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

作　　者：孙续 中拉毛草[2] 杨峰[3] 杨明俊[1] SUN Xu;ZHONGLA Maocao;YANG Feng;YANG Mingjun(College of Life Sciences and Engineering,Lanzhou University of Technology,Lanzhou Gansu 730050,China;Animal Disease Prevention and Control Center of Gannan Tibetan Autonomous Prefecture,Hezuo Gansu 747000,China;Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS,Lanzhou Gansu 730050,China)

机构地区：[1]兰州理工大学生命科学与工程学院,甘肃兰州730050 [2]甘肃省甘南藏族自治州动物疫病预防控制中心,甘肃合作747000 [3]中国农业科学院兰州畜牧与兽药研究所,甘肃兰州730050

出　　处：《中兽医医药杂志》2024年第3期1-9,F0002,共10页Journal of Traditional Chinese Veterinary Medicine

基　　金：国家重点研发计划项目(2022YFD1302101)。

摘　　要：分离并鉴定肺炎克雷伯菌裂解性噬菌体,分析所分离肺炎克雷伯菌噬菌体的生物学特性。以肺炎克雷伯菌ATCC700603标准株作为宿主菌,从甘肃、宁夏、青海、四川等地多个养殖场污水中分离裂解性肺炎克雷伯菌噬菌体;分析肺炎克雷伯菌噬菌体Yuri1的生物学特性,用透射电子显微镜观察其形态;通过酶切分析核酸类型;采用双层平板法测定最佳感染复数、一步生长曲线、温度和pH稳定性及紫外线敏感性;通过点滴试验测定裂解谱;采用平板计数法评价噬菌体体外抗菌活性;基于全基因组测序分析其安全性。结果显示,共分离裂解性噬菌体6株,其中KlebsiellaphagevB_KpnS_Yuri1属有尾目长尾噬菌体,核酸类型为双链DNA,最佳感染复数为0.0001;一步生长曲线显示,KlebsiellaphagevB_KpnS_Yuri1的潜伏期为15min,裂解期为15min;在-20℃~60℃或pH值为5.0~10.0时,KlebsiellaphagevB_KpnS_Yuri1仍具有较高的滴度,且呈现紫外线耐受;点滴试验表明,KlebsiellaphagevB_KpnS_Yuri1对68株奶牛乳腺炎肺炎克雷伯菌中的7株具有裂解活性,而对粪肠球菌、大肠杆菌和金黄色葡萄球菌均无裂解活性;在LB肉汤培养基和牛奶介质中,噬菌体在2h内的杀菌率均达到99.99%;基于全基因组分析,KlebsiellaphagevB_KpnS_Yuri1基因组全长50116bp,共有77个开放阅读框,未发现与耐药、毒力及溶原相关基因。分离的肺炎克雷伯菌噬菌体KlebsiellaphagevB_KpnS_Yuri1最佳感染复数低、潜伏期短,最适温度和pH范围广且耐受紫外线,体外抗菌活性强,在基因水平上具有安全性,可为进一步研究噬菌体鸡尾酒疗法及裂解酶治疗肺炎克雷伯菌引发的奶牛乳腺炎提供理论依据和技术支撑。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 0.0001.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 50116 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 theore

关 键 词：肺炎克雷伯菌 噬菌体 分离 鉴定 生物特性 

分 类 号：S855.1[农业科学—临床兽医学]