Atividade antibacteriana, antibiofilme e o efeito combinado a antibióticos de complexos de rutênio do tipo cis-[RuCl2(dppb)(NN-R)]2+ sobre bactérias resistentes e o efeito destes combinados aos antibióticos ampilicina e tetraciclina

Abstract

Antimicrobial resistance is a critical global public health problem, which is increasing the number of deaths caused by multiresistant bacteria. Bacterial infections are associated with the ability of these microorganisms to form biofilms, that is essential for colonization of the most diverse environments, in addition to conferring high levels of resistance. In the face of this problem, the discovery of new classes of antimicrobial molecules, constitutes a target to be reached, to overcome the current problems of resistance to antibiotics. Currently, studies based on ruthenium (II) complexes have shown the antimicrobial activity of this element when associated with specific ligands, thus representing an alternative for microbial control. The objective of this study was to assess the antibacterial activity, antibiofilme activity and the antibiotic-associated effect of three ruthenium complexes of the type cis-[RuCl2(dppb)(NNR)]2+ called RuNN, RuNN-F and RuNN-Br, against the strains Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 700698, Staphylococcus epidermidis ATCC 12228, Staphylococcus epidermidis ATCC 35984, Pseudomonas aeruginosa ATCC 10145 e Escherichia coli ATCC 11303. To determine the antibacterial activity, the compounds were diluted in concentrations ranging from 500 to 7,8 μg/ml and distributed into 96-well plates of polystyrene and flat bottom with each bacterial suspension adjusted (1 x 106 CFU/ml) in TSB medium and then incubated for 24 hours at 37 ° C. The susceptibility of the ruthenium complexes was evaluated by the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). CIM values were combined with antibiotics ampicillin and tetracycline and their effect was evaluated using the checkerboard technique, whereas the MBC values were used to determine the time of death of the bacteria treated with these complexes. The antibiofilm activity, evaluated by biomass quantification, metabolic activity and cell viability were evaluated by staining (CV), XTT reduction assay and enumeration of viable biofilm cells (CFU). The results showed that the three complexes exhibited antimicrobial activity against Gram-positive bacteria with MIC values ranging from 7.8 μg/ml to 62.5 μg/ml, and CBM from 31.25 μg/ml to 125 μg/ml , but not against Gram-negative bacteria. The synergistic effect against Staphylococcus spp., was only observed when these complexes were combined with ampicillin, exhibiting ICIF values of 0.187, which did not occur when associated with tetracylin. The time of death studies demonstrated bactericidal effect in the first hours of contact, both for S. aureus strains and S. epidermidis strains, with a maximum death time of 24 hours. In relation to the antibiofilm activity, the three complexes were able to reduce the biomass by more than 30% to the concentration of 7.8 μg/ml and almost 100% to the concentration of 500 μg/mL. The same result was observed when the metabolic activity was evaluated through reduction XTT, observing a low metabolic activity at concentrations above 62.5 μg/ml for all strains of Staphylococcus spp. In addition, the number of viable cells of the biofilm was reduced between 1.2 and 4.9 logs. In conclusion, the three cis- [RuCl2 (dppb)(NNR]2+ ruthenium complexes showed activity against Gram-positive bacteria, either alone or in combination with antibiotics. These findings are fundamental for the implantation of new therapies designed to treatment bacterial infections caused by Staphylococcus spp.