Stability and genetic insights of the co-existence of blaCTX-M-65, blaOXA-1, and mcr-1.1 harboring conjugative IncI2 plasmid isolated from a clinical extensively-drug resistant Escherichia coli ST744 in Shanghai

Abstract

Background: Co-existence of colistin, β-lactam and carbapenem in multidrug-resistant Enterobacteriaceae isolates poses a serious threat to public health. In this study, we investigated and characterized the co-occurrence of bla_CTX-M-65, bla_OXA-1, and mcr-1.1 strain isolated from a clinical extensively-drug-resistant Escherichia coli ST744 in Shanghai.

Methods: Antimicrobial susceptibility test was carried out by agar dilution methods. Whole genome sequencing was conducted, and resistance genes, and sequence types of colistin in E. coli isolates were analyzed. Plasmid stability and amino acid mutations were assessed in E. coli isolates.

Results: A colistin resistant E. coli ST744, named ECPX221, was identified out of 145 fecal samples collected. The strain carries a 60,168 IncI2 plasmid with the mcr-1.1 gene. The strain also has bla_CTX-M-65, bla_OXA-1, dfrA14, qnrS1, cmlA5, arr2, ampC, aph(4)-Ia, sul1, and aadA5 resistance genes. The plasmid pECPX221 was capable of conjugation with an efficiency of 2.6 × 10^-2. Notably, 45% of the transconjugants were determined as mcr-1.1-harboring in the colistin-free environment after 60 generation of passage. No mutations occurred in pmrB, mgrB, and phoPQ gene in the mcr-1.1-harboring transconjugants. Bioinformatic analysis indicated pECPX221 shared highly similar backbone with the previously reported mcr-1.1-harboring pAH62-1, pMFDS1339.1, pSCZE4, and p2018-10-2CC. Furthermore, sequencing and phylogenetic analyses revealed a similarity between other MCR-1-homolog proteins, indicating that ECPX221 was colistin resistant.

Conclusion: The stable transferable mcr-1.1-harboring plasmid found in the E. coli ST744 strain indicated the high risk to disseminate the extensively-drug-resistance phenotype among Enterobacteriaceae.

Keywords: Escherichia coli STstrain-744 (ST744); MCR-1; colistin; plasmid; stability.

Figure 1

The resistance colonies that harboring mcr-1.1 gene in the colistin (red) and antibiotic free environment (blue).

Figure 2

Comparison of pECPX221 and other mcr-1-harboring plasmids including pAH62-1, pMFDS1339.1, pSCZE4, and p2018-10-2CC by FastANI software.

Figure 3

Linear comparison of complete plasmid sequences of plasmid Escherichia coli pECPX221 (this study), pAH62-1 from E. coli AH62 (GenBank Accession No. CP055260), pMFDS1339.1 from E. coli MFDS1339 (GenBank Accession No. MK852553), pSCZE4 from E. coli SCZE5 (GenBank Accession No. CP051226), and p2018-10-2CC from E. coli 2018–10-2CC (GenBank Accession No. LC511662).

Figure 4

Map of mcr-1-harbouring plasmid pECPX221. The mcr-1 gene is marked in red.

Figure 5

Phylogenetic study of MCR-1 and MCR-1-like proteins reveals ancestral origins and diversification. Using amino acid sequences from MCR-1 protein of ECPX221 in this study, the BLAST search tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi, Accessed on 20 June 2023) was used to retrieve related sequences of MCR-1 and MCR-1-like proteins from the NCBI database. MCR-1 and MCR-1-like proteins of E. coli, Salmonella, and strains containing LptA and others were among the sequences categorized. Using aligned MCR-1 sequences from CLUSTALW, the maximum likelihood method of MEGA X was used to create a phylogenetic tree. (A) Phylogeny of 54 MCR-1 proteins of E. coli origin retrieved from NCBI database including sequenced MCR-1 gene of ECPX221 from this study. (B) Phylogeny of 26 MCR-1 proteins retrieved from NCBI database except E. coli strains.