Bactericidal Effect Of Cold Atmospheric Pressure Plasmas On Staphylococcus Aureus

In recent years,based on the characteristics of low temperature,high efficiency,environmental protection,safety and portability of cold atmospheric pressure plasmas(CAPPs),the research on its bactericidal effect and mechanism in medical equipment,skin,oral,food,environment and other fields have been the hot topics in plasma medicine.Although the killing effect of plasma on a variety of microorganisms is undoubted,due to the complexity of plasma composition and biological responses,its mechanism of cold plasma sterilization against microorganisms still needs to be further explored.Therefore,in this thesis,Staphylococcus aureus(S.aureus)was used as the experimental object to study the physicochemical properties and main reactive oxygen species of CAPPs,as well as the effect of CAPPs on bacterial morphology,cell wall,cell membrane,biomacromolecules on cell surface,intracellular reactive oxygen species,apoptosis and virulence factors for a further reveal about the sterilization mechanism of CAPPs.This paper mainly included the following four parts:(1)Plasma discharge parameters and bactericidal effect evaluation: This study used helium as working gas,and the voltage,current,temperature or humidity during CAPPs discharge were monitored in real time.In addition,the bactericidal effect of CAPPs on S.aureus was evaluated by plate colony counting method.The results indicated that helium gas can generate a stable voltage and current during plasma discharge,and the longer treatment time,the higher humidity in the discharge chamber.Besides,the survival rate of bacteria had a significant reduction with the plasma treatment time.(2)Plasma physicochemical properties evaluation: The amount of hydroxyl radicals and singlet oxygen in plasma processing solution were detected by adding a specific capture agent or scavenger,meanwhile the oxidation-reduction potential and pH value were measured.The results indicated that hydroxyl radicals and singlet oxygen generated by CAPPs accumulate in the solution with the prolongation of the treatment time,and the solution exhibited strong oxidation and acidic properties.(3)Effect of plasma on bacterial biological damage evaluation: In this study,bacterial cell apoptosis,cell membrane potential and intracellular reactive oxygen species were detected by flow cytometry,and the amount of intracellular protein and nucleic leakage was measured,in addition,the surface morphology of bacterial cells were observed by scanning electron microscope,finally the fourier transform infrared spectrum was used to analysis the biological macromolecules of the cells.The results showed that when the reactive substance generated by CAPPs acted on bacterial cells,it oxidatively attacks the biological macromolecules such as protein and fats,causing the cell membrane potential and surface morphology changed,resulting in more plasma active components entered the cells,and making a large amount of cell contents leaked,so that the cell's normal physiological functions are disordered,resulting in apoptosis or even death.This results revealed the mechanism of oxidative damage of S.aureus by CAPPs from the perspective of bacterial biological response.(4)Effects of plasma on bacterial virulence factors: Staphyloxanthin is one of the important virulence factors of S.aureus.In this study,Raman spectroscopy and high performance liquid chromatography(HPLC)were used to qualitatively and quantitatively measured the content of Staphyloxanthin in S.aureus' s cell membrane.The results showed for the first time that after CAPPs treated for 20 min,the content of Staphyloxanthin in the cell membrane of S.aureus was significantly reduced form 448.75 ?g/mL to 19.72 ?g/mL,thus reducing the virulence level of bacteria.This results provided a new idea for the bactericidal mechanism of CAPPs against S.aureus based on the virulence factors of the bacteria itself.