畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (11): 2785-2793.doi: 10.11843/j.issn.0366-6964.2020.11.017

• 预防兽医 • 上一篇    下一篇

犬源奇异变形杆菌噬菌体分离、鉴定及对生物被膜的作用

宋军, 徐若洋, 靳育琦, 陈立, 王宇辉, 阮红日, 郑家三*, 武瑞*   

  1. 黑龙江八一农垦大学动物科技学院, 大庆 163319
  • 收稿日期:2020-04-24 出版日期:2020-11-25 发布日期:2020-11-20
  • 通讯作者: 郑家三,主要从事小动物疾病诊疗研究,E-mail:zjs3399@aliyun.com;武瑞,主要从事动物疾病诊断和新兽药研发研究,E-mail:fuhewu@126.com
  • 作者简介:宋军(1983-),男,黑龙江佳木斯人,博士,讲师,主要从事人兽共患细菌性疾病防控研究,E-mail:songjun_2005@126.com
  • 基金资助:
    国家自然科学基金青年科学基金项目(31802226);黑龙江省青年科学基金项目(QC2017021);黑龙江省政府博士后资助(LBH-Z17185);黑龙江省农垦总局重点科研项目(HKKY190307);黑龙江八一农垦大学三横三纵支持计划(TDJH201903);黑龙江八一农垦大学引进人才启动项目(XYB201807)

Isolation and Identification of Lytic Phages Infecting Proteus mirabilis from Canine-derived and Their Effects on Biofilm

SONG Jun, XU Ruoyang, JIN Yuqi, CHEN Li, WANG Yuhui, RUAN Hongri, ZHENG Jiasan*, WU Rui*   

  1. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China
  • Received:2020-04-24 Online:2020-11-25 Published:2020-11-20

摘要: 以犬源奇异变形杆菌为宿主菌,从城市污水中分离出奇异变形杆菌噬菌体,研究其生物学特性及对生物被膜的作用,为治疗和控制奇异变形杆菌感染提供基础。采用双层平板培养法从污水中分离、纯化噬菌体,并观察噬菌斑特征。通过透射电镜观察纯化后噬菌体的形态特征。测定噬菌体的最佳感染复数、一步生长曲线、热稳定性、酸碱稳定性、核酸类型、最佳保存条件及对生物被膜的抑制和清除效果。成功分离出3株奇异变形杆菌的噬菌体,分别命名为vB_PmM_S、vB_PmM_W和vB_PmM_X。噬菌体噬菌斑透明、无晕且边缘整齐;电镜观察显示,噬菌体的头部呈二十面体的立体对称,直径(30±3)nm;噬菌体的最佳感染复数为0.001;一步生长曲线的结果表明,vB_PmM_S潜伏期约为20 min,爆发期约为35 min,平均裂解量为70 PFU·cell-1;3株噬菌体在10~60℃、pH 5.0~8.0滴度稳定;短期内可以将噬菌体(vB_PmM_S)置于SM液直接保存在4℃,长期保存可采用甘油(30%)或者DMSO(5%)置于-80℃。噬菌体鸡尾酒能够破坏奇异变形杆菌生物被膜,预防生物被膜形成。本文分离纯化的3株裂解性噬菌体为奇异变形杆菌及其引起的泌尿系统感染的治疗奠定了基础。

关键词: 犬, 奇异变形杆菌, 裂解性噬菌体, 生物学特性, 生物被膜

Abstract: The present study aimed to isolate phages that could lyse Proteus mirabilis from sewage in Daqing, Heilongjiang, and analyze their biological characteristics and effects on biofilm and provide the basic data for phage therapy as well as prevention. Phages were isolated and purified from sewage by double plate culture method, and their plaque morphology were observed. Morphological characteristics of purified phages were observed by transmission electron microscopy. The optimal multiplicity of infection (MOI), one-step growth curve, thermal stability, pH, nucleic acids analysis and the inhibition and removal effect of biofilm were determined. Three lytic of bacteriophages of Proteus mirabilis were successfully isolated and purified, and named as vB_PmM_S, vB_PmM_W and vB_PmM_X. The phage plaques were circular, clear and transparent with smooth edge. The electron microscope observation showed that the phage particles, those head was twenty-side, head length was about (30±3) nm. The optical MOI was 0.001, the incubation period was 20 min, the burst period was 35 min, and the burst size was 70 PFU·cell-1. The phages could withstand the temperature range from 10 to 60 ℃ and keep stable titer under pH 5.0-8.0. The phage vB_PmM_S can be stored at 4 ℃ in SM solution for a short time, and stored at -80 ℃ in glycerin (30%) or DMSO (5%) for a long time. The mixture of three bacteriophages can inhibit the biofilm formation and remove the biofilm. The results laid the foundation for the treatment of Proteus mirabilis and urinary tract infections.

Key words: canine, Proteus mirabilis, lytic bacteriophage, biological characteristics, biofilm

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