Effect of Dietary Sugars on Bacterial-Fungal Cross-Kingdom Biofilm Formation and Virulence

Yilan Miao

Miao, Yilan1, Arthur, Rodrigo A.2, Negrini, Thais C.3
Faculty / Advisor: Koo, Hyun1
1University of Pennsylvania School of Dental Medicine, Department of Orthodontics
2Dental School - Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
3School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, SP, Brazil, Department of Clinical Analysis


Dental caries is a polymicrobial biofilm disease driven by the diet and microbiota-matrix interactions on tooth surface. Sugars fuel the emergence of pathogens, the assembly of matrix and the acidification of the biofilm microenvironment, causing ecological changes and enamel demineralization. Growing evidences suggest that cross-kingdom interactions occur within biofilm communities. For example, Streptoccous mutans and Candida albicans have been frequently detected in plaque-biofilms from toddlers with early childhood caries, resulting in increased biofilm mass and cell densities. However, the interaction process and disease mechanism remain unclear. We hypothesized that bacterial-fungal coaggregates adheres to tooth surface to develop biofilm. Given the critical role of dietary sugars in the pathogenesis of dental caries, the aim of current study is to explore the dynamics of S. mutans-C. albicans cross-kingdom coaggregation and to assess the effects of distinct dietary sugars on S. mutans-C. albicans coaggregation-mediated biofilm formation.


S. mutans UA159 and C. albicans SC5314 were grown in ultrafiltered tryptone-yeast extract broth (UFTYE) to mid-exponential phase. Aliquots of each microbial suspension were mixed with human clarified saliva (CLS) supplemented with different sugars as follows: a) 0.5% glucose + 0.5% fructose, b) 1% starch, c) 1% starch + 0.5% glucose + 0.5% fructose, d) 1% sucrose or e) 1% starch + 1% sucrose, and no sugar was used as control. Suspensions were incubated at 37°C under rocking for 120 min and then bind to saliva-coated hydroxyapatite (sHA) discs for 60 min at 37°C. Each disk was then transferred into UFTYE supplemented with each sugar combination described above for 19 h. Coaggregates and biofilms were imaged using confocal laser scanning microscopy (CLSM) with quantitative computational image analysis. Biochemical/microbiological analysis were employed to assess biofilm properties.


The cross-kingdom coaggregate formation in human saliva is time-dependent and facilitated by sucrose, where more complex coaggregate structures such as clusters were found in CLS+sucrose group. The average surface areas were 80x and 18x greater than pure CLS group at 60 min and at 120 min, respectively. Sucrose-mediated coaggregates adhered to sHA as aggregates of S. mutans interacting with yeast chains/pseudohyphae in a specific 3D structure mediated by the EPS-matrix. Other dietary sugars also affect cross-kingdom co-aggregate formation in a sugar-dependent manner.


The present data showed that bacterial-fungal coaggregation is an important event that occurs before microorganisms adhering to sHA surfaces. In the presence of sucrose or starch+sucrose, biofilm growth and its acidogenic potential was boosted, posing an increased risk for caries development.