The Journal of Antibiotics (2010) 63, 271–274 & 2010 Japan Antibiotics Research Association All rights reserved 0021-8820/10 $32.00 www.nature.com/ja NOTE Identification and characterization of bacteria with antibacterial activities isolated from seahorses (Hippocampus guttulatus) Jose´ L Balca´zar1,2, Sara Loureiro1, Yolanda J Da Silva1, Jose´ Pintado1 and Miquel Planas1 The Journal of Antibiotics (2010) 63, 271–274; doi:10.1038/ja.2010.27; published online 26 March 2010 Keywords: antagonism; marine bacteria; seahorses; Vibrio species Seahorse populations have declined in the last years, largely due to To test the ability of the isolates to inhibit growth of pathogenic overfishing and habitat destruction.1 Recent research efforts have, there- Vibrio strains (Table 1), we grew all isolates on suitable agar media at fore, been focused to provide a better biological knowledge of these 20 1C for 2–3 days. After incubation, we spotted a loop of each isolate species. We have recently studied, as part of the Hippocampus project, onto the surface of marine agar previously inoculated with overnight wild populations of seahorse (Hippocampus guttulatus)insomeareasof cultures of the indicator strain. Clear zones after overnight incubation the Spanish coast and established breeding programs in captivity.2 at 20 1C indicated the presence of antibacterial substances. With the development of intensive production methods, it has Bacterial isolates showing antagonistic activity against pathogenic become apparent that diseases can be a significant limiting factor. Vibrio strains were identified using the 16S rRNA gene, amplified from Vibrio species are among the most important bacterial pathogens of extracted genomic DNA with primers 27F and 907R and Taq DNA marine fish. They are responsible for several diseases, and high polymerase (Invitrogen, Carlsbad, CA, USA).8 PCR (95 1C for 10 min; mortalities due to vibriosis have been reported.3,4 Thus, we have 30 cycles of 94 1C for 30 s, 50 1C for 1 min and 72 1Cfor2min;and screened marine bacteria isolated from the intestinal content and 72 1C for 10 min) yielded products of approximately 0.9 kb, which cutaneous mucus of seahorses (H. guttulatus) for production of underwent sequencing. The sequences obtained were compared to antibacterial compounds against pathogenic Vibrio species. This those available in the GenBank, EMBL and DDBJ databases with the mechanism of competition offers the possibility of using these BLAST program (NCBI, Bethesda, MD, USA).9 Sequences were antagonistic microorganisms as biological control agents.5–7 subsequently integrated within the ARB program (Munich, Germany) Adult seahorses (n¼8) were collected from the coast of Galicia (NW package and analyzed with its alignment tools.10 The phylogenetic tree Spain). The culturable microbiota was isolated from the intestinal was constructed by the neighbor-joining method with Jukes Cantor content and cutaneous mucus as follows. Intestinal content from each correction. The robustness of the tree topology was verified through seahorse was collected, weighed homogenized using tissue grinders, calculating bootstrap values for the neighbor-joining tree and through and vortexed vigorously in sterile saline solution (8.5 g lÀ1 NaCl), comparison with the topology of a maximum likelihood tree, calcu- whereas the cutaneous mucus was collected from the dorsal surface lated by using the default settings in ARB.10 with a sterile cotton swab into a small amount of sterile saline All antagonistic isolates were also studied for the characterization of solution. Tenfold serial dilutions of samples were prepared and plated antibacterial substances. Antagonistic isolates were grown in 100 ml of on marine agar (Difco, Detroit, MI, USA), tryptic soy agar supple- marine broth without agitation at 20 1Cfor2days.Afterincubation, mented with 15 g lÀ1 NaCl (Cultimed, Barcelona, Spain) and we removed the bacteria by centrifugation (2000 g), and cell-free Cytophaga agar prepared with 50% seawater (0.5 g lÀ1 tryptone, culture supernatants were recovered by passage through 0.22 mm 0.5 g lÀ1 yeast extract, 0.2 g lÀ1 sodium acetate, 15 g lÀ1 agar and pore size filters. adjusted to pH 7.2). All plates were incubated for 3–7 days at 20 1C. All cell-free culture supernatants were adjusted to pH 6.5 with 5 M Colonies with different morphological characteristics from each sam- NaOH to eliminate the inhibitory effects produced by organic acids. ple were selected, subcultured in suitable media and stored in sterile Moreover, sensitivity of cell-free culture supernatants to trypsin and glycerol (15% v/v) at À80 1C. proteinase K (Sigma Chemical Co., St Louis, MO, USA) at a final 1Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Cientı´ficas, Vigo, Spain and 2Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, Girona, Spain Correspondence: Dr JL Balca´zar, Instituto de Investigaciones Marinas, Consejo Superior de Investigaciones Cientı´ficas, Eduardo Cabello 6, Vigo 36208, Spain. E-mail: [email protected] Received 24 December 2009; revised 18 February 2010; accepted 8 March 2010; published online 26 March 2010 Antagonism among marine bacteria JL Balca´zar et al 272 Table 1 Effect of proteolytic enzymes on the activity of the cell-free culture supernatants Indicator strainsa Enzyme V. alginolyticus V. harveyi V. ichthyoenteri V. parahaemolyticus V. splendidus Bacterial strain producers treatment N26-1 HT351 HT21 HT352 HT29 Brachybacterium sp. strain HG-2F Controlb +++ + + Proteinase K + + + + + Trypsin + + + + + Vibrio sp. strain HG-3F Control – ÀÀ ++ ++ Proteinase K ÀÀÀ À À Trypsin ÀÀÀ ++ ++ Pseudoalteromonas ruthenica strain HG-4F Control + ++ ++ ++ ++ Proteinase K + ++ ++ ++ ++ Trypsin + ++ ++ ++ ++ Neptunomonas naphthovorans strain HG-6F Control + + + + ++ Proteinase K + + + + ++ Trypsin + + + + ++ Ruegeria sp. strain HG-8F Control + ++ ++ ++ ++ Proteinase K + ++ ++ ++ ++ Trypsin + ++ ++ ++ ++ Aquimarina sp. strain HG-9F Control + + + + ++ Proteinase K + + + + ++ Trypsin + + + + ++ Pseudoalteromonas sp. strain HG-10F Control ++ ++ ++ ++ ++ Proteinase K ++ ++ ++ ++ ++ Trypsin ++ ++ ++ ++ ++ Jannaschia donghaensis strain HG-11F Control + + + + + Proteinase K + + + + + Trypsin + + + + + Aliivibrio fischeri strain HG-12F Control ÀÀ ++++ Proteinase K ÀÀÀ À À Trypsin ÀÀÀ À À Ruegeria sp. strain HG-13F Control + ++ ++ ++ ++ Proteinase K + ++ ++ ++ ++ Trypsin + ++ ++ ++ ++ Vibrio sp. strain HG-14F Control + + + + + Proteinase K ÀÀÀ À À Trypsin ÀÀÀ À À Pseudomonas sp. strain HG-15F Control + + + + + Proteinase K + + + + + Trypsin + + + + + Shewanella sp. strain HG-17F Control + + + + + Proteinase K + + + + + Trypsin + + + + + Diameter of inhibition zone: +, 6À10 mm; ++, 410 mm; À, no inhibition zone. aVibrio species isolated from disease processes in seahorses. bControl samples consisting of cell-free supernatants without enzyme treatment. concentration of 1.0 mg mlÀ1 was also tested in buffers recommended sterile Pasteur pipette, and cell-free culture supernatants (10 ml) from the by the supplier. Samples with and without enzymes were incubated at isolates were placed into each well. The plates were incubated aerobically 37 1C for 2 h and residual activity was determined. To exclude at 20 1C for 1–2 days and then examined for zones of inhibition. potential inhibition by hydrogen peroxide, we added catalase (Sigma In all, 250 bacterial isolates from the intestinal content and Chemical Co.) at a final concentration of 0.5 mg mlÀ1 and incubated cutaneous mucus were analyzed. Only 13 of these produced zones at 37 1C for 30 min. All assays were independently repeated at least two of inhibition, ranging from 15 to 20 mm, against at least one of the times for reproducibility. indicator strains. Comparative 16S rRNA gene sequence analysis Indicator bacteria (V. alginolyticus N26-1, V. harveyi HT351, placed the antagonistic isolates in the Gammaproteobacteria V. ichthyoenteri HT21, V. parahaemolyticus HT352 and V. splendidus (61.5%), Alphaproteobacteria (23.1%), in the CFB group of HT29) were grown in 5 ml of tryptic soy broth supplemented with Bacteroidetes (7.7%) and Actinobacteria (7.7%). Bacterial isolates 1.5% NaCl at 20 1C for 24 h. The cells were harvested by centrifugation belonging to the Gammaproteobacteria were obtained exclusively (2000 g), washed twice with sterile saline solution and resuspended in from the intestinal content. In particular, the genera Aliivibrio, 5 ml of the same solution. The bacterial suspensions were transferred Neptunomonas, Pseudoalteromonas, Pseudomonas, Shewanella and to marine agar plates. Four wells were made in each agar plate with a Vibrio were identified (Figure 1). The Alphaproteobacteria were The Journal of Antibiotics Antagonism among marine bacteria JL Balca´zar et al 273 HG-3F (FN356948) Vibrio parahaemolyticus (AF388386) Vibrio harveyi (AY750575) Vibrio alginolyticus (X56576) Vibrio rotiferianus (AJ316187) HG-14F (FN356957) Vibrio fortis (AJ514916) Vibrio agarivorans (AJ310647) Aliivibrio fischeri (X74702) HG-12F (FN356955) Aliivibrio salmonicida (X70643) Aliivibrio wodanis (AJ132227) Pseudoalteromonas peptidolytica (AF007286) Pseudoalteromonas rubra (X82147) HG-10F (FN356953) Pseudoalteromonas spongiae (AY769918) HG-4F (FN356949) Gammaproteobacteria Pseudoalteromonas ruthenica (AF316891) Pseudoalteromonas haloplanktis (X67024) Pseudoalteromonas tunicata (Z25522) Shewanella marinintestina (AB081757) Shewanella benthica (X82131) HG-17F (FN356959) HG-15F (FN356958)