Rev. bras. farmacogn. vol.25 número6

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w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Biofilm

inhibition

activity

of

compounds

isolated

from

two

Eunicea

species

collected

at

the

Caribbean

Sea

Yenny

Martínez

Díaz

a

_,

_Gina

_Vanegas

_Laverde

b

_,

_Luis

_Reina

_Gamba

b

_,

Humberto

Mayorga

Wandurraga

c,∗

,

Catalina

Arévalo-Ferro

d

,

Freddy

Ramos

Rodríguez

b

,

Carmenza

Duque

Beltrán

b

_,

_Leonardo

_Castellanos

_Hernández

b

a_Universidad_Nacional_de_Colombia,_Sede_Bogotá,_Facultad_de_Ciencias,_Posgrado_{Interfacultades}_de_{Microbiología,}_Bogotá_D.C.,_Colombia

b_Universidad_Nacional_de_Colombia,_Sede_Bogotá,_Facultad_de_Ciencias,_Departamento_de_Química,_Grupo_de_Estudio_y_{Aprovechamiento}_de_Productos_Naturales_Marinos_y_Frutas_de

Colombia,BogotáD.C.,Colombia

c_Universidad_Nacional_de_Colombia,_Sede_Bogotá,_Facultad_de_Ciencias,_Departamento_de_Química,_Grupo_de_Productos_Naturales_Vegetales_Bioactivos_y_Química_Ecológica,

BogotáD.C.,Colombia

d_Universidad_Nacional_de_Colombia,_Sede_Bogotá,_Facultad_de_Ciencias,_Departamento_de_Biología,_Grupo_de_{Comunicación}_y_Comunidades_Bacterianas,_Bogotá_D.C.,_Colombia

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30April2015 Accepted9August2015

Availableonline26September2015

Keywords:

Anti-biofilm

Euniceafusca Euniceasp. Lipidcompounds Terpenoids

Marinenaturalproducts

a

b

s

t

r

a

c

t

Biofilmhasaprimaryroleinthepathogenesisofdiseasesandintheattachmentofmulticellularorganisms toafouledsurface.Becauseofthat,thecontrolofbacterialbiofilmshasbeenidentifiedasanimportant target.Inthepresentstudy,fivelipidcompoundsisolatedfromsoftcoralEuniceasp.andthreeterpenoids togetherwithamixtureofsterolsfromEuniceafuscacollectedattheColombianCaribbeanSeashowed differenteffectivenessagainstbiofilmformationbythreemarinebacteriaassociatedwithimmersed fouledsurfaces,Ochrobactrumpseudogringnonense,AlteromonamacleodiiandVibrioharveyi,andagainst twoknownbiofilmformingbacteria,PseudomonasaeruginosaATCC27853andStaphylococcusaureus

ATCC25923.ThepurecompoundswerecharacterizedbyNMR,HRESI-MS,HRGC-MSandopticalrotation. Themosteffectivecompoundswerebatylalcohol(1)andfuscosideEperacetate(6),actingagainstfour strainswithoutaffectingtheirmicrobialgrowth.Compound1showedbiofilminhibitiongreaterthan30% againstA.macleodii,andupto60%againstO.pseudogringnonense,V.harveyiandS.aureus.Compound 6inhibitedO.pseudogringnonenseandV.harveyibetween25and50%,andP.aeruginosaorS.aureusup to60%at0.5mg/ml.Theresultssuggestthatthesecompoundsexhibitspecificbiofilminhibitionwith lowerantimicrobialeffectagainstthebacterialspeciesassayed.

Introduction

Allimmersedsurfacesaresubjecttocolonizationby microor-ganismsasbacteria,microalgaeandthenmacroorganismssuchas invertebrates,ina complexlayercalledbiofouling(Vianoetal., 2009).Bacteria arethefirst colonizersand theirbiofilm forma-tiontakesplaceinmultiplestages(Jadhavetal.,2013).Biofilms areabundantinnatureandaredevelopedwhenplanktonic bacte-riaadheretoasurfaceandinitiatetheformationofamicrocolony thatexistsasacommunityencasedinanextracellularmatrixthat confersahighdegreeofprotection(O’tooleetal.,2000;Huigens etal.,2007).

∗ Correspondingauthor.

E-mail:hmayorgaw@unal.edu.co(H.MayorgaWandurraga).

In marine environments, biofilms are formed by bacteria, diatomsandprotozoa(Vianoetal.,2009;Dobretsovetal.,2013). Bacterialbiofilmsareknownforbeingimplicatedinthe regula-tionofa subsequentsettlementbyinvertebratelarvae,mediate larval metamorphosis, provide suitable food source to newly metamorphosed juveniles,and serve asbiofilterstoabsorband biodegradeexcessivenutrients(Yang etal.,2014).Ontheother hand, biofilms are prevalent in medical, dental, industrial and marineenvironmentalsettings,wheretheyareundesirabledueto theirpathogenicity,andresistancetowardantimicrobialagentsor anti-biofoulingtechnologies(Dusaneetal.,2011;Rautetal.,2013). Inthissense,inhibitionofbiofilmsmayplayanimportantrolein biofoulingprevention(Quinnetal.,2012;Dobretsovetal.,2013).

Althoughadiversityofstrategieshasbeendevelopedtocontrol biofouling,thesearchfornovelandeffectivetechnologies contin-ues(Dusane etal.,2011).Oneofthemostecologicallyrelevant antifoulingstrategiesistoapplythechemicaldefensesofsessile

http://dx.doi.org/10.1016/j.bjp.2015.08.007

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marineorganisms that keep theirbody surfacesfree of fouling (Jadhavetal.,2013;MajikandParvatkar,2014).Those antifoul-ingcompoundscouldbeisolatedfromsponges,algae,bryozoans, andcorals,whichproducesuchcompoundspresumablyasameans ofprotectionfrompredationorfoulingortoreducecompetition forspace(Mora-Cristanchoetal.,2011).Theuseofthesemarine naturalproductsismore favoredthancommonly usedbiocides becauseof theirbiocompatibility, stability, biodegradabilityand lowtoxicityunderdifferentenvironmentalconditions(Qietal., 2008;Dusaneetal.,2011;Telloetal.,2011).Therefore,continuous searchesforantifoulingcompoundsfrommarineinvertebratesof theColombianCaribbeanSeahavebeenreported(Mayorgaetal., 2011;Telloetal.,2011,2012;Cuadradoetal.,2013).However,an anti-biofilmpossibilityofthecompoundsassayedinthepresent investigationhasnotbeenstudiedbefore.

Theaimofthepresentstudywastoevaluatetheanti-biofilm activityoflipidsandterpenoidcompoundsisolatedfromEunicea sp.andEuniceafuscaDuchassaingandMichelotti(phylumCnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea, family Plexauridae),collectedatSantaMartaBay(Colombia),againstthree marinebacteriaandtwoknownbiofilmformingbacteriabythe microtiterplatemethod.

Materialsandmethods

Generalexperimentalprocedures

Forhigh-resolutionelectrospraymassspectrometry(HRESIMS), a LC-MS-IT-TOF was used on positive mode, and for ESIMS a LCMS-2010-ESI(Shimadzu,Tokyo,Japan)waslikewiseused. Opti-calrotation wasmeasuredona Polax-2L(Atago,Japan)and an ADP440(Bellingham+Stanley,USA).TheNMRspectraat400MHz for1_H_and₁₀₀_MHz_for13_C_and_DEPT_in_CDCl

3 wererecordedon aBrukerAvance400spectrometer,withTMS.Highperformance liquidchromatographyHPLCwasperformedonaMerck-Hitachi (L-4250UV/Visdetector,USA)at210nm,andonaLunaC18column 250mm×4.60mmi.d.×5␮m(Phenomenex).Forgas

chromatog-raphy mass spectrometry HRGCMS, Agilent equipment model 7890A-5975C with a DB-1 (30m×250␮m×0.25␮m) capillary

columnwasemployed.Allsolventsusedwereanalyticalgrade.

Coralmaterial

Euniceasp.andEuniceafuscaDuchassaingandMichelotti, sam-pleswerecollectedbyhandatSantaMartaBay(Latitude11.25N, Longitude74.2W)in2007,throughscubadivingat6–14mdeepby Prof.Dr.S.Zea,andvoucherspecimensweredepositedatthe Insti-tutodeCienciasNaturalesdelaUniversidadNacionaldeColombia codedasICN-MHN-PONo.250andNo.252respectively.The col-lectionof freshcoral colonies wassuperficiallyair-driedin the shade,immediatelyfrozenandwasthentransportedbyairplane tolaboratoryinBogota,wherewaskeptinfreezeruntilextraction.

Extractionandisolationofcompounds

ThesoftcoralEuniceasp.wascutintosmallpiecesandwas partiallyair-dried(wetweight850g,600gdryweight),extracted threetimeswithdichloromethane:methanol(1:1)atroom tem-perature.Thecombinedextractswereconcentrated byrotatory evaporation,andthecrudeextract(25g)waspartitionedbetween dichloromethane:water(1:1).Theorganicfraction(15g)was sub-jectedtosilicagel,columnchromatography(CC)withsolventsof increasingpolarityhexane,benzene,ethylacetateandmethanol, toobtainfifteenfractions(F1–F15)inabioguidedmannerusingan antimicrobialtestwithseveralmarinebacteriaisolatedfromheavy

colonizedsurfaces byMora-Cristancho etal. (2011).The bioac-tivefractionswerefurtherfractionatedoverSiO2CCasfollows.F4 (0.3g),elutedwithhexane-benzene(1:1),waschromatographed using an elution gradient with hexane:benzene (10:0–0:10) to givecompound 3 (60mg).Fraction F7(0.8g), eluted with ben-zene:ethylacetate(9:1),wassubjectedtoCCwithbenzene-ethyl acetate(10:0–2:8)andsubfractionF7.1waspurifiedbyHPLCusing methanoltoobtaincompounds 4(5mg)and 5(7mg).Fraction F10 (1.1g), eluted with benzene:ethylacetate (2:8),was sepa-ratedonCCusingbenzene-ethylacetate(5:5–0:10).Subsequently, subfractionF10.6,elutedwithbenzene:ethylacetate(4:6), con-tainscompound1(150mg)andsubfractionF10.9,waspurifiedby reverse-phaseCC(LichroprepRP-18,Merck)andfinallybyHPLCto affordcompound2(9mg).

FromE.fusca,thefollowingproductswerepreviouslyisolated: fuscosideE,fuscosideB(7),(+)-germacreneD(8),themixtureof sixsterols(9–14),andthecompoundfuscosideEperacetate(6)was semi-synthesized(Reinaetal.,2011).

Compoundsidentificationandfattyacidanalysis

To determinethe structure of compounds from Euniceasp., opticalrotation,NMR, ESIMSand HRESIMSdatawererecorded. Compounds2,4and5weresubjectedtomethanolysisaspreviously reported(Ramosetal.,2006),andthemixtureoffattyacidmethyl esterswasanalyzedbyHRGCMSasdescribedearlier(Castellanos etal.,2010).

Bacterialstrains

Two reference gram-positive strains, Staphylococcus aureus (ATCC 25923)and Staphylococcus epidermidis (ATCC 51400310) and two gram-negative Pseudomonas aeruginosa (ATCC 27853) andKlebsiellapneumoniae(206),commonlyreportedasbiofilms forming strains, were used (Quinn et al., 2012). They were suppliedbythemolecularepidemiologylaboratoryofthe Biotech-nologyInstitute(UniversidadNacionalde Colombia).Wildtype marinebacteriaassociated withfouledsurfaceswerealsoused; thesegram-positivestrains:Kokuriasp.(10-4DEP),Bacillus mega-terium(16-Ains5)andOceanobacillusiheyensis(31-C),andthese gram-negative strains: Vibrio campbellii (6-8PIN),Ochrobactrum pseudogringnonense(4-4DEP),Alteromonamacleodii(29-C)and Vib-rioharveyi(Phy-2A),aswasreportedforMora-Cristancho etal. (2011).

Microtiterplatebiofilmproductionassay

Inordertoselectthemostsuitablereferencebacteriaas con-trolfortheassaywithmarinestrains,thebiofilmformationwas testedbytheirabilitytoadheretowellsofpolystyrene(353072-BD FalconTM_Clear_96-well)_using_a_modification_of_protocol_by_O’toole

etal.(2000).Pre-inoculumsweregrownintrypticasesoy broth

(TSB)toanopticaldensityat600nm(OD600)of0.2–0.3.Fromeach culturedbroth,20␮lwasinoculatedtothewellsandincubatedfor

24hand48hat37◦_C._After_removal_of_the_planktonic_cells_by

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inordertoestablishwhetherthechangefrom37◦_C_to₂₅◦_C_had

affectedtheirbiofilmformation,forthesubsequentassayunder thoseconditionswithmarinestrains.

Biofilminhibitionactivityassay

Threemarinestrains:O.pseudogringnonense,A.macleodiiand V.harveyi,andtworeferencestrains:P.aeruginosaandS.aureus, selectedbecauseoftheirstrongbiofilm-formingabilityconfirmed in this study,were usedin the assayof compounds (1–14), as previouslyreported(Telloetal.,2011),andfollowingthebiofilm productionassaydescribedabove.Pre-inoculumsweregrownto anOD600of0.1–0.2inmarinebroth(MB)andTSBrespectively. Twenty␮lofinoculated cultureweremixed withtwodifferent

aliquot, 100␮g/wellor 5␮g/well ofpurecompound completed

withmediumtoconcentrationsat0.5mg/mlor0.025mg/ml,and incubatedfor48hat25◦_C._Planktonic_cells_were_discarded,_and

finallythedifferentialstainingwasquantifiedusingaCVassay.The valuesareexpressedintermsofthepercentageofbiofilminhibited incomparisontotheuntreatedcontrolbiofilms(positivecontrol). Thepercentageofgrowthinhibitionwascalculatedforeachassay asacontrol.Additionally,toevaluatethecompounds’effectonthe remainingviablebacteria,aftertheassay,10␮lofthesuspension

fromeachwellwasinoculatedintoLuriaBertani(LB)agarplate solidmedium,andbacterialgrowthwastested.

Antimicrobialactivityassay

BioguidedseparationofcompoundsfromEuniceasp.was per-formedagainstthesevenmarinestrainssetupasdescribedby

Mora-Cristancho et al. (2011). Moreover, the concentration at

which compounds(1–14)couldbebactericidalwasestablished throughamodificationoftheKirby-Bauerdiskdiffusion suscep-tibilitytesting(Wilkinsetal.,1972),usingtworeferencestrains, K.pneumoniaeandS.aureus,andtwomarinestrains,A.macleodii andV.harveyi.LBsolidagarplateswereinoculatedwiththetested microorganisms.Thenfilterpaperdisksof5mmdiameter impreg-natedwithdifferentamountsofcompounds(3␮g/disk,7.5␮g/disk,

15␮g/diskand30␮g/disk)wereplacedontheinoculatedplates

andincubatedat37◦_C_for_reference_strains_or_at₂₅◦_C_for_marine

strains.Thegrowthinhibitionhalosweremeasuredfor24and48h ofincubation.

Statisticalanalyses

Biofilminhibitionandgrowthinhibitiondatawerestatistically analyzedbyANOVA,andallexperimentswereconductedin trip-licateforeachstrain.Todetectdifferencesbetweenandamong thegroupsofdataorwhensignificantdifferencesweredetected, pairwisecomparisonsweremadeamongallthegroupsusingthe Turkey’smethodtoadjustformultiplecomparisons(Montgomery, 2007).

Results

IdentificationofcompoundsisolatedfromEuniceasp

CompoundsfromEuniceasp.wereidentifiedbycomparingthe spectroscopicdata withthose previously published. Compound

1 (176mg/kg yield from the coral) had the molecular formula C21H44O3,asdeducedfromHRESIMSwitha[M+Na]+peakatm/z 367.3194 (calcd. for C21H44O3Na+, 367.3183). Optical rotation [␣]D25+3.2(c1.5,CHCI3)andits1Hand13CNMRspectrashowed acloseresemblancetothosepublishedbyQuijanoetal.(1994)

and Sun et al. (2005) for (S)-(+)-3-octadecyloxy-l,2-propane

diol,analkylglycerol alsoknownasbatylalcohol. Compound2

(10.6mg/kg)hadthemolecularformulaC34H65NO3asestablished byHRESIMS.ItsmethanolysisandanalysisbyHRGCMSdisplayed a singlevolatilecompound identifiedas methylhexadecanoate. The negative optical rotation [␣]D25 −9.7 (c 0.3, CHCl3) and NMR spectra suggest that 2, (2S,3R,4E,8E)-N -hexadecanoyl-2-amino-4,8-octadecadiene-1,3-diol, was a ceramide as reported

by Shin and Seo (1995) and Han et al. (2005). Compound 3

(70mg/kg) was recognized as octadecyl (9Z)-9-octadecenoate (stearyl oleate), onthe basis of HRESIMS, and NMR data were coincident tothose of thewax previously synthetized (Vieville etal.,1995).Compound4(5.9mg/kg)wasopticallyinactiveandits methanolysisgaveamixtureofmethylhexadecanoateandmethyl (9Z)-9-octadecenoateinproportion2:1byHRGCMS.Accordingto ESIMS,theNMRdatawereidenticaltothoseofthesynthesized glycerolipid 1,3-dihexadecanoyl-2-(9Z-octadecenoyl)-glycerol as reportedbyStamatovandStawinski(2007).NMRspectroscopic featuresof5(8.2mg/kgyieldfromthecoral)weresimilartothose of 4exceptfor thelackof doublebond signalsand it wasalso optically inactive. It revealed the presence of methyl hexade-canoateandmethyloctadecanoateina1:2ratioasperHRGCMS. InagreementwithESIMSanalysis,compound5waspostulatedas 1,3-dioctadecanoyl-2-hexadecanoyl-glycerol.

1

2

3

4

5

6 7

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0.00 0.20 0.40 0.60 0.80 1.00

48h 24h

OD

621

nm

P. aeruginosa

ATCC 27853

K. pneumoniae

206

S. aureus

ATCC 25923

S. epidermidis

ATCC 51400310

Fig.1.Biofilmformingabilityofreferencestrainsat37◦_C._Microtiter_plate_method wasusedforquantifyingbiofilmformation,dyedwithcrystalviolet.Thebarson thegraphrepresentmean±SDofthebiofilmformationfromthreeindependent experiments.TheX-axisgivestheincubationtime(h).

0.0 0.4 0.8 1.2 1.6 2.0

O. pseudogringnonense 4-4DEP

A. macleodii 29-C

V. harveyi Phy-2A

P. aeruginosa ATCC 27853

OD

621

nm

Fig.2. Biofilmformingabilityofmarinestrainsduring48hat25◦_C._Microtiter_plate methodandCVstainingtoquantifywereused.Betweenthereferencestrains,P. aeruginosawasusedaspositivecontrolinthepresentstudyduetoitshigherbiofilm formationlevel.Thebarsonthegraphrepresentmean±SDofbiofilmformation fromthreeindependentexperiments.TheANOVAtestshowedastaticallysignificant differencerelativetocontrol(p<0.05).

Biofilmproductionassaybymicrotiterplatetest

The results showed that the biofilm was higher after 48h ofincubation for three referencestrains. From these,P. aerugi-nosaexhibitedthehighest biofilm formationlevel, significantly more than other strains (Fig. 1), followed by S. aureus being the selected reference strains. On the other hand, the assay withthose two strains at 25◦_C _determined _that _the _S. _aureus_’

biofilmdecreasedwhileP.aeruginosashowedhigherlevelsinits biofilmformation.Thus, P.aeruginosawasusedaspositive con-trolin biofilmsformation assay of marine strains. Then, those resultsshowedthatthemarine strainsO. pseudogringnonese, A. macleodii and V. harveyi had higher biofilmformation, suchas shownbythecontrolstrainP.aeruginosa(Fig.2),whiletheother marine bacteria practically did not show any biofilm produc-tion.

Biofilminhibitionactivityassayofcompounds

Theanti-biofilm activityofcompounds1–8andsterols9–14

wasdeterminedtowardthreemarinestrains,andtworeference strains,allselectedinthepresentstudyduetotheirstrong biofilm-formingability.Asaresult,O.pseudogringnonense wasinhibited over50%onlybycompound1,upto61%at0.5mg/ml,whileat 0.025mg/ml,compounds 1,7,8 hadan inhibition closeto30% (Fig.3).Thestrainexhibitedlowresponse toothercompounds. Onlycompound1wasupto33%andthemixture9–14decreased thebiofilmformationofA.macleodiiat0.5mg/ml(Fig.3).Other compoundsslightlyreduced microbialgrowthwithouteffecton biofilms.InV.harveyi,biofilminhibitionwasshownby1upto64%

at0.5mg/ml; andat0.025mg/mlcompounds7and8 werethe mostactive.ResultswiththereferencestrainP.aeruginosa,shown ascompounds 6,(9–14) and2 at0.5mg/mlintheirorder, had thehighestlevelofeffectivenessreaching6upto66%,whereas at 0.025mg/ml, 2 and (9–14) showed effectiveness as well. Additionally,S.aureusastheonlygram-positive strainincluded wasthemostsusceptibleandcompounds1,3,6and7showed highpercentagesofinhibitionupto100%,at0.5mg/ml(Table1) andat0.025mg/mltheseexhibitedlowactivity.Inallcases,the compounds were less active against bacterial growth and the assessmentofbacterialviabilitybyplatingonagarshowedthat eachstraingrewagain.

9

12

R= R=

13 14

10 11

Biofilmsformationwassignificantlyreducedascomparedto untreated biofilmsof each of thesebacteria at thetested con-centrationsofcompounds.ThiswasvalidatedbyANOVA,witha significancelevelof95%andameancomparisonTukeytest,with an␣errorof0.05.

AntimicrobialactivityinextractsandcompoundsfromEunicea softcorals

Inthisstudy,thecrudeextractofEuniceasp.evidenced mod-erategrowthinhibitionagainstsixofthesevenmarinebacteria. Theorganicfractionandisolatedcompounds1to5showedmild antimicrobialactivitytowardO.pseudogringnonense,Kokuriasp., andV.harveyi.Thedeacetylatedof6,theterpene8andthesterols (9–14)fromEuniceafuscaalsohadlowactivityagainstBacillussp., O.pseudogringnonense andV. harbeyi asdetailedby Reinaetal. (2011).Finally,thediskdiffusionsusceptibilitytestdidnot evi-dence that compounds 1–14 couldbe bactericide, even at the highestassayedconcentration(30␮g/disk),confirmingtheirlow

antibacterialactivity.

Discussion

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0 20 40 60 80 100 5 4 3 2 1 0.5 mg/ml 5 4 3 2 1 9-14 8 7 6 0.025 mg/ml

8 9-14 7 6

Inhibition (%)

B Alteromona macleodii 29-C

Growth inhibition % Biofilm inhibition %

0 20 40 60 80 100 5 4 3 2 1 0.5 mg/ml 5 4 3 2 1 9-14 8 7 6 0.025 mg/ml 9-14 8 7 6 Inhibition (%)

C Vibrio harveyi Phy-2A Growth inhibition (%)

Biofilm inhibition (%)

D Pseudomonas aeruginosa ATCC 27853

Growth inhibition (%) Biofilm inhibition (%) 0 20 40 60 80 100 5 4 3 2 1

0.5 mg/ml

5 4 3 2 1 9-14 8 7 6

0.025 mg/ml

9-14 8 7 6

Inhibition (%)

A Ochrobactrum pseudogringnonense 4-4DEP

Growth inhibition (%) Biofilm inhibition (%)

E Staphylococcus aureus ATCC 25923

Growth inhibition (%) Biofilm inhibition (%)

Fig.3.Biofilminhibitionandgrowthinhibitioneffectofisolatedcompounds1tomixture9–14fromEuniceasp.andEuniceafuscaagainstmarineandreferencebacteria. Biofilmwasquantifiedusingthemicrotiterplatetestandcrystalvioletassay.TheX-axisgivesthenumberoftheisolatedcompoundanditsconcentrationat0.5mg/ml or0.025mg/ml.Thebarsonthegraphrepresentmean±SDasapercentageofbiofilminhibitionoftriplicateexperiments.Biofilmsformationwassignificantlyreducedas comparedtountreatedbiofilms(control)ofeachofthesebacteriaatthetestedconcentrationsofthecompounds(ANOVAp<0.05)withasignificancelevelof95%anda meancomparisonTukeytestwithan␣errorof0.05.

acylglycerols: 1,3-dihexadecanoyl-2-(9Z-octadecenoyl)-glycerol (4), and 1,3-dioctadecanoyl-2-hexadecanoyl-glycerol (5). For decades,thefamilyof bioactivelipidshasgrowntremendously

(McAnoy et al., 2005; Deniau et al., 2010; Blunt et al., 2014).

Therefore, the possibilities of biofilm inhibition of lipids from Euniceasp.,togetherwithterpenoids,fuscosideEperacetate(6), fuscoside B(7), (+)-germacrene D (8), and sterols (9–14), from Eunicea fusca (Reina et al., 2011), were essayed by microtiter

Table1

Biofilminhibition(valuesfrom25%)ofcompoundsisolatedfromEuniceasp.andEuniceafuscaat0.5or0.025mg/ml,againstthreemarinestrainsandtworeferencebacteria.

Meanpercentageinhibitionofbiofilm*

Compound O.pseudogringnonense

4-4DEP A.macleodii 29-C V.harveyi Phy-2A P.aeruginosa ATCC27853 S.aureus ATCC25923

0.5 0.025 0.5 0.025 0.5mg/ml 0.025 0.5 0.025 0.5 0.025

1 61±6.2 39±5.8 33±6.0 64±4.2 38±1.3 85±10.3

2 63±9.2 42±1.8

3 91±9.0

4 32±3.7 35±4.6 26±4.8

5

6 29±6.5 33±6.4 44±9.0 66±14.3 100±0.0

7 32±6.1 26±5.3 47±2.9 69±8.4

8 38±6.8 32±5.0 34±0.0 49±1.6 30±3.4

9–14 29±7.0 45±10.0 65±1.4 37±5.4

Biofilmwasquantifiedbymicrotiterplatetestandcrystalviolet.Inthistable,theeffectivenessofmorethan25%ofinhibitionwasconsideredincomparisontotheuntreated controlbiofilms(positivecontrol).

*_Data_represent_mean

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platetests,a frequentlyused techniquefor quantifying biofilm formation(O’tooleetal.,2000;Al-SohaibaniandMurugan,2012).

Accordingtotheresultsobtainedinthisstudy,outofthefour referencestrains,P.aeruginosaevidencedthebestbiofilm forma-tion,followedbyS.aureusandtheywereusedforfurtheressays (Fig.1).P.aeruginosaandS.aureusaremodelorganisms,knownfor theircapacityofbiofilmformationandtheirinherentresistance toantimicrobialagents(O’tooleetal.,2000;Quinnetal.,2012). Ontheotherhand,theresultsaboutmarinestrainsshowedthat O.pseudogringnonense,A.macleodiiandV. harveyihad the high-estbiofilmformation(Fig.2),asthecontrolstrainP.aeruginosaat thesameculturecondition.AspublishedbyVikrametal.(2011), biofilmformationandbioluminescenceproductioninV.harveyiare aknownquorumsensing-controlledprocess,anditwasrecently studiedalsoinO.pseudogringnonense(Cuadradoetal.,2013),while A.macleodiihasbeencommonlyfoundinbiofilmsfromdifferent depthsofoceans(Ivars-Martinezetal.,2008).Thesemarine bacte-riatogetherwithreferencestrainsaremore efficient,andwere selectedaspositivecontrolforthefurtherbiofilminhibitiontests. Consequently,compounds1–8andsterols9–14weretestedfor assessingtheanti-biofilmability,andhaddifferenteffectiveness dependingonthebacteria,andsomewereactiveagainstvarious microorganisms(Fig.3).Compounds1and6werethemost effec-tiveastheyactedagainstfourstrains.Compound1inhibitedA. macleodii,O.pseudogringnonense,V.harveyiandS.aureustoa per-centagegreaterthan30%againstthefirst,andabove60%forthe lastthree.Compound6affectedO.pseudogringnonenseandV. har-veyiwitheffectivenessbetween25and50%,andP.aeruginosaand S.aureusabove60%ofbiofilminhibition(Table1).Compounds7

and8hadaneffectgreaterthan25%againstthreemicroorganisms. Compound4andmixtureofsterols9–14 wereeffectiveagainst twostrains,withapercentagehigherthan25%.Compounds2and

3showedeffectivenessonlytoonestrainbutabove50%.Incontrast, compound5wasinactivetobiofilminhibitionagainstallstrains.

Additionally, compounds 1–5, 8 and sterols 9–14 had low antibacterial activity by the disk-diffusion test against marine bacteria.Results,consistentwiththoseofthediskdiffusion suscep-tibilitytest,showedthatnoneofthecompounds1–8and(9–14) displayedabactericidaleffectevenatthehighestassayed concen-trationandthattheyonlyevidencedtheirlowantibacterialactivity. Interestingly,theresults confirmedthat thecompounds exhib-itedgrowthinhibitionmuchlowerthantheanti-biofilmactivity, andthataproportionofthebiofilmbacteriastillremainedviable, suggestingthatthereisbiofilminhibitionwithoutaffectingtheir growth,andthatthesecompoundsarelittletoxic.

Inthepresent study,thealkylglycerol– batylalcohol 1– is describedforthefirsttimeasananti-biofilmcompound. Alkyl-glycerolsarefoundinmarineinvertebratesinhigherlevelsthanin landvertebrates(WatschingerandWerner,2013).Theirbiological activitiescomprisestimulationofhematopoiesis,immunological defenses,antitumorandantimetastasis(Deniauetal.,2010;Iannitti andPalmieri,2010).Batylalcohol1waspreviouslyisolatedfrom sharkliver oil(Myers and Crews, 1983), from theinvertebrate spongeDesmapsamnaanchorata(Quijanoetal.,1994),fromthesoft coralPlexauraflexuosa(KindandBergmann,1942),fromCladiella species(Radhika,2006),andfromthegorgoniaScirpeariagracilis, showingmoderategrowthinhibitionformarinebacterialspecies asPseudoalteromonaspiscida,Ruegeriasp.,Vibrioalginolyticus,V. furnissiiandV.harveyi(Qietal.,2008).Onlydodecylglycerolwas foundasaneffectiveantibacterial againstStreptococcus faecium (Vedetal.,1984).Inpreviousstudies,otherstructurallyrelated naturallipidsshowedpotentialanti-biofilm,suchasaglycolipid composedofglucoseandpalmiticacid,whichdisruptedbiofilms ofCandidaalbicans,P.aeruginosaandBacilluspumilus(Dusaneetal., 2011),andthelinoleicacidthatreducedbiofilmaccumulationof Streptococcusmutans(Jungetal.,2014).

Inearlierworks,ceramide2wasobtainedfromanemone Para-condylactisindicus,gorgonianAcabariaunhlata(ShinandSeo,1995), softcoralDendronephthyagigantean(Hanetal.,2005),and zoan-thids, Palythoa caribaeorum and Protopalyhtoa variabilis. Other ceramideshavebeenrecognizedfor creatinganepidermal per-meability barrier, and showedcytotoxic or antitumoractivities (Almeidaetal.,2012;Bluntetal.,2014).Waxesandglycerolipids havebeencommonlydetectedbyanalyticaltechniques(Vieville

etal.,1995;McAnoyetal.,2005).Wax3waspreviously

biosyn-thetizedby Corynebacteriumsp.(Seoetal.,1982)and hasbeen detected in the eyelids of humans and mammalians (Butovich etal.,2007).Thepurificationofglycerolipids4and5hasnotbeen reported from natural sources. Nevertheless, another regioiso-merof4,called1,2-dihexadecanoyl-3-(9Z-octadecenoyl)-glycerol, whichisquiral,wasdetectedinbovineheart(Shinzawa-Itohetal., 2007).Inthesameway,theregioisomerof5, 1,2-dioctadecenoyl-3-hexadecanoyl-glycerol,wasdetectedinrapeseedoil(Beermann etal.,2007).

EuniceafuscaisthesourceoffuscolandfuscosidesA–E hav-inganti-inflammatoryactivity,aswellas(+)-germacreneD8and thesterolsmixture(9–14)(Shin andFenical,1991;Reinaetal., 2011).The(−)-germacreneDisa commoncompound ofplants (Reinaetal.,2011),andtheessentialoilthatcontainsgermacrene fromAchilleamillefoliuminhibitsbiofilmsofListeriamonocytogenes andListeriainnocua(Jadhavetal.,2013).Rautetal.(2013) demon-stratedthebiofilminhibitionofotherterpenoidsagainstCandida albicans.Moreover,bioactivesterolsmixtureorpurecompounds exhibitantibacterialorantifoulingactivities(Al-Lihaibietal.,2010;

Castellanos et al., 2010; Blunt et al., 2014). In the meanwhile,

otherditerpenoidssuchas xenicanes,dolabelanesand cembra-noids frommarinesources possess anti-biofilm activityagainst marine bacterialisolates (Viano et al., 2009; Tello et al., 2011, 2012).Notwithstanding,previousworkshavenotyetreportedthe possibilityof lipidsand terpenoidshereassayedasanti-biofilm compounds.

Inthepresentstudy,batylalcohol1andfuscosideEperacetate6

werethemosteffectivecompoundsagainstthebestbiofilm forma-tionbythebacteriatestedhere,andduetotheirlowantibacterial effect theycouldbe consideredas less toxic anti-biofilm com-poundstotheenvironment.Theresultsarestimulatingforfurther researchandbetterunderstandingthestructure-activity relation-shiportheactionmodeofthemostactivecompoundsfoundin thisstudy.In thissense,furtherstudiestodeterminethe mini-malinhibitoryconcentration(MIC)withsomeofthesesynthesized compoundsareunderdevelopmentinourlaboratory.

Conclusion

The present study showed the anti-biofilm capacity of five lipid compounds isolated from soft coral Eunicea sp., together with three terpenoids and a mixture of sterols from E. fusca collectedatCaribbeanSea,againstthreemarinebacteria,O. pseu-dogringnonense,A.macleodiiandV.harveyi,andtwoknownbiofilm formingbacteria, P.aeruginosaand S. aureus.They showed dif-ferenteffectiveness andthemostactivecompoundswerebatyl alcoholandfuscosideEperacetate.Thesecompoundsexhibit spe-cificbiofilminhibitionandcouldbeconsideredlesstoxictothe environmentbyhavinglowerantimicrobialactivity.

Author’scontributions

(7)

workandcontributedtothebiologicalstudies.FARRsupervised the laboratory work and contributed to critical reading of the manuscript. CDBcontributed withstudydesign and supervised theresearchproject.LCHsupervisedtheresearchprojectashead oftheresearch team,and contributedtocritical reading ofthe manuscript.Alltheauthorshave read thefinal manuscript and approveditssubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

ThisworkwassupportedbyUniversidadNacionalde Colom-bia[grantnumber5815/2012and 20406/2014],andColciencias [grantnumber1101-521-28468/2011].TheMinisteriode Ambi-ente, Vivienda y Desarrollo Territorial granted permission for researchonmarineorganisms[No.4of10/02/2010,andNo.0306 of22/02/2011].TheauthorsaregratefultoprofessorsSvenZea, MonicaPuyanaandEdissonTello.

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