Volume 4, Issue 2, June 2019, Page: 29-37
Isolation and Characterization of a Bacillus spp. Against Vibrio Parahaemolyticus from Shrimp Culture Ponds
Mengfan Peng, National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China
Ye Zhang, National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China; Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Aquaculture, Shanghai Ocean University, Shanghai, China; National Pathogen Collection for Aquatic Animals, Shanghai Ocean University, Shanghai, China
Zengfu Song, National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, China; Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Aquaculture, Shanghai Ocean University, Shanghai, China; National Pathogen Collection for Aquatic Animals, Shanghai Ocean University, Shanghai, China
Received: Feb. 23, 2019;       Accepted: May 6, 2019;       Published: Jun. 4, 2019
DOI: 10.11648/j.ijmb.20190402.11      View  29      Downloads  17
Pathogenic Vibrio species is one of the major factors affecting the development of aquaculture and the safety of seafood. Using the antagonistic activity of probiotics against pathogens offers a promising alternative to fish and shrimp aquaculture. In the present study, nine strains of bacteria were isolated from the shrimp culture ponds and screened for their directly antimicrobial activity against pathogenic Vibrio parahaemolyticus Vp1. Strain G, showing significant antimicrobial and non-hemolytic activity, was selected for further assays. The results of biochemical and 16S rRNA sequence analysis indicated that strain G highly related to Bacillus licheniformis. The present study also evaluated the in vitro and in vivo antagonistic effect of strain G against the Vibrios. Strain G exhibited significant inhibitory activity of Vibrio fluvialis FX-2, Vibrio parahaemolyticus K, and V. parahaemolyticus Vp1 in vitro. The inhibition diameter of strain G against Vibrio spp. ranged from 16 to 20 mm on Nutrient Agar. Under in vivo conditions, strain G was non-toxic to zebrafish and effectively protected zebrafish against V. parahaemolyticus Vp1. The non-toxicity of strain G showed final survival rate of 100% in zebrafish at inoculation densities up to 5.6×1010 CFU/ml at 96 h postchallenge. A significant reduction in mortality (P<0.001) was found by addition of 1.5×108 CFU/ml or 1.5×107 CFU/ml strain G in zebrafish against V. parahaemolyticus Vp1. In conclusion, the present study result reveals that strain G is a promising probiotic candidate and has potential applications for controlling pathogenic Vibrios in aquaculture practices.
Antagonist, Aquaculture, Bacillus. spp, Vibrio parahaemolyticus, Probiotic
To cite this article
Mengfan Peng, Ye Zhang, Zengfu Song, Isolation and Characterization of a Bacillus spp. Against Vibrio Parahaemolyticus from Shrimp Culture Ponds, International Journal of Microbiology and Biotechnology. Vol. 4, No. 2, 2019, pp. 29-37. doi: 10.11648/j.ijmb.20190402.11
Copyright © 2019 Authors retain the copyright of this article.
This article is an open access article distributed under the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Fernandez-Piquer J, Bowman JP, Ross T, Tamplin ML (2011) Predictive models for the effect of storage temperature on Vibrio parahaemolyticus viability and counts of total viable bacteria in Pacific oysters (Crassostreagigas). Appl Environ Microbiol. 77:8687–8695.
Xu HM, Rong YJ, Zhao MX, Song B, Chi ZM (2014) Antibacterial activity of the lipopetides produced by Bacillus amyloliquefaciens M1 against multidrug-resistant Vibrio spp. isolated from diseased marine animals. Appl Microbiol Biotechnol. 98:127–136.
Aranda CP, Valenzuela C, Barrientos J, Paredes J, Leal P, Maldonado M, Godoy FA, Osorio CG (2012) Bacteriostatic anti-Vibrio parahaemolyticus activity of Pseudoalteromonas sp. Strains DIT09, DIT44 and DIT46 isolated from Southern Chilean intertidal Perumytiluspurpuratus. World J Microbiol Biotechnol. 28:2365–2374.
Touraki M, Karamanlidou G, Karavida P, Chrysi K (2012) Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarumbioencapsulated in Artemianauplii against vibriosis in European sea bass larvae (Dicentrarchuslabrax, L.). World J Microbiol Biotechnol. 28:2425–2433.
Zarei M, Borujeni MP, Jamnejad A, Khezrzadeh M (2012) Seasonal prevalence of Vibrio species in retail shrimps with an emphasis on Vibrio parahaemolyticus. Food Control. 25:107–109.
Yu WT, Jong KJ, Lin YR, Tsai SE, Tey YH, Wong HC (2013) Prevalence of Vibrio parahaemolyticusin oyster and clam culturing environments in Taiwan. Int J Food Microbiol. 160:185–192.
Wu YN, Wen J, Ma Y, Ma XC, Chen Y (2014) Epidemiology of foodborne disease outbreaks caused by Vibrio parahaemolyticus, China, 2003–2008. Food Control. 46:197–202.
Nithya C, Aravindraja C, Pandian SK (2010) Bacillus pumilus of Palk Bay origin inhibits quorum-sensing-mediated virulence factors in Gram-negative bacteria. Res Microbiol. 161:293–304.
Sinhaseni P, Limpoka M, Samatiwat O (2000) Human health aspects of the use of chemicals in aquaculture, with special emphasis on food safety and regulations. In: Arthur JR, Lavilla-Pitogo CR, Subasinghe RP (eds) Use of chemicals in aquaculture in Asia. SEAFDEC, Iloilo, pp 55–60.
Cabello FC (2006) Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol. 8:1137–1144.
Esposito A, Fabrizi L, Lucchetti D, Marvasi L, Coni E, Guandalini E (2007) Orally administered erythromycin in rainbow trout (Oncorhynchus mykiss): residues in edible tissues and withdrawal time. Antimicrob Agents Chemother. 51:1043–1047.
Bacon CW, Hinton DM, Mitchell TR, Snook ME, Olubajo B (2012) Characterization of endophytic strains of Bacillus mojavensis and their production of surfactin isomers. Biol Control. 62:1–9.
Chahad OB, El Bour M, Calo-Mata P, Boudabous A, Barros Vela `zquez J (2012) Discovery of novel biopreservation agents with inhibitory effects on growth of food-borne pathogens and their application to seafood products. Res Microbiol. 163:44–54.
Makino K, Oshima K, Kurokawa K (2003) Genome sequence of Vibrio parahaemolyticus: a pathogenic mechanism distinct from that of V cholera. The Lancet. 9359: 743-749.
Planas M, Pe ´rez-Lorenzo M, Hjelm M, Gram L, Fiksdal IU, Bergh Ø, Pintado J. (2006) Probiotic effect in vivo of Roseobacter strain 27-4 against Vibrio (Listonella) anguillarum infections in turbot (Scophthalmusmaximus L.) larvae. Aquaculture. 255:323–333.
Zokaeifar H, Balca ´zar JL, Saad CR, Kamarudin MS, Sijam K, Arshad A, Nejat N (2012) Effects of Bacillus subtilis on the growth performance, digestive enzymes, immune gene expression and disease resistance of white shrimp, Litopenaeusvannamei. Fish Shellfish Immunol. 33:683–689.
Robertson PAW, Dowd CO, Burrells C, Williams P, Austin B (2000) Use of Carnobacterium sp. as a probiotic for Atlantic salmon_Salmosalar L. and rainbow trout, Oncorhynchusmykiss, Walbaum. Aquaculture. 185:235–243.
Villamil L, Figueras A, Planas M, Novoa B (2003) Control of Vibrio alginolyticus in Artemia culture by treatment with bacterial robiotics. Aquaculture. 219:43–56.
Duc, H. L., Hong, H. A., Barbosa, T. M., Henriques, A. O., & Cutting, S. M. (2004). Characterization of Bacillus probiotics available for human use. Appl Environ Microbiol, 70(4), 2161-2171.
Liu, X. F, Li, Y, Li, J. R, Cai, L. Y, Li, X. X, & Chen, J. R. (2015). Isolation and characterisation of Bacillus spp. antagonistic to Vibrio parahaemolyticus for use as probiotics in aquaculture. World J Microb Biot, 31(5), 795-803.
Kamiso HN, Isnansetyo A, Triyanto, Istiqomah I, Murdjani M (2005) Isolation, identification and characterization of pathogenic Vibrio spp. causative agents of vibriosis in grouper at brackishwater Aquaculture Development Centre, Situbondo. J Fish Sci VII: 80–94.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876–4882.
Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenic trees. Mol Biol Evol. 4:406–425.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 10:2731–2739.
M. Westerfield (2000) The Zebrafish Book: a Guide for the Laboratory Use of Zebrafish (Danio rerio), fourth ed., Oregon, OR.
Xie FJ, Chen HZ, Zhao YY (1992) Water quality-Determination of the acute toxiclty of substance to freshwater fish (Brachydanio rerio Hamilton-Buchanan). National Standard of the People’s Republic of China, GB/T13267-91.
Reed LJ and Munench H (1938) A simple method of estimating fifty percent endpoints. Am J Hyg. 27: 493–497.
Ravi A. V, Musthafa K. S, Jegathammbal G, Kathiresan K, Pandian S. K (2007) Screening and evaluation of probiotics as a biocontrol agent against pathogenic Vibrios in marine aquaculture. Lett Appl Microbiol. 45:219–223.
Aunpad R, Na-Bangchang K (2007) Pumilicin 4, a novel bacteriocin with anti-MRSA and anti-VRE activity produced by newly isolated bacteria Bacillus pumilus strain WAPB4. Curr Micobiol. 55:308–313.
Aunpad R, Panbangred W (2012) Evidence for two putative holin-like peptides encoding genes of Bacillus pumilus strain WAPB4. Curr Microbiol. 64:343–348.
Li GG, Liu BS, Shang YJ, Yu ZQ, Zhang RJ (2012) Novel activity evaluation and subsequent partial purification of antimicrobial peptides produced by Bacillus subtilis LFB112. Ann Microbiol. 62:667–674.
Sugita H, Ohta K, Kuruma A, Sagesaka T (2008) An antibacterial effect of Lactococcus lactis isolated from the intestinal tract of the Amur catfish, Silurus asotus Linnaeus. Aquac Res. 38:1002–1004.
Kennedy, S. B, Tucker, J. W, Neidig, C. L, Vermeer, G. K, Cooper, V. R, Jarrell, J. L. and Sennett, D. G. (1998) Bacterial management strategies for stock enhancement of warm water marine fish: a case study with common snook (Centropomus undecimalis). Bulletin of Marine Sciences. 62, 573–588.
Isnansetyo, A, Istiqomah, I, Muhtadi, Sinansari, S, Hernawan, R. K, & Triyanto. (2009). A potential bacterial biocontrol agent, strain S2V2 against pathogenic marinevibrioin aquaculture. World J Microb Biot, 25(6), 1103-1113.
Vinoj, G, Vaseeharan, B, Thomas, S, Spiers, A. J, & Shanthi, S. (2014). Quorum-quenching activity of the AHL-lactonase from Bacillus licheniformis DAHB1 inhibits Vibrio biofilm formation in vitro and reduces shrimp intestinal colonisation and mortality. Mar Biotechnology, 16(6), 707.
Xu D, Wang Y, Sun L. Inhibitory activity of a novel antibacterial peptide AMPNT-6 from Bacillus subtilis against Vibrio parahaemolyticus in shrimp. Food Control, 2013, 30(1):58-61.
Chan, W. C, Bycroft, B. W, Leyland, M. L, Lian, L.-Y, and Roberts, G. C. K (1993) A novel post-translational modification of the peptide antibiotic subtilin: isolation and characterization of a natural variant from Bacillus subtilis A. T. C. C. 6633. Biochem. J. 291:23−27.
Phelan, R. W, Barret, M, Cotter, P. D, O’Connor, P. M, Chen, R., Morrissey, J. P, Dobson, A. D. W, O’Gara, F, and Barbosa, T. M. (2013) Subtilomycin: a new lantibiotic from Bacillus subtilis strain MMA7 isolated from the marine sponge Haliclona simulans. Mar. Drugs. 11:1878−1898.
Wang, J, Zhang, L, Teng, K, Sun, S, Sun, Z, and Zhong, J (2014) Cerecidins, novel lantibiotics from Bacillus cereus with potent antimicrobial activity. Appl. Environ. Microbiol. 80: 2633-2643.
Lawton, E. M, Cotter, P. D, Hill, C, and Ross, R. P (2007) Identification of a novel two-peptide lantibiotic, haloduracin, produced by the alkaliphile Bacillus halodurans C-125. FEMS Microbiol. Lett. 267: 64−71.
Barbosa, J, Caetano, T, and Mendo, S. J (2015) Class I and Class II Lanthipeptides Produced by Bacillus spp. Nat. Prod. 78: 2850–2866.
Browse journals by subject