Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Purication and characterization of antibacterial substances produced by a marine bacterium Pseudoalteromonas haloplanktis strain
G. Hayashida-Soiza1, A. Uchida2, N. Mori3, Y. Kuwahara4 and Y. Ishida5
1 2 3 4 5 Departamento de Ciencias Qumicas y Farmaceuticas, Universidad Catolica del Norte, Antofagasta, Chile Department of Applied Biosciences, Faculty of Agriculture, Kyoto University, Sakyo Kyoto, Japan Department of Applied Lifesciences, Faculty of Agriculture, Kyoto University, Kyoto, Japan Department of Bioenvironmental Science, Kyotogakuen University, Kameoka, Kyoto, Japan Department of Marine Biotechnology, Faculty of Engineering, Fukuyama University, Fukuyama, Hiroshima, Japan

Keywords 2-methylbutyric acid, antimicrobial activity, isovaleric acid, marine bacteria, Pseudoalteromonas haloplanktis. Correspondence G. Hayashida-Soiza, Departamento de Ciencias Qumicas y Farmaceuticas, Universidad Catolica del Norte, Avenida Angamos 0610, Antofagasta, Chile. E-mail: ghayashi@ucn.cl

Abstract Aims: To purify and characterize compounds with antimicrobial activity from Pseudoalteromonas haloplanktis inhibition (INH) strain. Methods and Results: The P. haloplanktis isolated from a scallop hatchery was used to analyse antibacterial activities. Crude extracts were obtained with ethyl acetate of the cultured broth, after separation of bacterial cells, and assays against six strains of marine bacteria and nine clinically important pathogenic bacteria. The active compounds were puried from ethyl acetate extracts, by a combination of SiO2 column and thin layer chromatography. Two active fractions were isolated, and chemical structures of two products from the major one were unambiguously identied as isovaleric acid (3-methylbutanoic acid) and 2-methylbutyric acid (2-methylbutanoic acid), by comparing their mass spectra and 1H- and 13C-nuclear magnetic resonance spectra to those of authentic compounds. Conclusions: In the antibacterial activity of P. haloplanktis INH strain, extra cell compounds are involucred, mainly isovaleric and 2-methylbutyric acids. Signicance and Impact of the Study: Production of antimicrobial compounds by marine micro-organisms has been widely reported; however, the efforts not always are conducted to purication and applications of these active compounds. This study is a signicant contribution to the knowledge of compounds unique from marine bacteria as potential sources of new drugs in the pharmacological industry.

2007 1617: received 5 October 2007, revised 23 April 2008 and accepted 7 May 2008
doi:10.1111/j.1365-2672.2008.03878.x

Introduction Because so many toxic molecules have been known from marine organisms, it has become evident that ocean is a likely source of pharmaceuticals, and interesting biochemicals useful to biotechnology and its researches (Colwell 2002; Rajeev and Xu 2004; Faulkner and Fenical 2005). In fact, marine organisms, such as fungi, microalgae and bacteria, are recognized as an important resources of bioactive compounds (Manzi and Mayz 2003; Oranday et al. 2004; Fortman and Sherman 2005).
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In medical and pharmacological areas, the signicance of the bioactive nature in metabolites is supported not only on direct pharmacological and chemotherapeutic effects but also on the potential source of new drug development by modifying from their molecular structures. Antibiotics are receiving special attention among natural bioactive compounds not only because of their economic signicance and their effectiveness but also of increasing incidents of drug-resistant causing difculties in the pharmacological eld (Finch 1987; Leiva et al. 2004; Garateix 2005). Therefore, continuous search of

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bioactive compounds with microbial growth inhibition capacity should be promoted to develop new antibiotic drugs. Also, micro-organisms with antimicrobial ability could be potentially used as probiotics in aquaculture (Imada et al. 1985; Nogami and Maeda 1992; Olsson et al. 1992; Riquelme et al. 1996 and Abraham 2004). The antimicrobial activity among marine bacteria has been known for a long time (Gauthier 1976; Gauthier and Breittmayer 1979; Sakata et al. 1982; Oclarit et al. 1994). Marine Alteromonas-like bacteria are known as rich source of active antimicrobial metabolites. Examples of such metabolites are either heat labile substances, macromolecules of protein nature or brominated compounds (Wratten et al. 1977; Sakata et al. 1982; Imada et al. 1985; Nair and Simidu 1987; Mc Carthy et al. 1994; Longeon et al. 2004). However, the molecular structure of the active compounds is not always elucidated in several studies evidencing that bacteria contains microbial growth inhibitor (Sugahara et al. 1992; Manage et al. 2000; Abra ham 2004; Avendano-Herrera et al. 2005). A bacterial strain previously isolated from a commercial Argopecten purpuratus scallop hatchery is one of such species showing antimicrobial activity, and it was identied by 16SRNA gene sequence as Pseudoalteromonas haloplanktis inhibition (INH) strain (Riquelme et al. 1996). In this study, purication, isolation and characterization of the active substances from this Pseudoalteromonas haloplanktis INH strain are carried out to identify those antibiotic compounds. Also, its growth inhibition spectrum was investigated. Materials and methods Bacterial culture Pseudoalteromonas haloplanktis INH strain kept in our laboratory as a slant culture of Marine Trypticase Soya Agar (MTSA) medium in a test tube, was transferred to a Erlenmeyer asks (300 ml) containing Marine Trypticase Soya Broth (MTSB) medium (100 ml), and incubated at 20C for 48 h to prepare subculture. The subculture (20 ml) was transferred to an Erlenmeyer asks (2 l) containing 1 l of MTSB medium, and incubated at 20C for 48 h. Release of antibiotic substances to the culture medium Forth-eight hours incubated cultures were centrifuged at 8000 g for 30 min, then, pellet and supernatant were recovered separately. To remove remaining cells, the supernatant was ltered through 02-lm lters; meanwhile, the pellet was suspended in 10 mmol l)1 Tris buffer. Antibiotic activity of the cell-free supernatant and

pellet was evaluated by the disc-diffusion method (Uchida et al. 1988), against Vibrio alginolyticus VAR strain, previously isolated from a commercial scallop hatchery (Riquelme et al. 1995). Sterile lter paper discs (6 mm in diameter) were charged with 40 ml of cell-free supernatant of the P. haloplanktis cultures and others were charged with 40 ml of pellet of P. haloplanktis cultures. All charged discs were placed on the seeded MTSA medium with test culture of V. alginolyticus, and incubated at 20C for 48 h. Paper discs impregnated with cell-free culture medium were used as control. Diameters of the growth inhibition areas were measured. Extraction of active compounds Active substances were extracted with ethyl acetate as following, Cell-free supernatant of the cultured broth was partitioned with equal volume of ethyl acetate CH3COOCH2CH3. The extract was concentrated using a rotary evaporator. The resulting crude extract was suspended in chloroform and stored at 5C. Antibacterial spectrum and minimal inhibitory concentration The minimal inhibitory concentration (MIC) was determined by the discs-diffusion method against Vibrio ordalii strain, using lter paper discs (6 mm in diameter). They were impregnated with the following concentration of CHCl3 solution (40 ml) of the crude extract; 01, 1, 5, 10 and 25 mg ml)1. Paper discs impregnated with chloroform and dried for 3 h were used as control. Antibacterial spectrum of the crude extract at 10 mg ml)1 concentration was examined by the method described above against the following marine and clinical pathogenic bacteria; V. ordalii ATCC 33509, V. alginolyticus ATCC 17749, Vibrio anguillarum IFO 13266, V. anguillarum (VAR strain), Pseudomonas uorescens IFO 3903, Aeromonas hydrophila IFO 13287, Escherichia coli IFO 3366, Salmonella thyphimunium LT-2, Morganella morganii ATCC 25830, Schewanella putrefaciens IFO 1349, Achromobacter sp. IFO 13495, Bacillus subtilis ATCC 6051, Staphylococcus aureus IFO 13276 and Micrococcus luteus IFO 3333. Purication of active compounds The INH crude extract (9 g) obtained from 10 l of culture was subjected to four steps of column chromatographies packed with silica Wakogel C-200 (Wako Pure Chemical Industry) and or orisil (100200 mesh; Floridiin Co.). Solvents such as benzene (C6H6), chloroform (CHCl3), methanol (CH3OH) and ethyl acetate (CH3COOCH2CH3) were used as eluents. In addition, plates of
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silica gel (thickness 025 mm; Kieselgel Merck 60F 254, Darmstadt, Germany) were used for thin layer chromatography (TLC) to examine the components of each column fraction by visualizing under UV light. Identication of active compounds H-nuclear magnetic resonance (NMR) and 13C-NMR spectra were recorded by an AC300 spectrometer (300 MHz; Bruker) in CDCl3 CD3OD with TMS as internal standard. Gas liquid chromatography (GLC) was performed by a Chromatograph (Hewlett Packard 5890A Plus), equipped with a ame ionization detector at a splitless mode, using a HP-5 chemical bonded capillary column (032 mm i.d. 25 m). The carrier gas was helium at 123 ml min)1 ow rate, and the oven temperature was programmed from 60C (2 min) to 290C at a rate of 10C min)1. Injection and detector temperatures were 200C and 300C respectively. Chromatograms were processed by a Hewlett Packard Integrator model 3396 series II. Mass spectra were obtained by a Hewlett Packard 5989B GC MS spectrometer operated at 70 eV and a low-resolution mode, using the same capillary column and at the same condition as stated above. Determination of isovaleric acid production in the culture The P. haloplanktis INH strain was inoculated in Erlenmeyer asks (2 l) containing 1 l of MTSB medium and shake-incubated at 20C. Cell-free MTSB medium was used as control. Ethyl acetate extracts from each litre of broth were prepared at 6, 24, 48, 72 and 96 h of incubation intervals, following the method indicated above. Each extract was subjected to GLC, and the time-course data of isovaleric acid production were obtained to x the incubation span for its optimum production. Results Cells and cell-free culture medium of P. haloplanktis INH strain showed antibiotic activity against V. anguillarum VAR strain on MTSA medium (Fig. 1), while the growth of V. ordalii was clearly inhibited by the ethyl acetate extract from P. haloplanktis INH strain at 10 mg ml)1 (Fig. 2). Chloroform used as control did not show any inhibitory activity, and the fact also indicated that chloroform has completely evaporated from the assay discs. In the antibacterial spectrum of the INH, all tested bacteria were inhibited by the crude extract at 10 mg ml)1 concentration (Table 1). One might to emphasize the clear growth inhibition of Morganella morganii ATCC 25830 strain by INH crude extract, which in a previous
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1

R 3/4 VA Pell

Cells (Pellet) INH

334

INH

Cells-free supernatant Sup

Figure 1 Cells and cells-free broth of Pseudoalteromonas haloplanktis inhibiting the Vibrio anguillarum (VAR) growth.

or

da

lii

( c ru d
e

c 5 1

4 3

Figure 2 Inhibition activity of INH crude extract against Vibrio ordalii. 1, 01 mg ml)1; 2, 1 mg ml)1; 3, 1 mg ml)1; 4, 10 mg ml)1; 5, 25 mg ml)1; c, control.

Table 1 Antibacterial spectrum of the crude extract of Pseudoalteromonas haloplankti

Tested bacteria Marine strains Vibrio ordalii ATCC 33509 Vibrio alginolycticus ATCC 17749 Vibrio anguillarum IFO 13266 Vibrio anguillarum (VAR) Pseudonomonas uorescens IFO 3903 Aeromonas hydrophila IFO 13287 Pseudoalteromonas haloplanktis (INH) Clinical strains Escherichia coli IFO 3366 Salmonella thyphimurium LT-2 Morganella morganii ATCC 25830 Shewanella putrefaciens IFO 1349 Achromobacter sp. IFO13495 Bacillus subtilis ATCC 6051 Staphylococcus aureus IFO 13276 Micrococcus Iuteus IFO 3333 +Inhibition zone 510 mm; ++Inhibition zone > 10 mm. Inhibition

+ + + + + + ++ + + ++ + ++ ++ + ++

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study (Riquelme et al. 1996) showed resistance to cell-free supernatant of INH strain culture. Activity after the rst SiO2 column chromatography was detected in two fractions; (A) eluted with benzene and (B) with CHCl3 MeOH (9 : 1 v v). The structure elucidations of compounds present in the B fraction are in progress, therefore the nature and numbers of compounds present in this fraction are unknown. The A fraction (23 g) was puried by Florisil column loaded with CHCl3 MeOH, and the activity was detected in the fraction A-1 (15 g) eluted with CHCl3 MeOH (99 : 1 v v). Then this fraction was again puried by Florisil column and the activity was detected in the fraction A-1-1 (11 g) eluted with C6H6. Furthermore, it was chromatographed on orisil column and the activity was detected in the fraction A-1-1-1 (140 mg) eluted with CHCl3. The GC MS analysis of A-1-1-1 showed two peaks (Fig. 3). Peak 1 (tR 575 min) gave M+ ion at m z 102 (056%), and the base ion at m z 60 (100%) with the following diagnostic ions at m z 41 (257%), 43 (297%), 45 (143%), 69 (57%), 74 (43%) and 87 (243%). Although M+ ion at m z 102 was not observed in peak 2
Peak 1 (a) Peak 2

10 Absorbance (660 nm)

10 8 Iso-valeric acid (mg ml1)

1 6 4 01 2 001 0 20 40 60 Incubation time (h) 80 0 100

Figure 4 Production of isovaleric acid during the incubation of Pseudoalteromonas haloplanktis. s, INH growth; d ISO-Val-acid.

Relative abundance

(tR 616 min), the base ion and all of the other diagnostic ions at m z 41 (373%), 43 (4%), 45 (61%), 69 (4%), 74 (71%) and 87 (202%) were the same to those of peak 1. From 1H-NMR and 13C-NMR spectra, the compounds present in A-1-1-1 were concluded to be isovaleric acid (80%) and 2-methylbutyric acid (20%). 1H-NMR and 13 C-NMR shift data were in concordance with those of the authentic isovaleric acid and 2-methylbutyric acid (Aldrich Chemical Co. 1993). The GC MS spectra were also identical to authentic samples. The production curve of isovaleric acid by INH strain showed an increment from 047 to 901 mg ml)1 during the incubation (Fig. 4). In addition, after 6 h of incubation, the ratio of isovaleric acid and 2-methylbutyric acid was 3 : 1, while at 48 h the ratio was 6 : 1 (data not shown). Discussion The antibiotic activity showed by cells and cell-free culture medium of P. haloplanktis (Fig. 1) indicates the occurrence of extra cellular antibacterial substances in INH strain. Bacterial secondary metabolites with antimicrobial activity have been detected which may act as an effective measure to control its populations and maintaining the microbial diversity, preventing colonization of competitors to the adjacent space (Lemos et al. 1985 and Long et al. 2003). It was reafrmed to detect antibiotic activity present in the crude extract of P. haloplanktis (Fig. 2). Chloroform used as control did not show any inhibitory activity, and the fact also indicated that chloroform has completely evaporated from the assay discs. In relation to the antibacterial spectrum of the crude extract (Table 1), one might to emphasize is the clear growth inhibition of M. morganii ATCC 25830 strain by
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5 (b) 43 45 60

7 min 60

11

41 40

87 69 74 80 m/z

+ M 102 100

(c) 43 45 60

60 87 69 74 80 m/z + M 102 100

41 40

Figure 3 Total ion chromatogram (TIC) of the active fraction A-1-1-1 by GC MS and their mass spectra. (a) TIC, (b) mass spectrum of peak 1(tR 575 min) and (c) mass spectrum of peak 2 (tR 616 min).

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INH crude extract, which in a previous study (Riquelme et al. 1996) showed resistance to cell-free supernatant of INH strain culture. From the production curve of isovaleric acid (Fig. 4), the results allowed conclude that INH strain produces isovaleric acid more abundant and more quickly than 2-methylbutyric acid. Also, the antibiotic activity of the fraction A-1-1-1 was due to the presence of these both compounds. The results obtained by this work evidence the production of at least two compounds involved in the antibacterial activity of P. haloplanktis INH strain. The synthesis of several antibiotics types by marine bacteria has been previously described (Lemos et al. 1985; Sakata et al. 1986). These reports evidence that substances differ in their chemical nature and their physiological action on bacterial cells. Particularly, Pseudoalteromonas species had been described to produce a diverse range of biologically active compounds that specically target a wide variety of mar ine fouling organisms (Holmstrom and Kjelleberg 1999; Holmstrom et al. 2002; Franks et al. 2006). They could act as a control to maintain the species diversity of microbial ecosystems (De Freitas and Fredickson 1978; Nair and Simidu 1987). Recently, it has demonstrated that epibiotic bacteria from the surface of the soft coral Dendronephthya sp. inhibit the growth of bacteria commonly found in marine natural biolms (Dobretsov and Quian 2004). On the other hand, the production of short chain fatty acid by micro-organisms seems to be frequent and some brominated fatty acids with antibacterial activity have been reported (Elyakov et al. 1994; Dembitsky and Srebnik 2002). However, as far as we know, this is the rst report on antibacterial activity of isovaleric acid produced by a Pseudoalteromonas strain. Likewise, Uchida et al. (1988) reported antibacterial activity of long-chain fatty acid, but it was produced by a Dinoagellate. In relation to the isovaleric acid, it is cited as an inhibitor of human lymphocytes and cell of periodontal tissues proliferation (Scragg et al. 1994; Kurita-Ochiai et al. 1995), but none environmental control functions have been described. There is no doubt that the sea possesses plenty of metabolites, and constitutes a potential source of new drugs for ghting antibiotics-resistant infections and other deadly diseases. In fact, great efforts of scientists at different parts of the world have became extracted various kinds of drugs for several diseases in recent years. Therefore, every discovery of metabolites is considered important, because it adds to the knowledge base of compounds unique to the marine environment. Furthermore when as <10% of the biodiversity in the world has been tested for biological activity (Dembitsky
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and Srebnik 2002), many more useful natural lead compounds are awaiting discovery. Acknowledgements We thank to Dr Paula Daz-Palma, Dr Carlos Contreras, Dr Victor Kesternich and Dr Marta Hengst for their constructive comments on the manuscript. This study was carried out as postdegree research supported by the Japan Government MOMBUSHO scholarship. References
Abraham, T.J. (2004) Antibacterial marine bacterium deter luminous vibriosis in shrimp larvae. NAGA Worldsh Cent Q 27, 2831. Aldrich Chemical Co. (1993) Aldrich Library of 13C NMR and 1 H NMR Spectra. Milwaukee, WI: Aldrich Chemical Co., QC462.85. A44 1993. Avendano-Herrera, R., Lody, M. and Riquelme, C. (2005) Produccion de substancias inhibitorias entre bacterias de biopelculas en substratos marinos. Rev Biol Mar Oceanogr 40, 117125. Colwell, R. (2002) Fullling the promise of biotechnology. Biotechnol Adv 20, 215228. De Freitas, M.J. and Fredickson, A.G. (1978) Inhibition as a factor in the maintenance of microbial ecosystems. J Gen Microbiol 106, 307320. Dembitsky, V.M. and Srebnik, M. (2002) Natural halogenated fatty acids: their analogues and derivates. Prog Lipid Res 41, 315367. Dobretsov, S. and Quian, P-Y. (2004) The role of epibotic bacteria from the surface of the soft coral Dendronephthya sp. in the inhibition of larval settlement. J Exp Mar Biol Ecol 299, 3550. Elyakov, G.B., Kuznetsova, T.A., Stonik, V.A. and Mikhailov, V.V. (1994) New trends of marine biotechnology development. Pure Appl Chem 66, 811818. Faulkner, J. and Fenical, W. (2005) The Biomedical Potential of California Marine Organisms. San Diego, CA: University of California, California Sea Grant Program. Research Proles, PPNMP05_02. Finch, R.G. (1987) Clinical uses of antimicrobial drugs. In Pharmaceutical Microbiology ed. Hugo, W.B. and Russel, A.D. pp. 130150. London: Blackwell Scientic Publications. Fortman, J.L. and Sherman, D.H. (2005) Utilizing the power of microbial genetics to bridge the Gap between the promise and the application of marine natural products. Chembiochem 6, 960978. Franks, A., Egan, S., Holmstrom, C., James, S., Lappin-Scott, H. and Kjelleberg, S. (2006) Inhibition of fungal colonization by Pseudoalteromonas tunicata provides a competitive advantage during surface colonization. Appl Environ Microbiol 72, 60796087.

2008 The Authors Journal compilation 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 16721677

G. Hayashida-Soiza et al.

Antibacterial substances by P. haloplanktis

Garateix, A. (2005) El mar: fuente de nuevos farmacos. Elementos 58, 3947. Gauthier, M.J. (1976) Alteromonas rubra sp. nov., a new marine antibiotic-producing bacterium. Int J Syst Bacteriol 26, 459466. Gauthier, M.J. and Breittmayer, V.A. (1979) A new antibioticproducing bacterium from seawater: Alteromonas aurantia sp. nov. Int J Syst Bacteriol 29, 366372. Holmstrom, C. and Kjelleberg, S. (1999) Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents. FEMS Microbiol Ecol 30, 285293. Holmstrom, C., Egan, S., Franks, A., McCloy, S. and Kjelleberg, S. (2002) Antifouling activities expressed by marine surface associated Pseudoalteromonas species. FEMS Microbiol Ecol 41, 4758. Imada, C., Simidu, U. and Taga, N. (1985) Isolation and characterization of marine bacteria producing alkaline protease inhibitor. Bull Jpn Soc Sci Fish 51, 799803. Kurita-Ochiai, T., Fukushima, K. and Ochiai, K. (1995) Volatile fatty acids, metabolic by-products of periodontopathic bacteria, inhibit lymphocyte proliferation and cytokine production. J Dent Res 74, 13671373. Leiva, S., Yanez, M., Zaror, L., Rodrguez, H. and GarcaQuintana, H. (2004) Antimicrobial activity of actinomycetes isolated from aquatic environments in Southern Chile. Rev Med Chile 132, 151159. Lemos, M.L., Toranzo, A.E. and Barja, J.L. (1985) Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds. Microb Ecol 11, 149163. Long, R.A., Qureshi, A., Faulkner, D.J. and Azam, F. (2003) 2-g-Pentyl-4-Quinolinol produced by a marine Alteromonas sp. and its potential ecological and biogeochemical roles. Appl Environ Microbiol 69, 568576. Longeon, A., Peduzzi, J., Barthelemy, M., Corre, S., Nicolas, J.L. and Guyot, M. (2004) Purication and partial identication of novel antimicrobial protein from marine bacterium Pseudoalteromonas species strain X153. Mar Biotechnol 6, 633641. Manage, P., Kawabata, Z. and Nakano, S. (2000) Algicidal effect of the bacterium Alcaligenes denitricans on Microcystis spp. Aquat Microb Ecol 22, 111117. Manzi, L.V. and Mayz, J.C. (2003) Evaluating microorganisms. Rev Soc Ven Microbiol 23, 8588. Mc Carthy, S.A., Johnson, R.M. and Kakimoto, D. (1994) Characterization of an antibiotic produced by Alteromonas luteoviolacea Gauthier 1982, 85 isolated from Kinko Bay, Japan. J Appl Bacteriol 77, 426432. Nair, S. and Simidu, U. (1987) Distribution and signicance of heterotrophic marine bacteria with antibacterial activity. Appl Environ Microbiol 53, 29572962.

Nogami, K. and Maeda, M. (1992) Bacteria as biocontrol agents for rearing larvae of the crab Portunus trituberculatus. Can J Fish Aquat Sci 49, 23732376. Oclarit, J.M., Ohta, S., Kamimura, K., Yamaoka, Y. and Ikegami, S. (1994) Production of an antibacterial agent, o-Aminophenol by a bacterium isolated from the marine sponge Adocia sp. Fish Sci 60, 559562. Olsson, C., Westerdahl, A., Conway, P. and Kjelleberg, S. (1992) Intestinal colonization potential of turbot (Scophthalmus maximus) and Dab (Limanda limanda) associated bacteria with inhibitory effects against Vibrio anguillarum. Appl Environ Microbiol 58, 551556. Oranday, M.A., Verde, M.J., Martnez-Lozano, S.J. and Waksman, N.H. (2004) Active fractions from four species of marine algae. Phyton 53, 165170. Rajeev, K. and Xu, Z. (2004) Biomedical compounds from marine organisms. Mar Drugs 2, 123146. Riquelme, C., Hayashida, G., Toranzo, A.E., Vilches, J. and Chavez, P. (1995) Pathogenicity studies of a Vibrio anguillarum-related (VAR) strain causing an epizootic in Argopecten purpuratus larvae cultured in Chile. Dis Aquat Org 22, 135141. Riquelme, C., Hayashida, G., Araya, R., Uchida, A., Satomi, M. and Ishida, Y. (1996) Isolation of a native bacterial strain from the scallop Argopecten purpuratus with inhibitory effects against pathogenic vibrios. J Shellsh Res 15, 369374. Sakata, T., Sakaguchi, K. and Kakimoto, D. (1982) Antibiotic production by marine pigmented bacteria-I. Mem Fac Fish Kagoshima Univ 31, 243250. Sakata, T., Sakaguchi, K. and Kakimoto, D. (1986) Antibiotic production by marine pigmented bacteria-II. Mem Fac Fish Kagoshima Univ 35, 2937. Scragg, M.A., Cannon, S.J. and Williams, D.M. (1994) Comparative cytotoxic effects of short-chain fatty acids produced by periodontal pathogens on two cultured broblast lines. Microb Ecol Health Dis 7, 8390. Sugahara, I., Kohhashi, K. and Kimura, T. (1992) Isolation and properties of a bacterium capable of inhibiting the growth of red tide plankton. In Abstracts of the 6th International Symposium on Microbial Ecology (ISME-6) ed. Guerrero, R. and Pedros-Alio, C. 173 pp. Barcelona: International Committee on Microbial Ecology. Uchida, A., Shimada, A. and Ishida, Y. (1988) Antibacterial and antialgal substances produced by the dinoagellate Peridinium bipes. Nippon Suisan Gakkaishi 54, 1941 1945. Wratten, S., Wolf, M., Anderson, R. and Faulkner, D. (1977) Antibiotic metabolites from a marine pseudomonad. Antimicrob Agents Chemother 11, 411414.

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