Candida albicans exhibits heterogeneous and adaptive cytoprotective responses to antifungal compounds.

Candida albicans, an opportunistic human pathogen, poses a significant threat to human health and is associated with significant socio-economic burden. Current antifungal treatments fail, at least in part, because C. albicans can initiate a strong drug tolerance response that allows some cells to grow at drug concentrations above their minimal inhibitory concentration. To better characterize this cytoprotective tolerance program at the molecular single-cell level, we used a nanoliter droplet-based transcriptomics platform to profile thousands of individual fungal cells and establish their subpopulation characteristics in the absence and presence of antifungal drugs. Profiles of untreated cells exhibit heterogeneous expression that correlates with cell cycle stage with distinct metabolic and stress responses. At 2 days post-fluconazole exposure (a time when tolerance is measurable), surviving cells bifurcate into two major subpopulations: one characterized by the upregulation of genes encoding ribosomal proteins, rRNA processing machinery, and mitochondrial cellular respiration capacity, termed the Ribo-dominant (Rd) state; and the other enriched for genes encoding stress responses and related processes, termed the Stress-dominant (Sd) state. This bifurcation persists at 3 and 6 days post-treatment. We provide evidence that the ribosome assembly stress response (RASTR) is activated in these subpopulations and may facilitate cell survival.

Many drugs currently used to treat fungal diseases are becoming less effective. This is partly due to the rise of antifungal resistance, where certain fungal cells acquire mutations that enable them to thrive and proliferate despite the medication. Antifungal tolerance also contributes to this problem, wherein certain cells can continue to grow and multiply, while other ­ genetically identical ones ­ cannot. This variability is partly due to differences in gene expression within the cells. The specific nature of these differences has remained elusive, mainly because their study requires the use of expensive and challenging single-cell technologies. To address this challenge, Dumeaux et al. adapted an existing technique to perform single-cell transcriptomics in the pathogenic yeast Candida albicans. Their approach was cost effective and made it possible to examine the gene expression in thousands of individual cells within a population that had either been treated with antifungal drugs or were left untreated. After two to three days following exposure to the antifungal treatment, C. albicans cells commonly exhibited one of two states: one subgroup, the 'Ribo-dominant' cells, predominantly expressed genes for ribosomal proteins, while the other group, the 'Stress-dominant' cells, upregulated their expression of stress-response genes. This suggests that drug tolerance may be related to different gene expression patterns in growing cell subpopulations compared with non-growing subpopulations. The findings also indicate that the so-called 'ribosome assembly stress response' known to help baker's yeast cells to survive, might also aid C. albicans in surviving exposure to antifungal treatments. The innovative use of single-cell transcriptomics in this study could be applied to other species of fungi to study differences in cell communication under diverse growth conditions. Moreover, the unique gene expression patterns in C. albicans identified by Dumeaux et al. may help to design new antifungal treatments that target pathways linked to drug resistance.

A Deep Learning Approach to Capture the Essence of Candida albicans Morphologies.

We present deep learning-based approaches for exploring the complex array of morphologies exhibited by the opportunistic human pathogen Candida albicans. Our system, entitled Candescence, automatically detects C. albicans cells from differential image contrast microscopy and labels each detected cell with one of nine morphologies. This ranges from yeast white and opaque forms to hyphal and pseudohyphal filamentous morphologies. The software is based upon a fully convolutional one-stage (FCOS) object detector, a deep learning technique that uses an extensive set of images that we manually annotated with the location and morphology of each cell. We developed a novel cumulative curriculum-based learning strategy that stratifies our images by difficulty from simple yeast forms to complex filamentous architectures. Candescence achieves very good performance (~85% recall; 81% precision) on this difficult learning set, where some images contain hundreds of cells with substantial intermixing between the predicted classes. To capture the essence of each C. albicans morphology and how they intermix, we used a second technique from deep learning entitled generative adversarial networks. The resultant models allow us to identify and explore technical variables, developmental trajectories, and morphological switches. Importantly, the model allows us to quantitatively capture morphological plasticity observed with genetically modified strains or strains grown in different media and environments. We envision Candescence as a community meeting point for quantitative explorations of C. albicans morphology. IMPORTANCE The fungus Candida albicans can "shape shift" between 12 morphologies in response to environmental variables. The cytoprotective capacity provided by this polymorphism makes C. albicans a formidable pathogen to treat clinically. Microscopy images of C. albicans colonies can contain hundreds of cells in different morphological states. Manual annotation of images can be difficult, especially as a result of densely packed and filamentous colonies and of technical artifacts from the microscopy itself. Manual annotation is inherently subjective, depending on the experience and opinion of annotators. Here, we built a deep learning approach entitled Candescence to parse images in an automated, quantitative, and objective fashion: each cell in an image is located and labeled with its morphology. Candescence effectively replaces simple rules based on visual phenotypes (size, shape, and shading) with neural circuitry capable of capturing subtle but salient features in images that may be too complex for human annotators.

Signal-mediated localization of Candida albicans pheromone response pathway components.

A MAPK cascade consists of three kinases, (MEKK, MEK and MAPK), that are sequentially activated in response to a stimulus and serve to transmit signals. In C. albicans and in yeast, an MAPK cascade is linked to the pheromone pathway through a scaffold protein (Cst5 and Ste5, respectively). Cst5 is much shorter and lacks key domains compared to Ste5, so in C. albicans, other elements, in particular the MEKK Ste11, play key roles in controlling the associations and localizations of network components. ABSTRACT: Candida albicans opaque cells release pheromones to stimulate cells of opposite mating type to activate their pheromone response pathway. Although this fungal pathogen shares orthologous proteins involved in the process with Saccharomyces cerevisiae, the pathway in each organism has unique characteristics. We have used GFP-tagged fusion proteins to investigate the localization of the scaffold protein Cst5, as well as the MAP kinases Cek1 and Cek2, during pheromone response in C. albicans. In wild-type cells, pheromone treatment directed Cst5-GFP to surface puncta concentrated at the tips of mating projections. These puncta failed to form in cells defective in either the Gα or ß subunits. However, they still formed in response to pheromone in cells missing Ste11, but with the puncta distributed around the cell periphery in the absence of mating projections. These puncta were absent from hst7Δ/Δ cells, but could be detected in the ste11Δ/Δ hst7Δ/Δ double mutant. Cek2-GFP showed a strong nuclear localization late in the response, consistent with a role in adaptation, while Cek1-GFP showed a weaker, but early increase in nuclear localization after pheromone treatment. Activation loop phosphorylation of both Cek1 and Cek2 required the presence of Ste11. In contrast to Cek2-GFP, which showed no localization signal in ste11Δ/Δ cells, Cek1-GFP showed enhanced nuclear localization that was pheromone independent in the ste11Δ/Δ mutant. The results are consistent with CaSte11 facilitating Hst7-mediated MAP kinase phosphorylation and also playing a potentially critical role in both MAP kinase and Cst5 scaffold localization.

Candida albicans Mrv8, is involved in epithelial damage and biofilm formation.

Candida albicans is the most common human fungal pathogen that can cause superficial and deep-seated infections in susceptible individuals. Despite its medical importance, the vast majority of C. albicans genes remain of unknown function. Here, we report a role for the lineage-specific gene, MRV8, in host pathogen interactions, mycelial microcolony maturation and biofilm formation. In silico analysis indicated that MRV8 encodes a four-pass transmembrane protein unique to the closely related pathogens C. albicans and Candida dubliniensis. Deletion of MRV8 did not affect C. albicans adherence to, or initial invasion into human oral epithelia, but inhibited mycelial development and strongly reduced epithelial damage. mrv8Δ/Δ cells exhibited a media-dependent defect in biofilm formation and mutant biofilm metabolic activity was enhanced by cyclosporin A. mrv8Δ/Δ biofilms were more tolerant to treatment with caspofungin, but not to fluconazole or amphotericin B. Co-stimulation with calcium chloride and calcofluor white rescued biofilm growth in the presence of caspofungin, and this rescue-effect was Mrv8-dependent. Together, our data demonstrate an important role for a lineage-specific gene (MRV8) in C. albicans biofilm formation, drug tolerance and host-pathogen interactions.

Screening of Candida albicans GRACE library revealed a unique pattern of biofilm formation under repression of the essential gene ILS1.

Candida albicans biofilm formation is governed by a regulatory circuit comprising nine transcription factors which control a network of target genes. However, there are still unknown genes contributing to biofilm features. Thus, the GRACE library was screened to identify genes involved in mature biofilm development. Twenty-nine conditional mutants were selected for a second screening revealing three groups of genes: twenty- two conditional mutants were defective for normal growth and unable to form biofilms; six strains, conditionally defective in genes ARC40, ARC35, ORF19.2438, SKP1, ERG6, and ADE5,7 that are likely essential or involved in general cell processes, grew normally as free-floating cells but produced less biofilm; finally, the conditional strain for a putative essential isoleucyl- tRNA synthetase gene, ILS1, was unable to grow as yeast-phase cells but was capable of producing a tridimensional biofilm structure in spite of reduced metabolic activity. This unique biofilm still relied on the classical biofilm genes, while it differentially induced groups of genes involved in adhesion, protein synthesis, cell wall organization, and protein folding. Although the conditional mutant repressed genes annotated for morphology and homeostasis processes affecting morphology and metabolism, the dynamic cell growth enabled the formation of a complex biofilm community independent of ILS1.

Photodynamic inactivation of virulence factors of Candida strains isolated from patients with denture stomatitis.

Candida species are major microorganisms isolated in denture stomatitis (DS), an inflammatory process of the mucosa underlying removable dental prostheses, and express a variety of virulence factors that can increase their pathogenicity. The potential of Photodynamic inactivation (PDI) in planktonic culture, biofilms and virulence factors of Candida strains was evaluated. A total of 48 clinical Candida isolates from individuals wearing removable maxillary prostheses with DS were included in the study. The effects of erythrosine (ER, 200 µM) and a green LED (λ 532 ± 10 nm, 237 mW/cm(2) and 42.63 J/cm(2)) in a planktonic culture were evaluated. The effect of the addition of ER at a concentration of 400 µM together with a green LED was evaluated in biofilms. The virulence factors of all of the Candida strains were evaluated before and after the PDI process in cells derived from biofilm and planktonic assays. All of the Candida species were susceptible to ER and green LED. However, the biofilm structures were more resistant to PDI than the planktonic cultures. PDI also promoted slight reductions in most of the virulence factors of C. albicans and some of the Candida tropicalis strains. These results suggest that the addition of PDI is effective for reducing yeasts and may also reduce the virulence of certain Candida species and decrease their pathogenicity.

Estudo de genes de Candida albicans com função desconhecida quanto à formação de biofilme, características biológicas e interação patógeno- hospedeiro / Study of unknown function genes of Candida albicans as to biofilm formation, biological characteristics, and host-pathogen interaction

Candida albicans é um fungo oportunista capaz de causar infecções superficiais e até sistêmicas. A maioria das infecções é mediada pela formação de biofilme que confere resistência aos agentes antifúngicos e ao sistema imune, porém os mecanismos de desenvolvimento do biofilme e patogenicidade ainda não foram completamente elucidados. No presente estudo foram selecionados 9 genes de C. albicans com função desconhecida, dentre 34 cepas mutantes que apresentaram fenótipo alterado para formação de biofilme. Os biofilmes foram formados em placas de 96 poços ou discos de poliestireno e avaliados em diferentes tempos de desenvolvimento. A seguir foram construídas 4 cepas complementadas que foram avaliadas quanto à susceptibilidade a agentes estressantes, crescimento sob limitação de nutrientes e testes de filamentação. A arquitetura dos biofilmes foi analisada por microscopia eletrônica de varredura (MEV). Os biofilmes também foram avaliados quanto à quantidade de β-1,3-glucana e quitina. Para os modelos de infecção, células epiteliais bucais (TR-146) foram utilizadas para análise de aderência, invasão e dano. A patogenicidade das cepas foi avaliada em ovos embrionados de galinha durante 7 dias, após a inoculação das cepas. Células planctônicas e biofilmes foram submetidos a testes antifúngicos com os agentes fluconazol, anfotericina B e caspofungina. A reação em cadeia da polimerase quantitativa foi realizada para verificar a expressão dos genes MRV8 e NDT80 em células fúngicas em interação com células epiteliais e MRV8, MRV1 e MRV6 em células crescidas em biofilme. Os resultados foram analisados por teste t de Student, ANOVA, Tukey e testes de Log-rank (Mantel-Cox) (p < 0,05). Foram construídas 4 cepas complementadas para os genes selecionados ORF19.823, ORF19.7170, ORF19.6847 e MRV8. A função de ORF19.823 ainda permanece desconhecida, pois não foi observado fenótipo significativo para a cepa mutante quanto aos testes realizados. A cepa mutante para ORF19.7170 ...

Candida albicans is an opportunistic fungi capable of causing superficial and systemic infections. Most of infections are mediated by biofilm formation which confers resistance to antifungal agents and immune system, but the mechanisms of biofilm development and pathogenicity were not thoroughly elucidated yet. In the presente study, 9 unknown function genes of C. albicans were selected among 34 mutant strains that presented altered phenotype for biofilm formation. The biofilms were formed on 96-well microtitle plates or on polystyrene disks and evaluated in different time intervals. Next, 4 complemented strains were constructed and evaluated for susceptibility to stressor agents, growth under nutrient limitation and filamentation tests. The biofilm architecture was analyzed by scanning electron microscopy (SEM). The biofilms were also assessed as the quantity of β-1,3-glucan and chitin. For the infection models, buccal epithelial cells (TR-146) were used for adherence, invasion and damage assays. The pathogenicity of the strains was evaluated in embrionated chicken eggs for 7 days, after inoculation of the strains. Planktonic cells and biofilms were submitted to antifungal tests with fluconazole, amphotericin B, and caspofungin. Quantitative polymerase chain reaction was performed to verify the expression of MRV8 and NDT80 genes in fungal cells in interaction with epithelial cells and MRV8, MRV1, and MRV6 genes in cells grown in biofilm. The results were submitted to the Student t test, ANOVA, Tukey's test, and Log-rank test (Mantel-Cox) (p < 0.05). Four complemented strains were constructed for the selected genes ORF19.823, ORF19.7170, ORF19.6847, and MRV8. The function of the ORF19.823 is still unknown, because the mutant strain did not show any significative phenotype for the tests performed. The mutant strain for the ORF19.7170 caused less damage on epithelial cells, but the result was not significant and the gene was dispensable for biofilm formation. ...

Estudo de genes de Candida albicans com função desconhecida quanto à formação de biofilme, características biológicas e interação patógeno- hospedeiro / Study of unknown function genes of Candida albicans as to biofilm formation, biological characteristics, and host-pathogen interaction

Candida albicans é um fungo oportunista capaz de causar infecções superficiais e até sistêmicas. A maioria das infecções é mediada pela formação de biofilme que confere resistência aos agentes antifúngicos e ao sistema imune, porém os mecanismos de desenvolvimento do biofilme e patogenicidade ainda não foram completamente elucidados. No presente estudo foram selecionados 9 genes de C. albicans com função desconhecida, dentre 34 cepas mutantes que apresentaram fenótipo alterado para formação de biofilme. Os biofilmes foram formados em placas de 96 poços ou discos de poliestireno e avaliados em diferentes tempos de desenvolvimento. A seguir foram construídas 4 cepas complementadas que foram avaliadas quanto à susceptibilidade a agentes estressantes, crescimento sob limitação de nutrientes e testes de filamentação. A arquitetura dos biofilmes foi analisada por microscopia eletrônica de varredura (MEV). Os biofilmes também foram avaliados quanto à quantidade de β-1,3-glucana e quitina. Para os modelos de infecção, células epiteliais bucais (TR-146) foram utilizadas para análise de aderência, invasão e dano. A patogenicidade das cepas foi avaliada em ovos embrionados de galinha durante 7 dias, após a inoculação das cepas. Células planctônicas e biofilmes foram submetidos a testes antifúngicos com os agentes fluconazol, anfotericina B e caspofungina. A reação em cadeia da polimerase quantitativa foi realizada para verificar a expressão dos genes MRV8 e NDT80 em células fúngicas em interação com células epiteliais e MRV8, MRV1 e MRV6 em células crescidas em biofilme. Os resultados foram analisados por teste t de Student, ANOVA, Tukey e testes de Log-rank (Mantel-Cox) (p < 0,05). Foram construídas 4 cepas complementadas para os genes selecionados ORF19.823, ORF19.7170, ORF19.6847 e MRV8. A função de ORF19.823 ainda permanece desconhecida, pois não foi observado fenótipo significativo para a cepa mutante quanto aos testes realizados. A cepa mutante para ORF19.7170...

Candida albicans is an opportunistic fungi capable of causing superficial and systemic infections. Most of infections are mediated by biofilm formation which confers resistance to antifungal agents and immune system, but the mechanisms of biofilm development and pathogenicity were not thoroughly elucidated yet. In the presente study, 9 unknown function genes of C. albicans were selected among 34 mutant strains that presented altered phenotype for biofilm formation. The biofilms were formed on 96-well microtitle plates or on polystyrene disks and evaluated in different time intervals. Next, 4 complemented strains were constructed and evaluated for susceptibility to stressor agents, growth under nutrient limitation and filamentation tests. The biofilm architecture was analyzed by scanning electron microscopy (SEM). The biofilms were also assessed as the quantity of β-1,3-glucan and chitin. For the infection models, buccal epithelial cells (TR-146) were used for adherence, invasion and damage assays. The pathogenicity of the strains was evaluated in embrionated chicken eggs for 7 days, after inoculation of the strains. Planktonic cells and biofilms were submitted to antifungal tests with fluconazole, amphotericin B, and caspofungin. Quantitative polymerase chain reaction was performed to verify the expression of MRV8 and NDT80 genes in fungal cells in interaction with epithelial cells and MRV8, MRV1, and MRV6 genes in cells grown in biofilm. The results were submitted to the Student t test, ANOVA, Tukey's test, and Log-rank test (Mantel-Cox) (p < 0.05). Four complemented strains were constructed for the selected genes ORF19.823, ORF19.7170, ORF19.6847, and MRV8. The function of the ORF19.823 is still unknown, because the mutant strain did not show any significative phenotype for the tests performed. The mutant strain for the ORF19.7170 caused less damage on epithelial cells, but the result was not significant and the gene was dispensable for biofilm formation...

Essential oil of Melaleuca alternifolia for the treatment of oral candidiasis induced in an immunosuppressed mouse model.

BACKGROUND: The search for alternative therapies for oral candidiasis is a necessity and the use of medicinal plants seems to be one of the promising solutions. The objective of this study was to evaluate the in vitro and in vivo effects of the essential oil of Melaleuca alternifolia on Candida albicans. METHODS: The minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) of M. alternifolia were determined by the broth microdilution assay. For the in vivo study, twelve immunosuppressed mice with buccal candidiasis received topical applications of M. alternifolia with MBEC. After treatment, yeasts were recovered from the mice and quantified (CFU/mL). Mice were killed for morphologic analysis of the tongue dorsum by optical and scanning electron microscopy. Data were analyzed using Student's t test or Mann-Whitney test. RESULTS: The MIC of M. alternifolia was 0.195% and the MBEC was 12.5%. Treatment with M. alternifolia achieved a 5.33 log reduction in C. albicans and reduced the microscopic lesions of candidiasis. CONCLUSIONS: M. alternifolia oil at a 12.5% was effective to eradicate a C. albicans biofilm formed in vitro and to reduce yeasts of C. albicans in an immunosuppressed mouse model.

Comparison of the effect of rose bengal- and eosin Y-mediated photodynamic inactivation on planktonic cells and biofilms of Candida albicans.

Candida albicans is an opportunistic yeast that can cause oral candidosis through the formation of a biofilm, an important virulence factor that compromises the action of antifungal agents. The objective of this study was to compare the effect of rose bengal (RB)- and eosin Y (EY)-mediated photodynamic inactivation (PDI) using a green light-emitting diode (LED; 532 ± 10 nm) on planktonic cells and biofilms of C. albicans (ATCC 18804). Planktonic cultures were treated with photosensitizers at concentrations ranging from 0.78 to 400 µM, and biofilms were treated with 200 µM of photosensitizers. The number of colony-forming unit per milliliter (CFU/mL) was compared by analysis of variance and Tukey's test (P ≤ 0.05). After treatment, one biofilm specimen of the control and PDI groups were examined by scanning electron microscopy. The photosensitizers (6.25, 25, 50, 200, and 400 µM of EY, and 6.25 µM of RB or higher) significantly reduced the number of CFU/mL in the PDI groups when compared to the control group. With respect to biofilm formation, RB- and EY-mediated PDI promoted reductions of 0.22 log10 and 0.45 log10, respectively. Scanning electron microscopy showed that the two photosensitizers reduced fungal structures. In conclusion, EY- and RB-mediated PDI using LED irradiation significantly reduced C. albicans planktonic cells and biofilms.

Comparison of disinfection protocol of irreversible hydrocolloid (alginate) impressions through plastic and metallic trays / Comparação do protocolo de desinfecção de moldes com hidrocolóide irreversível (alginato) em moldeiras plásticas e metálicas

Introduction: Many studies have evaluated the disinfection of irreversible hydrocolloid impressions through different disinfecting agents. However, impression trays can be source of cross-infection requiring disinfection. This study aimed to determine which would be the most suitable tray (metallic or plastic), available in dental market, and the ideal time to achieve disinfection by using 1% sodium hypochlorite poured into the alginate impression. Material and method: Thirty dental impressions from the patients aged from 7-12 years and treated in the Discipline of Orthodontics of the institution were divided into two groups according to the impression tray type: 15 impressions through plastic tray (Morelli) and 15 impressions through metallic tray (Tecnodent). The material collection was performed before and after the application of 1% sodium hypochlorite for 3, 5 and 10 min. After the incubation period of 48 h at 37 ºC, the microorganism colonies were counted on the plates presenting from 30 to 300 colonies to determine the colony-forming unit (CFU) per mL. CFU/ mL results were transformed into logarithm and submitted to statistical analysis by applying ANOVA and Tukey test (p ≤ 0.05). Results: Greater CFU percentage reduction occurred in alginate after three min, in both tray types. Concerning to tray types, it could be observed that the plastic tray showed 100% of reduction after 5 min while the metallic tray exhibited 81.49% of reduction after 3 min. Conclusion: 1 – The plastic tray showed the most effective disinfection after 5 min, with 100% of CFU reduction; 2 – The most effective time of disinfection with 1% sodium hypochlorite poured into the impression was 5 min, for both tray types.

Introdução: Muitos são os trabalhos que avaliam a desinfecção das moldagens com hidrocolóide irreversível e com diferentes agentes desinfetantes, mas as moldeiras também podem ser vetores de infecção cruzada e é necessária a sua desinfecção. A proposta do trabalho foi determinar qual a melhor moldeira (metálica ou plástica), disponíveis no mercado para a utilização do cirurgião dentista, e o tempo ideal para se obter a desinfecção utilizando o hipoclorito de sódio a 1% vertido na moldagem de com alginato. Material e método: Foram obtidas 30 moldagens de pacientes em tratamento na Disciplina de Ortodontia, do Curso de Odontologia, do ICT-UNESP-SJC, com idades entre 7 e 12 anos, divididas em dois grupos de acordo com o tipo de moldeira empregada: 15 moldagens com moldeiras de plástico (Morelli) e 15 com moldeiras de metal (Tecnodent). A coleta de material foi realizada antes e após a aplicação do hipoclorito de sódio a 1% durante 3, 5 e 10 min. Após período de incubação de 48 h a 37 ºC foram contadas colônias de microorganismos nas placas que apresentaram de 30 a 300 colônias para determinação de Unidades Formadoras de Colônias (UFC) por mL. Os resultados em UFC/ mL foram transformados em logaritmo e submetidos à análise estatística Anova e teste de Tukey (p ≤ 0,05). Resultados: No alginato ocorreu uma maior redução percentual de UFC após 3 min, em ambas as moldeiras. Em relação a maior redução nas moldeiras, pudemos observar que a moldeira plástica ocorreu 100% de redução após 5 min e 81,49% de redução na moldeira metálica após 3 min. Conclusão: 1 - A moldeira plástica apresentou desinfecção mais eficiente, após 5 min, com redução de 100% de UFC; 2 - O tempo mais eficaz de desinfecção da moldagem com hipoclorito de sódio a 1% vertido na moldagem foi de 5 min, para moldeira de plástico e de metal.

Photodynamic inactivation of Streptococcus mutans and Streptococcus sanguinis biofilms in vitro.

The purpose of this study was to evaluate specific effects of photodynamic inactivation (PDI) using erythrosine (ER) and Rose Bengal (RB) photosensitizers and a blue light-emitting diode (LED) on the viability of Streptococcus mutans and Streptococcus sanguinis biofilms. Biofilms were grown in acrylic disks immersed in broth to production of biofilms, inoculated with microbial suspension (10(6) cells/mL) and incubated for 48 h. After the formation of biofilms, the effects of the photosensitizers ER and RB at a concentration of 5 µM for 5 min and blue LED (455 ± 20 nm) for 180 s, photosensitizers alone and conjugated were evaluated. Next, the disks were placed in tubes with sterile physiological solution (0.9 % sodium chloride) and sonicated for to disperse the biofilms. Tenfold serial dilutions were carried and aliquots seeded in brain heart infusion agar which were then incubated for 48 h. Then the numbers colony-forming units per milliliter (CFU/mL; log10) were counted and analyzed statistically (ANOVA, Tukey test, P ≤ 0.05). Significant decreases in the viability of all microorganisms were observed for biofilms exposed to PDI mediated by both photosensitizers. The reductions with RB and ER were, 0.62 and 0.52 log10 CFU mL(-1) for S. mutans biofilms (p=0.001), and 0.95 and 0.88 log10 CFU mL(-1) for S. sanguinis biofilms (p=0.001), respectively. The results showed that biofilms formed in vitro by S. mutans and S. sanguinis, were sensitive to PDI using a blue LED associated with photosensitizers ER or RB, indicating its use in the control of caries and periodontal diseases.

Effect of erythrosine- and LED-mediated photodynamic therapy on buccal candidiasis infection of immunosuppressed mice and Candida albicans adherence to buccal epithelial cells.

OBJECTIVE: This study evaluated the effects of photodynamic therapy (PDT) on buccal candidiasis in mice and on the adherence of yeast to buccal epithelial cells (BECs) in vitro. STUDY DESIGN: A total of 56 immunosuppressed mice with buccal candidiasis were subjected to PDT, consisting of treatment with erythrosine (400 µmol/L) followed by exposure to a green LED (14.34 J cm(-2)). After treatment, the yeasts recovered from the mice were quantified (CFU/mL) and analyzed for the effects of PDT on their adherence to BECs. The data were analyzed using ANOVA, the Tukey test, Kruskal-Wallis test and Student t test. RESULTS: PDT significantly reduced the amount of yeast present in the lesions by 0.73 log(10) (P = .018) and reduced C. albicans adherence to BECs by 35% without damaging adjacent tissues (P = .045). CONCLUSIONS: 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.

The effects of rose bengal- and erythrosine-mediated photodynamic therapy on Candida albicans.

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.

Susceptibility of Candida albicans and Candida dubliniensis to erythrosine- and LED-mediated photodynamic therapy.

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.

Effect of photodynamic therapy on clinical isolates of Staphylococcus spp

Staphylococcus spp. are opportunistic microorganisms known for their capacity to develop resistance against antimicrobial agents. The objective of this study was to evaluate the effect of photodynamic therapy (PDT) on 20 Staphylococcus strains isolated from the human oral cavity, including S. aureus, S. schleiferi, S. epidermidis, S. capitis, S. haemolyticus, and S. lentus. A suspension of each Staphylococcus strain (10(6) cells/mL) was submitted to PDT using methylene blue and a low power laser. The isolated effects of methylene blue, laser treatment and ciprofloxacin were also evaluated. After the experimental treatments, 0.1 mL aliquots of the suspensions were seeded onto BHI agar for determination of the number of colony-forming units (CFU/mL). The results were analyzed by analysis of variance and Tukey's test (p < 0.05). The mean reduction in bacterial counts of the strains submitted to PDT ranged from 4.89 to 6.83 CFU (log10)/mL, with the observation of a decreasing susceptibility to treatment of S. schleiferi, S. haemolyticus, S. epidermidis, S. capitis, S. aureus, and S. lentus. The results showed that PDT was effective in reducing the number of viable cells of all clinical Staphylococcus isolates studied.

Effect of photodynamic therapy on clinical isolates of Staphylococcus spp.

Staphylococcus spp. are opportunistic microorganisms known for their capacity to develop resistance against antimicrobial agents. The objective of this study was to evaluate the effect of photodynamic therapy (PDT) on 20 Staphylococcus strains isolated from the human oral cavity, including S. aureus, S. schleiferi, S. epidermidis, S. capitis, S. haemolyticus, and S. lentus. A suspension of each Staphylococcus strain (10(6) cells/mL) was submitted to PDT using methylene blue and a low power laser. The isolated effects of methylene blue, laser treatment and ciprofloxacin were also evaluated. After the experimental treatments, 0.1 mL aliquots of the suspensions were seeded onto BHI agar for determination of the number of colony-forming units (CFU/mL). The results were analyzed by analysis of variance and Tukey's test (p < 0.05). The mean reduction in bacterial counts of the strains submitted to PDT ranged from 4.89 to 6.83 CFU (log10)/mL, with the observation of a decreasing susceptibility to treatment of S. schleiferi, S. haemolyticus, S. epidermidis, S. capitis, S. aureus, and S. lentus. The results showed that PDT was effective in reducing the number of viable cells of all clinical Staphylococcus isolates studied.

Enzymatic activity, sensitivity to antifungal drugs and Baccharis dracunculifolia essential oil by Candida strains isolated from the oral cavities of breastfeeding infants and in their mothers' mouths and nipples.

Candida strains can cause oral candidosis, as well as nipples candidosis and lead to premature weaning or yeast transmission. The aim of this study was to evaluate 51 Candida isolates obtained from the oral cavities of infants during breastfeeding and mothers' oral cavities and nipples, their enzymatic activity and their sensitivity to amphotericin B, fluconazole and Baccharis dracunculifolia essential oil. Among the studied strains, 96.1% produced phospholipase and 78.4% produced proteinase. The antifungal resistance was only observed among isolates of C. albicans, for which three strains showed a resistant activity to fluconazole and one showed a resistant activity to amphotericin B. All strains were sensitive to B. dracunculifolia essential oil with MIC between 0.2 and 6.25 mg/ml. It was concluded that most of the strains showed significant enzymatic activity and were sensitive to amphotericin B and fluconazole. B. dracunculifolia essential oil inhibited the growth of all strains, including the ones resistant to commercial antifungal agents.

Susceptibility of Candida albicans, Staphylococcus aureus, and Streptococcus mutans biofilms to photodynamic inactivation: an in vitro study.

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.

Avaliação da terapia fotodinâmica em candida albicans in vitro e in vivo / In vitro and in vivo evaluation of Photodynamic Therapy on Candida albicans

Os objetivos do presente estudo foram avaliar a ação antifúngica da Terapia Fotodinâmica (TFD) em culturas planctônicas e biofilmes de Candida albicans formados in vitro e em modelo de candidose experimental em camundongos, bem como sua interferência na aderência de C. albicans às células epiteliais bucais humanas in vitro. Foi utilizada cepa padrão de C. albicans (ATCC 18804) para os ensaios. Como fonte de luz foi utilizado o Diodo Emissor de Luz (LED) com emissão de luz verde (532±10 nm) com potência de 90 mW e fotossensibilizador eritrosina nas concentrações variando de 200 a 0,39 μM para os ensaios em cultura plactônica. O biofilme foi formado em placas de 96 poços e tratado com o fotossensibilizador eritrosina na concentração de 400 μM e luz LED. 56 camundongos machos e adultos foram imunossuprimidos e inoculados com suspensões contendo 108 células/mL de C. albicans. Os animais foram submetidos a TFD mediada pelo corante eritrosina (400 μM) e irradiados por LED. Antes e após os tratamentos, foram recuperadas leveduras da cavidade bucal dos animais. As leveduras recuperadas após a TFD e grupo controle foram avaliadas quanto a interferência da TFD na aderência de C. albicans às células do epitélio bucal humano. Em seguida, os animais foram sacrificados e as línguas retiradas para análise macroscópica e histológica. Os biofilmes, línguas e lâminas de aderência foram também analisados por Microscopia Eletrônica de Varredura (MEV). A análise dos dados foi feita por ANOVA e Teste de Tukey, teste t de Student e Kruskal-Wallis (P < 0,05). A TFD aplicada à cultura planctônica de C. albicans foi dose-dependente com redução significativa a partir da menor concentração testada (0,39 μM) e 100% de morte das células a partir de 3,12 μM. A TFD em biofilme formado in vitro reduziu 0,74 log10 de C. albicans com redução de leveduras e hifas verificada por MEV. In vivo, ocorreu redução de C. albicans de 0,73 log10 e 35% de aderência às células epiteliais...

The aims of the present study were evaluate the antifungal action ofPhotodynamic Therapy (PDT) on Candida albicans planktonic culturesand biofilms formed in vitro and experimental candidosis model in mice, as well as its interference on C. albicans adherence on humans buccal epithelial cells in vitro. Standard strain of C. albicans (ATCC 18804) was used for assays. Green Light Emitting Diode (LED- 532 ± 10 nm) was used as light source with an output power of 90 mW and erythrosine photosensitizer at concentrations range from 200 to 0.39 μM for the assays on plaktonic cultures. The biofilm was formed on plates of 96- wells and treated with the erythrosine photosensitizer at a concentration of 400μM and LED light. 56 adults and males mice were immunosuppressed andinoculated with suspensions containing 108 cells/ mL of C. albicans. Theanimals were submitted to erythrosine dye- mediated PDT (400 μM) andirradiated by LED. Before and after the treatments, yeasts from the animals´ oral cavities were recovered. The yeasts recovered after PDT and control group were evaluated for interference of PDT on C. albicans adherence to humans buccal epithelial cells. Following, the animals were sacrified and their tongues taken away for macroscopic and histological analysis. The biofilms, tongues and adherence slices were also analized by Scanning Electron Microscopy (SEM). The analysis of the dates were done by ANOVA and Tukey test, Student t test and Kruskal-Wallis (P < 0.05). PDT applied on C. albicans planktonic culture was concentration dependent with significant reduction from minor concentration tested (0.39μM) and 100% of cells death from 3.12 μM. C. albicans biofilm formed invitro was reduced 0.74 log10 by PDT with reduction of yeasts and hyphaesverified by SEM. In vivo, 0.73 log10 of C. albicans and 35% adherence onbuccal epithelial cells were reduced, but there was not significant reduction of macroscopic and histological lesions. SEM revealed...