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Cellular basis in the inhibitory activity of non-thermal plasma against biofilm-forming pathogenic bacteria

  • 서혜미 (아주대학교 일반대학원)

초록/요약

Inactivation of bacterial growth is essential for a healthy human life, and some of the most important advances in agriculture, medicine, and food science have been created by the application of biochemical, cellular, and molecular knowledge of bacterial control. However, inactivation of biofilms formed by various pathogenic bacteria, including Staphylococcus, Pseudomonas, and Pectobacterium bacterial genus, remains to be one of the most unmanageable tasks in the control of bacterial diseases. In this context, the effective inhibition of a biofilm-forming pathogenic bacteria using non-thermal atmospheric plasma has recently emerged as an attractive antimicrobial technique. To study the antimicrobial nature of plasma, various non-thermal atmospheric-pressure plasmas were generated using types of plasma devices and a number of gas sources such as Air, N2, and Helium. Plasma generated by our micro-jet devices using N2 gas effectively inhibited planktonic bacteria, and the applications of PBS pretreated with plasma (PBS/N2Plasma) were found to be more convenient and efficient as compared to direct applications of the plasma. Scavenger assays using various antioxidants revealed that ROS were involved in the inhibitory cellular actions of PBS/N2Plasma, with H2O2 and singlet oxygen being the major active components essential for bacterial death. The subsequent intensive analysis of PBS/N2Plasma, stored at different temperatures and periods, showed that the bactericidal efficacy was well maintained at -80℃ for three months, or at -4℃ for three weeks. The underwater plasma device from a bubbler with 30-50% porosity was shown to effectively inhibit growth of pathogenic bacteria such as Staphylococcus and Pseudomonas. Reactive oxygen species (ROS) are biological molecules that play an important role in bacterial death. The highest number of ROS was detected in the underwater plasma of the bubbler with the porosity of 50%. ROS signaling molecules—in particular, singlet oxygen and H2O2—could play a role in plasma-mediated damage to bacteria. The underwater plasma solution of the bubbler with the porosity of 50% was found to have a strong antimicrobial effect with a considerably low cytotoxic effect on cells, implying that the higher bubbler porosity is more promising in terms of its practical use in plasma medicine. Further studies demonstrated that PBS/N2Plasma effectively inhibited not only the growth of planktonic bacteria, but also the biofilm they formed. The remarkable inhibition on the biofilm was visualized and analyzed using the LIVE/DEAD viability assay and confocal laser scanning microscopy (CLSM) imaging. The 3D CLSM imaging data revealed that the antimicrobial activity of PBS/N2Plasma was sufficiently permeable to affect the cells embedded inside the biofilm. The prominent permeability could be the crucial feature of PBS/N2Plasma that contributes to the effectiveness of biofilm inactivation. Disease control by non-thermal plasma was evaluated using a semi-in vivo assay system with the GFP-tagged crop pathogen, and the efficacy was confirmed by the results showing a significant reduction in disease symptoms.

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목차

ABSTRACT ⅰ
TABLE OF CONTENTS ⅲ
CHAPTER I 1
A. INTRODUCTION 2
B. MATERIALS & METHODS 5
1. Structure of the micro-plasma jet and plasma discharge system 5
2. Structure and manufacturing of underwater plasma device 5
3. Bacterial strain and treatment of plasma 5
4. LIVE/DEAD bacterial viability assay 6
5. Scanning electron microscopy (SEM) 7
6. Measurement of ROS 7
7. Scavenger assay using antioxidants 8
8. Measuring Cell Viability/Cytotoxicity: WST 8
9. Statistical analysis 9
C. RESULTS 10
1. Experimental set up of non-thermal atmospheric plasma 10
2. Anti-microbial effects of PBS/N2 and PBS/Air Plasma 10
3. LIVE/DEAD Bacterial Viability assay 11
4. Duration test of non-thermal PBS/N2Plasma and PBS/Air Plasma activity 12
5. Anti-bacterial effects depending on the storage temperature of the PBS/N2Plasma 12
6. Scanning electron microscopy (SEM) 13
7. Involvement of ROS in bacterial inhibition of PBS/N2Plasma against S. aureus 14
8. Structure and manufacturing of underwater plasma device 15
9. Antimicrobial effects of underwater plasma devices equipped with bubblers 16
10. Comparison of antimicrobial effects according to underwater plasma treatment time 17
11. Duration of underwater plasma according to bubbler porosity 17
12. Observation of underwater plasma treated bacterial cells with scanning electron microscopy (SEM) 18
13. Evaluation of Cytotoxic Effects by the WST assay 19
14. Involvement of ROS in bacterial inhibition of underwater plasma against S. aureus 19
D. DISCUSSION 21
E. LIST OF FIGURES 26
CHAPTER II 47
A. INTRODUCTION 48
B. MATERIALS & METHODS 51
1. Bacterial strain and biofilm formation 51
2. Swimming motility 51
3. Swarming motility 52
4. Confocal Laser Scanning Microscopy (CLSM) 52
C. RESULTS 54
1. Motility assays; swimming and swarming motility 54
2. Inhibition efficacy of non-thermal plasma against Pst DC3000 under various conditions. 55
3. Scanning electron microscopy (SEM) 56
4. Optical density of the dissolved biofilm 57
5. Inhibitory efficacy of non-thermal atmospheric plasma on the biofilm of Pst DC3000 according to the application methods 57
6. 3D analysis on anti-biofilm efficacy of PBS/N2Plasma against Pst DC3000 using CLSM 58
7. Inhibitory efficacy of non-thermal atmospheric plasma on the biofilm of P. aeruginosa PA01 59
8. Combined treatment of P. aeruginosa PA01 with the antibiotic and PBS/N2Plasma 60
D. DISCUSSION 62
E. LIST OF FIGURES 66
CHAPTER III 83
A. INTRODUCTION 84
B. MATERIALS & METHODS 87
1. Growth conditions of Bacterial strain and biofilm formation 87
2. Bacterial strain and GFP tagging 87
3. Semi-in vivo assay 88
C. RESULTS 89
1. Inhibition efficacy of non-thermal plasma against Pseudomonas and Pectobacterium under various conditions. 89
2. Inhibitory efficacy of PBS/N2Plasma on vegetative cell and biofilm of soft-rot bacteria. 90
3. Semi-in vivo evaluation of inhibitory activity 90
D. DISCUSSION 92
E. LIST OF FIGURES 94
F. REFERENCES 103
국문요약 108

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