Dental Biofilms – Software Model of Structure and Function of Bacilli

Objectives: Biofilm is commonly defined as accumulation of microbes, embedded in self-secreted extra-cellular matrix, formed on various surfaces. Dental biofilm is a complex, multi-species plaque, associated with caries and periodontal diseases. Bacillus subtilis, a common inhabitant of the oral cavity, which presumably plays a role in dental biofilm development, forms distinct three-dimensional biofilms on solid surfaces. This study investigates structural characteristics of B. subtilis biofilms, using a software model to quantitatively analyze morphological structures within.
Methods: B. subtilis colonies were grown to maturity on Lysogeny broth agars supplemented with glycerol and manganese (biofilm-promoting medium). Three-dimensional structure of formed biofilms was examined under CLSM and analyzed with specially-developed image processing software. Specifically, we modeled a network of channels that crisscross the full thickness of the biofilm. In order to assess functional characteristics of the channels, fluorescent analog of glucose, a regularly consumed carbohydrate (2-NBDG), was utilized to monitor glucose uptake by cells at different locations within the biofilm.
Results: Our results reveal the non-uniform nature of B. subtilis biofilms – at the colony center, equal-sized clusters of cells are surrounded by a meshwork of channels that protrude in a number of different directions. In contrast, at the colony periphery, the formed channels assume a specific directionality and exhibit different structural characteristics – longer, thicker channels. Monitoring of the native uptake of glucose further demonstrates the functional role of channels with regards to nutrient absorption/distribution.
Conclusions: Understanding the processes behind bacterial organization in dental biofilm, pathogenic or otherwise, is important for a number of reasons, not least of which is to identify structural “weak links” that can be targeted for purposes of biofilm inhibition/eradication. Quantitative software-based models demonstrate the link between structural and functional characteristics of undisrupted B. subtilis biofilms. Our approach reveals the different sub-regions of the colonies, and specifically, the unique organization of channels within.