Abstract
Candida dubliniensis is a recently identified yeast species primarily associated with oral carriage and infection in individuals infected with the human immunodeficiency virus. The species can be divided into at least four genotypes on the basis of the nucleotide sequence of the internal transcribed spacer region of the rRNA operon. Previous studies have shown that a small number of clinical isolates belonging to genotype 1 are resistant to the commonly used antifungal drug fluconazole. The aim of the present study was to investigate the molecular mechanisms responsible for reduced susceptibility to azole drugs in C. dubliniensis genotype 3 isolates obtained from a patient with fluconazole-recalcitrant oral candidiasis. Four isolates from a single clinical sample, one susceptible, the other three exhibiting reduced susceptibilities to fluconazole, itraconazole, ketoconazole, voriconazole, and posaconazole, were examined. Results showed that reduced susceptibility to azole drugs was associated with an increase in the expression of the multidrug transporters CdCDR1 and CdCDR2 which correlated with reduced intracellular accumulation of radiolabeled fluconazole and an increase in the activity of energy-dependent efflux mechanisms. In contrast to observations made in previous studies, overexpression of the multidrug transporter CdMDR1 was not observed. Despite a thorough investigation of all commonly encountered mechanisms of azole resistance, no other mechanism could be associated with reduced susceptibility to azole drugs in the clinical isolates studied. This is the first report of CdCDR2 involvement in azole resistance in C. dubliniensis.
While the introduction of azole antifungal agents has improved the outcome of many fungal infections, treatment failure due to resistance to these agents has become a clinical problem. In particular, resistance to azole drugs has been associated with oral candidiasis relapses in human immunodeficiency virus (HIV)-infected and AIDS patients following extensive therapeutic and prophylactic use of fluconazole (23, 36, 37).
A variety of molecular mechanisms by which Candida cells can develop resistance to azole drugs have been described previously. Azole resistance can be caused by increased expression of the target enzyme (cytochrome P450 lanosterol 14α-demethylase), resulting in increased levels of the enzyme in cells (18, 34), or by point mutations which reduce its affinity for azole drugs (11, 13, 16, 17, 24, 29). Reduced accumulation of the drug due to increased efflux is another mechanism commonly involved in azole resistance in clinical Candida isolates (1, 15, 21, 25, 31, 35). In addition, resistance to azole drugs has also been associated with modifications of the ergosterol biosynthetic pathway, such as defects in the sterol C5,6-desaturation step (10, 12, 19, 26).
Candida dubliniensis, a recently described species closely related to Candida albicans, has been documented as a significant cause of oral disease in HIV-infected patients, particularly those who routinely receive fluconazole therapy for the treatment of oral candidiasis (3, 14, 27, 33). Recently, four distinct genotypes were identified among C. dubliniensis isolates on the basis of sequence variations in the ITS1 and ITS2 regions of the rRNA operon (8). Gee et al. found that isolates belonging to genotype 1 were predominant worldwide and were recovered mainly from HIV-infected patients, while isolates belonging to genotypes 2, 3, and 4 were recovered mainly from HIV-negative individuals. Resistance to fluconazole in clinical isolates of C. dubliniensis belonging to genotype 1 has been observed previously (22, 25, 28). In addition, we have previously shown that in vitro exposure of C. dubliniensis to fluconazole can result in the development of stable resistance (21, 22). Previous molecular studies have shown that fluconazole-specific resistance in azole-resistant C. dubliniensis genotype 1 clinical isolates and in vitro-generated derivatives is associated primarily with overexpression of the major facilitator CdMdr1p (21, 39). Although upregulation of CdCDR1 has been observed in fluconazole-resistant clinical isolates and in vitro-generated derivatives (21), it has been shown that CdCdr1p is not essential for fluconazole resistance (20). This is in contrast to the findings for C. albicans, where almost all isolates with reduced susceptibility to azoles examined to date show upregulation of CaCDR1 (24). Moreover, Moran et al. found that 58% of C. dubliniensis genotype 1 isolates harbor mutated alleles of CdCDR1 that encode a truncated, nonfunctional CdCdr1p protein.
In the present study, we describe the first case of reduced susceptibility to azole drugs in C. dubliniensis isolates belonging to genotype 3. Using a matched susceptible isolate, an in-depth investigation of all molecular mechanisms previously associated with azole resistance showed that reduced azole susceptibility was associated exclusively with overexpression of CDR (Candida drug resistance) efflux pumps, a mechanism not previously described for genotype 1 C. dubliniensis isolates.
MATERIALS AND METHODS
C. dubliniensis clinical isolates.
An AIDS patient showing persistent symptoms of oral candidiasis despite fluconazole therapy attended the Dublin Dental Hospital in November 1997. A single oral swab sample taken from the dorsum of the tongue yielded both C. albicans and C. dubliniensis when plated on CHROMagar Candida (Paris, France) medium (without fluconazole). All 12 single-colony isolates of C. dubliniensis present on the isolation plate were recovered simultaneously from this sample. On the basis of their susceptibilities to azole drugs, four of the clinical C. dubliniensis isolates (one susceptible and three with reduced susceptibility) were selected for detailed molecular analysis. In addition, four single-colony C. albicans isolates were selected and subcultured for further study. The C. dubliniensis isolates were originally identified on the basis of their dark green color on CHROMagar Candida medium, and their identities were confirmed by using phenotypic and molecular techniques, including biotyping using the API ID 32C yeast identification system (bioMérieux, Marcy l'Etoile, France) and PCR identification using the method developed by Donnelly et al. (6). The C. albicans isolates were originally selected on the basis of their light green color on CHROMagar Candida medium and were identified by using the API ID 32C yeast identification system.
C. dubliniensis and C. albicans clinical isolates were routinely cultured on potato dextrose agar (Oxoid, Basingstoke, United Kingdom) medium (pH 5.6) at 37°C. For liquid culture, the isolates were grown in yeast extract-peptone-dextrose (YEPD) broth at 37°C in an orbital shaker (Gallenkamp, Leicester, United Kingdom) at 200 rpm.
Susceptibility testing.
Broth microdilution (BMD) susceptibility testing with azole drugs was carried out by the EUCAST BMD method (5). All tests were conducted in duplicate.
DNA and RNA extractions and Southern and Northern blot analyses.
Total genomic DNAs from the clinical isolates were prepared as described previously by Gallagher et al. (7). Fingerprinting and karyotype analyses were carried out as described by Gee et al. (8). Similarity coefficients (SAB), based on band positions obtained with the C. dubliniensis-specific fingerprinting probe Cd25, were calculated as 2E/(2E + a + b), where E is the number of bands shared by isolates A and B, a is the number of bands unique to A, and b is the number of bands unique to B. An SAB of 0.00 represents totally different patterns with no correlated bands; an SAB of 1.00 represents identical patterns (32).
RNA extraction, electrophoresis, transfer, and hybridization were carried out as outlined by Moran et al. (21). Membranes were then exposed to BioMax MS film (Eastman Kodak Company, Rochester, N.Y.) for 24 to 72 h. For normalization purposes, all membranes were hybridized with a probe homologous to the C. dubliniensis gene encoding translation elongation factor 3 (CdTEF3). Hybridization levels were analyzed by scanning densitometry (GelWorks 1D Intermediate; Ultra-Violet Products Ltd., Cambridge, United Kingdom) and normalized to CdTEF3 expression levels by dividing the intensity obtained for the gene of interest by the signal obtained for CdTEF3.
PCR amplification and cloning of C. dubliniensis DNA sequences.
CdERG11 sequences were amplified by using the primer pair ERG11F-ERG11R (26). The CdMDR1 promoter sequences (positions −882 to +40 with respect to the CdMDR1 start codon) were amplified by PCR with primers PROF (5′-CAAAACGTGTTAGAATTGCGC-3′) and PROR (5′-CTCTACCAACAAAACTATCTC-3′). Primer pair CdMDR1F-CdMDR1R (21) was used to amplify the CdMDR1 open reading frame (ORF) sequences. In order to reduce PCR errors, all PCRs were carried out using a proofreading polymerase (Expand High Fidelity PCR System; Roche, Lewes, East Sussex, United Kingdom). The amplimers were used directly in sequencing reactions carried out by Lark, Inc. (Saffron Walden, Essex, United Kingdom).
Biochemical analyses.
Accumulation of [3H]fluconazole in C. dubliniensis was assessed by the method of Sanglard et al. (31) using cells grown to mid-log phase in drug-free yeast nitrogen base broth. A time point of 20 min was used, because this has been shown previously to represent steady-state conditions (21).
For gas chromatography-mass spectrometry (GC-MS), nonsaponifiable sterols were extracted and analyzed by using the method described by Pinjon et al. (26).
Glucose-induced efflux of rhodamine 6G (R6G) was determined in the absence of fluconazole by using the method described by Pinjon et al. (26).
RESULTS
Twelve single-colony isolates of C. dubliniensis were recovered from the same clinical specimen obtained from a patient with oral candidiasis receiving fluconazole treatment. All were found to belong to C. dubliniensis genotype 3 on the basis of nucleotide sequence analysis of the internal transcribed spacer (ITS) region of the rRNA operon (data not shown). The isolates exhibited a range of susceptibilities to fluconazole (MIC range, 0.25 to 8 μg/ml), itraconazole (MIC range, 0.06 to 0.5 μg/ml), ketoconazole (MIC range, 0.07 to 0.125 μg/ml), voriconazole (MIC range, 0.07 to 0.125 μg/ml), and posaconazole (MIC range, 0.01 to 0.25 μg/ml) (Table 1). In addition, four C. albicans isolates were selected from the initial isolation plate. All four were found to be susceptible to fluconazole (MIC range, 0.25 to 0.5 μg/ml) and itraconazole (MIC, 0.03 μg/ml) (Table 1). Of the 12 C. dubliniensis isolates, 4 representative isolates were selected for further study on the basis of their susceptibilities to azole drugs. One isolate, CD519-8, was susceptible to all the azole drugs tested, while the other three isolates (CD519-1, CD519-7, and CD519-14) showed reduced susceptibility to these drugs (Table 1). Although resistance to azole drugs is frequently associated with resistance to other antifungal drugs and metabolic inhibitors, the four clinical isolates were all susceptible to amphotericin B (MIC, 0.06 μg/ml), and all had similar susceptibilities to 4-nitroquinoline N-oxide, flucytosine, crystal violet, and 1,10-phenanthroline (data not shown). To determine the relatedness between these four isolates, they were analyzed by Southern hybridization with the C. dubliniensis-specific fingerprinting probe Cd25 (9). The four isolates yielded very similar Cd25-generated fingerprint profiles that were significantly different from the fingerprint profile obtained for the C. dubliniensis type strain, CD36 (SAB, 0.24 to 0.26), which was used as a reference in fingerprinting procedures (Fig. 1A). However, the Cd25-generated fingerprint profile of CD519-14 (SAB, 0.93) contained three polymorphic bands (Fig. 1A) which were not present in the fingerprint profiles of isolates CD519-1, CD519-7, and CD519-8 (SAB, 1.00). This finding suggested that, although isolate CD519-14 was closely related to the other three isolates, it had undergone minor genetic reorganization suggestive of microevolution, which has been observed previously for C. dubliniensis (8, 9). The karyotypes of the four isolates were analyzed by pulsed-field gel electrophoresis. The four clinical isolates yielded identical karyotype profiles which were clearly distinct from the karyotype profile of the C. dubliniensis type strain, CD36, used as a reference (Fig. 1B). Taken together, the results of the Southern blot and karyotype fingerprinting experiments showed that the four isolates examined were clonally related.
TABLE 1.
Susceptibilities to azole drugs of the C. dubliniensis and C. albicans isolates recovered from the same clinical sample
| Isolatea | MIC (μg/ml)b
|
||||
|---|---|---|---|---|---|
| Fluconazole | Itraconazole | Ketoconazole | Voriconazole | Posaconazole | |
| C. dubliniensis | |||||
| CD519-1 | 8 | 0.5 | 0.125 | 0.125 | 0.25 |
| CD519-2 | 4 | ND | ND | ND | ND |
| CD519-3 | 4 | ND | ND | ND | ND |
| CD519-4 | 0.25 | ND | ND | ND | ND |
| CD519-5 | 4 | ND | ND | ND | ND |
| CD519-6 | 8 | 0.125 | ND | ND | ND |
| CD519-7 | 8 | 0.25 | 0.125 | 0.125 | 0.125 |
| CD519-8 | 0.5 | 0.06 | <0.07 | 0.07 | <0.01 |
| CD519-10 | 4 | ND | ND | ND | ND |
| CD519-11 | 2 | 0.125 | ND | ND | ND |
| CD519-13 | 4 | ND | ND | ND | ND |
| CD519-14 | 8 | 0.5 | 0.125 | 0.125 | 0.125 |
| C. albicans | |||||
| CA1197-1 | 0.25 | 0.03 | ND | ND | ND |
| CA1197-2 | 0.25 | 0.03 | ND | ND | ND |
| CA1197-3 | 0.25 | 0.03 | ND | ND | ND |
| CA1197-4 | 0.5 | 0.03 | ND | ND | ND |
Boldfaced isolates were selected for more-detailed phenotypic and molecular analysis.
MICs were determined by BMD as described in Materials and Methods. ND, not determined.
FIG. 1.
DNA fingerprint patterns of C. dubliniensis clinical isolates CD519-1, CD519-7, CD519-8, and CD519-14 and C. dubliniensis type strain CD36. (A) Cd25 hybridization patterns of EcoRI-digested genomic DNA. The relative positions of molecular size reference markers are indicated on the left. The positions of three polymorphic bands in the fingerprinting profile of CD519-14 are indicated by arrows (B) Electrophoretic karyotype patterns of C. dubliniensis clinical isolates CD519-1, CD519-7, CD519-8, and CD519-14 and C. dubliniensis type strain CD36. The relative positions of molecular size reference markers are indicated on the left.
In order to elucidate the mechanism(s) responsible for reduced susceptibility to azole drugs in these isolates, we carried out a thorough analysis investigating the mechanisms commonly associated with azole resistance in C. dubliniensis (20, 21, 25, 26).
We have recently shown that resistance to azole drugs in C. dubliniensis can be associated with defective sterol C5,6-desaturation, resulting in a lack of ergosterol synthesis and the accumulation of atypical sterol precursors (26). In order to determine whether the decreased susceptibilities to azole drugs observed in clinical isolates CD519-1, CD519-7, and CD519-14 could be due to a defect in the enzyme sterol C5,6-desaturase, the nonsaponifiable sterols present in their membranes were analyzed by GC-MS. This analysis showed that all four clinical isolates accumulated ergosterol in their membranes, which indicated that the ergosterol synthesis pathway, and more particularly the sterol C5,6-desaturation function, was intact in all four clinical isolates (Table 2).
TABLE 2.
Sterols accumulated by C. dubliniensis clinical isolates in order of retention time
| Isolate | Accumulated sterolsa (%) |
|---|---|
| CD519-1 | Ergosta-tetraenol (15.3) |
| Ergosterol (61.2) | |
| Episterol (8.3) | |
| Obtusifoliol (2.8) | |
| Unknown sterols (12.4) | |
| CD519-7 | Ergosta-tetraenol (17.8) |
| Ergosterol (61.5) | |
| Fecosterol (3.7) | |
| Episterol (7.5) | |
| Obtusifoliol (3.0) | |
| Unknown sterols (6.7) | |
| CD519-8 | Ergosta-tetraenol (16.8) |
| Ergosterol (55.8) | |
| Fecosterol (6.7) | |
| Episterol (6.0) | |
| Obtusifoliol (1.8) | |
| Unknown sterols (12.8) | |
| CD519-14 | Ergosta-tetraenol (21.3) |
| Ergosterol (63.2) | |
| Fecosterol (2.9) | |
| Episterol (5.0) | |
| Obtusifoliol (3.1) | |
| Unknown sterols (4.5) |
Sterols were extracted from cells grown overnight in YEPD broth at 30°C and were analyzed by GC-MS as described in Materials and Methods.
In order to investigate whether overexpression of the enzyme lanosterol demethylase (CdErg11p) was involved in mediating reduced susceptibility to azole drugs in clinical isolates CD519-1, CD519-7, and CD519-14, expression levels of the CdERG11 gene in these isolates and the azole-susceptible clinical isolate CD519-8 were examined by Northern blot analysis. There was no significant elevation of CdERG11 mRNA levels in any of the four clinical isolates examined (Fig. 2).
FIG. 2.
Expression of CdCDR1, CdCDR2, CdMDR1, and CdERG11 analyzed by Northern blotting of total RNAs from isolates CD519-1, CD519-7, CD519-8, and CD519-14, C. dubliniensis type strain CD36, and CM2, a fluconazole-resistant genotype 1 C. dubliniensis isolate characterized by Moran et al. (21). Total RNA was extracted from cells growing exponentially in YEPD in the absence of azole drugs, and 15 μg was electrophoresed on a denaturing agarose gel. Following transfer to a nylon membrane, the blots were sequentially probed with radiolabeled DNA probes homologous to CdMDR1, CdCDR1, CdCDR2, CdERG11, and the constitutively expressed CdTEF3 gene. Expression of CdTEF3 was used as an internal control for RNA loading.
In addition, the CdERG11 genes from all four isolates were amplified by PCR and sequenced directly. All four isolates had identical CdERG11 nucleotide sequences which contained two homozygous polymorphisms (I188V and R499K) and one heterozygous polymorphism (V402G) relative to the recently published CdERG11 sequence (25). To confirm this, the CdERG11 genes from two isolates with differing susceptibilities to azole drugs (i.e., CD519-1 and CD519-8) were expressed in the azole-hypersusceptible Saccharomyces cerevisiae strain YKKB-13 as described by Sanglard et al. (29). The susceptibilities to itraconazole and fluconazole of the S. cerevisiae transformants expressing CdERG11 genes from clinical isolates CD519-8 and CD519-1 were determined by BMD. There was no difference in susceptibility to azole drugs between transformants expressing the CdERG11 gene from the azole-susceptible isolate CD519-8 and transformants expressing the CdERG11 gene from the isolate with reduced susceptibility to azoles, CD519-1 (data not shown).
In order to determine if reduced accumulation of drug contributed to the difference in MICs observed between the CD519 clinical isolates, levels of [3H]fluconazole accumulation in all four isolates were examined. The isolates and the type strain, CD36, were examined at a single time point following a 20-min exposure to [3H]fluconazole (Fig. 3). The control strain, CD36, which harbors a defective CdCDR1 gene (20), accumulated 518 ± 0.7 cpm/107 cells. The three isolates with reduced susceptibility to azoles (CD519-1, CD519-7, and CD519-14) showed lower levels of [3H]fluconazole accumulation than the azole-susceptible isolate CD519-8. The azole-susceptible isolate CD519-8 yielded an average of 471 ± 29 cpm/107 cells following a 20-min exposure to [3H]fluconazole, while isolates CD519-1, CD519-7, and CD519-14, which had reduced susceptibilities to azoles, yielded averages of 253 ± 3, 272 ± 30, and 308 ± 57 cpm/107 cells, respectively. This showed that in these three isolates, reduced susceptibility to azoles was associated with reduced intracellular accumulation of drug, suggesting an increased efflux of fluconazole in these isolates.
FIG. 3.
Accumulation of [3H]fluconazole in C. dubliniensis clinical isolates with reduced susceptibility to azole drugs (CD519-1, CD519-7, and CD519-14) and in a clonally related azole-susceptible isolate (CD519-8). Accumulation levels were determined following a 20-min incubation in the presence of [3H]fluconazole. The C. dubliniensis type strain CD36 was used as a control in this experiment.
In order to more closely examine energy-dependent efflux mechanisms, glucose-mediated efflux of rhodamine 6G was measured in the clinical isolates with reduced susceptibility to azole drugs. The method used in the present study directly assessed the efflux of R6G by measuring extracellular R6G concentrations following the addition of glucose to energy-starved cells in the absence of fluconazole. Efflux of R6G from C. dubliniensis type strain CD36, harboring a nonfunctional CdCDR1 gene, was lower than R6G efflux from the clinical isolates (Fig. 4). In the absence of glucose, the extracellular R6G concentrations were similar in all four isolates, reflecting similar levels of R6G uptake (data not shown). However, in the presence of glucose, R6G efflux from the azole-susceptible isolate CD519-8 was lower than that from isolates CD519-1, CD519-7, and CD519-14 (Fig. 4). This showed an increase in energy-dependent efflux in the three isolates with reduced susceptibility to azole drugs.
FIG. 4.
Glucose-induced rhodamine 6G efflux from clinical isolates with reduced susceptibility to azole drugs (CD519-1, CD519-7, and CD519-14) and from a clonally related azole-susceptible isolate (CD519-8). The C. dubliniensis type strain CD36 was used as a control in this experiment. Each bar represents the mean from three sets of experiments. Error bars, standard errors.
All C. dubliniensis isolates with a defective CdCDR1 gene identified to date belong to genotype 1 (17). Therefore, since the CD519 isolates belong to C. dubliniensis genotype 3, they were expected to harbor a functional CdCDR1 gene. In order to investigate the functionality of this gene, PCR amplification followed by restriction fragment length polymorphism analysis was carried out. Moran et al. have shown that the presence of a stop codon at position 756 in the CdCDR1 ORF removes a restriction site for the enzyme SspI (20). As expected, none of the CD519 isolates harbored the stop codon at position 756 of the CdCDR1 ORF.
Northern blot analysis was carried out in order to determine if the observed increase in energy-dependent efflux in the clinical isolates with reduced susceptibility to azole drugs correlated with increased expression of multidrug resistance genes. Isolates CD519-1, CD519-7, and CD519-14, with reduced susceptibility to azole drugs, showed higher mRNA levels of the two multidrug resistance genes CdCDR1 and CdCDR2 than the azole-susceptible isolate CD519-8. However, this was not the case for the multidrug resistance gene CdMDR1, expression of which was not detectable in any of the four clinical isolates (Fig. 2). CdCDR1 expression was increased approximately five-, two-, and sixfold in isolates CD519-1, CD519-7, and CD519-14, respectively. Expression of CdCDR2 was increased >10-fold in isolates CD519-1, CD519-7, and CD519-14. Since RNA was extracted from cultures grown in the absence of fluconazole, it can be assumed that upregulation of CdCDR1 and CdCDR2 is constitutive.
In contrast to previously established mechanisms of resistance to fluconazole in C. dubliniensis, reduced susceptibility to azole drugs in C. dubliniensis clinical isolates CD519-1, CD519-7, and CD519-14 did not correlate with overexpression of CdMDR1. For this reason, it was decided to study the CdMDR1 gene from the four C. dubliniensis CD519 isolates. The ORF and promoter sequences from these isolates were amplified by PCR using primer pairs CdMDR1F-CdMDR1R and PROF-PROR, respectively, and were sequenced. Fragments of the expected sizes (approximately 1.7 kb for the CdMDR1 ORF and 920 bp for the CdMDR1 promoter) were obtained in each case. Sequence analysis of the four ORFs showed that the sequences obtained for the four clinical isolates were identical and contained five polymorphisms (D32G, T68S, A105, T307I, and E415K) which affected the amino acid sequence of the CdMdr1p protein relative to the published sequence of CdMDR1 obtained from the C. dubliniensis type strain CD36 (EMBL accession no. AJ227752) (21).
Sequence analysis of the CdMDR1 promoter region showed that promoter sequences obtained from clinical isolates CD519-1, CD519-7, CD519-8, and CD519-14 were identical. However, there were 13 nucleotide differences between the sequence previously obtained from the type strain, CD36, and the sequence data obtained from the four isolates CD519-1, CD519-7, CD519-8, and CD519-14.
Finally, in order to determine if the CdMDR1 gene from the CD519 clinical isolates was functional, it was heterologously expressed in the azole-hypersusceptible S. cerevisiae strain YKKB-13. The transformant expressing the CdMDR1 gene from isolate CD519-1 exhibited a susceptibility pattern identical to that of the transformant expressing the CdMDR1 gene from the C. dubliniensis type strain, CD36. Both transformants showed lower susceptibilities to fluconazole, benomyl, cycloheximide, and 1,10-phenanthroline than the transformant harboring the empty plasmid vector (Fig. 5).
FIG. 5.
Susceptibilities of S. cerevisiae YKKB-13 (Δpdr5) transformants harboring cloned CdMDR1 genes to fluconazole and metabolic inhibitors. CdMDR1 alleles from C. dubliniensis isolates were amplified by PCR, cloned into the pAAH5 expression vector (2), and transformed into the S. cerevisiae strain YKKB-13. The transformants harbor the pAAH5 plasmid alone (YP5) or pAAH5 carrying the CdMDR1 gene from isolate CD36 (YGM3) or CD519-1 (EPY84). Each transformant was grown to the exponential-growth phase (density, 2 × 107 cells/ml), and 4 μl was spotted in a dilution series onto minimal agar medium plates containing fixed concentrations of fluconazole or metabolic inhibitors as indicated. Plates were incubated for 48 h at 30°C.
DISCUSSION
It is now well established that long-term treatment of Candida infections with azole drugs can result in the development of antifungal resistance. In particular, fluconazole treatment of oral candidiasis in HIV-infected and AIDS patients has been associated with treatment failures (23, 36, 37). The recently described species C. dubliniensis has been shown to be particularly prevalent in this patient cohort, and the recovery of C. dubliniensis genotype 1 isolates exhibiting reduced susceptibility or resistance to fluconazole has been described previously (22, 25, 28). The present study concentrated on the investigation of the molecular mechanisms involved in mediating reduced susceptibility to azoles in four clonally related C. dubliniensis genotype 3 oral isolates recovered from an AIDS patient with recurrent oral candidiasis recalcitrant to fluconazole treatment. One isolate (CD519-8) was susceptible to azole drugs, while the other three (CD519-1, CD519-7, and CD519-14) showed decreased susceptibilities to the azole drugs fluconazole, itraconazole, ketoconazole, voriconazole, and posaconazole (Table 1).
The decreased susceptibility to azole drugs of the three isolates was not associated with overexpression or mutation of the CdERG11 gene (Fig. 2). There was no indication of membrane permeability alterations in these isolates. Indeed, all four isolates accumulated ergosterol in their membranes (Table 2), suggesting that defects in the enzyme sterol C5,6-desaturase were not involved in mediating reduced susceptibility to azole drugs in these clinical isolates.
Reduced susceptibility to azole drugs was, however, associated with reduced intracellular fluconazole accumulation (Fig. 3). This association was confirmed by Northern blot analysis, which showed a correlation between overexpression of the multidrug resistance genes CdCDR1 and CdCDR2 and a reduction in azole susceptibility (Fig. 2). In contrast to previous studies of fluconazole resistance mechanisms in C. dubliniensis, overexpression of the multidrug resistance gene CdMDR1 was not observed in the three clinical isolates exhibiting reduced susceptibility to azole drugs. Although resistance to fluconazole in C. dubliniensis clinical isolates has been associated with combinations of different molecular mechanisms (25), the primary mechanism of resistance to fluconazole in this species has been shown previously to be due to reduced intracellular drug accumulation mediated by the overexpression of CdMDR1.
While overexpression of CDR2 has been observed in azole-resistant isolates of C. albicans (30), overexpression of its C. dubliniensis homologue, CdCDR2, had never been reported previously and was therefore thought not to be implicated in azole drug resistance in this species. However, heterologous expression of CdCDR2 in S. cerevisiae has previously shown that it is able to mediate resistance to fluconazole and itraconazole (20). Because both CdCDR1 and CdCDR2 were found to be upregulated in the present study, it is not possible to establish the exact contribution of CdCDR2 overexpression to the phenotype. In order to investigate this, it would be necessary to disrupt the CdCDR2 and/or the CdCDR1 gene.
Although five amino acid-altering polymorphisms (D32G, T68S, A105, T307I, and E415K) were identified in the sequence of the CdMDR1 ORF obtained from the four CD519 isolates, heterologous expression in S. cerevisiae YKKB13 showed that the CdMDR1 gene was functional, since it was able to mediate resistance to fluconazole and metabolic inhibitors with the same efficiency as the CdMDR1 gene from the C. dubliniensis type strain, CD36 (Fig. 5). While several polymorphisms were identified in the promoter sequences of the CdMDR1 genes from the CD519 clinical isolates relative to the promoter sequence obtained from the C. dubliniensis type strain, CD36 (EMBL accession no. AJ227752), it is not known whether these could affect the expression of CdMDR1 in these isolates (38). A comparative functional analysis of the CdMDR1 promoter from the CD519 isolates and the C. dubliniensis type strain, CD36, should be carried out to find out the relevance of these polymorphisms.
While our data strongly imply that the observed reduced susceptibility to azole drugs was due to upregulation of CdCDR1 and CdCDR2 expression, the possibility still remains that additional resistance mechanisms could be involved. This possibility is currently being investigated using microarray analysis. In C. albicans, a transcriptional activator of the ABC transporter genes CDR1 and CDR2, named TAC1 (for transcriptional activator of CDR genes), has recently been identified (4). TAC1 alleles from azole-resistant strains, reintroduced in a TAC1 homologous mutant, were able to confer constitutive CDR1 and CDR2 upregulation, thus showing that azole resistance in the clinical strains had evolved from mutations in TAC1. Recently, the complete sequence of the C. dubliniensis genome has become available and a homologue of the TAC1 gene has been identified. It is likely that this homologue could be involved in the upregulation of CdCDR1 and CdCDR2 observed in the clinical isolates analyzed in the present study.
In conclusion, the analysis of susceptibility of multiple C. dubliniensis colonies from a single clinical sample revealed a significant degree of variation in susceptibility to azole drugs. The thorough analysis of matched clinical isolates belonging to C. dubliniensis genotype 3 showed that reduced susceptibility to azole drugs appeared to be associated only with increased energy-dependent efflux mechanisms mediated by the overexpression of the CdCDR1 and CdCDR2 genes. These results are in contrast to the mechanisms of azole resistance described to date for C. dubliniensis genotype 1, and they highlight the complexity and diversity of mechanisms by which C. dubliniensis isolates can develop resistance to azole drugs. Our previous studies were based on the analysis of the most common C. dubliniensis genotype, genotype 1. Since the majority of these isolates possess a defective CdCDR1 gene, we suggested that azole resistance mechanisms in C. dubliniensis were different from those in C. albicans. However, based on the data of the present study, it would appear that resistance mechanisms in C. dubliniensis genotypes 2, 3, and 4 may be more similar to those found in C. albicans.
Acknowledgments
This study was supported by the Irish Health Research Board (grants 04-97 and RP/2002/6), by the Dublin Dental School and Hospital, and by the BBSRC (S.L.K. and C.J.J.).
REFERENCES
- 1.Albertson, G. D., M. Niimi, R. D. Cannon, and H. F. Jenkinson. 1996. Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrob. Agents Chemother. 40:2835-2841. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Ammerer, G. 1983. Expression of genes in yeast using the ADC1 promoter. Methods Enzymol. 101:192-201. [DOI] [PubMed] [Google Scholar]
- 3.Coleman, D. C., D. J. Sullivan, D. E. Bennett, G. P. Moran, H. J. Barry, and D. B. Shanley. 1997. Candidiasis: the emergence of a novel species, Candida dubliniensis. AIDS 11:557-567. [DOI] [PubMed] [Google Scholar]
- 4.Coste, A., M. Karababa, F. Ischer, J. Bille, and D. Sanglard. 2004. Presented at the 2004 Yeast Genetics and Molecular Biology Meeting, Seattle, Wash.
- 5.Cuenca-Estrella, M., W. Lee-Yang, M. A. Ciblak, B. A. Arthington-Skaggs, E. Mellado, D. W. Warnock, and J. L. Rodriguez-Tudela. 2002. Comparative evaluation of NCCLS M27-A and EUCAST broth microdilution procedures for antifungal susceptibility testing of Candida species. Antimicrob. Agents Chemother. 46:3644-3647. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Donnelly, S. M., D. J. Sullivan, D. B. Shanley, and D. C. Coleman. 1999. Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT1 intron and exon sequences. Microbiology 145:1871-1882. [DOI] [PubMed] [Google Scholar]
- 7.Gallagher, P. J., D. E. Bennett, M. C. Henman, R. J. Russell, S. R. Flint, D. B. Shanley, and D. C. Coleman. 1992. Reduced azole susceptibility of Candida albicans from HIV-positive patients and a derivative exhibiting colony morphology variation. J. Gen. Microbiol. 138:1901-1911. [DOI] [PubMed] [Google Scholar]
- 8.Gee, S. F., S. Joly, D. R. Soll, J. F. Meis, P. E. Verweij, I. Polacheck, D. J. Sullivan, and D. C. Coleman. 2002. Identification of four distinct genotypes of Candida dubliniensis and detection of microevolution in vitro and in vivo. J. Clin. Microbiol. 40:556-574. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Joly, S., C. Pujol, M. Rysz, K. Vargas, and D. R. Soll. 1999. Development and characterization of complex DNA fingerprinting probes for the infectious yeast Candida dubliniensis. J. Clin. Microbiol. 37:1035-1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Kelly, S. L., D. C. Lamb, A. J. Corran, B. C. Baldwin, and D. E. Kelly. 1995. Mode of action and resistance to azole antifungals associated with the formation of 14α-methylergosta-8,24(28)-dien-3β,6α-diol. Biochem. Biophys. Res. Commun. 207:910-915. [DOI] [PubMed] [Google Scholar]
- 11.Kelly, S. L., D. C. Lamb, and D. E. Kelly. 1999. Y132H substitution in Candida albicans sterol 14α-demethylase confers fluconazole resistance by preventing binding to haem. FEMS Microbiol. Lett. 180:171-175. [DOI] [PubMed] [Google Scholar]
- 12.Kelly, S. L., D. C. Lamb, D. E. Kelly, J. Loeffler, and H. Einsele. 1996. Resistance to fluconazole and amphotericin in Candida albicans from AIDS patients. Lancet 348:1523-1524. [DOI] [PubMed] [Google Scholar]
- 13.Kelly, S. L., D. C. Lamb, J. Loeffler, H. Einsele, and D. E. Kelly. 1999. The G464S amino acid substitution in Candida albicans sterol 14α-demethylase causes fluconazole resistance in the clinic through reduced affinity. Biochem. Biophys. Res. Commun. 262:174-179. [DOI] [PubMed] [Google Scholar]
- 14.Kirkpatrick, W. R., S. G. Revankar, R. K. McAtee, J. L. Lopez-Ribot, A. W. Fothergill, D. I. McCarthy, S. E. Sanche, R. A. Cantu, M. G. Rinaldi, and T. F. Patterson. 1998. Detection of Candida dubliniensis in oropharyngeal samples from human immunodeficiency virus-infected patients in North America by primary CHROMagar candida screening and susceptibility testing of isolates. J. Clin. Microbiol. 36:3007-3012. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Lamb, D. C., D. E. Kelly, N. J. Manning, and S. L. Kelly. 1997. Reduced intracellular accumulation of azole antifungal results in resistance in Candida albicans isolate NCPF 3363. FEMS Microbiol. Lett. 147:189-193. [DOI] [PubMed] [Google Scholar]
- 16.Lamb, D. C., D. E. Kelly, T. C. White, and S. L. Kelly. 2000. The R467K amino acid substitution in Candida albicans sterol 14α-demethylase causes drug resistance through reduced affinity. Antimicrob. Agents Chemother. 44:63-67. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Marichal, P., L. Koymans, S. Willemsens, D. Bellens, P. Verhasselt, W. Luyten, M. Borgers, F. C. Ramaekers, F. C. Odds, and H. Vanden Bossche. 1999. Contribution of mutations in the cytochrome P450 14α-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans. Microbiology 145:2701-2713. [DOI] [PubMed] [Google Scholar]
- 18.Marichal, P., H. Vanden Bossche, F. C. Odds, G. Nobels, D. W. Warnock, V. Timmerman, C. Van Broeckhoven, S. Fay, and P. Mose-Larsen. 1997. Molecular biological characterization of an azole-resistant Candida glabrata isolate. Antimicrob. Agents Chemother. 41:2229-2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Miyazaki, Y., A. Geber, H. Miyazaki, D. Falconer, T. Parkinson, C. Hitchcock, B. Grimberg, K. Nyswaner, and J. E. Bennett. 1999. Cloning, sequencing, expression and allelic sequence diversity of ERG3 (C-5 sterol desaturase gene) in Candida albicans. Gene 236:43-51. [DOI] [PubMed] [Google Scholar]
- 20.Moran, G., D. Sullivan, J. Morschhauser, and D. Coleman. 2002. The Candida dubliniensis CdCDR1 gene is not essential for fluconazole resistance. Antimicrob. Agents Chemother. 46:2829-2841. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Moran, G. P., D. Sanglard, S. M. Donnelly, D. B. Shanley, D. J. Sullivan, and D. C. Coleman. 1998. Identification and expression of multidrug transporters responsible for fluconazole resistance in Candida dubliniensis. Antimicrob. Agents Chemother. 42:1819-1830. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Moran, G. P., D. J. Sullivan, M. C. Henman, C. E. McCreary, B. J. Harrington, D. B. Shanley, and D. C. Coleman. 1997. Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected subjects and generation of stable fluconazole-resistant derivatives in vitro. Antimicrob. Agents Chemother. 41:617-623. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Muller, F.-M. C., M. Weig, J. Peter, and T. J. Walsh. 2000. Azole cross-resistance to ketoconazole, fluconazole, itraconazole and voriconazole in clinical Candida albicans isolates from HIV-infected children with oropharyngeal candidosis. J. Antimicrob. Chemother. 46:323-342. [DOI] [PubMed] [Google Scholar]
- 24.Perea, S., J. L. Lopez-Ribot, W. R. Kirkpatrick, R. K. McAtee, R. A. Santillan, M. Martinez, D. Calabrese, D. Sanglard, and T. F. Patterson. 2001. Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob. Agents Chemother. 45:2676-2684. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Perea, S., J. L. Lopez-Ribot, B. L. Wickes, W. R. Kirkpatrick, O. P. Dib, S. P. Bachmann, S. M. Keller, M. Martinez, and T. F. Patterson. 2002. Molecular mechanisms of fluconazole resistance in Candida dubliniensis isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis. Antimicrob. Agents Chemother. 46:1695-1703. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Pinjon, E., G. P. Moran, C. J. Jackson, S. L. Kelly, D. Sanglard, D. C. Coleman, and D. J. Sullivan. 2003. Molecular mechanisms of itraconazole resistance in Candida dubliniensis. Antimicrob. Agents Chemother. 47:2424-2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Ponton, J., R. Ruchel, K. V. Clemons, D. C. Coleman, R. Grillot, J. Guarro, D. Aldebert, P. Ambroise-Thomas, J. Cano, A. J. Carrillo-Munoz, J. Gene, C. Pinel, D. A. Stevens, and D. J. Sullivan. 2000. Emerging pathogens. Med. Mycol. 38(Suppl. 1):225-236. [DOI] [PubMed] [Google Scholar]
- 28.Ruhnke, M., A. Schmidt-Westhausen, and J. Morschhauser. 2000. Development of simultaneous resistance to fluconazole in Candida albicans and Candida dubliniensis in a patient with AIDS. J. Antimicrob. Chemother. 46:291-295. [DOI] [PubMed] [Google Scholar]
- 29.Sanglard, D., F. Ischer, L. Koymans, and J. Bille. 1998. Amino acid substitutions in the cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) from azole-resistant Candida albicans clinical isolates contribute to resistance to azole antifungal agents. Antimicrob. Agents Chemother. 42:241-253. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Sanglard, D., F. Ischer, M. Monod, and J. Bille. 1997. Cloning of Candida albicans genes conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene. Microbiology 143:405-416. [DOI] [PubMed] [Google Scholar]
- 31.Sanglard, D., K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille. 1995. Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob. Agents Chemother. 39:2378-2386. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Soll, D. R. 2000. The ins and outs of DNA fingerprinting the infectious fungi. Clin. Microbiol. Rev. 13:332-370. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Sullivan, D., and D. Coleman. 1998. Candida dubliniensis: characteristics and identification. J. Clin. Microbiol. 36:329-334. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Vanden Bossche, H., P. Marichal, F. C. Odds, L. Le Jeune, and M. C. Coene. 1992. Characterization of an azole-resistant Candida glabrata isolate. Antimicrob. Agents Chemother. 36:2602-2610. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Venkateswarlu, K., D. W. Denning, N. J. Manning, and S. L. Kelly. 1995. Resistance to fluconazole in Candida albicans from AIDS patients correlated with reduced intracellular accumulation of drug. FEMS Microbiol. Lett. 131:337-341. [DOI] [PubMed] [Google Scholar]
- 36.White, T. C. 1997. Increased mRNA levels of ERG16, CDR, and MDR1 correlate with increases in azole resistance in Candida albicans isolates from a patient infected with human immunodeficiency virus. Antimicrob. Agents Chemother. 41:1482-1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.White, T. C., M. A. Pfaller, M. G. Rinaldi, J. Smith, and S. W. Redding. 1997. Stable azole drug resistance associated with a substrain of Candida albicans from an HIV-infected patient. Oral Dis. 3(Suppl. 1):S102-S109. [DOI] [PubMed] [Google Scholar]
- 38.Wirsching, S., S. Michel, G. Köhler, and J. Morschhäuser. 2000. Activation of the multiple drug resistance gene MDR1 in fluconazole-resistant clinical Candida albicans strains is caused by mutations in a trans-regulatory factor. J. Bacteriol. 182:400-404. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Wirsching, S., G. P. Moran, D. J. Sullivan, D. C. Coleman, and J. Morschhauser. 2001. MDR1-mediated drug resistance in Candida dubliniensis. Antimicrob. Agents Chemother. 45:3416-3421. [DOI] [PMC free article] [PubMed] [Google Scholar]