Abstract
We characterized site-specific organization of the oral microbiome in 59 healthy, non-smoking women in their twenties using 16S rRNA (V3-V4) sequencing of dental plaque (DQ), mouthwash (MW), and tongue coating (TC) samples (177 libraries; rarefied to 29,853 reads). The order of alpha diversity was DQ > MW > TC (p < 0.001), with mean Observed Features of 227.6, 208.7, and 142.4, respectively, consistent with differences in local niche structure within the oral cavity. Beta diversity (Bray-Curtis; weighted/unweighted UniFrac) showed clear separation by site (PERMANOVA overall p < 0.001), with DQ most distinct from MW and TC. At the phylum level, DQ was enriched for Actinobacteriota and Proteobacteria, whereas Firmicutes predominated in MW and TC, indicating compositional shifts across oral microenvironments. Dominant genera included Streptococcus (20.6%), Neisseria (11.7%), Haemophilus (10.1%), Prevotella (7.1%), Veillonella (6.0%), Rothia (5.1%), and Lactobacillus (4.4%). Site-associated taxa included Cardiobacterium, Corynebacterium, and Campylobacter in DQ; Actinobacillus in MW; and Absconditabacteriales (SR1) and Lactobacillus in TC. TC exhibited the lowest alpha diversity but the highest genus richness (n = 340), indicating an uneven community with many low-abundance taxa. LEfSe identified discriminant taxa, including Rothia, Actinomyces, Fusobacterium (DQ), Streptococcus, and Gemella (MW); and Lactobacillus, Prevotella, Veillonella, and Haemophilus (TC). These results demonstrate pronounced site specificity within the oral cavity of a well-defined healthy cohort and indicate that multi-site resolution is essential for advancing mechanistic and translational oral microbiome research.