鲍曼不动杆菌噬菌体的分离鉴定及生物学特性的研究

发布时间：2018-03-27 07:04

本文选题：鲍曼不动杆菌　切入点：噬菌体　出处：《吉林大学》2013年硕士论文

【摘要】：鲍曼不动杆菌（Acinetobacter baumannii，Ab），广泛存在于自然界的土壤、水和医院的环境中，是一种需氧非发酵糖类，氧化酶阴性，硝酸盐还原阴性的革兰氏阴性杆菌，在分类上属需氧非发酵革兰阴性杆菌中的不动杆菌属（包括鲍曼不动杆菌、醋酸钙不动杆菌、琼氏不动杆菌、鲁菲不动杆菌、溶血不动杆菌和约翰逊不动杆菌）。随着抗生素的广泛应用，各种致病菌和条件致病菌（如鲍曼不动杆菌）的耐药率不断的攀升，特别是有关多重耐药的“超级细菌”包括鲍曼不动杆菌的报道，已引起医学界的高度关注。由于细菌的耐药性已成为全球医学领域关注的焦点，特别是“超级细菌”的频频出现，人类正重新步入“前抗生素”时代的可能正成为现实。噬菌体是一种侵袭细菌的病毒，特别是毒性噬菌体裂解细菌的特异性和高效性，使得各国研究机构重新开始探索利用噬菌体治疗细菌感染（特别是耐药菌株引起的感染）的可行性问题。本项工作以鲍曼不动杆菌作为宿主菌，从污水中分离出3株针对鲍曼不动杆菌的毒性噬菌体，分别命名为ФAb-1, ФAb-2和ФAb-3，并对噬菌体ФAb-1的生物学特性进行了初步分析。（1）通过电镜观察发现：3株噬菌体的头部呈二十面体立体对称，直径约为50nm，有一短尾，属于有尾病毒目，足尾噬菌体科；（2）噬菌体ФAb-1核酸电泳和限制性酶切图谱显示：噬菌体ФAb-1的核酸为双链DNA，其基因组大小约为40kb，且含有Hind III、EcoR V、Nde I、BamH I和Afl III的多个酶切位点，进一步的酶切初步证实：噬菌体ФAb-1的基因组为环状DNA；（3）通过噬菌体ФAb-1的SDS-PAGE分析，可观察到8-9个蛋白条带，分子量在29～116kDa之间；（4）最佳感染复数(Multiplicity of Infection，MOI)的测定表明：噬菌体ФAb-1感染其宿主菌的MOI是10-2：（5）噬菌体ФAb-1的一步生长曲线揭示：潜伏期为20min，，爆发期为30min，爆发量约为190PFU/cell；（6）在宿主菌裂解谱的试验中，噬菌体ФAb-1能裂解3株来源不同的鲍曼不动杆菌；（7）采用不同的保存方法，测得噬菌体的滴度变化不明显。在完成噬菌体的分离和鉴定的基础上，本项研究选择鲍曼不动杆菌作为致病菌，以BALB/c小鼠为感染对象，在氨苄青霉素和环磷酰胺的作用下，经口感染建立肠源性脓毒血症的动物模型。通过给予噬菌体治疗，评价噬菌体疗法在肠源性细菌性脓毒血症中的疗效。结果表明；接受噬菌体治疗的动物，其存活率明显高于对照组，说明通过口服噬菌体对于由鲍曼不动杆菌引起的肠源性脓毒血症是有效的。我们期待有更多的进一步研究和噬菌体疗法在抗感染中的应用。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R378

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