Prevalence of Carriage of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among Slaughtered Adult Cattle in France

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these “top five” STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples. O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P < 0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover, simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.     

Evaluation of a Loop-Mediated Isothermal Amplification Suite for the Rapid,Reliable, and Robust Detection of Shiga Toxin-Producing Escherichia coli in Produce

Shiga toxin-producing Escherichia coli (STEC) strains are a leading cause of produce-associated outbreaks in the United States. Rapid, reliable, and robust detection methods are needed to better ensure produce safety. We recently developed a loop-mediated isothermal amplification (LAMP) suite for STEC detection. In this study, the STEC LAMP suite was comprehensively evaluated against real-time quantitative PCR (qPCR) using a large panel of bacterial strains (n = 156) and various produce items (several varieties of lettuce, spinach, and sprouts). To simulate real-world contamination events, produce samples were surface inoculated with a low level (1.2 to 1.8 CFU/25 g) of individual STEC strains belonging to seven serogroups (O26, O45, O103, O111, O121, O145, and O157) and held at 4°C for 48 h before testing. Six DNA extraction methods were also compared using produce enrichment broths. All STEC targets and their subtypes were accurately detected by the LAMP suite. The detection limits were 1 to 20 cells per reaction in pure culture and 10⁵ to 10⁶ CFU per 25 g (i.e., 10³ to 10⁴ CFU per g) in produce, except for strains harboring the stx_2c, eae-β, and eae-θ subtypes. After 6 to 8 h of enrichment, the LAMP suite achieved accurate detection of low levels of STEC strains of various stx₂ and eae subtypes in lettuce and spinach varieties but not in sprouts. A similar trend of detection was observed for qPCR. The PrepMan Ultra sample preparation reagent yielded the best results among the six DNA extraction methods. This research provided a rapid, reliable, and robust method for detecting STEC in produce during routine sampling and testing. The challenge with sprouts detection by both LAMP and qPCR calls for special attention to further analysis.     

Prevalences of Shiga Toxin Subtypes and Selected Other Virulence Factors among Shiga-Toxigenic Escherichia coli Strains Isolated from Fresh Produce

Shiga-toxigenic Escherichia coli (STEC) strains were isolated from a variety of fresh produce, but mostly from spinach, with an estimated prevalence rate of 0.5%. A panel of 132 produce STEC strains were characterized for the presence of virulence and putative virulence factor genes and for Shiga toxin subtypes. About 9% of the isolates were found to have the eae gene, which encodes the intimin binding protein, and most of these belonged to known pathogenic STEC serotypes, such as O157:H7 and O26:H11, or to serotypes that reportedly have caused human illness. Among the eae-negative strains, there were three O113:H21 strains and one O91:H21 strain, which historically have been implicated in illness and therefore may be of concern as well. The ehxA gene, which encodes enterohemolysin, was found in ∼60% of the isolates, and the saa and subAB genes, which encode STEC agglutinating adhesin and subtilase cytotoxin, respectively, were found in ∼30% of the isolates. However, the precise roles of these three putative virulence factors in STEC pathogenesis have not yet been fully established. The stx_1a and stx_2a subtypes were present in 22% and 56%, respectively, of the strains overall and were the most common subtypes among produce STEC strains. The stx_2d subtype was the second most common subtype (28% overall), followed by stx_2c (7.5%), and only 2 to 3% of the produce STEC strains had the stx_2e and stx_2g subtypes. Almost half of the produce STEC strains had only partial serotypes or were untyped, and most of those that were identified belonged to unremarkable serotypes. Considering the uncertainties of some of these Stx subtypes and putative virulence factors in causing human illness, it is difficult to determine the health risk of many of these produce STEC strains.     

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.     

Diversity of Shiga Toxin-Producing Escherichia coli (STEC) O26:H11 Strains Examined via stx Subtypes and Insertion Sites of Stx and EspK Bacteriophages

Shiga toxin-producing Escherichia coli (STEC) is a food-borne pathogen that may be responsible for severe human infections. Only a limited number of serotypes, including O26:H11, are involved in the majority of serious cases and outbreaks. The main virulence factors, Shiga toxins (Stx), are encoded by bacteriophages. Seventy-four STEC O26:H11 strains of various origins (including human, dairy, and cattle) were characterized for their stx subtypes and Stx phage chromosomal insertion sites. The majority of food and cattle strains possessed the stx_1a subtype, while human strains carried mainly stx_1a or stx_2a. The wrbA and yehV genes were the main Stx phage insertion sites in STEC O26:H11, followed distantly by yecE and sbcB. Interestingly, the occurrence of Stx phages inserted in the yecE gene was low in dairy strains. In most of the 29 stx-negative E. coli O26:H11 strains also studied here, these bacterial insertion sites were vacant. Multilocus sequence typing of 20 stx-positive or stx-negative E. coli O26:H11 strains showed that they were distributed into two phylogenetic groups defined by sequence type 21 (ST21) and ST29. Finally, an EspK-carrying phage was found inserted in the ssrA gene in the majority of the STEC O26:H11 strains but in only a minority of the stx-negative E. coli O26:H11 strains. The differences in the stx subtypes and Stx phage insertion sites observed in STEC O26:H11 according to their origin might reflect that strains circulating in cattle and foods are clonally distinct from those isolated from human patients.     

Emerging Shiga Toxin-Producing Escherichia coli Serotypes in Europe: O100:H- and O127:H40

Novel and as yet rare non-O157 Shiga toxin (Stx)-producing Escherichia coli (STEC) serotypes are emerging in Europe. Two different sorbitol-fermenting STECs, O100:H- carrying the virulence gene stx2 and O127:H40 carrying stx1 and eae genes (found in two related subjects), were isolated from patients’ stool samples. Non-O157 STEC infections in humans are currently under-diagnosed. This report highlights the need for, and importance of, screening for Shiga toxins or serotypes other than just O157.     

Isolation of Potentially Pathogenic Escherichia coli O157:H7 from the Ganges River

Escherichia coli serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for stx₁, stx₂, and eae gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.     

Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity

We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espPα, espPβ, espPγ, and espPδ), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPβ, espPγ, and espPδ genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espPα, espPβ, espPγ, and espPδ in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspPα (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspPγ cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPβ and EspPδ either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.     

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

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

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

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

Heterogeneity of Shiga Toxin-Producing Escherichia coli Strains Isolated from Hemolytic-Uremic Syndrome Patients, Cattle, and Food Samples in Central France

A detailed analysis of the molecular epidemiology of non-O157:H7 Shiga toxin-producing Escherichia coli (STEC) was performed by using isolates from sporadic cases of hemolytic-uremic syndrome (HUS), animal reservoirs, and food products. The isolates belonged to the O91 and OX3 serogroups and were collected in the same geographical area over a short period of time. Five typing methods were used; some of these were used to explore potentially mobile elements like the stx genes or the plasmids (stx₂-restriction fragment length polymorphism [RFLP], stx₂ gene variant, and plasmid analyses), and others were used to study the whole genome (ribotyping and pulsed-field gel electrophoresis [PFGE]). The techniques revealed that there was great diversity among the O91 and OX3 STEC strains isolated in central France. A close relationship between strains of the same serotype having the same virulence factor pattern was first suggested by ribotyping. However, stx₂-RFLP and stx₂ variant analyses differentiated all but 5 of 21 isolates, and plasmid analysis revealed further heterogeneity; a unique combination of characteristics was obtained for all strains except two O91:H21 isolates from beef. The latter strains were shown by PFGE to be the most closely related isolates, with >96% homology, and hence may be subtypes of the same strain. Overall, our results indicate that the combination of stx₂-RFLP, stx₂ variant, and plasmid profile analyses is as powerful as PFGE for molecular investigation of STEC diversity. Finally, the non-O157:H7 STEC strains isolated from HUS patients were related to but not identical to those isolated from cattle and food samples in the same geographical area. The possibility that there are distinct lineages of non-O157:H7 STEC, some of which are more virulent for humans, should be investigated further.     

Prevalence of Hemolysin Genes and Comparison of ehxA Subtype Patterns in Shiga Toxin-Producing Escherichia coli (STEC) and Non-STEC Strains from Clinical,Food, and Animal Sources

Shiga toxin-producing Escherichia coli (STEC) belonging to certain serogroups (e.g., O157 and O26) can cause serious conditions like hemolytic-uremic syndrome (HUS), but other strains might be equally pathogenic. While virulence factors, like stx and eae, have been well studied, little is known about the prevalence of the E. coli hemolysin genes (hlyA, ehxA, e-hlyA, and sheA) in association with these factors. Hemolysins are potential virulence factors, and ehxA and hlyA have been associated with human illness, but the significance of sheA is unknown. Hence, 435 E. coli strains belonging to 62 different O serogroups were characterized to investigate gene presence and phenotypic expression of hemolysis. We further investigated ehxA subtype patterns in E. coli isolates from clinical, animal, and food sources. While sheA and ehxA were widely distributed, e-hlyA and hlyA were rarely found. Most strains (86.7%) were hemolytic, and significantly more hemolytic (95%) than nonhemolytic strains (49%) carried stx and/or eae (P < 0.0001). ehxA subtyping, as performed by using PCR in combination with restriction fragment length polymorphism analysis, resulted in six closely related subtypes (>94.2%), with subtypes A/D being eae-negative STECs and subtypes B, C, E, and F eae positive. Unexpectedly, ehxA subtype patterns differed significantly between isolates collected from different sources (P < 0.0001), suggesting that simple linear models of exposure and transmission need modification; animal isolates carried mostly subtypes A/C (39.3%/42.9%), food isolates carried mainly subtype A (81.9%), and clinical isolates carried mainly subtype C (66.4%). Certain O serogroups correlated with particular ehxA subtypes: subtype A with O104, O113, and O8; B exclusively with O157; C with O26, O111, and O121.     

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 and Genetic Characterization of Shiga Toxin-Producing Escherichia coli Isolates from Slaughtered Animals in Bangladesh

To determine the prevalence of Shiga toxin (Stx)-producing Escherichia coli (STEC) in slaughter animals in Dhaka, Bangladesh, we collected rectal contents immediately after animals were slaughtered. Of the samples collected from buffalo (n = 174), cows (n = 139), and goats (n = 110), 82.2%, 72.7%, and 11.8% tested positive for stx₁ and/or stx₂, respectively. STEC could be isolated from 37.9%, 20.1%, and 10.0% of the buffalo, cows, and goats, respectively. STEC O157 samples were isolated from 14.4% of the buffalo, 7.2% of the cows, and 9.1% of the goats. More than 93% (n = 42) of the STEC O157 isolates were positive for the stx₂, eae, katP, etpD, and enterohemorrhagic E. coli hly (hly_EHEC) virulence genes. STEC O157 isolates were characterized by seven recognized phage types, of which types 14 (24.4%) and 31 (24.4%) were predominant. Subtyping of the 45 STEC O157 isolates by pulsed-field gel electrophoresis showed 37 distinct restriction patterns, suggesting a heterogeneous clonal diversity. In addition to STEC O157, 71 STEC non-O157 strains were isolated from 60 stx-positive samples from 23.6% of the buffalo, 12.9% of the cows, and 0.9% of the goats. The STEC non-O157 isolates belonged to 36 different O groups and 52 O:H serotypes. Unlike STEC O157, most of the STEC non-O157 isolates (78.9%) were positive for stx₁. Only 7.0% (n = 5) of the isolates were positive for hly_EHEC, and none was positive for eae, katP, and etpD. None of the isolates was positive for the iha, toxB, and efa1 putative adhesion genes. However, 35.2% (n = 25), 11.3% (n = 8), 12.7% (n = 9), and 12.7% (n = 9) of the isolates were positive for the lpf_O113, saa, lpfA_O157/01-141, and lpfA_O157/OI-154 genes, respectively. The results of this study provide the first evidence that slaughtered animals like buffalo, cows, and goats in Bangladesh are reservoirs for STEC, including the potentially virulent STEC strain O157.     

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.     

Serotyping, stx2 Subtyping, and Characterization of the Locus of Enterocyte Effacement Island of Shiga Toxin-Producing Escherichia coli and E. coli O157:H7 Strains Isolated from the Environment in France

Twenty-seven Shiga toxin-producing Escherichia coli (STEC) strains were isolated from 207 stx-positive French environmental samples. Ten of these strains were positive for stx₁, and 24 were positive for stx₂ (10 were positive for stx_2vh-a or stx_2vh-b, 19 were positive for stx_2d, and 15 were positive for stx_2e). One strain belonged to serotype O157:H7, and the others belonged to serogroups O2, O8, O11, O26, O76, O103, O113, O121, O141, O166, and O174. The environment is a reservoir in which new clones of STEC that are pathogenic for humans can emerge.     

Heterogeneity in Induction Level,Infection Ability,and Morphology of Shiga Toxin-Encoding Phages (Stx Phages) from Dairy and Human Shiga Toxin-Producing Escherichia coli O26:H11 Isolates

Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria are foodborne pathogens responsible for diarrhea and hemolytic-uremic syndrome (HUS). Shiga toxin, the main STEC virulence factor, is encoded by the stx gene located in the genome of a bacteriophage inserted into the bacterial chromosome. The O26:H11 serotype is considered to be the second-most-significant HUS-causing serotype worldwide after O157:H7. STEC O26:H11 bacteria and their stx-negative counterparts have been detected in dairy products. They may convert from the one form to the other by loss or acquisition of Stx phages, potentially confounding food microbiological diagnostic methods based on stx gene detection. Here we investigated the diversity and mobility of Stx phages from human and dairy STEC O26:H11 strains. Evaluation of their rate of in vitro induction, occurring either spontaneously or in the presence of mitomycin C, showed that the Stx2 phages were more inducible overall than Stx1 phages. However, no correlation was found between the Stx phage levels produced and the origin of the strains tested or the phage insertion sites. Morphological analysis by electron microscopy showed that Stx phages from STEC O26:H11 displayed various shapes that were unrelated to Stx1 or Stx2 types. Finally, the levels of sensitivity of stx-negative E. coli O26:H11 to six Stx phages differed among the 17 strains tested and our attempts to convert them into STEC were unsuccessful, indicating that their lysogenization was a rare event.     

Early Attachment Sites for Shiga-Toxigenic Escherichia coli O157:H7 in Experimentally Inoculated Weaned Calves

Weaned 3- to 4-month-old calves were fasted for 48 h, inoculated with 10¹⁰ CFU of Shiga toxin-positive Escherichia coli (STEC) O157:H7 strain 86-24 (STEC O157) or STEC O91:H21 strain B2F1 (STEC O91), Shiga toxin-negative E. coli O157:H7 strain 87-23 (Stx⁻ O157), or a nonpathogenic control E. coli strain, necropsied 4 days postinoculation, and examined bacteriologically and histologically. Some calves were treated with dexamethasone (DEX) for 5 days (3 days before, on the day of, and 1 day after inoculation). STEC O157 bacteria were recovered from feces, intestines, or gall bladders of 74% (40/55) of calves 4 days after they were inoculated with STEC O157. Colon and cecum were sites from which inoculum-type bacteria were most often recovered. Histologic lesions of attaching-and-effacing (A/E) O157⁺ bacteria were observed in 69% (38/55) of the STEC O157-inoculated calves. Rectum, ileocecal valve, and distal colon were sites most likely to contain A/E O157⁺ bacteria. Fecal and intestinal levels of STEC O157 bacteria were significantly higher and A/E O157⁺ bacteria were more common in DEX-treated calves than in nontreated calves inoculated with STEC O157. Fecal STEC O157 levels were significantly higher than Stx⁻ O157, STEC O91, or control E. coli; only STEC O157 cells were recovered from tissues. Identifying the rectum, ileocecal valve, and distal colon as early STEC O157 colonization sites and finding that DEX treatment enhances the susceptibility of weaned calves to STEC O157 colonization will facilitate the identification and evaluation of interventions aimed at reducing STEC O157 infection in cattle.     

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.     

Localization of the Insertion Site and Pathotype Determination of the Locus of Enterocyte Effacement of Shiga Toxin-Producing Escherichia coli Strains

Of 220 Shiga toxin-producing Escherichia coli (STEC) strains collected in central France from healthy cattle, food samples, and asymptomatic children, 12 possessed the eae gene included in the locus of enterocyte effacement (LEE) pathogenicity island. Based on gene typing, we observed 7 different eae espA espB tir pathotypes among the 12 STEC strains and described the new espAβv variant. As previously observed, the O157 serogroup is associated with eaeγ, O26 is associated with eaeβ, and O103 is associated with eae. However, the unexpected eaeζ allele was detected in 5 of the 12 isolates. PCR amplification and pulsed-field gel electrophoresis using the I-CeuI endonuclease followed by Southern hybridization indicated that the LEE was inserted in the vicinity of the selC (three isolates), pheU (two isolates), or pheV (six isolates) tRNA gene. Six isolates harbored two or three of these tRNA loci altered by the insertion of integrase genes (CP4-int and/or int-phe), suggesting the insertion of additional foreign DNA fragments at these sites. In spite of great genetic diversity of LEE pathotypes and LEE insertion sites, bovine strains harbor alleles of LEE genes that are frequently found in clinical STEC strains isolated from outbreaks and sporadic cases around the world, underscoring the potential risk of the bovine strains on human health.