1株鲤源致病性铜绿假单胞菌的分离鉴定及药敏试验

doi:10.16431/j.cnki.1671-7236.2023.11.037

摘要/Abstract

摘要： 【目的】分离鉴定导致鲤大量急性死亡的病原菌，确定其毒力基因和药物敏感性，为后续病鲤的临床治疗提供参考。【方法】从病鲤的内脏中分离培养病原菌，并对该菌株进行形态学观察、革兰染色、生理生化鉴定及16S rRNA分子鉴定；采用毒力基因检测、K-B药敏纸片法及人工回归感染试验分析该病原菌的药物敏感性和致病性。【结果】分离菌在LB固体培养基上呈现圆形或椭圆形菌落，镜检为短棒状的革兰阴性菌，具有一定的运动能力。生理生化试验结果显示，分离菌株在D-葡萄糖、明胶、脲酶、氧化酶、精氨酸、鸟氨酸、赖氨酸及柠檬酸试验中均呈阳性反应，其余指标皆呈阴性反应，符合铜绿假单胞菌的生理生化特征。16S rRNA基因序列系统进化树显示，分离菌与铜绿假单胞菌NR_114471.1呈现高度同源，相似性为99%。综合以上试验结果，将分离菌鉴定为铜绿假单胞菌，命名为HB2210。通过毒力基因检测发现，该菌株含有鞭毛蛋白结构基因fla基因、气溶素aer基因、细胞毒性肠毒素act基因、外膜蛋白ompA基因及热稳定性肠毒素ast基因等多种毒力基因。通过药敏试验发现，分离菌株对头孢他啶、头孢曲松、头孢哌酮、诺氟沙星、氧氟沙星、环丙沙星、多黏菌素B、左氧氟沙星及哌拉西林9种抗菌药敏感。通过人工回归感染试验发现，分离菌对鲤具有急性毒性，高浓度组死亡率高达85%。【结论】本试验从病鲤组织中分离的铜绿假单胞菌HB2210具有极强的致病性，药敏试验筛选出的头孢他啶、头孢曲松、头孢哌酮、诺氟沙星、氧氟沙星、环丙沙星、多黏菌素B、左氧氟沙星及哌拉西林9种抗菌药素可为鲤养殖企业精准用药提供理论基础。

关键词: 鲤; 铜绿假单胞菌; 药敏试验; 毒力基因

Abstract: 【Objective】 The purpose of this study was to isolate and identify the pathogenic bacteria that caused a large number of acute deaths in carp, determine their virulence genes and drug sensitivity, and provide reference for the subsequent clinical treatment of sick carp.【Method】 The pathogenic bacteria were isolated and cultured from the viscera of diseased carp.Morphological observation, Gram staining, physiological and biochemical identification and 16S rRNA molecular identification were performed.The drug sensitivity and pathogenicity of the pathogen were analyzed by virulence gene detection, K-B drug sensitive disk method and artificial regression infection test.【Result】 The isolated bacteria showed round or oval colonies on LB solid medium.Microscopic examination showed short rod-like Gram-negative bacteria with certain locomotor ability.The results of physiological and biochemical tests showed that the isolated strains showed positive reactions in D-glucose, gelatin, urease, oxidase, arginine, ornithine, lysine and citric acid tests, while the other indexes showed negative reactions, which was consistent with the physiological and biochemical characteristics of Pseudomonas aeruginosa.The phylogenetic tree of 16S rRNA gene sequence showed that the isolated bacteria showed high homology with Pseudomonas aeruginosa NR_114471.1, with 99% similarity.Based on the above test results, the isolated bacteria was identified as Pseudomonas aeruginosa and named HB2210.Virulence gene detection showed that the strain contained several virulence genes, such as flagellin structure gene fla gene, aerolysin aer gene, cytotoxic enterotoxin act gene, outer membrane protein ompA gene and heat stable enterotoxin AST gene. The strain was found to be sensitive to nine antibiotics including ceftazidime, ceftriaxone, cefoperazone, norfloxacin, ofloxacin, ciprofloxacin, polymyxin B, levofloxacin and piperacillin by drug sensitivity testing.And it was found to be acutely virulent to common carp by artificial regression infection tests, with mortality rates of up to 85% in the high concentration group.【Conclusion】 In this experiment, Pseudomonas aeruginosa HB2210 isolated from diseased common carp tissues had strong virulence, and nine antibiotics such as ceftazidime, ceftriaxone, cefoperazone, norfloxacin, ofloxacin, ciprofloxacin, polymyxin B, levofloxacin, and piperacillin, etc.screened out by sensitization test, and could provide the theoretical basis of the precise medication for common carp aquaculture enterprises.

Key words: common carp; Pseudomonas aeruginosa; drug sensitivity test; virulence gene

中图分类号:

S852.61

邹昊博, 薛淑群, 吕晓楠, 高聪婷, 邴辉, 古今朝, 韩英, 夏邦华. 1株鲤源致病性铜绿假单胞菌的分离鉴定及药敏试验[J]. 中国畜牧兽医, 2023, 50(11): 4703-4713.

ZOU Haobo, XUE Shuqun, LYU Xiaonan, GAO Congting, BING Hui, GU Jinzhao, HAN Ying, XIA Banghua. Isolation, Identification and Drug Sensitivity Test of a Pathogenic Strain of Pseudomonas aeruginosa from Common Carp[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(11): 4703-4713.

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