Microscale spatial fragmentation promotes bacterial survival under antibiotics

Most bacteria inhabit fragmented microscale environments rather than well-mixed systems in which antibiotic efficacy is typically defined. Here, we show that microscale spatial fragmentation alone can deterministically promote bacterial survival under antibiotic exposure. A coarse-grained theoretical model predicts that, at fixed bulk cell density and antibiotic concentration, fragmentation generates refuges through two coupled mechanisms: slower growth, which reduces susceptibility to growth-dependent antibiotics, and a finite antibiotic-per-cell constraint that lowers the effective intracellular antibiotic concentration. Using a microdroplet platform spanning five orders of magnitude in volume, we experimentally confirm these predictions for {beta}-lactams; we further demonstrate that the same fragmentation-mediated protection extends to antibiotics with distinct modes of action. Spatial fragmentation thus creates robust, non-genetic antibiotic refuges governed by physical constraints rather than resistance or collective protection, with implications for bacterial persistence in natural and clinical environments and for antibiotic design.