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Updated: Sep 21, 2026

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Published on: October 15, 2019
Unraveling microbial heterogeneity in post-treatment endodontic infections via 16S rRNA gene profiling
Brenda P F A Gomes1, Priscila A Francisco1, Augusto R Lima1
1Division of Endodontics, Department of Restorative Dentistry, Piracicaba Dental School, State University of Campinas, UNICAMP, Piracicaba, SP, Brazil.
Objectives:
This study aimed to characterize the intraradicular bacteriome of post-treatment endodontic infections using high-throughput 16S rRNA gene sequencing and to assess inter-individual microbial heterogeneity through a disaggregated ecological analysis.
Design:
Endodontic samples were collected from single root canals of 10 female patients requiring retreatment. The V3-V4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Amplicon sequence variants were generated using DADA2-based processing. Microbial communities were characterized using relative-abundance profiles, Bray-Curtis, Hellinger, and CLR-transformed Aitchison distances, together with Local Contribution to Beta Diversity (LCBD), Species Contribution to Beta Diversity (SCBD), and Baselga's abundance-based beta-diversity partitioning.
Results:
Proteobacteria and Firmicutes predominated, although their relative contributions varied substantially among individuals, with greater heterogeneity at genus and species levels. Desulfobulbus predominated in R3, R10, R17, and R19, Enterococcus in R5 and R11, Haemophilus in R15 and R22, and Streptococcus in R21, whereas R20 showed a more distributed polymicrobial profile. Streptococcus oralis subsp. dentisani clade 398 showed the highest mean relative abundance, while Parvimonas micra had the highest prevalence despite relatively low abundance. R21, R22, and R20 showed the greatest beta-diversity contributions. Community differences were driven predominantly by balanced changes in taxon abundance rather than abundance gradients. The species-level core was limited and threshold-dependent.
Conclusion:
Post-treatment endodontic infections exhibited highly individualized microbial profiles and substantial inter-individual variation, with a limited species-level core. These findings highlight the ecological complexity of microbial communities present after endodontic treatment and support comprehensive infection control during retreatment.
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