The apical root canal system microbial communities determined by next-generation sequencing

www.nature.com/scientificreports OPEN The apical root canal system microbial communities determined by next‑generation sequencing Luciana Carla Neves de Brito1, Janet Doolittle‑Hall2, Chun‑Teh Lee 3, Kevin Moss2, Wilson Bambirra Júnior4, Warley Luciano Fonseca Tavares4, Antônio Paulino Ribeiro Sobrinho4* & Flávia Rocha Fonseca Teles5 The aim of this study was to explore the microbial communities of endodontic infections at their apical portion by 16S rRNA Illumina sequencing and delineate the core microbiome of root canal infections and that of their associated clinical symptomatology. Samples were collected from fifteen subjects presenting one tooth with a root canal infection, and their associated symptoms were recorded. Samples were collected from the apical third of roots using a #10 K file and then amplified using multiple displacement amplification and PCR-amplified with universal primers. Amplicons were sequenced (V3–V4 hypervariable region of the 16S rRNA gene) using MiSeq (Illumina, CA). The microbial composition of the samples was determined using QIIME and HOMINGS. Data were analyzed using t tests and ANOVA. A total of 1,038,656 good quality sequences were obtained, and OTUs were assigned to 10 bacterial phyla, led by Bacteroidetes (51.2%) and Firmicutes (27.1%), and 94 genera were represented primarily by Prevotella (17.9%) and Bacteroidaceae G-1 (14.3%). Symptomatic teeth were associated with higher levels of Porphyromonas (p < 0.05) and Prevotella. P. endodontalis and P. oris were present in both cores. The present study demonstrated the complexity of the root canal microbiome and the “common denominators” of root canal infections and identified taxa whose virulence properties should be further explored. The polymicrobial etiology of endodontic infections has long been established. However, few studies have focused on expanding the breadth and depth of coverage of microbiome-infected root canals at their apical portion. Microbiological evaluations of infected root canals have expanded our knowledge on the topic1–4, confirmed the predominance of anaerobic species, revealed previously unrecognized bacterial diversity1–3,5,6 and showed that the complexity of the microbial consortium influences the pathogenesis of periradicular conditions4,7. In infected root canals, microbial communities remain as surface-associated b iofilms8,9. The bacterial biofilm requires treating root canals to prevent and/or heal apical periodontitis10. There is overwhelming evidence to support that an unspecific microbial community is able to induce periapical lesion development4,7,11. However, as stated by Tatikonda et al.12, there are a few studies that focus on analyzing “the pulp canal segments”. The infected apical third of root canals maintains a distinct array of microorganisms from its coronal segment13,14. Nevertheless, few studies have focused on the analysis of the most apical portion of endodontic infections. This gap in the current literature has precluded a better picture of the root canal core microbiome and its associated clinical parameters. The cloistered root canal space interferes in its microbial colonization (10, 12). The microbial status of infected root canals was demonstrated by studies that have progressed depending on the evolution of microbiological methods. Recently, culture-independent strategies, such as next-generation sequencing, have improved this knowledge. Additionally, molecular studies have revealed significant differences in the prevalence of certain pathogens, demonstrating that geographical and individual characteristics may influence bacterial community 1 School of Dentistry, University of Itauna, Itaúna, MG, Brazil. 2Dental Research/Center for Oral Systemic Diseases, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. 3Department of Periodontics and Dental Hygiene, School of Dentistry, The University of Texas Health Science Center at Houston, Houston, TX, USA. 4Department of Operative Dentistry, School of Dentistry, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil. 5School of Dental Medicine, School of Engineering and Applied Sciences, Center for Innovation and Precision Dentistry, University of Pennsylvania, Philadelphia, USA. *email: Scientific Reports | (2020) 10:10932 | https://doi.org/10.1038/s41598-020-67828-3 1 Vol.:(0123456789) www.nature.com/scientificreports/ profiles1,2,5,15–18. Next-generation sequencing (NGS) has revolutionized genomic r esearch19,20. The ultramodern MiSeq platform is ideal for rapid and cost-effective genetic a nalysis21. However, MiSeq sequencing may not always reach species-level taxonomic resolution. To improve this limitation, H OMINGS22–24 utilized the speed and efficiency of next-generation sequencing combined with the refinement of bacterial species-level identification based on 16S rDNA comparisons17. Therefore, knowing that the apical portion of the root canal infection harbors a distinct microbiome from its coronal segment, this study aimed to explore the microbiome of the apical portion of the root canal through metagenetics approaches and its association with clinical symptomatology. Materials and methods Study population. Study participants were 15 patients referred to the dental school to receive endodontic care. The exclusion criteria for this study was antibiotic therapy up to 3 months before starting endodontic therapy, systemic diseases, and pregnancy. All participants signed the Free Agreement Formulary. The Ethics Committee of the FUMG approved this study (ETIC 122⁄08). Clinical samples were taken from 15 teeth (single and multirooted) with pulp necrosis and apical periodontitis that were diagnosed by clinical (presence of tissue swelling, percussion sensitivity, symptomatic or asymptomatic) and radiographic analyses, in addition to pulp sensibility tests. Additionally, if the tooth was symptomatic, the following parameters were recorded: onset of pain (time and duration) and quality of pain (throbbing, stabbing, dull) and whether teeth were single or multirooted. The final diagnosis was made based on those findings. All teeth selected for sampling presented pulpal necrosis, as well as no history of trauma, periodontal involvement, or previous root canal treatment. The clinical parameter of each tooth is described in Supplemental Table 1. Root canal samples. The selection and preparation of the teeth, as well as the sample collection, was per- formed, as previously d escribed1,2, by the same experienced endodontist. Briefly, the tooth was cleaned with pumice and isolated with a rubber dam. The teeth were decontaminated and disinfected with a 30% hydrogen peroxide solution (H2O2) and then with 2.5% sodium hypochlorite solution (NaOCl). The access cavity was prepared with a high speed sterile carbide bur, and before the pulp chamber was exposed, the cleaning of the tooth and rubber dam was repeated as previously described. The sa (...truncated)