Antimicrobial profile of Arthrobacter kerguelensis VL-RK_09 isolated from Mango orchards

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Microbial

Physiology

Antimicrobial

profile

of

Arthrobacter

kerguelensis

VL-RK

09

isolated

from

Mango

orchards

Rajesh

Kumar

Munaganti

_,

_{Vijayalakshmi}

_Muvva

_,

_Saidulu

_Konda

_,

Krishna

Naragani

_,

_Usha

_Kiranmayi

_Mangamuri

_,

_Kumar

_Reddy

_Dorigondla

_,

Dattatray.

M.

Akkewar

a_{AcharyaNagarjunaUniversity,DepartmentofBotanyandMicrobiology,Guntur,India}

b_{CSIR-IndianInstituteofChemicalTechnology,OrganicChemistryDivision-II(CPC),Hyderabad,India}

c_{CSIR-IndianInstituteofChemicalTechnology,OrganicChemistryDivision-I(NaturalproductsLaboratory),}

Hyderabad,India

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received30April2015 Accepted14March2016 Availableonline29July2016 AssociateEditor:LuisHenrique SouzaGuimarães Keywords: Mangoorchard Arthrobacterkerguelensis S,S-dipropylcarbonodithioate

a

b

s

t

r

a

c

t

AnactinobacterialstrainVL-RK09havingpotentialantimicrobialactivitieswasisolated fromamangoorchardinKrishnaDistrict,AndhraPradesh(India)andwasidentifiedas

Arthrobacterkerguelensis.ThestrainA.kerguelensisVL-RK09exhibitedabroadspectrumof

invitroantimicrobialactivityagainstbacteriaandfungi.Productionofbioactive

metabo-litesbythestrainwasthehighestinmodifiedyeastextractmaltextractdextrosebroth,as comparedtoothermediatested.Lactose(1%)andpeptone(0.5%)werefoundtobethemost suitablecarbonandnitrogensources,respectively,fortheoptimumproductionofthe bioac-tivemetabolites.Themaximumproductionofthebioactivemetaboliteswasdetectedinthe culturemediumwithaninitialpHof7,inwhichthestrainwasincubatedforfivedaysat 30◦_{Cundershakingconditions.Screeningofsecondarymetabolitesobtainedfromthe}

cul-turebrothledtotheisolationofacompoundactiveagainstawidevarietyofGram-positive andnegativebacteriaandfungi.Thestructureofthefirstactivefractionwaselucidated usingFouriertransforminfraredspectroscopy,electrosprayionizationmassspectrometry,

1_Hand13_{Cnuclearmagneticresonancespectroscopy.Thecompoundwasidentifiedas}

S,S-dipropylcarbonodithioate.Thisstudyisthefirstreportoftheoccurrenceofthiscompound inthegenusArthrobacter.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

The naturalproducts of microbial origin appearto be the mostpromisingsourceoffutureantibiotics.1,2 _{Asearch for}

∗ _{Correspondingauthor.}

E-mail:lfhraji@gmail.com(V.Muvva).

new antibiotics or newmicrobial strains producing antibi-otics continues to be of utmost importance in research programs around the world because of the increases in resistantpathogensandtoxicityofsomeknownantibiotics. Nevertheless,thereisanalarmingscarcityofnewantibiotics

http://dx.doi.org/10.1016/j.bjm.2016.07.010

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

in the pharmaceutical industry. Therefore, to ensure the availability ofeffective drugs in the future, it isnecessary to improve the antimicrobial use patterns and to devise strategiesfor the identification of newantibiotics through previouslyunexploredtargets.3,4_{Developmentofnoveldrugs} withabroad-spectrummodeofactionagainstpathogensis theneedofthehour.

Actinobacteriaareaerobic,Gram-positiveandfilamentous innature,whichformasexualsporesandmyceliaandhave ahighguanineandcytosine(GCcontent)intheDNA.They are the most economicallyand biotechnologicallyvaluable prokaryoteswiththeunparalleledabilitytoproducebioactive secondarymetabolites,notably,antibiotics,antitumoragents, immunosuppressiveagents, and enzymes.In view oftheir excellenttrackrecordinthisregard,extensiveattemptshave beenmadeinthepast50yearstoisolateactinobacteriafrom terrestrial sourcesfordrug screening. In the present days, anexploration ofrare actinobacteria forbioactive metabo-liteproductionhasdramaticallyincreased,particularlydue to their unique and diverse metabolic pathways against life-threateningresistantpathogens.5_{Arelativelylow} occur-renceofnon-streptomycetespeciesasproducersofsecondary metabolites is due tothe absence of novel techniques for theisolationofthesestrainsfromtheenvironment.Another problem is their complicated preservation and cultivation methods,whichfrequentlyrequirespecificandunusual con-ditions. These are the main reasons why these microbes are considered rare organisms and why investigation and manufacturingoftheir productsare difficult.Most isolates recoveredonagarplatesbyconventionalisolationtechniques havebeenidentifiedasspeciesofStreptomyces,whicharethe dominantactinobacteriapresentinsoil.2,6–8

Rare actinobacteria are widely distributed in terrestrial andmarineecosystemshavingdifferentenvironmental con-ditions such as soil type, pH, humus content and humic acidcharacteristicsofthesoil.9_{Theyarediverseandbelong} to many suborders, including Micrococcineae and to a variety of genera.10,11 _{The best-known genus of the} fam-ily Micrococcaceae is Arthrobacter, which includes aerobic, catalase-positive,cocciorcoryneformbacteriawithlysinein theircellwalls.Arthrobacterspp.exhibitgreatmetabolic versa-tilityandareabletoproduceanumberofvaluablesubstances, suchasaminoacids,vitamins,enzymes,specificgrowth fac-tors,andpolysaccharides.12_{Howevertheseorganismsarenot} wellexplored assecondary metaboliteproducers, although therehavebeenreportsofproductionofbioactivecompounds bysomespecies.13–16_{Whilerareactinobacteriamayprovide} increasedchancesofdiscoveringnovelstructures,their genet-icsandphysiologyarepoorlyunderstood.

Asapartofourongoingresearchonrareactinobacteria,a promisingstrainVL-RK09,withagoodantimicrobial poten-tialwasidentifiedasArthrobacterkerguelensis.Thestrainwas isolatedfromasoilsamplecollectedatamangoorchardin Vissannapet,KrishnaDistrict,AndhraPradesh(India),andits DNA sequence was deposited toGenbank under accession numberKJ787652.Sincethecomponentsofculturemediaand culturalconditionsofteninfluencetheproductionofbioactive metabolitesbymicroorganisms.17–21_{Anattemptwasmadeto} enhancetheproductionofbioactivemetabolitesandto char-acterizethemetabolitesofA.kerguelensisVL-RK09.

Materials

and

methods

Chemicals

All solvents,reagents,and mediacomponentsused inthis study were of extra-pure grade and procured from Merck (Mumbai,India).

Strainisolation

The strain, A. kerguelensisVL-RK09, was isolated on yeast extract-malt extract-dextrose (YMD) agar by asoil dilution techniquefromasoilsamplecollectedatamangoorchardin Vissannapet,AndhraPradesh,India.Themediumconsistedof maltextract(1%),yeastextract(0.4%),dextrose(0.4%),CaCO3 (0.2%),andagar(2.0%),pH7.0±0.2.Thestrainwasstoredon YMDagarmediumat4◦C.22

Antimicrobialprofileofbioactivemetabolitesproducedby thestrain

Tostudytheantimicrobialprofile,athree-day-oldcultureof thestrainwascultivatedinYMDbroth(seedmedium)at30◦C for24h.Oftheseedculture,10%wastransferredtothesame medium formass fermentation. The fermentation process wasallowedtocontinueat30◦Cforeightdays.The produc-tion ofbioactivemetaboliteswasdetermined bymeasuring diametersofinhibitionzonesagainsttestmicrobes.Culture filtrates harvested atregular intervals were extracted with ethylacetateandevaporatedtodryundervacuumat35◦C. Theresiduethusobtainedwasdissolvedindimethyl sulfox-ide(DMSO)andtestedforantimicrobialactivityagainsttest bacteria suchas Staphylococcus aureus(MTCC 3160),Bacillus

megaterium (NCIM 2187),Shigella flexneri (MTCC 1457),

Xan-thomonascampestris(MTCC2286),Proteusvulgaris(ATCC6380),

Pseudomonasaeruginosa(ATCC9027)andEscherichiacoli(ATCC

35218)andfungusCandidaalbicans(MTCC183)byanagar dif-fusionassay.23

Mediaoptimization

Attempts were madeto enhancethe antimicrobialactivity by optimizing the culture conditions such as pH, temper-ature, carbon sources,nitrogen sources and minerals. The bioactive metabolite production was determined after five daysofincubation.Fermentationwascarriedoutin100mL ofmediain250-mLErlenmeyerflaskswithconstantshaking at180rpm.Theeffect ofinitialpH onbioactivemetabolite productionbythestrainwasdeterminedbyadjustingpHof theproductionmediumintherangefrom4to10.The opti-malpH achievedatthisstep wasusedforfurther study.24 Similarly,theoptimumtemperaturewasdeterminedby incu-bating thestrainattemperatures rangingfrom20 to40◦C, whilemaintainingallother conditionsatoptimumlevels.25 Carbonsourcessuchasmaltose,lactose,fructose,galactose, sucrose,glucose,starch,cellulose,mannitol,andsorbitolwere supplementedseparatelyata1%concentrationintothe fer-mentation medium.26 _{Theeffect ofvarying concentrations} (0.5–4%) ofthe best carbon sourceon bioactive metabolite

productionwasalsoinvestigated.Similarly,variousnitrogen sources,such aspeptone, methionine,tryptophan, sodium nitrate,proline,leucine,tyrosine,valine,asparticacid,urea, yeastextract,andargininewereindividuallysupplemented at0.4%intothefermentationmedium.Further,theimpactof differentconcentrations(0.1–1.5%)oftheoptimumnitrogen sourcewasstudiedtoenhancetheproductionof antimicro-bialmetabolites.27_{Toevaluatetheeffectofmineralsalts,the} optimizedmediumcontainingthesuperiorcarbonand nitro-gensourcewassupplementedseparatelywithvariousmineral supplements,suchasK2HPO4,KH2PO4,KCl,MgSO4,FeSO4, MnCl2,andNaClataconcentrationof0.05%(w/v).28

Extractionofthemetaboliteandantimicrobialactivity assay

Thestrain A. kerguelensis VL-RK09 was grown under opti-mizedconditionsforfivedays.Theculturebrothwasobtained bycentrifugationat6000rpmfor20min,thenextractedwith ethylacetate, and dried ina rotary evaporator at 40◦C to obtain a crude extract. Theantimicrobial metabolites pro-duced bythe strain were tested by an-agar well diffusion assayagainstbacteria,including S.aureus,B.megaterium,S.

flexneri,X.campestris,P.vulgaris,P.aeruginosa,E.coli,Salmonella

typhi,VibriocholeraandStreptococcusmutans(MTCC497),grown

overnightat37◦C,C.albicans(ATCC10231),Aspergillusniger,

A.flavus,Fusariumsolani,F.oxysporum(MTCC3075),Penicillium

citrinumandAlternariasp.wereusedastestfungi.

Purificationoftheantimicrobialcompound

A seed culture (10%, v/v) prepared by culturing A.

kergue-lensis VL-RK09 in ISP-2 (International Sterptomyces Project)

medium was transferred to fermentation broth contain-ingmalt extract-(1%), lactose(1%), peptone(0.5%),K2HPO4 (0.05%),andCaCO3(0.2%),withpHadjustedto7.0.Atotalof40 conicalflasksof2-Lcapacitycontaining1Lofthebroth,were inoculatedandincubatedonarotaryshaker(180rpm)at30◦C for120hunderoptimizedconditions.Theculturebroth(40L) obtainedafterfiltrationwasextractedtwicewithethylacetate and concentrated under reduced pressure toyield acrude extract(4.2g),whichwasthensubjectedtosilicagelcolumn (80×2.5cm) chromatography. The separation of the crude extractwasconductedbygradientelutionwithhexane/ethyl acetate.Theeluentwasrunoverthecolumn,andsmall vol-umesoftheeluatecollectedintesttubeswereanalyzedby thin-layerchromatography(TLC)usingsilicagelplatesanda hexane/ethylacetatesolventsystem.Compoundswith iden-tical retention factors (Rf)were combined and assayed for

antimicrobialactivity against Gram-positive (B.megaterium) andGram-negative(E.coli)bacteriaandtheyeast(C.albicans) usinganagarplatediffusionassay.23_{Amongthefractions,the} firstactivefraction(200mg)withgoodantimicrobialactivity was re-chromatographed on a silicagel column using hex-ane/ethylacetate(80:20)asanelutingsolventsystemtoobtain 30mgofapurecompound(S1),whichwasselectedforfurther studies.Thestructureoftheactivefractionwasdeducedby Fouriertransforminfraredspectroscopy(FT-IR),electrospray

ionizationmassspectrometry(ESI-MS)and1_Hand13_Cnuclear magneticresonancespectroscopy(NMR)spectroscopy.

Biologicalassays

Minimuminhibitoryconcentration

The antimicrobial spectra of the bioactive compound pro-ducedbythestrainweredeterminedintermsofminimum inhibitoryconcentration(MIC)byusingtheagarplate diffu-sionassay.TheMICofactivecompoundS1wasinvestigated in comparison with tetracycline. Muller-Hinton agar and Sabourauddextroseagar mediawere preparedtogrowthe bacteria and fungi, respectively. Dilutions ofthe test com-poundS1andthereferencedrugwerepreparedinDMSOat concentrationsrangingfrom0to1000␮g/mLandwereadded tocups.ThePetridisheswereinoculatedwith1.5×104_colony forming units (cfu/mL) and incubated at 37◦C for 24–48h forbacteria and48–72hat28◦C forfungi.Thelowest con-centrationofthebioactivecompoundsexhibitingsignificant antimicrobialactivityagainstthetestmicrobeswastakenas theMICofthecompound.

TheMICsofthebioactivecompound(S1)producedbythe strainwasdeterminedagainstseveralpathogenicbacteriaand fungi.S.aureus,B.megaterium,B.subtilis(ATCC6633),B.cereus (MTCC430),Serratiamarcescens(MTCC1457),X.campestris,P.

vulgaris(MTCC7299),P.aeruginosa(ATCC9027),E.coli(ATCC

35218),Enterococcusfaecalis(MTCC439),S.mutans,Lactobacillus

casei(MTCC1423),andL.acidophilus(MTCC495)wereusedfor

theantibacterialassay.C.albicans,A.niger,A.flavus,F.solani,

F.oxysporumandP.citrinumwereusedfortestingantifungal

activity.

Statisticalanalysis

TheresultsofbioactivemetaboliteproductionbyA.

kergue-lensis under different cultural conditions were statistically

analyzedwithone-wayanalysisofvariance(ANOVA).

Results

and

discussion

Antimicrobialprofileofbioactivemetabolitesproducedby thestrain

The growth pattern of A. kerguelensis VL-RK09 was stud-iedinYMDbroth.Thestationaryphaseofthestrainlasted from 96 to 120h of incubation (Fig. 1). The crude extract obtainedfromafive-day-oldcultureexhibitedhigh antimi-crobialactivityagainstthetestmicroorganisms.Aprevious report demonstrated high antimicrobial activity of crude extracts of a five-day-old culture of Rhodococcus erythropo-lis VL-RK05.29 _{Naragani et al.}30 _{reported the presence of} antimicrobialcompoundsinfive-day-oldcultureextractsof

StreptomycesviolaceoruberVLK-4.

Influenceofculturemediaonantimicrobialactivity

Antimicrobial activity of the strain was studied by grow-ing it in different culture media (Fig. 2). ISP-2 supported the production of bioactive compounds with the highest

0 5 10 15 20 25 Zo ne of inhibition (mm) 24 S.aureus P.vulgaris 48 72 96 120 B.megaterium P.aeruginosa Incubation time (h) 144 S.faecalis E.coli 168 X.campestris C.albicans 192

Fig.1–TimecourseofbioactivemetaboliteproductionbyArthrobacterkerguelensisVL-RK09.Thedatawerestatistically analyzedandfoundtobesignificantata5%level.

antimicrobialactivityamongtheeightmediatested,followed bymaltose–tryptone broth(ISP-6)andISP-4.Modified yeast extract–maltextract–dextrosebroth(ISP-2)favoredthe max-imumproductionofbioactivemetabolitesbyPseudonocardia sp.VUK-10isolatedfrommangrovesoils.31_{Similarly,Naragani} etal.30_{reportedthatyeastextract–maltextract–dextroseagar} supportedthemaximumbioactivemetaboliteproductionby amarineisolate,R.erythropolisVLK-12.

ImpactofpHandtemperatureonantimicrobialactivity

ThestrainwasabletogrowoverawiderangeofpHvaluesbut showedthemaximumantimicrobialactivityatpH7(Fig.3). Naraganietal.30_{reportedhighantimicrobialactivityofR.}

ery-thropolisVLK-12grownatpH7.Munagantietal.29_andDeepa

etal.32_{reportedmaximalyieldsofantimicrobialmetabolites} produced byR.erythropolis VL-RK05 and Streptomyces

cellu-losaeVJDS-1whentheywereincubatedatpH7.Antimicrobial

activityofthestrainwasalsorecordedwhenitwasgrownat temperaturesof20–40◦C,andtheoptimumwasobservedat 30◦C(Fig.4).Astheincubationtemperatureincreasedfrom20 to30◦C,therewasanincreaseinbioactivemetabolite produc-tion.However,afurtherincreaseoftemperature(above30◦C)

led toadeclineintheproductionofbioactivemetabolites. SeveralspeciesofStreptomyceswerereportedtoproducehigh levelsofbioactivemetabolitesatanincubationtemperature of30◦C.33–36

Effectofcarbonandnitrogensourcesonantimicrobial activity

Tofindasuitablecarbonsourceforsecondarymetabolite pro-duction bythe strain, dextroseinYMD brothwasreplaced withvarious carbon sourcessupplementedata concentra-tionof0.4%.Theeffectsofcarbonsourcesonproductionof bioactivemetabolitesbyA.kerguelensisVL-RK09arepresented in Fig. 5. Sincelactose supported ahigh yieldofbioactive metabolites,differentconcentrationsoflactose(0.5–4%)were testedtodeterminetheoptimalconcentration.Lactoseata concentrationof1%supportedthehighestyieldofbioactive metabolites(Fig.6).

Theseresultsarecomparablewiththoseobtainedfor

Strep-tomyceshygroscopicusstrainsAK-111-81andCH-7,which

uti-lizedlactoseasacarbonsourceforantibioticproduction.37,38 Inordertoobtainaneffectivecompositionofthegrowth medium, variousnitrogensourceswere evaluated fortheir

0 5 10 15 20 25 Zo ne of inhibition (mm) ISP-1 S.aureus P.vulgaris ISP-3 ISP-2 ISP-4 B.megaterium P.aeruginosa ISP-5 Media ISP-6 S.faecalis E.coli ISP-7 X.campestris C.albicans Starch casein broth

Fig.2–InfluenceofdifferentculturemediaonantimicrobialactivityofArthrobacterkerguelensisVL-RK09.Thedatawere statisticallyanalyzedandfoundtobesignificantata5%level.

0 5 10 15 20 25 30 Zone of inhibition (mm) 4 5 6 7 pH range 8 9 10 S.aureus P.vulgaris B.megaterium P.aeruginosa S.faecalis E.coli X.campestris C.albicans

Fig.3–TheeffectofpHontheantimicrobialactivityofArthrobacterkerguelensisVL-RK09.Thedatawerestatistically analyzedandfoundtobesignificantata5%level.

0 5 10 15 20 25 30 Zo ne of inhibition (mm) 20 25 30 Temperature (ºC) 355 40 S.aureus P.vulgaris B.megaterium P.aeruginosa S.faecalis E.coli X.campestris C.albicans

Fig.4–TheeffectoftemperatureontheantimicrobialactivityofArthrobacterkerguelensisVL-RK09.Thedatawere statisticallyanalyzedandfoundtobesignificantata5%level.

influenceonantimicrobialactivityofthestrain.Amongthe nitrogensourcestested, peptone wasfound tobethe best forantimicrobialmetabolite, followed byyeast extractand tryptophan (Fig. 7). Since peptone enhanced the antimi-crobial activity of strain VL-RK09, the effects of different

concentrationsofpeptoneweretested,and0.5%wasfoundto bethebestforimprovedantimicrobialactivity(Fig.8).Peptone wasalsoreportedasasuitablenitrogensourceforobtaining optimalyieldsofbioactivemetabolitesproducedby

Strepto-mycesrocheiG16439_{andS.scabiesPK-A41.}40

0

Maltose Sucrose Mannitol Lactose Starch _Cellulose

Galactose Sorbitol Fructose

5 10 15 20 25 30 35 Zo ne of inhibition (mm)

S.aureus B.megaterium S.faecalis X.campestris

Carbon source

P.vulgaris P.aeruginosa E..coli C.albicans

Fig.5–TheeffectofdifferentcarbonsourcessupplementedinthemodifiedYMDbrothontheantimicrobialactivityof ArthrobacterkerguelensisVL-RK09.Thedatawerestatisticallyanalyzedandfoundtobesignificantata5%level.

0 5 10 15 20 25 30 35 Zo ne of inhibition (mm) 0.5 1 2 Lactose, % 3 4

S.aureus B.megaterium S.faecalis X.campestris P.vulgaris P.aeruginosa E..coli C.albicans

Fig.6–TheeffectofthedifferentconcentrationsoflactoseasasolecarbonsourceonantimicrobialactivityofArthrobacter kerguelensisVL-RK09.Thedatawerestatisticallyanalyzedandfoundtobesignificantata5%level.

0 5 10 15 20 25 30 35 Zo ne of inhibition (mm) Nitrogen sources Peptone

Methionine Tryptophan Arginine_{Aspartic acid} L-proline

Leucine Tyrosine Valine Urea

Sodium nitrate Yeast extract

S.aureus B.megaterium S.faecalis X.campestris P.vulgaris P.aeruginosa E..coli C.albicans

Fig.7–TheeffectofvariousnitrogensourcessupplementedinthemodifiedYMDbrothontheantimicrobialactivityof ArthrobacterkerguelensisVL-RK09.Thedatawerestatisticallyanalyzedandfoundtobesignificantata5%level.

Theeffectofmineralsaltsonantimicrobialactivity

The effects of mineral salts on secondary metabolite pro-ductionbystrain VL-RK09 areshowninFig.9.Amongthe mineralsalts tested,K2HPO4 supportedthehighest antimi-crobialactivity.SimilarresultswerereportedforStreptomyces

albidoflavus.41 _{Ripa et al.}31 _{and Usha et al.}42 _{reported that}

K2HPO4showedpositiveeffectsonantibioticproductionby

Streptomycessp.RUPA-08PRandPseudonocardiasp.

Thesecondarymetabolitesobtainedfromthe strain VL-RK09under optimized culturalconditions,which included maltextract(1%),lactose(1%),peptone(0.5%),K2HPO4(0.05%),

0 5 10 15 20 25 30 35 40 Zo ne of inhition (mm) 0.1 00.25 Peptone, % 0.5 11 1.5

S.aureus B.megaterium S.faecalis X.campestris P.vulgaris P.aeruginosa E..coli C.albicans

Fig.8–Theeffectofthedifferentconcentrationsofpeptoneasasolenitrogensourceontheantimicrobialactivityof ArthrobacterkerguelensisVL-RK09.Thedatawerestatisticallyanalyzedandfoundtobesignificantata5%level.

0 5 10 15 20 25 30 35 40 Zo ne of inhibition (mm) Mineral salts KH2PO4 K2HPO4 NaCl MgSO4 FeSO4 MnCl2 KCl

S.aureus B.megaterium S.faecalis X.campestris P.vulgaris P.aeruginosa E..coli C.albicans

Fig.9–TheeffectofthedifferentmineralsaltsontheantimicrobialactivityofArthrobacterkerguelensisVL-RK09.Thedata werestatisticallyanalyzedandfoundtobesignificantata5%level.

Table1–AntimicrobialactivityofArthrobacter

kerguelensisVL-RK09underoptimizedculturing

conditions.

Zoneofinhibition(mm)

Testorganisms(bacteria)

1 Staphylococcusaureus 34 2 Bacillusmegaterium 39 3 Shigellaflexneri 27 4 Xanthomonascampestris 32 5 Proteusvulgaris 30 6 Pseudomonasaeruginosa 33 7 Escherichiacoli 24 8 Corynebacteriumdiphtheriae 26 9 Salmonellatyphi 18 10 Streptococcusmutans 25 11 Vibriocholerae 24 Fungi 12 Candidaalbicans 29 13 Aspergillusniger 25 14 A.flavus 22 15 Fusariumsolani 21 16 F.oxysporum 20 17 Penicilliumcitrinum 18 18 Alternariasp. 21

CaCO3(0.2%),pH7.0±0.2,temperature30◦C,andthe

incuba-tionperiodoffivedays,showedstrongantimicrobialactivity againstbacteriaandfungi(Table1).Amongthebacteriatested,

B. megaterium, S. aureus, P. aeruginosa, X. campestris, and P.

vulgarisappeared tobe highlysensitive tothe metabolites

producedbythestrain.

Theisolation,purification,andstructuralelucidationofan activemetabolite

Theculturefiltrateobtainedafter120hoffermentationwas extractedwithethylacetateandconcentratedinvacuumto yieldadarkbrownresidue,whichwasthensubjectedtosilica gelcolumnchromatographyusingagradientsolventsystem ofhexane/ethylacetate.

Amongthefractions,thefirstactivefraction(200mg)with goodantimicrobialactivitywasre-chromatographedona sil-ica gel column using hexane: ethyl acetate (80:20) as the eluting solventsystemtoobtain apurecompound(30mg). ThestructureofthepurifiedcompoundS1waselucidatedand characterizedbyFT-IR,mass,andNMRspectroscopy.

CompoundS1wasisolatedasayellow-orangeliquid, sol-ubleinCHCl3,methanol,dichloromethane,andDMSO.TheIR absorptionmaximums(Vmax)at2924,2856,1739,1629,1380 and1095cm−1suggestedthepresenceofcarbonylfunctional groups(Fig.S1).InESI–MS,thecompoundshowedmolecular ionsatm/z178,inferringthemolecularformulaofC7H14OS2 (Fig.S2).TheprotonNMRofthecompounddisplayedproton signals at2.33 (t,J=7.3Hz,2H), 1.66 (q,J=7.3,7.47Hz, 2H), and0.97(t,J=7.47Hz,3H)(Fig.S3).13_{CNMRdepictedpeaks} andshowedsignalsat180.12,35.97,17.73,and13.34(Fig.S4). Basedonthesespectraldata,thefirstactivecompoundS1was identifiedasS,S-dipropylcarbonodithioate(Fig.10).Thisisthe firstreportoftheoccurrenceofthiscompoundinthegenus Arthrobacter.

Arthrobacterisacommongenusofsoilbacteria,whichare metabolicallyversatileandproducedifferentenzymes allow-ingthebacteriatogrowonawiderangeofsubstrates.The members of thegenus are not well exploredas secondary metabolite producers although there had been reports of productionofbioactivecompoundsbysomespecies. Antimi-crobialmetaboliteshavebeenstudiedinsomeaquaticand polarstrainsofArthrobacter.43,44_{Hence,anattemptwasmade} in the present study toevaluatethe productionof antimi-crobial metabolites by a terrestrial isolate, A. kerguelensis VL-RK09,isolatedfromamangoorchard.

O

S S

Fig.10–MolecularstructureofS,S-dipropyl carbonodithioate(S1).

Table2–MICofS,S-dipropylcarbonodithioateisolated

fromArthrobacterkerguelensisVL-RK09againstdifferent

pathogenicbacteriaandfungi.

Testorganisms MICa_(␮g/mL)

S1 Positivecontrolb Bacteria Staphylococcusaureus 75 25 Streptococcusmutans 80 30 Bacillussubtilis 90 40 Lactobacilluscasei 85 25 Lactobacillusacidophilus 90 25 Xanthomonascampestris 75 40 Bacillusmegaterium 75 30 Bacilluscereus 90 30 Escherichiacoli 90 25 Pseudomonasaeruginosa 70 40 Serratiamarcescens 90 25 Proteusvulgaris 125 50 Enterococcusfaecalis 150 25 Yeast Candidaalbicans 150 50 Molds Aspergillusniger 100 5 A.flavus 75 10 Fusariumoxysporum 90 10 Fusariumsolani 95 10 Penicilliumcitrinum 125 10

a _{MIC,minimuminhibitoryconcentration.}

b _{Tetracyclineagainstbacteria,Griseofulvinagainstyeastand}

Car-bendazimagainstmolds. S1,S,S-dipropylcarbonodithioate.

Biologicalassay

Theantimicrobialprofileofthebioactivecompoundbasedon theMICvaluesisshowninTable2.B.megateriumwashighly sensitivetothecompound,followedbyS.aureus,S.mutans,

B.subtilis,andB.cereusamongtheGram-positivebacteria.P.

aeruginosawashighlysensitivetothecompoundfollowedbyX.

campestris,E.coli,S.marcesens,P.vulgaris,andE.faecalisamong

theGram-negativebacteria.

Amongthefungitested, A.flavuswasfoundtobemore sensitivethantheothers.TheMICvaluesofS,S-dipropyl car-bonodithioateagainstthetestbacteriaandfungivariedfrom 75to150␮g/mL.

Conclusions

Inthepresentstudy,arareactinobacteriumwasisolatedon ISP-2agarmediumasapredominantstrainfromsoilcollected atamangoorchard.Screeningofthestrainforantagonistic activityrevealeditspotentialtoinhibitseveralopportunistic pathogens in vitro. The strain was identified as A.

kergue-lensis bygenomic analysis and designated as VL-RK09.In

ordertoimprovetheproductivityofthestrain,an optimiza-tionofnutritionalandphysiologicalfactorswascarriedout. Thestrainexhibitedhighantimicrobialactivitywhengrown inanoptimizedculturemedium(lactose,1%;peptone,0.5%; K2HPO4,0.05%)atpH7.0andincubatedat30◦Cfor120h.An

attemptwasalsomadetocharacterizeantimicrobial metabo-litesbyculturingthestraininalargevolumeofthemedium. Extraction,followedbybioactivity-guidedisolationand purifi-cationofacrudeethylacetateextract,revealedthepresence ofS,S-dipropyl carbonodithioate. Thisisthe first reporton S,S-dipropylcarbonodithioateisolatedfromA.kerguelensis VL-RK09.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

WewouldliketothanktheDepartmentofBotanyand Microbi-ologyforprovidingthelaboratoryfacilities.RKMthanksUGC forprovidingthefinancialassistancetocarryouttheresearch work.

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2016.07.010.

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e

s

1.ThumarJT,DhuliaK,SinghSP.Isolationandpartial

purificationofanantimicrobialagentfromhalotolerant

alkaliphilicStreptomycesaburaviensisstrainKut-8.WorldJ

MicrobiolBiotechnol.2006;26:2081–2087.

2.KaltenpothM.Actinobacteriaasmutualists:general

healthcareforinsects?TrendsMicrobiol.2009;17:529–535.

3.NanZ,ChangpoS,ZhenS,etal.Isolation,purificationand

characterizationofantifungalsubstancesfromStreptomyces

hygroscopicusBS-112.JActaMicrobiolSin.2011;2:14–20.

4.SmithTL,JarvisWR.Antimicrobialresistancein

Staphylococcusaureus.MicrobesInfect.1999:795–805.

5.MikamiY.BiologicalworkonmedicallyimportantNocardia

species.Actinomycetologica.2007;21:46–51.

6.LechevalierHA,LechevalierMP.Biologyofactinomycetes.

AnnRevMicrobiol.1967;21:71–100.

7.IwaiY,TakahashiY.Selectionofmicrobialsourcesof

bioactivecompounds.In:OmuraS,ed.TheSearchforBioactive

CompoundsFromMicroorganisms.NewYork:Springer-Verlag;

1992:281–302.

8.AnupamaM,NarayanaKJP,VijayalakshmiM.Screeningof

Streptomycespurpeofuscusforantimicrobialmetabolites.ResJ

Microbiol.2007;2:992–994.

9.HayakawaM.Studiesontheisolationanddistributionof

rareactinomycetesinsoil.Actinomycetologica.2008;22:12–19.

10.EuzebyJP.Listofbacterialnameswithstandingin

nomenclature:afolderavailableontheInternet.IntJSyst

Bacteriol.1999;47:590–592.

11.GoodfellowM,FiedlerHP.Aguidetosuccessful

bioprospecting:informedbyantibacterialsystematics.

AntonieVanLeeuwenhoek.2010;98:119–142.

12.LiJQ,TanBP,MaiKS,etal.Comparativestudybetween

probioticbacteriumArthrobacterXE-7andchloramphenicol

onprotectionofPenaeuschinensispost-larvaefrom

13.O’BrienA,SharpR,NicholasJ,RollerS.Antarcticbacteria

inhibitgrowthoffood-bornemicroorganismsatlow

temperatures.FEMSMicrobiolEcol.2004;48:157–167.

14.FondiM,OrlandiniV,EmilianiG,etal.Draftgenome

sequenceofthehydrocarbon-degradingand

emulsan-producingstrainAcinetobactervenetianusRAG-1T_.J

Bacteriol.2012;194:4771–4772.

15.Velázquez-BecerraC,Macías-RodríguezL,López-BucioJ,

etal.TherhizobacteriumArthrobacteragilisproduces

dimethylhexadecylamine,acompoundthatinhibitsgrowth

ofphytopathogenicfungiinvitro.Protoplasma.

2013;250:1251–1262.

16.LiY,LiQ,HaoD,etal.Production,purification,and

antibiofilmactivityofanovelexopolysaccharidefrom

Arthrobactersp.B4.PrepBiochemBiotechnol.2015;45:192–204.

17.BasakK,MajumdarSK.Utilizationofcarbonandnitrogen

sourcesbyStreptomyceskanamyceticusforkanamycin

production.AntimicrobAgentsChemother.1973;4:6–10.

18.RabahFL,ElshafeiA,SakerM,CheikhB,HocineH.

Screening,isolationandcharacterizationofanovel

antimicrobialproducingactinomycetestrainRAF10.

Biotechnology.2007;6:489–496.

19.RizkM,TahanyAM,HanaaM.Factorsaffectinggrowthand

antifungalactivityofsomeStreptomycesspeciesagainst

Candidaalbicans.IntJFoodAgricEnviron.2007;5:446–449.

20.SinghV,TripathiCKM,BihariV.Production,optimizationand

purificationofanantifungalcompoundfromStreptomyces

capoamusMTCC8123.MedChemRes.2008;17:94–102.

21.BangaJ,PraveenV,SinghV,TripathiCKM,BihariV.Studies

onmediumoptimizationfortheproductionofantifungal

andantibacterialantibioticsfromabioactivesoil

actinomycete.MedChemRes.2008;17:425–436.

22.ShirlingEB,GottliebD.Methodsforcharacterizationof

Streptomycesspecies.IntJSystBacteriol.1966;16:313–340.

23.CappuccinoJG,ShermanN.Microbiology,LaboratoryManual.

NewDelhi:PearsonEducation,Inc.;2004:282–283.

24.SrinivasanMC,LaxmanRS,DeshpandeMV.Physiologyand

nutritionalaspectsofactinomycetes:anoverview.WorldJ

MicrobiolBiotechnol.1991;7:171–184.

25.SauravK,KannabiranK.Diversityandoptimizationof

processparametersforthegrowthofStreptomycesVITSVK9

sp.IsolationfromBayofBengal,India.JNatEnvironSci.

2010;1:56–65.

26.ElliahP,SrinivasuluB,AdinarayanaK.Optimizationstudies

onNeomycinproductionbyamutantstrainofStreptomyces

marinensisinsolidstatefermentationprocess.Biochemistry.

2000;39:529–534.

27.KathiresanK,BalagurunathanR,SelvamMM.Fungicidal

activityofmarineactinomycetesagainstphytopathogenic

fungi.IndianJBiotechnol.2005;4:271–276.

28.FaridMA,El-EnshasyHE,Ei-DiwanyAI,El-sayedEA.

OptimizationofthecultivationmediumforNatamycin

productionbyStreptomycesnetalensis.JBasicMicrobiol.

2000;40:157–166.

29.MunagantiRK,NaraganiK,MuvvaV.Antimicrobialprofileof

RhodococcuserythropolisVL-RK05isolatedfromMango

Orchards.IntJPharmSciRes.2015;6:1805–1812.

30.NaraganiK,RajeshkumarM,UshaKiranmayiM,

VijayalakshmiM.Optimizationofcultureconditionsfor

enhancedantimicrobialactivityofRhodococcuserythropolis

VLK-12isolatedfromSouthCoastofAndhraPradesh,India.

BrMicrobiolResJ.2014;4:63–79.

31.UshaKiranmayiM,SudhakarP,SreenivasuluK,

VijayalakshmiM.Optimizationofculturingconditionsfor

improvedproductionofbioactivemetabolitesby

Pseudonocardiasp.VUK-10.Mycobiology.2011;39:174–181.

32.DeepaI,VijayalakshmiM,RajeshkumarM.Streptomyces

cellulosaeVJDS-1,apromisingsourceforpotentialbioactive

compounds.IntJPharmPharmSci.2015;7:57–61.

33.MustafaO.Antifungalandantibacterialcompoundsfrom

Streptomycesstrains.AfrJBiotechnol.2009;8:3007–3017.

34.GhoshUK,PrasadB.Optimizationofcarbon,nitrogen

sourcesandtemperatureforhypergrowthofantibiotic

producingstrainStreptomyceskanamyceticusMTCC324.

Bioscan.2010;5:157–158.

35.AttaHM,BahobailAS,El-SehrawiMH.Studiesonisolation,

classificationandphylogeneticcharacterizationof

antifungalsubstanceproducedbyStreptomyces

albidoflavus-143.NYSciJ.2011;4:40–53.

36.UshaKiranmayiM,SudhakarP,KrishnaN,VijayalakshmiM.

Influenceofculturalconditionsforimprovedproductionof

bioactivemetabolitesbyStreptomycescheonanensisVUK-A

isolatedfromcoringamangroveecosystem.CurrTrends

BiotechnolPharm.2012;6:99–111.

37.GeshevaV,IvanovaV,GeshevaR.Effectofnutrientsonthe

productionofAK-111-81macrolideantibioticbyStreptomyces

hygroscopicus.MicrobiolRes.2005;160:243–248.

38.IlicSB,KonstantinovicSS,VeljkovicVB,SavicDS,GordanaÐ,

CvijovicG.Theimpactofdifferentcarbonandnitrogen

sourcesonantibioticproductionbyStreptomyces

hygroscopicusCH-7.CurrResTechnolEducTopApplMicrobiol

MicrobBiotechnol.2010;2:1337–1342.

39.ChattopadhyayD,SenSK.Optimizationofcultural

conditionsforantifungalantibioticaccumulationby

StreptomycesrocheiG164.HindustanAntibiotBull.

1997;39:64–71.

40.HanWC,LeeJY,ParkDH,LimCK,HwangBK.Isolationand

antifungalandantioomyceteactivityofStreptomycesscabie

strainPK-a41,thecausalagentofcommonscabdisease.

PlantPatholJ.2004;20:115–126.

41.NarayanaKJP,VijayalakshmiM.Optimizationof

antimicrobialmetabolitesproductionbyStreptomyces

albidoflavus.ResJPharmacol.2008;2:4–7.

42.RipaFA,NikkonF,ZamanS,KhondkarP.Optimalconditions

forantimicrobialmetabolitesproductionfromanew

Streptomycessp.RUPA-08PRisolatedfromBangladeshisoil.

Mycobiology.2009;37:211–214.

43.LoGiudiceA,BrunV,MichaudL.Characterizationof

Antarcticpsychrotrophicbacteriawithantibacterial

activitiesagainstterrestrialmicroorganisms.JBasicMicrobiol.

2007;47:496–505.

44.RojasJL.BacterialdiversityfrombenthicmatsofAntarctic

lakesasasourceofnewbioactivemetabolites.MarGenomics.