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Stability of the Fecal and Oral Microbiome over 2 Years at -80°C for Multiple Collection Methods.

Author(s): Zouiouich S, Byrd DA, Hua X, Karwa S, Wan Y, Shi J, Humphrey GC, Ackermann GL, Knight R, Abnet CC, Vogtmann E, Sinha R

Publication: Cancer Epidemiol Biomarkers Prev, 2023, Vol. 32, Page 444-451

PubMed ID: 36649143 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the stability of the microbial profile over a 2 year period for saliva stored in OMNIgene ORAL tubes and Scope mouthwash and fecal specimens stored without additive, in 95% ethanol, in RNAlater stabilization solution, on fecal occult blood test (FOBT) cards, or in fecal immunochemical test (FIT) tubes. Fecal and saliva specimens were self-collected at the clinic by 51 healthy volunteers. Fecal specimens were immediately mixed and aliquoted into Sarstedt feces tubes (no additive), tubes containing 2.5 mL of 95% ethanol, tubes containing 2.5 mL of RNAlater Stabilization Solution, FOBT cards, and FIT tubes. Fecal specimens were stored at room temperature until they were placed in frozen storage at -80°C on day 0 or day 4.  Matched saliva specimens were collected using OMNIgene ORAL tubes and frozen immediately or collected with Scope mouthwash and frozen after 0 or 4 days. DNA was isolated using the O-BIO PowerMag Soil DNA Isolation Kit after 0, 1, and 2 years of storage at -80°C. The V4 region of the 16S rRNA genes were amplified before sequencing on a Illumina HiSeq. Bioinformatics analysis was performed in QIIME2 2019.1 and diversity measures were calculated using the R phyloseq, vegan, and GUniFrac packages.

   Summary of Findings:

   In fecal and saliva specimens, most of the variability in beta-diversity metrics (Bray-Curtis and weighted and unweighted UniFrac distances) was attributed to individual differences (55-65% and 51-78%, respectively), with small percentages attributed to preservative (4-13% and 3-8%, respectively) and storage duration (<1-2%, both). When specimens stored without additive were removed from the analysis, the percentage of variability in beta-diversity metrics in fecal specimens attributable to individual differences increased to 65-79%, with only 2-8% attributable to preservation method. The intraclass correlation coefficient (ICC) among the preserved fecal specimens over the course of two years of storage was high (0.73-0.99), but fecal specimens that were frozen without preservation had lower ICCs for Bacteroidetes (0.23), Firmicutes (0.32), Shannon Diversity index (0.42), and the beta-diversity metrics (Bray-Curtis, 0.43; unweighted UniFrac 0.83; and weighted UniFrac, 0.45). Similarly, the ICC for the top 20 genera in fecal specimens were high (0.66-1.0), with the exception of Streptococcus, which depended on the preservation method (0.05-0.93) and whether specimens were frozen in the absence of preservative (0.18-0.78). Overall, the relative abundance of the bacteria at the phyla and genus levels were similar among the preservation methods examined, with the exception of specimens that were frozen without preservation. A total of 27 genera were significantly affected by storage without preservation, resulting in large differences at the phyla and genus level from preserved specimens at those timepoints. For saliva specimens, the ICCs also were generally high (≥0.74), indicating stability over the storage durations examined, but lower ICCs were observed for two of the alpha-diversity metrics (Faith’s phylogenetic diversity and the observed OTUs) in saliva specimens collected using Scope mouthwash (0.23, both). At the genera level, ICCs for Bacteroides, Faecalibacterium, and Roseburia were low in saliva specimens collected in both types of tubes: Scope (0.11, 0.11, and 0.15, respectively) and OMNIgene (0.19, 0.08, and 0.09, respectively). The authors report similar ICC in specimens stored for 4 days at room temperature before freezing than those frozen immediately in the same preservative. Like fecal specimens, the relative abundance of taxa at the genus and phyla level was similar among saliva Scope and OMNIgene specimens, but some changes were noted with storage, particularly in Scope mouthwash specimens. 

   Biospecimens

   • Bodily Fluid - Saliva
   • Bodily Fluid - Feces

   Preservative Types

   • Frozen
   • RNAlater
   • Ethanol
   • Other Preservative

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	0 days 4 days
   Biospecimen Preservation	Type of fixation/preservation	Ethanol Frozen RNAlater FOBT Card Fecal immunochemical test (FIT) OMNIgene oral collection kit Scope mouthwash
   Storage	Storage in ethanol (duration/condition)	0 years 1 year 2 years

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