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Antimicrobial Agents and Chemotherapy, November 1998, p. 3018-3021, Vol. 42, No. 11
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Organism-Dependent Fungicidal Activities of
Azoles
Elias K.
Manavathu,*
Jessica L.
Cutright, and
Pranatharthi H.
Chandrasekar
Division of Infectious Diseases, Department
of Medicine, Wayne State University School of Medicine, Detroit,
Michigan 48201
Received 8 December 1997/Returned for modification 10 March
1998/Accepted 13 August 1998
 |
ABSTRACT |
We investigated the antifungal activities of itraconazole and
voriconazole on Aspergillus species by time kill studies,
and the results were compared with those obtained for
Candida species. Exposure of Aspergillus
fumigatus conidia to varying concentrations (1.25 to 10 µg/ml)
of itraconazole and voriconazole resulted in cellular death; the
cytocidal effect was time and concentration dependent. In contrast, no
killing of Candida albicans occurred in the presence of
itraconazole and voriconazole at concentrations as high as 10 µg/ml,
although candidal growth was inhibited compared to the drug-free
control. Amphotericin B (1.25 to 10 µg/ml), on the other hand, killed
both A. fumigatus and C. albicans. Similar results were obtained for non-A. fumigatus aspergilli and
non-C. albicans Candida species. These observations
indicate that both itraconazole and voriconazole are cytocidal agents
for Aspergillus species but not for Candida
species, suggesting that azoles possess organism-dependent fungicidal activities.
| |
TEXT |
Amphotericin B and certain members
of the azole family of antifungals are the most commonly used
antibiotics for the treatment of systemic fungal infections (3-6,
14). Amphotericin B acts on the cytoplasmic membrane, in
particular the ergosterol component (1, 13), leading to
irreversible damage of the membrane and consequent leakage of essential
nutrients. The lack of a permeability barrier to essential nutrients
and ions is believed to be responsible for the fungicidal activity of
amphotericin B (2, 11, 12). In contrast, the azoles are
known to be fungistatic agents. These heterocyclic compounds inhibit
the synthesis of sterol in fungi by inhibiting cytochrome
P-450-dependent 14
-lanosterol demethylase (P-45014DM),
which specifically removes the methyl group on C-14 of lanosterol
(5, 16-18). This demethylation is an essential intermediate
step in the synthesis of ergosterol, the major sterol found in fungi.
The action of azoles on P-45014DM of yeasts appears to be
reversible; once the drug is removed, the organism recovers rapidly and
functions normally. As a result of the static effect of azoles on
yeasts, it is difficult to obtain clearly defined endpoints during
susceptibility studies, and the so-called "trailing phenomenon" is
very common (9, 15). On the other hand, the trailing
phenomenon is uncommon during susceptibility tests with fungicidal
agents such as amphotericin B and nystatin. During our recent studies
of the susceptibility of Aspergillus species to
voriconazole, we observed clearly defined endpoints, and prolonged incubation of the MIC tubes rarely resulted in higher MICs
(8). Moreover, the minimum fungicidal concentrations were
only two- to fourfold higher than the MICs. These findings suggested
that voriconazole acts possibly as a fungicidal agent for
Aspergillus fumigatus and prompted us to examine the
activity of voriconazole against pathogenic yeasts and
Aspergillus species by kill curve experiments and to compare
the results with those obtained for itraconazole and amphotericin B.
Clinical isolates of A. fumigatus and non-A.
fumigatus species were obtained from the Microbiology Laboratory,
Detroit Medical Center, Wayne State University. Working cultures were
maintained on peptone-yeast extract-glucose (PYG) (peptone, 1 g;
yeast extract, 1 g; glucose, 3 g [per liter of distilled
water]) agar at room temperature. The primary cultures obtained from
the Detroit Medical Center were subcultured on PYG agar to assure the
purity of the cultures. Long-term storage of the cultures was done as
conidial suspensions in 25% glycerol at 
70°C.
Working cultures of Candida species used in this study were
grown for 48 h at 30°C on Sabouraud dextrose agar from stock
cultures stored at 70°C in litmus milk (Becton Dickinson
Microbiology Systems, Cockeysville, Md.). Single colonies from the
2-day-old cultures were used as the source of the inoculum for all
subsequent experiments.
Conidial suspensions of various Aspergillus species were
prepared, and the MICs of various antifungals were determined as described previously (7, 10). Briefly, fresh conidia were resuspended in PYG medium at a density of 2 × 104
conidia/ml. Twice the required concentrations of the drugs were prepared in PYG medium (0.5 ml) by serial dilution in sterile 6-ml
polystyrene tubes (Falcon 2054) and inoculated with an equal volume
(0.5 ml) of the conidial suspension. The tubes were incubated at 35°C
for 48 h and scored for visible growth after gentle vortexing of
the tubes or scraping of the walls of the tubes followed by vortexing.
The MIC was defined as the lowest concentration of drug at which no
visible growth occurred. The MIC determination for each isolate was
repeated at least once, and the data were within ±1 dilution.
The MICs of amphotericin B, itraconazole, and voriconazole for various
Candida species used in this study were determined by the
broth microdilution method, as recommended by the National Committee
for Clinical Laboratory Standards (9). The MIC was defined
as the lowest concentration of drug that inhibited growth by 80%
compared to the drug-free control after 48 h of incubation at
35°C.
Aspergillus spp.
Five milliliters of conidial
suspension prepared in PYG broth (106 conidia/ml) was
incubated at 35°C in the presence of various concentrations of
amphotericin B, itraconazole, and voriconazole (0 to 10 µg/ml). At
various time intervals, 0.1-ml aliquots of the conidial suspension were
removed and diluted appropriately to obtain 10- to 104-fold
dilutions, and 0.1-ml aliquots were spread in duplicate on PYG agar
plates. The plates were incubated at 35°C for 48 h, and the
numbers of CFU per milliliter of conidial suspension were determined.
Candida spp.
Fresh 24-h-old cultures of
Candida species prepared in PYG broth were diluted
approximately 1,000-fold to obtain a cell density of 106
CFU per ml. One-milliliter aliquots of the diluted cultures were incubated with 0 to 10 µg of the antifungals per ml at 35°C. At various time intervals, aliquots (0.05 ml) of the drug-treated cell
suspensions were removed and serially diluted (10- to
106-fold), and 0.1-ml amounts were spread on Sabouraud
dextrose agar plates in duplicate. The plates were incubated at 35°C
for 24 h, and the numbers of CFU/ml of culture were determined. A
similar treatment without the drug was used as a growth control where applicable.
Itraconazole (R 51,211, batch no. STAN-9304-005-1), voriconazole, and
amphotericin B (batch no. 20-914-29670) were obtained from Janssen
Pharmaceutica, Beerse, Belgium; Pfizer Pharmaceuticals, New York, N.Y.;
and Squibb Institute for Medical Research, Princeton, N.J.;
respectively. All antifungals were dissolved in dimethyl sulfoxide at
concentrations of 1 mg/ml and stored as 0.25-ml aliquots at 20°C.
The frozen stocks were thawed at room temperature and gently vortexed
several times to ensure that any remaining crystals were completely
dissolved before use. Drug concentrations ranging from 0 to 16 µg/ml
were used for MIC determinations. Comparable concentrations of dimethyl
sulfoxide were tested to examine its effect on the growth of A. fumigatus. No detectable inhibition of growth occurred at the
concentrations used.
The MICs of amphotericin B, itraconazole, and voriconazole obtained for
various
Aspergillus and
Candida species are shown
in Table
1. As shown, all of the isolates
used in this study
were susceptible to low concentrations of
amphotericin B (MIC
range, 0.02 to 4 µg/ml), itraconazole (MIC range,
0.031 to 4 µg/ml),
and voriconazole (MIC range, 0.015 to 2 µg/ml).
The effects of amphotericin B, itraconazole, and voriconazole over a
24-h period on the ability of
A. fumigatus W73355 conidia to
produce colonies are shown in Fig.
1. The
concentrations
(1.25 to 10 µg/ml) of the drugs used for the kill
curve studies
were 2.5- to 20-fold higher than the MICs of the drugs
for efficient
killing. All three compounds at the concentrations used
reduced
the number of CFU with time in a dose-dependent manner,
compared
to the initial inoculum. For example, approximately 99% of
the
conidia were killed by amphotericin B at 5 µg/ml (Fig.
1A) within
24 h. Under the same conditions, itraconazole at 5 µg/ml (Fig.
1B) provided 85% killing. Approximately 95% killing was obtained
with
voriconazole at 5 µg/ml (Fig.
1C), suggesting that it has
slightly
better fungicidal activity than itraconazole but is not
as efficient as
amphotericin B.
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|
FIG. 1.
Comparison of the fungicidal activities of amphotericin
B (A), itraconazole (B), and voriconazole (C) against A. fumigatus W73355. Each point represents the mean of two
independent determinations. Experiments were repeated three times with
similar results; the data shown here are from a typical experiment.
|
|
Figure
2 shows the effects of exposure of
Candida albicans to amphotericin B, itraconazole, and
voriconazole over a 24-h period.
In contrast to the fungicidal
activities of the three drugs against
Aspergillus, only
amphotericin B showed a reduction in CFU per
milliliter with time
against
Candida species. Amphotericin B at
5 and 10 µg/ml
provided
99.99 and 100% killing, respectively
(Fig.
2A) within
24 h, whereas no killing was obtained at either
5 or 10 µg of
itraconazole (Fig.
2B) or voriconazole (Fig.
2C)
per ml. With both
azoles, the growth of the organism after 24
h was inhibited
approximately 80 to 85% compared to the drug-free
control. However,
there was an increase in CFU per milliliter
compared to the initial
inoculum.
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|
FIG. 2.
Comparison of the fungicidal or fungistatic activities
of amphotericin B (A), itraconazole (B), and voriconazole (C) against
C. albicans 90028. Each point represents the mean of two
independent determinations. Experiments were repeated twice with
similar results; the data shown here are from a typical experiment.
|
|
In addition to
A. fumigatus and
C. albicans, we
investigated the fungicidal activities of amphotericin B, itraconazole,
and
voriconazole on other clinically important species of
Aspergillus and
Candida. As shown in Table
2, non-
A. fumigatus aspergilli
examined showed
87% reduction in CFU per milliliter within 24
h
of drug exposure. Hence, itraconazole and voriconazole were
fungicidal
against
A. fumigatus and other
Aspergillus
species
tested. All seven non-
C. albicans Candida species
examined were
killed (
95%) within 24 h by amphotericin B,
whereas no killing
was obtained with voriconazole and itraconazole.
This finding
demonstrated that the azoles tested have only fungistatic
activity
against
C. albicans and the other yeasts examined.
View this table:
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|
TABLE 2.
Fungicidal or fungistatic activities of amphotericin B,
itraconazole, and voriconazole against Aspergillus and
Candida speciesa
|
|
Our present observations show that itraconazole and voriconazole, like
amphotericin B (albeit to a lesser degree), have fungicidal
activity
against
A. fumigatus and the other
Aspergillus
species
examined. Shorter incubation times of conidia in the presence
of higher concentrations of drugs were not very effective. This
finding
is not surprising considering that any killing of cells
via inhibition
of the sterol synthetic pathway would take longer
than production of a
leaky cytoplasmic membrane by direct action
on ergosterol (e.g.,
amphotericin B). Furthermore, our studies
support previous findings
that the azoles have fungistatic activity
against
Candida
species. Varying concentrations (including levels
achievable in humans)
of itraconazole and voriconazole showed
time-dependent cytocidal
activities against
Aspergillus species
but only cytostatic
effects against
Candida species. The exact
reason(s) for
such differential activities of azoles against fungi
is not clear. It
is possible that the sterol synthetic pathway
in
Aspergillus
is essential and that inhibition of ergosterol
synthesis may lead to
cell death in
Aspergillus but not in
Candida.
As
fungal infections due to filamentous fungi are seen increasingly
in
patients with immunocompromised states, the cytocidal activities
of
azoles against
Aspergillus species may be of clinical
significance
and hold promise in the treatment of these frequently
fatal
infections.
| |
ACKNOWLEDGMENTS |
We thank William Brown and Joe Vazquez (Detroit Medical Center,
Wayne State University) for kindly providing the various isolates of
Aspergillus and Candida species used in this study.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine, Wayne State University, 427 Lande Building, 550 E. Canfield, Detroit, MI 48201. Phone: (313) 577-1931. Fax: (313) 993-0302. E-mail:
aa1388{at}wayne.edu.
| |
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Antimicrobial Agents and Chemotherapy, November 1998, p. 3018-3021, Vol. 42, No. 11
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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