Magnetic Resonance Reveals Radical Stability in Native Biofilms and Its Role in Bacterial Resistance

Bacterial cells embedded in biofilms exhibit enhanced antimicrobial resistance compared with planktonic cells, contributing to persistent infections and failure of conventional antibiotic treatments. This resistance is influenced by the extracellular matrix (ECM), a heterogeneous network of polysaccharides, proteins, and lipids surrounding embedded cells. However, how the ECM regulates the accessibility of redox‐active small molecules remains poorly understood. Here, we combine electron paramagnetic resonance (EPR), solid‐state NMR (ssNMR), and dynamic nuclear polarization (DNP) ssNMR to determine how native Pseudomonas fluorescens Pf0‐1 colony biofilms differentially reduce nitroxide radicals across morphologies. EPR reveals that radical reduction depends on biofilm morphology, hydration, and ECM composition. Wild‐type biofilms exhibit slower nitroxide reduction than planktonic cells, while isolated ECM and dehydrated biofilms show negligible reduction, highlighting the importance of cellular accessibility. ssNMR identifies preferential spatial proximity of nitroxides to ECM polysaccharides and lipids, while DNP ssNMR reveals increasing polysaccharide content across biofilm morphologies that correlates with slower reduction kinetics. These findings support a model of protection in which the ECM provides the barrier to nitroxide access, and cellular reduction is the final step. Our integrated magnetic resonance approach provides molecular insight into how biofilm composition and structure determine the fate of therapeutically relevant redox‐active small molecules.