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【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】 To isolate a bacteriophage capable of lysing multidrug-resistant Enterococcus faecalis from hospital effluent, analyze the biological characteristics of the bacteriophage, and perform genome-wide sequencing and analysis to provide a basis for the treatment and control of multi-resistant Enterococcus faecalis infection . 【Method】 Bacteriophage-resistant Enterococcus faecalis was used as a host to isolate phage from hospital effluent. The phage titer, MOI and one-step growth curve were detected by double-layer plate assay. The phage morphology was observed by negative staining electron microscopy after purification. Protease The whole genome of phage was extracted by K / SDS method. After electrophoresis analysis, agarose gel electrophoresis analysis was performed. Whole genome sequencing was performed using Ion Torrent sequencing platform. Sequencing, phage genome-wide sequence assembly, annotation, phylogenetic analysis and comparative analysis were performed. 【Result】 One Enterococcus faecalis phage was isolated and named as v B_E.faecalis_IME196 (IME196). The optimal multiplicity of infection was 0.01. The one-step growth curve showed that the incubation period of IME196 was 30 min and the outbreak was 50 PFU. The phage was a phage plague phage, and it was identified by BLASTp analysis as belonging to the Sarcomastigma phagemid. The gene sequencing showed that the phage IME196 was DNA with 38 895 bp in full length and 33.9% G + C. 【Conclusion】 Isolation and identification of one Enterococcus faecalis bacteriophage and its genome-wide sequencing and analysis provided a new approach for the prevention and control of Enterococcus faecalis infection in the future, which laid the foundation for bacteriophage treatment of multi-drug resistant bacteria.