THE GENETICS AND EPIDEMIOLOGY OF SHIGELLA SONNEI AND SHIGELLA FLEXNERI IN VIETNAM Benjamin Robert Sobkowiak A thesis submitted for the degree of Doctorate of Philosophy Department of Genetics, Evolution and Environment University College London 1 I, Benjamin Robert Sobkowiak, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. ………………………………………………………………………………… 2 Abstract Shigella sonnei is rapidly emerging as the primary agent of bacillary dysentery, or shigellosis, in many developing countries, replacing the historically more prevalent species, S. flexneri, in these regions. There have been various theories proposed to explain this phenomenon, including environmental changes and increased antimicrobial use, though the precise reasons for this shift are still uncertain. Here I present four studies investigating key ecological and genetic differences between S. sonnei and S. flexneri from a region that has undergone this pattern of species replacement, Vietnam. This work combines experimental and bioinformatics techniques with the aim of identifying the extent that differences in disinfectant sensitivity, chromosomal antimicrobial resistance profiles and gene content will contribute to the successful spread of S. sonnei over S. flexneri. Firstly, I conducted in vitro experimental work to characterise differences between species with respect to resistance to chlorine disinfection and tolerance to the detergent SDS. The mechanisms by which the bacteria respond to this treatment, in particular the role of efflux pumps, were then explored to determine whether any informative variation in these systems will explain any differences in disinfectant sensitivity. The availability of high quality whole genome sequences for ~150 of each Shigella species allowed for robust bioinformatics work to describe genomic variation between species. These sequences are used to detect key resistance mutations in each species and look for associated fitness compensating mutations. Finally, the complete genome sequence of all coding regions in each strain was de novo assembled to look for species-level gene content variation that might contribute to functional differences between S. sonnei and S. flexneri. The results of these studies show that there are clear biological differences between S. sonnei and S. flexneri, though more work is necessary to fully elucidate the reasons for the species replacement in developing countries. 3 Acknowledgments I am very thankful to everyone that has contributed to the completion of this thesis, both in the production of its content and the emotional support afforded to me throughout, by friends, family and colleagues. Firstly, I would like to thank my Ph.D. supervisor Francois Balloux for giving me the opportunity to undertake this research and guiding me throughout the process by constantly challenging me to develop my ideas and to never lose sight of the biological significance of my work. The lessons I have learnt under his supervision have already enabled me to advance my scientific endeavours and no doubt will continue to do so throughout my career. All members of the Balloux group, past and present, have helped in many ways to develop this work. In particular, I would like to thank Adrien Rieux, Matteo Fumagalli, Matteo Spagnoletti, Stephen Price, Liam Shaw and Florent Lassalle for their academic and personal support throughout this project. I would also like to thank Vegard Eldholm for teaching me so much in a short time about microbial pathogens and microbiology. In addition, I am grateful to Stephen Baker for imparting his knowledge of bacterial pathogens and always being quick to share data and ideas. I also thank my current supervisor Taane Clark for providing me the time to finish this work. I thank the Natural Environmental Research Council - NERC and the Department of Genetics, Evolution and Environment at UCL for funding this work. On a personal level, all my friends and family that have provided support and distraction since I began this degree have been extremely important in allowing me to complete this research. Notably, my close family, friends and girlfriend that have all helped to inspire and keep me going through the hardest times. Finally, I would like to dedicate this thesis to my mum, Deborah Fielder, who has always done everything to enable me accomplish my goals. I appreciate everything that she has done and this would not have been possible without her. Thank you all. Ben 4 Table of contents 1. Introduction………………………………………………………………………... p.7-35 1.1. Bacterial pathogens 1.2. Antibiotic resistance and compensatory mutations 1.3. The genus Shigella 1.4. Shigella in Vietnam 1.5. The shifting dominance of Shigella species in developing countries 1.6. Thesis outline References 2. Chlorine and detergent tolerance of Shigella sonnei and Shigella flexneri………………………………………………………………………………..p.36-69 2.1. Abstract 2.2. Introduction 2.3. Materials and methods 2.4. Inactivation assays 2.4.1. Results 2.5. Competition assays 2.6. Discussion References 3. The role of efflux pumps and antibiotic resistance on chlorine and detergent tolerance in Shigella spp………………………………………………………. p. 70-80 3.1. Abstract 3.2. Introduction 3.3. Materials and methods 3.4. Results 3.5. Discussion References 5 4. The characterisation of antibiotic resistance genes and compensatory mutations in Shigella sonnei and Shigella flexneri in Vietnam………….p. 81-101 4.1. Abstract 4.2. Introduction 4.3. Methods 4.4. Results & Discussion 4.5. Conclusions References 5. Environmental persistence and niche adaptation in Vietnamese Shigella sonnei and Shigella flexneri – insights from the pan genome………………..…p. 102-143 5.1. Abstract 5.2. Introduction 5.3. Methods 5.4. Results 5.5. Discussion References 6. General discussion……………………………………………….…………… p. 144-150 Conclusions References Supplementary materials…………………………………………….………. p. 151-200 6 Chapter 1. General introduction Chapter 1 General Introduction 1.1 Bacterial Pathogens Infectious disease epidemiology Bacteria are among the most diverse and abundant organisms on Earth, with the sum of bacteria living on a single human individual outnumbering human cells 10 to 11. Many bacterial species are human pathogens, with a number of important diseases caused by microbial agents, including tuberculosis, meningitis and pneumonia. As a consequence, bacterial infections continue to cause a great number of deaths every year in humans. Discovering treatments for these diseases and preventing their transmission is of the upmost importance for global health. At the beginning of the 20th century many bacterial diseases were almost untreatable, contributing to low life expectancies and high infant mortality rates2. With the success of antibiotics around the mid-century, as well as a general improvement in human health and the scientific understanding of the biology of these organisms, the threat from such infections was declining in the developed world, though they remained a cause for concern in developing countries, notably with diarrheal diseases in children2. The end of the 20th century brought global cases of newly emerging microbial infections and the re-emergence of many diseases that were thought to have been eradicated in the developed world, such as tuberculosis, cholera and infections from Streptococcus aureus3,4. The awareness that infectious diseases have been increasing globally and understanding the reasons for the rise in the number of cases has led to an emphasis on research to address this growing concern. Major threats to human populations also include viral and fungal diseases such as human Immunodeficiency virus (HIV) and Aspergillosis, though for the purposes of this report I will be concentrating only on the transmission of bacterial pathogens. The main factors that influence the present-day transmission of bacterial pathogens are shown in Figure 1. A rise in human migration and recreational travel has changed the accessibility of susceptible individuals to certain diseases, as the chance increases of transmission of novel bacterial infections that would not have been able to pass between previously disparate groups. There can also be different genetic and social risk factors associated with disease transmission that may be influencing the prevalence of particular infections in a more connected and diverse populace5. 7 Chapter 1. General introduction Figure 1. Factors influencing the emergence and spread of human infectious bacterial diseases. Illustration taken from Cohen2. Human population demographics, notably in developed nations, are transforming due to societal and behavioural changes. Many of these countries have an aging population as people wait longer to have children and as advances in healthcare leads to higher average life expectancies. This can increase the number of individuals who are susceptible to particular illnesses through weakening immune systems with age or can increase the transmission and reactivation of chronic or latent infections as individuals have a greater chance of harbouring a disease for a longer time2,6. The risk of bacterial disease transmission has also increased through exposure to particular infections with changes in human behaviour, such as the increase in people living in close proximity in densely populated cities, dietary changes and changes in sexual behaviours. The increases in HIV both in men who have sex with men (MSM) and heterosexual