Introduction
Piscirickettsiosis is a severe disease that nowadays causes major economic losses in the aquaculture industry, especially in Chilean salmon production. Piscirickettsia salmonis is the etiological agent responsible of this disease. Antibiotics have been extensively used against infections produced by this pathogen, what has led to the appearance of resistant phenotypes to antibiotic treatments. In addition, existing vaccines show low efficacy.
Natural products, especially from plants, have been investigated for their therapeutic effects on several fish diseases (Rattanachaikusopon et al, 2009) as an alternative to antibiotics. Aliium species, such as garlic and onion, are a promising tool for this, as they have been widely used as medicinal plants because of the antimicrobials properties of their organosulfur compounds, including alk(en)yl thiosulfinates and thiosulfonates (Kyung, 2012). This study aims to evaluate the antimicrobial effect of an organosulfur compound from Allium plants against Piscirickettsia salmonis.
Materials & Methods
The organosulfur compound was obtained from DOMCA S.A.U. (Granada, Spain) in a commercial and standardized product (Aquagarlic).
P. salmonis type strain was obtained from LGC Standars-ATCC culture collection (Piscirickettsia salmonis ATCC VR-1361). P. salmonis was grown in monolayers on Chinook salmon embryo (CHSE-214) cell line. Cells were cultured with Earle`s Minimal Essential Medium supplemented with 10% of foetal bovine serum, 2mM of glutamine and 1% of non-essential amino acids. Cells were grown in dark and at dark, with a 5% CO2 atmosphere.
For antimicrobial activity evaluation, Minimal Inhibitory Concentration (MIC) was assessed according to the recommendation of the guide M49-A (CLSI 2006), using the AUSTRAL-SRS broth culture medium (Yañez et al., 2012) for P. salmonis growth.
MTT essay was performed to determine the viability of CHSE-214 cells exposed to the organosulfur compound, according to Mossman (1983). Cells were seeded in 96-well plates and exposed to the antimicrobial substance at different concentrations (0.1 to 100 mg ml-1) for 24 h.
Results
Our results points out that the organosulfur compound has a significant antimicrobial activity against P. salmonis with an average value of MIC of 20 mg l-1. This data show that the product tested is active against the target bacteria, although new studies will be necessary to determine the MICs against wild isolates of P. salmonis, in order to set the adequate dose of Aquagarlic in fed.
Results also show that the organosulfur compound did not have cytotoxicity on CHSE-214 cell line for concentrations from 0.1 up to 100 mg ml-1, suggesting the lack of in vivo toxicity and its safe use in fish nutrition.
Conclusion
An organosulfur compound from Allium spp. was found to be an effective antimicrobial product against P. salmonis without any cytotoxicity effect on CHSE-214 cell line. Therefore, this compound might be a safe, promising and effective solution to control this pathogen replacing antibiotics on Salmon Industry.
References
Clinical and Laboratory Standards Institute. 2006. Methods for Broth Dilution Susceptibility Testing of bacteria isolated from aquatic animals; approved guideline. CLSI document M49-A. 1-56238-612-3j, Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, USA.
Kyung KH. 2012. Antimicrobial properties of allium species. Curr Opin Biotechnol 23:142-147.
Mossman, T. 1983. Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. Journal of Immunological Methods 65: 55-63.
Rattanachaijunsopon P. and Phumkhachorn P. 2009. Prohylactic effect of Andrographis paniculata extracts against Streptococcus agalactiae infection in Nile tilapia (Oreochromis niloticus). J. Biosc. Bioeng., 107:579-582.
Yañez A.J., Valenzuela K., Silva H., Retamales J., Romero A., Enriquez R., Figueroa J., Claude A., González J., Avendaño-Herrera R. and Carcamo J.G. 2012. Broth medium for the successful culture of the fish pathogen Piscirickettsia salmonis. Diseases of Aquatic Organisms 97:197-205.