Opening doors for broader participation in oral microbiome studies: the stability and comparability of ethanol-free mouthwash

By Pooja Toshniwal PahariaReviewed by Lily Ramsey, LLMMay 1 2023

In a recent study published in PLOS ONE, researchers compared the use of ethanol-free mouthwash and ethanol-containing mouthwash for oral microbiome studies and assessed the stability of the mouthwash samples for ≤10 days before processing.

Study: Evaluation of alcohol-free mouthwash for studies of the oral microbiome. Image Credit: JuJae-young/Shutterstock.com

Background

Bacteria in the mouth or oral cavity play critical roles in preventing human disease. Oral cavity samples obtained using mouthwashes that contain ethanol are commonly utilized in oral microbiota research.

Ethanol is commonly used as a solvent for active substances and tastes, as well as a preservative due to its antibacterial characteristics. However, the alcohol is combustible and inappropriate for large-scale transportation and storage.

Individuals might refrain from using ethanol-containing mouthwashes because of the associated burning-type sensation and/or other medical, personal, cultural, and/or religious concerns.

Moreover, individuals with contraindications, such as current or recovering alcoholics, oral mucosal injuries, and immunosuppressive conditions, should avoid ethanol exposure. However, whether they are appropriate for oral microbiota investigations is not certain.

About the study

In the present study, researchers compared oral washes obtained using mouthwashes with or without ethanol for microbiome analysis. They also investigated whether the mouthwashes were stable for ≤10.0 days to assess their sensitivity to delayed processing.

The study included 40 volunteers, 20 men, and 20 women, recruited from the Frederick National Cancer Research Laboratory’s Research Donor Program for the analysis. Each participant provided two self-obtained samples of oral washes, one using an ethanol-free mouthwash and another using an ethanol-containing mouthwash.

The two samples were obtained on consecutive days, wherein 50% of the participants provided ethanol-free mouthwash samples on the initial day and ethanol-containing mouthwash samples on a subsequent day, and vice versa for the remaining half of the study participants. At the laboratory, three aliquots of 3.0 mL each were created from all samples.

Among the aliquots, one was processed immediately and frozen, whereas the other two aliquots were stored at 4^◦C for five days and at room temperature (22^◦C) for ten days to simulate delays in shipping before freezing. The team extracted deoxyribonucleic acid (DNA) following amplification of the 16S ribosomal ribonucleic acid (rRNA) gene variable 4 (V4) region using polymerase chain reaction (PCR), amplicon sequencing, and processing using bioinformatics analysis.

The two types of mouthwashes were compared using multiple microbiota metrics. In addition, the participants filled out questionnaires to provide data on demographics, oral health, hygiene practices, oral health, and recent history of antibiotic use.

Only healthy individuals aged 40 to 65 with no history of chronic medical conditions such as cardiovascular, respiratory, renal, hematological, or infectious diseases were included.

In addition, the included individuals had a full set of teeth, worked at the Frederick National Laboratory or were members of the Fort Detrick community, and weighed ≥110.0 pounds. The ethanol-free mouthwash comprised cetylpyridinium chloride (0.07%), glycerin, water, flavor, sodium saccharin, poloxamer 407, sucralose, methylparaben, Blue 1, and propylparaben.

The constituents of the ethanol-containing mouthwash were ethanol (15% by weight), glycerin, water, polysorbate 80, flavor, cetylpyridinium chloride, benzoic acid, sodium benzoate, Yellow 5, Blue 1, and sodium saccharin.

Results and conclusions

The mean participant age was 52.0 years; 88% were white, 90% documented their oral health status as good to excellent; 80% documented no gingival disease; 55% were completely dentulous, and 93% of individuals had reported tooth decay. Among the participants, 53% used mouthwashes ≥1.0 times weekly in the month before the samples were obtained.

Microbiota metrics were comparable for the two types of mouthwash; the intraclass correlation coefficient (ICC) values were above 0.85 for alpha (α) diversity and beta (β) diversity metrics, irrespective of the mouthwash type.

High ICCs, above 0.75, were observed for the most abundant genera (Prevotella, Streptococcus, Veillonella, Gemella, and Haemophilus) and phyla (Patescibacteria, Firmicutes, and Proteobacteria), concerning the comparability of the two types of mouthwashes.

However, significant differences were observed in the abundances of a few microbial taxa, in relative terms, likely due to the differences in the chemical composition of the mouthwashes. Further, both mouthwash types were highly stable despite processing delays, based on the measures of α and β diversity and the abundance of the 4.0 most numerous genera and species, in relative terms.

The findings indicated that the performance of the ethanol-lacking mouthwash was comparable to that of the ethanol-comprising mouthwash in collecting oral washes and analyzing the oral microbiota, and both mouthwash types were stable without freezing for 10.0 days before processing.

The antimicrobial property of cetylpyridinium chloride, present in both types of mouthwashes, probably inhibited bacterial growth in the sampled oral washes, conferring high stability to the mouthwashes during delays in processing.

The median DNA yields for the mouthwashes with and without ethanol were 1,803.0 ng and 1,343.0 ng, respectively. The median DNA yields for samples immediately processed, stored for five days, and stored for ten days were 1,517.0 ng, 1,636.0 ng, and 1,539.0 ng, respectively. The median DNA yields on the first and second days of sample collection were 1,375 ng and 1,723 ng, respectively.

Significant differences were observed concerning amplicon sequence variants (ASVs) between the two types of mouthwash; median ASVs for ethanol-free and ethanol-containing mouthwashes were median 116 and 124, respectively.

The median values for the Shannon index in the corresponding samples were 4.46 and 4.50, respectively. However, Faith’s phylogenetic diversity (PD) values for the two types of mouthwashes did not differ significantly. Similar findings were observed for samples stored for ten days before processing.

Based on the study findings, ethanol-free mouthwashes are appropriate for obtaining and transporting oral wash specimens to analyze the oral microbiota.