この作品は、アメリカの心臓協会[助成金SDG 0630219NとAID 09GRNT2180065にYQX] [ASBに助成R01AI-39108]米国国立衛生研究所によってサポートされていました。

1。感染のMRSA菌株を準備する<ol><li>大豆寒天（TSA）平板トリプチケースヒツジ血液（特定の試薬や機器の表を参照）、-80の在庫管からのMRSAの文化の白金耳°Cを接種し、37℃で一晩インキュベートします。 </li><li>全く汚染がないことを確認する血液寒天プレート（同じようなコロニー表現型）に文化の純度を確認してください。 </li><li>ヒツジ血液TSAプレートから1コロニーを採取し、15ミリリットルスナップキャップチューブに5ミリリットルトリプチケースソイブロス（TSB）にコロニーを接種する。 </li><li> 200 rpmで振盪しながら37℃で一晩インキュベートします。 </li></ol> 2。手術カテーテルの準備 10cmの長さに、滅菌鉗子で先端を押すことによって、一方の端を溶かし、ポリエチレンチューブ（Becton Dickinson社製、注文番号427401。PE10）を切り取ります。カテーテルの一端を封止する目的は、カテーテル検査中に出血を避けるためです。 3。事前のurgeryの準備と麻酔<ol><li>イソフルランと酸素の混合ガス（50％：50％）を含むチャンバー内で行われたSprague-Dawleyラット（ハーラン、インディアナポリス、インディアナ州の女性、250〜300グラム）は、麻酔薬が有効になるまで（例えば、筋肉が緩和され、ペダルの反射は存在しない）、およびガス混合物と、手術中に麻酔状態の動物を維持します。 </li><li>顎からちょうどBetadine、70％エタノールで胸骨下の首のエリアを清掃してください。 </li></ol> 4。外科的処置<ol><li>手術中無菌テクニックを使用しています。胸骨上に首だけの皮膚層を介して垂直に切開（1〜1.5 cm）を確認します。 </li><li>鈍的切開を使用して、湾曲した歯鉗子の2ペアを使用して、右頸動脈を露出させる筋膜を分離します。 </li><li>そっと首キャビティの動脈をプルアップ、シルク縫合糸の代わりに2つの10センチの長さ動脈の下に、露出した頭部の端に動脈を縛る、アルテにクリップを配置出血を防ぐために、RY。 </li><li>カテーテルイントロデューサー（Becton Dickinson社製、注文番号406999）を使用して、動脈の上部に小さな穴を作り、動脈の穴から鉗子でカテーテルを挿入し、クリップを削除し、心臓に向かってカテーテルをプッシュダウンするまでの抵抗満たされている。 </li><li>動脈の尾側端周りに緩い縫合糸を結ぶとによって決定されるカテーテルが所定の位置にあるときに絹縫合糸で固定カテーテルを固定します。i）カテーテルの長さ（4-5センチ）を挿入し、ⅱ）更なる発展への抵抗及びiii）のハートビートとカテーテルの脈動。実験の残りの場所にカテーテルを残す。 </li><li>過剰な縫合糸、カテーテルの端をカットし、カテーテルからの出血がないことを確認してください。首の皮膚の下に緩い端を押し込むと皮膚のクリップで皮膚を閉じます。 </li><li>麻酔から回復するまで暖かい場所にケージにラットを入れ、食料や水を提供しています。中に頻繁にラットをチェックして、後、麻酔からの回復。 </li><li>この手術は &quot;カテゴリE&quot;の手順です。痛みを生成手順は、動物に苦痛を伴うMRSA感染以来、鎮痛薬によって単調です。 </li></ol> 5。 MRSA感染<ol><li>感染症は、1と7日後に手術との間で行われるが、実験内での一貫性を保つことができます。 </li><li>クリーンラットの70％エタノールで尾、所望の細胞数（4月 10日から6月 10日までCFUほとんどの黄色ブドウ球菌株のために/動物）で0.5ミリリットルMRSAを注入します。尾静脈から静注し27 G 1/2インチの針で注意：MRSA培養はバイオセーフティーレベル2（BSL2）に属しており、人員や環境への適度な潜在的な危険を持っています。 </li><li>止血は、ケージにラットを返す前に発生するまで、サイトでの圧力を保持します。 </li></ol> 6。ラットおよび文化標的組織を生け贄に捧げる<ol><li>ペントバルビタールナトリウム（200 mgの腹腔内注射によってラットを犠牲にする/ kg）を1から6日後に感染後。 </li><li>彼女の背中にラットを置き、70％エタノールで胸を拭いてください。 </li><li>胸骨下の胸のV字型の切開を行い、心臓を露出させる胸骨の両側に肋骨の軟骨を切った。 </li><li>優しく心をプルアップし、心臓組織を介してクリップに近い大動脈へと心を解放するまで解剖する。 </li><li> 4x4のインチガーゼ内部に滅菌ペトリ皿に心を置き、左心室内壁カットスルーを行い、左サイド室を開きます。 </li><li>カテーテル留置のために視覚的に確認してください。調べ、ハサミとピンセットでバルブから植生を削除します。 </li><li>重さと均質化植生し、定量培養のためにPBSで段階希釈を行います。 </li></ol> 7。代表的な結果すぐに動脈にカテーテルを挿入し、心臓に向かって4〜5センチメートル約カテーテルを押し下げた後、抵抗値がexperienになりますCED。全く抵抗が検出されていない場合は、カテーテルが正常にカテーテルの配置に影響を与える可能性が心腔の左側に挿入されていない可能性があります。以前に9を公開し、適切なカテーテル留置は、 図1に示されています。 MRSA感染のサンプルの一部は、感染サンプルの感染率と純度の正確な生菌数を確認して定量的に培養する必要があります。さらに、植生から回収された生物は、接種で使用されているものと同じである必要があります。 表1は、Sの病原性の例を示し9以前に発行されたラットの心内膜炎モデルにおける黄色ブドウ球菌株 。感染症は、高Sでendocarditsを開発した後、すべての動物は10 5 10 6 CFUの接種で挑戦して3から6日の間に犠牲に心臓の疣贅と同様に、腎臓および脾臓における黄色ブドウ球菌の密度（ <st栄&gt;表1）。無菌バルブ培養したラットは感染していないと見なされます。 <img alt="図1" src="/files/ftp_upload/3863/3863fig1.jpg" /> 図1。カテーテルは正しい場所（心腔の左側）にあり、多数の疣贅は、大動脈弁9の周りに表示されます。 <table border="1"><tbody><tr><td> 接種（アニマス数） </td><td colspan="3"> 組織の10 CFU / gをログに記録平均値±SDで： </td></tr><tr><td></td><td> 植生 </td><td> 腎臓 </td><td> 脾臓 </td></tr><tr><td> 10 6 CFU /動物（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 /動物（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 /動物（7） </td><td> 3.46±0.50 * </td><td> 1.81±0.74 * </td><td> 1.58±0.59 * </td></tr></tbody></table> SD、標準偏差、P &lt;0.001 10 5 10 6 CFU-挑戦の動物に関して。 表1。S.ラットの心内膜炎モデル9の異なる接種と心臓の植生における黄色ブドウ球菌の密度。

<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. 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利害の衝突が宣言されません。

ラットの心内膜炎は、細菌感染症9-11の治療における病態と抗菌薬のin vivo試験のために重要で、十分に特徴付けられた動物モデルである。さらに、ラットの心内膜炎モデルでは、急性および亜急性感染症の複合を表しており、密接に人間相手と人間の自然弁心内膜炎に似ています。また、住居内のカテーテルを伴って、それは古典的なバイオ関連感染症、臨床12一般的?...

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

心内膜炎を含む血管内感染症は、生命を脅かす感染症症候群は1-3である。 黄色ブドウ球菌でも、適切な抗菌剤の治療4-6容認できないほど高い罹患率と死亡率とそのような症候群の最も一般的なワールドワイドな原因となっています。メチシリン耐性S.による感染の増加黄色ブドウ球菌 （MRSA）は、バンコマイシンの臨床治療の障害とリネゾリドおよびダプトマイシン抵抗の増大の問題率の高さは、このような感染症患者のそれ以降のすべての複雑な管理を持っており、高い医療費の7、8に導いた。さらに、それは抗生物質による治療の成果と最新の研究が臨床現場で基づいているため、よく患者への患者から様々な宿主因子によって影響を受ける可能性があることが強調されるべきである。したがって、宿主因子は、動物から動物から類似しているでは血管内感染症の適切な動物モデルは、より多くのです。微生物の病原性と同様に、新規の抗菌剤の有効性を検討することが重要。ラットにおける心内膜炎は、密接に人間の自然弁心内膜炎を近似する十分に確立された実験動物モデルである。このモデルは、特定のブドウ球菌病原性因子の役割とブドウ球菌性心内膜炎のため抗生物質の治療レジメンの有効性を調べるために使用されています。本稿では、細菌の病原性と抗生物質治療に対する反応を調査するために使用することができる。MRSAによる実験的心内膜炎モデルを説明します。

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

メチシリン耐性による実験的ラットの心内膜炎モデル<em&gt; S.黄色ブドウ球菌</em&gt;。

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 ...

experimental-endocarditis-model-methicillin-resistant-staphylococcus

Research

JoVE Journal

Immunology and Infection

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

15.2K ビュー.  Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center. メチシリン耐性による実験的ラットの心内膜炎モデル S.黄色ブドウ球菌。

ビデオ: 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.

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

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.

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 ...

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.

移入、抗原チャレンジにより実験的自己免疫性脳脊髄炎（EAE）抵抗性マウス系統の応答がないことを克服する

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.

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. ...

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 ...

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.

マウスのモデルを評価する生得の免疫反応黄色ブドウ球菌感染症

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