DNase enhances photodynamic therapy against fluconazole-resistant Candida albicans biofilms.

OBJECTIVE: This study evaluated the effectiveness of DNase I combined with antimicrobial photodynamic therapy, mediated by Photodithazine® and light-emitting diode light, against biofilms formed by a fluconazole-resistant Candida albicans strain (ATCC 96901) and two clinical isolates (R14 and R70). MATERIALS AND METHODS: Biofilms were grown for 48 h and exposed to DNase for 5 min, followed by application of a photosensitizer (P) and light (L), either singly or combined (P+L+, P-L+, P+L-, P-L-, P-L-DNase, P+L+DNase, P+L-DNase, and P-L+DNase; n = 12). Biofilm analysis included quantification of extracellular matrix components (water-soluble and insoluble polysaccharides, proteins and extracellular DNA), and biomass (total and insoluble), as well as the enumeration of colony-forming units. The data were analyzed using three-way analysis of variance with Bonferroni's post hoc test. RESULTS: The DNase treatment combined with aPDT showed a reduction of 1.92, 1.65, and 1.29 log10 of cell viability compared with untreated controls for ATCC 96901, R14, and R70 strains, respectively. It also reduced extracellular matrix contents of water-soluble polysaccharides (36.3%) and extracellular DNA (72.3%), as well as insoluble biomass content (43.3%). CONCLUSION: The three strains showed similar behavior when treated with DNase, and the extracellular matrix components were affected, improving the effectiveness of antimicrobial photodynamic therapy.

Distinct Agents Induce Streptococcus mutans Cells with Altered Biofilm Formation Capacity.

Dental caries is a multifactorial biofilm- and sugar-dependent disease. This study investigated the influence of different agents on the induction of surviving Streptococcus mutans cells after successive treatment cycles and characterized the biofilms formed by these cells recovered posttreatment. The agents (with their main targets listed in parentheses) were compound 1771 (lipoteichoic acids), 4' hydroxychalcone (exopolysaccharides), myricetin (exopolysaccharides), tt-farnesol (cytoplasmatic membrane), sodium fluoride (enolase-glycolysis), chlorhexidine (antimicrobial), and vehicle. Recovered cells from biofilms were generated from exposure to each agent during 10 cycles of consecutive treatments (modeled on a polystyrene plate bottom). The recovered cell counting was different for each agent. The recovered cells from each group were grown as biofilms on saliva-coated hydroxyapatite discs (culture medium with sucrose/starch). In S. mutans biofilms formed by cells recovered from biofilms previously exposed to compound 1771, 4' hydroxychalcone, or myricetin, cells presented higher expression of the 16S rRNA, gyrA (DNA replication and transcription), gtfB (insoluble exopolysaccharides), and eno (enolase-glycolysis) genes and lower quantities of insoluble dry weight and insoluble exopolysaccharides than those derived from other agents. These findings were confirmed by the smaller biovolume of bacteria and/or exopolysaccharides and the biofilm distribution (coverage area). Moreover, preexposure to chlorhexidine increased exopolysaccharide production. Therefore, agents with different targets induce cells with distinct biofilm formation capacities, which is critical for developing formulations for biofilm control. IMPORTANCE This article addresses the effect of distinct agents with distinct targets in the bacterial cell (cytoplasmatic membrane and glycolysis), the cell's extracellular synthesis of exopolysaccharides that are important for cariogenic extracellular matrix construction and biofilm buildup in the generation of cells that persisted after treatment, and how these cells form biofilms in vitro. For example, if preexposure to an agent augments the production of virulence determinants, such as exopolysaccharides, its clinical value may be inadequate. Modification of biofilm formation capacity after exposure to agents is critical for the development of formulations for biofilm control to prevent caries, a ubiquitous disease associated with biofilm and diet.

Compounds with Distinct Targets Present Diverse Antimicrobial and Antibiofilm Efficacy against Candida albicans and Streptococcus mutans, and Combinations of Compounds Potentiate Their Effect.

Candida albicans and Streptococcus mutans interact synergistically in biofilms associated with a severe form of dental caries. Their synergism is driven by dietary sucrose. Thus, it is necessary to devise strategies to hinder the development of those biofilms and prevent cavities. Six compounds [tt-farnesol (sesquiterpene alcohol that decreases the bacterium acidogenicity and aciduricity and a quorum sensing fungal molecule), myricetin (flavonoid that interferes with S. mutans exopolysaccharides production), two 2'-hydroxychalcones and 4'-hydroxychalcone (intermediate metabolites for flavonoids), compound 1771 (inhibitor of lipoteichoic synthase in Gram-positive bacteria)] with targets in both fungus and bacterium and their products were investigated for their antimicrobial and antibiofilm activities against single-species cultures. The compounds and concentrations effective on single-species biofilms were tested alone and combined with or without fluoride to control initial and pre-formed dual-species biofilms. All the selected treatments eliminated both species on initial biofilms. In contrast, some combinations eliminated the bacterium and others the fungus in pre-formed biofilms. The combinations 4'-hydroxychalcone+tt-farnesol+myricetin, 4'-hydroxychalcone+tt-farnesol+fluoride, and all compounds together with fluoride were effective against both species in pre-formed biofilms. Therefore, combinations of compounds with distinct targets can prevent C. albicans and S. mutans dual-species biofilm build-up in vitro.

Dual-species biofilms of Streptococcus mutans and Candida albicans exhibit more biomass and are mutually beneficial compared with single-species biofilms.

Background: Streptococcus mutans (Sm) and Candida albicans (Ca) are found in biofilms of early childhood caries. Objective: To characterize in vitro dual- and single-species biofilms of Sm and Ca formed on saliva-coated hydroxyapatite discs in the presence of sucrose. Design: Evaluation of biofilms included biochemical [biomass, proteins, matrix's water-soluble (WSP) and alkali-soluble (ASP) polysaccharides, microbiological, 3D structure, gene expression, and stress tolerance analyses. Results: Biomass and proteins were higher for dual-species and lower for Ca (p = 0.001). Comparison of Sm single- and dual-species biofilms revealed no significant difference in Sm numbers or quantity of WSP (p > 0.05). Dual-species biofilms contained a higher population of Ca (p < 0.001). The quantity of ASP was higher in dual-species biofilms (vs Ca single-species biofilms; p = 0.002). The 3D structure showed larger microcolonies and distinct distribution of Sm-derived exopolysaccharides in dual-species biofilms. Compared with dual-species biofilms, expression of gtfB (ASP) and nox1 (oxidative stress) was higher for single-species of Sm whilst expression of BGL2 (matrix), PHR1 (matrix, acid tolerance) and SOD1 (oxidative stress) was higher in single-species of Ca. There was no difference for acid tolerance genes (Sm atpD and Ca PHR2), which was confirmed by acid tolerance challenge. Dual-species biofilms were more tolerant to oxidative and antimicrobial stresses (p < 0.05). Conclusions: Dual-species biofilms present greater 3D complexity, thereby, making them more resistant to stress conditions.

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

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.