1株羊源多重耐药肺炎克雷伯菌噬菌体的生物学特性及全基因组分析

Biological Characteristics and Whole Genome Analysis of a Multidrug-resistant Klebsiella pneumoniae Phage of Sheep Origin

【目的】多重耐药病原菌的出现对公共卫生安全构成严重威胁,本试验拟对1株羊源多重耐药肺炎克雷伯菌裂解性噬菌体进行生物学特性和基因组特征分析,为寻找可用于防治肺炎克雷伯菌感染的噬菌体制剂提供参考。【方法】以羊源多重耐药肺炎克雷伯菌分离株YH-2为宿主菌,采用双层琼脂平板法从粪污样品中分离纯化噬菌体,通过透射电镜观察其形态;测定噬菌体宿主谱、最佳感染复数(MOI)、一步生长曲线、热稳定性、酸碱耐受性等生物学特性;基于全基因组测序对其基因组特点进行分析。【结果】试验成功分离到1株肺炎克雷伯菌裂解性噬菌体,命名为vB_KpnP_PYH-2(简称PYH-2),电镜观察发现PYH-2属于短尾噬菌体科,其噬菌斑中心透亮,周围有晕圈。随着培养时间的延长,晕圈会向四周扩散。噬菌体PYH-2不仅可以裂解1株羊源肺炎克雷伯菌,还能裂解5株奶牛乳房炎源肺炎克雷伯菌。噬菌体PYH-2的最佳MOI为0.001,潜伏期为15 min,爆发期为10 min,裂解量为63 PFU/cell;在40~50℃和pH 4.0~12.0的环境下均能保持稳定活性。噬菌PYH-2基因组全长45958 bp,GC含量为51.86%,不含耐药基因及毒力基因;全基因组中包含52个开发阅读框(ORFs),只有21个为已知功能基因,其中尾部相关蛋白有6个。【结论】本研究分离到1株潜伏期短、生物特性稳定、能高效杀菌的噬菌体,可作为裂解动物源肺炎克雷伯菌的噬菌体候选毒株,为噬菌体防治多重耐药性肺炎克雷伯菌感染奠定了基础。

【Objective】The emergence of multidrug-resistant pathogens poses a serious threat to public health security.The biological and genomic characteristics of a lytic phage of multidrug-resistant Klebsiella pneumoniae from sheep were analyzed in order to provide reference for the search of phage preparations for the prevention and treatment of Klebsiella pneumoniae infection.【Method】Multidrug-resistant Klebsiella pneumoniae strain YH-2 from sheep was used as host bacteria to isolate and purify phage from fecal samples by double agar plate method,then observed the morphology by transmission electron microscope.The host spectrum,optimum multiplicity of infection(MOI),one-step growth curve,thermal stability,acid and base tolerance of phage were measured.The genome characteristics were analyzed based on whole genome sequencing.【Result】A lytic phage from Klebsiella pneumoniae,named vB_KpnP_PYH-2(PYH-2),was successfully isolated,which belong to the Podoviridae family.There was a halo around it,the halo spread around as the culture time went on.Phage PYH-2 could not only lyse the Klebsiella pneumoniae strains of sheep origin,but also lyse Klebsiella pneumoniae strains derived from cow mastitis.The optimal MOI was 0.001,the latent period was 15 min,the burst period was 10 min,and the amount of cleavage was 63 PFU/cell.Phage PYH-2 could maintain stable activity at 40-50℃and pH 4.0-12.0.The full length of the phage PYH-2 genome was 45958 bp,with 51.86%GC content,and it did not contain any drug resistance genes and virulence genes.There were 52 open reading frames(ORFs)in the genome of PYH-2,only 21 ORFs were known functional genes,including 6 tail-associated proteins.【Conclusion】In this study,a phage with short incubation period,stable biological characteristics and high bactericides was isolated,which could be used as a candidate phage strain of cracking animal derived Klebsiella pneumoniae,laying a foundation for the prevention and treatment of multi-drug resistant Klebsiella pneumoniae infection.