f u n g a l b i o l o g y 1 1 5 ( 2 0 1 1 ) 1 0 5 1e1 0 6 5

journa l homepage : www.e lsev ier . com/ loca te / funb io

Elucidation of distribution patterns and possible infectionroutes of the neurotropic black yeast Exophiala dermatitidisusing AFLP

Montarop SUDHADHAMa,b, A. H. G. GERRITS VAN DEN ENDEa, P. SIHANONTHc,S. SIVICHAId, R. CHAIYARATe, S. B. J. MENKENb, A. VAN BELKUMf, G. S. DE HOOGa,b,*aCentraalbureau voor Schimmelcultures Fungal Biodiversity Centre, Utrecht 3584 CT, The NetherlandsbInstitute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam 1098 XH, The NetherlandscDepartment of Microbiology, Chulalongkorn University, Bangkok 10800, ThailanddBiotec-Mycology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), Pathumthani 12120, ThailandeDepartment of Biology, Faculty of Science, Mahidol University, Bangkok 10400, ThailandfErasmus University Medical Centre, Department of Medical Microbiology and Infectious Diseases, Rotterdam 3015 GD, The Netherlands

a r t i c l e i n f o

Article history:

Received 14 May 2009

Received in revised form

14 June 2010

Accepted 17 July 2010

Available online 24 July 2010

Corresponding Editor: Kentaro Hosaka

Keywords:

AFLP

Black yeasts

Infection

Neurotropism

* Corresponding author. Centraalbureau voorE-mail address: de.hoog@cbs.knaw.nl

1878-6146/$ e see front matter ª 2010 Britisdoi:10.1016/j.funbio.2010.07.004

a b s t r a c t

Distribution of populations of the opportunistic black yeast Exophiala dermatitidis was stud-

ied using AFLP. This fungus has been hypothesized to have a natural habitat in association

with frugivorous birds and bats in the tropical rain forest, and to emerge in the human-

dominated environment, where it occasionally causes human pulmonary or fatal dissem-

inated and neurotropic disease. The hypothesis of its natural niche was investigated by

comparing a set of 178 strains from natural and human-dominated environments in Thai-

land with a worldwide selection of 107 strains from the reference collection of the CBS Fun-

gal Biodiversity Centre, comprising 75.7 % clinical isolates. Many isolates had unique AFLP

patterns and were too remote for confident comparison. Eight populations containing mul-

tiple isolates could be distinguished, enabling determination of geographic distributions of

these populations. Some of the populations were confined to Thailand, while others oc-

curred worldwide. The local populations from Thailand contained strains from natural

and urban environments, suggesting an environmental jump of the fungus. Strains from

human brain belonged to widely dispersed populations. In some cases cerebral isolates

were identical to isolates from the human intestinal tract. The possibility of cerebral infec-

tion through intestinal translocation was thus not excluded.

ª 2010 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction most stressful parts of their life cycle. Such fungi are often re-

Many fungi living in hostile (micro)niches or showing other

types of extremotolerant behavior are heavily melanized

and frequently show reduced morphology, with budding cells

or with clumpy, meristematic growth expressed during the

Schimmelcultures Fung

h Mycological Society. Pu

ferred to with the umbrella term ‘black yeasts’. One of the

main groups of black yeasts and relatives phylogenetically

are members of the ascomycete order Chaetothyriales. This

order comprises extremotolerant fungi, but also numerous

species that are regularly encountered as etiologic agents of

al Biodiversity Centre, Utrecht 3584 CT, The Netherlands.

blished by Elsevier Ltd. All rights reserved.

1052 M. Sudhadham et al.

subcutaneous and systemic infections in humans and other

vertebrates (Gueidan et al. 2008). The evolutionary origin of

virulence is not known, but has been supposed to have an eco-

logical trigger in their preference for environments that are

unsuitable for most competing microorganisms. One of the

major research questions in the Chaetothyriales is whether

the species causing infection are accidental opportunists,

i.e., having their natural niche in the environment, or whether

they are truly pathogenic, i.e., showing increased fitnesswhen

any part of their life cycle makes use of an animal vector

(de Hoog et al. 2000). Understanding of the natural habitat of

black yeast-like fungi is essential for answering this question.

Vitality factors enhancing survival in environmental habitats

may explain fungal behavior in human-made environments,

or may recur as virulence factors determining their potential

to cause infection.

One of the examples of black yeasts with a complex life cy-

cle supporting an unusual type of ecology is Exophiala dermati-

tidis. This species is a very frequent colonizer of public Turkish

steam baths (Matos et al. 2002), an ecology probably deter-

mined by a combination of thermotolerance (i.e., surviving

the high temperature of the bath; Padhye et al. 1978;

Sudhadham et al. submitted for publication), oligotrophism

(Satow et al. 2008; i.e., coping with low nutrient levels on

bath tiles), and presence of an extracellular polysaccharide

(EPS) capsule enhancing adhesion to smooth surfaces of the

tiles (Yurlova & de Hoog 2002). This combination of properties

hints at a natural life cycle in association with frugivorous an-

imals in the tropical rain forest, which is one of the rare natu-

ral habitats where the fungus is isolated at a frequency

slightly above a very low base line (Sudhadham et al. 2008).

According to this hypothesis, adhesion to surfaces and oligo-

trophism are adaptations to an epiphytic part of the life cycle

of the fungus on fruits and berries, where nutritional condi-

tions are limiting, and sugar levels require a certain degree

of osmotolerance (de Hoog & Haase 1993). Thermotolerance

is necessary after ingestion by frugivorous animals while

passing their intestinal tracts. Also in humans intestinal

colonization has been reported, although at low frequency

(de Hoog et al. 2005); this is a potential source of systemic

and disseminated infection (Hiruma et al. 1993).

If this assumption is correct, the zoonotic fungus E. derma-

titidis has gone through a considerable environmental shift,

from a defined natural habitat in the tropical rain forest to

prevalence in human-made environments, and ultimately to

humans themselves. The transition of the fungus from its nat-

ural habitat may reflect emergence of a novel human patho-

gen, and the question is which genes are involved in this

ecological transition. As the fungus is able to cause fatal dis-

seminated, neurotropic infections in humans without known

immune disorder (Li et al. 2010), this poses a significant public

health risk. In the present paper, we will analyze geographic

distribution patterns and natural substrates of E. dermatitidis

using strain typing, in order to test the hypothesis of an eco-

logical shift and subsequent adaptation to the human-domi-

nated environment.

For genotyping we used Amplified Fragment Length Poly-

morphisms (AFLP) analysis, a molecular fingerprinting tech-

nique that was introduced by Vos et al. (1995). The technique

has been widely applied in studies of e.g. phylogenetic

relationships among plants (Schmidt-Lebuhn et al. 2005), ge-

netic variation of fungal plant pathogens (Kothera et al.

2003), emergence of pathogenic clones (Boekhout et al. 2001),

and inmany other ecological and evolutionary studies. Our in-

vestigation concerned an analysis of strains of E. dermatitidis

from Thailand. South-East Asia was chosen because the se-

vere disseminated human cases are confined to this region.

The set of isolates was compared with a worldwide selection

of clinical and environmental isolates from a reference collec-

tion, comprising isolates from fatal cases reported in the

literature.

Materials and methods

Fungal strains and culture conditions

A total of 178 strains were acquired after selective isolation as

described by Sudhadham et al. (2008). Samples were taken

mainly from bathing facilities, from railway ties, from feces

of frugivorous animals, and from healthy tropical fruits. The

isolation protocol was described in detail in Sudhadham

et al. (2008). Briefly, samples were taken repeatedly from the

same sampling location at distances of one to several meters.

Samples were incubated in 5.5 mL Raulin’s solution and

shaken gently in a near horizontal position at 10 r.p.m. at

25 �C for 2e3 d. Suspensions were then vortexed for 10 s and

0.5 mL aliquots were spread onto (selective) Erythritol Chlor-

amphenicol Agar (ECA) and (non-selective) Sabouraud’s Glu-

cose Agar (SGA) plates and dispersed with a Drigalski

spatula. Plates were sealed with Parafilm and incubated for

up to 30 d at 40 �C. Small black colonies were purified in

0.1 % Tween80 by single-spore isolation on Potato Dextrose

Agar (PDA). Stock cultures were maintained on Malt Extract

Agar (MEA) and PDA agar slants at 4 �C. Prior to DNA extrac-

tion, fungi were subcultured on MEA at 24 �C for 3e5 d. Refer-

ence strains included 81 clinical and 26 environmental

isolates from the reference collection of the CBS Fungal Biodi-

versity Centre, Utrecht, The Netherlands, showing a world-

wide distribution. Strain data of the isolates grouped with

�3 members in populations out of a total of 286 isolates are

given in Table 1.

Geographic mapping

Distances between sampling locations were measured using

ARCVIEW version 3.1. Coordinates were tracked by a GPS device

(Garmin, U.S.A.).

DNA extraction and identification

About 1 cm2 of myceliumwas transferred to a 2 mL Eppendorf

tube containingw300 mgglass beads (SigmaG9143) and400 mL

TE-buffer pH 9.0 (100 mM Tris, 40 mM Na-Ethylenediaminete-

traacetic acid (EDTA)). Samples were homogenized for 1 min

using a MoBio vortex (Bohemia, NewYork, U.S.A.) adding

120 mL 10 % SDS. Samples were incubated in a water bath at

55 �C for 30 min with an additional 10 mL proteinase K (10 mg/

mL), mixed well, and vortexed for 3 min in 120 mL 5 M NaCl,

while 65 mL of 10 % CTAB (cetyltrimethylammoniumbromide)

Table 1 e Strains analysed limited to strains with ‡3 strains per population. Main genotypes are derived from ITSsequencing data. Attributions to AC-A main groups are listed. Population numbering is based on AC-A profiles.

Strain number ITS genotype Source Country AC-A population

dH 13134¼Mont KH 18/3 B Soil in feeding area,

Chonburi

Thailand 2

dH 13172¼MTH CH348/1 B Flying fox’s feces (Pteropus

scapulatus), Chonburi

Thailand 2

dH 13133¼MKH3 B Common myna’s feces,

Chonburi

Thailand 2

dH 17540¼MTH 822/3-3S B Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 2

dH 14130¼MTH 582 s/6 B Mango’s surface, Bangkok Thailand 1

dH 14131¼MTH 582 S/7 B Mango’s surface, Bangkok Thailand 1

dH 13173, dH 13227 B Flying fox’s feces (Pteropus

scapulatus), Chonburi

Thailand 1

dH 14412¼MTH 552 S/31 B Pineapple surface

(Ananas comosus), Rayong

Thailand 1

dH 13183¼MTH BL 95 B Rhinoceros Hornbill,

Hala-bala, Narathiwat

Thailand 1

dH 17524¼MTH 822/2-7S B Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 1

dH 17575¼MTH 822/7-3e B Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 1

dH 17580¼MTH 822/7-12E B Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 1

dH 17979¼MTH 821/5-2s B Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 1

dH 9814¼CBS 100341 B Blood Germany 1

dH 17531¼MTH 822/2-14S B Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 1

dH 17578¼MTH 822/7-8.1S B Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 1

dH 17579¼MTH 822/7-8.2S B Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 1

dH 14152¼MTH 919/4 B Steam room, Chiangrai Thailand 3

dH 14174¼MTH 923/8 B Steam room, Chiangrai Thailand 3

dH 14175¼MTH 923/9 B Steam room, Chiangrai Thailand 3

dH 13722¼MTH 919/1 B Steam room, Chiangrai Thailand 3

dH 13723¼MTH 920/1 B Steam room, Chiangrai Thailand 3

dH 14154¼MTH 920/2 B Steam room, Chiangrai Thailand 3

dH 14156 ¼MTH 920/4 B Steam room, Chiangrai Thailand 3

dH 14164¼MTH 921/7 B Steam room, Chiangrai Thailand 3

dH 13719¼MTH 890/1 B Steam room, Chiangrai Thailand 3

dH 12984 B Respiratory tract Qatar 3

dH 14137¼MTH 890/7 B Steam room, Chiangrai Thailand 3

dH 14138¼MTH 891/2 B Steam room, Chiangrai Thailand 3

dH 14139¼MTH 891/3 B Steam room, Chiangrai Thailand 3

dH 13699¼CBS 120472 B Lesion on leg USA 3

dH 11835¼CBS 109144 B Steam bath in sauna

complex, Amsterdam

The Netherlands 3

dH 13139¼MS 5/2 B Steam room wall, Bangkok,

Thailand

Thailand 3

dH 13310¼Astrid 28 B Steam room floor Austria 3

dH 13174¼MTH MS 8/1; MS 8/1 B Steam room, Bangkok Thailand 3

dH 14184¼MTH 925/1 B Steam room, Chiangrai Thailand 3

dH 14187¼MTH 925/4 B Steam room, Chiangrai Thailand 3

(continued on next page)

Distribution patterns and possible infection routes of E. dermatitidis 1053

Table 1 e (continued)

Strain number ITS genotype Source Country AC-A population

dH 13182¼MTH 697¼CBS 120548 A Bonobo feces, zoo

Apeldoorn

The Netherlands 4

dH 12922¼ det 188/2002 938-II A3 Citrus pectin Denmark 4

dH 13585¼Astrid Q A2 Steam room seat Austria 4

dH 17537¼MTH 822/3-1S A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 4

dH 9883¼ det. 128 A Fruit Germany 4

dH 11380 A Cystic Fibrosis Finland 4

dH 13106 A Meningitis USA 4

dH 13693¼ Sutton 93-228 A Stool USA 4

dH 15533¼CBS 207.35 A Facial lesion Japan 4

dH 15696¼CBS 292.49 A Feces Brasil 4

dH 12929¼ det 205 III A Citrus pectin Denmark 4

dH 11830¼CBS 109140¼T-12 A Public Finnish sauna, Laren The Netherlands 4

dH 12771¼Tadeja 508 A Human feces Slovenia 4

dH 12924¼ det M217/2002 A Nail Denmark 4

dH 15434¼CBS 156.90 A Sputum of patient with

Cystic Fibrosis, Aachen

Germany 4

dH 12921 A Citrus pectin Denmark 4

dH 14102¼MTH 983 S/10 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 5

dH 14191¼MTH 983 E/3 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 5

dH 14219¼MTH 984 S/6 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 5

dH 14195¼MTH 983 E/7 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 5

dH 14201¼MTH 983 s/4 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 5

dH 14228¼MTH 987 E/3 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 5

dH 14226¼MTH 987 E/1 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 5

dH 14430¼M 812/3 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 5

dH 17543¼MTH 822/3-5S A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 5

dH 17552¼MTH 822/4-1S A3 Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 5

dH 17553¼MTH 822/4-2S A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 5

dH 17565¼MTH 822/5-2E A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 5

dH 13228¼MTH CH344 A3 Flying fox’s feces (Pteropus

scapulatu), Chachaengsao

Thailand 5

dH 15416¼CBS 149.90 A Sputum of patient with

Cystic Fibrosis, Aachen

Germany 5

dH 13138¼MS 7/2 B Steam room, furrow in wall,

Bangkok

Thailand 5

dH 13725¼MTH 983e/1

¼CBS 116726

B Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 5

1054 M. Sudhadham et al.

Table 1 e (continued)

Strain number ITS genotype Source Country AC-A population

dH 14270¼MTH 991 S/1 B Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 5

dH 15135¼CBS 100340 B Bat liver, Manaus,

Amazonia

Brazil 5

dH 17520¼MTH 822/1-2S A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 5

dH 17525¼MTH 822/2-9S A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 5

dH 17566¼MTH 822/5-3E A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 5

dH 17743¼MTH 826/9-7s C Railway station, stone

stained with petroleum oil,

Pitsanulok

Thailand 5

dH 17981¼MTH 821/6-2s A Railway station, stone

stained with petroleum oil,

Pitsanulok

Thailand 5

dH 17988¼MTH 820/5-8e A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 5

dH 17989¼MTH 820/8-4e A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 5

dH 17993¼MTH 826/9-1s A Railway station, stone

stained with petroleum oil,

Pitsanulok

Thailand 5

dH 17994¼MTH 826/9-2s A Railway station, stone

stained with petroleum oil,

Pitsanulok

Thailand 5

dH 17998¼MTH 826/9-5e A Railway station, stone

stained with petroleum oil,

Pitsanulok

Thailand 5

dH 17999¼MTH 826-9/5s A Railway station, stone

stained with petroleum oil,

Pitsanulok

Thailand 5

dH 18000¼MTH 826/9-6s A Railway station, stone

stained with petroleum oil,

Pitsanulok

Thailand 5

dH 14300¼MTH 992 S/5 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 7

dH 14301¼MTH 992 S/6 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 7

dH 14305¼MTH 992 S/9 B Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 7

dH 14212¼MTH 984 E/6 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 7

dH 14414¼M 811/3 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 14443¼M 819/3 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 17549¼MTH 822/3-11E A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 7

(continued on next page)

Distribution patterns and possible infection routes of E. dermatitidis 1055

Table 1 e (continued)

Strain number ITS genotype Source Country AC-A population

dH 14208¼MTH 984 E/2 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 7

dH 14207¼MTH 984 E/1 A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 7

dH 14210¼MTH 984 E/4 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 7

dH 14213¼MTH 984 E/7 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 7

dH 14119¼MTH 552 S/29-2 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 7

dH 14211¼MTH 984 E/5 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 7

dH 14227¼MTH 987 E/10 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 7

dH 14415¼M 811/4 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 14416¼M 811/5 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 14419¼M 811/8 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 14420¼M 811/9 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 14426¼M 811/14 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 14427¼M 811/16 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 14428¼MTH 811/17 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 14432¼M 813/2 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 14440¼M 815/4 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 7

dH 17526¼MTH 822/2-8S A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 8

dH 17982¼MTH 821/6-3s A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 8

dH 17990¼MTH 826/2s A Railway station, stone

stained with petroleum oil,

Pitsanulok

Thailand 8

dH 18001¼MTH 824/1-7s A Railway station, stone

stained with petroleum oil,

Pitsanulok

Thailand 8

dH 14194¼MTH 983 E/6 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 8

1056 M. Sudhadham et al.

Table 1 e (continued)

Strain number ITS genotype Source Country AC-A population

dH 14397¼M 811/1 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 8

dH 14417¼MTH 811/6 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 8

dH 14418¼M 811/7 A1 Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 8

dH 14421¼M 811/10 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 8

dH 14422¼M 811/11 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 8

dH 14424¼M 811/13 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 8

dH 14429¼M 812/2 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 8

dH 14431¼M 812/4 A Railway station, stone

stained with petroleum oil,

Srakaew

Thailand 8

dH 17581¼MTH 822/7-13E A Railway station, stone

stained with petroleum oil,

Nakornsawan

Thailand 8

dH 13726¼MTH 983e/9 A1 Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 8

dH 14193¼MTH 983 E/5 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 8

dH 14196¼MTH 983 E/8 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 8

dH 14222¼MTH 986 S/2 A Railway station, wood

stained with petroleum oil,

Prachinburi

Thailand 8

Distribution patterns and possible infection routes of E. dermatitidis 1057

were added. After incubation at 55 �C for 60 min and 3 min

MoBio vortexing, 700 mL SEVAG (chloroform:isoamylalcohol

24:1) was added, mixed carefully by hand, and spun for 5 min

at 4 �C at 20400g. After transferring the aqueous supernatant

to a new Eppendorf tube (w600 mL), 225 mL 5 M NH4Ac was

added and gently mixed. Samples were kept on ice for at least

30 min, spun again, and subsequently the supernatant

(w800 mL) was transferred to a clean, sterile Eppendorf tube.

510 mL isopropanolwas added to the supernatant,mixed, incu-

bated at �20 �C for 30e60 min, and spun down for 5 min. The

supernatant was decanted and pellets were washed with

100 mL ice-cold 70 % ethanol. Pellets were dried at room tem-

perature and resuspended in 100 mL TE-buffer pH 9.0 (100 mM

Tris, 40 mM Na-EDTA), incubated at 37 �C for 30 min, and re-

frigerated. DNA concentration was tested with Nanodrop and

adjusted to 50 ng/mL; quality of samples was verified on gel.

Confirmation of species identity and attribution tomain infra-

specific genotypes was based on sequencing the rDNA ITS

region (Sudhadham et al. 2008) or on RFLP screening

(Sudhadham et al. 2009).

AFLP fingerprinting

For AFLP fingerprinting, the protocol provided by PE Applied

Biosystems (Applied Biosystems, Nieuwerkerk aan de IJssel,

The Netherlands) was followed, with some modifications.

Analysis was carried out with 100 ng DNA. Digestion and liga-

tion were done in 1-tube reactions. The adaptor master mix

containing 1H T4 DNA ligase buffer with ATP, 60 pmol MseI

adaptor, 6 pmol EcoRI adaptor, 0.5 mg BSA (Applied Biosystems,

Nieuwerkerk aan de IJssel, TheNetherlands), 50 mMNaCl, and

the enzyme master mix containing 1� T4 DNA ligase buffer,

2 U MseI, 10 U EcoRI, 40 U T4 DNA ligase (Westburg, Leusden,

The Netherlands), 50 mM NaCl were prepared separately.

Both were mixed afterwards and the volume was adjusted to

9 mL with dH2O. Genomic DNA (50 ng/mL) was added to the to-

tal mix resulting in a reaction volume of 11 mL. After vortexing

for 10 s the digestioneligation mix was incubated at 37 �C for

at least 2 h, but not longer than 3 h because of possible star ac-

tivity of the EcoRI endonuclease. Subsequently, the mixture

was diluted approx. 3� by adding 25 mL dH2O and 2 mL were

1058 M. Sudhadham et al.

taken for preselective amplification with 0.3 mL (50 ng) EcoR1

Coreseq (50-GAC TGC GTA CCA ATT CA-30), 0.3 mL (50 ng) MseI

Coreseq (50-GAT GAG TCC TGA GTA AC-30) (Table 2), and

7.5 mL CoreMix (Applied Biosystems). PCR was as follows: ini-

tial elongation at 72 �C for 2 min followed by 20 cycles of dena-

turation at 94 �C (20 s), annealing at 56 �C (30 s), and

elongation at 72 �C (2 min). Preselective PCR products were di-

luted two times by adding 10 mL dH2O; 1.5 mLwere taken for se-

lective PCR. Selective amplification was done with 5 ng EcoRI

CoreseqþAC labeled with NED/30 ng MseI CoreseqþA (AC-A

system) and 5 ng EcoRI CoreseqþC labeled with FAM/30 ng

MseI CoreseqþA (Applied Biosystems) (¼C-A system). The to-

tal volume of selective PCR was 10 mL. PCR conditions were as

follows: 2 min at 94 �C, followed by 10 cycles consisting of 20 s

at 94 �C, 30 s at 66 �C decreasing 1 �C with every cycle, and

2 min at 72 �C, followed by 20 cycles consisting of 20 s at

94 �C, 30 s at 56 �C, and 2 min at 72 �C.There were two ways of preparation for acrylamide elec-

trophoresis: (1) PCR products from the [NED] combination

(C-A system) were prepared for ABI PRISM 310 Genetic Ana-

lyzer (PE Biosystems, CA, U.S.A.) as follows: 2.0 mL of selective

PCR product, 24.8 mL of deionized formamide, and 0.3 mL of

GeneScan-500 [ROX] size standard (Applied Biosystems). De-

naturationwas done at 95 �C for 5 min and then put on icewa-

ter to stop the reaction. (2) PCR products from the [FAM]

combination (AC-A system) were prepared for ABI PRISM 377

Genetic Analyzer (PE Biosystems, CA, U.S.A.) as follows: the

selective PCR products were cleaned with Sephadex G-50. Pi-

pette 1.0 mL of selective PCR products with 0.3 mL LIZ 500 size

standard (Applied Biosystems) into a new plate. The total vol-

ume was adjusted to 10 mL with dH2O. Denaturation was done

at 95 �C for 5 min and then the reaction was stopped on ice

water. Reproducibility was dependent on DNA quality and ab-

sence of shearing, and then was usually >95 %. The data

obtained from both machines were analysed with BIONUMERICS

software package (version 4.61, Applied Maths, Kortrijk, Bel-

gium). Main criterion was optimal separation of groups rather

than coherence between groups; this was achieved using

Ward’s averaging. Groups were recognized using the auto

cut-off option set at <90 % and including �3 members.

Results

Profiles of 175 strainswere generatedwith C-AAFLP and of 180

strains with AC-A AFLP. Overlapping data including 24.1 % of

the strains were generated for control, isolates being analysed

with both systems. The moderately selective C-A system,

which was mainly used for large sets of isolates from the

Table 2 e Primer and adaptor sequences used for AFLP analys

EcoRI

Adaptors 50-CTCGTAGACTGCGTACC CATCTGAC

Core primers 50-GACTGCGTACCAATTCPreselective primers (Coreseq) Core primerþA

Selective primers Preselective primerþACþNED

Preselective primerþCþ FAM

same environment, the data set being minimized after clone

correction, yielded a large number (50e70) of bands (Fig 3).

The resulting profiles were sometimes difficult to interpret vi-

sually, but facilitated automated recognition of isolates hav-

ing unambiguously the same profile using BIONUMERICS.

Banding patterns were grouped with Ward’s averaging using

the auto cut-off option at <50 % similarity, which resulted in

seven (C-A) or eight (AC-A) approximate clusters (Fig 1). C-A

clusters 1 and 2 and AC-A cluster 6 in Fig 1 consisted of highly

deviating singular profiles. Profiles that showed near-identity

with auto cut-off (>90 %) in BIONUMERICS were interpreted to

represent populations (Table 1), representatives of which

may differ by individual bands. Some visible differences in

profile and banding intensities occurred but were disregarded

by the program.

In most strains, the highly selective AC-A AFLP showed

about 30 major bands (Fig 2), and profiles could be grouped in-

tuitively. The diversity of the AC-A data set was higher than

that of C-A. This was expected, because strains analysed

with AC-A concerned primarily the more widely distributed

strains, while C-A data mainly focused on larger sets of iso-

lates originating from a small number of relatively rich sam-

pling sites. Within the main the clusters recognized with

auto cut-off above, total of 10 unambiguously identical popu-

lations (i.e., without unique bands and containing more than

two isolates) were recognizable with C-A, but 22 such popula-

tions with AC-A (overlaid in grey in Fig 1; strains attributed are

listed in Table 1). This was due to the larger number of bands

generated with C-A, making unambiguous clone attribution

more difficult and thus many profiles remained uninter-

preted/unidentified. Distribution patterns were calculated

from isolates grouped within populations only, whereas the

remaining singular or ambiguously attributed profiles were

disregarded for further analysis. GPS data were available for

strains fromThailand only, while geographic data of reference

strains mostly were only approximate, mostly not allowing

differentiationwithin countries; many of the strainswere sev-

eral decades old and had been deposited without adequate

geographic information.

Frequencies of populations were corrected for repeated

growth from a single sample within a sampling site. Isolates

originating from different samples within the same site were

maintained in subsequent calculations. As explained above,

a relatively large number of profiles could not unambiguously

be matched with any other profile, while in contrast some of

the populations showedhigh frequencies. Largest abundances

of populations were noted in a steam bath in Chiangrai, Thai-

land, analysed with C-A (Fig 3), and on petroleum oil and fecal

grease-contaminated railway ties in Nakornsawan, Thailand

is.

MseI

GCATGGTTAA-50 50-GACGATGAGTCCTGAG TACTCAGGACTCAT-50

50-GATGAGTCCTGAGTAACore primerþC

Preselective primerþA

Fig 1 e A, B. Trees based on banding patterns grouped with Ward’s averaging using the auto cut-off option at <50 % simi-

larity, resulting in seven (C-A; Fig 1A) and eight (AC-A; Fig 1B) approximate clusters. Profiles that showed near-identity with

auto cut-off (>90 %) in BIONUMERICS, had no unique band bands and contained ‡3 members were listed as genetically nearly

identical populations for biogeographic purposes (Table 1). Scale bars represent percentages overall similarity. Dotted ver-

tical lines are auto cut-off levels at 50 and 90 % similarity. (A) and (B) indicate ITS genotypes of the respective groups.

Distribution patterns and possible infection routes of E. dermatitidis 1059

Fig 2 e Example of profiles based on AFLP AC-A. A population of strains from two railway-sampling locations in Thailand.

1060 M. Sudhadham et al.

analysed with AC-A (Fig 2). These intensively sampled loca-

tions contained, respectively, two (in each of the AFLP data

sets) and eight populations within distances of a few meters.

Some of these populations were closely similar, i.e., differing

qualitatively in 1e2 bands. Nearly all populations were also

foundoutside the twosampling locations, someevenbeingen-

countered on different continents (Table 1, Fig 4). Six popula-

tions were confined to relatively restricted geographic areas

Fig 3 e Example of profiles based on AFLP C-A. Two population

Thailand and Austria.

in Thailand (Fig 4). Populations matched with earlier subdivi-

sions of the species in genotypes A and B based on sequence

differences in rDNA ITS-1.

Population 1a (AC-A; ITS genotype B) comprised four

strains, three of which originated from the surface of fresh

and healthy fruits (mango and pineapple) in Thailand. The

fourth isolate came from feces of flying foxes in Thailand at

a distance of about 90 and 80 km from the sampling locations

s of strains each from two steam bath-sampling locations in

Fig 4 e Global distribution of clone-corrected populations containing ‡3 members. Populations in Thailand are shown in

insert.

Distribution patterns and possible infection routes of E. dermatitidis 1061

ofmango and pineapple, respectively. The population was not

found outside this area.

Populations 1b (AC-A; ITS genotype B) and 1c (ITS genotype

B) originated from a single set of samples from a concrete sup-

port of a railway tie which was polluted with petroleum oil

and fecal remains in Nakornsawan province, Thailand. AFLP

banding patterns showed only minute variation. One of the

isolates (dH 13183) of population 1b originated from hornbill

feces at the Hala-Bala wildlife sanctuary, Narathiwat prov-

ince, Thailand, at a distance of 1100 km from the Nakornsa-

wan railway site.

Population 2 (AC-A; ITS genotype B) showed slight varia-

tion in minor bands. The group contained strains from con-

crete, grease-stained railway ties at Nakornsawan, Thailand,

as well as isolates from feces of tropical animals such as flying

fox and common myna, and from a bird feeding place in an

open zoo in Chonburi, Thailand, at a distance of 250 km.

Population 3a (AC-A; ITS genotype B) comprised a cluster of

Thai strains, mainly from the Chiangrai steam bath. Popula-

tion 3b (AC-A; ITS genotype B) matched with a group of 20

strains of the (C-A) data set from a steam bath in Chiangrai,

Thailand. Profiles were very similar to each other (Fig 3), and

all isolates belonged to ITS genotype B (Table 1). Isolates

emerged from different samples taken at several meters dis-

tance inside the same steam room. Strain dH 12984 with the

same AFLP banding pattern originated from a respiratory

patient in Qatar. Population 3c (AC-A; ITS genotype B) showed

massive proliferation in the same steam bath in Chiangrai

(compare 3b). The strainswere also recognized inmain cluster

4 with C-A. Strict congruence of both typing systems was

achieved. Population 3e (AC-A; ITS genotype B) was found in

steam baths in Chiangrai and also in Bangkok, Thailand, at

a distance of nearly 700 km. Population 3d (AC-A; ITS genotype

B) had a worldwide distribution, as it originated from a steam

bath in Thailand butwas also encountered in similar locations

in The Netherlands and Austria; a further strain was isolated

from a cutaneous lesion in the U.S.A.

Population 4a (AC-A; ITS genotype A) comprised four iso-

lates, showing the characteristic genotype A polymorphisms

at positions 162, 184, and 196 in ITS-1. Strains showed aworld-

wide distribution and were found in different environments

including railway ties and steam baths, as well as in feces of

a bonobo with diarrhea in a zoo in The Netherlands.

Population4b (AC-A; ITSgenotypeA)was identical toanum-

ber of isolates recognized in the C-A data set, which also were

ITS genotype A. This group contained systemic and dissemi-

nated isolates from the U.S.A. and Japan, strains from human

feces in Brazil, Slovenia and France, as well as strains from

lungs of patientswith cystic fibrosis. The group also comprised

an isolate from a flying fox in Chonburi, Thailand.

Population 5a (AC-A; ITS genotype A) was found on con-

crete and oak wood railway ties at Nakornsawan, Prachinburi

1062 M. Sudhadham et al.

and Srakhaew in Thailand, at a maximum distance of 390 km.

Cluster 5bec contained six strains showing highly similar

banding patterns, but six bands varied qualitatively, thus

not allowing geographic interpretation. Population 5d (AC-A;

ITS genotype A) was found on the concrete railway ties of

Nakornsawan and Pitsanulok.

In group 6 no populations with �3 members were detected

(Fig 1).

Population 7a (C-A; ITS genotype A) contained strains from

human brain, feces and sputum of patients originating from

different continents. Isolate dH 13227 originated from flying

fox feces in Thailand. Populations 7b (C-A; ITS genotype B)

and 7c (AC-A; ITS genotype A) had similar wide deviations in

geography and predilection.

Population cluster 8 (AC-A; ITS genotype A) entirely origi-

nated from tropical railway samples in Nakornsawan, Pra-

chinburi, Pitsanulok and Srakhaew. The closely similar

subtypes 8aec, differing quantitatively only, are not found

outside this environment. Population 8c, with one qualitative

band difference, was restricted to Prachinburi.

In summary, populations 1aec, 2, 3aec, and 5aed had

a limited distribution in Thailand, and often contained iso-

lates from the (semi)natural environment in addition to

strains from urban sources. Most of these isolates came

from temple parks and zoos within urban influence, while

the hornbill isolates from Hala-Bala Sanctuary (1b) concerned

undisturbed tropical rain forest. The remaining populations

were found at a worldwide scale. In two occasions an isolate

from a (semi)natural environment in Thailand had the same

profile as strains originating from other continents. Steam

baths and tropical railway ties proved to be successfully colo-

nized by the fungus, with mostly several populations being

present at the same sampling site. Among the (semi)natural

environments, feces of flying foxes at Chachoengsao con-

tained three populations, 1a, 2, and 5c. Population 1a was

also found on the surface of healthy fruit. Pineapple samples

at Rayong contained populations 1a and 7c.

Discussion

AFLP is a useful method for population genetic studies be-

cause it requires only small amounts of DNA (w10e1000 ng),

profiles being highly reproducible, and providing information

that reflects the entire genome (Vos et al. 1995). However, good

DNA quality is compulsory, which is problematic in black

yeasts and particularly in Exophiala dermatitidis due to the

abundance of EPS capsules in exponential phase-grown cell

cultures (Yurlova & de Hoog 2002). For that reason, strains

were cultured no longer than 4 d at 25 �C and were harvested

just before massive EPS formation started. Cellular biomass

was increased by inoculating plates densely with suspensions

and intensive spread during transfer.

In a preliminary experiment using different adapters it was

found that the combination C-A and AC-A gave the best re-

sults (data not shown), yielding readable profiles with well-

separated bands. The degree of variation within E. dermatitidis

appeared to be considerable. Main groups were separated at

<50 % similarity, with significant numerous unique bands.

In Aspergillus fumigatus, Warris et al. (2003) and de Valk et al.

(2007), using TGA- and T-TGAA AFLP, respectively, noted

that most of the major bands were consistently present in

>95 % of the strains analysed. With AC-G AFLP in Cryptococcus

neoformans a diversity comparable to our data in E. dermatitidis

was obtained (Barreto de Oliveira et al. 2004).

Exophiala dermatitidis was previously known as a colonizer

of public bathing facilities (Matos et al. 2002) and railway ties

(Sudhadham et al. 2008) in the urban environment. Its natural

niche involves epiphytic, asymptomatic growth on sugary

fruit surfaces, determining its slightly osmotic character (de

Hoog & Haase 1993). Pineapple samples at Rayong contained

populations 1a and 6c, demonstrating that a suitable habitat

is concerned. But the species does not have the ubiquitous dis-

tribution of, for example, the phyllosphere-colonizing black

yeast Aureobasidium pullulans (Andrews et al. 1994; Zalar et al.

2008). In addition, the species is consistently thermotolerant.

This indicates that this habitat can only represent part of

a complex life cycle. The missing link has been hypothesized

to be ingestion of the E. dermatitidis-colonized fruit by frugivo-

rous animals (Sudhadham et al. 2008). Feces of these animals

were repeatedly found to contain several genotypes of the

fungus, which suggests that this environment is highly suit-

able for growth; Aureobasidium was not recovered from such

samples. Association with frugivorous bats and birds in the

tropical rain forest has been observed in Thailand

(Sudhadham et al. 2008), Brazil (Reis & Mok 1979) and Nigeria

(Muotoe-Okafor & Gugnani 1993). In addition to the occur-

rence of local, natural populations of the fungus, our data

show that some genotypes have a worldwide distribution.

This may have originated by human ingestion of contami-

nated fruits and berries, intestinal colonization, and subse-

quent distribution outside the original endemic areas. The

present study demonstrates that the fungus we know in the

clinic as an emerging opportunist on humans originally may

have been a zoonotic fungus in the tropical rain forest. With

human-made, unexpected options for proliferation, the or-

ganism expands quickly and effectively in urban settings on

a worldwide scale.

Taking into account the supposed slow natural dispersal of

the fungus due to the absence of airborne propagules, local

structuring of populations might be expected, but this

appeared to be the exception rather than the rule. Population

1awas found on the feces of flying foxes in Thailand, and also

occurred asymptomatically on fruits within 80e90 km dis-

tance. Flying foxes are known to eat mangoes, which is an

abundant fruit species in natural and domesticated forests

and parks in Thailand. Individual animals have a foraging ra-

dius of about 10e20 km (Palmer &Woinarski 1999; McDonald-

Madden et al. 2005). Population 1a thus may represent a local

focus of E. dermatitidis, the hypothesized original condition

of the species, with a life cycle of animal ingestion and local

dispersal via feces. Population 1bwas distributed over a larger

distance: it was found abundantly at a grease-polluted railway

tie in Nakornsawan, Thailand, at a distance of about 1100 km

from a site with feces of a rhinoceros hornbill (Fig 4), which is

supposed to be an original focus. This distance can easily be

explained by overlapping foraging radius of E. dermatitidis-car-

rying vectors. Population 2 also consisted of strains from

grease-stained railway ties and isolates from frugivorous ani-

mal feces, at a distance of 254 km. The fungus may have been

Distribution patterns and possible infection routes of E. dermatitidis 1063

transported by birds and found a suitable place to proliferate

in the petroleum oil-polluted environment. From these data

wemay conclude that natural populationsmay have had a rel-

atively wide geographic distribution within suitable climate

zones. In Thailand, all isolates from feces of frugivorous ani-

mals were ITS genotype B. In general, AFLP populations had

a single ITS genotype, either A or B. Only in cluster 7 subtypes

were associated with different ITS types (Fig 1B) suggesting

different genetic backgrounds of DNA fragments with the

same mobility.

Global distribution of populations also occurs, in addition

to focal association with frugivorous tropical animals. This

is particularly obvious in population 3d, all isolates being ITS

genotype B and showing worldwide distribution in steam

baths in The Netherlands, Austria, and Thailand, while the

same population was also found to be involved in a cutaneous

lesion in the U.S.A. Amechanism other than animal-vectoring

must be at work, enabling long-distance dispersal. The fungus

is known to occur in the human intestinal tract in 5.2 % of

mostly non-impaired individuals and was suggested to main-

tain as a colonizer over prolonged periods, as the fungus was

repeatedly recovered from an individual during several epi-

sodes of diarrhea (de Hoog et al. 2005). Humans may thus pro-

vide a mechanism of worldwide dispersal. After ingestion of

contaminated fruits humans may carry the fungus asymp-

tomatically and spread it into urban environments, where it

proliferates in habitats that share essential ecological factors

with the natural habitat of the fungus. This environmental

shift matches the concept of ecological fitting (Agosta &

Klemens 2008).

Population 3b showed two features which were recurrent

in our data set: (1) rapid colonization of suitable habitats,

and (2) occurrence of several genotypes at a single, apparently

suitable habitat. As the species is very rare in randomly sam-

pled environments, both features strongly indicate that a suit-

able habitat is concerned. Cluster 3b comprised a large

number of identical strains, originating from separate sam-

ples taken within a single steam room in Chiangrai, Thailand.

Thus, populations were able to colonize efficiently once they

had reached a suitable environment. Similarly, population

5a from polluted railway ties at Prachinburi, Thailand was lo-

cally present with many isolates. Here again we witness the

very rapid and effective colonization of a suitable environ-

ment. Further sampling of most railway sites revealed multi-

ple populations of the species. Occurrence of divergent

populations at a single sampling site was also seen at the rail-

way ties in Nakornsawan, Thailand. In the (semi)natural envi-

ronment this was the case in the small, focal sampling sites of

frugivorous bird and flying fox feces in Chonburi and Cha-

choengsao, Thailand (Sudhadham et al. 2008; Table 1). We

therefore conclude that frugivorous animal feces is (part of)

the natural habitat of E. dermatitidis. The same may hold true

for pineapple and mango samples in Thailand, where on

two occasions two populations were encountered at the

same sampling site (Table 1), indicating that fruit surfaces rep-

resent at least a part of the natural niche of the organism.

Population 4b is remarkable in suggesting a truly world-

wide distribution, representing cerebral systemic and dis-

seminated isolates from the U.S.A. and Japan, strains from

human feces in Brazil, Slovenia and France, and European

strains from CF lungs. A similar distribution pattern of iso-

lates is observed in population 7a, containing strains from

human brain, feces, and sputum from different continents.

Pulmonary strains of E. dermatitidis usually concerned pa-

tients suffering from cystic fibrosis. Since CF patients often

have an increased immune response due to their chronic

lung colonization with microorganisms, dissemination from

a pulmonary source seems less likely. To our knowledge, de-

spite the regular colonization of CF lungs with E. dermatitidis

(Haase et al. 1991; Horre et al. 2003), never any cerebral case

in a CF patient was reported. Instead, Hiruma et al. (1993)

suggested possible translocation from the intestines, e.g.

resulting from an intestinal wound. The finding of regular

asymptomatic colonization and the occurrence of intestinal

and cerebral strains with the same population underline

this hypothesis. Contamination of the blood circulation by

E. dermatitidis may rapidly lead to cerebral infection (Li et al.

2010). Similar phenomena are also known to occur in Candida

albicans, which may be found in the brain after enterocolitis

(Cimbaluk et al. 2005).

Several populations, suchas5a and5d, originated fromrail-

way ties in Thailand. These ties were made of different mate-

rials: in Nakornsawan and Pitsanulok the ties consisted of

concrete, while in Prachinburi and Srakhaew they were made

of creosote-impregnated oak wood. Both types of railway ties

are apparent niches for E. dermatitidis, despite the fact that

they differ extremely as environments for growth. The sup-

portingmaterial thusseemstoplayonlya limitedrole.Thespe-

cies was never encountered in the numerous isolations from

rock in hot climates (e.g. Wollenzien et al. 1995; Sterflinger

et al. 1999; Ruibal 2004). Characteristic for railway ties is pollu-

tionwith petroleum oil and human feces, which probably pro-

mote growth of E. dermatitidis. The fungus was found between

ties, where fecal contamination was combined with machine

oil, as well as outside the rails, where pollution predominantly

consisted of oily debris (Sudhadham et al. 2008). Petroleum oil-

likecomponents thusseemtoprovide the fungusacompetitive

advantage over other microorganisms. An ecological role of

alkylbenzenes in black yeasts has been discussed by

Prenafeta-Boldu et al. (2006) and Vicente et al. (2008).

All strains of systemic infections, irrespective of popula-

tion attribution, were ITS genotype A (Li et al. 2010). Also in

fecal strains genotype A is preponderant (de Hoog et al.

2005). Population 7a discussed above also comprised isolate

dH 13227 originating from flying fox feces in Thailand. In the

(semi)natural habitat of tropical frugivorous animals, ITS ge-

notypes A and Bwere found next to each other, at comparable

frequencies (Sudhadham et al. 2008). But genotype A is much

more often encountered as an etiologic agent of systemic

and disseminated infections and as colonizer of the human

intestinal tract (de Hoog et al. 2005). Strains of ITS genotype

B were mostly environmental and were rarely involved in

deep infection (Table 1). Thus it seems that the cluster of pop-

ulations with ITS genotype A on average displays a higher de-

gree of virulence to humans. Strains belonging to genotype A

seem to have shifted to the human-dominated environmental

more successfully than strains of the B cluster.

Successfully colonized environments (steam baths, pol-

luted railway ties, animal feces) comprised different popula-

tions, but this was mostly not a truly random selection.

1064 M. Sudhadham et al.

Regional structuring can be observed in part of the popula-

tions (Fig 4). This makes preponderance of rapid, random (air-

borne) dispersal of the fungus less likely. Rather than air- or

waterborne transport, suitable vectors in outdoor environ-

ments are likely to be frugivorous animals, while in case of in-

door contamination asymptomatic intestinal transport by

humans may be an option, which is in line with our hypothe-

ses explained above. Nearly all etiologic agents of systemic

disease in immunocompetent humans belonged to ITS geno-

type A, whereas those with ITS genotype B, even when having

a comparably wide distribution, lack such invasive behavior.

The two genotypes are known to show limited recombination

(Sudhadham et al. submitted for publication). Virulence prob-

ably differs at the population level, the A-cluster containing

more virulent populations. In the natural habitat, both A and

B had comparable frequencies, but those in the A-cluster

seem to be promoted in the human-made environment, lead-

ing to a higher frequency of virulent A populations. On aver-

age, the virulence of the species E. dermatitidis is thus likely

to go up, leading to emergence of a truly opportunistic fungus.

r e f e r e n c e s

Agosta SJ, Klemens JA, 2008. Ecological fitting by phenotypicallyflexible genotypes: implications for species associations,community assembly and evolution. Ecology Letters 11:1123e1134.

Andrews JH, Harris RF, Speaer RN, Lau GW, Nordheim EV, 1994.Morphogenesis and adhesion of Aureobasidium pullulans.Canadian Journal of Microbiology 40: 6e17.

Barreto de Oliveira MT, Boekhout T, Theelen B, Hagen F,Baroni FA, Lazera MS, Lengeler KB, Heitman J, Rivera ING,Paula CR, 2004. Cryptococcus neoformans shows a remarkablegenotypic diversity in Brazil. Journal of Clinical Microbiology 42:1356e1359.

Boekhout T, Theelen B, Diaz M, Fell JW, Hop WCJ, Abeln ECA,Dromer F, Meyer W, 2001. Hybrid genotypes in the pathogenicyeast Cryptococcus neoformans. Microbiology 147: 891e907.

Cimbaluk D, Scudiere J, Butsch J, Jakate S, 2005. Invasive candidalenterocolitis followed shortly by fatal cerebral hemorrhage inimmunocompromised patients. Journal of Clinical Gastroenter-ology 39: 795e797.

Gueidan C, Ruibal Villase�nor C, de Hoog GS, Gorbushina A,Untereiner WA, Lutzoni F, 2008. An extremotolerant rock-in-habiting ancestor for mutualistic and pathogen-rich fungallineages. Studies in Mycology 61: 111e119.

de Hoog GS, Guarro J, Gene J, Figueras MJ, 2000. Atlas of ClinicalFungi, second edn. Centraalbureau voor Schimmelcultures/Universitat Rovira i Virgili, Utrecht/Reus.

de Hoog GS, Haase G, 1993. Nutritional physiology and selective iso-lation of Exophiala dermatitidis.Antonie van Leeuwenhoek 64: 17e26.

de Hoog GS, Matos T, Sudhadham M, Luijsterburg KF, Haase G,2005. Intestinal prevalence of the neurotropic black yeastExophiala dermatitidis in healthy and impaired individuals.Mycoses 48: 142e145.

Haase G, Skopnik H, Groten T, Kusenbach G, Posselt H-G, 1991.Long-term fungal cultures from patients with cystic fibrosis.Mycoses 34: 373e376.

Hiruma M, Kawada A, Ohata H, Ohnishi Y, Takahashi H,Yamazaki M, Ishibashi A, Hatsuse K, Kakihara M, Yoshida M,1993. Systemic phaeohyphomycosis caused by Exophialadermatitidis. Mycoses 36: 1e7.

HorreR,SchrTtelerA,MarkleinG,BreuerG,SiekmeierR,SterzikB,deHoog GS, Schnitzler N, Schaal KP, 2003. Vorkommen von Exo-phiala dermatitidis bei Patientenmit zystischer Fibrose in Bonn.Atemwegs-und Lungenkrankheiten 29: 373e379.

Kothera RT, Keinath AP, Dean RA, Farnham MW, 2003. AFLPanalysis of a worldwide collection of Didymella bryoniae.Mycological Research 107: 297e304.

Li DM, Li RY, de Hoog GS, Sudhadham M, Wang DL. 2010. FatalExophiala infections in China: a syndrome endemic to eastAsia. Mycoses (PMID 20337943).

Matos T, de Hoog GS, de Boer AG, de Crom I, Haase G, 2002. Highprevalence of the neurotrope Exophiala dermatitidis and relatedoligotrophic black yeasts in sauna facilities. Mycoses 45:373e377.

McDonald-Madden E, Schreiber ESG, Forsyth DM, Chooquenot D,Clancy TF, 2005. Factors affecting Grey-headed flying-fox(Pteropus poliocephalus: Pteropodidae) foraging in the Melbournemetropolitan area, Australia. Australian Ecology 30: 600e608.

Muotoe-Okafor FA, Gugnani HC, 1993. Isolation of Lecythophoramutabilis and Wangiella dermatitidis from the fruit eating bat,Eidolon helvum. Mycopathologia 122: 95e100.

Padhye AA, McGinnis MR, Ajello L, 1978. Thermotolerance ofWangiella dermatitidis. Journal of Clinical Microbiology 8: 424e426.

Palmer C, Woinarski JCZ, 1999. Seasonal roosts and foragingmovements of the black flying fox (Pteropus alecto) in theNorthern Territory: resource tracking in a landscape mosaic.Wildlife Research 26: 823e838.

Prenafeta-Boldu FX, Summerbell RC, de Hoog GS, 2006. Fungigrowing on aromatic hydrocarbons: biotechnology’s unex-pected encounter with biohazard. FEMS Microbiological Reviews30: 109e130.

Reis NR, Mok WY, 1979. Wangiella dermatitidis isolated from batsin Manaus, Brazil. Sabouraudia 17: 213e218.

Ruibal C. 2004. Isolation and characterization of melanized, slow-growing fungi from semiarid rock surfaces of central Spain andMallorca. Dissertation, Universidad Autonoma de Madrid.

Satow MM, Attili-Angelis D, de Hoog GS, Angelis DF, Vicente VA,2008. Selective factors involved in oil flotation isolation ofblack yeasts from the environment. Studies in Mycology 61:157e163.

Schmidt-Lebuhn AN, Kessler M, Mhller J, 2005. Evolution of Su-essenguthia (Acanthaceae) inferred from morphology AFLP data,and ITS rDNA sequences. Organisms, Diversity & Evolution 5:1e13.

Sterflinger K, de Hoog GS, Haase G, 1999. Phylogeny and ecologyof meristematic ascomycetes. Studies in Mycology 43: 5e22.

Sudhadham M, Haase G, Menken SBJ, van Belkum A, Gerrits vanden Ende AHG, Sihanonth P, de Hoog GS. Molecular diversityof the black yeast Exophiala dermatitidis, a neurotropic oppor-tunist in humans, submitted for publication.

Sudhadham M, de Hoog GS, Menken SBJ, Gerrits van denEnde AHG, Sihanonth S, 2009. Rapid screening for genotypesas possible markers of virulence in the neurotropic black yeastExophiala dermatitidis using PCR-RFLP. Journal of MicrobiologicalMethods 80: 138e142.

Sudhadham M, Sihanonth P, Sivichai S, Chaiyarat R,Dorrestein GM, Menken SBJ, de Hoog GS, 2008. The neuro-tropic black yeast Exophiala dermatitidis has a possible origin inthe tropical rain forest. Studies in Mycology 61: 145e155.

de Valk HA, Meis JFGM, De Pauw BE, Donnelly PJ, Klaassen CHW,2007. Comparison of two highly discriminatory molecularfingerprinting assays for analysis of multiple Aspergillus fumi-gatus isolates from patients with invasive aspergillosis. Journalof Clinical Microbiology 45: 1415e1419.

Vicente VA, Attili-Angelis D, Pie MR, Queiros-Telles F, Cruz LM,Najafzadeh MJ, de Hoog GS, Zhao JS, Pizzirani-Kleiner A, 2008.Environmental isolation of black yeast-like fungi involved inhuman infection. Studies in Mycology 61: 137e144.

Distribution patterns and possible infection routes of E. dermatitidis 1065

Vos P, Hogers R, Bleeker M, Reijans M, Lee Tvan de, Hornes M,Frijters J, Pot J, Peleman J, Kuiper M, Zabeau M, 1995. AFLP:a new technique for DNA fingerprinting. Nucleic Acids Research23: 4407e4414.

Warris A, Klaassen CHW, Meis JFGM, De Ruiter MT, De Valk HA,Abrahamsen TG, Gaustad P, Verweij PE, 2003. Molecular epi-demiology of Aspergillus fumigatus isolates recovered fromwater, air, and patients shows two clusters of geneticallydistinct strains. Journal of Clinical Microbiology 41: 4101e4106.

Wollenzien U, de Hoog GS, Krumbein WE, Urzı C, 1995. On theisolation of microcolonial fungi occurring on and in marbleand other calcareous rocks. Science of the Total Environment 167:287e294.

Yurlova NA, de Hoog GS, 2002. Exopolysaccharides and capsulesin human pathogenic Exophiala species. Mycoses 45: 443e448.

Zalar P, Gostincar C, de Hoog GS, Ur�si�c V, Sudhadham M, Gunde-Cimerman N, 2008. Redefinition of Aureobasidium pullulans andits varieties. Studies in Mycology 61: 21e38.