中国兽医杂志2023，Vol.59Issue(12)：1-10,10.
铜绿假单胞菌噬菌体的分离鉴定和全基因组序列分析
Isolation,Identification and Whole Genome Sequence Analysis of Pseudomonas aeruginosa Phage
摘要
Abstract
Pseudomonas aeruginosa(P.aeruginosa)is an opportunistic pathogen causing zoonotic diseases and substantial economic losses in the livestock industry.Phages show promising applications in preventing and treating infectious diseases caused by P.aeruginosa.In this study,the clinical isolate PA31 of P.aeruginosa was used as the host bacterium to isolate and purify phages from water samples using the drop method and double-layer agar plate method.The lysis spectrum of the phages was analyzed using the drop method with PA31 and clinical isolates of Escherichia coli,Salmonella,and Enterococcus faecalis as host bacteria.The morphology of the phages was observed under transmission electron microscopy using negative staining.The optimal multiplicity of infection(MOI),one-step growth curve,and the stability of the phages under different pH and temperature were determined using double-layer agar plates.The whole genome sequence of the phages was obtained using the Illumina NovaSeq platform,and genome assembly,annotation,and comparative genomics analysis were performed using A5-MiSeq,SPAdes,GeneMarkS,Rfam,Diamond,BLASTp,and MEGA 11.0 bioinformatics software.The results revealed the isolation of a novel P.aeruginosa phage named vB_PaeM_Ty,which only lysed the host strains PA31,Escherichia coli-39,and Enterococcus faecalis-8a,exhibiting a certain host specificity.The purified vB_PaeM_Ty had a titer of 1 × 1012 PFU/mL,and belonged to the family Myoviridae,with an MOI of 1.0.The one-step growth curve showed a latentcy period of 20 minutes,a burst period of 40 minutes,and a burst size of approximately 142 PFU/mL when infecting the PA31 host bacteria.The phage remained active within pH 3-12 and at temperatures ranging from 45 to 70 ℃.The nucleic acid of vB_PaeM_Ty was identified as double-stranded DNA(dsDNA)with a genome size of 50 051 bp,a GC content of 47.23％,and 74 open reading frames(ORFs).Comparative genomic analysis revealed the highest homology between vB_PaeM_Ty and Pseudomonas phage K4.The genome sequence of vB_PaeM_Ty has been submitted to GenBank with accession number OQ555403.These findings indicate that the newly isolated phage vB_PaeM_Ty is a virulent phage,resistant to high-temperature and pH extremes,and harbors various functionally relevant proteins,suggesting its potential for treating multidrug-resistant strains of P.aeruginosa.关键词
铜绿假单胞菌/噬菌体/分离纯化/生物学特性/基因组分析Key words
Pseudomonas aeruginosa/phage/isolation and purification/biological characteristics/genome analysis分类
农业科技引用本文复制引用
谢丹莹,谢景昊,黄若超,陆鑫,杨晨,郭爽,李和平,王月影..铜绿假单胞菌噬菌体的分离鉴定和全基因组序列分析[J].中国兽医杂志,2023,59(12):1-10,10.基金项目
河南省科技攻关计划(212102110355,222102110402) （212102110355,222102110402）
