一株屎肠球菌烈性噬菌体的分离鉴定及基因组测序分析

Isolation, identification and genome sequencing of a virulent phage infecting Enterococcus faecium

摘要:

【背景】屎肠球菌为ESKAPE (由屎肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和肠杆菌属六大超级细菌的拉丁学名首字母组成)病原体之一，对多种抗菌药物具有耐药性，严重威胁全球人类健康，被世界卫生组织列入亟须研发新抗菌药的病原体名单。【目的】分离针对屎肠球菌的烈性噬菌体，测定其基本生物学特性并进行基因组测序分析，为屎肠球菌噬菌体疗法提供原料。【方法】从牧场污水中分离筛选出一株烈性屎肠球菌噬菌体，命名为Enterococcus phage 1A11，通过透射电镜观察噬菌体的形态，测定其最佳感染复数、一步生长曲线和裂解谱，并进行全基因组的测序和分析，以阐释该噬菌体的基本生物学特性。【结果】电镜下可观察到屎肠球菌噬菌体1A11具有典型的正二十面体头部结构和较长的尾部结构，属于有尾病毒目长尾病毒科，而且测得其最佳感染复数为0.01，裂解周期为70 min，潜伏期为30 min，暴发期为40 min，并特异性地对部分屎肠球菌产生裂解作用。噬菌体1A11的基因组大小为42 750 bp，GC含量为34.71%，含有70个推定的开放阅读框(open reading frame, ORF)，不含抗生素抗性基因和毒力基因，可作为屎肠球菌噬菌体疗法的原料。【结论】本研究分离到一株新的屎肠球菌噬菌体1A11，具有一定的研究价值和应用潜力，为屎肠球菌噬菌体疗法研究奠定了基础。

关键词:噬菌体;屎肠球菌;生物学特性;全基因组测序

Abstract:

[Background] Enterococcus faecium is one of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. Due to the resistance to a variety of antimicrobial agents, E. faecium seriously threatens human health and is included in the WHO priority pathogens list for R&D of new antibiotics. [Objective] To isolate, characterize, and sequence the genome of the virulent phage against E. faecium, so as to provide raw materials for the phage therapy of E. faecium. [Methods] A phage strain 1A11 against E. faecium was isolated from pasture sewage. The phage was observed in a transmission electron microscope, and its optimal multiplicity of infection, one-step growth curve, and lysis spectrum were determined. The biological characteristics of the strain were analyzed and the whole genome was sequenced. [Results] E. faecium phage 1A11 had a typical icosahedral head and a long tail, belonging to the Siphoviridae of the Caudovirales. The strain showed the optimum multiplicity of infection of 0.01, a lysis cycle of 70 min, an incubation period of 30 min, and an outbreak period of 40 min. It exerted specific lysis effects on several strains of E. faecium. Phage 1A11 had a genome of 42 750 bp with the GC content of 34.71%, 70 putative open reading frames (ORFs), and no antibiotic resistance genes or virulence genes. It can be used for the phage therapy of E. faecium. [Conclusion] A new strain of E. faecium phage 1A11 was isolated, which has certain research value and application potential, and laid a foundation for the study of E. faecium phage therapy.

Key words:phage;Enterococcus faecium;biological characteristics;whole genome sequencing