Syracuse University SURFACE Biomedical and Chemical Engineering - Dissertations College of Engineering and Computer Science 8-2013 Controlling Bacterial Persister Cells and Biofilms yb Synthetic Brominated Furanones Jiachuan Pan Follow this and additional works at: https://surface.syr.edu/bce_etd Part of the Chemical Engineering Commons Recommended Citation Pan, Jiachuan, "Controlling Bacterial Persister Cells and Biofilms yb Synthetic Brominated Furanones" (2013). Biomedical and Chemical Engineering - Dissertations. 68. https://surface.syr.edu/bce_etd/68 This Dissertation is brought to you for free and open access by the College of Engineering and Computer Science at SURFACE. It has been accepted for inclusion in Biomedical and Chemical Engineering - Dissertations by an authorized administrator of SURFACE. For more information, please contact [email protected]. ABSTRACT Bacteria are well known to obtain tolerance to antibiotics by forming multicellular structures, known as biofilms, and by entering dormancy and forming persister cells. Both mechanisms allow bacteria to tolerate antibiotics at concentrations hundreds to thousands of times higher than the lethal dose for regular planktonic cells of the same genotype. Persister formation increases in biofilms; thus, effective control of persister cells, especially those in biofilms is critically important to infection control. Over the past decades, a bacterial signaling system based on cell density, named quorum sensing (QS), has been found to regulate biofilm formation and, in Pseudomonas aeruginosa , the level of persistence. In this study, we characterized the effects of synthetic brominated furanones, a group of QS inhibitors, on the persistence of P. aeruginosa and Escherichia coli . Our results revealed that (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5 H)-one (BF8) can reduce persister formation in P. aeruginosa PAO1 and E. coli RP437 and restore the antibiotic susceptibility of isolated persister cells at growth non-inhibitory concentrations. In addition to planktonic persister cells, BF8 was also found to reduce persister formation in the biofilms of P. aeruginosa PAO1 and E. coli RP437. Study at the genetic level using DNA microarrays demonstrated that BF8 induced the gene mdaB in both strains and 7 genes encoding oxidoreductases in P. aeruginosa . In E. coli RP437 persister cells, BF8 was also found to repress the genes for synthesizing indole, a signaling molecule reported to induce persister formation in E. coli . Interestingly, although BF8 is a QS inhibitor, the QS signal 3-oxo-C12 - acyl homoserine-lactone also rendered the persister cells of P. aeruginosa PAO1 and its mucoid mutant PDO300 more sensitive to antibiotics. Furthermore, BF8 was found to have cidal effects on PDO300. Besides BF8, some other BFs were also found to restore the susceptibility of P. aeruginosa PAO1 persister cells to ciprofloxacin. Collectively, these results indicate that this group of QS inhibitors has promising activities to control multidrug tolerant persister cells; and there might be other bacterial targets of BFs in addition to QS inhibition. Key words: quorum sensing, biofilm, persister, antibiotic tolerance, brominated furanones. CONTROLLING BACTERIAL PERSISTER CELLS AND BIOFILMS BY SYNTHETIC BROMINATED FURANONES By Jiachuan Pan B.E. China Agriculture University, P. R. China, 2005 M.S. Chinese Academy of Sciences, P. R. China, 2008 DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemical Engineering in the Graduate School of Syracuse University AUGUST 2013 Copyright 2013 Jiachuan Pan All Rights Reserved ACKNOWLEDGEMENTS First of all, I sincerely appreciate the guidance of Dr. Dacheng Ren for leading me to this fascinating research and supporting my work throughout my Ph. D study. I appreciate his instruction with patience, knowledge and talent, which made me able to accomplish this work. I also thank our excellent collaborators, Dr. Jing An, Dr. Rebecca Bader and Dr. Yan-Yeung Luk for their kind help and access to facilities. I am grateful to Dr. Thomas K. Wood at Pennsylvania State University, Dr. Michael Givskov at University of Copenhagen, Dr. Matthew Parsek at University of Washington, Dr. John Parkinson at University of Utah and Dr. Robert Austin at Princeton University for sharing bacterial strains. I also thank Professor Frank Middleton and Ms. Karen Gentle at SUNY Upstate Medical University for helping with DNA microarray hybridization. I also want to thank the U.S. National Science Foundation (CAREER-1055644 and EFRI- 1137186) and Syracuse University for supporting my study. I wish to express my appreciation to my colleagues in the Ren lab, Dr. Shuyu Hou, Miao Duo, Huiqing Zheng, Xi Chen, Xiangyu Yao and Tagbo Herman Roland Niepa, Geetika Choudhary, Ali Adem Bahar, Fangchao Song, Shuyuan Ma, Li Zhang, Jing Wang, Henry Lars Peterson, Ginger Star Gunnip as well as Fei Cheng, Dr. Sijie Yang, Dr. Debjyoti Bandyopadhyay in Luk lab. It was a great pleasure to work with them. I wish to express my thankfulness to Jacques Lewalle, Dawn M. Long, Sabina Redington, Lynore de la Rosa, Kristin Lingo, Kathleen M. Joyce, Terrie M Monto, Neil Jasper and Karen Low for their help and their kindness. In my struggling days, their lovely and smiling faces are really encouraging. I also want to thank my friends: Camil Hodzic, Elizabeth Hughes, Burrell Jardin, Bill Dossert, John Kotlarz, Dick Chave and John Banas for their kindness to me. I wish to give my greatest appreciation to my family: Qin Li and Zheng Pan. I didn’t find chance to visit them back in China within 5 years, but their understanding is the biggest support during my study. Dedication: To my beloved late grandmother Shubin Wang and my late great grandfather Ru Zhu. v Table of Contents CHAPTER 1 ................................................................................................................................... 1 LITERATURE REVIEW ............................................................................................................... 1 1.1 Antibiotic resistance on the rise ............................................................................................ 2 1.2 Antibiotic resistance vs. antibiotic tolerance......................................................................... 3 1.2.1 Acquired resistance to antibiotics................................................................................... 3 1.2.2 Intrinsic tolerance in individual cells........................................................................ 4 1.3 Intrinsic tolerance based on multicellular behaviors............................................................. 7 1.3.1 Bacterial quorum sensing systems.................................................................................. 8 1.3.2 Quorum sensing and biofilm formation........................................................................ 11 1.3.3 Biofilm control by QS inhibitors.................................................................................. 12 1.4 Persister cells and control.................................................................................................... 18 1.4.1 Persister control ............................................................................................................ 18 1.4.2 Potential methods to reduce persisters.......................................................................... 19 References ................................................................................................................................. 22 Chapter 2....................................................................................................................................... 44 REVERTING ANTIBIOTIC TOLERANCE OF Pseudomonas aeruginosa PAO1 PERSISTER CELLS BY ( Z)-4-BROMO-5-(BROMOMETHYLENE)-3-METHYLFURAN-2(5 H)-ONE..... 44 2.1 Abstract ............................................................................................................................... 45 2.2 Introduction ......................................................................................................................... 46 2.3 Materials and Methods........................................................................................................ 48 2.3.1 Furanone synthesis. ...................................................................................................... 48 2.3.2 Bacterial strain and growth media................................................................................ 48 2.3.3 Persister isolation.......................................................................................................... 49 2.3.4 Effect of BF8 on AHL-mediated QS in the reporter strain V. harveyi BB886............. 50 2.3.5 Effect of BF8 on QS in PAO1...................................................................................... 50 2.3.6 Effects of BF8 on persistence of PAO1........................................................................ 50 2.3.7 Effects of D-glucose and D-mannitol........................................................................... 51 2.3.8 Effects of BF8 on antibiotic susceptibility of isolated persister cells........................... 51 2.3.9 Synergy with other antibiotics...................................................................................... 52 2.3.10 Effects of N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12 -HSL)................ 52 vi 2.3.11 Effects of BF8 on persister cells in established