NIAID的赠款K22AI081748。

1。滴流生物膜反应器<ol><li>滴流生物膜反应器（Biosurface技术或定制设计的版本，通常可以由大学机械厂，看到图1）是组装和​​灭菌。大会涉及商会和保护室盖张贴券。随着生物膜的介质（胰蛋白酶大豆肉汤2克/ L和葡萄糖2克/升），和营养管进水室，高压灭菌消毒。 </li><li>接种的滴流式反应器是预制反应器放置在一个平面上，夹紧污水管线，灌装用10 mL的胰蛋白酶大豆肉汤每个分庭，加入10μLA S. 金黄色葡萄球菌文化过夜胰蛋白酶大豆肉汤。接种反应器，然后放置在37℃培养箱中培养18小时。 </li><li>经过18个小时的孵化，松开污水管反应器上放置一个木块切成10 °角。 </li><li>无菌连接进水每瓶含连续流动的营养肉汤的营养管。饲料油管线运行最高速度（会有所不同取决于泵模型）泵通过泵和总理管。 </li><li>一旦进水管路催芽停泵和附加连接针（22号，1英寸），每管结束。用乙醇擦拭，并在无菌条件下通过入口塞插入针室入口塞。 </li><li>打开泵，并允许媒体缓慢滴注（流量〜125μL/分钟）以上的优惠券。从入口塞端口的出水端口，媒体应该沿着票面向下流动。工作在连续流动反应器为2-5天（取决于应用），偶尔会检查适当的排水反应堆。 </li><li>收获的滴流式反应器生物膜，停泵，并小心取出针头从反应堆。该反应堆可以被放置在一个平面上的优惠券，可在无菌条件下使用无菌镊子去除。如果需要显微镜，优惠券现在可以处理（图2B是滴生物膜反应器中生长的一种金黄色葡萄球菌生物膜的扫描电子显微镜）。如果生物膜的生物量或生理学研究的量化研究的目的，生物膜可以免去券使用细胞刮刀。虽然持有优惠券，用血管钳轻轻刮去票面生物膜的一个锥形管含有磷酸盐缓冲液使用细胞刮刀。注：量化生物膜的菌落形成单位，这是必要的同质化与组织匀浆分解团块，形成一个均匀的悬浮生物膜的收获。组织均质机的各种型号都适合这种应用。我们利用1分钟均质生物膜样品全速费希尔科学Tissuemiser均质机（产品＃15-338-420）。不均质生物膜会导致低估样品中菌落形成单位。 </li></ol> 2。旋转盘生物膜反应器<ol><li>旋转盘生物膜反应器（可从Biosurface技术，或可定制，参见图3）的组装和灭菌。反应堆组装第一名纺成转盘的插槽磁盘券，并放入1升的玻璃烧杯与溢流口。被用作反应堆的第一个孔钻，让媒体流和曝气15胶塞。生物膜反应器，媒体（胰蛋白酶大豆肉汤为2 g / L和葡萄糖2克/升）和入口管再经高压灭菌消毒。 </li><li>生物膜反应器的旋转盘接种无菌培养基中放置250毫升到反应堆，并加入0.5毫升的一个通宵的S. 金黄色葡萄球菌培养胰蛋白酶大豆肉汤。然后放置在反应堆是不小的轰动板设置到250转和孵育过夜所需的温度。 </li><li>孵化后16小时内的端口连接的入口管中型水库，将其连接到蠕动泵。然后打开泵和流量设置为〜0.25毫升/分（取决于所需的增长率可以改变流）。 </li><li> 24小时后停止反应器，并在无菌条件下删除不接触生物膜券光盘。使用无菌镊子取出券和浸入每一个磷酸缓冲液，以消除任何松散附着细菌。 </li><li>对于抗菌化合物测试，该芯片可以被放置到96盘感兴趣的化合物以及包含的个别井。孵育后，转入1 mL含磷酸缓冲液1.5 ml离心管芯片，并与组织匀浆分散完整的生物膜匀浆。 </li><li>细胞，然后串行稀释和镀营养琼脂培养基上，以确定可行的殖民地formi吴单位。 </li><li>注：很多变化，可以对上述协议。例如，可涂有旋转盘优惠券或潜在的抗生物膜化合物的疗效进行测试。生物膜分散剂，也可以被孵化券分散化合物和量化附加与分离的细菌进行评估。 </li></ol> 3。代表性的成果一套加快滴流式反应器的一个例子是，如果图1所示。经过三天流会积累丰富的生物膜上的券面，图2A。总生物量会有所不同取决于菌株和精确的生长条件。甲A S.扫描电子显微镜滴流式反应器中生长的金黄色葡萄球菌生物膜如图2b所示。 旋转盘反应器是在图3a所示。描述的协议，可适应一般的任何微生物形成生物膜的能力的具体要求。图3b显示18贴塑料磁盘旋转的光盘。特别适合于这些磁盘生长的生物膜的抗菌测试和产量高可重复性的结果 11 。 <img src="/files/ftp_upload/2470/2470fig1.jpg" alt="图1" /> 图1。滴流式反应器设置。标记为重要组成部分。 <img src="/files/ftp_upload/2470/2470fig2.jpg" alt="图2" /> 图2的滴流的例子s 金黄色葡萄球菌生物膜。 A）以下的协议描述这种生物膜的生长三天。从反应堆的前两个商会的盖子被删除，显示黄南金黄色葡萄球菌生物膜的生物量。二）Sacnning电子显微镜下的A S.金黄色葡萄球菌生物膜滴流式反应器中生长。 <img src="/files/ftp_upload/2470/2470fig3.jpg" alt="图3" /> 图3。旋转盘堆。一个正在运行的旋转盘反应堆）例。关键部件标记。 b）关闭了一个旋转的磁盘。

<ol>
	<li>Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R., Lappin-Scott, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+Biofilms.">Microbial Biofilms.</a> Annu. Rev. Microbiol. 49, 711-745 (1995).</li><li>Costerton, J. W., Cheng, K. J., Gessey, G. G., Ladd, T. I., Nickel, J. C., Dasgupta, M., Marrie, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+biofilms+in+nature+and+disease.">Bacterial biofilms in nature and disease.</a> Ann. Rev. Microbiol. 41, 435-464 (1987).</li><li>O'Toole, G. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+biofilm+formation+in+Pseudomonas+fluorescens+WCS365+proceeds+via+multiple,+convergent+signaling+pathways:+a+genetic+analysis.">Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis.</a> Mol. Micro. 28, 449-461 (2002).</li><li>Boles, B. R., Horswill, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agr-mediated+dispersal+of+Staphylococcus+aureus+biofilms.">Agr-mediated dispersal of Staphylococcus aureus biofilms.</a> PLoS Pathog. 4, e1000052-e1000052 (2008).</li><li>Goeres, D. M., Haamilton, M. A., Beck, N. A., Buckingham-Meyer, K., Hilyard, J., Loetterle, L. A., Walker, D. K., Stewart, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+growing+a+biofilm+under+low+shear+at+the+air-liquid+interface+using+the+drip+flow+biofilm+reactor.">A method for growing a biofilm under low shear at the air-liquid interface using the drip flow biofilm reactor.</a> Nature Protocols. 4, 783-788 (2009).</li><li>Fu, W., Forster, T., Mayer, O., Curtin, J. J., Lehman, S. M., Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+cocktail+for+the+prevention+of+biofilm+formation+by+Pseudomonas+aeruginosa+on+catheters+in+an+in+vitro+model+system.">Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system.</a> Antimicrob Agents Chemother. 54, 397-404 (2010).</li><li>Xu, K. D., McFeters, G. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhamnolipids+mediate+detachment+of+Pseudomonas+aeruginosa+from+biofilms.">Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms.</a> Mol. Microbiol. 57, 1210-1223 (2005).</li><li>Hentzer, M., Teitzel, G. M., Balzer, G. J., Heydorn, A., Molin, S., Givskov, M., Parsek, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+overproduction+affects+Pseudomonas+aeruginosa+biofilm+structure+and+function.">Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function.</a> J. Bacteriol. 183, 5395-5401 (2001).</li><li>Lin, H. Y., Chen, C. T., Huang, C. 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在不同的反应堆生长的生物膜，往往会产生不同的特点和每个反应器具有不同的应用。在这项工作中，我们描述了两个生物膜反应器的使用：滴流生物膜反应器和一个旋转的磁盘反应堆。滴流电抗器是有用和越来越低气 - 液界面剪切生物膜，适应各种条件。我们发现大量的生物膜生物量是可取的研究极为方便。这种设置可以很容易地适应对涉及的微传感器的监测和测试潜在的antibiofilm表面研究。 <...

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

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<td>Drip Flow Reactors</td>
<td>&nbsp;</td>
<td>BioSurface Technologies Corporation</td>
<td>DFR 110</td>
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the use of drip flow and rotating disk reactors for staphylococcus aureus biofilm analysis

被认为是自然界中最微生物在生物膜表面相关的社区存在。细菌生物膜 1矩阵内的包裹和连接表面生物膜的形成和发展，通常是在实验室使用批处理系统，如微孔板或流量的研究系统，如流细胞。这些方法，筛选突变体和化学库（微孔板）可视化（流动池）4 3或生长生物膜。在这里，我们提出两种其他类型的流量系统生物膜生长在金黄色葡萄球菌的详细协议：滴流生物膜反应器和生物膜反应器的旋转盘。 滴流生物膜反应器是专为低剪切条件下生长的生物膜的研究，5滴流式反应器由四个平行测试通道，每持有一个标准玻璃显微镜玻片大小的优惠券，或导管或限制的长度。滴流式反应器是理想的微传感器监测，一般生物膜研究，生物膜cryosectioning样品，高生物量生产，医用材料的评价，并留置医疗器械检测 6,7,8,9 。 旋转圆盘式反应器由聚四氟乙烯包含可移动优惠券的凹槽的磁盘。10可拆卸的优惠券可以由任何可加工材料。旋转盘的底部有一个条形磁铁，让创建跨磁盘旋转液体表面剪切表面冲洗券。 1000毫升的玻璃侧臂的反应堆压力容器是摆在整个磁盘，其中包含18券。液体增长媒体是通过船只的分发，而磁盘是由磁力搅拌器旋转。优惠券是从反应堆压力容器，然后刮下收集生物膜样本作进一步研究或显微成像。旋转圆盘反应器是专为实验室生物杀灭剂的功效，去除生物膜和防污材料的性能评价。9,11,12,13

Watch this Scientific Journal Video about The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis at JoVE.com

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

详细介绍了利用开放系统流生物膜与滴流反应器和旋转磁盘反应堆的协议。

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 ...

the-use-drip-flow-rotating-disk-reactors-for-staphylococcus-aureus

Research

JoVE Journal

Immunology and Infection

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

17.0K 次观看.  University of Michigan. 详细介绍了利用开放系统流生物膜与滴流反应器和旋转磁盘反应堆的协议。

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

铜绿假单胞菌和酿酒酵母流细胞生物膜

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

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 ...

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

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

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 ...

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 ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...