Continuous real-time monitoring of cationic porphyrin-induced photodynamic inactivation of bacterial membrane functions using electrochemical sensors

We analysed the porphyrin-induced photodynamic inactivation of the membrane functions of bacteria through the in situ monitoring of changes in respiration rates, membrane permeability and membrane potential, using electrochemical sensors, such as oxygen, K+ and tetraphenylphosphonium (TPP+) electrodes. We used two cationic porphyrins, tetrakis(4-N, N, N-trimethylammoniumphenyl) porphyrin (TTMAPP) and tetrakis(4-N-methylpyridinium) porphyrin (TMPyP), along with an anionic porphyrin, tetrakis(4-sulfonatophenyl) porphyrin (TSPP), as a negative control. TTMAPP and TMPyP inhibited the respiration of bacteria within minutes of photo-irradiation at a concentration of 1 mu M, where the survival of bacteria decreased, while TSPP did not affect the bacteria. The respiration of Staphylococcus aureus cells (Gram-positive bacterium) was more strongly inhibited than that of Escherichia coli cells (Gram-negative bacterium). Increasing the concentration of porphyrin strengthened the respiratory inhibition. Although TTMAPP increased the permeability to K+ of the cytoplasmic membranes of bacteria, the change was relatively slow. Cationic porphyrins, showing the strong respiratory inhibition of S. aureus cells, induced the dissipation of membrane potential within minutes of photo-irradiation, in accord with the time traces of respiratory inhibition. Such a correlation strongly supported that porphyrin-induced photo-inactivation of bacteria involved rapid damage to the energy-producing system of bacteria induced by inhibition of the respiratory chain, leading to a dissipation of membrane potential. These results are discussed in connection with the ability of porphyrins to generate singlet oxygen and bind to the bacterial cell envelope.