A quantitative comparison of oral microbiota using stimulated and unstimulated saliva samples: a pilot study

Objectives: To quantitatively compare the oral microbial profiles of unstimulated saliva (US) and stimulated saliva (SS) in community-dwelling older people, and to evaluate the influence of the saliva collection method on microbiological outcome.Methods: A pilot study was conducted with 28 older people.Paired US and SS samples were collected from each participant.Oral bacteria, including 14 selected oral bacterial species and total bacterial load, were quantified using multiplex real-time polymerase chain reaction targeting 16S ribosomal DNA and species-specific genes.DNA was extracted using a standardized protocol, and bacterial counts were calculated based on cycle threshold (Ct) values and standard curves.The Wilcoxon signed-rank test was used for paired comparisons, and Cohen's d was calculated to assess effect size.Results: There were no statistically significant differences in the number of individual oral bacteria or total bacterial counts between the US and SS groups.Although the SS group had a higher mean total bacterial count, the difference was not significant (P=0.137).Effect size analysis indicated generally small differences, with moderate effects observed for Fusobacterium nucleatum, Streptococcus mutans, and total bacteria.The Ct values of the individual bacterial species did not differ significantly between the two methods.However, the total bacterial Ct value was significantly higher in the US group than in the SS group (P=0.044),suggesting a relatively lower microbial concentration in the US group.Conclusions: US and SS samples provided comparable quantitative results for oral microbiome analysis in older people.However, differences in total bacterial Ct values indicate that the saliva collection method may influence microbial concentration measurement.These findings suggest that both methods are complementary; however, methodological consistency should be considered in study design and interpretation.