金黄色葡萄球菌裂解性噬菌体的分离及生物学特性鉴定

Isolation and biological characterization of a lytic bacteriophage against Staphylococcus aureus

以金黄色葡萄球菌CVCC 546为宿主菌,从粪便中分离出1株噬菌体,命名为4086-1。采用双层平板法分离并纯化噬菌体,观察噬斑形态,通过透射电镜观察噬菌体形态,同时测定一步生长曲线、热稳定性、pH稳定性,并分析有机试剂、去污剂及渗透压对噬菌体活性的影响。结果显示,噬斑呈圆形、透亮,边界清晰、有晕环,直径1~3 mm。电镜观察结果显示噬菌体为短尾噬菌体。一步生长曲线结果显示,潜伏期为20min,裂解量为222 PFU/cell。噬菌体在-20~42℃范围可稳定生存,在pH为4~12范围稳定存在。用750m L/L乙醇处理30 min后,噬菌体完全失活;氯仿、500 m L/L DMSO及800 m L/L丙酮分别处理30 min,噬菌体活性无明显变化。1 g/L SDS处理30 min后,噬菌体完全失活;1 g/L CTAB和1 m L/L月桂酰胺氨酸钠处理30 min后,噬菌体效价分别降低0和2个数量级。在不同盐浓度(氯化钠浓度为0~4.5 mol/L)的环境中作用15 min后,噬菌体效价无明显变化,表明该噬菌体对渗透压有一定的耐受性。综上,通过对噬菌体4086-1生物学特性及理化特性的研究,可为其进一步应用奠定基础。

A Staphylococcus aureus phage was isolated from the feces using S.aureus CVCC 546 as the host strain and named 4086-1.The double-layer agar method was used for isolation and purification of phage.Phage plaque was observed.Morphology of phage was observed by transmission electron microscopy.The onestep growth curve was detected.Temperature and pH sensibility were analyzed.Effects of organic reagents,detergents and osmotic pressure on the activity of phage were also determined.The results showed that the morphology of plaques was round,transparent with clear boundary.Each plaque was from 1 mm to 3 mm in diameter.Electron microscopy showed phage 4086-1 was belonged to Podoviridae family.The result of one-step growth curve showed that the latency period was 20 min and burst size was 222 PFU per infected cell.The activity of phage was stable at temperature range from-20℃to 42℃and p H range from 4 to 12.Phages were completely inactivated when treating with 750 m L/L ethanol for 30 min.The activity did not changed insignificantly when treating with chloroform,500 m L/L DMSO,800 m L/L acetone for 30 min respectively.Phages were completely inactivated when treatment with 1 g/L SDS for 30 min.After treatment with 1 g/L CTAB or 1 m L/L sarkysol respectively,the titer of phage decreased by 0 and 2 orders of magnitude,respectively.When treating in different salt concentrations(sodium chloride concentration of 0 to 4.5 mol/L)for 15 min,the phage titers did not change significantly,indicating that it had certain tolerance to osmotic pressure.In conclusion,the study on the biological and physicochemical characteristics of phage 4086-1 can lay the foundation for its further application.