Comparative analysis of bacterial profiles in unstimulated and stimulated saliva samples

Daniel Belstrøm, Palle Holmstrup, Allan Bardow, Alexis Kokaras, Nils-Erik Fiehn, Bruce J Paster, Daniel Belstrøm, Palle Holmstrup, Allan Bardow, Alexis Kokaras, Nils-Erik Fiehn, Bruce J Paster

Abstract

Background and objective: The microbial profiles of stimulated saliva samples have been shown to differentiate between patients with periodontitis, patients with dental caries, and orally healthy individuals. Saliva was stimulated to allow for easy and rapid collection; however, microbial composition may not reflect the more natural, unstimulated state. The purpose of this study was to validate whether stimulated saliva is an adequate surrogate for unstimulated saliva in determining salivary microbiomes.

Design: Unstimulated (n=20) and stimulated (n=20) saliva samples were collected from 20 orally and systemically healthy, non-smoking participants. Salivary bacterial profiles were analyzed by means of the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS), and statistical analysis was performed using Mann-Whitney test with Benjamini-Hochberg's correction for multiple comparison, cluster analysis, principal component analysis, and correspondence analysis.

Results: From a total of 40 saliva samples, 496 probe targets were identified with a mean number of targets per sample of 203 (range: 146-303), and a mean number of probe targets of 206 and 200 in unstimulated and stimulated saliva samples, respectively (p=0.62). Based on all statistical methods used for this study, the microbial profiles of unstimulated and stimulated saliva samples collected from the same person were not statistically significantly different.

Conclusions: Analysis of bacterial salivary profiles in unstimulated and stimulated saliva samples collected from the same individual showed comparable results. Thus, the results verify that stimulated saliva is an adequate surrogate of unstimulated saliva for microbiome-related studies.

Keywords: HOMINGS; bacteria; saliva.

Figures

Fig. 1
Fig. 1
Relative abundance of predominant species-level and genus-level probe targets. (a) Relative abundance of the 20 most predominant species-level probe targets in each group. (b) Relative abundance of the 10 most predominant genus-level probe targets in each group.
Fig. 2
Fig. 2
Cluster analysis based on Spearman Rank Correlation. Unstimulated saliva samples, blue; stimulated saliva samples, red. Sample denotation: 1–20 (Individual 1–Individual 20). US, unstimulated saliva sample; S, stimulated saliva sample.
Fig. 3
Fig. 3
Principal component analysis visualized two-dimensionally with axes expressed as the two most crucial components accounting for 59.8% of the variation of the dataset. Sample denotation: 1–20 (Individual 1–Individual 20). Unstimulated saliva samples (blue) and stimulated saliva samples (red).
Fig. 4
Fig. 4
Correspondence analysis visualized two-dimensionally with axes expressed as the two most crucial inertia values accounting for a cumulative inertia of 34.92%. Sample denotation: 1–20 (Individual 1–Individual 20). Unstimulated saliva samples (blue) and stimulated saliva samples (red).
Fig. 5
Fig. 5
Shannon index displaying alpha diversity between microbial profiles of unstimulated and stimulated saliva samples. (a) Collectively among subjects. (b) Within individual subjects.

References

    1. Dewhirst FE, Chen T, Izard J, Paster BJ, Tanner AC, Yu WH, et al. The human oral microbiome. J Bacteriol. 2010;192:5002–17.
    1. Aas JA, Paster BJ, Stokes LN, Olsen I, Dewhirst FE. Defining the normal bacterial flora of the oral cavity. J Clin Microbiol. 2005;43:5721–32.
    1. Jenkinson HF, Lamont RJ. Oral microbial communities in sickness and in health. Trends Microbiol. 2005;13:589–95.
    1. Teles R, Teles F, Frias-Lopez J, Paster B, Haffajee A. Lessons learned and unlearned in periodontal microbiology. Periodontol 2000. 2013;62:95–162.
    1. Wade WG. The oral microbiome in health and disease. Pharmacol Res. 2013;69:137–43.
    1. Curtis MA, Zenobia C, Darveau RP. The relationship of the oral microbiotia to periodontal health and disease. Cell Host Microbe. 2011;10:302–6.
    1. Lazarevic V, Whiteson K, Gaia N, Gizard Y, Hernandez D, Farinelli L, et al. Analysis of the salivary microbiome using culture-independent techniques. J Clin Bioinforma. 2012;2:4.
    1. Segata N, Haake SK, Mannon P, Lemon KP, Waldron L, Gevers D, et al. Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples. Genome Biol. 2012;13:R42.
    1. Belstrøm D, Holmstrup P, Nielsen CH, Kirkby N, Twetman S, Heitmann BL, et al. Bacterial profiles of saliva in relation to diet, lifestyle factors, and socioeconomic status. J Oral Microbiol. 2014;6 23609. doi: .
    1. Giannobile WV, McDevitt JT, Niedbala RS, Malamud D. Translational and clinical applications of salivary diagnostics. Adv Dent Res. 2011;23:375–80.
    1. Yoshizawa JM, Schafer CA, Schafer JJ, Farrell JJ, Paster BJ, Wong DT. Salivary biomarkers: toward future clinical and diagnostic utilities. Clin Microbiol Rev. 2013;26:781–91.
    1. Belstrøm D, Fiehn NE, Nielsen CH, Kirkby N, Twetman S, Klepac-Ceraj V, et al. Differences in bacterial saliva profile between periodontitis patients and a control cohort. J Clin Periodontol. 2014;41:104–12.
    1. Belstrøm D, Fiehn NE, Nielsen CH, Holmstrup P, Kirkby N, Klepac-Ceraj V, et al. Altered bacterial profiles in saliva from adults with caries lesions: a case-cohort study. Caries Res. 2014;48:368–75.
    1. Belstrøm D, Fiehn NE, Nielsen CH, Klepac-Ceraj V, Paster BJ, Twetman S, et al. Differentiation of salivary bacterial profiles of subjects with periodontitis and dental caries. J Oral Microbiol. 2015;6 27429. doi: .
    1. Yakob M, Fuentes L, Wang MB, Abemayor E, Wong DT. Salivary biomarkers for detection of oral squamous cell carcinoma – current state and recent advances. Curr Oral Health Rep. 2014;1:133–41.
    1. Schafer CA, Schafer JJ, Yakob M, Lima P, Camargo P, Wong DT. Saliva diagnostics: utilizing oral fluids to determine health status. Monogr Oral Sci. 2014;24:88–98.
    1. Simón-Soro A, Tomás I, Cabrera-Rubio R, Catalan MD, Nyvad B, Mira A. Microbial geography of the oral cavity. J Dent Res. 2013;92:616–21.
    1. Dasanayake AP, Caufield PW, Cutter GR, Roseman JM, Köhler B. Differences in the detection and enumeration of mutans streptococci due to differences in methods. Arch Oral Biol. 1995;40:345–51.
    1. Asikainen S, Alaluusua S, Saxén L. Recovery of A. actinomycetemcomitans from teeth, tongue, and saliva. J Periodontol. 1991;62:203–6.
    1. Ebersole JL, Schuster JL, Stevens J, Dawson D, III, Kryscio RJ, Lin Y, et al. Patterns of salivary analytes provide diagnostic capacity for distinguishing chronic adult periodontitis from health. J Clin Immunol. 2013;33:271–9.
    1. Kongstad J, Ekstrand K, Qvist V, Christensen LB, Cortsen B, Gronbaek M, et al. Findings from the oral health study of the Danish Health Examination Survey 2007–2008. Acta Odontol Scand. 2013;71:1560–9.
    1. Gomes BP, Berber VB, Kokaras AS, Chen T, Paster BJ. Microbiomes of endodontic-periodontal lesions before and after chemomechanical preparation. J Endod. 2015;41:1975–84.
    1. Belstrøm D, Paster BJ, Fiehn NE, Bardow A, Holmstrup P. Salivary bacterial fingerprints of established oral disease revealed by the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS) technique. J Oral Microbiol. 2016;8 30170. doi: .
    1. Belstrøm D, Holmstrup P, Bardow A, Kokaras A, Fiehn NE, Paster BJ. Temporal Stability of the Salivary Microbiota in Oral Health. PLoS One. 2016;11:e0147472.
    1. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci USA. 2011;108(Suppl 1):4516–22.
    1. Hochberg Y, Benjamini Y. More powerful procedures for multiple significance testing. Stat Med. 1990;9:811–18.
    1. Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, Howe EA, et al. TM4 microarray software suite. Methods Enzymol. 2006;411:134–93.
    1. Kinney JS, Morelli T, Braun T, Ramseier CA, Herr AE, Sugai JV, et al. Saliva/pathogen biomarker signatures and periodontal disease progression. J Dent Res. 2011;90:752–8.
    1. Rathnayake N, Akerman S, Klinge B, Lundegren N, Jansson H, Tryselius Y, et al. Salivary biomarkers of oral health: a cross-sectional study. J Clin Periodontol. 2013;40:140–7.
    1. Giannobile WV, Beikler T, Kinney JS, Ramseier CA, Morelli T, Wong DT. Saliva as a diagnostic tool for periodontal disease: current state and future directions. Periodontol 2000. 2009;50:52–64.

Source: PubMed

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