biofilm - forming bacteria are often seen on the surfaces of tissues ( 1 - 3 ) and biomaterials at sites of persistent infection ( 4 , 5 ) .
medical implants and catheters are particularly susceptible to biofilm formation because immune responses are significantly reduced in proximity to foreign bodies ( 6 , 7 ) . in fact , biofilm formation is a main cause of implant failure and often limits the lifetime of many indwelling medical devices ( 8) . biofilms protect microorganisms from opsonization by antibodies , phagocytosis , and removal via the ciliary action of epithelial cells ( 5 ) .
in addition , bacterial populations in biofilms are considerably more resistant than free - living planktonic cells are to antibacterial agents ( 9 , 10 ) .
therefore , treatment of an infection after a biofilm has been established is frequently futile with the current therapy options ( 11 ) .
klebsiella pneumoniae is frequently found in a variety of environmental niches , such as soil , vegetation , and water , including the drinking - water distribution system .
it is also an important nosocomial pathogen involved in urinary tract infections , hospital - acquired pneumonia ( hap ) , ventilator - associated pneumonia ( vap ) , surgical - wound infection , bacteremia , and septicemia ( 12 - 14 ) .
interestingly , previous studies reported a high frequency of opportunistic infections caused by k. pneumoniae among persons with certain debilitating medical conditions , such as diabetes mellitus or bladder neuropathy ( 15 , 16 ) . the most important virulence factors contributing to k. pneumoniae pathogenesis with regard to the severity of its infections are capsular polysaccharides , type 1 and type 3 pili , which can contribute to biofilm formation ( 14 , 17 , 18 ) .
the first biofilm - forming k. pneumoniae strain was described at the end of the 1988s ( 19 ) .
these bacteria can produce a thick layer of extracellular biofilm as a virulence factor that helps the organism attach to living or abiotic surfaces , preventing the effects of antimicrobial agents ( 20 ) .
for these reasons , detailed knowledge about biofilm formation and biofilm - producing bacteria is necessary for the management and treatment of related infections .
in addition , the determination of genetic diversity for dominant species identification can be useful for preventing the spread of infection in hospitals .
knowing the dominant genotype of isolates can be helpful for the identification of the source of an infection , and for implementing preventive procedures and applying infection control .
the present study was designed to evaluate biofilm development among k. pneumoniae clinical isolates , and to perform molecular genotyping using the enterobacterial repetitive intergenic consensus - polymerase chain reaction ( eric - pcr ) method .
in the present study , 94 k. pneumoniae strains were isolated at two hospitals in tehran from february to july 2014 .
the k. pneumoniae strains were recovered from blood , sputum , surgical wound swabs , and urine samples of hospitalized patients .
after recognition , the k. pneumoniae strains were stored in tryptic soy broth ( tsb ) ( merck co. , germany ) with 10% glycerol at -70c .
all isolates were freshly subcultured on brain - heart infusion ( bhi ) agar ( merck co. , germany ) prior to every analysis .
fresh colonies of k. pneumoniae isolates were used to inoculate 5 ml of brain - heart infusion ( bhi ) broth medium ( merck co. , germany ) .
following incubation , the amount of cells in each culture was quantified and adjusted to 0.5 mcfarland ( 1.5  10 cfu / ml ) , and 200 l of each bacterial suspension was transported into eight wells of a 96-well microtiter plate containing bhi broth .
staphylococcus epidermidis rp62a and pseudomonas aeruginosa pao1 , both of which are well - recognized as biofilm - forming strains , were used as two positive controls in the biofilm assays .
was then located in the wells of the microtiter plate , allowing the pegs to be submerged within the bacterial culture .
the inoculated peg plate was relocated to a 96-well microtiter plate containing fresh bhi broth .
the inoculated microtiter plates were incubated on a shaking platform at 37c for two days , permitting mature biofilms to be established .
the biofilm biomass assay was semi - quantified after the modification of a procedure described by mowat et al .
after incubation , the peg plate was removed from the microtiter plate , then washed with pbs to remove planktonic free - floating cells that were weakly attached to the surface .
each replicate peg was stained with 0.5% ( w / v ) crystal violet for 5 min , and surplus crystal violet was eliminated by carefully rinsing the peg plate under running distilled water .
the stained biofilms were decolorized by the addition of 100 ml of 95% ethanol to each well for 1 min .
the ethanol was relocated to a clean 96-well microtiter plate and the optical density ( od ) was measured using a microtiter plate reader .
the biofilm - forming capacity of each test isolate was compared with the positive and negative controls by analyzing the absorbance of the crystal violet stain obtained for each biofilm .
the strains were classified into four groups : non - adherent ( < 25% absorbance compression with positive control ) , weakly adherent ( 25% - 50% ) , moderately adherent ( 51% - 75% ) , or highly adherent ( 76% - 100% ) , depending on the od of the bacterial biofilm ( table 1 ) . to evaluate the genetic relatedness among the isolates ,
an isolate was considered a different type if its banding pattern differed by two or more bands . in other words , isolates were classified as identical types when their banding patterns were different in one band only ( 24 ) .
the k. pneumoniae genotypes were named by k1 to kn . to determine the similarity rate among the obtained types , they were analyzed using the unweighted pair - group method with arithmetic mean ( upgma ) .
chi - square analysis was used for comparisons between the capacity of biofilm production and the type of clinical specimen .
in the present study , 94 k. pneumoniae strains were isolated at two hospitals in tehran from february to july 2014 .
the k. pneumoniae strains were recovered from blood , sputum , surgical wound swabs , and urine samples of hospitalized patients .
after recognition , the k. pneumoniae strains were stored in tryptic soy broth ( tsb ) ( merck co. , germany ) with 10% glycerol at -70c .
all isolates were freshly subcultured on brain - heart infusion ( bhi ) agar ( merck co. , germany ) prior to every analysis .
fresh colonies of k. pneumoniae isolates were used to inoculate 5 ml of brain - heart infusion ( bhi ) broth medium ( merck co. , germany ) .
the cultures were incubated for 18 hours at 37c with shaking at 200 rpm . following incubation ,
the amount of cells in each culture was quantified and adjusted to 0.5 mcfarland ( 1.5  10 cfu / ml ) , and 200 l of each bacterial suspension was transported into eight wells of a 96-well microtiter plate containing bhi broth .
staphylococcus epidermidis rp62a and pseudomonas aeruginosa pao1 , both of which are well - recognized as biofilm - forming strains , were used as two positive controls in the biofilm assays .
a 96-peg plate was then located in the wells of the microtiter plate , allowing the pegs to be submerged within the bacterial culture .
the inoculated peg plate was relocated to a 96-well microtiter plate containing fresh bhi broth .
the inoculated microtiter plates were incubated on a shaking platform at 37c for two days , permitting mature biofilms to be established .
the biofilm biomass assay was semi - quantified after the modification of a procedure described by mowat et al .
, the peg plate was removed from the microtiter plate , then washed with pbs to remove planktonic free - floating cells that were weakly attached to the surface .
each replicate peg was stained with 0.5% ( w / v ) crystal violet for 5 min , and surplus crystal violet was eliminated by carefully rinsing the peg plate under running distilled water .
the stained biofilms were decolorized by the addition of 100 ml of 95% ethanol to each well for 1 min .
the ethanol was relocated to a clean 96-well microtiter plate and the optical density ( od ) was measured using a microtiter plate reader .
the biofilm - forming capacity of each test isolate was compared with the positive and negative controls by analyzing the absorbance of the crystal violet stain obtained for each biofilm .
the strains were classified into four groups : non - adherent ( < 25% absorbance compression with positive control ) , weakly adherent ( 25% - 50% ) , moderately adherent ( 51% - 75% ) , or highly adherent ( 76% - 100% ) , depending on the od of the bacterial biofilm ( table 1 ) .
to evaluate the genetic relatedness among the isolates , the eric - pcr method was performed as described previously ( 23 ) .
an isolate was considered a different type if its banding pattern differed by two or more bands . in other words , isolates were classified as identical types when their banding patterns were different in one band only ( 24 ) .
the k. pneumoniae genotypes were named by k1 to kn . to determine the similarity rate among the obtained types , they were analyzed using the unweighted pair - group method with arithmetic mean ( upgma ) .
chi - square analysis was used for comparisons between the capacity of biofilm production and the type of clinical specimen .
of 94 k. pneumoniae clinical isolates , 58 ( 61.7% ) were obtained from urine specimens , 17 ( 18.1% ) from surgical wounds , 11 ( 11.7% ) , from sputum , and 8 ( 8.5% ) from blood .
the strains were divided into four categories as described above . according to the biofilm analysis , 52.1% ( n = 49 ) of the 94 tested k. pneumoniae isolates were categorized as moderately biofilm - producing strains , 33% ( n = 31 ) formed fully established biofilms , 8.5% ( n = 8) produced negligible biofilms , and 6.4% ( n = 6 ) of the strains were non - biofilm producers .
the k. pneumoniae strains that were isolated from sputum , surgical - wound swabs , and urine samples had a remarkably greater ability to form fully and moderately established biofilms , compared with isolates obtained from blood samples ( table 2 ) .
high ( + + + ) biofilm - formation ability among the isolates obtained from sputum was more than other isolates ( p < 0.001 ) significantly .
genotyping of k. pneumoniae revealed 31 different eric types , while 11 isolates were non - typeable because they had no banding pattern after pcr reaction and electrophoresis .
the k1 type was the most common and included 26 isolates , followed by type k2,which comprised 19 patterns .
the highest similarity coefficient was associated with the k05 and k06 types ( 90% similarity ) .
of 94 k. pneumoniae clinical isolates , 58 ( 61.7% ) were obtained from urine specimens , 17 ( 18.1% ) from surgical wounds , 11 ( 11.7% ) , from sputum , and 8 ( 8.5% ) from blood .
the strains were divided into four categories as described above . according to the biofilm analysis , 52.1% ( n = 49 ) of the 94 tested k. pneumoniae isolates were categorized as moderately biofilm - producing strains , 33% ( n = 31 ) formed fully established biofilms , 8.5% ( n = 8) produced negligible biofilms , and 6.4% ( n = 6 ) of the strains were non - biofilm producers .
the k. pneumoniae strains that were isolated from sputum , surgical - wound swabs , and urine samples had a remarkably greater ability to form fully and moderately established biofilms , compared with isolates obtained from blood samples ( table 2 ) .
high ( + + + ) biofilm - formation ability among the isolates obtained from sputum was more than other isolates ( p < 0.001 ) significantly .
genotyping of k. pneumoniae revealed 31 different eric types , while 11 isolates were non - typeable because they had no banding pattern after pcr reaction and electrophoresis .
the k1 type was the most common and included 26 isolates , followed by type k2,which comprised 19 patterns .
the highest similarity coefficient was associated with the k05 and k06 types ( 90% similarity ) .
in the present study , we assayed the capacity for biofilm production in isolates of k. pneumoniae obtained from clinical specimens .
our findings indicated that an enormous proportion ( more than 93% ) of isolates were biofilm - producing strains .
biofilm - forming bacteria cause infections , such as catheter- and implant - related infections , as well as life - threatening disorders in patients with cystic fibrosis , chronic wounds , and chronic otitis media .
these bacteria affect millions of people around the world every year , with a high mortality rate ( 25 ) .
the ability of nosocomial opportunistic microorganisms such as k. pneumoniae to produce biofilms on host - tissue surfaces is a critical stage in the development of infection .
biofilm formation affects the efficacy of antimicrobial therapies and the outcomes of subsequent infections ( 18 , 26 ) .
yang and colleagues conducted a study in 2008 on biofilm formation by k. pneumoniae strains isolated from urine samples , sputum , wound swabs , and blood ( 27 ) .
they found that 62.5% of the isolates generated biofilms , which is less than the results obtained in our study .
saeed and colleagues evaluated biofilm formation in three k. pneumoniae strains , and showed that all of them produced biofilms ( 28 ) .
the reason for the discrepancy in our results compared to their findings may be due to their small sample size ( n = 3 ) .
some studies , such as one by saxena et al . , evaluated biofilm production in other bacteria by assessing biofilm formation in p. aeruginosa with the qualitative tube method ( 29 ) .
the findings revealed that 20% , 21.25% and 58.75% of the isolates , respectively , were highly potent biofilm producers , moderate producers , or weak / non - biofilm producers . according to these results
, the ability of k. pneumoniae strains to form biofilms may be higher than that of p. aeruginosa strains .
clinical observations and experimental investigations have clearly confirmed that in most cases , antibiotic therapy alone is inadequate to eradicate biofilm - forming infections ( 30 ) . consequently , the successful treatment of biofilm - associated infections with currently accessible antibiotics , and the evaluation of outcomes , has become important and urgent for clinicians .
pcr - based fingerprinting techniques , such as eric - pcr , are quick methods for typing the strains of k. pneumoniae .
the eric - pcr method is most reliable when two or more band differences are used as the cutoff ( 24 ) . in the present study , this method was used for molecular typing of k. pneumoniae isolates from two hospitals in tehran .
most of the type k1 strains ( 20 out of 26 ) were isolated from urinary infections at one hospital , where the most prevalent nosocomial type was k1 .
strong genetic similarity was seen between the k5 and k6 types , which were both obtained from the same hospital .
indicated that eric - pcr has a discriminatory power similar to that of pulsed - field gel electrophoresis ( pfge ) .
therefore , itis an acceptable technique for molecular classification compared to pfge , as a gold standard method ( 24 ) . in a previous study performed on various clinical samples , a high level of genetic diversity among k. pneumoniae strains
was reported , which is similar to the results of the present study ( 31 ) .
this heterogeneity may be due to the survival of k. pneumoniae on dry surfaces in the hospital environment , making accidental contamination of patients possible . in our study
, we did not observe any correlation between eric type and biofilm formation ; in other words , the ability of biofilm formation in a specific eric type was not seen .
our results were in contrast with those of diago - navarro et al . , who showed that the k. pneumoniae isolates with a strong capacity for biofilm formation were classified in a particular sequence type ( 32 ) .
therefore , according to diago - navarro et al.s study , multilocus sequence typing can be helpful in the determination of the dominant types of biofilm - producer isolates .
biofilm formation is an important cause of bacterial resistance to the immune system and to antibacterial agents ( 5 , 9 ) .
new approaches , such as photodynamic therapy and the utilization of medicinal plants , have recently been introduced against bacterial biofilms ( 33 , 34 ) .
we suggest the use of these strategies to eliminate biofilm - producing bacteria from the hospital environment and to treat infections . in conclusion
, our results showed that an enormous proportion of k. pneumoniae isolates from sputum and surgical - wound swabs produced fully established biofilms .
in contrast , k. pneumoniae strains isolated from blood exhibited weaker tendencies to form massive biofilms . given these findings
, there is reason to assume a relationship between infection and biofilm formation . in molecular genotyping , no correlation between eric types and biofilm - formation ability was observed .
supplementary research into the mechanisms of biofilm formation in k. pneumoniae , as well as the design and use of new and effective approaches , will ultimately aid in the treatment of biofilm - mediated infections and in the reduction of morbidity and mortality in patients suffering from life - threatening nosocomial infections .
 background : klebsiella pneumoniae is among the most frequently recovered etiologic agents from nosocomial infections . 
 this opportunistic pathogen can generate a thick layer of biofilm as one of its important virulence factors , enabling the bacteria to attach to living or abiotic surfaces , which contributes to drug resistance.objectives:the resistance of biofilm - mediated infections to effective chemotherapy has adverse effects on patient outcomes and survival . 
 therefore , the aim of the present study was to evaluate the biofilm - formation capacity of clinical k. pneumoniae isolates and to perform a molecular characterization using enterobacterial repetitive intergenic consensus - polymerase chain reaction ( eric - pcr ) to determine the dominant biofilm - producing genotype.patients and methods : in the present study , 94 k. pneumoniae isolates were obtained from two hospitals in tehran , iran . 
 biofilm formation was assayed by a modified procedure , then eric - pcr was carried out.results:the distributions of the clinical specimens used in this study were 61.7% from urine , 18.1% from wounds , 11.7% from sputum , and 8.5% from blood . among these isolates , 33% formed fully established biofilms , 52.1% were categorized as moderately biofilm - producing , 8.5% formed weak biofilms , and 6.4% were non - biofilm - producers . 
 genotyping of k. pneumoniae revealed 31 different eric types . 
 biofilm - formation ability in a special eric type was not observed.conclusions:our results indicated that an enormous proportion of k. pneumoniae isolated from sputum and surgical - wound swabs produced fully established biofilms . 
 it is reasonable to assume the existence of a relationship between the site of infection and the formation of biofilm . a high level of genetic diversity among the k. pneumoniae strains was observed . 
1. Background
2. Objectives
3. Patients and Methods
3.1. Bacterial Isolation and Identification
3.2. Biofilm Model
3.3. Semi-quantification of Biofilm Biomass
3.4. Molecular Genotyping
3.5. Statistical Analysis
4. Results
4.1. Distribution of Samples
4.2. Biofilm Formation
4.3. ERIC-PCR Typing
5. Discussion