Abstract

Objective The objective of this study was to investigate the microbial signature of the oral cavity in caries-free adolescents without orthodontic treatment from the general population. We also aimed to identify lifestyle factors shaping oral microbiota composition and assess the impact of technical covariates related to sample collection and analysis. Methods For the cross-sectional analysis, 750 Dutch adolescents without caries or orthodontic treatment (16.1%) were selected from all participants from the Generation R Study with oral microbiota profiling (n = 4,646). Oral microbiota data was obtained from supragingival biofilm swabs using 16S rRNA sequencing (V3-V4 region), and analyzed at four levels; core microbiota, alpha diversity, beta diversity and differential abundance. Results Participants (13.6 ± 0.29 years, 56% male) shared a large core microbiota consisting of 23 different genera, with the five most abundant and prevalent—Streptococcus, Rothia, Haemophilus, Gemella, and Neisseria—being well-known early colonizers of the dental biofilm. Among participant characteristics, tooth brushing frequency emerged as the strongest determinant of microbiota composition. Compared to participants who brushed twice or more a day, those who brushed once or less, exhibited higher bacterial richness (Chao1; β:15.04, 95% CI:1.78;28.31) and higher Porphyromonas abundance (LFC:0.63, SD:0.16, q-value:0.002). The sampling time, identified as the most impactful technical covariate, showed a negative association with bacterial richness (early-morning vs late-afternoon: Chao1; β:-57.04, 95% CI:-77.44;-36.64). Conclusion This study revealed a high similarity in oral microbiota of adolescents from the general population at the genus level, while highlighting the influence of tooth brushing frequency, sex, and sugar consumption on bacterial composition. Clinical relevance The findings provide a valuable reference for future studies exploring the link between adolescents' oral microbiota and both oral and systemic health.