Exogenous glutamate potentiates gentamicin to kill multidrug- and carbapenem-resistant Pseudomonas aeruginosa by activating the biosynthesis of unsaturated fatty acids.

Multidrug- and carbapenem-resistant Pseudomonas aeruginosa (MDR-PA and CR-PA) are difficult to control due to the predicament caused by their limited membrane permeability. The metabolic reprogramming approach is an effective strategy to promote membrane permeability. In this study, a gas chromatography-mass spectrometer-based metabolomics identified decreased abundance of glutamate as the most characteristic feature in gentamicin-resistant P. aeruginosa (PA-R_GEN). Exogenous glutamate enhanced gentamicin killing to lab-evolved PA-R_GEN as well as clinical MDR-PA and CR-PA isolates. By applying a multi-faceted approach, including glutamate-reprogramming metabolomics, isotope-tracing analysis, glutamate-reprogramming lipidomics, membrane permeability measurement, and oleic acid replacement test, we demonstrated that the glutamate metabolic flux increases the biosynthesis of unsaturated fatty acids and decreases the biosynthesis of saturated fatty acids. This change in lipid composition promotes membrane permeability and enhances gentamicin uptake in the presence of glutamate. However, the opposite phenotypes were exhibited in MDR- and CR-PA in the absence of glutamate. These results identify an effective reprogramming metabolite to combat MDR- and CR-PA with gentamicin and reveal a resistance mechanism of membrane permeability that limits drug uptake and its reversal approach in MDR- and CR-PA.

Importance: Antibiotic-resistant Pseudomonas aeruginosa is a major clinical challenge due to limited drug uptake. This study shows that exogenous glutamate restores gentamicin efficacy by reprogramming bacterial metabolism to enhance membrane permeability. The effect is mediated through increased biosynthesis of unsaturated fatty acids, which is further confirmed by oleic acid supplementation. These findings reveal a novel metabolic approach to overcome multidrug and carbapenem resistance, offering a promising adjunct strategy to improve antibiotic treatment outcomes.