VTT SCIENCE26 .Withspecial... fecalmicrobiota Characterization ofthehumanpredominant Characterization of the human predominant fecal IENCE C • •S T microbiota S E N C With special focus on the Clostridial clusters IV and XIVa O H I N S O I V Dissertation L • O S The human gut microbiota is considered to be a complex fermentor with a metabolic G T 26 Y H potential rivaling that of the liver. In addition to its primary function in digestion, the • R G I E indigenous microbial community has an important infl uence on host physiological, L S H E G A I R H C nutritional and immunological processes. H Molecular tools were developed for rapid, sensitive, and highly specifi c characterization of the human predominant fecal and salivary microbiota. Predominant bacterial, Eubacterium rectale – Blautia coccoides group (Erec), Clostridium leptum group (Clept), and Bacteroides spp. populations of healthy adults were temporally rather stable, showing intra-individual diversity and inter-individual variability. However, rRNA-based denaturing gradient gel electrophoresis (DGGE) profi les showed more temporal instability than DNA-based profi les. Moreover, the diversity of predominant bacteria and Erec-group bacteria was signifi cantly higher in elderly subjects as compared to younger adults. Clostridial populations represented the dominant fecal microbiota of most of the studied subjects. However, the proportion of Erec-group was signifi cantly lower in the constipation type IBS subjects than in the healthy adults. Altogether, the observations indicated that in addition to temporal instability of the active predominant fecal bacterial population, clostridial microbiota may be involved in IBS. Thereafter, the similarity of the salivary and fecal microbiota was studied to assess whether the upper gastrointestinal tract microbiota infl uence the results obtained with DNA-based methods from feces. The predominant bacteria, bifi dobacteria, and Erec-group bacteria of the oral cavity and feces showed more diversity in feces than in saliva and different species compositions for the two sampling sites. However, 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 -20°C the numbers of Bacteroides spp. were signifi cantly lower. Furthermore, the numbers of predominant bacteria, Erec-group, Clept-group, bifi dobacteria, and Atopobium-group were 0.5–4 log-units higher after mechanical Characterization of the DNA-extraction than after enzymatic DNA-extraction, regardless of the storage. Moreover, the bacterial composition of Erec-group differed signifi cantly depending human predominant on the DNA-extraction protocol applied. 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. fecal microbiota With special focus on the Clostridial clusters ISBN 978-951-38-7954-9 (soft back ed.) IV and XIVa ISBN 978-951-38-7955-6 (URL: http://www.vtt.fi /publications/index.jsp) ISSN 2242-119X (soft back ed.) ISSN 2242-1203 (URL: http://www.vtt.fi /publications/index.jsp) Johanna Maukonen VTT SCIENCE 26 Characterization of the human predominant fecal microbiota With special focus on the Clostridial clusters IV and XIVa Johanna Maukonen Doctoral dissertation for the degree of Doctor of Science in Technology to be presented with due permission of the School of Chemical Technology for public examination and debate in Auditorium KE2 (Komppa Auditorium) at the Aalto University School of Chemical Technology (Espoo, Finland) on the 7th of December, 2012, at 12 noon. ISBN 978-951-38-7954-9 (soft back ed.) ISSN 2242-119X (soft back ed.) ISBN 978-951-38-7955-6 (URL: http://www.vtt.fi/publications/index.jsp) ISSN 2242-1203 (URL: http://www.vtt.fi/publications/index.jsp) Copyright © VTT 2012 JULKAISIJA – UTGIVARE – PUBLISHER VTT PL 1000 (Tekniikantie 4 A, Espoo) 02044 VTT Puh. 020 722 111, faksi 020 722 7001 VTT PB 1000 (Teknikvägen 4 A, Esbo) FI-02044 VTT Tfn +358 20 722 111, telefax +358 20 722 7001 VTT Technical Research Centre of Finland P.O. Box 1000 (Tekniikantie 4 A, Espoo) FI-02044 VTT, Finland Tel. +358 20 722 111, fax + 358 20 722 7001 Kopijyvä Oy, Kuopio 2012 Characterization of the human predominant fecal microbiota With special focus on the Clostridial clusters IV and XIVa Ihmisen vallitsevan ulostemikrobiston karakterisointi. Painopiste erityisesti klostridien fylogeneettisissä ryhmissä IV ja XIVa. Johanna Maukonen. Espoo 2012. VTT Science 26. 161 p. + app. 62 p. Abstract The human gut microbiota is considered to be a complex fermentor with a meta- bolic potential rivaling that of the liver. In addition to its primary function in diges- tion, it affects the human host in numerous ways: maturation and modulation of the immune system, production of short-chain fatty acids and gases, transfor- mation of bile acids, formation of vitamins, and also potential formation of muta- genic, toxic, and carcinogenic substances. Commensal bacteria are able to modu- late the expression of host genes that regulate diverse and fundamental physio- logical functions. Thus the indigenous microbial community has an important influ- ence 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 rapid, sensitive, and highly specific charac- terization 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. popula- tions of healthy adults were temporally rather stable, showing intra-individual di- versity and inter-individual variability. The rRNA-based profiles showed more tem- poral instability than DNA-based profiles. The enumerated clostridial groups (Erec, Clept, C. lituseburense, and C. histolyticum) represented the dominant fecal mi- crobiota 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 proportion of Erec-group was significantly lower in the constipation type IBS subjects than in 3 the healthy controls. The rRNA-based DGGE-profiles of the fecal microbiota of the IBS subjects further indicated higher temporal instability of their microbiota com- position as compared to the control subjects. Our 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. Therefore, the GOS-yoghurt may have a stabilizing effect on the predominant fecal microbiota of elderly people. Thereafter, the similarity of the salivary and fecal microbiota was studied to as- sess whether the upper gastrointestinal tract microbiota influence the results ob- tained with DNA-based methods from feces. The predominant bacteria, bifidobac- teria, and Erec-group bacteria of the oral cavity and feces were generally stable during probiotic consumption, showing more diversity in feces than in saliva and different species compositions for the two sampling sites. Lactobacilli, however, showed temporal instability in both feces and saliva. Furthermore, fecal and salivary samples contained identical indigenous Lctobacillus genotypes (L. amnosus, L. i, L. racasei, L. antar group, and a Lobacillus sp.)