Emergence of New ST301 Shiga Toxin-Producing Escherichia coli Clones Harboring Extra-Intestinal Virulence Traits in Europe.

O80:H2 enterohemorrhagic Escherichia coli (EHEC) of sequence type ST301 is one of the main serotypes causing European hemolytic and uremic syndrome, but also invasive infections, due to extra-intestinal virulence factors (VFs). Here, we determined whether other such heteropathotypes exist among ST301. EnteroBase was screened for ST301 strains that were included in a general SNP-phylogeny. French strains belonging to a new heteropathotype clone were sequenced. ST, hierarchical clusters (HC), serotype, resistome, and virulome were determined using EnteroBase, the CGE website, and local BLAST. The ST301 general phylogeny shows two groups. Group A (n = 25) is mainly composed of enteropathogenic E. coli, whereas group B (n = 55) includes mostly EHEC. Three serotypes, O186:H2, O45:H2 and O55:H9, share the same virulome as one of the O80:H2 sub-clones from which they derive subsequent O-antigen switches. The O55:H9 clone, mainly present in France (n = 29), as well as in the UK (n = 5) and Germany (n = 1), has a low background of genetic diversity (four HC20), although it has three Stx subtypes, an H-antigen switch, and genes encoding the major extra-intestinal VF yersiniabactin, and extended-spectrum beta-lactamases. Diverse heteropathotype clones genetically close to the O80:H2 clone are present among the ST301, requiring close European monitoring, especially the virulent O55:H9 clone.

EHEC O111:H8 strain and norovirus GII.4 Sydney [P16] causing an outbreak in a daycare center, Brazil, 2019.

BACKGROUND: This study describes the investigation of an outbreak of diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) at a daycare center in southeastern Brazil, involving fourteen children, six staff members, six family members, and one nurse. All bacterial and viral pathogens detected were genetically characterized. RESULTS: Two isolates of a strain of enterohemorrhagic Escherichia coli (EHEC) serotype O111:H8 were recovered, one implicated in a case of HUS and the other in a case of uncomplicated diarrhea. These isolates had a clonal relationship of 94% and carried the stx2a and eae virulence genes and the OI-122 pathogenicity island. The EHEC strain was determined to be a single-locus variant of sequence type (ST) 327. EHEC isolates were resistant to ofloxacin, doxycycline, tetracycline, ampicillin, and trimethoprim-sulfamethoxazole and intermediately resistant to levofloxacin and ciprofloxacin. Rotavirus was not detected in any samples, and norovirus was detected in 46.7% (14/30) of the stool samples, three of which were from asymptomatic staff members. The noroviruses were classified as the recombinant GII.4 Sydney [P16] by gene sequencing. CONCLUSION: In this outbreak, it was possible to identify an uncommon stx2a + EHEC O111:H8 strain, and the most recent pandemic norovirus strain GII.4 Sydney [P16]. Our findings reinforce the need for surveillance and diagnosis of multiple enteric pathogens by public health authorities, especially during outbreaks.

Genetic characterization of Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 from diarrhoeic and septicaemic calves and relatedness to human Shigatoxigenic E. coli O80:H2.

AIM: The purpose of this work was to identify and genetically characterize enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) O80:H2 from diarrhoeic and septicaemic calves in Belgium and to comparing them with human EHEC after whole genome sequencing. METHODS AND RESULTS: Ten EHEC and 21 EPEC O80 identified by PCR between 2009 and 2018 from faeces, intestinal content and a kidney of diarrhoeic or septicaemic calves were genome sequenced and compared to 19 human EHEC identified between 2008 and 2019. They all belonged to the O80:H2 serotype and ST301, harboured the eaeξ gene, and 23 of the 29 EHEC contained the stx2d gene. Phylogenetically, they were distributed in two major sub-lineages: one comprised a majority of bovine EPEC whereas the second one comprised a majority of stx2d bovine and human EHEC. CONCLUSIONS: Not only EPEC but also EHEC O80:H2 are present in diarrhoeic and septicaemic calves in Belgium and are genetically related to human EHEC. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings support the need to assess cattle as potential source of contamination of humans by EHEC O80:H2 and to understand the evolution of bovine and human EHEC and EPEC O80:H2.

Enterohaemorrhagic Escherichia coli O121:H19 acquired an extended-spectrum ß-lactamase gene during the development of an outbreak in two nurseries.

Enterohaemorrhagic Escherichia coli (EHEC) is an important human pathogen worldwide. Although serotype O157 is currently the most dominant and important EHEC strain, serotypes O26, O111, O91, O103 and O121 are also recognized as serious pathogens that affect public health. EHEC outbreaks often occur in nurseries and elderly care facilities. In 2012, a nursery outbreak of EHEC O121 occurred during which the bacterium acquired a plasmid-borne extended-spectrum ß-lactamase (ESBL) gene. ESBL-producing E. coli O86 was concurrently isolated from one of the EHEC patients. Therefore, we investigated the isolates by whole-genome sequence (WGS) analysis to elucidate the transmission dynamics of the EHEC strains and the ESBL plasmid. According to WGS-based phylogeny, all 17 EHEC O121 isolates were clonal, while E. coli O86 was genetically distant from the EHEC O121 isolates. The complete sequence of an ESBL plasmid encoding the CTX-M-55 ß-lactamase was determined using S1-PFGE bands, and subsequent mapping of the WGS reads confirmed that the plasmid sequences from EHEC O121 and E. coli O86 were identical. Furthermore, conjugation experiments showed that the plasmid was capable of conjugative transfer. These results support the hypothesis that EHEC O121 acquired an ESBL-producing plasmid from E. coli O86 during the outbreak. This report demonstrates the importance of implementing preventive measures during EHEC outbreaks to control both secondary infection and the spread of antimicrobial resistance factors.

Attack of the clones: whole genome-based characterization of two closely related enterohemorrhagic Escherichia coli O26 epidemic lineages.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O26:H11/H-, the most common non-O157 serotype causing hemolytic uremic syndrome worldwide, are evolutionarily highly dynamic with new pathogenic clones emerging rapidly. Here, we investigated the population structure of EHEC O26 isolated from patients in several European countries using whole genome sequencing, with emphasis on a detailed analysis of strains of the highly virulent new European clone (nEC) which has spread since 1990s. RESULTS: Genome-wide single nucleotide polymorphism (SNP)-based analysis of 32 EHEC O26 isolated in the Czech Republic, Germany, Austria and Italy demonstrated a split of the nEC (ST29C2 clonal group) into two distinct lineages, which we termed, based on their temporal emergence, as "early" nEC and "late" nEC. The evolutionary divergence of the early nEC and late nEC is marked by the presence of 59 and 70 lineage-specific SNPs (synapomorphic mutations) in the genomes of the respective lineages. In silico analyses of publicly available E. coli O26 genomic sequences identified the late nEC lineage worldwide. Using a PCR designed to target the late nEC synapomorphic mutation in the sen/ent gene, we identified the early nEC decline accompanied by the late nEC rise in Germany and the Czech Republic since 2004 and 2013, respectively. Most of the late nEC strains harbor one of two major types of Shiga toxin 2a (Stx2a)-encoding prophages. The type I stx2a-phage is virtually identical to stx2a-phage of EHEC O104:H4 outbreak strain, whereas the type II stx2a-phage is a hybrid of EHEC O104:H4 and EHEC O157:H7 stx2a-phages and carries a novel mutation in Stx2a. Strains harboring these two phage types do not differ by the amounts and biological activities of Stx2a produced. CONCLUSIONS: Using SNP-level analyses, we provide the evidence of the evolutionary split of EHEC O26:H11/H- nEC into two distinct lineages, and a recent replacement of the early nEC by the late nEC in Germany and the Czech Republic. PCR targeting the late nEC synapomorphic mutation in ent/sen enables the discrimination of early nEC strains and late nEC strains in clinical and environmental samples, thereby facilitating further investigations of their geographic distribution, prevalence, clinical significance and epidemiology.

Long-Term Survival and Thermal Death Kinetics of Enterohemorrhagic Escherichia coli Serogroups O26, O103, O111, and O157 in Wheat Flour.

Wheat flour has been associated with outbreaks of enterohemorrhagic Escherichia coli (EHEC), but little is known on EHEC's survival during storage and thermal processing. The objective of this study was to determine long-term viability and thermal inactivation kinetics of EHEC serogroups O26, O103, O111, and O157. Wheat flour samples were inoculated with a cocktail of five strains of a single serogroup and stored at 23 and 35°C. Inoculated samples were heated at 55, 60, 65, and 70°C. Viability was determined by plate counting. Decimal reduction time (D) and first decimal reduction time (δ) values were calculated with log-linear and Weibull models, respectively. At 23°C, EHEC counts declined gradually for 84 days and samples tested positive from 84 to 280 days. The thermal resistance (D and δ) values ranged from 7.5 to 8.2 and 3.1 to 5.3 days, respectively, but there were no significant differences among serogroups (P ≤ 0.05). At 35°C, no EHEC was quantifiable by day 7 and no positive samples were detected after 49 days. Heating at 55 and 65°C resulted in δ-value ranges of 15.6 to 39.7 min and 3.0 to 3.9 min, respectively, with no significant difference among serogroups either. Z values were 12.6, 6.7, 10.2, and 13.4°C for O26, O103, O111, and O157, respectively. Thermal death kinetics of EHEC in flour were better described using the Weibull model. Survival and inactivation rates of four serogroups were remarkably similar. These findings indicated that all EHEC serovars tested remained viable for at least 9 months at room temperature and survived for up to 60 min at 70°C in wheat flour.IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) and Salmonella have recently caused several gastroenteritis outbreaks and recalls of wheat flour. Because EHEC can cause illness with very low doses and there is very scarce information regarding their ability to survive storage and heating in flour, the present study was undertaken to assess the long-term survival of EHEC serogroups O26, O103, O111, and O157 in flour. These findings are relevant, as we report that EHEC can survive for more than 9 months in wheat flour during storage. In addition, results obtained suggest that thermal inactivation at 65°C for 30 min or 2 months of storage at 35°C may be feasible strategies to mitigate the risk of most EHEC serovars in wheat flour.

A four-season longitudinal study of enterohaemorrhagic Escherichia coli in beef cow-calf herds in Mississippi and Nebraska.

Our objective was to describe the probability of detecting seven serogroups of enterohaemorrhagic Escherichia coli (EHEC-7) of public health importance in faecal samples from beef cow-calf herds and to test for factors associated with their detection. Fresh faecal samples (n = 85) from two Mississippi and two Nebraska herds were collected in each of four seasons. Samples were tested for each EHEC-7 serogroup by a molecular screening assay. Separate management groups within herds were sampled, and group-level factors were recorded. To measure the effects of factors on faecal shedding of EHEC-7, separate multivariable logistic regression models were used, accounting for the random effect of clustering by group within farm. Statistical significance was set α = 0.05. Fifty-nine samples (4.3%) were positive for EHEC O26, and Nebraska samples were more likely to be positive than Mississippi samples (OR = 12.4, 95% CI: 1.1, 139.2). Forty-four samples (3.2%) were positive for EHEC O45. Odds for detection were greater in the summer than all other seasons combined (OR = 4.2, 95% CI: 1.3, 14.0), and odds decreased if a precipitation event occurred (OR = 0.07, 95% CI: 0.006, 0.8). EHEC O103 was detected in 66 samples (4.9%) with increased probability to be detected at increased temperature. EHEC O111 was detected in 71 samples (5.2%), and 43 samples (3.2%) were positive for EHEC O145. Both EHEC O111 and O145 were associated separately with season, with greater probability for detection in the summer. Eighteen (1.3%) and 68 (5.0%) samples were positive for EHEC O121 and EHEC O157, respectively. We failed to detect significant explanatory factors associated with probability to detect EHEC O121 or O157. Factors that vary by time and place, such as precipitation, ambient temperature, region and season, are uniquely associated with the probability to detect EHEC-7 in fresh faeces collected from cow-calf herds.

Enterohemorrhagic Escherichia coli O157 subclade 8b strains in Chiba Prefecture, Japan, produced larger amounts of Shiga toxin 2 than strains in subclade 8a and other clades.

Enterohemorrhagic Escherichia coli O157 (O157) strains can be classified into clades (one of several phylogenetic groups) by single nucleotide polymorphisms (SNPs): these are clade 1, clade 2, clade 3, descendant and ancestral clades 4/5, clade 6, clade 7, clade 8, clade 9, and clade 12. Some recent studies showed that some O157 strains in clade 8 produced a larger amount of Shiga toxin (Stx) 2 than other strains. In this study, 1121 epidemiologically unlinked strains of O157 isolated in Chiba Prefecture, Japan were classified into clades during 1996-2014. Clade 8 strains were further classified into subclade 8a (67 strains) and subclade 8b (48 strains) using SNP analysis. In the absence of mitomycin C (MMC), subclade 8a strains in this study produced significantly greater amounts of Stx2 than subclade 8b strains. However, in the presence of MMC, the levels of Stx2 production in subclade 8b strains were significantly greater than subclade 8a strains. On the other hand, a recent study reported that the Stx2 production level in O157 strains was determined mainly by the subtypes of Stx2a phage (ϕStx2_α, ß, γ, δ, ε, and ζ). Using O157 strains in this study, the Stx2a phages were classified into these subtypes. In this study, all strains of subclades 8a and 8b carried ϕStx2a_γ and ϕStx2a_δ, respectively. Some strains in clade 6 also carried ϕStx2a_δ. In the presence of MMC, subclade 8b strains produced significantly greater amounts of Stx2 than clade 6 strains carrying ϕStx2_δ. In this study, we propose that Stx2 production in subclade 8b strains in the presence of MMC might be enhanced due to genetic factors other than ϕStx2_δ.

Culture-Based Quantification with Molecular Characterization of Non-O157 and O157 Enterohemorrhagic Escherichia coli Isolates from Rectoanal Mucosal Swabs of Feedlot Cattle.

Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens carried in the intestinal tracts of ruminants and shed in the feces. High concentrations (≥104 colony-forming units [CFU]/g) of EHEC in cattle feces are associated with contamination of hides, and subsequently, carcasses and beef. Several studies using agar media have quantified O157 but few have quantified non-O157 EHEC in samples from cattle. Thus, the objective of this study was to determine the concentration of O157 and non-O157 EHEC in cattle, and to characterize the associated EHEC isolates for their virulence potential. Two hundred feedlot steers were sampled by rectoanal mucosal swab (RAMS) every 35 days over four sampling periods, and a spiral plating method using modified Possé differential agar was used to quantify EHEC organisms in these samples. Bacterial colonies from agar plates were tested by multiplex PCR for Shiga toxin and intimin genes (stx and eae, respectively), and confirmed EHEC isolates (i.e., positive for both stx and eae) were serotyped and characterized for virulence genes using a microarray. Organisms detected in this study included O26, O101, O103, O109, O121, O145, O157, and O177 EHEC, with all except O121 quantifiable and measuring within a range from 9.0 × 102 to 3.0 × 105 CFU/g of RAMS sample. Organisms of the same EHEC serogroup were not detected in quantifiable concentrations from a single animal more than once. EHEC organisms most commonly detected at quantifiable levels were O26, O157, and O177. Interestingly, O26 EHEC isolates tested negative for stx1 but positive for stx2a. High concentrations of EHEC were detected in 11 (5.5%) of the steers at least once over the sampling period. These results indicate that in addition to O157, non-O157 EHEC are transiently present in high concentrations in the rectoanal mucosal region of cattle.

Population structure of Escherichia coli O26 : H11 with recent and repeated stx2 acquisition in multiple lineages.

A key virulence factor of enterohaemorrhagic Escherichia coli (EHEC) is the bacteriophage-encoded Shiga toxin (Stx). Stxs are classified into two types, Stx1 and Stx2, and Stx2-producing strains are thought to cause more severe infections than strains producing only Stx1. Although O26 : H11 is the second most prevalent EHEC following O157 : H7, the majority of O26 : H11 strains produce Stx1 alone. However, Stx2-producing O26 strains have increasingly been detected worldwide. Through a large-scale genome analysis, we present a global phylogenetic overview and evolutionary timescale for E. coli O26 : H11. The origin of O26 has been estimated to be 415 years ago. Sequence type 21C1 (ST21C1), one of the two sublineages of ST21, the most predominant O26 : H11 lineage worldwide, emerged 213 years ago from one of the three ST29 sublineages (ST29C2). The other ST21 lineage (ST21C2) emerged 95 years ago from ST21C1. Increases in population size occurred in the late 20th century for all of the O26 lineages, but most remarkably for ST21C2. Analysis of the distribution of stx2-positive strains revealed the recent and repeated acquisition of the stx2 gene in multiple lineages of O26, both in ST21 and ST29. Other major EHEC virulence genes, such as type III secretion system effector genes and plasmid-encoded virulence genes, were well conserved in ST21 compared to ST29. In addition, more antimicrobial-resistance genes have accumulated in the ST21C1 lineage. Although current attention is focused on several highly virulent ST29 clones that have acquired the stx2 gene, there is also a considerable risk that the ST21 lineage could yield highly virulent clones.

Genome sequencing and comparative genomics of enterohemorrhagic Escherichia coli O145:H25 and O145:H28 reveal distinct evolutionary paths and marked variations in traits associated with virulence & colonization.

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) O145 are among the top non-O157 serogroups associated with severe human disease worldwide. Two serotypes, O145:H25 and O145:H28 have been isolated from human patients but little information is available regarding the virulence repertoire, origin and evolutionary relatedness of O145:H25. Hence, we sequenced the complete genome of two O145:H25 strains associated with hemolytic uremic syndrome (HUS) and compared the genomes with those of previously sequenced O145:H28 and other EHEC strains. RESULTS: The genomes of the two O145:H25 strains were 5.3 Mbp in size; slightly smaller than those of O145:H28 and other EHEC strains. Both strains contained three nearly identical plasmids and several prophages and integrative elements, many of which differed significantly in size, gene content and organization as compared to those present in O145:H28 and other EHECs. Furthermore, notable variations were observed in several fimbrial gene cluster and intimin types possessed by O145:H25 and O145:H28 indicating potential adaptation to distinct areas of host colonization. Comparative genomics further revealed that O145:H25 are genetically more similar to other non-O157 EHEC strains than to O145:H28. CONCLUSION: Phylogenetic analysis accompanied by comparative genomics revealed that O145:H25 and O145:H28 evolved from two separate clonal lineages and that horizontal gene transfer and gene loss played a major role in the divergence of these EHEC serotypes. The data provide further evidence that ruminants might be a possible reservoir for O145:H25 but that they might be impaired in their ability to establish a persistent colonization as compared to other EHEC strains.

Computational Analysis of Host-Pathogen Protein Interactions between Humans and Different Strains of Enterohemorrhagic Escherichia coli.

Serotype O157:H7, an enterohemorrhagic Escherichia coli (EHEC), is known to cause gastrointestinal and systemic illnesses ranging from diarrhea and hemorrhagic colitis to potentially fatal hemolytic uremic syndrome. Specific genetic factors like ompA, nsrR, and LEE genes are known to play roles in EHEC pathogenesis. However, these factors are not specific to EHEC and their presence in several non-pathogenic strains indicates that additional factors are involved in pathogenicity. We propose a comprehensive effort to screen for such potential genetic elements, through investigation of biomolecular interactions between E. coli and their host. In this work, an in silico investigation of the protein-protein interactions (PPIs) between human cells and four EHEC strains (viz., EDL933, Sakai, EC4115, and TW14359) was performed in order to understand the virulence and host-colonization strategies of these strains. Potential host-pathogen interactions (HPIs) between human cells and the "non-pathogenic" E. coli strain MG1655 were also probed to evaluate whether and how the variations in the genomes could translate into altered virulence and host-colonization capabilities of the studied bacterial strains. Results indicate that a small subset of HPIs are unique to the studied pathogens and can be implicated in virulence. This subset of interactions involved E. coli proteins like YhdW, ChuT, EivG, and HlyA. These proteins have previously been reported to be involved in bacterial virulence. In addition, clear differences in lineage and clade-specific HPI profiles could be identified. Furthermore, available gene expression profiles of the HPI-proteins were utilized to estimate the proportion of proteins which may be involved in interactions. We hypothesized that a cumulative score of the ratios of bound:unbound proteins (involved in HPIs) would indicate the extent of colonization. Thus, we designed the Host Colonization Index (HCI) measure to determine the host colonization potential of the E. coli strains. Pathogenic strains of E. coli were observed to have higher HCIs as compared to a non-pathogenic laboratory strain. However, no significant differences among the HCIs of the two pathogenic groups were observed. Overall, our findings are expected to provide additional insights into EHEC pathogenesis and are likely to aid in designing alternate preventive and therapeutic strategies.

Feedlot- and Pen-Level Prevalence of Enterohemorrhagic Escherichia coli in Feces of Commercial Feedlot Cattle in Two Major U.S. Cattle Feeding Areas.

The objective of this study was to determine feedlot- and pen-level fecal prevalence of seven enterohemorrhagic Escherichia coli (EHEC) belonging to serogroups (O26, O45, O103, O111, O121, O145, and O157, or EHEC-7) in feces of feedlot cattle in two feeding areas in the United States. Cattle pens from four commercial feedlots in each of the two major U.S. beef cattle areas were sampled. Up to 16 pen-floor fecal samples were collected from each of 4-6 pens per feedlot, monthly, for a total of three visits per feedlot, from June to August, 2014. Culture procedures including fecal enrichment in E. coli broth, immunomagnetic separation, and plating on selective media, followed by confirmation through polymerase chain reaction (PCR) testing, were conducted. Generalized linear mixed models were fitted to estimate feedlot-, pen-, and sample-level fecal prevalence of EHEC-7 and to evaluate associations between potential demographic and management risk factors with feedlot and within-pen prevalence of EHEC-7. All study feedlots and 31.0% of the study pens had at least one non-O157 EHEC-positive fecal sample, whereas 62.4% of pens tested positive for EHEC O157; sample-level prevalence estimates ranged from 0.0% for EHEC O121 to 18.7% for EHEC O157. Within-pen prevalence of EHEC O157 varied significantly by sampling month; similarly within-pen prevalence of non-O157 EHEC varied significantly by month and by the sex composition of the pen (heifer, steer, or mixed). Feedlot management factors, however, were not significantly associated with fecal prevalence of EHEC-7. Intraclass correlation coefficients for EHEC-7 models indicated that most of the variation occurred between pens, rather than within pens, or between feedlots. Hence, the potential combination of preharvest interventions and pen-level management strategies may have positive food safety impacts downstream along the beef chain.

Enterohemorrhagic Escherichia coli pathogenesis: role of Long polar fimbriae in Peyer's patches interactions.

Enterohemorrhagic Escherichia coli (EHEC) are major food-borne pathogens whose survival and virulence in the human digestive tract remain unclear owing to paucity of relevant models. EHEC interact with the follicle-associated epithelium of Peyer's patches of the distal ileum and translocate across the intestinal epithelium via M-cells, but the underlying molecular mechanisms are still unknown. Here, we investigated the involvement of Long polar fimbriae (Lpf) in EHEC pathogenesis. Of the 236 strains tested, a significant association was observed between the presence of lpf operons and pathogenicity. In sophisticated in vitro models of the human gastro-intestinal tract, lpf expression was induced during transit through the simulated stomach and small intestine, but not in the colonic compartment. To investigate the involvement of Lpf in EHEC pathogenesis, lpf isogenic mutants and their relative trans-complemented strains were generated. Translocation across M-cells, interactions with murine ileal biopsies containing Peyer's patches and the number of hemorrhagic lesions were significantly reduced with the lpf mutants compared to the wild-type strain. Complementation of lpf mutants fully restored the wild-type phenotypes. Our results indicate that (i) EHEC might colonize the terminal ileum at the early stages of infection, (ii) Lpf are an important player in the interactions with Peyer's patches and M-cells, and could contribute to intestinal colonization.

Identification of a New Virulent Clade in Enterohemorrhagic Escherichia coli O26:H11/H- Sequence Type 29.

Enterohemorrhagic Escherichia coli (EHEC) O26 infections cause severe human diseases such as hemolytic uremic syndrome and encephalopathy, and is the predominant serogroup among non-O157 EHEC in many countries. Shiga toxin (Stx), which consists of two distinct types (Stx1 and Stx2), plays a central role in EHEC pathogenesis. The major stx gene type in EHEC O26 strains is stx1, although isolates with only stx2 have emerged in Japan since 2012 and have been reported in Europe. In this study, we selected 27 EHEC O26 strains isolated in Japan and identified a distinct genetic clade within sequence type (ST) 29, designated ST29C1, that carried only stx2 and had the plasmid gene profile ehxA+/katP-/espP+/etpD-. We showed that ST29C1 strains produced higher Stx2a levels, and greater virulence in Vero cells and in germ-free mice than other lineages. We also showed that ST29C1 was a distinct phylogenetic clade by SNP analysis using whole genome sequences and clearly differed from the major European EHEC O26 virulent clone, which was designated ST29C2 in this study. The combination of toxin production analysis, virulence analysis in Vero cells and germ-free mice, and phylogenetic analysis identified a newly emerging virulent EHEC clade.

Comparison of Enterohemorrhagic Escherichia coli (EHEC) O157 and EHEC Non-O157 Isolates from Patients with Diarrhea in Korea.

We compared 47 enterohemorrhagic Escherichia coli (EHEC) O157 isolates with 184 EHEC non-O157 isolates from Korean patients with diarrhea. In the O157 group, the strains harboring both Shiga toxin genes (stx1 and stx2) were detected with highest frequency, whereas the strains harboring only stx1 gene were most frequently detected in the non-O157 group. Eight virulence genes (eaeA, hlyA, ehx, iha, efa1, tir, toxB, and espA) were found to show a higher frequency of occurrence in the O157 group than in the non-O157 group. In addition, the symptom of bloody diarrhea was exhibited at a higher rate in the O157 group (51.1%) than in the non-O157 group (16.8%). Our findings demonstrate that EHEC O157 strains are more frequently implicated in cases of bloody diarrhea in the Korean population than EHEC non-O157 strains.

Are Escherichia coli Pathotypes Still Relevant in the Era of Whole-Genome Sequencing?

The empirical and pragmatic nature of diagnostic microbiology has given rise to several different schemes to subtype E.coli, including biotyping, serotyping, and pathotyping. These schemes have proved invaluable in identifying and tracking outbreaks, and for prognostication in individual cases of infection, but they are imprecise and potentially misleading due to the malleability and continuous evolution of E. coli. Whole genome sequencing can be used to accurately determine E. coli subtypes that are based on allelic variation or differences in gene content, such as serotyping and pathotyping. Whole genome sequencing also provides information about single nucleotide polymorphisms in the core genome of E. coli, which form the basis of sequence typing, and is more reliable than other systems for tracking the evolution and spread of individual strains. A typing scheme for E. coli based on genome sequences that includes elements of both the core and accessory genomes, should reduce typing anomalies and promote understanding of how different varieties of E. coli spread and cause disease. Such a scheme could also define pathotypes more precisely than current methods.

Fitness of Enterohemorrhagic Escherichia coli (EHEC)/Enteroaggregative E. coli O104:H4 in Comparison to That of EHEC O157: Survival Studies in Food and In Vitro.

In 2011, one of the world's largest outbreaks of hemolytic-uremic syndrome (HUS) occurred, caused by a rare Escherichia coli serotype, O104:H4, that shared the virulence profiles of Shiga toxin-producing E. coli (STEC)/enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC). The persistence and fitness factors of the highly virulent EHEC/EAEC O104:H4 strain, grown either in food or in vitro, were compared with those of E. coli O157 outbreak-associated strains. The log reduction rates of the different EHEC strains during the maturation of fermented sausages were not significantly different. Both the O157:NM and O104:H4 serotypes could be shown by qualitative enrichment to be present after 60 days of sausage storage. Moreover, the EHEC/EAEC O104:H4 strain appeared to be more viable than E. coli O157:H7 under conditions of decreased pH and in the presence of sodium nitrite. Analysis of specific EHEC strains in experiments with an EHEC inoculation cocktail showed a dominance of EHEC/EAEC O104:H4, which could be isolated from fermented sausages for 60 days. Inhibitory activities of EHEC/EAEC O104:H4 toward several E. coli strains, including serotype O157 strains, could be determined. Our study suggests that EHEC/EAEC O104:H4 is well adapted to the multiple adverse conditions occurring in fermented raw sausages. Therefore, it is strongly recommended that STEC strain cocktails composed of several serotypes, instead of E. coli O157:H7 alone, be used in food risk assessments. The enhanced persistence of EHEC/EAEC O104:H4 as a result of its robustness, as well as the production of bacteriocins, may account for its extraordinary virulence potential. IMPORTANCE: In 2011, a severe outbreak caused by an EHEC/EAEC serovar O104:H4 strain led to many HUS sequelae. In this study, the persistence of the O104:H4 strain was compared with those of other outbreak-relevant STEC strains under conditions of fermented raw sausage production. Both O157:NM and O104:H4 strains could survive longer during the production of fermented sausages than E. coli O157:H7 strains. E. coli O104:H4 was also shown to be well adapted to the multiple adverse conditions encountered in fermented sausages, and the secretion of a bacteriocin may explain the competitive advantage of this strain in an EHEC strain cocktail. Consequently, this study strongly suggests that enhanced survival and persistence, and the presumptive production of a bacteriocin, may explain the increased virulence of the O104:H4 outbreak strain. Furthermore, this strain appears to be capable of surviving in a meat product, suggesting that meat should not be excluded as a source of potential E. coli O104:H4 infection.

Enterohemorrhagic Escherichia coli Hybrid Pathotype O80:H2 as a New Therapeutic Challenge.

We describe the epidemiology, clinical features, and molecular characterization of enterohemorrhagic Escherichia coli (EHEC) infections caused by the singular hybrid pathotype O80:H2, and we examine the influence of antibiotics on Shiga toxin production. In France, during 2005-2014, a total of 54 patients were infected with EHEC O80:H2; 91% had hemolytic uremic syndrome. Two patients had invasive infections, and 2 died. All strains carried stx2 (variants stx2a, 2c, or 2d); the rare intimin gene (eae-ξ); and at least 4 genes characteristic of pS88, a plasmid associated with extraintestinal virulence. Similar strains were found in Spain. All isolates belonged to the same clonal group. At subinhibitory concentrations, azithromycin decreased Shiga toxin production significantly, ciprofloxacin increased it substantially, and ceftriaxone had no major effect. Antibiotic combinations that included azithromycin also were tested. EHEC O80:H2, which can induce hemolytic uremic syndrome complicated by bacteremia, is emerging in France. However, azithromycin might effectively combat these infections.

Basic Reproduction Number and Transmission Dynamics of Common Serogroups of Enterohemorrhagic Escherichia coli.

UNLABELLED: Understanding the transmission dynamics of pathogens is essential to determine the epidemiology, ecology, and ways of controlling enterohemorrhagic Escherichia coli (EHEC) in animals and their environments. Our objective was to estimate the epidemiological fitness of common EHEC strains in cattle populations. For that purpose, we developed a Markov chain model to characterize the dynamics of 7 serogroups of enterohemorrhagic Escherichia coli (O26, O45, O103, O111, O121, O145, and O157) in cattle production environments based on a set of cross-sectional data on infection prevalence in 2 years in two U.S. states. The basic reproduction number (R0) was estimated using a Bayesian framework for each serogroup based on two criteria (using serogroup alone [the O-group data] and using O serogroup, Shiga toxin gene[s], and intimin [eae] gene together [the EHEC data]). In addition, correlations between external covariates (e.g., location, ambient temperature, dietary, and probiotic usage) and prevalence/R0 were quantified. R0 estimates varied substantially among different EHEC serogroups, with EHEC O157 having an R0 of >1 (∼1.5) and all six other EHEC serogroups having an R0 of less than 1. Using the O-group data substantially increased R0 estimates for the O26, O45, and O103 serogroups (R0 > 1) but not for the others. Different covariates had distinct influences on different serogroups: the coefficients for each covariate were different among serogroups. Our modeling and analysis of this system can be readily expanded to other pathogen systems in order to estimate the pathogen and external factors that influence spread of infectious agents. IMPORTANCE: In this paper we describe a Bayesian modeling framework to estimate basic reproduction numbers of multiple serotypes of Shiga toxin-producing Escherichia coli according to a cross-sectional study. We then coupled a compartmental model to reconstruct the infection dynamics of these serotypes and quantify their risk in the population. We incorporated different sensitivity levels of detecting different serotypes and evaluated their potential influence on the estimation of basic reproduction numbers.