Role of starch and sucrose in Streptococcus mutans biofilms formation

The combination of starch and sucrose, the main dietary carbohydrates, in the presence of surface-adsorbed salivary amylase modulate biofilm formation by Streptococcus mutans in situ by enhancing the synthesis of the extracellular polysaccharide (EPS) matrix and acidogenicity. Objectives: To examine the structural and molecular basis of the role of starch and sucrose in S. mutans biofilm formation on saliva-coated hydroxyapatite (HA) surface. Methods: S. mutans UA159 biofilms were formed on saliva-coated HA discs in batch culture for 5 days in the presence of 1% (w/v) starch, 1% (w/v) sucrose, or in combinations. Amylase activity of adsorbed saliva on HA was detected up to 72 h after pellicle formation. The biofilms were removed at specific time points and subjected to biochemical, structural (GC-MS and confocal fluorescence microscopy with COMSTAT software) and molecular (real-time PCR and cDNA microarrays) analyses. Results: The combination of starch+sucrose produced biofilms with more biomass and a higher content of insoluble polysaccharides with 3-linked branching (3,4-, 3,6-, and 3,4,6-linked glucose) than those grown in sucrose (P<0.05); the presence of starch alone resulted in little biofilm formation. The 3-D structural organization of starch+sucrose-biofilms was markedly distinct from sucrose-grown biofilms; the middle and outer layers of starch+sucrose-biofilms presented higher polysaccharide to bacteria ratio while biofilms grown in sucrose showed more bacteria than polysaccharides. Furthermore, biofilms formed in the presence of starch+sucrose expressed significantly higher levels of gtfB mRNA than those grown in sucrose (P<0.05). Lastly, transcriptome analyses of the biofilms revealed that specific pathways in the phosphoenolpyruvate-phosphotransferase and two-component systems may be modulating the metabolism and EPS development in biofilms grown in starch+sucrose. Conclusion: The combination of sucrose with starch has profound effects on the metabolism, structure and gene expression profile of S. mutans biofilms formed on saliva-coated HA, which may enhance the cariogenic potential of dental biofilms.