EFFECTS OF ANTIMICROBIAL PHOTODYNAMIC THERAPY IN BACTERIAL BIOFILMS

Antimicrobial resistance (AMR) is a major global health threat, further complicated by the ability of Staphylococcus aureus to form biofilms with an extracellular polymeric substance (EPS) matrix that protects embedded cells and contributes to antimicrobial tolerance. Antimicrobial photodynamic therapy (aPDT) has emerged as a promising biophotonic approach to target biofilm-associated bacteria. This study evaluated the effects of curcumin-mediated aPDT combined with blue light (450 nm) on the viability, biomass, biochemical composition, and antimicrobial susceptibility of S. aureus biofilms. Biofilms of S. aureus strain CBAM0026 were developed for 48 h for viability and antimicrobial susceptibility assays and for 72 h for spectroscopic analysis. Curcumin was evaluated at 40, 80, and 160 µM following 30 min of dark incubation. Biofilms were irradiated using a Biotable^® device at 450 nm, with an irradiance of 27 mW/cm² for 24 min 41 s, corresponding to a radiant exposure of 40 J/cm². Biofilm viability was assessed by CFU/mL counting, while total biomass was evaluated using crystal violet staining. Fourier-transform infrared (FTIR) spectroscopy coupled with hierarchical cluster analysis (HCA) was used to investigate biochemical changes. The antimicrobial susceptibility of cells recovered after treatment was determined by minimum inhibitory concentration (MIC) assays. The highest curcumin concentration (160 µM) combined with blue light produced a 2.5-log¹⁰ reduction in bacterial viability. Crystal violet staining indicated that residual biofilm biomass remained after treatment, despite the marked reduction in viable cells. FTIR analysis revealed biochemical alterations in lipid (2800–3100 cm⁻¹), protein (1500–1800 cm⁻¹), and carbohydrate (900–1200 cm⁻¹) regions. HCA showed distinct spectral clustering of the aPDT-treated group, particularly in the carbohydrate region, suggesting alterations in EPS-associated biochemical components. Moreover, aPDT increased the antimicrobial susceptibility of recovered cells, with reductions in MIC values for oxacillin, gentamicin, and erythromycin. Notably, erythromycin MIC decreased from 512 to 8 µg/mL. These findings indicate that curcumin-mediated aPDT can substantially reduce S. aureus biofilm viability and induce biochemical alterations in the biofilm matrix, while increasing the susceptibility of recovered cells to conventional antibiotics. Thus, aPDT represents a promising adjuvant approach for targeting biofilm-associated S. aureus.