新疆阿克苏地区奶牛粪源大肠杆菌分型与耐药性分析

Genotyping and drug resistance analysis of Escherichia coli from cow feces in Aksu area,Xinjiang

参考USDA检测法,对新疆阿克苏地区101头荷斯坦奶牛的新鲜肛拭子样品选择性增菌,分别利用麦康凯和伊红美蓝琼脂培养基对大肠杆菌进行分离纯化;通过PCR方法鉴定,对这些分离株进行致病性大肠杆菌血清型鉴定、系统进化群及多位点序列分型(multilocus sequence typing,MLST)分析;利用goeBURST软件进行聚类,采用K-B纸片法进行分离株药物敏感试验,并对耐药基因进行PCR检测。结果显示:从101份奶牛肛拭子样品中分离获得51株大肠杆菌分离株,检出率为50.5%;鉴定出8种与致病性相关的血清型,检出率为62.75%,其中肠致病性大肠杆菌(EPEC)检出率最高(33.33%),优势血清型为O125:K70(B15)(15.69%);B1群所占比例最高,为68.63%;存在41个ST型,其中ST-154和ST-1727为优势ST型,均占5.88%;ST聚类分析显示有4个ST克隆群。51株大肠杆菌分离株对青霉素、头孢噻吩等表现出耐药性,耐药率均在60.0%以上;多重耐药率为78.43%;PCR检测出TEM、qnrS、oqxA、aac(6’)-Ib-cr、sul1、sul2及sul3这7种耐药基因。结果表明,新疆阿克苏地区奶牛粪源大肠杆菌多样性丰富,不仅存在多种致病性大肠杆菌血清型,而且大肠杆菌菌株之间还存在一定的克隆关系;另外大肠杆菌分离株多重耐药严重,提示规模化奶牛场应具备科学合理的粪便处理设施,来避免将携带耐药菌的粪便直接暴露于外界环境中,这可能引起这些耐药菌二次感染动物,致使耐药菌在动物间的不断传播。

Referring to USDA detection method,the fresh anal swab samples of 101 Holstein cows in Aksu region of Xinjiang were selectively enriched.E.coli was isolated and purified by MacConkey and Eosin Methylene Blue agar medium respectively.The isolates were identified by PCR.The pathogenic E.coli serotypes,phylogenetic groups and multilocus sequence typing(MLST)were analyzed.The drug sensitivity test of the isolates was carried out by K-B disk method,and the drug resistance genes were detected by PCR.The results showed that 51 strains of E.coli were isolated from 101 cow anal swab samples,and the detection rate was 50.5%;8 serotypes related to pathogenicity were identified,and the detection rate was 62.75%.The detection rate of enteropathogenic E.coli(EPEC)was the highest(33.33%),and the dominant serotypes were O125:K70(B15)(15.69%).The proportion of B1 group was the highest(68.63%).There were 41 ST types,of which ST-154 and ST-1727 were dominant ST types,accounting for 5.88%.ST cluster analysis showed that there were 4 ST clones.Fifty-one strains of E.coli showed resistance to penicillin and cephalothin,and the resistance rates were more than 60.0%.The rate of multidrug resistance was78.43%.Seven drug resistance genes including bla TEM,qnrs,oqxa,aac(6’)-Ibcr,sul1,sul2 and sul3 were detected by PCR.The results showed that the fecal E.coli diversity of dairy cows in Aksu region of Xinjiang was rich.There are not only a variety of pathogenic E.coli serotypes,but also a certain clonal relationship among E.coli strains.In addition,the multiple drug resistance of E.coli isolates is serious,suggesting that large-scale dairy farms should have scientific and reasonable fecal treatment facilities to avoid directly placing the feces carrying drug-resistant bacteria in the external environment,which may cause these drug-resistant bacteria to infect animals again,resulting in the continuous spread of drug-resistant bacteria among animals.