本文選題：等離子體　切入點(diǎn)：非平衡　出處：《華中科技大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】： 目的初步探討大氣壓非平衡等離子體對(duì)淋球菌的殺滅作用及滅菌機(jī)制。 方法淋球菌(Neisseria gonorrhoeae)菌株FA1090、FA19和MS11經(jīng)大氣壓非平衡等離子體照射處理后,用菌落計(jì)數(shù)法(CFU)檢測(cè)其存活率,進(jìn)而利用激光共聚焦顯微鏡觀察淋球菌存活能力,透射電鏡觀察其細(xì)胞壁、細(xì)胞膜等結(jié)構(gòu)的變化。 結(jié)果大氣壓非平衡等離子體對(duì)三株淋球菌均有較強(qiáng)的殺滅作用。大氣壓非平衡等離子體對(duì)淋球菌液體培養(yǎng)基中的淋球菌作用5min, MS11平均存活率為60.65%,FA19平均存活率為92.60%,FA1090平均存活率為96.40%。用該等離子體對(duì)淋球菌作用6min, MS11平均存活率為21.13%,FA19平均存活率為31.60%,FA1090平均存活率為91.00%。激光共聚焦顯微鏡觀察見(jiàn)等離子體處理淋球菌6min時(shí),MS11有30%的細(xì)菌為染成紅色的死菌,而FA1090、FA19僅有20%的細(xì)菌為死菌。透射電鏡觀察見(jiàn)淋球菌的細(xì)胞壁在大氣壓非平衡等離子體作用下有明顯破損。 結(jié)論大氣壓非平衡等離子體可快速有效地殺滅淋球菌浮游菌,在消毒滅菌、某些局部淋球菌感染的治療等領(lǐng)域有廣闊的應(yīng)用前景,其滅菌機(jī)制可能與淋球菌細(xì)胞壁、膜的破裂有關(guān)。 目的比較淋球菌浮游菌和生物膜菌對(duì)大氣壓非平衡等離子體照射敏感性的差異。 方法用大氣壓非平衡等離子體分別照射蓋玻片上的淋球菌浮游菌和生物膜菌。細(xì)菌存活率用菌落計(jì)數(shù)及SYTO9、碘化吡啶(PI)雙染色后激光共聚焦顯微鏡觀察存活能力,透射電鏡觀察其細(xì)胞壁、膜等結(jié)構(gòu)的變化。 結(jié)果菌落計(jì)數(shù)及SYTO9、PI雙染色,均顯示生物膜中的淋球菌比浮游菌對(duì)大氣壓非平衡等離子體有更強(qiáng)的抵抗力。大氣壓非平衡等離子體對(duì)淋球菌浮游菌和生物膜分別作用6min,浮游菌存活細(xì)菌數(shù)的對(duì)數(shù)(log10N)平均約為3.42,生物膜中存活細(xì)菌數(shù)的對(duì)數(shù)(log10N)平均約為3.71。用該等離子體對(duì)淋球菌浮游菌和生物膜分別作用12min,浮游菌存活細(xì)菌數(shù)的對(duì)數(shù)(log10N)約為0,生物膜中存活細(xì)菌數(shù)的對(duì)數(shù)(log10N)平均約為2.36。激光共聚焦顯微鏡觀察見(jiàn)等離子體處理淋球菌8min時(shí),浮游菌有92%為染成紅色的死菌,8%為染成綠色的活菌。而生物膜細(xì)胞有90%為死菌。處理時(shí)間為8min至12min時(shí),浮游細(xì)菌中染成綠色的活菌數(shù)逐漸下降至接近零。而生物膜細(xì)胞中染成紅色的死菌數(shù)量隨照射時(shí)間延長(zhǎng)逐漸增多。透射電鏡觀察見(jiàn)等離子體處理相同的時(shí)間后,浮游菌細(xì)胞壁、膜受損情況較生物膜內(nèi)細(xì)胞明顯。 結(jié)論淋球菌浮游菌比生物膜對(duì)大氣壓非平衡等離子體敏感,在消毒滅菌、淋病預(yù)防中需考慮生物膜存在的影響而需相應(yīng)延長(zhǎng)處理時(shí)間。 目的研究淋球菌生物膜形成過(guò)程中相關(guān)基因的表達(dá)情況及大氣壓非平衡等離子體作用下淋球菌生物膜基因的差異表達(dá)。 方法制備淋球菌生物膜模型,熒光定量PCR檢測(cè)培養(yǎng)第1,2,4,6天pilC1、pilE、mntC mRNA的表達(dá)水平。培養(yǎng)2天的標(biāo)本分別用大氣壓非平衡等離子體照射2,4,8,10min后接著培養(yǎng)6h,熒光定量PCR檢測(cè)等離子體照射不同時(shí)間后pilC1、pilE基因表達(dá)的變化。培養(yǎng)4天的標(biāo)本分別用大氣壓非平衡等離子體照射2,4,8,10min后接著培養(yǎng)6h,熒光定量PCR檢測(cè)等離子體照射不同時(shí)間后mntC mRNA的表達(dá)水平的變化。 結(jié)果在淋球菌培養(yǎng)的第1、2天,pilC1、pilE轉(zhuǎn)錄水平逐漸增加,從培養(yǎng)的第4天開(kāi)始,pilC1、pilE轉(zhuǎn)錄水平逐漸降低,至第6天,pilC1、pilE轉(zhuǎn)錄水平明顯下降。在培養(yǎng)的第1、2、4天,mntC基因轉(zhuǎn)錄水平呈不斷上升趨勢(shì),但在第6天,其轉(zhuǎn)錄水平明顯下降。用大氣壓非平衡等離子體照射培養(yǎng)第2天的標(biāo)本,隨照射時(shí)間的延長(zhǎng),pilC1、pilE mRNA不斷降低。用等離子體照射培養(yǎng)第4天的標(biāo)本,在照射的4min內(nèi),mntC mRNA值隨照射時(shí)間的延長(zhǎng)呈上升趨勢(shì),而照射8min后,mntC mRN值隨照射時(shí)間的延長(zhǎng)呈明顯的下降趨勢(shì)。 結(jié)論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過(guò)程中起一定的作用。大氣壓非平衡等離子體作用下pilC1、pilE表達(dá)下調(diào),mntC基因表達(dá)在一定照射劑量?jī)?nèi)上調(diào),超過(guò)一定照射劑量則下調(diào)。 目的研究大氣壓非平衡等離子體對(duì)淋球菌抗生素敏感性的影響。 方法穩(wěn)定期生長(zhǎng)的淋球菌菌株MS11和FA1090用大氣壓非平衡等離子體照射。等離子體照射不同時(shí)間后,用MIC法和殺滅曲線體外測(cè)定淋球菌對(duì)五種抗生素的敏感性。 結(jié)果大氣壓非平衡等離子體照射8min后,淋球菌對(duì)多種抗生素(青霉素、四環(huán)素、環(huán)丙沙星、大觀霉素、頭孢曲松)的MIC減少4-8倍。殺滅曲線的研究顯示等離子體照射8min后,當(dāng)使用1/4MIC濃度的抗生素(青霉素、四環(huán)素、環(huán)丙沙星、大觀霉素、頭孢曲松)時(shí),淋球菌菌落計(jì)數(shù)較未經(jīng)等離子體照射的含抗生素的對(duì)照組減少4-6個(gè)log10CFU/mL。 結(jié)論大氣壓非平衡等離子體能提高淋球菌對(duì)多種抗生素的敏感性。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R759.2

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本文編號(hào)：1643854