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

CERVINI BÖHM G, DI VENOSA G, SAENZ D, CALVO G, VALLECORSA P, ORLANDO C, BUZZOLA F, CASAS A.

Villa Carlos Paz

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

Bacterial Photo-Inactivation (BPI) is an antimicrobial treatment that uses a photosensitizing molecule (FS), which is then irradiated with visible light generating reactive oxygen species and the consequent damage to the microorganisms. On the other hand, antibiotic-resistant infections represent an important clinical problem, most due to the formation of biofilms. Consequently, the need arises for new alternative therapies to the use of antibiotics. In this work we evaluated δ-aminolevulinic acid (ALA) action as a precursor of photosensitive porphyrins [1-2]] in planktonic cultures and biofilms of Staphylococcus aureus ATCC 25923 [4], Staphylococcus epidermidis, Escherichia coli [3] and Pseudomonas aeruginosa ATCC 27853. We worked with 24-h biofilms grown over polystyrene plates and planktonic cultures with an OD600 0.7. After incubation in the dark at different doses of ALA, we irradiated the bacteria with a non-coherent white light source. The determination of the effectiveness of the treatments was performed counting colony forming units (CFU/ml). In addition, the amount and type of porphyrins synthesized in the different treatments were determined fluorimetrically and by HPLC. The BPI with concentrations between 1 and 2 mM of ALA, reduces 6 logs the viability of the planktonic 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 sensitivity to ALA-BPI in planktonic culture, reducing its viability by 5 logs.Porphyrins from Gram positive bacteria peaks at concentrations of 1 mM or 2 mM for planktonic or biofilm cultures respectively, concentrations leading to the maximal phototoxicity. In addition, in the case of S. aureus, a change in the pattern of porphyrins synthesized can be observed upon increasing ALA concentrations, which is in line with a decrease of the photoinactivation rate. In conclusion, the results obtained here indicate that BPI employing ALA is an effective treatment, alternative to antibiotics, for Gram positive bacteria in culture and biofilms. In addition, the sensitivity of this treatment is directly related 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. 2008 Apr;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