Occurrence of Virulence Genes Associated with Diarrheagenic Pathotypes in Escherichia coli Isolates from Surface Water

Escherichia coli isolates (n = 300) collected from six sites in subtropical Brisbane, Australia, prior to and after storm events were tested for the presence of 11 virulence genes (VGs) specific to diarrheagenic pathotypes. The presence of eaeA, stx₁, stx₂, and ehxA genes specific for the enterohemorrhagic E. coli (EHEC) pathotype was detected in 56%, 6%, 10%, and 13% of isolates, respectively. The VGs astA (69%) and aggR (29%), carried by enteroaggregative (EAEC) pathotypes, were frequently detected in E. coli isolates. The enteropathogenic E. coli (EPEC) gene bfp was detected in 24% of isolates. In addition, enteroinvasive E. coli (EIEC) VG ipaH was also detected in 14% of isolates. During dry periods, isolates belonging to the EAEC pathotype were most commonly detected (23%), followed by EHEC (11%) and EPEC (11%). Conversely, a more uniform prevalence of pathotypes, EPEC (14%), EAEC (12%), EIEC (10%), EHEC (7%), and ETEC (7%), was observed after the storm events. The results of this study highlight the widespread occurrence of potentially diarrheagenic pathotypes in the urban aquatic ecosystems. While the presence of VGs in E. coli isolates alone is insufficient to determine pathogenicity, the presence of diarrheagenic E. coli pathotypes in high frequency after the storm events could lead to increased health risks if untreated storm water were to be used for nonpotable purposes and recreational activities.     

Diarrheagenic Escherichia coli Carrying Supplementary Virulence Genes Are an Important Cause of Moderate to Severe Diarrhoeal Disease in Mexico

Diarrheagenic Escherichia coli (DEC) cause acute and persistent diarrhoea worldwide, but little is known about their epidemiology in Mexico. We determined the prevalence of bacterial enteropathogens in 831 children with acute diarrhoea over a four-year period in Yucatan, Mexico. Six DEC supplementary virulence genes (SVG), mainly associated with enteroaggregative E. coli (EAEC), were sought in 3100 E. coli isolates. DEC was the most common bacterial enteropathogen (28%), surpassing Salmonella (12%) and Shigella (9%). Predominant DEC groups were diffusely adherent E. coli (DAEC) (35%), EAEC (24%), and enteropathogenic E. coli (EPEC) (19%). Among children with DEC infections, 14% had severe illness mainly caused by EPEC (26%) and DAEC (18%); 30% had moderate diarrhoea mainly caused by DAEC (36%), mixed DEC infections (33%) and EAEC (32%). DAEC was most prevalent during spring, while ETEC, EAEC and EPEC predominated in summer. EAEC was more frequent in children 6–24 months old than in those younger than 6 months of age (P = 0.008, OR = 4.2, 95% CI, 1.3–13.9). The presence of SVG dispersin, (aatA), dispersin-translocator (aatA), enteroaggregative heat-stable toxin 1 (astA), plasmid encoded toxin (pet), cytolethal distending toxin (cdt) was higher in DEC than non-DEC strains, (36% vs 26%, P <0.0001, OR = 1.5, 95% CI, 1.3–1.8). 98% of EAEC-infected children harboured strains with SVG; 85% carried the aap-aatA gene combination, and 33% of these also carried astA. 28% of both EPEC and ETEC, and 6% of DAEC patients had strains with SVG. 54% of EPEC patients carried pet-positive strains alone or in combination with astA; only this DEC group harboured cdt-positive isolates. All ETEC patients carried astA- or astA-aap-positive strains. astA and aap were the most common SVG in DAEC (3% and 2%) and non-DEC strains (21% and 13%). DEC carrying SVG are an important cause of moderate to severe bacterial diarrhoea in Mexican children.     

Identification of Potentially Diarrheagenic Atypical Enteropathogenic Escherichia coli Strains Present in Canadian Food Animals at Slaughter and in Retail Meats

This study identified and characterized enteropathogenic Escherichia coli (EPEC) in the Canadian food supply. Eighteen of 450 E. coli isolates from food animal sources were identified as atypical EPEC (aEPEC). Several of the aEPEC isolates identified in this study possessed multiple virulence genes, exhibited adherence and attaching and effacing (A/E) lesion formation, disrupted tight junctions, and were coclassified with the extraintestinal pathogenic E. coli (ExPEC) and enterotoxigenic E. coli (ETEC) pathotypes.     

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.     

Exhaustive isolation of diarrhoeagenic Escherichia coli by a colony hybridization method using hydrophobic grid-membrane filters in combination with multiplex real-time PCR

Aims: The present study aimed to develop a colony hybridization method for the exhaustive detection and isolation of diarrhoeagenic Escherichia coli (DEC) from samples containing numerous coliform bacteria. Methods and Results: Digoxigenin‐labelled DNA probes were designed to detect seven pathotypes of DEC based on type‐specific genes. A total of 615 meat, food and faeces samples identified as DEC‐positive by multiple real‐time PCR for the virulence genes (eae, stx, elt, est, virB, aggR, afaB and astA) were analysed by a colony hybridization method, which involved filtering enrichment cultures through hydrophobic grid‐membrane filters. DEC were isolated from 72·5% (446/615) of samples by the colony hybridization method but were only detected in 26·3% (162/615) of samples by a conventional culture method. The hybridization method was particularly effective for isolating low‐level contaminants, such as enterotoxigenic and Shiga toxin‐producing E. coli, which were isolated from 51·8% (58/112) of samples identified as positive by PCR for the enterotoxin genes, in contrast to only 4·5% (5/112) of samples analysed by the conventional method. Conclusions: The developed colony hybridization system allows for the efficient and simultaneous isolation of all DEC pathotypes. Significance and Impact of the Study:  The colony hybridization system described here permits the sensitive isolation of DEC and represents a suitable tool for ecological investigations of DEC.     

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.     

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.     

The traditional enteropathogenic Escherichia coli (EPEC) serogroup O125 comprises serotypes which are mainly associated with the category of enteroaggregative E. coli

Genotypic and phenotypic virulence markers of the different categories of diarrheagenic Escherichia coli were investigated in 76 strains of the enteropathogenic E. coli (EPEC) serogroup O125. The most frequent serotype found was O125ac:H21. None of the serotypes behaved as EPEC, i.e. carried the eaeA, bfpA, and EAF DNA sequences simultaneously and presented localized adherence to HeLa cells. All strains of O125ac:H6 were atypical EPEC since they carried eaeA only, and presented an indefinite pattern of adherence. All strains of O125ab:H9, O125ac:H9, O125?:H16, and O125ab:H21 and 79% of the O125ac:H21 strains were enteroaggregative E. coli, since they carried a specific DNA sequence and presented the typical aggregative adherence pattern.     

Detection of diarrhoeagenic Escherichia coli in clinical and environmental water sources in South Africa using single-step 11-gene m-PCR

Escherichia coli (E. coli) consists of commensal (ComEC) and diarrhoeagenic (DEC) groups. ComEC are detected using traditional culture methods. Conformational steps are performed after culturing if it is required to test for the presence of DEC, increasing cost and time in obtaining the results. The aim of this study was to develop a single-step multiplex polymerase chain reaction (m-PCR) that can simultaneously amplify genes associated with DEC and ComEC, with the inclusion of controls to monitor inhibition. A total of 701 samples, taken from clinical and environmental water sources in South Africa, were analysed with the optimised m-PCR which targeted the eaeA, stx1, stx2, lt, st, ial, eagg, astA and bfp virulence genes. The mdh and gapdh genes were included as an internal and external control, respectively. The presence of the external control gapdh gene in all samples excluded any possible PCR inhibition. The internal control mdh gene was detected in 100 % of the environmental and 85 % of the clinical isolates, confirming the classification of isolates as E. coli PCR positive samples. All DEC types were detected in varying degrees from the mdh positive environmental and clinical isolates. Important gene code combinations were detected for clinical isolates of 0.4 % lt and eagg. However, 2.3 % of eaeA and ial, and 8.7 % of eaeA and eagg were reported for environmental water samples. The E. coli astA toxin was detected as positive at 35 and 17 % in environmental isolates and clinical isolates, respectively. Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested. In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types. The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.     

Identification of Unconventional Intestinal Pathogenic Escherichia coli Isolates Expressing Intermediate Virulence Factor Profiles by Using a Novel Single-Step Multiplex PCR

Intestinal pathogenic Escherichia coli represents a global health problem for mammals, including humans. At present, diarrheagenic E. coli bacteria are grouped into seven major pathotypes that differ in their virulence factor profiles, severity of clinical manifestations, and prognosis. In this study, we developed and evaluated a one-step multiplex PCR (MPCR) for the straightforward differential identification of intestinal pathotypes of E. coli. The specificity of this novel MPCR was validated by using a subset of reference strains and further confirmed by PCR-independent pheno- and genotypic characterization. Moreover, we tested 246 clinical E. coli isolates derived from diarrhea patients from several distinct geographic regions. Interestingly, besides strains belonging to the defined and well-described pathotypes, we identified five unconventional strains expressing intermediate virulence factor profiles. These strains have been further characterized and appear to represent intermediate strains carrying genes and expressing factors associated with enteropathogenic E. coli, Shiga toxin-producing E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli alike. These strains represent further examples of the extraordinary plasticity of the E. coli genome. Moreover, this implies that the important identification of specific pathotypes has to be based on a broad matrix of indicator genes. In addition, the presence of intermediate strains needs to be accounted for.     

Occurrence of Virulence and Antimicrobial Resistance Genes in Escherichia coli Isolates from Different Aquatic Ecosystems within the St. Clair River and Detroit River Areas

Although the number of Escherichia coli bacteria in surface waters can differ greatly between locations, relatively little is known about the distribution of E. coli pathotypes in surface waters used as sources for drinking or recreation. DNA microarray technology is a suitable tool for this type of study due to its ability to detect high numbers of virulence and antimicrobial resistance genes simultaneously. Pathotype, phylogenetic group, and antimicrobial resistance gene profiles were determined for 308 E. coli isolates from surface water samples collected from diverse aquatic ecosystems at six different sites in the St. Clair River and Detroit River areas. A higher frequency (48%) of E. coli isolates possessing virulence and antimicrobial resistance genes was observed in an urban site located downstream of wastewater effluent outfalls than in the other examined sites (average of 24%). Most E. coli pathotypes were extraintestinal pathogenic E. coli (ExPEC) pathotypes and belonged to phylogenetic groups B2 and D. The ExPEC pathotypes were found to occur across all aquatic ecosystems investigated, including riverine, estuarine, and offshore lake locations. The results of this environmental study using DNA microarrays highlight the widespread distribution of E. coli pathotypes in aquatic ecosystems and the potential public health threat of E. coli pathotypes originating from municipal wastewater sources.     

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.     

Frequency of indicator bacteria,Salmonella and diarrhoeagenic Escherichia coli pathotypes on ready‐to‐eat cooked vegetable salads from Mexican restaurants

The presence of coliform bacteria, faecal coliforms, Escherichia coli, diarrhoeagenic E. coli pathotypes (DEP) and Salmonella were determined in ready‐to‐eat cooked vegetable salads (RECS) from restaurants in Pachuca city, Mexico. The RECS were purchased from three types of restaurants: national chain restaurants (A), local restaurants (B) and small restaurants (C). Two restaurants for each A and B, and three for C, were included. Forty RECS samples were purchased at each A and B restaurant and 20 at each C restaurant. Of the overall total of 220 analysed samples, 100, 98·2, 72·3, 4·1 and 4·1% had coliform bacteria, faecal coliforms, E. coli, DEP and Salmonella, respectively. Identified DEP included enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and Shiga toxin‐producing E. coli (STEC). The EPEC, ETEC and STEC were isolated each from 1·4% of samples. No E. coli O157:H7 were detected in any STEC‐positive samples. The analysis of Kruskal–Wallis anova and median test of microbiological data showed that the microbiological quality of RECS did not differ between the different restaurants (P > 0·05).

Significance and Impact of the Study

This is the first report regarding microbiological quality and Salmonella, enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and Shiga toxin‐producing E. coli (STEC) isolation from ready‐to‐eat cooked vegetable salads from Mexican restaurants. Ready‐to‐eat cooked vegetable salads could be an important factor contributing to the endemicity of EPEC, ETEC and STEC, and Salmonella caused gastroenteritis in Mexico.     

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.     

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.     

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.     

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.     

Distribution, functional expression, and genetic organization of Cif, a phage-encoded type III-secreted effector from enteropathogenic and enterohemorrhagic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) inject effector proteins into host cells via a type III secretion system encoded by the locus of enterocyte effacement (LEE). One of these effectors is Cif, encoded outside the LEE by a lambdoid prophage. In this study, we demonstrated that the Cif-encoding prophage of EPEC strain E22 is inducible and produces infectious phage particles. We investigated the distribution and functional expression of Cif in 5,049 E. coli strains of human, animal, and environmental origins. A total of 115 E. coli isolates from diverse origins and geographic locations carried cif. The presence of cif was tightly associated with the LEE, since all the cif-positive isolates were positive for the LEE. These results suggested that the Cif-encoding prophages have been widely disseminated within the natural population of E. coli but positively selected within the population of LEE-positive strains. Nonetheless, 66% of cif-positive E. coli strains did not induce a typical Cif-related phenotype in eukaryotic cells due to frameshift mutations or insertion of an IS element in the cif gene. The passenger region of the prophages carrying cif was highly variable and showed various combinations of IS elements and genes coding for other effectors such as nleB, nleC, nleH, nleG, espJ, and nleA/espI (some of which were also truncated). This diversity and the presence of nonfunctional effectors should be taken into account to assess EPEC and EHEC pathogenicity and tropism.     

Biological and Physicochemical Wastewater Treatment Processes Reduce the Prevalence of Virulent Escherichia coli

Effluents discharged from wastewater treatment plants are possible sources of pathogenic bacteria, including Escherichia coli, in the freshwater environment, and determining the possible selection of pathogens is important. This study evaluated the impact of activated sludge and physicochemical wastewater treatment processes on the prevalence of potentially virulent E. coli. A total of 719 E. coli isolates collected from four municipal plants in Québec before and after treatment were characterized by using a customized DNA microarray to determine the impact of treatment processes on the frequency of specific pathotypes and virulence genes. The percentages of potentially pathogenic E. coli isolates in the plant influents varied between 26 and 51%, and in the effluents, the percentages were 14 to 31%, for a reduction observed at all plants ranging between 14 and 45%. Pathotypes associated with extraintestinal pathogenic E. coli (ExPEC) were the most abundant at three of the four plants and represented 24% of all isolates, while intestinal pathogenic E. coli pathotypes (IPEC) represented 10% of the isolates. At the plant where ExPEC isolates were not the most abundant, a large number of isolates were classified as both ExPEC and IPEC; overall, 6% of the isolates were classified in both groups, with the majority being from the same plant. The reduction of the proportion of pathogenic E. coli could not be explained by the preferential loss of one virulence gene or one type of virulence factor; however, the quinolone resistance gene (qnrS) appears to enhance the loss of virulence genes, suggesting a mechanism involving the loss of pathogenicity islands.     

Characterization of Escherichia coli virulence genes,pathotypes and antibiotic resistance properties in diarrheic calves in Iran

Background

Calf diarrhea is a major economic concern in bovine industry all around the world. This study was carried out in order to investigate distribution of virulence genes, pathotypes, serogroups and antibiotic resistance properties of Escherichia coli isolated from diarrheic calves.

Results

Totally, 76.45% of 824 diarrheic fecal samples collected from Isfahan, Chaharmahal, Fars and Khuzestan provinces, Iran were positive for E. coli and all of them were also positive for cnf2, hlyA, cdtIII, f17c, lt, st, stx1, eae, ehly, stx2 and cnf1 virulence genes. Chaharmahal had the highest prevalence of STEC (84.61%), while Isfahan had the lowest (71.95%). E. coli serogroups had the highest frequency in 1–7 days old calves and winter season. Distribution of ETEC, EHEC, AEEC and NTEC pathotypes among E. coli isolates were 28.41%, 5.07%, 29.52% and 3.49%, respectively. Statistical analyses were significant for presence of bacteria between various seasons and ages. All isolates had the high resistance to penicillin (100%), streptomycin (98.25%) and tetracycline (98.09%) antibiotics. The most commonly detected resistance genes were aadA1, sul1, aac[3]-IV, CITM, and dfrA1. The most prevalent serogroup among STEC was O26.

Conclusions

Our findings should raise awareness about antibiotic resistance in diarrheic calves in Iran. Clinicians should exercise caution when prescribing antibiotics.