Frequent convergence of mcr-9 and carbapenemase genes in Enterobacter cloacae complex driven by epidemic plasmids and host incompatibility

Abstract

Convergence of mcr and carbapenemase genes has been sporadically detected in Enterobacter cloacae complex (ECC) with an upward trend. However, the state of the epidemic and underlying mechanism of such convergence has been poorly understood. In this study, the co-occurrence of MCR and carbapenemases was systematically analyzed in 230 clinical ECC isolates collected between 2000 and 2018 together with a global dataset consisting of 3,559 ECC genomes compiled from GenBank. We identified 48 mcr-9/mcr-10-positive isolates (MCR-ECC) (20.9%) in our collection, and a comparable ratio of MCR-ECC (720/3559, 20.2%) was detected in the global dataset. A high prevalence of carbapenemase-producing MCR-ECC (MCR-CREC) was further identified in the MCR-ECC of both datasets (16/48, 33.3%; 388/720, 53.9%), demonstrating a frequent convergence of mcr-9/10 and carbapenemase genes in ECC worldwide. An epidemic IncHI2/2A plasmid with a highly conserved backbone was identified and largely contributed to the dissemination of mcr-9 in ECC worldwide. A highly conserved IncX3-type NDM-1-carrying plasmid and IncN-type IMP-4-carrying plasmid were additionally detected in MCR-CREC isolated in China. Our surveillance data showed that MCR-CREC emerged (in 2013) much later than MCR-ECC (in 2000), indicating that MCR-CREC could be derived from MCR-ECC by additional captures of carbapenemase-encoding plasmids. Tests of plasmid stability and incompatibility showed that the mcr-9/mcr-10-encoding plasmids with the NDM-1-encoding plasmids stably remained in ECC but incompatible in Escherichia coli, suggesting that the convergence was host-dependent. The findings extend our concern on the convergence of resistance to the last resort antibiotics and highlight the necessity of continued surveillance in the future.

Keywords: Enterobacter cloacae complex; antibiotic resistance; carbapenemase; epidemic plasmid; mcr-9.

Figure 1.

Phylogenetic analysis and characteristics of the 48 MCR-ECC isolates collected in this study. A core-genome phylogenetic tree including the 48 MCR-ECC isolates was constructed and mid-point rooted. The isolates clustered into four clades corresponding to four species (blue: E. hormaechei, yellow: E. kobei, green: E. roggenkampii, purple: E. mori). The ST of each isolate is shown, and the novel ST is indicated by a dash. The absence (in pink) or presence (in red) of colistin and carbapenem resistance genes is diaplayed by a heatmap, and a truncated mutant of mcr-9 was marked with an asterisk. Clinical information of the isolates and patients are listed. The sterile-site specimens were in bold.

Figure 2.

Comparative genomic analysis on eight complete sequenced mcr-9-harboring MCR-CRE isolates. (A) Core-genome phylogenetic tree accompanied by a similarity matrix of mcr-9 plasmids. The isolates are coloured according to the STs (blue: ST133; red: ST418; green: a novel ST). The pairwise similarity between plasmids is shown by a heatmap, and is defined as the coverage of homology regions for query plasmid (row-wise) and subject plasmid (column-wise). (B) Synteny analysis of eight circularized mcr-9 plasmids. Identical regions (i.e. 100% similarity) are highlighted by grey rectangles. Arrows with direction indicate the sense of transcription of genes. Δ represents truncated genes.

Figure 3.

Detection of pECC116-MCR-9, pECC47-MCR-9, and pECC65-MCR-9 in 44 mcr-9-carrying MCR-ECC. The percentage length of virulence plasmid sequences are obtained by mapping short reads of the 44 isolates to the three mcr-9 plasmids (pECC116-MCR-9, pECC47-MCR-9, and pECC65-MCR-9) used as references. The existence of plasmid is defined by that isolates having short reads mapped to ≥90% of the reference plasmid length. The isolates are clustered according to coverages using the Pearson method. STs of isolates are indicated, and NA represents the novel ST.

Figure 4.

Pairwise comparison of bla_NDM-1-carrying plasmids. Twelve bla_NDM-1-carrying plasmids are included in the analysis, of which 11 are circularized, and the other one (Genome of ECC189) is analyzed using the draft genome. The similarity between sequences is defined as the coverage of homology regions for query plasmid (row-wise) and subject sequences (column-wise).

Figure 5.

Synteny analysis for genetic environments of (A) mcr-9, (B) mcr-10, and (C) bla_IMP-4. Identical regions are highlighted by dark grey rectangles (i.e. 100% similarity). Arrows with direction indicate the sense of transcription of genes, and Δ represents truncated sequences.