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Method Development for the in Situ Screening of Uncultivable Bacteria for Antibiotic Production

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Method Development for the in situ Screening of Uncultivable Bacteria for Antibiotic Production Daniel Blicher Holst Hansen Department of Life Sciences, Imperial College London A thesis submitted for the degree of Doctor of Philosophy December 2018 1 Acknowledgements I would like to thank my two supervisors Harry Low and Martin Buck for their continued guidance and support during the past three years. Your guidance made me a better scientist and gave me a skill set that I hope to use for the rest of my professional career - and for that I am deeply grateful. Also thank you to my two examiners Dr Thomas Bell and Dr Mark Paget. I hope you enjoy reading my thesis. I would like to acknowledge the students I have supervised during my studies. Thank you, Belen Sola Barrado, for your help, especially in developing a M. smegmatis luminescence strain. Thank you to Bernard Cassar Torreggiani and Daniel Corredera Nadal for your help screening for novel antibiotic producers. Finally, a special thank you to Andrew Morrison, who on multiple occasions went above and beyond on a project that needed all the hands it could get. Andrew’s biggest contributions included helping me develop the B. subtilis reporter strains and optimising the microscopy images. The facility managers involved in this project also deserve recognition and gratitude. This includes Jane, Catherine, and Jess in flow cytometry and Paul Brown from physics. Steve Nelson and Stephen Rothery both contributed with unique skill sets that would have been hard to be without. I am as always grateful to my family for their continued support, without which this would not have been possible: my mum, dad, brother and sisters, especially. In that regard, I would like to tell Dagmar Scholes and Sönke Mehrgardt that I truly appreciate their help and the degree to which they made me feel part of the family. I would also like to tell all my colleagues at Imperial College London how much I appreciated their help and support. This is especially true of Eli Cohen, Cian Duggan, Josie Ferreira, and, of course, Olga Bohuszewicz. I am grateful to the anonymous donors that made an applied, yet dreamy, project possible. I hope this thesis shows that significant progress has been made, and that the money given might therefore end up helping a lot of people. Finally, a heartfelt thank you to Natalie Scholes who keeps helping me be better. 2 Abstract Multidrug-resistant bacteria are one of the most critical threats facing human health. Despite an urgent need for novel treatments, antibiotic discovery has stalled. Consequently, no novel antibiotic class has been discovered in the last 40 years. Most existing antibiotics were discovered by screening the 1 % of bacteria that can be cultivated in standard laboratory conditions. The 99 % residual bacteria represent a bioresource that could yield novel antibiotics; but at present this bioresource remains largely unscreened. Here I present the development of a novel high throughput in situ screening array (“ISSA” platform), which screens soil bacteria for antibiotic production in their native environment. The developed platform can isolate a known number of soil strains and screen them against specific clinically relevant pathogens. The platform consists of 4080 discrete compartments, with each compartment containing an average of one soil bacterium and an adjacent luxCDABE bioreporter strain that turns off when antibacterial compounds are secreted. The in situ incubation in the ISSA platform significantly boosts cultivation rates of previously uncultivable bacteria, resulting in the isolation and cultivation of novel bacterial strains. The ISSA platform overall leads to an efficient identification process of soil bacteria that secrete antibacterial compounds. So far, the platform has identified seven antibacterial compound producing strains. Three of these strains are novel antibacterial compound producers, which demonstrates that the developed ISSA platform can be a valuable tool in the race to discover novel antibiotics. 3 Declaration of originality I declare that this thesis titled “Method Development for the in situ Screening of Uncultivable Bacteria for Antibiotic Production” is original work conducted by myself for the degree of Doctor of Philosophy. Work performed by others has been acknowledged and credited. All resources and ideas from the work of others has been appropriately referenced. Copyright declaration The copyright of this thesis rests with the author. Unless otherwise indicated, its contents are licensed under a Creative Commons. Attribution Non Commercial 4.0 International Licence (CC BY- NC). Under this licence, you may copy and redistribute the material in any medium or format. You may also create and distribute modified versions of the work. This is on the condition that: you credit the author and do not use it, or any derivative works, for a commercial purpose. When reusing or sharing this work, ensure you make the licence terms clear to others by naming the licence and linking to the licence text. Where a work has been adapted, you should indicate that the work has been changed and describe those changes. Please seek permission from the copyright holder for uses of this work that are not included in this licence or permitted under UK Copyright Law. 4 Contents Acknowledgements ........................................................................................................................... 2 Abstract ............................................................................................................................................. 3 Declaration of originality................................................................................................................... 4 Copyright declaration ........................................................................................................................ 4 List of figures ..................................................................................................................................... 9 List of tables .................................................................................................................................... 12 Abbreviations .................................................................................................................................. 13 Chapter 1. Introduction .................................................................................................................. 14 1.1 Defining antibiotics ........................................................................................................... 14 1.2 The historic importance of antibiotics .............................................................................. 14 1.3 Increase in bacterial resistance to antibiotics .................................................................. 15 1.4 Most urgent threats posed by bacterial pathogens ......................................................... 16 1.5 The history of antibiotics discovery .................................................................................. 17 1.6 Re-evaluating bacteria as a bioresource for novel antibiotics ......................................... 22 1.7 Uncultivable bacteria - metagenomic analysis of bacterial diversity in soil ..................... 22 1.8 Uncultivable bacteria - metagenomic functional analysis of secondary metabolites in soil ................................................................................................................................................. 25 1.9 Cultivating unculturable bacteria – understanding the required growth conditions ...... 26 1.10 Cultivating unculturable bacteria – existing methods .................................................... 27 1.11 Using uncultivable bacteria as a resource for novel antibiotics ..................................... 31 1.12 Biosensors as in vivo antibiotic secretion detectors ....................................................... 32 Chapter 2. Aims ............................................................................................................................... 33 Chapter 3. Materials and methods ................................................................................................. 36 3.1 Materials ............................................................................................................................... 36 3.1.1 Media ............................................................................................................................. 36 3.1.2 Bacterial strains .............................................................................................................. 37 3.1.3 Vectors ........................................................................................................................... 38 3.1.4 Primers ........................................................................................................................... 38 3.1.5 General lab reagents and kits ........................................................................................ 38 3.1.6 General materials ........................................................................................................... 38 3.1.7 Machines and microscopes ............................................................................................ 39 3.1.8 Software ......................................................................................................................... 39 3.3 Methods ...............................................................................................................................
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