Influência de Streptococcus mutans na construção da matriz durante o desenvolvimento de biofilmes microcosmos expostos a desafio nutricional
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2021-03-30
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Universidade Estadual Paulista (Unesp)
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A cárie é a doença crônica mais prevalente no mundo. Sua fisiopatologia está associada a fatores como: biofilmes (microrganismos e matriz extracelular); dieta rica em carboidratos; tecidos dentários e tempo de interação. Embora muitos modelos laboratoriais tenham simplificado o estudo da cárie focando apenas no entendimento em microrganismos específicos, mimetizar a cavidade bucal para o controle das variáveis que influenciam a cárie ainda é um dos maiores desafios. O objetivo do estudo foi avaliar de forma longitudinal a importância de Streptococcus mutans na construção da matriz extracelular e na estruturação de biofilmes polimicrobianos associados a cárie. Foram desenvolvidos biofilmes microcosmos para avaliar o desenvolvimento destas comunidades. Para isso a microbiota salivar humana foi inoculada sobre discos de esmalte bovino em quatro desafios nutricionais ou modelos de biofilme in vitro. Foram avaliados: pH dos meios de cultura; microdureza e rugosidade dos discos de esmalte; biomassa total e dinâmica populacional (via UFC. mL-1) dos biofilmes. Entre os modelos estudados o modelo 3 (M3) – ‘Três refeições diárias’ apresentou características mais próximas da cavidade bucal, uma vez que os valores de pH permaneceram próximos do neutro, apresentaram desmineralização sutil e progressiva do esmalte, não favoreceram grupos microbianos específicos e permitiram o acúmulo do biofilme, o que foi evidenciado pelo aumento da biomassa e da microbiota total. M3 foi utilizado para testar duas estratégias de inoculação única (UNI) e múltipla (MULTI), a segunda visando simular a colonização secundária dos microrganismos. Foram avaliados: pH dos meios de cultura; os biofilmes (arquitetura; dinâmica populacional microbiana via UFC. mL-1, sequenciamento de DNA e qPCR; biomassa insolúvel e componentes da matriz extracelular); e desmineralização do esmalte (via microdureza). As flutuações observadas no pH foram semelhantes as relatadas na cavidade bucal, observando-se uma acidificação após ingestão de carboidratos e a ação tamponante da saliva, simulada com o retorno do caldo de cultura de saliva com um pH neutro. A configuração espacial foi semelhante a encontrada em biofilmes orais associados com cárie, representada por arquiteturas do tipo ouriços (hedgehog) e espigas de milho (corncob). A representatividade dos estreptococos do grupo mutans foi ≤ 2 % da microbiota total cultivável. O gênero Streptococcus foi o mais abundante, porém, S. mutans não foi detectado em nenhuma amostra via sequenciamento. Na estratégia MULTI foi detectada uma maior diversidade microbiana. Ao quantificar o número de células bacterianas via qPCR observou-se que S. mutans representa < 0,01 % da saliva e < 0,005 % nos biofilmes. Conforme o envelhecimento do biofilme, houve um aumento da biomassa, dos exopolissacarídeos e do DNA extracelular na matriz nas duas estratégias. O acúmulo destes componentes está associado a uma estrutura coesa que pode favorecer a formação de nichos ácidos. O amadurecimento do biofilme e a desmineralização do esmalte aconteceu de forma independente à baixa proporção de S. mutans e da estratégia de inoculação, diminuindo o protagonismo de certas espécies em uma doença que possuí caráter multifatorial.
Dental caries is the most prevalent chronic disease in the world. Its pathophysiology is associated with factors such as biofilms (microorganisms and extracellular matrix), carbohydrate-rich diet, dental tissues, and interaction time. Although many laboratory models have simplified the study of caries by focusing only on understanding specific microorganisms, mimicking the oral cavity to control the variables that influence caries is still one of the greatest challenges. The study aimed to evaluate longitudinally the importance of Streptococcus mutans constructing the extracellular matrix and structuring polymicrobial biofilms associated with dental caries. Microcosm biofilms were developed to assess the dynamics of these communities. For this purpose, the human salivary microbiota was inoculated on bovine enamel discs in four biofilm models in vitro. The following were assessed: pH culture media; microhardness and roughness of enamel discs; total biomass and population dynamics (via CFU mL-1) of biofilms. Among the models studied, model 3 (M3) - 'Three meals a day' presented characteristics closest to the oral cavity since the pH values remained close to neutral, subtle and progressive enamel demineralization, did not favor specific microbial groups, and allowed the accumulation of biofilm, which was evidenced by the increase in biomass and total microbiota. M3 was used to test two single (UNI) and multiple (MULTI) inoculation strategies, the second simulating the secondary colonization of microorganisms. The following were assessed: pH of the culture media; biofilms (architecture; microbial population dynamics via CFU mL-1, DNA and qPCR sequencing; insoluble biomass and matrix components); and enamel demineralization (via microhardness). The fluctuations observed in the pH were similar to those reported in the oral cavity; there was acidification after exposure to carbohydrates, and the buffering action of the saliva was simulated by returning biofilms to a saliva culture broth with a neutral pH. The spatial configuration was similar to that found in oral biofilms associated with caries, represented by hedgehogs and corn cobs architectures. The representativeness of streptococci from the mutans group was ≤ 2% of the total cultivable microbiota. The genus Streptococcus was the most abundant; however, S. mutans was not detected in any sample via sequencing. In the MULTI strategy, a greater microbial diversity was detected. When quantifying the number of bacterial cells via qPCR, it was observed that S. mutans represents <0.01% of saliva and <0.005% in biofilms. As the biofilm aged, there was an increase in biomass, exopolysaccharides, and extracellular DNA in the extracellular matrix in both inoculation strategies. The accumulation of these components is associated with a cohesive structure that can favor the formation of acidic niches. The maturation of the biofilm and enamel demineralization occurred independently of the low proportion of S. mutans and the inoculation strategy, reducing the role of specific species in the multifactorial disease.
Dental caries is the most prevalent chronic disease in the world. Its pathophysiology is associated with factors such as biofilms (microorganisms and extracellular matrix), carbohydrate-rich diet, dental tissues, and interaction time. Although many laboratory models have simplified the study of caries by focusing only on understanding specific microorganisms, mimicking the oral cavity to control the variables that influence caries is still one of the greatest challenges. The study aimed to evaluate longitudinally the importance of Streptococcus mutans constructing the extracellular matrix and structuring polymicrobial biofilms associated with dental caries. Microcosm biofilms were developed to assess the dynamics of these communities. For this purpose, the human salivary microbiota was inoculated on bovine enamel discs in four biofilm models in vitro. The following were assessed: pH culture media; microhardness and roughness of enamel discs; total biomass and population dynamics (via CFU mL-1) of biofilms. Among the models studied, model 3 (M3) - 'Three meals a day' presented characteristics closest to the oral cavity since the pH values remained close to neutral, subtle and progressive enamel demineralization, did not favor specific microbial groups, and allowed the accumulation of biofilm, which was evidenced by the increase in biomass and total microbiota. M3 was used to test two single (UNI) and multiple (MULTI) inoculation strategies, the second simulating the secondary colonization of microorganisms. The following were assessed: pH of the culture media; biofilms (architecture; microbial population dynamics via CFU mL-1, DNA and qPCR sequencing; insoluble biomass and matrix components); and enamel demineralization (via microhardness). The fluctuations observed in the pH were similar to those reported in the oral cavity; there was acidification after exposure to carbohydrates, and the buffering action of the saliva was simulated by returning biofilms to a saliva culture broth with a neutral pH. The spatial configuration was similar to that found in oral biofilms associated with caries, represented by hedgehogs and corn cobs architectures. The representativeness of streptococci from the mutans group was ≤ 2% of the total cultivable microbiota. The genus Streptococcus was the most abundant; however, S. mutans was not detected in any sample via sequencing. In the MULTI strategy, a greater microbial diversity was detected. When quantifying the number of bacterial cells via qPCR, it was observed that S. mutans represents <0.01% of saliva and <0.005% in biofilms. As the biofilm aged, there was an increase in biomass, exopolysaccharides, and extracellular DNA in the extracellular matrix in both inoculation strategies. The accumulation of these components is associated with a cohesive structure that can favor the formation of acidic niches. The maturation of the biofilm and enamel demineralization occurred independently of the low proportion of S. mutans and the inoculation strategy, reducing the role of specific species in the multifactorial disease.
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Palavras-chave
Cárie dental, Biofilme, Microbiota, Desmineralização, Carboidratos, Dental caries, Biofilm, Microbiota, Desmineralization, Dietary carbohydrates