WWeesstteerrnn UUnniivveerrssiittyy SScchhoollaarrsshhiipp@@WWeesstteerrnn Electronic Thesis and Dissertation Repository 8-5-2014 12:00 AM CChheemmiiccaall CCoommmmuunniiccaattiioonn ooff AAnnttiibbiioottiicc RReessiissttaannccee bbyy HHiigghhllyy RReessiissttaanntt BBaacctteerriiaa.. Omar M. El-Halfawy, The University of Western Ontario Supervisor: Dr. Miguel A. Valvano, The University of Western Ontario A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Microbiology and Immunology © Omar M. El-Halfawy 2014 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Bacteriology Commons RReeccoommmmeennddeedd CCiittaattiioonn El-Halfawy, Omar M., "Chemical Communication of Antibiotic Resistance by Highly Resistant Bacteria." (2014). Electronic Thesis and Dissertation Repository. 2187. https://ir.lib.uwo.ca/etd/2187 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]. CHEMICAL COMMUNICATION OF ANTIBIOTIC RESISTANCE BY HIGHLY RESISTANT BACTERIA (Thesis format: Integrated-Article) by Omar M. El-Halfawy Graduate Program in Microbiology and Immunology A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada © Omar M. El-Halfawy 2014 Abstract The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. I use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, I report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of PmB than the rest of the cells in the culture, and protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to PmB. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. I propose that putrescine and YceI resemble "danger" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species. Putrescine protects from antibiotics through its ability to compete with PmB for surface binding and protection against antibiotic-induced oxidative stress. YceI proteins are conserved bacterial lipocalins or “bacteriocalins”. Bacteriocalins from different Gram-positive and Gram- negative bacteria are involved in the response to hydrophobic or amphiphilic antibiotics (PmB, rifampicin, norfloxacin and ceftazidime) but not hydrophilic ones (such as gentamicin). This effect is achieved by their preferential binding affinity to hydrophobic moieties. Together, my findings uncover a novel, non-genetic and cooperative mechanism of transient increase in resistance chemically communicated from more resistant members of heterogeneous populations to less resistant bacteria of the same or other species. This multifactorial mechanism of communication of antibiotic resistance offers novel targets for antimicrobial intervention. ii Keywords Antibiotic resistance; Heteroresistance; Communication of antibiotic resistance; Antimicrobial peptides; Polymyxin B; Bactericidal antibiotics; Polyamines; Putrescine; Population analysis profiling; YceI; Lipocalins; Reactive oxygen species; ROS; Burkholderia cenocepacia; Pseudomonas aeruginosa; Mycobacterium tuberculosis; Salmonella typhi; Shigella flexneri; Escherichia coli. iii Dedication To my father, my mother, my brother, my wife Somiraa and my daughter Nada for their unconditional support throughout my academic career. iv Acknowledgements First and foremost I praise and thank God for giving me the capability, patience and will to accomplish this work. I would like to record my sincerest gratitude for my Supervisor Dr. Miguel Valvano whose support and advice has guided me through my Ph. D. studies and allowed me to develop my skills whilst allowing me the room to work in my own way. His truly scientist intuition exceptionally inspired and enriched the work. His leadership and mentorship has created an environment that fostered inquiry, creativity, independence and teamwork. One simply could not wish for a better mentor. I would like to thank the members of my advisory committee; Dr. Carole Creuzenet and Dr. Martin McGavin. Thank you for the valuable suggestions and recommendations; I really benefited from our discussions and greatly appreciate spending your time to make our meetings as productive as they were. During the course of my studies I had the privilege to serve as a Teaching Assistant for 3 years; I thank Dr. Susan Koval for this wonderful experience. I would also like to thank all the members of the Valvano lab, past and present, for their help and support. I valued their ideas and comments at our weekly lab meetings and enjoyed having the opportunity to interact with such a diverse group of people. Special thanks to Slade Loutet, Daniel Aubert, Soledad Saldias, and Mohamed Hammad for their valuable discussions and advice. Thank you also to Katie Bain, Roberto Rosales, Angel Andrade, Sarah Furlong, Crystal Schmerk, Maryam Khodai-Kalaki, Faviola Tavares, Yasmine Fathy, Xiang Ruan, Anna Hanuszkiewicz, Kinnari Patel, Jennifer Tolman, Cristobal Mujica, Stephanie Lamothe, Janet Torres, Andrea Valderrey, Maha Al-Zayer, Chelsea Clarke, Hanna Ostapska, and Lorena Albarnez. I would like to thank Dr. Luke Alderwick, Institute of Microbiology and Infection, University of Birmingham for providing M. tuberculosis H37Rv genomic DNA; and Dr. Sameer Elsayed, London Health Science Centre, the University of Western Ontario for providing clinical isolates. I also acknowledge the Ontario Graduate Scholarship Program for International students. v Finally, I cannot thank my family enough; without their support throughout the years, I would not be the same person that I am now. Special thanks to my wife Somiraa Said and daughter Nada, I could not have achieved this without you. Omar El-Halfawy vi Table of Contents Abstract ......................................................................................................................................................... ii Keywords ..................................................................................................................................................... iii Dedication ................................................................................................................................................... iiv Acknowledgements ....................................................................................................................................... v Table of Contents ........................................................................................................................................ vii List of Tables .............................................................................................................................................. xii List of Figures .............................................................................................................................................xiii Chapter 1 ....................................................................................................................................................... 1 Introduction ................................................................................................................................................... 1 1.1. Introduction ........................................................................................................................................ 2 1.2. Antibiotics and the dilemma of antibiotic resistance ......................................................................... 2 1.2.1. Classes of antibiotics ................................................................................................................... 3 1.2.2. Intrinsic versus acquired resistance to antibiotics ....................................................................... 8 1.2.3. Antibiotic resistant bacteria ...................................................................................................... 10 1.3. Antimicrobial Heteroresistance: an emerging field in need of clarity ............................................. 11 1.3.1. Multiple definitions of heteroresistance .................................................................................... 12 1.3.2. Measuring heteroresistance ....................................................................................................... 16 1.3.3. Reports of heteroresistance in different bacteria ....................................................................... 19 1.3.4. Mechanisms of heteroresistance ............................................................................................... 25 1.3.5. Significance and relevance of heteroresistance ......................................................................... 25 1.4. Non-genetic mechanisms communicating antibiotic resistance ....................................................... 28 1.4.1. Chemical signals modulating antibiotic resistance ................................................................... 29 1.4.2. New targets for drug discovery ................................................................................................. 40 1.5. Hypothesis and general objectives ................................................................................................... 41 1.6. Chapter 1 references......................................................................................................................... 42 Chapter 2 ..................................................................................................................................................... 63 Chemical communication of antibiotic resistance by a highly resistant subpopulation of bacterial cells .. 63 vii 2.1. Introduction ...................................................................................................................................... 64 2.2. Materials and Methods ..................................................................................................................... 65 2.2.1. Strains and reagents .................................................................................................................. 65 2.2.2. Population analysis profiling (PAP) ......................................................................................... 65 2.2.3. Co-culture ................................................................................................................................. 68 2.2.4. Volatile-mediated protection ..................................................................................................... 68 2.2.5. RNA extraction ......................................................................................................................... 68 2.2.6. qRT-PCR ................................................................................................................................... 69 2.2.7. Ornithine decarboxylase (ODC) assay ...................................................................................... 69 2.2.8. Thin-layer chromatography analyses of polyamines ................................................................ 69 2.2.9. Competition between putrescine and fluorescent PmB on surface binding .............................. 70 2.2.10. Cloning, expression, and purification of YceI ........................................................................ 70 2.2.11. Binding assay of YceI to PmB ................................................................................................ 71 2.2.12. Statistical analyses .................................................................................................................. 71 2.3. Results and Discussion .................................................................................................................... 71 2.3.1. Heteroresistance of B. cenocepacia to PmB ............................................................................. 71 2.3.2. A more resistant subpopulation of B. cenocepacia protects naïve bacteria from PmB ............ 74 2.3.3. The more resistant subpopulation releases higher amounts of a subset of proteins upon exposure to PmB ................................................................................................................................. 81 2.3.4. A role for putrescine in PmB resistance .................................................................................... 83 2.3.5. The role of YceI protein ............................................................................................................ 92 2.3.6. B. cenocepacia is heteroresistant to other bactericidal antibiotics ............................................ 92 2.4. Conclusions ...................................................................................................................................... 94 2.5. Chapter 2 References ....................................................................................................................... 98 Chapter 3 ................................................................................................................................................... 102 Putrescine reduces antibiotic-induced oxidative stress as a mechanism of modulation of antibiotic resistance in Burkholderia cenocepacia ................................................................................................... 102 3.1. Introduction .................................................................................................................................... 103 3.2. Materials and Methods ................................................................................................................... 105 3.2.1. Strains and reagents. ............................................................................................................... 105 3.2.2. General molecular techniques. ................................................................................................ 105 3.2.3. Fluorometric determination of ROS. ...................................................................................... 105 viii 3.2.4. Antibiotic susceptibility testing. ............................................................................................. 106 3.2.5. In vitro antioxidant activity assay. .......................................................................................... 106 3.2.6. Transcriptional fusions to luxCDABE. .................................................................................... 108 3.2.7. Luminescence expression assays. ........................................................................................... 108 3.2.8. Construction of a conditional mutant. ..................................................................................... 108 3.2.9. Thin-layer chromatography analyses of polyamines. ............................................................. 109 3.2.10. Catalase enzyme activity assay. ............................................................................................ 109 3.2.11. Statistical Analyses. .............................................................................................................. 110 3.3. Results And Discussion ................................................................................................................. 110 3.3.1. Putrescine reduces ROS production induced by PmB. ........................................................... 110 3.3.2. Expression of the putrescine synthesis enzymes in response to PmB. .................................... 117 3.3.3. ROS production in response to other bactericidal antibiotics. ................................................ 123 3.4. Conclusions .................................................................................................................................... 129 3.5. Chapter 3 References ..................................................................................................................... 133 Chapter 4 ................................................................................................................................................... 137 A novel mechanism of resistance and protection from the action of hydrophobic antibiotics mediated by secreted bacterial lipocalins ...................................................................................................................... 137 4.1. Introduction .................................................................................................................................... 138 4.2. Materials and methods ................................................................................................................... 139 4.2.1. Strains and reagents ................................................................................................................ 139 4.2.2. General molecular techniques ................................................................................................. 139 4.2.3. Protein analysis and Western Blotting .................................................................................... 142 4.2.4. Antibiotic susceptibility testing. ............................................................................................. 142 4.2.5. Fluorometric binding assays ................................................................................................... 143 4.2.6. Galleria mellonella larvae in vivo infection models ............................................................... 144 4.2.7. Statistical Analyses ................................................................................................................. 144 4.3. Results and discussion ................................................................................................................... 144 4.3.1. Secretion of B. cenocepacia bacteriocalins ............................................................................. 144 4.3.2. The role of the different B. cenocepacia bacteriocalins in response to antibiotics ................. 144 4.3.3. Fluorometric assays of binding interaction of B. cenocepacia bacteriocalins ........................ 146 4.3.4. Luciferase expression assays of B. cenocepacia bacteriocalins .............................................. 150 4.3.5. Bacteriocalins from different bacterial species are involved in antibiotic resistance ............. 150 ix