Bacterial community shift in treated periodontitis patients revealed by ion torrent 16S rRNA gene amplicon sequencing

Abstract

Periodontitis, one of the most common diseases in the world, is caused by a mixture of pathogenic bacteria and inflammatory host responses and often treated by antimicrobials as an adjunct to scaling and root planing (SRP). Our study aims to elucidate explorative and descriptive temporal shifts in bacterial communities between patients treated by SRP alone versus SRP plus antibiotics. This is the first metagenomic study using an Ion Torrent Personal Genome Machine (PGM). Eight subgingival plaque samples from four patients with chronic periodontitis, taken before and two months after intervention were analyzed. Amplicons from the V6 hypervariable region of the 16S rRNA gene were generated and sequenced each on a 314 chip. Sequencing reads were clustered into operational taxonomic units (OTUs, 3% distance), described by community metrics, and taxonomically classified. Reads ranging from 599,933 to 650,416 per sample were clustered into 1,648 to 2,659 non-singleton OTUs, respectively. Increased diversity (Shannon and Simpson) in all samples after therapy was observed regardless of the treatment type whereas richness (ACE) showed no correlation. Taxonomic analysis revealed different microbial shifts between both therapy approaches at all taxonomic levels. Most remarkably, the genera Porphyromonas, Tannerella, Treponema, and Filifactor all harboring periodontal pathogenic species were removed almost only in the group treated with SPR and antibiotics. For the species T. forsythia and P. gingivalis results were corroborated by real-time PCR analysis. In the future, hypothesis free metagenomic analysis could be the key in understanding polymicrobial diseases and be used for therapy monitoring. Therefore, as read length continues to increase and cost to decrease, rapid benchtop sequencers like the PGM might finally be used in routine diagnostic.

Figure 1. Rarefaction analysis for each sample based on clustering of high quality de-noised reads.

In total samples from four patients were analyzed two times, i.e., before (continuous lines) and two months after intervention (dashed lines). Two control group patients (Co) were treated by SRP alone whereas two experimental group patients (Ex) received SRP plus adjunctive antibiotics. Lines are colored according to the sample origin.

Figure 2. Relative abundance of the most prevalent genera (> = 1% relative abundance within one sample) in subgingival plaque samples before and after intervention.

Each bar displays the normalized relative abundance (individual relative abundance divided by the number of analyzed samples at the given time point) of the corresponding genera pre and post intervention. Each bar is further partitioned into four stacked parts for each of the four analyzed patients. The color indicates the type of intervention (blue for control, red for experimental group).