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.

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.

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.

The aim of this study was to isolate, quantify, identify, and compare opportunistic microorganisms (Candida and Staphylococcus genera and Enterobacteriaceae/Pseudomonadaceae families) from prosthesis-fitting surfaces, the hard palate, and mouth rinses of individuals wearing removable maxillary prosthesis with (50) and without (50) lesions of denture stomatitis (DS). The strains were collected and identified using phenotypic, biochemical and molecular tests. The counts of microorganisms were significantly higher in the group of individuals with DS (P < 0.05). C. albicans was the most frequently isolated yeast species in both groups, following by C. tropicalis and C. glabrata. Six isolates were identified as C. dubliniensis. S. aureus and S. epidermidis were the most frequent Staphylococcus species in both groups. Klebsiella pneumoniae was the predominant species in both groups. The association between Candida spp. and bacteria isolated in this study with DS suggests that these microorganisms may play important roles in the establishment and persistence of this disease.

The aim of this study was to evaluate the effects of photodynamic therapy (PDT) using rose bengal or erythrosine with light emitting diode (LED) on Candida albicans planktonic cultures and biofilms. Seven C. albicans clinical strains and one standard strain (ATCC 18804) were used. Planktonic cultures and biofilms of each C. albicans strain were submitted to the following experimental conditions: (a) treatment with rose bengal and LED (RB+L+); (b) treatment with erythrosine and LED (E+L+); and (c) control group, without LED irradiation or photosensitiser treatment (P-L-). After irradiation of the planktonic cultures and biofilms, the cultures were seeded onto Sabouraud dextrose agar (37 °C at 48 h) for counting of colony-forming units (CFU ml(-1) ) followed by posterior anova and Tukey's test analyses (P < 0.05). The biofilms were analysed using scanning electron microscopy (SEM). The results revealed a significant reduction of planktonic cultures (3.45 log(10) and 1.97 log(10) ) and of biofilms (<1 log(10) ) for cultures that were subjected to PDT mediated using either erythrosine or rose bengal, respectively. The SEM data revealed that the PDT was effective in reducing and destroying of C. albicans blastoconidia and hyphae. The results show that erythrosine- and rose bengal-mediated PDT with LED irradiation is effective in treating C. albicans.

Photodynamic therapy exhibited antifungal effects against C. albicans biofilms formed in vivo and reduced the capacity of C. albicans to adhere to BECs in vitro.

Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH-dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2Δ growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrt1/Zrt2-cellular import, followed by Zrc1-dependent intracellular compartmentalisation.

BackgroundCandida can cause mucocutaneous and/or systemic infections in hospitalized and immunosuppressed patients. Most individuals are colonized by Candida spp. as part of the oral flora and the intestinal tract. We compared oral and systemic isolates for the capacity to form biofilm in an in vitro biofilm model and pathogenicity in the Galleria mellonella infection model. The oral Candida strains were isolated from the HIV patients and included species of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. norvegensis, and C. dubliniensis. The systemic strains were isolated from patients with invasive candidiasis and included species of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. lusitaniae, and C. kefyr. For each of the acquired strains, biofilm formation was evaluated on standardized samples of silicone pads and acrylic resin. We assessed the pathogenicity of the strains by infecting G. mellonella animals with Candida strains and observing survival.ResultsThe biofilm formation and pathogenicity in Galleria was similar between oral and systemic isolates. The quantity of biofilm formed and the virulence in G. mellonella were different for each of the species studied. On silicone pads, C. albicans and C. dubliniensis produced more biofilm (1.12 to 6.61 mg) than the other species (0.25 to 3.66 mg). However, all Candida species produced a similar biofilm on acrylic resin, material used in dental prostheses. C. albicans, C. dubliniensis, C. tropicalis, and C. parapsilosis were the most virulent species in G. mellonella with 100% of mortality, followed by C. lusitaniae (87%), C. novergensis (37%), C. krusei (25%), C. glabrata (20%), and C. kefyr (12%).ConclusionsWe found that on silicone pads as well as in the Galleria model, biofilm formation and virulence depends on the Candida species. Importantly, for C. albicans the pathogenicity of oral Candida isolates was similar to systemic Candida isolates, suggesting that Candida isolates have similar biofilm-forming ability and virulence regardless of the infection site from which it was isolated.