这项工作是由美国国立卫生研究院的支持[授予R01AI-39108上海贝尔]美国心脏协会补助SDG的0630219N和援助09GRNT2180065到YQX]。

1。 MRSA菌株感染的准备<ol><li>接种MRSA的文化loopful从股票管在-80°C到胰酶大豆琼脂（TSA）板的羊血（见特定的试剂和设备表），并于37℃过夜。 </li><li>检查的文化上的血琼脂平板上（类似殖民地的表型），以确保有是没有污染的纯度。 </li><li>从绵羊血TSA的板接一个殖民地和SNAP-皑皑管15毫升到5毫升胰酪胨大豆肉汤（TSB）在接种殖民地。 </li><li>在37°C间在200 rpm摇过夜。 </li></ol> 2。准备手术导管聚乙烯管材（PE10;碧迪，订单没有427401）削减至10厘米长，按无菌镊子尖的一端融化。导管的一端密封的目的是为了避免导尿过程中的出血。 3。前-Surgery准备和麻醉<ol><li>地方SD大鼠在含有isofluorane氧混合气体（50％：50％）分（哈伦，印第安纳州印第安纳波利斯，女，250-300克），直到麻醉生效（如肌肉放松和脚蹬反射缺席），并保持在与气体混合物的手术在麻醉状态下的动物。 </li><li>从下巴正下方的胸骨与优碘和70％的乙醇清洁颈部。 </li></ol> 4。外科手术<ol><li>整个手术使用的无菌技术。请通过颈部的胸骨皮肤层以上垂直切口（1-1.5厘米）。 </li><li>使用钝性分离，分离筋膜，暴露右颈内动脉，用2双曲面齿镊。 </li><li>轻轻一拉动脉颈腔，和地方两个10厘米丝线长度下动脉和配合，在裸露的头年底动脉，放置在ARTE剪辑RY，以防止出血。 </li><li>在使用动脉导管导引（Becton Dickinson公司，订单号406999）。上方的一个小孔，插入导管通过动脉孔与镊子，删除剪辑和倒推往心脏导管，直到阻力得到满足。 </li><li>配合周围动脉尾端宽松的缝合，并在地方用丝线缝合固定导管，当导管到位的决定：我是）抵抗，以进一步推进长度的导管插入（4-5厘米）; II）及iii）与心跳导管的脉动。离开导管在其余实验的地方。 </li><li>削减多余的缝线和导管两端，并确保有没有从导管出血。掖下颈部皮肤有始有终，并关闭皮肤与皮肤剪辑。 </li><li>放置在老鼠笼在一个温暖的地方，直至从麻醉中恢复，并提供食物和水。经常检查大鼠期间和从麻醉后恢复。 </li><li>这种手术是一个“E类”的程序。长此以往的程序产生的疼痛，​​是因为MRSA的陪同窘迫的动物感染的止痛药。 </li></ol> 5。 MRSA感染<ol><li>感染可以进行手术后1至7天，但​​在实验中保持一致。 </li><li>用70％乙醇的清洁鼠尾，所需的细胞计数（10 4 - 10 6 CFU /动物大多数的金黄色葡萄球菌菌株）注入0.5毫升MRSA的27 G的1/2寸毫针从尾静脉注射静脉注射注意：MRSA的文化，属于生物安全2级（BSL2），并具有适度的人员和环境的潜在危险。 </li><li>在现场举行的压力，直到止血，然后返回笼子的老鼠发生。 </li></ol> 6。牺牲大鼠和文化靶组织<ol><li>牺牲大鼠腹腔注射戊巴比妥钠（200毫克/公斤）后，感染后1至6天。 </li><li>将大鼠在她的背上，并用70％乙醇擦拭胸部。 </li><li>在下面的胸骨胸部V形切口，切断胸骨端在每个暴露心脏的肋骨软骨。 </li><li>轻轻拉起来的心，通过主动脉靠近心脏组织的剪辑和剖析释放的心。 </li><li>放在一个无菌的Petri菜的4x4英寸纱布内心脏，通过左心室内壁切割，打开左侧室。 </li><li>目视检查导管的位置。检查和清除阀用剪刀和镊子的植被。 </li><li>权衡和同质化的植被，并用PBS连续稀释，定量培养。 </li></ol> 7。代表结果后立即插入动脉导管向下推约4-5厘米向心导管，阻力会experienCED。检测，如果没有阻力，导管可能没有成功插入左侧心腔，这可能会影响导管置入。此前公布的9正确的导管放置在图1所示。 MRSA感染样本的一部分，需要定量培养“确保感染和感染样品纯度的确切可行的细菌总数。此外，从植被恢复的生物应该是为那些在接种所使用的相同。 表1列出了一个与毒力的例子金黄色葡萄球菌菌株在大鼠感染性心内膜炎模型被公布之前9。 10 5 10 6 CFU接种所有动物的挑战和牺牲后3至6天之间感染高学发展endocardits 金黄色葡萄球菌在心脏赘生物，以及肾脏和脾脏的密度（ <st荣毅仁表1）。用无菌阀培养的大鼠被视为感染。 <img alt="图1" src="/files/ftp_upload/3863/3863fig1.jpg" /> 图1。导管是在正确的位置（左侧心脏腔室），和大量的植被是可见主动脉瓣9左右。 <table border="1"><tbody><tr><td> 接种（阿尼马斯的号） </td><td colspan="3"> 平均登录10 CFU /克组织±SD 在 ： </td></tr><tr><td></td><td> 植被 </td><td> 肾脏 </td><td> 脾 </td></tr><tr><td> 10 6 CFU /动物（9） </td><td> 10.36±0.85 </td><td> 7.30±0.64 </td><td> 6.70±0.57 </td></tr><tr><td> 10 5 CFU /动物（8） </td><td> 9.93±0.53 </td><td> 7.14±0.53 </td><td> 6.44±0.63</td></tr><tr><td> 10 4 CFU /动物（7） </td><td> 3.46±0.50 * </td><td> 1.81±0.74 * </td><td> 1.58±0.59 * </td></tr></tbody></table> 一个 SD，标准差，P &lt;0.001 10 5 10 6 CFU挑战动物。 表1。S。 金黄色葡萄球菌在心脏植被密度与不同菌剂在大鼠感染性心内膜炎模型9。

<ol>
	<li>Petti, C. A., Fowler, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+aureus+bacteremia+and+endocarditis.">Staphylococcus aureus bacteremia and endocarditis.</a> Cardiol. Clin. 21, 219-233 (2003).</li><li>Bashore, T. M., Cabell, C., Fowler, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+infective+endocarditis.">Update on infective endocarditis.</a> Curr. Probl. Cardiol. 31, 274-352 (2006).</li><li>Fowler, V. G., Justice, A., Moore, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+hematogenous+complications+of+intravascular+catheter-associated+Staphylococcus+aureus+bacteremia.">Risk factors for hematogenous complications of intravascular catheter-associated Staphylococcus aureus bacteremia.</a> Clin. Infect. Dis. 17, 313-320 (1993).</li><li>Wisplinghoff, H., Bischoff, T., Tallent, S. M., Seifert, H., Wenzel, R. P., Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+bloodstream+infections+in+US+hospitals:+analysis+of+24,179+cases+from+a+prospective+nationwide+surveillance+study.">Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study.</a> Clin. Infect. Dis. 39, 309-317 (2004).</li><li>Hoen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+issues+in+the+management+of+infective+endocarditis+caused+by+gram-positive+cocci.">Special issues in the management of infective endocarditis caused by gram-positive cocci.</a> Infect Dis. Clin. North Am. 16, 437-452 (2002).</li><li>Moise, P. A., Hershberger, E., Amodio-Groton, M. I., Lamp, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+clinical+outcomes+when+utilizing+high-dose+(&gt;+or+=8+mg/kg)+daptomycin+therapy.">Safety and clinical outcomes when utilizing high-dose (&gt; or =8 mg/kg) daptomycin therapy.</a> Ann. Pharmacother. 43, 1211-1219 (2009).</li><li>Sakoulas, G., Brown, J., Lamp, K. C., Friedrich, L. V., Lindfield, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+outcomes+of+patients+receiving+daptomycin+for+the+treatment+of+Staphylococcus+aureus+infections+and+assessment+of+clinical+factors+for+daptomycin+failure:+a+retrospective+cohort+study+utilizing+the+Cubicin+Outcomes+Registry+and+Experience.">Clinical outcomes of patients receiving daptomycin for the treatment of Staphylococcus aureus infections and assessment of clinical factors for daptomycin failure: a retrospective cohort study utilizing the Cubicin Outcomes Registry and Experience.</a> Clin. Ther. 31, 1936-1945 (2009).</li><li>Xiong, Y. Q., Willard, J., Kadurugamuwa, J. L., Yu, J., Francis, K. P., Bayer, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+in+vivo+bioluminescent+imaging+for+evaluating+the+efficacy+of+antibiotics+in+a+rat+Staphylococcus+aureus+endocarditis+model.">Real-time in vivo bioluminescent imaging for evaluating the efficacy of antibiotics in a rat Staphylococcus aureus endocarditis model.</a> Antimicrob. Agents Chemother. 49, 380-387 (2005).</li><li>Peerschke, E. I., Bayer, A. S., Ghebrehiwet, B., Xiong, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gC1qR/p33+blockade+reduces+Staphylococcus+aureus+colonization+of+target+tissues+in+an+animal+model+of+infective+endocarditis.">gC1qR/p33 blockade reduces Staphylococcus aureus colonization of target tissues in an animal model of infective endocarditis.</a> Infect. Immun. 74, 4418-4423 (2006).</li><li>Ganesh, V. K., Rivera, J. J., Smeds, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+structural+model+of+the+Staphylococcus+aureus+ClfA-fibrinogen+interaction+opens+new+avenues+for+the+design+of+anti-staphylococcal+therapeutics.">A structural model of the Staphylococcus aureus ClfA-fibrinogen interaction opens new avenues for the design of anti-staphylococcal therapeutics.</a> PLoS Pathog. 4, e1000226 (2008).</li><li>Boles, B. R., Horswill, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcal+biofilm+disassembly.">Staphylococcal biofilm disassembly.</a> Trends Microbiol. , (2011).</li><li>Tseng, C. W., Sanchez-Martinez, M., Arruda, A., Liu, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcutaneous+Infection+of+Methicillin+Resistant+Staphylococcus+Aureus+(MRSA).">Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA).</a> J. Vis. Exp. (48), e2528 (2011).</li></ol>

没有利益冲突的声明。

大鼠感染性心内膜炎是一个重要和良好的特点的动物模型，为在体内研究中的发病机制和抗菌药物在治疗细菌感染9-11。此外，在大鼠感染性心内膜炎模型代表一种急性，亚急性感染的复合材料，并密切模仿人类的​​对口和人天然瓣膜心内膜炎。此外，在住宅导管的陪同下，它代表一个典型的生物膜相关感染常见和棘手的问题，在12个临床的设置。大鼠感染性心内膜炎模型...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>血琼脂平板上，5％的胰酶大豆琼脂羊血液（TSA） </td><td>哈迪诊断</td><td> A10BX </td><td></td></tr><tr><td>胰酪胨大豆肉汤（TSB）， </td><td>碧迪</td><td> 211825 </td><td></td></tr><tr><td>摇床</td><td> labnet </td><td> I5311-DS </td><td></td></tr><tr><td>聚乙烯管材</td><td>碧迪</td><td> 427401 </td><td></td></tr><tr><td>导管导引</td><td>碧迪</td><td> 6999 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> isofluorance </td><td>西方医学增刊Y，公司</td><td> 2147 </td><td>按照安全和处理信息</td></tr><tr><td>手术器械</td><td>精细的科学工具和生物医学研究仪器公司</td><td></td><td>从两家​​公司找到您需要的文书</td></tr></tbody></table>

experimental endocarditis model of methicillin resistant staphylococcus aureus (mrsa) in rat

血管内感染，包括感染性心内膜炎，危及生命的传染病综合征1-3。 金黄色葡萄球菌是最常见的世界性不可接受的高发病率和死亡率甚至与适当的抗菌剂治疗4-6综合征的原因。在感染的增加，由于耐甲氧西林S.金黄色葡萄球菌 （MRSA），万古霉素的临床治疗失败和利奈唑胺和达托霉素抵抗日益严重的问题的高利率有此类感染患者都进一步复杂化的管理，并导致高的医疗费用7，8。此外，应强调用抗生素治疗效果的最近期的研究已根据临床的设置，因此很可能被主机从病人到门诊不同因素的影响。因此，有关的腔内感染的动物模型，在宿主因素是相似的，从动物到动物更探讨微生物的发病机制，以及新型抗菌药物的疗效的关键。在大鼠感染性心内膜炎是一种行之有效的实验动物模型，接近人天然瓣膜心内膜炎。该模型已被用于研究，尤其是金黄色葡萄球菌致病因素的作用，并为金黄色葡萄球菌感染性心内膜炎的抗生素治疗方案的疗效。在这份报告中，我们描述的实验性心内膜炎模型，由于MRSA的，可以用来研究细菌致病和抗生素治疗。

Watch this Scientific Journal Video about Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat at JoVE.com

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

大鼠实验性心内膜炎模型由于耐甲氧西林<em&gt; S金黄色葡萄球菌</em&gt;。

耐甲氧西林实验性心内膜炎模型<em&gt;金黄色葡萄球菌</em&gt;（MRSA）的大鼠

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

experimental-endocarditis-model-methicillin-resistant-staphylococcus

Research

JoVE Journal

Immunology and Infection

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

15.0K 次观看.  Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. 大鼠实验性心内膜炎模型由于耐甲氧西林 S金黄色葡萄球菌。

视频: Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

科研

免疫与感染

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the United States. Among soft-tissue infections, myositis, pyomyositis, and necrotizing fasciitis have been increasingly reported in association with MRSA arising from the community. To understand the interplay between MRSA and host immunity leading to more severe infection, the availability of animal models is critical, permitting the study of host and bacterial factors. Several infection models have been introduced to assess the pathogenesis of S. aureus during superficial skin infection. Here, we describe a subcutaneous infection model that examines the skin, subcutaneous, and muscle pathologies.

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

Murine skin and soft tissue infection model is utilized for assessing the virulence function of methicillin resistant Staphylococcus aureus (MRSA) and the host immunological responses. Here, we presented a subcutaneous infection model for skin and soft tissue infection.

皮下感染耐甲氧西林金黄色葡萄球菌（MRSA）的

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis (EAE) Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for study of the human demyelinating disease, multiple sclerosis (MS). EAE is characterized by pathologic infiltration of mononuclear cells into the CNS and by clinical manifestation of paralytic disease. Similar to MS, EAE is also under genetic control in that certain mouse strains are susceptible to disease induction while others are resistant. Typically, C57BL/6 (H-2b) mice immunized with myelin basic protein (MBP) fail to develop paralytic signs. This unresponsiveness is certainly not due to defects in antigen processing or antigen presentation of MBP, as an experimental protocol described here had been used to induce severe EAE in C57BL/6 mice as well as other reputed resistant mouse strains. In addition, encephalitogenic T cell clones from C57BL/6 and Balb/c mice reactive to MBP had been successfully isolated and propagated.
The experimental protocol involves using a cellular adoptive transfer system in which MBP-primed (200 &mu;g/mouse) C57BL/6 donor lymph node cells are isolated and cultured for five days with the antigen to expand the pool of MBP-specific T cells. At the end of the culture period, 50 million viable cells are transferred into naive syngeneic recipients through the tail vein. Recipient mice so treated normally do not develop EAE, thus reaffirming their resistant status, and they can remain normal indefinitely. Ten days post cell transfer, recipient mice are challenged with complete Freund adjuvant (CFA)-emulsified MBP in four sites in the flanks. Severe EAE starts to develop in these mice ten to fourteen days after challenge. Results showed that the induction of disease was antigenic specific as challenge with irrelevant antigens did not induce clinical signs of disease. Significantly, a titration of the antigen dose used to challenge the recipient mice showed that it could be as low as 5 &mu;g/mouse. In addition, a kinetic study of the timing of antigenic challenge showed that challenge to induce disease was effective as early as 5 days post antigenic challenge and as long as over 445 days post antigenic challenge. These data strongly point toward the involvement of a "long-lived" T cell population in maintaining unresponsiveness. The involvement of regulatory T cells (Tregs) in this system is not defined.

Overcoming Unresponsiveness in Experimental Autoimmune Encephalomyelitis EAE Resistant Mouse Strains by Adoptive Transfer and Antigenic Challenge

Certain mouse strains are able to resist induction of experimental autoimmune encephalomyelitis (EAE) with myelin basic protein. Described here is a simple immunization protocol that reverses the unresponsiveness and induces paralytic disease in several typical EAE resistant mouse stains.

在实验性自身免疫性脑脊髓炎（EAE）抗性品系小鼠继转移和抗原挑战克服反应迟钝

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) and has been used as an animal model for ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

金黄色葡萄球菌生长使用人血红蛋白作为铁源

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

针对相关生物膜<em&gt;金黄色葡萄球菌</em&gt;使用基于刃天青药物敏感性测定

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

方法基因水平转移的研究<em&gt;金黄色葡萄球菌</em

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...