Rev. bras. farmacogn. vol.27 número1

(1)

w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Anti-biofilm

activity

against

Staphylococcus

aureus

MRSA

and

MSSA

of

neolignans

and

extract

of

Piper

regnellii

Lara

Z.S.

Brambilla

a

_,

_Eliana

_H.

_Endo

a

_,

_Diógenes

_A.G.

_Cortez

b

_,

_Benedito

_P.

_Dias

_Filho

a,∗

a_Laboratório_de_Inovac¸_ão_Tecnológica_no_{Desenvolvimento}_de_Fármacos_e_Cosméticos,_Departamento_de_Farmácia,_Universidade_Estadual_de_Maringá,_Maringá,_PR,_Brazil

b_Laboratório_de_Pesquisas_em_Produtos_Naturais_e_{Biotecnologia,}_Departamento_de_Farmácia,_Universidade_Estadual_de_Maringá,_Maringá,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received16February2016 Accepted9August2016 Availableonline13October2016

Keywords:

Biofilm Conocarpan

Dichloromethaneextract Eupomathenoid-5

Staphylococcusaureus

a

b

s

t

r

a

c

t

Manyinfectionsworldwideareassociatedwithbacterialbiofilms.Theeffectsofisolatedneolignans (conocarpanandeupomathenoid-5)andthedichloromethaneextractofPiperregnellii(Miq.)C.DC., Piperaceae,weretestedagainstisolatesofmethicillin-resistantStaphylococcusaureusand methicillin-sensitiveS.aureusbiofilmsandS.aureusplanktoniccells.Thedichloromethaneextractpresentedbetter resultsthanisolatedneolignansagainstallofthebiofilmstested,withaminimuminhibitory concen-tration<400␮g/mlforpreformedbiofilmsandminimalbiofilminhibitoryconcentrationof15.6␮g/ml forbiofilmformation.Theminimuminhibitoryconcentrationtoplanktoniccellswas<12.5␮g/ml.These resultsindicateagoodeffectofthedichloromethaneextractagainstmethicillin-resistantS.aureusand methicillin-sensitiveS.aureusbiofilmsandefficientprophylaxis.

Introduction

Infectionsthatareassociatedwithantimicrobialresistanceare a primary challengein publichealth, resulting in highrates of morbidityandmortality,increasedlengthofhospitalization,and higher healthcare costs (Neidellet al., 2012). According tothe

NationalInstitutesofHealth(2002),approximately80%ofall

infec-tionsworldwideareassociatedwithbiofilms,especiallythosethat involvebiomaterials.

Biofilmsarecommunitiesofmicroorganismsthatare embed-dedinanextracellularmatrixthatiscomposedofproteins,lipids, polysaccharides,andnucleicacids.Themembersofabiofilmare protectedfromenvironmentalfactors (e.g., ultravioletlight and dehydration)andhostimmunecells(e.g.,neutrophilsandother phagocytes)(Hall-Stoodleyetal.,2004).Biofilm-associated bacte-riaarealsomuchmoreresistanttoantimicrobialagents(Stewart

andCosterton,2001).

Studies that investigate biofilm physiology are important. Searchingfornewstrategiestocontrolthiscomplexmodeof bac-teriallifeisextremelychallenging(Trentinetal.,2013).Plantswith antibacterialactivity,suchasthegenusPiper,haveshownpromise.

∗ _{Corresponding}_author.

E-mail:bpdfilho@uem.br(B.P.DiasFilho).

Piperregnellii(Miq.)C.DC.,popularlyknowninBrazilas “pari-paroba,” is an herbaceous plant found to exist in tropical and subtropicalregionsoftheworld(Cronquist,1981).Leavesandroots areusedascrudeextracts,infusionsorplaterstotreat wounds, reductionofswellingsandskinirritations(Corrêa,1984).Extracts oftheleavesofP.regnelliihaveshown goodantibacterial activ-ityagainstStaphylococcusaureusandBacillussubtilis(Pessinietal., 2003)andantifungalactivityagainsttheyeastsCandidaalbicans,

Candidakrusei,andCandidaparapsilosis(Pessinietal.,2005).The extract of the leavesand neolignan eupomatenoid-5have also shownactivityagainstmethicillin-resistantS.aureus(Marc¸aletal., 2010).NeolignansthatareisolatedfromtheleavesofP.regnelliialso haveactivityagainsttheparasitesTrypanosomacruzi(Luizeetal., 2006)andLeishmaniaamazonensis(Vendramettoetal.,2010).

BecauseofthereportedeffectofP.regnelliiagainstS.aureus,the aimofthepresentstudywastotestadichloromethaneextractofP. regnelliiandisolatedneolignans(conocarpanand eupomathenoid-5)againstmethicillin-resistantS.aureusbiofilmsand methicillin-sensitive S. aureus biofilms that were obtained from clinical isolates.

Materialsandmethods

Plantmaterial

Leaves from Piper regnellii (Miq.) C. DC., Piperaceae, were collectedinJune2012intheProfa.IreniceSilvaMedicinalPlant

http://dx.doi.org/10.1016/j.bjp.2016.08.008

(2)

GardenonthecampusoftheUniversidadeEstadualdeMaringá andidentifiedbyMariliaBorgooftheBotanyDepartmentofthe UniversidadeFederaldoParaná.Avoucherspecimen(HUM8392) wasdepositedintheHerbariumoftheUniversidadeEstadualde Maringá,Paraná,Brazil.

HPLCanalysis

The analyses were carried out using a Waters Binary HPLC Pump1525,equippedwithUV-VISdetector2489,anautosampler 2707witha20␮lloop,andcontrolledbyBreeze2Software.

Chro-matographicseparationswerecarriedoutinaPhenomenexODS (C18)Lunacolumn,5␮m,250×4.6mm,maintainedatroom

tem-perature,inanisocraticsystem,usingacetonitrile–wateracidified with2%aceticacid(70:30,v/v)withaflowrateof1ml/min.The detectionwascarriedoutat280nmandtheruntimewas20min. Conocarpan(1),eupomathenoid-6(2)and eupomathenoid-5(3) werequantifiedbyexternalstandardization,inmethodpreviously validatedbeinglinear,preciseandaccurate.Sampleswerediluted inmethanol,1000␮g/ml.

HO HO

2 R=H 3 R=OCH₃

O

O R

1

Isolationoftheconstituents

DriedandpowderedleavesofP.regnellii(300g)wereextracted bymacerationwithethanol:water(9:1)atroomtemperaturein aleaf:solventratioof1:10(w/v).Thesolventwasthenremoved undervacuumat40◦_C_to_give_an_aqueous_extract_and_dark_green

residuethatwaswashedwithdichloromethane,yielding24gof thedichloromethane extract (DE), and dried atroom tempera-ture,withyieldof8.2%.TheDE(12g)wassubjectedtovacuum column chromatography with silica gel (230–400 mesh) and elutedwithhexane(1000ml),dichloromethane(1400ml),ethyl acetate(1000ml),acetone(700ml),andmethanol(1000ml).The hexanefractionresultedintheisolationofeupomathenoid-5(3, 1.05g).Thedichloromethanefraction(6g)waschromatographed bycolumnchromatographyonsilicagel60(230–400mesh)with hexane:dichloromethane:ethyl acetate (12:7:1, v/v/v) to yield conocarpan(1,0.53g)andeupomathenoid-6(2),butinverysmall

quantity, 0.002g. The column chromatography procedure was monitoredbythinlayerchromatography (TLC)andusedasthe mobilephase,hexane:dichloromethane:ethylacetate(12:7:1)and vanillin sulfuric 2%. Structureswere identified by spectroscopy (UV,1_H_NMR,13_C_NMR,_H–H_COSY,_gNOE,_HETCOR,_HMBC) _and

comparisonswiththeliterature(Achenbachetal.,1987;Chauret

etal.,1996;Snideretal.,1997).

Bacterialstrains

Theorganismsusedinthisstudywereobtainedoflaboratory collectionandwereoriginatedandidentifiedinUniversityHospital ofMaringá.Tenclinicalisolatesofmethicillin-resistantS.aureus

(MRSA),strains72,73,74,76,77,78,79,81,83and90(withstrains 72and73obtainedfrombloodandstrains74,76,77,78,79,81, 83and90obtainedfromsecretion),andthreeclinicalisolatesof andmethicillin-sensitiveS.aureus(MSSA),strains97,170,and212, obtainedfromurine(Marc¸aletal.,2010).ItwasalsousedS.aureus

ATCC25923.Teststrainswerepreservedinglycerol10%at−80◦_C

andwereculturedonnutrientagarandincubatedfor24hat37◦_C

priortodeterminationoftheminimuminhibitoryconcentration (MIC).

Antibacterialsusceptibilitytesting

The MICof conocarpan,eupomathenoid-5, and theDE were determined by a microdilution method in sterile flat-bottom microplatesaccordingtoCLSI(2012)usingMueller-Hintonbroth (MerckS.A.,SãoPaulo,Brazil).Inoculateswerepreparedinthesame mediumatadensitythatwasadjustedtoa0.5McFarlandturbidity standard(108_{colony-forming}_units_[CFU]/ml)_and_diluted_1:10_for

thebrothmicrodilutionprocedure.Conocarpan, eupomathenoid-5 and DE were dilutedand transferredinto the first well,and serialdilutions1:2wereperformedsothatconcentrationsinthe rangeof100–1.56␮g/mlwereobtained.Vancomycinwasusedas

thereferenceantibacterialcontrolintherangeof50–0.8␮g/ml.

Positivecontrolofstrains(withoutpresenceofdrugs)and nega-tivecontrol(withmediumsolely)wasperformed.Microtitertrays wereincubatedat37◦_C, _and_the_MICs_were_recorded_after₂₄_h

of incubation. Threesusceptibilityendpoints wererecorded for each isolate. TheMIC was defined asthe lowest concentration of thecompounds atwhich the microorganismdidnot exhibit visible growth. Theminimum bactericidal concentration (MBC) wasperformedinMueller-Hintonagar,incubatedat37◦_C_during

24h. MBCwasdefinedasthelowest concentrationthatyielded negativesubculturesoronlyonecolony.Theinvitroresultsfor thedrugswereclassifiedasthefollowing.MIC<100␮g/ml(good

antibacterialactivity),MIC=100–500␮g/ml(moderate

antibacte-rialactivity),MIC=500–1000␮g/ml(weakantibacterialactivity),

andMIC>1000␮g/ml(noactivity)(Pessinietal.,2003).

Invitropreformedbiofilmassay

BiofilmsofS.aureusATCC25923,10clinicalisolatesofMRSA, andthree clinicalisolates ofMSSAwereformedonpolystyrene 96-well microtiter plates. A 100␮l suspension that contained

108_cells/ml_in_TSB_medium_with_1%_glucose_was_seeded_in_wells

andincubatedat37◦_C_for₂₄_h._The_well_content_was_discharged,

andthewellswerewashedwithphosphate-bufferedsaline(PBS). Severaldilutions of theDE,conocarpan, eupomathenoid-5, and vancomycin (standard reference drug) (15.6–1000␮g/ml) were

then addedtoeach well.Afterincubationat 37◦_C _for ₂₄_h, _the

wells wererinsedwithPBS. TheMTT reductionassaywas per-formedtoevaluatetheviabilityofthebiofilms.Threeindependent assayswereperformed.TheMTTreductionassaywasaslight mod-ification of themethod reported by Schillaci et al. (2008).The MTTsolution(50␮l;2mg/mlinPBS)wasaddedtoeachwell,and

theplateswereincubatedat37◦_C_for₂_h._After_staining,_the_MTT

solutionwasremovedfromeachwell,and100␮lof

dimethylsulf-oxide(DMSO)wasaddedtodissolvetheMTTformazanproduct. TheDMSO(100␮l)wastransferredtoanewplate,andthe

opti-caldensity wasmeasuredat 570nmusinga microplatereader. Positivecontrolofbiofilmformation(withouttreatment)and neg-ativecontrol(withmediumsolely)wasperformed.Theresultsare expressedastheMIC50,atwhich50%ofthesessileS.aureuscells

wereinhibitedcomparedwiththecontrol(withouttreatment)for theMTTassays.

Inhibitingbiofilmformationinvitro

This assay investigated the drugs ability to preventbiofilm formation. Briefly, 100␮l of drug (DE, conocarpan and

eupomathenoid-5)atdifferentconcentrations(15.6–1000␮l/ml)

andstandarddrug,vancomycininconcentration7.8–500␮l/mlin

(3)

a96-wellmicrotiterplate,seededwith100␮lofasuspensionthat

contained108_cells/ml, _and_incubated _for₂₄_h_at ₃₇◦_C _to_allow

biofilmformation.Thecontentsofthewellswereaspiratedand washedthreetimesinsterilePBS.Theextentofbiofilmformation wasassessedbytheMTTreductionassayasdescribedabove.The resultsareexpressedastheminimalbiofilminhibitory concen-tration(MBIC)comparedwiththecontrol(withouttreatment)for theMTTassays.

Scanningelectronmicroscopy

Scanningelectronmicroscopy(SEM)wasperformedonglass coverslipsbydispensing400␮lofthestandardizedcell

suspen-sionsthatcontained1.0×108cells/mlofTSBsupplementedwith

1%glucose intothe wellsof 24-well flat-bottomedpolystyrene plates.Toviewpreformedbiofilm,theplateswereincubatedat 37◦_C _for ₂₄_h. _After_washing_with_PBS,_conocarpan ₍₁₁₀

␮g/ml)

andtheDE(370␮g/ml)wereaddedtopreformedbiofilms,andthe

plateswereincubatedfor24hat37◦_C._To_view_biofilm_formation,

standardizedcellsuspensionsanddrug(15.6␮g/ml conocarpan,

31.5␮g/mleupomathenoid-5,15.6␮g/mlDE,and7.8␮g/ml

van-comycin)wereaddedandincubatedat37◦_C_for₂₄_h._The_coverslips

werethenwashedtwicewithPBSandfixedwith2.5% glutaralde-hydeovernightat4◦_C._The_coverslips_were_then_washed_twice_with

0.1Mcacodylatebufferfor15minanddehydratedbyreplacingthe bufferwithincreasingconcentrationsofethanol (30,50, 70,80, 90,95,and100%)for10mineach.Aftercritical-point-dryingand coatingwithgoldsputter,thesampleswereexaminedwitha scan-ningelectronmicroscope,inShimadzuSS-550(Probe4.0andAccV 15.0V).

Resultsanddiscussion

Isolationoftheconstituents

TheextractionofP.regnellii(Miq.)C.DC.resultedinan8.2% yield of dichloromethane extract. From this extract, theuse of silicagelchromatographiccolumnsand agradient systemwith increasingdegreesofpolarityallowedtheisolationoftwomajor compoundsthatwerepresentintheextract:conocarpan(1)and eupomathenoid-5(3),andeupomathenoid-6(2)inminoramount,

notallowing usein biological assays. Conocarpan,with stereo-chemistry(+)-conocarpanisthemostabundantinnature,produced naturallyinplantsofseveralfamilies,andthesemoleculesexhibit atrans-dihydrobenzofuranheterocycleasakeystructuralelement

(ChenandWeisel,2013).Thesesubstanceswereisolatedand

iden-tifiedbycomparingspectroscopicdataof1_H_and13_C_NMR_spectra

withthosepreviouslypublished(Achenbachetal.,1987;Chauret

etal.,1996;Snideretal.,1997).

HPLCanalysis

Fig. 1 shows dichloromethane extract chromatogram. HPLC analysisallowedtheneolignansquantification.Dichloromethane extract DE was standardized in 135.32␮g/ml of conocarpan

(1), 91.90␮g/ml of eupomathenoid-6 (2) and 106.98␮g/ml of

eupomthenoid-5(3).

Antibacterialeffectinplanktoniccells

TheMICresultsobtainedinthestudyareshown inTable1. ThedichloromethaneextracthadgoodactivityagainstMRSAand MSSA, with MIC <15␮g/ml and MBC <30␮g/ml. These values

wereverysimilartothosereportedbyMarc¸aletal.(2010). Cono-carpanalsohadgoodactivityagainstMRSAandMSSA(Table1),

withamaximumMIC=50␮g/mlandmaximumMBC=100␮g/ml.

Eupomathenoid-5hadaMIC≤6.25␮g/mlandMBC<25␮g/ml.

TheDEandisolatedneolignansweretestedagainstthestandard strain of S. aureus, with a MIC <15␮g/ml and MBC <30␮g/ml

(Table1).Thesevaluesaresimilartothosereportedintheliterature

(Pessinietal.,2003;Felipeetal.,2008).

Antibacterialeffectinpreformedbiofilm

Resistance mechanisms of biofilms are multifactorial and dependoneach organism.Althoughnotfullyunderstood,these mechanismscanbeattributedtosuchfactorsasareduction of the penetration of antibiotics through the biofilm matrix, the presenceofslow-growingornon-growingcells inthebiofilm,a heterogeneousbacterialpopulationwiththepresenceof pheno-typicsubpopulations withdifferentlevelsofresistance,andthe persistentpresenceofcells(MahandO’Toole,2001;Harrisonetal.,

2005).

Table1shows goodactivityoftheisolateneolignansagainst

planktoniccells,buttheactivityagainstthebiofilmwasnotthe

same(Table2).Euphomatenoid-5showednoactivityagainstS.

aureus ATCC 25923 biofilm. However, conocarpan and the DE hadasatisfactoryMIC50againstthebiofilm,110and370␮g/ml,

respectively(Table2).TheseMTTassayresultsdemonstratethe metabolicactivityofthecellsinthebiofilm,whichwasconfirmed bySEM(Fig.2).

ThephotomicrographsinFig.2Ashowthecontrolbiofilmwith alargeamountofadheredcellsandanextracellularmatrix.The treatedbiofilms(Fig.2BandC)presentedcellularlysisregionsand biofilmdetachment,suggestingthegoodactionofconocarpanand theDEagainstpreformedbiofilms.

Althoughtheconcentrationofconocarpan thatwasrequired tocombatthebiofilmwaslessthantheconcentrationoftheDE, the isolated substance showed no activity against clinical iso-latesofMRSAandMSSAbiofilms(Table2).Thedichloromethane extractwaseffectiveagainstallofthetestedclinicalisolatesof MRSAand MSSAbiofilms(MIC50<400␮g/ml). Inmany partsof

theworld, MRSA is responsible for a highrate of community-and hospital-acquired S. aureus infections. The most important classesofantibioticsthatareusedtopreventandtreatS.aureus

infectionareineffective(Köcketal.,2010).Inthepresentstudy, dichloromethaneextractofP.regnelliihadbetteractivityagainstS. aureusbiofilms.

Antibacterialeffectininhibitionbiofilmformation

Analternativetothecontrolofbiofilmformationistoprevent colonization.Thus,we evaluatedtheabilityofdifferent concen-trationsoftheDE,conocarpan, andeupomathenoid-5toinhibit theformationofbiofilmswhenaddedtothemediumatthesame timeasthecells.Theresultswerecomparedwithvancomycin.Ifan agentisaddedatthebeginningoftheexperiment,thentheagent mightactbeforethebiofilmisformedandinhibititsdevelopment. Thiscouldbeofinterestforcombatingrecalcitrantinfections(Khan

andAhmad,2012).Concentrationsaslowas15.6␮g/mlresultedin

asignificantreductionofthemetabolicactivityofadherentcells, with>95%inhibitionofMRSAandMSSAbiofilmformation com-paredwithuntreatedcontrolcells(Table3).TheDEhad results thatwereveryclosetovancomycinfortheinhibitionofS.aureus

biofilmformation.

TheseresultswereconfirmedbySEM.Fig.3showsthepositive controlwithalargenumberofcellsthatadheredtoeachotherand thepresenceanextracellularmatrix(Fig.3A).Photomicrographs ofcellsthatweretreatedwithconocarpan(15.6␮g/ml;Fig.3B),

(4)

0.00 0.10 0.20 0.30

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 1

2

3

Minutes

AU

11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 0.40

0.50 0.60

Fig.1. ChromatographicprofileofdichloromethaneextractofPiperregenelliat1000␮g/ml.(1)Conocarpan;(2)eupomatenoid-6;(3)eupomatenoid-5.Conditions:mobile

phase–acetonitrile:waterwith2%aceticacid(70:30);flow:1ml/min,detectionat280nm.

Table1

MICandMBCofPiperregnelliidichloromethaneextract,eupomathenoid-5,conocarpanandVancomycininS.aureusplanktoniccells.

MIC(MBC)␮g/ml

Strains Dichloromethaneextract Eupomathenoid-5 Conocarpan Vancomycin

72 12.5(12.5) 6.25(25) 50(100) 1.56(1.56)

73 12.5(12.5) 6.25(25) 50(100) 1.56(1.56)

74 6.25(12.5) 6.25(6.25) 25(25) 1.56(1.56)

76 6.25(25) 6.25(12.5) 25(25) 1.56(1.56)

77 12.5(25) 6.25(12.5) 25(25) 1.56(1.56)

78 6.25(12.5) 3.13(6.25) 12.5(100) 1.56(1.56)

79 12.5(25) 3.13(6.25) 25(25) 1.56(1.56)

81 12.5(25) 3.13(6.25) 25(50) 1.56(1.56)

83 6.25(12.5) 3.13(6.25) 25(100) 1.56(1.56)

90 6.25(25) 3.13(6.25) 25(100) 1.56(1.56)

97 12.5(25) 6.25(6.25) 12.5(12.5) 1.56(1.56)

170 12.5(25) 6.25(12.5) 12.5(12.5) 1.56(1.56)

212 12.5(25) 6.25(12.5) 12.5(25) 1.56(1.56)

ATCC25923 6.25(12.5) 6.25(12.5) 12.5(25) 1.56(1.56)

TheMICresultswereconsideredgoodantibacterialactivity(MIC<100␮g/ml).

Table2

MIC50ofPiperregnelliidichloromethaneextract,eupomathenoid-5,conocarpanandvancomycintoS.aureuspreformedbiofilms.

MIC50(␮g/ml)

72 300 – >1000 –

73 170 – – –

74 50 – – –

76 360 – – –

77 390 – >1000 –

78 180 – >1000 –

79 190 – >1000 –

81 210 – >1000 –

83 230 – >1000 –

90 130 – >1000 –

97 260 – >1000 –

170 260 – >1000 –

212 370 – >1000 –

ATCC25923 370 – 110 –

MIC50,minimalinhibitoryconcentrationatwhich50%ofthesessileS.aureuscellswereinhibited.

Fig.2.ScanningelectronmicrographsofrandomlychosenareasofS.aureusATCC25923biofilmat24h.(A)Positivecontrol.(B)Biofilmtreatedwith370␮g/mlof

(5)

Table3

Minimalbiofilminhibitoryconcentration(MBIC)valuesforthePiperregnelliidichloromethaneextract,eupomathenoid-5,conocarpanandvancomycintoS.aureusbiofilms formation.

MBIC(␮g/ml)

72 15.6 15.6 15.6 7.8

73 15.6 15.6 15.6 7.8

74 15.6 31.2 15.6 7.8

76 15.6 31.2 15.6 7.8

77 15.6 250 125 7.8

78 15.6 15.6 15.6 7.8

79 15.6 15.6 15.6 15.6

81 15.6 15.6 15.6 7.8

83 31.2 31.2 31.2 62.5

90 15.6 15.6 15.6 7.8

97 15.6 15.6 15.6 7.8

170 15.6 15.6 15.6 7.8

212 15.6 15.6 15.6 7.8

ATCC25923 15.6 31.2 15.6 7.8

Fig.3. ScanningelectronmicrographsofrandomlychosenareasofS.aureusATCC25923biofilmformation.(A)Positivecontrol.(B)Biofilmtreatedwithconocarpan (15.6␮g/ml).(C)Biofilmtreatedwitheupomathenoid-5(31.25␮g/ml).(D)Biofilmtreatedwithdichloromethaneextract(15.6␮g/ml),D Biofilmtreatedwithvancomycin

(7.8␮g/ml).3000magnification.Thisstudywasperformedaccordingtotheinternational,nationalandinstitutionalrulesconsideringclinicalstudiesandbiodiversityrights.

Fig. 3D) showed isolated and fewer cells compared with van-comycin(Fig.3E).

Biofilm-relatedinfectionsareanimportantcauseof healthcare-associatedinfections(Darouiche,2004).Biofilm-embedded bacte-ria are challenging to treat because they display tolerance to antibioticsandthehost’simmunesystem(Johnetal.,2011;Leite etal., 2011).Traditionalantibiotics thatwere developed tokill planktonicbacteriaoftenhavelimitedeffectsonsessilebacteria thatareencasedwithinabiofilm.Additionally,thedevelopment ofantimicrobial resistanceiscommon insessile bacteria. Thus, thereisanurgentneedtodevelopnon-antimicrobialtreatment strategiestopreventortreatbiofilm-associatedinfections(Kuehn,

2011).

Conclusion

Goodresultswereachievedwiththedichloromethaneextract of P.regnellii (Miq.) C. DC. and isolated neolignans conocarpan andeupomathenoid-5againstplanktoniccellsofS.aureusMRSA andMSSA,similartothestandarddrugvancomycin.Similargood

effectswerefoundagainstbiofilmformation.Thedichloromethane extractandisolatedneolignansalsoshowedactivitythatwasclose to vancomycin and appeared to be effective as a prophylactic alternative.TheDEwasalsoeffectiveagainstS.aureusMRSAand MSSApreformedbiofilms.Theseresultssupporttheapplicationof thisextractofP.regnelliiasanalternativeagentagainst biofilm-associatedbacterialinfections.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata

appearinthisarticle.

Righttoprivacyandinformedconsent. Theauthorsdeclarethat

(6)

Author’scontribution

LZSB responsible for isolation of substances and performed inhibitionsassays,biofilmformationandpreformedbiofilm.EHE performedinhibitions assays, biofilm formation and preformed biofilm.DAGCsupervisedthelaboratoryworkandcontributedto theisolationandidentificationofsubstances.BPDFsupervisedthe laboratoryworkandcontributedtothebiologicalstudies.Allthe authorshavereadthefinalmanuscriptandapprovedits submis-sion.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

References

Achenbach,H.,Grob,J.,Xorge,A.D.,Cano,G.,Verde,J.S.,Brussolo,L.D.C.,Mu˜noz,G., Salgado,F.,López,L.,1987.Lignans,neolignansandnorneolignansfromKrameria cystisoides.Phytochemistry26,1159–1166.

Chauret,D.C.,Bernad,C.B.,Arnason,J.T.,Durst,T.,1996.Insecticidalneolignansfrom Piperdecurrens.J.Nat.Prod.59,152–155.

Chen,C.,Weisel,M.,2013.Conciseasymmetricsynthesisof(+)-conocarpanand obtusafuran.Synlett24,189–192.

CLSI,2012.MethodsforDilutionAntimicrobialSusceptibilityTestforBacteriathat GrowAerobically.ApprovedStandard,9thed.M07-A9,ClinicalandLaboratory StandardsInstitute,Wayne,PA.

Corrêa,M.P.,1984.DicionáriodasPlantasÚteisdoBrasiledasExóticasCultivadas. MinistériodaAgricultura,InstitutoBrasileirodeDesenvolvimentoFlorestal, Brasília,687pp.

Cronquist,A.,1981.AnIntegratedSystemofClassification.ColumbiaUniversity, NewYork,670pp.

Darouiche,R.O.,2004.Treatmentofinfectionsassociatedwithsurgicalimplants.J. Med.350,1422–1429.

Felipe,D.F.,DiasFilho,B.P.,Nakamura,C.V.,Cortez,D.A.G.,2008.Evaluationofthe antimicrobialactivityofPiperregnellii(Miq.)C.DC.var.pallescens(C.DC.)Yunck. Lat.Am.J.Pharm.27,618–620.

Hall-Stoodley,L.,Costerton,J.W.,Stoodley,P.,2004.Bacterialbiofilms:fromthe naturalenvironmenttoinfectiousdiseases.Nat.Rev.Microbiol.2,95–108.

Harrison,J.J.,Turner,R.J.,Marques,L.R.,Ceri,H.,2005.Biofilms:anewunderstanding ofthesemicrobialcommunitiesisdrivingarevolutionthatmaytransformthe scienceofmicrobiology.Am.Sci.93,508–551.

John,A.K.,Schmaler,M.,Khanna,N.,Landmann,R.,2011.Reversibledaptomycin toleranceofadherentstaphylococciinanimplantinfectionmodel.Antimicrob. AgentsChemother.55,3510–3516.

Khan,M.S.A.,Ahmad,I.,2012.Antibiofilmactivityofcertainphytocompoundsand theirsynergywithfluconazoleagainstCandidaalbicansbiofilms.J.Antimicrob. Chemother.67,618–621.

Köck,R.,Becker,K.,Cookson,B.,vanGemert-Pijnen,J.E.,Harbarth,S.,Kluytmans, J.,Mielke,M.,Peters,G.,Skov,R.L.,Struelens,M.J.,Tacconelli,E.,Navarro,A., Witte,W.,Friedrich,A.W.,2010.Methicillin-resistantStaphylococcusaureus (MRSA):burdenofdiseaseandcontrolchallengesinEurope.EuroSurveill.15, 19688–19690.

Kuehn,B.M.,2011.Scientistsseekantibioticadjuvants.JAMA306,2203–2204.

Leite,B.,Gomes,F.,Teixeiras,P.,Souza,C.,Pizzolitto,E.,Oliveira,R.,2011.Invitro activityofdaptomycin,linezolidandrifampicinonStaphylococcusepidermidis biofilms.Curr.Microbiol.63,313–317.

Luize, P.S.,Ueda-Nakamura,T., Dias-Filho,B.P.,Cortez, D.A.G.,Nakamura,C.V., 2006.ActivityofneolignansisolatedfromPiper regnellii(MIQ.)C.DC.var. pallescens(C.DC.) YunckagainstTrypanosomacruzy. Biol.Pharm.Bull. 29, 2126–2130.

Mah,T.F.,O’Toole,G.A.,2001.Mechanismsofbiofilmresistancetoantimicrobial agents.TrendsMicrobiol.9,34–39.

Marc¸al, F.J.B., Cortez, D.A.G., Ueda-Nakamura, T., Nakamura, C.V., Dias-Filho, B.P., 2010. Activity of the extracts and neolignans from Piper regnellii against methicillin-resistant Staphylococcus aureus (MRSA). Molecules 15, 2060–2069.

NationalInstitutesofHealth,2002.Availablefrom:http://grants.nih.gov/grants/ guide/pa-files/PA-03-047.html(accessedOctober2013).

Neidell,M.J.,Cohen,B.,Furuya,Y.,Hill,J.,Jeon,C.Y.,Glied,S.,Larson,E.L.,2012.Costsof healthcare-andcommunity-associatedinfectionswithantimicrobial-resistant versusantimicrobial-susceptibleorganisms.Clin.Infect.Dis.55,807–815.

Pessini,G.L.,Dias-Filho,B.P.,Nakamura,C.V.,Cortez,D.A.G.,2003.Antibacterial activ-ityofextractsandneolignansfromPiperregnellii(Miq.)C.DC.var.pallescens(C. DC.)Yunck.Mem.I.OswaldoCruz98,1115–1120.

Pessini,G.L.,Dias-Filho,B.P.,Nakamura,C.V.,Cortez,D.A.G.,2005.Antifungalactivity oftheextractsandneolignansfromPiperregnellii(Miq.)C.DC.var.pallescens(C. DC.)Yunck.J.Braz.Chem.Soc.16,1130–1133.

Schillaci, D., Arizza, V., Dayton, T., Camarda, L., Stefano, V.D.,2008. In vitro anti-biofilmactivityofBoswelliaspp.oleogumresinessentialoils.Lett.Appl. Microbiol.47,433–438.

Snider,B.B.,Han,L.,Xie,C.,1997.Synthesisof2,3-dihydrobenzofuransby Mn(Oac)3-basedoxidativecycloadditionof2-cyclohexenoneswithalkenes.Synthesisof (±)-conocarpan.J.Org.Chem.62,6978–6984.

Stewart,P.S.,Costerton,J.W.,2001.Antibioticresistanceofbacteriainbiofilms. Lancet358,135–138.

Trentin,D.S.,Giordani,R.B.,Macedo,A.J.,2013.Biofilmesbacterianospatogênicos: aspectosgerais,importânciaclínicaeestratégiasdecombate.Rev.Liberato14, 113–238.