Blue-light-activated berberine-gentamycin inhibits and eradicates Staphylococcus aureus biofilms by promoting oxidative stress and structural disruption

Abstract

Diabetic foot ulcers (DFUs) remain a major therapeutic challenge due to persistent biofilms, with Staphylococcus aureus as the predominant pathogen. Antimicrobial photodynamic inactivation (aPDI) offers an alternative to antibiotics but shows limited efficacy against biofilms. This study introduces a novel combinative strategy in which berberine (Ber) acts simultaneously as a photosensitiser and an adjuvant to restore gentamicin (Gen) activity, enabling enhanced aPDI efficacy against S. aureus biofilms. Two clinical strains, a methicillin-resistant (MRSA, MJMC568-B) and a methicillin-susceptible (MSSA, CECT 976), were used. The Ber-Gen interaction was first evaluated by checkerboard assay, followed by blue light activation (420 nm, 30 mW/cm2, 10 min, 18 J/cm2) to test biofilm prevention and eradication. In pre-formed biofilms, single or triple irradiation cycles (every 24 h) were applied, and regrowth was monitored for up to 72 h, allowing assessment of post-treatment biofilm recovery dynamics. Quantitative analyses included biomass (crystal violet), metabolic activity (resazurin), and culturability (CFU/cm2). Mechanistic insights were obtained through reactive oxygen species (ROS) quantification, flow cytometry, confocal microscopy, and optical coherence tomography. Ber restored Gen susceptibility, showing additive effects in MRSA and synergism in MSSA. Photoactivated Ber-Gen prevented biofilm formation with over 90% reduction in biomass, metabolic activity, and culturability (6-log CFU/cm2). In mature biofilms, reductions reached 7-log CFU/cm2 with over 90% reduction in biomass and metabolic activity. While single irradiation allowed partial regrowth, triple cycles prevented recovery for 72 h. Mechanistic analyses confirmed enhanced ROS generation, membrane disruption, and biofilm matrix disruption. These findings demonstrate that Ber-Gen-mediated aPDI suggests a potential light-activated therapeutic strategy, supporting a dual-functionally integrated approach that combines antibiotic potentiation with photodynamic activity to overcome biofilm tolerance and effectively suppress post-treatment regrowth in chronic DFUs.