山东地区乳房炎牛奶中大肠杆菌的分离鉴定及耐药性分析  

Isolation,Identification and Drug Resistance Analysis of E.coli from Mastitis Milk in Shandong Province

作　　者：苑晓萌 赵效南 李璐璐[2] 张庆[2] 胡明[2] 骆延波[2] 张印[2] 齐静[2] 刘玉庆 YUAN Xiaomeng;ZHAO Xiaonan;LI Lulu;ZHANG Qing;HU Ming;LUO Yanbo;ZHANG Yin;QI Jing;LIU Yuqing(College of Life Science, Shandong Normal University, Jinan 250014,China;Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan 250100, China)

机构地区：[1]山东师范大学生命科学学院,济南250014 [2]山东省农业科学院畜牧兽医研究所,济南250100

出　　处：《中国畜牧兽医》2021年第1期312-323,共12页China Animal Husbandry & Veterinary Medicine

基　　金：山东省农业科学院高层次人才及创新团队引进项目;2019年度国家重大科技专项子课题“病原菌耐药监测技术集成与示范应用研究”(2018ZX10733402-013)。

摘　　要：试验旨在了解山东地区乳房炎牛奶中大肠杆菌的污染状况及耐药情况。选择山东省3个地区的规模化奶牛场共采集227份牛奶样品,采用细菌学方法对大肠杆菌进行分离鉴定,用微量肉汤稀释法检测分离菌对11种常规抗菌药物的敏感性,采用PCR方法对常见的13种耐药基因、8种毒力基因和Ⅰ类整合子基因盒结构进行分析。结果显示,从227份牛奶样品中共分离出71株大肠杆菌;大肠杆菌对1种及1种以上抗菌药耐药的菌株达到77.5%,多重耐药率为15.5%,其中对多黏菌素耐药率为52.2%,对阿莫西林-克拉维酸耐药率为39.4%,而所有菌株均对新霉素表现为敏感。PCR检测耐药基因、毒力基因和Ⅰ类整合子结果显示,β-内酰胺类耐药基因中blaTEM基因携带率为100%,其中全部为blaTEM-1基因,blaCTX-M基因携带率为32.4%,其中主要为blaCTX-M-15基因,没有检测到blaSHV、blaOXA基因;多黏菌素的耐药基因mcr-1携带率为29.6%;喹诺酮类耐药基因中aac(6’)-Ⅰb-cr基因携带率为29.6%,qnrB基因携带率为20.8%,没有检测到qnrA和qnrC耐药基因;对8种毒力基因检测分析结果显示,仅Hly毒力基因没有被检出,Ecs3703、Irp2基因的检出率较高,分别为90.1%和63.4%,71株大肠杆菌中共有11株携带Ⅰ类整合子,检出率为15.5%,11株大肠杆菌携带6种耐药基因盒结构,最主要的耐药基因盒排列为dfr17-aadA5。本研究结果表明,山东地区乳房炎牛奶中大肠杆菌的耐药现象严重,携带毒力基因Ecs3703、Irp2的大肠杆菌可能是引起奶牛乳房炎的致病菌,Ⅰ类整合子的检测在细菌耐药性与基因携带率方面发挥着关键作用,可为临床预防和治疗奶牛乳房炎大肠杆菌病提供理论依据。This experiment was aimed to estimate the contamination and drug resistance of E.coli in mastitis milk in Shandong.The large-scale dairy farms located in three regions of Shandong province were selected as the sampling sites for this study.A total of 227 milk samples were collected,the bacteriological method was used to isolate and identify E.coli,the micro-broth dilution method was used to detect 11 conventional antibacterial drugs,the 13 common drug resistance genes,8 virulence genes and classⅠintegron were screened by PCR.The results showed that a total of 71 E.coli strains were isolated from 227 milk samples.Antibiotic susceptibility testing of E.coli strains showed that strains which were resistant to one or more antibacterial drugs reached 77.5%,the multi-drug resistance rate was 15.5%,of which the resistance rate to polymyxin was 52.2%,and the amoxicillin-clavulanic acid resistance rate was 39.4%.However,all strains were sensitive to neomyci.Of the 71 E.coli strains,all of them carried blaTEM gene,and they were all blaTEM-1 gene,32.4%of the strains carried blaCTX-M gene,and most of them were blaCTX-M-15 gene.In addition,blaSHV and blaOXA genes were not detected;29.6%of the isolates carried mobile colistin resistance genes mcr-1;29.6%of the strains carried aac(6’)-Ⅰb-cr gene,and 20.8%carried qnrB gene,qnrA and qnrC genes were not detected.For the analysis of 8 virulence genes,only Hly gene were not detected,and the detection rates of Ecs3703 and Irp2 genes were higher,which were 90.1%and 63.4%,respectively.ClassⅠintegrons were detected in 11 strains,with a rate of 15.5%,and carried 6 types of gene cassettes,the prevalence of the gene cassette was dfr17-aadA5.The results indicated that the drug resistance of E.coli in mastitis milk in Shandong was serious.E.coli that carrying virulence genes Ecs3703 and Irp2 might be the pathogenic bacteria causing mastitis in dairy cow.The detection of classⅠintegrin played a key role in bacterial resistance and gene carrying rate.This study could provide a theore

关 键 词：大肠杆菌 耐药性 耐药基因 毒力基因 Ⅰ类整合子 

分 类 号：S852.612[农业科学—基础兽医学]