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.     

Low-Density Macroarray Targeting Non-Locus of Enterocyte Effacement Effectors (nle Genes) and Major Virulence Factors of Shiga Toxin-Producing Escherichia coli (STEC): a New Approach for Molecular Risk Assessment of STEC Isolates

Rapid and specific detection of Shiga toxin-producing Escherichia coli (STEC) strains with a high level of virulence for humans has become a priority for public health authorities. This study reports on the development of a low-density macroarray for simultaneously testing the genes stx₁, stx₂, eae, and ehxA and six different nle genes issued from genomic islands OI-122 (ent, nleB, and nleE) and OI-71 (nleF, nleH1-2, and nleA). Various strains of E. coli isolated from the environment, food, animals, and healthy children have been compared with clinical isolates of various seropathotypes. The eae gene was detected in all enteropathogenic E. coli (EPEC) strains as well as in enterohemorrhagic E. coli (EHEC) strains, except in EHEC O91:H21 and EHEC O113:H21. The gene ehxA was more prevalent in EHEC (90%) than in STEC (42.66%) strains, in which it was unequally distributed. The nle genes were detected only in some EPEC and EHEC strains but with various distributions, showing that nle genes are strain and/or serotype specific, probably reflecting adaptation of the strains to different hosts or environmental niches. One characteristic nle gene distribution in EHEC O157:[H7], O111:[H8], O26:[H11], O103:H25, O118:[H16], O121:[H19], O5:H−, O55:H7, O123:H11, O172:H25, and O165:H25 was ent/espL2, nleB, nleE, nleF, nleH1-2, nleA. (Brackets indicate genotyping of the flic or rfb genes.) A second nle pattern (ent/espL2, nleB, nleE, nleH1-2) was characteristic of EHEC O103:H2, O145:[H28], O45:H2, and O15:H2. The presence of eae, ent/espL2, nleB, nleE, and nleH1-2 genes is a clear signature of STEC strains with high virulence for humans.Since the early 1980s, Shiga toxin-producing Escherichia coli (STEC) has emerged as a major cause of food-borne infections (17, 30). STEC can cause diarrhea in humans, and some STEC strains may cause life-threatening diseases, such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). On the basis of its human pathogenicity, this subset of STEC strains was also designated enterohemorrhagic E. coli (EHEC) (22, 25). Numerous cases of HC and HUS have been attributed to EHEC serotype O157:H7 strains, but it has now been recognized that other serotypes of STEC belong to the EHEC group. The STEC seropathotype classification is based upon the serotype association with human epidemics, HUS, and diarrhea and has been developed as a tool to assess the clinical and public health risks associated with non-O157 EHEC and STEC strains (18). Only a few serotypes of STEC have been reported as most frequently associated with severe disease in humans. Besides E. coli O157:[H7], five other serotypes, namely O26:[H11], O103:H2, O111:[H8], O121:[H19], and O145:[H28], account for the group of typical EHEC (25). (Brackets indicate genotyping of the flic or rfb genes; the absence of brackets indicates data obtained with the conventional serotyping approach using specific antisera, as described in Materials and Methods.) Atypical EHEC group strains of serotypes O91:[H21], O113:H21, and O104:H21 are less frequently involved in hemorrhagic diseases than typical EHEC but are a frequent cause of diarrhea (8, 12, 25). Recent data from Enter-Net, a global surveillance consortium of 35 countries that tracks enteric infectious diseases, showed that the number of human cases of illness caused by non-O157 EHEC increased globally by 60.5% between 2000 and 2005, while at the same time the number of cases linked to EHEC O157 increased by only 13% (1). In the past few years, new serotypes of EHEC that differ from those previously known as typical and atypical EHEC have emerged (6, 8, 23, 24, 31). These EHEC strains were identified as important causes of food-borne infections in humans and were described as “new emerging EHEC.”The production of Shiga toxin (Stx) by EHEC is the primary virulence trait responsible for HUS, but many E. coli non-O157:H7 strains that produce Stx do not cause HUS. Identification of human-virulent STEC by detection of unique stx genes may be misleading, since not all STEC strains are clinically significant for humans (11). Besides the ability to produce one or more types of Shiga toxins, typical EHEC strains harbor a genomic island called the “locus of enterocyte effacement” (LEE). Atypical EHEC strains are negative for the LEE but may carry other factors for colonization of the human intestine (6, 25). The LEE carries genes encoding functions for bacterial colonization of the gut and for destruction of the intestinal mucosa, thus contributing to the disease process (25). The LEE eae gene product intimin is directly involved in the attaching and effacing (A/E) process (37). The LEE includes regulatory elements, a type III secretion system (TTSS), secreted effector proteins, and their cognate chaperon (13, 29). In addition to the intimin, most of the typical EHEC strains harbor the plasmid-borne enterohemolysin (ehxA), which is considered an associated virulence factor (6, 25).A number of other pathogenicity island (PAI) candidates, including O island 122 (OI-122) and O island 71 (OI-71), have been found in EHEC and EPEC strains, but their role in disease is not fully clear. Within the EHEC group, both O157:H7 strains (19, 34) and non-O157 strains (18, 35) present a variable repertoire of virulence determinants, including a collection of non-LEE-encoded effector (nle) genes that encode translocated substrates of the type III secretion system (9, 20). Our objective was to identify type III secreted virulence factors that distinguish EHEC O157 and non-O157 strains constituting a severe risk for human health from STEC strains that are not associated with severe and epidemic disease, a concept called “molecular risk assessment” (MRA) by Coombes et al. (9). Supporting the MRA approach requires the development of diagnostic tests based on multiplex nucleic acid amplification and microfluidics-based detection using standardized platforms applicable in hospital service or public health laboratories. It is now feasible to develop low-density DNA arrays that can be used to examine the gene inventory from isolated strains, offering a genetic bar coding strategy. A recent innovation in this field is the introduction of the GeneSystems PCR technology (5, 36). In this study, we have developed a GeneDisc array designed for simultaneous detection of genes encoding Shiga toxins 1 and 2 (stx₁ and stx₂), intimins (eae), enterohemolysin (ehxA), and six different nle genes derived from genomic islands OI-71 and OI-122. We focused our efforts on the detection of the OI-122 genes, ent/espL2 (Z4326), nleB (Z4328), and nleE (Z4329), and the OI-71 genes, nleF (Z6020), nleH1-2 (Z6021), and nleA (Z6024). The macroarray presented here was evaluated for its specificity and ability to discriminate between STEC causing serious illness in humans and other E. coli strains.     

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.     

Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

Using colony blot hybridization with stx₂ and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx₂-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx₂-positive isolates fliC_H7, non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx₂-positive strains by loss of stx₂ during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx₂-positive and stx-negative isolates demonstrated that yecE was occupied in stx₂-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx₂-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx₂-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx₂-harboring phages. The ability to recycle stx₂, a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.     

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.     

Molecular and Phenotypic Characterization of Escherichia coli O26:H8 among Diarrheagenic E. coli O26 Strains Isolated in Brazil

Escherichia coli strains of serogroup O26 comprise two distinct groups of pathogens, characterized as enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among the several genes related to type III secretion system-secreted effector proteins, espK was found to be highly specific for EHEC O26:H11 and its stx-negative derivative strains isolated in European countries. E. coli O26 strains isolated in Brazil from infant diarrhea, foods, and the environment have consistently been shown to lack stx genes and are thus considered atypical EPEC. However, no further information related to their genetic background is known. Therefore, in this study, we aimed to discriminate and characterize these Brazilian O26 stx-negative strains by phenotypic, genetic, and biochemical approaches. Among 44 isolates confirmed to be O26 isolates, most displayed flagellar antigen H11 or H32. Out of the 13 nonmotile isolates, 2 tested positive for fliC_H11, and 11 were fliC_H8 positive. The identification of genetic markers showed that several O26:H11 and all O26:H8 strains tested positive for espK and could therefore be discriminated as EHEC derivatives. The presence of H8 among EHEC O26 and its stx-negative derivative isolates is described for the first time. The interaction of three isolates with polarized Caco-2 cells and with intestinal biopsy specimen fragments ex vivo confirmed the ability of the O26 strains analyzed to cause attaching-and-effacing (A/E) lesions. The O26:H32 strains, isolated mostly from meat, were considered nonvirulent. Knowledge of the virulence content of stx-negative O26 isolates within the same serotype helped to avoid misclassification of isolates, which certainly has important implications for public health surveillance.     

Genomic Diversity and Virulence Profiles of Historical Escherichia coli O157 Strains Isolated from Clinical and Environmental Sources

Escherichia coli O157:H7 is, to date, the major E. coli serotype causing food-borne human disease worldwide. Strains of O157 with other H antigens also have been recovered. We analyzed a collection of historic O157 strains (n = 400) isolated in the late 1980s to early 1990s in the United States. Strains were predominantly serotype O157:H7 (55%), and various O157:non-H7 (41%) serotypes were not previously reported regarding their pathogenic potential. Although lacking Shiga toxin (stx) and eae genes, serotypes O157:H1, O157:H2, O157:H11, O157:H42, and O157:H43 carried several virulence factors (iha, terD, and hlyA) also found in virulent serotype E. coli O157:H7. Pulsed-field gel electrophoresis (PFGE) showed the O157 serogroup was diverse, with strains with the same H type clustering together closely. Among non-H7 isolates, serotype O157:H43 was highly prevalent (65%) and carried important enterohemorrhagic E. coli (EHEC) virulence markers (iha, terD, hlyA, and espP). Isolates from two particular H types, H2 and H11, among the most commonly found non-O157 EHEC serotypes (O26:H11, O111:H11, O103:H2/H11, and O45:H2), unexpectedly clustered more closely with O157:H7 than other H types and carried several virulence genes. This suggests an early divergence of the O157 serogroup to clades with different pathogenic potentials. The appearance of important EHEC virulence markers in closely related H types suggests their virulence potential and suggests further monitoring of those serotypes not implicated in severe illness thus far.     

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.     

Extensive genomic diversity and selective conservation of virulence-determinants in enterohemorrhagic Escherichia coli strains of O157 and non-O157 serotypes

Background

Enterohemorrhagic Escherichia coli (EHEC) O157 causes severe food-borne illness in humans. The chromosome of O157 consists of 4.1 Mb backbone sequences shared by benign E. coli K-12, and 1.4 Mb O157-specific sequences encoding many virulence determinants, such as Shiga toxin genes (stx genes) and the locus of enterocyte effacement (LEE). Non-O157 EHECs belonging to distinct clonal lineages from O157 also cause similar illness in humans. According to the 'parallel' evolution model, they have independently acquired the major virulence determinants, the stx genes and LEE. However, the genomic differences between O157 and non-O157 EHECs have not yet been systematically analyzed.

Results

Using microarray and whole genome PCR scanning analyses, we performed a whole genome comparison of 20 EHEC strains of O26, O111, and O103 serotypes with O157. In non-O157 EHEC strains, although genome sizes were similar with or rather larger than O157 and the backbone regions were well conserved, O157-specific regions were very poorly conserved. Around only 20% of the O157-specific genes were fully conserved in each non-O157 serotype. However, the non-O157 EHECs contained a significant number of virulence genes that are found on prophages and plasmids in O157, and also multiple prophages similar to, but significantly divergent from, those in O157.

Conclusion

Although O157 and non-O157 EHECs have independently acquired a huge amount of serotype- or strain-specific genes by lateral gene transfer, they share an unexpectedly large number of virulence genes. Independent infections of similar but distinct bacteriophages carrying these virulence determinants are deeply involved in the evolution of O157 and non-O157 EHECs.     

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.     

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.     

Adherence of Escherichia coli O157:H7 Mutants In Vitro and in Ligated Pig Intestines

There are contradictory literature reports on the role of verotoxin (VT) in adherence of enterohemorrhagic Escherichia coli O157:H7 (O157 EHEC) to intestinal epithelium. There are reports that putative virulence genes of O island 7 (OI-7), OI-15, and OI-48 of this pathogen may also affect adherence in vitro. Therefore, mutants of vt2 and segments of OI-7 and genes aidA₁₅ (gene from OI-15) and aidA₄₈ (gene from OI-48) were generated and evaluated for adherence in vitro to cultured human HEp-2 and porcine jejunal epithelial (IPEC-J2) cells and in vivo to enterocytes in pig ileal loops. VT2-negative mutants showed significant decreases in adherence to both HEp-2 and IPEC-J2 cells and to enterocytes in pig ileal loops; complementation only partially restored VT2 production but fully restored the adherence to the wild-type level on cultured cells. Deletion of OI-7 and aidA₄₈ had no effect on adherence, whereas deletion of aidA₁₅ resulted in a significant decrease in adherence in pig ileal loops but not to the cultured cells. This investigation supports the findings that VT2 plays a role in adherence, shows that results obtained in adherence of E. coli O157:H7 in vivo may differ from those obtained in vitro, and identified AIDA-15 as having a role in adherence of E. coli O157:H7.Escherichia coli O157:H7 is the prototypical enterohemorrhagic E. coli (EHEC) strain and is the most common serotype associated with large outbreaks and sporadic cases of hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS) (25). It is well established that EHEC O157:H7 can colonize the intestine of humans and animals and that adherence to intestinal epithelial cells occurs through the formation of attaching-and-effacing (AE) lesions, which is a critical early step in infection. Some researchers have suggested that EHEC uses fimbriae to make the initial contact with epithelial cells, prior to intimate attachment mediated by locus of enterocyte effacemen-encoded proteins (15). Several potential adherence factors of EHEC O157:H7 have been described, but only the outer membrane protein intimin has been demonstrated to play a role in intestinal colonization in animal models (16). Intimin mediates the intimate adherence component of the AE lesion by binding to the translocated intimin receptor Tir, resulting in close attachment of the bacteria to the host cell membrane (17). Intimin can also bind to β1 integrins and nucleolin on host cells (9, 36). Severe damage due to infection with EHEC is attributable to the cytotoxic verotoxin (VT), which damages epithelial and endothelial cells, leading to bloody diarrhea and HUS (16). Several investigators have reported that VT does not play a role in colonization of the intestine (2, 4, 34). However, Robinson et al. (30) reported recently that VT enhances adherence to epithelial cells and colonization of the mouse intestine by E. coli O157:H7. Therefore, the present study examined the involvement of VT in adherence in vitro and in vivo.Several putative virulence genes have been identified in O islands (OIs) in EHEC O157:H7 strain EDL 933 (26), including those encoding Iha and AIDA-I in OI-43/48, AIDA-I in OI-15, and a ClpB chaperone protein and a putative macrophage toxin in OI-7 (26, 27). OI-7 also contains many unknown open reading frames (ORFs) whose function in the pathogenesis of EHEC O157:H7 has not been investigated. Iha, an adherence-conferring outer membrane protein similar to IrgA (the product of iron-regulated gene A) (38), is a virulence factor in uropathogenic E. coli strain CFT073 (14). AIDA-I, encoded by aidA, was first identified in EPEC and confers the capacity for diffuse adhesion of the bacteria to epithelial cells (1). AIDA-I-like adhesins from OI-15 and OI-43/48 show 55% and 68% homology, respectively, to the AIDA-I of EPEC (26, 27). All three AIDA proteins show characteristics of an autotransporter membrane protein with a β-barrel structure (20), which is exposed at the surface of the bacteria (13). These observations suggest that the two homologs of AIDA-I may also function as adhesins in EHEC O157:H7; however, the roles of the AIDA-I-like adhesins in EHEC have yet to be determined.EHEC O157:H7 has been isolated from pigs, and conventional pigs are a permissive host and therefore a potential reservoir for human infection with EHEC O157:H7 (8). One recent family outbreak was associated with pork salami (3). Pigs are highly relevant models for the study of virulence of EHEC O157:H7 in humans and have been extensively used to characterize putative virulence factors and to investigate the pathogenesis of EHEC O157:H7 and other verotoxigenic E. coli strains (6, 11, 21). The present study was designed to examine VT2-negative mutants, OI-7 deletions, and aidA knockouts from OI-15 and OI-48 of EHEC O157:H7 in vitro and in the pig intestines for their roles in adherence.     

First isolation and further characterization of enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EPEC) O157:H45 strains from cattle

Background  

Enteropathogenic Escherichia coli (EPEC), mainly causing infantile diarrhoea, represents one of at least six different categories of diarrheagenic E. coli with corresponding distinct pathogenic schemes. The mechanism of EPEC pathogenesis is based on the ability to introduce the attaching-and-effacing (A/E) lesions and intimate adherence of bacteria to the intestinal epithelium. The role and the epidemiology of non-traditional enteropathogenic E. coli serogroup strains are not well established. E. coli O157:H45 EPEC strains, however, are described in association with enterocolitis and sporadic diarrhea in human. Moreover, a large outbreak associated with E. coli O157:H45 EPEC was reported in Japan in 1998. During a previous study on the prevalence of E. coli O157 in healthy cattle in Switzerland, E. coli O157:H45 strains originating from 6 fattening cattle and 5 cows were isolated. In this study, phenotypic and genotypic characteristics of these strains are described. Various virulence factors (stx, eae, ehxA, astA, EAF plasmid, bfp) of different categories of pathogenic E. coli were screened by different PCR systems. Moreover, the capability of the strains to adhere to cells was tested on tissue culture cells.     

Characterization of Shiga Toxin Gene (stx)-Positive and Intimin Gene (eae)-Positive Escherichia coli Isolates from Wastewater of Slaughterhouses in France

Wastewater samples from 12 slaughterhouses located in different regions in France were tested for the presence of stx-positive and eae-positive Escherichia coli isolates, and characteristics of the isolates obtained were determined. A total of 224 wastewater samples were collected in wastewater treatment plants at different stages of wastewater processing. Altogether, 5,001 E. coli isolates were obtained by colony counting and screened for the presence of stx and eae genes by multiplex PCR. stx-positive and eae-positive E. coli isolates were detected in 25% of the samples collected; they were found in 13% and 3% of the samples obtained from treated effluent and sludge, respectively, suggesting that they could be spread into the environment. Screening of the samples collected by immunomagnetic separation allowed us to isolate 31 additional E. coli serogroup O157 isolates. Four of these isolates harbored stx and eae genes. All stx-positive and eae-positive E. coli isolates were analyzed for eae and stx genetic variants, as well as for additional virulence factors and serotypes. Our results suggest that the majority of the stx- and eae-positive E. coli isolates from wastewater have low virulence for humans. However, the diversity of the enterohemorrhagic E. coli-associated virulence factors in the strains indicates that the environment may play an important role in the emergence of new pathogenic enterohemorrhagic E. coli strains.     

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.     

Intimin Gene (eae) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae, and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.     

Persistence of Escherichia coli O157:H7 and its mutants in soils

The persistence of Shiga toxin-producing E. coli O157:H7 in the environment poses a serious threat to public health. However, the role of Shiga toxins and other virulence factors in the survival of E. coli O157:H7 is poorly defined. The aim of this study was to determine if the virulence factors, stx ₁, stx ₂, stx _1–2, and eae in E. coli O157:H7 EDL933 play any significant role in the growth of this pathogen in rich media and in soils. Isogenic deletion mutants that were missing one of four virulence factors, stx ₁, stx ₂, stx _1–2, and eae in E. coli O157:H7 EDL933 were constructed, and their growth in rich media and survival in soils with distinct texture and chemistry were characterized. The survival data were successfully analyzed using Double Weibull model, and the modeling parameters of the mutant strains were not significantly different from those of the wild type. The calculated T_d (time needed to reach the detection limit, 100 CFU/g soil) for loamy sand, sandy loam, and silty clay was 32, 80, and 110 days, respectively. It was also found that T_d was positively correlated with soil structure (e.g. clay content), and soil chemistry (e.g. total nitrogen, total carbon, and water extractable organic carbon). The results of this study showed that the possession of Shiga toxins and intimin in E. coli O157:H7 might not play any important role in its survival in soils. The double deletion mutant of E. coli O157:H7 (stx ₁ ⁻ stx ₂ ⁻) may be a good substitute to use for the investigation of transport, fate, and survival of E. coli O157:H7 in the environment where the use of pathogenic strains are prohibited by law since the mutants showed the same characteristics in both culture media and environmental samples.     

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.