Detection of mcr-1 Gene in Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae From Human Urine Samples in Pakistan

Abstract

Background: Colistin is the last-resort antibiotic available to date against Multiple-drug-resistant (MDR) bacteria, particularly carbapenem-resistant Enterobacteriaceae (CRE) harboring the NDM 1 and KPC 2 genes. Objectives: The current study was designed to investigate extended-spectrum β-lactamase (ESBL) production, colistin resistance, and the presence of mcr-1 in Klebsiella pneumoniae isolated from urine samples. Methods: A total of 298 clinical isolates of K. pneumoniae were collected for seven months in 2017 from the main labs of three government tertiary care hospitals in Pakistan. The ESBL activity of the isolates was assessed by the Double Disc Synergy test (DDST). All the ESBL-producing isolates were phenotypically screened for colistin resistance by dilution methods. Colistin-resistant isolates were subjected to PCR for mcr-1 detection. The confirmation was done by the Sanger sequencing method. Results: Out of 298 K. pneumoniae isolates, 35 (11.7%) isolates showed ESBL activity. They were phenotypically screened for colistin resistance. Four (11.4%) colistin-resistant isolates out of 35 (11.7%) showed the minimum inhibitory concentrations (MICs) ranging from 4 mg/L to 8 mg/L. The mcr-1 gene was detected in all four colistin-resistant isolates via specific primers/PCR and confirmed by Sanger sequencing, showing 99% sequence similarity with the mcr-1 gene in GenBank. The sequence was submitted to NCBI GenBank, and an accession number was assigned. Conclusions: The presence of the mcr-1 gene in ESBL-producing bacteria isolated from human urine samples highlights the urgent need for surveillance studies on a larger scale to overcome the inappropriate use of colistin-containing formulations and prevent further spread of resistance to this antibiotic.