Formation of Biofilms Containing P. gingivalis Using Static Culture Method

Objective: The aim of this project was to characterize oral bacterial biofilms formed under conditions of no media flow. This investigation was undertaken to help confirm and improve the knowledge of current biofilm growth techniques.

Method: Streptococcus gordonii, Streptococcus mutans, Fusobacterium nucleatum and Porphyromonas gingivalis were chosen to investigate the no flow biofilm method. Cultures were grown anaerobically in 30 mm petri dishes for 7 days in TYHK broth with 1 or 10 µg/ml hemin. Select biofilms were treated with Erythromycin; formed biofilms were stained using one of the following: Live Dead stain, Acridine orange, or anti-P. gingivalis antibody. The three dimensional structure of formed biofilms was visualized with a confocal microscope.

Results: Pure culture biofilms revealed that S. gordonii and the S. mutans each formed a thin relatively flat layer of cells with occasional mound formations. All of the P. gingivalis strains formed a thicker fairly uniform layer of cells and F. nucleatum formed more of a tertiary structure. P. gingivalis specific staining of P. gingivalis F. nucleatum mixed biofilms revealed that P. gingivalis was not distributed uniformly, but rather associated with F. nucleatum in distinct locations. Antibiotic treatment of completely formed P. gingivalis biofilms revealed bacterial killing only at the biofilm surface/liquid interface.

Conclusion: The static culture method employed in this study confirmed S. gordonii and S. mutans form thin layered biofilms, confirmed limited antibiotic penetration of biofilms, and showed specific location of P. gingivalis in P. gingivalis F. nucleatum biofilms. This method can be used to study P. gingivalis F. nucleatum interaction and pure culture biofilms.

Supported by: T32 DE007132