青藏高原4省牦牛源肠球菌分离鉴定及其毒力耐药基因与生物被膜检测  

Isolation and identification of yak-derived Enterococcus from four provinces of Tibetan plateau and detection of virulence resistance genes and biofilms

作　　者：辛娇娇 高桂琴 赵小龙 娄永志 李晶 常攀 宋仁德 孔祥颖 石红梅 罗晓林[8] 格桑卓玛 索朗斯珠 贡嘎 XIN Jiaojiao;GAO Guiqin;ZHAO Xiaolong;LOU Yongzhi;LI Jing;CHANG Pan;SONG Rende;KONG Xiangying;SHI Hongmei;LUO Xiaolin;GESANG Zhuoma;SUOLANG Sizhu;GONG Ga(Collge of Animal Science,Xizang Agriculture Animal Husbandry University,Linzhi,Xizang 860000,China;Baiyin District Agricultural Com prehensive Administrative Law Enforcement Team,Baiyin,Gansu 730900,China;Changdu Animal Husbandry Station,Changdu,Xizang 854000,China;Animal Epidemic Prevention and Control Center of Tibet Autonomous Region,Lasa 850000,China;Livestock and Veterinary Workstation,Yushu Tibetan Autonomous Prefecture,Yushu,Qinghai 815400,China;Animal Husbandry and Scientific Research of Qinghai Province,Haibei,Qinghai 810200,China;Gannan Animal Husbandry Workstation,Gannan,Xizang 747000,China;Sichuan Grassland Science Research Institute,Chengdu 610097,China)

机构地区：[1]西藏农牧学院动物科学学院,西藏林芝860000 [2]甘肃省白银市白银区农业综合行政执法队,甘肃白银730900 [3]西藏自治区昌都市畜牧总站,西藏昌都854000 [4]西藏自治区动物疫病预防控制中心,西藏拉萨850000 [5]青海玉树藏族自治州畜牧兽医工作站,青海玉树815400 [6]青海省海北藏族自治州畜牧兽医科学研究所,青海海北810200 [7]甘南州畜牧工作站,西藏甘南747000 [8]四川省草原科学研究院,四川成都610097

出　　处：《中国兽医学报》2025年第2期289-297,共9页Chinese Journal of Veterinary Science

基　　金：国家现代农业产业技术体系资助项目(CARS-37);西藏牦牛产业技术体系资助项目(XZARS-MN-2023-04);国家重点研发计划资助项目(2023YFD1801301)。

摘　　要：为了解青藏高原牦牛源肠球菌流行情况及其耐药性、毒力基因特点、生物被膜黏附能力情况,从青藏高原4省区(西藏自治区、四川省、甘肃省、青海省)采集新鲜牦牛粪样346份,奶样311份,共计657份,对其进行细菌分离鉴定,16S rDNA基因检测并构建系统进化树。使用PCR方法对分离菌株进行耐药、毒力基因检测,利用18种抗菌药物进行药敏试验;采用改良半定量结晶紫染色法确定分离菌株生物被膜黏附能力。结果显示,肠球菌总分离率为32.27%,其中四川省最高,达60.23%,其次甘肃省、青海省和西藏自治区,分别为42.70%、23.47%和18.31%。从样品种类来看,粪样分离率为36.71%,奶样为27.33%。经PCR扩增得到约1 400 bp目的条带,选取5株菌株进化分析单独聚成一簇。212株分离菌对克林霉素、庆大霉素、利奈唑胺、利福平、链霉素产生较高耐药性,并存在多种耐药现象,占60.85%。12种耐药基因只有5种被检测出,分别为erm(B)、tet(L)、tet(O)、tet(M)和ant(6)-Ia。13种毒力基因均有在肠球菌中检测出,其中cpd检出率为95.75%,gelE为91.98%,efaA为86.79%,asal为86.32%,其余的在5.19%~55.66%之间。而奶源肠球菌中暂未检测出fsr毒力基因。分离菌中强黏附能力占3.30%,弱黏附能力占48.58%,无黏附能力占48.11%。综上研究揭示了牦牛源肠球菌流行情况、耐药基因和毒力基因携带情况、生物被膜表型相关性,为掌握青藏高原牦牛源肠球菌研究数据奠定了基础。This study aims to understand the epidemic distribution characteristics,antimicrobial resistance,virulence genes,and biofilm adhesion ability of Enterococcus in yaks on the Tibetan plateau.Three hundred and forty-six fresh yak fecal samples and 311 milk samples were collected from four provinces on the Tibetan plateau(Xizang,Sichuan,Gansu,Qinghai),totaling 657 sam-ples.Bacterial isolation and identification were conducted,followed by 16S rDNA gene detection and the construction of a systematic evolutionary tree.The isolated strains were tested for antimi-crobial resistance and virulence genes using PCR,and sensitivity tests were performed using 18 types of antibiotics.The biofilm adhesion ability of the isolated bacteria was determined using an improved semi-quantitative crystal violet staining method.The results showed that the total isola-tion rate of Enterococcus was 32.27%,with Sichuan having the highest at 60.23%,followed by Gansu,Qinghai,and Tibet autonomous region at 42.70%,23.47%,and 18.31%respectively.In terms of sample types,the isolation rate in fecal samples was 36.71%,and in milk samples,it was 27.33%.Through PCR amplification,bands of approximately 1400 bp were obtained,and 5 strains were selected for evolutionary analysis,forming a separate cluster.Among the 212 isolated strains,a high resistance to clindamycin,quinupristin-dalfopristin,linezolid,levofloxacin,and erythromycin was observed,with various resistance phenomena,accounting for 60.85%.Only 5 out of 12 resist-ant genes were detected,namely erm(B),tet(L),tet(O),tet(M),and ant(6)-Ia.All 13 virulence genes were detected in Enterococcus,with detection rates in the range of 5.19%to 95.76%,where cpd was 95.75%,gelE was 91.98%,efaA was 86.79%,asal was 86.32%,and the rest ranged from 5.19%to 55.66%.The fsr virulence gene was not detected in Enterococcus from milk sources.Among the isolated strains,3.30%showed medium adhesive ability,48.58%showed weak adhesive ability,and 48.11%showed no adhesive ability.The above research revealed the preva-lence o

关 键 词：青藏高原 牦牛 肠球菌 毒力基因 耐药性 生物被膜 

分 类 号：S852.61[农业科学—基础兽医学]