腹泻奶牛源大肠杆菌的分离鉴定及中药的体外抑菌效果  

Identification of Escherichia coli from dairy cattle and the antibacterial effect of two Chinese medicines in vitro

作　　者：吴道义 马金萍 王明进 王霞 曾继晶 周礼扬 罗耀 李坤 WU Daoyi;MA Jinping;WANG Mingjin;WANG Xia;ZENG Jijing;ZHOU Liyang;LUO Yao;LI Kun(Bijie Institute of Animal Husbandry and Veterinary Science,Bijie 551799,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]毕节市畜牧兽医科学研究所,贵州毕节551799 [2]南京农业大学动物医学院,江苏南京210095

出　　处：《畜牧与兽医》2024年第2期116-123,共8页Animal Husbandry & Veterinary Medicine

基　　金：威宁黄牛营养调控及粗饲料高效利用关键技术研究与示范贵州省科技支撑计划项目(黔科合支撑[2021]一般153);喀斯特山区粮改饲技术集成示范与产业化(黔科合服企[2021]12-03号);农业农村部国家肉牛牦牛产业技术体系毕节试验站(CARC-37);贵州省威宁县威宁黄牛繁育基地贵州省第六批人才基地项目(黔人领发[2018]3号)。

摘　　要：为了研究贵州毕节某奶牛场荷斯坦奶牛发生腹泻的原因,从该奶牛场采集50份新鲜粪便样本,通过平板划线法对大肠杆菌进行分离纯化和PCR鉴定,采用K-B纸片法进行药物敏感试验,并对多种耐药基因进行PCR检测。针对多重耐药菌,选取虎耳草和杜鹃花2种中草药进行体外抑菌效果研究。结果显示:从腹泻奶牛粪便中分离出31株细菌,其菌落形态和生化特性与大肠杆菌相符;16S rRNA基因测序比对发现该牛场分离的菌株与NCBI数据库中大肠杆菌参考菌株的同源性达到99.65%~99.86%,确定该奶牛场的腹泻病原为大肠杆菌;药敏试验发现该牛场来源的大肠杆菌对多种抗生素(强力霉素、红霉素、青霉素G和四环素)耐药,而对磺胺异恶唑、头孢曲松、环丙沙星、庆大霉素、多黏菌素B、磷霉素等10种抗生素敏感;PCR扩增相关耐药基因发现,导致该牛场腹泻的大肠杆菌携带2种β-内酰胺类(blaCTX-M、blaTEM),4种四环素类(TetD、TetB、TetK、TetD、TetA),2种氨基糖苷类(ant(3″)-Ia、rmtB)和3种喹诺酮类(qnrS、qepA、oqxA)耐药基因。体外抑菌研究发现,杜鹃花(3.125 mg/mL)的水提物有显著的抑菌效果。试验结果可为该奶牛场防治大肠杆菌性腹泻提供用药指导,同时为开发替抗中草药产品提供了依据。In order to identify the cause of diarrhea of Holstein cows in a dairy farm in Guizhou Province,this study collected fresh fecal samples(n=50)from the dairy farm,and the samples were separated and purified by plate scribing and PCR identification.Next,drug sensitivity test was conducted using the K-B paper method,and multiple drug resistance genes were detected by PCR.Finally,two Chinese herbs of rhododendron and Saxifraga stolonifera were used to determine the bacteriostatic effect against multiple resistant bacteria in vitro.The results showed that 31 strains of bacteria were isolated from the feces of the dairy cows suffering from diarrhea,the colony morphology and biochemical characteristics of the strains were consistent with those of Escherichia coli.Sequences alignment showed that the isolates from the dairy farm(16S rRNA gene)had 99.65%-99.86%homology with the reference Escherichia coli strains in the NCBI database,which confirmed that the diarrhea pathogen on the dairy farm was Escherichia coli.The results of drug sensitivity test showed that the isolated strains from this dairy farm were resistant to several antibiotic including doxycycline,erythromycin,penicillin G and tetracycline,but sensitive to sulfamethoxazole,ceftriaxone,ciprofloxacin,gentamycin,polymyxin B,fosfomycin,etc.PCR amplification of related drug-resistant genes found that the E.coli strains causing diarrhea in the cattle farm carried drug-resistant genes of twoβ-Lactam resistance genes of blaCTX-M and bla TEM,four tetracycline resistance genes of TetD,TetB,TetK,TetD and TetA,two aminoglycoside resistance genes of ant(3″)-Ia and rmtB,and three quinolone resistance genes of qnrS,qepA and oqxA.In vitro bacteriostatic study showed that water extract of rhododendron(3.125 mg/mL)had significant bacteriostatic effect.These results provided guidance for the dairy farm in prevention and treatment of coliform diarrhea,and might serve as the basis for development of anti-bacterium Chinese herbal medicine products.

关 键 词：腹泻 大肠杆菌 分离鉴定 耐药性 耐药基因 

分 类 号：S852[农业科学—基础兽医学]