Atividade de porfirinas tetra-catiônicas frente a isolados e biofilmes de Moraxella spp. envolvidas na ceratoconjuntivite infecciosa bovina

Abstract

Infectious keratoconjunctivitis (IBK) is the most important eye disease in ruminant worldwide and, although it is not fatal, it can cause large losses in animal production. The primary agent of the disease is Moraxella bovis, but M. ovis and M. bovoculi are commonly isolated from animals with IBK. The therapeutic measures available for its control have limited success and generate high costs, justifying the importance of searching for new treatment alternatives. Photodynamic therapy is a method that uses photoactive molecules, such as porphyrins, that interact with light generating reactive oxygen species that cause cell death. Thus, this dissertation aimed to evaluate the antibacterial activity of tetra-cationic metalloporphyrins (H2TMeP and ZnTMeP) against isolates and reference strains of Moraxella spp., in vitro, in an ex vivo model and on sessile cells. In experiment 1 (chapter 1) the antibacterial activity of porphyrins against planktonic cells of Moraxella spp. in vitro and ex vivo. For in vitro analysis, each porphyrin (4.0 μM) was incubated with ~1x104 colony forming units (CFU) per mL of each Moraxella spp. (n=22). This solution (200 μL) was exposed to artificial light for 0; 2.5; 5.0 and 7.5 min, or kept in the dark (control) and then plated and incubated for 24 h for CFU quantification. All clinical isolates and reference strains of Moraxella spp. were completely inactivated by ZnTMeP porphyrin within 7.5 min of irradiation. H2TMeP did not show antibacterial activity against Moraxella spp., therefore, the other experiments were performed only with the porphyrin ZnTMeP. For the ex vivo assay, corneas excised from the eyeballs of slaughtered cattle were irrigated with a culture of Moraxella spp. (~1x104 CFU/mL) followed by addition of porphyrin. The pieces were irradiated by 0; 7.5 and 30 min, or kept in the dark. Corneal swabs were collected and seeded for CFU quantification. ZnTMeP promoted a significant reduction (p<0.05) in the concentration of Moraxella spp. after 30 min of irradiation. In the second experiment (chapter 2) the antibacterial activity of porphyrin ZnTMeP and oxytetracycline hydrochloride (OXY) against sessile cells of M. bovis, M. bovoculi and M. ovis (during and after the consolidation of the biofilm) was evaluated. ZnTMeP (4.0 μM) and OXY (20 μg/mL) were used alone and in association during and after biofilm consolidation. ZnTMeP had no effect in both phases. OXY was able to reduce biofilm formation of all strains. Against consolidated biofilm, OXY reduced the number of viable cells of M. bovoculi and M. ovis, but did not change the viability of M. bovis in consolidated biofilm. The combination of ZnTMeP and OXY against Moraxella spp. in biofilm showed no superior effect on cell destruction compared to the application of each compound alone. These results encourage the performance of future in vivo experiments using ZnTMeP in order to inactivate planktonic cells of Moraxella spp. causing IBK. On the other hand, we reiterate that the active search for new compounds and therapies that have an effective action on Moraxella spp. it's fundamental.