Quantitative analysis of the effects of essential oil mouthrinses on clinical plaque microbiome: a parallel-group, randomized trial

Abstract Background The rich diversity of microorganisms in the oral cavity plays an important role in the maintenance of oral health and development of detrimental oral health conditions. Beyond commonly used qualitative microbiome metrics, such as relative proportions or diversity, both the species-level identification and quantification of bacteria are key to understanding clinical disease associations. This study reports the first-time application of an absolute quantitative microbiome analysis using spiked DNA standards and shotgun metagenome sequencing to assess the efficacy and safety of product intervention on dental plaque microbiome. Methods In this parallel-group, randomized clinical trial, essential oil mouthrinses, including LISTERINE® Cool Mint Antiseptic (LCM), an alcohol-containing prototype mouthrinse (ACPM), and an alcohol-free prototype mouthrinse (AFPM), were compared against a hydroalcohol control rinse on clinical parameters and the oral microbiome of subjects with moderate gingivitis. To enable a sensitive and clinically meaningful measure of bacterial abundances, species were categorized according to their associations with oral conditions based on published literature and quantified using known amounts of spiked DNA standards. Results Multivariate analysis showed that both LCM and ACPM shifted the dysbiotic microbiome composition of subjects with gingivitis to a healthier state after 4 weeks of twice-daily use, resembling the composition of subjects with clinically healthy oral conditions recruited for observational reference comparison at baseline. The essential oil-containing mouthrinses evaluated in this study showed statistically significant reductions in clinical gingivitis and plaque measurements when compared to the hydroalcohol control rinse after 6 weeks of use. Conclusions By establishing a novel quantitative method for microbiome analysis, this study sheds light on the mechanisms of LCM mouthrinse efficacy on oral microbial ecology, demonstrating that repeated usage non-selectively resets a gingivitis-like oral microbiome toward that of a healthy oral cavity. Trial registration The trial was registered on ClinicalTrials.gov on 10/06/2021. The registration number is NCT04921371.