铜绿假单胞菌噬菌体vB_PaeS_HP1的分离鉴定和基因组学初步研究

Isolation and identification of bacteriophage vB_PaeS_HP1 from Pseudomonas aeruginosa and preliminary research in genomics

目的研究一株从医院未经处理的污水中筛选出的铜绿假单胞菌噬菌体的生物学特性和基因组成,为临床治疗铜绿假单胞菌感染提供新选择。方法利用16S rDNA技术对宿主菌进行菌种鉴定,并以其为模板采用双层平板法筛选出铜绿假单胞菌噬菌体,并测定其噬菌斑、形态特征、最佳感染复数等生物学特性;提取噬菌体DNA样品,进行全基因组检测,使用在线网站对功能基因进行预测,Mauve软件对噬菌体的共线性进行分析。结果菌种鉴定结果显示宿主菌Pae-01为铜绿假单胞菌,成功分离得到一株铜绿假单胞菌噬菌体,命名为vB_PaeS_HP1,其噬菌斑直径为7 mm,边缘整齐,清晰透明,无晕环;透射电镜观察属短尾噬菌体,多面体头部直径约为75 nm,尾部长度约为53 nm;最佳感染复数为1;潜伏期为30 min,裂解期为50 min;对不同的温度和pH具有较强的适应能力;NCBI数据库比对后确定其为一株全新的噬菌体;预测出43个功能基因,包括与噬菌体的结构相关基因、DNA复制和调控相关的基因、DNA包装蛋白相关的基因和其他功能基因;共线性分析显示其共线性良好,无基因突变情况。结论vB_PaeS_HP1是一株短尾噬菌体,生物学特性明确,功能基因完整,可为噬菌体疗法治疗铜绿假单胞菌感染提供新方案。

Objective To study the biological characteristics and genetic make up of a bacteriophage of Pseudomonas aeruginosa screened from untreated sewage from a hospital and provide a new option for treating Pseudomonas aeruginosa infections.Methods 16S rDNA technology was used to identify the host bacteria,and the bilayer plate method was used as a template to screen out Pseudomonas aeruginosa phages,and their biological characteristics such as plaque,morphological characteristics,and optimal infection complex were determined.Bacteriophage DNA samples were extracted,genome-wide testing was performed,functional genes were predicted using online websites,and collinear analysis of phages was performed with Mauve software.Results The identification results of the strain showed that the host bacterium Pae-01 was Pseudomonas aeruginosa,and a strain of Pseudomonas aeruginosa phage was successfully isolated as vB_PaeS_HP1,with a plaque diameter of 7 mm,neat edges,clear and transparent,and no halo rings.The observations from transmission electron microscopy showed that it was a short-tailed bacteriophage with a polyhedral head diameter of about 75 nm and a tail length of about 53 nm.The optimal number of infection is 1;the incubation period was 30 min and the lysis period was 50 min.The phage possessed strong adaptability to different temperatures and pH.After comparison of the NCBI database,it was determined to be a new bacteriophage.43 functional genes were predicted and they could be divided into four modules:bacteriophage structure,DNA replication and regulation,DNA packaging proteins,and other functional genes.Collinearity analysis showed that it was collinear and had no genetic mutations.Conclusion vB_PaeS_HP1 is a short-tailed phage with clear biological characteristics and intact functional genes that provides a new protocol for phage therapy for the treatment of Pseudomonas aeruginosa infection.