Palmitoleic acid drives gentamicin influx to outpace aminoglycoside-modifying enzymes and overcome resistance

Rising antibiotic resistance causes >700,000 deaths annually and could reach ~10 million by 2050. Because the development of new antibiotics lags behind resistance, strategies that restore the activity of approved drugs are urgently needed. Gentamicin efficacy against Staphylococcus aureus and Enterococcus spp. is limited by widespread aminoglycoside-modifying enzymes (AMEs). Here, we show that palmitoleic acid, a host-derived monounsaturated fatty acid, re-sensitizes resistant gram-positive pathogens to gentamicin by accelerating intracellular drug influx rather than inhibiting AMEs. Single-cell flow cytometry and high-resolution mass spectrometry demonstrate rapid gentamicin accumulation that outpaces enzymatic inactivation. The combination lowers MICs across clinical isolates and significantly enhances killing of mature biofilms at clinically relevant concentrations. These findings establish a framework in which resistance reflects the balance between antibiotic entry and enzymatic inactivation. By shifting this balance toward drug influx, resistance can be functionally reversed without targeting resistance genes, supporting permeability-based adjuvant strategies to expand the utility of existing antibiotics.