纳米银对多重耐药鲍曼不动杆菌的抗菌机制研究

发布时间：2018-01-23 09:33

本文关键词： 鲍曼不动杆菌多重耐药纳米银细菌凋亡细菌增殖　出处：《天津医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:多重耐药鲍曼不动杆菌(MDRAB)引起的感染日益增多,给临床抗感染治疗带来极大困难。纳米银作为一种新型抗菌剂,具有广泛的抗菌活性和强大的抑菌、杀菌作用,且无耐药性,安全性高,但其确切的抗菌机制目前尚不十分明确。本课题组前期研究表明,纳米银可促进大肠埃希菌的中晚期凋亡,抑制大肠埃希菌的增殖,且凋亡的程度与纳米银的浓度呈正相关,这是首次从细菌凋亡和增殖的角度阐述了纳米银的抗菌机制。本文旨在探讨纳米银是否对临床分离的多重耐药鲍曼不动杆菌有抗菌作用,并探索其是否通过诱导细菌产生凋亡和抑制细菌增殖,抵抗多重耐药细菌的生长,从而为纳米银抵抗多重耐药菌的机制研究提供一个新的方向,对纳米银未来的抗菌应用具有重要意义。方法:收集38株临床分离的多重耐药鲍曼不动杆菌,进行来源病区和标本种类的统计分析;应用Vitek 2 Compact全自动微生物分析系统对多重耐药鲍曼不动杆菌进行鉴定及药敏试验,并分析抗生素耐药率;使用多重聚合酶链反应(PCR)法和单一PCR法检测鲍曼不动杆菌携带的耐药基因blaOXA-23、blaOXA-24、blaOXA-51、blaOXA-58、blaIMP、blaGIM、blaVIM、blaNDM-1、blaKPC、blaGES及插入序列ISAba1、ISAba4、ISAba125;利用ERIC2-PCR法对临床分离株进行基因分型及同源性分析;选取3株造成血流感染的多重耐药鲍曼不动杆菌,并参照参考文献确定纳米银的实验浓度,即10μg/ml和20μg/ml,通过计数CFU实验绘制多重耐药鲍曼不动杆菌的生长曲线,确定纳米银对此耐药菌株生长的影响;应用电子显微镜观察纳米银对菌株形态的影响;采用流式细胞术Annexin V-PI双染色法检测纳米银是否引起多重耐药鲍曼不动杆菌凋亡及凋亡的比例,并应用Flowjo和GraphPad Prism软件分析数据;应用BrdU ELISA方法检测纳米银对多重耐药鲍曼不动杆菌增殖的影响,并用GraphPad Prism软件统计分析数据。结果:1.本研究收集的多重耐药鲍曼不动杆菌菌株主要来源于重症监护病房,耐药性强,均携带耐药基因blaOXA-23和blaOXA-51及插入序列ISAba1,ERIC2-PCR基因分型结果显示来源于同一克隆株。2.分离自血流感染的3株多重耐药鲍曼不动杆菌经纳米银处理后生长速率降低,并且加入的纳米银浓度越高,这种降低的趋势越明显,推测其原因可能是纳米银诱导多重耐药鲍曼不动杆菌凋亡或者纳米银抑制多重耐药鲍曼不动杆菌增殖。3.通过透射电镜观察纳米银对多重耐药鲍曼不动杆菌菌体形态的影响,发现纳米银处理后的多重耐药鲍曼不动杆菌核酸发生了凝集,而菌体的形态保持完整,纳米银并未使多重耐药鲍曼不动杆菌的细胞壁和细胞膜发生物理性的破坏。4.应用Annexin V-PI双染法和流式细胞术检测纳米银对多重耐药鲍曼不动杆菌凋亡的影响,发现3株多重耐药鲍曼不动杆菌经纳米银处理后,Annexin V染色阳性率均增加,并且这种凋亡增加的趋势随着加入纳米银浓度的升高而愈发明显。进一步对早期和中晚期凋亡分别统计分析显示,纳米银诱导多重耐药鲍曼不动杆菌中晚期凋亡具有统计学意义。5.采用BrdU ELISA方法检测经纳米银处理后的多重耐药鲍曼不动杆菌新合成DNA的情况,结果显示纳米银处理后,3株多重耐药鲍曼不动杆菌新生DNA的合成均减少,细菌增殖呈下降趋势,且这种下降趋势与加入纳米银的浓度成正相关,具有统计学意义。结论:本研究表明纳米银可以抵抗多重耐药鲍曼不动杆菌的生长,促进多重耐药鲍曼不动杆菌发生凋亡并抑制多重耐药鲍曼不动杆菌的增殖,为纳米银抵抗多重耐药菌的机制研究提供了一个新的方向,对纳米银未来的抗菌应用具有重要意义。
[Abstract]:Objective: multidrug resistant Acinetobacter Bauman (MDRAB) caused by infection is increasing, for clinical anti infection treatment has brought great difficulties. As a new type of nano silver antibacterial agent, has a wide range of antibacterial activity and strong antibacterial, bactericidal effect, and no drug resistance, high safety, but the exact mechanism is antibacterial not very clear. Ourprevious studies showed that silver nanoparticles can promote the apoptosis of Escherichia coli, inhibiting Escherichia coli proliferation and apoptosis and the degree of concentration of silver nanoparticles, this is the first death from bacterial proliferation and wither the perspective of the antibacterial mechanism of silver nanoparticles. This paper aims to explore the nano silver is Bauman multidrug resistance in clinical isolates of Acinetobacter have antibacterial effect, and to explore whether the bacteria produce by inducing apoptosis and inhibit the proliferation of bacteria resistance, multidrug resistant bacteria, so as to Provide a new direction of research on the mechanism of silver nanoparticles against multi drug resistant bacteria, has important significance for the future application of nano silver antibacterial. Methods: We collected 38 strains of multidrug-resistant clinical isolates of Acinetobacter Bauman, source and statistical analysis of species were endemic; the application of Vitek 2 Compact automatic microbial analysis system of multi drug resistance Bauman Acinetobacter were identified and drug sensitive test, and analysis of antibiotic resistance rate; using multiplex polymerase chain reaction (PCR) resistance gene blaOXA-23, detection of Bauman Acinetobacter carrying method and single PCR method blaOXA-24, blaOXA-51, blaOXA-58, blaIMP, blaGIM, blaVIM, blaNDM-1, blaKPC, blaGES and ISAba1 ISAba4 insertion sequence., ISAba125; on clinical isolates were genotyped by ERIC2-PCR and homology analysis method; selection of multiple drug resistance of 3 strains of Bauman Acinetobacter infection caused by blood flow, and refer to the paper Offer to determine the concentration of silver nanoparticles, 10 g/ml and 20 g/ml, by counting the CFU experiment draw multidrug resistant Bauman real growth curve of bacteria, to determine the influence of nano silver in resistant strains growth; application of electron microscope to observe the effect of silver nanoparticles on the morphology of strain; using flow cytometry Annexin V-PI staining. The detection method of nano silver is caused by multidrug-resistant Acinetobacter bacilli Bauman apoptosis and apoptosis ratio, and the data was analyzed by Flowjo and GraphPad Prism software; application of BrdU ELISA method to detect the real effect of silver nanoparticles on the proliferation of multidrug resistant bacillus Bauman, and GraphPad Prism software for statistical analysis of the data. Results: This study collected 1. multidrug resistant Bauman the Acinetobacter strains mainly originated from ICU, drug resistance, were carrying resistance genes blaOXA-23 and blaOXA-51 and the insertion sequence ISAba1, ERIC2-PCR genotyping. The results show that came from the same clone.2. isolated from bloodstream infection in 3 strains of multi drug resistant Acinetobacter Bauman by nano silver after the growth rate decreases, and the concentration of silver nanoparticles added more high, this decreasing trend is more obvious, it may be induced by silver nanoparticles of multi drug resistant Acinetobacter Bauman apoptosis or nano Bauman silver against multi resistant Acinetobacter.3. proliferation observed by transmission electron microscopy of silver nanoparticles of immobile Bacillus on morphology of multi drug resistance Bauman, Bauman showed multiple resistance of nano silver treated real happened agglutination coli nucleic acid, cell morphology and intact, silver nanoparticles did not make Bauman real multidrug resistant cell wall coli and cell membrane biological rational damage.4. using Annexin V-PI double staining and silver nanoparticles by flow cytometry real effects on apoptosis of multidrug resistant bacillus found Bauman. 3 strains of multidrug-resistant Acinetobacter Bauman by silver nanoparticles after treatment, Annexin V positive rate was increased, and the increasing trend with the increase of apoptosis with concentration of silver nanoparticles and becoming increasingly apparent. Further on the early and late apoptosis respectively. Statistical analysis showed that silver nanoparticles induced by multi drug resistant Acinetobacter Bauman in late apoptosis.5. had statistical significance using BrdU ELISA method to detect the multidrug resistance of Bauman nano silver after the new synthesis of DNA coli. Results showed that nano silver, 3 strains of multi drug resistant Acinetobacter Bauman synthesis of nascent DNA were decreased, the proliferation of bacteria showed a downward trend, and this decline was positively correlated with concentration the addition of nano silver, with statistical significance. Conclusions: This study indicates that silver nanoparticles can resist the multidrug resistant Acinetobacter Bauman growth, promote multi drug resistant Acinetobacter Bauman Apoptosis and inhibition of multidrug-resistant Acinetobacter baumannii proliferation, provide a new direction for the mechanism of nano silver resistance to multi drug resistant bacteria, and is of great significance for the future antibacterial applications of nano silver.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R440

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