一株肺炎克雷伯菌噬菌体的生物学特性及全基因组分析

【背景】随着抗生素的广泛使用甚至滥用,细菌耐药性问题日益显著,利用噬菌体治疗耐药致病菌的方法重新开始被人们关注。【目的】对一株烈性肺炎克雷伯菌噬菌体vB_KpnP_IME279进行生物学特性研究及生物信息学分析。【方法】以一株多重耐药的肺炎克雷伯菌为宿主菌,从医院污水中分离噬菌体,应用双层平板法检测噬菌体效价、最佳感染复数(Optimal MOI)、一步生长曲线以及裂解谱,纯化后通过透射电镜观察噬菌体形态;应用蛋白酶K/SDS法提取噬菌体全基因组,使用Illumina MiSeq测序平台进行噬菌体全基因组测序,测序后对噬菌体全基因组序列进行组装、注释、进化和比较基因组学分析。【结果】分离到一株新的肺炎克雷伯菌噬菌体,命名为vB_KpnP_IME279;其最佳感染复数为0.1,一步生长曲线显示潜伏期为20 min,平均裂解量140 PFU/cell,电镜观察显示该噬菌体属于短尾噬菌体科(Podoviridae)。基因组测序表明,噬菌体基因组全长为42 518 bp,(G+C)mol%含量为59.3%。BLASTn比对结果表明,该噬菌体与目前已知噬菌体的相似性较低,基因组仅70%区域与已知噬菌体有同源性。构建噬菌体主要衣壳蛋白的基因进化树,分析了噬菌体IME279与其他短尾科噬菌体的进化关系,结果表明该噬菌体是短尾科噬菌体的一名新成员。【结论】分离鉴定了一株新的肺炎克雷伯菌噬菌体,进行了生物学特性、全基因组测序和生物信息学分析,为研究肺炎克雷伯菌噬菌体与宿主之间的相互作用关系以及治疗多重耐药细菌感染奠定了基础。

Characterization and genome analysis of a novel Klebsiella pneumoniae phage

Background] With the widespread use and even abuse of antibiotics, the problem of bacterial resistance has become increasingly prominent. The use of phage to treat drug-resistant pathogens has begun to attract attention. Objective] Biological characteristics and bioinformatics analysis of a newly found phage vB_KpnP_IME279 of Klebsiella pneumoniae. Methods] A multi-drug resistant Klebsiella pneumoniae was used as the host strain to isolate phage from hospital sewage. We used the double-layer plate method to conduct the titer, optimal multiplicity of infection (optimal MOI), one-step growth curve and lysis spectrum of the phage. Phage morphology was observed by transmission electron microscopy after purification. Its genome was sequenced using the Illumina MiSeq sequencing platform. Complete genome sequence was used for genome annotation, comparative genomics and evolutionary analyses. Results] A novel phage vB_KpnP_IME279 was successfully isolated from infected host cells. The optimal MOI of IME279 is 0.1. One-step growth curve shows that IME279 has a burst size of 140 PFU/cell and a latent period of 20 min. The genome of IME279 is 42 518 bp and 59.3% (G+C)mol%. Electron microscopic observation showed that the phage was belongs to the family Podoviridae. The BLASTn alignment showed that the genome of the phage had limited similarity with the currently known phages. The evolutionary relationship between phage IME279 and other Podoviridae phage was analyzed by gene phylogenetic tree of phage major capsid protein, which suggests IME279 is a new member of the Podoviridae phage. Conclusion] Isolation and identification of the new phage of Klebsiella pneumonia and the biological characteristics, genome-wide sequencing and bioinformatics analysis will help study the relationship between Klebsiella pneumoniae phage and host and the treatment of multi-drug resistant bacteria with phages.