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)
Abstract:
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.
Poster
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