广西地区美国牛蛙源弗氏柠檬酸杆菌的分离鉴定、耐药性及组织病理

Isolation,identification,drug resistance and histopathology of Citrobacter freundii from bullfrog(Rana catesbeiana)in Guangxi

为了探明2022年5月在广西南宁某养殖基地引起美国牛蛙暴发疾病的原因,本实验从患病美国牛蛙的肝脏、脾脏、肾脏和肠道中分离出优势菌,采用形态学观察、生理生化实验、16S rRNA基因序列测定及系统发育学分析进行鉴定。为确定该菌株的致病机理,对该菌株进行了人工感染实验、组织病理学观察、毒力基因检测、药物敏感性实验以及耐药基因检测。结果显示,在患病濒死美国牛蛙的肝脏、脾脏、肾脏以及肠道均分离到单一的优势菌株,从肠道分离得到的一株优势菌被命名为NFCF-02,并经鉴定为弗氏柠檬酸杆菌。该菌株携带viaB、ompX、ureE、ureD、ureG和ureF等6种毒力基因,对美国牛蛙的半数致死浓度为3.12×10^(6)CFU/mL,发病临床症状包括肝脏变黄、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充血。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。药敏实验结果显示,分离菌株NFCF-02对新霉素、多黏菌素B、头孢拉定、多西环素等4种抗菌药物敏感,对林可霉素等14种抗菌药物已产生耐药性,携带gyrA、Sul1等7种耐药基因。研究表明,弗氏柠檬酸杆菌对美国牛蛙具有高致病性,可引起美国牛蛙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。本研究首次从组织病理学分析和毒力因子携带情况的角度确定了弗氏柠檬酸杆菌对美国牛蛙的致病力,为养殖美国牛蛙中弗氏柠檬酸杆菌病的诊断和药物防控提供了参考依据。

This study was conducted to investigate the cause of a disease outbreak in bullfrogs(Rana catesbeiana)at a breeding base in Nanning,Guangxi in May 2022.The dominant bacterial colonies were isolated from the liver,spleen,kidney and intestine tissues of diseased R.catesbeiana,and identified using morphological observation,physiological and biochemical experiments,16S rRNA sequencing and phylogenetic analysis.To determine the pathogenic mechanism of these dominant bacteria,artificial infection experiments,histopathological observations,drug sensitivity experiments,virulence gene and resistance gene testing were conducted.The results showed that a single dominant strain was isolated from the liver,spleen,kidneys,and intestines of the critically ill R.catesbeiana.The dominant strain isolated from the intestine was named NFCF-02 and identified as Citrobacter freundii.This strain carried six virulence genes,viaB,ompX,ureE,ureD,ureG,and ureF,with a half-lethal concentration of 3.12×10^(6) CFU/mL.The R.catesbeiana infected with C.freundii presented the symptoms of liver changing to yellow and necrosis,kidney congestion and redness,blood streaks on the surface of the stomach,and blood pus in intestines.Histopathological observation revealed degeneration and necrosis of liver cells,with disordered arrangement of liver cords.Meanwhile,swelling and necrosis of renal tubular epithelial cells,severe tubular collapse and necrosis in severe areas,and splenic central vein dilation and congestion with an increase in hemosiderin and pigment cells were also observed.Besides,intestinal mucosal detachment,necrosis and detachment of intestinal epithelial cells,and necrosis of goblet cells were also the symptoms in R.catesbeiana infected with C.freundii.C.freundii was sensitive to neomycin,polymyxin B,cefradine,and doxycycline,while it has developed resistance to 14 antibiotics,including lincomycin,and carries 7 resistance genes such as gyrA and Sul1.In summary,C.freundii has high pathogenicity to bullfrogs,causing pathological damage to multiple organ tissues and even death of the R.catesbeiana.This study is the first to determine the pathogenicity of C.freundii to R.catesbeiana from the perspective of histopathological analysis and virulence factor carrying,which can provide reference for the diagnosis and drug control of C.freundii disease in farmed R.catesbeiana.