Effect of Experimental CPC Mouthwash on Oral Multi-species Biofilm Architecture

With the advent of sophisticated microscopy techniques, and associated software and hardware, the ability to study the effect of antimicrobials on biofilms in detail is continually evolving. Objectives: The aim of this work was to develop and/or refine a model-system to study oral multi-species biofilms and examine their 3-dimensional architecture following exposure to two mouthwashes: a placebo or an experimental, containing 0.075% cetylpyridinium chloride (CPC). Methods: Saliva was suspended in 50% glycerol and stored at -80oC until required. LAB-TEK™ chamber slides were coated with poly-L-lysine, conditioned with filter-sterilized saliva, and inoculated with Schaedler media containing 50% thawed saliva. During incubation at 37oC in 5% CO2, Schaedler media was replaced every 24-hours for up to 10-days. Biofilms were then exposed to mouthwash solutions for 2-minutes: either one containing 0.075% CPC or a placebo mouthwash solution that lacked CPC. Biofilms were washed in PBS (pH7.4) and stained with LIVE/DEAD® stain and visualized using a LEICA SP5 confocal microscope. Three-dimensional images were generated using IMARIS installed on a 64bit IBM computer running a RADEON 2600XT graphics card. Results: Five-day multi-species biofilms displayed an archipelago-like structure containing a mixture of mostly live cell-types (inferred by LIVE/DEAD staining). Ten-day biofilms covered most of the surfaces and possessed a complex 3D-structure containing mostly live cells. Exposure to placebo mouthwash had a marginal effect on 5-day and 10-day biofilm architecture and proportions of live and dead cells. Conversely, the mouthwash containing 0.075% CPC appeared to increase the number of dead biofilm cells in 5-day biofilms and also caused removal of biofilm cells in 10-day biofilms. Conclusion: We have developed a model system to grow and computationally render multi-species oral biofilms in 3-dimensions to explore changes in architecture. Such an ability, complements existing methods to examine the effect of antimicrobials on oral bacterial biofilms. Funding provided by Colgate-Palmolive.