Histatins Influence on Candida Albicans and Streptococcus Mutans Dual-Species Biofilm

Objectives: This study assessed the effect of the antimicrobial peptides DR9-RR14, RR14, Histatin 3 and Histatin 5 in Candida albicans and Streptococcus mutans dual-species biofilm formed under acrylic resin and their extracellular matrix (ECM), as well, their cytotoxicity in oral gingival fibroblasts (FGH).
Methods: Initially, the biofilm inhibitory concentration (BIC-2) was evaluated (n=9/group). Biofilms containing C. albicans ATCC 10231 and S. mutans UA159 were formed (24h/37 °C /5% CO₂) and exposed to dilutions ranging from 0 to 4096 µM of DR9-RR14, RR14, Histatin 3 and Histatin 5. The BIC-2 quantification was performed by colony-forming units per milliliter (CFU/mL). Then, pellicle was formed under acrylic resins slabs with each peptide’s BIC-2 (200 μL/well/slab) and incubated (2h/37^oC/60 rpm): DR9–RR14 (2048 µM), RR14 (1024 µM), Histatin 3 (256 µM) and Histatin 5 (512 µM) or NaCl (pH 7.2). Dual-species biofilms were formed under each acrylic resin (48h/37^oC/10% CO₂). The biofilm cell viability it was assessed using CFU/mm² (n=9/group). The biofilm ECM were evaluated by biomass (dry-weight and insoluble weight), protein, alkali-soluble polysaccharides (ASP), water-soluble polysaccharides (WSP) and extracellular-DNA (eDNA) (n=9/group). Human gingival fibroblast cells (FGH) were quantified (1.2 X 10⁵ cells/well) for the experiments (37^oC/5%/CO₂) with each peptide BIC-2 using alamarBlue (n=12/group).
Results: Among the peptides evaluated, histatin 3 was the most promising, having the lowest BIC-2 (256 µM) against dual-biofilm (CFU/mL), followed by Histatin 5 (512 µM), RR14 (2048 µM) and DR9-RR14 (4096 µM). Histatin 3 reduced the CFU/mm² (46%), insoluble-dry weight (55%), protein (63%), eDNA (64%) and WSP (46%), affecting vital constituents of the ECM. Histatin 3 also was biocompatible with FGH cells (13.9% reduction) and classified as “non-cytotoxicity” (<25%).
Conclusions: Histatin 3 prevented C. albicans and S. mutans biofilm adhesion and colonization on the acrylic resin, representing a promising alternative therapeutic approach for denture stomatitis.