Development and validation of a multiplex polymerase chain reaction assay for detection of the five families of plasmid-encoded colistin resistance

Plasmid-mediated colistin resistance is increasingly described worldwide in Enterobacteriaceae from animal and human isolates. Diffusion of these resistance traits among carbapenem-resistant enterobacterial isolates is of particular concern as colistin has become the last resort antibiotic for treating human infections with these organisms. Therefore, being able to monitor the presence of these transferable colistin resistance genes (mcr-1 to mcr-5-variants) is crucial. This paper describes the development of a multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes in Enterobacteriaceae. Five primer pairs were designed to amplify mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 gene products in a multiplex PCR. This assay was validated retrospectively on colonies of 50 Escherichia coli, 44 Klebsiella pneumoniae and 12 Salmonella enterica isolates of animal and human origin, all well characterized, and validated prospectively on 450 carbapenem-resistant enterobacterial isolates received by the French National Reference Centre. In addition, 82 Aeromonas spp. and 10 Shewanella spp. known to be the progenitors of mcr-3 and mcr-4 alleles, respectively, were screened. Mcr-multiplex PCR assay displayed 100% specificity, sensitivity, negative predictive value and positive predictive value. The assay was able to detect all variants of the different mcr alleles, and was able to detect chromosomally encoded mcr-4-like variants present in two Shewanella bicestrii JAB-1 and Shewanella woodyi S539. In conclusion, a rapid and robust multiplex PCR assay able to detect all known mcr gene families described in Enterobacteriaceae was developed and validated. This type of test is critical for the epidemiological surveillance of plasmid-encoded resistance, especially in carbapenem-resistant bacteria.