Deep sequencing of the oral microbiome reveals signatures of periodontal disease

Abstract

The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (~2 lanes Illumina 76 bp PE) and high human DNA contamination (up to ~90%) we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.

Figure 1. Relative abundance of genera in the samples estimated from 16S rDNA sequencing. * - genus significantly enriched in cases; ∧- genus significantly enriched in controls (p< = 0.05, Metastats [ 30]).

Only genera with >1% abundance in at least one sample were included. Colors reflect relative abundance from low (red) to high (white). Sample H31 (control) clusters together with the diseased samples, consistent with clinical observations of early symptoms of periodontal disease.

Figure 2. Metabolic pathways present in our samples.

Dark blue – significantly enriched in healthy samples (p<0.05, MetaPath [84]); Dark red– significantly enriched in diseased samples (p<0.05, MetaPath). (Figure constructed with iPath [85]).

Figure 3. Systems-level analysis reveals the disease state to occupy a narrow region within the space of possible states for the microbiome.

A – Shannon diversity calculated from 16S rDNA data is significantly higher in diseased samples (community is more diverse). B – Principal Component Analysis of the taxonomic compositions from 16S rDNA (empty symbols) and pooled WGS data (filled symbols). Disease samples cluster together in the bottom left corner. Sample H31 (tooth with incipient periodontal disease from an otherwise healthy patient) clusters together with the disease samples. C – Principal component analysis of the enzyme content of samples based on metagenomic sequencing. The PCA graph shows a tighter clustering of disease samples (red) relative to the healthy ones (blue). This suggests that the disease state may be linked to a specific metabolic configuration, and that the space of disease configurations is more constrained than the healthy one. Replicates (forward/reverse reads from one or two instrument lanes) are shown separately as identical symbols, and exhibit minimal metabolic variation within each sample. D – Comparison of relative frequencies of tetramers (4 bp motifs) in metagenomic reads across disease cases (in red, P1 vs. P2) and across control cases (in blue, H1 vs. H2). Based on the relative frequencies of tetramers, disease samples are more similar to each other (points lie along the diagonal) than controls are to each other.

Figure 4. Schematic representation of the putative metabolic lifestyle shifts associated with the change in microbial flora around the tooth and gum tissue upon the transition from a healthy (A) to an advanced periodontal disease (B) state.

The healthy state is dominated by the bacterial genera Streptococcus, Fusobacterium, Actinomyces, and Corynebacterium, whereas the disease state is primarily dominated by pathogenic genera such as Prevotella, Leptotrichia, Treponema, and Fusobacterium.

Figure 5. Comparative analysis of the Actinomyces naeslundii MG1 reference genome (HOMD SeqID SEQF1063), and assemblies from samples H1 and H2.

Counting from outside, the first ring is the reference genome with genes annotated by colored bands: orange bands are conserved genes; green bands are genes involved in horizontal gene transfer; yellow bands are genes with known functions; black bands are genes with unknown functions. Only regions that are associated with genomic variations are colored and annotated. The second and sixth rings are the assembled contigs from sample H2 and H1. The heatmaps in the third and seventh rings represent the depth of coverage of the contigs with 5K bp window and 100 bp step for sample H2 and H1. The histograms in the fourth and eighth rings represent the scaled SNP rate with 5 Kbp window and 100 bp step for sample H2 (max = 0.08) and H1 (max = 0.03). The purple bands in the fifth ring represent regions with significantly higher theta values than average (p< = 0.05). Image generated with Circos .