Barcode Identifiers as a Practical Tool for Reliable Species Assignment of Medically Important Black Yeast Species

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of Medically Important Black Yeast Species

Guido Heinrichs,a_{G. Sybren de Hoog,}b_{and Gerhard Haase}a

Institut für Medizinische Mikrobiologie, RWTH Aachen University Hospital, Aachen, Germany,a_{and Centraalbureau voor Schimmelcultures KNAW Fungal Biodiversity}

Centre, Utrecht, and Institute of Biodiversity and Ecosystem Dynamics, Amsterdam, The Netherlandsb

Herpotrichiellaceous black yeasts and relatives comprise severe pathogens flanked by nonpathogenic environmental siblings. Reliable identification by conventional methods is notoriously difficult. Molecular identification is hampered by the sequence variability in the internal transcribed spacer (ITS) domain caused by difficult-to-sequence homopolymeric regions and by poor taxonomic attribution of sequences deposited in GenBank. Here, we present a potential solution using short barcode identifiers (27 to 50 bp) based on ITS2 ribosomal DNA (rDNA), which allows unambiguous definition of species-specific fragments. Start-ing from proven sequences of ex-type and authentic strains, we were able to describe 103 identifiers. Multiple BLAST searches of these proposed barcode identifiers in GenBank revealed uniqueness for 100 taxonomic entities, whereas the three remaining identifiers each matched with two entities, but the species of these identifiers could easily be discriminated by differences in the remaining ITS regions. Using the proposed barcode identifiers, a 4.1-fold increase of 100% matches in GenBank was achieved in comparison to the classical approach using the complete ITS sequences. The proposed barcode identifiers will be made

accessible for the diagnostic laboratory in a permanently updated online database, thereby providing a highly practical, reliable, and cost-effective tool for identification of clinically important black yeasts and relatives.

B

lack yeasts and their filamentous relatives that belong to the ascomycete familyHerpotrichiellaceae(orderChaetothyriales) comprise a large group of pathogenic fungi. The infections caused by these fungi frequently occur in immunocompetent humans and range from mild cutaneous to severe, deep, or disseminated. The mycoses are difficult to treat, and systemic cases have a high fatality rate. For example,Cladophialophora bantianaand Rhino-cladiella mackenzieiare neurotropic agents classified as biosafety level 3 organisms (5). Some neurotropic species, likeExophiala dermatitidis, are common in wet habitats of indoor environments, such as in bathrooms (14), in steam baths (15), and on the rubber seals of dishwashers (19). Other black yeast species are associated with infections in cold-blooded animals (6). In total, the order contains 44 species that have been proven to be involved in infec-tions of vertebrate hosts (5) and ranges among the most species-rich clinically relevant groups in the fungal kingdom.

Identification of black yeasts by morphological criteria alone is mostly impossible because of variable expression of morphologi-cal traits (synanamorphs) during complex, pleomorphic life cy-cles or low degrees of differentiation. Conversely, nearly identical morphological structures can be observed in phylogenetically dis-tantly related species (20). Since comparable physiological data are also lacking for most species and test results may be variable within the species, these traits do not allow reliable species iden-tification either.

Genetic identification of herpotrichiellaceous black yeasts is rou-tinely based upon sequences of the ribosomal DNA (rDNA) internal transcribed spacer (ITS) followed by a similarity search in public da-tabases. However, one should keep in mind that GenBank was not designed for this purpose. Nilsson et al. (16) noted that 27% of the ITS sequences deposited in the International Nucleotide Sequence Database (INSD; GenBank, EMBL, and DDBJ) had been incorrectly annotated to the species level, partly due to a general neglect of type materials defining the species. The need for a fast, easy, and reliable identification tool can be best illustrated by the sibling species

Clado-phialophora bantianaandCladophialophora psammophila:C. banti-anais an agent of fatal brain disease, whereasC. psammophilais as-sociated with aromatic hydrocarbons, proven to be avirulent in animal tests (1). With the molecular developments over the last de-cade, increasing numbers of novel species and reclassifications have been proposed, among which were numerous opportunistic patho-gens. In principal, usage of ITS sequence data is reliable for identifi-cation (20). Reference sequences have barcode status, i.e., sequences are open access and have been deposited with trace data and eventu-ally with original specimens. Of note, obvious sequence variability occurring mainly in homopolymeric regions due to formation of stutter products in the PCR (4) may interfere with unambiguous attribution of query sequences to a given species. Short barcode frag-ments are therefore needed which are invariable within the species and show stable indels or differences in nucleotide composition, e.g., due to compensatory base exchanges with neighboring species. The selection of barcode fragments with such properties may provide an elegant and straightforward approach to identify herpotrichiella-ceous black yeasts and their relatives.

MATERIALS AND METHODS

Strains and culture conditions. Sequences of ex-type and authentic strains of species with clinical relevance and closely related environmental

species ofHerpotrichiellaceaewere chosen for analysis. In addition, species

were included that had repeatedly been isolated from clinical samples. This resulted in a selection of 68 strains (Table 1), which are preserved at the CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands. In

Received1 March 2012Returned for modification29 March 2012

Accepted5 July 2012

Published ahead of print11 July 2012

doi:10.1128/JCM.00574-12

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TABLE 1 Compilation and description of 103 proposed barcode identifiers Species Strain

Conserved flanking region

Barcode

identifier

Conserved flanking region Accession no. Position Habitat No. of 100% D2 hits ITS identity with ex-type (%) Cladophialophora arxii 1 CBS 306.94 T TTGG ACGGTTTGGTGGAAGGGTGACACCCTTCCGCCCGT CCTAA EU103986.1 400–443 Man 3 100 Cladophialophora arxii 2 CBS 409.96 TTGG ACGGTTCGGTGGGGAAGGGGTACACCCTTCCACCCGT CCTAA EU103987.1 399–444 Man 1 99 Cladophialophora bantiana CBS 173.52 T TTGG ACGGTCTGGCGGAAGTGTCGTGCACCCCGCCCCT CCTAA EU103989.1 392–444 Man 14 100 Cladophialophora carrionii 1 CBS 160.54 LT TTGG ACGGTTTTGGTCGAGTGTTCTCGACCCCT CCTAA AF050262.1 391–428 Man 11 100 Cladophialophora carrionii 2 CBS 163.54 TTGG ACGGTCTTGGTCGAGTGTTCTCGACCCCT CCTAA EU137304.1 389–426 Man 73 98 Cladophialophora carrionii 3 CBS 260.83 TTGG ACGGTTTTGGTCGAGTGTTCTCGACCCCT CCTAA EU137292.1 389–426 Man, skin lesion 16 98 Cladophialophora carrionii 4 FMC.248 TTGG ACGGTCTTGGTCGAGTGTTCTCGACCCCC CCTAA AF397181.1 411–448 Man 1 97 Cladophialophora devriesii 1 CBS 147.84 T TTGG ACGGCTTGGTAGAGTCCCTACCCCT CCTAA EU103985.1 394–427 Man 1 100 Cladophialophora devriesii 2 IFM 51369 TTGG ACGGCTTGGTAAATTCCCTACCCCT CCTAA AB091212.1 416–449 1 99 Cladophialophora aff. devriesii CBS 119710 T TTGG ACGGCTTGGTAGAGAGTCTCTACCCCT CCTAA FJ385275.1 394–429 Crab 1 100 Cladophialophora emmonsii 1 CBS 979.96 T TTGG ACGGCCTGGCGGAGTGCACGACACTCTGCCCCT CCTAA EU103996.1 389–430 Man, subcutaneous lesion of right forearm 3 100 Cladophialophora emmonsii 2 CBS 640.96 TTGG ACGGCCTGGCGGAGTGCACGACACCCTGCCCCT CCTAA EU103995.1 349–390 Cat, subcutaneous lesion 1 99 Cladophialophora immunda 1 CBS 834.96 T TTGG ACGGTTTGGTAGGGCGCCTACCCCT CCTAA EU137318.1 396–429 Man 5 100 Cladophialophora immunda 2 CBS 102227 TTGG ACGGTTTGGTAGAGTGCCTACCCCT CCTAA EU137319.1 399–432 Syagrus romanzoffiania ,stem 2 96 Cladophialophora modesta CBS 985.96 T TTGG AATCTCAGTTGAGTGATCAACTGGTT CCCAA GU225939.1 383–416 Man 1 100 Cladophialophora mycetomatis 1 CBS 122637 T TTGG ACGGTTTGGTCGACGACACCCGACCCCT CCCAA FJ385276.1 380–416 Man 1 100 Cladophialophora mycetomatis 2 CBS 454.82 TTGG ACGGTTTGGTCGACGACATCCGACCCCT CCCAA EU137293.1 399–435 Culture contaminant 1 98 Cladophialophora psammophila CBS 110553 T TTGG ACGGTCTGGCGGAAGTGTCGTGCACGCCGCCCCT CCTAA AY857517.1 398–440 Gasoline-polluted soil 1 100 Cladophialophora samoensis CBS 259.83 T TTGG AGGGTCCCTGGTCGAGGCTCTACCTCGACCT CCTAA EU137291.1 393–432 Man, skin lesion 1 100 Cladophialophora saturnica CBS 118724 T TTGG ACGGCTTGGTAGAGTTCCCTCTACCCT CCTAA EU103984.1 398–433 Interdigital, tinea nigra-like skin lesion of a 4-year-old HIV-positive child 4 100 Cladophialophora subtilis CBS 122642 T TTGG ACGGCCTTGGTCGCGCACGCGCCGACCCCT CCTAA FJ385273.1 387–425 Iced tea 2 100 Cladophialophora yegresii CBS 114405 T TTGG ACGGTTTTGGTCGAGTGTCCCTCGACCCCT CCTAA EU137322.1 394–432 Stenocereus griseus 3 100 Cyphellophora laciniata CBS 190.61 T TTGG ACGTCGGCGGCGGCCTTTAGTGGCGTACCGCCCGT CTCAA EU035416.1 523–566 Man, skin 11 100 Cyphellophora pluriseptata CBS 286.85 T TTGG ACGCGGCGGCGGCGCACCCGCCCCCGCCCGT CTCAA FN908214.1 404–444 Man, skin of foot 1 100 Cyphellophora suttonii CBS 449.91 T TTGG ATGTTGGCGGCGGCCTTTAGTGGCGTACCGCCCGT CTCAA GU225946.1 382–425 Dog, ear 2 100 Cyphellophora vermispora CBS 228.86 T TTGG ACGTCGGCGGCGGCCTTTAGTGGCGTACCGCCCGT CTCAA GU225947.1 383–426 Triticum aestivum ,root 11 100 Exophiala alcalophila CBS 520.82 T TTGG ACGGCTTGGTTACGCTCCCCGCGTGACCCCT CCTAA JF747041.1 395–434 Soil 4 100 Exophiala angulospora 1 CBS 482.92 T TTGG ACGGCCTGGTCGAGTCCGCTCGACCCCT CCTAA JF747046.1 385–421 Water from drinking well 4 100 Exophiala angulospora 2 CBS 122264 TTGG ACGGCCTGGTCGAGTCCCCTCGACCCCT CCTAA JF747052.1 396–439 Hydrocarbon-polluted soil 5 98 Exophiala angulospora 3 CBS 109906 TTGG ACGGCTTGGTCGAGTCCCCTCGACCCCT CCTAA JF747047.1 395–439 Drinking water in home installation 59 8 Exophiala aquamarina CBS 119918 T TTGG ACGGCTTGGTGGACGCCCCGCACGGGGCGTCCACCCCT CCCAA JF747054.1 391–437 Phycodurus eques (seadragon), skin 8 100 Exophiala asiatica BMU 00015 T TTGG ACGGCCCGGTCGACGCGTCATTTAGCGCCGACCCCT CCTAA EU910265.1 400–444 Man 1 100 Exophiala attenuata CBS 110026 TTGG ACGGTTTGGTGTCTGGCAACGGTCACCCCT CCCAA AF549446.1 399–437 Man, cutaneous lesion of ring finger 4 100 Exophiala bergeri CBS 353.52 T TTGG ACGGTCTGGTTTAGGCACCGCCTAGACCCCT CCTAA EF551462.1 391–430 Man 6 100 Exophiala brunnea CBS 587.66 T TTGG ACGGTTTGGTGGAGGCCCCTCACGGGGCTCCTGCCCCT CCTAA JF747062.1 386–432 Acacia karro ,litter 2 100 Exophiala cancerae CBS 120532 T TTGG ACGGTTTGGTGGAGGGCCTTCGGGCGCCACCCCT CCCAA JF747063.1 383–425 Crab 10 100 Exophiala castellanii CBS 158.58 T TTGG ACGGTTTGGTGTCGGCAACGTCACCCCT CCCAA GU225940.1 394–430 Man, subcutaneous cyst, left wrist 1 100 Exophiala capensis CBS 128771 T TTGG ACGGCTCGGTCTAGGTACCGCCTAGACCCCT CCTAA JF499841.1 928–976 Leaf bracts of Phaenocoma prolifera 11 100 Exophiala dermatitidis 1 CBS 207.35 T TTGG ACGGTCTGGTCGAGCGTTTCCGCGCGACCCCT CCCAA AF050269.1 395–435 Man 40 100 Exophiala dermatitidis 2 CBS 100338 TTGG ACGGTATGGTCGAGCGTTTCCGCGCGACCT CCCAA AF147495.1 391–429 Humidifier 1 97 Exophiala dermatitidis 3 BMU 00035 TTGG ACCGGTCTGGTCAAGCGTTTCCGCGCGACCCT CCCAA EU910258.1 395–436 Man, cerebrospinal fluid 1 97 Exophiala equina 1 CBS 119.23 T TTGG ACGGTTTGGTGGAGGCCCCCTCGGGGGCTCCTGCCCCT CCCAA JF747094.1 385–431 Horse skin 63 100

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Exophiala equina 2 CBS 122263 TTGG ACGGTTTGGTGGAGGCCCCCTCGGGGGTTCCTGCCCCT CCCAA JF747099.1 385–431 9 98 Exophiala equina 3 CBS 122270 TTGG ACGGTTTGGTGGAGGTCCCCTCGGGGGCTCCTGCCCCT CCCAA JF747101.1 384–430 Human foot 9 98 Exophiala exophialae CBS 668.76 T TTGG ACGGTTTGGTCCCGGGACGCCCCTGGACCCCT CCCAA AY156973.1 385–425 Straw in burrow of Dasypus septemcinctus 3 100 Exophiala halophila CBS 121512 T TTGG ACGGCTTGGTCGCGCCCCCCGGGCACCGACCCCT CCTAA JF747108.1 394–436 Man, axillary 3 100 Exophiala heteromorpha CBS 232.33 T TTGG ACGGTCTGGTCGAGCCCGCCTCGACCCCT CCCAA AY857524.1 389–426 Wood pulp 6 100 Exophiala jeanselmei 1 CBS 507.90 T TTGG ACGGTTTGGTCTCGGGTCCGACCCCCCTTGACCCCT CCCAA AY156963.1 389–433 Man 14 100 Exophiala jeanselmei 2 CBS 677.76 TTGG ACGGTTTGGTCAAAGGGTCCGACCCCCCTAGACCCCT CCCAA AY163553.1 386–431 Skin, abscess of foot 2 96 Exophiala lacus CBS 117497 T TTGG ACGGTTTGGCGGAGACCTGTTACAGGCCTCCACCCCT CCCAA JF747110.1 384–429 Water 1 100 Exophiala lecanii-corni CBS 123.33 T TTGG ACGGCCTGGCGTCGGCGACGACCCCACCT CCCAA AY857528.1 409–446 Man 10 100 Exophiala mesophila 1 CBS 402.95 T TTGG ACGGCTTGGTGTCAGCGATGTCACCCCT CCTAA GU225941.1 396–432 Silicone seal, in shower room of hospital 16 100 Exophiala mesophila 2 CBS 121511 TTGG ACGGCTTGGTGTCAGCAATGTCACCCCT CCTAA JF747122.1 397–433 Nasal tissue 1 97 Exophiala moniliae CBS 520.76 T TTGG ACGGTCTGGTTAGGCGACTGACCCCT CCTAA GU225948.1 390–424 Quercus ,twig 6 100 Exophiala nishimurae CBS 101538 T TTGG ACGGCTTGGTCTAGGTCGTCGCCTAGACCCCT CCCAA AY156966.1 a 392–432 Bark 1 100 Exophiala oligosperma 1 CBS 725.88 T TTGG ACGGTCTGGTCCGGGGACCTCAAACCCCCTGGACCCCT CCCAA AY163551.1 397–443 Man, tumor of sphenoidal cavity 25 100 Exophiala oligosperma 2 UTHSC95-2041 TTGG ACGGTTTGGTTCGGGGACCTCAAACCCCCTGGACCCCT CCCAA EF025415.1 388–434 Man 2 96 Exophiala oligosperma 3 UTHSC 91–870 TTGG ACGGTTTGGTCCGGGGACCTCAAAGCCCCCTGGACCCCT CCCAA EF551458.1 354–401 Man, hand 8 99 Exophiala oligosperma 4 IFM 41701 TTGG ACGGTTTGGTCCGGGGACCTCAAAAACCCCCTGGACCCCT CCCAA AY163548.1 392–440 Soil 1 97 Exophiala opportunistica CBS 109811 TTGG ACGGTTTGGTGGAGACCCCCTTCGCGGGCTTCTACCCCT CCCAA JF747123.1 384–431 Drinking water 1 100 Exophiala pisciphila CBS 537.73 T TTGG ACGGTTTGGTGGAGGCCCCCTCGCGGGGCCACCGCCCCT CCCAA AF050272.1 390–437 Ictalurus punctatus 17 100 Exophiala salmonis CBS 157.67 T TTGG ACGGTTTGGTGGAGGCCCCTTGCGGGCATCTGCCCCT CCCAA AF050274.1 512–557 Salmo clarkii 4 100 Exophiala sideris CBS 121818 T TTGG ACGGTTTGGTCCAGGTCACCTGGACCCCT CCTAA HQ452311.1 391–428 Berry, Sorbus aucuparia 26 100 Exophiala spinifera CBS 899.68 T TTGG ACGGTTTGGTCCCGGGACCCCCCTGGACCCCT CCCAA AY156976.2 384–424 Man, nasal granuloma 37 100 Exophiala “siphonis” UTHSC88-471 TTGG ACGGTCTGGTCGAGCTGCTCGACCCCT CCCAA EF025401.1 391–426 Man 6 100 Exophiala xenobiotica 1 CBS 118157 T TTGG ACGGTTTGGTTTAGGTACCCCTAGACCCCT CCTAA DQ182587.1 383–421 Oil sludge 32 100 Exophiala xenobiotica 2 CBS 119306 TTGG ACGGTTTGGTTTAGGCACCCCTAGACCCCT CCTAA EF025409.1 377–415 Man 1 97 Exophiala xenobiotica 3 CBS 117665 TTGG ACGGTTTGGTTTAGGTACCCCTAGATCCCT CCTAA DQ182588.1 383–421 Man, tissue 7 99 Exophiala xenobiotica 4 CBS 117676 TTGG AGGTCTTGGTGTAGGGTTCCCCCCTCCACCCCT CCTAA DQ182592.1 388–429 Man, finger 1 93 Exophiala xenobiotica 5 CBS 117641 TTGG AGGTCTTGGTGTAGGGTCCCCCCTACACCCCT CCTAA DQ182591.1 388–428 Man, knee cyst 1 94 Fonsecaea erecta 1 dH 20513 TTGG ACGGTCCGGTGGAGAGTCATCCTTTCCACCCGT CCTAA 391–432 1 100 Fonsecaea erecta 2 dH 20502 TTGG ACGGTCCGGTGGAGAGTCATCCTTTTCACCCGT CCTAA 428–469 0 b 99 Fonsecaea minima dH 20511 TTGG ACGGTTCGGTGGAGAGTCACACCTCCCACCCGT CCTAA 396–437 2 100 Fonsecaea monophora 1 CBS 269.37 T TTGG ACGGCTTGGTGGAGTAAGTTCACACTTTCCACCCCT CCTAA AY857511.1 401–445 Man 20 100 Fonsecaea monophora 2 CBS 121732 TTGG ACGGCTTGGTGGAGCAAGTTCACACTTTCCACCCCT CCTAA EF513759.1 400–444 Man 20 99 Fonsecaea monophora 3 IFM 4889 TTGG ACGGCTTGGTGGAGTAAGTTCACGCTTTCCACCCCT CCTAA AB091204.2 423–467 Man 33 97 Fonsecaea monophora 4 IFM 54446 TTGG ACGGCTTGGTGGAGTAGGTTCACGCTTTCCACCCCT CCTAA AB251881.1 400–444 Cervical lymph nodes 1 97 Fonsecaea multimorphosa CBS 980.96 T TTGG ACGGCTCGGTGGACTCCTTCCACCCGT CCTAA JF267657.1 392–427 Cat 0 b 100 Fonsecaea nubica 1 CBS 269.64 T TTGG ACGGTTTGGTGGAGCGAGTTCACACTTTCCACCCCT CCTAA EU938592.1 400–444 Man, skin 6 100 Fonsecaea nubica 2 CBS 121733 TTGG ACGGCTTGGTGGAGCGAGGTCACACTTTCCACCCCT CCTAA EF513756.1 447–491 Man 2 98 Fonsecaea nubica 3 CBS 557.76 TTGG ACGGCTTGGTGGAGCGAGTTCACACTTTCCACCCCT CCTAA EU938593.1 399–443 52 99 Fonsecaea pedrosoi 1 CBS 271.37 T TTGG ACGGCTTGGTGGAGCGAGTTCACACTTTCCACCCCT CCTAA AB114127.1 423–467 Man 52 100 Fonsecaea pedrosoi 2 CBS 122741 TTGG ACGGCTTGGTGGAGCGAGTTCACACTTTCCACCCCCT CCTAA EU938589.1 400–445 Man, foot 1 98 Knufia epidermidis CBS 120353 T TTGG ACCCAAGTTTTTGGCTATTAAAAACTTGGT CCTAA EU730589.1 392–430 Man, foot 2 100 Phaeoannelomyces elegans CBS 110172 TTGG ACGGTTTGGTGGAGGCCCCCTCGGGGGCTCCTGCCCCT CCCAA EF551549.1 348–394 Railway tie 63 100 Phialophora americana CBS 840.69 TTGG ACGGATTTTGGTCGTGTAACAACGACCCCT CCTAA AF050283.1 387–425 Decaying timber 14 100 Phialophora europaea CBS 129.96 T TTGT AGCGGCCCCCCCGCTCTT CCCAA EF551553.1 386–412 Man, toe 12 100 Phialophora reptans CBS 113.85 T TTGG ACCCGGCGGGAGCTTCGCGCCCCCGCCCGT CTCAA EU514699.1 389–427 4 100 Phialophora verrucosa 1 CBS 273.37 TTGG ACGGATCTTGGTCGTGTGATCGCGACCCCT CCTAA AF050281.1 389–427 Man 2 100 Phialophora verrucosa 2 IMTSP.800 TTGG ACGGATCCTGGTCGTGTGATCGCGACCCCT CCTAA AF397135.1 410–448 1 99 Phialophora verrucosa 3 CBS 286.47 TTGG ACGGATTTTGGTCGTGTGATATCGACCCCT CCTAA AF050282.1 397–429 Fruit rachis, Musa sapientum 59 6 Phialophora verrucosa 4 CBS 839.68 TTGG ACGGATTTTGGTCGTGTGATACCGACCCCT CCTAA DQ404353.1 434–472 Kiwifruit elephantiasis 2 98 Rhinocladiella anceps 1 CBS 181.65 INT TTGG AAGGCCTGGTGTCCCCCCGGGGACACCCCT CCCAA EU041805.1 385–423 Soil under Thuja plicata 2 100 Rhinocladiella anceps 2 CBS 157.54 TTGG AAGGCCTGGTGTCTCC CCCCGGGGATACCCCT CCCAA EU041804.1 417–457 Fagus sylvatica ,stem 4 95 (Continued on following page)

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case of missing sequences, sequencing was performed. All sequences of the complete ITS regions of these strains have been deposited in GenBank. Fungi were cultured in 5 ml 2% Sabouraud’s glucose broth (Merck, Darmstadt, Germany) in cultivation tubes (Sarstedt, Nümbrecht, Ger-many) for at least 5 days at 28°C.

DNA extraction, PCR, sequencing.Tubes were centrifuged for 10

min at 3,180⫻gat 4°C. Aliquots of about 0.5 g were removed and washed

with 500␮l aqua dest. in 2-ml cups. One hundred microliters of lysis

buffer (50 mM Tris HCl [pH 7.2], 50 mM EDTA, 3% SDS) was added, and the biomass was crushed with a micropestle for 1 min. The cups were

incubated at 99°C for 2 min, 300␮l lysis buffer was added, and cups were

incubated at 80°C for an additional 10 min. After extraction with 400␮l

extraction mix (50:48:2 [percent volume]

phenol-chloroform-isoamylal-cohol) and 20 min of centrifugation at 20,000⫻g, DNA was precipitated

from the aqueous phase with 40␮l 3 M sodium acetate (pH 5.2) and 400

␮l isopropanol and separated by centrifugation with 20,000⫻gfor

an-other 20 min. The pellet was washed with 70% ethanol and dissolved in 50

␮l aqua dest. PCR was performed with 500 ng target DNA, ITS5 (5=-GG

AAGTAAAAGTCGTAACAAGG-3=) and NL-4 (5=-GGTCCGTGTTTCA

AGACGG-3=) as primers (Sigma, Taufkirchen, Germany), and

Taq-Poly-merase (Roche, Mannheim, Germany) using the following program: 94°C initially for 1 min, 55°C for 1 min, and 72°C for 3 min for 35 cycles, with a terminal elongation step at 72°C for 5 min (Labcycler, Sensoquest, Göt-tingen, Germany). Sequencing was done with 200 ng PCR product on an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Delaware) with ITS5 and NL-4 as sequencing primers.

BLAST searches and parameters.BLAST searches (BLASTN 2.2.25⫹) were performed at the NCBI website with the (i) 68 barcode identifier candidate sequences (range, 27 to 50 bp; strategy A), (ii) ITS2 sequence segments (range, 183 to 249 bp; strategy B), and (iii) complete ITS1, 5.8S, and ITS2 sequence segments (range, 477 to 595 bp; strategy C) using the GenBank database as of 31 October 2011. For all three search strategies, BLAST algorithm parameters were set as follows. General parameters were as follows: max target sequences, 100; short queries, automatically adjust parameters for short input sequences; expect threshold, 10; word size, 28. Scoring parameters were as follows: match/mismatch scores, 1,

⫺2; gap costs, linear. Filters and masking were as follows: filter, low

com-plexity regions; mask, mask for lookup table only. The search was re-stricted to fungi (taxid 4751). Revealed sequences with less than 100% query coverage were not taken into account.

RESULTS

Determination of the barcode candidates.ITS sequences (n⫽

68) were downloaded and processed using BioEdit (version 7.0.9.1) (9). The rDNA spacer ITS1 and ITS2 regions were delim-ited manually using a Web-based tool (11). Sequences were veri-fied for suitable regions meeting the requirements of a barcode fragment with low intraspecies variability and with a significant gap toward the neighboring species. This fragment is termed a “barcode identifier” since it contains only the signature sequence (hypervariable region) (10) of the barcode since the term “bar-code” is typically used in conjunction with an entire gene. It be-came evident that domain two (13), located in the ITS2 rDNA, was a promising candidate for this purpose. Within the Herpotrichiel-laceae, this region was 27 to 50 bp in length, had highly conserved flanking regions at the 5=and 3=ends (Table 1), and showed great variability in the central portion, even in case of closely related taxa. Therefore, this region was chosen for further analysis on the basis of assumed high interspecies and low intraspecies variability. To detect intraspecies variation within each of the 68 entities, a search with every species name as well as respective synonyms was performed in GenBank with restriction to internal transcribed spacer sequences. Resulting sequences covering at least 85% of the

TABLE

1

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Species

Strain

Conserved flanking region

Barcode

identifier

Conserved flanking region Accession no.

Position

Habitat

No.

of

100%

D2

hits

ITS

identity

with

ex-type

(%)

Rhinocladiella

aquaspersa

CBS

313.73

T

TTGG

ACGGTCGGCCTC

TTCACCGAGGCCCCT

CCCAA

GU017733.1

388–433

Man

4

100

Rhinocladiella

atrovirens

CBS

264.49

AUT

TTGG

ACGGCTGGTCGGGCGACGCCCGACCCCT

CCTAA

EU041812.1

396–432

Honey

6

100

Rhinocladiella

basitona

CBS

101460

T

TTGG

ACGGTTTGGUCTAGGGCCTCCCTAGACCCCT

CCCAA

AY163561.1

384–423

Man,

subcutaneous

lesion

2

100

Rhinocladiella

mackenziei

1

CBS

650.93

T

TTGG

ACGGCCTGGGTTTGGAAGTTTTCTCTCGAGGCCCCCT

CCCAA

AY857540.1

373–418

Man,

brain

1

100

Rhinocladiella

mackenziei

2

CBS

367.92

TTGG

ACGGCCTGGGTTTGGAAGTTTTCTCTCGAGCCCCT

CCCAA

EU041808.1

393–437

Man,

brain

6

99

Rhinocladiella

mackenziei

3

CBS

102590

TTGG

ACGGCCTGGGTTTGGAAGTTTTCTCTCAAGCCCCT

CCCAA

EU041810.1

380–424

Man,

brain

1

99

Rhinocladiella

similis

1

CBS

111763

T

TTGG

ACGGTTTGGTCCAGGGCCCCCCCTGGACCCCT

CCCAA

EF551461.1

343–383

Man,

chronic

cutaneous

ulcer

16

100

Rhinocladiella

similis

2

dH

13054

TTGG

ACGGTTTGGTCCAGGGTCCCCCCTGGACCCCT

CCCAA

AY857529.1

386–426

Water

1

99

Veronaea

botryosa

1

CBS

254.57

T

TTGG

ACGGTTTGGTGGAGGCCCCCTCGTCGGGGTCGCTGCCCCT

CCCAA

EU041816.1

394–442

Sansa

olive

slag

2

100

Veronaea

botryosa

2

CBS

350.65

TTGG

ACGGTTTGGTGGAGGCCCCCCTCGTCGGGGTCGCTGCCCCT

CCCAA

EU041817.1

408–457

Dung

of

goat

7

99

Veronaea

compacta

CBS

268.75

T

TTGG

ACGGTTTGGCGGAGGCCCCTCGCGGGGCTCTCGCCCCT

CCCAA

EU041819.1

407–453

1

100

Veronaea

japonica

CBS

776.83

T

TTGG

ACGGTTTGGCGGAGGCCCCCTCGGGGGCTCTCGCCCCT

CCCAA

EU041818.1

419–465

Dead

bamboo

culm

7

100

aDeposited

sequence

belongs

to

strain

CBS

110628.

bSequence

not

deposited

at

the

time

of

analysis.

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length of the respective ex-type strain’s entire ITS region were added to the initial sequence alignment. Deviating barcodes with the same species denotation (i.e., proven to be conspecific in the literature by genealogical concordance analysis) and exhibiting

ⱖ93% sequence identity (Table 1) in the remaining ITS region of the specific ex-type strain were treated as sequevars of the respec-tive species. The original ex-type or authentic strain’s name was then denoted with “1,” and all following sequevars were indicated by increasing numbers according to decreasing sequence similar-ity of the identifier region (e.g.,E. dermatitidis,Table 1). For 22 of the original 68 barcode identifiers, 38 tentative sequevars (2 to 5 per species) were revealed, resulting in a total of 106 barcode iden-tifier candidates (Table 1). A flow diagram of the procedure of barcode identifier selection is given inFig. 1.

Screen for uniqueness of the barcode identifiers.An NCBI

BLAST search with the barcode identifier candidates (n⫽106) of 27 to 50 bp in length (search strategy A) was performed using the parameters described above. Only hits with 100% query coverage were considered for further analysis. In 44 cases, the barcode identifier was found to be unique, providing only a single species denotation. In 28 cases, 100% hits with insuffi-ciently identified source organisms (e.g., “Exophiala unde-scribed species” or “uncultured ascomycete”) were obtained. Since all these exhibitedⱖ97% sequence identity of the entire ITS region with the ex-type isolate of the respective black yeast species, they were assigned to the species name of the particular barcode identifier. In two cases, viz. Fonsecaea erecta 1 and Fonsecaea multimorphosa, no match could be obtained because FIG 1Determination and analysis of the barcode identifiers.

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ITS sequences of these novel species had not been deposited in GenBank at the time of analysis.

In 29 cases, 100% hits were obtained with source organisms other than the species defined by the ex-type isolate. This was reevaluated by a thorough search of recent literature with eventual taxonomic novelties. All but three conflicts could be traced back to obsolete or simply erroneous species denotations of the respective GenBank entries, which had not been updated or corrected. In those cases, respective sequences were reassigned to the particular species or sequevars. The three remaining conflicting sequences with different species sharing the same barcode identifier were compared with the entire ITS domain. Of note,Cyphellophora laciniataandCyphellophora vermisporacould be distinguished by 1.3% ITS diversity,Fonsecaea pedrosoisequevar 1 andFonsecaea nubicasequevar 3 by 2.6%, andExophiala equinasequevar 1 and Phaeoannellomyces elegansby 13.0%, with differences occurring mainly in the ITS1 region.Figure 1illustrates the described ap-proach which led to 100 unique barcode identifiers and 3 barcode identifiers shared by two species or sequevars each (n⫽106 initial barcode identifier candidates).

Performance of BLAST search strategies. Three BLAST

searches were performed with the 103 barcode identifiers (strategy A), the ITS2 (strategy B), and the near-complete ITS domain (strategy C) using GenBank with the settings as described above. The first and second best hits were documented (data not shown). BLAST searches A, B, and C were compared for resulting 100% sequence matches. When the barcode identifiers (strategy A) were used, about four times more 100% hits were found than with the entire ITS domain (strategy C), underlining the diagnostic strength of the use of barcode identifiers. With increasing length of the fragment used in a BLAST search, the total number of 100% hits with concordant species names decreased, as well as the total number of 100% hits with ambiguous or inconsistent species names (A¡B¡C). Detailed values are given inTable 2. This phe-nomenon is due to accumulating sequence errors in longer se-quences occurring mainly in homopolymeric regions due to for-mation of stutter products in the PCR (4) (Table 3).

DISCUSSION

[image:6.585.334.512.52.730.2]

Black yeast fungi and their filamentous relatives in the Herpo-trichiellaceaecomprise harmless environmental species, some of which are closely related to severely pathogenic biosafety level 3 organisms (1). Pathogens and saprobes cannot be discriminated by conventional methods using micromorphology or physiology. A genetic approach using the ITS domain deposited in public databases for sequence analysis is regarded as the gold standard for routine identification, even though this may be hampered by poor quality of databases in terms of sequence quality, sequence length, TABLE 2Number of 100% sequence matches resulting from three different BLAST search strategies

BLAST search

strategy Segment blasted

Total no. of 100% matches

Total no. of 100% hits with concordant species name

Total no. of 100% hits with ambiguous or inconsistent species name

A Barcode identifier 944 496 448

B ITS2 410 243 167

C ITS1, 5.8S, ITS2 231 140 91

TABLE

3

Examples

of

homoploymeric

regions

in

the

domain

4

joining

fragment

and

the

resulting

sequence

divergence

referring

to

the

respective

ex-type

strain

of

three

Exophiala

species

Species

Strain

Accession

no.

Barcode

identifier

Domain

4

joining

region

a

%

difference

from

ITS2

E.

dermatitidis

CBS

207.35

T

FJ974060.1

TTGGACGGTCTGGTCGAGCGTTTCCGCGCGACCCCTCCCAA

TTGCT

CCCC

TGC

GGG

AGTGGGAGAGAA

CCCCCCTTTTT

-ATCAAGGTTGACC

0

E.

dermatitidis

CBS

149.90

AF050268.1

TTGCT

CCCC

TGC

GGG

AGTGGGAGAGA-CCCCCCTTTTT

-ATCAAGGTTGACC

1

E.

dermatitidis

IFM

4960

AF147492.1

TTGCT

CCC

-TGC

GG

-AGTGGGAGAGAA

CCCCC

-TTTTT

-ATCAAGGTTGACC

2

E.

dermatitidis

IFM

4826

AF147495.1

TTGGACGGTATGGTCGAGCGTTTCCGCGCGACCTCCCAA

TTGCT

CCC

-TGC

G

--AGTGGGAGAGAA

CCCC

--TTTTT

-ATCAAGGTTGACC

4

E.

dermatitidis

IFM

41479

AB087205.1

TTGCT

CCC

-TGC

G

--AGTGGGAGAAAA

CCCC

--TTTTT

-ATCAAGGTTGACC

5

E.

dermatitidis

IFM

4962

AF147491.1

TTGCT

CC

--TGC

G

--AGTGG-AGAGAA

CCCC

--TTT

---AT-AAGGTTGACC

8

E.

oligosperma

CBS

725.88

T

AY163551.1

TTGGACGGTCTGGTCCGGGGACCTCAAACCCCCTGGACCCCTCCCAA

ACCTATA

TTTTT

-CAAGG

TT

GACC

0

E.

oligosperma

dH

13364

ACCTATA

TTT

---CAAGG

T

-GACC

1

E.

oligosperma

dH

13367

ACCTATA

TTAT

--CAAGG

T

-GACC

1

E.

oligosperma

dH

13757

ACC--TA

TTTTT

-CAAGG

TT

GACC

2

E.

oligosperma

IFM

5386

ACCTATA

TTTTTT

CAAGG

TT

GACC

3

E.

oligosperma

dH

13446

TTGGACGGTTTGGTTCGGGGACCTCAATCCCCCTGGACCCCTCCCAA

ACCTGTA

TTTT

--CAAGGT-GACC

4

E.

jeanselmei

CBS

507.90

T

AY156963.1

TTGGACGGTTTGGTCTCGGGTCCGACCCCCCTTGACCCCTCCCAA

ATATC

TTTT

---A--CAAGGTTGACC

0

E.

jeanselmei

dH

13641

ATATC

TTTT

---AG-CAAGGT-GACC

1

E.

jeanselmei

CBS

116.86

AY163556.1

ATATC

TTTTTTT

A--CAAGGTTGACC

3

E.

jeanselmei

dH

13746

ATATC

TTTTTTT

A--CAAGGT-GACC

3

E.

jeanselmei

dH

13638

ATATC

TTTTTTT

AG-CAAGGT-GACC

4

aBolding

represents

variable

homopolymeric

regions.

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and taxonomic or other updates. Nilsson et al. (16) found that more than a quarter of the ITS sequences in general databases had been incorrectly annotated; 2.5% of all sequences had more than 1% IUPAC ambiguities, 82% lacked an explicit reference to a voucher specimen, and 99.2% of all fungal sequences had never been updated after first submission. The databases are not and were not intended to be appropriate for the identification of spe-cies. Smaller, dedicated, and validated databases are increasingly implemented (e.g.,Fusarium, http://www.cbs.knaw.nl/fusarium

/BioloMICS.aspx;Trichoderma,http://www.isth.info/), but these

as yet cover only a fraction of the fungal kingdom. Furthermore, the ITS2 region of theHerpotrichiellaceaecomprises homopoly-meric regions, particularly poly(T), which may result in artificial sequence variance of up to 8% of base positions (Table 3). The barcode identifier approach eliminates these uncertainties effec-tively (Table 2).

To overcome the obstacles described above, we here present an identification approach based on barcode identifiers validated by comparison with ex-type strains. A 27- to 50-bp fragment located in the ITS2 is selected. We have shown that 100 out of 106 species or sequevars could be distinguished unequivocally using the pro-posed 103 barcode identifiers. Three barcode identifiers were shared by the remaining six taxonomic entities with two entities each. Of note, they could be easily distinguished by sequence dif-ferences in the ITS1 region. The results of species diversities in the barcode identifier region are given inTable 1and are open-access available to professional users in diagnostic microbiology labs

(www.blackyeast.org). This highly practicable tool for

identifica-tion ofHerpotrichiellaceaeis an important step toward rapid on-line identification of black yeasts and relatives, which always have been regarded as a diagnostic nightmare (3).

The ITS2 region in fungi is regarded as relatively stable as shown by Krüger & Gargas (12). Domain 2 of the ITS2 region turned out as a promising candidate for barcode identifiers because it provides highly conserved flanking sequences with a highly variable section in between, which varies markedly in both length and nucleotide composition even between closely related taxonomic entities (Table 1). The sequevars outlined in this study in part may have a tentative character, because stud-ies on specstud-ies delimitation are ongoing. It is recommended to take the barcode identifier into account with the description of new black yeast species, in analogy to dermatophyte taxonomy and molecular identification (8). Of note, such identification approaches are artificial but straightforward and hence highly practical. The availability of an online, continuously updated platform with validated barcodes will contribute to stability in taxonomy and diagnostics of black yeasts and relatives. A pre-liminary analysis of domain 2 of the ITS2 region ofAspergillus, Candida, Cladosporium, Fusarium, and Trichophyton species revealed that the interspecies variance is too low to be used as a reliable barcode identifier (data not shown). Here, identifica-tion is achievable with other domains of the ITS region (17), ␤-tubulin, translation elongation factor 1-␣, and other genes. For this reason, the presented barcode identifier approach is restricted so far to members of the orderChaetothyriales. Main human pathogens are located in the familyHerpotrichiellaceae, but the recent reclassification ofConiosporium epidermidisto Knufia epidermidis(18), a genus belonging to the Chaetothyri-aceae, indicates that the proposed barcode identifier tool is also applicable to the familyChaetothyriaceae.

We here introduce a highly practicable tool for species assign-ment of black yeast isolates in a routine diagnostic laboratory to overcome problems associated by using the (nearly) entire ITS rDNA for identification. To achieve reliable species identification, only ITS2 region sequences or even shorter fragments comprising the barcode identifier segment are required. Thereby, the short length of the barcode, here introduced as the barcode identifier (27 to 50 bp), enables the usage of straightforward pyrosequenc-ing techniques (2,17). Furthermore, metagenomic pyrosequenc-ing approaches like tag-encoded FLX amplicon pyrosequencpyrosequenc-ing (TEFAP) (7), where the quality of identification benefits from short barcode identifiers, becomes feasible. Rapid and cost-effec-tive identification and analysis ofHerpotrichiellaceaepresent in biofilms can be achieved (unpublished results). Resulting short sequences can easily be matched with the barcode identifiers pro-vided inTable 1. A searchable curated online database will be implemented.

In conclusion, we present an easy-to-use, reliable, highly prac-ticable, and cost-effective approach for rapid molecular identifi-cation of clinically importantChaetothyriales(5).

ACKNOWLEDGMENT

G. Heinrichs was supported by a research grant from Rheinenergie AG.

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