The dynamics, control and virulence factor expression by periodontal pathogens in oral biofilm ---------------------------------------------------------------------------------Introduction Periodontitis is a biofilm-associated disease

that affects the connective tissue surrounding teeth.Fusobacterium nucleatum and Porphyromonas gingivalis are among subgingival bacterial species that are considered to play a major role in the dental biofilm, F.nucleatum acting as a bridge between early and late colonizers in dental biofilm. F.nucleatum coaggregates with almost all species that are considered putative periodontal pathogens. P. gingivalis which harbors many virulent factors is spatially arranging themselves during the course of chronic periodontitis in the top of dental biofilm facing the epithelium linings. Dental plaque is a true biofilm and the formation of bacterial biofilm is one example of group behavior that is coordinated through a quorum sensing. Bacteria in the biofilm may express different gene sets than their planktonic counterparts(8),the gene expression of virulence factors may be different according to their living conditions,For example,in the biofilm mode of growth, microorganisms exhibit increased resistance to antimicrobial agents, environmental stresses and host immune defense mechanisms (5). Extracellular Matrix of the biofilm Extracellular polysaccharides and proteins have been shown to be a key components of the matrix and recent studies indicate that extracellular DNA plays an important role in the establishment of biofilm structure (7). Some studies suggested that eDNA released mainly from cell lysis ,but several studies also revealed that some active secretion mechanism may exist.

Marwan Mohammed*, Audun Nerland*, Mohammed Al-Haroni and Vidar Bakken* *The Gade Institute - Oral Microbiology, University of Bergen Institute of Pharmacy, University of Troms

3- Confocal Laser Scanning Microscopy (CLSM) - CLSM (Zeiss LSM 510 Meta) will be used in the visualization of the biofilm with appropriate stains. 4- Randomly Amplified Polymorphic DNA (RAPD) - After isolation of the matrix RAPD will be used to compare external DNA with cellular DNA isolated from F. nucleatum bacterial biofilm. 5- Real time Polymerase Chain Reaction (real time PCR) - P. gingivalis has a number of putative virulence factors that contribute to its pathogenicity. The use of real-time PCR to study of the virulence factors expression is a suitable tool in this respect Project approach
The project will be done in four aspects according to the specific objectives. Study (1): Growing and studying dynamic F. nucleatum and P. gingivalis biofilm. Study (2): Role of extracellular DNA in the matrix of F. nucleatum biofilm. Study (3): Gene expression in multispecies biofilm Study (4): Evaluate the effects of antimicrobial agents on biofilm

The General Aim of The Study The main aim of the project is to study and characterize a dual species biofilm composed of F. nucleatum and P. gingivalis in vitro using molecular imaging techniques
Objectives The following aims will be recognized in the project:

-To study the F. nucleatum and P. gingivalis biofilm under different physiological conditions. (e.g carbon source , flow rate, pH, temperature, etc.) -To determine the role of external DNA (eDNA) in the matrix of F. nucleatum and P. gingivalis biofilm and its effect in the spatial arrangement of the cells in biofilm. -To study the expression of virulence factors in the mixed F. nucleatum and P. gingivalis biofilm compared to the expression levels of these genes when P. gingivalis cultured alone. -To study the antibacterial effect of selected antimicrobial agents on biofilm

Figure2:36 hour F.nucleatum biofilm growen in flow cells and stained with Cyto9 and Propodium Iodine.

Materials and methods 1- Bacteria and growth conditions F. nucleatum subsp. nucleatum, type strain (ATCC 25586). P. gingivalis type strain (ATCC53978) (W50). The media that will be used is chemostat liquid broth media. Fastidious Anaerobe Agar (FAA). 2- Flow Cells Biofilm System:

1.Slots J, Genco RJ. Black-pigmented Bacteroides species, Capnocytophaga species, and Actinobacillus actinomycetemcomitans in human periodontal disease: virulence factors in colonization, survival, and tissue destruction. J Dent Res. 1984 Mar;63(3):412-21. 2.Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL, Jr. Microbial complexes in subgingival plaque. J Clin Periodontol. 1998 Feb;25(2):134-44. 3.Bolstad AI, Jensen HB, Bakken V. Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. Clin Microbiol Rev. 1996 Jan;9(1):55-71. 4.Rosen G, Sela MN. Coaggregation of Porphyromonas gingivalis and Fusobacterium nucleatum PK 1594 is mediated by capsular polysaccharide and lipopolysaccharide. FEMS Microbiol Lett. 2006 Mar;256(2):304-10. 5.Rickard AH, Gilbert P, High NJ, Kolenbrander PE, Handley PS. Bacterial coaggregation: an integral process in the development of multi-species biofilms. Trends Microbiol. 2003 Feb;11(2):94-100. 6.Socransky SS, Haffajee AD. Dental biofilms: difficult therapeutic targets. Periodontol 2000. 2002;28:12-55. 7.Socransky SS, Haffajee AD. Periodontal microbial ecology. Periodontol 2000. 2005;38:135-87.

Figure2: A) The flow cell system setup, medium bottle(a) the pump(b) the bubble trap(c) the flow cell(d) and the effluent bottle(e). B) Dimensions of the flow cell.