Rapid detection of human origin colistin-resistance genes mcr-1, mcr-3, mcr-8, mcr-10 in clinical fecal samples.

Plasmid-mediated colistin-resistance genes have been reported in human origin clinical samples worldwide which raises its threats to human infections. Notably, mcr-1, mcr-3, mcr-8, and mcr-10 have been reported isolated directly from clinical samples which creates more seriously threaten to human health than other mcr gene types. A multiplex polymerase chain reaction (Multi-PCR) protocol was developed to detect and genotype mobile colistin-resistance genes (mcr-1, mcr-3, mcr-8, mcr-10) in Enterobacteria for clinical laboratory purposes. We first designed four pairs of new primers for the amplification of mcr-1, mcr-3, mcr-8, and mcr-10 gene respectively to achieve stepwise separation of amplicons between 216 and 241 bp, and complete this Multi-PCR system with the assistance of another pair of universal primer. Among which the forward primers for mcr-8 and mcr-10 amplicons were identical. The protocol was validated by testing 11 clinical isolates of Escherichia coli and 3 clinical isolates of Klebsiella from human origin, each well characterized and prospectively validated. The Multi-PCR assay showed full concordance with whole-genome sequence data and displayed higher sensitivity and 100% specificity. The assay could detect all variants of the various mcr alleles described. The Multi-PCR assay successfully genotyped of mcr alleles described in one test.