Anti Bacterian A

(2006) 79–84www.elsevier.com/locate/aqua-online

Aquaculture 252

Screening of cultivated seaweeds for antibacterial activity againstfish pathogenic bacteria

Anne Bansemir, Maja Blume, Susanne Schröder, Ulrike Lindequist ⁎

Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-Moritz-Arndt University Greifswald, Friedrich-Ludwig-Jahn-Strasse 17,D-17487 Greifswald, Germany

Received 6 April 2005; accepted 18 November 2005

Abstract

Because of the evolving resistance of microorganisms to existing antibiotics, there is an increasing need for new antibiotics notonly in human but also in veterinary medicine. Competition for space and nutrients led to the evolution of antimicrobial defencestrategies in the aquatic environment. Therefore, aquatic organisms, e.g., seaweeds, offer a particularly rich source of potential newdrugs. The aim of our studies was to identify seaweeds, which possess activities against fish pathogenic bacteria and could be analternative to the commonly used antibiotics in aquaculture.

Dichloromethane, methanole and water extracts of 26 species of cultivated seaweeds were screened for their antibacterialactivities against five fish pathogenic bacteria strains (Aeromonas salmonicida ssp. salmonicida, Aeromonas hydrophila ssp.hydrophila, Pseudomonas anguilliseptica, Vibrio anguillarum, Yersinia ruckeri). The dichloromethane extracts of Asparagopsisarmata, Ceramium rubrum, Drachiella minuta, Falkenbergia rufolanosa, Gracilaria cornea and Halopitys incurvus showed strongantibacterial activities. V. anguillarum and P. anguilliseptica were the two most susceptible bacteria strains. The screening resultsconfirm 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 fishculture has been applied for many years. The occurrenceof antibiotic resistant bacteria associated with fishdiseases is a worldwide problem in aquaculture, whichhas received considerable attention in the last years andcontinues to increase due to the absence of a moreeffective and safer use of antibiotics. The prevention andtreatment of these infectious diseases by applying

⁎ Corresponding author. Tel.: +49 3834 86 4868; fax: +49 3834 864885.

E-mail address: [email protected] (U. Lindequist).

0044-8486/$ - see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.aquaculture.2005.11.051

products from marine organisms appears as a possiblealternative. Hence, the interest in marine organisms as apotential and promising source of pharmaceutical agentshas increased during the last years (Lindequist andSchweder, 2001; Mayer and Hamann, 2002; Newman etal., 2003). Seaweeds are considered as such a source ofbioactive compounds as they are able to produce a greatvariety of secondary metabolites characterized by abroad spectrum of biological activities. Compoundswith cytostatic, antiviral, anthelmintic, antifungal, andantibacterial activities have been detected in green,brown and red algae (Lindequist and Schweder, 2001;Newman et al., 2003). There are numerous reportsconcerning the inhibiting activities from macroalgae

Table 1Classification and origin of studied algae

Species Class Location Origin

Anotrichumfurcellatum(J. Agardh)Baldock

Rhodophyceae Atlantic, Faro,Portugal

4

Asparagopsisarmata Harvey

Rhodophyceae Atlantic, Pleubian,France

3*

Ceramium rubrum(Hudson)C. Agardh

Rhodophyceae North Sea,Helgoland,Germany

1*

Chondrus crispusTurner

Rhodophyceae Atlantic, MindeloBeach, Portugal

2

Codium tayloriiP.C. Silva

Chlorophyceae Atlantic, Eastcoast Gran Canaria

5

Corallina elongataJ. Ellis andSolander

Rhodophyceae Atlantic, Eastcoast Gran Canaria

5

Cystoseiraabies-marina(Turn.) Agardh

Phaeophyceae Atlantic, Eastcoast Gran Canaria

5

Dictyotadichotoma(Hudson)J.V. Lamouroux

Phaeophyceae Atlantic, Faro,Portugal

4

Drachiella minuta(Kylin)Maggs andHommersand


5

Enteromorphacompressa(L.) Nees


5

Falkenbergiarufolanosa(Harvey)F. Schmitz

Rhodophyceae Atlantic,Villefrance, France

4

Fryeella gardneri(Setchell) Kylin

Rhodophyceae Pacific, Frydayharbour, Canada

1

Gracilaria bursa-pastoris Gmelin

Rhodophyceae Atlantiv, AveiroLagoon, Portugal

2

Gracilaria corneaJ. Agardh(red variant: r)

Rhodophyceae Carribean Sea 5

Gracilaria corneaJ. Agardh(greenvariant: g)

Rhodophyceae By cultivation ofred variant

5

Gracilaria corneaJ. Agardh (m)

Rhodophyceae Mixture of red andgreen variant

5

Gracilariaverrucosa(Hudson)Papenfuss


5

GrateloupiadichotomaJ. Agardh


5

Grateloupiadoryphora(Montagne)M.A. Howe


5

Table 1 (continued)

Species Class Location Origin

Halopitys incurvus(Hudson)Batters


5

Hypnea spinella(C. Agardh)Kützing


5

Hypoglossumhypo-glossoides(Stackhouse)F.S.Collins andHervey


4

Laminariasaccharina (L.)J.V. Lamouroux

Phaeophyceae North Sea, Sylt,Germany

1

LaurenciachondrioidesB?rgesen


5

Palmaria palmata(L.) Kuntze

Rhodophyceae Atlantic, Roscoff,France

1

Plocamiumcartilagineum(L.) P.S. Dixon


1

Ulva rigidaC. Agardh

Chlorophyceae Atlantic, Faro,Portugal

4

Ulva rigidaC. Agardh


5

Valonia utricularis(Roth)C. Agardh


5

1—Sylt, Germany; 2—Porto, Portugal; 3—Pleubian France; 4—Faro,Portugal; 5—Gran Canaria, Spain.*Collected from natural sources.

80 A. Bansemir et al. / Aquaculture 252 (2006) 79–84

against human pathogens, fungi and yeasts, but only fewcontain data about effects against fish pathogens(Sridhar and Vidyavathi, 1991; Mahasneh et al., 1995;de Val et al., 2001; Liao et al., 2003).

Therefore, the aim of the present study was toinvestigate the antimicrobial activity of extracts ofmarine algae against five fish pathogenic bacteria thatare often the cause of bacterial diseases in aquaculture.The investigated seaweeds were selected by seaweedspecialists of the EU project SEAPURA according totheir potential as component of integrated aquaculturesystems, as sources of pharmaceuticals, fine chemicalsor cosmetics and as fish feed. The possible use of activeseaweeds for prevention or treatment of the bacterialfish diseases should be discussed.

2. Materials and methods

2.1. Algal materials and preparation of the material

Seaweeds (19 Rhodophyceae, 3 Phaeophyceae, 4Chlorophyceae) were collected or cultivated at five

81A. Bansemir et al. / Aquaculture 252 (2006) 79–84

different places of Europe (Sylt, Germany; Porto,Portugal; Pleubian, France; Faro, Portugal; GranCanaria, Spain, Table 1). Asparagopsis armata wascollected at the Atlantic coast of France by the group ofCEVA, Pleubian (Dr. Patrick Dion). Ceramium rubrumwas collected in the North Sea by the group of theAlfred-Wegner-Institut (Prof. Klaus Lüning). All otherseaweeds were cultivated by the collaborating groups ofthe EU project SEAPURA (Sylt: Stiftung Alfred-Wegner-Institut, Prof. Klaus Lüning; Porto: Universi-dade de Porto, Instituto Botanico, Dr. Isabel SousaPinto; Faro: Universidade do Algarve, Centre of MarineSciences, Prof. Rui Santos; Gran Canaria: Universidadde Las Palmas de Gran Canaria, Instituto de AlgologiaAplicada, Prof. Guillermo Garcia Reina). Falkenbergiarufulanosa is the tetrasporangial phase of the species A.armata. Because both generations of this species wereinvestigated separatedly, A. armata and F. rufulanosaamounted separatedly. From Gracilaria cornea 3variants were used. The Ulva rigida samples camefrom 2 different locations. The fresh samples werewashed with seawater and fresh water to remove salts,epiphytes, microorganisms and other suspended materi-als. The clean algae were frozen and lyophilized. Thedry material was stored at −20 °C. Voucher specimensare deposited in the Institute of Pharmacy, Departmentof Pharmaceutical Biology, Ernst-Moritz-Arndt-Univer-sity Greifswald, Germany.

2.2. Preparation of seaweed extracts

Extracts of the freeze dried and powdered biomasswere prepared using different organic solvents withincreasing polarity (dichloromethane, methanole andwater). Each extraction was carried out in a Soxhletapparatus for 24 h and after evaporation in vacuum theextracts were stored at −20 °C until use.

2.3. Antibacterial tests

The agar diffusion assay was performed according toEuropean Pharmacopoe (1997). One loopful of each testorganism (Vibrio anguillarum DSMZ 11323, Pseudo-monas anguilliseptica DSMZ 12111, Yersinia ruckeriATCC 29493, Aeromonas salmonicida sp. salmonicidaATCC 51413, Aeromonas hydrophila sp. hydrophilaDSMZ 6173) was suspended in 3 ml 0.9% NaClsolution separately. Nutrient agar (Difco™ Tryptic SoyAgar, Becton Dickinson and Company, USA) wasinoculated with this suspension of the respectiveorganism and poured into a sterile petri dish. Sterilisedpaper discs, containing the extract of the alga (2 mg),

were transferred onto these prepared petri dishes.Oxytetracycline (Merck, Germany) was used as apositive, the solvent of each extract as a negativecontrol. A pre-diffusion for 3 h was guaranteed.Inhibition zones were measured after 18 h incubationat 26 °C. The inhibition zones were measured exceptingthe 6 mm paper disc. After pre-diffusion and incubationthe petri dishes were sprayed with a colouring solution(p-iodo nitrotetrazolium violet, 5% in 50% aqueousethanole). Living bacteria produce a red colouredcompound, the inhibition zone appears colourless(Brantner, 1997). Every experiment was carried out 10times. Inhibition zonesN15 mmwere declared as strong,from 8 to 15 mm as moderate and from 1 to 8 mm asweak activities. Minimal inhibitory concentration (MIC)values against V. anguillarum and P. anguillisepticawere determined by standard serial broth microdilutionassay (European Pharmacopoe, 1997).

2.4. Statistical analysis

The data were statistically analysed by applying anone-way ANOVA.

3. Results

The results of the antimicrobial screening assays aresummarized in Table 2. It can be seen that only thedichloromethane extracts showed significant inhibitoryeffects. Dichloromethane extracts of 6, 4 or 11 samplesexhibited strong, moderate or weak antimicrobialactivity, respectively. 8 dichloromethane extracts didnot display activity. Considering the methanole extracts,only 6 species showed moderate or weak antimicrobialactivity, and no water extract exhibited antibacterialactivity.

The two most susceptible organisms were V.anguillarum and P. anguilliseptica, which were inhib-ited by extracts of 17 and 15 species, respectively. Thestrongest antibacterial activities were obtained by A.armata with inhibition zones of 19 mm against V.anguillarum. Considerable antimicrobial activitiesagainst V. anguillarum were presented also by Falken-bergia rufolanosa and G. cornea (r, g, m). The MICvalues against V. anguillarum were b100 μg/ml for A.armata and b400 μg/ml for C. rubrum, F. rufolanosa,G. cornea and Halopitys incurvus. The MIC values ofthe other extracts were N400 μg/ml. The MIC value foroxytetracycline against V. anguillarum amounted to0.5 μg/ml.

The growth of P. anguilliseptica was stronglyinhibited by A. armata and G. cornea (g) (27 mm).

Table 2Antimicrobial activity of the investigated dichloromethane-extracts (2 mg/disc) of seaweed species in agar diffusion assay (inhibition zone wasmeasured without paper disc)

Species Inhibition zones (mm,) against

Vibrio anguillarum Pseudomonasanguilliseptica

Aeromonassalmonicida

Aeromonashydrophila

Yersinia ruckeri

Anotrichum furcellatum 5.6±2.2 2.5±2.3 3.7±2.0 0.4±0.7 0Asparagopsis armata 19.3±1.3 26.9±2.2 17.0±2.2 14.9±2.7 15.3±1.7Ceramium rubrum 6.1±1.5 17.0±3.8 2.6±3.4 0 0Chondrus crispus 1.4±0.4 0 0 0 0Codium taylorii 0 0.4±0.5 0 0 0Corallina elongata 0 0 0 0 0Cystoseira abies-marina 5.0±2.0 2.1±1.4 0 0 0Dictyota dichotoma 5.1±2.7 1.6±0.9 0 0 0Drachiella minuta 1.4±1.4 15.0±3.7 0 0 0Enteromorpha compressa 0 3.1±2.3 0 0 0Falkenbergia rufolanosa 14.5±1.7 15.1±1.0 7.6±1.7 12.5±0.8 7.3±1.9Fryeella gardneri 0 0 0 0 0Gracilaria bursa-pastoris 4.9±2.8 1.5±1.0 0 0 0Gracilaria cornea (r) 10.1±2.2 4.1±2.9 0.3±0.6 0 0Gracilaria cornea (g) 13.1±2.0 27.2±9.9 3.3±1.8 2.5±1.2 3.0±2.6Gracilaria cornea (m) 12.4±3.6 5.1±1.7 1.1±0.8 0 0Gracilaria verrucosa 2.2±1.5 0 2.4±1.7 0 0Grateloupia dichotoma 0.6±1.8 0.7±1.2 0.3±0.9 0 0Grateloupia doryphora 0 0 0 0 0Halopitys incurvus 6.3±2.2 17.2±3.9 5.4±4.4 12.5±1.6 8.9±3.0Hypnea spinella 0 0 0 0 0Hypoglossumhypoglossoides

7.6±4.1 3.1±3.6 0 0 0

Laminaria saccharina 3.7±1.9 0 0 0 0Laurencia chondrioides 6.9±2.2 8.8±7.3 0 0.4±1.2 0Palmaria palmata 0 0 0 0 0Plocamium cartilagineum 2.8±1.2 6.3±1.2 2.8±1.9 0.2±0.6 0Ulva rigida 2.3±1.8 0.8±1.1 0 0 0Ulva rigida 3.0±1.4 3.1±0.7 0 0 0Valonia utricularis 0 0 0 0 0Oxytetracycline 20 μg/disc 20.0 27.0 31.0 14.5

Inhibition zonesN15 mm were declared as strong (bold), from 8 to 15 mm as moderate and from 1 to 8 mm as weak activities.


Inhibition zones between 14 mm and 17 mm weremeasured for C. rubrum, Drachiella minuta, F. rufola-nosa and H. incurvus. MIC valuesb400 μg/ml for P.anguilliseptica were only attained by G. cornea (g) andF. rufolanosa extracts. The MIC value for oxytetracy-cline against P. anguilliseptica was 0.08 μg/ml.

An influence on the growth of Aeromonas salmoni-cida was detected for 8 seaweed species. Asparagosisarmata showed the strongest activity (17 mm). H.incurvus and F. rufolanosa developed inhibition zonesbetween 5 and 10 mm.

Only 3 extracts (A. armata, H. incurvus, F.rufolanosa) showed antimicrobial activities againstAeromonas hydrophila that are worth mentioning.

Antimicrobial effects against Y. ruckeri were pre-sented by A. armata, F. rufolanosa and H. incurvus.

Among the methanole extracts only those of C.rubrum, F. rufolanosa and H. incurvus demonstrated

weak inhibiting activities against V. anguillarum, P.anguilliseptica and A. salmonicida.

Summarizing the results it can be conducted that themost effective seaweeds were A. armata, C. rubrum, D.minuta, F. rufolanosa, G. cornea (g) and H. incurvus.

4. Discussion

The main objective of this study was to evaluatethe ability of different cultivated seaweed speciesfrom several location to inhibit the growth of fishpathogenic bacteria with the aim to use them in thefuture as alternatives to common antibiotics inaquaculture.

Most active species were A. armata, C. rubrum, D.minuta, F. rufolanosa, G. cornea and H. incurvus. Theybelong to the Rhodophyceae. The high efficiency ofseaweeds belonging to the Rhodophyceae agrees with

83A. Bansemir et al. / Aquaculture 252 (2006) 79–84

the results of previous studies using other test micro-organisms (Mahasneh et al., 1995; Padmakumar andAyyakkannu, 1997). The MIC values of the extracts aremuch higher than those of the positive control substanceoxytetracycline. This is not surprisingly because extractsare complex mixtures of many compounds and theportion of active compounds is very low.

Several solvents were used for the extraction offreeze-dried seaweed powder. Our data revealed thatconsiderable inhibition zones were only observed forthe dichloromethane extracts. Therefore, the com-pounds responsible for the antimicrobial activity arelipophilic. We assume, that the active compoundscould be, at least partly, lipophilic halogenatedcompounds. Halogen-containing terpenoids, acetylensand phenols have been identified in several seaweedspecies as biologically active compounds (König andWright, 1997; Carvalho and Roque, 2000; Vairappanet al., 2001). The two most active dichloromethaneextracts were those of A. armata and F. rufolanosa.Both algae represent different generations of the samespecies of red algae. Whereas A. armata represents thegametangial phase, F. rufolanosa is the tetrasporangialphase. They are morphological dissimilar (Knappe,1980). A series of small molecular volatile halogenat-ed compounds (halomethanes, haloether, haloacetales)accounts for the antimicrobial action of A. armata(McConnell and Fenical, 1977). They could also beresponsible for the observed effects against fishpathogenic bacteria. Considering Laurencia chon-drioides, possessing moderate activity, we found twohalogenated sesquiterpenes as responsible metabolitesfor the observed activity against fish pathogenicbacteria (Bansemir et al., 2004). Besides halogenatedcompounds, fatty acids have been identified asantimicrobial substances in algae (Rosell and Srivas-tava, 1987). We showed that C. rubrum containsseveral fatty acids with antimicrobial activities (Ban-semir, 2004). The structure elucidation of furtheractive compounds is in progress.

Some halogenated compounds from algae possesscytotoxic and mutagenic properties (Teuscher andLindequist, 1994). Before starting the use of seaweedsfor prophylaxis and therapy of bacterial fish diseases invitro and in vivo toxicity studies with seaweeds,fractions and purified compounds have to be done.Besides, investigations about the stability of thematerials in an aquatic environment, about digestabilityfor fish and about the metabolism of the activecompounds in fishes would be necessary. Onlydependent on these results a decision can be madewhich material would be the most suitable for a possible

practical use. Toxicological tests using fish and humancell cultures and investigations for digestability in fishesare currently undertaken.

5. Conclusions

The results clearly show that seaweeds are aninteresting source for biologically active compoundsthat may be applied for prophylaxis and therapy ofbacterial fish diseases additionally or instead ofcommercial antibiotics. An alternative approach to theuse of extracts, fractions or purified compounds fromalgae as drugs might be to employ seaweeds as fish feedcomponents. However, prior further investigationsregarding toxicity, stability and metabolism of seaweedsand seaweed components must be undertaken.

Acknowledgement

This work is supported by EU grant Q5RS-2000-31334 (SEAPURA) in the specific programme ‘Qualityof Life and Management of Living Resources’.

Supply of algae biomass by our SEAPURA projectpartners is gratefully acknowledged.

References

Bansemir A., 2004. Phytochemische und biologische Untersuchungenausgewählter Makroalgen unter besonderer Berücksichtigung ihrerWirksamkeit gegen die Erreger bakterieller Fischkrankheiten. PhDthesis, University Greifswald.

Bansemir, A., Just, N., Michalik, M., Lalk, M., Lindequist, U., 2004.Sesquiterpene derivatives from the red alga Laurencia chon-drioides with antibacterial activity against fish and humanpathogenic bacteria. Chem. Biodiv. 1, 463–467.

Brantner, A., 1997. Influence of various parameters on the evaluationof antibacterial compounds by the bioautographic tlc assay. Pharm.Pharmacol. Lett. 7, 152–154.

Carvalho, L.R., Roque, N.F., 2000. Halogenated and/or sulphatedphenols from marine macroalgae. Quimica Nova 23, 757–765.

de Val, A.G., Platas, G., Basilio, A., Cabello, A., Gorrochategui, J.,Suay, I., Vicente, F., Portilllo, E., del Rio, M.J., Reina, G.G.,Peláez, F., 2001. Screening of antimicrobial activities in red, greenand brown macroalgae from Gran Canaria (Canary Islands, Spain).Int. Microbiol. 4, 35–40.

European Pharmacopoe, 1997. Mikrobiologische Wertbestimmungvon Antibiotika, Diffusionsmethode, 3. Ausgabe, Deutscher–Apotheker–Verlag, Stuttgart, 1997, Kapitel 2.7.2.

Knappe, J., 1980. Physiological studies on Asparagopsis falkenbergia.Ber dtsch bot Ges 92, 473–482 (in German).

König, G.M., Wright, A.D., 1997. Sesquiterpene content of theantibacterial dichlormethane extract of the red alga Laurenciaobtusa. Planta Med. 63, 186–187.

Liao, W.-R., Lin, J.-Y., Shieh, W.-Y., Jeng, W.-L., 2003. Antibioticactivity of lectins from marine algae against marine vibrios. J. Ind.Microbiol. Biotech. 30, 433–439.


Lindequist, U., Schweder, T., 2001. Marine biotechnology. In: Rehm,H.J., Reed, G. (Eds.), Biotechnology, vol. 10. Wiley-VCH,Weinheim, pp. 441–484.

Mahasneh, I., Jamal, M., Kashashneh, M., Zibdeh, M., 1995.Antibiotic activity of marine algae against multiantibiotic resistantbacteria. Microbios 83, 23–26.

Mayer, A.M.S., Hamann, M.T., 2002. Marine pharmacology in 1999:compounds with antibacterial, anticoagulant, antifungal, anthel-mintic, anti-inflammatory, antiplatelet, antiprotozoal and antiviralactivities affecting the cardiovascular, endocrine, immune andnervous systems, and other miscellaneous mechanism of action.Comp. Biochem. Physiol., Part C 132, 315–339.

McConnell, O., Fenical, W., 1977. Halogen chemistry of the red algaAsparagopsis. Phytochemistry 16, 367–374.

Newman, D.J., Cragg, G.M., Snader, K.M., 2003. Natural products assource of new drugs over the period 1981–2002. J. Nat. Prod. 66,1022–1037.

Padmakumar, K., Ayyakkannu, K., 1997. Seasonal variation ofantibacterial and antifungal activities of the extracts of marinealgae from southern coast of India. Bot. Mar. 40, 507–515.

Rosell, K.-G., Srivastava, L.M., 1987. Fatty acids as antimicrobialsubstances in brown algae. Hydrobiologia 151/152, 471–475.

Sridhar, K.R., Vidyavathi, N., 1991. Antimicrobial activity ofseaweeds. Acta Hydrochim. Hydrobiol. 5, 455–496.

Teuscher, E., Lindequist, U., 1994. Biogene Gifte. Gustav FischerVerlag, Stuttgart. 681 pp. (in German).

Vairappan, C.C., Daitoh, M., Suzuki, M., Abe, T., Masuda, M., 2001.Antibacterial halogenated metabolites from the Malaysian Lau-rencia sp. Phytochemistry 58, 291–297.

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Anti Bacterian A