Measuring Biofilm Penetration Effect of Mouthrinses Using Multiple In-vitro Analyses

Objective: Determine the penetration effect of mouthrinses containing LISTERINE® Antiseptic essential-oils (EO), cetylpyridinium chloride (CPC) or chlorhexadine (CHX) versus negative control on statically grown mixed-species biofilm (MSSB) or under flow conditions (MSFB). Method: For both MSSB and MSFB, stimulated saliva was collected from donors, pooled and homogenized.  MSSB-Saliva pellicle was formed in polystyrene plates.   Saliva was then replaced with saliva/media mixture and incubated shaking at 35^OC for 48 hours with media changes twice daily. MSFB-Saliva was dispensed into a 12-channel bio-cassette to form a pellicle on HA disks suspended from a fitted lid into the 12-channel bio-cassette. The bio-cassette was attached to a 24-channel continuous flow peristaltic pump system in a 30.5ºC chamber.  Media was added, allowing biofilms to form for 24 hours. Biofilms were treated over the course of 60 hours following label instructions. Biofilms subjected to twice daily “rinsing” had 5 hours between treatments.  Biofilms were harvested after the final treatment and analyzed for ATP bioluminescence. Results were reported as mean log₁₀RLU (relative light units) as an expression of ATP concentration. Samples were stained using a Live/Dead stain cocktail (Invitrogen). Viability was analyzed using a Leica-SP5 confocal microscope. 3D images were generated and analyzed using Volocity software to visualize penetration. Stains are reported as percent Live/Dead.  

Results:  

	%Live	%Dead	Log10 ±95%CI
Multi Species Static Biofilm(MSSB)
EO	7	93	-
CPC	23	77	-
CHX	13	87	-
Multi Species Flow Biofilm(MSFB)
EO	1	99	5.58±0.20
CPC	13	87	5.70±0.17
CHX	8	92	5.57±0.17

Conclusion: These results indicate that in the MSSB and the MSFB models, the EO mouthrinse shows the deepest penetration via live/dead stain over CPC and CHX mouthrinses. In the MSFB, the EO mouthrinse exhibited superior biofilm kill to CPC and similar kill to CHX, further supporting the mechanism of EOs to control plaque and gingivitis.