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Notes: Background: Bacteria contained in biofilms have been shown to have a decreased susceptibility to antimicrobial agents compared to those in planktonic form. Thus, in vitro biofilm models have been developed for screening oral antimicrobial formulations in an effort to produce findings more predictive of clinical activity. This study compared the antimicrobial activity of three mouthrinse formulations when tested against isogenic strains of Actinobacillus actinomycetemcomitans (Aa), one of which was a clinical isolate which forms tenacious biofilms in vitro and the other of which was a spontaneous variant which always grows planktonically.Method: Biofilm-forming Aa strains CU1000 and NJ4300, obtained as clinical isolates, and their respective spontaneous planktonic variants, CU1060 and NJ4350, were grown under standard laboratory conditions and exposed for 15 s to either a negative control (phosphate buffered saline [PBS]), an essential-oil containing mouthrinse (Listerine® Antiseptic [LA]), an amine fluoride/stannous fluoride-containing mouthrinse (Meridol®[M]), or a triclosan and PVM/MA copolymer-containing mouthrinse (Plax®[P]). The cells were then washed, serially diluted, plated, and incubated for enumeration of viable bacteria. Colony-forming units (CFU)/ml were log10 transformed and the mouthrinse groups were compared to the PBS group using analysis of variance.Results: All 3 mouthrinses produced statisically significant 99.99% reductions (p〈inlineGraphic alt="leqslant R: less-than-or-eq, slant" extraInfo="nonStandardEntity" href="urn:x-wiley:03036979:JCPE280714:les" location="les.gif"/〉0.0001) in both planktonic strains compared to the PBS control. Effects on the biofilm forms of the organisms were more variable. Exposure to LA produced statistically significant (p〈inlineGraphic alt="leqslant R: less-than-or-eq, slant" extraInfo="nonStandardEntity" href="urn:x-wiley:03036979:JCPE280714:les" location="les.gif"/〉0.0001) reductions in strains CU1000 and NJ4300 of 98.20% and 96.47%, respectively, compared to PBS. M and P produced much smaller reductions which were not statistically significant.Conclusions: The results of this study, in which antimicrobial mouthrinses were tested against biofilm-forming and planktonic strains of the same organism, provide a clear demonstration of the resistance to antimicrobial agents conferred by biofilm formation and provide additional support for employing tests using biofilms to more accurately assess the relative activities of antiplaque agents in vitro.