Phenotypic Antimicrobial Susceptibility Testing Based on Nucleic Acid Analysis Thesis by Nathan Garrett Schoepp In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2019 (Defended May 10, 2019) ii ã 2019 Nathan Garrett Schoepp ORCID: 0000-0002-2406-3693 iii ACKNOWLEDGEMENTS There have been countless people who have helped me learn and mature until this point in my life. Nothing is done in isolation and I am extremely grateful for everyone who has helped, supported, and taught me. I am thankful for all the individuals (named and not named below) who I have learned from. I have been privileged in so many ways, and especially to be able to do work that I enjoy and I try to remember that every day. First and foremost, I would like to thank my family for their love and constant support. My mom Julie, my dad Scott, and my brother Tim have given me so much even though I take them for granted far too often. They have provided me with the foundation to succeed, and have made so many parts of my life easy. I am forever grateful for this and hope in the future I can provide even a fraction of the support I was given. My family has helped me through stressful times and hard decisions. They have taught me the values I hold today, and they are as much responsible for my drive and motivation as anyone. I hope they feel some ownership over this work as well. I would also like to thank the partners and friends who have supported me and given me much needed breaks from the difficulties of lab. These were important and memorable moments that kept me going. All members of the Ismagilov lab also deserve a special thanks. Without them there would be no lab environment, no lab jokes, and no lab discussion. I value all of these, and my PhD would have been much less enjoyable without them. I have learned many technical and personal skills from each of you. Specifically, I would like to thank Travis Schlappi, Justin Rolando, Said Bogatyrev, Asher Preska Steinberg, Erik Jue, Emily Savela, Eric Liaw, Jesus Rodriguez Manzano, Daan Witters, and Natasha Shelby. I would like to especially acknowledge Travis Schlappi, for setting the bar for a model colleague and friend. I hope to get to work with more people like you. iv I would like to thank the mentors in my life up to now. I have had the incredible fortune of working with many fabulous scientists and advisors. It seems the role of advisor/mentor is often underappreciated and thankless, and there is no way this paragraph does them justice. First, I would like to thank my advisor Rustem and my committee. Thank you Rustem for teaching me scientific rigor and excellence, providing truly exceptional resources, and for always being available. I would also like to thank my committee Jim Heath, Dave Tirrell, and Jared Leadbetter for their continued guidance and support throughout my PhD and their willingness to always take time when I need advice. I would also like to thank some exceptional individuals not on my committee but who have never hesitated to offer guidance or support: Mikhail Shapiro and Justin Bois. Both of you have been model mentors and I have the utmost respect for you. Collaborations significantly increase the speed of scientific progress and I have seen this first-hand during my PhD. I would like to thank the UCLA microbiology lab (specifically Romney Humphries, Shelley Miller, and Janet Hindler) who provided clinical samples and participated in countless discussions. You made collaboration easy and smooth. I would also like to thank S.O. at the University of Washington for his passion and involvement in the ongoing work with Neisseria gonorrhoeae. v ABSTRACT Antimicrobial resistance (AMR) is one of the most widely recognized threats to global health, and one that continues to grow as new mechanisms of resistance evolve and resistant pathogens spread. Antibiotics are a cornerstone of modern medicine, but their misuse and overuse has constantly and consistently reduced their efficacy to the critically low levels we observe today. As a result, the rate of mortality as a direct result of AMR is approaching over a million deaths annually, with 20-year projections in the ten-millions. Rapid, phenotypic antimicrobial susceptibility testing (AST) that could be performed at the point of care (most notably in ≤ 30 min) would decrease the overuse of antimicrobials, allow physicians to make informed choices about which antimicrobials to prescribe, and improve patient outcomes. Today no such method exists. The ultimate goal of the below work is to allow physicians to choose, instead of guess, which antibiotics to use. We envision that development of these tests into distributable diagnostics will drastically improve patient outcomes, curb the spread of resistance, strengthen global antibiotic stewardship, and forestall the post-antibiotic era. vi PUBLISHED CONTENT AND CONTRIBUTIONS Chapter II: Nathan G. Schoepp†, Eugenia M. Khorosheva†, Travis S. Schlappi, Matthew S. Curtis, Romney M. Humphries, Janet A. Hindler, and Rustem F. Ismagilov. “Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure.” Angewandte Chemie 2016, 55 (33), 9557-9561. DOI: 10.1002/anie.201602763 This chapter describes the use of digital quantification of nucleic acids to shorten the required antibiotic exposure time needed to determine the antibiotic susceptibility phenotype of E. coli clinical isolates. Nathan G. Schoepp: 1. Major contributor to selecting DNA replication as AST marker, contributed knowledge on AST state of the art and effects of antibiotics on replication, contributed to digital resolution hypothesis, contributor to chromosome segregation hypothesis 2. Optimized antibiotic exposure protocols 3. Performed all antibiotic exposures 4. Maintained bacterial isolates 5. Performed all bulk and digital quantification experiments 6. Contributed all data to figures 1, 2, 3, S1, S2, S3, and table S2, contributed all non-sheared data to figure 4 7. Drew figures 1, 2, 3, 4, S1, S2, S3, and constructed table S2 8. Contributed to writing abstract, introduction, results/discussion, and conclusion sections of manuscript 9. Contributed to writing of supplemental information Eugenia Khorosheva: 1. Major contributor to selecting DNA replication as AST marker, major contributor of knowledge on AST state of the art and effects of antibiotics on replication, contributed to selecting 23S gene as a target of choice, contributed to digital resolution hypothesis, contributed to chromosome segregation hypothesis 2. Selected experimental protocols for maintenance and growth of isolates 3. Established initial AST protocols and experimental workflow from exposure to extraction 4. Selected and optimized protocols for amplification with Enterobacteriaceae specific 23S primers. 5. Contributed to optimizing DNA shearing experiments. 6. Contributed to writing introduction, results/discussion, and conclusion sections of manuscript Travis S. Schlappi: 1. Contributed knowledge on AST statistics, contributed to digital resolution hypothesis 2. Connected FDA guidelines for establishing new antimicrobial susceptibility determination methods to statistical hypothesis testing for both qPCR and dPCR. 3. Performed statistical analysis (p- vii values and error bars) for all data presented in the manuscript and supplemental information. 4. Performed preliminary digital PCR experiments showing that dPCR can resolve differences in concentration after 15min exposure that qPCR cannot. 5. Contributed to writing of supplemental information. Matthew S. Curtis: 1. Contributed to selecting DNA replication as AST marker, contributed knowledge on AST state of the art and effects of antibiotics on replication, contributed to digital resolution hypothesis, major contributor to chromosome segregation hypothesis 2. Selected and optimized shearing protocols on extracted DNA for the analysis of chromosome structure. 3. Performed shearing experiments to generate data for figure 4. 4. Contributed to statistical analysis 5. Contributed to writing of the introduction, results/discussion, and conclusion of the manuscript Chapter III: Nathan G. Schoepp†, Travis S. Schlappi†, Matthew S. Curtis, Slava S. Butkovich, Shelley Miller, Romney M. Humphries, and Rustem F. Ismagilov. “Rapid pathogen-specific phenotypic antibiotic susceptibility testing using digital LAMP quantification in clinical samples.” Science Translational Medicine 2017, 9 (410). DOI: 10.1126/scitranslmed.aal3693 This chapter describes the use of rapid digital LAMP assay to determine phenotypic antibiotic susceptibility of bacteria directly from clinical urinary tract infection samples in less than 30 min. The order of co-first authors was determined by coin toss. T.S.S., N.G.S., and R.F.I. contributed to the design and/or interpretation of the reported experiments or results. T.S.S., N.G.S., M.S.C., S.S.B., and R.F.I. contributed to the acquisition and/or analysis of the data. T.S.S., N.G.S., R.M.H., and R.F.I. contributed to the drafting and/or revising of the manuscript. M.S.C. was primarily responsible for real-time imaging acquisition and analysis. S.M. and R.M.H. were primarily responsible for acquiring clinical samples and performing gold standard broth microdilution ASTs. R.F.I. and R.M.H. contributed administrative, technical, and supervisory support. Chapter IV: Tahmineh Khazaei, Jacob T. Barlow, Nathan G. Schoepp,