耐碳青霉烯类肺炎克雷伯菌噬菌体vB_KpnP1的分离鉴定和生物学特性研究  

Isolation,identification and biological characteristics of phage vB_KpnP1 for carbapenem-resistant Klebsiella pneumoniae

作　　者：罗书泓 汪璐 徐朝霞 刘佳姣 张维维 冯健 李福祥 LUO Shuhong;WANG Lu;XU Zhaoxia;LIU Jiajiao;ZHANG Weiwei;FENG Jian;LI Fuxiang(Department of Respiratory Medicine,Affiliated Hospital of Southwest Medical University,Luzhou,Sichuan Province,646000;Department of Critical Care Medicine,the General Hospital of Western Theater Command,Chengdu,Sichuan Province,610083,China;Department of Clinical Laboratory,the General Hospital of Western Theater Command,Chengdu,Sichuan Province,610083,China;Department of Emergency,the General Hospital of Western Theater Command,Chengdu,Sichuan Province,610083,China)

机构地区：[1]西南医科大学附属医院呼吸内科,四川泸州646000 [2]西部战区总医院重症医学科,成都610083 [3]西部战区总医院检验科,成都610083 [4]西部战区总医院急诊科,成都610083

出　　处：《陆军军医大学学报》2023年第10期1030-1039,共10页Journal of Army Medical University

基　　金：四川省卫生健康委员会干部保健科研课题(川干研2022-1303);西部战区总医院军事医学科研项目(2019LH05)。

摘　　要：目的分离出1株裂解碳青霉烯类耐药肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumoniae,CRKP)的噬菌体,研究其生物学特性和全基因组信息。方法采用双层琼脂平板法从西部战区总医院污水中分离纯化噬菌体,通过透射电镜观察噬菌体的形态,测定噬菌体的生物学特性和体外抑菌活性,进行全基因组测序并分析其结构特征,系统发育进化树和比较基因组分析其进化关系。结果分离出1株烈性噬菌体,命名为vB_KpnP1,形成的噬菌斑为2~3 mm透明的圆形,周围有明显的晕环;电镜下发现该噬菌体的头部大小约68.2 nm×64.3 nm,尾部长约11 nm,其头部为正二十面体,有1条不可收缩的短尾,属于有尾噬菌体目,自复制短尾噬菌体科;其最佳感染复数(multiplicity of infection,MOI)为0.0001,潜伏期为10 min,裂解期为30 min,爆发量为26 PFU/cell;10 min吸附率约为80%;该噬菌体对氯仿不敏感,在温度为10~50℃、pH值为4~12条件下较稳定;其对碳青霉烯类耐药肺炎克雷伯菌裂解率为54.55%(12/22);体外抑菌实验表明其可抑制宿主菌的生长。噬菌体vB_KpnP1为双链DNA(dsDNA),基因组全长为40352 bp,GC含量为53.09%,含有50个编码序列(coding sequence,CDS)。结论成功分离出1株具有治疗碳青霉烯类耐药肺炎克雷伯菌感染潜力的毒性噬菌体vB_KpnP1。Objective To isolate a strain of lysing carbapenem-resistant Klebsiella pneumoniae phage,and explore its biological characteristics and whole genome information.Methods The phage was isolated and purified from waste water of the General Hospital of Western Theater Command Hospital by the double-layer agar plate method.The morphology of phage was observed by transmission electron microscopy,and its biological properties and in vitro antibacterial activity were determined.Whole-genome sequencing was used to analyze its structural characteristics,and a phylogenetic evolutionary tree and the comparative genomic analysis was conducted to analyze the evolutionary relationships of phage.Results A virulent phage was isolated and named vB_KpnP1,which formed a 2~3 mm transparent circular spot surrounded by a distinct halo;Electron microscopy showed that the head size of the phage was about 68.2 nm×64.3 nm,and the tail length was about 11 nm;The head of the phage was a regular icosahedron with a non-contractile short tail,which was thus identified as a member of the Autographiviridae family and the Caudovirales order;the optimal multiplicity of infection was 0.0001,the incubation period was 10 min,the lysis period was 30 min,and the amount of lysis was 26 PFU/cell;The adsorption rate was about 80%in 10 min.The phage was insensitive to chloroform and was stable at temperatures of 10~50℃and pH values of 4~12;The lysis rate of carbapenem-resistant Klebsiella pneumoniae was 54.55%(12/22),and the host spectrum was wide;In vitro antibacterial test showed that it could inhibit the growth of host bacteria.Phage vB_KpnP1 was double-stranded DNA(dsDNA)with a 40352 bp of full genome length,and 53.09%of GC content.It contained 50 CDS.Conclusion A virulent Klebsiella pneumoniae phage,vB_KpnP1,with the potential to treat carbapenem-resistant Klebsiella pneumoniae infections,is successfully isolated.

关 键 词：噬菌体 碳青霉烯类耐药肺炎克雷伯菌 生物学特性 基因组分析 

分 类 号：Q93-331[生物学—微生物学] Q939.48