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. 2023 Oct 3:14:1253570.
doi: 10.3389/fmicb.2023.1253570. eCollection 2023.

Processing and storage methods affect oral and gut microbiome composition

Dorothy K Superdock  1 , Wei Zhang  2 , Angela C Poole  1
Affiliations

Processing and storage methods affect oral and gut microbiome composition

Dorothy K Superdock et al. Front Microbiol. .

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.

Keywords: dental; freeze dried; frozen; gut; liquid nitrogen; lyophilized; microbiome; saliva.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Study design. From 10 donors, we collected three different sample types—dental swabs, saliva samples, and fecal samples—which were then subjected to several storage and processing methods. For each sample, each condition was tested in triplicate.
Figure 2
Figure 2
Microbial taxa differ at the phylum level for different fecal and oral sample storage and processing methods. (A) Relative abundances of the most prevalent microbial taxa in fecal samples categorized by condition (lyophilized, fresh, LN2, and LN2post72hr). (B) Relative abundances of the most prevalent microbial taxa in dental samples categorized by thawed fraction (composite, pellet, and supernatant). (C) Relative abundances of the most prevalent microbial taxa in saliva samples categorized by condition (fresh, composite, pellet, supernatant, and HMP). (A–C) All legends list taxa in order from most to least abundant. (A–C) Taxa that were not visible due to low relative abundances are not listed in the figure key. Microbial taxa grouped by donor can be found in Supplementary Figure 1.
Figure 3
Figure 3
Beta diversity of fecal samples. Principal coordinate analysis plot of (A) unweighted and (B) weighted UniFrac distances between fecal samples, colored by condition. Shapes represent different subjects. (C) Box plot representing pairwise weighted UniFrac distances between each condition and lyophilized fecal samples. Red asterisks (*) indicate significantly different distances from lyophilized samples as determined by PERMANOVA without stratification by donor.
Figure 4
Figure 4
Beta diversity of dental samples. Principal coordinate analysis plot of (A) unweighted and (B) weighted UniFrac distances between dental samples, colored by fraction. Shapes represent different subjects. (C) Box plots represent pairwise weighted UniFrac distances between each dental fraction and dental supernatant. Red asterisks (*) indicate significantly different distances from dental supernatant samples as determined by PERMANOVA without stratification by donor.
Figure 5
Figure 5
Beta diversity of saliva samples. Principal coordinate analysis plot of (A) unweighted and (B) weighted UniFrac distances between saliva samples, colored by condition. Shapes represent different subjects. (C) Box plots represent pairwise weighted UniFrac distances between samples in each condition and fresh saliva samples. Red asterisks (*) indicate significantly different distances from fresh saliva samples as determined by PERMANOVA without stratification by donor.
Figure 6
Figure 6
Alpha diversity of fecal samples. (A) Observed ASVs in fecal samples by condition. (B) Evenness of fecal samples by condition. (C) Phylogenetic diversity as determined by Faith's PD of fecal samples by condition. Statistically significant (p < 0.05) differences between groups as determined by pairwise comparisons are denoted by an asterisk (*) in (A–C). (D) Phylogenetic diversity in individual subjects, colored by condition.
Figure 7
Figure 7
Alpha diversity of dental samples. (A) Observed ASVs in dental samples by fraction. (B) Evenness of dental samples by fraction. (C) Phylogenetic diversity as determined by Faith's PD of dental samples by fraction. Statistically significant (p < 0.05) differences between fractions are denoted by an asterisk (*) in (A–C). (D) Phylogenetic diversity by individual subject, colored by dental fraction.
Figure 8
Figure 8
Alpha diversity of saliva samples. (A) Observed ASVs in saliva samples by condition. (B) Evenness in saliva samples by condition. (C) Phylogenetic diversity as determined by Faith's PD in saliva samples by condition. Statistically significant (p < 0.05) differences are denoted by an asterisk (*) in (A–C). (D) Phylogenetic diversity by individual subject for each condition.
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