Aspartate potentiates tobramycin against multidrug-resistant <i>Edwardsiella tarda</i> through enhancing proton motive force and membrane permeability

The antibiotic susceptibility/resistance of bacteria is influenced by their metabolic states. In tobramycin-resistant <i>Edwardsiella tarda</i> (LTB4-R_TOB), a dynamic metabolic state is observed, characterized by a general reduction in most metabolites as the minimum inhibitory concentration increases. Among these, aspartate downregulation emerges as the most critical biomarker. Exogenous aspartate enhances the efficacy of tobramycin, enabling it to effectively kill both lab-evolved and clinically isolated multidrug-resistant (MDR) <i>E. tarda</i>, as demonstrated in an animal model. Aspartate increases intracellular tobramycin levels in a concentration- and time-dependent manner by promoting drug uptake. Metabolic reprogramming analysis of LTB4-R_TOB and clinically isolated MDR strain WY28 reveals that exogenous aspartate restores downregulated metabolites, primarily enhancing alanine, aspartate, and glutamate metabolism; the pyruvate cycle; and glycine, serine, and threonine metabolism. These changes elevate the proton motive force and membrane permeability, thereby increasing tobramycin uptake. The resulting intracellular drug concentration surpasses the bactericidal threshold, effectively overcoming resistance in both LTB4-R_TOB and clinical MDR <i>E. tarda</i>. These findings identify aspartate as a potent metabolic reprogramming agent that potentiates aminoglycosides by boosting intracellular drug accumulation, offering a strategy to combat antibiotic-resistant <i>E. tarda</i>.IMPORTANCE<i>Edwardsiella tarda</i> is a significant pathogen that causes edwardsiellosis in aquaculture, resulting in substantial economic losses while also posing a global public health threat. The increasing prevalence of antibiotic-resistant <i>E. tarda</i> strains has further exacerbated this challenge. The present study used a metabolic state-reprogramming approach to identify an ideal biomarker as an antibiotic adjuvant to increase conventional antibiotics that are already resistant. Exogenous aspartate most effectively potentiated tobramycin to lab-evolved and clinical isolated MDR <i>E. tarda</i> in <i>vitro</i> and in <i>in vivo</i> models (fish and mice). Mechanistically, aspartate enhanced tobramycin uptake in MDR <i>E. tarda</i> by overcoming efflux pump activity. This effect was mediated through aspartate-induced activation of the pyruvate cycle, which increased the proton motive force via NADH generation and enhanced membrane permeability. Our findings demonstrate that the aspartate-tobramycin combination represents a promising therapeutic strategy against MDR <i>E. tarda</i> infections.