These experiments have been supported by research grants from Merck and Co. and Summit, PLC.

1.从不同的环境或临床来源分离艰难梭菌<ol><li>环境隔离物:使用预先消毒的棉布（轻轻地用0.85％NaCl浸湿），拭拭任何感兴趣区域的表面（地板，门，手柄，搁板等 ）。 8确保使用无菌手套，并在完成后将拭子放在灭菌管中。 </li><li>临床分离物（粪便）:使用接种环将头10至100mg临床粪便样品置于头孢西丁 - 环丝氨酸 - 果糖琼脂（CCFA）上，并在严格厌氧条件下孵育48-72小时。将分离出的艰难梭菌菌落的菌落储存在-80℃的低温条件下进一步分析。 7，9，10 </li><li>在含有0.05％牛磺胆酸钠的脑心脏输液（BHI）肉汤中浓缩环境拭子样品，并置于37℃的厌氧室5天。以10,000 xg离心1 mL培养物，并将沉淀重悬于100μL乙醇中。 </li><li>将重悬细胞（50μL）置于环丝氨酸果糖琼脂（CCFA）板上，并在37℃的厌氧室中孵育40-48小时。将分离出的艰难梭菌菌落的菌落储存在-80℃的低温条件下进一步分析。 </li><li>使用乳胶凝集试剂或PCR测试疑似艰难梭菌菌落。 </li></ol> 2.培养艰难梭菌和杀伤动力学程序<ol><li>在37℃的厌氧室中，在血液琼脂平板上生长纯化的环境或临床艰难梭菌菌株48小时。 </li><li>取一个分离的菌落与接种环，转移到5 mL BHI培养基在15 mL管中，并在37℃的厌氧室中生长24小时。 </li><li>将培养前1:100稀释至约10 6菌落形成单位新鲜预还原BHIS（BHI加5g / L酵母提取物和1％L-半胱氨酸）补充有0.1％牛磺胆酸钠和适当浓度的抗生素（T0）的s（CFU）/ mL。 </li><li>在每个时间点（T0，T6，T24，T48）用移液管收集1 mL样品，并将一小部分（连续稀释100μL）扩散/扩散到血琼脂平板上。让细胞在37℃的厌氧小室中在血琼脂平板上生长48小时，并计算所得的菌落数以确定CFU。 </li></ol> 3.准备样品进行扫描电子显微镜<ol><li>在微量离心管中从每个时间点收集1 mL细胞，并以10,000 xg离心10分钟。丢弃上清液并在PBS中洗涤细胞。 </li><li>将样品以10,000xg离心10分钟，弃去上清液。在1 mL 4％多聚甲醛中稀释细胞，并在室温下孵育1 h。 </li><li>离心机样品在1℃再次离心10分钟000 xg并丢弃上清液。用蒸馏水洗涤细胞两次，并在100μL蒸馏水中稀释。根据溶液的浊度调节体积。 注意:进行多次连续稀释是一个好主意。 </li><li>盖上盖子并加入40μL样品。在流动罩中孵育15分钟以蒸发液体，并允许细胞粘附到盖玻片上。如果仍然存在液体，请使用鼓风机去除液体。 </li><li>将标有盖玻片的地板放在台式溅射机和胶带中。在溅射机中安全纯金。打开机器，并在低压（50 mTorr）下开始溅射。涂层细胞在80mA下30秒，这转化为20nm的金涂层。 </li><li>将涂覆的细胞转移到扫描电子显微镜。 </li></ol> 4.在扫描电子显微镜上成像艰难梭菌细胞<ol><li>用扫描电子显微镜（SEM）正确放气在计算机软件上安装通风按钮。 </li><li>使用碳带，将涂覆的盖玻片固定在金属台上。扫描电镜放空后，门应轻松打开。通过拧入金属台进入SEM室。 </li><li>单击计算机软件上的PUMP按钮。当系统读取"Vac OK"时，SEM将可用。 </li><li>点击检测器选项卡下的SE检测器。通过点击显示电压的按钮打开光束。在增加电压（高达15 kV）之前，以较低的电压（5 kV）开始成像。光束打开后会出现图像。 </li><li>使用跟踪功能，在涂覆的盖玻片上找到一个区域进行图像。放大区域并找到杆状结构;这些是艰难梭菌 。 </li><li>放大并对焦图像以校准系统。这应该在多个工作距离上完成:15,9和5 mm。工作距离为5mm的图像。 </li><li>打开你ltra高分辨率成像模式，并开始集中和优化散光。 </li><li>开始以高放大倍数进行对焦。使用粗略和精细的焦点切换为此。调整散光以获得更清晰的图像。 <ol><li>以高放大倍率调节散光。要做到这一点，调整散光切换（它们看起来类似于细/粗焦点切换），并通过数字放大计算机软件来检查图像的清晰度。 </li></ol></li><li>扫描慢扫描功能，以收集高质量的图像。将收集的图像保存为.TIFF文件，将用于分析。如果在分析过程中进行测量，请确保选择了数据条。 </li><li>以不同的角度收集图像（通过在SEM上直接手动转动角度达52°）。有角度的图像倾向于显示更多的深度信息。 </li><li>根据正在成像的单元格改变光束电压。对于所有的实验，这主要保持在5 - 15 kV之间。 </li&gt; <li>成像完成后，关闭光束并将工作距离提高到20 mm。然后可以将腔室排空，并且可以移除该台。将所有图像复制到驱动器上进行进一步分析。 </li></ol>图像处理与分析<ol><li>将免费提供的软件FIJI（http://fiji.sc）下载并安装到计算机上。 </li><li>打开FIJI中的图像文件。 </li><li>使用线功能，精确跟踪比例尺。 </li><li>单击FIJI程序中的"分析"选项卡，最后选择"选择比例"功能。将出现一个窗口，需要根据比例尺设置已知距离。更改长度单位，然后单击确定。 </li><li>现在该程序已经校准了距离，使用线路功能测量单元长度。要获取长度，请使用线功能来完整地跟踪单元格。再次选择分析选项卡，然后单击测量。应该是应用程序的长度耳朵在以前表示的单位。 </li></ol>

<ol>
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KWG has received past and current research support from Merck &amp; Co. and Summit, PLC.

目前的研究目的是创建一种高通量的方法来分离艰难梭菌 ，并使用扫描电子显微镜（SEM）作为抗菌药物药理作用的更全面表征的手段。使用本文概述的方案，我们已经证明，成像细胞对抗生素治疗的表型反应可以揭示药物的药理作用的洞察力。总的来说，该方案的成像部分在收集细胞后的持续时间大约需要2小时，但比单独的典型的杀伤动力学研究更为歧视。在学习使用SEM的同时，技术上要求很高，样品的制备比较简单快捷。我们认为这里概述的这些方案将提供客观的方法来评估抗生素治疗的药理作用。 考虑到t之间的相似之处他的抗生素杀伤曲线（ 图2 ），我们试图确定细胞对药物治疗的反应是否有差异。使用SEM，我们确定甲硝唑对药物超高浓度的细胞长度有影响。虽然这些表型以前没有报道，但它们与抗生素的作用机制一致，并且暗示了对细胞代谢和生长的影响。相比之下，万古霉素对细胞壁有显着影响，也与其作用机制一致。虽然这些抗生素之间的杀伤动力学有相似之处，但从SEM图像中可以看出，有显着的表型差异。创建抗生素表型库将允许对可能没有确定的明确作用机制的药物进行更全面的鉴定，如吡啶立唑的情况。 因为...这种方法在技术上是有挑战性的，可能需要进行修改以解决科学问题，包括一致的细胞制备和金涂层溅射的改变。太多的涂层可能会对细胞的外部造成任何影响，但没有足够的涂层可能导致束的充电并使图像变形。为避免这种情况，请准备不同金镀层的样品以优化溅射时间。最后，研究之间的一致方法将允许进行实验间比较。 使用SEM的局限性在于限制观察细胞形态变化;因此，功能研究是确认任何可疑反应所必需的。因此，我们认为这种成像方案可以用作指导功能研究的手段。尽管有这种限制，免疫金标签可以使用SEM来确认蛋白质运输或聚集的任何可疑变化;然而，我们还没有进行这些实验。 由于艰难梭菌治疗的新型抗生素活跃，需要更全面地评估药物作用。如本文所述，SEM提供了独特的，高通量和可靠的表征药理作用的机会。通过在不同艰难梭菌菌株之间成像许多不同的抗生素效应，我们将能够了解为什么一些抗生素比其他针对特定致病菌株如027 / NAP1 / BI流行株更有效。此外，SEM分析可能有助于区分核型或菌株之间的抗生素效应，并可用于研究其广泛适用性的其他细菌种类。

艰难梭菌是一种革兰氏阳性，形成细菌的细菌，每年在美国造成大约50万感染，被疾病控制和预防中心（CDC）认定为威胁级紧急病原体，这是最高的风险。 1过去十年来，针对艰难梭菌活性的抗微生物药物开展了相当大的药物开发 。 2，3 体外研究是药物开发过程的必要组成部分。 4传统上， 体外易感性和时间杀死研究用于验证未来动物和其他体内研究。 虽然这些方法在评估杀伤作用方面起着重要作用，但它们不能捕获细胞对药理学治疗的表型反应。通过纳入扫描电子显微镜（SEM）与标准d杀死动力学研究，可以更全面地表征抗生素的直接作用。 5，6，7这里，我们提供一种方法，其中SEM用作描述抗生素治疗功效的手段。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>cotton gauze&#160;</td>
			<td>Caring</td>
			<td>PRM21408C</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Macron</td>
			<td>7532</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI)&#160;</td>
			<td>Criterion</td>
			<td>C5141</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Alfa Aesar</td>
			<td>A10389</td>
			<td></td>
		</tr>
		<tr>
			<td>yeast extract</td>
			<td>Criterion</td>
			<td>C741</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium taurocholate</td>
			<td>Alfa Aesar</td>
			<td>A18346</td>
			<td></td>
		</tr>
		<tr>
			<td>anaerobic chamber</td>
			<td>Coy</td>
			<td></td>
			<td>vinyl anaerobic chamber</td>
		</tr>
		<tr>
			<td>cycloserinecefoxitin fructose agar (CCFA) plates</td>
			<td>Anaerobe systems</td>
			<td>AS-213</td>
			<td></td>
		</tr>
		<tr>
			<td>blood agar plates</td>
			<td>Hardy diagnostics</td>
			<td>A-10</td>
			<td></td>
		</tr>
		<tr>
			<td>latex agglutination reagent</td>
			<td>Oxoid</td>
			<td>DR1107A</td>
			<td>C. diff test kit</td>
		</tr>
		<tr>
			<td>microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>222363204</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Fisher Scientific</td>
			<td>50-259-98</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>J. Melvin freed brand</td>
			<td>7525M</td>
			<td>75x25mm</td>
		</tr>
		<tr>
			<td>flow hood</td>
			<td>Labconco</td>
			<td>Class II type A2&#160;</td>
			<td>biosafety cabinet</td>
		</tr>
		<tr>
			<td>desk sputtering machine</td>
			<td>Denton Vacuum</td>
			<td>Desk II</td>
			<td></td>
		</tr>
		<tr>
			<td>tape</td>
			<td>Plastic Core</td>
			<td>05072-AB</td>
			<td>SPI Double Sided Adhesive Carbon Tape</td>
		</tr>
		<tr>
			<td>gold</td>
			<td>Denton Vacuum</td>
			<td>TAR001-0158</td>
			<td>2.375&#8221; Diameter x .002&#8221; Thick Gold foil</td>
		</tr>
		<tr>
			<td>scanning electron microscope</td>
			<td>FEI</td>
			<td>XL-30</td>
			<td></td>
		</tr>
	</tbody>
</table>

a protocol to characterize the morphological changes of clostridium difficile in response to antibiotic treatment

用针对厌氧菌的新药开发评估抗生素的作用是困难和技术上的要求。为了获得可能的MOA的了解，可以使用扫描电子显微镜（SEM）显现与抗生素暴露相关的形态学变化。将SEM成像与传统杀伤曲线整合可能会提高我们对药物作用的深入了解，推动药物开发过程。为了测试这个前提，用已知但不同的MOA（万古霉素和甲硝唑）的药物进行杀伤曲线和SEM研究。 艰难梭菌细胞（R20291）在有或没有抗生素存在下长达48小时生长。在48小时的间隔期间，在多个时间点收集细胞以确定抗生素的功效并在SEM上成像。与以前的报道一致，万古霉素和甲硝唑在通过菌落形成单位（CFU）计算的24小时治疗后具有显着的杀菌活性婷。使用SEM成像，我们确定甲硝唑对细胞长度有显着影响（与对照组和万古霉素相比，每种抗生素的细胞长度减少50％， P &lt;0.05）。虽然以前没有以这种方式记录对药物治疗的表型反应，但是它们与药物的MOA一致，证明了成像和测量的通用性和可靠性以及该技术对其他实验化合物的应用。

艰难梭菌是孢子形成细菌，因此在任何功能分析之前，必须确定营养和孢子细胞之间的形态差异。 图1显示了在生长曲线和孢子细胞的指数期捕获的营养细胞的代表性图像。如图所示，营养细胞是长而光滑的棒状结构，而孢子是具有粗糙外观的小的椭圆形结构。在功能上，营养细胞生长和分裂迅速，并通过分泌毒素来造成艰难梭菌感染的毒力，而孢子通常休眠，活性很小。因此，抗生素对营养细胞主要是活性的，而孢子通常对药物治疗有抗药性，这是由于几层使用DNA保护核心，并且缺乏生理活性。由于这些事实，大多数的形态分析集中于营养细胞。 11，12 抗生素杀伤曲线是证明对生长细菌的抗生素功效的标准方法。艰难梭菌菌株R20291使用的浓度基于MICs值，万古霉素为1μg/ mL，甲硝唑为0.51μg/ mL。 10 如图2所示 ，对照细胞生长并达到平台，而处理细胞的总菌落形成单位（CFU）降低到显示杀菌效果的检测限（LOD）。如图所示，万古霉素（ 图2A ）和甲硝唑（ 图2B ）在超高浓度（4xMIC）下杀死艰难梭菌是有效的。人们可能会注意到抗生素杀伤曲线看起来是相似的，但药物具有不同的ac机制（万古霉素防止细胞壁合成，而甲硝唑影响DNA复制）。因此，我们建议抗生素杀伤曲线可能没有足够的歧视力来提供这些抗生素之间的详细差异。我们使用显微镜来提供更多的歧视。 由于其微观尺寸， 艰难梭菌在高放大倍数下无法成像。这提供了使用扫描电子显微镜（SEM）的机会，该扫描电子显微镜（SEM）是可以获得纳米尺度的分辨率的成像方法，以评估直接对细胞的抗生素处理的详细影响。为了证明这种方法的实用性，在药物治疗之前和之后对细胞进行成像，以确定形态如何变化（ 图3A ）。如所证明的，一些细胞的壁受到影响，如万古霉素的情况，并且一些细胞的尺寸较小，如甲硝唑的情况。为了测试细胞大小是否受影响，我们使用程序FIJI分析细胞长度。植物细胞大小可以在对照情况下变化一些，但大部分长度大约为6μm（ 图3B ）。当考虑来自对照和治疗设置的许多细胞时，可以快速注意到细胞长度优先在甲硝唑中受到影响，但不受万古霉素处理的影响（ 图3C ）。 <img alt="图1" src="/files/ftp_upload/55383/55383fig1.jpg" /> 图1: 通过扫描电子显微镜来区分细胞类型。显示艰难梭菌营养和孢子细胞的代表性图像。营养细胞是长而鞭毛的杆状结构。相比之下，孢子细胞很小，并且具有粗糙和颠簸的外套。孢子是伪色红色的图像purpo只有ses比例尺显示在图像上。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55383/55383fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55383/55383fig2.jpg" /> 图2: 抗生素时间 - 杀伤曲线。对四种不同的抗生素:万古霉素（A） ，甲硝唑（B）进行SupraMIC杀伤曲线。这些抗生素对艰难梭菌菌株R20291具有显着的杀伤作用，接近用于计数菌落形成单位（CFU）的检测限（LOD）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55383/55383fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55383/55383fig3.jpg" /> 图3:检查抗生素治疗的药理作用。在整个杀伤动力学研究（A）中，在多个时间点拍摄经处理和未处理的细胞的图像。示出了测量长度（B）的对照营养细胞的代表性实例。治疗和未处理的细胞被测量并比较（C） 。至少重复进行实验，每组测量&gt; 17个细胞。 *与对照组和万古霉素组相比， P &lt;0.05。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55383/55383fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment at JoVE.com

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

表征形态变化的议定书<em&gt;艰难梭菌</em&gt;应对抗生素治疗

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into ...

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Immunology and Infection

A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

8.9K 次观看.  University of Houston College of Pharmacy. Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.

视频: A Protocol to Characterize the Morphological Changes of Clostridium difficile in Response to Antibiotic Treatment

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

使用人工痰介质测试抗生素药效试验<em&gt;绿脓杆菌</em&gt;更多相关的肺囊肿性纤维化的条件

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

科研

免疫与感染

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

比较方法来表征景观的宿主 - 病原体蛋白 - 蛋白相互作用

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

培养和维护<em&gt;难辨梭状芽孢杆菌</em&gt;在厌氧环境

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学:生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291

This protocol outlines the cefoperazone mouse model of Clostridium difficile infection (CDI) using a clinically-relevant and genetically-tractable strain, R20291. Emphasis on clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol.

头孢哌酮治疗小鼠模型的临床相关的<em&gt;艰难梭菌</em&gt;应变R20291

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

主机表型的考试<em&gt;食蚊鱼</em&gt;继抗生素治疗

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

用于炎症反应的新内皮调节因子的筛选测定

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...