Discovery of eucalyptus essential oil as a novel colistin adjuvant eradicate multidrug-resistant Gram-negative bacteria.

Multidrug-resistant (MDR) Gram-negative bacteria poses a significant public health risk due to its multidrug resistance mechanism. The discovery of novel antibiotic adjuvant to combat clinical superbugs is an urgent priority for addressing the antibiotic resistance crisis. Here, through checkerboard assays, time-kill analyses, disk diffusion tests, biofilm inhibition assays, and microscopic examinations, eucalyptus essential oil (EEO) was identified as a potent enhancer of colistin activity. Mechanistic analyses further demonstrated that EEO downregulated bacterial genes associated with iron homeostasis, leading to intracellular accumulation of ferrous iron. The resulting imbalance enhanced oxidative stress via the Fenton reaction, ultimately accelerating bacterial damage and death. The synergistic interaction between EEO and colistin was further verified in vivo using a murine Salmonella Typhimurium (S. Typhimurium) HYM2 infection model. This study demonstrated that EEO synergistically enhances the antibacterial, anti-infective, and anti-inflammatory effects of colistin, as evidenced by its superior bacterial clearance and ability to mitigate inflammation. Our findings identify EEO as a promising colistin adjuvant candidate, providing a powerful strategy eradicate MDR Gram-negative bacterial infections.