Esculetin restores colistin susceptibility in MCR-positive bacteria through multiple mechanisms, thereby combating drug-resistant bacterial infections in chickens.

Given the longstanding reliance of the poultry industry on antibiotics to control bacterial diseases, MCR-1 plasmid-mediated colistin-resistant Escherichia coli infections pose a significant threat to poultry health. Therefore, alternative strategies are urgently needed. Esculetin, a coumarin derivative with notable pharmacological potential, has emerged as a promising candidate for restoring colistin susceptibility. However, the mechanism by which esculetin restores the antibacterial activity of colistin and its potential in combination with colistin against resistant bacterial infections in chickens remain unclear. In this study, esculetin showed synergistic antibacterial activity with colistin against the tested MCR-1-positive E. coli and exhibited acceptable safety in cytotoxicity and hemolysis assays. Metabolomic analysis indicated that EL significantly affected glycerophospholipid metabolism, with lysophosphatidic acid identified as a key candidate metabolite. EL showed predicted binding affinity for glycerophospholipid metabolism-related enzymes and was associated with increased GPAT activity, decreased PLA2 activity, elevated lysophosphatidic acid levels, and reduced phosphoethanolamine content. Consistently, esculetin decreased phosphoethanolamine-modified lipid A in bacterial cells, whereas purified MCR-1 enzymatic assays suggested that esculetin did not directly inhibit MCR-1 activity. The EL combined with colistin altered bacterial membrane permeability, fluidity, and charge, promoted colistin-associated antibacterial effects, and induced downstream oxidative membrane damage, including ROS accumulation, iron-homeostasis disruption, lipid peroxidation, and reductions in phosphoethanolamine, phosphatidylglycerol, and cardiolipin. In a chicken infection model, the combined treatment improved survival, reduced bacterial loads, and alleviated tissue injury and inflammatory responses. Together, these findings suggest that esculetin may restore colistin susceptibility mainly by remodeling glycerophospholipid metabolism and the bacterial membrane environment, whereas oxidative and iron-dependent lipid damage may further contribute to bacterial injury. Esculetin therefore represents a potential membrane-metabolic adjuvant for improving colistin efficacy against MCR-positive E. coli infections in chickens.