Effect of and inhibitory of Plectranthus amboinicus (Lour.) Spreng essential oil on Klebsiella pneumoniae

(1)

ContentslistsavailableatSciVerseScienceDirect

Phytomedicine

j o ur na l h o me p a g e :w w w . e l s e v i e r . d e / p h y m e d

Effect

of

subinihibitory

and

inhibitory

concentrations

of

Plectranthus

amboinicus

(Lour.)

Spreng

essential

oil

on

Klebsiella

pneumoniae

Thially

Braga

Gonc¸

alves

a,b,∗

,

Milena

Aguiar

Braga

a

,

Francisco

F.M.

de

Oliveira

a,b

,

Gilvandete

M.P.

Santiago

c,d

,

Cibele

B.M.

Carvalho

b,e

,

Paula

Brito

e

Cabral

a,b

,

Thiago

de

Melo

Santiago

f

_,

_Jeanlex

_S.

_Sousa

f

_,

_Eduardo

_Bedê

_Barros

f

_,

Ronaldo

Ferreira

do

Nascimento

g

_,

_Aparecida

_T.

_Nagao-Dias

a,b

a_Departamento_de_Análises_Clínicas_e_{Toxicológicas,}_Faculdade_de_Farmácia,_Odontologia_e_Enfermagem,_Universidade_Federal_Ceará,_Rua_Capitão_Francisco_Pedro_1210,

CEP60430-370,Fortaleza,Ceará,Brazil

b_Curso_de_{Pós-Graduac¸}_ão_em_{Microbiologia}_Médica,_Faculdade_de_Medicina,_Universidade_Federal_do_Ceará,_Rua_Monsenhor_Furtado_S/N,_CEP_60430-270,_Fortaleza,_Ceará,_Brazil

c_Departamento_de_Farmácia,_Faculdade_de_Farmácia,_Odontologia_e_Enfermagem,_Universidade_Federal_do_Ceará,_Rua_Capitão_Francisco_Pedro_1210,_CEP_60430-370,_Fortaleza,

Ceará,Brazil

d_Curso_de_{Pós-Graduac¸}_ão_em_Química,_Centro_de_Ciências,_Universidade_Federal_do_Ceará,_Cx_Postal_12.200,_CEP_60021-970,_Fortaleza,_Ceará,_Brazil

e_Departamento_de_Patologia_e_Medicina_Legal,_Faculdade_de_Medicina,_Universidade_Federal_do_Ceará,_Rua_Monsenhor_Furtado_S/N,_CEP_60430-270,_Fortaleza,_Ceará,_Brazil

f_Departamento_de_Física,_Centro_de_Ciências,_Universidade_Federal_do_Ceará,_CEP_60451-970,_Fortaleza,_Ceará,_Brazil

g_Departamento_de_Química_Analítica_e_{Físico-Química,}_Universidade_Federal_do_Ceará,_Campus_do_Pici,_Bloco_940,_Fortaleza,_Ceará,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Keywords:

Antibioticmultiresistance Antimicrobialactivity Klebsiellapneumoniae Plectranthusamboinicus

a

b

s

t

r

a

c

t

Weevaluatedtheantimicrobialactivityandsomemechanismsusedbysubinhibitoryandinhibitory concentrationsoftheessentialoil,obtainedfromleavesofPlectranthusamboinicus,againstastandard strainofKlebsiellapneumoniaeand5multiresistantclinicalisolatesofthebacteria.Theminimalinhibitory concentration(MIC)andminimalbactericidalconcentration(MBC),therateofkillandthepHsensitivity oftheessentialoilweredeterminedbymicrodilutiontestsperformedin96-wellplates.Subinhibitory andinhibitoryconcentrationsoftheessentialoilweretestedinordertocheckitsactiononK.pneumoniae membranepermeability,capsuleexpression,ureaseactivityandcellmorphology.TheMICandMBCof theessentialoilwere0.09±0.01%.Acompleteinhibitionofthebacterialgrowthwasobservedafter2h ofincubationwithtwicetheMICoftheessentialoil.AbetterMICwasfoundwhenneutraloralkalinepH brothwasused.Alterationinmembranepermeabilitywasfoundbytheincreaseofcrystalvioletuptake whenthebacteriawereincubatedwithtwicetheMIClevelsoftheessentialoil.Theureaseactivitycould bepreventedwhenallthesubinhibitoryconcentrationsweretestedincomparisontotheuntreated group(p<0.001).Alterationofthebacterialmorphologybesidesinhibitionofthecapsuleexpression wasverifiedbyatomicforcemicroscopy,andAnthony’sstainmethod,respectively.Ourdataallowusto concludethattheessentialoilofP.amboinicuscanbeagoodcandidateforfutureresearch.

Introduction

Sincetheintroductionofantimicrobialagents,therehasbeen aselectivepressureofmicroorganisms, primarilycaused bythe indiscriminate useof antibiotics and chemotherapy,which has resultedinthedevelopmentofresistantspecies(Peres-Botaetal. 2003;Pittet2005).Oneofthemostseriouspublichealthproblems facedinthelastdecadeshasbeentheaggravationof antimicro-bialresistanceinbacterialpopulations,especially innosocomial

∗_{Corresponding}_author_at:_Departamento_de_Análises_Clínicas_e_{Toxicológicas,} FaculdadedeFarmácia,OdontologiaeEnfermagem,UniversidadeFederalCeará, RuaCapitãoFranciscoPedro1210,CEP60430-370,Fortaleza,Ceará,Brazil. Tel.:+558533668270;fax:+558533668292.

E-mailaddress:thially@gmail.com(T.B.Gonc¸alves).

outbreaks(Oliveiraetal.2006).Animportantaspectwhich fre-quently results in antibioticresistance resides on the bacterial enzymeproduction,suchas,beta-lactamases.Thebeta-lactamases ofclinicalinterestareextendedspectrumbeta-lactamase(ESBL), carbapenemases (KPC),metalobetalactamase (MBL) and class C beta-lactamase(AmpC).K.pneumoniaeESBLiscapableof inacti-vatingthirdgenerationcephalosporinsandmonobactams,except carbapenemsandareinhibitedbybeta-lactamaseinhibitorssuch asclavulanate,sulbactamandtazobactam.TheenzymeKPCwas firstlydescribedintheU.S.in2001andwasfurtheridentifiedin othercountriesaroundtheworld.Thisenzymehastheabilityto hydrolyzecephalosporinsandaztreonam(PattersonandBonomo 2005;Bertrandetal.2006).

ManyplantsareusedinBrazilintheformofcrudeextracts, infusionsfortreatmentofvariousconditions,includingbacterial

(2)

19 (2012) 962–968 963

andfungalinfections(Gurgeletal.2009).Essentialoilshavelong beenused due totheir many therapeutic properties, including antiviral,antispasmodic,analgesic,antimicrobial,woundhealing, expectorant,soothing,antisepticairway,larvicide, antihelmintic and antiinflammatoryactivities(Costa et al.2010; Oyedeji and Afolayan2006).

ThegenusPlectranthusinvolvesabout300speciesoccurring nat-urallyinAfrica,AsiaandAustralia(Lukhobaetal.2006).Speciesof thisgenusexhibitbiosyntheticsesquiterpenes,diterpenesand phe-noliccompounds,somewithprovenrelevantbiologicalproperties (Lukhobaetal.2006).

Plectranthusamboinicus(Lour.)Spreng(Labiatae),alsoknownas

ColeusamboinicusLour.,Coleusaromaticus,Plectranthusaromaticus

(Benth)(Murthyetal.2009),andpopularlyknownas“malvarisco”, isanaromaticperennialherb cultivatedallover theNorth and NortheastregionsofBrazil(Matos2000).Itiswidelyusedin Brazil-ianfolkmedicinefortherapeuticpurposesagainstcolds,asthma, constipation,headache,cough,feveranddigestivediseases(Morais etal.2005).

Thispresent workproposedtoinvestigate theantimicrobial activityoftheessentialoilagainstK.pneumoniaestandardstrains andmultiresistantclinicalisolates,andalsotoevaluatethe abil-ityofsubinhibitoryandinhibitoryconcentrationsofP.amboinicus

essentialoilinalteringthemembranepermeability,thecapsule expressionandtheureaseactivityofK.pneumoniae,itsrateofkill anditscapabilityongrowingindifferentpHconditions.

Materialsandmethods

Plantmaterial

LeavesofPlectranthusamboinicus(Lour.)Sprengwerecollected fromtheHorto de Plantas MedicinaisProfesssor FranciscoJosé de AbreuMatos (Fortaleza, Ceará, Brazil). A voucher specimen (EAC40080)has been deposited at the Herbário Prisco Bezerra (EAC),UniversidadeFederaldoCeará,Brazil.

Extractionoftheessentialoil

ThefreshleavesofP.amboinicusweresubjectedto hydrodistilla-tioninaClevenger-typeapparatusfor2htoaffordpaleyellowoil. Theisolatedoil,afterdryingoveranhydroussodiumsulfateand filtration,wasstoredinsealedglassvialsandmaintainedunder refrigerationuntilfurtheranalysis.Theyield(w/w)wascalculated basedonthefreshweightoftheleaves.

Theessentialoilwasobtainedinthreedifferentperiodsand ana-lyzedbygaschromatography(GC)andGC/MSinordertocompare thechemicalconstituentsfromeachextraction.Theessentialoils werenamedS1,S2andS3.

Chemicalanalysisoftheessentialoil

Gas chromatography (GC) analysis was performed on a Shimadzu GC-17A gas chromatograph equipped with a flame

ionizationdetectorusinganon-polarDB-5fusedsilicacapillary column(30m×0.25mm×0.25␮mfilmthickness).Hydrogenwas usedascarriergasataflowrateof1ml/minand30psiinlet pres-sure;splitratio1:30.Thecolumntemperaturewasprogrammed from35◦_C_to₁₈₀◦_C_at_a_rate_of₄◦_C/min,_then_heated_at_a_rate_of

17◦_C/min_to₂₈₀◦_C_and_held_isothermal_for₁₀_min;_both_injector

anddetectortemperatureswere250◦_C.

TheGC/MSanalysiswascarriedoutonaHewlett-PackardModel 5971GC/MSinstrument,usinganon-polarDB-5fusedsilica capil-larycolumn(30m×0.25mm×0.1␮mfilmthickness);carriergas helium,flowrate1ml/minandwithsplitmode.Theinjectorand detectortemperatureswere250◦_C_and₂₀₀◦_C,_{respectively.}_The

column temperature was programmed from 35◦_C _to ₁₈₀◦_C _at

4◦_C/min_and_then₁₈₀_to₂₅₀◦_C_at₁₀◦_C/min._Mass_spectra_were

recordedfrom30to450m/z.

Individualcomponentswereidentifiedbymatchingtheir70eV massspectrawiththoseofthespectrometerdatabaseusingthe WileyL-builtlibraryandtwoothercomputerlibraries.Mass spec-trometry(MS)searchesusingretentionindicesasapre-selection routine,aswellasbyvisualcomparisonofthefragmentation pat-ternswiththosereportedin theliterature(Alencar etal.1990; Adams2007).

Microbialstrains

TheKlebsiellapneumoniae(ATCC700603)wasobtainedfrom the Instituto Nacional de Controle de Qualidade em Saúde (INCQS),InstitutoOswaldoCruz,RiodeJaneiro,Brazil.Fiveclinical isolates were obtained from the Hospital Universitário Wal-ter Cantídio(Fortaleza, Ceará,Brazil). Theclinical isolateswere analyzed by standard methods for identification of Enterobac-teriaceae (Koneman 2008). Susceptibility profile was tested by disc-diffusionagar(CLSI2009)withthefollowingdrugs:amikacin, ampicillin,ampicillin+sulbactam,cephalothin,cefoxitin,cefpime, caeftazidime, ceftriaxone, gentamicin, imipenem, meropenem, piperacillin,quinolones,andsulfazotrimticarcillin+clavulanicacid (Table1).

Determinationoftheminimalinhibitoryconcentrationand minimalbactericidalconcentration

To determinethe minimal inhibitoryconcentration we per-formed microdilution tests (CLSI 2009) in 96-well microplates (Costar,USA).Eachwellcontained100␮lMueller-HintonInfusion (MHI)broth,10␮lofinoculum(1.5×108_CFU/ml)_and₁₀₀_␮_l_of_the essentialoil,whichrangedfrom0.009%to1.25%(v/v)inDMSO3%. Theinoculumbacterialconcentrationwasdeterminedby adjust-ingitsturbidity(at625nm)to0.5McFarlandscale.Theplateswere coveredandincubatedfor24hat37◦_C._The_lowest_{concentration}_of

thetestsampleinwhichnogrowthcouldbevisualizedwasdefined astheMIC.Weusedciprofloxacin(SigmaChemicalCo.,St.Louis, MO,USA), rangingfrom0.125to32␮g/ml,asthepositive con-trolanddimethylsulfoxide(Merck,Darmstadt,Germany),ranging from0.039to10%(v/v),asthenegativecontrol.Aftertheincubation

Table1

TableofpatternofdrugresistanceinK.pneumoniaeclinicalisolates:CE,clinicalspecimen;URO,uroculture;ATR,trachealaspirate;BLO,blood;NT,nottested;S,sensitive;R, resistant;I,intermediate;AMI,amikacin;AMP,ampicillin;ASB,ampicillin+sulbactam;ATM,aztreonam;CEP,cephalothin;FM,cefepime;CTX,cefotaxime;CFO,cefoxitin; CAZ,ceftazidime;CEN,ceftriaxone;CIP,ciprofloxacin;COL,colistin;ERT,ertapenem;GEN,gentamicin;IPM,imipenem;MER,meropenem;PPT,piperacillin+tazobactam; TIG,tigecycline.

Clinicalisolates AMI AMP/ASB/CEF ATM/ERT/IPM/MER COM/CFO/CAZ/PPT CTX CEN CIPRO COL GEN TIG

K2(uro) nt R R R R nt S S S S

K5(blood) S R R R R nt R S S R

K8(uro) nt R S R nt R S nt R R

K12(uro) I R R R R nt R S S S

(3)

964 19 (2012) 962–968

period,10␮lofsterile0.05%resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide)(Sigma,USA)inaqueoussolutionwereaddedto theplates.After2hofincubation,thereadingsweredone.Acolor changecaused bythereduction ofresazurintoresorfurin indi-catedpresence(pinkcolor)orabsence(bluecolor)ofcellviability (Palominoetal.2002).Theminimalbactericidalconcentrationwas determinedfortheessentialoilbysubculturing5␮lfromeachtube (MIC,2and4×MIC)ontoBHIagar.TheMBCwasdefinedasthe lowestconcentrationoftheessentialoilthatcouldinhibit99%of thebacterialgrowth.Thetestswererunintriplicatewiththethree samples(S1–S3)oftheessentialoil.

pHsensitivityassay

TheeffectofpHonantibacterialactivityofP.amboinicus essen-tialoil (S1) and ciprofloxacin against K. pneumoniae was done accordingtotheproceduredescribedbyDevietal.(2010).Cultures ofthestandardstrainweregrownonMuller-Hintonbroth previ-ouslypreparedandcalibratedtodifferentpHranges(from5.5to 9.0).TheMICwasdetermined.

Determinationofrateofkill

Onemilliliterofthestandardstrainwasadjustedtothe concen-trationof1.5×108_CFU/ml_and_incubated_at₃₅◦_C_for₂_h,_before_its

additiontotubescontaining0.16%(whichrepresentedtwotimes theMIC).Controltubeswerealsopreparedwithouttheessential oil(S1).ThetubesTestandControlwereincubatedat37◦_C_and

aliquotsof10␮lwerecollectedatintervalsof0,2,4,6,8and24h aftertheadditionoftheplantsamples.Serialdilutionswereplated ontoMacConkeyagartodeterminethenumberofviablecellswhich wasexpressedaslogofcolonyformingunitsperml(logCFU/mL). Thebactericidal(≥3-log-unitreductioninlog10CFU/ml)and bac-teriostaticactivities(<3-log-unitreductioninlog10CFU/ml)were determined(Devietal.2010).

Crystalvioletassay

Thealterationinmembranepermeabilitywasdetectedby crys-talvioletassayaccordingtotheproceduredescribedinliterature (Devietal.2010).Briefly,suspensionsofthebacteriawere pre-paredinBrainHeartInfusion(BHI)broth.Cellswereharvestedat 4500×gfor5minat4◦_C._The_cells_were_washed_twice_and

resus-pendedinPBSat pH7.4.Theessentialoil(S1)of P.amboinicus

(testedatMICandtwicetheMIC),theantibiotics(testedatMIC), besidesethylenediaminetetraaceticacidorEDTA(positivecontrol) andphosphatebufferedsalineorPBS(negativecontrol)wereadded tothecellsuspensionsandincubatedfor30minat37◦_C_(all_tests

weredoneinquintuplicate).Thecellswereharvestedat9300×g

for5min.Afterthisprocedure,thecellswereresuspendedinPBS containing0.010mgofcrystalviolet.Thecellsuspensionwasthen incubatedfor10minat37◦_C_and_centrifuged_at₁₃₄₀₀_×_g_for₂_h

andtheOD590ofthesupernatantwasmeasured.

Effectofsub-MICandMIClevelsofP.amboinicusessentialoilon ureaseactivity

Bacterial urease activity was determined by the procedure describedbyDerakhshanetal.(2008).FiveclinicalisolatesofK. pneumoniaeandtheATCCstandardweregrownovernightinurea brothcontainingsub-MICandMIClevelsoftheessentialoil(S1) (1/8to1/1×MIC).Untreatedcellswereusedasnegativecontrol.All tubeswereincubatedfor24hat37◦_C._Next,_the_suspensions_were

centrifugedat5000×gfor3minandthesupernatantswere recov-ered.Color intensitywasmeasuredat560nmusingauniversal

microplatereader(Biotek,China).Thesampleswererunin tripli-cate.

Effectofsub-MICandMIClevelsofP.amboinicusessentialoilon bacterialcapsuleexpression

TheK.pneumoniaecapsulewasstainedbytheAnthony’scapsule stain,which procedurewasdescribed inliterature(Derakhshan etal.2008).Briefly,fiveclinicalisolatesofthebacteriaandtheATCC standardstrainweregrowninmilkbroth(9.5gpowdermilk/l)for 24hinthepresence ofdifferentconcentrationsoftheessential oil(S1)(1/8to1/1×MIC).Smearswerepreparedonglassslides andallowedtoairdry.Thereafter,theslideswerecoveredwith1% crystalvioletfor2minandrinsedgentlywitha20%copper sul-fatesolution.Thebacterialcells(nottreatedcontrol)wouldappear purplishwhilethecapsuleswouldappeartransparentunderanoil immersionlensatopticalmicroscopy.

Atomicforcemicroscopy

Changesin themorphology of K.pneumoniaecaused by the essential oil of P. amboinicus were analyzed by atomic force microscopy(AFM)(BragaandRicci1998).Suspensionsofcultures wereincubatedinthepresenceofdifferentconcentrationsofthe essential oil (S1) (1/8 to1/1×MIC)for 18hat 37◦_C. _The _cells

werewashedandcentrifugedat4500×gfor5minat4◦_C,_washed

twiceandresuspendedinPBS.Avolumeof10␮lofthe suspen-sionwasplacedonaglassslideanddriedinairfor15min.Then thesamplewasexaminedbya MultimodeAtomicForce Micro-scope NanoscopeIII-a (Digital Instruments, SantaBarbara, CA). Scanswereperformedinairandamplitudeimageswereacquired byintermittentcontactmodeusingcrystallinesiliconcantilevers (Veeco-probes)withaspringconstantofapproximately40N/m, resonancefrequencyof242.38kHz andtipradiusof15nm.The amplitudeimagescanbeusedtobetterevidencecellbordersand theirshape.CulturesofnottreatedK.pneumoniaeweretakenas controls.

Statisticalanalysis

TheeffectofvariousconcentrationsofP.amboinicusessentialoil ontheK.pneumoniaeureaseactivityandontheuptakeofcrystal violetbythebacteriawereanalyzedbyone-wayanalysisof vari-ance(ANOVA)andTukey–Kramermultiplecomparisonstests.The statisticalsignificancewasconsideredatp-valueof0.05.

Results

Chemicalanalysisoftheessentialoilanddeterminationofthe minimalinhibitoryconcentrationandminimalbactericidal concentration

ThreedifferentsamplesofessentialoilfromtheleavesofP. amboinicuswereanalyzedbyGCandCG/MS.Table2showsthe chemicalcompositionofthethreesamplesoftheessentialoil. Car-vacrol(90–98%)isthemajorconstituentidentifiedinallsamples. Thesamplesofessentialoilobtainedindifferentperiodshavefew differencesintheirchemicalconstitutions.

Alltestedstrains(clinicalandthestandard)weresusceptibleto theessentialoil(MICmean±standarderror,thatis,0.09±0.01%,

(4)

19 (2012) 962–968 965

Table2

ChemicalcompositionofessentialoilfromleavesofP.amboinicus.

Compound RI RT S1 S2 S3

4-Terpineol 1177 16.80 1.34 – –

Carvacrol 1298 21.39 90.55 95.39 98.03

␤-Caryophyllene 1417 25.46 3.09 – 0.01

␣-Bergamotene 1433 25.97 1.78 – –

Caryophylleneoxide 1583 30.97 1.36 – 0.20

p-Cimene 1024 8.28 – – 1.65

␥-Terpinene 1059 9.55 – – 0.01

␣-trans-Bergamotene 1434 25.79 – – 0.02

␣-Caryophyllene 1454 25.49 – – 0.02

Totalidentified – – 98.12 95.39 99.94

RI:relativeretentionindex;RT:retentiontime;S:sampleofessentialoilofP.amboinicus.

Table3

EffectofpHonantibacterialactivityofP.amboinicusessentialoilagainstKlebsiella pneumoniae.

pH Essentialoil Cipro(␮g/ml)

% ␮g/ml

5.5 0.08 700.00 4.0

6.0 0.08 700.00 4.0

6.5 0.08 700.00 4.0

7.0 0.08 700.00 2.0

7.5 0.04 350.00 2.0

8.0 0.04 350.00 1.0

8.5 0.04 350.00 1.0

9.0 0.04 350.00 1.0

Determinationofratekill

TreatmentofK.pneumoniaewith0.16%ofessentialoil,which representedtwotimestheMIC,ofessentialoilwasenoughtokill allthebacteriaafter2hofincubationcomparedtocontrolandto ciprofloxacin.

pHsensitivityassay

WithrespecttothepHsensitivityassay,abetterMICwasfound when neutral or alkaline pHbrothwas used (Table 3).Similar resultswerefoundwhenciprofloxacinwastested.

Crystalvioletassay

Theuptakeof crystalviolet wasconsideredtobesignificant whentwicetheMICofessentialoilwasused(Table4),compared tothenottreatedgroup(p<0.01)ortociprofloxacin(p<0.01)and toEDTA(p<0.05).EDTAisachelatingagentwhichincreasesthe membranepermeabilityofthebacterialcells.

Effectofsub-MICandMIClevelsofP.amboinicusessentialoilon ureaseactivity

Alltheconcentrationsoftheessentialoiltestedcould signifi-cantlyreducetheureaseactivity,comparedtothenottreatedgroup (p<0.001),asshowninFig.1.ThedashedlineinFig.1isthebest exponentialfitofthedata.

Effectofsub-MIClevelsofP.amboinicusessentialoilonbacterial capsule

AsshowninFig.2,smallercolonieswithreducedexpression ofcapsulescouldbefoundwhenthebacteriaweretreatedwith 1/8×MICoftheessentialoilcomparedtothenottreatedcontrols (Fig.2aandb).However,whenthebacteriaweretreatedwith1/4

Fig.1. Effects ofsub-inhibitory(1/8 to 1/2)andinhibitory concentrationsof P.amboinicus essential oil onKlebsiella pneumoniae urease activity. Itshows the absorbance averaged over five samples vs the MIC. The dashed line is the best exponential decay fitting of the experimental data {optical den-sity=0.552+0.423exp(−MIC/0.251)}.Thisexpressionindicatesthatforsufficient highvaluesofMICtheabsorbancepresentsasaturationvalue,near0.552.

orhighersubinhibitoryconcentrationsoftheessentialoilalmost nobacteriacouldbefound,onlycellulardebris(Fig.2c–e).

Atomicforcemicroscopy

Accordingtotheatomicforcemicroscopy(Fig.3AandB),the morphologyofuntreatedK.pneumoniaewassmoothandregular withanintactcellmembraneandindividualbacteriawerereadily distinguishable.Fig.3Ashowsafter24hoftreatmentwith1/8and 1/4×MICessentialoil(Fig.3AbandAc),itwasobservedan increas-ing loss of the membrane integrity with a consequent altered morphologyofthebacteria.Finally,aseverecelldamagewas veri-fiedwhen1/2×MICandtheinhibitoryconcentrations(1×MICand 2×MIC)wereused(Fig.3Ad,AeandAf).Fig.3Bshowsthethree dimensionalaspectofthenottreatedbacteriacomparedtothose treatedwithtwicetheMICoftheP.amboinicusessentialoil.

Discussion

(5)

966 19 (2012) 962–968

Table4

CrystalvioletuptakebyKlebsiellapneumoniaepreviouslyincubatedwithPlectranthusamboinicusessentialoil.TheresultswereexpressedbymeanofOD590values±standard error.Thesamplesweretestedinquintuplicate.

Groups PBS EDTA CIPRO Essentialoil

MIC 2×MIC

xAbs±EM 0.412–0.013 0.383–0.019 0.412–0.037 0.315–0.029 0.254–0.016 CI95 0.371–0.454 0.329–0.437 0.308–0.517 0.234–0.396 0.209–0.299

p N.S. N.S. N.S. N.S. <0.01(vsPBSandCIPRO)<0.05(vsEDTA)

Fig.2.Effectsofsub-inhibitory(1/8to1/2),inhibitoryandtwicetheinhibitoryconcentrationsofP.amboinicusessentialoilonKlebsiellapneumoniaecapsuleexpression.The bacteriawerestainedwiththeAnthonycapsularstainingmethod.Thecolonymorphologyandcapsulevisualizationwasdoneunderlightmicroscopy(magnification1000× with3×opticalzoom).

Carvacrolisamonoterpenephenolwhichisknowntobeeither bactericidal or bacteriostatic agent, depending on the concen-trationused(Nostroetal.2004).Previousstudiesreportedthat carvacrolhadthepropertyofdestroyingthecellularmembraneof

Escherichiacoli,Salmonellatyphimurium,Shigellaflexneri(Dorman andDeans2000).

P.amboinicusessentialoilpresentsbactericidaleffectwhena concentrationoftwicetheMICisaddedtoK.pneumoniae suspen-sion.After2hofincubation,thereisacompletelossofbacterial viability.The datashown is relevantby thefact K.pneumoniae

isoneofthemainpathogensassociatedwithnosocomial infec-tions;also,asthepHsensitivitytestshowed,theessentialoilwas

(6)

19 (2012) 962–968 967

moreactivein higherpHranges.These dataarevaluable, once

K.pneumoniaecangrowinthesmallintestine,wherepancreatic juiceandbilemaketheenvironmentpHhighlyalkaline(Koneman 2008).

Klebsiellapneumoniaeisanonmotileentericorganismthat fer-mentslactose,decarboxylateslysine,hydrolyzesurea,andforms largemucoidcolonies,duetothepresenceofcapsule(Koneman 2008).ThecapsuleofK.pneumoniaeconsistsofthickanddense bundles of fine fibers, which contributes to its high virulence capacity.

Generally, subinhibitory concentrations of an essential oil, althoughnotabletokillbacteria,canmodifythearchitectureof theiroutermostsurface and may interferewith somebacterial functions(Derakhshanetal.2008).In ourstudy,whenK. pneu-moniaewastested in thepresence of1/8 MICof P.amboinicus

essentialoil,analterationofitsmorphologybesidesreductionof capsuleexpressionwasfound.Whenhighersubinhibitory concen-trations(1/4and1/2)oftheessentialoilweretested,almostno bacteriacouldbevisualized,but onlycellulardebris. Thiscould meanthatsubinhibitoryconcentrationof1/8×MICcouldreduce thebacterialcapacity ofcapsule expression.Besidesthis obser-vation,alterationinmembranepermeabilitywasdetectedbythe increaseofcrystalvioletuptakewhenthebacteriawereincubated withtwicetheMIClevelsofP.amboinicusessentialoil.Lossof mem-braneintegrityandleakageofintracellularcomponentscouldbe demonstratedbytheatomicforcemicroscopy,when1/2×MIC lev-elsoftheessentialoilwasused,andacompletedestructionofthe cellsattheinhibitoryconcentrations(MICand2×MIC)oftheplant sample.

Manymicroorganismsneedtouseureaseinordertoobtain nitrogenfortheirgrowth(Maroncleetal.2006).Ureasecatalyzes thehydrolysisofureatoammoniaandcarbamateandhasbeen foundto beimplicated in kidneyand bladder stone formation and to the pathogenesis of several diseases. Urease inhibitors are potential alternatives for the prevention of K. pneumoniae

fromcolonizingthegastrointestinaltract,whereisplentyofurea (Koneman2008;Hostacká2000;Maroncleetal.2006).Imipenem andofloxacin,donotaffecttheenzymeactivity(Hostacká2000). Our data showed a strong inhibition of urease activity by the

P.amboinicusessentialoilinallconcentrationstestedcompared to the not treated bacteria (p<0.001). Although 1/8 MIC was only able to reduce 15.7% of the urease activity, 1/4×MIC to 1 MIC reduce from 30.5% to 42%, respectively. These percent-ages of the enzyme inhibition were superior to ␤-lactam and aminoglycoside actions (20% of inhibition), and equivalent or superior toacetohydroxamic acid(30% of inhibition), which is frequentlyusedtoinhibitureaseactivity(Maroncleetal.2006; Raufetal.2011).Ontheotherhand,ithasbeenrecentlyshown novelbarbituricandthiobarbituricacid-derivedsulfonamideswith ability to inhibit more than 88% of the urease activity when the drugs were tested at 500␮M concentrations (Rauf et al. 2011).

Conclusion

The results of our data clearly demonstrated that essential oilofPlectranthus amboinicuspossessesapotentialantibacterial activityagainst Klebsiella pneumoniae. Subinhibitory concentra-tions of the essential oil could affect the capsule expression, alterationsinmembraneintegrity,inhibitionoftheurease activ-ity.Atbactericidalconcentrations,acompletedestructionofthe bacteriawasfound.Ourdataallowustoconcludethatthe essen-tial oil of P. amboinicus could be a good candidate for future research.

Conflictofinterest

Noconflicttodisclose.

Acknowledgements

ThisresearchwasfinanciallysupportedbytheCNPq(Processes 579437/2008-6and554970/2010-4)andFUNCAP/PPSUS(Process 09100057-2).

References

Adams,R.P.,2007.IdentificationofEssentialOilComponentsbyGas Chromatogra-phy/MassSpectrometry.AlluredPublishingCorporation,CarolStream,IL. Alencar,J.W.,Craveiro,A.A.,Matos,F.J.A.,Machado,M.I.L.,1990.Kovatsindices

sim-ulationinessentialoilsanalysis.QuimicaNova13,282–284.

Bertrand,S.,Weill,F.-X.,Cloeckaert,A.,Vrints,M.,Mairiaux,E.,Praud,K., Dier-ick,K.,Wildemauve,C.,Godard,C.,Butaye,P.,Imberechts,H.,Grimont,P.A.D., Collard,J.-M.,2006.Clonalemergenceofextended-spectrumbeta-lactamase (CTX-M-2)-producingSalmonellaentericaserovarVirchowisolateswithreduced susceptibilitiestociprofloxacinamongpoultryandhumansinBelgiumand France(2000to2003).JournalofClinicalMicrobiology44,2897–2903. Braga,P.C.,Ricci,D.,1998.Atomicforcemicroscopy:applicationtoinvestigationof

Escherichiacolimorphologybeforeandafterexposuretocefodizime. Antimicro-bialAgentsChemotherapy42,18–22.

Castillo,R.A.M.,González,V.P.,1999.Plectranthusamboinicus(Lour.)Spreng.Revista CubanadePlantasMedicinales4,110–115.

CLSI,2009.PerformanceStandardsforAntimicrobialSusceptibilityTesting;18th InformationalSupplement.CLSIM07-A8.ClinicalandLaboratoryStandards Institute,Wayne,PA,pp.15–18.

Costa,J.G.,Campos,A.R.,Brito,S.A.,Pereira,C.K.B.,Souza,E.O.,Rodrigues,F.F.G.,2010. BiologicalscreeningofararipebasinmedicinalplantsusingArtemiasalinaLeach andpathogenicbacteria.PharmacognosyMagazine6(24),331–334. Derakhshan,S.,Sattari,M.,Bigdeli,M.,2008.Effectofsubinhibitoryconcentrations

ofcumin(CuminumcyminumL.)seedessentialoilandalcoholicextractonthe morphology,capsuleexpressionandureaseactivityofKlebsiellapneumoniae. InternationalJournalofAntimicrobialAgents32,432–436.

Devi,K.P.,Nisha,S.A.,Sakthivel,R.,Pandain,S.K.,2010.Eugenol(anessentialoil ofclove)actsasantibacterialagentagainstSalmonellatyphibydisruptingthe cellularmembrane.JournalofEthnopharmacology130,107–115.

Dorman,H.J.D.,Deans,S.G.,2000.Antimicrobialagentsfromplants:antibacterial activityofplantvolatileoils.JournalofAppliedMicrobiology88,308–316. Gurgel,A.,Silva,J.G.,Grangeiro,A.R.S.,Xavier,H.S.,Oliveira,R.A.G.,Pereira,M.S.V.,

Souza,I.A.,2009.AntibacterialeffectsofPlectranthusamboinicus(Lour.)Spreng (Lamiaceae)inmethicillinresistantStaphylococcusaureus(MRSA).Latin Amer-icanJournalofPharmacy28(3),460–464.

Hostacká,A.,2000.Theinvitroeffectofimipenemandofloxacinonenzymicactivity ofKlebsiellastrains.FoliaMicrobiologica45,387–390.

Koneman,E.W.,2008.DiagnósticoMicrobiológico–TextoeAtlasColorido. Guan-abaraKoogan,RiodeJaneiro,pp.209–290.

Lukhoba,C.W.,Simmonds,M.S.J.,Paton,A.J.,2006.Plectranthus:areviewof ethnob-otanicaluses.JournalofEthnopharmacology103,1–24.

Maroncle,N.,Rich,C.,Forestier,C.,2006.TheroleofKlebsiellapneumoniaeurease inintestinalcolonizationandresistancetogastrointestinalstress.Revistade Microbiologia157,184–193.

Matos,F.J.A.,2000.Plantasmedicinais:guiadeselec¸ãoeempregodeplantasusadas emfitoterapianoNordestedoBrasil,vol.2,pp.36–42.

Morais,S.M.,Dantas,J.D.P.,Silva,A.R.A.,Magalhães,E.F.,2005.Plantasmedicinais usadaspelosíndiosTapebasdoCeará.RevistaBrasileiradeFarmacognosia15, 169–177.

Murthy,P.S.,Ramalakshmi,K.,Srinivas,P.,2009.FungitoxicactivityofIndianborage (Plectranthusamboinicus)volatiles.FoodChemotherapy114,1014–1018. Nostro,A.,Blanco,A.R.,Cannatelli,M.A.,Enea,V.,Flamini,G.,Morelli,I.,Roccaro,S.,

Alonzo,V.,2004.Susceptibilityofmethicillin-resistantstaphylococcitooregano essentialoil,carvacrolandthymol.FEMSMicrobiologyLetters230,191–195. Oliveira,J.F.P.,Cipullo,J.P.,Burdmann,E.A.,2006.Nefrotoxicidadedos

aminogli-cosídeos.RevistaBrasileiradeCirurgiaCardiovascular21(4),444–452. Oyedeji,A.O.,Afolayan,A.J.,2006.Chemicalcompositionandantibacterialactivity

oftheessentialoilisolatedfromSouthAfricanMenthalongifolia(L.)L.subsp. capensis(Thunb.)Briq.TheJournalofEssentialOilResearch18,57–59. Palomino,J.C.,Martin,A.,Camacho,M.,Guerra,H.,Swing,J.,Portaels,F.,2002.

Resazurinmicrotiterassayplate:simpleandinexpensivemethodfor detec-tionofdrugresistanceinMycobacteriumtuberculosis.AntimicrobialAgentsand Chemotherapy46(8),2720–2722.

Patterson,D.L.,Bonomo,R.A.,2005.Extended-spectrumbeta-lactamases:aclinical update.ClinicalMicrobiology18,657–686.

Peres-Bota,D.,Rodriguez,H.,Dimopoulos,G.,2003.Areinfectionsduetoresistant pathogensassociatedwithaworseoutcomeincriticallyillpatients?Journalof Infection47,307–316.

(7)

968 19 (2012) 962–968

Pittet,D.,2005.Infectioncontrolandqualityhealthcareinthenewmillennium. AmericanJournalofInfectionControl33,258–267.

Rauf, A., Ahmed, F., Qureshi, A.M., Aziz-ur-Rehman, Khan, A., Qadir, M.I., Choudhary, M.I., Chohan, Z.H., Youssoufid, Z.M.H., Haddad, T.B., 2011. Synthesis and urease inhibition studies of barbituric and thiobarbituric acidderived sulphonamides. Journal ofthe Chinese Chemical Society58, 528–533.

Singh,G.,Singh,O.P.,Prasad,Y.R.,Lamposona,M.P.,Catalan,C.,2002.Studieson essentialoils.Part33.Chemicalandinsecticidalinvestigationsonleafoilof ColeusamboinicusLour.FlavourandFragranceJournal17,440–442.