本文選題：粘質沙雷菌 + 噬菌體�。� 參考：《吉林大學》2014年碩士論文

【摘要】：粘質沙雷菌(Serratia marcescens)屬腸桿菌科（Enterobacteriaceae）成員，一般存在于水和土壤中，亦可存在醫(yī)院的環(huán)境，對免疫功能低下、大面積燒傷、器官移植和腫瘤的患者可造成各種感染，如泌尿道和呼吸道感染、傷口感染、菌血癥、內毒素休克和心內膜炎等。特別是近年來由多重耐藥粘質沙雷菌所致的新生兒監(jiān)護病房的感染及爆發(fā)流行，已引起醫(yī)務工作者和醫(yī)院管理者的高度重視。多重耐藥粘質沙雷菌和其他耐藥性細菌引起的各種感染一方面降低了抗生素的療效，另一方面也增加了患者的經濟負擔。噬菌體是感染細菌的病毒，存在于自然環(huán)境中，由于噬菌體對細菌裂解的特異性和高效性，使得人們重新開始關注噬菌體在細菌性感染(特別是多重耐藥細菌和超級細菌所致的感染)中的重要作用。根據現有的資料，有關粘質沙雷菌噬菌體的研究很少，因此，深入和全面了解粘質沙雷菌噬菌體的生物學特性和觀察噬菌體療法的抗感染作用，具有重要的現實意義。 本項研究以10株粘質沙雷菌為宿主菌，采用雙層瓊脂法從醫(yī)院排出的污水中分離出7株裂解性粘質沙雷菌的噬菌體，分別命名為ФSM41Y、ФSM254Y、ФSM23Y、ФSM13Y、ФSM01Y、ФSM9-3Y和ФSM22Y。電鏡觀察顯示：這7株噬菌體的形態(tài)特征符合有尾病毒目(Ordr Caudovirale)、肌尾病毒科（Family Myoviridae）噬菌體。進一步對噬菌體ФSM9-3Y的生物學特性分析時發(fā)現：(1)頭部呈20面體立體對稱、直徑約70nm，尾鞘長約50nm，尾鞘的末端連接有尾板和尾絲；(2)噬菌體ФSM9-3Y基因組的限制性酶切圖譜提示，基因組為雙鏈DNA，大小約為54kb左右，且含EcoRV、BamHI、SacII和NdeI的酶切位點；(3)裂解試驗顯示，噬菌體ФSM9-3Y對10株宿主菌的裂解譜較窄，對非宿主菌無任何作用；(4)噬菌體ФSM9-3Y的最佳感染復數為1(MOI=1)；(5)一步生長曲線表明，噬菌體ФSM9-3Y的潛伏期為30min，暴發(fā)期的時間近40min，暴發(fā)量為629PFU/cell；(6) SDS-PAGE蛋白電泳分析發(fā)現，至少觀察到13種蛋白，相對分子質量范圍在25～130kDa之間，其中分子量為48kDa的主要蛋白帶可能是噬菌體的衣殼蛋白。 在初步完成噬菌體ФSM9-3Y的生物學特性分析后，將粘質沙雷菌經腹腔感染BLAB/c小鼠建立菌血癥動物模型。在感染后的20min通過腹腔給予噬菌體(1×109PFU/mL)治療,動物的存活率為100%，感染后40和60min給予噬菌體治療，其動物的存活率為60%，明顯高于對照組的存活率。說明通過腹腔途徑給予噬菌體能提高由粘質沙雷菌引起菌血癥動物的存活率。藥代動力學表明，將噬菌體(109PFU/mL)注射動物體內后，24h和48h噬菌體滴度穩(wěn)定在9.6×104PFU/mL和6.5×104PFU/mL的水平；如果將噬菌體(109PFU/mL)和細菌(108CFU/mL)同時注射動物體內，24h和48h噬菌體的滴度分別為6.9×106PFU/mL和7×105PFU/mL，細菌的濃度則降至102CFU/mL和2.1×10CFU/mL。說明體內如有宿主菌存在，由于不斷產生新的噬菌體，其滴度維持在較高的水平，故可持續(xù)發(fā)揮抗感染作用。 根據上述研究，可得出以下結論： (1)分離的這7株粘質沙雷菌噬菌體為毒性噬菌體（即具有裂解宿主菌的特性），其形態(tài)學特征符合有尾病毒目（Caudovirale）、肌尾病毒科（Myoviridae）噬菌體。 (2)生物學特性分析表明，噬菌體ФSM9-3Y感染宿主菌的潛伏期為30min；爆發(fā)期持續(xù)的時間約40min；暴發(fā)量為629PFU/cell。噬菌體ФSM9-3Y的最佳感染復數是1，即MOI=1。噬菌體ФSM9-3Y的基因組為雙鏈DNA，大小約54kb。 (3) BALB/c小鼠經腹腔感染最小致死量的粘質沙雷菌后，給予噬菌體(ФSM9-3Y)治療，能明顯提高動物的存活率。 (4)藥代動力學顯示：噬菌體ФSM9-3Y與宿主菌同時注射動物體內后的24h，噬菌體的滴度要高于單一注射噬菌體的滴度。
[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.
【學位授予單位】：吉林大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R378.2

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