BackgroundEnterohemorrhagic Escherichia coli (EHEC) O145 are among the top non-O157 serogroups associated with severe human disease worldwide. Two serotypes, O145:H25 and O145:H28 have been isolated from human patients but little information is available regarding the virulence repertoire, origin and evolutionary relatedness of O145:H25. Hence, we sequenced the complete genome of two O145:H25 strains associated with hemolytic uremic syndrome (HUS) and compared the genomes with those of previously sequenced O145:H28 and other EHEC strains.ResultsThe genomes of the two O145:H25 strains were 5.3 Mbp in size; slightly smaller than those of O145:H28 and other EHEC strains. Both strains contained three nearly identical plasmids and several prophages and integrative elements, many of which differed significantly in size, gene content and organization as compared to those present in O145:H28 and other EHECs. Furthermore, notable variations were observed in several fimbrial gene cluster and intimin types possessed by O145:H25 and O145:H28 indicating potential adaptation to distinct areas of host colonization. Comparative genomics further revealed that O145:H25 are genetically more similar to other non-O157 EHEC strains than to O145:H28.ConclusionPhylogenetic analysis accompanied by comparative genomics revealed that O145:H25 and O145:H28 evolved from two separate clonal lineages and that horizontal gene transfer and gene loss played a major role in the divergence of these EHEC serotypes. The data provide further evidence that ruminants might be a possible reservoir for O145:H25 but that they might be impaired in their ability to establish a persistent colonization as compared to other EHEC strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-017-1094-3) contains supplementary material, which is available to authorized users.

c Shiga toxin-producing Escherichia coli (STEC) belonging to certain serogroups (e.g., O157 and O26) can cause serious conditions like hemolytic-uremic syndrome (HUS), but other strains might be equally pathogenic. While virulence factors, like stx and eae, have been well studied, little is known about the prevalence of the E. coli hemolysin genes (hlyA, ehxA, e-hlyA, and sheA) in association with these factors. Hemolysins are potential virulence factors, and ehxA and hlyA have been associated with human illness, but the significance of sheA is unknown. Hence, 435 E. coli strains belonging to 62 different O serogroups were characterized to investigate gene presence and phenotypic expression of hemolysis. We further investigated ehxA subtype patterns in E. coli isolates from clinical, animal, and food sources. While sheA and ehxA were widely distributed, e-hlyA and hlyA were rarely found. Most strains (86.7%) were hemolytic, and significantly more hemolytic (95%) than nonhemolytic strains (49%) carried stx and/or eae (P < 0.0001). ehxA subtyping, as performed by using PCR in combination with restriction fragment length polymorphism analysis, resulted in six closely related subtypes (>94.2%), with subtypes A/D being eae-negative STECs and subtypes B, C, E, and F eae positive. Unexpectedly, ehxA subtype patterns differed significantly between isolates collected from different sources (P < 0.0001), suggesting that simple linear models of exposure and transmission need modification; animal isolates carried mostly subtypes A/C (39.3%/42.9%), food isolates carried mainly subtype A (81.9%), and clinical isolates carried mainly subtype C (66.4%). Certain O serogroups correlated with particular ehxA subtypes: subtype A with O104, O113, and O8; B exclusively with O157; C with O26, O111, and O121.

Most Shiga toxin-producing Escherichia coli (STEC) strains associated with severe disease, such as hemolytic-uremic syndrome (HUS), carry large enterohemolysin-encoding (ehxA) plasmids, e.g., pO157 and pO103, that contribute to STEC clinical manifestations. Six ehxA subtypes (A through F) exist that phylogenetically cluster into eae-positive (B, C, F), a mix of eae-positive (E) and eae-negative (A), and a third, more distantly related, cluster of eae-negative (D) STEC strains. While subtype B, C, and F plasmids share a number of virulence traits that are distinct from those of subtype A, sequence data have not been available for subtype D and E plasmids. Here, we determined and compared the genetic composition of four subtype D and two subtype E plasmids to establish their evolutionary relatedness among ehxA subtypes and define their potential role in pathogenicity. We found that subtype D strains carry one exceptionally large plasmid (>200 kbp) that carries a variety of virulence genes that are associated with enterotoxigenic and enterohemorrhagic E. coli, which, quite possibly, enables these strains to cause disease despite being food isolates. Our data offer further support for the hypothesis that this subtype D plasmid represents a novel virulence plasmid, sharing very few genetic features with other plasmids; we conclude that these plasmids have evolved from a different evolutionary lineage than the plasmids carrying the other ehxA subtypes. In contrast, the 50-kbp plasmids of subtype E (pO145), although isolated from HUS outbreak strains, carried only few virulence-associated determinants, suggesting that the clinical presentation of subtype E strains is largely a result of chromosomally encoded virulence factors. IMPORTANCEBacterial plasmids are known to be key agents of change in microbial populations, promoting the dissemination of various traits, such as drug resistance and virulence. This study determined the genetic makeup of virulence plasmids from rare enterohemolysin subtype D and E Shiga toxin-producing E. coli strains. We demonstrated that ehxA subtype D plasmids represent a novel E. coli virulence plasmid, and although subtype D plasmids were derived from nonclinical isolates, they encoded a variety of virulence determinants that are associated with pathogenic E. coli. In contrast, subtype E plasmids, isolated from strains recovered from severely ill patients, carry only a few virulence determinants. The results of this study reemphasize the plasticity and vast diversity among E. coli plasmids. This work demonstrates that, although E. coli strains of certain serogroups may not be frequently associated with disease, they should not be underestimated in protecting human health and food safety. Shiga toxin-producing Escherichia coli (STEC) strains of various serotypes can cause severe illnesses, such as hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). While E. coli O157:H7 represents the most prevalent serotype associated with severe human illness, non-O157 STEC strains are of...