摘要
目的明确肺炎克雷伯菌噬菌体PhF168的生物学特性及其对小鼠重症败血症的疗效。方法电镜观察PhF168的形态,调查其噬菌谱并研究其一步生长曲线、温度和酸碱稳定性等生物学特性;提取PhF168的基因组,酶切鉴定其核酸类型,并初步估算其基因组的大小;建立小鼠重症败血症感染模型,观察PhF168治疗效果。结果 PhF168可在宿主菌F168上形成直径3～5mm、完全透明并带有较宽晕环的噬菌斑,为裂解性噬菌体。电镜下其颗粒呈有尾噬菌体目长尾噬菌体科的形态特征。PhF168的基因组为dsDNA,〉40kb,含有EcoRⅠ和BamHⅠ限制性内切酶切位点。PhF168在宿主菌F168中的潜伏期为15min,裂解量为（64±5）PFU/细胞,在pH 4～9和〈50℃下具良好稳定性。用PhF168治疗由其宿主菌F168感染所致的重症败血症小鼠,所有小鼠的生存期均在3d以上,7d存活率达40%。未治疗对照组小鼠1d内全部死亡。结论 PhF168杀菌作用高效,具有较好的热稳定性和酸碱稳定性,治疗宿主菌引起的小鼠重症败血症疗效显著,可作为治疗细菌感染的噬菌体候选株。
Objective The purpose of this work was to characterize a phage, PhF168, that infects clinical isolates of Klebsiella pneurnoniae. The efficacy of PhF168 in alleviating a serious systemic infection induced by K. pneumoniae was also investigated. Methods The morphology of the PhF168 phage was observed using electron microscopy, and the phage was characterized based on its host range, one-step growth curve, temperature, and pH sensitivity. The restriction pattern of the PhF168 genome was investigated. Moreover, PhF168 was used to treat mice with fatal septicemia induced by K. pneurnoniae. Results PhF168 exhibited lytic activity on the host strain （K. pneumoniae clinical isolate F168） as indicated by formation of a distinguishable clear spot with a wide halo （3-5 mm in diameter）. Morphologically, PhF168 can be classified as a member of the Styloviridae family and the Caudovirales order. The PhF168 genome is longer than 40 kb in length and it can be cleaved by the restriction enzymes EcoR I and BamH I. In the sensitive strain F168, PhF168 proliferated efficiently, with a short latency period （15 rain） and a large burst size （64_＋5 PFU/ml）. The phage is stable over a wide pH range （4 to 9） and at high temperatures （50~C）. Treatment with the PhF168 phage significantly improved the survival of mice. Without treatment, all mice died within 24 h of inoculation via intraperitoneal injection of 106 CFU of the F168 strain of K. pneumoniae. In contrast, 107 PFU of PhF168 intravenously provided 2 hours after inoculation resulted in a 100% survival rate after 3 days and a 40% survival rate after 7 days （P〈0.01）. Conclusion This study provides experimental evidence that the PhF168 phage is highly effective in treating a serious systemic infection caused by the F168 strain of K. pneumoniae in mice. The characteristics of PhF168 greatly enhs ice its utility as a biological bactericide.
出处
《中国病原生物学杂志》
CSCD
北大核心
2016年第6期495-499,共5页
Journal of Pathogen Biology
基金
河南省科技厅重点科技攻关项目(No.142102310440)
河南省医学科技攻关计划项目(No.201303199)
