Genomic Characterization of ESBL- and Carbapenemase-Positive Enterobacteriaceae Co-harboring mcr-9 in Japan

Abstract

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 <i>mcr-1</i> to <i>mcr-9</i> of ESBL-producing <i>Escherichia coli</i> (ESBL-Ec) and CPE in Japan and clarify the genomic structure of strains harboring <i>mcr</i> genes (especially <i>mcr-9</i>). This study included 168 ESBL-Ec and 126 CPE strains isolated at Japanese medical facilities. Colistin susceptibility testing and multiplex PCR targeting <i>mcr-1</i> to <i>mcr-9</i> 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 <i>mcr</i> gene-carrying strains. Two CPE strains showed a MIC ≥ 4 μg/ml in colistin susceptibility testing, with no known resistance mechanism detected. However, PCR conducted on all target strains detected three <i>mcr-9</i>-carrying strains showing colistin susceptibility. The <i>bla</i> _{<i>CTX-M-62</i>} -positive <i>E. coli</i> THUN648 strain simultaneously carried <i>bla</i> _{<i>CTX-M-62</i>} and <i>mcr-9</i> on a 275-kbp plasmid. Besides, <i>bla</i> _<i>IMP-6</i> + <i>bla</i> _{<i>CTX-M-2</i>} -positive <i>Klebsiella pneumoniae</i> THUN262 and <i>bla</i> _{<i>GES-24</i>} -positive <i>Enterobacter kobei</i> THUN627 had <i>mcr-9</i> encoded on the chromosome. Only THUN627 encoded <i>qseB/C</i>, which is suggested to be a regulatory gene for <i>mcr-9</i>, downstream of <i>mcr-9</i>. 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 μg/ml, which is below the epidemiological cutoff (ECOFF) value (https://eucast.org/) or clinical breakpoint (CB) (CLSI M100-S30) reported for colistin, indicating neither "microbiological" nor "clinical" resistance. Several colistin-susceptible Enterobacteriaceae carrying silent <i>mcr-9</i> encoded on plasmids and chromosomes have already spread worldwide along with other antimicrobial resistance genes. However, the mechanism of colistin resistance by <i>mcr-9</i> remains unclear.