一株绒山羊源大肠杆菌噬菌体φPTK的分离鉴定及生物学特性分析
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摘要
以从患羔羊痢疾的羔羊粪便中分离的1株大肠杆菌为宿主菌,从污水中分离纯化1株烈性大肠杆菌特异性噬菌体φPTK,并对其生物学特性进行研究,为研制一种替代抗生素的有效生物防治方法提供资源。双层琼脂平板培养后,噬菌斑呈现清晰透明、边缘光滑的圆形,直径2~3 mm。噬菌体φPTK生物学特性试验结果表明:φPTK对大肠杆菌的最佳感染复数为0.000 1;φPTK吸附和感染宿主菌的潜伏期为15 min,裂解期约195 min,裂解量93;噬菌体浓缩液经SDS-PAGE分析可以观察到至少6条蛋白质条带,相对分子质量为2.5×10~4~1.8×10~5,说明其蛋白质外壳至少含有6个结构蛋白;在30~90℃范围内φPTK的裂解活性均较强,30~40℃时活性最高,90℃处理90 min后仍然具有较强的活性;对酸碱的耐受力较强,p H值在5至9范围内均有较强的裂解活性,当p H值超过9后活性较低;对紫外线的耐受能力较强,经紫外线照射8 h后仍然具有较强的裂解能力。这株烈性大肠杆菌特异性噬菌体φPTK最佳感染复数较小,吸附和感染宿主菌的潜伏期短,裂解能力强,杀菌效果好,有较强的环境适应能力,具有研发成为有效防治羔羊大肠杆菌病的生物杀菌制剂的潜力。
In order to provide an alternative antibiotics for the prevention of lamb dysentery,a lamb-associated Escherichia coli virulent specific bacteriophage,named φPTK,was isolated and purified from sewage,and its biological characteristics were determined. The plaque of the phage φPTK showed circular,clear and transparent with smooth edge,showing 2-3 mm of diameter,using the double-layer agar plate method. The analysis results of its biological properties showed that the optimal multiplicity of infection( MOI) was 0.000 1. The determination of one-step growth kinetics showed a short latency period of about 15 min and a burst phase time of 195 min and a relatively larger burst size was 93. The structural proteome of phage φPTK was analyzed by SDS-PAGE,at least six protein bands could be observed with a relative molecular mass of 2.5×10~4-1.8×10~5,indicating that the phage φPTK had at least six structural proteins. In the range of 30-90 ℃,φPTK had a strong lytic activity,and the activity was highest at 30-40 ℃. After treated 90 minutes at 90 ℃,it still had a strong activity. Phage φPTK was quite stable in a wide range of p H values( p H 5-9). Moreover,phageφPTK was resistant to ultraviolet ray treatment,and the titer was still remained a high level after ultraviolet irradia-tion for eight hours. In the present study,an E. coli-specific virulent phage was isolated,the phage φPTK had a short latency period,strong lytic activity and ability of sterilization. It has the potential to be developed as a new approach to control the lamb-associated E. coli disease.
In order to provide an alternative antibiotics for the prevention of lamb dysentery,a lamb-associated Escherichia coli virulent specific bacteriophage,named φPTK,was isolated and purified from sewage,and its biological characteristics were determined. The plaque of the phage φPTK showed circular,clear and transparent with smooth edge,showing 2-3 mm of diameter,using the double-layer agar plate method. The analysis results of its biological properties showed that the optimal multiplicity of infection( MOI) was 0.000 1. The determination of one-step growth kinetics showed a short latency period of about 15 min and a burst phase time of 195 min and a relatively larger burst size was 93. The structural proteome of phage φPTK was analyzed by SDS-PAGE,at least six protein bands could be observed with a relative molecular mass of 2.5×10~4-1.8×10~5,indicating that the phage φPTK had at least six structural proteins. In the range of 30-90 ℃,φPTK had a strong lytic activity,and the activity was highest at 30-40 ℃. After treated 90 minutes at 90 ℃,it still had a strong activity. Phage φPTK was quite stable in a wide range of p H values( p H 5-9). Moreover,phageφPTK was resistant to ultraviolet ray treatment,and the titer was still remained a high level after ultraviolet irradia-tion for eight hours. In the present study,an E. coli-specific virulent phage was isolated,the phage φPTK had a short latency period,strong lytic activity and ability of sterilization. It has the potential to be developed as a new approach to control the lamb-associated E. coli disease.
引文
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[21]彭勇,丁云娟,林洪,等.一株副溶血弧菌噬菌体VPp1的分离鉴定及裂解性能[J].海洋科学,2013,37(1):96-101.
[22]刘悦,李菁华,史红艳,等.肠出血性大肠埃希菌O157噬菌体的生物学特性[J].吉林大学学报(医学版),2012,38(1):79-83.
[23]赵俊杰.大肠杆菌噬菌体LZZ-17生化特性及全基因组学研究[D].兰州:甘肃农业大学,2016.
[24]彭勇,王静雪,丁云娟,等.两株副溶血弧菌噬菌体的分离鉴定及裂解性能[J].水产科学,2012,31(11):645-650.
[25]何觅之.大肠杆菌K88、k99广谱噬菌体的分离与生物学特性鉴定[D].武汉:华中农业大学,2012.
[26]代保英.大肠杆菌K88噬菌体的分离、分类、初步鉴定和生物学特性的测定[D].扬州:扬州大学,2009.
[27]张培东,孙岩,任慧英,等.大肠杆菌噬菌体的分离及其生物学特性[J].中国兽医杂志,2008,44(4):10-12.
[28]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.
[2]宋亚雄,王丽丽,李晓宇,等.噬菌体在畜禽细菌性疾病控制中应用研究进展[J].动物学进展,2016,36(3):91-94.
[3]张培东.大肠杆菌噬菌体的分离鉴定与初步应用[D].青岛:青岛农业大学,2007.
[4]姚玉峰.后抗生素时代的耐药防控对策[J].世界科学,2014(12):14.
[5]LI L,ZHANG Z.Isolation and characterization of a virulent bacteriophage SPW specific for Staphylococcus aureus isolated from bovine mastitis of lactating dairy cattle[J].Molecular Biology Reports,2014,41(9):5829-5838.
[6]COFFEY B,RIVAS L,DUFFY G,et al.Assessment of Escherichia coli O157∶H7-specific bacteriophages e11/2 and e4/1c in model broth and hide environments[J].International Journal of Food Microbiology,2011,147(3):188-194.
[7]WANG L,QU K,LI X,et al.Use of Bacteriophages to Control Escherichia coli O157∶H7 in domestic ruminants meat products,and fruits and vegetables[J].Foodborne Pathog Dis,2017,14(9):483-493.
[8]HUFF W E,HUFF G R,RATH N C,et al.Prevention of Escherichia coli respiratory infection in broiler chickens with bacteriophage(SPR02)[J].Poultry Science,2002,81(4):437-441.
[9]HUFF W E,HUFF G R,RATH N C,et al.Prevention of Escherichia coli infection in broiler chickens with a bacteriophage aerosol spray[J].Poultry Science,2002,81(10):1486-1491.
[10]RAYA R R,OOT R A,MOORE-MALRY B,et al.Naturally resident and exogenously applied T4-like and T5-like bacteriophages can reduce Escherichia coli O157∶H7 levels in sheep guts[J].Bacteriophage,2011,1(1):15-24.
[11]CALLAWAY T R,EDRINGTON T S,BRABBAN A D,et al.Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157∶H7 populations in ruminant gastrointestinal tracts[J].Foodborne Pathog Dis,2008,5(2):183-191.
[12]NIU Y D,XU Y,MCALLISTER T A,et al.Comparison of fecal versus rectoanal mucosal swab sampling for detecting Escherichia coli O157∶H7 in experimentally inoculated cattle used in assessing bacteriophage as a mitigation strategy[J].Journal of Food Protection,2008,71(4):691-698.
[13]STANFORD K,MCALLISTER T A,NIU Y D,et al.Oral delivery systems for encapsulated bacteriophages targeted at Escherichia coli O157∶H7 in feedlot cattle[J].Journal of Food Protection,2010,73(7):1304-1312.
[14]KUTAELADZE M,ADAMIA R.Bacteriophages as potential new therapeutics to replace or supplement antibiotics[J].Trends Biotechnol,2010,28(12):591-595.
[15]BRUSSOW H.Targeting the gut to protect the bladder:Oral Phage therapy approaches against urinary Escherichia coli infections?[J].Environmental Microbiology,2016,18(7):2084-2088.
[16]BRUSSOW H.Phage therapy:the Escherichia coli experience[J].Microbiology,2005,151(7):2133-2140.
[17]CASEY E,MAHONY J,NEVE H,et al.Novel phage group infecting Lactobacillus delbrueckii subsp.lactis,as revealed by genomic and proteomic analysis of bacteriophage Ldl1[J].Applied and Environmental Microbiology,2015,81(4):1319-1326.
[18]宽太吉.羔羊大肠杆菌病的诊断方法及防治措施[J].中兽医学杂志,2015(8):30.
[19]MIEDZYBRODZKI R,FORTUNA W,WEBER-DABROWSKA B,et al.Phage therapy of staphylococcal infections(including MRSA)may be less expensive than antibiotic treatment[J].Postepy Hig Med Dosw,2007,61:461-465.
[20]吴伟胜,李玉保,王守荣,等.大肠杆菌噬菌体的研究进展[J].江苏农业科学,2015,43(8):8-11.
[21]彭勇,丁云娟,林洪,等.一株副溶血弧菌噬菌体VPp1的分离鉴定及裂解性能[J].海洋科学,2013,37(1):96-101.
[22]刘悦,李菁华,史红艳,等.肠出血性大肠埃希菌O157噬菌体的生物学特性[J].吉林大学学报(医学版),2012,38(1):79-83.
[23]赵俊杰.大肠杆菌噬菌体LZZ-17生化特性及全基因组学研究[D].兰州:甘肃农业大学,2016.
[24]彭勇,王静雪,丁云娟,等.两株副溶血弧菌噬菌体的分离鉴定及裂解性能[J].水产科学,2012,31(11):645-650.
[25]何觅之.大肠杆菌K88、k99广谱噬菌体的分离与生物学特性鉴定[D].武汉:华中农业大学,2012.
[26]代保英.大肠杆菌K88噬菌体的分离、分类、初步鉴定和生物学特性的测定[D].扬州:扬州大学,2009.
[27]张培东,孙岩,任慧英,等.大肠杆菌噬菌体的分离及其生物学特性[J].中国兽医杂志,2008,44(4):10-12.
[28]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.