粪肠球菌噬菌体IME-EF3的分离及其治疗优势的发掘

Isolation of a novel Enterococcus faecalis phage IME-EF3 and its potential therapeutic advantages

目的利用医院污水分离到临床耐药粪肠球菌的噬菌体IME-EF3，分析其生物学特性，发掘其治疗特性，为噬菌体制剂个体化治疗耐药粪肠球菌感染及利用噬菌体消除耐药粪肠球菌污染提供应用基础。方法利用医院污水,经污水富集法分离噬菌体,以粪肠球菌临床耐药分离株为宿主菌。经双层平板法确定噬菌斑形态及大小，纯化后测定其宿主谱范围、最佳感染复数（MOI）、一步生长曲线，并检测温度、紫外线照射对IME-EF3活性的影响。取适量噬菌体液，利用蛋白酶K俗DS法提取其基因组,对其进行酶切处理．确定其遗传物质的组成及形态。用酶切法进行了随机克隆，测序结果进行在线Blastx，并构建系统进化树分析与已知噬菌体的进化关系。结果成功分离到一株噬菌体斑．边缘清晰，斑体透明的裂解性粪肠球菌噬菌体IME-EF3热稳定性较好．对紫外线敏感。一步生长曲线显示IME-EF3的潜伏期为10～20min，呈爆发性增长，裂解量为75。其遗传物质为双链DNA,通过Blastx结果初步确定IME-EF3为尾病毒目，长尾噬菌体科。通过系统进化树可看出IME—EF3与其他噬菌体的亲缘关系较远。结论从医院废水成功分离到裂解性噬菌体IME-EF3，其爆发力强、潜伏期短、高裂解性、较好的热稳定性、与已知噬菌体远缘性等一系列的优良特点，为其噬菌体个体化治疗及与药物的联合使用提供了依据。

Objective To isolate bacteriophage and analyze its biological characteristics, and explore its potential in phage therapy against antibiotics resistant Enterococcus faecalis infections. Methods The bacteriophage IME-EF3 was isolated from the hospital sewage by using clinically isolated Enterococcus faeca!is as host. A double-layer agar plate method was used to determine the shape of plaque and the titer of phage preparations, the best multiplicity of infection（MOI）, as well as the one-step growth curve. The heat and ultraviolet irradiation stability of IME-EF3 was analyzed by incubating the phage preparations at different temperatures and radiation durations. The phage genomic DNA was extracted using the method of K/SDS protease and characterized by nuclease digestion and agarose gel analysis. The DNA was then inserted into the cloning vector, and the sequenced result was blasted online. The phylogenetic tree of the major capsid proteins of IME-EF3 was formed by Neighbor- Joining method of MEGA5. Results Bacteriophage IME-EF3 was isolated successfully from hospital sewage, which was relatively heat-stable, but sensitive to ultraviolet irradiation. The one-step growth curve showed that IME-EF3 demonstrated explosive growth with an incubation period of 10 - 20 minutes and cracking capacity of 75. IME-EF3 genome was circular dsDNA. The Blastx sequencing results of a random fragment indicated that it belonged to the Siphoviridae. The relationship among IME-EF3 and other phages was distantly related. Conclusion It is demonstrated that the lyric bacteriophage IME-EF3 owns explosive growing strength with short incubation period, high cracking resistance and good thermal stability. The biological characterization of IME-EF3 provides useful information for the application of IME-EF3 as an alternative therapeutics to treat antibiotics-resistant Enterococcus faecalis infection.