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The Development of Bacterial Magnetic Resonance Imaging for Microbiota Analyses Western University Scholarship@Western Electronic Thesis and Dissertation Repository 9-11-2020 12:00 PM The development of bacterial magnetic resonance imaging for microbiota analyses Sarah C. Donnelly, The University of Western Ontario Supervisor: Burton, Jeremy P., The University of Western Ontario Co-Supervisor: Goldhawk, Donna E., The University of Western Ontario A thesis submitted in partial fulfillment of the equirr ements for the Master of Science degree in Microbiology and Immunology © Sarah C. Donnelly 2020 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Bacteria Commons, Medical Biophysics Commons, and the Medical Microbiology Commons Recommended Citation Donnelly, Sarah C., "The development of bacterial magnetic resonance imaging for microbiota analyses" (2020). Electronic Thesis and Dissertation Repository. 7381. https://ir.lib.uwo.ca/etd/7381 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 Current microbial analyses to assess either the commensal microbiota or microorganism infection and disease typically require ex vivo techniques that risk contamination and are not undertaken in real time. The possibilities for employing imaging techniques in the microbiology field is becoming more prominent as studies expand on the use of positron emission tomography, ultrasound and numerous microscopy techniques. However, magnetic resonance imaging (MRI), a non-invasive in vivo modality that can produce real-time results is falling behind. Here, we examined the feasibility of detecting bacteria using clinical field strength MRI. Commensal, probiotic and uropathogenic Escherichia coli were scanned by 3 Tesla MRI where signal was related to the number of colony-forming units as well as total cellular iron and manganese content as determined by mass spectrometry. Various microbes commonly found in the human gut and urogenital tract were also assessed by MRI. Lactobacillus spp. displayed significantly higher transverse relaxation rates than other species, despite their low iron usage, potentially due to high manganese content. High MR relaxivity may enable detection of lactobacilli amidst host tissues, therefore, we assessed the potential to distinguish the MR signatures of two distinct cell types within mixed samples. With clinical field strength MRI, we were able to detect as few as ~26 x 106 colony-forming units of Lactobacillus crispatus ATCC33820 per mm3. In the future, MRI may allow for non-invasive evaluation of health or dysbiosis in the human microbiota as well as potential applications in tracking the dispersion and persistence of bacteria in broad applications from probiotic use, infection to tumour tracking. Keywords Magnetic resonance imaging (MRI), bacterial detection, bacterial tracking, iron homeostasis, manganese homeostasis, microbiota, urinary tract infection, E. coli, Lactobacillus crispatus, MagA ii Summary (lay abstract) Background: Bacteria inhabit various niches throughout the human body. These bacteria can promote health, but in some cases specific bacteria can overgrow and be harmful to the host. We would like to use magnetic resonance imaging (MRI) to visualize and track these bacteria within their preferred environments to learn more about human-bacterial and bacterial-bacterial interactions in health and disease. Hypotheses: Different bacterial types will appear differently on images from MRI. Adding the iron uptake gene magA to E. coli will make it easier to detect by MRI. Methods: We imaged various bacteria and strains of E. coli found in the human gut and urogenital tract using MRI. We then cloned a gene (magA) which incorporates magnetic properties into an E. coli strain and then assessed its influence on bacterial MRI detection. We measured the concentrations of iron and manganese in all our bacteria since these metals can impact MRI. Finally, we imaged dilutions of Lactobacillus crispatus, a bacterium that we found to be very visible by MRI and mixed it with human bladder cells to see if we could distinguish the two cell types within a mixed sample. Results and Significance: Different E. coli strains and bacteria have unique magnetic resonance signatures due to differences in metal content. In the future we hope to use MRI to identify and track bacteria within the human host. iii Co-Authorship Statement All portions of this thesis were exclusively written by S. C. Donnelly with advice and editing from D. E. Goldhawk, J. P. Burton and N. Gelman. In Chapter 2, magA was cloned into pET28a+ by R. S. Flannagan. D. E. Heinrichs provided Staphylococcus aureus iron acquisition mutants and bacterial iron handling and cloning expertise. N. Gelman and F. S. Prato provided software and expertise on magnetic resonance physics. K. Quiaoit prepared the pcDNA3.1MycHisA+/Ha- magA construct. iv Acknowledgments I would like to thank everyone who has helped along the way in completing my M.Sc. and throughout my educational career. Therefore, I would like to briefly acknowledge some of the people who helped me achieve this academic milestone. First, I would like to thank my supervisors Dr. Jeremy Burton and Dr. Donna Goldhawk. Jeremy, thank you for always doing your best to keep my research moving forward and reminding me to look at the big picture. Donna, thank you for always answering my questions and helping me think through unexpected results. Thank you both for your patience, support and guidance along the way. Next, I would like to thank my advisory committee, Dr. David Heinrichs and Dr. Neil Gelman for all the advice and resources you have provided me to help in my study. Dr. Heinrichs, thank you for providing me with the iron acquisition mutants to help expand my study. Neil, thank you for helping me through the physics portions of my project and for always contributing to the discussion when I obtained unexpected results. I would also like to thank the members of the Burton, Goldhawk and Reid labs (past and present) who have helped to create a great environment to conduct research. Thanks for your advice on my research, and for suggesting alternative methods various experiments, I truly appreciate it. Thank you to Dr. Ron Flannagan for his advice, technical expertise and helping troubleshoot when I experienced cloning difficulties. I’d like to thank Dr. Frank Prato and Dr. Gregor Reid for their out of the box ideas and constant feedback on how to improve my research. Shannon Seney, thank you for always being around to answer my questions, train me on new techniques and keep the lab running smoothly. Thanks to Praveen Dassanayake, Daisy Sun, Emiley Watson, Scarlett Puebla Barragán, Hannah Wilcox, Johnny Chmiel and many others in the lab who have acted as my sounding boards and made research life more enjoyable. Many thanks to John Butler, Heather Biernaski, Lynn Keenliside and Jeff Warner for their technical assistance during this project. Finally, thank you to my family and my husband for their unconditional love and support throughout my life. Mike, thank you for always being around to cheer me up. Thank you for v being understanding and driving me to and from the lab at all hours when I had to start or induce a culture or move a partially solidified phantom to the fridge. Thank you for pretending to listen to me explain my project and new findings as well as practicing for presentations. Overall, thank you for believing in me and always being there to listen, support and encourage me; I could not have achieved this without you! vi Table of Contents Abstract ..................................................................................................................................... ii Lay Abstract ............................................................................................................................. iii Co-Authorship Statement ........................................................................................................ iv Acknowledgments .................................................................................................................... v Table of Contents .................................................................................................................... vii List of Tables .......................................................................................................................... xi List of Figures ......................................................................................................................... xii List of Appendices ................................................................................................................ xiv List of Abbreviations ............................................................................................................ xvi Chapter 1: Introduction ............................................................................................................. 1 1.1 Bacteriology .................................................................................................................. 1 1.1.1 Human microbiota ................................................................................................ 1 1.1.2 Urinary tract infection .......................................................................................... 2 1.1.3 Bacterial metal homeostasis ................................................................................
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