rfbE Gene Research Articles

Escherichia coli O157:H7 and Non‐O157 Shiga Toxin‐producing E. coli in Healthy Cattle, Sheep and Swine Herds in Northern Spain

Three-hundred and forty-five herds (17 swine, 122 dairy sheep, 124 beef and 82 dairy cattle) were investigated for prevalence of Shiga toxin-producing Escherichia coli (STEC). Rectal faecal samples were selectively enriched and then examined by immunodetection techniques (Immunomagnetic Separation with anti-E. coli O157 Dynabeads, ImmunoMagnetic cell Separation (IMS) and automated enzyme-linked fluorescent immunoassay using VIDAS) and polymerase chain reaction (PCR) (rfbE and fliC genes) to assess the prevalence of E. coli O157:H7. Prevalence of non-O157 STEC was estimated by PCR screening for stx genes of 10 lactose-positive colonies grown on MacConkey agar after enrichment. PCR was used on all STEC isolates to detect stx(1), stx(2), eaeA and E-hlyA genes. Both immunodetection methods showed a moderate-good level of agreement (kappa = 0.649) but IMS showed 87.5% complementary sensitivity. Prevalence of positive herds for E. coli O157:H7 was estimated at 8.7% for sheep and 3.8% for cattle, whereas all the porcine herds tested negative. Non-O157 STEC were also absent from swine, but were isolated more frequently from ovine (50.8%) than bovine herds (35.9%). Within-herd prevalences of excretion of E. coli O157:H7 established by individual testing of 279 sheep (six herds) and 30 beef cattle (one herd) were 7.3% and 6.7% respectively. PCR analysis of 49 E. coli O157:H7 and 209 non-O157 isolates showed a different distribution of virulence genes. All E. coli O157:H7 were stx(2) gene-positive, eaeA was detected in 95.9%, and the toxigenic profile stx(2)/eaeA/E-hlyA was present in 75.5% of the isolates. Among the non-O157 STEC, prevalence of eaeA was significantly lower (5.3%) and E-hlyA was present in 50.2% of the isolates but only sporadically associated with eaeA. stx(2) was predominant in non-O157 isolates from cattle, whereas in sheep the combination stx(1)/stx(2) was more prevalent. This study demonstrated the wide distribution of STEC in ruminant herds, which represent an important reservoir for strains that pose a potential risk for human infections.

Molecular Characterization of Escherichia coli O157:H7 Isolates Obtained From Bovine and Porcine Carcasses

ISEE-808 Objective: The objectives of this study were to determine the presence and/or absence of Escherichia coli serotype O157:H7 in bovine and porcine carcasses, and molecular characterization of the isolated E. coli by means of PCR. Materials and Methods: The study was carried out between February and October 2004. Two slaughterhouses were sampled, 1 of porcine (I) and 1 of bovine (II) from La Comarca Lagunera, Mexico. Samplings were made in 2 dates for each slaughterhouse, February 20, 2004, and March 15, 2004, for site I and March 02, 2004, and March 24, 2004, for site II. The isolates of E. coli were obtained using the NOM-030 z00, 1995, but we also used the NOM-109-SSAI, 1994. For the analysis in life surfaces 15 carcasses were sampled randomly on each slaughterhouse, using a sterile sponge (NASCO) in 3 different places. The characteristic colonies were confirmed with biochemical tests using API 20 E (Biomereux, France) and the serotype using the serology kit (DIFCO). From the samples in which presence of E. coli O157:H7 was confirmed, DNA was isolated for the molecular characterization by PCR. Results: Of the 18 isolates, 12 were positive by PCR to rfbE and fliC genes, which determined O157:H7 serotype. Of those 12, 1 bovine, and 3 porcine carcasses samples were positive by PCR to stx1, stx2, and eaeA genes; therefore, they were considered enterohemorrhagic strains. The differences found in the positive carcasses number to any of the genes were not statistically significant. Conclusions: E. coli O157:H7 was found in both carcasses types, representing a risk for the human health; so strict hygienic and handling practices should be taken to avoid consumption of contaminated meat by final consumers.

Incidence of Escherichia coli O157:H7 and E. coli Virulence Factors in US Bulk Tank Milk as Determined by Polymerase Chain Reaction

Samples of bulk tank milk from dairies across the United States, taken as part of the National Animal Health Monitoring Dairy 2002 survey, were analyzed for the presence of several genes encoding virulence factors associated with enterohemorrhagic forms of Escherichia coli (EHEC) using real-time and conventional PCR assays. Samples from 859 farms in 21 states were collected and enriched in EC medium at 42.5°C to amplify any E. coli present, and DNA was isolated from the resulting biomass. The eaeA gene encoding intimin, a virulence factor associated with enteropathogenic forms of E. coli and EHEC, was detected in 199 (23%) of the samples. Thirty-six samples (4.2%) were positive for eaeA, the gamma allele of the translocated intimin receptor (γ-tir), found in EHEC strains of O157:H7, and one or both shiga-like toxin genes (stx1 and stx2), a combination that may be indicative of the presence of O157:H7 EHEC. Testing these 36 samples with a commercially available real-time PCR kit for detection of O157:H7 indicated that 5 samples could be contaminated with O157:H7. A multiplex PCR to detect the presence of fliC, rfbE, and hlyA genes found in O157:H7 reduced to 2 (0.2% of all samples) the number of samples likely to be contaminated with this organism. A strain of O157:H7 (eaeA+, γ-tir+, stx2+, rfbE+, fliC+, hlyA+) was subsequently isolated from one sample. Thirty-four eaeA-positive samples did not contain detectable γ-tir but did contain one or both of the stx genes suggesting the presence of EHEC strains other than O157:H7. These results indicate a low incidence of O157:H7 in bulk tank milk but suggest that a risk from other enteropathogenic and EHEC forms of E. coli may exist and that PCR targeting virulence factors associated with highly pathogenic forms of E. coli may be an effective means of detecting potential dangers in raw milk.

Prevalence of Shiga toxin-producing Escherichia coli stx1, stx2, eaeA, and rfbE genes and survival of E. coli O157:H7 in manure from organic and low-input conventional dairy farms.

Manure samples were collected from 16 organic (ORG) and 9 low-input conventional (LIC) Dutch dairy farms during August and September 2004 to determine the prevalence of the STEC virulence genes stx(1) (encoding Shiga toxin 1), stx(2) (encoding Shiga toxin 2), and eaeA (encoding intimin), as well as the rfbE gene, which is specific for Escherichia coli O157. The rfbE gene was present at 52% of the farms. The prevalence of rfbE was higher at ORG farms (61%) than at LIC farms (36%), but this was not significant. Relatively more LIC farms were positive for all Shiga toxin-producing E. coli (STEC) virulence genes eaeA, stx(1), and stx(2), which form a potentially highly virulent combination. Species richness of Enterobacteriaceae, as determined by DGGE, was significantly lower in manure positive for rfbE. Survival of a green fluorescent protein-expressing E. coli O157:H7 strain was studied in the manure from all farms from which samples were obtained and was modeled by a biphasic decline model. The time needed to reach the detection limit was predominantly determined by the level of native coliforms and the pH (both negative relationships). Initial decline was faster for ORG manure but leveled off earlier, resulting in longer survival than in LIC manure. Although the nonlinear decline curve could theoretically be explained as the cumulative distribution of an underlying distribution of decline kinetics, it is proposed that the observed nonlinear biphasic pattern of the survival curve is the result of changing nutrient status of the manure over time (and thereby changing competition pressure), instead of the presence of subpopulations differing in the level of resistance.

Evaluation of enrichment-free PCR-based detection on the rfbE gene of Escherichia coli O157—Application to municipal wastewater

Escherichia coli O157 strains have emerged as important human enteric pathogens. Strains that express the O-antigen 157 are commonly associated with severe clinical manifestations, including bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. E. coli O157 strains may be transmitted in a variety of ways, including food, water and person-to-person or animal-to-person contact. Fecal contamination is one source of environmental contamination and is responsible for the presence of these pathogens in the environment. We used a specific and sensitive PCR assay based on the rfbE gene to detect low levels of these pathogens in wastewater. The set of primers used was designed to amplify an intragenic segment of the rfbE gene. The amplification assay detected 200 CFU of E. coli O157 in pure water. The prevalence of E. coli O157 in the effluents of 44 wastewater treatment plants was determined (7%).

Phage types, virulence genes and PFGE profiles of Shiga toxin-producing Escherichia coli O157:H7 isolated from raw beef, soft cheese and vegetables in Lima (Peru)

The present study was conducted in Lima Metropolitana to evaluate the prevalence of Shiga toxin-producing Escherichia coli (STEC) O157:H7 in raw beef, raw ground beef, soft cheese and fresh vegetables, sampled at different markets in the city. Between October 2000 and February 2001, 407 food samples were collected from different markets in the 42 districts of Lima Metropolitana. Samples were assayed for E. coli O157 by selective enrichment in modified Tryptic Soy Broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Fifty (12.3%) of 407 food samples resulted positive for E. coli O157 isolation (23 of 102 ground beef; 15 of 102 beef meat; eight of 102 soft cheese and four of 101 fresh vegetables). Thirty-five E. coli O157 isolates were further analysed for the presence of virulence genes. All 35 were positive by PCR for O157 rfbE, fliCh7, eae-γ1 and ehxA genes. In addition, genes encoding Shiga toxins were detected in 33 of 35 isolates, five isolates (14%) encoded stx 1, stx 2, and 28 (80%) stx2 only. The isolates were of seven different phage types (PT4, PT8, PT14, PT21, PT34, PT54, and PT87) with three phage types accounting for 80% of isolates: PT4 (15 isolates), PT14 (8 isolates), and PT21 (5 isolates). Interestingly, the majority (31 of 35; 89%) of E. coli O157:H7 isolates characterized in this study belonged mainly to the phage types previously found in STEC O157:H7 strains associated with severe human disease in Europe and Canada. Pulsed-field gel electrophoresis (PFGE) of 32 isolates revealed 14 XbaI-PFGE groups (I to XIV) of similarity > 85%, with 23 (72%) isolates grouped in five clusters. Some isolates from different districts presented a high clonal relatedness. Thus, PFGE group VIII clustered eleven strains from nine different districts. The broad range of PFGE subtypes found in this study demonstrates the natural occurrence of many genetic variants among STEC O157:H7 spread in Lima.

Occurrence of virulence genes associated with enterohemorrhagic Escherichia coli in raw municipal sewage

Municipal sewage influent was screened for the presence of the virulence genes encoding Shiga-like toxins SLT-I and SLT-II ( slt-I and slt-II) and intimin ( eaeA) and those involved in biosynthesis of O157 ( rfbE) and H7 ( fliC) antigens by multiplex PCR to simultaneously identify the enterohemorrhagic Escherichia coli (EHEC) O157:H7 and its virulence factors in a single reaction. The screening was carried out monthly from October 2004 to September 2005. Direct PCR analysis using total DNA from sewage concentrate showed the presence of at least one virulence gene in 100% samples ( n = 12). Sixty six percent of these samples were also positive for rfbE (O157) gene and fliC (H7) gene. The PCR amplification of these genes was possible when the concentration was above 20 cells ml −1. From the multiplex PCR of the isolates following plating on Cefixime-Tellurite Sorbitol MacConkey (CT-SMAC) agar to detect non-sorbitol fermenting (NSF) colonies ( n = 600), one E. coli strain carrying slt-II gene and two strains of E. coli O157:H7 carrying slt-I were detected. The results show that municipal sewage represents a potential reservoir of EHEC. CT-SMAC agar was proved to have limited E. coli O157:H7 selectivity and only 0.005% (3/600) sensitivity for sewage samples due to the high frequency (43%) of NSF strains in sewage. The enrichment of sewage sample in modified E. coli broth (mEC) increased the sensitivity of PCR resulting in the clearer amplification of five genes. Amplification of target cell type in mEC broth implied that EHEC were present in sewage in a culturable and hence potentially infectious state. However, pre-enrichment did not affect the selectivity of CT-SMAC because frequency of NSF colonies remained the same as that obtained without enrichment. The study, therefore, underscores the need for more sensitive screening techniques that can be routinely employed for the regular monitoring of sewage influent.

Evaluation of Immunomagnetic Separation and PCR for the Detection of Escherichia coli O157 in Animal Feces and Meats

Series of animal feces and meat samples artificially contaminated with strains of Escherichia coli O157 isolated from different sources were tested by both an immunomagnetic separation (IMS)–based method and a PCR method using primers specific for a portion of the rfbE gene of E. coli O157. IMS is laborious and time consuming but ends up with the isolation of the pathogen. PCR is fast and less laborious, but it can only be used for screening purposes, so a further culture step is required to isolate the organism. For both fecal and meat samples, the IMS method was found to be more sensitive than the PCR. Furthermore, the detection efficiency of the PCR was influenced by the origin of the fecal sample and the type of meat. For sheep feces, the efficiency of the PCR appeared to be systematically lower than for cattle feces. And the efficiency of the PCR in detecting E. coli O157 in spiked samples of raw minced beef and dry-fermented sausages was systematically lower than in samples of filet americain. Based on this study, it can be concluded that both for animal feces and meat, IMS can be used more successfully to detect E. coli O157 than PCR, because IMS showed to be more sensitive and the outcome was not influenced by the type of animal feces or meat.

Real-time reverse transcription-multiplex PCR for simultaneous and specific detection of rfbE and eae genes of Escherichia coli O157:H7

A real-time reverse transcription multiplex polymerase chain reaction (rRT-MPCR) was developed for detection of mRNA encoded by rfbE and eae genes of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A 129-bp and a 106-bp sequence specific to rfbE and eae, respectively, were targeted for reverse transcription, amplification, and real-time detection. A single-step RT-PCR kit containing a mixture of reverse transcriptases converted mRNA into cDNA, which was subsequently amplified by Taq polymerase included in the same kit. The real-time detection of amplification products was achieved by incorporating rfbE(O157)- and eae(O157:H7)-specific TaqMan probes in rRT-MPCR. The ability of two sets of primers and probes for specific detection of rfbE(O157) and eae(O157:H7) was initially verified by screening RNA of eight E. coli serotypes possessing different O antigens and eae alleles. These two sets of primers and probes were also tested in a standard real-time PCR (rPCR) using DNA prepared from several E. coli and non-E. coli strains to verify that only rfbE(O157)- and eae(O157:H7)-specific sequences were amplified and detected. The rRT-MPCR was then evaluated for detecting low-level contamination of EHEC O157:H7 in feces. When RNA prepared from bovine feces, which were artificially seeded with EHEC O157:H7 cells and cultured for five hours, was tested in rRT-MPCR as low as 1cfu/g of feces could be detected. The detection range for the two genes in fecal cultures was 5.1 x 10(-1)-5.1 x 10(4) cfu/g of feces. Thus, the described procedure could be applied to rapid detection of very low levels of EHEC O157:H7 using total RNA as a template. Since the presence of rfbE(O157)- and eae(O157:H7)-specific mRNA is dependent on replicating cells, rRT-MPCR could provide important information about the viability of EHEC O157:H7 in feces.

Detection of Escherichia coli O157 using equal-length double-stranded fluorescence probe in a real-time polymerase chain reaction assay

Background Enterohemorrhagic Escherichia coli ( E. coli) O157 is a dangerous pathogen, which causes bloody diarrhea and severe hemolytic uremic syndrome (HUS). Although several assay systems based on real-time polymerase chain reaction (PCR) have been integrated to detect this pathogen, most of them are not specific. We report a real-time quantitative PCR method targeting rfbE, a gene specifically expressed in E. coli O157. This method can therefore be used to diagnose enterohemorrhagic Escherichia coli ( E. coli) O157. Methods A nucleic acid based diagnostic assay system, combining equal-length double-stranded fluorescence probe technique and real-time PCR, was developed to detect E. coli O157. This assay system take advantage of the highly conserved rfbE O-antigen synthesis gene, and a pair of fluorescence-quenching probes complementary to rfbE gene were used in a real-time PCR to quantify the presence of the pathogen. Results The specificity of the diagnostic method was assessed by comparing test results on 14 different related pathogens including common E. coli, enteroinvasive Escherichia coli (EIEC), Salmonella, Shigella and E. coli O157. The detection limit of the method was determined using 10-fold serial dilutions of an E. coli O157 standard sample, and as few as 1.49 × 10 3 CFU/ml could be detected. All E. coli with serotype O157, which expresses rfbE gene, were positive in this assay, while all other species without rfbE gene expression were negative. Conclusions By combining equal-length double-stranded fluorescence probe technique and real-time PCR, we have developed a simple, rapid, specific and sensitive method to detect E. coli O157.

A reverse transcriptase-polymerase chain reaction assay for detection of viable Escherichia coli O157:H7: investigation of specific target genes.

To determine suitable target genes for detection of the pathogen Escherichia coli O157:H7 by reverse transcriptase-polymerase chain reaction (RT-PCR). Potential genes used as indicators for viability included rfbE, fliC, stx1, stx2, mobA, eaeA, hly and 16S rRNA. Under normal growth conditions, rfbE, stx1, hly and 16S rRNA amplicons were detected in association with all growth phases. The products of 16S rRNA, mobA, rfbE and stx1 were readily visualized in RNA isolated from viable but non-culturable cells. The 16S rRNA gene was not amplified following heat treatment of cells at 121 degrees C for 15 min and mRNA targets were not amplified after treatment at 60 degrees C for 20 min. In this instance, genes that are not amplified are good targets for determining viability. The results of RT-PCR amplification indicate that, under the conditions examined, the rfbE gene is the most appropriate target for detection of viable E. coli O157:H7. Prior to detection or identification from an environmental or food sample E. coli O157:H7 may be exposed to many harsh conditions that influence nucleic acid (RNA and DNA) stability. This study gives an insight into the effects of temperature and nutrient deprivation on identification of viable cells using RT-PCR. It also suggests that, if RT-PCR is to be used for detection of live cells in a sample without enrichment, 10(7) cfu of the target organism are required.

Use of Real-Time Polymerase Chain Reaction and Molecular Beacons for the Detection of Escherichia coli O157:H7

Molecular beacons (MBs) are oligonucleotide probes that fluoresce upon hybridization. In this paper, we described the development of a real-time PCR assay to detect the presence of Escherichia coli O157:H7 using these fluorogenic reporter molecules. MBs were designed to recognize a 26-bp region of the rfbE gene, coding for an enzyme necessary for O-antigen biosynthesis. The specificity of the MB-based PCR assay was evaluated using various enterohemorrhagic (EHEC) and Shiga-like toxin-producing (STEC) E. coli strains as well as bacteria species that cross-react with the O157 antisera. All E. coli serotype O157 tested was positively identified while all other species, including the closely related O55 were not detected by the assay. Positive detection of E. coli O157:H7 was demonstrated when >10 2 CFU/ml was present in the samples. The capability of the assay to detect E. coli O157:H7 in raw milk and apple juice was demonstrated. As few as 1 CFU/ml was detected after 6 h of enrichment. These assays could be carried out entirely in sealed PCR tubes, enabling rapid and semiautomated detection of E. coli O157:H7 in food and environmental samples.

Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR.

Cattle are considered the major reservoir for Escherichia coli O157:H7, one of the newly emerged foodborne human pathogens of animal origin and a leading cause of haemorrhagic colitis in humans. A sensitive test that can accurately and rapidly detect the organism in the food animal production environment is critically needed to monitor the emergence, transmission, and colonization of this pathogen in the animal reservoir. In this study, a novel multiplex polymerase chain reaction (PCR) assay was developed by using 5 sets of primers that specifically amplify segments of the eaeA, slt-I, slt-II, fliC, rfbE genes, which allowed simultaneous identification of serotype O157:H7 and its virulence factors in a single reaction. Analysis of 82 E. coli strains (49 O157:H7 and 33 non-O157:H7) demonstrated that this PCR system successfully distinguished serotype O157:H7 from other serotypes of E. coli and provided accurate profiling of the shiga-like toxins and the intimin adhesin in individual strains. This multiplex PCR assay did not cross-react with the background bacterial flora in bovine faeces and could detect a single O157:H7 organism per gram of faeces when combined with an enrichment step. Together, these results indicate that the multiplex PCR assay can be used for specific identification and profiling of E. coli O157:H7 isolates, and may be applied to rapid and sensitive detection of E. coli O157:H7 in bovine faeces when combined with an enrichment step.

ＰＣＲ法による大腸菌Ｏ１５７およびＨ７抗原遺伝子の検出

A PCR method was developed for the simultaneous detection of Escherichia coli O157 and H7 antigens. Two PCR primer pairs for amplification of both E. coli O157 rfbE and H7 fliC genes, which are necessary for the expression of the O157 and H7 antigen respectively, were performed for the detection of E. coli O157: H7. All Shiga toxin-producing E. coli (STEC) O157: H7 and STEC O157: NM strains were positive for both E. coli O157 rfbE and H7 fliC genes.Non-STEC O157 strains were positive only for E. coli O157 rfbE genes and H7 strains except O157 were positive only for H7 fliC genes . Some of the nonmotile strains were positive for H7 fliC genes. No cross-reaction was observed with other E. coli serotypes (except O157 and H7) and other bacterial species, like Salmonella O30 and Citrobacter freundii which react with E. coli O157 antiserum. It is recommended that PCR amplification of both E. coli O157 rfbE and H7 fliC genes is one of the most specific methods for E. coli O157: H7 identification.

Detection of verotoxin-producing Escherichia coli O157:H7 by multiplex polymerase chain reaction.

We constructed primers for multiplex polymerase chain reaction (PCR) to detect verotoxin-producing Escherichia coli (VTEC) O157:H7. The multiplex PCR primers were designed from the sequence of the flagellin structural gene of Escherichia coli flagellar type H7 (GenBank under accession number L07388), and from the sequence of the rfbE gene of Escherichia coli O157:H7 (GenBank under accession number S83460). In addition to these primers, we used a primer pair reported by Karch and Meyer (J. Clin. Microbiol. 27: 2751-2757, 1989) to amplify various VT genes from VTEC. All of the examined specimens (18 isolates) of VT-producing E. coli O157:H7 showed a positive result by the multiplex PCR test with the three sets of primers. The sensitivity of detection for VT-producing E. coli O157:H7 was shown to be at least 3,000 cells per PCR tube.

Identification of Escherichia coli O157 antigen by polymerase chain reaction

An assay was developed for the specific detection of Escherichia coli O157 using PCR, because O serological cross-reactivities have been reported between E. coli O157 and some E. coli, other bacterial species. PCR amplification of E. coli O157 rfbE (Ec O157:H7) gene that is necessary for the expression of the O157 antigen, was performed for the identification of E. coli O157. All Shiga toxin-producing Escherichia coli (STEC) O157:H7 and O157:H, non-STEC O157 strains were positive, and other non-O157 E. coli strains were negative by PCR. All tested strains of other bacterial species, like Salmonella O30 and Citrobacter freundii which gave positive results with O157 detection kits, were negative by PCR. It is recommended that PCR amplification of O157 rfbE gene is one of the most specific method for E. coli O157 identification.

Genetic organisation and evolution of Yersinia pseudotuberculosis 3,6-dideoxyhexose biosynthetic genes

3,6-dideoxyhexose (DDH) sugars occur in some of the O antigens of Salmonella enterica and Yersinia pseudotuberculosis, but are otherwise rarely found in nature. Y. pseudotuberculosis DDH biosynthetic genes rfbS (encoding CDP-paratose synthetase) and rfbE (encoding CDP-tyvelose epimerase) were amplified and cloned, and their sequences determined. Comparisons with the equivalent genes of S. enterica show that the genetic arrangement of DDH genes is very similar; however, in Y. pseudotuberculosis there is no suggestion that paratose producing strains are derived from tyvelose-producing strains by inactivation of rfbE, which is the case in S. enterica. The previously determined DNA sequence of the rfb region of an abequose-producing strain was re-examined. It contains the remnants of an insertion sequence (IS) adjacent to a truncated and non-functional rfbE gene. This suggests that the IS was involved in recombination events contributing to O-antigen antigenic diversity in Y. pseudotuberculosis.

Occurrence of Escherichia coli in Wildlife from Different Habitats of Sarawak, Malaysia

This study was carried out to assess the occurrence of Escherichia coli (E. coli) in the bats, birds and rodents asrepresentative of wildlife from different habitats in Sibu and Kapit, Sarawak, Malaysia. A total of 682 swabsamples were collected from wildlife hosts and screened for the bacteria E. coli and E. coli O157:H7 usingstandard microbiological methods and molecular techniques. The overall occurrence rates of E. coli among thesehosts were 14%, 17% and 54% for bats, birds and rodents, respectively. The occurrence of E. coli was thehighest in rodents regardless of the habitats. Isolated E. coli were then screened for E. coli O157:H7 by using amultiplex PCR with four primer pairs targeting for Shiga toxin producing genes (slt-I and slt-II), and the genesinvolved in biosynthesis of O157 antigen (rfbE) and H7 antigen (fliCH7 ). slt-I, slt-II and rfbE genes were notdetected in any of the E. coli isolates. However, the gene encoding for H7 antigen was detected in 23 E. coliisolates. This indicated that E. coli O157:H7 strain was not detected in the wildlife studied. Absence of E. coliO157:H7 in the wildlife studied indicated these wild animals do not serve as an important reservoir of E. coliO157:H7. However, precautions have to be taken as other group of pathogenic E. coli may pose a zoonotic riskfor humans and other animals.