Analysis of the Genome of theEscherichia coliO157:H7 2006 Spinach-Associated Outbreak Isolate Indicates Candidate Genes That May Enhance Virulence

Abstract

In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx 1 . The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx 1 , indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx 1 and a propensity for HUS.

Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157 : H7

“…Stx2 has been associated with severe disease more frequently than Stx1 (Boerlin et al, 1999;Ethelberg et al, 2004), and was shown to be 400 times more potent than Stx1, in mice (Tesh et al, 1993). Mitomycin C treatment also led to increased induction of stx2 expression in TW14359 compared to Sakai (Kulasekara et al, 2009). Here we show that in the absence of DNA-damaging agents, baseline expression of stx2 is markedly higher in TW14359 relative to Sakai before and after epithelial cell exposure, suggesting that increased Stx2 production in this strain may play a role in HUS development.…”

“…This could be due in part to an unrecognized gene or set of genes unique to TW14359. Indeed, the recent comparison of TW14359 and Sakai genomes identified several genes exclusive to TW14359, including putative TTSS effectors and an intact norV gene, the deletion of which is hypothesized to decrease colonization of the large intestine (Kulasekara et al, 2009). Future work should focus on comparing TTSS and effector protein levels between these strains.…”

“…Strain TW14359 belongs to an ancestral lineage compared to that of Sakai (Leopold et al, 2009). TW14539 also differs by 10 % of nucleotide sequence in 2741 conserved genes (Manning et al, 2008) and contains more unique sequence (70 kb) than both Sakai and EDL-933 outbreak strains (Kulasekara et al, 2009). Given that EDL-933 is so closely related to Sakai (Laing et al, 2009;Leopold et al, 2009;Manning et al, 2008), we chose to make comparisons between the two strains representing the most divergent phylogenetic lineages.…”

“…Indeed, the Sakai and TW14359 genomes differ by 10 % in nucleotide sequence among 2741 conserved genes, while the difference between Sakai and EDL-933 (Perna et al, 2001), another genome strain associated with the first O157 : H7 outbreak in 1982 (Riley et al, 1983), was only 3 % (Manning et al, 2008). TW14359 also contains a variant of the Shiga toxin 2 gene (stx2c) and the lysogenic bacteriophage 2851 that carries it (Strauch et al, 2008), as well as several putative virulence factor genes absent in the other two genomes (Kulasekara et al, 2009). In addition, TW14359 is a member of clade 8, an O157 : H7 subpopulation that was more commonly identified from patients with HUS in one study (Manning et al, 2008), while another study demonstrated that TW14359 caused more severe disease in germ-free mice than Sakai (Eaton et al, 2008).…”

Phylogenetic characterization of Escherichia coli O157 : H7 based on IS629 distribution and Shiga toxin genotype

“…Genome analysis by octamer-based genomic scanning and microarraybased comparative genomic hybridization (Kim et al, 1999;Zhang et al, 2007) has shown that E. coli O157 : H7 can be assigned to three main phylogenetic lineages: termed lineages I, II and I/II. Typically, lineage I isolates originate from human clinical and bovine sources; whereas, lineage II strains predominantly have a bovine origin, while I/II strains are associated with human infection and include a hyper-virulent group consisting of the multistate 'spinach' outbreak strains Kulasekara et al, 2009;Laing et al, 2009). A key genetic signature of the three E. coli O157 : H7 lineages lies in the possession of different shiga toxin (stx) genes, carried by shiga toxin-encoding prophages.…”