中华鳖3种细菌的鉴定及致病性研究
|
摘要
针对一例中华鳖(Trionyx sinensis)爆发性死亡疾病,本研究旨在寻找其致病因子,明确分离菌的生物学地位、致病性和耐药特征。从患病中华鳖体内获得菌株C1、C2和C3,经人工回归感染健康鳖,测定分离菌的致病作用;经形态学观察、生理生化鉴定及16S rRNA测序,结合系统发育学分析,做菌种的归类判定;用琼脂扩散(K-B)法测定菌株对抗生素药物的敏感性。分离纯培养的3株菌分别为弗氏柠檬酸杆菌(Citrobacter freundii)、摩氏摩根氏菌(Morganella morganii)、肺炎克雷伯氏菌(Klebsiellapneumoniae);能引起中华鳖急性感染性疾病,3种菌共同致病死亡率最高,可达100%。药敏试验结果显示:弗氏柠檬酸杆菌对头孢他啶、头孢曲松、左氧氟沙星高度敏感,对万古霉素等17种药物耐药;摩氏摩根氏菌对头孢他啶和头孢曲松高度敏感,对克林霉素等17种药物耐药;肺炎克雷伯氏菌对恩诺沙星等13种药物敏感,对青霉素等6种药物耐药。本研究所检出的弗氏柠檬酸杆菌、摩氏摩根氏菌和肺炎克雷伯氏菌为该例中华鳖爆发性死亡疾病的病原菌,3菌混合致病力强于单一菌液。弗氏柠檬酸杆菌和摩氏摩根氏菌呈现高度多重耐药,肺炎克雷伯氏菌耐药趋势异于前两者,表现为弱耐药株。
Due to the outbreak of a deadly disease on Chinese turtle(Trionyx sinensis),this study aims to find out the pathogenic factor,and identify the biological status,the etiology and drug resistance characteristics of isolated bacteria.Three pathogenic bacteria assigned as C1,C2,C3 were isolated from the diseased Chinese turtle(Trionyx sinensis).The pathogenic effect of the isolated bacteria was determined through artificial regression on healthy Chinese turtle.Species of bacteria were classified through morphological observation,physiological and biochemical identification,16 S rRNA sequencing and phylogenetic analysis.Kirby-Bauer agar diffusion method was used for the drug sensitivity test.The three isolated bacteria strains were classified as Citrobacter freundii,Morganella morganii,and Klebsiella pneumoniae.Isolated bacteria could cause acute infectious disease on healthy Chinese turtle,and the mix of 3 species of bacteria could lead to almost 100%mortality.Drug-sensitivity tests showed that Citrobacter freundii were highly sensitive to Ceftazidime,Ceftriaxone and Levofloxacin,while extremely resistant to 17 kinds of drugs such as Vancomycin;Morganella morganii were very sensitive to Ceftriaxone,Ceftriaxone and highly resistant to 17 kinds of drugs such as Clindamycin;Klebsiella pneumoniae were hypersensitive to 13 kinds of drugs such as Enrofloxacin and extremely resistant to 6 kinds of drugs such as Penicillin.This study showed that Citrobacter freundii,Morganella morganii and Klebsiella pneumoniae were the pathogenic bacteria which caused the deadly disease outbreak on the Chinese turtle.The pathogenicity of the 3 mixed isolate bacteria was stronger than any single bacteria strain.Citrobacter freundii and Morganella morganii showed a high degree of multi-drug resistance,while resistant tendency of Klebsiella pneumoniae is different from the others,which was a weak resistant strain.
Due to the outbreak of a deadly disease on Chinese turtle(Trionyx sinensis),this study aims to find out the pathogenic factor,and identify the biological status,the etiology and drug resistance characteristics of isolated bacteria.Three pathogenic bacteria assigned as C1,C2,C3 were isolated from the diseased Chinese turtle(Trionyx sinensis).The pathogenic effect of the isolated bacteria was determined through artificial regression on healthy Chinese turtle.Species of bacteria were classified through morphological observation,physiological and biochemical identification,16 S rRNA sequencing and phylogenetic analysis.Kirby-Bauer agar diffusion method was used for the drug sensitivity test.The three isolated bacteria strains were classified as Citrobacter freundii,Morganella morganii,and Klebsiella pneumoniae.Isolated bacteria could cause acute infectious disease on healthy Chinese turtle,and the mix of 3 species of bacteria could lead to almost 100%mortality.Drug-sensitivity tests showed that Citrobacter freundii were highly sensitive to Ceftazidime,Ceftriaxone and Levofloxacin,while extremely resistant to 17 kinds of drugs such as Vancomycin;Morganella morganii were very sensitive to Ceftriaxone,Ceftriaxone and highly resistant to 17 kinds of drugs such as Clindamycin;Klebsiella pneumoniae were hypersensitive to 13 kinds of drugs such as Enrofloxacin and extremely resistant to 6 kinds of drugs such as Penicillin.This study showed that Citrobacter freundii,Morganella morganii and Klebsiella pneumoniae were the pathogenic bacteria which caused the deadly disease outbreak on the Chinese turtle.The pathogenicity of the 3 mixed isolate bacteria was stronger than any single bacteria strain.Citrobacter freundii and Morganella morganii showed a high degree of multi-drug resistance,while resistant tendency of Klebsiella pneumoniae is different from the others,which was a weak resistant strain.
引文
[1]杨先乐,贺路.鳖病研究的现状及其展望[J].中国水产科学,1995,2(4):78-85.
[2]黄琪琰.中国水产动物疾病学研究进展[J].水产学报,1996,20(1):51-57.
[3]钱明明,宋杰,武斌,等.中华鳖常见疾病的诊断及其综合防治[J].动物医学进展,2009,30(4):103-107.
[4]胡广洲,李登峰,苏秀榕.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(4):859-868.
[5]吴惠仙,王淑霞.中华鳖疾病研究进展[J].水产科学,1999,18(5):38-41.
[6]潘晓艺,郝贵杰,姚嘉赟,等.中华鳖爱德华菌病病原菌的分离鉴定及致病因子研究[J].淡水渔业,2010,40(6):40-45.
[7] Saitou N, Nei M. The neighbor-joining method:A new method forreconstructing phylogenetic trees[J].Molecular Biological Evolution,1987,4(4):406-425.
[8]沈萍,范秀容,李广武.微生物学实验[M].第三版.北京:高等教育出版社,1999:62-64.
[9] National Committee for Clinical Laboratory Standards.Performance standards for antimicrobial susceptibility testing[S].Eighteen information supplement NCCLS document M100-S18,2008:25-61.
[10]陈永亮,高晓华,李怡,等.鳖源摩氏摩根菌分离鉴定及药敏特性分析[J].南方农业学报,2015,46(11):2046-2052.
[11]徐海圣,舒妙安.中华鳖肺炎克雷伯氏菌病的病原研究[J].浙江大学学报:理学版,2002(6):702-706.
[12]沈锦玉,潘晓艺,余旭平,等.中华鳖白底板病病原的分析[J].中国水产科学,2007,14(5):815-822.
[13]陈晓岚,周常义,青新.鳖“白板病”致病细菌的研究[J].水产学报,1997,21(3):309-315.
[14]田飞焱,欧阳敏,张晓燕,等.中华鳖弗氏柠檬酸杆菌的分离、鉴定及药物敏感性试验[J].水产学杂志,2013,26(4):42-46.
[15]林启存,朱丽敏,李忠全,等.中华鳖弗氏柠檬酸杆菌败血症病原分离鉴定与药敏试验[J].水产科学,2008,27(1):42-43.
[16]童桂香,吴建坤,吴祥庆,等.山瑞鳖源肺炎克雷伯菌的分离鉴定及其药敏特性分析[J].中国兽医学报,2016(5):750-755.
[17]陶锦华,李康然,韦平.石龟肺炎克雷伯菌感染的诊断与防治[J].广西畜牧兽医,2002,18(6):20-21.
[18]邓国成,罗霞,江小燕,等.鳗肺炎克雷伯菌的分离与鉴定[J].上海海洋大学学报,2009,18(2):2193-2197.
[19]唐毅,张芬,孙翰昌,等.白鲢肺炎克雷伯菌的分离鉴定[J].西南大学学报:自然科学版,2007,29(6):73-76.
[20]孔蕾,朱凝瑜,贝亦江,等.中华鳖(Pelodiscus sinensis)摩氏摩根菌(Morganella morganii)的鉴定及致病性研究[J].海洋与湖沼,2013(3):722-727.
[21]马有智,舒妙安.一种中华鳖穿孔病病原菌的分离和特性研究[J].浙江大学学报:农业与生命科学版,2000,26(4):414-416.
[22]郑曙明,陈章宝,贺蓉.中华鳖皮肤溃烂病的病原研究[J].四川畜牧兽医学院学报,1999,13(2):1-5.
[23]陈春山,郭明磊,魏凯,等.天蚕素和溶菌酶对中华鳖腐皮病预防效果的研究[J].四川动物,2017,36(3):311-316.
[2]黄琪琰.中国水产动物疾病学研究进展[J].水产学报,1996,20(1):51-57.
[3]钱明明,宋杰,武斌,等.中华鳖常见疾病的诊断及其综合防治[J].动物医学进展,2009,30(4):103-107.
[4]胡广洲,李登峰,苏秀榕.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(4):859-868.
[5]吴惠仙,王淑霞.中华鳖疾病研究进展[J].水产科学,1999,18(5):38-41.
[6]潘晓艺,郝贵杰,姚嘉赟,等.中华鳖爱德华菌病病原菌的分离鉴定及致病因子研究[J].淡水渔业,2010,40(6):40-45.
[7] Saitou N, Nei M. The neighbor-joining method:A new method forreconstructing phylogenetic trees[J].Molecular Biological Evolution,1987,4(4):406-425.
[8]沈萍,范秀容,李广武.微生物学实验[M].第三版.北京:高等教育出版社,1999:62-64.
[9] National Committee for Clinical Laboratory Standards.Performance standards for antimicrobial susceptibility testing[S].Eighteen information supplement NCCLS document M100-S18,2008:25-61.
[10]陈永亮,高晓华,李怡,等.鳖源摩氏摩根菌分离鉴定及药敏特性分析[J].南方农业学报,2015,46(11):2046-2052.
[11]徐海圣,舒妙安.中华鳖肺炎克雷伯氏菌病的病原研究[J].浙江大学学报:理学版,2002(6):702-706.
[12]沈锦玉,潘晓艺,余旭平,等.中华鳖白底板病病原的分析[J].中国水产科学,2007,14(5):815-822.
[13]陈晓岚,周常义,青新.鳖“白板病”致病细菌的研究[J].水产学报,1997,21(3):309-315.
[14]田飞焱,欧阳敏,张晓燕,等.中华鳖弗氏柠檬酸杆菌的分离、鉴定及药物敏感性试验[J].水产学杂志,2013,26(4):42-46.
[15]林启存,朱丽敏,李忠全,等.中华鳖弗氏柠檬酸杆菌败血症病原分离鉴定与药敏试验[J].水产科学,2008,27(1):42-43.
[16]童桂香,吴建坤,吴祥庆,等.山瑞鳖源肺炎克雷伯菌的分离鉴定及其药敏特性分析[J].中国兽医学报,2016(5):750-755.
[17]陶锦华,李康然,韦平.石龟肺炎克雷伯菌感染的诊断与防治[J].广西畜牧兽医,2002,18(6):20-21.
[18]邓国成,罗霞,江小燕,等.鳗肺炎克雷伯菌的分离与鉴定[J].上海海洋大学学报,2009,18(2):2193-2197.
[19]唐毅,张芬,孙翰昌,等.白鲢肺炎克雷伯菌的分离鉴定[J].西南大学学报:自然科学版,2007,29(6):73-76.
[20]孔蕾,朱凝瑜,贝亦江,等.中华鳖(Pelodiscus sinensis)摩氏摩根菌(Morganella morganii)的鉴定及致病性研究[J].海洋与湖沼,2013(3):722-727.
[21]马有智,舒妙安.一种中华鳖穿孔病病原菌的分离和特性研究[J].浙江大学学报:农业与生命科学版,2000,26(4):414-416.
[22]郑曙明,陈章宝,贺蓉.中华鳖皮肤溃烂病的病原研究[J].四川畜牧兽医学院学报,1999,13(2):1-5.
[23]陈春山,郭明磊,魏凯,等.天蚕素和溶菌酶对中华鳖腐皮病预防效果的研究[J].四川动物,2017,36(3):311-316.