Investigation of photodynamic inactivation of bacteria using the detection of singlet oxygen luminescence

Jürgen Baier, Tim Maisch, Barbara Franz, Max Maier, Michael Landthaler, Rolf-Markus Szeimies and Wolfgang Bäumler
(J. Baier, T. Maisch, B. Franz, M. Maier, M. Landthaler, R. M. Szeimies and W. Bäumler)

In view of the increasing resistance of bacteria to antibiotics, photodynamic inactivation of bacteria is a promising new technique. It is known, that Gram(+) and Gram(-) bacteria can be killed by antibacterial photodynamic inactivation depending on the used photosensitizer. The objective was to evaluate localisation of the photosensitizer Photofrin® in Gram(+) S. aureus and Gram(-) E. coli by detection of singlet oxygen time-resolved by its luminescence at 1270 nm directly. Singlet oxygen was generated by energy transfer from the photoexcited Photofrin, dissolved in aqua dest. After incubation of S. aureus or E. coli with Photofrin and subsequent irradiation, the viability of S. aureus decreased yielding 99.9% dead bacteria, whereas the viability of E. coli was hardly affected. Sodium azide, quencher of singlet oxygen, inhibited the killing of S. aureus. Fluorescence microscopy showed an uptake of Photofrin by S. aureus but not by E. coli. Due to the limited resolution of the microscope, the subcellular localization of Photofrin in bacteria failed and therefore a detailed insight into the mechanisms of action was not possible. However, the localization of Photofrin is correlated to the localization of singlet oxygen, which is correlated to luminescence decay time of singlet oxygen measured. The resolution of this method is given by the diffusion length of singlet oxygen, which is very short in a biological environment. When incubating E. coli with 300 µg/ml Photofrin for 90 min no singlet oxygen luminescence was detected confirming the results of cell viability experiment. When incubating S. aureus with Photofrin, a singlet oxygen luminescence decay time of 6 µs ± 2 µs was measured. Adding the quencher Sodium azide the luminescence decay time was shortened (3 µs ± 1 µs). Obviously, the decay time of luminescence is an intermediate time of singlet oxygen decaying in phospholipids (14 µs ± 2 µs) of membranes and in the surrounding water (3.5 µs ± 0.5 µs). Thus, singlet oxygen seems to decay in outer cell wall areas of S. aureus, which is then the subcellular localization of Photofrin. The luminescence decay time in large agglomerates of bacteria was much longer (40 µs ± 16 µs) than in the suspension with single bacteria. This is the first time that singlet oxygen was measured directly by its luminescence inside living bacteria.

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