Vibrio Pelagius Research Articles

Anti-Osteoclastogenic and Antibacterial Effects of Chlorinated Polyketides from the Beibu Gulf Coral-Derived Fungus Aspergillus unguis GXIMD 02505.

One new depsidone derivative, aspergillusidone H (3), along with seven known biosynthetically related chlorinated polyketides, were obtained from the Beibu Gulf coral-derived fungus Aspergillus unguis GXIMD 02505. Their structures were determined by comprehensive physicochemical and spectroscopic data interpretation. Notably, the X-ray crystal structure of 2 and the previously unknown absolute configuration of 8, assigned by ECD calculations, are described here for the first time. Compounds 1–5, 7 and 8 exhibited inhibition of lipopolysaccharide (LPS)-induced NF-κB in RAW 264.7 macrophages at 20 μM. In addition, the two potent inhibitors (2 and 7) dose-dependently suppressed RANKL-induced osteoclast differentiation without any evidence of cytotoxicity in bone marrow macrophages cells (BMMs). This is the first report of osteoclastogenesis inhibitory activity for the metabolites of these kinds. Besides, compounds 1, 2, 4, and 6–8 showed inhibitory activity against marine biofilm-forming bacteria, methicillin-resistant Staphylococcus aureus, Microbulbifer variabilis, Marinobacterium jannaschii, and Vibrio pelagius, with their MIC values ranging from 2 to 64 μg/mL. These findings provide a basis for further development of chlorinated polyketides as potential inhibitors of osteoclast differentiation and/or for use as anti-fouling agents.

Diets containing shrimp protein hydrolysates provided protection to European sea bass (Dicentrarchus labrax) affected by a Vibrio pelagius natural infection outbreak

This study was conducted to investigate the effects of the dietary supplementation of shrimp protein hydrolysate (SPH) on somatic growth performance, innate immune response in juvenile European sea bass (Dicentrarchus labrax) and their differential cumulative mortality when affected by a Vibrio pelagius natural infection outbreak. A diet containing 20% fish meal (FM) was used as a control, whereas three other diets differing in the level of FM inclusion (75 and 25% FM replacement by plant protein sources) and the inclusion of the additive (5% FM, 5% FM + 5% SPH and 15% FM + 5% SPH) were tested. After 110 days, there were no statistically significant differences in somatic growth parameters nor proximate composition in fish fed different experimental diets (P > 0.05), while the humoral non-specific immune responses (lysozyme, bacteriolytic and complement activities) were significantly enhanced by the inclusion of SPH in diets (P < 0.05). Additionally, an outbreak of the pathogenic bacteria V. pelagius, a bacterial species previously described as producer of the virulence factor hemolysin, occurred in all experimental tanks (4 replicates per diet) due to crowding and repeated handling stress for fish sorting. Survival rates among different experimental groups ten days after the bacterial epizootic differed depending on the diets, with groups containing SPH showing the best results (P < 0.05). In particular, fish fed the 15% FM + 5% SPH diet showed the highest survival rate (96.4 ± 5.0%), followed by those fed the 5% FM5 + 5% SPH5 (61.8 ± 16.3%). In contrast, survival rates in fish fed diets deprived of the additive (20% FM and 5% FM5 diets) were the lowest ones (32.0 ± 6.7% and 38.2 ± 13.5%, respectively). The present study showed that SPH can be incorporated in aquafeeds with high levels of FM substitution by PP sources without detrimental impact on the somatic growth performance of fish. In addition, the non-specific humoral immunity in seabass and their survival when affected by an epizootic outbreak of V. pelagius were positively affected, which showed the immunomodulatory benefits of shrimp protein hydrolysate to promote health and prevent diseases in fish.

The genus Listonella MacDonell and Colwell 1986 is a later heterotypic synonym of the genus Vibrio Pacini 1854 (Approved Lists 1980) – a taxonomic opinion

We analysed the taxonomic position of the genus Listonella based on phylogenetic, genomic and phenotypic data. The species of the genus Listonella were nested within the genus Vibrio according to the 16S rRNA gene sequence-based phylogenetic tree. The closest neighbour of Vibrio (Listonella) anguillarum strains LMG 4437(T) and ATCC 68554 (=strain 775) was Vibrio ordalii LMG 13544(T), with more than 99.5% 16S rRNA gene sequence similarity. Furthermore, Vibrio (Listonella) pelagius is highly related to Vibrio splendidus. According to average amino acid identity (AAI), multilocus sequence analysis (MLSA) and Karlin genome signature, the closest neighbour of L. anguillarum ATCC 68554 is V. ordalii LMG 13544(T), with 95% AAI, 98% MLSA and 5 in Karlin. V. anguillarum ATCC 68554 and Vibrio cholerae N16961 had 77% similarity in AAI, 85% in MLSA and 14 in the Karlin signature. Phenotypic analyses of previously published data for V. (L.) anguillarum and V. (L.) pelagius revealed that the genus Listonella is extremely similar to the genus Vibrio. V. ordalii and L. anguillarum strains yielded up to 67% DNA-DNA hybridization. There are only a few phenotypic features that might be used to discriminate these two species: L. anguillarum is positive for the Voges-Proskauer reaction, citrate utilization, starch hydrolysis, lipase activity and acid production from glycerol, sorbitol and trehalose, whereas V. ordalii is negative for these traits. We suggest that the genus Listonella is a later heterotypic synonym of the genus Vibrio and propose to use the names Vibrio anguillarum and Vibrio pelagius in place of Listonella anguillarum and Listonella pelagia, respectively.

Defences against oxidative stress in vibrios associated with corals

Bacteria colonizing healthy coral tissue may produce enzymes capable of overcoming the toxic effects of reactive oxygen species, including superoxide dismutase (SOD) and catalase. Significant differences in the activities of these enzymes were observed in cultures of Vibrio campbellii, Vibrio coralliilyticus, Vibrio harveyi, Vibrio mediterranei, Vibrio pelagius, Vibrio rotiferanus, Vibrio tasmaniensis, and Photobacterium eurosenbergii isolated from healthy, bleached or necrotic tropical and cold water corals. Levels of SOD in exponential phase cultures of V. coralliilyticus grown at 28 degrees C were only slightly higher than those grown at 16 degrees C whereas the levels in stationary phase cultures at 28 degrees C were 7.3 x higher than those at 16 degrees C. The increase in catalase activity of V. coralliilyticus and V. harveyi upon entry to stationary phase conferred protection against killing by oxidative stress. Increased temperature affected up-regulation of enzymes in stationary phase cultures, but pretreatment of cultures with hydrogen peroxide had no significant effect on induction of catalase or SOD. The increased activities appear to be due to up-regulation of gene expression rather than induction of different forms of the enzymes. We suggest that SOD and catalase are unlikely to be major factors in the virulence of these bacteria for corals and that their main function may be to protect against endogenous superoxide.

Antibiotic activity of lectins from marine algae against marine vibrios.

Saline and aqueous ethanol extracts of marine algae and the lectins from two red algal species were assayed for their antibiotic activity against marine vibrios. Experimental studies were also carried out on the influence of environmental factors on such activity, using batch cultures. The results indicated that many of the saline extracts of the algal species were active and that the activity was selective against those vibrios assayed. The algal extracts were active against Vibrio pelagius and the fish pathogen V. vulnificus, but inactive against V. neresis. Algal lectins from Eucheuma serra (ESA) and Galaxaura marginata (GMA) strongly inhibited V. vulnificus but were inactive against the other two vibrios. The antibacterial activity of algal extracts was inhibited by pretreatment with various sugars and glycoprotein. Extracts of the two red algae, E. serra and Pterocladia capillacea, in saline and aqueous ethanol, inhibited markedly the growth rate of V. vulnificus at very low concentrations. Culture results indicated that metabolites active against V. vulnificus were invariably produced in P. capillacea over a wide range of temperature, light intensity, and nutritional conditions. Enhanced antibacterial activity occurred when P. capillacea was grown under higher irradiance, severe nutrient stress and moderate temperature (20 degrees C), reflecting the specific antibiotic characteristics of this alga. The strong antibiotic activity of lectins towards fish pathogenic bacteria reveals one of the important roles played by algal lectins, as well as the potential high economic value of those marine algae assayed for aquaculture and for biomedical purposes.

Non-specific immune response of turbot, Scophthalmus maximus (L.), experimentally infected with a pathogenic Vibrio pelagius.

The effect of a pathogenic Vibrio pelagius, isolated during a mass mortality of turbot larvae, on the non-specific immune response of turbot, Scophthalmus maximus (L.), macrophages was studied both in vitro and in vivo. The in vitro treatment of head kidney (HK) macrophages with viable V. pelagius caused a significant inhibition of the chemiluminescence (CL) response in comparison with untreated macrophages, while incubation with heat-killed bacteria did not affect this response. In vivo, the intraperitoneal injection of V. pelagius resulted in a significant inhibition of the CL response in infected fish at days 1 and 4 post-infection compared with the control fish response. The HK macrophage nitric oxide (NO) production was enhanced by in vitro incubation with intermediate doses of viable V. pelagius (5 x 10(3) and 5 x 10(4) bacteria mL(-1)) and higher doses of the heat-killed bacteria (5 x 10(4)-5 x 10(6) bacteria mL(-1)). In both cases, the NO inhibitor N-omega -nitro-L-arginine was capable of down-regulating the specific NO induction caused by incubation with the bacterial treatments. In contrast, incubation with ECPs at higher doses caused a reduction in NO production. In vivo, a significant enhancement in NO production was also observed in macrophage supernatants at day 10 post-infection. Lysozyme concentration in the serum was also significantly increased in the experimentally infected fish at days 4 and 10 post-injection. In addition, viable V. pelagius and its ECPs significantly reduced HK macrophage viability in vitro, whereas no significant differences in viability were observed during the incubation with heat-killed bacteria. As NO production was enhanced in the experimentally infected fish, the inhibitory effect of the NO donor, S-nitroso-acetyl-penicillamine (SNAP), was tested in vitro in a cell-free assay. The results showed that growth of V. pelagius was significantly inhibited using SNAP at a high concentration (1 mM).

Isolation of a highly pathogenic Vibrio pelagius strain associated with mass mortalities of turbot, Scophthalmus maximus (L.), larvae.

A bacterial strain, characterized as Vibrio pelagius (Hq 222), was isolated from a turbot, Scophthalmus maximus (L.), larvae mass mortality in a commercial fish farm in Spain. Turbot larvae, post-larvae (0.2 g) and juveniles (5 and 15 g) were experimentally infected. The bacterium appeared to be very virulent for larvae and post-larvae, LD50 being < 5 bacteria mL(-1) for larvae 1 week post-infection and 3.9 x 10(5) bacteria mL(-1) in post-larvae at day 12 post-infection. The bacterial strain was recovered in pure culture from the internal organs of infected fish. Histological lesions in post-larvae exhibited swelling and necrosis of gill secondary lamellae, sloughing of intestinal mucosa and necrosis of haematopoietic tissue in the kidney. Vibrio pelagius (Hq 222) was able to grow in sterile sea water when incubated at room temperature or at 15 degrees C. Vibrio pelagius (Hq 222) was more adherent to the turbot cell lines TV-1 and TF than Escherichia coli. In both cell lines, the number of adhered bacteria increased with incubation time.

Purification and partial characterisation of a fish lethal extracellular protease from Vibrio pelagius

The purification, characterisation and lethal effect of an extracellular protease present in the extracellular products (ECPs) of a pathogenic Vibrio pelagius (7P) strain are described. The extracellular protease was purified by size-exclusion high-performance liquid chromatography and characterised by enzymatic assays. The lethal effect was evaluated by injection into fish. The native protease had a molecular mass of 39 kDa, was active on casein and l-leucyl-β-naphthylamide (LNA) and its metalloprotease nature was shown by the LNA inhibition profile. Kinetic studies on the hydrolysis of casein and LNA confirmed a competitive inhibition of one substrate with respect to the other. The temperature assays showed that both aminopeptidolytic and caseinolytic activities were labile at 70 °C for 3 min. The N-terminal amino acid sequence of 7P protease revealed a high degree of homology with other metalloproteases of Vibrio species that are implicated in virulence. The purified 7P protease showed an LD 50 of 1.77 μg protein/g fish for turbot. The quick lethal effect (<24 h) and the macroscopic damage (external haemorrhagic areas, principally on fins and mouth, petechial haemorrhages in internal organs, but with no external or internal apparent necrotic areas) detected in the host were similar to those obtained by injection of total ECP and live cells of 7P strain. An extracellular protease with endopeptidolytic and exopeptidolytic activities, responsible for the lethal effect of ECP and clinical signs of vibriosis in turbot was purified from a pathogenic V. pelagius (7P) strain.

Structures of ribonuclease P RNAs of Vibrio core species

The structures of an RNA component of ribonuclease P (RNase P RNA) were examined for Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio natriegens, Vibrio campbellii, Vibrio proteolyticus, Vibrio pelagius and Vibrio harveyi to clearly determine their genetic differences. The RNase P RNAs ranged from 382 to 454 nucleotides (nt) in size, and were remarkably different from each other in the structure of two helices, P3 and P12. The P3 helices were comprised of tandem repeats of a palindromic sequence (24 nt), resulting in the longitudinal repetition of a stem structure. The number of repetitions ranged from four in V. harveyi, to one in both V. alginolyticus and V. proteolyticus. The genes for the RNase P RNAs of all species were located between two open reading frames, the amino acid sequences of which were similar to the hypothetical proteins located at 70.92 and 1.94 min in the Escherichia coli chromosome.

Vibrio pelagius: Differences of the Type Strain Deposited at Various Culture Collections

A critical evaluation of published and own taxonomic and phylogenetic studies on Vibrio pelagius showed substantial diversity of strains received as type strains from various Culture Collections. The comparison of data based upon 16S rRNA sequence analyses, earlier genomic DNA-DNA similarity studies as well as physiological investigations and the original description indicate that Vibrio pelagius strains CECT 4202T and ATCC 25916T really represent the originally described type species whereas strains NCIMB 1900T and CIP 102762T highly likely are representatives of Vibrio natriegens.

Does Carnobacterium divergens isolated from Atlantic salmon, Salmo salar L., colonize the gut of early developing turbot, Scophthalmus maximus L., larvae?

It is well established from endothermic animals that the adhesion of lactic acid bacteria involves host speci®city (Fuller 1986). In juvenile ®sh, JoEborn, Olsson, Westerdahl, Conway & Kjellberg (1997) reported by in vitro tests that a Carnobacterium sp. isolated from Atlantic salmon, Salmo salar L., colonized the gastrointestinal tract of rainbow trout, Onchorynchus mykiss (Walbaum). However, no information from ®sh larvae is available on whether the adhesion of lactic acid bacteria involves host speci®city. This is relevant in the context of whether or not it is possible to colonize the gastrointestinal tract of larvae at early stages with probiotics, notably Gram-positive bacteria, such as lactic acid bacteria, or by a resident micro ora, which may contribute to nutrition. The following bacteria were used in the present study: Vibrio pelagius isolated from copepod-fed turbot, Scophthalmus maximus L., larvae (Munro, Barbour & Birkbeck 1994), which is unusual in that it contains the polyunsaturated fatty acid eicosapentaenoic acid (20:5 n-3) (Ringu, Sinclair, Birkbeck & Barbour 1992), and a lactic acid bacterium, Carnobacterium divergens, isolated from Atlantic salmon. The main reason for using this species was that it produced a substance that inhibited the growth of both Vibrio anguillarum and Proteus vulgaris (Strum 1988), and Aeromonas salmonicida (Ringu 1999). Recently, colonization of different bacterial strains in larval turbot was determined using an enzyme-linked immunosorbent assay (ELISA) (Ringu, Birkbeck, Munro, Vadstein & Hjelmeland 1996; Ringu & Vadstein 1998). The purpose of the current study, was to evaluate whether a C. divergens, orginally isolated from Atlantic salmon, was able to colonize the gut of turbot larvae by adding this bacterial species to the rearing water and to compare these results with those for larvae exposed to V. pelagius, by using the ELISA method. Furthermore, a previous study (Ringu et al. 1996) demonstrated that antisera used in ELISA showed high sensitivity against homologous bacterial antigens and very little crossreaction with heterologous bacteria. Turbot eggs were obtained from Tinfos Aqua, Norway, and 1000 eggs were incubated in 80-L circular tanks with at bottoms in static water. A detailed description of larval rearing conditions and cultivation of live feed (Brachionus plicatilis SINTEFstrain) is given by Ringu et al. (1996). The following bacterial strains were used: C. divergens, originally isolated from young Atlantic salmon juveniles (Strum 1988); and Vibrio pelagius, isolated from copepod-fed turbot larvae (Munro et al. 1994). Vibrio pelagius was identi®ed as described by Munro et al. (1994) using the `Bacterial Identi®er' program (Bryant, Lee, West & Colwell 1986). Carnobacterium divergens was identi®ed using carboR

Antibiotic Resistance and Plasmid Profiles of Vibrio Isolates from Cultured Silver Sea Bream, Sparus sarba

A total of 51 potential pathogenic vibrios were isolated from moribund silver sea bream ( Sparus sarba) collected from fish farms in Hong Kong. Using the API 20E system and the scheme of Alsina and Blanch (1994), 7 species were identified from all isolates. These species were Vibrio alginolyticus (24 strains), Vibrio vulnificus (12 strains), Vibrio parahaemolyticus (7 strains), Vibrio logei (4 strains), Vibrio pelagius II (2 strains), Vibrio fluvialis (1 strain) and Vibrio meditterranei (1 strain). The three dominant species ( V. alginolyticus, V. vulnificus and V. parahaemolyticus) were confirmed to be virulent to sea bream by experimental challenge. All isolates were screened for plasmid DNA by agarose gel electrophoresis and tested for susceptibility to 16 antibiotics by the agar dilution method. Of the 51 isolates examined, all strains were sensitive to ceftriaxone, streptomycin, nalidixic acid and rifampicin, and almost all were sensitive to ceftazidime, netilimicin, chloramphenicol and sulfamethoxazole. Most isolates were resistant to ampicillin (60.8%), cefuroxime (66.7%), amikacin (55%), kanamycin (58.8%) and trimethoprim (76.5%). Fifteen of the 51 isolates harboured 1–4 plasmids, with sizes ranging from 9 to 123 kb. Both the plasmids and the associated antibiotic resistance (ampicillin, cefuroxime and trimethoprim) of 9 isolates could be transferred to a recipient by single-step conjugation. However, the frequencies were very low, ranging from 10 −11 to 10 −9. The present results indicate that resistance to these antibiotics is chromosomal.

Vibrio halioticoli sp. nov., a non-motile alginolytic marine bacterium isolated from the gut of the abalone Haliotis discus hannai.

Six alginolytic, facultatively anaerobic, non-motile marine bacteria were isolated from the gut of abalone Haliotis discus hannai. DNA-DNA hybridization data showed that the six strains constituted a single genospecies. Phylogenetic analyses of 16S rDNA sequences indicated that the isolates should be assigned to the genus Vibrio. The phenotypic features of the isolates were closely related to Vibrio fischeri and Vibrio pelagius biovar I, but 13 traits (motility, luminescence, alginase production, lipase production, lysine decarboxylase, indole production, growth in 1 and 6% NaCl and assimilation of five carbon compounds) distinguished these strains from V. fischeri, and 17 traits (motility, growth at 37 degrees C, lipase production, indole production, growth in 1 and 6% NaCl, acid from sucrose and D-sorbitol, and assimilation of nine carbon compounds) distinguished these strains from V. pelagius. The G + C content of the isolates was 41.6-43.1 mol%. According to DNA-DNA hybridization data and 16S rDNA phylogenetic analyses, it was concluded that the six isolates constitute a new species different from any other Vibrio species. The name Vibrio halioticoli sp. nov. (type strain IAM 14596T) is proposed. A set of phenotypic features which enables differentiation of the new species from other species of the Vibrionaceae family is described.

The effect of early exposure to Vibrio pelagius on the aerobic bacterial flora of turbot, Scophthalmus maximus (L.) larvae

Vibrio pelagius was added to filtered sea water in experimental tanks containing newly‐hatched larvae of Scophthalmus maximus. The bacterial load of larvae increased from day 1 post‐hatch and by day 14 had reached 5 × 104 bacteria per larva. Vibrio pelagius dominated the aerobic bacterial flora of larvae exposed to this bacterial species but was not detected in larvae not exposed to exogenous bacteria. An enzyme‐linked immunosorbent assay using rabbit antiserum against V. pelagius allowed a specific quantitative assay for homologous bacterial antigens in individual larvae, with very little cross‐reaction against heterologous bacteria. The results of enzyme‐linked immunosorbent assays of V. pelagius antigens in larvae from tank water inoculated with this bacterial species correlated with the bacterial levels found in the larvae by culture on agar plates.

Isolation and Characterization of Halophilic Vibrio from Bivalves Bred in Nurseries at the Ebre Delta

A total of 380 strains of halophilic Vibrio and related bacteria were isolated from shellfish bred in the nurseries located in the Ebre river delta. Isolates were characterized using 24 phenotypic features and identified as different species of Vibrio. The most frequent species was Vibrio fluvialis followed by Vibrio pelagius, Vibrio tubiashii, and Vibrio splendidus . Several strains of Vibrio alginolyticus and Vibrio parahaemolyticus were isolated. No infectious diseases of bivalves have been described in the zone. The possible consequences of these findings for bivalve breeding and public health are discussed.

Production of Eicosapentaenoic Acid (20:5 n-3) by Vibrio pelagius Isolated from Turbot ( Scophthalmus maximus (L.)) Larvae

Fourteen bacterial strains isolated from turbot, Scophthalmus maximus (L.), larvae were screened for eicosapentaenoic acid (20:5 n-3) (EPA) production. Gas chromatography analysis revealed that one bacterial species, Vibrio pelagius, contained a high proportion of EPA in cellular lipid. This finding was confirmed by gas chromatography-mass spectrometry analysis. A higher concentration of EPA was detected when the bacterium was cultured at 4 degrees C.

Taxonomy of Four Marine Bacterial Strains That Produce Tetrodotoxin

Four strains of tetrodotoxin-producing bacteria isolated from a red alga and from pufferfish were characterized. Two of these strains are members of the genus Listonella MacDonell and Colwell. The phenotypic characteristics, guanine-plus-cytosine contents, and base sequences of the 16S rRNAs of these organisms indicated that they are members of Listonella pelagia (Vibrio pelagius) biovar II. The other two strains are members of the genus Alteromonas Baumann et al. and the genus Shewanella MacDonell and Colwell. These two strains are mutually distinct and distinct from the previously described Alteromonas and Shewanella species and therefore are placed in new species. The names Shewanella alga and Alteromonas tetraodonis are proposed for these organisms; the type strains are strains OK-1 and GFC, respectively.

Rapid serological identification of Vibrio vulnificus by anti-H coagglutination.

Staphylococcus aureus Cowan 1 cells were armed with anti-flagellar (anti-H) antibody produced in rabbits immunized with flagellar core protein prepared from Vibrio vulnificus. This reagent was assessed by coagglutination for its capacity to agglutinate and identify V. vulnificus. A species-specific H antigen is expressed in the core proteins of the polar flagella of V. vulnificus. Of 435 V. vulnificus isolates identified bacteriologically, 432 (99.3%) were agglutinated in the slide test within 2 min after the addition of the anti-V. vulnificus H coagglutination reagent. Other than Vibrio pelagius, the reagent did not agglutinate 19 heterologous Vibrio spp. tested, including 290 V. cholerae, 22 V. mimicus, 395 V. parahaemolyticus, and 16 V. fluvialis isolates recovered from seafood and the marine environment. The serological resolution of the coagglutination reaction was enhanced if the organism under test was suspended in 0.1 M Tris buffer-0.1 mM EDTA-1.0% Triton X-100 (TET) for 24 h before serological examination. The TET buffer also increased the sensitivity of the coagglutination reaction 100-fold over that for isolates suspended in 0.3% formalinized phosphate-buffered saline before testing. The anti-H coagglutination test is a rapid, serologically specific, and inexpensive procedure for identifying V. vulnificus one step beyond primary isolation.

NaCl concentration determines the outcome of competition experiments betweenVibrio pelagius andVibrio cholerae in chemostat cultures

The effect of NaCl concentration gradients on the growth of a marine species,Vibrio pelagius, and two freshwater and estuarine species,Vibrio cholerae andAeromonas hydrophila, was determined in chemostat cultures containing a mineral medium withd-glucose as the sole source of carbon and energy. Complementary cell density profiles were obtained with the marine and the freshwater-estuarine species at the extreme ends of the NaCl concentration gradients. At the lower NaCl concentrations,V. cholerae andA. hydrophila attained maximal cell densities, while no growth or submaximal cell yields were characteristic ofV. pelagius; the reverse was true at the higher NaCl concentrations. At the intermediate concentrations (100–400 mM) all three organisms were able to grow with cell densities ranging from 80%–100% of the maximum. Competition experiments betweenV. cholerae andV. pelagius within this region (100, 150, and 300 mM) indicated that the lower NaCl concentrations favoredV. cholerae while the higher concentrations favoredV. pelagius. The results suggest that the responses ofV. pelagius, V. cholerae, andA. hydrophila to different concentrations of NaCl are important in determining the environmental distribution of these species.