Background
The emergence of carbapenemase-producing
Enterobacteriaceae (CPE) has become a serious problem in medical settings worldwide [
1]. The most frequently detected and globally widespread carbapenemase produced by CPE between the Asian countries are the class B metallo-β-lactamases (MBLs), which include IMP-type, NDM-type, and VIM-type MBLs [
2].
Because of the emergence of multidrug-resistant Gram-negative pathogens and the lack of new antibiotics with efficient activities, colistin, a polymyxin-type antibiotic, has been the last resort used to treat CPE infections [
3,
4]. Bacteria acquire colistin resistance through chromosomal mutation(s) or plasmid transfer [
5]. Chromosome-mediated colistin resistance results from mutation(s) or deletion(s) of two component systems, such as
phoPQ and
pmrAB, altering the structure of lipopolysaccharides [
6,
7]. For example, colistin resistance has been associated with modification of the lipid A moiety in lipopolysaccharide, such as by the addition of 4-amino-4-deoxy-L-arabinose (L-Ara-4 N) and phosphoethanolamine (PEtN) to the anionic phosphate groups of lipid A. These additions reduce the anionic charges on lipid A and its affinity to the cationic colistin, inhibiting membrane destruction resulting from the binding of colistin to lipid A, followed by cell death [
8].
To date, various types of plasmid-mediated mobilized colistin-resistance genes,
mcr, have been identified, including
mcr-1 to
mcr-9, with several, including
mcr-1, − 2, − 3, − 4, and − 6, shown to have PEtN transferase activity [
6]. The
mcr-1 gene was initially detected in isolates of
Escherichia coli and
Klebsiella pneumoniae obtained from humans and animals in 2015 in China [
9], and
mcr-9 was initially identified in a clinical isolate of the colistin-susceptible bacterium,
Salmonella enterica serotype
typhimurium. The amino acid and nucleotide sequences of
mcr-9 are closest to those of
mcr-3 with similarities of 64.5 and 99.5%, respectively [
10]. In this study,
mcr-9 was detected in 335 genomes in multiple genera of
Enterobacteriaceae. The analysis of
mcr-9 promoter region in these genomes showed conserved regions which is likely a recognition sequence for transcription regulator, suggesting that other factors might be involved in full-expression of
mcr-9. Of the 335 genomes, 65 had at least one plasmid replicon indicating that
mcr-9 can be found extrachromosomally in different species of
Enterobacteriaceae [
10].
Isolates of
E. cloacae complex resistant to both carbapenem and colistin have been reported in several countries, including China [
11,
12], France [
13], India [
14], the USA [
15,
16] and Vietnam [
17]. One of these, an isolate of
E. cloacae complex (
Enterobacter hormaechies) co-harboring
blaVIM-4 and
mcr-9, was first reported in the United States in 2019 [
16]. In addition, a colistin-resistant
E. hormaechei isolate producing both MCR-9 and NDM-1 was isolated from a patient in China with bloodstream infection in 2019 [
11]. This emergence of colistin resistance, particularly in CPE, may result in significant clinical and public health concerns [
18,
19].
The study describes three clinical isolates of E. cloacae complex that were resistant to carbapenem but susceptible to colistin. To our knowledge, this is the first report of isolates of E. cloacae complex harboring both blaIMP-1 and mcr-9 in Japan.
Discussion
The
mcr-9 gene may be silently spreading in
Enterobacteriaceae throughout the world. The prevalence of
mcr-9 is unclear because this gene is not actually related to colistin resistance, as it may be silent or inducible in clinical isolates of
Enterobacteriaceae. For example, an isolate of
E. hormechei harboring
mcr-9 did not express its gene product [
16]. This isolate was susceptible to colistin, likely because the two-component system genes
qseCB were lacking from the region downstream of
mcr-9. In contrast, another isolate of
E. hormechei that harbored and expressed
mcr-9 was found to be resistant to colistin and to have the two-component system genes in the region downstream of
mcr-9 [
11]. The expression of
mcr-9 is mediated by the two-component system QseCB and can be induced by subinhibitory concentrations of colistin [
24]. At least 11
mcr-9-positive IncHI2 plasmids have been detected by Blast, with six having and five lacking the two-component system genes [
11].
The two-component QseCB system, consisting of a sensor (qseC) and a response regulator (qseB), plays an essential role in the expression of
mcr-9 [
24]. Our finding, that the isolate A2563 harbored
mcr-9 along with the two-component system genes
qseCB but was susceptible to colistin suggests that other, as yet undetermined, genes or molecules may regulate
mcr-9 expression. The pA2480mcr-9 and pA2504mcr-9 had similar structures to those of pME-1a and pCTXM9_020038, as they lacked
qseCB. This two-component system was transcribed as an operon, with the QseB promoter binding to low- and high-affinity binding sites located − 500 to − 10 bp at upstream of
qseB [
26]. The nucleotide sequence of this region in A2563 was 100% identical to that of the QseB promoter (− 500 to + 1 bp) in pMCR-SCNJ07, which confers resistance to colistin [
11], suggesting that the QseB promoter in A2563 may be repressed by an as yet undetermined mechanism [
26]. Four plasmids, pME-1a, pCTXM9_020038, pRH-R27 and pMCR-SCNJ07, had the conserved gene structure,
rcnR-
pcoS-
pcoE-IS-5, upstream of
mcr-9. Whereas, the chromosome of A2563 had the same conserved gene structure, but with a 53-bp deletion in
pcoE (Δ
pcoE), suggesting that the deleted region may be associated with
mcr-9 expression. Further studies are necessary to determine the mechanism for regulation of
mcr-9 expression in
Enterobacteriaceae.
To our knowledge, it is the first report describing a bacterial isolate harboring
mcr-9 on its chromosome, indicating that
mcr-9 may have been inserted into the chromosome by mobile elements. Several
Enterobacteriaceae isolates from animals and humans have reported the chromosomal location of
mcr-1 and
mcr-2 [
27‐
33]. The
mcr-1 was detected on the chromosomes of two colistin-resistant
E. coli strains isolated from swine in 2012 in China [
28], and on the chromosome of an
E. coli ST410 strain harboring
blaCTX-M-15 isolated from a sample of turkey meat in 2013 in Germany [
27]. The chromosomal integration of
mcr-1 was also detected in a clinical strain of
E. coli ST156 harboring
blaNDM-5 isolated from a bile sample in 2015 in China [
29], in
E. coli isolated from food production animals in 2011–2016 in Poland [
32], and in
E. coli isolated from veal calves in 2016 in the Netherlands [
30]. Chromosomes carrying
mcr-1 were detected in
Enterobacteriaceae from environmental water sources in 2017 in China [
33]. Moreover, the
mcr-2 gene (
mcr-6.1) was detected on the chromosome of a strain of
Moraxella isolated from a pig in 2014–2015 in Great Britain [
31]. These studies support the mobility characterization of
mcr genes across different genetic elements and insertion of the plasmid-variant of
mcr into chromosome could lead to higher prevalence of colistin resistance among
Enterobacteriaceae specious.
The direct origin of the
mcr-9 on the chromosome of A2563 is unclear. However, the genetic environments of the
mcr-9 and
qseCB genes in A2563 are similar to those of pMCR-SCNJ07 from
E. hormaechei in China in 2019 (GenBank accession no. MK933279), pRH-R27 from
Salmonella enterica Infantis in Germany in 2015 (GenBank accession no. LN555650), pT5282-mphA from
E. cloacae in China in 2012 (GenBank accession no. KY270852), pN1863-HI2 from
E. cloacae in China in 2017 (GenBank accession no. MF344583), pSE15-SA01028 from
S. enterica subsp.
enterica in Germany in 2018 (GenBank accession no. NZ_CP026661) and p707804-NDM from
Leclercia adecarboxylata in China in 2018 (GenBank accession no. MH909331). These 7 strains carrying plasmids with
mcr-9 in China and Germany did not harbored
blaIMPs, but
blaNDMs or
blaVIMs [
11].
The plasmids pA2483imp-1 and pA2504imp-1 had the same backbone as the plasmid pJJ1886_4 (GenBank accession no CP006788.1), which had been isolated in the USA. The 55,956 bp plasmid pJJ1886_4, which was smaller in length than the 61,594 bp plasmids pA2483imp-1 and pA2504imp-1, lacked a class I integron carrying blaIMP-1 (intl-blaIMP-1-aac (6′)-IIc-qacE ∆1-sul1). The E. cloacae EN3600 plasmid (GenBank accession no CP035638.1) carrying blaIMP-8 also had the same backbone as pJJ1886_4, with 83% coverage and 96.8% identity. These findings indicate that pJJ1886_4 has spread globally and captured drug-resistance genes and that this plasmid functions as a carrier of acquired drug-resistance genes.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.