A study on the changes and effects of the oral microbiome and the development of eco-friendly microbial preparations according to the use of chemical mouthwashes for oral health.

A total of 26 participants (16 males and 10 females) completed the study, contributing 78 oral swab samples collected at three time points. The overall mean age was 29.5 ± 5.2 years, and age differed significantly among the three groups, with the saline control group being the oldest (34.2 ± 4.8 years), followed by the CPC group (29.8 ± 2.3 years) and the essential oil (EO) group (24.2 ± 3.2 years; Kruskal–Wallis H = 15.09, p < 0.001). Sex distribution, smoking status, alcohol use, and most oral hygiene behaviors were similar among groups, although prior mouthwash use (no participant in the EO group reported any previous use) and caffeinated beverage intake differed significantly among groups. No intervention-related adverse events were reported, and no significant between-group differences were observed in salivary biochemical parameters (pH, calcium, potassium, etc.) at the recovery time point. 16S rRNA gene sequencing yielded approximately 1.55 million primer-trimmed reads across the 78 samples, with a mean of 88.2% of reads retained after quality filtering and denoising (1,295 amplicon sequence variants identified). All samples were retained for downstream analysis. No significant between-group differences were observed for any alpha-diversity metric (Shannon diversity, Simpson diversity, Pielou's evenness, or Chao1 richness) at baseline, after the 3-day mouthwash-use phase, or after the 3-day recovery phase. Results differed depending on the beta-diversity metric used. Weighted UniFrac distances differed significantly among groups at all three time points (baseline p = 0.002, among others); however, this significance was lost after adjustment for age, indicating that groups already differed in community composition at baseline and that the observed differences during the mouthwash-use and recovery phases likely reflected persistent baseline group differences rather than a direct mouthwash effect. In contrast, Bray–Curtis dissimilarity showed no significant between-group differences at any time point, regardless of age adjustment. At the phylum level, Bacillota, Pseudomonadota, Bacteroidota, and Actinomycetota together accounted for more than 90% of mean relative abundance, and Streptococcus was the most abundant genus; however, no phylum showed a consistent directional change across the mouthwash groups. Age-adjusted differential abundance analysis (ANCOM-BC) identified 106 significant genus-level results. In the EO group, GWA2-37-10 was consistently higher and Kingella consistently lower than in the control group across all three time points. In the CPC group, the recovery phase showed lower Kingella abundance and higher Haemophilus and HOT-345 abundance relative to control. However, because many of these differences were already present at baseline, they should be interpreted as age-adjusted between-group differences rather than direct evidence of mouthwash-induced change. In an exploratory analysis of recovery after mouthwash cessation, the EO group showed a significant decrease in Bray–Curtis distance to baseline between the mouthwash-use and recovery phases (p = 0.021), suggesting partial compositional recovery; however, this finding did not survive Bonferroni correction and was not significant in a sensitivity analysis using non-rarefied data, and should therefore be interpreted cautiously as exploratory. The Recovery Index was likewise positive in the EO group (median 0.15) but near zero or negative in the CPC (-0.02) and control (-0.14) groups, without a statistically significant between-group difference (Kruskal–Wallis p = 0.091). Overall, this study did not provide strong evidence that CPC or EO mouthwash use induces distinct or consistent changes in the oral microbiome relative to saline control; rather, pre-existing baseline differences among groups, particularly related to age, appear to account for much of the observed signal.