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)

Effect of Lactobacillus rhamnosus on the response of Galleria mellonella against Staphylococcus aureus and Escherichia coli infections.

This study evaluated the prophylactic effects of the live or heat-killed probiotic strain Lactobacillus rhamnosus ATCC 7469 in Galleria mellonella, inoculated with Staphylococcus aureus or Escherichia coli. L. rhamnosus suspension was prepared and a part of it was autoclaved to obtain heat-killed lactobacilli. The larvae were inoculated of these suspensions and pathogenic. The survival of the larvae was observed during 7 days and after 24 h of inoculation haemocytes counted, melanization and nitric oxide production were analyzed. Larvae survival rate increased in the group inoculated with heat-killed L. rhamnosus, however, with no statistical difference. There was a significant increase in total haemocyte counts in all test groups. Haemolymph melanization and nitric oxide production were higher in the group inoculated with L. rhamnosus and infected with S. aureus. It was concluded that, in this model of infection, heat-killed L. rhamnosus ATCC 7469 promoted greater protection in Galleria mellonella infected with S. aureus or E. coli.

Metodologia da criação de Galleria mellonella para uso como modelo de infecção e efeitos de Lactobacillus rhamnosus inativa do pelo calor in vivo e in vitro, desafiados por Staphylococcus aureuse Escherichia coli / Methodology for creation of Galleria mellonella for use as infection model and effects of inactivated Lactobacillus rhamnosus in vivo and in vitro challenged by Staphylococcus aureus and Escherichia coli

Galleria mellonella é utilizada para estudar a virulência de microorganismose a potência de antimicrobianos. Este estudo buscou estabelecer criação de lagartas utilizadas em ensaios in vivo e avaliar o efeito do probiótico Lactobacillus rhamnosus ATCC 7469, inativado pelo calor, no modelo e in vitro. Os objetivos foram: a) desenvolver uma metodologia de criação de G. mellonella, avaliando quatro dietas diferentes sobre crescimento larval, volume da hemolinfa, quantidade de hemócitos e resposta à infecção por meio da curva de sobrevivência b) avaliar os efeitos de L. rhamnosus sobre G. mellonella analisando: curva de sobrevivência; contagem de hemócitos, melanização da hemolinfa, produção de óxido nítrico na hemolinfa e os efeitos de L. rhamnosussobre macrófagos RAW 264.7 desafiados por S. aureus ou E. coli, analisando o perfil de indução de citocinas e óxido nítrico. Os resultados foram analisados estatisticamente (ANOVA e Tukey, 5%) e a curva de morte e estimativa das diferenças na sobrevivência foram determinadas por Log-rank (Mantel-Cox, 5%). As rações a base de fubá e pólen apresentaram os melhores resultados, sendo semelhantes entre si e diferentes das demais rações (p < 0,05), sendo a ração a base de fubá escolhida por apresentar resultados semelhante ao pólen e menor custo. Os resultados in vivo demonstraram diminuição na mortalidade das lagartas no grupo com inoculação de L. rhamnosus, entretanto, sem diferença estatística. Houve aumento na contagem de hemócitos quando G. mellonella foi inoculada com S. aureus e E. coli, com ou sem inoculação de L. rhamnosus, além de haver melanização da hemolinfa,demonstraram que o L. rhamnosus melhorou a resposta de G. mellonella quando desafiada por bactérias. Os resultados in vitro demonstram que L.rhamnosus induziu alta produção de TNF-α, igualmente aos demais grupos (p ≤ 0,05), não havendo produção de IL-1β e IL-6 no grupo estimulado apenas por L. rhamnosus. Nos grupos que receberam o segundo estímulo...

Galleria mellonella is used to study microorganisms virulence and antimicrobial power. This study aimed to standardize the creation of worms used in In vivo assays and evaluate the effect of heat-killed probiotic, Lactobacillus rhamnosus ATCC 7469, on this model and in in vitro studies. The objectives were: a) developing a methodology for breeding G. mellonella with four different diets influence on larval growth,hemolymph volume, quantity of hemocytes and infection response bymeans of survival curve; b) evaluating the effects of L. rhamnosus on G.mellonella by means of the following analyzes: survival curve, hemocytes counting, hemolymph melanization, nitric oxide release, and the effects ofL. rhamnosus on macrophages RAW 264.7 challenged by S. aureus or E.coli by means of cytokines and nitric oxide production. Results were statistically analyzed (ANOVA and Tukey, 5%). Death curve and estimation of differences in survival were determined by Log-rank (MantelCox, and pollen-based rations showed the best results, being similar to each other and different from the other ones (p <0.05).Cornmeal ration was chosen since it presents results similar topollen and lower cost. In vivo results showed reduction in mortality of caterpillars in the group inoculated with L. rhamnosus, with no statistical difference. Hemocyte counting increased when G. mellonella was inoculated with S. aureus and E. coli, with or with out inoculation of L. rhamnosus, add to that hemolymph melanization, showing that L.rhamnosus improved G. mellonella response challenged by bacteria. Invitro results show that L. rhamnosus induced high production of TNF-α,like other groups (p = 0.05), with no production of IL-1β and IL-6 in thegroup stimulated only by L. rhamnosus. The groups which received only the second stimulus or only the contact with S. aureus or E. coli, there wasIL-1β, IL-6 and IL-10 production. The highest nitric oxide production was observed in the groups challenged...

Metodologia da criação de Galleria mellonella para uso como modelo de infecção e efeitos de Lactobacillus rhamnosus inativa do pelo calor in vivo e in vitro, desafiados por Staphylococcus aureuse Escherichia coli / Methodology for creation of Galleria mellonella for use as infection model and effects of inactivated Lactobacillus rhamnosus in vivo and in vitro challenged by Staphylococcus aureus and Escherichia coli

Galleria mellonella é utilizada para estudar a virulência de microorganismose a potência de antimicrobianos. Este estudo buscou estabelecer criação de lagartas utilizadas em ensaios in vivo e avaliar o efeito do probiótico Lactobacillus rhamnosus ATCC 7469, inativado pelo calor, no modelo e in vitro. Os objetivos foram: a) desenvolver uma metodologia de criação de G. mellonella, avaliando quatro dietas diferentes sobre crescimento larval, volume da hemolinfa, quantidade de hemócitos e resposta à infecção por meio da curva de sobrevivência b) avaliar os efeitos de L. rhamnosus sobre G. mellonella analisando: curva de sobrevivência; contagem de hemócitos, melanização da hemolinfa, produção de óxido nítrico na hemolinfa e os efeitos de L. rhamnosussobre macrófagos RAW 264.7 desafiados por S. aureus ou E. coli, analisando o perfil de indução de citocinas e óxido nítrico. Os resultados foram analisados estatisticamente (ANOVA e Tukey, 5%) e a curva de morte e estimativa das diferenças na sobrevivência foram determinadas por Log-rank (Mantel-Cox, 5%). As rações a base de fubá e pólen apresentaram os melhores resultados, sendo semelhantes entre si e diferentes das demais rações (p < 0,05), sendo a ração a base de fubá escolhida por apresentar resultados semelhante ao pólen e menor custo. Os resultados in vivo demonstraram diminuição na mortalidade das lagartas no grupo com inoculação de L. rhamnosus, entretanto, sem diferença estatística. Houve aumento na contagem de hemócitos quando G. mellonella foi inoculada com S. aureus e E. coli, com ou sem inoculação de L. rhamnosus, além de haver melanização da hemolinfa,demonstraram que o L. rhamnosus melhorou a resposta de G. mellonella quando desafiada por bactérias. Os resultados in vitro demonstram que L.rhamnosus induziu alta produção de TNF-α, igualmente aos demais grupos (p ≤ 0,05), não havendo produção de IL-1β e IL-6 no grupo estimulado apenas por L. rhamnosus. Nos grupos que receberam o segundo estímulo ou ...

Galleria mellonella is used to study microorganisms virulence and antimicrobial power. This study aimed to standardize the creation of worms used in In vivo assays and evaluate the effect of heat-killed probiotic, Lactobacillus rhamnosus ATCC 7469, on this model and in in vitro studies. The objectives were: a) developing a methodology for breeding G. mellonella with four different diets influence on larval growth,hemolymph volume, quantity of hemocytes and infection response bymeans of survival curve; b) evaluating the effects of L. rhamnosus on G.mellonella by means of the following analyzes: survival curve, hemocytes counting, hemolymph melanization, nitric oxide release, and the effects ofL. rhamnosus on macrophages RAW 264.7 challenged by S. aureus or E.coli by means of cytokines and nitric oxide production. Results were statistically analyzed (ANOVA and Tukey, 5%). Death curve and estimation of differences in survival were determined by Log-rank (MantelCox, and pollen-based rations showed the best results, being similar to each other and different from the other ones (p <0.05).Cornmeal ration was chosen since it presents results similar topollen and lower cost. In vivo results showed reduction in mortality of caterpillars in the group inoculated with L. rhamnosus, with no statistical difference. Hemocyte counting increased when G. mellonella was inoculated with S. aureus and E. coli, with or with out inoculation of L. rhamnosus, add to that hemolymph melanization, showing that L.rhamnosus improved G. mellonella response challenged by bacteria. Invitro results show that L. rhamnosus induced high production of TNF-α,like other groups (p = 0.05), with no production of IL-1β and IL-6 in thegroup stimulated only by L. rhamnosus. The groups which received only the second stimulus or only the contact with S. aureus or E. coli, there wasIL-1β, IL-6 and IL-10 production. The highest nitric oxide production was observed in the groups challenged...

Live and Heat-Killed Lactobacillus rhamnosus ATCC 7469 May Induce Modulatory Cytokines Profiles on Macrophages RAW 264.7.

This study aimed to evaluate the capacity of Lactobacillus rhamnosus and/or its products to induce the synthesis of cytokines (TNF-α, IL-1ß, IL-4, IL-6, IL-10, and IL-12) by mouse macrophages (RAW 264.7). Three microorganism preparations were used: live L. rhamnosus (LLR) suspension, heat-killed L. rhamnosus (HKLR) suspension, and the supernatant of a heat-killed L. rhamnosus (SHKLR) suspension, which were cultured with macrophages (37°C, 5% CO2) for 2 h and 30 min. After that, cells were cultured for 16 h. The supernatants were used for the quantitation of cytokines, by ELISA. The results were compared with the synthesis induced by lipopolysaccharide (LPS) and analysed, using ANOVA and Tukey test, 5%. LLR and HKLR groups were able to significantly increase the production of TNF-α, IL-6, and IL-10 (P < 0.05). SHKLR also significantly increased the production of TNF-α and IL-10 (P < 0.05) but not IL-6 (P > 0.05). All the L. rhamnosus suspensions were not able to produce detectable levels of IL-1ß or significant levels of IL-4 and IL-12 (P > 0.05). In conclusion, live and heat-killed L. rhamnosus suspensions were able to induce the synthesis of different cytokines with proinflammatory (TNF-α and IL-6) or regulatory (IL-10) functions, suggesting the role of strain L. rhamnosus ATCC 7469 in the modulation or in the stimulation of immune responses.

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).