中华竹鼠源致病性大肠杆菌分离鉴定、耐药性及毒力基因检测  

Isolation,Identification,Drug Sensitivity,and Virulence Gene Analysis of Pathogenic Escherichia coli from Bamboo Rats

作　　者：阎朝华 张旭[1,2] 陈国权 周碧君 王开功[1,2,3] 赵大杰 文明[1,2,3] 张件军 YAN Chaohua;ZHANG Xu;CHEN Guoquan;ZHOU Bijun;WANG Kaigong;ZHAO Dajie;WEN Ming;ZHANG Jianjun(Guizhou University Animals College of Science,Guiyang,550025,China;Institute of Animal Diseases,Guizhou University,Guiyang,550025,China;Guizhou Key Laboratory of Animal Diseases and Veterinary Public Health,Guiyang,550025,China)

机构地区：[1]贵州大学动物科学学院,贵阳550025 [2]贵州大学动物疫病研究所,贵阳550025 [3]贵州省动物疫病与兽医公共卫生重点实验室,贵阳550025

出　　处：《野生动物学报》2020年第3期705-712,共8页CHINESE JOURNAL OF WILDLIFE

基　　金：贵州省科技支撑计划(黔科合支撑[2019]2282号);贵州竹馏规模养殖场育繁关键技术研究(黔科合支撑[2019]2282号)。

摘　　要：试验旨在查明贵州某竹鼠养殖场竹鼠死亡原因。通过细菌分离鉴定、形态学观察、生理生化特性定、16S rDNA基因序列分析及系统进化树构建等方法对分离菌种属进行确定,纸片扩散法进行药敏试验,PCR方法检测耐药基因,人工感染小鼠试验探究分离菌的致病性和PCR检测毒力基因。结果显示:经革兰氏染色镜检分离菌为革兰阴性短杆菌,其16S rDNA基因序列经测序及BLAST比对,显示其与大肠埃希氏菌序列相似度达98.2%—100%。在系统发育树上与大肠杆菌聚为一枝。结合生理生化鉴定结果确定分离菌为大肠埃希氏菌。药敏试验结果显示,分离菌对氟苯尼考、头孢曲松、头孢氨苄等药物敏感、对四环素、多西环素、青霉素等药物表现耐药,4种耐药基因检测结果显示Intl 1(146 bp)、tetA(831 bp)2种耐药基因,与药敏表型相符。人工感染试验结果显示,该菌对小鼠有致病性。8种毒力基因检测分离菌携带papA(761 bp)、UidA(264 bp)、afa(251 bp)3种毒力因子。上述结果表明本实验分离到一株对四环素类耐药的竹鼠源大肠埃希氏菌,为该竹鼠养殖场的竹鼠疾病防治与合理用药提供了科学依据。We aimed to identify the cause of bamboo rat death in a bamboo rat farm in Guizhou.The bacterial species were identified by bacterial isolation,morphological observation,determination of physiological and biochemical characteristics,16S rDNA gene sequence analysis,and phylogenetic tree construction.The paper diffusion method was used for drug sensitivity tests and the PCR method was used to detect drug-resistant genes.Pathogenicity was determined by artificially infecting mice and virulence genes were tested by PCR analysis.The isolate was a Gram-negative Brevibacterium.The 16S rDNA gene was sequenced and compared with BLAST,which showed sequence similarity to Escherichia coli of 98.2%to 100%,and clustered with Escherichia coli on a phylogenetic tree.Combined with the results of physiological and biochemical identification,the isolate was identified as Escherichia coli.The drug sensitivity test showed that the isolates were sensitive to florfenicol,ceftriaxone,and cephalexin,and were resistant to tetracycline,doxycycline,and penicillin.Test of the four resistance genes found Intl 1(146 bp)and tetA(831 bp)resistance genes,which are consistent with the drug-susceptible phenotype.The results of artificial infection tests showed that the bacteria were pathogenic to mice.Detection of eight virulence genes indicated that the isolates carried three virulence factors,viz papA(761 bp),UidA(264 bp),and afa(251 bp).We isolated a strain of Escherichia coli derived from bamboo rat that is resistant to tetracycline.This provides a scientific basis for disease prevention and rational drug treatment for bamboo rat at this bamboo rat farm.

关 键 词：中华竹鼠 大肠埃希氏菌 分离鉴定 致病力分析 

分 类 号：S852.61[农业科学—基础兽医学]