Sub-inhibitory tilmicosin promotes horizontal transfer of blaNDM via extracellular vesicles through activation of the zraS/zraR system.

The frequent use of macrolide antibiotics such as tilmicosin (TMS) in livestock production has raised increasing concerns about their potential role in the dissemination of antimicrobial resistance. Extracellular vesicles (EVs), nanoscale bilayered structures secreted by bacteria, have emerged as novel mediators of horizontal gene transfer (HGT), particularly under antibiotic-induced stress conditions. In this study, we investigated the effects of sub-inhibitory concentrations of TMS on EVs production and its contribution to the transfer of the blaNDM resistance gene in carbapenem-resistant Escherichia coli (CREC) isolated from swine. Exposure to 1/32 minimum inhibitory concentration (MIC) TMS significantly enhanced EVs secretion in CREC, accompanied by increased vesicle concentration and a dose-dependent elevation in the intra-species transfer frequency of blaNDM. Transcriptomic profiling revealed substantial changes in the expression of genes associated with signal transduction and membrane structure, and identified the zraS/zraR two-component system as a potential key regulator. Deletion of zraS and zraR using CRISPR/Cas9 led to marked reductions in EVs production and blaNDM transfer, confirming the central role of zraS/zraR in TMS-induced EVs biogenesis. Collectively, our findings demonstrate that TMS can promote EV-mediated dissemination of blaNDM by activating the zraS/zraR regulatory pathway, providing new insights into the molecular mechanisms underlying antibiotic-driven resistance spread in swine farms and supporting more prudent use of macrolides in animal husbandry.