Root Microbiota in Primary and Secondary Apical Periodontitis

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.

Keywords: 16S rRNA gene; Enterococcus faecalis; Fusobacterium nucleatum; apical periodontitis; community profiling; endodontics; oral microbiome.

FIGURE 1

Taxonomic composition and group average clustering of bacterial communities in root canal (A) and dentin (B). The heat map shows the relative abundance of OTUs across samples. OTUs with average relative abundance >0.5% in a given sample type are presented. The hierarchical clustering was based on square root-transformed proportions of OTUs and Bray–Curtis similarity matrix. PAP, primary apical periodontitis; SAP, secondary apical periodontitis.

FIGURE 2

Principal coordinate analysis of Bray–Curtis similarity of bacterial communities. The analysis was based on square root-transformed proportions of OTUs and included either all (A), root canal (B), or dentin samples (C). PAP, primary apical periodontitis; SAP, secondary apical periodontitis.

FIGURE 3

OTUs differentially represented between PAP and SAP root samples. OTUs with significant changes (Wilcoxon rank-sum test, P < 0.05) and a minimum average relative abundance ≥0.5%) are presented. Boxplots show the first and third quartile (top and bottom edges of the rectangle) divided by median. Whiskers correspond to the highest and lowest values within 1.5× the interquartile range. Outliers are shown as circles. ^∗0.01 < P < 0.05; ^∗∗0.001 < P < 0.01; ^∗∗∗P < 0.001. PAP, primary apical periodontitis; SAP, secondary apical periodontitis.

FIGURE 4

Alpha diversity of the microbiota from root canal of PAP and SAP teeth. Shannon diversity index was based on OTU relative abundance. ^∗∗0.001 < P < 0.01. PAP, primary apical periodontitis; SAP, secondary apical periodontitis.

FIGURE 5

Co-occurrence and exclusion patterns among most abundant OTUs in PAP (A) and SAP (B) in root canal samples. OTUs found in at least eight samples and with a minimal average relative abundance of 1% (considering all root canal samples) were analyzed. The edges represent positive (blue) and negative (red) Spearman correlations. Only correlations with Spearman R > 0.5 or R < –0.5 are presented. The line thickness is proportional to the absolute value of the Spearman’s correlation. Node sizes reflect average relative abundance of each OTU. PAP, primary apical periodontitis; SAP, secondary apical periodontitis.

FIGURE 6

Functional profiles of PAP and SAP-associated bacterial communities in root canal samples. The KEGG orthologs (predicted functions) inferred from the relative abundance of OTUs using PICRUSt were collapsed into level-3 KEGG ontology (KEGG pathways) and compared among PAP and SAP groups using STAMP. (A) Principal component analysis of KEGG pathways. (B) Significant differences (P < 0.05, two-sided Welch t-test) between PAP and SAP functions. STAMP was set to consider pathways represented by at least 10 sequences and an effect size filter (ratio of proportion) of 1.2. PAP, primary apical periodontitis; SAP, secondary apical periodontitis.