Elucidation of Radical Degradation in Native Biofilms by EPR Sheds Light on Bacterial Resistance and Efficient DNP...

Bacterial biofilms are complex communities protected in an extracellular matrix (ECM) composed of polysaccharides, extracellular DNA, proteins, lipids, and other molecules. These protected bacteria typically manifest enhanced antimicrobial resistance (AMR) which presents a major challenge in treating chronic infections. Here, we employ a combination of electron paramagnetic resonance (EPR), solid-state NMR (ssNMR), and dynamic nuclear polarization (DNP) ssNMR to investigate radical stability within native Pseudomonas fluorescens Pf0-1 colony biofilms towards efficient hyperpolarized DNP ssNMR applications. EPR measurements reveal that the native ECM is the primary contributor to radical reduction, whereas other biofilm components, such as planktonic cells, isolated ECM, and dried biofilms show minimal activity. Radical reduction rates vary with biofilm morphology and composition. ssNMR identifies both rigid and flexible polysaccharides and lipids within the ECM as primary radical interaction sites. These findings support a mechanism in which the ECM not only serves as a physical barrier but also has reductive activity that protects against xenobiotics. Importantly, we demonstrate that potassium ferricyanide preserves EPR signal intensity and radical lifetime in native biofilms, offering a promising biocompatible mitigation strategy. Our findings quantify and pinpoint the origins of the reductive nature of bacterial biofilms and provide a solid framework for improving radical stability in native biological systems for high efficiency structural studies. This work enables high efficiency DNP ssNMR on native biofilms and sets the stage for high-resolution measurements of structure-function relations in these medically relevant, complex biological assemblies.