[Enterohemorrhagic Escherichia coli as the cause of diarrhea in the Czech Republic, 1965-2013].
Enterohemorrhagic Escherichia coli (EHEC) is the cause of diarrhea, bloody diarrhea, and haemolytic uremic syndrome (HUS) worldwide. The role of EHEC in the etiology of HUS in the Czech Republic has recently been described, but the prevalence, characteristics, and epidemiology of EHEC causing diarrhea have not been fully known. Therefore, this study analyzed the serotypes, stx genotypes, and virulence factors in EHEC strains isolated in 1965-2013 from patients with diarrhea or bloody diarrhea and their family contacts. In addition, we characterized diagnostically relevant phenotypes of EHEC strains, their antimicrobial susceptibility, seasonal trends, and distribution by administrative region.
Serogrouped E. coli isolates from patients were referred to the National Reference Laboratory (NRL) for E. coli and Shigella for the detection of Stx. Specimens of both human and non-human origin were referred to the NRL for epidemiological investigation. Serotyping was performed by conventional and molecular methods, PCR was applied to stx genotyping and identification of non-stx virulence factors, and standard methods were used for phenotypic analysis and antimicrobial susceptibility testing. The epidemiological link between the human and animal isolates was confirmed using pulsed-field gel electrophoresis (PFGE).
Of 50 EHEC strains, 24 were recovered from patients with diarrhea without blood, 19 from patients with bloody diarrhea, six from family contacts, and one from an epidemiologically linked animal. EHEC cases were reported during the whole year, with peaks in May through October, most often in the Central Bohemian and Hradec Králové Regions. EHEC outbreaks occurred in three families: in one of them sheep-to-human transmission of EHEC was detected. The EHEC strains were assigned to five serotypes, with more than half of them being non-sorbitol fermenting (NSF) O157:H7/NM[fliCH7] and a third being strains O26:H11/NM[fliCH11]; serotypes O111:NM[fliCH8], O118:NM[fliCH25], and O104:H4, similarly to sorbitol-fermenting (SF) strains O157:NM[fliCH7], were rare. Of seven stx genotypes identified, all were present in NSF EHEC O157, two in each of EHEC O26 and O111, and one in each of EHEC O118, O104, and SF O157. All but one strain were Stx producers. Genes encoding other virulence factors including toxins (EHEC-hlyA, cdt-V, and espP) and adhesins (eae, efa1, iha, lpf, and sfpA) were detected in all strains and their occurrence was serotype specific. The most common of these genes were eae encoding adhesin intimin and EHEC-hlyA encoding EHEC hemolysin. All EHEC strains but SF O157 harboured terE encoding tellurite resistance. All strains except NSF EHEC O157 and EHEC O118 fermented sorbitol and produced ß-D-glucuronidase. Most (89.8%) EHEC strains were susceptible to all 12 antimicrobials tested.
EHEC strains cause diarrhea and bloody diarrhea in the Czech Republic. Nevertheless, only a systematic screening of the stool from patients with diarrhea can make it possible to elucidate their actual role in the etiology of diarrheal diseases (as well as HUS) in the Czech Republic and to consider the data in the European context. EHEC cases are reported to the European Centre for Disease Prevention and Control (ECDC) within the Food and Waterborne Diseases Surveillance Network.
Marejková M
，Petráš P
《EPIDEMIOLOGIE MIKROBIOLOGIE IMUNOLOGIE》
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
... -
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