Emergence of colistin-resistant
Enterobacter cloacae
and
Raoultella ornithinolytica
carrying the phosphoethanolamine transferase gene,
mcr-9
, derived from vegetables in Japan
Authors: Xedzro Christian , Shimamoto Toshi , Yu Liansheng , Zuo Hui , Sugawara Yo , Sugai Motoyuki , Shimamoto Tadashi 
Published: November 2023

Colistin, a last-line antimicrobial agent, is recommended for the treatment of severe infections caused by multidrug-resistant (MDR) microorganisms. However, reports on plasmid-mediated mobilized colistin resistance (
mcr
) genes have prompted the importance of routine colistin resistance surveillance. Herein, we report the emergence of
Enterobacter cloacae
CST17-2 and
Raoultella ornithinolytica
CST129-1 carrying an
mcr-9.1
gene in vegetables from Japan. A total of 308 colistin-resistant isolates were retrieved from 200 fresh vegetables in Hiroshima Prefecture, Japan. PCR detection of
mcr-1
to
mcr-9
was conducted. While none of the isolates detected positive for
mcr-1
to
mcr-8
genes, we found two (0.65%) positive strains,
E. cloacae
CST17-2 and
R. ornithinolytica
CST129-1, that harbored
mcr-9.1
allele. These isolates were subjected to phenotypic susceptibility testing, whole-genome sequencing (WGS), PCR-based replicon typing, and conjugation experiment. We found that both isolates had high colistin resistance [minimum inhibitory concentration (MIC) 16 or >64 µg/mL] and showed MDR phenotypes. WGS of both isolates revealed
mcr-9
on a plasmid of the IncHI2/HI2A backbone. The
mcr-9
-bearing plasmid, pCST17-2_1, was self-transferable, although the pCST129-1_1 plasmid was not. Despite being colistin-resistant, the so-called two-component regulatory operon,
qseBC
, which induces polymyxin resistance, was absent from the genetic arrangements downstream of
mcr-9
in
R. ornithinolytica
CST129-1. Nonetheless, a conjugation experiment demonstrated that
mcr-9
in a
Raoultella
-type background is capable of mediating colistin resistance.
In silico
genomic analysis and comparison revealed distinct genetic structures surrounding
mcr-9
, especially in the downstream vicinities. The
E. cloacae
CST17-2 strain is of sequence-type ST738, a sequence type that has emerged in
mcr-9.1
-containing
E. cloacae
. Remarkably, we report the first
mcr-9
-carrying colistin-resistant
Enterobacteriaceae
isolated from Japanese vegetables, which is a grave public health concern. Our findings highlight the importance of strict epidemiological monitoring to track and/or prevent further dissemination of
mcr
homologs across the vegetable industry.

IMPORTANCE

Plasmid-mediated mobile colistin-resistance genes have been recognized as a global threat because they jeopardize the efficacy of colistin in therapeutic practice. Here, we described the genetic features of two
mcr-9.1
-carrying Gram-negative bacteria with a colistin-resistant phenotype derived from vegetables in Japan. The colistin-resistant
mcr-9.1
, which has never been detected in vegetables, was located on a large plasmid in
Enterobacter cloacae
CST17-2 and
Raoultella ornithinolytica
CST129-1, suggesting a high chance of horizontal gene transfer. To the best of our knowledge, this is the first report of
mcr-9
in
R. ornithinolytica
. This study indicates that fresh vegetables might be a potential source for the transmission of
mcr-9
genes encoding resistance to frontline (colistin) and clinically relevant antimicrobials. The study also provides additional consideration for colistin use and the relevance of routine surveillance in epidemiological perspective to curb the continuous spread of
mcr
alleles.