低温等离子体灭活金黄色葡萄球菌生物膜及其抗感染效果研究

发布时间：2018-07-12 18:24

本文选题：等离子体 + 等离子体活性水　；参考：《塔里木大学》2017年硕士论文

【摘要】：微生物引起的慢性感染与细菌生物膜(bacterial biofilm,BF)的形成有直接关系,细菌生物膜引起的感染常常表现为反复发作且久治不愈。目前治疗微生物感染主要还是依靠抗生素,但是由于细菌生物膜的形成,机体免疫和抗生素治疗已不能够有效清除生物膜,而抗生素的滥用还有极大可能使耐药性继续增强,这使得微生物感染治疗成本急剧增加,且严重降低了患者生活质量,因此函待寻找有效灭活细菌生物膜的新方法。本实验通过在硅胶膜上建立金黄色葡萄球菌生物膜模型,研究等离子体及等离子体活性水(plasma active water,PAW)灭活细菌生物膜的能力。结果显示,等离子体处理金黄色葡萄球菌生物膜0、2、4、6、8 min,生物膜内细菌分别减少0.97、1.6、2.47、4.71个数量级。处理2、4、6 min的PAW处理金黄色葡萄球菌生物膜,生物膜内细菌分别减少了0.74、2.87、3.56个数量级。通过荧光显微镜和扫描电镜观察等离子体处理后的金黄色葡萄球菌生物膜发现,等离子体处理会引起生物膜内细菌细胞膜通透性增加,细菌皱缩变形,并最终破裂。检测到生物膜中胞外蛋白质含量持续增加,eDNA的含量先下降随后又逐渐增加。利用发射光谱仪和活性粒子检测试剂盒检测等离子体气相和液相中的主要活性成分,探究等离子体灭活细菌生物膜的机制。结果显示,等离子体气相中含有NO、N2、N+、N2+、He以及O原子的发射光谱带,液相中H2O2以及硝酸/亚硝酸的浓度随等离子体处理时间增加而上升。检测等离子体处理不同时间后,诺氟沙星(norfloxacin,Nor)、环丙沙星(ciprofloxacin,Cip)、利福平(rifampici,Rif)和万古霉素(vancomycin,Van)对金黄色葡萄球菌生物膜最低清除浓度(minimal biofilm eradicate concentration,MBEC)的变化,研究两者联合作用的效果。结果显示,等离子体处理金黄色葡萄球菌生物膜0、2、4、6 min,最多可使Nor、Cip、Rif和Van的MBEC降为原浓度的1/8-1/4。等离子体联合抗生素处理生物膜比单独使用等离子体或抗生素处理多下降2.59-3.71个数量级。通过等离子体及PAW分别处理金黄色葡萄球菌生物膜皮肤创口感染和腹腔膜感染模型,研究等离子体及PAW抗感染治疗效果。结果显示,等离子体可以使创口部位定值的细菌下降2.44个数量级,使皮肤感染创口愈合提前4-6天。PAW处理使小鼠肾脏中细菌数量下降了2.62个数量级,小鼠存活率提高了40%。本研究结果表明,等离子体及PAW能有效灭活金黄色葡萄球菌生物膜,为临床医学应用治疗皮肤创口感染和腹腔感染提供了重要实验依据。
[Abstract]:The chronic infection caused by microbes is directly related to the formation of bacterial biofilm BF. The infection caused by bacterial biofilm often appears to be recurrent and incurable. At present, the treatment of microbial infections mainly depends on antibiotics, but because of the formation of bacterial biofilm, the body immunity and antibiotic therapy can no longer effectively clear the biofilm, and the abuse of antibiotics is also very likely to make drug resistance continue to increase. As a result, the cost of treatment of microbial infection has increased dramatically, and the quality of life of patients has been seriously reduced. Therefore, a new method to effectively inactivate bacterial biofilm is proposed. The ability of plasma and plasma active water (plasma active) to inactivate bacterial biofilm was studied by establishing staphylococcus aureus biofilm model on silica gel membrane. The results showed that when the biofilm of Staphylococcus aureus was treated with plasma for 8 min, the bacteria in the biofilm decreased by 0.97U 1.6N 2.47 and 4.71 orders of magnitude, respectively. The bacteria in the biofilm of Staphylococcus aureus treated with PAW of 2 ~ 4 ~ 4 ~ 6 min decreased by 0.744 ~ 2.87 and 3.56 orders of magnitude respectively. The membrane of Staphylococcus aureus treated by plasma was observed by fluorescence microscope and scanning electron microscope. It was found that plasma treatment could increase the permeability of bacterial cell membrane, cause bacterial shrinkage and deformation, and eventually break down. The content of extracellular protein in biofilm decreased firstly and then increased gradually. The mechanism of plasma inactivation of bacterial biofilm was investigated by using emission spectrometer and active particle detection kit to detect the main active components in plasma gas and liquid phases. The results show that there are emission bands of no _ 2 N _ 2N _ 2 N _ 2 he and O atoms in the gas phase of the plasma. The concentrations of H _ 2O _ 2 and nitric acid / nitrite in liquid phase increase with the increase of plasma treatment time. The effects of norfloxacin nor, ciprofloxacin Cip, rifampiciripine and vancomycin Van on (minimal biofilm eradicate concentration of Staphylococcus aureus biofilm were measured after plasma treatment for different time. The results showed that the plasma treatment of Staphylococcus aureus biofilm 0 ~ (2) O ~ (2 +) ~ 4 ~ (4) min could reduce the Rif and Van's MBEC to 1 / 8 ~ (1 / 4) of the original concentration. The biofilm treated by plasma combined with antibiotics was 2.59-3.71 orders of magnitude lower than that treated with plasma or antibiotics alone. Plasma and PAW were used to treat Staphylococcus aureus biofilm wound infection and peritoneal membrane infection respectively. The results showed that plasma could reduce the number of bacteria in the wound site by 2.44 orders of magnitude, and the wound healing of skin infection could be prematurely treated 4-6 days. PAW treatment could decrease the number of bacteria in the kidney of mice by 2.62 orders of magnitude, and the survival rate of mice increased by 40%. The results show that plasma and PAW can effectively inactivate Staphylococcus aureus biofilm and provide an important experimental basis for clinical application in the treatment of skin wound infection and abdominal infection.
【学位授予单位】：塔里木大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378.1

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