h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Environmental
Microbiology
Analysis
of
bacterial
communities
and
characterization
of
antimicrobial
strains
from
cave
microbiota
Muhammad
Yasir
KingAbdulazizUniversity,KingFahdMedicalResearchCenter,SpecialInfectiousAgentsUnit,Jeddah,SaudiArabia
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received12October2016 Accepted15August2017 Availableonline18October2017 AssociateEditor:JohnMcCulloch
Keywords: Caves 16SribosomalRNA Microbiota Antimicrobial Sediments
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Inthisstudyforthefirst-timemicrobialcommunitiesinthecaveslocatedinthemountain rangeofHinduKushwereevaluated.Thesampleswereanalyzedusingculture-independent (16SrRNA gene amplicon sequencing) and culture-dependent methods.The amplicon sequencing results revealed a broad taxonomic diversity, including 21 phyla and 20 candidatephyla.Proteobacteriaweredominantinbothcaves,followedbyBacteroidetes, Acti-nobacteria,Firmicutes,Verrucomicrobia,Planctomycetes,andthearchaealphylumEuryarchaeota. RepresentativeoperationaltaxonomicunitsfromKoatMaqbariGhaarandSmasse-Rawo Ghaarweregroupedinto235and445differentgenera,respectively.Comparativeanalysis oftheculturedbacterialisolatesrevealeddistinctbacterialtaxonomicprofilesinthe stud-iedcavesdominatedbyProteobacteriainKoatMaqbariGhaarandFirmicutesinSmasse-Rawo Ghaar.MajorityofthoseisolateswereassociatedwiththegeneraPseudomonasandBacillus. Thirtystrainsamongtheidentifiedisolatesfrombothcavesshowedantimicrobial activ-ity.Overall,thepresentstudygaveinsightintothegreatbacterialtaxonomicdiversityand antimicrobialpotentialoftheisolatesfromthepreviouslyuncharacterizedcaveslocatedin theworld’shighestmountainsrangeintheIndiansub-continent.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Caves withsurface entrances represent oneof the unique andpoorlystudiedecosystemsonEarth.Theyinclude hydro-logicalsystemsthatarerelativelyisolatedfromthesurface, andsharebasicphysicochemicalconditions,including com-plete darkness, constanthumidity, and thermal stability.1,2 Caves comprised of unique underground communities of
E-mail:[email protected]
organisms,andcavemicroclimatesoftensupportdense pop-ulationsofextremophiles.3Multidisciplinarystudiesoncave microbiologyhaveimplicatedmicroorganismsinthe geolog-ical processes of caves, and have opened several avenues ofresearch,includingcavegeochemistry,cave environmen-talmicrobiologyandidentificationofnewmicrobialspecies andexplorationofnovelbiotechnologicalmoleculesfromcave sources.4,5Recently,severalnewmembersofthegenera Catel-latosporaandNonomuraeawerediscoveredincavesofMexico
https://doi.org/10.1016/j.bjm.2017.08.005
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
and NorthernThailand.6,7 In 2005,Agromyces subbeticussp. nov.,wasisolatedfromacaveintheCordobaareaof south-ernSpain.8 Jurado et al. reported Aurantimonasaltamirensis sp.nov., a Gram-negative member of the order Rhizobiales fromAltamiraCave,locatedintheCantabria,Spain.9These microorganismsnewlydiscoveredincavesdemonstratethe potentialforidentifyingsecondarymetabolitesthathaveyet tobefullyevaluatedandexploited.4,7
Cavesarefoundworldwide,andbiospeleologicalresearch hasbeen muchincreasedinthe lasttwentyyears.2,4 Avail-ableliteratureindicatesthe existenceofshortcavesinthe world’s highest mountains range called Karakoram, Hindu Kush,andPamir,whichincorporatesomeoftheworld’s high-estpeaks,includingtheK2(8610m)andNangaParbat(8125m) located inthe sub-continent.10 In the Chitral area of Pak-istan, the Hindu Kush mountain is surrounded by a high limestoneplateau,andtherearevariousunverifiedreportsof cavesinthisregion.11 TheNanga ParbathasRakhiotCave, whichis73.2mlongandthehighestknowncaveatan alti-tudeof 6644m.11 So far, no systematic studies have been conductedtoexplorethecavessysteminthosemountains. The caves identified by local people have never been sur-veyed formicroorganisms. In this study for the first time, microbialcommunitieswere investigatedinthe twocaves, KoatMaqbari Ghaar (KMG)and Smasse-Rawo Ghaar (SRG), locatedintheHinduKushMountainandsituatedinthe north-ernKhyberPakhtunkhwaprovinceofPakistan.Advancesin sequence-basedmetagenomicapproacheshavemadestudies onmicrobialdiversityandcommunitycompositionindiverse environmentsmorepossibleandinformative.However,using high-throughputsequencing techniquesalone tostudy the microbialcommunityinanenvironmenthasthepossibilities tomissthelowabundanttaxaaspreviouslyobserved.12Inthis study,anintegratedapproachofculture-basedand culture-independentpyrosequencing methodswere usedto gain a moreaccuraterepresentationofthemicrobialcommunities inthestudysites.Bacterialstrainsisolatedinthisstudywere screenedforantibacterialandantifungalactivities.
Materials
and
methods
Samplescollectionandchemicalanalysis
Thisstudyrepresentsapolyphasicanalysisofmicrobial com-munitiesfromtwocavesintheHinduKushmountainrange situatedintheNorth-WestregionofPakistan.Thelocalnames ofthosecavesareKoatMaqbariGhaar(KMG,34◦4913.06N, 72◦3041.81E) and Smasse-Rawo Ghaar (SG, 34◦3338.1N, 71◦5103.4E),andtheyarelocatedintheSwatandMalakand division,respectively.TheKMGcaveisabout2.5mwideand 15mdeep,andislocatedatabout1260mabovesealevel.The SRGcave hasanalmost horizontalorientationandaround 2mwide,10mlong,andislocatedat1062mabovesealevel. Bothcavesareofdriednatureandverylimitedlyinfluencedby anthropogenicactivities.Sedimentsamplesinthreereplicates werecollectedfromeachcaveusingautoclavedandsterilized bottles.Thesampleswerecollected inFebruary2013.Allof thesampleswerestoredat4◦Cforaround20hduring trans-portation,andwereimmediatelyprocessedforculturingafter
arrivinginthelaboratory.Apartofeachsamplewasstoredat −80◦CformetagenomicDNAextraction.Nospecific permis-sionwasrequiredforsamplingthestudiedcaves.Theselands were notprivatelyowned orprotectedinany wayand the cavesarenotpartofanationalparkorreserve.Oursampling didnotinvolveendangeredorprotectedspecies.
ThepHofeachsamplewasmeasuredusingSartoriuspH meters (Denver, Germany) ina 1/10 (w/v)saturated colloid solution ofsediment in deionized water. Temperaturewas measuredusingASTMthermometers(Gilson,USA)oneach site.Totalsoilorganicmatterandtotalnitrogenwere deter-minedusingthepartialoxidationmethodandmicroKjeldahl method.13Totalphosphoruswasmeasuredcolorimetrically.13 Physicochemicalanalysiswereperformedintriplicateforeach sample.
DNAextractionandpyrosequencing
TotalDNAwasextractedfrom eachhomogenizedcave sed-imentreplicatesusingtheprotocolforthePowerSoil® DNA extractionkit(MoBioLaboratories,Carlsbad).Amplification of the 16S rRNA gene hypervariable region V4 was per-formed using bar-coded 515F and 806R universal primers containing A and B sequencing adaptors, following the procedure previouslydescribed.14 PCRproductswere quan-tified using high-sensitivity Qubit technology (Invitrogen, USA), and were purified using Agencourt Ampure beads (Agencourt, USA). The 454 FLX–titanium pyrosequencing platform (Roche, Basel Switzerland) was used to perform high-throughput sequencing following the manufacturer’s protocol. Raw pyrosequencing data was processed using the analysis pipelineof MRDNA (Texas, USA).14 Sequence reads<150bpwereremoved,andtheremainingreadswere screened for homopolymer runs exceeding 6bp, chimeric sequences,and sequences containingNs;allofthesewere also excluded. Barcodes and primers were depleted from sequences.Highqualitysequencereadswereclusteredinto OTUsusingathresholdof97%sequencesimilarity.For single-tonreadsthedefaultvalueof2readsinQIIMEv1.9software was used to exclude from further analysis.15 OTUs were taxonomically classified using BLASTn against the curated databases GreenGenes, RDP (http://rdp.cme.msu.edu), and
NCBI(www.ncbi.nlm.nih.gov).16Thealphadiversityanalysis
wasperformedwithChao1andthenon-parametricShannon formulausingQIIMEv1.9software.15
Culture-dependentsamplesprocessing
Sediment sampleswere seriallydilutedfortheisolationof bacterialcolonieswithimprovedculturemethods,basedon anincreasednumberofinoculationplatesforeachdilution, longerincubationtimes,selectionofmicro-colonies,anduse ofmodifiedculturemediaaspreviouslydescribed.17Briefly, two different concentrations ofR2A medium(full strength 18g/Landhalfstrength9g/L)anddilutednutrientbroth(1/5 strength 3g/Land 1/10strength1.3g/L) supplementedwith 1.5% agar,20% aqueousextract ofthe collected cave sedi-ment,andtwoincubationconditions,17◦Cand37◦C,were usedtoculturebacteriafromthecavessamples.Theplates were incubated in aerobic condition for one week in seal
plasticbagscontainingwettissuepaperstoavoiddryingof culturemedia duringthis long incubation. Colony forming units(CFU)werecounted,andtheCFUofthethreereplicates were usedtoestimate thesizeofthe bacterialpopulation. Colonieswerepurifiedbysub-culturingandthenstoredusing a15%glycerolstockincryogenicvialsat−80◦C.Eighty-four isolateswereselectedfor16SrRNAgenesequencingbasedon thecolonymorphologyandgrowthcharacteristics.Genomic DNAwasextractedfromtheisolatedstrainsusing1%Triton X-100(Sigma)andboilingat95◦Cfor20min.The16SrRNAgene wasamplifiedusingtheuniversalbacterialprimers,27Fand 1492R,asdescribedpreviously.17PurifiedPCRproductswere sequencedwithABIPrismSequencer3730(Applied Biosys-tems,USA)accordingtomanufacturer’sprotocol.Ananalysis ofchimerasinthe16SrRNAgenesequenceswasperformed usingBellerophonsoftware.18Sequenceswereblastedatthe EzTaxon server (http://www.ezbiocloud.net/)to identifythe related typestrains.19 A multiplesequence alignment was performed,and aphylogenetic treewasconstructedbythe neighbor-joiningdistancemethodusingJukes-Cantormodel tocomputeevolutionarydistanceinMEGA6software.20 Boot-strapvalueswerecalculatedbasedon1000replications.The 16SrDNAsequencesweredepositedinGenBank(NCBI)under accessionnumbersKF747002–KF747085.
Antimicrobialactivity
Antimicrobial activities of the isolated strains on growth ofthe pathogenic bacteria Salmonellatyphi and Staphylococ-cusaureus,and the yeastCandidaalbicanswere determined by confrontation bioassay using paper discs. A concentra-tion of0.5 McFarland ofthe target strains were spread on media-agarplates.Twentymicrolitersofeachbacterial sus-pensioncontainingapproximately108cfu/mLwas addedto asterilepaperdisk(Advantec, Japan),and thendiscs were placed on the target strain plates. The plates were incu-batedfor72hat25◦C,and zonesofinhibitionaround the paperdiscs weremeasured.21 Chloramphenicol(30g/disk) andamphotericin-B(20g/disk)wereusedasreferenceto esti-matetheantimicrobialactivitiesofthetestedisolatesagainst bacteriaandyeastrespectively.
Statisticalanalysis
One-wayANOVAandTukey’sHSD(HonestlySignificant Differ-ence)testswereusedtostatisticallycomparephysicochemical parameters,and CFUobtainedatdifferentmedia composi-tionsandincubationtemperature.TheStatisticalPackagefor theSocialSciences(SPSS)version20wasusedforthe statisti-calanalysis.
Results
Physicochemicalanalysis
ThepHvaluesofKMG(7.6±0.21)and SRG(8.0±0.25)were notsignificantly(p=0.29)differentfromoneanother,andthe sedimentofSRGwasslightlybasic.TemperatureofKMGwas 14◦C and SRG17◦C.Chemical analysisindicatedthat both
caveswerenutritionallypoor.Totalorganicmatterand nitro-gencontentinKMG(1.7±0.08%and0.09±0.002%)andSRG (1.05±0.02%and0.05±0.004%)weresignificantly(p=0.01and p=0.001)differentbetweenthetwocaves.Phosphoruslevel intheKGMandSRGwere191.4±0.4ppmand82.6±0.6ppm, respectively.
Microbialtaxonomicdiversity
Taxonomicassignmentofabout44,015high-quality pyrose-quencing reads from KMG (23,838) and SRG (20,177) were obtained using the V4 region of the 16S rRNA gene. The taxonomic composition of the bacterial communities was highly diverse in both caves sediments. The majority of the identified operational taxonomic units (OTUs), defined by 97% sequence similarity, were affiliated with 21 phyla that included 19 bacterial phyla, 2 archaea, and 20 can-didate phyla. Among the identified phyla, 10 phyla and 5 candidates divisionswere detectedin bothcaves. The per-centagesequence readsfrom KMG andSRG were affiliated with 6 bacterial phyla detected at relatively higher abun-dance including; Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes,VerrucomicrobiaandPlanctomycetes(Fig.1).Among theProteobacteria,␣-Proteobacteria(45.6%KMGand34.1%SRG) and ␥-Proteobacteria(35.2% KMGand32.1% SRG)were dom-inantinbothcaves,followedby␦-Proteobacteria(12.6%KMG and16.9%SRG)and-Proteobacteria(9.2%KMGand16.7%SRG). Thephylum Deinococcus-ThermuswasdetectedonlyinKMG (6.8%),andWS3(candidatedivision,4.3%)wasdetectedinSRG. The15candidatedivisionsand10bacterialphyla,including 2.9%Acidobacteria,2.8%KSB1,and1.2%OP3(candidate divi-sion),weredetectedspecificallyinSRG.Thearchaealphylum Euryarchaeota wasdetectedat 3.4%and 0.2%abundancein KMGandSRG,respectively.Cyanobacteriaweredetectedat<1% abundanceinbothcaves.
Atotalof498differentgeneraweredetected.Thehighest numberofgenera(445)wasdetectedinSRG,while235genera werepresentinKMG.Amongthese,185generawerepresent inbothcaves,while53generawerespecificallydetectedin KMGand263inSRG.Moreover,17generainKMGand21 gen-era inSRG werepresent atanabundance≥1%,accounting for74%and58%ofthetotalsequencereadsintherespective samples(Fig.2).InKMG,thefollowingbacterialgenerawere detectedatrelativelyhighabundances:Cellvibrio(17.1%), Sac-charophagus(15.5%),Bacillus(7.6%),andDeinococcus(6.6%).In SRG,thedominantgenerawere Chthoniobacter(6.9%), Opitu-tus(6.6%),Chondromyces(3.4%),Rubrobacter(3.4%),Rhodoplanes (3.1%),andProsthecobacter(3%).OnlythegenusCytophagawas presentinbothcavesat≥1%abundance.
In thenext step,weanalyzedthe OTUsatspecieslevel inthe KMGand SRGcaves. Atotalof873differentspecies wereidentified,including384speciesinKMGand628species inSRG.Only139specieswerepresentinbothcaves.Ineach cave,morethan60%specieswereaffiliatedwithfourbacterial phyla:Proteobacteria,Firmicutes,Bacteroidetes,and Actinobacte-ria.Intotal,170(44.3%)identifiedspeciesfromKMGand309 (35.4%)from SRG were affiliatedwithProteobacteria.Among them,89specieswerecommonlypresentinbothcaves includ-ing Saccharophagus degradans and Lysobacter spongicola that werepresentatrelativelyhigherabundanceinKMG,andwere
KMG(%) 0 0.3 1.1 1.5 3.4 5.4 6.8 14.6 17.3 49.7 36.7 16.6 12.1 8.9 5.5 4.7 4.5 4.2 2.9 2.8 1.2 0.2 0 SRG(%) Acidobacteria Actinobacteria Bacteroidetes Deinococcus-Thermus Euryarchaeota Firmicutes
KSBI (candidate division) OP3 (candidate division) Others
Planctomycetes Proteobacteria Verrucamicrobia WS3 (candidate division)
Fig.1–DistributionofmicrobialphylaandcandidatedivisionsdetectedatrelativelyhigherabundanceintheKMGandSRG caves.PercentageabundancefromKMGareshownonthex-axisandfromSRGonthey-axis.Thecategory“others” representsmicrobialphylaandcandidatedivisionsthatwerepresentat<1%abundanceineachcave.KMG,KoatMaqbari Ghaar;SRG,Smasse-RawoGhaar.
18 15 12 9 6 5 4 3 2 1 0 % Cellvibr io L ysobacter T eredinibacter Bacillus De v osia Actinomadur a Saccharophagus Rhodother m u s
Ammoniphilus Virgibacillus Persicobacter Sporosarcina
Cytophaga Deinocooccus Ag robacter ir um Oceanobacillus Rhodocyclus Streptom yces Chthonibacter Acidimicrobium Br adyrhiz obium
Chitinophaga Segetibacter Rubrobacter Rhodoplanes
Prosthecobacter KSB1 WS3 Planctom yces Candidatus Haliangium Chondrom yces Thauer a Meth ylosin us Opitutus OP3 KMG SRG
Fig.2–PercentagedistributionoftherelativelydominantgeneradetectedintheKMGandSRGcaves.KMG,KoatMaqbari Ghaar;SRG,Smasse-RawoGhaar.
detectedat<0.01%abundanceinSRG(Fig.3).TheHaliangium spp.andBradyrhizobiumlupiniwerepresent athigher abun-dancesinSRGthaninKMG.ThespeciesCellvibrioostraviensis, Cellvibriojaponicus,andTeredinibacterturneraeweredominant and specific to KMG. While, Rhodoplanes spp., Methylosinus trichosporium, Chondromyces crocatus, Rhodocyclus tenuis, and ChondromycesapiculatuswerespecificallydetectedinSRG at ≥1%abundance. From the phylum Firmicutes, 250different
specieswereidentifiedincluding 27speciesthatwere com-monlypresentinbothcaves.While,79Firmicutesassociated species were specifically detected in KMG and 71 species OTUs were specificto SRG.Onlythe species;Ammoniphilus oxalaticus,Bacillusasahii,Sporosarcinaginsengisoli,and Virgibacil-lushalodenitrificansweredetectedat>1%abundanceinKMG. OtherFirmicutesspeciesweredetectedat<1%abundancein KMG and SRG.From the phylum Actinobacteria,Rubrobacter
Planc to my ces bras iliensis; Planctom ycetes KMG
% Abundance
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
SRG Prostheco bacter debon tii; Verrucomicrobia
Optitutus terrae; Verrucom icrobia Chthonio bacter flavus; Verrucomicrobia Halococcus hamelinensis; Eury archaeota Deinococcus papagonensis; Deinococcus-Thermus Chitinophaga flexibacter sancti; Bacteroidetes Riemerella anatipestifer; Bacteroidetes Flexibacter flexilis; Bacteroidetes Rhodothermus marinus; Bacteroidetes Persicobacter diffluens; Bacteroidetes Cytophaga hutchinsonii; Bacteroidetes Candidatus solibacter usitatus; Acidobacteria Actinom adura napierensis; Actinobacteria Acidimicrobium ferrooxidans; Actinobacteria Rubrobacter xylanophilus; Actinobacteria Virgibacillus halodenitrificans; Fimicutes Sporosarcina ginsengisoli; Fimicutes Bacillus asahii; Fimicutes Ammoniphilus oxalaticus; Fimicutes Chondromyces apiculatus; Proteobacteria Rhodocyclus tenuis; Proteobacteria Chondromyces crocatus; Proteobacteria Methy losinus trichosporium; Proteobacteria Rhodoplanes spp; Proteobacteria Bradyrhizobium lupini; Proteobacteria Haliangium spp; Proteobacteria Teredinibacter turnerae; Proteobacteria Cellvibrio japonicus; Proteobacteria Lysobacter spongicola; Proteobacteria Saccharophagus degradans; Proteobacteria Cellvibrio ostraviensis; Proteobacteria Segetibacter spp.; Bacteroidetes
Fig.3–PercentagedistributionofrelativelydominantOTUsatspeciesleveldetectedin≥1%atleastinonecave.KMG,Koat MaqbariGhaar;SRG,Smasse-RawoGhaar.
xylanophilusandAcidimicrobiumferrooxidansweredetectedin bothcavebutpresent atrelativehigherabundanceinSRG. ActinomaduranapierensisandCandidatussolibacterwerethetwo dominantspicesdetectedat>1%abundancespecifically in KMG and SRG,respectively. From the phylum Bacteroidetes, Cytophagahutchinsoniiwasdetectedatarelativelyhigh abun-danceinbothcaves.ThespeciesChitinophagaflexibacterand Segetibacterspp.were moreabundantintheSRG,and Persi-cobacterdiffluensand Rhodothermusmarinuswere detectedat >1%abundanceinKMG.ApartfromtheOTUsatspecieslevel from dominantphyla,the C.solibacter usitatus,Opitutus ter-rae and Planctomyces brasiliensis were detectedat relatively higher abundancein SRG sample.The Gram-negative bac-terium Deinococcus papagonensis and Halococcus hamelinensis fromarchaeaphylumEuryarchaeotawerespecificallydetected inKMG(Fig.3).
Chao1andShannonindex
Chao1 and Shannon indices were calculated to estimate alphadiversity.Rarefactioncurveswereestablishedata97% sequencesimilarityleveltodefineOTUs.Inrarefactioncurves, bothsamplesweretendedtoapproachsaturationplateau,and
thesamplesSRGwasplottedintheupperpartofthegraph (Fig.4A).TheChao1analysisdemonstratedadecreasedtrend ofrichnessinthesampleKMG(3622)comparedtoSRG(4701,
Fig.4B).TheShannondiversityindexforKMGwas9.0andfor SRGwas10.0,indicatinghighdiversityinbothcaves(Fig.4C). However,speciesdiversityinSRGwashigherthanthatofKMG.
Culturedbacterialdiversityandantimicrobialactivityof isolates
Incubationat37◦Candinfull-strengthR2Amediumyielded higher numbersofbacteria frombothcaves KMG(8.5) and SRG (8.4)(countsexpressedaslog10cfu/g)thatwere signifi-cantly(p<0.05)higherthantheCFUobtainedfromallother testedculturemediaandtemperatureusedinthisstudy.From incubationat17◦C,significantlyhighernumbersofbacteria were noticed onhalf-strength R2A from KMG(7.5), and on full-strength R2A from SRG (7.7) compared to other tested mediawithrespectivesamples.Overall,therecoveryofviable bacteriacolonieswashigherat37◦Cthanat17◦C.Initially, 350 strainswere purifiedonthe basisofcolony morpholo-gies and were screened for antimicrobialactivity. The 16S rRNAgenesof84isolatesfromthetwocavesweresequenced,
A
5000 Obser v e d O TUs Chaol estimated O TUs Shannon inde xSequences per sample
Sequences per sample KMG SRG KMG KMG SRG SRG 5000 4000 3000 2000 1000 10000 1500020000 25000 30000 15 12 9 6 3 0 0 5000 6000 4000 3000 2000 1000 0 0 50001000015000200002500030000 0
B
C
Fig.4–Alphadiversityofthesequencereads.(A)RarefactioncurvesoftheexperimentallyobservedOTUsversusdiversity (B)estimatedbyChao1.(C)BoxplotsoftheShannonindex.KMG,KoatMaqbariGhaar;SRG,Smasse-RawoGhaar.
andtheydisplayed96.2–100%identitytothesequences avail-ableinGenBank,andwereassignedtomajorbacterialgroups using phylogenetic analysis (Fig. 5). Three strains showed <97%sequencesimilaritywiththeclosestrelatedtypestrain, suggestingascandidatenovelspecies.Proteobacteriawasthe dominantphylumamongKMGisolates(52.6%),and represent-ing25.9%ofisolatesfromSRG.The␥-Proteobacteriasequences representedmajorityofisolatesfromKMG(43.5%)andSRG (11.1%).␣-Proteobacteriaand -Proteobacteriasequences were evidentin8.8%ofKMGisolates,and14.8%ofisolatesfromSRG wereaffiliatedwith␣-Proteobacteria.Firmicutesand Actinobac-teriaaccountedfor40.4%and7%,respectively,ofisolatesfrom KMG.FirmicuteswasthedominantphyluminSRG(71.4%),and noActinobacteria isolateswere detectedfromSRG.Cultured isolatesfrombothcaveswereclassifiedinto14distinct gen-era.Majorityoftheisolateswereassociatedwiththegenera Pseudomonas(40.3%KMGand11.1%SRG)andBacillus(22.8% KMGand44.4%SRG).Intotal39distinctspecieswere identi-fiedamongtheculturedisolatesfrombothcaves.Fourspecies werecommonbetweenthetwocaves.Twenty-sixspecieswere uniquetoKMG,and9specieswerespecificallydetectedinSRG. Thefollowingfourspecies,Exiguobacteriumundae,Pseudomonas koreensis,Sphingomonasmelonis,andPseudomonasmosseliiwere isolatedfromthesedimentofbothcaves.InKMG,the dom-inant cultured specieswas P. koreensis, followed byBacillus humi,Pseudomonaspeli,Pseudomonasbaetica,andPseudomonas cremoricolorata.InSRG,thedominantcultured specieswere Paenibacillus lautus, Bacillus timonensis, Bacillus simplex, and Caulobactervibrioides.
Thirty strains showed antimicrobial activity among the totalisolatesfrombothcaves(Table1).Fifteenstrainsshowed antibacterial activity against S. typhi, and 20 strains were activeagainstS.aureus.Sixstrainsshowedantibacterial activ-ityagainstbothbacterialpathogenstested. Isolatedstrains belongingtothegeneraPseudomonasandBacillusweremore antagonistic,as demonstrated strong antimicrobialactivity
againsttestedpathogenicbacteria. Onlytwostrains, which showed around 100% sequence similarity with Brevibacillus borstelensisandP.mosselii,inhibitedthegrowthofthehuman fungalpathogenC.albicans.
Discussion
Organic material inthe studied caveswas closeto starva-tion level; this is common in caves with limited external influence.22 Microorganisms residing insuch cave systems aremostlyoligotrophicorchemolithotrophicinnature,and requirespecificnutrientsfortheirgrowth.5Therefore,many bacteriaaredifficulttocultivatefromthesesources.Despite thestarvedcondition,phylogeneticallydiversebacterialtaxa colonizedbothcaves,andmainlycomprisedofProteobacteria followedbyFirmicutes,Bacteroidetes,Actinobacteria, Verrucomi-crobia, and Planctomycetes as observed in previous caves studies.23,24 Wefoundan importantdifference inthe rela-tive abundanceofdifferent Proteobacteria subdivisions.The ␣-Proteobacteria and ␥-Proteobacteria were dominant in the studiedcaves,followedby␦-Proteobacteriaand-Proteobacteria. Inthemicrobiologicallywell-studiedSpanishAltamiracave, cosmopolitanProteobacteriagroupsweredominantindripping watersandcavewalls.5,25Similarly,Proteobacteriarepresented halfofanentirewall’sbacterialcommunityintheTitoBustillo caveinSpain,andprobablyduetotheirversatilemetabolic capabilities survive on available ions in the rock contents and used themfor chemolithotrophicenergyproduction.26 TheGram-negativeProteobacteria,S.degradans thatdegrades anumberofcomplexpolysaccharidesasenergysource,was commonly identifiedin the studiedKMG and SRG caves.27 While,C.crocatusfrommyxobacteriathatpredominantlylives in the soil and feed on insoluble organic substances was specificallypresentathigherabundanceintheSRG.28
Fig.5–Phylogeneticanalysisbasedon16SrRNAgenesequencesofthebacterialisolates.Theneighbor-joiningclustering methodwasusedtoconstructthetree.Bootstrapvalueswerecalculatedbasedon1000replicationsandareshownat branchpoints.Thebarrepresents0.01sequencedivergence.SequencesderivedfromtheNCBIGenBankdatabaseare shownwiththeiraccessionnumbers.FilledcirclesfollowingtheexperimentalsequencenamescorrespondtoKMG,and non-filledcirclescorrespondtoSRG.KMG,KoatMaqbariGhaar;SRG,Smasse-RawoGhaar.
Overthepastdecade,anabundanceofActinobacteriahas been foundincavesand other subterraneanenvironments suggesting that caves are favored habitats for this group ofbacteria, and activelyinvolvedin theformation of crys-talsincavewallsand biomineralizationprocess.29,30 Inour study,severalActinobacteriawerecommonlyidentifiedinboth cavesincludingtheR.xylanophilusandA.ferrooxidans.Member fromRubrobacteriscommonlyfoundinbiodeteriorated mon-uments,inducecrystalformationincavesandformbiofilm onthelimestone.31ThephylotypesrelatedtoA.ferrooxidans identifiedinthisstudywerepreviouslydetectedincavewalls
oftheBudathermalkarstsystem.32Similarly,Firmicutesare frequently identifiedin moreextremeecosystems, and are comparativelymoreresistanttonutrientstress.2,33In particu-lar,thebacillusgroupretrievedfromthecavesinthisstudyhas theabilitytoformendospores.33Leeetal.constructeda phy-logenetictreeof16SrRNAgenesequencesretrievedfrom60 cavesaroundtheworld.23Thetreeshowedtherelative abun-dancesofdifferentclassesofbacteriaincaves,withthemost abundant groups being Proteobacteria, Chlorobi/Bacteroidetes, Actinobacteria,andChloroflexi.24However,proportionsofthese groupsdependedontheparticularfeaturesofeachcave.
Table1–Invitroantimicrobialactivityoftheculturedisolatesagainstpathogenicbacteriaandyeast.
Species Isolates S.typhi S.aureus C.albicans
Fictibacillusnanhaiensis MY-CB9 + + −
Microbacteriumoleivorans MY-CA97
Paenibacilluslautus MY-CB14
Pseudomonasgraminis MY-CA27
Pseudomonaskoreensis MY-CA28,MY-CA50
Bacillushumi MY-CA172 + − −
Bacillusmuralis MY-CB146
Bacillussimplex MY-CB11,MY-CB152
Bacillustequilensis MY-CA3
Bacillustimonensis MY-CB12
Brevibacillusborstelensis MY-CA2
Paenibacilluslautus MY-CB145
Bacilluseiseniae MY-CA109 − + −
Bacillushumi MY-CA167
Bacillusmycoides MY-CA84
Bacillusniacini MY-CB144
Bacillusthaonhiensis MY-CA168
Bacillustimonensis MY-CB128
Carnobacteriuminhibens MY-CA29
Caulobactervibrioides MY-CB65
Pseudomonasbaetica MY-CA85
Pseudomonascremoricolorata MY-CA66
Pseudomonaskoreensis MY-CA169
Pseudomonasmosselii MY-CA17
Pseudomonassimiae MY-CA99
Staphylococcuswarneri MY-CA92
Pseudomonasmosselii MY-CB149 + − +
Brevibacillusborstelensis MY-CA127 − − +
Intheisolatenames,CArepresentsKoatMaqbariGhaar(KMG)andCBrepresentsSmasse-RawoGhaar(SRG).Symbols:−,noactivity;+,activity.
Several functionally important strains were identified from genera that were reported in others cave studies, includingMethylobacterium, Rhizobiumandisolatesfrom the generaKocuria,Acinetobacter,RenibacteriumandBacillus.4,26,33,34 These bacteria can utilize a wide variety of carbon sub-strates, and play species-specific roles in nitrogenfixation andcalcification.5,25ThegenusThiothrix,detectedby pyrose-quencing were previously reported to make up thick mats of filamentous sulfur-oxidizing epsilon.23,35 Rapidly digestingcrystalline cellulosebacterium C.hutchinsonii and ionizing-radiationresistant bacteriasuchasD.papagonensis and R. xylanophilus were detected in both caves.36,37 Sev-eral oligotrophicand facultative oligortophic bacteria such as Nitrospira moscoviensis, Prochlorococcus spp., Sphingopyxis alaskensis, Sphingomonas oligophenolica, Bacillus cereus, Paeni-bacillus polymyxa, Streptomyces sp., Brevibacillus laterosporus, Bacillussubtilis,Arthrobacterspp.werefoundincultured inde-pendentpyrosequencinganalysisofthisstudy,andwere pre-viouslyreportedfromdifferentoligotrophicenvironments.38 In addition, mesophilic Crenarchaeota and particularly Eur-yarchaeota were detectedat relatively higher abundance in the KMG that were previously found in the Lechuguilla Cave (United States)33 and in the AltamiraCave (Spain).39 Legatzkietal.reportedthepresenceofanarchaeal commu-nityoncalcitespeleothemsfromKartchnerCaverns,Arizona, USA.40
Oneofthelimitationofthisstudywasthatonlyaerobic conditionalong withtwomediacompositionsand temper-atureconditionswere usedforculture-dependentanalysis.
Overall, lower diversity was observed among cultured iso-lates that cannot be compared to the diversity observed withthepyrosequencinganalysis.Theculturedisolatesfrom bothcaveswereclassifiedintothephylaProteobacteria, Acti-nobacteria,andFirmicutes.Amongthe13generaidentifiedby culture, a few ofthem were detectedin the pyrosequenc-ing analysis,including Bacillus,Microbacterium,Pseudomonas, andPsychrobacter.However,bacteriafromthegenera Carnobac-terium,Exiguobacterium,Paucisalibacillus,andFictibacilluswere not detected in pyrosequencing data. Interestingly,among the39distinctculturedbacterialspecies,onlysevenspecies, B. humi,Bacillusniacin, B.simplex,C.vibrioides,P. lautus, Psy-chrobacter alimentarius, and S. melonis were found in the pyrosequencingdataindicatethatthebacteriainlow abun-dance in a community can be only captured by cultured analysisaspreviouslyobserved.12Thephylogeneticanalysis and16SrRNAgenesequencesimilarityindicatedthatthree oftheculturedisolateshavenocloserelativeamongthe cul-turedtypestrains.Thesestrainsrepresentnovelcandidatesin cavesbiodiversity,indicatingthatculturetechniquesprovide uswiththeopportunitytodiscovernewmicroorganismsfrom cavesecosystem.30Theidentificationofnewpathogenic bac-teriaandtherapiddevelopmentofantimicrobialresistance highlight theimportanceofdiscovery ofnewantimicrobial agents.41Severalstrainsisolatedfromthecavesinthisstudy, includingActinobacteria,Myxobacteria,Pseudomonas,and Bacil-lusspp.showedantimicrobialactivityagainsttwoimportant pathogenicbacteria,S.typhiandS.aureus.Inrecentyears, sev-eral studieshighlightedthat rarelystudiedcave microbiota
couldbeapotentialsourceofnewmicroorganismandasource forfutureantimicrobialagents.42
In conclusion, diverse microbial communities were observed within the studied caves that were correspond-ing to other caves at higher taxonomic level. However, at lowertaxonomiclevelseveraldistincttaxawereidentified.In addition,nitrogenfixation,crystallinecellulosedigestingand ionizing-radiationresistantrelatedbacteriaweredetectedin theculture-independentanalysis.Cultivatedisolatesfromthe generaBacillusandPseudomonasweredominantinbothcaves andexhibitedantimicrobialactivityagainstpathogenic bac-teriaandyeast.Thereisaneedforfurtherstudiestoexplore virgincavessystemsintheworld’shighestmountainsrangein northernPakistan,andtoexploitthemicrobialcommunities ofthosecavesfornewbiomolecules.
Conflicts
of
interest
Theauthordeclaresnoconflictsofinterest.
Acknowledgments
ThisworkwasfundedbytheDeanshipofScientificResearch (DSR),KingAbdulazizUniversity,Jeddah,undergrantno. (141-003-D1434).Theauthor,therefore,acknowledgewiththanks DSRtechnicalandfinancialsupport.
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