Isolation and inhibitory effect of anti-Vibrio substances from Pseudoalteromonas sp. A1-J11 isolated from the coastal sea water of Kagoshima Bay Carmelo S del CASTILLO, 1 Md Iftekharul WAHID, 1 Takeshi YOSHIKAWA 2 AND Taizo SAKATA 2 * 1 The United Graduate School of Agricultural Sciences, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, and 2 Laboratory of Microbiology, Faculty of Fisheries, Kagoshima University, 4-50-20 Shimoarata, Kagoshima 890-0056, Japan ABSTRACT: Among several marine antagonistic bacteria isolated from the sea water of Kagoshima Bay, Pseudoalteromonas sp. A1-J11 was found to produce anti-Vibrio substances. The anti-Vibrio substances were extracted from the culture supernatant of the strain with chloroform and isolated using reverse-phase (Cosmosil 75C18-OPN) column chromatography followed by high-pressure liquid chromatography (Mightysil RP-18 GP Aqua). Purified substances, designated as AVS-03a, c and d showed similar ultraviolet absorption spectra with lmax at 215, 235, 315 and 327 nm in methanol. AVS-03d, the major anti-Vibrio substance, was thermostable up to 100°C and pH stable over a pH range higher than 4.0, and also showed strong inhibitory activities, specifically against Vibrio harveyi strains. KEY WORDS: anti-Vibrio substance, high-pressure liquid chromatography, Pseudoalteromo- nas, ultraviolet absorption spectrum. INTRODUCTION Members of the genus Vibrio have been implicated as major causative agents in diseases in fish, mollusks and crustaceans, causing mass mortali- ties worldwide. 1–6 One technique to alleviate this condition is the use of microorganisms as biocon- trol agents, either by antagonistic exclusion or by direct inhibition. This technique has been gaining popularity recently with an increase in the use of probiotic bacteria. 7,8 For this purpose, several bac- teria that may be potential biocontrol agents have been identified, and these bacteria are a potential source for new antibacterial substances. 9–14 Recently, several biologically active substances have been isolated from marine bacteria. 15–22 Taking these into consideration, and trying to look for potential agents that can control the growth of fish pathogenic bacteria, including Vibrio harveyi, several antagonistic strains were isolated by the authors in Kagoshima Prefecture, Japan, in 2001. 23 Some of these strains were studied for bacteriolytic activity resulting from the production of pro- teolytic enzymes. 23 In addition, the same strains were found to produce low molecular weight com- pounds that inhibit the growth of Vibrio strains. This study reports the isolation and character- ization of anti-Vibrio compounds from strain A1-J11 isolated from the coastal sea water of Kagoshima Bay, Japan. MATERIALS AND METHODS Bacterial strains and media Various antagonistic bacteria were previously isolated from several sources in and around Kagoshima Bay, Japan. 23 Vibrio harveyi strain ATCC 14126 and ATCC 35084 and Vibrio alginolyticus ATCC 17749 were obtained from the American Type Culture Collection (ATCC), Manassas, VA, USA. Vibrio parahaemolyticus IFO 12711 (NBRC 12711) and Micrococcus luteus IFO 12708 (present *Corresponding author: Tel: 81-099-286-4190. Fax: 81-099-286-4015. Email: sakata@fish.kagoshima-u.ac.jp Received 28 May 2007. Accepted 10 August 2007. FISHERIES SCIENCE 2008; 74: 174–179 © 2008 Japanese Society of Fisheries Science doi:10.1111/j.1444-2906.2007.01507.x