Collection media and delayed freezing effects on microbial composition of human stool.

Roberto Flores,Bing Ma,Rashmi Sinha,Jacques Ravel,James J Goedert,Guoqin Yu,Jianxin Shi

doi:10.1186/s40168-015-0092-7

Abstract

BackgroundDifferent bacteria in stool have markedly varied growth and survival when stored at ambient temperature. It is paramount to develop optimal biostabilization of stool samples during collection and assess long-term storage for clinical specimens and epidemiological microbiome studies. We evaluated the effect of collection media and delayed freezing up to 7 days on microbial composition. Ten participants collected triplicate stool samples each into no media as well as RNAlater® with and without kanamycin or ciprofloxacin. For each set of conditions, triplicate samples were frozen on dry ice immediately (time = 0) or frozen at −80 °C after 3-days and 7-days incubation at 25 °C. Microbiota metrics were estimated from Illumina MiSeq sequences of 16S rRNA gene fragments (V3–V4 region). Intraclass correlation coefficients (ICC) across triplicates, collection media, and incubation time were estimated for taxonomy and alpha and beta diversity metrics.ResultsRNAlater® alone yielded the highest ICCs for diversity metrics at time = 0 [ICC median 0.935 (range 0.89–0.97)], but ICCs varied greatly (range 0.44–1.0) for taxa with relative abundances <1 %. The 3- and 7-day freezing delays were generally associated with stable beta diversity for all three media conditions. Freezing delay caused increased variance for Shannon index (median ICC 0.77) and especially for observed species abundance (median ICC 0.47). Variance in observed species abundance and in phylogenetic distance whole tree was similarly increased with a 7-day delay. Antibiotics did not mitigate variance. No media had inferior ICCs at time 0 and differed markedly from any media in microbiome composition (e.g., P = 0.01 for relative abundance of Bacteroidetes).ConclusionBacterial community composition was stable for 7 days at room temperature in RNAlater® alone. RNAlater® provides some stability for beta diversity analyses, but analyses of rare taxa will be inaccurate if specimens are not frozen immediately. RNAlater® could be used as collection media with minimal change in the microbiota composition.

Highlights

Different bacteria in stool have markedly varied growth and survival when stored at ambient temperature
Reproducibility of microbiota metrics with no collection media and RNAlater-based collection media We first compared the microbial composition of stool replicates collected without collection media to those collected with different media at time zero
RNAlater® alone, the no media condition had lower Intraclass correlation coefficients (ICC) for reproducibility of relative abundance for Bacteroidetes (ICC 0.71), alpha diversity estimates, and Reproducibility of microbiota metrics with different collection media at time zero We first sought to identify differences at time zero among the three media (RNAlater® alone, RNAlater® with kanamycin, or with ciprofloxacin) by calculating ICCs for 14 microbiota metrics across triplicate samples from the 10 subjects. These metrics included relative abundances of the major phyla (Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria), four alpha-diversity metrics (Shannon, Chao-1, PD_WT, richness), and beta diversity represented by principal coordinate analysis (PCoA) scores (Table 1)

Summary

Introduction

Different bacteria in stool have markedly varied growth and survival when stored at ambient temperature. It is paramount to develop optimal biostabilization of stool samples during collection and assess long-term storage for clinical specimens and epidemiological microbiome studies. Ten participants collected triplicate stool samples each into no media as well as RNAlater® with and without kanamycin or ciprofloxacin. Intraclass correlation coefficients (ICC) across triplicates, collection media, and incubation time were estimated for taxonomy and alpha and beta diversity metrics. To move the field of human microbiome research forward both for clinical purposes and in epidemiological research, validated methods are needed for collecting specimens that represent, as closely as possible, the true in vivo parameters and to understand any technical variation that can be introduced. For large population-based microbiome studies, specimen collection methods must be acceptable to participants and, most importantly, tolerant of suboptimal field conditions.

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