Antimicrobial photodynamic inactivation of Pseudomonas aeruginosa biofilms by a multifunctional triple combination of natural photosensitisers, gentamicin, and colistin

Antimicrobial photodynamic inactivation (aPDI) is a promising strategy for combating chronic wound infections, particularly those involving biofilms. However, its efficacy against Pseudomonas aeruginosa biofilms is limited by poor light penetration and photosensitisers through the biofilm matrix. This study explores, for the first time, the combination of subinhibitory colistin (Col) concentrations with berberine/curcumin (Ber/Cur) and gentamicin (Gen) to enhance aPDI activity against P. aeruginosa biofilms, including a clinical strain from a diabetic foot ulcer. Membrane integrity changes induced by Col were assessed by flow cytometry with propidium iodide. Synergistic concentrations of Ber/Cur-Gen-Col were determined via checkerboard assay. The effects of blue light (420 nm, 30 mW/cm 2 , 10 min) on these combinations were evaluated for both biofilm prevention and eradication assays. For established biofilms, one or three irradiation cycles were applied at 24 h intervals, followed by an assessment of regrowth after 48 and 72 h. Biomass, metabolic activity, and culturability were quantified using crystal violet, resazurin, and colony-forming units (CFU). The mode of action was investigated via ROS production, membrane disruption, and biofilm structural changes using fluorometry, microscopy, and optical coherence tomography. Col at subinhibitory concentrations reduced membrane integrity (≈30 %), enhancing Ber/Cur-Gen efficacy. Photoactivated Ber/Cur-Gen-Col inhibited biofilm formation (>90% reduction in biomass and metabolic activity and 6-log reduction in CFU). Against pre-formed biofilms, this combination achieved a 7-log reduction in CFU and a 90% reduction in biomass and metabolic activity. Three irradiation cycles sustained inhibition for up to 72 h. Ber-Gen-Col showed superior performance, highlighting the synergistic potential of subinhibitory Col in enhancing aPDI as a promising strategy for managing persistent wound infections.