这项工作是由宝来惠康事业奖和国家卫生部授予AI074832研究院庚寅刘的支持。

1。准备葡萄球菌感染（感染前两天） <ol><li>接种的MRSA loopful从股市文化血液琼脂（胰酪胨大豆琼脂（TSA））板。 </li><li>检查血琼脂平板上溶血表型（每个殖民地周围明确区）。 </li><li>选择一个殖民地，与其他实验是一致的，具有溶血性表型。 </li><li>接种3毫升托德休伊特肉汤（THB）的殖民地，与适当的抗生素在必要时，在单元皑皑管15毫升。 </li><li>在37 ° C晃动在220转过夜。 </li></ol> 2。准备小鼠感染（感染的前一天） <ol><li>根据老鼠的大小，削去一个3 × 4厘米的面积上的小鼠背部的皮毛。 </li><li>剃光〜5毫米3头发卸妆霜（从当地药店购买）。 </li><li>让头发卸妆霜，在皮肤表面孵育约1分钟。 </li><li> DDH 2 O湿纸巾</li><li>用湿纸巾擦去头发卸妆霜。 </li></ol> 3。准备MRSA的感染（感染当天） <ol><li>过夜的细菌培养在1:500到1:1000稀释预热泰铢10毫升，50毫升的螺丝帽管。 </li><li>在37℃孵育，用颤抖的在220转， 直到 540到达2.5℃，约2.5小时。 </li><li> 10分钟在3225 XG收集离心MRSA在4 ° C。 </li><li>上清液。 </li><li>细菌沉淀重新悬浮在10毫升贝科磷酸盐缓冲液（DPBS）。 </li><li>重复步骤3和4。 </li><li>在DPBS细菌沉淀重悬在所需浓度。 </li><li>串行稀释的菌悬液从10 1 10 8。 </li><li>板到血琼脂平板稀释的菌悬液。 </li><li>孵育37℃过夜板。 </li><li>观察和记录溶血性表型的接种。 </li><li>计数菌落形成单位（CFU）数盘上。 </li><li>板CFU数为基础计算的接种。 </li></ol> 4。皮下的MRSA感染（感染当天，感染后3天） <ol><li>皮下注入100μL，剃光面积的菌悬液。 </li><li> 3至5个小时的观察动物注射后，以确保小鼠还活着。 </li><li>每天观察病变的动物的回，并记录病变区，每日需要的时候。应包括病变的观察记录病灶的大小和形态。皮肤和肌肉病变的定量乘以病变的长度和宽度。需要被分解成更小的对称件，每件用同样的方法测量不规则形的病灶。病变的测量，也可以使用电脑辅助组织形态学评估方案（ImageJ; <a href="http://rsb.info.nih.gov/ij">开源</a> http://rsb.info.nih.gov/ij/从美国国立卫生研究院提供）2。 </li><li>异氟醚吸入颈椎脱位，感染后第3天牺牲的动物。 </li><li>观察并记录在皮肤上的病变。 </li><li>尼克的皮肤，一双无菌剪刀。 </li><li>关闭仔细剥离皮肤，观察并记录肌肉病变。 </li><li>切除病灶及周边地区约2 - 5毫米。 </li><li>收集脾和肾。 </li><li>均质使用组织匀浆的组织或反复应用到离心管中含有的组织和100μL的DPBS，从1 mL注射器柱塞。 </li><li>以每管加入900μL的DPBS。 </li><li>涡5分钟的样品。 </li><li>串行稀释与DPBS 10 1至10月6日暂停。 </li><li>在临屋板的稀释悬浮板。 </li></ol> 5。代表性的成果： <ol><li>后立即皮下注射，将观察到的气泡在皮肤表面感染时做正确（图1A）。在皮肤表面观察到，如果没有气泡注射过深，这可能会影响感染的结果（病灶的大小，CFU）的。 </li><li>需要连续稀释的菌悬液的CFU需要血琼脂平板上，每个实验研究。这一步将产生重要的信息：1）接种是否有同质化的表型; 2）感染的确切可行的含菌量。 </li><li>可能被评定在不同时间点后感染病灶和细菌生存。在这里，我们审查了感染，感染后第3天。分析病灶大小在皮肤表面（图1b：1X 10 9;图1D：1 × 10 7 CFU）和肌肉表面（图1C：1X 10月9日 </s上涨;图1E：1 × 10 7 CFU）。病灶面积等于长度（毫米）x宽（mm）可用于评估组织损伤（图2 A和B）来衡量。这一结果可能有助于确定一个给定的致病因子引起的组织损伤的程度。此外，病灶切除，并在DPBS匀浆，并镀以定量的CFU（图2C），这表明，在感染部位的细菌的可行性。 </li></ol><img src="/files/ftp_upload/2528/2528fig1.jpg" alt="图1" /> 图1。皮肤和肌肉病变。CD1小鼠皮下接种于一个MRSA（LAC）的侧翼。照片拍摄时间为0，感染后第3天。 （一）时间后感染（PI），（二） - （G）的第3天PI，B，D和F：皮损，C，E和G：肌肉病变，B和C：1X 10月9日 CFU感染; D和E：1 × 10 7 CFU感染; F和G：模拟。 <img src="/files/ftp_upload/2528/2528fig2.jpg" alt="图2" /> 图2。在感染部位，感染后第3天，皮肤和感染MRSA（LAC）的CD1小鼠肌肉病变皮肤病灶的大小和活菌计数。 （一）皮肤病灶的大小。 （二）肌肉病变的大小。 （c）总组织的CFU。 H：1 × 10 9 CFU接种，L：1 × 10 7 CFU接种。 *：P &lt;0.05，Mann - Whitney检验。

<ol>
	<li>Ahn, J. Y., Song, J. Y., Yun, Y. S., Jeong, G., Choi, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+of+Staphylococcus+aureus-infected+septic+mice+by+suppression+of+early+acute+inflammation+and+enhanced+antimicrobial+activity+by+ginsan.">Protection of Staphylococcus aureus-infected septic mice by suppression of early acute inflammation and enhanced antimicrobial activity by ginsan.</a> FEMS Immunol Med Microbiol. 46, 187-197 (2006).</li><li>Girish, V., Vijayalakshmi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affordable+image+analysis+using+NIH+Image/ImageJ.">Affordable image analysis using NIH Image/ImageJ.</a> Indian J Cancer. 41, 47-47 (2004).</li><li>Hahn, B. L., Onunkwo, C. C., Watts, C. J., Sohnle, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+dissemination+and+cutaneous+damage+in+a+mouse+model+of+staphylococcal+skin+infections.">Systemic dissemination and cutaneous damage in a mouse model of staphylococcal skin infections.</a> Microb Pathog. 47, 16-23 (2009).</li><li>Hruz, P., Zinkernagel, A. S., Jenikova, G., Botwin, G. J., Hugot, J. P., Karin, M., Nizet, V., Eckmann, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD2+contributes+to+cutaneous+defense+against+Staphylococcus+aureus+through+alpha-toxin-dependent+innate+immune+activation.">NOD2 contributes to cutaneous defense against Staphylococcus aureus through alpha-toxin-dependent innate immune activation.</a> Proc Natl Acad Sci U S A. 106, 12873-12878 (2009).</li><li>Ji, Y., Zhang, B., Van, S. F., Horn, P. W. a. r. r. e. n., Woodnutt, G., Burnham, M. K., Rosenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+critical+staphylococcal+genes+using+conditional+phenotypes+generated+by+antisense+RNA.">Identification of critical staphylococcal genes using conditional phenotypes generated by antisense RNA.</a> Science. 293, 2266-2269 (2001).</li><li>Somerville, G. A., Beres, S. B., Fitzgerald, J. R., DeLeo, F. R., Cole, R. L., Hoff, J. S., Musser, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+serial+passage+of+Staphylococcus+aureus:+changes+in+physiology,+virulence+factor+production,+and+agr+nucleotide+sequence.">In vitro serial passage of Staphylococcus aureus: changes in physiology, virulence factor production, and agr nucleotide sequence.</a> J Bacteriol. 184, 1430-1437 (2002).</li><li>Tseng, C. W., Kyme, P., Low, J., Rocha, M. A., Alsabeh, R., Miller, L. G., Otto, M., Arditi, M., Diep, B. A., Nizet, V., Doherty, T. M., Beenhouwer, D. O., 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=Staphylococcus+aureus+Panton-Valentine+leukocidin+contributes+to+inflammation+and+muscle+tissue+injury.">Staphylococcus aureus Panton-Valentine leukocidin contributes to inflammation and muscle tissue injury.</a> PLoS One. 4, e6387-e6387 (2009).</li><li>von Kockritz-Blickwede, M., Rohde, M., Oehmcke, S., Miller, L. S., Cheung, A. L., Herwald, H., Foster, S., Medina, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+mechanisms+underlying+the+genetic+predisposition+to+severe+Staphylococcus+aureus+infection+in+the+mouse+model.">Immunological mechanisms underlying the genetic predisposition to severe Staphylococcus aureus infection in the mouse model.</a> Am J Pathol. 173, 1657-1668 (2008).</li></ol>

<ol><li>小鼠的皮肤及软组织感染模型是一个强大的工具，在体内的病原体毒力评估。 s 的致病性皮肤及软组织感染的金黄色葡萄球菌可能会有所不同，取决于一些参数。这些措施包括：接种量，细菌的生长阶段，接种的深入，对小鼠的年龄，和小鼠的遗传背景 7 ，8。研究毒力的功能，使用这种模式时，关键是这些参数控制，以确保结果将是一致的。我们已经研究了几株小鼠，包括SKH1（?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>THB</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>95025-314</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>21-031-CV</td>
<td>&nbsp;</td>
<tr>
<td>1 ml syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>309602</td>
<td>&nbsp;</td>
<tr>
<td>27G1/2 needle</td>
<td>&nbsp;</td>
<td>BD</td>
<td>305109</td>
<td>&nbsp;</td>
<tr>
<td>Sheep Blood Agar (TSA)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>90004-328</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

subcutaneous infection of methicillin resistant staphylococcus aureus (mrsa)

MRSA是一种全球范围内对公众健康构成威胁，现在占一半以上在美国的所有软组织感染MRSA的皮肤及软组织感染。在软组织感染，肌炎，pyomyositis，坏死性筋膜炎已被越来越多的报道在协会与MRSA从社会中产生的。要了解MRSA和宿主免疫导致更严重的感染之间的相互作用，动物模型的可用性是至关重要的，允许主机和细菌因素的研究。几个感染模型已被引入到评估的S.发病在浅表皮肤感染的金黄色葡萄球菌。在这里，我们描述，检查皮肤，皮下和肌肉病理的皮下感染模型。

Watch this Scientific Journal Video about Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA at JoVE.com

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

利用小鼠的皮肤及软组织感染模型，以评估耐甲氧西林金黄色葡萄球菌（MRSA）和宿主免疫反应的毒性作用。在这里，我们提出了一个皮肤及软组织感染，皮下感染模型。

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 ...

subcutaneous-infection-methicillin-resistant-staphylococcus-aureus

Research

JoVE Journal

Immunology and Infection

Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus (MRSA)

22.8K 次观看.  Cedars-Sinai Medical Center. 利用小鼠的皮肤及软组织感染模型，以评估耐甲氧西林金黄色葡萄球菌（MRSA）和宿主免疫反应的毒性作用。在这里，我们提出了一个皮肤及软组织感染，皮下感染模型。

视频: Subcutaneous Infection of Methicillin Resistant Staphylococcus Aureus MRSA

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

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.

??PCR?????????????????????MEC??????V<em&gt;???????</em

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. ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

Establishment and Optimization of a High Throughput Setup to Study Staphylococcus epidermidis and Mycobacterium marinum Infection as a Model for Drug Discovery

This video article describes the high throughput pipeline that has been successfully established to infect and analyze large numbers of zebrafish embryos providing a new powerful tool for compound testing and drug discovery using a whole animal vertebrate organism.

建立和高通量安装程序研究优化<em&gt;表皮葡萄球菌</em&gt;和<em&gt;分枝杆菌</em&gt;感染作为模型药物发现

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening ...

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 ...