作者要感谢宾夕法尼亚大学的杰弗瑞·韦瑟博士赠送肺炎 链球菌临床菌株。这项工作由加拿大卫生研究所资助。简历由M.G.德格罗特奖学金和加拿大胸科学会的奖学金资助。这项工作由安大略肺脏协会和加拿大卫生研究所（CIHR）资助。鲍迪什实验室的工作部分得到了迈克尔·德格罗特传染病研究中心和麦克马斯特免疫学研究中心的支持。

在开始之前：除非另有说明，否则所有步骤均在生物危害 2 级 （BSL2） 生物安全柜 （BSC） 中完成。请确保在开始实验之前，您已获得适当的生物危害批准，以便根据机构指南使用传染性细菌病原体。此外，请确保您拥有进行事先准备的程序所需的所有材料和试剂。这些实验中使用的老鼠包括杰克逊实验室、查尔斯河或塔科尼奇的雌性C57BL/6小鼠，年龄在10-14周（尽管我们尚未发现鼻腔结肠间隙或感染的动能存在任何性别依赖性显著差异）。这些实验中使用的所有小鼠都是在无病原体条件下繁殖和维持的，并且没有常见的病毒（LCMV、MNV、MPV、病毒ECTV和其他）细菌（如幽转杆菌）和寄生虫（如针虫、异位虫），通过粪便样本检测，以及经常对其设施内的哨点小鼠进行解剖评估。在进行这些实验时，我们建议使用年龄不小于10-12周、年龄不小于6个月的对照小鼠。小鼠比这个年龄范围小或大，更容易受到更长的鼻咽运输持续时间和传播感染的可能性增加的影响。小鼠背景是另一个重要的考虑因素，可能会影响殖民化实验的结果，因为几个小组已经证明，不同遗传背景的小鼠有不同的易感性，以S.肺炎D39（血清型2）株9，10。肺炎不是天然存在的穆林病原体，它唯一的天然储液库是人类鼻咽。传播通过呼吸液滴发生，由于小鼠不产生呼吸分泌物，个别小鼠不能将细菌传染给其他小鼠，因此对小鼠对小鼠的传播没有顾虑。有关本手稿中描述的程序的视觉概述，请参阅图1。
1. 准备肺炎文化
<ol><li>接种5毫升的试用大豆，用于 肺炎链球菌的暂停生长。</li>
	<li>静态条件下的培养在 37 °C 的 5% CO2 中，直到细菌接种达到日志阶段增长，相应的液体密度为 108 CFU/ml，由设定为 600 nm 的里程表确定。与此 CFU 对应的确切读数将因特定选定的细菌菌株而异：对于大多数 S.肺炎 菌株，这相当于OD600 范围0.45-0.55。通常，在建议条件下，液体培养中的 S.肺炎 菌株将在1.5-2.5小时内增长到这种密度，无需亚文化。不应允许该培养物过度生长（超出 0.75 的 OD 读数），因为这意味着细菌不再处于日志阶段生长并正在经历广泛的自动分析。</li>
	<li>每只老鼠将接种约107 种细菌。因此，每9只小鼠被殖民，移液器1毫升的接种进入埃彭多夫管和旋转在15，000 x 克1分钟。应该可以看到白丸。去除超标物，小心不要干扰颗粒，并补充磷酸盐缓冲盐水 （PBS） 100 μl 中的细菌，从而将浓度提高到 109 CFU/ml。在这个阶段，细菌应该保持存活，但不会轻易复制。</li>
	<li>如果使用多个单引，则组合成一个管子，以控制细菌密度的轻微样本间变化。</li>
	<li>将细菌保存在冰上，直到准备好接种，最长1小时。</li>
	<li>要获得精确的细菌计数，请执行从整洁的细菌接种开始的日志串行稀释系列。连续稀释 10 倍，将 10μl 添加到 90μl 的无菌 PBS 中。</li>
	<li>将 10 μl 稀释样本 10-5 - 10-9的 3 滴，加上 PBS 专用污染控制，放到单独标记的试用大豆 agar （TSA） 板上，并辅以 5% 羊血（图 2）。确保每一步从较高的 CFU 浓度稀释到较低的 CFU 浓度时，移液器提示都会更改，以避免携带过量的细菌并增加结果的变异性。人血阿加 （HBA） 板也可以用来代替 TSA 。由于许多 肺炎 菌株对新霉素（从5-20微克/毫升）有耐药性，这种抗生素也可能在制片阶段添加到首选的阿加介质中。这有助于列举，因为它消除了非耐受细菌。必须事先测试每种菌株的抗生素易感性，以确定每个细菌菌株使用抗生素的最佳浓度。</li>
	<li>允许干燥15-30分钟，然后覆盖板，倒置在细菌孵化器设置为37°C和5%CO2。在盘子里长出细菌菌落24小时。</li>
	<li>确定殖民地形成单位的数量及其相应的浓度。根据OD600值的确定，浓度应在1-4 x 109 CFU/ml范围内。 S. 肺炎菌落应以小而圆形的殖民地出现，呈黄色米色，中心为小凹陷，使其外观类似甜甜圈（图3）。</li>
</ol>2. 穆林内纳萨尔殖民化
<ol><li>将小鼠放入小鼠约束装置（经过改装的 50 毫升猎鹰管，尖端被切断以创建光圈）用拇指固定它们的身体底部，使其鼻子从约束器装置的锥形末端（图 4）中浮现出来。使用这种装置可以固定老鼠的头部和隔离其鼻孔的方式，尽量减少运动，以及阻碍动物试图粘乳移液器尖端，允许完全交付的接种。或者，小鼠可以通过颈部擦伤和手动约束来固定。我们不建议在进行内接种之前对动物进行麻醉。在麻醉下给动物接种接种导致一些接种剂扩散到肺部12，13。</li>
	<li>使用P10或P20移液器，通过沉积10微克的预制培养物给每只小鼠接种疫苗，在两个鼻孔之间均匀分布（允许接种者通过逐渐脉动接种滴入鼻子，使小鼠需要时间吸入接种）。为了实现接种的完全交付，暂停管理在任何时候，老鼠开始过度移动它的鼻子。整个接种不能注射到鼻腔中，因为老鼠在呼气时可能会通过鼻子排出一些疫苗：然而，由于被驱逐的量往往是微不足道的，而且接种中含有极高的细菌量，这不会显著影响殖民细菌的负荷。此外，可用于鼻咽粘膜殖民化的表面积有限，因此我们和其他人发现，建议的107 剂量足以获得所有小鼠的细菌水平一致，导致最初殖民细菌14，15 的变异性最小。</li>
	<li>如果将体重指标用作端点监控的一部分，则称量小鼠。每 12-24 小时监测一次小鼠的临床症状，包括嗜睡、毛皮褶皱和体重减轻。小鼠通常不会表现出疾病的症状，直到3-5天后殖民化，这些将之前减肥，可能平均每天约5%的总体重。随着老鼠病得越来越重，它们会采取驼背姿势，表现出活动减少和对刺激（包括处理）的反应能力下降。在这个阶段，疾病通常表明败血症和/或肺炎，并有可能是绝症，虽然小鼠可以每天用1毫升皮下盐水治疗，以改善结果。幸存的小鼠在殖民化后的第7天之后应该会开始出现好转，体重稳定后体重增加就证明了这一点，尽管不同株的S.肺炎可能会更快地诱发疾病，并导致临床症状沿着不同的时间线发展。有关在与 P1547 菌株殖民的小鼠中跟踪的体重的代表性结果，请参阅图 5。</li>
</ol>3. 鼻腔拉瓦奇样品收集
开始前： 使用1毫升注射器准备罐装针头，盖有26个3/8G斜针。切开2.5厘米的PE20聚乙烯管，内径0.38毫米，确保每端有一个斜尖。使用钳子，将一块2.5厘米长的PE20聚乙烯管（内径0.38毫米）滑到针尖上，避免刺穿管子侧。罐头可以保存在70%乙醇，直到需要。
<ol><li>安乐死实验鼠。由于颈椎脱位可能会损坏气管，因此必须避免这种安乐死方法。我们首选的方法是异氟乙烯麻醉，然后是脱盐，但是，在选择安乐死模式时，确保您遵循机构指南。</li>
	<li>使用70%的水乙醇，消毒动物的超安特里奥皮毛，特别是颈部，小心防止乙醇进入鼻孔。</li>
	<li>沿着动物颈部的中线进行单次纵向切割，在两端进行两次水平切口，从而创建一个开口来设想气管。</li>
	<li>小心地剥回皮肤到两侧，露出下面的颈部组织。</li>
	<li>气管应该可见，两侧的纵向肌肉包围。小心地剪下这些，以提供气管本身的清晰视图，注意不要切断周围的血管。</li>
	<li>如果血管被切断，血液存在，在进行之前，允许出血停止，然后通过分配无菌PBS和使用无菌纱布轻轻地吸收该地区多余的水分来清洁该地区几次。</li>
	<li>气管适当暴露后，在气管中半乳形切口约半向上（图6）。</li>
	<li>将 1，000μl 的消毒 PBS 绘制到先前准备的罐头中。</li>
	<li>将管插入气管朝向鼻子，保持斜边向下指向，以方便插入（图 7）。针头到位后，旋转180°，轻轻向上探针，直到感觉有光阻力。</li>
	<li>将埃彭多夫指定用于采集样本，就在鼠标鼻子下方。</li>
	<li>通过分配最少（+20微l）的PBS熔岩液来测试针头的正确位置 - 应在小鼠的鼻孔周围形成液滴;如果是这样的话，继续步骤3.13）。</li>
	<li>如果测试PBS直接从动物的嘴里出现，拉回管子，然后通过再次轻轻地向前探测，直到感觉非常轻微的阻力重新定位 - 小心不要推动坎努拉过这个阻力太远，因为你会移动它过去的鼻腔，并通过口腔。</li>
	<li>迅速分配针头的内容，以帮助取代和收集最大数量的细胞 - 内含物应流出通过鼠标的鼻孔和收集管。立即将样品放在冰上。</li>
	<li>要收集用于RNA分析的样本，使用包含同一小鼠上 500 μl RNA 裂解缓冲器的罐装针头重复步骤 3.8-3.13）。这将允许从剩余细胞群中收集利萨酸盐样本，这些样本主要由鼻咽粘膜上皮组成，因为非附属细胞应在初始 PBS 厕所后被移除。请注意，RNA裂解缓冲器会破坏上皮并破坏周围的组织，因此，如果需要保留这些组织，必须小心避免与肺等器官接触。收集后，将样品直接放在干冰上的RNA裂解缓冲器中，以捕捉冻结。一旦进入裂解缓冲区，样品可以按照制造商的说明存储，并且通常稳定在 -70 °C 几个月。</li>
</ol>4. 纳索芬克斯细菌负荷的确定
<ol><li>通过为每个粘膜鼻腔样本准备一个系列稀释系列来量化细菌。一般来说，细菌负荷可预期在0-104 CFU之间，因此进行三次10倍的连续稀释。将 10 μl 的整洁鼻腔熔岩样本 （100 CFU/ml） 添加到第一根管中，浓度为 10-1 CFU/ml。</li>
	<li>将细菌板分成象限，并分别用稀释系列（10 0 -10-3 CFU/ml）的成员标记象限。盘出3滴10μl样品的3稀释和整洁的样品试用大豆糖板补充5%羊的血液，如图2。</li>
	<li>允许干燥15-30分钟，然后覆盖板，倒置在细菌孵化器中，具有最佳的细菌生长条件（通常为37°C和5%CO2）。</li>
	<li>在盘子里长出细菌菌落18-24小时。</li>
	<li>通过平均每个稀释板上形成的菌落来确定殖民细菌的数量（图3）。图8显示，在不同时间点，由鼻熔岩培养决定的细菌密度，在3种不同菌株的S.肺炎中殖民，最长可达21天。</li>
</ol>5. 为流测定量的样品准备
开始之前： 准备抗体组合。为了量化白细胞种群，我们建议在指定稀释时进行以下组合：PE-Ly6G（克隆 1A8， 1 μg/ml）、FITC-Ly6C（克隆 AL-21，1 μg/ml）、eFluor 450-CD45（克隆 30-F11， 2.67 微克/毫升），APC-F4/80（克隆 PM8 RUO，0.67 微克/毫升），PerCP-Cy5.5-CD11c（克隆 N418 RUO， 0.5 微克/毫升）、PE-Cy7-CD11b（克隆 M1/70、0.33 μg/ml）、亚历克萨氟 700-CD3（克隆 1782、4 μg/ml）、eFluor 605NC-CD4（克隆 GK1.5、6.67 微克/毫升）。请注意，此组合的浓度为 2 倍（参见步骤 5.5）。所有抗体应稀释在FACs洗缓冲器（0.5%胎儿小腿血清，2mM EDTA，0.1%钠阿齐德在PBS），这也应事先准备。在等型匹配对照抗体的混合物中，最好是来自与标记抗体相同的供应商，并且与特定抗体的浓度相同。用同型控制抗体处理的样品将起到负对照作用。在用同型控制抗体治疗的样本中观察到的任何荧光应被视为背景。
<ol><li>预切离心机，可旋转 1.5 毫升埃彭多夫管到 4 °C。</li>
	<li>离心鼻拉瓦奇样品在 2，000 x g 10 分钟在 4 °C。 小心地移出超自然和储备。注意：由于鼻咽内细胞量小，除非有不受欢迎的红血球污染，否则细胞颗粒将看不见，而红血球会呈鲜红色。如果看到此问题，应丢弃样品。</li>
	<li>在50μl的Fc中重新补充样品？RIIb/CD16-2 （2.4G2） 抗体 （结合 Fc 受体并减少非特异性抗体结合） 在 FAC 洗涤缓冲区中，浓度为 4μg/ml。</li>
	<li>在冰上孵化样品30分钟。</li>
	<li>加入50微升预先准备的2倍浓缩荧光抗体混合物到样品中。从每个实验组中留出一个具有代表性的样本，作为等型控制。将同型抗体混合物添加到此样品中，以代替污渍混合。</li>
	<li>在冰上孵化样品1小时。</li>
	<li>离心机样品在2，000 x g 10分钟在4°C。 丢弃超自然物，并在 200μl 的 PBS 中重新使用。</li>
	<li>重复步骤5.7。</li>
	<li>第二次洗涤后，离心机样品在 4 °C 下 10 分钟内再次以 2，000 x g 的速度进行。</li>
	<li>在PBS（如果立即运行样品）或2%的甲醛（如果在染色后1-3天运行样品）中再吸收。</li>
	<li>进行流细胞测量时，收集每个样本的最大事件量，或直到整个样本被吸气。在未受感染、健康、幼鼠中，这一事件总数将少至1 000至2 000只：在细菌殖民化事件期间，这个数字可能会增加2到5倍以上，这取决于动物的疾病状况以及年龄和遗传背景等因素。 图9 显示了从3激光贝克顿·迪肯森LSRII流细胞仪中收集的代表性流量细胞测量结果，该测速仪使用450的向前散射和300的侧散射，尽管我们建议在样品收集之前优化您打算使用的特定流细胞仪的参数。注意：如果样本中甚至含有微量的血液污染，则收集的总事件将明显高于预期，并且样本应从分析中打折扣。</li>
</ol>6. 鼻熔岩的定量PCR（qPCR）分析
<ol><li>从步骤 3.14 室温解冻细胞。</li>
	<li>按照推荐的协议，选择首选的RNA提取。</li>
	<li>按照指示完成RNA提取程序后，使用光谱仪或基于电泳的方法（图10）量化RNA的量。我们通常以 260/280 nm 的比例获得每个样本 975 至 3，250 ng 的总 RNA，>1.7 或 RNA 完整性编号 （RIN） 左右，约为 8.1±0.13。</li>
	<li>根据制造商的协议，使用 M-MULV 反向转录酶使用 1，000 ng RNA（最大 13 μl）转录 cDNA。</li>
	<li>稀释生成的 cDNA 样品 8 倍，并同样引用到 4 个单独的管中，在 -20 或 -80 °C 的长期存储中。</li>
	<li>要通过 qPCR 测量基因表达，请在冰块或冷块上的三脚架中准备 25 个 μl 样本反应，其中包含： 您选择的 qPCR 套件中的 2 倍 qPCR 主组合中的 12.5 μl， 0.25 μl 参考染料，2 μl 稀释的 cDNA（步骤 7.5），1 μl 的混合前进和反向引物（400 nM 最终），9.25 μl 的 RNAse-DNAS 无水。该协议是以前公布的方法16的改编。</li>
	<li>一般来说，我们发现两步 qPCR 放大（95 °C 10 分钟，然后最多 40 个周期 x [95 °C x 15 秒，60 °C x 1 分钟]） 是有效的 （图 11a）;但是，必须优化每个引号对。放大后必须执行分离（熔化）曲线，以确保不会发生非特定放大。放大 （图 11b）</li>
	<li>我们通常运行每个分析的基因的标准曲线，以及每个96井板分析的标准校准器（源自肺或脾脏同质性）。通过参考染料首先使原始周期阈值 （Ct） 值正常化并通过相应的标准曲线转换结果值来获得相对成绩单量。这些相对量随后根据适用情况标准化为标准校准器和家政基因。</li>
</ol>

<ol>
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作者没有什么可透露的。

在这项研究中，我们提出了使用 肺炎链球菌 临床分离菌株的小鼠的鼻内结肠的详细方法，以及随后针对细菌招募到鼻咽的免疫细胞的隔离和特征。我们演示了如何在营养丰富的介质中培养细菌，并用于在小鼠中建立殖民活动，最初仅限于鼻咽。然后，我们展示了如何通过使用罐装针头，在气管暴露、切口和鼻腔熔洗后分离出招募到鼻咽的免疫细胞类型。鼻腔熔岩样本可在PBS中采集，以分?...

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 <td>M-MuLV Reverse Transcriptase</td>
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 <td>GoTaq qPCR Master Mix</td>
 <td>Promega</td>
 <td>A6001</td>
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</table>

characterization of inflammatory responses during intranasal colonization with streptococcus pneumoniae

肺炎链球菌的鼻咽结肠是侵入肺部或血液的先决条件。这种生物体能够殖民鼻咽的粘膜表面，在那里它可以居住，繁殖，并最终克服宿主防御入侵到宿主的其他组织。在通常的下呼吸道中发现感染会导致肺炎。或者，细菌可以传播到血液中，导致细菌血症，这与高死亡率2有关，否则直接导致肺炎球菌脑膜炎的发展。了解鼻咽结肠的动能和免疫反应是S.肺炎感染模型的一个重要方面。
我们的小鼠内殖民化模型改编自人类模型3， 并被多个研究小组用于鼻咽4-7宿主病原体反应的研究。在模型的第一部分，我们使用 肺炎 的临床分离建立一个自我限制的细菌殖民化，类似于人类成人的运输事件。此处详细说明的程序包括准备细菌接种，然后通过通过鼻内管理途径传递接种库建立殖民化事件。在稳定状态下，居民巨噬细胞是鼻咽的主要细胞类型。通常，在未受感染的小鼠8中，淋巴细胞很少，但粘膜结肠会导致低至高档炎症（取决于细菌物种和菌株的毒性），从而产生免疫反应，并随后招募宿主免疫细胞。这些细胞可以通过气管内含物的厕所进行分离，并与殖民化细菌的密度相关，以更好地了解感染的动能。

图 1 表示概述示意图，总结了协议的主要步骤。 图2-3 提供了本文所述协议所固有的微生物方法的可视化。 图4 表示小鼠进行鼻内殖民化的正确定位，而 图5 则描绘了与 S.肺炎 菌株P1547结肠的小鼠体重的典型变化。 图6-7 表示过程鼻腔熔洗部分的特定阶段，用于辅助可视化这两种技术。 图8-11 包括对从鼻腔清洗后从...

Watch this Scientific Journal Video about Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae at JoVE.com

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

描述了 肺炎链球菌 的鼻咽结肠化以及随后对附体或招募细胞的提取。该技术涉及冲洗鼻咽和收集流体通过鼻孔，并适应各种读数，包括微分细胞定量和分析的mRNA表达 原位。

肺炎链球菌在鼻内殖民期间炎症反应的特征

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

characterization-inflammatory-responses-during-intranasal

Research

JoVE Journal

Immunology and Infection

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

12.1K 次观看.  McMaster University. 描述了 肺炎链球菌 的鼻咽结肠化以及随后对附体或招募细胞的提取。该技术涉及冲洗鼻咽和收集流体通过鼻孔，并适应各种读数，包括微分细胞定量和分析的mRNA表达 原位。

视频: Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

科研

免疫与感染

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

在无菌无鼠标进定植评估肝代谢的变化 1 H核磁共振光谱

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

监测改变膜极性，膜完整性和细胞内离子浓度<em&gt;肺炎链球菌</em&gt;使用荧光染料

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

荚膜血清型<em&gt;肺炎链球菌</em&gt;使用荚膜肿胀反应

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

荚膜血清型<em&gt;肺炎链球菌</em&gt;乳胶凝集

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

B组的小鼠模型<em&gt;链球菌</em&gt;阴道定植

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

增加了恢复时间，并减少LPS管理研究迷走神经刺激机制的有限炎症反应

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

IL-8瞬时转化的小鼠模型<em&gt;体内</em&gt;长期监测炎症反应

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.