应用裂解性噬菌体治疗多重耐药肺炎克雷伯氏菌引起的小鼠肺炎

发布时间：2018-05-17 13:58

  本文选题：噬菌体 + 多重耐药　； 参考：《大连理工大学》2014年硕士论文

【摘要】：肺炎克雷伯氏菌是医院获得性肺炎主要的条件致病菌。近年来抗菌药物的不合理使用,导致肺炎克雷伯氏菌耐药株增多、耐药性增强,给用药及治疗造成了极大的困扰,严重威胁人的生命健康。噬菌体是一类细菌性病毒,在自然环境中普遍存在,因其具有特异性、高效性、安全性、低成本、开发周期短等优点,被认为是最具前景的抗生素替代品。本研究探索应用裂解性噬菌体防治多重耐药肺炎克雷伯氏菌引起的小鼠肺炎,为多重耐药菌的治疗提供一条新途径。 从医院患肺炎病人的痰液中分离纯化病原菌,经形态学观察、生理生化实验、16SrRNA序列分析、药敏实验分析、生物被膜形成实验对病原菌进行分类鉴定,分离获得一株产生物被膜的多重耐药肺炎克雷伯氏菌(K. pneumoniae),命名为KP1513。将已分离的病原菌作为宿主菌,采用双层平板法从医院处理前污水中分离裂解性噬菌体。该噬菌体纯化后,通过透射电镜观察,考察其氯仿敏感性、pH稳定性、温度稳定性、宿主谱、潜伏期、裂解量等噬菌体生物学特性,将筛选获得的裂解性噬菌体命名为phage1513。该噬菌体属于有尾噬菌体目、长尾噬菌体科,宿主专一性强,对氯仿不敏感,因而不含脂类物质,且在小于40℃,pH为5-10范围内较稳定。感染宿主菌的潜伏期约30min,裂解量约264PFU/cell。phage1513基因组测序及功能基因分析表明,该噬菌体基因组大小为49462bp,编码72个基因,含有4个功能模块,与Tunalinkevirus噬菌体属亲缘关系最近,不含抗生素抗性基因及毒素基因,且其裂解机制与穿孔素及裂解酶相关。 通过测定细菌与噬菌体共培养物在650nm处的吸光值来考察噬菌体对病原菌的体外裂解效果。体外抑菌实验表明,phage1513具有快速感染并裂解宿主菌的能力,能够抑制生物被膜的形成。体内实验采用滴鼻的给药方式。小鼠致死模型中,通过对比PBS组、噬菌体预防实验组(攻毒24h前给药)和噬菌体治疗实验组(攻毒2h后给药)小鼠的存活率,评价噬菌体防治小鼠肺炎的效果。结果表明,治疗组中不同感染复数的噬菌体均能一定程度上降低小鼠死亡率,且感染复数越高,死亡率越低,但噬菌体不能对小鼠肺炎起到预防作用。在小鼠亚致死模型中,通过考察小鼠肺部组织病理切片,小鼠体重变化,以及肺组织匀浆液中菌数、噬菌体数以及炎症因子(IL-6,TNF-α)的变化,证明phage1513在体内能高效裂解病原菌,改善肺炎症状,减轻炎症反应。 本研究筛选了针对多重耐药肺炎克雷伯氏菌的裂解性噬菌体,研究其生物学性质,进行基因组测序及功能基因分析,并对体外体内裂解宿主的能力和效果进行评价,为噬菌体应用于细菌性肺炎的防治提供了依据。
[Abstract]:Klebsiella pneumoniae is a major opportunistic pathogen of hospital acquired pneumonia. In recent years, the irrational use of antimicrobial agents has led to the increase of resistant strains of Klebsiella pneumoniae and the increase of drug resistance, which has caused great distress to drug use and treatment, and has seriously threatened the life and health of human beings. Bacteriophage is a kind of bacterial virus, which is widely used in natural environment. Because of its specificity, efficiency, safety, low cost and short development cycle, phage is considered as the most promising substitute for antibiotics. This study explored the application of lytic phage in the prevention and treatment of mouse pneumonia induced by multidrug resistant Klebsiella pneumoniae and provided a new approach for the treatment of multidrug resistant bacteria. The pathogenic bacteria were isolated and purified from the sputum of patients with pneumonia in hospital. The pathogens were classified and identified by morphological observation, physiological and biochemical experiments, 16s rRNA sequence analysis, drug sensitivity analysis and biofilm formation experiment. A strain of multidrug resistant Klebsiella pneumoniae was isolated and named KP1513. The lytic bacteriophage was isolated from pre-hospital sewage by double-layer plate method using isolated pathogenic bacteria as host bacteria. The bacteriophage was purified by transmission electron microscope. The pH stability, temperature stability, host spectrum, incubation period and cleavage amount of chloroform sensitive phage were investigated. The selected lytic phage was named phage1513. The bacteriophage belongs to the order Phage with tail. It has strong host specificity, is not sensitive to chloroform, so it does not contain lipids, and is stable in the range of pH 5-10 at less than 40 鈩，

本文编号：1901645