Piperidine-derived resensitizers revive aminoglycoside capacity against persister cells of Pseudomonas aeruginosa in vivo.

Bacterial persister cells represent a major barrier to antimicrobial therapy by surviving lethal antibiotic exposure in a transient, non-genetic state and serving as a reservoir for recurrent infection. Although persister resensitization has emerged as a promising therapeutic concept, its mechanistic basis and in vivo applicability remain poorly defined. Here, we demonstrate that the piperidine-derived compound C10 functions as an antibiotic-resensitizing agent. In persister-state Escherichia coli, C10 increased ribosomal RNA promoter B-driven green fluorescent protein expression and chemical uptake, suggesting partial transcriptional and metabolic reactivation in persister cells. In clinically relevant persister cells of Pseudomonas aeruginosa, C10 restored aminoglycoside susceptibility and revived bactericidal capacity resulting in improved therapeutic efficacy in multiple murine infection models such as systemic sepsis, neutropenic thigh infection, and pneumonia. To improve translational feasibility, medicinal chemistry optimization identified KP35, a derivative with reduced mammalian cytotoxicity while retaining resensitization activity. Transcriptomic profiling revealed selective activation of nitrate respiration-associated pathways, consistent with respiratory and metabolic priming rather than global growth reactivation. Collectively, our findings establish chemical persister resensitization as a therapeutically actionable strategy for restoring antibiotic efficacy against bacterial persister cells and provide mechanistic insight into respiratory rewiring-mediated aminoglycoside resensitization.