Genomic Characterization Of Esbl- And Carbapenemase-Positive Enterobacteriaceae Co-Harboring Mcr-9 In Japan

Worldwide spread of Enterobacteriaceae resistant to colistin, a polypeptide antibacterial drug for last-resort treatment of carbapenemase-producing Enterobacteriaceae (CPE) infections, is concerning. This study aimed to elucidate colistin MICs and molecular characteristics of mcr-1 to mcr-9 of ESBL-producing Escherichia coli (ESBL-Ec) and CPE in Japan and clarify the genomic structure of strains harboring mcr genes (especially mcr-9). This study included 168 ESBL-Ec and 126 CPE strains isolated at Japanese medical facilities. Colistin susceptibility testing and multiplex PCR targeting mcr-1 to mcr-9 were performed for all strains with S1-nuclease pulsed-field gel electrophoresis, Southern blot hybridization, and whole-genome sequencing (WGS) with hybrid assembly performed for mcr gene-carrying strains. Two CPE strains showed a MIC >= 4 mu g/ml in colistin susceptibility testing, with no known resistance mechanism detected. However, PCR conducted on all target strains detected three mcr-9-carrying strains showing colistin susceptibility. The bla(CTX-M-62)-positive E. coli THUN648 strain simultaneously carried bla(CTX-M-62) and mcr-9 on a 275-kbp plasmid. Besides, bla(IMP-6) + bla(CTX-M-2)-positive Klebsiella pneumoniae THUN262 and bla(GES-24)-positive Enterobacter kobei THUN627 had mcr-9 encoded on the chromosome. Only THUN627 encoded qseB/C, which is suggested to be a regulatory gene for mcr-9, downstream of mcr-9. However, this strain showed no increased expression of these genes in mRNA quantitative analysis under colistin exposure. Colistin MICs of ESBL-Ec and CPE in Japan were all below 2 mu g/ml, which is below the epidemiological cutoff (ECOFF) value () or clinical breakpoint (CB) (CLSI M100-S30) reported for colistin, indicating neither "microbiological " nor "clinical " resistance. Several colistin-susceptible Enterobacteriaceae carrying silent mcr-9 encoded on plasmids and chromosomes have already spread worldwide along with other antimicrobial resistance genes. However, the mechanism of colistin resistance by mcr-9 remains unclear.