Martínez Sol R, Palacios Yohana B, Heredia Daniel A, Agazzi Maximiliano L, Durantini Andrés M
Departamento de Química, Facultad de Ciencias Exactas, Físico-Químicas y Naturales , Universidad Nacional de Río Cuarto , Ruta Nacional 36 Km 601 , X5804BYA Río Cuarto , Córdoba , Argentina.
IDAS-CONICET, Departamento de Química, Facultad de Ciencias Exactas, Físico-Químicas y Naturales , Universidad Nacional de Río Cuarto , Ruta Nacional 36 Km 601 , X5804BYA Río Cuarto , Córdoba , Argentina.
ACS Infect Dis. 2019 Sep 13;5(9):1624-1633. doi: 10.1021/acsinfecdis.9b00185. Epub 2019 Jul 19.
Herein we report a simple fluorescence microscopy methodology that, jointly with four photosensitizers (PSs) and a cell viability marker, allows monitoring of phenotypic bacterial resistance to photodynamic inactivation (PDI) treatments. The PSs, composed of BODIPY dyes, were selected according to their ability to interact with the cell wall and the photoinactivating mechanism involved (type I or type II). In a first approach, the phenotypic heterogeneity allowing bacteria to persist during PDI treatment was evaluated in methicillin-resistant (MRSA) and as Gram-positive and Gram-negative models, respectively. By means of propidium iodide (PI), we monitored with spatiotemporal resolution cell viability at the single bacterium level. All the PSs were effective at inactivating pathogens; however, the cationic nonhalogenated PS (compound ) surpassed the others and was capable of photoinactivating even under optimal growth conditions. Compound was further tested on two other Gram-negative strains, and , with outstanding results. All bacterial strains used here are well-known ESKAPE pathogens, which are the leading cause of nosocomial infections worldwide. Thorough data analysis of individual cell survival times revealed clear phenotypic variation expressed in the cell wall that affected PI permeation and thus its intercalation with DNA. For the same bacterial sample, death times may vary from seconds to hours. In addition, the PI incorporation time is also a parameter governed by the phenotypic characteristics of the microbes. Finally, we demonstrate that the results gathered for the bacteria provide direct and unique experimental evidence that supports the time-kill curve profiles.
在此，我们报告一种简单的荧光显微镜方法，该方法与四种光敏剂（PSs）和一种细胞活力标记物相结合，可用于监测细菌对光动力灭活（PDI）治疗的表型抗性。由硼二吡咯染料组成的PSs是根据它们与细胞壁相互作用的能力以及所涉及的光灭活机制（I型或II型）来选择的。在第一种方法中，分别以耐甲氧西林金黄色葡萄球菌（MRSA）作为革兰氏阳性模型，以及其他革兰氏阴性菌作为模型，评估了在PDI治疗期间允许细菌持续存在的表型异质性。通过碘化丙啶（PI），我们在单细菌水平上以时空分辨率监测细胞活力。所有的PSs都能有效地灭活病原体；然而，阳离子非卤代PS（化合物 ）表现优于其他PSs，甚至在最佳生长条件下也能够进行光灭活。化合物 在另外两种革兰氏阴性菌株 和 上进一步测试，结果出色。这里使用的所有细菌菌株都是著名的ESKAPE病原体，它们是全球医院感染的主要原因。对单个细胞存活时间的全面数据分析揭示了细胞壁中明显的表型变异，这种变异影响了PI的渗透，从而影响了其与DNA的嵌入。对于相同的细菌样本，死亡时间可能从几秒到几小时不等。此外，PI的掺入时间也是一个受微生物表型特征控制的参数。最后，我们证明，针对细菌收集的结果提供了直接且独特的实验证据，支持时间 - 杀灭曲线图谱。
