Effect of tt-farnesol and myricetin on in vitro biofilm formed by Streptococcus mutans and Candida albicans

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.

Keywords: Candida albicans; Cariogenic biofilm; Myricetin; Streptococcus Mutans; Topical treatment; tt-farnesol.

Fig. 1

Treatment regimens used for topical application. a Depicts the regimen one used to accesses the effect on pre-formed biofilms (treatments application starts at 19 h of biofilm development). b Depicts the regimen two used to evaluate the effect on biofilm assembly and accumulation (treatments application on saliva-coated hydroxyapatite discs before microbial inoculation)

Fig. 2

pH of spent media of treated biofilms. a Represents the data generated for regimen one, while (b) for regimen two. The pH values were measured when the culture media was changed. V: vehicle; F: tt-farnesol; M: myricetin; MF: tt-farnesol + myricetin; MF250: fluoride + tt-farnesol + myricetin; 250: fluoride (250 ppm); CHX: chlorhexidine. The data shown are averages, and error bars indicate the standard deviation. Statistical analysis is not depicted in the graphs

Fig. 3

Biomass of treated biofilms. a represents the data generated for regimen one, while (b) for regimen two. V: vehicle; F: tt-farnesol; M: myricetin; MF: tt-farnesol + myricetin; MF250: fluoride + tt-farnesol + myricetin; 250: fluoride (250 ppm); CHX: chlorhexidine. The data shown are averages, and error bars indicate the standard deviation

Fig. 4

Microbial population of S. mutans and C. albicans after treatment of biofilms. Data are presented as colony forming unit (CFU) per biofilm. a and c represent data generated for regimen one, while (b) and (d) correspond to regimen two. V: vehicle; F: tt-farnesol; M: myricetin; MF: tt-farnesol + myricetin; MF250: fluoride + tt-farnesol + myricetin; 250: fluoride (250 ppm); CHX: chlorhexidine. The data shown are averages, and error bars indicate the standard deviation

Fig. 5

Exopolysaccharides quantities in the ECM after treatments. Both water soluble (WSP) and water insoluble (ASP) exopolysaccharides in the extracellular matrix are represented in milligrams (mg). (a) and (c) represent data generated for regimen one, while (b) and (d) correspond to regimen two. Panels a and b depict WSP data and panels c and d show ASP data. V: vehicle; F: tt-farnesol; M: myricetin; MF: tt-farnesol + myricetin; MF250: fluoride + tt-farnesol + myricetin; 250: fluoride (250 ppm); CHX: chlorhexidine. The data shown are averages, and error bars indicate the standard deviation