この作品は、バロウズ、ウェルカムキャリア賞でとGY劉への保健助成金AI074832の国立研究所によってサポートされていました。

1。感染のためにMRSAを準備する（感染の2日前） <ol><li>株式の培養液から血液寒天（トリプチケースソイ寒天培地（TSA））プレートへのMRSAの白金耳を接種する。 </li><li>血液寒天プレート上に溶血の表現型を（それぞれのコロニーの周りをクリアゾーン）を確認してください。 </li><li>他の実験との整合性溶血性表現型を持つコロニーを選択してください。 </li><li> 15 mLのスナップキャップチューブに、充当抗生物質、必要に応じて、3 mLのトッドヒューイットブイヨン（THB）にコロニーを接種。 </li><li> 220rpmで振盪しながら37℃で一晩インキュベートする。 </li></ol> 2。感染のためにマウスを準備する（感染前日） <ol><li>マウスのサイズに応じて、マウスの裏面に3 × 4 cmのエリアから毛皮を剃る。 </li><li>剃毛エリアに〜5 mm 3の脱毛剤クリームを（ローカルドラッグストアから購入）を適用します。 </li><li>脱毛剤クリームは約1分間皮膚表面にインキュベートすることができます。 </li><li>のddH 2 Oのウェットペーパータオル</li><li>ぬれた紙タオルで脱毛​​剤クリームをふき取ってください。 </li></ol> 3。 （感染の日に）感染のためにMRSAの準備<ol><li> 50mLのスクリューキャップチューブに、予め温めておいたTHB 10mLで1:500で1:1000に一晩細菌培養液を希釈する。 </li><li> ° Cで約2.5時間540が 2.5に達するまで、220rpmで振盪しながら37℃でインキュベートする。 </li><li> 4℃で10分間3225 × gで遠心分離によってMRSAを収集℃に</li><li>上清を捨てる。 </li><li> 10mLのダルベッコのリン酸塩バクテリアペレットを再懸濁さ生理食塩水（DPBS）はバッファ。 </li><li>手順3と4を繰り返します。 </li><li>所望の濃度でDPBSでバクテリアペレットを再懸濁します。 </li><li>シリアル10 1 10 8に細菌懸濁液を希釈する。 </li><li>血液寒天プレート上にプレート希釈細菌懸濁液を。 </li><li> 37℃で一晩培養する。 </li><li>接種材料の溶血性表現型を観察し、記録する。 </li><li>プレート上でコロニー形成単位（CFU）の数カウント。 </li><li>プレート上のCFUの数に基づいて接種を計算する。 </li></ol> 4。 MRSAの皮下感染（感染日と3日後の感染症について） <ol><li>剃毛面積の皮下100μL細菌懸濁液を注入する。 </li><li>マウスが生きていることを確認するため、注入後3〜5時間のために動物を観察。 </li><li>毎日の動物の背面にある病変を観察し、それが必要なときに毎日の病変面積を記録。病変の観察は、病変のサイズと形態の記録を含める必要があります。皮膚と筋肉の病変の両方は、病変の長さと幅を乗じて定量される。不規則な形状の病変は、より小さな対称の部分に分割し、各部分が同一の方法で測定する必要があります。 、病変の測定には、コンピュータ支援組織形態評価プログラム（でNIHから入手できるオープンソースのImageJを使用することができます<a href="http://rsb.info.nih.gov/ij">http://rsb.info.nih.gov/ij/</a> 2）。 </li><li>頸椎脱臼が続くイソフルランの吸入により3日目、感染後に動物を生け贄に捧げる。 </li><li>皮膚病変を観察し、記録する。 </li><li>滅菌はさみでニックは皮膚。 </li><li>注意深くオフ皮膚の皮と筋肉で病変を観察し、記録する。 </li><li>消費税​​病変と約2 - 周辺地域から5 mm。 </li><li>脾臓と腎臓を収集する。 </li><li>組織ホモジナイザーを使用して組織をホモジナイズまたは組織とDPBSの100μLを含むマイクロチューブに1 mLのシリンジからプランジャーを繰り返し適用し。 </li><li>各チューブにDPBS 900μLを加える。 </li><li> 5分間ボルテックスのサンプルを。 </li><li>シリアル10 6にDPBS 10 1との希薄懸濁液。 </li><li> THAプレート上にプレートに希釈懸濁。 </li></ol> 5。代表的な結果： <ol><li>すぐにそれぞれの皮下注射後、ブレブは感染が（図1A）が正しく行われているときに皮膚表面に見られます。何ブレブが皮膚表面で観測されていない場合は、注射が深すぎるかもしれない、とこれは（病変サイズ、CFU）感染症の転帰に影響を与える可能性があります。 </li><li>細菌懸濁液は希釈する必要があるとCFUは、各実験のための血液寒天平板上で検討する必要があります。接種材料は、均質な表現型を持っているかどうかを1）;感染症2）正確な生菌数：このステップでは、重要な情報が得られます。 </li><li>病変と細菌の生存には様々な時点で、感染後に評価することができる。ここで、我々は3日、感染後に感染を調べた。と筋肉の表面（図1C上：1 × 10 9：;：病変サイズは皮膚の表面（図1D 1 × 10 9 1 × 10 7 CFU図1B）で分析されています</s上&gt;、図1E：1 × 10 7 CFU）。病変面積は、（mm）×幅（mm）の組織の損傷（図2、図B）を評価するために測定される長さと等しくなります。この結果は、特定の病原性因子による組織の損傷の程度を判断するのに役立ちます。さらに、病変を切除し、DPBS中でホモジナイズし、感染部位での細菌の生存率を示すCFUの数を（図2C）、定量するためにメッキされています。 </li></ol><img src="/files/ftp_upload/2528/2528fig1.jpg" alt="図1" /> 図1。皮膚と筋肉の病変。CD1マウスは、MRSA（LAC）を持つ複数脇腹の皮下に接種した。写真は、時間0と3日目、感染後で撮影したもの。 （）時間0感染後（PI）、（B） - （G）3日目π、B、D.とF：皮膚病変、C、E、およびG：筋肉病変、BとC：1 × 10 9 CFU感染、DとE：1 × 10 7 CFU感染、FとG：モック。 <img src="/files/ftp_upload/2528/2528fig2.jpg" alt="図2" /> 図2。皮膚病変の大きさと生菌は3日、感染後に感染部位の数でカウント。MRSA（LAC）に感染したCD1マウスの皮膚と筋肉の病変。 （A）皮膚病変の大きさ。 （B）筋病変の大きさ。 （C）全組織CFU。 H：1 × 10 9 CFUの接種、L：1 × 10 7 CFUの接種。 *：P &lt;0.05、Mann - Whitney検定。

<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>

利害の衝突は宣言されません。

<ol><li>マウスの皮膚および軟部組織感染症モデルでは、病原体の生体毒性評価のための強力なツールです。 S.の病原性皮膚および軟部組織感染症の黄色ブドウ球菌は、パラメータの数によって異なります。これらは、接種量、細菌の増殖期、接種の深さ、マウスの年齢、およびマウスの遺伝的背景7、8が含まれています。このモデルを用いて病原性の機能を調べると?...

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subcutaneous infection of methicillin resistant staphylococcus aureus (mrsa)

MRSAは、公衆衛生の世界的な脅威であり、MRSAの皮膚および軟部組織感染症は現在、より多くの米国内のすべての軟部組織感染症の半分以上を占めている。間の軟部組織感染症、筋炎、化膿性筋炎、および壊死性筋膜炎は、ますますMRSAが地域社会から生じると関連して報告されている。 MRSAと、より重篤な感染症につながる宿主免疫間の相互作用を理解するために、動物モデルの可用性は、宿主と細菌の要因の調査を可能にする、非常に重要です。いくつかの感染症のモデルがSの病因を評価するために導入されている表在性皮膚感染症の間に黄色ブドウ球菌 。ここで、我々は、皮膚、皮下、および筋肉の病理を調べる皮下感染モデルを説明します。

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

マウスの皮膚および軟部組織感染症のモデルは、メチシリン耐性黄色ブドウ球菌（MRSA）およびホストの免疫応答の病原性の機能を評価するために利用されている。ここで、我々は、皮膚および軟部組織感染症のための皮下感染モデルを発表した。

メチシリン耐性黄色ブドウ球菌（MRSA）の皮下感染

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 ビュー.  Cedars-Sinai Medical Center. マウスの皮膚および軟部組織感染症のモデルは、メチシリン耐性黄色ブドウ球菌（MRSA）およびホストの免疫応答の病原性の機能を評価するために利用されている。ここで、我々は、皮膚および軟部組織感染症のための皮下感染モデルを発表した。

ビデオ: 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.

用ドリップ流れと回転円板の原子炉の使用黄色ブドウ球菌バイオフィルム分析

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

免疫・感染学

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.

メチシリン耐性の実験的心内膜炎モデル<em&gt;黄色ブドウ球菌</em&gt;（MRSA）ラットにおける

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.

鉄源としてヒトヘモグロビンを用いた黄色ブドウ球菌の成長

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.

の相互作用を研究<em&gt;黄色ブドウ球菌</em&gt;フローサイトメトリーとタイムラプス顕微鏡による好中球と

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.

多剤耐性を実証<em&gt;結核菌</em&gt;増幅マイクロアレイ

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.

設立し、研究するために、高スループットの設定の最適化<em&gt;表皮ブドウ球菌</em&gt;と<em&gt;マイコバクテリウムmarinumの</em創薬のためのモデルとして&gt;感染症

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.

バイオフィルム関連をターゲット<em&gt;黄色ブドウ球菌</em&gt;レサズリン基づく薬剤感受性アッセイを使用して

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.

のジェノタイピング<em&gt;黄色ブドウ球菌</em&gt;によってリボソームスペーサー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.

で水平遺伝子伝達の研究のための方法論<em&gt;黄色ブドウ球菌</em

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