Reorganization of mcr-1-bearing large MDR plasmids resolved by nanopore sequencing

Sir,

The emergence of plasmid-borne mcr genes among pathogens poses great public concern.¹ Different mcr variants have been identified from various sources and the plasmids encoding mcr genes are diverse and evolve constantly. Plasmid reorganization accelerates the evolution of MDR plasmids and facilitates the generation of large MDR plasmids.^2,3 Here, we report the isolation of an mcr-1-bearing Escherichia coli strain from a Père David’s deer in China in 2018. We have characterized the underlying molecular mechanism of the mcr-1-bearing plasmid reorganization responsible and highlight the role of mobile elements such as ISCR2 in the generation of hybrid and cointegrate plasmids.

The mcr-1-bearing E. coli strain LD91-1 was isolated from the faeces of a Père David’s deer and isolate identification was confirmed by PCR and 16S rDNA sequencing.¹ MICs of several antimicrobials were determined by the broth microdilution method. E. coli ATCC 25922 was used as the quality control. LD91-1 exhibited resistance to colistin (4 mg/L), florfenicol (>64 mg/L), amoxicillin (>128 mg/L), ceftiofur (>64 mg/L), streptomycin (32 mg/L) and doxycycline (16 mg/L), reduced susceptibility to enrofloxacin (1 mg/L) and susceptibility to meropenem (≤0.125 mg/L). To investigate the transferability of mcr-1, a conjugation assay was performed with E. coli J53 Azi^R as the recipient. Thirty-seven transconjugants selected were positive for mcr-1 and S1-PFGE was conducted to examine the plasmid profiles of the donor strain and transconjugants [Figure 1a and Figure S1 (available as Supplementary data at JAC Online)]. Three plasmids (∼245 kb, ∼140 kb and ∼78 kb) were observed in LD91-1. However, the plasmids in transconjugants were of different sizes (∼280 kb, ∼350 kb and ∼390 kb) (Figure S1). To determine the genomic basis of the reorganization of the mcr-1-bearing plasmids, WGS of LD91-1 was completed with a hybrid strategy combining Illumina short-read and nanopore long-read data.⁴ Plasmids of three representative transconjugants with different plasmid sizes (Figure 1a) were extracted with the QIAGEN Plasmid Midi Kit, subjected to nanopore MinION sequencing and de novo assembled into complete circular sequences with the Flye tool.⁵ The complete sequences of LD91-1 have been deposited in the NCBI database under the following accession numbers: chromosome (CP042585), pLD91-1-76kb (CP042584), pLD91-1-146kb (CP042586) and pLD91-1-MCR1 (CP042587). The sequences of the three fused plasmids have been deposited in figshare (https://figshare.com/s/ae5aded17a16498587f1). Tools including RAST, ISFinder, ResFinder, PlasmidFinder, BRIG and Easyfig were utilized to analyse the plasmids in detail.

Figure 1

S1-PFGE of transconjugant representatives harbouring large fused plasmids and the schematic diagrams depicting the linear structures of the parent plasmids and three fused plasmids. (a) S1-PFGE of the donor strain and transconjugant representatives. M, Salmonella serotype Braenderup strain H9812 universal size marker. Three transconjugant representatives were selected from the 37 transconjugants (Figure S1); comparisons between the parental plasmids (pLD91-1-MCR1 and pLD91-1-146kb) and (b) hybrid plasmid pTLD91-1-297kb generated by recombination in ISCR2 and IS1; (c) hybrid plasmid pTLD91-1-352kb generated by ISCR2 and a Tn2 variant segment; and (d) cointegrate plasmid pTLD91-1-390kb generated by ISCR2. The red arrows indicate the recombination sites. For the detailed structures and annotations of the fused plasmids in circular form, refer to Figures S3, S4 and S5. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Strain LD91-1 has a 4 733 770 bp chromosome (ST162) and three plasmids, pLD91-1-MCR1 (246 716 bp), pLD91-1-146kb (146 133 bp) and pLD91-1-76kb (76 292 bp). The mcr-1 gene is in pLD91-1-MCR1, which is a typical IncHI2 mcr-1-bearing plasmid carrying various resistance genes such as mcr-1, mph(A), bla_CTX-M-14, fosA3, aac(3)-IVa, aph(4)-Ia, sul2, dfrA12, aadA2, cmlA1, aadA1, sul3, aph(3′)-Ia and sul1 dispersed among ISs (Figure S2a). pLD91-1-MCR1 is most similar to the mcr-1-bearing plasmids pSLK172-1(CP017632) and pSH15G2194 (MK477611) (Figure S2). In pLD91-1-MCR1, ISApl1-mcr-1-pap2 is inserted in the same place as in other mcr-1-bearing plasmids from different bacteria. Another MDR plasmid, pLD91-1-146kb (IncFIB), carries tet(A), bla_TEM-135, dfrA14, qnrS1 and floR. A similar plasmid, pL65-2 (CP034739), with nearly 100% identity to pLD91-1-146kb was found in E. coli derived from a goose (Figure S2b), which suggests that this kind of plasmid has spread between bacteria from different sources. No resistance genes, ISs or replicons were found in pLD91-1-76kb.

The three fused plasmids in representative transconjugants were completed with long-read data and the underlying reorganization mechanisms were investigated (Figure 1). The sizes of plasmids were 390 532 bp (pTLD91-1-390kb), 352 115 bp (pTLD91-1-352kb) and 297 916 bp (pTLD91-1-297kb). Comparisons between the plasmids in LD91-1 and in transconjugants suggested different recombination events between pLD91-1-MCR1 and pLD91-1-146kb involving ΔISCR2, a fragment of a Tn2 variant and IS1 (Figure 1). pTLD91-1-297kb is a hybrid plasmid consisting of parts of the parental plasmids apparently generated by recombination in both ISCR2 and IS1 (Figure 1b). pTLD91-1-352kb is a hybrid plasmid consisting of parts of the parental plasmids apparently generated by recombination in both ISCR2 and a fragment of the Tn2 variant (Figure 1c). pTLD91-1-390kb is a cointegrate of the two parental plasmids apparently generated by recombination in △ISCR2 (Figure 1d). Intriguingly, ISCR2 was the universal recombination site during the generation of the three fused plasmids (Figure 1 and Figures S3 to S5). ISCR2 (part of which corresponds to ISVsa3 in ISfinder),⁶ belonging to the IS91 family of rolling circle elements, has played a pivotal role in the transmission of various resistance genes.^7,8 Our study demonstrates that ISCR2 and its truncated version can take part in the generation of fused plasmids. Considering the ratios of the fused plasmids in the 37 transconjugants, pTLD91-1-390kb generated by recombination in ISCR2 was the dominant fused plasmid (29 out of 37 transconjugants, Figure S1). Compared with the role of the segment of Tn2 variant and IS1, ISCR2 was the most important mobile element facilitating recombination of MDR plasmids in this study. Recently, ISCR2 (ISVsa3) was found to be associated with plasmid-mediated tigecycline resistance genes tet(X3) and tet(X4) and can facilitate their transposition in vivo.^9,10 In view of the existence of ISCR2 in mcr-1-bearing and tet(X)-bearing plasmids, the convergence of mcr-1 and tet(X) genes in the same conjugative MDR plasmid would be a possible evolutionary pathway under the recombination and transposition activity of ISCR2, which would constitute a potential public health risk. One of the 37 transconjugants carried pLD91-1-MCR1, suggesting that this plasmid may have a functional conjugation region. Analysis with oriTfinder (http://bioinfo-mml.sjtu.edu.cn/oriTDB/index.php) suggests that both pLD91-1-MCR1 and pLD91-1-146kb may have a functional conjugation region. The reason why most of the transconjugants harbour hybrid or cointegrate plasmids warrants further studies.

To conclude, ISCR2 was found to be responsible for mcr-1-bearing large fused MDR plasmids. ISCR2 appears to facilitate generation of large fused MDR plasmids carrying mcr-1. As ISCR2 is also associated with plasmid-borne tet(X) genes, possible co-occurrence of mcr-1 and tet(X) genes in conjugative MDR plasmids warrants investigation.

Funding

This work was supported by the National Natural Science Foundation of China (grants no. 31872523 and no. 31872526), the Natural Science Foundation of Jiangsu Province (grant no. BK20180900) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Transparency declarations

None to declare.

Supplementary data

Figures S1 to S5 are available as Supplementary data at JAC Online.

Author notes

Ruichao Li and Xiaoyu Lu contributed equally to the work.