Structural Analysis of a Subgingival Biofilm Model System

Objectives: The aim of this project was to analyze both the development and structure of 10-species ‘subgingival' biofilms grown in vitro. This presentation addresses the abundance of the organisms in dependency of time and nutritional conditions and discusses the structure of the corresponding biofilms. Methods: Our experimental system was a modified version of a recently described ‘subgingival' biofilm model (Guggenheim et al., BMC Microbiol, 9:280, 2009). Biofilms were generated anaerobically on pellicle-coated hydroxyapatite discs in batch cultures over 64.5 h in mFUM medium supplemented with various concentrations of glucose and human serum but not saliva. Harvested biofilms were processed using standard techniques for cultural analysis or confocal laser scanning microscopy (CLSM). For structural analyses individual species were labeled by fluorescent in situ hybridization (FISH). Results: Microscopical analyses showed that after 4 h discs were populated mainly by streptococci. On top of this layer, Fusobacterium nucleatum expanded rapidly but later decreased parallel to the expansion of the other organisms. Delayed addition of streptococci lead to a predominance of Actinomyces naeslundii (CFUs increased by one order of magnitude) and was accompanied by a decrease of F. nucleatum, Veillonella dispar and streptococci. The addition of human serum altered the community by promoting growth of Campylobacter rectus, Treponema denticola and Prevotella intermedia and restricting growth of streptococci and V. dispar. In contrast, the amount and kind of sugars provided altered the bacterial community only marginal. Conclusions: The data indicate that this biofilm community is mainly affected by the influence of distinct species and blood serum, whereas changes in the composition of the available sugars have only minor impact. Streptococci are the initially most rapidly expanding population in our model biofilms thereby building a mandatory ground for the up-coming of F. nucleatum. Funding: SSO Forschungsfonds (242 – 09).