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Effects of Stool Sample Preservation Methods on Gut Microbiota Biodiversity: New Original Data and Systematic Review with Meta-Analysis.

Author(s): Li XM, Shi X, Yao Y, Shen YC, Wu XL, Cai T, Liang LX, Wang F

Publication: Microbiol Spectr, 2023, Vol. 11, Page e0429722

PubMed ID: 37093040 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the microbial profile of fecal specimens from healthy individuals that were analyzed fresh with those that were stored at-80°C or in liquid nitrogen or preserved with absolute ethanol and stored at room temperature, -80°C, or in liquid nitrogen for up to 12 months. The authors also conducted a meta-analysis of studies investigating the effects of preservatives and storage on the stability of the microbial profile. Feces were collected by three healthy volunteers and immediately aliquoted.  Aliquots (200 mg) were: (i) frozen at -80°C, (ii) snap-frozen in a liquid nitrogen, (iii) preserved with absolute ethanol stored at -80°C, (iv) preserved with absolute ethanol and stored in liquid nitrogen, (v) preserved with absolute ethanol and stored at room temperature or (vi) analyzed immediately. The final concentration of ethanol was not specified. DNA was extracted using a kit from the Beijing Kangwei Century Biotechnology Co and bacterial 16S rRNA sequencing was performed using the Illumina MiSeq kit and instrument. Sequences were trimmed and quality filtered with USEARCH. Operational taxonomic unit (OTU) clustering was performed with UPARSE and taxonomy of the OTUs was assigned with the Ribosomal Database Project (RDP) classifiers. Alpha diversity (observed OTU number, Shannon index, and Simpson index) and beta diversity (unweighted UniFrac distances and weighted UniFrac distances) were calculated based on rarefied OTU counts and used for principal-coordinate analysis (PCoA). A meta-analysis of 30 papers investigating storage durations and conditions of fecal specimens was also conducted.

   Summary of Findings:

   The most abundant phyla were Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, with some changes in relative abundance observed among the storage timepoints for each storage condition. A significant increase in levels of Proteobacteria was reported in specimens preserved with ethanol after 12 months of room temperature relative to unpreserved specimens that were immediately analyzed (P<0.5). The authors report that the relative phyla abundance was most stable in specimens preserved with ethanol and stored in liquid nitrogen. Bacteroidaceae, Lachnospiraceae, Ruminococcaceae, Veillonellaceae, and Clostridiaceae were the most represented families that were detected in all specimens.  The relative abundance of Lachnospiraceae was unaffected by storage under all conditions tested, and the relative abundance of Clostridiaceae was unaffected by storage at -80°C with or without ethanol preservation. However, the abundance of Veillonellaceae was reduced in ethanol-preserved specimens after 12 months of room temperature storage relative to immediately analyzed unpreserved specimens (P<0.5). Alpha diversity (Species richness, Shannon index and Simpson index) decreased over time in ethanol-preserved specimens stored at room temperature. Similarly, in PcoA of Beta diversity measures (unweighted and weighted UniFrac distances), specimens preserved with ethanol and stored at room temperature showed the largest spread among storage timepoints and those that were frozen in liquid nitrogen were the most tightly clustered. Ethanol-preserved specimens that were stored at room temperature had a significant increase in Eubacterium after 3 months (P<0.01); decreased counts of Eubacterium (P<0.05), Desulfovibrio (P<0.01), Prevotella (P<0.01), and Megamonas (P<0.05) after 6 months; decreased Eubacterium (P<0.05), Megamonas (P<0.01), Clostridium (P<0.05), and Adlercreutzia (P<0.05) after  9 months; and a decrease of Eubacterium (P<0.05), Desulfovibrio (P<0.01), Clostridium (P<0.05), and Adlercreutzia (P<0.05) after 12 months compared to fresh specimens. In contrast, frozen specimens stored in liquid nitrogen or at -80°C only displayed non-significant differences in bacterial counts relative to fresh specimens. Based on literature meta-analysis, the authors conclude that for most preservatives the magnitude of differences in the microbial profile following storage for ≤5 days were comparably small relative to the variability observed between donors. Nevertheless, effects of storage were observed in many of the reviewed studies and depended on preservation method and storage duration. 

   Biospecimens

   • Bodily Fluid - Feces

   Preservative Types

   • Frozen
   • None (Fresh)
   • Ethanol

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	None (fresh) Frozen Ethanol
   Storage	Storage duration	0 days 3 months 6 months 9 months 12 months
   Storage	Storage temperature	Room temperature -80°C Liquid nitrogen

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