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

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.