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Keywords:

  • anti-Vibrio substance;
  • high-pressure liquid chromatography;
  • Pseudoalteromonas;
  • ultraviolet absorption spectrum

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

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 λmax 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.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Members of the genus Vibrio have been implicated as major causative agents in diseases in fish, mollusks and crustaceans, causing mass mortalities worldwide.1–6 One technique to alleviate this condition is the use of microorganisms as biocontrol 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 bacteria 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 proteolytic enzymes.23 In addition, the same strains were found to produce low molecular weight compounds that inhibit the growth of Vibrio strains.

This study reports the isolation and characterization of anti-Vibrio compounds from strain A1-J11 isolated from the coastal sea water of Kagoshima Bay, Japan.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Bacterial strains and media

Various antagonistic bacteria were previously isolated from several sources in and around Kagoshima Bay, Japan.23Vibrio 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 name Kocuria rhizophila NBRC 12708) were obtained from the Institute for Fermentation (IFO), Osaka, Japan. Vibrio harveyi 9S-F4 and FF-P1 and luminous bacteria, such as Vibrio spp. 9M-P5-1, 9M-B9 and 9J-F4, were isolated from black tiger shrimp (Penaeus monodon) culture ponds in the Philippines. Edwardsiella tarda strains E22, E381 and SU226, and Vibrio anguillarum strains NUF113 and NUF691, isolated from diseased fish, were kindly supplied by Dr Atsushi Yamamoto, Kagoshima University. Pseudomonas spp. 55b-11 and 56a-1 were isolated from sea water in Kagoshima Bay. Escherichia coli JM109 was purchased from Takara Biochemicals, Tokyo, Japan.

Bacterial strains were cultured in a modified ZoBell 2216E medium, Z-CII,24 containing polypeptone (Nippon Seiyaku, Tokyo, Japan) 5 g/L and yeast extract (Nippon Seiyaku) 1 g/L in 3/4-strength artificial sea water (ASW; Herbst's formula composed of 30.0 g NaCl, 7.0 g KCl, 10.8 g MgCl2·6H2O, 5.4 g MgSO4·7H2O and 1.0 g CaCl2·2H2O/L). The bacterial strains were stored at −80°C in Z-CII containing 20% glycerol and subcultured to the same agar (1.5%) slants and maintained at 4°C. Target strains used for experiments were seeded from the agar slants and grown in 10 mL Z-CII broth at 25°C for 2 days in a rotating L-shaped test tube.

Potentially antagonistic bacteria were characterized according to standard methods used in previous papers.23,24 Strain A1-J11, which showed the highest potential for isolation of anti-Vibrio substances, was further characterized and identified by comparing its 16S rDNA sequence to database sequences.

Organic solvent extraction of the active substances

Culture supernatant of strain A1-J11 was separated by centrifugation at 10 000 ×g for 15 min. A 3-day culture (300 mL) of antagonistic strains was centrifuged and the supernatant (30 mL) was extracted with equal volume of chloroform (CHCl3) or ethyl acetate (EtAc). Crude extracts were evaporated, dissolved in methanol (MeOH) and used for determination of growth inhibition using the disk diffusion method.

Antibiotic activity assay

A growth inhibition assay using the disk diffusion method was conducted by applying the test substances dissolved in MeOH onto a paper disk (diameter 8 mm), drying and placing on a double-layer Z-CII agar plate (1.5% agar bottom layer/0.5% agar top layer seeded with 10% v/v of the bacterial culture). Inhibitory activity was determined by subtracting the radius of the paper disk from that of the inhibition zone.

For the microdilution assay, 10 μL of test substance dissolved in MeOH was dried in a sterile 96-well plate before adding 100 μL from 10 mL of 2-day culture of the target strain. The plate was incubated at 25°C overnight. Growth of the strain was assessed by optical density (OD) at 655 nm using a plate reader (MPR-A4i; Tosoh, Tokyo, Japan).

Purification of anti-Vibrio substances from A1-J11

The culture supernatant of 5 L of A1-J11 grown at 25°C for 5 days was extracted with CHCl3. The crude extract was applied to a normal-phase open column (Silica Gel 60, 30 mm × 250 mm; Merck, Darmstadt, Germany) and eluted with CHCl3 : EtAc : acetonitrile (12:1:1 by volume). Four-milliliter fractions were tested for activity and monitored at 210 nm and 325 nm. The pooled active fractions from the first column chromatography were applied to a reverse-phase open column (Cosmosil 75C18-OPN 20 mm × 200 mm; Nacalai Tesque, Kyoto, Japan) and eluted with 50% MeOH. An aliquot of the fractions (4 mL) was tested for activity and monitored at 210 nm and 325 nm. The pooled active fractions from the second column chromatography was applied to reverse phase-high-pressure liquid chromatography (RP-HPLC) (Mightysil RP-18 GP Aqua 250-4.6, 4.6 mm internal diameter × 250 mm; Kanto Chemicals, Tokyo, Japan) and eluted with 25% acetonitrile/0.01% trifluoroacetic acid (TFA) at a flow rate of 0.8 mL/min and the elution profile was monitored at 325 nm.

Characterization of anti-Vibrio substances

The ultraviolet (UV) absorption spectra of the substances were measured from 200 nm to 400 nm in MeOH. The minimum inhibitory concentration (MIC) was measured using a microdilution assay. The test substances from 64.0 to 0.25 μg/mL were examined for inhibitory activity relative to a negative control (solvent only).

Thermostability of the substances was measured in triplicate using a microdilution assay. The test substance dissolved in MeOH (1.0 μg/mL) was dried completely and redissolved in dimethyl sulfoxide (DMSO). The sample solutions were exposed to various temperatures ranging from 30°C to 100°C by 10°C increments for 10 min. In addition, autoclave treatments (120°C for 20 min) were tested. After cooling, the samples were dried completely and redissolved in MeOH prior to the assay.

The pH stability of the substances was measured in triplicate using a microdilution assay. A sample (1.0 μg/mL) dissolved in MeOH was dried completely and redissolved in the appropriate buffer system, including McIllvaine buffer (pH 2.5–7.0), Tris-HCl (pHs 7.0–9.0) and glycine-NaOH (pHs 8.0–12.5). After 30 min exposure to each pH, the samples were dried completely and redissolved in MeOH prior to the assay.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Identification of strain A1-J11

As reported previously, strain A1-J11 was a gram negative motile rod with oxidase and catalase positive, and glucose oxidative reaction.23 It required NaCl for growth and hydrolyzed casein, starch, chitin, lecithin and Tween 80. It was tentatively identified as Pseudomonas sp. by morphological and biochemical characterization; however, 16S rDNA homology revealed a 99% sequence similarity to Pseudoalteromonas piscicida in European Molecular Biological Laboratory (EMBL), GenBank and DNA Database of Japan (DDBJ) databases (data not shown).

Extraction of anti-Vibrio substances from A1-J11 culture

Several strains isolated from coastal sea water in Kagoshima Bay were previously reported to form an inhibition zone on double-layer agar plates containing Vibrio spp.23 Ethyl acetate extracts of the culture supernatant were also found to inhibit growth of Vibrio strains (Table 1). Representative strains, A1-J11 and A1-J17 demonstrated an inhibitory activity profile that peaked at 3 days culture and remained stable up to 7 days (Fig. 1).

Table 1.  Growth inhibition of Vibrio strains with ethyl acetate extract from antagonistic bacteria culture on double-layer agar plates
Antagonistic strainsTarget strains
V. harveyi9M-P5-1
  • Vibrio harveyi ATCC 14126.

  • Vibrio sp. isolated from a shrimp culture pond.

  • –, no inhibition; +/–, weak inhibition; +, moderate inhibition; ++, strong inhibition.

A1-M1
A1-M2
A1-J1
A1-J10++++
A1-J11++++
A1-J17+++
A1-J25a-1++/–
A1-J25a-2
image

Figure 1. Inhibitory activity of crude extracts from strains A1-J11 and A1-J17 against Vibrio harveyi ATCC 14126 and their growth measured by optical density (OD) at 540 nm. Thirty milliliters from a 300 mL of modified ZoBell 2216E culture was sampled at 1, 2, 3, 5 and 7 days, and extracted with ethyl acetate. The crude extracts were determined for growth inhibition using the disk diffusion method. ●, activity of A1-J11; ▴, activity of A1-J17; ○, growth of A1-J11; ▵, growth of A1-J17.

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Purification of anti-Vibrio substances from strain A1-J11

The culture supernatant of strain A1-J11 was extracted with chloroform and applied to an open column of Silica Gel 60. Each fraction (4 mL) was monitored at 325 nm and tested for inhibitory activity. The most active fractions were eluted using a CHCl3 : EtAc : acetonitrile mixture (12:1:1 by volume). The active fractions were pooled and applied to an open column of Cosmosil 75C18-OPN. Each fraction (4 mL) from the second column chromatography was also monitored at 325 nm and tested for inhibitory activity. The active fractions were combined and applied to a RP-HPLC column (Mightysil RP-18 GP Aqua). Four major compounds were detected when eluted with 25% acetonitrile/0.01% aqueous TFA mixture and named AVS-03ad (Fig. 2). Their UV absorption spectra were measured and their inhibitory activities were tested. The substance AVS-03b did not exhibit any inhibitory activity and had a different UV spectrum compared to the others. The three other compounds (AVS-03a, c and d) exhibited inhibitory activities against V. harvayi ATCC 14126 and had similar UV absorption spectra (Fig. 3). The major substance, AVS-03d, showed inhibitory activity against Vibrio spp., especially V. harveyi (Table 2).

image

Figure 2. Reverse phase-high-pressure liquid chromatography (RP-HPLC) profile of the pooled fractions obtained from reverse-phase (Cosmosil) column chromatography. The sample was eluted using 25% acetonitrile/0.1% aqueous trifluoroacetic acid with a flow rate of 0.80 mL/min on a RP-18 GP Aqua RP-HPLC column. (a) AVS-03a, (b) AVS-03b, (c) AVS-03c and (d) AVS-03d. OD, optical density.

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image

Figure 3. Ultraviolet absorption spectra of isolated fractions with reverse phase-high-pressure liquid chromatography. ——, AVS-03d; ·······, AVS-03a; –·–, AVS-03c.

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Table 2.  Inhibitory activity of AVS-03d on the growth of various bacterial strains using the disk diffusion method
SpeciesStrainsInhibition
  1. +, inhibition; –, no inhibition on double-layer agar plates.

Vibrio harveyiATCC 14126+
ATCC 35084+
9SF4+
FFP1+
Vibrio fischeriVF-74+
Vibrio parahaemolyticusIFO 12711
Vibrio alginolyticusATCC 17749
Vibrio anguillarumNUF113+
NUF691+
Vibrio sp.9M-P5-1
Edwardsiella tardaE22
E381
SU226
Pseudomonas sp.55b-11
56a-1
Escherichia coliJM109
Micrococcus luteusIFO 12708

Minimum inhibitory concentration of purified substances

The purified substances AVS-03a and AVS-03d were tested for antibacterial activity against several strains of Vibrio spp., including V. harveyi ATCC 14126 and V. alginolyticus ATCC 17749. AVS-03d possessed stronger activity than AVS-03a and showed very high activity against V. harveyi ATCC 14126 (Table 3). AVS-03d and AVS-03a were comparatively less effective against V. alginolyticus ATCC 17749 at more than 64 μg/mL.

Table 3.  Minimum inhibitory concentration of AVS-03a and d in liquid cultures of the target strains
Target strainsMinimum inhibitory concentration (μg/mL)
AVS-03aAVS-03d
Vibrio harveyi ATCC 141264.00.5
Vibrio alginolyticus ATCC 17749>6464.0
Vibrio parahaemolyticus 9J-F432.08.0
Vibrio fischeri 9M-B916.08.0
Vibrio sp. 9M-P5-164.08.0

pH and thermal stability of the purified substances

The active substances were subjected to various temperature and pH treatments. All three substances were stable up to 100°C for 10 min, but the inhibitory activities declined after autoclaving at 120°C for 20 min (Fig. 4). AVS-03d was stable at pH higher than 4.0, whereas the activity was lost at pH lower than 3.0 (Fig. 5).

image

Figure 4. Thermal stability of the purified substances. Each sample solution (n = 3) was exposed to various temperatures for 10 min or autoclaved at 120°C for 20 min. ◆, AVS-03a; ●, AVS-03c; ▴, AVS-03d.

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image

Figure 5. pH stability of AVS-03d dissolved in various buffer solutions (n = 3). pH 2.5–7.0, McIllvaine buffer; pH 7.0–9.0, Tris-HCl; pH 8.0–12.5, glycine-NaOH.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

Pseudoalteromonas piscicida is closely associated with red tides and is a well-known producer of a fish toxin. Recently a cytotoxic compound, norharman,22 which also has an apoptotic effect on cancer cells was reported. A close relative, Pseudoalteromonas rubra, is known to produce a polyanionic molecule that has been found to inhibit bacterial respiration.20 The same substances have been found in Pseudoalteromonas citrea and Pseudoalteromonas luteoviolacea.21 Other pseudoalteromonads have been reported to produce biologically active substances: Pseudoalteromonas F-420 produces korormicin, Pseudoalteromonas phenolica produces MC21-A, and Pseudoalteromonas KP20-4 produces pseudoalterobactin A and B 40.17–19 Other strains belonging to the same genus have been reported to show antibacterial activity, agarolytic activity and antiviral activities.16 The list is a bit dubious because some of the previously reported Alteromonas and Pseudomonas spp. are now recognized to be Pseudoalteromonas spp. The UV absorption spectra of three anti-Vibrio substances isolated from the strain in this study point to a similarity in chemical structure. By comparing reported UV absorption spectra, they may not bear any resemblance to other products reported for pseudoalteromonads (e.g. norharman, korormicin, MC21-A, pseudoalterobactins). The elucidation of the complete chemical structure of these compounds is of very high interest and is the subject of further studies.

The highly specific inhibitory activity of Pseudoalteromonas strain A1-J11 against V. harveyi is a bit of an enigma, by virtue of its mode of action, because antibacterial substances would usually be effective over a considerably broader range. An intragenus specificity would indicate different metabolic pathways between species and antibacterial targeting specifically in intracellular metabolism. The chemical structure and highly specific activity against V. harveyi of the substance are interesting subjects for further study.

ACKNOWLEDGMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES

The authors are deeply grateful to Dr Yuto Kamei of the Coastal and Highland Bioresource Center of Saga University for helpful guidance and comments on the chemical analysis. Bacterial strains belonging to E. tarda and V. anguillarum were supplied by Dr Atsushi Yamamoto, Faculty of Fisheries, Kagoshima University.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
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