Enhanced antibacterial metabolite production through the application of statistical methodologies

21 Pages·2017·3.06 MB·English

RESEARCHARTICLE
Enhanced antibacterial metabolite production
through the application of statistical
methodologies by a Streptomyces nogalater
NIIST A30 isolated from Western Ghats forest
soil
JubiJacob1,2,ReshmaUmaRajendran1,SyamaHariPriya1,JayamurthyPurushothaman1,2,
a1111111111 DileepKumarBhaskaranNairSaraswathyAmma1,2*
a1111111111
a1111111111 1 Agro-ProcessingandTechnologyDivision,CSIR-NationalInstituteforInterdisciplinaryScienceand
Technology(NIIST),Thiruvananthapuram,Kerala,India,2 AcademyofScientificandInnovativeResearch
a1111111111
(AcSIR),CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology(NIIST),Thiruvananthapuram,
a1111111111
Kerala,India
*[email protected]
OPENACCESS Abstract
Citation:JacobJ,RajendranRU,PriyaSH,
PurushothamanJ,SaraswathyAmmaDKBN StreptomycesstrainsisolatedfromNelliyampathyforestsoilofWesternGhats,Kerala,
(2017)Enhancedantibacterialmetabolite Indiawereevaluatedfortheirantibacterialefficacyagainsttwoindicatorpathogenicbacteria
productionthroughtheapplicationofstatistical
(EscherichiacoliandStaphylococcusaureus).Among140strainstested,sixteenrecorded
methodologiesbyaStreptomycesnogalaterNIIST
potentantibacterialpropertiesandwerefurtherscreenedagainstelevenbacterialpatho-
A30isolatedfromWesternGhatsforestsoil.PLoS
ONE12(4):e0175919.https://doi.org/10.1371/ gens.AstrainidentifiedasStreptomycesnogalateranddesignatedasNIISTA30exhibited
journal.pone.0175919 maximuminhibitionagainstallthetestpathogens.Amongtheeightfermentationmedia
Editor:Marie-JoelleVirolle,UniversiteParis-Sud, tested,inorganicsaltsstarchbrothrecordedthebestforantibacterialproduction.Theethyl
FRANCE acetatecrudeextractexhibitedantioxidantpropertieswithIC valueof30μg/mLandhad
50
Received:November28,2016 nocytotoxicitytowardsL6,H9c2andRAW264.7celllinesuptoaconcentrationof50μg/
mL.MaximummetaboliteproductionwasachievedinpH7.0at35˚Cafter7daysincubation.
Accepted:March14,2017
Thesignificantmediacomponentsformaximummetaboliteproductionwereoptimized
Published:April24,2017
throughresponsesurfacemethodologyemployingPlackett-BurmanandBox-Behnken
Copyright:©2017Jacobetal.Thisisanopen
designs.Thecompositionofthefinaloptimizedmediumwassolublestarch,14.97g;
accessarticledistributedunderthetermsofthe
(NH ) SO ,2.89g;K HPO ,2.07g;MgSO .7H O,1g;NaCl,1g,CaCO ,2g;FeSO .7H O,
CreativeCommonsAttributionLicense,which 4 2 4 2 4 4 2 3 4 2
permitsunrestricteduse,distribution,and 1mg;MnCl .7H O,1mg;andZnSO .7H O,1mgperlitreofdistilledwater.Theoptimization
2 2 4 2
reproductioninanymedium,providedtheoriginal resultedanantibacterialactivityof28±1.5mmagainstS.epidermidiswhichwasinclose
authorandsourcearecredited.
accordancewiththepredictedvalueof30mm.Itisalsoevidentfromtheresultthatan
DataAvailabilityStatement:Allrelevantdataare increaseof86.66%antibacterialproductionwasrecordedinoptimizedmedia.Thechosen
withinthepaperanditsSupportingInformation
methodwaseconomical,efficientandusefulforfutureantibacterialdrugdiscoveryfroma
files.
broadspectrummetaboliteproducerlikeStreptomycesnogalaterNIISTA30.
Funding:Financialsupportwasreceivedfrom
KeralaStateCouncilforScience,Technologyand
Environment(KSCSTE),Thiruvananthapuramand
theDepartmentofScienceandTechnology,New
DelhiforInspireFellowship(IF130648)toJJ.The
fundershadnoroleinstudydesign,datacollection
PLOSONE|https://doi.org/10.1371/journal.pone.0175919 April24,2017 1/21
EnhancedantibacterialmetaboliteproductionfromStreptomycesnogalaterNIISTA30
andanalysis,decisiontopublish,orpreparationof Introduction
themanuscript.
Thedemandandsearchfornovelantibioticsisincreasingduetotheemergenceofmultiple
Competinginterests:Theauthorshavedeclared
drugresistanceinpathogensaroundtheglobeassociatewiththediscoveryofverylessnumber
thatnocompetinginterestsexist.
ofnewpotentialmolecules.Forthis,Streptomycesspeciesareoneofthemajortargetsformin-
ingnewantibiotics.Thehistoryofantibioticsfromthesefilamentous,Gram-positivebacteria
gotattentionamongtheresearcherswiththediscoveryofstreptothricinin1942followedby
streptomycinaftertwoyears[1,2].ThegenusStreptomycesbelongingtothegroupactinobac-
teriawhosespeciesarewidelydistributedinterrestrialandmarinehabitats,havebeen
regardedasabowlofnaturalproductswithvariousinterestingbiologicalactivitiesincluding
antibacterialproperties.Themetabolitesincludeantibiotics,antitumoragents,immunosup-
pressants,antihelminthicsandplantgrowthhormones[3].Byemployingmanyimprovedtech-
niquesundervariousscreeningstrategies,therateofdiscoveryofnaturalproductsexceededto
amillionsofar,outofwhich22,250bioactivecompoundsarefrommicroorganismsinwhich
45%areproducedbyactinobacteria,38%byfungiand17%fromotherbacteria.Itwasalso
recordedthatamongtheactinobacteria,Streptomycesspeciesproducesnearly80%ofallthe
reportedmetabolites[4].
Amajorchunkofnaturalproductsfrombacterialoriginhavebeenidentifiedfromorgan-
ismsthatinhabitthesoil.Sincesoilitselfisahighlyporousmixtureofmineralsandorganic
matter,ithasbeenshownthatfilamentousbacteriamightbeabletobridgeair-filledgaps
betweensoilparticlesbetterthantheirunicellularcounterparts.Thegenusactinomycetespos-
sessanoutstandingpositionastargetsinvariousscreeningprogramsfortheirprovenability
toproducenovelmetabolitesviz.antibioticsandotherleadmoleculesofpharmaceuticalinter-
est.Currently,variousscientificstudieshaveproventheirbiologicalpropertiessuchasantimi-
crobial,antioxidant,anti-inflammatoryandanti-cancerproperties[5].
Thecompositionofafermentationmediumisasignificantfactorthatinfluencesthelevel
ofantimicrobialproductionbyamicroorganism[6].Thesourceandconcentrationofnutri-
entsalongwiththeculturalconditionsareknowntohavemysteriouseffectsonbioactive
metaboliteproductioninStreptomycestoo,sincetheantibioticproductionisnotastaticprop-
erty[7].Therefore,designinganappropriatemediumandconditionsforcultivationhasprime
importanceinimprovingtheantibioticyield[8].Singleparameterpertrialusedinconven-
tionalmediaoptimizationtechniqueisalaboriousandtimeconsumingprocessinvolvinga
largenumberofexperimentaltrialstodeterminetheoptimumlevelsofallvariables.Inaddi-
tion,itmaynotprovidethecorrelationbetweendifferentparametersandoftenfailstoidentify
thevariablesthatgiveanoptimumresponse.Aviablealternativeforthisistodevelopastrat-
egywhichinvolvesstatisticaloptimizationthatcanresolvethoseissuesrelatedtoconventional
optimization[9].
Responsesurfacemethodology(RSM),acollectionofmathematicalandstatisticaltech-
niquesforbuildingempiricalmodels,hasbeenrecognizedasafascinatingapproachfor
enhancedproductionofcommerciallyimportantbioactivemetabolitesandenzymes.Ithas
beensuccessfullyappliedinvariousfacetsviz.improvementofbiofuelandbiomassproduc-
tion[10],andidentifyingthefactorsenhancingenzymeproductionaswellasassessmentof
carbonmineralizationfromsewagesludges[11].Henceinthepresentstudy,RSMwaspre-
ferredtooptimizethemediacomponentsinculturemediumformaximumantibacterial
metaboliteproduction.
Inthepresentstudy,astraindesignatedasStreptomycesnogalaterNIISTA30,wasinvesti-
gatedforenhancedantibacterialmetaboliteproductionthroughRSMmethodologywiththe
applicationofPlackett-Burmandesign(PBD)andBox-Behnkendesign(BBD).
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EnhancedantibacterialmetaboliteproductionfromStreptomycesnogalaterNIISTA30
Materialsandmethods
Ethicsstatement
ThisistoconfirmthatthesampleswerecollectedfromNelliyampathyforesthills,apartof
WesternGhatsareawherenospecificpermissionwasrequiredforthecollection.Wealsocon-
firmthatthepresentstudydidnotinvolveanyendangeredorprotectedspecies.
Chemicalsandreagents
ThechemicalsandreagentsusedformicrobiologicalworkwereprocuredfromHi-Media,
Mumbai,Indiawhereasthesolventsusedforextractionandthinlayerchromatography
(TLC)werepurchasedfromMerck,Mumbai,India.NormalcelllinessuchasL6ratmyoblast,
H9c2ratcardiacmyoblastandRAW264.7murinemacrophagewereobtainedfromNational
CentreforCellSciences(NCCS),Pune,India.
Site,samplecollectionandisolationofactinomycetestrains
SoilsampleswerecollectedfromNelliyampathyforesthills(10˚32’20.2@N76˚41’37.1@E)ofthe
WesternGhatsregioninKerala,Indiaandbroughttothelaboratoryundersterileconditions.
Thesampleswerethendriedinanoven(Equitron,Mumbai,India)at45˚Candprocessed
within36hfortheisolationofactinomycetestrains.Forthis,10gofsoilsamplesweresus-
pendedin90mLsteriledistilledwaterandshakenvigorouslyfor1hinashakingincubator
(LabCompanion,Korea)at160rpmat28±2˚C.Sampleswerethenallowedtosettleandserial
dilutionsupto10−3werepreparedandaliquotof100μlfromeachdilutionwasspreadevenly
onactinomyceteisolationagar(AIA)andPotatodextroseAgar(PDA)platesintriplicatesand
incubatedat28±2˚Cfor14to28days.Theemergingcolonieswithdifferentmorphological
characterswereselectedandthepurifiedstrainsweremaintainedonAIA.Theviabilityofthe
strainswerecheckedinAIA,PDAandISP4,wherePDAwasthebest,hencefurtherstorage
andmaintenanceoftheculturewasdoneinPDA.
Testpathogens
ThebacterialpathogensusedforantibacterialstudieswereBacilluscereusMTCC1305,B.sub-
tilisMTCC2756,MycobacteriumsmegmatisMTCC993,StaphylococcusaureusMTCC902,S.
epidermidisMTCC435,S.simulansMTCC3610,(allGrampositive)andEscherichiacoli
MTCC2622,KlebsiellapneumoniaeMTCC109,ProteusmirabilisMTCC425,Pseudomonas
aeruginosaMTCC2642,SalmonellatyphiMTCC3216,(allGramnegative).Allthebacterial
strainswereprocuredfromMicrobialTypeCultureCollectionandGeneBank(MTCC),
CSIR-InstituteofMicrobialTechnology(IMTECH),Chandigarh,India.
ThepreliminaryantibacterialstudiesusingliveStreptomycesspecies
Theprimaryscreeningof140Streptomycesisolateswasdonebyagaroverlaytechnique.For
this,StreptomycesisolateswerespotinoculatedonPDAplatesandgrownat28±2˚Cfor7days.
Thentheplateswerecoveredwith0.6%ofnutrientagarmediumpreviouslyseededindividu-
allywithtwotestindicatorbacterialpathogens(EscherichiacoliandStaphylococcusaureus)to
evaluatetheirantibacterialactivity.Theactivitywasrecordedafter24hofgrowthat37˚Cand
expressedaszoneofinhibition(inmm).
Inthesecondaryscreening,16Streptomycesstrainswhichexhibitedsignificantantibacterial
activityagainstthetwoindicatororganismswerefurthertestedagainstelevenbacterialpatho-
gensbyagaroverlaymethodasmentionedabove,tocheckthebroadspectrumantibacterial
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EnhancedantibacterialmetaboliteproductionfromStreptomycesnogalaterNIISTA30
properties.Thestrainwhichrecordedbestzoneofinhibitionwasselectedforfurtherdetailed
studies.
Morphological,culturalandphysiologicalcharacterization
ThestrainNIISTA30,whichrecordedthebestantibacterialactivityinthescreeningasmen-
tionedabovewascharacterizedonthebasisofmorphologicalfeatures,includingcolonychar-
acteristicsandpigmentproductiononvariousmediaviz.Nutrientagar(NA);Bennet’sagar
(BA);Czapekdextroseagar(CDA);Kustner’sagar(KA);International StreptomycesProject
(ISP)agarsuchasISP1(Tryptoneyeastextractagar),ISP2(Maltextractagar),ISP3(Oat
mealagar),ISP4(Inorganicsaltstarchagar),ISP5(Glycerolasparagineagar),ISP6(Peptone
yeastextractagar)andISP7(Tyrosineagar);Potatodextroseagar(PDA)andStarchcasein
agar(SCA).ColourformationwasdeterminedaccordingtoNBS/IBCCColourSystem.
(http://www.dodomagnifico.com/Colors/Cent.html).
MorphologicalcharacteristicsofisolateNIISTA30wereassessedbylightmicroscopyand
scanningelectronmicroscopy(SEM).ForSEManalysis,sterilizedcircularaluminiumstubs
wereinsertedintothePDAplateatanangleofabout45˚Candsterilecoverslipswereinserted
atthesameangleonthesampleplateforviewinggrownculture.Theplateswithstubsandcov-
erslipwereincubatedat37˚Cfor24htocheckanycontaminationduringthehandlingproce-
dure.After24h,isolateNIISTA30wasintroducedalongthelinewherethesurfaceofthestub
mettheagarmediumandincubatedat28±2˚Cfor7days.Thestubswerethencarefully
removedandcoatedundervacuum,withafilmofgoldfor15–20minutesandviewedonthe
scanningelectronmicroscope(ZeissEvo40EP,Germany).Similarly,thecoverslipwasalso
mountedontheglassslidehavingonedropofmethyleneblue(0.3gin10mLdistilledwater)
andfixedslideswereobservedunderlightmicroscope(OlympusCX41,Japan).
Biochemicaltestsviz.Gramstaining,catalase,oxidase,hydrolysisofcellulose,gelatin,lipids,
pectin,protein,starchandureaweredoneusingthestandardprocedures[12].Antibioticsus-
ceptibilityofthestrainwasdoneusingvariousantibioticdiscs(Hi-Media,India).Cultural
characteristicssuchaspHrange(0.5to12)andtemperaturerange(25to45˚C)weredoneby
inoculatingthestrainininorganicsaltstarchbroth.Theutilizationofcarbonsourceswas
determinedbygrowingtheisolateinBasalliquidmedium(BLM)supplementedwith1%of
variouscarbonsources(fructose,galactose,glycerol,lactose,maltose,mannitol,starch,
sucrose,andxylose).BLMaloneinoculatedwiththeteststrainservedasacontrol.Nitrogen
utilizationofthestrainwastestedinBLMwith1%ofvariousnitrogensourcesviz.ammonium
chloride,ammoniumsulphate,beefextract,biopeptone,casein,maltextract,meatextract,
meatinfusionpowder,meatpeptone,peptone,potassiumnitrate,soybeanmeal,ureaand
yeastextract.BLMsupplementedwithglucoseservedasacontrol[13].Alltheresultswere
notedafter7daysofincubation.
MolecularidentificationofNIISTA30using16SrRNAsequencingand
phylogeneticanalysis
ThetotalgenomicDNAwasisolatedfromNIISTA30usingNucleoSpin1TissueKit
(Macherey-Nagel).PCRamplificationwerecarriedoutusing16SrRNAprimers(forward
primer,16S-RS-F-5’CAGGCCTAACACATGCAAGTC3’andreverseprimer,16S-RS-R-
5’GGGCGGWGTGTACAAGGC3’)andamplificationprofileconsistsofaninitialdenaturation
at95˚Cfor5minutesfollowedby35amplificationcyclesof95˚Cfor30sec,60˚Cfor40sec
and72˚Cfor60sec.
ThePCRproductwascheckedin1.2%agarosegelsandvisualizedinaUVtransilluminator
andthegelimagingwasdoneusingGeldocumentationsystem.Theamplifiedproductwas
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EnhancedantibacterialmetaboliteproductionfromStreptomycesnogalaterNIISTA30
purifiedbyExoSAP-ITTreatmentandsequencedinABI3730DNAAnalyzer(AppliedBiosys-
tems,USA).
Sequenceanalysisandphylogenetictreeconstruction
ThesequencequalitywastestedusingSequenceScannerSoftwarev1(AppliedBiosystems,
USA).Sequencealignmentandrequirededitingoftheobtainedsequenceswerecarriedout
usingGeneiousProv5.6[14].Thehigh-quality16SrRNApartialsequencewasdepositedin
GenBankdatalibrary(AccessionNo.KX190774.1).Thesequencewascomparedwithother
relatedspeciesdownloadedfromthepublicdatabaseusingEzTaxontool[15]andaphyloge-
netictreewasconstructedwithMEGAversion6.Sequenceswerealignedusingthecomputer
packageClustalWandwereanalyzedtodeterminetherelationshipsbetweenisolatesbythe
neighbor-joiningmethod.Bootstrapvaluesweregeneratedusing1000replicates.
Standardizationoffermentationmediumformaximumantibacterial
metaboliteproduction
ThestrainwascultivatedinvariousmediasuchasGlucosesoybeanmealbroth,Tryptone
yeastextractbroth,Yeastmaltbroth,Inorganicsaltstarchbroth,Kuster’sbroth,Nutrient
broth,SabourauddextrosebrothandStarchcaseinbroth(seeTableEinS1File).Allthe
mediawereinoculatedwiththestrainNIISTA30andincubatedfor7daysat28±2˚C.After
fermentation,theculturebrothwascentrifuged(Remicoolingcentrifuge,Mumbai,India)at
10,000rpmfor15mintoobtaincellfreeculturefiltrate.Theculturefiltratewasthenextracted
withanequalamountofethylacetateandagitatedfor45min.Thesolventlayerwasconcen-
tratedusingarotaryevaporator(Buchi,Switzerland)at40˚Candthecrudeextractwascol-
lectedbyrinsingwith5mlofmethanol,dried,andwerekeptat4˚Cforfurtherstudies.
Antibacterialstudyofthecrudeextractsfromdifferentfermentation
media
Theantibacterialactivityofthecrudeextractfromdifferentfermentationmediawasexamined
usingagardiscdiffusionmethod[16].Thetestbacteriawasfirstinoculatedintonutrientbroth
tubesandincubatedat37˚Cfor24h.Eachoftheinoculumswerethenadjustedto0.5McFar-
landturbiditystandardsandswabbedintoMueller—Hintonagar(MHA)plates.Sixmmster-
ilefilterpaperdiscs(WhatmanNo.3,Hi-Media,India)impregnatedwithethylacetatecrude
extract(25μl)wasplacedontheswabbedMHA.Asterilediscwithmethanolservedasthe
control.Plateswerethenincubatedat37˚Candthezoneofinhibitionwasmeasured(inmm)
after24hincubation.
Antioxidantactivitiesofcrudeethylacetateextract(scavengingoffree
radicals)
DPPHfreeradicalscavengingassay. Theantioxidantpotentialofcrudeethylacetate
extractwasdeterminedbyfreeradicalDPPH(2,2-diphenyl-1-picrylhydrazyl)scavenging
activity[17].AmethanolDPPHsolution(0.16%)wasmixedwithserialdilutions(10–100μg/
mL)ofcrudeextractandafter30min,theabsorbancewasreadat515nm.Theradicalscav-
engingactivitywasexpressedasIC (μg/mL),(thedoserequiredtocausea50%inhibition).
50
VitaminCwasusedasthestandardandtheabilitytoscavengetheDPPHradicalwas
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EnhancedantibacterialmetaboliteproductionfromStreptomycesnogalaterNIISTA30
calculatedbythefollowingformula:
A (cid:0) A
DPPHradicalscavengingactivity%¼ 0 1(cid:2)100
A
0
WhereA istheabsorbanceofthecontrolat30minandA istheabsorbanceofthesampleat
o 1
30min.Theexperimentwasperformedintriplicates.
ABTSfreeradicalscavengingassay. AnethanolicABTS[2,20-Azino-bis(3-ethyl-
benzthiazoline-6-sulfonicacid)]solutionwasmixedwithserialdilutions(10–100μg/mL)of
crudeextractandafter5min;theabsorbancewasrecordedat734nm[18].Theradicalscav-
engingactivitywasexpressedasIC (μg/mL),(thedoserequiredtocausea50%inhibition).
50
Troloxwasusedasthestandard.TheabilitytoscavengetheABTSradicalwascalculatedby
thefollowingformula:
A (cid:0) A
ABTSradicalscavengingactivity%¼ 0 1(cid:2)100
A
0
WhereA istheabsorbanceofthecontrolat5minandA istheabsorbanceofthesampleat
o 1
5min.Theexperimentwasperformedintriplicates.
Effectofcrudeextractonnormalcellviability
ViabilityofL6,H9c2andRAW264.7wereassessedby3-(4,5-dimethythiazol-2-yl)-2,
5-diphenyltetrazoliumbromide(MTT)assay[19].Eachcellswereseeded(1×104cells/
well)ina96-wellplate.Thecellsweretreatedwithvariousconcentrationsofcrudeextracts.
After24hincubation,cellswerewashedand100μlofMTT(5mg/mL),dissolvedinDul-
becco’smodifiedEagle’smedium(DMEM),wasaddedtoeachwellandincubatedat37˚C
inaCO incubator.After4hincubation,DMSOwasaddedtoeachwellandtheplatewas
2
keptonashakerat12rpmfor45min.Thechangeincolourwasmonitoredusingamicro-
platereader(BIOTEK,USA)at570nm.Resultswereexpressedaspercentageofcytotoxic-
ity:
Absorbance of Control(cid:0) Absorbance of Sample
Percentage of Toxicity¼ (cid:2)100
Absorbance of Control
OptimizationofpH,temperatureandincubationtimeformaximum
antibacterialmetaboliteproduction
Optimizationoftemperature. Theeffectofcultureconditionsontheproductionof
enhancedantibacterialmetaboliteproductionwasstudiedonISP4againstS.epidermidis
(Pathogenicbacteriawhichexhibitedmaximumactivityinscreeningstudies).Theoptimum
temperatureforthemaximumantibacterialcompoundproductionwasinvestigatedonISP4.
TenmLbacterialsuspensionwasintroducedin250mLErlenmeyerflaskscontaining100mL
ofbrothandincubatedatdifferenttemperatures(25,30,35,40and45˚C)atpH7.0.Theanti-
bacterialactivitywasassayedafter7daysbydiskdiffusionmethodasmentionedabove.
OptimizationofpH. TheimpactofpHonantibacterialmetaboliteproductionwasstud-
iedatdifferentpH,rangesfrom5.0to12.0at35˚Candfor7days.
Optimizationofincubationperiod. Theoptimizationofincubationperiodwasalsocar-
riedoutbyincubatingfor3–15daysat35˚CandpH7.Theantibacterialactivitywasmoni-
toredatevery24hintervalsbydiskdiffusionmethod.
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EnhancedantibacterialmetaboliteproductionfromStreptomycesnogalaterNIISTA30
OptimizationofmediumcomponentsinISP4formaximumproductionof
antibacterialcompoundsbyresponsesurfacemethodologyusing
Plackett-Burmandesign(PBD)
ThePlackett—Burmanexperimentaldesign[20]waschosenforinitialevaluationofISP4
mediumcomponentstoshort-listcomponentshavingsignificanteffectonantibacterialactiv-
itybyNIISTA30.ThefactorsconsideredweremacronutrientsinISP4mediumsuchasstarch,
K HPO ,(NH ) SO ,CaCO ,MgSO andNaCl,eachanalysedattwolevels,low(-)andhigh
2 4 4 2 4 3 4
(+).Themicronutrientsweretakenasperstandardmediumandthetotalnumbersofexperi-
mentsasperthePBDwere13.Theexperimentswerecarriedoutintriplicatestoassessthecon-
sistencyofresultsandtheaverageantibacterialactivityagainstS.epidermidiswasdetermined
astheresponse.Theexperimentaldesignwasdevelopedusingthestatisticalsoftwarepackage,
MINITAB17.
Optimizationofsignificantcomponentsbyresponsesurface
methodology(RSM)
BasedonthepreliminaryPlackett-Burmananalysis,accordingtothelowp-values(<0.05)and
highconfidencelevels(>90%),thesignificantmediacomponentswereselectedasthevari-
ablestotestinthe15-runexperimentoftheBox-Behnkendesign[21].Thevariableswere
studiedatlow,middleandhighconcentrationlevelsandweredesignatedas−1,0and+1
(codedvalues),respectively.TheMINITABstatistical(MINI-TAB17)softwarewasusedto
computetheresultsandgenerateresponsesurfacegraphs.
Inthissystem,theregressionanalysiswasperformedtoestimatetheresponsefunctionasa
secondorderpolynomialequation,Y=β +∑βX +∑β XX +∑β X2[22].Where,Ydenotes
0 i i ij i j ii i
thepredictedresponse,β istheinterceptterm,β isthelinearcoefficient,β isthequadratic
0 i ij
coefficientandβ istheinteractioncoefficient,andXX denotesindependentvariablesunder
ii i j
study.Thestatisticalcompetenceofthemodelwasresolvedthroughanalysisofvariance
(ANOVA).Theexcellenceofthepolynomialmodelequationwasconcludedstatistically
throughcoefficientofdetermination(R2)andadjustedR2.Three-dimensionalresponsesurface
plotswereproducedtoelucidatetherelationshipbetweentheresponsesandtheexperimental
levelsofeachindependentvariable.Theoptimumlevelofthevariablesformaximumantibac-
terialactivitywasresolvedbyresponseoptimizertoolofthesoftware.
Validationofstatisticalmodelandoptimizationthroughwetlab
Themathematicalmodelandtheoptimizationwereexperimentallyvalidatedbyculturing
NIISTA30underunoptimizedandoptimizedlevelsofvariablesatpH7.0and35±2˚Cfor7
days.Afterincubation,theantimicrobialmetabolitewasextractedtwicewithequalvolumeof
ethylacetateanddriedextract(350mg/L)wassuspendedin100μlmethanolandassayedas
aboveforantibacterialactivity.
Autobiographictest
Theethylacetatecrudeextractwasevaluatedforantibacterialactivitythroughautobiographic
agaroverlaymethod[23].Forthis,thedevelopedTLCplatewasairdriedandencasedina
sterilePetriplateandoverlaidwithnutrientagarmediumcontaining0.6%agarinoculated
withtestbacterialsuspension.Theplateswerethenincubatedat37˚Candzoneofinhibition
aroundthespotswereobservedafter24h.
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EnhancedantibacterialmetaboliteproductionfromStreptomycesnogalaterNIISTA30
Statisticalanalysis
AlldataregardingantibacterialactivityweresubjectedtostatisticalanalysisusingSPSSsoft-
warepackage(Windowsversion20.0;IBMSPSS)andexpressedasmean±SD.Graphswere
plottedusingacomputerprogramOriginPro8(OriginLab,Corporation,USA)
Results
Site,samplecollectionandisolationofStreptomycesstrains
FromthesoilsamplescollectedfromthedifferentpartsofNelliyampathyForestsite,140mor-
phologicallydifferentStreptomycesstrainswereisolated.Thestrainswerecheckedfortheir
purityinAIAandPDAplatesandstoredonPDAslants,asPDAslantsshownbettershelflife
organismsthanAIAandISP4.
AntibacterialstudiesusingliveStreptomycesstrains
Inprimaryscreening,16strainsrecordedactivityagainstboththeindicatortestorganisms,
S.aureusandE.coli.Sixty-eightstrainsexhibitedantibacterialactivityagainstS.aureus
whereas56strainsshowedactivityagainstE.coli.Outofthesestrains,NIISTA30displayed
maximumactivityagainstbothindicatorpathogens(44and40mmagainstS.aureusand
E.colirespectively).
Secondaryscreeningwasdonewith16strainsagainstelevenpathogenicbacteriaofwhich
NIISTA30exhibitedmaximumzoneofinhibitionagainstallthetestpathogens(datanot
shown).S.epidermidiswasthemostinhibited(56mm)whereasS.typhi(33mm)wastheleast
inhibited(Table1).Thus,thestrainNIISTA30wasselectedforfurtherdetailedinvestigations.
Morphological,culturalandphysiologicalcharacterization
Thedataregardingmorphological,culturalandphysiologicalcharacterizationwasasshownin
supplementaryattachments(seeTableA,TableB,TableCandTableDinS1File).Scanning
electronmicroscopicexaminationshowedthatthestrainformedextensivelybranched,non-
fragmentedsubstrateandarialmycelia.Thesporechainmorphologyofthestrainwasstraight
andrectiflexiblewithsmoothsurfacewithlessthan50sporesinachain(Fig1).
Table1. InvitroantibacterialactivityofNIISTA30againsttestpathogens.
Testpathogen Zoneofinhibition(mm)
Bacilluscereus 41±1.15*
Bacillussubtilis 38±0.57
Escherichiacoli 42±2
Klebsiellapneumoniae 43±1
Mycobacteriumsmegmatis 32±2.5
Pseudomonasaeruginosa 49±1.15
Proteusmirabilis 53±1.15
Salmonellatyphi 29±1.15
Staphylococcusaureus 49±1.15
Staphylococcusepidermidis 59±1.15
Staphylococcussimulans 54±1.73
*Valuesareaveragefromthreereadings.
https://doi.org/10.1371/journal.pone.0175919.t001
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EnhancedantibacterialmetaboliteproductionfromStreptomycesnogalaterNIISTA30
Fig1.Scanningelectronmicroscopicimageofsporearrangement.
https://doi.org/10.1371/journal.pone.0175919.g001
MolecularcharacterizationNIISTA30
GenomicDNAisolatedfromthestrainwasamplifiedusinguniversalprimersfor16SrRNA.
ThePCRproductshowed1500bpbandongelelectrophoresiswassequenced.Thehighqual-
itysequencewasalignedusingClustalWsoftware.SequencesimilaritywasdonebyusingEz
taxondatabase.The16SrRNApartialsequencewasdepositedinGenBankdatalibrarywithan
accessionNo.KX190774.1.ThephylogenetictreewasdrawnwiththehelpofMega6.0Version
softwareusingneighbourjoiningmethod.Phylogeneticanalysisshowedthatthestrainisiden-
tifiedasStreptomycesnogalaterwith100%similarity(Fig2).
Standardizationofoptimalfermentationmediaforsignificant
antibacterialmetaboliteactivity
Amongtheeightmediatested,maximummetaboliteproduction(180mg/L)andantibacterial
activitywasrecordedinISP4followedbyglucosesoybeanmealandyeastmaltbroth(Table2).
Antioxidantactivitiesofcrudeethylacetateextract(scavengingoffree
radicals)
CrudeextractexhibitedsignificantdosedependentinhibitionofDPPHactivity,witha50%
inhibition(IC )at30μg/mL.TheIC valueforvitaminCwas295μg/mL.ABTSscavenging
50 50
activitydemonstrates50%inhibition(IC )at20μg/mL(Fig3).
50
PLOSONE|https://doi.org/10.1371/journal.pone.0175919 April24,2017 9/21
EnhancedantibacterialmetaboliteproductionfromStreptomycesnogalaterNIISTA30
Fig2.PhylogenetictreeshowingtheevolutionaryrelationshipofthestrainStreptomycesnogalaterNIIST
A30withitsrelatedspecies.
https://doi.org/10.1371/journal.pone.0175919.g002
Effectofcrudeextractonnormalcellviability
Theconcentrationofcrudeextractupto50μg/mLwasfoundtobelessthan20%toxicinall
thecelllinesforaperiodof24h(Fig4).However,aconcentrationof100μg/mLandabove
wasfoundtobetoxic(40%)inRAW264.7celllineswhereasinL6andH9c2celllinesthe
extractexhibited20and23percentagestoxicity,respectively.
Table2. AntibacterialactivityofNIISTA30crudeextractsfromdifferentbroths.
Testpathogens Zoneofinhibition(mm)
GSMB ISP1 ISP2 ISP4 KB NB SDB SCB
B.cereus 9±0.57* 8±0.57 7.6±0.57 13.6±0.57 11±0 7±1 8±0 8±0.57
B.subtilis 10±0 10±0 17.6±0.57 12±1 16±0 0 17±0 12.3±0.57
E.coli 7.6±0.28 7.5±0.5 9±0 10±0 8±0.57 9.3±0.57 6±0 10±0
K.pneumoniae 6.5±0.5 8.3±0.57 9±1 12±0 11±0 10.3±0.57 7±0 10.3±0.57
M.smegmatis 11±1 10±1 10±1 11±0 11.3±0.28 6±0 6±0 9.16±0.28
P.aeruginosa 9.3±0.57 0 7.6±0.57 10±1 6.8±0.28 0 8±0.57 9±1
P.mirabilis 7.5± 0 8±0.57 11±0 10±0 10±0 11±0 10.3±0.28
S.aureus 9.3±0.28 10±0 10±0 9±0.57 8±0.57 7±0 8±0 10±0
S.epidermidis 13.3±0.28 12.3±0.28 11±1 15±1.52 14±0.57 12±0 13.3±0.28 14±0.57
S.simulans 10±0 11±0 6±1 12±1 10±0 12±0 7±0 8±0.57
S.typhi 10±1 5±0 9±0 12±1 8±0 10±0 7.6±0.57 5±0
*Valuesareaveragefromthreereadings.
https://doi.org/10.1371/journal.pone.0175919.t002
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