Origanum vulgare L. Essential Oil as a Potential Anti-Acne Topical Nanoemulsion—In Vitro and In ...

16 Pages·2017·1.77 MB·English

molecules
Article
Origanum vulgare L. Essential Oil as a Potential
Anti-Acne Topical Nanoemulsion—In Vitro and In
Vivo Study
MohammedH.Taleb1,2,NourtanF.Abdeltawab1,* ID,RehabN.Shamma3,
ShereinS.Abdelgayed4,SarahS.Mohamed5 ID,MohamedA.Farag6,7and
MohammedA.Ramadan1
1 DepartmentofMicrobiologyandImmunology,FacultyofPharmacy,CairoUniversity,Cairo11562,Egypt;
[email protected](M.H.T.);[email protected](M.A.R.)
2 DepartmentofPharmaceuticsandPharmaceuticalTechnology,FacultyofPharmacy,Al-Azhar
University–Gaza,POBox1277,Gaza79702,Palestine
3 DepartmentofPharmaceuticsandIndustrialpharmacy,FacultyofPharmacy,CairoUniversity,Cairo11562,
Egypt;[email protected]
4 DepartmentofPathology,FacultyofVeterinaryMedicine,CairoUniversity,Cairo12211,Egypt;
[email protected]
5 DepartmentofPharmacologyandToxicology,FacultyofPharmacy,CairoUniversity,Cairo11562,Egypt;
[email protected]
6 DepartmentofPharmacognosy,FacultyofPharmacy,CairoUniversity,Cairo11562,Egypt;
[email protected]
7 DepartmentofChemistry,SchoolofSciences&Engineering,TheAmericanUniversityinCairo,
Cairo11853,Egypt
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:18July2018;Accepted:20August2018;Published:28August2018
Abstract: Antibiotics are often prescribed in acne treatment; however, Propionibacterium acnes
andStaphylococcusepidermidis,thetwoofthemajoracne-associatedbacteria,developedantibiotic
resistance. Essentialoils(EOs)presentanatural,safe,efficaciousandmultifunctionalalternative
treatment. Thisstudyaimedtoassessthepotentialanti-acneactivityofselectedsevenEOscommonly
usedinMediterraneanfolkmedicine. Antimicrobialactivityscreeningoftheseoilsshowedoregano
to exhibit the strongest antimicrobial activity with minimum inhibitory concentration (MIC) of
0.34mg/mLandminimumbactericidalconcentration(MBC)of0.67mg/mLagainstP.acnes;andMIC
of0.67mg/mLandMBCof1.34mg/mLagainstS.epidermidis. Thecompositionofthemosteffective
EOs(oreganoandthyme)wasdeterminedusinggaschromatography-massspectrometry(GC-MS).
MonoterpenoidphenolspredominatedoreganoandthymeEOwiththymolpercentile99and72,
respectively. ThymolshowedMIC0.70mg/mLagainstbothP.acnesandS.epidermidiswhereasMBC
was1.40and2.80mg/mLagainstP.acnesandS.epidermidis,respectively.Moreover,oreganoexhibited
thestrongestanti-biofilmeffectagainstS.epidermidiswithMBIC1.34mg/mLandkillingdynamic
timeof12and8hagainstP.acnesandS.epidermidis,respectively. Oregano,themosteffectiveEO,
wasformulatedandtestedasananoemulsioninanacneanimalmousemodel. Theformulation
showedsuperiorhealingandantimicrobialeffectscomparedtothereferenceantibiotic. Collectively,
ourdatasuggestedthatoreganooilnanoemulsionisapotentialnaturalandeffectivealternativefor
treatingacneandovercomingtheemergingantibioticresistance.
Keywords:acnevulgaris;essentialoils;oregano;thymol;S.epidermidis;P.acnes;antimicrobialactivity;
nanoemulsion;antibioticresistance
Molecules2018,23,2164;doi:10.3390/molecules23092164 www.mdpi.com/journal/molecules
Molecules2018,23,2164 2of16
1. Introduction
Acne vulgaris is the 8th most prevalent disease and second top skin disease globally [1].
Adolescentsofbothgendersaretypicallyaffectedbyacne.Acneinadolescentscancausepsychological
disordersandinseverecasescanleadtodepression[2]. Acneisamultifactorialdiseasecharacterized
by pathological alteration in pilosebaceous units of the neck and upper trunk. It results in the
formation of non-inflammatory comedones and inflammatory lesions such as papules, pustules,
and nodules [3]. Two bacteria are associated with acne pathogenesis: Propionibacterium acnes and
Staphylococcus epidermidis [4]. These bacteria are part of normal human skin microbiota, but if
dysbiosis occurs, infection of pilosebaceous units can lead to acne. Accordingly, most anti-acne
drugsaredirectedagainstP.acnesandS.epidermidisinfectionandassociatedinflammatoryresponses.
Anti-acne therapy includes systemic and topical therapies. Topical therapies include comedolytic
agents,anti-inflammatoryagentsandantibiotics[5]. Bacterialresistanceaccompaniestopicalanti-acne
antibiotics. Resistancetotopicalanti-acneantibioticsisattributedtomultiplefactors,includinguseof
repeatedsingleantibiotic,sub-inhibitoryconcentrations,oruseoverextendedtime[6,7].
Toovercometheemergingresistancetoconventionalantibiotics,alternativenaturalantimicrobial
agentshavebeeninvestigated. Essentialoils(EOs)ofaromaticplantssuchasoregano,teatreeoil,
lemongrass, and thyme have antimicrobial activities that can be used as a natural alternative [8].
The antimicrobial activity of these EOs is attributed to their major constituents: Monoterpenoid
phenols. Inaddition,EOs’minorconstituents,suchasthemonoterpenehydrocarbons,γ-terpinene,
and p-cymene, may contribute to the antibacterial activity of these oils [9,10]. Some studies show
thatafewEOshaveanti-acneactivity; teatreeoiliscommerciallyused[11]. Therefore,thisstudy
testedtheanti-acnepotentialofsevenEOsusedinMediterraneanfolkmedicine: Oregano(Origanum
vulgare),thyme(Thymusvulgaris),lemongrass(Cymbopogoncitratus),teatree(Melaleucaalternifolia),
mentha (Mentha piperita), lavender (Lavendula anguestifolia), and chamomile (Matricaria recutita).
The Mediterranean region is one of the largest producers of these aromatic plants. Its favorable
climatic and cultivation conditions enhance the quality of EO constitutes [12]. We assessed the
antimicrobialactivityoftheabove-selectedEOsagainstacne-causingbacteriainvitro.
AnotheraimofthisstudywastodevelopapharmaceuticalformulationoftheEOwiththehighest
antimicrobial effect. Based on our invitro antibacterial results of tested EOs against acne-causing
bacteria, we formulated oregano EO as a nanoemulsion formula. Nanoemulsions offer enhanced
solubilizationcapacityforhydrophobic,poorlysolubledrugsastypicalinthecaseofEOs[13]. Inthis
study,weassessedthehealingandantimicrobialactivityofthedevelopednanoemulsionofthemost
effectiveEOinvivoinanacnemousemodelasapotentialnewformulationforacnetreatment.
2. Results
2.1. ScreeningofAntibacterialActivityofTestedEOsUsingAgarDiscDiffusionMethod
TheselectedsevenEOshavebeenscreenedfortheirantibacterialactivitiesagainstP.acnesand
S.epidermidisusingthediscdiffusiontest. Theresultswererepresentedasthediameterofinhibition
zone(Figure1andTableS1). P.acnesandS.epidermidisweresusceptibletooreganoandthymeEOs
with a zone of inhibition (ZOI) ranging from 16 to 32 mm larger than those of erythromycin and
clindamycinreferenceantibiotics(Figure1). WhilelemongrassandteatreeshowedmoderateZOI
from9–17mm,lavender,mentha,chamomileEOsfailedtoinhibitthegrowthofbothbacteria(Figure1
andTableS1). Thetwostudies’positivecontrols(erythromycinandclindamycin)showedZOIranging
from 10 to 20 mm. The negative control vehicle dimethylsulfoxide (DMSO) showed no inhibition
againstbothbacteria.
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FFiigguurree 11.. OOrreeggaannooE EOOe xehxihbiitbeidtetdh ethlaer glaersgteinsht iibnihtiiobnitzioonn ezaomneo nagmthonegte sttheed toeislste.dA notiilbsa. cAtenrtiiablaacctteivriiatyl
aocfttihveitysc orefe tnheed sEcOresenuesidn gEaOgsa rudsiinscg daigffaurs idoinscm deitfhfuosdioang aminestth(oad)S a.gepaiidnesrtm (iad)i sSw. eipthidEerOmsiadtisc ownicthen EtrOasti oant
coofn0c.e7n%tr;a(bti)oSn. oepf i0d.e7r%m;i d(ibs)w Si.t hepEidOersmaitdciso nwciethn tEraOtiso nato cfo1n.4ce%n;t(rca)tiPo.na confe s1.w4%ith; (Ec)O Ps. aatccnoens cwenitthr aEtiOons aotf
c0o.7n%ce;n(tdr)aPti.oanc noefs 0w.7i%th; E(dO) sPa. taccnoensc wenitthra EtiOons aotf c1o.4n%ce.nDtraattaioisn roefp 1re.4s%en. tDedataas ism reeapnrsesoefnitnehdi baist imoneaznosn oefs
i(nmhmib)it±ionS Dzo.nCeosn (tmromls)u ±s eSdDw. Ceorentcrloinlsd aumseydc iwnearne dcleinrydtahmroymciync ainndas epryotshitriovmeycocinnt raosl ,pwoshiitlieveD cMonSOtroals,
wnehgilaet iDveMcSoOnt aros ln.egative control.
22..22.. AAnnttiimmiiccrroobbiiaall AAccttiivviittyy ooff tthhee TTeesstteedd EEOOss UUssiinngg MMiinniimmaall IInnhhiibbiittoorryy CCoonncceennttrraattiioonn ((MMIICC)) MMeetthhoodd
Minimal inhibitory concentration (MIC) test results confirmed that the most potent EOs against
Minimalinhibitoryconcentration(MIC)testresultsconfirmedthatthemostpotentEOsagainst
PP.. aaccnneess aanndd SS.. eeppiiddeerrmmiiddiiss wweerree oorreeggaannoo,, tthhyymmee,, lleemmoonnggrraassss aanndd tteeaa ttrreeee ooiillss ((TTaabbllee 11)).. OOrreeggaannoo EEOO
ddiissppllaayyeedd lloowweesstt MMIICC vvaalluueess ooff 00..3344,, 00..6677 mmgg//mmLL aaggaaininsst tPP.. aaccnneess aanndd SS.. eeppidideerrmmididisis, ,rreessppeecctitviveelyly. .
SSeeccoonndd ttoo oorreeggaannoo wwaass ththyymmee EEOO, ,exehxhibiibtiitning gana nMMICIC ofo 0f.605.6, 51,.310.3 m0 gm/gm/Lm aLgaaignasitn Pst. aPc.naecsn aesnda nSd.
eSp.ideperimdeirdmisi,d irse,srpeescpteivcetilvye. lyC.oCnsoenqsueeqnuteianltliya,l lyo,reogreagnaon, ot,htyhmyem, el,emlemonognrgarsas,s sa,nadn dtetae atrtereee EEOOss wweerree
selected for determining minimum bactericidal concentration (MBC), demonstrating the most
selectedfordeterminingminimumbactericidalconcentration(MBC),demonstratingthemosteffective
einffheicbtiivtieo ninahgibaiintisotnP a.gacaninessta Pnd. aScn.eeps iadnerdm Sid. iespbidyerdmisidcids ibffyu dsiioscn dainffduMsioICn aanssda yMsI.C assays.
Molecules2018,23,2164 4of16
Table1.Minimalinhibitoryconcentration(MIC)oftheseventestedEOsagainstS.epidermidisandP.acnes.
MIC
EssentialOils
S.epidermidis(mg/mL) P.acnes(mg/mL)
Oregano 0.67 0.34
Thyme 1.30 0.65
Lemongrass 1.22 1.22
Teatree 1.27 1.28
Lavender 2.52 2.52
Mentha 5.28 2.60
Chamomile 6.22 3.18
2.3. TheBactericidalandAnti-BiofilmActivitiesoftheTestedEOs
The bactericidal activity of the four EOs with the top ZOIs and MICs were tested against
acne-causingbacteria. OreganoEOataconcentrationof0.67and1.34mg/mLinhibitedthegrowthof
P.acnesandS.epidermidis,respectively(Table2). TeatreeEOexhibitedtheleastbactericidalactivityat
2.558and5.116mg/mLagainstP.acnesandS.epidermidis,respectively. Next,thefour-selectedEOs
wereassayedforanti-biofilmactivityagainstS.epidermidis(Table3). OreganoEOinhibitedbiofilm
formationbyS.epidermidisatitsbactericidalconcentration(1.34mg/mL).Incontrast,theleasteffective
EOwasteatreeoilwasunabletoinhibitbiofilmformationofS.epidermidisatconcentrationupto
2.56mg/mLequivalentto0.28%v/v(Table3).
Table2.Minimalbactericidalconcentrations(MBC)oftheselectedEOsagainstS.epidermidisandP.acnes.
MBC
EssentialOils
S.epidermidis(mg/mL) P.acnes(mg/mL)
Oregano 1.34 0.672
Thyme 2.60 1.30
Lemongrass 2.44 2.44
Teatree 5.10 2.55
Table3.Minimumbiofilminhibitoryconcentration(MBIC)oftheselectedEOsagainstS.epidermidis.
MBIC
EssentialOils
mg/mL
Oregano 1.344
Thyme 2.60
Lemongrass 2.44
Teatree 2.55
2.4. KillingDynamicsofOreganoEOagainstS.epidermidisandP.acnes
Sinceourresultsofagardiscdiffusion,MIC,MBCandMBICassaysshowedthatoreganoEO
wassuperior,thedynamicsoforeganoEO’skillingactivitywasdetermined. MIC,2MICand4MICof
oreganoEOshowedsignificantreductioninthenumberofbothP.acnesandS.epidermidisafter4hof
application(Figure2). OreganoEOkillingcapacityagainstS.epidermidiswashigherthanP.acnesat8
and12h(Figure2).OreganoEOvolatileconstituentsweredeterminedusinggaschromatography-mass
spectrometry(GC-MS)analysistopinpointactiveagentsinoreganoEOlikelytomediatetheobserved
anti-acneeffects.
Molecules2018,23,2164 5of16
Molecules 2018, 23, x FOR PEER REVIEW 5 of 16

FFiigguurree 22.. OOrreeggaannoo EEOO iinnhhiibbiitteedd tthhee ggrroowwtthh ooff SS.. eeppiiddeerrmmiiddiiss ffaasstteerr tthhaann PP.. aaccnneess.. OOrreeggaannoo EEOO aatt
ccoonncceennttrraattiioonnss ooff 00..667722,, 11..3344,, 22..6688 mmgg//mmLL ((11,, 22,, 44 MMIICC)) aanndd nnoo EEOO ccoonnttrrooll wwaass uusseedd ffoorr aassssaayyiinngg tthhee
kkiilllliinngg rraateteo fobf abcatecrteiarliacle lclselalts 0a,t2 ,04, ,28, a4n,d 81 2anhda n1d2 ehx parnedss eedxparsetshseedsu arvs ivthineg sbuarcvtievriinag(l obga1c0teCrFiaU /(lmogL10)
aCtFdUif/fmerLen) taetx pdoifsfuerreentitm eexs.pWoseuures edtimsteasr.t inWgeb aucsteerdia lstsaurstpinegn siboanctceorniacle nsturasptieonns1io08n CcFoUn/cemnLtr.aOtiroeng a1n0o8
ECOFUk/imlliLn.g Oraretegaangoai EnsOt (kai)llSin.gep riadteer magidaiisnasnt d(a()b S). Pe.paidcnerems.idis and (b) P. acnes.
2.5. Chemical Composition of Oregano and Thyme EOs
2.5. ChemicalCompositionofOreganoandThymeEOs
Both oregano and thyme belong to the same family (Lamiaceae), producing aromatics as major
Bothoreganoandthymebelongtothesamefamily(Lamiaceae),producingaromaticsasmajor
essential oil constituents. Our GC‐MS analysis of oregano and thyme EO volatile constitutes showed
essentialoilconstituents. OurGC-MSanalysisoforeganoandthymeEOvolatileconstitutesshowed
that the major volatile compound was thymol (Table 4 and Figure S1). Thymol represented 99.44% of
thatthemajorvolatilecompoundwasthymol(Table4andFigureS1). Thymolrepresented99.44%of
oregano EO and 72% of thyme volatile compounds. Other volatiles identified were at much‐lower
oreganoEOand72%ofthymevolatilecompounds. Othervolatilesidentifiedwereatmuch-lower
lleevveellss iinn bbootthh ooiillss aanndd iinncclluuddee pp-‐ccyymmeennee,, cciinneeoollee aanndd γγ-‐tteerrppiinneennee ((TTaabbllee 44))..
Table 4. GC‐MS analysis of volatile compounds in oregano and thyme EOs.
Table4.GC-MSanalysisofvolatilecompoundsinoreganoandthymeEOs.
Compound Oregano EO Content % Thyme EO Content % Retention Time Retention Index
Compound OreganoEOContent% ThymeEOContent% RetentionTime RetentionIndex
Thymol a 99.44 72.08 12.81 1280
p‐cyTmheynmeo al a 09.92.4 4 7225.0.188 12.881.79 12810005
p-cymenea 0.2 25.18 8.79 1005
γ‐terpinene 0.03 2.52 9.31 1038
γ-terpinene 0.03 2.52 9.31 1038
α‐Tαh-uTjheunjeen e 0.00.011 00.0.011 7.175.15 911911
CinCeionleeo ale a 0.00.066 −− 8.986.96 10112012
ThyTmhyoml iosloimsoemr er 0.00.077 00.0.000 121.628.68 12714274
Isocaryophyllenea 0.04 − 14.34 1428
Isocaryophyllene a 0.04 − 14.34 1428
Carvacrol − 0.07 12.67 1272
Carvacrol − 0.07 12.67 1272
arepresentsvolatilescomparedwithauthenticstandard.
a represents volatiles compared with authentic standard.
TheantimicrobialactivitydatashowedthatoreganoEOexhibitedthelargestinhibitionzones
The antimicrobial activity data showed that oregano EO exhibited the largest inhibition zones
andMICamongthetestedsevenoils. OreganoEOdisplayedthebestMBCandMBICamongselected
and MIC among the tested seven oils. Oregano EO displayed the best MBC and MBIC among
topfourEOs,alongwithrapidkillingratesofbothtestedbacteria. Moreover,ourGC-MCanalysis
selected top four EOs, along with rapid killing rates of both tested bacteria. Moreover, our GC‐MC
showedthatthymolcontentinoreganoEOwashigherthanthymeEO,suggestingthatthymolmight
analysis showed that thymol content in oregano EO was higher than thyme EO, suggesting that
bethereasonfororeganosuperiorantimicrobialactivities. Therefore,wenextaimedtoassaythymol
thymol might be the reason for oregano superior antimicrobial activities. Therefore, we next aimed
antimicrobialactivitycomparedtooreganoandthymeEOs.
to assay thymol antimicrobial activity compared to oregano and thyme EOs.
Molecules 2018, 23, x FOR PEER REVIEW 6 of 16
2.6. Antimicrobial Activity of Thymol Using In Vitro Disc Diffusion, MIC, MBC and MBIC Assays
Molecules2018,23,2164 6of16
The antimicrobial activity of thymol, the main volatile constituent of oregano and thyme EOs,
was assayed. Using the disc diffusion method, thymol at 0.7% and 1.4% concentration inhibited the
2.6. AntimicrobialActivityofThymolUsingInVitroDiscDiffusion,MIC,MBCandMBICAssays
growth of S. epidermidis and P. acnes significantly higher than erythromycin (inhibition zone > 19
mm) (TFhigeuarnetsim 1 iacnrodb 3ia, lTaacbtilve iSty1)o. fOthny amssoaly,tinheg mofa tihnev MolIaCti loefc tohnysmtitoule angtaoinfsotr eSg. aepniodearnmdidthisy amned EPO. asc,nweas,s
oarsesgaaynedo .aUndsi nthgytmheed wisecrde iffofuusniodn tom beeth soudp,etrhiyomr tool tahty0m.7%ol a(Tnadb1le.4s% 1 caonndc e5n).t rSaitmioinlairnlyh,i bMitBedC tohfe tghryomwothl
aogfaSin.espt idPe.r macidniess awndasP .laocwneesrs itghnainfi coarnetglyanhoig hanerdt htahnymerey.t hMroBmCy coifn t(hinyhmiboilt ioangazionnset >S.1 9epmidmer)m(iFdiigsu wreass1
ianntedrm3,eTdaibatlee Sb1et)w. Oeenna osrseagyainngo oafntdh ethMymICe oEfOths y(Tmaoblleasg 2a iannsdtS 5.)e. pFiidnearmllyid, itshaynmdoPl .aanctnibesio,ofirlemg aanctoivaintyd
atghayimnset wS.e erpeidfoeurmndiditso wbeass ulopwereiro rthtaont hoyrmegoaln(oT,a tbhlyesm1ea, nledm5o)n. Sgirmasisla arnlyd, MteBa Ctreoef tEhOyms (oTlaabgleasin 3s taPn.da c5n).e s
wasDloewsperitteh tahne oprreegdaonmoiannadncthe yomf eth.yMmBoCl ionf othreygmaonloa EgOai,n isnt Sv.iterpoid aenrmtimidiiscrwoabsiailn atessrmayesd sihatoewbeedtw theaent
oorreeggaannoo EanOd wthaysm seupEeOrsio(rT atbol ethsy2manodl a5l)o.nFein. aTllhye,rtehfyomreo, lwane tisbeiloefictlemd aocrtievgiatynoa gEaOin sftoSr. enpaindeoremmiduilsswioans
floorwmeurltahtiaonno arnedga tneost,etdh yitm ine ,vlievmoo ansg ar atosspaicnadl atenatit‐raeceneE Otrsea(Ttmabelnets. 3and5).

FFiigguurree 33.. TThhyymmooll eexxhhiibbiitteedd lloowweerr iinnhhiibbititioionnz zoonneet hthanano roergeagnaonoE OE.OA. nAtinbtaibctaecrtiearliaacl taivcittiyviotyf thofy mthoylmanodl
atnhde mthoes tmboiosat cbtiivoeacEtOivse uEsOinsg udsisincgd idffiuscs iodniffmuestiohnod m(zeothnoedo f(zinohnieb iotifo innihnibmitmio)na gina imnsmt()a )agSa.einpisdte (ram)i dSi.s
ewpiidthermthiydmis owliatnhd thEyOmsoalt acnodnc EenOtsr aatti ocnonocfe0n.7tr%a;ti(obn) So.f e0p.i7d%er;m (bid)i sSw. eipthidtehrmymidoisl wanidthE tOhysmatoclo anncden EtrOasti oant
coofn1c.e4n%tr;a(cti)oPn. aocfn 1es.4w%i;t h(ct)h Py.m aocnleasn dwEitOh sthaytmcoonlc aenntdr aEtiOons aotf c0o.7n%ce;n(dtr)aPt.ioancn oesf w0.i7t%h;t h(dym) Po.l aacnndesE wOsithat
tchoynmceonl tarnadti oEnOosf a1t. 4c%on.cDenattraaitsiorne porfe 1se.4n%te.d Daastam ies arnesproefsiennhteibdi taios nmzeoanness o(fm imnh)ib±itSioDn. zCoonnetsr o(mlsmus)e ±d
SwDe.r Ceoclnintrdoalsm uysceidn awnedree rcylitnhdroammyycciinn aasnpdo esirtyivthercoomnytrcoinlw aist hpoDsMitiSvOe caosnnterogla twiviethc oDnMtrSoOl. as negative
control.
Molecules2018,23,2164 7of16
Table5.MIC,MBCandMBICofthymolagainstS.epidermidisandP.acnes.
Acne-CausingBacteria MIC(mg/mL) MBC(mg/mL) MBIC(mg/mL)
S.epidermidis 0.7 2.8 2.8
P.acnes 0.7 1.4 –
DespitethepredominanceofthymolinoreganoEO,invitroantimicrobialassaysshowedthat
oregano EO was superior to thymol alone. Therefore, we selected oregano EO for nanoemulsion
formulationandtesteditinvivoasatopicalanti-acnetreatment.
2.7. NanoemulsionFormulationofOreganoEO
EOs exhibit poor water solubility, hindering their application in aqueous formulations.
O/WnanoemulsionoforeganoEOwasformulatedtoimproveitspoorwatersolubility. Itcontained
lessthan5%(w/w)oforeganoEOandPluronicF127, exhibitingaclearstablesolution. Thenano
formulation stability was assessed for four weeks at room temperature. No signs of cloudiness,
creaming or phase separation were observed. The prepared nanoemulsion had a particle size of
39.54nm,withapolydispersityindexof0.285,indicatingitslowsizedistribution. FigureS2showsthe
resultsofmeandropletsizeandpolydispersityindexofthepreparednanoemulsion.
2.8. AssessmentofInVivoAntimicrobialandHealingActivitiesofOreganoEONanoemulsionUsingInVivo
AcneMouseModel
Oildosewasadjustedintheformulationat2MICas0.672mg/mLinordertoavoidpossible
irritation or hypersensitive skin reaction. We assessed the anti-acne activity of the formulated
nanoemulsion of oregano EO using invivo acne mouse model. We infected BALB/c mice ears
intradermallywithP.acnes. Aftertwodays,nanoformulationor2%erythromycinepicutanouslywas
applied on mice ears and measured anti-inflammatory and antimicrobial activity against P. acnes.
A third group of mice was injected with phosphate buffered saline (PBS) without P. acnes as a
healthycontrolgroup. Miceoftheuninfectedearcontrolgrouphadnodetectablebacterialgrowth.
Wealsoevaluatedhealingbyexamininghistopathologyofmouseeartissue. Wecomparedresults
to mice treated with 2% erythromycin (positive controls) and untreated mice (negative controls).
Nanoemulsion of oregano EO significantly reduced inflammation at the end of the experiment
(Figure 4). The percentage inhibition of inflammation for all animal groups was calculated using
thefollowingformula[14]:
%inhibition( )−%inhibition( )
%inflammationinhibition= control treatment
%inhibition( )
control
Inhibitionofinflammationattheendoftheexperimentwassignificantlylowerinmicetreated
with nanoemulsion compared to the positive control erythromycin (Figure 4). Moreover, the rate
of reduction of mice ear thickness, post treatment, was also superior to erythromycin positive
control(Figure5).
The invivo antimicrobial activity of oregano EO nanoemulsion was assessed where bacterial
countsofP.acnesdroppedfrom1×108to4.3×101CFU/mLpost-treatmentwith0.672mg/mLof
oreganonanoemulsion(Figure6). Incontrast,thebacterialnumberforthenegativecontrolgroup
(treatedwithPBSbuffer)wasmaintainedat5×105 CFU/mL,whereasthepositivecontroltreated
witherythromycinsolutionreducedthebacterialnumberto3.5×103CFU/mL.
Finally,weassessedthehealingeffectsoforeganoEOnanoemulsionusinghistopathologicaland
digitalphotography. Infectedmiceearstreatedwithnanoemulsionshowedhealingcomparabletothe
erythromycinpositivecontrol,resemblingnormaleartissue. Ourinvivoresultscollectivelysuggested
thatoreganonanoemulsioneffectivelykilledP.acnesandhealedeartissueinflammationbetterthan
erythromycin,thestandardantibioticforacne.
Molecules2018,23,2164 8of16
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Figure 4. Oregano EO nanoemulsion showed stronger inhibition of inflammation than erythromycin
Figure4.OreganoEOnanoemulsionshowedstrongerinhibitionofinflammationthanerythromycin
control in acne mouse model. The acne mouse model was induced by intraderm al injection of
controlinacnemousemodel.TheacnemousemodelwasinducedbyintradermalinjectionofBALB/c
FBiAgLuBre/ c4 m. Oicree’gsa lnefot EeOar sn waniothe m10u8 lCsiFoUn sihno 2w0 emd Ls torof nPg. earc ninesh. iTbhiteio mn iocfe i’ns frliagmhtm eaatriso nse trhvaend earsy uthnrionmfeyctceind
mice’sleftearswith108CFUin20mLofP.acnes.Themice’srightearsservedasuninfectedcontrol.
ccoonnttrrooll . iWn ea acpnpe limedo uespei cumtoandoelu. sTlyh eei tahcenre o rmegoaunsoe EmOo ndaenl owemasu ilnsidounc oerd 2 %by E irnyttrhardoemrmycailn isnojelucttiioonn oonf
We applied epicutanously either oregano EO nanoemulsion or 2% Erythromycin solution on the
BthAeL iBnf/ce cmteidce m’s ilceef te eaarsrs. Aw itthhi r1d0 8g CroFuUp inof 2 m0 imceL s oefr vPe. da canse su. nTthreea mteidc ec’os nrtigrohlt. e(aa)r sP seerrcveendt iansf luanminmfeactitoedn
infected mice ears. A third group of mice served as untreated control. (a) Percent inflammation
cwoanst raosls. eWssee dap ppoliset de pepiciucutatnaneoouuss layp epitlhiceart ioorne goafn tore EaOtm neannto aesm tuhels idoinff eorre 2n%ce E breyttwhreoemn yecaicnh s moluoutisoen eoanr
wasattshhseice kisnnsfeeedscst epodfo imsntficeeecp teeicadur ast.na Adn e utohnuiirnsdf eagcprteopduli pec aaortfsi .om Onicroeeg fsaetnrrevoa entdam naeos neumtnautrslesatiohtened sd hcioofnfwetrerodeln .h c(iaeg)h bPeeret rrwcaetenee to nifn refelaadcmuhcmtmiaotoniou inns e ear
thicknwineafslsas maosfmseianstsfieeodcn t pethodastna n e2pd%icu uenrtayinnthfeeroocumtse ydacpiepna lrdicsua.rtOiionrnge gothfa ent rtoeraentamatmneonenet mta tsiu mtlhseei o idnnitfesfrehvroaewnl. c(eebd )b ehOtiwrgehegeeannr ore aantcaehn omofeomrueusdelus iecoatniro nin
inflamtshhmiocwkanteiedos nss iotghfn iainfnfice2ac%tnetdley ar hnyidtgh hurenorim npfyeeccrtcienednd te uaorrfs in.i nOghritebhgietainotrnoe naoatf mnioneefnlmatmutlimsmioaenti oisnnht oe(w>r6ve0ad%l .h)i (gbaht) etOrh erra eetgena donf oorefn dtaruencatoitomenme inunt l sion
showipenedfrlaisomidgm tnhaiafitinco an2n% tth leayrny ht2hi%groh emeryrytcphinreo rcmcoenynctrtinool df(2ui0nr%ihn)gi.b Dtihtaieot atnr reeoaptfmreinesneflnta ttmeimdm ea sai ntmitoeernavna( l>±. 6s(bt0a)%n Od)raaergtda tdnheoev nieaantnidooneo moffu ttlrhseiroaentem ent
periosdinhdtohewapenedn2 ds%eignnet irefyixctpahenrrtoilmym ehynicgtishn, etcor otpanle trmrcoeiclne(t 2 po0ef%r ign).rhoDiubaiptti a1o5nr emopfir ceiens. feWlanmete umdsaeatdsio tmn‐t ee(s>at6n f0o%±r )c soatmta ntphdaera irendngd dg eroovf uitapretsia owtnmiteohnf ptt hree
indepp<e e0nr.i0do5ed cn tothnaesnxid p2ee%rr sieimgrynetinhfirtcosa,mnttyo. ctianl cmonictreopl (e2r0g%r)o. uDpata1 5rempriecsee.nWtede auss medeatn-t e± ssttafnodracrodm dpevairaitniogng orfo tuhrpese with
independent experiments, total mice per group 15 mice. We used t‐test for comparing groups with p
p<0.05considersignificant.
< 0.05 consider significant.

Figure 5. Oregano nanoemulsion as a potent anti‐acne agent tested in vivo model of P. acnes infection
Photo images of BALB/c mice ear skin at the end of the experiment showing (a) untreate d control
Fmigicuer, eo 5n.e O eraerg ainnjoe cntaendo wemituhl s2i0o nµ aLs ao fp 1o0te8 nCt FaUnt iP‐a. cancen easg esnuts pteesntesdio nin svhivoow minogd meli ocrf oPc.o amcneeds oinnfeesc tainond
Figure 5. Oregano nanoemulsion as a potent anti-acne agent tested invivo model of P. acnes
Pinhfolatmo mimaatgioens oinf BinAfLecBt/ecd m leicfte eeaarr sckoimn paatr tehde teon dth oe f uthnein efexcpteerdi mriegnht t sheaorw; i(nbg) (ian)f luamntmreaattieodn cionn tlreoflt
infectionPhotoimagesofBALB/cmiceearskinattheendoftheexperimentshowing(a)untreated
minifceec,t eodn ee aera rd iinsjaepcpteeda rwedit ha f2t0er µtLr eoaft m10en8 tC FwUit hP . earcyntehsr osmusypceinns isoonl usthioonw icnogm mpaicrraobcloem teod othnee s riagnhdt
controlmice,oneearinjectedwith20µLof108CFUP.acnessuspensionshowingmicrocomedones
iunnflianmfemcteadti oena ri;n (cin) faebctseedn clee fto fe ainr flcaommmpaarteodry toa ptpheea ruannicnef eicnte dle frti gihntf eecaterd; (eba)r intrfelaamtemd abtiyo no rieng alenfot
and inflammation in infected left ear compared to the uninfected right ear; (b) inflammation in
ifnofremctuelda ; e(da)r hdisistoappaptehaorleodg icaaftle ar ntarleyastims oefn tu nwinitfhec teerdy tmhriocem eyacri ns ksino lutitsisoune sctoaminpeadr awbilteh thoe mthaeto xriyglhint
left inuafnnedicn tfeeeodcsteienda rsehadori;w s(aicnp)g p aenbasorerenmdcael a ofhtfie sirtnoftllroaegmaytm moafet onmrtyo wuasipetph eeaearrra ynttichsesr uoienm wylecifttih n inbsfaoeslcautlet diloa nyeaerrc otamrnedapt aeerdpa ibbdlyee rmotroaelgt ahcneelolr ight
uninffmeocratmteuduraleata;i or(n;d( )cp h)rieassbteosrpevanetdche;o l(ooefg) iihncaiflslt aoampnaamtlyhasotiloso rgoyyf auopnfp inienfaeferccatetnedcd em uiinncetlr eeefaattrei nsdkf eicnco tnteitsdrsouel eam rsittcareien aeetaderd wsbhityohw ohirneemgg aannteoocxryfooltiirncm ula;
(d)hiadsnteodrmp eaaotthistioinsl oscghhioacwraalicntaegnr ;an lnoyrosmtiesa olt hfheui snstoeinvloefgeryec tedodfe rmmmoaicul esneee acerraorss kitsii ns(satuiresr sowuwei thhst eaabidans)e,a dla nlwadyi telhry mhanepmdh oaectpyoitdxicye rliminnfaiall tnrcadetlele osin
showm(ianragrtounwroa)rt;mi o(fna) l peharires stseohrlovowegdyin; og(ef a)m chuoitsuet osdepeaertamhroatltioistgsisyu ceohwfa riinathcfetebcrtae; sdna olutlena tytrheeera taceondnd gceoepnstitedrdoel r bmmloiaocldec eevalelrs mssehalsot uw(ariranrtgoiow nn ehpcerraoedtsi)ec, rved;
(e) hidasnetordmp saalittgihthiost lolcyghmayrpaohcofteciryn;t fince occtteeel ldst hiuen nfistletrrveaeatriteoe ndd (eacrormrnoatwlr o)n ltercmeraoitcseieds eb(aayrr reorsywhto hhwroeimandyg)c, innae nscdor loulyttiimconpd;h e(ogrcm)y etaiactr i tisinhsfoiclwthriaantrgea cter;
note(atahpreproaswreev)n; et(lrfye) nedoaerrm rsmhaola whliinsntegocl oraogcusyit seo fd( amerrormuoasweti tehiase rca thdias)sr,uaacetn ewdr;i tnlhyo tmthee pt hahbeo scceoynntcigece osifnt eifidnl ftblralamotoemd (avatoersrrsyoe wrlse) a(;ca(trifro)onwe, a thrreesaahtdeo)d,w ing
awnidth s olirgehgta nlyom foprhmocuylati.c cells infiltration (arrow) treated by erythromycin solution; (g) ear showing
acutedermatitischaracter;notethecongestedbloodvessels(arrowhead),andslightlymphocyticcells
apparently normal histology of mouse ear tissue with the absence of inflammatory reaction, treated
infiltration(arrow)treatedbyerythromycinsolution;(g)earshowingapparentlynormalhistologyof
with oregano formula.
mouseeartissuewiththeabsenceofinflammatoryreaction,treatedwithoreganoformula.
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FFiigguurree 66.. OOrereggaannoo EEOO nnaannooeemmuulslisoionn sshhoowweedd hhigighheer raanntitmimicicrorobbiaial leefffefecct taaggaaininsst tPP.. aaccnneess tthhaann
eerryytthhrroommyycciinn ccoonnttrrooll iinn aaccnnee mmoouussee mmooddeell.. WWee iinndduucceedd aaccnnee iinn aa mmoouussee mmooddeell bbyy iinnttrraaddeerrmmaall
iinnjjeeccttiioonn ooff BBAALLBB//c cmmiciece leleftf teeaarrss wwitihth 11008 8CCFFUU inin 2200 µµLL ooff PP.. aaccnneess.. TThhee mmiiccee’’ss rriigghhtt eeaarrss sseerrvveedd aass
uunniinnffeecctteedd ccoonnttrrooll.. WWee aapppplliieedd eeppiiccuuttaannoouussllyy eeiitthheerr oorreeggaannoo EEOO nnaannooeemmuullssiioonn oorr 22%% EErryytthhrroommyycciinn
ssoolluuttiioonn oonn iinnffeecctteedd mmicicee eeaarsr.s .AA thtihridr dgrgoruopu pofo mfmiceic seesrevrevde dasa usnutrnetareteadte cdocnotrnotlr.o Vl.iaVbilaeb cleoucnoutsn otsf Po.f
aPc.naecsn leosaloda idn imnimceic eeaeras rast atthteh eenedn dofo tfhteh etrteraetamtmenetn tofo fvvarairoiouus smmicicee ggrroouuppss wweerree ppeerrffoorrmmeedd.. OOrreeggaannoo
nnaannooeemmuullssiioonn ssigignnifiifcicaanntltylyl olwowereerdedba cbtaecrtiearlilaola dloiandt reina tetrdemateicde emariscet heaanrs2 %thEarny th2%ro mEyrcyitnh.rBomacytecriina.l
Bloaacdterwiaals loeaxdp rwesasse dexapsreLsosegd10 aCs FLUog/1m0 CL.FDUa/mtaLr. eDpraetase rnetperdesaesnmtedea ans ±mesatann ±d asrtdanddeavrdia tdioenviaotfiotnh roeef
tihnrdeeep iennddeepnetnedxepnet reimxpeenrtism,etonttasl, mtoitcael mpeirceg rpoeur pgr1o5ump i1c5e .mTi-ctee.s tTw‐teasst uwsaeds ufoserdc ofmorp caorminpgagrirnogu pgsrowuipths
wpi<th0 .p0 5< c0o.0n5s icdoenrseiddesrigedn isfiicgannifti.cant.
33.. DDiissccuussssiioonn
OOrreeggaannoo EEOOe xehxihbiibteitdedth ethsetr osntrgoensgteasntt imanitcirmobiciarolbeifafel cteaffgeacitn satgtahienstet sttehde atcensete-cda uascinneg‐bcaauctseinriga .
bUascitnegriad. iUscsindgif fduissico dni,ffMusIiCon,, aMndICM, aBnCd MasBsCay ass,saoyrse,g oarneog,anthoy, mthey,maen, dantdh tyhmymolowl wereeret hthee ttoopp tthhrreeee
aannttiimmiiccrroobbiiaall aaggeennttss aaggaaiinnsstt PP.. aaccnneess.. MMeeaannwwhhiliele, ,teteaa trtreeee aanndd lleemmoonnggrraassss EEOOss eexxhhiibbiitteedd aann
iinntteerrmmeeddiiaattee aannttiimmiiccrroobbiiaall eeffffeecctt aaggaaiinnsstt PP.. aaccnneess aanndd SS.. eeppiiddeerrmmiiddiiss.. BBootthh tteesstteedd aaccnnee‐-ccaauussiinngg bbaacctteerriiaa
wweerree rreessiissttaanntt ttoo cchhaammoommiillee,, llaavveennddeerr,, aanndd mmeenntthhee EEOOss.. TThhuuss,, iinn tthhiiss ssttuuddyy,, tthhee aannttii‐-aaccnnee eeffffeeccttss ooff
oorreeggaannoo EEOO ssuurrppaasssseedd tthhaatt ooff ootthheerr EEOOss eevvaalluuaatteedd iinncclluuddiinngg ccoommmmeerrcciiaalliizzeedd oovveerr‐-tthhee‐-ccoouunntteerr aaccnnee
ttrreeaattmmeenntt tteeaa ttrreeee EEOO [[1155,,1166]].. MMoorreeoovveerr,, ppaarrtt ooff aaccnnee ppaatthhooggeenneessiiss iinncclluuddeess SS.. eeppiiddeerrmmiiddiiss bbiiooffiillmm
ffoorrmmaattiioonn.. BBaaccteteriraia ggrroowwiningg inin bbioioffiilmlmss aarree mmoorree rreessiissttaanntt ttoo aannttiibbiioottiiccss ccoommppaarreedd ttoo ppllaannkkttoonniicc
lliiffeessttyyllee [[1177]].. TThheerreeffoorree, ,wwee aasssseesssseedd tthhee aannttii‐-bbiiooffiillmm aabbiilliittyy ooff tthhee mmoosstt ppootteenntt aannttiimmiiccrroobbiiaall EEOOss
aanndd ffoouunndd tthhaatt oorreeggaannoo EEOO eexxhhiibbiitteedd ssttrroonnggeesstt aannttii‐-bbiiooffiillmm aaccttiivviittyy.. MMoorreeoovveerr,, oorreeggaannoo EEOO aatt 44 MMIICC
sshhoowweedd rraappiidd kkiilllliinngg ooff bbootthh tteesstteedd aaccnnee‐-ccaauussiinngg bbaacctteerriiaa..
TThhyymmooll eexxhhiibbiitteeddp optoetnetnat natinmtiimcriocbroiabliaacl tiavcittiyvaitgya iangstaiancsnte -accanues‐icnagubsainctge rbiaa,cbteurtilae,s sbtuhta nleossr etghaanno
oErOegiatnseol fE. OTh iytsmelofl. eTxhhyimbiotsl seixghniibfiictas nstigbnaicftiecraincti dbaalcatectriivciidtyal[ 1a8c–t2iv0i]tya n[d18r–e2d0u] caensdb arcetdeuricaelsr ebsaicsttearnicael
rteosiasntatinbcieo ttioc san[2t1ib].ioTtihcys m[2o1l].a Tnhtiymmicorlo abnitailmaicctrioobniaisl amctaiionnly ism meadiinaltye dmvediaiaintehdi bviitain ignhbiabcittienrgia blagcrtoewriathl
garnodwltahc taatnedp lraocdtautcet iponro,ddueccrteioansi,n dgeccerlelauslainrgg lcueclolusleaur pgtlaukceo,scea uuspitnagkely, sciasuosfinfugn lgyaslish yopf hfaulnwgaall lh[y22p,h2a3l] .
wThayllm [2o2l,2w3a].s Tthheymproiln cwipasa lthpeh epnroinliccipcaolm pphoenneonlitco cfoomrepgoanneontE oOf (o>r9e9g%an)oa nEdO t(h>y9m9%e)E aOnd(> t7h0y%m).e IEtOis
(t>h7u0s%s)u. gIgt eiss ttehdufso sruitgsgreoslteedin fmore idtsia rtoinleg itnh emEeOdsia’tainntgi mthicer EobOisa'l aenffteicmt.icHroobwiaelv eefrf,etchty. mHoolwaelovneer, wthaysmleosls
aplootneen twthasa nleosrse pgoanteonEt Oth.aInt cooruelgdanbeo tEhOat. oItt hceorumldi nboer tvhoalta otitlheesrp mreisneonrt avtolloawtileers lpevreeslsensut caht laoswpe-cry lmeveenles ,
sγu-ctehr pasin pe‐ncyem, αe-nthe,u γje‐nteerpainndencein, eαo‐ltehusyjenneer ganizde dcintheyomle osly’sneerfgfeicztedin thoyremgoanl’os eEfOfecatn idn omreagyaancoc oEuOn tanfodr
mdiaffye arecncocuenint faocrt diviiftfyerbenetcwe eine nacotirveigtayn boetawnedenth oyrmegoal.noC aonmdp tohsyimtioonl. oCfoEmOpsodsietipoenn odfs EoOnsa dnepuemnbdesr oonf
afa cntuomrs,bienrc loufd ifnagcthoarsr,v eisntcinlugdsienags ohnasr,vpelsatnintgcr ossesa-ssoecntsio, np,leaxnttr accrtoiosns‐mseectthioond,, aenxtdragcetoiognra pmheicthalosdo, uarnceds
geographical sources (reviewed in [24]). This can account for differences between results obtained
from different studies, where variation in thymol amount is significant [25,26]. The EOs used in this
Molecules2018,23,2164 10of16
(reviewedin[24]). Thiscanaccountfordifferencesbetweenresultsobtainedfromdifferentstudies,
wherevariationinthymolamountissignificant[25,26]. TheEOsusedinthisstudywereobtained
fromplantscollectedduringthefloweringstage,whichcouldexplainthehighphenoliccompound
levels. Collectively,oreganoEOwasthemostpotentantimicrobial. Consequently,itwasformulated
innanoemulsiontopicaldosageformtobeassessedasanti-acneformulainvivoonacne-induced
mousemodel.
Treatmentoftheacnemousemodelwithproposedoreganonanoemulsionresultedinreductionof
inflammation,bacterialloadandhealingoftissuesuperiortoerythromycin. EOs’antibacterialeffects
areimprovedthroughformulationasnanoemulsions[27–29]. Thepreparedemulsionhadaparticle
sizeof39.54nmandapolydispersityindexof0.285,indicatingitslowsizedistribution;itis,therefore,
consideredasananoemulsion[30]. PluronicF127surfactantwasusedataconcentrationof4.5%and
theoilat0.5%w/w.TheusedconcentrationisabovePluronicF127criticalmicelleconcentration(CMC),
whereCMCofPluronicF127rangesbetween0.26–0.8wt%. Oreganooilexhibitedanti-inflammatory,
anti-leishmanial, antioxidant, hepatoprotective and anti-tumor activities, reviewed in [31]. To the
bestofourknowledge,thisisthefirststudytoreporttheanti-acneeffectoforeganoEO.Oregano’s
anti-acneeffectssurpassthoseofthecommercializedover-the-counteracneteatreeEOandselectEOs
withdocumentedanti-microbialeffects[11]. Mostanimalsdonotproducesufficienttriglyceridesto
harborP.acnes[32]. Therefore,westandardizedouracnemousemodelusingBALB/cmicethrough
intradermalinjectionofP.acnesinmiceears[32,33]. Basedonourinvivoresults,weproposeBALB/c
miceasanotherpossiblemousestrainforthedevelopmentofananimalmodelofacne.
Theessentialoilsofaromaticplantsofferspasmolytic,mucolyticandcoughsoothingeffects[31,34].
Inaddition, antimicrobialeffectsofEOsagainstpathogensandfoodcontaminantshavealsobeen
favored due to the safety of natural-origin products, preferred by the public [35]. Little attention,
however,hasbeenpaidtoEO’santimicrobialeffectagainstacne-causingbacteria. Overall,ourresults
indicatethattheproposedoreganonanoemulsionexhibitshighantimicrobialandhealingeffectswith
fewer side-effects than anti-acne reference antibiotics. The potential use of our proposed oregano
nanoemulsionasananti-acneagentopensthefieldfornewalternativetreatmentsofnaturalorigin,
avoidingtheproblemsassociatedwiththeuseofantibioticsforacne. Furtherclinicalstudiesonour
proposedformulationcanincreaseitspotentialasadrugtobeusedclinicallyinhumans.
4. MaterialsandMethods
4.1. EssentialOils
SevenEOswereusedinthisstudy,viz.,oregano(Origanumvulgare),thyme(Thymusvulgaris),
lemongrass(Cymbopogoncitratus),teatree(Melaleucaalternifolia),mentha(Menthapiperita),lavender
(Lavendula anguestifolia) and chamomile (Matricaria recutita). The pharmaceutical grade EOs were
kindlyprovidedasagiftfromtheDepartmentofFoodScienceandTechnology,NebraskaUniversity,
Lincoln,NE,USA.WedeterminedthecompositionoforeganoandthymeEOsbyGC-MSanalysisat
theDepartmentofPharmacognosy,FacultyofPharmacy,CairoUniversity,asdetailedinSection4.8.
PurethymolwaspurchasedfromSigmaAldrich,St. Louis,MO,USA.
4.2. BacterialStrainsandCulturing
Propionibacterium acnes ATCC 6919 and Staphylococcus epidermidis ATCC 28319 were kindly
providedbyDr. MayriA.Diaz,ManchesterUniversity,UK.S.epidermidiswasculturedaerobically
on brain heart infusion (BHI) agar (LAB M limited, Lancashire, UK) and incubated at 37 ◦C for
18h. Wesub-culturedisolatedcoloniesofS.epidermidisinBHIbrothandincubatedat37◦Cfor18h
aerobically. For P. acnes, we cultured the bacteria anaerobically on reinforced clostridial medium
(RCM)agar(OxoidLimited,Basingstoke,UK)for48hat37◦Cand~5%CO usinganaerobicjarand
2
anaerobicatmospheregenerationbags(Sigma-Aldrich,St. Louis,MO,USA).Wesub-culturedisolated
coloniesofP.acnesinRCMbrothfor48hat37◦Cunderanaerobicconditions.