Role of Intestinal Microbiota in Ulcerative Colitis

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samples for all primer sets. Analysis of the standard curves allowed verification of PCR efficiency for the chosen PCR conditions. Additionally, all primers were tested to confirm sensitivity and specificity using DNA from pure bacterial species. Statistical analysis Statistical analysis of the qPCR data was performed with SAS JMP software (version 6.0.2; SAS Institute Inc., Cary, North Carolina, USA). Data was analyzed by a mixed model ANOVA, where microbiota source (healthy, UC patients in remission and UC patients in relapse) and community (lumen and mucus) were used as fixed effects, while individual subjects were entered as random effects. The ANOVA was used to determine significant differences among the two communities. Pair‐wise t‐test was used to compare significant difference between the two communities for each microbiota source. The PCR measurements were log transformed before statistical analysis to obtain normal distribution of the data. Normal distribution was assessed using D'Agostino & Pearson omnibus normality test. Univariate ANOVA was used to analyze difference in the age of the three groups using GraphPad Prism software (version 5.03; GraphPad Software Inc., La Jolla, California, USA). Tests were considered statistically significant if P‐values lower than 0.05 were obtained. Principal Component Analysis (PCA) was carried out using SAS JMP software (version 6.0.2) on the qPCR data to investigate the difference between luminal and mucosal community and to address which bacterial taxa may be important for colonization. Results Microbial community analysis using DGGE Comparison of DGGE profiles containing 16S ribosomal genes amplified from luminal and mucosal samples of healthy subjects and UC patients after 42h colonization revealed a distinct difference between the luminal and mucosal environment in terms of bacterial composition (Figure 2). The dendrogram from the Dice cluster analysis showed three clusters with five luminal samples in cluster I (53.89% similarity), all mucosal samples in cluster II (54.61% similarity) and seven luminal samples in cluster III (41.15% similarity). Clustering did not occur according to the health status of the human subjects (healthy, UC in remission and UC in relapse). 8
Microbial community analysis using qPCR After colonization (42 hours) of the luminal or the mucosal environment by fecal microbes from healthy subjects and UC patients (in remission or relapse), qPCR was applied to measure the density of gene targets encoding 16S rRNA gene content of selected bacterial taxonomic units. Principal component analysis (PCA) was performed on the relative quantities of bacterial levels to examine the main differences between all samples. While no obvious difference based on subject health status was observed, luminal and mucosal samples were separated from one another (Figure 3). The separation among all samples was explained by PC1 and PC2 (25.29% and 15.77% of explained variance, respectively). Especially PC2 explained the difference between the luminal and mucosal environment. The relative quantities of following bacterial taxa were higher in the luminal content: Bifidobacterium spp., B. bifidum, B. pseudocatenulatum, B. adolescentis, Lactobacillus spp., C. leptum subgroup, C. coccoides group, F. prausnitzii, Akk. muciniphila and Alistipes spp.. In contrast, Roseburia spp. and E. rectale rather correlated with the mucosal environment (Table 2). The preference of specific bacterial groups to colonize the mucosal and/or luminal compartment was different depending on the three microbiota sources (healthy subjects, UC patients in remission or UC patients in relapse) (Table 2). In the vessels with communities derived from UC patients in relapse, mucus was colonized by significantly lower levels of bifidobacteria, B. adolescentis, lactobacilli, C. coccoides group, C. leptum subgroup, and Alistipes spp.(P
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Role of Intestinal Microbiota in Ul
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Role of Intestinal Microbiota in Ul
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Preface Preface This thesis present
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Summary Summary The microbiota of t
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Dansk sammendrag Dansk sammendrag M
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Introduction and objectives Introdu
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List of Manuscripts Not included in
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List of contents List of Centents P
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List of Centents Methodology append
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1. The intestinal environment Theor
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Theoretical part 5 1. The intestina
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2. The colonic environment Theoreti
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Theoretical part 9 2. The colonic e
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Table 1: The presence of glycoside
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Theoretical part Figure 3: The colo
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3. Inflammatory Bowel disease Theor
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Theoretical part 17 3. Inflammatory
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Theoretical part 19 4. Modulation o
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Table 4: Clinical trials on the pre
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Page 60 and 61: Introduction Methodology part 42 Pa
Page 62 and 63: Abstract Background Detailed knowle
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Page 68 and 69: Statistics PCA were generated by SA
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Page 74 and 75: Acknowledgements The authors thank
Page 76 and 77: Table 2 ‐ 16S rRNA gene and 16S
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Page 82 and 83: Reference List 1. Ahmed S, Macfarla
Page 84 and 85: 32. Matsuki T, Watanabe K, Fujimoto
Page 87 and 88: Methodology part Paper 2 Fecal lact
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Page 91 and 92: Introduction The mucus layer lining
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Page 103 and 104: Table 1 ‐ 16S rRNA gene of phylum
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Appl Microbiol Biotechnol (2011) 90
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Methodology part Paper 6 Tailored e
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Process Biochemistry 46 (2011) 1039
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Table 1 List of enzymes. Enzyme Sou
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J. Holck et al. / Process Biochemis
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Intensity 50 0 C1 175.05 J. Holck e
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J. Holck et al. / Process Biochemis
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[29] Bauer S, Vasu P, Persson S, Mo
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Methodology part 150
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Methodology part 152 Appendix 1 hor
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Appendix 3 Methodology part Appendi
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Methodology part 156 Appendix 3
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7. Discussion and perspectives Disc
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Discussion and conclusion 161 7. Di
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References References Abreu,M.T., V
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References Derrien,M., Vaughan,E.E.
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References Haarman,M. and Knol,J. (
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References Lantz,P.G., Matsson,M.,
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References Matsuki,T., Watanabe,K.,
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References Pullan,R.D., Thomas,G.A.
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References Tannock,G.W. (2010). Ana
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National Food Institute Technical U
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