一株粘质沙雷氏菌烈性噬菌体污水分离及特性

Isolation and characterization of Serratia marcescens phage

【目的】以粘质沙雷氏菌(8039)为宿主菌从医院污水中分离噬菌体并对其基本生物学特点进行研究。【方法】四步法污水分离噬菌体;单、双层平板噬菌斑实验筛选烈性噬菌体并观察噬菌斑形态;纯化后2%磷钨酸染色电镜观察;手工法提取噬菌体核酸酶切后琼脂糖凝胶电泳分析;利用双层平板噬菌斑实验测定最佳感染复数和完成一步生长实验。【结果】从医院污水中成功分离出粘质沙雷氏菌烈性噬菌体一株(SM701),该噬菌体有一个正多面体立体对称的头部,头径约64nm,无囊膜,有一长尾,无收缩尾鞘,尾长约143nm;基因组核酸能被双链DNA内切酶BamHⅠ及HindⅢ切开,大小约57kb;噬菌斑圆形透明,直径1mm左右(培养12h),边界清楚;当感染复数(multiplicity of infection,MOI)为10时,子代噬菌体滴度较高;按照一步生长实验结果绘制出一步生长曲线,可知感染宿主菌的潜伏期是约为30min,爆发期约100min,平均爆发量约为63。【结论】按照国际病毒分类委员会分类标准,该噬菌体属于长尾噬菌体科(siphoviridae)烈性噬菌体,按照Bradley和Ackermann形态分类法属于B1亚群;噬菌斑与周围红色细菌生长区,颜色差异明显,非常便于观察和计数;噬菌体头部大小和形态与呼吸道病毒中的呼肠病毒和腺病毒最为接近;国内尚未见粘质沙雷氏菌噬菌体相关报道。

[Objective] To isolate the bacteriophage of Serratia marcescens（8039） from sewage, and to study on its biological characteristics. [Methods] We used Serratia marcescens（8039） as the host to isolate phage from raw sewage from the Sewage Treatment Center of Hospital. Phage SMT01 was examined in electron microscope. The genome of SMT01 was extracted and the size of its nucleic acid was identified with restriction enzyme analysis. Phage isolated was subjected to plaque forming unit （PFU） assay using double layer agar medium plate method and morphological properties of plaque was observed, and finally optimal MOI multiplicity of infection and one-step growth experiments were carried out. [Results] Phage SM701 specific to Serratia marcescens （8039） was isolated successfully from the raw sewage. SM701 had an isometric polyhedral head （about 64nm in diameter） and a long noncontractile tail （about 143nm long）.The nucleic acid could be cut off by dsDNA restriction enzyme BamH Ⅰ or Hind Ⅲ and its complete size was about 57kb. The plaque of SMT01 was transparent about lmm in diameter at the 12th hour. When MOI equaled 10, the number of phage offspring was higher. One-stepgrowth kinetics was determined according to the results of one-stepgrowth experiment, which showed that the latent period was about 30 min, the rise period was about 100 min, and the average bust size was about 63 pfu/cell. [Conclusion] Phage SM701 belonged to tailed family： siphoviridae and lyric bacteriophage. It was quite easy to observe and count the plaques due to existing color differences between lytic and nonlytic zone.