作者要感谢为这项研究提供资金的囊性纤维化基金会和MedImmune慷慨捐赠的抗Psl0096抗体。在这项研究中，成像是在多伦多大学的CAMiLoD成像设施进行的。

收集和储存人类受试者的痰液样本需要获得研究伦理委员会 （REB） 的批准。本文介绍的研究已获得病童医院 REB#1000058579 的批准。
1. 痰液收集
<ol><li>将咳出的痰液储存在无菌收集杯中，并在固定前立即在4°C下储存最多24小时。 注意:痰液在4°C下放置时间过长而不固定会导致细胞降解，特别是白细胞降解。最好尽快固定。</li>
	<li>将痰液样本转移到无菌的 15 mL 管中。</li>
	<li>向痰液样本中加入等体积的 4% 多聚甲醛 （PFA）。例如，如果痰液样本为 0.5 mL，则添加 0.5 mL 的 4% PFA。通过轻轻倒置混合。</li>
	<li>将痰液在4°C孵育过夜。</li>
	<li>通过向每 2 mL 固定痰液中加入 5 mL 磷酸盐缓冲盐水 （PBS） 来洗涤样品。 注意:此洗涤步骤不需要离心。</li>
	<li>用移液管小心地除去上清液。 注意: 避免用移液器吸痰，将尖端远离痰液，然后非常缓慢地吸出表面液体。洗涤步骤不涉及离心，以免干扰痰塞的结构完整性。</li>
	<li>再重复步骤 1.6-1.7 两次。</li>
	<li>将沉淀重悬于含有0.01%（w / v）叠氮化钠的PBS体积的2倍中。</li>
	<li>将痰液储存在4°C。</li>
</ol>2. MiPACT痰液的处理
<ol><li>将水凝胶组分（30%29:1丙烯酰胺:双丙烯酰胺，硬化剂和PBS）在含有厌氧包装的密封容器中脱气72小时。 注意:厌氧包装和密封容器是必要的，因为氧气会抑制丙烯酰胺聚合。或者，可以通过在厌氧罩中执行此步骤、对密封容器施加真空或通过丙烯酰胺混合物鼓泡 N2 气体来去除氧气。<ol><li>将 2 mL 的 30% 29:1 丙烯酰胺:双丙烯酰胺溶液加入 15 mL 试管中，在密封的厌氧容器内取下盖子。</li>
		<li>在 15 mL 试管中向 5 mL PBS 中加入 0.5 g 固化剂，制成 10% （w/v） 固化剂的浓缩原液。将盖子从管子上取下，存放在密封的厌氧容器中。</li>
		<li>将几毫升无菌 PBS 留在厌氧容器内的试管中，盖上盖子。</li>
	</ol></li>
	<li>在 15 mL 试管中制备 5 mL 水凝胶溶液，终浓度为 0.2% 固化剂和 4% 29:1 丙烯酰胺:双丙烯酰胺的 PBS 溶液。倒置混合并过滤灭菌。</li>
	<li>从冰箱中取出痰液样本，然后在无菌条件下用手术刀切成小段（直径约5毫米）。 注意:如果痰液非常流动，那么仍然可以执行此步骤，但是，在使用手术刀分离所需部分之前，需要增加耐心和练习，用镊子从储存溶液中取出痰液。</li>
	<li>将切割的痰液样品放入8室盖玻片的孔中。</li>
	<li>将步骤2.2中的300μL过滤灭菌水凝胶溶液加入到每个含有痰液的孔中。</li>
	<li>将8室盖玻片置于装有厌氧包的密封容器内，在37°C下放置3小时。 注:一旦聚合，嵌入痰液的水凝胶应为坚硬凝胶的稠度。</li>
	<li>将固化的水凝胶痰液样品转移到含有5mL的8%十二烷基硫酸钠（SDS），pH 8的15mL培养管中，并让样品在37°C下清除3-14天（有或没有摇动），直到痰液变得透明。 注意:清除时间取决于痰液成分，通常可以通过摇晃来减少。但是，请注意不要将振荡速度提高到破坏样品完整性的程度。与富含粘液的样品相比，富含 DNA 的样品需要更长的时间才能清除。</li>
	<li>将 8% SDS 溶液倒入废物收集容器中。使用无菌镊子将每个水凝胶包埋的样品转移到无菌 50 mL 锥形管中。向50mL锥形管中的每个管中加入10mL PBS以洗涤水凝胶，并在倾析前让溶液静置30分钟至1小时。重复此操作 2 次以上。 注意:此洗涤步骤不涉及离心。用移液管小心除去多余的液体和上清液。</li>
	<li>将洗涤的样品储存在含有0.01%（w / v）叠氮化钠和1x RNase抑制剂的PBS中，温度为4°C。</li>
</ol>3. 水凝胶荧光 原 位杂交（FISH）方案
<ol><li>使用无菌镊子从其储存溶液中取出水凝胶样品，并将样品放在无菌表面（如载玻片或培养皿）上。</li>
	<li>使用无菌手术刀将水凝胶切成~1毫米厚的切片。</li>
	<li>将 1 mm 水凝胶切片放入无菌 1.5 mL 管内。</li>
	<li>在 1.5 mL 试管中制备 1 mL 杂交缓冲液（25% 甲酰胺、0.9 M NaCl、20 mM Tris-HCl [pH 7.6]、0.01% SDS、纯化和去离子化 H2O）。</li>
	<li>将荧光标记的PseaerA探针（150.7nM12）加入杂交缓冲液中，并通过倒置混合。 注意:福尔马林溶液应远离明火。杂交缓冲液可以预先制备，并在-20°C13下以等分试样储存。</li>
	<li>向每个~1mm水凝胶切片中加入200-500μL杂交缓冲液，并确保整个水凝胶样品浸没。</li>
	<li>通过将PseaerA探针置于黑暗中~18-24小时，在46°C下不摇晃，使PseaerA探针与水凝胶样品杂交。</li>
	<li>将杂交缓冲液倒入废物收集容器中。</li>
	<li>通过向每个 1.5 mL 试管中加入 1 mL 洗涤缓冲液，然后将其取出，用过滤灭菌的洗涤缓冲液（337.5 mM NaCl、20 mM Tris-HCl、5 mM EDTA（乙二胺四乙酸）[pH 7.2]、0.01% SDS 和纯化和去离子的 H2O）冲洗样品一次。 注意:洗涤缓冲液可以提前制备并在室温 （RT） 下储存。</li>
	<li>向试管中加入1mL新鲜洗涤缓冲液，然后将样品在48°C下在黑暗中孵育6小时，不要摇晃。</li>
</ol>4. 水凝胶与Psl0096抗体结合
<ol><li>使用 1 mL 移液器无菌除去洗涤缓冲液。</li>
	<li>通过加入和除去 1 mL 的 2% BSA 溶液，用 PBS 溶液中的 2% BSA （w/v） 冲洗样品。</li>
	<li>向水凝胶样品中加入 500 μL 2% BSA/PBS 溶液以阻断非特异性蛋白结合。然后将样品在黑暗中在室温下孵育过夜，不要摇晃。</li>
	<li>使用 1 mL 移液管无菌除去封闭溶液。</li>
	<li>通过在 500 μL 新鲜的 2% BSA/PBS 中将抗体稀释至 0.112 μg/mL 的终浓度来制备 Psl0096-Texas Red 抗体溶液。</li>
	<li>将 500 μL 抗体溶液加入水凝胶样品中，并在室温下孵育 6 小时，避光，不要摇晃。</li>
</ol>5. DAPI（4′，6′-二脒基-2-苯基吲哚）染色
<ol><li>通过将 40 g 非离子密度梯度培养基、30 μL 吐温 20、3 μg 叠氮化钠和 30 mL PBS 加入到含有磁力搅拌棒的烧瓶中来制备折射率匹配溶液 （RIMS）。在磁力搅拌器上搅拌溶液15分钟，或直至完全溶解。</li>
	<li>过滤 使用 10 mL 注射器和无菌 0.2 μm 过滤器将溶液灭菌到 50 mL 锥形管中。 注意:溶液可以在4°C下储存数月。</li>
	<li>用无菌 1 mL 移液管取出 Psl0096-Texas Red 抗体溶液。</li>
	<li>通过加入然后除去 1 mL PBS 冲洗水凝胶样品。</li>
	<li>将水凝胶样品与 250 μL RIMS 溶液和 10 μg/mL DAPI 在室温下在室温下孵育，并在黑暗中轻轻摇动过夜。</li>
	<li>在进行共聚焦成像之前，将样品安装到0.9 mm或1.7 mm灌注室上，并用玻璃盖玻片密封。 注:在 FISH 和/或免疫组化之后以及浸入 RIMS 之前，如果需要痰粘液的可视化，可以应用荧光凝集素染色12。</li>
</ol>6. 影像学检查
<ol><li>使用标准技术以 25 倍、40 倍、63 倍或 100 倍放大倍率进行共聚焦激光扫描显微镜成像。</li>
</ol>

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该协议的目的是了解CF患者痰液中 铜绿假单胞菌 细胞的原位组织。痰液样本应储存在4°C，如果不能立即固定，则应进行处理。已经证明，如果在4°C下储存1小时，24小时或48小时处理，痰中的 铜绿假单胞菌 细胞数量不会显着变化，但如果在25°C下放置24或48小时，细菌细胞计数将由于细菌生长而显着增加14。对于这项研究，痰液样本在4°C下储存，吐痰后最多24小时。应该注意的是，如果保持在4°C并在9小时后处理超过15，则痰液中的炎症细胞计数已被证明会降低。因此，在决定样品处理的截止时间时，重要的是要考虑希望在痰液中可视化的特定细胞和标志物。
该方法中的样品处理始于将痰液样品固定在4%PFA中。多聚甲醛将交联细菌细胞及其细胞外基质，保留其结构以用于显微可视化和分析11,16。不幸的是，如果目的是获得痰液中某些炎症细胞的总细胞计数，PFA 已被证明会降低这些细胞的计数，因此应考虑使用其他固定剂17.这项研究的另一个局限性是它可能很耗时，可能需要两周以上的时间才能完成。因此，它可能不适合发展成一种需要时间敏感的治疗决策的诊断方法。此外，为了了解痰液样本中的总微生物多样性，该方法不适合，但可以与其他高通量微生物检测方法（如qPCR）配对使用。
丙烯酰胺水凝胶的组成可以根据组织类型和应用11而改变。对于不稳定的标本，如CF痰液，有必要用29:1的丙烯酰胺:双丙烯酰胺混合物（而不仅仅是丙烯酰胺）提供结构支撑。在水凝胶中加入多聚甲醛可以进一步稳定结构，但需要权衡更长的孵育时间，以允许探针和抗体扩散9。在水凝胶中添加甲醛还可以防止组织在清除过程中肿胀，如果这种效果是不希望的11.
目前的方法专门针对CF患者痰液中的铜绿假单胞菌细胞。可以考虑对该协议的替代方法和修改来指导其他细菌可视化的优化。通过应用物种和属特异性 FISH 和杂交链式反应 （HCR） 探针，可以鉴定其他 CF 病原体，例如金黄色葡萄球菌、链球菌属和木糖氧化无色杆菌12。在我们的研究中，我们靶向假单胞菌胞外多糖Psl。在未来的实验中，可以使用对这些胞外多糖具有特异性的荧光抗体来检查其他靶标，包括海藻酸盐或Pel。将 MiPACT 方法与 FISH 和抗体染色一起应用于 CLSM 需要几周时间才能完成。如果研究问题不涉及痰液的三维空间可视化，则有更快速的方法来可视化存在的细菌。以前用于可视化痰液样本中细菌的方法利用薄切片或涂片，包括:FISH18、革兰氏染色和免疫组织化学技术，这些技术应用一抗和复染剂来可视化生物膜胞外多糖和细菌细胞19,20。
这些类型的成像技术在未来有几种潜在的应用。可视化不同细菌生物体及其与宿主细胞（如吞噬细胞）相互作用的能力可能会进一步理解为什么某些 铜绿假单胞 菌菌株可以有效地从CF气道中清除，而其他菌株则不能。呼吸道标本中的细菌成像也可用作抗菌功效的衡量标准和新型抗生物膜药物的研究结果21。此外，可视化 铜绿假单胞菌 与CF肺微生物组内其他生物体（如 金黄色葡萄球菌）之间的空间关系可能有助于阐明合并感染/定植在肺部恶化发病机制中的作用，以及它们对抗生素治疗的反应。细菌的 体内 成像也可以应用于其他感染，包括呼吸机相关性肺炎或慢性伤口感染22。因此，所获得的见解可用于指导未来的治疗开发。

在这项研究中，设计了实验来可视化小儿囊性纤维化 （CF） 患者痰液中铜绿假单胞菌的体内结构。铜绿假单胞菌感染在 30-40% 的儿科 CF 人群中发展为慢性;慢性感染一旦形成，几乎不可能消除1.来自早期感染患者的铜绿假单胞菌分离株通常更易受到抗菌药物的影响，因此，这些药物用抗假单胞菌抗生素治疗，以防止慢性感染的建立2。不幸的是，并非所有铜绿假单胞菌分离株在抗生素治疗后都能从肺部有效清除。与抗生素失败相关的确切机制尚未完全阐明。先前的研究表明，生物膜细胞密度、聚集和多糖产生的变化会影响抗生素疗效3。铜绿假单胞菌产生三种细胞外多糖:Pel、Psl 和海藻酸盐4。大多数铜绿假单胞菌菌株具有表达每种胞外多糖的遗传能力，尽管通常一种多糖主要表达5.海藻酸外多糖与CF肺的慢性感染有关，导致粘液样表型6,7。多糖 Pel 和 Psl 具有多种功能，包括帮助初始附着和维持生物膜结构，以及赋予抗生素耐药性8。
已经为各种样品类型开发了旨在可视化组织体内结构的方法9,10,11。最近，它们被定制用于可视化 CF 患者痰液中的体内微生物群落12。DePas等人，2016年12开发了专门用于鉴定痰中微生物群落的组织清除方案的优化。术语 MiPACT 代表 Passive CLARITY technique 之后的 microbial i识别，用于清除 CF 痰液11,12。对于组织透明化技术，首先将标本固定，然后使其透明，同时保持其固有结构完好无损，以便进行染色和显微观察11。固定和清除CF痰液样本使研究人员能够回答与生物膜结构、细菌细胞密度、多种微生物关联以及病原体与宿主细胞之间的关联相关的问题。直接检查保存在痰液中的细菌的优点是可以在宿主特定的环境中对其进行分析和可视化。尽管在实验室中用于实验的临床分离株的体外生长可以提供非常丰富的信息，但这些方法无法完全重建CF肺环境，导致实验室结果与患者结果脱节。
这里介绍的方法可用于固定和清除痰液以可视化细菌，无论是来自 CF 患者还是患有其他呼吸道感染的患者。本文描述的染色和显微分析的具体类型是荧光 原位 杂交（FISH），然后在水凝胶内进行抗Psl抗体结合，然后通过共聚焦激光扫描显微镜（CLSM）进行分析。组织清除后，也可以应用其他免疫组化和显微镜方法。

<table><tbody><tr><td>29:1 acrylamide bisacrylamide, 30 % solution</td><td>BioRad</td><td>161-0146</td><td></td></tr><tr><td>8-Chambered Coverglass Nunc Lab-Tek</td><td>ThermoFischer Scientific</td><td>155411</td><td></td></tr><tr><td>Anaerogen2.5L</td><td>Oxid Inc.</td><td>35108</td><td></td></tr><tr><td>Coverwell perfusion chambers</td><td>Electron Microscopry Sciences</td><td>70326 -12/-14</td><td></td></tr><tr><td>HistoDenz</td><td>Sigma</td><td>D2158</td><td></td></tr><tr><td>Protect RNA Rnase Inhibitor</td><td>Sigma</td><td>R7387</td><td></td></tr><tr><td>PseaerA - GGTAACCGTCCCCCTTGC</td><td>Eurofins</td><td></td><td>Order Details: Product: Modified DNA Oligo; Name: PseaerA; Sequence: [Alexa488]GGTAACCGTCCCCCTTGC; Synthesis: 50 nmol; Purification: HPLC; Ship state: Full yield (dry)</td></tr><tr><td>Psl0096-Texas Red</td><td>Medimmune</td><td></td><td>The Psl0096-Texas red antibodies were a gift kindly provided by Medimmune and the company should be contacted for order inquiries.</td></tr><tr><td>VA-044 Hardener</td><td>Wako</td><td>27776-21-21</td><td></td></tr></tbody></table>

visualization of pseudomonas aeruginosa within the sputum of cystic fibrosis patients

及早发现和根除囊性纤维化患者肺部的铜绿假单胞菌可以减少发生慢性感染的机会。慢性铜绿假单胞菌感染的发展与肺功能下降和发病率增加有关。因此，对阐明抗生素治疗未能根除铜绿假单胞菌的原因非常感兴趣，铜绿假单胞菌发生在大约 10-40% 的儿科患者中。可能影响宿主对铜绿假单胞菌清除和抗生素敏感性的众多因素之一是空间组织（如聚集或生物膜形成）和多糖产生的变化。因此，我们有兴趣可视化CF患者痰液中铜绿假单胞菌的原位特征。将痰液样品包埋到水凝胶基质中后，将组织透明化技术应用于痰液样品，以保留相对于宿主细胞的 3D 结构。组织清除后，添加荧光标记物和染料以允许可视化。荧光原位杂交用于细菌细胞的可视化，荧光标记的抗Psl抗体的结合用于胞外多糖的可视化和DAPI染色对宿主细胞的染色以获得结构见解。这些方法允许通过共聚焦激光扫描显微镜对CF患者痰中的铜绿假单胞菌进行高分辨率成像。

实验的整体设计总结在图 1 和 图2中。 图 1 总结了痰液处理和痰液清除方案。痰液处理和清除可能需要长达 17 天的时间。但是，该方案可以停止，并且可以在用PFA固定（第2天）或组织清除后（第5-17天，取决于清除时间）后储存样品。 在图2中，总结了FISH和抗体结合方案。FISH 和抗体染色方案需要 4 天才能完成，但应在开始后完成并拍摄共聚焦图像。使用上述方案，可以获得 铜绿假单胞 菌细胞的高分辨率3D图像，并在痰液中可视化其原位结构。
清除痰液并随后应用该方案中使用的荧光染色剂允许在样品中详细观察 铜绿假单胞菌 。图 3 和 图 4 所示的痰液样本是从一名新发铜 绿假单胞 菌感染的小儿 CF 患者（17 岁）身上采集的。 在图3A中，在痰液样本中观察到细胞聚集体;黄绿色棒状物的出现是由于所有三个荧光团的重叠。虽然我们没有该痰液样本的细胞计数，但我们发现探针检测限为 104 个细胞/mL（参见 补充图 1）。 在图3B中，PseaerA-488探针与 铜绿假单胞菌 细胞的物种特异性结合以绿色显示单个杆状。红色显示的 Psl0096-Texas Red 抗体说明了假单胞菌胞外多糖在痰液中的位置;在这种情况下，Psl0096抗体似乎主要与 铜绿假单胞 菌细胞重叠（图3C）。该方法还允许可视化痰液中的假单胞菌细胞与其他细菌细胞和宿主结构的关系。 在图 4 中，可以看到一簇 铜绿假单 胞菌细胞在真核细胞内被吞噬，并在附近观察到其他小球菌细胞簇。已经证明，Psl0096抗体还可以与浮游铜 绿假单胞 菌产生的Psl结合（见 补充图2）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61631/61631fig01.jpg"> 图 1:说明痰液处理和 MiPACT 协议的流程图。 总之，痰液在包埋在聚丙烯酰胺溶液中之前是固定的。清除后，水凝胶可以储存在4 °C或与FISH和抗体结合方案一起使用。本矢量图使用 BioRender.com 创作。 <a href="https://www.jove.com/files/ftp_upload/61631/61631fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61631/61631fig02.jpg"> 图 2:表示荧光 原 位杂交和抗体染色方案的流程图。 一旦痰液在水凝胶基质中完全清除，就可以应用染色方法。本矢量图使用 BioRender.com 创作。 <a href="https://www.jove.com/files/ftp_upload/61631/61631fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61631/61631fig03.jpg"> 图 3:从嵌入水凝胶基质的早期 铜绿假单胞菌 感染患者身上采集的痰液样本的免疫荧光图像。 将水凝胶样品与 PsearA-Alexa488 探针（绿色）、Psl0096-Texas Red 抗体（红色）和 DAPI（蓝色）杂交。（A） 在所有 3 个通道下观察的痰液样本，（B） 仅在指示 PseaerA-488 探针结合位置的绿色通道下观察痰液，以及 （C） 仅在发生 Psl0096-Texas 红色结合的红色通道下的痰液。图像以 100 倍放大倍率拍摄。 <a href="https://www.jove.com/files/ftp_upload/61631/61631fig03large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61631/61631fig04.jpg"> 图 4:从嵌入水凝胶基质的早期 铜绿假单胞菌 感染患者身上采集的痰液样本的免疫荧光图像。 将水凝胶样品与 PsearA-Alexa488 探针（绿色）、Psl0096-Texas Red 抗体（红色）和 DAPI（蓝色）杂交。 <a href="https://www.jove.com/files/ftp_upload/61631/61631fig04large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
补充图 1:PAO1 浮游培养物与 PseaerA-488 探针的荧光原位杂交。<a href="https://cloudflare.jove.com/files/ftp_upload/61631/61631supfig01.jpg" target="_blank">请点击这里下载此图。</a>
补充图2:用DAPI和Psl0096-Texas Red抗体染色的铜绿假单胞菌。（A）菌株PAO1-Δpsl和（B）菌株PAO1。<a href="https://cloudflare.jove.com/files/ftp_upload/61631/61631supfig02.jpg" target="_blank">请点击这里下载此图。</a>

Watch this Scientific Journal Video about Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients at JoVE.com

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

该方案提供了囊性纤维化患者痰液中细菌细胞和多糖合成位点（Psl）多糖的可视化方法。

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

visualization-pseudomonas-aeruginosa-within-sputum-cystic-fibrosis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

3.0K 次观看.  Hospital for Sick Children. 该方案提供了囊性纤维化患者痰液中细菌细胞和多糖合成位点（Psl）多糖的可视化方法。

视频: Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

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 ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

长期慢性<em&gt;铜绿假单胞菌</em&gt;在呼吸道感染小鼠

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

伪多莫纳斯航空感染模型实时评估工具

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

<meta charset="utf8"></head><body>用于抗菌检测和微生物动力学分析的适应性 CF 模型

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

<meta charset="utf8"></head><body>开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

囊性纤维化患者痰液中铜绿假单胞菌的可视化

摘要

探索更多视频

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

使用人工痰介质测试抗生素药效试验

长期慢性

可视化痰生物膜发展影响使用腔室盖玻片型号

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

伪多莫纳斯航空感染模型实时评估工具

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

囊性纤维化患者痰液中 铜绿假单胞菌 的可视化

摘要

探索更多视频

共培养模型绿脓杆菌现场人类呼吸道细胞生长的生物膜

使用人工痰介质测试抗生素药效试验

长期慢性

可视化痰生物膜发展影响使用腔室盖玻片型号

P. 阿鲁吉诺萨 气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估

在囊性纤维化肺中的伪肌瘤和金黄色葡萄球菌生物膜的前体内模型中进行抗生素疗效测试

伪多莫纳斯航空感染模型实时评估工具

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

开发多微生物菌落生物膜模型以测试囊性纤维化中的抗菌剂

囊性纤维化患者痰液中铜绿假单胞菌的可视化

P. 阿鲁吉诺萨气液界面中支气皮细胞和巨噬细胞的感染三维共培养，用于抗感染的临床前评估