Long-term stability of microbiome diversity and composition in fecal samples stored in eNAT medium

Abstract

Fecal samples collected for microbiome analyses are typically frozen to avoid postcollection changes in microbial composition. eNAT is a guanidine thiocyanate-based medium that stabilizes microbial DNA and allows safe specimen handling and shipping by inactivating microorganisms. We collected fecal samples (n = 50) from children undergoing hematopoietic stem cell transplantation. We divided samples into three aliquots: (a) stored in RNAlater and immediately transferred to -80°C; (b) stored in eNAT medium and immediately transferred to -80°C; and (c) stored in eNAT medium at ambient temperature (~20°C) for 30 days prior to transfer to -80°C. Mean (standard deviation) Shannon diversity and Chao1 indices in sample aliquots were 2.05 (0.62) and 23.8 (16.6), respectively. Comparing samples frozen immediately in RNAlater to samples frozen immediately in eNAT, there were no differences in Shannon diversity (p = .51), Chao1 richness (p = .66), and overall microbiome composition (p = .99). Comparing eNAT samples frozen immediately to samples stored at ambient temperature, we identified no differences in Shannon diversity (p = .65), Chao1 richness (p = .87), and overall microbiome composition (p = .99). Storage of fecal samples in eNAT at ambient temperature for 30 days did not alter microbiome richness, diversity, or composition. eNAT may be a useful medium for fecal microbiome studies, particularly when cold chain storage is unavailable.

Keywords: 16S rRNA sequencing; bacterial inactivation; biological sample shipping; stabilization media.

Figure 1

Similar microbiome composition of matched fecal sample aliquots prepared in eNAT (−80°C), RNAlater (−80°C), and eNAT (20°C). The nonmetric multidimensional scaling distances of the Bray–Curtis matrix show no difference in global composition by sample preparation method (PERMANOVA; p = .99)

Figure 2

Matched fecal sample aliquots stored in eNAT and RNAlater and frozen immediately to −80°C do not have significantly different SDI, Chao1, global microbiome composition, and relative abundances of common bacterial genera. (a) A Bland–Altman plot of the Shannon diversity shows no proportional bias between sample preparation methods. Differences in the Shannon diversity index of matched sample aliquots are plotted on the y‐axis, and the mean of the Shannon diversity index of matched sample aliquots is plotted on the x‐axis; (b) A Bland–Altman plot of the natural log of the Chao1 index shows no proportional bias between sample preparation methods; (c) The nonmetric multidimensional scaling distances of the Bray–Curtis matrix show no difference in global microbiome composition by sample preparation method (PERMANOVA; p = .99); and (d) The median differences of relative abundances of the 20 most abundant bacterial genera are between 1.5% higher in RNAlater and 1.6% higher in eNAT

Figure 3

Matched fecal samples aliquots stored in eNAT and frozen immediately to −80°C and stored in eNAT at ambient temperature do not have significantly different SDI, Chao1, global composition, and genus relative abundance. (a) A Bland–Altman plot of the Shannon diversity shows no proportional bias between sample preparation methods; (b) A Bland–Altman plot of the natural log‐transformed Chao1 index shows no proportional bias by sample preparation method; (c) The nonmetric multidimensional scaling distances of the Bray–Curtis Matrix show no difference in global microbiome composition by sample preparation method (PERMANOVA; p = .99); and (d) The median differences of relative abundances of the 20 most abundant bacterial genera are between 1.6% higher in eNAT frozen immediately to −80°C and 2.1% higher in eNAT stored at ambient temperature

Figure A1