Identification of Intermediate in Evolutionary Model of Enterohemorrhagic Escherichia coli O157

Christian Jenke, Shana R. Leopold, Thomas Weniger, Jörg Rothgänger, Dag Harmsen, Helge Karch, and Alexander Mellmann

Author affiliations: Institute for Hygiene and National Consulting Laboratory on Hemolytic Uremic Syndrome, Münster, Germany (C. Jenke, S.R. Leopold, H. Karch, A. Mellmann); University Hospital Münster Periodontology, Münster (T. Weniger, D. Harmsen); Ridom GmbH, Münster (J. Rothgänger)

Abstract

Highly pathogenic enterohemorrhagic Escherichia coli (EHEC) O157 cause a spectrum of clinical signs that include diarrhea, bloody diarrhea, and hemolytic uremic syndrome. The current evolutionary model of EHEC O157:H7/H^– consists of a stepwise evolution scenario proceeding from O55:H7 to a node (hypothetical intermediate) that then branches into sorbitol-fermenting (SF) O157:H^– and non-SF (NSF) O157:H7. To identify this hypothetical intermediate, we performed single nucleotide polymorphism analysis by sequencing of 92 randomly distributed backbone genomic regions of 40 O157:H7/H^– isolates. Overall, 111 single nucleotide polymorphisms were identified in 75/92 partial open reading frames after sequencing 51,041 nt/strain. The EHEC O157:H7 strain LSU-61 from deer occupied an intermediate position between O55:H7 and both O157 branches (SF and NSF O157), complementing the stepwise evolutionary model of EHEC O157:H7/H^–. The animal origin of this intermediate emphasizes the value of nonhuman reservoirs in the clarification of the evolution of human pathogens.

Enterohemorrhagic Escherichia coli (EHEC) belongs to the Shiga toxin–producing E. coli group and causes clinical signs ranging from watery to bloody diarrhea for most symptomatically infected patients (1,2). EHEC serotypes O157:H7 and O157:H^– (nonmotile) are the most frequently isolated from patients with severe EHEC-associated diseases, such as bloody diarrhea and hemolytic uremic syndrome. Infections caused by EHEC O157:H7/H^– are major public health threats and require considerable resources for control and prevention (1,3). Sorbitol-fermenting (SF) EHEC O157:H^–, initially found in Germany and later in other countries such as Scotland, Finland, and Australia, are increasingly associated with severe disease (4). These strains can ferment sorbitol after overnight incubation on sorbitol MacConkey agar, unlike non-SF (NSF) EHEC O157:H7. Today, SF EHEC O157:H^– strains cause ≈20% of all hemolytic uremic syndrome cases in Germany (4–8). Classic NSF EHEC O157:H7 are of animal origin and have caused multiple outbreaks through contaminated food (4), but SF EHEC O157:H^– are almost exclusively isolated from humans, which suggests that humans are the main reservoir (5).
On the basis of multilocus enzyme electrophoresis and multilocus sequence typing (MLST) data (9,10), the evolutionary model of EHEC O157 suggests that EHEC O157 emerged from E. coli O55:H7 by loss and acquisition of virulence and phenotypic traits (10). To further explain the evolution from O55:H7, a hypothetical intermediate and putatively extinct clone (missing link) SF O157:H7 emerging from O55:H7 was introduced; theoretically, it is from this intermediate that the 2 branches (NSF O157:H7 and SF O157:H^–) diverged (9,10).
Shaikh and Tarr subdivided NSF O157:H7 into 3 clusters (11); in their analysis, the SF O157:H^– branch remained evolutionary conserved and clearly separated from NSF O157:H7, with additional data suggesting a hypothetical intermediate. Recent studies based on whole core genome single-nucleotide polymorphisms (SNPs) enabled precise reconstruction of this model (12). The E. coli O157:H^– strain LSU-61, which was isolated from a deer (10,13), had been previously discussed by Feng et al. as a potential intermediate, but that hypothesis was rejected because the strain lacked a gene encoding Shiga toxin (stx) and had a distinct MLST sequence type (10). We used an SNP-based approach to examine isolates from different sources of EHEC O157:H7/H^– to further elucidate the evolutionary model of emergence of this pathogen, paying particular attention to identifying the “missing link” hypothetical intermediate.