Rapid detection and differentiation of mobile colistin resistance (mcr-1 to mcr-10) genes by real-time PCR and melt-curve analysis

Background The emergence of multi-drug-resistant (MDR) micro-organisms prompted new interest in older antibiotics, such as colistin, that had been abandoned previously due to limited efficacy or high toxicity. Over the years, several chromosomal-encoded colistin resistance mechanisms have described; more recently, 10 plasmid-mediated mobile (mcr) genes identified. Spread these among MDR Gram-negative bacteria is a matter serious concern; therefore, reliable and timely mcr detection paramount. Aim To design validate multiplex real-time polymerase chain reaction (PCR) assay for differentiation genes. Methods All available alleles were downloaded from National Center Biotechnology Information Reference Gene Catalogue, aligned with Clustal Omega primers designed using Primer-BLAST. Real-time PCR monoplexes optimized validated panel 120 characterized strains carrying wide range genes, often combination. Melt-curve analysis was used confirm positive results. Findings In-silico enabled ‘screening’ mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 mcr-9/10, paired an internal control discount inhibition. A ‘supplementary’ subsequently differentiate mcr-1, mcr-2, mcr-6, mcr-9 mcr-10. Expected results obtained all (100% sensitivity specificity). showed consistent melting temperature Inhibition not observed. Conclusions rapid easy perform, enabling unequivocal even when than one variant present. Adoption by clinical veterinary microbiology laboratories would aid surveillance amongst bacteria.