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>
	<li>Lessa, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+Clostridium+difficile+infection+in+the+United+States.">Burden of Clostridium difficile infection in the United States.</a> N Engl J Med. 372 (9), 825-834 (2015).</li><li>Vickers, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ridinilazole:+a+novel+therapy+for+Clostridium+difficile+infection.">Ridinilazole: a novel therapy for Clostridium difficile infection.</a> Int J Antimicrob Agents. 48 (2), 137-143 (2016).</li><li>Shah, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection:+update+on+emerging+antibiotic+treatment+options+and+antibiotic+resistance.">Clostridium difficile infection: update on emerging antibiotic treatment options and antibiotic resistance.</a> Expert Rev Anti Infect Ther. 8 (5), 555-564 (2010).</li><li>Ambrose, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+EMA+guideline+for+antimicrobial+development.">New EMA guideline for antimicrobial development.</a> Lancet Infect Dis. 12 (4), 265-266 (2012).</li><li>Bass&#232;res, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+on+toxin+production+and+cell+morphology+in+Clostridium+difficile+by+ridinilazole+(SMT19969),+a+novel+treatment+for+C.+difficile+infection.">Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.</a> J Antimicrob Chemother. 71 (5), 1245-1251 (2016).</li><li>Endres, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+imaging+the+pharmacological+effects+of+antibiotic+treatment+on+Clostridium+difficile.">A novel method for imaging the pharmacological effects of antibiotic treatment on Clostridium difficile.</a> Anaerobe. 40, 10-14 (2016).</li><li>Endres, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+Effects+of+Surotomycin+Treatment+on+Clostridium+difficile+Toxin+A+and+B+Production,+Immune+Response,+and+Morphological+Changes.">Evaluating the Effects of Surotomycin Treatment on Clostridium difficile Toxin A and B Production, Immune Response, and Morphological Changes.</a> Antimicrob Agents Chemother. 60 (6), 3519-3523 (2016).</li><li>Alam, M. J., Anu, A., Walk, S. T., Garey, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+potentially+pathogenic+Clostridium+difficile+contamination+in+household+environs.">Investigation of potentially pathogenic Clostridium difficile contamination in household environs.</a> Anaerobe. 27, 31-33 (2014).</li><li>Aitken, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+the+Endemic+Setting,+Clostridium+difficile+Ribotype+027+Is+Virulent+But+Not+Hypervirulent.">In the Endemic Setting, Clostridium difficile Ribotype 027 Is Virulent But Not Hypervirulent.</a> Infect Control Hosp Epidemiol. , 1-6 (2015).</li><li>Basseres, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+on+toxin+production+and+cell+morphology+in+Clostridium+difficile+by+ridinilazole+(SMT19969),+a+novel+treatment+for+C.+difficile+infection.">Impact on toxin production and cell morphology in Clostridium difficile by ridinilazole (SMT19969), a novel treatment for C. difficile infection.</a> J Antimicrob Chemother. 71 (5), 1245-1251 (2016).</li><li>Walters, B. A., Roberts, R., Stafford, R., Seneviratne, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relapse+of+antibiotic+associated+colitis:+endogenous+persistence+of+Clostridium+difficile+during+vancomycin+therapy.">Relapse of antibiotic associated colitis: endogenous persistence of Clostridium difficile during vancomycin therapy.</a> Gut. 24 (3), 206-212 (1983).</li><li>Chilton, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effect+of+oritavancin+on+Clostridium+difficile+spore+germination,+outgrowth+and+recovery.">Evaluation of the effect of oritavancin on Clostridium difficile spore germination, outgrowth and recovery.</a> J Antimicrob Chemother. 68 (9), 2078-2082 (2013).</li><li>Ofosu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection:+a+review+of+current+and+emerging+therapies.">Clostridium difficile infection: a review of current and emerging therapies.</a> Ann Gastroenterol. 29 (2), 147-154 (2016).</li><li>McDonald, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+epidemic,+toxin+gene-variant+strain+of+Clostridium+difficile.">An epidemic, toxin gene-variant strain of Clostridium difficile.</a> New Eng J Med. 353 (23), 2433-2441 (2005).</li></ol>

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?...

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><tbody><tr><td>cotton gauze&amp;nbsp;</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>50 mL tubes</td><td>Falcon</td><td>352098</td><td></td></tr><tr><td>Brain Heart Infusion (BHI)&amp;nbsp;</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>75 x 25mm</td></tr><tr><td>flow hood</td><td>Labconco</td><td>Class II type A2&amp;nbsp;</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&amp;rdquo; Diameter x .002&amp;rdquo; 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?...

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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Research

JoVE Journal

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

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

科研

遗传学

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 ...

免疫与感染

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

环境科学

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

生物化学

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 ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

用于维持和区分生物型的实验室技术<em&gt;霍乱弧菌</em&gt;临床和环境隔离

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory bowel disease, asthma and arthritis. Thus, understanding the mechanisms underlying microbiota-immune system crosstalk is of crucial importance. Antibiotic administration, while aiding pathogen clearance, also induces drastic changes in the size and composition of intestinal bacterial communities which can have an impact on human health. Antibiotic treatment in mice recapitulates the impact and long-term changes in human microbiota from antibiotic treated patients, and enables investigation of the mechanistic links between changes in microbial communities and immune cell function. While several methods for antibiotic treatment of mice have been described, some of them induce severe dehydration and weight-loss complicating the interpretation of the data. Here, we provide two protocols for oral antibiotic administration which can be used for long-term treatment of mice without inducing major weight-loss. These protocols make use of a combination of antibiotics that target both Gram-positive and Gram-negative bacteria and can be provided either ad libitum in the drinking water or by oral gavage. Moreover, we describe a method for the quantification of microbial density in fecal samples by qPCR which can be used to validate the efficacy of the antibiotic treatment. The combination of these approaches provides a reliable methodology for the manipulation of the intestinal microbiota and the study of the effects of antibiotic treatment in mice.

Here we provide detailed protocols for the oral administration of antibiotics to mice, collection of fecal samples, DNA extraction and quantification of fecal bacteria by qPCR.

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

The gut microbiota has a central influence on human health. Microbial dysbiosis is associated with many common immunopathologies such as inflammatory ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光抗生素探针对细菌耐药性的可视化

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin.

大肠杆菌抗生素持久性的群体和单细胞分析

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...

表征形态变化的议定书

摘要

探索更多视频

此视频中的章节

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

培养和维护

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作

头孢哌酮治疗小鼠模型的临床相关的

用于维持和区分生物型的实验室技术

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

使用荧光抗生素探针对细菌耐药性的可视化

大肠杆菌抗生素持久性的群体和单细胞分析

表征形态变化的议定书

摘要

探索更多视频

此视频中的章节

设计与低成本的使用，自动Morbidostat细菌的适应性进化在抗生素药物的选择

培养和维护

水性抗生素耐药细菌的分离和鉴定及其抗生素耐药基因的分子表征

通过成像质谱分析 研究 感染期间琼脂和组织中生长的细菌菌落的微生物合作

头孢哌酮治疗小鼠模型的临床相关的

用于维持和区分生物型的实验室技术

检测梭菌感染后抗原特异性抗体应答的蛋白微阵列测定方法

口服抗生素治疗小鼠肠道微生物群的定量聚合酶链反应分析

使用荧光抗生素探针对细菌耐药性的可视化

大肠杆菌抗生素持久性的群体和单细胞分析

通过成像质谱分析研究感染期间琼脂和组织中生长的细菌菌落的微生物合作