罗氏沼虾豚鼠气单胞菌的分离鉴定

doi:10.11924/j.issn.1000-6850.casb20191200924

摘要/Abstract

摘要：

为了判断导致罗氏沼虾(Macrobrachium rosenbergii)发病死亡的原因,本研究从发病濒死的罗氏沼虾中,分离了一株具有生长优势的菌株,对分离菌株的溶血性、革兰氏染色、形态学、生理生化进行鉴定。结果表明,该分离菌株为革兰氏阴性,氧化酶、阿拉伯糖、葡萄糖产酸、蛋白胨水、精氨酸水解酶、明胶、苯丙氨酸为阳性,并对该分离菌株16S rRNA基因进行了测序以及系统进化树分析,该分离菌株与豚鼠气单胞菌聚为一支,其亲缘关系较近,综合该分离菌株理化性质和序列分析结果,可判断该分离菌株为豚鼠气单胞菌（Aeromonas caviae,登录号MN736843）。以注射法进行人工感染实验,证明了该分离菌株对罗氏沼虾具有较强的致病性,药物敏感试验结果表明,该菌株对头孢曲松、氯霉素、氟苯尼考、四环素、多西环素5种药物高度敏感,对卡那霉素1种药物中度敏感,对环丙沙星、诺氟沙星、红霉素、阿奇霉素、新生霉素、链霉素、复方新诺明和利福平8种药物耐药。本研究实验结果为罗氏沼虾豚鼠气单胞菌的病原鉴定及药物诊治提供一定的参考和理论基础。

关键词: 罗氏沼虾, 豚鼠气单胞菌, 分离鉴定, 革兰氏染色, 生化鉴定, 药敏试验

Abstract:

To identify the mortality cause of Macrobrachium rosenbergii, the strain with growth advantage was isolated from the dying M. rosenbergii, and its hemolysis, gram staining, morphological characteristics, physiology and biochemistry were determined. The results showed that, the isolated strain was a gram-negative while oxidase, l-arabinose, glucose (acid), peptone water, arginine dihydrolase, gelatin, and phenylalanine were positive. The 16S rRNA gene of this isolate was sequenced and the phylogenetic tree was constructed. The isolated strain was clustered with Aeromonas caviae and had a close genetic relationship. The isolated strain was confirmed as Aeromonas caviae (Accession number MN736843) based on its biochemical characters and 16S rRNA sequence analysis. The artificial infection experiment with the injection method confirmed that the isolated strain was highly pathogenetic to M. rosenbergii. The results of drug susceptibility tests revealed that the strain was highly sensitive to ceftriaxone, chloramphenicol, florfenicol, tetracycline and doxycycline; it was moderately sensitivity to kanamycin and resistant to ciprofloxacin, norfloxacin, erythromycin, azithromycin, novobiocin, streptomycin, compound sulfamethoxazole and rifampicin. This study provides a reference and theoretical basis for the pathogen identification, drug diagnosis and treatment of Aeromonas cavia in M. rosenbergii.

Key words: Macrobrachium rosenbergii, Aeromonas caviae, isolation and identification, gram staining, biochemical identification, drug sensitivity test

中图分类号:

S945.4+1

郭莹, 周淼, 李艳和, 王卫民. 罗氏沼虾豚鼠气单胞菌的分离鉴定[J]. 中国农学通报, 2020, 36(33): 147-153.

Guo Ying, Zhou Miao, Li Yanhe, Wang Weimin. Aeromonas caviae from Macrobrachium rosenbergii: Isolation and Identification[J]. Chinese Agricultural Science Bulletin, 2020, 36(33): 147-153.

图/表 8

参考文献 33

[1]	Dwivedi M, Mishra A, Prasad A, et al. Aeromonas caviae septicemia in immunocompetent gastrointestinal carriers[J]. Brazilian Journal of Infectious Diseases, 2008,12(6):547-8. doi: 10.1590/s1413-86702008000600023 URL pmid: 19287850
[2]	Al-Benwan K, Abbott S, Janda J M, et al. Cystitis Caused by Aeromonas caviae[J]. Journal of Clinical Microbiology, 2007,45(7):2348-2350. doi: 10.1128/JCM.00480-07 URL pmid: 17522269
[3]	Kumar A, Kanungo R, Jagdish S, et al. Aeromonas caviae as a cause of cholecystitis[J]. Indian Journal of Pathology and Microbiology, 2000,43(2):169-170. URL pmid: 11217277
[4]	何永龙, 黄锦炉, 乔晶鑫, 等. 草鱼致病性豚鼠气单胞菌的分离鉴定[J]. 中国预防兽医学报, 2015,37(12):20-24.
[5]	许信刚, 伊岚, 王高学, 等. 观赏金鱼豚鼠气单胞菌的分离鉴定及防治[J]. 西北农业学报, 2006,15(1):40-42.
[6]	李梅, 游卓霖, 陈锋, 等. 广东省南美白对虾豚鼠气单胞菌的分离鉴定[J]. 上海海洋大学学报, 2015(04):102-109.
[7]	余旭平, 马有智. 甲鱼病原性豚鼠气单胞菌的分离鉴定[J]. 中国兽医学报, 1997(5).
[8]	曹海鹏, 温乐夫, 杨移斌, 等. 克氏原螯虾源致病性豚鼠气单胞菌的分离及其生物学特性[J]. 水生生物学报, 2014,38(6):1047-1053. doi: 10.7541/2014.154 URL
[9]	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(09):123-126.
[10]	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):677-681.
[11]	Yang G, Frinsko M, Chen X, et al. Current status of the giant freshwater prawn (Macrobrachium rosenbergii) industry in China, with special reference to live transportation[J]. Aquaculture Research, 2012,43(7):1049-1055 doi: 10.1111/j.1365-2109.2011.03009.x URL
[12]	Cheng W, Chen J. Isolation and characterization of an Enterococcus-like bacterium causing muscle necrosis and mortality with Macrobrachium rosenbergii in Taiwan[J]. Diseases of Aquatic Organisms, 1998,34:93-101. doi: 10.3354/dao034093 URL pmid: 9828405
[13]	Chen S, Lin Y, Liaw L, et al. Lactococcus garvieae infection in the giant freshwater prawn Macrobranchium rosenbergii confirmed by polymerase chain reaction and 16S rDNA sequencing[J]. Diseases of Aquatic Organisms, 2001,45(1):45-52. URL pmid: 11411643
[14]	Qian D, Shi Z, Zhang S, et al. Extra small virus-like particles (XSV) and nodavirus associated with whitish muscle disease in the giant freshwater prawn, Macrobrachium rosenbergii[J]. Journal of Fish Diseases, 2003,26(9):521-527 doi: 10.1046/j.1365-2761.2003.00486.x URL pmid: 14575370
[15]	Liang T M, Li X L, Du J, et al. Identification and isolation of a spiroplasma pathogen from diseased freshwater prawns, Macrobrachium rosenbergii, in China: A new freshwater crustacean host[J]. Aquaculture, 2011,318(1-2):1-6. doi: 10.1016/j.aquaculture.2011.03.018 URL
[16]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001.
[17]	布坎南RE, 吉本斯NE. 伯杰细菌鉴定手册[M].8版. 北京: 科学出版社, 1984.
[18]	曹海鹏, 杨先乐, 王玉洁, 等. 鲟源致病性豚鼠气单胞菌的分离及其生长特性[J]. 动物学杂志, 2007,42(6):1-6.
[19]	余旭平, 马有智. 甲鱼病原性豚鼠气单胞菌的分离鉴定[J]. 中国兽医学报, 1997,17(5):460-462.
[20]	李小波, 黄文芳. 丰产鲫细菌性败血病的研究Ⅰ—病原分离与鉴定[J]. 微生物学通报, 2003,30(5):56-60.
[21]	安立国, 傅荣恕, 邢维贤, 等. 鲤竖鳞病病原及其疫苗的研究[J]. 水产学报, 1998,22(2):136-142.
[22]	樊海平, 吴斌, 曾占壮, 等. 日本鳗鲡体表溃疡病病原菌的分离、鉴定及单克隆抗体制备[J]. 中国水产科学, 2009,16(2):295-302.
[23]	吉莉莉, 汪开毓, 肖丹, 等. 南方鲇溃疡综合症病原菌的分离与鉴定[J]. 淡水渔业, 2008,38(3):68-72.
[24]	黄伟坚, 黄均, 秦爱珍, 等. 野生胡子鲶”腹水病“病原分离和鉴定[J]. 中国兽医科技, 2000,30(4):30-33.
[25]	樊海平, 孟庆显, 俞开康. 中国对虾败血病病原菌(气单胞菌)的致病性与生物学性状[J]. 水产学报, 1994,18(1):32-38.
[26]	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):677-681.
[27]	孔繁德. 红嘴鸥豚鼠气单胞菌病的分离鉴定及防治[J]. 福建畜牧兽医, 1999(3):8-8.
[28]	龚永平, 王洪永, 杨光友, 等. 致林麝猝死的病原豚鼠气单胞菌的分离与鉴定[J]. 中国兽医科学, 2017(02):86-90.
[29]	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 Journal of Microbiology and Biotechnology, 2012,28(5):1863-1870. doi: 10.1007/s11274-011-0915-z URL pmid: 22806009
[30]	刘春, 李凯彬, 王庆, 等. 杂交鳢(斑鳢♀x乌鳢♂)内脏类结节病病原菌的分离、定与特性分析[J]. 水产学报, 2012,36(7):1119-1125. doi: 10.3724/SP.J.1231.2012.27916 URL
[31]	Nagar V, Shashidhar R, Bandekar J R, et al. Characterization of Aeromonas strains isolated from Indian foods using rpoD gene sequencing and whole cell protein analysis[J]. World Journal of Microbiology and Biotechnology, 2013,29(4):745-752. URL pmid: 23255059
[32]	Pablos M, Huys G, Cnockaert M, et al. Identification and epidemiological relationships of Aeromonas isolates from patients with diarrhea, drinking water and foods[J]. International Journal of Food Microbiology, 2011,147(3):203-210. doi: 10.1016/j.ijfoodmicro.2011.04.006 URL pmid: 21550680
[33]	樊海平, 孟庆显, 俞开康. 由二种气单胞菌引起的中国对虾败血病的研究[J]. 海洋与湖沼, 1995,26(5):302-308.

生化项目	分离菌株	参考菌株
氧化酶	+	+
硫化氢	–	–
阿拉伯糖	+	+
丙二酸盐	–	–
赖氨酸脱羧酶	–	–
鸟氨酸脱羧酶	–	–
枸缘酸盐	–	+
葡萄糖产酸	+	+
蛋白胨水	+	–
精氨酸水解酶	+	+
V-P	–	–
明胶	+	+
苯丙氨酸	+	+
葡萄糖酸盐	–	–

生化项目	分离菌株	参考菌株
氧化酶	+	+
硫化氢	–	–
阿拉伯糖	+	+
丙二酸盐	–	–
赖氨酸脱羧酶	–	–
鸟氨酸脱羧酶	–	–
枸缘酸盐	–	+
葡萄糖产酸	+	+
蛋白胨水	+	–
精氨酸水解酶	+	+
V-P	–	–
明胶	+	+
苯丙氨酸	+	+
葡萄糖酸盐	–	–

药物	抑菌圈直径判断标准/mm			药物含量/ (ug/disc)	抑菌圈直径/mm	敏感性
药物	耐药	中度敏感	高度敏感	药物含量/ (ug/disc)	抑菌圈直径/mm	敏感性
环丙沙星	≤15	16-20	≥21	5.00	7.38	R
诺氟沙星	≤12	13-16	≥17	10.00	0.00	R
红霉素	≤13	14-22	≥23	15.00	0.00	R
阿奇霉素	≤13	14-17	≥18	15.00	0.00	R
头孢曲松	≤14	15-20	≥21	30.00	33.65	S
新生霉素	≤12	13-16	≥17	30.00	7.41	R
链霉素	≤11	12-14	≥15	10.00	0.00	R
卡那霉素	≤13	14-17	≥18	30.00	13.06	I
氯霉素	≤12	13-17	≥18	30.00	23.37	S
氟苯尼考	≤17	18-20	≥21	30.00	21.56	S
四环素	≤14	15-18	≥19	30.00	19.58	S
多西环素	≤12	13-15	≥16	30.00	19.59	S
复方新诺明	≤14	15-20	≥21	23.75	8.77	R
利福平	≤16	17-20	≥20	5.00	10.20	R

药物	抑菌圈直径判断标准/mm			药物含量/ (ug/disc)	抑菌圈直径/mm	敏感性
药物	耐药	中度敏感	高度敏感	药物含量/ (ug/disc)	抑菌圈直径/mm	敏感性
环丙沙星	≤15	16-20	≥21	5.00	7.38	R
诺氟沙星	≤12	13-16	≥17	10.00	0.00	R
红霉素	≤13	14-22	≥23	15.00	0.00	R
阿奇霉素	≤13	14-17	≥18	15.00	0.00	R
头孢曲松	≤14	15-20	≥21	30.00	33.65	S
新生霉素	≤12	13-16	≥17	30.00	7.41	R
链霉素	≤11	12-14	≥15	10.00	0.00	R
卡那霉素	≤13	14-17	≥18	30.00	13.06	I
氯霉素	≤12	13-17	≥18	30.00	23.37	S
氟苯尼考	≤17	18-20	≥21	30.00	21.56	S
四环素	≤14	15-18	≥19	30.00	19.58	S
多西环素	≤12	13-15	≥16	30.00	19.59	S
复方新诺明	≤14	15-20	≥21	23.75	8.77	R
利福平	≤16	17-20	≥20	5.00	10.20	R