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Contribution of Enterococcus Faecalis to Urinary Tract Infection

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Contribution of Enterococcus Faecalis to Urinary Tract Infection Western University Scholarship@Western Electronic Thesis and Dissertation Repository 3-29-2018 3:00 PM Contribution of Enterococcus faecalis to urinary tract infection Samantha Ann Whiteside The University of Western Ontario Supervisor Reid, Gregor The University of Western Ontario Co-Supervisor Burton, Jeremy P. The University of Western Ontario Graduate Program in Microbiology and Immunology A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Samantha Ann Whiteside 2018 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Bacterial Infections and Mycoses Commons Recommended Citation Whiteside, Samantha Ann, "Contribution of Enterococcus faecalis to urinary tract infection" (2018). Electronic Thesis and Dissertation Repository. 5270. https://ir.lib.uwo.ca/etd/5270 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract The purpose of this thesis was to increase understanding of enterococcal urinary tract infection (UTI), in particular, the response of Enterococcus to antibiotic prophylaxis in vitro and in vivo and enterococcal communication with the bladder. We studied the in vitro effects of trimethoprim-sulfamethoxazole (TMP/SMX) and nitrofurantoin, two of the most commonly used antibiotic treatments for the management of both UTI and recurrent UTI (RUTI), on Enterococcus faecalis attachment to urothelial cells. In doing so, we documented increases in bacterial attachment at growth inhibitory concentrations of nitrofurantoin, but not TMP/SMX. This increased virulence did not correlate with increased expression of virulence factors but was correlated with increased expression of three putative genes. We then explored whether this corresponded to alterations in bacterial communities throughout antibiotic prophylaxis for paediatric patients with RUTI. Bacterial culture results indicated uropathogens were present in the urine of children with and without a history of RUTI and that antibiotic prophylaxis induced a transient decrease in uropathogen load. Interestingly, none of our patients were experiencing symptomatic UTI at the time of urine sample collection, yet a significant proportion of midstream urine samples met the clinical threshold for UTI, indicating these patients had asymptomatic bacteriuria (ABU). Further, E. faecalis bacterial load was positively correlated with non-E. coli uropathogens, suggesting some patients may be pre-disposed to polymicrobial UTI. To elucidate mechanisms by which enterococci can mask the host’s perception of UTI, which may also contribute to the polymicrobial nature of E. faecalis UTI, we completed targeted metabolomics of neuroactive molecules in vitro under conditions mimicking the bladder environment. Our results suggest Enterococcus may produce tyramine in the bladder at concentrations that are likely to have a physiological effect on both urothelial cells and cohabiting bacteria. Our data raises questions about the application of nitrofurantoin to enterococcal UTI and the efficacy of antibiotic prophylaxis for RUTI. Further, our clinical and in vitro data suggest E. faecalis may contribute more to polymicrobial UTI than previously thought. Indeed, ii enterococcal production of tyramine may explain the high incidence of enterococci in not only polymicrobial UTI but also other infections. Keywords Enterococcus faecalis, urinary tract infection, recurrent urinary tract infection, antibiotic prophylaxis, paediatric urology. iii Co-Authorship Statement Samantha Whiteside predominantly carried out the experiments and data analysis within this thesis with the supervision of Jeremy Burton and Gregor Reid. Samantha Whiteside primarily wrote the chapters presented herein; exceptions are listed below: Chapter 1: General introduction The section on the microbiome of the urinary tract (Section 1.2) was conceived by Samantha Whiteside, Jeremy Burton, and Gregor Reid. Hassan Razvi and Sumit Dave provided clinical input. Chapter 2: Nitrofurantoin increases bacterial attachment to urothelial cells Samantha Whiteside, Jeremy Burton, and Gregor Reid conceived the experiments. Aidan Shair completed the PCR screen and ibuprofen growth curves; Samantha Whiteside carried out the remainder of the assays and performed data anaylsis. Chapter 3: Longitudinal study of the bacterial communities in urine from children with RUTI Samantha Whiteside, Jeremy Burton, Gregor Reid, and Sumit Dave conceived and designed the study. Sample collection, urinalysis, and patient follow-up was completed by the paediatric urology clinic staff. Patient clinical files were reviewed by both Samantha Whiteside and Peter Wang. Urine sample processing, including bacteriological culture and DNA extraction were performed by Samantha Whiteside. Kait Al completed the DNA amplification and submitted samples for sequencing. Microbiota analysis was performed by Samantha Whiteside, using the pipeline designed by Gregory Gloor and Jean Macklaim, whom also provided input. Shannon Seney performed cytokine multiplex assays, which were subsequently analysed by Samantha Whiteside. Chapter 4: Enterococcal production of neuroactive substances Samantha Whiteside, Jeremy Burton, and Gregor Reid conceived the experiments. Fatemah Mousavi and Janusz Pawliszyn optimised the SPME-LC-MS/MS methodology for microbial iv metabolomics. Samantha Whiteside performed the GABase experiments, all sample preparation, and metabolite extraction for exploratory metabolomics. Fatemeh Mousavi completed the exploratory LC-MS/MS and Samantha Whiteside analysed the data. Extraction and identification of metabolites for quantitative metabolomics was performed by Sofia Lendor and Janusz Pawliszyn, data analysis was completed by Samantha Whiteside. v Acknowledgments To my supervisors, Jeremy and Gregor, thank you for your guidance and support over the years. Together you foster an environment of scientific creativity, friendship, and instill within each of us the desire to explore the unknown. You gave even my most outrageous theories consideration and I cannot thank you enough for the opportunities I have had within your research group. To Jean and Greg, it’s not an understatement to say I would be lost without your advice and guidance. You are my “Bioinformatics Gods,” converting what often seems impossible to comprehend into something my mortal brain can understand. Thank you for your patience and helping me to locate my misplaced periods, quotations, and commas. To Sumit, Sandra, and all the staff of the paediatric urology clinic of Children’s Hospital at London Health Sciences Centre. Thank you for taking the time to answer my questions, for your patience in answering what must have been hundreds of phone calls, for sharing your limited space with my clinical study materials, and finally for going above and beyond to help me make this clinical study happen. Sumit, you have been a wonderful teacher and mentor over these last few years. I am incredibly grateful that you were willing to give me the responsibility of running a clinical study over the course of my graduate studies. To the members of the Reid, Burton, and Gloor labs, I cannot begin to thank you all for the friendship and love I have felt from each of you over the years. I will always cherish the shenanigans we got into, even the ones that ended in near disaster, including reporting a “gas leak” to Union Gas and replacing our front door after we had to kick it in. You are the best friends I could have asked for and I cannot wait to see what the future brings for all of us. To my family, you don’t always understand what I do, or why I do it, yet seem to appreciate telling people that over my research career I have developed a fascination for faeces in undergrad and urine in grad school. I am incredibly grateful for the love within our family and the times we’ve shared together. There is no one else I would want to argue with about “North African Trade Oranges,” torpedo trajectories through islands, or whose turn it is to host the next Family Scotch Club. You keep me honest and grounded. vi Brandon; I’m not sure how I tricked you into it, but you’ve committed to spending the rest of your life with me and I cannot wait to see where we go. You remind me to breathe, appreciate the moment, and push me to be a better person. I would probably be living in a gutter, if it were not for your, love, patience, support, compassion, sense of adventure, and ability to laugh both at me and with me. And finally, although they’ll never read this, Baxter and Remus, who remind me on a daily basis to get moving, and can put a smile on my face at the worst of times. vii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments .............................................................................................................. vi List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xiii Abbreviations .....................................................................................................................
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