Characterization of the global distribution and diversified plasmid reservoirs of the colistin resistance gene mcr-9

Abstract

The emergence and spread of mobilized colistin resistance (mcr) genes have triggered extensive concerns worldwide. Here, we characterized the global distribution of mcr-9, a newly-identified variant of mcr, by assembling the data set of mcr-9-positive isolates from GenBank database and the literature available. Genetic features of all the mcr-9-harboring plasmids were determined by bioinformatic analysis. We showed that mcr-9 is globally distributed in 21 countries across six continents, with a wide dissemination among various species of Enterobacteriaceae strains from human, animal, food and environment. IncHI2-ST1 plasmids were found to be the predominant replicon type carrying mcr-9. Comparative genomics highlighted that IncHI2-type plasmids may also serve as a critical reservoir of mcr-9, from which different types of circulating plasmids acquired the mcr-9. Results revealed that the rcnR-rcnA-pcoE-pcoS-IS903-mcr-9-wbuC structure was consistent in most mcr-9 cassettes, suggesting a relatively unitary model involved in the mobilization of mcr-9. It is most likely that the spread of mcr-9 was mainly attributed to the conjugation and recombination events of mcr-9-carrying plasmids. In summary, our results provide a comprehensive picture of the distribution and genetic environment of mcr-9, and demonstrate the central roles played by IncHI2 plasmids in the worldwide dissemination of mcr-9.

Figure 1

Overview of the distribution of mcr-9. (a) Worldwide distribution of mcr-9-positive isolates. The map was generated using the online software dituhui (https://g.dituhui.com/). (b) Distribution of host species harboring mcr-9. (c) Distribution of the isolation source of mcr-9-positive strains.

Figure 2

Phylogenetic analysis of IncHI2 mcr-9-harboring plasmids. Core-genome alignments and phylogenetic reconstruction of IncHI2 mcr-9-carrying plasmids were performed using Parsnp. The heatmap denotes the presence of ESBL or carbapenemase genes as determined by ABRicate. The annotation denotes (from left to right) locations, host species, isolation sources, and sequence types of plasmids. NA, not available. ND, not determined.

Figure 3

Genetic characterization of IncHI2-type mcr-9-harboring plasmids (hybrid plasmids are excluded). (a) Sequence alignment of 60 IncHI2 mcr-9 plasmids. pRH-R27(accession no. LN555650) was used as a reference to compare with other plasmids. Gaps indicate regions that were missing in the respective plasmid compared to the reference plasmid. The outer circle with dark blue arrows denotes annotation of reference plasmid. mcr-9 gene is highlighted by red arrows. Backbone (tra1 and tra2 region) and four accessory resistance regions (ter region, the sil–cop region, MDR region 1 and MDR region 2) are indicated by red curves. The mcr-9-harboring region is indicated by the dotted box. Information about the IncHI2 plasmids tested is provided in Table S1. (b) Linear comparison of the mcr-9-harboring region in pRH-R27, p17277A_477 (CP043927) and pMRVIM0813 (KP975077). The corresponding region on non-mcr-9-carrying plasmid R478 (BX664015, top) is shown for comparison. Grey shading denotes regions of shared homology among different plasmids ranging from 80% to100%. Colored arrows represent open reading frames, with brown, green, and red arrows representing heavy metal resistance genes, mobile elements, and the mcr-9 gene, respectively. Other plasmid backbone genes and hypothetical genes are colored gray.

Figure 4

Genetic characterization of mcr-9-harboring hybrid plasmids. (a) Comparison of the IncHI2-IncR hybrid plasmids, pMCR-SCNJ07, pT5282-mphA and pN1863-HI2, with the IncHI2 plasmid pRH-R27 and IncR plasmid pHN84KPC. pN1863-HI2 was used as a reference to compare with other plasmids. (b) Linear comparison of the mcr-9-harboring region in pRH-R27, pMCR-SCNJ07, pT5282-mphA and pN1863-HI2. (c) Comparison of the IncHI2-IncA/C₂ hybrid plasmid, pGMI14-002_1 with the IncHI2 plasmid pRH-R27 and IncA/C2 plasmid pR55. pGMI14-002_1 was used as a reference to compare with other plasmids. (d) Linear comparison of the mcr-9-harboring region in pRH-R27 and pGMI14-002_1. Gaps in the circular maps refer to plasmid regions that were missing in the respective plasmid compared to the reference plasmid. The outer circle with dark blue arrows denotes annotation of reference plasmid. mcr-9 gene is highlighted by red arrows. The foreign region that was proposed to be an insertion was indicated by red curves. The mcr-9-harboring region is indicated by the dotted box. Grey shading in the linear maps denotes regions of shared homology among different plasmids ranging from 80% to 100%. Colored arrows represent open reading frames, with brown, green, and red arrows representing heavy metal resistance genes, mobile elements, and the mcr-9 gene, respectively. Other plasmid genes are colored gray.

Figure 5

Genetic characterization of IncFII-type mcr-9-carrying plasmid p1_045523 and six untyped mcr-9-carrying plasmids. (a) Comparison of the p1_045523 with the IncHI2 plasmid pRH-R27 and IncFII plasmid pOZ172. p1_045523 was used as a reference to compare with other plasmids. (b) Linear comparison of the mcr-9-harboring region in pRH-R27 and p1_045523. (c) Comparative genome maps of pCAV1099-114 (accession no. CP011596), pCAV1335-115 (CP011617), pCAV1015-114 (CP017930), pSHV12-1301491 (CP031568), pCFSAN002050 (CP006057), pLEC-b38d (CP026168) and the IncHI2 plasmid pRH-R27. pCAV1099-114 was used as a reference to compare with other plasmids. (d) Linear comparison of the mcr-9-harboring region in six untyped mcr-9-carrying plasmids and pRH-R27. The outer circle with dark blue arrows denotes annotation of reference plasmid. Gaps in the circular maps refer to plasmid regions that were missing in the respective plasmid compared to the reference plasmid. mcr-9 gene is highlighted by red arrows. The mcr-9-harboring region is indicated by the dotted box. Colored arrows represent open reading frames, with brown, green, and red arrows representing heavy metal resistance genes, mobile elements, and the mcr-9 gene, respectively. The remaining genes are shown in gray. Grey shading in the linear maps denotes regions of shared homology among different plasmids ranging from 80% to 100%.

Figure 6

A summary of representative genetic contexts of mcr-9 from different types of plasmid and chromosomes available to date. The genetic contexts of mcr-9 were differed by the truncation status of neighboring genes (from rcnR to qseB) encompassing mcr-9. The ‘rcnR-rcnA-pcoE-pcoS-IS903-mcr-9-wbuC’ structure was revealed as the core structure of mcr-9 cassettes by comparison analysis. Colored arrows represent open reading frames, with brown, green, and red arrows representing heavy metal resistance genes, mobile elements, and the mcr-9 gene, respectively. The remaining genes are shown in gray. Grey shading denotes genetic regions that exhibit sequence homology among different segments.