This work was partly supported by Takeda Science Foundation, Pfizer Academic Contribution and JSPS Postdoctoral Fellowship for Foreign Researchers (FC).

注:在这项工作中使用的菌株和材料示于表1分别列出与材料的表 。在传输实验，N315和COL CFR的阳性衍生物被用作CFR的基因的供体（N315-45和COL-45）。这些菌株先前由缀合得到的，用作为供体的临床CFR的阳性金黄色表皮应变（ST2），按照标准缀合协议（见下面）。这株窝藏在pSCFS7般的质粒7 CFR基因。 1.共轭使用滤波器交配方法注意:一个金黄色葡萄球菌 N315菌株携带CFR的基因（N315-45）7（厘米R）被用作供体。 COL 8或Mu50 9株（四环素R，厘米S）被用作收件人。双-抗性菌落（四环素R，厘米R）能够以32毫克/氯霉素加8毫克L存在下生长/ L四环素被视为推定转导和分析通过菌落PCR来确定CFR的存在，并确定收件人易感性。 <ol><li> 以下先前描述的方案10提供了一些修改执行偶联: <ol><li>制备5毫升的胰蛋白酶大豆肉汤（TSB）培养基（有和没有32毫克/升氯霉素，分别）供体和受体的过夜培养物，在37℃振荡。 </li><li>通过使用新鲜TSB培养基调整过夜培养物以1（OD 600 = 1.0）的光密度（OD 600）。混合0.5mL的供体培养物（N315-45）的用0.5mL收件人培养的（COL或Mu50）。 1ml的PBS添加到混合物中。 </li><li>细菌的混合物转移到0.45μm的滤膜USI纳克真空泵系统。 </li><li>把滤膜上的绵羊血琼脂平板上。孵育在37℃下1天。 注:在这一步骤，富集介质是必要的，以允许抗双细胞的生长。 </li><li>从板中取出滤膜并在10ml磷酸盐缓冲盐水（PBS）的暂停。涡流以及收集附着在膜上的所有细菌。 </li><li>通过使用新鲜的TSB使细菌悬浮液的连续10倍稀释。板100微升上的TSB琼脂0-10 -4稀释的样品（TSA）平板上补充有32毫克/升氯霉素和8毫克/升四环素为转导的选择。板100微升稀释悬浮液（10 -5 -10 -6）到TSA平板单独用8毫克/升四环素的计数收件人的总数。孵育所述板在37℃下18-24小时。 </li><li>对于比照的存在分析抗性双菌落（推定的转导结合体）菌落PCR 7和敏感性图（抗菌谱）R基因。 </li><li>通过根据标准微量稀释法或纸片法11测定的适当的抗生素的最低抑菌浓度（MIC）测定抗菌素谱。在转导的易感性必须与接受者，除了氯霉素（和受CFR基因其他化合物）。这个决定是必要的，以排除由供体菌株开发的四环素抗性。 </li></ol></li></ol> 2.噬菌体转注:属于长尾噬菌体科家族（实验室股票）噬菌体MR83a在转导实验中使用。 N315-45用作噬菌体感染的CFR的供体。 N315，COL，或Mu50菌株用作收件人。 CFR的收购是由AB确定受体菌株的ility在32毫克/升氯霉素的存在下生长。菌落在此条件下生长进行了分析，以确定CFR的存在（通过菌落PCR），并确定易感性排除潜在的污染。 <ol><li> 捐助噬菌体放大 <ol><li>在5毫升营养肉汤中于37℃补充有3.6毫米的Ca 2+（NBCaCl 2）振荡（180转）制备N315-45的过夜培养。 注:钙需要噬菌体感染。请参阅材料表 。从其他公司的营养肉汤可能具有问题，如钙沉淀。 </li><li>在10mL NBCaCl 2的在50毫升玻璃烧瓶或玻璃小瓶，最终体积1000:通过稀释过夜培养1准备传代培养。振荡（180转）生长1小时的细菌在37℃。 </li><li>准备一系列的稀释噬菌体MR83a在NBCaCl 2 MEDI微米（10 -2，10 -3，10 -4，10 -5和10 -6）。加入20μl的稀释噬菌体到细菌培养。还制备无噬菌体感染的控制培养，其用作细菌细胞生长的阳性对照，可以被用来确定第二天噬菌体滴度。 </li><li>生长的培养物在过夜37℃温和振荡（100rpm）下。 注:细胞会生长到一定程度时，感染的噬菌体是不超过;然后，他们将进入裂解阶段由于通过裂解周期噬菌体扩增。无噬菌体的文化将完全显示的增长，而噬菌体的文化会呈现不同的透明度率。 </li><li>选择与添加的噬菌体的最高稀释一种或两种清零培养瓶中。 </li><li>转移培养到15ml离心管中。加入250微升的氯仿和反相管调匀。 </li><li>离心管在5000 <em&gt; xg离心在4℃下20分钟。 </li><li>转移上清液至新管，并将它们在4℃下储存直到使用。 注:噬菌体是稳定至少几个月，但存储噬菌体制备太久降低了效价和转导效率。 </li></ol></li><li> 测量噬菌体滴度（噬菌体斑块检测） <ol><li>制备营养肉汤琼脂（1.5％琼脂）培养基;保持水浴在55℃的温水。蒸压的0.5M的CaCl 2溶液在培养基中加入了3.6毫摩尔的最终浓度。倒入90毫米培养皿（NBCaCl 板 ）。 </li><li>准备N315的过夜培养在37℃5毫升NBCaCl 2的振荡;这可以用上述对照培养（步骤2.1.3）取代。 </li><li>添加10微升过夜培养物于200微升NBCaCl 2和均匀分布其上NBCaCl 2板。停止扩频时的表面板覆盖有液体。让板干燥。 </li><li>使用NBCaCl 2介质中预先准备的噬菌体（步骤2.1.8）的使串行1:10稀释（10 -5 -10 -10）。 </li><li>现货3微升每个稀释度噬菌体覆盖上细菌的板块。 </li><li>过夜孵育所述板在30℃。 </li><li>计数噬斑数量，并使用下列公式计算噬菌体滴度:空斑形成单位（PFU）/ ml的=斑块数×稀释因子/斑体积（3×10 -3毫升） </li></ol></li><li> 噬菌体转 注:在上一步（2.1.8）中制备的噬菌体池用于测试CFR基因转导到金黄色葡萄球菌菌株。在该实验中，菌株N315，COL，或Mu50用作收件人。 <ol><li>振荡（180转）制备N315，COL，或Mu50的5ml NBCaCl 2的过夜培养物在37℃。 </li><li>稀释ŧ他NBCaCl 2噬菌体〜10 9 PFU /毫升。 </li><li>在50ml玻璃小瓶中，混合500微升过夜培养物，将500μl新鲜NBCaCl 2，和1ml噬菌体（〜10 9 PFU / ml）的;感染的预期复数（MOI）不能在37℃超过1孵育的混合物轻轻摇动（100rpm）下进行30分钟。 注:热处理受体细胞之前，为了在加入噬菌体（52℃，2分钟以灭活内源性限制性内切酶）可以提高效率12。 </li><li>加入50微升20％的Na 3 -Citrate的。继续轻柔在37℃振荡30分钟。 注:na 3 -Citrate作为钙的中等螯合剂。 </li><li>制备熔融脑心脏浸液（BHI）琼脂（1.5％琼脂）培养基，并保持它在水浴在55℃。 </li><li>细菌噬菌体混合物转移到100毫升烧瓶中，加入50毫升温的BHI琼脂补充有32毫克/升chloramphenicol，拌匀。将混合物倒入90 25mm培养皿。 注意:此方法使少数生长的菌落（假定的转导）的检测。 </li><li>孵育在37℃的板24-48小时。 </li><li>由殖民地转移到补充32毫克/升氯霉素新的BHI琼脂平板上进一步测试阻力所产生的殖民地（转导）。确认CFR基因的菌落PCR 7的存在。 </li></ol></li></ol> 3.自然变换注:在金黄色葡萄球菌的天然转化测定以下在我们以前的研究2中记载的方法进行。的N315衍生物，N2-2.1 2,7，被用来作为收件人。在这株， 感叹轨迹被复制，从而组成性表达的叹息2。对于DET关于如何隔离感叹表达能力的变种ailed程序，请参见以前的说明2。如果要传输的抗性标记不氯霉素，PRIT叹息（厘米R）可以用来表达感叹，如前面所述的2。纯化的质粒或从CFR的整个DNA的提取-acquired 金葡菌 COL-45 7被用作供体DNA进行转化。 <ol><li> 供体DNA的制备 <ol><li>培育COL-45过夜在含有50毫升TSB的一个300ml的烧瓶中，在37℃振荡补充有32毫克/升氯霉素。文化的大肠杆菌 HST04携带pHY300 质粒（四环素 R）的提取为对照实验的质粒。 注:HST04（ 坝- / DCM - ）缺乏DNA甲基化酶基因13。用DNA甲基化酶其它常规菌株也可以是使用d，来制备DNA供体，因为DNA甲基化状态不影响转化效率。或者，从金黄色葡萄球菌 COL菌株纯化pT181的质粒也可以用作用于转化测定法2的阳性对照。 </li><li>通过离心（在4℃下8000×g离心 10分钟）收集细胞。 </li><li>提取使用质粒DNA提取试剂盒或常规DNA纯化方法以纯化整个DNA的质粒。 </li><li>通过光谱仪定量纯化的DNA，并保持它在4℃下直到使用。 注:使用的转化实验，通常不到一周龄一个新的DNA提取。古DNA制剂降低转化效率。 </li></ol></li><li> 转化试验 <ol><li>文化受体细胞（N2-2.1）隔夜在5毫升TSB在37℃振荡。 </li><li>转移0.5过夜培养毫升到1.5毫升管中。 Precipi通过离心（在4℃万xg离心 1分钟）优化射孔细胞。 </li><li>悬浮细胞用10mL CS2介质的在50毫升管中。 注:CS2介质是诱导能力为金黄色葡萄球菌 2天然转化（见材料表培养基组成）一个完整的合成培养基。其它标准实验室培养基，如TSB或BHI，不适合用于转化2,13。 </li><li>生长于37℃的细菌以摇动（180转）直到晚指数期（约8小时）。 </li><li>通过离心（在4℃下5000×g离心 5分钟）收获细胞。 </li><li>悬浮细胞在10毫升新鲜CS2介质。 </li><li>添加纯化的质粒或基因组DNA（步骤3.1.4）的10微克到细胞悬浮液。摇动在37℃和180rpm下2.5小时。 注:潜伏期较短时间与DNA（&lt;2.5小时），导致较低的TRANSFormation频率。 </li><li>通过离心（5,000×g离心 5分钟，4℃）收集细胞。 </li><li>悬浮细胞在10毫升BHI培养基的。混合细胞悬浮液用90毫升熔融BHI琼脂（55℃）的补充有32毫克/升氯霉素（在对照实验或5毫克/升四环素）。将混合物倒入90 25mm培养皿。迅速冷静，让琼脂凝固。 </li><li>孵育在37℃下将板2天。 </li><li>通过转移殖民地（使用牙签），以包含适当的抗生素，以确认其阻力特性的新BHI琼脂平板上复制产生的殖民地（转化）。确认所获取的抗性基因（CFR或tetM）通过菌落PCR 7。 </li></ol></li></ol>

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The authors have nothing to disclose.

<html>这个工作描述来研究的金黄色葡萄球菌的遗传决定的HGT三大方法。虽然转导，接合已经研究了数十年，自然转化的存在是最近才认可2。因此， 金黄色葡萄球菌配备所有HGT的三个主要模式的，和测试他们都需要澄清遗传决定的可能的传播途径。这项工作的目的是编制完成协议，并提供在以前发表的著作7采用的方法的实用信息。虽然共轭和转导?...

金黄色葡萄球菌是一种共生革兰氏阳性菌天然栖息人类和动物的皮肤和鼻腔。此细菌种类是在医院和医疗机构的医院感染的主要原因。此外，它的发展到不同抗微生物化合物抗性的能力已取得由这种细菌引起成全球关注的感染的管理。 参与性表型的扩散两个主要途径是已知的:抗性基因型的克隆传播和遗传因素的细菌池之间的传播。在金黄色葡萄球菌 ，不同的抗生素抗性基因（以及毒力决定）的情况下，已发现与移动遗传元件（MGEs）相关联的1。在金黄色葡萄球菌的基因组中这些元素的存在表明根的采集和传输细菌群体内客位材料可以发挥对金黄色葡萄球菌的适应和发展的重要作用。 转化，接合和转导噬菌体:遗传物质可通过HGT三个众所周知的机制在革兰氏阳性菌进行交换。变换涉及游离DNA的摄取。为了获得外源DNA，细菌细胞需要制定一个特殊的生理阶段:竞争力的阶段。当达到这个阶段，感受态细胞是能够传输的DNA进入细胞质，获得新的遗传决定的。在金黄色葡萄球菌的情况下，自然转化的存在最近已经证明2。本着这一精神，我们小组已经阐明的叹息因子的表达在发展的能力阶段，其组成型表达如何呈现能够reachin的金黄色葡萄球菌的相关性（一个神秘的二次转录σ因子）克竞争力的阶段，这允许通过自然转化2收购耐药表型的。 缀合是涉及从一个活细胞（供体）到另一种（受体）DNA的传输的处理。两个小区必须是直接接触，从而允许同时通过特殊的结构，如管或孔被保护要交换的DNA中。的DNA通过该方法转移需要接合机械。在金黄色葡萄球菌 ，原型接合质粒是PGO1，它怀有接合操纵TRAA 3。 噬菌体转导涉及DNA的细胞通过噬菌体感染转移到细胞并意味着细菌DNA的包装，而不是病毒DNA，进噬菌体衣壳。大多数金黄色葡萄球菌分离株被噬菌体1溶源化。在应力条件下，原噬菌体可以从细菌染色体组中切除e和移到裂解周期。 这些是在金黄色葡萄球菌的DNA传输三种公知的机制。还有一些附加的传输机制，例如"伪转变"2和致病岛4的转移噬菌体样系统。最近，一组报道，"纳米管"是参与细胞材料（包括质粒DNA）的相邻小区5,6之间的转移，但后续的研究还没有从其他组出现为止。 这项工作提供了必要的方法，通过解决三个主要途径转移研究HGT在金黄色葡萄球菌 :接合，转导和自然转化。用这些方法所获得的结果被用来研究之间的CFR的基因的发送（氯霉素/氟苯尼考电阻）金黄色葡萄球菌菌株7。这三种技术是MGE传输的金黄色葡萄球菌的调查多用途工具。

<table border="0" cellpadding="0" cellspacing="0" width="737">
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			<td height="21" style="height:21px;width:284px;">Tryptic Soy Broth (TSB)&#160;</td>
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			<td height="21" style="height:21px;">Brain Heart Infusion (BHI)</td>
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			<td height="21" style="height:21px;">Nutrient Broth No. 2</td>
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			<td height="21" style="height:21px;">Sheep blood agar</td>
			<td>Eiken Chemical Co.,Ltd.</td>
			<td>E-MR96</td>
			<td>Tryptic soy agar added with 5% (v/v) sheep blood according to the manufacturer.&#160;</td>
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			<td height="21" style="height:21px;">Agar powder</td>
			<td>Wako Pure Chemical Industries</td>
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			<td height="21" style="height:21px;">Sodium citrate (Trisodium citrate dihydrate)</td>
			<td>Wako Pure Chemical Industries</td>
			<td>191-01785</td>
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			<td height="21" style="height:21px;">Cellulose Ester Gridded 0.45 &#956;L HAWG filter</td>
			<td>Merck Milipore</td>
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</table>

methodology for the study of horizontal gene transfer in staphylococcus aureus

主要的机会人类病原体葡萄球菌的一个重要特点是其非凡的快速获得耐药性的能力。基因组学研究表明， 金黄色葡萄球菌携带位于移动遗传元件许多毒力和抗性基因，这表明水平基因转移（HGT）起着金黄色葡萄球菌发展的关键作用。然而，用于研究HGT在金黄色葡萄球菌仍然缺乏，特别是关于天然转化，这已在该细菌最近报道的方法的一个完整和详尽的描述。这部作品描述了三个协议，它们对金黄色葡萄球菌的HGT 体外调查有用:结合，噬菌体转导和自然转化。为了这个目标，CFR基因（氯霉素/氟苯尼考电阻），赋予了Phenicols，林可酰胺类，恶唑烷酮，截短侧耳素和链阳性菌素A（PhLOPSA）-resistance表型，使用。理解，通过该金黄色葡萄球菌的遗传物质转移给其它菌株的机制是理解的快速采集电阻的必要，有助于澄清报告监视计划或以进一步预测在未来的扩展模式传播的模式。

这里所代表的结果已经公布以前（改编自基准7与出版商的许可）。我们研究的CFR的基因，这会导致低级别利奈唑胺抗性和PhLOPSA-抗性表型14，15的中金黄色葡萄球菌菌株的表达，潜在传输通路通过调查HGT的三种机制。 图1示出了在接合试验?...

Watch this Scientific Journal Video about Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus at JoVE.com

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

我们在这里描述了用于在金黄色葡萄球菌接合，转导，和自然转化的体外研究三种不同的协议。

方法基因水平转移的研究<em&gt;金黄色葡萄球菌</em

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

methodology-for-study-horizontal-gene-transfer-staphylococcus

Research

JoVE Journal

Immunology and Infection

Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

16.4K 次观看.  University of Tsukuba. 我们在这里描述了用于在金黄色葡萄球菌接合，转导，和自然转化的体外研究三种不同的协议。 

视频: Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

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

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

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

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

滴流和旋转盘反应器使用金黄色葡萄球菌生物膜分析

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

科研

免疫与感染

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.

皮下感染耐甲氧西林金黄色葡萄球菌（MRSA）的

MRSA is a worldwide threat to public health, and MRSA skin and soft-tissue infections now account for more than half of all soft-tissue infections in the ...

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

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

耐甲氧西林实验性心内膜炎模型<em&gt;金黄色葡萄球菌</em&gt;（MRSA）的大鼠

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

金黄色葡萄球菌生长使用人血红蛋白作为铁源

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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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.

相互作用的研究<em&gt;金黄色葡萄球菌</em与中性粒细胞通过流式细胞仪和时间推移显微镜

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

有效生成荧光标记疫苗病毒蛋白的方法

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

Most bacterial infections produce a biofilm. By virtue of their environment, biofilm associated bacteria are often phenotypically drug resistant. Novel antibacterial molecules that kill bacteria in biofilms are thus a high priority. We establish an assay to quickly screen for antimicrobial compounds that are effective at eradicating biofilms.

针对相关生物膜<em&gt;金黄色葡萄球菌</em&gt;使用基于刃天青药物敏感性测定

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

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.

一种评价金黄色葡萄球菌感染先天免疫反应的小鼠模型

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