Phylogroup Determination of E. coli Isolates

The phylogroup of each isolate was determined based on E. coli phylogrouping method described by Clermont et al. (46 (link)). Briefly, this method assigns strains to phylogroups A, B1, B2, C, D, E, F that belong to E. coli sensu stricto, whereas the eighth is the Escherichia cryptic clade I. This technique has been designed based on extended quadruplex PCR and multilocus sequence typing (MLST) scheme (47 (link), 48 (link)). All PCR reactions were carried out in a 20 μl final volume containing 2 μl of 10X buffer, 2 μM of dNTPs, 2 U of Taq polymerase (Ampliqon, Denmark), 2 μl (100 ng) of DNA template and the appropriate primers. The amounts of primer used are 20 pmol, except for AceK.f (40 pmol), ArpA1.r (40 pmol), trpBA.f (12 pmol) and trpBA.r (12 pmol). PCR reactions were performed under the following conditions: denaturation 4 min at 94°C, 30 cycles of 5 s at 94°C and 20 s at 57°C (group E) or 59°C (quadruplex and group C), and a final extension step of 5 min at 72°C. The primers used for the allele-specific phylogroups E and C PCRs were ArpAgpE.f and ArpAgpE.r and trpAgpC.f and trpAgpC.r, respectively. In E- and C-specific PCR reactions, the primers trpBA.f and trpBA.r are added to provide an internal control.