Zhao Ke-Qing, Wu Yang, Yi Yu-Xi, Feng Si-Jia, Wei Ruo-Yan, Ma Ying, Zheng Chun-Quan, Qu Di
Department of Otorhinolaryngology-Head and Neck Surgery, Eye & ENT Hospital, Shanghai Key Clinical Disciplines of otorhinolaryngology, Fudan University;
Key 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.
J Vis Exp. 2018 Apr 16(134):57604. doi: 10.3791/57604.
Staphylococcus aureus (S. aureus) is a common human pathogen, which causes pyogenic and systemic infections. S. aureus infections are difficult to eradicate not only due to the emergence of antibiotic-resistant strains but also its ability to form biofilms. Recently, photodynamic therapy (PDT) has been indicated as one of the potential treatments for controlling biofilm infections. However, further studies are required to improve our knowledge of its effect on bacterial biofilms, as well as the underlying mechanisms. This manuscript describes an in vitro model of PDT with 5-aminolevulinic acid (5-ALA), a precursor of the actual photosensitizer, protoporphyrin IX (PpIX). Briefly, mature S. aureus biofilms were incubated with ALA and then exposed to light. Subsequently, the antibacterial effect of ALA-PDT on S. aureus biofilm was quantified by calculating the colony forming units (CFUs) and visualized by viability fluorescent staining via confocal laser scanning microscopy (CLSM). Representative results demonstrated a strong antibacterial effect of ALA-PDT on S. aureus biofilms. This protocol is simple and can be used to develop an in vitro model to study the treatment of S. aureus biofilms with ALA-PDT. In the future, it could also be referenced in PDT studies utilizing other photosensitizers for different bacterial strains with minimal adjustments.
金黄色葡萄球菌是一种常见的人类病原体，可引起化脓性感染和全身性感染。金黄色葡萄球菌感染难以根除，不仅是因为出现了抗生素耐药菌株，还因其具有形成生物膜的能力。最近，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，需要进一步研究以增进我们对其对细菌生物膜的作用及其潜在机制的了解。本手稿描述了一种使用5-氨基乙酰丙酸（5-ALA）的光动力疗法体外模型，5-ALA是实际光敏剂原卟啉IX（PpIX）的前体。简而言之，将成熟的金黄色葡萄球菌生物膜与ALA孵育，然后进行光照。随后，通过计算菌落形成单位（CFU）来量化ALA-PDT对金黄色葡萄球菌生物膜的抗菌作用，并通过共聚焦激光扫描显微镜（CLSM）进行活力荧光染色来可视化。代表性结果表明ALA-PDT对金黄色葡萄球菌生物膜具有强大的抗菌作用。该方案简单，可用于建立体外模型，以研究用ALA-PDT治疗金黄色葡萄球菌生物膜。未来，在利用其他光敏剂治疗不同细菌菌株的光动力疗法研究中，只需进行最小调整即可参考该方案。
