Molecular Detection of AmpC and ESBL Genes

All isolates testing positive for AmpC β-lactamase production on the CC-DDST were tested by polymerase chain reaction (PCR) for pAmpC gene carriage. Further, as pAmpC β-lactamase genes have also been detected in isolates with reduced susceptibility to third-generation cephalosporins, we tested isolates with inhibition zone diameters of ≤27 mm, ≤25 mm and ≤22 mm for cefotaxime, ceftriaxone and ceftazidime, respectively, for pAmpC gene carriage
^{17 ,
23 (link)}. In-house multiplex PCRs targeting AmpC β-lactamase genes
bla_CIT, bla_DHA, bla_MOX, bla_FOX, bla_EBC,
bla_ACCand
bla_CMY-2 were performed using published primers, Thermo-Fisher Taq DNA polymerase and PCR master-mixes, and conditions
^{6 (link),
26 (link)}. Amplification was performed in a 3Prime Mid-size thermocycler (Techne, UK) and the expected amplicon sizes were successfully generated. PCR amplification of ESBL genes
bla_CTX-M,
bla_TEM and
bla_SHV was performed with Taq DNA polymerase (Thermo-Fisher Inc.) using published primers
^{27 (link)}. Amplicons were sequenced (ACGT, Wheeling, IL, USA) by the chain termination method (Sanger sequencing) and sequences confirmed through
BLAST-searching at NCBI. Phylogenetic group typing of
E. coli was done according to the method of Clermont et al, in which PCR of a combination of two genes (
chuA &
yjaA) and an anonymous DNA fragment are used to classify strains
^{28 (link)}.