Salivary proteins-enhanced antimicrobial photodynamic therapy: Overcoming three distinct cultures of resistant mixed biofilms

BACKGROUND: Denture stomatitis is frequently associated with biofilm formation by Candida albicans, which can coexist with Streptococcus mutans. Current treatments face several limitations, including the emergence of resistant strains and the persistent impact of biofilm formation on antimicrobial efficacy. The salivary proteins Histatin 3 (His3) and Histatin 5 (His5) have demonstrated effectiveness against C. albicans single-species biofilms. However, their efficacy against mixed-species biofilms, particularly those involving S. mutans and antifungal-resistant C. albicans strains, remains poorly understood. OBJECTIVES: To investigate the efficacy of combining His3 and His5 with antimicrobial photodynamic therapy (aPDT) against mixed biofilms containing polyene-resistant (CaP+Sm), wild-type (CaW+Sm), and fluconazole-resistant (CaF+Sm) and S. mutans (Sm) on acrylic resins. METHODS: 48-hour mixed biofilms (37 °C/5 % CO₂) were formed on acrylic resin disks treated with His3 and His5 (2h/37 °C/120 rpm). Biofilms were subjected to aPDT using Photodithazine (200 mg/L) followed by 30 min of red LED irradiation (660 nm, 50 J/cm²). Viability was assessed by colony-forming units (CFU), while ECM components (proteins, alkali-soluble polysaccharides (ASP), water-soluble polysaccharides (WSP), and extracellular DNA (eDNA)) were analyzed (n = 6). RESULTS: Complete eradication of mixed biofilms was observed in CaW+Sm and CaF+Sm treated with His3+aPDT and His5+aPDT, while CaP+Sm showed a 98 % reduction in total microbiota. For CaP+Sm, combined His3+aPDT and His5+aPDT significantly reduced biofilm viability, achieving up to 99 % reduction in C. albicans and 80 % in S. mutans. ECM components, including proteins, ASP, WSP, and eDNA, were notably reduced, particularly in CaW+Sm and CaF+Sm cultures. CONCLUSION: Combining Histatins with aPDT demonstrated superior efficacy compared to individual treatments, disrupting mixed biofilms of C. albicans and S. mutans and significantly reducing viability. CLINICAL SIGNIFICANCE: Histatins with antimicrobial photodynamic therapy (aPDT) reduce biofilm viability and disrupt key components of extracellular matrix in resistant biofilm that contribute to the persistence of infections in denture stomatitis.