Virulence Genes and Molecular Typing of Different Groups of Escherichia coli O157 Strains in Cattle

Characterization of an Escherichia coli O157 strain collection (n = 42) derived from healthy Hungarian cattle revealed the existence of diverse pathotypes. Enteropathogenic E. coli (EPEC; eae positive) appeared to be the most frequent pathotype (n = 22 strains), 11 O157 strains were typical enterohemorrhagic E. coli (EHEC; stx and eae positive), and 9 O157 strains were atypical, with none of the key stx and eae virulence genes detected. EHEC and EPEC O157 strains all carried eae-gamma, tir-gamma, tccP, and paa. Other virulence genes located on the pO157 virulence plasmid and different O islands (O island 43 [OI-43] and OI-122), as well as espJ and espM, also characterized the EPEC and EHEC O157 strains with similar frequencies. However, none of these virulence genes were detected by PCR in atypical O157 strains. Interestingly, five of nine atypical O157 strains produced cytolethal distending toxin V (CDT-V) and carried genes encoding long polar fimbriae. Macro-restriction fragment enzyme analysis (pulsed-field gel electrophoresis) revealed that these E. coli O157 strains belong to four main clusters. Multilocus sequence typing analysis revealed that five housekeeping genes were identical in EHEC and EPEC O157 strains but were different in the atypical O157 strains. These results suggest that the Hungarian bovine E. coli O157 strains represent at least two main clones: EHEC/EPEC O157:H7/NM (nonmotile) and atypical CDT-V-producing O157 strains with H antigens different from H7. The CDT-V-producing O157 strains represent a novel genogroup. The pathogenic potential of these strains remains to be elucidated.Escherichia coli O157:H7 is a food- and waterborne zoonotic pathogen with serious effects on public health. E. coli O157:H7 causes diseases in humans ranging from uncomplicated diarrhea to hemorrhagic colitis and hemolytic-uremic syndrome (HUS) (30). Typically, enterohemorrhagic E. coli (EHEC) strains express two groups of important virulence factors: one or more Shiga toxins (Stx; also called verotoxins), encoded by lambda-like bacteriophages, and a pathogenicity island called the locus of enterocyte effacement (LEE) encoding all the proteins necessary for attaching and effacing lesions of epithelial cells (41). Comparative genomic studies of E. coli O157:H7 strains revealed extensive genomic diversity related to the structures, positions, and genetic contents of bacteriophages and the variability of putative virulence genes encoding non-LEE effector proteins (29, 43).Ruminants and, in particular, healthy cattle are the major reservoir of E. coli O157:H7, although the prevalence of O157:H7 strains in cattle may vary widely, as reviewed by Caprioli et al. (12). E. coli O157:H7 has been found to persist and remain infective in the environment for a long time, e.g., for at least 6 months in water trough sediments, which may be an important environmental niche.In Hungary, infections with E. coli O157 and other Shiga toxin-producing E. coli (STEC) strains in humans in cases of “enteritidis infectiosa” have been notifiable since 1998 on a case report basis. Up to now, the disease has been sporadic, and fewer than 100 (n = 83) cases of STEC infection among 2,700 suspect cases have been reported since 2001. However, until the present study, no systematic, representative survey of possible animal sources had been performed.In this study, our aim was to investigate healthy cattle in Hungary for the presence of strains of E. coli O157 and the genes encoding Shiga toxins (stx₁ and stx₂) and intimin (eae) and a wide range of putative virulence genes found in these strains. In addition, the phage type (PT) was determined, and pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to further compare the strains at the molecular level. Shiga toxin and cytolethal distending toxin (CDT) production was also examined, and phage induction experiments were conducted. The high incidence of enteropathogenic E. coli (EPEC; eae-positive) O157:H7 strains and atypical (eae- and stx-negative) O157 strains indicates that cattle are a major reservoir of not only EHEC O157 but also EPEC O157 and atypical E. coli O157 strains. These atypical, non-sorbitol-fermenting O157 strains frequently produced CDT-V and may represent a novel O157 clade as demonstrated by MLST and PFGE.     

Occurrence of Diarrheagenic Virulence Genes and Genetic Diversity in Escherichia coli Isolates from Fecal Material of Various Avian Hosts in British Columbia,Canada

Contamination of surface water by fecal microorganisms originating from human and nonhuman sources is a public health concern. In the present study, Escherichia coli isolates (n = 412) from the feces of various avian host sources were screened for various virulence genes: stx₁ and stx₂ (Shiga toxin-producing E. coli [STEC]), eae (enteropathogenic E. coli [EPEC]), est-h, est-p, and elt (encoding heat-stable toxin [ST] variants STh and STp and heat-labile toxin [LT], respectively) (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). None of the isolates were found to be positive for stx₁, while 23% (n = 93) were positive for only stx₂, representing STEC, and 15% (n = 63) were positive for only eae, representing EPEC. In addition, five strains obtained from pheasant were positive for both stx₂ and eae and were confirmed as non-O157 by using an E. coli O157 rfb (rfb_O157) TaqMan assay. Isolates positive for the virulence genes associated with ETEC and EIEC were not detected in any of the hosts. The repetitive element palindromic PCR (rep-PCR) fingerprint analysis identified 143 unique fingerprints, with an overall Shannon diversity index of 2.36. Multivariate analysis of variance (MANOVA) showed that the majority of the STEC and EPEC isolates were genotypically distinct from nonpathogenic E. coli and clustered independently. MANOVA analysis also revealed spatial variation among the E. coli isolates, since the majority of the isolates clustered according to the sampling locations. Although the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potentially pathogenic STEC and EPEC strains can be found in some of the avian hosts studied and may contaminate surface water and potentially impact human health.     

Estimating the Prevalence of Potential Enteropathogenic Escherichia coli and Intimin Gene Diversity in a Human Community by Monitoring Sanitary Sewage

Presently, the understanding of bacterial enteric diseases in the community and their virulence factors relies almost exclusively on clinical disease reporting and examination of clinical pathogen isolates. This study aimed to investigate the feasibility of an alternative approach that monitors potential enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) prevalence and intimin gene (eae) diversity in a community by directly quantifying and characterizing target virulence genes in the sanitary sewage. The quantitative PCR (qPCR) quantification of the eae, stx₁, and stx₂ genes in sanitary sewage samples collected over a 13-month period detected eae in all 13 monthly sewage samples at significantly higher abundance (93 to 7,240 calibrator cell equivalents [CCE]/100 ml) than stx₁ and stx₂, which were detected sporadically. The prevalence level of potential EPEC in the sanitary sewage was estimated by calculating the ratio of eae to uidA, which averaged 1.0% (σ = 0.4%) over the 13-month period. Cloning and sequencing of the eae gene directly from the sewage samples covered the majority of the eae diversity in the sewage and detected 17 unique eae alleles belonging to 14 subtypes. Among them, eae-β2 was identified to be the most prevalent subtype in the sewage, with the highest detection frequency in the clone libraries (41.2%) and within the different sampling months (85.7%). Additionally, sewage and environmental E. coli isolates were also obtained and used to determine the detection frequencies of the virulence genes as well as eae genetic diversity for comparison.     

Prevalence of Diarrhea-Associated Virulence Genes and Genetic Diversity in Escherichia coli Isolates from Fecal Material of Various Animal Hosts

In order to assess the health risk associated with a given source of fecal contamination using bacterial source tracking (BST), it is important to know the occurrence of potential pathogens as a function of host. Escherichia coli isolates (n = 593) from the feces of diverse animals were screened for various virulence genes: stx₁ and stx₂ (Shiga toxin-producing E. coli [STEC]), eae and EAF (enteropathogenic E. coli [EPEC]), STh, STp, and LT (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). Eleven hosts were positive for only the eae (10.11%) gene, representing atypical EPEC, while two hosts were positive for both eae and EAF (1.3%), representing typical EPEC. stx₁, stx₂, or both stx₁ and stx₂ were present in 1 (0.1%,) 10 (5.56%), and 2 (1.51%) hosts, respectively, and confirmed as non-O157 by using a E. coli O157 rfb (rfb_O157) TaqMan assay. STh and STp were carried by 2 hosts (2.33%) and 1 host (0.33%), respectively, while none of the hosts were positive for LT and ipaH. The repetitive element palindromic PCR (rep-PCR) fingerprint analysis identified 221 unique fingerprints with a Shannon diversity index of 2.67. Multivariate analysis of variance revealed that majority of the isolates clustered according to the year of sampling. The higher prevalence of atypical EPEC and non-O157 STEC observed in different animal hosts indicates that they can be a reservoir of these pathogens with the potential to contaminate surface water and impact human health. Therefore, we suggest that E. coli from these sources must be included while constructing known source fingerprint libraries for tracking purposes. However, the observed genetic diversity and temporal variation need to be considered since these factors can influence the accuracy of BST results.     

Pathogenic Potential,Genetic Diversity,and Population Structure of Escherichia coli Strains Isolated from a Forest-Dominated Watershed (Comox Lake) in British Columbia,Canada

Escherichia coli isolates (n = 658) obtained from drinking water intakes of Comox Lake (2011 to 2013) were screened for the following virulence genes (VGs): stx₁ and stx₂ (Shiga toxin-producing E. coli [STEC]), eae and the adherence factor (EAF) gene (enteropathogenic E. coli [EPEC]), heat-stable (ST) enterotoxin (variants STh and STp) and heat-labile enterotoxin (LT) genes (enterotoxigenic E. coli [ETEC]), and ipaH (enteroinvasive E. coli [EIEC]). The only genes detected were eae and stx₂, which were carried by 37.69% (n = 248) of the isolates. Only eae was harbored by 26.74% (n = 176) of the isolates, representing potential atypical EPEC strains, while only stx₂ was detected in 10.33% (n = 68) of the isolates, indicating potential STEC strains. Moreover, four isolates were positive for both the stx₂ and eae genes, representing potential EHEC strains. The prevalence of VGs (eae or stx₂) was significantly (P < 0.0001) higher in the fall season, and multiple genes (eae plus stx₂) were detected only in fall. Repetitive element palindromic PCR (rep-PCR) fingerprint analysis of 658 E. coli isolates identified 335 unique fingerprints, with an overall Shannon diversity (H′) index of 3.653. Diversity varied among seasons over the years, with relatively higher diversity during fall. Multivariate analysis of variance (MANOVA) revealed that the majority of the fingerprints showed a tendency to cluster according to year, season, and month. Taken together, the results indicated that the diversity and population structure of E. coli fluctuate on a temporal scale, reflecting the presence of diverse host sources and their behavior over time in the watershed. Furthermore, the occurrence of potentially pathogenic E. coli strains in the drinking water intakes highlights the risk to human health associated with direct and indirect consumption of untreated surface water.     

Bovine Feces from Animals with Gastrointestinal Infections Are a Source of Serologically Diverse Atypical Enteropathogenic Escherichia coli and Shiga Toxin-Producing E. coli Strains That Commonly Possess Intimin

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) cells were isolated from 191 fecal samples from cattle with gastrointestinal infections (diagnostic samples) collected in New South Wales, Australia. By using a multiplex PCR, E. coli cells possessing combinations of stx₁, stx₂, eae, and ehxA were detected by a combination of direct culture and enrichment in E. coli (EC) (modified) broth followed by plating on vancomycin-cefixime-cefsulodin blood (BVCC) agar for the presence of enterohemolytic colonies and on sorbitol MacConkey agar for the presence of non-sorbitol-fermenting colonies. The high prevalence of the intimin gene eae was a feature of the STEC (35 [29.2%] of 120 isolates) and contrasted with the low prevalence (9 [0.5%] of 1,692 fecal samples possessed STEC with eae) of this gene among STEC recovered during extensive sampling of feces from healthy slaughter-age cattle in Australia (M. Hornitzky, B. A. Vanselow, K. Walker, K. A. Bettelheim, B. Corney, P. Gill, G. Bailey, and S. P. Djordjevic, Appl. Environ. Microbiol. 68:6439-6445, 2002). Forty-seven STEC serotypes were identified, including O5:H−, O8:H19, O26:H−, O26:H11, O113:H21, O157:H7, O157:H− and Ont:H− which are known to cause severe disease in humans and 23 previously unreported STEC serotypes. Serotypes Ont:H− and O113:H21 represented the two most frequently isolated STEC isolates and were cultured from nine (4.7%) and seven (3.7%) animals, respectively. Fifteen eae-positive E. coli serotypes, considered to represent atypical EPEC, were identified, with O111:H− representing the most prevalent. Using both techniques, STEC cells were cultured from 69 (36.1%) samples and EPEC cells were cultured from 30 (15.7%) samples, including 9 (4.7%) samples which yielded both STEC and EPEC. Culture on BVCC agar following enrichment in EC (modified) broth was the most successful method for the isolation of STEC (24.1% of samples), and direct culture on BVCC agar was the most successful method for the isolation of EPEC (14.1% samples). These studies show that diarrheagenic calves and cattle represent important reservoirs of eae-positive E. coli.     

Cloning and characterization of the eae gene of enterohaemorrhagic Escherichia coli O157:H7

The eae (Escherichia coli attaching and effacing) gene from enteropathogenic Escherichia coli (EPEC) was previously shown to be essential for production of the 'attaching and effacing' histopathology characteristic of EPEC infections (Jerse et al., 1990). We have now cloned the eae gene from enterohaemorrhagic E. coli (EHEC) which, in addition to producing Shiga-like cytotoxins, also produces the attaching and effacing effect. The sequence homology between the EPEC and EHEC sequences was 86% and 83% at the nucleotide and amino acid levels, respectively. The predicted amino acid sequence of the EHEC eae gene shared 31% identity and 51% similarity with invasin of Yersinia pseudotuberculosis. Alignment of the EPEC and EHEC Eae proteins and the Y. pseudotuberculosis and Y. enterocolitica invasins shows striking regions of identity with the greatest divergence at the C-terminal end, the putative receptor-binding portion of invasin.     

Relationship between Phylogenetic Groups, Genotypic Clusters, and Virulence Gene Profiles of Escherichia coli Strains from Diverse Human and Animal Sources

Escherichia coli strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with E. coli strains present in various animal hosts are not well known. In this study, E. coli strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing E. coli (STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the stx_1c and/or stx_2d, ehxA, and saa genes and belonged to E. coli phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked eae. In contrast, 26 (1.7%) of the E. coli isolates examined were found to be potential enteropathogenic E. coli (EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among E. coli strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC ehxA-positive E. coli strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic E. coli strains. Our results revealed that potential human health hazards associated with pathogenic E. coli strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.     

Phylogenetic group distribution and prevalence of virulence genes in <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from food samples in South Korea

We analyzed the distribution of phylogenetic groups of foodborne Escherichia coli isolates. We also investigated the prevalence of virulence-associated genes of diarrheagenic E. coli. In total, 162 E. coli isolated from foods (raw meat, fish, and processed foods) were collected in Korea. Approximately 90% of the foodborne isolates belonged to phylogenetic groups A and B1, whereas 1.2% were allocated to group B2, and 9.3% to D. Multiplex polymerase chain reaction (PCR) assays were used to detect the following: stx ₁ and stx ₂ to identify Shiga toxin-producing E. coli (STEC), eae and bfpA to identify enteropathogenic E. coli (EPEC), ipaH for enteroinvasive E. coli, CVD432 for enteroaggregative E. coli, and lt and st for enterotoxigenic E. coli (ETEC). The presence of daaD in diffusely adherent E. coli was examined by singleplex PCR. Of the 162 foodborne E. coli isolates, three (1.9%) were confirmed to be pathogenic E. coli: STEC, ETEC, and atypical EPEC based on their possession of stx ₁, st, and eae, and the pathogenic strains were isolated in beef, rockfish, and pork, respectively. Molecular typing was conducted by multilocus sequence typing to investigate the genetic relationships among the pathogenic strains. All isolates positive for virulence genes had different mulilocus sequence typing profiles representing different sequence types (ST) of ST101, ST1815, and ST1820. These results indicate that some food samples were contaminated with pathogenic E. coli.     

Prevalence of Virulence Genes Associated with Pathogenic Escherichia coli Strains Isolated from Domestically Harvested Rainwater during Low- and High-Rainfall Periods

The possible health risks associated with the consumption of harvested rainwater remains one of the major obstacles hampering its large-scale implementation in water limited countries such as South Africa. Rainwater tank samples collected on eight occasions during the low- and high-rainfall periods (March to August 2012) in Kleinmond, South Africa, were monitored for the presence of virulence genes associated with Escherichia coli. The identity of presumptive E. coli isolates in rainwater samples collected from 10 domestic rainwater harvesting (DRWH) tanks throughout the sampling period was confirmed through universal 16S rRNA PCR with subsequent sequencing and phylogenetic analysis. Species-specific primers were also used to routinely screen for the virulent genes, aggR, stx, eae, and ipaH found in enteroaggregative E. coli (EAEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroinvasive E. coli, respectively, in the rainwater samples. Of the 92 E. coli strains isolated from the rainwater using culture based techniques, 6% were presumptively positively identified as E. coli O157:H7 using 16S rRNA. Furthermore, virulent pathogenic E. coli genes were detected in 3% (EPEC and EHEC) and 16% (EAEC) of the 80 rainwater samples collected during the sampling period from the 10 DRWH tanks. This study thus contributes valuable information to the limited data available regarding the ongoing prevalence of virulent pathotypes of E. coli in harvested rainwater during a longitudinal study in a high-population-density, periurban setting.     

Prevalence and Characteristics of Enteropathogenic Escherichia coli with the eae Gene in Diarrhoeic Rabbits

A field study was carried out with the objective of investigating the prevalence of enteropathogenic Escherichia coli (EPEC) with the eae gene in diarrhoeic rabbits. EPEC eae⁺ were isolated from 60 (74%) of 81 diarrhoeic rabbits sampled in 30 industrial fattening farms localized in the four provinces of Galicia (northwestern Spain). Attaching and effacing lesions were found in 44 of 50 animals processed for histology. The 111 E. coli strains identified belonged to 19 different O serogroups and 13 biotypes. However, 53 (48%) of the strains belonged to serogroup O103 and 36 (32%) showed the serobiotype O103:B14. The eae gene was significantly more frequent (100%; 47 of 47) among the highly pathogenic rhamnose-negative strains of serobiotypes O103:B6 and O103:B14 than among the E. coli strains belonging to other serobiotypes (36%; 23 of 64) (P < 0.001). In this first report about the prevalence of EPEC with the eae gene in rabbits, we conclude that the class of E. coli strains observed is a common cause of diarrhoea in Galician rabbit farms, and that highly pathogenic rhamnose-negative strains of serotype O103:K-:H2 and biotype B14 are specially predominant.     

Serotypes,intimin variants and other virulence factors of <Emphasis Type="Italic">eae</Emphasis> positive <Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae-η2)

Background  

Enteropathogenic Escherichia coli (EPEC) and Shigatoxin-producing Escherichia coli (STEC) share the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains.     

Attaching and Effacing Escherichia coli Downregulate DNA Mismatch Repair Protein In Vitro and Are Associated with Colorectal Adenocarcinomas in Humans

Background

Mucosa-associated Escherichia coli are frequently found in the colonic mucosa of patients with colorectal adenocarcinoma, but rarely in healthy controls. Chronic mucosal E. coli infection has therefore been linked to colonic tumourigenesis. E. coli strains carrying eae (encoding the bacterial adhesion protein intimin) attach intimately to the intestinal mucosa and are classed as attaching and effacing E. coli (AEEC). Enteropathogenic Escherichia coli (EPEC) are the most common form of AEEC identified in man. EPEC utilise a type III secretion system to translocate effector proteins into host cells and infection induces wide-ranging effects on the host cell proteome. We hypothesised that EPEC infection could influence molecular pathways involved in colorectal tumourigenesis.

Methodology/Principal Findings

When co-cultured with human colorectal cell lines, EPEC dramatically downregulated the expression of key DNA mismatch repair proteins MSH2 and MLH1 in an attachment specific manner. Cytochrome c staining and TUNEL analysis confirmed that this effect was not a consequence of apoptosis/necrosis. Ex vivo human colonic mucosa was co-cultured with EPEC and probed by immunofluorescence to locate adherent bacteria. EPEC entered 10% of colonic crypts and adhered to crypt epithelial cells, often in the proliferative compartment. Adenocarcinoma and normal colonic mucosa from colorectal cancer patients (n = 20) was probed by immunofluorescence and PCR for AEEC. Mucosa-associated E. coli were found on 10/20 (50%) adenocarcinomas and 3/20 (15%) normal mucosa samples (P<0.05). AEEC were detected on 5/20 (25%) adenocarcinomas, but not normal mucosa samples (P<0.05).

Significance/Conclusions

The ability of EPEC to downregulate DNA mismatch repair proteins represents a novel gene-environment interaction that could increase the susceptibility of colonic epithelial cells to mutations and therefore promote colonic tumourigenesis. The potential role of AEEC in colorectal tumourigenesis warrants further investigation.     

Transduction of Porcine Enteropathogenic Escherichia coli with a Derivative of a Shiga Toxin 2-Encoding Bacteriophage in a Porcine Ligated Ileal Loop System

In this study, we have investigated the ability of detoxified Shiga toxin (Stx)-converting bacteriophages Φ3538 (Δstx₂::cat) (H. Schmidt et al., Appl. Environ. Microbiol. 65:3855-3861, 1999) and H-19B::Tn10d-bla (D. W. Acheson et al., Infect. Immun. 66:4496-4498, 1998) to lysogenize enteropathogenic Escherichia coli (EPEC) strains in vivo. We were able to transduce the porcine EPEC strain 1390 (O45) with Φ3538 (Δstx₂::cat) in porcine ligated ileal loops but not the human EPEC prototype strain E2348/69 (O127). Neither strain 1390 nor strain E2348/69 was lysogenized under these in vivo conditions when E. coli K-12 containing H-19B::Tn10d-bla was used as the stx1 phage donor. The repeated success in the in vivo transduction of an Stx2-encoding phage to a porcine EPEC strain in pig loops was in contrast to failures in the in vitro trials with these and other EPEC strains. These results indicate that in vivo conditions are more effective for transduction of Stx2-encoding phages than in vitro conditions.     

Serotypes and virutypes of enteropathogenic and enterohaemorrhagic Escherichia coli strains from stool samples of children with diarrhoea in Germany

Aims: To investigate the prevalence of traditional and emerging types of enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) strains in stool samples from children with diarrhoea and to characterize their virulence genes involved in the attaching and effacing (A/E) phenotype. Methods and Results: Serological and PCR‐based methods were used for detection and isolation of EPEC and EHEC strains from 861 stool samples from diarrhoeic children. Agglutination with traditional EPEC and EHEC O‐group‐specific antisera resulted in detection of 38 strains; 26 of these carried virulence factors of EPEC or EHEC. PCR screening for the eae gene resulted in isolation of 97 strains, five carried genes encoding Shiga toxins (stx), one carried the bfpA gene and 91 were atypical EPEC. The 97 EPEC and EHEC strains were divided into 36 O‐serogroups and 21 H‐types, only nine strains belonged to the traditional EPEC O‐groups O26, O55, O86 and O128. In contrast, EPEC serotypes O28:H28, O51:H49, O115:H38 and O127:H40 were found in multiple cases. Subtyping the virulence factors intimin, Tir and Tir‐cytoskeleton coupling effector protein (TccP)/TccP2 resulted in further classification of 93·8% of the 97 strains. Conclusions: Our findings show a clear advantage of the eae‐PCR over the serological detection method for identification of EPEC and EHEC strains from human patients. Significance and Impact of the Study: Molecular detection by the eae‐PCR followed by serotyping and virutyping is useful for monitoring trends in EPEC and EHEC infections and to discover their possible reservoirs.     

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).     

Temporal Shedding Patterns and Virulence Factors of Escherichia coli Serogroups O26, O103, O111, O145, and O157 in a Cohort of Beef Calves and Their Dams

This study investigated the shedding of Escherichia coli O26, O103, O111, O145, and O157 in a cohort of beef calves from birth over a 5-month period and assessed the relationship between shedding in calves and shedding in their dams, the relationship between shedding and scouring in calves, and the effect of housing on shedding in calves. Fecal samples were tested by immunomagnetic separation and by PCR and DNA hybridization assays. E. coli O26 was shed by 94% of calves. Over 90% of E. coli O26 isolates carried the vtx₁, eae, and ehl genes, 6.5% carried vtx₁ and vtx₂, and one isolate carried vtx₂ only. Serogroup O26 isolates comprised seven pulsed-field gel electrophoresis (PFGE) patterns but were dominated by one pattern which represented 85.7% of isolates. E. coli O103 was shed by 51% of calves. Forty-eight percent of E. coli O103 isolates carried eae and ehl, 2% carried vtx₂, and none carried vtx₁. Serogroup O103 isolates comprised 10 PFGE patterns and were dominated by two patterns representing 62.5% of isolates. Shedding of E. coli O145 and O157 was rare. All serogroup O145 isolates carried eae, but none carried vtx₁ or vtx₂. All but one serogroup O157 isolate carried vtx₂, eae, and ehl. E. coli O111 was not detected. In most calves, the temporal pattern of E. coli O26 and O103 shedding was random. E. coli O26 was detected in three times as many samples as E. coli O103, and the rate at which calves began shedding E. coli O26 for the first time was five times greater than that for E. coli O103. For E. coli O26, O103, and O157, there was no association between shedding by calves and shedding by dams within 1 week of birth. For E. coli O26 and O103, there was no association between shedding and scouring, and there was no significant change in shedding following housing.     

Complete genome sequence and comparative genome analysis of enteropathogenic Escherichia coli O127:H6 strain E2348/69

Enteropathogenic Escherichia coli (EPEC) was the first pathovar of E. coli to be implicated in human disease; however, no EPEC strain has been fully sequenced until now. Strain E2348/69 (serotype O127:H6 belonging to E. coli phylogroup B2) has been used worldwide as a prototype strain to study EPEC biology, genetics, and virulence. Studies of E2348/69 led to the discovery of the locus of enterocyte effacement-encoded type III secretion system (T3SS) and its cognate effectors, which play a vital role in attaching and effacing lesion formation on gut epithelial cells. In this study, we determined the complete genomic sequence of E2348/69 and performed genomic comparisons with other important E. coli strains. We identified 424 E2348/69-specific genes, most of which are carried on mobile genetic elements, and a number of genetic traits specifically conserved in phylogroup B2 strains irrespective of their pathotypes, including the absence of the ETT2-related T3SS, which is present in E. coli strains belonging to all other phylogroups. The genome analysis revealed the entire gene repertoire related to E2348/69 virulence. Interestingly, E2348/69 contains only 21 intact T3SS effector genes, all of which are carried on prophages and integrative elements, compared to over 50 effector genes in enterohemorrhagic E. coli O157. As E2348/69 is the most-studied pathogenic E. coli strain, this study provides a genomic context for the vast amount of existing experimental data. The unexpected simplicity of the E2348/69 T3SS provides the first opportunity to fully dissect the entire virulence strategy of attaching and effacing pathogens in the genomic context.     

Molecular Subtyping and Genetic Analysis of the Enterohemolysin Gene (ehxA) from Shiga Toxin-Producing Escherichia coli and Atypical Enteropathogenic E. coli

Analyses of the distribution of virulence factors among different Escherichia coli pathotypes, including Shiga toxin-producing E. coli (STEC), may provide some insight into the mechanisms by which different E. coli strains cause disease and the evolution of distinct E. coli types. The aim of this study was to examine the DNA sequence of the gene for enterohemolysin, a plasmid-encoded toxin that readily causes the hemolysis of washed sheep erythrocytes, and to assess the distribution of enterohemolysin subtypes among E. coli isolates from various human and animal sources. The 2,997-bp ehxA gene was amplified from 227 (63.8%) of 356 stx- and/or eae-positive E. coli strains isolated from cattle and sheep and from 24 (96.0%) of 25 STEC strains isolated from humans with diarrheal disease. By using PCR and restriction fragment length polymorphism (RFLP) analysis of ehxA, six distinct PCR-RFLP types (A to F) were observed, with strains of subtypes A and C constituting 91.6% of all the ehxA-positive strains. Subtype A was associated mainly with ovine strains with stx only (P < 0.001), and subtype C was associated with bovine eae-positive strains (P < 0.001). Eleven ehxA alleles were fully sequenced, and the phylogenetic analysis indicated the presence of three closely related (>95.0%) ehxA sequence groups, one including eae-positive strains (subtypes B, C, E, and F) and the other two including mainly eae-negative STEC strains (subtypes A and D). In addition to being widespread among STEC strains, stx-negative, eae-positive strains (atypical enteropathogenic E. coli strains) isolated from cattle and sheep have similar ehxA subtypes and hemolytic activities.     

Presence and Characterization of Shiga Toxin-Producing Escherichia coli and Other Potentially Diarrheagenic E. coli Strains in Retail Meats

To determine the presence of Shiga toxin-producing Escherichia coli (STEC) and other potentially diarrheagenic E. coli strains in retail meats, 7,258 E. coli isolates collected by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) retail meat program from 2002 to 2007 were screened for Shiga toxin genes. In addition, 1,275 of the E. coli isolates recovered in 2006 were examined for virulence genes specific for other diarrheagenic E. coli strains. Seventeen isolates (16 from ground beef and 1 from a pork chop) were positive for stx genes, including 5 positive for both stx₁ and stx₂, 2 positive for stx₁, and 10 positive for stx₂. The 17 STEC strains belonged to 10 serotypes: O83:H8, O8:H16, O15:H16, O15:H17, O88:H38, ONT:H51, ONT:H2, ONT:H10, ONT:H7, and ONT:H46. None of the STEC isolates contained eae, whereas seven carried enterohemorrhagic E. coli (EHEC) hlyA. All except one STEC isolate exhibited toxic effects on Vero cells. DNA sequence analysis showed that the stx₂ genes from five STEC isolates encoded mucus-activatable Stx2d. Subtyping of the 17 STEC isolates by pulsed-field gel electrophoresis (PFGE) yielded 14 distinct restriction patterns. Among the 1,275 isolates from 2006, 11 atypical enteropathogenic E. coli (EPEC) isolates were identified in addition to 3 STEC isolates. This study demonstrated that retail meats, mainly ground beef, were contaminated with diverse STEC strains. The presence of atypical EPEC strains in retail meat is also of concern due to their potential to cause human infections.Escherichia coli is an important component of the intestinal microflora of humans and warm-blooded mammals. While E. coli typically harmlessly colonizes the intestinal tract, several E. coli clones have evolved the ability to cause a variety of diseases within the intestinal tract and elsewhere in the host. Those strains that cause enteric infections are generally called diarrheagenic E. coli strains, and their pathogenesis is associated with a number of virulence attributes, which vary according to pathotype (54). Currently, diarrheagenic E. coli strains are classified into six main pathotypes based on their distinct virulence determinants and pathogenic features, including enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC)/Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusively adherent E. coli (DAEC) (37).Among diarrheagenic E. coli strains, STEC strains are distinguished by the ability to cause severe life-threatening complications, such as hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (30). Other symptoms of STEC infection include watery diarrhea, bloody diarrhea, and hemorrhagic colitis (HC). STEC strains that cause HC and HUS are also called EHEC. Although individuals of all ages are at risk of STEC infection, children younger than 5 years of age and the elderly are more likely to suffer from severe complications (51). Outbreaks and sporadic cases of STEC infections have been reported frequently worldwide.The pathogenesis of STEC infection in humans is not fully understood. The major virulence factors implicated in STEC infection are potent Shiga toxins, which are classified into two groups: Stx1 and Stx2 (23). Additional factors that contribute to virulence have also been described, including intimin (encoded by the eae gene), an outer membrane protein involved in the attachment of E. coli to the enterocyte, and EHEC hemolysin (encoded by EHEC hlyA), which acts as a pore-forming cytolysin and causes damage to cells (41).The first STEC O157 infections were reported in 1982, when E. coli O157:H7 was involved in outbreaks associated with two fast food chain restaurants in the United States (44). Since then, ever-increasing numbers of cases and outbreaks due to STEC O157 have been reported worldwide. Although non-O157 STEC strains have also been associated with human cases and outbreaks, few laboratories have been looking for them, and their potential in causing human infections may be underestimated (2). Recently, though, the significance of non-O157 STEC strains as human pathogens has become more recognized. In the United States alone, there were 23 reported outbreaks of non-O157 STEC infection between 1990 and 2007 (10).Shiga toxin-producing E. coli can be transmitted through different routes, including food and water, person-to-person contact, and animal-to-person contact (9). Most human infections are caused by consumption of contaminated foods (16). Domestic and wild ruminant animals, in particular cattle, are considered the main reservoir of STEC and the main source for contamination of the food supply. Retail meats derived from animals could potentially act as transmission vehicles for STEC and other diarrheagenic E. coli strains. However, there is limited information about STEC contamination in retail meats, and fewer data exist about the presence of other diarrheagenic E. coli strains in retail meats. In the present study, we investigated 7,258 E. coli isolates from four types of meat samples (beef, chicken, pork, and turkey) collected during 2002 to 2007 to assess STEC contamination of retail meats. In addition, the presence of other potentially diarrheagenic E. coli strains was examined by detecting specific virulence determinants among E. coli isolates collected in 2006.