Objective To analyze the distribution and genomic characteristics of mcr-carrying IncX4 plasmids, quantitatively evaluate the effects of subinhibitory concentrations of polymyxin B on its conjugative transfer, and elucidate the underlying molecular mechanisms.

Methods Complete plasmid sequences carrying mcr gene family, which is associated with polymyxin resistance in Enterobacteriaceae, were retrieved from the NCBI database. After replicon typing and dereplication, 173 non-redundant IncX4 plasmid sequences were included. Their distribution, hosts, sources, and genomic characteristics were analyzed. A representative IncX4-type mcr-1 plasmid pSH16G2543 was used as study subject, a modified conjugation model was established by inserting mcr-1 into the chromosomes of the donor strain J53 and recipient strain MG1655 to reduce polymyxin B induced growth inhibition. Conjugation assay, growth curve analysis, membrane permeability testing, reactive oxygen species detection, and real-time quantitative PCR were performed to explore the mechanisms.

Results IncX4 plasmids carrying mcr genes were identified in 17 countries across 4 continents. They had broad host range and different sources. Most plasmids were approximately 33 kb in length and had relatively conserved backbone structures. Among them, 96.5% carried only mcr-1, and the pangenome remained open. After chromosomal insertion of mcr-1, the direct inhibitory effect of subinhibitory concentrations of polymyxin B on recipient growth was markedly reduced. By using this modified conjugation model, subinhibitory concentrations of polymyxin B promoted the conjugative transfer of mcr-carrying IncX4 plasmid. The most pronounced effect was observed at 1/4minimum inhibitory concentration , with aincreas of relative conjugation frequency by 1.24-fold compared with the control group. In addition, membrane permeability increased, reactive oxygen species levels rose, and multiple conjugation-related genes were upregulated in the conjugation system.

Conclusion IncX4 plasmids are important vehicles for the propagation of mcr genes and have the potential for persistent spread and adaptive evolution. Polymyxin B at subinhibitory concentrations can promote their conjugative transfer, which was closely associated with increased membrane permeability, enhanced oxidative stress, and activation of conjugation-related genes. These findings suggest that in the context of medical use, veterinary use, and environmental residues, low-dose polymyxin might promote the horizontal transfer of plasmids carrying mcr resistance genes, thereby facilitating the spread of drug resistance.