Taxonomy and Virulence Factors in the Root Canal Microbiome: Metagenomic Insights by Lesion Size and Clinical Factors in Primary Endodontic Infections

Introduction This study aimed to investigate the taxonomic and functional profiles of the root canal microbiome in teeth with large versus small periapical lesions, examining the influence of clinical variables on microbial composition and functional pathways. Methods Samples from 25 teeth with large (>8 mm) and 20 with small periapical lesions (<2 mm) were analyzed. Quantitative polymerase chain reaction, 16S next-generation and whole genome sequencing were used to assess microbial load, diversity, and composition. Functional predictions were performed using the Kyoto Encyclopedia of Genes and Genomes and MetaCyc databases. Alpha diversity was calculated using Shannon and Chao1 indices. Beta diversity was assessed using ANOSIM and PERMANOVA. Significant variables were explored using MaAsLin3. Kruskal–Wallis tests were used for univariate comparisons. Results Teeth with large lesions exhibited significantly higher bacterial load (P = .011), but comparable alpha diversity and number of species per group in 16S and whole genome analysis (P > .05). Lesion size showed significance by ANOSIM (P = .04) but not in PERMANOVA (P = .36). Age was significant in both beta diversity tests, but the effect size only explained 3.6% of the variance. All clinical variables were not significant in 16S analysis for beta diversity. MetaCyc pathway analysis identified percussion sensitivity as the most influential clinical variable in both tests (ANOSIM R = 0.182, P = .012; PERMANOVA R ² = 0.063, P = .046). MaAsLin3 modeling revealed enrichment of enzymatic pathways involved in methionine and cysteine-related metabolism. Conclusions Large periapical lesions contain significantly higher bacterial load, but similar diversity compared to small lesions. Functional predictions suggest bacterial metabolic activity may contribute to mechanical allodynia in endodontic infections.