Multicohort external validation of Anaeroglobus as a candidate salivary biomarker for childhood caries

Objective To evaluate the cross-cohort reproducibility of Anaeroglobus as a candidate salivary microbiome signal for childhood caries and to place this signal within plaque community, functional, and host-response contexts. Methods Public pediatric salivary 16S rRNA cohorts were analyzed using a discovery–primary external validation–second validation framework. Genus-level features were assessed after CLR transformation and covariate adjustment, with plaque microbiome, plaque transcriptome, and deep-caries dental pulp single-cell datasets used as supporting evidence layers. Results In the discovery cohort, Lactobacillus was associated with caries status and Anaeroglobus with total caries burden. In the primary external cohort, Anaeroglobus and Lactobacillus remained positively associated with caries [OR = 3.452 (95% CI: 1.851–6.449) and OR = 2.118 (95% CI: 1.049–4.282), respectively]. In the second validation cohort, Anaeroglobus remained directionally consistent [OR = 3.009 (95% CI: 1.241–7.312)]. The primary external microbiome model showed moderate discrimination (AUC = 0.782; approximate 95% CI: 0.703–0.861). Exploratory pooled-effect synthesis supported positive directional consistency, while the supporting plaque and multi-omics layers indicated broader community restructuring, carbohydrate/oxidative-stress adaptation, and host immune–stromal remodeling. Conclusions Anaeroglobus represents a directionally reproducible candidate salivary risk-related signal rather than a direct mechanistic or immediately diagnostic biomarker. The 36-sample second validation cohort should be interpreted as supportive age-extrapolation evidence, and the pooled synthesis should be interpreted as exploratory; further prospective, paired saliva–plaque, and multimodal validation is required.