CONICET | Buscador de Institutos y Recursos Humanos

Bacterial photodynamic inactivation (PDI) employing endogenous production of porphyrins from 5-aminolevulinicacid (ALA) ? named ALA-PDI-, is a new promising tool to achieve bacteria control in non-spread infections.The technique combines the action of the porphyrins acting as photosensitisers with light, to producereactive oxygen species to target the pathogen. To date, some clinical applications of ALA-PDI have been reportedalthough variable responses ranging from total eradication to absence of photokilling were found. ALAPDIconducted at suboptimal conditions may lead to misleading results and the complexity of haem synthesis inbacteria hinders the optimization of the treatment.The present work aimed to gain insight on the variables affecting ALA-PDI in Gram-positives and Gramnegativesbacteria growing on planktonic and biofilm cultures and to correlate the degree of the response withthe amount and type of porphyrin synthesised.Staphylococcus epidermidis and Escherichia coli clinical isolates and Pseudomonas aeruginosa ATCC27853 andStaphylococcus aureus ATCC25923 strains were utilised, and the optimal conditions of concentration and timeexposure of ALA, and light dose were set.In both Gram-positive species analysed, a peak of porphyrin synthesis was observed at 1?2 mM ALA in biofilmand planktonic cultures, which fairly correlated with the decrease in the number of CFU after PDI (5 to 7 logs)and porphyrin content was in the same order of magnitude. In addition, ALA-PDI was similarly effective forplanktonic and biofilm S. aureus cultures, and more effective in S. epidermidis planktonic cultures at low lightdoses. Beyond a certain light dose, it was not possible to achieve further photosensitization. Similarly, a plateauof cell death was attained at a certain ALA incubation time. Accumulation of hydrophilic porphyrins at longerincubation periods was observed.The proportion of porphyrins changed as a function of ALA concentration and incubation time in the Grampositivebacteria, though we did not find a clear correlation between the porphyrin type and PDI response. As asalient feature was the presence of isococroporphyrin isoforms in both Gram-positive and Gram-negative bacteria.Gram-negative bacteria were quite refractory to the treatment: P. aeruginosa was slightly inactivated (4-logsreduction) at 40 mM ALA, whereas E. coli was not inactivated at all. These species accumulated high ALAquantities and the amount of porphyrins did not correlate with the degree of photoinactivation. Our microscopystudies show that porphyrins are not located in the envelopes of Gram-negative bacteria, reinforcing the hypothesisthat endogenous porphyrins fail to attack these structures.

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