Flufenamic acid resensitizes MRSA to gentamicin through synergistic ion modulation

BACKGROUND AND OBJECTIVES: The increasing prevalence of MRSA challenges current therapeutic options and underscores the need for strategies that restore the efficacy of existing antibiotics. To evaluate the ability of flufenamic acid to enhance gentamicin activity against MRSA and to elucidate its mechanism of action. METHODS: The antibacterial activity of flufenamic acid combined with gentamicin was assessed by broth microdilution, time-kill assays and biofilm inhibition tests. Mechanistic studies included ion flux assays, membrane potential and charge measurements, metabolic profiling, reactive oxygen species (ROS) detection, and molecular docking against the biofilm-associated protein Bap. Efficacy was further examined in a Galleria mellonella infection model. RESULTS: Flufenamic acid reduced the MIC of gentamicin against MRSA from 32 mg/L to 1 mg/L and accelerated bactericidal killing. It disrupted Ca2+ and Cl- influx, leading to membrane depolarization and increased anionic surface charge, thereby promoting gentamicin uptake. Flufenamic acid also induced metabolic perturbation, enhanced ROS generation, and bound to Bap (binding energy: -7.678 kcal/mol), reducing biofilm amyloid content and increasing membrane permeability. In Galleria mellonella, the flufenamic acid/gentamicin combination significantly improved survival compared with gentamicin alone. CONCLUSIONS: Flufenamic acid acts as a synergistic ion modulator that resensitizes MRSA to gentamicin via disruption of membrane integrity, ion homeostasis and biofilm structure, supporting its potential as an adjunctive therapy against difficult-to-treat MRSA infections.