一株新型裂解K63荚膜型肺炎克雷伯菌的噬菌体分离鉴定和生物学特性研究及全基因组分析
Biological characteristics and complete genomic analysis of a novel virulent bacteriophage that infects Klebsiella pneumoniae capsular type K63
  
DOI:  10.13344/j.microbiol.china.190585
中文关键词:肺炎克雷伯菌,噬菌体,生物学特性,全基因组分析
英文关键词:Klebsiella pneumonia, Bacteriophage, Biological characteristics, Genomic analysis
基金项目:首都临床特色项目(Z161100000516181);国家自然科学基金(81572045)
作者单位E-mail
高明明 1 河北北方学院研究生部 河北 张家口 075000  
王灿 2 解放军总医院第五医学中心呼吸与危重症医学科 北京 100071  
李璞媛 2 解放军总医院第五医学中心呼吸与危重症医学科 北京 100071  
刘慧莹 2 解放军总医院第五医学中心呼吸与危重症医学科 北京 100071  
裴广倩 3 军事科学院军事医学研究院微生物流行病研究所 病原微生物安全国家重点实验室 北京 100071  
范航 3 军事科学院军事医学研究院微生物流行病研究所 病原微生物安全国家重点实验室 北京 100071  
张湘莉兰 3 军事科学院军事医学研究院微生物流行病研究所 病原微生物安全国家重点实验室 北京 100071  
米志强 3 军事科学院军事医学研究院微生物流行病研究所 病原微生物安全国家重点实验室 北京 100071  
童贻刚 3 军事科学院军事医学研究院微生物流行病研究所 病原微生物安全国家重点实验室 北京 100071
4 北京化工大学生命科学与技术学院 北京 100029 
tong62035@gmail.com 
柏长青 1 河北北方学院研究生部 河北 张家口 075000
2 解放军总医院第五医学中心呼吸与危重症医学科 北京 100071 
mlp1604@sina.com 
摘要点击次数: 234
全文下载次数: 193
中文摘要:
      【背景】噬菌体具有特定的杀菌能力,对生态和细菌的进化具有重要影响。近年来由于多重耐药细菌的全球出现,噬菌体疗法逐渐引起了人们的关注。【目的】对一株新型裂解K63荚膜型肺炎克雷伯菌的噬菌体vB_KpnP_IME308进行生物学特性研究、测序和比较基因组学的分析。【方法】以一株从临床分离到的肺炎克雷伯菌为宿主菌分离噬菌体,应用双层平板法进行噬菌体最佳感染复数(optimal multiplicity of infection)、一步生长曲线(one-step growth curve)、温度以及pH敏感性实验测定,纯化噬菌体并通过透射电镜观察噬菌体形态;应用标准的苯酚-氯仿提取方案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】分离到一株新型的肺炎克雷伯菌噬菌体,命名为vB_KpnP_IME308;其最佳感染复数为0.001,一步生长曲线结果显示,其感染宿主菌的潜伏期约为20 min,裂解期约为80 min,平均裂解量330 PFU/cell;噬菌体vB_KpnP_IME308在4?50 °C和pH 5.0?10.0范围内稳定;电镜观察该噬菌体属于短尾噬菌体科(Podoviridae)。基因组测序结果表明,噬菌体基因组全长为43 091 bp,(G+C)mol%含量为53.9%,(A+T)mol%含量为46.1%。BLASTn比对结果表明,该噬菌体与目前已知噬菌体基因组仅84%区域有相似性。噬菌体进化树结果表明该噬菌体属于Autographivirinae亚科的Drulisvirus属的成员。【结论】从医院污水中分离鉴定了一株新型的肺炎克雷伯菌噬菌体,表征并分析了噬菌体全基因组序列,这些结果均表明该噬菌体具有开发为抗肺炎克雷伯菌制剂的潜力,为噬菌体治疗多重耐药细菌感染奠定了基础。
英文摘要:
      [Background] Bacteriophages have a specific bactericidal ability to influence the evolution of bacteria and ecology. In recent years, due to the global emergence of multidrug-resistant bacterial strains, phage therapy has received renewed interest. [Objective] Biological characteristics and complete genome sequence of a novel virulent bacteriophage vB_KpnP_IME308 that infects Klebsiella pneumoniae capsular type K63. [Methods] A novel lytic phage was isolated from sewage of Chinese general Hospital of the PLA using a clinical Klebsiella pneumoniae strain as an indicator. The double-layer plate method was conducted the titer, optimal multiplicity of infection (MOI), one-step growth curve, thermostability and pH stability. Phage morphology was observed by transmission electron microscopy after purification. Phage 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, designated vB_KpnP_IME308, isolated from hospital sewage using a clinical Klebsiella pneumoniae strain as a host. The optimal MOI of phage vB_KpnP_IME308 is 0.001. One step growth curve showed that the latent period and the burst period of vB_KpnP_IME308 were 20 min and 80 min, respectively. The burst size was about 330 PFU/cell. The genome of vB_KpnP_IME308 is 43 091 base pairs (bp) with (G+C)mol% content of 59.3% and (A+T)mol% content of 46.1%. Electron microscopic observation showed that the phage 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 vB_KpnP_IME308 and other Podoviridae phages was analyzed by major capsid protein and large terminase protein of phage vB_KpnP_IME308, which suggest that phage vB_KpnP_IME308 was a member of the genus Drulisvirus of subfamily Autographivirinae. [Conclusion] A lytic phage, vB_KpnP_IME308, was isolated successfully from hospital sewage, characterization and genome analysis of a novel bacteriophage vB_KpnP_IME308, which shows its potency to be developed as a novel alternative for multi-drug resistant Klebsiella pneumoniae infects control and treatment.
高明明,王灿,李璞媛,刘慧莹,裴广倩,范航,张湘莉兰,米志强,童贻刚,柏长青.一株新型裂解K63荚膜型肺炎克雷伯菌的噬菌体分离鉴定和生物学特性研究及全基因组分析[J].微生物学通报,2020,47(1):210~221
HTML  查看全文  查看/发表评论  下载PDF阅读器