用体外模型研究 5-氨基乙酰丙酸介导光动力疗法对金黄色葡萄球菌生物膜的影响
1Department of Otorhinolaryngology-Head and Neck Surgery, Eye & ENT Hospital, Shanghai Key Clinical Disciplines of otorhinolaryngology,Fudan University, 2Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Department of Medical Microbiology and Parasitology, School of Basic Medical Science,Shanghai Medical College of Fudan University, 3Department of Dermatology, Huashan Hospital,Fudan University
Summary
本手稿描述了一种研究 5-氨基乙酰丙酸介导光动力疗法 (ALA)对金黄色葡萄球菌生物膜的抗菌作用的协议。该协议可用于开发一种体外模型来研究细菌生物膜与 PDT 在未来的治疗。
Abstract
金黄色葡萄球菌(金黄色葡萄球菌) 是一种常见的人类病原体, 引起化脓性和系统性感染。金黄色葡萄球菌感染很难根除, 这不仅是由于抗药性菌株的出现, 而且是它形成生物膜的能力。近年来, 光动力疗法 (PDT) 已被指出是控制生物膜感染的潜在治疗方法之一。然而, 需要进一步的研究, 以提高我们对其对细菌生物膜的影响的认识, 以及潜在的机制。这篇手稿描述了一个体外模型的 PDT 与 5-氨基乙酰丙酸 (5-阿拉), 一个前兆的实际光敏剂, 原卟啉 IX (PpIX)。简单地, 成熟的金黄色葡萄球菌生物膜与阿拉巴马一起孵化, 然后暴露在光中。随后, 通过共聚焦激光扫描显微镜 (CLSM) 计算菌落形成单元 (CFUs), 并通过活性荧光染色可视化方法对金黄色葡萄球菌生物膜的抗菌作用进行了量化。具有代表性的结果表明, 对金黄色葡萄球菌生物膜具有较强的抗菌作用。该协议很简单, 可用于开发一种体外模型, 用于研究用 ALA PDT 治疗金黄色葡萄球菌生物膜的方法。在未来的研究中, 也可以借鉴其他光敏对不同菌株的光动力分析, 并进行最小的调整。
Introduction
金黄色葡萄球菌是一种重要的革兰氏阳性病原体, 殖民了人类寄主的皮肤和粘膜。它形成生物膜的能力被认为是其发病机制的一个重要方面1。细菌生物膜是一种植入自产基质中的细菌群落, 由胞外高分子物质组成, 包括多糖、DNA 和蛋白质。这种基质在细菌感染的持续性中起着重要作用, 对人体免疫系统和目前的抗微生物治疗有很大的抵抗力,2。尽管抗生素对生物膜的影响有限, 但抗生素仍然是生物膜感染的主要治疗方法。先前已经表明, 生物膜中的细胞比它们的浮游相对物3更耐抗生素 10-1000 倍。因此, 需要采取替代战略来克服这一问题。
光动力疗法是一种治疗细菌感染的替代疗法, 它利用适当波长的光线激活光敏。这导致了活性氧种类 (ROS) 的生产, 这是致命的靶细胞通过扰乱细胞壁, 失活酶, 并损害 DNA4。这种多目标特性使得细菌难以对 PDT 治疗产生抗药性。
在以前的报告5、6中, 研究了 PDT 对细菌和真菌生物膜的抗菌作用, 其中包括甲苯胺蓝、孔雀石绿、亚甲基蓝、氯 e6 和卟啉等多种光敏. 7,8,9,10,11,12,13。5-阿拉, 药的实际光敏剂, PpIX, 其特点是其小分子量和快速清除12,14。这些优势给了阿拉 PDT 的主要潜力作为治疗应用。虽然对浮游细菌的影响已经研究了许多组12, 但对细菌生物膜的抗菌作用尚未阐明。同时, 比较以往研究的结果是很难的。其中一个原因是不同的协议被各种组使用。因此, 本协议描述了一种基于我们以前的工作15的一种用于 PDT 系统的体外模型。通过 CFU 计算和 CLSM 活性染色证实了该模型的有效性。
Protocol
1. 生物膜形成 96井微型生物膜的形成 检索金黄色葡萄球菌应变 USA300 和3生物膜形成的临床菌株 (C1-C3), 存储在-80 摄氏度。注: 临床菌株形成生物膜的能力由前述的微量滴定板检测方法确定(15)。 在5毫升的胰蛋白胨大豆汤中接种细菌, 并在一个孵化器中培养, 一夜之间以37摄氏度的温度抖动到固定的阶段。 将隔夜细菌培养在25摄?…
Representative Results
在 USA300 和三株临床菌株中, 与对照组 (以阿拉巴马为首的、以阿拉为主导的 led 和阿拉巴马为首的 +) 相比, 生物膜中细菌的生存能力下降了 (图 1)。 为证实 CFU 测定的结果, 观察其对金黄色葡萄球菌生物膜的抗菌作用, USA300 生物膜通过 CLSM 与活性染色进行可视化. 存活和死亡细胞分别染色绿色?…
Discussion
PDT 一直是一个良好的研究治疗癌症治疗, 因为它是发明100年前的18。在过去的十年中, PDT 已被应用作为一种抗菌策略, 并显示了对某些耐药性致病菌的有效性12。与浮游状态相比, 细菌生物膜似乎对抗生素治疗的抗药性更有3, 而对生物膜的影响还没有得到充分的研究。
本文介绍了一种体外PDT 系统, 并对该模型对金?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作由中国国家自然科学基金 (81300810 号)、上海市青年医生培训计划 (20141057 号) 和中国国家自然科学基金 (81671982、81271791和 81571955) 资助。我们感谢 LetPub (www.letpub.com) 在编写本手稿期间提供语言援助。
Materials
| Tryptone Soya Broth (TSB) | OXOID | CM0129B | |
| Tryptone Soya Agar (TSA) | OXOID | CM0131 | |
| SYTO9 | Thermo Fisher Scientific | L7012 | The LIVE/DEAD BacLight Bacterial Viability Kits |
| Propidium iodide (PI) | Thermo Fisher Scientific | L7012 | The LIVE/DEAD BacLight Bacterial Viability Kits |
| Pancreatin | Sigma-Aldrich | P3292 | |
| 5-aminolevulinic acid (ALA) | Fudan Zhangjiang Bio-Pharm | 3.1 | |
| Staphylococcus aureus strain USA300 | / | / | The source of USA 300 references “Tenover FC, Goering RV. J Antimicrob Chemother. 2009 Sep; 64(3):441-6”. |
| Staphylococcus aureus clinical strains (C1-C3) | / | / | All clinical strains were isolated from patients with chronic rhinosinusitis in the Department of Otorhinolaryngology-Head and Neck Surgery, Eye and ENT Hospital of Fudan University [Zhang QZ, Zhao KQ, Wu Y, et al. PLoS One. 2017 Mar; 12(3): e0174627]. |
| 96-well microplate | Corning Inc | 3599 | Clear Flat Bottom Polystyrene TC-Treated Microplates, Individually Wrapped, with Lid, Sterile |
| Fluorodish | NEST Biotechnology | 801001 | Glass bottom, Non-pyrogenic |
| Eppendorf Safe-Lock Tubes, 1.5 mL | Eppendorf | 0030120086 | |
| Eppendorf microcentrifuge 5417 | Eppendorf | Z365998 | SIGMA | |
| Incubator | Thermo Fisher Scientific | SHKE4000 | MaxQ 4000 Benchtop Orbital Shakers |
| Light emitting diode (LED) | Wuhan Yage Optic and Electronic Technique CO | LED-IB | |
| Leica TCS SP8 confocal laser-scanning microscope | Leica Microsystems | ||
| Leica LAS AF software | Leica Microsystems | ||
| IMARIS software | Bitplane |
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Citar este artículo
Zhao, K., Wu, Y., Yi, Y., Feng, S., Wei, R., Ma, Y., Zheng, C., Qu, D. An In Vitro Model to Study the Effect of 5-Aminolevulinic Acid-mediated Photodynamic Therapy on Staphylococcus aureus Biofilm. J. Vis. Exp. (134), e57604, doi:10.3791/57604 (2018).