Comparative analysis of endodontic microbiota in apical root thirds using cryopulverization and paper point sampling techniques: a pilot exploratory next generation sequencing study

This study aimed to compare the effectiveness of paper point (PP) and cryopulverization (CP) microbial sampling techniques for characterizing the bacterial communities in the apical third of teeth with post-treatment apical periodontitis using 16 S rDNA sequencing. No previous study has compared these sampling methods for characterizing of apical root canal microbiota. Apical 5-mm root segments from twenty extracted molar teeth diagnosed with post-treatment apical periodontitis were sectioned. Two distinct microbial samples were obtained from each apical root canal: the first using sterile paper points, and the second via cryogenic pulverization of the apical root segment.The V3–V4 hypervariable regions of 16 S rDNA from both sample types were amplified and sequenced using the Illumina MiSeq platform. Amplicon sequence variants (ASVs), generated via the DADA2 pipeline, were analyzed in the QIIME2 environment and taxonomically assigned using the SILVA database. Statistical analysis incorporated Linear Mixed-Effects Models (LMM) to evaluate alpha diversity metrics. For beta diversity and differential abundance, analyses were strictly tailored to our paired within-tooth design. This was achieved by utilizing blocked permutation PERMANOVA, along with Centered Log-Ratio (CLR) transformations and paired DESeq2 modeling to fully adjust for intra-tooth biological dependencies. Due to low-biomass amplification thresholds, high-quality bacterial reads were successfully recovered from 21 samples (12 CP and 9 PP), which included 5 strictly paired within-tooth sample sets ( n = 10). CP samples demonstrated greater observed taxonomic diversity than PP samples. Notably, candidate phyla including TM6, TM7 ( Saccharibacteria ), GN02, FBP, and Planctomycetes were exclusively identified in the CP group. LMM analysis indicated a significantly higher microbial richness (Chao1 index) and Observed ASV count in the CP group than in the PP group (respectively, p < 0.001 and p < 0.001). While the abundance profiles of dominant core taxa exhibited no significant shifts in Weighted UniFrac ( p = 0.625), Unweighted UniFrac metrics indicated a strong trend toward significance ( p = 0.0625), reflecting structural variations driven primarily by the presence or absence of low-abundance peripheral organisms. Cross-package differential abundance testing revealed a robust consensus: Corynebacterium , Stenotrophomonas , Geobacillus , Brevibacillus , Pullulanibacillus , and Veillonella were significantly enriched in the CP group under a comprehensive discovery threshold (padj < 0.10). Conversely, PP samples demonstrated significant enrichment of Rothia , Lacticaseibacillus , Barnesiella , Lachnoanaerobaculum , Lentimicrobium , Deinococcus , and Parascardovia (FDR < 0.10). Across both methodologies, core biomarkers maintained strict statistical validation after multiple-testing corrections. Both sampling methods successfully characterized the core apical microbiota. However, within the limitations of this study, by capturing greater microbial richness, exclusive candidate phyla, and method-specific biomarker families, the CP method offers a more comprehensive representation of refractory endodontic infections than traditional paper points.