Journal article
Next-Generation Sequencing to Assess Potentially Active Bacteria in Endodontic Infections.
- Nardello LCL Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil.
- Amado PPP Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
- Franco DC Institute of Environmental Sciences, Jagiellonian University, Krakow, Poland.
- Cazares RXR Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil.
- Nogales CG Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil.
- Mayer MPA Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil.
- Karygianni L Clinic of Conservative and Preventive Dentistry, Center of Dental Medicine, University of Zurich, Zürich, Switzerland.
- Thurnheer T Clinic of Conservative and Preventive Dentistry, Center of Dental Medicine, University of Zurich, Zürich, Switzerland.
- Pinheiro ET Department of Dentistry, School of Dentistry, University of São Paulo, São Paulo, Brazil; Clinic of Conservative and Preventive Dentistry, Center of Dental Medicine, University of Zurich, Zürich, Switzerland. Electronic address: erickapinheiro@usp.br.
- 2020-06-05
Published in:
- Journal of endodontics. - 2020
16S ribosomal RNA sequencing
apical periodontitis
endodontic infection
next-generation sequencing
Actinobacteria
Bacteria
Bacterial Infections
Clostridiales
DNA, Bacterial
High-Throughput Nucleotide Sequencing
Humans
RNA, Ribosomal, 16S
English
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.
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.
- Language
-
- English
- Open access status
- closed
- Identifiers
-
- DOI 10.1016/j.joen.2020.05.004
- PMID 32497654
- Persistent URL
- https://sonar.ch/global/documents/271239
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