α-Terpineol reverses mcr-mediated colistin resistance and potentiates colistin’s antibacterial activity in multidrug-resistant Escherichia coli

Amid the growing antibiotic resistance crisis, colistin remains a last-line therapeutic option for multidrug-resistant (MDR) Gram-negative bacterial infections. However, the emergence and rapid dissemination of plasmid-mediated colistin resistance gene ( mcr ) have markedly reduced its clinical effectiveness. Utilizing colistin adjuvants to restore its antibacterial potency is a promising strategy to combat this threat. In this study, we aimed to investigate the synergistic effects of α-terpineol in combination with colistin against colistin-resistant MDR Escherichia coli ( E. coli ) and to elucidate the underlying mechanisms of this synergy. In vitro synergistic activity was evaluated using checkerboard microdilution assays, time-kill curve analyses, and resistance development studies. Mechanistic insights were obtained through transcriptomic analysis and fluorescence-probe based assays. In vivo efficacy was validated using Galleria mellonella and mouse acute peritonitis infection models. The results showed that the combination of α-terpineol and colistin exhibited synergistic bactericidal activity and suppressed the development of colistin resistance. α-Terpineol increased inner- and outer-membrane fluidity in E. coli , thereby enhancing colistin uptake. Furthermore, it inhibited superoxide dismutase (SOD) activity, preventing the conversion of O 2 ·– to H 2 O 2 and causing superoxide accumulation. Elevated O 2 ·– levels damaged iron–sulfur clusters and impaired respiratory function. Together with the proton motive force (PMF) dissipation, these effects severely impaired ATP synthesis, ultimately sensitizing MDR E. coli to colistin. The combination also showed significant therapeutic efficacy in animal models. Our findings identify α-terpineol as a promising colistin adjuvant that restores antibacterial activity by disrupting membrane integrity and impairing antioxidant defenses, offering a viable strategy for treating MDR Gram-negative bacterial infections.