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 μM, where the survival of bacteria decreased, while TSPP did not affect the bacteria. The respiration of Staphylococcus aureuscells (Gram-positive bacterium) was more strongly inhibited than that of Escherichia colicells (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. aureuscells, 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.