Modified Drip Flow Reactor: In-vitro Biofilm Method for Evaluating Anti-plaque Agents

Objectives: To develop an in-vitro biofilm method to grow mixed species biofilms in a modified drip flow reactor (MDFR) and to validate this method by testing the performance of Colgate Total® (CT) and Colgate Cavity Protection® (CCP).  Background:  The MDFR is derived from ASTM method E2647.  It delivers a flow of medium over biofilms that are continuously cultured on saliva-conditioned hydroxyapatite coated slides, which allows researchers to grow biofilms representative of oral plaque on tooth like surfaces.  Methods: A mixed species inoculum containing equal levels of Actinomyces viscosus (ATCC#43146), Fusobacterium nucleatum (ATCC#10953), Lactobacillus casei (ATCC#334), Veilonella parvula (ATCC#17745) and Streptococcus oralis (ATCC#35037) from a continuous culture chemostat was used to establish the biofilm for 2 hours in the static phase followed by incubation in a continuous flow phase with Modified Marsh Medium at a flow rate of 10mls/hour/chamber for 24 hours at 25ºC under aerobic conditions.  Supernatants of dentifrice slurries (1:2 w/v) in water were then added to the biofilm for 2 minutes treatment followed by a second treatment at a 6-hr interval followed by a 24 hour continuous flow phase.  Biofilms were harvested and quantified using both total anaerobic plate counts and quantitative real-time PCR analysis (qPCR) for total DNA.  Results:  The results showed a 1.29 log difference between CT and CCP as measured by plate counts analysis while the qPCR analysis provided a 1.54 log difference between the toothpastes.  Conclusion: Both toothpastes CT and CCP were effective at reducing total anaerobes.  CT showed statistically higher performance using both analytical methods (p<0.05).  qPCR results showed greater separation since it measures total bacterial DNA of viable and non-viable bacteria.  Additionally, qPCR allows for determination of effects on individual species providing a robust method for discriminating between the performances of a dentifrice containing a broad-spectrum antimicrobial versus a regular fluoride dentifrice.