微生物学通报 Microbiology China tongbao@im.ac.cn Dec. 20, 2016, 43(12): 2686 2692 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.160029 研究报告鳜源致病性维氏气单胞菌的鉴定及药敏试验 高金伟 1,2 梁利国 2* 王亚冰 2,3 谢骏 (1. 201306) (2. 214081) (3. 214081) 2* 摘要 : 目的 通过对从患病的鳜(Siniperca chuatsi) 肝脏中分离得到的一株优势菌 WJ2014-1 进行鉴定, 旨在确定病因并筛选出敏感药物, 为今后鳜维氏气单胞菌 (Aeromonas veronii) 的防治提供参考 方法 从患病鳜肝脏分离致病菌, 通过对其生理生化特征与 16S rrna 基因序列分析进行鉴定, 并通过纸片扩散法进行药敏试验 结果 用菌株 WJ2014-1 进行人工回归感染试验后, 鳜发病症状与自然发病症状相似 根据该菌株的形态特征 生化特性 16S rrna 基因序列分析结果鉴定为维氏气单胞菌 该菌株对复方新诺明 强力霉素 罗红霉素 头孢噻肟 哌拉西林等 21 种抗生素敏感, 对氯霉素 诺氟沙星 萘啶酸等 9 种抗生素耐药 结论 分离得到的菌株 WJ2014-1 对鳜有致病性, 生产中可选用复方新诺明 强力霉素 罗红霉素等药物进行防治 关键词 : 鳜, 维氏气单胞菌, 鉴定, 药敏试验 Identification and susceptibility test of pathogenic Aeromonas veronii isolated from Siniperca chuatsi GAO Jin-Wei 1,2 LIANG Li-Guo 2* WANG Ya-Bing 2,3 XIE Jun 2* (1. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China) (2. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture, Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, Jiangsu 214081, China) (3. Wuxi Fisheries College, Nanjing Agricultural University, Wuxi, Jiangsu 214081, China) Abstract: [Objective] To determine the cause of death for Siniperca chuatsi and screen sensitive drugs, a dominant bacteria WJ2014-1 was isolated from the liver of sick S. chuatsi. This study will provide reference for further prevention and treatment of Aeromomas veronii in S. chuatsi. Foundation item: China Agriculture Research System (No. CARS-46-10); Three new Projects of Agricultural Aquaculture Program of Jiangsu Province (No. D2015-13) *Corresponding authors: LIANG Li-Guo: Fax: 86-510-85556566; E-mail: lianglg@zju.edu.cn XIE Jun: E-mail: xiej@ffrc.cn Received: January 09, 2016; Accepted: March 31, 2016; Published online (www.cnki.net): April 18, 2016 基金项目 : (No. CARS-46-10) (No. D2015-13) * 通讯作者 : Fax 86-510-85556566 E-mail:lianglg@zju.edu.cn E-mail xiej@ffrc.cn 收稿日期 :2016-01-09 接受日期 :2016-03-31 优先数字出版日期 (www.cnki.net):2016-04-18
: 2687 [Methods] The pathogenic bacteria were isolated and purified from the liver of S. chuatsi. And the identification of phenotypic information of the strain WJ2014-1 was conducted, including the biochemical identification and 16S rrna gene sequence determination. Antimicrobial susceptibility test was conducted by disk diffusion technique. [Results] The results of artificial infection tests by using WJ2014-1 to infect S. chuatsi displayed that the prevalence of symptoms and spontaneous onset symptoms were the same. According to morphological and biochemical characteristics as well as the result of 16S rrna gene sequence analysis, the isolated strain WJ2014-1 was A. veronii. WJ2014-1 was susceptible to sulfamethoxazole, doxycycline, roxithromycin, cefotaxime, piperacillin and other 21 kinds of antibiotics. Meanwhile, it showed resistance to chloramphenicol, norfloxacin, nalidixic acid and other 9 kinds of antibiotics. [Conclusion] The isolated bacterial strain WJ2014-1 was pathogenic to S. chuatsi, and prevented by administering drugs such as sulfamethoxazole, doxycycline and roxithromycin in fisheries production. Keywords: Siniperca chuatsi, Aeromonas veronii, Identification, Susceptibility test (Siniperca chuatsi) (Osteichthyes) (Perciformes) 鮨 (Serranidae)(Siniperca) [1-2] (Infectious spleen and kidney necrosis virus ISKNV) [3-4] (Flavobacterium cloumnare) [5] (Aeromonas hydrophila) [6] (Saprolegnia sp.) [7] (Ancyrocephalus mogurndae)(henneguya weishanensis) (Trichodina) (Ichthyophthirius multifiliis) [7-8] 2014 6 16S rrna 1 材料与方法 1.1 实验样品及主要试剂 120±5 g (TSB) (TSA) (RS) LB 5% BD Ezup DNA ( )
2688 微生物学通报 Microbiol. China 2016, Vol.43, No.12 1.2 细菌分离与纯培养 TSA 28 C 180 r/min 18 h TSA 4 C 5 WJ2014-1 WJ2014-5 WJ2014-1 1.3 人工感染试验 WJ2014-1 28 C 180 r/min 18 h 5 10 8 5 10 7 5 10 6 5 10 5 5 10 4 CFU/mL 50±5 g 7 d 10 0.1 ml 0.1 ml 2 10 1.4 细菌形态特征观察和理化特性检查 WJ2014-1 LB RS 28 C 18 h () [9] 1.5 细菌的分子鉴定 1.5.1 DNA 模板的制备 : ( ) Ezup DNA WJ2014-1 DNA 20 C 1.5.2 16S rrna 基因序列的扩增及测序 :16S rrna [10] PCR ( ) 1.5.3 系统发育树的构建 : NCBI BLASTn (http://blast.ncbi.nlm.nih.gov/blast.cgi) 16S rrna GenBank ClusterX 1.83 GenBank MEGA 5.0 Neighbour-Joining Kimura 2-parameter 1 000 (Bootstrap) 1.6 药物敏感试验 (K-B) WJ2014-1 39 28 C 24 h ( ) [11] 2 结果与分析 2.1 病原菌菌落和形态观察及其培养特性 2 28 C 18 h TSA RS LB β- 2.2 回归感染试验结果 WJ2014-1 18 h 24 h 3 d 100% 2.3 病原菌生理生化特性检测 WJ2014-1 1
: 2689 Table 1 表 1 分离菌株 WJ2014-1 主要生化特性检测结果 Main biochemical characteristics test results of the isolate WJ2014-1 Characteristics WJ2014-1 ATCC35624 Characteristics WJ2014-1 ATCC35624 Glucose + + Malonate Lactose + Simmons citrate + Maltose + + Phenylalanine + Mannitol + + Melibiose + Saccharose + + () Peptone water + + Sorbitol + Ornithine decarboxylase Dulcitol Lysine decarboxylase + + Rhamnose + Arginine decarboxylase + + Inositol Amino acid decarboxylase control Hydrothion + Arginine dihydrolase + + Acetamide Arginine dihydrolase control Salicin ( ) Nitrate (Reduction) + + Urea +. Note: +: Positive; : Negative; : Not record. [9] [12] WJ2014-1 2.4 16S rrna 基因序列分析 (WJ2014-1) 16S rrna 1 451 bp GenBank KF413415 NCBI BLASTn WJ2014-1 (KJ650080 FJ940798 KF358430) 99% ( 1) 16S rrna (Aeromonas) (A. veronii) 2.5 药敏试验 39 2 21 图 1 分离菌 WJ2014-1 16S rrna 基因序列发育进化树 Figure 1 The phylogenetic analysis based on 16S rrna gene sequence of WJ2014-1 GenBank Bootstrap. Note: Numbers in parenthesis represented GenBank accession number; Numbers at the branch points indicated the bootstrap values.
2690 微生物学通报 Microbiol. China 2016, Vol.43, No.12 Table 2 表 2 分离菌株 WJ2014-1 药敏试验结果 Drug susceptibility test results of the isolate WJ2014-1 Inhibition zone Inhibition zone Antibacterials Sensitivity Antibacterials Sensitivity (mm) (mm) Amikacin 19±0.01 I Doxycycline 25±0.31 S Gentamicin 13±0.03 R Norfloxacin 7±0.05 R Tobramycin 12±0.21 R Nalidixic acid 7±0.10 R Neomycin 17±0.06 I Acetylspiramycin 7±0.09 R Novobiocin 19±0.26 I Spectinomycin 7±0.02 R Tetracycline 16±0.08 I Cefradine 15±0.21 I Chloramphenicol 7±0.15 R Ofloxacin 17±0.18 I Erythrocin 16±0.23 I Ciprofloxacin 26±0.38 S Medemycin 16±0.31 I Minocyline 25±0.24 S Lincomycin 18±0.18 I Lomefloxacin 27±0.19 S Sulfamethoxazole 28±0.43 S Aztreonam 31±0.46 S Furazolidone 27±0.39 S Fleroxacin 30±0.39 S Vancomycin 10±0.27 R Cefoperazone 29±0.28 S Cefalotin 12±0.24 R Clarithromycin 21±0.20 S Enoxacin 26±0.35 S Levofloxacin 27±0.25 S Roxithromycin 21±0.26 S Cefalexin 28±0.32 S Enrofloxacin 27±0.26 S Fosfomycin 38±0.40 S Teicoplanin 27±0.35 S Cefamandole 31±0.37 S Cefotaxime 28±0.19 S Polymyxin 28±0.28 S Piperacillin 26±0.28 S S(d 20) I (14<d<20) R(0 d 14). Note: S: Denotes high sensitivity (d 20); I: Denotes moderate sensitivity (14<d<20); R: Denotes low or no sensitivity (0 d 14). 9 9 3 讨论 (Aeromonas veronii) (Aeromonadaceae) (Aeromonas) [13] 1987 [14] - - 鮰 [15] [16] [17] [18] [19] [20] [9] Garrity [12] 16S rrna
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