UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE ANTIBIOTIC COMBINATION THERAPY AGAINST MULTIDRUG-RESISTANT STAPHYLOCOCCUS EPIDERMIDIS BIOFILMS AND BROADENING ANTIBIOTIC SPECTRUM USING POLYETHYLENIMINE A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By ANH KIM LAM Norman, Oklahoma 2020 ANTIBIOTIC COMBINATION THERAPY AGAINST MULTIDRUG-RESISTANT STAPHYLOCOCCUS EPIDERMIDIS BIOFILMS AND BROADENING ANTIBIOTIC SPECTRUM USING POLYETHYLENIMINE A DISSERTATION APPROVED FOR THE DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY BY THE COMMITTEE CONSISTING OF Dr. Charles V. Rice, Chair Dr. Si Wu Dr. Zhibo Yang Dr. Yihan Shao Dr. Mark Nanny © Copyright by ANH KIM LAM 2020 All Rights Reserved. Dedicated to my mom and all teachers, scientists, researchers, health care providers, policy makers, and others who are supporting science. Acknowledgements The completion of this graduate-school journey could not have been possible without the guidance and support of many people. Although my words are fallen short in expressing boundless gratitude for them whose names may not all be enumerated, I am immensely grateful! The most special thankfulness goes to my family, Quen Nguyen (Mẹ/Mother), Tai Lam (Ba/Father), Nhan Bunn (Cô-2/Aunt), and Travis Bunn (Daddle) for their strong and rooted support. My sincere thanks also arise for my cherished and valued friends who have become my adopted family: Melissa Hill, Anita Ly, Hannah Panlilio, and Dai & Ninh Nguyen! I feel deeply grateful to all the colleagues and members in the Rice Lab for each of them helped me in ways that I could never fully express: Erika Moen, Casey Wouters, Jennifer Pusavat, Beatrice Wilson, Andrew Neel, Tristan Haight, and Neda Heydarian. A special word of appreciation also goes to Prof. Bob (Robert) Brennan from the University of Central Oklahoma (UCO) for his assistance, guidance, and intellectual suggestions during my last project with human cells. I extend my thankfulness to my graduate committee members: Prof. Mark Nanny, Prof. Yihan Shao, Prof. Zhibo Yang, and Prof. Si Wu, for their thoughtful and valuable advice and suggestions. iv Thanks are also due to Dr. Preston Larson, Dr. Phil Bourne, Dr. Erwin Abucayon, Dr. Naga Rama Kothapalli, and Dr. Steven Foster for their help and co- operation in their research expertise. I will fail in my duty if I do not thank my undergraduate professors: Prof. Fakhrildeen Albahadily, Prof. Wei Chen, Prof. Hari Kotturi, and Prof. Lilian Chooback for seeding/nurturing the love of science in me and providing me every help to further reach my dreamed-goal. My heartfelt appreciation also goes to my partner, Sam Wood, for always being there, putting up with me, and encouraging/supporting me. Finally and most importantly, I immensely thank my idol and respectful mentor, Prof. Charles V. Rice, for his mentorship, guidance, support, encouragement, and constructive criticism at every stage of this journey. His keen personal interest (and dad jokes) inspired me even more for a positive work attitude in science and research and to work more diligently and efficiently. I cannot conclude my acknowledgements without my heartful gratitude to his wife, Toni Rice, for her priceless advice and encouragements beyond academic subjects. They – my lab parents – both have welcomed and treated me like family, and their generous supports have made my time here incredibly joyous! v Table of Contents Acknowledgements ........................................................................................................ iv List of Tables .................................................................................................................. xi List of Figures ............................................................................................................... xii Abstract ....................................................................................................................... xxii Chapter 1: Staphylococcus epidermidis, an opportunistic pathogen .......................... 1 Antibiotic Resistance .................................................................................................. 2 The Emergence of MRSE ........................................................................................... 5 The Cell Wall and The Gram Stain Characteristics ................................................... 7 β-Lactam Antibiotic Resistance ............................................................................... 11 The Need for New Strategies ................................................................................... 15 Chapter 2: 600-Da Branched Polyethylenimine (BPEI) and Its Effect on Human Inflammatory Responses .................................................................................. 17 Background ............................................................................................................... 17 Chemistry and Applications of BPEI ....................................................................... 18 600-Da BPEI’s Cytotoxicity and Biocompatibility .................................................. 19 Purpose of Experiment ............................................................................................. 20 Experimental Procedures .......................................................................................... 21 High Performance Liquid Chromatography (HPLC) ......................................... 21 Mass Spectrometry (MS) .................................................................................... 21 Fourier-transform infrared spectroscopy (FTIR) ................................................ 21 Effects of BPEI on human epithelia’s inflammatory cytokines ......................... 22 Results and Discussion ............................................................................................. 23 vi Instrumental Analyses ........................................................................................ 23 Inflammatory Response of Human Epithelial Keratinocytes exposed to BPEI . 26 Therapeutic Potentials of 600-Da BPEI ............................................................. 32 Conclusions .............................................................................................................. 33 Chapter 3: Synergy between β-lactam antibiotics and BPEI against planktonic MRSE ................................................................................................................. 34 Background ............................................................................................................... 34 Purpose of Experiment ............................................................................................. 36 Experimental Procedures .......................................................................................... 36 Materials ............................................................................................................. 36 In vitro Checkerboard Assays ............................................................................ 36 Growth Curves .................................................................................................... 37 Scanning Electron Microscopy (SEM) ............................................................... 37 Transmission Electron Microscopy (TEM) ........................................................ 38 Results and Discussion ............................................................................................. 39 Checkerboard assays indicate synergistic effects between BPEI and β-lactams 39 Growth curves confirm antimicrobial activity of oxacillin was restored ........... 42 Scanning and transmission electron microscopies confirm BPEI’s mechanism of action ...................................................................................................... 44 Conclusions .............................................................................................................. 49 Chapter 4: BPEI potentiates β-lactam antibiotics against MRSE biofilms ........... 52 Background ............................................................................................................... 52 Biofilms and Multidrug Resistance .......................................................................... 52 vii Purpose of Experiment ............................................................................................. 56 Experimental Procedures .......................................................................................... 56 Materials ............................................................................................................. 56 MBEC Assay ...................................................................................................... 57 Biofilm Disrupting Assay ................................................................................... 59 Minimum Biofilm Inhibitory Concentration (MBIC) ........................................ 60 Biofilm Kill Curve .............................................................................................. 60 Results and Discussion ............................................................................................. 61 Confirmation of MRSE Biofilms ....................................................................... 62 Efficacy of BPEI and β-lactams Against MRSE Biofilms ................................. 64 BPEI Possesses Biofilm-Disrupting Potential ...................................................