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Applied and Environmental Microbiology, March 1999, p. 969-973, Vol. 65, No. 3
Danish Institute for Fisheries Research,
Received 29 September 1998/Accepted 22 December 1998
To study the possible use of probiotics in fish farming, we
evaluated the in vitro and in vivo antagonism of antibacterial strain
Pseudomonas fluorescens strain AH2 against the
fish-pathogenic bacterium Vibrio anguillarum. As iron is
important in virulence and bacterial interactions, the effect of
P. fluorescens AH2 was studied under iron-rich and
iron-limited conditions. Sterile-filtered culture supernatants from
iron-limited P. fluorescens AH2 inhibited the growth of
V. anguillarum, whereas sterile-filtered supernatants from
iron-replete cultures of P. fluorescens AH2 did not.
P. fluorescens AH2 inhibited the growth of V. anguillarum during coculture, independently of the iron
concentration, when the initial count of the antagonist was 100 to
1,000 times greater that of the fish pathogen. These in vitro results
were successfully repeated in vivo. A probiotic effect in vivo was
tested by exposing rainbow trout (Oncorynchus mykiss
Walbaum) to P. fluorescens AH2 at a density of
105 CFU/ml for 5 days before a challenge with V. anguillarum at 104 to 105 CFU/ml for
1 h. Some fish were also exposed to P. fluorescens AH2
at 107 CFU/ml during the 1-h infection. The combined
probiotic treatment resulted in a 46% reduction of calculated
accumulated mortality; accumulated mortality was 25% after 7 days at
12°C in the probiotic-treated fish, whereas mortality was 47% in
fish not treated with the probiont.
An increasing amount of the world's
fish resources is being supplied by farmed fish. While catches of wild
fish have stagnated at approximately 90 million metric tons, the amount
of farmed fish has increased from 10 million metric tons in 1984 to
more than 20 million metric tons in 1996 (11, 22). Disease
is a major problem for the fish farming industry. Although vaccines are
being developed and marketed, they generally cannot be used as a
universal disease control measure in aquaculture. Juvenile fish are not
fully immunocompetent and do not always respond to vaccination.
Vaccination by injection, sometimes the only effective route of
administration, is impractical when applied to small fish or larger
numbers of fish. The addition of substantial amounts of antibiotics and
chemotherapeutics remains the method of choice for disease control in
many parts of the aquaculture industry. Increased concern about
antibiotic-resistant microorganisms (1) has led to several
alternative suggestions for disease prevention, including the use of
nonpathogenic bacteria as probiotic biocontrol agents (3, 6, 36,
39). Lactic acid bacteria have been tested as probiotics in
warm-blooded animals, and attempts have also been made to use lactic
acid bacteria as antagonists of fish pathogens (14, 24, 25).
Fluorescent pseudomonads have been used as biocontrol agents in several
rhizosphere studies (31), where their inhibitory activity
has been attributed to a number of factors, such as the production of
antibiotics (29, 34), hydrogen cyanide (38), or
iron-chelating siderophores (26, 28). Pseudomonads
constitute a large part of the microflora of the gills, skin, and
intestinal tracts of live fish (9, 37) and are only rarely
reported as pathogens of fish (20). As with their
terrestrial counterparts, aquatic pseudomonads are often antagonistic
against other microorganisms (15, 27), including
fish-pathogenic bacteria (36) and fish-pathogenic fungi
(6, 20). One study demonstrated that bathing Atlantic salmon
presmolts in a strain of Pseudomonas fluorescens reduced subsequent mortality from stress-induced furunculosis (36).
When tested in vitro, iron limitation has been found to facilitate the
antibacterial activity of fluorescent pseudomonads (15, 36).
Thus, inhibition may be due to the production of siderophores, which
deprive the fish pathogen of iron. Production of siderophores is a
virulence factor in many microorganisms, such as members of the family
Enterobacteriaceae, Pseudomonas aeruginosa, and
Vibrio anguillarum (10), as reviewed by
Wooldridge and Williams (40). An efficient salmon
furunculosis vaccine elicits antibodies against iron-repressible outer
membrane proteins of Aeromonas salmonicida (21).
Iron available in the serum of fish, as in mammals (19), is
crucial for infection, and fish with iron overload are more prone to
attack by V. anguillarum than are fish with low serum iron
concentrations (30).
To further study the potential of pseudomonads as biocontrol agents in
fish farming, we investigated the inhibitory activity of a P. fluorescens strain of aquatic origin (AH2) against the fish-pathogenic bacterium, V. anguillarum in vitro and in
vivo. The influence of iron on the inhibitory activity was assessed because the availability of iron is important in virulence and disease.
(Part of this paper has been presented as posters at the General
Meeting of the American Society for Microbiology, 3 to 8 May 1997, and
at the Fifth European Marine Microbiology Symposium, 11 to 15 August
1996.)
Bacterial strains.
A virulent strain of the fish pathogen of
V. anguillarum (90-11-287; serotype O1) that carries the
pJM1 plasmid was obtained from K. Pedersen, Royal Veterinary and
Agricultural University, Copenhagen, Denmark (35). P. fluorescens AH2, which produces several siderophores
(2), was isolated from iced freshwater fish (Lates
niloticus) (18) and is antagonistic toward several gram-positive and gram-negative bacteria, particularly when iron limited (15, 17).
Media.
M9 salts (32) supplemented with 0.4%
glucose and 0.3% Casamino Acids (M9GC) or M9GC plus 3% NaCl
(M9GC+NaCl) was used as low-iron culture medium. The total iron
concentration, as determined by the phenanthroline method optimized for
low concentrations, was estimated to be 0.6 µM (24a).
Iron-replete conditions were obtained by adding 100 µM
FeCl3. In experiments with pure cultures, bacteria were
counted by surface plating on tryptone soy agar plates (Oxoid catalog
no. CM131) incubated at 25°C. V. anguillarum was routinely
cultured on LB medium (Difco 0446) (5) with a total of 2% NaCl.
Siderophore production.
Siderophore production was assayed
on chrome azurol S (CAS) agar (33) based on M9GC
(16). In liquid medium, siderophores were detected by the
CAS assay (33). Equal volumes of sterile-filtered culture
supernatant and CAS assay solution were mixed and left for 30 min at
room temperature. The A630 was measured with
sterile medium and CAS assay solution (33) as a blank. A
negative value indicated the presence of iron-chelating substances such
as siderophores (33).
Agar antagonism assay.
Initial screening of antagonism by
P. fluorescens AH2 was done in a plate assay. V. anguillarum (100 µl precultured in M9GC+NaCl for 5 days at
15°C) was spread on M9GC+NaCl agar plates with and without additional
iron. Wells 3 mm in diameter were punched into the solidified agar, and
10 µl of a 24-h culture of P. fluorescens AH2 was added.
The plates were incubated at 15°C, and zones of inhibition around the
wells were measured after 3 to 5 days.
Effect of P. fluorescens AH2 supernatants.
P.
fluorescens AH2 was precultured in M9GC with and without NaCl and
with or without iron (four combinations) and then used to inoculate 50 ml of M9GC in the same four combinations at an initial cell density of
103 to 104 CFU/ml. The flasks were incubated at
12 to 13°C with agitation (150 rpm), and samples were withdrawn
daily. One milliliter was used for serial dilutions and estimation of
colony counts on tryptone soy agar, and 2 ml was sterile-filtered
(0.2-µm pore size; Sartorius no. 16534). The possible inhibitory
activity of the sterile-filtered supernatant was tested by adding 100 µl of supernatant to 100 µl of fresh medium in microtiter wells
(Nunc microwell 96F) and inoculating it with 10 µl of a dilution of
V. anguillarum yielding approximately 104
CFU/ml. Controls were done by inoculating V. anguillarum in
200 µl of M9GC. Each combination was tested in triplicate, and the growth of the fish pathogen was monitored by recording the optical density at 600 nm (OD600) with a Labsystems Multiscan RC
microtiter plate reader.
Coculture experiments.
P. fluorescens AH2 and V. anguillarum were precultured separately in M9GC+NaCl at 13°C
with aeration for 3 to 5 days. Appropriate dilutions were prepared in
physiological saline, and V. anguillarum was inoculated in
M9GC+NaCl at an initial cell density of approximately 103
CFU/ml, whereas the initial levels of P. fluorescens AH2
were 104, 105, 106,
107, and 108 CFU/ml. All combinations were done
in duplicate. The flasks were incubated at 12 to 13°C with aeration,
and samples were withdrawn daily for determination of bacterial cell
densities. Numbers of V. anguillarum bacteria were estimated
by preparing 10-fold serial dilutions using 1 ml from each dilution to
inoculate tubes with 5 ml of H&L medium (23). The tubes were
covered with paraffin and incubated at 25°C. The fermentative growth
of V. anguillarum caused a change in the pH indicator of the
medium. The highest dilution still showing growth was used to calculate
the number of V. anguillarum bacteria present. This
procedure was chosen because low numbers of V. anguillarum
bacteria had to be estimated in a high background of P. fluorescens AH2, which did not grow in the anaerobic H&L tubes.
Inoculation of H&L medium with 109 P. fluorescens AH2 bacteria did not affect the color of the medium.
Probiotic treatment and infection of fish.
P.
fluorescens AH2 was grown for 6 days at 13°C (150 rpm) in M9GC
without NaCl addition, and V. anguillarum 90-11-287 was grown for 16 h in tryptone soy broth. A total of 644 rainbow trout (Oncorhynchus mykiss Walbaum) weighing approximately 40 g were divided into eight 600-liter tanks, and fish from four of the tanks were exposed for 5 days to P. fluorescens AH2 at a
level of 105 CFU/ml at 12°C (long-term treatment) by
adding the bacteria to the water. All fish were exposed to V. anguillarum at a level of 104 to 105
CFU/ml for 1 h in 50% seawater (i.e., a total of 1.5% NaCl) at 12°C. Half of the probiotically treated fish and half of the
untreated fish were also treated with P. fluorescens AH2
(107 CFU/ml) during exposure to V. anguillarum
(short-term treatment). The P. fluorescens AH2 bacteria used
for this short-term treatment were cultured at 13°C for 7 days in
M9GC with NaCl added. All fish, independently of probiotic treatment,
were physically handled in the same manner. After infection, the fish
were kept at 12°C in fresh water in eight 600-liter tanks with a
water flow of 50 liters/h and fed in accordance with the BioMar A/S
Ecolife 19 feeding table by automatic feeders during a 10-h daily
feeding period. Dead fish were collected and recorded daily. V. anguillarum was isolated from deceased fish by inoculation of head
kidney smears on blood agar plates, and its identity was verified by Mono VaR (BioNor Aqua, Skien, Norway) serum agglutination. Data were
recorded as accumulated mortality. The calculated accumulated mortality
was derived by adding the effect of a particular treatment to the
average mortality of all of the eight tanks and subtracting half of the
effect of the two individual factors (7) (no significant two-factor interactions between the two treatments were recorded). The
statistical significance of the two individual probiotic treatments, as
well as that of the combined effect, was calculated by the analysis of
variance program in the statistical software package Statgraphics
(Statistical Graphics Corporation, Princeton, N.J.).
Agar antagonism assay.
P. fluorescens AH2 caused a
clearing zone with a radius of 3 to 5 mm in lawns of V. anguillarum in the absence of iron. No inhibition zones were
observed when the medium was supplemented with iron.
Effect of P. fluorescens AH2 supernatants.
Strain
AH2 grew well and produced siderophores in Fe-limited M9GC with and
without 3% NaCl (Fig. 1). A weak CAS
reaction (A630, Coculture experiments.
The growth of V. anguillarum
was inhibited under iron-limited conditions by strain AH2 inoculated at
an initial level of 106 to 107 CFU/ml (Fig.
3a). Lower concentrations of strain AH2
(104 to 105 CFU/ml) allowed initial growth of
V. anguillarum, but cell densities never reached the level
of the control. High inoculum concentrations (105 to
107 CFU/ml) of strain AH2 under iron-replete conditions
allowed an initial increase of V. anguillarum followed by a
decrease in the viable count (Fig. 3b). Growth of P. fluorescens AH2 was not affected by coculturing with V. anguillarum (data not shown).
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Inhibition of Vibrio anguillarum by
Pseudomonas fluorescens AH2, a Possible Probiotic Treatment
of Fish
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
0.25) was measured when counts
of strain AH2 reached 106 cells/ml. A significant CAS
reaction (A630, ~0.6), indicating high
levels of siderophores, was not seen until strain AH2 reached 109 CFU/ml (Fig. 1). Addition of iron caused more-rapid
growth and a slightly higher maximum cell density, whereas 3% NaCl
caused a prolonged lag phase and a slightly lower growth rate (Fig. 1). Sterile-filtered strain AH2 supernatants with a strong CAS reaction were inhibitory to V. anguillarum (Fig. 1 and
2). In contrast, supernatants from
iron-replete cultures of strain AH2 or supernatants obtained from
iron-limited cultures at low cell density did not inhibit the growth of
the fish pathogen (Fig. 2b). The iron-replete cultures showed no sign
of iron limitation, as the CAS reaction remained negative. Addition of
iron to inhibitory sterile-filtered supernatants with a strong CAS
reaction eliminated the inhibitory effect. Growth curves of V. anguillarum in M9GC+NaCl (controls) were identical to curves
obtained with supernatants from low counts of strain AH2 that were not
CAS positive (data not shown).
View larger version (22K):
[in a new window]
FIG. 1.
Growth (a) and CAS reaction (b) of P. fluorescens AH2 grown in M9GC with or without 3% NaCl with or
without 0.1 mM FeCl3 at 13°C. The arrows indicate
sampling points where sterilely filtered supernatants were inhibitory
to V. anguillarum.
View larger version (25K):
[in a new window]
FIG. 2.
Growth of V. anguillarum at 25°C in
M9GC-3% NaCl with spent supernatants from P. fluorescens
AH2 cultured without (a) or with (b) 100 µM FeCl3.
View larger version (28K):
[in a new window]
FIG. 3.
Growth of V. anguillarum at 13°C in
M9GC-3% NaCl with and without P. fluorescens AH2 at
different initial cell densities without (a) or with (b) 100 µM
FeCl3.
Probiotic treatment and infection of fish. The accumulated mortality of infected fish not treated with strain AH2 reached 50% 9 days after infection with the onset of mortality at day 3. The mortality leveled off after the 7th day, at which the accumulated mortality was 47% (Fig. 4). No dead fish were found in control tanks not exposed to V. anguillarum. Both the long-term and short-term treatments with strain AH2 caused a decrease in accumulated mortality, to 44 and 35%, respectively. Combining the treatments caused a further reduction in accumulated mortality to 32% (Fig. 4). No significant two-factor interactions between the treatments was seen, and the effect of the two treatments combined was therefore regarded as additive (Table 1). The effect of probiotic treatment was most pronounced during the first days of infection, but all probiotic treatments were also significantly different from the infection control when the mortality stabilized (Fig. 4 and Table 1).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The concept of biological disease control, particularly using nonpathogenic bacterial strains for disease prevention, has received widespread attention during the last decade. Fuller (13) defined a probiotic as "a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance." However, as the skin and gill microflora of fish must be assumed also to contribute to disease prevention, we have used a broader definition here, namely, a live microbial supplement which beneficially affects the host animal by improving its microbial balance. A strain of P. fluorescens was successfully used to reduce the frequency of stress-induced infections by A. salmonicida (36). Austin et al. (3) reported that exposure to a nonpathogenic Vibrio alginolyticus strain reduced subsequent mortality due to vibriosis. To our knowledge, our data are the first to demonstrate a reduction in vibriosis-caused mortality in trout by the use of a probiotic P. fluorescens strain.
In agreement with other studies (15, 26, 41), we have seen that under iron-limited conditions, P. fluorescens AH2 is inhibitory to V. anguillarum when tested in vitro in a well diffusion assay or when sterile-filtered supernatants were tested. Similar to Smith and Davey (36), we found that addition of iron to sterile-filtered supernatants of strain AH2 eliminated the inhibitory activity. The appearance of inhibitory activity in spent supernatants from iron-limited P. fluorescens AH2 coincided with a strong CAS reaction, indicating the presence of siderophores. However, our data do not allow us to specifically implicate siderophores in the active mechanism.
Despite the importance of iron limitation seen in the deferred end point assays, iron was not as important during coculture, when inhibition was more a function of the density of the antagonist than of the iron concentration (Fig. 3). Due to its fast growth, P. fluorescens AH2 may compete for other nutrients, occupy colonization sites, or excrete antibacterial substances (29, 34). However, such substances, if produced, were not present in sufficient concentrations to allow detection in vitro in supernatants from iron-enriched cultures.
High levels of P. fluorescens AH2 were required before inhibition of V. anguillarum could be detected in coculture assays. In agreement with earlier studies of interactions between fish spoilage bacteria (17), sterile-filtered supernatant from P. fluorescens AH2 did not inhibit the growth of V. anguillarum until an antagonist level of 108 CFU/ml was reached. A number of studies have assessed the numbers of pseudomonads required to protect against various plant diseases. Xu and Gross (41) found that compared to infection with 104 Erwinia carotovora bacteria per potato seed, 106 antagonist bacteria per potato seed increased emergence 32% and 1010 antagonist bacteria per potato seed increased emergence 96%. It has been reported similarly that 106 CFU of a fluorescent pseudomonad per seed was required to protect sunflower seeds against Sclerotinia wilt, and protection increased with increasing amounts of the pseudomonad (12). Our studies also show that the antagonist must be present at significantly higher levels than the pathogen, and the degree of inhibition increases with the level of the antagonist. Thus, during coculture, 107 to 109 CFU/ml was required to inhibit the growth of the pathogen (Fig. 3). Therefore, a potential probiotic culture must either be supplied on a regular basis or be able to colonize and multiply on or in the host.
Rhizosphere studies often have great difficulties moving from in vitro to in vivo situations (8). In preliminary in vivo studies with P. fluorescens AH2 (data not shown), we found that short-term exposure, even to high numbers of the bacterium, had no effect on subsequent fish mortality. However, with more-constant exposure to AH2, a significant reduction in mortality was obtained (Fig. 4 and Table 1). The fact that the reduction in mortality obtained by the two different treatments was additive indicated that further reduction in mortality may be obtained by optimizing the procedure. Probiotic treatment of fish thus offers a very promising alternative to the use of antibiotics and chemotherapy.
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ACKNOWLEDGMENTS |
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This study was partly financed by the Danish Biotechnology Program 1991-1995, the Danish Food Technology Program, the Danish Ministry for Food, Agriculture and Fisheries, and the Academy for the Technical Sciences (T.F.N.).
We thank Søren S. Jørgensen for determination of the iron concentration in the defined medium. Valuable comments from two reviewers and Einar Ringø are appreciated.
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FOOTNOTES |
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* Corresponding author. Mailing address: Danish Institute for Fisheries Research, Department of Seafood Research, Technical University of Denmark, Bldg. 221, DK-2800 Lyngby, Denmark. Phone: 45 4525 2586. Fax: 45 4588 4774. E-mail: gram{at}dfu.min.dk.
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REFERENCES |
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Discussion
References
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Applied and Environmental Microbiology, March 1999, p. 969-973, Vol. 65, No. 3
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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[Abstract] [Full Text] -
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