粪肠球菌噬菌体vB_E.faecalis_IME196的生物学特性及其全基因组分析

Characterization and complete genome sequence analysis of Enterococcus faecalis bacteriophage v B_E. faecalis_IME196

【目的】从医院污水中分离一株能裂解多耐药粪肠球菌的噬菌体,分析该噬菌体的生物学特性,并进行全基因组测序和分析,为治疗和控制多耐药粪肠球菌感染提供基础。【方法】以耐药粪肠球菌为宿主,从医院污水分离噬菌体,双层平板法检测噬菌体效价、最佳感染复数(MOI)和一步生长曲线,纯化后负染法电镜观察噬菌体形态;蛋白酶K/SDS法提取噬菌体全基因组,酶切处理后琼脂糖凝胶电泳分析,使用Ion Torrent测序平台进行噬菌体全基因组测序,测序后进行噬菌体全基因组序列组装、注释、进化分析和比较分析。【结果】分离到一株粪肠球菌噬菌体,命名为v B_E.faecalis_IME196(IME196);其最佳感染复数为0.01,一步生长曲线显示IME196的潜伏期为30 min,暴发量为50 PFU,电镜观察该噬菌体为长尾噬菌体,结合BLASTp分析确定其属于尾病毒目长尾噬菌体科,基因测序表明,噬菌体IME196核酸类型为DNA,基因组全长为38 895 bp,G+C含量为33.9%。【结论】分离鉴定一株粪肠球菌噬菌体,进行了全基因组测序和分析,为以后预防和控制粪肠球菌的感染提供了一个新的途径,为噬菌体治疗多重耐药细菌奠定了基础。

[Objective] We isolated a novel virulent bacteriophage specifically infecting antibiotics-resistant Enterococcus faecalis from hospital sewage, to study its morphology and to analyze its genome so as to provide basis for treatment and infection control of antibiotics-resistant E. faecalis. [Methods] We used E. faecalis as the host to isolate phage from raw sewage from the sewage treatment center of a hospital. Transmission electromicroscopy, optimal multiplicity of infection（optimal MOI） and one-step growth curve were conducted. The genome DNA was extracted and sequenced by high-throughput sequencing technology. The feature of whole genome, and evolutionary relationship were analyzed. [Results] A lytic bacteriophage designated as v B_E. faecalis_IME196（IME196） was isolated. The optimal multiplicity of infection（optimal MOI） of IME196 is 0.01. One-step growth curve shows that IME196 has a burst size of 50 PFU and a latent period of 30 min. The genome of IME196 is a 38 895 bp in length, linear, terminally non-redundant double-stranded DNA and has a G＋C content of 33.9%. [Conclusion] Isolation and characterization of a lytic Enterococcus faecalis phage will help supply a new way to prevent and control the infection of E. faecalis, and laid a foundation to the treatment of multiple drug resistant bacteria in the future.