Two sporulated Bacillus enhance immunity in Galleria mellonella protecting against Candida albicans.

The aim of this study was to evaluate the effects of Bacillus subtilis and Bacillus atrophaeus on Galleria mellonella immunity challenged by Candida albicans. Firstly, we analyzed the susceptibility of G. mellonella to bacilli (vegetative and sporulating forms). It was found that both vegetative and sporulating forms were not pathogenic to G. mellonella at a concentration of 1 × 104 cells/larva. Next, larvae were pretreated with two species of Bacillus, in the vegetative and sporulating forms, and then challenged with C. albicans. In addition, the gene expression of antimicrobial peptides (AMPs) such as Gallerimycin, Gloverin, Cecropin-D and Galiomicin was investigated. Survival rates increased in the Bacillus treated larvae compared with control larvae inoculated with C. albicans only. Cells and spores of Bacillus spp. upregulated Gloverin, Galiomicin and Gallerimycin genes in relation to the control group (PBS + PBS). When these larvae were infected with C. albicans, the group pretreated with spores of B. atrophaeus and B. subtilis showed a greater increase in expression of Galiomycin (49.08-fold and 13.50-fold) and Gallerimycin (27.88-fold and 68.15-fold), respectively, compared to the group infected with C. albicans only (p = 0.0001). After that, we investigated the effects of B. subtilis and B. atrophaeus on immune system of G. mellonella evaluating the number of hemocytes, quantification of melanization, cocoon formation and colony forming units (CFU) count. Hemocyte count increased in response to stimulation by Bacillus, and a higher increase was achieved when larvae were inoculated with B. subtilis spores (p = 0.0011). In the melanization assay, all groups tested demonstrated lower production of melanin compared to that in the phosphate-buffered saline (PBS) group. In addition, full cocoon formation was observed in all groups analyzed, which corresponded to a healthier wax worm. Hemolymph culture revealed higher growth of B. atrophaeus and B. subtilis in the groups inoculated with spores. We concluded that spores and cells of B. atrophaeus and B. subtilis stimulated the immune system of G. mellonella larvae and protected them of C. albicans infection.

Effects of Bacillus subtilis on Candida albicans: biofilm formation, filamentation and gene expression / Efeitos de Bacillus subtilis sobre Candida albicans: formação de biofilme, filamentação e expressão gênica

Objective: The aim of this study was evaluate the effect of Bacillus subtilis on Candida albicans biofilm formation and filamentation by evaluating the gene expression of ALS3, HWP1, BCR1, EFG1 and TEC1. Material and Methods: Mixed (C. albicans / B.subtilis) and monotypic biofilms were cultured in plates at 37°C for 48 h under shaking for counting viable cells (CFU / mL) and analysis of gene expression by real-time PCR. The C. albicans filamentation assay was performed in medium containing 10% fetal bovine serum at 37°C for 6 hours. Data was analysed by t-Student and Mann­ Whitney tests. Results: B. subtilis reduced the biofilm formation of C. albicans in 1 log when cultured in the same environment (p<0.0001). In addition, it significantly reduced the yeast - hypha transition affecting the morphology of C. albicans. Among all of the analyzed genes, the ALS3 and HWP1 genes were the most affected, achieving 111.1- and 333.3- fold decreases in the C. albicans biofilms associated with B. subtilis, respectively. Conclusion: B. subtilis reduced the biofilm formation and filamentation of C. albicans by negatively regulating the ALS3, HWP1, BCR1, EFG1 and TEC1 genes that are essential for the production of biofilm and hyphae. (AU)

Objetivo: O objetivo deste estudo foi avaliar o efeito de Bacillus subtilis sobre a formação de biofilme e filamentação de Candida albicans através da avaliação da expressão dos genes ALS3, HWP1, BCR1, EFG1 and TEC1. Material e métodos: Biofilmes monotípicos e mistos (C. albicans / B.subtilis) foram cultivados em placas a 37°C por 48 h sob agitação, para a contagem de células viáveis (UFC/mL) e para a análise da expressão gênica por PCR em tempo real. O ensaio de filamentação de C. albicans foi realizado em meio contendo 10% de soro fetal bovino a 37°C por 6 h. Os dados obtidos foram analisados por testes t-Student e Mann­Whitney. Resultados: B.subtilis reduziu em 1 log a formação de biofilme por C. albicans quando cultivados no mesmo ambiente (p<0.0001). Além disso, reduziu significantemente a transição de levedura para hifa, afetando assim, a morfologia de C. albicans. Em relação aos genes analisados, os genes ALS3 e HWP1 foram os mais regulados negativamente, com uma diminuição de 111,1 e 333,3 vezes, respectivamente, na sua expressão em biofilmes de C. albicans associados a B. subtilis. Conclusão: B. subtilis reduziu a filamentação e a formação de biofilme de C. albicans através da regulação negativa dos genes ALS3, HWP1, BCR1, EFG1 e TEC1, que são essenciais na produção de hifas e de biofilme. (AU)

Action of antimicrobial photodynamic therapy on heterotypic biofilm: Candida albicans and Bacillus atrophaeus.

The increase in survival and resistance of microorganisms organized in biofilms demonstrates the need for new studies to develop therapies able to break this barrier, such as photodynamic therapy, which is characterized as an alternative, effective, and non-invasive treatment. The objective was to evaluate in vitro the effect of antimicrobial photodynamic therapy on heterotypic biofilms of Candida albicans and Bacillus atrophaeus using rose bengal (12.5 µM) and light-emitting diode (LED) (532 nm and 16.2 J). We used standard strains of B. atrophaeus (ATCC 9372) and C. albicans (ATCC 18804). The biofilm was formed in the bottom of the plate for 48 h. For the photodynamic therapy (PDT) experimental groups, we added 100 µL of rose bengal with LED (P+L+), 100 µL of rose bengal without LED (P+L-), 100 µL of NaCl 0.9 % solution with LED (P-L+), and a control group without photosensitizer or LED (P-L-). The plates remained in agitation for 5 min (pre-irradiation) and were irradiated with LED for 3 min, and the biofilm was detached using an ultrasonic homogenizer for 30 s. Serial dilutions were plated in BHI agar and HiChrom agar and incubated at 37 °C/48 h. There was a reduction of 33.92 and 29.31 % of colony-forming units per milliliter (CFU/mL) for C. albicans and B. atrophaeus, respectively, from the control group to the group subjected to PDT. However, statistically significant differences were not observed among the P+L+, P+L-, P-L+, and P-L- groups. These results suggest that antimicrobial photodynamic therapy using rose bengal (12.5 µM) with a pre-irradiation period of 5 min and LED for 3 min was not enough to cause a significant reduction in the heterotypic biofilms of C. albicans and B. atrophaeus.

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.

Influence of artificial saliva in biofilm formation of Candida albicans in vitro.

Due to the increase in life expectancy, new treatments have emerged which, although palliative, provide individuals with a better quality of life. Artificial saliva is a solution that contains substances that moisten a dry mouth, thus mimicking the role of saliva in lubricating the oral cavity and controlling the existing normal oral microbiota. This study aimed to assess the influence of commercially available artificial saliva on biofilm formation by Candida albicans. Artificial saliva I consists of carboxymethylcellulose, while artificial saliva II is composed of glucose oxidase, lactoferrin, lysozyme and lactoperoxidase. A control group used sterile distilled water. Microorganisms from the oral cavity were transferred to Sabouraud Dextrose Agar and incubated at 37 °C for 24 hours. Colonies of Candida albicans were suspended in a sterile solution of NaCl 0.9%, and standardisation of the suspension to 106 cells/mL was achieved. The acrylic discs, immersed in artificial saliva and sterile distilled water, were placed in a 24-well plate containing 2 mL of Sabouraud Dextrose Broth plus 5% sucrose and 0.1 mL aliquot of the Candida albicans suspension. The plates were incubated at 37 °C for 5 days, the discs were washed in 2 mL of 0.9% NaCl and placed into a tube containing 10 mL of 0.9% NaCl. After decimal dilutions, aliquots of 0.1 mL were seeded on Sabouraud Dextrose Agar and incubated at 37 °C for 48 hours. Counts were reported as CFU/mL (Log10). A statistically significant reduction of 29.89% (1.45 CFU/mL) of Candida albicans was observed in saliva I when compared to saliva II (p = 0.002, considering p≤0.05).

Influence of artificial saliva in biofilm formation of Candida albicans in vitro

Due to the increase in life expectancy, new treatments have emerged which, although palliative, provide individuals with a better quality of life. Artificial saliva is a solution that contains substances that moisten a dry mouth, thus mimicking the role of saliva in lubricating the oral cavity and controlling the existing normal oral microbiota. This study aimed to assess the influence of commercially available artificial saliva on biofilm formation by Candida albicans. Artificial saliva I consists of carboxymethylcellulose, while artificial saliva II is composed of glucose oxidase, lactoferrin, lysozyme and lactoperoxidase. A control group used sterile distilled water. Microorganisms from the oral cavity were transferred to Sabouraud Dextrose Agar and incubated at 37°C for 24 hours. Colonies of Candida albicans were suspended in a sterile solution of NaCl 0.9%, and standardisation of the suspension to 106 cells/mL was achieved. The acrylic discs, immersed in artificial saliva and sterile distilled water, were placed in a 24-well plate containing 2 mL of Sabouraud Dextrose Broth plus 5% sucrose and 0.1 mL aliquot of the Candida albicans suspension. The plates were incubated at 37°C for 5 days, the discs were washed in 2 mL of 0.9% NaCl and placed into a tube containing 10 mL of 0.9% NaCl. After decimal dilutions, aliquots of 0.1 mL were seeded on Sabouraud Dextrose Agar and incubated at 37°C for 48 hours. Counts were reported as CFU/mL (Log10). A statistically significant reduction of 29.89% (1.45 CFU/mL) of Candida albicans was observed in saliva I when compared to saliva II (p = 0.002, considering p≤0.05).

Terapia fotodinâmica em esporos de bacillus atrophaeus e bacillus subtilis: estudos com LASER, LED, azul de metileno, rosa bengala e verde malaquita / Photodynamic therapy of spores of Bacillus atrophaeus and Bacillus subtilis: studies with laser, LED, methylene blue, rose bengal and malachite green

Os esporos de Bacillus spp. são encontrados amplamente distribuídos na natureza, podendo ocasionar contaminação do meio ambiente e, eventualmente, doenças ao homem e animais. Em resposta a crescente resistência microbiana, a terapia fotodinâmica (PDT) surge para atuar como um tratamento alternativo e eficaz. O presente estudo teve como objetivo comparar e avaliar a ação exercida pelo LASER de baixa intensidade (vermelho visível) e pelo diodo emissor de luz verde (LED) em esporos de Bacillus atrophaeus e Bacillus subtilis na PDT, com o uso dos fotossensibilizadores azul de metileno (37,5 M), rosa bengala (12,5 M) e verde malaquita (300 M). Utilizou-se cepa padrão de Bacillus atrophaeus (ATCC 9372) e Bacillus subtilis (ATCC 19659). As cepas foram cultivadas, durante 7 dias, em Ágar Nutriente acrescidas de 0,003% de sulfato de manganês e analisadas quanto a formação de esporos (coloração de Wirtz-Conklin). Os esporos foram suspensos em água destilada esterilizada e centrifugados por 10 min a 653 Xg. As suspensões receberam choque térmico de 70 °C por 30 min. As suspensões foram padronizadas com 106 células/mL. Em placas de 96 poços adicionou-se 0,1 mL de suspensão dos esporos de Bacillus atrophaeus ou de Bacillus subtilis e 0,1 mL do fotossensibilizador ou de solução de NaCl a 0,9%, sendo agitadas durante 5, 10 e 30 minutos e irradiadas. Realizaram-se diluições seriadas. Alíquotas de 0,1 mL das diluições foram semeadas em placas com Ágar Infusão Cérebro-Coração e incubadas a 37 °C por 48 horas. Os resultados foram analisados estatisticamente (ANOVA, teste de Tukey, p<0,05). As maiores reduções observadas em UFC/mL (Log10) para os esporos de Bacillus atrophaeus foram 0,71 Log10 para azul de metileno (10 min); 2,49 Log10 para rosa bengala (30 min) e 0,42 Log10 para verde malaquita (10 min). Em relação aos esporos de Bacillus subtilis as maiores reduções foram 0,82 Log10 para azul de metileno (10 min); 3,86 Log10 para rosa bengala (30 min) e 0,63 Log10 para ...

The spores of Bacillus spp. are found widely distributed in nature, which may cause contamination of the environment and eventually diseases to humans and animals. In response to increasing microbial resistance, photodynamic therapy (PDT) appears to act as an alternative treatment and effective. The present study aimed to compare and evaluate the action exerted by low intensity laser and green light emitting diode (LED) in spores of Bacillus atrophaeus and Bacillus subtilis in PDT, with the use of photosensitizers blue methylene (37.5 M), rose bengal (12.5 M) and malachite green (300 M). It was used a standard strain of Bacillus atrophaeus (ATCC 9372) and Bacillus subtilis (ATCC 19659). The strains were cultivated for 7 days in Nutrient Agar added to 0.003% of manganese sulphate and analyzed for the formation of spores (Wirtz-Conklin staining). The spores were suspended in sterile distilled water and centrifuged for 10 min at 653 Xg. The suspensions were heat shock at 70 °C for 30 min. The suspensions were standardized to 106 cells / mL. In 96-well plates were added 0.1 ml of Bacillus subtilis or Bacillus atrophaeus and 0.1 ml of the photosensitizer or NaCl 0.9%, stirred for 5, 10 and 30 minutes and irradiated. Serial dilutions were performed. Aliquots of 0.1 mL of the dilutions were plated on Brain Heart Infusion Agar and incubated at 37 °C for 48 hours. The results were analyzed statistically (ANOVA, Tukey test, p<0.05). The highest observed reductions in CFU/ml (Log10) for Bacillus atrophaeus were 0.71 Log10 for methylene blue (10 minutes), 2.49 Log10 for rose bengal (30 min) and 0.42 Log10 for malachite green (10 min). In relation to Bacillus subtilis the largest reductions were 0.82 Log10 for methylene blue (10 minutes), 3.86 Log10 for rose bengal (30 min) and 0.63 Log10 for malachite green (10 min). The conclusion was that the PDT was effective against Bacillus subtilis and Bacillus atrophaeus the parameters tested