Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL^-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms.

Curcumin (CUR) has been used as photosensitizer in antimicrobial Photodynamic Therapy (aPDT). However its poor water solubility, instability, and scarce bioavalibility hinder its in vivo application. The aim of this study was to synthesize curcumin in polymeric nanoparticles (NP) and to evaluate their antimicrobial photodynamic effect and cytoxicity. CUR in anionic and cationic NP was synthesized using polylactic acid and dextran sulfate by the nanoprecipitation method. For cationic NP, cetyltrimethylammonium bromide was added. CUR-NP were characterized by physicochemical properties, photodegradation, encapsulation efficiency and release of curcumin from nanoparticles. CUR-NP was compared with free CUR in 10% dimethyl sulfoxide (DMSO) as a photosensitizer for aPDT against planktonic and biofilms (mono-, dual- and triple-species) cultures of Streptococcus mutans, Candida albicans and Methicillin-Resistant Staphylococcus aureus. The cytotoxicity effect of formulations was evaluated on keratinocytes. Data were analysed by parametric (ANOVA) and non-parametric (Kruskal-Wallis) tests (α = 0.05). CUR-NP showed alteration in the physicochemical properties along time, photodegradation similar to free curcumin, encapsulation efficiency up to 67%, and 96% of release after 48h. After aPDT planktonic cultures showed reductions from 0.78 log₁₀ to complete eradication, while biofilms showed no antimicrobial effect or reductions up to 4.44 log₁₀. Anionic CUR-NP showed reduced photoinactivation of biofilms. Cationic CUR-NP showed microbicidal effect even in absence of light. Anionic formulations showed no cytotoxic effect compared with free CUR and cationic CUR-NP and NP. The synthesized formulations improved the water solubility of CUR, showed higher antimicrobial photodynamic effect for planktonic cultures than for biofilms, and the encapsulation of CUR in anionic NP reduced the cytotoxicity of 10% DMSO used for free CUR.

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.

Antimicrobial photodynamic therapy (aPDT) has been proposed as an alternative method for oral candidiasis (OC), while nanocarriers have been used to improve the water solubility of curcumin (CUR). The aim of this study is to encapsulate CUR in polymeric nanoparticles (NPs) and to evaluate its photodynamic effects on a murine model of OC. Anionic and cationic CUR-NP is synthesized using poly-lactic acid and dextran sulfate and then characterized. Female mice are immunosuppressed and inoculated with Candida albicans (Ca) to induce OC. aPDT is performed by applying CUR-NP or free CUR on the dorsum of the tongue, followed by blue light irradiation for five consecutive days. Nystatin is used as positive control. Afterward, Ca are recovered and cultivated. Animals are euthanized for histological, immunohistochemical, and DNA damage evaluation. Encapsulation in NP improves the water solubility of CUR. Nystatin shows the highest reduction of Ca, followed by aPDT mediated by free CUR, which results in immunolabelling of cytokeratins closer to those observed for healthy animals. Anionic CUR-NP does not show antifungal effect, and cationic CUR-NP reduces Ca even in the absence of light. DNA damage is associated with Ca infection. Consecutive aPDT application is a safe treatment for OC.

The ability to manipulate the structure and function of promising systems via external stimuli is emerging with the development of reconfigurable and programmable multifunctional materials. Increasing antifungal and antitumor activity requires novel, effective treatments to be diligently sought. In this work, the synthesis, characterization, andin vitrobiological screening of pure α-Ag₂WO₄, irradiated with electrons and with non-focused and focused femtosecond laser beams are reported. We demonstrate, for the first time, that Ag nanoparticles/α-Ag₂WO₄composite displays potent antifungal and antitumor activity. This composite had an extreme low inhibition concentration againstCandida albicans, cause the modulation of α-Ag₂WO₄perform the fungicidal activity more efficient. For tumor activity, it was found that the composite showed a high selectivity against the cancer cells (MB49), thus depleting the populations of cancer cells by necrosis and apoptosis, without the healthy cells (BALB/3T3) being affected.