This study evaluated the ecological effects of 0.06% chlorhexidine toothpaste on the salivary microbiota of healthy individuals, focusing on taxonomic composition, functional shifts, and prevalence of predicted antimicrobial resistance genes. Saliva samples were collected from eleven healthy participants at three specific time points: baseline (following a 4-week run-in period using a control toothpaste), intervention (after 12 weeks of using 0.06% chlorhexidine digluconate toothpaste), and washout (after a subsequent 12-week period using the control toothpaste). Sequencing targeted the 16S rRNA V3-V4 region.
Study Type
INTERVENTIONAL
Allocation
NA
Purpose
HEALTH_SERVICES_RESEARCH
Masking
NONE
Enrollment
11
Participants used the toothpaste twice daily for 12 weeks.
Department of Public Health, Faculty of Medicine, Masaryk University
Brno, Czechia
Skydent s.r.o.
Brno, Czechia
Change in Salivary Microbial Alpha Diversity (Shannon and Simpson Indices)
Evaluation of microbial richness and evenness within individual saliva samples. Alpha diversity is quantified using the Shannon and Simpson indices based on 16S rRNA gene sequencing data. Differences between study phases (baseline, intervention, washout) are assessed using the Kruskal-Wallis test or Mann-Whitney U test, as appropriate, with a significance threshold of p \< 0.05.
Time frame: Baseline (week 0), after intervention (week 12), and after washout (week 24).
Change in Salivary Microbial Beta Diversity (Community Structure)
Evaluation of differences in microbial community composition between study phases. Calculated using Bray-Curtis dissimilarity on Centered Log-Ratio (CLR) normalized data and assessed via PERMANOVA to detect significant shifts (p \< 0.05).
Time frame: Baseline (week 0), after intervention (week 12), and after washout (week 24).
Differential Abundance of Salivary Bacterial Taxa
Evaluation of shifts in the relative abundance of bacterial taxa (at phylum, family, genus, and species levels) using 16S rRNA sequencing (V3-V4 region). Statistical analysis is performed using DESeq2 and edgeR to identify significant differences between study phases, with multiple testing corrections applied using the Benjamini-Hochberg (FDR) method (FDR \< 0.05).
Time frame: Baseline (week 0), after intervention (week 12), and after washout (week 24).
Changes in Predicted Antimicrobial Resistance Genes (ARGs)
Functional prediction of the salivary microbiota conducted using PICRUSt2. Assessment of metabolic potential and antimicrobial resistance pathways with significance assessed using adjusted p-values (p-adj \< 0.05).
Time frame: Baseline (week 0), after intervention (week 12), and after washout (week 24).
Gingival Index (GI) for Clinical Stability Assessment
Assessment of gingival health using the Silness and Löe Gingival Index (scale 0-3). This measure is used to verify that participants maintain optimal oral health throughout the study. Eligibility is strictly limited to participants with a maximum GI of 0.143 at screening to ensure a healthy baseline and to monitor that no gingival inflammation occurs during the study period.
Time frame: Screening (Week -4) and after washout (Week 24).
Plaque Index (PI) for Clinical Stability Assessment
Assessment of dental plaque accumulation using the Silness and Löe Plaque Index (scale 0-3). This measure is used to ensure consistent oral hygiene. Eligibility is strictly limited to participants with a maximum PI of 0.2 at screening. The index is monitored to confirm that participants adhere to the required oral hygiene protocol throughout the study.
Time frame: Screening (Week -4) and after washout (Week 24).
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