Evaluation of alcohol-free mouthwash for studies of the oral microbiome

Oral bacteria play important roles in human health and disease. Oral samples collected using ethanol-containing mouthwash are widely used for oral microbiome studies. However, ethanol is flammable and not ideal for transportation/storage in large quantities, and some individuals may avoid ethanol due to the burning sensation or due to various personal, medical, religious, and/or cultural factors. Here, we compared ethanol-free and ethanol-containing mouthwashes using multiple microbiome metrics and assessed the stability of the mouthwash samples stored up to 10 days before processing. Forty volunteers provided oral wash samples collected using ethanol-free and ethanol-containing mouthwashes (one mouthwash type collected per day from each individual over two consecutive days, with the order of mouthwash type randomized). Microbiome metrics measured in the two types of mouthwashes were very similar, with intraclass correlation coefficients (ICCs) for alpha and beta diversity metrics greater than 0.85. Relative abundances of some taxa were significantly different, but ICCs of the top four most abundant phyla and genera were high for the comparability of the mouthwashes. Stability during delayed processing of up to 10 days was also high for both mouthwash types based on alpha and beta diversity measures and relative abundances of the top four phyla and genera. These results demonstrate that ethanol-free mouthwash performs similarly to ethanol-containing mouthwash for microbial analyses, and both mouthwashes are stable for at least 10 days without freezing prior to laboratory processing. Ethanol-free mouthwash is suitable for collecting and shipping oral wash samples in epidemiologic studies of the oral microbiome.