Endogenous porphyrin production in bacteria by δ-aminolaevulinic acid and subsequent bacterial photoeradication

In the present study we examined the effects of the accumulation of endogenous porphyrins on the microorganisms Staphylococcus aureus and Escherichia coli. delta-Aminolaevulinic acid (delta-ALA) 50 mu g/ml can induce production of large amounts of porphyrins when incubated with the bacteria in the dark. This porphyrin production within the cells was examined directly by a fluorescence activated cell sorter (FACS) instrument and a significant increase in red fluorescence was observed. Incubation of F-ALA with these microorganisms did not slow down their growth. delta-ALA or delta-ALA methyl ester induced accumulation of uroporphyrin in S. aureus cells and excretion of coproporphyrin into the growth medium. In E. coli, these inducers resulted in the accumulation of uroporphyrin and protoporphyrin within the cells and excretion of coproporphyrin and protoporphyrin fi om the cells. Photoinactivation of the porphyrin-loaded bacteria can be achieved by various light sources, depending on the dose. The most effective photokilling of S. aureus and of E. coli by its endogenic porphyrins was achieved by blue light (400-450 nm) at approximately 50 J/cm(2). With red light (600-100 nm), a 10-fold higher light dose was required to get a similar result for S. aureus killing. With a laser beam (632.8 nm), 50 J/cm(2) was necessary for 3 orders of decrease in the viability of S. aureus. With white light, 75 J/cm(2) was needed for 2-3 orders of decrease of S. aureus viability. With the last two light sources eradication of E. coli required at least 10 times higher doses of light. It seems that S. aureus is more susceptible than E. coli to photosensitisation when it is loaded with endogenous porphyrins. Ultrastructural alterations were observed in the bacteria incubated with F-ALA in the dark and photosensitised by blue light (400-450 nm). Filamentous chromosomes were observed in E. coli, whereas honeycomb mesosomes were observed in S. aureus, with a destruction of the area around these mesosomes in the chromosomal area. The method described here is an additional approach to the photoinactivation of bacteria by porphyrins, with photoinactivation being accomplished by endogenous porphyrins.