Glutamate-Mediated Metabolic Rewiring Boosts CpxA/CpxR-OmpF and Proton Motive Force to Resensitize Antibiotic-Resistant <i>Escherichia coli</i> to Ceftazidime

Ceftazidime (CAZ) is a critically important broad-spectrum antibiotic that is widely used in clinical practice. However, the rapid emergence of bacterial resistance to CAZ poses a significant challenge in treating infections caused by multidrug-resistant pathogens. In this study, we employed a metabolism-reprogramming approach to characterize key features of laboratory-evolved CAZ-resistant <i>Escherichia coli</i> K12 and identified repressed glutamate metabolism as a reprogrammable target. Exogenous glutamate effectively resensitized both lab-evolved and clinically isolated multidrug-resistant <i>E. coli</i> strains to CAZ. The resensitization mechanism operates through two synergistic pathways driven by glutamate metabolic flux. First, glutamate conversion to inosine activates the inosine-CpxA/CpxR-OmpF regulatory axis, increasing outer membrane permeability. Second, glutamate entry into the pyruvate cycle restores the proton motive force (PMF), energizing the inner membrane. Together, increased outer membrane permeability and a restored PMF synergistically enhance intracellular accumulation of CAZ─by facilitating its entry through the widened OmpF porin and promoting its active uptake across the cytoplasmic membrane. This dual-mechanism strategy provides a novel two-pronged approach to overcoming CAZ resistance. Our findings underscore the potential of targeting bacterial metabolic pathways to restore susceptibility and extend the utility of existing antibiotics against resistant pathogens.