伪多蒙多尼亚小殖民地文化及其藻酸盐的定量
Summary
在这里，我们描述了一个生长条件，以培养伪多蒙多尼亚小殖民地变种。我们还描述了使用传统的尿酸卡巴佐尔测定法和基于藻酸盐的单克隆抗体（mAb）的ELISA检测和定量由P.aeruginosa产生的外糖藻酸盐的两种独立方法。
Abstract
假单组动物，一种机会性革兰氏阴性细菌病原体，可以过度产生外多糖藻酸盐，从而产生一种称为粘凝的独特表型。藻酸盐与慢性肺部感染有关，导致囊性纤维化 （CF） 患者预后不佳。了解调节藻酸盐生产的途径有助于制定针对藻酸盐形成的新型治疗策略。另一种与疾病相关的表型是小菌群变异（SCV）。SCV 是由于细菌生长缓慢，并且经常与抗菌素耐药性增加有关。在本文中，我们首先展示了一种由于苯丙胺生物合成突变而具有遗传定义的P.aeruginosa SCV的培养方法。补充氮碱，尿素或细胞氨酸，返回这些突变体的正常生长，表明存在一个拯救途径，从环境中清除自由碱。接下来，我们将讨论两种测量细菌藻酸盐的方法。第一种方法依靠多糖的水解到其尿酸单体，然后衍生与色原试剂，卡巴佐尔，而第二种方法使用ELISA基于市售的，藻酸盐特异性mAb。两种方法都需要一个标准曲线进行定量。研究还表明，该免疫法是藻酸盐定量的特异性，可用于临床标本的藻酸盐测量。
Introduction
慢性肺部感染与伪单多尼亚龙是囊性纤维化 （CF） 患者发病率和死亡率的主要原因。在幼儿期，患者被多种细菌病原体殖民，包括P.aeruginosa1，2的非粘胶分离物。小菌群变异（SCV）分离物的出现以及粘水井分离物是慢性感染发病的标志。SCV分离物具有很强的抗药性3，因为它们的生长速度缓慢4，这使得他们在治疗团和其他慢性感染5由P.aeruginosa严重威慑。Al Ahmar等人6号的工作表明，SCV与通过新丙胺生物合成所联系的粘体之间存在联系。由于与苯丙胺生产有关的基因突变，苯丙胺饥饿导致非粘凝参考菌株PAO1和粘尿衍生物PAO581（PAO1mucA25）中的SCV表型。
尽管藻酸盐过度生产是CF慢性肺部感染的重要疾病标志物，但尚不清楚藻酸盐量与肺病理学之间是否存在直接相关性，也不清楚藻酸盐能否用作治疗7的预后标志物。藻酸盐生产主要由两个操作器调节，一个调节操作体（algUmucABCD）8，9和生物合成操作子（阿尔格Doperon）10，11。藻酸盐生产受到西格玛因子AlgU9、12（也称为AlgT）和反西格玛因子MucA13的降解的严格调节。从患者的痰样原位监测藻酸盐的产生的能力有助于开发新的治疗方案。
在这里，我们描述了一个生长条件，它检测由不能合成新热胺的突变体引起的SCV的存在。补充尿素和/或细胞氨酸，苯丙胺核苷核苷酸的氮碱，到介质激活抢救途径，从而恢复突变体的正常生长。这种针对这些特定SCV突变体的生长方法可用作筛选方法，以识别患者样本中的丙酰胺突变。此外，我们还讨论了两种检测和测量由P.aeruginosa产生和分泌的藻酸盐的方法。第一种是使用高浓度酸降解多糖的传统方法14、15、16，然后添加一个色度指标来定量样品中的浓度。第二种方法，在我们的实验室开发，利用酶链接免疫吸附测定（ELISA）使用QED生物科学开发的抗藻酸盐单克隆抗体（mAb）。ELISA 方法证明比尿酸测定更具体、更敏感，由于避免了高浓度硫酸，因此可以更安全地使用。ELISA 能够直接用于患者痰样测量藻酸盐，因此可以开发为监测诊断工具，以跟踪感染不同时期肺部存在的藻酸盐量。
Protocol
Representative Results
Discussion
SCV 和藻酸盐都是与多种慢性感染相关的重要疾病标志物。因此，能够生长SCV以及研究由P.aeruginosa调节和生产的藻酸盐是发现这些慢性疾病的新疗法不可或缺的。
SCV菌株是出了名的难以生长，由于其缓慢的生长速度4相比，其他P.aeruginosa菌株，这有助于他们的抗微生物药物耐药性3。我们的工作确定了一种特定形式的P.aeruginosa<…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家卫生研究院（NIH）授予R44GM113545和P20GM103434的支持。
Materials
| 1-Step Ultra TMB-ELISA | Thermo Scientific | 34028 | via Fisher Scientific |
| Absolute Ethanol (200 Proof) | Fisher Scientific | BP2818-4 | Molecular Bio-grade |
| Accu Block Digital Dry Bath | Labnet | NC0205808 | via Fisher Scientific |
| Assay Plates 96-well | CoStar | 2021-12-20 | |
| Bench Top Vortex-Genie 2 | Scientific Industries | G560 | |
| Boric Acid | Research Products International Corp. | 10043-35-3 | |
| Cabinet Incubator | VWR | 1540 | |
| Carbazole | Sigma | C-5132 | |
| Carbonate-Bicarbonate Buffer | Sigma | C3041 | |
| Centrifuge Tubes (50 ml) | Fisher Scientific | 05-539-13 | via Fisher Scientific |
| Culture Test Tubes | Fisher Scientific | 14-956-6D | via Fisher Scientific |
| Cuvette Polystyrene (1.5 ml) | Fisher Scientific | 14955127 | via Fisher Scientific |
| Cytosine | Acros Organics | 71-30-7 | |
| Diposable Inoculation Loops | Fisher Scientific | 22-363-597 | |
| D-Mannuronic Acid Sodium | Sigma Aldrich | SMB00280 | |
| FMC Alginate | FMC | 2133 | |
| Glycerol | Fisher Scientific | BP906-5 | For Molecular Biology |
| Mouse Anti-Alginate Monoclonal Antibody | QED Biosciences | N/A | Lot # :15725/15726 |
| Phosphate Buffered Saline Powder (PBS) | Sigma | P3813 | |
| Pierce Goat Anti-Mouse Poly-HRP Antibody | Thermo Scientific | 32230 | via Fisher Scientific |
| Potassium Hydroxide | Fisher Scientific | 1310-58-3 | via Fisher Scientific |
| Prism 7 | GraphPad | ||
| Pseudomonas Isolation Agar (PIA) | Difco | 292710 | via Fisher Scientific |
| Pseudomonas Isolation Broth (PIB) | Alpha Biosciences | P16-115 | via Fisher Scientific |
| Round Toothpicks | Diamond | Any brand | |
| Seaweed alginate (Protanal CR 8133) | FMC Corporation | ||
| Skim Milk | Difco | 232100 | via Fisher Scientific |
| SmartSpec Plus Spectrophotometer | BioRad | 170-2525 | or preferred vendor |
| Sodium Chloride (NaCl) | Sigma | S-5886 | |
| SpectraMax i3x Multi-mode MicroPlate Reader | Molecular Devices | i3x | or preferred vendor |
| Sterile Petri Dish 100mm x 15mm | Fisher Scientific | FB0875713 | via Fisher Scientific |
| Sulfuric Acid | Fisher Scientific | A298-212 | Technical Grade |
| Sulfuric Acid (2 Normal -Stop Solution) | R&D Systems | DY994 | |
| Tween 20 | Sigma | P2287 | |
| Uracil | Acros Organics | 66-22-8 |
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