Exploring and Enhancing Context-Dependent Beta-Lactam Antibiotic Efficacy by Sarah Christine Bening B.Bm.E., University of Minnesota (2015) Submitted to the Department of Biological Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Engineering at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2021 © Massachusetts Institute of Technology 2021. All rights reserved. Author................................................................ Department of Biological Engineering December 29, 2020 Certified by. James J. Collins Professor of Biological Enginneering Thesis Supervisor Accepted by . Katharina Ribbeck Professor of Biological Engineering Chair of Graduate Program, Department of Biological Engineering 2 Exploring and Enhancing Context-Dependent Beta-Lactam Antibiotic Efficacy by Sarah Christine Bening Submitted to the Department of Biological Engineering on December 29, 2020, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biological Engineering Abstract Antibiotics, such as beta-lactams, are essential medical tools for the treatment of bacterial infections. Unfortunately, clinical treatment efficacy is declining over time as bacteria adapt to and evade antibiotic treatment through mechanisms called an- tibiotic resistance, tolerance, and persistence. Antibiotic tolerance and persistence, in particular, are often context-dependent phenotypes: environmental factors can in- fluence bacterial physiology and alter antibiotic efficacy. Optimal antibiotic use,as well as strategies to enhance antibiotic efficacy, can therefore be informed by studies of context-dependent antibiotic action. In this thesis, I present three vignettes about beta-lactam antibiotic efficacy and how environmental context alters in vitro treatment outcomes. First, I explore bac- terial killing in multi-drug contexts, focusing on how different beta-lactams can have different effects in combination with antibiotics of other classes. Second, Ipresent a new counter-tolerance method using metabolic stimulation to sensitize tolerant, stationary phase bacteria to beta-lactam antibiotics. Third, I present an extension of this metabolic counter-tolerance strategy, now combining metabolic and target- specific stimulation to further enhance beta-lactam efficacy. I demonstrate that this combined approach, when coupled with beta-lactamase inhibitors, restores beta- lactam sensitivity to simultaneously tolerant and resistant cultures of clinically rele- vant pathogens. I conclude by discussing opportunities for future study into antibiotic context-dependence and the application of counter-tolerance approaches such as the one described in this thesis. Thesis Supervisor: James J. Collins Title: Professor of Biological Enginneering 3 4 Acknowledgments My parents made sure I loved reading. There were always books in the house for me to read. While I read a lot of books growing up, somewhere around high school or undergrad I stopped finding the time to read for fun. Thankfully, shortly after my thesis proposal, I started reading books again. There have been so many good books that have helped me relax and get through the tough parts of graduate school, so many opportunities to go on someone else’s adventure for a while. Unlike when I was younger, my reading habits during graduate school always started with a flip to the acknowledgments section. I wanted to see what circles the author ran in (and maybe find some new books to read). More importantly, the acknowledgments were a chance to hear the author’s voice, unfiltered by their characters. A chance to hear from the real actual human that created each story. You’d think after reading all those acknowledgment sections writing this would’ve been a little easier. Anyways, here’s my thank you to all the people who helped me on my graduate school adventure: To Jim – when I started graduate school I knew next to nothing about how to study bacteria or about how antibiotics work. Thank you for giving me the opportunity to learn, the support and resources to make mistakes, and the freedom to pursue the projects that interested me. To my committee, Katharina Ribbeck and Mike Laub – thanks for your support and encouragement, and thank you for taking the time and energy to really be present at my committee meetings. I always enjoyed talking science with you. To the Collins lab – with no exceptions that I can think of (though there may very well be some), everything I know about microbiology research at the bench I learned from you all. I’m grateful for this massive and varied group of researchers that I got to work with and learn from every day. To the group from my early days in the Collins lab – Prerna Bhargava, Rebecca Shapiro, Caroline Porter, Saloni Jain, and Meagan Hamblin – thank you for helping me get started. Thank you for helping me learn how to be a scientist, how to work hard, ask questions, and celebrate the good experiments. And thank you for your 5 friendship, for all the lunch chats, snack breaks, trips to the Muddy, and recently the never ending group chats. To the Collins lab graduate students – especially those at the Broad: Ian Andrews, Bernardo Cervantes, Meagan Hamblin (honorary graduate student), and Erica Zheng – you’ve been a great group to struggle through graduate school with. And I mean struggle in the best way: a good struggle’s okay. You’ve been a great group to learn with, work hard with, and fail miserably at making random dinner restaurant decisions with. To the group that are coauthors on the core of my thesis work – Ian Andrews, Meagan Hamblin, and Allison Lopatkin – thank you for being my closest collabora- tors. Thank you for the experiments you did, but more importantly, thank you for all the time we spent talking science and for helping me learn how to present this story. To the MIT BE community – especially BE 2015, the BE Grad Board, the BE Communication Lab, and the BE department staff – I don’t know how to say it better than "community." The different groups I’ve been part of have given methe opportunity to get to know and work with so many more people than the typical graduate student might, and I’ve benefitted so much from getting to know you all. I’m grateful to be part of a community that works hard and knows how to have fun. To my friends – classmates, roommates, coworkers, friends from before MIT and outside of MIT – thank you for all the time we had fun and weren’t stressed about science. For all the juggling, cookie balling, moth fighting, Bopping to the Top, Muddy-ing, Naco Taco-ing, and (unfortunately) Zooming times. Thanks for giving me the space to just be a human and stop thinking about PCR for a while. Thanks for your friendship and helping me get across the finish line. To my sister, Dr. Bening – thank you for always setting the bar high. You’re right: I definitely had it easy never having to take the bus to high school. Thankyou for being my best and longest teammate and friend. To my parents – thank you for everything. Growing up you made sure I had the opportunity to do everything I wanted to do. And all those things – all the sports, (recovering from) all the knee injuries, all the piano lessons and everything trombone, 6 and all the people I’ve gotten to know along the way – have made me who I am today. Thank you for your example of loving to read, learn, and work hard. Thank you for the environment you created at home, and for making sure school was never a scary thing. It’s because you encouraged me to keep chasing what I was interested in that I’m writing my PhD thesis acknowledgments. 7 8 Contents 1 Introduction 13 1.1 Overview of Thesis Chapters . 13 1.2 Antibiotic Failure Modes . 14 1.3 Antibiotic Efficacy and Context-Dependence . 15 1.4 The Beta-Lactam Antibiotics and Bacterial Peptidoglycan . 17 2 Characterization of Killing by Bactericidal Antibiotic Combinations 21 2.1 Introduction . 21 2.2 Synergy with Aminoglycosides is Common . 23 2.3 Concentration-Dependent Interactions Between Beta-Lactams and Amino- glycosides . 25 2.4 Exploring Non-Beta-Lactam Cell Shape Perturbations . 27 2.5 Discussion . 29 2.6 Methods . 31 3 A Metabolic Counter-Tolerance Strategy for Beta-Lactams 33 3.1 Introduction . 33 3.1.1 Targeting Metabolism as a Counter-Tolerance Strategy . 34 3.1.2 Peptidoglycan Activity in Stationary Phase . 35 3.2 Stationary Phase Bacteria are Highly Tolerant to the Beta-Lactam Ampicillin . 35 3.3 Many Carbon Sources Restore Killing by High Ampicillin Concentrations 37 3.4 Cell Growth May Contribute to but Does Not Explain Sensitization . 40 9 3.5 Discussion . 45 4 Enhancing Beta-Lactam Counter-Tolerance Using D-Amino Acids 49 4.1 Introduction . 49 4.1.1 Stationary Phase Peptidoglycan . 50 4.1.2 L,D-Transpeptidases and Beta-Lactam Efficacy . 50 4.1.3 D-Amino Acids and Beta-Lactam Efficacy . 51 4.2 Testing Single D-Amino Acids . 52 4.3 Combining D-Amino Acids with Metabolic Stimulation by Carbon Sources . 55 4.4 Application to Other Organisms and 100% LB . 60 4.5 Discussion . 64 5 Future Directions and Conclusions 69 5.1 Antibiotic Discovery and Resistance . 69 5.2 Context-Dependence and Clinical Relevance . 70 5.3 Application of Counter-Tolerance Approaches . 72 5.3.1 Context-Dependent Carbon Source Efficacy . 72 5.3.2 Delaying the Evolution of Antibiotic Resistance . 73 5.3.3 Failure Modes of Counter-Tolerance Strategies . 74 A Methods for Sensitizing Tolerant Bacteria to Beta-Lactam Antibi- otics 91 10 List of Figures 1-1 Antibiotic Efficacy is a Systems-Level Process that is Sensitive to External Cues. ........................... 16 2-1 Killing by Low Concentrations of Quinolones or Beta-Lactams in Combination with Sublethal Gentamicin. 23 2-2 Synergy of Select Beta-Lactams at Low Concentrations with Additional Aminoglycosides.