Microbial Ecology

, Volume 60, Issue 3, pp 677–690 | Cite as

Analysis of Oral Microbiota in Children with Dental Caries by PCR-DGGE and Barcoded Pyrosequencing

  • Zongxin Ling
  • Jianming Kong
  • Peng Jia
  • Chaochun Wei
  • Yuezhu Wang
  • Zhiwen Pan
  • Wujing Huang
  • Lanjuan Li
  • Hui ChenEmail author
  • Charlie XiangEmail author


Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p < 0.05). The bacterial diversity in oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p < 0.05). The results showed that there was no one specific pathogen but rather pathogenic populations in plaque that significantly correlated with dental caries. The enormous diversity of oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.


Oral Cavity Dental Caries Bacterial Diversity Dental Plaque Actinomyces 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This present work was funded by the grant of National Basic Research Program of China (973 Program) No. 2007CB513001, and partly supported by grants from China’s National Science and Technology Major Project (No. 2008ZX10002-009, No. 2008ZX10004-002 and 2009ZX10004-105). We thank Dr. Michael Brownstein and Dr. Liliana Losada for critical reading and useful suggestions.

Supplementary material

248_2010_9712_MOESM1_ESM.doc (53 kb)
Table S1 List of the 112 8-bp barcodes used to tag each PCR product analyzed as part of the study (DOC 53 kb)
248_2010_9712_MOESM2_ESM.jpg (783 kb)
Fig. S1 (JPEG 783 kb)
248_2010_9712_MOESM3_ESM.jpg (73 kb)
Fig. S2 (JPEG 72 kb)


  1. 1.
    Gill SR, Pop M, Deboy RT, Eckburg PB, Turnbaugh PJ, Samuel BS, Gordon JI, Relman DA, Fraser-Liggett CM, Nelson KE (2006) Metagenomic analysis of the human distal gut microbiome. Science 312:1355–1359CrossRefPubMedGoogle Scholar
  2. 2.
    Friedrich MJ (2008) Microbiome project seeks to understand human body's microscopic residents. JAMA 300:777–778CrossRefPubMedGoogle Scholar
  3. 3.
    Dethlefsen L, McFall-Ngai M, Relman DA (2007) An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 449:811–818CrossRefPubMedGoogle Scholar
  4. 4.
    Cash HL, Whitham CV, Behrendt CL, Hooper LV (2006) Symbiotic bacteria direct expression of an intestinal bactericidal lectin. Science 313:1126–1130CrossRefPubMedGoogle Scholar
  5. 5.
    Ley RE, Peterson DA, Gordon JI (2006) Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124:837–848CrossRefPubMedGoogle Scholar
  6. 6.
    Ley RE, Turnbaugh PJ, Klein S, Gordon JI (2006) Microbial ecology: human gut microbes associated with obesity. Nature 444:1022–1023CrossRefPubMedGoogle Scholar
  7. 7.
    Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL (2005) An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122:107–118CrossRefPubMedGoogle Scholar
  8. 8.
    Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:1027–1031CrossRefPubMedGoogle Scholar
  9. 9.
    Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE (2005) Defining the normal bacterial flora of the oral cavity. J Clin Microbiol 43:5721–5732CrossRefPubMedGoogle Scholar
  10. 10.
    Paster BJ, Olsen I, Aas JA, Dewhirst FE (2006) The breadth of bacterial diversity in the human periodontal pocket and other oral sites. Periodontol 2000 42:80–87CrossRefPubMedGoogle Scholar
  11. 11.
    Ruby J, Goldner M (2007) Nature of symbiosis in oral disease. J Dent Res 86:8–11CrossRefPubMedGoogle Scholar
  12. 12.
    Lockhart PB, Durack DT (1999) Oral microflora as a cause of endocarditis and other distant site infections. Infect Dis Clin North Am 13:833–850, viCrossRefPubMedGoogle Scholar
  13. 13.
    Paju S, Scannapieco FA (2007) Oral biofilms, periodontitis, and pulmonary infections. Oral Dis 13:508–512CrossRefPubMedGoogle Scholar
  14. 14.
    Boggess KA, Beck JD, Murtha AP, Moss K, Offenbacher S (2006) Maternal periodontal disease in early pregnancy and risk for a small-for-gestational-age infant. Am J Obstet Gynecol 194:1316–1322CrossRefPubMedGoogle Scholar
  15. 15.
    Offenbacher S, Boggess KA, Murtha AP, Jared HL, Lieff S, McKaig RG, Mauriello SM, Moss KL, Beck JD (2006) Progressive periodontal disease and risk of very preterm delivery. Obstet Gynecol 107:29–36PubMedGoogle Scholar
  16. 16.
    Beck JD, Eke P, Heiss G, Madianos P, Couper D, Lin D, Moss K, Elter J, Offenbacher S (2005) Periodontal disease and coronary heart disease: a reappraisal of the exposure. Circulation 112:19–24CrossRefPubMedGoogle Scholar
  17. 17.
    Beck JD, Eke P, Lin D, Madianos P, Couper D, Moss K, Elter J, Heiss G, Offenbacher S (2005) Associations between IgG antibody to oral organisms and carotid intima-medial thickness in community-dwelling adults. Atherosclerosis 183:342–348CrossRefPubMedGoogle Scholar
  18. 18.
    Anusavice KJ (2002) Dental caries: risk assessment and treatment solutions for an elderly population. Compend Contin Educ Dent 23:12–20PubMedGoogle Scholar
  19. 19.
    Selwitz RH, Ismail AI, Pitts NB (2007) Dental caries. Lancet 369:51–59CrossRefPubMedGoogle Scholar
  20. 20.
    Loesche WJ (1992) The specific plaque hypothesis and the antimicrobial treatment of periodontal disease. Dent Update 19(68):70–72Google Scholar
  21. 21.
    Marsh PD (1994) Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 8:263–271PubMedGoogle Scholar
  22. 22.
    Theilade E (1986) The non-specific theory in microbial etiology of inflammatory periodontal diseases. J Clin Periodontol 13:905–911CrossRefPubMedGoogle Scholar
  23. 23.
    Jenkinson HF, Lamont RJ (2005) Oral microbial communities in sickness and in health. Trends Microbiol 13:589–595CrossRefPubMedGoogle Scholar
  24. 24.
    Kuramitsu HK, He X, Lux R, Anderson MH, Shi W (2007) Interspecies interactions within oral microbial communities. Microbiol Mol Biol Rev 71:653–670CrossRefPubMedGoogle Scholar
  25. 25.
    Hill JE, Goh SH, Money DM, Doyle M, Li A, Crosby WL, Links M, Leung A, Chan D, Hemmingsen SM (2005) Characterization of vaginal microflora of healthy, nonpregnant women by chaperonin-60 sequence-based methods. Am J Obstet Gynecol 193:682–692CrossRefPubMedGoogle Scholar
  26. 26.
    Schellenberg J, Links MG, Hill JE, Dumonceaux TJ, Peters GA, Tyler S, Ball TB, Severini A, Plummer FA (2009) Pyrosequencing of the chaperonin-60 universal target as a tool for determining microbial community composition. Appl Environ Microbiol 75:2889–2898CrossRefPubMedGoogle Scholar
  27. 27.
    Zaura E, Keijser BJ, Huse SM, Crielaard W (2009) Defining the healthy “core microbiome” of oral microbial communities. BMC Microbiol 9:259CrossRefPubMedGoogle Scholar
  28. 28.
    Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, Galvin JL, Boches SK, Dewhirst FE, Griffen AL (2002) Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol 40:1001–1009CrossRefPubMedGoogle Scholar
  29. 29.
    Peterson J, Garges S, Giovanni M, McInnes P, Wang L, Schloss JA, Bonazzi V, McEwen JE, Wetterstrand KA, Deal C, Baker CC, Di Francesco V, Howcroft TK, Karp RW, Lunsford RD, Wellington CR, Belachew T, Wright M, Giblin C, David H, Mills M, Salomon R, Mullins C, Akolkar B, Begg L, Davis C, Grandison L, Humble M, Khalsa J, Little AR, Peavy H, Pontzer C, Portnoy M, Sayre MH, Starke-Reed P, Zakhari S, Read J, Watson B, Guyer M (2009) The NIH human microbiome project. Genome Res 19:2317–2323CrossRefPubMedGoogle Scholar
  30. 30.
    Keijser BJ, Zaura E, Huse SM, van der Vossen JM, Schuren FH, Montijn RC, ten Cate JM, Crielaard W (2008) Pyrosequencing analysis of the oral microflora of healthy adults. J Dent Res 87:1016–1020CrossRefPubMedGoogle Scholar
  31. 31.
    Meyer M, Stenzel U, Hofreiter M (2008) Parallel tagged sequencing on the 454 platform. Nat Protoc 3:267–278CrossRefPubMedGoogle Scholar
  32. 32.
    Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700PubMedGoogle Scholar
  33. 33.
    Li M, Wang B, Zhang M, Rantalainen M, Wang S, Zhou H, Zhang Y, Shen J, Pang X, Zhang M, Wei H, Chen Y, Lu H, Zuo J, Su M, Qiu Y, Jia W, Xiao C, Smith LM, Yang S, Holmes E, Tang H, Zhao G, Nicholson JK, Li L, Zhao L (2008) Symbiotic gut microbes modulate human metabolic phenotypes. Proc Natl Acad Sci USA 105:2117–2122CrossRefPubMedGoogle Scholar
  34. 34.
    Watanabe K, Kodama Y, Harayama S (2001) Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting. J Microbiol Methods 44:253–262CrossRefPubMedGoogle Scholar
  35. 35.
    Roh SW, Kim KH, Nam YD, Chang HW, Park EJ, Bae JW (2010) Investigation of archaeal and bacterial diversity in fermented seafood using barcoded pyrosequencing. ISME J 4:1–16CrossRefPubMedGoogle Scholar
  36. 36.
    Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer ML, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, Lohman KL, Lu H, Makhijani VB, McDade KE, McKenna MP, Myers EW, Nickerson E, Nobile JR, Plant R, Puc BP, Ronan MT, Roth GT, Sarkis GJ, Simons JF, Simpson JW, Srinivasan M, Tartaro KR, Tomasz A, Vogt KA, Volkmer GA, Wang SH, Wang Y, Weiner MP, Yu P, Begley RF, Rothberg JM (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376–380PubMedGoogle Scholar
  37. 37.
    Hamady M, Walker JJ, Harris JK, Gold NJ, Knight R (2008) Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat Methods 5:235–237CrossRefPubMedGoogle Scholar
  38. 38.
    Parameswaran P, Jalili R, Tao L, Shokralla S, Gharizadeh B, Ronaghi M, Fire AZ (2007) A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing. Nucleic Acids Res 35:e130CrossRefPubMedGoogle Scholar
  39. 39.
    Sogin ML, Morrison HG, Huber JA, Mark Welch D, Huse SM, Neal PR, Arrieta JM, Herndl GJ (2006) Microbial diversity in the deep sea and the underexplored “rare biosphere”. Proc Natl Acad Sci USA 103:12115–12120CrossRefPubMedGoogle Scholar
  40. 40.
    Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541CrossRefPubMedGoogle Scholar
  41. 41.
    Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267CrossRefPubMedGoogle Scholar
  42. 42.
    Lozupone C, Hamady M, Knight R (2006) UniFrac—an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinform 7:371CrossRefGoogle Scholar
  43. 43.
    Binladen J, Gilbert MT, Bollback JP, Panitz F, Bendixen C, Nielsen R, Willerslev E (2007) The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing. PLoS ONE 2:e197CrossRefPubMedGoogle Scholar
  44. 44.
    Corby PM, Lyons-Weiler J, Bretz WA, Hart TC, Aas JA, Boumenna T, Goss J, Corby AL, Junior HM, Weyant RJ, Paster BJ (2005) Microbial risk indicators of early childhood caries. J Clin Microbiol 43:5753–5759CrossRefPubMedGoogle Scholar
  45. 45.
    Li Y, Ge Y, Saxena D, Caufield PW (2007) Genetic profiling of the oral microbiota associated with severe early-childhood caries. J Clin Microbiol 45:81–87CrossRefPubMedGoogle Scholar
  46. 46.
    Aas JA, Griffen AL, Dardis SR, Lee AM, Olsen I, Dewhirst FE, Leys EJ, Paster BJ (2008) Bacteria of dental caries in primary and permanent teeth in children and young adults. J Clin Microbiol 46:1407–1417CrossRefPubMedGoogle Scholar
  47. 47.
    Li Y, Ku CY, Xu J, Saxena D, Caufield PW (2005) Survey of oral microbial diversity using PCR-based denaturing gradient gel electrophoresis. J Dent Res 84:559–564CrossRefPubMedGoogle Scholar
  48. 48.
    Yu Z, Morrison M (2004) Comparisons of different hypervariable regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis. Appl Environ Microbiol 70:4800–4806CrossRefPubMedGoogle Scholar
  49. 49.
    Ercolini D (2004) PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. J Microbiol Methods 56:297–314CrossRefPubMedGoogle Scholar
  50. 50.
    Roesch LF, Fulthorpe RR, Riva A, Casella G, Hadwin AK, Kent AD, Daroub SH, Camargo FA, Farmerie WG, Triplett EW (2007) Pyrosequencing enumerates and contrasts soil microbial diversity. ISME J 1:283–290PubMedGoogle Scholar
  51. 51.
    Tringe SG, Hugenholtz P (2008) A renaissance for the pioneering 16S rRNA gene. Curr Opin Microbiol 11:442–446CrossRefPubMedGoogle Scholar
  52. 52.
    Liu Z, DeSantis TZ, Andersen GL, Knight R (2008) Accurate taxonomy assignments from 16S rRNA sequences produced by highly parallel pyrosequencers. Nucleic Acids Res 36:e120CrossRefPubMedGoogle Scholar
  53. 53..
    Engelbrektson A, Kunin V, Wrighton KC, Zvenigorodsky N, Chen F, Ochman H, Hugenholtz P (2010) Experimental factors affecting PCR-based estimates of microbial species richness and evenness. ISME J (in press)Google Scholar
  54. 54.
    Yang X, Xie L, Li Y, Wei C (2009) More than 9,000,000 unique genes in human gut bacterial community: estimating gene numbers inside a human body. PLoS ONE 4:e6074CrossRefPubMedGoogle Scholar
  55. 55.
    Andersson AF, Lindberg M, Jakobsson H, Backhed F, Nyren P, Engstrand L (2008) Comparative analysis of human gut microbiota by barcoded pyrosequencing. PLoS ONE 3:e2836CrossRefPubMedGoogle Scholar
  56. 56.
    Papaioannou W, Gizani S, Haffajee AD, Quirynen M, Mamai-Homata E, Papagiannoulis L (2009) The microbiota on different oral surfaces in healthy children. Oral Microbiol Immunol 24:183–189CrossRefPubMedGoogle Scholar
  57. 57.
    Tanner AC, Milgrom PM, Kent R Jr, Mokeem SA, Page RC, Riedy CA, Weinstein P, Bruss J (2002) The microbiota of young children from tooth and tongue samples. J Dent Res 81:53–57CrossRefPubMedGoogle Scholar
  58. 58.
    Chalmers NI, Palmer RJ Jr, Cisar JO, Kolenbrander PE (2008) Characterization of a Streptococcus sp.–Veillonella sp. community micromanipulated from dental plaque. J Bacteriol 190:8145–8154CrossRefPubMedGoogle Scholar
  59. 59.
    Egland PG, Palmer RJ Jr, Kolenbrander PE (2004) Interspecies communication in Streptococcus gordoniiVeillonella atypica biofilms: signaling in flow conditions requires juxtaposition. Proc Natl Acad Sci USA 101:16917–16922CrossRefPubMedGoogle Scholar
  60. 60.
    Al-Ahmad A, Follo M, Selzer AC, Hellwig E, Hannig M, Hannig C (2009) Bacterial colonisation of enamel in situ investigated with fluorescence in situ hybridization (FISH). J Med Microbiol (in press)Google Scholar
  61. 61.
    Choi EJ, Lee SH, Kim YJ (2009) Quantitative real-time polymerase chain reaction for Streptococcus mutans and Streptococcus sobrinus in dental plaque samples and its association with early childhood caries. Int J Paediatr Dent 19:141–147CrossRefPubMedGoogle Scholar
  62. 62.
    Arif N, Sheehy EC, Do T, Beighton D (2008) Diversity of Veillonella spp. from sound and carious sites in children. J Dent Res 87:278–282CrossRefPubMedGoogle Scholar
  63. 63.
    Takeshita T, Nakano Y, Kumagai T, Yasui M, Kamio N, Shibata Y, Shiota S, Yamashita Y (2009) The ecological proportion of indigenous bacterial populations in saliva is correlated with oral health status. ISME J 3:65–78CrossRefPubMedGoogle Scholar
  64. 64.
    Ohara-Nemoto Y, Kishi K, Satho M, Tajika S, Sasaki M, Namioka A, Kimura S (2005) Infective endocarditis caused by Granulicatella elegans originating in the oral cavity. J Clin Microbiol 43:1405–1407CrossRefPubMedGoogle Scholar
  65. 65.
    Frank DN, St Amand AL, Feldman RA, Boedeker EC, Harpaz N, Pace NR (2007) Molecular-phylogenetic characterization of microbial community imbalances in human inflammatory bowel diseases. Proc Natl Acad Sci USA 104:13780–13785CrossRefPubMedGoogle Scholar
  66. 66.
    Chhour KL, Nadkarni MA, Byun R, Martin FE, Jacques NA, Hunter N (2005) Molecular analysis of microbial diversity in advanced caries. J Clin Microbiol 43:843–849CrossRefPubMedGoogle Scholar
  67. 67.
    Dige I, Raarup MK, Nyengaard JR, Kilian M, Nyvad B (2009) Actinomyces naeslundii in initial dental biofilm formation. Microbiology 155:2116–2126CrossRefPubMedGoogle Scholar
  68. 68.
    Preza D, Olsen I, Aas JA, Willumsen T, Grinde B, Paster BJ (2008) Bacterial profiles of root caries in elderly patients. J Clin Microbiol 46:2015–2021CrossRefPubMedGoogle Scholar
  69. 69.
    Ready D, D'Aiuto F, Spratt DA, Suvan J, Tonetti MS, Wilson M (2008) Disease severity associated with presence in subgingival plaque of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Tannerella forsythia, singly or in combination, as detected by nested multiplex PCR. J Clin Microbiol 46:3380–3383CrossRefPubMedGoogle Scholar
  70. 70.
    Yilmaz O (2008) The chronicles of Porphyromonas gingivalis: the microbium, the human oral epithelium and their interplay. Microbiology 154:2897–2903CrossRefPubMedGoogle Scholar

Copyright information

© mSpringer Science+Business Media, LLC 2010

Authors and Affiliations

  • Zongxin Ling
    • 1
  • Jianming Kong
    • 2
  • Peng Jia
    • 3
  • Chaochun Wei
    • 3
  • Yuezhu Wang
    • 5
  • Zhiwen Pan
    • 4
  • Wujing Huang
    • 4
  • Lanjuan Li
    • 1
  • Hui Chen
    • 4
    Email author
  • Charlie Xiang
    • 1
    Email author
  1. 1.State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, the First Affiliated Hospital, College of MedicineZhejiang UniversityHangzhouChina
  2. 2.Zhejiang-California International Nanosystems Institute (ZCNI)Zhejiang UniversityHangzhouChina
  3. 3.Shanghai Center for Bioinformation TechnologyShanghaiChina
  4. 4.Affiliated Hospital of Stomatology, College of MedicineZhejiang UniversityHangzhouChina
  5. 5.Chinese National Human Genome Center at ShanghaiShanghaiChina

Personalised recommendations