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.