Keywords: Multiple drug resistance; Colistin resistance; mcr-1; Acinetobacter baumannii; Pseudomonas aeruginosa

The New Delhi Metallo-β-lactamase (NDM) is among the most threatening forms of carbapenemases produced by K. pneumoniae, well-known to cause severe worldwide infections. The molecular epidemiology of bla_NDM-1-harboring K. pneumoniae is not well elucidated in Pakistan. Herein, we aim to determine the antibiotics-resistance profile, genes type, molecular type, and plasmid analysis of 125 clinically isolated K. pneumoniae strains from urine samples during July 2018 to January 2019 in Pakistan. A total of 34 (27.2%) K. pneumoniae isolates were carbapenemases producers, and 23 (18.4%) harbored the bla_NDM-1 gene. The other carbapenemases encoding genes, i.e., bla_IMP-1 (7.2%), bla_VIM-1 (3.2%), and bla_OXA-48 (2.4%) were also detected. The Multi Locus Sequence Typing (MLST) results revealed that all bla_NDM-1-harboring isolates were ST11. The other sequence types detected were ST1, ST37, and ST105. The cluster analysis of Xbal Pulsed Field Gel Electrophoresis (PFGE) revealed variation amongst the clusters of the identical sequence type isolates. The bla_NDM-1 gene in all of the isolates was located on a 45-kb IncX3 plasmid, successfully transconjugated. For the first time, bla_NDM-1-bearing IncX3 plasmids were identified from Pakistan, and this might be a new primary vehicle for disseminating bla_NDM-1 in Enterobacteriaceae as it has a high rate of transferability.

Introduction: Enterobacteriaceae such as Escherichia coli and Klebsiella pneumoniae are the most prominent bacterial species resistant to almost all commonly used antibiotics. Carbapenem is one of the last resort drugs for treating such emerging multidrug-resistant bacteria. This study aimed to detect carbapenem-resistant bla_NDM-1 gene in ESBL producing E. coli and K. pneumoniae isolates. Methodology: A total of 190 E. coli and 350 K. pneumoniae isolates were screened for extended spectrumβ-lactamase (ESBL), carbapenemase and metallo β-lactamase (MBL) production via double-disk synergy test (DDST), modified Hodge test and combined-disk diffusion method. The bla_NDM-1 gene was detected by PCR and confirmed via Sanger sequencing method. Results: Of the 540 isolates tested, 71.8% were found to be multidrug-resistant. Overall rate of ESBL-positive isolates were 57.89% E. coli and 31.42% K. pneumoniae. Among ESBL positive isolates, 49.09% E. coli and 40% K. pneumoniae were positive for carbapenemase production whereas MBL production was detected in 29% E. coli and 22% K. pneumoniae isolates. In MBL positive isolates, (37%) E. coli and (40%) K. pneumoniae isolates harboured bla_NDM-1 gene. The pair-wise DNA was aligned with the NDM-1 sequence from GenBank. The alignment score was 243 and the blast nucleotide sequencing results showed 97% sequence similarity with the sequences in GenBank for the bla_NDM-1 gene. Conclusions: The bla_NDM-1 gene was found to be the most prevalent in urine samples. There is a dire need to conduct screening tests in hospitals and communities to find out the exact prevalence of the bla_NDM-1 spread in our population.

Incidence of blaIMP and blaVIM Genes among Carbapenemase Producing Escherichia coli in Lahore, Pakistan