2.6. Characterization of Phylogenetic Groups

Phylogenetic grouping of E. coli can give insight to their pathogenic potential. Currently, eight different phylogroups including seven E. coli sensu stricto and one Escherichia cryptic clade I are now recognized [31]. In particular, E. coli belonging to the phylogroups B2 and D are associated with extra-intestinal infection in humans ([31,42]), while the commensal and intestinal pathogenic strains belong to groups A, B1, and D, as reviewed in [42].The E. coli in phylogroups E are related to group D (of which some are 0157:H7), and group F is related to group B2 [39,41,42]. E. coli belonging to the latter phylogroups E and F could, thus, have potential pathogenic significance. Each confirmed STEC isolate in this study was subjected to the new Clermont quadruplex PCR, following the previously described protocol and the primers (Table 1) to determine the phylogenetic grouping. The quadruplex PCR detects four sequences: arpA (400 bp), chuA (288 bp), yjaA (211 bp), and TspE4.C2 (152 bp). On the basis of the results of the quadruplex PCR, subsequent PCRs were carried out to further assign the STEC into the eight currently known phylogroups. The amplified products were electrophoresed in a 2% ethidium bromide agarose gel and visualized under UV light in a gel imager. The isolates were grouped into A, B1, B2, C, D, E, F, or unknown based on the presence or absence of the genes, as described in the protocol.