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Probing genomic diversity and evolution of Escherichia coli O157 by single nucleotide polymorphisms

Wei Zhang1,5,1, Weihong Qi2, Thomas J. Albert3, Alifiya S. Motiwala4, David Alland4, Eija K. Hyytia-Trees1, Efrain M. Ribot1, Patricia I. Fields1, Thomas S. Whittam2 and Bala Swaminathan1

1 Foodborne and Diarrheal Diseases Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA; , 2 Microbial Evolution Laboratory, National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824, USA; , 3 NimbleGen Systems Inc., Madison, Wisconsin 53711, USA; , 4 Division of Infectious Diseases, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA

   ABSTRACT

Infections by Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) are the predominant cause of bloody diarrhea and hemolytic uremic syndrome in the United States. In silico comparison of the two complete STEC O157 genomes (Sakai and EDL933) revealed a strikingly high level of sequence identity in orthologous protein-coding genes, limiting the use of nucleotide sequences to study the evolution and epidemiology of this bacterial pathogen. To systematically examine single nucleotide polymorphisms (SNPs) at a genome scale, we designed comparative genome sequencing microarrays and analyzed 1199 chromosomal genes (a total of 1,167,948 bp) and 92,721 bp of the large virulence plasmid (pO157) of eleven outbreak-associated STEC O157 strains. We discovered 906 SNPs in 523 chromosomal genes and observed a high level of DNA polymorphisms among the pO157 plasmids. Based on a uniform rate of synonymous substitution for Escherichia coli and Salmonella enterica (4.7 x 10–9 per site per year), we estimate that the most recent common ancestor of the contemporary -glucuronidase-negative, non-sorbitolfermenting STEC O157 strains existed ca. 40 thousand years ago. The phylogeny of the STEC O157 strains based on the informative synonymous SNPs was compared to the maximum parsimony trees inferred from pulsed-field gel electrophoresis and multilocus variable numbers of tandem repeats analysis. The topological discrepancies indicate that, in contrast to the synonymous mutations, parts of STEC O157 genomes have evolved through different mechanisms with highly variable divergence rates. The SNP loci reported

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here will provide useful genetic markers for developing high-throughput methods for fine-resolution genotyping of STEC O157. Functional characterization of nucleotide polymorphisms should shed new insights on the evolution, epidemiology, and pathogenesis of STEC O157 and related pathogens.

Shiga toxin-producing Escherichia coli O157:H7 (STEC O157) can cause severe enteric infections. Symptoms may include abdominal pain, bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS) (Griffin and Tauxe 1991; Feng 1995). Numerous sporadic infections and outbreaks caused by STEC O157 have been reported in the United States and worldwide (Griffin and Tauxe 1991; Mead and Griffin 1998). The majority of STEC O157 infections are foodborne; many are associated with bovine sources (Feng 1995; Kim et al. 1999). STEC O157 was first linked to outbreaks of severe bloody diarrhea in 1982, and is often referred to as a "recently emerged" human pathogen (Griffin and Tauxe 1991; Bitzan et al. 1993).

Strains of STEC O157 from clinical and bovine sources have been shown to be genotypically diverse by a variety of molecular subtyping methods, including pulsed-field gel electrophoresis (PFGE) (Noller et al. 2003b), octamer-based genome scanning (Kim et al. 1999), and multilocus variable number of tandem repeats analysis (MLVA) (Noller et al. 2003a). Studies of prophage and prophage remnants in STEC O157 strains have suggested that such genotypic diversity is largely attributable to bacteriophage-related insertions, deletions, and duplications of variable sizes of DNA fragments (Kudva et al. 2002; Ohnishi et al. 2002; Shaikh and Tarr 2003). In contrast to phageborne diversity, orthologous protein-coding genes in STEC O157 populations exhibit extremely low levels of polymorphism (overall nucleotide sequence identity >99.9%) as shown by in silico comparison of the two complete STEC O157 genomes (Hayashi et al. 2001; Perna et al. 2001) and multilocus sequence typing (MLST) analysis of diverse sets of STEC O157 strains (Noller et al. 2003b; Foley et al. 2004). Based on the limited sequence data available, it is not clear to what extent single nucleotide polymorphisms (SNPs) in protein-coding genes contribute to the overall genomic diversity of STEC O157 population.

Previous phylogenetic studies have inferred a stepwise evolutionary model that STEC O157 strains descended from an O55:H7-like enteropathogenic ancestor by acquisition of bacteriophage-derived Shiga toxins (Stx1 and/or Stx2), a large virulence plasmid (pO157), and the

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