Potentially human-pathogenic Escherichia coli O26 in Norwegian sheep flocks

A national survey of Escherichia coli O26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, and E. coli O26 was detected in 17.9% of the flocks. One hundred forty-two E. coli O26 isolates were examined for flagellar antigens (H typing) and four virulence genes, including stx and eae, to identify possible Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC). Most isolates (129 out of 142) were identified as E. coli O26:H11. They possessed eae and may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between the E. coli O26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all the E. coli O26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing on E. coli O26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.     

Identification of genetic markers for differentiation of Shiga toxin-producing, enteropathogenic, and avirulent strains of Escherichia coli O26

Shiga toxin-producing Escherichia coli (STEC) O26 is one of the top five enterohemorrhagic E. coli (EHEC) O groups most often associated with hemorrhagic colitis and hemolytic uremic syndrome (HUS) worldwide. STEC O26 is considered to have evolved from enteropathogenic (EPEC) O26 strains through the acquisition of Shiga toxin (Stx)-encoding genes. Our PCR data identified several STEC-like strains expressing all features of STEC except Stx production and carrying remnants of Stx phages that were probably derivatives of EHEC O26. EHEC and EPEC O26 strains phenotypically resemble O26 EHEC-like and apathogenic E. coli O26 strains and are therefore undistinguishable by cultural methods. A clear discrimination between the different O26 groups is required for diagnostics in patients and for control of food safety. To develop an assay for specific detection of EHEC and EHEC-like O26 strains, we used a high-throughput PCR approach for selection of discriminative genetic markers among 33 tested genes mostly encoding type III secretion system effector proteins. The genes ECs1822, nleH1-2, nleA, nleC, nleH1-1, nleG, nleG2, nleG6-1, nleG6-2, espJ, espM2, nleG8-2, espG, ent (or espL2), nleB, nleE, efa1, and espB were detected at different frequencies in O26 EHEC, EHEC-like, and EPEC strains, indicating the possible role of these genes in virulence of human pathogenic O26 strains. The espK and espN genes were detected only in EHEC and EHEC-like O26 strains. espK was present in 99.14% of EHEC and 91.14% of EHEC-like O26 strains and was hence the best candidate as a genetic marker for characterizing these pathogroups. These data were corroborated by a genotyping real-time PCR test based on allelic discrimination of the arcA (aerobic respiratory control protein A) gene. The results indicate that a combination of molecular detection tools for O26 wzx (wzx(O26)), eae-beta, stx, espK, and arcA genotyping is highly discriminative for clear identification of EHEC and EHEC-like E. coli O26 strains. This simple diagnostic test might be applicable in hospital service laboratories or public health laboratories to test strains isolated from stools of patients suffering from diarrhea.     

Shiga toxin gene loss and transfer in vitro and in vivo during enterohemorrhagic Escherichia coli O26 infection in humans

Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx2 loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.     

Detection of Shiga toxin-producing Escherichia coli serotypes O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7 in raw-milk cheeses by using multiplex real-time PCR

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are a diverse group of food-borne pathogens with various levels of virulence for humans. In this study, we describe the use of a combination of multiple real-time PCR assays for the screening of 400 raw-milk cheeses for the five main pathogenic STEC serotypes (O26:H11, O103:H2, O111:H8, O145:H28, and O157:H7). The prevalences of samples positive for stx, intimin-encoding gene (eae), and at least one of the five O group genetic markers were 29.8%, 37.3%, and 55.3%, respectively. The H2, H7, H8, H11, and H28 fliC alleles were highly prevalent and could not be used as reliable targets for screening. Combinations of stx, eae variants, and O genetic markers, which are typical of the five targeted STEC serotypes, were detected by real-time PCR in 6.5% of the cheeses (26 samples) and included stx-wzx(O26)-eae-β1 (4.8%; 19 samples), stx-wzx(O103)-eae-ε (1.3%; five samples), stx-ihp1(O145)-eae-γ1 (0.8%; three samples), and stx-rfbE(O157)-eae-γ1 (0.3%; one sample). Twenty-eight immunomagnetic separation (IMS) assays performed on samples positive for these combinations allowed the recovery of seven eaeβ1-positive STEC O26:H11 isolates, whereas no STEC O103:H2, O145:H28, or O157:H7 strains could be isolated. Three stx-negative and eaeβ1-positive E. coli O26:[H11] strains were also isolated from cheeses by IMS. Colony hybridization allowed us to recover STEC from stx-positive samples for 15 out of 45 assays performed, highlighting the difficulties encountered in STEC isolation from dairy products. The STEC O26:H11 isolates shared the same virulence genetic profile as enterohemorrhagic E. coli (EHEC) O26:H11, i.e., they carried the virulence-associated genes EHEC-hlyA, katP, and espP, as well as genomic O islands 71 and 122. Except for one strain, they all contained the stx1 variant only, which was reported to be less frequently associated with human cases than stx2. Pulsed-field gel electrophoresis (PFGE) analysis showed that they displayed high genetic diversity; none of them had patterns identical to those of human O26:H11 strains investigated here.     

Evaluation of the 'GeneDisc' real-time PCR system for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigen-associated genes

Aims:  To evaluate the GeneDisc multiplex real-time PCR assay for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains.
Methods and Results:  GeneDiscs for detection of genes encoding Shiga toxins ( stx ), intimins ( eae ), E. coli O157 ( rfbE _O157) and H7 ( fliC _H7) antigens as well as genes specific for EHEC O26 ( wzx _O26), O103 ( wzx _O103), O111 ( wbd1 _O111), O145 ( ihp1 _O145) and O157 ( ihp1 _O157) were evaluated. The assay was run with native bacteria in 1 h in a GeneDisc Cycler. All genotypes of stx and eae , except stx _2f and eae -rho, were identified. Escherichia coli strains belonging to O-groups O26, O103, O111, O157 as well as EHEC O145:[H28] strains were specifically detected with this assay. The ihp1 _O157 gene was not found specific for EHEC O157. O-rough mutants of EHEC and non-motile EHEC O157 strains were reliably identified with the GeneDisc assay. Two to three colonies of EHEC strains were still detectable in a lawn of 50 000 apathogenic E. coli from agar plates.
Conclusions:  The GeneDisc assay is a specific and reliable assay for detection of major EHEC strains. It is robust enough to detect few EHEC colonies in mixed cultures of bacteria.
Significance and Impact of the Study:  The assay is promising for its use in EHEC diagnostics and for EHEC monitoring with different kinds of samples.     

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.     

Prevalence of Escherichia coli strains in the canteens

The prevalence of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) E. coli strains in stool specimens from asymptomatic human carriers working in the canteens and also in the kitchen and sanitary facilities was evaluated. The E. coli genes coding for the following virulence markers: intimin (eae), enterohaemolysin (hlyA), and verotoxins type I and II (stx1 and stx2) were sought by multiplex PCR assay. E. coli isolates were obtained from 144 stool specimens, 295 swabs taken from kitchen hardware and surrounding facilities, and from 33 meat specimens. Only 66 (8.5%) of total 777 E. coli isolates belonged to O44, O18, O25, O127, O55, O114, O125, and O142 serogroups, the prevalent serogroups in Poland. None of the strains was classified as serogroup O157. The serogroups O44 and O18 were present most often among all typeable strains and their incidence was 51.5% and 25.8% respectively. Among 363 isolates assayed for the presence of the genes encoding virulence markers only 10 isolates (2.8%) carried eae gene. None of the isolates possessing eae gene belonged to the serogroups tested. The hlyA, stx1 and stx2 genes were absent in all E. coli isolates tested.     

Phylogenetic grouping and virulence potential of extended-spectrum-β-lactamase-producing Escherichia coli strains in cattle

In line with recent reports of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates of highly virulent serotypes, such as O104:H4, we investigated the distribution of phylogroups (A, B1, B2, D) and virulence factor (VF)-encoding genes in 204 ESBL-producing E. coli isolates from diarrheic cattle. ESBL genes, VFs, and phylogroups were identified by PCR and a commercial DNA array (Alere, France). ESBL genes belonged mostly to the CTX-M-1 (65.7%) and CTX-M-9 (27.0%) groups, whereas those of the CTX-M-2 and TEM groups were much less represented (3.9% and 3.4%, respectively). One ESBL isolate was stx(1) and eae positive and belonged to a major enterohemorrhagic E. coli (EHEC) serotype (O111:H8). Two other isolates were eae positive but stx negative; one of these had serotype O26:H11. ESBL isolates belonged mainly to phylogroup A (55.4%) and, to lesser extents, to phylogroups D (25.5%) and B1 (15.6%), whereas B2 strains were quasi-absent (1/204). The number of VFs was significantly higher in phylogroup B1 than in phylogroups A (P = 0.04) and D (P = 0.02). Almost all of the VFs detected were found in CTX-M-1 isolates, whereas only 64.3% and 33.3% of them were found in CTX-M-9 and CTX-M-2 isolates, respectively. These results indicated that the widespread dissemination of the bla(CTX-M) genes within the E. coli population from cattle still spared the subpopulation of EHEC/Shiga-toxigenic E. coli (STEC) isolates. In contrast to other reports on non-ESBL-producing isolates from domestic animals, B1 was not the main phylogroup identified. However, B1 was found to be the most virulent phylogroup, suggesting host-specific distribution of virulence determinants among phylogenetic groups.     

Presence of virulence and fitness gene modules of enterohemorrhagic Escherichia coli in atypical enteropathogenic Escherichia coli O26

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O26 cause hemolytic-uremic syndrome (HUS) whereas atypical enteropathogenic E. coli (aEPEC) O26 typically cause uncomplicated diarrhea but have been also isolated from HUS patients. To gain insight into the virulence of aEPEC O26, we compared the presence of O island (OI) 122, which is associated with enhanced virulence in EHEC strains, among aEPEC O26 and EHEC O26 clinical isolates. We also tested these strains for the high pathogenicity island (HPI) which is a fitness island. All 20 aEPEC O26 and 20 EHEC O26 investigated contained virulence genes located within OI-122 (efa1/lifA, nleB, nleE, ent). In both aEPEC O26 and EHEC O26, OI-122 was linked to the locus for enterocyte effacement, forming a mosaic island which was integrated in pheU. Moreover, strains of these two pathotypes shared a conserved HPI. These data support a close relatedness between aEPEC O26 and EHEC O26 and have evolutionary implications. The presence of OI-122 in aEPEC O26 might contribute to their pathogenic potential.     

Shiga toxin-producing Escherichia coli and atypical enteropathogenic Escherichia coli strains isolated from healthy sheep of different populations in São Paulo, Brazil

Aims:  Sheep are important carriers of Shiga toxin-producing Escherichia coli (STEC) in several countries. However, there are a few reports about ovine STEC in American continent.
Methods and Results:  About 86 E. coli strains previously isolated from 172 healthy sheep from different farms were studied. PCR was used for detection of stx ₁, stx ₂, eae, ehxA and saa genes and for the identification of intimin subtypes. Restriction fragment length polymorphism (RFLP)–PCR was performed to investigate the variants of stx ₁ and stx ₂, and the flagellar antigen ( fli C) genes in nonmotile isolates. Five isolates were eae ⁺ and stx ⁻, and belonged to serotypes O128:H2/β-intimin (2), O145:H2/γ, O153:H7/β and O178:H7/ε. Eighty-one STEC isolates were recovered, and the stx genotypes identified were stx _1c stx _2d-O118 (46·9%), stx _1c (27·2%), stx _2d-O118 (23·4%), and stx _1c stx _2dOX3a (2·5%). Pulsed-field gel electrophoresis (PFGE) revealed 27 profiles among 53 STEC and atypical enteropathogenic Escherichia coli (EPEC) isolates.
Conclusions:  This study demonstrated that healthy sheep in São Paulo, Brazil, can be carriers of potential human pathogenic STEC and atypical EPEC.
Significance and Impact of the Study:  As some of the STEC serotypes presently found have been involved with haemolytic uraemic syndrome (HUS) in other countries, the important role of sheep as sources of STEC infection in our settings should not be disregarded.     

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.     

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

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.     

Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium

We identified Shiga toxin gene (stx)-negative Escherichia coli O26:H11 and O26:NM (nonmotile) strains as the only pathogens in the stools of five patients with hemolytic-uremic syndrome (HUS). Because the absence of stx in E. coli associated with HUS is unusual, we examined the strains for potential virulence factors and interactions with microvascular endothelial cells which are the major targets affected during HUS. All five isolates possessed the enterohemorrhagic E. coli (EHEC)-hlyA gene encoding EHEC hemolysin (EHEC-Hly), expressed the enterohemolytic phenotype, and were cytotoxic, in dose- and time-dependent manners, to human brain microvascular endothelial cells (HBMECs). Significantly reduced cytotoxicity in an EHEC-Hly-negative spontaneous derivative of one of these strains, and a dose- and time-dependent cytotoxicity of recombinant E. coli O26 EHEC-Hly to HBMECs, suggest that the endothelial cytotoxicity of these strains was mediated by EHEC-Hly. The toxicity of EHEC-Hly to microvascular endothelial cells plausibly contributes to the virulence of the stx-negative E. coli O26 strains and to the pathogenesis of HUS.     

Analysis of the clonal relationship of shiga toxin-producing Escherichia coli serogroup O165:H25 isolated from cattle

Variations in time and space of a clonal group of Escherichia coli O165:H25 on a cattle farm were monitored. The virulence marker pattern (stx genes, eae gene, hly(EHEC) gene, katP gene, espP gene, efa gene) suggests that E. coli O165:H25 of bovine origin may represent a risk for human infection.     

Shiga Toxin Gene Loss and Transfer In Vitro and In Vivo during Enterohemorrhagic Escherichia coli O26 Infection in Humans

Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx₂ loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2- and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.     

Isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) from calves and lambs with diarrhoea in India

AIMS: To determine the prevalence and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) in calves and lambs with diarrhoea in India. METHODS AND RESULTS: Faecal samples originating from 391 calves and 101 lambs which had diarrhoea were screened for presence of E. coli. A total number of 309 (249 bovine and 60 ovine) E. coli strains were isolated. A total of 113 bovine and 15 ovine strains were subjected to multiplex polymerase chain reaction (m-PCR) for detection of stx1, stx2, eaeA and EHEC hlyA genes. STEC and EPEC belonging to different serogpoups were detected in 9.73% of calves studied. Six per cent and 26.66% of lambs studied were carrying STEC and EPEC, respectively. Majority of the STEC serogroups isolated in this study did not belong to those which have been identified earlier to be associated mainly with diarrhoea and enteritis in cattle and sheep outside India. The most frequent serogroup among bovine and ovine EPEC was O26 (40%). One of the most important STEC serogroup O157, known for certain life-threatening infections in humans, was isolated from both bovine and ovine faecal samples. CONCLUSIONS: A high percentage of STEC and EPEC belonging to different serogroups are prevalent in calves and lambs with diarrhoea in India and could be the cause of disease in them. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports, for the first time, the isolation and characterization of STEC and EPEC serogroups associated with diarrhoea in calves and lambs in India. Many STEC and EPEC strains belonged to serogoups known for certain life-threatening diseases in humans.     

Computing TaqMan probes for multiplex PCR detection of E. coli O157 serotypes in water

Diarrheagenic E. coli strains contribute to water related diseases in urban and rural environment in developing and developed world. E. coli pathotype and pathogenicity varies due to complex multifactorial mechanism involving a large number of virulence factors. Rapid assessment of the virulence pattern of E. coli isolates is possible by Real-Time PCR probes like TaqMan. For designing TaqMan probes and primers for multiplex PCR selected E. coli gene sequences: stx1, stx2, hlyA, chuA, eae, lacZ, lamB and fimA were retrieved from NCBI's GenBank database. The alignment of the multiple sequences and analysis of conserved sequences was carried out using ClustalW and BLAST programs. The primers and Taqmen probes were designed using Beacon Designer software version 2.1 for two multiplexed PCR assays. In silico PCR simulation of these assays showed PCR products for stx2 (248bp) stx1 (102 bp), lacZ (228bp) and lamB (86 bp) in multiplex #1 and eae (200bp), chuA (147 bp), hlyA (141bp) and fimA (79 bp) in multiplex #2, respectively. These multiplexed PCR amplification products and probes can be used to identify and confirm presence of O157:H7/ H7-, O157:H43/45 and O26:H-/H11 serotypes. In conclusion, multiplex Real-Time Polymerase Chain Reaction oligomers and TaqMan probes designed and validated in silico will be helpful in management of water quality and outbreaks, by improving specificity and minimizing time needed for in vitro verification work.     

Verotoxin-producing Escherichia coli in Spain: prevalence,serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants,raw beef products,and humans

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H- [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H-, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O146:H8, O146:H21, O156:H-, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.     

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.     

Virulence genes and intimin types of Shiga-toxin-producing Escherichia coli isolated from cattle and beef products in Argentina.

A total of 153 Shiga-toxin-producing Escherichia coli (STEC) isolates from feces of cattle and beef products (hamburgers and ground beef) in Argentina were characterized in this study. PCR showed that 22 (14%) isolates carried stx1 genes, 113 (74%) possessed stx2 genes and 18 (12%) both stx1 and stx2. Intimin (eae), enterohemolysin (ehxA), and STEC autoagglutinating adhesin (saa) virulence genes were detected in 36 (24%), 70 (46%) and in 34 (22%) of the isolates, respectively. None of 34 saa-positive isolates carried the gene eae, and 31 were ehxA-positive. Fourteen (7 of serotype O26:H11 and 4 of serotype O5:H-) isolates had intimin b1, 16 isolates possessed intimin g1 (11 of serotype O145:H- and 5 of serotype O157:H7), 5 isolates had intimin type e1 (4 of serotypes O103:H- and O103:H2), and one isolate O111:H- showed intimin type q/g2. Although the 153 STEC isolates belonged to 63 different seropathotypes, only 12 accounted for 58% of isolates. Seropathotype ONT:H- stx2 (18 isolates) was the most common, followed by O171:H2 stx2 (12 isolates), etc. The majority (84%) of STEC isolates belonged to serotypes previously found in human STEC and 56% to serotypes associated with STEC isolated from patients with hemolytic uremic syndrome (HUS). Thus, this study confirms that cattle are a major reservoir of STEC pathogenic for humans. To our knowledge, this is the first study that described the presence of saa gene in STEC of serotypes O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, and ONT:H21. The serotypes O120:H19 and O185:H7 were not previously reported in bovine STEC.