牦牛隐性乳房炎主要病原菌及耐药和毒力基因分布情况

Main Pathogenic Bacteria Causing Subclinical Mastitis in Yak and the Distribution of Antibiotic Resistance and Virulence Genes

为探明牦牛隐性乳房炎(SCM)主要病原菌及其耐药和毒力基因的分布情况,本研究自甘肃省甘南州夏季牧场收集无乳房炎临床症状牦牛乳样,通过兰州乳房炎试验(LMT)筛选SCM乳样,从中分离病原菌并纯化培养,利用16S rDNA鉴定主要病原菌,通过纸片扩散法判定其药物敏感性,并采用PCR方法对相关耐药及毒力基因进行检测。结果显示,共筛选出牦牛SCM乳样324份,检出率14.43%;主要病原菌为葡萄球菌属、埃希氏菌属和肠球菌属,其中葡萄球菌分离株对青霉素和四环素耐药率最高,分别为59.57%和47.52%;大肠埃希氏菌分离株对四环素和氨苄西林耐药率最高,分别为43.40%和20.75%;粪肠球菌分离株对四环素和红霉素耐药率最高,分别为25.00%和16.67%;59株耐青霉素金黄色葡萄球菌中共检出MRSA 12株,其中7株携带mecA基因,5株含mecC基因;四环素外排泵基因tetK、tetA携带率最高(85.45%、56.36%),核糖体保护基因tetM携带率最低(34.55%);毒力基因中,clfA、clfB、fib、coa基因检出率较高(87.64%、84.27%、83.15%、82.02%)。研究表明,牦牛SCM的主要病原菌为金黄色葡萄球菌和大肠埃希氏菌,均对青霉素类和四环素类抗生素耐药性较高,其中金黄色葡萄球菌的主要毒力因子为黏附因子和凝固酶。

To investigate the main pathogenic bacteria causing subclinical mastitis(SCM)in yak and the distribution of antibiotic resistance genes and virulence genes,yak milk samples were collected from the summer pasture in Gannan Tibetan Autonomous Prefecture,Gansu.The Lanzhou mastitis test(CMT)was carried out to grade SCM.16S rDNA was used to identify the main pathogenic bacteria isolated from SCM samples,antibiotic resistance was determined by Kirby-Bauer method,and related antibiotic resistance and virulence genes were detected by PCR.The results showed that a total of 324 SCM samples were found,with the detection rate of 14.43%;The main pathogenic bacteria were Staphylococcus,Escherichia and Enterococcus;Staphylococcus were highly resistant to penicillin and tetracycline with the rate of 59.57%and 47.52%respectively,43.40%and 20.75%E.coli were resistant to tetracycline and ampicillin respectively,as well as 25.00%and 16.67%E.faecalis were resistant to tetracycline and erythromycin respectively;12 strains of methicillin-resistant S.aureus(MRSA)were detected in 59 strains of penicillin-resistant S.aureus,inclouding 7 strains of mecA MRSA and 5 strains of mecC MRSA;For tetracycline-resistant S.aureus,efflux pumps genes tetK and tetA had the highest carrying rate(85.45%,56.36%)and ribosomal protection gene tetM had the lowest frequency(34.55%);Among virulence genes,the detection rates of clfA,clfB,fib and coa genes were higher than others with the ratio of 87.64%,84.27%,83.15%,82.02%,respectively.In conclusion,the main pathogenic bacteria causing SCM in yaks were S.aureus and E.coli which were both highly resistant to penicillins and tetracyclines,and the main virulence factors of S.aureus isolates were clumping factors and coagulase.