Efeito do hexametafosfato de sódio, associado ou não ao fluoreto, no biofilme misto contendo Streptococcus mutans e Candida albicans
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Data
2018-10-26
Autores
Hosida, Thayse Yumi [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O presente estudo teve como objetivo verificar o efeito do hexametafosfato de sódio (HMP), associado ou não ao fluoreto (F), sobre a composição orgânica, inorgânica e no pH do biofilme mistos de S. mutans e C. albicans formados in vitro. Para todos os estudos, os biofilmes foram formados em poços de placas de microtitulação, colocando uma suspensão (1x107 células/mL C. albicans + 1x108 células/mL S. mutans) em saliva artificial suplementada com sacarose (0,4%), a qual tinha metade de seu conteúdo renovada a cada 24 horas. Os biofilmes foram tratados três vezes (72, 78 e 96 horas de formação), por um minuto, com soluções contendo HMP (0.25, 0.5 ou 1%) com ou sem 500 ppm F, além de soluções contendo 500 e 1100 ppm F. A saliva artificial foi utilizada como controle negativo. Para o estudo microbiológico, após o terceiro tratamento foram realizados os testes de quantificação de células cultiváveis (CFU), biomassa total (teste colorimétrico de cristal violeta – CV), atividade metabólica (redução de XTT) e quantificação dos componentes da matriz extracelular (proteína, carboidrato e ácidos nucleicos). Todos os ensaios foram realizados em triplicata, em três ocasiões diferentes. Os resultados foram submetidos à análise de variância a um critério, seguida pelo teste Fisher LSD (p <0.05). O HMP apresentou efeito redutor principalmente na biomassa, metabolismo e nos componentes da matriz extracelular do biofilme. Biofilmes formados por 96 h formam expostos a três diferentes concentrações de sacarose (10, 20 ou 30%) durante 1, 3 ou 5 min. O pH foi medido antes da exposição à sacarose, imediatamente após sua remoção e após 1, 3, 5 e 10 min após a retirada da sacarose. Os resultados foram submetidos à análise de variância a três critérios, seguida pelo teste Fisher LSD (p <0.05). O biofilme exposto a solução de sacarose a 20% por 3 min exibiu padrão de alteração de pH semelhante ao observado in vivo. Para o estudo da concentração de F, Ca, e Pi, após o período de tratamento, estes foram analisados no biofilme total e no fluido do biofilme após a mensuração do pH do biofilme. Em outro conjunto de experimentos, após o terceiro tratamento (96 h de formação de biofilme) o biofilme foi exposto, por 3 minutos, à solução de sacarose a 20%. Esta foi removida e, após 1 minuto, analisou-se o pH do meio e as concentrações de F, Ca, e Pi tanto na biomassa como no fluido do biofilme. Os dados foram submetidos à análise de variância a dois critérios, seguida pelo teste de Fisher LSD (p <0.05). O tratamento com HMP aumentou a concentração de F e Pi no fluido do biofilme antes da exposição à sacarose, além de manter o pH do meio mais próximo do neutro, mesmo após a exposição do biofilme à sacarose. Assim, é possível concluir que o HMP interfere na biomassa, metabolismo, composição orgânica e inorgânica, bem como no pH do biofilme testado
The aim of the present study was to verify the effect of sodium hexametaphosphate (HMP), associated or not to fluoride (F), on the inorganic, organic composition and pH of the mixed biofilm of S. mutans and C. albicans, formed in vitro. For all studies, the biofilms were formed in wells of microtiter plates by placing a suspension (1 x 107 cells/mL C. albicans + 1x108 cells/mL S. mutans) in artificial saliva supplemented with sucrose (0,4%), which had half of its content renewed every 24 hours. Biofilms were treated three times (72, 78 and 96 hours of formation), for one minute, with solutions containing HMP (0.25, 0.5 or 1%) with or without 500 ppm F, as well as solutions containing 500 and 1100 ppm F. Artificial saliva was used as a negative control. For the microbiological study, the following tests were performed: quantification of cultivable cells (UFC), total biomass (colorimetric crystal violet test - CV), metabolic activity (XTT reduction) and quantification of matrix components (protein, carbohydrate and nucleic acid). All assays were performed in triplicate on three different occasions. The results were submitted to one-way analysis of variance, followed by the Fisher LSD’s test (p <0.05). HMP showed a reducing effect mainly on the biomass, metabolism and components of the extracellular matrix of the biofilm. Biofilms formed for 96 h were exposed to three different concentrations of sucrose (10, 20 or 30%) for 1, 3 or 5 min. The pH was measured before exposure to sucrose, immediately after its removal and after 1, 3, 5 and 10 min after removal of the sucrose. The results were submitted to 3- way analysis of variance, followed by the Fisher LSD test (p <0.05). The biofilm exposed to 20% sucrose solution for 3 min exhibited a pattern of pH change similar to that observed in vivo. For the study of the concentrations of F, Ca, and Pi, these ions were analyzed in the total biofilm and in the biofilm fluid after treatment with the test solutions and after the pH measurement of the biofilm. In another set of experiments, after the third treatment (96 h of biofilm formation), the biofilms were exposed for 3 minutes to a 20% sucrose solution. This was removed and after 1 minute the biofilms were collected, and the pH of the medium and F, Ca, and Pi concentrations were determined both in the biomass and in the biofilm fluid. The data were submitted to two-way analysis of variance, followed by Fisher LSD's test (p <0.05). Treatment with HMP increased F and Pi concentration of the biofilm fluid prior sucrose exposition, and maintained the pH of the medium close to neutral values even after exposure of the biofilm to sucrose. Thus, it is possible to conclude that HMP interferes in the biomass, metabolism, organic and inorganic composition and the pH of the biofilm tested
The aim of the present study was to verify the effect of sodium hexametaphosphate (HMP), associated or not to fluoride (F), on the inorganic, organic composition and pH of the mixed biofilm of S. mutans and C. albicans, formed in vitro. For all studies, the biofilms were formed in wells of microtiter plates by placing a suspension (1 x 107 cells/mL C. albicans + 1x108 cells/mL S. mutans) in artificial saliva supplemented with sucrose (0,4%), which had half of its content renewed every 24 hours. Biofilms were treated three times (72, 78 and 96 hours of formation), for one minute, with solutions containing HMP (0.25, 0.5 or 1%) with or without 500 ppm F, as well as solutions containing 500 and 1100 ppm F. Artificial saliva was used as a negative control. For the microbiological study, the following tests were performed: quantification of cultivable cells (UFC), total biomass (colorimetric crystal violet test - CV), metabolic activity (XTT reduction) and quantification of matrix components (protein, carbohydrate and nucleic acid). All assays were performed in triplicate on three different occasions. The results were submitted to one-way analysis of variance, followed by the Fisher LSD’s test (p <0.05). HMP showed a reducing effect mainly on the biomass, metabolism and components of the extracellular matrix of the biofilm. Biofilms formed for 96 h were exposed to three different concentrations of sucrose (10, 20 or 30%) for 1, 3 or 5 min. The pH was measured before exposure to sucrose, immediately after its removal and after 1, 3, 5 and 10 min after removal of the sucrose. The results were submitted to 3- way analysis of variance, followed by the Fisher LSD test (p <0.05). The biofilm exposed to 20% sucrose solution for 3 min exhibited a pattern of pH change similar to that observed in vivo. For the study of the concentrations of F, Ca, and Pi, these ions were analyzed in the total biofilm and in the biofilm fluid after treatment with the test solutions and after the pH measurement of the biofilm. In another set of experiments, after the third treatment (96 h of biofilm formation), the biofilms were exposed for 3 minutes to a 20% sucrose solution. This was removed and after 1 minute the biofilms were collected, and the pH of the medium and F, Ca, and Pi concentrations were determined both in the biomass and in the biofilm fluid. The data were submitted to two-way analysis of variance, followed by Fisher LSD's test (p <0.05). Treatment with HMP increased F and Pi concentration of the biofilm fluid prior sucrose exposition, and maintained the pH of the medium close to neutral values even after exposure of the biofilm to sucrose. Thus, it is possible to conclude that HMP interferes in the biomass, metabolism, organic and inorganic composition and the pH of the biofilm tested
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Palavras-chave
Fosfatos, Flúor, Biofilmes, Streptococcus mutans, Candida albicans, Phosphates, Fluoride, Biofilms