囊性纤维化患者痰液中 铜绿假单胞菌 的可视化
概要
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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。?…
開示
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 29:1 acrylamide bisacrylamide, 30 % solution | BioRad | 161-0146 | |
| 8-Chambered Coverglass Nunc Lab-Tek | ThermoFischer Scientific | 155411 | |
| Anaerogen2.5L | Oxid Inc. | 35108 | |
| Coverwell perfusion chambers | Electron Microscopry Sciences | 70326 -12/-14 | |
| HistoDenz | Sigma | D2158 | |
| Protect RNA Rnase Inhibitor | Sigma | R7387 | |
| PseaerA – GGTAACCGTCCCCCTTGC | Eurofins | Order Details: Product: Modified DNA Oligo; Name: PseaerA; Sequence: [Alexa488]GGTAACCGTCCCCCTTGC; Synthesis: 50 nmol; Purification: HPLC; Ship state: Full yield (dry) | |
| Psl0096-Texas Red | Medimmune | The Psl0096-Texas red antibodies were a gift kindly provided by Medimmune and the company should be contacted for order inquiries. | |
| VA-044 Hardener | Wako | 27776-21-21 |
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