Phenotypic Resistance in Photodynamic InactivationUnravelled at the Single Bacterium Level

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 Staphylococcus aureus (MRSA) and Escherichia coli 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 1) surpassed the others and was capable of photoinactivating E. coli even under optimal growth conditions. Compound 1 was further teste...