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Abstract
Because of the evolving resistance of microorganisms to existing antibiotics, there is an increasing need for new antibiotics not
only in human but also in veterinary medicine. Competition for space and nutrients led to the evolution of antimicrobial defence
strategies in the aquatic environment. Therefore, aquatic organisms, e.g., seaweeds, offer a particularly rich source of potential new
drugs. The aim of our studies was to identify seaweeds, which possess activities against fish pathogenic bacteria and could be an
alternative to the commonly used antibiotics in aquaculture.
Dichloromethane, methanole and water extracts of 26 species of cultivated seaweeds were screened for their antibacterial
activities against five fish pathogenic bacteria strains (Aeromonas salmonicida ssp. salmonicida, Aeromonas hydrophila ssp.
hydrophila, Pseudomonas anguilliseptica, Vibrio anguillarum, Yersinia ruckeri). The dichloromethane extracts of Asparagopsis
armata, Ceramium rubrum, Drachiella minuta, Falkenbergia rufolanosa, Gracilaria cornea and Halopitys incurvus showed strong
antibacterial activities. V. anguillarum and P. anguilliseptica were the two most susceptible bacteria strains. The screening results
confirm the possible use of seaweeds as a source of antimicrobial compounds or as a health-promoting food for aquaculture.
2005 Elsevier B.V. All rights reserved.
Keywords: Algae; Screening; Antimicrobial activity; Fish pathogenic bacteria
1. Introduction
Antibiotic treatment of bacterial diseases in fish
culture has been applied for many years. The occurrence
of antibiotic resistant bacteria associated with fish
diseases is a worldwide problem in aquaculture, which
has received considerable attention in the last years and
continues to increase due to the absence of a more
effective and safer use of antibiotics. The prevention and
treatment of these infectious diseases by applying
Corresponding author. Tel.: +49 3834 86 4868; fax: +49 3834 86
4885.
E-mail address: lindequi@uni-greifswald.de (U. Lindequist).
0044-8486/$ - see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2005.11.051
80
Table 1
Classification and origin of studied algae
Table 1 (continued)
Species
Class
Location
Origin
Anotrichum
furcellatum
(J. Agardh)
Baldock
Asparagopsis
armata Harvey
Ceramium rubrum
(Hudson)
C. Agardh
Chondrus crispus
Turner
Codium taylorii
P.C. Silva
Corallina elongata
J. Ellis and
Solander
Cystoseira
abies-marina
(Turn.) Agardh
Dictyota
dichotoma
(Hudson)
J.V. Lamouroux
Drachiella minuta
(Kylin)
Maggs and
Hommersand
Enteromorpha
compressa
(L.) Nees
Falkenbergia
rufolanosa
(Harvey)
F. Schmitz
Fryeella gardneri
(Setchell) Kylin
Gracilaria bursapastoris Gmelin
Gracilaria cornea
J. Agardh
(red variant: r)
Gracilaria cornea
J. Agardh
(green
variant: g)
Gracilaria cornea
J. Agardh (m)
Gracilaria
verrucosa
(Hudson)
Papenfuss
Grateloupia
dichotoma
J. Agardh
Grateloupia
doryphora
(Montagne)
M.A. Howe
Rhodophyceae
Atlantic, Faro,
Portugal
Rhodophyceae
Rhodophyceae
Rhodophyceae
Chlorophyceae
Rhodophyceae
Atlantic, Pleubian,
France
North Sea,
Helgoland,
Germany
Atlantic, Mindelo
Beach, Portugal
Atlantic, East
coast Gran Canaria
Atlantic, East
coast Gran Canaria
3*
1*
2
5
5
Phaeophyceae
Atlantic, East
coast Gran Canaria
Phaeophyceae
Atlantic, Faro,
Portugal
Rhodophyceae
Chlorophyceae
Rhodophyceae
Rhodophyceae
Rhodophyceae
Rhodophyceae
Atlantic, East
coast Gran Canaria
Atlantic, East
coast Gran Canaria
Atlantic,
Villefrance, France
Pacific, Fryday
harbour, Canada
Atlantiv, Aveiro
Lagoon, Portugal
Carribean Sea
1
2
5
Species
Class
Location
Origin
Halopitys incurvus
(Hudson)
Batters
Hypnea spinella
(C. Agardh)
Ktzing
Hypoglossum
hypo-glossoides
(Stackhouse)
F.S.Collins and
Hervey
Laminaria
saccharina (L.)
J.V. Lamouroux
Laurencia
chondrioides
B?rgesen
Palmaria palmata
(L.) Kuntze
Plocamium
cartilagineum
(L.) P.S. Dixon
Ulva rigida
C. Agardh
Ulva rigida
C. Agardh
Valonia utricularis
(Roth)
C. Agardh
Rhodophyceae
Atlantic, East
coast Gran Canaria
Rhodophyceae
Atlantic, East
coast Gran Canaria
Rhodophyceae
North Sea,
Helgoland,
Germany
Phaeophyceae
Rhodophyceae
Atlantic, East
coast Gran Canaria
Rhodophyceae
Atlantic, Roscoff,
France
North Sea,
Helgoland,
Germany
Atlantic, Faro,
Portugal
Atlantic, East
coast Gran Canaria
Atlantic, East
coast Gran Canaria
Rhodophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
4
5
5
Rhodophyceae
By cultivation of
red variant
Rhodophyceae
Rhodophyceae
Atlantic, East
coast Gran Canaria
Rhodophyceae
Atlantic, East
coast Gran Canaria
Rhodophyceae
81
82
Table 2
Antimicrobial activity of the investigated dichloromethane-extracts (2 mg/disc) of seaweed species in agar diffusion assay (inhibition zone was
measured without paper disc)
Species
Anotrichum furcellatum
Asparagopsis armata
Ceramium rubrum
Chondrus crispus
Codium taylorii
Corallina elongata
Cystoseira abies-marina
Dictyota dichotoma
Drachiella minuta
Enteromorpha compressa
Falkenbergia rufolanosa
Fryeella gardneri
Gracilaria bursa-pastoris
Gracilaria cornea (r)
Gracilaria cornea (g)
Gracilaria cornea (m)
Gracilaria verrucosa
Grateloupia dichotoma
Grateloupia doryphora
Halopitys incurvus
Hypnea spinella
Hypoglossum
hypoglossoides
Laminaria saccharina
Laurencia chondrioides
Palmaria palmata
Plocamium cartilagineum
Ulva rigida
Ulva rigida
Valonia utricularis
Oxytetracycline 20 g/disc
Aeromonas
salmonicida
Aeromonas
hydrophila
Yersinia ruckeri
5.6 2.2
19.3 1.3
6.1 1.5
1.4 0.4
0
0
5.0 2.0
5.1 2.7
1.4 1.4
0
14.5 1.7
0
4.9 2.8
10.1 2.2
13.1 2.0
12.4 3.6
2.2 1.5
0.6 1.8
0
6.3 2.2
0
7.6 4.1
2.5 2.3
26.9 2.2
17.0 3.8
0
0.4 0.5
0
2.1 1.4
1.6 0.9
15.0 3.7
3.1 2.3
15.1 1.0
0
1.5 1.0
4.1 2.9
27.2 9.9
5.1 1.7
0
0.7 1.2
0
17.2 3.9
0
3.1 3.6
3.7 2.0
17.0 2.2
2.6 3.4
0
0
0
0
0
0
0
7.6 1.7
0
0
0.3 0.6
3.3 1.8
1.1 0.8
2.4 1.7
0.3 0.9
0
5.4 4.4
0
0
0.4 0.7
14.9 2.7
0
0
0
0
0
0
0
0
12.5 0.8
0
0
0
2.5 1.2
0
0
0
0
12.5 1.6
0
0
0
15.3 1.7
0
0
0
0
0
0
0
0
7.3 1.9
0
0
0
3.0 2.6
0
0
0
0
8.9 3.0
0
0
3.7 1.9
6.9 2.2
0
2.8 1.2
2.3 1.8
3.0 1.4
0
20.0
0
8.8 7.3
0
6.3 1.2
0.8 1.1
3.1 0.7
0
0
0
0
2.8 1.9
0
0
0
27.0
0
0.4 1.2
0
0.2 0.6
0
0
0
31.0
0
0
0
0
0
0
0
14.5
Inhibition zones N 15 mm were declared as strong (bold), from 8 to 15 mm as moderate and from 1 to 8 mm as weak activities.
the results of previous studies using other test microorganisms (Mahasneh et al., 1995; Padmakumar and
Ayyakkannu, 1997). The MIC values of the extracts are
much higher than those of the positive control substance
oxytetracycline. This is not surprisingly because extracts
are complex mixtures of many compounds and the
portion of active compounds is very low.
Several solvents were used for the extraction of
freeze-dried seaweed powder. Our data revealed that
considerable inhibition zones were only observed for
the dichloromethane extracts. Therefore, the compounds responsible for the antimicrobial activity are
lipophilic. We assume, that the active compounds
could be, at least partly, lipophilic halogenated
compounds. Halogen-containing terpenoids, acetylens
and phenols have been identified in several seaweed
species as biologically active compounds (Knig and
Wright, 1997; Carvalho and Roque, 2000; Vairappan
et al., 2001). The two most active dichloromethane
extracts were those of A. armata and F. rufolanosa.
Both algae represent different generations of the same
species of red algae. Whereas A. armata represents the
gametangial phase, F. rufolanosa is the tetrasporangial
phase. They are morphological dissimilar (Knappe,
1980). A series of small molecular volatile halogenated compounds (halomethanes, haloether, haloacetales)
accounts for the antimicrobial action of A. armata
(McConnell and Fenical, 1977). They could also be
responsible for the observed effects against fish
pathogenic bacteria. Considering Laurencia chondrioides, possessing moderate activity, we found two
halogenated sesquiterpenes as responsible metabolites
for the observed activity against fish pathogenic
bacteria (Bansemir et al., 2004). Besides halogenated
compounds, fatty acids have been identified as
antimicrobial substances in algae (Rosell and Srivastava, 1987). We showed that C. rubrum contains
several fatty acids with antimicrobial activities (Bansemir, 2004). The structure elucidation of further
active compounds is in progress.
Some halogenated compounds from algae possess
cytotoxic and mutagenic properties (Teuscher and
Lindequist, 1994). Before starting the use of seaweeds
for prophylaxis and therapy of bacterial fish diseases in
vitro and in vivo toxicity studies with seaweeds,
fractions and purified compounds have to be done.
Besides, investigations about the stability of the
materials in an aquatic environment, about digestability
for fish and about the metabolism of the active
compounds in fishes would be necessary. Only
dependent on these results a decision can be made
which material would be the most suitable for a possible
83
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