A clinical next-generation sequencing study on the microbial profiles of asymptomatic apical periodontitis in type 2 diabetic and systemically healthy individuals following adjuvant antimicrobial photodynamic therapy

The aim was to evaluate the effect of type 2 diabetes mellitus on the root canal microbiota and its response to antimicrobial photodynamic therapy (aPDT) combined with chemomechanical preparation in asymptomatic apical periodontitis (AP), by next-generation sequencing. A total of 22 teeth with a single root and single canal, diagnosed with asymptomatic AP, were included in this study: 11 teeth from patients with T2D (T2D group) and 11 from systemically healthy individuals (Control group). Root canal samples were collected before root canal treatment and after aPDT combined with chemomechanical preparation. Following chemomechanical preparation, canals were incubated with methylene blue for 5 min and photoactivated with a 630 nm LED for 60 s. Root canal samples were collected at two time points—before treatment (Control.Pre, T2D.Pre) and after treatment (Control.aPDT, T2D.aPDT). Following propidium monoazide (PMA) treatment, genomic DNA was isolated using a silica column method and quantified fluorometrically. The V3–V4 regions of the 16S rRNA gene were amplified and sequenced using the Illumina MiSeq platform. Data were processed in QIIME2 with DADA2 for denoising and classified taxonomically using Human Oral Microbiome Database (HOMD). Diversity analyses and statistical evaluations (PERMANOVA and multivariable association analysis using MaAsLin3; FDR-corrected) were performed in R. A total of 44 root canal samples (Control.Pre, Control.aPDT, T2D.Pre, T2D.aPDT; n = 11) were analyzed via 16S rRNA V3–V4 sequencing, yielding 4.6 million high-quality reads and 2,745 Amplicon Sequence Variants (ASVs). Alpha diversity did not differ between pre and post disinfection procedure samples in healthy individuals (p > 0.05), whereas a significant reduction in observed ASV richness was detected in the T2D group after the disinfection procedures (p < 0.05). The number of ASVs shared between pre and post disinfection procedure samples was lower in the T2D group than in controls. Beta diversity based on unweighted UniFrac distances showed significant shifts after the disinfection procedures in both groups (p < 0.05), while weighted UniFrac analyses showed no significant differences (p > 0.05). Taxonomic analysis revealed a post disinfection increase in oxygen tolerant taxa and a reduction in obligate anaerobes in both groups, with more pronounced changes in the T2D group. Following chemomechanical preparation and aPDT, a pronounced restructuring of the root canal microbiota was observed in both healthy individuals and individuals with T2D. In individuals with T2D, the post-disinfection period was characterized by reduced alpha diversity and a lower number of shared ASVs, indicating more limited microbial continuity. The reduced microbial stability observed in individuals with diabetes suggests that systemic metabolic status may affect microbial responses to chemomechanical disinfection combined with aPDT.