牛粪源金黄色葡萄球菌的分离鉴定及体外抑菌试验

为了掌握呼伦贝尔奶牛养殖地区奶牛肠道金黄色葡萄球菌的分布及危害,试验对该地区不同奶牛户共206头奶牛的新鲜粪便进行了致病性金黄色葡萄球菌的分离鉴定及药敏试验.结果表明:共筛选出34株疑似金黄色葡萄球菌,通过生化试验、血浆凝固酶试验和动物致病性试验,鉴定出致病性金黄色葡萄球菌7株,致病菌检出率为20.59%(7/34);...     

牛源金黄色葡萄球菌的分离培养及鉴定

本研究对新疆石河子奶牛场的23份奶样进行了金黄色葡萄球菌的分离,并通过触酶试验、凝固酶试验以及API对其进行了鉴定,23份奶样中检出金黄色葡萄球菌3份,阳性率为13.04%。药物敏感试验表明,分离的菌株对青霉素、庆大霉素、氨卡青霉素、林可霉素和多黏菌素B产生耐药性。     

牛奶中金黄色葡萄球菌的分离与鉴定

对西宁市某奶牛场７３份奶样进行了金黄色葡萄球菌的分离,并通过血浆凝固酶试验、ＤＮＡ酶试验和甘露醇发酵试验对其进行了鉴定,检出阳性样品２０份,阳性率为２７．４％（２０／７３）。     

奶牛乳房炎金黄色葡萄球菌的分离鉴定及药敏试验

[目的] 了解泰州市某奶牛场乳房炎中金黄色葡萄球菌（Staphylococcus aureus）感染情况及其对常用药物的敏感性。[方法] 试验选用Baird-Parker培养基分离、生化鉴定、PCR检测等方法,对采集的9份奶牛乳房炎奶样进行分离鉴定和药敏试验。[结果] 共分离到6株葡萄球菌,进一步通过凝固酶试验和16S rDNA测序等方法鉴定其中3株为金黄色葡萄球菌。药敏试验结果显示,3株金黄色葡萄球菌对青霉素G均耐药,2株对四环素耐药,2株对红霉素和苯唑西林表现为中介。[结论] 该次分离的金黄色葡萄球菌主要对青霉素G和四环素耐药,对其他常用抗生素表现为敏感。     

奶牛乳房炎金黄色葡萄球菌的分离鉴定

为检测江苏省某奶牛场中乳房炎是否为金黄色葡萄球菌所致,采取4份患病乳汁样本进行试验分离和鉴定,并针对金黄色葡萄球菌采用分离后鉴定,结果显示,分离到的2株致病菌为金黄色葡萄球菌,其他菌株还包括大肠杆菌、肠球菌等,为多种致病病原菌同时引起,主要为金黄色葡萄球菌感染。     

北京地区奶牛隐性乳房炎金黄色葡萄球菌的分离鉴定及药敏试验

为了解北京地区奶牛隐性乳房炎中金黄色葡萄球菌的感染情况及对常用药物的敏感性,用科玛嘉显色培养基和16SrRNA PCR检测方法对5个奶牛场的100份隐性乳房炎奶样进行金黄色葡萄球菌的分离鉴定,采用K-B纸片扩增法进行药敏试验。结果表明,显色培养基分离到24株疑似金黄色葡萄球菌,经16SrRNA PCR鉴定,15株为金黄色葡萄球菌;药敏试验结果显示,15株金黄色葡萄球菌均对β-内酰胺类中的氨苄西林产生普遍耐药性,对克林霉素和环丙沙星的耐药率较高,分别为53.33%和40%;对氧氟沙星、庆大霉素、头孢噻肟和头孢曲松的耐药性较低,耐药率为6.67%。说明15株金黄色葡萄球菌分离株对7类9种抗菌药物都有不同程度的耐药性。     

牛源金黄色葡萄球菌的分离鉴定及ClfA基因的克隆和真核表达载体的构建

以研制安全有效的奶牛乳腺炎金黄色葡萄球菌基因工程疫苗为目的,采集380份隐性和临床型乳房炎奶样,经高盐肉汤、过氧化氢酶、甘露醇发酵、三糖铁发酵试验以及Baird-Parker平板对金黄色葡萄球菌进行分离和生化鉴定,PCR扩增分离株凝集因子基因(Clf A)并连接TA克隆载体。经测序确定无误后,连接pc DNA3.1His B以构建真核表达载体pc DNA 3.1His B-Clf A,转化Trans T1TM感受态细胞,HindⅢ和XhoⅠ双酶切分析,同时进行测序和序列分析。结果显示,通过高盐肉汤、过氧化氢酶、甘露醇、三糖铁试验和Baird-Parker平板从380份隐性及临床型奶牛乳房炎奶样中共分离到35株金黄色葡萄球菌,成功构建了真核表达载体(pc DNA3.1His B-Clf A),为研制金黄色葡萄球菌基因工程疫苗奠定基础。     

牛奶金黄色葡萄球菌分离鉴定与耐药性分析

从某奶牛场随机采集26份奶样,通过奶样浓缩、细菌分离培养、染色镜检、H2O2酶试验及PCR鉴定,并进行菌株致病性和药物敏感性试验。结果从24株疑似菌株中鉴定出10株金黄色葡萄球菌,其中致病性金黄色葡萄球菌2株。药物敏感试验显示,10株金黄色葡萄球菌对多种药物有耐药性和多重耐药性,尤其对青霉素类药物100%耐药,对头孢唑林、左氧氟沙星、优孢欣均敏感。金黄色葡萄球菌为该奶牛场乳腺炎主要致病菌,其对抗生素耐药性和多重耐药性普遍,治疗应以体外药物敏感试验为参考,有目的性地选择敏感药物进行治疗。     

鸡金黄色葡萄球菌的分离鉴定

本试验对从病鸡分离到的病原进行微生物经细菌形态学观察、鉴别培养、生化试验、血浆凝固酶试验、过氧化氢酶试验,结果鉴定为金黄色葡萄球菌。     

锦州地区某奶牛场乳房炎金黄色葡萄球菌的分离鉴定及抗菌肽NZ2114对分离菌株细胞膜和耐药性的研究

自2017～2019年采集锦州地区某规模化牛场患有乳房炎的奶牛乳样150份,进行金黄色葡萄球菌的分离鉴定,并使用抗菌肽NZ2114对金黄色葡萄球菌进行抑菌活性检测。结果显示:试验共分离得到金黄色葡萄球菌48株,总分离率为32%;抗菌肽NZ2114对于金黄色葡萄球菌具有较强的抑菌活性,对于葡萄球菌细胞膜有一定的破坏作用;经过抗菌肽NZ2114处理后相关病菌体对于部分抗生素敏感性有所提高。     

猪肉和牛奶源葡萄球菌耐药性调查

2010年从贵州省猪肉和牛奶样品中分离鉴定出73株葡萄球菌,其中金黄色葡萄球菌50株,凝固酶阴性葡萄球菌23株。采用琼脂稀释法检测其对18种抗菌药物的敏感性,运用统计学方法比较分析金黄色葡萄球菌与凝固酶阴性葡萄球菌的耐药性差异。结果显示,动物性食品源葡萄球菌耐药较严重,对临床上常用药物耐药率较高,且为多重耐药,其中乳源葡萄球菌的多重耐药现象较猪肉源葡萄球菌严重;猪肉源金黄色葡萄球菌主要对青霉素类、四环素和大环内酯类耐药,乳源金黄色葡萄球菌主要对青霉素类、头孢菌素类、大环内酯类、四环素、克林霉素、泰妙菌素及利福平耐药;猪肉源凝固酶阴性葡萄球菌主要对氨苄西林、苯唑西林和泰妙菌素耐药,乳源凝固酶阴性葡萄球菌的耐药谱与乳源金黄色葡萄球菌类似。     

奶牛乳腺炎的细菌学研究

在对山东7个地区14个奶牛场临床型和隐性乳腺炎调查的基础上采集234头临床乳腺炎病牛乳样、241个隐性乳腺炎乳样并分别做了细菌学检查,结果表明:泌乳期临床型乳腺炎病原菌以凝固酶阴性葡萄球菌、金黄色葡萄球菌、链球菌、酵母菌和棒状杆菌为主;干奶期临床型乳腺炎病原菌以大肠杆菌、链球菌、金黄色葡萄球菌、凝固酶阴性葡萄球菌和酵母菌为主;隐性乳腺炎病原菌以凝固酶阴性葡萄球菌、金黄色葡萄球菌链球菌、酵母菌、假单胞菌和棒状杆菌为主;厌氧菌在隐性乳腺炎、干奶期乳腺炎和干奶期乳腺炎乳样的捡出率分别为 5．82％,4．17%,10．16％;隐性乳腺炎、干奶期乳腺炎细菌的共感染率较高,与泌乳期乳腺炎病原菌的差异极显著(P<0．01),隐性乳腺炎与干奶期乳腺炎病原菌共感染率差异不显著(P >0．05)。     

Isolation,Indentification and Antibiotic Resistance Analysis of Methicillin-resistant Staphylococcus aureus (MRSA) from Raw Cow Milk

In this study,we collected 502 california mastitis test (CMT) positive milk samples from several cattle farms and then the bacteria were isolated and identified from them.After that,we used multiplex-PCR for screening out methicillin-resistant Staphylococcus aureus (MRSA).Then we investigated the MRSA's antibiotics susceptibility to 19 kinds of antibiotics by discdiffusion method.The results showed that 338 strains of Staphylococci including 89 strains of MRSA were detected out from the 452 milk samples with bacteria.Additionally,most MRSA showed high sensitivity to vancomycin,fosfomycin,chloramphenicol and clarithromycin,while resistant to penicillin,polymyxin,spectinomycin and ceftazidime.The results provided certain factual basis and method for guiding the use of antibiotics in bovine mastitis,rapid identification of MRSA strains and prevention or control of MRSA infection.     

奶牛源MRSA菌株的分离鉴定和耐药性分析

本研究对部分奶牛场采集的502份隐性乳房炎试验（california mastitis test,CMT）检测呈阳性的奶样进行细菌分离、鉴定,运用多重PCR方法筛选出耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus,MRSA）,并对分离出的MRSA用纸片扩散法进行药敏试验,检测其对19种抗菌药物的敏感性。结果显示,分离到细菌的奶样有452份,其中检出葡萄球菌338株,MRSA 89株;MRSA分离株主要对万古霉素、磷霉素、氯霉素和克拉霉素敏感,对青霉素、多黏菌素、大观霉素及头孢他啶具有较强的耐药性。本研究为指导奶牛乳房炎用药、MRSA菌株的快速鉴定及制定MRSA感染的防控措施提供参考。     

一个高体细胞数牧场奶牛隐性乳腺炎病原菌的调查

对安徽某规模牧场随机采集的314份奶样进行细菌分离鉴定、牛奶体细胞计数(SCC),并对分离比例较高的凝固酶阴性葡萄球菌(CNS)进行亚种鉴定及药敏试验。结果显示120个低体细胞数奶样(SCC25×104/mL)的细菌检出率只有8.3%,分离细菌为CNS;而194个高体细胞数奶样(SCC≥25×104/mL)细菌检出率高达72.2%,分离出的细菌主要为金黄色葡萄球菌、CNS、大肠杆菌、停乳链球菌等。其中CNS感染比例最高、为28.8%,主要包括松鼠葡萄球菌、表皮葡萄球菌、人型葡萄球菌等。乳腺内细菌感染可引起患病乳区SCC显著升高(P0.05),松鼠葡萄球菌和表皮葡萄球菌感染也可引起SCC显著升高(P0.05),不同亚种的CNS对临床常用抗菌药物青霉素、庆大霉素、红霉素有较高的耐药率,分别为71.2%、53.8%和82.7%。     

Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates

Biofilm-forming ability has been increasingly recognized as an important virulence factor in Staphylococci, facilitating their persistence in the host, evading its defences and allowing bacterial survival at high antimicrobial concentrations. Staphylococcus aureus remains a major pathogen of chronic mastitis, but in the last years Staphylococcus epidermidis has emerged as a relevant mastitis pathogen. The present work aimed at the evaluation of the biofilm-forming ability of Staphylococci field isolates from bovine subclinical mastitis and at the development of a fluorescent in situ hybridisation (FISH) protocol that would allow the direct observation of biofilm formation in milk samples. The analysis of phenotypic expression in Congo Red Agar (CRA) and by FISH, showed that 37.5% of the S. aureus isolates produced biofilm, while by optical density measurement only 18.75% isolates revealed this phenotype. The results showed a fair agreement according to the kappa coefficient test (kappa = 0.259). Regarding S. epidermidis mastitis isolates, 37.5% revealed the ability to produce biofilm, but only four isolates were positive by all methods. This agreement was moderate (kappa = 0.467). The application of FISH to artificially contaminated milk samples allowed the direct observation of biofilm production by 37.5% isolates, showing total agreement with the CRA results. This method better mimics the in vivo conditions, especially in terms of the presence of calcium and iron, which in high concentrations, respectively, are known to inhibit or induce biofilm production.     

奶牛隐性乳房炎的病原检测与小白鼠致病性研究

本研究以新疆乌鲁木齐地区的2个奶牛场隐性乳房炎患牛为试验对象,共分离鉴定出21种91株引起奶牛乳房炎的主要病原菌,其中葡萄球菌28株（30.78%）,链球菌21株（23.07%）,杆菌25株（27.48%）,其他细菌17株（18.67%）。对主要病原菌通过动物试验确定致病力较强的菌为金黄色葡萄球菌、克雷伯氏菌和大肠杆菌。同时对17大类主要病原菌共30种药物的药敏试验结果表明,在奶牛隐性乳房炎中应用较多的青霉素、四环素等抗生素对常见致病菌几乎无效,而左氧氟沙星、呋喃妥因、头孢噻肟、阿米卡星则显示出较好的抑菌作用。     

多重PCR快速检测奶牛乳房炎3种主要病原体

奶牛乳房炎是引起奶牛业经济损失的一种重要疫病,目前还没有快速、特异检测奶牛乳房炎主要致病原的方法。本试验根据金黄色葡萄球菌、无乳链球菌、大肠杆菌各自保守的16S或23S rRNA基因序列,合成了3对特异性引物,建立了三重PCR检测方法。特异性试验表明,该方法对所有参与测试的金黄色葡萄球菌、无乳链球菌和大肠杆菌都能扩增出各自的阳性条带,而对所有参与测试的对照菌株则不能扩增出任何条带。敏感性试验表明该方法能检测到4个菌的金黄色葡萄球菌、无乳链球菌和2个菌的大肠杆菌。对送检的乳房炎奶样36份直接进行PCR检测,金黄色葡萄球菌阳性7份,无乳链球菌阳性2份,大肠杆菌阳性6份。     

Prevalence of udder infections and mastitis in 50 California dairy herds

The California mastitis test (CMT) and bacteriologic culture were performed on samples of bulk-tank milk and cow-composite milk (n = 23,138 cows) from 50 California dairies, 19 of the 50 with known mastitis problems. Thirty-eight (76.0%) bulk-tank milk samples and 12,334 (53.3%) cows were positive by results of the CMT. Potential mastitis agents were isolated from 5,085 (22%) cows. Staphylococcus aureus was isolated from all 50 herds, Streptococcus agalactiae was isolated from 47 herds, and Mycoplasma sp was isolated from 24 herds. For cow-composite milk samples, the prevalences were 9.3% for Str agalactiae, 9.1% for S aureus, 0.9% for Mycoplasma sp, 1.2% for coliform bacteria, 0.9% for other streptococci, 0.8% for coagulase-negative staphylococci, and 1.3% for other organisms. The relative sensitivity and the relative specificity of the CMT performed on cow-composite milk samples were 83.4% and 55.2%, respectively, and the predictive value of positive CMT results was 34.2%.     

Performance studies of an enzyme-linked immunosorbent assay for detecting Staphylococcus aureus antibody in bovine milk

An enzyme-linked immunosorbent assay (ELISA) for detecting Staphylococcus aureus antibody in bovine milk samples was examined for repeatability. A set of 51 bovine milk samples from 4 universities with confirmed culture results was assembled, and a panel of 30 milk samples was randomly selected. When the selected panel was tested at the collection laboratory, there was 97% agreement between the ELISA and the culture test. The panel was tested with the ELISA by the 4 university laboratories. Results were scored by both visual and optical density reader methods. When compared to reference ELISA results, the university laboratory ELISA results showed an agreement of 99.8% for negative samples, 98% for positive samples, and 99% for all samples. Additional studies on 19 milk samples that cultured positive for bacteria other than S. aureus showed 100% specificity. Overall comparison of ELISA and culture results showed high agreement between the 2 techniques. Disagreement appeared to result from explainable differences in antibody and bacterial levels and not from errors in either of the 2 techniques.