本文選題：鮑曼不動桿菌　切入點：噬菌體　出處：《吉林大學》2013年碩士論文

【摘要】：鮑曼不動桿菌（Acinetobacter baumannii，Ab），廣泛存在于自然界的土壤、水和醫(yī)院的環(huán)境中，是一種需氧非發(fā)酵糖類，氧化酶陰性，硝酸鹽還原陰性的革蘭氏陰性桿菌，在分類上屬需氧非發(fā)酵革蘭陰性桿菌中的不動桿菌屬（包括鮑曼不動桿菌、醋酸鈣不動桿菌、瓊氏不動桿菌、魯菲不動桿菌、溶血不動桿菌和約翰遜不動桿菌）。隨著抗生素的廣泛應用，各種致病菌和條件致病菌（如鮑曼不動桿菌）的耐藥率不斷的攀升，特別是有關多重耐藥的“超級細菌”包括鮑曼不動桿菌的報道，已引起醫(yī)學界的高度關注。由于細菌的耐藥性已成為全球醫(yī)學領域關注的焦點，特別是“超級細菌”的頻頻出現(xiàn)，人類正重新步入“前抗生素”時代的可能正成為現(xiàn)實。噬菌體是一種侵襲細菌的病毒，特別是毒性噬菌體裂解細菌的特異性和高效性，使得各國研究機構重新開始探索利用噬菌體治療細菌感染（特別是耐藥菌株引起的感染）的可行性問題。 本項工作以鮑曼不動桿菌作為宿主菌，從污水中分離出3株針對鮑曼不動桿菌的毒性噬菌體，分別命名為ФAb-1, ФAb-2和ФAb-3，并對噬菌體ФAb-1的生物學特性進行了初步分析。（1）通過電鏡觀察發(fā)現(xiàn)：3株噬菌體的頭部呈二十面體立體對稱，直徑約為50nm，有一短尾，屬于有尾病毒目，足尾噬菌體科；（2）噬菌體ФAb-1核酸電泳和限制性酶切圖譜顯示：噬菌體ФAb-1的核酸為雙鏈DNA，其基因組大小約為40kb，且含有Hind III、EcoR V、Nde I、BamH I和Afl III的多個酶切位點，進一步的酶切初步證實：噬菌體ФAb-1的基因組為環(huán)狀DNA；（3）通過噬菌體ФAb-1的SDS-PAGE分析，可觀察到8-9個蛋白條帶，分子量在29～116kDa之間；（4）最佳感染復數(shù)(Multiplicity of Infection，MOI)的測定表明：噬菌體ФAb-1感染其宿主菌的MOI是10-2：（5）噬菌體ФAb-1的一步生長曲線揭示：潛伏期為20min，，爆發(fā)期為30min，爆發(fā)量約為190PFU/cell；（6）在宿主菌裂解譜的試驗中，噬菌體ФAb-1能裂解3株來源不同的鮑曼不動桿菌；（7）采用不同的保存方法，測得噬菌體的滴度變化不明顯。 在完成噬菌體的分離和鑒定的基礎上，本項研究選擇鮑曼不動桿菌作為致病菌，以BALB/c小鼠為感染對象，在氨芐青霉素和環(huán)磷酰胺的作用下，經口感染建立腸源性膿毒血癥的動物模型。通過給予噬菌體治療，評價噬菌體療法在腸源性細菌性膿毒血癥中的療效。結果表明；接受噬菌體治療的動物，其存活率明顯高于對照組，說明通過口服噬菌體對于由鮑曼不動桿菌引起的腸源性膿毒血癥是有效的。我們期待有更多的進一步研究和噬菌體療法在抗感染中的應用。
[Abstract]:Acinetobacter Baumannii Aban, widely found in natural soils, water and hospital environments, is an aerobic non-fermentative sugar, oxidase negative, nitrate reduction-negative gram-negative bacillus. Acinetobacter (including Acinetobacter baumannii, Acinetobacter calcium Acetate, Acinetobacter Joan, Acinetobacter lufi) in aerobic nonfermentative Gram-negative bacilli. Acinetobacter haemolyticus and Acinetobacter Johnson. With the widespread use of antibiotics, resistance to various pathogenic and conditioned pathogens, such as Acinetobacter baumannii, has been increasing. In particular, reports of multidrug resistant "superbacteria", including Acinetobacter baumannii, have attracted great attention from the medical community. In particular, with the frequent emergence of "super bacteria," the possibility that humans are entering the era of "pre-antibiotics" is becoming a reality. Phage is a virus that invades bacteria, especially the specificity and efficiency of toxic bacteriophage lytic bacteria. The feasibility of using bacteriophages to treat bacterial infections, especially those caused by drug-resistant strains, has been restarted. In this work, Acinetobacter baumannii was used as host bacterium, and three strains of toxic bacteriophages against Acinetobacter baumannii were isolated from sewage. The biological characteristics of Ab-1 were preliminarily analyzed. (1) the heads of the three strains were found to be icosahedral stereosymmetric, with a diameter of about 50 nm, and a short tail, belonging to the order Tendroviridae. Ab-1 nucleic acid electrophoresis and restriction endonuclease analysis showed that the nucleic acid of Ab-1 was a double-stranded DNA, and its genome size was about 40kb, and it contained several restriction sites of Hind IIII-EcoR VCV-Nde Ab-1 BamHI and Afl III. Further restriction endonuclease analysis confirmed that the genome of Ab-1 was circular DNA3) by SDS-PAGE analysis of Ab-1, 8-9 protein bands could be observed. The determination of the best complex infection plural of fectivity of moi) showed that the MOI of the host strain infected by Ab-1 was 10-2: 5) the one-step growth curve of Ab-1 revealed that the incubation period was 20 min, the outbreak time was 30 min, and the amount of outbreak was about 190PFU / cell 6) in the host. In the test of bacteria cleavage spectrum, Ab-1 could cleavage three strains of Acinetobacter baumannii from different sources. On the basis of the isolation and identification of phage, Acinetobacter baumannii was selected as pathogenic bacteria, BALB/c mice were infected with ampicillin and cyclophosphamide. Animal model of enterogenic sepsis was established by oral infection. The efficacy of bacteriophage therapy in enterogenic bacterial sepsis was evaluated by phage therapy. The survival rate of Phage was significantly higher than that of control group, indicating that oral bacteriophage was effective for enterogenous sepsis caused by Acinetobacter baumannii. We look forward to further study and application of bacteriophage therapy in anti-infection.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R378

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