弗氏柠檬酸杆菌与迟钝爱德华氏菌拮抗菌的筛选与鉴定
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摘要
鉴于弗氏柠檬酸杆菌和迟钝爱德华氏菌是"人-兽-鱼"共患性条件致病菌,因此利用微生物拮抗原理研发微生态制剂进行细菌病害的防治对环境具有友好性.分别以从患病中华鳖分离的弗氏柠檬酸杆菌和迟钝爱德华氏菌作为指示菌,初步筛选获得对这2种菌具有拮抗作用的菌株6-1-1M和7-1-1M.用点种法和纸片法进一步验证了其拮抗活性,测定了2株菌930 bp的16S r DNA基因片段序列,发现二者序列完全一致,系统进化分析表明其属于希瓦氏菌属(Shewanella sp.).安全性验证实验结果表明其无致病性,具有开发应用前景.
Citrobacter freundii and Edwardsiella tarda are the pathogens of human, Terrestrial animals and fish. The use of microbial antagonistic to develop microecological agents for the prevention and control of bacterial diseases is environmentally friendly. In this study, Citrobacter freundii and Edwardsiella tarda isolated from diseased Trionyx sinensis were used as indicator bacterial to screen antagonistic bacteria. Antagonistic bacteria strains 6-1-1 M and 7-1-1 M from healthy T. Sinensis were found to produce inhibitory effects on the two pathogens respectively. Further study verified the antagonistic activity of the two strains by point method and paper method. A total of 930 bp 16 S r DNA genes of the two bacteria strains were amplified by PCR and sequenced. The two strains were identical. Phylogenetic tree based on16 S rDNA illustrated that 6-1-1 M(7-1-1 M) belongs to Shewanella sp. Safety verification results showed that 6-1-1 M and 7-1-1 M have no pathogenicity, which shows a great potential of application.
Citrobacter freundii and Edwardsiella tarda are the pathogens of human, Terrestrial animals and fish. The use of microbial antagonistic to develop microecological agents for the prevention and control of bacterial diseases is environmentally friendly. In this study, Citrobacter freundii and Edwardsiella tarda isolated from diseased Trionyx sinensis were used as indicator bacterial to screen antagonistic bacteria. Antagonistic bacteria strains 6-1-1 M and 7-1-1 M from healthy T. Sinensis were found to produce inhibitory effects on the two pathogens respectively. Further study verified the antagonistic activity of the two strains by point method and paper method. A total of 930 bp 16 S r DNA genes of the two bacteria strains were amplified by PCR and sequenced. The two strains were identical. Phylogenetic tree based on16 S rDNA illustrated that 6-1-1 M(7-1-1 M) belongs to Shewanella sp. Safety verification results showed that 6-1-1 M and 7-1-1 M have no pathogenicity, which shows a great potential of application.
引文
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[2]薛巧,赵战勤,刘会胜,等.弗氏柠檬酸杆菌对动物和人致病性研究进展[J].动物医学进展,2015,36(7):81-85.
[3]Barnes L A,Cherry W B.A group of paracolon organisms having apparent pathogenicity[J].American Journal of Public Health,1946,36(5):481-483.
[4]Austkettis A,Habte D,Holmgren J,et al.Enterotoxinproducing bacteria and parasites in stools of Ethiopian children with diarrhoeal disease[J].Archives of Disease in Childhood,1976,51(11):865-870.
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[6]Ortega J,Corpa J M,Orden J A,et al.Acute death associated with Citrobacter freundii infection in an African elephant(Loxodonta africana)[J].Journal of Veterinary Diagnostic Investigation,2015,27(5):632-636.
[7]Suzuki K,Yanai M,Hayashi Y,et al.Edwardsiella tarda bacteremia with psoas and epidural abscess as a food-borne infection:A case report and literature review[J].Internal Medicine,2018,57(6):893-897.
[8]刘双凤.迟钝爱德华氏菌在水产动物中致病性及致病因子的若干研究[J].黑龙江水产,2014(1):35-37.
[9]陈言峰,卓孝磊,王超,等.鲮鱼致病性迟钝爱德华氏菌的鉴定及药敏分析[J].水产科学,2017,36(1):48-53.
[10]王斌.迟钝爱德华氏菌粘附及侵袭特性研究[C]//中国科学技术协会学会,福建省人民政府.经济发展方式转变与自主创新--第十二届中国科学技术协会年会(第三卷),2010:8.
[11]杜迎春,杨菊凤,时晓,等.迟钝爱德华氏菌的分离鉴定及体外抑菌效果研究[J].河南农业,2017(8):51-52.
[12]傅松哲.弧菌拮抗菌的筛选及其对凡纳滨对虾的抑菌防病作用[J].中国环境科学,2009,29(8):867-872.
[13]王娟,封永辉,蔡立胜,等.来自大黄鱼(Pseudosciaena crocea)肠道的弧菌拮抗菌的筛选与鉴定[J].海洋与湖沼,2010,41(5):707-713.
[14]李海兵,宋晓玲,李,等.水产动物益生菌研究进展[J].动物医学进展,2008(5):94-99.
[15]Quintana G,Niederle M V,Minahk C J,et al.Nisin Zproduced by Lactococcus lactis from bullfrog hatchery is active against Citrobacter freundii,a red-leg syndrome related pathogen[J].World Journal of Microbiology and Biotechnology,2017,33(10):186.
[16]胡广洲,李登峰,苏秀榕,等.患暴发性败血症中华鳖体内细菌的分离与鉴定[J].中国水产科学,2010,17(4):859-868.
[17]张新明,李健,刘淇.弧菌拮抗菌的筛选及其效果[J].中国水产科学,2004,11(4):325-332.
[18]郑善坚.弗氏柠檬酸杆菌引起鱼、蛙溃烂病的防治案例[J].科学养鱼,2017(2):68-69.
[19]Bae J Y,Kang C K,Choi S J,et al.Sudden deaths of neonates receiving intravenous infusion of lipid emulsion contaminated with Citrobacter freundii[J].Journal of Korean Medical Science,2018,33(10):e97.
[20]Liu H,Zhao Z,Xue Y,et al.Fatal cases of Citrobacter freundii septicemia and encephalitis in sheep[J].Journal of Veterinary Diagnostic Investigation,2018,30(2):245-248.
[21]倪春山.重要海洋病原细菌迟钝爱德华氏菌感染小鼠及细胞的研究模型建立[D].上海:华东理工大学,2015.
[22]唐德,陈晓于.迟钝爱德华氏菌败血症1例[J].湛江医学院学报,1993(S1):64.
[23]孔杰,李小义,周洲,等.鲟源迟钝爱德华氏菌的分离鉴定与药敏特性研究[J].水产科学,2018(2):187-192.
[24]王岚,王印庚,张正,等.养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性分析[J].渔业科学进展,2017,38(4):17-24.
[25]赵健,兰成忠,陈庆河,等.抗番茄青枯病内生拮抗细菌的筛选及室内防效测定[J].武夷科学,2006,22(1):49-54.
[26]周晓梅,刘强,王瑛璐.拮抗菌生物防治农作物病害的研究进展[J].吉林师范大学学报(自然科学版),2010,31(4):36-39.
[27]王静,郑永华.拮抗菌在果蔬采后病害生物防治中的应用[J].生物技术进展,2013,3(6):393-398.
[28]李敏,胡美姣,高兆银,等.5株拮抗菌抑菌能力及其对香蕉采后病害的防治效果[J].浙江农业科学,2010(1):130-132.
[29]张书俊,杨先乐,李聃,等.水霉拮抗菌的筛选及其拮抗作用的初步研究[J].水生生物学报,2008(3):301-307.
[30]Riquelme C,Araya R,Vergara N,et al.Potential probiotic strains in the culture of the Chilean scallop Argopecten purpuratus,(Lamarck,1819)[J].Aquaculture,1997,154(1):17-26.
[31]于淑池,徐亚茹.拮抗细菌产生的活性物质及拮抗机理研究进展[J].承德职业学院学报,2006(1):67-69.
[32]许志诚,洪义国,罗微,等.厌氧条件下希瓦氏菌腐殖质还原对偶氮还原的影响[J].微生物学报,2006,46(4):591-597.
[33]许玫英,郭俊,钟小燕,等.一个降解染料的希瓦氏菌新种--中国希瓦氏菌[J].微生物学报,2004,44(5):561-566.
[34]商宝娣,杨星,李正友,等.希瓦氏菌的研究进展[J].福建农业,2015(7):152-154.
[35]Esteve C,Merchán R,Alcaide E.An outbreak of Shewanella putrefaciens group in wild eels Anguilla anguilla L.favoured by hypoxic aquatic environments[J].Journal of Fish Diseases,2017,40(7):929-939.
[36]肖康,彭云峰,罗玲,等.产EPA海洋细菌Shewanella baltica 6-42全基因组测序及比较基因组分析[J].中国油料作物学报,2017,39(3):404-413.
[37]Dailey F E,McGraw J E,Jensen B J,et al.The microbiota of freshwater fish and freshwater niches contain Omega-3 fatty acid-producing Shewanella species[J].Applied and Environmental Microbiology,2015,82(1):218-231.
[38]Valdivia-González M A,Díaz-Vásquez W A,Ruiz-León D,et al.A comparative analysis of tellurite detoxification by members of the genus Shewanella[J].Archives of Microbiology,2017,200(2):1-7.
[39]陈慧,郑平,谢作甫,等.Shewanella燃料电池的原理、组成和性能[J].环境科学与技术,2014(10):37-41.
[2]薛巧,赵战勤,刘会胜,等.弗氏柠檬酸杆菌对动物和人致病性研究进展[J].动物医学进展,2015,36(7):81-85.
[3]Barnes L A,Cherry W B.A group of paracolon organisms having apparent pathogenicity[J].American Journal of Public Health,1946,36(5):481-483.
[4]Austkettis A,Habte D,Holmgren J,et al.Enterotoxinproducing bacteria and parasites in stools of Ethiopian children with diarrhoeal disease[J].Archives of Disease in Childhood,1976,51(11):865-870.
[5]Guarino A,Capano G,Malamisura B,et al.Production of Escherichia coli STa-like heat-stable enterotoxin by Citrobacter freundii isolated from humans[J].Journal of Clinical Microbiology,1987,25(1):110-114.
[6]Ortega J,Corpa J M,Orden J A,et al.Acute death associated with Citrobacter freundii infection in an African elephant(Loxodonta africana)[J].Journal of Veterinary Diagnostic Investigation,2015,27(5):632-636.
[7]Suzuki K,Yanai M,Hayashi Y,et al.Edwardsiella tarda bacteremia with psoas and epidural abscess as a food-borne infection:A case report and literature review[J].Internal Medicine,2018,57(6):893-897.
[8]刘双凤.迟钝爱德华氏菌在水产动物中致病性及致病因子的若干研究[J].黑龙江水产,2014(1):35-37.
[9]陈言峰,卓孝磊,王超,等.鲮鱼致病性迟钝爱德华氏菌的鉴定及药敏分析[J].水产科学,2017,36(1):48-53.
[10]王斌.迟钝爱德华氏菌粘附及侵袭特性研究[C]//中国科学技术协会学会,福建省人民政府.经济发展方式转变与自主创新--第十二届中国科学技术协会年会(第三卷),2010:8.
[11]杜迎春,杨菊凤,时晓,等.迟钝爱德华氏菌的分离鉴定及体外抑菌效果研究[J].河南农业,2017(8):51-52.
[12]傅松哲.弧菌拮抗菌的筛选及其对凡纳滨对虾的抑菌防病作用[J].中国环境科学,2009,29(8):867-872.
[13]王娟,封永辉,蔡立胜,等.来自大黄鱼(Pseudosciaena crocea)肠道的弧菌拮抗菌的筛选与鉴定[J].海洋与湖沼,2010,41(5):707-713.
[14]李海兵,宋晓玲,李,等.水产动物益生菌研究进展[J].动物医学进展,2008(5):94-99.
[15]Quintana G,Niederle M V,Minahk C J,et al.Nisin Zproduced by Lactococcus lactis from bullfrog hatchery is active against Citrobacter freundii,a red-leg syndrome related pathogen[J].World Journal of Microbiology and Biotechnology,2017,33(10):186.
[16]胡广洲,李登峰,苏秀榕,等.患暴发性败血症中华鳖体内细菌的分离与鉴定[J].中国水产科学,2010,17(4):859-868.
[17]张新明,李健,刘淇.弧菌拮抗菌的筛选及其效果[J].中国水产科学,2004,11(4):325-332.
[18]郑善坚.弗氏柠檬酸杆菌引起鱼、蛙溃烂病的防治案例[J].科学养鱼,2017(2):68-69.
[19]Bae J Y,Kang C K,Choi S J,et al.Sudden deaths of neonates receiving intravenous infusion of lipid emulsion contaminated with Citrobacter freundii[J].Journal of Korean Medical Science,2018,33(10):e97.
[20]Liu H,Zhao Z,Xue Y,et al.Fatal cases of Citrobacter freundii septicemia and encephalitis in sheep[J].Journal of Veterinary Diagnostic Investigation,2018,30(2):245-248.
[21]倪春山.重要海洋病原细菌迟钝爱德华氏菌感染小鼠及细胞的研究模型建立[D].上海:华东理工大学,2015.
[22]唐德,陈晓于.迟钝爱德华氏菌败血症1例[J].湛江医学院学报,1993(S1):64.
[23]孔杰,李小义,周洲,等.鲟源迟钝爱德华氏菌的分离鉴定与药敏特性研究[J].水产科学,2018(2):187-192.
[24]王岚,王印庚,张正,等.养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性分析[J].渔业科学进展,2017,38(4):17-24.
[25]赵健,兰成忠,陈庆河,等.抗番茄青枯病内生拮抗细菌的筛选及室内防效测定[J].武夷科学,2006,22(1):49-54.
[26]周晓梅,刘强,王瑛璐.拮抗菌生物防治农作物病害的研究进展[J].吉林师范大学学报(自然科学版),2010,31(4):36-39.
[27]王静,郑永华.拮抗菌在果蔬采后病害生物防治中的应用[J].生物技术进展,2013,3(6):393-398.
[28]李敏,胡美姣,高兆银,等.5株拮抗菌抑菌能力及其对香蕉采后病害的防治效果[J].浙江农业科学,2010(1):130-132.
[29]张书俊,杨先乐,李聃,等.水霉拮抗菌的筛选及其拮抗作用的初步研究[J].水生生物学报,2008(3):301-307.
[30]Riquelme C,Araya R,Vergara N,et al.Potential probiotic strains in the culture of the Chilean scallop Argopecten purpuratus,(Lamarck,1819)[J].Aquaculture,1997,154(1):17-26.
[31]于淑池,徐亚茹.拮抗细菌产生的活性物质及拮抗机理研究进展[J].承德职业学院学报,2006(1):67-69.
[32]许志诚,洪义国,罗微,等.厌氧条件下希瓦氏菌腐殖质还原对偶氮还原的影响[J].微生物学报,2006,46(4):591-597.
[33]许玫英,郭俊,钟小燕,等.一个降解染料的希瓦氏菌新种--中国希瓦氏菌[J].微生物学报,2004,44(5):561-566.
[34]商宝娣,杨星,李正友,等.希瓦氏菌的研究进展[J].福建农业,2015(7):152-154.
[35]Esteve C,Merchán R,Alcaide E.An outbreak of Shewanella putrefaciens group in wild eels Anguilla anguilla L.favoured by hypoxic aquatic environments[J].Journal of Fish Diseases,2017,40(7):929-939.
[36]肖康,彭云峰,罗玲,等.产EPA海洋细菌Shewanella baltica 6-42全基因组测序及比较基因组分析[J].中国油料作物学报,2017,39(3):404-413.
[37]Dailey F E,McGraw J E,Jensen B J,et al.The microbiota of freshwater fish and freshwater niches contain Omega-3 fatty acid-producing Shewanella species[J].Applied and Environmental Microbiology,2015,82(1):218-231.
[38]Valdivia-González M A,Díaz-Vásquez W A,Ruiz-León D,et al.A comparative analysis of tellurite detoxification by members of the genus Shewanella[J].Archives of Microbiology,2017,200(2):1-7.
[39]陈慧,郑平,谢作甫,等.Shewanella燃料电池的原理、组成和性能[J].环境科学与技术,2014(10):37-41.