes from whole cells or tissue  samples.   However, there are also a number of limitations to the  use of PCR. The designing of primers for this technique  requires some knowledge of the DNA sequence to be  amplified. Although there are new genetically engineered  DNA polymerases that can synthesize large fragments of  DNA, there are still some restrictions with regards to the  maximum length of DNA that can be amplified. Ideally,  fragments of 0.1-3 kb can be easily amplified although  this technique, under the appropriate conditions, would  amplify larger fragments (up to 20 kb). In addition, the  slightest sample contamination can lead to false positive  results, which can have detrimental effects when this  technique is used in diagnostics. Finally, sometimes there  is a risk of non-specific amplification when the primers  anneal to sequences similar to the targets, leading to the  amplification of the wrong DNA.   The development of PCR has revolutionized not only  basic research but also different areas of medicine. Table  25.4 lists some of the most important clinical applica-  tions of PCR. These types of analysis were practically  impossible without PCR owing to the large amount of  samples that needed handling, the amount of time  required to obtain results or the lack of sensitivity of the  available tests.   One of the first and most important commercial applica-  tions of genetic engineering was the introduction of  genes coding for clinically important proteins into bacte-  ria. Because bacterial cells are cheap to grow on a large  scale in fermenters, they can synthesize vast amounts of  protein from the recombinant genes they carry. This  results in a significant reduction in cost and an increase  in the availability of these proteins. There are currently a  large number of important recombinant drugs available  on the market, examples of which are provided in Table  25.5. This chapter covers only the genetic manipulation  strategies used to produce some well-known drugs.   Recombinant human insulin, used for the treatment of  diabetes, was the first drug produced using genetic engi-  neering, in 1982. Before this, animals, notably pigs and  cattle, were the only non-human sources of insulin.  Animal insulin differs slightly from the human form and,  consequently, it can potentially elicit an immune response  in humans, making it ineffective. Additionally, the use of  recombinant insulin prevents the risks resulting from  potential contamination of animal insulin with other  hormones or viruses. To understand how insulin is pro-  duced using recombinant DNA techniques we first need  to review its structure. Figure 25.8 illustrates how insulin  is initially synthesized as preproinsulin, a single polypep-  tide which is processed during export into proinsulin and  finally into active insulin, once proteolytic cleavage of the  connecting sequence for the two A and B insulin chains  has occurred. These two chains remain joined through  disulphide bonds.   Currently, different approaches are employed to  produce recombinant insulin, one of which is shown in  Figure 25.8. First of all, two DNA fragments coding for  the A or the B insulin chains are synthesized chemically.    Figure 25.7 The polymerase chain reaction (PCR). A single  PCR reaction involves the following steps: ( 1 ) Denaturation of  the target ds DNA by heating at 95 °C and addition of an  excess of two oligonucleotide primers, each complementary to  a different strand of the target sequence, Taq DNA polymerase  and dNTPs. (2) Annealing of the primers to the DNA strands  by decreasing the temperature to 55 °C. (3) Elongation (or  synthesis) of the new strand of DNA by the Taq DNA  polymerase after increasing the temperature to 72 °C. This  cycle can be repeated up to 30 times. In each cycle the number  of DNA copies is doubled, hence, at the end of a PCR reaction,  we could have 2 30 copies of an original target DNA molecule.    Each of these synthetic fragments is then individually  inserted in a plasmid after the E. coli gene coding for (3-  galactosidase. This enables this bacterium to produce  large fusion proteins with the insulin chains tacked on to  the end of the (3-galactosidase enzyme. These fusion pro-  teins can then be purified from bacterial extracts and the  insulin chains released upon treatment with cyanogen  bromide, which cleaves peptide bonds following methio-  nine residues. As methionine was inserted at the bounda-  ries between the [3-galactosidase and the insulin chains,  and there are no methionines present internally within  the insulin molecule, treatment with cyanogen bromide  results in the cleavage of intact insulin chains from the  fusion proteins. The purified A and B insulin chains can  be mixed and reconstituted into an active insulin mole-  cule. Currently there are also other methods used to  produce recombinant insulin which are based on the gen-  eration of single (3-galactosidase fusions to the full-length  insulin gene containing the coding sequences for both A  and B chains. These alternative methods can simplify the  manufacturing of this drug.   Somatostatin, also known as the ‘antigrowth hormone’,  modulates the action of growth hormone and is fre-  quently used to treat acromegaly (uncontrolled bone  growth). Being a very small peptide, the gene coding for  it can easily be chemically synthesized and cloned into a  suitable expression vector. As E. coli tends to degrade  small peptides, the generation of a (3-galactosidase-  somatostatin fusion protein prevents this degradation.  Furthermore, as with insulin, the absence of methionine  residues in the somatostatin amino acid sequence allows  the insertion of a methionine codon in the junction  between (3-galactosidase and the somatostatin gene. This  enables the subsequent cleavage with cyanogen bromide  of the recombinant hormone purified from E. coli. The   Duchenne muscular dystrophy  Fragile X syndrome  Lesch-Nyhan syndrome  Tay-Sachs disease  Kennedy’s disease  Cystic fibrosis  Haemophilia B   Mutations in gene coding for angiotensin-converting enzyme (ACE)  Mutation in the angiotensinogen gene  Mutation in the apolipoprotein CII gene  Mutation in the LDL receptor    strategy used to generate recombinant somatostatin is  shown in Figure 25.9.   Somatotropin, also known as human growth hormone  (hGH), consists of 191 amino acids; it is produced in the  pituitary gland and regulates growth and development.  Regular injections of hGH are given to children with  dwarfism caused by the lack of this hormone so that they  can reach near normal heights. In this case, unlike with  insulin, animal-derived hormones are ineffective and    only the human protein works. Because of continuous  shortages of pituitaries from human cadavers, the pro-  duction of recombinant hGH has been imperative.  Furthermore, the contamination of children with fatal  viruses from the cadavers has been an additional reason  for moving away from this source of hormone. As the  gene for the hGH is 573 nucleotides long, it was in prac-  tice too large to be synthetically produced to generate the  recombinant hormone as achieved with insulin and  somatostatin. Hence there have been two practical ways  of generating recombinant hGH, one of which resulted   Two peptide chains:  A = 21 amino acids  B = 30 amino acids    in the generation of this hormone with an added methio-  nine at the N-terminus. Figure 25.10 shows these two  strategies.   Initially the coding region for hGH was isolated from  a cDNA library. The DNA fragment coding for the mam-  malian signal peptide can then be excised by a restriction  enzyme that also removes the first 24 codons of the  mature protein. A chemically synthesized DNA fragment  containing a methionine codon, to enable translation in  E. coli, followed by these first 24 codons was therefore  ligated to the DNA fragment coding for the remaining  amino acids 25-191 of the hGH (Figure 25.10A). The  resulting DNA was cloned into an expression vector and  transformed into E. coli where the recombinant hGH  accumulated in the cytoplasm. The recombinant hormone  can be isolated front bacterial cell extracts and, in contrast  to the non-recombinant protein, carries a methionine  residue at the N-terminus.   Figure 25.10B shows an alternative method consisting  of the replacement of the mammalian signal peptide for  a secretion signal peptide functional in bacteria. This  enables the purification of the recombinant hGH from  the periplasm of the bacterial cell, reducing the difficul-  ties associated with the purification of recombinant pro-  teins from the cytoplasm. To achieve this, once the  mammalian signal peptide has been removed as above, a  synthetic DNA molecule containing the missing 24  codons of the hGH, without an added methionine codon,  is ligated to the DNA fragment coding for the 25-191  remaining residues. The resulting DNA molecule is then  inserted in an expression vector that codes for a bacterial  signal peptide in fusion with the hGH gene. Once trans-  formed into E. coli, the recombinant hGH is produced  and the signal sequence will target the protein for secre-  tion into the periplasmic space where it will accumulate.  The periplasmic proteases release the signal peptide,  leaving hGH without additional amino acids. The protein  can then be easily purified from the periplasmic space  after release by hypotonic disruption of the outer  membrane.   Before the development of recombinant DNA technol-  ogy, two main strategies were employed for vaccine pro-  duction: the generation of inactivated vaccines consisting  of chemically killed derivatives of the infectious agent,  and of attenuated vaccines, which are altered viruses and  bacteria that are avirulent and can no longer cause  disease. However, these vaccines were potentially danger-  ous as they could be contaminated with infectious organ-    isms or revert to a virulent form. To avoid these problems,  recombinant DNA technology has enabled the produc-  tion of subunit vaccines consisting solely of immunogenic  surface proteins which can elicit immune responses  without the risk of infection.   The hepatitis B virus (HBV) vaccine was the first suc-  cessful subunit vaccine developed. This virus infects the  liver and can cause serious damage. This virus has a  surface antigen, HBsAg, which is found in blood of  infected patients and has been found to elicit a significant  immune response. The gene coding for this antigen has  been isolated from the virus and cloned into a vector that  allows high expression of the HBsAg protein in yeast cells.  Figure 25.11 shows the strategy currently used for the  generation of recombinant HBV vaccine. In this case, as  the 3.2-kb genomic sequence of the HBV virus was  known, the gene coding for the HBsAg was directly  cloned into a shuttle expression vector that replicates in  both, E. coli for the genetic manipulation steps, and in  yeasts such as Saccharomyces cerevisiae for the production  of the recombinant antigen. Transcription of the gene  encoding HBsAg is driven from a strong yeast promoter  and is stopped by a transcriptional terminator present in  the vector. The vector also has a leucine biosynthesis  marker for selection in yeasts and a tetracycline resistance  marker for selection in bacteria. The yeast harbouring  this plasmid can grow in fermenters in the absence of  leucine, generating large amounts of the antigen that can  subsequently be extracted from the cells.   Influenza viruses type A, B and C cause flu in mammals  and birds. The influenza virus type A has been the cause  of several pandemics in the past, causing the deaths of  millions of people. Its 13.6-kb genome consists of 8ss  linear RNA molecules that code for 1 1 proteins. Because  of the segmented nature of its genome, alleles can easily  be swapped between different influenza viruses strains  during the co-infection of a host. This makes the virus  extremely adaptable and able to escape the immune  system by rapidly acquiring novel combinations of its  immunogenic proteins. Two of these proteins are located  on the surface of the viral particles and define their anti-  genicity: a hemagglutinin (H or HA) and a neuramini-  dase (N or NA). To date there are 16 different H antigens  (H1-H16) and 9 different N antigens (N1-N9) known,  thus the influenza A viruses are classified accordingly as  H1N1 (swine flu or Spanish flu), H5N1 (bird flu), H7N7  (horse flu), etc. Seasonal flu recurs annually and since  2003 it has been caused mostly by variants of the H3N2,    Figure 25.8 Production of recombinant insulin. (A) Insulin consists of two polypeptide chains (chains A and B). It is initially  synthesized as part of a larger peptide called preproinsulin. The transport across the cell membrane of preproinsulin results in the  cleavage of the signal peptide and the formation of disulphide bridges to generate proinsulin. Finally, the connecting peptide is  cleaved generating the mature insulin. (B) One of the strategies used to make recombinant insulin consists of the cloning of the  DNA fragment coding for the A chain and the B chain into two separate expression vectors as (3-galactosidase fusions in E. coli.   The fusion proteins are then purified and the insulin is cleaved with cyanogen bromide (CNBr) after a methionine incorporated at  the P-galactosidase and insulin junction. The presence of several methionines in the P-galactosidase results in multiple cleavage of  this molecule by CNBr. Finally, the resulting insulin A and B chains are refolded and the cysteines are oxidized for the generation  of the active insulin.   Figure 25.9 Production of recombinant somatostatin. The  small size of somatostatin allows the chemical synthesis of the  gene coding for it. This gene can be cloned into an expression  fused to p-galactosidase. The fusion protein generated in E.  coli is then purified and the somatostatin polypeptide is  released by treatment with cyanogen bromide. RBS, ribosome  binding site; TT, transcriptional terminator.   H1N1 and influenza B viruses, the exact types changing  over time with developing acquired resistances through  the human population by exposure and vaccinations.  Therefore, the evolving prevalence of the different sero-    types is constantly being monitored with the aim of  anticipating the serotype composition of potential pan-  demic influenza viruses. Because of the short time frame  between the identification of a novel strain and the need  for a vaccine against it, recombinant biotechnology is  essential. As the hemagglutinin is the most rapidly evolv-  ing gene product and is crucial for viral attachment and  evasion from the immune system recombinant influenza  virus vaccines are developed using the gene coding this  protein. To achieve this, the RNA is first converted into  ds cDNA, cloned and sequenced. This allows the com-  plete identification and genotyping of the virus. Secondly,  the gene coding for the hemagglutinin is subcloned into  a bacmid expression vector and the recombinant glyco-  protein (rHA) is produced in large quantities by trans-  fecting Spodoptera frugiperda insect cells growing in  fermenters as for the hepatitis B vaccine.   A large number of the antibiotics currently used have  been isolated from the Gram-positive soil bacterium  Streptomyces, although many other microorganisms have  also been used as sources of antibiotics. The biosynthesis  of an antibiotic can sometimes include 10-30 separate  enzyme-catalysed reactions, which makes the cloning of  all the genes coding for these enzymes very difficult. A  strategy used to isolate the complete set of antibiotic  biosynthetic genes consists of the transformation of a  recombinant gene library from an organism producing  the antibiotic into a mutant strain of the same organism  unable to produce it. The transformants can be screened  for the production of the antibiotic by plating them on  to agar plates that have been seeded with a sensitive bac-  terium. The appearance of halos of growth inhibition  around the recombinant colonies indicates the successful  cloning of the antibiotic biosynthetic gene cluster. This  strategy was successfully used for the cloning and pro-  duction of the antibiotic undecylprodigiosin from  Streptomyces coelicolor, as illustrated in Figure 25.12.    Figure 25.10 Two strategies to produce recombinant hGH. These two strategies use as the starting material the recombinant cDNA  for hGH which contains the mammalian signal sequence required for the secretion of this protein from mammalian cells. This  signal peptide is first removed using a restriction enzyme that cleaves after the nucleotides coding for the first 24 amino acids of  the hGH. From this stage two strategies can be followed. (A) A chemically synthesized fragment containing the genetic information  for the first 24 amino acids of the hGH, plus a methionine codon in the 5' end, is ligated to the remaining cDNA fragment coding  for amino acids 25-191 and introduced into an expression vector. The hGH expressed from this vector in E. coli accumulates in the  cytoplasm and is extracted from whole cell lysates. (B) A chemically synthesized fragment also containing the genetic information  for the first 24 amino acids of the hGH, but without an added methionine codon, is ligated to the remaining cDNA fragment and  cloned into an expression vector immediately after the sequence for a bacterial signal peptide. Consequently, when expressed in E.  coli , the hGH is tagged on its N-terminus with this signal peptide that drives the export of this protein to the periplasm. Once in  the periplasm, the signal peptide is cleaved by proteases and the mature hGH can be released and purified upon hypotonic  disruption of the outer membrane. TT, transcriptional terminator.    In some instances, recombinant DNA technology has  been successfully used to generate novel antibiotics by  introducing in the same organism the genes responsible  for the synthesis of two closely related antibiotics. By  cross-feeding antibiotic precursors between two close  metabolic pathways, novel antibiotics can be generated.  This strategy has been very successful in the cross-feeding  of antibiotic pathways between different Streptomyces spp.   For many years clinical diagnostic laboratories had limi-  tations in the detection of pathogenic bacteria and para-  sites due to time constraints in the identification of these  agents, as they required to be cultured, a time-consuming  procedure detrimental to the patients’ health. In addition,  many inherited genetic disorders could not be identified  owing to the lack of appropriate techniques and the una-  vailability of the human genome sequence. The rapid  developments in molecular biology have enabled modern  medicine to overcome some of these problems. Currently,  a good diagnostic test must be specific for a target mole-  cule, sensitive enough to detect minute levels of that mol-  ecule, rapid and technically simple.   This section introduces some molecular diagnostic  techniques, based on the detection of specific nucleic acid  sequences, currently used in the clinic.   Each pathogenic microorganism contains genetic mate-  rial that distinguishes it from its host and from other  microorganisms. This specific material constitutes a sig-  nature that allows the identification of a particular    microorganism from a complex mixed population. In the  diagnosis of infectious diseases, identification of specific  sequences from microbial pathogens will allow appropri-  ate treatment at an early stage as well as prevention of the  spread of disease.   The two main techniques used for the diagnosis of  infectious disease targeting nucleic acids are hybridiza-  tion and PCR-based amplification. There are currently  specific primers and probes for the detection of more  than 100 infectious disease. Table 25.6 shows just a few  examples.   Nucleic acid hybridization is based on the precise nucle-  otide base pairing and hydrogen bonding between one  strand of nucleotides and a complementary nucleotide  sequence. Any diagnostic nucleic acid hybridization  consists of three essential elements: a DNA probe, the  target DNA and the signal detection system. Recent  developments in detection systems and improvements  in safety have enabled the use of highly sensitive non-  radioactive methods. Figure 25.13 illustrates the  general steps required for DNA hybridization using  chemiluminescent-based detection. This non-radioactive  system achieves signal amplification by enzymatic con-  version of a chemiluminescent substrate. First of all, DNA  from the biological sample including the target DNA  from the pathogen to be identified needs to be extracted.  A diagnostic biotin-labelled probe is then mixed with the  target DNA bound to a membrane support. The hybrid-  ized probe is then incubated with streptavidin, which has  multiple sites that avidly bind biotin. Subsequent incuba-  tion with biotin conjugated to alkaline phosphatase  results in the enzymatic labelling of the bound streptavi-  din. Finally, addition of a special substrate for the alkaline    Figure 25.11 Production of hepatitis B subunit vaccine. The gene for the hepatitis B surface antigen (HBsAg) was isolated from a  genomic library and cloned into a shuttle vector that promotes high expression levels of this gene in yeast cells. The presence of a  LEU2 marker allows the selection of yeasts containing this plasmid by growing them in the absence of leucine. Recombinant yeast  cells expressing the HBsAg are grown in large fermenters and the antigen is purified from whole cell extracts for further  formulation into the hepatitis B subunit vaccine.    Figure 25.12 Isolation of genes responsible for antibiotic  production. For the isolation of the genes required for  undecylprodigiosin biosynthesis a Streptomyces strain, unable  to make this antibiotic, was transformed with a genomic  library from a Streptomyces undecylprodigiosin-producing  strain. The resulting cells were plated onto agar seeded with a  bacterium sensitive to undecylprodigiosin. The recombinant  genes required for undecylprodigiosin biosynthesis could be  isolated from colonies showing a halo of cell lysis around them  resulting from the production of this antibiotic.    phosphatase results in the conversion of this substrate  into a product which emits light and which can be  detected after exposure to X-ray film or by using sensitive  imaging cameras.   The procedure described above can also be scaled  down, automated and redesigned to use thousands of  sequence-specific probes at once in what are called micro-  arrays. In this case non-labelled DNA probes are synthe-  sized and minute droplets are spotted at high density on  to glass slides where they will remain bound. The DNA  from the biological sample is then labelled at the extremi-  ties with a fluorescent dye and after denaturation into  single strands it is allowed to anneal with the probes  bound on the slide. After washing, labelled DNA frag-  ments which annealed to specific probes are visualized by  illuminating the slide with light of the appropriate wave-  length to cause the dyes to fluoresce. As microarrays can  carry thousands of probes per square centimetre, this is  performed by automated scanning devices and dedicated  software which identifies the probes producing positive  signals. This technique allows the screening at once of a  very large number of pathogens or defined genetic  markers.   As in many other fields, PCR has brought a revolutionary  change in nucleic acid-based diagnosis. In a clinical  setting, PCR has many desirable features such as the sen-   ' ' ' ' ' ' ' ' ' ' ' ' 1 J I'''''''    Figure 25.13 Diagnosis using DNA  hybridization with biotin-labelled  probes. The DNA from the pathogen to  be identified is first purified and bound  to a membrane. The membrane is then  incubated with a diagnostic biotinylated  probe. The biotin from the probe will be  recognized by streptavidin which has  several biotin recognition sites. Hence,  subsequent incubation with biotin-  labelled alkaline phosphatase results in  the recognition of the bound  streptavidin. As several molecules of  biotin-labelled alkaline phosphatase will  bind to a single streptavidin molecule,  incubation of the membranes with  chemiluminescence substrate for this  enzyme will lead to an amplified reaction  and the generation of light-emitting  products.    sitivity to work with tiny amounts of DNA samples from  blood or tissue to achieve a specific and significant ampli-  fication of target sequences. Furthermore, the rapidity of  this process, as explained previously in this chapter, pro-  vides a significant advantage in the early diagnosis and  treatment of infectious diseases.   A PCR fluorescence-based technique has been used  successfully in the diagnosis of infectious diseases. In this  case the PCR primers are labelled with a fluorescent dye  that is bound to the 5' end of each primer. Two main  types of fluorescent dyes are normally used: one is fluo-  rescein, which appears green under certain light wave-  lengths, and the other is rhodamine, which appears red.  After PCR amplification of the target sequence with the  fluorescent-labelled primers, the primers are removed by  chromatographic separation, and the presence of the  labelled PCR product is detected. The absence of labelled  PCR product is interpreted as the absence of the target  DNA sequence.   The procedure can also be applied to detect specific  RNAs, for example to detect RNA viruses. In this case the  extracted RNA has to be converted first to ds cDNA with  reverse transcriptase before the PCR amplification. The  procedure is then called reverse transcriptase PCR or  RT-PCR.   The use of new diagnostic techniques has allowed indi-  viduals to discover whether they or their offspring are at  risk of suffering from specific inherited diseases. DNA  analysis using PCR has been used for the identification  of carriers of hereditary disorders, for prenatal diagnosis  of deleterious genetic conditions and for the early diag-  nosis of these disorders before the manifestations of any  symptoms. Table 25.7 shows some examples of genetic  human disorders that are currently identified by PCR.   Bradley, J.R., Johnson, D. & Pober, B. (2006) Lecture Notes:  Medical Genetics, 3rd edn. Blackwell, Oxford.   Bruns, D.E., Ashwood, E.R. & Burtis, C.A. (2007) Fundamentals  of Molecular Diagnostics. Elsevier Science, Philadephia, PA.   Crommelin, D.J.A. & Sindelar, R.D.. (2005) Pharmaceutical  Biotechnology: Ait Introduction for Pharmacists and Pharma-  ceutical Scientists, 2nd edn. Taylor & Francis, London.   Crommelin, D.J.A., Sindelar, R.D. & Meibohm, B. (2008)  Pharmaceutical Biotechnology: Fundamentals & Applications,  3rd edn. Informa Healthcare, London.   Glick, B.R. & Pasternak, J.J. (2003) Molecular Biotechnology:  Principles & Applications of Recombinant DNA, 3rd edn.  American Society for Microbiology Press, Washington, DC.   Groves, M.J. (2006) Pharmaceutical Biotechnology, 2nd edn.  Taylor & Francis, Boca Raton, FL.   Ho, R.J.Y. & Gibaldi, M. (2003) Biotechnology and  Biopharmaceuticals. John Wiley & Sons, New York.   Madigan, M.T., Martinko, J.M., Dunlap, P.V. & Clark, D.P.  (2009) Brock Biology of Microorganisms, 12th edn. Benjamin/  Cummings, San Francisco, CA.   Mathews, C.K., van Holde, K.E. & Ahern, K.G. (2000)  Biochemistry, 3rd edn. Benjamin/Cummings, San Francisco,  CA.   Primrose, S.B. & Twyman, R.M. (2006) Principles of Gene  Manipulation and Genomics, 7th edn. Blackwell, Oxford.   Walsh, G. (2007) Pharmaceutical Biotechnology: Concepts and  Applications. John Wiley & Sons, Chichester.   Watson, J.D. (2007) Recombinant DNA: Genes and Genomes, a  Short Course, 3rd edn. W.H. Freeman, New York.   3 Applications of microorganisms in the partial synthesis of  pharmaceuticals 469   6 Use of microorganisms as models of mammalian drug  metabolism 476   There has long been a tendency, particularly in the  medical and pharmaceutical fields, to regard microbes as  harmful entities to be destroyed. However, as will be    described in this chapter, the exploitation of microorgan-  isms and their products has assumed an increasingly  prominent role in the diagnosis, treatment and preven-  tion of human diseases. Non-medical uses are also of  significance, e.g. the use of bacterial spores ( Bacillus thur-  ingiensis) and viruses (baculoviruses) to control insect    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    pests, the fungus Sclerotinia sclerotiorum to kill some  common weeds, and improved varieties of Trichoderma  harzianum to protect crops against fungal infections, and  these will also be explored.   The earliest uses of microorganisms to treat human  disease can be traced to the belief that the formation of  pus in some way drained off noxious humours responsi-  ble for systemic diseases. Although the spontaneous  appearance of pus in their patients’ wounds satisfied  most physicians, deliberate contamination of wounds  was also practiced. Bizarre concoctions of bacteria such  as ‘ointment of pigs’ dung’ and ‘herb sclerata’ were par-  ticularly favoured during the Middle Ages. Early central  European and South American civilizations cultivated  various fungi for application to wounds. In the 19th  century, sophisticated concepts of microbial antagonism  were developed following Pasteur’s experiments demon-  strating inhibition of anthrax bacteria by ‘common bac-  teria’ simultaneously introduced into the same culture  medium. Patients suffering with diseases such as diphthe-  ria, tuberculosis and syphilis were treated by deliberate  infection with what were then thought to be harmless  bacteria such as staphylococci, Escherichia coli and lacto-  bacilli. Following their discovery in the early part of the  last century, bacterial viruses (bacteriophages) were con-  sidered as potential antibacterial agents — an idea that  soon fell into disuse but has recently been revived  (Chapter 27).   Some of the most important and widespread uses of  microorganisms in the pharmaceutical sciences are in  the production of antibiotics and vaccines and the use  of microorganisms in the recombinant DNA industry.  These are described in Chapters 11, 24 and 25. However,  there are a variety of other medicinal agents derived  from microorganisms including vitamins, amino acids,  dextrans, iron-chelating agents and enzymes. Microor-  ganisms as whole or subcellular fractions, in suspension  or immobilized in an inert matrix are employed in a  variety of assays. Microorganisms have also been used  in the pharmaceutical industry to achieve specific  modifications of complex drug molecules such as ster-  oids, in situations where synthetic routes are difficult and  expensive to carry out, and more recently microorgan-  isms have been employed in their own right as platforms  for the discovery of novel therapeutic peptides and  proteins.   Dextrans are polysaccharides produced by lactic acid bac-  teria, in particular members of the genus Leuconostoc (e.g.  L. dextranicus and L. mesenteroides), following growth on  sucrose. These sugar polymers first came to the attention  of industrial microbiologists in sugar refineries where  large gummy masses of dextran clogged pipelines. Dextran  is essentially a glucose polymer consisting of (l-6)-a-  links of high but variable molecular weight (15 000-  20 000 000; Figure 26. 1 ) . Growth of the dextran producing  strain is carried out in large fermenters using media with  a low nitrogen but high carbohydrate content. The average  molecular weight of the dextrans produced will vary with  the strain used. This is important, since the laboratory or  clinical utility of the dextran is dependent on a defined  molecular weight. Two main methods are employed for  obtaining dextrans of a suitable molecular weight. The  first involves acid hydrolysis of very high molecular weight  polymers, while the second uses small preformed dex-  trans as templates for the polymerization process. These  templates are added to the culture fluid to produce dex-  trans of shorter chain length. Once formed, dextrans of  the required molecular weight are obtained by precipita-  tion with organic solvents prior to formulation.   Dextrans are produced commercially for use as plasma  substitutes (plasma expanders), which can be adminis-  tered by intravenous injection to maintain or restore the  blood volume, and for application to ulcerated wounds  or to burns where they form a hydrophilic layer that  absorbs fluid exudates.   A summary of the properties of the different types  of dextrans available is presented in Table 26.1. Dextrans  for clinical use as plasma expanders require a molecular  weight between 40000 and 300000. Polymers below  40 000 molecular weight are excreted too rapidly from the  kidneys, while those above 300000 molecular weight are  potentially dangerous because of retention in the body.  In practice, infusions containing dextrans of average  molecular weights of 40 000, 70 000 and 1 10 000 are com-  monly encountered.   Iron dextran injection, containing a complex of iron  hydroxide with dextrans of average molecular weight  between 5000 and 7000, is used for the treatment of iron-  deficiency anaemia in situations where oral therapy is  either ineffective or impractical. The sodium salt of sulphu-  ric acid esters of dextran, i.e. dextran sodium sulphate, has    anticoagulant properties comparable with those of heparin  and is formulated as an injection for intravenous use.   Several chemicals used in medicinal products are pro-  duced by fermentation.   Vitamin B 2 (riboflavin), one of the B group of vitamins,  is present in milk, liver, kidneys, cereals and vegetables  and is also synthesised by intestinal flora in carbohydrate-  rich diets. Vitamin B 2 deficiency, although rare, is char-  acterized by symptoms that include an inflamed tongue,  dermatitis and injury to the bone marrow. In genuine  cases of malnutrition these symptoms will accompany  those induced by other vitamin deficiencies. Riboflavin is  produced commercially in significant yields by the  moulds Eremothecium ashbyii and Ashbya gossypii and  some bacteria including B. subtilis (Table 26.2).   Pernicious anaemia is a fatal disease first reported in  1880, but it was not until 1926 that it was discovered that  eating raw liver effected a remission. The ‘anti-pernicious’  ingredient was subsequently isolated and called vitamin  B 12 or cyanocobalamin. Vitamin B 12 was initially obtained  from liver but during the 1960s it was determined that it  could also be obtained as a by-product of microbial  metabolism (Table 26.2). Hydroxycobalamin is the form   10% w/v in 5% w/v glucose  injection or 0.9% w/v  sodium chloride injection   IV infusion: improves blood flow and  tissue function in burns and conditions  associated with local ischaemia   6% w/v in 5% w/v glucose  injection or 0.9% w/v  sodium chloride injection   IV: used to produce an expansion of  plasma volume in conditions associated  with loss of plasma proteins   6% w/v in 5% w/v glucose  injection or 0.9% w/v  sodium chloride injection   Deep IM or IV (slow infusion): iron-  deficiency anaemia (oral therapy ineffective  or impractical)   The current British Pharmacopoeia and British National Formulary should be consulted for further information, including toxic  manifestations.   “In the USA, dextran injections with average molecular weights of about 75 000 are also available.   Effervescent products; sodium citrate used as an  anticoagulant; potassium citrate used to treat cystitis   Calcium lactate is a convenient source of Ca 2+ for  oral administration; constituent of intraperitoneal  dialysis solutions   Calcium gluconate is a source of Ca2+ for oral  administration; gluconates are used to render bases  more soluble, e.g. chlorhexidine gluconate    of choice for therapeutic use and can be derived either by  chemical transformation of cyanocobalamin or directly  as a fermentation product.   Biotin, formerly known as vitamin H, is now regarded  as another member of the vitamin B family and is found  in similar food types. Biotin acts as an essential cofactor  in chemical reactions that maintain normal metabolic  function. It is also an essential growth factor for some  bacteria. Its chemical structure was established in the  early 1940s and a practical, highly stereospecific, chemical  synthesis enabled D-biotin, identical to that found in  yeasts and other cells, to be produced.   Amino acids find applications as ingredients of infusion  solutions for parenteral nutrition and individually for the    treatment of specific conditions. They are obtained either  by fermentation processes similar to those used for anti-  biotics or in cell-free extracts employing enzymes isolated  from bacteria (Table 26.2). Details of the many and varied  processes reported in the literature will be found in the  appropriate references in the ‘Further reading’ section at  the end of the chapter.   Examples of organic acids (citric, lactic, gluconic) pro-  duced by microorganisms, together with pharmaceu-  tical and medical uses, are given in Table 26.2. Citric and  lactic acids also have widespread uses in the food and  drink and plastics industries. Gluconic acid is also used  as a metal-chelating agent in, for example, detergent  products.   The growth of many microorganisms in iron-deficient  growth media results in the secretion of low molecular  weight iron-chelating agents called siderophores, which  are usually phenolate or hydroxamate compounds. The  therapeutic potential of these compounds has generated  considerable interest. Uncomplicated iron deficiency can  be treated with oral preparations of iron(II) (ferrous)  sulphate but such treatment is not without hazard and  iron salts remain a common cause of poisoning in chil-  dren. The accidental consumption of around 3g of  ferrous sulphate by a small child leads to acidosis, coma  and heart failure which, if untreated, are fatal.  Desferrioxamine B (Figure 26.2), the deferrated form of  a siderophore produced by Streptomyces pilosus, is a  highly effective antidote for the treatment of acute iron  poisoning. Desferrioxamine owes its effectiveness both to  its high affinity for ferric iron (its binding constant is in  excess of 10 30 ) and because the iron-desferrioxamine  complex is highly water-soluble and is readily excreted  through the kidneys. In haemolytic anaemias such as  thalassaemia, desferrioxamine is used together with  blood transfusions to maintain normal blood levels of  free iron and haemoglobin. Desferrioxamine is prepared  as a sterile powder for use as an injection, but it is also    administered orally in acute iron poisoning to remove  unabsorbed iron from the gut.   Patients with iron overload disorders treated with des-  ferrioxamine may, however, have increased susceptibility  to infections. The important role played by iron availability  during infections in vertebrate hosts has only been recog-  nized relatively recently. The ability of the host to withhold  growth-essential iron from microbial and, indeed, neo-  plastic invaders while retaining its own access to this metal  has led to suggestions that microbial iron chelators or  their semisynthetic derivatives may be of use in antimicro-  bial and anticancer chemotherapy. Preliminary work  has shown some encouraging results. The bacterial  siderophores parabactin and compound II secreted by  Paracoccus denitrificans have been shown to inhibit the  growth of leukaemia cells in culture and in experimental  animals. They also appear capable of inhibiting the replica-  tion of RNA viruses. Siderophores such as desferrioxamine  may therefore find increasing applications not only in the  treatment of iron poisoning and iron-overloaded disease  states but also as chemotherapeutic agents.   Several enzymes have important therapeutic, medical  or pharmaceutical uses (Table 26.3). In this section,    Figure 26.2 Structure of desferrioxamine B and its corresponding iron chelate.    those enzymes used therapeutically will be described.  Applications of microbially derived enzymes for antibi-  otic inactivation in sterility testing and diagnostic assays  are discussed in section 5.   Mammalian blood will clot spontaneously within minutes  if allowed to stand, but if left to stand longer the clot  begins to dissolve as a result of the action of a proteolytic  enzyme called plasmin. Plasmin is normally present as its  inactive precursor, plasminogen. Certain strains of strep-  tococci were found to produce a substance that is capable  of activating plasminogen (Figure 26.3), a phenomenon  that suggested a potential use in liquefying clots, i.e. fibri-  nolysis. This substance, called streptokinase, was isolated  and determined to be an enzyme.   Streptokinase is administered by intravenous or intra-  arterial infusion in the treatment of thromboembolic  disorders, e.g. pulmonary embolism, deep vein thrombo-  sis and arterial occlusions. It is also used in emergency  medicine for acute myocardial infarction.   A second enzyme, streptodornase, present in strepto-  coccal culture filtrates, was observed to liquefy pus.  Streptodornase is a deoxyribonuclease that breaks down  deoxyribonucleoprotein and DNA, both constituents of  pus, resulting in a reduction in pus viscosity. Streptokinase  and streptodornase together have been used to facilitate    drainage by liquefying blood clots and/or pus in the chest  cavity. The combination can also be applied topically to  wounds that have excessive suppuration.   Streptokinase and streptodornase are isolated follow-  ing growth of non-pathogenic streptococcal producer  strains in media containing excess glucose. They are  obtained as a crude mixture from the culture filtrate and  can be prepared relatively free of each other. They are  commercially available as either streptokinase injection  or as a combination of streptokinase and streptodornase.   L-Asparaginase, an enzyme derived from E. coli or Erwinia  chrysanthemi, converts L-asparagine to aspartic acid and  ammonia. In contrast to normal tissue, some tumours  have an essential requirement for L-asparagine and l-  asparaginase has therefore been investigated as a selective  cancer chemotherapeutic. Although L-asparaginase  showed early promise in a variety of experimentally  induced tumours, it has primarily found utility in humans  for the treatment of acute lymphoblastic leukaemia and  occasionally for myeloid leukaemia.   Many tumours escape immune surveillance through  mechanisms including the masking of cell surface anti-  gens by, for example, N-acetylneuraminic (sialic) acid    residues. Neuraminidase, derived from Vibrio cholerae,  has been used experimentally to increase the immuno-  genicity of tumour cells by stripping sialic acid residues  from the outer surface of certain tumour cells resulting  in presentation of tumour-specific antigens to the host  immune system. In laboratory animals, administration  of neuraminidase-treated tumour cells was found to  be effective against a variety of mouse leukaemias.  Preliminary investigations in acute myelocytic leukaemia  patients have suggested that treatment of tumour cells  with neuraminidase in combination with conventional  chemotherapy may increase remission rates.   (3-Lactamase enzymes, whilst presenting a considerable  therapeutic challenge due to their ability to confer bacte-  rial resistance by inactivating penicillins and cepha-  losporins (Chapter 13), are nevertheless useful in the  sterility testing of certain antibiotics (see section 5.5) and,  prior to culture, in inactivating various (Tlactams in  blood or urine samples in patients undergoing therapy  with these drugs. One other important therapeutic appli-  cation is in the rescue of patients presenting symptoms  of a severe allergic reaction following administration of  a (3-lactamase-sensitive penicillin. In such cases, a highly  purified penicillinase obtained from B. cereus has been  administered either intramuscularly or intravenously and  in combination with other supportive measures such as  adrenaline and antihistamines.   Microorganisms and microbially derived enzymes con-  tinue to play a significant role in the production of novel  antibiotics. The potential of microorganisms as chemical  catalysts, however, was a later development and first    realized in the synthesis of industrially important ster-  oids. These reactions assumed increasing importance  following the discovery that certain steroids could be for-  mulated as potent therapeutics, e.g. hydrocortisone has  anti-inflammatory activity, and derivatives of the  steroidal sex hormones are useful as oral contraceptive  agents. More recently, chiral inversion of non-steroidal  anti-inflammatory drugs (NSAIDs) has also been  demonstrated.   In the antibiotics industry, the hydrolysis of benzylpeni-  cillin to give 6-aminopenicillanic acid by the enzyme  penicillin acylase is an important stage in the synthesis of  many clinically useful penicillins (Chapters 11). The com-  bination of genetic engineering techniques to produce  hybrid microorganisms with significantly higher acylase  levels, together with their entrapment in gel matrices,  which appears to improve the stability of the hybrids, has  resulted in considerable increases in 6-aminopenicillanic  acid yields.   A second example is provided by the production by  fermentation of cephalosporin C, which is used solely for  the subsequent preparation of semisynthetic cepha-  losporins (Chapter 11).   Furthermore, antibiotics produced by fermentation of  various moulds and particularly Streptomyces spp., can be  utilized by medicinal chemists as starting blocks in the  production of what might be more effective antimicro-  bial compounds.   Previously, steroid hormones could only be obtained in  small quantities directly from mammals and therefore  attempts were made to synthesize them from plant sterols,  which can be obtained economically in large quantities.  However, adrenocortical steroids are characterized by the  presence of an oxygen molecule at position 11 in the    steroid nucleus and although it is relatively easy to  hydroxylate a steroidal compound it is extremely difficult  to achieve site-specific hydroxylation, such that many of  the routes used for synthesizing the desired steroid are  lengthy, complex and consequently expensive. This  problem was overcome when it was realized that many  microorganisms are capable of performing limited oxida-  tions with both stereo- and regio-specificity. By simply  adding a steroid to growing cultures of the appropriate  microorganism, specific site-directed chemical changes  can be introduced into the molecule. In 1952, the first  commercially employed process involving the conversion  of progesterone to lla-hydroxyprogesterone by the  fungus Rhizopus nigricans was introduced (Figure 26.4).  This reaction is an important stage in the manufacture of  cortisone and hydrocortisone from more readily available  steroids. Table 26.4 gives several other examples of micro-  bially directed oxidations that have been or are employed  in the manufacture of steroidal drugs.   More recently, microorganisms utilized for biotrans-  formation reactions have been immobilized by entrap-  ment in a polymer gel matrix to avoid the often costly    and time-consuming enzyme extraction steps that can  result in enzyme inactivation. Immobilization also serves  to increase the stability of membrane-associated enzymes  that are unstable in the solubilized state, as well as permit-  ting the conversion of water-insoluble compounds like  steroids in two-phase water-organic solvent systems.   Several clinically relevant drugs including salbutamol (a  (3-adrenoceptor agonist), propanolol (a (3-adrenoceptor  antagonist) and the 2-arylpropionic acids (NSAIDs) are  administered in their racemic form but undergo in vivo  chiral inversion through metabolic transformations by  microorganisms that mimic phase I metabolic processes,  i.e. functionalization reactions. For example, the activity  of NSAIDs (e.g. ibuprofen), resides almost exclusively in  the S isomers. However, unidirectional chiral inver-  sion from R to S (Figure 26.5) occurs in vivo over  a 3-hour period. The S form is a more effective inhibi-  tor of prostaglandin synthesis, and enzymes from some  fungal species (e.g. Verticillium lecanii) convert a racemic  mixture into the S isomer in vitro. Another example    is the biotransformation of ±propranolol to S pro-  pranolol by R. arrhizus and Geotrichum candidum with  around 83% efficiency.   Microbial display platforms expressing recombinant  polypeptides (peptides, antibodies, enzymes) on their  surface are emerging as invaluable tools for the investiga-  tion of protein-protein interactions and can serve as bio-  logical combinatorial libraries for the discovery of new  therapeutics. To date three microbial display platforms  have been described: phage, bacterial, and yeast display.  All three technologies share the common principle of a  direct link between genotype and phenotype affording  the identification of the displayed polypeptide by gene  sequencing. Of the three technologies described, it is  probably phage display that has witnessed the most wide-  spread application.   The filamentous bacteriophage readily accepts relatively  large insertions of additional genetic material into its  genome, which allows for the display of polypeptides as  fusions with bacteriophage coat proteins. Phage libraries  containing a repertoire of many billions of viral particles  that each display a unique polypeptide sequence can be  subjected to an affinity selection process against a target  of interest. Those phage clones that display a polypeptide  that strongly interacts with the target can be recovered  and amplified for further rounds of selection before the  direct link between genotype and phenotype is exploited  to identify the polypeptide displayed.   Although to date no phage-derived product has  reached market, a number of clinical trials are currently  under way using polypeptides derived from phage  display: DX-88 (Ecallantide, Dyax Corp) is a highly spe-  cific and potent inhibitor of kallikrein (K { -20-40 pM).  Kallikrein is a key molecule in the regulation of inflam-  matory and blood clotting processes and plays a role in a  number of autoimmune and inflammatory conditions.    DX-88 has successfully completed phase III trials in  the treatment of hereditary angiodema and phase I/II  trials for the prevention of blood loss in on-pump coro-  nary artery bypass graft surgery. Compstatin (Potentia  Pharmaceutics), a cyclic 13-mer peptide originally identi-  fied from phage display, is a selective and potent inhibitor  (X d ~0.13pM) of C3 protein in the complement cascade  and will shortly enter clinical trials for the treatment of  age-related macular degeneration.   • determining the concentration of compounds (e.g.  amino acids, vitamins and some antibiotics) in complex  chemical mixtures or in body fluids   Although antibiotics may be assayed by a variety of  methods, the following section will only take into consid-  eration microbiological and radioenzymatic assays.   In microbiological assays the response of a growing pop-  ulation of microorganisms to the antimicrobial agent  under investigation is measured. The usual methods  involve agar diffusion assays in which the drug diffuses  into agar seeded with a susceptible microbial population  producing a zone of growth inhibition.   In the commonest form of microbiological bioassay  used today, samples to be assayed are applied in some  form of reservoir (porcelain cup, paper disc or well) to  a thin layer of agar seeded with indicator organism.  The drug diffuses into the medium and after incubation  a zone of growth inhibition forms, in this case as a  circle around the reservoir. All other factors being  constant, the diameter of the zone of inhibition is essen-  tially related to the concentration of antibiotic in the  reservoir.   During incubation the antibiotic diffuses from the res-  ervoir and that part of the microbial population distant    to the influence of the antibiotic increases by cell division.  The edge of the area of microbial growth is formed when  the minimum concentration of antibiotic that will inhibit  the growth of the organism on the plate (critical concen-  tration) reaches, for the first time, a population density  too great for it to inhibit. The position of the zone edge  is thus determined by the initial population density, the  growth rate of the organism and the rate of diffusion of  the antibiotic.   In situations where the likely concentration range of  the tests will lie within a relatively narrow range (e.g. in  determining potency of pharmaceutical preparations)  and maximal precision is sought, then a Latin square  design with tests and calibrators at two or three levels  of concentration may be used. For example, an 8 x 8  Latin square can be used to assay three samples and one  calibrator, or two samples and two calibrators at two  concentrations each (over a twofold or fourfold range),  with a coefficient of variation of around 3%. Using  this technique, parallel dose-response lines should be  obtained for the calibrators and the tests at the two  dilutions (Figure 26.6). Using such a method, potency  can be computed or determined from carefully prepared  nomograms.   Conventional plate assays require several hours’ incu-  bation and consequently the possibility of using rapid   Figure 26.6 Graphical representation of a 2 X 2 assay  response. X is the horizontal distance between the two lines.  The antilog of X gives the relative potency of the standard  and test.   • Urease assay. When Proteus mirabilis grows in a urea-  containing medium it hydrolyses the urea to ammonia  and consequently raises the pH of the medium. This  production of urease is inhibited by aminoglycoside  antibiotics (inhibitors of protein synthesis; Chapter 11).  In practice, it is difficult to obtain reliable results by this  method.   • Luciferase assay. In this technique, firefly luciferase (or  similar enzyme) is used to measure small amounts of ATP  in a bacterial culture, ATP levels being reduced by the  inhibitory action of aminoglycoside antibiotics.   Radioenzymatic assays depend on the fact that bacterial  resistance to aminoglycosides, such as gentamicin,  tobramycin, amikacin, netilmicin, streptomycin, spec-  tinomycin, and chloramphenicol is frequently associated  with the presence of specific enzymes (often coded for  by transmissible plasmids), which either acetylate,  adenylylate or phosphorylate the antibiotics, thereby ren-  dering them inactive (Chapter 13). Aminoglycosides  may be susceptible to attack by aminoglycoside acetyl-  transferases (AAC), aminoglycoside adenylyltransferases  (AAD) or aminoglycoside phosphotransferases (APH).  Chloramphenicol is attacked by chloramphenicol  acetyltransferases (CAT). Acetyltransferases attack sus-  ceptible amino groups and require acetyl coenzyme  A, while AAD or APH enzymes attack susceptible  hydroxyl groups and require ATP (or another nucleotide  triphosphate).   Several AAC and AAD enzymes have been used for  assays. The enzyme and the appropriate radio-labelled  cofactor ( [ 1- 14 C] acetyl coenzyme A, or [2- 3 H] ATP) are  used to radiolabel the drug being assayed. The radiola-  belled drug is separated from the reaction mixture  after the reaction has been allowed to go to completion;  the amount of radioactivity extracted is directly propor-  tional to the amount of drug present. Aminoglycosides  are usually separated by binding them to phosphocellu-  lose paper, whereas chloramphenicol is usually extracted  using an organic solvent.   These types of assay are rapid, taking approximately 2  hours, show good precision and are much more specific  than microbiological assays.   The principle of microbiological bioassays for growth  factors such as vitamins and amino acids is quite simple.    Unlike antibiotic assays (see section 5.1) that are based  on studies of growth inhibition, these assays are based on  growth exhibition. All that is required is a culture medium  that is nutritionally adequate for the test microorganism  in all essential growth factors except the one being  assayed. If a range of limiting concentrations of the test  substance is added, the growth of the test microorganism  will be proportional to the amount added. A calibration  curve of concentration of substance being assayed against  some parameter of microbial growth, e.g. cell dry weight,  optical density or acid production, can be plotted. One  example of this is the assay for pyridoxine (vitamin B 6 ),  which can be assayed using a pyridoxine-requiring  mutant of the mould Neurospora. Using elegant study  designs, it is possible to assay a variety of different growth  factors with a single test organism simply by preparing a  basal medium with different growth-limiting nutrients.  Table 26.5 summarizes some of the vitamin and amino  acid bioassays currently available. In practice however,  high performance liquid chromatography (HPLC) has  replaced bioassays as the method of choice for most  amino acids and several B group vitamins.   Phenylketonuria (PKU) is an inborn error of metabolism  in which the body is unable to convert surplus phenyla-  lanine (PA) to tyrosine for use in the biosynthesis of, for  example, thyroxine, adrenaline and noradrenaline. This  results from a deficiency in the liver enzyme phenyla-  lanine 4-mono-oxygenase (phenylalanine hydroxylase).    A secondary metabolic pathway comes into play in which  there is a transamination reaction between PA and a-  ketoglutaric acid to produce phenylpyruvic acid (PPVA),  a ketone and glutamic acid. Overall, PKU may be defined  as a genetic defect in PA metabolism such that there are  elevated levels of both PA and PPVA in blood and exces-  sive excretion of PPVA (Figure 26.7).   Control of PKU can be achieved simply by resorting  to a low PA-containing diet. However, failure to diagnose  PKU will result in mental deficiency, and thus early diag-  nosis is essential. In 1968, the UK Medical Research  Council Working Party on PKU recommended the adop-  tion of the Guthrie test as a convenient method for  screening newborn infants. This assay employs Bacillus  subtilis as the test organism. In minimal culture medium,  growth of this bacterium is inhibitedby (3-2-thienylalanine  (Figure 26.8a) and is competitively reversed in the pres-  ence of PA (Figure 26.8b) or PPVA. The use of filter-paper  discs impregnated with blood or urine permits the detec-  tion of elevated levels of PA and PPVA. The test can be  quantified by the measurement of the diameter of the  growth zone around the filter-paper disc and comparing  it with a calibration curve constructed from known con-  centrations of PA or PPVA (Figure 26.8c). If positive, the  Guthrie test provides presumptive evidence for the pres-  ence of PKU. This is then confirmed by other chemical  means. The test is still in widespread use but is gradually  being phased out in favour of modern genetic testing  techniques.   A carcinogen is a substance that causes living tissues to  become carcinomatous, i.e. to produce a malignant  tumour. A mutagen is a chemical (or physical) agent that  induces mutation in a human (or other) cell.   Mutagenicity tests are used to screen a wide variety of  chemicals for their ability to cause a mutation in the DNA  of a cell. Such mutations can occur at either the gene level  (cl point mutation) at individual chromosomes, or at the  level of a chromosome set, i.e. a change in the number of  chromosomes ( aneuploidy ). Some compounds are only  mutagenic or carcinogenic after metabolism (often in the  liver). This aspect must, therefore, be considered in  designing a suitable test method for such agents (see  section 5.4.2).   Forward mutation refers to mutation of the natural  (‘wild-type’) organism to a more stringent organism. By  contrast, reverse (backward) mutation is the return of a   Figure 26.7 (a) Normal metabolism, in which phenylalanine is converted by phenylalanine 4-monooxygenase to tyrosine.   (b) Phenylketonuria, in which there is a transamination reaction between phenylalanine and a-ketoglutaric acid. Phenylalanine  4-monooxygenase is absent in about 1 in every 10000 human beings because of a recessive mutant gene.    mutant strain to the wild-type form, i.e. it is a heritable  change in a previously mutated gene that restores the  original function of that gene.   • Frame-shift. In these mutants, the gene is altered by the  addition or deletion of one or more bases so that the  triplex reading frame for RNA is modified;   • Base-pair. In these mutants, a single base is altered so  that the triplex reading frame is again modified.    These principles of reverse mutation are utilized in one  important method, the Ames test (section 5.4.2), which  is used to detect compounds that act as mutagens or  carcinogens (most carcinogens are mutagens).   The Ames test is used to screen a wide variety of chemi-  cals for potential carcinogenicity or conversely for  their potential as cancer chemotherapeutic agents. The    test enables a large number of compounds to be screened  rapidly by examining their ability to induce mutagenesis  in several specially constructed bacterial mutants  derived from Salmonella enterica serovar Typhi. The test  strains contain mutations in the histidine operon such  that they cannot synthesize the amino acid histidine. Two  additional mutations increase further the sensitivity of  the system. The first is a defect in their lipopolysaccharide  structure (Chapter 3) such that they are in fact deep  rough mutants possessing only 2-keto-3-deoxyoctonate  (KDO) linked to lipid A. This mutation increases the  permeability of the mutants to large hydrophobic mole-  cules. The second mutation concerns a DNA excision  repair system, which prevents the organism repairing its  damaged DNA following exposure to a mutagen.   The assay method involves treatment of a large popu-  lation of these mutant tester strains with the test com-  pound. Histidine-requiring mutants are used to detect  mutagens capable of causing base-pair substitutions (in  some strains) or frame-shift mutations (other strains).  This can be carried out by incorporating both the test  strain and test compound in molten agar (at 45 °C),  which is then poured on to a minimal glucose agar plate.  Alternatively, the suspected mutagens can be applied to  the surface of the top agar as a liquid or as a few crystals.  The medium used for the top agar contains a limited  concentration of histidine, which permits the bacteria on  the plate to undergo several divisions, since for many  mutagens some growth is a necessary prerequisite for  mutagenesis to occur. After incubation for 2 days at 37 °C  the number of ‘revertant’ colonies can be counted and  compared with control plates from which the test com-  pound has been omitted. Each revertant colony is  assumed to be derived from a cell that has mutated back  to the wild-type and thus can now synthesize its own  histidine: see Figure 26.9 for a summary.   A further refinement to the Ames test permits screen-  ing of agents that require metabolic activation before  their mutagenicity or carcinogenicity is apparent. This is  achieved by incorporating into the top agar layer, along  with the bacteria, homogenates of liver (commonly rat or  human) whose activating enzyme systems have been  induced by exposure to polychlorinated biphenyl mix-  tures. This test is sometimes referred to as the Salmonella/  microsome assay because the fraction of liver homoge-  nate used, called the S9 fraction, contains predominantly  liver microsomes.   It is important to realize that this test is flexible and is  still undergoing modification and development. Almost  all the known human carcinogens have been tested and    shown to be positive. These include agents such as (3-  naphthylamine, cigarette smoke condensates, aflatoxin B  and vinyl chloride, as well as drugs used in cancer treat-  ment such as adriamycin, daunomycin and mitomycin C.  Although the test is not perfect for the prediction of  mammalian carcinogenicity or mutagenicity and for  making definitive conclusions about potential toxicity or  lack of toxicity in humans, it nevertheless provides useful  screening information rapidly and cheaply. The Ames test  remains an important part of a battery of tests, the others  of which are non-microbial in nature, for detecting muta-  genicity or carcinogenicity.   Sterile pharmaceutical preparations must be tested for  the presence of fungal and bacterial contamination before  use (see Chapter 21). Of course if the preparation con-  tains an antibiotic it must be removed or inactivated  and this is generally achieved by membrane filtration.  However, the technique has certain disadvantages includ-  ing accidental contamination and retention of the anti-  biotic on the filter followed by subsequent liberation into  the nutrient medium.   Enzymatic inactivation of the antibiotic (see also  Chapter 13) before testing would provide an elegant solu-  tion to this problem. Currently, the only pharmacopoeial  method permitted is that of using an appropriate 13-  lactamase to inactivate penicillins and cephalosporins.  Other antibiotics that are susceptible to inactivating  enzymes are chloramphenicol (by chloramphenicol  acetyltransferase) and the aminoglycosides, e.g. gen-  tamicin, which can be inactivated by phosphorylation,  acetylation or adenylylation. A method for acetylating    and consequently inactivating aminoglycosides prior to  testing using 3-N-acetyltransferase (an enzyme with wide  substrate specificity) in combination with acetyl coen-  zyme A has been described, but this method has yet to be  adopted.   The therapeutic uses of microbially derived enzymes have  already been examined (section 2.4). However, enzymes  also form the basis of many diagnostic tests used in clini-  cal medicine. For example, glucose oxidase, an enzyme  used in blood glucose analysis, is obtained commercially  from Aspergillus niger. Although the design of glucose  blood monitoring test strips has become more refined,  the basic design remains the same. Glucose oxidase is  sandwiched between polycarbonate and cellulose acetate  membranes. When a pinprick volume of blood is applied,  the polycarbonate membrane allows the diffusion of  glucose into the sandwich but limits entry of larger mol-  ecules thus reducing background contaminants. The  enzyme oxidizes the glucose, resulting in the formation  of hydrogen peroxide. The hydrogen peroxide diffuses  through the cellulose membrane where it is measured  amperometrically following interaction with a platinum  electrode. More recently, several types of implantable  glucose oxidase electrodes have been developed experi-  mentally for continuous monitoring of blood glucose  levels in diabetics. While there are challenges relating to  enzyme inactivation in vivo, glucose calibration, and  immune response there are, nevertheless, a number of  major research efforts in this area and indeed the US  Food and Drug Administration (FDA) has recently  approved a wireless subcutaneous sensor that continu-  ously monitors patient glucose levels for 7 days. It is likely  that biosensors employing immobilized enzymes which  are potentially useful for monitoring many substances of  clinical importance will become readily available in the  not-too-distant future.   The safety and efficacy of a drug must be exhaustively  evaluated before its approval for use in the treatment of  human diseases. Investigations of the manner in which a  drug is metabolized are extremely valuable as they poten-  tially provide information on its mode of action, why it  exhibits toxicity and how it is distributed, excreted and  stored in the body. Traditionally, drug metabolism studies    have relied on the use of animal models and, to a lesser  extent, liver microsomal preparations, tissue culture and  perfused organ systems. Each of these models has certain  advantages and disadvantages. Animals in particular are  expensive to purchase and maintain, and there is consid-  erable pressure from animal welfare groups to curb the  use of animals in scientific research.   The many similarities between certain microbial  enzyme systems and mammalian liver enzyme systems has  led to the utilization of various microbial models for the  exploration of mammalian drug metabolism. The major  advantages of using microorganisms are their ability to  produce significant quantities of metabolites that would  otherwise be difficult to obtain from animal systems or by  chemical synthesis, and the considerable reduction in  operating costs compared with animal studies.   Microbial drug metabolism studies are usually carried  out by firstly screening a large number of microorgan-  isms for their ability to metabolize a drug substrate.  The organism is usually grown in a medium such as  peptone glucose in flasks that are shaken to ensure good  aeration. Drugs as substrates are generally added after 24  hours of growth and are then sampled for the presence  of metabolites at intervals up to 14 days after substrate  addition. Once it has been determined that a microor-  ganism can metabolize a drug, the whole process can be  scaled up for the production of large quantities of metab-  olites for the determination of their structure and bio-  logical properties.   As an example of this the metabolism of the antide-  pressant drug imipramine can be considered. In mam-  malian systems, this is metabolized to five major  metabolites: 2-hydroxyimipramine, 1 0-hydroxyimipramine,  iminodibenzyl, imipramine-N-oxide and desipramine  (Figure 26.10).   For microbial metabolism studies, a large number of  fungi are screened, from which several are chosen for the  preparative scale production of imipramine metabolites.  Cunninghamella blakesleeana produces the hydroxylated  metabolites2-hydroxyimipramineandl0-hydroxyimipramine;  Aspergillus flavipes and Fusarium oxysporum f. sp. cepae  yield the N-oxide derivative and iminodibenzyl, respec-  tively; while the pharmacologically active metabolite  desipramine is produced by Mucor griseocyanus together  with the 10-hydroxy and N-oxide metabolites. By scaling  up this procedure, significant quantities of the metabo-  lites that are formed during mammalian metabolism can  be obtained.   Microorganisms thus have considerable potential as  tools in the study of drug metabolism. Although they    R 1 = (CH 2 ) 3 N(CH 3 )2; R 2 = R 3 =H  R 1 = (CH 2 ) 3 NHCH 3 ; R 2 = R 3 =H  R 1 = (CH 2 ) 3 N(CH 3 ) 2 ; R 2 =0H; R 3 =H  R 1 = (CH 2 ) 3 N(CH 3 ) 2 ; R 2 =0H; R 3 =H  R 1 =R 2 =R 3 =H   R 1 = (CH 2 ) 3 N(CH 3 ) 2 ; R 2 = R 3 =H    cannot completely replace animals, they are extremely  useful as predictive models for initial studies.   In the early 1900s, before the discovery of penicillin, Felix  D’Herelles observed that patients with high titres of bac-  teriophages in their faeces recovered from dysentery and  typhoid fever more rapidly. This paved the way for the  commercialization of bacteriophage preparations for a  variety of bacterial infections by, for example, the Societe  Fran^aise de Teintures Inoffensives pour Cheveux (The  French Society for Safe Flair Colouring) or L’Oreal as it  is known today. Following the advent of modern antibi-  otic therapy in the 1930s the science of phage therapy was  all but bankrupted, but the emergence of antibiotic resist-  ance has led to a resurgent interest in research and devel-  opment of phage therapeutics (Chapter 27) and at least  eight commercial enterprises are involved in the develop-  ment of clinically relevant phage medicines. Most phages  have a specific affinity for only a small group of bacteria,  predicated by the interaction of phage components with  bacterial surface receptors. Upon interaction, the viral  DNA is translocated into the bacterial cell for transcrip-  tion where lytic or lysogenic replication may occur  (Chapter 5). Lytic phages replicate and assemble and then  ‘burst’ from the host cell, resulting in cell death. In the  lysogenic lifecycle, bacteriophage DNA becomes inte-  grated in the host bacterium’s genome. This newly gener-    ated material termed a prophage is replicated during  cell division. The lysogenic lifecycle is shifted to a lytic  one when exposed to some external trigger, such as UV  radiation.   Lytic phage are in many ways an ideal antibacterial  agent. They are target specific and the existence of more  than 1 x 10 8 species of phage suggests there may be a  phage therapeutic for every bacterial species; they kill  bacteria rapidly and amplify at the site of infection, and  are relatively inexpensive to produce. In addition, the  FDA recognizes that humans ingest vast quantities of  phages on a daily basis and tacitly accept that they are safe  for oral administration. It is perhaps topical administra-  tion that has seen the most interest, however, with the  application of cocktails of phage to chronic wounds  either as simple suspensions or incorporated into some  form of dressing system such as a biodegradable polymer  infused with phage and antibiotics. The systemic admin-  istration of phage therapeutics is complicated by an  inadequate knowledge of the pharmacokinetic and phar-  macodynamic properties of most phage species, with  many studies indicating that the timing of administration  is critical for infection control. Recent reports suggest,  however, that phages, which are naturally immunostimu-  latory, may be useful as vaccine delivery vehicles either by  vaccinating with phages displaying the antigen or by uti-  lizing phages to deliver a DNA expression cassette inte-  grated into the phage genome.   The bacterial microflora that colonizes the gastrointesti-  nal (GI) tract is an essential feature of normal human  physiology and represents a symbiotic relationship where  the bacteria both protect the host against pathogenic  microbes and aid in the digestion of food, contributing  to the production of essential host nutrients. Under  certain conditions (e.g. illness, infection, antibiotic  therapy), the bacterial population in the GI tract may be  diminished, contributing to disease states. Probiotics are  live cultures of ‘good’ bacteria that are purported to  survive transit through the stomach, subsequently colo-  nizing the intestinal mucosa and replacing the dimin-  ished natural microflora or displacing pathogenic  microorganisms. Bifidobacteria and Lactobacillus spp. are  the most commonly encountered probiotic bacteria, pri-  marily because they are reported to survive the harsh  environment of the upper GI tract more readily than  other species. Of note, Bifidobacteria spp. are amongst the  first colonizers of the neonate intestine, as a consequence  of both Bifidobacteria and prebiotic content in breast    milk, and contribute to defence against pathogenic invad-  ers and maturation of the immune system. Probiotics are  generally formulated as capsules or as food supplements  particularly as dairy products such as yoghurts. Often  probiotic formulations are combined with prebiotics —  indigestible oligosaccharides that are fermented by anaer-  obic bacteria in the gut, yielding metabolic substrates that  promote probiotic growth.   Probiotics have been investigated for efficacy in a  range of conditions that may be associated with dimin-  ished bacterial microflora. Several probiotic species  including Lactobacillus spp. have shown utility in both  the prevention and treatment of nosocomial, antibiotic  and traveller’s diarrhoea. Long-term treatment with E.  coli Nissle 1917 (>12 months) is reported to be at least  equivalent to mesalazine therapy in preventing relapse in  ulcerative colitis. In irritable bowel syndrome (IBS) the  results are equivocal but hopeful, with some studies  reporting a reduction in symptoms such as abdominal  discomfort.   A cautionary note, however: probiotics are increas-  ingly marketed as a ‘lifestyle’ nutrient to healthy individu-  als to promote general GI and immune health, despite  limited evidence of any significant effect. Nevertheless,  research into the benefits of probiotics in both healthy  and diseased individuals is ongoing, using recognized and  novel probiotic species, and may in the future reap sig-  nificant reward.   In the late 19th century, a Belgian professor of microbiol-  ogy, van Ermengem, conducted a serious of experiments  to identify the cause of a fatal outbreak of food poisoning,  the clinical symptoms of which had been described over  a century before by the German physician Justinus  Kerner. van Ermengem’s endeavours resulted in the iden-  tification of botulinum toxin, a potent exotoxin produced  by the Gram-positive anaerobic bacterium Clostridium  botulinum. It is this toxin that is responsible for what is  now widely recognized as botulism food poisoning. The  symptoms of botulism, which remains a common cause  of fatal food poisoning, include GI disturbances, dys-  phagia, facial paralysis and, depending on the ingested  dose, more widespread muscle weakness resulting in pos-  sible respiratory paralysis and subsequent death.   In the mid-20th century the work of Burgen and col-  leagues established that the basic mechanism of action  for botulinum toxin is neuromuscular blockade. Now,    some 60 years later, we understand that the toxic compo-  nent of the protein complex is a 150kDa single-chain  polypeptide, consisting of a 100 kDa heavy chain linked  to a 50 kDa light chain by a disulphide bridge, which  temporarily inhibits acetylcholine release from the presy-  naptic membrane of cholinergic nerve terminals. Seven  different serotypes of the botulinum neurotoxin (A-G),  have been identified. The 150 kDa toxic component of  these macromolecular protein complexes is relatively  homologous, conferring only subtle differences between  the mechanisms of action of the serotypes. However the  non-toxic proteins within the bacterium-derived botuli-  num toxin protein complex also differ, depending on  the strain of Cl. botulinum. Botulinum toxin serotypes  therefore possess molecular weights between 300 and  900 kDa.   In the late 1970s and 1980s nanogram quantities of the  botulinum toxin were being locally injected, by clinical  researchers, into the muscles of human volunteers to  induce local paralysis in an attempt to treat various  movement disorders. In 1989, after more than a decade  of clinical development, the FDA approved the first botu-  linum toxin therapy. This commercial product contained  the botulinum toxin A serotype and was used for the  treatment of strabismus, blepharospasm and hemifacial  spasm. In 1991 Allergan obtained both the license and the  manufacturing facilities to become the sole supplier of  botulinum toxin A for clinical therapy and they branded  their product Botox.   There are currently five licensed pharmaceutical forms  of the botulinum toxin A: Botox, Vistabel, Dysport,  Xeomin and Azzalure. These pharmaceutical prepara-  tions contain different forms of the toxin, are formulated  differently and/or are licensed for different therapeutic  indications. For example, Xeomin contains only the  150 kDa light chain region of the toxin and contains  human albumin and sucrose as excipients, whereas Botox  contains the 900 kDa macromolecular protein complex  and contains human albumin and sodium chloride as  excipients. Doses of commercial botulinum toxin A prep-  arations are therefore not interchangeable and specific  brands should be prescribed for specific clinical indica-  tions. Doses are significantly less than the lethal dose for  a human, but systemic side effects of the toxin have been  observed, albeit rarely. Clinical administration of the  toxin relies on multiple localized injections, often in the  secondary care setting, directly into the target tissue.  However, the therapeutic effect of botulinum toxin is  transient, typically 6-12 months, and patients therefore  return for treatment at regular intervals.    In the past two decades the use of botulinum toxin A  in clinical practice has increased almost exponentially  and it is now used to treat a diversity of medical condi-  tions. Specific licensed indications include blepharos-  pasm, cervical dystonia, hemifacial spasm, glabellar lines  and hyperhidrosis. However, it has been used more  widely, often unlicensed, for a range of clinical indica-  tions related to movement disorders, spasticity, ophthal-  mic disorders, GI disorders, genitourinary disorders,  surgical interventions, tendon release in the Ponseti treat-  ment of talipes, and more recently pain. The toxin is even  better known for its widespread use in the cosmetic  industry. A significant population of patients and cos-  metic clients have therefore now been treated with the  neurotoxin, and in general it appears to be a safe and  effective addition to the therapeutic armoury. Botulinum  toxin type B is also available commercially, as Neurobloc,  but at present this serotype is used less widely in therapy.  The clinical use of botulinum toxin is primarily restricted  to conditions that are associated with superficial/  accessible tissues, to minimize the risk of systemic uptake,  but there is no doubt that the toxin is emerging as a useful  therapeutic entity and has already revolutionized the  treatment of some conditions including hyperhidrosis.   Vibrio cholerae is a bacterial pathogen that colonizes the  small intestine leading to cholera, an infection character-  ized by life-threatening acute diarrhoea. Cholera is  endemic in developing countries and in areas where  hygiene and sanitary conditions are poor, and even with  supportive therapy that includes rehydration and restora-  tion of electrolytes, morbidity and mortality rates remain  high. An oligomeric protein (87 kDa) secreted by V. chol-  erae was confirmed as the causative agent of cholera in  1963 by Finkelstein and colleagues and has been termed  cholera toxin (CT). CT is a member of the superfamily  of AB toxins comprised of a catalytic heterodimeric A  subunit (A1 and A2 chains) and a glycolipid receptor  binding homopentameric B-subunit connected by a  disulphide bond. When V. cholerae colonizes the small  intestine it secretes CT which subsequently interacts, via  the B-subunit, with an enterocyte membrane receptor  GM1 (monosialotetrahexosylganglioside) localized in  lipid raffs. The CT is then internalized into early endo-  somes and trafficked to the trans-Golgi network, eventu-  ally ending up in the endoplasmic reticulum where  protein disulphide isomerase dissociates and unfolds the  A1 chain from CT. The A1 chain is then translocated by  the Sec61 channel into the cytosol where it interacts with    proteins that regulate adenylate cyclase (AC) leading to  the constitutive activation of AC. This is accompanied by  an increase in intracellular cAMP concentration resulting  in phosphorylation of the cystic fibrosis transmembrane  conductance regulator (CFTR). The net consequence is  extracellular secretion of chloride ions into the small  intestine, producing an osmotic gradient that draws  water into the lumen, resulting in diarrhoea.   CT, despite its pathogenicity, has significant immuno-  logical properties and has been proposed as a mucosal  adjuvant for subunit vaccines where the toxin is co-  administered or complexed with the antigen. Mucosally  administered vaccines, i.e. oral, nasal, rectal or vaginal,  have a number of advantages over the more traditional  intravenous vaccines, not least the ability to stimulate  mucosal and systemic protection and may perhaps  enhance vaccine uptake rates given no needles are  involved. The mechanism of adjuvanticity of cholera  toxin is controversial and remains to be fully elucidated  but evidence suggests the A-subunit, when administered  orally, enhances antigen presentation following complex  interactions with mucosal cells and cells of the immune  system(see Chapter 9). The role of the B-subunit in  adjuvanticity is interesting; CT-B does not appear to  survive transit through the GI tract but there is growing  evidence of autoimmune stimulation following nasal or  intravenous administration and therefore the B-subunit  may offer utility in the treatment of autoimmune dis-  eases. A number of preclinical in vivo studies have  been undertaken to ascertain the effectiveness of CT as  a mucosal adjuvant for antigens derived from (for  example) Helicobacter pylori, influenza, tetanus, HIV and  Streptococcus pneumoniae, and for the treatment of dia-  betes mellitus. To date, however, it appears that oral doses  of CT that elicit adjuvanticity are similar to those that  induce diarrhoea amongst other adverse reactions,  although efforts to ‘detoxify’ CT through genetic engi-  neering are under way.   Like animals, insects are susceptible to infections, which  may be caused by viruses, fungi, bacteria or protozoa. The  use of microorganisms to spread diseases to particular  insect pests offers an attractive method of biological  control, particularly in view of the ever-increasing inci-  dence of resistance to chemical insecticides. However, any  microorganism used in this way must be highly virulent,  specific for the target pest but non-pathogenic to animals,    humans or plants. It must be economical to produce,  stable on storage and preferably rapidly acting. Bacterial  and viral pathogens have so far shown the most promise.   Perhaps the best studied, commercially available insec-  ticidal agent is B. thuringiensis. This insect pathogen con-  tains two toxins of major importance. The 8-endotoxin  is a protein present inside the bacterial cell as a crystalline  inclusion within the spore case. This toxin is primarily  active against the larvae of lepidopteran insects (moths  and butterflies). Its mechanism of action is summarized  in Figure 26.11. Commercially available preparations of  B. thuringiensis are spore-crystal mixtures prepared as  dusting powders. They are used primarily to protect  commercial crops from destruction by caterpillars and  are surprisingly non-toxic to humans and animals.  Although the currently available preparation has a rather  narrow spectrum of activity, a variant B. thuringiensis  strain has been isolated and found to produce a different  8-endotoxin with activity against coleopteran insects  (beetles) rather than lepidopteran or dipteran (flies and  mosquitoes) insects.   The second B. thuringiensis toxin, the (3-exotoxin, has  a much broader spectrum encompassing the Lepidoptera,  Coleoptera and Diptera. It is an adenine nucleotide,  probably an ATP analogue that acts by competitively  inhibiting enzymes that catalyse the hydrolysis of ATP  and pyrophosphate. However, this compound is toxic  when administered to mammals, so commercial prepara-    tions of the B. thuringiensis 8-endotoxin are obtained  from strains that do not produce the (3-exotoxin.   Strains of B. sphaericus pathogenic to mosquitoes were  isolated several years ago. Strains of this organism with  increased toxicity to mosquitoes have been isolated and  might have considerable potential as control agents.  Commercially available formulations of B. sphaericus are  now available as larvicides.   Other insect pathogens are being evaluated for activity  against insects that are vectors for diseases such as sleep-  ing sickness, as well as those that cause damage to crops.  Viruses may well have the greatest potential for insect  control as they are host-specific and highly virulent, and  one infected insect can release vast numbers of virus par-  ticles into the environment. They have already been used  with considerable success against the spruce sawfly and  pine moth.   Bioterrorism, or biological terrorism, involves the delib-  erate release of biological agents such as viruses, bacteria  and toxins, to intentionally cause terror, illness or death  in a target population. In a climate of increasing political  instability and radical fundamentalism there are many  concerns over the likelihood of exposure to, and the  ability to protect civilians from, such attacks. There are a    variety of biological agents that could potentially be used  as biological weapons. The US Centers for Disease  Control and Prevention (CDC) and the UK Health  Protection Agency (HPA) have defined bio terrorism  agents into three categories (A, B and C) based largely on  their lethality, ease of transmission and ability to cause  panic. Category A agents, i.e. those that pose the greatest  threat, are B. anthracis (anthrax), Cl. botulinum toxin  (botulism), Yersinia pestis (plague), variola major (small-  pox), Francisella tularensis (tularemia) and the filovirus  (Ebola, Marburg) and arenavirus (Lassa, Machupo)  strains that cause viral haemorrhagic fevers.   In the event of a biological attack governmental organ-  izations would invoke a preparedness plan that would  include environmental decontamination, e.g. hypochlo-  rite solution, and pharmaceutical prophylaxis and/or  treatment. Although it is practically, ethically, politically,  socially and economically difficult to vaccinate against  known bioterror pathogens, it may be possible to stock-  pile and thereafter disseminate pharmaceutical prophy-  lactics and therapeutics. For example, in the UK the HPA  would follow guidance from the Advisory Group for  Medical Countermeasures and respond with short-term  antibiotic cover with, for example, ciprofloxacin or doxy-  cycline (anthrax, plague) or doxycycline/rifampicin or  co-trimoxazole (brucella). Botulinum antitoxin would  also be provided for treatment of botulism. Clearly, the  effectiveness of this plan in preventing illness and death  would depend on the ability to detect the threat and  respond rapidly.   In the future, the problem may help to provide the  solution as advances in genetic medicine, a science that  exploits bacteria and viruses, produce vaccine platforms  that can be efficiently stockpiled to protect susceptible  populations against bioweapons.   Microorganisms are not always the killers they are por-  trayed to be. In fact, humankind has been remarkably  adept at harnessing microbes for a variety of purposes.  In many instances — e.g. antibiotics by whole or partial  synthetic production (Chapter 11) and various forms  of vaccines — products have been obtained to turn the  tables on infecting organisms. Other products have been  used for a variety of purposes (including many non-  pharmaceutical or non-medical ones, outside the scope  of this chapter). Microorganisms have also been  employed for specific assay purposes and different types    of chemical transformations, as well as in genetic engi-  neering (Chapter 25). Immobilized microorganisms have  now been used with considerable success in the partial  synthesis of steroids and antibiotics and in the produc-  tion of the antiviral compound adenine arabinoside  (Chapter 5). In a changing scientific landscape where  modern recombinant and molecular technologies are  being rapidly adopted, there remains significant scope for  the practice of pharmaceutical microbiology and the  beneficial harnessing of microbes is likely to continue for  many years.   The authors wish to both acknowledge and pay tribute to  the original author of this chapter and indeed this text,  A.D. Russell. Denver Russell was a pharmaceutical micro-  biologist par exemplar. Over a long and distinguished  career Denver witnessed the inception of the field of  pharmaceutical microbiology and contributed exten-  sively to the fledgling research community producing a  body of work that is testament to his international repu-  tation. 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(1975) Microbial models of mam-  malian metabolism. / Pharm Sci, 64, 1737-1759.   Truong, D., Dressier, D. 8c Hallett, M. (2009) Manual of  Botulinum Toxin Therapy. Cambridge University Press,  Cambridge.   Verall, M.S. (1985) Discovery and Isolation of Microbial Products.  Ellis Horwood, Chichester.   White, L.O. 8c Reeves, D.S. (1983) Enzymatic assay of aminogly-  coside antibiotics. In: Antibiotics: Assessment of Antimicrobial  Activity and Resistance (eds A.D. Russell 8c L.B. Quesnel), pp.  199-210. Society for Applied Bacteriology Technical Series  No. 18. Academic Press, London.   White, R. J. (1982) Microbiological models as screening tools  for anticancer agents: potentials and limitations. Annu Rev  Microbiol, 36, 415-433.   Zhang, L. 8c Demain, A.L. (2005) Natural Products: Drug Discovery  and Therapeutic Medicine. Humana Press, New York.   Most of us have no notion of what life would be like  without antibiotics. Penicillin was discovered in 1928 and  after a lengthy developmental period became available for  clinical use in the 1940s. Then followed what is often  referred to as the golden era of antibiotic discovery which  brought us the cephalosporins, the tetracyclines, the mac-  rolides, the aminoglycosides, the 4-quinolones, etc. (Table  27.1). It is interesting to note that the impact of antibiotic  discovery was sufficient to halt the development of virtu-  ally all other antimicrobial therapies being pursued at  that time. The fact that Alexander Fleming in the early  years had flagged up the probability of resistance develop-  ment in bacterial pathogens was largely ignored because  of the constant stream of new agents on to the market.  There is anecdotal evidence that in 1967 the US Surgeon  General stated ‘. . . it is time to close the book on infec-  tious disease, declare the war against pestilence won . . .’.  Whether or not he actually said this is debatable, but it    represented a widely held view at the time that the stream  of antibiotic discovery would never dry up. However, we  now know that is precisely what has happened.   At present we are faced with two major issues — we  have seen an inexorable rise in the incidence of antibiotic  resistance among the most important bacterial pathogens  (dealt with in Chapter 13), while at the same time the  development of novel antibacterial antibiotics has  all but stopped. Some clinical isolates from species such  as Pseudomonas aeruginosa, Acinetobacter baumannii.  Enterococcus faecium and Mycobacterium tuberculosis are  resistant to virtually every antibiotic we have at our dis-  posal. It is not appropriate here to discuss the issue of  antibiotic resistance, other than to say it is an almost  inevitable event occurring as a consequence of the bacte-  rial cell’s remarkable adaptability to environmental  change. Without this ability to adapt to their environ-  ment bacteria would not still be thriving 3.5 billion years  after first emerging on to what was at that time an  extremely hostile earth. Suffice it to say there is very little  we can do to stop this process.    Hugo and Russell’s Pharmaceutical Microbiology, Eighth Edition. Edited by Stephen P. Denyer, Norman Elodges, Sean P. Gorman,  Brendan F. Gilmore.   © 2011 Blackwell Publishing Ltd. Published 2011 by Blackwell Publishing Ltd.    Table 27.1 Time line for the discovery and use of naturally  occurring and synthetic antibiotics    The second issue of antibiotic discovery and develop-  ment is, of course, under our control. We have the scien-  tific ingenuity to devise novel approaches to circumvent  the resistance mechanisms put in place by these bacteria.  Unfortunately, the driving force behind drug discovery  is not based on altruism but has a financial imperative.  The cost of bringing a new chemical entity to the market    has been estimated as $800 million or more, while the  process of development takes about 8 years. Antibio-  tics simply do not generate sufficient revenue to justify  this initial outlay of time and cost, and not surprisingly  the focus for the large pharmaceutical companies has  switched to those medicines which patients will take  long-term such as statins, antidepressants and antihyper-  tensives. The market for antiviral agents, particularly  those to treat HIV/AIDS, remains buoyant simply because  the patients will be taking the medicines for the remain-  der of their lives.   All this poses the question, ‘How in the near future will  clinicians be able to treat patients with infections caused  by multiresistant bacterial pathogens?’ In reality this is a  question which is relevant today but will only become  more pressing in the future. Some respected authorities  in the field have already expressed the view that we are  on the verge of entering a postantibiotic era.   This chapter examines a range of alternative therapeu-  tic strategies available for the management of infectious  disease (Table 27.2). It is not the intention to cover each  of these in any depth but merely to give an overview of  the general situation; the reader is referred to the further  reading listed at the end of the chapter for more details.  As we will see, a number of the techniques to be described  have a very long history, some of them going back many  centuries. Some of these have very little chance of ever  moving from the fringes of medicine, some may have the  potential to be useful but in reality suffer from the same  developmental issues as conventional antibiotics, some  are niche approaches which may find relevance in specific  clinical areas, while a few may offer a realistic alternative,  or at least an adjunct, to antibiotics.   Essential oils, sometimes termed ethereal or volatile oils,  are contained within many plants and impart their char-  acteristic odour. They can be obtained from the plant  material by distillation, expression or solvent extraction  and have been used throughout the centuries for medici-  nal and other purposes. In recent times attention has  focused on a few of these oils, particularly tea tree oil,  because of their reported antimicrobial activity and this  will be used as an exemplar of the group as a whole. As  the name suggests, they are hydrophobic, which makes  them difficult to work with, and being natural products  they frequently contain a large number of chemical  components.    Table 27.2 Potential alternatives to antibiotics for the  treatment of bacterial infections    Tea tree oil (TTO) is the essential oil obtained from  the Australian plant Melaleuca alternifolia (although  other species may be used). Of its numerous chemical  constituents (mainly monoterpenes, sesquiterpenes and  their associated alcohols) terpinen-4-ol has been shown  to have the major antimicrobial activity. It has general  antimicrobial activity against viruses, bacteria and fungi  but the main interest in this compound is centred on its  activity against meticillin-resistant Staphylococcus aureus  (MRSA). TTO has been suggested as a potential agent for  the nasal and skin decolonization of MRSA in carriers. It  has been formulated in a variety of topical preparations  and is available over the counter for the treatment of a  range of conditions.   One of the many issues surrounding the use of any  natural material, especially multicomponent substances  such as plant extracts, is that of batch variability and  quality control. In the case of TTO there is a British  Standard which regulates the composition of the 15 com-  ponents of the oil and this is identical to ISO 4730:2004  (Table 27.3). The standard demands a lower limit of 30%  for the main antimicrobial compound terpinen-4-ol, and  other minor components are included in order to make  the formulation of an artificial oil more difficult. Unless    Table 27.3 Composition of tea tree oil as determined by  BS ISO 4730:2004    all preparations conform to the required standards of  composition it is very difficult to judge the value of any  in vitro or in vivo tests. The situation is further confused  because the nature of the oil makes it problematic to  evaluate. Oils are only sparingly soluble in the media used  to test antimicrobial activity and when mixed with  aqueous solutions result in turbid suspensions; conse-  quently, end-points based on turbidity are difficult to  determine, and the hydrophobic nature of the com-  pounds limits their diffusion in agar. Thus in the litera-  ture a range of methodologies have been described to  evaluate antimicrobial activity, and comparison between  them is difficult.   Those in vitro studies which have been conducted  indicate activity against a broad range of bacteria with  similar minimum inhibitory concentrations (MICs)  reported regardless of whether the isolate was antibiotic-  sensitive or resistant. Generally bacteria are susceptible  to concentrations below 1% (v/v) with the main excep-  tion being Ps. aeruginosa which requires concentrations    approaching 8% (v/v). The mechanism of action of TTO  is thought to involve disruption of cellular membranes  which will result in loss of intracellular constituents and  inhibition of enzyme function. The more complex outer  membranes of Ps. aeruginosa render that organism more  resistant to the effects of TTO.   Clinical studies have evaluated TTO for the treatment  of a range of bacterial and fungal infections. There is no  doubt that it is effective, although in many cases the treat-  ment was not found to be superior to conventional  therapy. Despite being used for many years TTO has  exhibited safety issues and is toxic if ingested. It has also  been shown to have irritant effects on the skin, although  these may be reduced if the oil is properly formulated.  Essential oils such as TTO cannot be directly compared  to conventional antibiotics since they are used primarily  as topical antiseptics. However, in this regard they cer-  tainly have some value although more extensive, properly  controlled trials are necessary to fully confirm their  potential use.   Bees collect nectar (a weak natural sugar solution) and  pollen from flowers in their locality, and in their hives it  is ultimately transformed into honey. As a consequence  of the processing in the hive, sucrose in the honey is  converted into fructose and glucose and the enzyme  glucose oxidase converts glucose into gluconic acid and  hydrogen peroxide. As water evaporates from the honey  the sugar concentration increases until it is of the order  of 79% w/v. These features act to preserve the honey from  microbial degradation. Other components of the honey  are dependent on the geographical source of the nectar,  and much interest has focused recently on honey col-  lected in the vicinity of the Australian tea tree ( Melaleuca  alternifolia) mentioned above, and the New Zealand  manuka ( Leptospermum scoparium). The honey collected  from these areas has been studied widely because of its  reported antibacterial and wound-healing properties.  These have been attributed to a number of factors,  including the following.   • pH. This ranges from 3.2 to 4.5, which is below the  minimum pH values required for the growth of many  common bacteria.   • Osmotic pressure and water activity. Honey is a  supersaturated sugar solution with a water content of  between 15 and 21% w/w. The water availability (A„)    ranges from 0.56 to 0.62, which again is below that  required for the growth of most common bacteria.   • Hydrogen peroxide. This is an effective antibacterial  agent and, although it is rapidly neutralized, the presence  of glucose oxidase ensures a constant replenishment.   • Non-peroxide antibacterial activity. Many workers have  shown that the antimicrobial activity of honey persists  even when the hydrogen peroxide has been removed.   The nature of the substance responsible for the non-  peroxide activity has been elusive, but recent studies  suggest that it may be methylglyoxal, found in some  highly active forms of manuka honey. However, many  other candidate materials are under investigation.   Results front animal studies, some clinical trials and  much anecdotal evidence over centuries suggests that  honey may have a role to play in the management of  infected wounds due to its antibacterial and wound-  healing properties. However, a recent Cochrane review  examined 19 published clinical trials in which honey was  evaluated in the treatment of acute or chronic wounds.  The conclusion reached was that although honey may  improve healing times in mild to moderate wounds there  was insufficient evidence to support its use in other areas.   Garlic ( Allium sativum) has been used as a culinary ingre-  dient for thousands of years, but it has also been shown  to possess a range of pharmacological activities leading  to uses in cardiovascular disease and cancer. Its use in  these conditions is outside the remit of this chapter;  however, of interest here is the fact that it has also been  reported to have antimicrobial activity against a broad  spectrum of bacterial pathogens.   The chemistry of garlic is highly complex and like  other plant extracts the material is made up of a large  number of compounds. The main sulphur-containing  components of intact garlic are y-glutamyl-S-allyl  cysteines and S-allyl-L-cysteine sulphoxides (of which the  main one is known as alliin). When garlic is crushed or  damaged, the enzyme alliinase, which is normally con-  tained within vacuoles, comes into contact with the  cytosolic cysteine sulphoxides and converts them to thio-  sulphinates such as allicin. This process is thought to  represent a protective effect for the plant because assault  by predators (worms, bacteria, etc.) would result in a  localized high concentration of these toxic products.  Allicin was the component that was first shown to be    responsible for the marked antimicrobial activity of fresh  garlic, and on the basis of this a number of garlic-  containing products were marketed for their antibacterial  effects. However, allicin was subsequently found to be  highly unstable and to degrade rapidly within a matter of  days to a variety of sulphides in both aqueous and alco-  holic solutions. In the context of the plant’s protective  strategy this makes sense, because having generated a  toxic metabolite to counter an insult it is in the plant’s  best interest to neutralize that toxin as rapidly as possible  after the invader has been repelled.   Orally administered allicin is degraded in the stomach  acid and is not absorbed from the gut, thus demonstrat-  ing that it cannot be responsible for any of the reported  in vivo antibacterial effects. Attention has thus switched  to other components, particularly the allicin breakdown  products including diallyl sulphide and diallyl disulphide.  Studies in mice have shown that oral dosing of these  agents can reduce MRSA viability in blood, liver, kidney  and spleen. They also provide immunological protective  properties. However, the number of studies conducted is  small and the methodologies variable. More comprehen-  sive clinical trials are required together with a rigorous  study of the role the individual components play in the  overall biological activity.   Garlic suffers from many of the issues raised above for  TTO in that the composition of the product varies mark-  edly depending on the source of the raw material, how it is  processed to extract the active ingredients, how it is formu-  lated and how it is stored. Inconsistencies reported for the  activity of garlic preparations are primarily due to a lack of  standardization of the product. As things currently stand,  garlic is unlikely to contribute significantly to the resolu-  tion of the issue of diminishing antibiotic availability.   Probiotics are defined as living microorganisms that  confer some health benefits when utilized in sufficient  numbers. The use of harmless bacteria to replace patho-  genic ones (bacteriotherapy) is not a new concept but has  gained renewed interest in recent years. Particularly, they  have been strongly marketed as a prophylactic approach  to the promotion of well-being. In addition, bacteriot-  herapy has been used in the treatment of a variety of  gastrointestinal conditions (including diarrhoea), respi-  ratory infections, recurrent bacterial vaginitis, recurrent  otitis media, streptococcal tonsillitis and infected burns.    The microorganisms most frequently used are normal  components of the gut microflora and include the lactic  acid bacteria such as lactobacilli, lactococci, bifidobacte-  ria, enterococci and streptococci (see Table 27.4). Other  bacteria such as Bacillus species and yeasts such as  Saccharomyces may also be utilized. Since each of these  organisms possesses different characteristics and require-  ments for growth it is likely that they do not have a  common mechanism of action.   Probiotics for oral administration are marketed as dairy  products such as yoghurt drinks, but also as powders,  tablets or capsules. Irrespective of the nature of the  product, there is a need to consume in the region of  10 9 -10 n microorganisms in order to bring about any  effect. A number of studies have highlighted issues of  inconsistency in the content of viable microorganisms  within various products. The precise manner in which  probiotics bring about their health benefits is unclear, but  a prerequisite property is the ability to colonize the gut.  The orally administered organisms must first, therefore,   Bacillus subtilis  Bifidobacterium bifidum  B. breve  B. infantis  B. lactis  B. longum   Enterococcus faecium  Escherichia coli Nissle 1917  Lactobacillus acidophilus  L. casei immunitas  L. johnsonii    be able to survive passage through the stomach acid and  then compete with the indigenous flora in the intestine.  A competitive edge will be provided by the ability to colo-  nize the gut wall and the capacity to produce bacteriocins.  Probiotics have been administered rectally to overcome  the necessity for acid tolerance, but there is a risk of  spreading faecal pathogens.   Probiotics have been found to be of benefit in the  treatment of infectious diarrhoea in children. When used  as a supplement to breast milk in premature infants they  were found to reduce the incidence and severity of necro-  tizing enterocolitis. A recent review summarizing the  available clinical trial data has suggested value in the  treatment of ulcerative colitis, but the results for Crohn’s  disease were disappointing and the authors stated that the  use of probiotics could not be recommended on the avail-  able evidence.   Clostridium difficile is a Gram positive, spore-forming  anaerobic rod which, in certain conditions, can overgrow  in the intestinal tract and produce toxins causing diar-  rhoea and damage to the epithelial lining of the gut. This  condition is a particular problem in elderly hospitalized  patients who have received courses of broad-spectrum  antibiotics. Since the colitis is caused by overgrowth of a  pathogenic bacterium in the intestinal tract it would  seem to be potentially amenable to probiotic treatment.  However, a recent Cochrane review of the current clinical  trial data found that only treatment with Saccharomyces  boulardii had any significant effect. Many hospitals use a  range of commercially available probiotic yoghurt prepa-  rations but the effectiveness of these is currently  unproven.   Burn wounds are devoid of microorganisms immediately  after the injury has been sustained, but fairly quickly  thereafter they will become colonized with microflora  from the adjacent intact skin and also by environmental  microbes. These bacteria are mainly Gram-positive  coagulase-negative staphylococci, but they may be gradu-  ally replaced by more harmful Gram-negative bacteria  such as Ps. aeruginosa and Acinetobacter spp. over the  following few days. These bacteria are notorious for being  highly antibiotic resistant and they flourish, often forming  biofilms, because the usual defensive properties of intact  skin are no longer present. Colonization with these  organisms delays healing, can inhibit grafting and may  lead to systemic infection. Studies have shown that appli-  cations of Lactobacillus plantarum were as effective as  silver sulphadiazine in the management of infected burn    wounds. This may be of relevance in those countries  where the costs of conventional antibiotic treatment are  prohibitive or where there is a high level of antibiotic  resistance.   Many women suffer from vaginitis, caused by either bac-  teria or Candida albicans, which may lead on to urinary  tract infections. In these cases the normal vaginal micro-  flora which comprises mainly lactobacilli is overrun by  various other, sometimes pathogenic, bacteria or fungi.  The usual approach to treatment is the administration of  antibiotics, but recurrence is commonplace. The vaginal  administration of probiotic cultures has proved to be  beneficial in these cases; however, strains of bacteria iso-  lated from dairy products are not always suitable. The  lactobacilli should be those that have the capacity to bind  specifically to vaginal epithelial cells.   This is a very common condition in young children in  which bacteria normally present in the nasopharynx  transfer via the eustachian tube into the middle ear. The  predominant bacteria are Streptococcus pneumoniae and  Haemophilus influenzae. Antibiotic therapy is frequently  unsuccessful and recurrences are common. As we have  seen in the examples outlined above, the picture is once  again one of undesirable bacteria replacing normal com-  mensal flora and causing disease. Studies have been  reported in which children were treated with suspensions  of a-haemolytic streptococci sprayed into the ear to  inhibit the growth of the invading pathogens and recolo-  nize the infected site. The results showed that the treated  patients had significantly better outcomes than the  placebo group.   Some workers have highlighted the fact that probiotics  should be used with caution in those patients whose  immune system is impaired because there have been  reported incidences of invasive infections occurring in  immunocompromised adults. In addition, Lactobacillus  bacteraemia has been reported following probiotic  therapy in two children who had received multiple  courses of antibiotics and who were suffering from other  serious underlying conditions.   The currently available data suggest that probiotics  may have some value in certain selected conditions, but  a great deal more background scientific information is  required before this can be moved forward substantially.  The approach of using a single culture for a whole spec-  trum of conditions is untenable. It is clear that the pro-    biotic bacteria will act in a variety ways, producing a  range of interfering substances and having different abili-  ties to adhere to epithelial cells. Individual cultures may  therefore need to be developed for use in specific condi-  tions and once this has been achieved large-scale trials are  needed to verify their value.   Chronic infected wounds arising from conditions such  as diabetes mellitus and peripheral vascular disease are  a major problem, and their incidence is increasing.  Whereas normal wounds go through well-defined phases  of healing, chronic wounds do not heal because of the  presence of necrotic tissue, slough, debris and infection.  In order to allow the wound to begin to heal it is neces-  sary to physically remove the necrotic burden, in a process  known as debridement, and also deal with the infection.  Clearing the wound of necrotic and devitalized tissue is  normally carried out surgically before application of anti-  microbial agents to remove the infecting pathogens. The  mechanical techniques used in debridement are varied  and can lead to pain and damage to underlying healthy  tissue.   The use of maggots to facilitate wound debridement  and subsequent healing has been known for several cen-  turies, but its association with modern medicine goes  back approximately 70 years. However, the clinical use of  maggots dwindled with the advent of antibiotics and has  only now experienced a resurgence of interest. The main  focus of attention has been in the area of wounds infected  with MRSA. The larvae of the blow fly Lucilia sericata are  grown under sterile conditions and used when they are  less than 8 hours old, at which point they are only 1-2 mm  in length. Up to 1000 maggots are placed in the wound,  covered to prevent escape and left for 1-3 days. The  maggots require a moist environment for optimal activity  and their secretions contain multiple proteolytic enzymes  which degrade necrotic tissue. This digested material is  subsequently consumed by the maggots as a source of  nutrients, but it has been suggested that there is also a  mechanical component to the clearing of necrotic tissue.  One of the characteristics of the larvae is that they try to  avoid light and as a result of their small size they tend to  burrow into deep crevices which may have proved diffi-  cult to debride mechanically. The larval secretions contain  ammonia which increases the local pH producing an  antimicrobial effect, and in addition the maggots ingest  the bacteria, thus clearing the site of infection. The pres-    ence of the maggots also leads to stimulation of granula-  tion tissue formation and hence promotes healing.   There is now abundant literature which appears to  affirm the view that maggot therapy can play an impor-  tant role in the treatment of chronic infected wounds.  However, a large (267 patients) multicentre trial was  carried out in the UK recently comparing maggot therapy  with a standard debridement technique (hydrogel) for  the treatment of chronic infected leg ulcers. The authors  found that there was no difference in healing times  between the two groups and the larval group experienced  more pain. Nonetheless, the larvae did reduce the time to  debridement. Some workers have shown that the antimi-  crobial efficacy of the larvae is dependent on the species  of bacterium under investigation. Gram-positive bacteria  such as Staph, aureus are very susceptible, whereas Gram-  negative bacteria such as Ps. aeruginosa are more resist-  ant. The precise reasons for this are unclear. Additional  hurdles to the widespread use of maggot therapy include  patient acceptability and resistance of medical staff to  apparently ‘old-fashioned’ techniques. Hence the case for  maggot therapy is equivocal, and more basic research is  required to understand why it appears to be effective in  some circumstances and not in others.   The therapeutic effects of light have been known for  thousands of years and the combination of light with  various chemicals again dates back to ancient times. It  was only at the beginning of the 20th century, however,  that the scientific basis of this phenomenon began to be  explored. The use of photosensitizing agents to kill  microorganisms was first shown in 1900 and it was  shortly after this that the term photodynamic therapy  (PDT) was coined. This chapter only discusses the use of  this technique for the killing of microorganisms, although  it should be pointed out that PDT has also been used to  treat tumours using a combination of photosensitizing  porphyrin compounds and laser light.   The most common photosensitizers used for their  antimicrobial effects are the thiazine dyes methylene blue  and toluidine blue O (also known as tolonium chloride;  Figure 27.1). Both of these molecules carry a positive  charge and this enables them to interact with the nega-  tively charged outer surfaces of bacteria. In particular,  they accumulate at the cytoplasmic membrane and when  activated cause lethal damage to that target site. It has   Figure 27.1 Structure of photosensitizers of the thiazine  family of dyes: (a) toluidine blue O, (b) methylene blue.   been found that Gram-positive bacteria are more sensi-  tive to PDT than Gram-negative bacteria, because of the  different nature of their cell walls (Chapter 3).   The light sources used in the recent past have been  lasers which produce light of a defined waveband designed  to match the peak excitation wavelength of the photosen-  sitizer used. Lasers also have the advantage of being able  to be passed through thin, flexible fibres for use in  difficult-to-access areas. More recently, advances in the  development of light-emitting diodes (LEDs), particu-  larly in respect of their increasing power, has enabled  these to be used under some circumstances in place of  lasers with obvious benefits of safety and cost. Toluidine  blue O has an absorbance peak of ±11 nm around the  maximum and so is suitable for use in conjunction with  these systems.   A detailed discussion of the mechanism of antimicro-  bial action of light-activated photosensitizers is outside  the scope of this chapter and the reader is referred to  Wainwright (1998) for further information. However, in  brief, the photosensitizer which is adsorbed on to the  bacterial cell will, when illuminated, give rise to the pro-  duction of toxic singlet oxygen which is very short-lived  but highly destructive. This singlet oxygen is present only  when the photosensitizer is excited by light and does not  persist once the light is extinguished. The toxic effect is  confined to bacteria and the photosensitizer does not  appear to have any adverse effects on normal tissue.   PDT is essentially a highly efficient disinfection process  which is harmless to surrounding tissues. The limitations  of the system are associated with the ability of both the  dye and the light to penetrate the matrix in which the  bacteria are located. The main use for the technology is  therefore to treat localized, surface infections which are    not particularly amenable to systemic therapy. It has been  used extensively for infections in dentistry, particularly  root canal infections, periodontitis and caries, and devices  are commercially available for use in dental surgeries.  Applications have also been found in dermatology, oph-  thalmology and ENT infections. It seems likely that the  range of conditions appropriate for this technology will  grow in the future, although the nature of the process is  such that it will be a niche market treating localized,  difficult-to-manage infections.   Vaccines to prevent both bacterial and viral diseases have  been available for many years and in some cases their use  predates that of antibiotics. In fact, for some diseases such  as whooping cough and diphtheria they are still the most  important form of infection control and highlight the  saying that prevention is better than a cure. Table 27.5  illustrates the range of bacterial vaccines currently avail-  able in the UK and some of these such as diphtheria,  tetanus, pertussis, meningococcal, haemophilus and BCG  vaccines (alongside viral vaccines) are an integral part of  the healthcare programme for the early years of life  (Chapters 10 and 24).   The recent explosion of knowledge in the area of  molecular biology has opened up new avenues for the  development of effective vaccines. DNA and viral vector  vaccines are examples of novel approaches currently  being studied. A number of vaccine technologies have  been explored recently aimed at Ps. aeruginosa which  causes severe opportunistic infections of wounds, the res-  piratory tract, the cornea and the urinary tract. Some  patients can be identified as being at particular risk of  contracting infections caused by this pathogen and they  include those with cystic fibrosis, burns patients and  those who are in intensive care, especially if they are  mechanically ventilated. These, and others, may therefore  benefit from an effective vaccine with a view to prevent-  ing infection from occurring. A recent review has argued  that from a scientific standpoint it is not a problem to  design vaccines containing specific bacterial antigens and  demonstrate their effectiveness in animals and preclinical  trials. There have however, been issues with conducting  meaningful clinical trials, due, among other things, to a  lack of patients available at a given time to provide appro-  priate comparisons. Similar arguments have been put  forward about the development of better vaccines to  replace BCG for protection against tuberculosis. In this    Table 27.5 Examples of bacterial infections for which vaccines or immunotherapy are currently available (BNF 58, 2009)   Acellular vaccine containing highly purified components of  B. pertussis. Whole cell vaccine used previously   Cell- free purified toxin adsorbed onto aluminium  hydroxide or aluminium phosphate. Immunoglobulin also  available   Heat and formaldehyde inactivated whole cells and  recombinant cholera toxin-B subunit    case the outcome of the investigation would not be  known until the clinical trial had been in progress for  many years.   It is perhaps not strictly appropriate to discuss the use of  silver as an alternative to antibiotics, because silver func-  tions really as an antiseptic biocide more like chlorhexi-  dine or povidone-iodine. However, some reviews have  discussed it in this context and so it is included here for  completeness. Silver has of course been used over centu-  ries to treat and prevent infections, and like some of the  other forms of treatment described in this chapter, its use  decreased with the advent of antibiotics. In the form of  silver sulphadiazine, however, it has been a mainstay in  the treatment of wound, particularly burn, infections  for the last 40 years and this is likely to continue. In addi-  tion, the number of wound dressings containing silver as  an antimicrobial component is increasing. Despite this,    there are a limited number of conclusive clinical trials  attesting to their efficacy and this situation certainly  needs to be addressed.   Silver can generate a number of different ions, i.e. Ag°,  Ag + , Ag 2+ and Ag 3+ . Elemental silver has no antibacterial  activity and the principal active component is the Ag +  cation which forms when in contact with aqueous solu-  tions. This ion has wide-ranging detrimental effects on  the bacterial cell by disrupting membrane function and  enzyme activity. There is activity against a broad range  of bacterial pathogens including the important Staph,  aureus and Ps. aeruginosa. Since the silver ion can be  quenched by the presence of chloride ions, continued  activity relies on the continuous release of further silver  ions. Improved activity has been reported for the use of  nanocrystalline silver which also appears to have an anti-  inflammatory action. Despite its widespread use there are  limited reports on the toxicity of silver and the incidence  of resistance is currently low.   The worsening situation with regard to antibiotic  resistance has brought all potential therapies into sharper    focus and silver is a candidate for increasing use, for  example as a coating on medical devices such as urinary  catheters and endotracheal tubes to prevent biofilm  formation.   Bacteriophages (phages) are viruses which specifically  infect and kill bacteria, but they are unable to attack  mammalian cells. In fact, their specificity is even more  profound, in that a particular bacteriophage will only  infect a given species of bacterium and often is even  strain-specific. There are an estimated 10 31 bacteriophages  on the planet, and viral predation accounts for a signifi-  cant part of carbon recycling in the oceans. These viruses  have existed on earth in conjunction with bacteria for  nearly 4 billion years and so are just as adept as their prey  at changing with their environment.   The structure of a typical bacteriophage is shown in  Figure 27.2. The genome is normally double-stranded  DNA and this is enclosed within a protein capsid. At the  tips of the tail fibres are receptors which recognize attach-  ment sites on the bacterial cell surface. When the phage  encounters a host bacterium it attaches via the tail fibres  and injects its DNA into the cell. This DNA then takes  over the cell’s metabolic machinery and the cell devotes 
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