Enhancing antimicrobial photodynamic inactivation through natural alkaloid-antiseptic synergy against...

Chronic wound infections are a major healthcare challenge, especially with the continued rise of antibiotic resistance. Antibacterial photodynamic inactivation (aPDI) is a localized, non-antibiotic approach in which light-activated photosensitizers (PSs) generate reactive oxygen species (ROS) that damage essential bacterial structures. Natural PSs, including harmine (HA), riboflavin 5'-phosphate (RB), palmatine (PM), and ferulic acid (FA), have gained attention for their bioactivity and photoreactivity. This study investigated for the first time the potential of combining these natural PSs with the membrane-active antiseptics, octenidine dihydrochloride (OCT), polyhexamethylene biguanide (PHMB), and hydrogen peroxide (H₂O₂), to improve aPDI efficacy against a clinical Staphylococcus aureus strain (MJMC568-B) isolated from a diabetic foot ulcer. Antibacterial activity of PSs and antiseptics, alone and in combination, was assessed by broth microdilution (minimum inhibitory and bactericidal concentration (MIC/MBC) determination), checkerboard, and disk diffusion assays. All PSs, except RB, demonstrated intrinsic antibacterial activity, and most combinations showed indifference under non-irradiated conditions. Photodynamic activity was evaluated using LED irradiation at 420 nm (30 mW cm^-2; 9.23-27.68 J cm^-2) for RB and PM, and 365 nm (5.5 mW cm^-2; 4.95-19.90 J cm^-2) for HA and FA. After photoactivation, specific PS-antiseptic combinations achieved complete bacterial inactivation (≈8 log (CFU mL^-1) reduction in culturability) at concentrations below individual MBCs: HA (100 µg mL^-1)-OCT (0.489-0.978 µg mL^-1) and HA (25 µg mL^-1)-PHMB (7.81 µg mL^-1) at 18.45 J cm^-2, and PM (200 µg mL^-1)-OCT (1.955 µg mL^-1) at 19.90 J cm^-2. FA-based combinations did not achieve bactericidal effects when irradiated. ROS generation capacity was evaluated using 2'7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and singlet oxygen production was evaluated using 9,10-anthracenediyl-bis (methylene) dimalonic acid (ADMA), which indicated an increase in oxidative stress when bacterial suspensions were irradiated as well as the capacity to produce singlet oxygen. Overall, HA and PM substantially enhanced the antibacterial performance of OCT and PHMB under photodynamic activation, enabling complete S. aureus inactivation. These findings highlight PS-antiseptic combinations as a promising non-antibiotic strategy for chronic wound management while reducing the required PS and antiseptic concentrations.