18AA Reprograms the Pyruvate Cycle and Enhances Proton Motive Force to Potentiate Ceftazidime-Avibactam against Drug-Resistant Escherichia coli

Abstract The rising threat of carbapenem-resistant Enterobacteriaceae demands novel strategies to restore antibiotic efficacy. Here we show that compound amino acid injection (18AA), a clinically approved parenteral nutrition formulation, markedly potentiates ceftazidime-avibactam (CZA) against multidrug-resistant and carbapenem-resistant Escherichia coli. Checkerboard and Bliss independence analyses demonstrated strong synergy. Time-kill kinetics confirmed dose-dependent bactericidal enhancement across susceptible, multidrug-resistant, and carbapenem-resistant strains. In a panel of 113 clinical isolates, the combination significantly improved population-based killing efficacy (KE50 and KE90) compared to CZA alone. Moreover, 18AA prolonged the post-antibiotic effect, reduced mutation frequency, and delayed resistance development during serial passage over 15 cycles. Untargeted metabolomics revealed that 18AA induces profound metabolic reprogramming, with robust activation of the pyruvate cycle as the most prominent signature common to all tested strains; key biomarkers pyruvate, ketoglutarate, and succinate were identified. Mechanistically, 18AA boosted the pyruvate cycle, increased NADH production, and elevated proton motive force (PMF), thereby promoting intracellular CZA accumulation, which was abrogated by the PMF uncoupler CCCP. Collectively, our findings establish 18AA as a promising metabolism-based adjuvant that rejuvenates CZA activity via the pyruvate cycle and PMF-dependent uptake, offering a readily translatable strategy against hard-to-treat E. coli infections.