JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1988, p. 301-3070095-1137/88/020301-07$02.00/0Copyright © 1988, American Society for Microbiology

Evaluation of Proteolytic Activity To Differentiate SomeDematiaceous Fungi

ANA ESPINEL-INGROFF,l* PEGGY R. GOLDSON,2 MICHAEL R. MCGINNIS,3 AND THOMAS M. KERKERING'

Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-05041; North CarolinaMemorial Hospital, Chapel Hill, North Carolina 275142; and Department ofPathology, University of Texas

Medical Branch, Galveston, Texas 775503

Received 17 August 1987/Accepted 10 November 1987

A total of 123 isolates of Cladosporium spp., Exophiala spp., Fonsecaea spp., Lecythophora hoffmannii,Phaeoannellomyces werneckii, Phialophora spp., WangieUa dernmtitidis, and Xylohypha bantiana were tested forproteolytic activity by using 26 different formulations of gelatin, milk, casein, and Loeffler media. OtheFphysiological properties examined included hydrolysis of tyrosine and xanthine, sodium nitrate utilization inCzapek Dox agar, and thermotolerance. Isolates of Exophiala jeanselmei, Fonsecaea compacta, Fonsecaeapedrosoi, W. dermatitidis, and X. bantiana lacked proteolytic activity. Proteolytic activity was variable amongthe remaining species, depending on the type of medium used. Thermotolerance had value in distinguishingsome taxa.

Proteolytic activity was first used to distinguish the patho-gens Cladosporium carrionii and Xylohypha bantiana (16)from nonpathogenic species which possessed similar mor-

phology (1, 2, 10-14, 19, 21, 23). The test was expanded toinclude numerous other species of dematiaceous fungi (1, 5,15, 17, 22, 24), some of which are occasional opportunisticpathogens. A variety of media, incubation periods, condi-tions, and temperatures have been used for the study ofproteolytic activity as well as other physiologic properties.The absence of standardization for these methods has re-

sulted in conflicting results and conclusions regarding thistype of approach to differentiating pathogenic from sa-

prophytic isolates.The purpose of this study was to determine if proteolytic

activity could be useful in distinguishing pathogenic fromnonpathogenic dematiaceous fungi. A second purpose wasto see if proteolytic activity could be used to differentiateamong the dematiaceous fungi. We also examined otherphysiological properties and thermotolerance to determinetheir potential usefulness.

MATERIALS AND METHODS

Cultures. A total of 123 isolates of dematiaceous moldsand yeasts maintained in the culture collections of theMedical College of Virginia, Virginia Commonwealth Uni-versity, Richmond, Va., North Carolina Memorial Hospital,Chapel Hill, N.C., University of Alberta, Edmonton, Al-berta, Canada, the State University of New York, Syracuse,N.Y., the Centers for Disease Control, Atlanta, Ga., andLoyola College, Baltimore, Md., were used in this study(Table 1). The purity and identity of each isolate was

confirmed, and the cultures were then maintained at 25°C on

potato dextrose agar by serial transfer until they were

needed for testing.Proteolytic activity. Several media obtained from multiple

sources were evaluated (Table 2). The pH for media in group

* Corresponding author.

A ranged between 6.9 and 7.2, except for one lot of aqueousgelatin (BBL Microbiology Systems, Cockeysville, Md.),which had a pH of 5.6. Portions (5 to 6 ml) of each mediumwere aseptically dispensed into sterile 16-by-125-mm plasticscrew-cap tubes. The tubes were inoculated with a smallportion of a colony (approximately 3 to 4 mm) that had beengrowing for 10 days on modified Sabouraud glucose agar

(Difco Laboratories, Detroit, Mich.) at 25°C. An uninocu-lated tube was included as a negative control. Cultures were

incubated at 28 to 30°C for 4 weeks and read three times a

week for proteolytic activity after being placed in a refriger-ator at 4°C for periods of 15, 30, and 60 min (until thenoninoculated gelatin solidified). Each tube was observedfor liquefaction of the gelatin, determined by the failure ofthe substrate to solidify at 4°C.The media in group B (Table 2) were prepared in sterile

100-by-15-mm plastic petri plates with four compartments bypipetting 6 to 7 ml of each medium into each compartment.The plates were inoculated by using pinpoint inocula (ap-proximately 1 to 2 mm) from 10-day-old colonies growing onmodified Sabouraud. glucose agar at 25°C. Four isolates weretested per plate. Cultures were incubated at 28 to 30°C for 4weeks and observed daily for enzymatic activity. Positiveenzymatic activity was read as a clearing in the agar aroundthe colonies. This was confirmed, when needed, by floodingthe cultures with an aqueous-saturated solution of ammo-nium sulfate to enhance the reaction before the cultures werediscarded as negative.

Hydrolysis of other substrates. (i) Casein agar. Commer-cially prepared casein agar (Remel, Linexa, Kans.) was

prepared by aseptically pipetting 6 ml of medium into each ofthe four compartments of 100-by-15-mm plastic petri plates.The medium was inoculated by using pinpoint inocula (asdescribed for group B). Cultures were incubated at 28 to30°C for 4 weeks and read daily for the presence of clearingaround or under the colonies.

(ii) Slants of Loeffler blood serum and medium. Commer-cially prepared slants of Loeffler blood serum (Difco) andLoeffler medium (BBL) were inoculated with a small inocu-

lum (approximately 3 to 4 mm) from 10-day-old coloniesgrowing on modified Sabouraud glucose agar at 25°C on the

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302 ESPINEL-INGROFF ET AL.

TABLE 1. Isolates studiedNo.~ ~of

Fungus No. of Clinical source (isolate no.) Environmental source (isolate no.)isolates

Cladosporium carrionii 8 MCV (53.35, 53.36, 53.37), NCMH (779, 781, None784, 791, 1451)

Cladosporium cladosporioides 2 MCV (53.25), NCMH (122) NoneCladosporium elatum 2 MCV (53.39) MCV (53.21)Cladosporium resinae 4 MCV (53.22) SUNY (ED-66, ED-199, P-137)Cladosporium 6 MCV (19.7, 53.16, 53.23), NCMH (1402, 1199) MCV (53.43)sphaerospermum

Exophiala jeanselmei 9 MCV (29.26, 29.21, 29.18), NCMH (123, 135) MCV (29.19, 29.28), NCMH (1051, 1346)Exophiala spinifera 7 MCV (29.49, 29.34), NCMH (1361, 152) NCMH (819, 820), DMD (45)Fonsecaea compacta S NCMH (10, 684, 904, 1000, 1271)Fonsecaea pedrosoi 8 NCMH (11, 707, 899, 925, 1032, 1302) NCMH (1475, 1490)Lecythophora hoffmannii 4 NCMH (1039) NCMH (19), UAMH (1371, 4383)Phaeoannellomyces werneckii 9 NCMH (1050), UAMH (3683, 3696, 4981, 4984) NCMH (18, 75, 137, 765)Phialophora parasitica 10 UAMC (4034, 5053, 5054, 5055, 5059) NCMH (134), UAMC 5024, 5192), SUNY

(P710, P754)Phialophora repens 3 NCMH (227, 184) MCV (29.16)Phialophora richardsiae il MCV (29.25), NCMH (490, 491, 728, 1231) NCMH (112,144,1359), SUNY (P752,

ED-117), CDC (B3764)Phialophora verrucosa 12 NCMH (102, 1060) MCV (29.15, 29.14, 29.57, 29.58), NCMH

UAMH (188, 2509, 2989), CDC (B3733) (488, 489)Wangiella dermatitidis 12 MCV (29.31, 29.32), NCMH (147, 1218, 1219, MCV (29.27), NCMH (941, 1070, 1076)

1223), DMD (368, 369)Xylohypha bantiana il MCV (53.12, 19.10, 53.34), NCMH (113, 115, MCV (53.20, 53.29, 53.40, 53.41), NCMH

121) (634)a Abbreviations: MCV, Medical College of Virginia, Virginia Commonwealth University; NCMH, North Carolina Memorial Hospital; UAMH, University of

Alberta Microfungus Collection and Herbarium; DMD, Loyola College; SUNY, State University of New York; CDC, Centers for Disease Control.

upper portion of the slant, as recommended by the manu-facturers. Cultures were incubated at 28 to 30°C for 4 weeksand read daily for hydrolysis or liquefaction of the mediumaround the colony.

(iii) Skim milk and litmus milk. Skim milk (Difco) andlitmus milk (BBL) (5 to 6 ml of each medium) were preparedin 16-by-125-mm plastic screw-cap tubes. Tubes Were inoc-ulated with a small amount of inoculum (as described above)

and incubated at 28 to 30°C for 4 weeks. Cultures were readdaily for the presence of coagulation (clot formation) orprecipitation of casein.For the testing of hydrolysis of xanthine and tyrosine,

commercially prepared xanthine and tyrosine agars (Remel)were used. These two media were tested and read asindicated above for the casein agar.

Thermotolerance tests. Slants of modified Sabouraud glu-

TABLE 2. Gelatin-based media used for determining proteolytic activityMedium % Gelatin (pH) Manufacturer Réference(s)

Group AGelatin (in water) 12 (7.1) Difco 10

12 (7.1) BBL 1012 (5.6) BBL 10

Nutrient gelatin 12 (6.9-7.0) Difco 10, 11, 2212 (6.9-7.0) BBL 10, 11, 22

Brain heart infusion broth 12 (6.9-7.0) BBL 1212 (6.9-7.0) Difco 1212 (7.0-7.2) Difco/BBLa 1212 (6.9-7.0) BBL/Difcob 12

Heart infusion broth 12 (7.0-7.2) Difco 1, 212 (6.9-7.0) Difco/BBL 1, 2

Thiogel 5 (6.9) BBL ilThioglycolate gelatin 5 (7.0-7.2) Difco

Group BNutrient agar (pH 6.0) 0.4 (6.7) Difco 24

0.4 (6.6) Difco/BBL 24Tryptone glucose extract agar 0.4 (6.9-7.0) Difco

0.4 (6.9) Difco/BBLStandard method agar 0.4 (7.0) BBL 22

0.4 (7.0) BBL/DifcoNocardia gelatin agarc 3 (6.6-6.7) Difco

3 (6.6-6.7) BBLa Medium (Difco)/gelatin (BBL).b Medium (BBL)/gelatin (Difco).C K2HPO4 (1 g), KCL (0.2 g), MgSO4(7H20) (0.2 g), agar (Difco) (10 g), and gelatin (30 g) in 1 liter of distilled water.

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PROTEOLYTIC ACTIVITY OF DEMATIACEOUS FUNGI 303

TABLE 3. Proteolytic activity of 123 dematiaceous fungi determined by using gelatin media

No. of positive reactions in the following media'Fungus Sourcea (no. Water NBc BHId BHIe HIBf

tested) __- Thiog Thio'D B B' D B D B D B D B

Cladosporium carrionii E (0)C (8) 0 0 0 0 0 0 0 0 0 0 0 0 0

Cladosporium spp. (14) 7 6 7 7 8 10 7 10 4 6 6 12 12(saprophyticy

Exophiala jeanselmei E (4) 0 0 0 0 0 0 0 0 0 0 0 0 0C (S) O O O O O O O O O O O O O

Exophiala spinifera E (3) 0 0 0 0 0 0 0 0 0 0 0 O OC (4) 0 0 0 0 0 0 0 0 0 0 0 0 0

Fonsecaea compacta E (0)C (S) O O O O O O O O O O O O O

Fonsecaea pedrosoi E (2) 0 0 0 0 0 0 0 0 0 0 0 0 0C (6) O O O O O O O O O O O O O

Lecythophora E (3) 1 2 3 1 2 1 1 1 2 2 2 2 3hoffmanni' C (1) 1 1 1 1 1 1 1 1 0 1 1 1 1

Phaeoannellomyces E (4) 0 0 0 0 0 1 1 1 0 0 0 0 0werneckii C (5) 0 0 0 0 0 1 0 0 0 0 0 0 1

Phialophora parasitica E (5) 4 4 4 4 4 4 3 4 2 3 3 4 4C(5) 1 2 3 2 2 2 1 3 3 3 3 4 5

Phialophora repens E (1) 1 1 1 0 1 1 1 1 1 1 1 1 1C (2) 1 2 1 1 1 1 2 1 1 1 1 2 2

Phialophora E (6) 1 2 2 1 2 0 0 2 0 2 1 2 3richardsiae' C (5) 0 5 3 0 3 0 0 1 1 1 0 1 5

Phialophora verrucosa E (6) 0 0 0 0 1 1 1 0 1 1 1 0 1C (6) 0 0 0 0 0 0 0 0 0 0 0 0 0

Wangiella dermatitidis E (5) 0 0 0 0 0 0 0 0 0 0 0 0 0C(7) 0 0 0 0 0 0 0 0 0 0 0 0 0

Xylohypha bantiana E (5) 0 0 0 0 0 0 O O O O O O OC (6) 0 0 0 0 0 0 0 0 0 0 0 0 0

a E, Environmental source; C, clinical source.b Media were placed in tubes and contained 12% gelatin except for thiogel and thioglycolate media, which contained 5% gelatin. D, Difco gelatin; B, BBL

gelatin. With the exception of thiogel and thioglycolate, all other 11 media showed a variable response with at least one of the isolates when the test was repeated.C Nutrient gelatin broth.d Difco brain heart infusion.e BBL brain heart infusion.f Difco heart infusion broth.g BBL thiogel.h Difco thioglycolate gelatin medium.'BBL gelatin, adjusted to pH 7.1.i Some reactions were positive at 15 min and negative at 30 min.

cose agar (Difco) were used for the thermotolerance tests. A solidify at 4°C, in contrast to 15 to 30 min for the otherheavy inoculum (approximately 5 to 6 mm) was used. Tests controls.were conducted at 30, 35, and 40 to 41°C; Slants were read Clear reactions were observed on gelatin-based nutrienttwice a week for 2 weeks, and the presence ofgrowth at each agar and Nocardia gelatin agar after 24 h. Tryptone glucosetemperature was recorded. extract agar (Difco) (0.4% gelatin) gave faster reactions than

Nitrate utilization. Czapek Dox agar from Difco and BBL standard methods agar did (BBL). Reactions on all of thesewere used for nitrate utilization. These media were prepared media (group B), especially the latter two, occasionallyas indicated above for casein agar. Cultures were read daily required the addition ofammonium sulfate for clarification offor the presence of growth which indicated that nitrate was the test results. Brain heart infusion broth (Difco and BBL)used as a sole source of nitrogen. supplemented with Difco gelatin (Difco), thiogel medium

(BBL), thioglycolate gelatin medium (Difco), and the groupRESULTS B media provided the most consistent results with 10 of the

Proteolytic activity. The 0.4 and 3% concentrations of 14 isolates of saprophytic Cladosporium spp. However, onegelatin in plates supported more rapid and stronger reactions of the latter isolates (Cladosporium elatum) failed to dem-than higher concentrations of S and 12% in test tubes did. onstrate proteolytic activity in all ofthe media tested (groupsReactions were easier to read by using media in plates. The A and B and other sources). Three isolates of Cladosporium5 and 12% gelatin media in tubes (group A) could be read resinae showed very little proteolytic activity regardless ofbetween 2 to 30 days, with the thiogel medium (BBL) and the medium used. The absence of proteolytic activity wasthioglycolate gelatin medium (Difco) having the most rapid also observed with isolates of Phialophora parasitica andreactions (4 to 20 days). These latter media required careful Phialophora richardsiae when group A media were used,reading against their corresponding uninoculated controls especially with the latter species. These two species werebecause of their lower concentrations of gelatin (5%). The highly positive on the group B media (low concentrations ofuninoculated controls typically required up to 60 min to gelatin). Reactions with the other nine media in group A

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304 ESPINEL-INGROFF ET AL.

TABLE 4. Proteolytic activity of 123 dematiaceous fungi determined by using miscellaneous media

No. of positive reactions in the following media"Source'

Fungus (no. NAc SA(D)d SA(B)e NGAf Milkh Loefflertested) Caseing

D B D B D B D B D B D B

Cladosporium E (0)carrionii C (8) 5 3 0 0 Q O O O 2 2 0 0 0

Cladosporium spp. (14) 13 13 12 il il il 10 il il il 10 6 7(saprophytic)

Exophiala E (4) 0 0 0 0 0 0 0 0 0 0 0 0 0jeanselmei C (5) 0 0 0 0 0 0 0 0 0 0 0 0 0

Exophiala E (3) 0 0 0 0 0 0 0 0 0 0 0 0 0spiniferak C (4) 0 0 0 0 0 0 0 1 O 1 1 0 0

Fonsecaea E (0)compacta C (5) 0 0 0 0 0 0 0 0 0 0 0 0 0

Fonsecaea pedrosoi E (2) 0 0 0 0 0 0 0 0 0 0 0' O OC (6) 0 0 0 0 0 O O O O 0' o' O O

Lecythophora E (3) 3 3 2 2 3 3 3 3 3 3 3 0 0hoffmannii C (1) 1 1 1 1 1 1 1 1 1 1 1 1 1

Phaeoannellomyces E (4) 1 1 1 1 1 1 1 1 3 1 0 0 0werneckii C (5) 2 1 2 1 0 i 2 2 4 1 2 0 0

Phialophora E (5) 5 4 4 4 4 4 4 4 3 5 5 1 4parasitica C (5) 4 4 4 4 3 3 4 4 3 5 5 3 3

Phialophora repens E (1) 0 1 0 0 0 0 0 0 0 1 1 0 0C (2) 2 2 2 2 2 2 2 2 1 2 2 0 0

Phialophora E (6) 6 6 6 5 5 4 6 6 6 6 6 0 1richardsiae C (5) 5 5 5 5 5 5 5 5 5 5 5 0 0

Phialophora E (6) 1 1 1 1 0 1 2 1 1 O0 o0 O Overrucosa C (6) 0 0 0 0 0 0 0 0 0 0 0 0 0

Wangiella E (S) O O O O O O O O O O O O Odermatitidisk C (7) 0 0 0 0 0 0 0 0 0 0 0 0 0

Xylohypha E (5) 0 0 0 0 0 0 0 0 0 0 0 0 Obantiana C (6) 0 0 0 0 0 0 0 0 0 0 0 0 0

aE, Environmental source; C, clinical source.b Media were prepared in plates except for skim milk, litmus milk, and Loeffler media. D, Difco gelatin; B, BBL gelatin.C Difco nutrient agar (pH 6.0) with 0.4% gelatin.d Difco tryptone glucose extract agar (standard plate count agar) with 0.4% gelatin.e BBL standard method agar with 0.4% gelatin.f Nocardia gelatin agar (formulation given by J. Kane) with 3% gelatin.8 Remel prepared casein agar.h Difco skim milk or BBL litmus milk.Difco prepared Loeffler blood serum (slants) and BBL prepared Loeffler medium (slants).Variable reactions.Presence of diffusible pigment made it difficult to assess these media with these species.

'Some isolates showed curdling of the milk but not coagulation.

were variable among the different species as well as amongisolates of the same species. Among the known pathogenicfungi, five of the eight isolates of C. carrionii demonstratedproteolytic activity on nutrient agar. Proteolytic activity(especially on group B media and other substrates) was alsoseen with all four isolates of Lecythophora hoffmannii,seven of the nine isolates of Phaeoannellomyces werneckii,all 10 isolates of P. parasitica, all three isolates of Phialo-phora repens, and all 11 isolates of P. richardsiae (Tables 3and 4). There were no differences between isolates originat-ing from environmental or clinical sources representing thesame species or between different species with mediumcontaining gelatin or other substrates. The exception wasone of the six isolates of Phialophora verrucosa isolatedfrom the environment. However, the other five isolates ofthis species gave the same results as the six isolates recov-ered from clinical specimens (Tables 3 and 4).

Hydrolysis of other substrates. Skim milk and especiallyLoeffler media gave more variable results than the media ingroups A and B did when tested with isolates of thesaprophytic Cladosporium spp. Loeffler medium (BBL) pro-vided faster (3 to 6 days) and clearer reactions than the Difcomedium did (3 to 16 days). The reaction rates for litmus milk

(BBL) and skim milk (Difco) varied from 24 h to 21 days,depending on the species tested. Clear, stable, and moreintense reactions were seen on casein agar in 48 h to 10 days.Casein agar was as sensitive as the media in group B fordetecting proteases produced by C. carriondi (variable reac-tions), L. hoffmannùi, Phaeoannellomyces werneckii, P.parasitica, P. repens, P. richardsiae, and one isolate of P.verrucosa. When Loeffler (Difco or BBL), skim milk, orlitmus milk medium was used, we found that the latter twowere more sensitive for detection of proteolytic enzymes(similar to casein agar or group B media), especially withisolates of L. hoffmannii, P. parasitica, P. repens, and P.richardsiae. However, negative results also were obtainedwith isolates of the Cladosporium spp. (Table 4).None of the 123 isolates demonstrated hydrolysis of

xanthine; reactions on tyrosine agar were variable, with theexception of 11 isolates of P. richardsiae, which wereconsistently negative (Table 5).

Thermotolerance. Ail 123 isolates grew well at 30°C, andthe majority grew at 35°C. Nine of 14 isolates of thesaprophytic Cladosporium spp., eight of the nine isolates ofExophiala jeanselmei, four of the nine isolates of Phaeoan-nellomyces werneckii, and one of the three isolates of P.

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PROTEOLYTIC ACTIVITY OF DEMATIACEOUS FUNGI 305

TABLE 5. Results of physiologic reactions for 123 dematiaceous fungi

Source' No. of isolates showing thermotoleranceb No. of positive reactions in:Fungus (no.

tested) 30°C 35°C 40°C Xc Td Cze

Cladosporium E (0)carrionii C (8) 8 8 1 0 2 8

Cladosporium spp. (14) 14 5 4 0 10 14(saprophytic)

Exophiala E (4) 4 0 0 0 4 4jeanselmei C (S) 5 1 0 0 3 5

Exophiala spinifera E (3) 3 3 0 0 3 3C(4) 4 4 0 0 1 4

Fonsecaea E (0)compacta C (5) 5 5 0 0 3 5

Fonsecaea pedrosoi E (2) 2 2 0 0 2 2C (6) 6 6 0 0 2 6

Lecythophora E (3) 3 3 2 0 2 3hoffmannii C (1) 1 1 0 0 1 1

Phaeoannellomyces E (4) 4 2 0 0 1 4werneckii C (5) 5 3 0 0 1 5

Phialophora E (5) 5 5 1 0 4 5parasitica C (5) 5 5 0 0 2 5

Phialophora repens E (1) 1 0 0 0 1 1C (2) 2 2 0 0 1 2

Phialophora E (6) 6 6 0 0 0 6richardsiae C (5) 5 5 0 0 0 5

Phialophora E (6) 6 5 0 0 0 6verrucosa C (6) 6 6 0 0 2 6

Wangiella E (S) 5 S 5 0 4 Sdermatitidis C (7) 7 7 7 0 6 7

Xylohypha E (S) S S S 0 2 Sbantiana C (6) 6 6 6 0 2 6a E, Environmental source; C, clinical source.b Thermotolerance tests were performed on modified Difco Sabouraud dextrose agar.C Remel xanthine medium.d Remel tyrosine medium.e Difco and BBL Czapek Dox agar.

repens did not grow at 35°C. Four isolates of C. resinae, twoisolates of L. hoffmannii, one isolate of P. parasitica, allisolates of Wangiella dermatitidis, and all isolates of X.bantiana grew at 40°C (Table 5).

Nitrate utilization. All 123 isolates grew on Czapek Doxagar, utilizing sodium nitrate as the sole source of nitrogen(Table 5).

DISCUSSION

In 1947, Carrion and Silva (4) reemphasized the observa-tion of E. M. Medlar (in 1915) that P. verrucosa does notcoagulate or peptonize milk. In the same publication, theyreviewed the biochemical activities observed by J. Meriin (in1930) for Fonsecaea pedrosoi and its inability to liquefygelatin and to coagulate milk. In 1949, Montemayor (18)reported that P. verrucosa, Fonsecaea compacta, F. pedro-soi, and E. jeanselmei had no proteolytic activity whengrown on gelatin, Loeffier serum, or milk. He also observedthat two isolates of Cladosporium spp. (as Hormodendrumspp.) possessed proteolytic activity when tested on thesemedia. De Vries (6) credited Planchon and Weigmann withfirst demonstrating the ability of Cladosporium herbarum toliquefy gelatin. He also showed that all nonpathogenicCladosporium spp. were able to utilize ammonium andnitrate as the sole sources of nitrogen and to liquefy gelatin.

Subsequent attempts to use proteolytic activity for thedifferentiation of dematiaceous fungi are well documented(1, 2, 5, 7, 10-15, 17, 19, 20-24). A variety of media,

substrates, incubation times, and temperatures have beenused in physiological studies to characterize dematiaceousfungi. The most widely used protein source has been gelatin,formulated in various concentrations, which has been usedas a supplement to many media (1, 2, 10-14, 17, 18, 22-24).Gelatin-based media, Loeffler medium (2, 5, 10, 12, 13, 18,19, 21, 23), skim milk (2, 4, 5, 10, 15, 18), and casein agar (3,7, 14, 17, 22, 23) have been widely recommended by variousauthors.

Brain heart infusion broth and thiogel containing 12%gelatin have been recommended to distinguish pathogenicdematiaceous fungi from saprophytic fungi like Cladospo-rium spp. (11, 13). In the present study, Difco brain heartinfusion (12% gelatin) failed to detect proteolytic activitywhen tested with four of the isolates of the saprophyticCladosporium spp., whereas the BBL medium gave thesame results with 10 of the 14 isolates. Our results withthiogel and thioglycolate were more in agreement withprevious observations (11, 12). However, one of the fourisolates of C. resinae and one isolate of C. elatum hadnegative reactions on the two latter media (Table 3).Among the other substrates, casein provided the most

reproducible, easy-to-read, and clear reactions. Previousreports regarding this substrate have been contradictory forC. carrionfi (7, 14, 23), P. parasitica (22), and Phaeoannel-lomyces werneckii (17). Our data showed variable resultswith these three species with this substrate. Our results arein disagreement with previous reports of the use of Loefflermedium (2, 10, 12, 13, 18, 19, 21, 23) and skim milk (2, 4, 10,18, 23). Other authors have also stated that the proteolytic

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306 ESPINEL-INGROFF ET AL.

activity on Loeffler agar (5) and the coagulation of milk (5,15) are not good differential criteria for the black yeasts ofmedical interest. Our results confirm these conclusions.

Despite previous reports or recommendations of proteo-lytic activity as a useful method to differentiate nonpatho-genic from pathogenic isolates, we found that only isolates ofE. jeanselmei, Fonsecaea spp., W. dermatitidis, and X.bantiana failed to have proteolytic activity on the mediatested. Dematiaceous fungi known to be opportunisticpathogens, such as C. carrionfi, L. hoffmannii, P. parasitica,P. repens, P. richardsiae, P. verrucosa, and Phaeoannel-lomyces werneckii, had variable proteolytic activities whichdid not correlate with isolates causing disease (Table 3 and4).

In this study, no significant differences in proteolyticactivity (gelatin-based media or other substrates) were ob-served between known pathogenic and nonpathogenic iso-lates. Fuentes and Bosch (10) found no proteolytic differ-ences between one environmental isolate of P. verrucosa(from palm leaves) and 11 isolates recovered from cases ofchromoblastomycosis. The same results were reported byConti-Difaz et al. (5) between isolates from the environmentof either E. jeanselmei or W. dermatitidis and isolates fromhuman sources. We have previously demonstrated (8, 9) byuse of the exoantigen test that environmental and clinicalisolates of the same species could not be differentiated.Serological studies (19) have shown that there are no differ-ences between isolates of C. carrionii from the environmentand those causing human disease.

It has been demonstrated (17) that W. dermatitidis appearsto be the most thermotolerant species among the blackyeasts. All of the isolates of W. dermatitidis that we testedgrew well at 40 to 41°C. The low thermotolerance of E.jeanselmei has been previously reported (5). Only oneisolate of E. jeanselmei grew at 35°C, but none grew at 40 to41°C. Moreover, none of the isolates of Exophiala spiniferaand Phaeoannellomyces werneckii grew at 40 to 41°C despiteprevious reports of the thermotolerance of these two species(5, 15, 17). Thermotolerance is useful in differentiatingisolates of X. bantiana from most isolates of commonlyencountered Cladosporium spp. (2, 7, 13, 14, 16, 19, 23).However, our four isolates of C. resinae also grew well at 40to 41°C (Table 5). Thermotolerance is a helpful auxiliary testthat must be used in conjunction with morphologic criteria.Only P. richardsiae failed to hydrolyze tyrosine. Reac-

tions with the other 114 isolates were variable, as previouslyreported (5, 14, 15). Earlier studies have shown that thetyrosine test has no diagnostic value in characterizing E.jeanselmei, E. spinifera, and W. dermatitidis (5, 15), C.carrionii (14), and P. parasitica (22). However, Mok (17)found that Phaeoannellomyces werneckii lacked the abilityto hydrolyze tyrosine. Weitzman et al. (22) had similarresults with P. richardsiae. Our results agreed with those ofthe latter authors for P. richardsiae but contradicted those ofthe former authors for Phaeoannellomyces werneckii (Table5). This disagreement may be due to differences in media orin the organisms used. The inability of dematiaceous fungi tohydrolyze xanthine has been previously reported (5, 14, 15,17, 22).

All 123 isolates grew well on Czapek Dox agar (Table 5),as expected. However, it is known that sodium nitrate, whenadded to Czapek Dox medium, does not support goodgrowth of W. dermatitidis (5, 17). Trejos (21) concluded thatdifferent carbon and nitrogen sources are not importantwhen dealing with the dematiaceous fungi.

In conclusion, results of the gelatin liquefaction and other

proteolytic tests that we evaluated showed that they varydepending on their composition and the fungus tested. Thereis no significant difference between known pathogenic andnonpathogenic isolates. Thermotolerance has value for dis-tinguishing isolates of X. bantiana and C. carrionii and forisolates of E. jeanselmei and W. dermatitidis (2, 5, 7, 13, 14,16, 17, 19, 23). Thermotolerance should be used as acomplement to a careful morphological assessment of theisolates being studied. We do not recommend the use ofproteolytic and similar physiological tests for characterizingthe dematiaceous fungi.

ACKNOWLEDGMENTS

We thank Julie Rhodes and Joan F. Peters for secretarial assis-tance in the preparation of this manuscript and Stanley White for histechnical help.

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