大气压非平衡等离子体对淋球菌的作用研究

发布时间：2018-03-21 12:29

本文选题：等离子体　切入点：非平衡　出处：《华中科技大学》2010年博士论文　论文类型：学位论文

【摘要】： 目的初步探讨大气压非平衡等离子体对淋球菌的杀灭作用及灭菌机制。方法淋球菌(Neisseria gonorrhoeae)菌株FA1090、FA19和MS11经大气压非平衡等离子体照射处理后,用菌落计数法(CFU)检测其存活率,进而利用激光共聚焦显微镜观察淋球菌存活能力,透射电镜观察其细胞壁、细胞膜等结构的变化。结果大气压非平衡等离子体对三株淋球菌均有较强的杀灭作用。大气压非平衡等离子体对淋球菌液体培养基中的淋球菌作用5min, MS11平均存活率为60.65%,FA19平均存活率为92.60%,FA1090平均存活率为96.40%。用该等离子体对淋球菌作用6min, MS11平均存活率为21.13%,FA19平均存活率为31.60%,FA1090平均存活率为91.00%。激光共聚焦显微镜观察见等离子体处理淋球菌6min时,MS11有30%的细菌为染成红色的死菌,而FA1090、FA19仅有20%的细菌为死菌。透射电镜观察见淋球菌的细胞壁在大气压非平衡等离子体作用下有明显破损。结论大气压非平衡等离子体可快速有效地杀灭淋球菌浮游菌,在消毒灭菌、某些局部淋球菌感染的治疗等领域有广阔的应用前景,其灭菌机制可能与淋球菌细胞壁、膜的破裂有关。目的比较淋球菌浮游菌和生物膜菌对大气压非平衡等离子体照射敏感性的差异。方法用大气压非平衡等离子体分别照射盖玻片上的淋球菌浮游菌和生物膜菌。细菌存活率用菌落计数及SYTO9、碘化吡啶(PI)双染色后激光共聚焦显微镜观察存活能力,透射电镜观察其细胞壁、膜等结构的变化。结果菌落计数及SYTO9、PI双染色,均显示生物膜中的淋球菌比浮游菌对大气压非平衡等离子体有更强的抵抗力。大气压非平衡等离子体对淋球菌浮游菌和生物膜分别作用6min,浮游菌存活细菌数的对数(log10N)平均约为3.42,生物膜中存活细菌数的对数(log10N)平均约为3.71。用该等离子体对淋球菌浮游菌和生物膜分别作用12min,浮游菌存活细菌数的对数(log10N)约为0,生物膜中存活细菌数的对数(log10N)平均约为2.36。激光共聚焦显微镜观察见等离子体处理淋球菌8min时,浮游菌有92%为染成红色的死菌,8%为染成绿色的活菌。而生物膜细胞有90%为死菌。处理时间为8min至12min时,浮游细菌中染成绿色的活菌数逐渐下降至接近零。而生物膜细胞中染成红色的死菌数量随照射时间延长逐渐增多。透射电镜观察见等离子体处理相同的时间后,浮游菌细胞壁、膜受损情况较生物膜内细胞明显。结论淋球菌浮游菌比生物膜对大气压非平衡等离子体敏感,在消毒灭菌、淋病预防中需考虑生物膜存在的影响而需相应延长处理时间。目的研究淋球菌生物膜形成过程中相关基因的表达情况及大气压非平衡等离子体作用下淋球菌生物膜基因的差异表达。方法制备淋球菌生物膜模型,荧光定量PCR检测培养第1,2,4,6天pilC1、pilE、mntC mRNA的表达水平。培养2天的标本分别用大气压非平衡等离子体照射2,4,8,10min后接着培养6h,荧光定量PCR检测等离子体照射不同时间后pilC1、pilE基因表达的变化。培养4天的标本分别用大气压非平衡等离子体照射2,4,8,10min后接着培养6h,荧光定量PCR检测等离子体照射不同时间后mntC mRNA的表达水平的变化。结果在淋球菌培养的第1、2天,pilC1、pilE转录水平逐渐增加,从培养的第4天开始,pilC1、pilE转录水平逐渐降低,至第6天,pilC1、pilE转录水平明显下降。在培养的第1、2、4天,mntC基因转录水平呈不断上升趋势,但在第6天,其转录水平明显下降。用大气压非平衡等离子体照射培养第2天的标本,随照射时间的延长,pilC1、pilE mRNA不断降低。用等离子体照射培养第4天的标本,在照射的4min内,mntC mRNA值随照射时间的延长呈上升趋势,而照射8min后,mntC mRN值随照射时间的延长呈明显的下降趋势。结论pilC1、pilE基因主要在淋球菌生物膜形成初期起作用,mntC基因在淋球菌生物膜成熟过程中起一定的作用。大气压非平衡等离子体作用下pilC1、pilE表达下调,mntC基因表达在一定照射剂量内上调,超过一定照射剂量则下调。目的研究大气压非平衡等离子体对淋球菌抗生素敏感性的影响。方法稳定期生长的淋球菌菌株MS11和FA1090用大气压非平衡等离子体照射。等离子体照射不同时间后,用MIC法和杀灭曲线体外测定淋球菌对五种抗生素的敏感性。结果大气压非平衡等离子体照射8min后,淋球菌对多种抗生素(青霉素、四环素、环丙沙星、大观霉素、头孢曲松)的MIC减少4-8倍。杀灭曲线的研究显示等离子体照射8min后,当使用1/4MIC浓度的抗生素(青霉素、四环素、环丙沙星、大观霉素、头孢曲松)时,淋球菌菌落计数较未经等离子体照射的含抗生素的对照组减少4-6个log10CFU/mL。结论大气压非平衡等离子体能提高淋球菌对多种抗生素的敏感性。
[Abstract]:Objective to investigate the killing effect and sterilization mechanism of atmospheric pressure nonequilibrium plasma on Neisseria gonorrhoeae.
Methods Neisseria gonorrhoeae (Neisseria gonorrhoeae) strain FA1090, FA19 and MS11 by atmospheric pressure non-equilibrium plasma irradiation, by colony counting method (CFU) to detect the survival rate and viability of Neisseria gonorrhoeae observed by confocal microscopy, transmission electron microscope to observe the cell wall, changes of cell membrane structure.
The atmospheric pressure non equilibrium plasma sterilization effects on three strains of Neisseria gonorrhoeae were strong. The atmospheric pressure non gonococcal 5min medium for gonococcal liquid equilibrium plasma, the survival rate of MS11 was 60.65% FA19, the average survival rate was 92.60%, the survival rate of FA1090 was 6min 96.40%. in the plasma of Neisseria gonorrhoeae, the survival rate of MS11 was 21.13%, the survival rate of FA19 was 31.60% FA1090, the average survival rate for 91.00%. laser scanning confocal microscope observation showed the plasma treatment of gonococcal 6min, MS11 30% of the bacteria were dyed red and dead bacteria, FA1090 FA19, only 20% of the bacteria for dead bacteria. Under transmission electron microscope, cell wall spray aureus in atmospheric pressure non equilibrium plasma under any obvious damage.
Conclusion atmospheric pressure non-equilibrium plasma can quickly and effectively kill Neisseria gonorrhoeae. It has broad application prospects in disinfection, sterilization and some local Neisseria gonorrhoeae treatment, and its sterilization mechanism may be related to the rupture of cell wall and membrane of Neisseria gonorrhoeae.
Objective to compare the sensitivity of Neisseria gonorrhoeae and biofilm bacteria to atmospheric pressure nonequilibrium plasma exposure.
Methods using atmospheric pressure non-equilibrium plasma were irradiated on glass coverslips gonorrhoeae planktonic bacteria and biofilm bacteria. The bacteria survival rate by colony counting and SYTO9, propidium iodide (PI) double staining after laser confocal microscopy to observe the survival ability, cell wall TEM, changes of membrane structure.
The results of colony count and SYTO9, PI double staining showed gonococcal biofilm than planktonic bacteria in the atmospheric pressure non-equilibrium plasma has stronger resistance. The non 6min effect respectively for gonococcal planktonic bacteria and biofilm equilibrium plasma at atmospheric pressure, the number of viable bacteria on planktonic bacteria number (log10N) average 3.42, the logarithmic number of viable bacteria in the biofilm (log10N) with an average of about 3.71. in the plasma of gonococcal planktonic bacteria and biofilm were 12min, planktonic bacteria viable bacteria number (log10N) is about 0, the logarithmic number of viable bacteria in the biofilm (log10N) with an average of about 2.36. confocal laser microscopic observation showed that plasma treatment of gonococcal 8min, planktonic bacteria 92% dead bacteria dyed red, 8% for dyed green. Bacteria biofilm cells have 90% dead cells. The treatment time is 8min to 12min, planktonic bacteria in dyed green The number of live bacteria gradually decreased to near zero. However, the number of dead red dye in the biofilm cells increased gradually with the increase of irradiation time. Transmission electron microscopy showed that the cell wall and membrane damage of planktonic bacteria were more obvious than those in the biofilm after the same treatment time.
Conclusion the planktonic bacteria of Neisseria gonorrhoeae are more sensitive to atmospheric non-equilibrium plasma than biofilms. In the disinfection and gonorrhea prevention, the influence of biofilm should be considered, and the corresponding prolongation time is needed.
Objective to study the expression of related genes in Neisseria gonorrhoeae biofilm formation and differential expression of Neisseria gonorrhoeae biofilm genes under atmospheric pressure non-equilibrium plasma.
Gonococcal biofilm model preparation method, fluorescence quantitative PCR detection training day 1,2,4,6 pilC1, pilE, mntC expression level of mRNA. The samples were cultured for 2 days using the atmospheric pressure non-equilibrium plasma irradiation for 2,4,8,10min and then cultured 6h, fluorescence quantitative PCR to detect the plasma irradiation at different time after pilC1, expression of pilE culture. The 4 day of the specimens were treated with atmospheric pressure non-equilibrium plasma irradiation for 2,4,8,10min and then cultured for 6h, the changes of mntC expression of mRNA in different time after detection of plasma irradiation of fluorescent quantitative PCR.
Results cultured in Neisseria gonorrhoeae 1,2 days, pilC1, the transcription level of pilE pilC1 increased gradually from the fourth day of culture, and the transcription level of pilE decreased to sixth days, pilC1, the transcription level of pilE decreased significantly. In cultured 1,2,4 days, mntC gene transcription levels were rising trend, but in the sixth day, the transcription level was decreased. With the high pressure of second days were equilibrium plasma radiation culture, pilC1 with prolonged irradiation time, pilE mRNA decreased. After fourth days of specimen by plasma irradiation, irradiation in 4min, mntC, mRNA value increases with the irradiation time increased, and irradiation after 8min, mntC extended mRN value decreased with increasing irradiation time.
Conclusion pilC1 and pilE play a role in the formation of the early gene of Neisseria gonorrhoeae biofilm, mntC gene plays a role in gonococcal biofilm maturation. Non equilibrium plasma under atmospheric pressure pilC1, pilE expression, mntC gene expression in a certain dose, dose is over a certain cut.
Objective to study the effect of atmospheric pressure nonequilibrium plasma on antibiotic sensitivity of Neisseria gonorrhoeae.
Methods the Neisseria gonorrhoeae strains MS11 and FA1090 in stable phase were irradiated by atmospheric pressure non-equilibrium plasma. The sensitivity of Neisseria gonorrhoeae to five kinds of antibiotics was detected by MIC and killing curves at different time after plasma irradiation.
The atmospheric pressure non equilibrium plasma after 8min irradiation, Neisseria gonorrhoeae to antibiotics (penicillin, tetracycline, ciprofloxacin, spectinomycin, cephalosporin Qu Song) MIC decreased 4-8 times. To study the killing curves show the plasma after 8min irradiation, when using the 1/4MIC concentration of antibiotics (penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone Qu Song), gonococcal colony count than untreated control group containing antibiotics reduce plasma irradiation of 4-6 log10CFU/mL.
Conclusion the non balanced plasma body of atmospheric pressure can improve the sensitivity of gonococcus to a variety of antibiotics.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R759.2

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