Salivary Microbiota and Antimicrobial Resistance in Relation to Chlorhexidine Use

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.

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.