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1 Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands
2 Institute for Biodiversity and Ecosystem Dynamics, University of
Amsterdam, Amsterdam, The Netherlands
3 Department of Microbiology, Chulalongkorn University, Bangkok,
Thailand
4 Biotec-Mycology Laboratory, National Center for Genetic Engineering and
Biotechnology (BIOTEC), Pathumthani, Thailand.
5 Department of Biology, Faculty of Science, Mahidol University, Bangkok,
Thailand
6 Dutch Research Institute for Avian and Exotic Animals (NOIVBD), Veldhoven,
The Netherlands
*
Correspondence: G.S. de Hoog,
de.hoog{at}cbs.knaw.nl
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Abstract |
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 TOP Abstract INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONReferences |
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Keywords Black yeasts / Exophiala dermatitidis / frugivorous animals / human faeces / intestinal colonization / neurotropism
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONReferences |
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The route of infection is still a mystery. The species is known to occur in the environment, but is not among the commonly encountered saprobes. It is practically absent from dead plant material or soil, and has never been reported from outdoor air (Matos et al. 2002). The somewhat odd spectrum of main sources of isolation of strains presently available in culture collections (fruit surfaces, steam baths, faeces, and human tissue) suggests that a hitherto unknown, quite specific natural niche must be concerned.
Particularly the occurrence in steam rooms of public bathing facilities is consistent and with high colony counts (Nishimura et al. 1987, Matos et al. 2002). The artificial environment of the steam bath apparently provides a novel environmental opportunity for this fungus. The transition from the hitherto unknown natural niche to the human-dominated environment may be accompanied by selection and/or adaptation to the new habitat, facilitated by the stress protection provided by melanin. Given the nature of the fungus as an opportunistic agent of potentially fatal infections in humans, this process may have clinically relevant consequences. The present article documents a possible natural habitat of the fungus and on processes taking place during transition from nature to the domestic environment.
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MATERIAL AND METHODS |
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TOPAbstractINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONReferences |
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1 g) from small zoo animals from the
Netherlands and autopsied at the Veterinary Faculty of the University of
Utrecht, The Netherlands were included. Also large numbers of wild fruits and
berries from the Netherlands and Thailand were analyzed
(Table 3). Solid specimens were
incubated in 5.5 mL Raulin's solution
(Booth 1971) in test tubes for
2-3 d at 25 ° C in nearly horizontal position and shaken at 10 r.p.m.
Subsequently 0.5 mL was transferred using a glass Drigalski spatula to
Erythritol Chloramphenicol Agar (ECA) (de
Hoog & Haase 1993) and Sabouraud's Glucose Agar (SGA)
(de Hoog et al. 2000)
– both media containing the same concentration of chloramphenicol
– and incubated for up to 40 d at 40 °C. Small, blackish-brown
colonies were transferred to new growth media on PDA using a loop, or were
separated from contaminating yeasts and aspergilli by repeatedly washing agar
blocks with black yeast cells in 0.1 % Tween 80 in sterile water followed by
dispersing the visually clean blocks over fresh agar plates.
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Validation of isolation procedure
Three strains were used to test the efficiency of recovery by the above
method: CBS 207.35
(genotype A, capsular), CBS
116014 (genotype B, capsular) and
CBS 109143
(genotype B, non-capsular). Strains were pre-cultured in Potato Dextrose Broth
(PDB) for 3 d at 30 °C. Suspensions were adjusted to 102
cells/mL and recovery verified by plating on ECA and SGA at 25 °C and 40
°C. Aureobasidium pullulans,
CBS 584.75 was used
as positive control at 25 °C. Suspensions in Raulin's solution with a
final concentration of 106 cells/mL were incubated for 3 d under
conditions specified above and the recovery rate was counted on ECA, SGA and
Potato Dextrose Agar (PDA) at 25 °C and 40 °C.
Identification of accompanying biota
White yeasts were identified physiologically by testing fermentation and
carbon and nitrogen assimilation. C-assimilative capabilities were tested
using API-ID 32 C strips (bioMérieux, Marcy-l'Étoile, France).
Single colonies were grown at 25 °C for 3 d as a maximum. Suspensions were
made and the densities were scored by McFarland turbidity standard point 2.
Strips were inoculated according to specifications provided by the
manufacturer and incubated at 25 °C. For nitrogen assimilation the
substrates nitrate, ethylamine, L-lysine, cadaverine,
D-glucosamine, HCl and tryptophane were used. Peptone was used as
positive control. Suspensions were applied in N-auxanograms in culture plates.
After the medium was cooled and solidified, a small amount of each N-source
was put on the medium. For fermentation, D-glucose,
D-galactose, maltose, sucrose, lactose and raffinose were tested.
Sugars solutions were 2 %, except for raffinose which 4 % solution was used.
These solutions were sterilized in tubes with Durham inserts and suspensions
of McFarland turbidity standard point 2 were added. The identification
software BIOLOMICS was used to score combined physiological
results. Morphology was investigated to confirm the identifications
(Kurtzman & Fell
1998).
Diagnostics
Strains were recognised as black yeasts were provisionally identified at
the species level by colony appearance, morphology, and temperature tolerance.
Species-identification and ITS-genotype attribution
(Matos et al. 2002)
was done on the basis of ITS rDNA, either by restriction length polymorphism
(Sudhadham et al.
2009a) or by sequencing ITS1 and 2
(Sudhadham et al.
2009b) using standard methodology
(de Hoog & Gerrits van den Ende
1998). The ITS-genotypes were recognized on the polymorphic sites
listed in Table 2. A small
number of isolates was genotype-attributed by similarity of patterns of
amplified fragment length polymorphism (AFLP) to strains of which to
ITS-genotype was known.
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RESULTS |
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TOPAbstractINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONReferences |
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The isolation protocol applied to animal faeces and to fruits and berries collected in temperate and tropical climates proved to be highly selective, judging from the very few ubiquitous saprobes that were recovered. The rarity of E. dermatitidis was proven by selective isolation from a large diversity of environments in temperate (The Netherlands) and tropical (Thailand) climates, supplementing data of Matos et al. (2002) which involved leaves, fruits and berries, animal faeces and soil in a temperate climate.
Genotyping
Nuclear rDNA ITS sequencing of 86 reference strains confirmed the existence
of two major genotypes differing in three positions in ITS1
(Table 2). Genotype A was more
common than B (A:B = 57:29). Two out of nine animal faeces samples from
Thailand belonged to genotype A, while seven were B. Two sets of samples from
bird guano in Khao Khaew Zoo (where occasional mechanic cleaning is carried
out) and flying fox faeces at a temple complex in Thailand contained genotypes
A and B, of which the latter was isolated more frequently.
Isolation in temperate climate
Fruits and berries
Two areas in The Netherlands were chosen for isolation from berries, namely
Boswachterij Noordwijk, in a dune area near the Northsea coast near Leiden,
and a lane planted with shrubs in a rural area in Maartensdijk in the central
part of the country. In Noordwijk, samples were taken in Autumn, when berries
were predomantly eaten by migratory frugivorous birds such as Turdus
pilaris (fieldfare). Twenty-one samples were taken from berries of
Rosa pimpinellifolia, 345 samples from Hippophae rhamnoides
and 187 samples from Ligustrum vulgare
(Table 3). The shrubs near
Maartensdijk were predominantly frequented by sedentary birds such as
Corvus monedula (jackdaw) and Sturnus vulgaris (European
starling). Thirty samples were taken from Crataegus monogyna, 92
samples from Viburnum opulus, 61 samples from Ilex
aquifolium, 22 samples from Rosa canina, 20 samples Rosa
rubiginosa, 36 samples Prunus spinosa, 19 samples Ligustrum
vulgare and 51 samples from Taxus baccata. With our isolation
protocol plates mostly remained blank, or white yeasts were encountered. No
black yeast was isolated.
Faeces of omnivorous birds
Faeces mixed with soil under Thuja conifers harbouring a large
combined roosting site of Corvus monedula (jackdaw) and Sturnus
vulgaris (European starling) in a park near Hilversum, The Netherlands,
was sampled, as well as a roosting site of jackdaw alone. All samples were
negative for E. dermatitidis
(Table 4).
Intestinal contents from sectioned zoo animals
A total of 731 samplings from dead animals originating from different zoos
in the Netherlands, were received at the Veterinary Faculty at Utrecht for
autopsy. In addition to routine analysis, the contents of the intestines with
visible disorders such as discoloration or halfway digested food was subjected
to selective isolation for E. dermatitidis. Results are available
from 16 reptiles, 406 birds, 183 mammals, 8 fishes, 3 turtles, 2 amphibians, 5
lizards and 11 snakes; these data will not be included in this article, but
are available as attachment at
www.cbs.knaw.nl.
Culture plates mostly remained blank. White yeasts were common in the
intestinal tract of frugivorous animals. A single strain of E.
dermatitidis (genotype A) was obtained from a bonobo monkey (Pan
paniscus) with diarrhoea in the Apeldoorn Zoo, The Netherlands. Bonobo's
are omnivorous with a marked preference of fruit.
Isolation in tropical climate
Fruits and berries
On grapes (Vitis vinifera) numerous white yeasts were isolated,
which were not identified down to the species level. The only black fungus
obtained was a Cladosporium species. On green papaya fruits
(Carica papaya) mainly white yeasts occurred. Few filamentous fungi
were obtained, mainly biverticillate Penicillium species, among which
was Eupenicillium cinnamopurpureum (anamorph: P.
phaeniceum). Three types of fruit were found positive on isolation for
E. dermatitidis: papaya, pineapple (Ananas comosus) and
mango (Mangifera indica). In papaya and pineapple, genotypes A and B
were found, while in mango only genotype B was encountered. Mango fruits
further contained white yeasts, a Rhizopus species, a recurrent
Aspergillus species, a biverticillate Penicillium and
Talaromyces intermedius (dH 13728). On lemon fruits (Citrus
sp.) white yeasts were common, and a single colony of a white filamentous
fungus was obtained. On fruits of tamarind (Tamarindus indica),
Queen's flower (Lagerstroemia calyculata) and Ficus lacor,
only Aspergillus species were obtained. In contrast, fruits of
Ficus annulata contained much more white yeasts in addition to
Aspergillus species; three times a hitherto undescribed species of
Munkovalsaria species was isolated. On fruits of yellow santol
(Sandoronicum indicum) the main filamentous fungi acquired were
Aspergillus species, and infrequently white yeasts were encountered.
Fruits of rambutan (Nephelium lappaceum), pomelo (Citrus
maxima) and mangrove palm (Nypa fruticans) contained many
white yeasts only. Rose apple fruits (Syzygium jambos) contained
biverticillate Penicillium species
(Table 3).
Faeces of frugivorous birds
Samples were taken from faeces of a number of frugivorous birds from two
zoos in Thailand. Bird species are described as follows. Columbiformes:
Ducula bicolor (pied imperial-pigeon); Coraciiformes: Aceros
undulatus (wreathed hornbill), Anorrhinus galeritus
(bushy-crested hornbill), Anorrhinus tickelli (rusty-cheeked
hornbill), Anthracoceros albirostris (oriental pied-hornbill),
Anthracoceros malayanus (black hornbill), Berenicornis comatus
(white-crowned hornbill), Buceros bicornis (great hornbill),
Buceros hydrocorax (rufous hornbill), Buceros rhinoceros
(rhinoceros hornbill), Rhinoplax vigil (helmeted hornbill);
Passeriformes: Acridotheres tristis (common myna), Sturnus
burmannicus (vinous-breasted starling), Pycnonotus finlaysoni
(stripe-throated bulbul); and Piciformes: Megalaima virens (great
barbet). White yeasts were predominant on isolation media. Some of these were
selected to be identified by ID32C and they invariably turned out to be
Candida tropicalis, while a single strain was identified as
Trichosporon loubieri. Exophiala dermatitidis (genotype B) was
isolated from fresh faeces of Acridotheres tristis (common myna). In
a feeding area for birds by fruits of papaya (Carica papaya), banana
(kluai namwa; Musa `ABB'), two isolates of Exophiala
dermatitids, genotype B were found (dH 13132, dH 13134) and genotype A
(dH 13135) (Fig. 2 D,E).
Frugivorous bird faeces were also analyzed in the national park HalaBala
Wildlife Sanctuary, established in 1996. Samples were taken in the Hala
portion of the Sanctuary, approximately 22 km west from the Malaysian border,
comprising a mix of forest broadly classified as tropical lowland evergreen
forest, and small-scale agricultural land. The area is particularly famous for
harbouring nine species of hornbill birds being one of richest areas for these
birds in Southeast Asia (Fig H). Exophiala dermatitidis (dH 13148
genotype B) was found from the faeces of Buceros rhinoceros
(rhinoceros hornbill) (Table
4).
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Faeces of granivorous birds
Faeces samples were collected from two zoos in Thailand and involved the
bird species Galliformes: Pavo cristatus (Indian peafowl);
Psittaciformes: Cacatua ducorpsii (Ducorps' cockatoo), Cacatua
goffini (Goffin's cockatoo), Cacatua moluccensis (Moluccan
cockatoo), Cacatua sulphurea (yellow-crested cockatoo), Cacatua
tenuirostris (long-billed corella), Calyptorhynchus magnificus
(red-tailed black cockatoo), Electus auratus (eclectus parrot),
Probosciger aterrimus (palm cockatoo), Psittacula alexandri
(red-breasted parakeet), Psittacula eupatria (Alexandrine
parakeet), Psittacus erithacus (African grey parrot), and
Psittrichas fulgidus (Pesquet's parrot). Very few white yeasts were
encountered, but filamentous fungi were relatively common. No
Exophiala was isolated (Table
4).
Faeces of insectivorous bats
Thirteen mostly limestone caves were chosen for sampling of insectivorous
bat faeces from different geographical regions in Thailand. Most of them were
located in montane rain forest, about 1 000–1 900 meters above sea
level. Others were touristic places surrounded by agricultural land, or were
part of a temple complex. Numerous filamentous fungi were obtained
(Table 4), most of these being
rapidly growing Aspergillus species, biverticillate penicillia and
zygomycetes. No black yeasts were detected
(Table 4)
(Fig. 2 F, G).
Public toilets
Public toilets of gas stations can be found along many highways in
Thailand. They differ only slightly with the company with respect to building
structure, hygienic level and intensity of warding. Most of them are pedestal
squat toilets, which means that there is no flushing system installed to clean
the toilet after use. Instead, water is collected from a tap into a bucket
placed close to the squat toilet. Due to this situation, most of the time
floors in the toilets were wet, and the hygienic level was low. Samples were
taken at different points, such as the floor in the toilet room, the wall, as
well as swabs taken from the bowl above the water level. The occurrence of
fungi which grew after incubation of swabs in Raulin's solution and incubation
on ECA at 40 °C was similar in all toilets: we found Aspergillus
species and some white yeasts, but no Exophiala
(Table 5)
(Fig. 2 J, K).
Sauna facilities
The saunas in Thailand that were chosen for this experiment were dry sauna
with continuous heating. The room was located inside the building complex with
other steam baths and Gyms for sport entertainment. Inside the sauna room,
seats and walls were made of wood. The steam rooms were also included in this
experiment; these were mostly located on the same floor as the sauna room. The
walls and floors were made of tiles while the seats were made from polyvinyl
chloride. Black yeasts appeared abundantly in the isolation step. Only
Exophiala dermatitidis could be found in these samples, no other
fungi were encountered. Sequencing results showed that E.
dermatitidis could be assigned to both genotypes A and B
(Table 6).
Railway ties
Six creosote-treated oak railways ties in Thailand were chosen for this
experiment. One of these was located at a fresh market, where heavy
contamination and regular cleaning lead to nutritional enrichment, and yielded
negative results. The remaining ties showed a line of blackish debris,
probably a mixture of faeces and machine oil. These samples contained an
enormous amount of Exophiala dermatitidis presenting both genotypes
from all locations. Srakhaew railway ties yielded 31 isolates of Exophiala
dermatitidis genotype A, Prachinburi railway ties had 108 genotype A and
5 genotype B, Nakornsawan railways had 176 genotype A, 29 genotype B and 2
genotype C, Pitsanulok railway yielded 61 genotypes A and 1 genotype C
(Table 7)
(Fig. 2 L).
Hot springs
In ten litre-samples of water from eleven hot springs, a few biverticillate
Penicillium species were found: dH 13743 = P. mineoluteum,
dH 13790 = P. pinophilum and dH 13744 = P. funiculosum. No
white yeasts were detected, but one sample yielded a colony of Exophiala
dermatitidis, genotype A. In the soil samples from the same hot springs,
after incubation in Raulin's solution, analyzed with a dilution series and
plated on ECA, few filamentous fungi were observed, but there was no evidence
of black yeasts (Table 8;
Fig. 2 I).
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DISCUSSION |
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TOPAbstractINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONReferences |
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During the present research we selected environments which are likely to be positive. Samples taken on the basis of strains already available in the reference collection of CBS, and on the basis of physiology. The species had previously recurrently been isolated from human sputum, particularly from CF patients, from deep infections, particularly from brain, from stool, from bathing facilities, from fruits and berries, and from creosote-treated wood (http://www.cbs.knaw.nl). This remarkably discontinuous spectrum of sources of isolation of E. dermatitidis suggests that the organism might be adapted to a particular, hitherto undiscovered habitat, rather than being a saprobe on dead plant material, as is frequently suggested in the literature (Gold et al. 1994). Based on its physiology, we hypothesized a niche containing a number of key elements. First, it must be dynamic, with simultaneously or consecutively occurring phases differing in environmental conditions, since the organism itself is polymorphic, exhibiting yeast-like, filamentous and meristematic phases (de Hoog et al. 1994). Second, the phases are likely to be nutritionally diverse, as is concluded from the fungus' consistent occurrence as an epiphyte on low-nitrogenous substrates such as fruit surfaces – promoted by its moderate osmotolerance – and bath tiles (Mayr 1999) combined with equally consistent occurrence in faeces (de Hoog et al. 2005). Third, the latter environment, combined with a consistent tolerance of E. dermatitidis of 40 °C (Padhye et al. 1978) and of very low pH values (de Hoog et al. 1994) led to the supposition of passage of the digestive tract of warm-blooded animals (G.S. de Hoog unpublished data). Fourth, the organism shows strong adhesion to artificial surfaces (Mayr 1999), probably promoted by production of sticky extracellular polysaccharides (Yurlova et al. 2002). An occurrence on wild fruits and berries that are subsequently ingested by frugivorous animals and dispersed via their faeces thus seems to provide a possible connection of the divergent sources of isolation. The hypothesis led to the successful development of a selective protocol used in this study, which involved an acidic enrichment step (Booth 1971) followed by a high temperature step on a nutritionally specific medium (de Hoog & Haase 1993). The protocol enabled the isolation of minute quantities of the fungus. Massive isolation studies further underlined that E.dermatitidis is a rare species in most outdoor environments. We believe our recovery data broadly reflect the actual presence of E. dermatitidis in the environment, for two reasons. (1) Environments known to harbor the species were indeed found to be positive at rates comparable to those published earlier (Matos et al. 2002). (2) Recently Zhao et al. (2008) applied a new, Chaetothyriales-specific isolation method based on toluene-enrichment at ambient temperature to the same shrubs near Maartensdijk and indeed found numerous mesophilic Exophiala species, but never the thermophilic species E. dermatitidis.
The recovery rate was tested experimentally and on average found to reflect the number of cells present prior to incubation in acid. However, slight differences were noted among the three strains analyzed. The representative of ITS-genotype A (CBS 207.35) was stimulated with a factor 4.3 by incubation in Raulin's solution, whereas genotype B (CBS 1160124) remained practically unaltered. The non-capsular strain (CBS 109143) was inhibited tenfold, but since such strains are extremely rare in the natural environment (Matos et al. 2002, Yurlova et al. 2002) we believe that this more vulnerable phenotype has little effect on the recovery rate of the species.
We analyzed a large diversity of substrates using a highly selective protocol, with accent on substrates bearing similarity to origins of reference strains (Table 2). Despite that, isolation was mostly unsuccessful outdoors in the temperate climate of The Netherlands (Table 3). Only a single strain from a berry of Sorbus aucuparia had been found (CBS 109142, genotype B) by Matos et al. (2002). The negative samples included berries commonly eaten by migratory and sedentary birds such as Turdus pilaris, Corvus monedula and Sturnus vulgaris, as well as faeces from several of these birds. From these extended environmental studies including 944 samples it may be concluded that E. dermatitidis does not occur naturally in temperate climates.
In contrast, the fungus was confirmed to reside consistently and abundantly from known foci in several artificial, indoor environments, such as steam rooms (Matos et al. 2002), in Slovenia, Austria, Thailand as well as in The Netherlands. Several of the negative berry-sampling locations were only a few kilometers away from steam baths that proved to be highly positive. The species was recovered at high frequency (about 1 000 CFU.cm-2) from thirteen bathing facilities. Steam baths (and not the adjacent sauna's) of public bathing facilities represent an artificial environment with conditions thought to be similar to parts of the natural habitat of E. dermatitidis, where high (body) temperature and epiphytie adhesion to (fruit) surfaces play a significant role.
In tropical Thailand, most fruits and berries were also negative (Table 3), although the species was encountered a few times on mango and pineapple. Positive samples were more regularly derived at low frequencies from animal faeces (Table 4). The consistent presence of E. dermatitidis in bat faeces and bird guano analyzed is demonstrated by the sample from guano-littered soil in the Khao Khaew Zoo in Chonburi, Thailand, and from flying fox faeces at the temple complex in Chachongsao, Thailand, where both genotypes A and B were recovered (Table 4), despite the overall environmental scarcity of E. dermatitidis (Fig. 2 A–C). Recovery rates were low, with a maximum of three colonies per culture plate. The isolation method used reflects the real frequency of genotype B in the original samples (Table 2), which means that positive samples contain maximally 3 CFU per gram faeces. Positive samples were obtained from birds as well as mammals such as flying foxes.
Sampling of autopsied zoo animals was almost always negative for black yeasts. The great majority of these animals fed on corn and seeds, and also yielded very few white yeasts or filamentous fungi. The single black yeast-positive animal intestinal sample (www.cbs.knaw.nl) was a bonobo monkey, which is a largely frugivorous animal. A common factor linking this sample with positive samples elsewhere in the study is the diet of the animals: E. dermatitidis was almost exclusively found in animals that fed partially or entirely on wild fruits and berries. Herbivores have a large caecum, where digestion of food is enhanced by fermentation aided by a resident bacterial flora and white yeasts. Frugivorous animals, as those that feed on honey and nectar, may have problematic yeast overgrowth due to a high sugar content in the intestinal tract. Exophiala dermatitidis has a slight preference for osmotic environments. In clinical practice this was noted with its occurrence in the lungs of patients with cystic fibrosis, a disease characterized by an elevated salt content of tissues (de Hoog & Haase 1993).
De Hoog et al. (2005) found that E. dermatitidis occurs at a low incidence in the intestinal tract of humans. This matches with the abundant presence of E. dermatitidis on railway ties in Thailand, which are heavily contaminated by faeces (Fig. 2 L). Similar samples were taken in The Netherlands (data not shown), and these were positive for black yeasts other than E. dermatitidis. This situation is comparable with our isolation data from berries, which were negative in temperate but positive in tropical climates. Apparently the environmental temperature plays a significant role in the life cycle of E. dermatitidis. Public toilets in Thailand (Fig. 2 J, K) were, somewhat against expectations, also negative. This may be explained by competition of other, rapidly growing saprobes in this environment, such as white yeasts and Aspergillus species.
Exophiala dermatitidis, similar to other Exophiala species, is an oligotroph, as shown in vitro by Satow et al. (2008) on the basis of the ability of growth utilizing inoculum cells only. The property may be useful for growth on fruit surfaces, but is particularly expressed, in combination with thermotolerance, in abundant replication on the smooth surface of tiles and plastics of steam bath walls. The fungus was detected in large numbers in nearly all bathing facilities investigated located in temperate as well as in tropical climates. The species was also occasionally encountered in natural hot springs, which may be somewhat more difficult to colonize for the fungus due to their relative richness in nutrients. A single strain of E. dermatitidis was found in one of the hot springs but its presence might be explained by local people using the spring to clean and boil bamboo shoots after harvest from the forest. The sugary shoots may have been contaminated by E. dermatitidis and the fungus may have survived for a short period without significant colonization. A second sampling one year later without human activities was negative (Fig. 2 I).
Combining thermotolerance of the species with the knowledge that E. dermatitidis tolerates very acidic conditions (de Hoog et al. 1994), it is hypothesised that the fungus is able to pass through the intestinal tract of warm-blooded animals. About 80 % of positive hosts had diarrhoea at the moment of isolation of E. dermatitidis, a condition also encountered in positive bonobo. This suggests that the fungus may be present at a higher frequency but can only be isolated when the host has diarrhoea. De Hoog et al. (2005) reported isolation of the fungus at 3-wk-intervals from the faeces of a single hospitalized patient with diarrhoea, which indicates that maintenance in the intestines is possible. As a route of infection, translocation from the intestines may thus be supposed. For pulmonary cases an inhalative route would be more logical, but the apparent absence of E. dermatitidis from air remains in conflict with this explanation. Other similar phenomenon which support this hypothesis was the strain which was successful isolated from the bonobo from the zoo in the Netherlands. At that time, the sample was taken from the bonobo with the presence of diarrhoea (G. Dorrestein, personal information). Unfortunately, further sampling could not be continued due to the fact that the monkey had returned to the mother.
Reis & Mok (1979) and Muotoe-Okafor & Gugnani (1993) repeatedly found the species in internal organs of tropical frugivorous bats (Phyllostomus discolor, Sturnira lilium and Eudolon helvum) in American as well as African tropical rain forests. Representative isolates were verified to be E. dermatitidis genotype B (Table 4). Despite isolating the fungus from bat organs, Mok (1980) failed to isolate it from roosting sites. This may have been due to the use of inadequate isolation procedures. Reis & Mok (1979) also reported the species from two insectivorous bats (Myotis albescens and M. molossus), but the identity of these strains could not be verified.
Since in the reference set genotype A is about twice as common as genotype B and genotype A shows a higher recovery rate with the used isolation method (Table 2), our technique would expect to yield an A: B ratio of 10.4: 1 would be expected in the fruit-eating animal faeces from Thailand. However, these samples showed a ratio A: B = 2: 7, which would mean that the frequency of genotype B in tropical fruit-eating animal faeces samples deviates with a factor 36.4 from the average of all other sources of isolation.
This model study aims to prove the supposition that aspects of human behaviour i.e. the creation of environment that are extreme from a fungal perspective, by being hot, poor in nutrients, or poisonous, lead to the emergence of new, potentially virulent genotypes. The fungus under study causes a potentially fatal brain disease in otherwise healthy humans; this clinical picture is known in eastern Asia only (Horré & de Hoog 1999). Understanding the origin and course of this evolution may eventually lead to the development of measures which canalize speciation processes into a direction which is less harmful to humanity. The human community creates opportunities for adaptation and the emergence of pathogenic host races. Artificial, human-made environments may stimulate evolution and generate pathogenic genotypes which otherwise would not have evolved. The source of contamination of a potentially harmful microorganism and its routes on infection and transmission will be studied, potentially leading to protocols for hygiene and prevention. This may be particularly significant in bathing facilities connected to hospitals, where susceptible populations of patients with cystic fibrosis or, in Asia, with immunosuppression are warded. Though the disease under study is extremely rare, our approach can be viewed as a model study for understanding emergence of new microbial pathogens in general and their translocation from the tropical rain forest to the human environment.
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Acknowledgments |
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