This document discusses the antibacterial activity of honey. It begins by providing background on honey's increasing acceptance in medicine to treat infections. It then summarizes key points from the document, including that there is significant variation in the antibacterial potency of different honeys depending on factors like floral source and handling. The document also notes that honey is a complex substance with various chemical, physical and biological properties that contribute to its antibacterial effects and potential medical applications.

1. The Antibacterial Activity of Honey
Introduction
Honeyisgainingacceptance bythe medical professionforuse asan antibacterial agentforthe
treatmentof ulcersandbedsores,and othersurface infectionsresultingfromburnsandwounds. In
manycases itis beingusedwithsuccessoninfectionsnotrespondingtostandardantibioticand
antiseptictherapy.Itseffectivenessinrapidlyclearingupinfectionandpromotinghealingisnot
surprisinginlightof the large numberof researchfindingsonitsantibacterialactivity.
None of the reportsin the medical literature,however,mentionanyselectionof the honeyusedforthe
treatmentof infections.Althoughitisrecognizedthathoneyhasantibacterial activity,itisnotgenerally
realizedthatthere isa verylarge variationinthe antibacterial potencyof differenthoneys,andthatthe
antibacterial propertiescanbe easilylostbyinappropriate handlingandstorage of honey.Part2 of this
reviewcoversthe researchthathasbeendone onthese aspects:givingregardtothese findingsshould
resultina more rational usage of honeyinmedicineandallow itsfull potentialasan antibacterial agent
be achieved.
History of the use of honey in Medicine
Today honeyismainlyknownforits sweeteningcapacityandas a desirable natural foodproduct;
howeverithasnot alwaysbeenso.Inancientcommunitiesitwasregardedasan importantmedical
treatmentforall kindsof healthproblems(Zaghloul etal., 2001). Honeyandotherbee products have
beenimportanttoMankindfor manymillennia,where ithasbeenusedbymaneitherforfood,or for
medical purposes.Consequentlybeesgainedanearsacred place insome religions.Itisdifficulttobe
accurate aboutwhenthe relationbetweenManandbee firststarted,butsince Man startedto express
himself throughcave paintings,beesare picturedandthe act of honeyhuntingdepicted(Crane,2001).
The characteristics of honey
Honeyisthe productof the honeybeesprocessingof the nectaror honeydewfromfloweringplants.
Nectaris a sugar solutionproducedbythe glandsof flowers(the nectaries) thathasfunctionsinthe
attractionof insectsandbirdsto visitthe flowertoallow cross-pollination.Once the honeybee collects
the nectar itis transportedbackto the hive inthe bee'snectarsack where itundergoesseveral
transformations.Whenthe bee arrivesinthe hive the nectaristransferredtothe honeysacof a worker
bee that will be responsible fortakingitintothe honeycombs.Here therewillbe aseriesof
regurgitationsandingestions,wherebythe bee will addsome enzymestothe nectarandmoisture
contentisreduced.The honeyproducedfromhoneydew (asugarsolutionexcretedbyinsectsfromthe
orderRhynchota) (Crane 1975) has differentcharacteristicstonectarhoneyas italreadycontains
enzymesaddedbythe insect.
Chemical characteristics
Honeyisa complex substance,made upof at least181 differentsubstancesknownatpresent,
(Jones,2001);withsome researchersbelieving thatthe numberisactuallycloserto600 different
substances (Bogdanov et al., 2004). Honey is a saturated or supersaturated sugar solution,

2. meaningthatit possessesahigh concentrationof sugar(withapproximately17% wateronaverage).
The main sugarspresentinhoneyare fructose (anaverage of 38%), glucose (-31%) anddisaccharides
like maltose (7.3%) andsucrose (1.3%),andhighersugars(1.5%) (White,Jr.1979). The presence of
fructose andglucose inhoneyisdue to the actionof the bee enzyme invertase onthe sucrose molecules
containedinnectar,producingaratio betweenglucoseandfructose of 1.2: 1 (Anklam, 1998).Starch has
alsobeenrecoveredfromhoney,and thisissolelyaproductof the processingof the nectar,as it isnot
presentinthe raw nectar.
Honeyisalsorich in organicacids,withat least30 differentorganicacidsbeingrecoveredfromthis
product,among the mostcommonare gluconicacid,aceticacid, citricacid,lactic acid,succinicacidand
formicacid (Mato et al., 2003). These acidsare the resultsof the actionof enzymeslike glucoseoxidase
on the sugars presentinhoneyandmakeupanaverage of 0.50% of the honeybyweight(Mato et
al.2003). The organic acidspresentinhoneyare believedtocontribute tothe organolepticproperties
such as flavourandcoloras well contributingtophysical andchemical characteristicssuchaspH, acidity
and electrical conductivity(Mato etal., 2003). Inthe 1930s citricand malicacidwere thoughtto be the
predominantacidsinhoney,butin1960 gluconicacidwas shownto be the predominantform.This
organicacid isderivedfromtwosources,the actionof the enzyme glucose oxidaseandthe metabolic
activityof certain Glucobacterspp bacteria(presentinthe bee'sgut).The concentrationof thisorganic
acid dependsonthe time neededforthe manufacture of the honey,strengthof the bee colonyandthe
qualityof the nectarto be transformed.Other organicacidsare eitherintermediatesof the Krebscycle
or productsof enzymaticpathways,andassuch can differsignificantlyfromhoneytohoney.Hence
variationsinthe organicacidspresentinhoneycan be usedas indicatorsof deterioration,authenticity
and purity(Mato et al., 2003).
Anotherimportantcharacteristicof honeyisitslow pH,the normal pH rangesbetween3-6.Thisacidity
isthoughtto be causedbythe presence of the differentorganicacidsinthe honey,andisone of the
factors limitingthe growthof microorganisms(CeyhanandUgur2001).
Honeypossessesalowamountof nitrogen(0.041% w/v) thatispart of proteins,enzymesandfree
aminoacids.The amount of nitrogenouscompoundspresentinhoneymayaffectitscharacteristics; the
highproteinconcentration(2%w/v) inheatherhoney,forexample isresponsible foritsviscous
characteristics.Estimationof proteininhoneybythe volume of precipitate withtanninhasbeenusedin
the past to distinguishbetweenhoneyandartificialblends(White,Jr.andRud),1978).
The presence of enzymesinhoneyisimportant,asthese aidthe transformationof the nectarinto
honey,some of the mostcommonenzymesrecoveredinhoneyare invertase,catalase,phosphatase
glucose Oxidase anddiastase ( Crane 1975). The mineral componentof honeyisreferredtoasthe cash
portionandmakesup about0.1% of all the componentsinhoney.Ashismore abundantindarker
honeysandmonofloral honeystendtohave lowerashcontent(Crane 1975). Potassiummakesup
aroundhalf of the total ash contentinhoney,andothermineralsfoundare calcium, copper,sodium,
magnesium,manganese andchlorine salts.Another30 differentmineralcomplexesmaybe usedto
determine floral originof the honeysastheyare characteristicforeachplant(Anklam1998).

3. Honeycontainstrace amount of vitamins,flavonoids,antioxidantcomponentsandunidentifiedplant
derivedelements(phytochemical components) (SatoandMiyata,2000). It also possessesrace amounts
of otherbeehiveproductslikepropolis,royal jellyandwax of whichthe firsttwoare recognisedas
antimicrobial agentsaswell (Anklam1998).
The actual chemical profile variesconsiderablyfromhoneytohoney,dependinguponthe floral originof
the nectar and evenonthe yearand time of year inwhichthe honeyiscollected(White,Jr.andRudA
1978). The type of bee producingthe honeyalsochangesitschemical characteristics(DeMeraand
Angert2004), and there are certainchemical markersthatare usedto determineif the honeyis
authentic(thatisif it hasbeenalteredinanyway,like the additionof extrasugaror water),like protein,
moisture,sugar,andhydroxyrnethylfurfural (HMF) content(MateoandBosch-Reig,1998).HMF isa
productof sugar breakdowninhoneyasa resultof heatingorstorage,so thissubstance isuseful to
determine if the honeywasheatprocessedoraged.
Usuallyhoneysusedformedical purposes,since the firstrecordings,tendedtobe local honeysthat
were producedmainlyfromthe nectarcollectedfromone predominantflowersource.Analysisof the
pollencontentof honeyismainlyusedtodeterminethe predominantfloralspeciesthatthe bee has
foranged,howeverasthe compositionof honeysisfurthercharacterisedchemically,othermarkers,like
specificproteinsororganicacidsare beingusedforhoneyclassification(Mato etal., 2003). Unifloral
honeysare largelyderivecdfromasingle floral source anduseful becausetheircharacteristicscanbe
betterdefinedthanthose of honeysthatcome frommore than one floral source (multifloral donot
demonstrate apredominantfloral source whenthe pollenisanalysed).
Physical characteristics
Honeyisa supersaturatedsugarsolutionwithahighosmolarity(Wahdan1998); itwill exertahigh
osmoticpressure onbacteriabecause watermoleculeswill be largelyboundtosugarmolecules,making
themunavailable forthe growthof mostmicroorganisms(one of the reasonswhyhoneyisnoteasily
spoiled) (CeyhanandUgur2001). Hence honeyhasa broadspectrumantimicrobial activity.Honeyis
usuallyaviscoussubstance (viscosityrangesfrom3.10 poise foralfalfahoneyto4.11 poise forsumac
honey),varyingwiththe temperature atwhichitismeasured(White,Jr. etal., 1963). This isdue to its
highsugar concentrationandproteinprofile(Apimondia2001), whichgivesitpropertiesthatmake it
desirable foruse inthe topical treatmentof infections.Itwill actas a barrierwhenappliedtowounds
(Molan2001a), protectingthe woundfromexternal contamination,aswell aslimitingthe releaseof
microbes.Otherphysical characteristicsthatcan helpinthe identificationof the differenthoneytypes
are colour(coloursrange fromwhite toalmostblack),the thennal conductivity(helpsdistinguish
betweenfloraland honeydewhoneys) andhygroscopicity(the capacityof eachhoneytoabsorb
humidity) (Crane 1975).
Honeyand the searchfor newantimicrobial agents, when antibiotics were first discovered,theywere
regardedas the cure for all infections.Forsome time thisseemedtobe the case,withdeathsfrom
infectiondrasticallybeingreduced,butearlyonthere were some dangersignswiththe firstpenicillin
resistantbacteria,more preciselyStaphylococcusaureus,beingisolated in1944 by Kirbyandby the
1950's penicillinresistance wasalreadycommoninUKhospitals(Gosbell,2003).In orderto fightthe

4. emergence of penicillinresistance,semisyntheticpenicillinswere introducedinthe UK,the firstbeing
benzy1penicillinfollowedafterbymethicillinin1959 but thiswassoonfollowed(1980s) byreportsof
methicillinresistance(Perez-Rothetal., 2001) and whenvancomycinwasintroducedtoeveryday
practice,as an antibioticof lastresort,reportssoonfollowedaboutthe emergence of resistance tothis
as well in1998 (Gosbell,2003). Withinone yearof the approval of oxazolidinones(anew classof
syntheticantimicrobials) bythe FDA resistance hasemerged,makingtreatmentof Staphylococcus
infectionsdifficult(Schmitz etal., 2000). The increasingprevalence of differentantibioticresistant
bacteriainboth hospital andcommunity,andthe decreasedrate of discovery/developmentof new
antibiotics(Greenwood1995), makesthe discoveryof alternativestreatmentsnecessarytocombat
resistantbacteria(DzidicandBedekovic2003).
Multiresistant strains or superbugs
"Superbugs"ishowthe mediareferstoantibiotic-resistantbacteria.Althoughithasbecome agreat
concernrecentlytothe general public,withmediatitles like "Superbughitsthe healthy"(Le Page,2003)
or "Superbugcrackdownislaunched"(Collignon,2002),the truthisthat antibiotic-resistantbacteria
alwaysexistedinnature butthattheywere discoveredsoonafterthe introductionof antibioticsinto
clinical practice (Gosbell2003). Some of the bacterial specieswithantibioticresistancecausingthe
greatestconcernsare methicillin-resistantStaphylococcusaureus(MRSA) andmultiresistant
Pseudomonasaeruginosa.There isanatural variance amongall communitiesof livingorganisms,and
thismeansthat ina populationof bacteriathere will alwaysbe those whichare more resistanttosome
antibioticsthanothers(Felmingham,2002);thisphenomenonisinnate resistance.Whenbacteriaare
facedwithan antimicrobial agent,the onesthatare more resistanttendtosurvive betterthan
susceptiblecellsandare able topasstheircharacteristicsonwardto theirprogeny.
Hence laterpopulationsof bacteriaappeartohave an increasedresistanceincomparison tothe initial
population.The processof natural selectionisparticularlyeffective inbacteria,andbecause theyhave
short generationtimes,the resultscanbe observedinonlyrelativeshorttime (Ang etal., 2004a).
Anotherimportantfactoristhat bacterial cellshave ahighspontaneousmutationrate (about10-7per
cell division) (Schmitz etal., 2000). This meansthattheycan change theircharacteristicsrapidly,thus
providingagreatervariationonwhichnatural selectioncanact,whichhelps themtosurvive inever
changingenvironmental conditions.
The indiscriminateuse of antimicrobial agentsthatistheiruse forconditionsthatdonot require their
prescription,andthe lackof compliance withpropertreatmentregimes,hasledtothe emergence by
natural selectionof resistantbacteriainEurope andaroundthe world(Schmidt2004). One of the main
problemsfacedinthe treatmentof antibioticresistantbacteriaisthe factthatsome have become
multiresistant,forexample vancomycin-resistantStaphylococcusaureus (VRSA),sothatinadditionto
beingresistantto vancomycin Propcqlion of MRSA igolatesinparticipatirgocuntriesin2004
Wound care problems
Woundscan simplybe dividedintotwocategories:acute orchronic.Acute woundstend toheal with
minimal medical intervention,while chronicwoundswill notheal withinpredictedtimeframes.Withan
ageingpopulation,especiallyindevelopedcountries,there isaneedtofocuson the problemsthatthis

5. kindof populationentails(Howell-Jonesetal., 2005). One such problemisthe increase inthe incidence
of chronicwoundsdue toa varietyof factors:bad circulation,diabetes andothertypesof insufficiencies
(Collier, 2004). One of the biggestproblemsin woundcare ishow to treat woundsinfected with
antibioticresistantbacteriainpatients thatare usuallyfragile.The optionsatthe momentare the use of
antibioticsand radical surgeryeitherforthe debridement of the woundoramputationof the affected
area. Howeverinpatients thathave a fragile healthstate like the elderlythesemightnotbe viable
options,thus alternativesare required(Eron etal., 2003).
Howeveraggressivetherapyisnotalways well receivedandcompliancemaybe anissue.Undeveloped
countriesalsoface woundmanagementproblemsasaresultof poor livingconditionsandwars.Insome
these of countrieswhere healthresources are limited,andAIDSprevalence high, mortalitydue to
infectedwoundsishighandthe mainmedical resourcesusedare those fromtraditional medicine asthe
cost of treatments ishighand the distributionof drugsis poor(Ryan,2000).This ismade more seriousby
the mobilityof bacterial resistance,whichhasresultedinincreased incidence of antibioticresistance in
countrieswhere antibioticuse isnot widespread.(Schmidt,2004). These two aspectsof woundcare
combinedshowthe needforeffective,cheapalternativeantimicrobial agents.
Factors influencing wound healing
There are manyreasonswhya wound mightnot progresstocomplete healing withinthe expectedtime.
Conditionsthataffecthealingtimesare nutritional status, diabetes,cardiacorrespiratory insufficiency,
ischaernia,infections, antimicrobial therapy,mobility,hydration, age,underlyingillnessesandprevious
immunosuppressivetherapies,suchas chemotherapyandradiotherapy.Thesefactorswill all contribute
to the way in whichthe patient'sbodyrespondstoa woundandwill influence itscapabilityfor healing
(Collier,2004). Althoughsome maybelievethatif a woundhasbacteriait will notheal,mostwounds
supportpolyrnicrobialcommunities(Bowler etal., 2001). The actual presence of bacteriamay
contribute tothe stimulationof the immune responseforarapid woundhealing (Edward-Jonesand
Greenwood,2003).
Furthermore,nowadaysthere isan increasingunderstandingof the difference betweencolonizationand
infection.A woundmay be colonizedbutnotinfected. Infectionstatuswill depend onmany factors, not
onlyon the microbiological resultsbutalsothe actual symptoms presented,asthere are speciesof
bacteria that can existinwoundsandstill allow itto heal (Cooper,2005).
CuttingandWhite (2005) have described the symptomsrequiredforthe diagnosisof aninfection as
being:localisederythrema, localisedpain,localisedheat,cellulitis,oederna,abscessed,ischarge,
delayedhealing,discoloration,friableandbleeding tissue andbadodor.To assesswhethera woundis
infectedornot,besidesthe visualanalysissamplesare collectedeitherby swabsorby woundbiopsy
and the bacteriapresentidentifiedandquantified(Bowler etal., 2001). The interpretationof the
microbiological resultsrequirescaution,as the species/strainsof bacterianeedtobe identified,
numbers,virulence andpossible interactionsbetweenthemneedtobe determined inordertoassess
the needforantimicrobial therapy,andtobe able to provide the righttype of therapyfor the situation
at hand (Howell-Jones etal., 2005; O'Meara et al., 2000). The range of organismsthatcan be recovered
froman infectedwoundislarge andmayinclude anynumberof the following:betahaernolytic

6. streptococci (like Streptococcus pyogenes), enterococci(like Enterococcus faecium orEnterococcus
faecalis),staphylococci(like Staphylococcusaureus, MRSA orStaphylococcusepidermidis), Gram
negative aerobic(like Pseudomonas aeruginosa) andfacultativerods(likeAcinetobacter,Enterobacter,
Escherichiacoli,Klebsiella,ProteusandSerratia species).Anaerobesare alsofound(usually indeeper
wounds) aswell asyeastsand fungi (Cooperetal., 2002). Thismeansthat inmost casesbroad-spectrum
antimicrobial agentsare requiredtoresolve the infection. Infectedwoundstendtohave acomplex
mixedpopulationof microorganisms.This populationiscapable of forming communities,alsoknownas
biofilms, whichallow themtothrive inthe wound environment.Biofilmsare threedimensional
structuresof bacteriaattached to a surface (inthiscase a woundbed) and encapsulatedinslime that
communicate amongeachotherusingchemical signals (quorumsensing).Thisarrangementprotects
themfromphagocytosisandthe actionof antimicrobial agents,makingtheireradicationmore difficult
(Cooper2005). Traditionallymedicinalhoneywouldhave beenof local origin,butfromcarefully
selectedfloral sources(Molan2000). Nowadays, one of the mostpopularhoneysforwound
managementismanukahoney,derivedfromthe manukashrub (Leptospermumscoparium),asithas
beenshowntopossessahighantibacterial activity.Today,honeyscanbe dividedinto twomaintypes:
those withhydrogen peroxide derivedantimicrobial activity,and those withnon-hydrogenperoxide
derivedantimicrobialactivity.All honeyspossess highsugarcontent,low watercontentand low pH
whichhelpstolimitthe growthof microorganisms.Since the recentpublicity of the non-hydrogen
peroxide activityof manukahoney,ithasbeenexportedfrom New Zealandtomanycountriesand
formulatedintoseveral medical products foruse inwoundmanagement.These include non-adherent
impregnated dressings;alginate andhoneywound dressings,ointmentsandsterilizedhoneyin tubesfor
use withindividual patients. Honeys formedical purposeshave been recommendedtopossessatleasta
nonhydrogen peroxide activityequivalentto 10% (w/v) phenol equivalent(UniquemanukaFactor,or
UMF 10). This standard has beenproposedbecausesome wound environmentspossesslarge volumes
of exudate thatamongothercomponentspossesseshydrogenperoxide-degradingenzymessoinorder
to assure a high antibacterial activityonwoundsof this type, honeyswithahighnon-hydrogen peroxide
activityare preferredasthe antibacterial activityisguaranteed(Molan, 2000).The workpresentedin
thisthesishas demonstratedthathoneyswithnonhydrogen peroxidederivedantimicrobial activity
above UMF 10 have beenfoundin Europe.
The nature of the non-hydrogenperoxide derivedantimicrobialactivityof manuka honeyisstill
unknown.Attemptshave been made atfractionation,andactive fractions werefound,the compositions
of which were neverfullydetermined(Snow and Manley-Harris2004).The complexityof the honey
compositionhasbeenrecognized asone of the factorspreventingafull characterizationof itschemical
composition.There are manycomponents presentatlow concentrationsthatcan interfere withassays
for chemical definitionsandanincreasingnumberof new more sensitive techniquesthatare able to
identifynew components.The wayin whichthese componentsinteractinhoney tocreate the
antibacterial activitythatcan be observedinvitroandinvivois alsonotwell understood.The
productionof honey isnota sterile process.Inthe hive environmentthere are bacteria,yeastsand
mouldthatreadilycontaminate honeybut whichdonotgenerallygrow onhoney,due toitssugar
concentrationandpH (Snowdon andCliver1996).

7. Honey-basedproductsavailableforwound managementare generallymade of gammairradiatedhoney
aftermanufacture and before use, soas to be safe for use inall patients,includingimmuno-
compromised patients,neverthelessthere are reportsof the use of non-sterilehoneybeingas effective
(Molanand Allen,1996).It is possible thatthe bacteriathatare presentin the raw honey(non-sterilised)
can secrete, some of the antimicrobial compoundsthat theyproducedinordertoelicitthe
inhibitionof the growthof the wound pathogenstestedinthiswork,thus contributingtothe overall
antimicrobial activityof the honey.The benefitof honeyforwoundmanagementdoesnotseemsto be
limitedtoitsantimicrobialproperties,as ithas beendescribedasastimulatorof the immune response
inwounds,leadingtoan improvedwoundhealing(Tonks etal.,2003). Thisactivityseemstobe distinct
fromthe honey'santimicrobial activity,as honeywithaweakantibacterial activityhas beenshownto
stimulate the release of cytokinesimportantinthe woundhealing processinvitro(Wheat,2004). In
orderto betterunderstandthe mechanismof actionof honey,inparticular honey,asanantimicrobial
agentin wound management,itsmechanismof actionin twoimportantwoundpathogens,
StaphylococcusaureusandPseudomonas aeruginosa,was investigated,withthe use of kineticsstudies,
resistance training, electronmicroscopy,leakage,oxygen consumptionandmutantandproteome
analysis.All the assayshave revealedthat the mechanismof actionof honeyonGram positive bacteria
isdifferenttothaton Gram negative.Inthe kineticstudieshoney wasshowntobe bactericidal forboth
S.aureus andP.aeruginosa inthe presence orabsence of catalase andalsointhe presence of serum,
whose proteinsare knowntointerferewiththe activityof some antimicrobialagents.The susceptibility
of S.aureus and Pseud.aeruginosatohoney wasdecreasedinthe presence of serum, but bactericidal
activitywasstill recordedat concentrationsthatcouldbe achievable ina clinical setting.
It ispossible toconclude thatthere are manyareas relatingtothe use of honeyin woundmanagement
that require furtherinvestigation,neverthelessthisworkhas attemptedtoshedsome lightintosome of
these.The mainconclusiontobe drawn fromthis work,isthat honey'santimicrobial activityislikelyas
complex asitsowncomposition,andthatitsactivity isdue to a combinationand synergisticactionof
more than one antimicrobial component, ratherthanasingle substance. Mostof all,althoughthisisa
broad spectrumantimicrobialagent,the mechanismsof antibacterial actionamong Grampositive and
negative bacteriaare different.Care mustbe takeninitsuse for medical purposesjustasforany other
agent,so that itremainsa useful wound remedyforanother4000 years. The pilotstudyof the
Portuguese honeys demonstratedthe potentialformedical grade honeytoexist inEurope.Thiswork
needstobe furtherdevelopedwiththe collection,analysisandcharacterizationof abiggernumberof
Portuguese honeys. AlsoaEuropeanNetworkforhoney research,where ideascouldbe sharedanda
biggervolume of samplestestedandcharacterizedcouldbe valuable.The developmentof the results
obtainedin
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