Skip to main content

Advertisement

SpringerLink
Log in

Comparative evaluation of microbial profiles of oral samples obtained at different collection time points and using different methods

  • Original Article
  • Published:
Clinical Oral Investigations Aims and scope Submit manuscript

Abstract

Objectives

Recently, the oral microbiome has been found to be associated with oral and general health status. Although various oral sample collection protocols are available, the potential differences between the results yielded by these protocols remain unclear. In this study, we aimed to determine the effects of different time points and methods of oral sample collection on the outcomes of microbiome analysis.

Materials and methods

Oral samples were collected from eight healthy individuals at four different time points: 2 h after eating, immediately after teeth brushing, immediately after waking up, and 2 h after eating on the subsequent day. Four methods of saliva collection were evaluated: spitting, gum chewing, cotton swab, and oral rinse. Oral microbiomes of these samples were compared by analyzing the bacterial 16S rRNA gene sequence data.

Results

The oral microbial composition at the genus level was similar among all sample collection time points and methods. Alpha diversity was not significantly different among the groups, whereas beta diversity was different between the spitting and cotton swab methods. Compared with the between-subject variations, the weighted UniFrac distances between the groups were not minor.

Conclusions

Although the oral microbiome profiles obtained at different collection time points and using different methods were similar, some differences were detected.

Clinical relevance

The results of the present study suggest that although all the described protocols are useful, comparisons among microbiomes of samples collected by different methods are not appropriate. Researchers must be aware of the issues regarding the impact of saliva collection methods.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Metzker ML (2010) Sequencing technologies - the next generation. Nat Rev Genet 11:31–46. https://doi.org/10.1038/nrg2626

    Article  PubMed  Google Scholar 

  2. Quintana FJ, Prinz M (2017) A gut feeling about multiple sclerosis. Proc Natl Acad Sci U S A 114:10528–10529. https://doi.org/10.1073/pnas.1714260114

    Article  PubMed  PubMed Central  Google Scholar 

  3. Qin J, Li Y, Cai Z, Li S, Zhu J, Zhang F, Liang S, Zhang W, Guan Y, Shen D, Peng Y, Zhang D, Jie Z, Wu W, Qin Y, Xue W, Li J, Han L, Lu D, Wu P, Dai Y, Sun X, Li Z, Tang A, Zhong S, Li X, Chen W, Xu R, Wang M, Feng Q, Gong M, Yu J, Zhang Y, Zhang M, Hansen T, Sanchez G, Raes J, Falony G, Okuda S, Almeida M, LeChatelier E, Renault P, Pons N, Batto JM, Zhang Z, Chen H, Yang R, Zheng W, Li S, Yang H, Wang J, Ehrlich SD, Nielsen R, Pedersen O, Kristiansen K, Wang J (2012) A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490:55–60. https://doi.org/10.1038/nature11450

    Article  PubMed  Google Scholar 

  4. Ley RE, Turnbaugh PJ, Klein S, Gordon JI (2006) Microbial ecology: human gut microbes associated with obesity. Nature 444:1022–1023. https://doi.org/10.1038/4441022a

    Article  PubMed  Google Scholar 

  5. Dinan TG, Cryan JF (2017) Gut instincts: microbiota as a key regulator of brain development, ageing and neurodegeneration. J Physiol 595:489–503. https://doi.org/10.1113/jp273106

    Article  PubMed  Google Scholar 

  6. Lira-Junior R, Akerman S, Klinge B, Bostrom EA, Gustafsson A (2018) Salivary microbial profiles in relation to age, periodontal, and systemic diseases. PLOS ONE 13:e0189374. https://doi.org/10.1371/journal.pone.0189374

    Article  PubMed  PubMed Central  Google Scholar 

  7. Yamashita Y, Takeshita T (2017) The oral microbiome and human health. J Oral Sci 59:201–206. https://doi.org/10.2334/josnusd.16-0856

    Article  PubMed  Google Scholar 

  8. Zhang X, Zhang D, Jia H, Feng Q, Wang D, Liang D, Wu X, Li J, Tang L, Li Y, Lan Z, Chen B, Li Y, Zhong H, Xie H, Jie Z, Chen W, Tang S, Xu X, Wang X, Cai X, Liu S, Xia Y, Li J, Qiao X, Al-Aama JY, Chen H, Wang L, Wu QJ, Zhang F, Zheng W, Li Y, Zhang M, Luo G, Xue W, Xiao L, Li J, Chen W, Xu X, Yin Y, Yang H, Wang J, Kristiansen K, Liu L, Li T, Huang Q, Li Y, Wang J (2015) The oral and gut microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment. Nat Med 21:895–905. https://doi.org/10.1038/nm.3914

    Article  PubMed  Google Scholar 

  9. Abe K, Takahashi A, Fujita M, Imaizumi H, Hayashi M, Okai K, Ohira H (2018) Dysbiosis of oral microbiota and its association with salivary immunological biomarkers in autoimmune liver disease. PLOS ONE 13:e0198757. https://doi.org/10.1371/journal.pone.0198757

    Article  PubMed  PubMed Central  Google Scholar 

  10. Peters BA, Wu J, Pei Z, Yang L, Purdue MP, Freedman ND, Jacobs EJ, Gapstur SM, Hayes RB, Ahn J (2017) Oral microbiome composition reflects prospective risk for esophageal cancers. Cancer Res 77:6777–6787. https://doi.org/10.1158/0008-5472.can-17-1296

    Article  PubMed  PubMed Central  Google Scholar 

  11. Fan X, Alekseyenko AV, Wu J, Peters BA, Jacobs EJ, Gapstur SM, Purdue MP, Abnet CC, Stolzenberg-Solomon R, Miller G, Ravel J, Hayes RB, Ahn J (2018) Human oral microbiome and prospective risk for pancreatic cancer: a population-based nested case-control study. Gut 67:120–127. https://doi.org/10.1136/gutjnl-2016-312580

    Article  PubMed  Google Scholar 

  12. Said HS, Suda W, Nakagome S, Chinen H, Oshima K, Kim S, Kimura R, Iraha A, Ishida H, Fujita J, Mano S, Morita H, Dohi T, Oota H, Hattori M (2014) Dysbiosis of salivary microbiota in inflammatory bowel disease and its association with oral immunological biomarkers. DNA Res 21:15–25. https://doi.org/10.1093/dnares/dst037

    Article  PubMed  Google Scholar 

  13. Bajaj JS, Betrapally NS, Hylemon PB, Heuman DM, Daita K, White MB, Unser A, Thacker LR, Sanyal AJ, Kang DJ, Sikaroodi M, Gillevet PM (2015) Salivary microbiota reflects changes in gut microbiota in cirrhosis with hepatic encephalopathy. Hepatology 62:1260–1271. https://doi.org/10.1002/hep.27819

    Article  PubMed  Google Scholar 

  14. Atarashi K, Suda W, Luo C, Kawaguchi T, Motoo I, Narushima S, Kiguchi Y, Yasuma K, Watanabe E, Tanoue T, Thaiss CA, Sato M, Toyooka K, Said HS, Yamagami H, Rice SA, Gevers D, Johnson RC, Segre JA, Chen K, Kolls JK, Elinav E, Morita H, Xavier RJ, Hattori M, Honda K (2017) Ectopic colonization of oral bacteria in the intestine drives TH1 cell induction and inflammation. Science 358:359–365. https://doi.org/10.1126/science.aan4526

    Article  PubMed  PubMed Central  Google Scholar 

  15. Takayasu L, Suda W, Takanashi K, Iioka E, Kurokawa R, Shindo C, Hattori Y, Yamashita N, Nishijima S, Oshima K, Hattori M (2017) Circadian oscillations of microbial and functional composition in the human salivary microbiome. DNA Res 24:261–270. https://doi.org/10.1093/dnares/dsx001

    Article  PubMed  PubMed Central  Google Scholar 

  16. Collado MC, Engen PA, Bandin C, Cabrera-Rubio R, Voigt RM, Green SJ, Naqib A, Keshavarzian A, Scheer F, Garaulet M (2018) Timing of food intake impacts daily rhythms of human salivary microbiota: a randomized, crossover study. FASEB J 32:2060–2072. https://doi.org/10.1096/fj.201700697RR

    Article  PubMed  PubMed Central  Google Scholar 

  17. Khurshid Z, Zohaib S, Najeeb S, Zafar MS, Slowey PD, Almas K (2016) Human saliva collection devices for proteomics: an update. Int J Mol Sci 17:846. https://doi.org/10.3390/ijms17060846

    Article  PubMed Central  Google Scholar 

  18. Krishnan K, Chen T, Paster BJ (2017) A practical guide to the oral microbiome and its relation to health and disease. Oral Dis 23:276–286. https://doi.org/10.1111/odi.12509

    Article  PubMed  Google Scholar 

  19. Bhattarai KR, Kim HR, Chae HJ (2018) Compliance with saliva collection protocol in healthy volunteers: strategies for managing risk and errors. Int J Med Sci 15:823–831. https://doi.org/10.7150/ijms.25146

    Article  PubMed  PubMed Central  Google Scholar 

  20. Segata N, Haake SK, Mannon P, Lemon KP, Waldron L, Gevers D, Huttenhower C, Izard J (2012) Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples. Genome Biol 13:R42. https://doi.org/10.1186/gb-2012-13-6-r42

    Article  PubMed  PubMed Central  Google Scholar 

  21. Belstrom D, Holmstrup P, Bardow A, Kokaras A, Fiehn NE, Paster BJ (2016) Temporal stability of the salivary microbiota in oral health. PLOS ONE 11:e0147472. https://doi.org/10.1371/journal.pone.0147472

    Article  PubMed  PubMed Central  Google Scholar 

  22. Stahringer SS, Clemente JC, Corley RP, Hewitt J, Knights D, Walters WA, Knight R, Krauter KS (2012) Nurture trumps nature in a longitudinal survey of salivary bacterial communities in twins from early adolescence to early adulthood. Genome Res 22:2146–2152. https://doi.org/10.1101/gr.140608.112

    Article  PubMed  PubMed Central  Google Scholar 

  23. Zhou Y, Gao H, Mihindukulasuriya KA, La Rosa PS, Wylie KM, Vishnivetskaya T, Podar M, Warner B, Tarr PI, Nelson DE, Fortenberry JD, Holland MJ, Burr SE, Shannon WD, Sodergren E, Weinstock GM (2013) Biogeography of the ecosystems of the healthy human body. Genome Biol 14:R1. https://doi.org/10.1186/gb-2013-14-1-r1

    Article  PubMed  PubMed Central  Google Scholar 

  24. Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R (2011) UniFrac: an effective distance metric for microbial community comparison. ISME J 5:169–172. https://doi.org/10.1038/ismej.2010.133

    Article  PubMed  Google Scholar 

  25. van der Meulen TA, Harmsen H, Bootsma H, Spijkervet F, Kroese F, Vissink A (2016) The microbiome-systemic diseases connection. Oral Dis 22:719–734. https://doi.org/10.1111/odi.12472

    Article  PubMed  Google Scholar 

  26. Lazarevic V, Whiteson K, Hernandez D, François P, Schrenzel J (2010) Study of inter- and intra-individual variations in the salivary microbiota. BMC Genomics 11:523. https://doi.org/10.1186/1471-2164-11-523

    Article  PubMed  PubMed Central  Google Scholar 

  27. David LA, Materna AC, Friedman J, Campos-Baptista MI, Blackburn MC, Perrotta A, Erdman SE, Alm EJ (2014) Host lifestyle affects human microbiota on daily timescales. Genome Biol 15:R89. https://doi.org/10.1186/gb-2014-15-7-r89

    Article  PubMed  PubMed Central  Google Scholar 

  28. Cameron SJ, Huws SA, Hegarty MJ, Smith DP, Mur LA (2015) The human salivary microbiome exhibits temporal stability in bacterial diversity. FEMS Microbiol Ecol 91:fiv091. https://doi.org/10.1093/femsec/fiv091

    Article  PubMed  Google Scholar 

  29. Wake N, Asahi Y, Noiri Y, Hayashi M, Motooka D, Nakamura S, Gotoh K, Miura J, Machi H, Iida T, Ebisu S (2016) Temporal dynamics of bacterial microbiota in the human oral cavity determined using an in situ model of dental biofilms. Npj Biofilms Microbiomes 2:16018. https://doi.org/10.1038/npjbiofilms.2016.18

    Article  PubMed  PubMed Central  Google Scholar 

  30. Belstrom D, Holmstrup P, Bardow A, Kokaras A, Fiehn NE, Paster BJ (2016) Comparative analysis of bacterial profiles in unstimulated and stimulated saliva samples. J Oral Microbiol 8:30112. https://doi.org/10.3402/jom.v8.30112

    Article  PubMed  Google Scholar 

  31. Gomar-Vercher S, Simon-Soro A, Montiel-Company JM, Almerich-Silla JM, Mira A (2018) Stimulated and unstimulated saliva samples have significantly different bacterial profiles. PLOS ONE 13:e0198021. https://doi.org/10.1371/journal.pone.0198021

    Article  PubMed  PubMed Central  Google Scholar 

  32. Lim Y, Totsika M, Morrison M, Punyadeera C (2017) The saliva microbiome profiles are minimally affected by collection method or DNA extraction protocols. Sci Rep 7:8523. https://doi.org/10.1038/s41598-017-07885-3

    Article  PubMed  PubMed Central  Google Scholar 

  33. Jo R, Nishimoto Y, Umezawa K, Yama K, Aita Y, Ichiba Y, Murakami S, Kakizawa Y, Kumagai T, Yamada T, Fukuda S (2019) Comparison of oral microbiome profiles in stimulated and unstimulated saliva, tongue, and mouth-rinsed water. Sci Rep 9:16124. https://doi.org/10.1038/s41598-019-52445-6

    Article  PubMed  PubMed Central  Google Scholar 

  34. Fan X, Peters BA, Min D, Ahn J, Hayes RB (2018) Comparison of the oral microbiome in mouthwash and whole saliva samples. PLOS ONE 13:e0194729. https://doi.org/10.1371/journal.pone.0194729

    Article  PubMed  PubMed Central  Google Scholar 

  35. Yu G, Phillips S, Gail MH, Goedert JJ, Humphrys M, Ravel J, Ren Y, Caporaso NE (2017) Evaluation of buccal cell samples for studies of oral microbiota. Cancer Epidemiol Biomarkers Prev 26:249–253. https://doi.org/10.1158/1055-9965.epi-16-0538

    Article  PubMed  Google Scholar 

  36. Vogtmann E, Chen J, Kibriya MG, Amir A, Shi J, Chen Y, Islam T, Eunes M, Ahmed A, Naher J, Rahman A, Barmon B, Knight R, Chia N, Ahsan H, Abnet CC, Sinha R (2019) Comparison of oral collection methods for studies of microbiota. Cancer Epidemiol Biomarkers Prev 28:137–143. https://doi.org/10.1158/1055-9965.epi-18-0312

    Article  PubMed  Google Scholar 

  37. Yano Y, Hua X, Wan Y, Suman S, Zhu B, Dagnall CL, Hutchinson A, Jones K, Hicks BD, Shi J, Abnet CC, Vogtmann E (2020) Comparison of oral microbiota collected using multiple methods and recommendations for new epidemiologic studies. mSystems 5. https://doi.org/10.1128/mSystems.00156-20

  38. Zhou X, Nanayakkara S, Gao JL, Nguyen KA, Adler CJ (2019) Storage media and not extraction method has the biggest impact on recovery of bacteria from the oral microbiome. Sci Rep 9:14968. https://doi.org/10.1038/s41598-019-51448-7

    Article  PubMed  PubMed Central  Google Scholar 

  39. Teng F, Darveekaran Nair SS, Zhu P, Li S, Huang S, Li X, Xu J, Yang F (2018) Impact of DNA extraction method and targeted 16S-rRNA hypervariable region on oral microbiota profiling. Sci Rep 8:16321. https://doi.org/10.1038/s41598-018-34294-x

    Article  PubMed  PubMed Central  Google Scholar 

  40. Vesty A, Biswas K, Taylor MW, Gear K, Douglas RG (2017) Evaluating the impact of DNA extraction method on the representation of human oral bacterial and fungal communities. PLOS ONE 12:e0169877. https://doi.org/10.1371/journal.pone.0169877

    Article  PubMed  PubMed Central  Google Scholar 

  41. Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glockner FO (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41:e1. https://doi.org/10.1093/nar/gks808

    Article  PubMed  Google Scholar 

Download references

Funding

The work was supported and conducted at the Department of Dentistry and Oral Surgery, Osaka Medical College, Osaka, Japan.

Author information

Authors and Affiliations

  1. Department of Dentistry and Oral Surgery, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki City, Osaka, 569-8686, Japan

    Michi Omori, Nahoko Kato-Kogoe, Nozomu Fukui, Kayoko Yamamoto, Yoichiro Nakajima, Kazuya Inoue, Hiroyuki Nakano & Takaaki Ueno

  2. Department of Microbiology and Infection Control, Osaka Medical College, Takatsuki, Osaka, Japan

    Shoichi Sakaguchi & Takashi Nakano

  3. Department of Infection Metagenomics, Genome Information Research Center, Research Institute for Microbial Diseases, Osaka University, Suita, Japan

    Daisuke Motooka & Shota Nakamura

Authors
  1. Michi Omori
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nahoko Kato-Kogoe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shoichi Sakaguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nozomu Fukui
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kayoko Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yoichiro Nakajima
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kazuya Inoue
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hiroyuki Nakano
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Daisuke Motooka
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Takashi Nakano
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Shota Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Takaaki Ueno
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the study conception and design. Material preparation was conducted by Michi Omori, Nozomu Fukui, Kayoko Yamamoto, Yoichiro Nakajima, Kazuya Inoue, and Hiroyuki Nakano. The data collection and analysis were conducted by Nahoko Kato-Kogoe, Shoichi Sakaguchi, Daisuke Motooka, and Shota Nakamura. The first draft of the manuscript was written by Nahoko Kato-Kogoe and review and editing by Takashi Nakano, Shota Nakamura, and Takaaki Ueno, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Nahoko Kato-Kogoe.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All procedures conducted in studies involving human participants were according to the ethical standards of the Ethics Committee of Osaka Medical College, approval no. 2145, and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the present study.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Omori, M., Kato-Kogoe, N., Sakaguchi, S. et al. Comparative evaluation of microbial profiles of oral samples obtained at different collection time points and using different methods. Clin Oral Invest 25, 2779–2789 (2021). https://doi.org/10.1007/s00784-020-03592-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00784-020-03592-y

Keywords

Search

Navigation