Rapid biosynthesis of silver nanoparticles from< i> Bacillus megaterium(NCIM 2326) and their antibacterial activity on multi drug resistant clinical pathogens

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The notion to fight against multi drug resistant pathogens is a great deal in the field of nanomedicine. The identifiable antimicrobial action of metal bionanoparticles on many microorganisms is reported earlier. As silver bionanoparticles (Ag-BNPs)
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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/234102104 Rapid biosynthesis of silver nanoparticles fromBacillus megaterium (NCIM 2326)and theirantibacterial activity on multi drug...  Article   in  Colloids and surfaces B: Biointerfaces · November 2011 DOI: 10.1016/j.colsurfb.2011.07.009 · Source: PubMed CITATIONS 59 READS 143 3 authors , including:Saravanan MuthupandianMekelle University 46   PUBLICATIONS   342   CITATIONS   SEE PROFILE Sisir Kumar BarikUniversity of Aberdeen 3   PUBLICATIONS   58   CITATIONS   SEE PROFILE All content following this page was uploaded by Saravanan Muthupandian on 04 December 2016. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  ColloidsandSurfacesB:Biointerfaces 88 (2011) 325–331 ContentslistsavailableatScienceDirect Colloids   and   Surfaces   B:   Biointerfaces  journalhomepage:www.elsevier.com/locate/colsurfb Rapid   biosynthesis   of    silver   nanoparticles   from   Bacillus   megaterium   (NCIM   2326)and   their   antibacterial   activity   on   multi   drug   resistant   clinical   pathogens M.   Saravanan ∗ ,   Anil   Kumar   Vemu,   Sisir   Kumar   Barik DepartmentofBiotechnology,FacultyofScienceandHumanities,SRMUniversity,SRMNagar,Kattankulathur,Chennai,Tamilnadu603203,India a   r   t   i   c   l   e   i   n   f   o  Articlehistory: Received18January2011Receivedinrevisedform3May   2011Accepted5July2011 Available online 12 July 2011 Keywords: Biosynthesis Bacillusmegaterium AtomicForceMicroscopyAntibacterialMultiDrugResistant a   b   s   t   r   a   c   t The   notion   to   fight   against   multi   drug   resistant   pathogens   is   agreat   deal   in   the   field   of    nanomedicine.   Theidentifiable   antimicrobial   action   of    metal   bionanoparticles   on   many   microorganisms   is   reported   earlier.Assilver   bionanoparticles   (Ag-BNPs)   are   known   to   have   efficient   antibacterial   properties   they   are   synthe-sizedinecofriendly   and   biocompatible   way.   The   present   study   is   focused   on   the   extracellular   biosynthesisof    highly   stable   Ag-BNPs   from   bacterial   strain   Bacillus   megaterium   (NCIM   2326)   bybio-reduction   of    silverionusing   the   culture   supernatant,   and   to   determine   the   antibacterial   efficacy   onmulti   drug   resistant   clin-ical   pathogens   such   as Streptococcus    pneumoniae   and   Salmonella   typhi .   The   biosynthesis   process   israpidand   Ag-BNPs   are   formed   within   few   minutes   if    AgNO 3  comes   to   contact   with   cell   filtrate.   Furthermore   thesynthesized   Ag-BNPs   are   characterized   byUV–vis   spectroscopy,   Atomic   Force   Microscopy   (AFM),   ThinLayer   Chromatography   (TLC)   and   Fourier   Transform   Infrared   Spectroscopy   (FTIR). © 2011 Elsevier B.V. All rights reserved. 1.Introduction Nanotechnologyistheemergingfieldin21stcenturywhichistherootcausefornextindustrialrevolution.Synthesis,char-acterization,manipulationandapplicationofnanomaterialsarebeingrapiddevelopmentinnanotechnology.Nanoparticlesarethebuildingblocksofnanotechnologyastheyplaymajorroleintheirapplications.Theapplicationofnanoscalematerialsandstructuresmayprovidesolutionstotechnologicalandenvironmentalchal-lengesintheareasofsolarenergyconversion,catalysis,medicineandwatertreatment[1,2].Metalnanoparticlesareofmuchimportanceduetotheirhighspecificsurfaceareaandahighfractionofsurfaceatoms,havebeenstudiedingreatextentbecauseoftheiruniquephysio-chemicalcharacterizationwhichincludescatalyticactivity,opticalproperties,electronicproperties,antimicrobialactivity,magneticproperty[3,4]andbiomedicine[5–8].Theexploringbactericidal effectofmetalnanoparticlesistheleadingresearchfieldinthisdecade,butitwasalsoacceptedthatnanoparticlesmay   havedetrimentaleffectsintheecosystem[9,10].   Amongmetalnanopar-  Abbreviations: AFM,AtomicForceMicroscopy;Ag-BNPs,SilverBionanoparti-cles;AgNO 3 ,silvernitrate;FTIR,FourierTransformInfraredSpectroscopy;MDR,MultiDrugResistant;MHA,MullerHintonAgar;NAWD,Nathan’sAgarWellDif-fusion;NCIM,NationalCollectionofIndustrialMicroorganisms;NCL,NationalChemicalLaboratories;TLC,ThinLayerChromatography. ∗ Correspondingauthor.Tel.:+9104427452270;fax:+9104427453903. E-mailaddresses: bioinfosaran@gmail.com,saravanan@sh.srmuniv.ac.in(M.   Saravanan). ticles(cadmiumsulfide,goldandsilver)[11],silvernanoparticles (Ag-NPs)haveeffectiveantimicrobialactivity[12–16],   especiallybactericidaleffect[17–21].   Inthismodernworldthepathogensareresistanttomultipleantimicrobialagents[22]isbeingamajor problem,e.g. Staphylococcusaureus resistanttoMethicillinand Can-didaalbicans resistanttoFluconazole[23]andmanypathogenicbacteria[24,25].Silvernanoparticlescanbeproducedbyphysicalandchemicalmethods[26,38]theycanalsobeproducedinbiologicalmethods specificallybybio-reduction[27–29].   Asthephysicalandchemi-calmethodusestoxicchemicalsintheirsynthesis,itraisesgreatconcernforenvironmentalreasons[30],nowitbecameimmense researchstudyfortheproductionofMetalNanoparticles(MeNPs)insafe,reliable,clean,ecofriendlyway.Sothebiosynthesisof theMeNPsfrommicroorganismsandplantsisbeingdeveloped.ThesynthesisandassemblyofNPsfromofcleannontoxicandenvironmentallyacceptable‘Greenchemistry’proceduresinvolvesmicroorganismsandplants[31].Bothunicellularandmulticellular organismsareknowntoproduceinorganicmaterialseitherintra-cellularlyorextracellularly[32–35].   Biologicalproductionsystemsareofspecialinterestduetotheireffectivenessandflexibility.Thebactericidaleffectofsilvernanoparticlesisduetotheirsmallsizeandhighsurfaceareatovolumeratio,whichallowsthemtointeractcloselywithmicrobialmembranes[36].Smallerparticles withalargersurfaceareahaveeffectiveantibacterialactivity[37]anditisalsoknownthatbactericidaleffectofsilvernanoparticlesdecreasesasthesizeincreasesandisalsoaffectedbytheshape[38].Effectoftheshapeontheantibacterialactivityofhasrecentlybeenreported[39]. 0927-7765/$–seefrontmatter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.07.009  326  M.Saravananetal./ColloidsandSurfacesB:Biointerfaces 88 (2011) 325–331 Fig.1. Purecultureof  Bacillusmegaterium (NCIM2326)usedforsynthesisofAg-BNPs. SeveralstudiesproposeAg-NPsmay   attachtothesurfaceof thecellmembranedisturbingandalteringthefunctionsofthecell[40].Thedamageofthecellmay   becausedbyinteractionof Ag-NPswithphosphorousandsulfur-containingcompoundssuchasDNAandproteins.Silvertendstohaveahighaffinityforsuchcompounds[41,42].Silverionsstronglyinteractwiththeavail- able–SHgroupsofthebiomoleculestoinactivatethebacteria[43].Furthermore,theantibacterialactivityofsilverionunderanaerobicconditionswasfoundlesspotentthaninoxygenrichenvironment[44].Suchinteractionsinthecellmembranewould preventDNAreplications[45]whichwouldleadtobacterialdeath [46,47].Thepresentinvestigationhasfocusedonextracellularbiosyn-thesisofhighlystablesilverbionanoparticles(Ag-BNPs)andtodeterminetheantibacterialefficacyagainstmultidrugresistantclinicalbacterialpathogen.ThesestableAg-BNPswerebesyn-thesizedbyusingabacterium Bacillusmegaterium (NCIM2326)toaccesstheirantimicrobialactivityagainstMultiDrugResis-tant(MDR)clinicalpathogenssuchas Streptococcuspneumoniae and Salmonellatyphi .Thestudyalsoincludesthecharacteriza-tionsofAg-BNPsbyUV–visspectrophotometer,TLC,AtomicForceMicroscopy(AFM)andFTIRspectralanalysis.Basedontheprob-lemofemergingantibioticresistantstrains,thepresentworkwouldbeamajorresearchefforttodiscoveralternativestrategies(Nanomedicine)forthetreatmentofmultidrugresistantbacterialinfections. 2.Experimentaldetails  2.1.Mediaandchemicals Allthemediacomponentsandanalyticalreagentswerepur-chasedfromHi-MediaLaboratoriesPvtLtd.(Mumbai,India)andSigmaAldrichChemicals(St.Louis,USA).  2.2.Microorganisms Thebacterialstrain B.megaterium (NCIM2326)obtainedfromNCL,PuneandthemultidrugresistantBacterialpathogenicstrainsobtainedfromSRMmedicalcollegehospitalandresearchcentre,Kattankulathur,Chennai.Thestrainsweremaintainedonnutri-entagarmediumat37 ◦ Candpreservedat4 ◦ Cforfurtherstudy.Thesestrainsweresubculturedtimetotimetoregulateviabilityinthemicrobiologylaboratory,DepartmentofBiotechnology,SRMUniversity,Chennai,Indiaduringthestudyperiod.  2.3.Extracellularsynthesisofsilverbionanoparticles The B.megaterium (NCIM2326)strainwasfreshlyinoculatedinanErlenmeyerflaskcontainingMullerHintonbroth.Theflakswereincubatedinorbitalshakerat37 ◦ Candagitatedat200rpmfor Fig.2. (a)Cellfiltrateof  Bacillusmegaterium (NCIM2326)withoutsilvernitrate(control),(b)cellfreeextractwithAgNO 3  after24hincubation.  M.   Saravananetal./ColloidsandSurfacesB:Biointerfaces 88 (2011) 325–331 327 Fig.3. UV–visspectroscopicanalysisofsynthesizedAg-BNPsandshowedtheplasmonresonancepeaknotedaround390nm. 24h.Afterincubation,thecellfiltrateswereobtainedbycentrifuga-tionat10,000rpmfor7min   andfollowedbydecantation.Thefinalconcentrationof1mM   AgNO 3  (0.017gAgNO 3 /100ml)   wasaddedinto100ml   ofcellfiltratein250ml   Erlenmeyerflask.Theflaskswereincubatedindarkroomconditionupto48h.ThecontrolwasmaintainedwithoutadditionofAgNO 3  withtheexperimentalflaskcontainingcellfiltrate.Thedarkbrowncoloredsolutionofsilverbionanoparticleswasstoredinscrewcappedvialsunderambientconditionforfurthercharacterizationandapplications.  2.4.Characterizationofsilverbionanoparticles Thesynthesizedsilverbionanoparticleswerefirstcharacter-izedbyUV–visspectrophotometerintherangeof250–650nm.(Perkin–Elmer,Germany),isusingaquartzcuvettewithcontrolasthereference.Thesilverbionanoparticleswerekeptatroomtemperatureforthreemonthstotesttheirstabilityandcharacteri-zationofthestabilizedAg-BNPswascarriedoutusingTLC.Thiswasbasedontheprinciplethatbionanoparticlesgetseparatedonbasisofdifferentialadsorptionontosilicagel.Thestandardsolventsys-temN-butanol,aceticacidanddistilledwater(8:2:2v/v)wasusedasamobilephase.ThebionanoparticleswasspottedonTLCsheetcoatedwiththinlayerofsilicagel(Macherey-Nagel,Germany)anditwaskeptinsidethechromatographicchambercontainingsolventandallowedtodevelopchromatogramfor20min.TheTLCsheetremovedfromthechamberwasallowedtodryafter20min   bykeepinginhotairovenat60 ◦ Cfor10min,andthen0.3%Ninhydrinwassprayedasdeveloperanddriedat45 ◦ Cfor5min.TheactivatedTLCsheetisobservedtoelucidatethepresenceofAg-BNPs.ThesizeandmorphologicalcharacterizationofthesynthesizedAg-BNPswerestudiedusingAtomicForceMicroscopyincontactmodebydissolvinglyophilizedAg-BNPssampleswithacetoneandspincoatingthesampleusingapexinstrumentsspincoateratamaximumspeedof9000rpmandthendriedthesamplefor10min.FurthercharacterizationofAgbionanoparticlesinvolvedFourierTransformInfraredSpectroscopy(FTIR)(Perkin–Elmer,Germany)byscanningthespectrumintherange450–4000cm − 1 atresolutionof4cm − 1 .  2.5.AnalysisofantimicrobialactivityofAgbionanoparticles(Ag-BNPs)welldiffusionmethod TheantibacterialactivityoftheAgbionanoparticleswasexam-inedbyfollowingthestandardNathan’sAgarWellDiffusion(NAWD)technique.Wellsof6mmdiameterweremadeonthepre-pouredMullerHintonAgar(MHA)plates.TheseMHA   plateswereinoculatedbyswabbingthe18–24holdmultidrugresis-tantBacterialpathogenstocreateaconfluentlawnofbacterialgrowth.TheAg-BNPsinvariousconcentrations(5  l,10  l,15  land20  l)wereloadedineachwell.WellinthecentreoftheplatewithouttheAg-BNPswas   maintainedascontrol.Theplateswereincubatedat37 ◦ Ctemperaturefor24h.Afterincubationthesusceptibilityofthetestorganismswas   determinedbymeasur-ingthediameterofthezoneofinhibitionaroundeachwelltothenearestmm. Fig.4. ThinLayerChromatographicseparationofAg-BNPs.  328  M.Saravananetal./ColloidsandSurfacesB:Biointerfaces 88 (2011) 325–331 Fig.5. FTIRspectralanalysisofAg-BNPssynthesizedusing Bacillusmegaterium (NCIM2326). Fig.6. AtomicForceMicroscopyimageofAg-BNPssynthesizedfrom Bacillusmegaterium (NCIM2326)anditscorrespondinglineprofileforsizedetermination(80–98.56nm)(a)   agglomerationofAg-BNPssizedeterminationusing360nmscale,(b)magnifiedimageofagglomeratedAg-BNPsusing89nmscale.
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