Photoinactivation of planktonic and biofilm bacterial cultures employing delta-aminolevulinic acid.

GABRIELA CERVINIBÖHM, GABRIELA DI VENOSA, DANIEL SÁENZ, GUSTAVO CALVO, PABLO VALLECORSA, GABRIEL ORLANDO, FERNANDA BUZZOLA*, ADRIANA CASAS.

Encuentro; XIII Encuentro Latinoamericano de Fotoquímica y Fotobiología; XIII ELAFOT; 2017

BacterialPhoto-Inactivation (BPI) is an antimicrobial treatment that uses aphotosensitizing molecule (FS), which is then irradiated with visible lightgenerating reactive oxygen species and the consequent damage to themicroorganisms. On the other hand, antibiotic-resistant infections represent animportant clinical problem, most due to the formation of biofilms.Consequently, the need arises for new alternative therapies to the use ofantibiotics. In this work we evaluatedδ-aminolevulinic acid (ALA) action as a precursor of photosensitiveporphyrins [1-2]] in planktonic cultures and biofilms of Staphylococcus aureus ATCC 25923 [4], Staphylococcusepidermidis, Escherichiacoli[3] and Pseudomonas aeruginosaATCC 27853. We worked with 24-h biofilms grown over polystyrene plates andplanktonic cultures with an OD600 0.7. After incubation in the darkat different doses of ALA,we irradiated the bacteria with a non-coherent white light source. Thedetermination of the effectiveness of the treatments was performed countingcolony forming units (CFU/ml). In addition, the amount and type of porphyrinssynthesized in the different treatments were determinedfluorimetricallyand byHPLC. The BPI with concentrations between 1 and 2 mM of ALA, reduces 6 logs the viability of theplanktonic and biofilms cultures of the two Gram positive strains analyzed,relative to the untreated controls. In the case of the Gram negative strains,only P. aeruginosa showed sensitivityto ALA-BPI in planktonic culture, reducing its viability by 5 logs.Porphyrinsfrom Gram positive bacteria peaks at concentrations of 1 mM or 2 mM for planktonic or biofilmcultures respectively, concentrations leading to the maximal phototoxicity. Inaddition, in the case of S. aureus, achange in the pattern of porphyrins synthesized can be observed upon increasingALAconcentrations, which is in line with a decrease of the photoinactivation rate.Inconclusion, the results obtained here indicate that BPI employing ALA is an effectivetreatment, alternative to antibiotics, for Gram positive bacteria in cultureand biofilms. In addition, the sensitivity of this treatment is directlyrelated to the pattern and amount of porphyrins synthesized. However,optimization of the BPI treatment in Gram negative bacteria is necessary.[1] Fotinos N,Convert M, Piffaretti JC, Gurny R, Lange N. Antimicrob Agents Chemother. 2008Apr;52(4):1366-73. doi: 10.1128/AAC.01372-07.[2]Harris F, Pierpoint L. Med Res Rev. 2012, 32(6):1292-1327.doi:10.1002/med.20251.[3] Szocs K,Gabor F, Csik G and Fidy J. B, J. Photochem. Photobiol., B, 1999, 50, 8?17.[4] Zhang QZ,Zhao KQ, Wu Y, Li XH, Yang C,Guo LM, Liu CH, Qu D, Zheng CQ. PLoS One. 2017, 12(3):e0174627