NIAIDの助成金K22AI081748。

1。ドリップフローバイオフィルムリアクター<ol><li>ドリップフローバイオリアクターが組み立てとオートクレーブされた（Biosurface技術またはカスタム設計されたバージョンから利用可能な通常の大学のマシンのお店で作ることができる、図1を参照してください）​​。アセンブリは、チャンバーと確保室の蓋に貼るクーポンを伴います。バイオフィルム培地（トリプシン大豆ブロス2グラム/ Lとグルコース2グラム/ L）、および流入栄養チューブと一緒にチャンバーは、オートクレーブによって滅菌されています。 </li><li>ドリップフローリアクターの接種は、平らな面に原子炉を配置排水配管ラインをクランプ、10 mLのトリプシン大豆ブロスでそれぞれのチャンバーに充填し、S.の10μLを加えることによって予備成形され黄色ブドウ球菌の培養には、トリプシン大豆ブロス中で一晩増殖。接種した原子炉は、18時間、37℃インキュベーター内の場所です。 </li><li>インキュベーションの18時間後、排水チューブがアンクランプされ、反応器は10 °の角度にカット木のブロックに配置されます。 </li><li>無菌的に連続的なフロー栄養ブロスを含む瓶に流入栄養チューブを接続してください。最大速度（ポンプのモデルによって異なります）でポンプを実行することにより、ポンプやプライムチューブを介してチューブラインを通します。 </li><li>一度流入管は、ポンプを停止し、各チューブの端に接続針（22ゲージ、1インチ）を取り付ける準備され。エタノールワイプと無菌的に口栓に針を挿入するとチャンバーの入口ストッパーを拭きます。 </li><li>ポンプの電源を入れ、クーポンを介して（流量〜125μL/分）メディアは徐々に滴下することができます。メディアは入口ストッパーのポートからの排水ポートにクーポンに沿って下方に流れる必要があります。時折、適切な排水のために原子炉をチェックし、2〜5日（アプリケーションに応じて）連続的な流れで原子炉を操作してください。 </li><li>ドリップ流反応器のバイオフィルムを回収するために、ポンプを停止し、慎重に反応器から針を取り除く。次いで反応器を平らな面に置くことができ、クーポンを無菌的に滅菌ピンセットを使用して削除することができます。顕微鏡が必要な場合は、クーポンが今それに応じて処理することができる（図2Bは、ドリップのバイオリアクターで成長させた黄色ブドウ球菌のバイオフィルムの走査型電子顕微鏡写真である）。バイオフィルムのバイオマスや生理学の研究の定量化は、研究の目的である場合、バイオフィルムは、セルスクレーパーを使用してクーポンから削除することができます。鉗子でクーポンを押しながら、静かにセルスクレイパーを用いてリン酸緩衝生理食塩水を含むコニカルチューブにクーポンからバイオフィルムをこすり落とす。注：バイオフィルムのコロニー形成単位を定量化するためには、それは、塊を分解し、均一な懸濁液を形成するために組織ホモジナイザーで収穫されたバイオフィルムを均一にする必要があります。組織ホモジナイザーの様々なモデルがこのアプリケーションに適しています。我々は、バイオフィルム試料を均質化するため1分間、フルスピードでフィッシャーサイエンティフィックTissuemiserホモジナイザー（製品＃15-338-420）を利用。バイオフィルムを均一に失敗すると、サンプル中に存在するコロニー形成単位の過小評価になります。 </li></ol> 2。回転円板のバイオフィルムリアクター<ol><li>回転ディスクのバイオリアクターは（作られた、図3を参照Biosurface Technologies社から入手できるから入手するか、カスタムすることができます）組み立てとオートクレーブされている。回転するディスクのスロットに最初の場所で回転するディスクのクーポンをリアクタを組み立て、オーバーフローのポートを備えた1リットルのガラスビーカーに置きます。メディアフローと曝気を許可するようにあけられた穴を持つ数15ゴム栓は、原子炉のキャップとして使用されます。リアクター、バイオフィルムメディア（トリプシン大豆ブロスを2g / L、グルコース2g / Lの）、およびインレットチューブはその後オートクレーブによって滅菌されています。 </li><li>回転ディスクのバイオリアクターは、リアクターに無菌培地250 MLSを配置することにより接種し、一晩S.の0.5 mLを加えている黄色ブドウ球菌の培養には、トリプシン大豆ブロス中で増殖させた。次いで反応器を250回転に設定されて撹拌プレート上に配置し、所望の温度で一晩インキュベートする。 </li><li>インキュベーションの16時間は、メディアタンクのポートにインレットチューブを接続し、蠕動ポンプに接続した後。ポンプは、システムの電源をオンにします。されて​​おり、流れは〜0.25 mL /分（フローが望ま成長率に応じて変更可能）に設定します。 </li><li> 24時間後に原子炉を停止し、無菌的にバイオフィルムのクーポンを触れることなく、ディスクを取り出します。滅菌ピンセットを使ってクーポンを削除し、リン酸にそれぞれを浸すにはどんな緩く付着細菌を除去するために緩衝生理食塩水。 </li><li>抗菌性化合物のテストのために、チップは、関心のある化合物を含有する96ウェルプレートの個々のウェルに配置することができます。インキュベーション後、チップを1 mLのリン酸緩衝生理食塩水を含む1.5 mlのマイクロチューブに移し、そのままバイオフィルムを分散させるためにホモジナイザー組織でホモジナイズ。 </li><li>次いで、細胞をシリアル希釈し、実行可能なコロニーformiを決定するために栄養寒天培地上にプレーティングされていますngの単位。 </li><li>注：多くの変化が上記のプロトコルで行うことができます。有効性をテストする例については、回転するディスクのクーポンをコーティングすることができるまたは潜在的な抗バイオフィルムの化合物から作ら。バイオフィルムの分散剤は、分散剤の化合物と接続されている対切り離さ定量化する細菌のクーポンをインキュベートすることを評価することができる。 </li></ol> 3。代表的な結果ドリップ流反応器までセットの例を図1の場合は示されています。流れの三日後にバイオフィルムのおびただしい量は、クーポンの表面に図2Aを蓄積することになります。総バイオマスは菌株と正確な成長条件によって異なります。 S.の走査型電子顕微鏡写真ドリップフロー反応器内で栽培ブドウ球菌のバイオフィルムは、図2Bに示されています。 回転するディスクの原子炉は、図3Aに示されています。記述されたプロトコルは、バイオフィルムを形成することが可能な一般に任意の微生物の特定の要件に適合させることができます。図3Bは、18プラスチック製のディスクが添付さで回転するディスクを示しています。これらのディスクに成長したバイオフィルムは、特に抗菌性試験に適しており、再現性の高い結果が11を得ています。 <img src="/files/ftp_upload/2470/2470fig1.jpg" alt="図1" /> 図1。ドリップ流反応器のセットアップ。重要なコンポーネントが表示されています。 <img src="/files/ftp_upload/2470/2470fig2.jpg" alt="図2" /> 図2。ドリップフローの例S.黄色ブドウ球菌のバイオフィルム。 A）このバイオフィルムは、記述されているプロトコルは、次の三日間成長させた。原子炉の最初の二つのチャンバーからのふたは黄色S.を表示するために削除されます。 黄色ブドウ球菌のバイオフィルムのバイオマス。 S.のB）Sacnning電子顕微鏡写真黄色ブドウ球菌のバイオフィルムは、ドリップフロー反応器内で栽培。 <img src="/files/ftp_upload/2470/2470fig3.jpg" alt="図3" /> 図3。回転ディスク型原子炉。 A）実行されている回転ディスクの原子炉の例。キーコンポーネントが表示されています。 B）回転するディスクのビューをクローズアップ。

<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. 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利害の衝突は宣言されません。

別の炉で成長させたバイオフィルムは、多くの異なる特性を持つことになりますし、各反応器は、異なるアプリケーションを持っています。ドリップフローバイオリアクターと回転ディスク型原子炉：この作品では、我々は2つ​​のバイオフィルムリアクターの使用を説明しています。フロー炉ドリップ空気 - 液体界面での低せん断バイオフィルムを成長させるために有用であり、様々な条...

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

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

自然の中で最も微生物が1細菌のバイオフィルムは、マトリックス内に収められています。バイオフィルムの表面に関連したコミュニティとして存在すると考えられ、表面2バイオフィルム形成と発展に添付されるかは、一般的にこのようなマイクロタイタープレートやフローなどのバッチシステムを使用して実験室で検討されていますこのようなフローセルなどのシステム、。これらの手法は、可視化（フローセル）4の（マイクロタイタープレート）3または成長するバイオフィルムの変異体、化学ライブラリーをスクリーニングするのに便利です。ドリップフローバイオリアクターと回転するディスクのバイオリアクター：ここでは、フローシステムのバイオフィルムの追加の2種類で成長している黄色ブドウ球菌の詳細なプロトコールを提示する。 ドリップフローバイオリアクターは、低剪断条件下で栽培したバイオフィルムの研究のために設計されています。5ドリップ流反応器は、4つの並列テストチャネル、一つの標準ガラスの顕微鏡スライドサイズのクーポンを持ってそれぞれの能力、またはカテーテルまたはスティントの長さで構成されています。ドリップ流反応器はマイクロセンサのモニタリング、一般的なバイオ研究、バイオフィルムcryosectioningサンプル、高いバイオマス生産、医療材料の評価、および留置医療機器のテストに最適です。6,7,8,9は 回転するディスクの原子炉は、取り外し可能なクーポンのための凹部を含むテフロンディスクで構成されています。10リムーバブルクーポンは、任意の被削材から作らできます。回転ディスクの底面は、ディスクの回転が表面フラッシュクーポンを越え液体表面のせん断を作成できるように棒磁石が含まれています。 18クーポンを含むディスク全体を1000mLのガラスサイドアームの原子炉容器内に配置されます。ディスクは、マグネチックスターラーで回転させながら液体増殖培地は、血管を通して循環される。クーポンは、原子炉容器から除去し、さらに研究や顕微鏡イメージングのためのバイオフィルムのサンプルを収集するようにこすりされています。回転するディスクの原子炉は、殺生物剤の有効性、バイオフィルムの除去、及び防汚材料の性能の実験評価用に設計されています。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

ドリップ流反応器と回転するディスクの原子炉とオープンシステムの流れのバイオフィルムを利用するためのプロトコルを詳細に表示されます。

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 ...

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 ビュー.  University of Michigan. ドリップ流反応器と回転するディスクの原子炉とオープンシステムの流れのバイオフィルムを利用するためのプロトコルを詳細に表示されます。

ビデオ: 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).

フローセル内の緑膿菌と出芽酵母のバイオフィルム

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

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 ...

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. ...

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 ...

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).

全血によって人間の Monocyte のサブセットの解析フローのフローサイトメトリー解析

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