Ultrasensitive Characterization of Native Bacterial Biofilms via Dynamic Nuclear Polarization-Enhanced Solid-State...

Bacterial biofilms are major contributors to persistent infections and antimicrobial resistance, posing significant challenges to treatment. However, obtaining high-resolution structural information on native bacterial biofilms has remained elusive due to the methodological limitations associated with analyzing complex biological samples. Solid-state NMR (ssNMR) has shown promise in this regard, but its conventional application is hindered by sensitivity constraints for unlabeled samples. In this study, we utilized high-sensitivity Dynamic Nuclear Polarization (DNP) ssNMR to characterize native Pseudomonas fluorescens colony biofilms. The ~75-fold sensitivity enhancement provided by DNP enabled structural characterization without isotope labeling or chemical/physical modification. We successfully collected 1D ¹³C/¹⁵N, and 2D ¹H-¹³C, ¹H-¹⁵N and ¹³C-¹³C ssNMR spectra within seconds, minutes or hours, facilitating the identification and quantification of biofilm extracellular matrix (ECM) components. Additionally, DNP ssNMR allowed quantitative detection of both flexible and rigid biofilm components by favorable freezing conditions. This study represents the first application of ultrasensitive DNP ssNMR to characterize a native bacterial biofilm, significantly expanding the capabilities of ssNMR for analyzing the composition and structure of a wide array of in vitro and ex vivo biofilms. The versatility of this approach will accelerate structure-guided efforts to combat infections caused by biofilm-forming microbes.