Fecal sample collection methods and time of day impact microbiome composition and short chain fatty acid concentrations

Jacquelyn Jones,Stacey N Reinke,Claus T Christophersen,Alishum Ali,Debra J Palmer

doi:10.1038/s41598-021-93031-z

Abstract

Associations between the human gut microbiome and health outcomes continues to be of great interest, although fecal sample collection methods which impact microbiome studies are sometimes neglected. Here, we expand on previous work in sample optimization, to promote high quality microbiome data. To compare fecal sample collection methods, amplicons from the bacterial 16S rRNA gene (V4) and fungal (ITS2) region, as well as short chain fatty acid (SCFA) concentrations were determined in fecal material over three timepoints. We demonstrated that spot sampling of stool results in variable detection of some microbial members, and inconsistent levels of SCFA; therefore, sample homogenization prior to subsequent analysis or subsampling is recommended. We also identify a trend in microbial and metabolite composition that shifts over two consecutive stool collections less than 25 h apart. Lastly, we show significant differences in bacterial composition that result from collecting stool samples in OMNIgene·Gut tube (DNA Genotec) or Stool Nucleic Acid Collection and Preservation Tube (NORGEN) compared to immediate freezing. To assist with planning fecal sample collection and storage procedures for microbiome investigations with multiple analyses, we recommend participants to collect the first full bowel movement of the day and freeze the sample immediately after collection.

Highlights

Associations between the human gut microbiome and health outcomes continues to be of great interest, fecal sample collection methods which impact microbiome studies are sometimes neglected
short chain fatty acid (SCFA) are associated with dysbiosis, and other inflammatory d isorders[5]; and investigating the gut microbiome by combining microbial sequencing data and metabolomic approaches has been an important step in unraveling links between resident bacteria, SCFA, and health outcomes[9,10,11]
Analysis of the fecal microbiome is commonly complemented by an additional analysis of microbial metabolites such as SCFA

Summary

Introduction

Associations between the human gut microbiome and health outcomes continues to be of great interest, fecal sample collection methods which impact microbiome studies are sometimes neglected. To compare fecal sample collection methods, amplicons from the bacterial 16S rRNA gene (V4) and fungal (ITS2) region, as well as short chain fatty acid (SCFA) concentrations were determined in fecal material over three timepoints. As far as the authors are aware spatial and short-term temporal variability of bacterial and fungal communities has never been evaluated together with SCFA composition. To address this gap, this study will assess the effects of five fecal sample collection methods, as well as consecutively collected whole stool samples (less than 25 h apart), on the variability of the fecal microbiome. The comparisons will be drawn from bacterial and fungal community composition as well as SCFA profiling

Methods

Results

Conclusion