粘质沙雷菌噬菌体的分离鉴定及噬菌体疗法对粘质沙雷菌感染小鼠的疗效观察

发布时间：2018-06-05 16:54

本文选题：粘质沙雷菌 + 噬菌体　；参考：《吉林大学》2014年硕士论文

【摘要】：粘质沙雷菌(Serratia marcescens)属肠杆菌科（Enterobacteriaceae）成员，一般存在于水和土壤中，亦可存在医院的环境，对免疫功能低下、大面积烧伤、器官移植和肿瘤的患者可造成各种感染，如泌尿道和呼吸道感染、伤口感染、菌血症、内毒素休克和心内膜炎等。特别是近年来由多重耐药粘质沙雷菌所致的新生儿监护病房的感染及爆发流行，已引起医务工作者和医院管理者的高度重视。多重耐药粘质沙雷菌和其他耐药性细菌引起的各种感染一方面降低了抗生素的疗效，另一方面也增加了患者的经济负担。噬菌体是感染细菌的病毒，存在于自然环境中，由于噬菌体对细菌裂解的特异性和高效性，使得人们重新开始关注噬菌体在细菌性感染(特别是多重耐药细菌和超级细菌所致的感染)中的重要作用。根据现有的资料，有关粘质沙雷菌噬菌体的研究很少，因此，深入和全面了解粘质沙雷菌噬菌体的生物学特性和观察噬菌体疗法的抗感染作用，具有重要的现实意义。本项研究以10株粘质沙雷菌为宿主菌，采用双层琼脂法从医院排出的污水中分离出7株裂解性粘质沙雷菌的噬菌体，分别命名为ФSM41Y、ФSM254Y、ФSM23Y、ФSM13Y、ФSM01Y、ФSM9-3Y和ФSM22Y。电镜观察显示：这7株噬菌体的形态特征符合有尾病毒目(Ordr Caudovirale)、肌尾病毒科（Family Myoviridae）噬菌体。进一步对噬菌体ФSM9-3Y的生物学特性分析时发现：(1)头部呈20面体立体对称、直径约70nm，尾鞘长约50nm，尾鞘的末端连接有尾板和尾丝；(2)噬菌体ФSM9-3Y基因组的限制性酶切图谱提示，基因组为双链DNA，大小约为54kb左右，且含EcoRV、BamHI、SacII和NdeI的酶切位点；(3)裂解试验显示，噬菌体ФSM9-3Y对10株宿主菌的裂解谱较窄，对非宿主菌无任何作用；(4)噬菌体ФSM9-3Y的最佳感染复数为1(MOI=1)；(5)一步生长曲线表明，噬菌体ФSM9-3Y的潜伏期为30min，暴发期的时间近40min，暴发量为629PFU/cell；(6) SDS-PAGE蛋白电泳分析发现，至少观察到13种蛋白，相对分子质量范围在25～130kDa之间，其中分子量为48kDa的主要蛋白带可能是噬菌体的衣壳蛋白。在初步完成噬菌体ФSM9-3Y的生物学特性分析后，将粘质沙雷菌经腹腔感染BLAB/c小鼠建立菌血症动物模型。在感染后的20min通过腹腔给予噬菌体(1×109PFU/mL)治疗,动物的存活率为100%，感染后40和60min给予噬菌体治疗，其动物的存活率为60%，明显高于对照组的存活率。说明通过腹腔途径给予噬菌体能提高由粘质沙雷菌引起菌血症动物的存活率。药代动力学表明，将噬菌体(109PFU/mL)注射动物体内后，24h和48h噬菌体滴度稳定在9.6×104PFU/mL和6.5×104PFU/mL的水平；如果将噬菌体(109PFU/mL)和细菌(108CFU/mL)同时注射动物体内，24h和48h噬菌体的滴度分别为6.9×106PFU/mL和7×105PFU/mL，细菌的浓度则降至102CFU/mL和2.1×10CFU/mL。说明体内如有宿主菌存在，由于不断产生新的噬菌体，其滴度维持在较高的水平，故可持续发挥抗感染作用。根据上述研究，可得出以下结论： (1)分离的这7株粘质沙雷菌噬菌体为毒性噬菌体（即具有裂解宿主菌的特性），其形态学特征符合有尾病毒目（Caudovirale）、肌尾病毒科（Myoviridae）噬菌体。 (2)生物学特性分析表明，噬菌体ФSM9-3Y感染宿主菌的潜伏期为30min；爆发期持续的时间约40min；暴发量为629PFU/cell。噬菌体ФSM9-3Y的最佳感染复数是1，即MOI=1。噬菌体ФSM9-3Y的基因组为双链DNA，大小约54kb。 (3) BALB/c小鼠经腹腔感染最小致死量的粘质沙雷菌后，给予噬菌体(ФSM9-3Y)治疗，能明显提高动物的存活率。 (4)药代动力学显示：噬菌体ФSM9-3Y与宿主菌同时注射动物体内后的24h，噬菌体的滴度要高于单一注射噬菌体的滴度。
[Abstract]:Serratia marcescens is a member of the family Enterobacteriaceae (Enterobacteriaceae), commonly found in water and soil, and also in the hospital environment. Patients with low immune function, large area burns, organ transplantation and tumor can cause various infections, such as urinary and respiratory infections, wound infection, bacteremia, endotoxic shock and Endocarditis, especially in recent years, the infection and outbreak of the neonatal intensive care unit caused by multi drug resistant saleanella saleanella has attracted the attention of medical workers and hospital administrators. On the one hand, the various infections caused by multiple drug resistant saleb and other drug resistant bacteria have reduced the efficacy of antibiotics on the one hand, and on the other hand, the efficacy of antibiotics is reduced. It also increases the financial burden of the patients. The phage is a virus infected with bacteria and exists in the natural environment. Due to the specificity and efficiency of the bacteriophage to bacterial lysis, people begin to pay attention to the important role of the bacteriophage in bacterial infection (especially the infection caused by multidrug-resistant bacteria and superfine bacteria). There are few studies on phage phage, so it is of great practical significance to understand the biological characteristics of phage phage and to observe the anti infection effect of phage therapy.
In this study, 10 strains of Serratia mucaris were used as host bacteria to separate 7 lysate salebbacteria phages from the sewage discharged from the hospital by double agar method, named SM41Y, SM254Y, SM23Y, SM13Y, SM01Y, SM9-3Y and SM22Y. electron microscopy. The morphological characteristics of the 7 bacteriophages conformed to the tail virus (Ordr). Caudovirale), the phage of the myocutaneous virus family (Family Myoviridae). Further, the biological characteristics of phage SM9-3Y were found: (1) the head was stereosymmetric with 20 sides, the diameter was about 70nm, the tail sheath was about 50nm, the tail of the tail sheath was connected with the tail plate and the tail filament; (2) the genome of the phage SM9-3Y genome was indicated by the restriction endonuclease Atlas of the genome. The size of the double stranded DNA was about 54kb, and it contained EcoRV, BamHI, SacII and NdeI. (3) the lysis test showed that the cleavage spectrum of the phage SM9-3Y to the 10 host bacteria was narrow and had no effect on the non host bacteria; (4) the optimal number of infection was 1 (MOI=1) for phage SM9-3Y, and (5) the one step growth curve indicated the potential of phage SM9-3Y. The incubation period was 30min, the time of the outbreak was nearly 40min and the outbreak was 629PFU/cell; (6) the SDS-PAGE protein electrophoresis analysis found that at least 13 proteins were observed, the relative molecular weight range was between 25 and 130kDa, and the main protein band of the molecular weight of 48kDa might be the capsid protein of phage.
After preliminarily completing the biological characteristics of phage SM9-3Y, the bacteriemia animal model was established by intraperitoneal infection with BLAB/c mice by intraperitoneal infection. The 20min was treated with phage (1 x 109PFU/mL) after infection. The survival rate of the animals was 100%, 40 and 60min after infection, and the survival rate of the animals was 60%. The survival rate in the control group was higher than that in the control group. It was indicated that the survival rate of bacteremia caused by saleba mucilage was increased by the peritoneal pathway, and the pharmacokinetics showed that the phage titer of 24h and 48h phage was stable at 9.6 x 104PFU/mL and 6.5 x 104PFU/mL levels after the phage (109PFU/mL) was injected in the animal body; if the phage (109PFU) was used, the phage (109PFU) /mL) and bacteria (108CFU/mL) were injected at the same time in animals. The titers of 24h and 48h phages were 6.9 x 106PFU/mL and 7 x 105PFU/mL respectively. The bacterial concentration decreased to 102CFU/mL and 2.1 x 10CFU/mL. indicating the presence of host bacteria in the body. The droplets of the phage were maintained at a high level because of the continuous production of new phages, so the anti infection was sustained. Use.
According to the above study, we can draw the following conclusions:
(1) the 7 phage phages isolated from the phage were toxic phages (that is, the characteristics of the lysate host bacteria), and their morphological features conformed to the phage of the tail virus (Caudovirale) and the myocutaneous (Myoviridae).
(2) the biological characteristics analysis showed that the incubation period of the phage SM9-3Y infected host was 30min, the duration of the outbreak period was about 40min, and the optimal number of 629PFU/cell. phage SM9-3Y was 1, that is, the genome of the MOI=1. phage SM9-3Y is double chain DNA, and the size is about 54kb.
(3) BALB/c mice infected with Serratia marcescens by abdominal cavity infection with minimal lethal amount of phage (phage SM9-3Y) can significantly improve the survival rate of animals.
(4) pharmacokinetics showed that phage SM9-3Y and 24h were injected simultaneously into host animals, and the titer of phages was higher than that of single injected phages.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R378.2

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