Synergetic antimicrobial effect of chlorin e6 and hydrogen peroxide on multi-species biofilms

Antimicrobial photodynamic therapy (aPDT) has been considered as a potential alternative to antibiotics for the treatment of biofilm infections. There is evidence that an additional H₂O₂ enhances the antimicrobial efficacy of aPDT. However, the minimum H₂O₂ concentration to achieve this synergistic effect is unclear. A saliva-derived multi-species biofilm was treated with the photosensitizer chlorin e6 (Ce6, 50 µM), H₂O₂ (0.3, 3.3, 33.3 mM), or their combination for 5 min, followed by no irradiation or irradiation at 15 J (cm²)^-1 (λ = 450 nm or 660 nm), with or without oxygen. Biofilm viability and metabolic activity were evaluated. The combination of 33.3 mM H₂O₂ and Ce6-aPDT strongly enhanced antimicrobial efficacy compared with either component alone, irrespective of oxygen availability and irradiation wavelength. In particular, the combination resulted in a 6.6-log colony forming unit (CFU) reduction anaerobically under blue irradiation. This combination is a promising treatment for biofilm infections, especially those thriving in an anaerobic microenvironment.