Is Malassezia nana the main species in horses' ear canal microbiome?

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Veterinary

Microbiology

Is

Malassezia

nana

the

main

species

in

horses’

ear

canal

microbiome?

Ana

Lúcia

Aldrovandi

_,

_Lika

_Osugui

_,

_Selene

_Dall’

_Acqua

_Coutinho

a_{MolecularandCellularBiologyLaboratory,UniversidadePaulista,SãoPaulo,Brazil}

b_{DepartmentofMicrobiology,ImmunologyandParasitology,UniversidadeFederaldeSãoPaulo,SãoPaulo,Brazil}

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Articlehistory:

Received28July2015 Accepted24November2015 Availableonline29April2016 AssociateEditor:MilianeMoreira SoaresdeSouza Keywords: Malassezianana Malasseziaslooffiae Microbiome Horses Earcanal

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TheobjectiveofthisstudywastocharacterizegenotypicallyMalasseziaspp.isolatedfrom theexternalearcanalofhealthyhorses.Fifty-fivehorses,39(70.9%)malesand16(29.1%) females,fromdifferentbreedsandadultswerestudied.Externalearcanalswerecleanedand asterilecottonswabwasintroducedtocollectcerumen.Atotalof110sampleswerecultured intoDixonmediumandwereincubatedat32◦Cforupto15days.Macro-and micromor-phologyandphenotypicidentificationwereperformed.DNAwasextracted,strainswere submittedtopolymerasechainreactiontechnique,andtheproductsobtainedwere sub-mittedtoRestrictionFragmentLengthPolymorphismusingtherestrictionenzymesBstCI

andHhaI.Strainsweresentofftogeneticsequencingoftheregions26SrDNAD1/D2and ITS1-5.8S-ITS2rDNA.Malasseziaspp.wereisolatedfrom33/55(60%)animalsand52/110 (47%)earcanals.NogrowthonSabourauddextroseagarwasobserved,confirmingthelipid dependenceofallstrains.Polymerasechainreaction-Restrictionfragmentlength polymor-phismpermittedthemolecularidentificationofMalassezianana–42/52(81%)andMalassezia slooffiae–10/52(19%).SequencingconfirmedRFLPidentification.ItwassurprisingthatM. nanarepresentedover80%ofthestrainsandnoMalasseziaequinawasisolatedinthisstudy, differingfromwhatwasexpected.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

YeastsoftheMalasseziagenusareconsideredregular inhabi-tantsofthecutaneousmicrobiomeofanimalsandhumans, buttheymayalsobeinvolvedinseveraldiseases,from cuta-neoustosystemic.1–5

∗ _{Correspondingauthor.}

E-mail:selene@uol.com.br(S.D.AcquaCoutinho).

Inthelastfewyears,molecularstudieshaveincreasedthe numberofspeciesclassifiedinthegenusMalasseziato14:M. furfur,M.pachydermatis,M.sympodialis,M.globosa,M.obtusa, M. restricta,M.slooffiae,M. dermatis,M.japonica,M.nana, M. yamotoensis,M.caprae,M.equinaandM.cuniculi.6,7

All species are lipophilic and lipodependent, with the exception of M. pachydermatis. In veterinary medicine,

http://dx.doi.org/10.1016/j.bjm.2016.04.017

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

M.pachydermatisisthemostcommonlyisolatedspecies2,3,5,8_; however,studieshaveshownthatthelipodependentspecies mayalso constitutethe microbiomeand cause diseases in domesticandwildmammals.8–11

Surveyswithhorsesanddomesticruminantsshowedthat the occurrence of lipodependent species was significantly higherthan thatofM.pachydermatis,whenit wasreported thepresenceofM.furfur,M.slooffiae,M.obtusa,M.globosa,M. sympodialisandM.restricta.12

Phenotypicalcharacterizationisstillacceptedtodiagnose clinicalcases;however, lipodependentspeciesmay present similar biochemical and physiological behavior, leading to erroneousidentifications.Therefore,studieshavesuggested theusemolecularmethodsthatallowproperdifferentiation amongspecies.4,6,11

Externalotitisisanextremelyseriousdisease inhorses, with uni/or bilateral presentation and the potential to becomea chroniccondition. Asignificant portion ofthese cases are bacterial in origin, but some reports of mycotic otitis in equines caused by yeasts are available in the literature.8,13 _{However,publishedstudiesregardingthe} pres-enceofMalasseziaintheearcanalofhorsesarescarce.8,12

Itiscriticaltocarryoutnewsurveysinthisareathatwill permitbetterunderstandingofthedistributionofMalassezia

speciesinthemicrobiomeofequineexternalearcanals. Therefore,theaimofthisstudywastogenotypically char-acterizeMalasseziaspp.isolatedfromtheexternalearcanalof healthyhorses.

Materials

and

methods

Fifty-fiveadulthorsesfromdifferentbreeds,39(71%)males and 16 (29%) females were studied. This study evaluated stalledhorsesfrom anequestriansocietyinthecityofSão Paulo,Brazil.

Theexternalearcanalswerecleanedwithanalcohol–ether solution(1:1)andasterilecottonswabwasintroducedto col-lect cerumen from bothears. A total of110samples were culturedinmodifiedDixonandSabourauddextroseagar,and the plates were incubated at32◦C forup to15 days.6 _The macro-andmicromorphology ofthe isolateswere observed byGram stain.Thephenotypicidentification wasbasedon thephysiologiccharacteristics,suchastheassimilationand growth indifferent lipid sources, theTween 20,40,60, 80, andcremophor-EL.Acatalasereaction,splittingofesculinand growthat40◦Cwerealsotested.6

DNA was extracted by10min ebullition. After that, the strainswere submittedtopolymerasechainreaction (PCR), using the following primers: forward, 5 -TAACAAGGATT-CCCCTAGTA-3andreverse,5-ATTACGCCAGCATCCTAAG-3.14 AmplificationoftheDNAtargetsequencewasperformed withthe kitPlatinum® _{Taq DNA Polymerase(Invitrogen}TM_,

Carlsbad,CA,USA),infinalvolumereactionsof50␮L:34.3␮L DEPECsterilewater(dimetilpyrocarbonate);5.0␮Lof10×PCR buffer;1.0␮Lof10mMdNTP(deoxynucleosidetriphosphate, 0.2mMfinalconcentrationofeachdeoxynucleoside);1.5mM ofMgCl2;0.2␮Mofforwardprimer26S-Fw;0.2␮Mofreverse

primer26S-Rv;oneunitofPlatinum® _{TaqDNAPolymerase}

and 5.0␮Lof the extracted genomic DNA.The tubes were

incubated in an Eppendorf Mastercycler Gradient® ₅₃₃₃

ther-mocycler(Eppendorf,Hamburg,Germany) at94◦Cfor5min forinitialdenaturationofthegeneticmaterialandenzyme activation, followed by 30 amplification cycles: denatur-ation at94◦Cfor45s, annealingat55◦Cfor45s,extension at 72◦C for 1min and a final extension at 72◦C for 7min.14

The temperature was maintained at4◦C after PCR and the sampleswere storedat−20◦_{C.Afteramplification, the}

PCRproductsweresubmittedtoelectrophoresisonagarose gel(1.5%dilutedinTBEbuffer),programmedat100Vforan hour.Followingtheelectrophoresis,0.5␮g/mLethidium bro-midesolutionwasusedtostainthegelfor15minandthegel wasvisualizedonanUVtransilluminatorandphotographed usingtheGelLogic200KodaksystemTM _{(EastmanKodakCo.,}

Rochester, NY,USA).Thesizeoftheamplificationwas esti-matedaccordingtoa100pbmolecularweightmarker(100pb DNA Ladder; Norgen, CA, USA). M. pachydermatis CBS1696 (580bp)andMilli-Qwereusedaspositiveandnegative con-trols,respectively.

TheproductsobtainedbyPCRweresubmittedtorestriction fragment length polymorphism (RFLP)4 _{using the} restric-tion enzymesBstCI (BioLabs®, Ipswich,MA, USA)and HhaI

(InvitrogenTM_{,Carlsbad,CA,USA).}14

Avolumeof21.5␮LofPCRproductwasadded to3.5␮L (10units) of restriction enzyme (0.5␮L enzyme and 3.0␮L buffer),uptoatotalvolumeof25␮L.Tubeswerepreparedwith twodifferentenzymes foreach PCRsample,andincubated at37◦Cfor3h.14_{ProductsobtainedbytheRFLPwereapplied} on2.0%agarosegeldilutedinTBEbuffer.Electrophoresiswas setat100Vfor1hand30min.Markersofmolecularweight andbandvisualizationwereperformedaccordingtothesame methodpreviouslydescribed.

Speciesconfirmationwasbasedontherandomselection offourstrains:twowithM.nana-andtwowithM.slooffiae-like

patternsobtainedbyRFLP,whichweresubmittedforgenetic sequencing.ThePCRproductswerepurifiedwiththe ExoSAP-ITsystem(GEHealthcareBio-SciencesLtd–USBCorporation, Cleveland,OH,USA),accordingtothemanufacturer’s instruc-tionsandquantifiedwithThermoScientificNanoDropTM₁₀₀₀

Spectrophotometer(ThermoFisherScientificInc,Wilmington, DE,USA).

For thesequencing, 30ngofthe amplifiedproductwere addedto2␮Lof5×sequencingbuffer(AppliedBiosystems; Carlsbad, CA, USA), 3.2pmol of primer, 2␮L ofABI PRISM DyeTerminatorCycleSequencingReadyReactionkit(BigDye v3.1,AppliedBiosystems;Carlsbad,CA,USA)andasufficient volumeofDEPECwaterwasusedtoachieveafinalvolumeof 10␮L.

The primers ITS5 (5 -GGAAGTAAAAGTCGTAACAAGG-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3) were employed for sequencing the region ITS1-5.8S-ITS2 rDNA; NL1 (5-GCATATCAATAAGCGGAGGAAAAG-3) and NL4 (5 -GGTCCGTGTTTCAAGACGG-3)wereemployedforsequencing theregion26SrDNAD1/D2.

Sequencing was performed on an ABI-PRISM® _3130XL

(AppliedBiosystems,Carlsbad,CA,USA)automaticsequencer with the POP6 polymer. The nucleotide sequences were analyzedbytheBLASTprogram15_{(http://blast.ncbi.nlm.nih.}

Fig.1–PCR.1and16:ladder(100pb);2.positivecontrol–

M.pachydermatis(CBS1696)(580bp);3.negativecontrol–

Milli-Q;4,5,6,7,8,9,10,11,13,14and15:isolatesfrom horses’earcanals–positiveforMalasseziasp;12.Blank.

productspecificityandwerethenalignedwithBioEditClustal X program on the 26S and ITS regions of the CBS-Knaw referencesstrains16_{(http://www.cbs.knaw.nl/):CBS7956–M.}

slooffiaeeCBS9557–M.nana.

ThisprojecthasbeenapprovedbyResearchEthics Com-mitteen◦024/10CEP/ICS/UNIP.

Results

Malasseziaspp.wereisolated from33/55(60%)animalsand 52/110(47%)earcanals.NogrowthonSabourauddextroseagar wasobserved,confirmingthelipiddependenceofallstrains. All strains were positive for the catalase reaction and capableof splitting esculin. Tween 20,40, 60 and 80 were assimilated by 37/52 (71%) strains, but not cremophor-EL; someofthesestrainspresentedpoorassimilationofthelipids, andtheother15/52(29%)strainsshoweddifferentpatternsof assimilation,whichdidnotpermitphenotypicspeciation.

Bandsbetween500and 600pbwereobtainedinthePCR technique,confirmingallstrainsasMalasseziaspp.(Fig.1).

RFLPpermittedthemolecularidentificationofthe52 iso-latesfromtheearcanalintwospeciesM.nana(42/52–81%) andM.slooffiae(10/52–19%)(Figs.2and3).

Geneticsequencingof theregions 26S rDNA D1/D2and ITS1-5.8S-ITS2 rDNA of the random four strains selected, whencompared toCBS’ culturecollection, confirmed RFLP identification,twoofthemwereidentifiedasM.nanaandtwo asM.slooffiae.

Discussion

ThepresentstudyshowsahighpercentageofMalasseziaspp. isolated from animals(60%) andear canals(47%).The fre-quencyofMalasseziaspp.observedinthemicrobiomeofthe earcanalswassimilar,orevensuperior,tothosementioned

Fig.2–RestrictionenzymeHhaI.1and14:ladder(100pb);

M.nana:2,3,5,9,10and12;M.slooffiae:7,8,11and13;

blank:4and6.

Fig.3–RestrictionenzymeBstC1.1and16:ladder(100pb);

M.nana:2,5,6,8,9,10and11;M.slooffiae:3and4;blank:

7,12,13,14,and15.

byotherstudiesthatreportedthepresenceofthisyeastonthe cutaneoussurfaceofhorses12,17,18_{andothermammals.}2,19,20 Thehighlevelofisolatesinthisstudyshowsthat lipodepen-dentyeastsarepartofthefungalmicrobiomeintheearcanals ofthesehorses.

Note that M. pachydermatis, which is considered to be the most common speciesfrom the cutaneous surfacesof animals,8,11 _{was notisolated inthis study.Inrecentyears,} lipodependentspecieshavebeenisolatedfromtheskinand mucosaofhealthy,10,19,20_{aswellasdiseasedanimals.}8,11,19–21 Althoughtheincidenceofexternalotitisinhorsesislow, the presence of Malassezia spp. in the microbiome of the ear canal suggests that these yeasts may cause infections whenahost–parasiteimbalanceoccurs,ashasalreadybeen reportedinequinecutaneousinfections.22_{Thepredisposing} factorsrelatedtothetransitionofMalasseziaspp.froma com-mensalorganismtoapathogenare stillpoorlyunderstood; beingconsideredopportunisticfungi,theseyeastscouldover

multiplyandacquireapathogeniccharacteristic.1_Mycotic oti-tishasalreadybeenindicatedinhorses,13andtheincidence ofotitisinhorsescausedbyMalasseziaspp.couldbe under-estimatedbecausecommercialveterinarylaboratoriesdonot usemediawiththeadditionoflipidsforthedetermination ofMalasseziaspp.,whichmightyieldfalse-negativeresultsin veterinaryclinicalsamples.

Althoughphenotypictechniquesareroutinelyusedin clin-icalsamples,inthisstudy,thesetechniquesfailedtoidentify lipodependentMalasseziaspecies.Weobservedthatseveral strainsdidnotpresent theexpectedpatterns,whichmade theircharacterization,basedononlyphenotypictestsalot morechallenging.

IntheRFLPtechnique,anenzymecleavesthePCRproduct generatingfragmentsofseveralsizesandconsequently,bands withdifferentmolecularweights.1_{Inthisresearch,theprofiles} obtainedallowedtheidentificationofthestrainsintotwo dif-ferentspecies:M.nanaandM.slooffiaethatwereidentifiedby sequencingoftheregions26SrDNAD1/D2andITS1-5.8S-ITS2 rDNA.

In2007,anewspeciesnamedM.equina,wasdescribedand characterizedby26rRNAgenesequencing.23 _Inthepresent study,geneticsequencingofthestrainsidentifiedbyPCR-RFLP confirmedtheisolatesasM.slooffiaeandM.nana.

M. slooffiae has already been isolated from horses’ skin microbiomein asurvey with 50 animals, as well as other lipodependentspecies: M.obtusa, M. globosa,M. sympodialis

andM.restricta.12_{Surprisingly,M.nanarepresentedover80%} ofthestrains,andnoM.equinawasisolatedinthisstudy, dif-feringfromwhatwasexpected.Itcouldbespeculatedthat

M.nanaisamoreprevalentspeciesinthemicrobiomeofthe externalearcanalofhorses.However,toconfirmsuchdata, itisimportanttocarryout newsurveysinseveraldifferent areasandincreasethesampling;alloftheanimalsevaluated inthisstudywereplacedinthesameinstitutionandsubjected tosimilarhusbandrypractices.

M.nanahasbeenisolatedfromcatswithexternalotitisand bovineswithorwithoutotitis,suggestingthatitmaybepart ofthemicrobiomeinsomedomesticanimals;however,ithas neverbeenisolatedfromhumans.24

Funding

Capes-PROSUPfellowship,FederalGovernment,Brazil.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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