Comparison of PFGE, MLVA and SNP typing.

<p>Comparison of PFGE, MLVA and SNP typing.</p

Phylogeny of isolate set using 32 discriminatory SNPs.

<p>This is a phylogeny of all isolates used in the study with the exception of a few positive controls shared by other authors using the panel of 32 discriminatory SNPs. The 10 clusters with insufficient epidemiological resolution have been labeled. These clusters are completely different from the three mentioned by Leopold, <i>et al</i>. AM series isolates were considered epidemiologically sporadic and should have therefore had additional reticulation in this figure. Some grouping of S-series isolates was expected because some outbreaks were represented by multiple isolates.</p

Canonical Single Nucleotide Polymorphisms (SNPs) for High-Resolution Subtyping of Shiga-Toxin Producing <i>Escherichia coli</i> (STEC) O157:H7

<div><p>The objective of this study was to develop a canonical, parsimoniously-informative SNP panel for subtyping Shiga-toxin producing <i>Escherichia coli</i> (STEC) O157:H7 that would be consistent with epidemiological, PFGE, and MLVA clustering of human specimens. Our group had previously identified 906 putative discriminatory SNPs, which were pared down to 391 SNPs based on their prevalence in a test set. The 391 SNPs were screened using a high-throughput form of TaqMan PCR against a set of clinical isolates that represent the most diverse collection of O157:H7 isolates from outbreaks and sporadic cases examined to date. Another 30 SNPs identified by others were also screened using the same method. Two additional targets were tested using standard TaqMan PCR endpoint analysis. These 423 SNPs were reduced to a 32 SNP panel with the almost the same discriminatory value. While the panel partitioned our diverse set of isolates in a manner that was consistent with epidemiological data and PFGE and MLVA phylogenies, it resulted in fewer subtypes than either existing method and insufficient epidemiological resolution in 10 of 47 clusters. Therefore, another round of SNP discovery was undertaken using comparative genomic resequencing of pooled DNA from the 10 clusters with insufficient resolution. This process identified 4,040 potential SNPs and suggested one of the ten clusters was incorrectly grouped. After its removal, there were 2,878 SNPs, of which only 63 were previously identified and 438 occurred across multiple clusters. Among highly clonal bacteria like STEC O157:H7, linkage disequilibrium greatly limits the number of parsimoniously informative SNPs. Therefore, it is perhaps unsurprising that our panel accounted for the potential discriminatory value of numerous other SNPs reported in the literature. We concluded published O157:H7 SNPs are insufficient for effective epidemiological subtyping. However, the 438 multi-cluster SNPs we identified may provide the additional information required.</p></div

Workflow for final discriminatory SNP panel.

<p>Workflow for final discriminatory SNP panel.</p

Panel of 32 informative SNPs.

<p>*These SNPs are reported to break up cluster 1, according to SNP discovery. Major and minor frequencies are based on 177 isolates.</p><p>** One of Manning, <i>et al</i>. 32 SNPs</p><p>*** One of Leopold, <i>et al</i>. cluster one SNPs.</p><p>Panel of 32 informative SNPs.</p