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JOURNAL OFCLINICALMICROBIOLOGY,Oct. 1994,p. 2528-2532 0095-1137/94/$04.00+0

Copyright ©D1994, American SocietyforMicrobiology

Monitoring

Spread

of

Malassezia

Infections

in

a

Neonatal

Intensive Care Unit

by

PCR-Mediated Genetic

Typing

ALEXVAN

BELKUM,1*

TEUN BOEKHOUT,2ANDRONBOSBOOM3

Department ofMolecularBiology, DiagnosticCenterSSDZ,2600 GA

Delft,

1 YeastDivision, Centraalbureau voor

Schimmelcultures, 2628 BCDelft2andDepartment ofMedicalMicrobiology,AcademicHospitalLeiden,

2300RC

Leiden,3

TheNetherlands

Received 4 March 1994/Returned formodification 20 June1994/Accepted 14July 1994

Malassezia furfur and Malassezia pachydermatis were isolated from newborn children and incubators in a

neonatalintensivecareunit. Toassesswhetherpersistenceorfrequentimportof theorganismswasthecause

of the elevatedincidence, genetic typingof the strainswasperformedbyPCR-mediated DNAfingerprinting. By usingPCRprimersaimed atrepeatconsensusmotifs,six differentgenotypescouldbedetected inacollection

of six M.furfurreference strains. In thecaseof 10 M.pachydermatisreferencestrains,nine differentgenotypes weredetected bythree different PCRassays.Noneoftheseassays coulddocumentgeneticdifferences among

the clinical isolates of eitherM.

furfiur

orM.pachydermatis. On the basis of these results it is concludedthat within the neonatal intensive care unit the longitudinal persistence of both an M. furfur and an M. pachydermatisstrain has occurred and thatMalassezia speciescanpersistonincubator surfaces forprolonged

periods of time. It canbe concluded that PCRfingerprinting isaMalassezia typingprocedure that is tobe preferredovertheanalysis ofchromosomal polymorphisms by pulsed-fieldgel electrophoresis in thisgenus.

Thebasidiomycetous yeastgenusMalassezia presently con-sists of three species (19). Malasseziafurfur and Malassezia

sympodialis are obligatory lipophilic skin flora yeasts of

hu-mans, whereas Malassezia pachyderinatis is a nonobligatory skin flora yeast also encountered in several other mammals

(12). M. furfurcan causearelativelybroad spectrum of clinical

phenomena, varyingfrompityriasisversicolor in adults (1)to

life-threatening invasive disease in neonates (7, 11, 13, 16). There is also an increasing number of reports discussing the relevance and potentialclinicalproblems of Malassezia

infec-tions incancer(8, 18)orAIDSpatients(19).SinceMalassezia

species are being encountered in clinical diagnostics more frequently, the need for identification and typing assays will

increasein thecomingyears.

For M.furfur, an immunological assaydiscriminating three serovarshasbeen described (6). It isclear that thisdegreeof

resolution is insufficient fordetailedepidemiological compar-isonsamongclinicalandenvironmental isolates. For epidemi-ological typingofyeasts in general various molecular proce-dures have beendeveloped (15), butwithrespect toMalassezia typing only a small numberof reportshave appeared inthe literature. Thesemainly elaborate on karyotyping by pulsed-fieldgelelectrophoresis (3, 10),whichagaindoesnotallow for

the highly efficient discrimination of Malassezia strains. In ordertodiscriminate thepersistence ofMalassezia strains from increased importation, novel procedures must be

imple-mented. InthisrespectPCRmayprovetobe valuable (14, 20). PCR can be used for the detection ofsubtle DNA

polymor-phisms by application of primers that aim at hypervariable

DNA loci(23, 24).This laboratory techniquehas already been

usedfrequently in ordertodetermineinterisolate relationships among fungal and protozoal species (9, 20-22). The present

reportdescribes the applicationofPCRfingerprinting for the

resolutionof Malasseziaepidemics inaneonatal intensivecare

*Corresponding author. Mailing address: Department of Clinical

Microbiology, Academic Hospital Dijkzigt, Dr. Molewaterplein 40,

3015GD Rotterdam,TheNetherlands.

unit. Recently, within a neonatal intensive care unit at the Academic Hospital Leiden (Leiden, The Netherlands), an increase in the number of M.

furfur

and M. pachydermatis infectionswas encountered(5). Severalofthe environmental andpatient isolatesweretypedby PCR, and the epidemiolog-ical implications will be discussed. Since some of the strains have also been karyotyped by pulsed-field electrophoretic separation of chromosomes (3),acomparisonbetween

karyo-typingand PCRgenotyping could be performed.

MATERIALSANDMETHODS

Yeast strains and pediatric patients.In thepresentstudy M.

furfur,

M. pachydermatis, and M sympodialis strains were analyzed. M furfur and M pachydernatis were grown on Leeming and Notmanagar(1% peptone,0.5%glucose, 0.1%

yeast extract, 0.4% desiccated ox bile, 0.1% glycerol, 0.05% glycerol monostearate, 0.05% Tween 60, 1% whole-fat cow's milk, 1.5% agar). In the case of M furfur cultivation, this mediumwassupplementedwithasmallamountof olive oil. M sympodialiswas grownon 1%yeast extract,0.5%peptone,4% glucoseagar(YPGAmedium). All strains studiedwere culti-vated at 35°C (Table 1). Table 1 lists the reference isolates from the CentraalbureauvoorSchimmelcultures (CBS; Yeast Division, Delft, The Netherlands) and the clinical isolates

collected in thedepartments of neonatologyordermatologyat

the Academic Hospital Leiden. Relevant patient data are included in Table 1.

DNAisolation and PCR. Toprevent contamination in the PCR assays, DNA isolation and DNA amplification were

performed inseparatelaboratoryrooms. Transport of

labora-toryequipment between theserooms wasprohibited.Also, the

PCRingredientswere stored in aseparateroom. Inorderto isolate DNA, cellswerescraped from the culture media and werecollected inanappropriatevolume of phosphate-buffered saline (PBS; pH 7.0). Cells were pelleted by centrifugation, washed with anothervolume ofPBS, and centrifuged again. FortheM.pachydermatis andM. sympodialisstrains, effective

lysiscouldbe achieved bytreatingcells with Novozym (Novo

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TYPING OF MALASSEZL4 SPP. BY PCR FINGERPRINTING 2529

TABLE 1. Overview of Malassezia strains examined in the study

Organism Straina Source

1. CBS 1878 2. CBS 7019 3. CBS6094

4. CBS4172

5. CBS 5333

6. CBS 6000 7. AZL113598 8. AZL 112926 9. AZL 115292 10. AZL 132977 11. AZL133102 12. AZL 112426 13. AZL113850

14. CBS 7222 15. CBS 7709 16. CBS 7705 17. CBS 7707 18. CBS 7044

19. CBS4165

20. CBS1891 21. CBS 1884 22. CBS 6534

23. CBS1919

24. CBS 6541

25. CBS6537 26. CBS 1885 27. CBS 6542 28. CBS6535 29. AZL J 30. AZL A4 31. AZL W 32. AZL D5 33. AZL KV 34. AZL107498

35. AZL RV 36. AZLSH

37. AZL 101992 38. AZL ST 39. AZLD3

40. AZLCM

41. AZLWS

42. AZLJV

R.W. Benham, type ofPityrosporum ovale

V. K.Hopsu, neotype, pityriasis versicolor, Finland F.Keddie, normal skin, United States

B. A.Custafson, skin of eland F.Flank, lesion of skin, Canada I. G. Murray, ex dandruff, India

R.Bosboom, urine of neonate B,11-5-90b

R.Bosboom,urine of neonate A, 6-5-90 R. Bosboom, urine of neonate B, 28-5-90 R.Bosboom, urine of neonate D, 26-11-90 R.Bosboom, urine of neonate E, 27-11-90 R.Bosboom,skin of neonate A, 30-4-90 R.Bosboom, urine of neonate B, 13-6-90 R. B.Simons,auditory tract, United States

E. Guley, skin withseborrheiceczema, The Netherlands E.Guley,pityriasis versicolor,The Netherlands E. Guley,pityriasis versicolor,The Netherlands R. van Breuseghem

J.Lodder,ear ofdog, The Netherlands B. A.Gustafson, ear of dog,Sweden

H.E. Rhoades, ear of dog, United States

P.W.C. Austwick,ulcerated ear of dog, United Kingdom H. E.Rhoades,earof dog

H.E.Rhoades, ear of dog, United States J.Lodder,ear ofdog,TheNetherlands H.E. Rhoades,earof dog

H. E.Rhoades,earof dog, United States

19-3-91

Incubator,22-1-91 15-1-91

Incubator,27-3-91 17-4-91

NKC

26-2-91 19-3-91 NK 22-1-94

Incubator, 6-3-91

17-4-91 5-2-91 17-1-91

aCBS, Yeast Division, CentraalbureauvoorSchimmelcultures,Delft,TheNetherlands; AZL, Academic Hospital Leiden, Leiden, The Netherlands. The numbers 1through 42correspondtothenumbering used in Fig. 1 and 2.

bIsolation datesaregiven inday-month-year.

NK,date of isolationnotknown.

Industries, AIS), subsequent spheroplast lysis,and DNA

puri-fication by a method with guanidinium isothiocyanate (4).

Since the clinical M. furfur isolates seemed to resist this

procedure, an alternative andmoregenerally applicable pro-cedure for the isolation ofDNA fromMalassezia specieswas developed. Cell pellets were washed once more in distilled water andlyophilized forat least 60 h. Theresulting powder wasresuspendedinabuffercontaining100 mMTris-HCl(pH 6.4), 4 M guanidine isothiocyanate, 10 mM EDTA, and 2%

(vol/vol)TritonX-100,and the mixturewasincubatedat37°C

for at least 2 h. Thereafter DNA was purified by affinity chromatography (22). The amounts of isolated DNA were estimated by electrophoresis in 1% agarose gels in 40 mM Tris-borate(pH 7)-i mM EDTA(0.5xTBE)andcomparison

with ethidium bromide-stained amounts of bacteriophage

lambdaDNA(Promega)inaparallel run.

For PCR, approximately 50 to 100 ng ofMalassezia DNA

was dissolved ina 100-pulvolume containing 10 mM Tris-HCl (pH 9), 50 mM KCI, 2.5 mM MgCl2, 0.01% gelatin, 0.1% Triton X-100, 0.2 mM (each) deoxyribonucleotide triphos-phate,and 0.5 U ofTaqDNApolymerase (SphaeroQ,Leiden,

The Netherlands). The following primers (sequences) were used for PCRfingerprinting ofMalassezia strains: ERIC IR

(5'-ATGTAAGCTCCTGGGGATfCAC-3'), ERIC2 (5'-AA GTAAGTGACTGGGGTGAGCG-3'), REP1R-I (5'-IIIICGI CGICATCTGGG-3'), REP2-I

(5'-ICGICTTATCIGGCCTA

C-3'),and BG-2(5'-TACATTCGAGGACCCCTAAGTG-3').

(I stands for the base derivative inosine). All primers have been described previously (22, 23). After the addition of 50

pmol ofone or moreof the PCRprimers, theaqueousphase wasoverlaidwith mineral oil andthermocyclingwasperformed

in a Biomed type 60 PCR machine. The PCR program consistedofa4-minpredenaturation stepat94°C;40cyclesof 1 minat 94°C, 1 minat 25°C, and 2 minat 74°C; anda final M.furfur

M.sympodialis

Mpachydermatis

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2530 vAN BELKUM ET AL.

extension step of 4 minat74°C.Amplimerswere

analyzed

by electrophoresis through 1 to 3% agarose gels in 0.5x TBE. Ethidium bromide-stained gels were

photographed

by using Polaroid Polapan T52 films.

PCR results werevalidated onlywhen the

negative

control

samples (in which no extraneous DNA was

included)

were

demonstrated nottocontain DNA after PCR.

RESULTS

All strains listed in Table 1 were

subjected

tovarious PCR assays. Initialexperimentswith the

arbitrary primer

BG2(22) appeared to be unsuccessful, although this

primer

has been veryusefulinelucidatingthe

interrelationships

among isolates ofother fungal species. Thegels showed

monomorphic

DNA banding patterns oflowcomplexitybecauseonly afew DNA fragments were synthesized (data not

shown).

Pilot

experi-mentsinvolvingasmallnumber ofMalasseziastrains revealed

thatthe application ofsingle REP orERIC

primers

(23)

was

nothighly

discriminatory.

However, thecombination of

prim-ers ERIC IR and ERIC2 in a single reaction revealed a

sufficiently high degree of DNA

diversity

among the various

strains. As can beseeninFig. 1,all six CBS strains ofMfurfur

could be unequivocally identified. In the group of 10 CBS strainsof M

pachydermatis,

nine differenttypesweredetected. In Table 2, the M pachydermatis genocodes constructed by

identifyinggiven DNA fingerprints with aletter ora number

show that the combined results oftwoPCR assays allowed 9 of 10 strainstobediscriminated. OnlyCBS 6541 and CBS6535,

both U.S. isolates

originating

from the

dog

auditory

tract, could bediscriminated. Inordertogaininsight intothegenetic

diversitythatcanbehighlighted byPCR

fingerprinting

of this

Malassezia species, more isolates,

preferably

from multiple

geographicorigins, mustbe analyzed.

All M sympodialis reference strains could be

distinguished

onthebasis of their DNA

fingerprints.

Itwas shown that the

bandingpattern of CBS7222,thetype strain ofM

sympodialis,

wasgrosslydifferent fromthoseof the other strains. All clinical

M pachydermatis strains appeared to be identical

(Fig. 1).

Interestingly, all incubator isolates (AZL A4, AZL D5, and

AZL D3) were genetically comparable. This implies that

despite regular cleaning of the

incubators,

M pachydermatis

persists on the glass surfaces. Since the isolation dateswere

over2months apart, thispersistence may beclinicallyrelevant.

Apparently, theclinical Mfurfur strains are clonallyrelated,

since strainsAZL 115292 and AZL 133102 displayed minor

differenceswithregard to the otherhospitalisolates whenthe

combination of the ERIC primers was used. These slightly

abberantstrains werederivedfrom twodifferent patients. The

differences among the clinical isolates of M

furfiur

aremuch

smaller than those among the CBS reference strains. The

combinationof REP1R-IandREP2-I primers generated

use-ful results, especially for the M

pachydernatis

strains.

How-ever, PCR fingerprints of M furfur and M sympodialis were

relatively vague, but the results described above were sup-ported. PCR withREP1-REP2 did not discriminate among the clinical M furfur strains. Figure 2 shows the results for M

pachydermatis type strainsandclinical isolates.

DISCUSSION

Whereas severalgenomic

typing

procedures forMalassezia

yeasts appeared to be insufficiently discriminatory, PCR

fin-gerprintingcanbesuccessfully used for the adequate typing of

Malassezia strains. Although the isolation of DNA from some

strainsmaybe troublesome and not all PCRprimers generate

0'lP Q In 11 19 M 1 2 mn 3 4A P

A.

B.

f/ / J/ /

29 30 31 32 33 34 30 37 38 39 40 41 42 40 36 M

18 2) 21 22 23 24 25 26 27 28 M 14 15 16 17

C.

-1018

_220

i_.1018

_220

_1.636

.22o

FIG. 1. Typing ofMalassezia strains by PCR fingerprintingwith

primers ERIC IR and ERIC2. (A) Comparison of clinical isolates

(strains7 to13)andreference strains(strains1to6)ofM.firfur.The

banding patterns indicate homogeneity among the clinical isolates versusgenetic heterogeneityamong the reference strains. Note that clinical isolates 9 and 11displayedminordifferences whentheywere

comparedwith theotherpatientstrains(seealsoResults). Theisolate

indicatedmp(arrow) representsM.pachydernatis (strain 28). (B)PCR

analysisofclinical M.pachydermatisstrains.Strains 30and 40 wererun in duplicate (arrows). All isolates display identical DNA banding patterns.(C)PCRanalysisof M.pachydermatis type strains (strains18

and 20 to28). DNAisolatedfromstrain 20 appearedtobe ofpoor quality.Strains 14 to 17 were M.sympodialis; strain 14 gave aclearly abberant DNA banding pattern. Molecular mass markers (M) are

indicated on the right (inbase pairs). Numbering of strains is asin

Table1.

sufficientlyvariable DNAbandingpatterns, several

epidemio-logicallyuseful assayscanbedeveloped by includingmultiple,

prokaryoticrepeat consensus primers like the ERIC or REP

motifs. DNAcanbe isolatedsuccessfully bycombining

lyoph-ilization with the lytic activity of thechaotropic saltguanidine

isothiocyanate. Sufficiently discriminative assays can be

de-signed.

As demonstrated in this report, on the basis of the

homo-geneityof thefingerprints of all clinical Malassezia isolates, as

opposed to the variation observed in groups of reference

strains, itmustbeconcluded that in the neonatology ward of theAcademic Hospital Leiden genuine nosocomial epidemics of both M.furfurand M.pachydermatis have occurred. This is

corroborated by the fact that the M. pachydermatis isolates

foundtocolonize theincubators were genetically indistinguish-able over time. Also, M. furfur could be found on incubator surfaces. For this species it has been demonstrated that genotypically homologous strains could be isolated from the

samesurfaces 3months after the original culture was obtained

(data not shown). This phenomenon indicates that regular

J.CLIN. MICROBIOL.

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TYPING OF AMLASSEZIA SPP. BY PCR FINGERPRINTING 2531

TABLE 2. PCRfingerprinting genocodes for CBS strains of M.pachydermatis

Strainno. in Type by PCR assaywitha:

CBS strain Table 1 and ERIC IR- REP1R-I

typeb

Fig.1and2 ERIC2 REP2-I

CBS 7044 18 A A I

CBS 1891 20 B B II

CBS 1884 21 C C III

CBS 6534 22 D D IV

CBS 1919 23 E E V

CBS6541 24 F F VI

CBS6537 25 D F VII

CBS 1885 26 G G VIII

CBS 6542 27 H H IX

CBS 6535 28 F F VI

aBoth assaysidentified eighttypes within the group of 10 strains. Combination

ofthe resultsleftonlystrainsCBS 6541 and CBS 6535 undiscriminated. b Ninetypeswereidentifiedoverall.

hygienic measuresdo not adequately remove or kill Malassezia yeasts.

Several ofthe strains described in this report were

karyo-typed bypulsed-field gel electrophoresis (3).Fromthe results

of these studies it appears that the speciesM.pachydermatis,

M.

fuirfr,

andM.sympodialiscould beidentifiedonthe basis of

their characteristic karyotypes, also showing that all species harbor a relatively small genome when compared with the genomesizes of other yeastspecies(25). Neither oftheclinical

M.

furfur

isolates could be discriminated on the basis of chromosomallengthpolymorphism,whereas the differentCBS

typestrainsofM

furfur

couldbedividedinto four

distinguish-able karyotypes. This type of DNA heterogeneity is in full

agreement with thePCR results. The resultsobtained for M

pachydermatis seem to be contradictory. On the basis of the

lengthpolymorphisms found in the smallest chromosomes, it has been suggested that the clinical isolates are diverse and

thatreinfection rather thananepidemichas takenplacein the

neonatology ward (3). The PCR fingerprinting results,

how-ever,seemtoindicateagenuine

epidemic.

Inorderto

explain

thiscontradiction, itcould be assumed that the chromosomal

length variation observed in the clinical M

pachydernatis

isolates is due to copy number variation in the

repetitive

ribosomal operons. This corroborates

previously

observed

phenomenawhich reflect onlyaminorsingle-locus variability,

which is

generally

considered a result of

growth

conditions.

A,

24 30 31 32 33234 35 36 37 36 39 40 41 42 NA .1636

_510

B. -1636

_ 5 1 0~~~~~~-1

FIG. 2. TypingofM.pachydermatis strainsbyPCRfingerprinting withprimersREP1R-I andREP2-I.All clinical isolatesappearedtobe

identical(A),whereasheterogeneitywasencounteredamongthetype

strains(B).Molecularmassmarkersareindicated ontheright (inbase

pairs).Forindexingofthebanding patternsforthe typestrains, see

Table 2.

Amplification

of ribosomal operons as a consequence of

environmental factors hasbeen demonstrated before (2, 17)

andcannotbe used as amarker for overall genetic variability.

The moredispersedtypeof variation(multilocus)detected by

PCR maygivea more global pictureof genomic evolution. As

aconsequence the resultsofPCRfingerprinting of Malassezia

DNA mayhavemoreextensiveepidemiologicalvalue than the detection of chromosomallength variationbypulsed-fieldgel

electrophoresis.

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