Growth in confinement promotes <i>Pseudomonas aeruginosa </i> tolerance to antibiotics

Bacteria often proliferate within confined spaces imposed by host tissues, extracellular matrices, or their own biofilms. In such environments, cells press against surrounding materials and experience elevated mechanical stress, but whether these forces influence pathogen physiology and fitness remains unclear. Here, we show that Pseudomonas aeruginosa adapts to mechanical confinement by increasing resilience to antibiotics. Using synthetic hydrogels of tunable stiffness that restrict physical expansion without limiting nutrient access, we demonstrate that growth in elastic materials reduces P. aeruginosa sensitivity to multiple clinically relevant antibiotics in a stiffness-dependent manner. Although slower growth contributes to this decreased susceptibility, Tn-seq under antibiotic treatment identified key regulators of mechanically induced tolerance. We find that active efflux mediated by sodium–proton Sha antiporters, together with protective remodeling of the bacterial membrane, enhances the resilience of confined populations without impacting growth. These findings reveal that P. aeruginosa adapts to mechanical stress in ways that may promote treatment failure even in the absence of intrinsic antibiotic resistance.