大气压低温等离子体射流对白色念珠菌生物膜的杀灭效果
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摘要
目的评价大气压低温等离子体射流对白色念珠菌生物膜的杀灭效果。方法将对数生长期的白色念珠菌悬液接种于24孔板上,培养白色念珠菌生物膜,通过活菌计数评价生物膜的培养稳定性;将大气压低温等离子体射流作用于白色念珠菌生物膜不同时间后,平板计数法计算残余活菌的量,荧光染色观察死亡真菌的情况,透射显微镜观察真菌形态变化。结果白色念珠菌培养72 h后,可在24孔板上形成典型的、成熟的、稳定的生物膜结构;大气压低温等离子体射流对白色念珠菌生物膜具有较好的杀灭效果,作用20 s可杀灭90%真菌,作用时间超过55 s,则无活菌检出;随着作用时间的增加,荧光素标记的死细胞面积也相应增加;透射显微镜结果显示大气压低温等离子体射流对白色念珠菌的细胞结构具有显著的破坏作用。结论大气压低温等离子体射流可破坏白色念珠菌结构,对白色念珠菌生物膜具有较强的杀灭作用。
Objective To evaluate the sterilization effect of new designed atmospheric low temperature plasma jet on Candida albicans(C.albicans) biofilm. Methods C.albicans was grown into the logarithmic phase, and then was added to polystyrene 24-well microtitre plate. The amount of germs were calculated by viable plate counting to determine the reproducibility of each biofilm well. The germs in biofilm were treated by plasma for different exposure time and then the survived germs were quantified by plate counting, the dead cells were determined by staining the biofilm with propidium iodide(PI), and the ultrastructural changes of the germs in biofilm were observed by transmission electron microscopy(TEM). Results When incubated for 72 h, germs tightly polymerized and classical mature biofilm were formed. This atmospheric low temperature plasma jet could inactivate C.albicans biofilm within a short exposure time. C.albicans were 90% inactivated when treated 20 s and 55 s of plasma treatment reduced bacteria populations to undetectable levels. With the increase of treatment time, enlarged fluorescent positive area appeared, and more bacteria died with the extending of exposure. The TEM scanning results showed that the new plasma jet inactivated C.albicans biofilm mainly via disrupting cell envelopes and then leading the release of cellular components, thus resulting in loss of cell viability. Conclusion Plasma generated from atmospheric low temperature plasma jet could damage the cell structure of C.albicans and efficiently sterilize C.albicans biofilm.
Objective To evaluate the sterilization effect of new designed atmospheric low temperature plasma jet on Candida albicans(C.albicans) biofilm. Methods C.albicans was grown into the logarithmic phase, and then was added to polystyrene 24-well microtitre plate. The amount of germs were calculated by viable plate counting to determine the reproducibility of each biofilm well. The germs in biofilm were treated by plasma for different exposure time and then the survived germs were quantified by plate counting, the dead cells were determined by staining the biofilm with propidium iodide(PI), and the ultrastructural changes of the germs in biofilm were observed by transmission electron microscopy(TEM). Results When incubated for 72 h, germs tightly polymerized and classical mature biofilm were formed. This atmospheric low temperature plasma jet could inactivate C.albicans biofilm within a short exposure time. C.albicans were 90% inactivated when treated 20 s and 55 s of plasma treatment reduced bacteria populations to undetectable levels. With the increase of treatment time, enlarged fluorescent positive area appeared, and more bacteria died with the extending of exposure. The TEM scanning results showed that the new plasma jet inactivated C.albicans biofilm mainly via disrupting cell envelopes and then leading the release of cellular components, thus resulting in loss of cell viability. Conclusion Plasma generated from atmospheric low temperature plasma jet could damage the cell structure of C.albicans and efficiently sterilize C.albicans biofilm.
引文
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[3] PETERS BM,WARD RM,RANE HS,et al.Efficacy of ethanol against Candida albicans and Staphylococcus aureus polymicrobial biofilms.Antimicrob Agents Chemother,2013,57(1):74-82.
[4] PARK SB,KIM B,BAE H,et al.Differential epigenetic effects of atmospheric cold plasma on MCF-7 and MDA-MB-231 breast cancer cells.PLoS One,2015,10(6):e0129931[2018-07-10].https://doi.org/10.1371/journal.pone.0129931.
[5] 熊紫兰,卢新培,鲜于斌,等.大气压低温等离子体射流及其生物医学应用.科技导报,2010,28(15):97-105.
[6] ZIUZINA D,PATIL S,CULLEN PJ,et al.Atmospheric cold plasma inactivation of Escherichia coli,Salmonella enterica serovar Typhimurium and Listeria monocytogenes inoculated on fresh produce.Food Microbiol,2014,42(12):109-116.
[7] RAMAGE G,SAVILLE SP,WICKES BL,et al.Inhibition of Candida albicans biofilm formation by farnesol,a quorum-sensing molecule.Appl Environ Microbiol,2002,68(11):5459-5463.
[8] DAVID BG.The emerging role of reactive oxygen and nitrogen species in redox biology and some implications for plasma applications to medicine and biology.J Appl Phys,2012,45(26):263001[2018-07-10].http://dx.doi.org/10.1088/0022-3727/45/26/263001.