Isolation and identification of Aeromonas veronii from diseased Micropterus salmoides
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摘要: 为探明广东省佛山市某养殖场人工养殖大口黑鲈 (Micropterus salmoides) 鱼苗出现暴发性死亡的病因，该研究对患病大口黑鲈鱼苗肝脏组织中分离的一株细菌GZXR2020进行了分子鉴定，PCR扩增获得该菌株的16S rRNA基因和gyrB基因全长序列，BLAST显示其与维氏气单胞菌 (Aeromonas veronii) 的相应序列高度同源，同源性分别高于99.5%和98.3%。根据VITEK2全自动细菌鉴定系统45个理化项目的鉴定结果、羊血琼脂平板溶血实验呈β-溶血的结果，进一步通过毒力基因检测并结合补充理化鉴定结果，可判断此分离菌株为维氏气单胞菌温和生物型 (A. veronii bv. sobria)。人工回归感染实验采用高、中、低3种菌液浓度 (3×107、1.5×108和3×108 CFU·mL−1) 进行腹腔注射攻毒，可分别导致7%、40%和100%的死亡率，证实维氏气单胞菌温和生物型是引起此次大口黑鲈暴发性死亡的致病原。药物敏感性实验显示，在所检测的17种抗菌药物中，该菌株对大观霉素、氟罗沙星、氟苯尼考等7种药物敏感。Abstract: Sudden death occurred in Micropterus salmoides larvae reared on a farm in Foshan, Guangdong Province of China. To investigate the etiology of this disease, we isolated a strain of bacteria GZXR2020 from the liver tissue of diseased M. salmoides larvae and conducted its molecular identification. We obtained the full-length sequences of the 16S rRNA gene and gyrB gene of the strain by PCR amplification. BLAST results show that the sequences were highly homologous to the corresponding sequence of Aeromonas veronii in GenBank, with homology higher than 99.5% and 98.3%, respectively. Using VITEK2 automatic bacterial identification system and according to the results of a total of 45 physical and chemical identification, sheep blood agar plate hemolysis test that showed β-hemolysis, detection of the virulence genes as well as the physicochemical identification, we identified the bacteria as A. veronii bv. sobria. In the artificial regression infection experiment, intraperitoneal injection of 3×107, 1.5×108 and 3×108 CFU·mL−1 resulted in 7%, 40% and 100% mortality, respectively. The drug sensitivity test of a total of 17 antimicrobial agents shows that the strain GZXR2020 was highly sensitive to 7 antibacterial drugs such as spectinomycin, fleroxacin, florfenicol and so on.
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图 1 人工感染后大口黑鲈出现的症状
a. 鳞片脱落、尾鳍基部出血；b. 病鱼肝脏、脾脏与肾脏肿大
Figure 1. Symptoms of M. salmoides after artificial infection
a. Scale exfoliation, bleeding of the base of caudal fin; b. Swelling of liver, spleen and kidney of the infected fish
图 2 根据细菌16S rRNA基因序列 (a) 与gyrB基因序列 (b) 构建的系统进化树
Figure 2. Phylogenetic trees constructed based on sequences of 16S rRNA (a) and gyrB (b)
图 3 分离菌株GZXR2020在羊血琼脂板上的β-溶血环
a. 分离菌株GZXR2020；b. 大肠杆菌；标尺显示单菌落直径
Figure 3. Beta hemolysis of isolated strain GZXR2020 on sheep blood agar
a. Isolated strain GZXR2020; b. E.coli. The bar shows the single colony diameter.
表 1 分离菌株GZXR2020对17种抗菌药物的敏感性
Table 1. Antibiotic sensitivities of isolated strain GZXR2020
抗菌药物
Antibiotic抑菌圈直径
Inhibition
zone/mm药物敏感性
Antibiotic
sensitivity大观霉素 Spectinomycin 36 S 卡那霉素 Amikacin 17 I 链霉素 Streptomycin 12 I 妥布霉素 Tobramycin 12 R 罗红霉素 Roxithromycin 14 R 乙酰螺旋霉素 Acetylspiramycin 21 R 磺胺异噁唑 Sulfisoxazole 16 R 复方新诺明 Cotrimoxazole 21 R 氟罗沙星 Fleroxacin 23 S 利福平 rifampicin 19 S 氟苯尼考 Florfenicol 30 S 阿莫西林 Amoxicillin 8 R 青霉素 Penicillin 13 R 氨苄西林 Ampicillin 13 R 四环素 Tetracycline 25 S 多西环素 doxycycline 24 S 头孢氨苄 Cefalexin 18 S 注：R. 耐药；I. 中等敏感；S. 敏感 Note: R. Low or no sensitivity; I. Moderate sensitivity; S. High sensitivity 
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表 2 分离菌株GZXR2020的生化鉴定结果
Table 2. Biochemical identification results of strain GZXR2020
测定项目 Determination item GZXR2020 测定项目 Determination item GZXR2020 丙氨酸-苯丙氨酸-脯氨酸芳胺酶 Alanine-phenylalanine-proline aromatase + 蔗糖 Sucrose + 谷氨酸-甘氨酸-精氨酸芳胺酶 Glutamic acid-glycine-arginine aromatase + D-塔格糖 D-tagatose − 吡咯烷基芳胺酶 Pyrrolidine arylaminase − D-海藻糖 D-trehalose + L-阿拉伯醇 L-arabinol − 柠檬酸盐(钠) Citrate (sodium) − D-纤维二糖 D-cellobiose − 丙二酸盐 Malonate − β -半乳糖苷酶 β-galactosidase + 5-酮-葡萄糖苷 5-Keto-glucoside − H2S产生 H2S production − 乳酸盐产碱 Alkali production by lactate − β-N-乙酰葡萄糖苷酶 β-N-acetyl glucosidase + α-葡萄糖 α-glucose − 谷氨酰芳胺酶 Glutamyl aromatase − 琥珀酸盐产碱 Alkali production by succinate + D-葡萄糖 D-glucose + N-乙酰- β-半乳 N-acetyl-β-galactosaminase − γ-谷氨酰转移酶 γ-glutamyltransferase − α-半乳糖苷酶 α-galactosidase − 葡萄糖发酵 Glucose fermentation + 磷酸酶 Phosphatase − β-葡萄糖苷酶 β-glucosidase − 氨基乙酸芳胺酶 Aminoacetic acid arylamidase − D-麦芽糖 D-maltose + 鸟氨酸脱羧酶 Ornithine decarboxylase − D-甘露醇 D-mannitol + 赖氨酸脱羧酶 Lysine decarboxylase − 脱羧酶阴性控制 Decarboxylase negative control − D-甘露糖 D-mannose + β-木糖苷酶 β-xylosidase − 组氨酸同化 Histidine assimilation + β-丙氨酸芳胺酶 β-alanine aromatase − β-葡萄糖苷酸酶 β-glucosidase − L-脯氨酸芳胺酶 L-proline aromatase + 0/129耐受 0/129 Resistance + 脂酶 Lipase − L-苹果酸盐同化 L-malate assimilation − 酪氨酸芳胺酶 Tyrosine aromatase + L-乳酸盐同化 L-lactate assimilation − 侧金盏花醇 Adonitol − D-山梨醇 D-sorbitol − 古老糖 Palatinose − 注： +. 阳性；−. 阴性 Note: +. Positive; −. Negative
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