Molecular characterization of bacterial communities in the human gastrointestinal tract

The human gastrointestinal (GI) tract is a complex ecosystem in which host and microbial cells live in close contact with each other. The microbial community in the human GI tract has an important nutritional and protective function and mainly consists of anaerobic bacteria. After birth, the germ-free GI tract is colonized by the first invading microbes and this community is successively changing in time, ultimately resulting in a stable climax community. One of the major problems that hampers studying the bacterial diversity in the GI tract is the inability to obtain the predominant bacteria in culture. Estimates of culturability vary between 10% and 50%. One way to overcome these culturability problems is the use of culture-independent approaches to detect, monitor, and quantify bacteria in the human GI tract. Approaches using the sequence variability of the 16S ribosomal (r)RNA gene have become widely applied, since the 16S rRNA gene is an ideal phylogenetic marker. In the year 2001 there were up to 20,000 nucleotide sequences of different 16S rRNA genes available in several databases, which is far more than for any other gene.In this thesis, the bacterial communities in the human GI tract were investigated using a culture-independent approach based on the sequence variability of bacterial 16S rRNA genes. Our main questions were related to the composition of the bacterial community in the GI tract, its stability in time, and the effect of the host on the GI tract community.Our first approach was based on direct DNA isolation from GI tract samples followed by PCR of 16S rDNA. Temperature and denaturing gradient gel electrophoresis (TGGE and DGGE, respectively) combined with cloning and sequence analysis of amplified 16S rDNA was performed to analyze and monitor the bacterial diversity in human GI tract samples. Before starting this culture-independent analysis of varying GI tract samples from humans, we optimized and validated carefully the nucleic acid isolation from feces and biopsy samples, the subsequent reverse transcriptase (RT) and regular PCR amplification, and the generation of TGGE and DGGE profiles from the bacterial communities. In addition, a specific primer was developed and validated to specifically detect Lactobacillus and related lactic acid bacteria in human GI tract samples.TGGE and DGGE analysis of the V6 to V8 regions of fecal 16S rRNA and rDNA revealed that the predominant bacterial communities were host-specific and stable in time. Only slight differences in band intensities were observed when DNA and rRNA derived profiles were compared, indicating that the predominant bacterial 16S rRNA genes are also predominantly expressed in the colon. Since TGGE profiles revealed the bacterial composition to be host-specific, the bands of a fecal profile from one individual were identified by a cloning and sequencing approach. More than half of the clones corresponded to one of the predominant amplicons in the TGGE profile and most of them appeared to originate from novel Gram-positive species. Bacteria related to Ruminococcus obeum were found frequently in DGGE and TGGE profiles and 16S rDNA clone libraries. A specific 16S rRNA targeted probe was developed to target this group in a PCR-independent approach. FISH analysis using this probe confirmed that these uncultured bacteria are predominant in feces.Since the predominant bacterial community in the human GI tract was found to be stable and host-specific, the impact of the host and of environmental factors on the GI tract community of human adults was studied. A significant positive relationship was found between the genetic relatedness of the hosts compared and the similarity between the bacterial communities in feces, while no significant effect was found for hosts sharing similar environmental conditions. These data suggest that host genotype-related factors have a major impact on the bacterial composition in the GI tract of human adults. In addition, it was observed that the predominant bacterial community attached to the colonic mucosa is equally distributed along the colon, is significantly different from the fecal community, and is also host-specific. These results indicate that host-related factors have a major impact on the bacterial composition in the human GI tract. The Lactobacillus group-specific DGGE profiles supported these results, although it was noteworthy that a Lactobacillus gasseri -like bacterium was observed as the predominant lactic acid bacterium of the mucosa-associated community in most individuals.Most cultivation studies have been performed by plating fecal bacteria on very rich media. A cultivation approach, in which anaerobic basal liquid medium with cellobiose as the main carbon source was used, was employed to isolate strain Cello T, which belongs to an unknown genus. The difficulty for strain Cello Tto grow on solid media and its limited substrate utilization are two explanations why this and related bacteria have never been isolated before. Therefore, it can be concluded that alternative cultivation approaches may result in the isolation of novel bacteria.The results obtained in this study provide valuable knowledge about the bacterial diversity in the human GI tract. The power of applying 16S rRNA-based techniques to study bacterial communities in the human GI tract has been demonstrated and ideas for follow-up research to study the ecology have been proposed.