一株裂解性奶牛乳房炎源屎肠球菌噬菌体的分离及其生物学特性分析

doi:10.11843/j.issn.0366-6964.2017.04.014

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (4): 706-713.doi: 10.11843/j.issn.0366-6964.2017.04.014

一株裂解性奶牛乳房炎源屎肠球菌噬菌体的分离及其生物学特性分析

张倩^1#, 于会举^1#, 孙耀强¹, 张湘莉兰², 张培生¹, 刘鸽¹, 屈勇刚^1*, 童贻刚^2*, 李岩³

1. 石河子大学动物科技学院, 石河子 832003;
2. 军事医学科学院微生物流行病研究所, 北京 100071;
3. 新疆生产建设兵团畜牧兽医工作总站, 乌鲁木齐 830063

收稿日期:2016-11-23 出版日期:2017-04-23 发布日期:2017-04-23
通讯作者: 屈勇刚，副教授，研究生导师，E-mail：quyonggang@shzu.edu.cn；童贻刚，教授，博士生导师，E-mail：tong.yigang@gmail.com
作者简介:张倩（1990-），女，河北保定人，硕士生，主要从事动物传染病诊断与防治研究，E-mail：1585705024@qq.com；于会举（1990-），男，河南平顶山人，硕士生，主要从事动物传染病诊断与防治研究，E-mail：609859455@qq.com
基金资助:
国家自然科学基金（31560699）；新疆生产建设兵团应用基础研究计划项目（2015AG012）

Isolation and Characterization of a Lytic Phage Infecting Enterococcus faecium of Bovine Mastitis

ZHANG Qian^1#, YU Hui-ju^1#, SUN Yao-qiang¹, ZHANGXIANG Li-lan², ZHANG Pei-sheng¹, LIU Ge¹, QU Yong-gang^1*, TONG Yi-gang^2*, LI Yan³

1. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China;
2. Beijing Institute of Microbiology and Epidemiology, The Academy of Military Medical Sciences, Beijing 100071, China;
3. The General Station of Animal Husbandry and Veterinary Medicine of Xinjiang Production and Construction Corps, Urumqi 830063, China

Received:2016-11-23 Online:2017-04-23 Published:2017-04-23

摘要/Abstract

摘要：

从新疆石河子地区奶牛粪样中分离裂解性屎肠球菌噬菌体，对其进行纯化及生物学特性分析，为治疗和控制细菌性感染提供基础。利用双层平板培养法从奶牛粪样中分离、纯化噬菌体，并观察噬菌斑特征，将纯化后的噬菌体浓缩液用磷钨酸负染，通过透射电子显微镜观察其形态特征。对该噬菌体进行全基因组测序和遗传进化分析，用双层平板培养法测定其宿主谱、最佳感染复数、一步生长曲线、热稳定性及pH稳定性。结果如下：分离并纯化出一株裂解性屎肠球菌噬菌体，命名为vB_EfaM_XJ3，其噬菌斑圆形透明、无晕环且边缘整齐，直径1.2~1.5 mm；电镜观察其头部呈廿面体，头长约53.15 nm，尾长约14.42 nm×199.62 nm；核酸类型为dsDNA，基因组全长为157.189 kb，GC含量为50.27%；其基因序列与沙门菌噬菌体SKML-39的相似性高达98%。生物学特性研究显示vB_EfaM_XJ3目前只裂解其宿主菌；最佳感染复数为0.001，潜伏期为15 min，爆发期为175 min，裂解量约为84；能耐受50℃左右高温，在pH 5.0~11.0范围内效价稳定。分离鉴定出一株裂解性肌尾科屎肠球菌噬菌体，对它进行了生物学特性和亲缘性分析，为沙门菌和屎肠球菌抑菌剂的研究与应用奠定了基础。

Abstract:

The aim of the present study was to isolate and characterize the phage that could lyse Enterococcus faecium from fecal samples of dairy cattle in Shihezi, Xinjiang, and provide the basic data for the phage therapy as well as prevention. The phage was isolated and purified from dairy cattle fecal samples by double-layer agar plate method. And its plaque characteristics was observed. The purified phage was concentrated, negatively stained with phosphotungstic acid and observed by transmission electron microscopy. The genome of the isolated phage was sequenced. Its genetic and evolutional history were analyzed. The host range, optimal multiplicity of infection (MOI), one-step growth curve, temperature, and pH stability of the phage were investigated by double-layer agar plate methods. A lytic phage of Enterococcus faecium was isolated and purified, and named vB_EfaM_XJ3. The phage plaques were circular, clear and transparent with smooth edge, and its diameter was 1.2-1.5 mm. The electron microscope observation showed that the phage particles, whose head was twenty-sided, head length was about 53.15 nm, tail length was about 14.42 nm×199.62 nm. The genome of the phage was comprised of double strand DNA, the size was 157.189 kb with GC content of 50.27%. The homology of amino acid sequence of phage vB_EfaM_XJ3 with Salmonella phage SKML-39 was 98%. The study of biological characteristics showed that the vB_EfaM_XJ3 only crack its host bacteria at present. The optimal MOI was 0.001, the incubation period was 15 min, the burst period was 175 min and the burst size was 84. The phage could withstand the temperature up to 50℃ and keep stable titer under pH 5.0-11.0. A lytic phage of Enterococcus faecium was isolated and identified. Its biological characteristics and genetic were analyzed. The results laid the foundation for the further research and application of the antibacterial agent for Salmonella and Enterococcus faecium.

中图分类号:

S852.61

张倩, 于会举, 孙耀强, 张湘莉兰, 张培生, 刘鸽, 屈勇刚, 童贻刚, 李岩. 一株裂解性奶牛乳房炎源屎肠球菌噬菌体的分离及其生物学特性分析[J]. 畜牧兽医学报, 2017, 48(4): 706-713.

ZHANG Qian, YU Hui-ju, SUN Yao-qiang, ZHANGXIANG Li-lan, ZHANG Pei-sheng, LIU Ge, QU Yong-gang, TONG Yi-gang, LI Yan. Isolation and Characterization of a Lytic Phage Infecting Enterococcus faecium of Bovine Mastitis[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(4): 706-713.

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参考文献

[1] 阚威. 奶牛乳房炎链球菌和肠球菌的分离鉴定及毒力基因的检测[D]. 兰州:甘肃农业大学, 2014.
KAN W. Isolation, characerization and virulence gene analysis of Streptococcus and Enterococcus collected from bovine mastitis[D]. Lanzhou:Gansu Agricultural University, 2014. (in Chinese)
[2] 董翔宸. 奶牛乳房炎肠球菌ESP基因真核重组质粒的构建与表达[D]. 兰州:甘肃农业大学, 2015.
DONG X C. Construction and expression of eukaryotic vector of ESP gene from bovine Enterococcus[D]. Lanzhou:Gansu Agricultural University, 2015. (in Chinese)
[3] KLIMIEN? I, RU?AUSKAS M, ŠPAKAUSKAS V, et al. Prevalence of gram positive bacteria in cow mastitis and their susceptibility to beta-lactam antibiotics[J]. Vet Med Zoot, 2011, 56(78):65-72.
[4] 齐亚银, 剡根强, 王静梅, 等. 致羔羊脑炎型粪肠球菌的分离及鉴定[J]. 石河子大学学报:自然科学版, 2005, 23(2):200-202.
QI Y Y, YAN G Q, WANG J M, et al. Isolation and identification of Enterococcus faecalis in lambs with meningitis[J]. Journal of Shihezi University (Natural Science), 2005, 23(2):200-202. (in Chinese)
[5] WALL S K, ZHANG J, ROSTAGNO M H, et al. Phage therapy to reduce preprocessing Salmonella infections in market-weight swine[J].Appl Environ Microbiol, 2010, 76(1):48-53.
[6] 张哲. 两株裂解性粪肠球菌噬菌体的分离鉴定及生物学特性分析[D]. 长春:吉林大学, 2015.
ZHANG Z. Isolation and biological characteristic of two lytic Enteroocus bacteriophages active against Enterococcus faecalis[D]. Changchun:Jilin University, 2015. (in Chinese)
[7] BISWAS B, ADHYA S, WASHART P, et al. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium[J]. Infect Immun, 2002, 70(1):204-210.
[8] 宫鹏娟, 张蕾, 夏翡翡, 等. 屎肠球菌噬菌体m5的分离鉴定及其对小鼠菌血症的治疗研究[J]. 中国兽医学报, 2016, 36(5):746-749,762.
GONG P J,ZHANG L, XIA F F, et al. Bacteriophage m5 therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium[J]. Chinese Journal of Veterinary Science, 2016, 36(5):746-749,762.(in Chinese)
[9] JAMALLUDEEN N,JOHNSON R P,FRIENDSHIP R, et al. Isolation and characterization of nine bacteriophages that lyse O149 enterotoxigenic Escherichia coli[J]. Vet Microbiol, 2007, 124:47-57.
[10] 萨姆布鲁克J, 拉塞尔D W. 分子克隆实验指南[M]. 3版.黄培堂,译.北京:科学出版社, 2002.
SAMBROOK J, RUSSELL D W. Molecular cloning:A laboratory manual[M]. 3rd ed. HUANG P T, trans. Beijing:Science Press, 2002. (in Chinese)
[11] 余静丹. 鲍曼不动杆菌噬菌体的分离鉴定及生物学特性的研究[D]. 长春:吉林大学, 2013.
YU J D. Isolation, characterization and biological features of lytic bacteriophages infecting Acinetobacter baumannii clinical isolates[D]. Changchun:Jilin University, 2013(in Chinese).
[12] NASU H, ⅡDA T, SUGAHARA T, et al. A filamentous phage associated with recent pandemic Vibrio parahaemolyticus O3:K6 strains[J]. J Clin Microbiol, 2000, 38(6):2156-2161.
[13] 冯烨, 刘军, 孙洋, 等. 噬菌体最新分类与命名[J]. 中国兽医学报, 2013, 33(12):1954-1958.
FENG Y, LIU J, SUN Y, et al. An introduction to current classification and nemenclature of bacterial viruses[J]. Chinese Journal of Veterinary Science, 2013, 33(12):1954-1958.(in Chinese)
[14] 包红朵, 张鹏禹, 周艳, 等. 沙门氏菌裂解性噬菌体的分离鉴定及其生物学特性[J]. 食品科学, 2015, 36(5):131-136.
BAO H D, ZHANG P Y, ZHOU Y, et al. Isolation and biological characteristics of lytic Salmonella bacteriophage[J]. Food Science, 2015, 36(5):131-136.(in Chinese)
[15] BUENO E, GARCÍA P, MARTÍNEZ B, et al. Phage inactivation of Staphylococcus aureus in fresh and hard-type cheeses[J]. Int J Food Microbiol, 2012, 158(1):23-27.
[16] FAUQUET C M, MAYO M A, MANILOFF J, et al. Virus taxonomy:Ⅷth report of the international committee on taxonomy of viruses[M]. Amsterdam:Elsevier Academic Press, 2005.
[17] KING A M Q, ADAMS M J, CARSTENS E B, et al. Virus taxonomy:ninth report of the international committee on taxonomy of viruses[M]. Amsterdam:Elsevier Academic Press, 2011.
[18] ANANY H, LINGOHR E J, VILLEGAS A, et al. A Shigella boydii bacteriophage which resembles Salmonella phage ViI[J]. Virol J, 2011, 8(1):242.
[19] 张文惠, 安小平, 范航, 等. 一株粪肠球菌噬菌体的分离及其生物学特性研究[J]. 生物技术通讯, 2013,24(4):484-487.
ZHANG W H, AN X P, FAN H, et al. Isolation and characterization of an Enterococcus faecalis phage[J]Letters in Biotechnology, 2013, 24(4):484-487. (in Chinese)
[20] 邢少贞, 张湘莉兰, 舒鹏, 等. 粪肠球菌噬菌体vB_E. faecalis_IME196的生物学特性及其全基因组分析[J]. 微生物学通报,2016, 43(9):2040-2048.
XING S Z, ZHANGXIANG L L, SHU P, et al. Characterization and complete genome sequence analysis of Enterococcus faecalis bacteriophage vB_E. faecalis_IME196[J]. Microbiology China, 2016, 43(9):2040-2048. (in Chinese)

一株裂解性奶牛乳房炎源屎肠球菌噬菌体的分离及其生物学特性分析

Isolation and Characterization of a Lytic Phage Infecting Enterococcus faecium of Bovine Mastitis

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