A new highly virulent clone of EHEC O26 has emerged in Europe. Its reservoirs and sources warrant identification.

Together with plague, smallpox and typhus, epidemics of dysentery have been a major scourge of human populations for centuries(1). A previous genomic study concluded that Shigella dysenteriae type 1 (Sd1), the epidemic dysentery bacillus, emerged and spread worldwide after the First World War, with no clear pattern of transmission(2). This is not consistent with the massive cyclic dysentery epidemics reported in Europe during the eighteenth and nineteenth centuries(1,3,4) and the first isolation of Sd1 in Japan in 1897(5). Here, we report a whole-genome analysis of 331 Sd1 isolates from around the world, collected between 1915 and 2011, providing us with unprecedented insight into the historical spread of this pathogen. We show here that Sd1 has existed since at least the eighteenth century and that it swept the globe at the end of the nineteenth century, diversifying into distinct lineages associated with the First World War, Second World War and various conflicts or natural disasters across Africa, Asia and Central America. We also provide a unique historical perspective on the evolution of antibiotic resistance over a 100-year period, beginning decades before the antibiotic era, and identify a prevalent multiple antibiotic-resistant lineage in South Asia that was transmitted in several waves to Africa, where it caused severe outbreaks of disease.

Outer membrane vesicles (OMVs) are important virulence tools of enterohaemorrhagic Escherichia coli (EHEC), but other biological functions of these nanostructures are unknown. We tested the hypothesis that modulation of OMV production enables EHEC to resist the intrahost environment during infection by investigating if simulated human gastrointestinal conditions affect OMV production in EHEC O157:H7 and O104:H4. All the conditions tested including a low pH, simulated ileal and colonic media, presence of mucin, intestinal epithelial cell lysate or antimicrobial peptides, as well as iron limitation, significantly increased OMV production by these pathogens. Accordingly, a maximum vesiculation in EHEC O104:H4 was observed immediately after its isolation from a patient's intestine, and rapidly decreased during passages in vitro. Most of the simulated intrahost conditions also upregulated the OMV-associated Shiga toxin 2a (Stx2a), the major EHEC virulence factor, and, as a result, OMV cytotoxicity. The data indicates that upregulation of OMV production by the human gastrointestinal milieu contributes to EHEC survival and adaptation within the host during infection. Moreover, the intrahost increase of vesiculation and OMV-associated Stx2a may augment EHEC virulence.

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BackgroundEnterohemorrhagic 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 FindingsVirulence 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 H25]. 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/SignificanceEHEC 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.

ABSTRACT:Pathogenic Vibrio cholerae non-O1/non-O139 was isolated from the fry of the Cardinal tetra, Paracheirodon axelrodi and in adult Raphael catfish, Platydoras costatus kept in aquarium conditions. Further, an outbreak of vibriosis occurred in the wild populations of common nase, Chondrostoma nasus; chub, Squalius cephalus; gudgeon, Gobio gobio; stone loach, Barbatula barbatula; barbel, Barbus barbus; European grayling, Thymallus thymallus; schneider, Alburnoides bipunctatus; and brown trout, Salmo trutta morpha trutta. Mortality was not observed in the Eurasian minnow, Phoxinus phoxinus. During 14 days the acute rather than the sub-acute-to-chronic process of the disease prevailed with severe gross lesions in common nase and chub (diffused and focal haemorrhages, erythema and hyperaemia located especially in the abdominal region, within the mouth and at the base of fins). Only in the infection of the common nase was the whole eye affected, causing rupture of the globe and destruction of ocular structures. Gross pathological lesions in experimentally infected fish manifested themselves as gasping, erratic swimming, congested capillaries on the wall of the air bladder and fluid and blood accumulation in the abdominal cavity. Injection of 2 × 10 4 bacteria (common carp, rainbow trout) and injection of 2 × 10 8 bacteria (common nase) via i.p. route resulted in mortalities within 120hrs and 16 h, respectively. Factors underlying the rise of infection are discussed, including an extraordinary increase in water temperature (20 °C to 23 °C), fluctuating oxygen content and low water level which contributed to increasing the concentration of the faecal contamination of water: these factors could stimulate the virulence of the vibrios, which survive in aquatic flora and fauna and in the biofilm on the surface of sediments.

Sorbitol-fermenting (SF) enterohemorrhagic (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 ,, and 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 ( and the -ABC operon) and adhesins (-γ, ,, and ) 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 operon, but neither of the plasmids contains the and 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 and operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic 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. Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic 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 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 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.

A large outbreak caused by a rare Shiga toxin-producing Escherichia coli serotype O104:H4 occurred in Germany in May to July 2011. The National Reference Laboratory for E. coli and Shigella investigated the stool sample from an American tourist with bloody diarrhea who arrived in the Czech Republic from Germany where she consumed salads with raw vegetable a week ago. Using culture of the enriched stool on extended-spectrum β-lactamase agar, we isolated E. coli strain which belonged to serotype O104:H4 as determined by conventional and molecular serotyping. The strain contained the major virulence characteristics of enterohemorrhagic E. coli (stx (2) encoding Shiga toxin 2) and enteroaggregative E. coli (aggA encoding aggregative adherence fimbriae I). This unique combination of virulence traits demonstrated that this strain belongs to the hybrid enteroaggregative hemorrhagic E. coli clone which caused the German outbreak. Using advanced culture and molecular biological approaches is the prerequisite for identification of new, unusual pathogens.