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Antimicrobial Agents and Chemotherapy, October 1998, p. 2645-2649, Vol. 42, No. 10
St. John's Cardiovascular Research Center,
Division of Infectious Diseases, Harbor-UCLA Research and Education
Institute, Torrance, California 905021;
Pfizer Central Research, Sandwich, Kent, United
Kingdom2; and the
UCLA School of
Medicine, Los Angeles, California 900243
Received 2 April 1998/Returned for modification 30 May
1998/Accepted 9 July 1998
The mechanisms of fluconazole resistance in three clinical isolates
of Candida krusei were investigated. Analysis of sterols of
organisms grown in the absence and presence of fluconazole demonstrated
that the predominant sterol of C. krusei is ergosterol and
that fluconazole inhibits 14 As the use of azole antifungal
agents has risen dramatically, there has been an increase in the number
of reports of azole-resistant isolates of Candida,
especially in patients with advanced AIDS. In addition to the
development of resistance by Candida albicans and
Candida glabrata, it has been reported that the frequent use of fluconazole can select for the emergence of Candida
krusei as a commonly isolated opportunistic pathogen in some
medical centers (1, 33). This finding is clinically
significant because C. krusei can cause serious infections
in susceptible patients (8, 19). Furthermore, this organism
is usually intrinsically resistant to fluconazole, both in vitro
(3) and in vivo (4).
Three general mechanisms of azole resistance have been described for
species of Candida. The first is an alteration in the target
enzyme, 14 To determine if fluconazole resistance in C. krusei is
mediated by one or more of these mechanisms, we analyzed the effects of
this antifungal agent on sterol synthesis by three strains of C. krusei. In addition, the fluconazole uptake and cytochrome P-450
content of these organisms were measured. Our results indicate that the
predominant mechanism of fluconazole resistance in these organisms is a
14 Fungal strains and susceptibility testing.
Three clinical
isolates of C. krusei, strains 91-1158, 91-1159, and
91-1161, were generously provided by Michael Rinaldi (San Antonio,
Tex.). A fourth isolate of C. krusei, ATCC 6258, was obtained from the American Type Culture Collection (Rockville, Md.).
Two clinical isolates of C. albicans, Y01.345 and SC5314, were supplied by Christopher Hitchcock and William Fonzi (Georgetown University School of Medicine, Washington, D.C.), respectively. The
susceptibilities of the organisms to fluconazole and itraconazole were
determined at 24 and 48 h by the National Committee for Clinical Laboratory Standards M27-A broth microdilution method at an inoculum of
103 organisms per well (20). The medium was RPMI
1640 (Irvine Scientific, Santa Ana, Calif.) buffered to pH 7.0 with
0.165 M morpholinepropanesulfonic acid (MOPS). The MICs were defined as
the concentrations of drug that reduced growth by 80% compared to that
of organisms grown in the absence of drug.
Sterol analysis.
For sterol extraction, each strain of
C. krusei was grown for 24 h at 37°C on a rotary
shaker. The medium was Sabouraud dextrose broth (Difco, Detroit,
Mich.), with and without fluconazole. The concentration of fluconazole
was 16 µg/ml, the highest concentration at which these organisms
would grow in this medium. The organisms were harvested by
centrifugation and washed twice in 0.85% saline, and their total
sterols were extracted by ethanolic KOH, as described previously
(6, 7). The resultant sterols were further purified by
thin-layer chromatography on PK6F silica gel 60-Å plates (Whatman, Clifton, N.J.) with a solvent system of petroleum ether-diethyl ether
(3:1, vol/vol) (27). The sterols were eluted from the silica
in chloroform-diethyl ether-ethanol (1:1:1). After being derivatized
with hexamethyldisilazane and trimethylchlorosilane (28),
the sterols were redissolved in hexane and analyzed by gas
chromatography-mass spectrometry. The sterols were identified by
comparison to known standards and published data (16, 17, 23).
Fluconazole accumulation.
A filter-based assay was used to
measure the accumulation of fluconazole by the organisms
(22). These organisms were grown to exponential phase in
Sabouraud dextrose broth and suspended in phosphate-buffered saline (pH
7.5) containing 5% glucose (wt/vol) at 108 organisms per
ml. Next, a mixture of [3H]fluconazole (specific
activity, 715 GBq/mmol) and unlabeled fluconazole was added to the
cells so that the final concentration of fluconazole was 100 nM (0.2 µCi/ml). At selected intervals, aliquots were removed and the
organisms were collected by filtration. Next, the organisms were washed
four times in phosphate-buffered saline containing 100 µM unlabeled
fluconazole. The amount of cell-associated radioactivity was determined
by scintillation counting. All experiments were performed in
triplicate.
Carbon monoxide difference spectra of microsomes.
The
cytochrome P-450 content of the organisms was analyzed by measuring
their carbon monoxide difference spectra. Organisms were grown to
exponential phase in Sabouraud dextrose broth at 37°C and harvested
by centrifugation. They were spheroplasted with lyticase (Sigma, St.
Louis, Mo.) in sorbitol buffer (1.5 M sorbitol in 10 mM Tris buffer, pH
7.4) and resuspended in 10 mM Tris buffer, pH 7.4, containing 0.65 M
sorbitol, 0.1 mM EDTA, 0.1 mM glutathione, and protease inhibitors
(Boehringer Mannheim, Indianapolis, Ind.) (10). All
subsequent steps were carried out at 4°C. The spheroplasts were
broken into microsomes by sonication, after which debris and unbroken
cells were removed by centrifugation at 1,500 × g for
10 min followed by 25,000 × g for 25 min. The microsomes were harvested with calcium chloride by the method of
Käppeli et al. (13), washed once in 10 mM Tris buffer,
pH 7.4, containing 150 mM potassium chloride, and resuspended in 100 mM
Tris containing 0.65 M glycerol, 0.1 mM EDTA, and 0.1 mM glutathione.
The carbon monoxide difference spectra of the microsomes were measured,
and their cytochrome P-450 content was calculated by using an
extinction coefficient of 91 liters/mmol/cm (21). The
protein concentration of each microsome suspension was determined by
the Bio-Rad protein assay (Bio-Rad, Hercules, Calif.). For each
organism, the cytochrome P-450 contents of at least three different
preparations of microsomes were measured.
Sterol biosynthesis by cell extracts.
To determine the
effects of fluconazole on the synthesis of ergosterol from
[14C]mevalonic acid, the organisms were grown in
Sabouraud dextrose broth until late exponential phase. The organisms
were broken by vortexing with glass beads in 0.1 M potassium phosphate,
pH 7.5. Debris and unbroken cells were removed by centrifugation at
2,000 × g for 5 min followed by 10,000 × g for 10 min. To measure sterol biosynthesis, 925 µl of
the resultant supernatant was added to 75 µl of cofactor buffer to
achieve the following final concentrations: 1 µM NADP, 1 µM NADPH,
1 µM NAD, 7 µM glucose-6-phosphate, 5 µM ATP, 3 µM reduced
glutathione, 2 µM MnCl2, and 0.25 µCi
[14C]mevalonic acid (10, 12). Selected
concentrations of fluconazole were added to the reaction mixtures
before the addition of the cell extracts. After incubation at 37°C
for 2 h, the reaction was stopped with ethanolic KOH. The samples
were saponified at 80°C for 45 min, after which the nonsaponifiable
lipids were extracted with petroleum ether (bp 40 to 60°C). The
samples were dried under nitrogen and redissolved in chloroform. The
sterols were separated by thin-layer chromatography on silica gel LK6D
(Whatman) with a solvent system of petroleum ether-diethyl ether (3:1,
vol/vol) (27). The sterols were visualized by iodine
staining and identified by comparison with commercially available
standards which were run in parallel. Next, the sterol-containing bands
were scraped from the plates and 14C incorporation was
determined by liquid scintillation counting. All experiments were
repeated at least three times with different cell extracts.
Growth in fluconazole reduced the ergosterol content of C. krusei.
Fluconazole MICs for C. krusei 91-1158, 91-1159, and 91-1161 were all high, whereas both strains of C. albicans were susceptible to this drug (Table
1). C. albicans Y01.345 was
slightly more susceptible to itraconazole than C. albicans
SC5314 or the three strains of C. krusei. Analysis of the
sterols of the organisms by gas chromatography-mass spectrometry
revealed that the predominant sterol in C. krusei was
ergosterol (Table 2). Growing all three strains of C. krusei in the presence of fluconazole caused a
decrease in their ergosterol content and a marked increase in
lanosterol. Other 14-methyl sterols that were increased in organisms
exposed to fluconazole were 14-methylfecosterol and eburicol. These
results suggest that fluconazole inhibits 14
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mechanism of Fluconazole Resistance in
Candida krusei
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-demethylase in this organism. The
14-demethylase activity in cell extracts of C. krusei
was 16- to 46-fold more resistant to inhibition by fluconazole than was
14-demethylase activity in cell extracts of two
fluconazole-susceptible strains of Candida albicans.
Comparing the carbon monoxide difference spectra of microsomes from
C. krusei with those of microsomes from C. albicans indicated that the total cytochrome P-450 content of
C. krusei is similar to that of C. albicans.
The Soret absorption maximum in these spectra was located at 448 nm for
C. krusei and at 450 nm for C. albicans.
Finally, the fluconazole accumulation of two of the C. krusei isolates was similar to if not greater than that of
C. albicans. Thus, there are significant qualitative differences between the 14-demethylase of C. albicans
and C. krusei. In addition, fluconazole resistance in these
strains of C. krusei appears to be mediated predominantly
by a reduced susceptibility of 14-demethylase to inhibition by this
drug.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-demethylase. Inhibition of this enzyme by azoles causes
an accumulation of C14 methylated sterols which likely disrupt membrane structure (9). In some resistant organisms, there is overexpression of the 14-demethylase gene and/or the enzyme
is less susceptible to azole inhibition (15, 24, 32). The
second mechanism is decreased drug accumulation, mediated by either
diminished uptake or increased efflux of the drug (22, 26).
The third mechanism of resistance is the presence of a deficiency in
C5(6) desaturase. Organisms deficient in this enzyme produce 14-methylfecosterol and remain viable when 14-demethylase activity is inhibited (5, 14).
-demethylase with reduced susceptibility to the inhibitory effects
of fluconazole.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-demethylase in C. krusei. No 14-methyl-ergosta-8,24(28)-dien-3,6-diol was detected,
although this sterol has been reported to accumulate when C. krusei is exposed to azoles (18, 31).
TABLE 1.
Antifungal susceptibilities of C. albicans
and C. krusei
TABLE 2.
Effect of fluconazole on the sterols
of C. krusei
The 14-demethylase from cell extracts of C. krusei
showed reduced susceptibility to inhibition by fluconazole.
Although fluconazole appeared to inhibit 14-demethylase in C. krusei, exposure to a relatively high concentration of drug was
required to produce this inhibition. Therefore, we examined whether the
enzyme itself was resistant to the inhibitory effects of fluconazole.
We determined the effects of this drug on the synthesis of ergosterol
from [14C]mevalonic acid in cell extracts of three
strains of C. krusei and two isolates of C. albicans. We found that the concentration of fluconazole required
to inhibit the synthesis of ergosterol by 50% (IC50) was
16- to 46-fold higher in cell extracts from C. krusei than
in extracts from either strain of C. albicans (Table 3). This difference indicates that
reduced fluconazole susceptibility of the 14-demethylase from
C. krusei is likely the predominant mechanism of fluconazole
resistance in these isolates.
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Carbon monoxide difference spectra also indicated that the
cytochrome P-450 of C. krusei is different from that of
C. albicans.
Next, we determined the carbon monoxide
difference spectra of microsomes from these organisms. This procedure
enabled us to evaluate further the possibility that fluconazole
resistance was mediated by quantitative and/or qualitative changes in
the 14-demethylase of the organism. We found that the cytochrome
P-450 content of the three strains of C. krusei was similar
to that of the two strains of C. albicans (Table 3). Since
14-demethylase accounts for the majority of cytochrome P-450 in
yeasts, these findings suggest that the mechanism of fluconazole
resistance in C. krusei is not mediated by overproduction of
the target enzyme.
-demethylase from the two different species of Candida.
Fluconazole accumulation by C. krusei and C. albicans was similar. To determine if reduced intracellular concentrations of the drug contributed to fluconazole resistance, the fluconazole accumulation of the three isolates of C. krusei was compared with that of the two strains of C. albicans. During the first 30 min, the accumulation of fluconazole by C. krusei was not consistently different from the accumulation of this drug by C. albicans (Fig. 1). However, at later time points, there was a trend towards greater accumulation of fluconazole by the strains of C. krusei. Of the organisms studied, C. albicans Y01.345 had the lowest fluconazole accumulation at 60 and 90 min. Therefore, the three isolates of C. krusei appear to be resistant to fluconazole by a mechanism that is independent of reduced fluconazole accumulation.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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By comparing the effects of fluconazole on the synthesis of
sterols by cell extracts of C. krusei and C. albicans, we determined that a major mechanism of fluconazole
resistance in the three strains of C. krusei studied appears
to be a reduced susceptibility of 14-demethylase to fluconazole. The
fluconazole IC50s for sterol synthesis by cell extracts of
C. krusei ranged from 0.495 to 0.795 µM. These values were
16- to 46-fold greater than the corresponding IC50s for
extracts of C. albicans. In the literature, there is substantial variability in the reported fluconazole IC50s
for cell extracts of C. krusei. These values range from
0.080 to 1.38 µM (18, 31). Possible explanations for these
differences include differences in methodology or strain-to-strain
variations in the mechanism of fluconazole resistance in C. krusei. It is noteworthy that no previous investigators have
reported the direct comparison of the fluconazole IC50 of
C. krusei with that of a fluconazole-susceptible species of
Candida. However, Venkateswarlu et al. (30) have reported that the fluconazole IC50 for cell extracts of
different strains of C. albicans ranged from 0.042 to 0.055 µM, and we have determined previously that the fluconazole
IC50 for 14-demethylase purified from C. albicans is 0.074 µM (11). These values are only
slightly higher than the ones reported here.
An additional finding was that the Soret maximum of microsomes of
C. krusei was located at 448 nm, whereas it was located at
450 nm when microsomes of C. albicans were analyzed. This
result also suggests that the 14-demethylase of C. krusei
is significantly different from that of C. albicans.
Venkateswarlu et al. (29, 31) reported that the Soret peak
of C. krusei microsomes was at 448 nm, which is similar to
our findings. However, other investigators have found that the Soret
peak of C. albicans microsomes is also located 448 nm, and
we have determined that purified 14-demethylase of C. albicans has a Soret peak at 447 (12, 15). It is
possible that the difference between these results and our present
findings is the result of differences in culture conditions and/or the methods used to purify the microsomes. For example, Sanglard et al.
(25) found that the Soret maximum of microsomes of
Candida tropicalis ranged from 447 to 450 nm, depending on
the conditions under which the organisms were grown.
Burgener-Kairuz et al. (2) have determined the sequence of a
1.2-kb fragment of the C. krusei 14-demethylase gene.
They found that the deduced amino acid sequence of this fragment was only 80% similar to the corresponding portion of the 14-demethylase of C. albicans. This finding also supports our conclusion
that the 14-demethylase of C. krusei differs
significantly from that of C. albicans. It is not known
which region(s) of the C. krusei 14-demethylase is
responsible for its reduced susceptibility to fluconazole, but this
question is currently being investigated.
The reduced carbon monoxide difference spectra also indicated that the cytochrome P-450 content of C. krusei was similar to that of C. albicans. Other investigators have reported that the cytochrome P-450 contents of C. krusei and C. albicans are approximately 0.03 nmol/mg of protein and 0.02 to 0.05 nmol/mg of protein, respectively (12, 29-31). These results support the conclusion that C. krusei is not resistant to fluconazole because of overproduction of the target enzyme.
When the different strains of C. krusei were grown in the presence of fluconazole, we found a decrease in the amount of ergosterol and an increase in 14-methyl sterols, mainly lanosterol. Unlike other investigators, we did not find any evidence of 14-methyl-ergosta-8,24(28)-dien-3,6-diol in the organisms exposed to fluconazole (18, 31). This sterol was not detected in cell extracts from C. krusei ATCC 6258, even though Venkateswarlu et al. (31) reported its presence when this strain was grown in the presence of itraconazole. A likely explanation for this difference is that even though we grew the organisms in the maximal concentration of fluconazole that did not completely inhibit fungal growth, there was only partial inhibition of ergosterol synthesis. Others have observed that a strain of C. krusei grown in subinhibitory concentrations of itraconazole had a reduction in ergosterol content without the accumulation of 14-methyl-ergosta-8,24(28)-dien-3,6-diol (18). Thus, it is likely that, if it had been possible to grow the organisms in higher concentrations of fluconazole, we would have found the accumulation of this sterol. Nevertheless, because we found that fluconazole caused an accumulation of 14-methylfecosterol which was not converted to 14-methyl-ergosta-8,24(28)-dien-3,6-diol, we cannot completely rule out the possibility that C5(6) desaturase contributes to fluconazole resistance in some strains of C. krusei.
An additional finding was that the fluconazole accumulations of the three strains of C. krusei were similar to those of C. albicans. These results are different from those of Marichal et al. (18), who concluded that C. krusei is resistant to fluconazole on the basis of diminished drug accumulation. However, these investigators found that the fluconazole accumulation by exponential-phase organisms was actually fourfold greater than that observed with the strains of C. krusei used in this study. It is possible that differences in either methodology or the strains of C. krusei account for these differences in results.
Venkateswarlu et al. (31) also reported that itraconazole resistance in C. krusei is mediated by reduced drug accumulation. They found that a strain of C. krusei that was resistant to itraconazole accumulated less drug than did an itraconazole-susceptible isolate of C. krusei. While the two organisms had different susceptibilities to itraconazole, both exhibited high-level fluconazole resistance. These results demonstrate that resistance to fluconazole in C. krusei is mediated by a different mechanism than is itraconazole resistance.
In conclusion, our data indicate that, in the strains of C. krusei studied, fluconazole resistance is largely the result of a
decreased susceptibility of 14-demethylase to the inhibitory effects
of fluconazole. Diminished accumulation of fluconazole did not appear
to contribute to fluconazole resistance in these isolates. However, it
is possible that reduced fluconazole accumulation is the predominant
mechanism of fluconazole resistance in other isolates of C. krusei. Future work will investigate whether different strains of
C. krusei have different mechanisms of resistance to this
drug and why the 14-demethylase of this organism is less susceptible
to inhibition by fluconazole.
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ACKNOWLEDGMENTS |
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We thank Michael Mador and Trang Phan for technical assistance. We also appreciate the assistance of W.-N. Paul Lee and Anne Bergener at the Stable Isotope Facility at Harbor-UCLA Research and Education Institute.
This work was supported by a grant from Pfizer, Inc.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Infectious Diseases, Harbor-UCLA Research and Education Institute, 1124 West Carson St., RB-2, Torrance, CA 90502. Phone: (310) 222-6426. Fax: (310) 782-2016. E-mail: Filler{at}HUMC.EDU.
Present address: Department of Dermatology, Center for Medical
Mycology, Case Western Reserve University, Cleveland, OH 44106.
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Abstract
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Materials & Methods
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Discussion
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Antimicrobial Agents and Chemotherapy, October 1998, p. 2645-2649, Vol. 42, No. 10
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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