研究辅助供氧对囊性纤维化气道微生物组影响的模型的设计与开发
1Division of Pulmonary and Critical Care Medicine,Massachusetts General Hospital, 2Department of Molecular Biology & Biochemistry,University of California, Irvine, 3Department of Medicine,Massachusetts General Hospital, 4Department of Microbiology & Immunology,Geisel School of Medicine at Dartmouth, 5Swiss i-research and teaching institute
Summary
该协议的目标是开发一个模型系统，用于高氧对囊性纤维化气道微生物群落的影响。 人工痰培养基模拟痰液的组成，高氧培养条件模拟了辅助氧气对肺微生物群落的影响。
Abstract
气道微生物群落被认为在囊性纤维化（CF）和其他慢性肺部疾病的进展中起重要作用。传统上，微生物是根据其使用或耐受氧气的能力进行分类的。辅助供氧是一种常见的药物治疗，用于囊性纤维化（pwCF）患者;然而，关于氧气和气道微生物组的现有研究集中在缺氧（低氧）而不是高氧（高氧）如何影响主要有氧和兼性厌氧肺微生物群落。为了解决这一关键的知识差距，该协议是使用模仿pwCF痰液组成的人造痰液培养基开发的。使用产生透明培养基的过滤器灭菌允许光学方法跟踪悬浮培养物中单细胞微生物的生长。为了创建高氧条件，该模型系统利用已建立的厌氧培养技术来研究高氧条件;不是除去氧气，而是通过每天用压缩氧气和空气的混合物喷射血清瓶将氧气添加到培养物中。来自50 pwCF的痰液每天进行72小时的喷射，以验证该模型维持差分氧条件的能力。对来自11 pwCF的培养和未培养的痰液样本进行霰弹枪宏基因组测序，以验证该培养基支持囊性纤维化痰中常见的共生和致病微生物生长的能力。从从pwCF获得的112株分离株中获得生长曲线，以验证这种人造痰培养基支持常见囊性纤维化病原体生长的能力。我们发现该模型可以在CF痰中培养各种病原体和共生菌，在正常氧条件下恢复与未培养痰高度相似的群落，并在不同的氧气条件下产生不同的培养表型。这种新方法可能会更好地理解在pwCF中使用氧气对气道微生物群落和常见呼吸道病原体引起的意外影响。
Introduction
囊性纤维化（CF）是一种遗传性疾病，其特征在于无法清除肺部的粘液，导致反复感染和进行性肺功能下降，通常导致需要肺移植或死亡。囊性纤维化（pwCF）患者的气道微生物组似乎跟踪疾病活动1，与不良长期结局相关的微生物多样性减少2，3。在pwCF的临床研究中，补充氧疗与更晚期的疾病4，5相关，尽管传统上，氧疗的使用仅被视为疾病严重程度的标志6 。最近一项针对呼吸衰竭患者的临床试验研究表明，较高的患者氧气水平与严重细菌感染的增加和更高的死亡率相矛盾7，这表明补充氧气可能有助于疾病的发病机制。补充氧气对囊性纤维化肺微生物组以及相关的肺和气道微生物群落的影响尚未得到很好的研究。
由于后勤困难以及与未知医疗益处或伤害的干预措施相关的潜在伦理问题，机械研究通常无法直接对人类受试者进行。在这些情况下，将人类生物标本整合到模型系统中的转化方法可以提供重要的生物学见解。虽然使用或耐受氧气的能力传统上是微生物分类的重要组成部分，但对于向环境引入辅助氧气的治疗性如何扰乱气道微生物群落知之甚少。为了阐明辅助供氧对pwCF气道微生物组的未知影响，我们需要解决两个主要挑战;首先，创建一种培养基，在生理上接近CF痰的组成;其次，创建一个模型系统，允许在较长的时间内保持培养物中升高的氧浓度。
人工痰培养基（ASM）被广泛用于模拟肺痰体外8、9、10，但对具体配方没有明确的共识。该协议描述了一种精心设计的人造痰液培养基配方和制备策略，以从生理上近似于来自pwCF的痰液。表1概述了基于已发表文献的所选配方值。基本化学成分和pH值与人类CF痰液研究确定的值相匹配11，12，13。使用蛋黄加入低浓度的生理营养素，其作为最终体积的0.25%加入，以及维生素和微量金属混合物14，15。粘蛋白是痰16的关键成分，包括在1%w / v14。虽然过滤器杀菌更费力，但过滤器灭菌被选择在更传统的热灭菌实践之上，以减少由热引起的基本介质组分变性的潜在问题10。过滤器灭菌的另一个好处是它产生透明的介质（由于盐和蛋白质的沉淀和凝固，热灭菌可以产生浑浊的介质），允许这种人造痰培养基用于根据浊度的增加来跟踪微生物生长。
该高氧培养模型系统基于厌氧培养技术，其中添加而不是去除氧气，从而为pwCF补充氧气的使用效果创建了模型。 图1 和相关氧气喷射方案概述了氧气喷射系统的组件，这些组件可以从一般实验室和医院供应商处以低成本获得。该系统能够将压缩氧气和空气混合到21%-100%氧气的固定浓度。氧气传感器的集成允许验证输出气体混合物的浓度，以及检查先前喷射的血清瓶的流出气体成分，以验证氧气条件是否保持在所需范围内。
该协议概述了创建人造痰液培养基的程序，氧气喷射系统的构建和使用，以及两者在差氧条件下用于培养CF痰液的应用。
Protocol
该研究获得了合作伙伴机构审查委员会的批准（协议编号2018P002934）。纳入标准包括为研究提供书面知情同意书的囊性纤维化成年患者。没有排除标准。根据方案指南，所有痰液样本都是在预定的门诊就诊期间从囊性纤维化患者那里收集的。 1. 人工痰培养基制备 注：此处列出的数量用于生产1 L最终人造痰液，并假设材料表中列出的特定试剂。?…
Representative Results
这些方案应用于来自pwCF的50个痰液样本，这些样本在马萨诸塞州波士顿马萨诸塞州总医院的门诊囊性纤维化诊所进行常规护理。使用人造痰培养基在21%，50%和100%氧气条件下培养每位患者的痰液，在24小时，48小时和72小时的培养时间从每种培养物中取出0.5mL等分试样进行测试。当提取以跟踪视觉变化时，对培养物进行拍摄。此外，在培养前取每个原发痰样本的0.5mL等分试样。这导致每位患者有10个离…
Discussion
在这项研究中，开发了一个 体外 模型来研究高氧对肺微生物群落的影响。该模型基于人工痰培养基和血清瓶的每日喷射，可维持升高的氧气浓度，并支持在pwCF痰液中鉴定出的微生物的生长。
这种方法有几个关键步骤。首先是选择使用过滤灭菌而不是人工痰媒的热灭菌。过滤器灭菌可防止粘蛋白和培养基中其他热敏组分变性，并产生可用于微生物生长光学测量的透明?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作的一部分是在海洋生物实验室进行的，得到了海洋生物实验室，美国能源部（DE-SC0016127），NSF（MCB1822263），HHMI（授权号5600373）以及西蒙斯基金会的礼物的支持。
Materials
| BME Vitamins (100x) Solution | MilliporeSigma | B6891 | Concentrated solution of supplemental vitamins. |
| Crimper, 30 mm | DWK Life Sciences | 224307 | Crimper for attaching aluminum seals to serum bottles. |
| D-(+)-Glucose | MilliporeSigma | G7021 | Solid glucose powder (dextrorotatory isomer). |
| Diaphragm Pump ME 2 NT | VACUUBRAND | 20730003 | Vacuum pump for vacuum filtration. |
| Egg Yolk Emulsion | HiMedia | FD045 | Sterile emulsion of 30% egg yolk in saline. |
| Ferritin, Cationized from Horse Spleen | MilliporeSigma | F7879 | Ferritin (iron-storage protein) solution. |
| FIREBOY plus Safety Bunsen Burner | Integra Biosciences | 144000 | Bunsen burner with user interface and safety features. |
| Hydrion pH Paper (1.0–14.0) | Micro Essential Laboratory | 94 | pH testing paper for the range of 1.0–14.0. |
| Hydrion pH Paper (4.0–9.0) | Micro Essential Laboratory | 55 | pH testing paper for the range of 4.0–9.0. |
| Hydrion pH Paper (6.0–8.0) | Micro Essential Laboratory | 345 | pH testing paper for the range of 6.0–8.0. |
| Hypodermic Needle-Pro EDGE Safety Device, 18 G | Smiths Medical | 401815 | 18 G needles with safety caps. |
| In-Line Pressure Gauge | MilliporeSigma | 20469 | Gas pressure gauge for monitoring bottle pressure. |
| Innova 42 Incubated Shaker | Eppendorf | 2231000756 | Combination incubator/orbital shaker. |
| Luer-Lok Syringe with Attached Needle | Becton Dickinson | 309580 | Combination 3 mL syringe and 18 G needle. |
| Luer Valve Assortment | World Precision Instruments | 14011 | Valves for gas flow tubing. |
| LSE Orbital Shaker | ThermoFisher Scientific | 6780-NP | Orbital shaker to agitate media during filtration. |
| Magnesium Sulfate Heptahydrate | MilliporeSigma | M2773 | Solid epsom salt (magnesium sulfate heptahydrate). |
| Medical Air Single Stage Regulator with Flowmeter | Western Enterprises | M1-346-15FM | Air flow rate regulator with 15 L/min meter. |
| MEM Amino Acids (50x) Solution | MilliporeSigma | M5550 | Concentrated solution of essential amino acids. |
| MEM Non-Essential Amino Acids (100x) Solution | MilliporeSigma | M7145 | Concentrated solution of non-essential amino acids. |
| Millex-GP Filter, 0.22 µm | MilliporeSigma | SLMP25SS | 0.22 µm polyethersulfone membrane sterile syringe filter. |
| Milli-Q Academic | MilliporeSigma | ZMQS60E01 | Milli-Q sterile water filtration system. |
| MiniOX 3000 Oxygen Monitor | MSA | 814365 | Gas flow oxygen percentage monitor. |
| MOPS Buffer (1 M, pH 9.0) | Boston BioProducts | BBM-90 | MOPS buffer for adjusting media pH. |
| Mucin from Porcine Stomach | MilliporeSigma | M2378 | Mucin (glycosylated gel-forming protein) powder. |
| Natural Polypropylene Barbed Fitting Kit | Harvard Apparatus | 72-1413 | Connectors for gas flow tubing. |
| Nextera XT DNA Library Preparation Kit | Illumina | FC-131-1096 | Library preparation for identification during sequencing. |
| NovaSeq 6000 Sequencing System | Illumina | 770-2016-025-N | Shotgun sequencing platform for generating sample reads. |
| Oxygen Single Stage Regulator with Flowmeter | Western Enterprises | M1-540-15FM | Oxygen flow rate regulator with 15 L/min meter. |
| Oxygen Tubing with 2 Standard Connectors | SunMed | 2001-01 | Tubing for connecting gas system components. |
| Phosphate buffered saline, 10x, pH 7.4 | Molecular Biologicals International | MRGF-6235 | Concentrated phosphate-buffered saline solution. |
| PC 420 Hot Plate/Stirrer | Marshall Scientific | CO-PC420 | Combination hot plate/stirrer. |
| Potassium Chloride | MilliporeSigma | P9541 | Solid potassium chloride salt. |
| PTFE Disposable Stir Bars | ThermoFisher Scientific | 14-513-95 | Disposable magnetic stir bars. |
| PTFE Thread Seal Teflon Tape | VWR | 470042-938 | Teflon tape for reinforcing gas system connections. |
| Q-Gard 2 Purification Cartridge | MilliporeSigma | QGARD00D2 | Purification cartridge for Milli-Q system. |
| Reusable Media Storage Bottles | ThermoFisher Scientific | 06-423A | Bottles for mixing and storing culture media. |
| Rubber Stopper, 30 mm, Gray Bromobutyl | DWK Life Sciences | 224100-331 | Rubber stoppers for serum bottles. |
| Serum Bottle with Molded Graduations, 500 mL | DWK Life Sciences | 223952 | Glass serum bottles for sealed culturing. |
| Small Bore Extension Set | Braun Medical | 471960 | Tubing extension with luer lock connectors. |
| Sodium Chloride | MilliporeSigma | S3014 | Solid sodium chloride salt. |
| Spike-in Control I (High Microbial Load) | ZymoBIOMICS | D6320 | Spike-in microbes (I. halotolerans and A. halotolerans) for absolute microbial load calculations |
| Stericup Quick Release Sterile Vacuum Filtration System | MilliporeSigma | S2GPU02RE | 250 mL 0.22 µm vacuum filtration chamber. |
| Super Sani-Cloth Germicidal Disposable Wipes | Professional Disposables International | H04082 | Disposable germicidal wipes for sterilization. |
| Trace Metals Mixture, 1000x | ThermoFisher Scientific | NC0112668 | Concentrated solution of physiological trace metals. |
| Unlined Aluminum Seal, 30 mm | DWK Life Sciences | 224187-01 | Aluminum seals crimped over top of rubber stoppers. |
| USP Medical Grade Air Tank | Airgas | AI USP200 | Compressed air tank for input to sparging system. |
| USP Medical Grade Oxygen Tank | Airgas | OX USP200 | Compressed oxygen tank for input to sparging system. |
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Citer Cet Article
Vieira, J., Gallagher, T., Sui, H., Jesudasen, S., Whiteson, K., O’Toole, G. A., Hanselmann, K., Lai, P. S. Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome. J. Vis. Exp. (174), e62888, doi:10.3791/62888 (2021).