Development and validation of a multiplex PCR assay for the detection of the five families of plasmid-encoded colistin resistance.

Plasmid-mediated colistin resistance is increasingly described worldwide in Enterobacteriaceae from animal and from human isolates. Diffusion of these resistance traits among carbapenem-resistant enterobacterial isolates is particularly worrisome, since 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. Here, we have developed a multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes in Enterobacteriaceae. We designed five primer pairs to amplify mcr-1, -2, -3, -4, -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 well characterized isolates of animal and human origin, and prospectively on 450 carbapenem-resistant enterobacterial isolates received by the French National Reference Centre. In addition, we also screened 82 Aeromonas spp. and 10 Shewanella spp. species known to be the progenitors of mcr-3 and mcr-4 alleles, respectively. Mcr-multiplex PCR assay displayed 100% specificity, sensitivity, negative 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 S. woodyi S539. We developed and validated a rapid and robust multiplex PCR assay able to detect all known mcr gene families described in Enterobacteriaceae. This kind of test is critical for the epidemiological surveillance of plasmid-encoded resistance, especially in carbapenem-resistant bacteria.