Inhibitory effects of benzyl isothiocyanate on widespread mcr-1-harbouring IncX4 plasmid transfer

Abstract

The global dissemination of mobile colistin resistance (mcr) genes represents a significant public health threat due to colistin's critical role in treating multidrug-resistant (MDR) bacterial infections. We identified high rates of carbapenem resistance in Escherichia coli (27.82%) and Klebsiella pneumoniae (57.98%) and colistin resistance in E. coli (7.52%) and K. pneumoniae (19.68%) among MDR clinical isolates in Thailand. We reported sequences of self-transferable IncX4 plasmids (~ 34 kb) that facilitated the spread of the mcr-1.1 gene among six diverse MDR strains, often co-transferring bla_CTX-M-55. Additionally, E. coli ST101 was found to co-transfer mcr-1.1, mcr-3.5, bla_CTX-M-55, and tet(X4) via three plasmids (~ 34-kb IncX4, ~ 84-kb IncFII, ~ 278-kb IncHI2), resulting in increases in MICs for colistin, ceftriaxone, and tigecycline. Core SNP analysis revealed that closely related IncX4 plasmids harbouring mcr-1 (< 35 SNP differences) were reported from at least 12 countries. We first demonstrated the inhibitory effects of benzyl isothiocyanate (BITC) on the conjugation of mcr-1-bearing IncX4 plasmids to 1.57 ± 1.00% to 48.86 ± 12.31% relative to control (100%), targeting VirB4 and VirB11 proteins, reducing ATPase activity by over 30%. This study highlights the widespread mcr-1-harbouring IncX4 plasmids and proposes BITC as a potential inhibitor to control the dissemination of colistin resistance.

Keywords: Escherichia coli; Klebsiella pneumoniae; Benzyl isothiocyanate; Colistin; Plasmids; Thailand.

Fig. 1

Comparison of the AMR observed between MDR-E. coli and MDR-K. pneumoniae isolates from patients in Thailand during 2017 to 2021. CRO ceftriaxone, CAZ ceftazidime, ETP ertapenem, IMP imipenem, MER meropenem, CIP ciprofloxacin, CHL chloramphenicol, TE tetracycline, GEN gentamicin, AK amikacin, SXT trimethoprim-sulfamethoxazole, COL colistin; *, p < 0.05; and **, p < 0.01.

Fig. 2

Dendrogram generated by PFGE-XbaI of (a) seven mcr-harbouring E. coli and (b) four mcr-harbouring K. pneumoniae, clinical isolates in Thailand. Isolate summary information showing antimicrobial susceptibility profiles, colistin MIC, AMR mechanisms, and pulsotypes. Black squares represent isolates that were resistant to antimicrobial agents. Antimicrobial agents are abbreviated as follows: ceftriaxone, CRO; ceftazidime, CAZ; chloramphenicol, CHL; tetracycline, TE; ciprofloxacin, CIP; gentamicin, GEN; trimethoprim-sulfamethoxazole, SXT; ertapenem, ERT; imipenem, IMP; meropenem, MER; amikacin, AK; and colistin, COL. CBR, Chonburi; BKK, Bangkok; CTB, Chanthaburi; PMQR, plasmid-mediated quinolone resistance; -, not found; and ND, not done.*Plasmid sizes and AMR mechanisms confirmed for the location on the plasmid by Southern blot and hybridisation, which are coloured by red, blue, and green for the mcr-1 gene, mcr-3 gene, and bla_CTX-M-15/55 gene, respectively.

Fig. 3

Genetic annotation of reconstructed MDR plasmids of co-habouring mcr-1.1 and mcr-3.5 E. coli EN-1077 clinical isolate in Thailand. (a) IncX4 type mcr-1.1-carrying plasmid pEc1077-34 and sequence comparison of reconstructed IncX4 type of mcr-1.1-carrying plasmids. (b) IncFII type mcr-3.5-carrying plasmid pEc1077-84. (c) IncHI2 type tetX4-carrying plasmid pEc1077-278. The outer circle with arrows signifies the annotation of a reference sequence. The plasmid pEc1077-34 (33.85 kb) was used as the reference to compare with the IncX4 plasmids as follows: the purple ring represents the plasmid pKp1230-34 (33.98 kb) from K. pneumoniae EN-1230, the orange ring represents the plasmid pEc1313-34 (33.98 kb) from E. coli EN-1313, and the green ring represents the plasmid pEc430-34 (33.98 kb) from E. coli EN-430.

Fig. 4

Midpoint-rooted maximum likelihood tree from core SNP alignment of four complete sequences of IncX4 plasmid carrying mcr-1 gene in this study and a total of 57 IncX4 plasmids carrying mcr-1 gene currently deposited in the NCBI GenBank database. Isolates obtained in this study are highlighted in red. The branch length represents the distance of the core plasmid sequence. Clades are coloured yellow and light blue. From the inside to the outside, each circle represents the organism, host, and country, respectively.

Fig. 5

The binding conformation with the best energy and full fitness values from the blind docking of BITC (PubChem CID: 2346) into the molecular model of VirB4 (PDB ID: 7O41) (a, c) and VirB11 (PDB ID: 2GZA) (b, d). The upper and bottom panels correspond to the protein–ligand interactions in surface and cartoon representations. The N-terminal domain (NTD) is purple, and the C-terminal domain (CTD) is dark blue, connected via an orange linker. The light blue regions represent ATP-binding sites. The interacting residues are shown in stick form with a wire ball. Green dotted lines represent hydrogen bonding.

Fig. 6

Effect of increasing BITC concentrations on the reductions (%) of ATPase activity and the reductions (%) of conjugation frequency observed in representative plasmid pEc430-34 (IncX4) relative to the control without BITC (100%). Data represent means ± SD.