The Journal of hospital infection | 2021
Rapid detection and differentiation of mobile colistin resistance, mcr-1 to mcr-10, genes by real time PCR and melt curve analysis.
Abstract
BACKGROUND\nEmergence of multidrug resistant (MDR) microorganisms prompted new interest in older antibiotics like colistin that were previously abandoned due to limited efficacy or high toxicity. Over the years, several chromosomal-encoded colistin resistance mechanisms were described; more recently, ten plasmid-mediated mobile colistin resistance (mcr) genes have also been identified. Spread of these genes among MDR Gram-negative bacteria is a matter of serious concern, therefore reliable and timely mcr detection is paramount.\n\n\nAIM\nTo design and validate a multiplex real-time PCR for detection and differentiation of mcr genes.\n\n\nMETHODS\nAll available mcr alleles were downloaded from the NCBI Reference Gene Catalog, aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized and validated using a panel of 120 characterised Gram-negative strains carrying a wide range of resistance genes, often in combination. Melt-curve analysis was used to confirm positive results.\n\n\nFINDINGS\nIn silico analysis allowed to design a screening assay for detection of mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 and mcr-9/10 paired with an internal control assay to discount inhibition. A supplementary assay was then designed to differentiate mcr-1, mcr-2, mcr-6, mcr-9 and mcr-10. Expected results were obtained for all strains (100% sensitivity and specificity). Melt-curve analysis showed consistent Tm results. Inhibition was not observed.\n\n\nCONCLUSIONS\nThe assay is rapid and easy to perform, enabling unequivocal mcr detection and differentiation even when more than one variant is simultaneously present. Adoption by clinical and veterinary microbiology laboratories would aid the surveillance of mcr genes amongst Gram-negative bacteria.