A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/129030 Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications Exploring the molecular mechanisms of antimicrobial resistance in Brachyspira hyodysenteriae using whole genome sequencing Ewart Jonathan Sheldon, BSc, MSc University of Warwick This thesis is submitted for the degree of Doctor of Philosophy May 2018 Declaration This thesis is submitted to the University of Warwick in support of my application for the degree of Doctor of Philosophy. It has been composed by myself and has not been submitted in any previous application for any degree The work presented (including data generated and data analysis) was carried out by the author except in the cases outlined below: 1. In chapter 3 twenty isolates were purified by Jon Rodgers at APHA Bury St Edmunds (js01, js21, js34, js35, js39, js41, js47, js51, js63, js64, js65, js68, js70, js78, js79, js83, js86, js89) The sequencing department at the APHA performed all sequencing conducted at the APHA, this involved one MiSeq metagenomic sequencing run and all NextSeq sequencing A Perl script written by Nicholas Dugget was used to automate the identification of SNPs by Snippy A python script designed by Richard Brown was used in chapter 3, 4 and 5 to automate processes Novel MLST sequence types were assigned by Tom La i Acknowledgements I would like to thank Warwick Medical School and the VMD for funding the work presented in this thesis. I would also like to thank my supervisors Chrystala Constantinidou, Muna Anjum and Mark Pallen for their help and support throughout my PhD. I would also like to thank Emma Stubberfield and Roderick Card for their help and advice which helped me learn the art of growing Brachyspira hyodysenteriae. I would also like to thank Gemma Kay for advice and encouragement with sequencing and Nabil-Fareed Alikhan for advice with bioinformatics. There are many people I would also like to thank in BAC3 at the APHA and MIU for all their advice and support. Unfortunately, there is not space to thank everyone individually, but I really appreciate all your support. My Mum and Dad have my enormous gratitude for reading through multiple copies of my thesis, especially the early drafts. Finally, I would like to thank my girlfriend Claire for her patience and support through the long process of writing the thesis. ii Abstract Brachyspira hyodysenteriae is the causative agent of swine dysentery, a disease characterised by bloody diarrhoea. It is endemic to the UK, and if untreated it can cause severe economic cost to farmers. Currently, it is treated with antibiotics including the pleuromutilin antibiotics tiamulin and valnemulin. B. hyodysenteriae has become more resistant to the antibiotics used to treat infections , and increasing levels of pleuromutilin resistance has been observed in some countries. In this study, 84 clinical isolates, from 2004 to 2015, were sequenced on an Illumina MiSeq. From this, the population structure of B. hyodysenteriae in the UK was constructed. In addition, the phenotypic resistance of 47 sequenced isolates was obtained using a commercial broth dilution assay. The use of sequenced isolates enabled detection of a recently identified pleuromutilin resistance gene and enabled prediction of the resistance phenotype of all sequenced isolates. The use of whole- genome sequencing has increased our knowledge of B. hyodysenteriae in the UK, highlighting potential regional differences and has created a reference database of all B. hyodysenteriae isolates from 2004 to 2015. This will improve surveillance and increase the power of outbreak analysis. Direct sequencing from clinical samples could further strengthen outbreak analysis and surveillance. This would improve the speed of identification and could provide useful information. To explore swine dysentery, positive samples were sequenced on an Illumina MiSeq and B. hyodysenteriae reads were extracted. From this, it was possible to identify the closest sequenced strains. This study highlights the potential uses of whole genome sequencing to analyse B. hyodysenteriae. iii Table of Contents Declaration ........................................................................................................... i Acknowledgements ............................................................................................. ii Abstract.............................................................................................................. iii Table of Contents ................................................................................................ iv List of Tables ..................................................................................................... viii List of Figures ...................................................................................................... x List of Abbreviations .......................................................................................... xii Chapter 1. Introduction ........................................................................................ 1 1.1. The domestic pig and pig farming ............................................................. 1 1.1.1. Historical overview .................................................................................... 1 1.1.2. World production and trade ....................................................................... 1 1.1.3. Role in UK agricultural economy ................................................................. 2 1.1.4. Current challenges in pig farming ............................................................... 3 1.1.5. The pig gut microbiota ............................................................................... 3 1.1.6. Infectious diseases of pigs ................................................................................. 3 1.2. Swine dysentery ............................................................................................ 4 1.2.1. Epidemiology and clinical features .................................................................... 4 1.2.2. Microbiology .................................................................................................... 5 1.2.3. Pathogenesis .................................................................................................... 6 1.2.4. Immune response ............................................................................................. 8 1.2.5. Diagnosis .......................................................................................................... 8 1.2.6. Infection control ............................................................................................. 13 1.2.7. Treatment ...................................................................................................... 14 1.3. The Challenge of antimicrobial resistance .................................................... 14 1.3.1. Antimicrobial use and resistance in human medicine ....................................... 17 1.3.2. Antimicrobial use and resistance in veterinary and agricultural practice ........... 17 1.3.3. Antimicrobial resistance in B. hyodysenteriae .................................................. 18 1.4. New opportunities: whole-genome sequencing ........................................... 21 1.4.1. Sequencing technologies ................................................................................. 21 1.4.2. Bacterial genome biology, evolution and epidemiology ................................... 22 1.4.3. Metagenomics ................................................................................................ 24 1.4.3.1. Applications in microbial ecology .................................................................................. 26 1.4.3.2. Applications in pathogen discovery and detection........................................................ 26 1.5. New opportunities: alternatives to antibiotics ............................................. 27 iv 1.5.1. Diet ................................................................................................................ 27 1.5.2. Bacteriotherapy .............................................................................................. 29 1.5.3. Phage therapy ................................................................................................ 29 1.6. Aims and Objectives of this Study ................................................................ 29 2. Material and Methods ................................................................................... 31 2.1 Samples ....................................................................................................... 31 2.2 Media .......................................................................................................... 31 2.2.1 Bacterial Media ............................................................................................... 32 2.2.2 Bacterial Growth Conditions ............................................................................ 32 2.3 Isolation of DNA ..........................................................................................