Fluoride varnish application and the temporal evolution of supragingival microbiota in children with differential caries risk

Background Fluoride varnish is used to prevent early childhood caries, but its longitudinal ecological impact on the supragingival microbiome beyond enamel remineralization remains poorly understood, especially long-term.Objective To characterize 24‑month supragingival plaque microbiome evolution in young children under biannual fluoride varnish, stratified by caries risk, and fluoride’s ecological effect on microbial structure, interactions, and metabolism.Design A total of 48 children were categorized into low‑, moderate‑, and high‑caries‑risk groups using the modified caries-risk assessment tool for preschool children (PSC-MCAT). All received fluoride varnish every six months. Supragingival plaque collected at five timepoints over 24 months. 16S rRNA sequencing assessed diversity, key taxa, co-occurrence networks, and PICRUSt2 metabolic pathways.Results Baseline alpha‑ and beta‑diversity differed significantly across risk groups (p<0.05). After sustained fluoride, intergroup differences diminished. High‑risk group retained caries‑associated genera (e.g. Leptotrichia, Prevotella, Veillonella) with suppressed abundance. Fluoride increased network complexity and negative correlations in moderate‑/high‑risk groups. Glycolysis, TCA cycle, and starch/sucrose metabolism were inhibited in the high‑risk group post‑intervention.Conclusions Regular fluoride varnish reduces ecological disparities across risk groups, suppresses cariogenic taxa, and alters microbial interactions and carbohydrate metabolism, promoting homeostasis. High‑risk children may require more frequent interventions.