Mechanisms Underlying Antimicrobial Efficacies of Non-Thermal Dielectric Barrier Discharge (DBD) Plasma-Treated N-Acetyl Cysteine (NAC) Solution A Thesis Submitted to the Faculty of Drexel University by Utku Kürşat Ercan in partial fulfillment of the requirements for the degree of Doctor of Philosophy July 2013 ! ii! © Copyright 2013 Utku Kürşat Ercan. All rights reserved ! iii! ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere gratitude to my advisor, Dr. Suresh G. Joshi for his valuable guidance, support, encouragement and patience throughout my studies. Without his help this thesis wouldn’t be possible. I am sincerely grateful to my thesis committee members, Dr. Kenneth A. Barbee, Dr. Ahmet Sacan, Dr. Yinghui Zhong, Dr. Ari Brooks and Dr. Haifeng Ji, for accepting the invitation to become a member of my committee. Their valuable suggestions and scientific criticism are respectfully acknowledged. I would like to extend my gratitude to Dr. Banu Onaral, director of School of Biomedical Engineering, Science and Health Systems for her support and help starting from my first day in Drexel University. I also would like to thank my advisor Dr. Suresh G. Joshi, my co-adviser Dr. Kenneth A. Barbee, and Dr. Frank Ferrone, from Drexel University Scientific Integrity Office, for teaching me importance of scientific ethics and their encouragement and support to defend my rights. I would like to acknowledge members of Surgical Infection Research Laboratory, Dr. Bhaswati Sen, Nachiket Vaze and Adam Yost for their help and discussions on my thesis study and for scientific collaboration. I would also like to thank to Dr. Boris Polyak from Drexel College of Medicine, Department of Surgery for his support whenever I needed. Special thanks to Dr. Thomas Edlind of Microbiology and Immunology, Drexel University College of Medicine, Dr. Garth James from Center for Biofilm Engineering, Montana State University, graduate students Arben Kojtari and Josh Smith from Department of Chemistry, Drexel University for their help and contributions on this project. ! iv! I am grateful to Republic of Turkey, Ministry of National Education for their financial support during the whole period of my studies in the United States of America. Last but not the least; I would like to express my heart-felt gratitude to my wonderful family members for their understanding, encouragement and unconditional love and support throughout my entire endeavor. ! v! This thesis is dedicated to my loving family. ! vi! TABEL OF CONTENTS LIST OF TABLES………………………………………………………………………….xii LIST OF FIGURES………………………………………………………………………...xiii LIST OF ABBREVIATIONS……………………………………………………………..xvii ABSTRACT…………………………………………………………………………...…...xxi 1 CHAPTER I: INTRODUCTION AND BACKGROUND………………………………....1 1.1 Hospital Acquired Infections…………………………………………………..…1 1.1.1 Catheter Related Infections…………………………………………………3 1.1.2 Ventilator Associated Pneumonia…………………………………………..7 1.1.3 Surgical Site Infections……………………………………………………..8 1.2 Antisepsis, Disinfection and Sterilization……………………………………….11 1.2.1 Antisepsis Methods……………………………………………………….12 1.2.2 Disinfection Methods……………………………………………………..13 1.2.3 Sterilization Methods……………………………………………………..18 1.3 Plasma…………………………………………………………………………...22 1.3.1 Non-Thermal Dielectric Barrier Discharge (DBD) Plasma………………23 1.3.2 Plasma Chemistry………………………………………………………...26 1.3.3 Plasma Medicine………………………………………………………….30 1.3.3.1 Plasma Assisted Blood Coagulation………………………………...31 1.3.3.2 Plasma Applications for Cancer Therapy…………………………...32 1.3.3.3Applications of Plasma in Dentistry…………………………………33 1.3.3.4 Plasma Applications in Dermatology and Wound Healing…………34 1.3.3.5 Plasma Assisted Cell Transfection………………………………….35 ! vii! 1.3.3.6 Plasma-Eukaryotic Cell Interactions………………………………..36 1.3.3.7 Plasma Disinfection…………………………………………………36 1.4 Thesis Organization……………………………………………………………..38 2 CHAPTER II: ANTIMICROBIAL PROPERTIES AND INACTIVATION KINETICS OF NON-THERMAL DBD PLASMA-TREATED N-ACETYL CYSTEINE (NAC) SOLUTION…………………………………………………………………………………41 2.1 Introduction……………………………………………………………………..41 2.2 Materials and methods………………...………………………………………...45 2.2.1 Preparation of N-Acetyl Cysteine (NAC) Solution……………....…...45 2.2.2 Isolates and Cultivation of Microorganisms………..…………....……46 2.2.3 Plasma Treatment of NAC Solution…………………………………..46 2.2.4 Temperature and pH Measurements of Plasma-Treated NAC Solution……………………………………………………………….48 2.2.5 Treatment Time Dependent Antimicrobial Effect of Plasma-Treated NAC Solution………………………………………………………….49 2.2.6 Holding (Contact) Time Kinetics.………………..…………………...50 2.2.7 Effect of Initial Bacterial Load on Antimicrobial Efficiency of Plasma- Treated NAC Solution…………...……………….…………………...50 2.2.8 Antimicrobial Effect of Plasma-Treated NAC Solution on Planktonic Forms of Different Pathogens………………………………….……..51 2.2.9 Antimicrobial Effect of Plasma-Treated NAC Solution on Biofilm Forms of Different Pathogens ………………………...………………52 2.3 Results…………………………………………………………………………...56 2.3.1 Temperature and pH Measurements of Plasma Treated NAC Solution………………………………………………………………..56 2.3.2 Antimicrobial Effect of Plasma-Treated NAC Solution………………58 2.3.3 Effect of Holding Time on Inactivation…………...…………………. 58 ! viii! 2.3.4 Cell Density Dependent Inactivation by Plasma-Treated NAC Solution………………………………………………………………..59 2.3.5 Bactericidal and Fungicidal Effect of Non-Thermal Plasma Treated NAC Solution…………………………………………………………60 2.4 Discussion………………………………………………………………………65 3 CHAPTER III: CHEMICAL CHANGES IN PLASMA TREATED N-ACETYL CYSTEINE (NAC) SOLUTION AND THEIR EFFECT ON MICROBIAL INACTIVATION………………………………………………………………………......71 3.1 Introduction……………………………………………………………………..71 3.2 Materials and Methods…………………………………..……………………...74 3.2.1 Hydrogen Peroxide Detection…………………………………………74 3.2.2 Nitrite and Nitrate Detection………………………………………….74 3.2.3 UV-Visible Spectrum Analysis of Plasma Treated NAC Solution..….75 3.2.4 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis of Plasma Treated NAC Solution………………………………………………...76 3.2.5 Nuclear Magnetic Resonance (NMR) Analysis of Plasma Treated NAC Solution…………………………………………………………76 3.2.6 Antimicrobial Effect of acidic pH and Plasma-Generated Species.......76 3.3 Results…………………………………………………………………………...78 3.3.1 Hydrogen Peroxide Detection…………………………………………78 3.3.2 Nitrite and Nitrate Detection………………………………………….79 ! ! 3.3.3 UV-Visible Spectrum Analysis……………………………………….80 3.3.4 FT-IR Analysis of Plasma Treated NAC Solution……………………82 3.3.5 NMR Analysis of Plasma Treated NAC Solution…………………….84 3.2.6 Antimicrobial Effect of acidic pH and Plasma Generated Species.......85 3.4 Discussion………………………………………………………………………88 ! ix! 4 CHAPTER IV: ANTIMICROBIAL STABILITY AND TOXICITY OF PLASMA- TREATED N-ACETYL CYSTEINE (NAC) SOLUTION…………………………………95 4.1 Introduction……………………………………………………………………...95 4.2 Materials and Methods…………………………………..……………………...96 4.2.1 Delayed Exposure and Aging of Plasma-Treated NAC Solution…………………………………………………………96 4.2.2 Antimicrobial Effect of Components of Plasma-Treated NAC Solution……………………………………………………………….98 4.2.3 Effect of Vitamin E on Antimicrobial Properties of Plasma-Treated NAC Solution………………………………………………………..100 4.2.4 Repeated Exposures of Plasma-Treated NAC Solution…...………...101 4.2.5 Cytotoxicity of Plasma-Treated NAC Solution……………………...102 4.2.6 Systemic Toxicity of Plasma-Treated NAC Solution in Rats….……103 4.3 Results………………………………………………………………………….105 4.3.1 Antimicrobial Effect of Plasma-Treated NAC Solution Over the Time………………………………………………………..105 4.3.2 Antimicrobial Effect of Components of Plasma-Treated NAC Solution………………………………………………………………107 4.3.3 Effect of Vitamin E on Antimicrobial Properties of Plasma-Treated NAC Solution………………………………………………………..110 4.3.4 Repeated Exposures of Plasma-Treated NAC Solution………...…...110 4.3.5 Cytotoxicity of Plasma-Treated NAC Solution……………………...112 4.3.6 Systemic Toxicity of Plasma-Treated NAC Solution in Rats…….…114 4.4 Discussion……………………………………………………………………...116 5 CHAPTER V: CELLULAR RESPONSES IN E. coli UPON EXPOSURE TO PLASMA- TREATED NAC SOLUTION ……….………………………..…………………………..123 5.1 Introduction…………………………………………………………………….123 ! x! 5.2 Materials and Methods………………………………………………………...126 5.2.1 TBARS Assay (MDA Quantification): Detection of Lipid Peroxidation………………………………………………………….…….126 5.2.2 Live/Dead BacLight Bacterial Viability Assay……………………...127 5.2.3 Intracellular Glutathione Levels……………………………………..128 5.2.4 Nitrotyrosine Detection……………………………………………...129 5.2.5 Detection of DNA Damage………………………………………….130 5.2.6 DNA Microarray..…………………………………………………...132 5.2.7 Differential Expression of Selected Genes………………...………...134 5.3 Results…………………………………………………………………………136 5.3.1 Membrane Lipid Peroxidation……………………………………….136 5.3.2 Live/Dead BacLight Bacterial Viability Assay……………………...137 5.3.3 Intracellular Glutathione Levels……………………………………..138 5.3.4 Intracellular Nitrotyrosine Levels……………………………………139 5.3.5 Detection of DNA Damage………………………………………….140 5.3.6 Gene Expression Analysis Through Microarray…………………….141 5.3.7 Differential Expression of Selected Genes………………….……….142 5.4 Discussion……………………………………………………………………...143 6 CHAPTER VI: CONCLUDING REMARKS…………………………………………...152 6.1 Summary of the Research and Conclusion……………………………………152 6.2 Research Contributions………………………………………………………..154 6.3 Suggested Future Work………………………………………………………..156 6.3.1 Bacterial Resistance Against Plasma-Treated Liquids………………156 6.3.2 Determination of Inactivation Dose of Fluid Mediated Plasma ! 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Treatment…………………………………………………………….157