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RESEARCHARTICLEOpenAccessInvitrosynergismofmagnololandhonokiolincombinationwithantibacterialagentsagainstclinicalisolatesofmethicillin-resistantStaphylococcusaureus(MRSA)Guo-YingZuo1*,Xin-JuanZhang1,2,JunHan3*,Yu-QingLi3andGen-ChunWang1AbstractBackground:Methicillin-resistantStaphylococcusaureus(MRSA)isaproblematicpathogenposingaserioustherapeuticchallengeintheclinic.
Itisoftenmultidrug-resistant(MDR)toconventionalclassesofantibacterialagentsandthereisanurgentneedtodevelopnewagentsorstrategiesfortreatment.
Magnolol(ML)andhonokiol(HL)aretwonaturallyoccurringdiallylbiphenolswhichhavebeenreportedtoshowinhibitionofMRSA.
Inthisstudytheirsynergisticeffectswithantibacterialagentswerefurtherevaluatedviacheckerboardandtime-killassays.
Methods:ThesusceptibilityspectrumofclinicalMRSAstrainswastestedbythediskdiffusionmethod.
Theminimalinhibitoryconcentrations(MICs)andminimalbactericidalconcentrations(MBCs)ofMLandHLwereassayedbybrothmicrodilution.
Thesynergywasevaluatedthroughcheckerboardmicrodilutionandtime-killingexperiments.
Results:MLandHLshowedsimilaractivityagainstbothMSSAandMRSAwithMIC/MBCat16~64mg/L,withpotencysimilartoamikacin(AMK)andgentamicin(GEN).
Whentheywereusedincombinationwithconventionalantibacterialagents,theyshowedbacteriostaticsynergywithFICIsbetween0.
25~0.
5,leadingtothecombinedMICsdecreasingtoaslowas1~2and1~16mg/LforML(HL)andtheagents,respectively.
MIC50ofthecombinationsdecreasedfrom16mg/Lto1~4mg/LforML(HL)and8~128mg/Lto2~64mg/Lfortheantibacterialagents,whichexhibitedabroadspectrumofsynergisticactionwithaminoglycosides(AMK,etilmicin(ETM)andGEN),floroquinolones(levofloxacin(LEV),ciprofloxacinandnorfloxacin),fosfomycin(FOS)andpiperacillin.
Thetimesofdilution(TOD,theextentofdecreasinginMICvalue)weredeterminedupto16forthecombinedMIC.
AmoresignificantsynergyaftercombiningwasdeterminedasML(HL)withAMK,ETM,GENandFOS.
ML(HL)combinedwithantibacterialagentsdidnotshowantagonisticeffectsonanyofthetenMRSAstrains.
ReversaleffectsofMRSAresistancetoAMKandGENbyMLandHLwerealsoobserved,respectively.
AllthecombinationsalsoshowedbetterdynamicbactericidalactivityagainstMRSAthananyofsingleML(HL)ortheagentsat24hincubation.
ThemoresignificantsynergyofcombinationsweredeterminedasHL(ML)+ETM,HL+LEVandHL+AMK(GENorFOS),withLC24of2.
02~2.
25.
Conclusion:MLandHLshowedsynergisticpotentiationofantibacterialagentsagainstclinicalisolatesofMRSAandwarrantfurtherpharmacologicalinvestigation.
Keywords:Magnolol,Honokiol,MRSA,Synergy,Antibacterialagent*Correspondence:zuoguoying@263.
net;hanzjn@126.
com1ResearchCenterforNaturalMedicines,KunmingGeneralHospitalofChengduMilitaryCommand,Kunming650032,China3SchoolofBasicMedicalSciences,YunnanTraditionalChineseMedicalCollege,Kunming650500,ChinaFulllistofauthorinformationisavailableattheendofthearticle2015Zuoetal.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://creativecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Zuoetal.
BMCComplementaryandAlternativeMedicine(2015)15:425DOI10.
1186/s12906-015-0938-3BackgroundTheopportunisticpathogenStaphylococcusaureus(SA)isaleadingcauseofbacterialinfectionsofpeople,caus-ingabroadspectrumofpathologyrangingfromcom-monskininfectionstodeep-seatedfataldisease[1].
AntibiotictreatmentofSAhasoncecontributedgreatlytohumanhealthfordecades.
However,duetolong,wideandirrationalapplicationsofantibacterialagentsintreatmentsinvariousfieldsotherthanintheclinic,methicillin-resistantStaphylococcusaureus(MRSA)hasevolvedasaproblematicpathogenandhasposedaser-ioustherapeuticchallengeinclinic[1,2].
Nowadays,MRSAinfectionscanbemonitoredbothinhospitals[healthcare-acquired/associated(HA)MRSA]andcom-munity[community-acquired/associated(CA)MRSA].
Thelivestock-associatedMRSA[(LA)MRSA]hasalsooccurred[3].
MRSAisabletoproduceresistancetonearlyallcommonclassesofantibioticsincludingβ-lactams,aminoglycosides,macrolides,tetracyclinesandquinolones,andeventhevancomycin-resistantS.
aureus(VRSA)hasalsobeenreported[4].
Thedecreasingef-fectivenessofconventionaldrugsiscontinuouslyhaunt-ingbothcliniciansanddrugresearchers,andthecriticalshortageofnewantibioticsindevelopmentagainstMRSAandothermultidrug-resistantbacteriaisofgreatconcernworldwide.
NewtargetswithnovelstrategyoftherapyandmechanismofactionfordevelopmentofantibacterialagentsagainstMRSAareurgentlyneeded[5].
Plantnaturalresourceshavebeendemonstratedtopos-sessgreatchemicalandbiologicaldiversitiesandpromisingfindingsofantibacterialphytochemicalswhichshowednotonlyanti-MRSAactivityalone,butalsosynergisticpoten-tialswhentheywereusedincombinationwithconven-tionalantibacterialagentsagainstMRSA[6–10].
Inrecentyears,wearedevotingeffortstosearchfornovelphyto-chemicalsthatshowedsynergywithconventionalantibac-terialagentsagainstMRSAfrommedicinalplantsources,especiallyfromthetraditionalChinesemedicines(TCM)[11–13].
Wefoundtwodiallylbiphenols,i.
e.
magnolol(ML)andhonokiol(HL)aresuchphytochemicalscontainedintheChinesecrudedrugHou-po,thestemorrootbarkofMagnoliaofficinalisRehd.
etWils.
(Magnoliaceae)[14].
Hou-poisanimportantspeciesinTCM[14].
Ithasbeentraditionallyusedforrespiration,digestionandin-fectionrelatedailmentslikecough,diarrhea,andallergicrhinitis[14].
Modernpharmacologicalreportsalsodem-onstrateditsantimicrobial,anti-inflammatoryandanal-gesic,antianxietyandantidepressant,antitumorandanticoagulanteffectsaswellasmyocardial/cerebralische-miaprotections[15].
MLandHLaretwomainphenolicconstituentsprimarilyisolatedfromHou-poandalsofoundinotherMagnoliasp.
,togetherwithothernon-phenolicconstituentssuchasalkaloidsandessentialoils[16–18].
TheantimicrobialeffectsofHou-poextractsonBacillusanthracis,S.
aureusandotherpathogenswerefoundasearlyassixdecadesago[19–21].
PreviousreportsonantimicrobialactivitiesofMLandHLandtheirsyn-theticderivativesincludeantibacterial[22–31],antifungal[32–34],antiviral[35]andnemicidal[36]activities.
Al-thoughtheirantibacterialactivitiesagainstMRSAandvancomycin-resistantenterococci(VRE)havebeenre-ported[37,38],theirpotentialforcombinedactiononconventionalantibacterialagentsagainstMRSAhasnotbeenstudied.
Onlythesynergyofhonokiolwithflucona-zoleagainstclinicalisolatesofazole-resistantCandidaalbicans[39],andsynergisticeffectoflysozymeonbactericidalactivityofmagnololandhonokiolagainstacariogenicbacteriumofStreptococcusmutansOMZ176[40]includingtheirpotentiationoftheantitumoragents[41–46]werereported.
Inthispaper,wewillshowthepotentialsynergisticeffectsofMLandHLincombinationwithconventionalantibacterialagentsagainstclinicalMRSAstrainsthroughthecheckerboardandtime-killcurvemethods.
MethodsAntimicrobialagentsanddisksTheeightantibacterialagents,i.
e.
amikacin(AMK)(JiangsuWuzhongPharmaceuticalGroupCo.
,Ltd.
,Suzhou,China);Etilmicin(ETM)(WuxiJiminkexinShanhePharmaceuticalCo.
,Ltd.
);gentamicin(GEN)(GuangzhouBaiyunshanTianxinPharmaceuticalCo.
,Ltd.
,Guangzhou,China);Piperacillin(PIP)(HarbinPharmaceuticalGroupCo.
,Ltd.
,Harbin,China);Norfloxacin(NOR)andCiprofloxacin(CIP)(SichuanKelunPharmaceuticalCo.
,Ltd.
,Chengdu,China);Levofloxacin(LEV)(YangzhijiangpharmaceuticalCo.
,Ltd.
,Taizhou,China);Fosfomycin(FOS)(NortheastPharma-ceuticalGroupCo.
,Ltd.
,Shenyang,China).
magnolol(ML)andhonokiol(HL)(HPLC>98%;XianXiaocaoScienceandTechnologyCo.
,Ltd.
,Xian,China)(Fig.
1).
Vancomycin(VAN)(EliLillyJapanK.
K.
,SeishinLaboratories)wasusedasthepositivecontrolagent.
Cefoxitin(cfx,0.
03mg)andotherantibioticimpregnateddiskswerepurchasedfromBeijingTiantanbiologicalproductsCo.
,Ltd.
,China.
Fig.
1Thestructuresofmagnolol(ML)andhonokiol(HL)Zuoetal.
BMCComplementaryandAlternativeMedicine(2015)15:425Page2of10BacterialstrainsandmediaTenMRSAstrains(MRSA01–10)wereisolatedandcharacterizedfromtheinfectioussputumsampleofcritic-allyillpatientsinKGHaspreviouslyreported[11–13].
Thestrainsweredeterminedwithzonediameter(ZD)≤21mmagainstcefoxitindiskandthepropertiesofsusceptible(S),intermediate(I)andresistant(R)toantibacterialagentsweredeterminedaccordingtotheZDInterpretiveCriteriaofTable2Cin2012CLSIbycomparisonwiththeZDofcorrespondingantibacterialagents(Table1)[47].
ThepresenceofmecAgeneandSCCmecgenotypesweredeterminedbymultiplexPCRmethodsinKunmingInstituteofVirology,PLA,China,aspreviouslyre-ported[48].
ThecontrolstrainforMRSAwasS.
aureus(ATCC25923;methicillin-susceptibleS.
aureus(MSSA))whichwaspurchasedfromtheBeijingTiantanPharmaceuticalandBiologicalTechnologyCo.
,Ltd.
,China.
StandardMueller-Hintonagarandbroth(MHAandMHB,TianheMicrobialAgentsCo.
,Hangzhou,China)wereusedasbacterialculturemedia.
ColonycountsweredeterminedusingMHAplates.
MHBwasusedforquantitativesusceptibilitytestinganddynamictime–killexperiments.
SusceptibilitytestingThetestofsusceptibilityspectrumofthetenclinicalMRSAstrainstoconventionalantibacterialagentswasperformedbydiskdiffusiontestfollowingtheCLSIguideline[47,49,50].
MICs/MBCsofMLandHLweredeterminedbystandardizedbrothmicrodilutiontechniqueswithinoculumsof5*105CFU/mLac-cordingtoCLSIguidelinesandincubatedat35°Cfor24h[51–53].
ThesolventusedfordissolvingthecompoundsandtheantibioticswasMHB(orcontainingthefinalconcentrationoflessthan5%ofdimethylsulphoxide).
SynergytestingPotentialinteractionsofMLandHLincombinationwithvariousantibioticsagainstMRSAwereevaluatedbydeterminationoffractionalinhibitoryconcentrationindices(FICIs)andtime-killcurvesthroughusingthechecker-boardanddynamictime-killmethodsasdescribedprevi-ously[12,13].
ThebacteriostaticinteractionmodewasjudgedbyFICIsasfollows:FICI≤0.
5,synergy;0.
51log10CFU/mL,antagonism;wheretheLC24wascalculatedthroughthekillingbyacombination(LC24(co.
))deductingthatbythemostactivesingledrug(LC24(si.
))inthecombination,i.
e.
LC24=LC24(co.
)-LC24(si.
)[56].
StatisticalanalysisAlltheexperimentswereperformedintriplicate.
Dataareexpressedasthemean±standarderror.
StatisticalanalyseswereperformedusingtheStatisticalPackagefortheSocialSciences(SPSS20.
0)software(SPSSInc.
,Chicago,IL,USA).
DatawereanalysedbyKruskal–WallistestandthesignificantdifferencesbetweengroupswereanalysedbyDunnett'stest.
StatisticalsignificancewasacceptedatalevelofpETM>HL>ML>GEN>AMK>LEV(PIP,FOS)>CIP(NOR)judgedbythevaluesofMIC90/MBC90.
Therefore,theantibacterialactivityofthetwocompoundsallshowedmorepotentagainstMRSAthanmostoftheconventionalantibacterialagentswiththeexceptionofETMandVAN(Table2).
SynergyofMLandHLincombinationwithantibacterialagentsagainstMRSATable3showsthedifferentdegreeofsynergisticinterac-tionsofMLandHLincombinationwiththeeightanti-bacterialagentsagainstthetenclinicalMRSAisolates.
TherearethreetotenMRSAstrainsthatshowedsynergywithFICIsbetween0.
25~0.
5,leadingtothecombinedMICsdecreasingtoaslowas1~2and1~16mg/LforML(HL)andtheagents,respectively.
MIC50ofthecom-binationsdecreasedfrom16mg/Lto1~4mg/LforML(HL)and8~128mg/Lto2~64mg/Lfortheantibacter-ialagents,whichexhibitedabroadspectrumofsynergisticactionwithaminoglycosides(amikacin,etilmicinandgentamicin),floroquinolones(levofloxacin,ciprofloxa-cinandnorfloxacin),fosfomycinandpiperacillin.
Thetimesofdilution(TOD,theextentofdecreasinginMICvalue)weredeterminedupto16forthecom-binedMIC.
Themoresignificantsynergyaftercom-biningwasdeterminedasML(HL)withAMK,ETM,GENandFOS.
Therefore,MLandHLshowedgenerallythesamesynergisticbacteriostaticeffectsonthetestedantibacterialagents.
Moreover,ML(HL)combinedwithantibacterialagentsdidnotshowantagonisticeffectsonanyofthetenMRSAstrains.
Therewereonly1~2strainsthatshowedindifference.
TofurtherevaluatethedynamicbactericidaleffectsofML(HL)incombinationwiththeantibacterialagents,thetime-killcurveexperimentswereperformedandthere-sultsareshowninTable4andFigs.
2and3.
Eightofthecombinationsshowedsynergyindynamickilleffects,withtheorderofpotencyasHL+ETM>HL+LEV>ML+ETM>HL+AMK>HL+FOS>HL+GEN>ML+AMK>ML+FOS.
TherestofthefourcombinationsML+LEV,ML+CIP,ML+GENandHL+CIPshowedadditiveeffects(Table4).
AllthecombinationsshowedbetterbactericidalactivityagainstMRSAthananyofsingleML(HL)ortheagentsat24hincubation.
Thebactericidalefficiencyofthecombinationsgener-allylastedlongerthanthatofthesingleagents(Figs.
2and3).
Themoresignificantcombinationsweredeter-minedasHL+ETM,HL+LEVandML+ETM,withLC24of2.
08~2.
25(Table4).
ThecombinationsofantibacterialagentswithHLshowedmoresignificantkillingeffectsthanthosewithMLforasameantibacterialagent.
Forexample,thecombinationofHL+LEVshowedsynergybutthecombinationofML+LEVshowedonlyadditively.
Therefore,HLisamoreoptimisticagentforbactericidalpotentiationoftheeffectofconventionalantibacterialagents(Table4).
ReversalofMRSAresistancetoamikacinandgentamicinbyMLandHLBesidesthesynergyeffectiveness,thecombinationofML(HL)withamikacin(AMK)ledMICs(mg/L)ofAMKtodecreasemarkedlyeventoreversetheMRSAresistancetoAMKbytheMICInterpretiveCriteriaofCLSIPerformanceStandards,i.
e.
MIC≤16mg/L(sus-ceptible),MIC=32mg/L(intermediate),MIC≥64mg/L(resistant)[47].
ThereisanequivalenteightstrainsofMRSA(n=10)thatshowedMICs≤16mg/LagainstAMKwhenitwasusedincombinationwithMLandHL,respectively(p0.
01Zuoetal.
BMCComplementaryandAlternativeMedicine(2015)15:425Page4of10Table3MICs(mg/L)ofmagnolol(ML)andhonokiol(HL)usedaloneandincombinationwithantibacterialagentsagainstthetenMRSAstrainsCombinationaEffectMIC(mg/L)FICIcInteraction(n)dAloneCombinedTODbSynAddIndML+AMK*Range8+32~32+1282+4~8+328+16~2+20.
375~0.
7582050%16+642+84+40.
590%16+644+324+40.
625ML+ETMRange8+4~32+161+1~8+416+8~2+40.
313~0.
7582050%16+82+24+40.
37590%16+164+22+40.
75ML+GEN*Range8+16~32+1281+4~16+3216+8~1+20.
25~1.
2572150%16+642+88+40.
590%16+644+322+21ML+FOSRange8+128~32+2562+16~16+1288+8~2+10.
25~1.
572150%16+1284+324+80.
37590%16+2568+642+40.
75ML+CIPRange8+256~32+5121+32~16+51216+8~2+10.
25~1.
554150%16+2564+644+80.
590%16+5128+2562+20.
75ML+LEVRange8+128~32+2562+16~16+1288+8~1+10.
25~1.
544250%16+1284+324+80.
62590%16+2568+1282+21.
25ML+NORRange8+128~32+2562+16~16+1288+8~1+10.
375~1.
544250%16+1284+644+40.
62590%16+2568+1282+21.
25ML+PIPRange8+128~32+2562+32~8+1284+8~2+10.
375~1.
544250%16+1284+324+40.
7590%16+2568+1282+11.
25HL+ETMRange8+4~16+161+1~4+28+8~4+40.
25~0.
5100050%16+82+28+40.
37590%16+164+24+40.
5HL+AMK*Range8+32~16+1281+4~4+328+8~4+20.
25~0.
7591050%16+642+88+40.
37590%16+644+324+40.
5HL+GEN*Range8+16~16+1281+2~4+328+8~4+20.
25~0.
7582050%16+642+168+40.
37590%16+644+324+20.
625HL+FOSRange8+128~16+2562+8~8+1288+3~21+20.
25~1.
2572150%16+1284+324+40.
590%16+2568+642+21HL+CIPRange8+256~16+5122+16~8+5128+16~2+10.
25~1.
563150%16+2562+644+40.
590%16+5128+2562+20.
75HL+LEVRange8+128~16+2562+16~16+648+8~1+40.
25~1.
2554150%16+1284+324+80.
5Zuoetal.
BMCComplementaryandAlternativeMedicine(2015)15:425Page5of10DiscussionInthepresentreport,weperformedtheantibacterialevaluationsofMLandHLagainstMRSAbothusedaloneandincombinationwithclinicalconventionalantibacterialagentsofbroadclasses.
ThesynergismofMLandHLonMRSAisfoundforthefirsttimesofartothebestofourknowledge[37,39].
WedeterminedtheantibacterialactivityofMLandHLagainstMRSAalonewithMIC50/MBC50(n=10)of16~64mg/LwhicharesimilartotheresultsofthepreviousreportwhichshowedMIC/MBCof12.
5/25mg/L[37].
ThedifferenceisreasonableforthevariedMRSAstrainsused.
FromtheresultsofcombinatoryeffectonMRSAandthereportedantifungalsynergism[39],thetwocompoundsaredemonstratedtopossessawidespectrumofantimicrobialpotentiationofconventionalchemo-therapy.
Itwouldbebeneficialforthetreatmentofmixedinfectionsandeventheco-infectionoftumordiseases,consideringML(HL)alsopotentiatinganti-tumoragents[41–46].
ItisimportantandvaluableofMLandHLthatpo-tentiatetheantimicrobialactivityofaminoglycosidesandotherantibacterialdrugsagainstMRSA,whichcouldpreventthedrugsfromdevelopmentofMDRtothetroublesomegerm.
Asaminoglycosidesareoneclassoftheimportantantimicrobialsforthetreatmentofin-fectiousdiseases,theMRSAresistancereversaleffectsofMLandHLtotheaminoglycosidesagents(AMK,ETMandGEN)areespeciallysignificantfortheirapplicationwithlargelyreducingtoxicreactionsofthehosts'liver,kidneyandneuralsystembyasubstantiallylowereddos-age.
Hence,greatersamplescalesareneededinordertodrawamorereliablesignificanceoftheeffectivenessonMRSAfromclinicalspecimensandantibiotics.
ThemechanismoftheactionofMLandHLagainstMRSAtogetherwiththeirsynergismwithantibacterialagentsisstillanunmetquestion.
Twopreviousreviewssummarizedfourmainresistancemechanismsfrombac-teria:(i)receptororactivesitemodification,(ii)enzymaticdegradationormodificationofantibiotic,(iii)decreasedpenetration,or(iv)increasedefflux[9,10].
ItwasreportedthattheantimicrobialmechanismsofMagnoliaofficinalisextractresultedmainlyincellmembraneandwalldam-age,causingincreasedpermeabilityofcellmembranesorlysisofcellwallsandlossofcellularconstituents,impair-mentofstructuralcomponentsandchangesinbacterialcellmorphology[21],whichcouldascribedtoMLandHL,thetwomainconstituentsoftheplant.
SomestudiesalsohavedemonstratedthatincreasedpermeabilityofTable3MICs(mg/L)ofmagnolol(ML)andhonokiol(HL)usedaloneandincombinationwithantibacterialagentsagainstthetenMRSAstrains(Continued)90%16+2568+322+41.
125HL+PIPRange8+128~16+2561+16~16+1288+8~1+10.
25~1.
553250%16+1282+328+40.
590%16+2568+1282+21.
25HL+NORRange8+128~16+2561+16~8+2568+8~1+10.
25~1.
535250%16+1284+644+40.
7590%16+2568+1282+21.
25aMLMagnolol,HLHonokiol,AMKAmikacin,FOSFosfomycin,LEVLevofloxacin,ETMEtilmicin,PIPPiperacillin,CIPCiprofloxacin,NORNorfloxacin,Alldataontheleftsideof"+"belongtoMLorHL,andthedataontherightsideof"+"belongtotheconventionalantibacterialagents,forexample,"ML+AMK"meansMLcombinedwithAMK.
bTODTimesofdilution=MICAlone/MICCombined,rangedfromthemaximumtotheminimum.
cFICIFractionalinhibitoryconcentrationindex,dAddAdditivity(0.
50.
01)Table4Collectedtime-killingassayresultsofvariouscombinationsofMLandHLwithantibacterialagentsat24hincubationagainstaclinicalMRSA144strainCombinationaMascbLC24(Int)cHL+ETMETM2.
25±0.
12(Syn)*HL+LEVHL2.
09±0.
09(Syn)*ML+ETMETM2.
08±0.
1(Syn)*HL+AMKAMK(~ML)2.
05±0.
07(Syn)*HL+FOSFOS2.
04±0.
03(Syn)HL+GENHL2.
02±0.
01(Syn)ML+AMKAMK(~ML)2.
02±0.
02(Syn)ML+FOSFOS(~ML)2.
00±0.
01(Add)ML+LEVLEV(=ML)1.
64±0.
04(Add)ML+CIPCIP1.
24±0.
05(Add)ML+GENGEN1.
24±0.
03(Add)HL+CIPHL1.
06±0.
04(Add)aSALSalvianolate,AMPAmpicillin,CAZCeftazidime,CFZCefazolin,CPSCefoperazone-sulbactam,PTZPiperacillin-tazobactam,AMKAmikacin,CLIClindamycin,ERYErythromycin,FOSFosfomycin,LEVLevofloxacinbMascMostactivesingledrugcLC24Log10CFU/mLat24h,IntInteraction,SynSynergy(LC24≥2),AddAdditivity(10.
01;a:alone;c:combined)Fig.
3Time-killingcurvesofsixcombinationsofHLwithAMK(a),GEN(b),ETM(c),CIP(d),LEV(e)andFOS(f)at1*MICand24hincubationagainstarepresentativeclinicalMRSA144strain.
Dataareexpressedasthemean±standarderrorZuoetal.
BMCComplementaryandAlternativeMedicine(2015)15:425Page8of10CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsGY-ZandJHwrotethepaper.
GY-ZandXJ-Zconceivedthestudyandperformedtheexperiments.
JHandYQ-Lparticipateinthedataanalyses.
GC-Wcontributedtomaterials/technicalsupport.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(NSFC81173504)andthesupportingfundofYunnanProvinceofChina(2008PY001).
Authordetails1ResearchCenterforNaturalMedicines,KunmingGeneralHospitalofChengduMilitaryCommand,Kunming650032,China.
2SchoolofPharmacy,KunmingMedicalUniversity,Kunming650032,China.
3SchoolofBasicMedicalSciences,YunnanTraditionalChineseMedicalCollege,Kunming650500,China.
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