*2.4. Amplification of Genes*

The total bacterial DNA from *E. coli* isolates was extracted using the alkaline lysis method [10]. The genetic confirmation of *E. coli* was done through PCR with genus-specific oligonucleotide primers [11]. β-lactamase-encoding (*bla*C*TXM*, *blaTEM*, and *blaSHV*); plasmid-mediated quinolone resistance (*qnrA*, *qnrS*, *qnrS*, *aac(6 )-Ib-cr*, and *qepA*), carbapenem resistant (*VIM*, *NDM*, *IMP*) and colistin resistant (*mcr-1*) genes were amplified and identified with previously-described primers [12,13] for all *E. coli* isolates. The phylogenetic grouping of all *E. coli* was performed based on *chuA*, *vjaA*, and *TspE4C2* genes which were amplified by multiplex PCR as described in detail elsewhere [14]. All of the amplified PCR products were visualized using a gel documentation system for all *E. coli* isolates.
