Contents lists available at ScienceDirect Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm Photodynamic inactivation mediated by 5-aminolevulinic acid of bacteria in planktonic and biofilm forms Gabriela Cervini Bohm a , Lautaro Gándara a , Gabriela Di Venosa a , Leandro Mamone a , Fernanda Buzzola b , Adriana Casas a, ⁎ a Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP), CONICET and Hospital de Clínicas José de San Martín, Universidad de Buenos Aires. Córdoba 2351 1er subsuelo, Ciudad de Buenos Aires CP1120AAF, Argentina b Universidad de Buenos Aires, CONICET, Instituto de Investigaciones en Microbiología y Parasitología Médica (IMPaM), and Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Buenos Aires, Argentina ARTICLE INFO Keywords: Bacteria Photoinactivation 5-Aminolevulinic acid Biofilms Antimicrobial ABSTRACT Bacterial photodynamic inactivation (PDI) employing endogenous production of porphyrins from 5-aminole- vulinic acid (ALA) – named ALA-PDI-, is a new promising tool to achieve bacteria control in non-spread in- fections. The technique combines the action of the porphyrins acting as photosensitisers with light, to produce reactive oxygen species to target the pathogen. To date, some clinical applications of ALA-PDI have been re- ported although variable responses ranging from total eradication to absence of photokilling were found. ALA- PDI conducted at suboptimal conditions may lead to misleading results and the complexity of haem synthesis in bacteria hinders the optimization of the treatment. The present work aimed to gain insight on the variables affecting ALA-PDI in Gram-positives and Gram- negatives bacteria growing on planktonic and biofilm cultures and to correlate the degree of the response with the amount and type of porphyrin synthesised. Staphylococcus epidermidis and Escherichia coli clinical isolates and Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC25923 strains were utilised, and the optimal conditions of concentration and time exposure 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 biofilm and 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 for planktonic and biofilm S. aureus cultures, and more effective in S. epidermidis planktonic cultures at low light doses. Beyond a certain light dose, it was not possible to achieve further photosensitization. Similarly, a plateau of cell death was attained at a certain ALA incubation time. Accumulation of hydrophilic porphyrins at longer incubation periods was observed. The proportion of porphyrins changed as a function of ALA concentration and incubation time in the Gram- positive bacteria, though we did not find a clear correlation between the porphyrin type and PDI response. As a salient feature was the presence of isococroporphyrin isoforms in both Gram-positive and Gram-negative bac- teria. Gram-negative bacteria were quite refractory to the treatment: P. aeruginosa was slightly inactivated (4-logs reduction) at 40 mM ALA, whereas E. coli was not inactivated at all. These species accumulated 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 hy- pothesis that endogenous porphyrins fail to attack these structures. https://doi.org/10.1016/j.bcp.2020.114016 Received 5 February 2020; Accepted 4 May 2020 Abbreviations: ALA, 5-aminolevulinic acid; CFU, colony forming units; Copro, Coproporphyrin III; EPS, exopolysaccharide; Hexa, Hexaporphyrin; Hepta, Heptaporphyrin; Isocopro, Isocoproporphyrin; Penta, Pentaporphyrin; PDI, Photodynamic inactivation; Proto, Protoporphyrin IX; PS(s), photosensitiser(s); Uro, Uroporphyrin III ⁎ Corresponding author at: Centro de Investigaciones sobre Porfirinas y Porfirias (CIPYP), CONICET and Hospital de Clínicas José de San Martín, University of Buenos Aires, Córdoba 2351 1er subsuelo, Ciudad de Buenos Aires CP1120AAF, Argentina. E-mail address: adriana@qb.fcen.uba.ar (A. Casas). Biochemical Pharmacology 177 (2020) 114016 Available online 07 May 2020 0006-2952/ © 2020 Elsevier Inc. All rights reserved. T