Effect of dietary sugars on dual-species biofilms of Streptococcus mutans and Streptococcus sobrinus – a pilot study

Original Articles Effect of dietary sugars on dual-species biofilms of Streptococcus mutans and Streptococcus sobrinus – a pilot study Efeito dos açucares da dieta em biofilme dupla espécie de Streptococcus mutans e Streptococcus sobrinus – um estudo piloto Rosa Virginia Dutra de OLIVEIRAa  Yasmin Etienne ALBUQUERQUEa  Denise Madalena Palomari SPOLIDORIOa  Cristiane Yumi KOGA-ITOb  Elisa Maria Aparecida GIROa  Fernanda Lourenção BRIGHENTIa  aFaculdade de Odontologia, UNESP – Universidade Estadual Paulista, Araraquara, SP, Brasil bFaculdade de Odontologia, UNESP – Universidade Estadual Paulista, São José dos Campos, SP, Brasil Abstract Introduction Frequent consumption of sugars and the presence of Streptococcus mutans and Streptococcus sobrinus are correlated with higher caries experience. Objective The aim of this pilot study was to elucidate the effect of different fermentable carbohydrates on biomass formation and acidogenicity of S. mutans and S. sobrinus biofilms. Material and method Single and dual-species biofilms of S. mutans ATCC 25175 and S. sobrinus ATCC 27607 were grown at the bottom of microtiter plates at equal concentrations for 24 h at 37 °C under micro-aerobic atmosphere. Carbohydrates were added at 2% concentration: maltose, sucrose, glucose and lactose. BHI Broth (0.2% glucose) was used as negative control. Acidogenicity was assessed by measuring the pH of spent culture medium after 24 h, immediately after refreshing the culture medium and for the next 1 h and 2 h. Crystal violet staining was used as an indicator of the total attached biofilm biomass after 24 h incubation. Data were analyzed by two-way ANOVA followed by Bonferroni post hoc test. Significance level was set at 5%. Result All carbohydrates resulted in higher biomass formation in single- and dual-species biofilms when compared to the control group. Sucrose, lactose and maltose showed higher acidogenicity than the control group in both single- and dual-species biofilms after 24 h. Conclusion These findings indicate that the type of biofilm (single- or dual-species) and the carbohydrate used may influence the amount of biomass formed and rate of pH reduction. Descriptors:  Biofilm; biomass; Streptococcus mutans; Streptococcus sobrinus Resumo Introdução O consumo frequente de açucares e a presença de Streptococcus mutans e Streptococcus sobrinus estão correlacionados com maior experiência de cárie. Objetivo Elucidar o efeito de diferentes carboidratos fermentáveis na biomassa e acidogenicidade de biofilmes formados por S. mutans e S. sobrinus. Material e método Biofilmes única e dupla- espécie de S. mutans ATCC 25175 e S. sobrinus ATCC 27607 em concentrações iguais cresceram no fundo de placas de microtitulação por 24 h a 37 °C em microaerofilia. Maltose, sacarose, glicose e lactose foram adicionados a 2%. BHI caldo (0.2% glicose) foi usado como controle negativo. Acidogenicidade foi avaliada por meio da medição do pH do meio de cultura após 24 h, imediatamente após troca de meio e nas próximas 1 h e 2 h. Coloração por cristal violeta foi usada como indicador do total de biomassa aderida, após 24 h de incubação. Os dados foram analisados por teste ANOVA two way e Teste de Bonferroni. O nível de significância foi de 5%. Resultado Todos os carboidratos resultaram em maior formação de biomassa em ambos os tipos de biofilme (única ou dupla- espécie), quando comparado ao grupo controle. Sacarose, lactose e maltose mostraram maior acidogenicidade que o grupo controle após 24 h nos biofilmes única ou dupla-espécie, apenas após 24 h. Conclusão Os achados indicam que o tipo de biofilme (única ou dupla- espécie) e o tipo de carboidrato usado podem influenciar tanto na quantidade de biomassa formada quanto na taxa de redução do pH. Descritores:  Biofilme; biomassa; Streptococcus mutans; Streptococcus sobrinus INTRODUCTION Epidemiological studies have shown a correlation between the presence of Streptococcus mutans and Streptococcus sobrinus with caries incidence1-4. In early childhood caries, the presence of both microorganisms promoted a significantly higher caries increment than S. mutans alone5. Although S. sobrinus has been isolated less frequently from carious lesions, it has been associated with active dental caries and may be considered a determinant of caries experience3, mainly early childhood caries2. It is believed that the cariogenic potential of these bacteria is directly related to their ability to generate acids and to tolerate acidic environments6. These features provide a competitive advantage over other biofilm bacteria during the periods of acidification6. S. mutans can grow and carry out glycolysis at pH values below 5.0 and can lower the pH to values below 4.07. At low pH levels, S. sobrinus is capable of sustain acid production, whereas other species tend to discontinue or reduce this production8. S. sobrinus can produce acid more rapidly than S. mutans at pH values between 6.5 and 5.0. Thus, S. sobrinus may be considered the most acidogenic of the oral streptococci9. An association between the consumption of sugar-containing beverages and the presence of mutans streptococci in infants was found10. Frequent consumption of sugar-sweetened snacks by school children was also correlated with higher caries experience and with higher isolation of S. mutans and S. sobrinus3. Animals fed with carbohydrates exhibited formation of higher amounts of coronal plaque on the smooth surfaces of the tooth11. The fermentation of sucrose produces large amounts of acids within biofilms and serves as a substrate for extracellular and intracellular polysaccharide synthesis12-14. Extracellular polysaccharides increase porosity of the biofilm matrix, allowing carbohydrate diffusion through the biofilm. At the tooth-plaque interface these carbohydrates are fermented to acids resulting in pH decrease14. Extracellular polysaccharides also increase microorganism adhesion and accumulation, mainly of S. mutans13,15. Intracellular polysaccharides are reservoirs of carbohydrates that promote pH drop during nutrient deprivation, prolonging the exposure of tooth surfaces to organic acids12. Sucrose, which is considered the most cariogenic carbohydrate, needs to be catabolized into glucose and fructose by sucrase before it can be metabolized by S. mutans. On the other hand, glucose can be directly metabolized by this microorganism15. Glucose also appears to be more efficiently metabolized by S. sobrinus, since a higher amount of acids is produced by this microorganism when compared to S. mutans16. Maltose, a starch derivative, is one of the most abundant carbohydrates in the human diet and is easily fermentable to potentially cariogenic acids by S. mutans17. Approximately one half of the total amount of acidic end products is produced from glucose and sucr (...truncated)