Role of Intestinal Microbiota in Ulcerative Colitis

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depending the level of disease activity. Finally, and for the first time, the prevalences of seven selected species of Bifidobacterium in healthy controls and UC patients were under study. Denaturing Gradient Gel Electrophoresis (DGGE) and quantitative PCR (qPCR), both of which are culture‐independent methods, were applied. Although not generating data as extensive as the costly metagenomic‐analysis [41], these methods have recently proven very useful for analyzing the qualitative and quantitative diversity of human fecal microbial communities [8,56]. Additionally, qPCR is a useful tool for quantifying very low concentrations of bacterial targets [56,60]. The primers chosen for qPCR analysis targeted a broad range of selected bacteria taxa, presumed to play a role in the homeostasis of the colonic microbial ecosystem. Our finding should help elucidate compositional differences in the fecal microbiota in patients with ulcerative colitis and in healthy controls. Methods Subjects and fecal sampling Fecal samples were obtained from 12 patients with UC and 6 healthy controls. Within the UC group, 6 patients were in clinical remission and 6 patients had active disease at the time of sampling according to clinical and endoscopical criteria [6]. The patients were previously diagnosed with UC according to standardised diagnostic criteria at the Department of Gastroenterology, Herlev Hospital [26]. The study was performed in accordance with the Second Helsinki Declaration, reported to the Danish Data Protection Agency and approved by the Regional Ethics Committee. Written, informed consent was obtained from each participant under a protocol approved by the Danish National Committee on Biomedical Research Ethics. Four of 6 patients with inactive UC received maintenance treatment with oral mesalazine in a dosage of 1.6‐ 2.4 gram daily and one also azathioprine 100 mg daily. One patient received oral olsalazine (1 gram daily) and one no treatment. All six patients with active UC were treated with oral mesalazine in a dosage of 2.4‐3.2 gram daily as well as topical mesalazine 1 gram daily either as an enema (n = 5) or as a suppository (n = 1). One patient also received azathioprine 100 mg daily. One patient had active extensive UC, one left sided colitis, and the rest either active proctitis or proctosigmoiditis. None of the participants had been treated with antibiotics for at least 2 months 4
efore enrolment and there was no significant difference (P > 0.10) in the mean age of the participants comparing the 3 groups. Sample collection and processing The stool samples were collected at home by the participants in airtight containers and kept at 4 °C (limited storage time was encouraged [35] ) until delivery to the laboratory, where they were processed immediately. 200 mg wet weight feces were collected in triplicates in the middle of each stool sample for DNA extraction. Extraction of bacterial DNA from fecal samples DNA was extracted from the feces samples using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) with an added bead‐beater step as described previously [28]. The purified DNA was stored at – 20 °C until use. PCR amplification for DGGE Aliquots (10 μL) of purified DNA were applied to the following to give a 50 μL PCR reaction mixture: 20 μL of 5 PRIME MasterMix (2.5×) (VWR & Bie & Berntsen) and 10 pmol of each of the primers (Eurofins MWG Synthesis GmbH, Ebersberg, Germany). Universal bacterial primers HDA1‐ GC/HDA2 [55] targeting 16S rRNA genes were used in a touchdown PCR. Initial denaturation was at 96 °C for 5 min, amplification was carried out using 20 cycles including denaturation at 94 °C for 1 min, annealing at 65°C for 1 min decreased by 0.5°C for each cycle, and extension at 72°C for 1 min. This was followed by additional 5 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. Analysis of fecal microbiota by DGGE DGGE was carried out as described previously [5] using a DcodeTM Universal Mutation Detection System instrument and gradient former model 475 according to the manufacturer’s instructions (Bio‐Rad Labs, Hercules, California). The 9% polyamidegels were made with denaturing gradients ranging from 25% to 65%. The 100% denaturant solution contained 40% formamide and 7M urea. Thirteen microlitres PCR products were mixed with 3 µL loading dye before loading. Gels were run 5
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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
Page 14 and 15: List of Manuscripts Not included in
Page 16 and 17: List of contents List of Centents P
Page 18 and 19: List of Centents Methodology append
Page 21 and 22: 1. The intestinal environment Theor
Page 23 and 24: Theoretical part 5 1. The intestina
Page 25 and 26: 2. The colonic environment Theoreti
Page 27 and 28: Theoretical part 9 2. The colonic e
Page 29 and 30: Table 1: The presence of glycoside
Page 31 and 32: Theoretical part Figure 3: The colo
Page 33 and 34: 3. Inflammatory Bowel disease Theor
Page 35 and 36: Theoretical part 17 3. Inflammatory
Page 37 and 38: Theoretical part 19 4. Modulation o
Page 43 and 44: Table 4: Clinical trials on the pre
Page 45 and 46: Theoretical part 5. Production of p
Page 51: Methodology part
Page 54 and 55: Methodology part 6. Methodology, co
Page 60 and 61: Introduction Methodology part 42 Pa
Page 62 and 63: Abstract Background Detailed knowle
Page 66 and 67: in 1 x TAE at 60 °C for 16 h at 36
Page 68 and 69: Statistics PCA were generated by SA
Page 70 and 71: The PCA of the Gram‐positive bact
Page 72 and 73: layer of UC patients and found that
Page 74 and 75: Acknowledgements The authors thank
Page 76 and 77: Table 2 ‐ 16S rRNA gene and 16S
Page 78 and 79: 1. Firmicutes phylum 2. Bacteroidet
Page 80 and 81: Supplementary Figure S1. Dice clust
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
Page 89 and 90: Fecal lactobacilli and bifidobacter
Page 91 and 92: Introduction The mucus layer lining
Page 93 and 94: efore enrolment and there was no si
Page 95 and 96: (Bio‐Rad Labs, Hercules, Californ
Page 97 and 98: Microbial community analysis using
Page 99 and 100: difference from the luminal microbi
Page 101 and 102: that C. coccoides group and C. lept
Page 103 and 104: Table 1 ‐ 16S rRNA gene of phylum
Page 105 and 106: Table 2 ‐ Preference of bacterial
Page 107 and 108: Figure 1. A) Schematic overview of
Page 109 and 110: A. B. Figure 3. Principal component
Page 111 and 112: 15. Fooks LJ, Gibson GR. (2002) In
Page 113 and 114: 47. Ouwehand AC, Suomalainen T, Tol
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Methodology part Paper 3 Paper 3 In
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APPLIED AND ENVIRONMENTAL MICROBIOL
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8338 VIGSNÆS ET AL. APPL. ENVIRON.
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Methodology part Introduction The a
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Journal of Agricultural and Food Ch
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Methodology part Paper 5 Paper 5 Ma
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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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Role of Intestinal Microbiota in Ulcerative Colitis

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