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Background: Staphylococcus aureus have a great ability to become rapidly resistant to conventional antimicrobial therapies. This study evaluated the efficacy of antimicrobial photodynamic therapy (aPDT) mediated by Curcumin (Cur) and light-emitting diode (LED) in the inactivation of biofilms of methicillin susceptible and resistant S. aureus (MSSA and MRSA, respectively)., Methods: Biofilms were treated with Cur (20, 40 or 80 μM) and illuminated with LED source (455 ± 3 nm; 5.28 J/cm 2 ) (aPDT groups), or treated either with Cur or LED only. Other samples were not exposed to Cur or LED (negative control). The biofilms viability after all experimental conditions were evaluated by counting the number of colonies (CFU/mL) and XTT assay. Additional samples were also evaluated by LIVE/DEAD® staining using confocal laser scanning microscopy (CLSM). Data were analyzed by ANOVAs followed by the Games-Howell post hoc test (α = 0.05)., Results: For both strains, all aPDT groups significantly reduced both CFU/mL and metabolic activity of biofilms compared to the negative control (p < 0.001). The results were enhanced when 80 μM of Cur was used. CLSM images showed that both bacteria biofilms submitted to aPDT had a large number of red-stained colonies, especially at aPDT80. In general, MRSA biofilms tended to be less susceptible to aPDT than MSSA biofilms., Conclusions: It can be concluded that aPDT mediated by Cur and LED was an efficient method to inactivate 48 -h biofilms of both S. aureus strains., Competing Interests: Declarations of interest All authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Photosensitizers in nanocarriers have been investigated for antimicrobial Photodynamic Therapy (aPDT). However, most studies are focused against microorganisms in planktonic or monospecies biofilm. Thus, this in vitro study evaluated the effect of aPDT using Chloroaluminium phthalocyanine (ClAlPc) in cationic nanoemulsion (NE) against Candida albicans, Candida glabrata and Streptococcus mutans grown as multispecies biofilm., Methods: Standard suspensions of each microorganism were added into wells of a microtiter plate for biofilm growth for 48 h in a candle jar. The biofilms were incubated with ClAlPc in cationic NE at 31.8 μM for 30 min and illuminated with red light fluence of 39.3 J/cm 2 (P+L+ group). Additional samples were treated only with photosensitizer (P+L-) or red light (P-L+) or neither (P-L-, control group). aPDT efficacy was assessed by colony quantification, biofilm's metabolic activity, total biomass, and confocal microscopy. Data were analyzed by ANOVA/Welch and post-hoc Tukey/Games-Howell tests (α = 0.05)., Results: aPDT (P+L+) reduced the colony count in 1.30 to 2.24 lg 10 and the metabolic activity in 53.7% compared with the control group (P-L-). The total biomass showed no statistical difference among the groups. The confocal microscopy analyzes showed uptake of the PS in the biofilm, and dead cells were observed in the biofilm treated with aPDT., Conclusion: aPDT mediated by ClAlPc in cationic NE promoted photoinactivation of the multispecies biofilm, which was confirmed by colony quantification, metabolic activity, and confocal microscopy. However, the total biomass of the biofilm was not affected by the treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The purpose of this study was to evaluate the effectiveness of anti-microbial photodynamic therapy (aPDT) mediated by curcumin (Cur) associated with LED light against biofilms of Candida dubliniensis, and further, investigate cellular uptake and drug penetration through the biofilms under confocal laser scanning microscopy (CLSM). Four C. dubliniensis strains were tested: three clinical isolates from HIV-positive patients and one reference strain (CBS 7987). Biofilms were treated with three Cur concentrations (20.0, 30.0, and 40.0 μM). All samples were incubated in the dark for 20 min and exposed to a 5.28 J/cm 2 of LED light fluence. Additional samples of each strain were treated either with Cur or LED light only. Control samples had neither Cur nor light. After aPDT, results were read using the XTT salt reduction method. The data were statistically analyzed by two-way ANOVA followed by Games-Howell post-hoc test (α = 0.05). Confocal laser scanning microscopy was used to verify both the uptake of Cur by yeast cells and its penetration through the biofilm. The results showed that aPDT promoted significant reduction on the metabolism of the biofilm-organized cells of C. dubliniensis. Further, while Cur was rapidly taken up by C. dubliniensis cells, a longer time interval was required to allow Cur penetration into biofilm cells. Based on these results, aPDT associating LED and Cur presents promising potential on fungal control of biofilms of C. dubliniensis.

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.

Methicillin-resistant Staphylococcus aureus (MRSA) can grow as structured biofilm in different surfaces, including oral mucosa and denture surfaces. Such biofilms can be released into the oral fluids and aspirated, causing systemic infections such as aspiration pneumonia. This study evaluated the efficacy of two disinfectant solutions and microwave irradiation in disinfecting acrylic specimens contaminated with MRSA biofilm. Thirty-six acrylic specimens were made, sterilized and contaminated with MRSA (107 cfu/mL). After incubation (37 °C/48 h), the specimens were divided into 4 groups: not disinfected (positive control); soaking in 1% sodium hypochlorite for 10 min; soaking in 2% chlorhexidine gluconate for 10 min; and irradiating by microwave for 3 min at 650 W. The viability of cells was evaluated by XTT reduction method. All specimens from the positive control group showed biofilm formation after 48 h incubation. The mean absorbance value of the control specimens was 1.58 (OD at 492 nm). No evidence of biofilm formation was observed on specimens after the disinfection methods. Disinfection by soaking in 1% sodium hypochlorite and 2% chlorhexidine gluconate and irradiating by microwaves resulted in 100% reduction of MRSA biofilm metabolism. The use of chemical solutions and microwave irradiation was shown to be effective for eradicating mature MRSA biofilms on acrylic resin specimens.

The adhesion of Candida albicans to surfaces is the prerequisite for occurrence of denture stomatitis, a common disease diagnosed among denture wearers. A routine of denture cleansing is essential to prevent biofilm formation and the onset of this infection. The aim of this study was to investigate the effectiveness of combining brushing and cleansing agents in killing C. albicans biofilm. Disks of acrylic resin were made, sterilized, and inoculated with C. albicans (10(7) cfu/mL). After incubation (37°C/48 h), specimens were randomly assigned to 10 experimental groups (n=9): 5 subjected to brushing with distilled water or cleansing agents - dentifrice slurry, 2% chlorhexidine gluconate (CHX), 1% sodium hypochlorite (NaOCl), and Polident fresh cleanse(®) (combined method) - and 4 exposed to the cleansing agents without brushing (immersion). Non-cleansed specimens were used as positive controls. The viability of cells was evaluated by XTT reduction method. Results were analyzed by Mann-Whitney and Kruskal-Wallis tests (α=0.05). The combined method was significantly more effective (p<0.0001) in reducing biofilm viability than the immersion. Brushing with CHX and NaOCl resulted in 100% removal of the biofilm. Immersion in the agents reduced significantly (p<0.0001) the biofilm viability, with CHX being the most effective (p<0.0001). The use of the combined method of brushing with cleansing agents is an effective method to reduce C. albicans biofilm, being CHX and NaOCl the most effective solutions.