The Synergy Between a Silver-Ruthenium Antimicrobial and Aminoglycosides Is Based on Severe Macromolecular Damage.

The rise of multidrug-resistant (MDR) bacterial pathogens, including uropathogenic Escherichia coli (UPEC), highlights the urgent need for alternative treatment strategies to restore antibiotic efficacy. The silver-ruthenium antimicrobial AGXX exerts potent bactericidal effects through the production of reactive oxygen species (ROS); however, its potential synergy with antibiotics has not been thoroughly investigated. Here, we show that sublethal concentrations of AGXX strongly enhance aminoglycoside-mediated killing across a diverse panel of Gram-negative and Gram-positive MDR clinical isolates, including highly aminoglycoside-resistant strains. Combinational treatments significantly reduced the effective concentrations of gentamicin, tobramycin, kanamycin, and amikacin required to kill bacteria. Mechanistic analyses revealed that AGXX/aminoglycoside co-treatments induce pronounced intracellular ROS accumulation, resulting in an imbalanced proteostasis due to extensive protein aggregation and DNA damage. Scavenging ROS abolished synergistic killing, establishing oxidative imbalance as the primary driver of the synergy between both antimicrobials. We further identified polyphosphate as a key bacterial defense mechanism that mitigates ROS accumulation, proteotoxicity, and genotoxic stress during combinational treatment. Moreover, AGXX-aminoglycoside synergy was preserved in an artificial urine medium and across clinical UPEC isolates, underscoring its relevance to urinary tract infections. Together, these findings position AGXX as a potent aminoglycoside adjuvant that restores antibiotic efficacy through ROS-driven macromolecular damage, supporting its development for combination therapies against MDR bacterial infections.