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

Two therapeutics regimens were performed (Fig. 1). In the first regimen, biofilms were topically treated after 21 h of development to evaluate the effect of agents on pre-formed biofilms (regimen one). In the second therapeutic regimen, the treatments were applied before microbial inoculation (i.e., the saliva-pellicle surface was treated) and after 8 h of biofilm growth to evaluate the preventive effect on biofilm assembly and accumulation (regimen two). The times of culture medium exchange and additional treatments were the same for both regimens until 67 h of each experiment.

The hydroxyapatite discs (surface area of 2.7 ± 0.2 cm², Clarkson Chromatography Products Inc., PA, USA) were placed vertically into custom made holders (two discs per holder) and sterilized by autoclaving. To generate saliva-coated hydroxyapatite (sHA), discs were hydrated during 20 min with sterilized MiliQ water transferred to 24-well plates containing human stimulated whole saliva filtered sterilized (0.22 μm low protein binding polyethersulfone membrane filter), and incubated (37 °C, 75 RPM, 1 h) [34]. Saliva collection was approved by the Institutional Ethical Committee (CAAE: 31,725,114.8.0000.5416). Each apparatus with discs was removed from saliva, dip-washed twice into adsorption buffer (AB buffer: 50 mM KCl, 1 mM KPO₄, 1 mM CaCl₂, 1 mM MgCl₂, in dd-H₂O, pH 6.5), being ready to for biofilm formation and/or treatment, depending on the treatment regimen.

The strains S. mutans UA159 and C. albicans SC5314 were used to prepare dual-species biofilms. The strains were grown on blood agar plates (48 h / 37 °C / 5% CO₂). Five to ten colonies of each microorganism were inoculated into 10 ml of culture medium: tryptone with yeast extract containing 1% of glucose (TYE+ 1% glucose) and incubated (37 °C / 5% CO₂). After 16 h, 1:20 dilutions of each starter culture were performed in TYE+ 1% glucose medium, and the cultures were grown until mid-log growth phase (OD_600nm 0.71 ± 0.27 for S. mutans, and 0.97 ± 0.03 for C. albicans). A dual-species inoculum was prepared with a defined population (S. mutans 2 × 10⁸ CFU/mL and C. albicans 2 × 10⁶ CFU/mL; CFU: colony forming units), in TYE with 1% sucrose [12]. In regimen one, sHA discs were placed into this inoculum, and discs were left undisturbed and incubated (37 °C/ 5% CO₂) to allow initial biofilm formation. In regimen two, sHA discs were topically treated, then incubated into biofilm inoculum (37 °C/ 5% CO₂), and after 8 h these biofilms were treated again. After 19 h of incubation for both regimens, the culture medium was replaced. This procedure was repeated twice daily (at 8 a.m. and 4 p.m.) and the pH of the spent medium was measured (Fig. 1). The biofilms were treated twice-daily 2 h after culture medium change (at 10 a.m. and 6 p.m.) until the end of the experiment (67 h-old biofilms).

The biofilms were exposed to the test agents four times in the regimen one and six times in the regimen two, by topically dripping 160–240 μL of each treatment during 1.5 min. Rinses of sHA discs/biofilms into saline solution (0.89% NaCl) were performed before and after treatments to remove any excess of culture medium or treatment. After treatments, discs were placed back into the culture medium in the 24-well plates. Biofilms assays were performed in duplicate in at least four different experiments (only CHX were done in quadruplicate in two experiments because there was less biofilm formation).

At the end of the experimental period (67 h), biofilms were dip-washed in saline solution. Each biofilm (disc) was transferred to a glass tube containing 1 mL of saline solution. Next, 1 mL of saline solution was used to wash the walls of each tube. The glass tubes with biofilms/discs were placed inside a Becker containing distilled water and subjected to water bath sonication (Kondortech Digital Ultrasonic Cleaner, Kondortech Indústria e Comércio Ltda., São Carlos, Brazil) during 10 min. A sterilized metal spatula was used to scrape off any remaining biofilm from each disc surface and the 2 mL of each biofilm suspension were transferred to a new 15 mL tube. Next, each glass tube was washed with 3 mL of saline solution, which were transferred to the tube containing the initial 2 mL, yielding a 5-mL total biofilm suspension per biofilm/disc. The 5 mL of each biofilm suspension were sonicated using a probe at 7 W during 30 s (Q125, Q Sonica). An aliquot of each suspension was used for a 10-fold serial dilution to determine the number of CFU by plating onto blood agar plates (37 °C/ 5%CO₂/ 48 h).

The remaining suspension volume of each biofilm was centrifuged (3942 rfc/20 min/4 °C), the supernatant was saved (supernatant 1) and each pellet was washed twice with sterile water generating supernatants 2 and 3. Three supernatants (totaling 10 mL) were used to quantify water soluble polysaccharides (WSP). Each biofilm pellet was suspended in 2.6 mL of MiliQ water. Each biofilm suspension was used to analyze insoluble dry weight (biomass) and alkali soluble or water-insoluble polysaccharides (ASP). The biofilm aliquot for ASP extraction was dried (Speed Vac Concentrator RVC 2–18 CD Plus, Christ). The resulting pellet was weighted and used to extract ASP using 1 N NaOH (0.3 mL of 1 N NaOH per 1 mg of biofilm dry weight). The quantification of WSP and ASP was performed using phenol-sulfuric acid colorimetric assay with glucose as standard [35].