Whole genome-based genetic insights of producing clinical isolates in hospital settings of Pakistan.

Carbapenem resistance among Enterobacterales has become a global health concern. Clinical isolates producing the metallo β-lactamase NDM have been isolated from two hospitals in Faisalabad, Pakistan. These strains were characterized by MALDI-TOF, PCR, antimicrobial susceptibility testing, XbaI and S1 nuclease pulsed-field gel electrophoresis (PFGE), conjugation assay, DNA hybridization, whole genome sequencing, bioinformatic analysis, and experiments. Thirty-four producing strains were identified among 52 nonduplicate carbapenem-resistant strains. More than 90% of the isolates were found to be multidrug resistant by antimicrobial susceptibility testing. S1 PFGE confirmed the presence of gene on plasmids ranging from 40 kbps to 250 kbps, and conjugation assays demonstrated transfer frequencies of harboring plasmids ranging from 1.59 × 10 to 6.46 × 10 per donor. Whole genome sequencing analysis revealed as the prominent NDM subtype with the highest prevalence of , , , , and ) associated resistant determinants. sequence types: ST405, ST361, and ST167 were prominent, and plasmid Inc types: FII, FIA, FIB, FIC, X3, R, and Y, were observed among all isolates. The genetic environment of region on IncF plasmids included partial IS, the bleomycin gene, and a class I integron. The virulence genes and were frequently observed, and experiments showed that virulence correlated with the number of virulence determinants. A strong infection control management in the hospital is necessary to check the emergence of carbapenem resistance in Gram-negative bacteria.IMPORTANCEWe describe a detailed analysis of highly resistant clinical isolates from two tertiary care centers in Pakistan including carbapenem resistance as well as common co-resistance mechanisms. South Asia has a huge problem with highly resistant . However, we find that though these isolates are very difficult to treat they are of low virulence. Thus the Western world has an increasing problem with virulent that are mostly of low antibiotic resistance, whereas, South Asia has an increasing problem with highly resistant that are of low virulence potential. These observations allow us to start to devise methodologies to limit both virulence and resistance and combat problems in developing nations as well as the Western world.