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Background: Ciprofloxacin (Cip) is the most commonly used quinolone in clinical practice; however largescale use has favored the increase of multiresistant pathogenic microorganisms. Antimicrobial peptides (AMPs) appear to be a promising alternative in potentiating these conventional drugs. Objective: The aim of this study was to evaluate the effect of the peptide Lys-[Trp6]hy-a1 (lys-a1) on the antimicrobial and antibiofilm activity of ciprofloxacin against clinically relevant gram-negative bacteria. Methods: The antimicrobial effects of Cip and lys-a1 were assessed by determining the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The synergistic action of Cip and lys-a1 was determined by checkerboard assay. The time-kill curve was constructed for the Cip/lys-a1 combination against Pseudomonas aeruginosa ATCC 9027. The antibiofilm activity of this combination was analyzed by crystal violet, colony-forming unit count and atomic force microscopy (AFM). Results: The data demonstrated that lys-a1 was able to inhibit planktonic growth of strains of P. aeruginosa and Klebsiella pneumoniae both at 125 µg/mL. The fractional inhibitory concentration index (FICi) showed a synergistic effect between Cip and lys-a1 against P. aeruginosa, decreasing the MICs of the individual antimicrobial agents by 4- and 8-fold, respectively. This effect was also observed for the death kinetics and antibiofilm activity. Analysis of the early biofilms (6 h) as well as isolated cells by AFM images evidenced the cell perturbation caused by Cip/lys-a1 treatment. Conclusion: These data suggest that lys-a1 has biotechnological potential as a therapeutic tool for the treatment of infections caused by clinically relevant microorganisms, especially P. aeruginosa.

Resumo: Duas formas de peroxidase (POX), denominadas PdPI e PdPII, foram parcialmente purificadas de arilos de Pithecellobium dulce, utilizando precipitação com sulfato de amônio e cromatografia de troca iônica. O zimograma das proteínas obtidas após precipitação com sulfato de amônio na faixa de 60 a 90% de saturação (fração F6090), mostrou duas bandas com atividade peroxidásica. Na coluna de celulose DE-52, a atividade peroxidásica foi detectada nos picos não adsorvido (PdPI) e adsorvido (PdPII) eluído com NaCl 0,2 mol L -1 , sugerindo que a PdPI (~114 KDa) e a PdPII (~77 KDa) são proteínas básicas e ácidas, respectivamente, sendo a PdPI estável a 80 ºC. Este é o primeiro relato de purificação parcial de POX obtida de arilos de P. dulce, indicando que esta espécie pode ser uma nova fonte de enzimas para utilização em biossensores e outras aplicações biotecnológicas. Palavras-chave:Enzimas; Atividade peroxidásica; P. dulce; Zimograma. AbstractTwo peroxidase (POX) forms, named PdPI and PdPII, were partially purified from the Pithecellobium dulce aril, using ammonium sulfate precipitation and anion exchange chromatography. Zymography of the proteins obtained after precipitation in the range of 60-90% ammonium sulfate (F6090 fraction) showed two bands with peroxidase activity. In DE-52 cellulose column, the peroxidase activity was detected in unadsorbed peak (PdPI) and adsorbed peak eluted with 0.2 mol L -1 NaCl (PdPII) suggesting that PdPI (~114 KDa) and PdPII (~77 KDa) are basic and acidic proteins, respectively, being PdPI stable at 80 ºC. This is the first report of partial purification of POX from P. dulce aril, indicating that this species may be a new source of enzymes for use in biosensors and other biotechnological applications.