PlEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Proj(!ct Report Sheet Surname or Family name: Grehan First name: Martin Othe£ name/s: James Abbreviation for degree as given in the University calendar: PhD School: Biote<;hnology & Faculty:· Science Title: Biomolecular Sciences Molecular studies of the human and murine intestinal micro biota. Studies of the diversity of bacterial species in the human colon have recently been based on 16S ribosomal RNA sequences using molecular methods such as PCR and denaturing gradient gel electrophoresis (DGGE). However due to the limited number of published primer sets, an incomplete representation of faecal bacteria is obtained with this approach. This thesis developed molecular methods for detecting Helicobacter species in faecal samples and examined whether Helicobacter spp. were associated with inflammatory bowel disease. A PCR-DGGE assay for the detection and speciation of lower bowel helicobacters was developed and validated in mice. In a human study of 43 controls and 27 inflammatory bowel disease cases with nested PCR, there was no significant difference in the prevalence of Helicobacter spp. DNA found in IBD cases and controls. In all positive subjects the amplified DNA was identical to Helicobacter pylori, suggesting that wash-down of gastric organisms was responsible for the positive results. In order to increase the number of bacterial species that were represented by PCR- DGGE, primer sets were also developed for the three major groups of anaerobes present in human faeces; the Bacteroides-prevotella group, Clostridium coccoides group and Clostridium leptum subgroup. Using these assays it was shown that the bacterial species present in murine faecal and caecal samples were nearly identical. The primers were also applied to show that preparation of mice and humans with antibiotics and cathartics reduced colonisation resistance such that the bacterial populations of the large intestine could be altered. In particular, the administration of a suspension of donated faeces to a human or murine subject that had been prepared with antibiotics and cathartics resulted in a hybrid microbiota in the colon of that individual. In humans, the hybrid microbiota was predominantly derived from the infused faecal suspension (rather than the subjects own baseline microbiota) and was largely stable over the following 24 weeks. The new PCR-DGGE primer sets in combination produced a more complete representation of bacterial species diversity than existing universal primers. Using these assays it was shown for the first time that the composition of the adult colonic microbiota can be altered. Declaration relating to disposition of project report/thesis 1 am. fully aware of the policy of the University relating to the retention and use of higher degree project· reports and thes_e7, namely that th~ UniversitY retains the copies submitted for examination and is free to allow them to be consulted or borrowed. Subject to the prov1s1ons of the Copynght Act 1968, the University may issue a proj.ect report or thesis in whole or in part, in photostat or microfilm or other copying medium. 1 also author-ise the publication by University Microfilms of 1- 350 word abstract in Dissertation Abstracts International (applicable to doctorates only). Signature .... ......... ......... ... ... I U I .. ..........?.12 .. t~ . Y: .. ........ .. U\ Witness Date The University recognises that there may be exceptional circumstances requiring restrictions on_copying o~ c_onditions on use_. Requ.ests for r~striction for a period of up to 2 years must be made in writing to the Registrar. Requests for a longer penod of restnct10n may be c.ons1de~ed m except10n?l circumstances if accompanied by a letter of support from the Supervisor or Head of School. Such requests must be subm1tted wtth the thests/project re art. FOR OFFICE USE ONLY Date of completion of requirements for Award: CJ 4 /I j{f) Re!!:istrar ann DPnutv Princioal THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS N: \ FlORENCE\ABSTRACT Molecular studies of the human and murine intestinal microbiota. Martin James Grehan A thesis submitted for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Sciences The University of New South Wales Sydney, Australia March2004 UNSW 11 FEB 2005 LIBRARY Acknowledgments I would like to sincerely thank each of the following people for their support and encouragement during this project. Most importantly, Associate Professor Hazel Mitchell for her support during the past four years and for being a particularly good listener. Your capacity for hard work and ability to balance this with family life is inspirational. Professor Adrian Lee for his enthusiasm and for generously offering me the opportunity to join his research team. Dr Stephen Danon for his patient teaching, friendship and good humour. Dr Jani O'Rourke for generously sharing her great wealth of experience and common-sense. To all of my co-workers in the lab in particular Andrew Harris, Gauri Tamotia, Martin Wiseman, Daniel Sieveking, Li Zhang, Thosapom Coldham and Mai Dung Ha- thank you for your friendship. John Wilson and Kathleen Kimpton for their friendship and animal handling expertise. And finally my wife Penny for her gentle persuasion and encouragement during these studies, and my father Peter for his anecdotes and perspective. Certificate of Originality I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, nor material which to a substantial extent has been accepted for publication for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged. (Signed) ABSTRACT Studies of the diversity of bacterial species in the human colon have recently been based on 16S ribosomal RNA sequences using molecular methods such as PCR and denaturing gradient gel electrophoresis (DOGE). However due to the limited number of published primer sets, an incomplete representation of faecal bacteria is obtained with this approach. This thesis developed molecular methods for detecting Helicobacter species in faecal samples and examined whether Helicobacter spp. were associated with inflammatory bowel disease. A PCR-DGGE assay for the detection and speciation of lower bowel helicobacters was developed and validated in mice. In a human study of 43 controls and 27 inflammatory bowel disease cases with nested PCR, there was no significant difference in the prevalence of Helicobacter spp. DNA found in IBD cases and controls. In all positive subjects the amplified DNA was identical to Helicobacter pylori, suggesting that wash-down of gastric organisms was responsible for the positive results. In order to increase the number of bacterial species that were represented by PCR­ DGGE, primer sets were also developed for the three major groups of anaerobes present in human faeces; the Bacteroides-prevotella group, Clostridium coccoides group and Clostridium leptum subgroup. Using these assays it was shown that the bacterial species present in murine faecal and caecal samples were nearly identical. The primers were also applied to show that preparation of mice and humans with antibiotics and cathartics reduced colonisation resistance such that the bacterial populations of the large intestine could be altered. In particular, the administration of a suspension of donated faeces to a human or murine subject that had been prepared with antibiotics and cathartics resulted in a hybrid microbiota in the colon of that individual. In humans, the hybrid microbiota was predominantly derived from the infused faecal suspension (rather than the subjects own baseline micro biota) and was largely stable over the following 24 weeks. The new PCR-DGGE primer sets in combination produced a more complete representation of bacterial species diversity than existing universal primers. Using these assays it was shown for the first time that the composition of the adult colonic microbiota can be altered. TABLE OF CONTENTS CHAPTER 1: Introduction 1.1 The anatomy and function of the human colon 1 1.2 Bacterial metabolism in the colon 2 1.3 Culture based assessment of human gut bacterial ecology 6 1.4 Molecular studies of colonic bacterial species diversity 11 1.5 The acquisition of the colonic microbiota 15 1.6 Overview of human colonic bacterial ecology 17 1.7 Colonic bacteria and disease 21 1.8 Molecular methods for assessing the colonic microbiota 29 1.8.1 Qualitative molecular methods 29 1.8.1.1 PCR-denaturing gradient gel electrophoresis 29 1.8.1.2 PCR-cloning 31 1.8.2 Quantitative molecular methods 33 1.9 Helicobacter species and human IBD 35 1.10 Colonisation Resistance 39 1.11 Probiotics and colonic disease 42 1.12 Overview, Hypotheses and Aims 45 CHAPTER 2: Materials and Methods 2.1 Methods 49 2.1.1 Bacterial Culture 49 2.1.1.1 Culture of reference and laboratory strains of Helicobacter and Campylobacter species 49 2.1.1.2 Culture of reference and laboratory strains of other bacterial species 50 2.1.1.3 Culture of Desulfovibrio