Molecular Epidemiology of mcr-1, bla(KPC-2,) and bla(NDM-1) Harboring Clinically Isolated Escherichia coli from Pakistan

PURPOSE: The multiple-drug resistant Escherichia coli are among the deadliest pathogens causing life-threatening infections. This study was planned to determine the molecular epidemiology of mcr-1, bla(KPC-2), and bla(NDM-1) harboring clinically isolated E. coli from Pakistan. METHODS: In total, 545 strains of E. coli from clinical samples were collected from June 2018 to September 2019. All the isolates were screened for colistin-resistance, extended-spectrum-β-lactamases (ESBL), and carbapenemases through the micro-dilution method, Double-Disk-Synergy-Test (DDST), and Modified-Hodge-Test (MHT). The detection, sequence-typing, conjugal transfer, S1-PFGE, plasmid-replicon-typing, and southern-blotting for mcr, ESBL, and carbapenemase-encoding genes were performed. FINDINGS: A total of four (0.73%) colistin-resistant strains carrying alongside mcr-1 and bla(CTX-M-15) genes, three of these strains also had the bla(TEM-1) gene. The presence of ESBL genes was detected in 139 (25.5%) isolates harboring bla(CTXM-15) (74.82%), bla(TEM) (34.53%), bla(SHV) (28.06%) and bla(OXA-1) (28.78%). In 129 carbapenemase-producers, 35.83% possessed bla(NDM-1), 26.67% bla(KPC-2), 8.3% bla(OXA-48), 25% bla(VIM-1), and 20.83% bla(IMP-1) genes. The sequence typing revealed that mcr-1 harboring isolates belonged to ST405, ST117, and ST156. Fifty percent of bla(KPC-2) and 48.83% of bla(NDM-1) were found on ST131 and ST1196, respectively. Two rare types of STs, ST7584, and ST8671 were also identified in this study. The mcr-1 gene was located on Incl2 (60-kb) plasmid. The bla(KPC-2) was present on (140-kb) IncH12, (100-kb) IncN, (90-kb) Incl1, while bla(NDM-1) was located on (70-kb) IncFIIK, (140-kb) IncH12, (100-kb) IncN, (60-kb) IncA/C, and (45-kb) IncFII plasmids, which were successfully trans-conjugated. Among the plasmid types, the Incl1 carrying bla(KPC-2), IncH12 harboring bla(KPC-2) and bla(NDM-1), and IncFIIK carrying bla(NDM-1) were for the first time detected in Pakistan. CONCLUSION: The mcr-1, bla(KPC-2), and bla(NDM-1) genes finding in various clonal and plasmids types indicate that a substantial selection of the resistance genes had occurred in our clinical strains.