中兽医医药杂志2024，Vol.43Issue(3)：1-9,封2,10.DOI:10.13823/j.cnki.jtcvm.2024.034
肺炎克雷伯菌噬菌体的分离鉴定及vB_KpnS_Yuri1的生物学特性研究
Isolation and identification of phage of Klebsiella pneumoniae and study on biological characteristics of vB_KpnS_Yuri1
摘要
Abstract
The lytic phage of Klebsiella pneumoniae was isolated and identified,and the biological characteristics of the phage was analyzed.Using the standard strain of K.pneumoniae ATCC700603 as host bacteria,phages of lytic K.pneumoniae were isolated from wastewater of many farms in Gansu,Ningxia,Qinghai and Sichuan.The biological characteristics of Klebsiella phage vB_KpnS_Yuri1 were analyzed,and the morphology was observed by transmission electron microscopy.Analysis of nucleic acid types by enzyme digestion;the optimal infection multiplicity,one-step growth curve,temperature and pH stability and UV sensitivity were measured by double-layer plate method.The cracking spectrum was determined by spot test.Plate counting method was used to evaluate antibacterial activity in vitro.Its safety was analyzed based on whole genome sequencing.A total of 6 lytic phages were isolated,including Klebsiella phage vB_KpnS_Yuri1,a long-tailed phage with double-stranded DNA,and the optimal infection complex number was 0.000 1.The one-step growth curve showed that the incubation period and cleavage period of Klebsiella phage vB_KpnS_Yuri1 were 15 min and 15 min,respectively.Klebsiella phage vB_KpnS_Yuri1 showed high titer and UV tolerance when the temperature was-20℃ to 60℃ or pH was 5.0 to 10.0.The drop test showed that Klebsiella phage vB_KpnS_Yuri1 had lytic activity against 7 of 68 strains of Klebsiella pneumoniae,but had no activity against Enterococcus faecalis,Escherichia coli and Staphylococcus aureus.In LB broth medium and milk medium,the bactericidal rate reached 99.99%within 2 h.Based on whole-genome analysis,the total length of Klebsiella phage vB_KpnS_Yuri1 genome was 50 116 bp,with 77 open reading frames,and no genes related to drug resistance,virulence and lysogen were found.The bacteriophage Klebsiella phage vB_KpnS_Yuri1 was isolated with low infection number,short incubation period,wide optimum temperature and pH range,UV resistance,strong antibacterial activity in vitro,and safety at the gene level.It can provide theoretical basis and technical support for further study of phage cocktail therapy and lyase in the treatment of cow mastitis caused by Klebsiella pneumoniae.关键词
肺炎克雷伯菌/噬菌体/分离/鉴定/生物特性Key words
Klebsiella pneumoniae/phage/isolation/identification/biological characteristics分类
农业科技引用本文复制引用
孙续,中拉毛草,杨峰,杨明俊..肺炎克雷伯菌噬菌体的分离鉴定及vB_KpnS_Yuri1的生物学特性研究[J].中兽医医药杂志,2024,43(3):1-9,封2,10.基金项目
国家重点研发计划项目(2022YFD1302101) （2022YFD1302101）
