Characterization of Bifidobacterium species in feaces of the Egyptian fruit bat: Description of B. vespertilionis sp. nov. and B. rousetti sp. nov.

ContentslistsavailableatScienceDirect

Systematic

and

Applied

Microbiology

j o ur na l h o me pa g e :h t t p : / / w w w . e l s e v i e r . c o m / l o c a t e / s y a p m

Characterization

of

Bifidobacterium

species

in

feaces

of

the

Egyptian

fruit

bat:

Description

of

B.

vespertilionis

sp.

nov.

and

B.

rousetti

sp.

nov.

Monica

Modesto

_,

_Maria

_Satti

_,

_Koichi

_Watanabe

_,

_Edoardo

_Puglisi

_,

Lorenzo

Morelli

_,

_Chien-Hsun

_Huang

_,

_Jong-Shian

_Liou

_,

_Mika

_Miyashita

_,

Tomohiko

Tamura

_,

_Satomi

_Saito

_,

_Koji

_Mori

_,

_Lina

_Huang

_,

_Piero

_Sciavilla

_,

Camillo

Sandri

_,

_Caterina

_Spiezio

_,

_Francesco

_Vitali

_,

_Duccio

_Cavalieri

_,

Giorgia

Perpetuini

_,

_Rosanna

_Tofalo

_,

_Andrea

_Bonetti

_,

_Masanori

_Arita

_,

Paola

Mattarelli

a_{DepartmentofAgriculturalSciences,UniversityofBologna,VialeFanin44,40127Bologna,Italy}

b_{DepartmentofGenetics,SOKENDAIUniversity(NationalInstituteofGenetics),Yata1111,Mishima,Shizuoka411-8540,Japan} c_{DepartmentofAnimalScienceandTechnology,NationalTaiwanUniversity,Taipei,Taiwan}

d_{BioresourceCollectionandResearchCenter,FoodIndustryResearchandDevelopmentInstitute,Hsinchu,Taiwan}

e_{DepartmentforSustainableFoodProcesses,FacultyofAgricultural,FoodandEnvironmentalSciences,Universita`CattolicadelSacroCuore,ViaEmilia} Parmense84,29122Piacenza,Italy

f_{BiologicalResourceCenter(NBRC),NationalInstituteofTechnologyandEvaluation(NITE),2-5-8,Kazusakamatari,Kisarazu,Chiba292-0818,Japan} g_{DepartmentofAnimalHealthCareandManagement,ParcoNaturaViva—GardaZoologicalPark,Bussolengo,Verona,Italy}

h_{DepartmentofBiology,UniversityofFlorence,Florence,Italy}

i_{FacultyofBioscienceandTechnologyforFood,AgricultureandEnvironment,UniversityofTeramo,Teramo,Italy} j_{RIKENCenterforSustainableResourceScience,1-7-22Suehiro,Tsurumi,Yokohama,Kanagawa230-0045,Japan} k_{BioinformationandDDBJCenter,NationalInstituteofGenetics,Yata1111,Mishima,Shizuoka411-8540,Japan}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received15May2019

Receivedinrevisedform18August2019 Accepted20August2019

Keywords: Newspecies Bifidobacterium

Bifidobacteriumvespertilionissp.nov. Bifidobacteriumrousettisp.nov. Rousettusaegyptiacus

a

b

s

t

r

a

c

t

FifteenbifidobacterialstrainswereobtainedfromfaecesofRousettusaegyptiacus;aftergroupingthem byRAPDPCRonlyeightwereselectedandcharacterized.Analysisof16SrRNAandoffivehousekeeping (hsp60,rpoB,clpC,dnaJ,dnaG)genesrevealedthattheseeightstrainswereclassifiedintofiveclusters: ClusterI(RST8andRST16T_{),ClusterII(RST9}T_{andRST27),ClusterIII(RST7andRST11),ClusterIV(RST}

19),ClusterV(RST17)wereclosesttoBifidobacteriumavesaniiDSM100685T_{(96.3%),Bifidobacterium}

callitrichosDSM23973T_{(99.2%and99.7%),BifidobacteriumtissieriDSM100201}T_{(99.7and99.2%),}

Bifi-dobacteriumreuteriDSM23975T_{(98.9%)andBifidobacteriummyosotisDSM100196}T_{(99.3%),respectively.}

StrainsinClusterIandstrainRST9inClusterIIcouldnotbeplacedwithinanyrecognizedspecieswhile theotheroneswereidentifiedasknownspecies.Theaveragenucleotideidentityvaluesbetweentwo novelstrains,RST16T_andRST9T_{andtheirclosestrelativeswerelowerthan79%and89%,respectively.}

InsilicoDNA–DNAhybridizationvaluesforthoseclosestrelativeswere32.5and42.1%,respectively. Phe-notypicandgenotypictestsdemonstratedthatstrainsinClusterIandRST9T_{inClusterIIrepresenttwo}

novelspeciesforwhichthenamesBifidobacteriumvespertilionissp.nov.(RST16T_=BCRC81138T_=NBRC

113380T_=DSM106025T_{;RST8=BCRC81135=NBRC113377)andBifidobacteriumrousettisp.nov.(RST}

9T_=BCRC81136T_=NBRC113378T_=DSM106027T_{)areproposed.}

©2019ElsevierGmbH.Allrightsreserved.

Ofalllivingmammals,batsareextraordinaryanduniquegiven theirpeculiaradaptations[28].Theyaretheonlymammalsthat

∗ Correspondingauthorat:DepartmentofAgriculturalSciences,Universityof Bologna,VialeFanin44,40127Bologna,Italy.

E-mailaddress:[email protected](P.Mattarelli). 1 _{Theseauthorscontributedequally.}

canperformrealactiveflight,whichisessentialfortheirbiology [14]andthischaracteristicgavethebatorderitsscientificname —Chiroptera,orhand-winginGreek[11,8].Theyhaveevolvedto thriveindiverseecologicalnichesacrosstheglobe[28]andcanfeed oninsects,smallmammals,fish,blood,nectar,fruit,andpollen[27].

https://doi.org/10.1016/j.syapm.2019.126017 0723-2020/©2019ElsevierGmbH.Allrightsreserved.

[1,28].Bats areabletomigrateover longdistances,creating opportunitiesfordiverseexposureandwidespreaddissemination ofmicrobes[29].

The Egyptian fruit-bat Rousettus aegyptiacus (body mass: 140–160g)iswidelydistributedintropicalandsubtropicalregions ofAfrica,andAsia.Itsnorthernmostdistributionreachessouthern Turkey.Thisbatconsumesavarietyofwildandcultivatedfruits suchasFicusretusaandF.sycomorus(fig),Meliaazedarach (chin-aberry),Phoenixdactylifera(date),mangoandbananaandtherefore isapotentialseeddisperser[13,5].

Fromtheanatomicalstandpoint,Rousettusaegyptiacushasa rel-ativelyshortintestine,notdifferentiatedintosmallandlargeparts andwithnoobservedcecumorappendix;consequently,the dura-tionoftheintestinalpassisapproximately40min[11,12].

Studiesonthemicrobialecologyofgutmicrobiotainbatsare limitedandsuchinformationisnecessaryindeterminingthe eco-logicalsignificanceofthesehosts[11].

Therearerelatively fewerstudiesinvestigating thebacterial microbiotainbatswithrespecttodietaryhabitsofbats.Except a few [11,12],majority of the studieshave used culture based approachtostudythebacterialcommunitiesassociatedwiththe batintestinaltract.Thesehaveledtotheidentificationof differ-entbacteriasuchasSalmonella,Shigella,Enterobacter,Yersiniaand manyotherentericpathogensfromthebatdigestivetract. Differ-entsourcesusedforbacterialisolationandidentificationincludes urine,guanoandintestinalcontentorintestine[1,7,11].

Atthetimeofwriting,thereisnostudyinvestigatingthe pres-enceandthedistributionofBifidobacteriumspeciesinfruitbats. Severalstudieshave suggested theimportanceof isolating and identifyingnovelstrainsofthegenusBifidobacteriumfrom vari-ousanimals,includinghumans,inordertounderstandhowthey are mostly distributed [17,19] and, especially, which are their phenotypicandgenotypiccharacteristics,thusallowingthe recon-structionofamorerobustbifidobacterialphylogeny.

Therefore,theobjectiveofthisstudywasthecharacterization ofbifidobacterialisolatesderivedfromthefaecesofEgyptianfruit bat(Rousettusaegyptiacus).

InMarch2017,freshguanosfromthecaptivecolonyofthe Egyp-tianfruitbathousedundersemi-naturalconditionsinParcoNatura Viva-GardaZoologicalPark(Bussolengo,Verona,Italy),were col-lectedfromthegroundusingasterilespoonbytheanimal-care staffs(keepers)duringtheirroutinecleaningoftheenclosure,put intoasterileplastictubeandstoredunderanaerobicconditionsin ananaerobicjar(Merck)at4◦C,andweretakenpromptlytothe laboratory(within2h).Theanaerobicatmospherewasobtained usingtheGasPakEZAnaerobicPouchsystem(BD).

Animalswerefreefromintestinalinfectionsanddidnotreceive antibioticsorprobioticsfortwomonthsbeforesampleswere col-lected.Theirdietconsistedmostlyoffruit.

For isolation and enumeration of bifidobacteria, aliquots of approximately 1–2g of faecal sample were serially 10-fold dilutedwithPeptoneWater(Merck)supplementedwithl-cysteine hydrochloride(0.5g/L).Aliquotsof1mlfromeachdilution(from 10−1downto10-9_{)wereinoculatedontoMRS(Difco)agar} supple-mentedwithl-cysteinehydrochloride(0.5g/L),aceticacid(1ml/L) (Merck)andmupirocin(100mg/L)(Applichem)[21].Plateswere incubatedinanaerobicconditions,at37◦Cfor48–72h.The anaer-obic atmosphere was obtained using the GasPak EZ Anaerobic Pouchsystem.Afterincubation, resultsshowedthepresence of bifidobacteria,withacolonycountnumberof4.6log10CFUg−1 fae-ces.Therefore,morphologicallydifferentcolonieswererandomly pickedandre-streakedforseveralgenerationsinordertoisolate purifiedindividualbacterialstrains.

A total of 15 strains were obtained, namely: RST 1, RST 2, RST 3, RST 4, RST 5, RST 7, RST 8, RST 9, RST 11, RST 12, RST 16, RST 17, RST 19, RST 26 and RST 27. For discrimina-tionof the isolates, RAPD-PCRfingerprintings werecarried out by using BOXA1R primer (5 -CTACGGCAAGGCGACGCTGACG-3), ERIC1 (5-ATGTAAGCTCCTGGGGATTCAC-3) and ERIC2 (5-AAGTAAGTGACTGGGGTGAGCG-3) primers, as previously described [18,19]. Based on the resulting BOX-PCR and ERIC bandingprofiles, theisolates werecategorized into sixgroups: GroupI(RST1,RST2,RST3,RST4,RST5,RST8,RST12,RST16and RST26),GroupII(RST9T_{),GroupIII(RST7andRST11),GroupIV} (RST19),GroupV(RST17)andGroupVI(RST27)(Supplementary Figs. S1 and S2). One or wherever possible two representative strainsforeach groupwereselectedandatotal ofeightstrains werechosen(RST8andRST16T_{forGroupI,RST9}T_forGroupII, RST7andRST11forGroupIII, RST19 forGroupIV,RST17for GroupVandRST27forGroupVI)for16SrRNAgenesequencing asdescribedbyMichelinietal.[16].

The16SrRNAgenesequences(1400bp)ofstrainsandoftheir closestrelativesretrievedfromtheDDBJ/GenBank/EMBLdatabases werealigned using CLUSTAL Omega ina CLCSequence Viewer (1328nt).Aphylogenetictreebasedona totalof81partial16S rRNAgenesequences,includingthoseofmembersofthegenus Bifidobacteriumwasreconstructedwiththemaximum-likelihood method [3] and theevolutionary distances were computed by nucleotide model of GTR CAT.The treewas constructed using RaxMLversion8.2.7[25]androotedwithScardoviainopinataJCM 12537T_{.Thestatisticalreliabilityofthetreewasevaluatedbythe} bootstrap analysis of 1000 replicateswith thealgorithm Boot-straprapidhillclimbingalgorithm.Thetreewasvisualizedusing FigTree[22].

Comparativeanalysisofthe16SrRNAgenesequencerevealed thattheseeightstrainswereclassifiedintofiveclusters:Cluster I(RST8,RST16T_{),ClusterII(RST9}T_{andRST27),ClusterIII(RST} 7,RST11),ClusterIV(RST19),ClusterV(RST17).Strainsin Clus-terI(RST8,RST16T_{)weretheclosesttoBifidobacteriumavesanii} (96.30%similarity),strainsinClusterII(RST9T_{andRST27)were} closesttoBifidobacteriumcallitrichosDSM23973T_{(99.2%and99.7%,} respectively),strainsinClusterIII(RST7andRST11)wereclosest toBifidobacteriumtissieriDSM100201T_{(99.7%and99.2%),strain} inClusterIV(RST19)wasclosesttoBifidobacteriumreuteriDSM 23975T _{(98.9%),straininClusterV(RST17)wasclosestto} Bifi-dobacteriummyosotisDSM100196T_{(99.3%)(Fig.1).}

In order to assess the genetic diversity of the eight strains as compared to the other currently recognized Bifidobacterium species,multilocussequenceanalysis(MLSA)basedonthe con-catenatedfivehousekeepinggenes(hsp60,rpoB,clpC,dnaJ,dnaG) wascarriedout.Thus,thephylogeneticlocationofthenovelstrains wasverifiedby theanalysisof geneswhich haveproven tobe discriminativefor theclassificationof thegenusBifidobacterium [10,30].

Forthispurpose,aphylogenetictreefor79bifidobacterialtype strainswasconstructedbyjoiningthefivecodingsequencesinthe followingorder:hsp60(662bp),rpoB(500bp),clpC(720bp),dnaJ (477bp)anddnaG(992bp).Theresultingin-frameconcatenated genesequences(3154bp)werealignedwiththeMAFFTprogram atCBRC(http://mafft.cbrc.jp/alignment/software/)[9].The evolu-tionarydistanceswerecomputedbynucleotidemodelGTRCAT, andthephylogenetictreewasconstructedbyRaxML(version8.2.7, maximum-likelihoodmethod)[25]withScardoviainopinataJCM 12537T_{asanoutgroup.Thestatisticalreliabilityofthetreewas} evaluatedbybootstrapanalysis(rapidhillclimbing)of1000 repli-cates.ThevisualizationwasperformedwithFigTree.

Fig.1.PhylogenetictreeoftheBifidobacteriumgenusbasedon16SrRNAgenesequences,showingtherelationshipbetweenstrainsisolatedfromEgyptianfruitbatsand othermembersoftheBifidobacteriumgenus.The16SrRNAgene-basedtreewasconstructedbythemaximumlikelihoodmethod;thecorrespondingsequenceofScardovia inopinataJCM12537T_{wasusedasoutgroup.Bootstrappercentagesabove70areshownatnodepoints,basedon1000replicatesofthephylogenetictree.}

TheMLSAanalysisconfirmedtheclosephylogeneticrelatedness withthenearestneighboursofallthestrains(Fig.2).

Furthermore,thelevelofsimilarityforthepartial housekeep-ing gene sequences of strains in relation to the type strains of their closest phylogenetic relatives was calculated using EMBOSSWaterweb-basedprogram(https://www.ebi.ac.uk/Tools/

psa/embosswater/nucleotide.html):thevaluesofsimilarityforthe hsp60,rpoB,clpC,dnaJanddnaGgenesequenceswerecalculated andreportedinSupplementaryTableS1.

Thegenomeofall8strainswasdecodedthroughanext genera-tionsequencing(NGS)approach,usingaMiSeqplatform(Illumina) atBioFabResearch(Roma,Italy).Thegenerateddataweredepleted

Fig.2. Phylogeneticthreebasedontheconcatenationofproteinssequencesdeducedfromthehousekeepinggeneshsp60,rpoB,clpC,dnaJanddnaG,showingthephylogenetic relationshipsbetweenstrainsisolatedfromEgyptianfruitbatsandmembersoftheBifidobacteriumgenus.Thehousekeepinggene-basedtreewasconstructedbythemaximum likelihoodmethod,withcorrespondingsequencesofScardoviainopinataJCM12537T_{beingusedasoutgroup.Bootstrappercentagesabove70areshownatnodepoints,} basedon1000replicatesofthephylogenetictree.

of low quality reads using FASTQ/A Trimmer in FASTX-toolkit (http://hannonlab.cshl.edu/fastxtoolkit/), assembled by SPADES version3.13.0[29]andannotatedthroughDFASTprogram[26].

ThedraftgenomesizeofstrainsRST8andRST16T_(ClusterI), RST9T_{andRST27(ClusterII),RST7andRST11(ClusterIII),RST} 19(ClusterIV)andRST17(ClusterV)rangedbetween2.80 and 3.28MbpasindicatedinTable1togetherwiththeirothergenomic features.Insilicoanalysisofallthesequencedgenomesallowed theestimationoftheirG+Ccontents,whichrangedfrom60.39to 64.55mol%,fallingintherangeindicatedforthegenus Bifidobac-terium,i.e.,52–67mol%.Theprojectoutlinehasbeensubmittedto theBioProjectPRJNA415181andtheGenBankaccessionnumbers werereportedinTable1.

Inordertoreconfirmtheabovephylogeneticanalysis,wealso constructedthephylogenetic treebased onthecoregenomeof Bifidobacteriumspp.Total76typestrainsofBifidobacteriumwere annotatedwiththeDFASTprogram[26],and355orthologousgenes

wereidentifiedasthecoreretainedinallgenomes.Theaminoacid sequencesofcoregenesfromeachgenomewereconcatenatedand alignedusingtheMAFFTprogram(version7.313)[9].The align-mentsweretrimmedusingtrimAlwith-automated1option[2].

The phylogenomic tree (Fig. 3) based on the core genome (355genes)confirmedthepositioningoftheeightisolatedstrains withinthegenusBifidobacteriumasobservedinthephylogenetic analysesbasedon16SrRNA and housekeepinggenesequences (Figs.1and2).

Inaddition,thegeneticsimilarityatgenomiclevelofRST16T andRST8 (ClusterI),RST9T _{and RST27(ClusterII),RST 7and} RST11 (Cluster III),RST 19 (ClusterIV) andRST 17 (ClusterV) isolateswithrespecttheothercurrentlyrecognized bifidobacte-rialspecieswasevaluatedbasedonaveragenucleotideidentity (ANI)analysisbyusingthewebserverJSpeciesWS(http://jspecies. ribohost.com/jspeciesws/).Thisanalysis(SupplementaryTableS2) showedthatthehighestsequenceidentityvaluebetweenRST16T

Table1

GenomicandphylogeneticfeaturesofnovelbifidobacterialstrainsisolatedfromtheEgyptianbats.

Strain RST16T _{RST8 RST9}T _{RST7 RST11 RST17 RST19 RST27}

Accessionnumber RZNZ00000000 RZOA00000000 PEBH00000000 RZUI00000000 RZUJ00000000 RZUH00000000 RZUG00000000 RZJP00000000

GCcontent 64.20% 64.20% 64.55% 60.96% 60.76% 63.16% 60.39% 63.58% Contigs 49 74 37 49 46 60 80 11 Length(bp) 3075992 3067389 3053799 3032244 2986510 3275217 2833112 2797830 No.ofORF 2577 2594 2712 2526 2540 2759 2436 2320 tRNA 57 62 64 61 62 62 59 61 rRNA 5 3 5 2 2 5 3 3 Coverage 118 165 189 107 145 209 140 155

andRST8was78.66%and78.49%whencomparedtothe chromo-somesequenceofBifidobacteriumreuteriDSM23975T_,thehighest sequenceidentityvaluebetweenRST9T_andRST27when com-paredtoBifidobacteriumcallitrichosDSM23973T_{were89.43%and} 96.06%respectively,thehighestsequenceidentityvaluebetween RST7andRST11was95.10%and94.78%whencomparedto Bifi-dobacteriumtissieriDSM100201T_{,thehighestsequenceidentity} valuebetweenRST19was96.94%whencomparedto Bifidobac-teriumreuteriDSM23975T_{andthatofRST17was95.98%when} comparedtoBifidobacteriummyosotisDSM100196T_.

ANIwithclosestneighbourssupportedanindependent phylo-geneticposition,i.e.ANIvalueof≤95%forstrainsinClustersI(RST 16T_{andRST8)andinClusterII(RST9}T_{).Therefore,strainRST16}T andstrainRST9T_{wereselectedasthetypestrainsofthetwonovel} species.However,RST9T_{showedhighsequencesimilaritywithB.} callitrichosDSM23973T_{fortheir16SrRNAgenesequences.}

ToverifytheirlowANIvalues,aninsilicoDDH(isDDH)wasalso carriedoutusingGenome-to-GenomeDistanceCalculator(GGDC) version2.1formula2,themostaccurateknowntoolforcalculating DDH-analogousvalues,developedatDSMZandavailableatwww. ggdc.dsmz.de.Thethresholdvalueof_≤70%isgenerallyaccepted forseparatedprokaryotespecies[15].WhencomparingstrainsRST 16T_andRST9T_{withtheirnereastneighbours,thevaluesachieved} were32.5and42.1%,respectively.Theanalysiswasalsoperformed amongthe8isolatedstrainsandobtainedresultswereintherange of65.2–79.5%(SupplementaryTableS3).

EvaluationoftheabovephylogeneticrelationshipsofRST16T andRST8,RST9T_{,RST7andRST11,RST19,RST17andRST27} isolateswithrespecttoother(sub)speciesclearlyrecognizedtwo putativenovelspecies,namelyRST16T_andRST9T_.

Furthermore,thisworkdescribethemorphological, biochemi-calandmolecularcharacterizationsoftheremainingstrainsplaced onClustersII,III,IVandVbelongingtotheknownspeciesB. callithri-cos,B.tissieri,B.myosotisandB.reuteri,respectively,allofwhich havebeendescribedasisolatesfromRousettusaegyptiacus.

Theeight strains,selected asrepresentativesfromidentified clusters,showedrod-shapedcells,frequentlyformingfilaments, withirregularcontractionsalongthecellsandbifurcations.They werecultivatedunderanaerobicconditionsandmaintainedinTPY broth[24]pH6.9,at37◦C,unlessindicatedotherwise.

Morphological, cultural and biochemical characterization of thestrainswereperformedat 37◦C unlessotherwise stated,as describedpreviously[18].

The morphology of cells of strains RST 16T _{and RST 9}T_{, as} revealedbyphase-contrastmicroscopy,isshowninFig.4.

Optimalgrowthconditionsofthestrainsweredeterminedin TPYbrothafter24hofincubationat37◦Cinanaerobiccondition. Growthat22,25,30,35,37,40,42,45,48◦Cwastested.Sensitivity tolowpHwasscreenedat3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,and7.5 valuesofpH.Theabilityofthestrainstogrowunderaerobicand microaerophilicconditions(CampyGen;Oxoid)wasalsoverifiedin TPYbrothafter48hofincubationat37◦C.Particularly,forstrains RST16T_,RST8andRST9T_{bestgrowthconditionswereobtainedin} TPYbrothpH7at37◦Candtheywereabletosurviveandgrowin

microaerophilicandinaerobicconditions.Allresultsareshowed inTable2.

Haemolyticactivitywas determinedin Columbiablood agar (Biolife)at37◦Cunderanaerobicconditionsfor48h[27].

Gramstaining,motilityassay,catalaseand oxidaseactivities wereperformedasdescribedpreviously[24].

StrainsRST16T_andRST8,RST9T_{,RST7andRST11,RST19,} RST17andRST27andrelatedspeciesB.avesaniiDSM100685T_,B. reuteriDSM23975T_{,B.myosotis100196}T_{,B.tissieriDSM100201}T_, andB.callitrichosDSM23973T_{werealsoinvestigatedforsubstrates} utilizationandenzymesproductionwithAPI50CHLandRapidID 32Atestkits(BioM ´erieux).ResultsaresummarizedinTable2.

Bifidobacteriaandmembersofrelatedgenerapossess fructose-6-phosphate phosphoketolase (F6PPK), the enzyme degrading hexoseviatheF6PPKpathway,whichisconsideredataxonomic markerforidentificationofBifidobacteriumandrelatedgenera[15]. DetectionofF6PPKactivitywascarriedoutaccordingtothemethod describedbyOrban&Patterson[26].Allstudiedstrainspossessed theF6PPKactivity.

For analysis of amino acid composition, the cell-wall pep-tidoglycan of strains RST 16T _{and RST 9}T _{was prepared and} hydrolysedasdescribedpreviously[6].Cell-wallaminoacidswere analysed by HPLC (model LC-20AB; Shimadzu) equipped with a Wakopak wakosil-PTC column (200×4.0mm i.d.; Wako Pure Chemical Industries),as theirphenylisothiocyanatederivatives (Wako). Amino acid isomers in the cell-wall hydrolysate were analysed asdescribed previouslyusingaliquid chromatograph-massspectrometer(modelLCMS-2020andLC-20AB;Shimadzu) equipped witha Shim-Pack FC-ODS column(150×2.0mm i.d.; Shimadzu)[20].ThepeptidoglycanofstrainRST16T_contained glu-tamic acid(Glu),serine (Ser), alanine(Ala)and ornithine (Orn) in a molarratio of4:1:2:1. Enantiomericanalysis ofthe pepti-doglycan amino acids revealed thepresence of l-Orn-l-Glu3-d -Ser-Glu3.

The peptidoglycan of strain RST 9T _{contained glutamic acid} (Glu),alanine(Ala)ornithine(Orn)andlysine(Lys)inamolarratio of2:2:1:1.Enantiomericanalysisofthepeptidoglycanaminoacids revealedthepresenceofl-Orn(l-Lys)-d-Glu.

Whole-cellfattyacidswereanalysedasfattyacidmethylesters (FAMEs)withSherlockphospholipidfattyacidsoftware(midi).The culturesofallstudiedstrainswereincubatedonMRSagaradded with0.05%cysteineplatesat37◦Cfor48hunderanaerobic condi-tions.FAMEswereextractedandpreparedasdescribedpreviously [23].ResultsareshowninTable3.

Onthebasisofthephenotypicandchemotaxonomic charac-terizationaswellasthemolecular-basedmethodsphylogenetic analysisbasedonthe16SrRNAgenesequences,MLSAbasedonthe concatenatedfivehousekeepinggenesequences,andthe whole-genome-basedsequencecomparisonsinsilico,strainsRST16T_and RST9T_{weregeneticallyandphenotypicallydiscerniblefromthe} currentlyrecognizedspeciesofbifidobacteria;thus,accordingto MinimalStandardguidelines[16],theyrepresenttwonoveltaxa forwhichthenameBifidobacteriumvespertilionissp.nov.and Bifi-dobacteriumrousettisp.nov.areproposed.

M. Modesto et al. / Systematic and Applied Microbiology 42 (2019) 126017 Glycerol – – – – – – – – + – – – – d-Arabinose – – – – – – – w – – – – – l-Arabinose + + + + + + + + + + + – + Ribose w + + – – w w w – – – – – d-Xylose – w + + + + + + + + + + + l-Xylose – – – – – – – – – – – – + Methyl-ˇ-d-xylopyranoside – – – – – w – – – w – – – Galactose + + + + w + + + + w + – w Fructose + w + + + + + + + w + + + Mannose + + + w w – + + + w + – + Sorbose – – – – – w – w – – – – – Rhamnose – w w – – – – w + – – – – Inositol – – – – – – – + – – – – – Mannitol + + + w + – + + w w + – – Sorbitol + – – – – – + + – – + – – Methyl-˛-d-mannopyranoside – – – – – – – + – – – – – Methyl-˛-d-glucopyranoside – + + w w + + + + – w – – N-Acetylglucosamine – w w – – – – + w – w – – Amygdalin – + + – – + + w – w – w – Arbutin – + + – – + + + – – + w – Aesculin + + + – – + + + + + + – – Salicin – + + – – + + + + + + + – Cellobiose – + + – – w w + + + + w – Maltose W W + + + + + + + + + + – Lactose W + + + + + + + + + + + – Melebiose + + + + + + + + – + + + – Sucrose – + + + + + + + + + + + + Trehalose – + + – – – + + + + + w – Inulin – – – – – – w – – – – – – Melezitose – + + – – – + – w w + w – Raffinose + + + + + + + + + – + + + Starch W w w w w + w w – – – + – Glycogen – – – – – – – – – – – + – Xylitol – – – – – – w w – – – – – Gentiobiose – + + – – + + + – w w + – Turanose + + + + + + + + – w w w – Lyxose – – – – – – – w – – + – – l-Fucose – – – – – – – + – – + – – Gluconate + – – + + + + + – – w w – 2-keto-gluconate – + + – – w – – + – – – w 5-keto-gluconate w + + w w w w w – – – – – Enzymaticactivity Urease + + + + + – – + + + + – – l-argininedihydrolase w + – w w + + + + + – – ˛-Glucosidase + + + + + + + + – w + + + ß-Glucosidase w + + w w + + + – w + + – ˛-Arabinosidase + – + w + + + – – – + – ß-Glucuronidase + – + – + – – + – – w – w N-Acetyl-ß-glucosaminidase – – – – + – – – – – – – – Glutamicacid decarboxylase + w w – – – + – – – + – –

M. Modesto et al. / Systematic and Applied Microbiology 42 (2019) 126017 7 Argininearylamidase + + + + + + + + – – + + +

Leucylglycinearylamidase – – – – + + + + + + w w +

Phenylalaninearylamidase + + + + + + + + + + + w + Leucinearylamidase + w + + + + + + + + + + + Pyroglutamicacid arylamidase – – – + + – – – – – – – + Tyrosinearylamidase + w + + + + – + + + + + + AlanineaArylamidase + – – – + + + + + + w – + Glycinearylamidase + + + w + + + + + + w – + Histidinearylamidase + + + + + + + + + + + + +

Glutamylglutamicacid arylamidase

– – – – W – – – – – – – w

Serinearylamidase + + + + + + + + – + w – +

Temperaturerangefor growth

22–48◦_{C 22–48}◦_{C 22–48}◦_{C 22–48}◦_{C 22–48}◦_{C 22–48}◦_{C 22–48}◦_{C 22–48}◦_{C 20–44}◦_{C 20–48}◦_{C 26–42}◦_{C 35–37}◦_{C 25–50}◦_C

Optimumtemperature 37◦_{C 37}◦_{C 37}◦_{C 37}◦_{C 37}◦_{C 37}◦_{C 37}◦_{C 37}◦_{C 35}◦_{C 42}◦_{C 37}◦_{C 37}◦_{C 40}◦_C

pHrangeforgrowth 3.5–7.5 3.5–7.5 3.5–7.5 3.5–7.5 3.5–7.5 3.5–7.5 3.5–7.5 3.5–7.5 5.5–7.5 4–7.5 5.0–8.0 5.0–7.0 4.0–7.5

OptimumpH 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 6.5 7.0 7.0 7.0 6.0

DNAGCcontent(mol%) 64.5a _60.96a _60.76a _64.20a _64.20a _63.16a _60.39a _63.58a _63.7a _65.1a _64.3a _61.3b _65.9c

Peptidoglycantype

l-Orn(l-Lys)-d-Glu l-Glu-l-Ala-l-Lys l-Glu-l-Ala-l-Lys l-Orn-l-Glu3-d-Ser l-Orn-l-Glu3-d-Ser l-Glu-l-Ala-l-Lys l-Lys-Gly l-Lys(l-Orn)-d-Asp l-Glu-l-Ala-l-Lys l-Glu-l-Ala-l-Lys l-Lys(l-Orn)-d-Asp l-Lys-Gly l-Orn(Lys)-d-Ser-d-Glu. +,positive;−,negative;w,weaklypositive.Allstrainsfermentglucose.Nostrainsfermenterytrol,l-xylose,d-adonitol,dulcitol,glycogen,tagatose,d-fucose,dandl-arabitol.Nostrainsshowˇ-galactosidase-6-phophatase, ˛-fucosidase,noreductionofnitratesorindoleproduction.

a_{Dataarefromthisstudy.} b _{DataarefromEndoetal.[4].} c _{dataarefromMichelinietal.[16].}

Fig.3.PhylogenetictreeoftheBifidobacteriumgenusbasedontheconcatenatedaminoacidsequencesof355coregenesofstrainsisolatedfromEgyptianfruitbatsand membersoftheBifidobacteriumgenus.Thecoregenes-basedtreeshowsthesubdivisionofthesevenphylogeneticgroupsoftheBifidobacteriumgenusrepresentedwith differentcolors.ThephylogenetictreewasbuiltbythemaximumlikelihoodmethodwithcorrespondingsequencesofScardoviainopinataJCM12537T_{beingusedasoutgroup.} Bootstrappercentagesabove70areshownatnodepoints,basedon1000replicatesofthephylogenetictree.

DescriptionofBifidobacteriumvespertilionissp.nov

Bifidobacteriumvespertilionis(ve.sper.ti.lio’nis.L.gen.n. vesper-tilionisofabat).

Cells are Gram-positive-staining, non-motile, asporogenous, non-haemolytic,F6PPK-positive,catalase- andoxidase-negative, indole-negative, and when growing in TPY broth are rods of various shapes forming a branched structure with ‘Y’ at both sides. The well isolated colonies grown on the surface of TPY

agarunderanaerobicconditionsarewhite,opaque,smoothand circularwithentireedges,whiletheembeddedcoloniesare lens-shapedorelliptical.Coloniesreach1.0–2.0mmindiameterafter 3 days of incubation. Cells can grow in the range 22–48◦C. CellsgrowatpH4.0–7.5.Optimalconditionsofgrowthoccurat pH 7 and 37◦C. Using API 50 CHL system acids are produced from d-glucose, l-arabinose, lactose, d-fructose, d-mannitol, d-mannose,maltose,sucrose,raffinose,turanose,d-xylose,gluconic acid,andproducedweaklyfromd-galactose,d-mannose,

Methyl-Fig.4. Phase-contrastphotomicrographsofcellsofB.rousettisp.nov.RST9T_{(A)andB.vespertilionissp.nov.RST16}T_{(B)growninTPYbrothshowingcellularmorphology.} Bar,10␮m.

Table3

CellularfattyacidprofilesofstrainsisolatedfromtheEgyptianfruitbat.

Fattyacid ClusterI ClusterII ClusterIII ClusterIV ClusterV

RST8 RST16T _RST9T _{RST27 RST7 RST11 RST19 RST17} C16:0 51.15±0.30 39.02±0.20 34.82±1.04 39.89±0.30 56.21±0.67 43.46±0.54 24.29±0.43 37.78±0.27 C14:0 5.80±0.12 16.3±0.47 24.96±1.75 23.07±0.72 10.57±0.39 11.80±0.45 21.68±0.62 19.68±0.17 C18:1␻9c 18.15±0.64 8.14±0.2 8.17±0.71 8.74±0.34 11.13±0.28 15.15±0.62 7.20±0.34 C16:1␻9c 2.45±0.10 6.07±0.56 2.83±0.10 2.15±0.03 1.62±0.31 1.54±0.16 8.18±0.23 3.30±0.10 C18:0 1.99±0.01 1.64±0.04 1.30±0.07 2.04±0.07 9.76±0.29 6.15±0.29 1.64±0.01 C12:0 1.35±0.02 1.85±0.30 1.74±0.12 1.84±0.10 2.84±0.20 1.87±0.09 C13:1at12-13 2.19±0.30 2.03±0.26 1.65±0.14 2.19±0.01 1.10±0.07 2.32±0.05 C19:0isoI 2.37±0.05 1.14±0.04 C20:0 1.91±0.23 C17:1␻9c 1.49±0.09 C10:0 1.26±0.08 Summedfeatures* 1 2.30±0.07 6.85±0.44 6.53±0.39 5.34±0.16 6.68±0.29 3.32±0.22 2.74±0.09 6.86±0.26 4 1.49±0.09 2.41±0.08 1.55±0.10 7 12.53±0.40 13.80±0.90 11.38±1.00 10.05±0.34 13.62±0.10 18.19±0.91 13.70±0.28 8 1.76±0.14 2..34±0.29 3.12±0.11 2.26±0.06 3.27±0.30 2.12±0.03 2.44±0.16

Inboldthemainfattyacidsproduced.

˛-d-glucopyranoside,starch,5-ketogluconatebutnotfromother carbohydrates.Activitywasobservedfor˛-andˇ-galactosidase, ˛-glucosidase,˛-arabinosidase,N-acetyl-ˇ-glucosaminidase, alka-linephosphatase,argininearylamidase,prolinearylamidase,leucyl glycinearylamidase,phenylalaninearylamidase,leucine arylami-dase,pyroglutamicacidarylamidase,tyrosinearylamidase,alanine arylamidase, glycine arylamidase, histidine arylamidase, serine arylamidase. Activity was also observed weakly for arginine dihydrolase,ˇ-glucosidase, glutamyl glutamic acidarylamidase. Aesculine is not hydrolysed. No reduction of nitrates was rec-ognized.Cellsarepositivefor urease.Thepeptidoglycantype is l-Orn-l-Glu3-d-Ser.

ThetypestrainRST16T_(=BCRC81138T_=NBRC113380T_=DSM 106025T_{)wasisolatedfromthefaecesof theEgyptian fruitbat} Rousettusaegyptiacus.TheDNAG+Ccontentofthetypestrainis 64.20mol%.

ThetaxonumberofdigitalprotologueisTA00874.

DescriptionofBifidobacteriumrousettisp.nov

Bifidobacteriumrousetti(rou.set’ti.N.L.gen.n.rousettiof Rouset-tusaegyptiacus,theEgyptianfruitbat).

Cells are Gram-positive-staining, non-motile, asporogenous, non-haemolytic,F6PPK-positive, catalase-and oxidase-negative, indole-negative,andwhengrowinginTPYbrotharerodsofvarious shapesformingabranchedstructurewith‘Y’atbothsides.Thewell isolatedcoloniesgrownonthesurfaceofTPYagarunder anaero-bicconditionsarewhite,opaque,smoothandcircularwithentire edges,whiletheembeddedcoloniesarelens-shapedorelliptical. Coloniesreach1.0–2.0mmindiameterafter3daysofincubation. Cellscangrowintherange22–48◦C. CellsgrowatpH4.0–7.5.

OptimalconditionsofgrowthoccuratpH7and37◦C.UsingAPI 50CHLsystemacidsareproducedfromd-glucose,l-arabinose, d-fructose,d-mannitol,d-mannose,raffinose,turanose,d-galactose, sorbitol,gluconicacidandproducedweaklyfromd-ribose, mal-tose, lactose, starch and 5-ketogluconate but not from other carbohydrates.Activitywasobservedfor␣-andˇ-galactosidase, ˛-glucosidase,˛-arabinosidase,glutamicaciddecarboxylase, argi-ninearylamidase,prolinearylamidase,phenylalaninearylamidase, leucine arylamidase, tyrosine arylamidase,alanine arylamidase, glycine arylamidase, histidine arylamidase, serine arylamidase. Activitywasalsoobservedweaklyforl-argininedihydrolase, ˇ-glucosidase.Aesculineishydrolysed.Noreductionofnitrateswas recognized.Cellsarepositiveforurease.Thepeptidoglycantypeis l-Orn(l-Lys)-d-Glu.

ThetypestrainRST9T _(=BCRC81136T_=NBRC113378T_=DSM 106027T_{)wasisolatedfromthefaeces oftheEgyptianfruitbat} Rousettusegyptiacus.TheDNAG+Ccontentofthetypestrainis 64.55mol%.

ThetaxonumberofdigitalprotologueisTA00875.

GenBankaccessionnumber

The GenBank accession number for the 16S rRNA par-tial gene sequence for the novel isolated strains RST 7, RST 8, RST 9T_{, RST 11, RST 16}T_{, RST 17, RST 19, RST 27} are MK722390, MK722393, MK722392, MK722391, MK722394, MK722395,MK722396,MK722397,respectively.

TheGenBankaccessionnumbersforthegenomesofthenovel isolated strainsRST 7,RST 8, RST9T_{,RST 11, RST 16}T_{,RST 17,} RST19,RST27areRZUI00000000,RZOA00000000,PEBH00000000,

following:RST7(BCRC81134,NBRC113376,DSM106022),RST8 (BCRC81135,NBRC113377);RST11(BCRC81136,NBRC113379, DSM106024);RST17(BCRC81139,NBRC113381,DSM106026); RST19(BCRC81140,NBRC113382,DSM106027);RST27(BCRC 81141,NBRC113383,DSM106028) Acknowledgements

ComputationalanalysiswasperformedontheNIG supercom-puteratTheResearchOrganization ofInformationand Systems (ROIS).Thetravelcost wassupportedbySOKENDAIShort-term ResearchAbroad&long-termInternshipProgram2018.Thiswork waspartiallysupportedbyJSPSKAKENHI(17K19248)inJapan. AppendixA. Supplementarydata

Supplementarymaterialrelated tothis article canbefound, intheonlineversion,atdoi:https://doi.org/10.1016/j.syapm.2019. 126017.

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