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Black yeast habitat choices and species spectrum on high altitude creosote-treated railway ties
f u n g a l b i o l o g y 1 1 7 ( 2 0 1 3 ) 6 9 2 e6 9 6
journal homepage: www.elsevier.com/locate/funbio
Black yeast habitat choices and species spectrum on high altitude creosote-treated railway ties € GEN a, Macit ILKITb,*, G. Sybren DE HOOGc,d,e,f,g Aylin DO a
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Mersin, Mersin, Turkey Division of Mycology, Department of Microbiology, Faculty of Medicine, University of C¸ukurova, Adana 01330, Turkey c CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands d Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands e Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China f Sun Yat-sen Hospital, Sun Yat-sen University, Guangzhou, China g Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China b
article info
abstract
Article history:
Polyextremotolerant black yeast-like fungi thrive in moderately hostile environments
Received 5 January 2013
where they are concomitantly subjected to several types of stress, such as toxicity, scarce
Received in revised form
nutrient availability, and high or low temperature extremes. Their ability to assimilate al-
20 July 2013
kylbenzenes (toxic environmental pollutants) enhances their growth in harsh conditions,
Accepted 23 July 2013
including on railway ties. Samples were collected using cotton swabs, premoistened with
Available online 11 August 2013
physiological saline, from 658 oak and concrete railway ties at six train stations in Turkey
Corresponding Editor:
at altitudes ranging between 1026 and 1427 m. The samples were inoculated on malt ex-
Andrew N. Miller
tract agar supplemented with chloramphenicol, and incubated at 26 C for 4 weeks.
Keywords:
tested positive for fungi. Exophiala crusticola was found to be the most common species
Twenty-four samples (3.6 %), 17 from oak and 7 from concrete (5.6 % vs. 2 %; P ¼ 0.02), Chaetothyriales
(n ¼ 13), followed by Exophiala phaeomuriformis (n ¼ 7) and Exophiala heteromorpha (n ¼ 4).
Exophiala crusticola
These results suggest that hydrocarbons, particularly creosote-treated oak woods, support
Exophiala phaeomuriformis
the growth of black yeasts, some of which are opportunists in humans.
Extremophily
ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Filamentous fungi
Introduction Members of the black yeast genus Exophiala (order Chaetothyriales) have been isolated from superficial and subcutaneous infections, as well as fatal systemic infections, in immunocompetent humans (de Hoog et al. 2000). Different species of black yeast-like fungi have distinct traits and are associated with various disease categories (Zeng et al. 2007; Saunte et al. 2011). They are ubiquitous in wet environments that differ in temperature and are consequently able to harbor different
species (Lian & de Hoog 2010; Zalar et al. 2011). Differing pathogenic potentials have been described among clades of Chaetothyriales (de Hoog et al. 2011a, 2011b). Examples of varying degrees of pathogenicity include: (i) species that are able to grow at temperatures well over 36e37 C (e.g., bantiana, dermatitidis, and jeanselmei clades) and may cause systemic or disseminated infections in humans; (ii) species that are able to tolerate a maximum of approximately 36e37 C (e.g., carrionii and europaea clades) and cause superficial or subcutaneous infections; and (iii) species of the salmonis clade, which survive
* Corresponding author. Tel.: þ90 532 286 0099; fax: þ90 322 457 3072. E-mail address: [email protected] (M. Ilkit). 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.07.006
Black yeast-like fungi on railways
at a maximal temperature of 27e33 (36) C and cause at most superficial infections (Li et al. 2009; de Hoog et al. 2011a, 2011b; Saunte et al. 2011). Consistent with these findings, Saunte et al. (2011) recovered mostly Phialophora europea, Exophiala lecaniicorni, and Knufia epidermidis from human superficial black yeast infections. The thermophilic species Exophiala dermatitidis, one of the most common systemic pathogens, is thought to have a natural niche on fruit surfaces, with a life cycle involving passage through the intestinal tracts of fruit-eating animals, such as flying foxes and hornbills in the tropics (Sudhadham et al. 2008). This species has also been found in hot water from natural springs. Black fungi are able to tolerate low and high pH values (Zalar €g en et al. 2013b) and can live in environments with et al. 2011; Do high salt concentrations (Gunde-Cimerman et al. 2009). Several man-made environments appear to serve as an artificial equivalent to hot springs, and these species have commonly been recovered from Turkish steam baths (Matos et al. 2002; 2003), €g en et al. 2013b), and bathrooms dishwashers (Zalar et al. 2011; Do (Lian & de Hoog 2010). Black yeasts have also been isolated from bathrooms using toluene enrichment (Lian & de Hoog 2010). Additionally, black yeasts are prevalent in environments that are contaminated by aromatic hydrocarbons, such as creosotetreated railway ties (Sudhadham et al. 2008; Vicente et al. 2008; €g en et al. 2013a). Zhao et al. 2010; Seyedmousavi et al. 2011; Do For present study, we sampled creosote-treated oak and concrete railway ties from six stations and two different lines, at altitudes ranging from 1026 to 1427 m above sea level. These samples were taken in four cities in the Central Anatolia re de, Kayseri, Konya, and Karaman) and one city in gion (Nig the Mediterranean region (Adana) in Turkey. We aimed to (i) determine the prevalence of opportunistic chaetothyrialean black fungi in creosote-rich environments under nontropical conditions, and (ii) discuss the diversity of these fungi in light of their possible pathogenicity in humans.
Materials and methods Sampling area While collecting samples in August 2012, we visited six different railway stations in five different cities in Turkey, including
693
de, Yes¸ilhisar in Kayseri, and Pozantı in Adana, Ulukıs¸la in Nig de, Konya, and Karaman. These stations the city centers of Nig were all situated at altitudes of over 1000 m and on two different railway lines (Fig 1). On the first line, Pozantı lies at 1249-m, de at 1208-m, while Yes¸ilhisar Ulukıs¸la at 1427-m, and Nig (1150-m) is 67-km from Mount Erciyes, a popular skiing destination. On the second railway line, Konya lies at 1026-m, and Karaman lies at 1250-m. The distance from Pozantı to Yes¸ilhisar is 166 km, and the distance from Konya to Karaman is 102 km. The trains running on these lines have open toilets, similar to most other trains.
Sampling method A total of 658 railway tie samples were collected, including 302 oak (Pozantı ¼ 45, Ulukıs¸la ¼ 116, Yes¸ilhisar ¼ 108, and de ¼ 120, Karaman ¼ 33) and 356 concrete (Pozantı ¼ 73, Nig Konya ¼ 100, and Karaman ¼ 63). In total, 462 samples were collected from the first line, and 196 were collected from the second line. The oak railway ties had been treated with arsenic creosote, and the concrete ties were stained black with petroleum that had leaked from the trains. Fecal contamination was not observed on either type of tie; thus, we collected only machine-oil-impacted samples. Cotton swabs moistened with physiological saline were used to collect the samples, which were then transported in sterile tubes and inoculated on malt extract agar (MEA; Oxoid, Basingstoke, UK) culture plates containing 100 mg ml1 chloramphenicol (Sigma, Steinheim, Germany). The plates were incubated at 26 C and monitored daily for up to 4 weeks for evi dence of growth. Slow-growing, olivaceous black colonies were selected and transferred to fresh MEA plates for purification.
DNA extraction, PCR, and molecular analysis DNA extraction and PCR amplification were performed as described by Turin et al. (2000). rDNA sequences spanning the internal transcribed spacer (ITS) 1 region were amplified using the universal fungal primers ITS1 and ITS4 on an ABI PRISM 3130XL genetic analyzer at Refgen Biotechnology (Ankara, Turkey). CAP Contig assembly software, which is included in BioEdit Sequence Alignment Editor 7.0.9.0, was used to edit the sequences (Hall 1999). Assembled DNA sequences were
Fig 1 e Map of the two railway lines showing the areas sampled.
€g en et al. A. Do
694
Table 1 e Distribution of Exophiala species on railway ties. Isolate
Oak (n ¼ 302)
Concrete (n ¼ 356)
7 3 7
6 1 e
17
7
E. crusticola (n ¼ 13) E. heteromorpha (n ¼ 4) E. phaeomuriformis genotype 2 (n ¼ 7) Total
Results During this investigation, we encountered oily black spots on both types of railway ties from the six stations but did not observe visible fecal contamination. An incubation temperature of 26 C was used and we obtained numerous contaminants on MEA plates. If any medium was entirely covered by contaminants, the tie samples were then re-inoculated onto another MEA plate and re-incubated under the same conditions. Twenty-four out of 658 samples (3.6 %), representing four stations and three cities from the first line, were found to be positive for black yeast-like fungi. Creosote-treated oak-wood railway ties harboured significantly more black yeast-like fungi than concrete ties (5.6 % vs. 2 %). The magnitude of this difference was 0.0366 (95 %, CI ¼ 0.068e0.0667), which was statistically significant (P ¼ 0.02). The fungal isolation rates in the central railway stations in Pozantı (13 oak, three concrete), de (four concrete), and Yes¸ilhisar Ulukıs¸la (two oak), Nig (two oak) were 13.5 %, 1.7 %, 3.3 %, and 1.9 %, respectively. No black yeast-like fungi were recovered from the 196 second line samples taken at Konya and Karaman. The most common black yeast-like fungus was Exophiala crusticola (54.1 %), followed by Exophiala phaeomuriformis genotype 2 (29.2 %) and Exophiala heteromorpha (16.7 %). Table 1 shows the species distributions according to sample type. The thermophilic species E. phaeomuriformis grew within 3e6 d, while the E. crusticola and E. heteromorpha strains required 10e15 d for growth. Isolates of E. phaeomuriformis (genotype 2) tolerated cycloheximide and were found to grow in MEA with pH levels ranging from 2.5 to 12.5. However, 2/7 of the E. phaeomuriformis strains were not viable at 47 C and 5e17 % salinity. Additionally, isolates of E. crusticola and E. heteromorpha exhibited poor growth in the presence of cycloheximide. Exophiala crusticola isolates grew at 5e37 C, but not at 42 C or 47 C. Exophiala heteromorpha also showed growth at 5e42 C, but not at 47 C. Moreover, these two species did not show growth at pH 12.5. Remarkably, E. crusticola tolerated only 5 % NaCl, while E. heteromorpha strains were found to be viable at 10 % salinity (Table 2). A selection of the studied isolates was deposited in the reference collection of the Centraalbureau voor Schimmelcultures (CBS)-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands. In addition, the ITS rDNA sequences of these isolates were deposited in GenBank (Table 3).
examined using the BLAST (nucleotideenucleotide) software in the National Center for Biotechnology Information (NCBI) database.
Genotyping Sequences were aligned using ClustalW (Tamura et al. 2011) and compared by neighbour-joining phylogenetic tree analyses using MEGA version 5.05 software (www.megasoftware.net). Genotypic indicators of Exophiala phaeomuriformis were taken from Uijthof et al. (1998), and the genotypes were compared with a local CBS research database (www.cbs.knaw.nl) containing w10 000 black yeast sequences for verification.
Physiology The Exophiala strains were tested for several growth characteristics. First, thermotolerance tests were performed in 96well microtiter plates, with each well containing 300 ml of malt extract broth (MEB) inoculated with 10 ml of a cell suspension. The plates were incubated at 5 C, 10 C, 33 C, 37 C, 42 C, or 47 C. All media were assessed for growth daily over 2 weeks. Visual and spectrophotometric readings were performed at 450 nm with a Thermo Scientific Multiskan spectrophotometer (Vantaa, Finland). The pH tolerances of 2.5, 4, 10, and 12.5 were assessed in MEB with the addition of appropriate amounts of 0.1 M HCl and 0.1 M NaOH. Halotolerance was assessed in MEB supplemented with 5 %, 10 %, or 17 % NaCl (w/v). Tolerance to MEA containing 100 mg ml1 cycloheximide (Sigma, Germany) was also tested (Sudhadham et al. €g en et al. 2013b). 2008; Zalar et al. 2011; Do
Statistical analyses
Discussion
The differences between the frequencies of black yeast-like fungi on the creosote-treated oak and concrete railway ties stained with petroleum oil were evaluated using chi-square analysis. A P-value of <0.05 was considered to be significant.
All the strains of black yeast-like fungi recovered in the present study were derived from the first line plates, where 24 of
Table 2 e Growth characteristics of Exophiala species at a range of temperatures, pH values, and salt concentrations. Isolates
E. crusticola (n ¼ 13) E. heteromorpha (n ¼ 4) E. phaeomuriformis genotype 2 (n ¼ 7)
Temperatures
pH tests
Growth on NaCl
5 C
10 C
28 C
33 C
37 C
42 C
47 C
2.5
4
10
12.5
5%
10 %
17 %
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ
5/7
11/13 þ þ
11/13 þ þ
þ þ þ
þ
þ þ 5/7
þ 5/7
5/7
Black yeast-like fungi on railways
695
Table 3 e CBS and GenBank accession numbers (ITS rDNA sequences) of selected study isolates. Isolate E. E. E. E. E. E. E.
crusticola crusticola crusticola heteromorpha heteromorpha phaeomuriformis genotype 2 phaeomuriformis genotype 2
Sampling area
Railway tie
GenBank accession no. (ITS rDNA)/CBS no
Pozantı, Adana Pozantı, Adana de Nig Yes¸ilhisar, Kayseri de Nig Pozantı, Adana Pozantı, Adana
Concrete Oak-wood Concrete Oak-wood Concrete Oak-wood Oak-wood
KC349857/CBS 134043 KC349858/CBS 134427 KC349859/CBS 134428 KC349855/CBS 134041 KC349856/CBS 134042 KC349861/CBS 134012 KC349860/CBS 134013
the 462 (5.2 %) isolation plates were positive. Although we took a comparably large set of samples (n ¼ 196) from the second line, all the samples were found to contain sterile fungi. Based on our current knowledge, we do not know why this line was found to sterile for black yeasts. Exophiala crusticola was the most commonly identified black yeast species, followed by Exophiala phaeomuriformis and Exophiala heteromorpha (Table 1). In this study, the presence of black yeast-like fungi was lower (3.6 %) in cold and arid habitats than that in subtropical regions (17.0 %) in Turkey reported in our previous pa€g en et al. 2013a). per (Do Black yeasts found on railway ties include Exophiala derma€g en et al. titidis (Sudhadham et al. 2008; Vicente et al. 2008; Do 2013a) and some species with significant affinities for hydrocarbons, such as Exophiala bergeri (Vicente et al. 2008; Zhao et al. 2010), Exophiala xenobiotica (Vicente et al. 2008; Zhao et al. 2010), and Exophiala sideris (Seyedmousavi et al. 2011). In €g en et al. 2013a) and addition, Exophiala phaeomuriformis (Do Veronaea botyrosa (Vicente et al. 2008), which belong to the same fungal group, have also been found. The present investigation adds that E. crusticola and E. heteromorpha have a niche in the creosoted environment of railway ties. We previously noted that creosote-treated oak-wood railway ties support the growth of black yeast-like fungi better than €g en et al. 2013a), those made of concrete (19.7 % vs. 13.6 %) (Do which was similar to the results found in the current study (5.6 % vs. 2 %). Black yeasts are oligotrophic, and their presence on nutrient-poor concrete railway ties may be enhanced by the presence of oil spilled from trains. The contamination was lower at high altitudes (11501427 m) in the mountains in Turkey than €g en at altitudes ranging from 4 to 23-m in subtropical Turkey (Do et al. 2013a). In our previous study, we used a highly selective method with an incubation temperature of 37 C and recovered only the thermophilic species E. dermatitidis (57.7 %) and E. phaeomuriformis (42.3 %). In the present study, however, the species spectrum of black yeasts at a high altitude is different, e.g., E. crusticola and E. heteromorpha (Table 1). In general, chaetothyrialean black yeasts are slow-growing microorganisms that require long incubation times of at least 3 weeks (de Hoog et al. 2011a, 2011b). Here, we observed the growth of E. phaeomuriformis within 3e6 d, while E. crusticola and E. heteromorpha produced visible colonies after 1015 d. We hypothesize that low degrees of microbial competition on creosote-covered railway ties led to a massive enrichment €g en et al. 2013a). of Exophiala species (Do To date, E. crusticola has been reported in biological soil crusts in the USA only. This black species is able to grow at temperatures of 5e30 C but not at or above 37 C (Bates
et al. 2006). In this study, we found this fungus to be the most commonly recovered fungal species on railway ties in Turkey. This species has never been isolated from human hosts, and it has no clinical significance. In contrast, E. phaeomuriformis has been recovered from cutaneous, subcutaneous, and deep tissue human infections in the USA, with a total rate of 6.4 % of all black yeasts (Zeng et al. 2007). This thermophilic species was also responsible for the long-term colonization of the lungs of a patient with cystic fibrosis (Packeu et al. 2012). Recently, we described the isolation of E. phaeomuriformis €g en et al. 2013b). E. phaeomurfrom dishwashers in Turkey (Do iformis is somewhat more dependent on the availability of moisture, which is virtually absent from concrete railway ties in this investigation (Table 1). Our study also identified E. heteromorpha, a rare human opportunist. This member of the E. dermatitidis group was found (0.5 % of black yeasts) in human clinical isolates in the USA (Zeng et al. 2007). A possible physiological connection was suggested between the ability to co-metabolize aromatic hydrocarbons and mammalian fungal infections (Prenafeta-Bold et al. 2006), and several chaetothyrialean fungi have been implicated in human superficial infections (Zeng et al. 2007; de Hoog et al. 2011a, 2011b; Saunte et al. 2011). Their prevalence is low (2.2 %; Saunte et al. 2011) but consistent, and is most likely underdiagnosed or underreported. Two cases of systemic phaeohyphomycosis caused by E. dermatitidis have € been reported in Turkey (Alabaz et al. 2009; Oztas ¸ et al. 2009). In the present study, this species was not recovered from temperate, arid, elevated environments, but it was found in the same habitat in subtropical Turkey. Species of Exophiala show differential maximal growth at temperatures that generally coincide with geography and clinical predilection.
Acknowledgements We wish to thank Professor Refik Burgut for statistical analyses and Engin Kaplan, M.Sc., for laboratory assistance. We are grateful to the staff of the CBS-KNAW Fungal Biodiversity Centre collection service for processing several of the fungal isolates.
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Exophiala (Wangiella) dermatitidis in an immunocompetent child. Medical Mycology 47: 653e657. Bates ST, Reddy GSN, Garcia-Pichel N, 2006. Exophiala crusticola anam. nov. (affinity Herpotrichillaceae), a novel black yeast from biological soil crusts in the Western United States. International Journal of Systematic Evolutionary Microbiology 56: 2697e2702. J, Figueras MJ, 2011a. Atlas of Clinical de Hoog GS, Guarro J, Gene Fungi. CD-ROM Version 3.1. CBS-KNAW Fungal Biodiversity Centre, Utrecht. de Hoog GS, Vicente VA, Najafzadeh MJ, Harrak MJ, Badali H, Seyedmousavi S, 2011b. Waterborne Exophiala species causing disease in cold-blooded animals. Persoonia 27: 46e72. de Hoog GS, Queiroz-Telles F, Haase G, Fernandez-Zeppenfeldt G, Attili Angelis D, Gerrits van den Ende AH, Matos T, PeltrocheLlacsahuanga H, Pizzirani-Kleiner AA, Rainer J, RichardYegres N, Vicente V, Yegres F, 2000. Black fungi: clinical and pathological approaches. Medical Mycology 38 (suppl. 1): 243e250. €g en A, Kaplan E, Ilkit M, de Hoog GS, 2013a. Massive contamiDo nation of Exophiala dermatitidis and E. phaeomuriformis in railway stations in subtropical Turkey. Mycopathologia 175: 381e386. €g en A, Kaplan E, Oksuz Z, Serin MS, Ilkit M, de Hoog GS, 2013b. Do Dishwashers are a major source of opportunistic yeast-like fungi in indoor environments in Mersin, Turkey. Medical Mycology 51: 493e498. Gunde-Cimerman N, Ramos J, Plemenitas A, 2009. Halotolerant and halophilic fungi. Mycological Research 113: 1231e1241. Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98 NT, vol. 41, Nucleic Acids Symposium Series, 958. Li DM, Li RY, de Hoog GS, Wang YX, Wang DL, 2009. Exophiala asiatica, a new species from a fatal case in China. Medical Mycology 47: 101e109. Lian X, de Hoog GS, 2010. Indoor wet cells harbour melanized agents of cutaneous infection. Medical Mycology 48: 622e628. Matos T, de Hoog GS, de Boer AG, de Crom I, Haase G, 2002. High prevalence of the neurotrope Exophiala dermatitidis and related oligotrophic black yeasts in sauna facilities. Mycoses 45: 373e377. Matos T, Haase G, Gerrits van den Ende AHG, de Hoog GS, 2003. Molecular diversity of oligotrophic and neurotropic members of the black yeast genus Exophiala, with accent on E. dermatitidis. Antonie van Leeuwenhoek 83: 293e303. € € Oztas ¸ E, Odemis ¸ B, Kekilli M, Kurt M, Dinc¸ BM, Parlak E, Kalkancı A, S¸as¸maz N, 2009. Systemic phaeohyphomycosis resembling primary sclerosing cholangitis caused by Exophiala dermatitidis. Journal of Medical Microbiology 58: 1243e1246.
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journal homepage: www.elsevier.com/locate/funbio
Black yeast habitat choices and species spectrum on high altitude creosote-treated railway ties € GEN a, Macit ILKITb,*, G. Sybren DE HOOGc,d,e,f,g Aylin DO a
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, University of Mersin, Mersin, Turkey Division of Mycology, Department of Microbiology, Faculty of Medicine, University of C¸ukurova, Adana 01330, Turkey c CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands d Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Amsterdam, The Netherlands e Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China f Sun Yat-sen Hospital, Sun Yat-sen University, Guangzhou, China g Shanghai Institute of Medical Mycology, Changzheng Hospital, Second Military Medical University, Shanghai, China b
article info
abstract
Article history:
Polyextremotolerant black yeast-like fungi thrive in moderately hostile environments
Received 5 January 2013
where they are concomitantly subjected to several types of stress, such as toxicity, scarce
Received in revised form
nutrient availability, and high or low temperature extremes. Their ability to assimilate al-
20 July 2013
kylbenzenes (toxic environmental pollutants) enhances their growth in harsh conditions,
Accepted 23 July 2013
including on railway ties. Samples were collected using cotton swabs, premoistened with
Available online 11 August 2013
physiological saline, from 658 oak and concrete railway ties at six train stations in Turkey
Corresponding Editor:
at altitudes ranging between 1026 and 1427 m. The samples were inoculated on malt ex-
Andrew N. Miller
tract agar supplemented with chloramphenicol, and incubated at 26 C for 4 weeks.
Keywords:
tested positive for fungi. Exophiala crusticola was found to be the most common species
Twenty-four samples (3.6 %), 17 from oak and 7 from concrete (5.6 % vs. 2 %; P ¼ 0.02), Chaetothyriales
(n ¼ 13), followed by Exophiala phaeomuriformis (n ¼ 7) and Exophiala heteromorpha (n ¼ 4).
Exophiala crusticola
These results suggest that hydrocarbons, particularly creosote-treated oak woods, support
Exophiala phaeomuriformis
the growth of black yeasts, some of which are opportunists in humans.
Extremophily
ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Filamentous fungi
Introduction Members of the black yeast genus Exophiala (order Chaetothyriales) have been isolated from superficial and subcutaneous infections, as well as fatal systemic infections, in immunocompetent humans (de Hoog et al. 2000). Different species of black yeast-like fungi have distinct traits and are associated with various disease categories (Zeng et al. 2007; Saunte et al. 2011). They are ubiquitous in wet environments that differ in temperature and are consequently able to harbor different
species (Lian & de Hoog 2010; Zalar et al. 2011). Differing pathogenic potentials have been described among clades of Chaetothyriales (de Hoog et al. 2011a, 2011b). Examples of varying degrees of pathogenicity include: (i) species that are able to grow at temperatures well over 36e37 C (e.g., bantiana, dermatitidis, and jeanselmei clades) and may cause systemic or disseminated infections in humans; (ii) species that are able to tolerate a maximum of approximately 36e37 C (e.g., carrionii and europaea clades) and cause superficial or subcutaneous infections; and (iii) species of the salmonis clade, which survive
* Corresponding author. Tel.: þ90 532 286 0099; fax: þ90 322 457 3072. E-mail address: [email protected] (M. Ilkit). 1878-6146/$ e see front matter ª 2013 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.funbio.2013.07.006
Black yeast-like fungi on railways
at a maximal temperature of 27e33 (36) C and cause at most superficial infections (Li et al. 2009; de Hoog et al. 2011a, 2011b; Saunte et al. 2011). Consistent with these findings, Saunte et al. (2011) recovered mostly Phialophora europea, Exophiala lecaniicorni, and Knufia epidermidis from human superficial black yeast infections. The thermophilic species Exophiala dermatitidis, one of the most common systemic pathogens, is thought to have a natural niche on fruit surfaces, with a life cycle involving passage through the intestinal tracts of fruit-eating animals, such as flying foxes and hornbills in the tropics (Sudhadham et al. 2008). This species has also been found in hot water from natural springs. Black fungi are able to tolerate low and high pH values (Zalar €g en et al. 2013b) and can live in environments with et al. 2011; Do high salt concentrations (Gunde-Cimerman et al. 2009). Several man-made environments appear to serve as an artificial equivalent to hot springs, and these species have commonly been recovered from Turkish steam baths (Matos et al. 2002; 2003), €g en et al. 2013b), and bathrooms dishwashers (Zalar et al. 2011; Do (Lian & de Hoog 2010). Black yeasts have also been isolated from bathrooms using toluene enrichment (Lian & de Hoog 2010). Additionally, black yeasts are prevalent in environments that are contaminated by aromatic hydrocarbons, such as creosotetreated railway ties (Sudhadham et al. 2008; Vicente et al. 2008; €g en et al. 2013a). Zhao et al. 2010; Seyedmousavi et al. 2011; Do For present study, we sampled creosote-treated oak and concrete railway ties from six stations and two different lines, at altitudes ranging from 1026 to 1427 m above sea level. These samples were taken in four cities in the Central Anatolia re de, Kayseri, Konya, and Karaman) and one city in gion (Nig the Mediterranean region (Adana) in Turkey. We aimed to (i) determine the prevalence of opportunistic chaetothyrialean black fungi in creosote-rich environments under nontropical conditions, and (ii) discuss the diversity of these fungi in light of their possible pathogenicity in humans.
Materials and methods Sampling area While collecting samples in August 2012, we visited six different railway stations in five different cities in Turkey, including
693
de, Yes¸ilhisar in Kayseri, and Pozantı in Adana, Ulukıs¸la in Nig de, Konya, and Karaman. These stations the city centers of Nig were all situated at altitudes of over 1000 m and on two different railway lines (Fig 1). On the first line, Pozantı lies at 1249-m, de at 1208-m, while Yes¸ilhisar Ulukıs¸la at 1427-m, and Nig (1150-m) is 67-km from Mount Erciyes, a popular skiing destination. On the second railway line, Konya lies at 1026-m, and Karaman lies at 1250-m. The distance from Pozantı to Yes¸ilhisar is 166 km, and the distance from Konya to Karaman is 102 km. The trains running on these lines have open toilets, similar to most other trains.
Sampling method A total of 658 railway tie samples were collected, including 302 oak (Pozantı ¼ 45, Ulukıs¸la ¼ 116, Yes¸ilhisar ¼ 108, and de ¼ 120, Karaman ¼ 33) and 356 concrete (Pozantı ¼ 73, Nig Konya ¼ 100, and Karaman ¼ 63). In total, 462 samples were collected from the first line, and 196 were collected from the second line. The oak railway ties had been treated with arsenic creosote, and the concrete ties were stained black with petroleum that had leaked from the trains. Fecal contamination was not observed on either type of tie; thus, we collected only machine-oil-impacted samples. Cotton swabs moistened with physiological saline were used to collect the samples, which were then transported in sterile tubes and inoculated on malt extract agar (MEA; Oxoid, Basingstoke, UK) culture plates containing 100 mg ml1 chloramphenicol (Sigma, Steinheim, Germany). The plates were incubated at 26 C and monitored daily for up to 4 weeks for evi dence of growth. Slow-growing, olivaceous black colonies were selected and transferred to fresh MEA plates for purification.
DNA extraction, PCR, and molecular analysis DNA extraction and PCR amplification were performed as described by Turin et al. (2000). rDNA sequences spanning the internal transcribed spacer (ITS) 1 region were amplified using the universal fungal primers ITS1 and ITS4 on an ABI PRISM 3130XL genetic analyzer at Refgen Biotechnology (Ankara, Turkey). CAP Contig assembly software, which is included in BioEdit Sequence Alignment Editor 7.0.9.0, was used to edit the sequences (Hall 1999). Assembled DNA sequences were
Fig 1 e Map of the two railway lines showing the areas sampled.
€g en et al. A. Do
694
Table 1 e Distribution of Exophiala species on railway ties. Isolate
Oak (n ¼ 302)
Concrete (n ¼ 356)
7 3 7
6 1 e
17
7
E. crusticola (n ¼ 13) E. heteromorpha (n ¼ 4) E. phaeomuriformis genotype 2 (n ¼ 7) Total
Results During this investigation, we encountered oily black spots on both types of railway ties from the six stations but did not observe visible fecal contamination. An incubation temperature of 26 C was used and we obtained numerous contaminants on MEA plates. If any medium was entirely covered by contaminants, the tie samples were then re-inoculated onto another MEA plate and re-incubated under the same conditions. Twenty-four out of 658 samples (3.6 %), representing four stations and three cities from the first line, were found to be positive for black yeast-like fungi. Creosote-treated oak-wood railway ties harboured significantly more black yeast-like fungi than concrete ties (5.6 % vs. 2 %). The magnitude of this difference was 0.0366 (95 %, CI ¼ 0.068e0.0667), which was statistically significant (P ¼ 0.02). The fungal isolation rates in the central railway stations in Pozantı (13 oak, three concrete), de (four concrete), and Yes¸ilhisar Ulukıs¸la (two oak), Nig (two oak) were 13.5 %, 1.7 %, 3.3 %, and 1.9 %, respectively. No black yeast-like fungi were recovered from the 196 second line samples taken at Konya and Karaman. The most common black yeast-like fungus was Exophiala crusticola (54.1 %), followed by Exophiala phaeomuriformis genotype 2 (29.2 %) and Exophiala heteromorpha (16.7 %). Table 1 shows the species distributions according to sample type. The thermophilic species E. phaeomuriformis grew within 3e6 d, while the E. crusticola and E. heteromorpha strains required 10e15 d for growth. Isolates of E. phaeomuriformis (genotype 2) tolerated cycloheximide and were found to grow in MEA with pH levels ranging from 2.5 to 12.5. However, 2/7 of the E. phaeomuriformis strains were not viable at 47 C and 5e17 % salinity. Additionally, isolates of E. crusticola and E. heteromorpha exhibited poor growth in the presence of cycloheximide. Exophiala crusticola isolates grew at 5e37 C, but not at 42 C or 47 C. Exophiala heteromorpha also showed growth at 5e42 C, but not at 47 C. Moreover, these two species did not show growth at pH 12.5. Remarkably, E. crusticola tolerated only 5 % NaCl, while E. heteromorpha strains were found to be viable at 10 % salinity (Table 2). A selection of the studied isolates was deposited in the reference collection of the Centraalbureau voor Schimmelcultures (CBS)-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands. In addition, the ITS rDNA sequences of these isolates were deposited in GenBank (Table 3).
examined using the BLAST (nucleotideenucleotide) software in the National Center for Biotechnology Information (NCBI) database.
Genotyping Sequences were aligned using ClustalW (Tamura et al. 2011) and compared by neighbour-joining phylogenetic tree analyses using MEGA version 5.05 software (www.megasoftware.net). Genotypic indicators of Exophiala phaeomuriformis were taken from Uijthof et al. (1998), and the genotypes were compared with a local CBS research database (www.cbs.knaw.nl) containing w10 000 black yeast sequences for verification.
Physiology The Exophiala strains were tested for several growth characteristics. First, thermotolerance tests were performed in 96well microtiter plates, with each well containing 300 ml of malt extract broth (MEB) inoculated with 10 ml of a cell suspension. The plates were incubated at 5 C, 10 C, 33 C, 37 C, 42 C, or 47 C. All media were assessed for growth daily over 2 weeks. Visual and spectrophotometric readings were performed at 450 nm with a Thermo Scientific Multiskan spectrophotometer (Vantaa, Finland). The pH tolerances of 2.5, 4, 10, and 12.5 were assessed in MEB with the addition of appropriate amounts of 0.1 M HCl and 0.1 M NaOH. Halotolerance was assessed in MEB supplemented with 5 %, 10 %, or 17 % NaCl (w/v). Tolerance to MEA containing 100 mg ml1 cycloheximide (Sigma, Germany) was also tested (Sudhadham et al. €g en et al. 2013b). 2008; Zalar et al. 2011; Do
Statistical analyses
Discussion
The differences between the frequencies of black yeast-like fungi on the creosote-treated oak and concrete railway ties stained with petroleum oil were evaluated using chi-square analysis. A P-value of <0.05 was considered to be significant.
All the strains of black yeast-like fungi recovered in the present study were derived from the first line plates, where 24 of
Table 2 e Growth characteristics of Exophiala species at a range of temperatures, pH values, and salt concentrations. Isolates
E. crusticola (n ¼ 13) E. heteromorpha (n ¼ 4) E. phaeomuriformis genotype 2 (n ¼ 7)
Temperatures
pH tests
Growth on NaCl
5 C
10 C
28 C
33 C
37 C
42 C
47 C
2.5
4
10
12.5
5%
10 %
17 %
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ þ
þ þ
5/7
11/13 þ þ
11/13 þ þ
þ þ þ
þ
þ þ 5/7
þ 5/7
5/7
Black yeast-like fungi on railways
695
Table 3 e CBS and GenBank accession numbers (ITS rDNA sequences) of selected study isolates. Isolate E. E. E. E. E. E. E.
crusticola crusticola crusticola heteromorpha heteromorpha phaeomuriformis genotype 2 phaeomuriformis genotype 2
Sampling area
Railway tie
GenBank accession no. (ITS rDNA)/CBS no
Pozantı, Adana Pozantı, Adana de Nig Yes¸ilhisar, Kayseri de Nig Pozantı, Adana Pozantı, Adana
Concrete Oak-wood Concrete Oak-wood Concrete Oak-wood Oak-wood
KC349857/CBS 134043 KC349858/CBS 134427 KC349859/CBS 134428 KC349855/CBS 134041 KC349856/CBS 134042 KC349861/CBS 134012 KC349860/CBS 134013
the 462 (5.2 %) isolation plates were positive. Although we took a comparably large set of samples (n ¼ 196) from the second line, all the samples were found to contain sterile fungi. Based on our current knowledge, we do not know why this line was found to sterile for black yeasts. Exophiala crusticola was the most commonly identified black yeast species, followed by Exophiala phaeomuriformis and Exophiala heteromorpha (Table 1). In this study, the presence of black yeast-like fungi was lower (3.6 %) in cold and arid habitats than that in subtropical regions (17.0 %) in Turkey reported in our previous pa€g en et al. 2013a). per (Do Black yeasts found on railway ties include Exophiala derma€g en et al. titidis (Sudhadham et al. 2008; Vicente et al. 2008; Do 2013a) and some species with significant affinities for hydrocarbons, such as Exophiala bergeri (Vicente et al. 2008; Zhao et al. 2010), Exophiala xenobiotica (Vicente et al. 2008; Zhao et al. 2010), and Exophiala sideris (Seyedmousavi et al. 2011). In €g en et al. 2013a) and addition, Exophiala phaeomuriformis (Do Veronaea botyrosa (Vicente et al. 2008), which belong to the same fungal group, have also been found. The present investigation adds that E. crusticola and E. heteromorpha have a niche in the creosoted environment of railway ties. We previously noted that creosote-treated oak-wood railway ties support the growth of black yeast-like fungi better than €g en et al. 2013a), those made of concrete (19.7 % vs. 13.6 %) (Do which was similar to the results found in the current study (5.6 % vs. 2 %). Black yeasts are oligotrophic, and their presence on nutrient-poor concrete railway ties may be enhanced by the presence of oil spilled from trains. The contamination was lower at high altitudes (11501427 m) in the mountains in Turkey than €g en at altitudes ranging from 4 to 23-m in subtropical Turkey (Do et al. 2013a). In our previous study, we used a highly selective method with an incubation temperature of 37 C and recovered only the thermophilic species E. dermatitidis (57.7 %) and E. phaeomuriformis (42.3 %). In the present study, however, the species spectrum of black yeasts at a high altitude is different, e.g., E. crusticola and E. heteromorpha (Table 1). In general, chaetothyrialean black yeasts are slow-growing microorganisms that require long incubation times of at least 3 weeks (de Hoog et al. 2011a, 2011b). Here, we observed the growth of E. phaeomuriformis within 3e6 d, while E. crusticola and E. heteromorpha produced visible colonies after 1015 d. We hypothesize that low degrees of microbial competition on creosote-covered railway ties led to a massive enrichment €g en et al. 2013a). of Exophiala species (Do To date, E. crusticola has been reported in biological soil crusts in the USA only. This black species is able to grow at temperatures of 5e30 C but not at or above 37 C (Bates
et al. 2006). In this study, we found this fungus to be the most commonly recovered fungal species on railway ties in Turkey. This species has never been isolated from human hosts, and it has no clinical significance. In contrast, E. phaeomuriformis has been recovered from cutaneous, subcutaneous, and deep tissue human infections in the USA, with a total rate of 6.4 % of all black yeasts (Zeng et al. 2007). This thermophilic species was also responsible for the long-term colonization of the lungs of a patient with cystic fibrosis (Packeu et al. 2012). Recently, we described the isolation of E. phaeomuriformis €g en et al. 2013b). E. phaeomurfrom dishwashers in Turkey (Do iformis is somewhat more dependent on the availability of moisture, which is virtually absent from concrete railway ties in this investigation (Table 1). Our study also identified E. heteromorpha, a rare human opportunist. This member of the E. dermatitidis group was found (0.5 % of black yeasts) in human clinical isolates in the USA (Zeng et al. 2007). A possible physiological connection was suggested between the ability to co-metabolize aromatic hydrocarbons and mammalian fungal infections (Prenafeta-Bold et al. 2006), and several chaetothyrialean fungi have been implicated in human superficial infections (Zeng et al. 2007; de Hoog et al. 2011a, 2011b; Saunte et al. 2011). Their prevalence is low (2.2 %; Saunte et al. 2011) but consistent, and is most likely underdiagnosed or underreported. Two cases of systemic phaeohyphomycosis caused by E. dermatitidis have € been reported in Turkey (Alabaz et al. 2009; Oztas ¸ et al. 2009). In the present study, this species was not recovered from temperate, arid, elevated environments, but it was found in the same habitat in subtropical Turkey. Species of Exophiala show differential maximal growth at temperatures that generally coincide with geography and clinical predilection.
Acknowledgements We wish to thank Professor Refik Burgut for statistical analyses and Engin Kaplan, M.Sc., for laboratory assistance. We are grateful to the staff of the CBS-KNAW Fungal Biodiversity Centre collection service for processing several of the fungal isolates.
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