加州鲈源弗氏柠檬酸杆菌的分离鉴定及耐药性分析

Isolation, identification and drug resistance analysis of Citrobacter freundii from Micropterus salmoides

  • 摘要: 【目的】明确引起佛山市三水某水产种苗有限公司育种基地加州鲈(Micropterus salmonoides)体表出血和溃烂的病原菌,并进行药物敏感性分析,为该病的临床诊断及科学防控提供科学依据。【方法】采用常规细菌分离方法从患病加州鲈的肝脏、脾脏和肾脏等组织中分离病原菌,经革兰氏染色和磷钨酸负染后,使用光学显微镜和透射电子显微镜观察菌体形态及其染色特性;通过ATB Expression生化鉴定仪和Biolog微生物自动鉴定系统进行生理生化特性鉴定,基于16S rRNA序列和gyrB基因序列进行分子生物学鉴定,经人工回归感染试验和组织病理学观察进一步确定病原菌的致病性及其对组织细胞的损伤,同时以K-B纸片扩散法测定其药敏特性。【结果】从患病加州鲈肝脏中分离获得1株优势菌株(180803bj_jzl),为周鞭毛的革兰氏阴性杆菌,Biolog微生物系统自动鉴定其为弗氏柠檬酸杆菌(Citrobacter freundii)的可能性高达99.4%;基于16S rRNA序列和gyrB基因序列相似性构建的系统发育进化树均显示,分离菌株180803bj_jzl与弗氏柠檬酸杆菌聚类为一支,与GenBank已公布弗氏柠檬酸杆菌参考菌株的16S rRNA序列相似性均在99.00%以上,gyrB基因序列相似性则在91.00%以上。人工回归感染7 d内的累积死亡率达60%,临床症状与自然发病加州鲈的症状基本相同;其肝脏组织中央静脉和肝窦有红细胞淤积,肝细胞质内有大量空泡,呈散在变性坏死;脾脏的白髓与红髓分界不清,脾窦和脾小血管内有嗜铁血红素中心;鳃小片上皮细胞与毛细血管分离,呈球拍状,部分上皮细胞坏死脱落,只残存少量鳃小片骨架。分离菌株180803bj_jzl对氟罗沙星、氧氟沙星、大观霉素、新霉素、庆大霉素、恩诺沙星和诺氟沙星等7种抗菌药物敏感,对多西环素、四环素、乙酰螺旋霉素、罗红霉素、头孢氨苄和阿莫西林等15种抗菌药物已产生耐药性。【结论】弗氏柠檬酸杆菌感染可引起加州鲈体表出血和溃疡,且具有较强的毒力,致使肝脏、脾脏和鳃等组织病理损伤,实际生产中可选用新霉素和恩诺沙星等渔用抗菌药物进行防治。

     

    Abstract: 【Objective】The California perch(Micropterus salmonoides) broke out with surface hemorrhage and ulceration at the breeding base of an aquatic seedling company in Sanshui, Foshan.The pathogenic bacteria and drug sensitivity were clarified, which provided scientific evidence for the clinical diagnosis and scientific prevention and control of the disease.【Method】The pathogenic bacteria were isolated from the tissues of diseased M.salmonoides, such as the liver, spleen, kidney, by using conventional bacterial isolation methods.After Gram staining, phosphotungstic acid staining, observation of morphology and staining characteristics of bacteria using optical microscope and transmission electron microscope was conducted.ATB Expression biochemical identification instrument and Biolog Microbes Automatic Identification System were used to identify the physiological and biochemical characteristics, and molecular biology identification was conducted based on 16S rRNA sequence and gyrB gene sequence.The artificial regression test and histopathology analysis were applied to further determine the pathogenicity of the bacteria and tissue damage, and its antibiotic susceptibility was tested by Kirby-Bauer disc diffusion.【Result】A dominant strain 180803 bj_jzl was isolated from the liver of diseased M.salmoides.It was gram-negative rod-shaped bacteria with periflagellum.The biochemical characteristics were identified as the same as Citrobacter freundii up to 99.4% by the Biolog Microbes Automatic Identification System.Phylogenetic tree based on homology of 16S rRNA and gyrB gene sequences revealed that isolated strain 180803bj_jzl clustered with the same branch of C.freundii.The 16S rRNA sequence similarity with the reference strains of C.freundii published by GenBank was more than 99.00%, and the similarity of the gyrB gene sequence was more than 91.00%.The cumulative mortality rate within 7 d of artificial recurrent infection was 60%, and its symptoms were basically the same as those of naturally-occurring M.salmoides.There were the erythrocytes congestion in the central vein and hepatic sinusoids of liver tissue, and a large number of vacuoles in the hepatocyte cytoplasm.The degeneration and necrosis were scattered in liver tissue.The white pulp and red pulp had unclear boundaries.And there were ferritinophilic hemoglobin centers in the splenic sinusoids and splenic small vessels.The epithelial cells of the gill lamellae were separated from the capillaries in a racket-shaped manner, and some epithelial cells of the gill lamellae were necrotic and detached, and only a small amount of gill lamellae skeleton remained.The drug sensitivity tests showed that 180803bj_jzl isolate was sensitive to 7 antimicrobial drugs such as fleroxacin, ofloxacin, macromycin, gentamicin, enrofloxacin and norfloxacin, and resistant to 15 antimicrobial drugs such as doxycycline, tetracycline, acetylcholine, roxithromycin, cefalexin and amoxicillin.【Conclusion】C.freundii can cause the body surface ulceration of M.salmoides and the histopathological damage to the liver, spleen and gills, and it has strong toxicity.The fishery antibiotics, such as neomycin and enrofloxacin, can be used to prevent and treat the disease in actual production,.

     

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