この研究は、国立アレルギー感染症研究所から米国公衆衛生局助成金AI69233およびAI073843によってサポートされていました。 EPSは、感染症の発症機序におけるバローズウェルカム·フェローでもある。 KPHは5 T32 A107611-10細胞·分子微生物学研修助成プログラムによって賄われていた。

1。新鮮な血液からヘモグロビンの精製<ol><li>抗凝固剤を補充した新鮮なヒト血液を取得します。浄化を通して氷上もしくは4℃で血液℃で保管してください。 </li><li> 1500×gで20分間血液を遠心分離します。赤血球は（赤血球）チューブの底になります。注意深く上清を吸引し、ゆっくりと氷冷0.9％（w / v）のNaCl溶液中でペレットを再懸濁します。遠心分離を繰り返し、3回洗浄します。 </li><li>氷冷10mMトリス-HCl（pH8.0）の1ボリュームにペレットを再懸濁します。これは浸透圧に起因する赤血球の溶解を誘導します。 〜20％の最終容量にトルエンを追加します。 </li><li>一晩ロティサリーで4℃でインキュベートする。 </li><li> 1時間20,000 xgでライセートを遠心分離します。手つかずのトルエン（上フローティング）とペレットを残して真ん中溶血画分を集める。中間留分を収集するために、長い首のピペットを使用しています。 </li><li> 0.44μmのシリンジフィルターを通過します。解決策は、微粒子が含まれている場合物質とは、ステップ1.5を繰り返して、フィルタを通過することはできません。 </li><li>高速液体クロマトグラフィー（HPLC）、陰イオン交換カラム（Varian、PL-SAX千オングストロームは8μm、150ミリメートル×4.6ミリメートル）とヘモグロビン（Hb）を浄化する。移動相Bは、10mMトリス-HCl（pH8.0）+0.5 M NaClで10mMのトリス-HCl（pH8.0）および移動相である。溶媒Bの0％-100％の勾配は2.0ミリリットル/分の流量で2分間にわたって実行されます。 （：410 nmと280 nmλ）の溶出が吸収に基づいて監視されています。鮮やかな赤い色と著名な吸収ピーク（ 図1）によって特徴付け一部のみを収集します。 </li><li>に対する溶出を透析するリン酸緩衝再び生理食塩水（PBS）で一晩してから数時間。 0.22μmのシリンジフィルターを通過することにより滅菌する。 </li><li> Hb濃度を測定するために、PBS中の既知濃度の標準ヘモグロビン溶液を調製する。標準溶液を混合することにより、試料中のヘモグロビンの濃度を測定（試薬達とテーブルを参照してください。ed）または1:1の比率で2倍Drabkin試薬（粉末から調製）と試料溶液。例えば、96ウェルプレートに100μlの2倍Drabkinの試薬を用いて、100μlのHb溶液を混ぜる。 540nmで15分間測定吸光度インキュベートする。標準曲線をプロットし、試料中のヘモグロビン濃度を測定します。 5〜15 mg / mlの収率は典型的なものである。 </li><li>重複の15％SDS-PAGE上で15から20μgの精製ヘモグロビンを実行します。ゲルの1を汚し、ヘモグロビンのニトロセルロース膜とイムノブロット（ 図2）の上に別のゲルからタンパク質を転送します。 </li><li>凍結し、液体窒素中でヘモグロビンの1mlのアリコートを格納します。 </li></ol> 2。鉄枯渇増殖培地の調製<ol><li>メーカーと1％cassamino酸（CA）（w / v）のが推奨するように重炭酸ナトリウムを加える、水にRPMI粉末を溶解することによりロズウェルパーク記念研究所（RPMI）ブロスを準備します。 0.2μmフィルターや店舗冷媒を通過させることにより殺菌erated。 </li><li> Chelex 100 7％（w / v）を追加し、撹拌プレート上で一晩混合することにより、金属が枯渇RPMI（NRPMI）を準備します。 0.2μmフィルターや店舗冷蔵を通過することによりChelex 100を削除します。 、25μMZnCl 2を 、無菌の1,000×溶液として予め用意し、25μMのMnCl 2、100 mM CaCl 2および1mMのMgCl 2：本質的な非鉄金属とメディアを補足するものです。再利用可能な電源からの鉄汚染を避けるために、このステップのための使い捨てのプラスチック容器を使用しています。 </li></ol>ソレ鉄源としてヘモグロビンを用いた3。 黄色ブドウ球菌の成長<ol><li>ストリークS.凍結ストックからトリプシン大豆寒天（TSA）は上の分離のための黄色ブドウ球菌 。 20〜24時間37℃でインキュベートする。 </li><li> 100mmに無水エタノールに再懸濁し、エチレンジアミン-N、N&#39;-ビス（2 - ヒドロキシフェニル酢酸）（EDDHA）。 EDDHAは解決策にはなりませんが、エタノールで滅菌される。 </li><li>最終濃度0.5mMにRPMIにEDDHAを追加します。許すEDDHAは、次のステップに進む前に、少なくとも30分間溶解する。バッチ間のばらつきにより、最終EDDHA濃度は、細菌の増殖を可能にする0.25 mMまで低下させることが必要になることがあります。 </li><li> Sの単一コロニーを植菌15ミリリットルのスクリューキャップコニカルチューブでRPMI含むEDDHAの5ミリリットルに黄色ブドウ球菌 。 37℃で16から20時間、毎分180回転（rpm）で振盪しながらCを。 </li><li> 7500×gで5分間、一晩培養を遠心NRPMIが0.5mMのEDDHAを含有するペレットを再懸濁します。 〜3のOD 600を正規化します 。 </li><li> NRPMIはミリリットルHbおよび0.1から1.0 mMのEDDHAあたり2.5μgを含む準備します。バッチ間のばらつきによる、NRPMI内遊離鉄をキレートするために必要なEDDHA濃度は変わる場合があります。 </li><li> 15ミリリットルのスクリューキャップコニカルチューブに1ミリリットルNRPMI EDDHA + + HBにステップ3.5からの細菌懸濁液10μlをサブカルチャー。 </li><li> 180rpmでまたはローリングドラムに振とうしながら、最大48時間まで37℃で培養する。 </li><li&gt;は6月12日すべての時間は、96ウェルプレートで培養液50μlを除去し、PBSを150μlと混合することにより、OD 600測定値を取る。 </li></ol>

<ol>
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我々は、開示することは何もない。

鉄は人生15のすべての国からの生物が必要とする必須栄養素である。脊椎動物では、鉄は、この要素によって引き起こされる毒性を避けるために隔離されている。この隔離はまた、栄養免疫16として知られている過程で微生物が侵入してくるから鉄を隠蔽する。応答では、病原体が栄養免疫を回避する戦略を進化させてきた。そのようなメカニズムの1つは、ホスト17内...

<table border="1"><tbody><tr><td> 試薬/材料の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td> HPLCによる陰イオン交換カラム</td><td>バリアン</td><td> PL1551-3802 </td><td></td></tr><tr><td> Drabkinの試薬</td><td>シグマ</td><td> D5941-6VL </td><td></td></tr><tr><td>ヘモグロビン標準</td><td>ポワント·サイエンティフィック</td><td> H7506-STD </td><td></td></tr><tr><td> RPMI </td><td> Hyclone社</td><td> SH30011.02 </td><td></td></tr><tr><td> Chelex 100ナトリウム型</td><td>シグマ</td><td> C7901 </td><td></td></tr><tr><td> EDDHA </td><td> LGC規格社</td><td> ANC 001 </td><td></td></tr><tr><td>ヘモグロビン抗体</td><td> Santa Cruz Biotechnology社株式会社</td><td> SC-21005 </td><td> &lt;/ TD&gt; </tr><tr><td>トリプシン大豆寒天</td><td> BD </td><td> 236920 </td><td></td></tr></tbody></table>

staphylococcus aureus growth using human hemoglobin as an iron source

黄色ブドウ球菌は、鉄が重要な代謝機能と原因疾患を遂行する必要があり、病原性細菌である。人間のホスト内の鉄の最も豊富な貯水池はヘモグロビンのヘム補因子である。ヘモグロビンから鉄を獲得するには、S.黄色ブドウ球菌は、鉄調節表面決定因子（ISD）システム1として知られている精巧なシステムを採用しています。 ISDシステム最初のバインド、ホスト·ヘモグロビンの構成要素は、次に抽出し、ヘムをインポートして、最終的に細菌の細胞質2,3にヘムから鉄を遊離する。この経路は、in vitro試験4-9 の数多く通って解剖されています。さらに、感染症へのISDシステムの寄与を繰り返しマウスモデル8,10-14で実証されている。ヘモグロビン由来の鉄獲得にISDシステムの寄与を確立し、成長がより困難であることが証明された。唯一の鉄源としてヘモグロビンを用いた増殖アッセイは、b複雑なものyの市販のヘモグロビンの不安定、フリー増殖培地中の鉄、鉄キレート剤に関連する毒性を汚染。ここでは、これらの制限を克服する方法を提案する。高品質のヘモグロビンは、新鮮な血液から調製されており、液体窒素中で保存されています。精製されたヘモグロビンは脊椎動物宿主病原体内部で発生した鉄 - 劣悪な環境を模倣した鉄枯渇培地に補充される。 Sを飢えによって遊離鉄の黄色ブドウ球菌 、我々は、ヘモグロビンを結合するヘムを抽出し、細菌の細胞エンベロープを通ってヘムを通過して細胞質内でヘムを分解する能力に完全に依存した方法で成長を誘発するヘモグロビンの低侵襲操作形で補う。このアッセイは、Sのhemoglobin-/heme-derived鉄獲得のメカニズムを解明しようとしている研究者にとって有用であろうブドウ球菌およびおそらく他の細菌性病原体。

我々は、HPLC（プロトコルステップ1.7）で溶血からヒトヘモグロビンを精製した。 図1は、280および410 nmの波長での溶出液の吸光度を記録した。フラクション5を回収し、他の画分を廃棄した。溶出液のミリリットル当たりのヘモグロビンの五から十五ミリグラムの収量は、一般的に取得される。精製されたヘモグロビンは、重複でSDS-PAGEで分析したところ、ゲルをどちらかのタン?...

Watch this Scientific Journal Video about Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source at JoVE.com

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

ここでは、のための増殖アッセイを記述<em&gt;黄色ブドウ球菌</em&gt;利用可能な栄養素鉄の唯一の供給源としてヘモグロビンを用いた。このアッセイは、ヘモグロビン由来の鉄獲得に関わる細菌因子の役割を確立します。

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 ...

staphylococcus-aureus-growth-using-human-hemoglobin-as-an-iron-source

Research

JoVE Journal

Immunology and Infection

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

16.4K ビュー.  Vanderbilt University Medical School. ここでは、のための増殖アッセイを記述黄色ブドウ球菌利用可能な栄養素鉄の唯一の供給源としてヘモグロビンを用いた。このアッセイは、ヘモグロビン由来の鉄獲得に関わる細菌因子の役割を確立します。

ビデオ: Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

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 ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

特定のヒトと動物のウイルスを用いた微生物ソースのトラッキングのための費用対効果の高い方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

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 ...

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 ...

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes (PMNs or neutrophils). This protocol describes both the purification of human PMNs and the quantification of S. aureus cytotoxicity against PMNs in three different sections. Section 1 details the isolation of PMNs and serum from human blood using density centrifugation. Section 2 tests the cytotoxicity of extracellular proteins produced by S. aureus against these purified human PMNs. Section 3 measures the cytotoxicity against human PMNs following the phagocytosis of live S. aureus. These procedures measure disruption of PMN plasma membrane integrity by S. aureus leukocidins using flow cytometry analysis of PMNs treated with propidium iodide, a DNA binding fluorophore that is cell membrane impermeable. Collectively, these methods have the advantage of rapidly testing S. aureus cytotoxicity against primary human PMNs and can be easily adapted to study other aspects of host-pathogen interactions.

Quantifying the Cytotoxicity of Staphylococcus aureus Against Human Polymorphonuclear Leukocytes

This protocol describes a method for the purification of polymorphonuclear leukocytes from whole human blood and two distinct assays that quantify the cytotoxicity of Staphylococcus aureus against these important innate immune cells.

ヒト多形核白血球に対する黄色ブドウ球菌の細胞毒性の定量

Staphylococcus aureus is capable of secreting a wide range of leukocidins that target and disrupt the membrane integrity of polymorphonuclear leukocytes ...