In vitro alterations of intestinal bacterial microbiota in fecal samples during storage

Abstract

The human gastrointestinal tract harbors an extremely diverse and complex microbial ecosystem. Most of the existent data about the enteric microflora have been generated using stool samples, but the collection and storage of fecal samples are often problematic. The influence of the storage of stool samples on the bacterial diversity and the degradation of bacterial DNA was analysed in this study. Stool samples from 5 healthy volunteers were exposed to different storage temperatures and durations. The bacterial diversity and the amount of intact bacterial DNA were analysed by single-stranded conformation polymorphism analysis (SSCP) and real-time polymerase chain reaction (PCR), both using a 16S rDNA approach. Additionally, biopsy specimens were taken from 3 of the 5 individuals to compare fecal and mucosal flora. The bacterial diversity of the fecal flora and the total number of bacteria were significantly reduced after 8 and 24 hours at both room temperature and 4°C. The mucosa-associated bacterial microflora showed substantial differences compared with the fecal flora. The observed alterations of fecal flora during storage point to the difficulty of the molecular analysis of the bacterial diversity and the enumeration of bacterial cells in fecal samples.

Introduction

The human intestine harbors an extremely complex microbial ecosystem constituting at least 400 to 500 different bacterial species. Bacterial concentrations vary from 10³/g in the stomach and duodenum to up to 10¹²/g in the feces (Simon and Gorbach, 1984). The total number of bacterial cells in the human gastrointestinal tract is estimated at 10¹⁴, equivalent to 10 times the number of cells in the entire body. The role of the gut flora in health and disease has long been underestimated; thus, the complex interactions between host and intestinal bacterial flora, along with its metabolic and protective functions, are just starting to be understood (Hooper et al., 2001, Lu and Walker, 2001).

The development of new molecular techniques in microbiology in recent years has resulted in renewed interest in the intestinal microflora. Molecular microbiology has contributed to a better understanding of the pathogenesis and pathophysiology of many diseases, including environmental diseases such as atopic dermatitis and allergies, and chronic inflammatory bowel disease (Bjorksten et al., 2001, Linskens et al., 2001, Farrell and LaMont, 2002, Huijsdens et al., 2002, Kalliomaki and Isolauri, 2002, Ott et al., 2004a). Positive results of medical trials using probiotics to control clinical symptoms and improve quality of life underline the crucial role of the intestinal flora for the host in maintenance of health and defence of diseases (Isolauri et al., 2001, Rautava and Isolauri, 2002, Saavedra and Tschernia, 2002, Van Niel et al., 2002).

For the most part, our current understanding of the composition of the enteric microflora is based on the selective cultivation and molecular analysis of fecal samples. Generally, stool samples are the preferred material to investigate the intestinal flora because of their overall availability and easy access. Most of the epidemiologic and interventional medical trials, including the therapeutical manipulation of the intestinal flora through antibiotics or probiotics, have been monitored by examination of the fecal flora. Such studies revealed that the predominant bacterial community in mammalian feces is stable in time, host specific, affected by aging, and not altered after consumption of certain probiotic strains (Heilig et al., 2002, Hopkins et al., 2001, Simpson et al., 2000, Tannock et al., 2000, Zoetendal et al., 1998, Zoetendal et al., 1998).

The microbiologic investigation of fecal samples is associated with several technical and methodological problems, however. Based on our own clinical experience, a delay of several hours might elapse between the collection of stool samples by patients and adequate storage by clinical or laboratory personnel. Because of hygienic considerations, patients often fail to cool stool samples during storage. The fecal flora constitutes an extremely dynamic ecologic system that is reliant on complex metabolic and environmental requirements such as anaerobic growth conditions or pH-values, and alteration of the fecal flora because of starvation or bacterial overgrowth is inevitable. To the best of our knowledge, no systematic investigation of the influence of storage conditions on the composition of the fecal flora has been published.

Molecular analysis of the enteric microflora by 16S rDNA-based genetic techniques circumvents the shortcomings of classic microbiologic cultivation of bacteria and is able to characterize 70% to 90% of the dominant fecal flora in healthy subjects (Alm et al., 1996, Dore et al., 1998, Langendijk et al., 1995, Suau et al., 1999, Zheng et al., 1996). Various experimental methods that are based on the sequence-specific separation of 16S rDNA amplicons, such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and single-stranded conformation polymorphism (SSCP) analysis of 16S rDNA, have been developed for the investigation of the intestinal bacterial diversity (Schwieger and Tebbe, 1998, Seksik et al., 2003, Tebbe et al., 2001, Zoetendal et al., 1998). Among them, SSCP analysis is a simple and reproducible method to separate mixtures of polymerase chain reaction (PCR) products amplified from complex ecologic samples without the application of special denaturing conditions or gradients (Schwieger and Tebbe, 1998, Tebbe et al., 2001).

Quantification of bacterial species and populations has long been a particular problem in microbiology because only 10% to 20% of bacteria present in a habitat are cultivable (Hugenholtz et al., 1998, McFarlene and Gibson, 1994, Wilson and Blitchington, 1996). Real-time PCR is a culture-independent tool for accurate and sensitive quantification of individual bacterial species as well as the total number of bacteria (Corless et al., 2000, Guiver et al., 2000, Higgins et al., 2000, Huijsdens et al., 2002, Lyons et al., 2000, Ott et al., 2004b, Sen, 2000). The design of specific probes binding to variable regions of the 16S rDNA allows quantification of both single bacteria and user-defined groups of bacteria. By the use of general primers and universal probes, the quantification of the total number of bacteria in a complex sample is possible (Lyons et al., 2000, Nadkarni et al., 2002, Ott et al., 2004b).

This study assessed the influence of the storage of stool samples on the composition of the fecal flora. Different storage conditions, varying temperature, and duration of storage were investigated. The mucosa-associated flora of biopsy specimens was examined to show differences between fecal samples and biopsies. A 16S rDNA-based approach was used to characterize the composition of the fecal bacterial microflora and to quantify the amount of intact bacterial DNA. Biodiversity and sequence profiles of dominant bacterial groups in stool and biopsy samples were displayed using SSCP analysis with general primers. Selected bands were sequenced to obtain taxonomic information. For quantification of the amount of intact bacterial DNA corresponding to the total number of cells, a 16S rDNA-based real-time PCR with general primers and a universal TaqMan probe was performed.