Inactivation And Related Mechanisms Of Atmospheric-pressure Low-temperature Plasmas On Staphylococcus Aureus

Up till now, atmospheric-pressure low-temperature plasmas have been extensively applied in many fields of industrial production due to unique advantages such as near room-temperature, advanced plasma chemistry, non-pollution, cost-effectiveness, etc.. In the process of its formation, low-temperature plasmas could generate a series of chemical reactive species and have been applied in bacterial sterilization/decontamination in recent years in daily production and life as well as clinical medicine. Researches have developed various types of low-temperature plasmas and studied the inactivation effects and related mechanisms of them on many kinds of microorganisms, but basically focusing on physical level which means changing plasma parameters to discover the plasma-inactivation mechanisms while taking microbes as a "black box". However, studies from the biological perspective, especially on the effects of plasma on bacterial gene expression changes as well as structural and functional changes of plasmids are rare. Therefore in this dissertation, comprehensive and systematical investigations of the treatment effects and related mechanisms by two types of atmospheric-pressure low-temperature plasmas on Staphylococcus aureus are carried out mainly through experiments and the details are presented as follows:1. Developments and diagnostics on characteristic parameters of atmospheric-pressure low-temperature plasma setups suitable for biomedical applicationsa) A noble gas single-electrode plasma jet setup driven by sinusoidal AC power supply:A thin copper rod is connected to the high voltage AC power supply as the high-voltage electrode and directly generate safe room-temperature plasma with the gas temperature at about300K, which is very mild and can be touched with no harm. Plasma discharged-generated reactive particles could directly exert effects on the targets.b) A large-area atmospheric-pressure multi-pins air discharge plasma setup driven by DC pulsed power supply:An array with fine copper rods is used as the high-voltage electrode. The discharge current is being restricted by the ballast resistors and the gas temperature of the plasma is within the range of290-300K and suitable for generating plasma on human skins for treatment without damage.c) Measurement of electric parameters of two types atmospheric-pressure low-temperature plasmas above.d) Optical emission spectrometry and mass spectrometry are measured to detect the multi-kinds of reactive oxygen/nitrogen species (RONS) generated in the plasma jet to convey more detailed information on the APPJ-generated representative reactive species (Neutral particles, positive ions, negtive ions, etc.). The emission intensities of various representative reactive species gradually change with distance. The emission intensities of above-mentioned reactive species at5mm below the jet nozzle are basically higher than those at10mm.e) Optical emission spectrometry is measured for the air discharge plasma, emissions representing a series of reactive species are observed. In the spectral analysis, the most intense emissions are observed between300-450nm and600-800nm, which are attributed to N2second positive system (N2(C-B)) and the second order emission of N2second positive system (N2(C-B)). The emission bands of the NOγ-system at200-300nm (NO(A-X)) as well as reactive atomic oxygen (O) at777.2nm and844.6nm are detected.f) Measurement of the concentrations of some representative long life-span reactive oxygen/nitrogen species (H2O2, O3, NO2-, NO3-) in bacteria suspension after treatment by different doses of air discharge using spectrophotometer.g) Concentration of ozone generated in the air plasma discharge is detected by method of UV absorption (about507ppm)2. Study on the inactivation of Staphylococcus aureus biofilms by APPJ treatment.In this dissertation, different doses of atmospheric-pressure helium plasma jet is applied on the pathogenic S. aureus biofilms formed on borosilicate slices and great deactivation effects are achieved. After10min of plasma treatment, the quantity of S. aureus biofilms reduces more than3log10CFU/cm3and the biofilms are obviously more resistant to the plasma treatment than adherent S. aureus. Result of resazurin-assay test shows that more than70%of biofilm S. aureus have lost their metabolic capacity and about30%of them have entered a viable-but-not-cultivable (VBNC) state at the same treatment time. The scanning electron microscopy (SEM) images reveal that plasma erodes EPS initially and then damages the extracellular structure (cell wall/membranes) and finally destroys the bacteria located in the top several layers of biofilms through the surface etching process. The SYTO9/PI stained images disclose that the membrane structures of69%total bacteria are damaged after plasma exposure for10min. The membrane integrity-dependent impact depth after5-min plasma treatment is nearly12.4μm and meanwhile, the DCFH-DA stained images disclose a depth/layer-related intra-bacterial ROS accumulation effect with an impact depth at about12.0μm. The plasma treatment may have triggered the production of intracellular ROS, inducing oxidative stress which possibly contributes to bacteria death.3. Study on the genetic effects of atmospheric-pressure pulsed DC air discharge plasma on Staphylococcus aureus at gene transcription levelIn this dissertation, quantitative real-time polymerase chain reaction (q-rt-PCR) is utilized to analyze the dynamics of gene expression on the transcription level with regard to several cellular responses of S. aureus by different doses of air plasma treatments. The plasma treatment influences the expression of genes which are associated with several important bio-molecular processes against averse environment in the bacteria, including oxidative stress response, biofilm formation, antibiotics resistance, and DNA damage protection/repair. Reactive oxygen/nitrogen species (RONS) generated in gas phase and induced by the plasma chemistry in liquid phase may account for these changes synergistically.4. Investigation on the treatment effects and related mechanisms by atmospheric-pressure pulsed DC air discharge plasma on the plasmid structure of planktonic S. aureus and E. coliAfter air plasma treatment on planktonic S. aureus and E. coli with plasmids, their plasmids are extracted and structures detected by method of DNA agarose gel electrophoresis. Results display that the concentrations of plasmid DNA (super-coil, linear, open circular) keep decreasing along with increasing plasma treatment dose, which indicate that the plasmid structures of planktonic bacteria have been destroyed. It proves from the side that large dose of plasma treatment could destruct the genetic information of planktonic bacteria almost completely which means thoroughly killing of bacteria. Plasma discharge at gas-phase can initiate a large number of plasma-chemical reactions in various types and then form large amounts of primary and secondary reactive species in the liquid-phase, which could exert destructing effects on bacterial plasmids via a series of cellular bio-chemical reactions.