Processing and Storage Methods Affect Oral and Gut Microbiome Composition

Overview

Journal Front Microbiol

Specialty Microbiology

Date 2023 Oct 19

PMID 37854339

Authors

Dorothy K Superdock

Wei Zhang

Angela C Poole

Affiliations

Soon will be listed here.

Abstract

In microbiome studies, fecal and oral samples are stored and processed in different ways, which could affect the observed microbiome composition. In this study, we compared storage and processing methods applied to samples prior to DNA extraction to determine how each affected microbial community diversity as assessed by 16S rRNA gene sequencing. We collected dental swabs, saliva, and fecal samples from 10 individuals, with three technical replicates per condition. We assessed four methods of storing and processing fecal samples prior to DNA extraction. We also compared different fractions of thawed saliva and dental samples to fresh samples. We found that lyophilized fecal samples, fresh whole saliva samples, and the supernatant fraction of thawed dental samples had the highest levels of alpha diversity. The supernatant fraction of thawed saliva samples had the second highest evenness compared to fresh saliva samples. Then, we investigated the differences in observed community composition at the domain and phylum levels and identified the amplicon sequence variants (ASVs) that significantly differed in relative abundance between the conditions. Lyophilized fecal samples had a greater prevalence of Archaea as well as a greater ratio of Firmicutes to Bacteroidetes compared to the other conditions. Our results provide practical considerations not only for the selection of storage and processing methods but also for comparing results across studies. Differences in processing and storage methods could be a confounding factor influencing the presence, absence, or differential abundance of microbes reported in conflicting studies.

Citing Articles

Processing and storage methods affect oral and gut microbiome composition.

Superdock D, Zhang W, Poole A Front Microbiol. 2023; 14:1253570.

PMID: 37854339 PMC: 10579807. DOI: 10.3389/fmicb.2023.1253570.

References

Cruz P, Mehretu A, Buttner M, Trice T, Howard K . Development of a polymerase chain reaction assay for the rapid detection of the oral pathogenic bacterium, Selenomonas noxia. BMC Oral Health. 2015; 15:95. PMC: 4536781. DOI: 10.1186/s12903-015-0071-1. View

Johnson E, Heaver S, Walters W, Ley R . Microbiome and metabolic disease: revisiting the bacterial phylum Bacteroidetes. J Mol Med (Berl). 2016; 95(1):1-8. PMC: 5187364. DOI: 10.1007/s00109-016-1492-2. View

Fiedorova K, Radvansky M, Nemcova E, Grombirikova H, Bosak J, cernochova M . The Impact of DNA Extraction Methods on Stool Bacterial and Fungal Microbiota Community Recovery. Front Microbiol. 2019; 10:821. PMC: 6479168. DOI: 10.3389/fmicb.2019.00821. View

Verbrugghe P, Brynjolfsson J, Jing X, Bjorck I, Hallenius F, Nilsson A . Evaluation of hypoglycemic effect, safety and immunomodulation of Prevotella copri in mice. Sci Rep. 2021; 11(1):21279. PMC: 8553810. DOI: 10.1038/s41598-021-96161-6. View

Scher J, Sczesnak A, Longman R, Segata N, Ubeda C, Bielski C . Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. Elife. 2013; 2:e01202. PMC: 3816614. DOI: 10.7554/eLife.01202. View

Kovatcheva-Datchary P, Nilsson A, Akrami R, Lee Y, De Vadder F, Arora T . Dietary Fiber-Induced Improvement in Glucose Metabolism Is Associated with Increased Abundance of Prevotella. Cell Metab. 2015; 22(6):971-82. DOI: 10.1016/j.cmet.2015.10.001. View

Zhang H, DiBaise J, Zuccolo A, Kudrna D, Braidotti M, Yu Y . Human gut microbiota in obesity and after gastric bypass. Proc Natl Acad Sci U S A. 2009; 106(7):2365-70. PMC: 2629490. DOI: 10.1073/pnas.0812600106. View

Magne F, Gotteland M, Gauthier L, Zazueta A, Pesoa S, Navarrete P . The Firmicutes/Bacteroidetes Ratio: A Relevant Marker of Gut Dysbiosis in Obese Patients?. Nutrients. 2020; 12(5). PMC: 7285218. DOI: 10.3390/nu12051474. View

Song S, Amir A, Metcalf J, Amato K, Xu Z, Humphrey G . Preservation Methods Differ in Fecal Microbiome Stability, Affecting Suitability for Field Studies. mSystems. 2016; 1(3). PMC: 5069758. DOI: 10.1128/mSystems.00021-16. View

10.

Torresyap G, Haffajee A, Uzel N, Socransky S . Relationship between periodontal pocket sulfide levels and subgingival species. J Clin Periodontol. 2004; 30(11):1003-10. DOI: 10.1034/j.1600-051x.2003.00377.x. View

11.

Fouhy F, Deane J, Rea M, OSullivan O, Ross R, OCallaghan G . The effects of freezing on faecal microbiota as determined using MiSeq sequencing and culture-based investigations. PLoS One. 2015; 10(3):e0119355. PMC: 4352061. DOI: 10.1371/journal.pone.0119355. View

12.

Poole A, Goodrich J, Youngblut N, Luque G, Ruaud A, Sutter J . Human Salivary Amylase Gene Copy Number Impacts Oral and Gut Microbiomes. Cell Host Microbe. 2019; 25(4):553-564.e7. DOI: 10.1016/j.chom.2019.03.001. View

13.

Basseri R, Basseri B, Pimentel M, Chong K, Youdim A, Low K . Intestinal methane production in obese individuals is associated with a higher body mass index. Gastroenterol Hepatol (N Y). 2012; 8(1):22-8. PMC: 3277195. View

14.

Bahl M, Bergstrom A, Rask Licht T . Freezing fecal samples prior to DNA extraction affects the Firmicutes to Bacteroidetes ratio determined by downstream quantitative PCR analysis. FEMS Microbiol Lett. 2012; 329(2):193-7. DOI: 10.1111/j.1574-6968.2012.02523.x. View

15.

Wade W . The oral microbiome in health and disease. Pharmacol Res. 2012; 69(1):137-43. DOI: 10.1016/j.phrs.2012.11.006. View

16.

Teng F, Darveekaran Nair S, Zhu P, Li S, Huang S, Li X . Impact of DNA extraction method and targeted 16S-rRNA hypervariable region on oral microbiota profiling. Sci Rep. 2018; 8(1):16321. PMC: 6218491. DOI: 10.1038/s41598-018-34294-x. View

17.

Selway C, Jensen E, Pena A, Smart G, Weyrich L . Type 1 diabetes, periodontal health, and a familial history of hyperlipidaemia is associated with oral microbiota in children: a cross-sectional study. BMC Oral Health. 2023; 23(1):15. PMC: 9832783. DOI: 10.1186/s12903-022-02625-0. View

18.

Abduweli Uyghurturk D, Lu Y, Urata J, Dvorak C, Den Besten P . Dental caries as a risk factor for bacterial blood stream infection (BSI) in children undergoing hematopoietic cell transplantation (HCT). PeerJ. 2022; 10:e14040. PMC: 9511999. DOI: 10.7717/peerj.14040. View

19.

Callahan B, McMurdie P, Rosen M, Han A, Johnson A, Holmes S . DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016; 13(7):581-3. PMC: 4927377. DOI: 10.1038/nmeth.3869. View

20.

Marotz C, Cavagnero K, Song S, McDonald D, Wandro S, Humphrey G . Evaluation of the Effect of Storage Methods on Fecal, Saliva, and Skin Microbiome Composition. mSystems. 2021; 6(2). PMC: 8092129. DOI: 10.1128/mSystems.01329-20. View