路德维希肠杆菌噬菌体的分离及生物学特性

Isolation and characterization of an Enterobacter ludwigii phage, GM20

【背景】噬菌体能够特异性杀死宿主细菌,特别是耐药性细菌,可以作为新型杀菌剂,而关于路德维希肠杆菌噬菌体的研究尚属空白。【目的】分离路德维希肠杆菌噬菌体,并对其生物学特性进行研究。【方法】通过双层平板法分离、纯化、鉴定噬菌体;通过SDS-PAGE电泳分析结构蛋白;通过透射电子显微镜分析噬菌体的形态;通过结晶紫染色法和刚果红平板法分析生物被膜。【结果】以路德维希肠杆菌X20为指示菌从环境样品中分离获得了噬菌体GM20,噬菌斑透明且有较小晕环,直径大小平均为0.47 mm。透射电镜观察显示,噬菌体GM20具有可伸缩尾部,属于肌尾噬菌体科(Myoviridae)。GM20的滴度为7.65×109^PFU/mL,为烈性噬菌体。一步生长曲线结果显示,噬菌体的潜伏期约为15 min,释放量约为164.3 PFU/infection center。进一步分析显示,GM20具有较好的抑菌效果,耐噬菌体菌株突变株平均突变率为1.06×10^-5。【结论】烈性噬菌体GM20能够杀死路德维希肠杆菌X20,有可能应用于路德维希肠杆菌感染的预防与控制。

[Background] Phage can specifically kill host bacteria especially the strains resistant to antibiotics. Phage can be a new antimicrobial agents alternative to conventional drugs. [Objective] No Enterobacter ludwigii phage has been reported until now. The purpose of this work is to isolate and characterize E. ludwigii phage. [Methods] The double-layer plate method was used for isolation, purification, and quantification of phage;SDS-PAGE was used for viral structural protein analysis;transmission electron microscopy(TEM) was used for phage morphology analysis;and the Crystal Violet staining and Congo Red agar methods were used for biofilm analysis. [Results] The phage GM20 was isolated from the environmental samples with the strain E. ludwigii GM20 as indicator. The plaques were transparent with the average diameter about 0.47 mm. TEM showed that GM20 has a contractile tail, and it was classified as a member of the Myoviridae family. The titer of phage is 7.65×109 PFU/mL, suggesting that it is a virulent phage. The one-step growth curve showed that GM20 had a latent period of 15 min and a burst size of 164.3 PFU/infection center. Further experiments showed that GM20 could inhibit bacteria growth effectively, and the average mutation frequency of phage-resistant mutants was about 1.06×10-5. [Conclusion] The virulent phage GM20 can effectively kill the host strain E. ludwigii X20 and may be used in the prevention and control of E. ludwigii infection.