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Pseudomonas Aeruginosa Pathogenesis in Urinary Tract Infections

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Pseudomonas Aeruginosa Pathogenesis in Urinary Tract Infections The University of Liverpool Pseudomonas aeruginosa Pathogenesis in Urinary Tract Infections Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by: Hussain Ebrahim, B.Sc. (Hons), M.Sc. November 2019 Abstract Urinary tract infections (UTIs) cause high morbidity, mortality and economic burden. Pseudomonas aeruginosa can cause persistent UTIs and it has many virulence factors including quorum sensing systems and iron acquisition mechanisms. Furthermore, this pathogen is highly resistant to antibiotics and has been highlighted by the World Health Organisation as a pathogen for which there is critical need for new antimicrobials. I have characterised P. aeruginosa UTI isolates from both the UK and Kuwait. The UTI isolates were variable in terms of their phenotypic characteristics and genomic composition. Multiple resistance genes linked to resistance to different classes of antibiotics including aminoglycosides, fluoroquinolones and β-lactams were identified from the sequence data using the CARD database, particularly in the isolates from Kuwait. These isolates were pan-resistance and multidrug resistance, with the ability to spread resistance via horizontal gene transfer. Laboratory media are commonly used to study bacteria in vitro. Nutrient rich media does not accurately reflect the physiological environment, prompting the development of multiple media mimicking human body fluids such as Artificial Urine Medium (AUM). My results show that bacterial responses in LB and AUM were highly different. AUM and urine showed similar responses with regards to the increase in proteins associated with iron acquisition mechanisms and of the decrease in proteins associated with the Type III secretion system. However, differences were observed in other important pathways such as those involved in phenazines production. This suggests that AUM is a more reflective media than LB to study P. aeruginosa UTI isolates, particularly when the environment needs to be highly controlled/consistent, such conditions cannot be controlled in urine. The difference in the protein abundance profile in AUM suggest that further optimisation of the medium could be performed for better resemblance of urine. Hormones are regulatory substances that are produced by the host and transported around the body. Sex-related hormones, oestrogen (oestradiol), progesterone and testosterone are examples of these. Previous studies have indicated that sex ii hormones such as oestradiol may modulate P. aeruginosa mucoidy and alter innate immune responses in females. In this study, treatment with all three hormones decreased the abundance of P. aeruginosa proteins associated with iron acquisition mechanisms. Furthermore, individual hormones also showed specific bacterial changes such as increased protein abundance in pqs quorum sensing mechanism by oestradiol compared to testosterone and progesterone. This study suggests that P. aeruginosa pathogenesis may be variable in UTI infections based on the host hormonal profile. Understanding the individual role of host factors could allow us to fully understand the contribution of host components and the impact these may have on infection susceptibility and outcome. Therefore, further research is required into the role of host components, in particular sex hormones, in host-pathogen interactions. iii Declaration This thesis is the result of work performed mainly within two institutions: The Institute of Infection & Global Health under the supervision of Dr Jo Fothergill and the Institute of Translational Medicine under the supervision of Dr Rachel Floyd, who now resides at the Institute of Integrative Biology. All of these institutions are part of the University of Liverpool. Some of the work presented in this dissertation was carried out in collaboration with others. Dr Christina Bronowski and John Newman assisted with the biofilm data in chapter 4. Sequencing data in chapter 3 was conducted by Dr Sam Haldenby in the Centre of Genomic Research at the University of Liverpool. Microscopy experiments was setup by the assistance of Dr Marco Marcello. Dr Dave Mason provided assistance with microscopy analysis and Jennifer Adcott with technical aspects of imaging at The Liverpool Centre for Cell Imaging. Dr Stuart Armstrong provided initial analysis of the proteomic data in chapter 4 and 5. iv Acknowledgments Undertaking this PhD has been a life-changing, thrilling and great experience. Without the constant support of many people whom I offer my deepest gratitude, studying for this degree would not have been possible. I would like to thank my supervisor Dr Joanne Fothergill who offered me this opportunity. Your kindness, patience and guidance in less than ideal circumstances have been one of the main driving forces behind achieving this PhD. I cannot think of any words that will do you any justice for the development of so many skills gained throughout my laboratory work and the write-up of this dissertation. Many thanks also to my supervisor Dr Rachel Floyd, whose active support, knowledge and positive demeanour ensured my progression throughout the ups and downs over the PhD years. I will be always grateful for the time spent out of your busy schedule to enhance my academic knowledge and reach the aims of this PhD. I would also like to offer much love and gratitude for my parents, who have been patient all over those years with me away from home, they never stopped supporting me throughout thick and thin in my academic journey in Scotland and England. Your love and encouragement are extremely difficult to pay back, I hope I am making you proud. I would also like to thank my wife and best friend Dr Zahra Amir, your unconditional support through the darkest of times allowed me to pursue my dreams and made my targets achievable, your success and encouragement are the ultimate sources of inspiration for which I will forever be grateful for. Many thanks also go out for pride and joy, my children Tasneem, Yaseen and Ali. Your presence in my life provided me with much needed purpose and desire to succeed. I am also grateful to so many friends and colleagues at the Institute of Infection & Global Health, particularly to the head of our research group Professor Craig Winstanley whose valuable insight during laboratory meetings helped shape my understating of all things Pseudomonas. Many thanks also go to the group members, namely, Dr Laura Wright for the constant help and console in the laboratory and Dr Hassan Chowdhury for his assistance. I would also like to thank John Newman and v Yasmin Hilliam for the all PhD chatter over those cold mornings over Coffee on Merseyside. My deepest thanks also go to the technical team including Wesley Reid and Nahida Mia for always answering my queries and providing advice to matters regarding living in Liverpool. vi Abstract ____________________________________________________________ ii Declaration ________________________________________________________ iv Acknowledgments ___________________________________________________ v List of tables ______________________________________________________ xiv List of figures______________________________________________________ xvi List of abbreviations ________________________________________________ xix 1 General introduction ________________________________________________ 1 1.1 Urinary tract: Anatomy and Function _____________________________________ 1 1.2 Urinary tract infections _______________________________________________ 1 1.2.1 Prevalence, classification and diagnosis _______________________________________ 1 1.2.2 Studying uropathogenic bacteria ____________________________________________ 3 1.2.3 UTI causing bacteria ______________________________________________________ 5 1.2.3.1 Escherichia coli ______________________________________________________ 5 1.2.3.2 Klebsiella pneumoniae ________________________________________________ 6 1.2.3.3 Proteus mirabilis _____________________________________________________ 6 1.2.3.4 Enterococci Spp ______________________________________________________ 7 1.2.3.5 Pseudomonas aeruginosa in UTIs ________________________________________ 7 1.3 Initiation of P. aeruginosa UTI __________________________________________ 8 1.3.1 Adhesion _______________________________________________________________ 8 1.3.2 Motility ________________________________________________________________ 9 1.3.3 Flagella ________________________________________________________________ 9 1.3.4 Pili ___________________________________________________________________ 10 1.3.5 Outer membrane proteins ________________________________________________ 11 1.3.6 Lipopolysaccharides _____________________________________________________ 12 1.4 Virulence Factors ___________________________________________________ 13 1.4.1 Quorum sensing associated virulence factors _________________________________ 13 1.4.1.1 Quorum sensing ____________________________________________________ 13 1.4.1.2 Biofilm formation ___________________________________________________ 15 1.4.1.3 Pyocyanin _________________________________________________________ 16 1.4.1.3 Elastase and LasA ___________________________________________________ 18 1.4.1.4 Alkaline protease____________________________________________________ 19 1.4.1.5 Phospholipase C ____________________________________________________ 19 1.4.1.6 Rhamnolipids_______________________________________________________ 20 vii 1.4.1.7 Hydrogen Cyanide ___________________________________________________
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