Abstract

The handling and treatment of biological samples is critical when characterizing the composition of the intestinal microbiota between different ecological niches or diseases. Specifically, exposure of fecal samples to room temperature or long term storage in deep freezing conditions may alter the composition of the microbiota. Thus, we stored fecal samples at room temperature and monitored the stability of the microbiota over twenty four hours. We also investigated the stability of the microbiota in fecal samples during a six month storage period at −80°C. As the stability of the fecal microbiota may be affected by intestinal disease, we analyzed two healthy controls and two patients with irritable bowel syndrome (IBS). We used high-throughput pyrosequencing of the 16S rRNA gene to characterize the microbiota in fecal samples stored at room temperature or −80°C at six and seven time points, respectively. The composition of microbial communities in IBS patients and healthy controls were determined and compared using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline. The composition of the microbiota in fecal samples stored for different lengths of time at room temperature or −80°C clustered strongly based on the host each sample originated from. Our data demonstrates that fecal samples exposed to room or deep freezing temperatures for up to twenty four hours and six months, respectively, exhibit a microbial composition and diversity that shares more identity with its host of origin than any other sample.

Highlights

  • The intestinal microbiota is a complex community of bacteria, archaea, and eukarya

  • This is important in studies investigating the association of the intestinal microbiota with irritable bowel syndrome (IBS), where fecal samples have been left at room temperature for up to six hours before isolation of fecal DNA [8]

  • Until the advent of DNA-based methods to characterize complex bacterial communities in biological samples, it was widely accepted that human fecal samples must be immediately frozen upon collection in order to preserve the composition of the microbiota [26]

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Summary

Introduction

The bacterial fraction is believed to contain more than 500 different species and can reach numbers of 1012– 1014 cells/ml of luminal contents [1,2], with the highest densities residing in the colon Given this complex microbial community’s association with disease [3,4], accurate characterization of the composition and diversity of the intestinal microbiota within biological samples is of the utmost importance. The handling and treatment of biological samples used to characterize the intestinal microbiota is critical when comparing the composition of this complex microbial community between different ecological niches or diseases This is important in studies investigating the association of the intestinal microbiota with irritable bowel syndrome (IBS), where fecal samples have been left at room temperature for up to six hours before isolation of fecal DNA [8]. A recent report highlighted the significant effect of ‘length of time’ at ‘room temperature’ (approximately 25uC)

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