The present in vitro study examined the effects of the quorum-sensing molecules farnesol and tyrosol on the development of Candida albicans biofilm in order to elucidate their role as novel adjuvants in oral hygiene. The investigation was conducted in C. albicans ATCC 10231 and C. albicans isolates from dentures and was performed in flat-bottomed 96-well polystyrene plates. Yeast growth and their capacity to form biofilms were evaluated following 24 and 48 h incubations at 37°C in Sabouraud broth supplemented with 0.001–3 mM farnesol and/or 1–20 mM tyrosol. Yeast growth was assessed by turbidimetry and biofilms were quantitated by crystal violet staining, under aerobic and anaerobic conditions. The viability of the fungal cells was controlled by the culture of planktonic cells and by examination of the biofilms using fluorescence microscopy following staining with fluorescein diacetate and ethidium bromide. Farnesol at 3 mM exerted a stronger action when added at the beginning of biofilm formation (>50% inhibition) than when added to preformed biofilms (<10% inhibition). Similarly, tyrosol at 20 mM had a greater effect on biofilm formation (>80% inhibition) than on preformed biofilms (<40% inhibition). Despite significant reductions in attached biomass, yeast growth varied little in the presence of the investigated molecules, as corroborated by the turbidimetry, culture of supernatants on solid culture medium followed by counting of colony-forming units and viability tests using fluorescence microscopy. At the highest tested concentration, the molecules had a greater effect during the initial phases of biofilm formation. The effect of farnesol during anaerobiosis was not significantly different from that observed during aerobiosis, unlike that of tyrosol during anaerobiosis, which exhibited slightly reduced yeast biofilm inhibition. In conclusion, the present study demonstrated the specific anti-biofilm effect, independent of fungicidal or fungistatic action, of farnesol and tyrosol, as tested in C. albicans ATCC 10231 and 6 strains isolated from dentures. Prior to suggesting the use of these molecules for preventive purposes in oral hygiene, further studies are required in order to clarify the metabolic pathways and cellular mechanisms involved in their antibiofilm effect, as well as the repercussions on the oral microbiome.

The present study investigated the in vitro effect of lysozyme (0–1,000 µg/ml) on Candida albicans (C. albicans) biofilm development. Investigations were conducted on C. albicans ATCC 10231 and on 10 clinical isolates from dentures. Strains were cultured aerobically at 37°C in Sabouraud broth. Yeast growth was evaluated by turbidimetry. Biofilm biomass was quantified on a polystyrene support by crystal violet staining and on acrylic surfaces by counts of colony forming units. Lysozyme affected biofilm formation to a greater extent than it affected growth. For the ATCC 10231 reference strain, lysozyme acted as a biofilm promotor on polystyrene at the highest concentration tested (1,000 µg/ml, non-physiological). When the reference strain was investigated on acrylic resin support, lysozyme acted as a significant biofilm promotor on rough resin, but less on smooth resin. The attached biomass in the presence of physiological concentrations of lysozyme (10–30 µg/ml) was significantly decreased compared with the hypothetical value of 100% using a one-sample t-test, but a comparison between the different lysozyme conditions using analysis of variance and post hoc tests did not reveal significant differences. In 10 wild strains, different patterns of biofilm formation on polystyrene were observed in the presence of lysozyme. Some strains, characterized by large amounts of biofilm formation in the presence of 1,000 µg/ml lysozyme, were poor biofilm producers at low concentrations of lysozyme. In contrast, some strains that were poor biofilm producers with a high lysozyme concentration were more inhibited by low concentrations of lysozyme. The present study emphasizes the need to develop strategies for biofilm control based on in vitro experiments, and to implement these in clinical trials prior to approval of hygiene products enriched with exocrine proteins, such as lysozyme. Further studies will extend these investigations to other Candida species, and to fungi and bacteria present in oral biofilms.

IntroductionUnder well-defined experimental conditions, and in the presence of hydrogen peroxide, lactoperoxidase produces stable iodine–thiocyanate complexes that have antimicrobial properties. A novel process was developed to short circuit the consumption of hydrogen peroxide by microbial catalases by producing iodine–thiocyanate complexes prior to contact with microorganisms, with the aim of being able to decontaminate the ex vivo dentures colonized by yeasts.Materials and methodsTeabags containing lactoperoxidase adsorbed on inert clay beads were immersed for 1 minute in phosphate buffer solution (0.1 M pH 7.4) containing 5.2 mM potassium iodide, 1.2 mM potassium thiocyanate, and 5.5 mM hydrogen peroxide. After removing the adsorbed lactoperoxidase, the stability and efficacy of iodine–thiocyanate complexes for Candida-colonized denture decontamination were verified. Investigations were performed in vitro on Candida albicans ATCC 10231 and on clinical isolates from 46 dentures. A Candida plate count was performed after a 24-hour incubation at 37°C on Sabouraud–chloramphenicol or CHROMagar solid media; then, the yeast growth was evaluated in Sabouraud broth by turbidimetry and biofilm biomass by crystal violet staining.ResultsIn vitro tests demonstrated the effectiveness of the oxidant solution in sterilizing a suspension of 106 Candida cells per milliliter after a 5-minute incubation. A single ex vivo immersion of contaminated dentures in a solution of iodine–thiocyanate complexes led to a decrease of at least 1 log unit in the number of colony-forming units in 58.3% of the tested dentures, while immersing in water alone had no effect on denture colonization (significant c2: p = 0.0006).ConclusionThese data suggest a promising new strategy for decontamination of dentures.