Abstract

Apical periodontitis is an inflammatory disease of the dental periradicular tissues triggered by bacteria colonizing necrotic root canals. Primary apical periodontitis results from the microbial colonization of necrotic pulp tissues. Secondary apical periodontitis results from a persistent infection of incorrectly treated root canals. The aim of this study was to characterize the microbiota present in primary and secondary intraradicular infections associated with apical periodontitis using 16S rRNA gene amplicon sequencing. Teeth exhibiting apical periodontitis with or without root canal treatment were extracted after informed consent. From each tooth, the intraradicular content as well as a dentin sample (control) were collected and subjected to DNA extraction. PCR amplicons of the V3–V4 region of the bacterial 16S rRNA gene were pooled and sequenced (2 × 300) on an Illumina MiSeq instrument. The bioinformatics analysis pipeline included quality filtering, merging of forward and reverse reads, clustering of reads into operational taxonomic units (OTUs), removal of putative contaminant OTUs and assigning taxonomy. The most prevalent and abundant OTU in both dentin and root canal samples was assigned to anaerobic bacterium Fusobacterium nucleatum. Multivariate analysis showed clustering of microbiota by sample type (dentin vs. intraradicular content) and, in root canals, by pathology (primary vs. secondary infection). The proportions of Enterococcus faecalis and F. nucleatum were, respectively, higher and lower when comparing secondary to primary infected root canals. Co-occurrence network analysis provided evidence of microbial interactions specific to the infection type. The identification of bacterial taxa differentially abundant in primary and secondary intraradicular infections may provide the basis for targeted therapeutic approaches aimed at reducing the incidence of apical periodontitis.

Highlights

  • Apical periodontitis is an inflammatory disease of dental periradicular tissues triggered by oral pathogens invading necrotic root canals

  • Treated roots associated with secondary apical periodontitis have microbial populations distinct from those of untreated roots and culture-based studies have rarely identified more than 1–3 genera of Gram-positive facultative anaerobes, including Streptococcus, Lactobacillus, and Enterococcus (Sundqvist et al, 1998)

  • Illumina sequencing of the 16S ribosomal RNA (rRNA) V3–4 amplicon libraries generated from 52 root canals, 52 dentin and 52 negative controls yielded 6,950,125 raw read pairs of which 6,058,205 were joined and passed the quality control steps

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Summary

Introduction

Apical periodontitis is an inflammatory disease of dental periradicular tissues triggered by oral pathogens invading necrotic root canals. Anaerobic bacteria were shown to dominate the microbial population of untreated necrotic root canals, with approximately 5–12 genera including Peptostreptococcus, Prevotella, Porphyromonas, Fusobacterium, Eubacterium, and Actinomyces along with facultative anaerobic streptococci The presence of such pathogens in necrotic root canals was shown to fairly correlate with the observation of apical inflammatory lesions on intraoral radiographs (Bergenholtz, 1974; Fabricious et al, 1982; Ando and Hoshino, 1990; Chávez de Paz, 2005). Treated roots associated with secondary apical periodontitis have microbial populations distinct from those of untreated roots and culture-based studies have rarely identified more than 1–3 genera of Gram-positive facultative anaerobes, including Streptococcus, Lactobacillus, and Enterococcus (Sundqvist et al, 1998) Some of these bacteria were shown to resist the action of conventional antimicrobial agents and to survive in root-filled teeth for many years. Clinical studies reported that 30–65% of root-filled teeth may show a radiographic evidence of secondary apical periodontitis and that apical periodontitis may exacerbate several systemic diseases (Khalighinejad et al, 2016)

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