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Deep sequencing of the oral microbiome reveals signatures of periodontal disease.

Liu B, Faller LL, Klitgord N, Mazumdar V, Ghodsi M, Sommer DD, Gibbons TR, Treangen TJ, Chang YC, Li S, Stine OC, Hasturk H, Kasif S, Segrè D, Pop M, Amar S - PLoS ONE (2012)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, United States of America.

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.

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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 [86].
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pone-0037919-g005: 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 [86].

Mentions: Close analysis of one of the most abundant organisms in our samples (present at 24- and 6-fold coverage in samples H2 and H1, respectively, Table 4), a relative of Actinomyces naeslundii MG1 (sequence ID SEQF1063 in the Human Oral Microbiome Database (HOMD) database), provides evidence for structural variations distinguishing this strain from the reference strain originally isolated from a patient with mild gingivitis [56]. The average similarity between the assembled metagenomic contigs from our project and the reference sequence is 96.2% and 95.2% for samples H1 and H2, respectively (second and sixth ring in Figure 5). A number of genomic deletions with respect to the reference strain are apparent in our samples, several of which contain potential virulence factors. These differences could be explained by the fact that the reference genome was isolated from a patient with gingivitis, while in our samples the Actinomyces strains are predominantly associated with healthy samples. Most striking is a deletion at 2120 kbp containing a putative mobile element encoding a mercury resistance locus (including a mercury resistance gene, a site-specific recombinase, and an integrase). Mercury resistance is commonly found in oral bacteria, frequently associated with antibiotic resistance [42]. Interestingly, gene set enrichment analysis of the entire metagenomic data-set reveals an enrichment of mercury resistance genes in the diseased samples (Table S2), possibly due to the association of these genes with virulence loci. Several other deletions also appear to encode virulence factors - a drug transporter (at position 580 kbp in the reference strain) and an alcohol dehydrogenase gene (at position 165 kbp) – further underscoring the difference between the pathogenic reference strain and the presumably commensal Actinomyces strains found in our samples. Another two deletions (at positions 20 kbp, and 1010 kbp) contain genes predicted to encode proteins involved in secretion and response regulation. These deletions occur at slightly different locations in the two samples we analyzed, suggesting they may be subject to rapid evolution.


Deep sequencing of the oral microbiome reveals signatures of periodontal disease.

Liu B, Faller LL, Klitgord N, Mazumdar V, Ghodsi M, Sommer DD, Gibbons TR, Treangen TJ, Chang YC, Li S, Stine OC, Hasturk H, Kasif S, Segrè D, Pop M, Amar S - PLoS ONE (2012)

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 [86].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366996&req=5

pone-0037919-g005: 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 [86].
Mentions: Close analysis of one of the most abundant organisms in our samples (present at 24- and 6-fold coverage in samples H2 and H1, respectively, Table 4), a relative of Actinomyces naeslundii MG1 (sequence ID SEQF1063 in the Human Oral Microbiome Database (HOMD) database), provides evidence for structural variations distinguishing this strain from the reference strain originally isolated from a patient with mild gingivitis [56]. The average similarity between the assembled metagenomic contigs from our project and the reference sequence is 96.2% and 95.2% for samples H1 and H2, respectively (second and sixth ring in Figure 5). A number of genomic deletions with respect to the reference strain are apparent in our samples, several of which contain potential virulence factors. These differences could be explained by the fact that the reference genome was isolated from a patient with gingivitis, while in our samples the Actinomyces strains are predominantly associated with healthy samples. Most striking is a deletion at 2120 kbp containing a putative mobile element encoding a mercury resistance locus (including a mercury resistance gene, a site-specific recombinase, and an integrase). Mercury resistance is commonly found in oral bacteria, frequently associated with antibiotic resistance [42]. Interestingly, gene set enrichment analysis of the entire metagenomic data-set reveals an enrichment of mercury resistance genes in the diseased samples (Table S2), possibly due to the association of these genes with virulence loci. Several other deletions also appear to encode virulence factors - a drug transporter (at position 580 kbp in the reference strain) and an alcohol dehydrogenase gene (at position 165 kbp) – further underscoring the difference between the pathogenic reference strain and the presumably commensal Actinomyces strains found in our samples. Another two deletions (at positions 20 kbp, and 1010 kbp) contain genes predicted to encode proteins involved in secretion and response regulation. These deletions occur at slightly different locations in the two samples we analyzed, suggesting they may be subject to rapid evolution.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, United States of America.

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.

Show MeSH
Related in: MedlinePlus