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.

To evaluate the GeneDisc multiplex real-time PCR assay for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains. 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. 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. The assay is promising for its use in EHEC diagnostics and for EHEC monitoring with different kinds of samples.

Beutin L ，Jahn S ，Fach P 《-》

Micro-array for the identification of Shiga toxin-producing Escherichia coli (STEC) seropathotypes associated with Hemorrhagic Colitis and Hemolytic Uremic Syndrome in humans.

A micro-array has been developed, based on the GeneDisc(R) array, for the genetic identification of 12 O-types and 7 H-types of Shiga toxin-producing Escherichia coli (STEC) including the most clinically relevant enterohemorrhagic E. coli (EHEC) serotypes. The genes selected for determination of the O antigens (rfbE(O157), wzx(O26), wzx(O103), wbd1(O111), ihp1(O145), wzx(O121), wzy(O113), wzy(O91), wzx(O104), wzy(O118), wzx(O45), and wbgN(O55)) and H-types (fliC(H2), fliC(H7), fliC(H8), fliC(H11), fliC(H19), fliC(H21), and fliC(H28)) showed a high specificity and concordance with serology. The micro-array also had a high specificity for EHEC-associated virulence factors, including Shiga toxins 1 and 2 (stx1 and stx2), intimin (eae), enterohemolysin (ehxA), serine protease (espP), catalase peroxidase (katP), the type II secretion system (etpD), subtilase cytotoxin (subA), autoagglutinating adhesin (Saa) and type III secreted effectors encoded in the genomic islands OI-122 (ent/espL2, nleB, and nleE) and OI-71 (nleF, nleH1-2, and nleA). The eae gene was detected in all typical EHEC strains, and the pattern of nle genes encoded in OI-71 and OI-122 was found to be closely associated with certain serotypes of typical EHEC and emerging EHEC strains. Virulence plasmid associated genes such as katP, espP, and etpD were more common in EHEC than in STEC strains; this supports their association with virulence. This array constitutes a valuable approach for the identification of STEC strains with a high potential for human virulence.

Bugarel M ，Beutin L ，Martin A ，Gill A ，Fach P ... -  《-》

Application of a real-time PCR-based system for monitoring of O26, O103, O111, O145 and O157 Shiga toxin-producing Escherichia coli in cattle at slaughter.

Faecal samples were collected from 573 slaughtered cattle aged between three and 24 months in seven abattoirs. After enrichment (mTSB with novobiocin), samples were screened by real-time PCR first for stx and if positive, tested for the top-five Shiga toxin-producing Escherichia coli (STEC) serogroups using PCR assays targeting genes specific for serogroups O26, O103, O111, O145 and O157. Of 563 samples with available results, 74.1% tested positive for stx genes. Amongst them, the serogroups O145, O103, O26, O157 and O111 were detected in 41.9%, 25.9%, 23.9%, 7.8% and 0.8%, respectively. From 95 O26, 166 O145 and 30 O157 PCR-positive samples, 17 O26, 28 O145 and 12 O157 strains were isolated by colony hybridization after immunomagnetic separation. The 17 O26 strains were eae-positive, but only nine strains harboured stx (eight possessing stx1 and one stx2). Of the 28 O145 strains, ten were eae-positive including four harbouring stx1 or stx2, whereas 18 were negative for stx and eae. Five of the 12 O157 strains harboured stx2 and eae, did not ferment sorbitol, and were identified as STEC O157:H7/H⁻. The other seven O157 strains were negative for stx and eae or positive only for eae. Shiga toxin genes and the top-five STEC serogroups were frequently found in young Swiss cattle at slaughter, but success rates for strain isolation were low and only few strains showed a virulence pattern of human pathogenic STEC.

Hofer E ，Stephan R ，Reist M ，Zweifel C ... -  《-》

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.

Madic J ，Vingadassalon N ，de Garam CP ，Marault M ，Scheutz F ，Brugère H ，Jamet E ，Auvray F ... -  《-》

Simplex and multiplex real-time PCR assays for the detection of flagellar (H-antigen) fliC alleles and intimin (eae) variants associated with enterohaemorrhagic Escherichia coli (EHEC) serotypes O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7.

To develop real-time PCR assays targeting genes encoding the flagellar antigens (fliC) and intimin subtypes (eae) associated with the five most clinically important serotypes of enterohaemorrhagic Escherichia coli (EHEC), i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7.   Primers and probes specific to fliC(H2) , fliC(H7) , fliC(H8) , fliC(H11) , fliC(H28) , eae-β1, eae-γ1, eae-ε and eae-θ were combined in simplex and multiplex 5'-nuclease PCR assays. The specificity of the assays was assessed on 201 bacterial strains and the sensitivity determined on serially diluted EHEC genomes. The developed PCR assays were found to be highly specific and detected as few as five EHEC genome equivalents per reaction. Furthermore, it was possible to detect the five major EHEC serotypes in cheese samples inoculated at concentration levels of ≤5CFU per 25g after overnight enrichment using the PCR assays. The PCR assays developed here were found to be sensitive and specific for the reliable detection of genes encoding the flagellar antigens and intimin variants belonging to the five most clinically relevant EHEC serotypes. Application of real-time PCR assays should improve the identification of foods contaminated by EHEC and facilitate the molecular typing of these organisms.

Madic J ，Peytavin de Garam C ，Vingadassalon N ，Oswald E ，Fach P ，Jamet E ，Auvray F ... -  《-》