PB1样铜绿假单胞菌噬菌体PHW2的生物学特性

Characterization of a PB1-like phage PHW2 infecting Pseudomonas aeruginosa

摘要:

[背景] 铜绿假单胞菌（Pseudomonasaeruginosa）是一种引起医院感染、急性感染和慢性感染的常见条件致病菌。多重耐药铜绿假单胞菌仍然是引起严重医院感染的常见病菌，其临床治疗面临严峻挑战。噬菌体具有特异性杀菌的能力，在防治铜绿假单胞菌耐药菌方面具有应用前景。[目的] 分离能裂解碳青霉烯类耐药的铜绿假单胞菌的噬菌体，分析其生物学特性和基因组特征，为噬菌体治疗储备资源。[方法] 采集环境水样，用双层琼脂平板法分离噬菌体，对其形态、一步生长曲线、感染复数等生物学特性进行研究；使用Illumina MiSeq平台测定噬菌体的全基因组序列，利用Newbler 3.0、GeneMarkS、BlASTp、Mauve 2.4.0等生物信息软件进行拼接、注释和比较基因组学分析。[结果] 分离到一株噬菌体PHW2，该噬菌体属肌尾病毒科成员，可裂解7株碳青霉烯类耐药的铜绿假单胞菌；其最佳感染复数为0.1。一步生长曲线结果显示，其感染宿主菌PA001的潜伏期为100 min，裂解期为360 min，裂解量为25 PFU/cell；噬菌体PHW2在温度25-50℃和pH 6.0-8.0范围内稳定；紫外照射7 min后PHW2活性明显下降；5%氯仿处理100 min内，PHW2仍保持较高的活性。噬菌体PHW2的基因组长65 984 bp，GC含量为55.69%，编码92个ORFs，不含tRNA。基因组比对结果显示PHW2与PB1样噬菌体高度相似。体外生物被膜抑制试验和清除试验结果显示，噬菌体PHW2能显著抑制宿主菌PA001形成的生物被膜并破坏24 h内形成的生物被膜。[结论] 分离鉴定了一株新的PB1样噬菌体PHW2，对碳青霉烯类耐药的铜绿假单胞菌裂解能力强。生物学特性、基因组特征、体外生物被膜抑制试验和清除试验结果表明该噬菌体具有治疗铜绿假单胞菌耐药菌的潜力。

Abstract:

[Background] Pseudomonas aeruginosa is one of the main opportunistic pathogen that has an important role in nosocomial, acute, and chronic infections. The multidrug resistant (MDR) P. aeruginosa remains a common cause of severe nosocomial infection. Phages are viruses that infect and can kill bacteria, and phage therapy is an alternative treatment to eradicate MDR P. aeruginosa infection. [Objective] To isolate virulent phages that infect carbapenem-resistant P. aeruginosa and characterize its biological and genomic properties of phages for future development as phage therapy. [Methods] Environmental water samples were collected, and phages were isolated using double agar overlay plaque assay. The morphology, one-step growth curve, optimal multiplicity of infection and other biological characteristics of phages were studied. Phage genome was sequenced using the Illumina MiSeq sequencing platform. Newbler 3.0, GeneMarkS, BlASTp, and Mauve 2.4.0 were used for genome annotation, comparative genomics and evolutionary analyses. [Results] A new phage, PHW2, which is a member of Myoviridae, was isolated from pond water. It is able to lyse seven carbapenem-resistant P. aeruginosa clinical isolates. The optimal multiplicity of infection (MOI) is 0.1. In addition, one step growth curve showed that the latent period and rise period of PHW2 was 100 min and 360 min, respectively. The burst size was about 25 PFU/cell. The phage PHW2 was stable in the temperature of 25-50℃ and pH 6.0-8.0. The activity of PHW2 decreased significantly after UV irradiation for 7 min. Moreover, it was shown that the activity of PHW2 is unaffected after treatment with 5% chloroform for 100 min. The genome of PHW2 is 65 984 bp, with GC content of 55.69% and comprises of 92 ORFs. tRNAscan-SE analysis indicated that phage PHW2 does not possess tRNAs. The genome of PHW2 had high similarity with other PB1-like phages. Phage PHW2 inhibited the biofilm formation of P. aeruginosa PA001 within 24 h. [Conclusion] We isolated PHW2, a new PB1-like phage that shows potent lytic effect against several carbapenem-resistant P. aeruginosa clinical isolates. Biological characteristics and the in vitro biofilm test indicated that the phage has the potential to be used as a biological agent to control nosocomial multidrug resistant P. aeruginosa infections.