Why does apical periodontitis persist? A shotgun metagenomic survey of the microbial community in primary and persistent periapical granulomas

Objective This cross‑sectional observational study used shotgun metagenomic sequencing to compare the microbiomes of periapical granulomas from primary and persistent apical periodontitis to identify microbial taxa and functional pathways associated with persistent lesions.Methods Seventeen patients with untreated primary apical periodontitis (UA), 19 with persistent apical periodontitis (PA), and 19 healthy gingival reference samples (HC) were enrolled. Periapical granuloma and healthy gingival samples were collected during tooth extraction for whole-metagenome shotgun sequencing. Alpha diversity was assessed by the Shannon and Chao1 indices. Beta-diversity was evaluated using PERMANOVA based on Bray‑Curtis distances. Differential abundance analysis of species and predicted functional pathways between UA and PA was performed using the Mann‒Whitney U test with Benjamini‒Hochberg false discovery rate (FDR) correction. Clinical associations were examined using Spearman’s correlation. The HC group served as a descriptive reference.Results Alpha diversity showed a significant difference in species richness (Chao1, p < 0.05) but not overall diversity (Shannon, p > 0.05). Beta diversity revealed a trend toward separation but did not reach statistical significance (PERMANOVA, R2 = 0.05085, p = 0.051). Metagenomic sequencing revealed differences in species composition and functional potential between UA and PA. Taxonomic profiling identified 503 species in UA versus 255 in PA. At the species level, several taxa showed nominal differences between UA and PA (p < 0.05), but none remained significant after FDR correction (q > 0.05). Functionally, several KEGG pathways showed nominal differences between UA and PA (p < 0.05, q > 0.05), with the top three pathways all below 3% relative abundance. Swelling duration correlated negatively with several taxa and pathways (p < 0.05), while lesion diameter showed little association with microbial abundance.Conclusions Persistent apical periodontitis granulomas showed lower species richness and abundance, but similar predicted functional capacity, compared with primary infections. Further longitudinal and multi‑omics studies are needed to investigate the associations.