Ecology of the microbiome of the infected root canal system: a comparison between apical and coronal root segments

Abstract

Aim: To evaluate the microbial ecology of the coronal and apical segments of infected root canal systems using a complete sampling technique and next-generation sequencing.

Methodology: The roots of 23 extracted teeth with apical periodontitis were sectioned in half, horizontally, and cryo-pulverized. Bacterial communities were profiled using tagged 454 pyrosequencing of the 16S rDNA hypervariable V5-V6 region.

Results: The sequences were classified into 606 taxa (species or higher taxon), representing 24 bacterial phyla or candidate divisions and one archaeal phylum. Proteobacteria were more abundant in the apical samples (P < 0.05), whilst Actinobacteria were in significantly higher proportions in the coronal samples. The apical samples harboured statistically significantly more taxa than the coronal samples (P = 0.01) and showed a higher microbial diversity. Several taxa belonging to fastidious obligate anaerobes were significantly more abundant in the apical segments of the roots compared with their coronal counterparts.

Conclusions: Endodontic infections are more complex than reported previously. The apical part of the root canal system drives the selection of a more diverse and more anaerobic community than the coronal part. The presence of a distinct ecological niche in the apical region explains the difficulty of eradication of the infection and emphasizes the need for new treatment approaches to be developed.

Figure 1

Prevalence of exclusive and shared OTUs (sequences that cluster within 97% similarity level) by the two root segments of 23 root pairs. Only those OTUs that contained at least 10 reads/sample were counted as present in the corresponding sample. The order of the samples (Root numbers) from left to right corresponds to the increasing difference in phylogenetic distance (Weighted Unifrac) between the corresponding apical and coronal segments. The distances are shown above the respective root numbers. The two segments from R10 were highly similar (Distance=0.04), while those from R27 differed the most (Distance=0.44) among the corresponding pairs of samples.

Figure 2

Principal coordinates analysis (PCoA) plot of the phylogenetic distances (Weighted UniFrac) among the samples from the corresponding pairs of segments (red dots – apical; blue dots – coronal segments) from 23 infected roots. Principal coordinates PC1, PC2 (not shown) and PC3 explained 24%, 17% and 13% of the overall variance among the samples, respectively. The sample pairs (apical and coronal) from the same root are connected with dashed lines. Arrows are pointing towards the apical sample of each pair. The pairs of the samples differ in their phylogenetic relatedness from being very adjacent or similar (roots nr 29 and 10) to very distant or phylogenetically different (roots nr 19, 44, 35 and 12). For the majority of the pairs (16 out of 23) the apical sample was positioned in the positive direction of the PC3 axis, with arrow pointing upwards, indicating the common direction of the microbial shift from coronal to apical microbiome in these samples.

Figure 3

Relative abundance of bacterial phyla per root segment (A; Apical, C; Coronal and Avg; average of all roots) in 23 cryo-pulverized roots. Other phyla represent the sum of the reads belonging to all minor phyla: Thermomicrobia, Cyanobacteria, Synergistes, OD1, Deinococcus Thermus, Chlamydiae, Acidobacteria, OP3, Planctomycetes, OP11, Nitrospira, Tenericutes, Verrucomicrobia, BRC1, Euryarchaeota, OP9 and unclassifiable reads. The order of the roots (Root number) from left to right corresponds to the increasing difference in phylogenetic distance (Weighted Unifrac) between the corresponding apical and coronal segments. The distances are shown in figure 1. * Statistically significant difference between apical and coronal segments (Wilcoxon Signed Ranks Test, p<0.05).