Antifungal Activity of Commercial Essential Oils and Biocides against Candida Albicans

12 Pages·2017·1.03 MB·English

pathogens
Article
Antifungal Activity of Commercial Essential Oils
and Biocides against Candida Albicans
ElisaSerra1,LiliaAraidaHidalgo-Bastida1,JoannaVerran2 ID,DavidWilliams3
andSladjanaMalic1,*
1 SchoolofHealthcareScience,ManchesterMetropolitanUniversity,ManchesterM15GD,UK;
[email protected](E.S.);[email protected](L.A.H.-B.)
2 SchoolofResearch,EnterpriseandInnovation,ManchesterMetropolitanUniversity,
ManchesterM15GD,UK;[email protected]
3 SchoolofDentistry,CardiffUniversity,CardiffCF144XY,UK;[email protected]
* Correspondence:[email protected]
Received:12December2017;Accepted:23January2018;Published:25January2018
Abstract: Managementoforalcandidosis,mostfrequentlycausedbyCandidaalbicans,islimiteddue
totherelativelylownumberofantifungaldrugsandtheemergenceofantifungaltolerance. Inthis
study,theantifungalactivityofarangeofcommercialessentialoils,twoterpenes,chlorhexidineand
triclosanwasevaluatedagainstC.albicansinplanktonicandbiofilmform. Inaddition,cytotoxicityof
themostpromisingcompoundswasassessedusingmurinefibroblastsandexpressedashalfmaximal
inhibitoryconcentrations(IC50). Antifungalactivitywasdeterminedusingabrothmicrodilution
assay. Theminimuminhibitoryconcentration(MIC)wasestablishedagainstplanktoniccellscultured
inarangeofconcentrationsofthetestagents. Theminimalbiofilmeradicationconcentration(MBEC)
wasdeterminedbymeasuringre-growthofcellsafterpre-formedbiofilmwastreatedfor24hwiththe
testagents. Alltestedcommercialessentialoilsdemonstratedanticandidalactivity(MICsfrom0.06%
(v/v)to0.4%(v/v))againstplanktoniccultures,withanoticeableincreaseinresistanceexhibitedby
biofilms(MBECs>1.5%(v/v)). TheIC50softhecommercialessentialoilswerelowerthantheMICs,
whileaonehourapplicationofchlorhexidinewasnotcytotoxicatconcentrationslowerthantheMIC.
Inconclusion, thetestedcommercialessentialoilsexhibitpotentialastherapeuticagentsagainst
C.albicans,althoughhostcellcytotoxicityisaconsiderationwhendevelopingthesenewtreatments.
Keywords: Candidaalbicans;oralcandidosis;commercialessentialoils;biocides;antifungalactivity;
minimuminhibitoryconcentration;minimalbiofilmeradicationconcentration;cytotoxicity
1. Introduction
Candida are commensal fungal microorganisms that can colonise the oral cavity, where they
are mainly found on the posterior part of the tongue and the oral mucosa. Changes in the oral
environmentthatleadtoincreasedCandidagrowthcaninstigateoralcandidosis[1]. Therisingnumber
ofimmunocompromisedandimmunodeficientpatientshasresultedinanincreasedincidenceoffungal
infections. Tohighlightthis,Candida-relatedinfectionsaffect65%ofHIVpositiveindividualsandover
80%ofAIDSpatients[2–4].Thehigherlifeexpectancyofthegeneralpopulationhasalsoledtoarisein
denturewearing,withaconcomitantincreaseinCandida-associatedstomatitis[5–7].Eventhoughmore
than17Candidaspeciescancausehumaninfection,oralcandidosisaremainlycausedbyC.albicans[8].
Inthemouth,Candidatypicallygrowsasbiofilms,whicharethree-dimensionalstructuresattachedto
surfacesincludinghumantissueorabioticsubstrates(e.g.,adenture). Biofilmcellsareembeddedin
aself-producedextracellularpolymericmatrixandimportantlyoftenexhibitanelevatedtoleranceto
antimicrobialagentsandhostdefences[5].
Pathogens2018,7,15;doi:10.3390/pathogens7010015 www.mdpi.com/journal/pathogens
Pathogens2018,7,15 2of12
Currenttherapiesfororalcandidosisincludeuseoftopicalorsystemicantifungalagents,suchas
polyenes and azoles. Polyenes (e.g., nystatin and amphotericin B) are fungicidal through binding
to ergosterol in the fungal cell membrane and inducing cell membrane damage. Azoles, such as
fluconazoleandmiconazole,arefungistaticbyinhibitingtheenzymelanosteroldemethylase,involved
inergosterolbiosynthesis[9]. Importantly,therangeofavailableantifungalsarelimitedcomparedto
antibiotics[9]andcoupledwiththeriseofCandidaresistance,especiallywithinbiofilms,thishasledto
aninterestinthediscoveryofnewantifungalcompounds[10].
Essential oils are natural products produced by aromatic plants and are mainly composed
by terpenes and terpenoids [11]. Being lipophilic, these oils typically integrate into membrane
structurescausingincreasedcellpermeability,leachingofintracellularcomponentsandinactivation
ofenzymes[12,13]. EssentialoilscanactagainstCandidabyinhibitingergosterolsynthesis[14–18],
alteringcellwallmorphology[15,17–19],inhibitingenzymesinvolvedincellwallsynthesis[18,20],
changingcellmembranepermeability[21,22]andproducingoxygenreactivespecies[23].Furthermore,
essential oils can also interact with the mitochondrial membrane leading to cidal effects [11].
Antimicrobial,anti-aseptic,anti-inflammationandanti-oxidantactivityofessentialoils,aloneandin
combinationwithcommercialagentsiswellknown[13,24–26]. However,limitedknowledgeexists
regardingessentialoilactivityagainstbiofilmsandalsohostcellcytotoxicity.
Theaimofthisstudywasthereforetoinvestigatetheantifungalpotentialoftwelvecommercial
essentialoilsandtwoterpenes(E-cinnamaldehydeandlinalool)againstC.albicansplanktonicand
biofilmgrowth. Thecytotoxicityofthemostactivecommercialessentialoilswasestablishedagainst
mousefibroblasts. Antifungalactivityofcommercialessentialoilswascomparedtochlorhexidine
(CHX) and triclosan. These two biocides have previously shown antimicrobial properties against
awiderangeoforalpathogensandarefrequentcomponentsinmouthwashesandtoothpastes[27,28].
2. Results
2.1. MinimumInhibitoryConcentration(MIC)80andMinimalLethalConcentration
Theminimuminhibitoryconcentration(MIC)80ofthetestagentsagainstC.albicansNCYC1363
and C. albicans 135BM2/94 are shown in Table 1. The commercial essential oils that inhibited the
growthatthelowestconcentrationsweremelissaandgeraniol,whilemyrtleandsagehadthelowest
fungistaticpotential(p<0.001).
Fungicidal activity was also expressed as the lowest concentration of antimicrobial agent
that killed the microorganism (minimal lethal concentration) (Table 2). All tested compounds,
with exception of triclosan, had minimal lethal concentrations against C. albicans at tested
concentrations. However, these lethal concentrations were generally higher than the previously
establishedMICs.
Table 1. Minimum inhibitory concentration 80 of commercial essential oils and biocides against
C.albicansNYCY1363andC.albicans135BM2/94intheplanktonicform.
MinimumInhibitoryConcentration80[%(v/v)][(g/L)]
Antimicrobial
C.albicansNYCY1363 C.albicans135BM2/94
Basil 0.1(0.9) 0.1(0.9)
Bergamot 0.3(2.6) 0.3(2.6)
Cinnamon 0.1(1.0) 0.1(1.0)
Citronella 0.1(0.9) 0.1(0.9)
Geranium 0.07(0.6) 0.06(0.5)
Lavender 0.2(1.8) 0.1(0.9)
Melissa 0.06(0.5) 0.06(0.5)
Myrtle 0.4(3.5) 0.3(2.7)
Peppermint 0.1(0.9) 0.1(0.9)
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Table1.Cont.
MinimumInhibitoryConcentration80[%(v/v)][(g/L)]
Antimicrobial
C.albicansNYCY1363 C.albicans135BM2/94
Sage 0.4(3.7) 0.3(2.7)
Spearmint 0.2(1.6) 0.1(1.1)
Teatreeoil 0.2(1.8) 0.2(1.8)
E-cinnamaldehyde 0.03(0.3) 0.01(0.1)
Linalool 0.1(0.9) 0.1(0.9)
CHX 2×10−3(2.1×10−2) 5×10−3(5.3×10−2)
Triclosan 5.66×10−4(8.4×10−3) 5.89×10−4(8.8×10−3)
Minimalinhibitoryconcentration80(MIC80)definedasthelowestconcentrationoftheantimicrobialagentthatled
to80%reductioninabsorbancecomparedtocontrolswithoutagent.MICvaluesarein%(v/v)andinbracketsare
theequivalentMICvaluesin(g/L).
Table2. MinimallethalconcentrationofcommercialessentialoilsandbiocidesagainstC.albicans
NYCY1363andC.albicans135BM2/94intheplanktonicgrowthmode.
MinimalLethalConcentration[%(v/v)][(g/L)]
Antimicrobial
C.albicansNCYC1363 C.albicans135BM2/94
Basil 0.5(4.5) 0.5(4.5)
Bergamot 0.5(4.4) 0.5(4.4)
Cinnamon 0.1(1.0) 0.1(1.0)
Citronella 0.1(0.9) 0.1(2.7)
Geranium 0.1(0.9) 0.1(0.9)
Lavender 0.5(4.4) 0.3(2.6)
Melissa 0.1(0.9) 0.1(0.9)
Myrtle 1(8.8) 1(8.8)
Peppermint 0.3(2.7) 0.1(0.9)
Sage 1(9.2) 1(9.2)
Spearmint 1(9.2) 1(9.2)
Teatreeoil 0.5(4.5) 0.3(2.7)
E-cinnamaldehyde 0.03(0.3) 0.03(0.3)
Linalool 0.3(2.6) 0.3(2.6)
CHX 2.5×10−3(2.7×10−2) 5×10−3(5.3×10−2)
Triclosan NA NA
MinimallethalconcentrationwasdefinedasthelowestconcentrationoftheantimicrobialagentthatkilledC.albicans.
MLCvaluesarein%(v/v)andinbracketsaretheequivalentMLCvaluesin(g/L).NA=noantimicrobialactivity
attestedconcentrations.
2.2. MinimalBiofilmEradicationConcentration80
TheantifungalactivityofbiocidesandcommercialessentialoilsagainstC.albicansbiofilmswas
expressedastheminimalbiofilmeradicationconcentration(MBEC)[29]. Mosttestagentswerenot
activeagainstbiofilmsattestedconcentrationsanddidnotpreventregrowthafterremovalofthe
antimicrobial (Table 3). The antimicrobials that exhibited an MBEC against both tested C. albicans
strainsweremelissageranium,E-cinnamaldehydeandlinalool(Table3).
Table3.Minimalbiofilmeradicationconcentration80ofcommercialessentialoilsandbiocidesagainst
C.albicansNCYC1363andC.albicans135BM2/94.
MinimalBiofilmEradicationConcentration80[%(v/v)][(g/L)]
Antimicrobial
C.albicansNYCY1363 C.albicans135BM2/94
Basil NA NA
Bergamot NA NA
Cinnamon NA NA
Citronella NA NA
Geranium 2.5(22.3) 2(17.9)
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Table3.Cont.
Pathogens 2018, 7, 15 4 of 12
MinimalBiofilmEradicationConcentration80[%(v/v)][(g/L)]
Antimicrobial
Melissa 1.5 (13.3) 1.5 (13.3)
C.albicansNYCY1363 C.albicans135BM2/94
Myrtle NA NA
Lavender NA NA
Peppermint NA NA
Melissa 1.5(13.3) 1.5(13.3)
SagMe yrtle NAN A NNAA
SpeaPremppinertm int NAN A NNAA
Tea treeS oagile NAN A NNAA
Spearmint NA NA
E-cinnamaldehyde 0.8 (8.4) 0.8 (8.4)
Teatreeoil NA NA
Linalool 1 (8.7) 1.5 (13.1)
E-cinnamaldehyde 0.8(8.4) 0.8(8.4)
CHLXin alool 01.0(78 .7) 1.5(N13A.1 )
TriclosCaHn X >5 × 10−3 (70..4057 × 10−2) >5 × 10−N3 A(7.45 × 10−2)
Triclosan >5×10−3(7.45×10−2) >5×10−3(7.45×10−2)
Minimal biofilm eradication concentration 80 (MBEC80) defined as the lowest antimicrobial
cMoninceimntarlabtiioofinl mthearta dpirceavtieonntceodn caetn lteraatsito n808%0( MreBgEroCw80t)hd oefif nCeadnadsidtha,e alofwteers tthaen tbimioicfirlombi awlcaosn tcreenattreadtio wnitthha t
preventedatleast80%regrowthofCandida,afterthebiofilmwastreatedwithantimicrobialsfor24h.MBECvalues
antimicrobials for 24 h. MBEC values are in % (v/v) and in brackets are the equivalent MBEC values
are in % (v/v) and in brackets are the equivalent MBEC values in (g/L). NA = no antimicrobial activity at
inte s(gte/dL)c.o NncAen =tr antoio anns.timicrobial activity at tested concentrations.
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wwhhiicchh hhaallvveedd pprroolliiffeerraattioionne evveenna attt htheelo lwowesetscto cnocnecnetnrattriaotniotnes tteesdt.eIdn.d Ienedde,eadc, oan ccoenntcreantitornatoiofn0 .o00f 30%.00(v3/%v )
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as 2u4ff ihc ieexnptotosuhrael vaet f7i b×r o1b0l−a4%st p(vr/ovl)if weraasti osun.fficient to halve fibroblast proliferation.

(A) (B)
Figure1.Cont.
Pathogens2018,7,15 5of12
Pathogens 2018, 7, 15 5 of 12

(C) (D)

(E)
Figure 1. Cytotoxicity of selected antimicrobials against murine fibroblasts. Fibroblast numbers
Figure 1. Cytotoxicity of selected antimicrobials against murine fibroblasts. Fibroblastnumbers
(normalised by the control (0% (v/v) antimicrobial) after a 1 h (red square) and 24 h application (blue
(normalised by the control (0% (v/v) antimicrobial) after a 1 h (red square) and 24 h application
circle) of CHX (A); cinnamon (B); E-cinnamaldehyde (C); geranium (D) and melissa (E).
(bluecircle)ofCHX(A);cinnamon(B);E-cinnamaldehyde(C);geranium(D)andmelissa(E).
Table 4. Half maximal inhibitory concentration (IC50) against fibroblasts after 1 h and 24 h application
Table4.Halfmaximalinhibitoryconcentration(IC50)againstfibroblastsafter1hand24happlication
of the antimicrobial.
oftheantimicrobial.
Half Maximal Inhibitory Concentration [% (v/v)] [(g/L)]
Antimicrobial HalfMaximalInhibitoryConcentration[%(v/v)][(g/L)]
Antimicrobial 1 h 24 h
Cinnamon 0.03 (0.316)h 0.0214 (h0.11)
GeranCiuinmn amon 0.01 (00.0.038()0 .36) 00.0.011( 0(0.1.10)7)
MelisGsaer anium 0.03 0(.00.13)( 0.08) 0.00.103(0 (.007.3))
Melissa 0.03(0.3) 0.03(0.3)
E-cinnamaldehyde 0.003 (0.03) 0.002 (0.02)
E-cinnamaldehyde 0.003(0.03) 0.002(0.02)
CHX CHX 0.01 (00.0.115()0 .15) 77.3.232× ×1 10−0−44 ((00.0.00088))
Half maximal inhibitory concentration (IC50) defined as the antimicrobial concentration that inhibits
Halfmaximalinhibitoryconcentration(IC50)definedastheantimicrobialconcentrationthatinhibitsthe50%ofcell
tphreo l5if0e%rat ioofn cceolml pparroeldifteoracotinotnro lcsowmitphaoruetda gteon tc.oICnt5r0ovlsa luweistharoeuint a%g(evn/tv.) IaCn5d0i nvbarlaucekse tasraer einth e%e q(uv/ivva) leanntdIC i5n0
valuesin(g/L).
brackets are the equivalent IC50 values in (g/L).
33.. DDiissccuussssiioonn
EEsssseennttiiaall ooiillss aarree nnaattuurraall pprroodduuccttss oofftteenn eexxttrraacctteedd ffrroomm ppllaannttss aanndd tthheeyy ffrreeqquueennttllyy eexxhhiibbiitt
aannttiimmiiccrroobbiiaall,, aanntit-ia-asespeptitci,ca, natni-tiin-filnafmlammamtoartyorayn danandt ia-onxtiid-oaxnitdaacntitv iatcietisv.iTtihees.p Trihmea rpyriamimaroyf tahimis roesfe tahrcish
rwesaesartoche wvaalsu taote evthaeluaanteti ftuhne gaanltiafcutnivgiatyl aocftiv12ityc oomf 1m2 ecrocmialmeesrsceinatli easlsoeinlstiaalg oaiilnss atgCa.inalsbti cCan. as.lbAiclalntse. sAteldl
tested commercial essential oils demonstrated antifungal activity against planktonic C. albicans, with
MICs ranging from 0.06% (v/v) to 0.4% (v/v) and MLCs from 0.1% (v/v) to 1% (v/v). Comparison of
Pathogens2018,7,15 6of12
commercial essential oils demonstrated antifungal activity against planktonic C. albicans, with MICs
rangingfrom0.06%(v/v)to0.4%(v/v)andMLCsfrom0.1%(v/v)to1%(v/v).Comparisonofresults
with those of other studies is problematic given differences in assay techniques [30,31]. In addition,
thebotanicalsource,climateandenvironmentalconditions,timeofharvestingandextractionmethodcan
affectbothcompositionandantimicrobialactivityofcommercialessentialoils[31–33].
Theeffectofplantoriginonantimicrobialpropertiescanbeappreciatedbycomparingtheactivity
ofcinnamonoilextractedfromCinnamomumzeylanicumleavesandCinnamomumaromaticumleaves.
BothtypesofcinnamonoilsarefromtheevergreencinnamomumplantbutCinnamomumaromaticum
extract contains a higher amount of E-cinnamaldehyde, which could explain the higher
antifungal activity (MICs 0.0006% (v/v)–0.0096% (v/v)) [32] compared to the present study using
Cinnamomumzeylanicum(MIC0.1%(v/v))extract. TheimpactthattheamountofE-cinnamaldehyde
hasonantifungalpropertiesofanessentialoilwasalsoevidentinthisstudy(MICsof0.03%(v/v)
and0.01%(v/v)). Geraniumandmelissaoilsexhibitedhighestantifungalpotential. Bothcommercial
oilscontaingeraniolandcitronellol,whichareantifungal[34]andlikelyresponsibleforthesimilar
antifungal activity of these oils (p > 0.90). However, the MIC of melissa oil was lower than that
previouslyreported[35,36]. Thispresentstudyrevealedantifungaleffectsforbergamotoil(MICof
0.3% (v/v) and MLC of 0.5% (v/v)) which has previously only had limited attention. The MIC of
basiloil0.1%(v/v)(0.9g/L)waslowerthanpreviouslyreported,namely0.5%(v/v)[30]and0.312%
(v/v)[32]butcomparabletotheMIC(1250µg/mL)foundagainstafluconazoleresistantC.albicans
strain[15]. Themaincompoundofbasilandlavenderoilsislinalool,whichpreviouslyhashadMICs
rangingfrom0.06%(v/v)to0.12%(v/v)[37]. Comparingactivityofpurelinalooltothoseofbasil
andlavenderoils,theanticandidalactivityofterpenewasnotsignificantlyhigherthanthatofbasil
(p>0.99). TeatreeoilhadanMICof0.2%(v/v)andthiswassimilartothatrecordedbyHammeretal.
against C. albicans [38]. Sage oil exhibited MICs of 0.3% (v/v) (2.7 g/L) and 0.4% (v/v) (3.7 g/L),
whichwerecomparabletotheMICof2.78g/Lreportedusingadiskdiffusionmethod[39]butlower
thantheMICof1.32mg/mLmeasuredbybrothmicrodilutionassay[40]. Despitetheirdifferencesin
composition,peppermintandspearmintoilshadsimilarantifungalactivitieswithMICsof0.1%(v/v)
and0.1%(v/v)–0.2%(v/v),respectively(p>0.07). However,whiletheMICsofspearmintoilwere
similartothosereportedbyHammeretal.[30],theMICofpeppermintoilwashigherthanthatfound
byThoseetal.[41]. Myrtleoilhadthelowestantifungalpotential,eventhoughitsMICswerelower
thanthosepreviouslyreportedbyMahboubietal. (MICof0.8–1.6%(v/v))[42]. CHXandtriclosan,
two biocides whose antimicrobial properties are widely recognised and both commonly added to
mouthwashes and toothpastes, were also evaluated in this study. Triclosan exhibited fungistatic
activityonlyatconcentrationshigherthanthoseusedintoothpasteformulations(0.3%(w/v)[43])
butdidnotexhibitfungicidaleffectsattestedconcentrations.
Themajorityofagentshadlimitedantibiofilmactivity. Bacteriainbiofilmscanbebetween10and
1000timesmoretoleranttoantibioticsthantheirplanktoniccounterpartsandsimilarfindingshave
beenreportedforCandida[44]. Themechanismsbywhichbiofilmcellshaveelevatedantimicrobial
tolerance are complex and likely multifactorial. These include altered gene expression following
surface attachment, reduced growth rates in biofilms, variable nutrient availability that induces
changesinphenotypeandthepresenceofextracellularpolymericsubstancesthatimpedespenetration
ofagentsintothebiofilm[45]. Fewstudieshavepreviouslyreportedactivityofcommercialessential
oilsorbiocidesagainstC.albicansbiofilms[46,47]. Inthepresentstudy,frommelissaoil,geranium
oil, E-cinnamaldehydeandlinaloolallhadanti-biofilmactivity, whilstCHXonlyhadanti-biofilm
activityagainstC.albicansNCYC1363. A3minapplicationofcinnamon(1mg/mL)andcitronella
(1mg/mL)oilshasbeenfoundtoreducebiofilmcellnumbersimmediatelyaftertreatmentbutthis
effectwasnotevident48hposttreatment[46]. Theseresultsconcurwiththecurrentstudy,whereno
antibiofilmactivitywasnotedforcinnamonandcitronellaoilsafter24h. AnMBECofteatreeoilof
12.5%(v/v)hadpreviouslybeenreported[47],whichisahigherconcentration(8%(v/v))thantested
Pathogens2018,7,15 7of12
inthisstudy,asdifficultieswereencounteredinformingastablesuspensionoftheoil-mediumusing
1%(v/v)Tween80.
Fewstudieshaveinvestigatedthecytotoxiceffectsoftheseoils. CytotoxicityofCHX,cinnamon,
E-cinnamaldehyde,geraniumandmelissaoilshadadose-andtime-dependentcytotoxicity. Overall,
thecommercialessentialoilshalvedfibroblastproliferationatconcentrationslowerthantheirMICs.
The IC50 values for E-cinnamaldehyde, geranium and cinnamon oils were actually 10-fold lower
than their MIC 80, while melissa oil had an MIC 80 of 0.06% (v/v) and an IC50 of 0.03% (v/v).
Althoughadifferent assay and cell type was used, the melissa oil results (IC50 0.3 g/L) were in
accordancewiththoseofPauletal.[48]whodidnotseeasignificantchangeinleukocytesviability
after3htreatmentwith150µg/mLmelissaoil. SeveralstudieshaveusedE-cinnamaldehydetoinhibit
proliferationofcancercellsandreportedIC50srangingfrom45.8to129.4mM[49],higherthanthose
obtainedinthisstudywithfibroblasts(0.16–0.26mM).Barrosetal. foundthatatconcentrationslower
than those evaluated in this study (5 µg/mL), Cinnamomum zeylanicum oil had cytoxicity towards
erythrocytes[50].A1hexposureoffibroblaststoCHX(0.01%(v/v))halvedcellproliferationcompared
tocontrols. However,thisconcentrationwaslowerthantheMICs(2.5×10−3%(v/v)and5×10−3%
(v/v))foundinthecurrentstudy. ThisfindingwassimilartothecytotoxiceffectofCHXpreviously
reportedusingmacrophages[51]andhumanalveolarbonecells[52]. Eveniftheseresultsshowed
thatcommercialessentialoilswerecytotoxic,itshouldbetakenintoaccountthatcytotoxicitywas
conductedin2Dculture,whichisnotablydifferentfrominvivoconditions. Furtherinvestigationon
mammaliancellscouldbeperformedin3Dcultureorex/invivomodelstobettermimicthebiological
structureofthetissues.
4. MaterialsandMethods
4.1. EssentialOilsandBiocidesPreparation
Twelvecommercialessentialoils(EssentialOilsDirectLtd.,Oldham,UK)(Table5),twoterpenes
(E-cinnamaldehydeandlinalool(Sigma-Aldrich,Gillingham,UK)),chlorhexidinedigluconate(CHX)
(Sigma-Aldrich, Gillingham, UK) and triclosan (Irgasan from Sigma-Aldrich, Gillingham, UK)
wereevaluated.
Table5.Listofcommercialessentialoilstested.
PlantSpecies EssentialOil Origin
Ocimumbasilicum Basiloil Leaves
Citrusbergamia BergamotFCFoil Peel
Cinnamomumzeylanicum Cinnamonleafoil Leaves
Cymbopogonwinterianus Citronellaoil Aerialparts
Pelargoniumgraveolens Geraniumoil Floweringherb
Lavandulaangustifolia Lavenderoil Floweringherb
Melissaofficinalis Melissaoil Leavesandtops
Myrtuscommunis Myrtleoil Leaves
Menthapiperita Peppermintoil Wholeplant
Salviaofficinalis Sageoil Leaves
Menthaspicata Spearmintoil Aerialparts
Melaleucaalternifolia Teatreeoil Leavesandtwigs
Thecommercialessentialoilsweretestedatarangeofconcentrationsagainstplanktonicgrowth(2%
(v/v)to0.007%(v/v)andbiofilms(8%(v/v)to0.125%(v/v)). AllagentswerepreparedinSabouraud
DextroseBroth(SDB;OxoidLtd,Basingstoke,UK).Toenhancedispersionofessentialoilsinthemedium,
1%(v/v)Tween80(Sigma-Aldrich,Gillingham,UK)wasadded. Inthecaseofbiofilmstudies,0.015%
(w/v)AgarBacteriological(LP0011Oxoid)wasaddedtoSDB[53].CHXwasusedinSDBatconcentrations
between 0.04% (v/v) to 3.1 × 10−4% (v/v) and from 0.08% (v/v) to 6.2×10−4%(v/v) for planktonic
andbiofilmgrowthexperiments,respectively. A20%(w/v)stocksolutionoftriclosanwaspreparedin
Pathogens2018,7,15 8of12
DimethylSulfoxide(DMSO)(FisherChemical,Loughborough,UK).Serialdoublingdilutionsofthestock
solutionwerepreparedinSDByieldingfinalconcentrationsfrom5.2×10−6%(v/v)to6.7×10−4%(v/v)
andfrom1.7×10−4%(v/v)to5×10−3(v/v)forplanktonicandbiofilmexperiments,respectively.
4.2. Microorganisms
CandidaalbicansNYCY1363andC.albicans135BM2/94wereusedtoassessantifungalactivity
of commercial essential oils and biocides. Candida albicans 135BM2/94 is a clinical strain from the
SchoolofDentistry(CardiffUniversity),whichhasbeendescribedasahighinvaderoftissues[54].
StrainsweresubculturedontoSabouraudDextroseAgar(SDA)(CM0041Oxoid)andgrownat37◦C
inanaerobicincubatorfor24h. AcolonyofC.albicanswasinoculatedin20mLofSDBandincubated
aerobicallywithshaking(150rev/min)overnightat37◦C.TheovernightculturewaspreparedinSDB
toaturbidityequivalenttoa0.5McFarlandStandardandusedforfurtherexperiments.
4.3. MinimumInhibitoryConcentrationandMinimalLethalConcentration
Theminimuminhibitoryconcentration(MIC)andtheminimallethalconcentration(MLC)were
determinedusingabrothmicrodilutionassay. Themethodwasadaptedfromthatpreviouslyreported
by Malic et al. [29]. Briefly, 100 µL of antimicrobial and 100 µL of overnight culture diluted to
1×105CFU/mL were added to the wells of 96-well microtitre plates (Thermo Fisher Scientific,
HemelHempstead,UK).ControlsincludedCandidasuspensionculturedinSDB,withorwithout0.5%
(v/v)ofTween80. Inaddition,whentriclosanwastested,SDBcontaining1%(v/v)DMSOwasusedas
control.Theplateswerecoveredwiththelidssuppliedbythemanufacturerandsprayedwith3%(v/v)
ofTriton100-X(Sigma-Aldrich,Gillingham,UK)inpureethanoltoreducecondensation. Theplates
were incubated aerobically at 37 ◦C with shaking at 110 rpm, for 24 h. Growth was estimated by
measuringturbidityofeachwellbyspectrophotometricabsorbanceat620nm(ThermoScientific™
Multiskan™ GO Microplate Spectrophotometer), shaking 3 s before the reading. The absorbance
readingswerestandardisedagainstmicrobial-freecontrols. Theminimalinhibitoryconcentration
80(MIC80)wasdefinedasthelowestconcentrationoftheantimicrobialagentthatshowedatleast
80%reductioninabsorbancecomparedtothecontrol. TheMLCwasdeterminedbyplatingselected
well contents (where no visible growth was evident) on to SDA and incubating for 24 h at 37 ◦C.
The MLC was defined as the lowest concentration of antimicrobial agent that killed the Candida
as shown by no colony growth on SDA. All concentrations were tested in quadruplicate and on
threeseparateoccasions.
4.4. MinimalBiofilmEradicationConcentration80
Theminimalbiofilmeradicationconcentration(MBEC)methodwasadaptedfromMalicetal.
(2013) [29]. Briefly, a 96-well microtitre plate containing 200 µL of an overnight culture diluted
at 1×105CFU/mL was incubated for 48 h at 37 ◦C without agitation to allow biofilm formation.
ControlsincludedCandidasuspensionculturedinSDB,withorwithout1%(v/v)ofTween80and
0.015% (w/v) Agar Bacteriological. When triclosan was tested, SDB containing 8% (v/v) DMSO
wasalsousedascontrol. After48h, theSDBwasremovedandthemicrotitreplateinvertedonto
tissuepapertoremoveresidualmedium. Thebiofilmwaswashedthreetimeswith100µLofPBS.
OnehundredµLoftestagentwasaddedtothebiofilmandtheplateincubatedstaticallyfor24hat
37◦C.Afterincubation,testagentwasremovedandthebiofilmwashedtwicewith100µLofPBS.
TwohundredµL of SDB was added to each well and the biofilm disrupted by repeated pipetting.
Thethreereplicateswerethenpipettedintoamicrocentrifugetubewhichwasthencentrifugedfor
3minat3000rev/min(HettichUniversalMikro12–24,Hettich,Tuttlingen,Germany).Thesupernatant
containingresidualtestagentwasdiscardedandthemicroorganismsresuspendedinfreshSDBand
threewellsofa96-wellplatewereinoculatedwiththesuspension. Theturbidityofthesuspension
wasmeasuredbyspectrophotometerabsorbanceat620nmpriortoandafterincubationfor24hat
37◦Cwithshakingat110rev/min. Theminimalbiofilmeradicationconcentration80(MBEC80)was
Pathogens2018,7,15 9of12
definedasthelowestantimicrobialconcentrationthatpreventedatleast80%regrowthofCandida.
Allexperimentswereconductedonthreeseparateoccasions.
4.5. HalfMaximalInhibitoryConcentration
Mouse fibroblasts (NIH 3T3; Sigma-Aldrich, Gillingham, UK) were cultured in Dulbecco
ModifiedEagleMedium(DMEM,Sigma)supplementedwith10%(v/v)foetalbovineserum(FBS)
(Gibco, BRL), 1% (v/v) penicillin/streptomycin (Sigma-Aldrich, Gillingham, UK) and 1% (v/v)
L-glutamine (Sigma-Aldrich, Gillingham, UK). Serial doubling dilutions of commercial essential
oils and biocides were prepared in the fibroblast culture medium at final concentrations ranging
from 0.25% to 0.007% (v/v) for the commercial essential oils and from 0.04% to 3 × 10−4% (v/v)
forchlorhexidine. FibroblastswereharvestedusingtrypsinEDTA(EDTA0.25%(w/v),Trypsin0.53
mM,ThermoFisherScientific,HemelHempstead,UK)anddilutedtoadensityof5×105cells/mL.
One-hundredµlofthecellsuspensionwasusedtoinoculatea96-wellplate(5×104 cellsperwell)
whichwasthenincubatedat37◦Cand5%CO for1.5h. A100-µLvolumeoftheantimicrobialwas
2
thenadded. After1and24h,themediumwasremovedandthecellswashedtwicewith100µLofPBS.
ThreehundredµLofDMEMcontaining10%(v/v)ofalamarBlue(AlamarBlueCellViabilityReagent,
Invitrogen)wasaddedtoeachwellandtheplateincubatedfor1.5h. Fluorescencewasreadwith
aSynergyHTplatereader(BioTek®Instruments,Winooski,VT,USA)withexcitationandemission
wavelengthsof545nmand590nm,respectively. Thehalfmaximalinhibitoryconcentration(IC50)
wasdefinedastheantimicrobialconcentrationthatinhibited50%cellproliferationcomparedtothe
control(i.e.,DMEMwithoutantimicrobialagent). Eachconditionwasstudiedintriplicateandon
threeseparateoccasions.
4.6. StatisticalAnalysis
StatisticalanalysiswasperformedusingGraphPadPrismVersion7.0(GraphPadSoftware,Inc.,
LaJolla,CA,USA).Datawerepresentedasarithmeticmean±SD.Thedifferencebetweentreatments
wasstatisticallyanalysedusingone-wayanalysisofvariance(ANOVA)followedbyTukeymultiple
comparisonstest. Statisticallysignificantdifferencesweresetatp<0.05.
5. Conclusions
This study showed that all the twelve commercial essential oils, two terpenes and triclosan
and CHX had antifungal activity against planktonic C. albicans. Six of these compounds (CHX,
cinnamon, E-cinnamaldehyde, linalool, geraniumandmelissa)werealsoactiveagainstC.albicans
biofilms,whichareusuallychallengingtoeffectivelyinhibit. Cytotoxicityscreeningrevealedthatthe
commercialessentialoilshalvedfibroblastproliferationatconcentrationslowerthanthoserequired
toinhibitC.albicansgrowth. Furtherinvestigationontheeffectoftheseagentsagainstmammalian
cellsishoweverwarrantedbeforeanyinvivoapplication. Theantifungalpotentialoftheseessential
oilscouldbeafuturetherapeuticfortopicalcandidosisasanoptiontoovercomeemergingantifungal
drugresistance.
AuthorContributions: ElisaSerraperformedalltheexperiments,analysedthedataandwroteadraftofthe
paper;AraidaHidalgo-Bastidadesignedtheexperimentsrelatedtocellculture(cytotoxicitywork),reviewed
andrevisedthedraftmanuscript;SladjanaMalic,JoannaVerranandDavidWilliamsdraftedthework,designed
theexperimentsrelatedtothemicrobiologywork,oversawthelaboratorywork,reviewedandrevisedthedraft
manuscript;Allauthorsapprovethefinalmanuscriptforsubmission.
ConflictsofInterest:Theauthorsdeclarenoconflictsofinterest.
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