五株华南虎源弗氏柠檬酸杆菌的分离鉴定及其生物学特性研究  

Isolation,identification and biological characteristics of five strains of Citrobacter fredii from South China tiger

作　　者：李雨琪 许静茹 朱智豪 蔡思思 周娴静 靳雨欣 刘璐 邓莹滋 陈芳婷 薛梦如 蓝佳萱 康娅莉 薛羽希 陈腾腾 林锡潘 林秋 谭聪灵 林开雄 范克伟 LI Yuqi;XU Jingru;ZHU Zhihao;CAI Sisi;ZHOU Xianjing;JIN Yuxin;LIU Lu;DENG Yingzi;CHEN Fangting;XUE Mengru;LAN Jiaxuan;KANG Yali;XUE Yuxi;CHEN Tengteng;LIN Xipan;LIN Qiu;TAN Congling;LIN Kaixiong;FAN Kewei(Fujian Provincial Key Laboratory for the Prevention and Control of Animal Infectious Diseases and Biotechnology,Engineering Research Center for the Prevention and Control of Animal Original Zoonosis of Fujian Province University,College of Life Sciences,Longyan University,Longyan 364000,China;College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China;Fujian Meihuashan Institute of South China Tiger Breeding,Longyan 364201,China;Chongqing Nanchuan District Animal Husbandry and Veterinary Fishery Center,Chongqing 408499,China)

机构地区：[1]龙岩学院生命科学学院福建省家畜传染病防治与生物技术重点实验室动物源性人兽共患病防控福建省高校工程研究中心,福建龙岩364000 [2]福建农林大学动物科学学院,福建福州350002 [3]福建梅花山华南虎繁育研究所,福建龙岩364201 [4]重庆市南川区畜牧兽医渔业中心,重庆408499

出　　处：《中国兽医科学》2024年第1期70-78,共9页Chinese Veterinary Science

基　　金：国家自然科学基金青年科学基金项目(31602048);福建省农业科技重大专项(2019NZ09005);福建省高校产学合作项目(2020N5012);福建省自然科学基金项目(2023J01990);国家级大学生创新创业训练计划项目(202011312012,202111312006,202311312009);新罗区产学研科技联合创新项目(2022XLXYZ009);动物源性人兽共患病防控福建省高校工程研究中心开放基金项目(2022K008)。

摘　　要：为了解福建省梅花山繁育研究所华南虎潜在病原菌的携带情况并分析相关生物学特性,本研究对2018—2021年从梅花山华南虎繁育研究所采集的104份华南虎组织、粪便、肛门拭纸、鼻拭纸进行细菌分离,并对分离菌进行生化鉴定、16S rRNA序列扩增、毒力基因检测、致病性试验、药敏试验。结果显示,通过细菌分离培养、形态学观察、生化试验、16S rRNA序列分析,确定从104份华南虎样本中分离到5株弗氏柠檬酸杆菌;5株分离菌16S rRNA序列的相似性为99.3%~99.7%,与其中18株不同来源的弗氏柠檬酸杆菌的相似性为98.3%~99.9%;遗传进化分析结果显示,5株分离菌与18株弗氏柠檬酸杆菌位于同一进化分支,亲缘关系较近;毒力基因检测结果显示,FJ/Tiger-1803、FJ/Tiger-1804、FJ/Tiger-1806、FJ/Tiger-1807株均携带ureD、ureE、ureABC基因,FJ/Tiger-1805株仅携带ureD、ureE基因;小鼠致病性试验结果显示,在相同浓度(1×10^(9) CFU/mL)下腹腔注射感染健康小鼠,FJ/Tiger-1803、FJ/Tiger-1806、FJ/Tiger-1804、FJ/Tiger-1807、FJ/Tiger-1805株对小鼠的致死率分别为100%、100%、80%、80%和0;药敏试验结果显示,分离菌株对14种抗生素具有不同的耐药性,其中对阿莫西林、青霉素G、万古霉素和红霉素的耐药性最强,且呈现多重耐药现象。综上,本研究从不同华南虎样本中分离获得了5株对小鼠具有较强致病性且耐药严重的华南虎源弗氏柠檬酸杆菌,临床上应引起重视。In order to understand the carrying status of potential pathogens of South China tiger in Meihuashan Breeding Institute of Fujian Province and analyze relevant biological characteristics,this study isolated bacteria from 104 tissues,feces,anus swab and nose swab of South China tiger collected from Meihuashan Breeding Institute of Fujian Province from 2018 to 2021.Biochemical identification,16S rRNA sequence amplification,virulence gene detection,pathogenicity test and drug susceptibility test were performed.The results showed that 5 strains of Citrobacter freundii were isolated from 104 South China tiger samples through isolation and culture,morphological observation,biochemical test and 16S rRNA sequence analysis.The sequence similarity of 16S rRNA among the five isolates was 99.3%—99.7%,and the similarity with the 18 strains of Citrobacter freundii from different sources was 98.3%—99.9%.Genetic analysis showed that the 5 isolates were closely related to the 18 strains of Citrobacter freundii in the same evolutionary branch.Virulence gene test results showed that FJ/Tiger-1803,FJ/Tiger-1804,FJ/Tiger-1806 and FJ/Tiger-1807 all carried genes ureD,ureE and ureABC;FJ/Tiger-1805 only carried genes ureD and ureE.The results of pathogenicity test in mice showed that healthy mice were infected by intrabitoneal injection at the same concentration(1×10^(9) CFU/mL).The mortality rates of FJ/Tiger-1803,FJ/Tiger-1806,FJ/Tiger-1804,FJ/Tiger-1807 and FJ/Tiger-1805 in mice were 100%,100%,80%,80%and 0,respectively.The results of drug sensitivity test showed that the isolates had different resistance to 14 kinds of antibiotics,among which amoxicillin,penicillin G,vancomycin and erythromycin were the most resistant,and showed multiple resistance.In conclusion,five strains of Citrobacter freundii with strong pathogenicity and severe drug resistance to mice were isolated from different samples of South China tiger in this study,which should be paid attention to clinically.

关 键 词：华南虎 弗氏柠檬酸杆菌 分离鉴定 毒力基因 耐药性 

分 类 号：S852.612[农业科学—基础兽医学]