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Background: The purpose of the present investigation was to use the oral cavity as an in-vivo model to study the impact of internal and external perturbations on bacterial biofilm communities. We tested the hypotheses that bacterial gene expression of the healthy microbiota reflects habituation to site-specific ecological perturbations, and that the perturbation effect of chronic inflammation, i.e. periodontitis, impacts bacterial gene expression not only locally, but also at other sites of the oral cavity. Oral microbial samples were collected from three oral sites – plaque, tongue and saliva from patients with periodontitis and healthy controls. Paired metagenomics and metatranscriptomics were used to perform concomitant characterization of taxonomic composition and to determine species specific bacterial activity as expressed by the ratio of messenger RNA to the corresponding genomic DNA.
Results: Here we show the impact of two perturbations – oral site and periodontitis - on bacterial gene expression of the oral microbiota. The oral site was the main determinant of taxonomic composition as well as bacterial gene expression. However, bacterial activity at the three oral sites (plaque, tongue, and saliva) was significantly impacted by periodontitis, with a reduction of the carbohydrate metabolism.
Conclusions: Data from the present study characterize the impact of two perturbations – oral site and periodontitis - on bacterial gene expression of the oral microbiota. The oral site was the main determinant of taxonomic composition as well as bacterial gene expression. However, presence of periodontitis had impact on bacterial activity of both plaque but also on tongue and the salivary microbiota. Collectively, data suggest that periodontitis associates with impaired carbohydrate metabolism of the oral microbiota. Future longitudinal and interventional studies are warranted to evaluate the potential pathogenic role of impaired bacterial carbohydrate metabolism not only in periodontitis, but also in other diseases with low grade inflammation, such as type 2 diabetes mellitus.
Keywords
Metagenomics, Metatranscriptomics, Periodontitis, Biofilm, Ecological perturbation
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This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigures.docx
Fig. S1. Species differentially abundant between: A) healthy patients and patients with periodontitis disease. B) oral sites. Statistical tests were run on all the samples. Sample denotation: Red: periodontitis, dark blue: oral health, light blue: plaque, yellow: tongue and grey: saliva. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Microbial taxonomy based on metaphlan v3.0.1 - default – ALDEx2::aldex.kw, denom="iqlr”. Only significant results are displayed FDRA adjusted p. value <0.05. Fig. S2. Pathway-level species metatranscriptomic activity: A) healthy patients and patients with periodontitis disease - overall, B) healthy patients and patients with periodontitis disease on each site, and C) among the different oral sites in healthy patients and patients with periodontitis disease. Each point represents the pathway transcriptional activity as measured by log10(RNA/DNA). Sample denotation: Red: periodontitis, dark blue: oral health, light blue: plaque, yellow: tongue and grey: saliva. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Significant differences were determined using Kruskall-Wallis tests. Significant FDR-adjusted p.values were indicated as followed. * 0.05<p<0.01 ** 0.01 <p<0.001 *** p<0.001. Fig. S3. Differentially expressed pathways and contributing species in Plaque and Saliva sites: Pathways exhibiting significant differential expression between healthy and periodontitis patients as identified using MaAsLin2 in A) plaque and C) saliva site. Colour indicates heath status (blue: healthy controls vs red: periodontitis group), and shape reflects oral site. Species relative contribution to the differentially expressed pathways (identified in Fig. S3C) in B) plaque and D) saliva. Species are coloured similarly as in Fig. 3. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Fig S4. Diversity of microbial genes associated with associated with KEGG’s Orthologs : A) Alpha-diversity of gene families found in metagenomes (DNA) and metatranscriptomes (RNA) in plaque, tongue biofilm and saliva expressed as number of Observed gene families (i.e., richness), Evenness and Shannon’s diversity index. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. B) Beta diversity of gene expression visualized as measured by log RNA/DNA ratio quantified using weighted Jaccard distance and visualized on PCoA. Sample denotation: Red: periodontitis, Blue: oral health, Circle: plaque, Triangle: tongue biofilm, Square: saliva. Significant differencies were assessed using Kruskall-Wallis and PERMANOVAS tests for alpha and beta-diversity, respectively. Significant FDR-adjusted p.values were indicated as followed : * 0.05>p>0.01 ** 0.01>p>0.001 *** p<0.001. Fig S5. Genes (mapping KEGG’s orthologs) differentially expressed (RNA/DNA ratio) between healthy patient and patient with periodontal diseases: A) overall, B) plaque, C) saliva and D) tongue site. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles.
- SupplementaryFigures.docx
Fig. S1. Species differentially abundant between: A) healthy patients and patients with periodontitis disease. B) oral sites. Statistical tests were run on all the samples. Sample denotation: Red: periodontitis, dark blue: oral health, light blue: plaque, yellow: tongue and grey: saliva. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Microbial taxonomy based on metaphlan v3.0.1 - default – ALDEx2::aldex.kw, denom="iqlr”. Only significant results are displayed FDRA adjusted p. value <0.05. Fig. S2. Pathway-level species metatranscriptomic activity: A) healthy patients and patients with periodontitis disease - overall, B) healthy patients and patients with periodontitis disease on each site, and C) among the different oral sites in healthy patients and patients with periodontitis disease. Each point represents the pathway transcriptional activity as measured by log10(RNA/DNA). Sample denotation: Red: periodontitis, dark blue: oral health, light blue: plaque, yellow: tongue and grey: saliva. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Significant differences were determined using Kruskall-Wallis tests. Significant FDR-adjusted p.values were indicated as followed. * 0.05<p<0.01 ** 0.01 <p<0.001 *** p<0.001. Fig. S3. Differentially expressed pathways and contributing species in Plaque and Saliva sites: Pathways exhibiting significant differential expression between healthy and periodontitis patients as identified using MaAsLin2 in A) plaque and C) saliva site. Colour indicates heath status (blue: healthy controls vs red: periodontitis group), and shape reflects oral site. Species relative contribution to the differentially expressed pathways (identified in Fig. S3C) in B) plaque and D) saliva. Species are coloured similarly as in Fig. 3. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Fig S4. Diversity of microbial genes associated with associated with KEGG’s Orthologs : A) Alpha-diversity of gene families found in metagenomes (DNA) and metatranscriptomes (RNA) in plaque, tongue biofilm and saliva expressed as number of Observed gene families (i.e., richness), Evenness and Shannon’s diversity index. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. B) Beta diversity of gene expression visualized as measured by log RNA/DNA ratio quantified using weighted Jaccard distance and visualized on PCoA. Sample denotation: Red: periodontitis, Blue: oral health, Circle: plaque, Triangle: tongue biofilm, Square: saliva. Significant differencies were assessed using Kruskall-Wallis and PERMANOVAS tests for alpha and beta-diversity, respectively. Significant FDR-adjusted p.values were indicated as followed : * 0.05>p>0.01 ** 0.01>p>0.001 *** p<0.001. Fig S5. Genes (mapping KEGG’s orthologs) differentially expressed (RNA/DNA ratio) between healthy patient and patient with periodontal diseases: A) overall, B) plaque, C) saliva and D) tongue site. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles.
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Background: The purpose of the present investigation was to use the oral cavity as an in-vivo model to study the impact of internal and external perturbations on bacterial biofilm communities. We tested the hypotheses that bacterial gene expression of the healthy microbiota reflects habituation to site-specific ecological perturbations, and that the perturbation effect of chronic inflammation, i.e. periodontitis, impacts bacterial gene expression not only locally, but also at other sites of the oral cavity. Oral microbial samples were collected from three oral sites – plaque, tongue and saliva from patients with periodontitis and healthy controls. Paired metagenomics and metatranscriptomics were used to perform concomitant characterization of taxonomic composition and to determine species specific bacterial activity as expressed by the ratio of messenger RNA to the corresponding genomic DNA.
Results: Here we show the impact of two perturbations – oral site and periodontitis - on bacterial gene expression of the oral microbiota. The oral site was the main determinant of taxonomic composition as well as bacterial gene expression. However, bacterial activity at the three oral sites (plaque, tongue, and saliva) was significantly impacted by periodontitis, with a reduction of the carbohydrate metabolism.
Conclusions: Data from the present study characterize the impact of two perturbations – oral site and periodontitis - on bacterial gene expression of the oral microbiota. The oral site was the main determinant of taxonomic composition as well as bacterial gene expression. However, presence of periodontitis had impact on bacterial activity of both plaque but also on tongue and the salivary microbiota. Collectively, data suggest that periodontitis associates with impaired carbohydrate metabolism of the oral microbiota. Future longitudinal and interventional studies are warranted to evaluate the potential pathogenic role of impaired bacterial carbohydrate metabolism not only in periodontitis, but also in other diseases with low grade inflammation, such as type 2 diabetes mellitus.
Figure 1
Figure 1
Figure 2
Figure 2
Figure 3
Figure 3
Figure 4
Figure 4
Figure 5
Figure 5
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigures.docx
Fig. S1. Species differentially abundant between: A) healthy patients and patients with periodontitis disease. B) oral sites. Statistical tests were run on all the samples. Sample denotation: Red: periodontitis, dark blue: oral health, light blue: plaque, yellow: tongue and grey: saliva. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Microbial taxonomy based on metaphlan v3.0.1 - default – ALDEx2::aldex.kw, denom="iqlr”. Only significant results are displayed FDRA adjusted p. value <0.05. Fig. S2. Pathway-level species metatranscriptomic activity: A) healthy patients and patients with periodontitis disease - overall, B) healthy patients and patients with periodontitis disease on each site, and C) among the different oral sites in healthy patients and patients with periodontitis disease. Each point represents the pathway transcriptional activity as measured by log10(RNA/DNA). Sample denotation: Red: periodontitis, dark blue: oral health, light blue: plaque, yellow: tongue and grey: saliva. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Significant differences were determined using Kruskall-Wallis tests. Significant FDR-adjusted p.values were indicated as followed. * 0.05<p<0.01 ** 0.01 <p<0.001 *** p<0.001. Fig. S3. Differentially expressed pathways and contributing species in Plaque and Saliva sites: Pathways exhibiting significant differential expression between healthy and periodontitis patients as identified using MaAsLin2 in A) plaque and C) saliva site. Colour indicates heath status (blue: healthy controls vs red: periodontitis group), and shape reflects oral site. Species relative contribution to the differentially expressed pathways (identified in Fig. S3C) in B) plaque and D) saliva. Species are coloured similarly as in Fig. 3. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Fig S4. Diversity of microbial genes associated with associated with KEGG’s Orthologs : A) Alpha-diversity of gene families found in metagenomes (DNA) and metatranscriptomes (RNA) in plaque, tongue biofilm and saliva expressed as number of Observed gene families (i.e., richness), Evenness and Shannon’s diversity index. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. B) Beta diversity of gene expression visualized as measured by log RNA/DNA ratio quantified using weighted Jaccard distance and visualized on PCoA. Sample denotation: Red: periodontitis, Blue: oral health, Circle: plaque, Triangle: tongue biofilm, Square: saliva. Significant differencies were assessed using Kruskall-Wallis and PERMANOVAS tests for alpha and beta-diversity, respectively. Significant FDR-adjusted p.values were indicated as followed : * 0.05>p>0.01 ** 0.01>p>0.001 *** p<0.001. Fig S5. Genes (mapping KEGG’s orthologs) differentially expressed (RNA/DNA ratio) between healthy patient and patient with periodontal diseases: A) overall, B) plaque, C) saliva and D) tongue site. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles.
- SupplementaryFigures.docx
Fig. S1. Species differentially abundant between: A) healthy patients and patients with periodontitis disease. B) oral sites. Statistical tests were run on all the samples. Sample denotation: Red: periodontitis, dark blue: oral health, light blue: plaque, yellow: tongue and grey: saliva. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Microbial taxonomy based on metaphlan v3.0.1 - default – ALDEx2::aldex.kw, denom="iqlr”. Only significant results are displayed FDRA adjusted p. value <0.05. Fig. S2. Pathway-level species metatranscriptomic activity: A) healthy patients and patients with periodontitis disease - overall, B) healthy patients and patients with periodontitis disease on each site, and C) among the different oral sites in healthy patients and patients with periodontitis disease. Each point represents the pathway transcriptional activity as measured by log10(RNA/DNA). Sample denotation: Red: periodontitis, dark blue: oral health, light blue: plaque, yellow: tongue and grey: saliva. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Significant differences were determined using Kruskall-Wallis tests. Significant FDR-adjusted p.values were indicated as followed. * 0.05<p<0.01 ** 0.01 <p<0.001 *** p<0.001. Fig. S3. Differentially expressed pathways and contributing species in Plaque and Saliva sites: Pathways exhibiting significant differential expression between healthy and periodontitis patients as identified using MaAsLin2 in A) plaque and C) saliva site. Colour indicates heath status (blue: healthy controls vs red: periodontitis group), and shape reflects oral site. Species relative contribution to the differentially expressed pathways (identified in Fig. S3C) in B) plaque and D) saliva. Species are coloured similarly as in Fig. 3. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. Fig S4. Diversity of microbial genes associated with associated with KEGG’s Orthologs : A) Alpha-diversity of gene families found in metagenomes (DNA) and metatranscriptomes (RNA) in plaque, tongue biofilm and saliva expressed as number of Observed gene families (i.e., richness), Evenness and Shannon’s diversity index. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles. B) Beta diversity of gene expression visualized as measured by log RNA/DNA ratio quantified using weighted Jaccard distance and visualized on PCoA. Sample denotation: Red: periodontitis, Blue: oral health, Circle: plaque, Triangle: tongue biofilm, Square: saliva. Significant differencies were assessed using Kruskall-Wallis and PERMANOVAS tests for alpha and beta-diversity, respectively. Significant FDR-adjusted p.values were indicated as followed : * 0.05>p>0.01 ** 0.01>p>0.001 *** p<0.001. Fig S5. Genes (mapping KEGG’s orthologs) differentially expressed (RNA/DNA ratio) between healthy patient and patient with periodontal diseases: A) overall, B) plaque, C) saliva and D) tongue site. Boxplots display first quartile, median, third quartile and whiskers represent 1.5 times the interquartile range from the first and third quartiles.
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