噬菌体纤维化 斑马鱼胚 胎中伪多多多阿韦诺萨感染的噬菌体治疗应用
Summary
这里介绍的是伪 多多多酸菌感染和 噬菌体治疗应用囊性纤维化（CF）斑马鱼胚胎的协议。
Abstract
抗微生物药物耐药性是诊断不确定性和抗菌药物过度处方的一个主要后果，是全世界日益公认的严重感染、并发症和死亡的原因，它在我们的社会和卫生系统上产生了巨大影响。特别是，免疫系统受损或预先存在的慢性疾病（如囊性纤维化 （CF））的患者经常接受抗生素治疗，以控制多药耐药分离物的出现和传播感染。因此，迫切需要解决替代疗法，以抵消细菌感染。使用细菌的天敌噬菌体可能是一种可能的解决方案。本文详述的规程描述了噬菌体疗法在CF斑马鱼胚胎中对伪多多尼亚亚亚罗吉诺萨感染的应用。斑马鱼胚胎感染了 P.aeruginosa， 以证明噬菌体疗法对 P.aeruginosa 感染是有效的，因为它减少了CF胚胎中的致命性、细菌负担和亲炎免疫反应。
Introduction
噬菌体疗法，利用细菌的天敌来对抗细菌感染，随着细菌对抗生素的耐药性变得广泛而引起,人们重新的兴趣。这种疗法在东欧使用了几十年，可被视为对治疗CF患者肺部感染的抗生素的补充疗法，以及一种可能的治疗方法，用于治疗目前使用的所有抗生素22、33的细菌。抗生素治疗的优点是噬菌体在感染部位繁殖，而抗生素被代谢，从体内消除4，5。,5事实上，不同实验室分离的毒性噬菌体鸡尾酒的产生已证明对治疗动物模型中的伪多莫纳斯亚鲁吉诺萨感染是有效的，就像,昆虫和哺乳动物6、7、87一样。6在随机临床试验9中，噬菌体疗法还证明能够减轻感染了P.aeruginosa和Escherichia大肠杆菌的烧伤伤口的细菌负担。
斑马鱼（Danio rerio）最近已成为研究几种病原体感染的宝贵模型，包括P.aeruginosa10，11，10,11分枝杆菌脓肿和布尔科达雷塞西亚12，13。12,13通过微缩细菌直接进入胚胎血液循环14，很容易建立一种系统感染，由斑马鱼先天免疫系统抵消，这是进化保存与嗜中性粒细胞和巨噬细胞生成类似人类对应。此外，在生命的第一个月，斑马鱼胚胎缺乏适应性免疫反应，是研究先天免疫的理想模型，是人类肺部感染的关键防御机制。斑马鱼最近成为一个强大的基因模型系统，以更好地了解CF发病，并开发新的药理治疗10，16，17。10,16,17在斑马鱼中用形态注射产生的cftr击倒的CFR斑马鱼模型呈现抑制性呼吸突发反应和减少嗜中性粒细胞迁移10，而cftr击倒导致内部器官位置受损和破坏外分泌胰腺，一种反映人类疾病16，17,的表型。最感兴趣的是发现，在cftr-功能丧失胚胎中，P.aeruginosa细菌负担明显高于感染后8小时对等对子，这与小鼠和人类支气管上皮细胞2、18,18的结果平行。
在这项工作中，我们证明噬菌体疗法 对斑马鱼胚胎中的P.aeruginosa 感染是有效的。
Protocol
来自AB菌株（欧洲斑马鱼资源中心EZRC）的成年斑马鱼（Danio rerio）根据国际（欧盟指令2010/63/EU）和国家指南（意大利2014年3月4th日法令，第26号法令）维护，保护用于科学目的的动物。标准条件设置在鱼设施与14小时光/10小时暗循环和水箱水温在28°C。 1. 准备解决方案和工具 为生长斑马鱼胚胎的 E3 胚胎介质准备 50 倍的库存溶液和 1 倍的工作解决方案（ <s…
Representative Results
此处显示的结果和数字是指通过注射cftr 形态异形体生成的 CF 胚胎，如前面10 和步骤 5 所述。为了验证CF表型，考虑了心脏、肝脏和胰腺等内脏器官的受损位置，如前面描述的17（图1）。在WT胚胎的情况下，也取得了类似的结果，正如我们在上一份出版物19中报道的。 感染PAO1的CF胚胎的噬菌体?…
Discussion
在这份手稿中，我们描述了在斑马鱼胚胎中实施 P.aeruginosa（PAO1） 感染的协议，以及如何使用以前被确定为能够感染PAO1的噬菌体鸡尾酒来应用噬菌体疗法来解决它。使用噬菌体作为抗生素治疗的替代品，自去年年底以来，人们越来越关注。这主要是由于耐多药（MDR）细菌感染的传播，这对公共卫生构成严重问题。当然，这项工作的范围仅限于将噬菌病疗法应用到动物模型，而不是人类。然…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了意大利囊性纤维化基金会（FFC#22/2017） 的支持;协会”Gli amici della Ritty”卡斯尼戈和FFC#23/2019;皮奥卢斯拉马诺特萨奥卢斯的 Un respiro） 。
Materials
| Bacto Agar | BD | 214010 | |
| Calcium chloride | Sigma-Aldrich | 10043-52-4 | |
| CsCl | Sigma-Aldrich | 289329 | |
| Dulbecco's phospate buffered saline PBS | Sigma-Aldrich | D8537 | |
| Ethyl 3-aminobenzoate methanesulfonate | Sigma-Aldrich | 886-86-2 | common name tricaine |
| Femtojet Micromanipulator | Eppendorf | 5247 | |
| Fleming/brown P-97 | Sutter Instrument Company | P-97 | |
| LE-Agarose | Sigma-Aldrich | 11685660001 | |
| Low Melting Agarose | Sigma-Aldrich | CAS 9012-36-6 | |
| Magnesium sulfate | Sigma-Aldrich | 7487-88-9 | |
| Methyl Blue | Sigma-Aldrich | 28983-56-4 | |
| Microinjection needles | Harvard apparatus | ||
| N-Phenylthiourea >=98% | Aldrich-P7629 | 103-85-5 | |
| Oligo Morpholino | Gene Tools | designed by the researcher | |
| PEG6000 | Calbiochem | 528877 | |
| Phenol Red Solution | Sigma-Aldrich | CAS 143-74-B | |
| Potassium chloride | Sigma-Aldrich | 7447-40-7 | |
| Pronase | Sigma-Aldrich | 9036-06-0 | |
| Sodium chloride ACS reagent, ≥99.0% | Sigma-Aldrich | S9888 | |
| Stereomicroscope | Leica | S9I | |
| Tris HCl | Sigma-Aldrich | T5941 | |
| Triton X | Sigma-Aldrich | T9284 | |
| Tryptone | Oxoid | LP0042B | |
| Yeast extract | Oxoid | LP0021B | |
| Z-MOLDS Microinjection | Word Precision Instruments |
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Cite This Article
Cafora, M., Forti, F., Briani, F., Ghisotti, D., Pistocchi, A. Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos. J. Vis. Exp. (159), e61275, doi:10.3791/61275 (2020).