一株感染铜绿假单胞菌的双链RNA噬菌体PaP6的分离和鉴定

Isolation and characterization of PaP6, a newly isolated Pseudomonas aeruginosa bacteriophage containing three dsRNA genomic segments

摘要:

【目的】鉴定一株新分离的铜绿假单胞菌噬菌体PaP6 的生物学特性。【方法】利用铜绿假单胞菌临床分离株PA038 为宿主,从西南医院污水中分离得到一株裂解性噬菌体PaP6,观察其噬斑特点;氯化铯密度梯度离心纯化噬菌体颗粒后,用透射电子显微镜观察噬菌体形态;提取PaP6 基因组,通过DNA 酶和RNA 酶酶切,做基因组酶切图谱分析;按照感染复数(MOI)分别为10、1、0.1、0.01、0.001 和0.000 1 加入噬菌体和宿主菌,裂解细菌后,测定噬菌体滴度;以MOI=10 的比例加入噬菌体和宿主菌,绘制一步生长曲线;用112 株铜绿假单胞菌临床分离株检测PaP6 宿主谱。【结果】PaP6 的噬斑直径约2 mm-4 mm,圆形透明,边缘清晰;PaP6噬菌体呈多面体立体对称的头部,直径约45 nm;酶切图谱表明PaP6 基因组对DNase 不敏感,对RNase 敏感,未酶切基因组具有3 节段双链RNA (dsRNA),长度分别约为9.0、4.5、3.5 kb,共约17 kb;当MOI 为0.1 时PaP6 感染其宿主菌产生的子代噬菌体滴度最高,达到3.4×10⁹ PFU/mL;用一步生长曲线描绘了其生长特性;PaP6 可以感染40.1%的临床分离株,是一株比较广谱的噬菌体。【结论】首次报道了一株铜绿假单胞菌的dsRNA 分节段噬菌体,分类学上属于囊病毒科,该噬菌体具有较广的宿主谱,在噬菌体治疗领域具有应用前景。

Abstract:

[Objective] To characterize a novel Pseudomonas aeruginosa phage PaP6 via its biological properties. [Methods] Double-layer agar culture method was applied to observe the plaques of PaP6. Phage particles were examined by electron microscopy after cesium chloride density gradient centrifugation. PaP6 genome was extracted and digested with either DNase or RNase. Phage and host were mixed with multiplicity of infection (MOI) 10, 1, 0.1, 0.01, 0.001 and 0.000 1 respectively. Phage titers were measured after bacteria lysis. For one step growth experiments, phage and host with an MOI of 10 were used. The host range of PaP6 was tested with 112 Pseudomonas aeruginosa clinical isolates strains. [Results] The plaque of PaP6 was transparent and the edge of the plaque was clear. Its diameter was about 2 to 4 mm. PaP6 phage had an isometric head approximately 45 nm in diameter. Enzyme digestion experiment shows that PaP6 contained three double-stranded RNA segments. Infecting host bacteria with MOI of 0.1 gave the highest phage offspring. Phage growth characteristics were determined by one step growth curve. PaP6 infects 40.1% of Pseudomonas aeruginosa clinical isolated strains. [Conclusion] PaP6 genome is composed of three dsRNA segments, and it is the first reported Cystoviridae phage that infects Pseudomonas aeruginosa. The host range of PaP6 is wide, which implies its potential in phage therapy.