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RESEARCHOpenAccessIsolation,identificationandcharacterizationofBurkholderiapseudomalleifromsoilofcoastalregionofIndiaArchanaPrakash1,DuraipandianThavaselvam1*,AshuKumar1,AjithKumar2,SoniaArora1,SapanaTiwari1,AnitaBarua1andKannusamySathyaseelan1AbstractMelioidosisisanemerginginfectiousdiseasecausedbyafreelivingsoildwellingGram-negativebacteriumBurkholderiapseudomallei.
ThediseaseisendemictomostpartsofSoutheastAsiaandnorthernAustraliaandtheorganismhasbeenisolatedfrommoistsoilandwater.
InIndiaclinicalcasesarerecentlyreportedfromthestatesofTamilnadu,Kerala,Karnataka,Maharashtra,Orissa,Assam,WestBengal,PondicherryandTripura.
ThisstudyisaimedtoconfirmtheprevalenceofthisimportantbacterialspeciesinsoilsamplescollectedfromcoastalareasofTamilnadu.
FortyfivesoilsamplesfromfivedifferentsiteswerecollectedfromParangipettai,TamilnaduandscreenedforthepresenceofB.
pseudomallei.
Thestudyconfirmed4isolatesasB.
pseudomalleiwiththehelpofconventionalbacteriologicalmethodsandmolecularmethodsthatinclude;16SrDNAsequencing,B.
pseudomalleispecificPCR,fliCgeneRFLPandMALDI-TOFmassspectrometrybasedbacterialidentification.
ThisstudyrevealstheprevalenceanddistributionofB.
pseudomalleiinthesoilenvironmentincoastalareasofsouthernIndiaandfurthernecessitatesstudiesfromotherpartsofthecountry.
ItwillalsobehelpfultounderstandthedistributionofB.
pseudomalleiandtoaccessitsepidemiologicalimportance.
Keywords:Burkholderiapseudomallei;Ashdownagar;Melioidosis;Parangipettai;SoilisolateIntroductionMelioidosisiscausedbysoildwellingGram-negativebacteriumBurkholderiapseudomalleiandisanemerginginfectiousdiseaseinIndia.
ThediseaseismainlyendemicinSoutheastAsiaandnorthernAustraliawithhighestnumberofmelioidosiscasesreportedfromThailand.
Theglobaldistributionboundariesofmelioidosiscontinuetoexpandwellbeyondthetraditionallyrecognizedendemicregions(Currieetal.
,2008).
InIndia,clin-icalcaseshavebeenreportedfromstatesofTamilnadu,Kerala,Karnataka,Maharashtra,Orissa,Assam,WestBengal,PondicherryandTripura.
Burkholderiapseudo-malleihasbeenisolatedfromclinicalsampleslikeblood,sputum,pus,urine,synovial,peritonealandpericar-dialfluidsmostlyfromtertiarycarehospitalslocatedatVellore,TamilNaduandMangalore,Karnataka(Raghavanetal.
,1991;Kavithaetal.
,2008).
ThetrueincidenceofmelioidosisisnotknowninIndiaandrecentlylargernumbersofcaseshavebeenreportedfromthewesterncoastalareas(Vidyalakshmietal.
,2007).
Melioidosisisreferredtoas"agreatimitator"becauseofitswidespectrumofclinicalpresentations,rangingfrommildsubclinicalinfectiontofatalsepticaemiathatcanbechronic,localizedordisseminated.
Theinfectionoccursthroughinhalation,orskinabrasionsthatcomeincontactwithcontaminatedsoilorwater.
Diabetesisthemostcommonriskfactorthatisassociatedwiththediseaseandotherriskfactorsincludethalassaemia,alcoholismandrenalimpairment.
IsolationoftheorganismfromsoilisrequiredtodefinetheepidemiologyanddistributionofB.
pseudomallei,andtheassociatedrisktohumansandlive-stock(White,2003;Leelarasamee,2004).
EarlierstudieshaveshownthepresenceofB.
pseudomalleiintheenvir-onmentbasedoncultureofsoilandwaterfromdifferentgeographicregions,particularlyfromSoutheastAsiaandnorthernAustralia(Straussetal.
,1969).
Thedescription*Correspondence:dtselvam@drde.
drdo.
in1DivisionofMicrobiology,DefenceResearch&DevelopmentEstablishment,JhansiRoad,Gwalior474002,IndiaFulllistofauthorinformationisavailableattheendofthearticleaSpringerOpenJournal2014Prakashetal.
;licenseeSpringer.
ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Prakashetal.
SpringerPlus2014,3:438http://www.
springerplus.
com/content/3/1/438ofB.
thailandensis,anon-virulentbutcloselyrelatedspe-ciespresentinthesoil,hasmadetheisolationandcharacterizationofB.
pseudomalleifromsoilverychallen-ging(Brettetal.
,1998).
ThisspecieshassimilarcolonymorphologycharacteristicstoB.
pseudomalleionsolidmediaandbiochemicalandmoleculartechniquesareneededtodistinguishbetweenthem.
TheisolationofB.
pseudomalleifromdifferentsoildepthsandduringdifferentseasonsoftheyearhasbeenstudied,andquanti-tativecultureofB.
pseudomalleifromsoilsampleshasalsobeendoneinmanycountriespreviously(Smithetal.
,1995;Brooketal.
,1997).
RecentlyareviewfortheglobalpresenceanddistributionofB.
pseudomalleiclearlyindi-catesthatisolationofthisspeciesfromsoilhasnotbeenreportedfromIndia,despiteitsisolationfromhumancases(Limmathurotsakuletal.
,2013).
ThepresentstudywasundertakentoattempttheisolationofB.
pseudomal-leifromthecoastalricecultivatingareasofTamilNadu,Indiatoconfirmtheidentityofisolatesbyconventionalandmolecularmethods.
MaterialsandmethodsStudysiteandcollectionofsamplesThesoutheastcoastofParangipettai,DistrictCuddalore,Tamilnadu,India(11°49′Nand79°76′E)wasselec-tedasthesamplingsiteforthisstudy.
Parangipettaiis30.
3kmfromthemaincityofCuddaloreand183kmfromChennai.
Theannualaveragerainfallofthisareaisapprox.
945.
0mm,meanrelativehumidity57%andave-ragetemperaturerangebetween28°Cto40°Cinsum-merand18°Cto26°Cintheshortlivedwinterseason.
FivepaddyfieldswerechosenasstudysitesandthesamplingwasdonejustaftertherainyseasonfromJulytoSept.
2010atadepthof25to30cm.
Amongthefivepaddyfieldssites,sites1and2belongtoPonnanthittuvillage;sites3and4belongtoPinnathurvillagesituatedontheleftsideoftheVellarriverandsite5issitu-atedrightsideoftheVellarriver,neartoMutlurroad(Figure1).
Tensamplingpointswereselectedfromsite1tosite4andfivesamplingpointsfromsite5.
Soilsampleswerecollectedat100mdistanceintervalsfromeachotherinastraightline.
Approximately100gmofmoistsoilwerecollectedfromeachsamplingpointsandimmedi-atelyplacedintoasterile50mlconicalcentrifugetubes.
Thetubesweresealedtoavoidcontaminationandtrans-portedtothelaboratoryforfurtherprocessing.
SoilprocessingandisolationThesoilsamplescollectedwereprocessedfortheisola-tionofBurkholderiapseudomalleiasperthefollowingprotocol.
Briefly3gmofeachsoilsamplewasvigorouslymixedwith3mlofsteriledistilledwaterandleftforovernight.
100μloftheuppersurfaceliquidwasthentransferredinto5mlAshdownbrothwithasterilepipetteandincubatedat37°Cfor48hrs(Figure2).
Ashdownse-lectiveagarwasmodifiedfromtherecipeofAshdown(1979)asfollows:tryptone1.
5g,glycerol4ml,crystalviolet(25mg/ml)150μl,neutralred(25mg/ml)100μl,gentamicin8μg/mlfinalconcentrationfor100mlofmedium.
Afterincubation100μlofbrothwasplatedontoAshdownselectiveagarplatesandincubatedat42°C.
Theplatesincubatedforfourdayswerevisuallyinspecteddailyuntiltypicalcoloniesformedaspreviouslydescribed(Chantratitaetal.
,2007).
ThecolonieswerepurifiedbyfurthersubcultureonAshdownagartoconfirmthepurityandpreservedin30%glycerolstockat20°Cuntilfurtheruse.
BiochemicalandphenotypicconfirmationInitialscreeningofisolateswasperformedaccordingtothestandardprotocolsfollowedfortheidentificationofB.
pseudomallei(SentinelLaboratoryguidelines,2003).
Twostandardstrains,NCTC1688andNCTC10274,wereusedasreferencetypestrainsalongwiththeisolates.
ThecommerciallyavailableAPI20NE(Biomerieux)wasalsousedforthegenerationofbiochemicalprofilesofallsoilisolatesalongwithstandardstrain(Danceetal.
,1989;Amornchaietal.
,2007).
Resultswererecordedafterincu-bationof24hrto48hrat28°CandinterpretedreferringtothedatainterpretationtablefromtheAPI20NEman-ual.
Invitroantibioticsusceptibilityofisolatesforpoly-myxinB(100units/disc)andcolistin(25mcg/disc)werealsotestedonMueller–HintonagarbytheKirbyBauerdiskdiffusionmethod(CLSI,2007).
AssimilationofL-arabinosewasalsotestedconventionallyasbecauseitplaysakeyroleinthediscriminationofvirulentB.
pseudomalleifromthenonvirulentspeciesB.
thailan-densis(Wuthiekanumetal.
,1996).
ThesuspectedisolateswerefurtherscreenedforyellowhazeproductiononFrancisagarforthediscriminationofB.
pseudomalleifromB.
cepacia(Francisetal.
,2006).
MolecularconfirmationIdentificationbySpecificPCRAlleightbiochemicallyidentifiedB.
pseudomalleiiso-latesand59nonB.
pseudomalleiisolateswerefurtheridentifiedbyPCRproceduresbasedonamplificationof23SrDNAgeneandtheputativevirulentdeterminantTTSSgene(Table1).
PCRwasstandardizedwithfor-wardandreversePCRprimersandperformedinavol-umeof25μl,thereactionmixturecontaining200mMofeachdNTP,1.
5mMMgCl2,1*PCRbuffer,10pmolofeachprimer,1UofTaqDNApolymerase(Fermentas)and10ngDNA.
ThePCRcycleprotocolconsistofini-tialdenaturationat95°Cfor6minand30cyclesofdenaturationat95°Cfor1min,primerspecificannea-lingfor1minandextensionat72°Cfor2minwiththefinalextensionat72°Cfor10min.
PCRproductswerePrakashetal.
SpringerPlus2014,3:438Page2of10http://www.
springerplus.
com/content/3/1/438electrophoresedon1%agarosegelandvisualisedunderUVinanAlphaInnotechGelImager(AmershamPhar-maciaBiotech).
16SrDNAbasedphylogenicanalysis16SrDNAsequencingwasusedtoconfirmPCRidenti-fiedisolatesand16SrDNAsequenceofeachisolatewasBLASTanalysed(Geeetal.
,2003).
ThePCRreactionmixturefortheamplificationofthe16SrDNAgeneconsistedof200mMofeachdNTP,1.
5mMMgCl2,1*PCRbuffer,10pmolofeachprimer,1UofTaqDNApolymerase(Fermentas)and10ngDNA.
Thereactionwasmadeupto25μlwithsteriledistilledwaterandthecycleconsistedofinitialdenaturationat95°Cfor6minand30cyclesofdenaturationat95°Cfor1min,anneal-ingat50°Cfor1minandextensionat72°Cfor2minwiththefinalextensionat72°Cfor10min.
PCRprod-uctswereelectrophoresedon1.
0%agarosegelandvis-ualisedunderUVinageldocumentationsystemasabove.
Amplified16SrDNAPCRproductswerese-quencedbythedideoxychainterminationmethodusingtheBigdyeTerminatorv3.
1cyclesequencingkitandBigdyeXTerminatorPurificationkitinanABI10se-quencer(AppliedBiosystems).
ThederivedsequenceswerealignedusingDNASTARlasergene9CoreSuitandBLASTanalysiswasperformedforcomparisonwithotherbacterialspecies,sequencesavailableintheNCBIdatabase.
Adendogrambasedon16SrDNAsequenceArrowindicatesSiteofthesamplecollectionCirclesindicatespositivesitesCirclesindicatesnegativesitesFigure1SoilsamplingandfivestudysitesofParangipettai,Tamilnadu,Indiaindicatedbyarrowasshowninmap.
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SpringerPlus2014,3:438Page3of10http://www.
springerplus.
com/content/3/1/438Table1PrimersusedinmolecularconfirmationstudyPrimernamePrimersequencesAnnealingtemperatureAmplificationsizePurpose/ReferenceVMP23-1-F5′CTTTTGGGTCATCCTRGA3′48°C1,051bpIdentification(Bauernfeindetal.
,1998)MP23-2-R5′TCCTACCATGCGAGACT3′BPTTS1-F5′CGTCTCTATACTGTCGAGCAATCG3′58°C548bpIdentification(Novaketal.
,2006)BPTTS1-R5′CGTGCACACCGGTCAGTATC3′F85′AGTTTGATCCTGGCTCAG3′50°C1,488bp16Ssequencing(Geeetal.
,2003)R14925′ACCTTGTTACGACTT3′fliC-F5′CTCGGATCCAACAGCAAC3′52°C1,167bpPCR-RFLP(Primerdesigned)fliC-RR-5′TATTGCAGGTACCTTCAG3′Figure2Schematicpresentationofisolationandidentificationprocedureusedinstudy.
Prakashetal.
SpringerPlus2014,3:438Page4of10http://www.
springerplus.
com/content/3/1/438wasalsoconstructedbytheneighborjoiningmethodusingMolecularEvolutionaryGeneticsAnalysisversion5.
0(MEGA5)analyticalsoftware(Tamuraetal.
,2011).
MALDI-TOFanalysisforbacterialidentificationEightbiochemicallyidentifiedisolateswerealsoMALDI-TOF/MSanalyzedfortheconfirmation(Table2).
FortheMALDI-TOF/MSanalysis,purifiedsinglecoloniesfroma24hrcultureofeachisolatewasdirectlydepos-itedonaMALDI-TOFMTP96targetplate(BrukerDaltonikGmbH),induplicateand1μlof96%formicacidtoeachspottoinactivatetheculture.
Thepreparationwasthenoverlaidwith1μlofmatrixsolutionthatcontainedasaturatedsolutionofα-cyano-4-hydroxycinnamicacidin50%acetonitrile,and2.
5%trifluoroaceticacid.
Atotalof18spots,twospotsfortheE.
colistandardand8*2fortheeightisolatesweremadeontargetplate.
Thismatrix-samplewascrystallizedbyair-dryingatroomtempe-raturefor5minutes.
MeasurementswereperformedwithaMicroflexIIImassspectrometer(BrukerDaltonik)equippedwitha337-nmnitrogenlaser.
Spectrawerere-cordedinthepositivelinearmode(delay,170ns;ionsource1voltage,20kV;ionsource2voltage,18.
5kV;lensvoltage,6kV;massrange,2–20kDa).
Eachspectrumwasobtainedafter240shotsinautomaticmodeatavariablelaserpower,andtheacquisitiontimerangedfrom30to60sperspot.
DatawereautomaticallyacquiredusingAutoXecutemethodofflexControlversion3.
3acquisitioncontrolsoftware.
The2firstrawspectraobtainedforeachisolatewereimportedintoBioTypersoftware,version3.
0(BrukerDaltonikGmbH),andwereanalyzedbystandardpatternmatchingagainstthe4110massspectraofdif-ferentbacterialspeciesasreferencefromtheBiotyperdatabase.
FlagellinC(fliC)genebasedrestrictionfragmentlengthanalysisRestrictionfragmentlengthanalysiswasperformedonthespecificPCR,16SrDNAsequencingandMALDI-TOF/MSconfirmedisolatestoobservethatwhethertheseisolatesbelongstosimilarstrains(cloneofasinglestrain)orbelongstodifferentstrains.
TheprimersfliCforward5′-CTCGGATCCAACAGCAAC-3′andfliCreverse5′-TATTGCAGGTACCTTCAG-3′wereusedfortheamplificationofthe1167bpfliCgene.
ThePCRamplificationwascarriedoutina50μlreactionTable2BiochemicalprofileofstandardstrainsandconfirmedsoilisolatesSubstrateStandardstrainsSoilisolatesBurkholderiapseudomalleiNTCC10274BurkholderiapseudomalleiNTCC1688DRDEBPS1001DRDEBPS1002DRDEBPS1003DRDEBPS1004NitrateTryptophanGlucose(acidification)ArginineUrea*Esculin*GelatinPNPGAssimilationofGlucoseArabinoseMannoseMannitolN-AcetylglucosamineMaltoseGluconateCaprateAdipateMalateCitratePhenylacetate*VariabletestsforB.
pseudomallei.
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SpringerPlus2014,3:438Page5of10http://www.
springerplus.
com/content/3/1/438Table3BiochemicalprofilesofAshdowngrownsuspectedsoilisolatesconfirmedbyAPI20NESpeciesnameNO3/N2TRPGLUADHUREESCGELPNGGLUARAMNEMANNAGMALGNTCAPADIMLTCITPACOchrobactrumanthropi(n=3)Achromobacterxylosoxidans(n=5)Chromobacteriumviolaceum(n=9)Stenotrophomonasmaltophilia(n=10)Rizobiumradiobacter(n=1)Burkholderiacepacia(n=4)Delftiaacidovorans(n=5)Ralstoniapickettii(n=3)Enterococcusfaecalis(n=2)Brevundimonasvesicularis(n=1)Pseudomonasluteola(n=2)Comamonastestosterone(n=1)Pseudomonasaeruginosa(n=5)Acinetobacterlwoffii(n=1)Acinetobacterdentrificans(n=2)Pseudomonasmendocina(n=1)Chryseobacteriumindologenens(n=4)Figure3VMPPCRfortheconfirmationofisolates-Lane1:100bpplusMarker,Lane2:B.
pseudomalleiNTCC10274,Lane3:B.
pseudomalleiNTCC1688,Lane4:SoilisolateDRDEBPS1001,Lane5:DRDEBPS1002,Lane6:DRDEBPS1003,Lane7:DRDEBPS1004,Lane8:Negativecontrol.
Prakashetal.
SpringerPlus2014,3:438Page6of10http://www.
springerplus.
com/content/3/1/438Figure4TTSSPCRfortheconfirmationofisolates-Lane1:100bpplusMarker,Lane2:B.
pseudomalleiNTCC10274,Lane3:SoilisolateDRDEBPS1001,Lane4:DRDEBPS1002,Lane5:DRDEBPS1003,Lane6:DRDEBPS1004,Lane7:100bpplusMarker.
DRDEBPS1003DRDEBPS1004DRDEBPS1002DRDEBPS1001B.
pseudomalleiK96243B.
pseudomalleiPasteur52237B.
pseudomallei1710aB.
pseudomalleiNTCC1688B.
pseudomallei668B.
malleiATCC10399B.
malleiPRL-20B.
pseudomallei1106aB.
pseudomalleiPakistan9BUHB.
pseudomalleiMSHR346B.
oklahomensisC6786B.
glumaestrainP1-22-1B.
plantariistrainNIAES1723B.
thailandensisstrainCIP106301B.
ubonensisstrainGTC-P3-415B.
ambifariaAMMDB.
pyrrociniastrain2327B.
cepaciastrain717B.
vietnamiensisstrainLMG10929B.
cenocepaciastrainLMG16656B.
multivoransCGD2MB.
thailandensisBt4PMP6xxBTHxxBt4-3200.
005Figure5Dendogrambasedon16SrDNAsequencingconstructedbyneighborjoiningmethod(Barrepresents0.
005substitutionpersite).
SoilisolatesDRDEBPS1001,DRDEBPS1002,DRDEBPS1003andDRDEBPS1004showingproximitytoB.
pseudomalleistandardstrains.
Prakashetal.
SpringerPlus2014,3:438Page7of10http://www.
springerplus.
com/content/3/1/438volumewiththefollowingPCRconditions:initialde-naturationat95°Cfor6minand30cyclesofdenatur-ationat95°Cfor1min,annealingat52°Cfor1minandextensionat72°Cfor2minwiththefinalextensionat72°Cfor10min.
PCRproductswerepurifiedusingagelextractionpurificationkit(Qiagen)andconcentrationwasdeterminedspectrophotometrically.
Restrictiondi-gestionwasperformedwiththreerestrictionenzymesDdeI,MspIandPstI(Fermentas,fastdigest).
Restric-tiondigestionreactionmixturecontained7μlofpuri-fiedfliCPCRproductsataconc.
of100ng/μland1Uofrestrictionenzymes.
Restrictiondigestionreactionwascarriedoutin0.
5mltubesincubatedat37°Cfor2hrsandtheninactivatedbykeepingtubesinadrybathat65°Cfor10minutes.
Thendigestedproductswereelec-trophoresedon1.
8%agarosegelandvisualizedunderUVinanAlphaInnotechGelImager.
Results&DiscussionFromthe45soilsamples67isolatesweresuspectedandselectedaspossibleB.
pseudomalleibasedoncharacteris-ticcolonialmorphologyanddyeabsorptiononAshdownagarafterincubationof48hrat42°C.
Conventionalbio-chemicalprofilesofeachofthese67isolateswastestedandcomparedwiththeprofilesofB.
pseudomalleiNCTC1688andNCTC10274.
Thebiochemicalprofilesofeightisolates,namelyDRDEBPS1001,DRDEBPS1002,DRDEBPS1003,DRDEBPS1004,DRDEBPS1018,DRDEBPS1019,DRDEBPS1020andDRDEBPS1021matchedwiththebio-chemicalprofileofB.
pseudomalleiwithnegativearabin-osesugarassimilation.
Interestinglyalltheenvironmentalisolateswereabletoassimilatemaltosebutthestandardstrainscouldnotassimilatethissugar,asalsoobservedwithmostclinicalisolatesofB.
pseudomallei.
Thebio-chemicalprofileofallthese67isolateswasdeterminedbyAPI20NEandtheeightisolatesthatwereidentifiedasB.
pseudomalleibyconventionalbiochemicalassayswerealsopositivebyAPI20NE.
Theidentificationoftheremaining59isolatesbyAPI20NEisgiveninTable3.
Theantibioticsusceptibilityofthe8isolatesthatarebio-chemicallyconsistentwithB.
pseudomalleiwastestedagainstantibiotics,polymyxin(B100units/disc)andcolis-tin(25mcg/disc)andthesusceptibilitypatternwasfoundsimilartothatoftheB.
pseudomalleistandardstrains.
Theseeightisolateswerefurtherprocessedformole-cularconfirmationandonlyfourDRDEBPS1001,DRDEBPS1002,DRDEBPS1003andDRDEBPS1004werefoundpositivebyB.
pseudomalleispecificPCRforthetruncatedregionof23SrDNA(VMPPCR)andTTSSgene(TTSSPCR)withtheamplificationof1,051bpand548bprespectively(Figures3and4).
BoththesePCRswerealsoperformedwiththeDNAof59isolatesthatwerebio-chemicallygroupedasnonB.
pseudomalleiandnoampli-ficationwasobservedintheseisolatesconfirmingthebiochemicalresults.
Sequencingof16SrDNAoftheseeightisolatesconfirmedthat,thefourisolatesshowed100%homologywiththeNCBIdatabasereferencestrainsofB.
pseudomallei.
IsolatesDRDEBPS1018,DRDEBPS1019wereidentifiedasCupriavidusnecatorandisolatesDRDEBPS1020,DRDEBPS1021asEnterobactercloacae.
Thealignedpartialsequencesof16SrDNAweresub-mittedtoGenBankandaccessionnumbersJN001986,JN001987,JN001988andJN001989wereobtained.
Aphylogenetictreebasedon16SrDNAsequencesofallfourconfirmedisolateswasalsoconstructedalongwiththestandardstrainsofB.
pseudomalleiandothercloselyrelatedspecies(Figure5).
ThephylogeneticanalysisalsorevealedthatallfourisolatescloselymatchedwiththestandardstrainsofB.
pseudomalleiandwereplacedinthesameclade.
Thefourisolatesconfirmedby16SrDNAsequencingweresubjectedtoPCR-RFLPforthefliClocusandre-strictionprofilesforthreedifferentrestrictionenzymesweregenerated.
TherestrictionprofileafterdigestionwithMspIproduced443,285,134,123,99,64,19bpsizeproducts,PstIdigestionproduced681,266,220bpsizeproductsandDdeIenzymedigestionproduced628,307,123,109bpsizeproductsandtherestrictionprofileFigure6fliCgeneRestrictionpatternsofisolatesLane:M100bpplusMarker,Lane1,7,13:B.
pseudomalleiNTCC10274,Lane2,8,14:B.
pseudomalleiNTCC1688,Lane3,9,15:SoilisolateDRDEBPS1001,Lane4,10,16:DRDEBPS1002,Lane5,11,17:SoilisolateDRDEBPS1003,Lane6,12,18:SoilisolateDRDEBPS1004.
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SpringerPlus2014,3:438Page8of10http://www.
springerplus.
com/content/3/1/438forallthesethreeenzymesmatchedwithB.
pseudo-malleistandardstrainsNCTC1688andNCTC10274,therebyconfirmingallfourisolatesasB.
pseudomalleiwithidenticalrestrictionprofiles(Figure6).
RecentlyMALDI-TOFmassspectrumanalysishasbeenconsideredaneasyanddiscriminatorytoolforidentificationofbacterialspecies(Listaetal.
,2011).
TheresultsofMALDI-TOFmassspectrumanalysisoftheeightsuspectedisolatesmatchedwiththatofthe16SrDNAsequenceanalysis.
ThefourisolatesDRDEBPS1001,DRDEBPS1002,DRDEBPS1003andDRDEBPS1004wereconfirmedasB.
pseudomalleionthebasisofscorevalues2.
601,2.
099,2.
362and2.
047.
TheotherfourisolateshadbeenbiochemicallysuspectedbutnotsupportedbyboththePCRswerealsoidentifiedbyMALDI-TOFspectrumanalysisasCupriavidusnecatorandEnterobactercloacaewithscorevalueof2.
112,2.
122and2.
341,2.
241respectively,confirmingtheresultsofthe16Sanalysis.
TheisolationofB.
pseudomalleifromsoilisverycom-plexasthepresenceoflargenumbersofcloselyrelatedsoilmicroflorainterfereswithitsrecoveryalthoughuseofAshdownbrothandagarfortheisolationofB.
pseu-domalleifromsoilsamplesprovidessomeselectivity.
Thisselectivitycomesfromcrystalviolet,neutralredandgentamicinpresentintheAshdownmedium.
ThevastmajorityofB.
pseudomalleiisolatesareresistanttoaminoglycosidesduetoexpressionofamultidrugeffluxpump,thusallowinguseofgentamicinforselection.
Thisstudyconfirmsthat,althoughthismediumisusefulforinitialscreening,manyotherspeciesalsogrowonthismediumthusinterferingtherecoveryofB.
pseudo-mallei.
Francisagarisamediumthatcanclearlydif-ferentiatebetweenB.
pseudomalleiandothercloselyrelatedspeciesincludingB.
cepacia(Francisetal.
,2006).
Wealsoanalyzedall8suspectedisolatesofB.
pseudo-malleionFrancisagartoobservetheyellowhazearoundcolonies24hr.
AlthoughFrancisAgarwasnotdevelopedforprimarysoilisolationofthisspeciesitcanbeusedtoconfirmsuspectedcoloniesisolatedfromAshdownagar.
Inourstudytherateofisolationwasfoundtobe5.
97%as4isolateswereconfirmedasB.
pseudomalleioutof67suspectedisolatesfromAshdownagarand2isolateseachwereobtainedfromsite1andsite2(Figure1).
InsimilarsoilisolationstudiesconductedinMalaysiaandThailand,highisolationrateswerefoundinwetricefieldsandotherclearedandcultivatedareas(Brettetal.
,1998;Smithetal.
,1995).
ThisstudyreportsthefirstisolationofB.
pseu-domalleifromsoilcollectedfrompaddyfieldsinthecoas-talregionofIndia.
Thestudysiteselectedinthisstudyfulfilledwithallcriteriaofsoilsamplingwhichwerecon-sideredasimportantfactorsintheisolationofB.
pseu-domallei.
ThepresenceofB.
pseudomalleidependsonvariousenvironmentalfactorsthatsupportthesurvivalofB.
pseudomalleiinsoil.
ThepresenceofB.
pseudomalleiinsoilishighduringwhenploughingandplantingofseedlingstakesplace(Palasatienetal.
,2008;Currieetal.
,2004).
Themaintenanceofviablebacteriainsoilsamplesduringcollection,transportandstoragebeforeprocessinginlaboratoryisalsoanimportantfactorfortheisolation.
ThepresenceofB.
pseudomalleineedstobeinitiallyde-terminedbyculturebasedmethodsinmicrobiologicalla-boratoriesthatrequireonlybasicequipmentandprovidesliveorganismsforfurtherconfirmationbyDNAbasedmolecularmethods.
Theisolatesrecoveredfromsoilneedtobecomparedwiththoserecoveredfromclinicalcasesasitprovidesvitalinformationonthepathogenicityandvirulenceofsoilisolates(Currieetal.
,2004).
Thepresenceofidenticalgeneticpatternsamongclinicalandenviron-mentalisolatessuggestsalinkbetweenthebacteriapre-sentincontaminatedsoilandtheemergenceofindigenousmelioidosis(Chenetal.
,2010).
Futurestudiesarealsorec-ommendedwithlargenumberofsamplescollectedfromdifferentgeographicalregionsofIndiatostudythepatternofdistributionofthisimportantbacterialspeciesandalsotocorrelatetheepidemiologicalrelevanceofsoilisolationtotheoccurrenceofmelioidosis.
ConclusionWereportthefirstisolationandmolecularconfirmationofB.
pseudomalleifromsoilofpaddyfieldsinthecoas-talregionofIndia.
Theseisolateswereinitiallyidentifiedbyconventionalbiochemicalmethodsandfurthercon-firmedbyadvancedmolecularbasedmethodsof16SrDNAsequencing,B.
pseudomalleispecificPCR,PCR-RFLPatfliClocusandMALDI-TOFbasedproteinprofiling.
ThisstudyconfirmsthepresenceofB.
pseudo-malleiinsoilinthecoastalregionofIndia.
TheisolationofthisimportantbacterialspeciesfromthispartofIndiashouldinitiatefurtherstudiesontheextentofenviron-mentalandclinicalimpactofmelioidosisinIndia.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionAPcarriedoutprocessingofsoilsamplesandmolecularconfirmation,DTcarriedoutdatainspection,analysisofresultsandwritingofmanuscript,AKprovidedcomputationalhelpforthealignmentandphylogenicanalysisofsequences,AKwasresponsibleforcollectionofsoilsamplesandtransportedtothelab,SA,STAB,KShelpedinexperimentsanddataanalysis.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementAuthorsarethankfultoDrM.
P.
Kaushik,Director,DRDEGwaliorandDr.
UrmilTutejaHeadofMicrobiologyDivision,DRDEGwaliorfortheirkindsupportsandprovidingfacilitiesforthiswork.
ThefinancialsupporttoArchanaPrakashbyUGC,Govt.
ofIndiaisalsoacknowledged.
Authordetails1DivisionofMicrobiology,DefenceResearch&DevelopmentEstablishment,JhansiRoad,Gwalior474002,India.
2CentreforAdvancedStudiesinMarineBiology,AnnamalaiUniversity,Parangipettai,TamilNadu.
Prakashetal.
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:Isolation,identificationandcharacterizationofBurkholderiapseudomalleifromsoilofcoastalregionofIndia.
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