Botanical Studies (2009) 50: 331-335. microbioloGY Fungal keratitis caused by a new filamentous hyphomycete Sagenomella keratitidis Huei-MeiHSIEH1,Yu-MingJU1,Po-RenHSUEH2,Hsiu-YiLIN3,Fung-RongHU3,andWei-Li CHEN3,* 1Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan 2Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan 3Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan (ReceivedOctober6,2008;AcceptedMarch4,2009) ABSTRACT. Apreviouslyundescribedhyphomycetousfunguswasisolatedfromkeratitisdevelopedina softcontact-lenswearer.Itgrowsextremelyslowlyonvariousculturemedia.Itsphialide-likeconidiophores lacking an abrupt inflation and catenate, hyaline ameroconidia lead us to consider the fungus a species of the genusSagenomella. Keywords:Keratitis;Hyphomycetes;Sagenomellakeratitidis;Taxonomy. INTRODUCTION Conidiophoresandconidiawereexaminedbylight microscopy(LM)andscanningelectronmicroscopy Useofsoftcontactlenseshasbeenassociatedwith (SEM).Materialwasmountedinwaterforexaminationby the potential risk of developing microbial keratitis LMwithaLEICA/LEITZDMRBmicroscopeequipped (Donzis et al., 1987;Wilhelmus, 1987; Gray et al., with differential interference contrast optics. SEM 1995;Fongetal.,2004).However,previousreports observationsweremadebyPHILIPS(FEI)QUANTA oncontactlensassociatedfungalkeratitisshowedlow 200fittedwithPolaronPP2000Tcryo-SEMsystem prevalence(Yamaguchietal.,1984;Donzisetal.,1987; (QuorumTechnologies,UK).SamplesforSEMwere Wilhelmus,1987;Wilhelmusetal.,1988;Kirschand preparedbycuttingblocksof5×5×5mmfromone- Brownstein,1993;Grayetal.,1995;Fongetal.,2004). week-oldcoloniesalongwiththeirgrowingagarmedia. Herewedescribeahyphomycetebelongingtothegenus Thesampleswereplacedonasampleholderanddeep- SagenomellaW.Gams,whichisresponsibleforthe freezedinaliquidnitrogenslusher.Theywerethenplaced keratitisdevelopedinbotheyesofa35-year-oldfemale inthepreparationchamberwheretheyweresubjected softcontact-lenswearer. totheprocessofsublimationat-90°Cfor15minin ordertoeliminateicecrystalsfromthesamplesandwere MATERIALS AND METHODS subsequentlycoatedwithgoldat-130°Cfor60seconds withtheamperageadjustedat9-10mA.Temperaturein Fungal isolation, culturing, and observations thepreparationchamberwasdecreaseduntilitreached Ahyphomycetewasrepeatedlyisolatedfromcorneal -160°C before the samples were sent into the SEM scraping,contactlens,andstoragesolutionofcontactlens chamberforobservations. ofa35-year-oldfemale.Thefunguswasinoculatedonto 6-cmplasticPetridishescontainingPDYA(39g/LDifco DNA extraction, sequencing of various DNA potatodextroseagar,10g/LDifcoyeastextract),from loci, and DNA sequence analyses whichtheculturedescriptionwasmade,andincubatedat Myceliaweregrownin100mLofmaltextractbroth 25°C under 12 h fluorescent light. Colony morphologies (20g/LDifcomaltextract)onarotaryshakerat120rpm onMEA(20g/LDifcomaltextract,20g/LDifcoagar) for one month. They were harvested by filtration through andMMCA(Andoetal.,1998;5g/LDifcomaltextract, WhatmanNo.1filterpaper,freeze-dried,andstoredat 5g/LDifcocornmealagar,1g/LDifcoyeastextract,2 -20°C.GenomicDNAextractionfrommyceliumwas g/Lglucose,5g/LDifcoagar),werealsorecordedand performedaccordingtoHsiehetal.(2005). comparedwiththatonPDYA.Culturewasdepositedat FournuclearDNAlociweresequenced,includingITS, BCRC(theBioresourceCollectionandResearchCenter, 18S-rDNA,28S-rDNA,andTUB2(seeTable1fortheir Hsin-chu,Taiwan). fullnameandPCRprimerpairs).PCRconditionforITS wasasfollows:aninitialdenaturationstepat94°Cfor *Correspondingauthor:E-mail:[email protected];Tel: 3min,30cyclesof94°Cfor30sec,55°Cfor30sec,72 +886-2-23123456,ext.5206. 332 Botanical Studies, Vol. 50, 2009 °Cfor2min,andafinalextensionat72°Cfor10min. Gené&Guarro,AB024592fromS.sclerotialisW.Gams PCRconditionsfor18S-rDNAandβ-tubulinfollowed &Breton,NW_001594105fromAspergillusnigerTiegh., Hsiehetal.(2005),withprimerannealingtemperatures andNW_00159417fromA.niger,respectively(Table1). at55°Cand50°C,respectively.PCRconditionfor28S rDNAfollowedSøchtingandLutzoni(2003).Reaction Taxonomy componentsforPCRincludedapproximately0.1-0.7ng/ Sagenomella keratitidisW.-L.Chen,Y.-M.Ju,H.-M. µLoftotalDNA,0.2µM(28S-rDNAandTUB2)or2µM Hsieh,H.-Y.Lin&F.-R.Hu,sp.nov.Figures1-2 (ITSand18S-rDNA)ofeachprimer,200µMdNTP,1.5 Coloniae in PDYA 5-6 mm diameter aetate quatuor he- mMMgCL ,0.025U/µLofTaqpolymerase(Invitrogen, 2 bdomadum attigentes, albida primo, azonata, irregulatim Carlsbad,CA),and1xstandardPCRbuffersupplied rugosa, rimosa ubi veta, coriacea, marginibus integris vel withtheTaqpolymerase.PCRproductswerecleaned leviter crenatis, exsudata nulli, cinerascentes ubi conidii- withPCR-MTMclean-upsystem(Viogene-BiotekCorp., ferae. Phialides hyalinae, laeves, plerumque simplices, Hsichih,TaipeiCo.,Taiwan)followingthemanufacturer’s ampulliformes, 14-30 × 2-2.5 µm, ad apicem angustum protocol.DNAcloningwasthencarriedoutessentiallyas gradatim attenuatae. Conidia hyalina, laevia, globosa vel SambrookandRussell(2001).Ligatedplasmidswereused subglobosa, 2.5-3.5 µm. Chlamydosporae nullae. to transform high efficiency competent cells ofEscherichia colistrainDH5(Inoueetal.,1990).Afterincubation ColoniesonPDYAattaining5-6mmdiamin4weeks, overnightat37°ConLB/carbenicillin/IPTG/X-Galplates, whitish first, velvety, azonate, irregularly buckled, cracked singletransformedwhitebacterialcolonieswerepicked inage,leathery,withentireorslightlycrenatemargins, andtransferredintotubeswith6mLLB/carbenicillinbroth lackingexudates,becominglightgraywithconidial andincubatedovernightat37°Cinshakingculture.The production;reverselightbrown.Sporulatingregionson plasmidswereextractedwithaplasmidDNAextractionkit theentiresurfaceofcolonies.Conidiogenous structures (Viogene-BiotekCorp).ABIBig-dyeprimersequencing mononematous,unbranched,upright,simple,mostly kit(AppliedBiosystems)wasusedforDNAsequencing, reducedtosinglephialidesonly.Phialideshyaline,smooth, andsequencingreactionswereelectrophoresedonanABI flask-shaped, 14-30 × 2-2.5 µm, ca. 1 µm broad at apex. Prism 377 model DNA sequencer. Purified PCR products Conidiaproducedenteroblasticallyinbasipetalsequence weredirectlysequencedusingthesameprimerpairsas andremainingindrychains,hyaline,smooth,globoseto inthePCRreactions,whereasextractedplasmidswere subglobose,2.5-3.5µm.Chlamydosporesabsent. sequencedfrombothdirectionsusingT7andRuniversal primers. SimilarityvaluesofhomologysearchesintheGenBank databasewereobtainedbyusingMEGABLAST,withthe scoresof“match”,“mismatch”,“existencegapcost”,and “extensiongapcost”setat1,-1,2and2,respectively. RESULTS DNA sequence analyses Theobtainedsequencesrevealedthehighestmaximum scoreswithspeciesofthefamilyTrichocomaceae,with theclosestsequencesofITS,18S-rDNA,28S-rDNA,and TUB2beingAJ519984fromSagenomellachlamydospora Figure 1.ColoniesonPDYAina6-cmPetridishatonemonth. Table 1.ThefournuclearDNAlociofS.keratitidissequencedinthecurrentstudy. Maximumidentitytothesequence GenBank DNAlocus Abbreviation Primerpairused inGenBankmatchedwithhighest accessionno. maximumscore Ribosomalinternal ITS ITS1/ITS4(Whiteetal., EU140821 89%withAJ519984fromSagenomella transcribedspacers 1990) chlamydospora Ribosomalsmall 18S-rDNA NS1/NS4(Whiteetal., EU140822 99%withAB024592fromSagenomella subunit 1990) sclerotialis Ribosomallarge 28S-rDNA LROR/LR7(Vilgalys EU140823 95%withNW_001594105from subunit andHester,1990) Aspergillus niger β-tubulin TUB2 T1/T22(O’Donnelland EU140824 86%withNW_001594179from Cigelnik,1997) Aspergillus niger HSIEH et al. — Fungal keratitis caused by a new filamentous hyphomycete Sagenomella keratitidis 333 Figure 2.A,Conidiogenousstructuresmostlyreducedintosinglephialides,whichproduceconidiainchains;B,Conidia.Scalebars,A =8µm,B=4µm.AandBbySEM. ColoniesonMEAandMMCAingeneralasonPDYA DISCUSSION exceptforgrowingslightlyslower,4-5mmdiamin4 weeksandappearingwhitishratherthangrayishdueto Fungalkeratitisisuncommoncomplicationsincontact lessconidialproduction. lenswearers(Yamaguchietal.,1984;Donzisetal.,1987; Etymology.Forthecapabilityofcausingkeratitis. Wilhelmus,1987;Wilhelmusetal.,1988;Kirschand Brownstein,1993;Grayetal.,1995;Fongetal.,2004). Holotype.TAIWAN.BCRC34221,obtainedfrom Recently,contactlenscleaningsolutionhasbeensuspected humankeratitis;withisotypesdepositedinHASTand tocauseFusariumoutbreakfrom2005to2006(Chang NationalTaiwanUniversityHospital. etal.,2006;Khoretal.,2006).Becausenopredisposing Notes.Althoughthiskeratitis-causingfungusdoes factors, such as ocular trauma, pre-existing ocular nothaveaknownteleomorph,thereisoflittledoubt diseases,topicalsteroidusagesoranexposuretoBausch thatithasacloseaffinitytotheascomycetousfamily &LombReNuwiththeMoistureLoccleaningsolution, Trichocomaceae,whereanamorphsproduceconidiafrom wereinvolvedinthiscase,poorcontactlenshygienewas phialidesandhaveconnectivesbetweenchainedconidiain verylikelythereasonforthisrarefungalinfection. mostcases.ThisisalsocorroboratedbyBLASThomology WhilethemajorityofSagenomellaspeciesareknown searchesperformedintheGenBankdatabasebyusing fromsoil,S.bohemica Fassatiová & PĕčkováandS. thefournuclearDNAloci—ITS,18S-rDNA,28S-rDNA, sclerotialiswereisolatedfrompeloidsofabalneological andTUB2—thatwesequenced.Thesesequenceshad sample (Fassatiová and Pĕčková, 1990) and fodder highestmaximumscoreswiththosefromspeciesofthe ofryegrassandlucerne(Gams,1978),respectively. Trichocomaceae.SagenomellaandTorulomycesDelitsch SagenomellaoligosporaW.Gams&Luiten(Gams,1978) arethetwohyphomycetousgenerawithaffinitiestothe hasbeenisolatedfromdiversifiedsubstrates,including TrichocomaceaeharboringcertainAcremonium-likefungi.