tir- and stx-positive Escherichia coli in stream waters in a metropolitan area

Diarrheagenic Escherichia coli, which may include the enteropathogenic E. coli and the enterohemorrhagic E. coli, are a significant cause of diarrheal disease among infants and children in both developing and developed areas. Disease outbreaks related to freshwater exposure have been documented, but the presence of these organisms in the urban aquatic environment is not well characterized. From April 2002 through April 2004 we conducted weekly surveys of streams in the metropolitan Baltimore, Md., area for the prevalence of potentially pathogenic E. coli by using PCR assays targeting the tir and stx(1) and stx(2) genes. Coliforms testing positive for the presence of the tir gene were cultured from 653 of 1,218 samples (53%), with a greater prevalence associated with urban, polluted streams than in suburban and forested watershed streams. Polluted urban streams were also more likely to test positive for the presence of one of the stx genes. Sequence analysis of the tir amplicon, as well as the entire tir gene from three isolates, indicated that the pathogenic E. coli present in the stream waters has a high degree of sequence homology with the E. coli O157:H7 serotype. Our data indicate that pathogenic E. coli are continually deposited into a variety of stream habitats and suggest that this organism may be a permanent member of the gastrointestinal microflora of humans and animals in the metropolitan Baltimore area.     

Dynamics of Escherichia coli Virulence Factors in Dairy Herds and Farm Environments in a Longitudinal Study in the United States

Pathogenic Escherichia coli or its associated virulence factors have been frequently detected in dairy cow manure, milk, and dairy farm environments. However, it is unclear what the long-term dynamics of E. coli virulence factors are and which farm compartments act as reservoirs. This study assessed the occurrence and dynamics of four E. coli virulence factors (eae, stx₁, stx₂, and the gamma allele of the tir gene [γ-tir]) on three U.S. dairy farms. Fecal, manure, water, feed, milk, and milk filter samples were collected from 2004 to 2012. Virulence factors were measured by postenrichment quantitative PCR (qPCR). All factors were detected in most compartments on all farms. Fecal and manure samples showed the highest prevalence, up to 53% for stx and 21% for γ-tir in fecal samples and up to 84% for stx and 44% for γ-tir in manure. Prevalence was low in milk (up to 1.9% for stx and 0.7% for γ-tir). However, 35% of milk filters were positive for stx and 20% were positive for γ-tir. All factors were detected in feed and water. Factor prevalence and levels, expressed as qPCR cycle threshold categories, fluctuated significantly over time, with no clear seasonal signal independent from year-to-year variability. Levels were correlated between fecal and manure samples, and in some cases autocorrelated, but not between manure and milk filters. Shiga toxins were nearly ubiquitous, and 10 to 18% of the lactating cows were potential shedders of E. coli O157 at least once during their time in the herds. E. coli virulence factors appear to persist in many areas of the farms and therefore contribute to transmission dynamics.     

A New Shiga Toxin 2 Variant (Stx2f) from Escherichia coli Isolated from Pigeons

We have isolated Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from the feces of feral pigeons which contained a new Stx2 variant gene designated stx_2f. This gene is most similar to sltIIva of patient E. coli O128:B12 isolate H.I.8. Stx2f reacted only weakly with commercial immunoassays. The prevalence of STEC organisms carrying the stx_2f gene in pigeon droppings was 12.5%. The occurrence of a new Stx2 variant in STEC from pigeons enlarges the pool of Stx2 variants and raises the question whether horizontal gene transfer to E. coli pathogenic to humans may occur.     

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.     

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.     

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.     

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.     

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.     

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.     

Prevalence of the stx2 Gene in Coliform Populations from Aquatic Environments

Shiga toxin-producing Escherichia coli strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. The major virulence factors of these strains are Shiga toxins Stx1 and Stx2. The majority of the genes coding for these toxins are borne by bacteriophages. Free Stx2-encoding bacteriophages have been found in aquatic environments, but there is limited information about the lysogenic strains and bacteria present in the environment that are susceptible to phage infection. The aim of this work was to study the prevalence and the distribution of the stx₂ gene in coliform bacteria in sewage samples of different origins. The presence of the stx₂ gene was monitored every 2 weeks over a 1-year period in a municipal sewage treatment plant. A mean value of 10² genes/ml was observed without significant variation during the study period. This concentration was of the same order of magnitude in raw municipal sewage of various origins and in animal wastewater from several slaughterhouses. A total of 138 strains carrying the stx₂ gene were isolated by colony hybridization. This procedure detected approximately 1 gene-carrying colony per 1,000 fecal coliform colonies in municipal sewage and around 1 gene-carrying colony per 100 fecal coliform colonies in animal wastewaters. Most of the isolates belonged to E. coli serotypes other than E. coli O157, suggesting a low prevalence of strains of this serotype carrying the stx₂ gene in the wastewater studied.     

Glutamate Decarboxylase Genes as a Prescreening Marker for Detection of Pathogenic Escherichia coli Groups

The enzyme glutamate decarboxylase (GAD) is prevalent in Escherichia coli but few strains in the various pathogenic E. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA and gadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagic E. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using a gadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR to gadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx₁ and stx₂ was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenic E. coli groups.     

Multilocus Genotype Analysis of Escherichia coli O157 Isolates from Australia and the United States Provides Evidence of Geographic Divergence

Escherichia coli O157 is a food-borne pathogen whose major reservoir has been identified as cattle. Recent genetic information has indicated that populations of E. coli O157 from cattle and humans can differ genetically and that this variation may have an impact on their ability to cause severe human disease. In addition, there is emerging evidence that E. coli O157 strains from different geographical regions may also be genetically divergent. To investigate the extent of this variation, we used Shiga toxin bacteriophage insertion sites (SBI), lineage-specific polymorphisms (LSPA-6), multilocus variable-number tandem-repeat analysis (MLVA), and a tir 255T>A polymorphism to examine 606 isolates representing both Australian and U.S. cattle and human populations. Both uni- and multivariate analyses of these data show a strong association between the country of origin and multilocus genotypes (P < 0.0001). In addition, our results identify factors that may play a role in virulence that also differed in isolates from each country, including the carriage of stx₁ in the argW locus uniquely observed in Australian isolates and the much higher frequency of stx₂-positive (also referred to as stx_2a) strains in the U.S. isolates (4% of Australian isolates versus 72% of U.S. isolates). LSPA-6 lineages differed between the two continents, with the majority of Australian isolates belonging to lineage I/II (LI/II) (LI, 2%; LI/II, 85%; LII, 13%) and the majority of U.S. isolates belonging to LI (LI, 60%; LI/II, 16%; LII, 25%). The results of this study provide strong evidence of phylogeographic structuring of E. coli O157 populations, suggesting divergent evolution of enterohemorrhagic E. coli O157 in Australia and the United States.     

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.     

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.     

Cytotoxicity Potential and Genotypic Characterization of Escherichia coli Isolates from Environmental and Food Sources

The presence of Escherichia coli isolates in the environment is a potential source of contamination of food and water supplies. Moreover, these isolates may harbor virulence genes that can be a source of new forms of pathogenic strains. Here, using multiplex PCR, we examined the presence of virulence gene markers (stx₁, stx₂, eaeA, hlyA) in 1,698 environmental isolates of E. coli and 81 isolates from food and clinical sources. The PCR analysis showed that ~5% (79 of 1,698) of the total environmental isolates and 96% (79 of 81) of the food and clinical isolates were positive for at least one of the genes. Of the food and clinical isolates, 84% (68 of 81 isolates) were positive for all four genes. Of the subset of environmental isolates chosen for further analysis, 16% (13 of 79 isolates) were positive for stx₂ and 84% (66 of 79 isolates) were positive for eaeA; 16 of the latter strains were also positive for hlyA. The pathogenic potentials of 174 isolates (81 isolates from food and clinical sources and 93 isolates from environmental sources) were tested by using a cytotoxicity assay based on lactate dehydrogenase release from Vero cells. In general, 97% (79 of 81) of the food and clinical isolates and 41% (39 of 93) of the environmental isolates exhibited positive cytotoxicity. High cytotoxicity values correlated to the presence of stx genes. The majority of hly-positive but stx-negative environmental isolates also exhibited a certain degree of cytotoxicity. Isolates were also tested for sorbitol utilization and were genotyped by ribotyping and by repetitive extragenic palindromic PCR (REP-PCR) as potential means of quickly identifying virulent strains from the environment, but none of these methods could be used to distinguish cytotoxic environmental isolates. Only 31% of the isolates were negative for sorbitol fermentation, and none of the isolates had common ribotypes or REP-PCR fingerprints. This study suggests that overall higher cytotoxicity values correlated with the production of stx genes, and the majority of hly-positive but stx-negative environmental isolates also exhibited a certain degree of cytotoxicity. This study demonstrated that there is widespread distribution of potentially virulent E. coli strains in the environment that may be a cause of concern for human health.     

Transduction of Enteric Escherichia coli Isolates with a Derivative of Shiga Toxin 2-Encoding Bacteriophage φ3538 Isolated from Escherichia coli O157:H7

We investigated the ability of a detoxified derivative of a Shiga toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx₂ gene of the patient E. coli O157:H7 isolate 3538/95 was replaced by the chloramphenicol acetyltransferase (cat) gene from plasmid pACYC184. Phage 3538(Δstx₂::cat) was isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and E. coli from the physiological stool microflora were infected with 3538(Δstx₂::cat), and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC strain, none of the E. coli strains supported the formation of plaques when used as indicators for 3538(Δstx₂::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of 3538(Δstx₂::cat) that formed plaques on a lawn of E. coli laboratory strain C600. The results of our study demonstrate that 3538(Δstx₂::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced infectious phage progeny. Stx-encoding bacteriophages are able to spread stx genes among enteric E. coli strains.     

Genetic Diversity and Pathogenic Potential of Attaching and Effacing Escherichia coli O26:H11 Strains Recovered from Bovine Feces in the United States

Escherichia coli O26 has been identified as the most common non-O157 Shiga toxin-producing E. coli (STEC) serogroup to cause human illnesses in the United States and has been implicated in outbreaks around the world. E. coli has high genomic plasticity, which facilitates the loss or acquisition of virulence genes. Attaching and effacing E. coli (AEEC) O26 strains have frequently been isolated from bovine feces, and there is a need to better characterize the relatedness of these strains to defined molecular pathotypes and to describe the extent of their genetic diversity. High-throughput real-time PCR was used to screen 178 E. coli O26 isolates from a single U.S. cattle feedlot, collected from May to July 2011, for the presence or absence of 25 O26 serogroup-specific and virulence-associated markers. The selected markers were capable of distinguishing these strains into molecularly defined groups (yielding 18 unique marker combinations). Analysis of the clustered regularly interspaced short palindromic repeat 1 (CRISPR1) and CRISPR2a loci further discriminated isolates into 24 CRISPR types. The combination of molecular markers and CRISPR typing provided 20.8% diversity. The recent CRISPR PCR target SP_O26-E, which was previously identified only in stx₂-positive O26:H11 human clinical strains, was identified in 96.4% (161/167 [95% confidence interval, 99.2 to 93.6%]) of the stx-negative AEEC O26:H11 bovine fecal strains. This supports that these stx-negative strains may have previously contained a prophage carrying stx or could acquire this prophage, thus possibly giving them the potential to become pathogenic to humans. These results show that investigation of specific genetic markers may further elucidate our understanding of the genetic diversity of AEEC O26 strains in bovine feces.     

Molecular characterization of Escherichia coli O157:H7 recovered from meat and meat products relevant to human health in Riyadh,Saudi Arabia

Raw meat can harbor pathogenic bacteria, potentially harmful to humans such as Escherichia coli O157:H7 causing diarrhea and hemolytic-uremic syndrome (HS). Therefore, the current study was carried out to evaluate the prevalence and the molecular detection characterization of E. coli serotype O157:H7 recovered from raw meat and meat products collected from Saudi Arabia. During the period of 25th January 2013 to 25th March 2014, 370 meat samples were collected from abattoirs and markets located in Riyadh, Saudi Arabia “200 raw meat samples and 170 meat products”. Bacteriological analysis of the meat samples and serotyping of the isolated E. coli revealed the isolation of 11 (2.97%) strains of E. coli O157:H7. Isolation of E. coli O157:H7 in raw beef, chicken and mutton were 2%, 2.5%, and 2.5%, respectively, however, there was no occurrence in raw turkey. The incidences of E. coli O157:H7 in ground beef, beef burgers, beef sausage, ground chicken and chicken burgers were 5%, 10%, 0.0%, 5% and 0.0%, respectively. The multiplex PCR assay revealed that 3 (27.27%) out of 11 E. coli O157:H7 isolates from raw beef, chicken and mutton had stx1, stx2, and eae while 5 (45.45%) E. coli O157:H7 isolates from ground beef, ground chicken, and raw beef had both stx1 and stx2. However, from beef burgers, only one E. coli O157:H7 isolate had stx1 while two were positive for hlyA gene. These results call for urgent attention toward appropriate controls and good hygienic practices in dealing with raw meat.     

Prevalence and Characterization of Shiga Toxin-Producing Escherichia coli in Swine Feces Recovered in the National Animal Health Monitoring System's Swine 2000 Study

A study was conducted to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in swine feces in the United States as part of the National Animal Health Monitoring System's Swine 2000 study. Fecal samples collected from swine operations from 13 of the top 17 swine-producing states were tested for the presence of STEC. After enrichment of swine fecal samples in tryptic soy broth, the samples were tested for the presence of stx₁ and stx₂ by use of the TaqMan E. coli STX1 and STX2 PCR assays. Enrichments of samples positive for stx₁ and/or stx₂ were plated, and colony hybridization was performed using digoxigenin-labeled probes complementary to the stx₁ and stx₂ genes. Positive colonies were picked and confirmed by PCR for the presence of the stx₁, stx₂, or stx_2e genes, and the isolates were serotyped. Out of 687 fecal samples tested using the TaqMan assays, 70% (484 of 687) were positive for Shiga toxin genes, and 54% (370 of 687), 64% (436 of 687), and 38% (261 of 687) were positive for stx₁, stx₂, and both toxin genes, respectively. Out of 219 isolates that were characterized, 29 (13%) produced stx₁, 14 (6%) produced stx₂, and 176 (80%) produced stx_2e. Twenty-three fecal samples contained at least two STEC strains that had different serotypes but that had the same toxin genes or included a strain that possessed stx₁ in addition to a strain that possessed stx₂ or stx_2e. The STEC isolates belonged to various serogroups, including O2, O5, O7, O8, O9, OX10, O11, O15, OX18, O20, O57, O65, O68, O69, O78, O91, O96, O100, O101, O120, O121, O152, O159, O160, O163, and O untypeable. It is noteworthy that no isolates of serogroup O157 were recovered. Results of this study indicate that swine in the United States harbor STEC that can potentially cause human illness.     

Shiga Toxin 2e-Producing Escherichia coli Isolates from Humans and Pigs Differ in Their Virulence Profiles and Interactions with Intestinal Epithelial Cells

Thirteen Escherichia coli strains harboring stx_2e were isolated from 11,056 human stools. This frequency corresponded to the presence of the stx_2e allele in 1.7% of all Shiga toxin-producing E. coli (STEC) strains. The strains harboring stx_2e were associated with mild diarrhea (n = 9) or asymptomatic infections (n = 4). Because STEC isolates possessing stx_2e are porcine pathogens, we compared the human STEC isolates with stx_2e-harboring E. coli isolated from piglets with edema disease and postweaning diarrhea. All pig isolates possessed the gene encoding the F18 adhesin, and the majority possessed adhesin involved in diffuse adherence; these adhesins were absent from all the human STEC isolates. In contrast, the high-pathogenicity island encoding an iron uptake system was found only in human isolates. Host-specific patterns of interaction with intestinal epithelial cells were observed. All human isolates adhered to human intestinal epithelial cell lines T84 and HCT-8 but not to pig intestinal epithelial cell line IPEC-J2. In contrast, the pig isolates completely lysed human epithelial cells but not IPEC-J2 cells, to which most of them adhered. Our data demonstrate that E. coli isolates producing Shiga toxin 2e have imported specific virulence and fitness determinants which allow them to adapt to the specific hosts in which they cause various forms of disease.