Available
online
at
journalhomepage:www.elsevier.com/locate/hermed
Antibacterial
activity
of
Lantana
camara
L.against
multidrugresistantpathogens
fromICUpatients
ofateaching
hospital
DebasmitaDubey
,RabindraN.Padhy
a
DepartmentofMicrobiology,IMS&SumHospitalMedicalCollege,SikshaOAnusandhanUniversity,KalingaNagar,Bhubaneswar751003,Odisha,India
b
CentralResearchLaboratory,IMS
&SumHospitalMedicalCollege,SikshaOAnusandhanUniversity,KalingaNagar,Bhubaneswar751003,Odisha,India
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received12July2012Receivedinrevisedform23September2012Accepted17December2012Availableonline4February2013
Keywords:
Antibacterialactivity
Lantanacamara
MinimumbactericidalconcentrationMinimuminhibitoryconcentrationMultidrug-resistantbacteriaPhytochemicalanalysis
a
b
s
t
r
a
c
t
Thescientificbasisfortheuseofthecommonshrub-weedplant
Lantanacamara
L.wasinves-tigatedbytestingleafextractsforantibacterialactivity.Driedleafpowderswereextractedusingahot-solventextractionmethodwitheightpolartonon-polarsolventsinsuccession.Crudeextractsweretestedforantibacterialactivityagainstthreemultidrug-resistant(MDR)Gram-positivebacteria:methicillin-resistant
Staphylococcusaureus
(MRSA),
Streptococcuspyo-genes
,
andvancomycin-resistant
Enterococcusfaecalis
(VRE);andfiveMDRextended-spectrum
-lactamase-producingGram-negativebacteria:
Acinetobacterbaumannii
,
Citrobacterfreundii
,
Proteusmirabilis
,
Proteusvulgaris
and
Pseudomonasaeruginosa
.TheMRSAstrainwasresis-tant
to16of18antibiotics,while
Streptococcuspyogenes
andVREwereresistantto15of18antibiotics.Similarly,
A.baumannii
and
P.aeruginosa
wereresistantto14of16antibiotics.Itwas
foundthatplantextractswithpetroleumetherandwaterhadtheleastantibacterialactivity.Leafextractswithdichloromethaneandmethanolregisteredthehighestantibac-terialactivityonallbacterialstrains.Theminimuminhibitoryconcentrationandminimumbactericidalconcentrationoftwoactiveleafextracts,obtainedwithdichloromethaneandmethanolweredetermined.Phytochemicalanalysisofdichloromethaneleafextractscon-firmedthepresenceofalkaloids,glycosides,terpenoids,saponins,flavonoids,andsteroids,butreducingsugarswerealsoabsent;and,inthemethanolicleafextract,alkaloids,ter-penoids,saponins,flavonoidsandsteroidswerepresent,butglycosides,reducingsugarsand
tanninswereabsent.Thesefindingspointtothepotentialoftheplantasaprobablesourceofbioactivecompoundsandprovideascientificbasisforitsfolklore/ethnomedicinalusesforinfectiousdiseases.©2012ElsevierGmbH.Allrightsreserved.
1.
Introduction
Lantanacamara
L.(redsage,Verbenaceaefamily)isashrub-weedcommonlyfoundinSouthAmerica,Africa,AsiaandAustralia,wheretherearetropicalandsubtropicalclimates,andisaprolificweedinKenya.InNigeria,
L.camara
leaf
∗
Correspondingauthor
.Formerlyat:BJBAutonomousCollege,Bhubaneswar,India.Tel.:+916746511205.E-mailaddress:rnpadhy54@yahoo.com(R.N.Padhy).
incinerationistraditionallyusedforrepellingmosquitoes(Egunyomietal.,2010).InIndian
Ayurveda
,
adecoctionoffreshrootoftheplantisusedasabeneficialgargleforodontal-gia.Foralltypesofdysentery,cuts,wounds,andswellings,infusionofpowderedleavesisusedbyhilltribes(Ghisalberti,2000),astheleaveshaveantimicrobialandtermiticidal
2210-8033/$–seefrontmatter©2012ElsevierGmbH.Allrightsreserved.http://dx.doi.org/10.1016/j.hermed.2012.12.002
66
activities(VermaandVerma,2006).Theanti-tubercularactiv-
ityofthisplantagainstmultidrug-resistant(MDR)tuberclebacillihasbeenreportedfromMexico( Jimenez-Arellanesetal.,2003).
Invitro
antibacterialactivityofamethanolicleaf extractof
L.camara
againstthenon-resistantGram-positive(GP)andGram-negative(GN)bacterialstrains
Bacillussub-tilis
,
Staphylococcusaureus
,
Klebsiellapneumoniae
,
Pseudomonasaeruginosa
and
Escherichiacoli
,
hasbeenreportedfromIndia(Udayprakashetal.,2011).Amongthephytochemicalsof
Lcamara
,
lantadines,anthraquinonesandtriterpeneshavebeenreportedtoalsohaveantifungalproperties(Kumaretal.,2006; Julianietal.,2002).However,teratologicaleffectsoftheleavesonratshavebeenrecorded(Melloetal.,2005).
L.camara
hasbeenusedinmany
partsoftheworldtotreatawidevarietyofdisorders(Ross,1999).Ithasbeenused
infolkremediesforcancersandtumours.Leavesandflow-ersoftheplantareusedforpreparingadecoctionthatisusedagainstfever,influenzaandstomach-ache.InCentralandSouthAmerica,leavesaremadeintoapoulticetotreatsores,chickenpoxandmeasles.Fevers,colds,rheumatism,asthmaandhighbloodpressurehavebeentreatedwithprepa-rationsoftheplant(Irvine,1961).InGhana,aninfusionof
thewholeplantwasusedforbronchitisandtherootpow-deredinmilkwasgiventochildrenforstomach-ache(Irvine,1961).InAsiancountries,leaveshavebeenusedtotreatcuts,rheumatism,ulcersandintestinalworms.Decoctionswereappliedexternallyforleprosyandscabies.Ithasbeenclaimedthatasteroid,lancamarone,fromtheleaveshasexhibitedcardiotonicproperties(Sharmaetal.,1992),andlantamine,
analkaloidfromthestem,barkandrootshowedantipyreticandantispasmodicpropertiescomparabletothoseofquinine(Ghisalberti,2000),butthevalidityoftheseclaimshasnot
beenconfirmed.Analkaloidfractionfromtheleaveshasbeenreportedtolowerbloodpressureandtoacceleratedeepres-piration,anditwassuggestedthatthosemightbeusefulinreducingfeversandasthmaattacks(Ghisalberti,2000).More-
over,sixphenoliccompoundsinan
L.camara
extractwereidentifiedbyhigh-pressureliquidchromatography(HPLC):sal-icylicacid,gentisicacid,
-resorcylicacid,coumarin,ferulicacidand6-Mecoumarin(Yietal.,2006).Theseandseveral
otherphytocompoundsoftheplantcouldbecontributing,
a priori
,totherecordedanecdotalhealthbenefits.Infectiousdiseasescausedbydrug/antibiotic-resistantbacterialstrainshavealwaysbeenamatterofclinicalcon-cern.Mortalityduetourinarytractinfections(UTIs)hasbeenofutmostclinicalconcerninthelast4decades,with81%in1973and50%in2006duetoMDR
P.aeruginosa
,forexample(Giamarellos-Bourboulisetal.,2006;Penningtonetal.,1973).Authors,fromtheInstituteofMedicalSci-ences(IMS)andSumHospital,havepreviouslyreportedMDR
P.aeruginosa
astheprimarycandidateforcommunityandhospital-acquiredUTIswithextended-spectrum
-lactamase(ESBL)production(Sahuetal.,2012).Indeed,shortcourses
ofempirictherapy(1–3days)areusuallycompletedforUTIsbeforetestresultsbecomeavailable,promotingmis-matchesduetodrugresistance.Here,theusualtreatmentwouldbetheuseofcefepime,acephalosporinthatistra-ditionallyusedtotreatclinicallyimportantstrainsof
P.aeruginosa
,
asempirictherapyforaUTIinanintensivecareunit(ICU),butwhentheinfectionisconfirmed,theantimicrobialstewardshipprogrammesubsequentlyusesacarbapenem.Notsurprisingly,80–90%ofuncomplicatedUTIcasesworldwideinotherwisehealthyadultsoccurwiththesecond-mostimportantcausativemicrobe,
Acine-tobacterspecies
.
Evencommunity-acquiredinfectionsbyESBL-producing
Acinetobacterbaumannii
werereportedin2007intheU.S.(DeBusscheretal.,2009).Furthermore,
A.bauman-nii
hasbeenapandrug-resistant(resistanttoallantibioticsinuseatpresent)bacterium,precipitatingnumerousglobalcatastrophicepisodeswithanever-increasingresistancepatterntocefepime,ceftazidime,ciprofloxacin,gentami-cin,levofloxacin,piperacillin/tazobactam,andtrimetho-prim/sulfamethoxazole,asreportedfromNorthAmerica,Europe,AsiaPacificandLatinAmerica(Perezetal.,2007).Moreover,inthepastdecade,reportsofoccurrencesof community-acquiredESBL-producing
E.coli
isolates,thethirdmostcommonUTIcausativehaveincreasedworldwide;butitsprevalenceisstilluncommonintheU.S.,exceptfromnoso-comialsources(Naasetal.,2007).Strainsof
E.coli
,
causing enteropathogenicepisodeswerefoundtoberesistantto16antibioticsfromfivegroupsintheauthor’slaboratory(Rathetal.,inpress).Furthermore,
Citrobacterfreundii
,whichisawidelyprevalentnosocomialpathogenassociatedwithmanyinfections,diarrhoea,septicaemia,meningitisandailmentsinurinaryandrespiratorytracts,wasreportedtoberesistanttocarbapeneminChina(Shenetal.,2009).
K.pneumoniae
carbapenemase(KPC)enzymeandmetallo-
-lactamasearethecommonarmamentariaofcarbapenemresistanceinEnterobacteriaceae(Kitcheletal.,2009;Cendejasetal.,2010).InThailand,
Proteusmirabilis
hadbeendemon-stratedtohavemultidrugresistance,forexample,tobroad-spectrum
-lactamderivatives,carbapenems,strep-tomycin,tetracycline,sulphonamides,trimethoprimandfluoroquinolones(Girlichetal.,2001).Morerecently,
P.mirabilis
NKU(NorthernKentuckyUniversity)strainhasbeenrecordedasshowingresistancetoalargenumberofantibiotics,there-fore,theircontrolisverydifficult(Doubletetal.,2010).Gram-negativebacteriaarenaturallytransformableandparticipateinDNAexchangesinnaturaluncleanwaterswhereotherGram-negativebacteriaarealsopresent,sometimeswithdrugresistancemarkers(LorenzandWackernagel,1994;Metzgaretal.,2004).The
A.baumannii
strainADP1hasaremarkablenaturalcompetency100-foldhigherthanCaCl
2
-induced
E.coli
(Metzgaretal.,2004).Pathogenicbacterialstrainsgainmultidrugresistanceduetotheirsimple/plasticgenomesandassociatedDNAexchangeswithotherstrains.Eventually,theMDR
strainsarespreadworldwide,causingdisproportionatemorbidityandmortality.ManyclinicalandsocialfactorsalsoenhancetheproblemofemergenceofMDRpathogens,asdiscussedelsewhere(Sahuetal.,2012).Obviously,thisproblemlargely
affectseconomicandsocial/publichealthfactors,leadingtothenecessityofanurgentsearchforantimicrobialsfromalter-natesources.Plantsprovideapalpablesourceofdrugs.Theefficacyofcrudeextractsofphytochemicalsincontrolling MDRbacteriahasbeendemonstratedby
invitro
studies(Dubeyetal.,2012a;DubeyandPadhy,2012;Arokiyarajetal.,2012).Phytocompoundsincrudeextractshavediversestructural
67
complexityandastheyareofnon-microbialorigin,nomicrobe,nomatterhowwellgeneticallyequippedanddevel-opedcaneverover-ridethesecoalescedchemicals
invitro
.Thesameistrue
invivo
whenthephytocompoundsareacces-sorizedwithregularchemotherapy,sincephytochemicalsincrudeextractsremainasunbreakablebarrierstomicrobialpathogens.Itwouldseemthataccumulatedethnomedicinalreportsofdifferentcountriesareidealisticwithoutanysci-entificverification,butshouldformthebasisoffurtherworkondrugtargetingagainstMDR
pathogens,ashasbeenstatedindetailelsewhere(Dubeyetal.,2012b).Takingrecourseto
plantsfornewchemicals,fromwell-knownandlesser-knownweedsasantimicrobialswouldbeaprudentalternative,notleastbecausethe
Streptomyces
sourceofantibioticsisexhausted,butalsobecausealargeamountofpurephyto-chemicalshasbeenservingthehealthdomainholistically.Indeed,themany
naturalphytochemicalsincrudeextractsencouragesthepreparationofcomplementarydrugsfortar-getingMDR
pathogens.SeveralcommonMDR
pathogens,methicillin-resistant
S.aureus
(MRSA),MDR
P.aeruginosa
,
MDR
A.baumannii
andMDR
tuberclebacillus,needtobecontrolledcarefully.Anon-committalattitudetowardsthesenaturalchemicalswouldbeamistake.Obviously,hosttoxicitytesting remainsanessentialcorollaryindesigningphytochemicalsascomplementarymedicines.Thereisalsoawidelyheldcon-sensusthatphytocompoundscanbeusedasdrugsanditislikelytobeheldinthefuture(see,MacLennanandPendry,2011),evenbytheWorldHealthOrganisation.
L.camara
,
withitsunusuallystrongunpleasantaroma,wasanticipatedtohavecontroloverMDR
bacterialstrains,emulatinganydove-tailedsyntheticdrug.Asareportoftheantibacterialactivityof
L.camara
uponresistantpathogenicstrainswasunavail-ableintheliterature,thisresearchonthreeGPandfiveGNMDR
bacteria,isolatedfromclinicalsampleswasinitiatedtoquantifythe
invitro
controlcapacityofcrudeleafextractsof
L.camara
usingseveralsolvents.
2.
Materials
and
methods
2.1.
Preparations
of
plant
extracts
Driedleavesof
L.camara
(Fig.1)werepowderedandthepow-
derwasstoredatroomtemperatureinair-tightpolythenepacksuntiluse.Leafextractswereobtainedbythehotextrac-tionmethodusingeightnon-polartopolarsolvents.Foreachofthesolvents(petroleumether,chloroform,ethylacetate,dichloromethane,acetone,methanol,ethanolandwater),40g oftheleafpowderwasextractedinasoxhletextractorfor40siphonsorcycles,withavolumeof400mL
ofsolvent.Indi-vidualsolventextractswerefilteredandconcentratedwitharotaryevaporator,runat40
◦
C.Thestickymassesobtainedwere5.67,4.12,3.33,3.56,3.6,3.77,2.32and2.8gofextract/40g ofdryleafpowderforpetroleumether,chloroform,ethylacetate,dichloromethane,acetone,methanol,ethanolandwater,respectively.Eachconcentratedstickymasswasdilutedwithanappropriatevolumeof10%(v/v)dimethylsulfoxide(DMSO)toproducea100mg/mL
stocksolutionthatwasstoredat
−
4
◦
Cuntiluse.
Fig.1–
Lantanacamara
.
2.2.
Isolation
and
identification
of
pathogenic
bacteria
StrainsofeightbacteriawereisolatedfromclinicalsamplesfrompatientsoftheICU,IMSandSumHospital.Sampleswereculturedonsuitablemediaandthebacterialisolateswereidentifiedbyusingstandardbiochemicalprocedures(Table1).CorrespondingstandardMicrobialTypeCultureCol-
lection(MTCC)strainsofeachbacterium(Table1)wereused
asreferencecontrols.ThreeGP[MRSA,
Streptococcuspyogenes
,vancomycin-resistant
Enterococcusfaecalis
(VRE)]andfiveGN(
A.baumannii
,
C.freundii
,
P.mirabilis
,
Proteusvulgaris
and
P.aeruginosa
)bacteriawereisolatedandusedinthestudy.Forpure-culturesofGPcocci,catalaseandcoagulasetestswereperformed.Thecatalasetestwascarriedoutwithadropof3%H
2
O
2
thatcausedeffervescence,indicatingthepres-enceofcatalaseenzyme.Forthecoagulasetest,alumpof testorganismwasemulsifiedwithadropofnormalsalinewater(0.89%)andadropofhumanbloodserumwasaddedtothesuspension;clumpingofcellswasobservedwithin10sinthepresenceofboundcoagulaseenzyme.WhenasampleofGPcoccirespondedpositivelytobothcatalaseandcoagulasetests,itwasconfirmedas
S.aureus
.
Catalase-negativecolonieswerealsoculturedonbloodagartocheckfortheirhaemolyticpatterns,andabacitracintest(Forbesetal.,2007),wasconducted.Catalase-negativeGPcolonieswith
-haemolysis(completehaemolysisoferythrocytes)onbloodagarandsimultaneouslysensitivetothebacitracinwereiden-tifiedasGroupAstreptococcior
S.pyogenes
.Catalase-negative,
-haemolytic(partialorgreenhaemolysisoferythrocytes)coloniesweresubjectedtoabile-esculintest.Thebile-esculinmediumcontainsesculinandpeptonefornutritionandbiletoinhibitgrowthofGPbacteria,otherthanGroupDstreptococciorenterococci.Ferriccitratewasaddedasacolourindicator.Organismswhichsplitesculinmoleculesandusetheliber-atedglucosetosupplyenergyreleaseesculinintothemedium.Thefreeesculinreactswithferriccitrateinthemediumtoformaphenolicironcomplexwhichturnstheagar-slantfromdarkbrowntoblack.Anagar-slantthatwasmore
thanhalfdarkenedwithin48hofincubationwasbile-esculinpos-itive,fortheconfirmationof
E.faecalis
;
butthealternative
Satisfaites votre curiosité
Tout ce que vous voulez lire.
À tout moment. Partout. Sur n'importe quel appareil.
Aucun engagement. Annulez à tout moment.