Antibacterial Activities of Bacteria Isolated from the Marine Sponges

19 Pages·2017·2.11 MB·English

marine drugs
Article
Antibacterial Activities of Bacteria Isolated from the
Marine Sponges Isodictya compressa and
Higginsia bidentifera Collected from Algoa Bay,
South Africa
RelebohileMatthewMatobole1,LeonardoJoaquimvanZyl1,*,ShirleyParker-Nance2,3,
MichaelT.Davies-Coleman4andMarlaTrindade1
1 InstituteforMicrobialBiotechnologyandMetagenomics(IMBM),DepartmentofBiotechnology,
UniversityoftheWesternCape,RobertSobukweRoad,Bellville7535,CapeTown,SouthAfrica;
[email protected](R.M.M.);ituffi[email protected](M.T.)
2 DepartmentofZoology,NelsonMandelaMetropolitanUniversity,UniversityWay,
PortElizabeth6031,SouthAfrica;[email protected]
3 SouthAfricanInstituteforAquaticBiodiversity(SAIAB),SomersetStreet,Grahamstown6139,SouthAfrica
4 DepartmentofChemistry,UniversityoftheWesternCape,RobertSobukweRoad,Bellville7535,
CapeTown,SouthAfrica;[email protected]
* Correspondence:[email protected];Tel.:[phone]
AcademicEditor:KeithB.Glaser
Received:8November2016;Accepted:30January2017;Published:17February2017
Abstract:Duetotheriseinmulti-drugresistantpathogensandotherdiseases,thereisrenewedinterest
inmarinespongeendosymbiontsasarichsourceofnaturalproducts(NPs).TheSouthAfricanmarine
environmentisrichinmarinebiotathatremainslargelyunexploredandmayrepresentanimportant
sourceforthediscoveryofnovelNPs.Wefirstinvestigatedthebacterialdiversityassociatedwithfive
SouthAfricanmarinesponges,whosemicrobialpopulationshadnotpreviouslybeeninvestigated,
andselectthetwosponges(IsodictyacompressaandHigginsiabidentifera)withhighestspeciesrichness
toculturebacteria. Byemploying33differentgrowthconditions415sponge-associatedbacterial
isolateswereculturedandscreenedforantibacterialactivity. Thirty-fiveisolatesshowedantibacterial
activity, twelve of which exhibited activity against the multi-drug resistant Escherichia coli 1699,
implyingthatsomeofthebioactivecompoundscouldbenovel. Genomesequencingoftwoofthese
isolatesconfirmedthattheyharbouruncharacterizedbiosyntheticpathwaysthatmayencodenovel
chemicalstructures.
Keywords: Isodictyacompressa;Higginsiabidentifera;naturalproducts;marinesponge;antimicrobial
1. Introduction
The marine environment has been identified as a rich source of bioactive compounds with
interesting chemical diversity, and in particular those produced by marine sponges [1]. Sponges
belong to the phylum Porifera, and are some of the oldest metazoans on Earth [2,3]. There are
fourclassesofmarinesponges,namelyCalcarea,Hexactinellida,HomoscleromorphaandDemospongiae
withtheDemospongiaebeingmostabundant,representing83%ofdescribedspecies[4]. Thesefilter
feedingsessileorganismsoccurprimarilyinmarineenvironmentswheretheysurviveunderimmense
competitionandpredation[5–7].
Thereisevidencethatmanyofthepreviouslyidentifiedmarinespongesecondarymetabolites
are of microbial origin [1,8–10]. Their ability to survive in this competitive environment could
be due to their own adaptation, in addition to or perhaps because of the microbial communities
Mar.Drugs2017,15,47;doi:10.3390/md15020047 www.mdpi.com/journal/marinedrugs
Mar.Drugs2017,15,47 2of19
they harbor, which can account for up to 40% of the sponge’s wet weight [5,7,11]. Furthermore,
these microbes are proven to produce medically relevant compounds including ecteinascidin-743
(Yondelis®), dolastatin-10 (Adcetris®) and halichondrin B (Halaven®) [12]. As a result, marine
sponge-associated microbes are sought after for natural product discovery [13–16] due to their
potentialfordeliveringnovelbioactivecompounds[17,18]. Ingeneral,thelargescaleproductionof
microbial-derivedsecondarymetabolitesforstudyanduseishamperedbythelackofthesebacteriain
culturecollections,eitherthroughaninabilitytoculturethemoridentifythemicrobesresponsiblefor
producinganobservedbioactivity[19].Itisthereforeimportanttodeveloptechniquestocultivatethese
marinespongesymbiontsinordertoaccessandexploittheirsecondarymetabolitecapabilities[20].
Theuseofnovelculturemedia,suchasveryminimalnutrientcontent[21],applicationofdiffusion
chambers[20,22]ormicroencapsulation[23],havesuccessfullyresultedintheisolationofpreviously
unculturedmarinebacteria.
SouthAfricahasauniquecoastlinethatisdominatedbytwomajorcurrentsystems: thecold
BenguelacurrentandthewarmAgulhascurrent[24,25].Asaresultofthephysico-chemicaldifferences
introducedbythetwocurrents,thecoastlinehasanexceptionallyrichandvariedmarinelifewhich
remainslargelyunexplored. ThefewreportedstudiesperformedonSouthAfricanmarinesponges
thus far do not take their associated microorganisms into account [26]. The latrunculid sponge,
Tsitsikammafavus,endemictothecoastofSouthAfrica,representsoneofthefewspecimenswhose
microbial diversity has been assessed and serves as an example of what South African sponges
may have to offer [8]. Unique to this sponge is the production of pyrroloiminoquinones, known
as tsitsikammamines, that mediate cytotoxicity through the cleavage of DNA due to inhibition of
topoisomerase I [27] and it is thought that these secondary metabolites are of microbial origin [9].
TheSouthAfricancoastthereforerepresentsapotentiallyresourcefulenvironmentfortheexploration
ofnovelbioactivecompounds.
ThisstudyassessesthebacterialdiversityassociatedwithfiveSouthAfricanmarinesponges,
namelyWaltherarndtiacaliculatum,Higginsiabidentifera,Isodictyacompressa,SpongiaandAxinellaspecies.
AllfivespongesbelongtotheclassDemospongiae. TheHigginsiagenuswasfirstdescribedbyHigginin
1877duringascientificexpeditionaboardtheshipArgo,withthespeciesHigginsiabidentiferadescribed
in1886andaccordingtotheWorldPoriferaDatabase,appearstobeendemictoSouthAfricancoastal
waters[28,29]. IsodictyacompressawasfirstdescribedbyEsperin1794,whileWaltherarndtiacaliculatum
was described by Kirkpatrick in 1903, both of which are endemic to South Africa [30–33] and the
bacteriaassociatedwiththesethreemarinespongeshavenotbeeninvestigated. AxinellaandSpongia
species have a global distribution and the microbial populations of some species of these sponge
generahavebeenstudied[34–37]. Wefirstdeterminedthebacterialdiversityassociatedwiththese
five sponges, then isolated bacteria from two of these and demonstrated their ability to produce
antibacterialcompounds. HerewereportanewcollectionofSouthAfricanmarinesponge-associated
bacteriathatshowantibacterialactivitywhensubjectedtoavarietyofcultureconditions.
2. MaterialsandMethods
Unless otherwise specified, chemicals and reagents used in this study were supplied by
SigmaAldrichChemicalCompany(Darmstadt,Germany),MerckChemicalandLaboratorySupplies
(Darmstadt, Germany) and Kimix Chemical and Laboratory Supplies (Cape Town, South Africa).
Theenzymes,DNAsizemarkersandpolymeraseswerepurchasedfromFermentasLifeSciencesLtd.
(Vilnius,Lithuania).
2.1. SampleCollection
Thefivemarinesponges(Waltherarndtiacaliculatum,PE05;Higginsiabidentifera,PE07;Isodictya
compressa,PE09;anAxinellaspecies,PE11;andaSpongiaspeciesPE08)werecollectedinJanuaryand
April2013inAlgoaBay,PortElizabeth,SouthAfrica(longitude34◦00.366Sandlatitude25◦43.209E;
15 ◦C), and kept on ice in sterile sea water. The sponge samples (Figure S1) were shipped to the
Mar.Drugs2017,15,47 3of19
InstituteforMicrobialBiotechnologyandMetagenomics,CapeTown,wherethespongematerialwas
storedat–20◦Cand–80◦Cin20%glyceroluntilfurtherprocessing. Voucherspecimensofallsponge
samplesarehousedintheSouthAfricanInstituteforBiodiversity(SAIAB)inGrahamstown.
2.2. TerminalRestrictionFragmentLengthPolymorphism(T-RFLP)Analysis
Thefivespongesampleswerethawedonanice/waterslurryandcutwithasterilebladeinto1g
aliquotsforfurtherprocessing. TheUltraCleanSoilDNAIsolationkit(MOBIOLaboratories,Carlsbad,
CA, USA) was used to isolate DNA according to the manufactures protocol. Metagenomic DNA
(mDNA)wasfurtherpurifiedasdescribedbyLilesetal.[38]. TheNucleoSpin®GelandPCRClean-up
kit(Macherey-Nagel,Düren,Germany)wasusedtoextractthemDNAfromtheagaroseplug.
ThePCRreactionmixturetogenerateampliconsforT-RFLPwasasfollows: 1×PhusionDNA
polymerase buffer, 0.2 mM dNTP mix, 0.5 µM of each forward primer E9F (5(cid:48)-GAGTTTGATCCT
GGCTCAG-3(cid:48)) labelled at the 5(cid:48) end with fluorescein amidite (FAM) and reverse primer U1510R
(5(cid:48)-GGTTACCTTGTTGTTACACTT-3(cid:48)),0.02U/µLofPhusionDNApolymerase(Fermentas,Lithuania),
100ngoftemplatemDNAandultraviolet(UV)treatedwater[39,40]. Thefollowingcyclingconditions
wereused: initialdenaturationof98◦Cfor2minfollowedby35cyclesof98◦Cfor20s,54◦Cfor30s,
72◦Cfor1minandfinalelongationat72◦Cfor5min. Duplicateamplificationswereconductedfor
eachsample,pooledandpurifiedusingtheNucleoSpin®GelandPCRClean-upkit(Macherey-Nagel).
Amplicons(1µg)weredigestedin30µLvolumescontaining3µLof10×Tangobuffer,2µLofHhaI
(10U/µL),4µLHaeIII(10U/µL),andUVtreatedwater. DigestionswereperformedinaBio-RadT100
ThermalCycler(Bio-Rad,Hercules,CA,USA)overnightat37◦C,followedbyinactivationat80◦C
for20min. Capillaryelectrophoresisandanalysisofthefluorescentlylabelledterminalrestriction
fragments(T-RFs)wascarriedoutonanautomatedABI3130XLgeneticanalyzer(AppliedBiosystems,
FosterCity,CA,USA)bytheCentralAnalyticalFacilityattheUniversityofStellenbosch,SouthAfrica.
The marker used for the analysis was GS500LIZ. T-RFLP profiles were analysed using freeware
PeakScannerTMSoftwareVersion1.0(AppliedBiosystems). Peakheightwasusedtocharacterize
uniqueT-RFs. Peaksshorterthan25bpandlargerthan1200bpwereexcludedfromfurtheranalysis.
ValidpeakswereidentifiedandalignedusingtheonlineT-REXsoftware[41]tocreateanoperational
taxonomicunit(OTU)datamatrix. Intheory, eachOTUrepresentsanindividualT-RFwhereone
T-RFrepresentsonedistinctribotype[42]. Itshouldbenotedhoweverthat,onepeakmayrepresent
twodifferentbacterialspeciesortwopeaksmaybeonebacterialspeciesdependingontherecognition
siteofrestrictionenzymesonthe16SribosomalRNA(rRNA)genesequence[43–45]. TheOTUmatrix
wasanalysedusingPrimer6,Version6.1.11(PrimerE,Plymouth,UK)softwareanddiversityindices
forallthesamplesweredetermined.
2.3. BacterialCulturing
Thawedspongematerial(1g)washomogenisedbygrindingwithasterilepestleandmortarin
9mLofsterileseawater. Thespongehomogenateswereseriallydilutedinsterileseawaterto10−6,
andfromeachdilution,100µLaliquotswereplatedon23generalmedia(TableS1). Thefullculturing
schemeisdepictedinFigureS2. Inaddition,aliquotsofthespongehomogenatesunderwentheat
treatmenttoselectforendosporeformingbacteria[15]andtoinhibitthegrowthofGramnegativefast
growingbacteria[46].TheheattreatmentprocesswasadaptedfromPhelanetal.(2012)[15]withminor
modifications. Analiquotof100µLofthe10−1dilutionofthespongehomogenatewasincubatedat
80◦Cfor10minandseriallydilutedto10−6. Aliquotsof100µLwereasepticallyplatedonthreemedia
ShivjiNutrientAgar(SNA),TrypticSoyAgar(TSA)andZobellAgar(ZBA).Liquidenrichmentwas
usedtoselectforcyanobacteria[47]andnitrogenfixingbacteria[48]. Forliquidenrichmentcultures,
100µLofthe10−1dilutionsofthespongehomogenatewereaddedto9mLofAshby’sNitrogenFree
Media(ANFA),MagMinSemi-SolidMedium(MMM)andBlueGreenAlgae(BG11)liquidmedia.
Theliquidenrichmentcultureswereincubatedat15◦Cforuptosixweeksonashakerat150rpm.
Subsequently,100µLoftheliquidenrichedcultureswereplatedonZBAplates,asZBAisoneofthe
Mar.Drugs2017,15,47 4of19
richmediawhichcangenerallysupportproliferationofarangeofdifferentbacteria. Aftertheinitial
isolations,threemediaGlucoseYeastMedia(GYM),TSAandZBAwereselectedasthe“standard/rich
media”forroutineculturingandcharacterizationofthebacterialisolates. IsolatesfromTrypticase
Soya Agar pH 4.5 (TS4), TSA and SNA, inclusive of those which underwent different treatments,
wereculturedonTSA.IsolatesfromMediumAAgar(MAA),GYM,Modified172Fmedium(172),
OatmealAgar(OMA),ActinomycesIsolationMedium(AIM),andPlanctomycetesMedium(PMM)
alongwiththeirdifferenttreatmentswereculturedonGYM.Isolatesfromthethirteenothermedia
withtheirrespectivetreatmentswerealsoculturedonZBA.Antibiotictreatedspongehomogenates
werepreparedbyadding20µg/mLofstreptomycin[49]tothehomogenate. Thehomogenateswere
agitatedonashaker(150rpm)for1hatroomtemperature. Thespongehomogenateswerecentrifuged
at 11,000× g for 3 min and the supernatant was discarded. Sterile sea water (1 mL) was used to
re-suspendthepelletandthesuspensionwasseriallydilutedto10−6. Aliquotsof100µLofthe10−1to
10−6dilutionswereplatedoneightmedia(OMA,OMA+NaCl(1.8%wt/vol),AIM,GYM,172,MAA,
YeastExtract-MaltExtract(YEME)andStarchCaseinNitrateAgar(SCN))containingcyclohexamide
(100µg/mL)andnalidixicacid(50µg/mL)[50]. Allplateswereincubatedat15◦Cforuptofour
weeks. Colonies were examined and picked on a daily basis selecting for colonies with different
morphologies(shape,colour,texture,andsize).
2.4. AntimicrobialScreening
Themarinesponge-associatedisolateswerescreened,usingagaroverlayassays,forantibacterial
activityagainsttheGram-positivestrainsMycobacteriumsmegmatisLR222,BacilluscereusATCC10702,
StaphylococcusepidermidisATCC14990andtheGramnegativestrainsPseudomonasputidaATCC27853
andthemultidrugresistantEscherichiacoli1699(Cubist,Lexington,MA,USA).Isolateswerecultivated
onfour media (ZBA,GYM,TSAand ActivatedCharcoalMedium (ACM))fortwoweeks atroom
temperature. On the day prior to carrying out the overlay assays, the test strains were cultured
overnightin10mLLuriaBroth(LB)at37◦Cwithaerationat250rpm. Toensurethatapproximately
thesameconcentrationofcells(usingopticaldensitymeasurements,OD )werepresentinthesloppy
600
agar,thefollowingformulaewereusedtocalculatethevolumeofculturetoaddto6mLofsloppy
agar (LB): OD × X µL = 4 (for E. coli). OD × X µL =160 (for other test strains). The sloppy
600 600
agarcontainingtheteststrainswaspouredoverthemarineisolategrowth,incubatedat30◦Cand
examinedforzonesofinhibitionafter16handagainafter48h.
For induction of antimicrobial activities, a one-strain-many-active-compounds (OSMAC)
approach was followed. A total of 29 isolates were selected; 12 that gave hits against the panel
ofteststrainsintheoverlayassaysandanadditional17isolatesthatdidnotshowantibacterialactivity
inthefirstroundofscreening(TableS2). Amatrixincorporatingthefollowinggrowthmodificationsor
treatmentswasdeveloped: heatorcoldshocktreatment,co-culturingwithsponge-associatedbacteria,
andvaryingcarbon(mannitol,succinicacidandstarchat5mM)andnitrogen(NH ClorNaNO at
4 3
0.02%w/v)sourcesorphosphateconcentrations(KH PO at0.1µMor0.5mM).Thebasemedium
2 4
employedwasaminimalmediacontainingperliter: 18gNaCl,2gMgCl ,0.525gKCl,0.075gCaCl
2
and2.38gHEPESbuffer. Theexperimentwassetupina96-wellformatonlargepetridishplates
(20mm × 150mm). Themethodofplatingin96-wellformatwassimilartothatdescribedin[51]
withsomemodification. Singlecoloniesfromtheselectedisolateswereinoculatedinto96-wellplates
containing70µLsterilewaterineachwell,andshakenat100rpmonashakerfor10minatroom
temperature(RT).Cultureswerethenplatedasepticallyonthedifferentmatrixmediausinga96-pin
hedgehogandallowedtogrowfor2weeksatRT.Theorderofinoculationofisolateswasinterchanged
betweenisolatesthatgavepositivehitsonstandardmediuminthefirstroundofscreeningandthose
whichdidnot. Allisolateswereinoculatedintriplicateoneach96-wellplateandthereforelocated
atdifferentpositionsresultingindifferentneighboringisolateswhichresultedinaco-culturesetup
(FigureS3). Onthedaypriortoperformingoverlays,coldandheatshocktreatmentswerecarried
out. Theheatshockmethodusedwasadaptedfrom[52]withminorchanges. Heatshockplateswere
Mar.Drugs2017,15,47 5of19
incubatedfor1hat42◦C,whereascoldshocktreatmentwascarriedoutat4◦Cfor1h. Theheat
andcoldshocktreatmentswererepeatedonthedayoftheantibioticoverlayassay. Thedaypriorto
carryingouttheoverlayassay,singlecoloniesofeachoftheteststrainswasinoculatedintoLuriaBroth
(10mL)andthecultureswereincubatedat37◦Covernightwhileshakingat250rpm. Theoptical
density(OD )oftheculturesweredeterminedafterwhichtheyweredilutedtoachievethesame
600
overallconcentrationofcells. Anoverlayassaysimilartothatdescribedabovewascarriedoutusing
24mLofagarforthelargeformatplates(20mm×150mm).
2.5. DNASequencingandPhylogeneticAnalysis
For16SrRNAidentificationofthebioactiveisolates,genomicDNAextractionwasperformed
usingtheGeneJETGenomicDNAPurificationKit(ThermoScientific,Waltham,MA,USA).Asingle
colony was inoculated in 10 mL TSA broth or 1⁄4 strength ZBA broth and incubated on a shaker
(150rpm)atroomtemperaturefor2to5daysdependingongrowthrate. DNAextractionwascarried
outaccordingtothemanufacturer’sinstructionsforGram-negativebacteria. Minormodifications
were made for Gram-positive bacteria where 200 µL of lysis buffer (25 mM Tris-HCl pH8, 50 mM
glucose,10mMEDTAand25mg/mLlysozyme)wasaddedtothepelletedbacterialcellsandthe
solutionswereincubatedat37◦Cfor30min.Thereafterthemanufacturer’sinstructionswerefollowed.
PCRamplificationwasperformedin50µLvolumeswhichconsistedof1×DreamTaqbuffer,0.2mM
ofdNTPmix,1µMofeachofthereverseandforwardprimers,100ngtemplateDNA,1.25UDreamTaq
DNApolymerase(Fermentas),andsteriledistilledwater. TheuniversalprimersE9FandU1510Rwere
usedtoamplifythe16SrRNAgenesequencesofthemarinesponge-associatedbacteria. Thefollowing
cyclingconditionswereused: initialdenaturation95◦Cfor5min,35cyclesof95◦Cfor45s,55◦Cfor
45s,72◦Cforoneandahalfminandfinalelongationof72◦Cfor10min.
TheDNAsequencedeterminationofpurified16SrRNAgeneproductswerecarriedoutbythe
CentralAnalyticalFacilityatStellenboschUniversityusinganABIPRISM377automatedsequencer
(Applied Biosystems). The data from the sequencing unit was processed using Chromas Pro 1.5a
software (Technelysium, South Brisbane, QLD, Australia) for alignment and manual editing of
sequences. The neighbor-joining phylogenetic tree was constructed using MEGA 7 software [53].
Bootstraptestswereperformed1000times.
2.6. GenomeSequencing
WholegenomesequencingofPE09-72andPE09-221wasperformedatthesequencingfacility
withintheInstituteforMicrobialBiotechnologyandMetagenomics,UniversityoftheWesternCape,
SouthAfrica. GenomicDNAwaspreparedusingstandardphenol:chloroformextraction. Sequencing
librarieswerepreparedusingtheIlluminaNexteraXTlibraryprepkitaccordingtothemanufacturer’s
instructions and sequencing was performed using the Illumina MiSeq Reagent kit V3 (Illumina,
SanDiego,CA,Country)whichincludeda10%phiXV3spike[54]. Thisresultedin2×300basepair
(bp)pairedendreads(forwardandreverse). AnalysisofthesequencedatawascarriedoutusingCLC
Genomicsversion7.5.1(CLC,Aarhus,Denmark). RawreadswerereferenceassembledtophiXV3
genomeusedinthesequencingreactionforqualitycontrol. Unmappedreadsweremappedtothe
humangenometoremoveanycontaminatingsequences. Adenovoassemblywascarriedouton
theremainingunmappedreadsusingdefaultparameters. Basicanalysisoftheassembledcontigs
were carried out using the RAST server [55] and BLAST analysis against the NCBI database [56].
ThegenomeshavebeendepositedintheGenbankdatabaseunderaccessionnumbersMIJA00000000
andMIJB00000000forPE09-72andPE09-221respectively.
Mar.Drugs2017,15,47 6of19
3. ResultsandDiscussion
3.1. BacterialDiversityAnalysisandCulturingofMarineSponge-AssociatedBacteria
Fivesponges(PE05,PE07,PE08,PE09,PE11)wereinvestigatedfortheirbacterialdiversity,using
16S rRNA based T-RFLP analysis to identify those with highest diversity and unique operational
taxonomicunits(OTUs)toimprovethechanceofisolatingnovelbacteria. T-RFLPprofilesrevealed
thattherewasatotalof179OTUsfromthefivemarinesponges,90ofwhichwereunique(Figure1).
StudieshaveclassifiedmicrobialOTUsaseitherspeciesspecificOTUs(definedasOTUsuniquetoa
certainspoMnagr.e Dsrupgse 2c0i1e7,s 1)5,o 4r7 spongespecificOTUs(definedasOTUsfoundinspongesbut6 onf o18t foundin
otherenvironments)[57,58]. Notably,fiveoftheOTUsweresharedamongallfivespongesindicating
other environments) [57,58]. Notably, five of the OTUs were shared among all five sponges indicating 
thattherepthreats tehnet raetpirveeseOntTatUivse OcoTuUlsd cobuelds bpeo snpgonegsep sepcecifiificc [[5599,6,600].] .

Figure 1. Venn diagram of species specific and shared operational taxonomic units (OTUs) (at genus 
Figure1.Vleenvenl) dini athger asemlecotefds mpaerciniees spsopnegceisfi. candsharedoperationaltaxonomicunits(OTUs)(atgenus
level)intheselectedmarinesponges.
The Simpson index of the bacterial species from PE05 and the PE11 was the least even, indicating 
the presence of more dominating OTUs in these samples, while the other sponges had a more even 
TheSimpsonindexofthebacterialspeciesfromPE05andthePE11wastheleasteven,indicating
distribution of species abundance (Table 1). PE07 and PE09 yielded the most OTUs with 43 and 44 
the presenrceespoecftimveloyr eandd ohmadi nthaet ihniggheOstT eUvesnnienss.t hAeltsheousgahm sepelmesin,gwly hloiwle, tthhee OoTtUhse rfrosmp othneg perseshenatd  a more
even distrisbtuudtyio wneroe fsimspilearc tioe sthea bnuumnbdearsn ocfe T‐(RTFaLbPl‐eba1se)d. OPTEU0s 7repaonrdtedP fEro0m9 oythieerl dsteuddietsh wehemreo 4s5t aOndT Us with
32 OTUs were identified in Hymeniacidon heliophila and 36 and 22 OTUs were identified in Haliclona 
43 and 44 respectively and had the highest evenness. Although seemingly low, the OTUs from
tubifera depending on the restriction enzymes used [61]. PE09 had the highest number of species 
thepresentstudyweresimilartothenumbersofT-RFLP-basedOTUsreportedfromotherstudies
specific OTUs (14) with the second highest being PE05 with 11 OTUs. Although PE07 had the second 
where45anhidgh3e2st OnuTmUbserw oefr OeTidUes notnifilye tdhriene Hwyerme esnpieaccifiidco tno hthelisio spahmilpalea nwdhi3ch6 iannddica2te2dO thTaUt fsewweerr eH.i dentified
inHaliclonabidteunbtiiffeerraa‐spdeecpifiecn OdTinUgs woneret hiesorlaetsetdr ifcrtoimon theen zsaymmplees. uGsiveedn [t6h1e ].hiPghEe0r 9dhivaedrsittyh eanhdi gehveenst  number
representation of species present in both PE07 and PE09, as well as the uniqueness of PE09 profiles 
ofspeciesspecificOTUs(14)withthesecondhighestbeingPE05with11OTUs. AlthoughPE07had
and the representative nature of PE07, these were selected for bacterial isolation studies. 
the second highest number of OTUs only three were specific to this sample which indicated that
fewerH.bidentifera-speciTfiacblOe 1T. UDisvewrsietyr eindisicoelsa otfe tdhe fsrpoonmget‐ahsesosciaamtedp mleic.roGbiiavl epnoptuhlaetiohnisg. herdiversityandeven
representationSoamfpsplee IcDie spreseMntariinneb SoptohnPgeE I0D7andPES0p9e,caiess wSpeelclifaics OthTeUusniqSu enes1s −o λf PE09profiles
andtherepresenPtaEt0i5v enatureofPWE. 0ca7l,ictuhlaetsuemwereselectedforb1a1cterialisolat3io6 nstu0.d69i2e3s.
PE07  H. bidentifera  3  43  0.9388 
PTEa0b8l e1.DSipvoenrgsiait (yspionndgiica)e sspo. f0t0h1eRSsApoSPnNge -associatedm7 icrobialpopu2l4a tion0s..8514 
PE09  I. compressa 14 44  0.9306
PE11  Axiinella sp. 007RSASPN  7  32  0.7706 
SampleID MarineSpongeID SpeciesSpecificOTUs S 1−λ
Index: species richness (S) = OTUs; Simpson index (1 − λ) = evenness. 
PE05 W.caliculatum 11 36 0.6923
PE07 A total of 415 uniquHe b.abcitdeerniatli fceoralonies, as judged by their morp3hology (size, shap4e3, colour a0n.9d3 88
PEte0x8ture), wereS ipsoonlagteiad (fsrpoomn gthiae) twspo. s0p0o1nRgSe AsaSmPpNles with PE09 yieldi7ng 273 isolates and2 1442 bacte0r.i8al5 14
PEis0o9lates from PE07, respecIt.ivceolmy.p Trheses faewest number of isolates were1 o4btained from sam4p4les grow0in.9g3 06
PEin1 1liquid enrichmeAnxt iimneeldliausmp .a0n0d7 fRroSmA SthPeN heat treated medium, w7hich yielded 5 an3d2 12 isola0t.e7s7 06
respectively. This could be due to the selectivity in the liquid enrichment medium which was meant 
Index:speciesrichness(S)=OTUs;Simpsonindex(1−λ)=evenness.
to select for nitrogen fixing bacteria. In the case of heat treatment, the low viable microbial numbers 
post treatment indicate that the number of spore‐forming bacteria are low in these marine sponges 
or that sporulation had not been induced in spore formers at the time of harvest [15]. The antibiotic
Mar.Drugs2017,15,47 7of19
Atotalof415uniquebacterialcolonies,asjudgedbytheirmorphology(size,shape,colourand
texture),wereisolatedfromthetwospongesampleswithPE09yielding273isolatesand142bacterial
isolatesfromPE07,respectively. Thefewestnumberofisolateswereobtainedfromsamplesgrowing
in liquid enrichment medium and from the heat treated medium, which yielded 5 and 12 isolates
respectively. Thiscouldbeduetotheselectivityintheliquidenrichmentmediumwhichwasmeant
toselectfornitrogenfixingbacteria. Inthecaseofheattreatment,thelowviablemicrobialnumbers
posttreatmentindicatethatthenumberofspore-formingbacteriaarelowinthesemarinesponges
ortMhaar.t Dsrpuogsr 2u0l1a7t, i1o5,n 47h adnotbeeninducedinsporeformersatthetimeofharvest[15]. Thean7 toifb 1i8o tic
treatmentyielded37isolateswhilethenon-treatedmediumresultedin361bacterialisolates. GYM
treatment yielded 37 isolates while the non‐treated medium resulted in 361 bacterial isolates. GYM 
wasthemediumwhichresultedinthehighestnumberofbacterialisolates(40)whichaccountedfor
was the medium which resulted in the highest number of bacterial isolates (40) which accounted for 
10%ofthetotalisolates. AIMandZBAresultedin39(10%)and37(9%)isolatesrespectively(Figure2).
10% of the total isolates. AIM and ZBA resulted in 39 (10%) and 37 (9%) isolates respectively (Figure 
ThemediumwhichresultedinthefewestbacterialisolateswereMMMandSeawaterAgar(SWA)since
2). The medium which resulted in the fewest bacterial isolates were MMM and Seawater Agar (SWA) 
eachcontributedonlyonebacterialisolate. Bothmediadiscouragegrowthoffastgrowingbacteria
since each contributed only one bacterial isolate. Both media discourage growth of fast growing 
thubsarcetesruialt itnhgusi nrefseuwlteinrgn iunm febweresr onfummbicerrosb oifa lmisicorloabteiasl. isolates. 

Figure 2: Total number (and percentage) of bacterial isolates cultured from the marine sponge 
Figure2.Totalnumber(andpercentage)ofbacterialisolatesculturedfromthemarinespongesamples
samples PE07 and PE09 per medium type. The number shown inside each pie section indicates the 
PE07andPE09permediumtype.Thenumbershowninsideeachpiesectionindicatestherawnumber
raw number of isolates from that medium type. Only 1 isolate was recorded from MMM and SWA 
ofisolatesfromthatmediumtype.Only1isolatewasrecordedfromMMMandSWArespectively.
respectively. 
3.2. AntimicrobialScreeningandInduction
3.2. Antimicrobial Screening and Induction 
TheantibacterialpropertiesofmicrobialisolatesfromPE07andPE09wereinvestigatedinan
The antibacterial properties of microbial isolates from PE07 and PE09 were investigated in an 
agaargaorv oervlearylaays assasyaya gaagianisntstfi fviveet etesstts sttrraaiinnss ((TTaabbllee 22)).. MMoosstt iissoolalatetes soonnlyl yshsohwowede da naanrraorwro wspescpteructmru m
ofaocf taivctiitvyi,tyb,e bineginagc aticvtievea gaagianinststo onnlyly oonnee tteesstt ssttrraaiinn wwhheenn ccuultluturerded ono nstsatnadnadradr mdemdeiad (iZaB(ZAB, AGY,GMY, M,
TSATSaAn danAd CAMCM).)H. oHwoweveevre,ri, siosloaltaetessP PEE0077-‐77,, PPEE0077-‐114444,, PPEE0077‐-220077, ,PPEE090‐98-78,7 P,EP0E90‐190-150 a5nadn PdEP09E‐0191-91 19
disdpilsapylaeydeadc aticvtiivtyitya gaagianinststt wtwooo orrm moorree tteesstt ssttrraaiinnss.. NNoottaabblyly, ,PPEE007‐77- 7anadn dPEP0E90‐191-191 o9nolyn ldyisdpilsapyleady ed
actaivcittiyviatyg aaignasitnGstr aGmr-apmo‐spitoisvietivtees ttesstrt asintrsa,inwsh, iwlehPilEe0 7P-E10474‐,1P44E,0 7P-E20077‐2a0n7d aPnEd0 9P-E10059‐h1a05d ahcatdiv iatcytiavgitayi nst
botahgaGinrasmt b-optohs iGtirvaema‐npdosGitrivaem a-nndeg Gatriavme‐tneesgtasttirvaein tse.stM sotrsatinasc.t iMviotystw aactsivreitcyo wrdaesd raecgoaridnesdt Ba.gcaeirnesuts Bw. ith
17oceurteuosf w32itsht r1a7i nosust hoofw 32in sgtraacitnisv isthyoawgianign satctthivisityte astgastinrasitn t,hwis htielset ssetvraeinn,i swohlailtee ssegvaevne iascotliavtietsy gaagvaei nst
theahctiigvhitlyy aangatiibnisott ticher ehsiigsthalnyt aEn.tciboiloit1i6c9 r9es(irsetsainstta En.t ctooli5 126k9n9 o(wrensisatnatnitb itoot 5ic2s )k.nIotwwna saanltsiobinootitcesd). tIht awtanso ne
also noted that none of the isolates displayed antibacterial activity on more than two growth media, 
suggesting inducible genes or pathways. It is also important to note that all isolates grew to some 
degree on the media used and lack of activity is not due to the inability of the isolate(s) to grow.
Mar.Drugs2017,15,47 8of19
oftheisolatesdisplayedantibacterialactivityonmorethantwogrowthmedia,suggestinginducible
genesorpathways. Itisalsoimportanttonotethatallisolatesgrewtosomedegreeonthemediaused
andlackofactivityisnotduetotheinabilityoftheisolate(s)togrow.
Table2.Antibacterialactivitiesagainstteststrainsinstandardmedium.
TestStrains
Isolate GramNegative GramPositive
E.coli1699 P.putida M.smegmatis B.cereus S.epidermidis
PE07-5 GYM GYM ZBA
PE07-7 TSA,ZBA TSA,ZBA
PE07-86 TSA
PE07-105 GYM
PE07-133 GYM
PE07-143 TSA,ZBA
PE07-144 GYM GYM
PE07-172 TSA
PE07-200 GYM
PE07-201 GYM
PE07-204 GYM
PE07-207 GYM GYM
PE09-7 TSA
PE09-73 ACM
PE09-87 GYM GYM GYM
PE09-100 ZBA
PE09-105 GYM,TSA TSA,GYM TSA
PE09-108 GYM
PE09-110 ZBA
PE09-119 ZBA,GYM TSA,ZBA ACM,TSA
PE09-124 ACM
PE09-140 ACM
PE09-142 ACM
PE09-168 ACM
PE09-197 ACM
PE09-210 ACM
PE09-213 ACM
PE09-221 ZBA
PE09-228 ACM
PE09-229 ACM
PE09-235 ACM
PE09-266 ZBA
Totalhits 7 5 8 17 6
Tofurtherinduceantimicrobialactivities, anOSMACapproachwastakenforasubsetofthe
isolates. Fromscreening29microbialisolatesinthematrixof36culture/treatmentconditionsand
co-culture,sixisolatesshowedinducedantibacterialactivities(Table3). Mostnotably,isolatePE09-72
producedactivityagainstallteststrainsinmannitolcontainingmedium,especiallywiththeaddition
of heat shock, whereas only one activity was produced with succinic acid as the carbon source.
Thisindicatesthatthepathway(s)responsiblefortheactivitiesarelikelysubjecttocarboncatabolite
repression(CCR)[62]. Althoughnomediumcompositioninducedactivitiesagainstallteststrains,
some medium compositions (NH Cl-LP, NH Cl-HP and NaNO -HP) induced activity in PE09-72
4 4 3
againstfouroftheteststrainswhenmannitolwasusedascarbonsourceandthecultureheatshocked.
Differential antimicrobial activities were observed in relation to the media the isolates were
culturedon. Forinstance,theisolatePE07-5showedantibacterialactivitiesagainstE.coli1699and
M. smegmatis in GYM medium and only against B. cereus on ZBA medium. This is a well-known
phenomenonregardingmicrobialsecondarymetaboliteproduction[8,63].
Mar.Drugs2017,15,47 9of19
Mar. Drugs 2017, 15, 47  9 of 18 
Table3.Antibacterialactivitiesofsixselectedsponge-associatedisolatesundermatrixscreeningconditions.Threeoftheisolatesdidnotdisplayactivityintheinitial
scTreaebnlein 3g. ,Awnhtiebraecatsertihael aoctthiverititehsr eoef issiox lsaetelescdteidd .spRoTn:gReo‐oasmsoTceiamtepde irsaotluartees( 2u7nd◦Cer) ;mHaStr:iHx esacrteSehnoincgk ctroenadtimtioennst.( 4T2hr◦eCe )o;fC tSh:eC iosoldlastehso dckidt rneoatt mdiesnptla(y4 a◦cCti)v.ity in the initial 
screening, whereas the other three isolates did. RT: Room Temperature (27 °C); HS: Heat Shock treatment (42 °C); CS: Cold shock treatment (4 °C). 
None‐Active in Primary Screening Active in Primary Screening

PE07‐103 PE09‐55 PE09‐72 PE07‐105 PE09‐140 PE09‐229
TSA                                                             
Standard  GYM                                                             
media  ZBA                                                             
ACM         
NH4Cl‐LP         
NH4Cl‐HP                                                             
RT 
NaNO3‐LP                                                             
m  NaNO3‐HP         
u
di NH4Cl‐LP                                                             
e
m NH4Cl‐HP                                                             
nitol  HS  NaNO3‐LP                                                             
n NaNO3‐HP                                                             
a
M NH4Cl‐LP         
NH4Cl‐HP         
CS 
NaNO3‐LP                                                             
NaNO3‐HP                                                              
NH4Cl‐LP                                                             
NH4Cl‐HP         
RT 
m  NaNO3‐LP         
diu NaNO3‐HP                                                              
me NH4Cl‐LP                                                             
d  NH4Cl‐HP         
ci HS 
c a NaNO3‐LP         
cini NaNO3‐HP                                                              
uc NH4Cl‐LP                                                             
S
NH4Cl‐HP                                                             
CS 
NaNO3‐LP         
NaNO3‐HP
Mar.Drugs2017,15,47 10of19
Mar. Drugs 2017, 15, 47 Table3.Cont. 10 of 18
None-Active in Primary Screening Active in Primary Screening

PE07-103 PE09-55 PE09-72 PE07-105 PE09-140 PE09-229
NH4Cl-LP
NH4Cl-HP
RT
NaNO3-LP
m NaNO3-HP
diu NH4Cl-LP
me HS NH4Cl-HP
h NaNO3-LP
c
Star NaNO3-HP
NH4Cl-LP
NH4Cl-HP
CS
NaNO3-LP
NaNO3-HP

Key
Test Strain Colour
E. coli1699
M. smegmatis
P. putida
B. cereus
S. epidermidis