Full text of DOI:10.1111/j.1348-0421.2002.tb02663.x

Microbiol. Immunol., 46(2), 89―93, 2002
Antifungal Pharmacodynamic Characteristics of
Amphotericin B against Trichosporon asahii,
Using Time-Kill Methodology
Yoshimi Toriumi1, Takashi Sugita*, 1, Masamitsu Nakajima2, Toshiharu Matsushima2,
and Takako Shinoda1
1Department of Microbiology, Meiji Pharmaceutical University, Kiyose, Tokyo 204 8588, Japan, and 2Division of Respiratory
―
Diseases, Department of Medicine, Kawasaki Medical School, Kurashiki, Okayama 701―0192, Japan
Received October 4, 2001; in revised form, October 30, 2001. Accepted November 14, 2001
Abstract: We determined the MIC of amphotericin B against 45 Trichosporon asahii isolates from various
clinical and environmental sources, and used in vitro time-kill methods to characterize the relationship
between amphotericin B concentrations and MIC for four representative T. asahii isolates. Amphotericin
B had concentration-dependent antifungal activity. MICs ranged from 0.5 to 16 ꢀg/ml, and most T.
asahii isolates (76%, 34/45) were inhibited at safely achievable amphotericin B serum concentrations (
2 ꢀg/ml). However, 40% (18/45) of isolates were not killed at these concentrations (MFCs from 1.0 to 32
ꢀg/ml). At concentrations 2×MIC, amphotericin B exhibited fungicidal activity (＜99.9% reduction in
CFU) over a 12-hr time-period; the maximal effect was achieved at 4×MIC. Susceptibility testing
confirmed the resistance of T. asahii to amphotericin B, and in vitro pharmacodynamic results also suggest
that amphotericin B is not suitable therapy for T. asahii infection.
Key words: Trichosporon asahii, Amphotericin B, MIC
Reports of disseminated fungal infection caused by
Trichosporon species in immunocompromised patients
have been increasing. Patients with hematologic malig-
nancies, cytotoxic chemotherapy-induced granulocy-
topenia, hemochromatosis, organ transplants, or those
receiving corticosteroids are at major risk for dissemi-
nated trichosporonosis in the event of prolonged neu-
tropenia (13, 22). Although most cases of fungemia
are due to Candida albicans or other Candida species,
Trichosporon species are implicated in approximately
10% of all confirmed cases (12). Trichosporon beigelii
was formerly considered the causative agent in trich-
osporonosis, but recent taxonomic findings revealed that
T. beigelii actually consists of more than 10 distinct
species (5, 18). According to the current taxonomy of the
genus Trichosporon, T. asahii is the major cause of dis-
seminated or deep-seated trichosporonosis (4, 6, 21).
Amphotericin B, azole agents (fluconazole and itra-
conazole), or amphotericin B plus an azole agent have
been considered as treatments for disseminated trich-
osporonosis (7, 23, 24). Animal studies suggest that
combining human granulocyte colony-stimulating factor
with amphotericin B or fluconazole is also effective
against disseminated trichosporonosis in neutropenic
mice (16). Amphotericin B has been widely used to treat
Trichosporon and other fungal infections. Strains resis-
tant to amphotericin B are very rarely isolated from
patients; only Trichosporon sp. seem resistant to ampho-
tericin B in in vitro susceptibility testing. Furthermore,
some authors report that whereas azole agents are effec-
tive against trichosporonosis, amphotericin B is not (1,
13, 26). Klepser et al. (9) standardized the time-kill
methodology used to elucidate antifungal pharmacody-
namic characteristics. The in vitro concentration-
response characteristics of several antifungal agents―
including fluconazole (2, 3, 8), itraconazole (2, 3),
amphotericin B (2, 3, 8, 17), echinocandin (11), and
voriconazole (10)―were subsequently described for
Candida albicans and Cryptococcus neoformans. The
concentration-effect relationship describes the rate and
extent of antifungal activity, and provides a more ratio-
nal basis for determining optimal dosing regimens.
This study reassessed the susceptibility of T. asahii, the
*Address correspondence to Dr. Takashi Sugita, Department of
Microbiology, Meiji Pharmaceutical University, Kiyose, Tokyo
204
―
8588, Japan. Fax:＋81
―
424
―
95
―
8762. E-mail: sugita
Abbreviations: MFC, minimal fungicidal concentration; MIC,
@my-pharm.ac.jp
minimum inhibitory concentration.
89

90
Y. TORIUMI ET AL
major causative agent of disseminated trichosporono-
sis, to amphotericin B, and used time-kill methodology
to describe the organism’s concentration-effect rela-
tionship for amphotericin B.
as the concentration that killed ＞99.9% CFU/ml.
Carryover effect. Before starting the time-kill study,
we evaluated the effect of solubilized antifungal agent on
colony count determinations by the method of Klepser et
al. (9). Briefly, a fungal suspension was added to ampho-
tericin B at a concentration equal to multiples (1/16,
1/4, 1, 2, 4, and 16) of the MIC. Samples were then
immediately plated onto Sabouraud dextrose agar for
colony count determination. Mean colony counts for
each amphotericin B concentration were compared to
those of corresponding growth controls. A difference
ꢀ25% was considered significant.
Time-kill study. Time-kill studies were performed
using the methodology of Klepser et al. (9). Briefly, a
suspension of fungus in physiological saline solution
was transferred to growth medium (RPMI 1640, MOPS,
Sigma) containing amphotericin B (range of 1/16, 1/4, 1,
2, 4, and 16 times the MIC). At 0, 2, 4, 8, 12, 24, 36, 48,
and 72 hr, samples were removed aseptically, serially
diluted, and plated onto Sabouraud dextrose agar for
colony counts.
Materials and Methods
Trichosporon asahii strains. Forty-five isolates of T.
asahii were selected for testing: 34 clinical isolates from
urine (15), blood (10), feces (4), pleural fluid (3), lung (1)
and sputum (1), plus 11 environmental isolates (8 from
soil, and 3 from the house of a patient with summer-type
hypersensitivity pneumonitis). All isolates were identi-
fied by PCR with T. asahii species-specific oligonu-
cleotide primers, or by determining the specific DNA
sequence of the species, as previously described by
Sugita et al. (19, 20).
Susceptibility testing. The MICs of amphotericin B
(Wako Pure Chemical, Osaka, Japan) for each isolate
were determined according to the M27-A method pub-
lished by the National Committee for Clinical Laboratory
Standards (14). Briefly, the growth medium consisted of
RPMI 1640 (Sigma, Mo., U.S.A.) adjusted to pH 7.0
with 0.165 M morpholinepropanesulfonic acid (MOPS,
Sigma). Fungal suspensions were standardized to a 0.5
McFarland turbidity standard, then diluted with growth
medium to yield an initial inoculum of approximately
1×103 to 5×103 CFU/ml. One hundred µl of the sus-
pension were added to each well of a microtray con-
taining 100 µl of the antifungal agent at a concentration
equal to two times the final concentration. The
microplate was incubated at 35 C for 48 hr. The ampho-
tericin B MIC was defined as the lowest drug concen-
tration that totally inhibited visible growth.
Results
MIC and MFC
The MICs and MFCs of amphotericin B against T.
asahii isolates ranged from 0.5 to 16 µg/ml (Fig. 1A) and
1.0 to 32 µg/ml (Fig. 1B), respectively. Although most
T. asahii isolates (76%, 34/45) were inhibited at safely
achievable serum concentrations (ꢁ2 µg/ml), these con-
centrations failed to kill 40% (18/45) of isolates. The
mean MFCs of blood isolates, non-blood isolates, and
environmental isolates were 8.6, 3.6, and 10.2 µg/ml,
respectively, and the differences were not statistically sig-
nificant. Notably, 80% of blood isolates (8/10) had an
MIC above 2 µg/ml. Therefore, T. asahii is relatively
resistant to amphotericin B.
Determination of minimal fungicidal concentration
(MFC). One hundred microliter samples from each
well showing growth inhibition were plated onto
Sabouraud dextrose agar. Colony counts were per-
formed after a 48-hr incubation. The MFC was defined
A
B
Fig. 1. MIC and MFC of amphotericin B against 45 isolates of T. asahii. ■, blood isolate; , non-blood isolate;
□, environmental isolate.

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PHARMACODYNAMIC CHARACTERISTICS OF AMPHOTERICIN B
Carryover Effect
16 µg/ml, four isolates were chosen as representatives of
different MICs (strain M 9492 for 1 µg/ml, strain M
9497 for 2 µg/ml, strain M 9402 for 4 µg/ml, and strain
M 9802 for 8 µg/ml) for the time-kill studies. The time-
kill curves of four T. asahii isolates are presented in
Fig. 2, A to D. For each isolate, amphotericin B had con-
centration-dependent fungicidal activity; the maximal
effect occurred at ꢀ4×MIC, and re-growth was noted
within 72 hr at concentrations ꢀ2×MIC. The antifun-
gal activity of amphotericin B after 12 and 24 hr of
A 10-µl sample was taken from solutions containing
amphotericin B, and then plated directly for colony
count determination. Antifungal carryover was not
observed at any of the amphotericin B concentrations; all
amphotericin B samples were within 25% of the mean
control colony count.
Time-Kill Study
As the MICs of T. asahii isolates ranged from 0.5 to
Fig. 2. Time-kill curves for amphotericin B against Trichosporon asahii isolates. A, M 9802 (MIC, 8 µg/ml); B, M 9402 (MIC, 4 µg/ml);
C, M 9497 (MIC, 2 µg/ml); D, M 9492 (MIC, 1 µg/ml). ●, growth control; ○, 1/16×MIC; ▲, 1/4×MIC; △, 1×MIC; ■, 2×MIC;
□, 4×MIC; ◆, 16×MIC.
A
B
Fig. 3. Relationship of amphotericin B concentration to antifungal activity against four T. asahii isolates. ●, M 9802 (MIC, 8 µg/ml);
○, M 9402 (MIC, 4 µg/ml); ▲, M 9497 (MIC, 2 µg/ml); △, M 9492 (MIC, 1 µg/ml).

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Y. TORIUMI ET AL
contact with the four isolates was plotted as the multiple
of the MIC (Fig. 3, A and B). Overall, the fungicidal
activity of amphotericin B appeared to plateau at con-
centrations exceeding 2×MIC (Fig. 3).
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