Abstract

Calcified dental plaque (dental calculus) preserves for millennia and entraps biomolecules from all domains of life and viruses. We report the first, to our knowledge, high-resolution taxonomic and protein functional characterization of the ancient oral microbiome and demonstrate that the oral cavity has long served as a reservoir for bacteria implicated in both local and systemic disease. We characterize (i) the ancient oral microbiome in a diseased state, (ii) 40 opportunistic pathogens, (iii) ancient human–associated putative antibiotic resistance genes, (iv) a genome reconstruction of the periodontal pathogen Tannerella forsythia, (v) 239 bacterial and 43 human proteins, allowing confirmation of a long-term association between host immune factors, 'red complex' pathogens and periodontal disease, and (vi) DNA sequences matching dietary sources. Directly datable and nearly ubiquitous, dental calculus permits the simultaneous investigation of pathogen activity, host immunity and diet, thereby extending direct investigation of common diseases into the human evolutionary past.

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References

  1. 1.

    Are dental diseases examples of ecological catastrophes? Microbiology 149, 279–294 (2003).

  2. 2.

    , & Periodontal diseases. Lancet 366, 1809–1820 (2005).

  3. 3.

    Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 486, 207–214 (2012).

  4. 4.

    et al. The human oral microbiome. J. Bacteriol. 192, 5002–5017 (2010).

  5. 5.

    Dietary carbohydrates and dental-systemic diseases. J. Dent. Res. 88, 490–502 (2009).

  6. 6.

    , & Associations between periodontal diseases and systemic diseases: a review of the inter-relationships and interactions with diabetes, respiratory diseases, cardiovascular diseases and osteoporosis. Public Health 122, 417–433 (2008).

  7. 7.

    , & Cardiovascular disease and the role of oral bacteria. J. Oral Microbiol. 2, 5781–5793 (2010).

  8. 8.

    & Supragingival calculus: formation and control. Crit. Rev. Oral Biol. Med. 13, 426–441 (2002).

  9. 9.

    et al. Starch granules, dental calculus and new perspectives on ancient diet. J. Archaeol. Sci. 36, 248–255 (2009).

  10. 10.

    , & Microfossils in calculus demonstrate consumption of plants and cooked foods in Neanderthal diets (Shanidar III, Iraq; Spy I and II, Belgium). Proc. Natl. Acad. Sci. USA 108, 486–491 (2011).

  11. 11.

    et al. Sequencing ancient calcified dental plaque shows changes in oral microbiota with dietary shifts of the Neolithic and Industrial revolutions. Nat. Genet. 45, 450–455 (2013).

  12. 12.

    , & DNA from human ancient bacteria: a novel source of genetic evidence from archaeological dental calculus. Archaeometry 55, 767–778 (2013).

  13. 13.

    , & Paleomicrobiological study in dental calculus: Streptococcus mutans. Scanning Microsc. 10, 1005–1013; discussion 1014 (1996).

  14. 14.

    et al. Metagenomic sequencing reveals microbiota and its functional potential associated with periodontal disease. Sci. Rep. 3, 1843 (2013).

  15. 15.

    , & Toward an ecological classification of soil bacteria. Ecology 88, 1354–1364 (2007).

  16. 16.

    & Oral health and care in the intensive care unit: state of the science. Am. J. Crit. Care 13, 25–34 (2004).

  17. 17.

    Infectious complications of dental and periodontal diseases in the elderly population. Clin. Infect. Dis. 34, 1215–1223 (2002).

  18. 18.

    et al. Detection of oral bacteria in cardiovascular specimens. Oral Microbiol. Immunol. 24, 64–68 (2009).

  19. 19.

    et al. PATRIC: the comprehensive bacterial bioinformatics resource with a focus on human pathogenic species. Infect. Immun. 79, 4286–4298 (2011).

  20. 20.

    et al. Metagenomic detection of phage-encoded platelet-binding factors in the human oral cavity. Proc. Natl. Acad. Sci. USA 108 (suppl. 1), 4547–4553 (2011).

  21. 21.

    & Periodontal microbial ecology. Periodontol. 2000 38, 135–187 (2005).

  22. 22.

    et al. Genome sequencing reveals widespread virulence gene exchange among human Neisseria species. PLoS ONE 5, e11835 (2010).

  23. 23.

    , & Management of pharyngeal gonorrhea is crucial to prevent the emergence and spread of antibiotic-resistant Neisseria gonorrhoeae. Antimicrob. Agents Chemother. 56, 4039–4040 (2012).

  24. 24.

    , , & Host genetic determinants of Neisseria meningitidis infections. Lancet Infect. Dis. 3, 565–577 (2003).

  25. 25.

    et al. Complete genome sequence of Olsenella uli type strain (VPI D76D-27CT). Stand. Genomic Sci. 3, 76–84 (2010).

  26. 26.

    Composition and development of oral bacterial communities. Periodontol. 2000 64, 20–39 (2014).

  27. 27.

    , , & Antigens of bacteria associated with periodontitis. Periodontol. 2000 35, 101–134 (2004).

  28. 28.

    , , & Variations of Porphyromonas gingivalis fimbriae in relation to microbial pathogenesis. J. Periodontal Res. 39, 136–142 (2004).

  29. 29.

    , & Functional characterization of the antibiotic resistance reservoir in the human microflora. Science 325, 1128–1131 (2009).

  30. 30.

    et al. Community and gene composition of a human dental plaque microbiota obtained by metagenomic sequencing. Mol. Oral Microbiol. 25, 391–405 (2010).

  31. 31.

    et al. Antibiotic resistance is ancient. Nature 477, 457–461 (2011).

  32. 32.

    & Tannerella forsythia, a periodontal pathogen entering the genomic era. Periodontol. 2000 42, 88–113 (2006).

  33. 33.

    Virulence mechanisms of Tannerella forsythia. Periodontol. 2000 54, 106–116 (2010).

  34. 34.

    et al. The S-layer of Tannerella forsythia contributes to serum resistance and oral bacterial co-aggregation. Infect. Immun. 81, 1198–1206 (2013).

  35. 35.

    et al. Identification and characterization of the genes encoding a unique surface (S-) layer of Tannerella forsythia. Gene 371, 102–111 (2006).

  36. 36.

    Antimicrobial peptides of the oral cavity. Periodontol. 2000 51, 152–180 (2009).

  37. 37.

    et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 39, D561–D568 (2011).

  38. 38.

    , & Cytokine-activated endothelial cells delay neutrophil apoptosis in vitro and in vivo. A role for granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 190, 923–934 (1999).

  39. 39.

    Comparison of neutrophil functions in aggressive and chronic periodontitis. Periodontol. 2000 53, 124–137 (2010).

  40. 40.

    & Neutrophil extracellular traps: is immunity the second function of chromatin? J. Cell Biol. 198, 773–783 (2012).

  41. 41.

    , & Porphyromonas gingivalis and Escherichia coli lipopolysaccharide causes resistin release from neutrophils. Oral Dis. 19, 479–483 (2013).

  42. 42.

    , & Role of resistin in obesity, insulin resistance and Type II diabetes. Clin. Sci. (Lond.) 109, 243–256 (2005).

  43. 43.

    Fat bodies and thin bodies. Cultural, biomedical and market discourses on obesity. Appetite 55, 219–225 (2010).

  44. 44.

    , , & Microbiota and dietary interactions: an update to the hygiene hypothesis? Allergy 67, 451–461 (2012).

  45. 45.

    , & The blossoming of plant archaeogenetics. Ann. Anat. 194, 146–156 (2012).

  46. 46.

    et al. Animal DNA in PCR reagents plagues ancient DNA research. J. Archaeol. Sci. 34, 1361–1366 (2007).

  47. 47.

    , , & Molecular preservation and isotopy of Mesolithic human finds from the Ofnet cave (Bavaria, Germany). Anthropol. Anz. 55, 121–129 (1997).

  48. 48.

    et al. Multi-isotopic analysis reveals individual mobility and diet at the early iron age monumental tumulus of Magdalenenberg, Germany. Am. J. Phys. Anthropol. 148, 406–421 (2012).

  49. 49.

    et al. Early Neolithic diet and animal husbandry: stable isotope evidence from three Linearbandkeramik (LBK) sites in Central Germany. J. Archaeol. Sci. 38, 270–279 (2011).

  50. 50.

    , , , & Diet, status and decomposition at Weingarten: trace element and isotope analyses on early mediaeval skeletal material. J. Archaeol. Sci. 26, 675–685 (1999).

  51. 51.

    in Advances in Human Paleopathology (ed. Pinhasi, R. & Mays, S.) Ch. 1, 29 (John Wiley & Sons, New York, 2008).

  52. 52.

    et al. Oral biofilm architecture on natural teeth. PloS One 5, e9321 (2010).

  53. 53.

    et al. Bayesian community-wide culture-independent microbial source tracking. Nat. Methods 8, 761–763 (2011).

  54. 54.

    , , & The osteological paradox—problems of inferring prehistoric health from skeletal samples. Curr. Anthropol. 33, 343–370 (1992).

  55. 55.

    et al. The metagenomics RAST server—a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinformatics 9, 386 (2008).

  56. 56.

    et al. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 41, D1063–D1069 (2013).

  57. 57.

    & Stable isotope evidence for similarities in the types of marine foods used by late mesolithic humans at sites along the Atlantic coast of Europe. J. Archaeol. Sci. 26, 717–722 (1999).

  58. 58.

    , , , & UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27, 2194–2200 (2011).

  59. 59.

    , & Infernal 1.0: inference of RNA alignments. Bioinformatics 25, 1335–1337 (2009).

  60. 60.

    et al. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J. 6, 610–618 (2012).

  61. 61.

    et al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 37, D141–D145 (2009).

  62. 62.

    , & FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26, 1641–1650 (2009).

  63. 63.

    et al. Using QIIME to analyze 16S rRNA gene sequences from microbial communities. Curr. Protoc. Bioinformatics Chapter 10, Unit 10.17 (2011).

  64. 64.

    & Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829 (2008).

  65. 65.

    & ARDB antibiotic resistance genes database. Nucleic Acids Res. 37, D443–D447 (2009).

  66. 66.

    , , & BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 12, 402 (2011).

  67. 67.

    et al. Resolution of the type material of the Asian elephant, Elephas maximus Linnaeus, 1758 (Proboscidea, Elephantidae). Zool. J. Linn. Soc. 170, 222–232 (2014).

  68. 68.

    , , & H-score, a mass accuracy driven rescoring approach for improved peptide identification in modification rich samples. J. Proteome Res. 9, 5511–5516 (2010).

  69. 69.

    et al. The Human Oral Microbiome Database: a web accessible resource for investigating oral microbe taxonomic and genomic information. Database (Oxford) 2010, baq013 (2010).

  70. 70.

    et al. In-silico human genomics with GeneCards. Hum. Genomics 5, 709–717 (2011).

  71. 71.

    , , & MEGAN analysis of metagenomic data. Genome Res. 17, 377–386 (2007).

  72. 72.

    et al. Functional analysis of metagenomes and metatranscriptomes using SEED and KEGG. BMC Bioinformatics 12 (suppl. 1), S21 (2011).

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Acknowledgements

We thank the Kantonale Ethik-Kommission Zürich, the Functional Genomics Center Zürich, the Center for Microscopy and Image Analysis, and the Institute of Oral Biology at the University of Zürich; the PRIDE Team; G. Akgül, K. Alt, D. Ashford, P. Ashton, H. Barton, A. Bouwman, C. Burger, D. Coulthard, J. Hublin, V. Meskenaite, F. Najar, M. Richards, K. Sankaranarayanan, R. Schlapbach, L. Shillito, T. Stöllner, O. Ullrich and H. Zbinden for assistance with data collection, analysis and management; and M. Carver, F. Dewhirst, A. Tanner, K. Hardy and A. Henry for helpful comments on early drafts and data analyses. This work was supported by the Mäxi Foundation Zürich, the Swiss Foundation for Nutritional Research, Danish Research Foundation grant 29396, Danish Council for Independent Research grant 10-081390, Lundbeck Foundation grants R52-A5062 and R44-A4384, US National Institutes of Health grants R01-HG005172, R01-GM089886, R01-DE018499 and R21-DE018310, European Research Council grant UMICIS/242870, Marie Curie grants EUROTAST FP7-PEOPLE-2010 MC ITN, PALIMPSEST FP7-PEOPLE-2011-IEF 299101 and ORCA FP7-PEOPLE-2011-IOF 299075, a C2D2 Research Priming Fund grant partly funded by Wellcome Trust 097829, Swiss National Science Foundation grant 31003A-135688, the Novartis Foundation, the Novo Nordisk Foundation, the Max Planck Society and the University of York.

Author information

Author notes

    • Frank Rühli
    •  & Enrico Cappellini

    These authors jointly directed this work.

Affiliations

  1. Centre for Evolutionary Medicine, Institute of Anatomy, University of Zürich, Zürich, Switzerland.

    • Christina Warinner
    • , Natallia Shved
    • , Roger Seiler
    •  & Frank Rühli
  2. Department of Anthropology, University of Oklahoma, Norman, Oklahoma, USA.

    • Christina Warinner
    • , Raul Y Tito
    •  & Cecil M Lewis Jr
  3. Institute of Molecular Life Sciences, University of Zürich, Zürich, Switzerland.

    • João F Matias Rodrigues
    • , Rounak Vyas
    •  & Christian von Mering
  4. Swiss Institute of Bioinformatics, Lausanne, Switzerland.

    • João F Matias Rodrigues
    • , Rounak Vyas
    •  & Christian von Mering
  5. Functional Genomics Center Zürich, University of Zürich/Swiss Federal Institute of Technology (ETH) Zürich, Zürich, Switzerland.

    • Christian Trachsel
    • , Jonas Grossmann
    • , Simon Barkow-Oesterreicher
    •  & Paolo Nanni
  6. BioArCh, Department of Archaeology, University of York, York, UK.

    • Anita Radini
    • , Sarah Fiddyment
    • , Camilla Speller
    • , Jessica Hendy
    • , Sophy Charlton
    • , Kai Yik Teoh
    •  & Matthew J Collins
  7. University of Leicester Archaeological Services (ULAS), School of Archaeology and Ancient History, University of Leicester, Leicester, UK.

    • Anita Radini
  8. Department of Physics, University of York, York, UK.

    • Y Hancock
  9. Centre of Dental Medicine, Institute of Oral Biology, University of Zürich, Zürich, Switzerland.

    • Hans Ulrich Luder
  10. Research Group on Plant Foods in Hominin Dietary Ecology, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.

    • Domingo C Salazar-García
  11. Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.

    • Domingo C Salazar-García
  12. Department of Prehistory and Archaeology, University of Valencia, Valencia, Spain.

    • Domingo C Salazar-García
  13. Research Group Neuro-Endocrine-Immune Interactions, Institute of Anatomy, University of Zürich, Zürich, Switzerland.

    • Elisabeth Eppler
  14. Zürich Center for Integrative Human Physiology, University of Zürich, Zürich, Switzerland.

    • Elisabeth Eppler
  15. Department of Biology, Microbiology, University of Copenhagen, Copenhagen, Denmark.

    • Lars H Hansen
  16. Department of Environmental Science, Aarhus Universitet, Roskilde, Denmark.

    • Lars H Hansen
  17. Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

    • José Alfredo Samaniego Castruita
    • , Eske Willerslev
    • , M Thomas P Gilbert
    •  & Enrico Cappellini
  18. Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

    • Christian D Kelstrup
    •  & Jesper V Olsen
  19. Department of Immunology and Infectious Diseases, Forsyth Institute, Cambridge, Massachusetts, USA.

    • Toshihisa Kawai
  20. Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Harvard University, Boston, Massachusetts, USA.

    • Toshihisa Kawai
  21. Ancient DNA Laboratory, Murdoch University, Perth, Western Australia, Australia.

    • M Thomas P Gilbert

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Contributions

C.W. conceived the project, with input from M.J.C. R.S. and F.R. contributed samples. C.W., E.C., M.J.C., M.T.P.G., C.v.M., A.R. and Y.H. designed the experiments. C.W., E.C., N.S., C.T., A.R., Y.H., D.C.S.-G., S.C., S.F., H.U.L., P.N., C.D.K., J.V.O., K.Y.T. and E.E. performed the experiments. J.F.M.R., R.V., C.W., C.v.M., J.G., A.R., Y.H., R.Y.T., S.F., C.S., S.C., D.C.S.-G., J.H., J.A.S.C., L.H.H. and T.K. analyzed the data. S.B.-O., Y.H., E.W., C.M.L., M.T.P.G., M.J.C. and F.R. contributed material support to the project. Y.H. wrote the supplementary Raman section. C.W. wrote the manuscript, with critical input from C.M.L., M.T.P.G., M.J.C., C.v.M., E.W., E.C. and the remaining authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Christina Warinner or Enrico Cappellini.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–23, Supplementary Tables 1 and 5–29, and Supplementary Note

Excel files

  1. 1.

    Supplementary Table 2

    Comparison of putative pathogens identified in ancient dental calculus and HMP healthy cohort dental plaque samples.

  2. 2.

    Supplementary Table 3

    Specific virulence factors and mobile elements identified within ancient dental calculus metagenomic and metaproteomic data.

  3. 3.

    Supplementary Table 4

    Putative antibiotic resistance genes identified from ancient dental calculus metagenomic data.

Text files

  1. 1.

    Supplementary Data Set 1

    Ancient dental samples BIOM file for 454 data.

  2. 2.

    Supplementary Data Set 2

    Ancient dental and HMP samples BIOM file for 454 data.

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DOI

https://doi.org/10.1038/ng.2906

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