Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H- strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H- strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfAO157OI-141, and lpfAO157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H- strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H- strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology.IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H- (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic-uremic syndrome in Europe. They account for 10 to 20% of sporadic cases of this disease and have caused several large outbreaks. The strains isolated throughout Europe share conserved chromosomal and plasmid characteristics. Here we identified novel sorbitol-fermenting enterohemorrhagic E. coli O157:H- patient isolates in the Czech Republic which differ from all such strains reported previously by their unique plasmid characteristics, including plasmid number, composition of plasmid-carried virulence genes, and plasmid origins. Our findings contribute substantially to understanding the evolution of E. coli O157 strains and their plasmids. In practical terms, they enable the identification of strains with these novel plasmid characteristics in patient stool samples and thus the investigation of their roles as human pathogens in other geographic areas.

• Klíčová slova
• EHEC O157, Sfp fimbriae, enterohemorrhagic E. coli, hemolytic-uremic syndrome, outbreaks, plasmid analysis,
• MeSH
• Escherichia coli O157 klasifikace   izolace a purifikace   metabolismus   MeSH
• faktory virulence genetika   metabolismus   MeSH
• fermentace MeSH
• infekce vyvolané Escherichia coli mikrobiologie   MeSH
• lidé MeSH
• plazmidy genetika   metabolismus   MeSH
• proteiny z Escherichia coli genetika   metabolismus   MeSH
• sorbitol metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Německo MeSH
• Názvy látek
• faktory virulence MeSH
• proteiny z Escherichia coli MeSH
• sorbitol MeSH

BACKGROUND: Enterohemorrhagic Escherichia coli (EHEC) cause diarrhea-associated hemolytic uremic syndrome (D+ HUS) worldwide, but no systematic study of EHEC as the causative agents of HUS was performed in the Czech Republic. We analyzed stools of all patients with D+ HUS in the Czech Republic between 1998 and 2012 for evidence of EHEC infection. We determined virulence profiles, phenotypes, antimicrobial susceptibilities and phylogeny of the EHEC isolates. METHODOLOGY/PRINCIPAL FINDINGS: Virulence loci were identified using PCR, phenotypes and antimicrobial susceptibilities were determined using standard procedures, and phylogeny was assessed using multilocus sequence typing. During the 15-year period, EHEC were isolated from stools of 39 (69.4%) of 56 patients. The strains belonged to serotypes [fliC types] O157:H7/NM[fliC(H7)] (50% of which were sorbitol-fermenting; SF), O26:H11/NM[fliC(H11)], O55:NM[fliC(H7)], O111:NM[fliC(H8)], O145:H28[fliC(H28)], O172:NM[fliC(H25)], and Orough:NM[fliC(H250]. O26:H11/NM[fliC(H11)] was the most common serotype associated with HUS (41% isolates). Five stx genotypes were identified, the most frequent being stx(2a) (71.1% isolates). Most strains contained EHEC-hlyA encoding EHEC hemolysin, and a subset (all SF O157:NM and one O157:H7) harbored cdt-V encoding cytolethal distending toxin. espPα encoding serine protease EspPα was found in EHEC O157:H7, O26:H11/NM, and O145:H28, whereas O172:NM and Orough:NM strains contained espPγ. All isolates contained eae encoding adhesin intimin, which belonged to subtypes β (O26), γ (O55, O145, O157), γ2/θ (O111), and ε (O172, Orough). Loci encoding other adhesins (efa1, lpfA(O26), lpfA(O157OI-141), lpfA(O157OI-154), iha) were usually associated with particular serotypes. Phylogenetic analysis demonstrated nine sequence types (STs) which correlated with serotypes. Of these, two STs (ST660 and ST1595) were not found in HUS-associated EHEC before. CONCLUSIONS/SIGNIFICANCE: EHEC strains, including O157:H7 and non-O157:H7, are frequent causes of D+ HUS in the Czech Republic. Identification of unusual EHEC serotypes/STs causing HUS calls for establishment of an European collection of HUS-associated EHEC, enabling to study properties and evolution of these important pathogens.

• MeSH
• antibakteriální látky farmakologie   MeSH
• enterohemoragická Escherichia coli klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• fenotyp MeSH
• fylogeneze MeSH
• genotyp MeSH
• hemolyticko-uremický syndrom diagnóza   epidemiologie   mikrobiologie   MeSH
• kojenec MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• multilokusová sekvenční typizace MeSH
• předškolní dítě MeSH
• roční období MeSH
• sérotypizace MeSH
• shiga toxin genetika   MeSH
• virulence genetika   MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• antibakteriální látky MeSH
• shiga toxin MeSH

Using the immunomagnetic separation procedure, we isolated sorbitol-fermenting (SF) Shiga toxin-producing Escherichia coli (STEC) O157:H(-) strains from two patients, one with hemolytic-uremic syndrome and the other with diarrhea, and from a dairy cow epidemiologically associated with the patients. The phenotypic and genotypic characteristics of all isolates were identical or closely related. Moreover, the bovine isolate showed a clonal relatedness to SF STEC O157:H(-) strains isolated from patients in Germany and the Czech Republic from 1988 to 1998. This is the first evidence that cattle can be a reservoir of SF STEC O157:H(-) and a source of human diseases.

• MeSH
• dítě MeSH
• Escherichia coli O157 klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• fermentace MeSH
• hemolyticko-uremický syndrom mikrobiologie   MeSH
• imunomagnetická separace MeSH
• infekce vyvolané Escherichia coli mikrobiologie   přenos   MeSH
• kojenec MeSH
• lidé MeSH
• průjem mikrobiologie   MeSH
• pulzní gelová elektroforéza MeSH
• shiga toxin 1 biosyntéza   MeSH
• shiga toxin 2 biosyntéza   MeSH
• skot mikrobiologie   MeSH
• sorbitol metabolismus   MeSH
• zdroje nemoci * MeSH
• zvířata MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• skot mikrobiologie   MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• shiga toxin 1 MeSH
• shiga toxin 2 MeSH
• sorbitol MeSH

Two sorbitol-fermenting (SF) Shiga toxin-producing Escherichia coli (STEC) O157:H- strains were isolated from patients with hemolytic-uremic syndrome in the Czech Republic in 1995. Their phenotypic and genotypic characteristics and genomic DNA fingerprints were identical or closely related to those of SF STEC O157:H- strains isolated in Germany in 1988 to 1997. This indicates that the Czech isolates belong to the SF STEC O157 clone which is widespread in Germany. It is the first finding of the clone outside Germany.

• MeSH
• bakteriální toxiny biosyntéza   MeSH
• DNA bakterií analýza   MeSH
• DNA fingerprinting MeSH
• Escherichia coli O157 klasifikace   izolace a purifikace   MeSH
• feces mikrobiologie   MeSH
• fenotyp MeSH
• fylogeneze MeSH
• genotyp MeSH
• hemolyticko-uremický syndrom mikrobiologie   MeSH
• infekce vyvolané Escherichia coli mikrobiologie   MeSH
• kojenec MeSH
• lidé MeSH
• polymerázová řetězová reakce metody   MeSH
• shiga toxin 1 MeSH
• sorbitol metabolismus   MeSH
• technika náhodné amplifikace polymorfní DNA MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• bakteriální toxiny MeSH
• DNA bakterií MeSH
• shiga toxin 1 MeSH
• sorbitol MeSH