狐源豚鼠气单胞菌的分离鉴定
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摘要
2015年辽宁鞍山某狐狸养殖场偶发病原菌感染。病死狐狸后肢肌肉液化,出现溃疡。为查明致死原因进行了实验室诊断,从病灶部位分离到1株优势菌株,命名为M12。综合细菌形态观察、生理特性研究、传统生化试验和VETEK 2全自动生化分析,结果显示,该株菌为豚鼠气单胞菌,革兰阴性短杆状,最适生长温度30℃,最佳p H 7~8,细菌生长曲线比较显示,M12在相同条件下增殖速度明显高于豚鼠气单胞菌模式菌株。通过测定M12的16S r RNA和管家基因gyr B进行了气单胞菌属种内分子水平鉴定,BLAST结果显示,16S r RNA和gyr B基因与Gen Bank上的豚鼠气单胞菌同源性达到100%。将16S r RNA测序结果提交到Gen Bank,获得登录号KX170836。M12含有细胞毒性肠毒素(cytotoxic enterotoxin,act)和热敏感细胞肠毒素(heat-Labie cytotonic enterotoxin,alt)两种毒力基因。小鼠感染试验得到M12的LD_(50)为8×10~8,具有很强的致病性。药敏试验结果显示,该菌对大部分的抗生素具有很强的敏感性,从而为该菌的防治提供依据。
In 2015,there was an outbreak of some bacterial pathogens in Anshan,Liaoning Province.The hind limbs of fox got ulcers which later caused the death to determine the cause of these deaths we performed further laboratory experiments and some biological characteristic analysis.We isolated a dominant strain from the center of lesion named M12.Comprehensive bacterial morphology observation,physiological characteristics,traditional biochemical test,VETEK 2 automatic biochemical analysis and 16 S rRNA identification showed that M12 is the strain of Aeromonas caviae,a gram negative rod-shaped;the optimum growth temperature was 30 ℃and pH was 7—8,respectively.Bacterial growth curve comparison showed M12 under the same proliferation rate was significantly higher than that of the Aeromonas caviae style strain.The homology of 16 S rRNA and Gyr B genes of M12,compared with Gen Bank Aeromonas caviae,was reached up to 100%.The sequencing results were submitted to the Gen Bank,whose accession number was KX170836.M12 contained act and alt of two virulence genes.The bacteria had a high pathogenic activity with 8×10~8LD_(50) to the mouse.The drug sensitivity test showed that the strain had strong sensitivity to a lot of antibiotics,so as to provide evidence for the prevention of the bacteria.
In 2015,there was an outbreak of some bacterial pathogens in Anshan,Liaoning Province.The hind limbs of fox got ulcers which later caused the death to determine the cause of these deaths we performed further laboratory experiments and some biological characteristic analysis.We isolated a dominant strain from the center of lesion named M12.Comprehensive bacterial morphology observation,physiological characteristics,traditional biochemical test,VETEK 2 automatic biochemical analysis and 16 S rRNA identification showed that M12 is the strain of Aeromonas caviae,a gram negative rod-shaped;the optimum growth temperature was 30 ℃and pH was 7—8,respectively.Bacterial growth curve comparison showed M12 under the same proliferation rate was significantly higher than that of the Aeromonas caviae style strain.The homology of 16 S rRNA and Gyr B genes of M12,compared with Gen Bank Aeromonas caviae,was reached up to 100%.The sequencing results were submitted to the Gen Bank,whose accession number was KX170836.M12 contained act and alt of two virulence genes.The bacteria had a high pathogenic activity with 8×10~8LD_(50) to the mouse.The drug sensitivity test showed that the strain had strong sensitivity to a lot of antibiotics,so as to provide evidence for the prevention of the bacteria.
引文
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[11]樊海平,余培建.由豚鼠气单胞菌引起的欧洲鳗鲡败血症[J].水产学报,1999,23(3):313-318.FAN Hai-ping,YU Pei-jian.The septicaemia of anguilia anguilla infected by aeromonas caviae[J].Journal of Fisheries of China,1999,23(3):313-318.(in Chinese)
[12]郭秀平,林明辉,何有根,等.虎纹蛙蝌蚪致病菌株鉴定及药物敏感性分析[J].广东农业科学,2012,39(9):114-115.GUO Xiu-ping,LIN Ming-hui,HE You-gen,et al.Rana rugulosa tadpole's pathogenic strain identification and drug sensitivity analysis[J].Guangdong Agricultural Sciences,2012,39(9):114-115.(in Chinese)
[13]丁雷,宋憬愚.虹鳟皮肤溃烂病的病原菌研究[J].淡水渔业,2002,32(3):28-30.DING Lei,SONG Jing-yu.Study on pathogenic bacteria of rainbow trout skin fester disease[J].Fresh Water Fisheries,2002,32(3):28-30.(in Chinese)
[14]邓梅葵,孙迎,韩雯晴.细菌鉴定方法[J].生物医学工程学进展,2014,35(2):84-88.DENG Mei-kui,SUN Ying,HAN Wen-qing.Methods of identification of bacteria[J].Progress in Biomedical Engineering,2014,35(2):84-88.(in Chinese)
[15]SOLER L,YANEZ M A,CHACON M R,et al.Phylogenetic analysis of the genus aeromonas based on two housekeeping genes[J].Int J Syst Evol Microbiol,2004,54(5):1511-1519.
[16]YANEZ M A,CATALAN V,APRAIZ D,et al.Phylogenetic analysis of members of the genus aeromonas based on gyr B gene sequences[J].Int J Syst Evol Microbiol,2003,53(3):875-883.
[17]NAGAR V,SHASHIDHAR R,BANDEKAR J R.Characterization of aeromonas strains isolated from Indian foods using rpo D gene sequencing and whole cell protein analysis[J].World J Microbiol Biotechnol,2013,29(4):745-752.
[18]OSMAN K,ALY M,KHEADER A,et al.Molecular detection of the aeromonas virulence aerolysin gene in retail meats from different animal sources in Egypt[J].World J Microbiol Biotechnol,2012,28(5):1863-1870.
[2]XU R,POLK R E,STENCEL L,et al.Antibiogram compliance in university Health System consortium participating hospitals with clinical and laboratory standards institute guidelines[J].Am J Health Syst Pharm,2012,69(7):598-606.
[3]秦玉广,陈秀丽,王春生,等.鲟源豚鼠气单胞菌分离与强弱毒株病原生物学比较研究[J].井冈山大学学报:自然科学版,2011,32(6):51-58.QIN Yu-guang,CHEN Xiu-li,WANG Chun-sheng,et al.Isolation and pathogenbiology compare research of virulent and avirulent aeromonas caviae strains from sturgeon[J].Journal of Jinggangshan University:Natural Science,2011,32(6):51-58.(in Chinese)
[4]阳涛,汪开毓,肖丹,等.南方鲇源豚鼠气单胞菌胞外产物活性与致病性研究[J].淡水渔业,2008,38(2):40-45.YANG Tao,WANG Kai-yu,XIAO Dan.Study on activity and pathogenicity of extracellular products of aeromonas caviae isolated from silurusmeriaionalis[J].Fresh Water Fisheries,2008,38(2):40-45.(in Chinese)
[5]王海娟,王利.鲈鱼豚鼠气单胞菌的鉴定及其药敏试验[J].动物医学进展,2014,35(9):123-126.WANG Hai-juan,WANG Li.Identification and drug susceptibility test of aeromonas caviae from Lateolabrax japonicus[J].Progress in Veterinary Medicine,2014,35(9):123-126.(in Chinese)
[6]徐海圣,黄立峰,王淑霞.中华绒螯蟹豚鼠气单胞菌的分离和鉴定[J].浙江大学学报:农业与生命科学版,2001,27(6):667-681.XU Hai-sheng,HUANG Li-feng,WANG Shu-xia.Isolation and identification of aeromonas caviae from Eriocheir sinensis[J].Journal of Zhejiang University:Agriculture&Life Science,2001,27(6):677-681.(in Chinese)
[7]LIM Y L,EE R,YIN W F,et al.Quorum sensing activity of Aeromonas caviae strain YL12,a bacterium isolated from compost[J].Sensors(Basel),2014,14(4):7026-7040.
[8]李任峰,何启盖,周锐,等.细菌鞭毛研究概况及进展[J].微生物学通报,2005,32(6):124-127.LI Ren-feng,HE Qi-gai,ZHOU Rui,et al.The research advances on the bacterial flagella[J].Microiology China,2005,32(6):124-127.(in Chinese)
[9]TUHELA L,ROBINSON J B,TUOVINEN O H.Characterization of chemotactic responses and flagella of Hyphomicrobium strain W1-1B[J].J Bacteriol,1998,180(11):3003-3006.
[10]谷海瀛.细菌鞭毛镀银染色法的创新[J].中华微生物学和免疫学杂志,2002,22(1):110-115.GU Hai-ying.A new silver-plating method for staining flagella[J].Chinese Journal of Microbiology and Immunology,2002,22(1):110-115.(in Chinese)
[11]樊海平,余培建.由豚鼠气单胞菌引起的欧洲鳗鲡败血症[J].水产学报,1999,23(3):313-318.FAN Hai-ping,YU Pei-jian.The septicaemia of anguilia anguilla infected by aeromonas caviae[J].Journal of Fisheries of China,1999,23(3):313-318.(in Chinese)
[12]郭秀平,林明辉,何有根,等.虎纹蛙蝌蚪致病菌株鉴定及药物敏感性分析[J].广东农业科学,2012,39(9):114-115.GUO Xiu-ping,LIN Ming-hui,HE You-gen,et al.Rana rugulosa tadpole's pathogenic strain identification and drug sensitivity analysis[J].Guangdong Agricultural Sciences,2012,39(9):114-115.(in Chinese)
[13]丁雷,宋憬愚.虹鳟皮肤溃烂病的病原菌研究[J].淡水渔业,2002,32(3):28-30.DING Lei,SONG Jing-yu.Study on pathogenic bacteria of rainbow trout skin fester disease[J].Fresh Water Fisheries,2002,32(3):28-30.(in Chinese)
[14]邓梅葵,孙迎,韩雯晴.细菌鉴定方法[J].生物医学工程学进展,2014,35(2):84-88.DENG Mei-kui,SUN Ying,HAN Wen-qing.Methods of identification of bacteria[J].Progress in Biomedical Engineering,2014,35(2):84-88.(in Chinese)
[15]SOLER L,YANEZ M A,CHACON M R,et al.Phylogenetic analysis of the genus aeromonas based on two housekeeping genes[J].Int J Syst Evol Microbiol,2004,54(5):1511-1519.
[16]YANEZ M A,CATALAN V,APRAIZ D,et al.Phylogenetic analysis of members of the genus aeromonas based on gyr B gene sequences[J].Int J Syst Evol Microbiol,2003,53(3):875-883.
[17]NAGAR V,SHASHIDHAR R,BANDEKAR J R.Characterization of aeromonas strains isolated from Indian foods using rpo D gene sequencing and whole cell protein analysis[J].World J Microbiol Biotechnol,2013,29(4):745-752.
[18]OSMAN K,ALY M,KHEADER A,et al.Molecular detection of the aeromonas virulence aerolysin gene in retail meats from different animal sources in Egypt[J].World J Microbiol Biotechnol,2012,28(5):1863-1870.