This project was partially funded by a FAST (Faculty and Student Team) Award to TPP and JMC from EURECA (Center for Enhancing Undergraduate Research Experiences and Creative Activities) at Sam Houston State University.

所有动物实验根据的IACUC协议批准进行的，编号为14-05-05-1018-3-01，13-04-29-1018-3-01和14-04-17-1018-3-01。 1.动物采集，处理和伦理关怀<ol><li>使用一个小抄网并放入19升桶野外现场（在http://www.sms.si.edu/irlspec/Gambusia_affinis.htm识别指南）收集食蚊鱼 。用目测来确定物种。 </li><li>在池塘水的桶2天 - 休息鱼1。此后，转成鱼76或189在25℃，充满了一半池塘水/半自来水水量L水族箱，通入起泡多孔石。处理鱼时，以最低限度地打扰他们的皮肤菌群使用小抄网。 注:鱼密度应不超过20尾/ 38升水族箱的水高。鱼驯化水族馆进行实验前至少5天。 </li><li>饲料在日常养生与5毫克每鱼糜食用鱼。何LD鱼12h光照/ 12小时黑暗循环。 </li></ol> 2.初始抗生素暴露在所有实验<ol><li>从同一水族馆每个实验绘制所有鱼类，放置到两个单独的组（处理:暴露于抗生素和控制:未曝光）在19升桶。以前的数据采集显示，鱼在水族箱一样均匀有皮肤微生物组是在群落组成变化不大。 12 </li><li>在实验过程中，保持鱼在2L人工池塘水（APW 0.33克/升的CaCl 2，0.33克/升MgSO 4上，0.19克/升碳酸氢钠 ）是，通过高压灭菌，使用前灭菌。 </li><li>加入50毫克/毫升到二甲基亚砜（DMSO）准备利福平抗生素原液。利福平是代表性的广谱抗生素，是无毒的鱼。在人类和小鼠中，其在组织中分布良好。 </li><li>从利福平股票管理的最终在用于处理的鱼组的抗生素暴露3天2升APW 25微克/毫升的浓度。请注意，3 d后，皮肤可培养细菌数返回类似于预处理鱼皮。 </li><li>平行地，保持一组中APW未处理的鱼和给予的DMSO等当量（每APW L的0.5毫升）到水柱作为对照组。 </li><li>作为实验的目的是测量鱼的表型的抗生素改变的微生物的作用，以避免直接从抗生素本身的影响，用2L以下三天抗生素曝光（或控制）期间，转印鱼成桶新鲜的APW。 <ol><li>所以抗生素是由鱼的身体除去使用10小时的休息期。基地在那里的鱼暴露于25微克/毫升抗生素三天这种淘汰时间的实验。通过剪断脊髓后跟脑脊髓刺毁安乐死鱼，然后用组织研磨机匀化整个身体。</li><li>由后0.2微米的过滤放置50微升此悬浮液到琼脂培养皿的中心确定抗生素的存在。按上下颠倒的无菌200μL枪头，到琼脂中，从而消除一个小插件让这个悬挂了一个洞。 </li><li>使用琼脂平板用20mL测定营养琼脂的体积，并用棉签与指示生物， 枯草芽孢杆菌 ，这是利福平敏感扩散均匀。获得25°CO / N培养营养琼脂枯草芽孢杆菌 ，并使用棉签取得殖民地。在25℃过夜温育后观察抑制区表示在鱼组织抗生素的存在。 </li></ol></li></ol> 3.微生物组提取<ol><li>通过将鱼放入装有2毫升无菌PBST（137毫米钠无菌的15毫升锥形管提取外表皮粘膜皮肤微生物样本氯，10mM磷酸盐，0.1％吐温-20，pH 7.4）中的溶液并涡旋以10℃1分钟以10秒的暂停增量设置。洗涤剂和盐在缓冲促进即使在溶液的细菌分散液。比较的结果发现用0.85％的生理盐水但下调细菌计数用纯净水。 </li><li>从锥形管转移鱼回收桶。皮肤提取的杀伤力通常低于10％。或者，分析在管中的细菌悬浮液在萃取立即或以15,000 xg离心和存储由一个2分钟的自旋在离心机中在室温下沉淀细菌在-80℃以供将来分析。 注:所有的文化和生化分析是立竿见影的; DNA或蛋白质提取可以从冷冻的样品。 </li><li>为了获得肠道微生物样品，首先将鱼在无菌培养皿。通过直接切断脊髓型颈椎病的脑袋手术剪背后安乐死鱼，其次是穿髓，然后外部消毒体用70％乙醇擦拭。 </li><li>解剖后，由食道后肛门前，切割移除整个肠道。 </li><li>切肠小段1 - 长度为2毫米，将所有的部分分成两个毫升PBST解决方案在锥形管。涡旋1分钟后，除去上清液到另一干净的试管（采取谨慎不制作肠道部分）。 注:上清液包含可通过对皮肤样品提及以前的方法用于直接分析或沉淀萃取肠道细菌。 </li></ol> 4.感染模型的制备和巴斯特定病原<ol><li>获得爱德华氏菌 （E.氏菌 ）的感染株，并保持在-80℃的文化。应变93 - 146本研究使用，从被感染的鲶鱼孤立的，从马克·劳伦斯，兽医学院，密西西比州立大学获得的。 </li><li> STREAK E.氏菌股票上称为E.氏菌介质（EIM）的选择性和差异介质琼脂19的细菌培养，并在27℃孵育2天。 </li><li>从文化传递一个纯粹的孤立菌落接种含有40毫升的营养肉汤的150毫升瓶（NB; 5g / L的蛋白胨，4g / l的牛肉膏）。在在27℃下设置在150rpm，保持2天振荡器地方烧瓶中。 </li><li>孵育后，在650nm处稀释在PBST中的培养物的样品，以0.2的OD分光计读数来校准大肠杆菌氏菌培养为期望的感染剂量卷。 </li><li>接下来两个转移处理（后抗生素暴露3 d）和未经处理的鱼类群体纳入为130毫升新鲜的人工池塘的水，或APW为鱼类组织药物清除约10小时个人塑料杯。 </li><li>然后，从两组，再每条鱼转移到为130毫升的清洁APW的8盎司塑料杯。 </li&gt; <li>给每个鱼片含2.8×10 6 / ml的大肠氏菌文化进入APW（浴感染模型）致死剂量，并保持在27℃培养箱中24小时。 </li><li> 大肠氏菌洗完澡后，单独传送鱼放入新杯130毫升的APW。 </li><li>以下水置换，记录鱼的死亡率在一个星期的时间。鱼不是美联储在此期间。在对比鲶鱼， 食蚊显示感染的任何外部的迹象，因此使用杀伤力作为实验端点它是必不可少的。 </li></ol> 5.多种微生物挑战粪便和土壤<ol><li> 粪污处理 <ol><li>从谁没有在此前两周使用无菌手套和无菌50 mL锥形管接到抗生素，健康志愿者主题获得新鲜人类粪便。 </li><li>一旦收集，收集排泄物在无菌塑料袋中，然后转移到无菌15毫升Çonical管。 800毫克/毫升 - 在PBST暂停粪便给500原液创建一个股票浓度。此厚混合物是最好使用1毫升或更大的塑料移液管尖端，已被切割的底部用无菌剪刀使得开口较大转移。 </li><li>处理和控制未处理鱼基的初始抗生素曝光后，分离成含有粪便悬浮液在任一15毫克/毫升或10毫克/毫升到APW的终浓度以130毫升的总体积个别塑料杯。在25℃的恒温箱中保持杯子。 </li><li>在2天的粪便通过接触密切观察期间的记录鱼类死亡。 </li></ol></li><li> 土壤处理 <ol><li>收集1 - 2千克富含有机质的表土（应包含微生物的密度最高和多样性）约7 - 深度下跌15厘米，放入干净的塑料容器中。需要注意的是土应在以下收集两天内使用。 </li><li>巨熊初步接触期后ctly，抗生素分开处理和未处理的鱼两组到含有18.2克土130毫升APW的个别塑料杯。加入鱼之前，混合用手水井的土壤。大部分的颗粒不挂起，并停留在杯底。 </li><li>暴露鱼在25℃下3天的持续时间，并记录任何死亡率。 </li></ol></li></ol> 6.渗透胁迫挑战<ol><li>处理，接着进行一个10小时的休息时间，如上所述后，个性化鱼放入含NaCl单独杯在150毫升APW 17.5毫克/毫升（300毫摩尔）。这是海水的典型盐度，这是不充分致死（&lt;50％），以控制鱼的一半，但强烈的压力，因此需要有效的渗透调节。 </li><li>请遵守鱼类死亡超过36小时的持续时间。 </li></ol> 7.硝酸盐毒性挑战<ol><li>休息鱼抗生素博览会后10小时内URE，然后分离鱼入含有的硝酸钠浓度个别杯为10毫克/毫升（118毫摩尔）或17.5毫克/毫升（206毫摩尔）在130毫升APW的。 </li><li>记录死亡率超过4天的低剂量组和1个D高剂量。 </li></ol> 8.个性化或分组鱼的生长分析<ol><li>完整的3天抗生素曝光或控制期间在水桶组。 </li><li>对于个人 ，填补8盎司泡沫塑料杯每150毫升APW，传送单个鱼放入杯中，并记录总体重初步评估。 <ol><li>重复这两个处理和未处理组中的所有鱼。使用白色泡沫塑料杯，而不是透明的塑料杯，因为鱼不会在清澈的杯子时吃。 </li></ol></li><li>每天每鱼2球团矿的标准饮食喂养的鱼。颗粒使用，而不是因为片状颗粒有个别质量低方差（3.53±0.42毫克每一个，或8％的变化），resultin克鱼接受食物量相似。通过监控录像直观吃剩的颗粒消耗。消耗率通常为80％以上。 </li><li>在每星期的超过4周结束时，确定鱼体重。准备一个新的杯具新鲜APW，放在一个平衡，然后记录重量。然后，从原来的杯子倒入鱼放进一个抄网，并转移到新的杯子，然后再次称重。鱼不处理，避免精神紧张。 </li><li> 团体 ，放置2升APW到一个19升桶和去皮规模。转移到新的APW桶时，然后记录总重量组两组养鱼在一起。每星期超过通过转移到一个新的水桶一个月的时间跨度记录鱼的重量组。 </li></ol> 9.肠道传输时间<ol><li>使用荧光素标记的70 kDa的阴离子葡聚糖。葡聚糖不显著消化，过大跨肠道层被吸收，从而通道将测量通过肠运送时间。标记的葡聚糖被并入鱼食。 </li><li>到无菌1.7毫升管中，60℃的加热块上添加的无菌去离子水360微升和52毫克明胶（13％溶液），并放置该管，直到明胶熔化，并完全溶解。 <ol><li>碾金鱼食品薄片成细粉使用研钵和杵，和40mg该粉末添加到管中，用鱼油的20μL的沿。混合后，添加1.6毫克的FITC葡聚糖。 </li></ol></li><li>分装热的液体混合物倒入20微升滴在封口膜的实验台上，让他们迅速冷却和固化。滴，可存储长达4天，他们变得太硬鱼吃了。通过将封口膜到陪替氏培养皿中，然后浮在无菌水密封的塑料容器，保持加湿的液滴内部的一半在冰箱商店下降。 </li><li>只是馈送之前，季落入使用刀片部分。适应鱼的泰锘个别2天泡沫杯不吃草（注:只使用了未曝光控制鱼）。放置食物的四个季度与鱼杯中，观察鱼30分钟，他们每吃的食物有多少件。 </li><li>要开始监测期，用80毫升APW的用抄网单独传送鱼入杯子。每2小时，用手轻轻摇动APW，然后在4℃取1毫升样品，并存储在一个标有1.7毫升管。取样品36小时。 </li><li>记录荧光，与测定完成之后的荧光分光光度计，在同一时间的所有样品。将整个1毫升样品放入试管，并记录使用在495nm处激发和发射在520nm处测得的荧光。 </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Laboratory+Module+for+Host-Pathogen+Interactions:+America's+Next+Top+Model.">A Laboratory Module for Host-Pathogen Interactions: America's Next Top Model.</a> J. Microbiol. Biol. Educ. 11, (2010).</li><li>Uliano, E., Cataldi, M., Carella, F., Migliaccio, O., Iaccarino, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+acute+changes+in+salinity+and+temperature+on+routine+metabolism+and+nitrogen+excretion+in+gambusia+(Gambusia+affinis)+and+zebrafish+(Danio+rerio).">Effects of acute changes in salinity and temperature on routine metabolism and nitrogen excretion in gambusia (Gambusia affinis) and zebrafish (Danio rerio).</a> Comp Biochem Physiol A. 157, 283-290 (2010).</li><li>Shotts, E. B., Waltman, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+medium+for+the+selective+isolation+of+Edwardsiella+ictaluri.">A medium for the selective isolation of Edwardsiella ictaluri.</a> J Wildl Dis. 26, 214-218 (1990).</li><li>Under animal toxicity studies, sodium chloride entry. TOXNET - Hazardous Substances Data Bank Available from: <a target="_blank" href="https://toxnet.nlm.nih.gov/">https://toxnet.nlm.nih.gov/</a> (2016)</li><li>Under animal toxicity studies, sodium nitrate entry. TOXNET - Hazardous Substances Data Bank Available from: <a target="_blank" href="https://toxnet.nlm.nih.gov/">https://toxnet.nlm.nih.gov/</a> (2016)</li><li>Under animal toxicity studies, sodium nitrite entry. TOXNET - Hazardous Substances Data Bank Available from: <a target="_blank" href="https://toxnet.nlm.nih.gov/">https://toxnet.nlm.nih.gov/</a> (2016)</li><li>Vilz, T. O., 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=Functional+Assessment+of+Intestinal+Motility+and+Gut+Wall+Inflammation+in+Rodents:+Analyses+in+a+Standardized+Model+of+Intestinal+Manipulation.">Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation.</a> J Vis Exp. (67), e4086 (2012).</li><li>Katoh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+Harmonization+of+Laboratory+Animals.">International Harmonization of Laboratory Animals.</a> National Research Council (US) International Committee of the Institute for Laboratory Animal Research. Microbial Status and Genetic Evaluation of Mice and Rats: Proceedings of the 1999 US/Japan Conference. , (2000).</li></ol>

The authors have nothing to disclose.

一些挑战需要抗生素治疗的药物在鱼组织被耗尽后，清洁APW休息时间。如果暂停时间段被跳过然后抗生素存在可混淆的结果，特别是当测定涉及暴露于细菌。为了研究而无需在主机上，初步实验监控微生物组成（16S分析或全基因组测序）和人口密度（通过定量PCR 16S定量）对微生物的总数大的变化抗生素暴露期间从改变肠道微生物组成的影响将是需要。而3天在本系统中是最优的，改变宿主和/或抗?...

它已经确定，抗生素可以破坏人类微生物导致生态失调，这意味着微生物群落的不平衡。抗生素治疗后的微生物群的组成的改变已经显示降低社会的多样性，减少主要成员，并改变社区代谢，特别是在肠道1,2。肠道微生物的抗生素干扰可以减少定植抗力艰难梭菌 3,4和沙门氏菌 5。 此外，该微生物群的破坏已与许多综合征和人类疾病的发展（ 例如，抗生素相关性小肠结肠炎，炎性肠病，代谢紊乱， 等等 ）。抗生素也被广泛的农业实现为在生长促进畜禽生产6。这些功能强大的工具的使用也不是没有附带影响，这是抗生素耐药性的迅速崛起，以及一个破坏微生物的作用明显有它的有人居住的主机。许多研究表明，广谱抗生素的使用具有长期持久的后果的微生物群的结构和功能，然而，从抗生素被破坏的微生物影响宿主生理副作用仅具有尚待支持推测。 主机，微生物和抗生素之间的相互作用是远远以简洁的方式理解。因此，一个简单而更容易处理模式有利于高度复杂的哺乳动物系统上脱落光。在人体黏膜表面，包括肠道，怀有最高的密度和微生物的多样性，也是最亲密的微生物 - 宿主相互作用。鱼提供S上的粘膜皮肤微生物everal优势作为模型系统。的真骨鱼类 （硬骨鱼）是最早谱系的脊椎动物的含义，硬骨鱼既有先天和后天已经共同发展与共生的细菌群落7的关系的免疫系统内的分歧之一。鱼皮股哺乳动物1型粘膜表面，如生理功能，免疫组件和黏液产生细胞8的排列许多特性。鱼皮粘膜表面的外部位置提供了一个微生物易于操纵实验和样品。 西方蚊， 食蚊（G. 慈），是已经在过去已经用于研究交配和毒理学9，10，11的模型的鱼。由于体积小，种群数量在野外为入侵物种，男<html> inimal保健费用，和顽强的本性，我们已经开发G.慈竹作为粘膜微生物模式。此外， 食蚊鱼分享生下活年轻，胎生哺乳动物，这是鱼的种类罕见的生理。我们完成了鱼皮正常菌群的时间用16S最广泛的研究， 食蚊鱼 12剖析。进一步的工作表明以下使用广谱抗生素13的皮肤和肠道菌群破坏宿主三个负面影响。 在鱼以下抗生素暴露检查五个不同的效果。该微生物的最完善的主机的好处是病原体竞争排斥。鱼类病原爱德华氏菌是已知会导致肠道败血症的爆发商业鲶鱼养殖场14。 E.氏菌也被证明致死感染斑马鱼类="外部参照"&gt; 15，16和17 食蚊鱼 。从水柱这种病菌的一个挑战可以作为排斥的措施。作为比较，以易感性的个体的病原体，暴露于混合生物的高密度时存活率也进行了。粪便和富含有机物的土壤被用作微生物群落的普遍遇到的来源。 细菌肠社区执行另一建立角色是养分处理和能量收获，从而影响该主机的整体营养摄取。作为营养的严重测量，鱼体重的被喂食标准饮食前一个月和后进行比较。抗生素治疗鱼的平均体重下降，而平均控制鱼的体重增加超过一个月。这种缺乏体重增加的机制尚不清楚。一个可能的因素是在肠的食物的运送时间。地理标志莫蒂lity方法从斑马鱼（Adam Rich在，纽约州立大学布鲁克波特，个人通信）适用于确定运输时间。它没有如抗生素治疗鱼类具有改变的过境时间尚未确定。 所有的生物体，特别是鱼类，在自然的环境中遇到的一个共同挑战是渗透胁迫。 食蚊鱼已被证明，当在高浓度的盐分18敏锐地强调快速适应。出人意料的是，鱼表现出抗生素的微生物改变生存下降到一个高盐胁迫。对于这种新颖的表型的机制正在调查中。对水生动物，尤其是在水族箱另一种常见的应力，是氮的毒性形式（氨，硝酸盐，亚硝酸盐和）。针对硝酸盐生存不是抗生素治疗和控制鱼之间显著不同。在这个手稿提出的方法可与食蚊或类似鱼模式生物，如斑马使用鱼和青鳉，测量下列实验操作中的鱼的表型。

<table border="0" cellpadding="0" cellspacing="0" width="568">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:184px;">Rifampicin</td>
			<td style="width:104px;">Calbiochem</td>
			<td style="width:127px;">557303-1GM</td>
			<td style="width:153px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Nitrate</td>
			<td>Sigma Aldrich</td>
			<td>S5506</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:184px;">Fluorescein-labeled 70 kDa anionic dextran</td>
			<td style="width:104px;">ThermoFisher Scientific</td>
			<td>D1823</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS tablets</td>
			<td>Calbiochem</td>
			<td>6500-OP</td>
			<td>tablets dissolve in water to make phosphate-buffered saline</td>
		</tr>
	</tbody>
</table>

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.

用于研究从抗生素曝光13宿主鱼效果实验系统的整体示意图如图1A所示，并且包括该技术，用于提取从鱼的皮肤（ 图1B）和肠（ 图1C）微生物组。因为先前的数据显示，而总的皮肤可培养数在治疗早期下降，它返回之后3天到治疗前水平三天被选为曝光的抗生素的时期。同时，群落​​组成，由16S分析确定，已经强烈?...

Watch this Scientific Journal Video about Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment at JoVE.com

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

这项研究涉及到的方法来揭示继抗生素皮肤和肠道微生物群落组成的改变模型鱼主机上的影响。

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 ...

examination-host-phenotypes-gambusia-affinis-following-antibiotic

Research

JoVE Journal

Immunology and Infection

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

7.6K 次观看.  Sam Houston State University. 这项研究涉及到的方法来揭示继抗生素皮肤和肠道微生物群落组成的改变模型鱼主机上的影响。 

视频: Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

审核端粒G -悬结构锥虫布氏</em

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

科研

免疫与感染

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 ...

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of innate immunity are conserved between insects and mammals, and since Drosophila can readily be genetically and experimentally manipulated, they are powerful for studying immune system function and the physiological consequences of disease. The procedure demonstrated here allows infection of flies by introduction of bacteria directly into the body cavity, bypassing epithelial barriers and more passive forms of defense and allowing focus on systemic infection. The procedure includes protocols for the measuring rates of host mortality, systemic pathogen load, and degree of induction of the host immune system. This infection procedure is inexpensive, robust and quantitatively repeatable, and can be used in studies of functional genetics, evolutionary life history, and physiology.

Systemic Bacterial Infection and Immune Defense Phenotypes in Drosophila Melanogaster

Drosophila melanogaster is an outstanding model organism for studying innate immune systems and the physiological consequences of infection and disease. This protocol describes how to deliver robust and quantitatively repeatable bacterial infections to D. melanogaster, and how to subsequently measure infection severity and quantify the host immune response.

全身细菌感染与免疫防御的表型<em&gt;果蝇</em

The fruit fly Drosophila melanogaster is one of the premier model organisms for studying the function and evolution of immune defense. Many aspects of ...

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

Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (&#62; 50% reduction in cell length for each antibiotic; P&#60; 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug&#39;s MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.

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 ...

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 ...

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining

A simple ATP-measuring assay and live/dead staining method were used to quantify and visualize Neisseria gonorrhoeae survival after treatment with ceftriaxone. This protocol can be extended to examine the antimicrobial effects of any antibiotic and can be used to define the minimal inhibitory concentration of antibiotics in bacterial biofilms.

atp 利用商业检测和染色法定量检测淋病奈瑟菌骨料的抗生素敏感性

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

人类舱内单聚物研究病原体、共脉和宿主肠道上皮细胞之间的相互作用

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Antibiotic Dereplication Using the Antibiotic Resistance Platform

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

使用抗生素耐药性平台进行抗生素脱除

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this ...

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 ...

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis.
This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Described here is an established method to determine the extent of HIV-1 restriction by the cellular inhibitory protein SAMHD1. Human myeloid lineage U937 cells are transduced with a SAMHD1 expression vector co-expressing YFP, differentiated and then challenged with HIV-RFP. The level of restriction is determined by flow cytometry analysis.

流式细胞术分析人髓系U937细胞中SAMHD1限制性

Sterile &#945;-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are ...

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