Dismantling the eDNA-mediated H₂S barrier with biohybrids against refractory biofilm infections

The spatiotemporal distribution of gaseous mediators is critical for maintaining physiological functions. Biofilms both contain gas-producing bacteria and possess complex architectures that reshape gaseous mediator distribution. However, whether biofilm pathogenicity is linked to such gaseous regulation remains unclear. In this study, we reveal that extracellular DNA (eDNA) within the biofilm matrix contributes to the local retention and enrichment of H₂S. This enrichment stabilizes eDNA, strengthens biofilm barriers, impedes gas clearance, promotes bacterial persistence, and induces macrophage immunosuppression, creating a reciprocally protective defensive loop. To dismantle this defense, we engineered a stepwise-responsive biohybrid system in which probiotic-derived engineered minicells actively target hypoxic biofilm regions, while H₂S-triggered nanoparticle degradation enables deeper matrix penetration and concurrent H₂S scavenging. The retained minicells then catalyze in situ glucose oxidation to generate H₂O₂, which, together with Fe²⁺-mediated Fenton-like reactions, degrades eDNA and collapses the biofilm. This strategy eradicates persistent bacteria, reverses immunosuppression, and provides a therapeutic strategy to reduce recurrence and optimize refractory infection treatments.