Characterization of the human predominant fecal microbiota: With special focus on the Clostridial clusters IV and XIVa: Dissertation

The human gut microbiota is considered to be a complex fermentor with a metabolic potential rivaling that of the liver. In addition to its primary function in digestion, the indigenous microbial community has an important influence on host physiological, nutritional and immunological processes. The primary aim of this study was to characterize human predominant fecal microbiota with a special focus on Clostridial clusters XIV (Lachnospiraceae, Eubacterium rectale - Blautia coccoides group) and IV (Ruminococcaceae, Clostridium leptum group). The specific aims were: 1) To develop molecular methods for characterization of the human predominant fecal microbiota; 2) To assess the specificity, practicality, and usability of the developed methods for human fecal samples in healthy adults, elderly people, and people having IBS; 3) To assess possible confounding factors in the analysis of human fecal samples. Molecular tools were developed for sensitive and highly specific characterization of the human predominant fecal and salivary microbiota. DNA- and rRNA-based denaturing gradient gel electrophoresis methods (DGGE) were developed for Eubacterium rectale - Blautia coccoides group (Erec), rRNA-based DGGE method for predominant bacteria, and DNA-based DGGE methods for Clostridium leptum group (Clept) and Bacteroides spp. In addition, quantitative real-time PCR (qPCR) methods targeting predominant bacteria, Erec-group, Clept-group, Bacteroides spp., bifidobacteria, and Atopobium group were developed. Predominant bacterial, Erec-group, Clept-group, and Bacteroides spp. populations of healthy adults were temporally rather stable, showing intra-individual diversity and inter-individual variability. The rRNA-based profiles showed more temporal instability than DNA-based profiles. The enumerated clostridial groups (Erec, Clept, C. lituseburense, and C. histolyticum) represented the dominant fecal microbiota of most of the studied subjects, comprising altogether 29-87% of the total bacteria. Erec-group was the dominant group, accounting on average for 43% of total bacteria in control subjects, and 30% and 50% in irritable bowel syndrome (IBS) subjects affected by constipation and diarrhea, respectively. The observations indicated that in addition to temporal instability of the active predominant fecal bacterial population, clostridial microbiota may be involved in IBS. Differences in the predominant fecal microbiota between elderly people and younger adults were also assessed. Temporal stabilities of the studied bacterial populations (predominant bacteria, Erec-group, bifidobacteria, and lactobacilli) were similar in both age groups. However, the diversity of predominant bacteria and Erec-group bacteria was significantly higher in elderly subjects as compared to younger adults. Consumption of probiotic yoghurt containing galacto-oligosaccharide (GOS) for three weeks did not significantly affect the diversity or temporal stability of the studied bacterial groups. However, the composite data set containing all DGGE analyses of the study showed that the microbial communities from the GOS-supplemented subjects were more similar to each other than those of the control subjects. The similarity of the salivary and fecal microbiota was studied to assess whether the upper gastrointestinal tract microbiota influence the results obtained with DNA-based methods from feces. The predominant bacteria, bifidobacteria, and Erec-group bacteria of the oral cavity and feces were generally stable during probiotic con-sumption, showing more diversity in feces than in saliva and different species composi-tions for the two sampling sites. Lactobacilli, however, showed temporal instability in both feces and saliva. Furthermore, fecal and salivary samples contained identical indigenous Lactobacillus genotypes in most subjects. The effects of storage conditions and DNA-extraction protocols of fecal samples on the results were also evaluated. The DNA-extraction did not affect the diversity, composition, or quantity of Bacteroides spp., whereas after one week's storage at -20oC the numbers of Bacteroides spp. were 1.6-2.5 log-units lower. Furthermore, the numbers of predominant bacteria, Erec-group, Clept-group, bifidobacteria, and Atopobium-group were 0.5-4 log-units higher after mechanical DNA-extraction than after enzymatic DNA-extraction as detected with qPCR, regardless of the storage. The bacterial composition of Erec-group differed significantly depending on the DNA-extraction protocol;. According to the results, rigorous mechanical lysis leads to the detection of higher bacterial numbers and diversity from human fecal samples than enzymatic DNA-extraction. Since it was shown that the results of Clostridial and Actinobacterial populations are highly dependent on the DNA-extraction methods applied, the use of different DNA-extraction protocols may partly explain the contradictory results previously obtained in regard of obesity related and infant microbiota.