Microbial dynamics in primary and refractory apical periodontitis: Next-generation 16S rRNA sequencing reveals selective shifts and reduced diversity

Context: Apical periodontitis (AP) is an inflammatory condition caused by root canal infection. Primary AP (PAP) arises from untreated necrotic pulp and is typically polymicrobial, whereas persistent or refractory AP (RAP) occurs after root canal treatment (RCT) and may reflect selective microbial survival. Aim: The study aimed to compare the root canal microbiomes in PAP and persistent AP using 16S rRNA gene sequencing in a standardized mandibular molar cohort. Settings and Design: Comparative, observational, cross-sectional study conducted at a tertiary care dental institution. Materials and Methods: Intracanal samples were collected from 45 systemically healthy adults (30 RAP, 15 PAP) with chronic asymptomatic mandibular molars (Periapical Index 2–4). Samples were processed using Illumina MiSeq sequencing targeting the V3–V4 region. Bioinformatics analysis was performed using QIIME 2 and DADA2 pipelines. Statistical Analysis Used: Alpha diversity was analyzed using the Mann–Whitney U -test. Beta diversity was assessed using permutational multivariate analysis of variance (PERMANOVA). Differential abundance was evaluated using DESeq2 with Benjamini–Hochberg false discovery rate (FDR) correction. Results: RAP exhibited significantly reduced alpha diversity ( P ≤ 0.003) and a distinct microbial community structure compared with PAP (PERMANOVA, P ≤ 0.003). Persistent cases showed enrichment of Gram-positive taxa, particularly Enterococcus faecalis (38.5% vs. 2.1%; Log2 fold change +4.2, FDR < 0.001), whereas primary cases demonstrated higher abundance of anaerobic Gram-negative taxa such as Fusobacterium nucleatum and Porphyromonas gingivalis . Conclusions: Persistent AP is associated with reduced microbial diversity and enrichment of Gram-positive organisms, particularly E. faecalis . These findings may reflect selective survival of resistant taxa and/or secondary colonization following RCT.