6株铜绿假单胞菌噬菌体的分离及其生物学特性

Identification and biological characteristics of six Pseudomonas aeruginosa phage strains

目的分离铜绿假单胞菌噬菌体,比较其形态、裂菌特性并测定其裂解谱,根据裂解谱筛选出裂解百分比最高的噬菌体,并测定其最佳感染复数和体内代谢动力学等基本生物学特性。方法取兰州市区污水样品经0.22μm滤膜过滤,用双层琼脂平板培养法分离、纯化铜绿假单胞菌噬菌体并测定其裂解谱;纯化后的噬菌体进行扫描电镜观察;评估裂解谱后筛选出裂解百分比最高的为PaP106,分别按照感染复数(MOI)=10^(-1)、10^(-2)、10^(-3)、10^(-4)、10^(-5)、10^(-6)、10^(-7)比例加入PaP106和铜绿假单胞菌共培养后测定噬菌体滴度,确定其最佳感染复数;不同途径(腹腔、肌肉、皮下)给小鼠注射1×10^(10)pfu/m L PaP106 100μL,分别于0.5、1、3、6、12、24、36 h测定小鼠血液、肝脏、脾脏组织中噬菌体的浓度,分析噬菌体体内代谢动力学变化。结果共分离到6株形态不同的铜绿假单胞菌噬菌体,对铜绿假单胞菌耐药菌株的裂解百分比为25%—75%,其中PaP106噬菌体对铜绿假单胞菌耐药菌株的裂解百分比最高(75%),PaP106噬菌体的最佳感染复数为1×10^(-6),腹腔注射PaP106噬菌体后,其在血液、肝脏、脾脏的达峰时间短于皮下注射和肌肉注射途径。结论在分离得到的6株铜绿假单胞菌噬菌体中,PaP106噬菌体裂解性强、裂解谱宽,腹腔注射噬菌体后血液、肝脏、脾脏的分布及达峰时间最快,为今后临床治疗感染多重耐药铜绿假单胞菌提供了理论基础。

Objective To identify the Pseudomonas aeruginosa phage （PAP） and analyze its basic biological characteristics, such as morphology, lysis character. To determinate the multiplicity of infection （MOI）, and to carry out pharmacokinetic studies of PaP106. Methods PaP was isolated from Lanzhou city sewage samples by using 0.22 pm filtration and purified by using a double-layer agar plate. Lytic capacity of six strains of phage was measured by clinical multi drug resistant Pseudomonas aeruginosa （MDRPa）. The morphology of phagel06 was determined by transmission electron microscope （TEM）. The host bacteria were infected with PAP106 at different MOI 10-1, 10-2, 10-3, 10-4, 10-5, 10-6, and 10-7）, and the optimal MOI of PaP106 was deter- mined by the concentration of PaP. To evaluate the difference between different injection pathways （intraperito- neal, intramuscular and subcutaneous injections）, and pharmacokinetic phage studies of PaP 106 were conduct- ed. The dose of infection was 1 x 101 pfu/mL PAP106 100 pL. The mice were dead on 0.5, 1, 3, 6, 12, 24, and 36 h after intervention. The concentration of phage was measured within blood, liver, and spleen. Results Six strains of PaP had different morphologies. The PaP specifically infected and lysed between 25%--75% bacte- rium isolates, and the lysis volume of PAP106 was highest （75%）. The optimal MOI of PAP106 was 1 ~ 10-6. The time to peak of intraperitoneal injection was earlier than intramuscular injection and subcutaneous injec- tion in blood, liver, and spleen by injecting PAP106 in vivo. Conclusion PAP106 had a powerful lysis of Pseu- domonas aeruginosa. The distribution and time to peak of PAP106 was earliest in blood, liver, and spleen by intraperitoneal injection. Our study has paved a way to the further study of PAP106, and support autophage as an antibiotic for treating patients with multi drug resistant Pseudomonas aeruginosa infection in the future.