半刺厚唇鱼源维氏气单胞菌的分离鉴定

doi:10.11924/j.issn.1000-6850.casb2022-0781

摘要/Abstract

摘要：

对典型症状为皮肤溃烂、体表出血、肝脏肿大的养殖半刺厚唇鱼(Acrossocheilus hemispinus)进行细菌的分离、鉴定和药物敏感性研究。从患病半刺厚唇鱼肝组织分离纯化细菌，并肌肉注射回归感染健康半刺厚唇鱼。PCR扩增获得分离菌株16S rDNA序列和gyrB基因全长序列，并开展分离菌株生理生化鉴定，毒力基因检测以及药物敏感性实验。分离菌株编号为AS0829L1，人工回归感染实验结果表明菌液浓度5×10¹⁰、5×10⁹、5×10⁸ CFU/mL可分别导致健康半刺厚唇鱼100%、100%和28.6%的死亡率，BLAST显示其16S rDNA序列和gyrB基因与维氏气单胞菌(Aeromonas veronii)的相应序列高度同源，同源性分别高达99.93%和99.34%。综合分子生物学、生理生化特征、毒力基因检测结果证明分离菌株AS0829L1为维氏气单胞菌温和生物型(A.veronii bv.sobria)。药物敏感性实验显示，分离菌株对头孢曲松、头孢派酮、庆大霉素、新霉素、恩诺沙星、氧氟沙星等20种药物高度敏感，对羧苄西林、链霉素、氟苯尼考等5种药物中度敏感，对青霉素、诺氟沙星、红霉素等12种药物不敏感。本研究确定了发病半刺厚唇鱼的致病病原，开展了分离菌株的药物敏感性试验，为养殖生产中疾病的防控提供研究基础。

关键词: 半刺厚唇鱼, 维氏气单胞菌温和生物型, 致病性, 药物敏感性

Abstract:

The bacterial isolation, identification and drug sensitivity of diseased Acrossocheilius hemispinus with skin ulceration, surface hemorrhage, swollen and bleeding liver were studied. The bacterial strain was isolated and purified from liver tissue of the diseased fish and injected intramuscularly into healthy Acrossocheilius hemispinus. Full-length sequences of 16S rDNA and gyrB gene were obtained by PCR amplification of pathogenic bacteria, while physiological and biochemical identification, virulent genes detection, and drug sensitivity study were also conducted. The isolated strain was numbered as AS0829L1, and the artificial infection experiments showed that pathogenic groups of 5×10¹⁰, 5×10⁹, 5×10⁸ CFU/mL could cause 100%, 100%and 28.6% mortality, respectively. The BLAST result indicated the sequences of 16S rDNA and gyrB were homologous to the corresponding sequence of Aeromonas veronii in Genbank, with homology as high as 99.93% and 99.34%, respectively. Comprehensive molecular biology, physiological and biochemical characteristics, virulence gene detection results demonstrated the separate strain was Aeromonas veronii bv.sobria. The drug sensitivity test showed the isolated strain was highly sensitive to 20 antibacterial drugs including cefuroxime, cefoperazone, gentamicin, neomycin, enrofloxacin and ofloxacin, moderate sensitive to 5 antibacterial drugs including carboxybenzillin, streptomycin, flufenicol, and resistant to 12 antibacterial drugs including penicillin, norfloxacin, erythromycin. In this study, the pathogen of the diseased Acrossocheilius hemispinus was identified and the drug sensitivity test of the isolated strain was carried out, which provided a research basis for the prevention and control of diseases in aquaculture production.

Key words: Acrossocheilius hemispinus, Aeromonas veronii bv. sobria, pathogenicity, drug sensitivity

张晶晶. 半刺厚唇鱼源维氏气单胞菌的分离鉴定[J]. 中国农学通报, 2023, 39(20): 138-146.

ZHANG Jingjing. Isolation and Identification of Aeromonas Veroni from Diseased Acrossocheilius Hemispinus[J]. Chinese Agricultural Science Bulletin, 2023, 39(20): 138-146.

图/表 10

参考文献 49

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目的基因	引物序列(5’-3’)	产物大小/bp	退火温度/℃	维氏气单胞温和型	维氏气单胞维罗纳型
act	AGAAGGTGACCACCAAGAACA AACTGACATCGGCCTTGAACTC	232	55	+	+
ast	TCTCCATGCTTCCCTTCCACT GTGTAGGGATTGAAGAAGCCG	331	55	-	+
alt	TGACCCAGTCCTGGCACGGC GGTGATCGATCACCACCAGC	442	55	+	+
aerA	CCCGCCGATCTGCAACCGGG CTGGTCTGGATAGACGGGCTCTGCC	489	68	+	+
hlyA	GGCCGGTGGCCCGAAGATACGGG GGCGGCGCCGGACGAGACGGG	597	62	+	+
tapA	ATGACCTCTAGCCCCAATA ACCCGATTGATTTCTGCC	550	55	+	-

目的基因	引物序列(5’-3’)	产物大小/bp	退火温度/℃	维氏气单胞温和型	维氏气单胞维罗纳型
act	AGAAGGTGACCACCAAGAACA AACTGACATCGGCCTTGAACTC	232	55	+	+
ast	TCTCCATGCTTCCCTTCCACT GTGTAGGGATTGAAGAAGCCG	331	55	-	+
alt	TGACCCAGTCCTGGCACGGC GGTGATCGATCACCACCAGC	442	55	+	+
aerA	CCCGCCGATCTGCAACCGGG CTGGTCTGGATAGACGGGCTCTGCC	489	68	+	+
hlyA	GGCCGGTGGCCCGAAGATACGGG GGCGGCGCCGGACGAGACGGG	597	62	+	+
tapA	ATGACCTCTAGCCCCAATA ACCCGATTGATTTCTGCC	550	55	+	-

测定项目	AS0829L1	测定项目	AS0829L1	测定项目	AS0829L1	测定项目	AS0829L1
丙氨酸-苯丙氨酸-脯氨酸芳胺酶	-	β-丙氨酸芳胺酶pNA	-	D-纤维二糖	-	5-酮基-D-葡萄糖酸盐	-
谷氨酸-甘氨酸-精氨酸芳胺酶	+	甘氨酸芳胺酶	-	D-塔格糖	-	柠檬酸盐(钠)	-
L-脯氨酸芳胺酶	+	α-葡糖苷酶	+	D-葡萄糖	+	丙二酸盐	-
L-吡咯烷酮芳胺酶	-	β-木糖苷酶	-	D-甘露糖	+	L-苹果酸盐同化	+
鸟氨酸脱羧酶	-	磷酸酶	-	海藻糖	+	L-乳酸盐同化	-
α-半乳糖苷酶	+	尿素酶	-	古老糖	-	L-组氨酸同化	-
β-葡萄糖醛酸酶	-	β-半乳糖苷酶	+	D-麦芽糖	+	L-乳酸盐碱化	-
β-N-乙酰氨基葡萄糖苷酶	+	β-葡糖苷酶	-	发酵/葡萄糖	+	0/129耐(comp.vibrio)	+
谷氨酰芳胺酶pNA	+	脂酶	-	侧金盏花醇	-	香豆酸	+
赖氨酸脱羧酶	-	酪氨酸芳胺酶	+	D-山梨醇	-	ELLMAN	-
γ-谷氨酰转移酶	+	蔗糖	+	D-甘露醇	+	H₂S产生	-

测定项目	AS0829L1	测定项目	AS0829L1	测定项目	AS0829L1	测定项目	AS0829L1
丙氨酸-苯丙氨酸-脯氨酸芳胺酶	-	β-丙氨酸芳胺酶pNA	-	D-纤维二糖	-	5-酮基-D-葡萄糖酸盐	-
谷氨酸-甘氨酸-精氨酸芳胺酶	+	甘氨酸芳胺酶	-	D-塔格糖	-	柠檬酸盐(钠)	-
L-脯氨酸芳胺酶	+	α-葡糖苷酶	+	D-葡萄糖	+	丙二酸盐	-
L-吡咯烷酮芳胺酶	-	β-木糖苷酶	-	D-甘露糖	+	L-苹果酸盐同化	+
鸟氨酸脱羧酶	-	磷酸酶	-	海藻糖	+	L-乳酸盐同化	-
α-半乳糖苷酶	+	尿素酶	-	古老糖	-	L-组氨酸同化	-
β-葡萄糖醛酸酶	-	β-半乳糖苷酶	+	D-麦芽糖	+	L-乳酸盐碱化	-
β-N-乙酰氨基葡萄糖苷酶	+	β-葡糖苷酶	-	发酵/葡萄糖	+	0/129耐(comp.vibrio)	+
谷氨酰芳胺酶pNA	+	脂酶	-	侧金盏花醇	-	香豆酸	+
赖氨酸脱羧酶	-	酪氨酸芳胺酶	+	D-山梨醇	-	ELLMAN	-
γ-谷氨酰转移酶	+	蔗糖	+	D-甘露醇	+	H₂S产生	-