Photoinactivation of Salmonella enterica exposed to 5-aminolevulinic acid: Impact of sensitization conditions and irradiation time.

The photodynamic inactivation (PDI) represents the potential alternative to traditional antibiotic therapy, and can be applied to treat various bacterial infections, including those caused by Gram-negative bacterial strains. One of the treatment modalities is based on the capacity of bacterial cells to synthesize the excess amounts of porphyrins after exposure to an externally applied 5-aminolevulinic acid (5-ALA), which makes them photosensitive and leads to reduced survival after irradiation with an appropriately selected light source. This study focuses on the sensitization and the photoinduced inactivation of Salmonella enterica cells in PBS containing 0.5 mM 5-ALA, incubated at 37 °C for 4 h or for 20 h and afterwards irradiated with violet LED light (11.1 mW/cm2, a peak at 400 nm). It has been found that both amounts and composition of endogenous porphyrins not only depended on the incubation duration, but also were affected by externally induced photo- and chemo-oxidation reactions. The application of different sensitization conditions has revealed that the increasing amounts of endogenously produced porphyrins do not ensure the proportional reduction of bacterial cell survival numbers. The comparative investigations also demonstrated that the presence of endogenously produced porphyrins in the medium results in secondary sensitization of bacterial cells and causes a notably stronger photoinactivation effect in comparison to their externally applied standards.

Sensitization of Salmonella enterica with 5-aminolevulinic acid-induced endogenous porphyrins: a spectroscopic study.

Photodynamic therapy (PDT) of bacterial strains presents an attractive potential alternative to antibiotic therapies in search of the solution for the chemoresistance problem. The efficacy of the treatment is dependent on the interaction of photochemically active substances called photosensitizers (PSs) with the bacterial cell wall or their intracellular accumulation. In addition to exogenous PSs, other molecules such as 5-aminolevulinic acid (5-ALA), a natural precursor of heme, are gaining interest. When provided exogenously to cells, 5-ALA uptake results in the overproduction of various photoactive porphyrins. The pattern of their intracellular accumulation and release to the surroundings depends on incubation conditions such as the applied 5-ALA concentration, cell density and incubation duration. The detection of endogenously synthesized porphyrins in samples of Salmonella enterica cells and supernatants was accomplished after 4 h and 20 h incubation periods by means of fluorescence spectroscopy. The relative proportions of different types of porphyrins were assessed by modeling the registered spectra with the fluorescence spectra of standard porphyrins. After the shorter incubation period, the dominant porphyrins in the supernatant medium were coproporphyrins. The longer incubation period shifted the relative proportion of intracellular porphyrins from protoporphyrin IX towards water-soluble porphyrins such as uroporphyrin I, which interfered with additional by-products. The time-dependent changes in compositions of both intracellular and extracellular porphyrins imply that 5-ALA-induced sensitization might have triggered a complex protective mechanism of bacterial cells. Thus, identification and evaluation of the relative amounts of porphyrins, which accumulate in bacterial cells and are extruded outside after different time periods, could provide access to valuable information, working towards more efficient applications of 5-ALA-based antibacterial PDT.