一株宽谱裂解性溶藻弧菌噬菌体ФV170的分离鉴定及生物学特性

摘要

[Background] Vibrio alginolyticus, one of the most important opportunistic pathogens, can cause significant morbidity, mortality and economic losses during shrimp aquaculture. At present, phage therapy acts as an alternative sanitizing agent to control V. alginolyticus. [Objective] To isolate and characterize phage ФV170 lysing V. alginolyticus for the application of the phage to control aquatic animal diseases. [Methods] V. alginolyticus V170 was used as the host strain to isolate phage from Penaeus vannamei aquaculture water. Characterization was done by transmission electro-microscopy, optimal multiplicity of infection, one-step growth curve, lysis spectrum detection, thermal stability, and sensitivity to chloroform. The analyses of phage genome were based on enzyme digestion methods. [Results] A broad-spectrum lytic bacteriophage ФV170 was isolated and the plaques were neat and transparent, with a diameter of 1.5 mm at 12 h. The identification results of transmission electron microscopy (TEM) showed that its head had a stereosymmetric structure of the quasi-diaphedron, with a diameter between 60 nm and 65 nm, and its tail was 65 nm to 75 nm long and 14 nm to 18 nm wide. The nucleic acid type was dsDNA and the genome about 45 kb. Besides, it was not sensitive to chloroform. In conclusion, bacteriophage ФV170 belongs to Caudovirales and Myoviridae. The bacteriophage V170 can lyse 7 of 15 strains of Vibrio alginolyticus, possessing a broad host spectrum within the species. The optimal multiplicity of infection (MOI) was 0.01. One-step growth curve shows that the incubation period of ФV170 was 10 min and the amount of lysis was 101.3. Phage ФV170 was sensitive above 65 °C. [Conclusion] A lytic bacteriophage was isolated with potential functions to treat alginolyticosis in marine aquaculture.%[背景]溶藻弧菌(Vibrio alginolyticus)是水产养殖中重要的条件致病菌,对海水养殖业造成了极大的危害.传统的抗生素疗法引发的耐药问题已经成为全球面临的严峻挑战之一,而作为可替代抗生素的噬菌体疗法已被证实能够有效治疗弧菌病.[目的]深入研究溶藻弧菌噬菌体ФV170的生物学特性,为该菌株在水产动物病害控制中的应用提供数据支持.[方法]以溶藻弧菌V170为宿主菌,采用斑点法从凡纳滨对虾养殖水体中筛选噬菌体,并以双层平板法对噬菌体进行纯化、生长、效价等方面的研究;利用电镜观察噬菌体形态;通过酶切方法分析噬菌体的基因组大小及其类型.[结果]分离得到一株宽谱裂解性噬菌体ФV170,其噬菌斑边缘整齐且通透,12 h直径达1.5 mm.鉴定结果显示,噬菌体ФV170头部为正廿面体的立体对称结构,直径为60 nm?65 nm,尾部长为65 nm?75 nm,宽14 nm?18 nm,核酸类型为dsDNA,基因组大小约为45 kb,对氯仿不敏感,属于有尾噬菌体目(Caudovirales)肌尾噬菌体科(Myoviridae).此外,噬菌体ФV170可裂解15株溶藻弧菌中的7株,属于种内宽谱;最佳感染复数为0.01;一步生长曲线显示潜伏期为10 min,裂解量为101.3;对65 °C以上温度敏感.[结论]分离得到一株宽谱裂解性溶藻弧菌噬菌体,该噬菌体具有治疗海水养殖过程中溶藻弧菌病的潜力.