Salivary Microbiota and Antimicrobial Resistance in Relation to Chlorhexidine Use (SMARCH)

Study Protocol for 16S rRNA Sequencing of Healthy Oral Microbiota

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 Overview

• Status: Completed
• Conditions: Chlorhexidine; Oral Microbiota; Saliva Microbiome; Antimicrobial Resistance (AMR); Oral Microbiome Health
• Intervention / Treatment: Drug: 0.06% Chlorhexidine Digluconate Toothpaste

• Study Type: Interventional
• Enrollment (Actual): 11
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Czechia
  • Brno, Czechia, 62500
    • Department of Public Health, Faculty of Medicine, Masaryk University
  • Brno, Czechia, 62500
    • Skydent s.r.o.

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

• Age: Between 25-44 years.
• General Health: Good systemic health (absence of chronic inflammatory diseases or conditions affecting oral tissues).
• Oral Hygiene Routine: Participants must perform toothbrushing twice a day and use interdental cleaning aids once a day.
• Plaque Index (PI): A score of 0.0-0.2 according to Silness and Löe.
• Gingival Index (GI): A score of 0.0-0.143 according to Silness and Löe.

Exclusion Criteria:

Systemic and Lifestyle Factors:

• Presence of any chronic systemic disease (e.g., diabetes mellitus, autoimmune disorders)
• Regular use of medications that may affect gingival status or saliva production.
• Use of antibiotics within the last 6 months prior to the study.
• Current pregnancy or lactation.
• Current smokers or users of other tobacco/nicotine products.

Oral and Dental Factors:

• Active dental disease: Presence of untreated tooth decay, gingivitis (GI > 0.143), or any form of periodontitis.
• Prosthetics and Implants: Presence of dental implants, dentures, or significant tooth loss (excluding extracted third molars).
• Ongoing fixed or removable orthodontic treatment.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Health Services Research
• Allocation: N/A
• Interventional Model: Single Group Assignment
• Masking: None (Open Label)

Number of Arms

1

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Experimental: Chlorhexidine group; A single group of 11 healthy volunteers. The study consists of three consecutive phases: baseline (4 weeks), intervention with 0.06% chlorhexidine toothpaste (12 weeks), and washout (12 weeks).	Drug: 0.06% Chlorhexidine Digluconate Toothpaste; Participants used the toothpaste twice daily for 12 weeks.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
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.	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).	Baseline (week 0), after intervention (week 12), and after washout (week 24).

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
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).	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).	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.	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.	Screening (Week -4) and after washout (Week 24).

Collaborators and Investigators

• Sponsor: Masaryk University

Study record dates

Study Major Dates

• Study Start (Actual): April 11, 2022
• Primary Completion (Actual): November 29, 2022
• Study Completion (Actual): November 29, 2022

Study Registration Dates

• First Submitted: April 30, 2026
• First Submitted That Met QC Criteria: April 30, 2026
• First Posted (Actual): May 6, 2026

Study Record Updates

• Last Update Posted (Actual): May 6, 2026
• Last Update Submitted That Met QC Criteria: April 30, 2026
• Last Verified: April 1, 2026

More Information

Terms related to this study

• Keywords: Antimicrobial Resistance Genes; 16S rRNA Sequencing; 0.06% Chlorhexidine; Salivary Microbiota; Taxonomic Composition; Long-term Effect; Washout Period
• Other Study ID Numbers: 68/2020; MUNI/A/1807/2025 (Other Grant/Funding Number: Masaryk University)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No