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.

Institute for Hygiene University of Münster Münster Germany

National Reference Center for Salmonella and Other Enteric Pathogens Robert Koch Institut Wernigerode Germany

National Reference Laboratory for E coli and Shigella National Institute of Public Health Prague Czech Republic

Zobrazit více v PubMed

Karch H, Wiss R, Gloning H, Emmrich P, Aleksić S, Bockemühl J. 1990. Hemolytic-uremic syndrome in infants due to verotoxin-producing PubMed DOI

Karch H, Tarr PI, Bielaszewska M. 2005. Enterohaemorrhagic PubMed DOI

Bielaszewska M, Schmidt H, Liesegang A, Prager R, Rabsch W, Tschäpe H, Cízek A, Janda J, Bláhová K, Karch H. 2000. Cattle can be a reservoir of sorbitol-fermenting Shiga toxin-producing PubMed PMC

Eklund M, Bielaszewska M, Nakari UM, Karch H, Siitonen A. 2006. Molecular and phenotypic profiling of sorbitol-fermenting PubMed DOI

Jakubczak A, Szych J, Januszkiewicz K. 2008. Characterization of first sorbitol-fermenting Shiga toxin-producing PubMed

Orth D, Grif K, Dierich MP, Würzner R. 2006. Sorbitol-fermenting Shiga toxin-producing PubMed DOI PMC

Buvens G, Piérard D, Hachimi-Idrissi S, Lauwers S. 2009. First sorbitol-fermenting verocytotoxin-producing PubMed DOI

Pollock KG, Locking ME, Beattie TJ, Maxwell H, Ramage I, Hughes D, Cowieson J, Allison L, Hanson M, Cowden JM. 2010. Sorbitol-fermenting PubMed DOI PMC

Brandal LT, Løbersli I, Stavnes TL, Wester AL, Lindstedt BA. 2012. First report of the Shiga toxin 1 gene in sorbitol-fermenting PubMed DOI PMC

Marejková M, Bláhová K, Janda J, Fruth A, Petráš P. 2013. Enterohemorrhagic PubMed DOI PMC

Mellmann A, Bielaszewska M, Köck R, Friedrich AW, Fruth A, Middendorf B, Harmsen D, Schmidt MA, Karch H. 2008. Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic PubMed DOI PMC

Ammon A, Petersen LR, Karch H. 1999. A large outbreak of hemolytic uremic syndrome caused by an unusual sorbitol-fermenting strain of PubMed DOI

Alpers K, Werber D, Frank C, Koch J, Friedrich AW, Karch H, An DER, Heiden M, Prager R, Fruth A, Bielaszewska M, Morlock G, Heissenhuber A, Diedler A, Gerber A, Ammon A. 2009. Sorbitol-fermenting enterohaemorrhagic PubMed DOI

Nielsen S, Frank C, Fruth A, Spode A, Prager R, Graff A, Plenge-Bönig A, Loos S, Lütgehetmann M, Kemper MJ, Müller-Wiefel DE, Werber D. 2011. Desperately seeking diarrhoea: outbreak of haemolytic uraemic syndrome caused by emerging sorbitol-fermenting Shiga toxin-producing PubMed DOI

Rosser T, Dransfield T, Allison L, Hanson M, Holden N, Evans J, Naylor S, La Ragione R, Low JC, Gally DL. 2008. Pathogenic potential of emergent sorbitol-fermenting PubMed DOI PMC

Haugum K, Lindstedt BA, Løbersli I, Kapperud G, Brandal LT. 2012. Identification of the anti-terminator qO111:H− gene in Norwegian sorbitol-fermenting PubMed DOI

King LA, Loukiadis E, Mariani-Kurkdjian P, Haeghebaert S, Weill FX, Baliere C, Ganet S, Gouali M, Vaillant V, Pihier N, Callon H, Novo R, Gaillot O, Thevenot-Sergentet D, Bingen E, Chaud P, de Valk H. 2014. Foodborne transmission of sorbitol-fermenting PubMed DOI

Jaakkonen A, Salmenlinna S, Rimhanen-Finne R, Lundström H, Heinikainen S, Hakkinen M, Hallanvuo S. 2017. Severe outbreak of sorbitol-fermenting PubMed DOI

Vygen-Bonnet S, Rosner B, Wilking H, Fruth A, Prager R, Kossow A, Lang C, Simon S, Seidel J, Faber M, Schielke A, Michaelis K, Holzer A, Kamphausen R, Kalhöfer D, Thole S, Mellmann A, Flieger A, Stark K. 2017. Ongoing haemolytic uraemic syndrome (HUS) outbreak caused by sorbitol-fermenting (SF) Shiga toxin-producing PubMed DOI PMC

Rangel JM, Sparling PH, Crowe C, Griffin PM, Swerdlow DL. 2005. Epidemiology of PubMed DOI PMC

Bielaszewska M, Sinha B, Kuczius T, Karch H. 2005. Cytolethal distending toxin from Shiga toxin-producing PubMed DOI PMC

Müsken A, Bielaszewska M, Greune L, Schweppe CH, Müthing J, Schmidt H, Schmidt MA, Karch H, Zhang W. 2008. Anaerobic conditions promote expression of Sfp fimbriae and adherence of sorbitol-fermenting enterohemorrhagic PubMed DOI PMC

Janka A, Bielaszewska M, Dobrindt U, Greune L, Schmidt MA, Karch H. 2003. Cytolethal distending toxin gene cluster in enterohemorrhagic PubMed DOI PMC

Janka A, Bielaszewska M, Dobrindt U, Karch H. 2002. Identification and distribution of the enterohemorrhagic PubMed DOI

Bielaszewska M, Tarr PI, Karch H, Zhang W, Mathys W. 2005. Phenotypic and molecular analysis of tellurite resistance among enterohemorrhagic PubMed DOI PMC

Friedrich AW, Köck R, Bielaszewska M, Zhang W, Karch H, Mathys W. 2005. Distribution of the urease gene cluster among and urease activities of enterohemorrhagic PubMed DOI PMC

Tarr PI, Bilge SS, Vary JC Jr, Jelacic S, Habeeb RL, Ward TR, Baylor MR, Besser TE. 2000. Iha: a novel PubMed DOI PMC

Shaikh N, Holt NJ, Johnson JR, Tarr PI. 2007. Fim operon variation in the emergence of enterohemorrhagic PubMed DOI

Uhlich GA, Keen JE, Elder RO. 2001. Mutations in the PubMed DOI PMC

Brunder W, Karch H, Schmidt H. 2006. Complete sequence of the large virulence plasmid pSFO157 of the sorbitol-fermenting enterohemorrhagic PubMed DOI

Rump LV, Meng J, Strain EA, Cao G, Allard MW, Gonzalez-Escalona N. 2012. Complete DNA sequence analysis of enterohemorrhagic PubMed DOI PMC

Burland V, Shao Y, Perna NT, Plunkett G, Sofia HJ, Blattner FR. 1998. The complete DNA sequence and analysis of the large virulence plasmid of PubMed DOI PMC

Makino K, Ishii K, Yasunaga T, Hattori M, Yokoyama K, Yutsudo CH, Kubota Y, Yamaichi Y, Iida T, Yamamoto K, Honda T, Han CG, Ohtsubo E, Kasamatsu M, Hayashi T, Kuhara S, Shinagawa H. 1998. Complete nucleotide sequences of 93-kb and 3.3-kb plasmids of an enterohemorrhagic PubMed DOI

Schmidt H, Beutin L, Karch H. 1995. Molecular analysis of the plasmid-encoded hemolysin of PubMed PMC

Bielaszewska M, Rüter C, Kunsmann L, Greune L, Bauwens A, Zhang W, Kuczius T, Kim KS, Mellmann A, Schmidt MA, Karch H. 2013. Enterohemorrhagic PubMed DOI PMC

Schmidt H, Henkel B, Karch H. 1997. A gene cluster closely related to type II secretion pathway operons of gram-negative bacteria is located on the large plasmid of enterohemorrhagic PubMed DOI

Brunder W, Khan AS, Hacker J, Karch H. 2001. Novel type of fimbriae encoded by the large plasmid of sorbitol-fermenting enterohemorrhagic PubMed DOI PMC

Friedrich AW, Zhang W, Bielaszewska M, Mellmann A, Köck R, Fruth A, Tschäpe H, Karch H. 2007. Prevalence, virulence profiles, and clinical significance of Shiga toxin-negative variants of enterohemorrhagic PubMed DOI

Yu J, Kaper JB. 1992. Cloning and characterization of the PubMed DOI

Torres AG, Giron JA, Perna NT, Burland V, Blattner FR, Avelino-Flores F, Kaper JB. 2002. Identification and characterization of PubMed DOI PMC

Torres AG, Kanack KJ, Tutt CB, Popov V, Kaper JB. 2004. Characterization of the second long polar (LP) fimbriae of PubMed

Friedrich AW, Nierhoff KV, Bielaszewska M, Mellmann A, Karch H. 2004. Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene ( PubMed DOI PMC

Stephens CM, Skerker JM, Sekhon MS, Arkin AP, Riley LW. 2015. Complete genome sequences of four PubMed DOI PMC

Leimbach A, Hacker J, Dobrindt U. 2013. PubMed DOI

Stephens CM, Adams-Sapper S, Sekhon M, Johnson JR, Riley LW. 2017. Genomic analysis of factors associated with low prevalence of antibiotic resistance in extraintestinal pathogenic PubMed DOI PMC

Frost LS, Ippen-Ihler K, Skurray RA. 1994. Analysis of the sequence and gene products of the transfer region of the F sex factor. Microbiol Rev 58:162–210. PubMed PMC

Wick LM, Qi W, Lacher DW, Whittam TS. 2005. Evolution of genomic content in the stepwise emergence of PubMed DOI PMC

Feng PC, Monday SR, Lacher DW, Allison L, Siitonen A, Keys C, Eklund M, Nagano H, Karch H, Keen J, Whittam TS. 2007. Genetic diversity among clonal lineages within PubMed DOI PMC

Mellmann A, Bielaszewska M, Karch H. 2009. Intrahost genome alterations in enterohemorrhagic PubMed DOI

Kossow A, Zhang W, Bielaszewska M, Rhode S, Hansen K, Fruth A, Rüter C, Karch H, Mellmann A. 2016. Molecular characterization of human atypical sorbitol-fermenting enteropathogenic PubMed DOI PMC

Prager R, Strutz U, Fruth A, Tschäpe H. 2003. Subtyping of pathogenic PubMed DOI

Nagano I, Kunishima M, Itoh Y, Wu Z, Takahashi Y. 1998. Detection of verotoxin-producing PubMed DOI

Zhang W, Mellmann A, Sonntag AK, Wieler L, Bielaszewska M, Tschäpe H, Karch H, Friedrich AW. 2007. Structural and functional differences between disease-associated genes of enterohaemorrhagic PubMed DOI

Toma C, Martínez Espinosa E, Song T, Miliwebsky E, Chinen I, Iyoda S, Iwanaga M, Rivas M. 2004. Distribution of putative adhesins in different seropathotypes of Shiga toxin-producing PubMed DOI PMC

Scheutz F, Teel LD, Beutin L, Piérard D, Buvens G, Karch H, Mellmann A, Caprioli A, Tozzoli R, Morabito S, Strockbine NA, Melton-Celsa AR, Sanchez M, Persson S, O'Brien AD. 2012. Multicenter evaluation of a sequence-based protocol for subtyping Shiga toxins and standardizing Stx nomenclature. J Clin Microbiol 50:2951–2963. doi: 10.1128/JCM.00860-12. PubMed DOI PMC

Zhang WL, Köhler B, Oswald E, Beutin L, Karch H, Morabito S, Caprioli A, Suerbaum S, Schmidt H. 2002. Genetic diversity of intimin genes of attaching and effacing PubMed DOI PMC

Karmali MA, Petric M, Lim C, Fleming PC, Arbus GS, Lior H. 1985. The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing PubMed DOI

Kado CI, Liu ST. 1981. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 145:1365–1373. PubMed PMC

Zhang W, Bielaszewska M, Kunsmann L, Mellmann A, Bauwens A, Köck R, Kossow A, Anders A, Gatermann S, Karch H. 2013. Lability of the pAA virulence plasmid in PubMed DOI PMC

Bielaszewska M, Prager R, Vandivinit L, Müsken A, Mellmann A, Holt NJ, Tarr PI, Karch H, Zhang W. 2009. Detection and characterization of the fimbrial PubMed DOI PMC

Barton BM, Harding GP, Zuccarelli AJ. 1995. A general method for detecting and sizing large plasmids. Anal Biochem 226:235–240. doi: 10.1006/abio.1995.1220. PubMed DOI

Mellmann A, Bletz S, Böking T, Kipp F, Becker K, Schultes A, Prior K, Harmsen D. 2016. Real-time genome sequencing of resistant bacteria provides precision infection control in an institutional setting. J Clin Microbiol 54:2874–2881. doi: 10.1128/JCM.00790-16. PubMed DOI PMC

Alikhan NF, Petty NK, Ben Zakour NL, Beatson SA. 2011. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 12:402. doi: 10.1186/1471-2164-12-402. PubMed DOI PMC

Rump LV, Strain EA, Cao G, Allard MW, Fischer M, Brown EW, Gonzalez-Escalona N. 2011. Draft genome sequences of six PubMed DOI PMC

Latif H, Li HJ, Charusanti P, Palsson BØ, Aziz RK. 2014. A gapless, unambiguous genome sequence of the enterohemorrhagic PubMed DOI PMC

Hayashi T, Makino K, Ohnishi M, Kurokawa K, Ishii K, Yokoyama K, Han CG, Ohtsubo E, Nakayama K, Murata T, Tanaka M, Tobe T, Iida T, Takami H, Honda T, Sasakawa C, Ogasawara N, Yasunaga T, Kuhara S, Shiba T, Hattori M, Shinagawa H. 2001. Complete genome sequence of enterohemorrhagic PubMed DOI

European Committee on Antimicrobial Susceptibility Testing. 2017. Breakpoint tables for interpretation of MICs and zone diameters, version 7.1. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_7.1_Breakpoint_Tables.pdf Accessed 22 June 2017.

Matuschek E, Brown DF, Kahlmeter G. 2014. Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. Clin Microbiol Infect 20:O255–O266. doi: 10.1111/1469-0691.12373. PubMed DOI

Threlfall EJ, Rowe B, Ferguson JL, Ward LR. 1986. Characterization of plasmids conferring resistance to gentamicin and apramycin in strains of PubMed DOI PMC

Karch H, Böhm H, Schmidt H, Gunzer F, Aleksic S, Heesemann J. 1993. Clonal structure and pathogenicity of Shiga-like toxin-producing, sorbitol-fermenting PubMed PMC

Riley LW, Remis RS, Helgerson SD, McGee HB, Wells JG, Davis BR, Hebert RJ, Olcott ES, Johnson LM, Hargrett NT, Blake PA, Cohen ML. 1983. Hemorrhagic colitis associated with a rare PubMed DOI

Watanabe H, Wada A, Inagaki Y, Itoh K, Tamura K. 1996. Outbreaks of enterohaemorrhagic PubMed DOI