耐碳青霉烯类肺炎克雷伯菌噬菌体F19的分离鉴定和全基因组分析

Biological characteristics and complete genomic analysis of a novel bacteriophage F19 for carbapenem-resistant Klebsiella pneumonia

目的分析1株耐碳青霉烯肺炎克雷伯菌噬菌体F19的生物学特性和全基因组。方法从肺炎患者痰液分离肺炎克雷伯菌为宿主菌分离噬菌体F19,电镜观察其形态,并绘制生长曲线,观察F19对温度及pH值的敏感性。分析F19全基因组序列,预测可能存在的开放读码框。根据F19全基因组序列构建系统进化树,进行比较基因组学分析。结果分离到1株新型的肺炎克雷伯菌噬菌体,命名为F19,F19可在其宿主菌菌苔上形成裂解性噬菌体所具有的完全透明的滴斑。电镜观察F19具典型的有尾噬菌体目短尾病毒科病毒形态特征。一步生长曲线显示F19的潜伏期为12 min,裂解量为(102±13)PFU/细胞。F19全基因组长43766 bp,(G+C)%的含量为53.8%。预测和注释F19全基因组51个开放读码框(ORF),按照功能分为DNA复制及调控模块,噬菌体结构基因模块,裂解宿主蛋白模块3大类。比较基因组学分析F19与短尾噬菌体科Drulisvirus属噬菌体基因组具有明显相似保守框架,一致性较强。噬菌体DNA酶基因进化分析表明F19属于Drulisvirus属成员。结论从污水中分离鉴定1株新的耐碳青霉烯肺炎克雷伯菌噬菌体F19,在生物学分类上属于Drulisvirus属噬菌体,为噬菌体治疗耐碳青霉烯肺炎克雷伯感染奠定了基础。

Objective To characterize the biological properties and complete genome of F19,a bacteriophage that infects clinical isolates of carbapenem-resistant Klebsiella pneumoniae.Methods A novel lytic phage,F19,against the KP-19 strain of Klebsiella pneumonia was isolated from a patient’s sputum sample.The morphology of the F19 phage was observed with electron microscopy,and its one-step growth curve,thermo stability,and pH stability were determined.The characteristics of the determined genomic sequence and ORF were analyzed using GeneMark.Comparative genomic and phylogenetic analyses of the F19 phage were performed.Results The F19 phage exhibited lytic activity as evinced by clear areas on bacterial lawns.Morphologically,F19 was classified as a member of the Podoviridae family and the Caudovirales order.The phage is highly infectious with a short latent period(12 min)and a large burst size(102±13 PFU/cell).The F19 genome is 43,766 bp in length,with G+C content of 53.8%.A total of 54 ORFs were identified in the genome.The genome was organized into three functional modules:a DNA replication and regulation module,a structural protein and packaging module,and a host lysis module.Comparative genomic analysis indicated that F19 has the same conserved framework as bacteriophages in the genus Drulisvirus;their genomes were highly similar.The evolutionary relationship between the F19 phage and other phages in the Drulisvirus genus was analyzed using a DNA polymerase phylogenetic tree,which suggested that the F19 phage was a member of the genus Drulisvirus.Conclusion A new member of the genus Drulisvirus,the phage F19,for carbapenem-resistant K.pneumonia was isolated.This finding has laid the foundation for greater use of the phage to control and treat infections with carbapenem-resistant K.pneumoniae in the future.