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Dissertation DISSERTATION FOLATE PATHWAY INHIBITOR RESISTANCE MECHANISMS IN BURKHOLDERIA PSEUDOMALLEI Submitted by Nicole L. Podnecky Department of Microbiology, Immunology and Pathology In partial fulfillment of the requirements For the Degree of Doctor of Philosophy Colorado State University Fort Collins, Colorado Fall 2013 Doctoral Committee: Advisor: Herbert P. Schweizer Steven W. Dow Richard A. Slayden Laurie A. Stargell ABSTRACT FOLATE PATHWAY INHIBITOR RESISTANCE MECHANISMS IN BURKHOLDERIA PSEUDOMALLEI Antimicrobials are invaluable tools used to facilitate the treatment of infectious diseases. Their use has saved millions of lives since their introduction in the early 1900’s. Unfortunately, due to the increased incidence and dispersal of antimicrobial resistance determinants, many of these drugs are no longer efficacious. This greatly limits the options available for treatment of serious bacterial infections, including melioidosis, which is caused by Burkholderia pseudomallei, a Gram-negative saprophyte. This organism is intrinsically resistant to many antimicrobials. Additionally, there have been reports of B. pseudomallei isolates resistant to several of the antimicrobials currently used for treatment, including the trimethoprim and sulfamethoxazole combination, co-trimoxazole. The overarching goal of this project was to identify and characterize mechanisms of trimethoprim and sulfamethoxazole resistance in clinical and environmental isolates, as well as in laboratory induced mutants. Prior to these studies, very little work has been done to identify and characterize the mechanisms by which B. pseudomallei strains are or could become resistant to folate-pathway inhibitors, specifically trimethoprim and sulfamethoxazole. During the initial phases of these studies, we determined the antimicrobial susceptibilities of a large collection of clinical and environmental isolates from Thailand and Australia (n = 65). A high frequency of naturally-occurring resistance to trimethoprim alone (40%) was observed. However these strains were susceptible to sulfamethoxazole and to the co-trimoxazole combination. Trimethoprim resistance in a subset of these strains was due to increased ii expression of an efflux pump belonging to the resistance nodulation and cellular division (RND) superfamily, BpeEF-OprC, in the presence of trimethoprim. This efflux pump had been previously shown to efflux trimethoprim, chloramphenicol and tetracyclines when expressed in surrogate bacterial strains. The molecular mechanism of increased BpeEF-OprC expression in these isolates remains unknown. Similarly, decreased susceptibility in laboratory mutants selected on trimethoprim were due to mutations leading to amino acid substitutions in BpeT, which caused overexpression of BpeEF-OprC, or FolA, the trimethoprim drug target. This is the first description of mutations to FolA conferring trimethoprim resistance in B. pseudomallei, though similar mutations have been observed in B. cenocepacia and Escherichia coli. A similar study to select for sulfamethoxazole resistance, instead suggested that B. pseudomallei may be able to tolerate high concentrations of the drug. Studies to characterize laboratory induced mutants selected on co-trimoxazole led to the identification of two novel resistance determinants. Mutations to BpeS, a newly named LysR- type regulator with high similarity to the cognate BpeEF-OprC efflux pump regulator, BpeT, cause increased BpeEF-OprC expression in these strains. Additionally mutations to Ptr1, an annotated pteridine reductase, partially contributed to the decreased co-trimoxazole susceptibility. However, it is unclear what function Ptr1 has in the folate synthesis pathway, as deletion of this gene also caused slight decreases in antimicrobial susceptibility. Finally, in a collection of co-trimoxazole resistant clinical isolates from Thailand, high-level expression of the BpeEF-OprC was found in the resistant isolates. A mutation to BpeS was also observed in two of the clinical isolates that had BpeT-independent BpeEF-OprC overexpression. Co-trimoxazole resistant isolates were each resistant to both trimethoprim and sulfamethoxazole individually. However, deletion of the bpeEF-oprC efflux pump structural genes in all isolates resistant to co- iii trimoxazole or isolates resistant to trimethoprim alone (except those with a mutant FolA) resulted in antimicrobial susceptibility to trimethoprim, co-trimoxazole and sulfamethoxazole. These data suggest that sulfamethoxazole is also a substrate of the BpeEF-OprC efflux pump and this RND pump is the major resistance determinant contributing to clinically relevant folate pathway inhibitor resistance in B. pseudomallei. To summarize, we have identified and described several resistance determinants in B. pseudomallei causing decreased susceptibilities to trimethoprim, sulfamethoxazole and/or co- trimoxazole; these include drug target and metabolic pathway modifications and overexpression of the BpeEF-OprC efflux pump. Further characterization of these mechanisms and the development of specific detection assays could allow for rapid determination of antimicrobial resistance and provide useful information for the development of novel antimicrobials against B. pseudomallei. iv ACKNOWLEDGMENTS I would like to thank my advisor Dr. Herbert Schweizer for giving me the opportunity to work in his laboratory. His patient guidance, support and expertise of the years has kept me on track and progressing forward on my research. Dr. Schweizer’s trust to allow me to work independently and oversee many of the laboratory management responsibilities has provided invaluable professional experience for me. I would also like to thank the members of my dissertation committee, Drs. Steven Dow, Richard Slayden and Laurie Stargell for their patience, insightful discussions and advice during the course of this research. Funding for this research was provided through the Rocky Mountain Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (NIH NIAID grant AI065357). I would like to thank Drs. Vanaporn Wuthiekanun (Mahidol-Oxford Tropical Medicine Research Unit) and Sharon Peacock (Mahidol-Oxford Tropical Medicine Research Unit, Department of Microbiology and Immunology, Faculty of Tropical Medicine, Bangkok, Thailand and Department of Medicine, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom) for providing the clinical and environmental B. pseudomallei isolates used in this research, and Drs. Bart Currie (Menzies School of Heath Research, Darwin, Australia), Apichai Tuanyok (University of Hawaii at Manoa) and Paul Keim (Northern Arizona University) for providing the Australian B. pseudomallei isolates and for their insightful comments. I acknowledge Linnell B. Randall’s and Rachel Braley’s contributions to the sulfamethoxazole selection experiments and the BpeS genetic knockout, respectively. Special thanks to James M. Schupp, John Gillece, Rebecca Colman and Jordan Buchagen from the Center for Dangerous Pathogens, Pathogen Genomics Division of the Translational Genomics v Research Institute for their efforts and insight to analyze the whole genome comparative sequencing. I am grateful to Dr. Carolina Lόpez who very patiently worked with me when I first started my rotation in the Schweizer lab. Dr. Takehiko Mima was an irreplaceable source of knowledge specific to my research project. I am grateful for the time, advice and encouragement he gave me. In addition, I want to thank Kate Rhodes and Linnell Randall for their friendship, support and review of this dissertation, as well as, Drs. RoxAnn Karkhoff-Schweizer, Drew Rholl, Natalia Orosz, and Britta Wood, Sarah Eck, Amber Rico, Bryna Fitzgerald, Jen Higgins, and Jason Cummings among others, including many soccer ladies and A-Basin ski patrollers, for their encouragement and friendship over the years. Other members of the Schweizer lab both past and present have provided advice, encouragement and support over the years: Drs. Lily Trunck, Nah (Nawarat) Somprasong, Brian Kvitko, Jay (Piyatip Khuntayaporn), and Ian McMillian, Rachel Braley, Erin Breland, Marissa Castillo, Katy Quinn, Steven Bruckbauer, Laura Kapke, Franciszka Schneider, Katie Mladinich, Stephanie Lehman, et al. I owe special thanks to my family and friends for their love and support, as well as several remarkable educators and mentors who helped me step in the right direction; including Mrs. Pejouhy, Ms. Fisher, Mr. Brennan, Brenda Tessmann, Stephanie Phelps, Kathleen Treadway, Cari Beesley, and Drs. Eunice Froeliger, Joyce Oetjen, Alex Hoffmaster, Jay Gee, Robyn Stoddard, and Sandra Quackenbush. Finally, I would like to thank my husband, Jesse Dunham-Friel, for his patient, unwavering support and encouragement during these incredibly stressful and exhausting five years. I would not have been able to get through this without you. vi DEDICATION To my parents Josef & Janet Podnecky who taught me how to solve problems, to not be intimidated by challenging tasks, to never accept anything but my best, and to strive to constantly improve the world around me. vii TABLE OF CONTENTS Abstract ........................................................................................................................................... ii Acknowledgments........................................................................................................................... v Dedication ....................................................................................................................................
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