Synergistic interactions between doxycycline and terpenic components of essential oils encapsulated within lipid nanocapsules against gram negative bacteria

Synergistic interactions between doxycycline and terpenic

components of essential oils encapsulated within lipid nanocapsules against gram negative bacteria

C. Valcourt

^a,

*, P. Saulnier

^a

, A. Umerska

^a

, M.P. Zanelli

^b

, A. Montagu

^a

, E. Rossines

^c

, M.L. Joly-Guillou

^b

aINSERMU1066,MicroetNanomédecinesBiomimétiques,IBSCHU,4RueLarrey,F-49933Angers,France

bDepartmentofBacteriology,UniversityHospitalofAngers,4RueLarrey,F-49933Angers,France

cEydoPharma,41RueNoelBallay,28000Chartres,France

ARTICLE INFO

Articlehistory:

Received30August2015

Receivedinrevisedform23November2015 Accepted24November2015

Availableonline2December2015

Keywords:

Doxycycline Synergy

Lipidnanocapsules Essentialoils Resistantbacteria

ABSTRACT

Thecombinationofessentialoils(EOs)withantibioticsprovidesapromisingstrategytowardscombating resistantbacteria.Wehaveselectedamixtureof3majorcomponentsextractedfromEOs:carvacrol (oregano oil), eugenol (clove oil) and cinnamaldehyde (cinnamon oil). These compounds were successfullyencapsulatedwithinlipidnanocapsules(LNCs).TheEOs-loadedLNCswerecharacterisedby a noticeably highdrug loadingof 20%and averysmallparticle diameterof 114nm.The invitro interactionsbetweenEOs-loadedLNCsanddoxycyclinewereexaminedviacheckerboardtitrationand time-kill assay against 5 Gram-negative strains:Acinetobacter baumannii SAN, A. baumannii RCH, Klebsiellapneumoniae,EscherichiacoliandPseudomonasaeruginosa.Nogrowthinhibitioninteractions werefoundbetweenEOs-loadedLNCsanddoxycycline(FICindexbetween0.7and1.30).However,when bactericidaleffectswereconsidered,asynergisticinteractionwasobserved(FBCindexequalto0.5) againstalltestedstrains.Asynergisticeffectwasalsoobservedintime-killassay(adifferenceofatleast 3logbetweenthecombinationandthemostactiveagentalone).Scanningelectronmicroscopy(SEM) wasusedtovisualisethechangesinthebacterialmembrane.Theholesinbacterialenvelopeandleakage ofcellularcontentswereobservedinSEmicrographsafterexposuretotheEOs–LNCsandthedoxycycline combination.

ã2015PublishedbyElsevierB.V.

1.Introduction

Antibioticresistanceisarapidlygrowingproblemaroundthe world.Thecurrentemergenceofmultiresistantbacteriasuchas Acinetobacter baumannii represents a serious public health problem(Dennesen et al.,1998; Song et al., 2009).Thisis the reason whymany researchers have focused theirwork on the searchfornewantibioticsbydevelopingsyntheticmoleculesand

small-moleculelibrariescustomisedforbacterialtargets.Someof themhaveintensifiedresearchonDNAsynthesisandhavestudied the correlation between the chemical structure of different molecules and their biological activity (Committee on new directionsinthestudyofantimicrobialtherapeutics).Eventhough many effective compounds have been discovered in the past, chemical variability is insufficient to prevent an escalation of antibiotic resistance (Rosamond and Allsop, 2000). During the 2000sresearcherswereinterestedingenomesinsearchfornew antibiotics (Rosamond and Allsop, 2000). Other researchers focused theirwork on derivativesof natural origin substances such as actinonin, pleuromutilin, ramoplaninand tiacumicinB (ButlerandBuss,2006)oressentialoils(Hemaiswaryaetal.,2008;

Rosato etal.,2008, 2009; WagnerandUlrich-Merzenich,2009;

Fadli et al., 2012). Essential oils are known to penetrate cell membranesandinteractwithproteintargets(StoneandWilliams, 1992).

We selected a mixture of three terpenic components of essential oils: carvacrol, eugenol, and cinnamaldehyde (Fig.1).

Abbreviations:ATP,adenosinetriphosphate;BHI,brain–heartinfusionbroth;

CFU,colonyformingunits;EF-G,elongationfactorG;EF-Tu,elongationfactor thermosunstable;EOs,essentialoils;FBCindex,fractionalbactericidalconcentra- tionindex;FICindex,fractionalinhibitoryconcentrationindex;HLB,hydrophilic–

lipophilicbalance;HMDS,hexamethyldisilazane;LNCs,lipidnanocapsules;LPS, lipopolysaccharide; MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; PDI, polydispersity index; PIT, phase inversion temperature;SCIAM,servicecommund’imageriesetd’analysesmicroscopiques.

* Correspondingauthor.

E-mailaddress:[email protected](C. Valcourt).

http://dx.doi.org/10.1016/j.ijpharm.2015.11.042 0378-5173/ã2015PublishedbyElsevierB.V.

ContentslistsavailableatScienceDirect

International Journal of Pharmaceutics

j o u r n a lh o m e p a g e : w w w . e l s e vi e r . c o m / l o c a t e/ i j p h a r m

These components of plant extracts exert their antibacterial activitybydisruptingcellmembraneandinhibitingATPaseactivity (Raybaudi-Massiliaetal.,2009;Negi,2012).

Eugenol(2-methoxy-4-(2-propenyl)phenol)isamajorcompo- nentofcloveoil(Faragetal., 1989).Eugenolisusedinmedicineasa localantisepticandasananaesthetic(Didryetal., 1994;Markowitz etal.,1992).Carvacrol(2-methyl-5-(1-methylethyl)-phenol)is a majorcomponent of oreganoand thymeoil. It is alsofoundin tequila(León-Rodríguezetal.,2008).Itisanisoprenylphenolthat hasstrongantimicrobialactivity(RollerandSheedhar2002)andis used in food preservation (BenArfa et al., 2005).trans-Cinna- maldehyde(3-phenyl-2-propenal)isamajororganiccomponentof cinnamon oil, used as a flavouring agent and in perfumery.

Cinnamaldehydeisalsousedasafungicide.Itexertshypoglycemic effect, which could be of great interest in diabetes treatment (MarlesandFarnsworth,1995;Ross,2001)

Inordertoadministerandtoprotectlipophilicsubstancessuch asessentialoils,wehavechosenlipidnanocapsules(LNCs)asa drugdelivery system. LNCs are prepared viaa phase inversion methodintroducedforthefirsttimebyShinodaandSaitoin1969 (Shinoda and Saito, 1969).It is a low energy and solvent-free methodbasedonthechangesinthehydrophilic–lipophilicbalance (HLB) of a polyoxyethylene surfactant caused by temperature modification (Huynh et al., 2009). This method enables the obtentionofsmall,homogenouslydispersedparticles.Byvarying themassratiooftheLNCscomponents:surfactants(ahydrophilic surfactant—Solutol¹, and optionally a lipophilic surfactant-lec- ithin)andtheoil(Labrafac¹), onecanyieldparticleswithsizes ranging between 25 and 100nm. These particles consist of a triglyceridecoresurroundedbyasurfactantshell.Oneofthemost importantcharacteristicsofthesenanocarriersisahighsurface area-to-volumeratiothatcanoffer aconsiderableadvantagein interactionswithbacteria.

Whentreatinginfections,theuseofasingleantimicrobialagent is normally sufficient to achieve a desired therapeutic effect.

However,thereareseveralinstancesinwhichtwoormoredrugs mustbegiventogether.Thesesituationsincludethetreatmentof serious infections before the identity of the microorganism is known,adesiredsynergisticeffectagainstcertainorganismsand thepreventionoftheemergenceofresistantstrains(Garrod,1953;

Levinson,2014). Indeed, combined antibiotic therapy hasbeen used against multiresistant bacteria (Song et al., 2009; Saballs etal.,2006;Yietal.,2005;Monteroetal.,2004).Owingtotheir actionmechanism,essentialoilsofferapromisingalternativetobe usedincombinationwithtraditionalantibiotics.

We have selected doxycycline (Fig. 1) tetracycline class antibioticwithbacteriostaticactivityagainstavarietyofGram- positiveandGram-negativebacteria,mycoplasma,chlamydiaeand rickettsiae. Doxycycline is used in the treatment of bacterial infections such as acne, urinary tract infections and protozoal infections suchasmalaria. It is theantibiotic of choice forthe treatmentofinfectionscausedbyChlamydiapsittaci,Vibriocholerae (cholera), Vibrio vulnificus, Mycobacterium marinum and

Mycoplasmepneumoniae (Bryskier, 2005).Doxycycline is useful in the treatment of respiratory tract infections because of its activityagainstintracellularatypicalmicroorganismsknowntobe resistanttoantibiotics(Bruntonetal.,2006),suchas

b

^-lactams.

Doxycyclinehasanexcellentoralabsorptionandasatisfyinghalf- life(Naidongetal.,1990).Itsbacteriostaticactivityand itsrapid resistancewhenusedaloneexplainwhydoxycyclineisfrequently given with other antibiotics like neomycine (Bryskier, 2005), ofloxacin,amoxicillineorantibacterialcompoundslikelysosomo- tropic agents (drugs capable of penetratinglysosomes) (Raoult etal.,1990).Moreover,incertaindiseasessuchasendocarditis,itis oftenfavourabletouseabactericidaldrug(Finbergetal.,2004), since doxycycline cannot be used in the treatment of such infections.Otherstudiessuggestthatacombinationofbactericidal andbacteriostaticagentsmayleadtoimprovedclinicaloutcomes, comparedwitheitheragentsalone(Klastersky, 1986;ChowandYu, 1999;Finbergetal.,2004).

Considering that no doxycycline/EOs combination has been reportedtodate,theaimofthis studywastoexaminein vitro interactions between essential oils encapsulated within lipid nanocapsulesanddoxycyclineusingacheckerboardmethodanda time-killassay.

2.Materialsandmethods 2.1.Materials

Labrafac¹ WL1349 (caprylic/capric acid triglycerides; IVth EuropeanPharmacopeia,2002)waskindlyprovidedbyGattefossé S.A.(France).Lipoid¹S75-3(hydrogenatedlecithin)andSolutol¹ HS15(macrogol15hydroxystearate,polyoxyl15hydroxystearate;

amixtureoffreepolyethyleneglycol660andpolyethyleneglycol 660hydroxystearate)(IVthEuropeanPharmacopeia,2002)were kindlyprovidedbyLipoidGmbh(Germany)andBASF(Germany), respectively.Cinnamaldehydeandeugenolwerepurchasedfrom Merck (Germany) and carvacrol from Sigma–Aldrich (UK).

Doxycyclinehyclate(Vibraveneuse¹)(100mg)powderfor solu- tionforintravenousinfusionwasfurnishedbySerbLaboratoires.

Allotherchemicalsandsolventswereofanalyticalgrade.Brain– heart infusion (BHI) broth was purchased from bioMérieux (France). Columbia agar supplemented with sheep blood (5%) wasobtainedfromOxoid(France).

2.2.PreparationofLNCs

LNCs were prepared following the procedure described by Montaguetal.(2014)withthecompositionpresentedinTable1.

The components of the LNCs (polyoxyl 15 hydroxystearate, hydrogenatedlecithin,andtriglycerides)andNaClwereweighed, mixedwithwaterandheatedto90C.Thesampleswerecooledto 60C.Thesamplesweretreatedwiththreeheating–coolingcycles, andduringthelastcoolingcycle,at80–90C(thetemperatureof the phase inversion) the systemwas dilutedwith cold (4C) Fig.1.Structuralformulasofdoxycyclinehyclate(1),carvacrol(2),cinnamaldehyde(3)andeugenol(3).

water.Inthecaseofessentialoils-loadedLNCseugenolwasadded at82Cduringthethirdcoolingcycle,carvacrolwasaddedat76C duringthefourthcoolingcycle,andcinnamaldehydewasaddedat 75Cduringthefifthcoolingcycle(Fig.2),followedbycoolingto 40C and heating to90C. Having reached the temperatureof 55C,thesystemwasdilutedwithcoldwater.

2.3.CharacterisationofLNCs

Theintensity-averagedparticlediameterandthepolydispersity indexesoftheLNCsweredeterminedbyDynamicLightScattering (DLS) with 173 backscatter detection. The electrophoretic mobility values measuredby Laser Doppler Velocimetry (LDV) wereconvertedtozetapotentialbytheSmoluchowskiequation.

BothDLSandLDVmeasurementswerecarriedoutonaZetasizer nanoseriesNano-ZSfittedwithahelium-neonlaseroperatingat 633nm (Malvern Instruments, UK). The measurements were performedafter60-folddilutionoftheprimaryLNCsdispersion with Milli-Q water. Each analysis was carried out at 25C in triplicate.

2.4.Bacterialstrains

Antibacterialactivitywastestedagainsttworeferencestrains:

Pseudomonasaeruginosa(ATCC27853)andEscherichiacoli(ATCC 25922) and three clinical isolates: Klebsiella pneumoniae, A.

baumannii RCH and A. baumannii SAN008 obtained from the UniversityHospitalofAngers(France).

2.5.Preparationoftheinoculum

The test strainsweregrown overnight onColumbia agarat 37C. 1–2coloniestakenfromtheplatesweresuspendedin2mLof 0.85% NaCl. The density of themicroorganism suspension was

adjustedtoequalthatofthe0.5McFarlandstandard(1.510⁸ CFUmL¹). The bacterial suspension was subsequently diluted 10timesinBHI.

2.6.Minimuminhibitoryconcentration(MIC)andminimum bactericidalconcentration(MBC)

MIC and MBC were determined via a broth microdilution method.TheformulationsweredilutedwithBHItoafinalvolume of2mLina poly-propylenetube.100

m

^{LofBHIwasaddedinto}

eachwellofasterile96-wellplate.Serialtwo-folddilutionsofthe sampleswereperformedinBHItoobtainthedesiredconcentra- tionrange.100

m

^{LofabacterialsuspensioninBHIwasaddedinto}

eachwell.Positivecontrolwells(containingBHIandthebacterial suspension)andnegativecontrolwells(containingtheBHIandthe tested sample without the bacterial suspension) were also prepared. The plates wereincubated at 37C for 24h without shaking.Anamountofapproximately10

m

^{Lwaswithdrawnfrom}

eachwell,transferredontoaplatecontainingMuellerHintonagar using anAQS multipoint inoculatorand incubatedovernight at 37C.Theminimuminhibitoryconcentration(MIC)wasdefinedas thelowestconcentrationofdrugthatinhibitedthevisiblegrowth of the bacteria (Fadli et al., 2012). The minimum bactericidal concentration(MBC)wasthelowestconcentrationofanantibac- terial agent that reduced the viability of the initial bacterial inoculumby99.9%(Taylor,1998;Andrews,2001;Petrusetal., 2011).

2.7.Checkerboardtitration

CombinationofEOsandantibioticweretestedviaachecker- boardtitrationmethod.Two-folddilutionsofEOsloadedLNCsand doxycyclinewerepreparedbeforemixing.Theconcentrationrange of each antimicrobial agent in combination ranged from 1/32MIC to 4MIC. The fractional inhibitory concentration (FIC)indexwascalculatedusingthefollowingequation:

FICINDEX¼MICA=B

MICA þMICB=A

MICB

whereMICAistheMICofthecompoundAalone,MICA/BistheMIC ofcompoundAincombination,MICBandMICB/AaretheMICofthe compoundBaloneandincombination,respectively.

The fractional bactericidal concentration (FBC) index was calculatedusingthefollowingequation:

FBCINDEX¼MBC_A=B

MBCA þMBC_B=A MBCB

whereMBCAistheMBCofthecompoundAalone,MBCA/Bisthe MBCofcompound Aincombination, MBCBandMBCB/Aarethe MBCofthecompoundBaloneandincombination,respectively.

SynergywasdefinedasanFICorFBCindexof0.5,additivity/

indifferenceasanFICorFBCindexof>5butof4.Antagonismwas definedasanFICorFBCindexof>4(Whiteetal.,1996).

2.8.Time-killstudies

Thetime-killstudieswereperformedwithafinalinoculumof approximately 1.510⁵CFU/mL in a final volume of 2mL.

Doxycycline was used at a concentration of 4

m

^{g/mL, whereas}

theconcentrationofEOs-loadedLNCsrangedfrom250

m

^g/mLto

937

m

^{g/mLdependingonthestrain,basedontheMIC/MBCresults}

and preliminary experiments which helped to establish the concentrationsatwhicheitheringredient,whenusedalone,did notshowbactericidalaction.Thesamples,containingthetested formulations/compounds and the control (the bacterial Fig.2.SchematicrepresentationoftemperaturecyclesintheformulationofEOs-

loadedLNCsandblankLNCs.

Table1

Composition of EOs–LNCs and blank LNCs formulations prepared via phase inversionmethod.

EOs-loadedLNCs BlankLNCs

Solutol 846mg 846mg

NaCl 89.6mg 89.6mg

Labrafac 1028mg 1028mg

Lipoid 75.2mg 75.2mg

Milli-Qwater 2.98g 2.98g

Milli-Qwater(quenching) 12.5mL 12.5mL

Eugenol 173m^L –

Carvacrol 173m^L –

Cinnamaldehyde 173m^{L –}

suspensioninBHIwithouttestedformulations/compounds)were incubatedat37C. Ateachsamplingtime (0,3,6 and24h)an amountof100

m

^{Lwaswithdrawnfromeachtube,andserial100-}

folddilutionswerepreparedindistilledwaterwhennecessary.A 100

m

^{L aliquot of the diluted and/or undiluted sample was}

deliveredontothesurfaceoftheagarandallowedtobeabsorbed intotheagar.Havingincubatedtheagarplatesfor24hat37C,the colonieswerecounted.

Theactivitywasconsideredasbactericidalwhentheoriginal inoculumwasreducedby3log10CFU/mL(99.9%),andbacterio- staticactivitywasdefinedasareductionintheoriginalinoculum by<3log10CFU/mL(Tenoveretal.,2004).Synergywasdefinedas a>2log10decreaseinthenumberofCFUpermLcomparedwiththe mostactivesingleagent(Singhetal.,2002).

2.9.Scanningelectronmicroscopy(SEM)

The experiments were performed by the SCIAM (Service Commund’Imageriesetd’AnalysesMicroscopiques)usingaJeol JSM-6301Fscanningmicroscope.

The samples were incubated at 37C for 10h at the same concentrationofmicroorganismsasinthetime-killstudies.Then, thesamples were centrifugedat 3000g for 10min (2) and washed with PBS. Then, the bacteria were fixed with 2%

glutaraldehydeand2%paraformaldehydeovernightundervacu- um,andwashedwithphosphatebuffer(pH7.4).Thepostfixation wasdoneusingwith1%osmiumtetroxide(OsO4).Thesamples weredehydratedwithgradedethanolseries(50%,70%,95%and 100%),desiccatedwithhexamethyldisilazane(HMDS)andcoated with platinum. Samples were viewed at 15,000, 20,000 or 30,000timesmagnificationat3.0kV.

2.10.Statisticalanalysis

Thestatisticalsignificanceofthedifferencesbetweensamples was determined using one-way analysis of variance (ANOVA).

Differenceswereconsideredsignificantatp<0.05.

3.Resultsanddiscussion 3.1.PropertiesoftheLNCs

Blank,60nmdiameterLNCsweresuccessfullyobtainedviathe phase inversionmethod.The slightly negativezetapotential of these nanocarriers can be attributed tolecithin. Lecithin is an ampholyticsurfactant containing phosphate and amino groups and has an isoelectric point of 4.15 (Petelska and Figaszewski, 2002).Thenegativevaluezetapotentialisconfirmationthatthe negativechargeofthephosphategroupspredominatedunderthe examinedconditions(pH6).Moreover,thepresenceofthedipoles ofthePEG-groupsinpolyoxyl15hydroxystearatemoleculesmay contribute to the negative charge (Vonarbourg et al., 2005).

Interestingly, after incorporation of the EOs the particle size increasedsignificantlyto114nm.Furthermore,EOs–LNCsexhib- itedamorenegativezetapotentialthanblankLNCs.LNCshave beenusedascarriersforavarietyoflipophiliccompounds.The influence of theencapsulatedactiveingredient dependsonthe ingredientandonthedrugloading—higherdrugloadingaffectsthe properties of the LNCs, whereas low drug loading does not influencethecharacteristicsoftheLNCs(Umerskaetal.,2015a).

MostoftheactiveingredientsencapsulatedwithintheLNCs to datewereinasolidstateandhadameltingpointmarkedlyhigher than37C.TheEOsloadingwasnoticeablyhighercomparedwith theotherdrugsencapsulatedwithinLNCs,reachingthevaluesof approximately20%,comparabletothatofpolyelectrolytecomplex nanoparticles(Umerskaet al., 2014a,b;Umerska et al., 2015b),

thereforetheirsignificantinfluenceonthepropertiesoftheLNCsis notsurprising.Ithasbeenshownthattheparticlediameterofthe LNCs is strongly dependent on the proportions of the LNCs components,particularlyontheSolutol¹/oilmassratio(Heurtault etal.,2002;Huynhetal.,2009;Umerskaetal.,2015a).Theaddition ofEOssignificantlyincreasedthemassoftheoilphase,leadingto anincrease inparticlesize. Moreover,incorporationof theEOs couldhavedecreasedthedensityofSolutol1moleculesatthe particle surface, therefore increasing the contact of lecithin moleculeswiththeexternal phase andresultingin anincrease intheabsolutevalueofthezetapotential.

Theadditionofdoxycyclinedidnotaffectthepropertiesofthe EOs-loadedLNCs.Itisdifficulttoconcludewhetherdoxycycline wasadsorbedontheparticlesurface,astheconcentrationofthe former is markedly lower than the latter and might not be sufficienttosignificantlyaffectthepropertiesoftheLNCs.Sizeand zetapotentialresultsareshowninTable2.

3.2.AntibacterialactivityofEOs-loadedLNCs,doxycyclineandtheir combination

TheMICandMBCvaluesofEOs-loadedLNCsanddoxycycline aloneandincombinationareshowninTable3.

TheMICandMBCoftheEOs-loadedLNCswerethesame,either 625

m

^{g/mL(A.baumannii, RCHand SAN,E. coli)or 1250}

m

^g/mL

(K.pneumoniae)or2500

m

^g/mL(P.aeruginosa).Thesamevaluesof MIC and MBC might suggest a bactericidal action of the EOs.

Indeed,ithasbeenshownthatcarvacrolhadbactericidalactivity;

althoughthebacteriacangrowatlowconcentrations(Ulteeetal., 1999;GillandHolley,2004)demonstratedthebactericidalaction ofcinnamaldehydeagainstListeriamonocytogenesandofeugenol againstL.monocytogenesandLactobacillussakei.Ontheotherhand, Montaguetal.(2014)haveshownthatat2MICEOs-loadedLNCs werebacteriostatic.

P. aeruginosa was less sensitive toEOs-loaded LNCs than A.

baumanniiorE.coli,astheMIC(MBC)differedbymorethanone serial dilution. P. aeruginosa is known to have low antibiotic susceptibilityandlowpermeabilityofthecellenvelope.Interest- ingly,thusfaronlystrainsofthegenusPseudomonashavebeen showntobeabletoovercomethetoxicityofhighconcentrationsof cyclichydrocarbons(Sikkemaetal.,1995).

TheMBCofdoxycyclinedependedonthestrain—A.baumannii wassignificantlymoresensitivetodoxycyclinethanP.aeruginosa (MBC of 320

m

^{g/mL and 1280}

m

^g/mL, respectively). High MBC valuesofdoxycyclinearenotsurprising,asthisantibioticisknown to bebacteriostatic. Indeed, the MIC values of doxycycline are 40–320timeslowerthantheMBC.TheFICindexvaluesofEOs- loadedLNCsanddoxycyclinecombinationwerebetween0.7and 1.3,dependingonthestrain,andthereforeintermsofinhibitionof bacterialgrowth theinteractionsbetweendoxycyclineandEOs- loadedLNCscanbeconsideredasadditiveorindifferent.However, when the bactericidal effect is taken into account, the LNCs– doxycyclinecombinationdemonstratedasynergisticeffectagainst alltestedstrains(FBCindexof0.5).

Blank LNCs did not display any bactericidal effect, and the combinationLNCs–doxycyclinedemonstratedthesameeffectas doxycycline alone (not shown). These results prove that the

Table2

PhysicalpropertiesofblankLNCs,EOs-loadedLNCsandEOs-loadedLNCswith doxycycline.*p<0.05,**p<0.01,and***p<0.001versusblankLNCs.

BlankLNCs EOs–LNC EOs–LNC+doxycycline

Size(nm) 561 996*** 1032***

PDI 0.0600.028 0.1200.024* 0.1250.021*

Zetapotential(mV) 10.71.89 17.72.12* 21.42.83**

Table3

Minimuminhibitoryconcentration(MIC)andminimumbactericidalconcentration(MBC)ofEOs-loadedLNCsanddoxycyclineusedaloneandincombination.Fractional inhibitoryconcentrationindex(FIC)andfractionalbactericidalconcentrationindex.MICandMBCvaluesaregiveninmg/mL.

MICEOs–LNCalone MICEOs–LNCincombination MICdoxycyclinealone MICdoxycyclineincombination FICindex

Klebsiellapneumoniae 1250 625 8 4 1

AcinetobacterbaumanniiRCH 625 312 4 1 0.8

AcinetobacterbaumanniiSAN 625 312 4 1 0.8

Pseudomonasaeruginosa 2500 1250 32 8 0.7

E.coliATCC25922 625 160 1 1 1.3

Klebsiellapneumoniae 1250 625 640 4 0.5

AcinetobacterbaumanniiRCH 625 312 320 1 0.5

AcinetobacterbaumanniiSAN 625 312 320 1 0.5

Pseudomonasaeruginosa 2500 1250 1280 4 0.5

E.coliATCC25922 625 312 640 4 0.5

Fig.3.TimekillcurvesofEOs-loadedLNCsanddoxycyclineusedaloneandincombinationagainst(a)A.baumanniiRCH(EOs-loadedLNCsat295m^g/mL,doxycyclineat4m^g/

mL),(b)A.baumanniiSAN(EOs-loadedLNCsat250m^g/mL,doxycyclineat4m^{g/mL),(c)Klebsiellapneumoniae}(EOs-loadedLNCsat625m^g/mL,doxycyclineat4m^g/mL),(d)E.

coliATCC25922(EOs-loadedLNCsat465mg/mL,doxycyclineat4mg/mL)and(e)Pseudomonasaeruginosa(EOs-loadedLNCsat937mg/mL,doxycyclineat4mg/mL).

Diamonds—control,squares—EOs-loadedLNCs,circles—doxycycline,triangles—EOs-loadedLNCsanddoxycyclinecombination.

synergistic bactericidal effect between EOs-loaded LNCs and doxycyclineareduetothepresenceoftheEOsintheformulations, andnotduetothenanocapsules.

Time-killcurvesforK.pneumoniae,A.baumanniiSANandRCH, E.coli and P. aeruginosa are depicted in Fig. 3. Doxycycline at 4

m

^{g/mLexhibiteda}bacteriostaticeffectagainstA.baumanniiRCH.

EOs-loadedLNCsshowedbacteriostaticeffectafter6h,andafter 24h an increase in the number of colony forming units was observed compared with the initial inoculum. However, when usedincombination, EOs-loadedLNCsand doxycyclineshowed synergistic bactericidal action. A 6 log differencebetween the combinationandtheLNCswasfound.Thebactericidaleffectofthe combinationwasobservedafter24h;after6hthecombination exhibited a bacteriostatic effect similar to that of each of the components alone. In another A. baumannii strain, a 7 log differencebetweenthecombinationandeachoftheingredients usedalonewasfound(Fig.3b).SimilarlytoA.baumanniiRCH,a bacteriostatic effect was observed in the case of each of the ingredientsaswellastheircombinationagainstA.baumanniiSAN.

Tobetterunderstandthekineticsofanyantibacterialeffectandto determineexactlywhenthebactericidaleffectofthecombination occurred,thecoloniesofA.baumanniiSANwerecountedevery3h.

Thebactericidaleffectoccurredafter21hofincubation.Moreover,

the difference between the combination and each ingredient becamesignificant(4log10CFU/mL)after21h.Fig.3cshowsthat, whenusedalone,EOs-loadedLNCshadabacteriostaticeffectand doxycyclineat4

m

^{g/mLdidnotexhibitan}antibacterialeffectafter 24h,neverthelessthecombinationofdoxycyclineandEOs-loaded LNCs showed a synergistic and bactericidal effect against K.pneumoniae.A5logdifferencebetweenthecombinationand theLNCswasfound.Thiseffectwasobservedafter24h;between 0 and 6h EOs-loaded LNCs usedalone or in combination with doxycyclinehadsimilarinfluenceonthenumberofcolonyforming units.Fig.3dshowsthatEOs-loadedLNCsaloneat465

m

^g/mLhad

a bacteriostaticeffect(a2logreduction inthenumber ofCFUs compared with starting inoculum) against E. coli, whereas doxycycline at 4

m

^{g/mL showed an increased number of CFU}

compared with the starting inoculum. The combination of doxycycline and EOs-loaded LNC showed a synergistic and bactericidaleffect.A2–3logdifferencebetweenthecombination and the LNCs was found. In contrast to A. baumannii or K.

pneumoniastrains,aconsiderablereductioninthenumberofCFUs wasobservedafter3hfortheEOs-loadedLNCsanddoxycycline combination.After6hfurtherreductionwasobserved,whichwas higherthan3log,thereforethecombinationcanbeconsideredas bactericidalafter6h.Fig.4eshowsthatEOs-loadedLNCsaloneat

Fig.4.SEMimagesofA.baumanniiSANafter10hofincubation.(a)Control-untreatedA.baumanniiSAN,(b)aftertreatmentwithdoxycycline(4mg/mL),(c)aftertreatment withEOs-loadedLNCs(250mg/mL),(d)aftertreatmentwithEOs-loadedLNCs(250mg/mL)+doxycycline(4mg/mL).

937

m

^g/mLhadabactericidaleffectagainstP.aeruginosaafter3 and6h, butafter24hthenumberofCFUs markedlyincreased.

Doxycyclineshowedabacteriostaticeffectafter3h,andtheCFUs number was increased by 2 log after 24hcompared with the startinginoculum.Thecombinationshowedabactericidaleffect after3hsimilartothatof theEOs-loadedLNCsalone, however furtherreductioninthenumberofCFUwasobservedafter6h,and thebactericidaleffectwasstillmaintainedafter24h.

3.3.Scanningelectronmicroscopy

The influence of doxycycline, EOs-loaded LNCs and their combination onA.baumannii SAN is shown ifFig. 4. A similar effect was observed in Klebsiella pneumonia (not shown). The untreatedA.baumanniicellsdisplayedasmoothandintactsurface (Fig.4a).Aftertreatmentwithdoxycyclinebacterialdebriscanbe observed next to the bacteria, the surface became corrugated (Fig. 4b). Similarly, after treatment with EOs-loaded LNCs the bacteriabecame roughand holes in thebacterialcell envelope couldbeobserved(Fig.4c).Likewise,holesinthebacterialcelland large quantities of debris were also observed in A. baumannii treatedwiththecombinationofEOs-loadedLNCsanddoxycycline (Fig.4d).

3.4.Discussion

The outer membrane of Gram-negative bacteria provides a hydrophilic surface due to the presence of lipopolysaccharide (Nikaido,1996),whichservesasapenetrationbarrierformacro- moleculesand hydrophobiccompounds, makingGram-negative bacteria relatively resistant to hydrophobic antibiotics. trans- Cinnamaldehyde and carvacrol gain access to the periplasmic spaceandtothedeeperpartsofthecellofGram-negativebacteria throughtheporinproteinsoftheoutermembrane(Helanderetal., 1998; Lambert et al., 2001). Tetracyclines cross the outer

membrane ofGram-negativebacteriathroughOmpFandOmpC porinchannelsascoordinationcomplexeswithpositivelycharged cations(probably magnesium).Themetalion-tetracycline com- plex probably dissociates in the periplasmic space releasing uncharged tetracycline, a weakly lipophilic molecule able to diffusethroughthelipidbilayerregionsoftheinnermembrane.

Theuptakeoftetracyclinesacrossthecytoplasmicmembraneis energydependentanddrivenbythepHgradientcomponentofthe protonmotiveforce(ChopraandRoberts,2001).

The EOs exerted their antibacterial effect due to their lip- ophilicity,which allowsthem topartition intothelipidsofthe bacterial membranes. Their accumulation within membranes disturbs the bacterial structures and leads to subsequent membrane associated events such as energy depletion and an increasedpermeability resulting inthe leakageof cell contents (Knoblochetal.,1986;Sikkemaetal.,1994,1995;Helanderetal., 1998).Theleakageofthecellularmaterialcanbeobservedinthe scanningelectronmicrographs (Fig.4).Carvacrolwas shownto disintegrate the outer membrane of Gram-negative bacteria, releasinglipopolysaccharides(LPS)andsensitizingittodetergents.

It inhibited bacterial growth at concentration similar to those requiredfortheoutermembranedisintergrationandincreasedthe permeabilityofthecellmembranetoATP(Helanderetal.,1998).

Carvacrolwasassumedtodissolveinthephospholipidbilayerand to align between the fatty acid chains causing membrane destabilisation(Ulteeetal.,2000).Ithasbeenshownthatinthe presenceofcarvacrolthemembranefluidityincreased,whichin turnwouldincrease thepassivepermeability.It was concluded that carvacrol caused the formation of channels through the membrane by pushing apart the fatty acid chains of the phospholipids, allowing ions to leave the cytoplasm (Ultee, 2000). Cinnamaldehyde inhibited the growth of E. coli and S.typhimuriumatsimilarconcentrationstocarvacrol,butopposite tocarvacrolitdidnotdisintegratetheoutermembraneordeplete theintracellularATPpool(Helanderetal., 1998).Itmightberelated

Fig.5.Mechanismofactionofcarvacrol,eugenol,trans-cinnamaldehydeanddoxycycline.

(a)CarvacroldisintegratesthecytoplasmicmembraneofGram-negativebacteria,releasinglipopolysaccharides andincreasingthepermeabilityofthecytoplasmic membrane(Lambertetal.,2001;Burt,2004).EOscausedamagetomembraneproteins.(b)Eugenolcausescellwalldeteriorationandcelllysis(Thoroskietal.,1989).(c)EOs causedepletionofprotonmotiveforce.(d)Doxycyclinebindstothe30sribosomalsubunitsinhibitingproteinsynthesis.Proteinsynthesisinvolvesthefollowingsteps:(1) fixingtheaminoacyltRNAtotheAsiteintheformofacomplexwithGTPandEF-Tu(elongationfactorthermounstable).(2)Formationofpeptidebond.(3)Translocationin thepresenceofEF-TuandEF-G(elongationfactorG).TetracyclinsinhibittheattachmentofaminoacyltRNAtothesite(1)ofribosome(Bryskier,2005).

tothephenoliccharacterofcarvacrol,asphenolsareknownfor their membrane-disturbing properties (Sikkema et al., 1995;

Helander et al., 1998). Eugenol caused cell wall deterioration and cell lysis (Thoroski et al., 1989). Both, eugenol and cinnamaldehydearethoughttobindtoproteins,preventingthe actionofaminoaciddecarboxylasesinE.aerogenes(Wendakoon andSakaguchi,1995).HoweverGram-negativebacteriaareknown toadapttolipophiliccompounds.Theseadaptationsinvolvethe alterationof thecellenvelopecomposition, andtheremovalof toxic compounds via active transport systems (Sikkema et al., 1995).The increase inthe numberof CFUs after initialgrowth inhibition(Fig.3)maybetheresultoftheadaptationofbacteriato EOs.Indeed,Ulteeetal. (2000)showedthatB.cereusadaptsto carvacrolinthepresenceofnon-lethalcarvacrolconcentrationsin the growth medium by lowering membrane fluidity and by changingthefattyacidandhead-groupcomposition.

Similarly to EOs–LNCs, at tested concentrations doxycycline exertedabacteriostaticeffect(Fig.3).Doxycyclinebindstothe30S subunits of bacterial ribosomes. This prevents the binding of aminoacyltRNAstotheacceptor(A) siteofribosomeinhibiting translation(Fig.5)(Chopraetal.,1992;ChopraandRoberts,2001;

KogawaandNunesSalgado,2012).Thebindingofdoxycyclinewith ribosomeisreversible,providinganexplanationofthebacterio- staticactionofthisantibiotic(Chopraetal.,1992).However,the selectiveactionoftetracyclinesonbacteriaisnotatthelevelof ribosomes,becauseinvitrotetracyclinesinhibitproteinsynthesis equallywellinpurifiedribosomesforbothbacterialandhuman cells (Levinson, 2014). Its selectivity is based on its greatly increaseduptake intosusceptiblebacterialcells compared with humancells(Levinson,2014).

Inthecaseofcombination,theeffectofEOswasstrengthened bytheaction of doxycycline.It ispossible thatdoxycycline,by inhibitingproteinsynthesis,impededtheadaptationofbacteriato lipophilicEOs.InthecaseofA.baumanniiandK.pneumoniaethe bactericidaleffectwasobservedafterarelativelylongtime(21h).

Althoughtheantibacterialagentsdidnotinitiallykillthebacteria, theyeffectivelyinhibitedtheirgrowthbycausingtheleakageof cellcontents.Eventhoughthebacteriamaytoleratetheleakageof acertainamountofcellcontentswithoutlossofviability,theexit ofcriticalmoleculesandionsorexcessivelossofcellcontentswill leadtodeath(DenyerandHugo,1991).Arapidbactericidaleffect was observed against P. aeruginosa, but in this case the concentrationoftheEOs-loadedLNCswasmarkedlyhigherthan inotherstrains.Anotherexplanationofthesynergisticeffectisthat the EOs increased the concentration of doxycycline inside the bacteriafacilitatingitsuptakeviathedisruptedcellenvelopesand/

ordecreasingitstransportoutofthebacterialcell.

4.Conclusions

EOsweresuccessfullyencapsulatedwithinlipidnanocaspules with noticeably high drug loading of 20%. Due to their small particlesize,lipidnanocapsulesmakeapromisingdrugdelivery systemfortheadministrationofessentialoils.

The MIC values of EOs-loaded LNCs were equal to MBCs, indicatingabactericidalactionoftheEOs.BlankLNCsdidnotshow anyantibacterialeffect.Incontrast,theMBCofdoxycyclinewas 40–320 times higher than the MIC, indicating a bacteriostatic modeofaction.Intermsoftheinhibitionofbacterialgrowththe EOs-loadedLNCsanddoxycyclinecombinationshowedadditiveor indifferenteffect,howeverwhenbactericidalactionwasconsid- eredasynergybetweenthesetwocomponentswasobserved.The synergistic bactericidal effect was confirmed by two methods:

checkerboard titration and time-kill assay. The holes in the bacterial envelope and the leakage of cellular contents were observedinSEmicrographs.

Acknowledgements

ThisprojectwasfundedbyEydoPharma.TheauthorAUwas fundedbytheEuropeanUnion’sSeventhFrameworkProgramme (FP7/2007-2013) under grant agreement No. 604182. http://ec.

europa.eu.research within the project FORMAMP—Innovative Nanoformulation of Antimicrobial Peptides to Treat Bacterial InfectiousDiseases.

TheauthorsthankMrRomainMalletandMs.FlorenceManero, members of SCIAM (Servicecommun d’imageries et d’analyses microscopiques),fortheirhelpandimplicationinthisproject.

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