Novel molecular markers of disease-association among strains of Streptococcus suis: a genomic approach Thomas Mathew Wileman This dissertation is submitted for the degree of Doctor of Philosophy January 2018 University of Cambridge Department of Veterinary Medicine i Novel molecular markers of disease-association among strains of Streptococcus suis: a genomic approach Thomas Mathew Wileman ii Abstract This thesis focuses on the use of a genomic approach to identify novel molecular markers to differentiate Streptococcus suis (S. suis) isolates into two populations, i) disease-associated and ii) non-disease associated. S. suis is a Gram-positive coccus that is considered one of the most important zoonotic bacterial pathogens of swine responsible for significant economic losses to swine production worldwide. Importantly, S. suis is not only an invasive pathogen but also a very successful coloniser of mucosal surfaces; often endemic in swine populations sampled. The widescale use of antibiotics to control and prevent the various clinical manifestations caused by S. suis has become unsustainable, due to increases in antibiotic resistance and government pressures. Other popular control strategies, such as the development of efficacious vaccines, are hindered by differences in virulence not only between but also within S. suis serotypes, as well as, the lack of a detailed understanding of the role in pathogenesis of many proposed virulence-factors. As a result, the detection of S. suis in asymptomatic swine herds is of little practical value in predicting the likelihood of future clinical relevance. This thesis aims to further understanding of the role the S. suis genome has in pathogenesis. The value of future surveillance and preventative health management lies in the detection of strains that genetically have increased potential to cause disease in presently healthy animals. The first results chapter of this thesis (chapter 3) describes the use of genome-wide associations studies, a so-far unexploited method for S. suis, to identify genetic markers associated with the observed clinical phenotype i) invasive disease or ii) asymptomatic carriage on the palatine tonsils of swine. Chapter 4 then describes the analyses used to select three genetic markers to pathotype S. suis - differentiate isolates of the same species based on their ability to cause disease; going on to describe the design and evaluation of a multiplex-PCR tool targeting the three newly defined "pathotyping markers" in comparison to existing methods used to characterise S. suis. iii These findings were taken further by using the pathotyping markers to screen material scrapped from the palatine tonsils of swine with no obvious signs of streptococcal disease. This produced an interesting result - the production of both invasive disease-associated and non- disease associated multiplex-PCR amplicons from the same experimental sample. Unsurprising in itself, what was surprising is the frequency with which this observation was found. Picking single colonies from solid agar plates is a crippling bottleneck of existing S. suis diagnostics, and its removal has the potential to improve the sensitivity of surveillance and preventive health management programs. Chapter 5 describes investigation of this surprising observation and indicates that classic culture-based methods of detection are not sensitive enough to confidently report the presence (or absence) of invasive disease-associated S. suis strains. This thesis concludes with the description of efforts to address the lack of a comprehensive understanding of S. suis virulence/'virulence-associated' factors. Chapter 6 describes the design of an isogenic mutant knocking out the invasive disease-associated pathotyping marker, SSU1589 (also known as virA). That is then evaluated in simple in vitro and in vivo experimental models in order to understand the role Type I restriction modification proteins have in S. suis pathogenesis. In conclusion, this thesis furthers our understanding that differences in the S. suis genome are an important factor in S. suis pathogenesis, and describes the identification and evaluation of novel genetic markers for the detection and control of invasive disease-associated S. suis strains in intensive pig production systems. iv Table of contents Abstract ……………………………………………………………………………………………………………………………….. ii Table of contents …………………………………………………………………………………………………………………. iv List of figures …………………………………………………………………………………………………………………….. xiii List of tables ………………………………………………………………………………………….………………………….. xvii Abbreviations ……………………………………………………………………………………………………………………... xx Declaration …………………………………………………………………………………….………………………………... xxiii Statement of length ………………………………………………………………………………………………………….. xxiv Acknowledgements ………………………………………………………………………………………………………….... xxv Fellowships ………………………………………………………………………………………………………….…………. xxvii Publications ……………………………………………………………………………………………………………………... xxix Conferences attended ……………………………………………………………………………………………………….. xxxi Training courses ……………………………………………………………………………………………………………… xxxii Chapter 1 …………………………………………………………………………………………………………………...……….. 1 Literature review ……………………………………………………………………………………………………………….. 1 1 Introduction ………………………………………………………………………………………………….. 2 1.1 Streptococcus suis ………………………………………………………………………………………….. 4 1.1.1 Current approaches for diagnosis and surveillance ………………………………………… 4 1.1.1.1 Biochemical typing ………………………………………………………………………………………... 6 1.1.1.2 Lancefield grouping ………………………………………………………………………………………. 8 1.1.1.3 Serotyping …………………………………………………………………………………………………..… 9 1.1.1.3.1 Molecular 'serotyping' …………………………………………………………………………………. 11 1.1.1.4 Molecular typing …………………………………………………………………………………………. 13 1.1.1.4.1 Multilocus sequence typing ………………………………………………………………………….. 14 1.1.1.4.2 Minimum core genome sequence typing …………………………………………………….… 17 1.1.2 Epidemiology ………………………………………………………………………………………........... 19 1.1.2.1 Infection in pigs ………………………………………………………………………………………....... 19 1.1.2.1.1 Clinical signs and pathology ………………………………………………………………………… 19 v 1.1.2.1.2 Transmission ………………………………………………………………………………………………. 20 1.1.2.1.3 Survival in the environment ………………………………………………………………………… 21 1.1.2.1.4 Pathogenesis of infection ……………………………………………………………………………... 21 1.1.2.1.5 Immunity ……………………………………………………………………………………………………. 22 1.1.2.1.6 Prevention, treatment and control ……………………………………………………………….. 23 1.1.2.1.6.1 Farm management practices ………………………………………………………………………... 23 1.1.2.1.6.2 Antibiotics ………………………………………………………………………………………………...… 24 1.1.2.1.6.3 Vaccination …………………………………………………………………………………………………. 26 1.1.2.1.6.3.1 Whole cell vaccines …………………………………………………………………………………...… 27 1.1.2.1.6.3.2 Subunit vaccines ……………………………………………………………………………………….… 28 1.1.2.1.6.4 Disinfectants ……………………………………………………………………………………………….. 29 1.1.2.2 Infection in humans …………………………………………………………………………………….. 30 1.1.2.2.1 The 2005 outbreak in China’s central Sichuan province ………………………………… 32 1.1.2.2.2 The 1998 outbreak in China’s eastern Jiangsu province ………………………………… 33 1.1.2.3 Infection in other animal species ………………………………………………………………….. 33 1.1.3 Virulence factors …………………………………………………………………………………………. 34 1.1.3.1 'Classic' virulence factors …………………………………………………………………………….. 39 1.1.3.1.1 Capsule ……………………………………………………………………………………………………….. 39 1.1.3.1.2 Suilysin ……………………………………………………………………………………………………….. 41 1.1.3.1.3 Extracellular factor and muramidase-released proteins …........................................... 43 1.1.3.1.3.1 Extracellular factor protein ………………………………………………………………………….. 43 1.1.3.1.3.2 Muramidase-released protein ……………………………………………………………………… 44 1.1.3.2 Other proposed virulence-associated factors of Streptococcus suis ………………… 45 1.1.4 Experimental infection models …………………………………………………………………….. 46 1.1.4.1 Swine experimental infection models …………………………………………………………... 46 1.1.4.2 Murine experimental infection models …………………………………………………………. 48 1.1.4.3 Zebrafish ……………………………………………………………………………………………..……… 49 1.1.4.4 Greater Wax Moth ……………………………………………………………………………………….. 51 vi 1.1.5 The genome ………………………………………………………………………………………………… 54 1.2 Thesis objectives ……………………………………………………………………………………….… 61 Chapter 2 ………………………………………………………………………………………………………………………….. 64 2 Materials and methods …………………………………………………………………………………………. 64 2.1 Materials ….…………………………………………………………………………………….…………… 65 2.1.1 Apparatus …………………………………………………………………………………………………… 65 2.1.2 Buffers and growth media …………………………………………………………………………… 66 2.1.3 Consumables …………………………………………………………………………………………….… 67 2.1.4 Kits ……………………………………………………………………………………………..……………… 68 2.1.5 Bacterial isolates and strains ……………………………………………………………………..… 69 2.1.6 Whole-genome assemblies from next generation sequencing data ………………… 76 2.1.7 Swabs of oral fluid and material scraped from the palatine tonsils of pigs ……… 78 2.2 Methods ………..…………………………………………………………………………………………….. 79 2.2.1 Statistical methods ……………………………………………………………………………………… 79 2.2.1.1 Chi-squared test for independence ………………………………………………………………. 79 2.2.1.2 Cochran-Mantel-Haenszel Chi-squared test for count data ………………………….… 79 2.2.1.3 Discriminant Analysis of Principal Components ………………………………………….… 80 2.2.1.4 Logistic regression analysis ……………………………………………………………………….… 80 2.2.1.5 Interpretation of the real-valued output of a logistic regression model ………….. 81 2.2.1.5.1 Receiver operating characteristic curves ……………………………………………………… 81 2.2.1.5.2 Selection of a cutoff to convert the real-valued output of a generalised