Of the 48 EHEC O157 strains studied, 10 were SF serotype O157:H^–. Subgrouping and cluster designation of the NSF O157 strains resulted in 18 cluster 1 strains, 1 cluster 2 strain, and 17 cluster 3 strains. For 2 strains, LSU-61 and SNPO157_36, no characteristic SNP pattern was determined (Table). Further characterization by MLST of prototype strains that defined subgroups and clusters resulted in identical STs for all SF and NSF O157 (ST11) and in closely related STs of the O55:H7 strains (ST335).

The 50 strains of serotypes O157:H7/H^– and O55:H7 were further characterized with respect to their SNP prevalence in the core genome. In total, 92 core genomic loci were analyzed, comprising 51,041 bp sequencing information (≈0.9% of the O157:H7 Sakai genome) (Table; Technical Appendix Table 2). Sequencing demonstrated 111 biallelic variants, an average of 1.2 variants per sequenced locus (Technical Appendix Table 3). Deletions or insertions were not detected.

Of the 111 SNPs, 53 (47.7%) were synonymous SNPs (sSNPs) and 58 (52.3%) were nonsynonymous SNPs (nsSNPs); 78 (70.3%) SNPs were transitions, and 33 (29.7%) were transversions. Concatenation of all loci resulted in an average of 1 SNP every 460 bp; sSNPs occurred every 963 bp, and nsSNPs every 880 bp. On the level of analyzed partial ORFs, 1 SNP was found in 45 partial ORFs, 2 SNPs in 25 partial ORFs, 3 SNPs in 4 partial ORFs, and 4 SNPs in 1 partial ORF.

To further elucidate the SNP distribution, we categorized the 92 loci into 3 functional groups. Most loci belonged to (putative) metabolism or housekeeping genes because these were chosen solely from backbone regions. If no KEGG assignment was possible, we estimated the function of the corresponding fragment on the basis of BLAST homologies. Defined annotation information regarding the function in metabolism or housekeeping was determined for 25 partial ORFs. A housekeeping/metabolism function was predicted for 58 loci. The remaining 9 loci were hypothetical proteins only (Technical Appendix).

On the basis of the 111 SNPs, the 50 strains were clustered into 27 SNP genotypes (Figure). The 111 SNPs were able to reconstruct the well-known evolutionary model (9–12) with the stepwise evolution from O55:H7 (subgroup A) to either SF O157:H^– (subgroup B) or NSF O157:H7/H^– (subgroup C, clusters 1–3). Strain LSU-61 interlinked all 3 subgroups, thereby substituting for the unknown intermediate (Figure). Moreover, the applied SNP scheme differentiated the O55:H7 strains and exhibited 5 genotypes within the 10 SF O157:H^– strains segregated from the SF O157:H7 strain LSU-61. The NSF O157:H7 subgroup C₁ strains comprising 18 isolates were differentiated in 9 SNP genotypes, and the subgroup C₃ strains (n = 17) exhibited 8 SNP genotypes.

To further validate the role of stx-negative LSU-61 as a potential intermediate, we investigated each known potential stx insertion site in silico to determine the presence or absence of a Shiga toxin–carrying bacteriophage. We conducted BLAST searches within the recently published draft genome sequence of LSU-61 (28) by using published primers for the different insertion sites (29,30). All insertion sites for stx1 (yehV) and stx2 (wrbA, yecE, sbcB) were intact.

To investigate the effect of selective pressure on some loci and potential selecting biases, we analyzed sSNP and nsSNP types separately. In each scenario, the phylogenetic reconstruction resulted in comparable branching, with distinct lineages for SF and NSF O157 and strain LSU-61 as an intermediate. Only the number of SNP genotypes differed slightly: 19 sSNP genotypes (13 NSF O157:H7, 3 SF O157:H^–, 2 O55:H7, and LSU-61) based on the 53 sSNPs and 22 nsSNP genotypes (16 NSF O157:H7, 3 SF O157:H^–, 2 O55:H7, and LSU-61) based on the 58 nsSNPs. This excludes strong selection bias of the different loci.

On the basis of SNP analysis of 92 chromosomal backbone regions of EHEC O157, we identified an SF O157:H7 strain that complements the current model of the stepwise evolution from O55:H7 to EHEC O157 in which the hypothetical intermediate between O55:H7 and SF and NSF O157:H7/H^– has been unknown (10,12). As with the highly human pathogenic O157:H7 lineage of EHEC, which is known to reside in cattle, deer, and other ruminants, this intermediate strain was isolated from a deer (13). These findings support previous observations (31,32) and suggest an evolution toward an animal reservoir for O157:H7 soon after O157:H^– and O157:H7 divergence. Strain LSU-61 is motile (H-phase 7) and enterohemolysin active (10), traits that are typical for NSF O157, further suggesting the intermediate character of LSU-61 between SF and NSF O157. In contrast to the MLST scheme applied from Feng et al. and Lacher et al. (10,33), our MLST analysis, using the scheme of Wirth et al. (26) that analyzes different genes, further corroborates the intermediate character of LSU-61 because it shares the same ST with SF (subgroup B prototype strain) and NSF O157 (subgroup C prototype strains).

Strain LSU-61 does not carry a stx gene, but this fact does not contradict our findings because these genes are encoded on bacteriophages that can be acquired and lost (30,34,35), and we do not have evidence of a progenitor to LSU-61 that contains stx genes. Although known potential stx phage integration sites in O157 were intact, the possibility of a previous stx bacteriophage carriage cannot be excluded. If the SF O157:H7 cluster emerged ≈3,000–4,000 years ago (12), certain genetic and phenotypic changes (10) occurred well before the first descendants of this cluster were isolated and characterized.

Two previous studies (31,32) reported isolated comparable strains to LSU-61 from (European) red deer, belonging to the same family (Cervidae) as white-tailed deer (North America), with comparable phenotypic and genotypic traits. Some of these were SF O157:H7 strains (stx negative or positive, β-glucuronidase positive activity) (31,32). The proof of the existence of SF O157:H7 in a ruminant (deer) host may indicate transfer into animals soon after the 2 (human pathogenic) O157 subgroups B and C emerged. On the basis of shared characteristics with both O157 branches, we suggest strain LSU-61 as a representative of the intermediate cluster complementing the stepwise evolutionary model of EHEC O157. The phylogeny based on either sSNPs or nsSNPs also resulted in a comparable phylogenetic tree with LSU-61 as a member of the progenitor node, underlining its intermediate role.

On the level of gene categories, a higher percentage of sSNPs, though fewer SNPs overall, were observed in the metabolism/housekeeping category compared with the putative metabolism/housekeeping category. The higher rate of nsSNPs in the latter category, resulting in a higher phenotypic diversity, might be explained by uncertain gene categorization because of currently limited knowledge of gene function. Therefore, SNP typing results may help to find genes involved in host–pathogen interactions rather than in metabolism or housekeeping only. SNP data for hypothetical proteins are difficult to interpret because information about their function is too imprecise to enable estimation of the effect of evolutionary pressure.

The fact that 35 of the 38 O157:H7 strains were subgrouped into either cluster 1 or 3 (17 and 18 strains, respectively) shows a certain persistence of these O157:H7 clusters (29), characterized by a successful pathogenicity, for example, outbreaks over a broad time frame (4). The preponderance of cluster 1 strains has been noted before, as have the paucity of cluster 2 and the diminished proportion of cluster 3 strains in North America (29). We observed a higher number of SNPs within the different NSF O157:H7 clusters compared with the few SNPs within restricted SF O157:H^– genotypes and a maximum pairwise distance of 2 SNPs (Figure). A reason for this phenomenon may be the different animal host origins for the NSF O157:H7 clade, whereas SF O157:H^– are considered to have only 1 main host, humans (5,19). This high conservation was similarly recognized when multilocus variable-number tandem-repeat analysis was applied (19). In this context, certain SNP genotypes may serve to illuminate several strain-specific characteristics, such as increased virulence and other phenotypic traits, as other studies have similarly observed for both SF and NSF O157 (36,37).

Our results could be interpreted as if C₂ strain 86–24 is an offshoot of cluster 3, which is in contrast to the established stepwise model of O157. However, we believe that this is an artifact caused by sampling bias of the investigated 92 loci because only 11 backbone SNPs have been found to differentiate cluster 2 and 3 within the whole chromosomal backbone (12). One strain (SNPO157_36) did not cluster into any known O157:H7 cluster (Figure).

In summary, our identification of an intermediate member of the EHEC 1 clade complements the current evolutionary model of EHEC O157 by using chromosomal backbone SNP data of a spatiotemporally diverse strain collection. The different levels of genotypic conservation within the subgroups and the animal origin of the intermediate underline the great effect of host–pathogen interaction on the evolution of bacterial species. Future studies should focus on this interaction within both human and animal hosts to understand the evolution and persistence in nature of such human pathogens. The survival of the ancestral pathogen until today suggests that its genetic attributes could be informative in identifying fitness and potentially pathogenic loci.