JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1990, p. 2823-2827 Vol. 28, No.

12
0095-1137/90/122823-05$02.00/0
Copyright © 1990, American Society for Microbiology

Patterns of Morphologic Variation among Isolates of

Trichosporon beigelii
JAMES W. LEE,' GREGORY A. MELCHER,2 MICHAEL G. RINALDI,34 PHILIP A. PIZZO,l
AND THOMAS J. WALSH'*
Infectious Disease Section, Pediatric Branch, National Cancer Institute, Bethesda, Maryland 208921; Section of
Infectious Diseases, Wilford Hall U.S. Air Force Medical Center, Lackland Air Force Base, San Antonio,
Texas 782362; and Department of Pathology, University of Texas Health Science Center at
San Antonio,3 and Laboratory Service, Audie L. Murphy Veterans Administration
Medical Center,4 San Antonio, Texas 78284

Downloaded from http://jcm.asm.org/ on February 29, 2020 by guest

Received 20 June 1990/Accepted 28 August 1990

We observed considerable variability in colony and microscopic morphology among isolates of Trichosporon
beigelii. Deeply invasive clinical isolates showed four distinct morphotypes and spontaneous conversions among
certain morphotypes and grew well at 37°C. In contrast, superficial clinical and environmental isolates did not
demonstrate such morphotypes or conversions, and most grew poorly at 37°C. Thus, the morphologic and
physiologic features of invasive clinical isolates of T. beigelii follow certain patterns distinct from those of
superficial clinical and environmental isolates.

Trichosporon beigelii (Kuchenmeister et Rabenhorst)
Vuillemin is an emerging fungal pathogen that causes dis-
seminated infections in immunocompromised patients (4,
10-12). Pathogenic isolates of T. beigelii may be isolated in
clinical microbiology laboratories from blood and urine or
from biopsies of skin, lungs, or kidneys.
The colony of this fungus is variably described in standard
reference laboratory mycology texts as being white to yel-
lowish, creamy or smooth to wrinkled or lacy, or powdery or
velvety to rugose (1-3, 6-9). Microscopically, T. beigelii is
characterized by the presence of blastoconidia, arthro-
conidia, hyphae, and pseudohyphae, but detailed studies
have also noted variable microscopic morphology among
isolates (2, 5).
We also observed this variability in colony and micro-
scopic morphology among isolates of T. beigelii recently
recovered from patients with disseminated infections. One
of these isolates (TSAS-87) showed four different morpho-
types, with distinct colony and microscopic morphologies.
Moreover, other deeply invasive clinical isolates of T.
beigelii appeared to follow a similar phenotypic pattern. To
better understand these morphologic patterns and improve
the clinical laboratory diagnosis of T. beigelii, we studied the
colony and microscopic features, as well as the physiologic
properties, of a panel of isolates of this emerging fungal
pathogen.
Eighteen of 24 isolates originally obtained were found to
be suitable for study. Isolates were maintained on potato
dextrose agar slants at -70°C until subcultured onto Em-
mons modified Sabouraud glucose agar (SGA) for further
study. All morphologic examinations were performed on
colonies grown from pinpoint inoculations on SGA plates at
37, 30, and 25°C for 72 h and on cornmeal agar with Tween
80 (Media Unit, National Institutes of Health, Bethesda,
Md.) at 25°C by the Dalmau technique (1, 8). Microscopic
*
Corresponding author.
2823
morphology was characterized from wet mounts examined
at x400 under a phase-contrast microscope.
Carbohydrate assimilation patterns were determined for
all isolates with API 20C strips (Analytab Products, Plain-
view, N.Y.) as directed by the manufacturer. The kinetics of
growth of the four morphotypes of TSAS-87 were deter-
mined in triplicate by inoculation of 5 x 103 CFU of each
organism per ml into Sabouraud glucose broth and incuba-
tion at 37°C. The concentrations of organisms were deter-
mined with a hemacytometer every 2 h for 12 h, and the
mean doubling times in the log phase were determined.
Two major morphotypes of TSAS-87, with different gross
colony and microscopic morphologies, were consistently
observed on SGA. One type, which we designated Rugose,
formed white, jagged, peaked colonies at both 37 and 25°C;
the other, which we designated Powdery, formed a more
finely granulated colony, especially at 25°C (Fig. 1). Micro-
scopically, these morphotypes differed strikingly in that
Rugose consisted of >99% hyphae but Powdery consisted of
>90% blastoconidia or arthroconidia and few hyphae (Fig.
2).
During subculturing of these two major morphotypes,
spontaneous conversions of Rugose to Rugose-Gray variants
and Powdery to Powdery-Gray variants (Fig. 1) were ob-
served (Fig. 3). Both types of variants were stably replicated
by further serial subculturing. These two types of variants
both formed gray, glabrous colonies. Microscopically, how-
ever, the variants shared the morphologic characteristics of
their parent morphotypes (Fig. 2). Microscopically, Rugose-
Gray, like Rugose, was >99% hyphae at both 25 and 37°C
and was indistinguishable from Rugose. Like Powdery,
Powdery-Gray consisted of over two-thirds blastoconidia or
arthroconidia, but unlike Powdery, Powdery-Gray consisted
of more hyphae, especially at 37°C.
The other deeply invasive clinical isolates showed features
similar to those of the index morphotypes of TSAS-87 (Table
1). Among these isolates, two (CDC 21-87 and UMSMT-1)
were indistinguishable from Powdery, while two others
(UMSMT-2 and UMSMT-3) were nearly identical to Rugose
2824 NOTES
FIG. 1. Colony morphologies of Rugose (A), Powdery (B), Rugose-Gray (C), and Powdery-Gray (D) morphotypes of TSAS-87 after 72 h
at 25°C on SGA.
in both colony and microscopic appearances at both 25 and
37°C. Four isolates (1135-88, 1181-82, 297-87, and 958-85)
also showed Powdery morphology at 25°C, but at 37°C they
showed a morphology similar to Powdery-Gray. Three iso-
lates (TCM-86, UM-1P, and UM-2P) were similar to Pow-
dery-Gray at both 25 and 37°C. All isolates with either
Powdery or Rugose morphologies at 25°C exhibited sponta-
neous conversions to corresponding Gray variants at both 25
and 37°C (as for TSAS-87; Fig. 3). TSAS-87 was the only
clinical isolate found to have both the Powdery and Rugose
major morphotypes and to express both types of Gray
variants.
In contrast to the deeply invasive clinical isolates, the two
clinical isolates (342-85 and 38300) obtained from superficial
sites (toenail and skin) produced creamy, wet-looking colo-
nies, with the creamy color and consistency especially
J. CLIN. MICROBIOL.
Downloaded from http://jcm.asm.org/ on February 29, 2020 by guest
notable after 3 days at 37°C or 5 to 7 days at 25°C (Fig. 4).
Both isolates grew more slowly at 37°C. Microscopically,
these isolates had a predominance of hyphae at 25°C, in
contrast to the deeply invasive clinical isolates, in which
more hyphae were seen at 37°C (Table 1). Many arthro-
conidia (>20%) were also present. No conversion to Gray
variants was ever observed.
None of the four environmental isolates showed any
similarity macroscopically to the four morphotypes seen
among the deeply invasive clinical isolates (Table 1). All
were gray at 25°C, grew poorly or not at all at 37°C, and
exhibited variable colony morphologies that were neither
Powdery nor Rugose nor creamy and wet looking (Fig. 4).
Microscopically, all except 14905 had >99% hyphae.
For all isolates, there was no appreciable difference in
colony morphology on SGA between colonies grown at 25
 . A

*`:r
VOL. 28, 1990

j_,Xs,

} }:s
,/. . .
..
..

B
-
..
fc

..,; ....

..
>.

;,
..

..

(
...
,

çr
4

0"`uF
(vYl

~crqî?r
t ;s
i
D

4
.~.. .....
+2 3 j w

J
NOTES 2825

z w

Downloaded from http://jcm.asm.org/ on February 29, 2020 by guest

FIG. 2. Microscopic morphologies of Rugose (A), Powdery (B), Rugose-Gray (C), and Powdery-Gray (D) morphotypes of TSAS-87 after
72 h at 25°C on SGA.

versus 30°C. However, colonies that consisted of evenly Rugose --------- > Rugose-Gray
proportioned mixtures of blastoconidia, arthroconidia, and ,
1

hyphae at 37°C but not at 25°C tended to retain this mixed

microscopic morphology at 30°C.
The morphology of isolates grown on cornmeal agar with
Tween 80 at 25°C for 24 to 72 h was consistent with that of
T. beigelii for all isolates. Deeply invasive clinical isolates,
however, showed nearly identical morphologic features of
blastoconidia and hyphae, with the only discernible differ-
Powdery ----------- > Powdery-Gray
FIG. 3. Spontaneous conversions from the predominant Rugose
and Powdery morphotypes of TSAS-87 to the variants Rugose-Gray
and Powdery-Gray.
2826 NOTES J. CLIN. MICROBIOL.

TABLE 1. Morphologic and physiologic characteristics of isolates of T. beigelii

Morphology at:
Isolated Sourceb 250C 370C Physiology
(A PI 20C code)
Gross Microscopicc Gross Microscopicc
Deeply invasive clinical
TSAS-87 Powderyd 1 Powdery Blastoconidia Powdery Blastoconidia 274477le
TSAS-87 Rugosed 1 Rugose Hyphae Rugose Hyphae 2746770f
TSAS-87 Powdery- 1 Powdery-Gray Mixed Powdery-Gray Mixed 274477le
Grayd
TSAS-87 Rugose- 1 Rugose-Gray Hyphae Rugose-Gray Hyphae 27467719
Grayd
CDC 21-87 2 Powdery Blastoconidia Powdery Blastoconidia 274477le
UMSMT-1 2 Powdery Blastoconidia Powdery Blastoconidia 274477Ve

Downloaded from http://jcm.asm.org/ on February 29, 2020 by guest

UMSMT-2 2 Rugose Hyphae Rugose Hyphae 2744730f
UMSMT-3 2 Rugose Hyphae Rugose Hyphae 2744730f
1135-88 3 Powdery Blastoconidia Powdery-Gray Mixed 274477Ve
1181-82 3 Powdery Blastoconidia Powdery-Gray Mixed 2744771e
297-87 3 Powdery Blastoconidia Powdery-Gray Mixed 274477Ve
958-85 3 Powdery Blastoconidia Powdery-Gray Mixed 274477Ve
TCM-86 4 Powdery-Gray Mixed Powdery-Gray Mixed 2744730f
UM-1P 5 Powdery-Gray Mixed Powdery-Gray Mixed 274477Ve
UM-2P 5 Powdery-Gray Mixed Powdery-Gray Mixed 274477Ve
Superficial clinical
342-85 3 Creamy, wet, Hyphae, many Creamy, white; slow Mixed, many arth- 6744771f
fuzzy edges arthroconidia growth roconidia
38300 6 Fuzzy, gray Hyphae, many Creamy, wet, gray, Blastoconidia, many 2776773g
arthroconidia furrowed; slow arthroconidia
growth
Environmental
10266 6 Filamentous, gray Hyphae No growth 2544730e
11115 6 Furrowed, gray- Hyphae Furrowed, gray; slow Mixed 274477Ve
white growth
14905 6 Furrowed, gray Mixed No growth 27447319
28574 6 Furrowed, gray- Hyphae Furrowed, gray-white; Hyphae 274477Ve
white slow growth
a Deeply invasive clinical isolates were all from blood, except for one from sputum and one from a stool sample. Superficial clinical isolates were from a toenail
and leg skin. Environmental isolates were from soil, water, unprocessed milk, etc. Rejected isolates (22164, 22375, 32902, 32967, 34148, and 34181 [from the
American Type Culture Collection]) were contaminated, failed to grow, or had carbohydrate assimilation patterns inconsistent with T. beigelii.
b 1, University of Texas at San Antonio; 2, University of Maryland School of Medical Technology; 3, New York State Department of Health; 4, Microbiology
Service, Warren Grant Magnuson Clinical Center; 5, University of Maryland; 6, American Type Culture Collection.
C Explanations: Blastoconidia, >90% mostly blastoconidia with arthroconidia, and <10% hyphae; Hyphae, >99% hyphae, with rare blastoconidia or
arthroconidia; Mixed, approximately equal proportions of blastoconidia or arthroconidia and hyphae; many arthroconidia, >20% arthroconidia also present.
d Index
morphotype.
e
Acceptable likelihood of T. beigelii identification.
f Excellent likelihood of T. beigelii identification.
g Good likelihood of T. beigelii identification, but low selectivity.

ence between the isolates being that the Rugose isolates
continued to have >99% hyphae. On the other hand, both
the superficial clinical and environmental isolates showed
marked variability in the size, shape, branching, and relative
proportions of blastoconidia, arthroconidia, and hyphae.
The carbohydrate assimilation patterns of all the isolates
were very similar and coded for T. beigelii with comments of
excellent, good likelihood but low selectivity, or acceptable
(Table 1). The kinetics of growth of the index morphotypes
(TSAS-87) revealed mean doubling times in the log phase of
1.0 + 0.1 h for Powdery, 0.9 ± 0.1 h for Powdery-Gray, 1.2
+ 0.2 h for Rugose, and 1.2 ± 0.1 h for Rugose-Gray. The
more blastoconidial Powdery and Powdery-Gray morpho-
types grew faster than did the hyphal Rugose and Rugose-
Gray morphotypes (P < 0.05, Student's paired t test).
We have described a pattern of phenotypic variations in
deeply invasive clinical isolates of T. beigelii and have
classified four morphotypes, Powdery, Rugose, Powdery-
Gray, and Rugose-Gray. In contrast, the superficial clinical
and environmental isolates studied demonstrated a much
greater variety of morphologic features that did not follow
any particular pattern.
These observations are particularly useful for clinical mi-
crobiology laboratories in the identification of T. beigelii
because they indicate that the marked morphologic variations
occurring both macroscopically and microscopically appear
to follow certain patterns in invasive clinical isolates and that
systemic pathogens differ in morphologic features from su-
perficial clinical and environmental isolates. In addition,
awareness that temperature-dependent morphologic changes
and spontaneous conversions to Gray variants can occur may
prevent the misinterpretation of cultures as being mixed or
contaminated. The recognition of the differences between
pathogenic and environmental forms of T. beigelii may also
aid in the interpretation of culture results as possibly repre-
senting invasive disease versus skin contamination.
VOL. 28, 1990
FIG. 4. Colony morphology of 342-85 (A), a superficial clinical isolate with a creamy, wet texture, and 10266 (B), an environmental isolate
with a filamentous, starlike appearance. Note that both colonies, grown on SGA for 72 h at 25°C, differed strikingly from TSAS-87.
We especially thank Janet W. B. Andrews of the Clinical Micro-
biology Laboratory, National Institutes of Health, for contributing
time and expertise, and we thank Dennis M. Dixon of the New York
State Department of Health, Andrew G. Smith of the University of
Maryland School of Medical Technology, and Marcia M. Moody of
the University of Maryland for contributions of isolates. We also
thank Joanne Peter, Vanessa Thomas, and Robert Schaufele for
technical assistance.
LITERATURE CITED
1. AI-Doory, Y. 1980. Laboratory medical mycology, p. 89, 278-
279. Lea & Febiger, Philadelphia.
2. Carmo-Sousa, L. D. 1970. Trichosporon Behrend, p. 1326-1333.
In J. Lodder (ed.), The yeasts. A taxonomic study. North-
Holland Publishing Co., Amsterdam.
3. Emmons, C. W., C. H. Binford, J. P. Utz, and K. J. Kwon-
Chung. 1977. Medical mycology, 3rd ed., p. 182-183. Lea &
Febiger, Philadelphia.
4. Hoy, J., K. Hsu, K. Ralston, M. Luna, and G. P. Bodey. 1986.
Trichosporon beigelii infection: a review. Rev. Infect. Dis.
8:959-967.
NOTES 2827
Downloaded from http://jcm.asm.org/ on February 29, 2020 by guest
5. King, D. S., and S. C. Jong. 1977. A contribution to the genus
Trichosporon. Mycotaxon 6:391-417.
6. Koneman, E. W., and G. D. Roberts. 1985. Practical laboratory
mycology, 3rd ed., p. 140-141. The Williams & Wilkins Co.,
Baltimore.
7. Kreger-van Rij, N. J. W. (ed.). 1984. The yeasts: a taxonomic
study, 3rd ed., p. 942-946. Elsevier Science Publishers B. V.,
Amsterdam.
8. McGinnis, M. R. 1980. Laboratory handbook of medical mycol-
ogy, p. 399-400. Academic Press, Inc., New York.
9. Rippon, J. W. 1988. Medical mycology: the pathogenic fungi
and pathogenic actinomycetes, 3rd ed., p. 166-167. The W. B.
Saunders Co., Philadelphia.
10. Walling, D. M., D. J. McGraw, W. G. Merz, J. E. Karp, and
G. M. Hutchins. 1987. Disseminated infection with Trichospo-
ron beigelii. Rev. Infect. Dis. 9:1013-1019.
11. Walsh, T. J. 1989. Trichosporonosis. Infect. Dis. Clin. North
Am. 3:43-52.
12. Walsh, T. J., K. A. Newman, M. Moody, R. Wharton, and J. C.
Wade. 1986. Trichosporonosis in patients with neoplastic dis-
ease. Medicine (Baltimore) 65:268-279.