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Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 2015 Aug 18;53(9):2990–3000. doi: 10.1128/JCM.01482-15

Cladosporium Species Recovered from Clinical Samples in the United States

Marcelo Sandoval-Denis a, Deanna A Sutton b, Adela Martin-Vicente a, José F Cano-Lira a, Nathan Wiederhold b, Josep Guarro a, Josepa Gené a,✉
Editor: D W Warnock
PMCID: PMC4540897  PMID: 26179305

Abstract

Cladosporium species are ubiquitous, saprobic, dematiaceous fungi, only infrequently associated with human and animal opportunistic infections. We have studied a large set of Cladosporium isolates recovered from clinical samples in the United States to ascertain the predominant species there in light of recent taxonomic changes in this genus and to determine whether some could possibly be rare potential pathogens. A total of 92 isolates were identified using phenotypic and molecular methods, which included sequence analysis of the internal transcribed spacer (ITS) region and a fragment of the large subunit (LSU) of the nuclear ribosomal DNA (rDNA), as well as fragments of the translation elongation factor 1 alpha (EF-1α) and actin (Act) genes. The most frequent species was Cladosporium halotolerans (14.8%), followed by C. tenuissimum (10.2%), C. subuliforme (5.7%), and C. pseudocladosporioides (4.5%). However, 39.8% of the isolates did not correspond to any known species and were deemed to comprise at least 17 new lineages for Cladosporium. The most frequent anatomic site of isolation was the respiratory tract (54.5%), followed by superficial (28.4%) and deep tissues and fluids (14.7%). Species of the two recently described cladosporiumlike genera Toxicocladosporium and Penidiella are reported for the first time from clinical samples. In vitro susceptibility testing of 92 isolates against nine antifungal drugs showed a variety of results but high activity overall for the azoles, echinocandins, and terbinafine.

INTRODUCTION

Cladosporium species are among the most common fungal inhabitants worldwide, being isolated from almost any environmental source and geographic location (1). The genus is characterized by the typical form of its conidiophores, which are erect, straight or geniculate, produce abundant branched acropetal chains of smooth to roughened dry conidia, and show a distinct darkened coronate hilum, i.e., conidial scar characterized by a thick rim surrounding a central convex dome (2, 3). The relatively small conidia are easily detached and disseminated by the wind, Cladosporium being one of the most frequently isolated airborne fungi (2, 4).

The most common Cladosporium species are primarily isolated from soil and plant material, where they are frequently encountered as saprobes or secondary invaders on follicular lesions, concomitant with other plant-pathogenic fungi (1, 5, 6). However, several species are important pathogens of plants and some are also able to affect animals, including humans (7–9). Cladosporium is usually associated with allergic rhinitis (10) or localized superficial or deep lesions (11–14) but, rarely, can cause disseminated infections (7, 15–17).

The genus Cladosporium has been shown to be both morphologically and phylogenetically heterogeneous (18). On the basis of molecular data, the true human-pathogenic species C. bantiana, C. carrionii, and C. devriesii, characterized by their thermotolerance and the absence of conidiophores with pigmented conidial scars, were transferred to Cladophialophora (1, 7, 18). More recently, Cladosporium underwent extensive revisions based on polyphasic approaches (1, 3, 19–21), which resulted in the delimitation of 169 species currently accepted in Cladosporium sensu stricto (Cladosporiaceae, Capnodiales). On the other hand, a great number of taxa were excluded from that genus, now being considered doubtful species or accommodated into several related new genera, such as Hyalodendriella, Ochrocladosporium, Rachicladosporium, Rhizocladosporium, Toxicocladosporium, and Verrucocladosporium (1, 3).

The diversity of Cladosporium species associated with human disease is currently reduced to four, i.e., C. cladosporioides, C. herbarum, C. oxysporum, and C. sphaerospermum (7). Most of these data, however, are based on a reduced number of clinical cases with the identification of the etiological agents not confirmed by reliable methods. Moreover, three of the clinically relevant species, C. cladosporioides, C. herbarum, and C. sphaerospermum, have been demonstrated to be species complexes (19–21) encompassing several morphologically sibling species that can only be distinguished by means of phylogenetic analyses (1, 7). The clinical significance of these phylogenetic species, however, has yet to be evaluated (22).

The objective of this work was to assess the diversity of Cladosporium species associated with human and animal disease by analyzing a large set of isolates from clinical specimens by means of phenotypic and DNA sequence data analyses. In addition, the in vitro susceptibility of these isolates was evaluated against nine clinically available antifungal drugs.

MATERIALS AND METHODS

Fungal isolates.

A total of 92 isolates tentatively identified as Cladosporium spp. were included in this study (Table 1). All of the isolates were obtained from human and animal clinical specimens, mostly from the United States, received in the Fungus Testing Laboratory at the University of Texas Health Science Center at San Antonio (UTHSC) from different parts of the country mainly for identification purposes.

TABLE 1.

Clinical isolates, type or reference strains, and sequences included in this study

Species Strain/isolate no.a Origin (country)b GenBank nucleotide accession no. for:
ITS LSU EF-1α Act
Cercospora beticola CBS 116456T Beta vulgaris (Italy) NR121315 GU214404 AY840494 AY840458
Cercospora olivascens CBS 253.67T Unknown NR111773
Cladosporium allicinum CBS 121624T Hordeum vulgare (Belgium) EF679350 EF679425 EF679502
UTHSC DI-13-173 Human, lung (USA) LN834353 LN834449 LN834537
UTHSC DI-13-176 Human, skin (USA) LN834354 LN834450 LN834538
UTHSC DI-13-266 Canine, skin (USA) LN834355 LN834451 LN834539
Cladosporium angustisporum CBS 125983T Alloxylon wickhamii (Australia) HM147995 HM148236 HM148482
UTHSC DI-13-240 Human, toe nail (USA) LN834356 LN834452 LN834540
Cladosporium asperulatum CBS 126339 Eucalyptus leaf litter (India) HM147997 HM148238 HM148484
CBS 126340T Protea susannae (Portugal) HM147998 HM148239 HM148485
UTHSC DI-13-216 Feline, nasal (USA) LN834357 LN834453 LN834541
Cladosporium cladosporioides CBS 101367 Soil (Brasil) HM148002 HM148243 HM148489
CBS 112388T Indoor air (Germany) HM148003 HM148244 HM148490
UTHSC DI-13-204 Human, abdomen (USA) LN834358 LN834454 LN834542
UTHSC DI-13-209 Human, pleural (USA) LN834359 LN834455 LN834543
UTHSC DI-13-215 Human, sputum (USA) LN834360 LN834456 LN834544
Cladosporium colocasiae CBS 386.64T Colocasia antiquorum (Taiwan) HM148067 HM148310 HM148555
CBS 119542 Colocasia esculanta (Taiwan) HM148066 HM148309 HM148554
Cladosporium cucumerinum CBS 171.52T Fruit of Cucumis sativus (Netherlands) HM148072 HM148316 HM148561
CBS 173.54 Fruit of Cucumis sativus (Netherlands) HM148074 HM148318 HM148563
Cladosporium flabelliforme CBS 126345T Melaleuca cajuputi (Australia) HM148092 HM148336 HM148581
UTHSC DI-13-267 Human, sputum (USA) LN834361 LN834457 LN834545
Cladosporium funiculosum CBS 122128 Unknown HM148093 HM148337 HM148582
CBS 122129T Leaf of Vigna umbellata (Japan) HM148094 HM148338 HM148583
UTHSC DI-13-175 Human, BAL fluid (USA) LN834362 LN834458 LN834546
UTHSC DI-13-223 Human, BAL fluid (USA) LN834363 LN834459 LN834547
UTHSC DI-13-242 Human, nasal wash (USA) LN834364 LN834460 LN834548
Cladosporium halotolerans CBS 119416T Hypersaline water (Namibia) DQ780364 JN906989 EF101397
FMR 13493 Human, unknown (Spain) LN834365 LN834461 LN834549
UTHSC DI-13-164 Human, bone marrow (USA) LN834366 LN834462 LN834550
UTHSC DI-13-182 Marine mammal, dermis (USA) LN834367 LN834463 LN834551
UTHSC DI-13-183 Human, bronchus (USA) LN834368 LN834464 LN834552
UTHSC DI-13-206 Human, BAL fluid (USA) LN834369 LN834465 LN834553
UTHSC DI-13-213 Human, lymph node (USA) LN834370 LN834466 LN834554
UTHSC DI-13-221 Human, bone marrow (USA) LN834371 LN834467 LN834555
UTHSC DI-13-231 Catheter tip (USA) LN834372 LN834468 LN834556
UTHSC DI-13-249 Human, nasal (USA) LN834373 LN834469 LN834557
UTHSC DI-13-250 Human, scalp (USA) LN834374 LN834470 LN834558
UTHSC DI-13-252 Human, toe nail (USA) LN834375 LN834471 LN834559
UTHSC DI-13-259 Human, BAL fluid (USA) LN834376 LN834472 LN834560
UTHSC DI-13-263 Human, BAL fluid (USA) LN834377 LN834473 LN834561
Cladosporium herbaroides CBS 121626T Hypersaline water (Israel) EF679357 EF679432 EF679509
Cladosporium herbarum CBS 121621T Hordeum vulgare (Netherlands) EF679363 EF679440 EF679516
UTHSC DI-13-220 Human, BAL fluid (USA) LN834378 LN834474 LN834562
Cladosporium iranicum CBS 126346T Leaf of Citrus sinensis (Iran) HM148110 HM148354 HM148599
Cladosporium iridis CBS 138.40T Leaf of Iris sp. (Netherlands) EF679370 EF679447 EF679523
Cladosporium macrocarpum CBS 121623T Spinacia oleracea (USA) EF679375 EF679453 EF679529
UTHSC DI-13-191 Human, face (USA) LN834379 LN834475 LN834563
Cladosporium oxysporum CBS 125991 Soil (China) HM148118 HM148362 HM148607
CBS 126351 Indoor air (Venezuela) HM148119 HM148363 HM148608
Cladosporium perangustum CBS 125996T Cussonia sp. (South Africa) HM148121 HM148365 HM148610
UTHSC DI-13-208 Canine, BAL fluid (USA) LN834380 LN834476 LN834564
Cladosporium pseudocladosporioides CBS 117153 Leaf of Paeonia sp. (Germany) HM148157 HM148401 HM148646
CBS 125993T Outside air (Netherlands) HM148158 HM148402 HM148647
UTHSC DI-13-187 Turtle, unknown (USA) LN834381 LN834477 LN834565
UTHSC DI-13-232 Human, shoulder (USA) LN834382 LN834478 LN834566
UTHSC DI-13-233 Human, BAL fluid (USA) LN834383 LN834479 LN834567
UTHSC DI-13-261 Human, sputum (USA) LN834384 LN834480 LN834568
Cladosporium ramotenellum CBS 121628T Hypersaline water (Slovenia) EF679384 EF679462 EF679538
UTHSC DI-13-166 Human, nasal tissue (USA) LN834385 LN834481 LN834569
UTHSC DI-13-222 Animal, Nasal (USA) LN834386 LN834482 LN834570
UTHSC DI-13-224 Animal, Nasal (USA) LN834387 LN834483 LN834571
Cladosporium sinuosum CBS 121629T Fuchsia excorticata (New Zealand) EF679386 EF679464 EF679540
Cladosporium sphaerospermum CBS 193.54T Human, nail (Netherlands) DQ780343 EU570261 EU570269
UTHSC DI-13-184 Frog, abscess (USA) LN834388 LN834484 LN834572
UTHSC DI-13-229 Human, BAL fluid (USA) LN834389 LN834485 LN834573
UTHSC DI-13-237 Human, BAL fluid (USA) LN834390 LN834486 LN834574
Cladosporium subinflatum CBS 121630T Hypersaline water (Slovenia) EF679389 EF679467 EF679543
UTHSC DI-13-189 Human, toe nail (USA) LN834391 LN834487 LN834575
Cladosporium subtilissimum CBS 113754T Grape berry (USA) EF679397 EF679475 EF679551
Cladosporium subuliforme CBS 126500T Chamaedorea metallica (Thailand) HM148196 HM148441 HM148686
UTHSC DI-13-171 Human, CSF (USA) LN834392 LN834488 LN834576
UTHSC DI-13-180 Human, BAL fluid (USA) LN834393 LN834489 LN834577
UTHSC DI-13-214 Human, BAL fluid (USA) LN834394 LN834490 LN834578
UTHSC DI-13-254 Human, BAL fluid (USA) LN834395 LN834491 LN834579
UTHSC DI-13-255 Human, toe nail (USA) LN834396 LN834492 LN834580
Cladosporium tenuissimum CBS 125995T Fruits of Lagerstroemia sp. (USA) HM148197 HM148442 HM148687
UTHSC DI-13-174 Marine mammal, lung (USA) LN834397 LN834493 LN834581
UTHSC DI-13-177 Human, skin (USA) LN834398 LN834494 LN834582
UTHSC DI-13-188 Human, BAL fluid (USA) LN834399 LN834495 LN834583
UTHSC DI-13-205 Human, BAL fluid (USA) LN834400 LN834496 LN834584
UTHSC DI-13-236 Human, nasal (USA) LN834401 LN834497 LN834585
UTHSC DI-13-239 Human, sputum (USA) LN834402 LN834498 LN834586
UTHSC DI-13-253 Human, BAL fluid (USA) LN834403 LN834499 LN834587
UTHSC DI-13-258 Human, thorancentesis fluid (USA) LN834404 LN834500 LN834588
UTHSC DI-13-274 Human, toe (USA) LN834405 LN834501 LN834589
Cladosporium sp. UTHSC DI-13-165 Human, arm drainage (USA) LN834406 LN834502 LN834590
UTHSC DI-13-168 Human, BAL fluid (USA) LN834407 LN834503 LN834591
UTHSC DI-13-169 Human, BAL fluid (USA) LN834408 LN834504 LN834592
UTHSC DI-13-170 Human, toe nail (USA) LN834409 LN834505 LN834593
UTHSC DI-13-178 Animal, abscess (USA) LN834410 LN834506 LN834594
UTHSC DI-13-179 Human, hand (USA) LN834411 LN834507 LN834595
UTHSC DI-13-190 Human, CSF (USA) LN834412 LN834508 LN834596
UTHSC DI-13-207 Human, CSF (USA) LN834413 LN834509 LN834597
UTHSC DI-13-210 Human, skin (USA) LN834414 LN834510 LN834598
UTHSC DI-13-211 Human, BAL fluid (USA) LN834415 LN834511 LN834599
UTHSC DI-13-212 Human, ethmoid sinus (USA) LN834416 LN834512 LN834600
UTHSC DI-13-217 Human, nasal (USA) LN834417 LN834513 LN834601
UTHSC DI-13-218 Human, BAL fluid (USA) LN834418 LN834514 LN834602
UTHSC DI-13-219 Human, foot (USA) LN834419 LN834515 LN834603
UTHSC DI-13-225 Animal, BAL fluid (USA) LN834420 LN834516 LN834604
UTHSC DI-13-226 Human, BAL fluid (USA) LN834421 LN834517 LN834605
UTHSC DI-13-227 Human, sputum (USA) LN834422 LN834518 LN834606
UTHSC DI-13-228 Human, foot skin (USA) LN834423 LN834519 LN834607
UTHSC DI-13-234 Human, sputum (USA) LN834424 LN834520 LN834608
UTHSC DI-13-235 Human, BAL fluid (USA) LN834425 LN834521 LN834609
UTHSC DI-13-238 Human, leg (USA) LN834426 LN834522 LN834610
UTHSC DI-13-241 Human, foot (USA) LN834427 LN834523 LN834611
UTHSC DI-13-244 Human, BAL fluid (USA) LN834428 LN834524 LN834612
UTHSC DI-13-245 Human, toe (USA) LN834429 LN834525 LN834613
UTHSC DI-13-246 Human, BAL fluid (USA) LN834430 LN834526 LN834614
UTHSC DI-13-247 Human, BAL fluid (USA) LN834431 LN834527 LN834615
UTHSC DI-13-251 Human, BAL fluid (USA) LN834432 LN834528 LN834616
UTHSC DI-13-257 Human, sputum (USA) LN834433 LN834529 LN834617
UTHSC DI-13-262 Dolphin, bronchus (USA) LN834434 LN834530 LN834618
UTHSC DI-13-265 Human, BAL fluid (USA) LN834435 LN834531 LN834619
UTHSC DI-13-268 Human, toe nail (USA) LN834436 LN834532 LN834620
UTHSC DI-13-269 Human, BAL fluid (USA) LN834437 LN834533 LN834621
UTHSC DI-13-270 Human, nail (USA) LN834438 LN834534 LN834622
UTHSC DI-13-271 Human, BAL fluid (USA) LN834439 LN834535 LN834623
UTHSC DI-13-273 Human, toe nails (USA) LN834440 LN834536 LN834624
Cladosporium variabile CBS 121636T Spinacia oleracea (USA) EF679402 EF679480 EF679556
Penidiella sp. UTHSC DI-13-256 Human, nail (USA) LN834441 LN834445
Toxicocladosporium banksiae CBS 128215T Leaf of Banksia emulata (Australia) HQ599598 HQ599599
Toxicocladosporium chlamydosporum CBS 124157T Leaf of Eucalyptus camaldulensis (Madagascar) FJ790283 FJ790301
Toxicocladosporium ficiniae CBS 136406T Leaf of Ficinia sp. (South Africa) KF777190 KF777241
Toxicocladosporium irritans CBS 185.58T Moldy paint (Suriname) EU040243 EU040243
UTHSC DI-13-181 Human, blood (USA) LN834442 LN834446
UTHSC DI-13-230 Human, finger nail (USA) LN834443 LN834447
Toxicocladosporium pini CBS 138005T Needles of Pinus sp. (China) KJ869160 KJ869217
Toxicocladosporium posoqueriae CBS 133583T Leaf of Posoqueria latifolia (Australia) NR121555 KC005803
Toxicocladosporium protearum CBS 126499T Leaf of Protea burchellii (South Africa) HQ599586 HQ599587
Toxicocladosporium pseudoveloxum CBS 128775T Leaf of Phaenocoma prolifera (South Africa) JF499847 JF499867
Toxicocladosporium rubrigenum CBS 124158T Leaf of Eucalyptus camaldulensis (Madagascar) FJ790287 FJ790305
Toxicocladosporium sp. UTHSC DI-13-172 Human, BAL fluid (USA) LN834444 LN834448
Toxicocladosporium strelitziae CBS 132535T Leaf of Strelitzia reginae (South Africa) NR111765 JX069858
Toxicocladosporium veloxum CBS 124159T Leaf of Eucalyptus camaldunensis (Madagascar) FJ790288 FJ790306
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a

CBS, CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands; FMR, Facultat de Medicina, Universitat Rovira i Virgili, Reus, Spain; UTHSC, Fungus Testing Laboratory at the University of Texas Health Science Center, San Antonio, TX, USA; T, ex-type strain.

b

BAL fluid, bronchoalveolar lavage fluid specimen; CSF, cerebrospinal fluid.

Phenotypic identification.

The isolates were morphologically characterized following the procedures outlined in Bensch et al. (1), Crous et al. (23), Schubert et al. (19), and Zalar et al. (20). Briefly, all of the isolates were grown on synthetic nutrient-poor agar (SNA) (1 g KH2PO4, 1 g KNO3, 0.5 g MgSO4 · 7H2O, 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose, 1 liter water) and potato dextrose agar (PDA) (Pronadisa, Spain) for 7 days at 25°C. Microscopic observations were made from cultures on SNA mounted in Shear's solution (23). Colony characteristics were recorded from cultures on SNA and PDA. For the estimation of cardinal growth temperatures, the isolates were grown on PDA agar for 14 days at temperatures ranging from 15°C to 35°C at intervals of 5°C, as well as at 32°C and 37°C.

DNA extraction, amplification, and sequencing.

Total genomic DNA was extracted from mycelia obtained from colonies growing on PDA, using FastPrep (MP Biomedicals, Santa Ana, CA) according to the manufacturer's protocol. DNA was quantified using the NanoDrop 3000 (Thermo Scientific, Madrid, Spain).

The primers ITS5 and ITS4 (24) were used to amplify a region spanning internal transcribed spacer 1 (ITS1) and ITS2 and the 5.8S gene of the ribosomal DNA (rDNA); the primer pair LR0R/LR5 (25, 26) was used to amplify a fragment of the large subunit (LSU) gene of the rDNA; and the EF-728F/EF-986R and ACT-512F/ACT-783R primer pairs (27) were used for the translation elongation factor 1α gene (EF-1α) and the actin gene (Act), respectively.

Sequencing was performed in both directions using the same PCR primers at Macrogen Europe (Macrogen, Inc., Amsterdam, the Netherlands). Consensus sequences were obtained using SeqMan version 7.0.0 (DNAStar Lasergene, Madison, WI).

Molecular identification and phylogenetic analyses.

An initial presumptive generic identification of the isolates was performed based on BLAST searches of ITS and LSU sequences in the GenBank (http://www.ncbi.nlm.nih.gov/) and CBS (http://www.cbs.knaw.nl/) databases. Multiple sequence alignments of each locus were performed in MEGA version 6 (28) using the ClustalW application (29), refined with MUSCLE (30), and manually adjusted if necessary. Phylogenetic reconstructions were made using maximum-likelihood (ML) and Bayesian Inference (BI) under MEGA version 6 and MrBayes version 3.1.2 (31), respectively. The best nucleotide substitution model (generalized time-reversible model with gamma distribution and a portion of invariable sites [GTR+G+I] for the three independent data sets) was estimated using MrModelTest version 2.3 (32) following the Akaike criterion. Phylogenetic analyses using ML were at first made individually for each locus and compared in order to assess for any incongruent results between nodes with high statistical support. As no incongruences were observed, the four loci were combined as follows: ITS, EF-1α, and Act for members of Cladosporium sensu stricto and ITS combined with LSU for members of other cladosporiumlike genera. For the ML analysis, nearest-neighbor interchange (NNI) was used as the heuristic method for tree inference. Support for the internal branches was assessed by a search of 1,000 bootstrapped sets of data. A bootstrap support value of ≥70 was considered significant. For BI analysis, two simultaneous runs of 10,000,000 generations were performed and samples were stored every 1,000 generations. The 50% majority-rule consensus tree and posterior probability values (PP) were calculated after discarding the first 25% of the samples. A PP value of ≥0.95 was considered significant.

The first combined phylogenetic analysis with ITS, EF-1α, and Act sequences of clinical isolates belonging to Cladosporium sensu stricto and all the available type and reference strains was carried out following the alignments of Bensch et al. (1; data not shown). Only sequences of those species closely related (>95% similarity) to the clinical isolates tested here were included in the final analysis.

Antifungal susceptibility.

The antifungal susceptibility test was performed according to the CLSI M38-A2 standard (33) with slight modifications. The incubation temperature was set to 25°C, given the optimal growth requirements of Cladosporium and related taxa (1, 33). Nine antifungal agents were tested: amphotericin B (AMB), 5-fluorocytosine (5FC), itraconazole (ITC), posaconazole (PSC), voriconazole (VRC), terbinafine (TRB), anidulafungin (AFG), caspofungin (CFG), and micafungin (MFG). The minimal effective concentration (MEC), defined as the lowest drug concentration at which short, stubby, highly branched hyphae were observed, was determined at 24 h for the echinocandins, and the MIC was determined at 48 h for the remaining drugs. The MIC was defined as the lowest concentration exhibiting 100% inhibition of visible growth for AMB, ITC, PSC, and VRC or 50% and 80% reduction in growth for 5FC and TRB, respectively. Paecilomyces variotii ATCC MYA-3630 and Aspergillus fumigatus ATCC MYA-3626 were used as quality control strains. Statistical analyses of the MIC/MEC data were performed using the Mann-Whitney test in Prism version 6.0 (GraphPad Software, San Diego, CA).

Nucleotide sequence accession numbers.

DNA sequences determined in this study were deposited in GenBank under accession numbers LN834353 to LN834448 (rDNA), LN834449 to LN834536 (EF-1α), and LN834537 to LN834624 (Act) (Table 1).

RESULTS

Analysis of ITS and LSU sequences showed that 88 isolates (96%) belonged to Cladosporium sensu stricto, three isolates (3%) to the genus Toxicocladosporium, and one isolate (1%) to the genus Penidiella.

The phylogenetic analysis of Cladosporium sensu stricto included 121 taxa and 1,002 bp (447 bp for ITS, 337 bp for EF-1α, and 218 bp for Act), of which 485 bp were constant (347 bp for ITS, 60 bp for EF-1α, and 78 bp for Act), 496 were variable (97 bp for ITS, 260 bp for EF-1α, and 139 bp for Act) and 328 were parsimony informative (24 bp for ITS, 197 bp for EF-1α, and 107 bp for Act) (Fig. 1). The majority of isolates (57, 65%) nested into the C. cladosporioides complex: 28 belonged to nine species (i.e., C. angustisporum, C. asperulatum, C. cladosporioides, C. flabelliforme, C. funiculosum, C. perangustum, C. pseudocladosporioides, C. subuliforme, and C. tenuissimum), while 29 isolates clustered into 12 terminal subclades genetically distant from any currently known species of the genus. A total of 14 isolates were related to the C. herbarum complex (16%), mostly corresponding to five species (i.e., C. allicinum, C. herbarum, C. macrocarpum, C. ramotenellum, and C. subinflatum), while five isolates clustered into four new lineages in the genus. Seventeen isolates were nested within the C. sphaerospermum complex (19%) and mostly belonged to two species (i.e., C. halotolerans and C. sphaerospermum), while a single isolate represented a new lineage.

FIG 1.

FIG 1

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Maximum-likelihood (ML) tree inferred from combined ITS, EF-1α, and Act sequences of Cladosporium isolates. Branch lengths are proportional to phylogenetic distance. ML bootstrap support (BS) values of ≥70% and posterior probability (PP) values of ≥0.95 are shown above the branches. Thickened branches indicate BS of 100% and PP of 1.00. Cercospora beticola (CBS 116456) was used to root the tree. Type strains are indicated in bold font. CBS, CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands; FMR, Facultat de Medicina, Reus, Spain; UTHSC, Fungus Testing Laboratory at the University of Texas Health Science Center, San Antonio, TX.

Distinct morphological features of isolates in the C. cladosporioides complex included the formation of mostly unbranched, cylindrical conidiophores, bearing ovoidal to ellipsoidal intercalary and terminal conidia, smooth or rarely showing fine ornamentation (Fig. 2a to c); the maximum temperatures for growth were 32°C for C. cladosporioides, C. flabelliforme, C. perangustum, and C. pseudocladosporioides and 35°C for C. angustisporum, C. funiculosum, C. subuliforme, and C. tenuissimum. Isolates of the C. herbarum complex exhibited mostly nodulose conidiophores, bearing distinctly ornamented globose to subglobose terminal conidia (Fig. 2d to f); none of the isolates of this complex were able to grow at temperatures above 32°C, and C. allicinum exhibited a maximum growth temperature of 30°C. Isolates of the C. sphaerospermum complex formed cylindrical and branched conidiophores, bearing globose to subglobose conidia, smooth or finely ornamented (Fig. 2g to i); the maximum temperatures for growth were 32°C for C. sphaerospermum and 35°C for C. halotolerans. None of the clinical isolates formed sexual morphs in culture.

FIG 2.

FIG 2

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Conidiophores and conidia of fungi belonging to the C. cladosporioides complex (a to c), C. herbarum complex (d to f), C. sphaerospermum complex (g to i), and Toxicocladosporium spp. (j to l). White bars, 10 μm; black bars, 5 μm.

Overall, the most commonly identified species was C. halotolerans (14.8%), followed by C. tenuissimum (10.2%) and C. subuliforme (5.7%). However, 39.8% of isolates did not match with any known taxa and represent at least 17 putative new Cladosporium species (Fig. 1). The most common anatomical site of isolation was the respiratory tract (54.5%), mainly from bronchoalveolar lavage (BAL) fluid and nasal specimens, followed by superficial sites (28.4%); these percentages were similar for all of the species and species complexes identified.

Phylogenetic analysis of the Toxicocladosporium isolates included 15 taxa and 984 bp (530 bp for LSU and 454 bp for ITS), of which 826 bp were constant (464 bp for LSU and 362 bp for ITS), 155 were variable (66 bp for LSU and 89 bp for ITS), and 129 were parsimony informative (56 bp for LSU and 73 bp for ITS) (Fig. 3). Two clinical isolates belonged to Toxicocladosporium irritans, while the isolate UTHSC DI-13-172 formed an independent lineage, genetically related to Toxicocladosporium strelitziae but showing distinctive morphological features and probably corresponding to a new species. The main morphological characteristics of members of Toxicocladosporium were the presence of nonnodulose conidiophores with dark and thickened cell walls and septa, producing conidia without the typical coronate scars of Cladosporium (Fig. 2j to l), and a maximum temperature for growth of 35°C.

FIG 3.

FIG 3

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Maximum-likelihood (ML) tree inferred from combined ITS and LSU sequences of Toxicocladosporium isolates. Branch lengths are proportional to phylogenetic distance. ML bootstrap support (BS) values of ≥70% and posterior probability (PP) values of ≥0.95 are shown above the branches. Thickened branches indicate BS of 100% and PP of 1.00. Cercospora beticola (CBS 116456) and Cercospora olivascens (CBS 253.67) were used to root the tree. Type strains are indicated in bold font. CBS, CBS-KNAW Fungal Biodiversity Centre, the Netherlands; UTHSC, Fungus Testing Laboratory at the University of Texas Health Science Center, San Antonio, TX.

According to the LSU sequence analysis, a single isolate (UTHSC DI-13-256), originally identified as C. sphaerospermum, was related to but distant (<98.2% sequence similarity) from members of the genus Penidiella (i.e., Penidiella aggregata and Penidiella drakensbergensis; sequence accession numbers JF499862 and KC005792, respectively) (data not shown). However, its final identification was not possible given the scarcity of DNA sequences of the latter species for comparison. This isolate was characterized by restricted growth (3 to 4 mm at 25°C for 7 days) and the production of solitary penicillate conidiophores, composed of chains of ramoconidia with slightly pigmented and thickened conidiogenous scars.

The results of the antifungal susceptibility testing are summarized in Table 2. The overall results for Cladosporium species showed a geometric mean (GM) MIC and MIC90 for AMB of 0.64 μg/ml and 2 μg/ml, respectively. Among the azoles, ITC and PSC were the most active, with both drugs having a GM MIC of 0.43 μg/ml and respective MIC90s of 0.5 μg/ml and 1 μg/ml, while VRC showed a GM MIC and MIC90 of 1.68 μg/ml and 4 μg/ml, respectively. Flucytosine showed variable activity and had a GM MIC and MIC90 of 1.37 μg/ml and 4 μg/ml, respectively. TRB exhibited the most potent activity, with a GM MIC and MIC90 of 0.09 μg/ml and 1 μg/ml, respectively. With the exception of CFG, the echinocandins exhibited strong in vitro activity, with GM MIC values of 0.19 μg/ml and 0.12 μg/ml for AFG and MFG. All of the Cladosporium species tested showed similar susceptibility patterns except for C. sphaerospermum, where the three isolates tested exhibited higher MIC and MEC values, especially for the azoles, AMB, AFG, and MFG (P < 0.001). Comparison of antifungal susceptibility by species complex (Table 2) showed that AMB exhibited more potent activity against members of the C. herbarum complex, with GM MIC and MIC90 values of 0.18 μg/ml and 1 μg/ml (P < 0.002), while members of the C. sphaerospermum complex exhibited higher GM MIC and MIC90 values for AMB, PSC, ITC, and CSP (P < 0.003). Toxicocladosporium and Penidiella isolates exhibited similar susceptibility patterns, with mostly low GM MIC and MIC90 values against all antifungals tested but without statistically significant differences.

TABLE 2.

Results of in vitro antifungal susceptibility testing of the 92 clinical isolates included in the study

Genus Species (no. of isolates tested) MIC/MEC parametera Result (μg/ml) forb:
AMB 5FC VRC PSC ITC TBF CFGc AFGc MFGc
Cladosporium C. cladosporioides complex (57) Range 0.06–2 0.06–>32 0.25–16 <0.03–1 <0.03–2 <0.03–4 0.125–8 0.03–0.5 0.03–0.5
GM 0.73 1.20 1.65 0.40 0.34 0.12 2.78 0.19 0.11
MIC90 1 4 4 0.5 0.5 1 8 0.5 0.25
C. tenuissimum (9) Range 0.5–1 1–>16 1–4 0.25–0.5 0.25–0.5 0.06–1 4–8 0.125–0.5 0.125–0.25
GM 0.93 2.72 1.85 0.37 0.29 0.18 4.67 0.37 0.15
MIC90 1.00 4.00 2.00 0.50 0.50 0.25 8.00 0.50 0.25
C. subuliforme (5) Range 1–2 0.25–2 0.25–2 0.25–0.5 0.25 0.06–1 4–8 0.06–0.5 0.06–0.25
GM 1.15 1.00 0.66 0.29 0.25 0.28 5.28 0.16 0.12
MIC90 1.00 2.00 1.00 0.25 0.25 1.00 8.00 0.25 0.13
C. pseudocladosporioides (4) Range 0.5–1 0.5–1 2–4 0.25–0.5 0.5–1 0.03–2 0.25–8 0.03–0.25 0.03–0.125
GM 0.59 0.71 2.38 0.42 0.59 0.21 2.38 0.15 0.07
MIC90
C. cladosporioides (3) Range 0.5–1 1–2 0.5–16 0.25–1 0.25–0.5 0.5–2 1–4 0.125–0.25 0.125
GM 0.79 1.26 1.59 0.40 0.31 1.00 2.00 0.16 0.13
MIC90
C. funiculosum (3) Range 0.06–1 0.5–1 0.5–2 0.25–0.5 0.125–0.25 0.03–0.06 4 0.06–0.25 0.06–0.125
GM 0.31 0.63 1.00 0.31 0.20 0.04 4.00 0.12 0.08
MIC90
C. angustisporum (1) Range 1.00 1.00 4.00 0.50 0.50 2.00 4.00 0.13 0.06
GM
MIC90
C. asperulatum (1) Range 1 0.25 2 0.25 0.5 0.03 8 0.25 0.125
GM
MIC90
C. flabelliforme (1) Range 2 2 2 0.5 0.25 0.03 4 0.25 0.125
GM
MIC90
C. perangustum (1) Range 0.5 4 4 0.5 0.5 0.03 4 0.125 0.06
GM
MIC90
Cladosporium sp. (29) Range 0.125–2 0.06–>16 0.25–8 <0.03–1 <0.03–2 <0.03–4 0.125–8 0.03–0.5 0.03–0.5
GM 0.67 1.10 1.73 0.43 0.36 0.09 2.00 0.18 0.10
MIC90 1.00 4.00 4.00 0.50 0.50 1.00 8.00 0.50 0.25
C. herbarum complex (14) Range <0.03–2 0.5–>16 0.5–8 0.06–0.5 0.06–1 <0.03–0.125 0.125–8 0.06–1 0.06–0.5
GM 0.18 2.97 1.81 0.37 0.35 0.05 0.67 0.23 0.15
MIC90 1 8 4 0.5 0.5 0.125 2 0.5 0.5
C. allicinum (3) Range <0.03–0.125 2–4 2–4 0.5 0.25–0.5 0.03–0.06 1.00 0.25–0.5 0.125–0.25
GM 0.05 2.52 3.17 0.50 0.40 0.05 1.00 0.31 0.20
MIC90
C. ramotenellum (3) Range 1–2 4–>16 1–2 0.5 0.5–1 <0.03–0.125 0.5–8 0.25–1 0.125–0.5
GM 1.26 4.00 1.59 0.50 0.63 0.05 2.00 0.40 0.20
MIC90
C. herbarum (1) Range 0.06 8 2 0.5 0.5 0.03 0.5 0.125 0.125
GM
MIC90
C. macrocarpum (1) Range 0.5 2 1 0.25 0.5 0.125 1 0.125 0.5
GM
MIC90
C. subinflatum (1) Range 0.5 0.5 4 0.5 0.5 0.03 0.5 0.5 0.25
GM
MIC90
Cladosporium sp. (5) Range 0.06–0.5 2–4 0.5–8 0.06–0.5 0.06–0.5 <0.03–0.125 0.125–0.5 0.06–0.5 0.06–0.125
GM 0.11 2.30 1.32 0.25 0.19 0.05 0.29 0.14 0.08
MIC90 1.00 4.00 4.00 0.50 0.50 1.00 8.00 0.50 0.25
C. sphaerospermum complex (17) Range 0.125–2 0.06–4 0.5–16 0.06–4 0.25–>16 <0.03–1 0.06–4 <0.03–1 0.06–1
GM 1.13 1.13 1.70 0.64 1.13 0.06 1.27 0.15 0.13
MIC90 2 2 8 2 32 0.5 4 0.25 0.125
C. halotolerans (13) Range 0.125–2 0.06–4 0.5–2 0.06–1 0.25–2 <0.03–1 0.06–4 <0.03–0.25 0.06–0.125
GM 1.00 0.90 1.11 0.47 0.56 0.08 1.00 0.11 0.10
MIC90 2.00 2.00 2.00 1.00 2.00 1.00 4.00 0.25 0.13
C. sphaerospermum (3) Range 1–2 2–4 8–16 2–4 >16 <0.03–0.125 2–4 0.25–1 0.125–1
GM 1.59 2.52 10.08 2.52 >16 0.03 3.17 0.50 0.40
MIC90
Cladosporium sp. (1) Range 2 2 2 0.5 0.5 <0.03 2 0.25 0.125
GM
MIC90
Overall (88) Range <0.03–2 0.06–>16 0.25–16 <0.03–4 <0.03–>16 <0.03–4 0.06–8 <0.03–1 0.03–1
GM 0.64 1.37 1.68 0.43 0.43 0.09 1.91 0.19 0.12
MIC90 2.00 4.00 4.00 0.50 1.00 1.00 8.00 0.50 0.25
Toxicocladosporium T. irritans (2) Range 0.5–1 0.25–2 0.25 0.25–1 0.5 <0.03–0.06 0.125–2 0.06–0.25 0.06–0.5
GM 0.71 0.71 0.25 0.50 0.50 0.03 0.50 0.12 0.17
MIC90
Toxicocladosporium sp. (1) Range 0.5 0.125 0.25 0.125 0.125 <0.03 1 0.125 0.06
GM
MIC90
Overall (3) Range 0.5–1 0.125–2 0.25 0.125–1 0.125–0.5 <0.03–0.06 0.125–2 0.06–0.25 0.06–0.5
GM 0.63 0.40 0.25 0.31 0.31 0.02 0.63 0.12 0.12
MIC90
Penidiella Penidiella sp. (1) Range 2 0.06 0.125 0.06 >0.03 >0.03 0.25 0.25 0.25
GM
MIC90
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a

GMs and MIC90s are shown only for species with ≥5 isolates. GM, geometric mean.

b

AMB, amphotericin B; 5FC, flucytosine; VRC, voriconazole; PSC, posaconazole; ITC, itraconazole; CFG, caspofungin; AFG, anidulafungin; MFG, micafungin; TRB, terbinafine.

c

These columns include MEC data.

DISCUSSION

Members of Cladosporium are relatively easy to identify to genus and species complex based on their typical conidiogenous structures. However, morphological identification of Cladosporium species is difficult given the high morphological similarity between closely related species. In light of our results, it is strongly recommended that phenotypic identifications be confirmed with DNA sequencing. Several authors have demonstrated the usefulness of the EF-1α and Act loci to allow a good species delimitation in Cladosporium (1, 19, 21). This is especially important for members of the C. cladosporioides complex, which demonstrated the greatest species diversity, the highest number of species associated with clinical samples, and also, the greatest number of undescribed species. Moreover, we found that C. cladosporioides, the species most frequently cited as being clinically relevant, was poorly represented in our set of isolates, while C. asperulatum, C. funiculosum, C. flabelliforme, C. pseudocladosporioides, C. subuliforme, and C. tenuissimum are described for the first time from clinical samples. Similarly, in the C. sphaerospermum complex, most of the isolates morphologically identified as C. sphaerospermum were genetically reidentified as belonging to the phenotypically similar species C. halotolerans, which according to our data, emerged as the most common species from clinical origins. The latter species has never been associated with human infection; however, some isolates had been reported from human or animal clinical samples (1). In the case of the C. herbarum complex, 13 of the 14 isolates morphologically identified as C. herbarum, also considered a clinically relevant species, were found to belong to other species of this complex (i.e., C. allicinum, C. macrocarpum, and C. ramotenellum). While C. macrocarpum has been identified as the causative agent of human infections (17), C. allicinum and C. ramotenellum have never been reported before in the clinical setting, although some isolates have been recorded as obtained from human samples (1). However, due to the lack of clinical histories and histopathological findings, it was impossible for us to confirm a pathogenic role of the species reported here for the first time from clinical specimens.

It is remarkable that our phylogenetic analysis was unable to provide species-level identification of a high number of Cladosporium isolates (39.8%) that were originally considered to belong to several common morphospecies. Instead, those unidentified isolates were grouped into 5 terminal clades and 12 monotypic lineages, representing a large variety of phylogenetic species. It is probable that many of these clades and monotypic lineages represent new species; however, further studies combining phenotypic and molecular data would be necessary to confirm these findings. We report also for the first time the isolation of Toxicocladosporium and Penidiella species from clinical specimens. Isolates of these recently proposed genera were only known from leaves of several plants and from environmental sources (3, 34). According to our data, the vast majority of isolates were obtained from respiratory specimens, including BAL fluid, nasal, and sputum samples. This is not rare, because Cladosporium is preponderant in the airborne mycobiota (35), being considered one of the most important respiratory allergenic fungi, after Alternaria (10, 34, 36).

Reports of invasive infections by Cladosporium are extremely rare. Bentz and Sautter (37) reported a mixed disseminated infection by Aspergillus fumigatus and C. cladosporioides in an immunocompromised patient. Cladosporium cladosporioides and C. macrocarpum have been reported from two clinical cases involving the central nervous system (15, 17), while C. sphaerospermum was isolated from an intrabronchial infection (38). However, in none of these cases was the etiology of the infection supported by histopathological studies. The isolation of Cladosporium species from deep tissues seems improbable considering the inability of these organisms to grow at temperatures exceeding 35°C, and thermotolerance being one of the most important virulence factors for invasive or disseminated infections (39). In our study, less than half of the isolates exhibited very limited growth at 35°C, while none was able to grow at 37°C. However, surprisingly, several of our isolates were obtained from deep tissue samples, including bone marrow, cerebrospinal fluid (CSF), and lung and lymphatic tissue samples, among others. Isolation of these fungi from invasive infections may have been due to environmental contamination of the samples; however, occasionally isolates that fail to grow in culture at 37°C have been reported to cause invasive disease in immunocompromised individuals (40).

There is a paucity of information regarding antifungal susceptibility patterns for Cladosporium species. Most data are from a few reported clinical cases (7, 13, 15, 41). Our study provides the first in vitro data for a large set of clinical isolates, including several species obtained from diverse anatomical sites and not previously reported from clinical samples. Case reports have shown a favorable outcome using azole-based therapies. ITC has shown efficacy in the treatment of superficial infections caused by C. cladosporioides, C. sphaerospermum, and C. oxysporum (8, 14, 37, 41–44), while VRC was effective against C. macrocarpum in a brain abscess (17). This agrees with our in vitro data, which demonstrated that the azoles, particularly ITC and PSC, have good activity against Cladosporium species, although VRC displayed variable activity. AMB has been shown to be ineffective against C. cladosporioides (41) and C. sphaerospermum (38) in cases of skin and intrabronchial infections, respectively. Our results, however, suggest that this drug might be effective, especially against members of the C. herbarum complex. Kantarcioğlu and Yücel (45) reported potent in vitro activity of TRB against a set of unidentified Cladosporium species. Our data confirmed the results of that study, with TRB showing significant activity against all of the species tested. Echinocandin activity against Cladosporium species has not been previously evaluated; however, we observed that both AFG and MFG exhibited notable in vitro activity against all of our isolates, indicating that they could represent an important alternative for the treatment of infections by these fungi pending further confirmatory studies.

In conclusion, our study has significantly expanded the diversity of Cladosporium species seen in clinical specimens as a result of the molecular characterization of these isolates. We were unable, however, to document these organisms as etiologic agents in human or animal disease due to the lack of clinical information and/or histopathological findings. It is also important to note that most reported cases of Cladosporium infections lack molecular confirmation, and in those cases where they have been so characterized, the strains are not available. Given that many journals require the public availability of DNA sequence data, we recommend that clinical strains be deposited in international culture collections, thereby making them available for reidentification and further study.

ACKNOWLEDGMENTS

This study was supported by the Spanish Ministerio de Economía y Competitividad, grants CGL2011-27185 and CGL2013-43789-P.

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