Phenotypic Resistance in Photodynamic Inactivation Unravelled 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 tested on two other Gram-negative strains, Pseudomonas aeruginosa and Klebsiella pneumoniae, 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.