一株分离自野鼠鼠疫疫源地污水中的鼠疫噬菌体特性研究

Study on characteristics of a Yersinia pestis phage isolated from sewage of wild rodent plague focus

目的阐明云南鹤庆野鼠疫源地污水中分离的1株鼠疫噬菌体的表型及基因组特征,为进一步探究该疫源地鼠疫微生态学提供更多依据。方法以鼠疫疫苗株EV76为宿主菌,自污水中分离纯化噬菌体,观察其电镜下的形态、噬菌谱、最适pH及温度稳定性、最佳感染复数、一步生长曲线等生物学特性以及基因组特性。结果从采集的20份污水中分离得到1株鼠疫噬菌体,命名为DMC18。该噬菌体具有二十面体头部和短小的尾部,头部直径(40±2)nm,尾部长(20.5±1.0)nm,属短尾噬菌体科。DMC18除可裂解鼠疫菌外,37℃下还可裂解假结核耶尔森菌I、II、V、VI型;pH2~13及温度4~37℃对其活性无明显影响,最佳感染复数为0.1;一步生长曲线显示其潜伏期为40min,暴发期为30min;其基因组全长为39048bp,G+C含量为47.1%,编码47种蛋白,不编码tRNA基因,未发现抗生素抗性基因和毒力基因。遗传进化分析显示DMC18与YePe2位于同一分支,相似度为99.15%。结论从云南鹤庆野鼠鼠疫疫源地污水中分离到1株鼠疫烈性噬菌体,属T7噬菌体家族,该株鼠疫噬菌体环境适应性较强,值得进一步研究。

Objective To elucidate the phenotypic and genomic characteristics of a Yersinia pestis phage isolated from sewage in Heqing wild plague focus in Yunnan Province,and to provide more evidence for further exploring the microecology of plague in this focus.Methods Plague vaccine strain EV76 was used as the host bacteria,and phages were isolated and purified from sewage.The biological characteristics such as morphology,phage spectrum,optimal pH and temperature stability,optimal multiplicity of infection,one-step growth curve and genomic characteristics were observed.Results One strain of Y.pestis phage was isolated from 20 parts of sewage collected and named DMC18.The bacteriophage has an icosahedral head and a short tail with a head diameter of(40±2)nm and a tail length of(20.5±1.0)nm,belonging to the Autographiviridae.DMC18 can not only lyse Y.pestis,but also lyse Y.pseudotuberculosis type I,II,V and VI at 37℃.The pH 2-13 and temperature 4-37℃had no significant effect on its activity,and the optimum multiplicity of infection was 0.1.The one-step growth curve showed that the incubation period was 40 min and the outbreak period was 30 min.Its genome was 39048 bp,the G+C content was 47.1%,it encoded 47 proteins,did not encode tRNA genes,and found no antibiotic resistance genes and virulence genes.Genetic evolutionary analysis showed that DMC18 and YePe2 were located in the same branch with 99.15%similarity.Conclusion A strain of Y.pestis virulent phage was isolated from the sewage of the plague foci in Heqing,Yunnan Province,which belongs to the T7 phage family.The Y.pestis phage has strong environmental adaptability and is worthy of further study.