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Development of Anaerobic Culturing Methods to Study the Evolution and Transmission of the Human Intestinal Micro- Biota

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Development of Anaerobic Culturing Methods to Study the Evolution and Transmission of the Human Intestinal Micro- Biota Browne, HP (2018) Development of anaerobic culturing methods to study the evolution and transmission of the human intestinal micro- biota. PhD thesis, London School of Hygiene & Tropical Medicine. DOI: https://doi.org/10.17037/PUBS.04647551 Downloaded from: http://researchonline.lshtm.ac.uk/4647551/ DOI: 10.17037/PUBS.04647551 Usage Guidelines Please refer to usage guidelines at http://researchonline.lshtm.ac.uk/policies.html or alterna- tively contact [email protected]. Available under license: http://creativecommons.org/licenses/by-nc-nd/2.5/ Development of anaerobic culturing methods to study the evolution and transmission of the human intestinal microbiota Hilary Patrick Browne Thesis submitted in accordance with the requirements for the degree of Doctor of Philosophy of the University of London March 2018 Department of Pathogen Molecular Biology Faculty of Infectious and Tropical Diseases LONDON SCHOOL OF HYGIENE & TROPICAL MEDICINE Funded by the Medical Research Council. Research group affiliation: Lawley Faculty. Host-Microbiota Interactions Laboratory. Wellcome Trust Sanger Institute 1 Declaration The work presented in this thesis is my own unless otherwise stated. It was carried out under the supervision of Dr. Trevor Lawley of the Wellcome Trust Sanger Institute (WTSI), Hinxton, Cambridgeshire, UK and Prof. Brendan Wren of the London School of Hygiene and Tropical Medicine, London, UK. The work was carried out at the Wellcome Trust Sanger Institute. All experiments and analysis were carried out by me apart from the following: Taxonomic classification of metagenomic sequence reads using Kraken, calculation of relative abundance of metagenomic sequence reads and generation of the sporulation signature gene list was carried out by Dr. Sam Forster (WTSI). David Goulding (WTSI) created the transmission electron microscopy images from the bacterial cultures provided. Library creation and DNA sequencing was performed by the DNA pipelines group (WTSI). Assembly and annotation of whole-genome sequences was performed by in-house pipelines developed by the Pathogen Informatics group (WTSI). Figures taken from the review article ‘Transmission of the gut microbiota: spreading of health’ published in Nature Reviews Microbiology, (Reference [1]), were drawn by a Nature Review Microbiology illustrator but were conceived and created by me in collaboration with Dr. Anne Neville, Dr. Sam Forster and Dr. Trevor Lawley. This thesis does not exceed 100,000 words in length, including tables, figures and footnotes in accordance with the school regulations. 2 Abstract The human gastrointestinal tract is colonised by a diverse range of health-associated bacteria, in addition to other microorganisms, termed the intestinal microbiota. Sequence-based, culture-independent approaches have revolutionised this field of study, however, due to the perception that these bacteria are largely unculturable, in vitro phenotypic analysis has been hindered. In this study, an anaerobic culturing workflow was developed which revealed that the majority of these bacteria can be cultured using one growth medium. In total, 137 characterised and novel bacterial species were isolated and whole-genome sequenced. Inter-host transmission of the intestinal microbiota may represent a means to maintain a diverse assortment of commensal bacteria within individuals, yet it remains a poorly understood process. Some anaerobic pathogens utilise resilient aero-tolerant spores to survive externally and to facilitate transmission to new hosts. To investigate if commensal spore- formers utilise similar mechanisms, a phenotypic screen was incorporated into the culturing workflow to target spore-forming bacteria. This resulted in the isolation of 66 phylogenetically diverse, spore-forming species which, through subsequent phenotypic characterisation are shown to be specialised for host-to-host transmission and intestinal colonisation. Further phylogenetic and evolutionary analysis revealed body-site associated, loss of sporulation has occurred in different taxa. Also, loss of sporulation is associated with features of host-adaptation that are not present in spore-formers such as a smaller genome size and loss of genetic redundancy. This suggests that the human intestinal microbiota is populated by commensal bacteria that have evolved to engage in opposing lifestyles, either orientated towards inter- host dispersal or within-host adaptation. This study demonstrates the intestinal microbiota is not unculturable. In addition, commensal microbial transmission may be more prevalent than once thought as a significant proportion of these bacteria can survive outside of a host through the use of spores that are intrinsically resistant to environmental stresses. 3 Acknowledgements I would like to thank my supervisors Trevor Lawley and Brendan Wren for guidance, encouragement and advice and for providing me with the opportunity to undertake this study. Thanks also to those at the Wellcome Trust Sanger Institute who I have been fortunate to work with and who have contributed through discussion, advice and training- members of the Host-Microbiota Interactions Laboratory, in particular Anne Neville, Sam Forster, Nitin Kumar, Mark Stares and Blessing Anonye and also, Sally Kay and Dave Goulding. Finally, thanks to Ann and Cillian for their support and motivation. 4 Contents Declaration ................................................................................................................... 2 Abstract ........................................................................................................................ 3 Acknowledgements ...................................................................................................... 4 Contents ....................................................................................................................... 5 List of Figures ............................................................................................................ 10 List of Tables.............................................................................................................. 13 Glossary ..................................................................................................................... 14 Chapter 1 Introduction ............................................................................................... 17 1.1 The human intestinal microbiota ...................................................................... 17 1.1.1 The microbiota of humans ......................................................................... 17 1.1.2 The human intestinal microbiota ............................................................... 20 1.1.3 Defining a healthy intestinal microbiota .................................................... 20 1.1.4 Host selection of commensal intestinal bacteria ........................................ 21 1.2 The host-microbiota interactions of other animals ........................................... 23 1.2.1 The rumen microbiota ................................................................................ 24 1.2.2 Extreme host adaptation- the insect microbiota ......................................... 25 1.2.3 Genome reduction of extremely host-adapted bacteria ............................. 28 1.3 Studying the intestinal microbiota .................................................................... 30 1.3.1 Culturing and sequence-based approaches ................................................ 30 1.3.2 The benefits of bacterial culturing ............................................................. 32 5 1.4 Spore-forming bacteria within the intestinal microbiota .................................. 34 1.4.1 Sporulation and germination cycle and genetics ....................................... 34 1.4.2 Clostridium difficile- the model enteric spore-former ............................... 39 1.5 Transmission of commensal bacteria ............................................................... 42 1.5.1 Shared transmission routes of commensal and pathogenic intestinal bacteria ................................................................................................................ 43 1.5.2 Distinguishing the routes of transmission of commensal and pathogenic intestinal bacteria ................................................................................................ 46 1.5.3 Survival in the environment ....................................................................... 47 1.5.4 Reservoirs of commensal bacteria ............................................................. 52 1.6 Microbiota perturbation and restoration ........................................................... 57 1.6.1 Microbiota perturbation ............................................................................. 57 1.6.2 Microbiota restoration................................................................................ 59 1.6.3 Understanding microbiota perturbation through ecological theory ........... 60 1.7 Therapeutics based on the intestinal microbiota .............................................. 61 1.8 Thesis aims ....................................................................................................... 63 Chapter 2 Materials and Methods .............................................................................. 65 2.1 Culturing of bacteria from the human intestinal microbiota ............................ 65 2.1.1 Acquisition of donor faecal samples .........................................................
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