In Vitro Antibacterial Activity of Chitosan-phytochemical Conjugates against Fish Pathogenic Bacteria

Abstract

The object of this study was to evaluate the antibacterial activity of chitosanphytochemical conjugates against the fish pathogenic bacteria (Edwardseilla tarda, Vibrio harveyi and Photobacterium damselae), which were isolated from olive flounder, Japanese eel and black rockfish. The phytochemicals (cafeic acid, ferulicacid and sinapic acid) were grafted onto the chitosan backbone by chemical conjugation, resulting in chitosan-caffeic acid (CCA), chitosan-ferulic acid (CFA) and chitosan-sinapic acid (CSA) conjugates. The antibacterial activity of chitosan and its conjugates were determined by the minimum inhibitory concentration (MIC) assay. The chitosan and its conjugates exhibited a broad spectrum antibacterial activity with MIC values ranging from128μg/mL to 2,048 μg/mL against the fish pathogenic bacteria. The antibiotic susceptibility test showed a resistance of E. tarda to erythromycin with an inhibitory zone of 6 – 12 mm. V. harveyi showed resistance to ampicillin and erythromycin with an inhibitory zone of 12 – 14 mm. P. damselae also showed resistance to oxytetracycline and erythromycin with an inhibitory zone of 6 -14 mm. These results indicate that commercial antibiotics used in this study are ineffective to control infections related with these fish pathogenic bacteria therefore new approaches are needed. An alternative way to overcome the problems of antibiotic resistance is restoring the antibacterial activity of antibiotics losing their effectiveness in combination with natural derived substances. Thus, the synergistic antibacterial effect between chitosan-phytochemical conjugates and the commercial antibiotics using the checkerboard method against fish pathogenic bacteria was also evaluated in this study and interpreted as the fractional inhibitory concentration (FIC) index. The combination of the conjugates with erythromycin against E. tarda showed a synergistic effect of ˂0.5 FIC index. The combination of ampicillin with CCA and CFA against V. harveyi showed a synergistic effect of 0.14 and 0.156 ΣFIC index for RFHW 9L strain and 0.256 ΣFIC index for RFHW 1KA strain. However, the combination with erythromycin showed an additive effect of 1.062 for RFHW 9L strain. Synergistic effect was also recorded between CFA and oxytetracycline against P. damselae, FP 2261 strain but FP 4137 strain showed an additive effect of >0.5 FIC index whereas that of erythromycin showed synergistic effect of 0.312 with CSA for FP 4137 stain as well as the unmodified chitosan for FP 2261 strain. These results show that the presence of the chitosan conjugates (CCA, CFA and CSA) can restore the antibacterial activities of ampicillin, erythromycin and oxytetracycline against the fish pathogenic bacteria and can serve as an alternative combination therapeutic treatment for these antibiotic-resistant pathogenic bacteria.