Bioactivity Assessment of Indian Origin—Mangrove Actinobacteria against Candida albicans

18 Pages·2017·7.1 MB·English

marine drugs
Article
Bioactivity Assessment of Indian Origin—Mangrove
Actinobacteria against Candida albicans
J.G.S.PavanKumar1,AjithaGomathi1,VitorVasconcelos2,3,*andK.M.Gothandam1 ID
1 DepartmentofBiotechnology,SchoolofBiosciencesandTechnology,VITUniversity,Vellore632014,India;
[email protected](J.G.S.P.K.);[email protected](A.G.);[email protected](K.M.G.)
2 CIIMAR/CIMAR—InterdisciplinaryCentreofMarineandEnvironmentalResearch,UniversityofPorto,
TerminaldeCruzeirosdoPortodeLeixões,4450-208Matosinhos,Portugal
3 DepartmentofBiology,FacultyofSciences,UniversityofPorto,4069-007Porto,Portugal
* Correspondence:[email protected];Tel.:[phone]
Received:27December2017;Accepted:9February2018;Published:12February2018
Abstract: Actinobacteriaisfoundtohaveapotentmetabolicactivityagainstpathogens. Thepresent
studyrevealstheassessmentofpotentantifungalsecondarymetabolitesfromactinobacteriaisolated
fromIndianmarinemangrovesediments. Thesampleswerecollectedfromthecoastalregionsof
Muthupet,AndamanandtheNicobarIslands.Identificationwascarriedoutusing16SrRNAanalysis
andbiosyntheticgenes(PolyketidesynthasetypeI/IIandNon-ribosomalpeptidesynthase)were
screened. Actinobacteriawereassayedfortheirantifungalactivityagainst16clinicalCandidaalbicans
andthecompoundanalysiswasperformedusinggaschromatography-massspectrometryGC-MS.
The 31 actinobacterial strains were isolated and 16S rRNA gene sequencing revealed that this
ecosystemisrichonactinobacteria,withStreptomycesasthepredominantgenus. ThePCRbased
screeningofbiosyntheticgenesrevealedthepresenceofPKS-Iinsixstrains,PKS-IIinfourstrains
and NRPS in 11 strains. The isolated actinobacteria VITGAP240 and VITGAP241 (two isolates)
werefoundtohaveapotentialantifungalactivityagainstallthetestedC.albicans. GC-MSresults
revealedthattheactinobacterialcompoundswerebelongingtoheterocyclic,polyketidesandpeptides.
Overall,thestrainspossessawidespectrumofantifungalpropertieswhichaffordstheproductionof
significantbioactivemetabolitesaspotentialantibiotics.
Keywords: actinobacteria; antifungal; polyketide synthase; non ribosomal peptide synthase;
Candidaalbicans
1. Introduction
Bacterial cell factories and their wide applications in search of added value products such as
moleculartherapeuticsisboomingcurrently; theyhaveapromisingfutureasthesafestandmost
efficientmedicinalproductdiscovery[1]. Floraandfaunaaswellasmicroorganismsaregoodsources
toobtainawidevarietyofnaturalmedicinalproducts[2,3]. Actinobacteriaareverysignificantinthis
regard[4],takenintoaccounttheirintrinsicpharmacologicalimportance[5].Thesecondarymetabolites
producedbythesegrampositivebacteria,haveawidespectrumofactivitiessuchasantimicrobial,
antiviral,immune-modulatoryandasanticanceragents[6]. Peptidesandpolyketidesarenoticeable
andtheyareapotentialgroupofnaturalproducts,mainlysynthesizedbytwomajortypesofenzymes:
non-ribosomalpeptidesynthases(NRPS)andpolyketidesynthases(PKS)[7]. Microbesharbouring
these biosynthetic gene clusters are probable source of novel natural products. Polyketides have
extensiveapplicationsasantibioticsinthepharmaceuticalindustry[8]. WithseveralclassesofPKS
genesknown,typeIPKSencompassesmultidomainenzymesandtypeIIPKSiscomprisedofmany
enzymes[9]. NRPSaremodularenzymeswithmultipledomains,namelyacetylation,condensation,
andthioesterase[10]. Analysingthebiosyntheticgenesdepictsthepotentialofthemicrobetoproduce
Mar.Drugs 2018,16,60;doi:10.3390/md16020060 www.mdpi.com/journal/marinedrugs
Mar. Drugs 2018, 15, x 2 of 18
acetylation, condensation, and thioesterase [10]. Analysing the biosynthetic genes depicts the
Mar.Drugs 2018,16,60 2of18
potential of the microbe to produce a particular type of natural product [11]. Moreover, gene guided
metabolite studies also help in the avoidance of the redundant discovery of known compounds [12].
Tahpeasret igcuenlaers thyapveeo sfenrvateudr atol pbreo da ugcrte[a1t 1s]o.uMrcoer eoofv ienrt,egreesnteinggu nidaetudrmal eptarobdoluitcetss twuidthie sphalasromhaecloploingitchael
raevloevidaannccee [1o]f. the redundant discovery of known compounds [12]. These genes have served to be
agreAatpsaorut rfcreoomf itnrtaedreitsitoinnagl ntaetcuhrnailqpureosd, uscutcshw aitsh ipdheanrtmifiaccaotiloong icoarl rseclreeveanninceg [o1]f. pharmacologically
impoArtpanart tmfricormobtersa idsiotliaotneadl ftreocmhn viqaruieosu,s seuccohsyassteimdesn, tthifiec 1a6tiSo rnRoNrAs cprheyenloingegnoetficp ahnaarmlysaicso sloeegmicsa ltloy
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aarne evffiecryie nptrmometihsiondg tosopuoricnets oouft amcetidniocbinaacltleyriiam [p1o4r]t. aTnhteasceti nmoimcryocoertgeasn[1is3m].sM haanvger obveeene ciodseynsttiefmiesd aarse
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wgriitshe manytciifnun[1g5a]l. Ipnrofapcet,rtoiuesr.c Rurerfeenrrtirnegs etaor cfhewai mpreedvtioouidse rnetpifoyratcst i[n1o6,m17y]c, etthees wpritehseanntt iwfuonrgka lfpocroupseesrt ioens .
iRdeefnetrirfiynigngt omfiecwropbiraelv lieoaudssr efopro ratnsti[f1u6n,1g7a]l, mtheetapbreoslietne tpwroodrkucftoiocun saensdo ntoi dsecnreteifny itnhge misoiclraotebsia flolre athdes
pforresaenntcifeu onfg abliomsyetnatbhoeltiitce gpernoedsu. cTtihoen raensdultto rescvreeaelns tthhee ioscocluatrersenfocre tohfe sperceosnednacreyo mfbeitoasbyonltihtee tgicengeesn eins .
tThhee mreasujolrtirtyev eoaf lsththee ioscocluatrerse nacnedo fesneuconncidaaterys mtheetiarb oglrieteatg epnreosbianbitlhitey mianj obriitoysyonftthheesiizsoinlagt ensoavnedl
meneutanbcoialtietesst,h heeinrcger epartioprriotibzaibnigli ttyhei nisboiloasteysn tfhore snizaitnugranl opvreoldmucett adbioscliotvese,rhye. nceprioritizingtheisolates
fornaturalproductdiscovery.
2. Results
2. Results
2.1. Isolation and Identification of Mangrove Actinobacteria
2.1. IsolationandIdentificationofMangroveActinobacteria
From 10 marine sediment samples collected, approximately 31 actinobacterial isolates were
isolatFerdo mand1 0chmaararicnteeriszeeddi.m Feignutrsea m1 pillluesstcraotlelesc tthede ,isaoplaptriooxni manadte mlyo3r1phaocltoingoyb oafc ttehrei asltriasoinlas.t eTsabwlee r1e
liissotlsa tthede astnrdaicnhsa orabctateinriezded i.nF tihgius rsetu1dilylu asltorantge swthitehi sthoela stiaomnpalnindgm loorcpathioolno.g Fyroomft heeacsthr aoifn sth.eT asbalme1plliinstgs
sthiteess,t rsaeidnismoebnttasi nferodmin dthififsersetundt yspaolotsn gwwerieth ctohlelescatemdp tloin sgtulodcya ttihone .dFirvoemrsietayc hanodf tahbeusnadmapnlcien gofs itthese,
osergdaimniesnmtss.f Irno mthidsi sffteurdeyn,t 3s1p sottrsawinesr heacvoell beceteend istoolsattueddy frtohmed Mivuetrhsuitpyeatn (d6 iasboulantdesa)n acnedo ffrtohme oArngadnaimsmans .
aInndth tihses tNudicyo,b3a1rs tIrsalainnsdsh a(v25e biseoelnatiesso)l.a ItdedenftriofimcaMtiount houf ptehte( 6stirsaoilnast ews)aasn ddofnroem byA n16dSa mrDanNaAn dgetnhee
sNeiqcuoebnacriInsgla nudsisn(g2 5thiseo plartiems)e.rIsd 2e7nFti fiacnadt io14n9o2fRt.h Mesotrsat ionfs twhae sadcotinneobbyac1t6eSriarD isNoAlatgeedn ewseerqeu feonucnindg tuos fianlgl
uthnedperr imSetrresp2t7oFmayncdes1 4g9e2nRu.sM, owsthoiflet hfeeawc tinofo btahcete rsitaraisionlsa tewdewree roeff otuhned gtoenfaelrlau nRdheordSotrceopcctoums y(c1e)s,
Cgeonryuns,ebwahctielerinfeewae o(f1)t haensdt rAaicntsinwomeryeceotfatlehse (g1e).n eSriagnRifhicoadnotclyo,c ctuhse (s1t)r,aiCnos rylinsteebda citne rTinaebalee (21 )waenrde
iAdcetnintiofimedy caest anleosv(e1l) .spSiegcnieifis cbaanstelyd, tohne tshtrea binlassltis steimdiilnarTiatyb loef2 1w6SerrDeiNdAen tgifienede saesqnuoevneclessp. eIcni eosubra ssteuddoyn,
athmeobnlga staslli mmileadriitay uosfe1d6,S rSDtaNrcAh gceanseeisne qaugeanrc ems.eIdniao uprrsotvueddy ,taom boen gefaflelcmtivede iafours etdhe, Sitsaorclahticoans eoinf
aacgtainrombeadctiearpiar.o vedtobeeffectivefortheisolationofactinobacteria.

Figure 1. (a) Sampling of mangrove sediments; (b) Pure culture of Actinobacteria: l. VITGAP080, 2.
Figure1. (a)Samplingofmangrovesediments;(b)PurecultureofActinobacteria: l. VITGAP080,2.
VVIITTGGAAPP224400,, 3.3V. ITVGITAGPA24P12,441., VI4T. GVAIPT2G58A;P(c2)5G8;r am(c)s taGinrainmg osftSatirneipntgom yocfe sSaltbriedpotflomavyucses(V IaTlbGidAoPfl2a4v1u)s.
(VITGAP241).
Mar.Drugs 2018,16,60 3of18
Table1.IsolatesobtainedinthisstudyandtheiraccessionID.
Sequence-ID GenbankAccessionNumbers Organism SamplingLocation LatitudeandLongitude
VITGAP080 KY608546 Streptomycesrochei Muthupet,Thiruvarur,TamilNadu 10.39N,79.49E
VITGAP095 KY608547 Streptomycesvariabilis Muthupet,Thiruvarur,TamilNadu 10.39N,79.49E
VITGAP103 KY608548 Streptomycessp. Muthupet,Thiruvarur,TamilNadu 10.39N,79.49E
VITGAP104 KY608549 Streptomycessp. Muthupet,Thiruvarur,TamilNadu 10.39N,79.49E
VITGAP105 KY608550 Streptomycessp. Muthupet,Thiruvarur,TamilNadu 10.39N,79.49E
VITGAP229 KY608585 Streptomycessp. PortMout,AndamanandNicobarIslands 11.40N,92.41E
VITGAP231 KY608586 Streptomycessp. PortMout,AndamanandNicobarIslands 11.40N,92.41E
VITGAP232 KY608587 Streptomycessp. PortMout,AndamanandNicobarIslands 11.40N,92.41E
VITGAP233 KY608588 Streptomycesclavuligerus PortMout,AndamanandNicobarIslands 11.40N,92.41E
VITGAP235 KY608589 Streptomycessp. PortMout,AndamanandNicobarIslands 11.40N,92.41E
VITGAP238 KY608590 Streptomycessp. PortMout,AndamanandNicobarIslands 11.40N,92.41E
VITGAP240 KY608591 Streptomyceslividans Corbyn,AndamanandNicobarIslands 11.38N,92.44E
VITGAP241 KY608592 Streptomycesalbidoflavus Corbyn,AndamanandNicobarIslands 11.38N,92.44E
VITGAP242 KY608593 Streptomycessp. Corbyn,AndamanandNicobarIslands 11.38N,92.44E
VITGAP244 KY608594 Rhodococcussp. Corbyn,AndamanandNicobarIslands 11.38N,92.44E
VITGAP245 KY608595 Streptomycesviolascens Corbyn,AndamanandNicobarIslands 11.38N,92.44E
VITGAP246 KY608596 Corynebacterineaebacterium Corbyn,AndamanandNicobarIslands 11.38N,92.44E
VITGAP247 KY608597 Streptomycessp. Sippighat,AndamanandNicobarIslands 11.36N,92.41E
VITGAP248 KY608598 Streptomycessp. Sippighat,AndamanandNicobarIslands 11.36N,92.41E
VITGAP250 KY608599 Streptomycessp. Sippighat,AndamanandNicobarIslands 11.36N,92.41E
VITGAP253 KY608600 Streptomycessp. WandoorJetty,AndamanandNicobarIslands 11.35N,92.37E
VITGAP255 KY608601 Actinomycetalesbacterium Burmanalla,AndamanandNicobarIslands 11.33N,92.43E
VITGAP256 KY608602 Streptomycessp. Burmanalla,AndamanandNicobarIslands 11.33N,92.43E
VITGAP257 KY608603 Streptomycessp. Burmanalla,AndamanandNicobarIslands 11.33N,92.43E
VITGAP258 KY608604 Streptomycessp. Burmanalla,AndamanandNicobarIslands 11.33N,92.43E
VITGAP259 KY608605 Actinomycetalesbacterium Burmanalla,AndamanandNicobarIslands 11.33N,92.43E
VITGAP261 KY608606 Streptomycessp. Burmanalla,AndamanandNicobarIslands 11.33N,92.43E
VITGAP263 KY608607 Streptomyceschumphonensis Burmanalla,AndamanandNicobarIslands 11.33N,92.43E
VITGAP270 KY608608 Streptomycessp. MundaPahad,AndamanandNicobarIslands 11.29N,92.42E
VITGAP271 KY608609 Streptomycessp. Kalapahad,AndamanandNicobarIslands 11.36N,92.40E
Mar.Drugs 2018,16,60 4of18
Table2.Novelactinobacterialstrainsobtainedinthisstudy;16SrDNAgenesequencesimilarities.
IsolateNo. GenbankAccessionNo.oftheIsolates SamplingLocationoftheIsolates ClosestOrganism GenebankNo. SimilarityPercentage
VITGAP080 KY608546 Muthupet,Thiruvarur,TamilNadu Streptomycesrocheistrain KP823705 96%
VITGAP095 KY608547 Muthupet,Thiruvarur,TamilNadu Streptomycesvariabilis KU981101 95%
VITGAP103 KY608548 Muthupet,Thiruvarur,TamilNadu Streptomycessp. CP013142 95%
VITGAP105 KY608550 Muthupet,Thiruvarur,TamilNadu Streptomycessp. JQ009379 96%
VITGAP235 KY608589 PortMout,AndamanandNicobarIslands Streptomycessp. KX279534 83%
VITGAP240 KY608591 Corbyn,AndamanandNicobarIslands Streptomycesviolascens KU973980 91%
VITGAP253 KY608600 WandoorJetty,AndamanandNicobarIslands Streptomycessp. KU884356 94%
VITGAP255 KY608601 Burmanalla,AndamanandNicobarIslands Actinomycetalesbacterium EU368818 88%
VITGAP257 KY608603 Burmanalla,AndamanandNicobarIslands Streptomycessp. JF736620 97%
VITGAP258 KY608604 Burmanalla,AndamanandNicobarIslands Streptomycessp. KR817750 87%
VITGAP261 KY608606 Burmanalla,AndamanandNicobarIslands Streptomycessp. KX928494 92%
VITGAP263 KY608607 Burmanalla,AndamanandNicobarIslands Streptomyceschumphonensis NR_126175 94%
VITGAP271 KY608609 Kalapahad,AndamanandNicobarIslands Streptomycessp. KT588654 92%
Mar.Drugs 2018,16,60 5of18
2.2. GeneticScreening
PCRscreeningoftheactinobacterialstrainsrevealedthepresenceofPKStypeI,PKStypeIIand
NRPSbiosyntheticgenes. Ampliconsofdesiredsize[PKStypeI(bp-1.2kb),PKStypeII(bp-500bp),
NRPS(bp-700bp)]showsthepresenceofthebiosyntheticgene. Table3liststhebiosyntheticgenetic
potentialoftheactinobacterialstrains. Amongthe31strainsobtained,only14strainsshowedthe
presenceoftypeIPKSgeneand6ofthestrainsshowedthepresenceoftypeIIPKSgene. Interestingly,
allstrainsharboringtypeIIPKSwerealsofoundtoharbortypeIPKS.
ThebiosyntheticpotentialoftheactinobacterialstrainsrevealsthattypeIPKSaremoreabundant
thanthatoftypeII.TypeIPKSwasfoundtobeharboredbyabout20.8%oftheactinobacterialstrains,
whiletypeIIPKSwasfoundonlywith8.3%. Remarkably,approximately34.7%ofstrainsshowed
thepresenceofNRPSgene. AlmostallthestrainsharboringthegenesbelongtoStreptomycesgenus,
whileoneofthestrainsrevealingthepresenceoftypeIPKSisRhodococcus. Interestingly,strains
belongingtoothergenera,namelyCorynebacterineae,didnotharboranyofthestudiedgenes.
Table3.Biosyntheticpotentialoftheactinobacterialisolates.
Strains PKS(TypeI) PKS(TypeII) NRPS
VITGAP080 + + +
VITGAP095 +
VITGAP105 +
VITGAP240 + +
VITGAP241 + + +
VITGAP242 + + +
VITGAP244 +
VITGAP248 +
VITGAP250 +
VITGAP253 +
VITGAP255 +
VITGAP257 +
VITGAP258 + +
‘+’indicatesthepresenceofbiosyntheticgene.
2.3. PhylogeneticAnalysisoftheActinobacteriStrains
Phylogenetic analysis was performed by constructing the neighbor joining phylogenetic tree
of all the strains to analyze the evolutionary relationship, and the distance was calculated by
maximum-parsimonymethodusingMEGAsoftware(Figure2). Basedonthequerycoverageand
the percentage of identity, the top aligned sequences were retrieved (Table 4). Significance of the
branchorderwasdeterminedbybootstrapanalysisof1000replicates,seeFigures3–7(Thesefigures
depicttheevolutionaryrelationshipsoftaxaofthequerysequencesNRPS(VITGAP241),TypeIPKS
(VITGAP-240,VITGAP-241)TypeIIPKS(VITGAP-240,VITGAP-241).Theoptimaltreewiththesumof
branchlengthsforeachsequenceare1.31645770,1.25726471,1.52648453,4.16276907,and16.20030581
respectively. Thepercentageofreplicatetreesinwhichtheassociatedtaxaclusteredtogetherinthe
bootstraptest(1000replicates)areshownnexttothebranches. Thetreeisdrawntoscale,withbranch
lengthsinthesameunitsasthoseoftheevolutionarydistancesusedtoinferthephylogenetictree.
Theanalysisinvolved16,16,14,11and7nucleotidesequences. Codonpositionsincludedwere1st+
2nd+3rd+Noncoding).
Mar.Drugs 2018,16,60 6of18
MMara.r .D Druruggs s2 2001188, ,1 155, ,x x 66 o of f1 188
Mar. Drugs 2018, 15, x 6 of 18

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aanncceesstotorr. .A Acctitninoommyycceteatalelse sb baacctetreiruiumm i sis f ofouunndd b bee t hthee n neeww d diviveerrssee s sppeeccieiess a ammoonngg t hthee e enntitriree g grroouupp o of ft hthee
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SStrterpeptotommyyccese sf afammiliyly w witihth m mininimimuumm b booootststrtraapp v vaaluluee. .S Strterpeptotommyyccese ss spp. .i nin t wtwoo c claladdeess a arere c coommmmoonn a alolonngg
fSatmreipltyomwyitchesm faimniimlyu wmitbho motisntirmapumva bluoeo.tsSttrraeppt vomalyucee.s Sstpre.pitnomtwycoesc lsapd. eins atwreoc colmadmeso anrae lcoonmgmwoitnh atlhoenigr
wwitihth t htheeirir c clolosseelyly r reelalatetedd s sppeeccieiess, ,S Strterpeptotommyyccese sv vioiolalascscenens ss strtarainin. .
cwloitshe ltyhreeirla ctleodseslpye rceielast,eSdtr seppteocmieysc, eSstvreiopltaosmceyncsess tvriaoilnas.cens strain.

FFFiiFigggiuugururreeer e3 33 ..3. DD.D Deeennendnddrdrrooorggogrrgraaarmmamm o oo ffof N fNN NRRRPRPPSPSS S( ((V VV(IVIITTTIGTGGGAAAAPPP2P2244241141))1.).. ) .

FFigiguurere 4 4. .D Deennddroroggraramm o of fT Tyyppee I IP PKKSS ( V(VITITGGAAPP224400).) .
FFiigguurree 44.. DDeennddrrooggrraamm ooff TTyyppee II PPKKSS ((VVIITTGGAAPP224400))..
Mar.Drugs 2018,16,60 7of18
Mar. Drugs 2018, 15, x 7 of 18
Mar. Drugs 2018, 15, x 7 of 18

Figure 5. Dendrogram of Type I PKS (VITGAP241).
FFiigguurree 55.. DDeennddrrooggrraamm ooff TTyyppee II PPKKSS ((VVIITTGGAAPP224411))..

FFiigguurree 66.. DDeennddrrooggrraamm ooff TTyyppeeI IIIP PKKSS( V(VITITGGAAPP224400).).
Figure 6. Dendrogram of Type II PKS (VITGAP240).
TTaabblele4 4..Q Quueerryyc coovveerraaggeea annddp peerrcceenntataggeeo offi dideenntittiytyo offt hthees seeqquueenncceess..
Table 4. Query coverage and percentage of identity of the sequences.
Sequence Query Coverage % of Identity No of Hits
SeqSueenqcueence QueQryuCeorvye Craogveerage%o%f Iodfe Indtietnytity NNoo ooff HHiittss
NRPS_VITGAP241 99% >83% 15
NRPS_VITGAP241 99% >83% 15
NRPS_VITGAP241 99% >83% 15
Type I PKS VITGAP-240 99% >89% 15
TypTeyIpPeK IS PVKIST GVAITPG-2A40P-240 99% 99% >89>%89% 1155
TypTTeyyIppPeeK IIS PPVKKISTS G VVAIITTPG-G2A4A1PP--224411 80% 8800%% >79>>%7799%% 111333
TypTeyIpIeP KIIS PVKITSG VAIPT-G24A0P-240 56% 56% >70>%70% 1100
Type II PKS VITGAP-240 56% >70% 10
TypTeyIpIeP KIIS PVKITSG VAIPT-G24A1P-241 68% 68% >67>%67% 66
Type II PKS VITGAP-241 68% >67% 6
Mar. Drugs 2018, 15, x 8 of 18
Mar. Drugs 2018, 15, x 8 of 18
Mar.Drugs 2018,16,60 8of18

Figure 7. Dendrogram of Type II PKS (VITGAP241).
FFiigguurree 77.. DDeennddrrooggrraamm ooff TTyyppee IIII PPKKSS ((VVIITTGGAAPP224411))..
2.4. Antifungal Activity Prospect Evaluations
22..44.. AAnnttiiffuunnggaall AAccttiivviittyy PPrroossppeecctt EEvvaalluuaattiioonnss
Actinobacterial strains harboring biosynthetic genes were tested for their antifungal activity
againAAscctt ti1inn6oo bcblaaicnctitecerarialia lilss otsrltaariatneisns sho afhr abCro.b raoinlrbgiincbagin osbs iyoonbsttyhaneintthiecedgt iefcnr ogemsenw edesir aewgtneeosrtese tditce fsocterendtht eefroisrr aitnnh teTifiuirr nuagcnahtliifrauacpntigpvaiatlly liaa, cgTtaiavimnitsyitl
1aN6gaacdilniuns,it c Ia1nl6di sicoali.ln aAtiecmasloo ifnsCogl. atathlebesi c 1oan4f sCbo.i boat-lapbiionctaeendnstf iroaoblm tastidrnaieaidgn nsf,or osotmnicl ycd ei2na gtoenfro stsihtniecmT cir eu(nVcthIeTirrsGa piAnpP aT2lil4ri,0u Tcaahnmirdai lpVNpIaaTdlGlui,A ,TIPna2dm4i1ail).
ANexmahdoiubni,g teItndhd eai1an4. ebAfifmoe-cpotinovtgee n tathinaetl i1-sct4ar anbidnioisd-,paooln tlaeycnt2tiivoaiflt ytsh tearamgiani(snV,s ItTo nGthlAye P2t2e 4sot0fe adtnh pdeamVth IT(oVGgIeATnGPs2.A 4IP1n)2te4erx0eh saitbninidtge dlVya,I TnthGeeAfsfeePc 2tti4wv1eo)
aebnxiothia-icbcaittniveddei d asantlr aaeciftnfives cittpyivoaesg saaeinsnstsiet-dctha enbdtoeitsdhtea dlt yappcaetti hvoIi tgyae nnadsg. aIIniInt esPrte KtshtSie n ggtleeysn,tetehsde, spheaetwtnhcooegb eiaontatsrc.at iIcvnteitnesgrtre asfitnuinsrtgphloyesr,s etahstseteseden bttiowotnho.
tbVyipIoTeaGcItAiavnPed2 4IsI1tr PaaKilnsSos gsephnooeswss,eehsdse entdhce e bapottrtrehas cettniyncpgee fo ufIr ttahhneerd Na tRItIeP nPSt iKogSne n.geV.eI nTVeGIsTA, GPhA2e4Pn12ce4a 1ls aoetxtsrhhaiocbtwiitneegdd tmfhueortrphere eprs reonantcteoeunontfictohened.
NVacIRtTiPvGSiAtgyPe (n21e48.1 Vm aIlmTsGo d AsihaPmo2w4e1teeedrx )ht htihbeia tpnedr VemsIeTonGrceAep Poro2f 4nt0ho ue(1 nN3c eRmdPmaSc tgdiveiaintmye.e( 1Vte8IrTm).G mFAigdPui2ar4me1 e 8et eixlrhl)uitbshtiarteandteV smI TtohGreAe b Ppi2or4oa0cnto(i1vu3intmyce modf
dathciateim vsiettrtyae ri(n)1.s8F V imgIuTmrGe dA8iPailm2lu4es0tt eraarn)t edtsh VtahInTe VGbiIAoTaPGc2tAi4v1Pi.t2 y4 0o f(t1h3e mstmra idnisaVmITetGerA).P F2i4g0uarned 8 VilIlTuGstAraPt2e4s 1t.he bioactivity of
the stEErfaffifiinccisiee VnntItT aaGnntAtiiffPuu2nn4gg0aa all naacdctt iVivvIiiTttyyG wwAaaPss2 e4ex1xh.h ii bbiitteedd bbyy tthhee aaccttiinnoobbaacctteerriiaall ssttrraaiinnss.. AAss ooff oouurr kknnoowwlleeddggee,,
tthhiiss iiEss fttfhhiceei efifnirrsts tta rnreetpipfouorrnttg aaannla aallycyztziivinnigtgy t thwheea asan nettxiifhfuuinbngigtaealdl p pbooytte etnnhtteiia aallicitttyyin oooffb aaaccctttiiennrooiabblaa csctttreearriiinaas oo. ffA IInsn ddoiifaa nonu oorr rikiggniinonw aaglgeaadiingnsest,t
CtChaainsnd diisidd taah aeal lbfbiiirccsaatnn rsse (p(ccollirinnt iiaccanalal lppyaazttihnhogog gteehnness a))..n tTTifhhuoonuugggahhl pmmoataennnyyt ioaolffi tttyhh eeo fss attrrcaatiiinnnoss bppaoocsstessreeissass iionnfgg I ntthhdeeia gnge eonnreeigss iwnw eaergreea innnoosttt
fCfuuannnccdttiiidooann aaallllblyyic aaancctstii vv(ceel,,i ntthhiceea tltw wpooa tsshttorraagiiennnsss e)e.xx hThihibboiitutiignnhgg bbmiiooaaanccytt iivovifitt yyt,h, heh aasrrtbbroaoiuunrrsee ddp ooosnnseees oosfifn tthgh eet hbbeiio ogssyeynnntethshe ewttiicec rggeee nnneoesst
sfsuuunppcpptoioorrtntiiannlggly oo uaucrrt hihvyyepp, ootththheee stswiiss.o. strains exhibiting bioactivity, harboured one of the biosynthetic genes
supporting our hypothesis.

Figure 8. VITGAP240 & VITGAP241 showing significant activity against: (a) Clinical isolate 1 of
Figure 8. VITGAP240 & VITGAP241 showing significant activity against: (a) Clinical isolate 1 of
FCiagnudried a8 a. lbVicITanGsA (bP)2 C40li n&ic aVlI iTsGolAatPe2 24 1o fs Chaonwdiindag aslibgicnainfisc.a nt activity against: (a) Clinical isolate 1 of
Candidaalbicans(b)Clinicalisolate2ofCandidaalbicans.
Candida albicans (b) Clinical isolate 2 of Candida albicans.
Mar.Drugs 2018,16,60 9of18
2.5. AntibioticSensitivityAssessmentofActinobacteriaStrains
Toassesstheantibioticsensitivity, thestrainswerescreenedagainstthefollowingantibiotics:
piperacillin,co-trimoxazole,ofloxacin,amikacin,erthyromicin,cefuroxime,tobramycin,ampicillin,
tetracycline, ceftriazone, polmycin, nitrofurantin, rifampicin, clindamycin, netillin, ceftrazidime,
novobiocin, aztreonam, cephotaxine, chloramphenicol, streptomycin, penicillin, methicillin,
jancomycin,gentamycin,andvancomycin(Table5). BasedonPCRscreening(Table3),thepotential
strainswhichharboredPKSandNRPSgeneandafewstrainswhichdidnotshowthepresenceofthe
geneswerealsochosenrandomlyfortheassaytocomparethestrains.
All the tested strains displayed significant sensitivity against most of the antibiotics used.
TheHighestsensitivitywasobtainedagainstnetillin,novobiocin,cephotaxine(60%each),andlowest
sensitivity was obtained against piperacillin, erthyromicin, nitrofurantin, chloramphenicol and
jancomycin (6.6%). The strains exhibited resistance against the following antibiotics: aztreonam,
streptomycin, penicillin, and methicillin. The strain VITGAP240 exhibited resistance to the
following antibiotics: piperacillin, amikacin, erthyromicin, cefuroxime, ampicillin, polmycin,
nitrofurantin, rifampicin, clindamycin, ceftrazidime, aztreonam, chloramphenicol, streptomycin,
penicillin, methicillin, jancomycin and gentamycin. The strain VITGAP241 exhibited resistance
topiperacillin, ofloxacin, erthyromicin, cefuroxime, tobramycin, ampicillin, ceftriazone, polmycin,
nitrofurantin,clindamycin,ceftrazidime,aztreonam,streptomycin,penicillinandmethicillin. Hence,
the strains VITGAP240 and VITGAP241 proved to be promising and pharmacological important
targetsforthefuture,especiallyforantifungalbasedsecondarymetaboliteprofilingstudies.
Table5.Antibiogramprofileofthestrains(R-resistant,S-sensitive).
Strainswithoutthe
Antibiotics StrainswiththeBiosyntheticGenes
BiosyntheticGenes
80 95 240 241 242 105 248 250 253 255 258 103 105 261 263
Piperacillin R R R R S R R R R R R R R R R
Co-Trimoxazole R S S S S S S S R R S R R R R
Ofloxacin S S S R S R S R R S R R S S R
Amikacin S S R S S R S R R R R R R S R
Erthyromicin R S R R R R R R R R R R R R R
Cefuroxime S S R R R R S R R R R R R R R
Tobramycin R S S R R R R R R R R R R R R
Ampicillin R S R R R S S R R R S R R R R
Tetracycline R S S S S R R S R R S R R S R
Ceftriazone S S S R S R S R R R S R R R S
Polmycin S S R R R R S R R R R R R R R
Nitrofurantin R R R S R R R R R R R R R R R
Rifampicin S S R S S R S R R R R R R S R
Clindamycin R R R R S R R R R R R R R R R
Netillin S S S S S R S R R R S R S S R
Ceftrazidime R S R R S R R R R R R R R R R
Novobiocin S S S S S R S R R S S R S R R
Aztreonam R R R R R R R R R R R R R R R
Cephotaxine S S S S S R S S R R S R R S R
Chloramphenicol R R R S R R R R R R R R R R R
Streptomycin R R R R R R R R R R R R R R R
Penicillin R R R R R R R R R R R R R R R
Methicillin R R R R R R R R R R R R R R R
Jancomycin R R R S R R R R R R R R R R R
Gentamycin S S R S R R S R R R S R S S R
Vancomycin R R S S R R R R R R R R R R R
2.6. GCMSAnalysisandScreeningofBioactiveCompounds
ThecrudeextractofsecondarymetaboliteswasanalyzedwiththeGCMStechniquetoidentify
thepossiblebioactivecompounds’availability. Figure9representativelyillustratesasample’sGCMS.
Figures10–12arethesegregatedcompounds(Heterocyclic,Peptides,andPolyketides). Maximumof
Mar. Drugs 2018, 15, x 10 of 18
2.6. GCMS Analysis and Screening of Bioactive Compounds
The crude extract of secondary metabolites was analyzed with the GCMS technique to identify
tMhaer .Dprousgssib20le18 ,b1i6o,a60ctive compounds’ availability. Figure 9 representatively illustrates a sam10polfe1’s8
GCMS. Figures 10–12 are the segregated compounds (Heterocyclic, Peptides, and Polyketides).
Maximum of these compounds were found as unidentified or unpublished. Identified compounds
thesecompoundswerefoundasunidentifiedorunpublished. Identifiedcompoundswerefoundwith
were found with structural clarity and mostly equipped with functionally important
structuralclarityandmostlyequippedwithfunctionallyimportantscaffolds/groups. Theconfirmed
scaffolds/groups. The confirmed nature allowed us to screen them as the most suitable medications
natureallowedustoscreenthemasthemostsuitablemedicationsagainstfungalinfections.
against fungal infections.

Figure 9. GCMS result of Actinobacteria secondary metabolite from marine mangrove sediment.
Figure9.GCMSresultofActinobacteriasecondarymetabolitefrommarinemangrovesediment.
2.7. Pharmacological Property Predictions of Screened Compounds
2.7. PharmacologicalPropertyPredictionsofScreenedCompounds
In order to find the most suitable antifungal medications, the compounds that were categorized
Inordertofindthemostsuitableantifungalmedications,thecompoundsthatwerecategorized
from GCMS studies were predicted for their possible biological activity potentials through
from GCMS studies were predicted for their possible biological activity potentials through
Molinspiration and PASS (Figure 13). Among the segregated Heterocyclic (Cpd1–Cpd8) (Figure 10),
MolinspirationandPASS(Figure13). AmongthesegregatedHeterocyclic(Cpd1–Cpd8)(Figure10),
peptides (Cpd9–Cpd12) (Figure 11), and polyketides (Cpd13–Cpd16) (Figure 12), a significant
peptides(Cpd9–Cpd12)(Figure11),andpolyketides(Cpd13–Cpd16)(Figure12),asignificantprotease
protease inhibition ability was found especially for peptide and polyketides. Particularly, the
inhibitionabilitywasfoundespeciallyforpeptideandpolyketides. Particularly,theMolinspiration
Molinspiration predictions revealed the strong Protease inhibitory potentials of all peptides (Cpd9–
predictions revealed the strong Protease inhibitory potentials of all peptides (Cpd9–Cpd12) and
polyketides(Cpd13–Cpd16)withapositivescorerangeof0.3to1. Apartfromthis,PASSprediction
possibleactivityscoreof0.922(asNADPHperoxidaseinhibitor)wasfoundtohavebeenestablishedto
theHeterocycliccompound;Cpd1indicateditspotentialantifungalmedicinalvalue.Theopportunistic