activity of specialized biomolecules against gram positive

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Antibioticsȱ2020ȱ9ȱ314ȱdoi103390antibiotics9060314ȱ wwwmdpicomjournalantibioticsȱ

Articleȱ

ActivityȱofȱSpecializedȱBiomoleculesȱagainstȱȱGramȬPositiveȱandȱGramȬNegativeȱBacteriaȱTacircniaȱDȱTavaresȱJoanaȱCȱAntunesȱJorgeȱPadratildeoȱAnaȱIȱRibeiroȱAndreaȱZilleȱȱMȱTeresaȱPȱAmorimȱFernandoȱFerreiraȱandȱHelenaȱPȱFelgueirasȱȱ

CentreȱforȱTextileȱScienceȱandȱTechnologyȱ(2C2T)ȱDepartmentȱofȱTextileȱEngineeringȱUniversityȱofȱMinhoȱCampusȱofȱAzureacutemȱ4800Ȭ058ȱGuimaratildeesȱPortugalȱtaniatav2c2tuminhoptȱ(TDT)ȱjoanaantunes2c2tuminhoptȱ(JCA)ȱpadraoj2c2tuminhoptȱ(JP)ȱafr2c2tuminhoptȱ(AIR)ȱazille2c2tuminhoptȱ(AZ)ȱmtamorimdetuminhoptȱ(MTPA)ȱfnunesdetuminhoptȱ(FF)ȱȱ Correspondenceȱhelenafelgueiras2c2tuminhoptȱTelȱ+351Ȭ253Ȭ510Ȭ283ȱFaxȱ+351Ȭ253Ȭ510Ȭ293ȱ

Receivedȱ11ȱMayȱ2020ȱAcceptedȱ8ȱJuneȱ2020ȱPublishedȱ9ȱJuneȱ2020ȱ

AbstractȱTheȱ increasedȱresistanceȱofȱbacteriaȱagainstȱconventionalȱpharmaceuticalȱsolutionsȱ theȱantibioticsȱhasȱraisedȱseriousȱhealthȱconcernsȱThisȱhasȱstimulatedȱinterestȱinȱtheȱdevelopmentȱofȱbioȬbasedȱtherapeuticsȱwithȱlimitedȱresistanceȱnamelyȱessentialȱoilsȱ(EOs)ȱorȱantimicrobialȱpeptidesȱ(AMPs)ȱThisȱstudyȱenvisagedȱtheȱevaluationȱofȱtheȱantimicrobialȱefficacyȱofȱselectedȱbiomoleculesȱnamelyȱLL37ȱpexigananȱteaȱtreeȱoilȱ(TTO)ȱcinnamonȱleafȱoilȱ(CLO)ȱandȱniaouliȱoilȱ(NO)ȱagainstȱfourȱbacteriaȱcommonlyȱassociatedȱ toȱnosocomialȱ infectionsȱStaphylococcusȱaureusȱStaphylococcusȱepidermidisȱ Escherichiaȱ coliȱ andȱ Pseudomonasȱ aeruginosaȱ Theȱ antibioticȱ vancomycinȱ andȱ silverȱnanoparticlesȱ (AgNPs)ȱ wereȱ usedȱ asȱ controlȱ compoundsȱ forȱ comparisonȱ purposesȱ Theȱbiomoleculesȱwereȱ initiallyȱ screenedȱ forȱ theirȱantibacterialȱefficacyȱusingȱ theȱagarȬdiffusionȱ testȱfollowedȱbyȱtheȱdeterminationȱofȱminimalȱinhibitoryȱconcentrationsȱ(MICs)ȱkillȬtimeȱkineticsȱandȱtheȱevaluationȱofȱ theȱcellȱmorphologyȱuponȱ24ȱhȱexposureȱAllȱagentsȱwereȱeffectiveȱagainstȱ theȱselectedȱbacteriaȱInterestinglyȱtheȱAgNPsȱrequiredȱaȱhigherȱconcentrationȱ(4000Ȭ1250ȱΐgmL)ȱtoȱinduceȱ theȱsameȱeffectsȱasȱ theȱAMPsȱ (500Ȭ78ȱΐgmL)ȱorȱEOsȱ (3652Ȭ197ȱΐgmL)ȱPexigananȱandȱCLOȱwereȱ theȱmostȱ effectiveȱ biomoleculesȱ requiringȱ lowerȱ concentrationsȱ toȱ killȱ bothȱGramȬpositiveȱandȱGramȬnegativeȱbacteriaȱ(625Ȭ78ȱΐgmLȱandȱ393Ȭ197ȱΐgmLȱrespectively)ȱwithinȱaȱshortȱperiodȱofȱtimeȱ(averagingȱ2ȱhȱ15ȱminȱforȱallȱbacteria)ȱMostȱbiomoleculesȱapparentlyȱdisruptedȱtheȱbacteriaȱmembraneȱstabilityȱdueȱtoȱtheȱobservedȱcellȱmorphologyȱdeformationȱandȱbyȱeffectingȱonȱtheȱintracellularȱspaceȱAMPsȱwereȱobservedȱtoȱinduceȱmorphologicalȱdeformationsȱandȱcellularȱcontentȱ releaseȱwhileȱEOsȱwereȱseenȱ toȱ splitȱandȱcompletelyȱenvelopeȱbacteriaȱDataȱunraveledȱmoreȱofȱtheȱpotentialȱofȱ theseȱnewȱbiomoleculesȱasȱreplacementsȱ forȱtheȱconventionalȱantibioticsȱandȱallowedȱusȱtoȱtakeȱaȱstepȱforwardȱinȱtheȱunderstandingȱofȱtheirȱmechanismsȱofȱactionȱagainstȱinfectionȬrelatedȱbacteriaȱ

Keywordsȱantimicrobialȱpeptidesȱessentialȱoilsȱminimumȱ inhibitoryȱconcentrationȱbactericidalȱnosocomialȱȱ

1ȱIntroductionȱ

Bacterialȱgrowthȱcanȱbeȱ inhibitedȱbyȱantimicrobialȱagentsȱcausingȱdisruptionȱofȱvitalȱcellularȱfunctionsȱ resultingȱ inȱ rapidȱ cellȱ deathȱ Typicallyȱ theseȱ agentsȱ actȱ atȱ theȱ levelȱ ofȱ theȱ bacterialȱmembraneȱwhichȱisȱaȱcrucialȱstructureȱforȱcellȱsurvivalȱTheȱarchitectureȱandȱmolecularȱcomponentsȱofȱtheȱcellȱperipheralȱwallȱdifferȱbetweenȱGramȬpositiveȱandȱGramȬnegativeȱbacteriaȱparticularlyȱinȱwhatȱconcernsȱmembraneȱandȱcellȱwallȱstructureȱandȱdispositionȱ[12]ȱTheȱlatterȱisȱmoreȱcomplexȱcontainingȱtwoȱdistinctȱlipidȱmembranesȱtheȱcytoplasmicȱcellȱmembraneȱandȱtheȱouterȱmembraneȱ

Antibioticsȱ2020ȱ9ȱ314ȱ 2ȱofȱ16ȱ

withȱ aȱ thinȱ layerȱ ofȱ peptidoglycansȱ inȱ betweenȱ Theȱ outerȱmembraneȱ worksȱ asȱ anȱ additionalȱcompoundȬselectiveȱbarrierȱ[2]ȱItȱisȱhighlyȱpermeableȱandȱcontainsȱ lipopolysaccharidesȱ(LPS)ȱtheȱmainȱ lipidȱcomponentȱandȱaȱperiplasmicȱ spaceȱwhereȱenzymesȱcapableȱofȱdegradingȱmoleculesȱintroducedȱ fromȱ theȱ extracellularȱmediumȱ areȱpresentȱ [12]ȱ InȱGramȬpositiveȱbacteriaȱ cytosolȱ isȱenvelopedȱ byȱ oneȱ bilayerȱ membraneȱ theȱ cytoplasmicȱ membraneȱ attachedȱ toȱ aȱ thickȱ layerȱ ofȱpeptidoglycansȱformedȱofȱlinearȱpolysaccharideȱchainsȱcrossȬlinkedȱbyȱshortȱpeptidesȱthatȱgenerateȱaȱ threeȬdimensionalȱ (3D)ȱ rigidȱ structureȱ Inȱ thisȱ caseȱ lipoteichoicȱ acidsȱ areȱ adheredȱ toȱ theȱpeptidoglycanȱ layerȱTheseȱcomponentsȱprovideȱtheȱbacterialȱmembraneȱwithȱanȱamphiphilicȱandȱanionicȱcharacterȱ[2ndash4]ȱȱ

Antimicrobialȱagentsȱareȱgenerallyȱlipophilicȱandȱcationicȱallowingȱtheirȱpositivelyȱchargedȱsideȱchainsȱ toȱ bindȱ toȱ theȱ negativelyȱ chargedȱ surfaceȱ ofȱ theȱ bacterialȱmembranesȱ Subsequentlyȱ theȱlipophilicȱmotifsȱinteractȱwithȱtheȱlipidȱbacterialȱmembraneȱleadingȱtoȱinstabilityȱandȱruptureȱofȱtheȱmembraneȱmatrixȱandȱeventuallyȱtoȱtheȱcellȱdeathȱ[156]ȱȱ

CurrentlyȱthereȱisȱaȱvastȱarrayȱofȱantimicrobialȱbiomoleculesȱForȱmanyȱyearsȱtheȱmostȱwidelyȱusedȱhaveȱbeenȱtheȱantibioticsȱHoweverȱtheirȱexcessiveȱconsumptionȱhasȱledȱtoȱanȱalarminglyȱhighȱresistanceȱdevelopmentȱbyȱbacterialȱpathogensȱraisingȱaȱseriousȱglobalȱpublicȬhealthȱproblemȱ[78]ȱHenceȱ theȱ interestȱ inȱ theȱresearchȱ forȱnovelȱalternativesȱ toȱantibioticsȱhasȱbeenȱgrowingȱNaturalȱproductsȱ areȱ becomingȱ veryȱ promisingȱ asȱ antimicrobialȱ agentsȱ beingȱ consideredȱ safeȱ andȱenvironmentallyȱfriendlyȱ[910]ȱAntimicrobialȱpeptidesȱ(AMPs)ȱofȱnaturalȱoriginȱhaveȱbeenȱtheȱfocusȱofȱgreatȱ interestȱasȱalternativesȱ toȱconventionalȱantibioticsȱTheyȱplayȱanȱ importantȱ roleȱ inȱ innateȱimmunityȱprotectingȱ theȱhostȱagainstȱ infectionsȱbyȱmicroorganismsȱAMPsȱareȱoftenȱcationicȱandȱamphiphilicȱmoleculesȱwithȱlowȱmolecularȱweightȱandȱcanȱbeȱobtainedȱfromȱaȱvarietyȱofȱorganismsȱ(egȱ humansȱ insectsȱ amphibianȱ bacteriaȱ etc)ȱ orȱ synthesizedȱ asȱ analogsȱ ofȱ thoseȱ naturallyȱoccurringȱ[11ndash13]ȱLL37ȱforȱinstanceȱisȱanȱAMPȱthatȱbelongsȱtoȱtheȱcathelicidinȱfamilyȱanȱimportantȱantimicrobialȱagentȱinȱhumansȱLL37ȱisȱessentialȱforȱnormalȱinnateȱimmuneȱresponsesȱwithinȱinfectedȱandȱinjuredȱtissuesȱdisplayingȱaȱbroadȱantimicrobialȱactivityȱagainstȱbothȱGramȬpositiveȱandȱGramȬnegativeȱbacteriaȱThisȱAMPȱisȱalsoȱaȱpromoterȱofȱtissueȱregenerationȱ[14ndash16]ȱPexigananȱisȱanȱanalogȱofȱtheȱmagaininȱpeptidesȱisolatedȱfromȱtheȱskinȱofȱtheȱAfricanȱclawedȱfrogȱLikeȱLL37ȱpexigananȱisȱalsoȱ reportedȱ toȱpossessȱaȱbroadȱspectrumȱofȱantibacterialȱactionȱdisplayingȱactivityȱagainstȱoverȱ3000ȱclinicalȱisolatesȱincludingȱmethicillinȬȱandȱgentamicinȬresistantȱStaphylococcusȱaureusȱ[1718]ȱOnȱtheȱ otherȱ handȱ essentialȱ oilsȱ (EOs)ȱ areȱ composedȱ ofȱ plantȱ secondaryȱmetabolitesȱ thatȱ possessȱantibacterialȱantiviralȱandȱantifungalȱactivityȱdefendingȱtheȱhostȱfromȱmicrobiologicalȱinvasionȱ[19]ȱTheseȱconsistȱofȱaȱcomplexȱmixtureȱofȱchemicalȱcompoundsȱincludingȱterpenesȱphenolsȱalcoholsȱaldehydesȱethersȱandȱketonesȱ[2021]ȱmostȱofȱwhichȱareȱhydrophobicȱorȱpartiallyȱsolubleȱinȱwaterȱUsuallyȱEOsȱhaveȱstrongȱlipophilicityȱandȱvolatilityȱ[9]ȱmakingȱthemȱsuitableȱantibacterialȱagentsȱforȱvariousȱapplicationsȱencompassingȱantiȬinflammatoryȱandȱantioxidativeȱpropertiesȱNowadaysȱEOsȱapplicationȱisȱfoundȱinȱmanyȱfieldsȱincludingȱfoodȱpreservationȱcosmeticsȱandȱbiomedicineȱ[22]ȱCinnamonȱ(Cinnamomumȱzeylanicum)ȱisȱaȱwellȬstudiedȱEOȱwithȱvariousȱbiologicalȱpropertiesȱplayingȱaȱ keyȱ roleȱ inȱmaintainingȱhumanȱ healthȱ [23]ȱTheȱ cinnamonȱ leafȱ oilȱ (CLO)ȱ isȱmainlyȱ formedȱ ofȱeugenolȱwhichȱisȱreportedȱasȱtheȱmainȱcompoundȱresponsibleȱforȱitsȱantimicrobialȱpropertiesȱ[2425]ȱLikeȱCLOȱteaȱtreeȱoilȱ(TTO)ȱisȱalsoȱknownȱforȱitsȱgoodȱantimicrobialȱpropertiesȱTTOȱcanȱbeȱobtainedȱfromȱMelaleucaȱ alternifoliaȱ anȱ Australianȱ nativeȱ plantȱ andȱ hasȱ beenȱ incorporatedȱ asȱ anȱ activeȱingredientȱinȱmanyȱtopicalȱformulationsȱtoȱtreatȱcutaneousȱinfectionsȱ[26]ȱTheȱmainȱcompoundȱofȱTTOȱ isȱ terpinenȬ4Ȭolȱbeingȱmainlyȱ responsibleȱ forȱ itsȱantimicrobialȱactivityȱ [2627]ȱNiaouliȱoilȱ isȱextractedȱ fromȱMelaleucaȱ viridifloraȱ aȱperennialȱ treeȱnativeȱ toȱAustraliaȱNewȱCaledoniaȱ andȱ theȱFrenchȱPacificȱIslandsȱThisȱEOȱisȱofȱcommercialȱimportanceȱdueȱtoȱitsȱapplicationsȱinȱaromatherapyȱandȱpharmaceuticalȱpreparationsȱ[2829]ȱItsȱantimicrobialȱactivityȱhasȱalsoȱbeenȱestablishedȱ[3031]ȱAlthoughȱ bothȱ AMPsȱ andȱ EOsȱ haveȱ beenȱ reportedȱ asȱ promisingȱ alternativesȱ toȱ antibioticsȱparticularlyȱ forȱ theirȱ quickȱ actionȱ andȱ lowȱ tendencyȱ toȱ induceȱ resistanceȱ thereȱ isȱ stillȱmuchȱ toȱunderstandȱaboutȱtheirȱmechanismsȱofȱactionȱagainstȱselectedȱmicroorganismsȱ

TheȱpresentȱstudyȱaimedȱtoȱscreenȱandȱfurtherȱdetailȱtheȱantibacterialȱpropertiesȱofȱbothȱAMPsȱandȱEOsȱagainstȱfourȱcommonȱnosocomialȱbacteriaȱSȱaureusȱStaphylococcusȱepidermidisȱEscherichiaȱ


coliȱandȱPseudomonasȱaeruginosaȱprovidingȱanȱoriginalȱandȱcriticalȱoverviewȱonȱtheȱdifferencesȱandȱsimilaritiesȱofȱtheseȱrelativelyȱnovelȱwideȬspectrumȱbactericidalȱcompoundsȱIndeedȱtheȱselectedȱEOsȱproducedȱfromȱFolhaȱdrsquoAacuteguaȱCompanyȱfromȱPortugalȱhaveȱneverȱbeenȱexaminedȱinȱsuchȱlightȱnorȱhaveȱtheirȱantimicrobialȱperformanceȱorȱmechanismsȱofȱactionȱbeenȱcomparedȱwithȱAMPsȱinȱsuchȱdetailȱTheirȱefficiencyȱwasȱaccessedȱinȱlightȱofȱtheirȱminimalȱinhibitoryȱconcentrationȱ(MIC)ȱandȱkillȬtimeȱkineticsȱandȱalsoȱcomprisesȱaȱcriticalȱdiscussionȱofȱtheȱexertedȱeffectȱonȱtheȱbacteriaȱmorphologyȱextrapolatingȱfromȱtheseȱfindingsȱtheirȱinherentȱmechanismsȱofȱactionȱagainstȱtheȱaforementionedȱmicroorganismsȱȱ

2ȱMaterialsȱandȱMethodsȱ

21ȱMaterialsȱ

Commercialȱnanopowderȱofȱsilverȱnanoparticlesȱ (AgNPs)ȱcoatedȱwithȱpolyvinylȱpyrrolidoneȱ(PVP)ȱpossessingȱ20Ȭ30ȱnmȱinȱdiameterȱwereȱacquiredȱfromȱSkySpringȱNanomaterialsȱIncȱ(USA)ȱTheȱantibioticȱvancomycinȱhydrochlorideȱwasȱpurchasedȱfromȱSigmaȱ(USA)ȱTheȱAMPsȱpexigananȱ(Mwȱ 247717ȱ Daȱ GIGKFLKKAKKFGKAFVKILKK)ȱ andȱ LL37ȱ cathelicidinȱ (Mwȱ 44933ȱ DaȱLLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES)ȱwereȱprovidedȱfromȱInnovagenȱABȱ(Sweden)ȱandȱIscaBiochemicalsȱ(UK)ȱrespectivelyȱEOsȱTTOȱ(Uȱ=ȱ0895ȱgcm3ȱextractedȱfromȱMȱalternifolia)ȱCLOȱ (Uȱ=ȱ1049ȱgcm3ȱextractedȱ fromȱCȱzeylanicum)ȱandȱNOȱ (Uȱ=ȱ0913ȱgcm3ȱextractedȱ fromȱMȱviridiflora)ȱwereȱpurchasedȱfromȱFolhaȱdrsquoAacuteguaȱCompanyȱ(Portugal)ȱTrypticaseȱsoyȱbrothȱ(TSB)ȱandȱtrypticaseȱsoyȱagarȱ(TSA)ȱwereȱacquiredȱfromȱVWRȱ(USA)ȱwhileȱMuellerȱHintonȱbrothȱ(MHB)ȱwasȱobtainedȱ fromȱCondaLabȱ (Spain)ȱAllȱ testedȱbacteriaȱwereȱsuppliedȱ fromȱAmericanȱTypeȱCultureȱCollectionȱ (ATCCȱ Spain)ȱ encompassingȱ GramȬpositiveȱ bacteriaȱ Sȱ aureusȱ (ATCCȱ 6538)ȱ andȱ Sȱepidermidisȱ(ATCCȱ35984)ȱandȱGramȬnegativeȱbacteriaȱEȱcoliȱ(ATCCȱ25922)ȱandȱPȱaeruginosaȱ(ATCCȱ25853)ȱȱ

22ȱAntimicrobialȱSolutionsȱPreparationȱ

WaterȱdispersionsȱofȱAgNPsȱwereȱpreparedȱatȱconcentrationsȱrangingȱfromȱ5000ȱtoȱ195ȱΐgmLȱbyȱsonificationȱforȱ30ȱminȱinȱaȱBransonȱ3510ȱbathȱ(355ȱWȱ50ndash60ȱHz)ȱfollowedȱbyȱanotherȱ30ȱminȱinȱanȱOpticȱIvymenȱSytemȱCYȬ500ȱtipȱ(450ȱWȱ20ȱHzȱ80)ȱDispersionȱofȱNPsȱwasȱassessedȱviaȱscanningȱtransmissionȱelectronȱmicroscopyȱ(STEM)ȱusingȱanȱultrahighȬresolutionȱfieldȱemissionȱgunȱscanningȱelectronȱmicroscopeȱ(FEGȬSEMȱNOVAȱ200ȱNanoȱSEMȱFEIȱCompany)ȱSecondaryȱelectronȱimagesȱwereȱobtainedȱatȱanȱ electronȱ acceleratingȱvoltageȱofȱ15ȱkVȱTheȱantibioticȱvancomycinȱ (2000Ȭ195ȱΐgmL)ȱwasȱpreparedȱinȱdistilledȱwaterȱ(dH2O)ȱLL37ȱ(1000Ȭ098ȱΐgmL)ȱwasȱpreparedȱinȱphosphateȱbufferedȱ salineȱ solutionȱ (PBSȱ pHȱ 74)ȱ whileȱ pexigananȱ wasȱ dissolvedȱ inȱ dH2Oȱ atȱ theȱ sameȱconcentrationȱrangeȱEOsȱwereȱdilutedȱinȱMHBȱatȱconcentrationsȱfromȱ500ȱtoȱ018ȱΐgmLȱMaximumȱandȱminimumȱconcentrationsȱwereȱdefinedȱbasedȱonȱtheȱliteratureȱforȱtheseȱorȱsimilarȱcompoundsȱ

Beforeȱcontactȱwithȱtheȱbacteriaȱsuspensionsȱallȱsolutionsȱwereȱvortexedȱforȱ30ȱsȱtoȱguaranteeȱtheȱhomogeneousȱdistributionȱofȱtheȱantimicrobialȱagentsȱ

23ȱAgarȬWellȱDiffusionȱAssayȱ

TheȱantibacterialȱactivityȱofȱtheȱAgNPsȱantibioticȱAMPsȱandȱEOsȱwasȱassessedȱagainstȱtheȱfourȱbacteriaȱafterȱaȱ24ȱhȱincubationȱperiodȱ90ȱmmȱdiameterȱPetriȱdishesȱwereȱpreparedȱwithȱ15ȱmLȱTSAȱandȱ leftȱ toȱsolidifyȱBacteriaȱsuspensionsȱwereȱdilutedȱ inȱTSBȱ toȱaȱconcentrationȱofȱ2ȱtimesȱ106ȱcolonyȱformingȱunitsȱ(CFUs)mLȱAfterwardsȱ200ȹΐLȱofȱeachȱinoculumȱwasȱuniformlyȱspreadȱonȱtheȱsolidȱplatesȱSterilizedȱpunchersȱwereȱusedȱtoȱgenerateȱ6ȱmmȱdiameterȱholesȱonȱtheȱagarȱ40ȱΐLȱofȱeachȱantimicrobialȱagentȱatȱtheȱhighestȱconcentrationȱstudiedȱwereȱ introducedȱ inȱtheȱrespectiveȱholesȱPlatesȱwereȱ incubatedȱ atȱ37ȱ degCȱ forȱ24ȹȱhȱThereafterȱ theȱ zonesȱofȱ inhibitionȱ (ZoI)ȱobservedȱwereȱmeasuredȱtoȱconfirmȱtheȱantimicrobialȱagentsrsquoȱefficacyȱ ȱ


24ȱMinimumȱInhibitoryȱConcentrationsȱ(MICs)ȱ

MICsȱwereȱdeterminedȱusingȱ theȱbrothȱmicrodilutionȱprocedureȱdescribedȱbyȱWiegandȱetȱalȱ[32]ȱwhichȱ isȱ anȱ adaptationȱ ofȱ theȱ standardȱmethodȱ publishedȱ byȱ theȱClinicalȱ andȱ LaboratoryȱStandardsȱ Instituteȱ (CLSI)ȱ andȱ theȱEuropeanȱCommitteeȱ onȱAntimicrobialȱ SusceptibilityȱTestingȱ(EUCAST)ȱ[33]ȱȱ

WorkingȱsolutionsȱwereȱpreparedȱforȱeachȱantimicrobialȱagentȱasȱdescribedȱinȱSectionȱ22ȱandȱaddedȱtoȱtheȱfirstȱcolumnȱofȱ96Ȭwellȱplatesȱinȱaȱvolumeȱofȱ100ȹΐLȱSerialȱdilutionsȱ(12)ȱwereȱmadeȱwithȱMHBȱinȱtheȱconsecutiveȱwellsȱtoȱaȱfinalȱvolumeȱofȱ50ȹΐLȱThenȱtoȱeachȱofȱtheseȱwellsȱ50ȹΐLȱofȱtheȱbacteriaȱ suspensionsȱpreparedȱ atȱ 2ȱ timesȱ 107ȱCFUsmLȱ inȱMHBȱwereȱ addedȱAgentȬfreeȱbacteriaȱsuspensionsȱandȱcultureȱmediaȱwereȱusedȱasȱcontrolsȱAbsorbanceȱreadingsȱatȱaȱwavelengthȱofȱ600ȱnmȱ(EZȱREADȱ2000ȱMicroplateȱReaderȱBiochromȱUK)ȱwereȱmadeȱbeforeȱandȱafterȱplateȱincubationȱforȱ24ȱhȱatȱ37ȱdegCȱandȱ100ȱrpmȱTheȱMICȱvalueȱforȱeachȱagentbacteriaȱcombinationȱwasȱestablishedȱasȱtheȱconcentrationȱatȱwhichȱbacteriaȱdidȱnotȱshowȱanyȱgrowthȱdeterminedȱvisuallyȱandȱconfirmedȱbyȱ theȱ differencesȱ inȱ absorbanceȱ readingsȱ Theȱ numberȱ ofȱ viableȱ cellsȱ atȱ theȱ MICsȱ andȱ theȱconcentrationsȱatȱitsȱvicinityȱ(concentrationȱhigherȱandȱlowerȱthanȱMICȱvalue)ȱwasȱdeterminedȱbyȱestimatingȱtheȱnumberȱofȱCFUȱsmLȱBrieflyȱaliquotsȱofȱ10ȱΐLȱofȱeachȱcellȱsuspensionȱdilutedȱfromȱ101ȱtoȱ106ȱinȱPBSȱwereȱculturedȱinȱTSAȱplatesȱforȱ24ȹhȱatȱ37ȱdegCȱandȱcoloniesȱwereȱcountedȱȱ

25ȱKillȬTimeȱAnalysisȱBacteriaȱViabilityȱ

Bacteriaȱsuspensionsȱwereȱpreparedȱatȱ2ȱtimesȱ107ȱCFUsmLȱinȱMHBȱandȱcombinedȱatȱaȱ50ȱ(vv)ȱwithȱtheȱantimicrobialȱagentsȱatȱMICsȱControlȱgroupsȱwereȱpreparedȱwithoutȱtheȱadditionȱofȱanyȱagentȱAgentbacteriaȱsolutionsȱwereȱincubatedȱatȱ37ȱqCȱandȱ100ȱrpmȱAfterȱ0ȱ(beforeȱagentȱaction)ȱ1ȱ2ȱ4ȱ6ȱ10ȱ14ȱ18ȱandȱ24ȱhȱofȱincubationȱbacteriaȱwereȱseriallyȱdilutedȱ(101ȱtoȱ106ȱinȱPBS)ȱculturedȱonȱTSAȱplatesȱandȱ furtherȱ incubatedȱ forȱanotherȱ24ȱhȱatȱ37ȱ qCȱColoniesȱofȱ survivingȱbacteriaȱwereȱcountedȱ andȱ reportedȱ asȱmeanȱ plusmnȱ standardȱ deviationȱ (SD)ȱDataȱwasȱ collectedȱ inȱ triplicateȱ andȱprocessedȱusingȱtheȱGraphPadȱPrismȱ70ȱsoftwareȱȱ

26ȱCellȱWallȱDisruptionȱScanningȱElectronȱMicroscopyȱ(SEM)ȱObservationsȱ

ToȱhaveȱanȱoverviewȱofȱtheȱantimicrobialȱagentsrsquoȱcapacityȱtoȱinterfereȱwithȱtheȱcellȱmorphologyȱofȱGramȬpositiveȱ andȱGramȬnegativeȱ bacteriaȱ visualȱ studiesȱ resortingȱ toȱ SEMȱwereȱ conductedȱBacteriaȱsuspensionsȱwereȱpreparedȱatȱ2ȱtimesȱ107ȱCFUsmLȱinȱMHBȱandȱcombinedȱatȱaȱ50ȱ(vv)ȱwithȱAgNPsȱantibioticȱAMPsȱandȱEOsȱatȱhalfȱofȱtheȱconcentrationȱofȱtheȱMICȱvalueȱinȱthisȱwayȱallowingȱforȱliveȱandȱdeadȱcellsȱtoȱbeȱobservedȱsimultaneouslyȱandȱdifferencesȱinȱmorphologyȱbeȱidentifiedȱmoreȱeasilyȱ500ȱΐLȱofȱeachȱsolutionȱ(250ȱΐLȱagentȱ+ȱ250ȱΐLȱbacteria)ȱwereȱleftȱinȱdirectȱcontactȱwithȱ12Ȭwellȱtissueȱcultureȱplatesȱ(TCPS)ȱandȱincubatedȱatȱ37ȱqCȱforȱ24ȱhȱatȱ100ȱrpmȱAfterwardsȱcultureȱmediaȱwasȱremovedȱandȱ500ȱΐLȱofȱ25ȱ(vv)ȱglutaraldehydeȱinȱPBSȱwereȱaddedȱtoȱeachȱsampleȱforȱ1ȱhȱatȱroomȱtemperatureȱtoȱpromoteȱcellȱfixationȱtoȱtheȱTCPSȱwellsȱPlatesȱwereȱgentlyȱrinsedȱwithȱdH2Oȱandȱsubmittedȱtoȱaȱdehydrationȱprocessȱusingȱserialȱethanolȱdilutionsȱofȱ55ȱ70ȱ80ȱ90ȱ95ȱandȱ100ȱ(vv)ȱandȱeachȱsolutionȱwasȱleftȱinȱtheȱTCPSȱforȱ30ȱminȱatȱroomȱtemperatureȱandȱthenȱcarefullyȱ discardedȱ Afterȱ theȱ lastȱ solutionȱ theȱ remainingȱ ethanolȱ wasȱ evaporatedȱ atȱ roomȱtemperatureȱ [34]ȱDiscsȱwereȱ cutȱ fromȱ eachȱ TCPSȱwellȱ containingȱ theȱ fixatedȱ andȱ dehydratedȱbacteriaȱusingȱaȱhotȱpressȬonȱapparatusȱandȱcoveredȱwithȱaȱthinȱfilmȱ(10ȱnm)ȱofȱAuȬPdȱ(80Ȭ20ȱwt)ȱinȱ aȱ 208HRȱ highȬresolutionȱ sputterȱ coaterȱ (Cressingtonȱ CompanyȱWatfordȱWD19ȱ 4BXȱ UnitedȱKingdom)ȱ coupledȱ toȱ aȱMTMȬ20ȱCressingtonȱ highȬresolutionȱ thicknessȱ controllerȱBacteriaȱ cellsrsquoȱmorphologyȱwasȱobservedȱviaȱFEGȬSEMȱ(NOVAȱ200ȱNanoȱSEMȱFEIȱCompany)ȱusingȱanȱelectronȱacceleratingȱvoltageȱofȱ10ȱkVȱ ȱ


3ȱResultsȱandȱDiscussionȱ

31ȱAgarȬWellȱDiffusionȱ

InitialȱscreeningȱofȱtheȱantimicrobialȱactivityȱofȱtheȱinvestigatedȱagentsȱwasȱstudiedȱagainstȱtheȱfourȱmicroorganismsȱusingȱtheȱagarȬwellȱdiffusionȱtestȱwhichȱshowedȱtheȱpresenceȱandȱabsenceȱofȱZoIȱ(Tableȱ1)ȱAllȱagentsȱrevealedȱdifferentȱdegreesȱofȱantibacterialȱactivityȱagainstȱtheȱtestedȱbacteriaȱTheȱantibacterialȱactionȱwasȱclassifiedȱfollowingȱtheȱRotaȱetȱalȱscaleȱwhichȱreportsȱweakȱactivityȱwithȱaȱZoIȱ(halo)ȱǂȱ12ȱmmȱmoderateȱactivityȱwithȱaȱZoIȱrangingȱbetweenȱgt12ȱandȱlt20ȱmmȱandȱstrongȱactivityȱwithȱaȱZoIȱzoneȱ ǃȱ20ȱmmȱ [3536]ȱAgNPsȱdisplayȱaȱ lowȬactivityȱZoIȱparticularlyȱagainstȱGramȬnegativeȱbacteriaȱ Itȱhasȱbeenȱ reportedȱ thatȱmetalȱnanoparticlesȱ tendȱ toȱ formȱagglomeratesȱwhenȱinȱcolloidalȱdispersionsȱwhichȱreducesȱtheirȱdiffusivityȱlimitingȱtheȱcontactȱwithȱtheȱbacteriaȱ[37]ȱHereȱtheȱpresenceȱofȱagglomeratesȱ isȱevidentȱ(Figureȱ1)ȱsupportingȱthisȱstatementȱTheȱagarȱtortuosityȱmayȱalsoȱ influenceȱ thisȱphenomenonȱbyȱhinderingȱ theȱAgNPsȱpermeationȱ throughȱ theȱcultureȱmediaȱVancomycinȱantibioticȱactionȱoccursȱatȱtheȱbacterialȱcellȱwallȱthroughȱtheȱdisruptionȱofȱtheȱsynthesisȱofȱitsȱmajorȱconstituentȱpeptidoglycanȱVancomycinȱbindsȱtoȱtheȱterminalȱcarboxylȱgroupȱofȱDȬalanylȬDȬalanineȱwithinȱnascentȱpeptidoglycansȱofȱtheȱcellȱwallȱpreventingȱtheȱformationȱofȱlipidȱIIȱaȱkeyȱldquoshuttleȱcarrierrdquoȱofȱtheȱpeptidoglycanȱmonomersȱ[3839]ȱThisȱmoleculeȱformsȱaȱseriesȱofȱhydrogenȱbondsȱwithȱtheȱpeptideȱbackboneȱblockingȱitsȱprocessingȱ[40ndash42]ȱItȱisȱtrueȱthatȱGramȬnegativeȱ bacteriaȱ outerȱ cellȱwallȱ isȱ fairlyȱ impermeableȱ toȱ largeȱ glycopeptideȱmoleculesȱ suchȱ asȱvancomycinȱ[2]ȱThereforeȱvancomycinȱwasȱreportedȱasȱweakȱagainstȱtheȱGramȬnegativeȱbacteriaȱbutȱstrongȱagainstȱtheȱGramȬpositiveȱbacteriaȱAsȱsuchȱtheȱabsenceȱofȱtheȱouterȱmembraneȱandȱperiplasmȱinȱtheȱGramȬpositiveȱbacteriaȱallowsȱaȱhigherȱpermeabilityȱofȱvancomycinȱthroughȱitsȱcellularȱwallȱwhichȱpossessȱaȱhydrophilicȱporousȱstructureȱ[43ndash45]ȱ

ȱFigureȱ 1ȱ Silverȱ nanoparticlesȱ (AgNPs)ȱ colloidalȱ dispersionȱ scanningȱ transmissionȱ electronȱmicroscopyȱ (STEM)ȱ micrographsȱ atȱ magnificationsȱ ofȱ x100000ȱ andȱ x200000ȱ withȱ evidenceȱ ofȱformationȱofȱNPsȱclustersȱ

AMPsȱLL37ȱandȱpexigananȱareȱwellȬknownȱeffectiveȱantimicrobialȱagentsȱHoweverȱdataȱfromȱTableȱ1ȱrevealedȱaȱweakȱ toȱmoderateȱZoIȱagainstȱ theȱselectedȱbacteriaȱrespectivelyȱTheȱactionȱofȱLL37ȱ isȱ suspectedȱ toȱ haveȱ beenȱ compromisedȱ byȱ theȱ presenceȱ ofȱ saltsȱwithinȱ theȱ solventȱ PBSȱ(recommendedȱ byȱ theȱmanufacturer)ȱ asȱ theȱ LL37ȱ structureȱ variesȱwithȱ theȱ ionicȱ chargeȱ ofȱ theȱsolventsȱ possiblyȱ reducingȱ itsȱ agarȱ diffusionȱ capacityȱ [46]ȱ Onȱ itsȱ turnȱ pexigananȱ actionȱwasȱconsiderablyȱ superiorȱ toȱ thatȱofȱLL37ȱparticularlyȱ againstȱSȱ epidermidisȱ andȱPȱ aeruginosaȱTheseȱfindingsȱareȱconsistentȱwithȱtheȱliteratureȱ[18]ȱȱ

Theȱ EOsȱ displayedȱ aȱ variableȱ degreeȱ ofȱ antibacterialȱ activityȱ againstȱ theȱ testedȱ bacteriaȱInterestinglyȱtheyȱwereȱallȱfoundȱtoȱbeȱmoreȱeffectiveȱagainstȱtheȱSȱaureusȱbacteriaȱwithȱaȱmoderateȱ(NO)ȱtoȱstrongȱ(TTOȱandȱCLO)ȱactivityȱTheseȱresultsȱwereȱcloselyȱfollowedȱbyȱtheȱSȱepidermidisȱandȱEȱcoliȱbacteriaȱPȱaeruginosaȱwasȱtheȱleastȱsusceptibleȱtoȱtheȱEOsȱactionȱwithȱweakȱ(NO)ȱtoȱmoderateȱ(TTOȱandȱCLO)ȱZoIsȱbeingȱformedȱOnceȱagainȱthisȱcanȱbeȱexplainedȱbyȱtheȱdifferencesȱinȱcellȱwallȱstructureȱ betweenȱ GramȬpositiveȱ andȱ GramȬnegativeȱ bacteriaȱ withȱ theȱ latterȱ beingȱ capableȱ ofȱrestrictingȱ diffusionȱ ofȱ hydrophobicȱ compoundsȱ throughȱ itsȱ LPSȱ envelopeȱ [36]ȱ Fromȱ theȱ threeȱ


examinedȱ oilsȱ NOȱ wasȱ theȱ leastȱ effectiveȱ (ƿ16Ȭfoldȱ lowerȱ thanȱ theȱ remainderȱ oils)ȱ Theȱ EOsȱantimicrobialȱactivityȱisȱattributedȱtoȱtheȱpresenceȱofȱseveralȱlowȱmolecularȱweightȱphenolsȱterpenesȱandȱaldoketonesȱwithinȱtheirȱcompositionȱ[47]ȱHenceȱtheȱhigherȱZoIȱformedȱbyȱTTOȱorȱCLOȱcouldȱbeȱ explainedȱ byȱ theȱ presenceȱ ofȱ volatileȱ compoundsȱ TTOȱ encompassesȱ terpinenȬ4Ȭolȱ (40)ȱ Ȭterpineneȱ(20)ȱ΅Ȭterpineneȱ (10)ȱ18Ȭcineoleȱ(3)ȱ΅Ȭpineneȱ (3)ȱandȱ limoneneȱ (1)ȱ(vv)ȱTTOȱcomprisesȱ inȱ itsȱ formulationȱ eugenolȱ (80)ȱ ΆȬcaryophylleneȱ (4)ȱ benzylȱ benzoateȱ (4)ȱcinnamaldehydeȱ(3)ȱlinaloolȱ(2)ȱandȱ΅Ȭterpineneȱ(1)ȱ(vv)ȱAllȱtheseȱcompoundsȱareȱknownȱtoȱcontributeȱ significantlyȱ forȱ theȱ EOsȱ antimicrobialȱ activityȱ [264748]ȱ NOȱ isȱ knownȱ toȱ possessȱterpinenȬ4Ȭolȱ Ȭterpineneȱ ΅Ȭterpineneȱ 18Ȭcineoleȱ ΅Ȭpineneȱ limoneneȱ ΆȬcaryophylleneȱ andȱlinaloolȱbutȱwithȱaȱmajorȱ contributionȱofȱ18Ȭcineoleȱ (60)ȱ (vv)ȱ toȱ itsȱantimicrobialȱactivityȱ18ȬcineoleȱisȱknownȱtoȱexertȱlowerȱantimicrobialȱactionȱthanȱterpinenȬ4Ȭolȱorȱeugenolȱ[2849ndash52]ȱȱ

Tableȱ1ȱZonesȱofȱinhibitionȱ(ZoI)ȱofȱselectedȱantimicrobialȱagentsȱagainstȱGramȬpositiveȱandȱGramȬnegativeȱbacteriaȱ(nȱ=ȱ3ȱmeanȱplusmnȱstandardȱdeviationȱ(SD))ȱTheȱdiameterȱofȱtheȱholesȱ(Oslashȱ=ȱ6ȱmm)ȱwasȱincludedȱ Imagesȱ wereȱ collectedȱ withoutȱ regardȱ forȱ sizeȱ proportionalityȱ onlyȱ beingȱ usedȱ asȱrepresentationsȱofȱtheȱhalosȱformedȱ

AntimicrobialȱAgentsȱ ZoIȱdiameterȱ(mm)ȱSȱaureusȱȱ Sȱepidermidisȱȱ Eȱcoliȱȱ Pȱaeruginosaȱ

AgNPsȱ

115ȱplusmnȱ17ȱ

ȱ

106ȱplusmnȱ06ȱ

ȱ

88ȱplusmnȱ05ȱ

ȱ

88ȱplusmnȱ30ȱ

ȱ

Vancomycinȱ

225ȱplusmnȱ05ȱ

ȱ

225ȱplusmnȱ05ȱ

ȱ

80ȱplusmnȱ01ȱ

ȱ

80ȱplusmnȱ02ȱ

ȱ

LL37ȱ

65ȱplusmnȱ01ȱ

ȱ

65ȱplusmnȱ05ȱ

ȱ

63ȱplusmnȱ01ȱ

ȱ

62ȱplusmnȱ01ȱ

ȱ

Pexigananȱ

90ȱplusmnȱ05ȱ

ȱ

122ȱplusmnȱ06ȱ

ȱ

80ȱplusmnȱ15ȱ

ȱ

120ȱplusmnȱ01ȱ

ȱ

TTOȱ

202ȱplusmnȱ01ȱ

ȱ

150ȱplusmnȱ05ȱ

ȱ

155ȱplusmnȱ05ȱ

ȱ

133ȱplusmnȱ03ȱ

ȱ

CLOȱ

215ȱplusmnȱ05ȱ

ȱ

150ȱplusmnȱ10ȱ

ȱ

150ȱplusmnȱ19ȱ

ȱ

150ȱplusmnȱ06ȱ

ȱ


NOȱ

147ȱplusmnȱ04ȱ

ȱ

100ȱplusmnȱ05ȱ

ȱ

115ȱplusmnȱ05ȱ

ȱ

68ȱplusmnȱ05ȱ

ȱ

32ȱMICsȱ

TheȱobtainedȱMICsȱofȱtheȱselectedȱantimicrobialȱagentsȱforȱeachȱbacteriumȱareȱshownȱinȱTableȱ2ȱMICsȱevaluationȱshowedȱthatȱtheȱselectedȱantimicrobialȱagentsȱwereȱactiveȱagainstȱallȱtestedȱbacteriaȱwhichȱfairlyȱagreesȱwithȱtheȱdataȱobtainedȱfromȱtheȱagarȬwellȱdiffusionȱstudiesȱ

FromȱtheȱentireȱlistȱofȱtestedȱagentsȱtheȱMICsȱofȱtheȱAgNPsȱwereȱtheȱhighestȱ(4000Ȭ1250ȱΐgmL)ȱTheȱmainȱmechanismȱofȱactionȱofȱAgNPsȱagainstȱbacteriaȱrequiresȱtheȱattachmentȱandȱinteractionȱofȱmultipleȱNPsȱtoȱtheȱcellȱsurfaceȱ[53]ȱThisȱinducesȱtheȱdisruptionȱofȱtheȱbacteriaȱmembraneȱfunctionsȱandȱdissipationȱofȱtheȱprotonȱmotiveȱforceȱDueȱtoȱtheirȱhighȱsurfaceȬtoȬvolumeȱratioȱsmallȱAgNPsȱofȱfewȱnanometersȱmayȱevenȱalterȱtheȱmorphologyȱofȱtheȱcellȱwallȱincreasingȱtheirȱdiffusionȱtowardsȱtheȱintracellularȱspaceȱultimatelyȱleadingȱtoȱtheȱcellȱdeathȱ[54]ȱHereȱevenȱthoughȱPVPȱwasȱusedȱasȱaȱdispersantȱ agentȱ toȱproduceȱAgNPsȱ thereȱwasȱ stillȱ aȱ largeȱ tendencyȱ toȱ formȱ agglomeratesȱ inȱcolloidalȱdispersionsȱ(Figureȱ1)ȱConsequentlyȱaȱlargeȱnumberȱofȱNPsȱwereȱexpectedȱtoȱbeȱattractedȱandȱimmobilizedȱalongȱtheȱmembraneȱofȱeachȱbacteriumȱtoȱinduceȱaȱbactericidalȱeffectȱBetweenȱtheȱtestedȱorganismsȱPȱaeruginosaȱwasȱtheȱmostȱsusceptibleȱtoȱtheȱAgNPsȱactionȱItȱhasȱbeenȱpostulatedȱthatȱGramȬnegativeȱbacteriaȱareȱmoreȱsusceptibleȱtoȱAgNPsȱbecauseȱAgNPsȱpositiveȱchargesȱinteractȱwithȱ theȱouterȱLPSȱmembraneȱwithȱmoreȱaffinityȱ thanȱwithȱ theȱGramȬpositiveȱcellȱwallȱwhichȱ isȱthoughtȱtoȱhaveȱfewerȱinteractionȱsitesȱ[55]ȱThisȱeffectȱhoweverȱwasȱnotȱverifiedȱonȱtheȱEȱcoliȱwhichȱMICȱwasȱequalȱtoȱSȱaureusȱandȱSȱepidermidisȱȱ

Asȱ expectedȱ vancomycinȱ wasȱ moreȱ effectiveȱ againstȱ GramȬpositiveȱ thanȱ GramȬnegativeȱbacteriaȱwithȱMICsȱbeingȱ120Ȭfoldȱlowerȱ[2]ȱTheȱpexigananȱMICsȱwereȱalsoȱconsistentȱwithȱtheȱZoIȱfindingsȱbeingȱinȱaȱrangeȱcloseȱtoȱthatȱreportedȱinȱtheȱliteratureȱ[1718]ȱOnȱtheȱcontraryȱtheȱLL37ȱactionȱwasȱfoundȱtoȱbeȱsuperiorȱagainstȱGramȬnegativeȱbacteriaȱevenȱthoughȱtheȱZoIsȱwereȱmoreȱevidentȱagainstȱ theȱGramȬpositiveȱonesȱ (Tableȱ1)ȱAgainȱ theseȱ resultsȱ implyȱ theȱdifficultyȱofȱ theȱpeptideȱinȱdiffusingȱthroughȱtheȱsolidȱmediaȱ[46]ȱRegardingȱtheȱtestedȱbacteriaȱtheȱLL37ȱabilityȱtoȱactȱmoreȱeffectivelyȱagainstȱtheȱEȱcoliȱandȱPȱaeruginosaȱbacteriaȱisȱrelatedȱtoȱitsȱelectrostaticȱinteractionȱandȱitsȱstructureȱItȱhasȱbeenȱreportedȱthatȱLL37sȱfirstȱinteractionȱstepȱisȱpromotedȱbyȱitsȱelectrostaticȱattractionȱtoȱlipidȱAȱandȱtoȱphosphateȱgroupsȱlinkedȱtoȱsugarȱresiduesȱofȱLPSȱ[56]ȱSubsequentlyȱ΅Ȭhelixȱpeptidesȱ suchȱasȱLL37ȱgenerallyȱactȱviaȱaȱmembranolyticȱmechanismȱTheȱhelixȱ formationȱallowsȱanȱoptimalȱspatialȱarrangementȱofȱtheȱaliphaticȱsideȱchainsȱforȱmembraneȱinsertionȱStrongȱhydrophobicȱ interactionsȱ areȱ formedȱbetweenȱ theseȱ chainsȱ andȱ theȱ lipidȱ layerȱofȱGramȬnegativeȱbacteriaȱstabilizingȱ theȱAMPȱhelicalȱconformationȱ thusȱreducingȱmainȬchainȱhydrophobicityȱandȱallowingȱforȱaȱdeeperȱandȱeasierȱinsertionȱintoȱtheȱbilayerȱ[5758]ȱ

AsȱshownȱinȱTableȱ2ȱtheȱEOsȱdisplayedȱvariableȱlevelsȱofȱMICsȱforȱeachȱtestedȱmicroorganismȱCLOȱhadȱtheȱlowestȱMICsȱ(197Ȭ393ȱΐgmL)ȱfromȱtheȱgroupȱwhileȱNOȱhadȱtheȱhighestȱ(1370Ȭ3652ȱΐgmL)ȱTheseȱfindingsȱcorroborateȱtheȱZoIȱexaminationsȱ(Tableȱ1)ȱTheȱEOsȱdifferencesȱinȱchemicalȱcompositionȱandȱpresenceȱofȱmoreȱeffectiveȱlowȱmolecularȱweightȱantibacterialȱcompoundsȱonȱCLOȱthanȱonȱNOȱ exertsȱaȱmajorȱ roleȱ inȱ theirȱantibacterialȱactivityȱ efficacyȱ [47ndash49]ȱToȱ theȱbestȱofȱ theȱauthorsrsquoȱknowledgeȱNOȱhasȱnotȱyetȱbeenȱtestedȱagainstȱtheseȱspecificȱstrainsȱStillȱ inȱotherȱcasesȱMICsȱhaveȱbeenȱreportedȱaroundȱ300ȱandȱ500ȱΐgmLȱ[28]ȱRegardingȱtheȱTTOȱMICsȱevenȱthoughȱtheyȱareȱ slightlyȱ superiorȱ toȱ thoseȱ reportedȱ inȱ theȱ literatureȱ theȱpatternȱofȱefficiencyȱ remainsȱPȱaeruginosaȱltȱSȱepidermidisȱltȱSȱaureusȱltȱEȱcoliȱ[26]ȱȱ ȱ


Tableȱ2ȱMinimalȱ inhibitoryȱconcentrationsȱ (MICs)ȱofȱselectedȱantimicrobialȱagentsȱagainstȱGramȬpositiveȱandȱGramȬnegativeȱbacteriaȱ(nȱ=ȱ3ȱSDȱltȱplusmn50)ȱ

AntimicrobialȱAgentsȱ MICsȱ(ΐgmL)ȱSȱaureusȱȱ Sȱepidermidisȱȱ Eȱcoliȱȱ Pȱaeruginosaȱ

AgNPsȱ 40000ȱ 40000ȱ 40000ȱ 12500ȱVancomycinȱ 78ȱ 78ȱ 10000ȱ 10000ȱLL37ȱ 5000ȱ 5000ȱ 1250ȱ 2500ȱPexigananȱ 313ȱ 78ȱ 625ȱ 313ȱTTOȱ 671ȱ 1790ȱ 336ȱ 2685ȱCLOȱ 262ȱ 262ȱ 197ȱ 393ȱNOȱ 1370ȱ 1826ȱ 1370ȱ 3652ȱ

33ȱKillȬTimeȱAnalysisȱȱ

TheȱkillȬtimeȱkineticsȱforȱeachȱagentȱwasȱdeterminedȱbyȱtheȱnumberȱofȱremainingȱviableȱcellsȱatȱspecificȱtimeȱpointsȱnamelyȱ1ȱ2ȱ4ȱ6ȱ10ȱ14ȱ18ȱ22ȱandȱ24ȱhȱ(Figureȱ2)ȱAlthoughȱMICȱvaluesȱareȱcommonlyȱusedȱ toȱpredictȱ theȱantibacterialȱactionȱofȱanyȱagentȱ suchȱdataȱdoesȱnotȱ considerȱ theȱexposureȱandȱactionȱtimeȱofȱtheȱagentȱagainstȱeachȱbacteriumȱAsȱsuchȱtheȱkillȬtimeȱkineticsȱwasȱusedȱtoȱunravelȱtheȱantibacterialȱpotencyȱofȱtheȱtestedȱcompoundsȱoverȱtimeȱȱ

Forȱ allȱ bacteriaagentȱ combinationsȱ bactericidalȱ actionȱwasȱ observedȱ fromȱ theȱ firstȱ hourȱ ofȱcontactȱInȱfactȱtheȱactionȱofȱtheȱpexigananȱTTOȱCLOȱandȱNOȱwasȱsoȱimmediateȱthatȱafterȱ2ȱhȱofȱcontactȱveryȱlittleȱbacteriaȱremainedȱ(ƿ3ȱtimesȱ104ȱCFUsmLȱofȱSȱaureusȱwithȱTTOȱƿ2ȱtimesȱ104ȱCFUsmLȱofȱPȱaeruginosaȱwithȱCLOȱandȱƿ3ȱtimesȱ104ȱCFUsmLȱofȱSȱaureusȱwithȱNOȱtheȱremainderȱwereȱallȱkilledȱatȱthisȱpoint)ȱTheȱmainȱbactericidalȱactionȱofȱtheȱAMPȱpexigananȱresultsȱfromȱirreversibleȱmembraneȬdisruptiveȱdamageȱwhichȱbasedȱonȱtheȱmechanismȱofȱactionȱofȱmagaininȱ(precursor)ȱisȱexpectedȱtoȱexertȱitsȱantimicrobialȱactionȱveryȱquicklyȱwithinȱtheȱfirstȱmomentsȱofȱinteractionȱ[59]ȱOurȱdataȱisȱconsistentȱwithȱthisȱinformationȱandȱwithȱpreviousȱreportsȱ[17]ȱInȱfactȱallȱbacteriaȱwereȱdeadȱafterȱ1ȱhȱ ofȱ contactȱwithȱ noȱ regrowthȱ beingȱ observedȱwithinȱ theȱ 24ȱ hȱ testedȱ Regardingȱ theȱ EOsȱ theȱsusceptibilityȱpatternȱforȱeachȱbacteriumȱdidȱnotȱappearȱtoȱpredictȱtheȱactivityȱofȱtheȱEOȱForȱinstanceȱevenȱthoughȱTTOȱrequiredȱaȱveryȱsmallȱconcentrationȱtoȱkillȱSȱaureusȱitȱtookȱ6ȱhȱforȱthisȱbacteriumȱtoȱbeȱeliminatedȱwhereasȱTTOȱonlyȱrequiredȱ1ȱhȱtoȱeradicateȱtheȱotherȱbacteriaȱTheȱsameȱoccurredȱwithȱNOȱOnȱitsȱturnȱCLOȱfollowedȱtheȱpatternȱofȱMICȱconcentrationsȱrequiringȱ6ȱhȱtoȱkillȱtheȱPȱaeruginosaȱ andȱ lessȱ thanȱ 1ȱhȱ forȱ theȱotherȱbacteriaȱTheȱEOsȱmechanismȱofȱ actionȱ reliesȱonȱ theirȱinherentȱhydrophobicityȱwhichȱenablesȱ themȱ toȱaccumulateȱ inȱ theȱ cellȱmembraneȱdisturbingȱ itsȱstructureȱandȱfunctionalityȱandȱcausingȱanȱincreaseȱofȱpermeabilityȱ[3660]ȱDespiteȱsharingȱaȱsimilarȱmembraneȱandȱcellȱwallȱstructureȱandȱdispositionȱitȱisȱknownȱthatȱEȱcoliȱandȱPȱaeruginosaȱpossessȱdistinctȱlipidȱandȱproteinȱcompositionȱandȱconcentrationȱ[61]ȱThisȱmostȱlikelyȱisȱtheȱmainȱfactorȱforȱtheȱobservedȱMICȱdifferencesȱbetweenȱtheseȱbacteriaȱEvenȱthoughȱitȱisȱnotȱyetȱclearȱatȱwhichȱstageȱofȱbacteriaȱdevelopmentȱtheȱEOsȱareȱtheȱmostȱeffectiveȱitȱisȱgenerallyȱacceptedȱthatȱtheyȱstimulateȱcellȱ autolysisȱ inȱ exponentialȱ andȱ stationaryȱ cellȱ phasesȱ [62]ȱ Ourȱ findingsȱ demonstrateȱ thatȱexponentiallyȱgrowingȱcellsȱareȱveryȱsusceptibleȱtoȱtheȱEOsȇȱactionȱȱ

InȱcaseȱofȱtheȱLL37ȱtheȱactionȱwasȱquickerȱagainstȱGramȬnegativeȱbacteriaȱcomparedȱtoȱGramȬpositiveȱ onesȱ Itȱ hasȱ beenȱ shownȱ thatȱ permeabilizationȱ ofȱ theȱ cytoplasmicȱmembraneȱ ofȱGramȬpositiveȱbacteriaȱbyȱLL37ȱisȱconsiderablyȱslowerȱthanȱagainstȱGramȬnegativeȱ[5758]ȱInterestinglyȱvancomycinȱalsoȱhadȱaȱfasterȱactionȱagainstȱGramȬnegativeȱbacteriaȱthanȱagainstȱGramȬpositiveȱevenȱthoughȱallȱavailableȱdataȱupȱuntilȱnowȱhasȱrevealedȱitsȱhigherȱeffectivenessȱtowardsȱtheȱformerȱThisȱmayȱhaveȱhappenedȱdueȱtoȱtheȱdifferencesȱinȱMICȱvaluesȱWhileȱSȱaureusrsquoȱandȱSȱepidermidisrsquoȱMICȱwasȱonlyȱ78ȱΐgmLȱ1000ȱΐgmLȱofȱtheȱantibioticȱwasȱnecessaryȱtoȱkillȱEȱcoliȱandȱPȱaeruginosaȱSeveralȱstudiesȱhaveȱshownȱthatȱtheȱbactericidalȱactionȱnamelyȱkillȬtimeȱkineticsȱofȱaȱgivenȱantimicrobialȱagentȱisȱdependentȱonȱtheȱconcentrationȱtoȱwhichȱtheȱmicroorganismsȱareȱexposedȱ[1763]ȱForȱtheȱfourȱbacteriaȱAgNPsȱwasȱtheȱagentȱthatȱtookȱtheȱlongestȱtimeȱtoȱeliminateȱtheȱentiretyȱofȱCFUsȱAsȱseenȱinȱFigureȱ1ȱAgNPsȱclustersȱpreventedȱitsȱhomogenousȱdispersionȱwithinȱtheȱsolutionȱwhichȱimpliesȱthatȱAgNPsȱwereȱnotȱevenlyȱavailableȱtoȱbindȱtoȱsitesȱatȱtheȱbacteriaȱmembraneȱAsȱAgNPsȱ


areȱ onlyȱ capableȱ ofȱ disruptingȱ theȱ bacteriaȱ cellȱ membraneȱ afterȱ properȱ bindingȱ subsequentlyȱinfiltratingȱtheȱcytosolȱtoȱinduceȱcellȱdeathȱ[54]ȱthisȱlimitedȱdistributionȱmayȱhaveȱrequiredȱadditionalȱtimeȱthanȱtheȱfreeȬstateȱnonȬclusteredȱmoleculesȱcharacteristicȱofȱtheȱotherȱtestedȱagentsȱ

ȱFigureȱ2ȱKillȬtimeȱcurvesȱofȱAgNPsȱvancomycinȱLL37ȱpexigananȱTTOȱCLOȱandȱNOȱatȱtheȱMICsȱconcentrationsȱagainstȱ(a)ȱSȱaureusȱ(b)ȱSȱepidermidisȱ(c)ȱEȱcoliȱandȱ(d)ȱPȱaeruginosaȱupȱtoȱ24ȱhȱcultureȱDataȱderivedȱfromȱthreeȱrepetitionsȱPositiveȱcontrolsȱforȱeachȱbacteriumȱ(growthȱwithoutȱagent)ȱwereȱalsoȱconductedȱreachingȱmaximumȱvaluesȱofȱƿ80ȱtimesȱ108ȱcolonyȱformingȱunitsȱ(CFUs)mLȱforȱSȱaureusȱƿ16ȱtimesȱ109ȱCFUsmLȱforȱSȱepidermidisȱƿ14ȱtimesȱ109ȱCFUsmLȱforȱEȱcoliȱandȱƿ87ȱtimesȱ108ȱCFUsmLȱforȱPȱaeruginosaȱafterȱ24ȱhȱcultureȱ(dataȱnotȱshown)ȱ

34ȱCellȬWallȱDisruptionȱMechanismsȱofȱActionȱ

Possibleȱmechanismsȱofȱmembraneȱinteractionȱandȱdisruptionȱofȱtheȱtestedȱbacteriaȱhaveȱbeenȱobservedȱ viaȱ SEMȱ imagingȱ throughȱ expositionȱ toȱ theȱ selectedȱ agentsȱ atȱ halfȱ ofȱ theȱ MICsȱconcentrationsȱforȱ24ȱhȱFigureȱ3ȱshowsȱ theȱmorphologyȱofȱtheȱbacteriaȱwithȱandȱwithoutȱcontactȱwithȱAgNPsȱvancomycinȱandȱselectedȱAMPsȱandȱEOsȱAsȱexpectedȱtheȱcontrolȱ(withoutȱagent)ȱofȱtheȱSȱaureusȱandȱSȱepidermidisȱbacteriaȱrevealedȱaȱcoccoidȬshapedȱconformationȱwithȱaȱsmoothȱandȱuninterruptedȱ surfaceȱ Bothȱ cellȱ typesȱ tendedȱ toȱ beȱ arrangedȱ inȱ grapeȬlikeȱ clustersȱ andȱ cellȱpropagationȱwasȱrecurrentlyȱobservedȱ[6465]ȱTheȱmorphologyȱofȱtheȱEȱcoliȱandȱPȱaeruginosaȱbacteriaȱwasȱalsoȱveryȱsimilarȱwithȱbothȱdisplayingȱaȱrodȬshapedȱarchitectureȱEȱcoliȱcellsȱareȱtypicallyȱ20ȱΐmȱlongȱandȱ025ndash10ȱΐmȱinȱdiameterȱPȱaeruginosaȱcellsȱpresentȱsimilarȱdimensionsȱandȱinȱmanyȱcasesȱpolarȱ flagellaȱwhichȱendowsȱ theȱbacteriaȱwithȱmotilityȱmayȱalsoȱbeȱevidentȱ [66ndash68]ȱHereȱhoweverȱ thatȱ wasȱ notȱ theȱ caseȱ GramȬnegativeȱ controlȱ cellsȱ presentedȱ anȱ evenȱ distributionȱdisplayingȱmultipleȱcellsȱundergoingȱpolarȱbinaryȱfissionȱ


ThereȱareȱtwoȱmechanismsȱofȱactionȱwidelyȱacceptedȱforȱAgNPsȱtheȱcontactȱkillingȱandȱtheȱionȬmediatedȱkillingȱContactȱkillingȱisȱclearlyȱevidencedȱinȱallȱtestedȱmicroorganismsȱ(Figureȱ3b)ȱTheȱpositivelyȱ chargedȱAgNPsȱ areȱ attractedȱ toȱ theȱ negativelyȱ chargedȱ bacteriaȱ surfaceȱ enablingȱNPȱattachmentȱalongȱtheȱcellȱsurfaceȱThisȱactionȱinducesȱphysicalȱchangesȱinȱtheȱbacterialȱmembraneȱcompromisingȱitsȱintegrityȱandȱinducingȱtheȱdiffusionȱofȱNPsȱtowardsȱtheȱintracellularȱspaceȱHereȱAgNPsȱspeciesȱsuchȱasȱAg+ȱionsȱareȱreleasedȱandȱinteractȱeffectivelyȱwithȱspecificȱfunctionalȱgroupsȱinȱ proteinsȱ consequentlyȱ inhibitingȱ intracellularȱ metabolicȱ functionsȱ andȱ causingȱ proteinȱdenaturationȱAtȱthisȱpointȱtheȱcellularȱcontentȱwillȱleakȱultimatelyȱleadingȱtoȱtheȱcellȱdeathȱ[6970]ȱThisȱeffectȱisȱparticularlyȱevidentȱagainstȱtheȱPȱaeruginosaȱbacteriaȱasȱtheȱrodȬshapedȱmorphologyȱisȱbarelyȱ evidentȱ inȱ mostȱ cellsȱ andȱ theȱ cellȱ contentȱ isȱ alreadyȱ fusedȱ withȱ theȱ AgNPsȱ clustersȱAdditionallyȱAgNPsȱareȱalsoȱcapableȱofȱproducingȱhighȱlevelsȱofȱreactiveȱoxygenȱspeciesȱ(ROS)ȱandȱfreeȱradicalȱspeciesȱthatȱmayȱinteractȱelectrostaticallyȱwithȱtheȱcellȱwallȱgeneratingȱaȱchargeȱsuperiorȱtoȱitsȱtensileȱstrengthȱthereforeȱalsoȱcompromisingȱitsȱintegrityȱ[71]ȱȱ

DirectȱinhibitionȱofȱtheȱAtlȱamidaseȱdomainȱ(majorȱdomainȱinȱstaphylococcusȱbacteriaȱcellȱwall)ȱdueȱtoȱvancomycinȬinducedȱinhibitionȱofȱcellȱwallȱsynthesisȱcausesȱdefectsȱinȱtheȱcellȱmorphologyȱandȱ altersȱ cellȱmembraneȱ permeabilityȱ (Sȱ aureusȱ inȱ Figureȱ 3c)ȱ ultimatelyȱ leadingȱ toȱ autolysinȬtriggeredȱcellȱ ruptureȱreleaseȱofȱcellȱcontentȱandȱdeathȱ (Sȱ epidermidisȱ inȱFigureȱ3c)ȱ [40ndash42]ȱEvenȱthoughȱ theȱmodeȱofȱ actionȱ againstȱSȱ aureusȱ andȱSȱ epidermidisȱ isȱ similarȱFigureȱ 3cȱ recordedȱ theȱalterationsȱinducedȱbyȱvancomycinȱatȱtwoȱstagesȱanȱearlierȱforȱSȱaureusȱandȱaȱmoreȱadvancedȱforȱSȱepidermidisȱThisȱoccursȱbecauseȱvancomycinȱ requiresȱmoreȱ timeȱ toȱkillȱ theȱ firstȱbacteriaȱ thanȱ theȱsecondȱ (Figureȱ 2)ȱ thusȱ allowingȱ forȱ theȱ extrusionȱ andȱ reductionȱ ofȱ cellȱ contentȱ toȱ occurȱmoreȱintensivelyȱ inȱ theȱSȱ epidermidisȱuponȱ24ȱhȱexposureȱTheȱ sameȱexplanationȱcanȱbeȱappliedȱ toȱ theȱGramȬnegativeȱ bacteriaȱ Theȱ largeȱ sizeȱ ofȱ thisȱ glycopeptideȱ precludesȱ itȱ fromȱ beingȱ capableȱ ofȱpenetratingȱtheȱouterȱmembraneȱofȱGramȬnegativeȱbacteriaȱandȱinducingȱmorphologyȱchangesȱandȱautolysinȬtriggeredȱ cellȱ ruptureȱasȱhappensȱ inȱGramȬpositiveȱbacteriaȱ [43ndash45]ȱWeȱpostulateȱ thatȱbecauseȱofȱtheȱsuperiorȱconcentrationȱofȱvancomycinȱ(1000ȱΐgmL)ȱrequiredȱtoȱkillȱtheseȱbacteriaȱandȱitsȱfastȱactionȱtheseȱmoleculesȱaccumulateȱalongȱtheȱsurfaceȱofȱtheȱbacteriumȱisolatingȱitȱfromȱtheȱmediaȱandȱrespectiveȱnutrientsȱandȱprovidingȱenoughȱstericȱhindranceȱ toȱpreventȱpeptidoglycanȱsynthesisȱHenceȱ starvingȱ theȱ bacteriaȱmayȱ triggerȱ aȱ setȱ ofȱ eventsȱ somewhatȱ similarȱ toȱ thoseȱcharacteristicsȱofȱtheȱGramȬpositiveȱbacteriaȱthatȱculminateȱinȱcellȱruptureȱreleaseȱofȱcellȱcontentȱandȱdeathȱAsȱtheȱkillȬtimeȱkineticsȱisȱsoȱfastȱafterȱ24ȱhȱexposureȱitȱwasȱonlyȱpossibleȱtoȱcaptureȱfragmentsȱofȱindividualȱcellȱmembranesȱandȱresiduesȱofȱcellȱcontentȱforȱEȱcoliȱandȱaȱveryȱadvancedȱdeformedȱmorphologyȱforȱPȱaeruginosaȱȱ

AMPsȱ areȱ uniqueȱ biomoleculesȱ whichȱmodeȱ ofȱ actionȱmayȱ beȱ dividedȱ intoȱ directȱ killingȱ(membraneȱandȱnonȬmembraneȱtarget)ȱandȱimmuneȱmodulationȱLL37ȬtreatedȱSȱaureusȱdisplayedȱclearȱ irregularȱ protrudingȱ structuresȱ toȱ anȱ extentȱ thatȱ atȱ leastȱ someȱ bacteriaȱ appearedȱ toȱ haveȱextrudedȱcytoplasmȱandȱbecomeȱembeddedȱbyȱexudateȱInȱtheȱcaseȱofȱSȱepidermidisȱmorphologicalȱchangesȱwereȱeasilyȱdistinguishedȱwithȱaȱsmallȱleakageȱofȱcytoplasmȱcontentȱalsoȱbeingȱperceivedȱwithinȱtheȱbacteriaȱclusterȱLL37ȱperformsȱitsȱbactericidalȱactionȱbyȱelectrostaticȱbindingȱofȱitsȱcationicȱmoleculesȱtoȱtheȱouterȱsurfaceȱofȱtheȱbacterialȱcellȱThisȱpeptideȱusesȱtheȱcarpetȬlikeȱmechanismȱinȱwhichȱ theȱAMPȱ coatȱ theȱmicrobialȱmembraneȱupȱ toȱ saturationȱafterȱwhichȱpointȱwormholesȱareȱformedȱorȱtheȱtoroidalȱmechanismȱofȱactionȱinȱwhichȱafterȱbindingȱtoȱtheȱphospholipidȱheadȱgroupsȱtheȱAMPȱalignsȱandȱinsertsȱintoȱtheȱmembraneȱandȱclusterȱintoȱunstructuredȱbundlesȱthatȱspanȱtheȱmembraneȱ andȱ generateȱ channelsȱ fromȱ eachȱ ofȱ theȱ intracellularȱ contentȱ leaksȱ [1214]ȱ LL37ȱ isȱamphiphilicȱinȱnatureȱwithȱhydrophobicȱandȱhydrophilicȱresiduesȱalignedȱonȱoppositeȱsidesȱofȱtheȱpeptideȱThisȱfacilitatesȱtheirȱpenetrationȱthroughȱtheȱcellȱmembraneȱ[72]ȱwhichȱresultsȱinȱinhibitionȱofȱ nucleicȱ acidȱ andȱ proteinȱ biosynthesisȱ followedȱ byȱ leakageȱ ofȱ theȱ cellȱ cytoplasmȱ intoȱ theȱextracellularȱspaceȱcausingȱbacteriaȱdeathȱ[73]ȱAlthoughȱtheȱactionȱofȱLL37ȱagainstȱGramȬnegativeȱbacteriaȱisȱveryȱsimilarȱtoȱthatȱdescribedȱagainstȱGramȬpositiveȱbacteriaȱtheȱrateȱatȱwhichȱcytoplasmicȱpermeabilizationȱ occursȱ isȱ superiorȱ Theȱ peptideȱ ΅Ȭhelixȱ structureȱ formsȱ strongȱ hydrophobicȱinteractionsȱwithȱ theȱ outerȱmembraneȱ andȱ itsȱ LPSȱ andȱ OȬantigenȱ layersȱ ofȱ theȱ GramȬnegativeȱbacteriaȱwhichȱquicklyȱsaturatesȱ thusȱallowingȱ forȱaȱdeeperȱandȱ fasterȱ insertionȱ intoȱ theȱbilayerȱ


[5758]ȱ Theȱ haltingȱ ofȱ growthȱ occursȱ shortlyȱ afterȱ theȱ translocationȱ ofȱ LL37ȱ acrossȱ theȱ outerȱmembraneȱintoȱtheȱperiplasmicȱspaceȱandȱisȱfollowedȱbyȱtheȱrapidȱinterferenceȱwithȱtheȱsynthesisȱofȱtheȱnascentȱcurvedȱcellȱenvelopeȱ(theȱouterȱmembraneȱcytoplasmicȱmembraneȱpeptidoglycanȱlayerȱandȱLPSȱlayer)ȱandȱitsȱintracellularȱorganellesȱ[74]ȱTheseȱphenomenaȱmayȱexplainȱtheȱmoreȱadvancedȱstateȱofȱdeformationdecompositionȱ registeredȱbyȱ theȱEȱ coliȱ andȱPȱ aeruginosaȱbacteriaȱ afterȱ 24ȱhȱexposureȱtoȱtheȱLL37ȱ(Figureȱ3d)ȱHereȱaȱsubstantialȱdecreaseȱinȱsignalȱintensityȱcorrelatesȱtoȱcellsȱbeingȱdepletedȱofȱintracellularȱorganellesȱinȱaȱbedȱofȱorganicȱmatterȱ(veryȱeasilyȱidentifiedȱforȱEȱcoli)ȱJustȱasȱLL37ȱpexigananȱalsoȱexertȱitsȱantibacterialȱeffectȱbyȱformingȱtoroidalȱporesȱinȱtheȱbacterialȱmembraneȱ[75]ȱTheȱcationicȱAMPȱwithȱdivalentȱcationȱbindingȱsitesȱdisruptsȱtheȱarrangementȱofȱtheȱhydrophobicȱandȱhydrophilicȱ sectionsȱofȱ theȱbilayerȱ inducingȱaȱ localȱ curvatureȱwhichȱaltersȱ theȱmorphologicalȱ appearanceȱ ofȱ theȱ cellȱ (evidentȱ inȱ allȱ bacteriaȱ fromȱ Figureȱ 3e)ȱAsȱ theȱ poresȱ areȱtransientȱuponȱdisintegrationȱpexigananȱcanȱtranslocateȱtoȱtheȱinnerȱcytoplasmicȱleafletȱenteringȱtheȱcytoplasmȱandȱpotentiallyȱtargetingȱintracellularȱcomponentsȱ[1776]ȱAsȱtheȱantimicrobialȱactionȱofȱAMPsȱincludingȱpexigananȱisȱrelatedȱtoȱitsȱavailabilityȱbacteriaȱexposedȱtoȱaȱhigherȱconcentrationȱofȱpexigananȱwereȱmoreȱproneȱ toȱdisintegrateȱ andȱ releaseȱ theirȱ cellularȱ contentȱduringȱ theȱ 24ȱhȱcontactȱThisȱwasȱparticularlyȱclearȱonȱtheȱEȱcoliȱforȱwhichȱMICȱwasȱtheȱhighestȱ(625ȱΐgmL)ȱAsȱobservedȱAMPȱkillsȱbacteriaȱveryȱquicklyȱwithinȱtheȱfirstȱ2ȱhȱofȱexposureȱ(Figureȱ2)ȱbyȱphysicallyȱdisruptingȱ theȱ cellȱmembraneȱwhichȱ isȱ aȱ highlyȱ conservedȱ structureȱ thusȱ theȱ developmentȱ ofȱresistanceȱmayȱ notȱ beȱ anȱ immediateȱ concernȱwhichȱ potentiatesȱ furtherȱ researchȱ intoȱ itsȱ clinicalȱapplicationȱ[7778]ȱInȱfactȱallȱmutagenesisȱattemptsȱtoȱinduceȱpexigananȱresistanceȱinȱEȱcoliȱandȱSȱaureusȱfailedȱ[79]ȱ

Itȱ isȱgenerallyȱacceptedȱ thatȱEOsȱactȱprimarilyȱagainstȱ theȱcellȱcytoplasmicȱmembraneȱofȱ theȱmicroorganismȱTheirȱ inherentȱhydrophobicityȱ isȱanȱ importantȱcharacteristicȱ thatȱenablesȱ themȱ toȱaccumulateȱwithinȱ theȱ cellȱmembraneȱdisturbingȱ itsȱ structureȱ andȱ functionalityȱ andȱ causingȱ anȱincreaseȱofȱpermeabilityȱLeakageȱofȱintracellularȱcomponentsȱandȱimpairmentȱofȱmicrobialȱfunctionsȱcanȱ thenȱoccurȱultimatelyȱ causingȱcellȱdeathȱ [3660]ȱEvenȱ thoughȱ inȱSȱaureusȱ theȱactionȱofȱTTOȱappearedȱ toȱ haveȱ onlyȱ compromisedȱ theȱ cellȱwallȱwithȱ littleȱ cellȱ contentȱ beingȱ releasedȱ inȱ Sȱepidermidisȱitsȱeffectȱisȱveryȱpronouncedȱwithȱtheȱcompleteȱdisintegrationȱofȱtheȱcellȱmembraneȱandȱsubstantialȱ leakageȱ toȱ theȱextracellularȱ spaceȱHereȱcellȱ lysisȱ isȱ clearȱThisȱdifferenceȱ inȱbehaviorȱbetweenȱstaphylococcusȱbacteriaȱmayȱbeȱcorrelatedȱwithȱ theȱkillȬtimeȱkineticsȱofȱ theȱEOȱWhileȱSȱaureusȱwithstandsȱviableȱcellsȱforȱ6ȱhȱtheȱotherȱtestedȱmicroorganismsȱwereȱallȱeliminatedȱwithinȱ1ȱhȱ Inȱ factȱ disintegrationȱ ofȱ theȱ cellȱwallȱ leakageȱ ofȱ intracellularȱ componentsȱ andȱ cellȱ lysisȱ areȱespeciallyȱnoticeableȱinȱEȱcoliȱandȱPȱaeruginosaȱ(Figureȱ3f)ȱAnotherȱexplanationȱreliesȱonȱtheȱTTOȱactionȱmodeȱ againstȱ Sȱ aureusȱDataȱ suggestsȱ thatȱ theȱ primaryȱmechanismȱ ofȱ actionȱ againstȱ thisȱbacteriumȱmayȱnotȱbeȱjustȱgrossȱcellȱwallȱdamageȱasȱitȱhappensȱwithȱtheȱotherȱbacteriaȱbutȱratherȱaȱcombinationȱofȱtheȱweakeningȱofȱtheȱcellȱwallȱandȱsubsequentȱruptureȱofȱtheȱcytoplasmicȱmembraneȱdueȱ toȱ osmoticȱ pressureȱ withȱ theȱ impairmentȱ ofȱ microbialȱ autolyticȱ enzymeȱ systemsȱ whichȱeventuallyȱ induceȱ aȱ delayedȱ deathȱ [5051]ȱ Obviousȱ detrimentalȱ effectsȱ onȱ theȱ cellȱ membraneȱmorphologyȱwereȱ alsoȱ shownȱwhenȱ bacteriaȱwereȱ treatedȱwithȱ CLOȱ andȱNOȱMicrostructuralȱobservationsȱ demonstratedȱ theseȱ EOsrsquoȱ capacityȱ toȱ increaseȱ cellȱ permeabilityȱ distortingȱ theȱ cellȱmembraneȱintegrityȱandȱgeneratingȱholesȱorȱwrinklesȱTheȱlatterȱwereȱparticularlyȱclearȱonȱtheȱGramȬnegativeȱ bacteriaȱ possiblyȱ dueȱ toȱ theirȱ outerȱ cellȱmembraneȱ andȱ thinȱ peptidoglycanȱwallȱ Anȱincompleteȱ andȱ deformedȱ shapeȱ wasȱ observedȱ inȱ someȱ Sȱ aureusȱ andȱ Sȱ epidermidisȱ cellsȱ Cellȱshrinkageȱ andȱ blebbingȬlikeȱ architecturesȱ wereȱ alsoȱ detectedȱ amongȱ theseȱ microorganismsȱInterestinglyȱintracellularȱleakageȱwasȱonlyȱobservedȱonȱSȱaureusȱevenȱthoughȱruptureȱandȱlysisȱofȱmembranesȱwithȱaȱldquobreakingȬinȬhalfrdquoȬlikeȱdeformationȱwasȱpredominantȱ inȱSȱepidermidisȱCLOȱ isȱcomposedȱofȱ80ȱeugenolȱandȱitsȱantibacterialȱactionȱcanȱbeȱattributedȱtoȱaȱdoubleȱbondȱinȱtheȱ΅ΆȱpositionsȱofȱtheȱsideȱchainȱandȱaȱmethylȱgroupȱlocatedȱinȱtheȱȱpositionȱTypicallyȱeugenolȱexhibitsȱhigherȱactivityȱagainstȱGramȬnegativeȱbacteriaȱthanȱGramȬpositiveȱbacteriaȱ[50]ȱDeformationȱofȱtheȱbacterialȱcellȱwallȱofȱGramȬnegativeȱbacteriaȱisȱevidentȱuponȱexposureȱtoȱCLOȱItȱappearsȱthatȱtheȱEOȱsurroundsȱtheȱcellsȱisolatingȱthemȱforȱanȱeffectiveȱpermeabilizationȱofȱtheȱmembraneȱIndeedȱsinceȱtheseȱ bacteriaȱ areȱ relativelyȱ moreȱ resistantȱ toȱ hydrophobicȱ biomoleculesȱ toȱ overcomeȱ theirȱ


impermeabilityȱEOsȱrelyȱonȱtheȱorganismsȱisolationȱtoȱslowlyȱtraverseȱthroughȱtheȱouterȱwallȱporinsȱ[5080]ȱThisȱphenomenonȱisȱalsoȱevidentȱonȱtheȱGramȬnegativeȱbacteriaȱtreatedȱwithȱNOȱStillȱtheȱdifferencesȱinȱconcentrationȱbetweenȱCLOȱandȱNOȱnecessaryȱtoȱaccomplishȱsuchȱaȱtaskȱ(Tableȱ2)ȱmayȱbeȱaccompaniedȱbyȱaȱdifferentȱmechanismȱofȱactionȱagainstȱtheseȱbacteriaȱAsȱtheȱconcentrationȱofȱphenolicȱcompoundsȱinȱNOȱisȱsmallerȱthanȱonȱCLOȱ[47ndash49]ȱtheȱfirstȱmayȱrelyȱonȱtheȱinterferenceȱwithȱ enzymesȱ involvedȱ inȱ theȱ productionȱ ofȱ energyȱ toȱ induceȱ cellȱ lysisȱwhileȱ theȱ secondȱmayȱdenatureȱproteinsȱpresentȱatȱtheȱintracellularȱspaceȱ

ȱFigureȱ3ȱMicrographsȱofȱSȱaureusȱ(Sa)ȱSȱepidermidisȱ(Se)ȱEȱcoliȱ(Ec)ȱandȱPȱaeruginosaȱ(Pa)ȱbacteriaȱuntreatedȱ (control)ȱ andȱ treatedȱ withȱ theȱ selectedȱ antibacterialȱ agentsȱ atȱ theȱ smallestȱ testedȱconcentrationȱjustȱbeforeȱestablishingȱtheȱMICȱvalueȱConcentrationsȱwereȱdefinedȱatȱ2000ȱΐgmLȱinȱSaȱSeȱandȱEcȱandȱ625ȱΐgmLȱinȱPaȱforȱAgNPsȱ39ȱΐgmLȱinȱSaȱandȱSeȱandȱ500ȱΐgmLȱinȱEcȱandȱPaȱforȱvancomycinȱ250ȱΐgmLȱinȱSaȱandȱSeȱ625ȱΐgmLȱinȱEcȱandȱ125ȱΐgmLȱinȱPaȱforȱLL37ȱ157ȱΐgmLȱinȱSaȱandȱPaȱ39ȱΐgmLȱinȱSeȱandȱ313ȱΐgmLȱinȱPaȱforȱpexigananȱ336ȱΐgmLȱinȱSaȱ895ȱΐgmLȱinȱSeȱ168ȱΐgmLȱinȱEcȱandȱ1343ȱΐgmLȱinȱPaȱforȱTTOȱ157ȱΐgmLȱinȱSaȱandȱSeȱ99ȱΐgmLȱinȱEcȱandȱ197ȱΐgmLȱinȱPaȱforȱCLOȱandȱ685ȱΐgmLȱinȱSaȱandȱEcȱ913ȱΐgmLȱinȱSeȱandȱ1826ȱΐgmLȱinȱPaȱforȱNOȱ Theseȱ concentrationsȱ allowedȱ forȱ liveȱ andȱ deadȱ cellsȱ toȱ beȱ observedȱ simultaneouslyȱwithȱmorphologicalȱdifferencesȱbeingȱmoreȱeasilyȱidentified4ȱConclusionsȱ

TheȱwideȬspectrumȱantibacterialȱefficacyȱofȱAMPsȱandȱEOsȱwasȱfurtherȱthoroughlyȱanalyzedȱinȱthisȱworkȱTheȱ testedȱAMPsȱLL37ȱ andȱpexigananȱdisplayedȱ efficacyȱ againstȱ theȱ testedȱ bacteriaȱNeverthelessȱ inȱ comparisonȱ toȱ LL37ȱ pexigananȱ exhibitedȱ aȱ considerablyȱ lowerȱMICȱ (rangingȱbetweenȱ 2Ȭȱ andȱ 64Ȭfoldȱ lower)ȱ inȱ additionȱ toȱ itsȱ impressiveȱ killȬtimeȱ againstȱ allȱ bacteriaȱ (ǂ1ȱ h)ȱwhereasȱLL37ȱonlyȱexhibitedȱsuchȱkillingȱrateȱagainstȱGramȬnegativeȱbacteriaȱInterestinglyȱtheȱLL37ȱantimicrobialȱactionȱwasȱapparentlyȱhinderedȱbyȱtheȱagarȱtortuosityȱthusȱdisplayingȱaȱlimitationȱinȱitsȱapplicationȱTheȱmostȱeffectiveȱEOȱwasȱCLOȱexhibitingȱaȱlowerȱMICȱthanȱTTOȱ(betweenȱ17Ȭȱandȱ68Ȭfold)ȱandȱNOȱ(betweenȱ52Ȭȱandȱ93Ȭfold)ȱandȱaȱfastȱkillȬtimeȱ1ȱhȱforȱGramȬpositiveȱbacteriaȱandȱEȱ coliȱandȱ6ȱhȱ forȱPȱaeruginosaȱTheȱmainȱ targetȱofȱmostȱofȱ theȱ testedȱagentsȱwasȱcellȱenvelopeȱhighlightingȱ bothȱ theirȱwideȬspectrumȱ potentialȱ andȱ thatȱ theyȱ areȱ notȱ proneȱ toȱ rapidlyȱ induceȱbacterialȱresistanceȱTheseȱpropertiesȱmakeȱthemȱhighlyȱvaluableȱbiomoleculesȱforȱtheȱurgentȱldquobiocideȱtransitionrdquoȱtoȱreduceȱtheȱneedȱandȱuseȱofȱnonȬeffectiveȱconventionalȱantibioticsȱThisȱisȱaȱfirstȱstepȱinȱaȱlargerȱinvestigationȱinȱwhichȱtheȱcompetitiveȱandȱsynergisticȱbehaviorȱofȱtheseȱbiomoleculesȱandȱtheirȱaffinityȱ towardsȱbiodegradableȱ fibrousȱconstructsȱwillȱbeȱ theȱenvisagedȱgoalsȱResearchȱwillȱsoonȱbeȱpublishedȱonȱtheseȱsubjectsȱ

AuthorȱContributionsȱConceptualizationȱTDTȱJCAȱJPȱAIRȱandȱHPFȱmethodologyȱTDTȱJPȱandȱHPFȱvalidationȱTDTȱ JCAȱ JPȱAIRȱandȱHPFȱdiscussionȱofȱresultsȱTDTȱ JCAȱ JPȱAIRȱAZȱMTPAȱFFȱandȱHPFȱwritingmdashoriginalȱdraftȱTDTȱsupervisionȱAZȱMTPAȱFFȱandȱHPFȱfundingȱ


acquisitionȱAZȱMTPAȱFFȱandȱHPFȱAllȱauthorsȱhaveȱreadȱandȱagreedȱtoȱtheȱpublishedȱversionȱofȱtheȱmanuscriptȱ

FundingȱThisȱresearchȱreceivedȱfundingȱfromȱtheȱPortugueseȱFoundationȱforȱScienceȱandȱTechnologyȱ(FCT)ȱunderȱ theȱ scopeȱ ofȱ theȱ projectsȱ PTDCCTMȬTEX280742017ȱ (POCIȬ01Ȭ0145ȬFEDERȬ028074)ȱ PTDCCTMȬTEX282952017ȱandȱUIDCTM002642020ȱ

AcknowledgmentsȱTheȱauthorsȱacknowledgeȱtheȱPortugueseȱFoundationȱforȱScienceȱandȱTechnologyȱ(FCT)ȱFEDERȱfundsȱbyȱmeansȱofȱPortugalȱ2020ȱCompetitiveȱFactorsȱOperationalȱProgramȱ(POCI)ȱandȱtheȱPortugueseȱGovernmentȱ(OE)ȱforȱfundingȱtheȱprojectsȱPEPTEXȱwithȱreferenceȱPTDCCTMȬTEX280742017ȱ(POCIȬ01Ȭ0145ȬFEDERȬ028074)ȱandȱPLASMAMEDȱwithȱreferenceȱPTDCCTMȬTEX282952017ȱTheȱauthorsȱalsoȱacknowledgeȱprojectȱUIDCTM002642020ȱofȱtheȱCentreȱforȱTextileȱScienceȱandȱTechnologyȱ(2C2T)ȱfundedȱbyȱnationalȱfundsȱthroughȱFCTMCTESȱ

ConflictsȱofȱInterestȱTheȱauthorsȱdeclareȱnoȱconflictȱofȱinterestȱ

Referencesȱ

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18 MonteiroȱCȱFernandesȱMȱPinheiroȱMȱMaiaȱSȱSeabraȱCLȱFerreiraȬdaȬSilvaȱFȱCostaȱFȱReisȱSȱGomesȱPȱMartinsȱMCLȱAntimicrobialȱpropertiesȱofȱmembraneȬactiveȱdodecapeptidesȱderivedȱ fromȱMSIȬ78ȱBiochimȱBiophysȱActaȱ2015ȱ1848ȱ1139ndash1146ȱ

19 TavaresȱTDȱAntunesȱJCȱFerreiraȱFȱFelgueirasȱHPȱBiofunctionalizationȱofȱNaturalȱFiberȬReinforcedȱBiocompositesȱforȱBiomedicalȱApplicationsȱBiomoleculesȱ2020ȱ10ȱ148ȱ

20 ManȱAȱSantacroceȱLȱIacobȱRȱMareȱAȱManȱLȱAntimicrobialȱactivityȱofȱsixȱessentialȱoilsȱagainstȱaȱgroupȱofȱhumanȱpathogensȱAȱcomparativeȱstudyȱPathogensȱ2019ȱ8ȱ15ȱ

21 SwamyȱMKȱAkhtarȱMSȱSinniahȱURȱAntimicrobialȱpropertiesȱofȱplantȱessentialȱoilsȱagainstȱhumanȱpathogensȱ andȱ theirȱmodeȱ ofȱ actionȱ anȱupdatedȱ reviewȱEvidȬBasedȱComplementaryȱAlternȱMedȱ 2016ȱdoi10115520163012462ȱ

22 KalembaȱDAAKȱKunickaȱAȱAntibacterialȱandȱantifungalȱpropertiesȱofȱessentialȱoilsȱCurrȱMedȱChemȱ2003ȱ10ȱ813ndash829ȱ

23 ShreazȱSȱWaniȱWAȱBehbehaniȱJMȱRajaȱVȱIrshadȱMȱKarchedȱMȱAliȱIȱSiddiqiȱWAȱHunȱLTȱCinnamaldehydeȱandȱitsȱderivativesȱaȱnovelȱclassȱofȱantifungalȱagentsȱFitoterapiaȱ2016ȱ112ȱ116ndash131ȱ

24 KesiciȱGuumllerȱHȱCengizȱCcedilallıoAcircluȱFȱSesliȱCcediletinȱEȱAntibacterialȱPVPcinnamonȱessentialȱoilȱnanofibersȱbyȱemulsionȱelectrospinningȱJȱTextȱInstȱ2019ȱ110ȱ302ndash310ȱ

25 CruzȬValenzuelaȱMRRȱTapiaȬRodriguezȱMRȱSilvaȬEspinozaȱBAȱTestaȬNavaȱAȱGutierrezȬPachecoȱMMȱGonzaacutelezȬAguilarȱGAȱAyalaȬZavalaȱ JFȱAntiradicalȱAntibacterialȱ andȱOxidativeȱ Stabilityȱ ofȱCinnamonȱLeafȱOilȱEncapsulatedȱinȱΆȬcyclodextrinȱJMPBȱ2019ȱ8ȱ115ndash123ȱ

26 CarsonȱCFȱHammerȱKAȱRileyȱTVȱMelaleucaȱalternifoliaȱ(teaȱtree)ȱoilȱaȱreviewȱofȱantimicrobialȱandȱotherȱmedicinalȱpropertiesȱClinȱMicrobiolȱRevȱ2006ȱ19ȱ50ndash62ȱ

27 SilveiraȱMPȱSilvaȱHCȱPimentelȱIClȱPoitevinȱCGȱdaȱCostaȱStuartȱAKȱCarpineacuteȱDȱdeȱMatosȱJorgeȱLMȱ JorgeȱRMMȱDevelopmentȱofȱactiveȱcassavaȱ starchȱcelluloseȱnanofiberȬbasedȱ filmsȱ incorporatedȱwithȱnaturalȱantimicrobialȱteaȱtreeȱessentialȱoilȱJȱApplȱPolymȱSciȱ2019ȱ48726ȱ

28 FuselliȱSRȱdeȱlaȱRosaȱBGȱEguarasȱMJȱFritzȱRȱInȱvitroȱantibacterialȱeffectȱofȱexoticȱplantsȱessentialȱoilsȱonȱtheȱhoneybeeȱpathogenȱPaenibacillusȱlarvaeȱcausalȱagentȱofȱAmericanȱfoulbroodȱSpanȱJȱAgricȱResȱ2010ȱ8ȱ651ndash657ȱ

29 IrelandȱBFȱHibbertȱDBȱGoldsackȱRJȱDoranȱJCȱBrophyȱJJȱChemicalȱvariationȱinȱtheȱleafȱessentialȱoilȱofȱMelaleucaȱquinquenerviaȱ(Cav)ȱSTȱBlakeȱBiochemȱSystȱEcolȱ2002ȱ30ȱ457ndash470ȱ

30 HuertaȱBȱBarreroȬDominguezȱBȱGalanȬRelantildeoȱAȱTarradasȱCȱMaldonadoȱAȱLuqueȱIȱEssentialȱoilsȱinȱtheȱcontrolȱofȱinfectionsȱbyȱStaphylococcusȱxylosusȱinȱhorsesȱJȱEquineȱVetȱSciȱ2016ȱ38ȱ19ndash23ȱ

31 Christophȱ Fȱ StahlȬBiskupȱ Eȱ Kaulfersȱ PȬMȱ Deathȱ kineticsȱ ofȱ Staphylococcusȱ aureusȱ exposedȱ toȱcommercialȱteaȱtreeȱoilsȱslȱJȱEssentȱOilȱResȱ2001ȱ13ȱ98ndash102ȱ

32 Wiegandȱ IȱHilpertȱKȱHancockȱREWȱAgarȱandȱbrothȱdilutionȱmethodsȱ toȱdetermineȱ theȱminimalȱinhibitoryȱconcentrationȱ(MIC)ȱofȱantimicrobialȱsubstancesȱNatȱProtocȱ2008ȱ3ȱ163ȱ

33 MicrobiologyȱECfASTotESoCȱDiseasesȱ IȱDeterminationȱofȱminimumȱ inhibitoryȱ concentrationsȱ(MICs)ȱofȱantibacterialȱagentsȱbyȱbrothȱdilutionȱClinȱMicrobiolȱInfectȱ2003ȱ9ȱixndashxvȱ

34 Padraoȱ JȱGonccedilalvesȱ Sȱ Silvaȱ JPȱ SencadasȱVȱLancerosȬMeacutendezȱ Sȱ PinheiroȱACȱVicenteȱAAȱRodriguesȱ LRȱDouradoȱ Fȱ Bacterialȱ celluloseȬlactoferrinȱ asȱ anȱ antimicrobialȱ edibleȱ packagingȱ FoodȱHydrocollȱ2016ȱ58ȱ126ndash140ȱ

35 RotaȱMCȱHerreraȱAȱMartiacutenezȱRMȱSotomayorȱJAȱJordaacutenȱMJȱAntimicrobialȱactivityȱandȱchemicalȱcompositionȱofȱThymusȱvulgarisȱThymusȱzygisȱandȱThymusȱhyemalisȱessentialȱoilsȱFoodȱControlȱ2008ȱ19ȱ681ndash687ȱ

36 LvȱFȱLiangȱHȱYuanȱQȱLiȱCȱInȱvitroȱantimicrobialȱeffectsȱandȱmechanismȱofȱactionȱofȱselectedȱplantȱessentialȱoilȱcombinationsȱagainstȱfourȱfoodȬrelatedȱmicroorganismsȱFoodȱResȱIntȱ2011ȱ44ȱ3057ndash3064ȱ

37 KourmouliȱAȱValentiȱMȱvanȱRijnȱEȱBeaumontȱHJEȱKalantziȱOȬIȱSchmidtȬOttȱAȱBiskosȱGȱCanȱdiscȱ diffusionȱ susceptibilityȱ testsȱ assessȱ theȱ antimicrobialȱ activityȱ ofȱ engineeredȱ nanoparticlesȱ JȱNanoparticleȱResȱ2018ȱ20ȱ62ȱ

38 ChugunovȱAȱPyrkovaȱDȱNoldeȱDȱPolyanskyȱAȱPentkovskyȱVȱEfremovȱRȱLipidȬIIȱformsȱpotentialȱldquolandingȱterrainrdquoȱforȱlantibioticsȱinȱsimulatedȱbacterialȱmembraneȱSciȱRepȱ2013ȱ3ȱ1678ȱ

39 LevineȱDPȱVancomycinȱAȱHistoryȱClinȱInfectȱDisȱ2006ȱ42ȱS5ndashS12ȱ


40 Congȱ Yȱ Yangȱ Sȱ Raoȱ XȱVancomycinȱ resistantȱ Staphylococcusȱ aureusȱ infectionsȱAȱ reviewȱ ofȱ caseȱupdatingȱandȱclinicalȱfeaturesȱJȱAdvȱResȱ2020ȱ21ȱ169ndash176ȱ

41 EirichȱJȱOrthȱRȱSieberȱSAȱUnravelingȱtheȱProteinȱTargetsȱofȱVancomycinȱinȱLivingȱSȱaureusȱandȱEȱfaecalisȱCellsȱJȱAmȱChemȱSocȱ2011ȱ133ȱ12144ndash12153ȱ

42 WatanakunakornȱCȱModeȱofȱactionȱandȱinȬvitroȱactivityȱofȱvancomycinȱJȱAntimicrobȱChemotherȱ1984ȱ14ȱ7ndash18ȱ

43 LawrenceȱRȱTripathiȱPȱJeyakumarȱEȱIsolationȱpurificationȱandȱevaluationȱofȱantibacterialȱagentsȱfromȱAloeȱveraȱBrazȱJȱMicrobiolȱ2009ȱ40ȱ906ndash915ȱ

44 Coelhoȱ Dȱ Sampaioȱ Aȱ Silvaȱ CJSMȱ Felgueirasȱ HPȱ Amorimȱ MTPȱ Zilleȱ Aȱ Antibacterialȱelectrospunȱpolyȱ(vinylȱalcohol)enzymaticȱsynthesizedȱpolyȱ(catechol)ȱnanofibrousȱmidlayerȱmembraneȱforȱultrafiltrationȱACSȱApplȱMaterȱSciȱInterfacesȱ2017ȱ9ȱ33107ndash33118ȱ

45 FelgueirasȱHPȱ TeixeiraȱMAȱ Tavaresȱ TDȱAmorimȱMTPȱNewȱmethodȱ toȱ produceȱ polyȱ (vinylȱalcohol)celluloseȱ acetateȱ filmsȱ withȱ improvedȱ antibacterialȱ actionȱ Materȱ Todayȱ Procȱ 2020ȱdoi101016jmatpr201912100ȱ

46 ChangȱTȬWȱLinȱYȬMȱWangȱCȬFȱLiaoȱYȬDȱOuterȱmembraneȱ lipoproteinȱLppȱ isȱGramȬnegativeȱbacterialȱcellȱsurfaceȱreceptorȱforȱcationicȱantimicrobialȱpeptidesȱJȱBiolȱChemȱ2012ȱ287ȱ418ndash428ȱ

47 BurtȱSȱEssentialȱoilsȱtheirȱantibacterialȱpropertiesȱandȱpotentialȱapplicationsȱ inȱfoodsmdashaȱreviewȱIntȱJȱFoodȱMicrobiolȱ2004ȱ94ȱ223ndash253ȱ

48 ParanagamaȱPAȱWimalasenaȱSȱJayatilakeȱGSȱJayawardenaȱALȱSenanayakeȱUMȱMubarakȱAMȱAȱ comparisonȱ ofȱ essentialȱ oilȱ constituentsȱ ofȱ barkȱ leafȱ rootȱ andȱ fruitȱ ofȱ cinnamonȱ (CinnamomumȱzeylanicumȱBlum)ȱgrownȱinȱSriȱLankaȱJȱNatlȱSciȱFoundȱSriȱ2001ȱ29ȱ147ndash153ȱ

49 RamanoelinaȱPARȱBianchiniȱJȬPȱAndriantsiferanaȱMȱVianoȱJȱGaydouȱEMȱChemicalȱcompositionȱofȱniaouliȱessentialȱoilsȱfromȱMadagascarȱJȱEssentȱOilȱResȱ1992ȱ4ȱ657ndash658ȱ

50 NazzaroȱFȱFratianniȱFȱDeȱMartinoȱLȱCoppolaȱRȱDeȱFeoȱVȱEffectȱofȱessentialȱoilsȱonȱpathogenicȱbacteriaȱPharmaceuticalsȱ2013ȱ6ȱ1451ndash1474ȱ

51 Carsonȱ CFȱMeeȱ BJȱ Rileyȱ TVȱMechanismȱ ofȱActionȱ ofȱMelaleucaȱ alternifoliaȱ (Teaȱ Tree)ȱOilȱ onȱStaphylococcusȱaureusȱDeterminedȱbyȱTimeȬKillȱLysisȱLeakageȱandȱSaltȱToleranceȱAssaysȱandȱElectronȱMicroscopyȱAntimicrobȱAgentsȱChemotherȱ2002ȱ46ȱ1914ȱ

52 JirovetzȱLȱBuchbauerȱGȱDenkovaȱZȱStoyanovaȱAȱMurgovȱIȱSchmidtȱEȱGeisslerȱMȱAntimicrobialȱtestinasȱ andȱ gasȱ chromatoaraphicȱ analvsisȱ ofȱ pureȱ oxvaenatedȱmonoterpenesȱ 18Ȭcineoleȱ ΅ȬterpineolȱterpinenȬ4Ȭolȱandȱcamphorȱasȱwellȱasȱtargetȱcomooundsȱinȱessentialȱoilsȱofȱpineȱ(Pinusȱpinaster)ȱrosemaryȱ(Rosmarinusȱofficinalis)ȱteaȱtreeȱ(Melaleucaȱalternifolia)ȱSciȱPharmȱ2005ȱ73ȱ27ndash39ȱ

53 SlavinȱYNȱAsnisȱJȱHaumlfeliȱUOȱBachȱHȱMetalȱnanoparticlesȱunderstandingȱtheȱmechanismsȱbehindȱantibacterialȱactivityȱJȱNanobiotechnologyȱ2017ȱ15ȱ65ȱ

54 MandalȱBKȱChapterȱ 9ȬScopesȱ ofȱGreenȱ SynthesizedȱMetalȱ andȱMetalȱOxideȱNanomaterialsȱ inȱAntimicrobialȱTherapyȱinȱNanobiomaterialsȱinȱAntimicrobialȱTherapyȱGrumezescuȱAMȱEdȱWilliamȱAndrewȱPublishingȱCambridgeȱMAȱUSAȱ2016ȱppȱ313ndash341ȱ

55 ShrivastavaȱSȱBeraȱTȱRoyȱAȱSinghȱGȱRamachandraraoȱPȱDashȱDȱCharacterizationȱofȱenhancedȱantibacterialȱeffectsȱofȱnovelȱsilverȱnanoparticlesȱNanotechnologyȱ2007ȱ18ȱ225103ȱ

56 XhindoliȱDȱPacorȱSȱBenincasaȱMȱScocchiȱMȱGennaroȱRȱTossiȱAȱTheȱhumanȱcathelicidinȱLLȬ37mdashAȱporeȬformingȱantibacterialȱpeptideȱandȱhostȬcellȱmodulatorȱBiochimȱBiophysȱActaȱ2016ȱ1858ȱ546ndash566ȱ

57 ZelezetskyȱIȱPacorȱSȱPagȱUȱPapoȱNȱShaiȱYȱSahlȱHȬGȱTossiȱAȱControlledȱalterationȱofȱtheȱshapeȱandȱconformationalȱstabilityȱofȱalphaȬhelicalȱcellȬlyticȱpeptidesȱeffectȱonȱmodeȱofȱactionȱandȱcellȱspecificityȱBiochemȱJȱ2005ȱ390ȱ177ndash188ȱ

58 ZelezetskyȱIȱPontilloȱAȱPuzziȱLȱAntchevaȱNȱSegatȱLȱPacorȱSȱCrovellaȱSȱTossiȱAȱEvolutionȱofȱtheȱPrimateȱCathelicidinȱCorrelationȱ betweenȱ StructuralȱVariationsȱ andȱAntimicrobialȱActivityȱ JȱBiolȱChemȱ2006ȱ281ȱ19861ndash19871ȱ

59 ZasloffȱMȱMagaininsȱaȱclassȱofȱantimicrobialȱpeptidesȱfromȱXenopusȱskinȱisolationȱcharacterizationȱofȱtwoȱactiveȱformsȱandȱpartialȱcDNAȱsequenceȱofȱaȱprecursorȱProcȱNatlȱAcadȱSciȱUSAȱ1987ȱ84ȱ5449ndash5453ȱ

60 Chouhanȱ Sȱ SharmaȱKȱGuleriaȱ SȱAntimicrobialȱActivityȱ ofȱ SomeȱEssentialȱOilsȬPresentȱ Statusȱ andȱFutureȱPerspectivesȱMedicinesȱ2017ȱ4ȱ58ȱ

61 Mizunoȱ Tȱ KageyamaȱMȱ Separationȱ andȱ characterizationȱ ofȱ theȱ outerȱmembraneȱ ofȱ PseudomonasȱaeruginosaȱJȱBiochemȱ1978ȱ84ȱ179ndash191ȱ


62 MayȱJȱChanȱCHȱKingȱAȱWilliamsȱLȱFrenchȱGLȱTimendashkillȱstudiesȱofȱteaȱtreeȱoilsȱonȱclinicalȱisolatesȱJȱAntimicrobȱChemotherȱ2000ȱ45ȱ639ndash643ȱ

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characteristicsȱandȱphageȱtypingȱActaȱPatholȱMicrobiolȱScandȱ1966ȱ67ȱ149ndash156ȱ66 NanningaȱNȱMorphogenesisȱofȱEscherichiaȱcoliȱMicrobiolȱMolȱBiolȱRevȱ1998ȱ62ȱ110ndash129ȱ67 KubitschekȱHEȱCellȱvolumeȱincreaseȱinȱEscherichiaȱcoliȱafterȱshiftsȱtoȱricherȱmediaȱJȱBacteriolȱ1990ȱ172ȱ

94ndash101ȱ68 KirisitsȱMJȱProstȱLȱStarkeyȱMȱParsekȱMRȱCharacterizationȱofȱColonyȱMorphologyȱVariantsȱIsolatedȱ

fromȱampltemampgtPseudomonasȱaeruginosaampltemampgtȱBiofilmsȱApplȱEnvironȱMicrobiolȱ2005ȱ71ȱ4809ȱ69 SondiȱIȱSalopekȬSondiȱBȱSilverȱnanoparticlesȱasȱantimicrobialȱagentȱaȱcaseȱstudyȱonȱEȱcoliȱasȱaȱmodelȱ

forȱGramȬnegativeȱbacteriaȱJȱColloidȱInterfaceȱSciȱ2004ȱ275ȱ177ndash182ȱ70 QuinterosȱMAȱVivianaȱCAȱOnnaintyȱRȱMaryȱVSȱTheumerȱMGȱGraneroȱGEȱParajeȱMGȱPaacuteezȱ

PLȱBiosynthesizedȱsilverȱnanoparticlesȱDecodingȱ theirȱmechanismȱofȱactionȱ inȱStaphylococcusȱaureusȱandȱEscherichiaȱcoliȱIntȱJȱBiochemȱCellȱBiolȱ2018ȱ104ȱ87ndash93ȱ

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72 Nooreȱ JȱNooreȱAȱLiȱBȱCationicȱAntimicrobialȱPeptideȱLLȬ37ȱ IsȱEffectiveȱAgainstȱBothȱExtraȬȱAndȱIntracellularȱStaphylococcusȱAureusȱAntimicrobȱAgentsȱChemotherȱ2013ȱ57ȱ1283ȱ

73 ShurkoȱJFȱGalegaȱRSȱLiȱCȱLeeȱGCȱEvaluationȱofȱLLȬ37ȱantimicrobialȱpeptideȱderivativesȱaloneȱandȱinȱcombinationȱwithȱvancomycinȱagainstȱSȱaureusȱJȱAntibiotȱ2018ȱ71ȱ971ndash974ȱ

74 SochackiȱKAȱBarnsȱKJȱBuckiȱRȱWeisshaarȱJCȱRealȬtimeȱattackȱonȱsingleȱEscherichiaȱcoliȱcellsȱbyȱtheȱhumanȱantimicrobialȱpeptideȱLLȬ37ȱProcȱNatlȱAcadȱSciȱUSAȱ2011ȱ108ȱE77ndashE81ȱ

75 GottlerȱLMȱRamamoorthyȱAȱStructureȱmembraneȱorientationȱmechanismȱandȱfunctionȱofȱpexigananȬȬaȱhighlyȱpotentȱantimicrobialȱpeptideȱdesignedȱfromȱmagaininȱBiochimȱBiophysȱActaȱ2009ȱ1788ȱ1680ndash1686ȱ

76 KumarȱPȱKizhakkedathuȱJNȱStrausȱSKȱAntimicrobialȱPeptidesȱDiversityȱMechanismȱofȱActionȱandȱStrategiesȱtoȱImproveȱtheȱActivityȱandȱBiocompatibilityȱInȱVivoȱBiomoleculesȱ2018ȱ8ȱ4ȱ

77 Garbaczȱ Kȱ KamyszȱWȱ Piechowiczȱ LȱActivityȱ ofȱ antimicrobialȱ peptidesȱ aloneȱ orȱ combinedȱwithȱconventionalȱantibioticsȱagainstȱStaphylococcusȱaureusȱisolatedȱfromȱtheȱairwaysȱofȱcysticȱfibrosisȱpatientsȱVirulenceȱ2017ȱ8ȱ94ndash100ȱ

78 HancockȱREWȱPeptideȱantibioticsȱLancetȱ1997ȱ349ȱ418ndash422ȱ79 ZasloffȱMȱAntimicrobialȱpeptidesȱofȱmulticellularȱorganismsȱNatureȱ2002ȱ415ȱ389ndash395ȱ80 PleacutesiatȱPȱNikaidoȱHȱOuterȱmembranesȱofȱGramȬnegativeȱbacteriaȱareȱpermeableȱtoȱsteroidȱprobesȱMolȱ

Microbiolȱ1992ȱ6ȱ1323ndash1333ȱ

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withȱ aȱ thinȱ layerȱ ofȱ peptidoglycansȱ inȱ betweenȱ Theȱ outerȱmembraneȱ worksȱ asȱ anȱ additionalȱcompoundȬselectiveȱbarrierȱ[2]ȱItȱisȱhighlyȱpermeableȱandȱcontainsȱ lipopolysaccharidesȱ(LPS)ȱtheȱmainȱ lipidȱcomponentȱandȱaȱperiplasmicȱ spaceȱwhereȱenzymesȱcapableȱofȱdegradingȱmoleculesȱintroducedȱ fromȱ theȱ extracellularȱmediumȱ areȱpresentȱ [12]ȱ InȱGramȬpositiveȱbacteriaȱ cytosolȱ isȱenvelopedȱ byȱ oneȱ bilayerȱ membraneȱ theȱ cytoplasmicȱ membraneȱ attachedȱ toȱ aȱ thickȱ layerȱ ofȱpeptidoglycansȱformedȱofȱlinearȱpolysaccharideȱchainsȱcrossȬlinkedȱbyȱshortȱpeptidesȱthatȱgenerateȱaȱ threeȬdimensionalȱ (3D)ȱ rigidȱ structureȱ Inȱ thisȱ caseȱ lipoteichoicȱ acidsȱ areȱ adheredȱ toȱ theȱpeptidoglycanȱ layerȱTheseȱcomponentsȱprovideȱtheȱbacterialȱmembraneȱwithȱanȱamphiphilicȱandȱanionicȱcharacterȱ[2ndash4]ȱȱ

Antimicrobialȱagentsȱareȱgenerallyȱlipophilicȱandȱcationicȱallowingȱtheirȱpositivelyȱchargedȱsideȱchainsȱ toȱ bindȱ toȱ theȱ negativelyȱ chargedȱ surfaceȱ ofȱ theȱ bacterialȱmembranesȱ Subsequentlyȱ theȱlipophilicȱmotifsȱinteractȱwithȱtheȱlipidȱbacterialȱmembraneȱleadingȱtoȱinstabilityȱandȱruptureȱofȱtheȱmembraneȱmatrixȱandȱeventuallyȱtoȱtheȱcellȱdeathȱ[156]ȱȱ

CurrentlyȱthereȱisȱaȱvastȱarrayȱofȱantimicrobialȱbiomoleculesȱForȱmanyȱyearsȱtheȱmostȱwidelyȱusedȱhaveȱbeenȱtheȱantibioticsȱHoweverȱtheirȱexcessiveȱconsumptionȱhasȱledȱtoȱanȱalarminglyȱhighȱresistanceȱdevelopmentȱbyȱbacterialȱpathogensȱraisingȱaȱseriousȱglobalȱpublicȬhealthȱproblemȱ[78]ȱHenceȱ theȱ interestȱ inȱ theȱresearchȱ forȱnovelȱalternativesȱ toȱantibioticsȱhasȱbeenȱgrowingȱNaturalȱproductsȱ areȱ becomingȱ veryȱ promisingȱ asȱ antimicrobialȱ agentsȱ beingȱ consideredȱ safeȱ andȱenvironmentallyȱfriendlyȱ[910]ȱAntimicrobialȱpeptidesȱ(AMPs)ȱofȱnaturalȱoriginȱhaveȱbeenȱtheȱfocusȱofȱgreatȱ interestȱasȱalternativesȱ toȱconventionalȱantibioticsȱTheyȱplayȱanȱ importantȱ roleȱ inȱ innateȱimmunityȱprotectingȱ theȱhostȱagainstȱ infectionsȱbyȱmicroorganismsȱAMPsȱareȱoftenȱcationicȱandȱamphiphilicȱmoleculesȱwithȱlowȱmolecularȱweightȱandȱcanȱbeȱobtainedȱfromȱaȱvarietyȱofȱorganismsȱ(egȱ humansȱ insectsȱ amphibianȱ bacteriaȱ etc)ȱ orȱ synthesizedȱ asȱ analogsȱ ofȱ thoseȱ naturallyȱoccurringȱ[11ndash13]ȱLL37ȱforȱinstanceȱisȱanȱAMPȱthatȱbelongsȱtoȱtheȱcathelicidinȱfamilyȱanȱimportantȱantimicrobialȱagentȱinȱhumansȱLL37ȱisȱessentialȱforȱnormalȱinnateȱimmuneȱresponsesȱwithinȱinfectedȱandȱinjuredȱtissuesȱdisplayingȱaȱbroadȱantimicrobialȱactivityȱagainstȱbothȱGramȬpositiveȱandȱGramȬnegativeȱbacteriaȱThisȱAMPȱisȱalsoȱaȱpromoterȱofȱtissueȱregenerationȱ[14ndash16]ȱPexigananȱisȱanȱanalogȱofȱtheȱmagaininȱpeptidesȱisolatedȱfromȱtheȱskinȱofȱtheȱAfricanȱclawedȱfrogȱLikeȱLL37ȱpexigananȱisȱalsoȱ reportedȱ toȱpossessȱaȱbroadȱspectrumȱofȱantibacterialȱactionȱdisplayingȱactivityȱagainstȱoverȱ3000ȱclinicalȱisolatesȱincludingȱmethicillinȬȱandȱgentamicinȬresistantȱStaphylococcusȱaureusȱ[1718]ȱOnȱtheȱ otherȱ handȱ essentialȱ oilsȱ (EOs)ȱ areȱ composedȱ ofȱ plantȱ secondaryȱmetabolitesȱ thatȱ possessȱantibacterialȱantiviralȱandȱantifungalȱactivityȱdefendingȱtheȱhostȱfromȱmicrobiologicalȱinvasionȱ[19]ȱTheseȱconsistȱofȱaȱcomplexȱmixtureȱofȱchemicalȱcompoundsȱincludingȱterpenesȱphenolsȱalcoholsȱaldehydesȱethersȱandȱketonesȱ[2021]ȱmostȱofȱwhichȱareȱhydrophobicȱorȱpartiallyȱsolubleȱinȱwaterȱUsuallyȱEOsȱhaveȱstrongȱlipophilicityȱandȱvolatilityȱ[9]ȱmakingȱthemȱsuitableȱantibacterialȱagentsȱforȱvariousȱapplicationsȱencompassingȱantiȬinflammatoryȱandȱantioxidativeȱpropertiesȱNowadaysȱEOsȱapplicationȱisȱfoundȱinȱmanyȱfieldsȱincludingȱfoodȱpreservationȱcosmeticsȱandȱbiomedicineȱ[22]ȱCinnamonȱ(Cinnamomumȱzeylanicum)ȱisȱaȱwellȬstudiedȱEOȱwithȱvariousȱbiologicalȱpropertiesȱplayingȱaȱ keyȱ roleȱ inȱmaintainingȱhumanȱ healthȱ [23]ȱTheȱ cinnamonȱ leafȱ oilȱ (CLO)ȱ isȱmainlyȱ formedȱ ofȱeugenolȱwhichȱisȱreportedȱasȱtheȱmainȱcompoundȱresponsibleȱforȱitsȱantimicrobialȱpropertiesȱ[2425]ȱLikeȱCLOȱteaȱtreeȱoilȱ(TTO)ȱisȱalsoȱknownȱforȱitsȱgoodȱantimicrobialȱpropertiesȱTTOȱcanȱbeȱobtainedȱfromȱMelaleucaȱ alternifoliaȱ anȱ Australianȱ nativeȱ plantȱ andȱ hasȱ beenȱ incorporatedȱ asȱ anȱ activeȱingredientȱinȱmanyȱtopicalȱformulationsȱtoȱtreatȱcutaneousȱinfectionsȱ[26]ȱTheȱmainȱcompoundȱofȱTTOȱ isȱ terpinenȬ4Ȭolȱbeingȱmainlyȱ responsibleȱ forȱ itsȱantimicrobialȱactivityȱ [2627]ȱNiaouliȱoilȱ isȱextractedȱ fromȱMelaleucaȱ viridifloraȱ aȱperennialȱ treeȱnativeȱ toȱAustraliaȱNewȱCaledoniaȱ andȱ theȱFrenchȱPacificȱIslandsȱThisȱEOȱisȱofȱcommercialȱimportanceȱdueȱtoȱitsȱapplicationsȱinȱaromatherapyȱandȱpharmaceuticalȱpreparationsȱ[2829]ȱItsȱantimicrobialȱactivityȱhasȱalsoȱbeenȱestablishedȱ[3031]ȱAlthoughȱ bothȱ AMPsȱ andȱ EOsȱ haveȱ beenȱ reportedȱ asȱ promisingȱ alternativesȱ toȱ antibioticsȱparticularlyȱ forȱ theirȱ quickȱ actionȱ andȱ lowȱ tendencyȱ toȱ induceȱ resistanceȱ thereȱ isȱ stillȱmuchȱ toȱunderstandȱaboutȱtheirȱmechanismsȱofȱactionȱagainstȱselectedȱmicroorganismsȱ

TheȱpresentȱstudyȱaimedȱtoȱscreenȱandȱfurtherȱdetailȱtheȱantibacterialȱpropertiesȱofȱbothȱAMPsȱandȱEOsȱagainstȱfourȱcommonȱnosocomialȱbacteriaȱSȱaureusȱStaphylococcusȱepidermidisȱEscherichiaȱ




21ȱMaterialsȱ


























AgNPsȱ

115ȱplusmnȱ17ȱ

ȱ

106ȱplusmnȱ06ȱ

ȱ

88ȱplusmnȱ05ȱ

ȱ

88ȱplusmnȱ30ȱ

ȱ

Vancomycinȱ

225ȱplusmnȱ05ȱ

ȱ

225ȱplusmnȱ05ȱ

ȱ

80ȱplusmnȱ01ȱ

ȱ

80ȱplusmnȱ02ȱ

ȱ

LL37ȱ

65ȱplusmnȱ01ȱ

ȱ

65ȱplusmnȱ05ȱ

ȱ

63ȱplusmnȱ01ȱ

ȱ

62ȱplusmnȱ01ȱ

ȱ

Pexigananȱ

90ȱplusmnȱ05ȱ

ȱ

122ȱplusmnȱ06ȱ

ȱ

80ȱplusmnȱ15ȱ

ȱ

120ȱplusmnȱ01ȱ

ȱ

TTOȱ

202ȱplusmnȱ01ȱ

ȱ

150ȱplusmnȱ05ȱ

ȱ

155ȱplusmnȱ05ȱ

ȱ

133ȱplusmnȱ03ȱ

ȱ

CLOȱ

215ȱplusmnȱ05ȱ

ȱ

150ȱplusmnȱ10ȱ

ȱ

150ȱplusmnȱ19ȱ

ȱ

150ȱplusmnȱ06ȱ

ȱ


NOȱ

147ȱplusmnȱ04ȱ

ȱ

100ȱplusmnȱ05ȱ

ȱ

115ȱplusmnȱ05ȱ

ȱ

68ȱplusmnȱ05ȱ

ȱ

32ȱMICsȱ



































Referencesȱ


















































































ȱ


ȱ





21ȱMaterialsȱ


























AgNPsȱ

115ȱplusmnȱ17ȱ

ȱ

106ȱplusmnȱ06ȱ

ȱ

88ȱplusmnȱ05ȱ

ȱ

88ȱplusmnȱ30ȱ

ȱ

Vancomycinȱ

225ȱplusmnȱ05ȱ

ȱ

225ȱplusmnȱ05ȱ

ȱ

80ȱplusmnȱ01ȱ

ȱ

80ȱplusmnȱ02ȱ

ȱ

LL37ȱ

65ȱplusmnȱ01ȱ

ȱ

65ȱplusmnȱ05ȱ

ȱ

63ȱplusmnȱ01ȱ

ȱ

62ȱplusmnȱ01ȱ

ȱ

Pexigananȱ

90ȱplusmnȱ05ȱ

ȱ

122ȱplusmnȱ06ȱ

ȱ

80ȱplusmnȱ15ȱ

ȱ

120ȱplusmnȱ01ȱ

ȱ

TTOȱ

202ȱplusmnȱ01ȱ

ȱ

150ȱplusmnȱ05ȱ

ȱ

155ȱplusmnȱ05ȱ

ȱ

133ȱplusmnȱ03ȱ

ȱ

CLOȱ

215ȱplusmnȱ05ȱ

ȱ

150ȱplusmnȱ10ȱ

ȱ

150ȱplusmnȱ19ȱ

ȱ

150ȱplusmnȱ06ȱ

ȱ


NOȱ

147ȱplusmnȱ04ȱ

ȱ

100ȱplusmnȱ05ȱ

ȱ

115ȱplusmnȱ05ȱ

ȱ

68ȱplusmnȱ05ȱ

ȱ

32ȱMICsȱ



































Referencesȱ


















































































ȱ


ȱ





















AgNPsȱ

115ȱplusmnȱ17ȱ

ȱ

106ȱplusmnȱ06ȱ

ȱ

88ȱplusmnȱ05ȱ

ȱ

88ȱplusmnȱ30ȱ

ȱ

Vancomycinȱ

225ȱplusmnȱ05ȱ

ȱ

225ȱplusmnȱ05ȱ

ȱ

80ȱplusmnȱ01ȱ

ȱ

80ȱplusmnȱ02ȱ

ȱ

LL37ȱ

65ȱplusmnȱ01ȱ

ȱ

65ȱplusmnȱ05ȱ

ȱ

63ȱplusmnȱ01ȱ

ȱ

62ȱplusmnȱ01ȱ

ȱ

Pexigananȱ

90ȱplusmnȱ05ȱ

ȱ

122ȱplusmnȱ06ȱ

ȱ

80ȱplusmnȱ15ȱ

ȱ

120ȱplusmnȱ01ȱ

ȱ

TTOȱ

202ȱplusmnȱ01ȱ

ȱ

150ȱplusmnȱ05ȱ

ȱ

155ȱplusmnȱ05ȱ

ȱ

133ȱplusmnȱ03ȱ

ȱ

CLOȱ

215ȱplusmnȱ05ȱ

ȱ

150ȱplusmnȱ10ȱ

ȱ

150ȱplusmnȱ19ȱ

ȱ

150ȱplusmnȱ06ȱ

ȱ


NOȱ

147ȱplusmnȱ04ȱ

ȱ

100ȱplusmnȱ05ȱ

ȱ

115ȱplusmnȱ05ȱ

ȱ

68ȱplusmnȱ05ȱ

ȱ

32ȱMICsȱ



































Referencesȱ


















































































ȱ


ȱ



NOȱ

147ȱplusmnȱ04ȱ

ȱ

100ȱplusmnȱ05ȱ

ȱ

115ȱplusmnȱ05ȱ

ȱ

68ȱplusmnȱ05ȱ

ȱ

32ȱMICsȱ



































Referencesȱ


















































































ȱ


ȱ
































Referencesȱ


















































































ȱ


ȱ



































































ȱ


ȱ


activity of specialized biomolecules against gram positive

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