广西牛蛙源弗氏柠檬酸杆菌的分离鉴定、耐药性及组织病理分析

【目的】探明引起广西南宁市某养殖基地牛蛙（Rana catesbeiana）爆发疾病的病原及组织病理特性，为养殖牛蛙感染弗氏柠檬酸杆菌的诊断和药物防治提供参考依据。【方法】本研究从患病牛蛙肝脏，脾脏、肾脏、肠道中分离优势菌，采用形态学观察、生理生化试验、16S rRNA基因序列测定、系统发育学分析进行鉴定。为确定该菌株的致病机理，我们对该菌株进行了人工感染试验、组织病理分析、毒力基因检测、药物敏感性试验及耐药基因检测。【结果】从患病牛蛙的肠道中分离获得一株优势菌株(NFCF-02)，经形态学观察、生理生化特性鉴定及16S rRNA序列分析，最终鉴定为弗氏柠檬酸杆菌。该菌株携带cfa、ompX、viaB等6种毒力基因，对牛蛙的半数致死浓度为3.09×106 CFU/mL，且临床症状与自然发病牛蛙的症状基本相同，如肝脏变黄、花肝、肝脏坏死；肾脏充血发红、胃表面有血丝以及肠道呈红色和血脓充塞肠道的症状。组织病理学观察发现其肝细胞变性坏死，肝索排列紊乱；肾小管上皮细胞肿胀坏死、严重区域肾小管崩解、坏死；脾脏中央静脉扩张淤血，含铁血黄素、色素细胞增多；肠黏膜脱落，肠上皮细胞坏死脱落，杯状细胞坏死。药敏试验结果表明分离菌株NFCF-02对新霉素、多粘菌素B、头孢拉定、多西环素4种抗菌药物敏感，对林可霉素和卡那霉素等14种抗菌药物已产生耐药性，携带gyrA、Sul1等7种耐药基因，表明该菌株已产生多重耐药性。【结论】弗氏柠檬酸杆菌对牛蛙具有高致病力，可引起肝脏和肠道等多器官组织的病理损伤最终引发机体的损伤甚至死亡。

Isolation, identification, drug resistance and histopathological analysis of Citrobacter freundii from bull frog, Rana catesbeiana

【Objective】The present study was conducted to investigate the bacterial pathogen that caused outbreak disease of bullfrogs (Rana catesbeiana) farmed in Nanning, Guangxi, and determined its drug resistance and histopathological characterization in order to provided a reference for clinical diagnosis and effective control of Citrobacter freundii infection in bull frogs aquaculture.【Methods】The dominant bacterial colonies were isolated from the liver, spleen, kidney and intestine tissues of naturally infected bullfrogs in this study. After being purified, the pathogen was identified based on morphological observation, biochemical characteristics, 16S rRNA sequence analysis together with phylogenetic analysis. Then we performed artificial infection test, histopathological analysis, virulence gene test, drug sensitivity test and drug resistance gene test to determined the pathogenesis of the strain NFCF-02. 【Results】A dominant strain (NFCF-02) was isolated from the intestine tissue of the diseased bull frogs.The pathogenic bacteria was finally identified as Citrobacter freundii after morphological observation. biochemical characteristics test, 16S rRNA sequence analysis together with phylogenetic analysis. The results of drug resistance gene testing indicate that strain NFCF-02 carried six virulence genes including cfa, ompX and viaB, etc, and the median lethal concentration of bullfrosg in artificial regression infection test is 3.09 × 106 CFU/mL. The infection symptoms of the bull frogs by artificial infection germs appeared the same as the natural infection, such as the liver is yellowish in color and had a mottled surface and had pathological changes of liver necrosis; symptoms of kidney was severity engorged. As for stomach, there are blood strands on the surface of it, as well as the intestinal tract is inflamed and congested with blood and pus. Histopathological observation revealed degeneration and necrosis of hepatocytes and disordered arrangement of hepatic cords; Swelling and necrosis of renal tubular epithelial cells, collapse and necrosis of renal tubules in severe areas; the central vein of the spleen is dilated and congested, hemosiderin increases, and there is a tendency for pigment cells to increase; the main histopathological features of the intestinal tract include intestinal mucosal exfoliation, necrosis and exfoliation of intestinal epithelial cells, and necrosis of goblet cells. The results of drug susceptibility test showed that the isolate NFCF-02 was sensitive to four antimicrobial drugs including neomycin, polymyxin B, cefradine and doxycycline, and has been developed resistance to 14 antimicrobial agents including lincomycin and kanamycin.Finally, we tested for resistance genes in strain NFCF-02, which was found to carried seven resistance genes including gyrA, sul1, etc, it indicated that this strain had developed multiple resistance. 【Conclusion】Citrobacter freundii has high pathogenicity to bullfrog, which can cause pathological damage to the liver and intestinal organ tissues and eventually lead to body injury and even death.