某鸡场大肠杆菌噬菌体的分离鉴定及其生物学特性研究

doi:10.16431/j.cnki.1671-7236.2021.06.034

摘要/Abstract

摘要： 为了探究大肠杆菌噬菌体的形态特征和生物学特性，本研究以实验室保存的55株禽致病性大肠杆菌为宿主菌，通过双层琼脂平板法从某鸡场污水和淤泥中分离纯化得到1株宽裂解谱的大肠杆菌噬菌体，命名为Bp16，用2%磷钨酸负染后于透射电镜观察其形态结构，采用双层琼脂平板法测定其温度和pH稳定性、最佳感染复数（MOI）及一步生长曲线等生物学特性。透射电镜观察结果显示，分离得到的宽裂解谱噬菌体Bp16的头部为正二十面体结构，有尾且有尾丝，体长约为220 nm，根据《病毒分类——国际病毒分类委员会第9次报告》的分类特征，该噬菌体为有尾噬菌体目、肌尾噬菌体科的成员；以实验室保存的55株禽致病性大肠杆菌为指示菌，其裂解效率仅为12.73%；温度稳定性结果显示，在37~60 ℃作用30 min或1 h噬菌体效价基本保持不变，且在80 ℃作用1 h后效价仍能达到1×10⁴ PFU/mL以上，说明该噬菌体对温度的变化不敏感，具有耐热的特性；pH稳定性结果显示，在pH 5.0~10.0之间效价基本保持不变，对酸碱较耐受；最佳感染复数为1；根据最佳感染复数来绘制一步生长曲线，测得其潜伏期为20 min，暴发期为50 min，裂解量为73 PFU/cell。综上所述，噬菌体Bp16具有热稳定性、酸碱稳定性及裂解能力强等优点，为后续试验研制大肠杆菌噬菌体制剂奠定了基础。

关键词: 噬菌体; 分离鉴定; 生物学特性; 大肠杆菌

Abstract: In order to explore the morphological and biological characteristics of coliphages,55 strains of avian pathogenic Escherichia coli stored in the laboratory were used as the host bacteria in this study.A coliphage with a wide lysis spectrum was obtained from the sewage and sludge of a chicken farm by the double-layer agar plate method,named Bp16,after negative staining with 2% phosphotungstic acid,the morphology and structure were observed by transmission electron microscope.The temperature and pH stability,optimal multiplicity of infection (MOI),and one-step growth curve were measured by the double-layer agar plate method.Transmission electron microscopy showed that the isolated broad lysis spectrum phage Bp16 had an icosahedral structure in the head,with tails and filaments,and the body length was about 220 nm.According to the classification characteristics of Virus Classification—The 9th Report of the International Committee on Classification of Viruses,the bacteriophage was a member of the Caudovirales and the Mycobacteriophages family.Using 55 avian pathogenic Escherichia coli stored in the laboratory as indicator bacteria,its cracking efficiency was only 12.73%.The results of temperature stability showed that the titer of the phage remained basically unchanged at 37-60 ℃ for 30 min or 1 h,and the titer could still reach more than 1×10⁴ PFU/mL after treatment at 80 ℃ for 1 h,indicating that the phage was not sensitive to temperature changes and had heat resistance.The results of pH stability showed that the titer remained basically unchanged between pH 5.0 to 10.0,and it was relatively tolerant to acid and alkali.The results of the best MOI showed that the best MOI was 1.One-step growth curve according to the best MOI was drew,and it was measured that the incubation period was 20 min,the outbreak period was 50 min,and the lysis amount was 73 PFU/cell.In summary,the bacteriophage Bp16 had thermal stability,acid-base stability and strong lysis ability,which laid the foundation for the development of coliphage preparations for subsequent experiments.

Key words: phage; isolation and identification; biological characteristics; Escherichia coli

中图分类号:

S852.61

赵尊福, 阳亭亭, 张焕容. 某鸡场大肠杆菌噬菌体的分离鉴定及其生物学特性研究[J]. 中国畜牧兽医, 2021, 48(6): 2204-2211.

ZHAO Zunfu, YANG Tingting, ZHANG Huanrong. Isolation,Identification and Biological Characteristics of Coliphage from a Chicken Farm[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 2204-2211.

参考文献

[1] 马兴树,朱美霞,石虎,等.禽致病性大肠杆菌的分离、O血清群鉴定及药物敏感试验[J].中国畜牧兽医,2016,43(12):3343-3348. MA X S,ZHU M X,SHI H,et al.Isolation,O serogrouping and drug sensitivity test of avian pathogenic Escherichia coli[J].China Animal Husbandry & Veterinary Medicine,2016,43(12):3343-3348.(in Chinese)
[2] 陈慧.饲料全面禁抗开启催生2000万吨级行业巨头[J].甘肃畜牧兽医,2020,50(9):7-11. CHEN H.A total ban on feed resistance has been launched,giving birth to 20 million ton industrial giants[J].Gansu Animal Husbandry and Veterinary,2020,50(9):7-11.(in Chinese)
[3] 魏炳栋,丛聪,于维,等.一株产肠毒素大肠杆菌短尾噬菌体分离与鉴定[J].东北农业大学学,2020,51(6):35-45. WEI B D,CONG C,YU W,et al.Isolation and identification of anenterotoxigenic E.coli Podoviridae phage[J].Journal of Northeast Agricultural University,2020,51(6):35-45.(in Chinese)
[4] XIE M X,YE S G,YANG X Y.Research progress in phage[J].Agricultural Science & Technology,2016,17(7):1709-1713.
[5] 陶程琳,王少辉,张耀,等.噬菌体疗法预防细菌性感染的研究进展及发展方向[J].中国兽医科学,2020,50(9):1167-1175. TAO C L,WANG S H,ZHANG Y,et al.Advances and development direction of bacteriophage therapy for preventing bacterial infection[J].Chinese Veterinary Science,2020,50(9):1167-1175.(in Chinese)
[6] BATINOVIC S,WASSEF F,KNOWLER S A,et al.Bacteriophages in natural and artificial environments[J].Pathogens,2019,8(3):100.
[7] CHRISTINE T,SCOTT W O,YONATAN H G,et al.Estimating the proportion of bystander selection for antibiotic resistance among potentially pathogenic bacterial flora[J].Proceedings of the National Academy of Sciences of the United States of America,2018,115(51):11988-11995.
[8] 崔自红,季秀玲.细菌－噬菌体对抗性共进化研究进展[J].中国生物工程杂志,2020,40(Z1):140-145. CUN Z H,JI X L.Advances in bacteria-phage antagonistic coevolution[J].China Biotechnology,2020,40(Z1):140-145.(in Chinese)
[9] 赵影,刘秀侠,李金敏,等.一株鼠伤寒沙门氏菌噬菌体的分离鉴定及对小鼠的防制效果研究[J].中国畜牧兽医,2018,45(1):234-241. ZHAO Y,LIU X X,LI J M,et al.Isolation and identification of a phage against Salmonella Typhimurium and its control effect on mice[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):234-241.(in Chinese)
[10] 李陇平,张智英.金黄色葡萄球菌烈性噬菌体的分离鉴定和最佳保存方法研究[J].中国畜牧兽医,2011,38(6):141-146. LI L P,ZHANG Z Y.Isolate virulent phage of Staphylococcus aureus and the study of the approach to optimal condition for preservation[J].China Animal Husbandry & Veterinary Medicine,2011,38(6):141-146.(in Chinese)
[11] 韦德源,陆泽宁,张聪,等.产志贺毒素多重耐药大肠杆菌噬菌体的生物学特性及全基因组分析[J].中国畜牧兽医,2020,47(2):352-362. WEI D Y,LU Z N,ZHANG C,et al.Biological characteristics and genome analysis of shiga toxin-producing and multidrug-resistant Escherichia coli phages[J].China Animal Husbandry & Veterinary Medicine,2020,47(2):352-362.(in Chinese)
[12] XIANG Y Y,LI W Y,SONG F,et al.Biological characteristics and whole-genome analysis of the Enterococcus faecalis phage PEf771[J].Canadian Journal of Microbiology,2020,66(9):505-520.
[13] WANG R H,XING S Z,ZHAO F Y,et al.Characterization and genome analysis of novel phage vB_EfaP_IME195 infecting Enterococcus faecalis[J].Virus Genes,2018,54(6):804-811.
[14] 宋新慧,商延,张庆,等.噬菌体vB_SalS_DZ的生物学特性及对鸡沙门氏菌病的疗效[J].山东农业科学,2018,50(7):42-47. SONG X H,SHANG Y,ZHANG Q,et al.Biological characteristics of Salmonella phage vB_SalS_DZ and its therapeutic effect on salmonellosis of chicken[J].Shandong Agricultural Sciences,2018,50(7):42-47.(in Chinese)
[15] 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.The latest classification and nomenclature of bacteriophages[J].Chinese Journal of Veterinary Science,2013,33(12):1954-1958.(in Chinese)
[16] FABIGAN A P,KHALID A,MADDOCKS S,et al.Phage therapy for severe bacterial infections:A narrative review[J].Medical Journal of Australia,2020,212(6):279-285.
[17] 陈可炎.鸡大肠杆菌病及其防治措施[J].吉林畜牧兽医,2020,41(9):52-53. CHEN K Y.Chicken colibacillosis and its control measures[J].Jilin Animal Husbandry and Veterinary Medicine,2020,41(9):52-53.(in Chinese)
[18] 路建彪,王俊丽,吴伟胜,等.一株鸡大肠杆菌噬菌体的分离鉴定及治疗试验[J].中国预防兽医学报,2019,41(5):530-533. LU J B,WANG J L,WU W S,et al.Isolation and identification of a strain of chicken coliphage and its treatment effect on avian colibacillosis[J].Chinese Journal of Preventive Veterinary Medicine,2019,41(5):530-533.(in Chinese)
[19] 钟文诗,宋鹏,王佳,等.驴源大肠杆菌噬菌体的分离鉴定与生物学特性研究[J].黑龙江畜牧兽医,2020,98(17):92-95. ZHONG W S,SONG P,WANG J,et al.Isolation,identification and biological characteristics of Escherichia coli phage from donkey[J].Heilongjiang Animal Science and Veterinary Medicine,2020,98(17):92-95.(in Chinese)
[20] 傅强,单文雅,王兆飞,等.一株强裂解性大肠杆菌T1样噬菌体新成员的分离与鉴定[J].微生物学通报,2017,44(12):2782-2794. FU Q,SHAN W Y,WANG Z F,et al.Isolation and characterization of an efficient lytic T1-like phage against Escherichia coli[J].Microbiology China,2017,44(12):2782-2794.(in Chinese)
[21] 杨慧敏,吴圆圆,屈勇刚,等.一株鸡致病性大肠杆菌裂解性噬菌体的生物学特性及其对肠道菌群影响分析[J].中国家禽,2018,40(23):18-22. YANG H M,WU Y Y,QU Y G,et al.Biological characteristics of a chicken pathogenic Escherichia coli lytic phage and its effect on intestinal flora[J].China Poultry,2018,40(23):18-22.(in Chinese)
[22] 廖馨涓,潘海龙,罗辉艳,等.生物制品公司周边环境中2株大肠杆菌噬菌体的分离鉴定[J].上海交通大学学报(农业科学版),2017,35(2):7-12. LIAO X J,PAN H L,LUO H Y,etal.Isolation and characterization of two Escherichia coli specific bacteriophages from surrounding environment of biological products company[J].Journal of Shanghai Jiaotong University (Agricultural Science),2017,35(2):7-12.(in Chinese)
[23] 张倩,张湘莉兰,孙耀强,等.一株裂解性奶牛乳房炎源大肠杆菌噬菌体的分离鉴定及生物学特性分析[J].微生物学通报,2017,44(6):1387-1394. ZHANG Q,ZHANG X L L,SUN Y Q,et al.Isolation and characterization of a lytic phage infecting Escherichia coli from bovine mastitis[J].Microbio-logy China,2017,44(6):1387-1394.(in Chinese)
[24] 钟希娜,何涛,葛展霞,等.一种对mcr-1阳性大肠杆菌具有裂解作用的噬菌体生物学特性[J].江苏农业学报,2018,34(2):452-458. ZHONG X N,HE T,GE Z X,et al.Characterization of a lytic bacteriophage infecting mcr-1-positive Escherichia coli[J].Jiangsu Journal of Agricultural Sciences,2018,34(2):452-458.(in Chinese)
[25] 杭小英,刘莉,施伟达.噬菌体生物制剂在草鱼细菌性疾病治疗中的应用实例[J].科学养鱼,2020,5:55. HANG X Y,LIU L,SHI W D.Application of bacteriophage biological agents in the treatment of bacterial diseases of grass carp[J].Scientific Fish Farming,2020,5:55.(in Chinese)
[26] MOYE Z D,WOOLSTON J,SULAKVELIDZE A.Bacteriophage applications for food production and processing[J].Viruses,2018,10(4):205.
[27] 邹秀月,蔡德周.噬菌体治疗细菌性疾病的研究进展及发展方向[J].中国感染控制杂志,2019,18(9):888-892. ZOU X Y,CAI D Z.Research progress and development direction of bacteriophage therapy for bacterial infection[J].Chinese Journal of Infection Control,2019,18(9):888-892.(in Chinese)