本文關(guān)鍵詞： 鮑曼不動(dòng)桿菌 多重耐藥 納米銀 細(xì)菌凋亡 細(xì)菌增殖　出處：《天津醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:多重耐藥鮑曼不動(dòng)桿菌(MDRAB)引起的感染日益增多,給臨床抗感染治療帶來極大困難。納米銀作為一種新型抗菌劑,具有廣泛的抗菌活性和強(qiáng)大的抑菌、殺菌作用,且無耐藥性,安全性高,但其確切的抗菌機(jī)制目前尚不十分明確。本課題組前期研究表明,納米銀可促進(jìn)大腸埃希菌的中晚期凋亡,抑制大腸埃希菌的增殖,且凋亡的程度與納米銀的濃度呈正相關(guān),這是首次從細(xì)菌凋亡和增殖的角度闡述了納米銀的抗菌機(jī)制。本文旨在探討納米銀是否對(duì)臨床分離的多重耐藥鮑曼不動(dòng)桿菌有抗菌作用,并探索其是否通過誘導(dǎo)細(xì)菌產(chǎn)生凋亡和抑制細(xì)菌增殖,抵抗多重耐藥細(xì)菌的生長,從而為納米銀抵抗多重耐藥菌的機(jī)制研究提供一個(gè)新的方向,對(duì)納米銀未來的抗菌應(yīng)用具有重要意義。方法:收集38株臨床分離的多重耐藥鮑曼不動(dòng)桿菌,進(jìn)行來源病區(qū)和標(biāo)本種類的統(tǒng)計(jì)分析;應(yīng)用Vitek 2 Compact全自動(dòng)微生物分析系統(tǒng)對(duì)多重耐藥鮑曼不動(dòng)桿菌進(jìn)行鑒定及藥敏試驗(yàn),并分析抗生素耐藥率;使用多重聚合酶鏈反應(yīng)(PCR)法和單一PCR法檢測(cè)鮑曼不動(dòng)桿菌攜帶的耐藥基因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法對(duì)臨床分離株進(jìn)行基因分型及同源性分析;選取3株造成血流感染的多重耐藥鮑曼不動(dòng)桿菌,并參照參考文獻(xiàn)確定納米銀的實(shí)驗(yàn)濃度,即10μg/ml和20μg/ml,通過計(jì)數(shù)CFU實(shí)驗(yàn)繪制多重耐藥鮑曼不動(dòng)桿菌的生長曲線,確定納米銀對(duì)此耐藥菌株生長的影響;應(yīng)用電子顯微鏡觀察納米銀對(duì)菌株形態(tài)的影響;采用流式細(xì)胞術(shù)Annexin V-PI雙染色法檢測(cè)納米銀是否引起多重耐藥鮑曼不動(dòng)桿菌凋亡及凋亡的比例,并應(yīng)用Flowjo和GraphPad Prism軟件分析數(shù)據(jù);應(yīng)用BrdU ELISA方法檢測(cè)納米銀對(duì)多重耐藥鮑曼不動(dòng)桿菌增殖的影響,并用GraphPad Prism軟件統(tǒng)計(jì)分析數(shù)據(jù)。結(jié)果:1.本研究收集的多重耐藥鮑曼不動(dòng)桿菌菌株主要來源于重癥監(jiān)護(hù)病房,耐藥性強(qiáng),均攜帶耐藥基因blaOXA-23和blaOXA-51及插入序列ISAba1,ERIC2-PCR基因分型結(jié)果顯示來源于同一克隆株。2.分離自血流感染的3株多重耐藥鮑曼不動(dòng)桿菌經(jīng)納米銀處理后生長速率降低,并且加入的納米銀濃度越高,這種降低的趨勢(shì)越明顯,推測(cè)其原因可能是納米銀誘導(dǎo)多重耐藥鮑曼不動(dòng)桿菌凋亡或者納米銀抑制多重耐藥鮑曼不動(dòng)桿菌增殖。3.通過透射電鏡觀察納米銀對(duì)多重耐藥鮑曼不動(dòng)桿菌菌體形態(tài)的影響,發(fā)現(xiàn)納米銀處理后的多重耐藥鮑曼不動(dòng)桿菌核酸發(fā)生了凝集,而菌體的形態(tài)保持完整,納米銀并未使多重耐藥鮑曼不動(dòng)桿菌的細(xì)胞壁和細(xì)胞膜發(fā)生物理性的破壞。4.應(yīng)用Annexin V-PI雙染法和流式細(xì)胞術(shù)檢測(cè)納米銀對(duì)多重耐藥鮑曼不動(dòng)桿菌凋亡的影響,發(fā)現(xiàn)3株多重耐藥鮑曼不動(dòng)桿菌經(jīng)納米銀處理后,Annexin V染色陽性率均增加,并且這種凋亡增加的趨勢(shì)隨著加入納米銀濃度的升高而愈發(fā)明顯。進(jìn)一步對(duì)早期和中晚期凋亡分別統(tǒng)計(jì)分析顯示,納米銀誘導(dǎo)多重耐藥鮑曼不動(dòng)桿菌中晚期凋亡具有統(tǒng)計(jì)學(xué)意義。5.采用BrdU ELISA方法檢測(cè)經(jīng)納米銀處理后的多重耐藥鮑曼不動(dòng)桿菌新合成DNA的情況,結(jié)果顯示納米銀處理后,3株多重耐藥鮑曼不動(dòng)桿菌新生DNA的合成均減少,細(xì)菌增殖呈下降趨勢(shì),且這種下降趨勢(shì)與加入納米銀的濃度成正相關(guān),具有統(tǒng)計(jì)學(xué)意義。結(jié)論:本研究表明納米銀可以抵抗多重耐藥鮑曼不動(dòng)桿菌的生長,促進(jìn)多重耐藥鮑曼不動(dòng)桿菌發(fā)生凋亡并抑制多重耐藥鮑曼不動(dòng)桿菌的增殖,為納米銀抵抗多重耐藥菌的機(jī)制研究提供了一個(gè)新的方向,對(duì)納米銀未來的抗菌應(yīng)用具有重要意義。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R440

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