Identification of human-pathogenic strains of Shiga toxin-producing Escherichia coli from food by a combination of serotyping and molecular typing of Shiga toxin genes.
ABSTRACT: We examined 219 Shiga toxin-producing Escherichia coli (STEC) strains from meat, milk, and cheese samples collected in Germany between 2005 and 2006. All strains were investigated for their serotypes and for genetic variants of Shiga toxins 1 and 2 (Stx1 and Stx2). stx(1) or variant genes were detected in 88 (40.2%) strains and stx(2) and variants in 177 (80.8%) strains. Typing of stx genes was performed by stx-specific PCRs and by analysis of restriction fragment length polymorphisms (RFLP) of PCR products. Major genotypes of the Stx1 (stx(1), stx(1c), and stx(1d)) and the Stx2 (stx(2), stx(2d), stx(2-O118), stx(2e), and stx(2g)) families were detected, and multiple types of stx genes coexisted frequently in STEC strains. Only 1.8% of the STEC strains from food belonged to the classical enterohemorrhagic E. coli (EHEC) types O26:H11, O103:H2, and O157:H7, and only 5.0% of the STEC strains from food were positive for the eae gene, which is a virulence trait of classical EHEC. In contrast, 95 (43.4%) of the food-borne STEC strains carried stx(2) and/or mucus-activatable stx(2d) genes, an indicator for potential high virulence of STEC for humans. Most of these strains belonged to serotypes associated with severe illness in humans, such as O22:H8, O91:H21, O113:H21, O174:H2, and O174:H21. stx(2) and stx(2d) STEC strains were found frequently in milk and beef products. Other stx types were associated more frequently with pork (stx(2e)), lamb, and wildlife meat (stx(1c)). The combination of serotyping and stx genotyping was found useful for identification and for assignment of food-borne STEC to groups with potential lower and higher levels of virulence for humans.
Project description:When Shiga toxin-producing Escherichia coli (STEC) strains emerged as agents of human disease, two types of toxin were identified: Shiga toxin type 1 (Stx1) (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and the immunologically distinct type 2 (Stx2). Subsequently, numerous STEC strains have been characterized that express toxins with variations in amino acid sequence, some of which confer unique biological properties. These variants were grouped within the Stx1 or Stx2 type and often assigned names to indicate that they were not identical in sequence or phenotype to the main Stx1 or Stx2 type. A lack of specificity or consistency in toxin nomenclature has led to much confusion in the characterization of STEC strains. Because serious outcomes of infection have been attributed to certain Stx subtypes and less so with others, we sought to better define the toxin subtypes within the main Stx1 and Stx2 types. We compared the levels of relatedness of 285 valid sequence variants of Stx1 and Stx2 and identified common sequences characteristic of each of three Stx/Stx1 and seven Stx2 subtypes. A novel, simple PCR subtyping method was developed, independently tested on a battery of 48 prototypic STEC strains, and improved at six clinical and research centers to test the reproducibility, sensitivity, and specificity of the PCR. Using a consistent schema for nomenclature of the Stx toxins and stx genes by phylogenetic sequence-based relatedness of the holotoxin proteins, we developed a typing approach that should obviate the need to bioassay each newly described toxin and that predicts important biological characteristics.
Project description:<h4>Background</h4>Shiga toxin-producing E. coli (STEC) are a group of common and potentially deadly intestinal pathogens expressing Shiga toxin (Stx) as a primary virulence factor. Of the two types of Stx, Stx2 is responsible for more severe symptoms during infection, while Stx1 is almost identical to the Shiga toxin from Shigella dysenteriae, a ubiquitous pathogen in developing countries. Although antibodies against Stx1 have been reported, few have reached the affinity needed for assembling highly sensitive immunoassays. Sensitive and affordable immunoassays for Stx1 and Stx2 could help improve detection of STEC in livestock, food, the environment, and in clinical samples resulting in improved food safety and human health.<h4>Method and findings</h4>Three new monoclonal antibodies (mAbs) against the B subunit of Stx1 were generated using recombinant toxoid Stx1E167Q and hybridoma technology. These new mAbs recognize all subtypes of Stx1, but do not cross-react with any subtype of Stx2. In addition, they exhibited the ability to neutralize Stx1 toxicity in Vero cell assays. An optimized sandwich ELISA using of a pair of these mAbs had a limit of detection of 8.7 pg/mL, which is superior to any existing assay of this kind. Using one of these Stx1 mAbs in concert with Stx2 mAbs, the presence of hybrid Stx1/Stx2 toxin in the culture media of STEC strains that express both Stx1 and Stx2 was demonstrated.<h4>Conclusions</h4>These new mAbs provide a mix of availability, utility, versatility, and most importantly, increased sensitivity for detection of Stx1. There are numerous potential applications for these mAbs, including low-cost detection assays and therapeutic use. Analysis of hybrid Stx1/2 could provide new insights on the structure, activity, and cellular targets of Shiga toxins.
Project description:BACKGROUND AND OBJECTIVES:Shiga toxin-producing Escherichia coli (STEC) strains are human pathogens linked to hemorrhagic colitis and hemolytic uremic syndrome. Shiga toxins (Stx1 and Stx2) are the major virulence factors of these strains. The aim of this work was to study the prevalence and distribution of stx(1) and stx(2) gene in E. coli O157:H7 and non-O157:H7 strains isolated from cattle in Shiraz, Iran. MATERIALS AND METHODS:Four hundred and twenty samples consisted of recto-anal mucosal swabs were collected from cattle. They were checked for the presence of the stx1 and stx2 gene using multiplex-PCR every 1 week over a 1-year period (2007-2008). RESULTS:A total of 146 strains carrying the stx1 and stx2 gene were isolated from 51 (12.14%) cattle. Overall, 15 (3.57%) were identified as O157:H7 and 131 (31.19%) revealed to be non-O157:H7. Both stx2 and stx1 genes were detected in 51 (34.93%) STEC isolates. Genotypes stx1 and stx2 were detected in 15 (10.27%) and 78 (53.42%) respectively. Seasonal distribution of stx genes revealed high percentage of positive animals in warm seasons. The gene sequence similarity ranged from 94 to 100%. CONCLUSION:Frequency of stx1 and stx2 in animals and its relation to human disease is not well understood in Iran. The high prevalence of STEC in cattle seems to parallel that which is usually observed in warm seasons and it also parallels occurrence of human STEC. The higher prevalence of the stx2 gene than stx1 in strain populations isolated from cattle indicates a risk alert of E. coli O157:H7 being shed by cattle in these populations. Appropriate measures are now needed to prevent the spread of this life-threatening foodborne disease in our country.
Project description:Shiga toxin-producing Escherichia coli (STEC) is a notorious foodborne pathogen containing stx genes located in the sequence region of Shiga toxin (Stx) prophages. Stx prophages, as one of the mobile elements, are involved in the transfer of virulence genes to other strains. However, little is known about the diversity of prophages among STEC strains. The objectives of this study were to predict various prophages from different STEC genomes and to evaluate the effect of different stress factors on Stx prophage induction. Forty bacterial whole-genome sequences of STEC strains obtained from National Center for Biotechnology Information (NCBI) were used for the prophage prediction using PHASTER webserver. Eight of the STEC strains from different serotypes were subsequently selected to quantify the induction of Stx prophages by various treatments, including antibiotics, temperature, irradiation, and antimicrobial agents. After induction, Stx1-converting phage Lys8385Vzw and Stx2-converting phage Lys12581Vzw were isolated and further confirmed for the presence of stx genes using conventional PCR. Phage morphology was observed by transmission electron microscopy. The prediction results showed an average of 8-22 prophages, with one or more encoding stx, were predicted from each STEC genome obtained in this study. Additionally, the phylogenetic analysis revealed high genetic diversity of Stx prophages among the 40 STEC genomes. However, the sequences of Stx prophages in the genomes of STEC O45, O111, and O121 strains, in general, shared higher genetic homology than those in other serotypes. Interestingly, most STEC strains with two or more stx genes carried at least one each of Stx1 and Stx2 prophages. The induction results indicated EDTA and UV were the most effective inducers of Stx1 and Stx2 prophages of the 8 selected STECs, respectively. Additionally, both Stx-converting phages could infect non-pathogenic E. coli (WG5, DH5?, and MG1655) and form new lysogens. The findings of this study confirm that Stx prophages can be induced by environmental stress, such as exposure to solar radiation, and lysogenize other commensal E. coli strains.
Project description:Strains of Shiga toxin-producing Escherichia coli (STEC) have been associated with outbreaks of diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome in humans. Most clinical signs of disease arise as a consequence of the production of Shiga toxin 1 (Stx1), Stx2 or combinations of these toxins. Other major virulence factors include enterohemorrhagic E. coli hemolysin (EHEC hlyA), and intimin, the product of the eaeA gene that is involved in the attaching and effacing adherence phenotype. In this study, a series of multiplex-PCR assays were developed to detect the eight most-important E. coli genes associated with virulence, two that define the serotype and therefore the identity of the organism, and a built-in gene detection control. Those genes detected were stx(1), stx(2), stx(2c), stx(2d), stx(2e), stx(2f), EHEC hlyA, and eaeA, as well as rfbE, which encodes the E. coli O157 serotype; fliC, which encodes the E. coli flagellum H7 serotype; and the E. coli 16S rRNA, which was included as an internal control. A total of 129 E. coli strains, including 81 that were O157:H7, 10 that were O157:non-H7, and 38 that were non-O157 isolates, were investigated. Among the 129 samples, 101 (78.3%) were stx positive, while 28 (21.7%) were lacked stx. Of these 129 isolates, 92 (71.3%) were EHEC hlyA positive and 96 (74.4%) were eaeA positive. All STEC strains were identified by this procedure. In addition, all Stx2 subtypes, which had been initially identified by PCR-restriction fragment length polymorphism, were identified by this method. A particular strength of the assay was the identification of these 11 genes without the need to use restriction enzyme digestion. The proposed method is a simple, reliable, and rapid procedure that can detect the major virulence factors of E. coli while differentiating O157:H7 from non-O157 isolates.
Project description:Shiga toxins Stx1 and Stx2 play a prominent role in the pathogenesis of Shiga toxin-producing Escherichia coli (STEC) infections. Several variants of the stx(2) gene, encoding Stx2, have been described. In this study, we developed a PCR-restriction fragment length polymorphism system for typing stx(2) genes of STEC strains. The typing system discriminates eight described variants and allows the identification of new stx(2) variants and STEC isolates carrying multiple stx(2) genes. A phylogenetic tree, based on the nucleotide sequences of the toxin-encoding genes, demonstrates that stx(2) sequences with the same PvuII HaeIII HincII AccI type generally cluster together.
Project description:A total of 140 Shiga toxin-producing Escherichia coli (STEC) strains from wildlife meat (deer, wild boar, and hare) isolated in Germany between 1998 and 2006 were characterized with respect to their serotypes and virulence markers associated with human pathogenicity. The strains grouped into 38 serotypes, but eight O groups (21, 146, 128, 113, 22, 88, 6, and 91) and four H types (21, 28, 2, and 8) accounted for 71.4% and 75.7% of all STEC strains from game, respectively. Eighteen of the serotypes, including enterohemorrhagic E. coli (EHEC) O26:[H11] and O103:H2, were previously found to be associated with human illness. Genes linked to high-level virulence for humans (stx(2), stx(2d), and eae) were present in 46 (32.8%) STEC strains from game. Fifty-four STEC isolates from game belonged to serotypes which are frequently found in human patients (O103:H2, O26:H11, O113:H21, O91:H21, O128:H2, O146:H21, and O146:H28). These 54 STEC isolates were compared with 101 STEC isolates belonging to the same serotypes isolated from farm animals, from their food products, and from human patients. Within a given serotype, most STEC strains were similar with respect to their stx genotypes and other virulence attributes, regardless of origin. The 155 STEC strains were analyzed for genetic similarity by XbaI pulsed-field gel electrophoresis. O103:H2, O26:H11, O113:H21, O128:H2, and O146:H28 STEC isolates from game were 85 to 100% similar to STEC isolates of the same strains from human patients. By multilocus sequence typing, game EHEC O103:H2 strains were attributed to a clonal lineage associated with hemorrhagic diseases in humans. The results from our study indicate that game animals represent a reservoir for and a potential source of human pathogenic STEC and EHEC strains.
Project description:Shiga toxin (Stx) types 1 and 2 are encoded within intact or defective temperate bacteriophages in Stx-producing Escherichia coli (STEC), and expression of these toxins is linked to bacteriophage induction. Among Stx2 variants, only stx(2e) from one human STEC isolate has been reported to be carried within a toxin-converting phage. In this study, we examined the O91:H21 STEC isolate B2F1, which carries two functional alleles for the potent activatable Stx2 variant toxin, Stx2d, for the presence of Stx2d-converting bacteriophages. We first constructed mutants of B2F1 that produced one or the other Stx2d toxin and found that the mutant that produced only Stx2d1 made less toxin than the Stx2d2-producing mutant. Consistent with that result, the Stx2d1-producing mutant was attenuated in a streptomycin-treated mouse model of STEC infection. When the mutants were treated with mitomycin C to promote bacteriophage induction, Vero cell cytotoxicity was elevated only in extracts of the Stx2d1-producing mutant. Additionally, when mice were treated with ciprofloxacin, an antibiotic that induces the O157:H7 Stx2-converting phage, the animals were more susceptible to the Stx2d1-producing mutant. Moreover, an stx(2d1)-containing lysogen was isolated from plaques on strain DH5alpha that had been exposed to lysates of the mutant that produced Stx2d1 only, and supernatants from that lysogen transformed with a plasmid encoding RecA were cytotoxic when the lysogen was induced with mitomycin C. Finally, electron-microscopic examination of extracts from the Stx2d1-producing mutant showed hexagonal particles that resemble the prototypic Stx2-converting phage 933W. Together these observations provide strong evidence that expression of Stx2d1 is bacteriophage associated. We conclude that despite the sequence similarity of the stx(2d1)- and stx(2d2)-flanking regions in B2F1, Stx2d1 expression is repressed within the context of its toxin-converting phage while Stx2d2 expression is independent of phage induction.
Project description:At least 11 Stx2 variants produced by Shiga toxin-producing Escherichia coli (STEC) isolated from patients and animals have been described. The Stx2 subtyping of STEC isolated from healthy cows positive for stx(2) (n = 104) or stx(2) and stx(1) (n = 63) was investigated. Stx2vh-b, Stx2 (renamed Stx2-EDL933), and Stx2vh-a were the subtypes mostly detected among the bovine isolates (39.5, 39, and 25.5%, respectively). Stx2e was not present, and subtypes included in the Stx2d group (Stx2d-OX3a, Stx2d-O111, and Stx2d-Ount) were found infrequently among the isolates examined (8.5%). A combination of two distinct Stx2 subtypes was observed among 23.5% of the strains. For the first time, a combination of three subtypes (Stx2-EDL933/Stx2vh-b/Stx2d and Stx2vh-a/Stx2vh-b/Stx2d) was detected (3.5% of the isolates). In addition, bovine STEC harboring stx(1) and one or two stx(2) genes appeared highly cytotoxic toward Vero cells. A new Stx2 subtype (Stx2-NV206), present among 14.5% of the isolates, showed high cytotoxicity for Vero cells. Two amino acid residues (Ser-291 and Glu-297) important for the activation of Stx2 by human intestinal mucus were conserved on the Stx2-NV206 A subunit. The gene encoding Ehx enterohemolysin was prominent among STEC harboring stx(2)-EDL933 alone (78%) or a combination of stx(2)-EDL933 and stx(2)vh-b (85%). In addition, Stx2-EDL933 and/or Stx2vh-b subtypes were highly associated with other putative virulence factors such as Stx1 and EspP extracellular serine protease, but not with EAST1 enterotoxin.
Project description:By using sequence analysis of Shiga toxin 1 (Stx 1) genes from human and ovine Stx-producing Escherichia coli (STEC) strains, we identified an Stx1 variant in STEC of human origin that was identical to the Stx1 variant from ovine STEC, but demonstrated only 97.1 and 96.6% amino acid sequence identity in its A and B subunits, respectively, to the Stx1 encoded by bacteriophage 933J. We designated this variant "Stx1c" and developed stxB(1) restriction fragment length polymorphism and stx(1c)-specific PCR strategies to determine the frequency and distribution of stx(1c) among 212 STEC strains isolated from humans. stx(1c) was identified in 36 (17.0%) of 212 STEC strains, 19 of which originated from asymptomatic subjects and 16 of which were from patients with uncomplicated diarrhea. stx(1c) was most frequently (in 23 STEC strains [63.9%]) associated with stx(2d), but 12 (33.3%) of the 36 STEC strains possessed stx(1c) only. A single STEC strain possessed stx(1c) together with stx(2) and was isolated from a patient with hemolytic-uremic syndrome. All 36 stx(1c)-positive STEC strains were eae negative and belonged to 10 different serogroups, none of which was O157, O26, O103, O111, or O145. Stx1c was produced by all stx(1c)-containing STEC strains, but reacted weakly with a commercial immunoassay. We conclude that STEC strains harboring the stx(1c) variant account for a significant proportion of human STEC isolates. The procedures developed in this study now allow the determination of the frequency of STEC strains harboring stx(1c) among clinical STEC isolates and their association with human disease in prospective studies.