上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

铜绿假单胞菌噬菌体D204的生物学特性和基因组学研究

徐彬1，高晶2，郭晓奎1，秦金红1   

  1. 1．上海交通大学 医学院免疫学与微生物学系， 上海 200025； 2.复旦大学附属妇产科医院， 上海  200011
  • 出版日期:2016-01-28 发布日期:2016-02-26
  • 通讯作者: 秦金红, 电子信箱: jinhongq@hotmail.com。
  • 作者简介:徐彬（1984—）， 男， 硕士生; 电子信箱: xubin1984hl@126.com。
  • 基金资助:

    国家科技部支撑计划（2012EP001004）

Study on the biological and genomic characteristics of Pseudomonas aeruginosa phage D204

XU Bin1, GAO Jing2, GUO Xiao-kui1, QIN Jin-hong1   

  1. 1.Department of Microbiology and Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China； 2.Obstetrics and Gynecology Hospital of Fudan University， Shanghai  200011, China
  • Online:2016-01-28 Published:2016-02-26
  • Supported by:

    National Key Technology Research and Development Program of the Ministry of Science and Technology of China， 2012EP001004

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摘要：

目的 针对铜绿假单胞菌多耐药菌的不断出现，寻找高效、广谱的铜绿假单胞菌噬菌体。方法 以医院临床上分离到的铜绿假单胞菌为宿主菌，从环境污水中分离纯化铜绿假单胞菌噬菌体，磷钨酸负染法电镜观察噬菌体的形态和大小，测定其效价、宿主谱、酸碱稳定性和热稳定性，绘制一步生长曲线、吸附曲线和感染曲线，并抽取噬菌体DNA进行测序及分析。结果 分离到1株铜绿假单胞菌噬菌体，并命名为D204。D204在pH值为4~11及温度37~50 ℃环境下具有较高活性，电子显微镜观察发现此噬菌体由直径约为50 nm的多面体头部和约15 nm的短尾组成。一步生长曲线表明噬菌体在裂解宿主菌时，潜伏期为30 min，爆发期为40 min，裂解量约为160 pfu/cell。吸附曲线显示50 min时，其吸附率达95%以上。感染曲线显示当感染复数为0.1和1时，对宿主菌生长有抑制作用，在180 min后可完全抑制宿主菌生长。其基因组为环状双链DNA，全长为50 609 bp，GC含量为44.8%。预测共有75个开放阅读框。结论 分离、筛选出的铜绿假单胞菌噬菌体D204具有较强的裂解宿主的能力，其对酸碱和热比较稳定，裂解宿主时潜伏期较短，可以作为铜绿假单胞菌的储备噬菌体。

关键词: 铜绿假单胞菌；噬菌体；基因组, 生物学特性

Abstract:

Objective To identify efficient and broad-spectrum phages aiming at multi-drug resistant Pseudomonas aeruginosa. Methods Clinical isolated P.aeruginosa strains were used as host bacteria. The P.aeruginosa phages were isolated and purified from sewage. The shape and size of phages were observed by transmission electron microscope (TEM) after phosphorus acid negative staining. Titer, host range, pH stability, and thermal stability were detected. One-step growth curve, adsorption curve, and infection curve were plotted. The DNA of phages was extracted for sequencing and analysis. Results One strain of P.aeruginosa phage was isolated and named D204. The activity of D204 was high at temperatures from 37 to 50 ℃ and pH values from 4 to 11. TEM observation showed that the phage consisted of a polyhedron head with the diameter of 50 nm and a 15 nm long tail. One step growth curve indicated that during the lysis of host bacteria, the latent period of phages was about 30 min, outbreak period was about 40 min, and the burst size was about 160 pfu/cell. The adsorption curve showed that the adsorption rate was more than 95% within 50 min. The infection curve indicated that the growth of host bacteria was inhibited when the multiplicities of infection were 0.1 and 1. The growth of host bacteria was totally inhibited after 180 min. The genome of phages was circular double-stranded DNA with total length of 50 609 bp and the content of GC was 44.8%. It was predicted that there were 75 open reading frames in the genome. Conclusion Isolated P.aeruginosa phages D204 have a strong lytic ability and wide ranges of thermal and pH stability and can serve as reserved phages of P.aeruginosa.

Key words: Pseudomonas aeruginosa, phages, genome, biological characteristic