Blue light-activated berberine-gentamicin combination breaks down biofilms in diabetic foot ulcers

Diabetic foot ulcers (DFUs) represent a significant global burden, associated with high morbidity and increased mortality. More than half of DFUs become infected by polymicrobial communities, in which Pseudomonas aeruginosa and Staphylococcus aureus form resilient biofilms. Antimicrobial photodynamic inactivation (aPDI) using blue light is promising, its efficacy against polymicrobial biofilms remains suboptimal in infected DFUs. This study evaluated, for the first time, the activity of a berberine-gentamicin (Ber-Gen) combination under blue light photoactivation against dual-species P. aeruginosa MJMC568-A and S. aureus MJMC568-B biofilms, both isolated from a DFU patient. First, the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) for each agent against pre-formed dual-species biofilms were determined. Ber and Gen alone did not reach MBIC or MBEC at concentrations <2000 µg mL^-1, but in combination, MBIC values decreased two-fold to 1000 µg mL^-1 for Ber and 1024 µg mL^-1 for Gen. The combinatorial effect was assessed by checkerboard (CKB), with Ber-Gen resulting in a synergistic effect for MBIC values. The optimised concentrations from CKB were tested under one, two, and three irradiation cycles (with a 24 h interval between irradiation cycles) of blue light at 30 mW cm^-2 for 10 min per cycle (18 J cm^-2). Antibiofilm activity was quantitatively assessed by biomass (crystal violet), metabolic activity (alamar blue), and culturability (colony-forming unit (CFU cm^-2) counts). Photoactivated Ber-Gen produced strong reductions in biomass, metabolic activity, and culturability after one cycle (≈50%, ≈70%, and ≈5 log CFU cm^-2, respectively), near-complete eradication after two cycles (≈60%, ≈80%, and ≈6 log CFU cm^-2, respectively), and a further effect after three cycles (≈90%, ≈95%, and ≈10 log CFU cm^-2, respectively). Regrowth assays showed full recovery after one cycle, about half recovery after two, and less than 10% recovery after three cycles. Mechanistic assays on the antibiofilm effect included measurement of reactive oxygen species (ROS) by fluorometry, membrane integrity by flow cytometry and confocal microscopy, matrix components by confocal microscopy, spectrophotometric and fluorometric assays, and architecture by optical coherence tomography. Biofilm structure was markedly disrupted, with strong reductions in thickness, extracellular matrix components such as proteins, polysaccharides, and eDNA. These structural changes coincided with a decrease in biofilm cells' membrane integrity and increased ROS production. Overall, Ber-Gen-mediated blue light aPDI exhibits strong activity against dual-species biofilms of P. aeruginosa and S. aureus.