Caracterização de exopolissacarídeos solúveis e insolúveis de biofilmes mistos de Streptococcus mutans e Candida albicans via análises espectroscópicas
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Data
2022-05-30
Autores
Constantino, Vanessa Coronato Nogueira
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A cárie dentária é um problema de saúde pública mundial gerada por um biofilme oral patogênico resultante de uma dieta rica em açúcares. A construção desse biofilme patogênico depende da formação da matriz extracelular (MEC). A partir da adesão e co-agregação de microrganismos na película salivar, os mesmos usam a sacarose da dieta para produção de ácidos e de diversos polímeros de carboidratos denominados exopolissacarídeos (EPS), que são os principais componentes da matriz extracelular. Os principais exopolissacarídeos são glucanos. Com o passar do tempo, os microrganismos presentes no biofilme produzem ácidos que levam à desmineralização da superfície dentária, ou seja, causam as lesões de cárie. Candida albicans tem sido encontrada em associação com S. mutans em biofilmes de lesões de cárie de rápida progressão. Um modelo in vitro demonstrou que a formação do biofilme com a associação destes microrganismos possui uma maior quantidade de MEC, principalmente os exopolissacarídeos insolúveis, quando comparados com um biofilme simples. Streptococcus mutans produz predominantemente glucanos com ligações glicosídicas do tipo α-glucanos, enquanto Candida albicans, β-glucanos. Dependendo do carbono utilizado para a ligação destes glucanos para formação dos exopolissacarídeos, observa-se características solúveis, com ligações α ou β do tipo 1,6-glucano, e insolúveis, com ligações α ou β do tipo 1,3-glucano principalmente. Porém, ainda são desconhecidas quais as características de exopolissacarídeos (proporção e tipos de ligações glicosídicas) na MEC de biofilme misto de S. mutans e C. albicans. Então, o objetivo desse trabalho foi caracterizar os exopolissacarídeos solúveis e insolúveis de biofilmes mistos de S. mutans e C. albicans via análises espectroscópicas. Foi realizada a formação dos biofilmes in vitro de S. mutans UA159 e C. albicans SC5314 sobre 24 discos de hidroxiapatita contendo película salivar (CEP: 31725114.8.0000.5416). O meio de cultura utilizado durante a formação do biofilme foi o triptona com extrato de levedura suplementado com 1% de sacarose, sobre discos em posição vertical em placas de 24 poços. O inóculo do biofilme foi semeado em ágar sangue como forma de controle. Foram realizadas trocas de meio de cultura nos tempos de 19 e 27 horas. Ao completar 43 horas de desenvolvimento, os biofilmes foram removidos e uma porção foi semeada em ágar sangue. Os biofilmes foram processados para extração dos exopolissacarídeos solúveis e insolúveis em água. O método colorimétrico usado para a quantificação desses polímeros da matriz foi o fenol-ácido sulfúrico. A produção de exopolissacarídeos insolúveis superou significantemente a dos solúveis. Foram realizados 4 testes, sendo encontrado uma média de 3,95 mg de exopolissacarídeos solúveis e 9,44 mg de exopolissacarídeos insolúveis. A proteção dos microrganismos embebidos na matriz favorece a maturação do biofilme, tornando-o um potencial foco de desmineralização dental. Reduzir de alguma forma a organização deste tipo de biofilme depende em ter ciência desta alta produção de matriz e sua característica insolúvel. Este conhecimento pode ser usado para focar em princípios ativos que diminuam ou dificultem a produção desse tipo de matriz. Uma etapa a ser realizada ainda é a identificação dos tipos de ligações glicosídicas predominantes nos exopolissacarídeos isolados da matriz dos biofilmes.
Dental caries is a worldwide public health problem generated by a pathogenic oral biofilm resulting from a diet rich in sugars. The construction of this pathogenic biofilm depends on the formation of the extracellular matrix (ECM). From the adhesion and co-aggregation of microorganisms in the salivary film, they use the sucrose in the diet to produce acids and various carbohydrate polymers called exopolysaccharides (EPS), which are the main components of the extracellular matrix. The main exopolysaccharides are glucans. Over time, the microorganisms present in the biofilm produce acids that lead to demineralization of the tooth surface, i.e. cause caries lesions. Candida albicans has been found in association with S. mutans in biofilms of rapidly progressing caries lesions. An in vitro model showed that biofilm formation with the association of these microorganisms has a higher amount of ECM, especially insoluble exopolysaccharides, when compared to a simple biofilm. Streptococcus mutans produces predominantly glucans with glycosidic linkages of the α-glucan type, while Candida albicans, β-glucans. Depending on the carbon used for binding these glucans to form exopolysaccharides, soluble features are observed, with α- or β-bonds of the 1,6-glucan type, and insoluble ones, with α- or β-bonds of the 1,3-glucan type mainly. However, the characteristics of exopolysaccharides (proportion and types of glycosidic bonds) in the MEC of mixed biofilm of S. mutans and C. albicans are still unknown. Therefore, the aim of this work was to characterize soluble and insoluble exopolysaccharides of mixed biofilms of S. mutans and C. albicans via spectroscopic analysis. The in vitro biofilm formation of S. mutans UA159 and C. albicans SC5314 was performed on 24 hydroxyapatite disks containing salivary film (ZIP code: 31725114.8.0000.5416). The culture medium used during biofilm formation was tryptone with yeast extract supplemented with 1% sucrose, on discs in vertical position in 24-well plates. The biofilm inoculum was seeded on blood agar as a control. The culture medium was changed at 19 and 27 hours. At 43 hours of development, the biofilms were removed and a portion was seeded on blood agar. The biofilms were processed for extraction of water soluble and insoluble exopolysaccharides. The colorimetric method used for the quantification of these polymers in the matrix was phenol-sulfuric acid. The production of insoluble exopolysaccharides significantly exceeded that of soluble exopolysaccharides. Four tests were performed, and an average of 3.95 mg of soluble exopolysaccharides and 9.44 mg of insoluble exopolysaccharides were found. The protection of microorganisms embedded in the matrix favors the maturation of the biofilm, making it a potential focus of tooth demineralization. To somehow reduce the organization of this type of biofilm depends on being aware of this high matrix production and its insoluble characteristic. This knowledge can be used to focus on active ingredients that decrease or hinder the production of this type of matrix. A further step to be taken is the identification of the types of glycosidic bonds predominant in exopolysaccharides isolated from the biofilm matrix.
Dental caries is a worldwide public health problem generated by a pathogenic oral biofilm resulting from a diet rich in sugars. The construction of this pathogenic biofilm depends on the formation of the extracellular matrix (ECM). From the adhesion and co-aggregation of microorganisms in the salivary film, they use the sucrose in the diet to produce acids and various carbohydrate polymers called exopolysaccharides (EPS), which are the main components of the extracellular matrix. The main exopolysaccharides are glucans. Over time, the microorganisms present in the biofilm produce acids that lead to demineralization of the tooth surface, i.e. cause caries lesions. Candida albicans has been found in association with S. mutans in biofilms of rapidly progressing caries lesions. An in vitro model showed that biofilm formation with the association of these microorganisms has a higher amount of ECM, especially insoluble exopolysaccharides, when compared to a simple biofilm. Streptococcus mutans produces predominantly glucans with glycosidic linkages of the α-glucan type, while Candida albicans, β-glucans. Depending on the carbon used for binding these glucans to form exopolysaccharides, soluble features are observed, with α- or β-bonds of the 1,6-glucan type, and insoluble ones, with α- or β-bonds of the 1,3-glucan type mainly. However, the characteristics of exopolysaccharides (proportion and types of glycosidic bonds) in the MEC of mixed biofilm of S. mutans and C. albicans are still unknown. Therefore, the aim of this work was to characterize soluble and insoluble exopolysaccharides of mixed biofilms of S. mutans and C. albicans via spectroscopic analysis. The in vitro biofilm formation of S. mutans UA159 and C. albicans SC5314 was performed on 24 hydroxyapatite disks containing salivary film (ZIP code: 31725114.8.0000.5416). The culture medium used during biofilm formation was tryptone with yeast extract supplemented with 1% sucrose, on discs in vertical position in 24-well plates. The biofilm inoculum was seeded on blood agar as a control. The culture medium was changed at 19 and 27 hours. At 43 hours of development, the biofilms were removed and a portion was seeded on blood agar. The biofilms were processed for extraction of water soluble and insoluble exopolysaccharides. The colorimetric method used for the quantification of these polymers in the matrix was phenol-sulfuric acid. The production of insoluble exopolysaccharides significantly exceeded that of soluble exopolysaccharides. Four tests were performed, and an average of 3.95 mg of soluble exopolysaccharides and 9.44 mg of insoluble exopolysaccharides were found. The protection of microorganisms embedded in the matrix favors the maturation of the biofilm, making it a potential focus of tooth demineralization. To somehow reduce the organization of this type of biofilm depends on being aware of this high matrix production and its insoluble characteristic. This knowledge can be used to focus on active ingredients that decrease or hinder the production of this type of matrix. A further step to be taken is the identification of the types of glycosidic bonds predominant in exopolysaccharides isolated from the biofilm matrix.
Descrição
Palavras-chave
Biofilmes, Polissacarídeos, Matriz extracelular, Biofilms, Polysaccharides, Extracellular matrix