【作者】 杨梅；

【导师】 韩文瑜；

【作者基本信息】 吉林大学 ， 预防兽医学， 2015， 硕士

【摘要】 铜绿假单胞菌是一种在环境中广泛存在的革兰氏阴性致病菌。该菌可以引起水貂严重的出血性肺炎，给水貂养殖业带来了巨大的损失。近些年，由于多重耐药铜绿假单胞菌的出现，使该菌引起感染的控制变得越来越困难。噬菌体是可以特异性感染细菌的病毒，可以作为一种新型的治疗制剂，在治疗多重耐药菌株感染方面具有很大的应用潜力。本研究以实验室前期在貂场患病水貂体内分离的致病性铜绿假单胞菌菌株D9作为宿主菌，从污水中分离获得了一株新的噬菌体，将其命名为YH6。对噬菌体YH6的生物特性进行了研究，表明该噬菌体是可以特异性的裂解铜绿假单胞菌的烈性噬菌体，噬菌斑清晰透亮、无晕环；该噬菌体属于有尾病毒目短尾病毒科，头部呈正二十面体，头部直径大约是65nm，尾部较短，大约是25nm；其潜伏期为30min，裂解周期为60min；最佳感染复数为0.01-0.001，噬菌体滴度能达到1010PFU/mL；该噬菌体对乙醚、氯仿不敏感；在温度40°C、50°C环境中和pH值6.0~10.0时保持稳定；YH6可以裂解82.61%的铜绿假单胞菌，表现出了广谱的裂解活性。对噬菌体YH6进行了全基因组序列测序和分析，表明该噬菌体的遗传物质为双链线性DNA，长度为73,050bp，具有90个开放阅读框（ORFs），G+C%含量为54.88%，其基因编码率为92.64%，与铜绿假单胞菌的噬菌体LUZ7，vB_PaeP_C2-10_Ab09，PA26和LIT1的基因组同源性较近。YH6基因组中不含有细菌毒力和溶源性相关的ORF，更利于该噬菌体作为治疗制剂应用。通过序列比对和分析确定了噬菌体YH6的裂解酶基因（LysYH6），对该基因进行表达和纯化，活性测定实验表明裂解酶LysYH6在EDTA（25mM/L）存在的条件下表现出显著的杀菌活性。动物实验表明，对小鼠进行鼻腔接种铜绿假单胞菌D9（2×108CFU），能够成功构建小鼠的出血性肺炎模型。治疗实验表明一次性鼻腔应用噬菌体YH6（2×107PFU，MOI=0.1）可以有效保护感染铜绿假单胞菌造成的出血性肺炎小鼠模型。对治疗组小鼠血液、脾脏、肺脏进行了菌落计数，表明噬菌体YH6应用10h以后可以使脏器内的菌含量降低1.5个对数单位，2d以内可以将体内的铜绿假单胞菌完全清除；与此相反地，未治疗组脏器内菌含量升高4个对数单位，并在2d内引起死亡。另外，对治疗组小鼠的肺脏进行了组织病理学切片观察，表明YH6能有效的改善铜绿假单胞菌造成的小鼠肺损伤。综上，本研究所分离到的噬菌体YH6在治疗铜绿假单胞菌引起的小鼠出血性肺炎模型方面表现出很大的应用潜力。为后续研究将该噬菌体应用到水貂感染铜绿假单胞菌的治疗奠定了坚实的基础。更多还原

【Abstract】 Pseudomonas aeruginosa is a common opportunistic pathogen that is widelypresent in the environment. The treatment of the infection caused by P. aeruginosahas become even more difficult because of the emergence of multidrug-resistantstrains. Phage therapy is expected to be useful as an alternative therapeutic measurefor treating multidrug-resistant P. aeruginosa infections.In this study, we used P. aeruginosa D9isolated from diseased mink in laboratoryearly as host strain and isolated a new phage from sewage, named YH6. Thebiological characteristics of YH6were studied, which indicated that the phage isspecific cleavage of P. aeruginosa, the plaque of YH6was clear, bright and no halo.The phage belongs to Podoviridae in Caudovirales, which has an isometricallyhexagonal head of about65nm in diameter and a about25nm short tail. The curve ofYH6showed a short latency period of about30min and a rise period of60min. Thephage titers were highest when the MOI was0.01~0.001, reaching approximately1010PFU/mL. YH6was resistant to chloroform and ether. YH6was relatively stable at40°C and50°C. The infectivity ability of YH6was relatively stable at pH6.0~10.0.Host spectrum analysis showed that the YH6has very broad spectrum, lysing82.61%of P. aeruginosa strains.The sequencing and analysis show that the complete genome of YH6is made of73,050bp of double-stranded DNA containing90open reading frames (ORFs). PhageYH6has an overall G+C content of54.88%and the gene-coding potential of theglobal genome is92.64%. YH6showed similar morphology with several other P.aeruginosa phages, such as LUZ7, vB_PaeP_C2-10_Ab09, PA26and LIT1. Moreimportantly, there is no bacterial-virulence or lysogenesis-related ORF in the YH6genome, which made it eligible for use in phage therapy. The genome of phage YH6was sequenced and the endolysin gene (LysYH6) of YH6was identified by blast analysis, then LysYH6was expressed and purified. LysYH6showed a significantinhibitory activity against P. aeruginosa at the exist of EDTA (25mM/mL).Animal experiments showed that using a mink source of P. aeruginosa D9(2×107CFU) infection in mice could also cause pneumonia in the model. A singleadministration intranasal of YH6(2×107PFU, MOI=0.1) was sufficient to protect themice from fatal P. aeruginosa infection. The ability of YH6to reduce bacterial countsin the blood, spleen, and lung was investigated. The mice that were treated with YH62h after the inoculation demonstrated a drop of1.5log units by10h after YH6treatment and ultimately became undetectable within2days. By contrast, the bacterialcounts in untreated mice showed approximately4log units increasement, reaching to108CFU/mL, and caused death within2days. Moreover, YH6greatly improvedpathological manifestations of lung injury from pneumonia. In view of the effectiveprotective efficacy in mice, phage YH6might be an adjuvant or alternative treatmentstrategy for infections caused by multidrug-resistant P. aeruginosa.In summary, phage YH6showed potential application for controlling thehemorrhagic pneumonia of mouse caused by P. aeruginosa infections. This study laida solid foundation for the follow-up application of phage therapy in mink model.更多还原