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This Electronic Thesis Or Dissertation Has Been Downloaded from the King’S Research Portal At This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ The Microbial Community Ecology of the Cystic Fibrosis Lung Cuthbertson, Leah Forbes Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 06. Oct. 2021 The Microbial Community Ecology of the Cystic Fibrosis Lung A thesis submitted for the degree of Doctor of Philosophy in the Institute of Pharmaceutical Science, Molecular Microbiology Research Laboratory, King's College London By Leah Cuthbertson Pharmaceutical Sciences Research Division Kings College London October 2014 PhD – Kings College London – 2014 Declaration “I declare that I have personally prepared this report and that it has not in whole or in part been submitted for any degree or qualification. The work described here is my own, carried out personally unless otherwise stated and written in my own words. All sources of information, including quotations, are acknowledged by means of reference.” Signature: Date: 2 Abstract Respiratory failure, due to infection and concomitant inflammation is the major cause of morbidity and mortality in people suffering from the genetic disorder, cystic fibrosis (CF). Consequently, the CF Foundation currently estimates that patients with CF have a median predicted life expectancy of only 41.1 years. Understanding the relationship between the complex and diverse bacterial community present within the lower respiratory tract and patient outcomes has therefore become a top priority. Through the use of next generation sequencing technologies (Roche 454 and Illumina MiSeq) and ecological statistics and modelling, the complex relationships between the bacterial community within the CF lung and host related clinical factors were investigated. By first establishing guideline methodologies for the reduction of bias in the collection, storage and treatment of respiratory samples, this thesis aimed to use large scale spatial and longitudinal studies to investigate key relationships between the bacterial community and clinical factors. It has been well established that a complex and diverse bacterial community exists within the CF lung. Spatial sampling revealed key relationships between the bacterial community and other diagnostic parameters including, FEV1, gender, and clinic location. Longitudinal sampling aimed principally to investigate CF pulmonary exacerbations (CFPE), implicated in the progressive loss of lung function associated with CF lung disease. Over the course of a CFPE the common bacterial taxa show resistance to perturbations while the rare taxa show resilience. Through this investigation, Veillonella parvula was identified as a potential bioindicator of CFPE, introducing the potential for a rapidly testable parameter for clinicians to identify a CFPE. This finding could provide one of the most important recent developments in CF therapy. 3 Acknowledgements Firstly I would like to express may sincerest thanks to all my supervisors; Dr Christopher van der Gast, Dr Anna Oliver, Dr Alan Walker and Dr Kenneth Bruce. Without the help and support you have provided over the last four years I could not have hoped to achieve so much. I would also like to make a special thanks to Dr Geraint Rogers, your help and advice throughout this project has been invaluable. I would like to acknowledge the contribution of the clinicians and medical staff, around the world, who have worked tirelessly to provide me with samples and metadata required to produce this work. In particular I would like to thank Dr Mary Carroll and Professor Lucas Hoffman for their contributions to this work. Thanks to all the past and present, staff and students at CEH Wallingford, in particular, Tom August, Rory O’Connor, Phil Martin, Will Hentley, Scott McKenzie, Carolin Schultz, Louise Barwell, Phill James, Lindsey Newbold, Dan Reid, and Bruce Thompson. It’s been quite an adventure guys, thanks for all your help, support and friendship. There are so many people out there who deserve thanks for their friendship and encouragement over the last few years. I wish I could thank you all, but in the interest of not writing another book I want to mention some key people. Please know that I am grateful to every single person who has been part of my life during this time. A massive thank you goes to the amazing Helen Waters, you’re a true friend and your dedication to everything you do is an inspiration. Tom Simpson and Sam Jones, you guys have been amazing, thank you for keeping life in perspective. Laura “weasel” Balfour, you always know when to show up when you are truly needed. Frankie Alcock, you are a star, couldn’t have done it without you dude. I am so grateful to have you all as friends. Finally, endless thanks go out to my wonderful family who have supported me tirelessly through everything I have set out to achieve. I would not be where I am today without you. 4 Contents Declaration ............................................................................................................................. 2 Acknowledgements ............................................................................................................... 4 Chapter 1: General Introduction ......................................................................................... 12 1.1 Introduction .................................................................................................................. 13 1.2 The microbial world ...................................................................................................... 14 1.3 Microbial ecology ......................................................................................................... 17 1.4 Identifying bacteria ................................................................................................... 18 1.4.1 Human microbiota ................................................................................................ 21 1.5 Challenges of the respiratory tract ............................................................................... 22 1.6 Lower respiratory tract infections ................................................................................. 26 1.7 Cystic fibrosis ............................................................................................................... 26 1.8 Cystic fibrosis history ................................................................................................... 27 1.9 The CF mutation .......................................................................................................... 28 1.10 The CF lung ............................................................................................................... 32 1.10.1 Mucus ................................................................................................................. 32 1.10.2 Inflammation ....................................................................................................... 33 1.11 CF lung infections ...................................................................................................... 33 1.11.1 Sampling ............................................................................................................ 34 1.11.2 Traditional microbiology ..................................................................................... 36 1.11.3 Culture independent technologies ...................................................................... 39 1.11.4 Propidium monoazide ......................................................................................... 41 1.12 Microbial ecology and CF lung infections .................................................................. 44 1.12 Aims ........................................................................................................................... 46 1.12 References ............................................................................................................. 48 Chapter 2: Materials and methods ..................................................................................... 59 2.1 Bacterial strains ........................................................................................................ 60 2.2 Clinical samples ....................................................................................................... 62 2.3 Sputum sample processing .....................................................................................
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