本文選題：廣泛耐藥鮑曼不動桿菌 + 秀麗隱桿線蟲��； 參考：《廣東藥科大學(xué)》2017年碩士論文

【摘要】：目的:鮑曼不動桿菌(Acinetobacter baumannii,A.baumannii)生命力強,耐藥性嚴(yán)重,呈現(xiàn)世界流行趨勢,已經(jīng)成為我國臨床最常見的致病菌之一。然而,抗生素的研發(fā)速度滯后于細菌的耐藥速度,出現(xiàn)無藥可用的局面,針對此情況,各國關(guān)于鼓勵開發(fā)抗生素的優(yōu)惠政策相繼出臺,希望通過此措施進一步加速抗生素研發(fā)的速度。因此抗生素研發(fā)不僅意義重大,也符合國際藥物研發(fā)趨勢。本課題擬構(gòu)建廣泛耐藥鮑曼不動桿菌(Extensively drug-resistant Acinetobacter baumannii,XDR A.baumannii)-秀麗隱桿線蟲感染模型,并利用該模型篩選清熱解毒中藥提取物,以期能夠?qū)ふ业娇乖摼闹兴幪崛∥�。方�?1.選用XDR A.baumannii臨床菌株GZLC-1作為致病菌,glp-4(bn2ts);sek-1(km4)秀麗隱桿線蟲(Caenorhabditis elegans,C.elegans)作為模型動物,構(gòu)建廣泛耐藥鮑曼不動桿菌-秀麗隱桿線蟲感染模型。通過比較線蟲在M9緩沖液、10%BHI-M9、20%BHI-M9、BHI等幾種液體培養(yǎng)基中存活時間,確定最適比例的液體培養(yǎng)基。為了增強A.baumannii的致病性,進一步優(yōu)化了鐵離子在液體培養(yǎng)基的加入量。最后確定該感染模型液體培養(yǎng)基的組成成分。2.比較不同感染時長下線蟲的存活時間,確定線蟲的最佳感染時長。在最佳感染時長下進一步確定最佳感染菌濃度和治療時長。3.分別用水,50%乙醇,95%乙醇三種溶劑對28味清熱解毒中藥進行回流提取,藥液比是1:10,提取兩次,每次一小時,提取液合并,放在水浴鍋上干燥后制成粉末后放置4℃冰箱待用。4.采用廣泛耐藥鮑曼不動桿菌-線蟲感染模型對中藥提取物進行篩選,每種提取物配成1000μg/ml,100μg/ml,10μg/ml,1μg/ml,0.1μg/ml五個濃度進行藥效篩選實驗,同時設(shè)陰性對照(1%DMSO)和陽性對照(1%DMSO溶解2μg/ml多粘菌素B)。5.選取體內(nèi)抗A.baumannii效果較好的中藥提取物進行體外藥敏實驗,比較中藥提取物在體內(nèi)與體外的抗A.baumannii效果。結(jié)果:1、廣泛耐藥鮑曼不動桿菌-秀麗隱桿線蟲感染模型培養(yǎng)基組成成分是10%BHI-M9、5μg/ml萘啶酸、10μM Fe Cl3、1%DMSO。2、廣泛耐藥鮑曼不動桿菌-秀麗隱桿線蟲感染模型感染時長是6h,感染菌濃度是5×105CFU/ml,治療時長是24h。3、線蟲體內(nèi)抗A.baumannii效果較好的提取物(與陰性對照組相比存活率提高到4.5倍以上)有黃芩、大青葉、牡丹皮、雞骨草、何首烏、山豆根等六味中藥的水提取物。4、黃芩、大青葉、牡丹皮、雞骨草、何首烏、山豆根等六味中藥的水提取物對XDR A.baumannii GZLC-1的MIC值皆大于2048μg/ml。結(jié)論:1、本實驗利用臨床XDR A.baumannii GZLC-1,建立了廣泛耐藥鮑曼不動桿菌-線蟲感染模型,并證實線蟲的死亡是由于XDR A.baumannii在線蟲體內(nèi)定殖且不斷繁殖導(dǎo)致。2、黃芩、大青葉、牡丹皮、雞骨草、何首烏、山豆根等六種中藥水提取物對感染了XDR A.baumannii的線蟲具有較好的治療作用。3、黃芩、大青葉、牡丹皮、雞骨草、何首烏、山豆根等六種中藥水提取物體外抗XDR A.baumannii GZLC-1的效果較差,但體內(nèi)藥效結(jié)果較好,說明這六種提取物可能不是直接殺菌,而是從提高免疫力等其他途徑提高抗菌效果。
[Abstract]:Objective: Acinetobacter baumannii (Acinetobacter, A.baumannii), which has strong vitality and serious resistance, presents a worldwide trend and has become one of the most common pathogenic bacteria in our country. However, the speed of research and development of antibiotics lags behind the rate of antibiotic resistance, and there is a situation of no drug availability. The preferential policies of antibiotics have been introduced in succession, hoping to further accelerate the speed of antibiotic research and development. Therefore, the research and development of antibiotics is not only significant, but also in line with the trend of international drug development. This topic is to construct Extensively drug-resistant Acinetobacter baumannii (XDR A.baumannii) - the beautiful hidden secret The model of nematode infection and the use of this model to screen the extract of Chinese herbal medicine for clearing heat and detoxification in order to find the extract of Chinese traditional medicine. Method: 1. XDR A.baumannii clinical strain GZLC-1 was selected as pathogenic bacteria, glp-4 (bn2ts); SEK-1 (km4) Caenorhabditis elegans (Caenorhabditis elegans, C.elegans) was used as model animal to construct extensive drug resistance. The infection model of Acinetobacter Bauman, Caenorhabditis elegans, determines the optimum proportion of liquid medium by comparing the survival time of nematodes in several liquid medium such as M9 buffer, 10%BHI-M9,20%BHI-M9, BHI and other liquid medium. In order to enhance the pathogenicity of A.baumannii, the addition of iron ions in liquid medium is further optimized. Finally, the infection is determined. The composition of the model liquid medium,.2., compared the survival time of the long line worm in different infections, and determined the best time for the nematode infection. At the time of the best infection, the optimum concentration of the infected bacteria and the treatment time of.3., 50% ethanol, and 95% ethanol, respectively, were reflued and extracted by three kinds of solvents. It is at 1:10, two times, two times, one hour each time, the extraction solution is merged, then dried in a water bath and then made into powder and then placed at 4 degrees centigrade. The.4. Acinetobacter - Acinetobacter - nematode infection model is selected to select the extract of traditional Chinese medicine. Each extract is matched with 1000 mu g/ml, 100 mu g/ml, 10 mu g/ml, 1 mu g/ml, 0.1 u g/ml five concentration. Screening test, simultaneously setting negative control (1%DMSO) and positive control (1%DMSO dissolving 2 g/ml polymyxin B).5. to select the in vivo anti A.baumannii effect of traditional Chinese medicine extracts in vitro drug sensitivity test, compare the anti A.baumannii effect of traditional Chinese medicine extracts in vivo and in vitro. Fruit: 1, widely resistant Acinetobacter Bauman - Caenorhabditis elegans sense The composition of the culture medium was 10%BHI-M9,5 mu g/ml naphthalene acid, 10 mu M Fe Cl3,1%DMSO.2, the widespread resistance of the widely resistant Acinetobacter spp. - Caenorhabditis elegans infection model was 6h, the concentration of infected bacteria was 5 * 105CFU/ml, the treatment time was 24h.3, and the anti A.baumannii effect of nematode was better than that of the negative control group. Up to 4.5 times more than 4.5 times) the water extracts of six Chinese herbs,.4, Scutellaria, peony, peony, peony, peony, Radix Polygoni Multiflori, pea root and other six Chinese herbs, are all more than 2048 mu g/ml. conclusion: 1. 1, this experiment uses clinical XDR A.baumannii. GZLC-1, a widely drug resistant Acinetobacter Bauman nematode infection model was established, and the death of the solid line worm was due to the colonization of XDR A.baumannii in the worm and the continuous propagation of.2, Scutellaria, peony skin, chicken bone grass, Polygonum multiflorum, pea root and so on, which had good treatment for the XDR A.baumannii infected nematodes. The effects of.3, Scutellaria, Radix Scutellariae, pony leaf, peony skin, chicken bone grass, Radix Polygonum multiflorum, Radix Sophorae root and other six kinds of aqueous extracts were less effective against XDR A.baumannii GZLC-1, but the results were good, indicating that these six kinds of extracts may not be directly germicidal, but improve the antibacterial effect from other ways.

【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R-332;R285

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