Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut microbiota

Abstract

Some compounds originating from the human gut microbial metabolism of exogenous and endogenous substrates may have properties that profoundly affect the host's physiological processes. The influence of these metabolites on differences in disease risk among individuals could be mediated by metabolism specific to the gut microbial community composition. In this study, we evaluated the effectiveness of terminal restriction fragment polymorphism (TRFLP) as a biomarker of the fecal microbial community (as a surrogate of gut microbiota) for application in human population-based studies. We tested the effects of experimental conditions on DNA quality, DNA quantity, and TRFLP patterns derived from gut bacterial communities. Genomic DNA was extracted from fecal slurries and the bacterial 16S rDNA genes were amplified and analyzed by TRFLP. We found that the composition of the TRFLP fingerprints varied by different extraction procedure. The best quality and quantity of community DNA extracted from fecal material was obtained by using the QIAamp DNA stool minikit (Qiagen, Valencia, CA) with 95 °C incubation and moderate bead beating treatment during the cell-lysis step. Homogenization of fecal samples reduced variation among replicates. Once the TRFLP procedure was optimized, we assessed the methodological and inter-individual variation in gut microbial community fingerprints. The methodological variation ranged from 4.5–8.1% and inter-individual variation was 50.3% for common peaks. In conclusion, standardized TRFLP is a robust, reproducible, and high-throughput method that will provide a useful biomarker for characterizing gut microbiota in human fecal samples.

Introduction

Many studies have shown that commensal gut microbes play a significant role in human health (Adlercreutz, 1998, Hart et al., 2002, Hooper and Gordon, 2001). The microorganisms in the adult human intestine, which include at least 800 species of bacteria, metabolize compounds that might otherwise be unavailable for human nutrition (Cummings and Macfarlane, 1997, Eckburg et al., 2005). Bacterial consortia, consisting of numerous species, have the potential to produce bioactive agents from the diet. However, certain conversions are only observed in part of the population suggesting that some people lack the necessary microbiota to convert these compounds to chemopreventive molecules (Adlercreutz, 1998, Atkinson et al., 2004, Atkinson et al., 2005). Thus, inter-individual variation in the gut microbial community may be linked to inter-individual variation in the risk of cancer or other diseases (Adlercreutz, 1998, Atkinson et al., 2005, Duncan et al., 2000).

Gut community fingerprinting techniques, such as terminal restriction fragment polymorphism (TRFLP) analysis (Liu et al., 1997), potentially offer a rapid overview of inter-individual differences in gut microbial communities. When comparing the TRFLP data generated from different communities, variation can be found in the number and size of peaks and can be evaluated by adapting community parameters such as richness and evenness (Dunbar et al., 2000, Margalef, 1958). These data provide quantitative information on the compositional differences of gut microbial communities (Osborn et al., 2000) with the potential to serve as a biomarker in high-throughput population-based studies.

To be useful as a biomarker, TRFLP data need to be highly reproducible and reflect gut microbial community composition. Methodological parameters such as sampling technique and DNA extraction, have the potential to influence the TRFLP fingerprint of microbial community (Burgmann et al., 2001). Therefore, obtaining microbial genomic DNA that accurately represents the gut microbial community is important (Osborn et al., 2000). When extracting genomic DNA from a complex matrix such as feces, not only is extraction efficiency of genomic DNA from a wide variety of bacteria a consideration but removal of contaminants that co-elute with the DNA that may interfere with further molecular analyses is important as well. Several studies have explored different DNA extraction and molecular typing methods for application to human fecal microflora characterization (Li et al., 2003, McOrist et al., 2002, Sicinschi et al., 2003, Subrungruang et al., 2004, Yu and Morrison, 2004), however, few studies have evaluated the TRFLP method for large-scale, human population-based study (Sakamoto et al., 2003, Wang et al., 2004).

Here, we report a study designed to optimize tRFLP analysis of the fecal microbial community associated with human population-based studies. We evaluated the efficiency of DNA extraction from human feces using two commercial kits. These kits were chosen because previous studies have shown that they lysed fecal bacterial cells efficiently resulting in a representative genomic community DNA and they both have been shown to remove environmental organic contaminants which otherwise interfere with down-stream molecular analyses (Li et al., 2003, Yu and Morrison, 2004). In addition, we analyzed the effect of homogenization on variation in DNA extraction and tRFLP fingerprints and the effect of temperature and physical disruption during the cell lysis procedure on TRFLP fingerprints. Once the TRFLP fingerprint was optimized, we applied the approach to evaluate methodological and inter-individual variation in fecal microbial community fingerprints that showed the reliability of this biomarker for use in human population-based studies. These data can be used to estimate the sample size needed to characterize the fecal microbiota in human-population based studies.