UC San Diego UC San Diego Electronic Theses and Dissertations Title Exploring Antibiotic and Innate Immune Synergies to Treat Multi-Drug Resistant Bacterial Infections Permalink https://escholarship.org/uc/item/8nw4n64p Author Lin, Leo Publication Date 2016 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Exploring Antibiotic and Innate Immune Synergies to Treat Multi-Drug Resistant Bacterial Infections A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Leo Lin Committee in charge: Professor Victor Nizet, Chair Professor Joe Pogliano Professor Sharon Reed Professor Stephen Spector Professor Elizabeth Winzeler 2016 Copyright Leo Lin, 2016 All rights reserved This Dissertation of Leo Lin is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2016 iii DEDICATION To my family Pannyun & Nathan, parents Athena & Brian, and siblings Clinton & Maya: thank you all for your love, care, and encouragement and to Professor Nizet: I will always remember your selfless mentoring and passion for science. iv TABLE OF CONTENTS Signature Page ................................................................................................................... iii Dedication .......................................................................................................................... iv Table of Contents .................................................................................................................v List of Figures ................................................................................................................... vii List of Tables ..................................................................................................................... ix Acknowledgements ..............................................................................................................x Vita .................................................................................................................................... xii Abstract of the Dissertation ............................................................................................. xiv Chapter 1 Introduction .........................................................................................................1 Specific Aims .........................................................................................................23 References ..............................................................................................................24 Chapter 2 Azithromycin Synergizes with Cationic Antimicrobial Peptides to Exert Bactericidal and Therapeutic Activity Against Highly Multidrug-Resistant Gram Negative Bacterial Pathogens ............................................................................................36 Preface....................................................................................................................37 Abstract ..................................................................................................................38 Introduction ............................................................................................................39 Materials and Methods ...........................................................................................40 Results ....................................................................................................................47 Discussion ..............................................................................................................58 Supplemental Materials and Methods ....................................................................60 Acknowledgments..................................................................................................73 References ..............................................................................................................74 v Chapter 3 Standard Susceptibility Testing Overlooks Potent Azithromycin Activity and Cationic Peptide Synergy Against Multidrug-Resistant Stenotrophomonas maltophilia ..79 Preface....................................................................................................................80 Synopsis .................................................................................................................81 Introduction ............................................................................................................82 Materials and Methods ...........................................................................................83 Results ....................................................................................................................86 Discussion ..............................................................................................................91 Acknowledgments..................................................................................................93 Supplemental Materials and Methods ....................................................................94 References ..............................................................................................................99 Chapter 4 Component Analysis of Multi-Purpose Contact Lens Solutions to Enhance Activity Against Pseudomonas aeruginosa and Staphylococcus aureus .........................101 Preface..................................................................................................................102 Abstract ................................................................................................................103 Introduction ..........................................................................................................104 Materials and Methods .........................................................................................105 Results ..................................................................................................................109 Discussion ............................................................................................................115 Acknowledgments................................................................................................115 Supplemental Materials and Methods ..................................................................116 References ............................................................................................................119 Chapter 5 Conclusions .....................................................................................................121 Summary of Results .............................................................................................122 Future Studies ......................................................................................................123 vi LIST OF FIGURES Figure 2.1. AZM bactericidal activity against GNRs in eukaryotic media and human serum ..................................................................................................................................49 Figure 2.2. Colistin synergizes with AZM by increasing GNR outer membrane permeability .......................................................................................................................52 Figure 2.3. Cationic AMP LL-37 synergizes with AZM by increasing the outer membrane permeability of MDR-GNRs ............................................................................54 Figure 2.4. AZM activity as monotherapy against MDR-GNRs in vivo ...........................57 Figure S 2.1. Serial passage for 10 days of MDR GNR isolates at sub-minimum inhibitory concentrations of azithromycin to test for resistance evolution ........................64 Figure S 2.2. Colistin synergizes with azithromycin, erythromycin, and clarithromycin to kill MDR GNR pathogens in vitro .....................................................................................65 Figure S 2.3. Treating MDR A. baumannii with sub-MIC LL-37 or colistin significantly increases outer membrane permeability to 1-N-phenylnaphthylamine (NPN) ................66 Figure S 2.4. Pre-treating MDR A. baumannii with AZM increases outer membrane binding of LL-37 ................................................................................................................67 Figure S 2.5. Αddition of low dose AZM to reduced dose colistin in treatment of MDR GNRs in vivo .....................................................................................................................68 Figure S 2.6. Confirmation of structure and purity of synthesized NBD-tagged AZM ....69 Figure 3.1. Azithromycin bactericidal activity against SM observed in media alone and in synergy with cationic antimicrobial peptides ....................................................................89 Figure 3.2. Bacterial cytological profiling showing that cationic peptides facilitate azithromycin entry into SM cells .......................................................................................90 Figure 3.3. Azithromycin sensitizes SM to neutrophil killing and increases SM clearance in vivo ................................................................................................................................91 Figure S 3.1 Growth of SM K279a in bacteriologic (CA-MHB) versus supplemented tissue culture (RPMI+10%LB) media ...............................................................................96