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
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© Copyright 2013
Utku Kürşat Ercan. All rights reserved
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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.
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This thesis is dedicated to my loving family.
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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 ! xi!
Treatment…………………………………………………………….157
6.3.3 Bacterial Persistence Against Plasma Generated Species…………...157
REFERENCES………………………………………………………………...………...159
VITA……………………………………………………………………………………….180
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LIST OF TABLES
Table 1: Examples of most widely used skin antiseptics………………………………...... 13
Table 2: Typical microdischarge parameters in a 1 mm discharge gap in atmospheric pressure air…………………………………………………………………………………..26
Table 3: Primary acid-base dissociation reactions in atmospheric pressure plasma-treated liquid system………………………………………………………………………………...30
Table 4: Combinations of detected concentrations of plasma-generated species that are tested on 107 CFU/ml E. coli. ………………………...…………………………………….86
Table 5: Colony-counting assay showing antimicrobial efficacy of accelerated aging of plasma-treated NAC solution. (Results are shown as log10(N) surviving bacteria)…………………………..………………………………………………………..107
Table 6: Acute toxicity assay: Findings on systemic administration of plasma-treated NAC solution. Comparative blood test results are shown with reference to normal values. H represents higher value of given parameter than the reference value and L represents the lower value of the given blood [parameter than the reference value. (AST: Aspartate transaminase, ALT: Alanine transaminase, ALP: Alkaline phosphatase, WBC: White blood cells)………………………………………………………………………………………..116
Table 7: Sequences of forward and revers primers used in this study…...………………..135
Table 8: Summary of microarray data, showing expression levels of some genes that play role in response to oxidative and nitrosative stress in E. coli...... 143
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LIST OF FIGURES
Figure 1: Potential routes of contamination in catheters. ………………….………………..4
Figure 2: CDC classification of surgical site infections……………………………………..9
Figure 3: Examples of natural and man made plasmas: (a) Lightning, (b) Aurora borealis (c) Dielectric Barrier Discharge (DBD) Plasma………………………………………………..23
Figure 4: Upper row depicts different planar arrangements and lower row depicts different cylindrical arrangements of DBD setup according to the position of dielectric material……………………………………………………………………………………...25
Figure 5: Process of microdischarge formation…………………………………………….26
Figure 6: Plasma assisted blood coagulation: (a) Untreated blood, (b) Blood droplet after 5 seconds of DBD plasma treatment………………………………………………………….31
Figure 7: Plasma assisted tooth whitening: Upper row; conventional method using 30% hydrogen peroxide, (a) before treatment and (b) after hydrogen peroxide treatment. Lower row; non- thermal plasma and 30% hydrogen peroxide combination, (c) before and (d) after plasma assited hydrogen peroxide treatment………………………………………………..33
Figure 8: Treatment of venous ulcer: with 2-minute daily treatment with MicroPlaSter®. (a) Before treatment, (b) 7th and (c) 11th treatment…………………………………….………..34
Figure 9: Chemical structure of N-acetyl cysteine molecule…………...………………….43
Figure 10: Technical drawing of custom made fluid holder. Sizes are given in inches………………………………………………………………………………………..47
Figure 11: Experimental set up and the schematic of quartz fluid holder………………….48
Figure 12: Zone of inhibition on bacterial lawn following the exposure of 1-minute (a), 2- minute (b) and 3-minute (c) plasma-treated NAC solutions treated with previously used smaller treatment chamber and newly designed scaled up treatment chamber. Arrows indicate inhibition zone caused by NAC solution, which is treated with scaled up treatment chamber………………………………………………………………….…………………..49
Figure 13: Biofilm growth on polyurethane catheter slices (24 hours of maturation)…………………………………………………………………………………..53
Figure 14: Reduction of XTT due to cellular respiration to form the orange formazan dye...... 55
Figure 15: Experimental design for different biofilm treatment approaches: (a) Drip flow biofilm reactor (DFR) set up, (b) Set up for biofilm treatment with plasma-treated, nebulized NAC solution………………………….…………………..………………….……………..56 ! xiv!
Figure 16: Temperature (a) and pH (b) change of non-thermal DBD plasma-treated NAC solution depends on the plasma treatment duration…………………..……………………..57
Figure 17: Plasma treatment time dependent antimicrobial efficiency of non-thermal plasma treated NAC solution on E. coli……………………………………………………………..58
Figure 18: Effect of holding (contact) time on antimicrobial efficacy of 3-minute DBD plasma-treated NAC solution ………………….....………………………………….……..59
Figure 19: Initial bacterial load dependent antimicrobial efficiency of 3-minute plasma- treated NAC solution………………………………………………………………….…….60
Figure 20: Plasma treatment time dependent antimicrobial efficiency of plasma-treated NAC solution on planktonic forms of different bacteria and fungus strains………………..61
Figure 21: Bactericidal effect of 3-minute plasma-treated NAC on antibiotic resistant bacteria strains……………………………………………………………………………....62
Figure 22: Plasma treatment time dependent antimicrobial efficiency of plasma-treated NAC solution on biofilm forms of different bacteria and fungus strains………………………………………………………………………………………..63
Figure 23: Antimicrobial efficiency of 3-minute non-thermal plasma-treated and nebulized NAC solution on E. coli biofilm…………………………………………………………….64
Figure 24: Biofilm maturation time dependent Antimicrobial efficiency of 3-minute plasma-treated NAC solution on P. aeruginosa mPA01 biofilms grown on drip flow reactor……………………………………………………………………………………….64
Figure 25: (a) Standard curve for hydrogen peroxide detection in plasma-treated NAC solution and (b) Treatment time dependent hydrogen peroxide concentration in plasma- treated NAC solution………………………………………………………………………..79
- - Figure 26: Treatment time dependent concentrations of (a) NO2 (Nitrite) and (b) NO3 (Nitrate) in plasma-treated NAC solution. Concentrations of nitrite and nitrate increase with plasma treatment time……………………………………………………………………….80
Figure 27: (a) UV-visible spectra of NAC solution treated for 1,2 and 3 minutes. (b) UV- visible spectra of NAC solution, showing the peaks depending on plasma treatment. Green dashed line shows the peak at 302 nm and red dashed line shows the peak at 332 nm..…...82
Figure 28: (a) FT-IR spectra of NAC solution before and after 3 minutes of plasma treatment. (b) Chemical bonds and groups on untreated NAC molecule that are indicated on FT-IR spectrum. (c) Chemical bonds and groups on cysteic acid that are indicated on FT-IR spectrum……………………………………………………………………………………..83
Figure 29: NMR Spectra of (a) untreated and (b) 3 minute plasma-treated NAC solution. (a) Peaks and corresponding H atoms in untreated NAC solution. Peak 1 corresponds to signal ! xv!
from D2O. (b) Arrows indicate increased multiplets correspond to cysteic acid…………………………………………………………………………………………..84
Figure 30: Antimicrobial effect of different acids at pH ~2.3 alone and in combination with
0.93 mM H2O2……………………………………………………………………………….85
Figure 31: Combination of nitrite and superoxide completely kills more than 6.5 log E. coli. Antimicrobial effect disappears when nitrate and hydrogen peroxide added nitrite- superoxide mixture………………………………………………………………………….87
Figure 32: Antimicrobial effect of different concentrations of peroxynitrite and corresponding concentrations of sodium hydroxide………………………………………...88
Figure 33: (a) Specific absorbance peak of S-nitroso-N-acetyl cysteine at 545 nm. (b) Structure of S-nitroso-N-acetyl cysteine. (c) Color change after 1 minute plasma treatment of NAC solution. Pinkish color formation is indicator of S-nitroso-N-acetyl cysteine (SNAC) in NAC solution after 1 minute non-thermal plasma treatment……….....………..91
Figure 34: Relationship between aging time and corresponding equivalent time for accelerated aging experiments performed at 37 and 50OC aging temperature……..……….98
Figure 35: Experimental method for separation experiment. Liquid part of plasma-treated NAC solution is evaporated and then condensed. Dried powder was reconstituted with both the equal and the half volumes of the evaporated volume of PBS and same volume of condensed liquid part. Antimicrobial effects of all reconstituted liquids were tested and their UV-vis spectra were collected……………………………...……………………...………100
Figure 36: Flow chart of the experimental procedure for repeated exposures of plasma- treated NAC solution………………………………………………………………………102
Figure 37: Antimicrobial effect of plasma-treated NAC solution that was stored at room temperature and +4OC…………………………………..………………………………….106
Figure 38: Antimicrobial activity and pH of plasma-treated NAC solution following drying and reconstitution process. Antimicrobial activity and pH of condensed liquid portion of plasma-treated NAC solution is also demonstrated………………………………………..108
Figure 39: pH and antimicrobial effect of NAC solution, where NAC powder first treated by itself and then dissolved in PBS. Final concentrations are 5 mM and 10 mM………....109
Figure 40: UV-visible spectra of dried and reconstituted plasma-treated NAC solution……...... 109
Figure 41: Effect of various concentrations of Vitamin E on inactivation rates of bacteria following exposure to plasma-treated NAC………………………………...……………..110
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Figure 42: Antimicrobial effect of 3-minute plasma-treated NAC solution following consecutive exposure to bacteria…………………………………………………………..111
Figure 43: UV-visible spectra of plasma-treated NAC solution in comparison with UV- visible spectra of ONOO- and ATL buffer after exposure to bacteria…………………….112
Figure 44: Cytotoxicity of plasma-treated NAC solutions on EA.hy926 human somatic umbilical vein endothelial cells after (a) 24 and (b) 48 hours of recovery period………………………………………………………………………………………113
Figure 45: Effect of the presence of blood on the antimicrobial effect of 3-minute plasma- treated NAC solution. Antimicrobial effect of 3-minute plasma-treated NAC solution diminishes in the presence of 25% blood………………………………………………….114
Figure 46: Comparison of UV-visible spectra of 3-minute plasma-treated NAC solution and reconstituted NAC powder. Note that, intensities of the peak at 302 nm are same for 3- minute plasma-treated NAC solution and 1.5:1 reconstituted NAC powder samples. Dashed line indicates the peak at 302 nm…………………………………………………………..119
Figure 47: MDA-TBA Adduct……………………………………………………………126
Figure 48: Cell membrane lipid peroxidation levels upon exposure of plasma-treated NAC solution and hydrogen peroxide solution to E. coli..………………………………………137
Figure 49: Cell membrane damage after plasma-treated NAC solution exposure to E. coli. Green cells are alive and red cells have leaky/compromised membrane. Scale bar shows 50 μm………………………………………………………………………………………….138
Figure 50: GSSG (oxidized form of glutathione) to GSH (reduced form of glutathione) ratio is shown as the indicator of intracellular oxidation. Note that GSSG/GSH increases drastically after exposure of 3-minute plasma-treated NAC solution…..…………………139
Figure 51: Intracellular nitrotyrosine concentration upon exposure to plasma-treated NAC solution and peroxynitrite solution as positive control. …………………………………..140
Figure 52: DNA extracted from bacteria, which were exposed to plasma-treated NAC solutions, was run on agarose gel. 3-minute plasma-treated NAC solution causes complete DNA disintegration...... 141
Figure 53: Differential expression of selected genes in E. coli after exposure to plasma- treated NAC solution…………………………………..………………….……..………..143
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LIST OF ABBREVIATIONS
HAI: Hospital acquired infection
HCAI: Healthcare-associated infection
ICU: Intensive care unit
MRSA: Methicillin-resistant Staphylococcus aureus
MSSA: Methicillin-sensitive Staphylococcus aureus or Methicillin-susceptible
Staphylococcus aureus
VAP: Ventilator associated pneumonia
IV: Intravenous
CVC: Central vein catheter
CR-BSI: Catheter related blood stream infection
CRB: Catheter related bacteremia
GI: Gastrointestinal
UTI: Urinary tract infection
SSI: Surgical site infection
CFU: Colony forming unit
CDC: Center for Disease Control and Prevention
PJI: Prosthetic join infection
VRE: Vancomycin resistant Enterococcus
UV: Ultraviolet
IR: Infrared
OPA: Orthophthaldehyde ! xviii! ppm: Parts-per million
PVPI: Povidone-iodide
AgSD: Silver sulfadiazine
PAA: Peracetic acid
ISO: International Organization for Standardization
LTSF: Low temperature steam formaldehyde
DNA: Deoxyribonucleic acid
EO: Ethylene oxide
DC: Direct current
AC: Alternate current
DBD: Dielectric barrier discharge
FE-DBD: Floating electrode dielectric barrier discharge
ROS: Reactive oxygen species
RNS: Reactive nitrogen species
APC: Argon plasma coagulation
RBC: Red blood cells
PIM: Powder injection molding
FGF: Fibroblast growth factor
MNC: Mononuclear cells
PCL: Poly(ε-caprolactone)
NAC: N-Acetyl Cysteine
ILD: Interstitial lung disease
ATCC: American Type Culture Collection ! xix!
NDM-1: New Delhi Metallo-Beta-Lactamase-1
TSB: Trypticase soy broth
TSA: Trypticase soy agar
YPD: Yeast extract-peptone-dextrose
ZOI: Zone of inhibition
XTT: (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)
PBS: Phosphate buffered saline rpm: Revolution per minute
DFR: Drip flow biofilm reactor
SOTS-1: Superoxide thermal source
UV-Vis: Ultraviolet – visible spectroscopy
NMR: Nuclear magnetic resonance
FT-IR: Fourier transform infrared spectroscopy
DMSO: Dimethyl sulfoxide
SNAC: S-nitroso-N-acetyl cysteine
FBS: Fetal bovine serum
DMEM: Dulbecco’s Modified Eagle Medium
IACUC: Institutional Animal Care and Use Committee
WBC: White blood cell
AST: Aspartate transaminase
ALT: Alanine transaminase
ALP: Alkaline phosphatase
PUFA: Polyunsaturated fatty acid ! xx!
GSH: Glutathione, reduced form
GSSG: Glutathione, oxidized form
8-OHdG: 8-hydroxydeoxyguanosine
TBARS: Thiobarbituric acid reactive substance
MDA: Malondialdehyde
3-NT: 3-Nitrotyrosine
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ABSTRACT
Mechanisms Underlying Antimicrobial Efficacies of Non-Thermal Dielectric Barrier Discharge (DBD) Plasma-Treated N-Acetyl Cysteine (NAC) Solution
Utku Kürşat Ercan Advisors: Suresh G. Joshi, MD, PhD and Kenneth A. Barbee, PhD
Hospital-acquired infections are a major challenge for healthcare professionals and patients, and have gained an increasing importance for overall public health. According to the Center for Disease Control and Prevention, hospital-acquired infections affect about 1.7 million people every year, and increase mortality, morbidity rates and healthcare costs.
Commercially available biocides used in medical practice are not sufficient to completely decontaminate sources of nosocomial infections, including sessile biofilms with embedded pathogens, and are cytotoxic. Furthermore, antibiotics remain ineffective against multidrug resistant virulent pathogens. Therefore, a new non-toxic, broad-spectrum antimicrobial agent is needed.
Plasma medicine is rapidly growing interdisciplinary field that explores novel solutions and applications in biology and medicine. Plasma is the fourth state of matter, consists of charged particles, ions, radicals, UV photons and excited molecules and atoms.
Antimicrobial effect of non-thermal plasma is characterized and the underlying mechanisms are investigated. Liquids, which are treated with non-thermal plasma acquire antimicrobial properties through the creation of plasma generated ROS and RNS and their interactions. N- acetyl cysteine (NAC) is an amino acid derivative and acquires antimicrobial property upon plasma-mediated chemical modifications.
In this thesis study, we introduce non-thermal, dielectric barrier discharge (DBD) plasma-treated NAC solution as a novel antimicrobial agent, which is non-toxic and has ! xxii! broad-spectrum activity against an array of multidrug resistant pathogens in their planktonic and biofilm forms. The present research also involves the detection of plasma-generated species and their products in NAC solution and their contribution in antimicrobial effect.
Damaging effects of plasma-treated NAC solution in E. coli cells are studied such as
DNA damage, cell membrane damage and intracellular oxidative and nitrosative stress. In addition, most commonly reported oxidative and nitrosative stress mediated gene activation/repression are studied in E. coli (a model organism). This study also investigates in vitro cytotoxicity and in vivo acute systemic toxicity in rats, and the stability of plasma- treated NAC solution as a part of development of novel antimicrobial solution.
This study provides a better understanding of non-thermal plasma activated NAC solution and the associated chemical changes, which lead to antimicrobial effect. Non- thermal plasma- treated NAC solution is thus being explored as a new antimicrobial compound for the control of the reservoirs of nosocomial pathogens. Thus this dissertation contributes new knowledge in the field of plasma medicine and infection control.
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1 CHAPTER I: INTRODUCTION AND BACKGROUND
1.1 Hospital-Acquired Infections:
Hospital-acquired infection (HAI), or healthcare-associated infection (HCAI) is nosocomial infection, which originates from any healthcare delivery setting such as; hospitals, retirement houses, ambulatory care setting, etc. An infection can be classified as hospital-acquired infection if it wasn’t present or in incubation period when the patient was admitted to a health care facility [1]. Most of the hospital-acquired infections are observed in patients before discharge from hospital. However depending on the duration of hospitalization it also can be observed after the discharge of the patient.
Hence infections that occur 48-72 hours after the admission of patient to a healthcare facility, and within 10 days after discharge from the hospital are referred as hospital acquired infections. However it further can be altered depending on type of infection agent, their incubation periods, etc. [2]. Hospital acquired infection, can be spread after the admission of an already infected patient to a health care facility, contact in between healthcare professionals and patients, contact with contaminated surfaces or medical devices [3]. The prevalence of hospital-acquired infections has increased over the past few decades in spite of utilization of several new infection control methods. In spite of number of patients hospitalized has decreased from 38 million to 36 million and the duration of hospitalization has reduced from 7.9 days to 5.3 days, the rate of hospital- acquired infections has increased from 0.72% to 0.98% in between 1970s and 2000s due to the higher average age of patients [4]. Also for pediatric patients numbers become more remarkable where the prevalence of hospital-acquired infections reaches up to 25% ! 2! in [5]. Hospital-acquired infection is a major challenge for clinicians and other health care providers and has assumed increasing importance for overall public health. It is one of the most common causes of health care complications and death in the United States.
According to the Center for Disease Control and Prevention, about 1.7 million infections occur annually, of which about 100,000 deaths are associated with hospital-acquired infection, and are responsible for increased rates of mortality, morbidity and medical costs [6]. Also hospital acquired infections increase average hospital stay and cost per patient, to existing $28-$36 billion burden annually [7]. With the development and widespread use of antimicrobial agents, including antibiotics, resistant microorganisms have emerged. Observations about antibiotic usage over the time have shown that, whenever a broader spectrum of an existing antibiotic or a new generation of antibiotic is introduced to for clinical use, either existing resistance increases or a new clinically important resistance arises. Increase in existing resistance or occurrence of new resistance limits the usage of currently available antibiotics and antimicrobial agents and makes infections a great challenge for clinicians and a threat for patients. Due to extensive and constant use of antibiotics and large population of microorganisms, hospitals and other health care stings are adequate microenvironments for developing resistance [8-9]. Antibiotic resistances has important influence on patient morbidity and mortality and in the United States more than 50% of all hospital-acquired infections are caused by antibiotic resistant bacteria [10-11]. Because of their high prevalence in hospitals, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, and
Staphylococcus epidermidis have become important members of the group of organisms that lead to nosocomial infections, and form biofilms on animate and inanimate surfaces ! 3! in hospitals. Biofilm is colonies of microorganisms, in which cells adhere to each other on a surface. In the biofilm form, cells excrete extracellular polysaccharides, which help to trap nutrients and prevent the detachment of cells [12]. The pathogen biofilms often act as a reservoir of contamination and infection and are likely disseminated by health care providers [13-15]. In hospitals, resistant bacterial strains favorably cause infections in particular sites such as urinary tract, respiratory tract, blood, surgical sites due to insertion of catheters and invasive medical interventions, joints following implantation of implants, and prosthesis [16-18]. One of the most remarkable agents of hospital- acquired infections is methicillin-resistant Staphylococcus aureus (MRSA). It is a common source of ventilator-associated pneumonia (VAP), catheter associated infections, joint infections and infective endocarditis. In addition to S. aureus, other
Staphylococcus species, E. coli, Enterococcus species, Pseudomonas aeruginosa,
Candida albicans, and Klebsiella pneumoniae are also resistant strains and important causative agents of hospital-acquired infections [16,19]
1.1.1 Catheter Related Infections: Intravenous (IV) catheters are the supplementary part of the patient care since 1935, when Dr. Forsmann inserted the first catheter. IV catheters are used to administrate IV drugs, nutrients, blood, to collect blood sample and to observe blood hemodynamics. While most IV catheters are relatively short (< 5 cm) and inserted to smaller veins, central vein catheters (CVC) are longer (> 15 cm) and are inserted to larger veins. Catheters are mostly inserted to patients who currently have an underlying health problem, which makes them more susceptible to infections [1].
Therefore catheter related blood stream infections (CR-BSI) (or also called as catheter related bacteremia (CRB)) are one of the most common challenges of catheter usage and ! 4! important part of HAIs. The source of the CR-BSI might be intrinsic, such as the catheter and/or catheter insertion equipment are already contaminated before the insertion, or might be extrinsic such as the catheter gets contaminated during insertion or during time it is placed on patient. Extrinsic contamination can occur due to natural flora of the patient or the spread of other microorganism from the hospital environment [20].
Figure 1 shows potential routes of contamination on IV catheter. Klebsiella,
Enterobacter and Pseudomonas species are most common sources of the intrinsic contamination and Staphylococcus species are the most common source of the extrinsic contamination.
Figure 1: Potential routes of contamination in catheters. (From: http://www.arrowintl.com/products/PressureCVC/help_prevent_infection/)
Not only IV catheters but also urinary catheters and gastrointestinal (GI) catheters may cause catheter related infections. However IV catheter-related infections are relatively more dangerous than GI and urinary catheter related infections because IV catheters are in direct contact with blood stream, and lead sepsis. It was shown that nutrition through ! 5! nasogastric tube is directly related with nosocomial infections especially in pediatric intensive care units [21]. Since the inserted catheter is a foreign material, the host reacts against the catheter by producing fibrinous film outer and inner surface of the catheter, which is an appropriate surface for microorganism to form biofilm and may lead to infection on the catheterization site. Sterile insertion technique, topical antimicrobial solutions and antimicrobial catheter lock solutions have shown efficiency against the catheter related infections. Citrate-based solutions are currently in use as catheter lock solution. A high concentration of citrate (~30%) is required for prevention of infection in catheters, and many times it leads to leakage in blood stream. Leakage of citrate solution in to bloodstream may cause calcium complexation and hypocalcaemia, which can be followed by ventricular arrhythmia and sudden death [22,23]. Another approach to prevent catheter related infections is to use antibiotic based lock solutions. However aminoglycoside lock solutions are not effective as desired levels against Staphylococcus species and may reach toxic serum levels in case of leakage to bloodstream. Also gentamicin heparin lock solutions are currently used but because of low solubility of gentamicin, it remains ineffective on biofilms and also leads the development of resistance. Minocycline can be considered as antimicrobial catheter lock solution but it is no longer available for IV use, and also leads bacterial resistance. Overall antibiotic- based lock solutions are not capable to eradicate catheter-related infections as anticipated since microbial colonization in catheters is in biofilm form and it is difficult to inactivate embedded biofilms [24]. Antimicrobial impregnated catheters (chlorhexidine, antibiotic, silver) are effective on prevention of catheter associated infection to some extend, nevertheless still there is a high rate of catheter related infection on patients with ! 6! catheterization for more than 5 days [25]. As the rate of urinary catheterization is relatively high (10% of all hospitalized patients), catheter related urinary tract infections
(UTI) are common type of hospital-acquired infections. The risk of catheter-related urinary tract infections increase proportional to duration of catheterization (daily 3-10% per day and 26% of patients catheterized for 2-10 days) and almost 100% of patients catheterized for 4 weeks end up with urinary tract infections (cystitis, pyelonephritis) and bacteriurira. 4% of such patients with catheter related UTI develop bacteremia and
13-30% die due to the infection [1,26]. Enteric Gram-negative organisms along with natural perineal flora including Enterococcus and Candida species are most common sources of UTIs. Under normal conditions urethral flora is tend to migrate into bladder, however these organisms are washed out due to urination. When the catheter is placed in to urinary tract, this flushing mechanism is not effective and natural flora causes colonization, which results in UTI. Also reflux from drainage bag increases the risk of infection [26,27]. In the urinary tract, microorganisms can be present in planktonic and/or biofilm forms. Even though planktonic forms of bacteria usually are not capable of starting infection, biofilm growth on the surface of the catheter is considered main risk factor for UTIs. Many antimicrobial agents are incapable to penetrate through biofilm and eradicate colonization and prevent UTIs. Prophylactic antibiotic treatment prior to catheter insertion may cause toxicity and doesn’t protect patients against antibiotic resistant bacteria. Because of resistant bacteria, there is no agreement on the duration of prophylactic and post infection treatment time, which makes UTIs a major challenge for health care professionals and threat for patients (like other HAIs) [28-29].
! 7!
1.1.2 Ventilator Associated Pneumonia: Nosocomial pneumonia is the infection of the parenchymal tissue of the lung and occurs within 48 hours of admission to hospital.
Even though there is no consensus for definition of the ventilator-associated pneumonia
(VAP), symptoms such as; progressive pulmonary infiltration with fever, leukocytosis and purulent tracheobronchial secretion within 48 hours following the endotracheal intubation is considered as VAP [30]. VAP comprises around 15% of HAIs, and it is the most common cause of deaths due to HAIs having > 75% of mortality rate [1]. Similar to catheters, since the endotracheal tube is a foreign material, patient’s body shows reaction that is followed with fibrinous development around the tube leading biofilm growth.
Also presence of the endotracheal tubing causes chemical and mechanical injury on the ciliated epithelium, which is the first-line of defense mechanism of the body in upper respiratory tract. Patients receiving mechanical ventilator support are severely ill, debilitated, and are susceptible to infections [5]. Consequently loss of protective mechanism and the presence of favorable environment for biofilm formation following aspiration of contaminated secretions, infection in the respiratory tract becomes inevitable. S. aureus, K. pneumonia, E. coli, Enterobacter species, P. aeruginosa,
Acinetobacter species and Candida species are common pathogens responsible for the
VAP [31]. Selective decontamination therapy that utilizes the administration of non- absorbable antibiotics through the oral cavity, with the short course of prophylactic IV antibiotic administration is capable to prevent VAP. But it is not preferentially in use except Netherlands due to potential risk of toxicity and development of resistance.
Nebulization of gentamicin has shown eradication of biofilm in endotracheal tube, and presented better prevention compared to systemic administration of cephalosporin but ! 8! with a risk of resistance [32]. Also effect of chlorhexidine oral rinse is less understood, and may lead to the colonization of gram-negative bacteria [33].
1.1.3 Surgical Site Infections: Surgical site infections (SSI) are the type of hospital- acquired infections that occur within 30 days of the surgery or within 1 year following implantation of a medical device, implant or prosthesis. Surgical site infections are identifiable 3 days following surgery, however identification of infections after implantation of a medical device takes longer time (weeks to months) [1,34]. It is determined that SSIs increase duration of hospital stay by 7-10 additional days, and increase mortality rate by 2-11 times and put additional average $3000 of cost per patient compared to other patients, who don’t have surgical site infections [35,36]. Skin incision eliminates the barrier function of the skin and makes the patients more susceptible to infections. Also presence of foreign materials such as suture, drainage tube on the surgery site, increases the risk of SSI. For instance, 105 CFU per gram of the tissue remarkably increase risk of SSI and the presence of silk suture on the surgery site makes 102 CFU per gram tissue capable to cause SSI [37]. Depending on the surgical procedure, SSIs can occur on skin, subcutaneous tissue, deep soft tissue (fascia and muscle) and organ. Also following joint replacement surgeries, prosthetic joint infection
(PJI) is an important part of SSIs with 0.5%-2% incidence, high mortality and morbidity rates and with additional cost [41]. Figure 2 depicts the CDC classification of surgical site infections [38]. ! 9!
Figure 2: CDC classification of surgical site infections. [38]
Depending on surgical procedure, usually isolates from surgical sites infections are polymicrobial and consist of both aerobic and anaerobic Gram-positive and Gram- negative microorganisms. S. aureus, S. epidermidis, Enterococcus species, E. coli, C. albicans from patient’s natural flora, are reported as common sources of surgical site infections. Due to excessive antibiotic use, MRSA and Vancomycin resistant
Enterococcus (VRE) are increasingly isolated from surgical site infections [39,40].
Prevention and control of surgical site infections can be classified as; preoperative patient care, intraoperative patient care and postoperative patient care. As apart of preoperative care, preoperative showers reduce bacterial load on patient’s skin however a definite correlation with reducing SSIs has not been established. Shaving surgery site is also common practice for SSI prevention [40,42]. Skin disinfection is the most common method of preoperative and intraoperative infection control. Chlorhexidine and povidone iodide solutions are used with or without alcohol for preoperative surgical site ! 10! preparation. Presence of alcohol has an ignition risk, where the electro surgery techniques are used and also chlorhexidine and povidone iodide are toxic to tissues.
Wound drains placed at surgical sites are used to expel accumulated blood and exudate, which are adequate environments for bacterial colonization. Drains also provide access to pathogens to surgery site, and therefore closed system wound drainage should be preferred [1,42,43]. Since early 1980s antibacterial surgical sutures are in consideration.
Triclosan coated surgical sutures are currently in use [44]. Triclosan [5-chloro-2 (2,4- dichclorophenoxyphenol)] is a broad-spectrum antimicrobial agent and has widespread usage in soaps, hand sanitizers, toothpastes, etc. Also its antimicrobial efficiency increases when it is combined with antibiotics [45]. Several in vitro studies have shown that triclosan coated sutures are capable to reduce methicillin susceptible and methicillin-resistant S. aureus as well as S.epidermidis and E. coli. Edmiston et al. have reported that triclosan coated sutures decrease adherence of MRSA, S. epidermidis, VRE and E. coli on the suture surface. However, excessive use of triclosan-coated suture caused emergence triclosan resistance pathogens such as P. aeruginosa. Animal studies revealed that antibacterial triclosan coated sutures can be effective against ~50% of pathogens responsible for SSIs [44,46,47]. Additionally triclosan has potential risk of toxicity. Fiss et al. has reported that triclosan sutures produce chloroform, which is a carcinogen, when it comes in contact with chlorine-treated tap water. Separate animal studies have shown that it has toxic effect on animals. For example triclosan exposure to tadpoles accelerated metamorphosis, and decreased sperm production in rats [48,49].
Even though in vitro studies showed efficient antimicrobial efficiency of triclosan coated sutures, several clinical studies reported that any of triclosan-coated sutures do not have ! 11! significant antimicrobial efficiency as compared to uncoated sutures and its antimicrobial efficiency remains unclear [50].
1.2 Antisepsis, Disinfection and Sterilization:
In the consideration of biocidal process, there is an important distinction between sterilization, disinfection and antisepsis. Therefore it is important to recall some fundamental definitions. The term decontamination is a general term that refers to either removal or inactivation of microorganisms that is combination of cleaning and disinfection or sterilization. Decontamination generally covers sterilization, disinfection and antisepsis. Antisepsis is defined as the inhibition of microorganisms in or on living tissues that is capable to reduce or prevent the risk of infection. Certain number of bacterial cells is required to cause infection. Disinfection is the process of reducing the number of viable pathogenic organisms, so they will not be able to cause infection.
Disinfection and antisepsis are used interchangeably, however antisepsis usually refers to the chemical disinfection of living tissue while disinfection is mostly applicable inanimate surfaces. Sterilization is the term that refers to destruction or removal of all living microbial forms of life (vegetative cells and spores) [1,8,12,51-53]. D-value is the indicator of the efficiency of an antimicrobial agent or disinfection or sterilization method. D-value is defined as the time required to achieve 1 log reduction (90%) of a given microbial population and expressed as;
! = ∆!!"#$/∆!
!!!"#$% = −1/! ! 12!
where as Δlog N is the log10 reduction of microbial population, ΔT is change in the time and m is the slope of the survival curve [8,12].
1.2.1 Antisepsis Methods: Living tissues are more delicate to be damaged compared to inanimate surfaces. Therefore antiseptics should fulfill the requirement of nontoxicity. In general antiseptics should have following features:
• Broad spectrum biocidal effect against bacteria, fungi and viruses
• Rapid inactivation effect
• Less or no irritation, damage or toxicity to the living tissue
• Persistency or residual biocidal effect [1,8,54].
Skin hygiene, pre-surgical disinfection of skin, treatment of wound infections and treatment of mucosal membranes are considered as the important applications for antisepsis. Pre-surgical disinfection of the skin is one of the most important steps in order to prevent surgical site infections [55,56]. Disinfection of skin prior to surgical procedure consists of cleaning of site of incision, followed by antiseptic application.
Currently alcohols, iodine tinctures, iodophors and chlorhexidine are the most widely used biocides for the purpose of skin disinfection. Also plain soaps and antimicrobial soaps provide physical removal and some inactivation of microorganisms [57,58]. Table
1 summarizes antimicrobial spectrum and toxicity of most widely used skin antiseptics.
! 13!
Table 1: Examples of most widely used skin antiseptics. [8]
Biocide Antimicrobial activity Persistence Toxicity
Alcohols Bactericidal, fungicidal, None Skin drying and virucidal irritancy
Chlorhexidine Bactericidal, fungistatic, some Yes Keratitis, irritancy, virucidal ototoxicity
Iodine and iodophors Bactericidal, fungicidal, Some Irritation and toxicity virucidal
Chloroxylenol Some bactericidal, fungistatic, Yes Mild irritancy, some virucidal allergic reactions
Triclosan Some bactericidal, fungistatic Yes Irritancy, potential carcinogenesis
1.2.2 Disinfection Methods: According to application of disinfection method, disinfection processes can be classified as physical and chemical disinfection [59].
Processes that utilize heat are the most widely used physical disinfection method.
In the heat disinfection method, the rate and the efficiency of the disinfection are directly dependent on applied temperature and exposure time [60]. Uniform exposure of heat to the material that is being disinfected is crucial. Heating can damage liquids, especially organic material containing heat sensitive materials. Also heat disinfection can cause the release of endotoxins, which are pyrogenic and resistant to disinfection temperatures
[61]. Nonionizing radiation (UV, IR radiation and microwaves) is another method of physical disinfection. UV lights are used for the disinfection of different materials including liquids and food. It is also cost effective method for area decontamination, such as biological safety cabinets, operating rooms, food processing areas and air conditioning systems [62]. Presence of organic and inorganic matter reduces the contact of UV radiation with surface and limits the efficiency of disinfection method. UV ! 14! irradiation doesn’t have any residual effect, which increases the risk of recontamination right after the application of UV irradiation. The adverse effects of UV involve burn and irreversible eye damage [63-64].
IR lamps are used as heat sources for the disinfection of heat sensitive materials.
Penetration issue is a remarkable limitation of nonionizing radiation disinfection methods [65]. Filtration provides physical removal of microorganisms rather than inactivation; however biocide integrated filters are also for more efficient disinfection
[66]. Filtration method can remove wide range of contaminants including microorganisms, biological molecules such as endotoxins and chemicals such as metals and salts, depending on the pore size of the filter. Filtration is considered as physical sterilization method as well [66].
Besides physical disinfection methods, a wide range of chemicals is currently in use for disinfection. Different acids are used as preservatives. Acetic acid, propionic acid, benzoic acid, citric acid and sorbic acid are acids used for antimicrobial purposes.
Acids possess mostly bacteriostatic and fungistatic properties at the concentration that they are used. At higher concentrations, which are needed for microbial inactivation, acids cause irritation and can cause allergic reactions, and corrosion of surfaces including metals. Some microorganisms are capable to use acids as carbon sources and able to degrade them [8,59]. Alkalis are able to break down proteins into peptides, and this property is utilized for the cleaning and disinfection of blood fractionating columns.
Strong alkalis are very harmful to skin and mucosal membranes, and corrosive to metal surface [8,59]. Reaction of alkalis with metal surfaces may lead to release of flammable gases. Glutaraldehyde, orthophthalaldehyde (OPA) and formaldehyde are the most ! 15! common aldehydes used as disinfectant, fixative and preservative [66]. Formaldehyde is used in fumigation process, and is sporicidal with extended contact time. Formaldehyde is environmental friendly; it breaks down to carbon dioxide and water through conversion to formic acid in few hours however it is highly toxic, irritant, mutagenic, carcinogenic, causes irreversible olfactory tissue damage, and even at very low concentrations (0.05 ppm) causes skin and eye irritation [52,67]. Glutaraldehyde is used for the disinfection of heat sensitive liquids and medical devices such as endoscopes.
Glutaraldehyde is highly irritant to skin, eye mucosal membranes and respiratory tract.
Even as low as 0.3 ppm concentration, glutaraldehyde causes respiratory tract irritation
[68,69]. Isopropanol, ethanol and n-propanol are common alcohols used as disinfectants.
Alcohols show the optimum antimicrobial effect in the presence of water, therefore 60-
70% alcohol solutions are in practice. Alcohols show rapid inactivation of bacteria (70% ethanol within 30 seconds) and relatively slower activity on fungus (70% ethanol >2 minutes). Even though alcohols are not sporicidal they have sporstatic activity [59,70].
Alcohols are flammable therefore precautions should be taken during handling and application, and cause drying and irritation on skin, and occasionally allergic reactions.
Extended use of alcohols may result cracking of particular delicate inanimate surfaces
[66]. Biguanides are the chemical compounds that contain C2H5N7 ligand, including chlorhexidine. Chlorhexidine is the most widely used biguanide not only in hospital environment, but also in antimicrobial wound dressings, antimicrobial soaps and mouth wash solutions. Biguanides are broad-spectrum bactericidal agents (except Pseudomonas species) but less virucidal [66]. Some bacteria have developed tolerance to biguanides through plasmid-mediated antibiotic resistance and it is speculated to trigger antibiotic ! 16! resistance. Even though not common, it is reported that chlorhexidine (also other biguanides) can cause anaphylactic shock and they are irritant to eyes and skin [71,72].
Halogens are group of elements that have high electronegativity and reactivity including, fluorine, chlorine, bromine and iodine. Iodine is the most widely used skin antiseptic, used as surgical scrubs, for the decontamination of skin prior to surgical interventions and for the treatment of wounded skin and burns. Chlorine is one of the most widely used chemical agent for water disinfection and responsible for the control and prevention of the transmission of earlier common diseases such as cholera and typhoid. Even though limitations, chlorine is used as wound and mucosal membrane antiseptics. One of the remarkable applications of chlorine is inanimate surface disinfection as in the form of hypochlorite, commonly known as bleach [74]. Chlorine is a reactive agent that has strong corrosion effect on metal surfaces. Repetitive application of chlorines cause skin irritation and hypersensitivity and high concentrations of chlorine is poisonous to humans. Reaction of chlorine with particular organic compounds leads to generation of carcinogenic by-products such as; chloroform [66]. Iodine solutions have toxic effect when their concentration exceeds 5% and they also cause irritation of skin and mucosal membranes. Iodine solutions have corrosive effect on metal and some plastic surfaces.
Disadvantages of iodine solutions are notably reduced with the use of iodophors (such as povidone-iodide (PVPI)) but there are some speculative studies reporting complications of PVPI such as; impairment of thyroid function [75]. Variety of metals (including heavy metals; such as mercury, lead, arsenic, cadmium) has been traditionally used for disinfection purposes, however accumulation of metals in the tissues, and high toxicity limited their applications. Currently copper and silver are mostly utilized metals for ! 17! disinfection [12]. Copper is bactericidal even at low concentrations and mostly fungistatic and fungicidal in to some extent, but it is not sporicidal (but have sporstatic effect at higher concentrations). Copper is used for water disinfection as an alternative to chlorine application and for the control of Legionella spread in the hospital pipelines.
Also copper is able to prevent biofilm formation, therefore it is used as preservative on
various surfaces including some medical devices [76]. Silver nitrate (AgNO3) and silver sulfadiazine (AgSD) are mostly used silver containing disinfection agents, in which silver is in Ag+2 active form. Silver sulfadiazine containing preparations are used to prevent infection on skin burns, mucosal membranes and eyes. Silver is also used for the recreational water disinfection. Silver impregnated dressings are currently used especially around catheters for the control and the prevention of HAIs. Silver has bactericidal and fungicidal effect at even low concentrations, but have very limited virucidal effect [74]. Overuse of silver may cause burns on skin, delayed wound healing, and although rarely reported severe toxicity. Also silver resistance in E. coli and some
Pseudomonas species has been reported [78]. Copper is toxic at higher concentrations and causes skin irritation. Some bacteria and fungus including E. coli and Candida species can generate resistance against copper [66]. Peroxygens are chemical compounds containing a single oxygen-oxygen bound or peroxide ion (O-O-2). Peroxides are important group of chemical disinfectants including hydrogen peroxide, chlorine dioxide and peracetic acid (PAA). Peroxygens are considered as environmental friendly since they decompose to water and oxygen. Hydrogen peroxide is used as antiseptic agent at
3% concentrations. Benzoyl peroxide is one of the most widely used organic peroxide for disinfection purpose and used for the treatment of acne [8,66]. Due to its rapid ! 18! biocidal activity, hydrogen peroxide is used as surface disinfectant with in combination of PAA. PAA is highly unstable; therefore it is used synergistically with acetic acid and/or hydrogen peroxide. In medicine, PAA is considered as an alternative to glutaraldehyde for low temperature disinfection of reusable medical devices such as endoscopes [79]. Hydrogen peroxide demonstrates bactericidal, fungicidal, virucidal and sporicidal effect at relatively higher concentrations. Hydrogen peroxide gas is reported effective against parasite eggs, prions, and bacterial endotoxins. PAA is more potent oxidizer compared to hydrogen peroxide and shows broad spectrum antimicrobial activity at relatively low concentrations (0.35%), however it is more irritating to skin, mucosal membranes and respiratory tract [59]. When hydrogen peroxide comes in contact with particular surfaces such as; brass, copper, cellulose-containing materials, degrades rapidly and loses its activity. High concentrations of hydrogen peroxide cause instantaneous burn on skin and mucosal membranes. Some studies are available reporting allergic reactions of hydrogen peroxide [75]. Other chemical disinfection agents include antimicrobial dyes, anilides and diamidines [8, 52].
1.2.3 Sterilization Methods: In contrast to disinfection, sterilization is the process that inactivates all living forms of microorganisms including bacterial spores. Theoretically, any disinfection method that is effective against broad range microorganisms and spores can be developed as sterilization method. But it is important to emphasize that sterilization is a validated process that provides totally alive microorganism-free product
[1,8,12]. Requirements for sterilization validation are determined in ISO 14937 standards, which are as follows: ! 19!
• Characterization of the biocidal agents(s), including safety,
antimicrobial efficacy and the effects of materials.
• Characterization of the sterilization process and any delivery
equipment.
• Definition of the sterilization process for a given application
• Definition of the product to be sterilized within the process
• Validation of the sterilization process for its intended use [80].
Physical sterilization methods utilize; steam sterilization, dry heat sterilization and ionizing radiation application and chemical sterilization methods utilize the application of epoxides, low temperature steam formaldehyde (LTSF), high temperature formaldehyde-alcohol, hydrogen peroxide, electrolyzed water and ozone.
Steam sterilization is one of the most widely used sterilization method that utilizes the gas form of water. In the stem sterilization process increased pressure in a fixed volume sterilization chamber, increases the boiling point of the water so higher sterilization temperatures can be achieved [8]. Steam sterilization process is made in controlled containers, which can resist high gas pressures required for sterilization, called autoclaves. In the steam sterilization process, steam is used as the source of heat, which causes rapid denaturation of proteins and nucleic acids. High pressure increases the penetration of steam through the protective layers of microorganisms, especially in bacterial spores. Steam sterilization is typically performed at 121OC and 15 psi for at least 15 minutes, but can also be performed at temperatures and pressures, where sterilization can be achieved faster. Steam sterilization method is mostly used for the ! 20! sterilization of reusable medical devices such as metal surgical equipment [66,81]. Dry heat sterilization methods include sterilization ovens and incineration. Incineration is performed by passing materials through a flame and used for the disposal of medical devices. Sterilization oven is basically a chamber where the heated air is fed during sterilization. Dry heat sterilization is an effective dehydrogenation method however it has limited application due to heat sensitivity of materials [81].
Radiation sterilization method utilizes ionizing radiation such as γ radiation which is capable to penetrate through all cellular protective layers.. 60Co and 137Cs are the most common sources of γ radiation for sterilization purposes. Ionizing radiation sterilization process can be applied to wide range of materials including, medical devices, prosthesis, pharmaceutical products, food and water. Nucleic acids, particularly
DNA, are the main target of ionizing radiation, where it causes mutations, DNA breaks causing cell death. Also ionizing radiation exposure results with intracellular free radical formation that enhances sterilization effect. Use of radiation sterilization is limited due to safety requirements and risks on human health [66,81].
Despite of the availability of various chemical disinfectants, limited amount of them have been developed for sterilization applications. Chemical sterilization methods are alternatives to physical sterilization methods, for the sterilization of materials that are sensitive to physical methods such as thermal sensitivity [8,66,81]. Ethylene oxide (EO or ETO) is a flammable gas that is used for the sterilization of heat sensitive medical equipment and pharmaceutical products. Because of its penetration ability, ethylene oxide is used for the sterilization of medical devices having long lumens such as endoscopes. Activity of EO is directly dependent on the presence of humidity and it ! 21! shows excellent sterilization features at 40-80% humidity. Its compatibility with wide range of materials makes ethylene oxide an appropriate solution for the sterilization of sensitive medical devices. Ethylene oxide shows its activity through alkylation process, where it is tend to replace any available hydrogen atom with hydroxylethyl radical that causes crosslinking of nucleic acids and proteins [66]. Ethylene oxide is reported as mutagenic, carcinogenic, teratogen and toxic at relatively low concentrations, and further exposure to ethylene oxide can cause skin, eye, mucosal membrane irritation, and sensitivity in the respiratory tract, which leads to allergic reactions and asthma [81].
Formaldehyde is used for sterilization in two different methods: low temperature steam formaldehyde (LTSF) and high temperature alcohol-formaldehyde sterilization. LTSF is used for the sterilization of reusable medical devices in health care facilities. High temperature alcohol-formaldehyde sterilization system combines the biocidal effect of steam penetration, formaldehyde and alcohols, and usually used for the sterilization of dental equipment [8,66,]. Sterilization of porous materials with formaldehyde requires further aeration due to formaldehyde absorbance, and formaldehyde can polymerize with the material. As previously described, formaldehyde is toxic, mutagenic and causes irritation in skin, mucosal membranes and eyes [67]. As previously mentioned, hydrogen peroxide is a potent oxidizing agent with a broad range of antimicrobial efficacy. Even though relatively low concentrations of hydrogen peroxide demonstrate bactericidal, fungicidal and virucidal effect, higher concentration (25-60%) is needed for sporicidal activity. In order to enhance sterilization effect, hydrogen peroxide can also be combined with different oxidizing agents such as PAA. In gaseous form lower concentration of hydrogen peroxide can provide sporicidal activity, therefore in heated or vacuum ! 22! chambers hydrogen peroxide is used as sterilization [81]. There is an increasing interest in hydrogen plasma gas sterilizers. In these systems, following the exposure of hydrogen peroxide in to the sterilization chamber, plasma discharge is generated which also generates ozone and reactive oxygen species contributing biocidal effects [83].
1.3 Plasma: Plasma is first described by Langmuir in 1930 as the fourth state of matter, which consists of ionized gas, charged particles, free electrons, electrically excited particles and other reactive species, and constitutes ~99% of the universe [84].
Depending on the gas temperature and electron temperature, plasmas can be classified as thermal and non-thermal or equilibrium or non-equilibrium plasma. In all plasmas that are generated in the presence of an electric field, electrons receive energy much faster than heavier particles. In non-thermal plasmas cooling of heavier particles, such as gas ions and uncharged atoms, is much faster than the energy transfer from heated electrons to their environment, which leads to the gas to remain at low temperature. As opposed to non-thermal plasmas, in thermal plasmas energy transfer from electrons to heavier particles is in equilibrium to energy transfer from heavier particles to the environment, where in heavier particle’s temperature is almost equal to electron temperature. In other
words, in non-thermal plasmas electron temperature is much higher than ambient gas (Te
>> Tg) and in thermal plasmas electron temperature is in equilibrium with ambient gas
(Te=Tg) [85]. Plasma can exist in nature or can be generated artificially under an electric field. Sun and lightning are examples of natural thermal plasmas, DC corona discharge is an example of thermal plasma generated by electric field, aurora borealis (also know as northern lights) is an example of natural non-thermal plasma, and plasma jet and ! 23! dielectric barrier discharge (DBD) plasma are examples of non thermal plasmas generated by electrical field (Figure 3) [85-88].
Figure 3: Examples of natural and man-made plasmas:(a) Lightning, (b) Aurora borealis (c) Dielectric Barrier Discharge (DBD) Plasma !
1.3.1 Non-Thermal Dielectric Barrier Discharge (DBD) Plasma: DBD is known for more than a century, and first introduced by Werner von Siemens for ozone production
[89]. DBD plasma setup employs one high voltage electrode and one grounding electrode, where at least one of the electrodes is insulated with a dielectric material.
Depending on the arrangement of electrodes, DBD plasma setup can be utilized as planar or cylindrical electrodes as shown in Figure 4 [94]. Most widely used dielectric materials in DBD plasma setup are glass, quartz and some ceramic materials. When AC is applied to DBD plasma setup, the dielectric material limits the current and prevents spark formation. When the generated electrical field is enough to break down gas, electron discharge starts between high voltage and grounding electrodes. The dielectric ! 24! constant, thickness of dielectric material and the derivative of applied voltage with respect to time determine gas breakdown point. Because of the low mean current in spite of high breakdown voltage in air at atmospheric pressure, DBD can be applied to living materials [90-91]. According to method of application, there are two types of plasma treatments: direct plasma treatment and indirect plasma treatment. In direct plasma treatment, material that is being treated acts as a grounding electrode, and electron discharge gets in direct contact with the material that is being treated. In indirect treatment a jet of plasma products (plasma after glow) get in contact with material that is being treated. For indirect treatment a metal mesh can be used in order to capture charged particles [92]. Besides direct and indirect plasma treatment, recently a new plasma treatment has been reported by our laboratory, which is called fluid-mediated plasma treatment. Fluid-mediated plasma treatment can be considered as a combination of direct and indirect plasma treatment, where the plasma treatment of liquid fits the definition of direct plasma treatment. In terms of transferring plasma generated product and species with fluid, it fits to the definition of indirect plasma treatment [93].
! 25!
Figure 4: Upper row depicts the different planar arrangements and lower row depicts the different cylindrical arrangements of DBD setup according to the position of dielectric material [94]
Floating electrode dielectric barrier discharge (FE-DBD) plasma is employed for the living tissue treatment, in which the living tissue serves as the counter, grounding electrode [95].
When an electrical field is applied between two electrodes, first an electron avalanche is initiated by a free electron, which produces a streamer. In few nanoseconds, the streamer forms a conducting channel of weakly ionized plasma. An intense electron current will flow through this channel till the local electric field is terminated due to the accumulation of charges on the dielectric surface. After the termination of electron current between the electrodes, high volume of vibrational and electronic excitation in the channel volume still exist along with charges deposited on the surface, which is called as microdischarge remnant Figure 5. The microdischarge remnant facilitates the formation of new microdischarge in the same location, when the polarity of the applied voltage changes. ! 26!
Figure 5: Process of microdischarge formation [96].
This phenomenon allows us to see single filaments in DBD. When microdischarges form at different location on all over the electrode surface, the discharge becomes uniform. In case of a microdischarge remnant is not totally dissipated before the formation of the next microdischarge is called memory effect [96]. Typical microdischarge parameters in a 1 mm discharge gap in atmospheric pressure air are shown in the Table 2.
Table 2: Typical microdischarge parameters in a 1 mm discharge gap in atmospheric pressure air. [97]
Lifetime 1-20 (100) ns Filament Radius 50-100 μm
Peak current 0.1 A Current Density 0.1-1kAcm-2
Electron density 1014-1015 cm-3 Electron energy 1-10eV
Total transported 0.1-1 nC Reduced electric field E/n=(1-2)(E/n)paschen charge
Total dissipated 5 μJ Gas temperature ~300 K energy
1.3.2 Plasma Chemistry: When an electrical field is applied to a gas, seed electrons are accelerated to obtain higher kinetic energy. Collision of these electrons with ambient gas ! 27! atoms and molecules leads to formation of positively charged atomic and molecular ions due to the electron loss from ambient gas. Electrons that unbound from ambient gas are rapidly accelerated by the applied electric field and take part in ionizing collisions with other atoms and molecules [88]. Chemical species generated by plasma treatment, play major role on the biological effects of non-thermal plasmas [85,98,99]. Plasma produces biologically active reactive oxygen species (ROS) such as hydrogen peroxide (H2O2),
! − 1 hydroxyl radical (OH ), ozone (O3), superoxide (O2 ), singlet oxygen (O2( Δg)) and
− − reactive nitrogen species (RNS) such as nitric oxide (NO), nitrite (NO2 ), nitrate (NO3 ), peroxynitrite (ONOO−) [93,100-102]. During the plasma generation, energy transfer from electron discharge region to ambient gas triggers various effects such as excitation and ionization. These processes cause O2, N2 molecules in the ambient air to raise their excited states and hemolytic braking of H-OH and O=O bonds resulting the formation of
H!, OH! and O! radicals. Chemical reactions induced by electron discharge can be summarized as follows:
Dissociation of oxygen molecules by electron impact leads to ozone formation:
- ! ! - e + O2 " O + O + e (1)
! M + O + O2 " O3 (2)
! O3 + M " O3 + M (3)
Reaction of free electrons in the plasma discharge is able to convert molecular oxygen to superoxide anion:
- - e + O2 " O2 (4) ! 28!
Electron impact on nitrogen molecules leads to formation of nitrogen atoms:
- - e + N2 " N + N + e (5)
Plasma discharge causes hydrolysis of water molecule and electron impact on excited water molecules leads to formation of hydroxyl radical:
+ - H2O " H + OH (6)
- ! ! e + H2O " H + OH (7)
Fixation of H! radicals on O2 and reaction of hydroxyl radical with ozone and hydrogen peroxide generate peroxyl radical:
! ! H + O2 + M " HO2 + M (8)
! ! OH + H2O2 " HO2 + H2O (9)
! ! OH + O3 " HO2 + O2 (10)
Reaction of two hydroxyl radicals and two peroxyl radicals separately yields to formation of hydrogen peroxide, also in thermal plasmas reaction of peroxyl radical with ozone yields to hydrogen peroxide and molecular oxygen:
! ! OH + OH " H2O2 (11)
! ! HO2 + HO2 " H2O2 + O2 (12)
In the discharge zone reaction between N2 and O2 yields the formation of NO with a series of reactions according to Zeldovich model: ! 29!
! N2 + O2 " 2 NO (13)
! ! ! N2 + O " NO + N (14)
! ! ! N + O2 " NO + O (15)
N! + O! " NO! (16)
In the gas phase dissociation of nitrogen and oxygen (reactions 1 and 5) yields to formation of nitric oxides, nitric acid nitrous acid:
+ - 3 NO2 + H2O " 2 H + 2 NO3 + NO (17)
2 NO " N2O4 + H2O " HNO3 + HNO2 (18)
Reaction between nitric oxide and peroxyl radical and/or the reaction nitrogen dioxide and hydroxyl radical give rise to the formation of peroxynitrous acid:
! NO + HO2 " ONOOH (19)
! NO2 + OH " ONOOH (20)
In aqueous media, dissociation of peroxynitrous acid and reaction between nitric oxide and superoxide form peroxynitrite:
ONOOH " ONOO- + H+ (21)
- - NO + O2 " ONOO (22)
Above mentioned reactions occur in gaseous media, however products of given reactions are capable to diffuse into liquid media [103-108]. After plasma treatment of a given ! 30! liquid in air, pH reduction is well known and documented consequence of plasma treatment. Lowering pH of liquid is attributed to H+ cation via dissociation of plasma generated acids in the liquid medium [86,93,109-111]. Table 3 shows the primary acid base dissociation reactions in atmospheric air plasma treated liquid systems.
Table 3: Primary acid-base dissociation reactions in atmospheric pressure plasma- treated liquid system[103]
Reaction pKa
- + HNO3 " NO3 + H <0
- + HNO2 " NO2 + H 3.3
HOONO " ONOO- + H+ 6.8
- + HO2 " O2 + H 4.7
- + H2O2 " HO2 + H 11.75
OH " O- + H+ 11.9
+ + H3O " H2O + H <0
- + H2O " OH + H 15.74
(Dissociation and electrolysis)
1.3.3 Plasma Medicine: Thermal plasmas are already in use in medicine for cauterization, tissue removal (ablation), cutting tissues, sterilization of thermally stable medical instruments [112-114]. Argon plasma coagulation (APC) technique is used to control bleeding during surgery and for the treatment of vascular abnormalities, such as hemangioma [115]. The Helium Thermal Ablation System (Helica) is used to ablate endometrial tissue in endometriosis. Another system called Coblation allows tissue ablation at lower temperatures with lesser damage on surrounding tissue [116-117]. Also ! 31! thermal plasma employing Argon gas is used as plasma scalpel that helps cutting tissue in a similar a way to cutting diathermy [118]. Thermal pulsing nitrogen plasma developed by Rhytec Inc. is used for cosmetic applications such as rejuvenation of skin, treatment of scars and wrinkles [119]. Besides thermal plasmas, there is an increasing interest on applications of non-thermal plasmas in medical field due to their possibility to use on living tissues. Following pages explain different applications of non-thermal plasma in medical field.
1.3.3.1 Plasma Assisted Blood Coagulation: Blood coagulation is a natural response of the body in order to establish homeostasis following blood vessel injury. Non-thermal
FE-DBD plasma treatment accelerates blood coagulation in vitro (Figure 5) [120]. It was reported that non-thermal FE-DBD plasma treatment enhances blood coagulation by activating some of coagulation proteins that leads rapid fibrinogen aggregation and by increasing Ca2+ ion concentration via a redox mechanism caused by hydrogen ions generated in plasma [120,121].
Figure 6: Plasma assisted blood coagulation: (a) Untreated blood, (b) Blood droplet after 5 seconds of DBD plasma treatment.
! 32!
In another study Kuo et al. reported enhanced blood coagulation following treatment with a portable non-thermal air plasma torch and the accelerated blood coagulation is attributed to stimulation of red blood cells (RBC) and platelets by plasma generated reactive atomic oxygen and NO. However, further studies are needed to confirm the contribution of NO to enhanced blood coagulation [122].
1.3.3.2 Plasma Applications for Cancer Therapy: Recent studies show that non- thermal plasma treatment can have a place in cancer therapy. Fridman et al. have reported that melanoma cells undergo necrosis at high doses (15 s at 1.4 W/cm2) of plasma treatment and apoptosis at lower plasma doses (5 s at 0.8 W/cm2) [123]. Also in a similar study Huang et al. reported deactivation of A549 human lung cancer cell line following 120 seconds of treatment with DBD plasma needle. Deactivation of A549 cancer cells is attributed to generation of ROS and RNS during plasma treatment [124].
Vandamme et al. have tested antitumor activity of non-thermal DBD plasma on U87MG malignant glioma and HCT-116 colorectal carcinoma. They have reported, accumulation of cancer cells in S phase, which is an indicator of decreased cancer cell proliferation and formation of DNA damage leading to apoptosis and multiphase cell cycle arrest due to plasma generated ROS. They have also reported reduction of tumor volume after in vivo plasma treatment of U87 human glioma xenograft on mouse [125,126]. Kim et al. have suggested a new cancer treatment method with non-thermal plasma, where they have shown that cultured TC-1 lung carcinoma cells are more sensitive to non-thermal
He (helium) microplasma jet treatment than cultured fibroblasts and undergo apoptosis
[127]. Furthermore in addition to deactivation of cancer cells due to apoptosis, it was shown that non-thermal helium plasma torch inhibits the migration and inhibition of ! 33!
SW480 colorectal cancer cells. Addition of O2 gas to He in the plasma jet improved the inhibition efficiency of migration and invasion of SW480, where these effects can be attributed to ROS generation during plasma treatment [128]. In brief, above-mentioned studies indicate that non-thermal plasma could be a promising method for cancer therapy in future.
1.3.3.3 Applications of Plasma in Dentistry: Conventional oral cavity and dental treatment methods have pitfalls such as pain, and long-term treatment duration [129].
Park et al. have demonstrated the use of non-thermal plasma helium jet combined with
30% H2O2 significantly increases tooth whitening compared to 30% H2O2 application by itself, which is conventional method for tooth whitening, without any thermal damage
(Figure 6) [130,131]. !
Figure 7: Plasma assisted tooth whitening: Upper row; conventional method with 30% hydrogen peroxide, (a) before, and (b) after hydrogen peroxide treatment. Lower row; non-thermal plasma and 30% hydrogen peroxide combination, (c) before, and (d) after plasma assited hydrogen peroxide treatment. [131] ! 34!
Microleakage of dental fillings reduces the stability of dental restorative treatments and invites repetitive filling procedures. Treatment of dental surface has demonstrated significant surface modification on dentin, which leads the deeper penetration and better mechanical stability via increased bonding strength of filling materials [132]. Treatment of powder injection molded zirconia (PIM) implants with non-thermal helium plasma jet has increased the hydrophilicity of implant surface without altering microtopoghraphy, which leads to the enhanced bone integration on implant surface, bone implant contact ratio, bone volume on implants and implant removal torque [133].
1.3.3.4 Plasma Applications in Dermatology and Wound Healing: In vitro studies have revealed that non-thermal plasma treatment has a potential to induce wound healing, by reducing the microbial colonization and direct effects on skin cells. The first clinical trial on wound healing by using non-thermal, indirect Argon plasma jet
(MicroPlaSter®), has shown that plasma treatment is capable to accelerate wound healing, by reduction of bacteria such as Klebsiella oxytoca and Enterobacter cloacae
[134,135]. Figure 7 shows the progress of venous ulcer wound with the treatment with
MicroPlaSter®
! 35!
Figure 8: Treatment of venous ulcer: with 2-minute daily treatment with MicroPlaSter®. (a) Before treatment, (b) 7th and (c) 11th treatment [134]
FDA has approved the plasma skin regeneration (PSR) technology, which is a thermal nitrogen plasma jet, for the treatment of wrinkles, superficial skin lesions, actinic keratosis, seborrheic keratosis and viral papilloma. Effects of PSR are attributed to controlled thermal damage due to rapid cooling of plasma that stimulates collagen, reduced elasticity production and the regeneration of photo-damaged skin [136,137].
Emmert et al. has reported that the DBD plasma treatment for 30 days, 1 min/day has significantly improved the healing of atopic eczema, where they have observed the reduction of S. aureus colonization on the skin with reduced itching [138].
1.3.3.5 Plasma Assisted Cell Transfection: The basis of the gene therapy is the introduction of the genetic material to the target cell via viral vectors, which may cause the virus-mediated adverse host response. Leduc et al. reported the transfection of hrGFP-II-1 plasmid in HeLa cells with the atmospheric pressure glow discharge torch by forming pores ranging between 4.8 to 6.5 nm without any DNA damage [139]. In a similar study Sakai et al. have transfected HeLa-S3, HT-1080 and MCF-7 cell lines with pEGFP-C1 plasmid using electric pulse-activated gas plasma generator, which increases membrane permeability by forming momentarily pores on the cell membrane [140]. ! 36!
1.3.3.6 Plasma-Eukaryotic Cell Interactions: Non-thermal DBD plasma treatment promotes cell proliferation at sub-lethal doses through the FGF-2 release from endothelial cells by creating sub-lethal membrane damage in a plasma dose dependent manner. Arjunan et al. have reported that plasma generated ROS and RNS contributes the sub-lethal membrane damage that leads to FGF-2 release, causing endothelial cell proliferation leading to angiogenesis [101,141]. Haertel et al. has demonstrated interaction of non-thermal plasma with immune cells. In this, study rat spleen mononuclear cells (MNC) were treated with surface DBD non-thermal plasma at atmospheric pressure in air and argon. They have shown that the treatment of MNC increases the ratio of B cells over the T cells and can alter surface characteristics and migration and proliferation of immune cells, which may trigger the sensitization of immune cells against tumors [142]. Furthermore oxygen DBD plasma treatment of poly(ε-caprolactone) (PCL) scaffold causes the surface modification. Plasma treatment of PCL scaffold increases the hydrophilicity of the scaffold, which improves the mouse osteoblast cell proliferation on the scaffold [143].
1.3.3.7 Plasma Disinfection: Plasma disinfection has become one of the most widely studied biological applications of non-thermal plasma since last decade. Recent studies have shown that different non-thermal plasma treatment arrangements including DBD, surface DBD, plasma jet, corona discharge, gliding arc discharges can inactivate wide range of microorganisms both in planktonic and biofilm forms in treatment time dependent manner [85,92,98,102,144-148]. Biofilm forms of microorganism are more resistant to disinfectants than their planktonic forms. However non-thermal plasma treatment shows excellent antimicrobial activity on biofilms as well. Alkawareek et al. ! 37! have shown total eradication of biofilms of Gram-positive bacteria (B. cereus and S. aureus) in 4 minutes and Gram-negative bacteria (E. coli and P. aeruginosa) in 10 minutes with non-thermal plasma jet containing 99.5% helium and 0.5% oxygen gas flow. [149] In another study, Joaquin et al. have reported eradication of C. violaceum biofilms in plasma treatment time dependent manner, where the total inactivation of biofilm was achieved after 60 minutes treatment with helium-nitrogen plasma jet [150].
Not only bacterial biofilms, but also fungal biofilms can be inactivated with plasma treatment. Direct treatment of C. albicans biofilms with surface microdischarge plasma for 10 minutes resulted with 6 log reduction of biofilm forming fungal cells [147]. In addition to bactericidal and fungicidal effects, also virucidal and prion inhibition effects of non-thermal plasma are demonstrated. Treatment of recombinant adenovirus
(AdeGFPLuc) with surface microdischarge plasma resulted 240 seconds resulted with the 6 log reduction of adenovirus. It was shown that plasma is able to both inhibit the replication of adenovirus after infecting HEK293 cells and inactivate adenovirus. The virucidal effect of plasma treatement is attributed to diffusion of plasma generated ROS and RNS to the medium, and not to the acidic pH or localized heating [151]. Elmoualij et al. have reported prion inactivation by treating stainless steel inserts contaminated with infectious 139A prion and non-infectious prion containing brain homogenate.
Following the 40 minutes treatment of steel inserts with N2-O2 cold plasma afterglow, treated and untreated steel inserts were implanted on mice brains and mice were observed over the time. After 555 days 4 out of 5 mice that were received plasma treated, infectious 139A prion containing inserts, survived. On the other hand, all mice that were received untreated, infectious 139A prion containing insert, died by the end of ! 38! the observation period [152]. In addition to plasma disinfection effect where the plasma or plasma after glow comes in direct contact with microorganisms, also antimicrobial effects through plasma treated materials is reported. Yorsaeng et al. have shown that
DBD plasma treatment of chitosan coated, natural rubber latex is able to inactivate S. aureus and E. coli. Their results have revealed that antimicrobial activity was obtained after 20 seconds DBD plasma treatment of 2% chitosan coated latex, followed by 3 hours exposure to bacteria [153]. Poor et al. have demonstrated that 3 minute of DBD plasma treated calcium alginate gels can efficiently inactivate wide range of microorganisms, including bacterial and fungal strains, in both planktonic and biofilm forms without any significant cytotoxicity on eukaryotic cells [154]. Plasma treatment of water and water-based solutions, including PBS and saline solution, demonstrated that liquid acquires antimicrobial properties. This method comprises treatment of the liquid with a plasma source, then exposure of the microorganism to treated liquid for particular time [93,102,110,155-157].
In general, we have revealed a method wherein liquid is first treated, then exposed to bacteria for antimicrobial efficacy as fluid mediated plasma treatment [93].
Details of antimicrobial effects of fluid-mediated plasma treatment and its mechanism of action will be discussed in following chapters of this thesis.
1.4 Thesis Organization: It is hypothesized that DBD plasma generates reactive oxygen and reactive nitrogen species in N-acetyl cysteine (NAC) solution, and also leads to chemical modification of N-acetyl cysteine molecule, which makes NAC solution a strong antimicrobial agent. ! 39!
The specific aims of this study are as follows:
• Determine antimicrobial efficacy and antimicrobial kinetics of DBD
plasma-treated N-acetyl cysteine (NAC) solution on representative
pathogens.
• Determine chemical species and products generated by non-thermal
DBD plasma in NAC solution, and their contributions on microbial
inactivation.
• Determine antimicrobial the stability and toxicity of non-thermal DBD
plasma-treated NAC solution.
• Understand the cellular mechanisms of microbial inactivation in E. coli
upon exposure to non-thermal DBD plasma treated NAC solution.
This thesis divided into 6 chapters as follows:
Chapter 2 describes the optimization of plasma system for treatment of NAC solution, its antimicrobial effect on most common sources of hospital acquired infections, including
Gram-positive organisms, Gram-negative organisms, multidrug resistant bacterial isolates and fungal pathogens in their planktonic and biofilm forms. The parameters such as holding time, and inactivation kinetics of plasma-treated NAC solution are also presented. Chapter 3 describes chemical analysis and the quantification of plasma- generated species in plasma treated NAC solution. In this chapter contribution of detected species toward antimicrobial effect is tested. The efforts are directed understand chemical mechanisms underlying antimicrobial properties of plasma-treated NAC solution. In Chapter 4 we have investigated the stability and retention of antimicrobial properties of plasma treated NAC solution over time. We have also presented both the ! 40! systemic toxicity (in vivo) and cytotoxicity (in vitro) of plasma-treated NAC solution to explore the possibility of clinical application as a an antimicrobial solution. Chapter 5 aims to describe effects of oxidative and nitrosative stress on E. coli cells caused by plasma treated NAC solution to understand their cellular responses. We investigated the status of protein oxidation and nitrosylation, lipid peroxidation on cell membrane and
DNA damage upon exposure to plasma-treated NAC solution. This chapter also presents the expression of some of the genes mediating oxidative and nitrosative stress management, which defend E. coli cells from inactivation. Chapter 6 concludes this thesis by giving a brief summary of results, which explain the both chemical and biological mechanisms of antimicrobial properties of plasma-treated NAC solution, provides original scientific contributions of, and the future directions to continue this research project. ! 41!
! 2 CHAPTER II: ANTIMICROBIAL PROPERTIES AND INACTIVATION KINETICS OF NON-THERMAL DBD PLASMA-TREATED N-ACETYL CYSTEINE (NAC) SOLUTION
2.1 Introduction:
Hospital-acquired infection (HAI) is a major challenge for clinicians and other health care providers and has assumed increasing importance for overall public health. HAI is one of the most common causes of health care complications and death in the United
States. According to the Center for Disease Control and Prevention, about 1.7 million infections occur annually, of which about 100000 deaths are associated with hospital- acquired infection, which increases rates of mortality and morbidity and medical costs
[6] and adds an estimated about $28–$336 billion in excess annual health care costs [7].
Because of their high prevalence in hospitals, Escherichia coli, Staphylococcus aureus,
Acinetobacter baumannii, and S. epidermidis have become important members of the group of agents leading to nosocomial infections, partly because they are able to form biofilms on animate and inanimate surfaces in hospitals. Biofilm is colonies of microorganisms, in which cells adhere to each other on a surface. In the biofilm form, cells excrete extracellular polysaccharides, which help to trap nutrients and prevents the detachment of cells [12]. Biofilm growth has a significant impact on human health.
Diseases such as cystic fibrosis, dental plaques, tuberculosis, etc. are directly related to biofilm formation. Medical devices, catheters, and implants have perfect surfaces for biofilm formation, which contribute to biofilms’ threat to human health [1,12]. As the pathogen biofilms often act as a reservoir of contamination and infection and are likely disseminated by health care providers. Soft tissue infections, catheter-related bacteremia, ! 42! urinary tract infections, hospital-acquired pneumoniaes, and open wound infections are often the result of the transmission of such nosocomial pathogens [13-15]. Although medically approved, commercially available surface disinfectants and hand washes are routinely used, many of them are not sufficient to disinfect surfaces, and they fail to inactivate a substantial percent of the pathogens found in biofilms [13,14,98]. Also contamination of surfaces with multidrug-resistant pathogens is one of the major risk factors in hospitals, and an effective disinfection agent is required to prevent the transmission of these pathogens [98,158,159]. It has been reported that recent biocides containing benzalkonium chloride, chlorhexidine gluconate, and triclosan used commercially in medicine, and powerful agents such as Acticide Bac-50 (Thor Group
Ltd, Canterbury, Kent, UK) and MediHex-4 (Medichem International, Sevenoaks, Kent,
UK), are not sufficient to completely decontaminate sources and reservoirs of nosocomial infections, including sessile biofilm with embedded pathogens [158, 159].
Therefore, a new compound or formulation is needed to prevent hospital-acquired infections that are more effective, rapid, less deleterious to surfaces being treated, and able to prevent transmission [98,158-162]. Recently, direct or indirect application of non-thermal, dielectric-barrier discharge (DBD) plasma is under investigation for its ability to disinfect and sterilize biomaterial or the surfaces likely to be damaged by thermal plasma [135]. Cold plasmas are mediated by an electric field; electrons receive energy faster than heavier ions, and reach thousands of degrees before the surrounding ions heat up. Consequently, ionized gas remains at a lower temperature (room temperature) instead of receiving energy from excited ions, and the heated ions can be efficiently cooled down [86]. It is also reported that plasma creates charged and ! 43! uncharged species, which in part are responsible for the disinfection and sterilization effects. The most notable species created by treatment with plasma that play a major role in the inactivation of bacteria are superoxide, hydrogen peroxide, singlet oxygen, OH- radicals, ozone, nitric oxide, peroxynitrite, ultraviolet light, and electrons [86,98].
N-acetyl cysteine (NAC) is an N-acetyl derivative of the amino acid L-cysteine and the precursor of the antioxidant glutathione. In NAC, the acetyl group is bound to
nitrogen atom of the cysteine group. Its chemical formula is C5H9NO3S and its molecular weight is 163.2 g/mol (Figure 9) [167]. NAC plays important roles in the biochemical pathways in cells such as reducing disulfide bonds in proteins, binding metals to form complexes and scavenging reactive species. From a chemical point of view, NAC is similar to cysteine, however the existence of the acetyl group reduces the reactivity of the thiol group. In contrast to cysteine, NAC is less toxic, more resistant to oxidation, and is more water-soluble [168]. It is used as a nutritional supplement and pharmacological drug. It is widely used as a mucolytic agent and as an antidote for paracetamol (acetaminophen) toxicity. Also NAC has preventive properties, and positive effect on the prognosis of interstitial lung disease (ILD), and radiocontrast induced nephropathy, and it is beneficial for reducing symptoms of schizophrenia and bipolar disorder [169-171].
Figure 9: Chemical structure of the N-acetylcysteine molecule ! 44!
In a previous study that was carried out in our laboratories the scavenging effect of different antioxidants including NAC along with Vitamin E, Histidine, Mannitol,
Pyruvate and Ascorbic Acid were tested on direct plasma treatment. All antioxidants have shown protection of bacteria except NAC. Bacteria that were treated in NAC solution showed higher inactivation rates than in negative control groups. These results directed our attention to the NAC solution since it has turned into a strong antimicrobial solution as a result of plasma treatment.
There are two types of plasma treatments: direct plasma treatment and indirect plasma treatment. In direct plasma treatment, the material that is being treated serves as a counter electrode and directly comes in contact with the electron discharge. In indirect plasma treatment, the plasma is generated remotely from the material that will be treated and the plasma-generated species are carried via flow of gas through a plasma discharge region [163-166]. In the present work, the treatment of NAC solution with non-thermal
DBD plasma is defined as “fluid mediated plasma treatment” that can be considered as a combination of direct and indirect plasma treatment. The fluid mediated plasma treatment can be considered as direct plasma treatment in terms of the treatment of NAC solution, where it comes to direct contact with electron discharge. At the same time, it can also be considered as indirect plasma treatment in terms of treatment of bacteria, where NAC solution acts as a vehicle that carries plasma-generated species to bacteria for disinfection purpose. Although, the physical and chemical compositions are different, and the exact differences are not yet known, both direct and indirect plasma treatment techniques are being investigated in infection control and surface sterilization. Joshi et ! 45! al. have previously demonstrated the unique technique of applying plasma to biological material, or to the surface to be sterilized, known as the dielectric barrier discharge
(DBD) plasma technique, wherein the temperature at the site of the plasma application is maintained close to room temperature and thus does not harm heat-sensitive or heat- labile surfaces, yet completely inactivates bacterial pathogens in both planktonic and embedded biofilm forms [98,144]. It was also demonstrated that the DBD plasma generates high amounts of reactive oxygen species (ROS), which rapidly inactivate pathogens in planktonic and embedded biofilm forms [144]. With the direct application of the DBD plasma treatment, the plasma effect is composed of physical and chemical species created in normal atmospheric air (no other gas or air under pressure) between the high voltage electrode and the surface contaminated with the biological material
(secondary electrode).
In the present study, we introduced the application fluid mediated plasma treatment and demonstrate how effectively the DBD plasma-treated NAC solution carries antimicrobial properties and inactivates bacterial and fungal pathogens. We evaluated the ability of the fluid-mediated, non-thermal plasma treatment to inactivate the most common nosocomial pathogens in their planktonic and biofilm (sessile) forms and its inactivation kinetics in terms of holding time, inactivation capacity, and temperature pH changes.
2.2 Materials and methods:
2.2.1 Preparation of N-Acetyl Cysteine (NAC) Solution: A 100 mM stock solution of
N-Acetyl cysteine (NAC) solution was prepared by dissolving NAC powder (Sigma, St ! 46!
Louise, MO) in 1X sterile phosphate buffered solution (PBS) and filter sterilized through a 0.2 μm filter; aliquots were stored at -20O C until they are used. A 5 mM working solution of NAC was freshly prepared by diluting 100 mM stock solution appropriately in 1X sterile PBS prior to subsequent experiments.
2.2.2 Isolates and Cultivation of Microroganisms: Escherichia coli (ATCC 25922),
Staphylococcus aureus (ATCC 25923), Acinetobacter baumannii (ATCC 19606),
Staphylococcus epidermidis (ATCC 12228), Enterococcus faecalis (ATCC 29212),
Pseudomonas aeruginosa (ATCC 484054), MRSA USA300 (BAA-1680) (Methicillin-
Resistant S. aureus), MRSA USA400 (BAA-1683) K. pneumoniae NDM-1 (New Delhi
Metallo-β-lactamase-1), and E. coli O157:H7 strains were purchased from American
Type Culture Collection (ATCC, Manassas, VA). Clinical isolate of A. baumannii was isolated from patients with cardiac device-related infections at Hahnemann University
Hospital. Reference strains of Candida albicans and Candida glabrata were kindly provided by Dr. Thomas Edlind of, Drexel University College of Medicine. All bacterial strains were grown overnight in trypticase soy broth (TSB) (Fischer Scientific
Inc., Pittsburgh, PA) medium at 37O C and stock culture plates were prepared on trypticase soy agar (TSA) (BD, Franklin Lakes, NJ) plates and stored at 4O C. Reference strains of Candida albicans and Candida glabrata were grown overnight in yeast extract-peptone-dextrose (YPD) medium (Fischer Scientific Inc., Pittsburgh, PA) at 37O
C, and stock culture plates were prepared on YPD agar plates and stored at 4O C.
2.2.3 Plasma Treatment of NAC Solution: To generate non-thermal plasma DBD plasma discharge, a custom made copper electrode (38 mm x 64 mm) was used. The surface of the copper electrode was covered with a 1- mm thick glass slide (50 mm x 75 ! 47! mm) as dielectric barrier (Fischer Scientific Inc., Pittsburgh, PA). For the electrical insulation, the electrode was covered with waterproof bath silicone. Also a custom- made quartz fluid holder (fluid contact area sizes: 32 mm x 57 mm) that is capable to hold up to 1.8 mL of fluid with maximum 1 mm of fluid column was designed (Figure
10 and 11).
Figure 10: Technical drawing of custom-made fluid holder. Sizes are given in inches.
However in the present study, the fluid treatment volume was fixed to 1 mL where the average liquid column was 0.6 mm. The discharge gap between the high voltage electrode and the fluid holder was fixed at 2 mm. 5mM NAC solution was treated with non-thermal plasma for 1, 2, and 3 minutes for the assessment of antimicrobial effect. ! 48!
Figure 11: Experimental set up and the schematic of quartz fluid holder
Non-thermal DBD plasma discharge was generated by applying alternating polarity pulsed voltage between an insulated high voltage electrode and quartz fluid holder filled with 1 ml NAC solution. The voltage and current were measured using a voltage probe (North Star PVM-4, 100 MHz bandwidth) and a current probe (Pearsons
Model 2878) respectively. The signals were recorded with an oscilloscope (TDS 5052B,
Tektronix Inc). The pulse waveform had a 5 μs pulse duration at 31.4 kV and 1.5 kHz where 0.29 W/cm2 power density was generated.
2.2.4 Temperature and pH Measurements of Plasma-Treated NAC Solution: In order to determine plasma treatment dependent temperature changes in NAC solution, temperature was measured before and after plasma treatment by using an ultrasensitive
K-type thermocouple (Thermo Works, Lindon, UT, USA). Similarly, to measure pH change in plasma treated NAC solution after plasma treatment, an ultrasensitive pH meter (Thermo Fisher Scientific, Waltham, MA) was used. ! 49!
2.2.5 Treatment Time Dependent Antimicrobial Effect of Plasma-Treated NAC
Solution: In order to determine the required plasma treatment time of NAC solution for the desired inactivation rate, 1 ml of NAC solution was treated with non-thermal DBD plasma at 31.4 kV and 1.5 kHz for 0.5, 1, 2, 3 and 5 minutes. E. coli (ATCC 25922) was used as the model organism and zone of inhibition (ZOI) assay was used to qualitatively evaluate bacterial inactivation. In the zone of inhibition assay, 1 ml of overnight grown culture was plated and spread on TSA plates and held under the biological safety hood until excess liquid evaporated. Once the plates are dried, 50 μl droplets of NAC solutions, treated for various time points, were dripped on plates. Plates were incubated at 37O C for 24 hours. Following incubation of plates we have compared the plasma treatment time dependent inhibition zones with the inhibition zones (Figure 11) that were obtained by the treatment of 100 μl NAC solution, with a smaller electrode in a smaller treatment chamber, that we previously utilized in our laboratory [144].
Comparison of inactivation zones was taken in consideration in order to determine required plasma treatment time points (1, 2 and 3 minutes) that were used for the quantification of inactivation rates with colony counting assay by exposing E. coli
Figure 12: Zone of inhibition on bacterial lawn following the exposure of 1 minute (a), 2 minute (b) and 3 minute (c) plasma-treated NAC solution with previously used smaller treatment chamber and newly designed scaled up treatment chamber. Arrows indicate inhibition zone caused by NAC solution, which is treated with scaled up treatment chamber. ! 50!
2.2.6 Holding (Contact) Time Kinetics: Holding time is defined as the time that microorganisms come in contact with plasma treated fluid. E. coli was used as model organism for optimization of holding time. To evaluate the effect of holding time on inactivation rate, 3-minute plasma-treated fluid was exposed to bacteria for 0, 1, 2, 3, 5,
10 and 15 minutes. Following given holding time periods bacteria were diluted in 1X sterile PBS solution and colony counting assay was performed as previously described.
2.2.7 Effect of Initial Bacterial Load on Antimicrobial Efficiency of Non-Thermal
Plasma-Treated NAC Solution: As opposed to previous experiments where 107
CFU/ml bacteria exposed to plasma treated NAC solution, 5x107, 108, 5x108 and 109
CFU/ml of initial concentrations of bacteria were exposed to plasma treated NAC solution. E. coli was used as model organism to determine initial concentration dependent inactivation rates. In order to obtain 5x107, 108, 5x108 and 109 CFU/ml concentrations of bacteria, overnight grown culture’s optical density was adjusted to 0.2 at 600 nm to have 107 CFU/ml bacterial concentration. Then bacterial suspension with
107 CFU/ml concentration was spun down at 8000 rpm for 10 minutes, supernatant was removed and concentrated appropriately by suspending bacteria pellet less the volume of the TSB medium to increase bacterial concentration. Bacteria suspension with different initial concentrations were mixed with the same volume of plasma treated NAC solution
(50μl: 50μl) and held for 15 minutes. Following holding time bacteria were diluted appropriately in 1X sterile PBS solution and plated on TSA plates and incubated at 37O
C for 24 hours in order to evaluate inactivation rates.
! 51!
2.2.8 Antimicrobial Effect of Plasma-Treated NAC Solution on Planktonic Forms of Different Pathogens: In order to determine antimicrobial efficacy of non-thermal
DBD plasma treated NAC solution on planktonic forms of various pathogens, colony counting assay was used. Bacteria colonies were collected with 10 μl inoculation loop from stock plates and inoculated into 10 ml of TSB medium, vortexed gently and incubated overnight at 37O C. Following incubation, the optical density of bacteria culture was adjusted to 0.2 at 600 nm, where the culture’s concentration becomes 107
CFU/ml, with appropriate dilution in TSB. Cultures with 107 CFU/ml concentration were exposed to equal volume of NAC solutions treated for 1,2, and 3 minutes (50 μl:50 μl) and held together for up to 15 minutes. MRSA USA300, MRSA USA400, K. pneumoniae NDM-1 and clinical isolate of A. baumannii were exposed to equal volume
of NAC solution treated only for 3 minutes. 3% H2O2 (Sigma Aldrich, St. Louise, MO) and 1X sterile PBS solution were used as positive and negative controls respectively.
Following 15 minutes of holding time, cultures were appropriately diluted in 1X sterile
PBS, plated on TSA plates and incubated at 37O C for 24 hours. After incubation surviving colonies on TSA plates were counted manually. All plates were incubated for
48 more hours in order to assure bacteria hadn’t developed dormancy. Similarly fungus colonies were collected with 10 μl inoculation loop from stock plates and inoculated into
10 ml of YPD medium, vortexed gently and incubated overnight at 37O C. Following incubation, the optical density of fungus culture was adjusted to 0.38 at 520 nm where, culture’s concentration becomes 107 CFU/ml, with appropriate dilution in YPD medium.
Fungus cultures with 107 CFU/ml concentration were exposed to equal volume plasma
treated NAC solutions (50 μl:50 μl) and held together for 15 minutes. 3% H2O2 (Sigma ! 52!
Aldrich, St. Louise, MO) and 1X sterile PBS solution were used as positive and negative controls respectively. Following 15 minutes of holding time, cultures were appropriately diluted in 1X sterile PBS, plated on YPD agar plates and incubated at 37O C for 24 hours. After incubation surviving colonies on YPD agar plates were counted manually.
All plates were incubated 48 hours more in order to assure bacteria hadn’t developed dormancy. Colony numbers acquired form colony counting assay were multiplied by dilution factor, which is the measure of dilution amount made from initial concentration to the plated concentration. Obtained results were converted to logarithmic scale on base
10.
2.2.9 Antimicrobial Effect of Plasma-Treated NAC Solution on Biofilm Forms of
Different Pathogens: The antimicrobial efficiency of plasma treated NAC solution biofilm forms of organisms were tested by using three different approaches. In the first approach biofilms of different strains, including fungal and bacterial strains, were grown on polyurethane catheter slices and exposed to plasma treated NAC solution. In the second approach biofilms of E. coli were grown on the bottom of the 12 well plate and treated with nebulized plasma treated NAC solution. In the third approach biofilms of P. aeruginosa PA01 were grown on air-liquid interface by using drip flow reactor system in order to mimic natural biofilm formation such as found in the respiratory tract and inside of catheters; and grown biofilms were treated by circulating plasma treated NAC solution through biofilm grown surface. In order to determine antimicrobial efficacy of non-thermal DBD plasma treated NAC solution in the first approach, XTT assay
(Molecular Probes Inc., Grand Island, NY) was used. Biofilms were grown on 1 mm thick polyurethane catheter slices. Catheters were cut to 1 mm thick slices and held in 10 ! 53!
ml of 70% ethanol for 30 minutes then washed in sterile dH2O and kept under UV light for 30 minutes for sterilization purposes. Overnight cultures of bacteria were prepared as previously explained. 100 μl of bacterial overnight culture was added into 10 ml of
TSB medium with 100 μl of 50% of glucose solution to yield 0.5% (w/v) final concentration in order to enhance biofilm formation. Sterilized catheter slices were transferred in 24 well plates. 700 μl of biofilm culture suspension was added on catheter slices and incubated at 37O C for 24 hours (Figure 13). Following incubation medium containing floating bacteria was removed and each well was washed with 1X sterile PBS solution in order to remove non-biofilm forming bacteria. Then 700 μl of plasma treated
NAC solution was added on biofilms that are grown on catheter slices and held for 15 minutes. Following holding time plasma treated NAC solution was removed, biofilms were washed with 1X sterile PBS solution and 700 μl of XTT reagent was added and incubated at 37O C for 2 hours. After incubation 100 μl of reacted XTT was transferred into 96 well plates and samples were read at 492 nm with a spectrophotometer (Thermo
Scientific, Waltham, MA).
Figure 13: Biofilm growth on polyurethane catheter slices. (24 h. maturation) ! 54!
For fungal biofilm experiments, overnight cultures of fungus strains were prepared as previously explained. 100 μl of fungal overnight culture was added into 10 ml of YPD medium with 100 μl of 50% of glucose solution to yield 0.5% (w/v) final concentration in order to enhance biofilm formation. Sterilized catheter slices were transferred in 24 well plates. 700 μl of biofilm culture suspension was added on catheter slices and incubated at 37O C for 72 hours by replacing medium at every 24 hours. Following incubation medium containing floating fungus was removed and each well was washed with 1X sterile PBS solution in order to remove non-biofilm forming fungus. Then 700
μl of plasma treated NAC solution was added to biofilms grown on catheter slices and held for 15 minutes. Following holding time plasma treated NAC solution was removed, biofilms were washed with 1X sterile PBS solution and 700 μl of XTT reagent was added and incubated at 37O C for 2 hours. After incubation 100 μl of reacted XTT was transferred in to 96 well plates and samples were read at 492 nm with a
spectrophotometer (Thermo Scientific, Waltham, MA). 3% H2O2 (Sigma Aldrich, St.
Louise, MO) and 1X sterile PBS solution were used as positive and negative controls respectively for both bacterial and fungal biofilm experiments. Negative control survival rates were fixed as 100% survival rate and survival of treated samples were normalized according to negative control groups.
XTT (2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay is a quantification method based on the respiratory metabolic activity of viable cells.
XTT is a tetrazolium salt, which is based on the modification of the yellow tetrazolium salt XTT to form an orange formazan dye by metabolic active cells (Figure14) [167]. ! 55!
Cellular! Respiration!
Figure 14: Reduction of XTT due to cellular respiration to form the orange formazan
dye.
The stock solution of 10 mg/mL XTT was prepared in 10 mL 1X sterile PBS solution.
Aliquots of 100μl XTT were stored at -20°C. The working solution was prepared by
adding 50 μl stock solution in 1 ml dH2O to yield a final concentration of 0.5 mg/mL, and 1μl of 50 mM menadione to yield a final concentration of 50 μM.
In the second approach biofilms of E. coli were grown in a similar manner as previously described, but 2 ml of biofilm culture suspension was added into 12 well plate instead of 700 μl. The grown biofilms were washed with 1X PBS as previously described. 2 ml of 3 minutes plasma treated NAC solution was nebulized on biofilms by using Invacare Reusable Jet Nebulizer (Elyria, OH) that was connected to sterile compressed air under 30 psi (Figure 15b). The nebulizer can generate 5 μm particles and nebulize 3 ml of liquid in 7 minutes. Bacterial viability following treatment was quantified by using XTT assay.
In the third approach P. aeruginosa PA01 biofilm culture suspension was prepared in TSB, as previously described, in the presence of 0.5% serum to enhance biofilm formation. In the batch phase of the experiment, 15 ml of medium was added ! 56! on to glass slides in reactor channels, and the reactor cover was tightened and incubated at 37O C for 6 hours. In the continuous phase, the reactor was beveled to achieve 10O downward slope. Nutrient inlet and outlet tubing lines were connected to reactor and fresh nutrient was circulated over the biofilm at 37O C for 24, 48 and 72 hours. In this system fresh nutrient drips on biofilm through a needle connected to nutrient inlet line and then flows through biofilm due to the slope and accumulates in a reservoir (Figure
15a) [172]. After desired time of incubation grown biofilms were treated by circulating 5 ml, 3 minutes plasma treated NAC solution over biofilm for 30 minutes. Following the treatment, glass slides with biofilms were placed into 50 ml conical tubes containing 10 ml PBS. Tubes were vortexed for 30 seconds, sonicated for 2 minutes, and vortexed for and additional 30 seconds and colony-counting assay was performed.
Figure 15: Experimental design for different biofilm treatment approaches: (a) Drip flow biofilm reactor (DFR) set up, (b): Set up for biofilm treatment with plasma-treated, nebulized NAC solution
2.3 Results:
2.3.1 Temperature and pH Measurements of Plasma Treated NAC Solution: The temperature measurement of untreated and plasma treated NAC samples were performed ! 57! to demonstrate whether non-thermal plasma treatment causes increase in temperature.
Results revealed that after 1,2, and even 3 minutes of plasma treatment, temperature of
NAC solutions remain around the room temperature, reaching to 25.33O C, 25.17O C and
26.13O C respectively. No linear relation was seen between temperature and different treatment times (Figure 16a). We also noted that the pH of the liquid decreased with plasma treatment time, moving toward acidity, when tested with pH strips. Therefore, we measured the pH using an ultrasensitive pH meter with a digital display system. Figure
16b shows that the pH of plasma treated NAC solution drastically drops after 1minute of treatment to pH 2.82 and, after this point, acidity keeps increasing slightly to pH 2.39 and 2.35 after 2 and 3 minutes of plasma treatment respectively.
30.00!
28.00! a! 26.23! 26.00! 25.33! 25.17! 23.93! 24.00!
Temperature)(C)) 22.00!
20.00! 0! 1! 2! 3! Plasma)Treatment)Time)(min))
7.00! 6.24! 6.00! 5.00! b! 4.00! 2.82! pH) 3.00! 2.39! 2.35! 2.00! 1.00! 0.00! 0! 1! 2! 3! Plasma)Treatment)Time)(min))
Figure 16: Temperature (a) and pH (b) change of non-thermal DBD plasma treated NAC solution depending on plasma treatment duration.
! 58!
2.3.2 Antimicrobial Effect of Plasma Treated NAC Solution: To determine treatment time dependent antimicrobial effect of plasma-treated NAC solution, preliminary experiments were done using 107 CFU/ml initial bacteria concentration, which is clinically significant bacterial load [12]. As it is shown on Figure 17, 1 and 2-minute plasma treated NAC solution can inactivate 1.3 and 1.9-log bacteria respectively; while
3-minute plasma treated NAC solution is capable 7-log complete inactivation of bacteria.
8.00! 7.13! 7.00! 5.82! 6.00! 5.16! 5.00!
4.00!
3.00!
log)Surviving)Bacteria) 2.00!
1.00! 0.00! 0.00! 0.00! 0! 1! 2! 3! 3%!H2O2! Plasma)Treatement)Time)(min)) Figure 17: Plasma treatment time dependent antimicrobial efficiency of non-thermal plasma-treated NAC solution on E. coli
2.3.3 Effect of Holding Time on Inactivation: It is important to know the required contact time of plasma treated NAC solution with bacteria (previously described as holding time) in order to understand inactivation properties of plasma treated NAC solution such as; inactivation kinetics and inactivation mechanism. For this reason 3- minute plasma treated NAC solution was held with bacteria for different time points.
Holding time experiments yielded that an increasing fashion of inactivation rate can be ! 59! achieved with increasing holding time. We have observed a slightly increasing inactivation rate from 1 to 5 minutes of holding time and complete inactivation was observed when 3-minute plasma treated NAC solution comes in contact with 107
CFU/ml E. coli for 15 minutes (Figure 18). Unless otherwise stated 15 minutes of holding time has been kept constant for all experiments.
Untreated! Treated!
8.00! 7.06!7.05! 7.04!6.88! 7.05! 7.06! 7.06! 7.07! 6.56! 7.00! 6.29! 6.00! 5.49!
5.00!
4.00!
3.00!
log)Surviving)Bacteria) 2.00!
1.00! 0.00! 0.00! 0!min! 1!min! 2!min! 3!min! 5!min! 15!min! Holding)Time)(min)) Figure 18: Effect of holding (contact) time on antimicrobial efficacy of 3-minute DBD plasma-treated NAC solution
2.3.4 Cell Density Dependent Inactivation by Plasma-Treated NAC Solution: In previously explained experiments 107 CFU/ ml initial bacterial load was exposed to plasma treated NAC solution. In order to better understand how much bacteria can be inactivated by 3 minutes non-thermal plasma treated NAC solution under given conditions such as 15 minutes of holding time and exposure to equal volumes of bacteria and plasma treated NAC solution, higher initial bacterial loads were exposed to 3-minute plasma treated NAC solution. Figure 19 depicts that 3-minute plasma treated NAC ! 60! solution is capable of inactivating up to 108 CFU/ ml E. coli when exposed at equal volume after 15 minutes of holding time. 3-minute plasma treated NAC solution was ineffective when it was exposed to 5x108 CFU/ ml and, 109 CFU/ ml of initial bacterial load. Presented results shows that 3-minute plasma treated NAC can achieve up to 8-log reduction. However 107 CFU/ ml initial bacterial load was kept constant for other experiments in this thesis unless otherwise stated.
Untreated! Treated! Control!(3%!H2O2)! 10.00! 9.10! 8.94! 8.77!8.65! 9.00! 8.06! 7.74! 8.00! 7.03! 7.00! 6.00! 5.00! 4.00! 3.00! log)Surviving)Bacteria) 2.00! 1.00! 0.00! 0.00! 0.00! 0.00! 0.00! 0.00! 0.00! 0.00! 0.00! 10^7! 5*10^7! 10^8! 5*10^8! 10^9! Initial)Concnetration)of)Bacteria)(CFU/ml))
Figure 19: Initial bacterial load dependent antimicrobial efficiency of 3-minute non- thermal plasma-treated NAC solution
2.3.2 Bactericidal and Fungicidal Effect of Non-Thermal Plasma Treated NAC
Solution: In the following experiments, plasma treatment time dependent antimicrobial effect of non-thermal plasma treated NAC solution was tested on planktonic forms of different microorganisms including 3 Gram positive, 3 Gram negative, and 2 fungus strains. As shown in Figure 20, we have observed less inactivation rates of tested organisms following 1 and 2-minute treatment of NAC solution compared to 3-minute ! 61! treated one. While all strains showed less than 1 log reduction after 1 and 2-minute treatment of NAC solution, 7 log of complete inactivation of P. aeruginosa was achieved even after 2-minute treatment of NAC solution. In fungal strains 7 log inactivation was observed only for C. albicans while 5.1 log reduction was achieved for
C. glabrata.
0!min! 1!min! 2!min! 3!min! Control!(3%!H2O2)! 8.00! 7.11! 7.06! 7.07!6.90! 7.05! 7.00! 7.00! 7.00!6.88! 6.79! 7.04! 6.85! 7.04!6.82! 6.82! 7.00! 6.64! 6.72! 6.63! 6.47! 6.21! 6.11! 5.82! 6.00! 5.16! 5.00!
4.00!
3.00! logSurviving)Cells)) 1.90! 2.00!
1.00! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0! 0.00! E.coli'' S.aureus' A.baumannii'' S.epidermidis'P.'aeruginosa' E.faecalis' C.albicans' C.glabrata' Organisms))
Figure 20: Plasma treatment time dependent antimicrobial efficiency of non-thermal plasma-treated NAC solution on planktonic forms of different bacteria and fungus strains
The bactericidal effect of plasma treated NAC was also tested on MRSA
USA300, MRSA USA400, K. pneumoniae NDM-1 and clinical isolate of A. baumannii that are highly antibiotic resistant bacterial strains. As shown in Figure 21, 3-minute plasma treated NAC solution achieves 7 log reduction of highly antibiotic resistant bacteria. ! 62!
Untreated NAC# 3 minutes plasma treated NAC# 3% H2O2#
8.00# 7.29# 7.10# 7.03# 7.09# 6.97# 7.00# ! 6.00# 5.00# 4.00# 3.00# 2.00# log Surviving Bacteria log 1.00# 0.00# MRSA USA300# MRSA USA400# K. pnemuniae# E. coli O157:H7# A. baumannii clinical Isolate# Treated Bacteria!
Figure 21: Bactericidal effect of 3-minute plasma-treated NAC on antibiotic resistant bacteria strains
In biofilm experiments 3-minute plasma treated NAC solution was capable of inactivating 100% of bacterial biofilms of various strains similar to planktonic form experiments. S. epidermidis showed the most biofilm activation after 2-minutes treatment of NAC solution with 98% reduction rate. However, the maximum reduction rate on fungus biofilms was around 85% for both two fungal strains even after 3 minutes plasma treatment of NAC solution (Figure 22).
! 63!
0!min! 1!min! 2!min! 3!min! Control!(3%!H2O2)!
120!
100! 100! 100! 100! 100! 100! 100! 100! 100!
80! 62.32! 62.15! 57.52! 58.97! 60! 46! 39.2! 40! 33.3! 29! 29.1! 27.7! 23.11! 20! Percentage)of)Surviving)Cells) 16! 20! 9.98! 10.5! 11.74! 6.02! 0! 0! 0! 0! 0! 0! 2.04!0! 0! 0! 0! 0! 0! 0! 0! 0! E.coli'' S.aureus' A.baumannii''S.epidermidis'P.'aeruginosa' E.faecalis' C.albicans' C.glabrata' Organism) Figure 22: Plasma treatment time dependent antimicrobial efficiency of non-thermal plasma-treated NAC solution on biofilm forms of different bacteria and fungus strains
Also nebulization of 3 minutes, non-thermal plasma treatment of NAC solution has shown a significant effect on E. coli biofilms. We have shown that up to 85% of E. coli biofilms can be eradicated by nebulization of 3-minute plasma treated NAC solution
(Figure 23).
! 64!
100! 100.00!
80.00!
60.00!
40.00!
%)of)Surviving)Bacteria) 13.43! 20.00! 1.79! 0.00! H2O2! Untreated!NAC! 3!min!NAC! Treatment)Conditions)
Figure 23: Antimicrobial efficiency of nebulized, 3-minute non-thermal plasma-treated NAC solution on E. coli biofilm
Furthermore, 3-minute non-thermal plasma treated NAC solution has shown antimicrobial activity on stronger biofilms of P. aeruginosa mPA01 strain, which are potent biofilms, grown under shear stress on a liquid-air interface by a drip flow reactor
(DFR) system for an extended period of time. Around to 3.5-log inactivation was observed for even 3-day-old biofilms grown with DFR system as depicted in Figure 24.
10.00! 9.11! 8.84! 8.55! 9.00! 8.00! 7.00! 5.84! 6.00! 5.00! 4.37! 4.16! 4.00! 3.00! 2.00! log)Surviving)Bacteria) 1.00! 0.00! Control!! Treated! Control!! Treated! Control!! Treated! 1!day! 2!days! 3!days! Treatment)Conditions)and)BioAilm)Growth)
Figure 24: Biofilm maturation duration dependent antimicrobial efficiency of 3-minute non-thermal plasma-treated NAC solution on P. aeruginosa mPA01 biofilms grown on a drip flow reactor ! 65!
2.4 Discussion:
It is widely reported that direct application of non-thermal plasmas such as plasma jets mediated by argon gas or gliding arc plasmas decontaminate bacteria
[150,157,173]. The gas and the type of application technique used largely determine plasma-generated reactive species; thus, the antimicrobial efficacy is also likely differ
[105,157,174,175]. Treatment of water with the gliding arc plasma and its bactericidal effect has been reported [157,175]. Direct current (DC) electrical discharges in atmospheric air has also been tried by exposing either static or flowing water to decontaminate bacteria in water, wherein three different types of discharges were tested such as streamer corona, transient spark, and glow discharge [145].
Our approach and, plasma device design is different than the previously reported corona, spark, or glow plasma discharge. We use alternate current (AC) dielectric barrier discharge (micro pulsed plasma), and treat NAC solution first, and then expose treated it to microorganisms as previously reported from our laboratories [144,171]. Our goal is to understand the uniquely different DBD plasma technique and evaluate the antimicrobial efficacies of the NAC solution treated by this technique against planktonic and biofilm forms of microorganisms.
Joshi et al. have previously reported how direct plasma treatment inactivates contaminating pathogens when the non-thermal DBD plasma treatment is used. That study suggests that the NAC solution, which turns into a powerful antimicrobial solution is highly biocidal against a range of pathogens, including pathogenic fungi, and is as effective as the direct plasma treatment previously reported [144]. In the present study, non-thermal DBD plasma electrode was larger than reported earlier, to cover a larger ! 66! treatment area. The fluid holder was also large enough to hold 1 ml of liquid but the discharge gap between the electrode and the surface of the fluid remained same as 2 mm.
There are two reasons to adopt fluid-mediated plasma application. Briefly, it is known that non-thermal (cold) plasma generates electric field, magnetic field, and certain physical species, such as charged particles, ultraviolet photons and electrons in addition to chemical species, such as reactive oxygen species (ROS) or reactive nitrogen species (RNS). Physical species are more detrimental and damaging to surfaces, so it is a challenge to apply them to delicate, contaminated surfaces and to biological materials
[176]. The antimicrobial effect of plasma treated fluids that permits one to sterilize or disinfect such surfaces without damage is an advantage. These fluids can be used to improve hospital hygiene, to treat bacterial skin diseases, and cosmetically, to control acne-causing organisms. The skin is not needed as a counter electrode, which significantly simplifies their practical use on patients. Furthermore, the risks of electric current, thermal damage of tissue, and UV irradiation can be avoided by using fluid- mediated plasma treatment. Also, the purpose of investigations of plasma-activated liquids is to search for a better option as an antimicrobial agent over existing chemical preparations (such as chlorhexidine and silver containing compounds), which have toxicity.
The concentration of NAC (5 mM) selected for this study is considerably lower than the clinically permitted amount of NAC for therapeutic purposes. Also, the bacterial load (107 CFU/ml and more) used for inactivation experiments is high and therefore covers the clinically significant number of bacterial cells. Most other studies have used ! 67!
<6-log, which is clinically less relevant to represent severe sepsis.
Therefore, we performed a cell density-dependent experiment with treated NAC.
A large quantity of CFUs (108 CFU/ml) was completely inactivated by the treated NAC solution, again indicating its strong antimicrobial effect.
The type and concentration of pathogen are important in deciding the outcome of the processes of disinfection, decontamination, or sterilization. Multidrug resistance is an additional hurdle in these processes and helps disseminate hospital-acquired infections, either by direct transmission or through biofilm-loaded surfaces [162,177]. Therefore, to investigate the antimicrobial efficacy of plasma-treated fluids, we chose multidrug- resistant nosocomial pathogens in moderately high concentration (7-log CFU) known to form biofilms [144,158]. Because these pathogens also contaminate the surfaces of stethoscopes, catheters, and the hands of health care providers they are ideal candidates with which to test the decontamination regime [158]. Our results clearly demonstrate the strong antimicrobial effect of the plasma-treated fluid. The effect was comparable to that of 3% hydrogen peroxide solution. The holding time of these fluids with given bacteria is also crucial for decontamination of surfaces. Different biocidal solutions that are generated by plasma treatment have variable holding times [178]. Plasma treated NAC solution presented in this study was as effective as the existing commercial disinfectants and required only 15 minutes of holding time to completely inactivate reference and antibiotic resistant strains of all of the pathogens in vitro (A. baumannii, C. albicans, C. glabrata, E. coli, E. faecalis, P. aeruginosa, S. epidermidis, and S. aureus, MRSA strains, E. coli O157:H7, K. pneumoniae NDM-1). Holding time dependent inactivation results are comparable to bacterial responses found in earlier reports, wherein fungus ! 68! was not inactivated even after 30 minutes of exposure [157,179]. In the present study, both species of fungus (C. albicans and C. glabrata) were inactivated in 15 minutes when exposed to 3-minute plasma-treated NAC solution, both in their planktonic form, and in biofilm forms suggesting that the plasma-treated NAC solution is a potent biocidal agent. Furthermore, clinical isolate of A. baumannii, MRSA strains, E. coli
O157:H7 and K. pneumoniae used in this study are multidrug resistant and strong producers of biofilms. Inactivation of K. pneumoniae NDM-1 by plasma treated NAC solution deserves a special interest. K. pneumoniae NDM-1 is a strain that carries transmissible genetic element encoding multiple resistance genes called NDM-1 (New
Delhi Metallo-β-lactamase). It was first isolated from a patient in India and has been spread around the world. NDM-1 carrying microorganisms are called “superbugs” due to their resistance ability against carbapenems [180]. Our results with K. pneumoniae
NDM-1, along with other multidrug resistant bacteria, MRSA300 and MRSA400, showed that plasma treated NAC solution can inactivate broad range of even multidrug resistant microorganisms. Therefore, their rapid inactivation by the non-thermal plasma- treated NAC solution supports its powerful biocidal effect.
Ventilator associated infection is major threat to patients that are hospitalized in intensive care units and assisted with mechanical ventilator with an increasing cost [30].
Treatment of E. coli biofilms with nebulized NAC solution suggest that, plasma treated
NAC solution has a potential to prevent or cure ventilator associated infections. Also biofilm formation in respiratory tract is a major problem and source of infection for cystic fibrosis patients. Nebulized, plasma-treated NAC solution can provide a new solution for the treatment of severe respiratory tract infections. ! 69!
Drip flow bioreactor (DFR) system provides an environment for the growth of biofilm on air-liquid interface under low shear stress. DFR system provides continuous more potent biofilm growth. It is an ideal system for modeling the hospital biofilm growth in lungs affected by cystic fibrosis, catheters and body cavities [172]. In the present study we showed that, plasma treated NAC solution is capable of inactivating significant amount of biofilms grown with DFR system. Our results suggest that plasma treated NAC solution can be considered as a potential antimicrobial agent for patients with severe biofilm infections.
During plasma treatment of NAC solution, pH decreased from neutral to acidic.
Several authors have reported such a reduction in pH, and the acidification of solutions, but the exact mechanism is still not fully understood [105,110,156]. It is anticipated that a drop in pH could be due in part to generation of nitric acid via interaction of RNS,
ROS, and hydrolysis of water that gives rise to proton and hydroxyl radical. The extent of acidification of solution being treated by plasma depends, upon the amount of plasma dose (depending on voltage and frequency) being deposited, duration of the treatment, amount of liquid being treated, height (column) of liquid, immediate environment around the high voltage electrode and the discharge gap. A relatively smaller amount of solution if treated with plasma for a relatively longer duration of time, with a narrow gap for plasma discharge, can make solution highly acidic, and saturated with acidifying species. The previous literatures that have reported a moderate reduction in pH of PBS or water solution always had a plasma discharge gap of more than 2 mm through 45 mm, and the amount of liquid more than 1.5 ml through 10 ml [105,110,156]. This drastically changes the chemical properties and pH equation of these solutions. We did not find any ! 70! significant temperature changes in these treated fluids; in fact, the temperature was close to room temperature even after 3 minutes of treatment and was not related to the change in pH. Acidification of treated fluid could be the result of factors other than H2O2, nitric acid and nitrous acid, such as hydroperoxyl (HO2) and peroxynitrous acid (HOONO), or a combination of all of these and other unknown species. Similar changes are reported in the gliding arc plasma treatment, which is different than the technique we used [178]. In brief, plasma treated NAC solution inactivates a wide range of multidrug-resistant bacteria and fungal pathogens, independently their gram staining classification in both their planktonic and biofilm forms faster than previously reported plasma treatment methods (within15 minutes of holding time). ! 71!
! 3 CHAPTER III: CHEMICAL SPECIES IN PLASMA-TREATED N-ACETYL CYSTEINE (NAC) SOLUTION AND THEIR CONTRIBUTION TO MICROBIAL INACTIVATION
3.1 Introduction:
The antimicrobial effect of direct application of non-thermal plasmas is a widely investigated and well-known phenomenon [98,176,148,152]. Different factors are effective on the antimicrobial effect on non-thermal plasmas. In brief, the most common mechanisms that are responsible for microbial inactivation are reported as: UVC photons generated during plasma discharge, plasma generated ROS and RNS in the gas phase and diffusion of these species through the bacterial cell wall and membrane, physical effect of electron discharge and electrical field that causes damage to the cell surface, and localized heating effect to cell surface. Given mechanisms are capable of inactivating microorganisms synergistically [106,148,181]. Furthermore, plasma treated liquids have an increasing interest due to their antimicrobial activity. Different groups have reported antimicrobial effect of liquids including water 0.9% saline solution and
PBS that are treated with different non-thermal plasma sources (fluid mediated plasma treatment). Acidic pH following plasma treatment of liquids is the most commonly reported chemical modification [93,105,108-111,155-157,182-184]. In fluid mediated plasma treatment bacteria don’t come in contact with UV and electron discharge.
Therefore diffusion of plasma generated ROS and RNS into treated liquid are thought to be the main cause for microbiocidal effect [93]. The decreased pH of plasma treated
+ liquid is attributed to generation of HNO2, HNO3 and, H3O [93,109,156]. The acidification of plasma treated liquid is critical for microbiocidal effect; however, it was ! 72! reported that acidic pH is not the main source of antimicrobial effect [105,184,187].
Ikawa et al. have reported that acidic pH is essential for antimicrobial effect through plasma treated liquids. They have demonstrated that below pH 4.7 the antimicrobial effect is significantly higher compared to pH of greater than 4.7. They claimed that pH
4.7 is the critical value for microbial inactivation and it is almost universal for different type of bacteria including Gram positive, Gram negative and anaerobic bacteria [111]. In
- - addition to decreased pH also nitrate (NO3 ), nitrite (NO2 ) and hydrogen peroxide (H2O2) have been detected in plasma treated liquids [156,185,186]. Plasma also generates other
ROS and RNS such as OH radical superoxide nitric oxide etc. Direct contact of liquid with electron discharge, reactions of plasma generated ROS and RNS, and their
- - diffusion into liquid are responsible for the presence of NO3 , NO2 and H2O2 in plasma treated liquids. Reactions of ROS and RNS lead to the formation of other species that might contribute to antimicrobial effect. It is well known that plasma generates NO and
- - superoxide (O2 ), and their reaction yields to peroxynitrite (ONOO ) formation. Another possible route for peroxynitrite formation in the plasma treated liquid is the formation of
+ + - nitrosooxidanium (H2NO2 ) via the reaction of H cation with NO2 anion in highly acidic
+ + environment. Nitrosooxidanium (H2NO2 ) is broken down into nitrosonium (NO ) cation that is highly reactive and can attack biomolecules in the cell, and form peroxynitrous acid (ONOOH) in the presence of hydrogen peroxide. Finally peroxynitrous acid is dissociated to peroxynitrite in aqueous medium [194,195]. Peroxynitrite is highly reactive and can easily diffuse to cell membrane due to its high permeability. It attacks various biomolecules in the cell and causes protein and lipid nitrosylation and also intracellular oxidation. Intracellular damage induced by peroxynitrite can’t be repaired ! 73! by cellular repair mechanisms and cells die [196-198]. Acidified nitrite and nitrate have been known for decades for their antimicrobial effect both in vitro and as a part of natural protection mechanism of the body [188-190]. Salivary nitrite comes in contact with acid in the stomach when swallowed and acts as a natural host defense mechanism through the formation of biocidal species [192]. Antimicrobial effect of acidified nitrite is closely related with RNS involving mechanisms. For example, NO release from skin has been reported. Also natural flora of the skin reduces nitrate to nitrite. In the acidic
nature of the skin RNS, including nitrous acid (HNO2), dinitrogen trioxide (N2O3) and peroxynitrite (ONOO-) are produced, via NO and nitrite and nitrate that act as non- specific protection against pathogens on the skin [191]. Various groups have demonstrated that acidified nitrite has antimicrobial effect on various skin and oral pathogens [191-193]. The composition of the liquid that is being treated should be considered in order to clarify the mechanism of antimicrobial effect. As opposed to water and PBS, Oehmigen et al. have reported the antimicrobial effect of 0.85% saline solution (NaCl). However they concluded that effect of chlorine species that arises from
Cl- ion could be neglected [155].
In brief, during plasma treatment of the liquid, ROS and RNS are generated both in gas phase and liquid-gas interface. Acidic pH is common consequence of liquid mediated plasma treatment. Even though acidic pH doesn’t contribute to antimicrobial effect, it is essential for microbial inactivation. Antimicrobial effects of plasma treated liquids originate from diffused ROS and RNS in to liquid as opposed to direct plasma treatment, where physical impacts such as UV, electrical field, and electron bombardment are contributors to the biocidal effect. ! 74!
3.2 Materials and Methods:
3.2.1 Hydrogen Peroxide Detection: Hydrogen Peroxide Assay Kit (National
Diagnostics, Atlanta, GA, USA) was used for the detection of hydrogen peroxide in plasma treated NAC solution. The assay is based on the formation of a complex between
Xylenol Orange and ferric iron, which is produced by the peroxide dependent oxidation of ferrous iron. This reaction is quantified colorimetrically, and the kit can detect as little as 15ng/ml of peroxide. The kit consists of Reagent A, which is an aqueous solution of
2% sorbitol and Xylenol orange, and Reagent B, which is 14% solution of sulfuric acid with 1% ammonium ferrous sulfate. 19.8 ml of Reagent A and 0.2 ml of Reagent B were mixed, and 180 µl of assay mixture was mixed with 20 µl of plasma treated NAC sample and was incubated at room temperature for 30 minutes to allow for complete color development; dilutions were made where they were necessary. Different dilutions of
30% hydrogen peroxide were used to generate a standard curve. After incubation for color development, absorbance of samples and standards were detected with a UV- visible spectrophotometer (Thermo Scientific, Hudson NH, USA) at 560 nm.
Concentration of hydrogen peroxide in plasma treated NAC solution was determined according to the standard curve.
3.2.2 Nitrite and Nitrate Detection: Nitrite-Nitrate Test Kit from HACH (Loveland,
CO, USA) was used for the detection of Nitrite and Nitrate in plasma treated NAC solution. Kit consists of two powder components; one is NitraVer 5 for Nitrate detection that contains Cadmium and Sulfanic Acid and NitriVer 3 for Nitrite detection that contains Potassium Pyrosulfate. Kit is able to detect 2 mg/l Nitrate and 0.01 mg/l Nitrite.
For Nitrate detection 5 ml of plasma treated NAC solution was mixed with NitraVer 5 ! 75! reagent and shaken vigorously for 1 minute; we then waited for 1 more minute to observe complete color development and color change was observed by using color comparator. Similarly, for Nitrite detection plasma treated NAC solution was mixed with
NitriVer 3 reagent and was shaken vigorously for 1 minute; we then waited for 10 more minute for complete color development and color change was observed by using color comparator. Samples were diluted with deionized water, when the dilution is needed.
Detection results were validated by using standard solutions of nitrite that was prepared
by dissolving NaNO2 (Sigma, St. Louise MO, USA) in deionized water and nitrate that
was prepared by dissolving HNO3 (Fischer Scientific, Pittsburgh, PA, USA) in deionized water.
3.2.3 UV-Visible Spectrum Analysis: UV-visible spectra was collected for NAC solution that was treated for different time points, peroxynitrite standard solution, bacterial cell suspension after treatment with plasma treated NAC solution, peroxynitrite solution and ATL buffer. UV-visible spectra were generated with a UV-visible spectrophotometer (Thermo Scientific, Hudson, NH, USA) between 190 nm and 1100 nm wavelengths with 1 nm interval. Untreated NAC solution, deionized water and bacterial suspension and substance of interest were used as blank for the UV-visible spectra of plasma treated NAC solution, peroxynitrite and bacterial suspension that was treated with the substance of interest as respectively. Also UV-visible spectrum of dried and rehydrated plasma treated NAC solution was generated in the same manner, where the dried and rehydrated, untreated NAC solution was used as blank. Findings regarding to dried and rehydrated plasma treated NAC solution will be given and discussed at
Chapter 4. ! 76!
3.2.4 Fourier Transform Infrared Spectroscopy (FT-IR) Analysis of Plasma-
Treated NAC Solution:!Untreated and plasma treated NAC solutions were evaporated with a rotary evaporator. Infrared spectra of remaining powder from the evaporation of untreated and 3-minute plasma treated NAC solution were obtained with an Olympus
BX51 microscopy system (Olympus, Japan) and modified with a Fourier-Transform infrared spectrometer IlluminatIR that is equipped with an ATR lens (Smith Detection,
USA).
3.2.5 Nuclear Magnetic Resonance (NMR) of Plasma-Treated NAC Solution: NAC
stock solution for NMR analysis was prepared in heavy water (D2O) (Sigma Aldrich, St.
Louise, MO, USA) in order to avoid signal that rises from hydrogen in water. 1 X PBS solution was prepared by dissolving PBS tablets (Sigma Aldrich, St. Louise, MO, USA)
in D2O and then NAC powder was dissolved in PBS solution that was prepared in D2O to have 100 mM of final concentration. NAC stock solution was stored at -20O C, and 5
mM of working solution was prepared by appropriately diluting stock solution in D2O.
NAC solution that was prepared in D2O was treated in the same manner as described in
Chapter 2. Proton nuclear magnetic resonance spectra were collected using a Varian
INOVA 500 MHz FT-NMR device (Palo Alto, CA, USA). Antimicrobial effect of plasma treated NAC solution, which was prepared in D2O was tested in order to assure that its antimicrobial property is same as NAC solution that was prepared in H2O.
3.2.6 Antimicrobial Effect of acidic pH and Plasma-Generated Species: In order to determine if low pH after plasma treatment is responsible for plasma disinfection effect,
acetic acid (CH3COOH), nitric acid (HNO3) (Fischer Scientific, Pittsburgh, PA, USA), ! 77!
sulfuric acid (H2SO4), hydrochloric acid (HCl) and phosphoric acid (H3PO4) (Sigma
Aldrich, St. Louise, MO, USA) solutions were prepared at pH ~2.3 by titration. Prepared acid solutions were exposed to the same volume of 107 CFU/ml E. coli and held for 15 minutes. Then colony-counting assay was performed for the quantification of surviving bacteria as described in Chapter 2. Antimicrobial effects of plasma-generated species were tested by preparing detected concentrations of each species in deionized water. For the combination experiments final concentrations of each species were adjusted accordingly. 30% hydrogen peroxide solution, 1 N nitric acid, glacial acetic acid (Fisher
Scientific, Pittsburgh, PA, USA), sodium nitrite and cysteic acid (Sigma Aldrich, St.
Louise, MO, USA) was used to prepare desired concentrations of tested substances.
Superoxide thermal source (SOTS-1) (Cayman, Ann Arbor, MI, USA) was used to prepare superoxide solution. SOTS-1 is provided as crystalline powder and its stock solution was prepared by dissolving in dimethyl sulfoxide (DMSO) and stored at -80O C.
Working solution was prepared by dissolving it in PBS. SOTS-1 is an azo compound that can be thermally decomposed in aqueous solution to generate superoxide radical anion at a constant controlled rate. SOTS-1 thermally decomposes to form an intermediate that reacts with oxygen at the diffusion-controlled limit to generate superoxide. The decay of SOTS-1 into the intermediate follows first order kinetics and exhibits a half-life of 4900 seconds at physiological pH and temperature. Since the stock solution of SOTS-1 was prepared in DMSO, antimicrobial effect of corresponding concentration of DMSO in working solution was also tested in order to assure that
DMSO doesn’t interfere with antimicrobial effect. ! 78!
Peroxynitrite (ONOO-) (Cayman, Ann Arbor, MI, USA) solution was diluted in deionized water just before exposure to bacteria to obtain, plasma generated concentration of it. Peroxynitrite solution was provided in 0.3 M sodium hydroxide
(NaOH). Therefore antimicrobial effects of corresponding concentrations of NaOH were tested in order to make sure that it doesn’t interfere with antimicrobial effect. All species and all combinations of them were exposed to same equal volume of 107 CFU/ml E. coli and held for 15 minutes. Then colony-counting assay was performed for the quantification of surviving bacteria as described in Chapter 2.
3.3 Results:
3.3.1 Hydrogen Peroxide Detection: Standard curve was plotted in between 0.2 and 4.1
μM concentrations of hydrogen peroxide (Figure 25a). Appropriate dilutions of NAC solution were done after plasma treatment to fit hydrogen peroxide concentrations in plasma treated NAC solution to the standard curve. Calculated concentrations were multiplied by dilution factor to obtain real concentration of hydrogen peroxide in plasma treated NAC solution. We detected 0.42 mM, 1.67 mM and 0.93 mM of hydrogen peroxide after 1-minute, 2-minute and 3-minute treatment of NAC solution respectively.
It is interesting that, hydrogen peroxide concentration reaches to saturation after 2 minutes of plasma treatment and then drops after 3 minutes of plasma treatment (Figure
25b).
! 79!
0.5" a!
0.4" ! y = 0.0266x + 0.3019" 0.3" R² = 0.95771"
0.2" Absorbance 0.1"
0" 0" 1" 2" 3" 4" 5"
Concentration of H2O2 (μM) !
! 2.00" b! 1.67" 1.50" 0.93" 1.00" 0.42" 0.50"
concetration (mM) concetration 0.00" 2
O 0.00" 2
H 0" 1" 2" 3" Treeatment Time (min)!
Figure 25: (a) Standard curve for hydrogen peroxide detection in plasma treated NAC solution and (b) Treatment time dependent hydrogen peroxide concentration in plasma- treated NAC solution.
3.3.2 Nitrite and Nitrate Detection: Both nitrite and nitrate concentrations in plasma treated NAC solution increase with plasma treatment time. Nitrite concentrations in plasma treated NAC solution were determined as 0.03 mM, 0.16 mM and 0.33 mM after
1-minute, 2-minute and 3-minute of plasma treatment respectively (Figure 26a). Nitrate concentrations in plasma treated NAC solution were measured as 1 order magnitude more than nitrite, where 2.07 mM, 5.46 mM, and 9.35 mM nitrate was generated after 1,
2 and 3 minutes of plasma treatment respectively (Figure 26b). ! 80!
0.40" a! 0.33" ! 0.35" 0.30" 0.25" 0.20" 0.16" 0.15" 0.10" concnetration (mM) concnetration - 0.03" 2 0.05" 0.00" NO 0.00" 0" 1" 2" 3" Treatment Time (min)!
10" 9.35"
! 9" b! 8" 7" 6" 5.46" 5" 4" concnetration (mM) concnetration - 3" 2.07" 3 2" NO 1" 0" 0" 0" 1" 2" 3" Treatment Time (min)!
- - Figure 26: Treatment time dependent concentrations of (a) NO2 (Nitrite), and (b) NO3 (Nitrate) in plasma-treated NAC Solution. Both concentrations increase with plasma treatment time.
3.3.3 UV-Visible Spectrum Analysis of Plasma Treated NAC Solution: UV-visible spectra of plasma treated NAC solutions were obtained after 1, 2 and 3-minute plasma treatement. In the spectrum of 1-minute plasma treated NAC solution, we have seen a specific peak at 332 nm, which belongs to S-nitroso-N-acetyl cysteine (SNAC) a type of
S-nitrosothiol. This peak disappears in the spectra of 2 and 3-minute plasma treated
NAC solution. In the spectrum of 2-minute plasma treated NAC solution a new peak ! 81! starts to appear at 302 nm and the absorbance value of this peak increases after 3 minutes of plasma treatment (Figure 27a). In order to better understand chemical modifications in the NAC solution during plasma treatment, we have also obtained UV- visible spectra after of NAC solution 30, 45, 75 and 90 seconds of plasma treatment.
Figure 27b shows that the peak at 332 nm that is specific for S-nitrosothiols and in our case specifically belongs to S-nitroso-N-acetyl cysteine (SNAC) first appears after 30 seconds of plasma treatment and increases with the plasma treatment duration until 75 seconds of plasma treatment. After 90 seconds of plasma treatment the peak at 332 nm disappears and the peak at 302 nm appears. The peak at 302 nm increases with plasma treatment time until the end of plasma treatment (3 minutes). Concentration of peroxynitrite in plasma treated NAC solution is calculated by using the Beer-Lambert
Law. The Beer-Lambert Law defines the correlation between concentration of a substance and its absorbance at particular concentration. It is expressed with following equation:
! = ! ∙ ! ∙ !
Where; A is absorbance, ε is extinction coefficient (M-1.cm-1), l is the path length that
- light passes through and c is the concentration of the substance. εONOO is given as 1670
M-1.cm-1. From the given equation, concentration of peroxynitrite in 3-minute plasma treated NAC solution was calculated as 0.28 mM.
! 82!
1.4!
1.2! a!
1!
0.8! Untreated!NAC!
0.6! 1!min.!treated!NAC! Absorbance* 2!min.!treated!NAC! 0.4! 3!min.!treated!NAC! 0.2!
0! 260! 280! 300! 320! 340! 360! 380! 400! 420! 440! Wavelength*(nm)*
1.4! b* 1.2!
1! NAC!30!sec.!
0.8! NAC!45!sec.! NAc!60!sec.! 0.6!
Absorbance* NAC!75!sec.!
0.4! NAC!90!sec.!
0.2! NAC!180!sec.!
0! 250! 270! 290! 310! 330! 350! 370! 390! 410! 430! 450! Wavelength*(nm)*
Figure 27: (a): UV-visible spectra of NAC solution treated for 1,2 and 3 minutes. (b): UV-visible spectra of NAC solution, showing the peaks depending on plasma treatment. The green dashed line shows the peak at 302 nm and the red dashed line shows the peak at 332 nm.
3.3.4 FT-IR Analysis of Plasma Treated NAC Solution: As it is shown in Figure 28
IR peaks of untreated NAC molecule at 3375 cm-1, 2547 cm-1, 1718 cm-1, and 1535 cm-1 correspond to the stretching motion of N–H in CONH group, S-H, C=O and CONH group, respectively. The absorptions at these peaks disappear after plasma treatment of
NAC solution. New peaks after plasma treatment appear at 3600-3000 cm-1 (broad), and
-1 1344 cm (narrow), which corresponds to the stretching motion of -NH2 and -SO3H, ! 83! respectively. FT-IR spectra of untreated and 3 minutes of plasma treated NAC solution suggest that the SH group was converted to -SO3H. Moreover the cleavage of!N-acetyl
group leads to formation of acetic acid (CH3COOH) and –NH2 formation due to the hydrogenation of NH group. These results suggest that NAC molecule was converted to
acetic acid (CH3COOH) and cysteic acid (C3H7NO5S) due to oxidation of thiol group as a result of plasma treatment. Peaks corresponding acetic acid can’t be seen on the spectrum because of the evaporation of acetic acid during the evaporation of water before FT-IR analysis.
0.5! Untreated!NAC! a! 1" 3!minutes!Treated! 0.4! NAC!
0.3! 1" 2" 3" 0.2! 4" Transmitance* 0.1! 2"
0! 500! 1000! 1500! 2000! 2500! 3000! 3500! 4000!
;0.1! Wavenumber*(cm^81)*
Figure 28: (a): FT-IR spectra of NAC solution before and after 3 minutes of plasma treatment. (b): Chemical bonds and groups on untreated NAC molecule that are indicated on FT-IR spectrum. (c): Chemical bonds and groups on cysteic acid that are indicated on FT-IR spectrum. ! 84!
3.3.5 Nuclear Magnetic Resonance (NMR) Analysis of Plasma Treated NAC
Solution: As depicted in the Figure 29, NMR data corresponds with spin coupling patterns of N-acetyl cysteine, which is being chemically converted to cysteic acid by cleavage of the N-acetyl group from the NAC molecule and oxidation of the thiol to cysteic acid. Increasing duration of plasma treatment of N-acetylcysteine resulted in increase in intensity of multiplets found in 3.54-3.61 and 3.24-3.42 ppm, as well as decrease in intensity of the multiplet located at 2.95-3.01 ppm. Proton shifts suggest that
~90% of N-acetyl cysteine is converted to cysteic acid via cleavage of the thiol group.
Figure 29: NMR Spectra of (a) untreated and (b) 3-minute plasma treated NAC solution. (a): Peaks and corresponding H atoms in untreated NAC solution. Peak 1 corresponds to signal form D2O. (b): Arrows indicate increased multiplets correspond to cysteic acid. ! 85!
3.3.6: Antimicrobial Effect of Acidic pH and Plasma-Generated Species: As demonstrated in Figure 30, different acids at pH ~2.3, except acetic acid, don’t show significant antimicrobial effect on 107 CFU/ ml E. coli. Acetic acid caused 3.5-log reduction. Acetic acid is a weaker acid compared to other acids that were used in this experiment. Therefore higher amount of acetic acid is needed to obtain pH ~2.3 solution
- of it that leads higher concentration of acetate ion (CH3CO2 ). When tested acids were combined with 0.93 mM H2O2, which is the detected concentration of H2O2 in NAC solution after 3-minute of plasma treatment no significant improvement of antimicrobial effect was not observed except in the HCl. The increased antimicrobial effect of HCl
when it is combined with H2O2 is attributed to the formation of hypochlorous acid that is known as halogenating agent [8]. In addition, acetic acid loses its antimicrobial activity
when it is combined with H2O2.
"w. 0.93 mM H2O2""
! 7.16"7.03" 7.20"6.99" 7.15" 7.17" 8.00" 7.09" 7.07" 7.08" 7.03" 7.00" 6.00" 5.00" 3.63" 4.00" 2.67" 3.00" 2.00"
N Surviving Bacteria 1.00"
10 0.00" 0.00" log
Acids tested!
Figure 30: Antimicrobial effect of different acids at pH ~2.3 alone and combined with
0.93 mM H2O2 Antimicrobial effects of detected concentrations of nitrite (0.33 mM), nitrate (9.35 mM), hydrogen peroxide (0.93 mM), acetic acid (~5 mM), and cysteic acid (~ 5 mM) in 3- ! 86! minute plasma treated NAC solution were tested on 107 CFU/ ml E. coli by itself and in
combination with each other. The pH of all tested mixtures were set to ~2.3-2.5. H2SO4 was used to set the pH in order to avoid possible interaction of HCl and HNO3 with tested species. Table 4 shows all tested species and their combinations.
Table 4: Combinations of detected concentrations of plasma-generated species that are tested on 107 CFU/ml E. coli Combination of 2 Combination of 3 Combination of 4 ------H2O2 H2O2 + NO2 + H2O2 + NO2 + NO3 NO2 + A.A. + H2O2 + NO2 + NO3 + - NO2 A.A C.A. A.A. ------NO2 H2O2 + NO3 + H2O2 + NO2 + A.A. NO3 + A.A. + H2O2 + NO2 + NO3 + - NO3 A.A C.A. C.A. - - - - - NO3 H2O2 + NO2 + H2O2 + NO2 + C.A. H2O2 + A.A. + NO2 + NO3 + A.A. + A.A C.A C.A. C.A. - - - - A.A. H2O2 + NO3 + NO2 + NO3 + A.A. H2O2 + NO3 + A.A. + C.A. + H2O2 + - C.A C.A A.A. NO2 - - - - C.A. NO2 + A.A. + NO2 + NO3 + C.A. H2O2 + NO3 + A.A. + C.A. + H2O2 + - - NO3 C.A. C.A. NO3 Combination of 5 - - H2O2 + NO2 + NO3 + A.A. + C.A. *Note that all of tested mixtures achieved < 1log reduction. (A.A. = Acetic acid, C.A. = Cysteic acid)
Any of tested species and combinations were not able to present significant
antimicrobial effect (< 1 log). In a further experiment, detected concentrations of H2O2,
- - NO3 , and NO2 were combined with arbitrarily selected concentrations (0.1, 0.5 and 1 mM) of superoxide that was generated by SOTS-1 (superoxide thermal source) compound. Since the SOTS-1 is dissolved in DMSO, antimicrobial effects of corresponding concentrations of DMSO were tested in order to make sure that DMSO doesn’t interfere with results. Superoxide in given concentrations and in combination
with detected concentration of H2O2 didn’t present any significant antimicrobial effect (<
1 log). Similarly 0.1 and 0.5 mM of superoxide and nitrate (9.35 mM) combinations have shown less than 1-log reduction. Combination of 1 mM superoxide with 9.35 mM ! 87! nitrate inactivated close to 1 log bacteria. Finally given concentrations of superoxide were combined with 0.33 mM nitrite. Results revealed that only combination of 1 mM superoxide with 0.33 mM nitrite was able to achieve more than 6.5-log reduction. This effect was disappeared when hydrogen peroxide and nitrate added to superoxide-nitrite mixture (Figure 31). These results led us to think that RNS might be primary reason for the antimicrobial effect of plasma treated NAC solution.
7.00" 6.61! 6.55! 6.44!
! 6.00"
5.00"
4.00"
3.00"
2.00"
Surviving Bacteria Log10N Surviving Bacteria 1.00" 0.00! 0.00! 0.00"
Tested**Species*and*Combinations*
Figure 31: Combination of nitrite and superoxide completely kills more than 6.5 log E. coli. Antimicrobial effect disappears when nitrate and hydrogen peroxide are added to nitrite-superoxide mixture.
Therefore we have tested antimicrobial effect of detected concentration of peroxynitrite
(ONOO-) on 107 CFU/ ml of E. coli. Since the peroxynitrite solution is provided in
NaOH solution, antimicrobial effect of corresponding concentrations of NaOH were also tested in order to ensure that NaOH doesn’t interfere with antimicrobial effect. As it is ! 88! demonstrated in Figure 32 0.36 mM and 0.18 mM of peroxynitrite has shown more than
5.5 and 3.5-log reduction respectively.
8.00" 7.06" 6.95" 6.73" 6.93" 6.73" ! 7.00"
6.00"
5.00"
4.00" 3.65" 3.63"
3.00" 1.77" 1.87" 2.00"
Log 10 N Survivng bacteria 1.00" 0.00" 0.00" 0.00" 0.00" PBS" 3% 1 mM 8.3 mM 0.5 mM 4.1 mM 0.36 mM 3 mM 0.18 m 1.5 mM 0.09 mM 0.75 mM H2O2" ONOO-" NaOH" ONOO-" NaOH" ONOO-" NaOH" M NaOH" ONOO-" NaOH" ONOO-" Concentration of Peroxynitrite!
Figure 32: Antimicrobial effect of different concentrations of peroxynitrite and corresponding concentrations of sodium hydroxide.
3.4 Discussion: According to our findings, antimicrobial effect of plasma treated NAC solution seems to be from chemical modifications in NAC solution during plasma treatment. The previous literatures, which have reported a moderate reduction in pH of
PBS or water always had a plasma discharge gap of more than 2 mm through 45 mm, and the amount of liquid more than 1.5 ml through 10 ml [105,156.157.184]. This drastically changes the chemical properties and pH equation of these solutions. Our findings show that acidic pH has no direct contribution on bacterial inactivation as has also been commonly reported by other groups [105,184,187]. However low pH seems to be a crucial for antimicrobial effect referring to antimicrobial effect of acidified nitrites
[188-190]. ! 89!
We have attempted to provide a recipe of plasma treated liquids that is able to inactivate the same amount of bacteria as plasma treated liquids do under same conditions. In our experiments detected concentrations of plasma-generated species haven’t shown any significant antimicrobial activity. That might be due to lack of other plasma-generated species. Plasma treated liquids are complex mixtures of ROS and RNS and carry various charged and uncharged molecules. Further detailed chemical analyses and chemical kinetic experiments are required to better understand exact roles of plasma-generated species on microbial inactivation. Oehmigen et al. have reported that the mixture of
- 0.074 mM H2O2 and 0.032 mM NO2 at pH 3 is able to inactivate 3.5-log E. coli [155].
However this effect is mostly related to exposure conditions of bacteria to hydrogen peroxide-nitrite mixture. In the mentioned study, bacteria was exposed 50 times more volume of mixture (50 μl bacteria: 2.45 ml mixture) as opposed to our 1:1 (50 μl bacteria: 50 μl mixture or plasma treated NAC) ratio and held for 60 minutes as opposed to our 15 minutes of holding time. Therefore our findings on nitrite-superoxide mixture, which demonstrates 7-log reduction under the same exposure conditions with plasma treated NAC, seems to be more reliable and relevant to our plasma treatment method. In addition, the inactivation rate of peroxynitrite in the detected range supports our approach on the more dominant contribution of RNS to microbial inactivation. However precise detection and quantification of superoxide is required for better understanding of interactions between oxygen and nitrogen species.
An interesting chemical modification was observed after 1-minute plasma treated
NAC solution. In the UV-visible spectrum of 1-minute plasma treated NAC solution we have seen a peak at 332 nm, which is specific for S-nitrosothiols (in our case S-nitroso- ! 90!
N-acetyl cysteine). Also S-nitrosothiols have another specific peak at 545 nm with 16
M-1 cm-1 molar extinction coefficient, which is more suitable to for concentration calculation due to lack of possible interference of other nitrogen species [199].
According to absorbance of the peak at 545 nm and the molar extinction coefficient concentration of S-nitroso-N-acetyl cysteine is calculated as 0.75 mM by using Beer-
Lambert equation (Figure 33a). S-nitrosothiols, with the general structure, RSNO (R is and organic group) are the S-nitrosylated products of thiols and are known as NO (nitric oxide) donors and carriers with characteristic pink color [199-201]. Our findings and observations on 1 minute plasma treated NAC solution regarding the formation of S- nitroso-N-acetyl cysteine is consistent and supported by previous literature; we have observed a pinkish color development in 1 minute plasma treated NAC solution as demonstrated in Figure 33b. ! 91!
0.2! 0.18! a! 0.16! 0.14! 0.12! 0.1! 0.08!
Absorbance* 0.06! 0.04! 0.02! 0! 274! 374! 474! 574! Wavelength*(nm)*
Figure 33: (a) Specific absorbance peak of S-nitroso-N-acetyl cysteine at 545 nm. (b) Structure of S-nitroso-N-acetyl cysteine. (c) Color change in 1 minute plasma treated NAC solution. Pinkish color formation is indicator of S-nitroso-N-acetyl cysteine (SNAC) in NAC solution after 1-minute non-thermal plasma treatment.
Formation of S-nitrosothiols (RSNO) involves the reaction of a thiol (RSH) with NO and
- NO derivatives such as NO2, NO2 , N2O3. By itself NO reaction with a thiol yield to disulfide formation rather than RSNO. However in the presence of oxygen or other oxygen species such as hydrogen peroxide, NO oxidation yields to formation of S- nitrosothiols [202]. Oxidation of thiols with plasma generated ROS involves the production of sulfanyl radical that will later react with NO to form S-nitrosothiol as shown in the equations below [206]. ! 92!
- " - RSH + O2 ! RS + HOO (23)
RSH + HOO- ! RS" + OH- + HO" (24)
" " RSH + HO ! RS + H2O (25)
Hence plasma generated ROS and RNS cause the modification of NAC to S-NAC by
- the end of 1-minute plasma treatment. It is well known that reaction of O2 and NO yields to peroxynitrite (ONOO-). S-nitroso-N-acetyl cysteine acts as NO donor via decomposition for peroxynitrite formation in plasma treated NAC solution. Not only plasma generated ROS and RNS but also UV light that is generated during plasma treatement and acidic pH as a result of plasma treatment of liquids have influence of the production and the decomposition of SNAC. UV light and the presence of thiols
(unreacted NAC) in acidic environment induce decomposition of S-nitrosothiols (in our case S-NAC) [201]. Nitric oxide (NO) is released via the decomposition of S-NAC and
- reacts with plasma-generated superoxide (O2 ) for peroxynitrite formation. Another mechanism for the S-nitrosothiol mediated peroxynitrite formation, which is relevant to plasma treated liquids, involves the reaction of hydrogen peroxide with S-NAC. The mentioned mechanism involves the dissociation of hydrogen peroxide and the reaction
- of hydroperoxyl (HO2 ) with S-NAC as given in the following equations [204].
+ - H2O2 ! H + HOO (26)
RSNO + HOO- ! RS- + ONOO- + H+ (27)
In addition to plasma-generated superoxide, by itself S-nitrosothiol formation
mechanism can lead superoxide production via reduction of O2 by RSNO-H, a radical intermediate, as given in following equations [205]. ! 93!
RSH + NO ! RSN"-O-H (28)
" - RSH + NO ! RSN -O-H + O2 ! RSNO + O2 (29)
Our FT-IR and NMR data suggests that about ~90% NAC is converted to cysteic acid.
This mechanism involves the oxidation of NAC as given in equations 23-25. Oxidation of thiols is resulted with formation of disulfides that later can be oxidized to sulfonic acid (in our case cysteic acid) [206,207]. In the reaction, where NAC is converted to cysteic acid, also acetic acid is formed due to cleavage of acetyl group. The overall reaction is shown in the equation below:
- C5H9NO3S + HOO ! C3H7NO5S + C2H4O2 (30) (NAC) (Cysteic acid) (Acetic acid)
It can be easily seen from equation 30 that, in order to fulfill the atomic balance in the reaction, NAC should react with HOO-, which might be presenting in the plasma treated
NAC solution due to the dissociation of H2O2 as shown in equation 26, or protonation of superoxide This possible reaction mechanism explains the decreased hydrogen peroxide concentration in 3 minute plasma treated of NAC solution compared to 2-minute treated one. Also formation of cysteic acid and acetic acid contributes to the rapid and drastic pH drop in NAC solution following plasma treatment.
In conclusion, our findings suggest that plasma treatment turns NAC solution into an acidic mixture of ROS and RNS that both contribute to bacterial inactivation.
NAC seems to be in the center of all reactions and interactions between ROS and RNS. ! 94!
NAC by itself serves as a source of RNS by releasing NO and also source of ROS through intermediates on its reactions with other ROS. Even though antimicrobial effect can be attributed to both ROS and RNS, based on our results we speculate that dominantly ROS play a role in the modification of NAC molecule and RNS seems contribute to antimicrobial effect more dominantly. The action of mechanism of ROS and RNS on microbial inactivation will be discussed in detail in Chapter 5.
! 95!
! ! 4 CHAPTER IV: ANTIMICROBIAL STABILITY AND TOXICITY OF PLASMA-TREATED N-ACETYL CYSTEINE (NAC) SOLUTION
4.1 Introduction:
Effects of non-thermal plasmas on eukaryotic cells and living tissues have been studied by various groups, and also discussed in the Chapter 1. Direct applications of non-thermal plasmas have various effects on eukaryotic cells such as; blood coagulation, living tissue sterilization, tooth whitening, wound healing, etc. [95,99,120,146,173].
Next to exciting applications and biological effects of non-thermal plasmas on tissues, its toxicity is correlated with the plasma dose that is applied [85,95,141,209]. In particular cytotoxic effects of non-thermal plasma applications were attributed to DNA and cell membrane damage that rise from plasma-generated species and UV generation during plasma treatment [134,135]. However, reports regarding toxicity of plasma treated liquids on eukaryotic cells and tissues are limited. Since effects of electron discharge,
UV generation and localized heating are excluded by fluid-mediated plasma treatment, toxicity of plasma treated liquids can be attributed plasma-generated species and chemical modifications in the plasma treated liquid. Kim et al. have investigated toxicity and antimicrobial effect of liquid phase discharge plasma. In their study, PBS was placed on human skin, which was harvested by biopsy and treated with the liquid plasma electrode where it is in direct contact with the PBS. In other words they have performed direct treatment of skin with plasma in the liquid interface and they have reported that their treatment method doesn’t cause any significant toxicity on the skin [210]. Even though this study doesn’t represent the fluid-mediated plasma treatment, it is a close ! 96! model that can be taken into consideration. We recently reported that non-thermal DBD plasma-treated calcium alginate gel isn’t cytotoxic to endothelial cells and induces cell proliferation after and transient slowed growth [154]. Plasma-generated species have effect on cell death in a concentration dependent manner. For instance, hydrogen peroxide either can stimulate or reduces cell proliferation or cell apoptosis depending on its concentration. Similarly nitric oxide can enrich or reduce hydrogen dependent cell apoptosis in concentration dependent manner [211-213].
Studies about antimicrobial effects of plasma treated liquids for extended period of time are limited in the literature. Traylor et al. have reported that indirect DBD plasma treated water can retain its antimicrobial activity for up to 7 days when exposed to bacteria for 3 hours. They have observed ~5 log and ~2.4 log reduction 2 and 7 days after plasma treatment respectively. However in case of 15-minute exposure of indirect
DBD plasma treated water to bacteria, the antimicrobial effect diminishes in 30 minutes and completely disappears in seven days after plasma treatment [110]. In a similar study
Julak et al. have reported that 1 ml water and PBS samples that are treated for 1 hour with positive corona discharge can preserve its antimicrobial activity for up to 1 month
[182].
4.2 Materials and Methods:
4.2.1 Delayed Exposure and Aging of Non-Thermal DBD Plasma-Treated NAC
Solution: The duration between the termination of plasma treatment and the exposure of plasma treated liquid to bacteria is defined as delay time. In order to see how long 3- minute plasma treated NAC solution can retain its antimicrobial activity, plasma treated ! 97!
NAC solution was kept at +4o C and room temperature from 0 to 28 days in microcentrifuge tubes with their caps sealed with parafilm. For each delay time point one microcentrifuge tube was opened, and 3-minute plasma treated NAC solution was exposed to 107 CFU/ml E. coli, held for 15 minutes and colony counting assay was performed as previously described. For 0 day delay time, plasma-treated liquid was exposed to bacteria immediately after plasma treatment. For aging experiments, protocol of the U.S. Food and Drug Administration for aging pharmaceutical compounds [208] was used. 1 ml of NAC solution was treated for 3 minutes and immediately transferred to glass vials, screw caps placed, and vials sealed with parafilm and kept in thermostatically controlled incubator at 37oC and 50oC for over the time. The protocol for accelerated aging (also known as 10 degree rule) was developed around the collision theory-based Arrhenius model. The equivalent aging time to actual incubation time is calculated from the following equation:
TimeRT TimeA = [(T1 −TRT ) 10] Q10
Where as; TimeA is the actual incubation time, TimeRT is the equivalent time at room temperature to aging time at storage temperature, Q10 is reaction-rate coefficient, T1 is € aging temperature and TRT is room temperature (ambient temperature, in which the compound is stored for practical application).
Following steps should be considered in order to apply the given equation for the accelerated aging test:
• The reaction rate coefficient should be selected Q10 =2 unless a specific
rate coefficient is determined for the compound experimentally. ! 98!
• The ambient temperature should be selected between 20 and 25oC
• The aging temperature shouldn’t be selected more than 60oC
Aging time points and corresponding equivalent time points for both 37 and 50OC aging temperatures are depicted in Figure 34.
300!
255! 250! Aging!Days!@!37oC! Aging!Days!@!50oC!
200! 191!
150! 127! Days%for%aging% 100! 103! 78! 64! 50! 52! 32! 21! 26! 11! 9! 13! 0! 2!5!4! 0! 60! 120! 180! 240! 300! 360! 420! 480! 540! 600! 660! 720! Equivalent%to%Actual%
Figure 34: Relationship between aging time and corresponding equivalent time for accelerated aging experiment for 37 and 50OC aging temperature.
In order to evaluate the antimicrobial effect of aged NAC solution at the indicated time point, one vial was removed, and the solution was exposed to equal volume of
(50μl:50μl) 107 CFU/ml E. coli, held for 15 minutes and colony counting assay was performed as descried previously.
4.2.2 Antimicrobial Effect of Components of Plasma-Treated NAC Solution: In brief, NAC solution is prepared by dissolving NAC powder (solute) in PBS (solution). ! 99!
As discussed in Chapter 3, plasma treatment induces chemical modifications both in whole solution and NAC molecule. In order to evaluate the contribution of solute (NAC molecule) and solution (PBS) parts of plasma treated NAC solution, a separation experiment was carried out as demonstrated in Figure 35. After 3-minute plasma treatment, NAC solution was immediately transferred to a glass beaker, the glass beaker was closed with a glass petri dish and heated on a hot plate by not exceeding 50OC until the whole liquid part was evaporated. The evaporated liquid part condensed and collected separately in a microfuge tube. Dried, plasma-treated NAC powder (solute) was reconstituted in three different ways:
1st with PBS, same volume evaporated liquid that yields to 5 mM final
concentration,
2nd with PBS, the half of the evaporated liquid volume that yields to 10 mM final
concentration and
3rd in the same volume of evaporated liquid. (for a final concentration of 5 mM).
Also the condensed liquid part of plasma treated NAC solution was exposed to bacteria in 1:1 (50μl bacteria: 50μl solution) and 1:2 (50μl bacteria: 100μl solution) ratios.
The separation process was repeated for pH measurement, antimicrobial tests and UV- vis spectra analyses separately. For antimicrobial tests, each liquid was exposed to equal volume of (50μl:50μl) 107 CFU/ml E. coli, held for 15 minutes and colony counting assay was performed as previously described.
! 100!
Figure 35: Experimental method for separation experiment. Liquid part of plasma- treated NAC solution is evaporated and then condensed. Dried powder was reconstituted with the same and half of the evaporated volume of PBS and same volume of condensed liquid part. Antimicrobial effects of all reconstituted liquids were tested and their UV-vis spectra were collected.
Also NAC powder was treated with DBD plasma for 3 minutes and than NAC solution was prepared by dissolving the treated NAC powder in PBS to have 5 and 10 mM of final concentration. These solutions were exposed to equal volume of 107 CFU/ ml E. coli and colony counting assay and pH measurements were performed.
4.2.3 Effect of Vitamin E on Antimicrobial Properties of Plasma-Treated NAC
Solution: As previously described, plasma generated ROS and RNS are diffused into
NAC solution during plasma treatment. Plasma treated NAC solution (1,2, and 3 minutes) was exposed to equal volume of 107 CFU/ml E. coli suspension, which was prepared in varying concentrations of Vitamin E (alpha tocopherol) solution (10, 25, 50 and 100 mM) in order to evaluate oxidative and nitrosative capacity of plasma treated ! 101!
NAC solution. Following exposure of plasma treated NAC solution, bacteria were held for 15 minutes and colony-counting assay was performed as previously described.
4.2.4 Repeated Exposures of Plasma-Treated NAC Solution: In order to assess how plasma treated NAC solution shows its antimicrobial activity on bacteria, we have repeatedly exposed plasma treated NAC solution on bacteria pellet. In other words we have tried to demonstrate if all plasma-generated species are consumed immediately following exposure to bacteria, or required amount of them are consumed and rest of the plasma treated NAC solution remained in plasma treated NAC solution. In repeated exposure experiments 100 μl of 107 CFU/ml bacteria suspension was spun down at 8000 rpm for 10 minutes and supernatant was removed. Then 100 μl of 3-minute plasma treated NAC solution was exposed on bacteria pellet, homogenized by vortexing and held for 5 minutes and spun down for 10 minutes at 8000 rpm. So total holding time was kept 15 minutes (5 minutes exposure to bacteria and 10 more minutes during centrifugation) similar to previous experiments. After centrifugation supernatant, which is plasma treated NAC exposed to bacteria was collected and exposed to another bacteria pellet (Figure 36). Same procedure was repeated for 4 times. ! 102!
Figure 36: Flow chart of the experimental procedure for repeted exposures of plasma- treated NAC solution.
After each exposure of plasma treated NAC to bacteria, reacted NAC was separated by centrifugation and treated bacterial pellet was resuspended in sterile PBS and colony counting assay was performed as previously described.
4.2.5 Cytotoxicity of Plasma-Treated NAC Solution: Cytotoxicity of plasma treated
NAC solution was tested on the EA.hy926 human somatic umbilical vein endothelial cell line. EA.hy926 cell line was kindly provided by Dr. Peter Lelkes’ laboratory, at
Drexel University, School of Biomedical Engineering, Science & Health Systems.
Frozen stocks of cell line were thawed in water bath at 37OC for 3 minutes. 1 ml of thawed cells was transferred to 15 ml centrifuge tube and diluted with 1% FBS (Fetal
Bovine Serum) containing 9 ml of DMEM (Dulbecco’s Modified Eagle Medium)
(Invitrogen, Grand Island, NY, USA). Afterwards cells were spun down at 1500 rpm for ! 103!
5 minutes and supernatant was removed. 10% FBS and 1% penicillin-streptomycin containing 10 ml of fresh DMEM was added to the cell pellet and cells were gently homogenized by pipetting and transferred in to T25 cell culture flask (Corning, Corning,
O NY, USA) then incubated in CO2 incubator at 37 C for 24 hours. Grown cells were trypsinized and transferred to T75 cell culture flasks and incubated in CO2 incubator at
37OC until cells become 90% confluent. Confluent cells were trypsinized and counted with hemocytometer in order to fix cell number to ~20000 to seed in 96 well plate. 10%
FBS and 1% penicillin-streptomycin containing 200 μl of fresh DMEM was added to
O cells in each well and cells were incubated in CO2 incubator at 37 C until they get 90% confluent (~ 30000 cells/well). Confluent cells were washed with 1X sterile PBS for twice and 100 μl of serum free DMEM added on cells. Next, 100 μl of untreated and plasma treated NAC solutions (for 1, 2, and 3 minutes) were added on cells and held for
5, 10, 15, and 30 minutes. Following holding time, media and plasma treated NAC solution was removed; cells were washed with 1X sterile PBS for twice and 10% FBS and 1% penicillin-streptomycin containing 200 μl of fresh DMEM was added. Cells
O were incubated in CO2 incubator at 37 C for 24 and 48 hours for recovery. Serum free
DMEM and 4% chlorhexidine solution were used as negative and positive controls respectively. Following recovery period cell viability was determined by XTT assay as previously described.
4.2.6 Systemic Toxicity of Plasma-Treated NAC Solution in Rats: One of the possible practical applications of plasma treated NAC solution is to use it as a catheter lock solution for the eradication of biofilms, grown in IV catheters that are one of the main causes of sepsis. In this application, there is always potential risk of leakage of ! 104! plasma treated NAC solution into the bloodstream. In order to evaluate the possible toxic effects of plasma treated NAC solution in the event of leakage, we have administrated different doses of 3-minute plasma treated NAC solution to 3-month old female rats
(Sprague-Dawley strain). Rat infusion experiments were carried out in accordance with the Guide for the Human Care and Use of Laboratory Animals. The experimental procedure (Protocol# 19512) was approved by the Institutional Animal Care and Use
Committee, Drexel University (IACUC). Sprague-Dawley rats were purchased from
Harlan Laboratories (Indianapolis IN, USA). Upon arrival, rats were acclimatized for 5 days. Rats were divided in to five groups; no injection was performed in the first group of rats, 1ml/day untreated NAC solution was administrated to the second group of rats for 3 days, 1ml/day plasma treated NAC solution was administrated to the third group of rats for 3 days, 3 ml of plasma treated NAC solution was administrated to the fourth group of rats for one time and 3 ml/day plasma treated NAC solution was administrated to the fifth group of rats for three days. Administration of plasma treated NAC solutions were performed using 20 gauge IV catheter via tail vain. Prior to catheter placement rats were anesthetized with 3% isoflurane inhalation. NAC solutions that are used in infusion experiments were prepared in 0.9% saline solution. Therefore antimicrobial effect of
NAC solution prepared in 0.9% saline solution was tested as previously described in
Chapter 2. Following the final injections, rats were closely observed for 1 week. After 1 week of observation rats were euthanized with overdose sodium pentobarbital and phenytoin solution injection via portal vein. Liver, kidney, tail vein, heart and lung tissue samples were collected from euthanized rats and sent to ANTECH Diagnostics (Lake
Succes, NY, USA) for histopathology evaluation. Also blood samples were collected ! 105! before scarification of rats and sent to ANTECH Diagnostics for analysis in order to evaluate alterations in blood chemistry.
Antimicrobial activity of plasma treated NAC solution can be considered as its oxidative and nitrosative capacity, which also cause toxicity in eukaryotic cells. In order to simulate the scavenging capacity of blood during infusion experiments, antimicrobial effect of plasma treated NAC solution (prepared in 0.9% saline solution for injection purpose) was tested in the presence of blood. For this aim 107 CFU/ml E. coli culture suspension was prepared in PBS containing 1%, 25% and 50% blood, equal volume of plasma treated NAC solution was added on bacteria suspension, held for 15 minutes and colony-counting assay was performed as previously described in Chapter 2.
4.3 Results:
4.3.1 Antimicrobial Effect of Plasma-Treated NAC Solution Over Time: As demonstrated in Figure 37, 3-minute plasma treated NAC solution can retain its antimicrobial activity for up to 28 days when it is stored at room temperature and +4OC separately. For clarity negative control results are represented for Day 0. However for each delay time point initial bacterial load was 107 CFU/ml (7-log surviving bacteria). ! 106!
8.00" 7.08"7.07"
! 7.00" NAC stored at room temperature" 6.00" NAC stored at 4°C" 5.00"
4.00"
3.00"
2.00" Log10N Surviving Bacteria 1.00" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0" 0.00"
Days of Storage!
Figure 37: Plasma-treated NAC solution can retain its antimicrobial activity when stored at both room temperature and +4OC.
Similarly, as demonstrated in Table 5, accelerated aging experiments showed the same occurrence, where all aged samples retained their antimicrobial activity for 255 and 103 days, which are equivalent to 2 years (720 days) at room temperature when stored at
37OC and 50OC respectively. pH measurement results suggest that pH of plasma treated
NAC solution remains stable independent of storage duration or temperature.
! 107!
Table 5: Colony-counting assay showing antimicrobial efficacy of accelerated aging of plasma-treated NAC solution. (Results are shown as log10(N) surviving bacteria.)
37O C 50O C Equivalent Days (-) (+) Plasma Days (-) (+) Plasma Days Incubated Ctrl. Ctrl. Treated Incubated Ctrl. Ctrl. Treated NAC NAC 15 5 7.05 0 0 2 7.03 0 0 30 11 7.03 0 0 4 7.09 0 0 60 21 7.03 0 0 9 7.07 0 0 90 32 7.04 0 0 13 7.05 0 0 180 64 7.03 0 0 26 7.03 0 0 360 127 7.02 0 0 52 7.06 0 0 540 191 7.05 0 0 78 7.02 0 0 720 255 7.04 0 0 103 7.03 0 0
4.3.2 Antimicrobial Effect of Separated Components of Plasma-Treated NAC
Solution: As explained in the Materials and Methods section of Chapter 4, powder of plasma treated NAC obtained by evaporation. Dried plasma-treated NAC powder was reconstituted in three different ways:
1st in PBS, using the same volume of evaporated liquid that yields to 5 mM final
concentration,
2nd in PBS, using the half of the evaporated liquid volume that yields to 10 mM
final concentration and
3rd in the same volume of evaporated liquid. (to yield a final concentration of 5
mM)
It is important to remember that given final concentrations don’t represent concentrations of plasma generated species and refers to the initial concentration of unreacted NAC. ! 108!
In the first case no antimicrobial effect was observed. In the second and third cases 7-log reduction was observed. When condensed liquid portion of plasma treated NAC solution was used, no inactivation was observed. pH of reconstituted solutions was increased slightly and measured as 4.2, 3.5, and 3 for 1st, 2nd, and 3rd case respectively. Also condensed liquid retained its acidity, where the pH was measured as 3. Figure 38 summarizes inactivation and pH measurement results of reconstitution experiments.
log!Surviving!Bacteria! pH!
8.00! 7! 7.08! 6.2! 6.67! 6.64! 7.00! 6.50! 6!
6.00! 5!
5.00! 4.2! 4! 4.00! 3.5! pH% 3! 3! 3.00! 2.3! 2.4! 2.4!
log%Surviving%Bacteria% 2! 2.00!
1.00! 1! 0.00! 0.00! 0.00! 0.00! 0.00! 0! Untreated! Treated! H2O2! Recons.!1:1! Recons.!2:1! Condensed! Condensed! Recons.!1:1! NAC! NAC! (Final!Conc.!(Final!Conc.! Liquid!(1:1! Liquid!(1:2!Cond.!Liquid! 5!mM)! 10!mM)! Bac.:CL)!! Bac.:CL)! (5!mM)! Conditions%
Figure 38: Antimicrobial activity and pH of plasma-treated NAC solution following drying and reconstitution process. Antimicrobial activity and pH of condensed liquid portion of plasma-treated NAC solution is also demonstrated.
In case of DBD plasma treatment of NAC powder by itself and preparation of
NAC solution with plasma-treated NAC powder, no inactivation effect was achieved with both 5 mM and 10 mM solutions. pH of prepared solutions were measured as ~6
(Figure 39). ! 109!
log!Surviving!Bacteria! pH!
8.00! 7! 7.08! 6.69! 6.72! 7.00! 6.2! 6! 6.1! 6! 6.00! 5! 5.00! 4! 4.00! pH% 3! 3.00! 2.3! 2! 2.00! log%Surviving%Bacteria% 1.00! 1! 0.00! 0.00! 0.00! 0! Untreated!NAC!! Treated!NAC! H2O2! Treated!NAC,! Treated!NAC,! Dissolved!(Final!Dissolved!(Final! conc.!5!mM)! conc.!10!mM)! Conditions%
Figure 39: pH and antimicrobial effect of NAC solution, where NAC powder first treated by itself and then dissolved in PBS. Final concentrations are 5 mM and 10 mM.
UV-visible spectra of reconstituted solutions showed a specific peak at 302 nm similar to plasma treated liquid. Peaks at 302 nm may represent ONOO- and concentrations of ONOO- were calculated as 0.18 and 0.36 mM for the above-mentioned
1st and 2nd reconstitution cases respectively (Figure 40).
1.4" 3!min.!treated!NAC! NAC!Recons.!1:1! 1.2" NAC!Recons.!2:1! 1" !
0.8"
0.6"
0.4" Absorbance (AU)
0.2"
0" 260" 280" 300" 320" 340" 360" 380" 400" 420" Wavelength (nm)!
Figure 40: UV-visible spectra of dried and reconstituted plasma-treated NAC solution. ! 110!
4.3.3 Effect of Vitamin E on Antimicrobial Properties of Plasma-Treated NAC
Solution: Scavenging effect of Vitamin E is closely related with its concentration and plasma treatment time. As represented in Figure 41, 100 mM of Vitamin E reduced inactivation of bacteria by ~1.3 log when 2 –minute plasma treated NAC was exposed.
However even 100 mM of Vitamin E was not able to scavenge plasma-generated species in 3-minute plasma treated NAC. 1-minute plasma treated NAC solution hasn’t shown any antimicrobial effect on bacteria even in the absence of Vitamin E. Therefore scavenging effect of Vitamin E on 1-minute plasma treated NAC solution was unclear.
0!mM! 10!mM! 25!mM! 50!mM! 100!mM! 8.00! 7.32" 7.31" 7.30"7.31" 7.31" 7.30" 7.32" 7.32" 7.06" 7.14" 7.21" 7.00! 6.51" 5.90"5.87"5.99" 6.00!
5.00!
4.00!
3.00!
2.00! log%10N%Surviving%Bacteria% 1.00! 0.00"0.00"0.00"0.00"0.00" 0.00! 0!min! 1!min! 2!min! 3!min! Plasma%Treatment%Time%(minutes)%%
Figure 41: Effect of various concentrations of Vitamin E on inactivation rates of bacteria following exposure to plasma-treated NAC.
4.3.4 Repeated Exposures of Plasma Treated-NAC Solution: Our findings reveal that plasma-generated species in plasma treated NAC solution aren’t consumed immediately, and the consumption amount is closely related to bacteria number that is exposed to ! 111! plasma treated NAC. As demonstrated in Figure 42, 3-minute plasma treated NAC solution possesses sufficient plasma-generated species in it even up to 4th repetitive exposure to bacteria pellet.
8.00$ 7.00$ 7.00$ ! 6.00$
5.00$
4.00$
3.00$ log Surviving Bacteria log 2.00$
1.00$ 0.00$ 0.00$ 0.00$ 0.00$ 0.00$ ctrl$[A]$ first$use$[B]$ second$use$[C]$$$ third$use$[D]$ forth$use$[E]$ Conditions!
Figure 42: Antimicrobial effect of 3-minute plasma-treated NAC solution following consecutive exposure to bacteria.
Furthermore, UV-visible spectrum of plasma treated NAC solution that is exposed to bacteria shows that 3-minute plasma treated NAC solution retains sufficient plasma- generated species. As it can be seen in Figure 43, after the exposure of 3-minute plasma treated NAC solution to 107 CFU/ml of bacteria the peak at 302 nm still exists, and a new peak starts appearing at 286 nm. However after exposure of 3-minute plasma treated NAC solution to bacteria pellet, which was obtained by centrifuging 109 CFU/ml bacteria, complete inactivation couldn’t be achieved at repeated exposures and the peak at 302 nm disappears and the intensity of the peak at 286 nm increases. Also the intensity of the new peak that appears at 286 nm increases as the holding time increases. ! 112!
Also UV-spectrum of ATL buffer (a kind of detergent used in DNA isolation process to break down the cell membrane) was also obtained after exposure to bacteria in order to investigate the peak at 286 nm, which will be discussed in detail in the discussion section of this chapter.
ONOO" 0.9! Used ONOO" 0.8! 3 min. Treated NAC"
0.7! 15 min. Held on 107 CFU/ml" 15 min. Held on 109 CFU/ml" 0.6! 45 min. Held on 107 CFU/ml" 0.5! 45 min. Held on 109 CFU/ml"
0.4! ATL Buffer on 109 CFU/ml"
Absorbance%(AU)% 0.3!
0.2!
0.1!
0! 260! 280! 300! 320! 340! 360! 380! 400! Wavelength%(nm)%
Figure 43: UV-visible spectra of plasma-treated NAC solution in comparison with UV- visible spectra of ONOO- and ATL buffer after exposure to bacteria.
4.3.5 Cytotoxicity of Plasma-Treated NAC Solution: Following the exposure of plasma treated NAC solutions (untreated, and 1,2, and 3-minute treatement), cells were incubated in 10% serum containing media for recovery for 24 and 48 hours separately.
As demonstrated in Figure 44, EA.hy926 human somatic umbilical vein endothelial cells that are exposed to 3-minute plasma treated NAC die after both 24 and 48 hours of recovery periods. ! 113!
24%hours%of%recovery% 160! a! 140!
120! 105! 100! 100! 100! 100! 95! 100! 91! 87! 85! 79! 72! 80! 68! 70! 60! 60!
%%of%Surviving%Cells% 42! 37! 40!
20! 1! 0! 0! 0! 0! 0! 0! 0! 0! 0! 1! 2! 3! SFM!1%! 0! 1! 2! 3! SFM!1%! 0! 1! 2! 3! SFM!1%! 0! 1! 2! 3! SFM!1%! min!min!min!min! CH! min!min!min!min! CH! min!min!min!min! CH! min!min!min!min! CH!
5!min! 10!min! 15!min! 30!min! Treatment%Time%&%Holding%Time%
48%hours%of%recovery% 200! b! 180!
160! 146!147! 140! 120! 121! 113! 116! 120! 109! 100! 103! 100! 100! 100! 97! 94! 100! 92!
80! 72! %%of%Surviving%Cells%% 60!
40!
20! 5! 0! 0! 0! 0! 0! 0! 0! 0! 0! 1! 2! 3! SFM!1%! 0! 1! 2! 3! SFM!1%! 0! 1! 2! 3! SFM!1%! 0! 1! 2! 3! SFM!1%! min!min!min!min! CH! min!min!min!min! CH! min!min!min!min! CH! min!min!min!min! CH!
5!min! 10!min! 15!min! 30!min! Treatment%Time%&%Holding%Time%%
Figure 44: Cytotoxicity of plasma-treated NAC solutions on EA.hy926 human somatic umbilical vein endothelial cells after (a) 24 and (b) 48 hours of recovery period.
However cells that are exposed to 2-minute plasma treated NAC solution for 15 minutes were able to recover to 95% and more than 100% viability after 24 and 48 hours of recovery period respectively. It is important to recall that in 96 well plate can ! 114! accommodate maximum ~3x104 cells when cells are confluent. Therefore, 2-minute plasma treated NAC solution was also exposed to 5x104 and 105 CFU/ml bacteria for 15 minutes in order to evaluate its antimicrobial activity on given bacterial concentrations.
Inactivation assay showed that 2-minute plasma treated NAC solution is able to inactivate 5x104 and 105 CFU/ml E. coli in 15 minutes of holding time.
4.3.6 Systemic Toxicity of Plasma-Treated NAC Solution in Rats: As depicted in
Figure 45, 3-minute plasma treated NAC solution lost its antimicrobial activity when it was exposed to bacteria suspension that was prepared in the presence of 25% and 50% blood. In other words, loss of the antimicrobial effect can be interpreted as the loss of the oxidation capacity of the plasma treated NAC. This in vitro result suggests that the administration of NAC solution to rats is likely to be safe.
8" 7.06" 6.93" 7.00" 7"
6"
5"
4"
3"
log$Surviving$Bacteria$ 2"
1" 0.00" 0.00" 0" 0% blood" 1% blood" 25% blood" 50% blood" 0 min" 3min" Plasma Treatement Time (min) & Blood Concnetration (%)!
Figure 45: Effect of the presence of blood on the antimicrobial effect of 3-minute plasma-treated NAC solution. Antimicrobial effect of 3-minute plasma treated NAC solution diminishes in the presence of at least 25% blood.
! 115!
After administration of 3-minute plasma treated NAC solution to rats histopathological examination was performed on liver, kidney, lung and tail vein tissue samples. According to the histopathological examination report provided by ANTECH
Diagnostics (Lake Succes, NY, USA) a general conclusion regarding the parenchymal organs (liver, lung and kidney) is absence of lesions, which are believed to be a direct consequence of the experimental manipulations. Special attention is directed to the kidney and liver, which are the concentrating and metabolizing organs most likely to be affected by toxic substances. Neither kidney nor liver in any of rats exhibits any lesions at the light microscopic examination, which are referable to toxicity. There is no hepatocellular degeneration/necrosis, inflammation, or hepatocellular vacuolation. Mild to moderate microvascular congestion is a feature shared in common by kidney, liver, and lung. It is either normal physiological congestion or perhaps artifactual congestion that is associated with terminal agonal cardiovascular collapse. Mild to moderate peribronchiolar lymphoid hyperplasia is observed in most of rats, a very common observation even in normal, healthy, non-experimental rats representing a cellular response to inhalant antigens and/or irritants, also most likely unrelated to the experimental protocol. This is a pulmonary arteriolar adaptation to vascular hypertension, which may be spontaneous or induced but generally not a sign of toxicity.
There are several instances of mild to moderate tail vein thrombosis with generally mild non-necrotizing vasculitis and peri-vasculitis in a few instances also mild perivascular fibrosis. However, the vascular lesion is not sufficient to have caused regional infarction.
The tail vein distal to the injection site is likely to be normal. ! 116!
Even though there are some variations, blood tests don’t represent any specific signs of toxicity due to plasma treated NAC solution. Table 6 summarizes results of the blood test compared to reference values. As seen on Table 6, none of these variations can be correlated to any specific experimental group.
Table 6: Acute toxicity assay: Findings on systemic administration of plasma-treated NAC solution. Comparative blood test results are shown with reference to normal values. Control Untreated 3 ml/day x 3 3 ml/day x 1ml/day x 3 NAC days 1day days AST H H H H H ALT H H H ALP H H H Glucose H H H H Creatinine L K+ H H WBC L L L -3 PO4 H H Na+ L Globulin L H represents higher value of given parameter than the reference value and L represents the lower value of the given blood [parameter than the reference value. (AST: Aspartate transaminase, ALT: Alanine transaminase, ALP: Alkaline phosphatase, WBC: White blood cells)
4.4 Discussion:
In our study plasma treated NAC solution retains its antimicrobial activity for longer period of time as compared to the limited existing literature [110,182]. It is important to consider the plasma treated liquid ratio to bacteria concentration and the exposure time while evaluating the antimicrobial effect over the time. For example
Traylor et al. have exposed their plasma treated water to bacteria pellet for 3 hours, as ! 117! opposed to our experiments with bacteria suspension at 15 minute of exposure [110].
Thus, in their experiment the plasma treated water isn’t diluted by the bacteria suspension and reacts directly with bacteria rather than interaction with impurities that rise from liquid (such as PBS or growth media) that bacteria are suspended. The type and the amount of liquid that is being treated, plasma source and the treatment method all influence the ability of the plasma treated liquid to retain its antimicrobial activity.
For example, in different studies with varying parameters mentioned above, plasma treated liquids lost their antimicrobial activities in a range from 30 minutes to 7 days after plasma treatment [110,156,178,182]. Furthermore in these studies, water, PBS or saline solution were treated. All these liquids are less complex as compared to NAC solution. Our accelerated aging experiment results revealed that plasma treated NAC solution can still inactivate 7-log bacteria 2 years after plasma treatment. Even though 2- year time period is a theoretical value, which corresponds to the storage for 255 days at
37OC (or 103 days at 50OC), it is still a way longer period than in the previous reports.
We attribute this effect to the complexity of NAC solution as compared to water, PBS or saline solution. During plasma treatment of NAC solution not only ROS and RNS are generated, but also NAC molecule is modified. Products such as SNAC after 1 minute, and cysteic acid and acetic acid after 3 minutes are produced and some part of NAC molecule (~10%) remaines unreacted. All these products following the plasma treatment make plasma treated NAC solution more complex than plasma treated water, PBS, and saline solution and may be the main reason for longer retained antimicrobial activity by stabilization of short living ROS and RNS with an unknown mechanism. ! 118!
The UV-visible spectra obtained from reconstitution experiments reveal that the intensity of the peak at 302 nm is directly related with concentration. In other words following heating some peroxynitrite diminishes. When the remaining powder is reconstituted with PBS in a 1:1 ratio (same as the evaporated liquid), the absorbance of the peak at 302 nm is measured as 0.31, and when the powder is reconstituted with PBS in a 2:1 ratio (half volume of the evaporated liquid), the peak at 302 nm is measured as
0.62. With a basic dilution calculation we calculated that if dried powder is reconstituted with PBS in a 1.5:1 ratio (0.75 times of evaporated volume), the peak intensity should be same as the 3-minute plasma treated NAC solution. Therefore, dried NAC powder was reconstituted with PBS in a 1.5:1 ratio, its antimicrobial effect was tested using colony counting assay, and nitrite, nitrate and hydrogen peroxide concentrations were measured using HACH nitrite/nitrate detection kit and hydrogen peroxide assay kit respectively and UV-visible spectrum was obtained. As demonstrated in Figure 46, intensity of the peak at 302 nm became 0.47 same as 3-minute plasma treated NAC solution. In addition, the dried powder that was reconstituted in 1.5:1 ratio achieved 7-log reduction and nitrate, nitrite and hydrogen peroxide concentrations were measured as 0.8 mM, 0 mM, and 0 mM respectively. Through these results we speculate that products of the NAC molecule somehow act similar to a spin trap and play a role in the stabilization of plasma generated reactive species. Spin trap is a molecule that reacts with a short living radical and forms a stable radical adduct [214]. However the stabilization process is unclear and further experiments are required in order to understand the mechanism of stabilization of plasma-generated species.
! 119!
1.4" 3 min. treated NAC" 1:1 recons. NAC powder" 2:1 recons. NAC powder" 1.5:1 recons. NAC powder" 1.2"
! 1"
0.8"
0.6" Absorbance (AU)
0.4"
0.2"
0" 250" 270" 290" 310" 330" 350" 370" 390" 410" 430" 450" Wavelength%(nm)%
Figure 46: Comparison of UV-visible spectra of 3-minute plasma-treated NAC solution and reconstituted NAC powder. Note that, intensities of the peak at 302 nm are same for 3-minute plasma treated NAC solution and 1.5:1 reconstituted NAC powder samples. The dashed line indicates the 302 nm.
NAC powder treatment experiments show that, presence of water in treatment medium is essential for acidic pH and antimicrobial activity.
We have shown that after 2-minute plasma treatment vitamin E (α-tocopherol) scavenges plasma-generated species and protects bacteria in to some extend. However after 3-minute plasma treatment vitamin E remains ineffective on scavenging plasma- generated species. In a previous study Joshi et al. have reported complete protection effect of vitamin E on microbial cells during direct plasma treatment [98]. In direct ! 120! plasma treatment ROS and RNS generated gradually and increases with time. During direct plasma treatment, generated ROS and RNS might be scavenged by vitamin E simultaneously. However when plasma treated NAC solution comes in direct contact with vitamin E, ROS and RNS would be scavenged to some extend and remaining ROS and RNS inactivate bacteria.
Similar to vitamin E scavenging capacity, species in plasma treated NAC solution are not completely consumed when exposed to 107 CFU cell pellet and plasma treated NAC solution retains its antimicrobial activity in subsequent exposures. However when the cell number is increased to 109 CFU, NAC solution completely inactivates bacteria and can’t retain its antimicrobial activity in further exposures. As it is demonstrated in Figure 43, when 3-minute plasma treated NAC solution is exposed to
107 CFU bacteria and held for 15 and 45 minutes, intensity of the peak at 302 nm decreases slightly and the NAC solution retains its antimicrobial activity. However when
3-minute plasma treated NAC solution is exposed to 109 CFU bacteria the peak at 302 nm disappears and it can’t inactivate bacteria anymore. Source of the peak at 286 nm is unknown. The peak at 286 couldn’t be correlated to any specific potential reaction products of plasma-generated species (such as nitro fatty acids, nitro tyrosine through the attack of peroxynitrite to proteins and lipids in the membrane or cellular oxidation products). Thus we hypothesized that the peak at 286 nm can be attributed to leakage of cellular content due to membrane damage. In order to test this hypothesis, cell pellet was exposed to ATL buffer (detergent based compound that is used for DNA isolation to break down the cell membrane) and following exposure the peak at 286 nm was ! 121! observed. The increase in the intensity of the peak at 286 nm depending on holding time is attributed to more cellular content leakage due to cell membrane damage.
In cytotoxicity experiments even after 48 hours of recovery period, cell that were exposed to 3-minute plasma treated NAC solution died. This might be direct consequence of the ratio of cell number to plasma treated NAC solution. In antimicrobial experiments, 107 CFU/ml bacteria was exposed to plasma treated NAC solution in contrast to cytotoxicity experiments where maximum ~3x104 cell number can be achieved in 96 well plates when cells are 90% confluent. Therefore 2-minute plasma treated NAC solution was exposed to 5x104 and 105 CFU/ml bacteria and 100% inactivation was achieved. So we can conclude that, plasma treated NAC solution can effectively inactivate bacteria without toxic effects on eukaryotic cells. This phenomenon can be expressed as selectivity of plasma treated NAC solution similar to previously reported study where the selectivity of direct plasma treatment was sought
[209]. There might be different reasons for the protection of eukaryotic cells compared to bacteria cells. First of all in cytotoxicity experiments cells were monolayer thus only some portion of cells come in contact with NAC solution. However in bacterial inactivation experiments, bacteria in suspension and the whole surface of bacteria come in contact with plasma treated NAC. In other words, surface area to volume ratio determines the damage on eukaryotic and bacterial cells. Also DNA damage repair mechanisms in eukaryotic cells are more advanced as compared to bacterial cells [209].
Cellular compartmentalization in eukaryotic cells can be considered to be another factor on protection against the stress induced by plasma treated NAC solution [215]. As provided in results section according to histopathological examination report provided ! 122! by ANTECH Diagnostics, plasma treated NAC solution, even high in high doses
(3ml/day for 3 days), doesn’t induce any systemic toxicity. Similarly according to blood test that was performed by ANTECH Diagnostics alterations summarized in Table 6 are independent of injection conditions.
In conclusion plasma treated NAC solution can retain its antimicrobial activity up to 2 years, which is significantly longer than shown in previous studies. Also with an unknown mechanism, plasma treated NAC solution remains stable. Products of NAC molecule generated during plasma treatment might have a role in the stability of the plasma treated NAC solution. Due to limited literature on the chemistry and related stability of NAC solution, a general conclusion can’t be drawn. Further detailed chemical analyses and in vitro and in vivo experiments are needed in order to enlighten antimicrobial stability and interactions with eukaryotic cells and related toxicity.
! 123!
! 5 CHAPTER V: CELLULAR RESPONSES IN E. coli UPON EXPOSURE TO PLASMA-TREATED NAC SOLUTION
5.1 Introduction:
Plasma treatment generates ROS (reactive oxygen species) and RNS (reactive nitrogen species) such as hydrogen peroxide, superoxide, hydroxyl radical, nitric oxide, peroxynitrite, etc. [85,93,98,102,184]. When various liquids and NAC solution are treated with non-thermal plasma generated ROS and RNS, both diffuse into liquid and reacts with the components of the treated liquids [93,184]. Treated liquids serve as a medium to transfer plasma-generated species to bacterial cells. Plasma-generated species leads oxidative and nitrosative stress in bacterial cells that induce various reactions with macromolecules in the cell. As previously reported direct plasma treatment causes membrane damage via lipid peroxidation and DNA damage [98]. Lipid peroxidation involves a series of reactions between polyunsaturated fatty acids (PUFA) in cell the membrane and ROS. Lipid peroxidation starts with the electron loss from fatty acids that leads to generation of fatty acid radicals. Fatty acid radicals react with molecular oxygen to form lipid peroxyl radical. As a result of the reaction between unreacted fatty acids and lipid peroxyl radical, lipid peroxide is generated [221].
Glutathione is a tripeptide abundantly present in both eukaryotic and prokaryotic cells and acts as the main antioxidant system in cells. Glutathione consists of glutamate, glycine and cysteine amino acids. Because of the sulfhydryl group of the cysteine, glutathione can directly react with ROS by acting as an electron donor in a nonenzymatic way [223,224]. In cells glutathione may exist bound to protein and free forms. In healthy cells up to 98% of glutathione is in its reduced form (GSH). When ! 124! cells are exposed to oxidative stress, reduced glutathione is oxidized by ROS and oxidized form of glutathione (GSSG) is formed. Therefore the ratio of oxidized glutathione to reduced glutathione increases as result of oxidative stress and considered to be the indicator of oxidative stress [225,226].
RNS cause membrane damage through lipid nitration process. Lipoproteins and unsaturated fatty acids are potential targets for the lipid nitration. Peroxynitrous acid and peroxynitrite can diffuse through lipid membranes by passive diffusion with a diffusion rate close to water. Therefore accumulation of RNS at the inner side of membrane strongly induces the reaction of RNS with fatty acids. Peroxynitrite induces nitration of both free and esterified unsaturated fatty acids via different pathways involving
! ! homolysis of peroxynitrous acid to OH and NO2, reaction of lipid peroxyl radical with
NO, oxidation of NO to NO2 [222,227-230].
DNA damage is another consequence of oxidative and nitrosative stress. Even
- though NO is not reactive with DNA, peroxynitrite that is product of NO and O2 causes
DNA damage through chemical modification of bases and strand breaks. DNA base modifications by RNS involve alkylation, deamination, oxidation and nitration reactions
[231-232]. Peroxynitrite causes single strand DNA brakes by attacking C4` and C5` of deoxyribose that leads to C3`-phosphate bond cleavage. RNS are not capable of inducing double strand brake. However, the presence of base modifications and single strand brakes that are caused by RNS, in vicinity of replication fork may lead to double strand brakes [232-233]. Hydroxyl radical is the most remarkable source of oxidative DNA damage. Due to its lowest redox potential, guanine is oxidation is more dominant compared to other bases. The main target of hydroxyl radical in DNA is guanine base. ! 125!
Hydroxyl radical (OH!) directly attacks to C8 of the guanine base and forms the oxidation product 8-hydroxydeoxyguanosine (8-OHdG). In addition, hydroxyl radical attacks other bases and yields to formation of thymine peroxide, thymine glycols, and hydroxymethyl uracil. Hydroxyl radical causes single and double DNA strand damage by breaking hydrogen and phosphodiester bonds [98,234-236]. Other ROS like hydrogen peroxide and superoxide are not as reactive with DNA as hydroxyl radical. However, their reactions with RNS and other ROS leads to DNA damage in a similar way to hydroxyl radical [217,234].
Tyrosine nitration is a chemical process induced by RNS, which leads to the loss of protein function and is considered as the indicator of nitrosative stress. Peroxynitrite is considered to be the main contributing agent to tyrosine nitration [237]. However, peroxynitrite doesn’t readily react with tyrosine residues. Instead protonation of peroxynitrite gives rise to its conjugated acid, peroxynitrous acid (ONOOH) and Lewis
- adduct formation with CO2 gives rise to ONOOCO2 , which are the main nitrosating sources of tyrosine. The tyrosine nitration process is triggered with the oxidation of
! tyrosine to tyrosine radical. When generated tyrosine radical reacts with NO2, which is a product of peroxynitrite homolysis, the reaction yields to 3-nitrotyrosine, the final product [195,237,238].
In Chapter 5 we evaluated effects of plasma treated NAC solution on bacterial cells. Therefore we have investigated membrane, DNA, and protein damage caused by oxidative and nitrosative stress following exposure of plasma treated NAC solution. In order to better demonstrate mechanisms of bacterial inactivation we also investigated ! 126! genetic response of bacterial cell to the exposure of plasma treated NAC solution that can provide clues on the various reactions induced in the cells.
5.2 Materials and Methods:
5.2.1 TBARS Assay (MDA Quantification): Detection of Lipid Peroxidation: Lipid peroxidation is a well-known indicator of oxidative stress dependent cell damage. Lipid peroxides are unstable and decompose to natural byproducts of lipid peroxidation such as malondialdehyde (MDA) and 4-hyrdoxynonenal (4-HNE). MDA forms adduct with thiobarbituric acid reactive substance (TBARS). The MDA-TBA adducts (Figure 45) can be measured colorimetrically for the quantification of lipid peroxidation.
Figure 47: MDA-TBA Adduct (from: http://www.cellbiolabs.com/tbars-assay)
OxiSelectTM TBARS Assay Kit for the detection of lipid peroxidation was purchased from Cell Biolabs, Inc. (San Diego, CA, USA). 200 μl, 107 CFU/ml E. coli cells were exposed to 200 μl of untreated and 1,2 and 3-minute plasma treated NAC solution and held for 15 minutes. Following holding time 600 μl 1X sterile PBS solution was added on cells to quench the reaction between cells and plasma treated NAC solution and spun down at 8000 rpm for 10 minutes and supernatant removed. Remaining cell pellet was resuspended with 200 μl of butylated hydroxytoluene (BHT), 100 μl of SDS lysis buffer was added and it was incubated at room temperature for 5 minutes. Then 250 μl of TBA reagent was added and samples were incubated at 95OC for 60 minutes. Next, samples ! 127! were cooled down in the ice bath for 5 minutes. After that samples were centrifuged at
3000 rpm for 15 minutes and 200 μl of supernatant was collected and transferred to 96 well plate and absorbance reading was performed at 532 nm. Different concentrations of hydrogen peroxide solution (0.3% to 6%) were used as positive control. Standard curve was prepared by using MDA standards (0-125 μM) that was provided with the kit.
5.2.2 Live/Dead BacLight Bacterial Viability Assay: LIVE/DEAD® BacLightTM
Bacterial Viability Kit was purchased from Invitrogen life Technologies. The
LIVE/DEAD BacLight Viability Assay Kit contains SYTO® 9 green-fluorescent nucleic acid stain and propodium iodide red-fluorescent nucleic acid stain. Spectral and penetration characteristics of these dyes are different. SYTO 9 dye can penetrate and stain all bacteria with both intact and damaged membranes. Adversely, propodium iodide can only penetrate to cells with damaged membranes. When SYTO 9 and propodium iodide present together, propodium iodide reduces SYTO9 fluorescence.
Thus, when bacteria are stained with the mixture of SYTO9 and propodium iodide, cells with intact membrane, whereas stained green and cells with damaged membrane, whereas stained red can be detected. Both dyes are provided as powders in sealed plastic pasteur pipets. Both dyes were dissolved in 5 ml of sterile deionized water in order to achieve 6 μM SYTO 9 and 30 μM propodium iodide solutions. 200 μl of 3-minute plasma treated NAC solution was added on 200 μl of 107 CFU/ml E. coli and held for 15 minutes. Following 15 minute of holding time, 600 μl of 1X sterile PBS was added on cells to quench the reaction between bacteria and plasma treated NAC solution. Then the bacteria suspension was centrifuged at 8000 rpm for 10 minutes and supernatant was removed. Remaining bacteria pellet was resuspended with 200 μl of dye mixture and ! 128! incubated at room temperature for 15 minutes in dark. After 15 minute incubation of samples, 10 μl of stained bacteria was trapped between a glass slide and a glass coverslip and viewed using AMG EVOS® FL cell imaging system. Images of bacteria samples were captured with green and red fluorescent filters and overlaid to distinguish live and dead cells. The images were captured from five randomly selected areas for three different sets of experiments. Untreated NAC solution was used as negative control.
5.2.3 Intracellular Glutathione Levels: Glutathione is a tripeptide thiol composed of glutamic acid, cysteine, and glycine. Glutathione protects cells from oxidative stress damage by acting as an antioxidant. In cells, more than 90% of total glutathione exist as its reduced form (GSH) and less than 10% exist as its oxidized disulfide (GSSG) form.
As a result of the oxidative stress in the cell GSH is converted to GSSG via oxidation.
The increased GSSG/GSH ration in cells is to be considered as the indicator of oxidative stress [217,218]. Glutathione flurometric assay kit for the detection of intracellular GSH and GSSG levels was purchased from BioVision (Milpitas, CA, USA). In the kit OPA probe (o-phthaldehyde) reacts only with GSH and generates fluorescent signal. For the specific detection of GSSG, a GSH quencher is used to remove GSH without affecting
GSSG. Then by adding a reducing agent, the excess quencher is removed and GSSG is converted to GSH. For the detection of intracellular GSH and GSSG, 100 μl of 107
CFU/ml E. coli was exposed to 100 μl untreated and 1,2 and 3-minute plasma treated
NAC solution and held for 15 minutes. Following 15 minutes of exposure, cell were homogenized in 200μl of ice cold Glutathione Assay Buffer. 100 μl of each homogenate was transferred in to pre-chilled tubes that are containing 30 μl of PCA (Perchloric acid) and was vortexed to have uniform emulsion and then incubated on ice for 5 minutes. ! 129!
Following incubation on ice, samples were centrifuged at 14000 rpm for 2 minutes at
+4O C and supernatant was collected for detection. 40 μl of collected supernatant was transferred to another tube and 20 μl of ice-cold 6N KOH (potassium hydroxide) was added to precipitate and neutralize samples. Samples were kept on ice for 5 minutes and then centrifuged at 14000 rpm for 2 minutes at +4O C. 10 μl of neutralized sample was transferred in to 96 well plate. For the detection of GSH, 80 μl of assay buffer was added to the samples. For the detection of GSSG, 60 μl of assay buffer was added to the samples. Then 10 μl of GSH quencher was added to the samples and incubated at room temperature to allow the GSH to quench. Next 10 μl of reducing agent was added to remove excess GSH and to convert GSH to GSSG. Standards of GSH were prepared by diluting GSH standard solution that was provided with the kit. Finally 10 μl OPA probe
(o-phthaldehyde) was added into each sample and standard and fluorescent reading was performed at Ex/Em = 340/420 nm. Standard samples were used to generate a standard curve and GSH and GSSG concentrations in test samples were calculated by using the standard curve. Hydrogen peroxide and peroxynitrite were used as positive control.
5.2.4 Nitrotyrosine Detection: Nitrotyrosine is a product of tyrosine nitration mediated by RNS (especially peroxynitrite) and is considered as to be indicator of nitrosative stress [219]. For the detection of intracellular nitrosative stress, 3-NT ELISA kit was purchased from Abcam (Cambridge, MA, USA). The kit contains a 96 well plate coated with 3-NT antibody. 200 μl of 107 CFU/ml E. coli was exposed to 200 μl untreated and
1,2, and 3-minute plasma treated NAC solution and held for 15 minutes. Following exposure, bacteria suspension was centrifuged at 8000 rpm for 10 minutes and cells were rinsed with 1X sterile PBS twice. Centrifugation was repeated after each rinsing step. ! 130!
After the last centrifugation, cell pellet was homogenized in 100 μl of Extraction Buffer that was provided with the kit and incubated at room temperature for 20 minutes.
Following incubation period, cells were centrifuged at 15000 rpm for 20 minutes at 4OC, supernatants collected and transferred into clean tubes and remaining pellet was discarded. 100 μl of supernatant collected for each sample was transferred into 96 well plate coated with 3-NT antibody and incubated on plate shaker at 300 rpm for 2 hours at room temperature. Following incubation each well was aspirated and washed twice with
300 μl of 1X wash buffer, provided with the kit. After washing step 50 μl of 1X detector antibody was added into each well and plate was incubated on plate shaker at 300 rpm for 1 hour at room temperature. After incubation aspiration and washing step was repeated. Following washing step 50 μl of 1X HRP label was added into each well and plate was incubated on plate shaker at 300 rpm for 1 hour at room temperature. Reacted
HRP label in each well was aspirated and wells were washed for three times as described above. After washing step, 100 μl of TMB development buffer was added into each well and incubated for 15 minutes at room temperature then after 100 μl of 1N HCl was added and absorbance of each well was read at 600 nm with a spectrophotometer. By using different dilutions of nitrotyrosine standard solution, a standard curve was plotted.
Nitrotyrosine concentration in each sample was quantified by using the standard curve.
5.2.5 Detection of DNA Damage: Agarose gel electrophoresis was used to demonstrate physical DNA disintegration as previously reported [220]. 1 ml of 107 CFU/ml E. coli was exposed to 1 ml of untreated and 1, 2, and 3-minute plasma treated NAC solutions and held for 15 minutes. Following holding time 2 ml of 1X sterile PBS was added on bacteria in order to quench reaction between bacteria and plasma treated NAC solution ! 131! then bacteria suspension was centrifuged at 8000 rpm for 10 minutes and supernatant was removed. Qiagen DNeasy Blood & Tissue Kit (Valencia, CA, USA) was used for
DNA extraction. Remaining cell pellet was resuspended in 180 µl of ATL buffer (a compound to lyse cell membrane that was provided with the kit) and 20 µl of proteinase
K was added, and samples were incubated for 1 hour at 56OC. Following incubation, 40
µl of RNase was added into samples and incubated for 2 minutes by vortexing rigorously. Next 200 µl of buffer AL and 200 µl of 100% ethanol was added and samples were incubated for 1 hour at -20OC. After incubation, samples were transferred into DNeasy Mini spin column placed in 2 ml microcentrifuge tube, centrifuged at 8000 rpm for 1 minute and flow-through was discarded. DNeasy Mini spin column was placed in a clean 2 ml microcentrifuge tube, 500 µl of Buffer AW1 was added on samples and centrifuged at 8000 rpm for 1 minute and flow-through was discarded. Again, DNeasy
Mini spin column was placed in a clean 2 ml microcentrifuge tube, 500 µl of Buffer
AW2 was added on samples and centrifuged at 14000 rpm for 3 minutes and flow- through was discarded. Finally DNeasy Mini spin column was placed in a clean 1.5 ml microcentrifuge tube, 20 µl of Buffer AE was added on samples, incubated for 1 minute at room temperature and centrifuged at 8000 rpm for 1 minute and extracted DNA was collected. Last step was repeated twice in total in order to increase DNA yield. The DNA concentration was determined using BioPhotometer plus nanodrop spectrophotometer
(Eppendorf AG, Hamburg, Germany). 20 µl of DNA extracted from each sample was loaded into wells on 0.1% agarose gel in TBE buffer and subjected to electrophoresis for
30 minutes at 100 V and 400 mA. Ethidium bromide stained gel was visualized with UV ! 132! transilluminator GelDoc system (Labnet International Inc., Edison, NJ, USA). 1 kb DNA ladder was used as control.
5.2.6 DNA Microarray: Microarray analysis was performed with Affymetrix GeneChip
E. coli Genome 2.0 Array 900551 (Santa Clara, CA, USA). Sublethal dose (1-minute treated) of plasma treated NAC solution was exposed to bacteria in order to extract intact mRNA. 1ml of 107 CFU/ml E. coli was exposed to 1-minute plasma treated and held for
15 minutes. Following holding time 4 ml of 1X PBS was added on bacteria suspension to quench the reaction between bacteria and plasma treated NAC solution and samples were centrifuged at 8000 rpm for 10 minutes and supernatant was removed. Bacteria pellet was resuspended in 500 µl of 1X sterile PBS solution and 1 ml of bacteria protect reagent (provided in the kit) was added on samples and incubated at room temperature for 5 minutes. Next samples were centrifuged at 6000 rpm for 10 minutes and supernatant was removed. 100 µl of lysozyme solution that was prepared by dissolving
12 mg of lysozyme enzyme powder and 15 µl of proteinase K in 750 µl TE buffer was added on bacteria pellet and samples were incubated at room temperature with frequent vortexing. After that 350 µl of RLT buffer that was prepared by dissolving 10 µl of βME
(beta-mercaptoethanol) and 250 µl of 100% ethanol was added on samples, mixed well by pipetting. Well-mixed samples were transferred in to spin columns placed in to 2 ml of collection tubes and centrifuged at 9000 rpm and flow-through was discarded. Next
700 µl of RW1 was added on samples in spin column, incubated at room temperature for
3 minutes and then centrifuged at 9000 rpm for 1 minute and flow-through was discarded. Spin columns were placed in to clean collection tubes and 500 µl of RPE buffer was added on samples and centrifuged at 9000 rpm for 1 minute. The last step ! 133! was repeated one more time and followed by a final centrifugation at 10000 rpm for 2 minutes. Next spin columns were placed in to 1.5 ml microcentrifuge tubes and 20 µl of nuclease free water was added, incubated at room temperature and centrifuged at 10000 rpm for 2 minutes. The last step was repeated one more time in order to increase mRNA yield. The extracted mRNA concentration was determined with BioPhotometer plus nanodrop spectrophotometer (Eppendorf AG, Hamburg, Germany). The extracted mRNA was purified with DNase treatment. Following DNase treatment 1μl of DNase inactivation buffer was added on samples and samples were centrifuged at 10000 rpm for 10 minutes. Supernatant was collected as DNA free mRNA samples. Amplification and labeling of mRNA was performed using the Ovation Pico WTA-system V2 RNA amplification system (NuGEN Technologies, Inc., San Carlos, CA, USA) Briefly 50 ng of total RNA was reverse transcribed using a chimeric cDNA/mRNA primer, and a second complementary cDNA strand was synthesized. Purified cDNA was then amplified with ribo-SPIA enzyme and SPIA DNA/RNA primers (NuGEN Technologies,
Inc.) Amplified cDNA was purified with Qiagen MinElute reaction clean up kit. The concentration of purified cDNA was measured using the nanodrop. 2.5 µg of cDNA was fragmented and chemically labeled with biotin to generate biotinylated cDNA using FL-
Ovation cDNA biotin module V2 (NuGEN Technologies, Inc.) Affymetrix gene chips were hybridized with fragmented and biotin labeled target in 110 µl of hybridization cocktail. Target denaturation was performed at 99oC for 2 minutes and then 45oC for 5 minutes, followed by hybridization for 18 hours. Arrays then washed and stained using gene chip Fluidic Station 450, and hybridization signals were amplified using antibody amplification with goat IgG and anti-streptavidin biotinylated antibody. Chips were ! 134! scanned on an Affymetrix Gene Chip Scanner 3000, using Command Console Software.
Background correction and normalization were done using Iterative Plier 16 with
GeneSpring V11.5 software (Agilent, Palo Alto, CA, USA). 2-fold differentially expressed gene list was generated.
5.2.7 Differential Expression of Selected Genes: Based on the data obtained from microarray experiment, differential expression levels of katG, grxA, ahpF, nrdE, nrdF, nrdI, and nrdH genes were examined and normalized according to 16s rRNA gene. The initial concentration of bacteria to be treated and holding time were optimized in order to determine the appropriate sublethal dose of plasma treated NAC solution and achieve sufficient RNA yield. For the optimization reason 107 and 108 CFU/ml E. coli was exposed to equal volumes of untreated, 1,2, and 3-minute plasma treated NAC solutions and peroxynitrite solution as positive control and 1X PBS as positive control
(background for normalization), held for 3 and 5 minutes and RNA concentration was measured with BioPhotometer plus nanodrop spectrophotometer (Eppendorf AG,
Hamburg, Germany). 108 CFU/ml initial bacterial concentration and 3 minutes of holding time were found to be appropriate treatment parameters for sufficient RNA yield.
In brief, 1 ml of 108 CFU/ml E. coli was exposed to 1 ml of PBS, peroxynitrite, untreated NAC, and 1, 2, and 3-minute plasma treated NAC solutions and held for 3 minutes. Following 3-minute holding time, 4 ml of 1X sterile PBS was added on samples immediately in order to quench reaction between bacteria and plasma treated
NAC solutions. Bacteria samples were centrifuged at 8000 rpm for 10 minutes and RNA isolation was performed as described in detail in previous section. Extracted RNA was ! 135! diluted to 50 ng/μl concentration. cDNA conversion master mix was prepared by adding
3.6 μl of RNase free water, 4 μl reaction buffer 6.4 μl dNTP, 4 μl random primer and 2
μl Taq polymerase enzyme in to 20 μl of diluted RNA. The extracted RNA was subjected to cDNA conversion using cMasterTM RTplusPCR system (Eppendorf,
Hamburg, Germany). qPCR was performed to analyze differential gene expression levels of the above-mentioned genes using Mastercycler® ep realplex2 S (Eppendorf,
Hamburg, Germany). Reaction mixture for gene expression level analysis was prepared by mixing 1.25 μl cDNA, 0.5 μl reverse and forward primers and 5 μl SYBR Green
(Applied Biosystems, Grand Island, NY, USA). Forward and revers primers for each gene was designed using web based, Primer BLAST, primer-designing tool (from http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The forward and reverse primers
(listed in Table 7) were synthesized by Integrated DNA Technologies (Coralville, IA,
USA).
Table 7: Sequences of forward and revers primers used in this study. Genes Froward Primer Reverse Primer katG 5`-TTCTAACTCCGTCCTGCGTG-3` 5`-ATGCCGCCACGAAGTCTTTA-3` grxA 5`-TGTGCGTGCAAAAGATCTGG-3` 5`-GTGATCCCTTCCGCACGAAT-3` ahpF 5`-AAACCAACGTGAAAGGCGTG-3` 5`-AGAGAGGCTTTGGCACCTTC-3` nrdE 5`-CGGTAAACCGTGCTAACCCT-3` 5`-GGCGCTGTTAACCTGCAAAA-3` nrdF 5`-TGAATCTGGGCTACGAACCG-3` 5`-ATTTTCATCGGCATTCGGCG-3` nrdI 5`-CTTCGCGGCGTTATTGCTTC-3` 5`-TTCCGGGCAATCACATCTCC-3` nrdH 5`-TGTCGATCGCGTTCCTGAAG-3` 5`-AGCAATCACTACCGGCAACT-3` 16s rRNA 5`-TGCCTGATGGAGGGGGATAA-3` 5`-CCGAAGGTACCCCTCTTTGG-3`
Quantification of gene expression level of each gene was based on the CT value, which is the number of cycles required for the qPCR fluorescent signal crosses the detection
threshold. The CT value was determined by using the Realplex software (Eppendorf, ! 136!
Hamburg, Germany). Fold increase in gene expression was calculated using 2!∆∆!! method. In order to apply 2!∆∆!! method following calculations were done:
• ∆!!!"#$%"&! = !!!"#"!!"!!"#$%$&#! −!!!!!!"#$!!""#$%&!!"#"!
• ∆!!!"#!! = !!!"#"!!"!!"#$%$&#! −!!!!!!"#$!!""#$%&!!"#"!
• ∆∆!! = ∆!!!"#!! −!∆!!!"#$%"&!
• !"#$!!"#$%&'% = ! 2!∆∆!!
Where ∆!!!"#$%"&!is the CT value of control group (bacteria exposed to PBS),
∆!!!"#!! is the CT value of each tested condition (including positive controls).
5.3 Results:
5.3.1 Membrane Lipid Peroxidation: Cell membrane lipid peroxidation was detected using TBARS assay kit. MDA concentration upon exposure to hydrogen peroxide and plasma treated NAC solution was calculated from the standard curve plotted using different concentrations of MDA standard solution As demonstrated in Figure 48 upon exposure to hydrogen peroxide solution lipid peroxidation was observed depending on hydrogen peroxide concentration. However no lipid peroxidation was observed after plasma treated NAC solution exposure.
! 137!
1.60" ! b! 1.19" 1.40" 1.20" 0.70" 1.00" 0.80" 0.60" 0.34" 0.40"
MDA concentration (uM) concentration MDA 0.20" 0.00" 0.00" 0.00" 0.00" 0.00" 0.00" 0.00" PBS" 6% 3% 1% 0.3% 0" 1" 2" 3" H2O2" H2O2" H2O2" H2O2" Plasma&Treatment&Time&(min)&
Figure 48: Cell membrane lipid peroxidation levels upon exposure to plasma-treated NAC solution and hydrogen peroxide solutions.
5.3.2 Live/Dead BacLight Bacterial Viability Assay: To demonstrate the cell membrane damage due to plasma treated NAC solution exposure, cells were stained with
Live/Dead BacLight dye. As demonstrated in Figure 49, number of dead cells increases gradually with the increasing duration of plasma treatment. The scale bar on the pictures shows 50 μm. Relatively less cell number after exposure to 3-minute plasma treated
NAC solution is probably due to complete disintegration of bacterial cells.
! 138!
Figure 49: Cell membrane damage after plasma-treated NAC solution exposure to cells. Green cell are alive and red cells have leaky/compromised cells. Scale bar shows 50 μm.
5.3.3 Intracellular Glutathione Levels: Intracellular levels of reduced form (GSH) and oxidized disulfide form (GSSG) of glutathione were measured, and GSSG/GSH ratio was demonstrated in Figure 50 as an indicator of the intracellular oxidation. The
GSSG/GSH ratio increases gradually till to the exposure of 2-minute plasma treated
NAC solution and drastically following the exposure of 3-minute plasma treated NAC
- solution. GSSG/GSH ratios after exposure of 1% H2O2 and 0.28 mM ONOO solutions to bacterial cells were shown as positive control groups. ! 139!
5.00"
4.50" 3.86" 4.00"
! 3.50"
3.00"
2.50"
2.00" 1.64" GSSG/GSH ratio GSSG/GSH 1.50" 0.73" 1.00" 0.52" 0.49" 0.15" 0.50"
0.00" 0 min" 1 min " 2 min " 3 min" 1% H2O2" 0.28 mM ONOO" Treatment Conditions!
Figure 50: GSSG (oxidized form of glutathione) to GSH (reduced form of glutathione) shown as the indicator of intracellular oxidation. Note that GSSG/GSH increases drastically after exposure of 3-minute plasma-treated NAC solution.
5.3.4 Intracellular Nitrotyrosine Levels: ELISA was performed for the quantification of intracellular nitrotyrosine following exposure of plasma treated NAC solutions to E. coli. Nitrotyrosine concentration, which is an indicator of intracellular nitrosative stress, increases in bacteria proportional to plasma treatment duration. As demonstrated in
Figure 51, the concentration of nitrotyrosine in cells upon exposure to 3-minute plasma treated NAC solution increases drastically as compared to other NAC solutions treated for shorter duration.
! 140!
6.00! 5.27!
5.00! 3.74! 4.00!
3.00! 2.30!
2.00! 1.77! 30NT&Concentration&(nM)& 1.00! 0.33!
0.00! 0!min! 1!min! 2!min!! 3!min!! 0.28!mM! ONOO3! Treatment&Conditions& Figure 51: Intracellular nitrotyrosine concentration upon exposure to plasma-treated NAC solution and peroxynitrite solution as positive control.
5.3.5 DNA Damage Detection: Figure 52 depicts an agarose gel after electrophoresis representing fragmented DNA damage upon exposure of cells to plasma treated NAC solution that are treated for various durations. After exposure of cells to 1 and 2-minute plasma treated NAC solutions DNA can remain intact, however 3-minute plasma treated
NAC solution causes complete disintegration of the genomic DNA in E. coli cells.
! 141!
Figure 52: DNA extracted from bacteria exposed to plasma treated NAC solutions were run on agarose gel. 3-minute plasma-treated NAC solution causes complete DNA fragmentation of DNA.
5.3.6 Gene Expression Analysis Through Microarray: Microarray analysis was performed after exposing E. coli cells to 1-minute plasma treated NAC solution.
Microarray was carried out not only to demonstrate gene expression levels but also identify genes to evaluate in detail after exposure of untreated NAC and 1, 2, and 3- minute plasma treated NAC solutions. Results of the microarray data analysis yielded
104 differentially expressed transcripts (59 upregulated and 45 downregulated).
Microarray results showed that, genes coding inner membrane proteins, genes that have function on iron-sulfur cluster pathway, oxidative stress, universal stress protein, peroxide resistance protein were upregulated. Table 8 summarizes expression levels of upregulated and down regulated genes, which can be considered as indicator of oxidative and nitrosative stress, and also shows cellular response to the overall stress caused by ! 142! plasma treated NAC solution. Mostly genes belong to fli gene family that code flagellar proteins are downregulated.
Table 8: Summary of microarray data, showing expression levels of some genes that play role in response to oxidative and nitrosative stress in E. coli. Gene Gene Function Fold increase/decrease oxyS Regulon, that induces response to oxidative stress 99.31 ybjM Inner membrane protein 63.39 yeaR Transcription of year increases in response to nitrite, nitrate 30.49 and nitric oxide yaaA Peroxide resistance protein 11.32 trxC Thioredoxin 10.99 soxS Transcription induced due to increased superoxide levels 8.14 uspD Universal stress protein, involves in resistance to UV 7.42 irradiation yjcB Inner membrane structural protein 7.08 gor Glutathione reductase 5.14 yfeO Ion channel protein 5.15 yedR Inner membrane protein 5.13 uraA Uracil permease -19.55 fliI Flagellum specific ATP synthase -13.63 fliJ Flagellar protein, one of three soluble components of -8.01 flagellar export system upp Uracil phosphoribosyltransferase -7.27 fliQ Flagellar biosynthesis protein -7.07 fliO Internal membrane component of flagellar export apparatus -6.27 fliG Flagellar motor component -6.26 fliR Flagellar export protein -5.07 nmpC Outer membrane porin -4.08 flgH Flagellar basal body protein -3.57
Through the data obtained from microarray analysis, katG, grxA, ahpF, nrdE, nrdF, nrdI, and nrdH genes were selected to evaluate expression levels versus plasma treatement time of NAC solution.
5.3.6 Differential Expression of Selected Genes: Based on the microarray data, expression levels of katG, grxA, ahpF, nrdE, nrdF, nrdI, and nrdH genes were investigated depending on plasma treatment duration of NAC solution using qRT-PCR. ! 143!
Expression levels were obtained following 3-minute holding time and normalized according to E. coli that was exposed to 1X sterile PBS. As depicted in Figure 53, antioxidant effect of untreated NAC solution was observed, where evaluated genes were downregulated following exposure of untreated NAC solution to E. coli. All genes were upregulated following exposure of plasma treated NAC solution and ONOO- solution with a decreasing pattern as the longer treatment duration. Only nrdI was downregulated after exposure of 3-minute plasma treated NAC solution.
100000.00!
10000.00!
katG! 1000.00! grxA! ahpF! 100.00! nrdE! nrdF! nrdI!
Fold&Increase/Decrease& 10.00! nrdH!
1.00! 0!min! 1!min! 2!min! 3!min! 0.28!mM! ONOO!
0.10! Treatment&Conditions& Figure 53: Differential expression of selected genes in E. coli after exposure to plasma- treated NAC solution
5.4 Discussion: In Chapter 5 we present extensive evaluation of interactions of plasma treated NAC solution with E. coli cells. As it is depicted in Figure 49, following exposure of plasma treated NAC solution to bacteria, cell membrane becomes leaky due ! 144! to cell membrane damage induced by NAC solution. Also as explained in detail in
Chapter 4 a new peak at 286 nm appears on UV-visible spectra that are collected after exposure of plasma treated NAC solution. This new peak was not attributed to any possible product that may arise after exposure of plasma treated NAC solution.
Furthermore to investigate the cell membrane leakage as a result of plasma treated NAC solution exposure, we have treated cells with buffer ATL, which is a reagent that is used to lyse cell membrane for DNA isolation. Following treatment of cells with buffer ATL we have observed the same peak at 286 nm on UV-visible spectrum. Thus UV-Vis spectra results also suggest cell membrane damage and leakage of cellular content.
However, according to our lipid peroxidation experiments, even though it has oxidative capacity, plasma treated NAC solution doesn’t initiate lipid peroxidation in cell membrane. Therefore we postulate that lipid nitration causes cell membrane damage, which is induced by plasma generated RNS in NAC solution. Also oxidative and nitrosative damage of membrane proteins may contribute cell membrane damage.
Furthermore as it was summarized in Table 8 according to microarray results, upregulation of genes coding membrane related proteins such as ybjM (inner membrane protein), yfeO (ion channel protein), and yedR (inner membrane protein) indicates that cell membrane damage is a collective destruction of both membrane lipids and proteins.
The main source of intracellular nitrosative stress is reported as peroxynitrite
[195,219,240]. When peroxynitrite is decomposed it yields to super oxide and nitric oxide. Even though peroxynitrite is capable of initiating lipid peroxidation, peroxynitrite derived NO inhibits lipid peroxidation [229,241]. Nitric oxide inhibits lipid peroxidation via radical chain termination by the abstraction of H atom from lipid peroxyl radical and ! 145! yields to secondary reactive nitrogen containing products [229,240-242]. Another possibility of not observing the lipid peroxidation may be the higher permeability of peroxynitrite compared to ROS in plasma treated NAC solution. Diffusion rate of peroxynitrite through phospholipid membrane is close to that of water and ~400 times more than superoxide [196]. Thus, cell wall of the bacteria acts as a barrier between
ROS and at the same time peroxynitrite diffuses into cell because of its higher diffusion rate and induces RNS originated membrane damage before lipid peroxidation starts.
Peroxynitrite is responsible not only for nitrosative stress, but also for oxidative stress. Superoxide, as the product of peroxynitrite homolysis product induces intracellular oxidation [195,219,243]. Plasma generated ROS in plasma treated NAC solution also has influence on intracellular oxidation [244]. Glutathione oxidation is considered to be the indicator of intracellular oxidation. In normal physiological circumstances most of the glutathione is present in its reduced GSH form, and GSH is converted to oxidized form of glutathione in case of oxidative stress [225,226].
Increased GSSG/GSH ratio indicates the oxidative stress and oxidative damage of proteins. As it was demonstrated in Figure 48, intracellular GSSG/GSH ratio increases slightly after exposure of 1 and 2-minute plasma treated NAC solution and drastically after exposure of 3-minute plasma treated NAC solution. The intracellular GSSG/GSH ratio, measured after exposure of 3-minute plasma treated NAC solution is significantly higher than the ratio measured after exposure 1% hydrogen peroxide, which suggests that plasma treated NAC solution is a mixture of various potent oxidizing compounds.
Similar to glutathione oxidation, peroxynitrite also oxidizes sulfur containing amino acids such as cysteine. Oxidation of sulfur containing amino acids leads to inactivation ! 146! of critical enzymes involved cellular respiration such as glyceraldehyde-3-phosphate dehydrogenase. Enzymes involving cellular respiration are also inactivated by nitration of tyrosine residues [195,245]. Thus intracellular oxidative and nitrosative stress induced by peroxynitrite, which has influence on bacterial death via protein damage.
As demonstrated in Chapter 2, for all tested strains, bacterial inactivation was insignificant when 1 and 2-minute plasma treated NAC solutions were exposed and 7- log reduction was observed after exposure of 3-minute plasma treated NAC solution.
This drastic bacterial reduction by 3-minute plasma treated NAC exposure can be attributed to intracellular oxidation level, which similarly increases drastically after exposure of 3-minute plasma treated NAC solution. Also a similar trend, which can be attributed to significant bacterial inactivation, has been observed at nitrotyrosine detection as well, where intracellular nitrotyrosine level increases significantly after exposure of 3-minute plasma treated NAC solution compared to 1 and 2-minute plasma treated NAC solutions.
DNA damage is also another consequence of plasma treated NAC solution exposure to bacteria. In a previous report Joshi et al. have reported DNA fragmentation caused by direct plasma treatment of E. coli cells using gel electrophoresis [98]. In the present study we have also observed complete DNA fragmentation with gel electrophoresis following exposure of 3-minute plasma treated NAC solution. As it is shown in Figure 52, when 1 and 2-minute plasma treated NAC solution exposed to E. coli cells, DNA is not compromised as oppose to 3-minute plasma treated NAC solution exposed sample, where there is no visible band on agarose gel electrophoresis due to complete disintegration of DNA. Similar to GSSG/GSH ratio and intracellular ! 147! nitrotyrosine formation results, also DNA damage becomes significant when 3-minute plasma treated NAC solution is exposed to E. coli, which suggest that 1 and 2-minute plasma treated NAC solution remains non-lethal while 3-minute plasma treated NAC causes complete inactivation of 107 CFU/ml E. coli. These results together with glutathione oxidation and tyrosine nitration results show that after 3-minute plasma treatment, plasma-generated species reaches a critical concentration that will cause lethal oxidative and nitrosative stress.
One of the remarkable responses of bacterial cells is to reduce their surface area and motility in order to reduce both exposure to stress factor and energy consumption
[98, 246]. Microarray results, as summarized in Table 8, show down regulation of various flagellar genes. This genetic response is attributed to tendency of bacterial cells to be protected from stress factors. Also universal stress proteins regulate expression of flagella associated genes by suppressing their expression levels as a response to stress
[247]. Further more it was reported that nitrosative stress induces the suppression of motility and adherence, of P. aeruginosa. Motility and adherence properties have influence on the virulence of bacteria [248]. Therefore down regulation of flagellar genes indicate that, even complete inactivation cannot be achieved, plasma treated NAC can still eradicate biofilm formation by compromising adhesion of biofilm. As it was described in Chapter 2 after plasma treatment pH of NAC solution drops significantly. It was demonstrated that acidic pH suppresses expression of flagellar genes [249,250].
Thus our results on down regulation of flagellar genes indicate how low pH, which is common consequence of plasma treatment of liquids and reported widely, affects ! 148! microbial physiology and reduces bacterial virulence, by suppressing bacterial motility and adherence.
Uracil metabolism related genes, uraA and upp, are other remarkable down regulated genes. Nitrosative stress induces deamination of cytosine to form uracil [251].
Therefore down regulation of upp and uraA indicates increasing uracil amount in bacterial cells and postulated as indicator of nitrosative stress that is induced by plasma treated NAC solution.
In the light of microarray data, particular genes were selected to investigate bacterial genetic response to plasma treated NAC solution using qRT-PCR.
Peroxynitrite was used as positive control for investigating nitrosative stress on differential gene expression levels. As it is depicted in Figure 53, untreated NAC solution shows its antioxidant property, where the all genes are down regulated. Also following exposure of 3-minute plasma treated NAC solution, gene expression levels were significantly less than 1 and 2-minute plasma treated NAC solution exposed conditions. This trend is similar to trends that were observed with GSSG/GSH ratio, intracellular nitrotyrosine concentration, DNA damage and microbial inactivation experiments. In all mentioned experiments significant changes of investigated parameters were observed after exposure of 3-minute plasma treated NAC solution.
Therefore less expression levels following exposure of 3-minute plasma treated NAC solution compared exposure of 1 and 2-minute treated ones is attributed to intense nucleic acid damage originated from ROS and RNS that reached critical concentration capable of causing destructive reactions in bacteria cells. ! 149!
Following exposure of plasma treated NAC solution oxidative stress genes such as katG and ahpF were upregulated. katG has been reported to act as peroxynitritase in
M. tuberculosis and S. enterica and similarly ahpF have been reported as peroxynitritase in S. enterica [252-254]. Furthermore these genes were upregulated when peroxynitrite solution was exposed to E. coli. Similar findings related the upregulation of katG and ahpF following peroxynitrite exposure to E. coli are also available in the literature
[243]. Thus, we postulate that katG and ahpF have important role in response to nitrosative stress arises from peroxynitrite in plasma treated NAC solution.
grxA, glutaredoxin, is under control of oxyR regulon and plays major role in reduction of oxidized glutathione as the part of oxidative stress along with other oxidative and nitrosative stress genes such as ahpF and katG [255]. In addition to activation of oxyR regulon by oxidative stress, it is reported as the sensory mechanism of nitrosative stress in E. coli and glutaredoxin has been reported as “S-nitrosyl sink” in defense against nitrosative stress [256,258]. Furthermore it was reported that grxA
expression is induced along with katG and ahpF by exposure of NaNO2 and S- nitrosoglutathione (GSNO), which acts as NO donor and induces nitrosative stress,
[257]. The activation of grxA expression as response to nitrosative stress in E. coli is induced by reaction of redox active cysteine residue of oxyR, with RNS in a similar way to nitrosylation [257]. Thus upregulation of grxA indicates potent oxidative and nitrosative stress induced by exposure of plasma treated NAC solution.
! All four genes of nrdHIEF operon are closely coregulated and activation of it by addition of NO to anaerobically grown E. coli culture suggests that nrdHIEF operon plays a role in the response to nitrosative stress in E. coli [259,264]. Also in aerobic ! 150! conditions, introduction of GSNO leads to significantly more upregulation of nrdHI compared to nrdEF [264]. In nrdHIEF operon nrdE and nrdF responsible for coding ribonucleotide reductase class Ib, while nrdI enhances the conversion of ribonucleotides to deoxyribonucleotides and nrdH functions as the electron donor to for nrdEF
[260,261]. Furthermore because of thioredoxin-like activity of nrdH, it also functions as scavenger and plays role in repair mechanisms of damaged proteins [262]. It was reported that nrdH could also be upregulated especially in response to nitrosative stress or during repair of nitrosative damage, which is consistent with our data [263].
Overall, gene expression results obtained from both microarray and qRT-PCR indicates that, plasma treated NAC solution is a mixture of plasma generated RNS and
ROS, which induce potent intracellular oxidative and nitrosative stress. As it is demonstrated in Figure 53, following 1 and 2-minute plasma treated NAC solution oxidative and nitrosative stress genes are vastly upregulated. Lesser expression levels of investigated genes, following exposure of 3-minute plasma treated NAC solution could be attributed to rapid nucleic acid damage originated from potent oxidative and nitrosative effect of 3-minute plasma treated NAC solution. Indeed lower expression levels after exposure of 3-minute plasma treated NAC solution is consistent with significant inactivation of bacteria, increase in GSSG/GSH ratio and an increase in intracellular nitrotyrosine concentration compared to 1 and 2-minute plasma treated
NAC solution exposure. Also as it is depicted in Figure 52, observing DNA damage only after 3-minute plasma treated NAC solution exposure supports the explanation for the less expression levels after 3-minute plasma treated NAC solution compared to 1 and 2- minute ones. ! 151!
Upregulation of above-mentioned genes following exposure of 0.28 mM peroxynitrite, indicate that those genes are effective in response to nitrosative stress.
Also lower differential expression levels of mentioned genes after exposure of peroxynitrite solution compared to expression levels after exposure of plasma treated
NAC solution suggests, that plasma treated NAC solution is way more complicated mixture of ROS and RNS. Only nrdI was downregulated after 3-minute plasma treated
NAC solution exposure. However this result is most likely due to severe nucleic acid damage induced by the 3-minute plasma treated NAC solution.
In conclusion, plasma treated NAC solution initiates a series of intracellular oxidative and nitrosative stress that are resulted as the damaged biomolecules including lipids, nucleic acids and proteins. Both oxidative and nitrosative stress are responsible on antimicrobial activity originated from plasma treated NAC solution. Differential gene expression experiments also suggest contribution of oxidative and nitrosative stress on bacterial inactivation effect of plasma treated NAC solution. However lack of lipid peroxidation especially suggests that plasma generated RNS seems to have more dominant effect on microbial inactivation as compared to ROS.
! 152!
! ! 6 CHAPTER VI: CONCLUDING REMARKS
6.1 Summary of the Research and Conclusion:
In the present study we have developed a novel antimicrobial compound that is effective against common sources of hospital-acquired infections. We have utilized non- thermal dielectric barrier discharge (DBD) plasma treatment method and treated N- acetyl cysteine (NAC) solution in order to develop an antimicrobial compound.
Hospital-acquired infections are an important challenge for health care professionals, hospitalized patients, and public health and cost millions of dollars every year. Bacterial resistance is the source of multidrug resistant bacteria that cause hospital acquired infections. Bacterial colonization on inanimate surfaces serves as the sources of spread of hospital-acquired infections. Bacteria colonize on inanimate surfaces in biofilm forms, which is more resistant to antibiotics and disinfectants. Currently used disinfectants are insufficient to eradicate bacterial biofilms and also have toxic effects.
Therefore a non-toxic, broad-spectrum effective an antimicrobial agent is required.
In Chapter 2, we have determined the optimum plasma treatment parameters and designed a new plasma treatment setup for the treatement of 1 ml NAC solution in contrast to the 100 μl of treatment volume that has been previously used in our laboratory. We have evaluated antimicrobial activity of plasma-treated NAC solution on planktonic and biofilm forms of different bacteria and fungus including multidrug resistant bacteria such as K. pneumonia NDM 1, MRSA300, MRSA 400 and A. baumannii clinical isolate. We have shown that 3-minute plasma treated NAC solution is ! 153! able to inactivate up to 108 CFU/ml initial bacterial load when exposed at least for 15 minutes.
In Chapter 3, we have evaluated chemical modifications in NAC solution due to plasma treatment and quantified plasma-generated species using various analytical chemistry methods and species-specific detection kits. We have determined occurrence of acidic pH after plasma treatment of NAC solution, which is widely reported result of plasma treatment of fluids. Following the detection and quantification of plasma- generated species we have tested their contribution to antimicrobial effect. Our data revealed that acidic pH does not contribute to antimicrobial effect. Further more, antimicrobial effects of detected amounts of plasma-generated species were tested by themselves and in combination with each other. However we didn’t see any antimicrobial activity. Thereupon the antimicrobial effect of superoxide was tested and we have demonstrated that the mixture of superoxide and nitrite could inactivate 7-log bacteria. Therefore we evaluated the contribution of reactive nitrogen species (RNS) on antimicrobial effect and found that peroxynitrite is the species most dominantly contributing to antimicrobial effect.
In Chapter 4, we assessed the stability, cytotoxicity and acute systemic toxicity of the plasma treated NAC solution. Our results show that 3-minute plasma treated NAC solution retains its antimicrobial activity for up 235 days when stored at 37OC, which is equivalent to 2 years at room temperature. Additionally, we presented that when the plasma treated NAC solution is dried and reconstituted, it can retain its antimicrobial activity, which suggests the plasma-generated species responsible for the antimicrobial activity can be stabilized by the products of NAC solution. Also we tested cytotoxicity ! 154! of plasma treated NAC solution and found that plasma treated NAC solution can inactivate bacteria while not damaging eukaryotic cells. In our systemic toxicity experiments, for the first time in the literature, we have injected the 3-minute plasma treated NAC solution in rats and demonstrated that plasma treated NAC solution doesn’t cause any systemic toxicity.
In Chapter 5, we have investigated intracellular mechanisms of bacterial inactivation following exposure to plasma treated NAC solution. Our data suggest that plasma treated NAC solution triggers series of reactions in the cells, which are mediated by nitrosative stress and oxidative stress. Overall when bacteria are exposed to plasma- treated NAC solution, it compromises the cell membrane integrity, induces intracellular oxidative and nitrosative stress and causes DNA fragmentation. Also our gene expression analysis that was carried out by using microarray and qPCR demonstrated the response of the bacterial cell to plasma treated NAC solution, which supports our findings related with nitrosative and oxidative stress.
6.2 Research Contributions:
The main research contribution of this thesis has been to introduce a new stable, broad spectrum, and non toxic antimicrobial agent that potentially can be used as catheter lock solution, to eradicate biofilms in respiratory tract, disinfectant for the disinfection of medical devices and for treatment of infected wounds. This thesis research will help to improve our understanding on the generation and diffusion of plasma generated reactive species in liquid-gas interface and the antimicrobial capabilities and mechanism of action of plasma treated solutions on their antimicrobial ! 155! effect. This study will improve the knowledge and novel explanations in plasma medicine field. We believe that we have presented extensive novel data that will make the plasma treated NAC solution a new tool for both infection control and disinfection purposes, especially against multidrug resistant bacteria, in clinical practice.
The specific contributions of this thesis are as follows:
• For the first time we have demonstrated that the depth of the liquid is a
crucial parameter of plasma treatment for diffusion of plasma-generated
species.
• For the first time we have demonstrated antimicrobial effect of plasma
treated NAC solution on planktonic and biofilm forms of wide range of
pathogens including fungus and multidrug resistant bacteria.
• We have demonstrated the antimicrobial effect of plasma-treated,
nebulized NAC solution on biofilm forms of pathogens. Our results
indicate that the plasma-treated NAC solution can be a new, promising
treatment method for mechanical ventilator associated infections and
upper respiratory tract infections.
• To the best of our knowledge, for the first time we have shown that a
plasma-treated liquid (NAC solution) can retain its antimicrobial activity
for years.
• We have studied the cytotoxicity of plasma-treated NAC solution and for
the first time, and presented that plasma-treated NAC solution can
inactivate bacteria at the same time doesn’t harm eukaryotic cells. ! 156!
• For the first time, we have assessed the acute systemic toxicity of a
plasma-treated liquid by administrating it to rats.
• For the first time, we have demonstrated cell membrane damage, DNA
damage and intracellular nitrosative stress upon exposure of plasma-
treated NAC solution to bacterial cells.
• For the first time we have demonstrated differential gene expression in
cells that are exposed to plasma treated liquid, which improves our
understanding of how plasma treated NAC solution inactivates bacteria.
6.3 Suggested Future Work:
6.3.1 Bacterial Resistance Against Plasma Treated Liquids: Antibiotic resistance is a well-known phenomenon that reduces the efficacy of antibiotics against particular bacteria. Bacteria can generate resistance against antibiotics via various routes such as enzymatic destruction of antibiotics, modification of antibiotic target, efflux pumps, etc.
[9]. Bacteria can generate resistance against not only antibiotics but also various disinfectants. One of the main motivations of this thesis is increasing number of resistant bacteria [8,9]. Therefore evaluation of resistance against plasma generated RNS and
ROS is as important as findings presented in this thesis. One way to evaluate resistance against plasma generated RNS and ROS is the consecutive exposure of non-lethal doses of plasma treated liquids (direct plasma treatment should be considered as well). After each treatment with non-lethal dose, a new culture should be started from the preceding culture for particular number of passages. Once desired passage number is achieved, each bacterial passage can be evaluated for its susceptibility against various doses of ! 157! plasma treated liquids (also direct plasma treatment). Moreover comparison of gene expression levels of specific genes such as oxidative and nitrosative stress response genes for each bacterial passage will provide information not only to determine potential resistance, but also to better understand interactions between plasma treated liquids (or direct plasma treatment) and bacteria.
6.3.2 Determination of Inactivation Dose of Fluid Mediated Plasma Treatment:
Fluid mediated plasma treatment and disinfection has become an emerging research field in plasma medicine. Different research groups have reported antimicrobial activity of plasma treated liquids that are treated by different plasma sources. As per data presented in this thesis, power supply settings (voltage, pulse duration, frequency), plasma treatment duration, discharge gap amount and depth of liquid being treated, holding time, initial bacterial load and the ratio of plasma treated liquid volume to bacteria amount influences desired antimicrobial effect. Therefore developing an empirical model would be beneficial for the comparison of various data presented by various groups. An empirical model would help us to better understand interactions between plasma and liquid being treated, diffusion kinetics of plasma-generated species to liquid and chemical modifications in liquid caused by plasma treatment. From a practical application point of view, an empirical model will be helpful for quality control purpose and to determine required plasma dose for various cases of infections.
6.3.3 Bacterial Persistence Against Plasma-Generated Species: Investigating bacterial persistence should be considered as another way to understand response of bacteria to plasma-generated species. Bacterial persistence is defined as epigenetic ability of bacteria that is developed against various stress factors and considered to be ! 158! the cause of chronic infections. Bacterial persistence cannot be inherited to future generations, but enhances current infection. Persistent bacteria present different physiological state compared to wild type bacteria [239]. Thus investigating bacterial persistence against various plasma doses and ROS and RNS, will enhance our knowledge on the action of mechanism of fluid mediated plasma treatment and how bacteria respond to plasma treated liquids. This novel information will be complementary to currently existing data and suggested future work mentioned above.
Interpretation of the novel data together with currently existing data, will eventually lead us to a better understanding of the entire process starting from plasma treatment and to bacterial inactivation. ! 159!
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VITA
Full Name: Utku Kürşat Ercan
Date and Place of Birth: 03.30.1984, Denizli, Turkey
Education:
• BSc: Department of Bioengineering, Ege University, Izmir, Turkey 2006 • PhD: School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA, Expected in 2013
Professional Experience:
• Research Assistant: Department of Biomechanics, Health Sciences Institute, Dokuz Eylul University, Izmir, Turkey, 2006-2008 • Research Assistant: Surgical Infection Laboratory, Department of Surgery, Drexel University College of Medicine, Philadelphia, PA, 2009-2013
Awards and Honors:
• Scholarship for Masters and Ph.D. in Foreign Countries, Republic of Turkey, Ministry of National Education, for 5 years (2008-2013) • First Place in the Drexel’s IEEE poster competition, Drexel University IEEE graduate forum’s 4th annual research symposium March 3, 2011, Philadelphia PA, US
Professional Memberships: • International Society of Plasma Medicine May 2012-Present • Institute of Electrical and Electronics Engineers January 2012-Present • International Society of Plasma Chemistry July 2011-Present
Publications:
• Neidrauer M, Ercan UK, Bhattacharyya A, Trikha R, Barbee KA, Joshi SG. Antimicrobial Efficacy of a Topical Ointment Containing Nitric Oxide-Loaded Zeolite. Antimicrobial Agents and Chemotherapy. Under Review • Kojtari A*, Ercan UK*, Smith J, Brooks AD, Ji H, Joshi SG, Chemistry for Antimicrobial Effects of Non-Thermal DBD Plasma Treated Water. Environmental Science and Technology. Under Review (* Equal Contribution) • Zhang Z, Nix CA, Ercan UK, Gerstenhaber JA, Joshi SG, Zhong Y. Calcium binding- mediated sustained release of minocycline from hydrophilic multilayer coatings to target medical device- related infection and inflammation. Advanced Materials. Under review • Poor A, Ercan UK, Yost A, Brooks AD, Joshi SG. Microbial and cellular responses to nonthermal plasma- treated alginate wound dressing: control of multidrug- resistant pathogens. Surgical Infections. In press • Ercan UK, Wang H, Ji H, Fridman G, Brooks AD, Joshi SG. Nonequilibrium plasma- activated antimicrobial solutions are broad- spectrum and retain their efficacies for extended period of time. Plasma Process and Polymers. 2013;10(6):544-55 • Celik EU, Ergucu Z, Turkun LS, Ercan UK, Tensile bond strength of an aged resin composite repaired with different protocols. Journal of Adhesive Dentsitry 2011 Aug;13(4):359- 66. • Joshi SG, Cooper M, Yost A, Paff M, Ercan UK, Fridman G, Friedman G, Fridman A, Brooks AD. Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid peroxidation in Escherichia coli. Antimicrobial Agents Chemotherapy 2011 Mar;55(3):1053-62. !