Exploring the Diversity and Antimicrobial Potential of Marine Actinobacteria from the Comau Fjord

16 Pages·2016·3.45 MB·English

by  Beatriz Cámara

ORIGINALRESEARCH
published:19July2016
doi:10.3389/fmicb.2016.01135
Exploring the Diversity and
Antimicrobial Potential of Marine
Actinobacteria from the Comau Fjord
in Northern Patagonia, Chile
AgustinaUndabarrena1,FabrizioBeltrametti2,FernandaP.Claverías1,MyriamGonzález1,
EdwardR.B.Moore3,4,MichaelSeeger1andBeatrizCámara1*
1LaboratoriodeMicrobiologíaMolecularyBiotecnologíaAmbiental,DepartamentodeQuímica&CentrodeBiotecnología
DanielAlkalayLowitt,UniversidadTécnicaFedericoSantaMaría,Valparaíso,Chile,2ActygeaS.r.l.,Gerenzano,Italy,3Culture
CollectionUniversityofGothenburg(CCUG),SahlgrenskaAcademy,UniversityofGothenburg,Gothenburg,Sweden,
4DepartmentofInfectiousDiseases,SahlgrenskaAcademy,UniversityofGothenburg,Gothenburg,Sweden
Bioprospectingnaturalproductsinmarinebacteriafromfjordenvironmentsareattractive
Editedby:
Learn-HanLee, due to their unique geographical features. Although, Actinobacteria are well known
MonashUniversityMalaysiaCampus,
for producing a myriad of bioactive compounds, investigations regarding fjord-derived
Malaysia
marine Actinobacteria are scarce. In this study, the diversity and biotechnological
Reviewedby:
AtteVonWright, potential of Actinobacteria isolated from marine sediments within the Comau
UniversityofEasternFinland,Finland fjord, in Northern Chilean Patagonia, were assessed by culture-based approaches.
PolpassArulJose,
The 16S rRNA gene sequences revealed that members phylogenetically related
CentralSaltandMarineChemicals
ResearchInstitute,India to the Micrococcaceae, Dermabacteraceae, Brevibacteriaceae, Corynebacteriaceae,
*Correspondence: Microbacteriaceae, Dietziaceae, Nocardiaceae, and Streptomycetaceae families were
BeatrizCámara
present at the Comau fjord. A high diversity of cultivable Actinobacteria (10 genera)
[email protected]
was retrieved by using only five different isolation media. Four isolates belonging to
Specialtysection: Arthrobacter,Brevibacterium,CorynebacteriumandKocuriagenerashowed16SrRNA
Thisarticlewassubmittedto geneidentity<98.7%suggestingthattheyarenovelspecies.Physiologicalfeaturessuch
Antimicrobials,Resistanceand
Chemotherapy, assalttolerance,artificialseawaterrequirement,growthtemperature,pigmentationand
asectionofthejournal antimicrobial activity were evaluated. Arthrobacter, Brachybacterium, Curtobacterium,
FrontiersinMicrobiology
Rhodococcus, and Streptomyces isolates showed strong inhibition against both
Received:23May2016
Gram-negativePseudomonasaeruginosa,EscherichiacoliandSalmonellaentericaand
Accepted:07July2016
Published:19July2016 Gram-positive Staphylococcus aureus, Listeria monocytogenes. Antimicrobial activities
Citation: in Brachybacterium, Curtobacterium, and Rhodococcus have been scarcely reported,
UndabarrenaA,BeltramettiF,
suggesting that non-mycelial strains are a suitable source of bioactive compounds. In
ClaveríasFP,GonzálezM,MooreERB,
SeegerMandCámaraB(2016) addition, all strains bear at least one of the biosynthetic genes coding for NRPS (91%),
ExploringtheDiversityand PKSI(18%),andPKSII(73%).OurresultsindicatethattheComaufjordisapromising
AntimicrobialPotentialofMarine
sourceofnovelActinobacteriawithbiotechnologicalpotentialforproducingbiologically
ActinobacteriafromtheComauFjord
inNorthernPatagonia,Chile. activecompounds.
Front.Microbiol.7:1135.
doi:10.3389/fmicb.2016.01135 Keywords:cultivableactinobacteria,antimicrobialactivity,Comaufjord,marinesediments,NorthernPatagonia
FrontiersinMicrobiology|www.frontiersin.org 1 July2016|Volume7|Article1135
Undabarrenaetal. MarineActinobacteriafromaChileanPatagonianFjord
INTRODUCTION Magarveyetal.,2004;Jensenetal.,2005;Bredholdtetal.,2007;
Gontang et al., 2007; León et al., 2007; Maldonado et al., 2008;
The increased prevalence of multi-drug resistance pathogens Duncan et al., 2014; Yuan et al., 2014) and also in deep sea
along with the rapid development of cross resistances with sediments (Colquhoun et al., 1998; Pathom-Aree et al., 2006).
new antibiotics is the driving force in the identification and Marineactinobacteriahavebeendescribedasanemergingsource
production of novel therapeutic agents (Livermore, 2009). for novel bioactive molecules (Lam, 2006; Joint et al., 2010;
All classes of antibiotics have seen emergence of resistance SubramaniandAalbersberg,2012;Zotchev,2012).Themajority
compromising their use; hence there is an urgent need for of these secondary metabolites are produced by polyketide
new bioactive compounds (Genilloud, 2014). The traditional synthases(PKS)andnon-ribosomalpeptidesynthetases(NRPS)
approach consisting of isolation and cultivation of new metabolicpathways(Salomonetal.,2004).Notably,itisreported
microorganisms of underexplored habitats is still rewarding that actinobacteria have a higher number of these biosynthetic
(Axenov-Gribanov et al., 2016), and has brought to the genes(Donadioetal.,2007).
identification,productionandcommercializationofmostofthe TheextensivecoastofChileisapromisingbiometoexplore
antibiotics (Newman and Cragg, 2012). Despite the chemically marine actinobacterial communities, and in this context, the
syntheticefforts,naturalenvironmentsarestillthemainsource bioprospecting of sediments of a marine protected area, the
forthediscoveryofnovelantibiotics(FenicalandJensen,2006; Comaufjord,intheChileanNorthernPatagoniawasproposed.
BullandStach,2007).Although,thediversityoflifeinterrestrial TheComaufjordisapristineareauniquebyitsgeologicalnature.
environmentsiswellreported,thehighestbiodiversityisinthe It is comparatively smaller than other fjords in Chile, and also
world’soceans(DoniaandHamann,2003).Oceansarestrongly one of the deepest (Ugalde et al., 2013); characterized by steep
complexhabitatsintermsofpressure,salinityandtemperature slopes, with surrounding mountains that have a height of up
variations(Fenical,1993),thereforemarinemicroorganismshave to 2000m with a dense extratropical rainforest covering from
to develop physiological traits including chemically complex the sea to the top (Lagger et al., 2009). The aim of this study
biosynthesizedmetabolitestoensuretheirsurvivalinthishighly wastoisolatemarineactinobacteriafromthisuniqueecosystem.
dynamic habitat. Research has taken advantage from these The cultivable diversity of actinobacterial strains along with
unique molecules to discover novel bioactive compounds with theirenvironmentaladaptationtraitswasinvestigated,andtheir
antibacterial,antifungaland/orantitumorproperties,andapply abilitytoproduceantibacterialactivityagainstmodelstrainswas
themincurrentclinicalchallenges(GulderandMoore,2010). assessed.
Inthisscenario,bacteriafromthephylumActinobacteriaarea
prominentsourceofbiologicallyactivenaturalcompounds,since
MATERIALS AND METHODS
theyarewellknownfortheircapacitytobiosynthesizeversatile
secondarymetabolites(KatzandBaltz,2016).Actinobacteriaare Environmental Samples
oneofthemajorphylaofthedomainBacteria(Goodfellowand SamplingwasperformedintheMarineProtectedAreaofHuinay
Fiedler, 2010). It encompasses high GC-content Gram-positive in January 2013, located in the Commune of Hualaihué, in the
bacteriathatincludes17orders(GaoandGupta,2005;Senetal., Los Lagos Region, Chile. Samples were collected from marine
2014). Surprisingly, the class Actinobacteria contains both the sediments within the Comau Fjord in the Northern Patagonia.
mostdeadlybacterialpathogen(i.e.,Mycobacteriumgenus)and FourdifferentcoastallocationsweresampledinfrontofLilihuapi
the microorganisms that are the most important for antibiotic Island (42◦20, 634′S; 72◦27, 429′W), Tambor Waterfall (42◦24,
production (i.e., Streptomyces genus) (Doroghazi and Metcalf, 161′S; 72◦25, 235′W), Punta Llonco (42◦22, 32′S; 72◦25, 4′W),
2013). Streptomyces are responsible for two-thirds of all known and in front of Lloncochaigua River mouth (42◦22, 37′S;
antibiotics. In addition, several other important biologically- 72◦27,25′W)(Figure1).Underwatersampleswerecollectedby
active compounds have been found, including antitumoral, HuinayScientificFieldStationscubadivers,dispensingsamples
antifungal, herbicidal, and antiparasitic compounds (Bérdy, directlyfrommarinesedimentsintosterile50mLtubes.Marine
2005).DuetotheextensivesamplingofsoilStreptomyces,therate sediments were taken from subtidal zones at different depths,
of discovery of novel metabolites is decreasing (Fenical, 1993), ranging from 0.25 to 26.2 m. Salinity was measured at each
which is the reason why bioprospecting efforts are currently sampling site, and ranged from 5 µg L−1 in the coast in front
beingdevelopedinmarineunderexploredecosystems. of Lloncochaigua River mouth, where there is a meaningful
Marineenvironmentsareanestablishedecologicalnichefor input of fresh water, to 31 µg L−1 in the coast of Lilihuapi
actinobacteria(Dasetal.,2006;WardandBora,2006).Cultivable Island,locatedfurtherawayfromcontinentalland.Sampleswere
actinobacteria from marine habitats have been characterized maintainedoniceuntiltransportedtothelaboratory,wherethey
frommangroveforests(Hongetal.,2009;Baskaranetal.,2011; werestoredat4◦C.
Leeetal.,2014a,b;Seretal.,2015,2016),marinesponges(Kim
et al., 2005; Montalvo et al., 2005; Zhang et al., 2006; Jiang Isolation of Actinobacteria
etal.,2007;Sunetal.,2015),corals(Hodgesetal.,2012;Kuang Samples were both plated directly or serially diluted (10−4
et al., 2015; Mahmoud and Kalendar, 2016; Pham et al., 2016), and 10−6) before plating on selective media for the isolation
sea cucumbers (Kurahashi et al., 2010), pufferfishes (Wu et al., of actinobacteria. Five selective media were used as previously
2005), and seaweed (Lee et al., 2008). Notably, actinobacteria reported (Claverías et al., 2015): M1 Agar (Mincer et al.,
are predominant in marine sediments (Mincer et al., 2002; 2002), ISP2 and NaST21Cx Agar (Magarvey et al., 2004),
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Undabarrenaetal. MarineActinobacteriafromaChileanPatagonianFjord
FIGURE1|GeographyofsamplingsitesforactinobacteriaisolationfromtheComaufjordinNorthernPatagonia,Chile.Mapofsamplinglocationswithin
theComaufjord(LosLagosRegion).Numbersindicatethesiteswheremarinesedimentswerecollectedatthecoastcloseto:LilihuapiIsland(1),PuntaLlonco(2),
LloncochaiguaRivermouth(3),andTamborWaterfall(4).BlackdotindicateslocationoftheHuinayScientificFieldStation.
R2A Agar (Difco), and Marine Agar (MA) 2216 (Difco). All and subsequently revealed with SYBR Green staining (E-gel,
media were amended with nalidixic acid (25 µg mL−1), as an Invitrogen).
inhibitorofprimarilyfast-growingGram-negativebacteria,and Positive isolates were selected for 16S rRNA gene
cycloheximide(100µgmL−1)forfungiinhibition[28].Allmedia amplification, using universal primers 27F and 1492R (Lane,
withtheexceptionofMarineAgar,werepreparedwithartificial 1991). The reaction mix (50 µL) contained 1 µL of genomic
sea water (ASW) (Kester et al., 1967). The agar media cultures DNA, 25 µL of GoTaq Green Master Mix (Promega) and
wereincubatedat30◦Cuntilvisiblecolonieswereobserved,up 0.2 µM of each primer. The reaction started with an initial
to1–2months.Forisolationpurposes,colonieswereindividually DNA denaturation at 95◦C for 5min, followed by 30 cycles
streaked out onto Tryptic Soy Agar medium (TSA) prepared of denaturation at 95◦C for 1min, primer-annealing at
withASW(TSA-ASW)andeventuallytransferredonnewplates 55◦C for 1min and primer-extension at 72◦C for 1.5min,
untilpure cultureswereobtained.Isolatedbacteriawerestored with a final extension at 72◦C for 10min. PCR products
at−20and−80◦C,in20%glycerol,TSBmediumandASWfor were sent to Macrogen Inc. (Seoul, Korea) for purification and
maintenance. sequencingusingtheconserveduniversalprimer800R.Retrieved
sequencesweremanuallyeditedandBLASTnucleotideanalyses
Detection and Identification of were performed with the National Center for Biotechnology
Actinobacteria Information server (NCBI) and actinobacteria were initially
identifieduptothegenuslevel.
APCR-assaywasconductedasascreeningmethodfordetecting
actinobacterialstrainsamongtheisolateswithprimerstargeting
the V3–V5 regions of the 16S rRNA gene of actinobacteria Antimicrobial Activity Tests
(S-C-Act-0235-a-S-20 and S-C-Act-0878-A-19) (Stach et al., Bioprospectingforantimicrobialactivitywasinitiallyperformed
2003).DNAextractionswereperformed,usingalysismethodby using the cross-streak method as described (Haber and Ilan,
cultureboilingsuspensionsofbacterialcells(Mooreetal.,2004). 2014), with slight modifications (Claverías et al., 2015). Fresh
EachPCRreactioncontained1µLofgenomicDNA,12.5µLof cultures of the isolated actinobacterial strains were inoculated
GoTaqGreenMasterMix(Promega)and0.6µMofeachprimer as a line in the middle of an agar medium plate and incubated
in a final reaction volume of 25 µL. The reaction started with at30◦Cuntilnotablegrowthwasobserved(7daysformycelial
aninitialdenaturation,at95◦Cfor5min,followedby35cycles strainsand5daysfornon-mycelialstrains).Strainsweregrown
of DNA denaturation, at 95◦C for 1 min, primer-annealing, at on TSA-ASW and ISP2-ASW media. Five reference bacteria
70◦C for 1 min and extension cycle, at 72◦C for 1.5 min, with werethetargetofinhibitiontests:StaphylococcusaureusNBRC
a final extension at 72◦C for 10 min (Claverías et al., 2015). 100910T (STAU); Listeria monocytogenes 07PF0776 (LIMO);
PCR-ampliconswerevisualizedin2%agarosegelelectrophoresis Salmonella enterica subsp enterica LT2T (SAEN); Escherichia
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Undabarrenaetal. MarineActinobacteriafromaChileanPatagonianFjord
coli FAP1 (ESCO) and Pseudomonas aeruginosa DSM50071T were analyzed performing BLAST with NCBI to determine
(PSAU). Cultures were incubated at 37◦C overnight and the closest type strain match using the 16S ribosomal RNA
inhibition zones were ranked qualitatively as: −, no inhibition; sequence of Bacteria and Archaea database. The Neighbor-
+/−, attenuated growth of test strain in the area closest to the Joiningalgorithm(SaitouandNei,1987)usingMEGAsoftware
actinobacterial line; +,<50% growth inhibition (less than half version 6.0 (Tamura et al., 2013) with bootstrap values
ofthebacteriallinewasinhibited);++,50%growthinhibition based on 1000 replications (Felsenstein, 1985) was used to
(halfofthebacteriallinewasinhibited);+++,>50%growth construct a phylogenetic tree based on the V1-V9 region of
inhibition (more than half of the bacterial line was inhibited). the 16S rRNA gene sequences. The 16S rRNA gene sequences
All experiments were performed in duplicate,using an internal were deposited in GenBank under the following accession
controlwithoneofthereferencestrains. numbers:Arthrobactersp.H-JH3(KT799841);Brachybacterium
Further antimicrobial tests were performed with selected sp.H-CG1(KT799842);Brevibacteriumsp.H-BE7(KT799843);
isolates Streptomyces sp. H-KF8, Arthrobacter sp. H-JH3, Corynebacterium sp. H-EH3 (KT799844); Curtobacterium sp.
Brevibacteriumsp.H-BE7,Kocuriasp.H-KB5andRhodococcus H-ED12 (KT799845); Kocuria sp. H-KB5 (KT799846); Dietzia
sp. H-CA8f. Strains were grown in a 50 mL liquid culture in sp. H-KA4 (KT799847); Micrococcus sp. H-CD9b (KT799848);
ISP2-ASW medium for 10 days for non-mycelial strains and Rhodococcus sp. H-CA8f (KT799849); Streptomyces sp. H-KF8
15 days for the mycelial strain, with continuous shaking at (KT799850)andStreptomycessp.H-CB3(KT799851).
30◦C.Crudeextractswereobtainedaftersolventextractionusing
Phenotypic Characterization of
hexane, methanol and ethyl acetate in a 1:1 ratio (v/v) for two
times.Evaporationofsolventwasperformedwithspeedvacuum, Actinobacterial Strains
andextractwasdissolvedin10%dimethylsulphoxide(DMSO) Forthemorphologicalandphysiologicalcharacterizationofthe
until a final concentration of 5mg mL−1. Antimicrobial assays representative strains, colony pigmentation, spore formation,
wereevaluatedusing10µLofeachextract,overLBagarplates growth temperatures, ASW requirement and NaCl tolerance
spread with the bacterial test strains STAU, PSAU, SAEN, and were evaluated. Optimal colony pigmentation was observed on
ESCO.Plateswereincubatedovernightat37◦Candinhibitions TSA-ASW after a 3-month incubation at 4◦C. To establish
zoneswerechecked.ISP2mediumand10%DMSOwereusedas the effects of temperature on growth, 10 µL of actinobacterial
negativecontrols. cultures were streaked onto TSA-ASW plates, and incubated
at 4, 20, 30, 37, and 45◦C. For NaCl tolerance, LB agar with
Detection of PKS and NRPS Biosynthetic
0, 1, 3.5, 5.0, 7.0, 10, and 20% (w/v) NaCl was prepared. As
Genes describedpreviously,10µLoftheactinobacterialcultureswere
Amplification of biosynthetic genes was carried out by PCR, streaked onto LB agar plates and incubated at 30◦C. To detect
using degenerate primers targeting the ketosynthase domain the requirement of seawater on growth, ISP2 was prepared as
in PKS type I with primers KS-F (5′CCSCAGSAGCGCSTS follows: medium with Milli-Q H2O; medium with ASW; and
YTSCTSGA3′)andKS-R(5′GTSCCSGTSCCGTGSGYSTCSA3′) mediumwithMilli-QH2Osupplementedwith3.5%(w/v)NaCl
(Gontang et al., 2010); and PKS type II with primers KSα (equivalenttoASWNaClconcentration).Incubationtimeswere
(5′TSGRCTACRTCAACGGSCACGG3′) and KSβ (5′TACSAG from10days(fornon-mycelialstrains)to14days(formycelial
TCSWTCGCCTGGTTC3′)(Ayusoetal.,2005).Theadenylation stains)at30◦C.Thereferencetimeforgrowthwasthatonwhich
domain in NRPS systems was detected with primers A3F growthwasobservedoncontrolplates.Resultswereinterpreted
(5′GCSTACSYSATSTACACSTCSGG3′) and A7R (5′SASGTCV as:+,ifthestraintestedwasabletogrowonmedium-ASWbut
CCSGTSCGGTAS3′)(Ayuso-SacidoandGenilloud,2005).PCR didnotgrowonmedium/Milli-QH2Oandonmedium/Milli-Q
programswereperformedaspreviouslydescribed(Ayusoetal., H2O supplemented with 3.5% NaCl; and −, if the strain tested
2005;Ayuso-SacidoandGenilloud,2005;Gontangetal.,2010). wasabletogrowonallthreemedia.
Productswerevisualizedin1%agarosegelselectrophoresis,and
Resistance to Model Antibiotics
stainedwithGelRed(Biotium).StreptomycesvioleaceoruberDSM
40783wasusedasacontrolforallPCRreactions.Detectionwas Representativestrainsofeachgenusweregrowntoexponential
determinedas+,iftheampliconwaslocatedattheexpectedsize phase(turbidityat600nmof0.3)andplatedonMueller-Hinton
(700bpforPKStypeI;800–900bpforPKStypeIIand700–800 agarplatesforantibioticsusceptibilitytesting.Antibioticdiscsfor
bpforNRPS);and−,ifampliconwasabsentoritwaspresentat Gram-positivebacteria(Valtek)wereplacedaboveandinhibition
anyothersize. grown zones as diameters were measured and compared with
values obtained from the Clinical and Laboratory Standards
Phylogenetic Analysis Institute (CLSI) from year 2016 to determine susceptibility (S),
Representative strains for each genus identified from partial orresistance(R)ofeachantibiotictested.
16S rRNA gene sequence analyses were selected for the nearly-
complete sequencing of this gene, as previously described RESULTS
(Claverías et al., 2015). PCR products were quantified and sent
Isolation and Identification of
toMacrogenInc.(Seoul,Korea)forpurificationandsequencing,
using primers 27F, 518F, 800R, and 1492R. Manual sequence Actinobacteria
edition,alignment,andcontigassemblingwereperformedusing Eleven marine sediment samples were collected from four
VectorNTIv10softwarepackage(Invitrogen).Sequencecontigs different sites in Comau fjord, Northern Patagonia, Chile
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Undabarrenaetal. MarineActinobacteriafromaChileanPatagonianFjord
(Figure1). Altogether 25 marine actinobacteria were isolated. actinobacterialstrainsinhibitedbothLIMOandSTAU;whereas
Their distribution according to the sampling site was: 40% ISP2-ASW-grownstrains,56%showedinhibitionforLIMOand
from Lilihuapi island coast, 28% from Punta Llonco, and 36%forSTAU(Figure3B).
16% from Loncohaigua river mouth and Tambor waterfall, Notably, 67% of the antimicrobial activities observed with
each. The majority (80%) of the isolates were from sediments the cross-streak method were retrieved with various solvent
situated approximately 10m deep. Only occasional isolates extractions from actinobacterial liquid cultures (Table2). Ethyl
were obtained from deeper sediments or from the shallow acetate was more effective in extracting active compounds, as
locations.TheActinobacteriaisolatedbelongtothreesuborders: crudeextractsfromRhodococcussp.H-CA8f,Kocuriasp.H-KB5
Streptomycineae, Micrococcineae, and Corynebcaterineae; and Brevibacterium sp. H-BE7 presented antimicrobial activity.
comprising eight different families. Relative abundances of the On the other hand, antimicrobial activity from Arthrobacter
strains according to the genera isolated (Figure2A) indicated sp. H-JH3 was effectively extracted from the cell pellet using
that most abundant genera were Kocuria and Brachybacterium. methanol.CrudeextractsfromRhodococcussp.H-CA8fshowed
The selective media had a major influence on the number an antimicrobial effect against all bacteria tested, confirming
of isolates obtained (Figure2B). M1-ASW medium was resultsobtainedfromthecross-streakmethod.
the most effective regarding the number and diversity of
Detection of PKS and NRPS Biosynthetic
isolates recovered. Interestingly, strains of Brachybacterium,
Brevibacterium, Micrococcus, and Rhodococcus genera were Genes
isolatedexclusivelywiththismedium(Figure2B). The presence of biosynthetic PKS (type I and II) and NRPS
genes were detected by PCR in representative actinobacterial
Antimicrobial Activity Assays
isolates (Table3). Interestingly, most isolates bear at least one
Our first approach was to screen all actinobacterial strains biosynthetic gene of PKS or NRPS. Among them, NRPS was
for antimicrobial activity, using the cross-streak method, thepredominantgeneobserved(91%),followedbyPKStypeII
against five reference strains: STAU, LIMO, PSAU, SAEN, and (73%).Only18%ofactinobacterialisolatesshowedthepresence
ESCO(Figure3A).Actinobacterialstrainsshowedantimicrobial ofPKStypeIgene.
activity,presentingabroadspectrumofinhibitionalthoughwith
different inhibition patterns (Table1). Inhibition of reference Phylogenetic Analysis
strains largely depended on the media where actinobacterial For phylogenetic analysis, the 16S rRNA gene was sequenced
strainswerecultivated,provingTSA-ASWtobegenerallybetter for selected actinobacterial isolates, representatives of each
forantimicrobialactivitythanISP2-ASWmedium.Arthrobacter, genus retrieved in sediment samples from Comau fjord. A
Brachybacterium, Curtobacterium, and Rhodococcus isolates dendogram of the estimated phylogenetic relationships is
showedpotentantimicrobialbioactivitytomorethanonetarget presented in Figure4 and the sequence similarities of selected
(Table1). Regarding the Gram-negative bacteria tested, TSA- actinobacterial strains to type strains of related species are
ASW-grown actinobacterial strains were able to inhibit ESCO given in Table3. Four of the actinobacterial isolates are
(84%) and PSAU (24%); whereas ISP2-ASW-grown isolates below the 98.7% sequence identity threshold and therefore
inhibited up to 76 and 48%, respectively. Concerning the may be potential candidates of new taxons. These isolates
Gram-positive reference strains, 64% of the TSA-ASW-grown belong to Arthrobacter and Kocuria genera (Micrococcaceae
FIGURE2|BiodiversityofactinobacteriafromtheComaufjordinNorthernPatagonia.(A)Distributionoftherelativeabundanceoftheactinobacterialgenera
isolated.(B)Numberofactinobacteriaofvariousgeneraisolatedusingdifferentculturemedia.
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Undabarrenaetal. MarineActinobacteriafromaChileanPatagonianFjord
FIGURE3|AntimicrobialactivityofactinobacterialstrainsfromtheComaufjordinNorthernPatagonia.(A)Cross-streakmethodofRhodococcussp.
H-CA8fshowingdifferentpatternsofinhibitionzoneswithseveralmodelbacteria.(B)Antimicrobialactivityofactinobacterialstrainsusingthecross-streakmethod.
STAU,Staphylococcusaureus;LIMO,Listeriamonocytogenes;PSAU,Pseudomonasaeruginosa;SAEN,Salmonellaenterica;ESCO,Escherichiacoli.I,ISP2-ASW
media;T,TSA-ASWmedia.
TABLE1|Antimicrobialactivityofactinobacterialstrainsagainstmodelpathogensusingthecross-streakmethod.
Strain Genus STAU LIMO PSAU SAEN ESCO
ISP2 TSA ISP2 TSA ISP2 TSA ISP2 TSA ISP2 TSA
H-CA8b Arthrobacter +/− +/− +++ +++ +++ +++ +/− +/− ++ ++
H-JH1 Arthrobacter − − ++ − + − +/− +/− + −
H-JH3 Arthrobacter − − ++ − + − +/− +/− + +/−
H-CA4 Brachybacterium − − +++ + − ++ − +/− +/− +++
H-CD1 Brachybacterium − +/− − +++ − − − +/− +/− +/−
H-CE9 Brachybacterium − +/− + ++ − − + + ++ +
H-CF1 Brachybacterium ++ ++ + ++ − − − +/− − +/−
H-CG1 Brachybacterium − +/− − +++ +/− − − − +/− +/−
H-BE7 Brevibacterium + +/− − − + +/− ++ + + −
H-EH3 Corynebacterium − +/− +/− +++ − − − − − +/−
H-KF5 Corynebacterium − − − − +/− − − +/− − +/−
H-BE10 Curtobacterium +++ +++ +/− +++ − − ++ +++ +/− ++
H-CD9a Curtobacterium + + +/− + − − +/− − +/− +/−
H-ED12 Curtobacterium ++ ++ + ++ − − +/− − +/− +/−
H-KA4 Dietzia − − − − − − − +/− − −
H-KA9 Kocuria − − − − +/− − − +/− − +/−
H-KA10 Kocuria − +/− − +/− − − − +++ +/− +/−
H-KB1 Kocuria − − − − − − − +/− +/− +/−
H-KB5 Kocuria − − − − +/− − − +/− +/− −
H-KB6 Kocuria − +/− +/− +++ +/− +/− + +/− +/− +
H-JH7 Kocuria − − − − − − − + − +/−
H-CD9b Microccocus − +/− − +++ + +/− +/− − +/− +/−
H-CA8f Rhodococcus ++ ++ +++ +++ − +++ +++ +++ +++ +++
H-CB3 Streptomyces +++ +++ +/− +/− +/− − − − + ++
H-KF8 Streptomyces +++ +++ +/− +/− +/− − − − + +
−,noinhibition;+/−,attenuatedgrowth;+,<50%growthinhibition;++,50%growthinhibition;+++,>50%growthinhibition.BothmediawerepreparedwithASW.
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Undabarrenaetal. MarineActinobacteriafromaChileanPatagonianFjord
TABLE2|Antimicrobialactivitiesofcrudeextractsusingvarioussolvents Brevibacteriumsp.H-BE7,showedimprovedgrowth withboth
forselectedactinobacterialisolatesgrowninISP2-ASWmedium. ASWand3.5%NaCl,ratherthanwithMilli-QH Oand0%NaCl,
2
suggestingaspecificsaltrequirementconfirmedbyitsgrowthin
Strain Solvent BacterialTestStrain
10%(w/v)NaCl(Figures5B–D).
STAU PSAU SAEN ESCO As the Comau fjord deep-waters reach temperatures below
10◦C, actinobacterial strains were tested for growth at different
H-KF8 Hexane − − − −
temperatures.Notably,73%ofstrainsbelongingtoArthrobacter,
Ethylacetate − − − −
Brachybacterium, Brevibacterium, Kocuria, Dietzia, and
Methanol + − − +
Rhodococcus, and to a lesser extent, Streptomyces, were able to
growat4◦C(Figure6).Moreover,pigmentationofthecolonies
H-CA8f Hexane − − − −
was more intense after growth at 4◦C, in comparison to 30◦C
Ethylacetate + + + +
(Figures6B–D). Colony pigmentation of all representative
Methanol − − − −
actinobacteria was visualized macroscopically and detailed in
H-KB5 Hexane − − − − Table3.
Ethylacetate − + + +
Resistance to Model Antibiotics
Methanol − − − −
Antibiogram experiments demonstrated that all isolated
H-JH3 Hexane − − − − actinobacterial strains are resistant to at least one of the
Ethylacetate − − − − antibioticstested.Furthermore,theseisolatesshowedresistance
Methanol − − + + to several antibiotics of different classes. Interestingly, strains
H-JH3,H-BE7,H-KA4,H-CD9,H-CG1,H-ED12,andH-CA8f
H-BE7 Hexane − − − − showed resistances to ≥6 antibiotics, wherein resistance to
Ethylacetate − − + − tetracycline, ciprofloxacin and oxacyllin were observed for all
Methanol − − − − theactinobacterialstrains.Strain H-KA4andH-ED12showed
resistance to all antibiotics tested, whereas strain H-BE7 was
susceptibleonlyforsulfonamides(Table4).
family), Brevibacterium genus (Brevibacteriaceae family), and
Corynebacterium genus (Corynebacteriaceae family) (Table3).
DISCUSSION
Interestingly,thepsychrotolerantisolateKocuriasp.H-KB5has
a96.97%sequenceidentitywiththetypestrainK.polarisCMS
Marine actinomycetes isolated from the National Marine
76 orT, a strain isolated from an Antarctic cyanobacterial mat
Protected Area of Huinay at the Comau fjord in Northern
sample (Reddy et al., 2003). Moreover, strain H-KB5 forms a
Patagonia were studied, along with their physiological
separate branch within the Kocuria group in the phylogenetic
and taxonomic properties, and their potential to produce
tree (Figure4). This isolate will be further characterized in a
antimicrobial compounds. Patagonian fjords are largely
polyphasicapproachtodetermineitstaxonomicposition.
unexplored, and may provide a rich source of microorganisms
producing novel anti-infective compounds. This is the first
Phenotypic Characterization of Isolated
bioprospection report of cultivable actinobacteria in this
Actinobacterial Strains unique ecosystem, where 25 actinobacteria were isolated and
TheComaufjordischaracterizedbydefinedzoningpatternsof characterized. Two studies report the isolation of marine
strong vertical and horizontal salinity gradients. The first 15m actinobacteria from sediments of Chile’s vast coast; one from
underwater are influenced by waters of low salinity (∼1.0%). ChiloéIsland(Hongetal.,2010)andarecentstudyperformed
Belowthisdepth,ahaloclineisfoundthatproducesaconstant in Valparaíso Central Bay (Claverías et al., 2015). Only a
watersalinityof3.2%(Castilloetal.,2012).Inordertoanalyze metagenomic study has been carried out with a microbial mat
howthesalinityaffectsthegrowthoftheactinobacterialisolates, located in the Comau fjord, revealing that 1% of community
NaCltolerancewasdeterminedforeachstrain(Table3).82%of reads was represented by the phylum Actinobacteria (Ugalde
the representative isolates were able to grow in the presence of etal.,2013).
1.0,3.5,5.0,and7.0%(w/v)NaCl(Figure5).45%ofthestrains, Inthisstudy,alowerabundanceofactinobacteriaassociated
belonging to Arthrobacter, Brachybacterium, Brevibacterium, to marine sediments was observed compared to Valparaíso
Curtobacterium, and Kocuria genera, were able to grow in Bay where actinobacterial strains belonging to 18 genera were
presence of 10% (w/v) NaCl (Table3). None of the isolated isolated, using the same cultivating conditions (Claverías et al.,
actinobacteriawasabletogrowwith20%w/vNaCl. 2015).Although,membersoftheRhodococcusandDietziagenera
Tostudyadaptationtomarineenvironments,actinobacterial were successfully isolated from the Comau fjord, they were
strains were tested for ASW requirement. Most strains (73%), less represented (8%) than in Valparaiso Bay (33%). The lower
belonging to Arthrobacter, Brachybacterium, Corynebacterium, actinobacterial abundance in Comau fjord could be due to the
Dietzia, Kocuria, Rhodococcus, and Streptomyces genera were lower content of organic matter in this microhabitat that can
positively influenced by sea water as they required ASW range between 0.5 and 3.4% of organic carbon content for
for growth, suggesting marine adaptation. Interestingly, strain Northern Chilean Patagonian fjords (Sepúlveda et al., 2011).
FrontiersinMicrobiology|www.frontiersin.org 7 July2016|Volume7|Article1135
Undabarrenaetal. MarineActinobacteriafromaChileanPatagonianFjord
S
s RP + + + + + + + − + + +
ne N
e
g
nthetic PKSII − + + + + + + − + + −
y
s
o I
Bi KS − + + − − − − − − − −
P
s Pigmentation Brightcream Brightyellow Brightorange Brightcream Palecream Brightpink Intenseorange Lightyellow Lightpink Whitemycelium Whitemycelium
c
sti nt
aracteri ASWquireme + + − + − + + − + + +
h e
C R
al
c
ysiologi Salinity%NaCl) 0–10 0–10 0–10 7 0–10 0–10 0–7 0–7 0 0–7 0–7
h (
P
e
ur
mperat◦(C) 4–37 4–37 4–37 20–37 20–45 4–37 4–37 20–37 4–30 4–37 4–37
e
T
cterialstrains. acteristics ySedimentcharacteristics Shellsandsponges Hardsediment Goodwatervisibility Lowtide Goodwatervisibility Lowtide Sand,musselsandseaurchins Shells,poorwatervisibility Shellsandoldnets Shells,poorwatervisibility Sand,musselsandseaurchins
ativeactinoba ographicChar DepthSalinit(m)(ppt) 11.328.5 6.130.5 25.129.5 0.255 25.129.5 0.255 15.628.5 14.529 22.931 14.529 15.628.5
eristicsofrepresent Bioge SamplingSite LilihuapiIsland TamborWaterfall PuntaLlonco LoncochaiguaRiver PuntaLlonco LoncochaiguaRiver TamborWaterfall PuntaLlonco LilihuapiIsland PuntaLlonco TamborWaterfall
Biogeographicandphysiologicalcharact ◦ClosestTypeStrain(AccessionN)(%Identity) TArthrobacteroxydansDSM20119(X83408)(98,26) BrachybacteriumparaconglomeratumJCMT17781(AB645761)(99.16) TBrevibacteriumoceaniBBH7(AM158906)(97.94) CorynebacteriumpilbarenseIMMIBTWACC-658(FN295567)(98.10) CurtobacteriumoceanosedimentumATCCT31317(GU269547)(99.02) TKocuriapolarisCMS76or(NR028924)(96.97) TDietzianatronolimnaeaDSM444860(FJ468329)(99.06) TMicrococcusluteusNCTC2665(CP001628)(99.15) TRhodococcusjianlingiaedjl-6-2(DQ185597)(98.84) TStreptomycesprasinusNRRLB-2712(DQ026658)(99.92) TStreptomycesprasinusNRRLB-2712(DQ026658)(99.86)
|
TABLE3 Strain H-JH3 H-CG1 H-BE7 H-EH3 H-ED12 H-KB5 H-KA4 H-jCD9b H-CA8f H-KF8 H-CB3
FrontiersinMicrobiology|www.frontiersin.org 8 July2016|Volume7|Article1135
Undabarrenaetal. MarineActinobacteriafromaChileanPatagonianFjord
FIGURE4|PhylogenetictreeofrepresentativeactinobacterialstrainsisolatedfromtheComaufjordinNorthernPatagonia,Chile.Neighbour-joining
treeof16SrRNAgeneshowingthethreesuborderswithinthephylumActinobacteria.Nodenumbersrepresentthepercentageofbootstrapreplicates(1000
resampling)whichsupportedtheproposedbranchingordershownatconsistentnodes(valuesbelow50%werenotshown).Genesequencepositions55–1410were
considered,accordingtotheEscherichiacoliK12(AP012306)16SrRNAgenesequencenumbering.ArrowpointstotheoutgroupE.coliK12.GenBankaccession
numbersof16SrRNAsequencesaregiveninparentheses.Scalebarcorrespondsto0.01substitutionspernucleotidepositions.
FrontiersinMicrobiology|www.frontiersin.org 9 July2016|Volume7|Article1135
Undabarrenaetal. MarineActinobacteriafromaChileanPatagonianFjord
FIGURE5|NaCleffectonactinobacterialgrowth.Upperpanel:(A)DistributionofactinobacterialisolatesandtheirabilitytogrowinLBmediumwithvarious
percentagesofNaCl.Bottompanel:Asanexample,thehalophilicBrevibacteriumsp.H-BE7growninLBmediumcontaining:(B)3.5%;(C)5%;and(D)10%NaCl
concentrations.
Gram-positivebacteriaaremorecommonlyobservedinorganic five genera were retrieved using 7 media, being dominant
rich habitats (Fenical, 1993). Water samples from Valparaíso the Micromonospora genus (Hong et al., 2010). Although, no
Bay are influenced by contamination with polycyclic aromatic Micromonospora members have been isolated in this work, this
hydrocarbons as well as with heavy metals (Campos et al., could be due to the different isolation media used. In this
1987; Palma-Fleming et al., 2008; Fuentes et al., 2015). It can report,24%ofisolateswereobtainedfromNaST21Cxmedium,
also be influenced by hydrographic features such as seasonal which isderived from ST21Cx medium byelimination of yeast
upwellingwhichcansupplynutrientstoshallowwaters(Capone extract and replacement of artificial sea water (Magarvey et al.,
and Hutchins, 2013). In contrast, the Comau fjord has a high 2004). It has been reported that media composed of relatively
precipitation rate that provides a fresh water input (Silva, simplenutrientsyieldedmoreculturedactinobacteriaindiverse
2006) which can affect microorganisms in marine sediments. environments (Zhang et al., 2006; Gontang et al., 2007; Qin
The four sites from Comau fjord have minimal anthropogenic et al., 2012). This is consistent with the negligible amount of
intervention,thereofchangesinmicrobialcommunitiesaregiven nutrients that are actually available for marine actinobacteria
almostexclusivelybynaturalprocesses. within hostile ocean ecosystems (Das et al., 2006). This is the
Despitethefactthatarelativelylownumberofactinobacterial caseforourstudysincemoreisolateswereobtainedwithmedia
strainswereretrievedfromComaufjord,aratherhighcultivable containing low nutrients or complex carbon sources rather
biodiversity (10 genera) was observed using 5 isolation media. than common media constituents such as peptone and simple
In comparison, the actinobacteria isolated using 11 selective sugars, which are proposed to be unrealistic marine nutrients
media from the Trondheim fjord (Norway) belonged to 12 (Kurtböke et al., 2015). In this study, the major abundance
genera (Bredholdt et al., 2007). Also, in a culture-dependent of actinobacteria was found in deeper samples, which is in
study using sediments collected near Chiloé Island, Chile, accordancewiththatobservedintheTrondheimfjord(Hakvåg
FrontiersinMicrobiology|www.frontiersin.org 10 July2016|Volume7|Article1135