Photodynamic inactivation mediated by 5-aminolevulinic acid of bacteria in planktonic and biofilm forms

BOHM, GABRIELA CERVINI; GÁNDARA, LAUTARO; DI VENOSA, GABRIELA; MAMONE, LEANDRO; BUZZOLA, FERNANDA; CASAS, ADRIANA

BIOCHEMICAL PHARMACOLOGY

PERGAMON-ELSEVIER SCIENCE LTD

Año: 2020 vol. 177

0006-2952

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 produce reactiveoxygen 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. ALA-PDI conductedat suboptimal conditions may lead to misleading results and the complexity of haem synthesis in bacteriahinders the optimization of the treatment.The present work aimed to gain insight on the variables affectingALA-PDI in Gram-positives and Gram-negatives bacteria growing on planktonic and biofilm cultures and to correlatethe degree of the response with the amount and type of porphyrin synthesised.Staphylococcus epidermidis andEscherichia coli clinical isolates and Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC25923strains were utilised, and the optimal conditions of concentration and time exposure of ALA, and light dose wereset.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 andincubation time in the Gram-positive bacteria, though we did not find a clear correlation between the porphyrintype and PDI response. As a salient feature was the presence of isococroporphyrin isoforms in both Gram-positiveand Gram-negative bacteria.Gram-negative bacteria were quite refractory to the treatment: P. aeruginosa wasslightly inactivated (4-logs reduction) at 40 mM ALA, whereas E. coli was not inactivated at all. These speciesaccumulated high ALA quantities and the amount of porphyrins did not correlate with the degree of photoinactivation.Our microscopy studies show that porphyrins are not located in the envelopes of Gram-negative bacteria,reinforcing the hypothesis that endogenous porphyrins fail to attack these structures.