Comparison of the bacterial composition and structure in symptomatic and asymptomatic endodontic infections associated with root-filled teeth using pyrosequencing

Abstract

Residual microorganisms and/or re-infections are a major cause for root canal therapy failure. Understanding of the bacterial content could improve treatment protocols. Fifty samples from 25 symptomatic and 25 asymptomatic previously root-filled teeth were collected from Sudanese patients with periradicular lesions. Amplified 16S rRNA gene (V1-V2) variable regions were subjected to pyrosequencing (FLX 454) to determine the bacterial profile. Obtained quality-controlled sequences from forty samples were classified into 741 operational taxonomic units (OTUs) at 3% dissimilarity, 525 at 5% dissimilarity and 297 at 10% dissimilarity, approximately corresponding to species-, genus- and class levels. The most abundant phyla were: Firmicutes (29.9%), Proteobacteria (26.1%), Actinobacteria (22.72%), Bacteroidetes (13.31%) and Fusobacteria (4.55%). Symptomatic patients had more Firmicutes and Fusobacteria than asymptomatic patients, while asymptomatic patients showed more Proteobacteria and Actinobacteria. Interaction of disease status and age was observed by two-way ANOSIM. Canonical correspondence analysis for age, tooth restoration and disease status showed a correlation of disease status with the composition and prevalence of different members of the microbial community. The pyrosequencing analysis revealed a distinctly higher diversity of the microbiota compared to earlier reports. The comparison of symptomatic and asymptomatic patients showed a clear association of the composition of the bacterial community with the presence and absence of symptoms in conjunction with the patients' age.

Figure 1. Abundance of the different bacterial phyla detected in clinical samples of root-canal treated teeth.

Figure 2. Abundance of the different bacterial phyla in symptomatic (black) and asymptomatic (grey) secondary endodontic infections.

The relative abundance of the different phyla according to the disease status is depicted as mean values with standard deviation.

Figure 3. Summarizes the distribution of the top 25 most abundant genera in symptomatic (red) and asymptomatic cases (blue) with their relative abundance data (depicted as bars) and prevalence values (given as numbers next to bars).

Figure 4. Depicts the mean percentage abundance of the top ten bacterial taxa contributing to more than 52.6% dissimilarity among the two types of samples (symptomatic = black, asymptomatic = grey) from previously root-filled teeth as identified by Bray-Curtis model of SIMPER analysis.

Figure 5. Canonical Correspondence Analysis of symptomatic and asymptomatic secondary root canal infections on species-level (A; 3% dissimilarity) and genus-level (B; 5% dissimilarity).

The samples group together according to the disease status (symptomatic – asymptomatic) with the factors ‘age’ and ‘filling status’ taken into account (red = symptomatic, green = asymptomatic).

Figure 6. SIMPER Analysis (Bray-Curtis model): Mean percentage abundance (square root transformed) values of species-level OTUs (A; 3% dissimilarity) and genus-level OTUs (B; 5% dissimilarity) contributing to differences in symptomatic versus asymptomatic cases of secondary and persistent root canal infections.