Abstract
BackgroundMultiple factors can influence stool sample integrity upon sample collection. Preservation of faecal samples for microbiome studies is therefore an important step, particularly in tropical regions where resources are limited and high temperatures may significantly influence microbiota profiles. Freezing is the accepted standard to preserve faecal samples however, cold chain methods are often unfeasible in fieldwork scenarios particularly in low and middle-income countries and alternatives are required. This study therefore aimed to address the impact of different preservative methods, time-to-freezing at ambient tropical temperatures, and stool heterogeneity on stool microbiome diversity and composition under real-life physical environments found in resource-limited fieldwork conditions.MethodsInner and outer stool samples collected from one specimen obtained from three children were stored using different storage preservation methods (raw, ethanol and RNAlater) in a Ugandan field setting. Mixed stool was also stored using these techniques and frozen at different time-to-freezing intervals post-collection from 0–32 h. Metataxonomic profiling was used to profile samples, targeting the V1–V2 regions of 16S rRNA with samples run on a MiSeq platform. Reads were trimmed, combined and aligned to the Greengenes database. Microbial diversity and composition data were generated and analysed using Quantitative Insights Into Microbial Ecology and R software.ResultsChild donor was the greatest predictor of microbiome variation between the stool samples, with all samples remaining identifiable to their child of origin despite the stool being stored under a variety of conditions. However, significant differences were observed in composition and diversity between preservation techniques, but intra-preservation technique variation was minimal for all preservation methods, and across the time-to-freezing range (0–32 h) used. Stool heterogeneity yielded no apparent microbiome differences.ConclusionsStool collected in a fieldwork setting for comparative microbiome analyses should ideally be stored as consistently as possible using the same preservation method throughout.
Highlights
Profiling faecal microbiota is routinely applied to explore relationships between microbiota and host health status (Young, 2017)
Ensuring samples collected are representative and consistent, in fieldwork situations where homogenisation of the stool sample may be difficult due to limited resources, is another sampling consideration. To address these crucial issues, we explored the influence of time-to-freezing, storage preservation methodology, and stool heterogeneity on microbiome profiles for stool specimens collected from three children within a Ugandan community representative of an low and middle-income countries (LMIC) fieldwork setting
Microbiome profiles vary between individual children Each child had a distinct microbiome signature (Figs. 1A and 1B) that was apparent at all taxonomic levels, from phylum (Fig. 2A; Table S2) to genus (Fig. 2B; Data S1) level regardless of the preservation method used and the time-to-freezing duration
Summary
Profiling faecal microbiota is routinely applied to explore relationships between microbiota and host health status (Young, 2017). Focusing on conditions more likely to be accessible in these settings, several studies have assessed the impact of storage under standard cold chain, that is, +4 C (Choo, Leong & Rogers, 2015; Lauber et al, 2010; Penington et al, 2018; Tedjo et al, 2015), and ‘room’ (i.e. 25 C) temperatures (Cardona et al, 2012; Guo et al, 2016; Lauber et al, 2010; Tal et al, 2017; Tedjo et al, 2015) prior to freezing These approaches appear to be sufficient to maintain a representative metataxonomic 16S rRNA microbiota community profile in the short-term (up to 14 days post collection). Conclusions: Stool collected in a fieldwork setting for comparative microbiome analyses should ideally be stored as consistently as possible using the same preservation method throughout
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