Antibacterial Free Fatty Acids and Monoglycerides

40 Pages·2017·27.63 MB·English

International Journal o f
Molecular Sciences
Review
Antibacterial Free Fatty Acids and Monoglycerides:
Biological Activities, Experimental Testing, and
Therapeutic Applications
BoKyeongYoon1,JoshuaA.Jackman1,2,ElbaR.Valle-González1andNam-JoonCho1,* ID
1 SchoolofMaterialsScienceandEngineeringandCentreforBiomimeticSensorScience,
NanyangTechnologicalUniversity,50NanyangDrive,Singapore637553,Singapore;
[email protected](B.K.Y.);[email protected]@stanford.edu(J.A.J.);
[email protected](E.R.V.-G.)
2 DivisionofGastroenterologyandHepatology,DepartmentofMedicine,StanfordUniversitySchoolof
Medicine,Stanford,CA94305,USA
* Correspondence:[email protected];Tel.:[phone];Fax:[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:26February2018;Accepted:5April2018;Published:8April2018
Abstract: Antimicrobial lipids such as fatty acids and monoglycerides are promising antibacterial
agentsthatdestabilizebacterialcellmembranes,causingawiderangeofdirectandindirectinhibitory
effects.Thegoalofthisreviewistointroducethelatestexperimentalapproachesforcharacterizinghow
antimicrobiallipidsdestabilizephospholipidmembraneswithinthebroaderscopeofintroducingcurrent
knowledgeaboutthebiologicalactivitiesofantimicrobiallipids,testingstrategies,andapplications
fortreatingbacterialinfections. Tothisend,ageneralbackgroundonantimicrobiallipids,including
structuralclassification,isprovidedalongwithadetaileddescriptionoftheirtargetingspectrumand
currentlyunderstoodantibacterialmechanisms. Buildingonthisknowledge,differentexperimental
approachestocharacterizeantimicrobiallipidsarepresented,includingcell-basedbiologicalandmodel
membrane-basedbiophysicalmeasurementtechniques.Particularemphasisisplacedondrawingout
howbiologicalandbiophysicalapproachescomplementoneanotherandcanyieldmechanisticinsights
intohowthephysicochemicalpropertiesofantimicrobiallipidsinfluencemolecularself-assemblyand
concentration-dependentinteractionswithmodelphospholipidandbacterialcellmembranes.Examples
of possible therapeutic applications are briefly introduced to highlight the potential significance of
antimicrobiallipidsforhumanhealthandmedicine, andtomotivatetheimportanceofemploying
orthogonalmeasurementstrategiestocharacterizetheactivityprofileofantimicrobiallipids.
Keywords:antimicrobiallipid;fattyacid;monoglyceride;antibacterial;therapy;phospholipidmembrane
1. Introduction
Molecular design principles underpin the structure and function of biological assemblies such as
cells,andamphiphilicmoleculesdrivethespontaneousself-assemblyofkeyarchitecturalelementslike
phospholipidmembranes[1–3].Understandingtheroleofmolecularself-assemblyindirectingtheformation
ofbiologicalmacromolecularstructures,includinglipidbilayers,proteins,andassembliesthereof,ispart
ofthenanoarchitectonicsfield[4,5],andsuchinsightscanhelpsolveoutstandingbiomedicalproblems.
Within this scope, one of the greatest public health problems in the world today is the growing rise
ofantibiotic-resistantbacteriaandtheassociatedchallengestotreatandpreventbacterialinfections[6].
Less than a century ago, the world’s first antibiotic, penicillin, was discovered, and the specificity of
antibioticstoinhibitbacterialenzymesandotherproteinsnecessaryforbacterialcellfunctionprovedhighly
effectiveandaremarkableexampleofmolecularpharmaceutics,asevidencedbymarkedimprovementsin
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healthcarecapabilitiestotreatbacterialinfections.Asaresult,manyformerlyfatalordebilitatingdiseases
causedbybacterialpathogensweresuddenlycurablewithantibiotictreatment[7].
Withhighpotencyandworkingagainstabroadspectrumofbacterialtargets,antibioticsbecame
the standard drug option to treat bacterial infections, and are also widely used as precautionary
measures to treat suspected infections, even when the microbial origin is unknown and could be
bacterial,fungal,orviralamongotherpossibilities. Antibioticsarealsoadministeredprophylactically
incaseswherebacterialinfectionsmightarise,suchasaftersurgicaloperations. Inaddition,antibiotics
arecommonlyusedintheagriculturalsectortonotonlytreatandpreventbacterialinfectionsamong
livestock,butalsoserveasgrowthpromoterstoacceleratethetimetoreachmaturityaswellasincrease
thebodymassofanimals. Forallthesereasons,antibioticshavebecomeubiquitousinsocietyand
haveplayedanoutsizedroleinshapingmodernlife.
However,despitenumerousbenefits,thedrawbacksofantibioticsbeingsowidelyprevalentare
nowbecomingapparentaswell. Withincreasingexposuretoantibioticsandcorrespondingselective
pressure,bacteriahaveevolvedtobecomeresistanttomanyantibiotics,andantibiotic-resistantbacteria
arewidespread.Asaresult,existingantibioticsarelosingtheireffectivenesstotreatbacterialinfections,
andtheproblemisfurthercompoundedbythedearthofnewantibioticsthathavebeendiscovered
inrecentyears. Inpart,theproblemiseconomicbecausepharmaceuticalcompanieshavehadweak
interestindevelopingnewantibioticsduetolowpricepoints,however,themorepressingscientific
issueisthatthechemicalspaceavailableforidentifyingandrefiningantibioticsislimited. Thereis
growingrecognitionthatsocietyfacesanimpendingpost-antibioticera[8], andhence, thereisan
urgentneedtodevelopnewclassesofantibacterialagentsthatworkagainstnovelmoleculartargets.
Toaddressthisproblem,antimicrobiallipids—single-chainlipidamphiphilesthatdestabilize
bacterialcellmembranes—areattractivecandidatestobecomenext-generationantibacterialagentsfor
treatingbacterialinfections. Curiously,theantibacterialpropertiesofantimicrobiallipidshavebeen
knownsinceearlyreportsbyDr. RobertKochandcolleaguesinthelate1880s,whenitwasshownthat
fattyacids,aprominentclassofantimicrobiallipid,inhibitedgrowthoftheBacillusanthracispathogen
thatcausesanthrax[9]. Afewdecadeslater,Burtenshawandcolleaguesshowedthatantimicrobial
lipidsareanimportantcomponentofhumanskin’sinnateimmunesystem[10,11],lendingcredenceto
thepossibilitythatexogenousadditionofantimicrobiallipidswouldbemedicallyopportune. Despite
strongpromiseanddemonstratedresults,theprospectsforantimicrobiallipidsfadedawaybythelate
1940sduetotheemergenceofantibiotics,buthavereceivedrenewedattentionamidstthegrowing
impact of antibiotic-resistant bacteria. Indeed, one attractive feature of antimicrobial lipids is that
itisdifficultforbacteriatomutatetobecomeresistanttothem. Assuch,bacterialcellculturescan
begrowninthepresenceofantimicrobiallipids(atsub-lethalconcentrations)foratleastoneyear,
withoutsignsofdrug-resistantstrainsemerging[12].
Particularattentionisdrawntotwoclassesofantimicrobiallipid,namelyfattyacids(hydrocarbon
chains with a carboxylic acid functional group [13]) and monoglycerides (esterified adducts of a
fatty acid and glycerol molecule). The motivation for studying these two classes of antimicrobial
lipid arose from pioneering studies by Kabara and colleagues in the 1970s, which systematically
investigated the antibacterial potency of medium-chain saturated fatty acids and monoglycerides
with different chain lengths [14–17]. It was discovered that lauric acid (LA),which possesses a 12
carbon-longchain,hadthemostpotentactivitytoinhibitgrowthofGram-positivebacteria,andits
monoglyceridederivative,glycerolmonolaurate(GML),exhibitedevenstrongerinhibitoryactivity
thanLA.Importantly,bothLAandGMLareGenerallyRecognizedAsSafe(GRAS)bytheUnited
StatesFoodandDrugAdministration[18]andabundantinnature. Thesefactorshaveledtowide
explorationofLAandGMLforanti-infectiveapplications[19–21],includingapplicationtopicssuch
as agriculture [22] and cosmetics [13,23,24]. Other antimicrobial lipids such as capric acid, which
possessesa10carbon-longchain,anditsmonoglyceridederivative,monocaprin,havealsoreceived
attention. While numerous studies have been conducted to empirically investigate the inhibitory
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propertiesofantimicrobiallipids,clarifyinghowthephysicochemicalpropertiesofantimicrobiallipids
influencebiologicalactivitiesremainsanoutstandinggoalinmanyrespects.
Todate,theprimarymeansofassessingtheactivityprofileofanantimicrobiallipidhasbeento
evaluatehowtreatmentaffectsbacterialcellgrowth,withtheminimuminhibitoryconcentration(MIC)of
atestcompoundbeingdefinedasthedrugconcentrationatwhichnovisiblegrowthofbacteriaoccurs.
Whilesuchinformationprovidesinsightintothescopeandpotencyofanantimicrobiallipid,itdoes
notrevealmechanisticinformationandthereisgrowinginteresttounderstandhowantimicrobiallipids
destabilizebacterialcellmembranes. Biologicalassayshaveidentifiedthatantimicrobiallipidsactas
bacteriostatic(growth-inhibiting)orbactericidal(killing)agentsdependingonthedrugconcentration,
targetbacteriumandotherenvironmentalfactors[13,25].Todirectlyobservemorphologicaleffectson
bacterialcellmembranes,electronmicroscopytechniqueshavebeenutilizedtoimagebacterialspecimens
aftertreatmentwithantimicrobiallipids[26–32].Whilethisapproachprovidesvisualizationofmembrane
damage,veryhighconcentrationsofantimicrobiallipidaretypicallyused(5–10mM)andthebacterial
cellscanonlybeexaminedaftertreatmentandsamplefixation. Similarissuesexistwithatomicforce
microscopyexperimentsforexaminingthemorphologyoftreatedbacterialcells.Ingeneral,itisdifficult
toresolvemolecular-levelinteractionkineticswhenworkingwithcomplexbiologicalsamplessuchas
wholebacterialcells,therebymotivatingthedevelopmentofmodelsystems.
To obtain insights into real-time interaction kinetics with a more focused approach, a variety
ofsolution-phasemodelmembraneplatformsbasedonsmallunilamellarvesicles(SUVs)andlarge
unilamellar vesicles (LUVs) have been employed in combination with measurement techniques
such as dynamic light scattering in order to detect how antimicrobial lipids cause membrane
destabilizationviapartialsolubilizationaswellasmembranefission[33–40]. Thesemodelsystems
allow detailed characterization of membrane morphological changes by using well-controlled,
simplifiedphospholipidcompositionsthatmimicmorecomplexbiologicalmembranes. Time-lapsed
opticalmicroscopyimagingofgiantunilamellarvesicles(GUVs)hasalsoenableddirectvisualization
of membrane morphological responses, including swelling, fusion, and fission behaviors [41,42].
Asanotheroption,supportedlipidbilayers(SLBs)aretwo-dimensionalphospholipidbilayersthat
haveemergedasaparticularlyusefulmodelmembraneplatformbecausetheycanbestudiedwitha
widerangeofsurface-sensitivemeasurementtechniques,revealinginsightsintothemass,viscoelastic,
fluidic, and morphological properties of SLB platforms. Indeed, one particular advantage of SLB
platformsisthatthetwo-dimensionalphospholipidbilayercanremodelinresponsetoanapplied
membranestrain,givingrisetothree-dimensionalmorphologicalresponses. Untilrecently,therewas
onlyscantattentiontoemploySLBplatformsforinvestigatingantimicrobiallipids, withonlyone
studyreportingtheinteractionsbetweenalong-chainfattyacidandsingle-component,zwitterionic
phospholipidSLBplatformandanotherstudyinvestigatingtheinteractionbetweenashort-chain
monoglyceride and SLBs derived from bacterial cell membrane extracts [43–45]. Extending such
approachestoinvestigatemedium-chainsaturatedfattyacidsandmonoglycerides—representingthe
subsetofantimicrobiallipidswiththehighestactivity—iswarrantedinordertodevelopmodelsystems
forprofilingthescopeandpotencyoftheseantimicrobiallipidsandasubjectofactiveinvestigation.
Movingbeyond particularexperimentalapproaches, thereisactive progress towardsestablishing
comprehensiveframeworksforcorrelatingthephysicochemicalpropertiesofantimicrobiallipidswith
theircorrespondingbiophysicalandbiologicalactivities.
Recognizingthesepossibilities,thegoalofthisreviewistointroducethelatestexperimentalapproaches
forcharacterizinghowantimicrobiallipidsdestabilizephospholipidmembraneswithinthebroaderscope
ofintroducingcurrentknowledgeaboutthebiologicalactivitiesofantimicrobiallipids,testingstrategies,
andapplicationsfortreatingbacterialinfections.Thecontentsofthereviewareorganizedintothreemajor
sections. First, a general background on antimicrobial lipids is provided that describes the structural
classification,spectrumofantibacterialactivity,andcurrentlyunderstoodantibacterialmechanismslinked
tomembranedestabilization.Then,differentexperimentalapproachestocharacterizeantimicrobiallipids,
including cell-based biological and model membrane-based biophysical measurement techniques, are
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presented. Themajorfindingsobtainedbyeachexperimentalapproacharecriticallysummarized,and
supplementedbydiscussionabouthowbiologicalandbiophysicalapproachescanbeintegratedtobetter
understandhowthephysicochemicalpropertiesofantimicrobiallipidsinfluencemolecularself-assembly
andconcentration-dependentinteractionswithmodelphospholipidandbacterialcellmembranes.Finally,
itisdescribedhowantimicrobiallipidsinfreeformandnanostructuredassembliescanbeemployedfor
therapeuticapplications,includingwhenadministeredviasystemicandtopicaladministrationroutes.Taken
together,theinsightspresentedinthisreviewunderscoretheutilityofemployingorthogonalmeasurement
strategiestocharacterizetheactivityprofileofantimicrobiallipids.
2. AntimicrobialLipids
2.1. Classifications
Antimicrobial lipids are defined as single-chain lipid amphiphiles that interact with bacterial cell
membranesandexhibitantibacterialactivity.Fattyacidsareawidelystudiedtypeofantimicrobiallipid,
andarecomposedofasinglesaturatedorunsaturatedhydrocarbonchainandacarboxylicacidgroup
ononeend. Assuch, fattyacidsareamphipathicmolecules, withthehydrocarbonchainconstituting
thehydrophobicpartwhilethecarboxylicacidgroupishydrophilic(eitherpolaroranionicinaqueous
solutions,dependingonpHconditions).Forexample,thecarboxylicacidgroupsofmedium-chainfatty
suchascapricacidandLA,havepK valuesaroundpH5[46],andthereforethefattyacidsareanionic
a
(deprotonatedcarboxylicacidgroups)aroundthephysiological(blood)pHconditionof7.4.Inadditionto
fattyacids,otherderivativeshavebeenreportedtohaveantibacterialactivity,withoneprominentclass
beingmonoglyceridesthatarecomposedofafattyacidconnectedwithaglycerolmoleculeviaanester
bond.Othersyntheticversionsarealsopossiblesuchasrelatedcompoundswithetherbonds,rendering
suchmoleculesimpervioustobacteriallipases.Comparedtofattyacids,monoglyceridesbearthedistinction
ofnothavingionizablefunctionalgroupsacrossrelevantpHconditions,andhence,arenonionicmolecules
withneutralelectricalchargepropertiesandsomedegreeofpolarity.
Antimicrobial lipids are classified based on their chain lengths and degrees of unsaturation.
RepresentativecompoundswithdifferentmolecularstructuresarepresentedinFigure1. Inbiological
systems,fattyacidstypicallypossessanevennumberofcarbonatomsbetween4and28,althoughother
odd-numberedversionsarepossibleinselectsystemsorsyntheticallyproduced. Fattyacidsthatare
lessthan8carbonatomslongaredefinedasshort-chain,whilethosewithgreaterthan12carbonatoms
arelong-chainfattyacidsandmedium-chainfattyacidshavebetween8and12carbonatoms[13,47,48].
Anotherimportantparameteristhenumberofdegreesofunsaturation. Insaturatedfattyacids,all
the carbon atoms are linked by single covalent bonds, while unsaturated fatty acids have one or
moredoublebonds(degreesofunsaturation)inthecarbonbackbone. Specifically,unsaturatedfatty
acidshavingmorethanonedoublebondareidentifiedaspolyunsaturatedfattyacids. Thepresence
of double bonds, including the number of them and their orientation (cis- or trans-) can lead to
significantlydifferentphysicochemicalpropertiesoffattyacids,evenamongcompoundshavingthe
samehydrocarbonchainlength. Thus,classifyingfattyacidsandtheirderivativesisanimportantpart
ofinvestigatingtrendsinantibacterialactivitywithrespecttomolecularstructureandshape. Tothis
end,extensivemicrobiologicalstudieshavebeenconductedandunsaturatedfattyacidswithmedium
andlongerchainstypicallyshowgreaterefficacyagainstGram-positivebacteriathanGram-negative
bacteria[49,50]. Somestudieshavealsobeenreportedthatfocusonassessingantibacterialpotencyin
thepresenceofcompoundswithdoublebondsandrelatedproperties[27,50–53].
In past works, the potency of saturated fatty acids has also been examined as a function of
hydrocarbonchainlength[14,30,50,53–55]. Bysystematicallyinvestigatingsaturatedfattyacidswith
hydrocarbon chains ranging between 6 and 18 carbons long, it was identified that LA, which has a
12-carbon long chain, exhibits the most potent activity to inhibit growth of Gram-positive bacteria,
includingStaphylococcusaureus,amajorcausativeagentofbacterialskininfectionsaswellassystemic
ones.Its1-monoglyceridederivative,GML,showedevengreaterpotencyagainstS.aureus,asindicated
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byalowervalueoftheminimuminhibitoryconcentration(MIC)incomparisontoLA[14].Inaddition,
capricacidanditsmonoglyceridederivative,monocaprin,havesaturatedhydrocarbonchainsthatare10
carbonslong,andalsohavehighantibacterialactivity,especiallyagainstGram-negativebacteriathatare
commonlyassociatedwithfoodborneinfectionssuchasCampylobacterjejuni[56].Theconnectionbetween
antimicrobiallipidandantibacterialspectrumandpotencyisfurtherdiscussedinthenextsection.
Figure1. Chemicalstructuresoffattyacidsandmonoglycerides. Saturatedfattyacids;Capricacid
(C10:0),LA(C12:0). Monoglycerides; Monocaprin(MGC10:0),Glycerolmonolaurate(MGC12:0).
Unsaturatedfattyacids; Oleicacid(C18:1),Elaidicacid(trans-C18:1). Polyunsaturatedfattyacids;
Linoleicacid(C18:2),Linolenicacid(C18:3).Cx:yisdefinedsuchthatxisthenumberofcarbonsinthe
primaryalkylchainandyisthenumberofdegreesofunsaturation.
2.2. SpectrumofAntibacterialActivity
Broad-spectruminhibitoryactivityofantimicrobiallipidsagainstalgae,bacteria,fungi,protozoa,
and virus has been reported for several decades [13–15,23,57–59]. The antibacterial potency of fatty
acidsandmonoglyceridederivativeshasbeeninvestigatedextensivelyagainstawiderangeofbacteria,
includingpathogenicstrainssuchasmethicillin-resistantStaphylococcusaureus(MRSA),aspresented
inTable1. ExtensivescreeningoftheantibacterialactivitiesoffattyacidswasconductedbyKabara
andcolleaguesinthe1970s,andhelpedtoestablishthemodern-dayfieldofantimicrobiallipidsfroma
chemicalperspective.Fattyacidswithhydrocarbonchainsrangingfrom6to18carbonslongandselected
derivativeswithdifferentfunctionalizedheadgroupswereevaluated,resultinginthecomprehensive
identificationofLA(C12:0)asthemostpotentantimicrobiallipidtoinhibitgrowthofGram-positive
bacteria,asmentionedabove.Asageneralprinciple,theesterificationofafattyacidtoitscorresponding
monoglyceridederivativeincreasesantibacterialactivity[14,15,17].Anothersupportivestudyforthehigh
antibacterialpotencyofLAwasconductedwithdifferenttypesofGram-positivebacteria,andfurther
demonstratedthatunsaturatedfattyacidswith18carbonlongchains—oleicacid(C18:1),linoleic(C18:2),
andlinolenicacid(C18:3)—havepotentantibacterialactivitiesaswell[50].
Basedonthefindingsfromthesepioneeringstudies,moreselectiveanddetailedstudiesfocused
on either specific bacterial type or particular antimicrobial lipids have been performed[27,52,60,61].
Specifically, interest on fatty acids and monoglycerides as potential therapeutic agents and/or
preservatives against medically important pathogens led to the following studies of biomedical and
foodsciencerelevance.Wangetal.investigatedtheefficacyoffattyacidsandmonoglyceridesagainst
Listeriamonocytogenes,whichisaGram-positivebacteriumthatcausesaseriesoffood-borneinfections.
Ofthetestedfattyacidsandmonoglycerides,LA,linolenicacid,andGMLhadthestrongestbactericidal
activityat10–20µg/mLinbrainheartinfusionbrothatpH5.Interestingly,thebactericidalactivityofthe
fattyacidsdependedonsolutionpH,showingincreasedactivityatpH5ascomparedtopH6,whilethe
activityofthemonoglyceridewasnotinfluencedbysolutionpHacrossthisrange[28].Thisisconsistent
withtheionizableheadgroupoffattyacidswhilemonoglyceridesarenonionicmolecules.
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Table1.Antibacterialactivityoffattyacidsandmonoglyceridesagainstdifferentbacteria.
Bacteria(*) FattyAcid[FAs]/Monoglycerides[MGs]† KeyFindings Ref.
B.megaterium(+)
B.mycoides(+)
B.subtilis(+)
Bacillussp.(+)
Strep.faecium(+) • Amongsaturatedfattyacids,LA(C12:0)wasthemostpotentagainst
Strep.lactis(+) [FAs‡]: Gram-positivebacteria.
Staphylococcussp.(+) C8:0,C10:0,C12:0,C14:0,C16:0,C18:0,C18:1, • Unsaturatedfattyacids,linoleic(C18:2)andlinolenicacid(C18:3),weremore [50]
Micrococcussp.(+) trans-C18:1,C18:2,C18:3 potentthanLA.
M.lysodeikticus(+)
Cl.butyricum(+)
Cl.sporogenes(+)
Cl.welchii(+)
Pneumococci(+)
StreptococcusgroupA(+)
[FAs]: • LAwasthemostpotentsaturatedfattyacidsagainstGram-positivebacteria.
Streptococcusbeta-hemolyticnon-A(+)
Corynebacteriumsp.(+) C6:0,C8:0,C10:0,C12:0,C14:0,C16:0,C18:0, • Monocaprin(MGC10:0)andGML(MGC12:0)hadgreaterantibacterial
N.asteroides(+) C14:1,C16:1,C18:1,trans-C18:1,C18:2, activitythanfattyacidequivalents. [14]
Micrococcussp.(+) trans-C18:2,C18:3,C20:4 • GMLhadmorepotentactivitywithalowerMICvaluethanLAagainstmost
[MGs§]:
S.aureus(+) Gram-positivebacteria.
C10:0,C12:0
S.epidermidis(+)
StreptococcusgroupD(+)
Strep.faecalis(+) [FAs]: • LAandGMLwerethemostpotentantibacterialcompoundsamongthose
Strep.pyogenes(+) C11:0,C12:0,C13:0 testedagainstGram-positivebacteria.
S.aureus(+) [MGs]: • Esterificationoffattyacidstomonoglycerideformgenerallyincreased [15]
Corynebacteriumsp.(+)
C11:0,C12:0,C13:0 antibacterialactivity.
N.asteroides(+)
• Mosttestedunsaturatedfattyacidsshowedpotentbactericidaleffectagainst
[FAs]: M.smegmatis.
M.smegmatis(+) C10:0,C12:0,C14:0,C16:0,C18:0,C20:0, • Amongsaturatedfattyacids,onlyLAandmyristicacid(C14:0)showedsome [60]
C16:1,C18:1,C18:2,C20:4 degreeofantibacterialactivityat0.2mMconcentration,whichismuchweaker
thanthatofunsaturatedfattyacids.
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Table1.Cont.
Bacteria(*) FattyAcid[FAs]/Monoglycerides[MGs]† KeyFindings Ref.
S.aureus(+) • AlltestedGram-positivespeciesweresusceptibletotreatmentwith0.01mM
L.acidophilus(+) arachidonicacid(C20:4).
B.megaterium(+) [FA]: • L.acidophiluswasmostsusceptibleamongGram-positivebacteria. [27]
H.influenzae(−) C20:4 • BactericidaleffectofarachidonicacidtreatmentonS.aureusdependedon
N.gonorrhoeae(−)
treatmenttimeanddrugconcentration.
E.coli(−)
[FAs]: • LA,linolenicacid,andGMLexhibitedstrongantibacterialactivityagainstL.
C12:0,C14:0,C16:0,C18:0,C18:1,C18:2, monocytogenesat10–20µg/mL.
L.monocytogenes(+) C18:3 • BactericidalactivityofLAandlinolenicacidinbrainheartinfusionbrothwas [28]
[MGs]
higheratpH5thanpH6.
C12:0,C14:0,C16:0
[FAs]: • LAandmyristoleicacid(C14:1)showedmostpotentactivityagainstB.larvae.
B.larvae(+) C10:0,C11:0,C12:0,C13:0,C14:1,C16:1, • Saturatedfattyacidswithgreaterthan14-carbonlongchainsdidnotinhibit [52]
C18:2,etc. bacterialgrowth.
[FAs]: • Saturatedmonoglycerideswith10–14carbonlongchainsshowedbactericidal
C4:0,C5:0,C6:0,C7:0,C8:0,C9:0,C10:0, activityagainstH.pyloriwithmorethan99.99%reductionupontreatment
C11:0,C12:0,C13:0,C14:0,C15:0,C16:0, with1mMcompound.
H.pylori(−) C17:0,C12:1 • LAwasuniqueamongtestedsaturatedmedium-chainfattyacids,tohave [62]
[MGs]: antibacterialactivity.
C4:0,C5:0,C6:0,C7:0,C8:0,C9:0,C10:0, • Medium-chainmonoglyceridesinactivatedH.pylorieffectivelyandtherewas
C11:0,C12:0,C13:0,C14:0,C15:0,C16:0,
lessspontaneousresistancedevelopment.
C17:0,C12:1
[FAs]: • LA,capricacid(C10:0),andmonocaprinhadpotentantibacterialactivity
C8:0,C10:0,C12:0,C14:0,C16:1,C18:1 againstC.trachomatisupontreatmentwith10mMcompound.
C.trachomatis(−) [MGs]: • MonocaprinwasmostpotenttokillC.trachomatis,assuggestedby [29]
C8:0,C10:0,C12:0,C16:1,C18:1 destabilizationofthebacterialmembraneofelementarybodies.
[FAs]: • MonocaprinwasthemostpotenttoeffectivelykillN.gonorrhoeae.
N.gonorrhoeae(−) C8:0,C10:0,C12:0,C14:0,C16:1,C18:1 • LAandpalmitoleicacid(C16:1)hadbactericidalactivityagainstN. [63]
[MGs]: gonorrhoeae,asdeterminedbytreatmentwith2.5mMtestcompound.
C8:0,C10:0,C12:0,C14:0,C16:1,C18:1
[FAs]: • LA,palmitoleicacidandmonocaprinshowedstrongantibacterialactivity
StreptococcusgroupA(+) C8:0,C10:0,C12:0,C14:0,C16:1,C18:1 againsttestedStreptococcusspp.with5mMcompoundtreatment.
StreptococcusgroupB(+) [MGs]: • MonocaprinhadsignificantantibacterialactivityagainstS.aureusaswellas [30]
S.aureus(+) C8:0,C10:0,C12:0,C14:0,C16:1,C18:1 theStreptococcusspp.
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Table1.Cont.
Bacteria(*) FattyAcid[FAs]/Monoglycerides[MGs]† KeyFindings Ref.
• Testedcompoundswith10–16carbonlongchainswereactiveagainstH.pylori
H.pylori(−) [FAs]: upontreatmentwith10mMcompound,whilelargelyinactiveagainst
E.coli(−) C8:0,C10:0,C12:0,C14:0,C16:1,C18:1 Salmonellaspp.andE.coli. [64]
Salmonellaspp.(−) [MGs]: • MonocaprinandGMLshowedhighestlevelsofinhibitoryactivityagainst
C8:0,C10:0,C12:0,C14:0,C16:1,C18:1 H.pylori.
• Amongsaturatedfattyacids,LAwasthemostpotentbactericidalcompound
[FAs]: againstH.pylori,withanMBCvalueof1mM.
C4:0,C6:0,C8:0,C10:0,C12:0,C14:0,C16:0, • GMLwasthemostpotentmonoglycerideandhadalowerMBCvalue
H.pylori(−) C14:1,C16:1,C18:1,C18:2,C18:3 thanLA. [54]
[MGs]: • Amongunsaturatedfattyacids,myristoleicandlinolenicacidhadthemost
C12:0,C14:0,C16:0
potentantibacterialactivity.
[FAs]: • Caprylic(C8:0)andcapricacidshowedantibacterialactivityagainstE.coli;
E.coli(−) C2:0,C3:0,C4:0,C5:0,C6:0,C8:0,C10:0, caprylicacidhadhighestactivity. [65]
C12:0,C14:0,C16:0,C18:0,C18:1,C18:2 • BactericidaleffectofthetwofattyacidswashigheratpH5.2.
S.enteritidis(−) [FAs]: • CaprylicacidaloneshowedantibacterialactivityagainstSalmonellaspecies
S.infantis(−) C2:0,C3:0,C4:0,C5:0,C6:0,C8:0,C10:0, underlowerpHconditionsaround5.2–5.3. [66]
S.typhimurium(−) C12:0,C14:0,C16:0,C18:0,C18:1,C18:2
S.aureus(+)
Methicillin-SusceptibleStaphylococcusaureus [FAs]: • LAwasmostpotentsaturatedfattyacidsagainstMSSAandMRSAstrains
(MSSA)(+) C8:0,C10:0,C12:0,C14:0,C16:0,C18:0 andinhibitedtheirgrowthat400µg/mLtestconcentration. [19]
Methicillin-ResistantStaphylococcusaureus
(MRSA)(+)
[FAs]: • LAwasthemostpotentfattyacidagainstC.perfringensandmaintainedits
C.perfringens(+) C2:0,C3:0,C4:0,C5:0,C6:0,C8:0,C10:0, activityatpH>6,whilecapricacidwasonlyactiveatpH5.0–5.3. [31]
C12:0,C14:0,C16:0,C18:0,C18:1,C18:2
L.garvieae(+)
V.harveyi(−) [FAs]: • Unsaturatedfattyacidsshowedgreaterbactericidaleffectontestedbacteria,
V.anguillarium(−) C15:0,C16:0,C17:0,C18:0,C22:0,C18:1, especiallyGram-negativeVibriospp.,thansaturatedfattyacids. [67]
C18:4,C20:4,C20:5,C22:4,C22:5
V.alginocolyticus(−)
Int.J.Mol.Sci.2018,19,1114 9of40
Table1.Cont.
Bacteria(*) FattyAcid[FAs]/Monoglycerides[MGs]† KeyFindings Ref.
B.cereus(+)
S.aureus(+) [FA]: • LinolenicacidhadthepotentantibacterialactivityagainstB.cereusand
E.coli(−) C18:3 S.aureus.
V.parahaemolyticus(−) [MGs]: • Combinationoflinolenicacidandmonoglyceridesshowedsynergistic [68]
S.typhimurium(−) C12:0,C14:0 antibacterialeffectcomparedtotreatmentwithlinolenicacidalone.
S.enteritidis(−)
Strep.iniae(+) [FA]: • Antibacterialacitityofcaprylicacidanditsmonoglyceride,monocaprylatews
E.ictaluri(−) C8:0 investgatedagainstbacterialfishpathogensandshowedpotentefficacy.
E.tarda(−) [MG]: • Monocaprylateshowedgreaterantibacterialacitivtyinthe2.5–5mMrange [69]
Y.ruckeri(−) C8:0 thanthatofcaprylicacid.
P.acnes(+) • LAhadgreaterantibacterialactivityagainstP.acnesthanbenzoyl
S.aureus(+) [FA]: peroxide(BPO). [20]
S.epidermidis(+) C12:0 • LAwasnotcytotoxicagainsthumansebocytes.
S.aureus(+) • GMLwasinvestigatedasapreservativeagainstfood-relatedpathogens.
B.subtilis(+) [MG]: • AntibacterialactivityofGMLwassynergisticwiththeadditionofnisinagainst [70]
C12:0
E.coli(−) testedbacteria.
[FA]: • GMLhad200-foldgreaterbactericidalpotencythanLAagainstS.aureus.
S.aureus(+) C12:0 • Strep.pyogeneswasmoresusceptibletoGMLtreatment,ascomparedto [21]
Strep.pyogenes(+) [MG]: S.aureus.
C12:0
S.aureus(+)
B.cereus(+) [MGs]: • MGC11:0andMGC11:1effectivelyinhibitedgrowthofS.aureusandB.cereus,
E.coli(−) C11:0,C11:1 butwereineffectiveagainsttestedGram-negativebacteria. [71]
P.aeruginosa(−)
[FAs]: • Caprylicacidandmonocaprylatehadsignificantantibacterialacitivtyagainst
C.sakazakii(−) C6:2,C8:0,C10:0,C12:0 Cronobacterstrains.
C.malonaticus(−) [MGs]: • Bactericidalactivitiesofmonocaprylatearedependentoncompoound [72]
C6:2,C8:0,C10:0,C12:0 concentrationandtemperature.
*(+)indicates“Gram-positivebacteria”and(−)indicates“Gram-negativebacteria”;†[numberofcarbonatomsinalkylchain:numberofdoublebonds];‡FAindicates“fattyacid”;§MG
indicates“monoglyceride”.
Int.J.Mol.Sci.2018,19,1114 10of40
Another target bacterium that is susceptible to fatty acids is Helicobacter pylori, which is a
Gram-negativebacteriumthatcanbepathogenicinthestomachorduodenumleadingtodiseases
such as chronic gastritis, gastric ulcers, and stomach cancer. Petschow et al. tested a wide range
ofsaturatedfattyacidsandcorrespondingmonoglycerideswithchainlengthsfrom4to17carbons
against H. pylori and observed the following trend in bactericidal activities. Monoglycerides with
10–14carbonlongchainsshowedappreciablebactericidalefficacyat1mMconcentrationandhad
a lower tendency of spontaneous resistance development, while LA was the only saturated fatty
acid that had bactericidal potency under the tested conditions [62]. Similarly, the anti-H. pylori
activity of LA and GML was demonstrated by Sun et al., in which case selected unsaturated
fatty acids were evaluated along with a range of saturated fatty acids (4–16 carbon chains) and
monoglycerides(12–16carbonchains)[54]. LAcompletelykilledH.pyloriatminimumbactericidal
concentration(MBC)of1mM,andGMLshowedevenmorepotentactivitythanLAwithalower
MBC value around 0.5 mM. Additionally, it was identified that, among unsaturated fatty acids,
myristoleic (C14:1) and linolenic acid have the most potent activity against this bacterium [54].
BergssonandcolleaguesalsoassessedinhibitoryactivityagainstGram-negativebacteria,including
H.pylori,forawiderangeoffattyacidsandcorrespondingmonoglycerides[64]. Interestingly,the
testedcompoundsthathadhydrocarbonchainsbetween10–16carbonslongexhibitedinactivation
of H. pylori at 10 mM concentration, however, there was no significant activity observed against
Salmonella species and Escherichia coli. Bergssonetal. conducted additional studies in order to
characterizetheantibacterialpotencyofselectedantimicrobiallipidsagainstdifferentbacteria,andit
wasdemonstratedthatmonocaprin(MGC10:0)isthemostpotentbactericidalagentagainstChlamydia
trachomatis and Neisseria gonorrhoeae [29,63]. Additional studies have been reported investigating
theeffectsoffattyacids(withchainsof2to18carbonlength)onGram-negativebacteria,including
E.coli[65]andSalmonellaspecies[66],anddemonstratedthatcaprylicacid(C8:0)hasparticularlyhigh
antibacterialactivityagainstthesebacteria.Antibacterialactivityofcaprylicacidanditsmonoglyceride,
monocaprylate,werealsoidentifiedagainstfishpathogensincludingEdwardsiellaspeciesbyKollanoor
and colleagues [69]. Of particular note, caprylic acid and monocaprylate have showed potent
inhibitoryactivityagainstimportantfoodbornepathogens,includingE.coliO157:H7[73–77],along
withSalmonellaspecies[78,79],L.monocytogenes[80,81],andmastitispathogens[82].
Ofrelevancetoskininfection, S.aureusandMRSAarealeadingcauseofskinandsoft-tissue
infections,includingacutebacterialskinandskinstructureinfections. Withinthisscope,Kitaharaetal.
investigatedarangeofsaturatedfattyacidsagainstconventionalS.aureus,methicillin-susceptible
Staphylococcusaureus(MSSA)andMRSA,anditwasidentifiedthatLAisthemostpotentsaturated
fattyacidagainstMSSAandMRSA[19]. PotentinhibitoryactivityofGMLhasalsobeenreported
against S. aureus in the following studies. Lin et al. demonstrated that GML killed the bacteria
effectivelybothinvitroandinvivoandreducedtoxicshocksyndrome[83]. Anotherinvivostudy
performedbyPreussetal. confirmedtheantibacterialactivityofGMLagainstS.aureusinfection[84].
Inaddition,Nakatsujietal. exploredthefeasibilityofemployingLAtotreatamousemodelinfection
causedbyanotherGram-positivebacterium,specificallyPropionibacteriumacnes[20]. Hence,multiple
antimicrobiallipidshaveshowninvivotherapeuticpotential,andthereisalsogrowinginterestto
investigatecombinationsofseveralantimicrobiallipidsthathaveantibacterialpotencyagainstspecific
bacteriainordertoachievesynergisticandbroad-spectrumeffects[85–89].
2.3. MechanismsofAntibacterialActivity
Themechanism(s)ofantibacterialactivityoffattyacidsandmonoglycerideshavebeenexplored
usingdiverseexperimentalmethodstoidentifythattheymainlytargetbacterialcellmembranesand
interruptcrucialprocessesinvolvedincellularprotectionandfunction. Themembrane-lyticbehavior
offattyacidsandmonoglyceridesstemfromtheiramphipathicproperties,leadingtooverlapping
setsofbiophysicalphenomenaincludingmembranedestabilizationandporeformation. Inparticular,