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 β-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. This study reveals that microscale habitat fragmentation can protect bacteria from antibiotics. In tiny droplets, confinement slows growth and limits the antibiotic available per cell, lowering the effective dose cells are exposed to.