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


Nardello, Laura C L; Amado, Pamela P P; Franco, Diego C; Cazares, Roberto X R; Nogales, Carlos G; Mayer, Márcia P A; Karygianni, Lamprini; Thurnheer, Thomas; Pinheiro, Ericka T (2020). Next-Generation Sequencing to Assess Potentially Active Bacteria in Endodontic Infections. Journal of Endodontics, 46(8):1105-1112.

Abstract

Introduction: Because active bacteria present a higher abundance of ribosomal RNA (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 complementary DNA and DNA samples from 5 root canals of teeth with apical periodontitis were subjected to polymerase chain reaction with bar-coded primers amplifying the 16S rRNA gene hypervariable regions V4-V5. High-throughput sequencing was performed using MiSeq (Illumina, San Deigo, CA), and data were analyzed using Quantitative Insights Into Microbial Ecology and Human Oral Microbiome Database. Statistical analysis was performed for relative abundance of bacteria in the DNA- and rRNA-based NGS data using the Mann-Whitney test, whereas differences in the diversity and richness indexes were assessed using a nonparametric 2-sample t test (P < .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 indexes of bacterial richness and diversity, the relative abundance of bacterial members varied in both analyses. Comparing rRNA with DNA data, there was a significant decrease in the relative abundance of Firmicutes (P < .05). The bacterial taxa 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.

Keywords: 16S ribosomal RNA sequencing; apical periodontitis; endodontic infection; next-generation sequencing.

Abstract

Introduction: Because active bacteria present a higher abundance of ribosomal RNA (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 complementary DNA and DNA samples from 5 root canals of teeth with apical periodontitis were subjected to polymerase chain reaction with bar-coded primers amplifying the 16S rRNA gene hypervariable regions V4-V5. High-throughput sequencing was performed using MiSeq (Illumina, San Deigo, CA), and data were analyzed using Quantitative Insights Into Microbial Ecology and Human Oral Microbiome Database. Statistical analysis was performed for relative abundance of bacteria in the DNA- and rRNA-based NGS data using the Mann-Whitney test, whereas differences in the diversity and richness indexes were assessed using a nonparametric 2-sample t test (P < .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 indexes of bacterial richness and diversity, the relative abundance of bacterial members varied in both analyses. Comparing rRNA with DNA data, there was a significant decrease in the relative abundance of Firmicutes (P < .05). The bacterial taxa 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.

Keywords: 16S ribosomal RNA sequencing; apical periodontitis; endodontic infection; next-generation sequencing.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Center for Dental Medicine > Clinic of Conservative and Preventive Dentistry
Dewey Decimal Classification:610 Medicine & health
Scopus Subject Areas:Health Sciences > General Dentistry
Uncontrolled Keywords:General Dentistry
Language:English
Date:1 August 2020
Deposited On:06 Jan 2021 13:29
Last Modified:24 May 2024 01:46
Publisher:Elsevier
ISSN:0099-2399
OA Status:Closed
Publisher DOI:https://doi.org/10.1016/j.joen.2020.05.004
PubMed ID:32497654