Outer membrane stiffness gates multidrug tolerance

Bactericidal antibiotics initiate killing through class-specific target damage, yet the cellular properties that determine whether antibiotic-induced damage remains reversible or progresses to irreversible death remains unclear. Here, using kanamycin-centered evolution in Escherichia coli , we identified a multidrug-tolerant mutant that exhibits increased survival across aminoglycosides, β-lactams, quinolones and polymyxins without altered minimum inhibitory concentrations. We demonstrate that mechanistically distinct antibiotics converge on outer membrane destabilization, revealing a shared downstream vulnerability during killing. Elevated outer membrane stiffness limits antibiotic-induced envelope destabilization, thereby gating multidrug tolerance. Orthogonal chemical and physical perturbations further established a quantitative relationship between outer membrane stiffness and antibiotic survival across drug classes. Our findings reveal outer membrane stiffness as a previously unrecognized physical basis of multidrug tolerance and suggest that modulating bacterial envelope mechanics may provide new opportunities for antimicrobial intervention.