Terpenoids from Platostoma rotundifolium (Briq.) AJ Paton Alter the Expression of Quorum Sensing

22 Pages·2017·6.93 MB·English

International Journal o f
Molecular Sciences
Article
Terpenoids from Platostoma rotundifolium
(Briq.) A. J. Paton Alter the Expression of Quorum
Sensing-Related Virulence Factors and the Formation
of Biofilm in Pseudomonas aeruginosa PAO1
TsiryRasamiravaka1,5,*,†,JérémieNgezahayo2,3,*,†,LaurentPottier2,SofiaOliveiraRibeiro2,
FlorenceSouard2,6,7,LéonardHari3,CarolineStévigny2,MondherElJaziri1andPierreDuez2,4
1 LaboratoryofPlantBiotechnology,UniversitéLibredeBruxelles,ruedesProfesseursJeeneretBrachet12,
6041Gosselies,Belgium;[email protected]
2 LaboratoiredePharmacognosie,BromatologieetNutritionHumaine,FacultédePharmacie,Université
LibredeBruxelles,CP205/09,BoulevardduTriomphe,1050Bruxelles,Belgium;
[email protected](L.P.);sofi[email protected](S.O.R.);
fl[email protected](F.S.);[email protected](C.S.);[email protected](P.D.)
3 CentredeRechercheUniversitaireenPharmacopéeetMédecineTraditionnelle(CRUPHAMET),Facultédes
Sciences,UniversitéduBurundi,BP2700Bujumbura,Burundi;[email protected]
4 UnitofTherapeuticChemistryandPharmacognosy,UniversitédeMons(UMONS),BâtimentVI,Chemin
duChampdeMars25,7000Mons,Belgium
5 LaboratoiredeBiotechnologieetMicrobiologie,DépartementdeBiochimieFondamentaleetAppliquée,
FacultédesSciences,Universitéd’Antananarivo(UA),BP906,Antananarivo101,Madagascar
6 DépartementdePharmacochimieMoléculaire,UniversitéGrenobleAlpes,38000Grenoble,France
7 DépartementdePharmacochimieMoléculaire,CentreNationaldeRechercheScientifique,
38000Grenoble,France
* Correspondence:[email protected](T.R.);[email protected](J.N.);Tel.:[phone](J.N.)
† Theseauthorscontributedequallytothiswork.
Received:7May2017;Accepted:7June2017;Published:14June2017
Abstract:Platostomarotundifolium(Briq.)A.J.PatonaerialpartsarewidelyusedinBurunditraditional
medicinetotreatinfectiousdiseases. Inordertoinvestigatetheirprobableantibacterialactivities,
crudeextractsfromP.rotundifoliumwereassessedfortheirbactericidalandanti-virulenceproperties
againstanopportunisticbacterialmodel,PseudomonasaeruginosaPAO1. Whereasnoneofthetested
extractsexertbacteriostaticand/orbactericidalproprieties,theethylacetateanddichloromethane
extracts exhibit anti-virulence properties against Pseudomonas aeruginosa PAO1 characterized by
analterationinquorumsensinggeneexpressionandbiofilmformationwithoutaffectingbacterial
viability.Bioguidedfractionationoftheethylacetateextractledtotheisolationofmajoranti-virulence
compoundsthatwereidentifiedfromnuclearmagneticresonanceandhigh-resolutionmolecular
spectroscopyspectraascassipourol,β-sitosterolandα-amyrin. Globally,cassipourolandβ-sitosterol
inhibitquorumsensing-regulatedand-regulatorygenesexpressioninlasandrhlsystemswithout
affectingtheglobalregulatorsgacAandvfr,whereasα-amyrinhadnoeffectontheexpressionofthese
genes. Theseterpenoidsdisrupttheformationofbiofilmsatconcentrationsdownto12.5,50and
50µMforcassipourol,β-sitosterolandα-amyrin,respectively. Moreover,theseterpenoidsreduce
theproductionoftotalexopolysaccharidesandpromoteflagella-dependentmotilities(swimming
and swarming). The isolated terpenoids exert a wide range of inhibition processes, suggesting
acomplexmechanismofactiontargetingP.aeruginosavirulencemechanismswhichsupportthewide
anti-infectioususeofthisplantspeciesintraditionalBurundianmedicine.
Keywords: anti-virulence; biofilm; Platostoma rotundifolium; Lamiaceae; Pseudomonas aeruginosa;
quorumsensing
Int.J.Mol.Sci.2017,18,1270;doi:10.3390/ijms18061270 www.mdpi.com/journal/ijms
Int.J.Mol.Sci.2017,18,1270 2of22
1. Introduction
Assumingthatthesuccessofbacterialinfectionreliesonanoptimalexpressionofvirulence[1],
anattractiveanti-pathogenicapproachconsistsintargetingthesemechanisms[2–4]. Indeed,virulent
bacteriaareabletocolonizetheirhostsbybuildingbiofilms,disseminatingwithdifferenttypesof
motilityandreleasingmultiplevirulencefactors[5]. Besides,thepersistenceofbacterialinfectionis
linkedtotheirabilitytoformstructuredbiofilmswhichrepresentaprotectivebarrieragainstantibiotics
andimmunedefense,allowingsurvivalandfurtherre-dissemination[6,7].Interestingly,thesefaculties
areintimatelyinterconnectedtoquorumsensing(QS),abacterialcell-to-cellcommunicationthatallows
bacteriatocoordinatetheirbehaviordependingontheirpopulationdensity,throughthereleaseand
perceptionofsmalldiffusiblemoleculescalled“auto-inducers”[8]whichfinallyinducetheproduction
ofseveralvirulencefactorsandmodulatebacterialbehaviors,includingbiofilmlifestylegrowth[9].
P. aeruginosa has become a model organism of studying this bacterial communication and
harbors several QS mechanisms that depend on the type of the released signaling molecules [9].
Theacylhomoserinelactones(AHLs)N-3-oxo-dodecanoyl-L-homoserinelactone(3-oxo-C12-HSL)
and N-butanoyl-L-homoserine lactone (C4-HSL) are produced and sensed, respectively, by the
LasI/R and RhlI/R QS systems. LasR and rhlR genes encode LasR and RhlR regulator proteins,
respectively,whilelasIandrhlIgenesencodetheLasIandRhlIsynthasesnecessaryforthesynthesis
of3-oxo-C12-HSLandC4-HSL,respectively[10]. ThePQSsystem(Pseudomonasquinolonesignal)
isbasedon2-alkyl-4-quinolones[11]andrequiresseveralenzymesencodedbypqsABCDE,phnAB
andpqsHoperonsandPqsRregulator[12]. Altogether,fullactivationofthesesQSsystemsenhances
theproductionofseveralvirulencefactorssuchasrhamnolipids,pyocyanin,LasBelastase,hydrogen
cyanideandcytotoxiclectins[9].
Many bioactive molecules have been reported to exert modulatory properties on bacterial
virulenceexpressionandmostofthemarenaturally-derivedcompounds,suchashalogenatedC-30and
C-56furanones,inspiredfromnaturalcompoundsproducedbythemarinemacroalgaDeliseapulchra,
whichreducebiofilmandtargettheQSsystemsinP.aeruginosa,animportanthuman,animalandplant
pathogen[13]. InviewoffurtherinvestigatingnaturalproductsforQS-modulatingproperties,African
medicinal plants were investigated as they represent a largely untapped source for the discovery
ofbioactivecompoundswithnovelmechanismsofaction[14]. Platostomarotundifolium(Briq.) A.J.
PatoniswidelyusedintraditionalBurundianmedicineagainstmicrobialdiseases[15]anditsaerial
parts were shown to contain triterpenic acids (e.g., ursolic and corrosolic acids) with direct and
indirectbactericidalpropertiesagainstEscherichiacoliaswellassensitiveandmethicillin-resistant
Staphylococcusaureus[16],contributingtoexplainitspurportedanti-infectiousproperties.Inthepresent
study, we report on antibacterial activities of P. rotundifolium extracts towards P. aeruginosa PAO1,
an opportunistic pathogen which particularly infects immunocompromised patients and describe
theisolation,theidentificationandthecharacterizationofantibacterialpropertiesofmajorisolated
bioactivecompounds.
2. Results
2.1. AntibacterialActivitiesoftheP.rotundifoliumExtracts
TheMIC(MinimumInhibitoryConcentration)andMBC(MinimumBactericidalConcentration)of
fiveP.rotundifoliumaerialpartextractswithdifferentdegreesofpolaritywerehigherthan4000µg/mL
onP.aeruginosaPAO1,indicatinganextremelyweakpotentialasadirectantibiotic;bycomparison,
theMICandMBCfortobramycin,anantibioticgenerallyusedtotreatpatientswithcysticfibrosis
infected by P. aeruginosa [17], were 1 and 2 µg/mL, respectively (data not shown). To investigate
anti-virulenceproperty,theeffectsoftheseP.rotundifoliumextractsontheexpressionoftwovirulence
factorsgenes(lasB,encodingforthevirulencefactorLasBelastase,andrhlA,encodingfortheprecursor
ofthevirulencefactorrhamnolipids,bothgenesareregulatedbyQSsysteminP.aeruginosa)aswell
asonbiofilmformationbyP.aeruginosaPAO1wereassessed. Theflavanonenaringeninknownas
Int.J.Mol.Sci.2017,18,1270 3of22
virulence factor gene inhibitors [18] and the triterpenoid oleanolic acid known to reduce biofilm
formaIntit.o Jn. Mionl. SPc.i.a 2e0r1u7,g 1i8n, o12s7a0 [ 19]wereusedaspositivecontrols. AsshowninFigure1,theethy3 lofa 2c2e tate
(EtOAc)anddichloromethane(DCM)extractsat100µg/mLinhibittheexpressionoflasB(34±3%
acid known to reduce biofilm formation in P. aeruginosa [19] were used as positive controls. As
and 30 ± 6% of inhibition, respectively; Figure 1A) and rhlA (30 ± 2% and 35 ± 3% of inhibition,
shown in Figure 1, the ethyl acetate (EtOAc) and dichloromethane (DCM) extracts at 100 µg/mL
respectively; Figure1B)geneswithoutaffectingbacterialgrowthandreducebiofilmformationby
inhibit the expression of lasB (34 ± 3% and 30 ± 6% of inhibition, respectively; Figure 1A) and rhlA (30
P.aeru±g 2in%o saandPA 35O ±1 3(%35 o±f in3h%ibaitniodn4, 3re±spe7c%tivoeflyin; Fhiigbuitrieo 1nB,)r egsepneecs twiviethlyo;uFt iagffuerceti1nDg )b.aBcteesriidale gs,rothwetshe aenxdt racts
hadnroedeuffceec tbioonfilamQ fSo-rimnadteiopne nbdy ePn. tagereungein(oissao cPiAtrOat1e (l3y5a ±s e3-%en acnodd i4n3g ±a c7e%A ogfe inneh;ibFiitgiounr,e r1eCsp)e,cstuivgeglye;s ting
thatthFeigoubres e1rDv)e. dBeesfifdeecst,s tohnesQe eSxgtreanctess heaxdp nreos esfifoenct iosns pa eQcSifi-icndanepdedndoeenstn goetnree (siusoltciftrroatme layagsleo-benacloindhinigb ition
ofP.aaecreuAg igneonsea; PFAigOur1e m1Ce)t,a sbuoglgicesaticntgiv tihtyat[ 2th0e] .oHbseexrvanede ,emffeectths aonno Ql(SM geeOneHs )exapnrdesasqioune iosu sspe(AcifQic) aenxdtr acts
havendooeesf fnecott erietshuelrt QfrSo-md eap egnlodbeanlt i(nlahsiBbiatinodn rohfl AP). gaeernuegsineoxspa rPeAssOio1n mnoetraobnolbici oaficltmiviftyo r[m20a]t.i oHne(xFaingeu, re1).
methanol (MeOH) and aqueous (AQ) extracts have no effect either QS-dependent (lasB and rhlA)
Toconclude,P.rotundifoliumextractsexhibitapotentialantibacterialpropertythatisnotassociated
genes expression nor on biofilm formation (Figure 1). To conclude, P. rotundifolium extracts exhibit a
withbacteriostaticand/orbactericidalactivitybutratherwithanti-QSandanti-biofilmactivitiesin
potential antibacterial property that is not associated with bacteriostatic and/or bactericidal activity
P.aeruginosamodel.
but rather with anti-QS and anti-biofilm activities in P. aeruginosa model.

Figure 1. Anti-virulence effects of Platostoma rotundifolium extracts in P. aeruginosa PAO1: (A) effect of
Figure1. Anti-virulenceeffectsofPlatostomarotundifoliumextractsinP.aeruginosaPAO1: (A)effect
P. rotundifolium extracts on the expression of QS-regulated lasB gene; (B) effect of P. rotundifolium
ofP.rotundifoliumextractsontheexpressionofQS-regulatedlasBgene;(B)effectofP.rotundifolium
extracts on the expression of QS-regulated rhlA gene; (C) effect of P. rotundifolium extracts on the
extracts on the expression of QS-regulated rhlA gene; (C) effect of P. rotundifolium extracts on the
expression of QS-independent aceA gene; and (D) effect of P. rotundifolium extracts in biofilm
expressionofQS-independentaceAgene;and(D)effectofP.rotundifoliumextractsinbiofilmformation.
formation. Extracts (H: n-hexane, DCM: dichloromethane, EtOAc: ethyl acetate, MeOH: methanol,
Extracts(H:n-hexane,DCM:dichloromethane,EtOAc:ethylacetate,MeOH:methanol,AQ:aqueous)
AQ: aqueous) were tested at 100 µg/mL; naringenin (Nar, 4 mM) is used as a reference quorum
weresteenstseindga tin1h0i0biµtogr/; moLle;annaorliicn gaecnidin ((NOAar, ,480m0 Mµ)Mis) uisse dusaesda raesf earenn caentqi-uboiorfuilmm secnonsitnrogl inahndib itor;
oleandoilmiceathciydls(uOlfAox,id80e 0(DµMMS)Ois, 1u%se) dasa as saonlvaenntti -cboinotfirollm. Tchoen cterlol ldaennsditdy iomf eththe yblascutelfroiax iwdaes( aDsMsesSsOed, 1a%t )as
asolv6e0n0t ncmon (tbroolld. Terhreorc eblalrds)e annsdit ygeonfet hexepbreascstieorni awwasa smaesasseusresded asa tth6e0 0β-ngmala(cbtoosliddaesrer oarctbivairtys )oaf nthdeg ene
exprelsasciZo ngewnea sfumsieoanssu arnedd eaxsptrheessβed- gianl aMctiollesrid uansietsa (cgtriveyit ybaorf). tBhieoflialmcZ fgoremneatfiouns iownass aqnudanetixfiperde sbsye din
Millercruysntiatls v(igolreety stbaianri)n.gB ainodfi mlmeafsourrmeda atiso An59w0nma.s Eqrruoar nbtairfise rdepbreysecnrty tshtae lstvainodleartds tearrinorisn gof athned mmeaenass;u red
all experiments were performed in quintuplicate with three independent assays and asterisks
asA .Errorbarsrepresentthestandarderrorsofthemeans; allexperimentswereperformed
590nm
indicate samples that are significantly different from the DMSO (One-way ANOVA followed by
inquintuplicatewiththreeindependentassaysandasterisksindicatesamplesthataresignificantly
Dunnett’s test of multiple comparisons; p < 0.01).
differentfromtheDMSO(One-wayANOVAfollowedbyDunnett’stestofmultiplecomparisons;p<0.01).
Int.J.Mol.Sci.2017,18,1270 4of22
Int. J. Mol. Sci. 2017, 18, 1270 4 of 22
SincebothEtOAcandDCMextractsshowedpracticallysimilarTLCchromatographicprofiles
Since both EtOAc and DCM extracts showed practically similar TLC chromatographic profiles
withhigherintensityspotareaforEtOAcextracts(datanotshown),thislatterwassubjectedtofurther
with higher intensity spot area for EtOAc extracts (data not shown), this latter was subjected to
chromatographicfractionations.
further chromatographic fractionations.
2.2. FractionationoftheEtOAcExtractandIsolationofMajorBioactiveCompounds
2.2. Fractionation of the EtOAc Extract and Isolation of Major Bioactive Compounds
P. rotundifolium EtOAc extract was fractionated by column chromatography using a gradient
P. rotundifolium EtOAc extract was fractionated by column chromatography using a gradient
mixture of DCM and EtOAc with increasing polarities (10% to 100% of EtOAc). Fractions were
mixture of DCM and EtOAc with increasing polarities (10% to 100% of EtOAc). Fractions were
momnoitnoitroerdedb ybyT LTCLCa nadndp pooooleledd( a(accccoorrddiinngg ttoo tthheeiirr cchhrroommaattooggrraapphhici cpprorfoifilelse)s t)ot ogigviev eeigehigt hfrtafcrtaicotnios ns
(F1(F–1F–8F),8)w, whihcihchw weereret htheenn eevvaalluuaatteedd ffoorr ththeeiri rppotoetnetniatil aelffeefcftesc otsn oQnSQ-reSg-ruelgatuelda tleasdB laansBd arhnldA rghelnAe gase ne
aswweelll laass oonn bbiiooffiilmlm ffoorrmmaatitoionn. .FrFarcaticotniosn Fs1F–1F–4F w4ewree raella lalcaticvteiv ien ininhinibhiitbinitgi nlgaslBa saBndan rdhlrAh lAgengee ne
expexrpesrseisosino,nw, whheerereaass oonnllyy FF22 aanndd F4F 4inihnibhiitb bitiobfiilomfi lfmormfoartmiona t(iFoingu(rFei gSu1)r.e F2S 1fr)a.ctFio2nf,r tahcet imono,stt hacetimveo st
actfirvaectiforanc (tFioignu(rFei gSu1)r ewSa1s) twheans sthubenmistutebdm tiot tefldastho cflharsohmcahtorgormapahtoyg arnapdh tywaelnvde tswube-lfvreacstuiobn-sf rwacetrieo ns
wecroelleccotlelde catnedd wanerde fwuretrheerf usurtbhjeecrtesdu tboje pcrteepdartaotivper eTpLaCra ftoivr etheT LisColaftoiornt hofe thiseo mlaatijoorn coomf pthoeunmdsa, jor
comyieploduinngd cso,mypieoldunindgs 1c o(m11p mougn) dfrsom1 (s1u1b-mfrga)ctfioronm F2s(u7)b, -2fr a(1c0ti omng)F f2r(o7m), 2su(b1-0framctgio)nf rFo2m(8)s uabn-df r3a c(t7i on
F2m(8)g)a fnrdom3 s(7ubm-fgra)cftrioonm Fs2u(4b)- (fFraigcutiroen 2)F. 2T(h4e) s(Ftriugcutruere2s) o.fT ihsoelastterud cctoumrepsooufndisso wlaetered ecluomcidpaotuedn dussinwge re
eluNcMidRat (e1dDu asnidn g2DN) ManRd h(1igDh-arnedso2luDt)ioann (dHRh)i gMhS-r eexspoeluritmioennt(sH, cRo)mMpaSrienxgp weritihm leitnetrsa,tucroem dpataar.i nTghewsei th
litecroamtuproeunddasta w. eTreh eidseenctiofimedp oaus ncadsssiwpoeureroild (e1n) t[i2fi1e],d β-assitocastsesrioplo (u2r) o[l22(,12)3][ 2a1n]d, βα--saimtoysrtienr o(3l) ([22)4–[2226,]2 3]
(Tables S2 and S3, Figure 2).
andα-amyrin(3)[24–26](TablesS2andS3,Figure2).

Figure2.StructuresofisolatedbioactiveterpenoidsfromP.rotundifolium.Cassipourol,β-sitosteroland
Figure 2. Structures of isolated bioactive terpenoids from P. rotundifolium. Cassipourol, β-sitosterol
α-amyrin(compounds1–3,respectively).
and α-amyrin (compounds 1–3, respectively).
2.32..3Is. oIlsaotleadteCd oCmompopuonudnsdsE ExxeretrtA Anntit-i-QQSS aanndd//oorr AAnnttii--BBiiooffiillmm PPrrooppeerrttieiess oonn PP. .aaeerruuggininosoas aPPAAOO1 1
CaCsassipsiopuoruorlo,l,β β--ssiittoosstteerrooll aanndd αα-a-mamyryirni nwewree rienviensvtiegsattiegda tfeodr tfhoerirt hefefierctesf ofenc tQsSo-rnegQulSa-treedg ulalsaBt ed
lasaBndan rdhlrAh lgAengeesn eesxperxepssrieosns,i oQnS,-QreSla-rteedla tveidruvleinrucele nfaccetofrasc tporrosdpurcotidounc tainodn bainodfilbmio ffiolrmmafotiromn aitni oPn. in
P.aaeerruuggiinnoossaa PPAAOO11.. IIssoollaatteedd ccoommppoouunnddss wweerere tetsetsetded ata t20200 0µµMM, a,na narabribtriatrrayr cyocnocnecnetrnattriaotnio pnrepvrieovuisoluys ly
usuedsedb ybyR Rasaasmamiriaravvaakkaa eett aall.. [[44]] toto inivnevsetsigtiagtae taenatin-vtii-rvuilreunlceen accetiavcittiievsi toife steorpfetneorpidesn cooimdspocoumndpso. uAns ds.
Assshhoowwnn ini nFiFgiugruer 3e, 3ca,scsaispsoipuoroulr aonlda nβd-siβto-sstiteorostle, arot l2,0a0t µ2M00, µsiMgn,ifsiicgannitfilyc arendtluycree tdhue ceexpthreesesixopnr eosf sliaosBn of
las(B37( 3±7 4±%4 a%nda n2d5 2±5 5±% 5o%f ionfhiibnihtiiobnit,i orens,preecstpiveecltyi;v eFliyg;uFreig 3uAre) a3nAd) arhnldA r(h4l6A ±( 446%± an4d% 3a2n d± 35%2 ±of5 %
inhibition, respectively; Figure 3B), without affecting P. aeruginosa growth and the expression of the
of inhibition, respectively; Figure 3B), without affecting P. aeruginosa growth and the expression
Int.J.Mol.Sci.2017,18,1270 5of22
Int. J. Mol. Sci. 2017, 18, 1270 5 of 22
oQfSth-iendQeSp-einnddeepnet ngdenene tagceeAn e(FaicgeuAre( F3iCgu),r we h3Cer)e,aws hαe-raemasyrαin-a hmaydr inno heaffdecnto onef ftehcet eoxnprthesesieoxnp roefs tshioense
ogfetnheess. eBegseindeess., wBheesind Pes. ,aewruhgeinnoPs.a aPeAruOg1in wosaas PgAroOw1nw ina sthger opwrenseinncet hoef cparsessiepnocueroolf ocra βss-sipitooustreorlolo, ra
βd-rsaitsotisct eirnohl,ibaitdiorans toifc tihneh ipbritoidonucotifotnh eopf rpoydouccytaionnino (f7p5y ±o c4y%a nainnd( 7454 ±± 43%% aonf din4h4ib±iti3o%n,o rfeisnpheicbtiitvioelny,;
rFesigpuecrtei v3eDly); Faingdu rreh3aDm)naonldiprihdasm (4n5o li±p i4d%s (4a5nd± 344% ±a n3d%3 4of± in3%hiboiftiionnh,i brietisopne,crteivspeleyc;t ivFeiglyu;rFei g3uEr)e w3Ea)s
wreacsorredceodr.d eTdh.isT hefisfeecftf ewctaws ansont ootbosbesrevrevde dini npprreesseennccee ooff αα--aammyyrriinn.. IInntteerreessttiinnggllyy,, CCaassssiippoouurrooll,,
ββ--ssiittoosstteerrooll aannddα α-a-ammyyrrinini ninhhibibitieteddb bioiofiflimlmf oformrmataitoionn( 5(252± ±4 4%%,,4 444± ± 33%% aanndd5 555± ± 22%%,, rreessppeeccttiivveellyy;;
FFigiguurree3 3FF).).T oToe nesnusruerteh athtatht ethdee cdreecarseeaisne binio bfiilomfilbmio mbiaosmsaasnsd avnidru vleinrucleefnaccet ofrasctporrosd purcotidouncitsionno tisd nuoet
toduaen tion haibni tiinohniboiftigorno wofth g,rgorwowtht,h gkrionwettihc skainndetiCcsF Uanmde aCsFuUre mmeenatssurweemreenrtesc owrdereed rinectohredepdre sine ntchee
opfrceassesnicpeo oufr ocla,sβsi-psoituorsotel,r βol-soitrosαt-earmoly orri nα-aatm2y00rinµ Mat .2A00s µshMo.w Ans bshyotwunrb bidyi ttyur(bFiidgiutyr e(F4iAgu)raen 4dAC) aFnUd
(FCiFgUur e(F4iBg,uCr)e m4Bea,Csu) rmemeaesnutsr,etmheenctesl,l gthreo wcethll agnrdowvitahb ialintyd ovfiaPb.aileirtuyg oinf oPsa. PaAerOug1inwoesare PnAoOta1f fwecetreed nboyt
aanfyfeoctfetdh ebsye acnomy opfo tuhnedses ocovmerpaollusntdags eosvoefr baallc stetarigaelsg orof wbatcht.erial growth.

Figure 3. Anti-virulence effects of isolated compounds from P. rotundifolium EtOAc extracts in P.
Figure 3. Anti-virulence effects of isolated compounds from P. rotundifolium EtOAc extracts in
aeruginosa PAO1: (A) effect of isolated compounds on the expression of QS-regulated lasB gene; (B)
P.aeruginosaPAO1: (A)effectofisolatedcompoundsontheexpressionofQS-regulatedlasBgene;
effect of isolated compounds on the expression of QS-regulated rhlA gene; (C) effect of isolated
(B)effectofisolatedcompoundsontheexpressionofQS-regulatedrhlAgene;(C)effectofisolated
compounds on the expression of QS-independent aceA gene; (D) effect of isolated compounds in
compounds on the expression of QS-independent aceA gene; (D) effect of isolated compounds in
biofilm formation; (E) effect of isolated compounds on pyocyanin production; and (F) effect of
biofilmformation;(E)effectofisolatedcompoundsonpyocyaninproduction;and(F)effectofisolated
ciosmolpatoeudn cdosmonporuhnamdsn oolnip rihdasmpnroodliupcidtiso np.rEodacuhctiisoonla. tEeadccho misoploautendd cwomasptoeustnedd awta2s0 0teµstMed; naatr i2n0g0e µniMn;
(Nnaarri,n4gmenMin) (iNsuars,e 4d masMa)r eisf eurseendc easQ aS rienfheirbeintocer; QolSe ainnhoilbicitaocri;d o(leOaAno,8li0c0 aµciMd )(OisAu,s 8e0d0a µsMan) iasn utis-ebdio afisl man
caonnttir-oblioafnildmd cimonettrhoyl lasnudlf odxiimdeet(hDyMlsuSlOfo,x1i%de) a(DsMasSoOlv, e1n%t)c oans tar oslo.lTvhenetc ecollndtreonls. iTtyheo fctehlle dbeancsteitryia owf tahse
abssaecsteserida awt6a0s0 ansmse(sbsoedld aert ro6r00b anrsm)a n(bdoglde neereroxpr rbesasriso)n awnads mgeenaes uerxepdraessstihoenβ w-gaasla cmtoesaisduarseeda catsiv itthye
oβf-tghaelalacctZosgideanseef uascitoivnistya nodf ethxep rleascsZe dgeinneM fiullseirounns iatsn(dg reexypbreasrs).edB ioinfi lMmiflolerrm uantiiotsn (wgraesyq ubaanr)t.i fiBeiodfiblym
cfroyrsmtaaltvioionl ewtsatsa inqiunagnatinfidedm ebays ucrreydstaatl Av59io0nlemt .Pstyaoinciynagn inanwda smeexatrsaucrteedd ,aqtu aAn5t9i0finme.d Pbyyoacbysaonribna nwcaes
mexetarsaucrteemd, eqnutsanatti3fi8e0dn bmy aanbdsocrablacnuclaet emdeaassuthreemraetniotsb aett w38e0e nnAm and caanldcuAlated a.sT thhee rrhaatmio nboeltiwpiedesn
380nm 600nm
pAro38d0numc tainodn wA6a0s0nmm. eTahsue rrehdamusninoglipmidesth pyrloednue-cbtiloune- wbaasse dmmeaestuhroedd aunsdinegx mpreetshsyeldenine-µbglu/em-bLa.sEedrr omrebtharosd
raenpdre esexnptretshseedst iann µdagr/mdLer. rEorrrsoor fbtahrse rmeperaensse;natl tlheex sptearnimdaerndt serwroerrse opf etrhfeo rmmeeadnsi;n aqllu eixnptuerpimliceantets wwiethre
thpreerefoinrmdeepde nind eqnutinastusapylsicaanted wasittehr itshkrseien dinicdaetpeesnadmepnlte sasthsaaytsa raensdig nasifitecrainsktlsy idnidffiecraetnet sfarommpltehse tDhMat SaOre
(Osignne-ifwicaayntAlyN dOifVfAerefnoltl ofrwoemd tbhye DDuMnnSeOt t’(sOtnees-twoafym AulNtiOplVeAco fmolploawriseodn bs;yp D<u0n.n01e)tt.’sA tMesYt :oαf -mamuyltriipnl;e
CcAomS:pcaarsissiopnosu; rpo l<; 0a.n0d1)S. IATM:βY-s: iαto-astmeryorli.n; CAS: cassipourol; and SIT: β-sitosterol.
Int.J.Mol.Sci.2017,18,1270 6of22
Int. J. Mol. Sci. 2017, 18, 1270 6 of 22

Figure4.Effectofcassipourol,β-sitosterolandα-amyrinonthegrowthandviabilityofP.aeruginosa
Figure 4. Effect of cassipourol, β-sitosterol and α-amyrin on the growth and viability of P. aeruginosa
PAO1.(A)GrowthkineticsofPAO1inpresenceofcassipourolorβ-sitosterolorα-amyrinat200µMor
PAO1. (A) Growth kinetics of PAO1 in presence of cassipourol or β-sitosterol or α-amyrin at 200 µM
DMSO1%overaperiodof22h.ThecelldensityofthebacteriawasassessedasA andcolony
or DMSO 1% over a period of 22 h. The cell density of the bacteria was assessed as6 0A0n6m00nm and colony
formingunits(CFU)werequantifiedafter: 6h(B);and18h(C).Thestatisticalsignificanceofeach
forming units (CFU) were quantified after: 6 h (B); and 18 h (C). The statistical significance of each
test(n=3)wasevaluatedbyconductingOne-wayANOVAfollowedbyDunnett’stestofmultiple
test (n = 3) was evaluated by conducting One-way ANOVA followed by Dunnett’s test of multiple
comparisons(i.e.,eachtestwascomparedwiththecontrolcondition,DMSO),andapvalueof<0.01
comparisons (i.e., each test was compared with the control condition, DMSO), and a p value of <0.01
wasconsideredassignificant.
was considered as significant.
2.4. Cassipourolandβ-SitosterolAffecttheExpressionoflasI/R,rhlI/RinP.aeruginosaPAO1butNotthe
2.4. Cassipourol and β-Sitosterol Affect the Expression of lasI/R, rhlI/R in P. aeruginosa PAO1 but Not the
GlobalActivatorGenesgacAandvfr
Global Activator Genes gacA and vfr
As cassipourol and β-sitosterol impact on QS-dependent rhlA and lasB gene expression,
weAesv aclausastiepdotuhreoilm apnadc tβo-fsibtootshtecrooml pimoupnadcts oonn QQSS-r-edgeupleantodreynlta srIh/RlAa nadndrh llIa/sRBg genenese thexaptpreosssiitoivne,l ywe
evacolunatrtoedlt htheee ximprpesascito nofo bforhtlhA caonmdplaosuBngdesn eosn[ 2Q7]S.-Sreimguillaartloyr,yw elaesIv/aRl uaantedd rthhleI/iRr imgepnaecst othnatth peogsloitbivaelly
conactrtiovla tthoers egxapcAreasnsidonvf rotfh rahtlAex earntdp olassitBiv geeenffeesc t[s27o]n. tShiemtirlaanrslycr, iwpteio envaallruegauteldat othrseiLra simRpanadctR ohnlR th[2e8 g,2lo9]b.al
actCivaastsoiprso ugraoclAa nadndβ -vsiftro tshtearto elxaetr2t0 0poµsMitisvieg neiffifceacntst lyonre tdhuec etrtahnesecxripprteisosnioanl roefgtuhelaltaosrIsa nLdaslRas Rangde nRehslR
[28(,5209]±. C3%assainpdou43ro±l a5n%d oβf-sinithoisbtietrioonl ,arte 2s0p0e cµtiMve slyig;nFiifgiucarent5lAy )readnducoef tthhee erhxlpIraensdsiorhnl Rofg tehnee sla(s4I4 a±nd3 l%asR
genanesd (3590 ±± 33%% aonfdi n4h3i b±i 5ti%on o,fr einspheibctiitvioenly, ;reFsigpuercetiv5Bel)yb; uFtignuorteo 5fAth) eangdlo boaf lthaect rivhalIt oarndg ernhelRs ggaecnAesa n(4d4 ±
3%v farn(dF i3g9u r±e 35%C) o.Tf hinishsibuigtigoens,t srethspatecbtoivthelcyo;m Fpigouurned 5sBi)m bpuati rnQotS oaft tthhee glelovbelaol fatchtievlaatsoarn gdenrhelss gyascteAm asnd
vfrc (iFrciuguitrrye i5nCP).. aTehruisg isnuogsagePsAtsO t1h,awt hbiocthhc coonmsepqouuenntdlys rimedpuacier tQheS parto tdhuec lteiovnelo offp tyhoec ylaasn ainnd(F righul rseys3tDem) s
andrhamnolipids(Figure3E).
circuitry in P. aeruginosa PAO1, which consequently reduce the production of pyocyanin (Figure 3D)
and rhamnolipids (Figure 3E).
Int.J.Mol.Sci.2017,18,1270 7of22
Int. J. Mol. Sci. 2017, 18, 1270 7 of 22
FFiigguurree 55.. EEffffeecctto offc acsassispiopuoruorloaln adnβd- sβi-tsoisttoesrtoelroonl oQnS QgeSn gesen(leass I(/RlasaIn/Rd rahnldI/ Rr)halIn/Rd)g alonbda lgaloctbiavla taocrtigveanteosr
(ggeanceAs a(ngdacvAf ra)nedxp vrfers)s ieoxnprinesPs.ioaner uing inPo. saaePruAgOin1o:sa(A P)AeOffe1c: t(oAf)c eafsfseicpto oufr ocla,sβs-ipsiotousrtoelr, oβl-asnitdosαte-aroml yarnind
oαn-almasyRri(ng roeny lbaasrR) a(gnrdeyla bsIa(rc) leaanrdb laars)I e(cxlperaers sbiaorn) feoxlplorewsisniogn1 8fohlloowfginrgo w18th h; (Bof) gefrfoewcttho;f (cBas) seipffoeuctr oolf,
βca-ssistiopsotuerrooll,a βn-dsiαto-satmeryorli annodn αr-halRmy(grriney obna rrh)laRn (dgrrehylI b(calre)a arnbda rr)helIx (pcrleeassri obnarf)o elxlopwreinssgio1n8 fhololofwgrionwg t1h8;
ahn odf (gCr)oewfftehc;t aonfdc a(sCs)i peoffuercotl o,βf c-saistsoispteoruorloaln, dβ-αsi-taomstyerrionl oanndg aαc-Aam(gyreriynb oanr) gaancdA v(fgrr(ecyle baarrb)a arn)dex vpfrre (scslieoanr
fboalrlo) wexipnrge1ss8iohno ffogllroowwitnhg. E18a chh oisf oglraotewdthc.o Emapcohu isnodlawteads cteosmtepdoautn2d0 w0µasM te.sNteadri antg 2e0n0in µ(MN.a Nr,a4rminMge)noinr
A(Nziatrh, ro4m myMcin) (oAr ZAMz,it2hµrogm/mycLin= 2(AµZMM)a, r2e uµsge/dmaLs a= q2u oµrMum) asreen suinsgedin ahsi bait oqrucoornutmro l.seGnesninege xipnrheisbsiitoonr
wcoanstrmole.a Gsuerneed eaxsptrheessβio-ng awlaacst omsiedaassuereadct iavsi ttyheo fβt-hgeallaacctZosgiednaesef uascitoivnistya nodf tehxep lraecsZs egdenine Mfuisliloernsu naintds.
Eexrrporresbsaerds rienp rMesiellnetr thuenisttsa. nEdrarrodr ebrarorsrs roefptrheesemnet atnhse; asltlaenxdpaerrdim eernrotsrsw oefr ethpee rmfoermanesd; ainll qeuxipneturipmliecnattes
wwietrhet phreerefoirnmdeedp einnd qeunitnatsuspalyicsaaten dwaitshte trhisrkese iinnddiecpaetensdaemntp alesssatyhsa taanrde saisgtenriifisckasn itnlydidciaftfee rseanmtfprloems tthhaet
DarMe sSiOgn(iOfincaen-wtlayy dAifNferOeVnAt frfoolmlo wtheed DbMySDOu n(Onentet’-swtaeyst AofNmOuVlAtip floellcoowmepda rbiyso Dnus;npne<tt0’.s0 t1e)s.t of multiple
comparisons; p < 0.01).
2.5. IsolatedTerpenoidsDisrupttheP.aeruginosaPAO1BiofilmFormationinaDose-DependentManner
2.5. Isolated Terpenoids Disrupt the P. aeruginosa PAO1 Biofilm Formation in a Dose-Dependent Manner
Asthreecompoundsinhibitbiofilmformation,wefurthercharacterizethisanti-biofilmproperty
byprAims atrhilryeein vceosmtipgaotuinngdst hienirheibffiet ctbsioinfilamd ofsoer-mdeaptieonnd, ewntem faunrntheer.r Acshaarraecstuelrti,zaen ttih-bisio fialnmti-abcitoifviiltmy
ipsrogpreearttlyy ebnyh apnrcimedarbilyy inincrveeasstiingagtitnhge ctohneicre netfrfaetcitosn sino fat esdtoesde-cdoemppenoduenndts m(Faignunreer.6 )A, ss uag geresstiunlgt,
aandtoi-sbei-odfielpme nadcetinvtitayc tiisv igtyrefaotrlyc aesnshipaonucerodl (biyn tihnecrreaansigneg1 2th.5e– 8c0o0ncµeMnt;raICtionos fo1f8 0teµstMed) ,cβo-msitpoosutenrdosl
50
((5F0ig–u80re0 6µ)M, s;uIgCg5e0stoinfg2 0a0 )doasned-dαe-paemnydreinnt (a5c0t–iv8i0ty0 fµoMr ;caICss5i0pooufr1o9l 0()i.nA thded irtaionngael l1y2,.5in–8t0h0e µteMst; iInCg50f oorf
s1y8n0e µrgMie)s, βb-ysictohsetcekreorlb (o5a0r–d80m0 eµthMo;d I,Cc5o0 mofb 2in00a)t iaonnds oαf-aamctiyvreinc o(5m0–p8o0u0n µdMsr;e IvCe5a0 loifn 1d9if0f)e. rAendtdFitIiConinadlleyx, eins
(tFhIeC Itecsatsisnipgo uforor ls(1y0n0erµgMie)s/ αb-ya mcyhreicnk(e1r0b0oµaMrd) =m1e;tFhIoCdI, cacsosmipboiunraotlio(1n0s0 oµfM a)/ctβiv-sei tocsotmerpolo(u1n00dsµ Mre)v=ea1l;
FinICdiIffβe-rseintot sFteIrCo lin(1d0e0xµesM ()F/IαC-Ia mcaysrsiinpo(1u0r0olµ (M10)0= µ1M).)/α-amyrin (100 µM) = 1; FICI cassipourol (100
µM)/β-sitosterol (100 µM) = 1; FICI β-sitosterol (100 µM)/α-amyrin (100 µM) = 1).
Int.J.Mol.Sci.2017,18,1270 8of22
Int. J. Mol. Sci. 2017, 18, 1270 8 of 22
Figure 6. Dose-dependent anti-biofilm activity of: cassipourol (A); β-sitosterol (B); and α-amyrin (C).
Figure6.Dose-dependentanti-biofilmactivityof:cassipourol(A);β-sitosterol(B);andα-amyrin(C).
The biofilm formation of P. aeruginosa PAO1 grown in minimal medium supplemented with DMSO
ThebiofilmformationofP.aeruginosaPAO1growninminimalmediumsupplementedwithDMSO1%,
o1l%ea,n oolleiacnaocliidc 8a0ci0dµ 8M00( OµMA) (oOrAd)i fofer rdeinftfecroennct ecnotnracetinotnrsatoiofnpsu oriffi peudrcifoiemdp cooumnpdosu(fnrdosm (f6r.o2m5t 6o.2850 0toµ 8M0)0
aµftMer) ianfctuerb ainticounbwatiitohno uwtitahgoituatt iaognitaatti3o7n◦ aCt f3o7r °2C4 fho.rB 2i4o fihl.m Bifoofrimlma tfioornmwaatisoqnu wanasti fiqeudanbtyificerdy sbtayl cvriyoslteatl
svtaioinleint gstaanidnimnge aasnudre dmaesaAsured a.sA All5e9x0npme. riAmlle netxspwereirmeepnetrsfo wrmereed pinertrfioprlmiceadte iwni tthritphlrieceatien dweipthen tdhernete
590nm
aisnsdaeypse.nEdrernotr absasrasyrse. pErrersoern tbathrse rsetapnredsaerndt etrhreo rsstaonfdtahredm eerraonrss; oafll tehxep emreimanesn; tsalwl eexrepepreimrfoenrmtse wdeinre
qpueirnftourpmliecda tien wqiutihnttuhpreliecainted wepiethn dthenretea isnsdayepseannddenats taesrsiaskyss iannddi caastteersiasmksp ilnedsitchaatet asraemspiglensi fithcaatn talrye
dsiifgfenriefnictafnrotlmy tdhieffDerMenStO f(rOomne -twhaey DAMNSOOV A(Ofonlelo-wweady bAyNDOuVnnAe ttf’oslltoeswteodfm buyl tDipulencnoemtt’psa rteissotn os;f pm<u0l.t0ip1)l.e
comparisons; p < 0.01).
2.6. IsolatedTerpenoidsDisrupttheP.aeruginosaPAO1BiofilmPhenotype

Fluorescence microscopy indicated that P. aeruginosa PAO1 grown for 24 h in static control
conditionformsathickandhomogenousbiofilmlayeroncoverslipswithgoodcell-to-cellconnections
interspaced by uncolonized surfaces (Figure 7; DMSO). By contrast, cassipourol-, β-sitosterol-,
Int. J. Mol. Sci. 2017, 18, 1270 9 of 22
2.6. Isolated Terpenoids Disrupt the P. aeruginosa PAO1 Biofilm Phenotype
Fluorescence microscopy indicated that P. aeruginosa PAO1 grown for 24 h in static control
condition forms a thick and homogenous biofilm layer on coverslips with good cell-to-cell
Int.J.Mol.Sci.2017,18,1270 9of22
connections interspaced by uncolonized surfaces (Figure 7; DMSO). By contrast, cassipourol-,
β-sitosterol-, α-amyrin-treated P. aeruginosa PAO1 cells failed to establish compact cell-to-cell
attαac-ahmmyernint -rterseuatletidnPg. iane rau griendousactPiAonO o1fc melliscrfaoicleodlotnoieess tcaobnlifslhueconmcep (aFcitgcuerlle- t7o)-.c Aells atthtaec thhmreeen tcoremsuplotiunngds
imipnaairr etdhuec tfioornmoaftmioincr oocfo lPon. iaeesrcuogninflousean cPeA(FOig1u rbeio7f)i.lmAss,t hweteh rfeuercthoemr peoxuanmdsinimedp atihrethires foimrmpaatciot non
preo-ffoPr.maeerdu gPin.o saaerPuAgiOn1osab ioPfiAlmOs1, wbieoffiulmrth. eArse xsahmoiwnend itnh eFirisguimrep a8c,t tohne pared-dfoitrimone doPf . caaersusigpinoousraol,
β-sPiAtoOst1erboilo fialnmd. αA-asmshyoriwn ntoin oFnieg-udraey8-o,ltdh epared-dfoitriomnedo fbciaosfsiilpmosu rroels,uβlt-ss itions tae rololsasn odf αc-oammpyaricnt taond
one-day-oldpre-formedbiofilmsresultsinalossofcompactandheterogeneousstructures,leading
heterogeneous structures, leading to biofilms mainly composed by isolated bacterial clumps and
tobiofilmsmainlycomposedbyisolatedbacterialclumpsanddisorganizedmicrocoloniesstructure.
disorganized microcolonies structure. This remarkable modification is confirmed by a quantitative
TThisremarkablemodificationisconfirmedbyaquantitativereductionofbiofilmasmeasuredby
reduction of biofilm as measured by crystal violet method (for cassipourol-, β-sitosterol and
crystalvioletmethod(forcassipourol-,β-sitosterolandα-amyrin,47±3%,34±3%and38±2%of
α-amyrin, 47 ± 3%, 34 ± 3% and 38 ± 2% of reduction, respectively; Figure 8). Conversely, a two-fold
reduction,respectively;Figure8). Conversely,atwo-foldincreaseofplanktonicbacteriapopulation
increase of planktonic bacteria population is recorded for treated cultures by CFU quantification
isrecordedfortreatedculturesbyCFUquantification(Figure8),suggestingthatthesecompounds
(Figure 8), suggesting that these compounds induce bacterial dispersion out of a pre-formed biofilm.
inducebacterialdispersionoutofapre-formedbiofilm.

FigFuigreu re7.7 .PP.. aaeerruuggiinnoossaaP APAOO1b1i obfiilomfilpmh enpohteynpoetsyapseasf feacst eadffbeyctceadss ibpyo ucroalssoirpβo-usritools toerro lβo-rsiαto-asmteyroriln .or
α-aPm.ayerruing.i nPo.s aaePrAugOin1ocseal lPsAwOer1e cienlclus bwaeterde sintactuicbaalltyeda ts3ta7t◦icCalfloyr a2t4 3h7f o°Cr bfioorfi 2lm4 hfo fromr abtiioonfilimnp froersmenacteion
in opfreDsMenScOe o1f% D,oMrScOas s1i%po, uorro cla(CssAipSo)uorroβl -(sCitAosSt)e roorl β(S-sITit)oostreαro-al m(SyITri)n o(rA αM-aYm)aytri1n0 0(AµMMY.C) aeltl s10w0e µreM.
visualizedafterstainingwithSYTO-9(greenfluorescenceforlivingbacteria)andpropidiumiodide
Cells were visualized after staining with SYTO-9 (green fluorescence for living bacteria) and
(redfluorescencefordeadbacteria)furnishedintheLIVE/DEADBacLightkit.Fluorescencemicroscopy
propidium iodide (red fluorescence for dead bacteria) furnished in the LIVE/DEAD BacLight kit.
wasachievedbyusingaLeicaDMIRE2invertedfluorescencemicroscopeusinga40×objectivelens
Fluorescence microscopy was achieved by using a Leica DM IRE2 inverted fluorescence microscope
andimageswerefalse-coloredandassembledusingAdobePhotoshop.
using a 40× objective lens and images were false-colored and assembled using Adobe Photoshop.
Int.J.Mol.Sci.2017,18,1270 10of22
Int. J. Mol. Sci. 2017, 18, 1270 10 of 22

FFiigguurree 88.. EEffffeecctt ooff ccaassssiippoouurrooll,, ββ-s-sitiotossteterorol lanandd αα-a-mamyryirni nono na aonoen-ed-adya-yo-lodl dprperfoefromremde dbiobfiiolmfil mbyb Py.

Pa.eareurguignionsoas aPPAAOO11. .PP.. aaeerruuggiinnoossaa PPAAOO11 cceellllssw weererei nicnucbuabtaetdedfo fro2r4 2h4 ahn danthde nthternea ttreedaoterdn ootr fnorot2 4fohr w24it hh
DwMithSO D1M%S,Oor 1c%as,s iopro ucarsosli(pCoAuSro)lo r(CβA-sSit)o sotre rβo-ls(iStoITst)eororlα (-SaImTy) roinr (αA-aMmYy)raint 1(0A0MµMY). Batio 1fi0l0m µpMhe. nBoitoyfpilems
wphereenvoitsyupaelsiz wederaes vinisFuiagluizreed6 .asA idnd Fitiigounrael l6y,. bAidofidlimtiofnoarlmlya, tbioionfwilmas fqourmanattiifioend wbaysc qryusatnaltivfiieodle btyst carinyisntagl
avniodlemt esatsauinreindga sanAd5 90mnmeaasnudredC FaUs mAe5a90snumr eamnedn tCoFfUp lamnkeatosunricembaecntte roiaf apnldanbkiotofinlmic -ebnaccatpersiual aatendd
bbaioctfeilrmia-etrnecaatpedsufloarte2d4 hbawctiethriaD MtreSaOte1d% foorr 2c4a shsi pwoiuthro Dl(MCASOS) 1o%r βo-rs ictoasstseiproolu(rSoIlT )(CoArαS)- aomr yβr-isnit(oAstMerYo)l
a(StI1T0)0 oµr Mα-aomnyarionn (eA-dMayY-)o aldt 1c0u0l tµuMre .onQ ua aonnteifi-dcaayti-oonldo cfublitoufirelm. Qfuoarnmtiaftiicoantiobny oPf. baieorufiglmin ofsoarmgraotwionn biny
iPn. maeirnuigminaolsam gerdoiwan(g irne yinb mari)niamnadl CmFeUdiam (egarseuyr bemare) natnd(c lCeaFrUb mare)aosufrpelmanekntto (ncilceabra bcaterr) ioaf apfltaenrksttoantiicc
ibnaccutbearitaio anftaetr3 s7ta◦tCicf oinrc2u4bha.tiBoino fialtm 37f o°rCm faotrio 2n4w ha. sBqioufainlmti fifeodrmbyatcioryns twalasv iqouleatnsttiafiiendin bgya ncdrymstaeal svuioreledt
asttaAin5i9n0ngm anadn dmpelaasnukrteodn aict Aba5c90tnemr iaanbdy pClaFnUktmoneiac sbuarcetmereian tb.yE CrrFoUr bmaerassruerpermeseenntt. Ethrerosrt abnadrsa rrdeperrersoernst
othfeth setamnedaanrsd; aelrlreoxrsp eorfi mthene tsmweaenres; paelrlf oerxmpeerdiminenqtusi nwtuepreli cpaeterfowrimthedth riene qinudinetpuepnldiceantet awssiathy sthanrede
ainstdeeripseknsdinedntic aastesasyasm apnleds athstaetraisrkess iignndiificcaaten tslaymdpiflfeesr etnhtaftr oamre tshiegnDifMicSaOntl(yO ndeif-fweraeynAt fNroOmV Athfeo lDloMwSeOd
b(OynDeu-wnnaeyt tA’sNteOstVoAf mfoulllotiwpeledc boym Dpuarninsoentts's; pte<st0 o.0f 1m).ultiple comparisons; p < 0.01).
22..77.. IIssoollaatteedd TTeerrppeennooiiddss EExxhhiibbiittS SyynneerrggisistticicA Accttivivitityyw witithhT Toobbrarammyycicnina aggaianinststB Bioifioflimlm-E-Enncacpaspusulaltaetded
PP..a aeerruuggininoossaaP PAAOO11
GGiivveenn tthhee ddiissrruuppttiioonn ooff bbioiofifilmlm stsrtuructcuturer einidnudcuecde dbyb tyheth iesoilsaotleadt ePd. rPo.turontduinfodliiufomliu tmerpteernpoeidnso,i dwse,
whyephoytphoesthizeesdiz ethdatth abtiobfiiolmfil-men-ceanpcsauplsauteladt ebdabctaecrtiear iwaowuoludl dbebceocmome emmoorer eaacccceessssibiblele ttoo aann aannttiibbiioottiicc
ttrreeaattmmeenntt.. AAccccoorrddiinnggllyy,, tthhee eeffffeeccttiivveenneessss ooff ttoobbrraammyycciinn ccoommbbiinneedd wwiitthh ppuurriififieedd ccoommppoouunnddss wwaass
eevvaalluuaatteedd oonn oonnee--ddaayy--oolldd bbiioofifillmmss.. TThhee aaddddiittiioonn ooff ccaassssiippoouurrooll,, ββ--ssiittoosstteerrooll oorr αα--aammyyrriinn ((110000 µµMM))
ccoonnssiiddeerraabbllyyi mimpprorovvededth teheef feefcfeticvteivneenssesosf toofb troabmraymcinyc(i5n0 (µ5g0/ µmgL/m=L1 0=7 1µ0M7 )µaMga)i nasgtaPi.nasetr uPg. ianeorsuagPiAno0s1a
(PFAig0u1r e(F9i)gwurieth 9a) wdritahs taic drreadsuticct iroenduinctcieolnl vinia cbeillli tvyiaobfibliitoyfi olmf b-eionfcialmps-uenlactaepdsbualactteedri aba(8c9te±ria2 (%89, 6±9 2±%,2 6%9
a±n 2d%7 1an±d2 7%1, ±r e2s%pe, crteisvpeelyctviveerslyu sv4e0rs±us5 4%0 f±o r5%DM foSrO DtMreSaOtm terneat)t.mAesnstu).c Ahse fsfueccths emffaeyctasr imseafyr oamrisae sfirmompl ea
ssyimneprlgei sstyicneerffgeicsttibc eetfwfeecetn bteetswteedenc otmespteodu ncodmsapnoduntodbs raanmdy ctoinb,rathmuyscwine, tehvuaslu watee devthaleuFaIteCdi nthdee xFoICf
tienrdpeexn ooidf -tteorbpreanmoyidci-ntocbormambiyncainti ocnosminbiPn.aateiroungsi nions aPp.l aanerkutoginniocssat apgleanbkytochneicc ksetrabgoea rbdym cehtehcokde,ribnotahred
cmonetcheondtr,a itnio tnhser caonngcee6n.t2r5a–t8io0n0sµ rMan.Rgee s6u.2lt5s–s8h0o0w µtMha. tRtehseureltiss snhooswy ntheragt itshtiecreef fise cntob usytnraetrhgeirstiincd eiffffeecret nbcuet
(rFaItChIert obinradmiffyecrienn/cαe -a(mFIyCrIi nt=ob1r;aFmICyIctinob/αr-aammyycriinn/ β=- si1to; stFeIrCoIl =to1;bFraICmIytcoibnr/βam-siytocisnte/rαo-la m= yr1i;n =FI1C)I,
stuobgrgaemstyincgin/tαha-atmthyeriinn cr=e as1e),d esfufgecgteisvteinnges sthofatt obthraem iynccirneainsebdi ofieflfmec-teinvceanpessus laotef dtboabcrtaemriayciisn noint
absisooficlimat-eedncwaipthsualnatiendc rbeaacsteeroifa tihse niontt raisnssoicciaantetidb iwotiitch aacnti vinitcyreoafsteo obfr atmhey icnintr.insic antibiotic activity of
tobramycin.