Atividade antibacteriana in vitro de porfirinas tetracatiônicas frente às bactérias multirresistentes isoladas de infecções tegumentares de animais

Abstract

Otitis, pyodermitis and contaminated wounds are common pathologies in company animals and are frequently infected and/or coinfected with bacteria resistant to conventional antibiotics. The treatment of those lesions is a challenge at veterinary clinics; therefore, new therapeutic options are needed to deal with those pathogens. In this context, the photodynamic therapy (PDT) emerges as an alternative once it has been used efficiently on different microorganisms’ inactivation. The PDT consists of the use of a photosensitizer that, when irradiated with light, react with molecular oxygen, generating reactive oxygen species (ROS), which are toxic to bacterial cell, promoting its inactivation. The tetracationic porphyrin 3H2TMeP and 4-H2TMeP are photosensitizer that stand out for being water soluble, positively charged (favoring its biding with the bacterial cell, negatively charged), do not stain the animal skin, and are commercially available. This dissertation aimed to evaluate the PDT in vitro efficacy utilizing tetracationic porphyrins against to multidrug resistant bacteria isolated from superficial lesions of dogs, cats, and horses, both in monoculture and in bacteria associations (polyculture). To evaluate the antibacterial activity front to monocultures, was used isolates of Staphylococcus pseudointermedius, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens with multidrug resistant profile. Solutions of those bacteria were incubated with each one of porphyrins at a non-cytotoxic concentration for eukaryotic cells (40µM), and exposed to light for 0, 15 and 30 minutes for Gram-positive, and 0, 30, 60 and 90 minutes for Gram-negative bacteria, or maintained at dark (control). Then, the solution was plated in culture media, which were incubated at 37ºC for 24 hours, when was realized the plate counting of colonies former units (CFU) and compared to the control plates. The porphyrin 4H2TMeP was able to totally inactivate S. pseudointermedius e P. aeruginosa isolates and significantly reduced the concentration of E. coli, K. pneumoniae, e S. marcescens. So, it was chosen for polyculture assays and evaluation of atomic force assays. Polycultures of multidrug resistant of S. pseudointermedius and P. aeruginosa; E. coli and Proteus mirabilis; P. aeruginosa and P. mirabilis; S. pseudointermedius and E. coli; Staphylococcus pseudointermedius and P. mirabilis; was incubated with 40µM of 4-H2TMeP porphyrin and irradiated with the same conditions as in monocultures, for 0, 30 and 90 minutes. There was a significant reduction of bacteria concentration, with totally inactivation of E. coli and S. pseudointermedius, P. aeruginosa and S. pseudointermedius, P. aeruginosa and Proteus mirabilis associations. PDT effect at adhesion force on the bacteria wall, indicated weakness at Gram-positive bacteria, and a hardness at Gram-negative bacteria related to control. Both alterations may cause difficult the bacteria survive and multiplication. The porphyrin 4H 2 TMeP i n vitro results demonstrated that the PDT utilizing the was effective against multidrug resistant bacteria, both in pure cultures (monocultures) as in polycultures and validate this therapy for animal´s t permit to plan future studies to reatment of integumentary lesions.