The purpose of this study was to evaluate specific effects of photodynamic inactivation (PDI) using methylene blue as photosensitizer and low-power laser irradiation on the viability of single-, dual-, and three-species biofilms formed by C. albicans, S. aureus, and S. mutans. Biofilms were grown in acrylic discs immersed in sterile brain heart infusion broth (BHI) containing 5% sucrose, inoculated with microbial suspension (10(6) cells/ml) and incubated for 5 days. On the fifth day, the effects of the methylene blue (MB) photosensitizer at a concentration of 0.1 mg/ml for 5 min and InGaAlP laser (660 nm) for 98 s, alone and conjugated were evaluated. Next, the discs were placed in tubes with sterile physiological solution [0.9% sodium chloride (NaCl)] and sonicated for to disperse the biofilms. Ten-fold serial dilutions were carried and aliquots seeded in selective agar, which were then incubated for 48 h. Then the numbers CFU/ml (log(10)) were counted and analyzed statistically (ANOVA, Tukey test, p < 0.05). Scanning electron microscopy (SEM) on discs treated with PDI and control biofilms groups was performed. Significant decreases in the viability of all microorganisms were observed for biofilms exposed to PDI mediated by MB dye. Reductions (log(10)) of single-species biofilms were greater (2.32-3.29) than the association of biofilms (1.00-2.44). Scanning electron microscopy micrographs suggested that lethal photosensitization occurred predominantly in the outermost layers of the biofilms. The results showed that PDI mediated by MB dye, might be a useful approach for the control of oral biofilms.

This study was to evaluate specific effects of photodynamic therapy (energy density 15.8 J/cm(2), 26.3 J/cm(2) and 39.5 J/cm(2)) using methylene blue, toluidine blue and malachite green as photosensitizers and low-power laser irradiation on the viability of Candida albicans. Suspensions of C. albicans containing 10(6) cells/ml were standardized in a spectrophotometer. For each dye, 120 assays, divided into four groups according to the following experimental conditions, were carried out: laser irradiation in the presence of the photosensitizer; laser irradiation only; treatment with the photosensitizer only; no exposure to laser light or photosensitizer. Next, serial dilutions were prepared and seeded onto Sabouraud dextrose agar for the determination of the number of colony-forming units per milliliter (CFU/ml). The results were subjected to analysis of variance and the Tukey test (P < 0.05). Photodynamic therapy using the photosensitizers tested was effective in reducing the number of C. albicans.. The number of CFU/ml was reduced by between 0.54 log(10) and 3.07 log(10) and depended on the laser energy density used. Toluidine blue, methylene blue and malachite green were effective photosensitizers in antimicrobial photodynamic therapy against C. albicans, as was low-power laser irradiation alone.

INTRODUCTION: In HIV-infected patients, colonization of the oral cavity by potential pathogenic yeast may lead to development of systemic fungemia. We evaluated the prevalence of yeast in the oral cavity of Brazilian HIV-positive patients and verified whether or not the species characterized were enzymatically active. Furthermore, the species identified were tested for their susceptibility to antifungal treatment. METHODS: Patient saliva and oropharyngeal candidiasis samples were collected from 60 seropositive HIV patients and identified by the API20C system. Enzymatic activity was evaluated by the production of proteinase and phospholipase. Susceptibility to antifungal treatments were determined using the broth microdilution method. RESULTS: the most commonly isolated species were C. albicans (51.56%) followed by non-albicans Candida species (43.73%), Trichosporon mucoides (3.12%) and Kodamaea ohmeri (1.56%). Oral colonization by association of different species was observed in 42% of the patients. Enzymatic activity was verified in most of species isolated, except for C. glabrata, C. lusitaniae and C. guilliermondii. Resistance to Fluconazole and Amphotericin B was observed in isolates of C. albicans, C. glabrata, C. parapsilosis, C. krusei, and K. ohmeri. CONCLUSION: HIV-positive patients are orally colonized by single or multiple species of yeast that are occasionally resistant to Fluconazole or Amphotericin B.

(2015) Lactobacillus acidophilus ATCC 4356 inhibits biofilm formation by C.albicans and attenuates the experimental candidiasis in Galleriamellonella, Virulence, 6:1, 29-39, DOI: 10.4161/21505594.2014 Probiotic strains of Lactobacillus have been studied for their inhibitory effects on Candida albicans. However, few studies have investigated the effect of these strains on biofilm formation, filamentation and C. albicans infection. The objective of this study was to evaluate the influence of Lactobacillus acidophilus ATCC 4356 on C. albicans ATCC 18804 using in vitro and in vivo models. In vitro analysis evaluated the effects of L. acidophilus on the biofilm formation and on the capacity of C. albicans filamentation. For in vivo study, Galleria mellonella was used as an infection model to evaluate the effects of L. acidophilus on candidiasis by survival analysis, quantification of C. albicans CFU/mL, and histological analysis. The direct effects of L. acidophilus cells on C. albicans, as well as the indirect effects using only a Lactobacillus culture filtrate, were evaluated in both tests. The in vitro results showed that both L. acidophilus cells and filtrate were able to inhibit C. albicans biofilm formation and filamentation. In the in vivo study, injection of L. acidophilus into G. mellonella larvae infected with C. albicans increased the survival of these animals. Furthermore, the number of C. albicans CFU/mL recovered from the larval hemolymph was lower in the group inoculated with L. acidophilus compared to the control group. In conclusion, L. acidophilus ATCC 4356 inhibited in vitro biofilm formation by C. albicans and protected G. mellonella against experimental candidiasis in vivo.

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The study objective was to evaluate the effects of photodynamic therapy on buccal candidiasis in rats. After experimental candidiasis had been induced on the tongue dorsum, 72 rats were distributed into four groups according to treatment: treated with laser and methylene blue photosensitizer (L+P+); treated only with laser (L+P-); treated only with photosensitizer (L--P+); not treated with laser or photosensitizer (L-P-). The rats were killed immediately, 1 day, or 5 days after treatment, for microscopic analysis of the tongue dorsum. Observation verified that the photodynamic therapy group (L+P+) exhibited fewer epithelial alterations and a lower chronic inflammatory response than the L-P- group. The group L+P- presented more intense epithelial alterations and chronic inflammatory response than the remaining groups. The L-P+ group showed tissue lesions similar to those of the L-P- group. In conclusion, rats treated with photodynamic therapy developed more discrete candidiasis lesions than did the remaining groups.

The effect of erythrosine- and LED-mediated photodynamic therapy (PDT) on planktonic cultures and biofilms of Candida albicans and Candida dubliniensis was evaluated. Planktonic cultures of standardized suspensions (10(6)cells/mL) of C. albicans and C. dubliniensis were treated with erythrosine concentrations of 0.39-200 μM and LEDs in a 96-well microtiter plate. Biofilms formed by C. albicans and C. dubliniensis in the bottom of a 96-well microtiter plate were treated with 400 μM erythrosine and LEDs. After PDT, the biofilms were analysed by scanning electron microscopy (SEM). The antimicrobial effect of PDT against planktonic cultures and biofilms was verified by counting colony-forming units (CFU/mL), and the data were submitted to analysis of variance and the Tukey test (P<0.05). C. albicans and C. dubliniensis were not detectable after PDT of planktonic cultures with erythrosine concentrations of 3.12 μM or higher. The CFU/mL values obtained from biofilms were reduced 0.74 log(10) for C. albicans and 0.21 log(10) for C. dubliniensis. SEM revealed a decrease in the quantity of yeasts and hyphae in the biofilm after PDT. In conclusion, C. albicans and C. dubliniensis were susceptible to erythrosine- and LED-mediated PDT, but the biofilms of both Candida species were more resistant than their planktonic counterparts.

Galleria mellonella is a well-accepted insect model for the study of pathogen-host interactions and antimicrobial compounds. The main advantages of this model include the low cost of maintenance, the fast life cycle, the possibility of using a large number of caterpillars and the innate immune system, which is evolutionarily conserved relative to mammals. Because of these advantages, different research groups have been working to implement the rearing of G. mellonella in laboratory conditions. This protocol describes our experience in the rearing of G. mellonella caterpillars for experimental infection models and the influence of different artificial diets on developmental and physiological parameters. Here, we suggest a diet composition that benefits the life cycle of G. mellonella by accelerating the larval phase length and increasing the caterpillar weight. This diet also stimulated the immune system of G. mellonella by increasing the hemolymph volume and hemocyte concentration. In addition, our rearing protocol generated caterpillars that are more resistant to infection by Staphylococcus aureus, Escherichia coli and Candida albicans. A standard G. mellonella rearing protocol is fundamental to minimize external influences on the results, and this simple and easy protocol can support researchers starting to rear G. mellonella.