Analysis of the Genome of the Escherichia coli O157:H7 2006 Spinach-Associated Outbreak Isolate Indicates Candidate Genes That May Enhance Virulence

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 stx₁. 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 stx₁, 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 stx₁ and a propensity for HUS.Escherichia coli O157:H7 is a human pathogen that infects more than 73,000 North Americans per year (39). Although infection by this organism typically causes symptoms such as watery or bloody diarrhea, it may also lead to the development of hemolytic-uremic syndrome (HUS), an infection sequela characterized by hemolysis and renal failure that can result in long-lasting kidney damage. Variables that contribute to the development of HUS include host factors, such as age (51), and the genetic background of the enterohemorrhagic E. coli (EHEC) isolate. Currently, no effective prophylaxis exists for HUS (45). Antibiotic treatment of E. coli O157:H7 infections is contraindicated as it is associated with increased infection sequelae (45, 58).Humans become infected with EHEC by consuming contaminated food. EHEC are noninvasive pathogens that primarily colonize the human colon. Serotype O157:H7 is the predominant EHEC serotype in North America. The other commonly isolated EHEC serotypes include O26:H11, O103:H2, O111:NM, and O113:H21 (34). The systemic absorption of Shiga toxins produced by intestinal EHEC is thought to damage endothelial cells and to cause HUS (31). Shiga toxins are A-B-type toxins that inhibit protein synthesis. The genes encoding these potent toxins are borne on prophages that are related to phage λ. There are two main variants of Shiga toxin, Stx1 and Stx2. Stx2 is more cytotoxic than Stx1 in cell culture and animal models (27, 46, 48), and epidemiologic observations have revealed that the risk of developing HUS following an EHEC infection is heightened if the isolate produces Stx2 (4). Several variants of Stx2 exist, and Stx2c is the variant most commonly found in O157:H7 strains. Stx2 and Stx2c have the same biological function and possess identical A subunits and B subunits that share at least 97% identity (10).Although important for virulence, Stx2 does not appear to be the only EHEC factor that significantly influences whether patients infected with EHEC develop HUS. A comparison of statistics for several outbreaks caused by Stx2-producing O157:H7 strains showed that the rate of HUS can vary from less than 1% to 26% (23), indicating that strain-specific factors of stx₂-carrying O157:H7 strains are involved in determining clinical outcomes. To date, the most significant factor identified as a factor contributing to the variability is the presence of the stx₁ gene. O157:H7 strains that lack stx₁ but carry one or two stx₂ alleles are more likely to cause infections resulting in HUS (11, 35, 36).A comparison of the genome sequences of O157:H7 outbreak isolates that have resulted in different HUS rates may provide further insight into genetic factors that contribute to this severe sequela of EHEC infection. The genome sequences of two O157:H7 strains that caused low frequencies of HUS are available. The Sakai strain, the cause of the 1996 outbreak in Japan, caused ∼8,000 infections in people, the majority of whom were children, and the rate of HUS was 1.2% (32). In 1982, EDL933 caused the first diarrhea outbreak linked to the O157:H7 serotype and involved 44 individuals but no recorded HUS cases (41).Sakai shares 4.1 Mb of DNA with the commensal E. coli K-12 strain MG1655 and has 296 novel DNA segments more than 19 bp long, termed S-loops, that account for 1.39 Mb. EDL933 shares 4.1 Mb with E. coli K-12 strain MG1655 and has 177 unique sequence segments more than 50 bp long, termed O-islands, that account for 1.34 Mb (19). For both the Sakai and EDL933 genomes there is significant evidence of horizontal transfer due to the presence of numerous prophage-related elements and the pO157 virulence plasmid. The virulence factors carried on the O157:H7-specific DNA segments, as well as pO157, include stx₁, stx₂, the locus of enterocyte effacement (LEE), which confers the ability to cause attaching and effacing lesions on enterocytes and, notably, encodes a type III secretion system (TTSS) (22), at least 39 TTSS effectors encoded either on the LEE or at other chromosomal locations (49), numerous fimbrial and nonfimbrial adhesins, and more than one hemolysin (56).No genome sequence is available yet for an O157:H7 outbreak isolate that has caused an outbreak resulting in a significantly higher HUS rate. One O157:H7 isolate, TW14359, caused an outbreak associated with contaminated spinach that sickened 205 individuals in September and October of 2006. A total of 15% of the afflicted individuals developed HUS (5, 28). This rate is significantly higher than the average annual rate of 4.1% for O157:H7 cases that develop HUS (39). The relatively high percentage of adults, ∼8%, who developed HUS in the TW14359 outbreak also likely reflects the greater virulence of this strain (6). Furthermore, Manning et al. performed a phylogenetic analysis of TW14359 utilizing 96 single-nucleotide polymorphisms (SNPs) and demonstrated that this isolate belongs to a more virulent clade of O157:H7 strains (clade 8); the majority of these isolates lack stx₁ and carry stx₂ (28). A partial genome sequence consisting of 200 contigs of the TW14359 genome was also reported by Manning et al., which was found to contain stx₂ and stx_2c. While an analysis of these sequence data identified the genes of the two reference isolates that were also present in TW14359 and identified backbone SNPs, it did not provide a list of novel genetic features or provide assembled DNA segments containing repetitive DNA elements, such as phage-like elements. Here we describe the entire genome sequence of this isolate and, focusing on novel genetic material, identify potential genetic features of TW14359 that may promote this strain''s outstanding pathogenicity.