Journal of Dermatological Science (2006) 43, 214—217

www.intl.elsevierhealth.com/journals/jods

LETTER TO THE EDITOR

Verifiable single nucleotide polymorph-isms of the internal transcribed spacer 2region for the identification of 11 Malas-sezia species

To the Editor,

KEYWORDSMalassezia;Identification;ITS;SNP;PCR-RFLP

Malassezia yeasts are the globally distributed agentsof pityriasis versicolor and are implicated in thepathogenesis of seborrheic and atopic dermatitis[1]. The taxonomic status of the genus has relativelyrecently expanded to include seven species [2].Moreover, in the last 4 years Malassezia dermatis[3],M. japonica [4],M. nana [5] andM. yamatoensis[6] have been appended to the genus and theirspecies status as well as their contribution to skindisease is under investigation [1].

The current conventional identification schemesof the lipophilic Malassezia species [2,7] has inbuiltdifficulties as the results are not always unequivocaldue to the variable Tween assimilation profiles of M.slooffiaeandM.nana strains [5]. The identificationofthe new species was based on comparison of the%G + C content and on sequence analysis of the LargeSubunit rDNA and Intergenic Spacer regions [2—6].

Our aims were to upgrade the ITS 3/4 PCR-RFLPsystem developed previously [8] and assess its capa-city for reliable identification of 11 Type, referenceand clinical Malassezia species.

DNA was extracted from pure cultures of 120strains of 11 Malassezia species (Table 1) and theInternal Transcribed Spacer 2 (ITS 2) region wasamplified by PCR employing the ITS 3 (50-GCATCGAT-CAAGAACGCAGC-30) and ITS 4 (50-TCCTCCGCTTATT-GATATGC-30) primers (INTERACTIVA BiotechnologieGmbH, Ulm, Germany) as previously described

0923-1811/$30.00 # 2006 Japanese Society for Investigative Dermadoi:10.1016/j.jdermsci.2006.03.013

[8]. The ITS 3/4 PCR products of the novel speciesM. dermatis andM. nanawere selectively recoveredfrom the agarose gel (QIAEX1 II Gel ExtractionKit, QIAGEN, Hilden, Germany), sequenced bi-direc-tionally (Biogenomica, Athens, Greece) and theconsensus sequence for each strain was submittedto the GenBank (Acc. nos.: AY390283-5, DQ083032).Selection of the restriction endonucleases wasaccomplished comparing the expected ITS 3/4Restriction Fragment Length Polymoprhism (RFLP)profiles through alignment and comparison (Seq-man, DNA Star Inc., USA) of the published sequencesfor M. furfur (AF246896, AF522058-9, AY387100-131, AB105150-4), M. obtusa (AF521960,AB105155-8, AY387137-8, AY743631), M. pachyder-matis (AB118937-41, AY387139-42, AY743637,AF522061), M. japonica (AB105199), M. slooffiae(AY743633, AY387146-56, AF522063), M. globosa(AF522060, AY387132-6, AY743630, AJ437693,AY267224), M. restricta (AF522062, AJ437694-5,AY267225, AY387143-5, AY743636), M. yamatoensis(AB125261-2), M. nana (DQ083032), M. sympodialis(AB070366, AF522064 and AY387157-195) and M.dermatis (AY390283-5, AB070356-60). The relativelysmall number of the clinically important M. globosaand M. restricta species tested (15 and 3, respec-tively out of 120 strains) is due to the difficultyin maintaining them in culture and is depicted intothe small number of deposited sequences in theGenBank (9 and 8, respectively out of 123sequences).

The restriction endonucleases AluI, BanI andMspAI (New England Biolabs, Beverly, MA, USA) wereselected for producing distinct RFLP patterns andwere used for digestion of the PCR products [8].Single nucleotide polymorphisms that would alterthe predicted restriction profile were recorded andtested for interference with the identification pro-cedure. Electrophoresis of the PCR products andtheir restriction fragments was performed in 6%29:1 acrylamide:bis-acrylamide non-denaturatingacrylamide gels at 180 V for 3 h and 30 min or,alternatively, in 10 cm long 4% standard agarose gelsat 100 V for 4 h. The gels were stained with ethidium

tology. Published by Elsevier Ireland Ltd. All rights reserved.

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Table 1 Malassezia type, reference and clinical strains of 11 Malassezia species tested for actual nucleotide polym phisms in the ITS 2 amplified region by RFLP analysis

Group Malassezia species(total, n = 120)

Strains ITS 3/4 PCRproduct (bp

AluI BanI MspAI

I M. furfur(n = 35)

CBS1878, CBS7019, CBS9576, CBS9580, CBS9596,GS2Aa, GS2Ba, GS4Aa, GS9Aa, GS9Ba, B1a, B2a, B3a,B5a, B10a, B13a, B14a, B15a, B22a, B412a, B522a, CS3a

557 306, 251 389, 168 525, 32

CBS9375, CBS9569, CBS9572, CBS9575, CBS9577,CBS9578, CBS9581, CBS9582, CBS9584, CBS9587,CBS9589, CBS9590, CBS9597

NRSb

I M. obtusa(n = 2)

CBS7876, CBS7968 554 NRSb 396, 158 NRS

I M. japonica(n = 2)

CBS9431, CBS9432 528 394, 134 183, 199,146

498, 30

I M. pachydermatis(n = 10)

CBS1879, CBS1880, CBS1881, CBS1882, CBS1883,CBS1884, CBS1885, CBS6540, CBS7925, CBS9552

529 412, 117 NRS 499, 30

II M. slooffiae(n = 4)

CBS7956, CBS7971, CBS7973, CBS8738 505 385, 120 NRS 472, 33

II M. globosa(n = 15)

CBS7866, CBS7874, EM5635aa, EM5635ba, GS13Ba,GS19A2a, GS39Ba, GS40Ba, AA2Ba, AA5Ba,XG1Aa, XG1Ba, XG2Aa, XG2Ba, EM6279a

477 221, 16,240

NRS 447, 30

II M. restricta(n = 3)

CBS7877, AA6Ca, AA9Aa 463 NRS 186, 277 432, 31

II M. yamatoensis(n = 1)

CBS9725 470 NRS NRS NRS

III M. sympodialis(n = 44)

CBS7222, CBS9570, CBS9593, CBS9594,CBS9974, CS17a, GS3Ba,GS8Ba, GS10Ba, GS12Ba, GS15Ba, GS16Ba,GS18Aa, GS18Ba,GS19A1a,GS19Ba, GS20Ba,GS22Ba, GS24B a, GS25Ba,GS26Ba, GS29Ba, GS33Aa,GS34Ba, GS37Ba, AA1Ba, AA4Ba, AA13Ba,EM6154a, EM6265a, AA3Ca,AA14Ba, AA16Ba, B437a, B439a, B5a, B17a,B821a, B822a, B829a, B850a, B880a, B881a, XG3a

420 NRS NRS 281,109, 30

III M. dermatis(n = 3)

CBS9145, CBS9169, CBS9170 416 NRS 186, 230 192, 85,109, 30

III M. nana(n = 1)

CBS9557 428 NRS 188, 240 286, 110,32

The lengths reported (in base pairs) are approximate as small intraspecies variations exist.a Clinical isolates.b NRS: no restriction site.

or

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216 Letter to the Editor

bromide and visualized under UV illumination (Her-olab, EASY, Weisloch, Germany).

The 11 Malassezia species were clustered accord-ing to the size of the ITS 3/4 PCR product in threegroups (Table 1; Fig. 1). M. furfur, M. obtusa, M.pachydermatis and the novel species M. japonicacomprised group I and were differentiated by AluIrestriction digestion (Table 1; Fig. 1). All 35M. furfurstrains tested presented with a single AluI ITS 3/4RFLP pattern. Only in 2 (AY387104 and AY387124) outof the 40 GenBank retrieved sequences no AluIrestriction site could be identified. However, thesetwo strains could be differentiated from M. obtusa,which also does not possess anAluI restriction site, byrestriction digestion withMspAI (Table 1; Fig. 1). The12M. furfur strains, which were isolated from Greekpityriasis versicolor and seborrheic dermatitispatients, did not have a BanI ITS 2 restriction site

Fig. 1 ITS 3/4 PCR product of 11Malassezia species (panel AandMspAI (panel D) in 6% acrylamide gel. Lane 1, molecular sizobtusa; lane 4, M. japonica; lane 5, M. pachydermatis; lane 6,9,M. yamatoensis; lane 10,M. sympodialis; lane 11,M. dermat(NEB). ITS 3/4 PCR product size clustered the 11 MalasseziacomprisedM. furfur,M. obtusa,M. pachydermatis andM. japoM. restricta andM. yamatoensis (panel A: lanes 6—9). Group II10—12). In group I differentiation to species level was achieved40 GenBank retrieved sequences no AluI restriction site couldfrom M. obtusa by restriction digestion with MspAI (panel D: ladifferentiated by the AluI restriction profiles (Table 1; Fig. 1through the BanI restriction digestion (panel C: lane 8) while tdigested by any of the enzymes used in this study (panels B—Drestriction of the ITS3/4 product with BanI andMspAI. No BanI r4 PCR product (panel C: lane 10) while MspAI recognized twodermatis (panel D: lanes 11 and 12).

in contrast to the remaining 22 isolates and to theGenBank predicted BanI restriction profiles (Table 1;Fig. 1). Only oneM. fufur (CBS 9375) strain isolated inCanada did not possess a BanI restriction site. As thisis the first observation on a possible geographicalclustering of a given M. furfur subtype, the resultshave to be assessed by testing and sequencing iso-lates from different geographical regions.

In group II M. globosa and M. slooffiae weredifferentiated by the AluI restriction profiles(Table 1; Fig. 1). One of the M. globosa publishedsequences (AJ437693) did not possess the commonAluI restriction site at position 237. However, thetwo AluI predicted restriction fragments (221 and256 bp) did not exclude AluI from use in the identi-fication procedure. M. restricta was differentiatedthrough the BanI restriction digestion (Table 1;Fig. 1) while the single available strain of M. yama-

) and restriction profiles with AluI (panel B), BanI (panel C)e marker pBR322MspI digest; lane 2,M. furfur; lane 3,M.M. slooffiae; lane 7,M. globosa; lane 8, M. restricta; laneis; lane 12,M. nana; lane 13, molecular size marker bp100species in three groups (panel A: lanes 2—12). Group I

nica (panel A: lanes 2—5), group IIM. slooffiae,M. globosa,I included M. sympodialis, M. dermatis and M. nana (lanesby restriction with AluI (panel B: lanes 2—5). In 2 out of thebe identified and these two strains could be differentiatednes 2 and 3). In group II M. globosa and M. slooffiae were: panel B: lanes 6 and 7). M. restricta was differentiatedhe novel species M. yamatoensis was the only species not: lane 9). Group III species identification was achieved byestriction site was recognized on theM. sympodialis ITS 3/restriction sites in M. nana and one restriction site in M.

Letter to the Editor 217

toensis was the only species not digested by any ofthe enzymes used in this study (Table 1; Fig. 1).

Group III included the phylogenetically close spe-cies M. nana, M. dermatis and M. sympodialis [5].Within this group identification to species level wasachieved by restriction of the ITS3/4 product withBanI and MspAI. No BanI restriction site was recog-nized in the M. sympodialis ITS 3/4 PCR amplifica-tion product (Table 1; Fig. 1) whileMspAI recognizedtwo restriction sites in M. nana and one restrictionsite in M. dermatis (Table 1; Fig. 1).

Recently, aPCR-RFLPmethod for the identificationof 11 Malassezia species was applied on 35 strainsof the genus [10]. Themethod used primers designedto amplify part of the Malassezia 26S rDNA andsubsequently the PCR product was digested withtwo restriction endonucleases. The methodologydescribed was simple and reliable from pure culturematerial. Following specificity testing using otherskin pathogen DNA [8], the applicability for this assayon direct identification of Malassezia species fromskin scales will be assessed. The present study is thefirst upgrade of one of the current PCR-based identi-fication systems for the differentiation of 11 Malas-sezia species. Contrary to the Terminal FragmentLength Polymorphismanalysis [9] or the use of nestedPCR with species specific primers and subsequentsequencing of often ambiguous PCR products [4],theproposedPCR-RFLP systemneedsminimal labora-tory infrastructure, has low investment and runningcosts and it can be utilized for the identification ofMalassezia species from DNA extracted directly fromskin scales [8]. Thus, it could be used in epidemiolo-gical studies and routine diagnosis highlightingaspects of the complex biology and host—fungusinteractions of this skin commensal and pathogen.

Acknowledgements

This study was supported by the University of AthensMedical School grant no. 4121-10/2004. We thank M.Kambouris for his comments and S. Kritikou forlaboratory assistance.

References

[1] Gupta AK, Batra R, Bluhm R, Boekhout T, Dawson Jr TL. Skindiseases associated with Malassezia species. J Am AcadDermatol 2004;51(5):785—98.

[2] Gueho E, Midgley G, Guillot J. The genus Malassezia withdescription of four new species. Antonie Van Leeuwenhoek1996;69:337—55.

[3] Sugita T, Takashima M, Shinoda T, Suto H, Unno T, Tsuboi R,et al. New yeast species,Malassezia dermatis, isolated frompatients with atopic dermatitis. J Clin Microbiol 2002;40:1363—7.

[4] Sugita T, Takashima M, Kodama M, Tsuboi R, Nishikawa A.Description of a new yeast species,Malassezia japonica, andits detection in patients with atopic dermatitis and healthysubjects. J Clin Microbiol 2003;41:4695—9.

[5] Hirai A, Kano R, Makimura K, Duarte ER, Hamdan JS,Lachance MA, et al. Malassezia nana sp. nov., a novellipid-dependent yeast species isolated from animals. Int JSyst Evol Microbiol 2004;54:623—7.

[6] Sugita T, Tajima M, TakashimaM, Amaya M, Saito M, Tsuboi R,et al. A new yeast species,Malassezia yamatoensis, isolatedfrom a patient with seborrheic dermatitis, and its distribu-tion in patients and healthy subjects. Microbiol Immunol2004;48:579—83.

[7] Mayser P, Haze P, Papavassilis C, Pickel M, Gruender K, GuehoE. Differentiation of Malassezia species: selectivity for cre-mophor El, castor oil and ricinoleic acid for M. furfur. Br JDermatol 1997;137:208—13.

[8] Gaitanis G, Velegraki A, Frangoulis E, Mitroussia A, TsigoniaA, Tzimogianni A, et al. Identification of Malassezia speciesfrom patient skin scales by PCR-RFLP. Clin Microbiol Infect2002;8:162—73.

[9] Gemmer C, De Angelis Y, Theelen B, Boekhout T, Dauson TL.Fast noninvasive method for molecular detection and differ-entiation of Malassezia yeast species on human skin andapplication of the method to dandruff microbiology. J ClinMicrobiol 2002;40:3350—7.

[10] Mirhendi H, Makimura K, Zomorodian K, Yamada T, Sugita T,Yamaguchi H. A simple PCR-RFLP method for identificationand differentiation of 11 Malassezia species. Microbiol Meth2004;61(2):281—4.

George Gaitanis*Vincent Robert

Centraalbureau voor Schimmelcultures,Uppsalalaan 8, 3584 CT Utrecht,

The Netherlands

Aristea VelegrakiMycology Laboratory,

Department of Microbiology, Medical School,University of Athens, Greece

*Corresponding author. Tel.: +30 210 7462 146;fax: +30 210 7462 147

E-mail address: ggaitanis@med.uoa.gr(G. Gaitanis)

14 November 2005