The authors would like to acknowledge the University of Lille and the Pasteur Institute of Lille, especially Thierry Chassat and Jean-Pierre Decavel, responsible for animal housing breeding safety and husbandry. This work was supported by the "Soci&#233;t&#233; de Pathologies Infectieuses de Langue Fran&#231;aise" (SPILF).

动物实验的区域伦理区域委员会已经批准了这个方法，按照在研究性研究指导原则与各国和国际动物护理和使用。 1.样品采集<ol><li>样品存储<ol><li>收集所有的样品，并立即在存放 - 20℃或在冰上直到冷冻储存以避免恶化。置于冰上的无菌磷酸盐缓冲盐水（PBS），以改善支气管肺泡灌洗（BAL）的性能。 </li></ol></li><li>手术<ol><li>灭菌用高压灭菌器的所有手术设备。 注意:如果可能，建议使用两套不同仪器对于腹部和胸部的步骤，以避免交叉污染。需要解剖设备在图4A中详述</li></ol></li></ol> 2.鼠标，细菌和酵母菌株<ol><li>家鼠符合在当地再利用动物在通风机架遴选委员会的指导方针不超过5只小鼠笼子，食物和水即兴 ，在生物安全2级的住房设施，由于使用生物安全2级微生物:P。铜绿假单胞菌和C.白色念珠菌。 </li><li>保持细菌菌株在-80℃下在40％甘油中。 <ol><li>直接从冷冻股票添加到细菌含有使用10微升接种圈3毫升无菌卢里亚BERTANI肉汤培养管。离开O / N在37℃轨道振荡（400转）滴注的前一天。 </li><li>收获细菌离心2000×g离心5分钟。 </li><li>吸出上清液到一个合适的封闭的生物危险废物处置。观察白色沉淀粘附在培养管中的底部。 </li><li>洗净并暂停用5毫升的PBS的沉淀。 </li><li>重复步骤2.2.2至2.2.3第二次进行第二次洗涤。 </li><li>悬浮用1ml的PBS的沉淀的细菌和短暂的震荡。 </li><li>使用光学密度计在600nm处使用光学密度计确定接种密度。 0.9的密度相当于10 9 CFU /毫升PAO1，淡化相应。 注意:这个结果对于每个使用的菌株通过确定一个校准接种物的连续稀释液的密度来获得。 </li><li>验证接种通过连续对数稀释和板100微升每个稀释度的溴甲酚紫琼脂（BCP）板和O / N培养。管理每个小鼠经鼻内将50μl含1×10 8到2×10 8 CFU每毫升（5×10 6到1×10 7每鼠CFU）的溶液中。 </li></ol></li><li>使用C.白色念珠菌 SC5314作为参考压力。保存在40％的甘油培养基中的菌株在-80℃下。 <ol><li>补充酵母蛋白胨 - 葡萄糖肉汤0.015％，丁胺卡那霉素，避免细菌污染，有利于进一步计数。 </li><li>添加酵母使用10＃181; l接种环成准备YPD肉汤补充阿米卡星O / N在37℃。 </li><li>通过离心收获酵母在2000×g离心5分钟。 </li><li>除去上清液到适当的bioharzard废物处理。白粘附粒料应培养管的底部被观察到。 </li><li>洗净，暂停用5毫升的PBS和短暂的震荡的沉淀细菌。 </li><li>重复步骤2.2.2至2.2.3第二次进行第二次洗涤。 </li><li>悬浮用1ml的PBS和短暂的震荡的沉淀。 </li><li>通过使用标准的显微镜放大40倍于一个Mallassez血细胞计数计数确定所述种菌的大小。 注:（数酵母×10 5）/（计数网格的矩形的对Mallassez血细胞计数数）:浓度（以CFU / ml）的，使用下式求出。 </li><li>通过连续对数稀释验证至10 -5和 10 -6，确认溶液含有2×10 6 CFU /毫升。 </li><li>板块上YPD琼脂平板上添加了0.015％，丁胺卡那霉素。 </li></ol></li></ol> 3.航空公司定植C.白色念珠菌 注:环境适应后，小鼠每天称重两次。 <ol><li>在通风橱下，存款500微升七氟醚的在一块4厘米×4厘米的纱布（开滴技术）14。 <ol><li>立即将纱布上的约750 ml的感应腔室的地板上。立即将纱布上述网状凸起的平台，以避免动物和纱布之间的直接接触。 </li><li>关闭气密盖，并等待1至2分钟，以允许七氟烷扩散在腔室。 </li><li>传输鼠标从笼网平台，并盖上盖子。浅麻醉用保守的自主呼吸应在30至45秒来实现。 </li><li>通过观察翻正反射消失在这一点鼠标可以F为拆除监视肌张力低下光盘盒子和灌输。 </li></ol></li><li>滴鼻（可在10秒由受过训练的人员进行）。 <ol><li>按住鼠标单手依偎在它的背上直立举行（ 图4B）。 </li><li>使用食指，支撑头部和用拇指以保持钳口闭合，以避免咳痰（图4C）。 </li><li>由于在步骤2.3.8中所述，确保准备C.白色念珠菌液中含有每毫升2×10 6 CFU为50微升灌输量。 注:第二个关键点是滴注量。体积小于50微升，可能会导致不充分的殖民统治或气道滴入不均匀，较大的体积可能诱发溺水/窒息而死亡。 </li><li>灌输鼠标鼻内由接近吸管鼻孔。 </li><li>吸管50微升下降形成含有鼻孔溶液中的泡沫，由spontaneousl随后吸入Ÿ呼吸鼠标。 </li><li>在恢复区位鼠标（ 例如，一个大的良好的通气裸笼顶置加热灯）。小鼠必须直至完全苏醒进行监控。不要让动物无人看管，直到它重新获得足够的意识，以保持胸骨斜卧和翻正反射。在这一点上，鼠标可以返回到正常壳体笼。 </li></ol></li></ol> 4. P.铜绿假单胞菌诱导的急性肺部感染注:小鼠在以下四个天称重。通常情况下，老鼠C.期间长胖白色念珠菌介导的气道定植（图2A）。 <ol><li>制备含P.暂停铜绿假单胞菌滴注后，O / N增长（2.2节）的一天。 </li><li>简要麻醉使用七氟烷吸入以上（3.1节）中的说明。 注:要执行急性肺部感染，建议细菌负担的建议中表1中。 </li><li>灌输鼠标上面（3.2节）所描述的特别注意后滴入恢复。 </li></ol> 5.测量肺损伤指数<ol><li>制备含有FITC-标记的白蛋白溶液。注入人道毁灭之前，这个解决方案2小时。 <ol><li>称量0.2毫克白蛋白-FITC与适当的设备。 </li><li>加0.2毫克到1毫升的PBS。简单地说旋涡。如果不立即使用，将解决方案箔以避免暴露于环境光。 </li><li>注入腹膜内200μl的FITC标记的白蛋白溶液到每只小鼠。 </li></ol></li><li>安乐死<ol><li>称重小鼠最后重量数据。 </li><li>安乐死按照使用一个腹腔注射苯巴比妥致死的过量研究委员会指导当地动物使用一个单一的鼠标:300微升5.47％戊巴比妥。 </li><li>从笼中取出鼠标和接收升ethal注射操作员。 </li><li>注射后，独自一人转移鼠标移动到另一个笼子，瞒过任何其他动物。观察鼠标，直到不存在运动。确认死亡是由缺乏运动，特别是呼吸运动，缺少脉的。 </li><li>执行对动物尸体样本的采集手术，因此无需麻醉剂，也不镇痛药。 </li></ol></li><li>手术标本采集:胸阶段。 注:为了保持无菌条件下，用消毒设备在生物安全2级的环境中进行的所有手术。 <ol><li>适用乙醇到皮肤上。从胸骨到用剪刀中间腹部进行中线切开皮肤。从沿两边胸腔中线裁。折回上胸部两侧的皮肤以可视化的肋笼。 </li><li>两侧往上朝着锁骨按顺序执行胸腔的垂直切口，以便能够与船尾放倒整个前胸壁嗯让心脏和肺（ 图5A，5B）的完美可视化。 </li><li>使用预heparined注射器通过打孔旁室间动脉的心脏采集血液。撤回至少500微升，以获得至少100微升血浆。置于冰上血样。 </li><li>执行中线颈部切开以可视化的气管（图5B 和5C）。仔细解剖周围的气管筋膜。放置气管（图5C 和5D）后面的缝合线。随后，缝合线将围绕cannulating针封闭，以确保适当灌洗。 </li><li>使用20-G的改性灌胃针（图5D 和图4A）导尿气管。领带外科结周围的气管插管与先前放置缝合。 </li><li>要执行支气管肺泡灌洗（BAL），轻轻地，并逐步注入和吸取500微升冰冷的PBS到/从肺。放在我的样本CE避免细胞裂解。 </li><li>重复步骤5.3.6，3次，得到总1500微升BAL液和池灌洗样品到2ml离心管（图5E）。 </li><li>从胸部取出肺。放置一个肺段（大小应该对应于一个肺或一个波瓣的一半）到1.5毫升离心管中，并在快速存储 - 80℃。 </li><li>放置一个肺段在含PBS中，以确定细菌负荷，并将其放置在冰上预称重溶血管。 </li></ol></li><li>手术标本采集:腹部阶段。 <ol><li>上腹部的左侧执行另一个切口。通过观察腹膜脾。 </li><li>取出脾并放入第二溶血管含1毫升的PBS置于冰上。 </li></ol></li><li>肺损伤指数 注:肺泡 - 毛细血管膜渗透性是通过测量评估FITC标记白蛋白漏出从血管腔隙的肺泡 - 刘晓丹itial舱。 <ol><li>离心血样和BAL液在1500×g的10分钟。收集上清液到新的离心管中。粒料对应招募血浆或BAL细胞和应放置在冰上。 </li><li>加入100微升每血液上清液（血浆，黄色）或BAL上清液至96孔透明板（300微升孔）。放置的箔上盘如果不立即使用。 </li><li>测定血浆荧光水平，并用荧光酶标仪（激发，487纳米，发射520纳米）BAL上清液。 </li><li>通过计算荧光比率确定肺损伤指数[（BAL上清液/血液上清液）×100]。 </li></ol></li><li>支气管肺泡灌洗（BAL）差异细胞计数。 注:使用从BAL液离心分离得到的细胞沉淀步骤5.5.1。 <ol><li>如果需要的话，可以使用红细胞裂解缓冲液。加入500微升红细胞裂解缓冲液入含有CEL离心管升沉淀。简言之涡和置于冰上10分钟。加入500μlPBS阻止红细胞溶解。 </li><li>收获的细胞离心，在1500×g下10分钟。除去上清，悬浮细胞沉淀在1ml无菌PBS中。枚举在Mallassez血细胞计数细胞，利用血球计数器来计数细胞。专注于一个带离心涂片幻灯片细胞。 </li><li>使用着色工具包，允许细胞识别和计数（巨噬细胞，淋巴细胞，中性粒细胞）染色的细胞。 </li></ol></li><li>肺部细菌负担和细菌传播 注:为了评估肺部细菌负担和细菌传播，肺和脾，分别收集并存储到含1毫升的PBS（步骤5.3.9）的预先称重溶血管。 <ol><li>称重含有1ml PBS中并任肺或脾溶血管。使样品均质化用组织匀浆器以获得肺匀浆和脾匀浆。 </li><li>存入100微升组织同源的genates到含有900微升无菌PBS，得到串行数稀释离心管中。 </li><li>板最后两个适当稀释的样品（10 -3和10 -2）在任BCP琼脂为P.绿脓杆菌或YPD，丁胺卡那霉素，补充琼脂C.白色念珠菌肺和脾的负担的决心。 </li><li>孵育板O / N在37℃。次日，列举在平板上的菌落。 </li><li>指数的结果肺重量，得到每克肺的CFU。肺样本大小是不一样的，结果应表示为每克肺的CFU。 公式索引:[CFU]×[重溶血管和肺] - [重溶血管。 </li></ol></li></ol>

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The authors have nothing to disclose.

动物模型，特别是哺乳动物，是有用的阐明宿主 - 病原体相互作用的复杂机制的免疫的领域。当然，对信息的需求只能从动物模型一定是必不可少的获得;否则，使用动物必须由体外模型来代替。该动物模型示出了只能由动物模型来提供自病原体之间的相互作用是由多组分宿主反应介导的洞察力。目前用来研究这种宿主 - 病原体相互作用小鼠是6岁到10周与成熟，不变的免疫反应的年轻人。当...

动物模型，特别是小鼠，已被广泛用来研究对病原体的免疫应答。虽然啮齿动物和人7之间先天和获得性免疫不同，容易在育种和击倒的发展，为众多的基因，使小鼠极好模型来研究免疫应答8。免疫反应是复杂的，来自病原体的相互作用的结果，驻地微生物菌群和多种免疫（淋巴细胞，中性粒细胞，巨噬细胞）和非免疫（上皮细胞，内皮细胞）的细胞类型2。 体外研究不允许观察这些复杂的相互作用，并主要集中在唯一的小区 - 病原体相互作用。而动物模型必须谨慎使用，并仅限于非常具体和相关问题，小鼠模型提供了良好的洞察体内的哺乳动物的免疫反应，并且可以解决重要的临床问题，7份 。 6。而什么构成一个"正常"的气道微生物组仍有待确定，居民社区经常多种微生物，并从不同的生态源产生。化脓性慢性肺疾病（囊性纤维化，bronchectasis）的患者或机械通气患者由环境获得微生物9显示出一个特定的菌群，由于气道的定植。 绿脓杆菌 和白色念珠菌都是问题病原体5，经常分离一起从气管样品，和负责在这些患者严重的机会感染，特别是在重症监护病房（ICU）4。 这些微生物的急性肺炎在ICU的结果对铜绿抗微生物治疗期间隔离铜绿 bUT酵母通常不会在这个网站5考虑致病。P.之间的体外相互作用铜绿假单胞菌和C.白色念珠菌得到了广泛的报道，并表明，这些微生物可以影响生长和彼此的生存但研究不能断定，如果C的存在下白色念珠菌是有害还是有益的主机 10。小鼠模型被开发来解决这个P的相关性铜绿假单胞菌和C. 白色念珠菌在 体内 ，但微生物之间的相互作用不是重点。事实上，建立模型以评价C的参与白色念珠菌在宿主免疫应答，和结果。 通过Roux等建立了以前的模式已经使用初始定植C.白色念珠菌其次是一种急性肺部感染诱发P.铜绿假单胞菌，利用他们的模型中，作者发现P的有害作用rior C.白色念珠菌定植11。然而Roux等使用℃的高负荷期间连续3天白色念珠菌在其用2×10 6 CFU /小鼠模型。我们建立了C的 4天模式白色念珠菌呼吸道定植，或至少没有持续性肺损伤，在这个模型C.白色念珠菌被检索长达4天的每只小鼠（图2B）12,13 10 5 CFU单个滴注后。 4天后，没有观察到证据炎性细胞募集，炎症性细胞因子的产生，也没有上皮损伤。在24 - 48小时在C的峰值出现白色念珠菌 ，即使在蜂窝和细胞因子的先天免疫反应，观察，没有任何证据表明肺损伤。出人意料的是，这样的小鼠定植C.白色念珠菌 48小时前鼻内P的滴注绿脓杆菌已经减弱感染相比，小鼠P.绿脓杆菌感染孤单。一世ndeed，小鼠表现出较少的肺损伤和减少细菌负荷12,13。 有几个假说可以解释前定植C.这种有益作用白色念珠菌的P.铜绿假单胞菌介导的急性肺部感染。首先，将种间串扰涉及每个微生物群体感应系统，所述homoserinelactone基P.假单胞菌系统和金合欢醇系C.白色念珠菌系统，进行了评价。其次，C。白色念珠菌作为"诱饵"目标绿脓杆菌转向从肺上皮细胞中的病原体进行了研究。这两种假设都无效（未公布数据）。第三假设是一个由C"启动"先天免疫系统的白色念珠菌负责对铜绿增强后续的先天响应铜绿假单胞菌 。这最后一种假设得到证实。事实上C.白色念珠菌定植导致先天免疫的启动THROUGH，IL-22，主要由分泌的先天淋巴样细胞，导致增加细菌的间隙并减小肺损伤12。 最后，该宿主是微生物调节先天免疫反应和涉及不同炎性细胞类型之间的相互作用的中心角色。虽然这些复杂的免疫相互作用可以在体外被解剖的初始假设只能通过适当的体内模型提供。以下协议提供的体内研究宿主介导的病原体相互作用的可适于其他微生物的一个例子。

<table border="0" cellpadding="0" cellspacing="0" width="729">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sevorane, Sevoflurane</td>
			<td style="width:185px;">Abott</td>
			<td style="width:161px;">05458-02</td>
			<td style="width:167px;">250 mL plastic bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluorescence Reader Mithras&#160; LB940</td>
			<td style="width:185px;">Berthold Technologies</td>
			<td style="width:161px;">reference in first column</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromo-cresol purple agar</td>
			<td style="width:185px;">Biomerieux</td>
			<td align="right" style="width:161px;">43021</td>
			<td>x20 per unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pentobarbital sodique 5,47%</td>
			<td style="width:185px;">CEVA</td>
			<td align="right" style="width:161px;">6742145</td>
			<td>100 mL plastic bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-headed valve&#160;</td>
			<td style="width:185px;">Distrimed</td>
			<td align="right" style="width:161px;">92831</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile inoculation loop 10 &#181;L</td>
			<td style="width:185px;">Dutscher</td>
			<td align="right" style="width:161px;">10175</td>
			<td>x1000 conditioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insuline syringes 1 mL</td>
			<td style="width:185px;">Dutscher</td>
			<td align="right" style="width:161px;">30003</td>
			<td>per 100 conditioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2 positions Culture tube 8 mL</td>
			<td style="width:185px;">Dutscher</td>
			<td align="right" style="width:161px;">64300</td>
			<td>no comment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrospec 10&#160;</td>
			<td style="width:185px;">General Electric life sciences</td>
			<td style="width:161px;">80-2116-30</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemolysis tubes 13 x 75 mm&#160;</td>
			<td style="width:185px;">Gosselin</td>
			<td style="width:161px;">W1773X</td>
			<td>per 100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS - Phosphate-Buffered Saline</td>
			<td style="width:185px;">Life technologies</td>
			<td align="right" style="width:161px;">10010023</td>
			<td>packaged in 500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">amikacin 1g</td>
			<td style="width:185px;">Mylan</td>
			<td align="right" style="width:161px;">62516778</td>
			<td>per 10&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heparin 10 000 UI in 2 mL</td>
			<td style="width:185px;">Pan pharma</td>
			<td align="right" style="width:161px;">9128701</td>
			<td>x 10 per unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RAL 555 coloration kit</td>
			<td style="width:185px;">RAL Diagnostics</td>
			<td align="right" style="width:161px;">361550</td>
			<td>3 flacons of 100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,5 mL microcentrifuge tube</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:161px;">55.526.006</td>
			<td>x&#160; 1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transparent 300 &#181;L 96-well plate</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:161px;">82 1581500</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yest-peptone-Dextrose Broth</td>
			<td style="width:185px;">Sigma</td>
			<td align="right" style="width:161px;">95763</td>
			<td>in powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-albumin</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:161px;">A9771</td>
			<td>in powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Luria Bertani Broth</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:161px;">L3022</td>
			<td>in powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25-gauge needle</td>
			<td style="width:185px;">Terumo or unisharp</td>
			<td style="width:161px;">A231</td>
			<td>x100 conditioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytocentrifuge</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:161px;">A78300003</td>
			<td>no comment</td>
		</tr>
	</tbody>
</table>

studying microbial communities in vivo: a model of host-mediated interaction between candida albicans and pseudomonas aeruginosa in the airways

研究宿主 - 病原体相互作用使我们能够理解的致病性的基本机制微生物感染时。的主机的预后取决于抗病原菌1的适于免疫应答的参与。免疫反应是复杂的，结果从的病原体和几个免疫或非免疫细胞类型2的相互作用。 体外研究不能表征这些相互作用并集中于细胞-病原体相互作用。此外，在气道3中，特别是在患者的化脓性慢性肺疾病或机械通气患者中，多种微生物群落是本和复杂化宿主-病原体的相互作用。 绿脓杆菌 和白色念珠菌都问题病原体4，从气管样品经常分离，并相关的严重感染，尤其是在重症监护病房5。微生物相互作用这些病原体之间被报道在体外但是这些相互作用的临床影响仍不清楚6。研究℃。白色念珠菌和P之间的相互作用假单胞菌，C 的鼠模型白色念珠菌呼吸道定植，随后是P.进行aeruginosa-介导的急性肺部感染。

作为协议的描述中之前看到的，实验需要5个一天就能完成（ 图1:实验时间表）。在实验的整个运行过程中的一个操作员请求的和可处理的处理最多10只小鼠。如果更多的动物是必需的，需要特别用于外科样品收集两个人。实际上，所有样本必须收集在下2小时，以避免在最后的小鼠FITC标记的白蛋白的增加的被动肺泡 - 毛细血管渗漏。 第一步是C的制备白?...

Watch this Scientific Journal Video about Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways at JoVE.com

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

研究微生物群落<em&gt;在体内</em&gt;:主机之间介互动的典范<em&gt;白色念珠菌</em&gt;和<em&gt;铜绿假单胞菌</em&gt;在航空

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

studying-microbial-communities-vivo-model-host-mediated-interaction

Research

JoVE Journal

Immunology and Infection

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

8.4K 次观看.  Université Lille. While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

视频: Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

RNA的分离绿脓杆菌殖民小鼠胃肠道

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

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 ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

绿脓杆菌引起的肺损伤模型

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

慢性假单位病伤口感染的延迟接种模型

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

澄清和成像念珠菌生物膜

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

前体内分析研究胃 肠道的 念珠菌血脂磷

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

研究微生物群落

摘要

探索更多视频

此视频中的章节

RNA的分离绿脓杆菌殖民小鼠胃肠道

使用人工痰介质测试抗生素药效试验

绿脓杆菌引起的肺损伤模型

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

慢性假单位病伤口感染的延迟接种模型

澄清和成像念珠菌生物膜

前体内分析研究胃肠道的念珠菌血脂磷

囊性纤维化患者痰液中铜绿假单胞菌的可视化

研究微生物群落

摘要

探索更多视频

此视频中的章节

RNA的分离绿脓杆菌殖民小鼠胃肠道

使用人工痰介质测试抗生素药效试验

绿脓杆菌引起的肺损伤模型

来到光明的一面: 体内糖尿病无毛小鼠慢性伤口生物膜感染的监测

在研究隐球菌-阿米巴相互作用中,显微技术和荧光读数的补充应用

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

慢性假单位病伤口感染的延迟接种模型

澄清和成像念珠菌生物膜

前体内分析研究胃 肠道的 念珠菌血脂磷

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

前体内分析研究胃肠道的念珠菌血脂磷

囊性纤维化患者痰液中铜绿假单胞菌的可视化