Plaque Biofilm Penetration by Essential-Oil and Cpc Containing Mouthrinses

Objective: To compare the killing activity of the antiseptic mouthrinses LISTERINE^® Antiseptic Mouthwash (LMW) (Morris Plains, NJ, USA) and PLAX^® Overnight^® (PO) (Colgate-Palmolive, Sao Paulo, SP, Brazil) containing 0.05% CPC and alcohol in a previously established saliva-derived in vitro biofilm model. Methods: Unstimulated saliva was collected, pooled, diluted in BHI, centrifuged (10min @ 2,000rpm), inoculated into cover glasses and incubated overnight in anaerobic chamber (N₂ 85%, H₂ 10%, CO₂ 5%, 37°C).  After incubation growth medium was exchanged for fresh medium and every 12 hrs thereafter until the biofilms reached maturity. Growth medium was removed and biofilms were washed than treated with respective mouthrinses (100µl for 30s or 60s).  25% microorganism-free saliva in BSA (bovine serum albumin)-saline (0.25% BSA, 0.15M NaCl) and 70% EtOH served as negative and positive controls respectively. Biofilms were washed (3x), grown anaerobically (1hr) and washed again (3x) every hour for 6hrs and then treated again as described above. All 8 possible treatments were performed on the same slide. Biofilms were labeled and fluorescence was observed with a confocal laser scanning microscope.  Treatment effectiveness was quantified using image analysis software and proportion of damaged/dead cells was calculated. Results: LMW demonstrated statistically significantly greater killing activity than PO for both 2x30s and 2x60s exposures with respective rinses with p<0.0001. Additionally, in accordance with label recommendations 2x30s treatment with LMW demonstrated statistically significantly greater killing than 2x60s with PO with p<0.0001.

 

Listerine (2x30s)	Plax (2X60s)	EtOH (2X30s)	EtOH (2X60s)	Buffer (2X30s)	Buffer (2X60s)
20.8%±6.2	10.8%±3.7*	29.0%±17.9*	43.3%±9.3*	3.4%±2.9*	3.5%±2.6*

*p<0.0001

Conclusions: Both confocal imaging and quantitative analyses via live-dead staining of biofilms demonstrated that LMW was up to two times more effective in biofilm killing than PO when following label recommendations. The present study replicates results from other studies using similar and other biofilm kill kinetics methodology.