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 Available
 
online
 
at
 
Antibacterial
 
activity
 
of 
Lantana
 
camara
 
L.agains
 
multidrugresistantpathogens
 
fromICUpatients
 
ofateaching
 
hospital
DebasmitaDubey
a
,RabindraN.Padhy
b
,
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,andisaprolicweedinKenya.InNigeria,
L.camara
leaf 
Correspondingauthor
.Formerlyat:BJBAutonomousCollege,Bhubaneswar,India.Tel.:+916746511205.E-mailaddress:rnpadhy54@yahoo.com(R.N.Padhy).
incinerationistraditionallyusedforrepellingmosquitoes(Egunyomietal.,2010).InIndian
 Ayurveda
,
 
adecoctionoffreshrootoftheplantisusedasabenecialgargleforodontal-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
antibacterialactivityofamethanolicleaextractof 
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(13days)areusuallycompletedforUTIsbeforetestresultsbecomeavailable,promotingmis-matchesduetodrugresistance.Here,theusualtreatmentwouldbetheuseofcefepime,acephalosporinthatistra-ditionallyusedtotreatclinicallyimportantstrainso
P.aeruginosa
,
 
asempirictherapyforaUTIinanintensivecareunit(ICU),butwhentheinfectionisconrmed,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,AsiaPacicandLatinAmerica(Perezetal.,2007).Moreover,inthepastdecade,reportsofoccurrencesocommunity-acquiredESBL-producing 
E.coli
isolates,thethirdmostcommonUTIcausativehaveincreasedworldwide;butitsprevalenceisstilluncommonintheU.S.,exceptfromnoso-comialsources(Naasetal.,2007).Strainsof 
E.coli
,
 
causing enteropathogenicepisodeswerefoundtoberesistantto16antibioticsfromfivegroupsintheauthorslaboratory(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.TheefficacyofcrudeextractsofphytochemicalsincontrollinMDRbacteriahasbeendemonstratedby
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,hosttoxicitytestinremainsanessentialcorollaryindesigningphytochemicalsascomplementarymedicines.Thereisalsoawidelyheldcon-sensusthatphytocompoundscanbeusedasdrugsanditislikelytobeheldinthefuture(see,MacLennanandPendry,2011),evenbytheWorldHealthOrganisation.
L.camara
,
 
withitsunusuallystrongunpleasantaroma,wasanticipatedtohavecontroloverMDR
 
bacterialstrains,emulatinganydove-tailedsyntheticdrug.Asareportoftheantibacterialactivityof 
L.camara
uponresistantpathogenicstrainswasunavail-ableintheliterature,thisresearchonthreeGPandveGNMDR
 
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),40oftheleafpowderwasextractedinasoxhletextractorfor40siphonsorcycles,withavolumeof400mL
 
ofsolvent.Indi-vidualsolventextractswerefilteredandconcentratedwitharotaryevaporator,runat40
C.Thestickymassesobtainedwere5.67,4.12,3.33,3.56,3.6,3.77,2.32and2.8gofextract/40ofdryleafpowderforpetroleumether,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)]andveGN(
 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

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