ABSTRACT
Candida albicans and Streptococcus mutans interaction in the presence of dietary sucrose yields a complex biofilm with an organized and structured extracellular matrix that increases the tolerance to environmental stress, including antimicrobials. Both species are found in severe early childhood caries lesions. Thus, compounds 4'-hydroxychalcone (C135) (flavonoid intermediate metabolites), tt-farnesol (Far) (terpenoid), and sodium fluoride (F) were tested either isolated or combined as topical treatments (5 min twice daily) against C. albicans and S. mutans dual-species biofilms grown on saliva-coated hydroxyapatite discs. The biofilms were evaluated for gene expression, microbial population, biochemical components, and three-dimensional (3D) structural organization via confocal microscopy and scanning electron microscopy (SEM). The cytotoxicity of formulations was tested on the keratinocyte monolayer. C135 + Far + F promoted lower gene expression of fungal genes associated with ß-glucan synthesis (BGL2, FKS1) and remodeling (XOG1, PHR1, PHR2), oxidative stress (SOD1), and drug tolerance (CDR1, ERG11) and higher expression of bacterial nox1 (oxidative and acidic stress tolerance). C135 + Far yielded less insoluble exopolysaccharides, biomass, and proteins (insoluble portion) and lower expression of BGL2, ERG11, SOD1, and PHR2. C135 + F, C135 + Far + F, and C135 rendered lower biomass, thickness, and coverage percentage (confocal microscopy). C135 + Far and C135 + Far + F maintained C. albicans as yeast morphology (SEM). Therefore, the formulations with C135 affected fungal and bacterial targets but exerted a more pronounced effect against fungal cells.
ABSTRACT
BACKGROUND: Dental caries is considered a multifactorial disease, in which microorganisms play an important role. The diet is decisive in the biofilm formation because it provides the necessary resources for cellular growth and exopolysaccharides synthesis. Exopolysaccharides are the main components of the extracellular matrix (ECM). The ECM provides a 3D structure, support for the microorganisms and form diffusion-limited environments (acidic niches) that cause demineralization of the dental enamel. Streptococcus mutans is the main producer of exopolysaccharides. Candida albicans is detected together with S. mutans in biofilms associated with severe caries lesions. Thus, this study aimed to determine the effect of tt-farnesol and myricetin topical treatments on cariogenic biofilms formed by Streptococcus mutans and Candida albicans. METHODS: In vitro dual-species biofilms were grown on saliva-coated hydroxyapatite discs, using tryptone-yeast extract broth with 1% sucrose (37 °C, 5% CO2). Twice-daily topical treatments were performed with: vehicle (ethanol 15%, negative control), 2 mM myricetin, 4 mM tt-farnesol, myricetin + tt-farnesol, myricetin + tt-farnesol + fluoride (250 ppm), fluoride, and chlorhexidine digluconate (0.12%; positive control). After 67 h, biofilms were evaluated to determine biofilm biomass, microbial population, and water-soluble and -insoluble exopolysaccharides in the ECM. RESULTS: Only the positive control yielded a reduced quantity of biomass and microbial population, while tt-farnesol treatment was the least efficient in reducing C. albicans population. The combination therapy myricetin + farnesol + fluoride significantly reduced water-soluble exopolysaccharides in the ECM (vs. negative control; p < 0.05; ANOVA one-way, followed by Tukey's test), similarly to the positive control. CONCLUSIONS: Therefore, the combination therapy negatively influenced an important virulence trait of cariogenic biofilms. However, the concentrations of both myricetin and tt-farnesol should be increased to produce a more pronounced effect to control these biofilms.
