Standardizing oral microbiome sampling for qPCR: methodological and exploratory insights into nutritional status

Area of Science:

• Microbiology
• Human Physiology
• Molecular Biology

Background:

• Accurate oral microbiota quantification requires standardized sample collection.
• Quantitative polymerase chain reaction (qPCR) is a key method for bacterial enumeration.

Purpose of the Study:

• To evaluate and identify the most consistent oral sample collection method for bacterial quantification in adolescents using qPCR.
• To assess the biological applicability of the optimal method by comparing bacterial profiles and body composition in adolescents.

Main Methods:

• Collected unstimulated saliva, cheek swabs, and biofilm samples from adolescents.
• Utilized qPCR to quantify total bacteria (16S rRNA gene), Bacillota, and Bacteroidota phyla.
• Performed exploratory analysis correlating bacterial abundance with body composition parameters.

Main Results:

• Unstimulated saliva yielded the lowest variability in bacterial quantification compared to cheek swabs and biofilm (p < 0.05).
• Moderate correlations were found between saliva and biofilm, with weak associations between saliva and cheek swabs.
• While bacterial counts trended higher in overweight/obese adolescents, differences were not statistically significant.

Conclusions:

• Unstimulated saliva is a validated, reproducible, non-invasive, and cost-effective biofluid for oral microbiota quantification by qPCR.
• The findings support saliva's utility for large-scale, translational, or point-of-care microbiome assessments in nutritional and metabolic health studies.
• Group-specific associations between oral bacteria and body composition highlight saliva's potential in health research.