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><tbody><tr><td>Tryptic Soy Broth (TSB)&amp;nbsp;</td><td>Becton Dickinson&amp;nbsp;</td><td>211825</td><td></td></tr><tr><td>Brain Heart Infusion (BHI)</td><td>Becton Dickinson&amp;nbsp;</td><td>211059</td><td></td></tr><tr><td>Nutrient Broth No. 2</td><td>Oxoid</td><td>CM0067</td><td></td></tr><tr><td>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.&amp;nbsp;</td></tr><tr><td>Agar powder</td><td>Wako Pure Chemical Industries</td><td>010-08725</td><td></td></tr><tr><td>Sodium citrate (Trisodium citrate dihydrate)</td><td>Wako Pure Chemical Industries</td><td>191-01785</td><td></td></tr><tr><td>Cellulose Ester Gridded 0.45 &amp;mu;L HAWG filter</td><td>Merck Milipore</td><td>HAWG 02500</td><td></td></tr><tr><td>QIAfilter Plasmid Midi kit</td><td>QIAGEN</td><td>12243</td><td></td></tr></tbody></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

Universidad de Cartagena UDEC

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

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

科研

遗传学

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

生物化学

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

细菌结合频率的高分辨率比较

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

基于荧光的感染组织细菌基因调控方法

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

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. ...

免疫与感染

Heuristic Mining of Hierarchical Genotypes and Accessory Genome Loci in Bacterial Populations

Routine and systematic use of bacterial whole-genome sequencing (WGS) is enhancing the accuracy and resolution of epidemiological investigations carried out by Public Health laboratories and regulatory agencies. Large volumes of publicly available WGS data can be used to study pathogenic populations at a large scale. Recently, a freely available computational platform called ProkEvo was published to enable reproducible, automated, and scalable hierarchical-based population genomic analyses using bacterial WGS data. This implementation of ProkEvo demonstrated the importance of combining standard genotypic mapping of populations with mining of accessory genomic content for ecological inference. In particular, the work highlighted here used ProkEvo-derived outputs for population-scaled hierarchical analyses using the R programming language. The main objective was to provide a practical guide for microbiologists, ecologists, and epidemiologists by showing how to: i) use a phylogeny-guided mapping of hierarchical genotypes; ii) assess frequency distributions of genotypes as a proxy for ecological fitness; iii) determine kinship relationships and genetic diversity using specific genotypic classifications; and iv) map lineage differentiating accessory loci. To enhance reproducibility and portability, R markdown files were used to demonstrate the entire analytical approach. The example dataset contained genomic data from 2,365 isolates of the zoonotic foodborne pathogen Salmonella Newport. Phylogeny-anchored mapping of hierarchical genotypes (Serovar -&#62; BAPS1 -&#62; ST -&#62; cgMLST) revealed the population genetic structure, highlighting sequence types (STs) as the keystone differentiating genotype. Across the three most dominant lineages, ST5 and ST118 shared a common ancestor more recently than with the highly clonal ST45 phylotype. ST-based differences were further highlighted by the distribution of accessory antimicrobial resistance (AMR) loci. Lastly, a phylogeny-anchored visualization was used to combine hierarchical genotypes and AMR content to reveal the kinship structure and lineage-specific genomic signatures. Combined, this analytical approach provides some guidelines for conducting heuristic bacterial population genomic analyses using pan-genomic information.

This analytical computational platform provides practical guidance for microbiologists, ecologists, and epidemiologists interested in bacterial population genomics. Specifically, the work presented here demonstrated how to perform: i) phylogeny-guided mapping of hierarchical genotypes; ii) frequency-based analysis of genotypes; iii) kinship and clonality analyses; iv) identification of lineage differentiating accessory loci.

细菌种群中分层基因型和辅助基因组位点的启发式挖掘

Routine and systematic use of bacterial whole-genome sequencing (WGS) is enhancing the accuracy and resolution of epidemiological investigations carried ...

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

大肠杆菌中天然偶联质粒介导的水平基因转移检测

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

<meta charset="utf8"></head><body>可视化细菌病原体中的抗生素持久性

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to&#160;underlie clinical treatment failure.&#160;Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited.
This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus)&#160;during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.

<meta charset="utf8"></head><body>铜绿假单胞菌和金黄色葡萄球菌异质表型的延时落射荧光显微镜成像

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize&#160;phenotypic variants, such as persisters, by time-lapse epifluorescence&#160;microscopy.

铜绿假单胞菌和金黄色葡萄球菌异质表型的延时落射荧光显微镜成像

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment ...

方法基因水平转移的研究

摘要

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此视频中的章节

接合交配测定在接合介入转移蛋白的序列特异性分析

对于代谢物分析基于法 - 串联液相色谱 - 质谱法

细菌结合频率的高分辨率比较

基于荧光的感染组织细菌基因调控方法

相互作用的研究

细菌种群中分层基因型和辅助基因组位点的启发式挖掘

大肠杆菌中天然偶联质粒介导的水平基因转移检测

铜绿假单胞菌和金黄色葡萄球菌异质表型的延时落射荧光显微镜成像

铜绿假单胞菌和金黄色葡萄球菌异质表型的延时落射荧光显微镜成像

铜绿假单胞菌和金黄色葡萄球菌异质表型的延时落射荧光显微镜成像