Characterization of Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H - 1 from Czech Patients Identifies Novel Plasmid Composition Not Previously Seen in German 2 Isolates 3

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H - strains, 25 first identified in Germany, have emerged as important pathogens throughout Europe. Besides 26 chromosomally encoded Shiga toxin 2a, the major virulence factor, several putative virulence 27 loci including the hly , etp , and sfp operons encoding EHEC hemolysin, type II secretion system, 28 and Sfp fimbriae, respectively, are located on a 121 kb plasmid pSFO157 in German strains. 29 Here, we report novel SF EHEC O157:H - strains isolated from patients in the Czech Republic. 30 These strains share the core genomes and chromosomal virulence loci encoding toxins ( stx 2a , 31 cdt V- ABC operon) and adhesins ( eae γ, efa1 , lpfA O157OI-141 , lpfA O157OI-154 ) with German strains, 32 but essentially differ by their plasmids. In contrast to all previously detected SF EHEC O157:H - 33 strains, the Czech strains carry two plasmids of 79 kb and 86 kb. The 79 kb plasmid harbors the 34 sfp operon, but none of them contains the hly and etp operons. Sequence analyses demonstrated 35 that the 79 kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by 36 deletion of a 41,534 bp region via homologous recombination, resulting in the loss of hly and etp 37 operons. The 86 kb plasmid (pSFO157 258/98-2) displays 99% sequence similarity to a 92.7 kb 38 plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our findings of this novel 39 plasmid composition in SF EHEC O157:H - strains extend the evolutionary history of EHEC 40 O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification 41 of such strains, thereby facilitating further investigations of their geographic distribution, clinical threatening hemolytic uremic syndrome in Europe. They account for 10-20% of sporadic cases of this disease and 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 - patients´ isolates in the Czech Republic, which 51 differ from all such strains reported previously by their unique plasmid characteristics including 52 the plasmid number, composition of plasmid-encoded virulence genes, and plasmid origins. Our 53 findings substantially contribute to understanding the evolution of E. coli O157 and their 54 plasmids. In practical terms, they enable to identify strains with these novel plasmid 55 characteristics in patients´ stool samples and thus investigate their roles as human pathogens in 56 other geographic areas. SF EHEC O157:H - isolated in Germany and other countries (2, 4, 6, 7, 18, 38). We isolated SF EHEC O157:H - strains from patients in the Czech Republic that differ from the typical German SF EHEC 99 O157:H - by their plasmid profiles and plasmid virulence genes composition. Here, we compared the phylogeny, molecular characteristics and the corresponding phenotypes of these unusual SF EHEC O157:H - strains with those of prototype German SF EHEC O157:H - . We provide sequences of the novel plasmids harbored by the Czech SF EHEC O157:H - strains and portray their evolutionary history.