A simple osmotic intervention suppresses nanoparticle-induced bacterial persistence during environmental disinfection

Persister cells that survive disinfection can recover and drive bacterial regrowth, thereby limiting long-term treatment efficacy and remaining a challenge even for nano-enabled water treatment. Here, we subjected Escherichia coli to 14 cycles of high-dose exposure to silver nanoparticles (AgNPs) and found that repeated treatment increased persister levels by nearly 1,000-fold after recovery and regrowth. During the NP challenge, evolved cells displayed altered K⁺ flux, reduced cell volume, softened envelope mechanics, and envelope remodeling, consistent with an osmotic-stress-associated adaptive state. Following AgNP removal and regrowth, survivors from evolved populations exhibited lower ATP levels, longer lag times, and elevated guanosine tetraphosphate (ppGpp) levels compared with survivors from the wildtype population, indicating that AgNP adaptation drives cells into a persistence-prone recovery state rather than simply improving acute survival. Similar osmotic stress-associated responses were also induced by cyclic exposure to Fe₃O₄, CeO₂, and SiO₂ NPs, indicating convergence on a common envelope-interface mechanism across chemically distinct nanomaterials. Importantly, imposing an osmotic downshift during various NP exposure markedly reduced subsequent persister formation, bringing it close to baseline levels. This intervention effectively reduced persister enrichment in tap water and river water, but was much less effective in artificial seawater, highlighting osmolarity as a key determinant of NP-induced persistence. Together, these findings show that nano-enabled disinfection can unintentionally promote persistence through challenge-phase osmotic adaptation and identify osmotic-context manipulation as a simple and scalable strategy to limit residual survival and post-treatment regrowth.