Next Generation Sequencing to Assess Potentially Active Bacteria in Endodontic Infections.

INTRODUCTION As active bacteria present higher abundance of ribosomal rRNA than DNA (rRNA gene), the rRNA/DNA ratio of Next Generation Sequencing (NGS) data was measured to search for active bacteria in endodontic infections. METHODS Paired cDNA and DNA samples from 5 root canals of teeth with apical periodontitis were subjected to PCR with barcoded primers amplifying the 16S rRNA gene hypervariable regions V4-V5. High-throughput sequencing was performed using Illumina MiSeq, and data was analyzed using QIIME and HOMD. Statistical analysis was performed for relative abundance of bacteria in the DNA- and rRNA-based NGS data using Mann-Whitney test, whereas differences in the diversity and richness indices were assessed using a nonparametric two-sample t-test (p < 0.05). For bacterial taxa detected in both approaches, the rRNA/DNA ratios were calculated by dividing the average abundance of individual species in the respective analysis. RESULTS Although no significant difference was found in the indices of bacterial richness and diversity, the relative abundance of bacterial members varied in both analyses. Comparing rRNA to DNA data, there was a significant decrease in the relative abundance of Firmicutes (p < 0.05). Bacteroidales [G-2] bacterium HMT 274, Porphyromonas endodontalis, Tannerella forsythia, Alloprevotella tannerae, Prevotella intermedia, Pseudoramibacter alactolyticus, Olsenella sp. HMT 809, Olsenella sp. HMT 939, Olsenella uli, and Fusobacterium nucleatum subsp. animalis were both dominant (DNA ≥ 1%) and active (rRNA/DNA ≥ 1). CONCLUSIONS The integrated DNA- and rRNA-based NGS strategy was particularly important to disclose the activity of as-yet-uncultivated or difficult-to-culture bacteria in endodontic infections.