【摘要】 为了解湖北省部分地区奶牛场凝固酶阴性葡萄球菌(coagulase negative Staphylococcus,CNS)的流行情况和耐药特点，试验于2019—2020年采集武汉市江夏区(A场)、黄冈市溪水县(B场)、黄冈市(C场)、武汉市东西湖区(D场)和宜昌市(E场)的规模化奶牛场的健康奶牛、患亚临床型乳房炎(subclinical mastitis, SCM)奶牛和患临床型乳房炎(clinical mastitis, CM)奶牛的乳头乳、乳头皮肤拭子样本及环境样本共计887份，进行葡萄球菌的分离与纯化，对分离到的葡萄球菌进行触媒试验，并选取触酶阳性的菌株进行凝固酶试验，再对筛选到的CNS进行16S rDNA基因的PCR扩增、测序及菌种鉴定，统计各类型样本中不同种类CNS的分离率、各地区奶牛养殖场CNS的分离率，最后对CNS分离株进行耐药性分析。结果表明：从887份样本中共分离到234株葡萄球菌，这些分离株均为触酶阳性菌株，其中有134株菌株为CNS。134株CNS中，共鉴定到16种葡萄球菌，包括45株鸡葡萄球菌(占比为33.6%)、13株人葡萄球菌(占比为9.7%)、11株腐生葡萄球菌(占比为8.2%)、17株产色葡萄球菌(占比为12.67%)、12株未分类葡萄球菌(占比为9.0%)、10株表皮葡萄球菌(占比为7.5%)、5株模仿葡萄球菌(占比为3.7%)、5株木糖葡萄球菌(占比为3.7%)、3株阿尔莱特葡萄球菌(占比为2.2%)、2株克氏葡萄球菌(占比为1.5%)、4株路邓葡萄球菌(占比为3.0%)、2株溶血葡萄球菌(占比为1.5%)、2株肉质葡萄球菌(占比为1.5%)、1株伪中间型葡萄球菌(占比为0.8%)、1株琥珀葡萄球菌(占比为0.8%)和1株猪葡萄球菌(占比为0.8%)。鸡葡萄球菌在健康奶牛各类型样本、患SCM奶牛的乳头乳和乳头皮肤拭子样本及环境样本中分离率均最高。患CM奶牛的乳头乳样本中人葡萄球菌、模仿葡萄球菌和阿尔莱特葡萄球菌的分离率最高(1.4%),乳头皮肤拭子样本中鸡葡萄球菌、人葡萄球菌和模仿葡萄球菌的分离率最高(2.6%),肛门拭子样本中鸡葡萄球菌、产色葡萄球菌和人葡萄球菌的分离率最高(3.8%);患SCM奶牛的肛门拭子样本中鸡葡萄球菌、产色葡萄球菌和腐生葡萄球菌的分离率最高(3.0%)。CNS在E场的分离率最高(30.7%),然后依次为A场(13.8%)、C场(12.1%)、B场(8.9%)和D场(5.6%)。CNS分离株对磺胺异口恶唑、复方新诺明、头孢噻呋、恩诺沙星、氧氟沙星、青霉素、阿莫西林/克拉维酸、头孢西丁、克林霉素的耐药率较高，分别为100%、100%、94.8%、99.3%、63.4%、70.1%、53.0%、75.4%和51.5%;对万古霉素、替米考星、庆大霉素、氟苯尼考、多西环素、利奈唑胺、苯唑西林的耐药率较低，分别为7.5%、12.7%、7.5%、15.7%、17.9%、32.8%和36.6%。134株CNS均为多重耐药菌株，包括97种耐药表型。耐药重数最低为3重，占比为4.5%(6/134);耐药重数最高为11重，占比为2.2%(3/134);5重耐药菌株占比最大，为19.4%(26/134)。青霉素-头孢西丁-头孢噻呋-泰妙菌素-磺胺异口恶唑-复方新诺明-恩诺沙星和青霉素-利奈唑胺-头孢噻呋-泰妙菌素-磺胺异口恶唑-复方新诺明-恩诺沙星-氧氟沙星为优势耐药表型。说明湖北省部分地区奶牛场CNS普遍存在，且种类较多，鸡葡萄球菌占比较高；分离到的CNS均为多重耐药菌株，对大多数抗生素耐药率较高，对万古霉素和庆大霉素的耐药率较低。更多还原

【Abstract】 In order to understand the prevalence and drug resistance characteristics of coagulase-negative Staphylococcus(CNS) on dairy farms in some regions of Hubei Province, a total of 887 samples of teat milk, teat skin swabs, and environmental samples were collected from healthy cows, cows with subclinical mastitis(SCM), and cows with clinical mastitis(CM) in large-scale dairy farms in Jiangxia District, Wuhan City(farm A), Qishui County, Huanggang City(farm B), Huanggang City(farm C), Dongxihu District, Wuhan City(farm D), and Yichang City(farm E). These samples were used for the isolation and purification of Staphylococcus from 2019 to 2020. The catalase test was carried out on the isolated Staphylococcus, and the strains with positive catalase test results were selected for the coagulase test. Then, the 16S rDNA gene of the screened CNS was amplified by PCR, sequenced, and identified. The isolation of different types of CNS in various types of samples and the isolation of CNS in dairy farms in different regions were statistically analyzed. Finally, the drug resistance of the CNS isolates was analyzed.The results showed that a total of 234 strains of Staphylococcus were isolated from the 887 samples. All 234 Staphylococcus isolates were catalase-positive, among which 134 strains were CNS. Among the 134 CNS isolates, 16 species of Staphylococcus were identified, including 45 strains of Staphylococcus gallinarum(accounting for 33.6%), 13 strains of Staphylococcus hominis(accounting for 9.7%), 11 strains of Staphylococcus saprophyticus(accounting for 8.2%), 17 strains of Staphylococcus chromogenes(accounting for 12.7%), 12 strains identified only as unclassified Staphylococcus spp.(accounting for 9.0%), 10 strains of Staphylococcus epidermidis(accounting for 7.5%), 5 strains of Staphylococcus simulans(accounting for 3.7%), 5 strains of Staphylococcus xylosus(accounting for 3.7%), 3 strains of Staphylococcus arlettae(accounting for 2.2%), 2 strains of Staphylococcus kloosii(accounting for 1.5%), 4 strains of Staphylococcus lugdunensis(accounting for 3.0%), 2 strains of Staphylococcus haemolyticus(accounting for 1.5%), 2 strains of Staphylococcus carnosus(accounting for 1.5%),1 strain of Staphylococcus pseudintermedius(accounting for 0.8%), and 1 strain of Staphylococcus hyicus(accounting for 0.8%). The isolation rates of Staphylococcus gallinarum were the highest in various types of samples from healthy dairy cows, in nipple milk and nipple skin swab samples from cows with SCM, and in environmental samples. The isolation rates of Staphylococcus hominis, Staphylococcus xylosus and Staphylococcus arlettae in nipple milk samples from cows with CM were the highest(1.4%). The isolation rates of Staphylococcus gallinarum, Staphylococcus hominis and Staphylococcus xylosus in the nipple skin swab samples of cows with CM were the highest(2.6%). The isolation rates of Staphylococcus gallinarum, Staphylococcus chromogenes and Staphylococcus hominis in the anal swab samples of cows with CM were the highest(3.8%). The isolation rates of Staphylococcus gallinarum,Staphylococcus chromogenes and Staphylococcus saprophyticus in the anal swab samples of cows with SCM were the highest(3.0%). The isolation rate of CNS was the highest in farm E(30.7%), followed by farm A(13.8%), farm C(12.1%), farm B(8.9%) and farm D(5.6%). The CNS isolates showed high resistance to sulfisoxazole, cotrimoxazole, ceftiofur, enrofloxacin, ofloxacin, penicillin, amoxicillin/clavulanic acid, cefoxitin, and clindamycin, with resistance rates of 100%, 100%, 94.8%, 99.3%, 63.4%, 70.1%, 53.0%, 75.4%, and 51.5%, respectively. They showed low resistance to vancomycin, tilmicosin, gentamicin, florfenicol, doxycycline, linezolid, and oxacillin, with resistance rates of 7.5%, 12.7%, 7.5%, 15.7%, 17.9%, 32.8%, and 36.6%, respectively.A total of 134 CNS strains were multi-drug resistant, with a multi-drug resistance rate of 100%, including 97 different drug resistance phenotypes. The minimum number of drug resistances was 3, accounting for 4.5%(6/134); the maximum number of drug resistances was 11, accounting for 2.2%(3/134); the proportion of 5-drug resistance was the largest, at 19.4%(26/134). Penicillin-cefoxitin-ceftiofur-tiamulin-sulfamethoxazole-cotrimoxazole-enrofloxacin and penicillin-linezolid-ceftiofur-tiamulin-sulfamethoxazole-cotrimoxazole-enrofloxacin-ofloxacin were the dominant drug resistance phenotypes. It indicated that CNS was prevalent in some dairy farms in Hubei, with a large variety; the proportion of Staphylococcus gallinarum was relatively high, and all the isolated CNS strains were multi-drug resistant, with a relatively high resistance rate to most antibiotics and a relatively low resistance rate to VAN and GEN.更多还原