RAPID DETECTION AND CHARACTERIZATION OF MYCOBACTERIA USING MICROCHANNEL ELECTRICAL IMPEDANCE SPECTROSCOPY A Dissertation presented to the Faculty of the Graduate School at the University of Missouri - Columbia In Partial Fulfillment of the requirements for the Degree Doctor of Philosophy by ROLI KARGUPTA Dr. Shramik Sengupta, Dissertation Supervisor May 2017 The undersigned, appointed by the dean of the Graduate School, have examined the Dissertation entitled RAPID DETECTION AND CHARACTERIZATION OF MYCOBACTERIA USING MICROCHANNEL ELECTRICAL IMPEDANCE SPECTROSCOPY presented by Roli Kargupta, A candidate for the degree of Doctor of Philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. Dr. Shramik Sengupta, Department of Bioengineering Dr. Azlin Mustapha, Department of Food Science Dr. Shubhra Gangopadhyay, Department of Bioengineering Dr. Raghuraman Kannan, Department of Bioengineering Dr. Caixia Wan, Department of Bioengineering I dedicate this work to my family and friends who have been there alongside me. Special thanks to my parents, husband, brother, and my in-laws for all the guidance and support they have offered me through all these years. ACKNOWLEDGEMENTS I would like to express my heartfelt gratitude and appreciation to all the people who have helped me both professionally and personally to achieve my goals. My parents have been my pillar of strength. Without their inspiration and support, I would not have been able to reach my aim. I would like to thank my husband Dr. Sagnik Basuray for his constant support and guidance. My brother has always been my inspiration and played a crucial role in shaping my life. I would also like to thank my in-laws for their endless enthusiasm and encouragement. Also, I would like to thank my lab members and friends who have always been there for me. A special thanks to my advisor, Dr. Shramik Sengupta, for guiding and encouraging me. He has been an excellent mentor both on professional and personal fronts. He has been extremely patient and been a constant support during my doctoral studies. I would also like to thank all my committee members for agreeing to serve on my committee and providing me advice and guidance regarding my research work. The constructive discussions I had with my committee members helped me to complete my research studies. I will always be thankful to the University of Missouri for providing me the opportunity to conduct my research and would like to express my gratitude to all the professors, mentors and administrative staffs who have helped me through my journey of doctoral studies. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS……… ................................................................................................ ii LIST OF ILLUSTRATIONS ........................................................................................................ vii LIST OF TABLES ....................................................................................................................... xiv LIST OF ABBREVIATIONS ........................................................................................................xv ABSTRACT ................................................................................................................................ xvii Chapter 1 INTRODUCTION ....................................................................................................................1 1.1 MOTIVATION ..................................................................................................................... 1 1.2 HEALTHCARE-ASSOCIATED INFECTIONS (HAIS) ............................................................... 2 1.3 EMERGING INFECTIOUS DISEASES ..................................................................................... 5 1.4 CURRENT GOLD STANDARD FOR MICROORGANISM DETECTION ......................................... 8 2 COATINGS AND SURFACE MODIFICATIONS IMPARTING ANTIMICROBIAL ACTIVITY TO THE ORTHOPEDIC IMPLANTS ......................................................................10 2.1 INTRODUCTION ................................................................................................................ 11 2.2 BIOLOGY OF ORTHOPEDIC INFECTIONS ........................................................................... 13 2.3 APPROACHES TO PREVENT AND MANAGE ORTHOPEDIC INFECTIONS .............................. 15 2.4 ANTIMICROBIAL COATINGS ............................................................................................. 21 2.4.1 Coatings that prevent bacterial adhesion ................................................................... 22 2.4.1.1 Sacrificial coatings .............................................................................................. 22 iii 2.4.1.2 Adhesion-resistant coatings................................................................................. 23 2.4.2 Coatings that kill adherent bacteria ........................................................................... 26 2.4.2.1 Photoactive coatings ............................................................................................ 28 2.4.2.2 Metal-impregnated surface coatings ................................................................... 28 2.4.2.3 Surface-anchored antimicrobial peptides ............................................................ 31 2.4.2.4 Surface quaternary ammonium salts ................................................................... 32 2.4.2.5 Nitric Oxide ......................................................................................................... 32 2.4.3 Coatings that release antimicrobial agents ................................................................ 33 2.4.3.1 Antibiotic-loaded bone cement ........................................................................... 33 2.4.3.2 Polyhydroxyalkanoates ....................................................................................... 35 2.4.3.3 Mesoporous materials ......................................................................................... 36 2.4.3.4 Hydrogels ............................................................................................................ 36 2.4.3.5 Drug-eluting degradable coatings ....................................................................... 37 2.4.4 Coatings that accelerate osteocyte adhesion .............................................................. 43 2.4.4.1 Incorporation of growth factors........................................................................... 43 2.4.4.2 Bioglass ............................................................................................................... 44 2.4.4.3 Silicon carbide ceramics...................................................................................... 44 2.4.4.4 Coating with extracellular bone matrix (ECM)................................................... 45 2.4.5 Coatings with multiple modes of action ..................................................................... 45 2.5 CONCLUSIONS ................................................................................................................. 50 3 FOAMING BETADINE SPRAY AS A POTENTIAL AGENT FOR NON-LABOR- INTENSIVE PREOPERATIVE SURGICAL SITE PREPARATION .........................................52 3.1 BACKGROUND ................................................................................................................. 53 iv 3.2 MATERIALS AND METHODS ............................................................................................ 55 3.2.1 Overview ..................................................................................................................... 55 3.2.2 Bacterial cell culture .................................................................................................. 57 3.2.3 Selection of substrates ................................................................................................ 57 3.2.4 Selection of antimicrobial agents for testing .............................................................. 57 3.2.5 Evaluation of bactericidal activity ............................................................................. 58 3.2.6 Statistical analysis ...................................................................................................... 61 3.3 RESULTS .......................................................................................................................... 61 3.4 DISCUSSION ..................................................................................................................... 65 3.5 CONCLUSION ................................................................................................................... 65 4 MICROCHANNEL ELECTRICAL IMPEDANCE SPECTROSCOPY ...............................67 5 MYCOBACTERIA ................................................................................................................76 5.1 BACKGROUND ................................................................................................................. 76 5.2 TRANSMISSION ................................................................................................................ 77 5.3 STAINING ........................................................................................................................ 78 5.4 STRUCTURE ..................................................................................................................... 80 5.5 DIGESTION AND DECONTAMINATION .............................................................................