Antibacterial Activity of Endophytic Actinomycetes Isolated from the Medicinal Plant Vochysia

17 Pages·2017·2.48 MB·English

ORIGINALRESEARCH
published:06September2017
doi:10.3389/fmicb.2017.01642
Antibacterial Activity of Endophytic
Actinomycetes Isolated from the
Medicinal Plant Vochysia divergens
(Pantanal, Brazil)
FranciellyM.W.R.Gos1†,DaianiC.Savi2†,KhaledA.Shaaban3,4†,JonS.Thorson3,4,
RodrigoAluizio2,YveliseM.Possiede5,JürgenRohr3*andChirleiGlienke2*
1DepartmentofBasicPathology,FederalUniversityofParaná,Curitiba,Brazil,2DepartmentofGenetics,FederalUniversity
ofParaná,Curitiba,Brazil,3DepartmentofPharmaceuticalSciences,CollegeofPharmacy,UniversityofKentucky,Lexington,
KY,UnitedStates,4CenterforPharmaceuticalResearchandInnovation,CollegeofPharmacy,UniversityofKentucky,
Lexington,KY,UnitedStates,5DepartmentofBiology,FederalUniversityofMatoGrossodoSul,CampoGrande,Brazil
Editedby:
ElizabethM.H.Wellington, Endophyticactinomycetesfrommedicinalplantsproduceawidediversityofsecondary
UniversityofWarwick, metabolites (SM). However, to date, the knowledge about endophytes from Brazil
UnitedKingdom
remains scarce. Thus, we analyzed the antimicrobial potential of 10 actinomycetes
Reviewedby:
isolated from the medicinal plant Vochysia divergens located in the Pantanal
D.IpekKurtboke,
UniversityoftheSunshineCoast, sul-mato-grossense,anunexploredwetlandinBrazil.Strainswereclassifiedasbelonging
Australia
to the Aeromicrobium, Actinomadura, Microbacterium, Microbispora, Micrococcus,
AtteVonWright,
UniversityofEasternFinland,Finland Sphaerisporangium, Streptomyces, and Williamsia genera, through morphological and
*Correspondence: 16SrRNAphylogeneticanalyzes.Asusceptibilityanalysisdemonstratedthatthestrains
JürgenRohr were largely resistant to the antibiotics oxacillin and nalidixic acid. Additionally, different
[email protected]
culture media (SG and R5A), and temperatures (28 and 36◦C) were evaluated to
ChirleiGlienke
[email protected] select the best culture conditions to produce the active SM. All conditions were
†Theseauthorshavecontributed analyzed for active metabolites, and the best antibacterial activity was observed from
equallytothiswork. metabolites produced with SG medium at 36◦C. The LGMB491 (close related to
Aeromicrobium ponti) extract showed the highest activity against methicillin-resistant
Specialtysection:
Thisarticlewassubmittedto Staphylococcusaureus(MRSA),withaMICof0.04mg/mL,anditwasselectedforSM
Antimicrobials,Resistanceand
identification.StrainLGMB491produced1-acetyl-β-carboline(1),indole-3-carbaldehyde
Chemotherapy,
asectionofthejournal (2),3-(hydroxyacetyl)-indole(4),brevianamideF(5),andcyclo-(L-Pro-L-Phe)(6)asmajor
FrontiersinMicrobiology compoundswithantibacterialactivity.Inthisstudy,weaddtotheknowledgeaboutthe
Received:12May2017 endophytic community from the medicinal plant V. divergens and report the isolation of
Accepted:14August2017
rareactinomycetesthatproducehighlyactivemetabolites.
Published:06September2017
Citation: Keywords:actinomycetes,endophytes,Vochysiadivergens,pantanal,MRSA,secondarymetabolites
GosFMWR,SaviDC,ShaabanKA,
ThorsonJS,AluizioR,PossiedeYM,
RohrJandGlienkeC(2017) INTRODUCTION
AntibacterialActivityofEndophytic
ActinomycetesIsolatedfromthe
Endophytes are microorganisms that inhabit the internal tissues of plants without causing any
MedicinalPlantVochysiadivergens
negativeeffects,andactinomycetesisolatedfromplantshavebeenwidelystudiedduetheirability
(Pantanal,Brazil).
Front.Microbiol.8:1642. to produce active metabolites (Kim et al., 2000; Zhao et al., 2011; Kadiri et al., 2014; Golinska
doi:10.3389/fmicb.2017.01642 etal.,2015;Savietal.,2015a,b).Actinomyceteshavebeenusedfordrugdiscoveryformorethan
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Gosetal. AntibacterialActivityofEndophyticActinomycetes
fivedecades,producingmorethan10,000bioactivecompounds. The plates were incubated at 28◦C for 30 days, and were
Of these ∼75% are produced by Streptomyces, the by far examineddailyforthepresenceofcolonies.Theactinomycetes
mostly explored actinomycete genus. The remaining 25% isolates were deposited in the Laboratório de Genética de
bioactive compounds were isolated from “rare actinomycetes”, Microrganismos(LabGeM)culturecollection,FederalUniversity
i.e.,actinomycetesisolatedinlowerfrequencythanStreptomyces ofParaná,Brazil(http://www.labgem.ufpr.br/).
(Rong and Huang, 2012; Tiwari and Gupta, 2012). Since,
the rare actinomycetes are an underexplored group, the use Identification
of these organisms, and their compounds have gained great MorphologicalAnalysis
importanceindrugdiscoveryprograms(RongandHuang,2012; Four different culture media were used to access the macro-
Tiwari and Gupta, 2012), mainly to combat infections caused morphological characteristics, ISP2—Agar yeast-malt extract;
by resistant microorganisms. The widespread use of broad- ISP3—OatAgar;ISP4—AgarStarchandinorganicsalts;ISP5—
spectrum antibiotics has created a strong selective pressure, Glycerol Asparagine Agar (Shirling and Gottlieb, 1966). The
resulting in survival, and spread of resistant bacteria (Davies isolates were streaked on the plates and incubated at 28◦C for
andDavies,2010).Theincreaseinbacterialresistanceisamajor 21 days. The characteristics evaluated were growth rate, the
concernforpublichealth(Ventola,2015).Unfortunately,many formationandcolorofaerialsporemassandsubstratemycelia.
pharmaceutical companies have reduced or eliminated their
searchfornewantibiotics,duetoeconomicreasons,exasperating MolecularTaxonomy
the problem further (Borrero et al., 2014). In order to find Total genomic DNA was extracted from 3 day old cultures
microorganisms with potential to produce active metabolites using the method described by Raeder and Broda (1985).
ourgrouphasbeensearchingendophyticmicroorganismsfrom Partial sequence of the 16S rRNA gene was amplified using
medicinal plants located in underexplored environments, such primers 9F (5′GAGTTTGATCCTGGCTCAG3′) and 1541R
as the Brazilian wetland regions (Savi et al., 2015a,b; Hokama (5′AAGGAGGTGATCCAGCC3′), as described by Savi et al.
et al., 2016; Peña et al., 2016; Santos et al., 2016; Tonial et al., (2016). The PCR product was purified using Exo1 and FastAP
2017).TheBrazilianPantanalisthelargestwetlandintheworld, enzymes (GE Healthcare, USA), and sequenced using the
and it is characterized by two seasons: flooding and the dry. BigDye(cid:13)R Terminator v3.1 Kit. The products were purified
Hence,thePantanalhasdevelopedapeculiarbiologicaldiversity with SephadexG50 and submitted to an ABI3500(cid:13)R automated
regardingitsfaunaandflora(Alho,2008).AccordingtoArieira sequencer (Applied Biosystems, Foster City, CA, USA).
and Cunha (2006), only 5% of the species of plants of the ConsensussequenceswereanalyzedandalignedusingMega6.0
Pantanal can survive the stress caused by drought and flood (Tamura et al., 2013) and BioEdit, and compared to sequences
periods.AmongthemisthemedicinalplantVochysiadivergens, available in the GenBank database (http://www.ncbi.nlm.nih.
which is commonly used in form of syrups and teas for the gov/BLAST/).Typestrainsequenceswerefoundthroughsearch
treatmentofcolds,coughs,fever,pneumonia,andotherdiseases in the List of Prokaryotic Names with Standing Nomenclature
(Pottetal.,2004).Inastudycarriedoutwithendophytesfrom database(http://www.bacterio.net/).Allsequencesobtainedwere
V. divergens, Savi et al. (2015a) identified actinomycetes able deposited in the GenBank, the accession numbers are listed in
to produce highly active metabolites. However, the study was Table1.ForBayesianinferenceanalysis,aMarkovChainMonte
performed with a small number of isolates, and the diversity Carlo (MCMC) algorithm was used to generate phylogenetic
of V. divergens remained little explored. Thus, the focus of trees with posterior probabilities using MrBayesv3.2.6 x86
this study is to identify endophytic actinomycetes from the (Ronquistetal.,2011).GRTevolutionarymodelwasdetermined
medicinal plant V. divergens and to assay their secondary usingtheAkaikeInformationCriterion(AIC)inRsoftware(R
metabolites, dependent on different culture conditions, against Core Team, 2017) and the phangorn package (Schliep, 2011).
clinicalpathogensassociatedwithantibioticresistance. Comparisons of sequences with respect to their percentile
similarity were estimated using the R software (R Core Team,
MATERIALS AND METHODS 2017)andthepegaspackage(Paradis,2010).
Sample Collection AntibioticSensitivity
V. divergens leaves with no marks or injuries were collected Thesusceptibilityoftheendophytesto11antibiotics,oxacilin(a
from21plantslocatedinthePantanalsul-mato-grossense/Brazil, penicillin), vancomycin (a glycopeptide), chloramphenicol (an
specifically in two regions of the Pantanal of Miranda, Abobral amphionicol), meropenem (a carbapenem), streptomycin (an
(19◦30′09.5′′S, 57◦02′32.2′′W) and São Bento (19◦28′53.9′′S, aminoglycoside), tetracycline (a tetracycline), gentamicin
57◦02′36.9′′W). (another aminoglycoside), rifampicin (a macrolactam),
ampicillin (another penicillins), ceftazidime (a third generation
Isolation of Actinomycetes cephalosporin),andnalidixicacid(aquinolone)wereevaluated
The leaves from V. divergens were subjected to surface asdescribedbyPassarietal.(2015).Theanalysiswasperformed
sterilizationaccordingtoaprotocoldescribedbyPetrini(1986). considering the isolate sensitive (S) with an inhibition zone
Theleaveswerefragmented(8×8mm)anddepositedonpetri > 20mm, intermediate (I) with an inhibition zone of 10–19.9
dishescontainingstarchcaseinagar(SCA)(Mohsenietal.,2013), mmandresistant(R),iftheinhibitionzonewasbetween0.0–9.9
with nalidixic acid (50µg/mL) and cycloheximide (50µg/mL). mm(Williamsetal.,1989).
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Gosetal. AntibacterialActivityofEndophyticActinomycetes
diaat neagar Grown ++ ++ +++ +++ +++ ++ +++ ++ +++ +++
entcultureme erolasparagi Substrate mycelium Pink Ivory-white Yellow Ivory-white White Ivory-white White Ivory-white Brown Orange
oninfourdiffer ISP5—Glyc Aerial sporemass Low:White Low:White None None Abundant:White Abundant:White None Abundant:White Abundant:White None
ati
hologicalcharacteristics21daysafterinocul ISP4—Agarstarchandinorganicsalts AerialSubstrateGrown sporemassmycelium +Low:WhiteYellow +Low:WhiteYellow +++NoneYellow ++NoneIvory-white +Abundant:WhiteIvory-white +Abundant:WhiteIvory-white +++NoneWhite +++Abundant:PinkRed/Ivory-white +++Abundant:WhiteGray +++NoneOrange
p
s,mor Grown +++ +++ ++ +++ +++ +++ +++ +++ +++ +++
e
cactinomycetesisolat ISP3—Oatagar Substrate massmycelium ant:Yellow ant:Yellow Yellow Yellow Ivory-whiteant: ant:Ivory-white White Pinkant: Ivory-whiteated: Lightorange
dophyti Aerial spore AbundWhite AbundWhite None None AbundWhite AbundWhite None AbundWhite ModerWhite None
n
sticofe xtract Grown +++ +++ ++ +++ +++ +++ +++ +++ +++ +++
andmorphologicalcharacteri ISP2—Agaryeast-malte AerialSubstrate sporemassmycelium Moderated:BrownWhite Moderated:Ivory-whiteWhite NoneYellow NoneYellow Ivory-whiteAbundant:White Moderated:Ivory-whiteWhite NoneWhite BrownAbundant:White GrayAbundant:Gray NoneOrange
n e
o c
n,place,andsourceofisolati NCBIPlace/Sourgenbankofisolation◦accessionn KY458125AbobralLeaf KY421547AbobralLeaf KY411896AbobralLeaf KY423334SãoBentoLeaf KY411900SãoBentoStem KY411898SãoBentoStem KY423496AbobralLeaf KY458126AbobralStem KY423333AbobralStem KY421546AbobralStem Moderated;,low.+
o
TABLE1|Identificati◦28C. Straingenera Actinomadurasp.LGMB466 Actinomadurasp.LGMB487 AeromicrobiumpontiLGMB491 Microbacteriumsp.LGMB471 Microbisporasp. LGMB461 Microbisporasp.LGMB465 Micrococcussp.LGMB485 Sphaerisporangiumsp.LGMB482 S.thermocarboxydusLGMB483 WilliamsiaserinedensLGMB479 ,Abundant;,+++++
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Gosetal. AntibacterialActivityofEndophyticActinomycetes
Biological Activity elutedwithagradientofH O-MeOH(100:0–0:100)toproduce
2
ScreeningofCultureConditions fractions FI-FV. The single fractions were subjected to HPLC
Isolateswereinoculatedin50mLofSGmedium(Shaabanetal., andSephadexLH-20(MeOH;1×20cm)purificationstoyield
2011),incubatedfor3daysat36◦Cand180rpm.Subsequently,1 compounds1–9inpureform(Figure9,FigureS9).NMRspectra
mL from the pre-culture was inoculated in SG and R5A media were measured using a Varian (Palo Alto, CA) Vnmr 400 (1H,
(100 mL) (Fernandez et al., 1998), and incubated for 10 days 400MHz;13C,100MHz)spectrometer,δ-valueswerereferenced
at two different temperatures, 28 and 36◦C, and 180 rpm. The to the respective solvent signals (CD3OD, δH 3.31 ppm, δC
culturewasfiltered-offonWhatmann4filters,thewaterfraction 49.15 ppm; DMSO-d6, δH 2.50 ppm, δC 39.51 ppm). HPLC-
wasextractedwithEtOAc(3×100mL).Thecombinedorganics MS analyses were accomplished using a Waters (Milford, MA)
wereevaporatedinvacuoat40◦Canddilutedinmethanolat10 2695 LC module (Waters Symmetry Anal C18, 4.6 × 250 mm,
mg/mL. 5µm;solventA:H2O/0.1%formicacid,solventB:CH3CN/0.1%
formicacid;flowrate:0.5mLmin−1;0–4min,10%B;4–22min,
AntibacterialActivity–DiskDiffusionAssays 10–100%B;22–27min,100%B;27–29min,100–10%B;29–30
The antibacterial activity of crude extracts and the isolated min,10%B).HPLCanalyseswereperformedonanAgilent1260
compounds 1–9 was evaluated against methicillin-sensitive systemequippedwithaphotodiodearraydetector(PDA)anda
Staphylococcus aureus (MSSA) (ATCC 25923), methicillin- Phenomenex C18 column (4.6 × 250 mm, 5µm; Phenomenex,
resistant S. aureus (MRSA) (BACHC-MRSA), Pseudomonas Torrance,CA).Semi-preparativeHPLCwasaccomplishedusing
aeruginosa (ATCC 27853), Candida albicans (ATCC Phenomenex(Torrance,CA)C18column(10×250mm,5µm)
10231), Acinetobacter baumannii (BACHC-ABA), Klebsiella onaVarian(PaloAlto,CA)ProStarModel210equippedwitha
pneumoniae, the producer of the enzyme KPC (K. pneumoniae photodiodearraydetectorandagradientelutionprofile(solvent
carbapenemase) (BACHC-KPC), Stenotrophomonas maltophilia A:H2O,solventB:CH3CN;flowrate:5.0mLmin−1;0–2min,
(BACHC-SMA), and Enterobacter cloacae a producer of the 25%B;2–15min,25–100%B;15–17min,100%B;17–18min,
enzyme VIM (Verona integron-encoded metallo-β-lactamase) 100–25%B;18–19min,25%B).AllsolventsusedwereofACS
(BACHC-VIM). The bacteria were cultivated for 12 h at 37◦C, grade and purchased from the Pharmco-AAPER (Brookfield,
anddilutedaccordingtotheMcFarlandstandard0.5scale.Each CT).SizeexclusionchromatographywasperformedonSephadex
testorganismwasstreakedonasterileMueller-Hintonagarplate LH-20(25–100µm;GEHealthcare,Piscataway,NJ).
with acottonswab.Extractswerealiquotedin100µgamounts
per6mmsterilefilterdisc.Thediscswereplacedonplatesand RESULTS
incubated for 24h at37◦C. Thediameterhaloswere measured
in millimeters. As a positive control, a disc with a standard Isolation of Endophytic Actinomycetes
antibioticwithactivityagainsteachofthebacteriawasused,and From 2,988 fragments analyzed, 10 endophytic actinomycetes
pure methanol was used as negative control (CLSI, 2015; Savi wereisolated(Table1),thustheisolationfrequencywas0.34%.
etal.,2015b). Fromthe10isolates,70%(n=7)wereisolatedfromtheAbobral,
and30%(n= 3)from theSão Bentoregion. Fiveisolateswere
MIC–MinimumInhibitoryConcentrationand obtained from stems, and five from leaf tissues of the plant
MBC–MinimumBactericidalConcentration (Table1).
Extracts from strain LGMB491 that showed high antibacterial
activity were selected to determine the minimum inhibitory Morphological Identification
concentration.TheMICofextractsagainsttheclinicalpathogens Agreatmacro-morphologicaldiversitywasobserved,withwhite,
wasperformedasdescribedbyOstroskyetal.(2008)andCLSI. ivory-white,pink,brown,gray,orange,andyellowcolonycolors.
The minimum bactericidal concentration was determined as Most of isolates showed abundant to moderate growth after 21
describedbySoltaniandMoghaddam(2014). daysofincubation,andsixisolatesshowedabundanttomoderate
sporeformationonISP2andISP3media.IsolatesLGMB461and
StatisticalAnalyses LGMB465showedhighmorphologicalsimilarity,andprobably
Thestatisticalanalysiswasperformedusinganalysisofvariance representthesamespecies(Table1).
(ANOVA)tocompareextracteffectstotheirrespectivecontrols.
WealsoperformedPost-hoctestsusingTukey’shonestsignificant Molecular Analysis
difference.Alltestspremiseswerefulfilled;thesignificancelevel Using a BLAST analysis in the GenBank database, the isolates
usedwas0.05(α). were classified as eight genera: Aeromicrobium, Williamsia,
Microbacterium,Sphaerisporangium,Micrococcus,Microbispora,
Large-ScaleFermentation,ExtractionandIsolation Actinomadura,andStreptomyces.Eachgenuswasanalyzedina
A large-scale fermentation (10 L) of strain LGMB491 was separatephylogenetictreebasedonBayesianinference.
performed using SG culture medium at 36◦C for 10 days. The
culture was subjected to extraction with EtOAc (3 × v/v), and Actinomadura(LGMB466andLGMB487)
thecombinedorganiclayerswereevaporatedinvacuoat40◦Cto The alignment consisted of strains LGMB466 and LGMB487,
yield653mgofcrudeextract.Thecrudeextractwassubjectedto 55 type strains representative of Actinomadura genus, and
reversephaseC columnchromatography(20×8cm,250g), Streptomyces glauciniger (AB249964) as out group taxa. The
18
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Gosetal. AntibacterialActivityofEndophyticActinomycetes
analysis comprises of 1,402 characters, 1,011 of these were this phylogenetic analysis, strain LGMB491 is close related
conserved, 124 were parsimony informative and 131 were to Aeromicrobium ponti (Figure2), sharing high sequence
uninformative. Strains LGMB466 and LGMB487 showed high similarity,99.25%(TableS2).
similarity among themselves (98.86%), and in the phylogenetic
analysistheseisolatesdidnotclusterwithanyspeciesfromthe Microbacterium(LGMB471)
Actinomaduragenus(Figure1,TableS1),andprobablyrepresent Strain LGMB471 was aligned with type strains from the
anewspecies. Microbacteriumgenus,andAgrococcusjenensis(X92492)asout
group taxa. The alignment comprised of 1,314 characters, of
Aeromicrobium(LGMB491) those721conservedsites,122wereparsimonyinformative,and
Strain LGMB491 was aligned with all type strains from the 57 uninformative. In the phylogenetic tree, isolate LGMB471
Aeromicrobium genus (12 species), and Nocardioides albus ended up in a single branch related to species Microbacterium
(X53211) was used as out group taxa. The alignment consisted liquefaciens, Microbacterium maritypicum, Microbacterium
of 1,336 characters, 1,164 of these were conserved, 89 were oxydans, Microbacterium luteolum, Microbacterium saperdae,
parsimony informative and 68 were uninformative. Based on andMicrobacteriumparaoxydans(Figure3,TableS3).
FIGURE1|Bayesianphylogenictreebasedon16SrRNAgeneofLGMB466,LGMB487,andthe53typestrainofActionomaduragenus.Valuesonthenode
indicateBayesianposteriorprobabilities.ThespeciesStreptomycesglaucinigerwasusedasoutgroup.Scalebarindicatesthenumberofsubstitutionspersite.
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Gosetal. AntibacterialActivityofEndophyticActinomycetes
FIGURE2|Bayesianphylogenictreebasedon16SrRNAgeneofLGMB491andthe12typestrainofAeromicrobiumgenus.ValuesonthenodeindicateBayesian
posteriorprobabilities.ThespeciesNocardioidesalbuswasusedasoutgroup.Scalebarindicatesthenumberofsubstitutionspersite.
TABLE2|Antibioticsensitivitypatternofendophyticactinomycetes.
Antibioticsensitivity
Strain/Genera Oxa1µg Van30µg Clo30µg Mer10µg Est10µg Tet30µg Gen10µg Rif5µg Amp10µg Caz30µg Nal30µg
Actinomadurasp. R S I S S S S I R R R
LGMB466
Actinomadurasp. S I R S S S S S I S S
LGMB487
Aeromicrobiumponti R S R S S S S S R S S
LGMB491
Microbacteriumsp. R S I S S S S I R R R
LGMB471
Microbisporasp. R S R R S S S I R R R
LGMB461
Microbisporasp. R S R R S S I I R R R
LGMB465
Micrococcussp. R I R S I I I I R S R
LGMB485
Sphaerisporangiumsp R S I S S S S I R R R
LGMB482
Streptomyces R S R S S I S R R R R
thermocarboxydus.
LGMB483
Williamsiaserinedens. R S R S S I S I I S R
LGMB479
Degreeofsusceptibility:>20mm—Sensitive;10–19.9mm—intermediate;0.0–9.9mmresistant.Oxa,Oxacillin(1µg/disc);Van,Vancomycin(30µg/disc);Clo,Chloramphenicol
(30µg/disc);Mer,Meropenem(10µg/disc);Est,Streptomycin(30µg/disc);Tet,Tetracycline(30µg/disc);.Gen,Gentamicin(10µg/disc);Rif,Rifampicin(5µg/disc);Amp,Ampicillin
(10µg/disc);Caz,Ceftazidime(30µg/disc);Nal,Nalidixicacid(30µg/disc).
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Gosetal. AntibacterialActivityofEndophyticActinomycetes
FIGURE3|Bayesianphylogenictreebasedon16SrRNAgeneofLGMB471andthe94typestrainofMicrobacteriumgenus.ValuesonthenodeindicateBayesian
posteriorprobabilities.ThespeciesAgrococcusjenensiswasusedasoutgroup.Scalebarindicatesthenumberofsubstitutionspersite.
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Gosetal. AntibacterialActivityofEndophyticActinomycetes
Microbispora(LGMB461andLGMB465) Antibiotic Sensitivity Test
The analysis comprises of strains LGMB461 and LGMB465, In order to characterize the susceptibility profiles of the
10 species accepted in Microbispora genus, and the isolates endophytes, 11 antibiotics with different mechanisms-of-
previouslyreportedasMicrobisporasp.1,Microbisporasp.2,and action were utilized. Isolates were susceptible to vancomycin
Microbispora sp.3 (Savi et al., 2016). Actinomadura echinospora (80% sensitive and 20% intermediate), streptomycin (90%
(AJ420135) was used as out group taxa. The alignment sensitive and 10% intermediate), tetracycline (70% sensitive
consists of 1,371 characters, 1,309 of these were conserved, and30%intermediate),andgentamicin(80%sensitiveand20%
33 were parsimony informative, and 29 uninformative. In the intermediate). The two isolates of Microbispora sp. (LGMB461
phylogeneticanalysisstrainsLGMB461andLGMB465presented and LGMB465) showed resistance to meropenem, and 90% of
similarity with Microbispora sp.1 (LGMB259) with 99.84 and the isolates showed resistance to oxacillin, and nalidixic acid
100%fsimilarity,respectively(Figure4,TableS4). (Table2).
Micrococcus(LGMB485) Antibacterial Activity of Crude Extracts
The Bayesian analysis comprised of all Micrococcus type All strains and culture conditions analyzed produced active
strains, strain LGMB485 and Citricoccus parietis (FM9923367) extracts(Table3,TableS9),however,theextractfromLGMB491
as out group taxa (Figure5). The alignment consisted of (close related to A. ponti) cultured in SG medium at 36◦C
1,340characterswith452conservedsites,ninewereparsimony showed great antibacterial activity against S. aureus (22 mm)
informativeand19uninformative.Sincethesequenceswerevery and MRSA (19.8 mm), and moderate activity against others
similar (Table S5) and the alignment had only nine parsimony clinicalpathogens(Table3,FiguresS1–S8).TheMICandMBC
informativesites,aspeciesdesignationcannotbeassigned,and of extract from LGMB491 against S. aureus and methicillin-
isolateLGMB485wasidentifiedasMicrococcussp. resistant S. aureus were 0.02, and 0.04 mg/mL, respectively,
and the MBC was 5 mg/mL for both bacteria (Table4). In
addition,thecrudeextractfromLGMB491hadanMICof0.63
Sphaerisporangium(LGMB482)
mg/mLagainstgram-negativebacteriaassociatedwithantibiotic
For the Bayesian analysis, the sequence from LGMB482 was
resistance, K. pneumoniae KPC, S. maltophilia, and E. cloacae
aligned with strains of the Sphaerisporangium genus, and
VIM, and a MIC of 0.31 mg/mL against A. baumannii and
Actinomadura madurae (X97889) was used as out group taxa.
P.aeruginosa,respectively(Table4).
The alignment consisted of 1,320 characters, 886 of these
were conserved, 51 were parsimony informative and 47 were
uninformative.StrainLGMB482iscloselyrelatedtoS.melleum Structure Determination of Secondary
AB208714(99.4%similarity)andS.viridalbumX89953(97.89%
Metabolites from Strain LGMB491
similarity),however,itisinanisolatedbranchandmayrepresent
Scale-up fermentation of strain LGMB491 (10 L) using SG
a new species of the Sphaerisporangium genus (Figure6,
medium, followed by extraction afforded 653mg of crude
TableS6).
extract.Fractionation,isolationandpurificationoftheobtained
extract using various chromatographic techniques resulted
Streptomyces(LGMB483) in compounds 1–9 in pure forms (Figure S9). Thorough
The phylogenetic analysis was performed using 23 type strains analyses of the HPLC/UV, ESIMS and NMR spectroscopy
closely related with LGMB483; including Streptomyces albus data (FigureS10–S43), and by comparison with literature data
subsp. albus (X53163) as out group taxa. The alignment (Laatsch, 2012), the compounds were identified as 1-acetyl-β-
consisted of 1,391 characters, with 1,291 conserved sites, 45 carboline (1) (Shaaban et al., 2007; Savi et al., 2015b), indole-
were parsimony informative, and 39 uninformative. In the 3-carbaldehyde (2) (Zendah et al., 2012; Savi et al., 2015b),
phylogenetic tree, isolate LGMB483 grouped with Streptomyces tryptophol (3) (Rayle and Purves, 1967), 3-(hydroxyacetyl)-
thermocarboxydus, sharing 99.86% of similarity (Figure7, indole (4) (Zendah et al., 2012), brevianamide F (5) (Shaaban,
Table S7), and thus we suggest this isolate may belongs to this 2009), cyclo-(L-Pro-L-Phe) (6) (Barrow and Sun, 1994), cyclo-
species. (L-Pro-L-Tyr) (7) (Barrow and Sun, 1994), cyclo-(L-Pro-L-
Leu) (8) (Yan et al., 2004), and cyclo-(L-Val-L-Phe) (9)
(Pickenhagen et al., 1975) (Figure9). In order to determine
Williamsia(LGMB479) the compounds responsible for the biological activity observed
Theanalysisconsistsof11sequences,includingalltypestrainsof for the crude extract of strain LGMB491, we evaluated the
theWilliamsiagenus,thestrainLGMB479,andMycobacterium antibacterial activity of compounds 1–9 against S. aureus and
tuberculosis(X58890)wasusedasoutgrouptaxa.Thealignment methicillin-resistant S. aureus. 1-Acetyl-β-carboline (1) showed
comprisesof1,346characters,ofthese1,185wereconserved,81 an equivalent activity as the antibiotic methicillin against
wereparsimonyinformativeand56wereuninformative.Strain S. aureus, however, different from this antibiotic, compound
LGMB479 was in the same clade with Williamsia serinedens 1 also showed activity against MRSA (Table5). In addition,
(AM283464) (Figure8) and share 99.85% sequence similarity compounds 2, 4–6 also showed moderate activity against both
(TableS8),andmaybelongstothisspecies. MSSAandMRSA.
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Gosetal. AntibacterialActivityofEndophyticActinomycetes
FIGURE4|Bayesianphylogenictreebasedon16SrRNAgeneofLGMB461,LGMB465,the10typestrainofMicrobisporagenus,and7strainspreviouslyreported
bySavietal.(2016).ValuesonthenodeindicateBayesianposteriorprobabilities.ThespeciesCitricoccusparietiswasusedasoutgroup.Scalebarindicatesthe
numberofsubstitutionspersite.
FIGURE5|Bayesianphylogenictreebasedon16SrRNAgeneofLGMB485andthe9typestrainofMicrococcusgenus.ValuesonthenodeindicateBayesian
posteriorprobabilities.ThespeciesCitricoccusparietiswasusedasoutgroup.Scalebarindicatesthenumberofsubstitutionspersite.
FrontiersinMicrobiology|www.frontiersin.org 9 September2017|Volume8|Article1642
Gosetal. AntibacterialActivityofEndophyticActinomycetes
FIGURE6|Bayesianphylogenictreebasedon16SrRNAgeneofLGMB482andthe10typestrainofSphaerisporangiumgenus.Valuesonthenodeindicate
Bayesianposteriorprobabilities.ThespeciesActinomaduramaduraewasusedasoutgroup.Scalebarindicatesthenumberofsubstitutionspersite.
FIGURE7|Bayesianphylogenictreebasedon16SrRNAgeneofLGMB483andthe33typestrainofStreptomycesgenus.ValuesonthenodeindicateBayesian
posteriorprobabilities.ThespeciesStreptomycesalbussubsp.albuswasusedasoutgroup.Scalebarindicatesthenumberofsubstitutionspersite.
FrontiersinMicrobiology|www.frontiersin.org 10 September2017|Volume8|Article1642