Histatin–Photodynamic Therapy for Polyene-Resistant Biofilms in a 3D Model

Oral candidiasis is primarily caused by <i>Candida albicans</i>; however, bacteria such as <i>Streptococcus mutans</i> can also coexist and cooperate within the same biofilm. This study evaluated the combined effects of Histatin 3 (His3) and Histatin 5 (His5) with antimicrobial photodynamic therapy (aPDT) using a 3-dimensional oral epithelium model infected with mixed biofilms of <i>C. albicans</i> (polyene-resistant strains) and <i>S. mutans</i>. The tissues, divided into groups (His3 + aPDT, His5+aPDT, His3, His5, or aPDT), were first treated with the salivary proteins His3 or His5 (2 h, 37 °C, 60 rpm/min) followed by infection with mixed biofilms. After 24 h of biofilm formation, aPDT was applied (PDZ: 200 mg/L; LED light: 660 nm with 50 J/cm²) using intermittent irradiation cycles (<i>n</i> = 9/group). Biofilm viability (CFU/mL), total proteins (intracellular and extracellular), and proteomic analysis were performed. Biocompatibility and tissue invasion were also assessed by MTT assays and histological analysis, respectively. His3+aPDT and His5+aPDT reduced mixed biofilm viability by 67% (<i>P</i> = 0.0038) and 65% (<i>P</i> = 0.0062), respectively. His3 and His5 alone promoted a 35% to 45% (<i>P</i> = 0.018; <i>P</i> = 00.24) reduction, while aPDT alone reduced by 50% to 55% (<i>P</i> = 0.022; <i>P</i> = 0.037). Combined treatments enhanced efficacy by ~30% compared with individual treatments. Histological evaluation also confirmed reduced biofilm penetration and hyphal formation, particularly with His5+aPDT. Proteomic analysis of the proteins from <i>C. albicans</i> revealed the downregulation of structural and transport proteins, which can likely weaken the biofilm. The combination of His3 and His5 with aPDT enhances antimicrobial efficacy against resistant multispecies biofilms while maintaining biocompatibility. By reducing biofilm viability and compromising proteins, this approach provides a promising alternative for treating oral candidiasis associated with polyene-resistant <i>C. albicans</i> species.