JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1982, P. 714-719 Vol. 15, No.40095-1137/82/040714-06$02.00/0

Isolation of Dematiaceous Pathogenic Fungi from a Feed andSeed Warehouse

ANA ESPINEL-INGROFF, THOMAS M. KERKERING,I* AND H. JEAN SHADOMY'Department of Medic ine, Division of Infectious Diseases, ' and Departtlent of Microbiology, Medical

College of Virginia, Virginia Commonwealth Unii'ersity, Riclmnond, Vir-giniia 23298

Received 1 September 1981/Accepted 25 November 1981

In an epidemiological study, nine isolates of dematiaceous fungi were recoveredfrom the interior of a local feed and seed warehouse. Sample sites included brickwalls and floors. Air samples also were included. Samples were collected in salineand plated on Mycobiotic and Sabouraud agar. The nine dematiaceous fungirecovered from these samples were identified with microscopic morphology,thermotolerance, biochemical reactions, and animal virulence tests. Four isolateswere identified as nonpathogens on the basis of positive gelatin tests. Theidentified pathogens included Fonsecacea pedrosoi, Cladosporiium bantianuimin (C.trichoides), Wangiella dermatitidis (Dixon et al., Mycopathologia 70:153-161,1980), and Exophiala jeanselmei. These five organisms were injected into NCI/ALB mice. Only the isolate of C. bantianlum was neurotropic, as demonstratedhistopathologically and by the recovery of the organism from brain tissue. Noneof the remaining four isolates were seen or cultured from any of the mouse tissuesanalyzed. The recovery of pathogenic dematiaceous fungi from environmentalsites is not uncommon. However, this study is noteworthy in that it representsonly the second reported isolation of C. hantianwmn and the first isolation of F.pedrosoi from the environment in North America and suggests that these fungimay be more ubiquitous in this region than previously believed.

Dematiaceous fungi consist of a large heterog-enous group of organisms that are characterizedby a light to dark brown pigmentation of theirhyphal or conidial elements or both. The dema-tiaceous fungi are widely distributed among theAscomycetes, Basidiomycetes, and Zygomy-cetes. However, most of the strains consideredpathogenic for humans are part of a fourthgroup, the Deuteromycetes, and are consideredto be saphrophytes living in soil and vegetativematerial. Among the human and animal fungaldiseases caused by the pathogenic dematiaceousfungi are chromoblastomycosis (chromomyco-sis), keratomycosis, eumycotic mycetomas,phaeohyphomycosis, and superficial infections.With the exception of cerebral cladosporiosis,the route of infection for the dematiaceouspathogens is traumatic inoculation of the skin.

MATERIALS AND METHODSTwenty-four samples were collected from a feed and

seed warehouse. These included sphagnum moss, peatmoss, pine bark, dirt from brick walls, and airbornematerial collected on air settling plates. Samplesweighing 2 to 3 g were collected in sterile plastic tubes(16 by 125 mm) containing a small crystal of naptha-lene. Sterile distilled water (3 to 5 ml) was added toeach tube, and the samples were maintained at roomtemperature for 1 h, after which they were shaken by a

vortex mixer. Each sample was then cultured byplating 0.1-ml portions of the mixture on Sabouraudmodified agar (Difco Laboratories) supplemented withchloramphenicol and on Mycobiotic agar (BBL Micro-biology Systems). Each plate was then streaked forisolation and incubated between 28 and 30°C.

Identification. All plates were incubated for 48 h andthen observed daily for the presence of colonies.Colonies with macroscopic characteristics of dematia-ceous black yeasts or molds were observed micro-scopically by using clear lactophenol, which enabledobservation of the pigmentation. Colonies that ap-peared as black yeasts were repeatedly subcultureduntil hyphal phases were obtained. For final morpho-logical studies, slide cultures were prepared with pota-to dextrose agar (PDA) and Mycobiotic agar (4). Thesecultures were incubated in the dark at room tempera-ture (25°C) for 10 to 14 days. Microscopic examinationof the slide cultures was performed by using bright-field and phase microscopy (40 and 100x objectives).

Biochemical reactions. Biochemical studies on purecultures of the nine dematiaceous fungi isolated wereas follows: (i) hydrolysis of 12% gelatin, and theutilization of tyrosine, xanthine, and casein; (ii) gelatintubes were incubated at room temperature for 4 weeks(other tests were incubated for 2 to 4 weeks asrecommended); (iii) appropriate negative and positivecontrol cultures (Nocardia asteroides, Streptomycessp., and Cladosporium sp.) were included (4, 5).Thermotolerance. Thermotolerance tests (14) were

performed in triplicate on all nine isolates. Eachfungus was inoculated onto each of three Sabpuraud

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TABLE 1. Source of isolation of nine dematiaceousstrains

Source iNolatsof Fungi isolated

Moss from floor 3 W. dermatitidisE. jeanselmeiCladosporiiin sp.

Brick wall 3 C. bantianirmCladosporiuim sp. (2)

Air settling plate 2 W. dermatitidisF. pedrosoi

Bag of sphagnum moss 1 Cladosporiium sp.a Of 24 samples that were cultured. 9 isolates were

identified.

modified agar plates. One set was incubated at roomtemperature, the second was incubated at 37°C andthe third was incubated at 41°C. Plates were observedperiodically and considered negative when growth wasabsent at the end of 2 weeks.Animal inoculation. Saline (0.85%) suspensions con-

taining conidia and hyphal elements were obtainedfrom mature, 10-day-old colonies grown on Sabouraudmodified agar, and 0.5-ml suspensions (viable countsof 106 elements per ml) were inoculated intravenouslyinto each of three NCI/ALB mice (2). The mice weresacrificed at the end of 5 weeks. Each animal wasautopsied; the liver, brain, spleen, and lung tissueswere removed, portions were homogenized, and thematerial was cultured on Sabouraud modified agar andMycobiotic agar. The remaining tissue materials werefixed and examined histologically.

RESULTSIsolation. From the 24 samples collected, 9

isolates were selected as probable dematiaceouspathogens based on macroscopic observation ofthe colonies and preliminary morphologicalstudies. Three of the nine fungi (18.3, 23.1, and23.2) appeared first as black, mucoid, yeastlikecolonies; the other six were observed as brownto black, heaped, hyphal colonies. None of thesefungi showed visible cultural growth before 6days of incubation.

Table 1 summarizes the sites where the differ-ent fungi were isolated. Three isolates wererecovered from moss samples collected from thefloor, three were isolated from scrapings of abrick wall, two were found on an air settlingplate, and the ninth isolate was isolated fromsphagnum moss.

Biochemical studies. The results of the proteo-lytic activity of the fungi, based on the 12%gelatin test, are shown in Table 2. Five of thenine cultures showed no proteolytic activity.Based on this preliminary biological characteris-tic, the five isolates were considered pathogenicdematiaceous fungi (4, 5, 13, 16). This wasconfirmed further by thermotolerance testing,animal inoculation, and other biological reac-tions, as well as the morphology of the isolates.The remaining four isolates used gelatin andwere therefore considered saprophytic dematia-ceous fungi.Table 2 also shows the results of utilization or

hydrolysis of tyrosine, xanthine, and casein. Allisolates were negative for the hydrolysis ofxanthine. Four isolates were positive for caseinhydrolysis; with the exception of isolate 18.4, allisolates hydrolyzed tyrosine.

Thermotolerance. Table 2 shows the thermo-tolerance results for the nine fungi. Only three ofthe nine isolates were able to grow at all threetemperatures. All isolates grew well at room

temperature, but only four grew at 37°C; three ofthese four isolates also grew at 41°C.Animal inoculation. Fourteen days after inocu-

lation of the mice, one of the three animalsinjected with culture 21.3 died. On the same daythe other two mice began to convulse and theywere sacrificed. A direct microscopic KOHpreparation of the brain showed abundant, thin,lightly pigmented hyphal elements. The funguswas recovered from cultures of the brain tissue,whereas cultures prepared from all other organswere negative. The histopathological study ofthe tissue showed the presence of hyphae thatwere thin, septate, and branching (Fig. 1). Of theremaining eight isolates, none were seen orcultured from any of the mouse tissues ana-

lyzed.

TABLE 2. Results of biochemical and thermotolerance studies

Culture 12% Gelatin Tyrosine Xanthine Casein 41°C 37TC 25°C18.3 - + - - + + +18.4 -+12.0 + + - + - - +21.1 + + - + - _ +21.2 + + - + - + +21.3 - + + + +23.1 - + - - + + +23.2 - + - - - - +23.3 + + - + - - +

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716 ESPINEL-INGROFF, KERKERING, AND SHADOMY

FIG. 1. Bright microscopy of C. bantianums? culture 21.3 showing septate-branching hyphal elements in mousebrain tissue 14 days postinfection. H and E stain x 1.000.

Morphological studies. Culture 18.4 showedabundant acrotheca-like structures, numerous

phialides with collarettes, and scarce cladospor-ium-like structures on darkly pigmented septatehyphae (Fig. 2A and B). This fungus was identi-fied as Fonsecaea pecdrosoi.

Cultures 18.3 and 23.1 generally revealed thepresence of flask-shaped or cylindrical phialideswithout collarettes on scarce hyphal structures(Fig. 3). Yeast forms were observed mainly inthe original culture but also were seen in slidecultures. These two fungi were observed first as

black, mucoid yeastlike colonies, which afterseveral passages on PDA and Mycobiotic agardeveloped hyphal growth. These two isolateswere identified as Wangiella dermatitidis on thebasis of thermotolerance tests (growth at 41°C)and biochemicals (positive tyrosine test).

Culture 23.2 showed the presence of abundantpigmented septate hyphae and short and longannellides with the presence of rings or annella-tions on the conidiogenous cell (Fig. 4A). Therewere abundant annelloconidia arranged in clus-ters on top of annellides or cascading down thisstructure (Fig. 4B). This culture, identified as

Exophiala jeanselinei, was also seen first as a

black yeast, and the thermotolerance tests (no

growth at 41°C) were used for its final identifica-tion (Table 2).

Culture 21.3 revealed the presence of onlyhighly pigmented oval blastoconidia in longchains on short conidiophores and abundantseptate brown-colored hyphae (Fig. 5). Thisfungus was identified as Cladosporiwn ban-tianum (9). Thermotolerance (growth at 410C)and animal virulence tests also were used for itsidentification.

In the four isolates considered saprophyticbecause of proteolytic activity, only cladospor-ium-like structures were observed. These iso-lates were identified as Cladosporiirn.

DISCUSSIONPathogenic dematiaceous fungi are the etio-

logical agents of a variety of systemic or local-ized fungal infections. They have been known tocause eumycotic mycetomas, subcutaneous ab-scesses, phaeohyphomycosis (E. jeanselmei, W.dermatitidis), chromoblastomycosis (F. pedro-soi), and cerebral phaeophyphomycosis (C. ban-tianum).

This paper represents the second report of theisolation of C. bantianiirn from nature and the

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FIG. 2. Phase microscopy of F. pedrosoi culture 18.4. (A) 'Acrotheca' type of sporulation. Slide culturewas done on PDA. x1,000. (B) Phialides with collarette (arrow) type of sporulation. Slide culture was done onPDA. x 1000.

first isolation of F. pedrosoi from the environ-ment in North America. The first recovery of C.bantianirm in the United States was reported inVirginia from tree bark (1). This fungus previ-ously had been isolated from soil in Panama (7).There have been reports of brain abscessescaused by this fungus in cats (6) and dogs (12) aswell as in humans (10). F. pedrosoi has beenisolated from soil in Uruguay (3), Peru (15), andVenezuela (17) and reported as the cause ofchromomyblastomycosis in toads in Colombia(18). Reports of the recovery of W. dernaititidisand E. jeanselmei from the environment havebeen rather rare. W. dermatitidis has been iso-lated in Uruguay (3) and in the United States (2).The latter study also reported the first isolationof E. jeanselmei from nature (2). From thepresent study, it would appear that these fungimay readily be found in temperate climates andnot only in the tropical zones as was previouslybelieved.

It should be noted that the criteria we used forthe identification of the isolates are the standardprocedures used for the identification of dema-tiaceous fungi from clinical specimens. The pro-teolytic activity of dematiaceous fungi has beenwidely used as a method to screen or separatethe saprophytic species (by using a 12% gelatintest) from the pathogenic species which give anegative test (4, 5, 13, 16). Based on the resultsof this test and the morphological characteristicsof these isolates, we identified four of the nineorganisms as saprophytic Cladosporiiin sp. andthe other five as pathogenic dematiaceous fungi.The four saprophytic species also gave a posi-

tive reaction for the hydrolysis of casein. How-ever, there have been reports of isolates ofCladosporium carrionii, a pathogenic dematia-ceous fungus that was capable of hydrolyzingcasein (10). The results of xanthine and tyrosineutilization were not helpful in the differentiationof any of the present isolates. It has been

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FIG. 3.Phase microscopy of W. dermnatitides cul-ture 18.3 showing a phialide (without collarette) typeof sporulation. Slide culture was done on PDA.x 1,000.

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718 ESPINEL-INGROFF, KERKERING, AND SHADOMY

B

FIG. 4. Phase microscopy of E. jeanselmnei culture23.2. (A) annellide type of sporulation with annellationor ring (arrow). Slide culture was done on PDA.x1,000. (B) Annelloconidia cascading down on annel-lide. Slide culture was done on PDA. xl.OOO0.

reported that the tyrosine test in combinationwith growth at 41°C could be used to separateisolates of W. dermatitidis from E. jeanselmiiei(14), fungi which may under bright microscopyresemble each other morphologically. However,the three isolates of those fungi isolated duringthis study hydrolized tyrosine, and for this rea-

son morphological characteristics and thermo-tolerance were used as the primary criteria inestablishing their identity. Thus, W. der,natitidis

McGinnis 1977 (8) was distinguished from E.

jeanselmei principally by the lack of distinctconidiophores, the presence of flask-shaped or

cylindrical phialides without collarettes, the pro-duction of oval-shaped phialoconidia accumulat-ing in a ball at the apex of the phialides (Fig. 3),and the ability to grow at 41°C.The isolate characterized as E. jeanselmei, on

the other hand, had cylindrical, lightly pigment-ed, percurrent annellides, with the production of

FIG. 5. Phase microscopy of C. baintianzuin culture 21.3. Blastoconidia can be seen in chains. Slide culturewas done on PDA. x 1O000.

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abundant annelloconidia accumulating on top ofor cascading along the annellides (9, 11) (Fig.4B). The accumulation of annelloconidia was attimes so dense that it did not permit an easyobservation of the apex of the annellides. Theobservation and differentiation of annellides andtheir associated structures from phialides werenot readily performed without the aid of phasemicroscopy. The elongation of the annellidesand their narrow apices were quickly observed,but the annellations, or rings, were not clearlyseen until the preparations were placed under aphase microscope at high magnification (1000x).The isolate identified as F. pedrosoi had three

of the four types of conidial development associ-ated with this species. The culture primarilyshowed sympodial conidial development (astructure previously called acrotheca: Fig. 2A)similar to that seen in Rliinoladiella (9). Twodifferent sizes of conidia were observed: thesmaller ones were produced from phialides withcuplike collarettes (Fig. 2B), and the larger oneswere developed on the upper portion of theslightly swollen and pigmented conidiogenouscells. Cladosporium-like structures were rarelyseen. None of the other tests performed wereneeded to identify this isolate: this was the onlyculture negative for tyrosine, but more isolatesmust be tested before this procedure can be usedas a diagnostic tool in conjunction with themorphological criteria.

Culture 21.3 was identified as C. bwititianoiiion the basis of both morphological and clinicalcriteria. It showed the presence of typical chainsof lightly pigmented, oval blastoconidia on shortconidiophores and abundant, darkly pigmentedseptate hyphae (Fig. 5). The fact that this isolatealso grew at 41°C and that neurotropism wasobserved in experimentally inoculated miceclassified this isolate as C. bainitianuilm and not asC. (arrion ii. This latter fungus neither grows attemperatures higher than 36°C nor is neurotropicfor mice; however, both fungi morphologicallyare similar.

Thus, it seems that the recovery of dematia-ceous pathogens from the environment can bereadily accomplished. These fungi can be identi-fied with a combination of biochemical, morpho-logical, thermotolerance, and in some instances.animal virulence tests.

LITERATURE CITED

1. Dixon, D. M., H. J. Shadomv, and S. Shadomv. 1977.Isolation of Cladlosporiiim trichloides from nature. Myco-pathologia 62:125-127.

2. Dixon, D. M., H. J. Shadomy, and S. Shadomy. 1980.Dematiaceous fungal pathogens isolated from nature. Mv-copathologia 70:153-161.

3. Gezuele, E., J. E. MacKinnon, and I. A. Conti-Diaz. 1972.The frequent isolation of Phiailopihor-a verrucosa andPlii(ilopl)ot-a pedr-osoi from natural sources. Sabouraudia10:266-273.

4. Haley, L. D., and C. S. Calloway. 1978. Laboratory meth-ods in medical mycology. 4th ed. Center for- DiseaseControl. Atlanta.

5. Haley, L. D., and P. G. Standard. 1973. Laboratory meth-ods in medical mycology. 3rd ed. Center for DiseaseControl. Atlanta.

6. Jang, S. S., E. L. Biberstein, M. G. Rinaldi, A. NI. Hen-ness, G. A. Boorman, and R. F. Taylor. 1977. Feline brainabscesses due to Clolo.sporioiz trichoide.s. SaboLuraLidia15:115-123.

7. Klite, P. B., H. B. Kelley, Jr., and F. N. Diercks. 1965. Anew soil sampling technique for pathogenic fungi. Am. J.Epidemiol. 31:124-130.

8. McGinnis, M. R. 1977. Wangiella. a new genus to accom-modate Hormisciuom der-tl(ititindis. Mvcotaxon 5:353-363.

9. McGinnis, M. R. 1980. Taxonomic developments andchanges in medical mycology. Annu. Rev. Microbiol.34:109-135.

10. McGinnis, M. R. 1980. Dematiaceous fungi. p. 6(12-610.In E. H. Lennette. A. Balows. W. Hausler. Jr.. and J. P.Truant (ed.). Manual of clinical microbiology. 3rd ed.American Society for Microbiology. Washington. D.C.

11. McGinnis, M. R., and A. A. Padhye. 1977. Exop/iiulajeotlseletii. A new combination for Phiailophl oro jeansel-nzeii. Mvcotaxon 5:341-352.

12. Newsholme, S. J., and M. J. Tvrer. 1980. Cerebral myco-ses in a dog caused by Cladosporiumo trwoihdes Emmons1952. Onderstepoort J. Vet. Res. 47:47-49.

13. Nielsen, H. S., Jr. 1974. Dematiaceous fungi. p. 528-540.In E. H. Lennette. E. H. Spaulding. and J. P. Truant(ed.). Manual of clinical microbiology. 2nd ed. AmericanSociety for Microbiology. Washington. D.C.

14. Padhve, A. A., NI. R. McGinnis, and L. Ajello. 1978.Thermotolerance of WtitVgiellIo d(etitnatitisti.v. J. Clin. Mi-crobiol. 8:424-426.

15. Rieth, H. 1965. 1963. Isolierung Von HoronodendrumOped-o.soi aus erdborden des uriwaldgebiete bei Pucallpa inPeru. Proc. Int. Symp. Med. Mycol. X:89-90.

16. Rogers, A. L. 1980. Opportunistic and contaminating sap-rophytic fungi. p. 654-668. In E. H. Lennette. A. Balows.W. Hausler. Jr.. and J. P. Truant (ed.). Manual of clinicalmicrobiology. 3rd ed. American Society for Microbiolo-gy. Washington, D.C.

17. Salfelder, K., J. Schwarz, A. Romero, T. R. De Liscano, Z.Zambrano, and I. Diaz. 1968. Habitat of Nocardia astcr-oides, P. pedr-osoi and Crvptococcus nft'Of asoiIX in Ven-ezuela. Mycopathol. Mycol. Appl. 34:144-154.

18. Valasquez, L. F., and A. Restrepo. 1975. Chromomycosisin the toad. (Bufo marinus) and a comparison of theetiologic agent with fungi causing human chromycosis.Sabouraudia 13:1-19.

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