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 (stx(2)-restriction fragment length polymorphism [RFLP], stx(2) 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, stx(2)-RFLP and stx(2) 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 stx(2)-RFLP, stx(2) 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 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.     

Development of a Robust Method for Isolation of Shiga Toxin-Positive Escherichia coli (STEC) from Fecal,Plant, Soil and Water Samples from a Leafy Greens Production Region in California

During a 2.5-year survey of 33 farms and ranches in a major leafy greens production region in California, 13,650 produce, soil, livestock, wildlife, and water samples were tested for Shiga toxin (stx)-producing Escherichia coli (STEC). Overall, 357 and 1,912 samples were positive for E. coli O157:H7 (2.6%) or non-O157 STEC (14.0%), respectively. Isolates differentiated by O-typing ELISA and multilocus variable number tandem repeat analysis (MLVA) resulted in 697 O157:H7 and 3,256 non-O157 STEC isolates saved for further analysis. Cattle (7.1%), feral swine (4.7%), sediment (4.4%), and water (3.3%) samples were positive for E. coli O157:H7; 7/32 birds, 2/145 coyotes, 3/88 samples from elk also were positive. Non-O157 STEC were at approximately 5-fold higher incidence compared to O157 STEC: cattle (37.9%), feral swine (21.4%), birds (2.4%), small mammals (3.5%), deer or elk (8.3%), water (14.0%), sediment (12.3%), produce (0.3%) and soil adjacent to produce (0.6%). stx1, stx2 and stx1/stx2 genes were detected in 63%, 74% and 35% of STEC isolates, respectively. Subtilase, intimin and hemolysin genes were present in 28%, 25% and 79% of non-O157 STEC, respectively; 23% were of the “Top 6″ O-types. The initial method was modified twice during the study revealing evidence of culture bias based on differences in virulence and O-antigen profiles. MLVA typing revealed a diverse collection of O157 and non-O157 STEC strains isolated from multiple locations and sources and O157 STEC strains matching outbreak strains. These results emphasize the importance of multiple approaches for isolation of non-O157 STEC, that livestock and wildlife are common sources of potentially virulent STEC, and evidence of STEC persistence and movement in a leafy greens production environment.     

Non-O157:H7 Shiga toxin (verocytotoxin)-producing Escherichia coli strains: epidemiological significance and microbiological diagnosis

Shiga toxin (Stx)-producing Escherichia coli (STEC) are important causes of diarrhoea and the haemolytic uremic syndrome (HUS). The most common STEC serotype implicated worldwide is E. coli O157:H7 that is diagnosed using procedures based on its typical phenotypic feature, the lack of sorbitol fermentation. In addition to E. coli O157:H7, a variety of non-O157:H7 STEC strains that usually ferment sorbitol and are thus missed by using the diagnostic protocol for E.coli O157:H7 have been isolated from patients. Among these sorbitol-fermenting (SF) non-O157:H7 STEC, SF E. coli O157:H⁻ and non-O157 STEC strains of serogroups O26, O103, O111 and O145 have emerged as significant causes of HUS and diarrhoea in continental Europe and have been associated with human disease in other parts of the world. Microbiological diagnosis of non-O157:H7 STEC strains is difficult due to their serotype diversity and the absence of a simple biochemical property that distinguishes such strains from the physiological intestinal microflora. Screening for non-O157:H7 STEC and their isolation from stools is presently based on the detection of Stx production or stx genes that are common characteristics of such strains. Molecular subtyping of the most frequent non-O157 STEC demonstrated that strains of serogroups O26, O103 and O111 belong to their own clonal lineages and show unique virulence profiles. SF STEC O157:H⁻ strains that have been isolated mostly in Central Europe represent a new clone within E. coli O157 serogroup which has its own typical combination of virulence factors. This revised version was published online in November 2006 with corrections to the Cover Date.     

Characterization of Shiga Toxin-Producing Escherichia coli Isolates Associated with Two Multistate Food-Borne Outbreaks That Occurred in 2006

Shiga toxin-producing Escherichia coli isolates from two 2006 outbreaks were compared to other O157:H7 isolates for virulence genotype, biofilm formation, and stress responses. Spinach- and lettuce-related-outbreak strains had similar pulsed-field gel electrophoresis patterns, and all carried both stx₂ and stx_2c variant genes. Cooperative biofilm formation involving an E. coli O157:H7 strain and a non-O157:H7 strain was also demonstrated.     

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.     

Characterization of Shiga toxin-producing Escherichia coli isolated from healthy pigs in China

Background

Shiga toxin-producing Escherichia coli (STEC) is recognized as an important human diarrheal pathogen. Swine plays an important role as a carrier of this pathogen. In this study we determined the prevalence and characteristics of STEC from healthy swine collected between May 2011 and August 2012 from 3 cities/provinces in China.

Results

A total of 1003 samples, including 326 fecal, 351 small intestinal contents and 326 colon contents samples, was analyzed. Two hundred and fifty five samples were stx-positive by PCR and 93 STEC isolates were recovered from 62 stx-positive samples. Twelve O serogroups and 19 O:H serotypes including 6 serotypes (O100:H20/[H20], O143:H38/[H38], O87:H10, O172:H30/[H30], O159:H16, O9:H30/[H30]) rarely found in swine and ruminants were identified. All 93 STEC isolates harbored stx ₂ only, all of which were stx _2e subtype including 1 isolate being a new variant of stx _2e. 53.76%, 15.05% and 2.15% STEC isolates carried astA, hlyA and ehxA respectively. Four STEC isolates harbored the high-pathogenicity island. Of the 15 adherence-associated genes tested, 13 (eae, efa1, iha, lpfA _O113, lpfA _O157/OI-154, lpfA _O157/OI-141, toxB, saa, F4, F5, F6, F17 or F41) were all absent while 2 (paa and F18) were present in 7 and 4 STEC isolates respectively. The majority of the isolates were resistant to tetracycline (79.57%), nalidixic acid (78.49%), trimethoprim-sulfamethoxazole (73.12%) and kanamycin (55.91%). The STEC isolates were divided into 63 pulsed-field gel electrophoresis patterns and 21 sequence types (STs). Isolates of the same STs generally showed the same or similar drug resistance patterns. A higher proportion of STEC isolates from Chongqing showed multidrug resistance with one ST (ST3628) resistant to 14 antimicrobials.

Conclusions

Our results indicate that swine is a significant reservoir of STEC strains in China. Based on comparison by serotypes and sequence types with human strains and presence of virulence genes, the swine STEC may have a low potential to cause human disease.     

Presence and Characterization of Shiga Toxin-Producing Escherichia coli and Other Potentially Diarrheagenic E. coli Strains in Retail Meats

To determine the presence of Shiga toxin-producing Escherichia coli (STEC) and other potentially diarrheagenic E. coli strains in retail meats, 7,258 E. coli isolates collected by the U.S. National Antimicrobial Resistance Monitoring System (NARMS) retail meat program from 2002 to 2007 were screened for Shiga toxin genes. In addition, 1,275 of the E. coli isolates recovered in 2006 were examined for virulence genes specific for other diarrheagenic E. coli strains. Seventeen isolates (16 from ground beef and 1 from a pork chop) were positive for stx genes, including 5 positive for both stx₁ and stx₂, 2 positive for stx₁, and 10 positive for stx₂. The 17 STEC strains belonged to 10 serotypes: O83:H8, O8:H16, O15:H16, O15:H17, O88:H38, ONT:H51, ONT:H2, ONT:H10, ONT:H7, and ONT:H46. None of the STEC isolates contained eae, whereas seven carried enterohemorrhagic E. coli (EHEC) hlyA. All except one STEC isolate exhibited toxic effects on Vero cells. DNA sequence analysis showed that the stx₂ genes from five STEC isolates encoded mucus-activatable Stx2d. Subtyping of the 17 STEC isolates by pulsed-field gel electrophoresis (PFGE) yielded 14 distinct restriction patterns. Among the 1,275 isolates from 2006, 11 atypical enteropathogenic E. coli (EPEC) isolates were identified in addition to 3 STEC isolates. This study demonstrated that retail meats, mainly ground beef, were contaminated with diverse STEC strains. The presence of atypical EPEC strains in retail meat is also of concern due to their potential to cause human infections.Escherichia coli is an important component of the intestinal microflora of humans and warm-blooded mammals. While E. coli typically harmlessly colonizes the intestinal tract, several E. coli clones have evolved the ability to cause a variety of diseases within the intestinal tract and elsewhere in the host. Those strains that cause enteric infections are generally called diarrheagenic E. coli strains, and their pathogenesis is associated with a number of virulence attributes, which vary according to pathotype (54). Currently, diarrheagenic E. coli strains are classified into six main pathotypes based on their distinct virulence determinants and pathogenic features, including enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (EHEC)/Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusively adherent E. coli (DAEC) (37).Among diarrheagenic E. coli strains, STEC strains are distinguished by the ability to cause severe life-threatening complications, such as hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) (30). Other symptoms of STEC infection include watery diarrhea, bloody diarrhea, and hemorrhagic colitis (HC). STEC strains that cause HC and HUS are also called EHEC. Although individuals of all ages are at risk of STEC infection, children younger than 5 years of age and the elderly are more likely to suffer from severe complications (51). Outbreaks and sporadic cases of STEC infections have been reported frequently worldwide.The pathogenesis of STEC infection in humans is not fully understood. The major virulence factors implicated in STEC infection are potent Shiga toxins, which are classified into two groups: Stx1 and Stx2 (23). Additional factors that contribute to virulence have also been described, including intimin (encoded by the eae gene), an outer membrane protein involved in the attachment of E. coli to the enterocyte, and EHEC hemolysin (encoded by EHEC hlyA), which acts as a pore-forming cytolysin and causes damage to cells (41).The first STEC O157 infections were reported in 1982, when E. coli O157:H7 was involved in outbreaks associated with two fast food chain restaurants in the United States (44). Since then, ever-increasing numbers of cases and outbreaks due to STEC O157 have been reported worldwide. Although non-O157 STEC strains have also been associated with human cases and outbreaks, few laboratories have been looking for them, and their potential in causing human infections may be underestimated (2). Recently, though, the significance of non-O157 STEC strains as human pathogens has become more recognized. In the United States alone, there were 23 reported outbreaks of non-O157 STEC infection between 1990 and 2007 (10).Shiga toxin-producing E. coli can be transmitted through different routes, including food and water, person-to-person contact, and animal-to-person contact (9). Most human infections are caused by consumption of contaminated foods (16). Domestic and wild ruminant animals, in particular cattle, are considered the main reservoir of STEC and the main source for contamination of the food supply. Retail meats derived from animals could potentially act as transmission vehicles for STEC and other diarrheagenic E. coli strains. However, there is limited information about STEC contamination in retail meats, and fewer data exist about the presence of other diarrheagenic E. coli strains in retail meats. In the present study, we investigated 7,258 E. coli isolates from four types of meat samples (beef, chicken, pork, and turkey) collected during 2002 to 2007 to assess STEC contamination of retail meats. In addition, the presence of other potentially diarrheagenic E. coli strains was examined by detecting specific virulence determinants among E. coli isolates collected in 2006.     

Prevalence and Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli on Carcasses in Commercial Beef Cattle Processing Plants

Beef carcass sponge samples collected from July to August 1999 at four large processing plants in the United States were surveyed for the presence of non-O157 Shiga toxin-producing Escherichia coli (STEC). Twenty-eight (93%) of 30 single-source lots surveyed included at least one sample containing non-O157 STEC. Of 334 carcasses sampled prior to evisceration, 180 (54%) were found to harbor non-O157 STEC. Non-O157 STEC isolates were also recovered from 27 (8%) of 326 carcasses sampled after the application of antimicrobial interventions. Altogether, 361 non-O157 STEC isolates, comprising 41 different O serogroups, were recovered. O serogroups that previously have been associated with human disease accounted for 178 (49%) of 361 isolates. Although 40 isolates (11%) carried a combination of virulence factor genes (enterohemorrhagic E. coli hlyA, eae, and at least one stx gene) frequently associated with STEC strains causing severe human disease, only 12 of these isolates also belonged to an O serogroup previously associated with human disease. Combining previously reported data on O157-positive samples (R. O. Elder, J. E. Keen, G. R. Siragusa, G. A. Barkocy-Gallagher, M. Koohmaraie, and W. W. Laegreid, Proc. Natl. Acad. Sci. USA 97:2999-3003, 2000) with these data regarding non-O157-positive samples indicated total STEC prevalences of 72 and 10% in preevisceration and postprocessing beef carcass samples, respectively, showing that the interventions used by the beef-processing industry effected a sevenfold reduction in carcass contamination by STEC.     

An Environmental Shiga Toxin-Producing Escherichia coli O145 Clonal Population Exhibits High-Level Phenotypic Variation That Includes Virulence Traits

Shiga toxin-producing Escherichia coli (STEC) serotype O145 is one of the major non-O157 serotypes associated with severe human disease. Here we examined the genetic diversity, population structure, virulence potential, and antimicrobial resistance profiles of environmental O145 strains recovered from a major produce production region in California. Multilocus sequence typing analyses revealed that sequence type 78 (ST-78), a common ST in clinical strains, was the predominant genotype among the environmental strains. Similarly, all California environmental strains belonged to H28, a common H serotype in clinical strains. Although most environmental strains carried an intact fliC gene, only one strain retained swimming motility. Diverse stx subtypes were identified, including stx_1a, stx_2a, stx_2c, and stx_2e. Although no correlation was detected between the stx genotype and Stx1 production, high Stx2 production was detected mainly in strains carrying stx_2a only and was correlated positively with the cytotoxicity of Shiga toxin. All environmental strains were capable of producing enterohemolysin, whereas only 10 strains were positive for anaerobic hemolytic activity. Multidrug resistance appeared to be common, as nearly half of the tested O145 strains displayed resistance to at least two different classes of antibiotics. The core virulence determinants of enterohemorrhagic E. coli were conserved in the environmental STEC O145 strains; however, there was large variation in the expression of virulence traits among the strains that were highly related genotypically, implying a trend of clonal divergence. Several cattle isolates exhibited key virulence traits comparable to those of the STEC O145 outbreak strains, emphasizing the emergence of hypervirulent strains in agricultural environments.     

Shiga Toxin-Producing Escherichia coli in Yaks (Bos grunniens) from the Qinghai-Tibetan Plateau,China

Shiga toxin (Stx)-producing Escherichia coli (STEC) are recognized as important human pathogens of public health concern. Many animals are the sources of STEC. In this study we determined the occurrence and characteristics of the STEC in yaks (Bos grunniens) from the Qinghai-Tibetan plateau, China. A total of 728 yak fecal samples was collected from June to August, 2012 and was screened for the presence of the stx ₁ and stx ₂ genes by TaqMan real-time PCR after the sample was enriched in modified Tryptone Soya Broth. Of the 138 (18.96%) stx ₁ and/or stx ₂-positive samples, 85 (61.59%) were confirmed to have at least 1 STEC isolate present by culture isolation, from which 128 STEC isolates were recovered. All STEC isolates were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE) and characterized for the presence of 16 known virulence factors. Fifteen different O serogroups and 36 different O:H serotypes were identified in the 128 STEC isolates with 21 and 4 untypable for the O and H antigens respectively. One stx ₁ subtype (stx _1a) and 5 stx ₂ subtypes (stx _2a, stx _2b, stx _2c, stx _2d and stx _2g) were present in these STEC isolates. Apart from lpfA _O157/OI-141, lpfA _O157/OI-154, lpfA _O113, katP and toxB which were all absent, other virulence factors screened (eaeA, iha, efa1, saa, paa, cnf1, cnf2, astA, subA, exhA and espP) were variably present in the 128 STEC isolates. PFGE were successful for all except 5 isolates and separated them into 67 different PFGE patterns. For the 18 serotypes with 2 or more isolates, isolates of the same serotypes had the same or closely related PFGE patterns, demonstrating clonality of these serotypes. This study was the first report on occurrence and characteristics of STEC isolated from yaks (Bos grunniens) from the Qinghai-Tibetan plateau, China, and extended the genetic diversity and reservoir host range of STEC.     

Serotypes and virulence profiles of non-O157 Shiga toxin-producing Escherichia coli isolates from bovine farms

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are clinically significant food-borne pathogens. However, there is a dearth of information on serotype prevalence and virulence gene distribution, data essential for the development of public health protection monitoring and control activities for the meat and dairy industries. Thus, the objective of this study was to examine the prevalence of non-O157 STEC on beef and dairy farms and to characterize the isolates in terms of serotype and virulence markers. Bovine fecal samples (n = 1,200) and farm soil samples (n = 600) were collected from 20 farms throughout Ireland over a 12-month period. Shiga toxin-positive samples were cultured and colonies examined for the presence of stx₁ and/or stx₂ genes by PCR. Positive isolates were serotyped and examined for a range of virulence factors, including eaeA, hlyA, tir, espA, espB, katP, espP, etpD, saa, sab, toxB, iha, lpfA_O157/OI-141, lpfA_O113, and lpfA_O157/OI-154. Shiga toxin and intimin genes were further examined for known variants. Significant numbers of fecal (40%) and soil (27%) samples were stx positive, with a surge observed in late summer-early autumn. One hundred seven STEC isolates were recovered, representing 17 serotypes. O26:H11 and O145:H28 were the most clinically significant, with O113:H4 being the most frequently isolated. However, O2:H27, O13/O15:H2, and ONT:H27 also carried stx₁ and/or stx₂ and eaeA and may be emerging pathogens.     

Highly Virulent Non-O157 Enterohemorrhagic Escherichia coli (EHEC) Serotypes Reflect Similar Phylogenetic Lineages,Providing New Insights into the Evolution of EHEC

Enterohemorrhagic Escherichia coli (EHEC) is the causative agent of bloody diarrhea and extraintestinal sequelae in humans, most importantly hemolytic-uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). Besides the bacteriophage-encoded Shiga toxin gene (stx), EHEC harbors the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions. Currently, the vast majority of EHEC infections are caused by strains belonging to five O serogroups (the “big five”), which, in addition to O157, the most important, comprise O26, O103, O111, and O145. We hypothesize that these four non-O157 EHEC serotypes differ in their phylogenies. To test this hypothesis, we used multilocus sequence typing (MLST) to analyze a large collection of 250 isolates of these four O serogroups, which were isolated from diseased as well as healthy humans and cattle between 1952 and 2009. The majority of the EHEC isolates of O serogroups O26 and O111 clustered into one sequence type complex, STC29. Isolates of O103 clustered mainly in STC20, and most isolates of O145 were found within STC32. In addition to these EHEC strains, STC29 also included stx-negative E. coli strains, termed atypical enteropathogenic E. coli (aEPEC), yet another intestinal pathogenic E. coli group. The finding that aEPEC and EHEC isolates of non-O157 O serogroups share the same phylogeny suggests an ongoing microevolutionary scenario in which the phage-encoded Shiga toxin gene stx is transferred between aEPEC and EHEC. As a consequence, aEPEC strains of STC29 can be regarded as post- or pre-EHEC isolates. Therefore, STC29 incorporates phylogenetic information useful for unraveling the evolution of EHEC.     

Identification and Characterization of Shiga Toxin Type 2 Variants in Escherichia coli Isolates from Animals, Food, and Humans

There is considerable heterogeneity among the Shiga toxin type 2 (Stx2) toxins elaborated by Shiga toxin-producing Escherichia coli (STEC). One such Stx2 variant, the Stx2d mucus-activatable toxin (Stx2dact), is rendered more toxic by the action of elastase present in intestinal mucus, which cleaves the last two amino acids of the A2 portion of the toxin A subunit. We screened 153 STEC isolates from food, animals, and humans for the gene encoding Stx2dact by using a novel one-step PCR procedure. This method targeted the region of stx_2dact that encodes the elastase recognition site. The presence of stx_2dact was confirmed by DNA sequencing of the complete toxin genes. Seven STEC isolates from cows (four isolates), meat (two isolates), and a human (one isolate) that carried the putative stx_2dact gene were identified; all were eae negative, and none was the O157:H7 serotype. Three of the isolates (CVM9322, CVM9557, and CVM9584) also carried stx₁, two (P1332 and P1334) carried stx₁ and stx_2c, and one (CL-15) carried stx_2c. One isolate, P1130, harbored only stx_2dact. The Vero cell cytotoxicities of supernatants from P1130 and stx₁ deletion mutants of CVM9322, CVM9557, and CVM9584 were increased 13- to 30-fold after treatment with porcine elastase. Thus, Stx2dact-producing strains, as detected by our one-step PCR method, can be isolated not only from humans, as previously documented, but also from food and animals. The latter finding has important public health implications based on a recent report from Europe of a link between disease severity and infection with STEC isolates that produce Stx2dact.     

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.     

Genotypic Analyses of Shiga Toxin-Producing Escherichia coli O157 and Non-O157 Recovered from Feces of Domestic Animals on Rural Farms in Mexico

Shiga toxin-producing Escherichia coli (STEC) are zoonotic enteric pathogens associated with human gastroenteritis worldwide. Cattle and small ruminants are important animal reservoirs of STEC. The present study investigated animal reservoirs for STEC in small rural farms in the Culiacan Valley, an important agricultural region located in Northwest Mexico. A total of 240 fecal samples from domestic animals were collected from five sampling sites in the Culiacan Valley and were subjected to an enrichment protocol followed by either direct plating or immunomagnetic separation before plating on selective media. Serotype O157:H7 isolates with the virulence genes stx2, eae, and ehxA were identified in 40% (26/65) of the recovered isolates from cattle, sheep and chicken feces. Pulse-field gel electrophoresis (PFGE) analysis grouped most O157:H7 isolates into two clusters with 98.6% homology. The use of multiple-locus variable-number tandem repeat analysis (MLVA) differentiated isolates that were indistinguishable by PFGE. Analysis of the allelic diversity of MLVA loci suggested that the O157:H7 isolates from this region were highly related. In contrast to O157:H7 isolates, a greater genotypic diversity was observed in the non-O157 isolates, resulting in 23 PFGE types and 14 MLVA types. The relevant non-O157 serotypes O8:H19, O75:H8, O111:H8 and O146:H21 represented 35.4% (23/65) of the recovered isolates. In particular, 18.5% (12/65) of all the isolates were serotype O75:H8, which was the most variable serotype by both PFGE and MLVA. The non-O157 isolates were predominantly recovered from sheep and were identified to harbor either one or two stx genes. Most non-O157 isolates were ehxA-positive (86.5%, 32/37) but only 10.8% (4/37) harbored eae. These findings indicate that zoonotic STEC with genotypes associated with human illness are present in animals on small farms within rural communities in the Culiacan Valley and emphasize the need for the development of control measures to decrease risks associated with zoonotic STEC.     

Comparative Genomics to Delineate Pathogenic Potential in Non-O157 Shiga Toxin-Producing Escherichia coli (STEC) from Patients with and without Haemolytic Uremic Syndrome (HUS) in Norway

Shiga toxin-producing Escherichia coli (STEC) cause infections in humans ranging from asymptomatic carriage to bloody diarrhoea and haemolytic uremic syndrome (HUS). Here we present whole genome comparison of Norwegian non-O157 STEC strains with the aim to distinguish between strains with the potential to cause HUS and less virulent strains. Whole genome sequencing and comparisons were performed across 95 non-O157 STEC strains. Twenty-three of these were classified as HUS-associated, including strains from patients with HUS (n = 19) and persons with an epidemiological link to a HUS-case (n = 4). Genomic comparison revealed considerable heterogeneity in gene content across the 95 STEC strains. A clear difference in gene profile was observed between strains with and without the Locus of Enterocyte Effacement (LEE) pathogenicity island. Phylogenetic analysis of the core genome showed high degree of diversity among the STEC strains, but all HUS-associated STEC strains were distributed in two distinct clusters within phylogroup B1. However, non-HUS strains were also found in these clusters. A number of accessory genes were found to be significantly overrepresented among HUS-associated STEC, but none of them were unique to this group of strains, suggesting that different sets of genes may contribute to the pathogenic potential in different phylogenetic STEC lineages. In this study we were not able to clearly distinguish between HUS-associated and non-HUS non-O157 STEC by extensive genome comparisons. Our results indicate that STECs from different phylogenetic backgrounds have independently acquired virulence genes that determine pathogenic potential, and that the content of such genes is overlapping between HUS-associated and non-HUS strains.     

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.     

Multiplex real-time PCR assay for detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and screening for non-O157 Shiga toxin-producing <Emphasis Type="Italic">E. coli</Emphasis>

Background

Shiga toxin-producing Escherichia coli (STEC), including E. coli O157:H7, are responsible for numerous foodborne outbreaks annually worldwide. E. coli O157:H7, as well as pathogenic non-O157:H7 STECs, can cause life-threating complications, such as bloody diarrhea (hemolytic colitis) and hemolytic-uremic syndrome (HUS). Previously, we developed a real-time PCR assay to detect E. coli O157:H7 in foods by targeting a unique putative fimbriae protein Z3276. To extend the detection spectrum of the assay, we report a multiplex real-time PCR assay to specifically detect E. coli O157:H7 and screen for non-O157 STEC by targeting Z3276 and Shiga toxin genes (stx1 and stx2). Also, an internal amplification control (IAC) was incorporated into the assay to monitor the amplification efficiency.

Methods

The multiplex real-time PCR assay was developed using the Life Technology ABI 7500 System platform and the standard chemistry. The optimal amplification mixture of the assay contains 12.5 μl of 2 × Universal Master Mix (Life Technology), 200 nM forward and reverse primers, appropriate concentrations of four probes [(Z3276 (80 nM), stx1 (80 nM), stx2 (20 nM), and IAC (40 nM)], 2 μl of template DNA, and water (to make up to 25 μl in total volume). The amplification conditions of the assay were set as follows: activation of TaqMan at 95 °C for 10 min, then 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 60 s.

Results

The multiplex assay was optimized for amplification conditions. The limit of detection (LOD) for the multiplex assay was determined to be 200 fg of bacterial DNA, which is equivalent to 40 CFU per reaction which is similar to the LOD generated in single targeted PCRs. Inclusivity and exclusivity determinants were performed with 196 bacterial strains. All E. coli O157:H7 (n = 135) were detected as positive and all STEC strains (n = 33) were positive for stx1, or stx2, or stx1 and stx2 (Table 1). No cross reactivity was detected with Salmonella enterica, Shigella strains, or any other pathogenic strains tested.

Conclusions

A multiplex real-time PCR assay that can rapidly and simultaneously detect E. coli O157:H7 and screen for non-O157 STEC strains has been developed and assessed for efficacy. The inclusivity and exclusivity tests demonstrated high sensitivity and specificity of the multiplex real-time PCR assay. In addition, this multiplex assay was shown to be effective for the detection of E. coli O157:H7 from two common food matrices, beef and spinach, and may be applied for detection of E. coli O157:H7 and screening for non-O157 STEC strains from other food matrices as well.

     

Molecular analysis of shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients and dairy samples in France

Shiga toxin-producing Escherichia coli (STEC) has been associated with food-borne diseases ranging from uncomplicated diarrhea to hemolytic-uremic syndrome (HUS). While most outbreaks are associated with E. coli O157:H7, about half of the sporadic cases may be due to non-O157:H7 serotypes. To assess the pathogenicity of STEC isolated from dairy foods in France, 40 strains isolated from 1,130 raw-milk and cheese samples were compared with 15 STEC strains isolated from patients suffering from severe disease. The presence of genes encoding Shiga toxins (stx(1), stx(2), and variants), intimin (eae and variants), adhesins (bfp, efa1), enterohemolysin (ehxA), serine protease (espP), and catalase-peroxidase (katP) was determined by PCR and/or hybridization. Plasmid profiling, ribotyping, and pulsed-field gel electrophoresis (PFGE) were used to further compare the strains at the molecular level. A new stx(2) variant, stx(2-CH013), associated with an O91:H10 clinical isolate was identified. The presence of the stx(2), eae, and katP genes, together with a combination of several stx(2) variants, was clearly associated with human-pathogenic strains. In contrast, dairy food STEC strains were characterized by a predominance of stx(1), with a minority of isolates harboring eae, espP, and/or katP. These associations may help to differentiate less virulent STEC strains from those more likely to cause disease in humans. Only one dairy O5 isolate had a virulence gene panel identical to that of an HUS-associated strain. However, the ribotype and PFGE profiles were not identical. In conclusion, most STEC strains isolated from dairy products in France showed characteristics different from those of strains isolated from patients.