Real‐time imaging of photodynamic action in bacteria

Abstract

Fluorescence imaging studies of the processes leading to photodynamic inactivation of bacteria have been limited due to the small size of microorganisms as well as by the faint fluorescence of most photosensitizers. A versatile method based on highly-sensitive fluorescence microscopy is presented which allows to study, in real time, the incorporation of photosensitizers inside S. aureus upon photodynamic action. The method takes advantage of the fluorescence enhancement of phenothiazine and porphyrin photosensitizers upon entering the bacterial cytosol after the cell wall has been compromised. In combination with typical assays, such as the addition of specific enhancers of reactive oxygen species, it is possible to extract mechanistic information about the pathway of photodynamic damage at the single-cell level. Imaging experiments in deuterated buffer strongly support a Type-I mechanism for methylene blue and a very minor role of singlet oxygen.

Antimicrobial photodynamic therapy – what we know and what we don’t

“…(Preuß et al 2013). In accordance to the latter study, Gollmer et al revealed by highly-sensitive fluorescence microscopy that accumulation of TMPyP in the cytoplasm and the cell wall is minor in S. aureus and that this PS may only enter the cell after preceding cell wall damage (Gollmer et al 2017). On the contrary, the phenothiazinium dye Methylene Blue seems to locate intracellularly because its photodynamic efficacy can be increased by addition of efflux pump inhibitors (Tegos et al 2008).…”

“…For this purpose, mostly biochemical methods and transmission electron microscopy have been employed so far because the use of fluorescence microscopy was limited by the small size of bacteria and the faint fluorescence properties, i.e. photo-bleaching, of many PS (Alves et al 2014;Gollmer et al 2017). However, as stated above, very recently Gollmer et al were the first to apply novel highly-sensitive fluorescence microscopy to study the incorporation of PS inside bacteria (in this case: S. aureus) taking advantage of fluorescence enhancement of Methylene Blue and TMPyP upon entering the bacterial cytoplasm after aPDT-mediated oxidative deterioration of the cell wall and cytoplasmic membrane (Gollmer et al 2017).…”

“…photo-bleaching, of many PS (Alves et al 2014;Gollmer et al 2017). However, as stated above, very recently Gollmer et al were the first to apply novel highly-sensitive fluorescence microscopy to study the incorporation of PS inside bacteria (in this case: S. aureus) taking advantage of fluorescence enhancement of Methylene Blue and TMPyP upon entering the bacterial cytoplasm after aPDT-mediated oxidative deterioration of the cell wall and cytoplasmic membrane (Gollmer et al 2017). This technique merits further investigations on distinct classes of PS, but also on more bacterial species with different Gram-staining characteristics.…”

Light‐driven photosensitizer uptake increases Candida albicans photodynamic inactivation

“…In the opposite direction, Gollmer et al. used methylene blue as PS for bacteria and reported that PS fluorescence intensity increased along time up to stabilize when illuminated. However, the authors correlated this behavior a fluorescence quantum yield improvement.…”

Factors Determining the Susceptibility of Bacteria to Antibacterial Photodynamic Inactivation

“…While remaining outside, however, it could provide significant phototoxicity by singlet oxygen generation ( 106 ). Additionally, Gollmer et al showed that TMPyP accumulates minimally in the cytoplasm and the cell wall of S. aureus but only after cell wall damage ( 107 ). Nevertheless, these results are not in accordance with previous studies on the localization of TMPyP ( 108 ), so further investigation of the external action of PSs is important.…”