AUTHOR=Nakamura Akihiro , Nakamura Tatsuya , Niki Makoto , Kuchibiro Tomokazu , Nishi Isao , Komatsu Masaru 

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

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.665432

DOI=10.3389/fmicb.2021.665432

ISSN=1664-302X

ABSTRACT=<p>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 <italic>mcr-1</italic> to <italic>mcr-9</italic> of ESBL-producing <italic>Escherichia coli</italic> (ESBL-Ec) and CPE in Japan and clarify the genomic structure of strains harboring <italic>mcr</italic> genes (especially <italic>mcr-9</italic>). This study included 168 ESBL-Ec and 126 CPE strains isolated at Japanese medical facilities. Colistin susceptibility testing and multiplex PCR targeting <italic>mcr-1</italic> to <italic>mcr-9</italic> 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 <italic>mcr</italic> 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 <italic>mcr-9</italic>-carrying strains showing colistin susceptibility. The <italic>bla</italic><sub><italic>CTX–M–62</italic></sub>-positive <italic>E. coli</italic> THUN648 strain simultaneously carried <italic>bla</italic><sub><italic>CTX–M–62</italic></sub> and <italic>mcr-9</italic> on a 275-kbp plasmid. Besides, <italic>bla</italic><sub><italic>IMP–6</italic></sub> + <italic>bla</italic><sub><italic>CTX–M–2</italic></sub>-positive <italic>Klebsiella pneumoniae</italic> THUN262 and <italic>bla</italic><sub><italic>GES–24</italic></sub>-positive <italic>Enterobacter kobei</italic> THUN627 had <italic>mcr-9</italic> encoded on the chromosome. Only THUN627 encoded <italic>qseB/C</italic>, which is suggested to be a regulatory gene for <italic>mcr-9</italic>, downstream of <italic>mcr-9</italic>. 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 (<ext-link ext-link-type="uri" xlink:href="https://eucast.org/" xmlns:xlink="http://www.w3.org/1999/xlink">https://eucast.org/</ext-link>) or clinical breakpoint (CB) (CLSI M100-S30) reported for colistin, indicating neither “microbiological” nor “clinical” resistance. Several colistin-susceptible Enterobacteriaceae carrying silent <italic>mcr-9</italic> encoded on plasmids and chromosomes have already spread worldwide along with other antimicrobial resistance genes. However, the mechanism of colistin resistance by <italic>mcr-9</italic> remains unclear.</p>