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AIM Evaluate the root canal microbiome composition and bacterial functional capability in cases of primary and secondary apical periodontitis utilizing whole-metagenome shotgun sequencing. METHODOLOGY Twenty-two samples from patients with primary root canal infections, and eighteen samples obtained from previously treated teeth currently diagnosed with apical periodontitis were analyzed with whole metagenome shotgun sequencing at a depth of 20M reads. Taxonomic and functional gene annotations were made using MetaPhlAn3 and HUMAnN3 software. The Shannon and Chao1 indices were utilized to measure alpha diversity. Differences in community composition were evaluated utilizing analysis of similarity (ANOSIM) using Bray-Curtis dissimilarities. The Wilcoxon rank sum test was used to compare differences in taxa and functional genes. RESULTS Microbial community variations within a community were significantly lower in secondary relative to primary infections (alpha diversity P = 0.001). Community composition was significantly different in primary versus secondary infection (R = 0.11, P = 0.005). The predominant taxa observed among samples (>2.5%) were Pseudopropionibacterium propionicum, Prevotella oris, Eubacterium infirmum, Tannerella forsythia, Atopobium rimae, Peptostreptococcus stomatis, Bacteroidetes bacterium oral taxon 272, Parvimonas micra, Olsenella profusa, Streptococcus anginosus, Lactobacillus rhamnosus, Porphyromonas endodontalis, Pseudoramibacter alactolyticus, Fusobacterium nucleatum, Eubacterium brachy and Solobacterium moorei. The Wilcoxon rank test revealed no significant differences in relative abundances of functional genes in both groups. Genes with greater relative abundances (top 25) were associated with genetic, signaling and cellular processes including the iron and peptide/nickel transport system. Numerous genes encoding toxins were identified: exfoliative toxin, hemolysins, thiol- activated cytolysin, phospholipase C, cAMP factor, sialidase, and hyaluronic glucosaminidase. CONCLUSIONS Despite taxonomic differences between primary and secondary apical periodontitis, the functional capability of the microbiomes was similar.