Introduction to Listeria and Taxonomy. Bacteria belonging to the genus Listeria are Gram-positive, non-spore forming, motile rods characterized in part by their capability for growth over a wide range of temperatures (-0.4.degree. C. to 45.degree. C.) (J. M. Farber and P. I. Peterkin, Microbiol. Rev., 55:476 (1991)) and pH (.ltoreq.5.5 to 9.5) (J. Bille and M. P. Doyle, in Manual of Clinical Microbiology, pp. 287-295 (A. Balows et al., eds) (American Society for Microbiology, Washington, D.C., 1991)).
The taxonomic relationships between the genus and allied Gram-positive taxa are not clear. Listeria were previously included within the family Corynebacteriaceae, but are now taxonomically included in the Clostridium-Lactobacillus-Bacillus branch of the gram-positive bacteria phylogeny. Table 1 contains a list of Listeria species. One organism, previously named L. denitrificans, has been reclassified into a new genus, Jonesia. The new name of this organism is J. denitrificans; this organism is not included in the table.
TABLE 1 ______________________________________ SPECIES OF THE GENUS LISTERIA ______________________________________ L. grayi L. innocua L. ivanovii subspecies ivanovii L. ivanovii subspecies londoniensis L. monocytogenes L. murrayi* L. seeligeri L. welshimeri ______________________________________ *Some researchers include L. murrayi within L. grayi.
Because all Listeria isolates show a high degree of DNA relatedness and very close similarity in biochemical, phenotypic and protein characteristics, there is disagreement in the field regarding relationships between the identified species within the genus. B. J. Wilkinson and D. Jones, J. Gen. Microbiol., 98:399 (1977). J. McLauchlin, J. Appl. Bacteriol., 63:1 (1987).
Although there are disagreements between taxonomists, there remains the necessity of identifying members of the Listeria genus to the species level, in order to facilitate assessment of an isolate's pathogenic potential for humans as well as other animals. L. monocytogenes is a well-documented opportunistic pathogen of humans and other animals. L. ivanovii is most commonly pathogenic for animals other than humans, while L. grayi, L. innocua, L. murrayi and L. welshimeri are generally considered nonpathogenic. Thus, if the isolate from silage is identified as L. monocytogenes, it is a significant cause for concern to people such as ranchers and dairy farmers. If on the other hand, the isolate is L. murrayi, it is much less significant.
Natural History
Listeria species are ubiquitous in nature and are widely present in soil, plants, and surface waters. L. monocytogenes, the species most commonly associated with human disease, has been isolated from silage, sewage, slaughterhouse waste, milk from normal and mastitic cows, feces from humans and other, domestic animals (e.g., cattle, sheep, goats, and poultry), insects, and various wild animals. J. M. Farber and P. I. Peterkin, Microbiol. Rev., 55:476-511 (1991); M. L. Gray and A. H. Killinger, Bacteriol. Rev., 30:309-382 (1966); S. A. McCarthy, p. 25-29, in A. J. Miller et al. (eds), Foodborne Listeriosis, (Society for Industrial Microbiology, Elsevier Science Publishing, Inc., New York, 1990); J. Weis and H. P. R. Seeliger, Appl. Microbiol., 30:29-32 (1975). L. monocytogenes has also been recognized as a gastrointestinal tract transient, present in the stool of 5% of the human population. M. N. Swartz, in Microbiology, 4th edition, pp. 717-726 (B. D. Davis et al., eds) (J. B. Lippincott, Philadelphia, 1990).
In ruminants, the central nervous system is primarily affected (causing "circling disease"); septic abortion is a common result of infection of the female reproductive tract. Septicemia and multiple visceral abscesses also occur. As in humans, sporadic cases and epidemic outbreaks have been reported. Thus, while human listeriosis garners the most media attention and is of concern to medical practitioners, the ability to differentiate between Listeria species is also of great importance to veterinary practitioners and those who work to ensure a safe food supply.
Clinical Significance
Because the majority of human listeriosis cases (approximately 70%) occur in individuals with underlying conditions which cause suppression of T-cell mediated immunity, improved characterization of the genus would have important medical consequences. Predisposing conditions often associated with listeriosis include neoplastic disease, immunosuppression, pregnancy, extremes of age (neonates as well as the elderly), diabetes mellitus, cirrhosis, alcoholism, cardiovascular and renal collagen diseases, hematochromatosis, administration of drugs which reduce gastric acidity, frequent transfusions, and hemodialysis failure. R. E. Nieman and B. Lorber, Rev. Infect. Dis., 2:207-227 (1980); Swartz, supra. In a recent study of listeriosis in the United States, researchers estimated a minimum case rate of 90 per 100,000 AIDS patients, a rate 150 times greater than that of the general population in the same age group. B. G. Gellin et al., Am. J. Epidemiol. 133:392-401 (1991).
Although in most immunologically normal adults, the symptoms of listeriosis are relatively mild and flu-like, clinical syndromes include central nervous system infections, primary bacteremia and endocarditis, and infection of various organ systems. J. M. Farber and P. I. Peterkin, Microbiol. Rev. 55:476-511 (1991). Central nervous system infection with L. monocyogenes is typically meningitic or encephalitic, usually presenting with prodromal symptoms of headache, vomiting, fever, and malaise, prior to the appearance of central nervous system infection. Meningitis cases in adults and the elderly are generally associated with a high mortality rate (20-50%) or neurological sequelae in survivors. Bille and Doyle, supra. Due to the strong tropism of L. monocytogenes for the meninges, this organism should be included in the differential diagnosis of meningitis in high-risk groups. In the United States, L. monocytogenes is the fifth most common cause of bacterial meningitis; in the past several decades, it has increased four to five-fold in relative frequency underscoring the emerging importance of this organism. Swartz, supra.
In pregnant women, L. monocytogenes often causes an influenza-like bacteremic illness, which if unrecognized and untreated may progress to amnionitis and infection of the fetus, resulting in abortion, stillbirth or premature birth of an infected fetus. See Bille and Doyle, supra. Transplacental infection results in disseminated abscessed or granulomas in multiple organs (granulomatosis infantiseptica). Neonatal meningitis and/or bacteremia may result from perinatal bacteremia or from infection acquired during vaginal delivery.
While most human infections are sporadic, there have been several food-borne listeriosis epidemics reported from various countries. Some human listeriosis epidemics have resulted from epizootic outbreaks. For example, one outbreak involved cabbage which had been fertilized with sheep manure obtained from a flock in which many of the animals had died due to listeriosis. Schlech et al., New Eng. J. Med., 308:203-209 (1983). Other listeriosis outbreaks in the North America have been reported from Listeria-contaminated food, including a 1983 milk-associated outbreak (Fleming et al., New Eng. J. Med., 312:404-407 (1985)), and a 1985 outbreak associated with contaminated soft cheese (James et al., Morbid. Mortal. Wkly Rept. 34:357-359 (1985)). There has even been a recent suggestion that listeriosis may be the leading fatal food-borne infection in the United States. B. G. Gellin, supra.
With the need to improve the capability to identify all members of the Listeria genus to the species level, there is also the need to better understand the pathogenicity, if any, associated with each of these particular species. As indicated above, nearly all of the reported cases of human Listeria infections have been caused by L. monocytogenes. See McLauchlin, J. Appl. Bacteriol. 63:1 (1987). However, instances have been reported in which L. ivanovii, L. innocua and L. seeligeri have caused disease in humans. See, E. A. Szabo and P. M. Desmarchelier, Epidemiol. Infect. 105:245 (1990); Bille and Doyle, supra.
Isolation and Identification of Listeria From Clinical Specimens
A tentative diagnosis of listeriosis may be made by direct examination of Gram-stained sediment from normally sterile fluids such as cerebrospinal fluid (CSF) or amniotic fluid. Direct examination of Gram-stained clinical specimens is of limited diagnostic value, as Listeria may be present in such low numbers as to go undetected in a stained smear, resulting in a false negative result. Listeria in CSF may be confused with other organisms such as streptococci or corynebacteria, and excessively decolorized organisms may be confused with Haemophilus influenzae. Thus, cultures and biochemical tests are required for definitive identification of Listeria.
Blood, CSF, amniotic fluid and tissue biopsies specimens are commonly submitted for Listeria isolation and identification. Clinical specimens obtained from unsterile sites (or any sample which may contain competing flora) should be processed in the same manner as environmental and food specimens. These procedures involve the use of enrichment steps and selective media. Both the Food and Drug Administration (FDA) and the Centers for Disease Control (CDC), have developed enrichment and selective protocols for Listeria isolation and identification.
Tests commonly used in conventional Listeria species identification include observation of beta hemolysis, the CAMP test with S. aureus and R. egui, nitrate reduction, the Voges-Proskauer test, hydrolysis of cellulose, hippurate and starch, production of lecithinase and phosphatase, acid production (within 1 week) from L-arabinose, dextrin, galactose, glycogen, lactose, D-xylose, mannitol, melezitose, melibiose, L-rhamnose, sorbitol, soluble starch. sucrose, D-xylose, and pathogenicity for mice. Bille and Doyle, supra; H. P. R Seeliger and D. Jones, in Bergey's Manual of Systematic Bacteriology, Vol. 2, pp. 1235-1245 (P. H. A. Sneath et. al., eds)(Williams and Wilkins, Baltimore, 1986); Skalka et. al., J. Clin. Microbiol. 15:503 (1982). Other biochemical tests have been utilized, but have been reported as unsuitable for differentiation between Listeria species, such as fermentation of methyl-D-glucoside. V. H. Seiler and M. Busse, Berl. Munch. Tierarztl. Wsch., 102:166 (1989).
Isolation and Identification of Listeria Food Isolates
Given the ubiquitous nature of this organism and the dire consequences associated with listeriosis in debilitated populations, a major focus has been on disease prevention. An important consideration is the non-existence of a human or veterinary Listeria vaccine. Thus, preventive measures must be undertaken without the safeguards provided by immunization protocols helpful in preventing zoonotic disease outbreaks. As a safe food supply is of utmost concern, methods for the rapid screening of food samples for Listeria are of great importance to the food industry. Under current regulations, the presence of viable cells any Listeria species in foods is cause for alarm.
The FDA and comparable agencies in other countries have promulgated standard laboratory methods to detect the presence of Listeria in environmental or food specimens (e.g., milk). The FDA method is a 7-day enrichment method, (Lovett et al., P17, p. 253, Abstracts of the Annual Meeting of the American Society for Microbiology, 1985), while the Centers for Disease Control method is a cold enrichment procedure (Hayes et al., Appl. Environ. Microbiol. 51:438-440 (1986)). These methods involve culturing an appropriately prepared sample on microbiological media under conditions favorable for growth of these organisms and unfavorable for other bacteria. Detection of Listeria species is attempted by examining the morphological and biochemical characteristics of the resultant colonies. This process is typically started 48 hours after acquisition of the sample and requires 9-19 days for completion (if enrichment techniques are required, it will take much longer). See R. L. Buchanan et al., J. Assoc. Off. Anal. Chem., 71:651 (1988); J. Lovett, J. Assoc. Off. Anal. Chem. 71:658 (1988); D. McClain and W. H. Lee, J. Assoc., Off. Anal. Chem. 71:660 (1988); and M. T. Knight et. al., J. Assoc. Off. Anal. Chem. 71: 682 (1988).
Newer methods of detecting Listeria include (a) nucleic acid probes capable of binding to the nucleic acid of particular species, and (b) antibodies capable of reacting with antigens specific to particular species. See e.g., G. Comi et al., Zbl. Hyg. 192:134 (1991); A. R. Datta et. al., Appl. Environ. Microbiol. 54:2933 (1988) (probes to a fragment of a presumptive hemolysin gene of L. monocytogenes); J. Klinger, J. Assoc. Off. Anal. Chem. 71:669 (1988) (nucleic acid hybridization assay for Listeria in foods); PCT No. 0355147 (corresponding to U.S. Ser. No. 227,402 and 143,490) (a probe directed to the hemolysin gene in L. monocytogenes); European Patent Application No. 0314294 (corresponding to U.S. Ser. No. 965,510) and U.S. Pat. No. 5,089,386 (probes to rRNA of L. monocytogenes); U.S. Pat. No. 4,950,589 (monoclonal antibodies directed to a Listeria antigen); G. R. Siragusa and M. G. Johnson, Appl. Environ. Microbiol. 56:1897 (1990) (monoclonal antibodies to an antigen shared with three Listeria species); J. A. Mattingly, J. Assoc. Off. Anal. Chem. 71: 679 (1988) (antibody-based assay for detection of L. monocytogenes); European Patent Application No. 0303309 (corresponding to U.S. Ser. No. 836,619) (antibodies to heat-treated extracts of Listeria).
Due to the need for reliable and rapid tests, suitable for a wide range of settings, the suitability of various test kits for Listeria identification has been evaluated. Indeed, the number of test kits and evaluators is testament to the need for the development of a simple, affordable, rapid and reliable method to identify Listeria species. The majority of these kits were originally developed for the identification of other genera. Thus, they are not tailored to the particular needs unique to Listeria identification to the species level. Some require additional tests even to identify a possible Listeria isolate to the genus level.
A. P. MacGowan et al., J. Clin. Pathol. 42:548 (1989), found that API 20 STREP (developed by API-bioMerieux, La Balme des Grottes, France, to identify Streptococcus isolates) identified Listeria in four hours and may be useful for identification of L. monocytogenes implicated in human infection. However, they determined that this test kit would not be useful to identify environmental isolates, where identification to the species level is important.
API 50CH (API-bioMerieux, La Balme des Grottes, France) has also been used to identify Listeria and was compared with the API 20 STREP. The utility of API 20 STREP was criticized by Kerr et al., Appl. Environ. Microbiol. 56:657 (1990), for its lack of tests for xylose and rhamnose fermentation, and a-methyl mannosidase activity. These authors were also critical of the large number of substrates used in the API 50CH and the relatively small number of substrates actually relevant in Listeria species identification. The same authors also evaluated the Mast ID system produced by Mast Laboratories (Bootle, United Kingdom). This system utilizes agar plates incorporated with substrates and indicators to determine .alpha.-methyl-mannosidase activity, esculin hydrolysis, acetoin production (the Voges-Proskauer test), and fermentation of mannitol, rhamnose, trehalose, salicin and xylose. They found this system to be less expensive than the API 50CH, yet as reliable and rapid (24 hours for results, as opposed to conventional carbohydrate fermentation tests which may require up to 5 days of incubation). However, agar plates containing the appropriate substrates must be prepared prior to inoculation with putative Listeria isolates.