Antimicrobial Mechanism of Phenalen-1-one-mediated Antimicrobial Photodynamic Therapy in Gram-positive Bacteria

Objectives: In view of increasing resistance against antibiotics, antimicrobial photodynamic therapy (aPDT) may be a promising alternative in dentistry. The aim of this study was to evaluate the effects of aPDT with the phenalene-1-one derivative SAPYR on membrane potential and metabolic activity when applied to biofilms of Actinomyces naeslundii and Streptococcus mutans. Furthermore, the effects of aPDT treatment on the intracellular formation of reactive oxygen species (ROS) were investigated.
Methods: Monospecies biofilms (Actinomyces naeslundii DSM43013; Streptococcus mutans DSM2052) were cultured under aerobic conditions for 48h followed by treatment with the photosensitizer SAPYR at three concentrations (50, 100, 500μM) and chlorhexidine (CHX, 0.2%) as a positive control for incubation periods 5, 10, 20 and 30min and subsequent irradiation for 10min (Waldmann PIB 3000; l_em = 360-600nm; 50mW/cm²; 30J/cm²). Control samples were treated with dH₂O and kept in dark for the same time. Antibacterial efficacy was determined by CFU assay. Membrane potential was investigated with flow cytometry using DiBAC₄(3). Metabolic activity was evaluated by XTT-assay. The formation of intracellular ROS was investigated fluorometrically using H₂DCF-DA. All experiments were performed at least 3 times in duplicates.
Results: CHX and aPDT showed an antimicrobial killing of ≥5log₁₀ CFU. Changes in membrane potential were found after treatment with CHX but not with aPDT. XTT-assay showed a reduction in metabolic activity following aPDT (60-80%) and CHX (100%). Fluorometric assay showed increased formation of intracellular ROS in both species after aPDT, but only for A. naeslundii after CHX treatment.
Conclusions: APDT was successful in antimicrobial killing, reduced bacterial metabolic activity and increased intracellular ROS, but did not result in changes of membrane potential. For deeper understanding of antimicrobial mechanism of aPDT, further investigations on changes in protein and gene expression after treatment with aPDT are necessary.