ARTICLE IN PRESS

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h tt p : / / 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 Availableonlinexxx

AssociateEditor:JohnMcCulloch

Keywords:

Caves

16SribosomalRNA Microbiota Antimicrobial Sediments

a bs t r a c t

Inthisstudyfortheﬁrst-timemicrobialcommunitiesinthecaveslocatedinthemountain rangeofHinduKushwereevaluated.Thesampleswereanalyzedusingculture-independent (16S rRNA 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 oftheculturedbacterialisolatesrevealeddistinctbacterialtaxonomicproﬁlesinthestud- iedcavesdominatedbyProteobacteriainKoatMaqbariGhaarandFirmicutesinSmasse-Rawo Ghaar.MajorityofthoseisolateswereassociatedwiththegeneraPseudomonasandBacillus.

Thirtystrainsamongtheidentiﬁedisolatesfrombothcavesshowedantimicrobialactiv- ity.Overall,thepresentstudygaveinsightintothegreatbacterialtaxonomicdiversityand antimicrobialpotentialoftheisolatesfromthepreviouslyuncharacterizedcaveslocatedin theworld’shighestmountainsrangeintheIndiansub-continent.

licenses/by-nc-nd/4.0/).

Introduction

Caves withsurface entrances represent oneof the unique andpoorlystudiedecosystemsonEarth.Theyincludehydro- logicalsystemsthatarerelativelyisolatedfromthesurface, andsharebasicphysicochemicalconditions,includingcom- pletedarkness, constant humidity, and thermal stability.^1,2 Caves comprised of unique underground communities of

E-mail:[email protected]

organisms,andcavemicroclimatesoftensupportdensepop- ulationsofextremophiles.³Multidisciplinarystudiesoncave microbiologyhaveimplicatedmicroorganismsinthegeolog- ical processes of caves, and have opened several avenues ofresearch,includingcavegeochemistry,caveenvironmen- talmicrobiologyandidentiﬁcationofnewmicrobialspecies andexplorationofnovelbiotechnologicalmoleculesfromcave sources.^4,5Recently,severalnewmembersofthegeneraCatel- latosporaandNonomuraeawerediscoveredincavesofMexico

https://doi.org/10.1016/j.bjm.2017.08.005

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Pleasecitethisarticleinpressas:YasirM.Analysisofbacterialcommunitiesandcharacterizationofantimicrobialstrainsfromcavemicrobiota.

and NorthernThailand.^6,7 In 2005,Agromyces subbeticussp.

nov.,wasisolatedfromacaveintheCordobaareaofsouth- ernSpain.⁸ Juradoet al. reported Aurantimonasaltamirensis sp. nov., a Gram-negative member of the order Rhizobiales fromAltamiraCave,locatedintheCantabria,Spain.⁹These microorganismsnewlydiscoveredincavesdemonstratethe potentialforidentifyingsecondarymetabolitesthathaveyet tobefullyevaluatedandexploited.^4,7

Cavesarefoundworldwide,andbiospeleologicalresearch hasbeenmuchincreasedinthe lasttwentyyears.^2,4 Avail- ableliteratureindicatesthe existenceofshortcavesinthe world’s highest mountains range called Karakoram, Hindu Kush,andPamir,whichincorporatesomeoftheworld’shigh- 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 TheNangaParbathasRakhiotCave, whichis73.2mlongandthehighestknowncaveatanalti- tude of6644m.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, microbial communitieswere investigatedinthe twocaves, KoatMaqbari Ghaar (KMG)and Smasse-Rawo Ghaar (SRG), locatedintheHinduKushMountainandsituatedinthenorth- ernKhyberPakhtunkhwaprovinceofPakistan.Advancesin sequence-basedmetagenomicapproacheshavemadestudies onmicrobialdiversityandcommunitycompositionindiverse environmentsmorepossibleandinformative.However,using high-throughputsequencing techniquesalone tostudy the microbialcommunityinanenvironmenthasthepossibilities tomissthelowabundanttaxaaspreviouslyobserved.¹²Inthis study,anintegratedapproachofculture-basedandculture- independentpyrosequencing methodswere usedto gain a moreaccuraterepresentationofthemicrobialcommunities inthestudysites.Bacterialstrainsisolatedinthisstudywere screenedforantibacterialandantifungalactivities.

Materials and methods

Samplescollectionandchemicalanalysis

Thisstudyrepresentsapolyphasicanalysisofmicrobialcom- 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 SRG cavehasanalmost horizontalorientationandaround 2mwide,10mlong,andislocatedat1062mabovesealevel.

Bothcavesareofdriednatureandverylimitedlyinﬂuencedby anthropogenicactivities.Sedimentsamplesinthreereplicates werecollectedfromeachcaveusingautoclavedandsterilized bottles.Thesampleswere collectedinFebruary2013.Allof thesampleswerestoredat4^◦Cforaround20hduringtrans- portation,andwereimmediatelyprocessedforculturingafter

arrivinginthelaboratory.Apartofeachsamplewasstoredat

−80^◦CformetagenomicDNAextraction.Nospeciﬁcpermis- 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.Totalsoilorganicmatterandtotalnitrogenweredeter- minedusingthepartialoxidationmethodandmicroKjeldahl method.¹³Totalphosphoruswasmeasuredcolorimetrically.¹³ Physicochemicalanalysiswereperformedintriplicateforeach sample.

DNAextractionandpyrosequencing

TotalDNAwasextractedfrom eachhomogenizedcavesed- imentreplicatesusingtheprotocolforthePowerSoil^® DNA extractionkit(MoBioLaboratories,Carlsbad).Amplification of the 16S rRNA gene hypervariable region V4 was performed using bar-coded 515F and 806R universal primers containing A and B sequencing adaptors, following the procedure previouslydescribed.¹⁴ PCRproductswerequan- 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<150bpwere removed,andtheremainingreadswere screened for homopolymer runs exceeding 6bp, chimeric sequences,and sequences containingNs;all ofthese were also excluded. Barcodes and primers were depleted from sequences.Highqualitysequencereadswereclusteredinto OTUsusingathresholdof97%sequencesimilarity.Forsingle- tonreadsthedefaultvalueof2readsinQIIMEv1.9software was used to exclude from further analysis.¹⁵ OTUs were taxonomically classified using BLASTn againstthe 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 1/10strength 1.3g/L) supplementedwith 1.5% agar, 20% aqueousextract ofthe collected cave sediment,andtwoincubationconditions, 17^◦Cand 37^◦C,were usedtoculturebacteriafromthecavessamples.Theplates were incubated in aerobic condition for one week in seal

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plasticbagscontainingwettissuepaperstoavoiddryingof culturemediaduring this long incubation. Colonyforming units(CFU)werecounted,andtheCFUofthethreereplicates were usedtoestimatethe sizeofthe bacterialpopulation.

Colonieswerepurifiedbysub-culturingandthenstoredusing a15%glycerolstockincryogenicvialsat−80^◦C.Eighty-four isolateswereselectedfor16SrRNAgenesequencingbasedon thecolonymorphologyandgrowthcharacteristics.Genomic DNAwasextractedfromtheisolatedstrainsusing1%TritonX- 100(Sigma)andboilingat95^◦Cfor20min.The16SrRNAgene wasamplifiedusingtheuniversalbacterialprimers,27Fand 1492R,asdescribedpreviously.¹⁷PurifiedPCRproductswere sequencedwithABIPrismSequencer3730(AppliedBiosys- tems,USA)accordingtomanufacturer’sprotocol.Ananalysis ofchimerasinthe16SrRNAgenesequenceswasperformed usingBellerophonsoftware.¹⁸Sequenceswereblastedatthe EzTaxon server(http://www.ezbiocloud.net/)to identifythe related typestrains.¹⁹ A multiplesequence alignment was performed,andaphylogenetic treewasconstructedbythe neighbor-joiningdistancemethodusingJukes-Cantormodel tocomputeevolutionarydistanceinMEGA6software.²⁰Boot- 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 confrontationbioassay using paper discs. A concentra- tion of0.5 McFarland ofthe target strains were spread on media-agarplates.Twentymicrolitersofeachbacterialsus- pensioncontainingapproximately10⁸cfu/mLwasadded to asterilepaperdisk(Advantec, Japan),and thendiscs were placed on the target strain plates. The plates were incu- batedfor72h at25^◦C,andzones ofinhibitionaround the paperdiscs weremeasured.²¹ Chloramphenicol(30␮g/disk) andamphotericin-B(20␮g/disk)wereusedasreferencetoesti- matetheantimicrobialactivitiesofthetestedisolatesagainst bacteriaandyeastrespectively.

Statisticalanalysis

One-wayANOVAandTukey’sHSD(HonestlySigniﬁcantDiffer- ence)testswereusedtostatisticallycomparephysicochemical parameters,and CFUobtainedatdifferentmediacomposi- tionsandincubationtemperature.TheStatisticalPackagefor theSocialSciences(SPSS)version20wasusedforthestatisti- 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.Totalorganicmatterandnitro- 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-qualitypyrose- 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 candidate phyla. Among the identified phyla, 10 phyla and 5 candidates divisionsweredetectedinboth caves.Theper- centagesequence readsfrom KMG andSRG were affiliated with 6 bacterial phyla detected at relatively higher abundance including; Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes,VerrucomicrobiaandPlanctomycetes(Fig.1).Among theProteobacteria,␣-Proteobacteria(45.6%KMGand34.1%SRG) and ␥-Proteobacteria(35.2%KMG and32.1% SRG)weredom- 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(candidatedivision),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,17generainKMGand21gen- 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,andActinobacte- ria.Intotal,170(44.3%)identifiedspeciesfromKMGand309 (35.4%)from SRG wereaffiliated withProteobacteria.Among them,89specieswerecommonlypresentinbothcavesinclud- ing Saccharophagus degradans and Lysobacter spongicola that werepresentatrelativelyhigherabundanceinKMG,andwere

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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

%

Cellvibrio Lysobacter Teredinibacter Bacillus Devosia Actinomadura Saccharophagus Rhodothermus Ammoniphilus Virgibacillus Persicobacter Sporosarcina Cytophaga Deinocooccus Agrobacterirum Oceanobacillus Rhodocyclus Streptomyces Chthonibacter Acidimicrobium Bradyrhizobium Chitinophaga Segetibacter Rubrobacter Rhodoplanes Prosthecobacter KSB1 WS3 Planctomyces Candidatus Haliangium Chondromyces Thauera Methylosinus Opitutus OP3 KMG SRG

Fig.2–PercentagedistributionoftherelativelydominantgeneradetectedintheKMGandSRGcaves.KMG,KoatMaqbari Ghaar;SRG,Smasse-RawoGhaar.

detectedat<0.01%abundanceinSRG(Fig.3).TheHaliangium spp.andBradyrhizobiumlupiniwerepresent athigherabun- 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

specieswereidentifiedincluding27speciesthatwerecom- monlypresentinbothcaves.While,79Firmicutesassociated species were specifically detected in KMG and 71 species OTUs were specificto SRG.Onlythe species;Ammoniphilus oxalaticus,Bacillusasahii,Sporosarcinaginsengisoli,andVirgibacil- lushalodenitrificansweredetectedat>1%abundanceinKMG.

OtherFirmicutesspeciesweredetectedat<1%abundancein KMG and SRG.From the phylum Actinobacteria, Rubrobacter

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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, Cytophagahutchinsoniiwasdetectedatarelativelyhighabun- danceinbothcaves.ThespeciesChitinophagaflexibacterand Segetibacterspp.were moreabundantinthe SRG,andPersi- cobacterdiffluensand Rhodothermusmarinusweredetectedat

>1%abundanceinKMG.ApartfromtheOTUsatspecieslevel from dominantphyla,the C.solibacter usitatus, Opitutuster- rae and Planctomyces brasiliensis were detectedat relatively higher abundancein SRG sample.The Gram-negativebac- 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)thatweresignifi- 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 toother tested mediawithrespectivesamples.Overall,therecoveryofviable bacteriacolonieswashigherat37^◦Cthanat17^◦C.Initially, 350 strainswere purifiedonthe basisofcolonymorpholo- gies and were screened for antimicrobialactivity. The 16S rRNAgenesof84isolatesfromthetwocavesweresequenced,

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A

5000

Observed OTUs Chaol estimated OTUs

Shannon index

Sequences per sample

Sequences per sample KMG KMG SRG

KMG SRG

SRG 5000

4000 3000 2000 1000

10000150002000025000 30000

15 12 9 6 3 0 0

5000 6000

4000 3000 2000 1000 0 0

5000 100001500020000 25000 30000 0

B

C

Fig.4–Alphadiversityofthesequencereads.(A)RarefactioncurvesoftheexperimentallyobservedOTUsversusdiversity (B)estimatedbyChao1.(C)BoxplotsoftheShannonindex.KMG,KoatMaqbariGhaar;SRG,Smasse-RawoGhaar.

andtheydisplayed96.2–100%identitytothesequencesavail- ableinGenBank,andwereassignedtomajorbacterialgroups using phylogenetic analysis (Fig. 5). Three strains showed

<97%sequencesimilaritywiththeclosestrelatedtypestrain, suggestingascandidatenovelspecies.Proteobacteriawasthe dominantphylumamongKMGisolates(52.6%),andrepresent- ing25.9%ofisolatesfromSRG.The␥-Proteobacteriasequences representedmajorityofisolatesfromKMG(43.5%)andSRG (11.1%).␣-Proteobacteriaand ␤-Proteobacteriasequences were evidentin8.8%ofKMGisolates,and14.8%ofisolatesfromSRG wereafﬁliatedwith␣-Proteobacteria.FirmicutesandActinobac- teriaaccountedfor40.4%and7%,respectively,ofisolatesfrom KMG.FirmicuteswasthedominantphyluminSRG(71.4%),and noActinobacteria isolatesweredetectedfrom SRG.Cultured isolatesfrombothcaveswereclassiﬁedinto14distinctgen- era.Majorityoftheisolateswereassociatedwiththegenera Pseudomonas(40.3%KMGand11.1%SRG)andBacillus(22.8%

KMGand44.4%SRG).Intotal39distinctspecieswereidenti- ﬁedamongtheculturedisolatesfrombothcaves.Fourspecies werecommonbetweenthetwocaves.Twenty-sixspecieswere uniquetoKMG,and9specieswerespeciﬁcallydetectedinSRG.

Thefollowingfourspecies,Exiguobacteriumundae,Pseudomonas koreensis,Sphingomonasmelonis,andPseudomonasmosseliiwere isolatedfromthesedimentofbothcaves.InKMG,thedom- 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.Sixstrainsshowedantibacterialactiv- ityagainstbothbacterialpathogenstested. Isolatedstrains belongingtothegeneraPseudomonasandBacillusweremore antagonistic,as demonstrated strong antimicrobialactivity

againsttestedpathogenic bacteria.Onlytwostrains,which showed around 100% sequence similarity with Brevibacillus borstelensisandP.mosselii,inhibitedthegrowthofthehuman fungalpathogenC.albicans.

Discussion

Organic material inthe studied caveswas close tostarva- 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 abundanceof differentProteobacteria 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 them forchemolithotrophicenergyproduction.²⁶ TheGram-negativeProteobacteria,S.degradansthatdegrades 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

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Fig.5–Phylogeneticanalysisbasedon16SrRNAgenesequencesofthebacterialisolates.Theneighbor-joiningclustering methodwasusedtoconstructthetree.Bootstrapvalueswerecalculatedbasedon1000replicationsandareshownat branchpoints.Thebarrepresents0.01sequencedivergence.SequencesderivedfromtheNCBIGenBankdatabaseare shownwiththeiraccessionnumbers.FilledcirclesfollowingtheexperimentalsequencenamescorrespondtoKMG,and non-ﬁlledcirclescorrespondtoSRG.KMG,KoatMaqbariGhaar;SRG,Smasse-RawoGhaar.

Overthepastdecade,anabundanceofActinobacteriahas beenfound incavesand other subterraneanenvironments suggesting that caves are favored habitats for this group ofbacteria, andactively involvedinthe formation ofcrys- talsincavewallsandbiomineralization process.^29,30 Inour study,severalActinobacteriawerecommonlyidentifiedinboth cavesincludingtheR.xylanophilusandA.ferrooxidans.Member fromRubrobacteriscommonlyfoundinbiodeterioratedmon- uments,inducecrystalformationincavesandformbiofilm onthelimestone.³¹ThephylotypesrelatedtoA.ferrooxidans identifiedinthisstudywerepreviouslydetectedincavewalls

oftheBudathermalkarstsystem.³²Similarly,Firmicutesare frequently identiﬁedin moreextremeecosystems, andare comparativelymoreresistanttonutrientstress.^2,33Inparticu- lar,thebacillusgroupretrievedfromthecavesinthisstudyhas theabilitytoformendospores.³³Leeetal.constructedaphy- logenetictreeof16SrRNAgenesequencesretrievedfrom60 cavesaroundtheworld.²³Thetreeshowedtherelativeabun- dancesofdifferentclassesofbacteriaincaves,withthemost abundant groups being Proteobacteria, Chlorobi/Bacteroidetes, Actinobacteria,andChloroﬂexi.²⁴However,proportionsofthese groupsdependedontheparticularfeaturesofeachcave.

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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, Rhizobiumand isolatesfrom 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,detectedbypyrose- 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.werefoundinculturedinde- pendentpyrosequencinganalysisofthisstudy,andwerepre- viouslyreportedfromdifferentoligotrophicenvironments.³⁸ In addition, mesophilic Crenarchaeota and particularly Eur- yarchaeota were detected atrelatively higher abundance in the KMG that were previously found in the Lechuguilla Cave (United States)³³ and inthe AltamiraCave (Spain).³⁹ Legatzkietal.reportedthepresenceofanarchaealcommu- nityoncalcitespeleothemsfromKartchnerCaverns,Arizona, USA.⁴⁰

Oneofthelimitationofthisstudywasthatonlyaerobic conditionalong withtwomediacompositionsand temper- atureconditionswere usedforculture-dependentanalysis.

Overall, lower diversity was observed among cultured isolates that cannot be compared to the diversity observed withthepyrosequencinganalysis.Theculturedisolatesfrom bothcaveswereclassifiedintothephylaProteobacteria,Acti- nobacteria,andFirmicutes.Amongthe13generaidentifiedby culture, a few ofthem were detectedin the pyrosequencing analysis,including Bacillus, Microbacterium, Pseudomonas, andPsychrobacter.However,bacteriafromthegeneraCarnobac- 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 pyrosequencingdataindicatethatthebacteriainlowabun- dance in a community can be only captured by cultured analysisaspreviouslyobserved.¹²Thephylogeneticanalysis and16SrRNAgenesequencesimilarityindicatedthatthree oftheculturedisolateshavenocloserelativeamongthecul- turedtypestrains.Thesestrainsrepresentnovelcandidatesin cavesbiodiversity,indicatingthatculturetechniquesprovide uswiththeopportunitytodiscovernewmicroorganismsfrom cavesecosystem.³⁰Theidentificationofnewpathogenicbac- teriaandtherapiddevelopmentofantimicrobialresistance highlight theimportanceofdiscovery ofnewantimicrobial agents.⁴¹Severalstrainsisolatedfromthecavesinthisstudy, includingActinobacteria,Myxobacteria,Pseudomonas,andBacil- lusspp.showedantimicrobialactivityagainsttwoimportant pathogenicbacteria,S.typhiandS.aureus.Inrecentyears,several studieshighlightedthat rarelystudiedcave microbiota

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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 andexhibitedantimicrobialactivityagainstpathogenicbac- 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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