INTRODUCTION

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The development of fluconazole resistance in Candida albicans after long exposures to the drug is well documented for patients with AIDS and recurrent oropharyngeal candidiasis (28). Phenotypically stable resistance to azole antifungals in C. albicans can result from mutations in or increased expression of genes involved in the ergosterol synthesis pathway (including the target enzyme 14- demethylase) and increased expression of ATP-binding cassette transporter and major facilitator efflux pumps (28). Recently, several groups have noted that resistance can develop in C. albicans after only short exposures to the drug, both in vitro (2) and in vivo (10, 14). One example of this rapid development of resistance was in a strain of C. albicans that caused disseminated infection in a patient after receipt of bone marrow transplantation (BMT) (10). The susceptible isolate became resistant to azole drugs, with an associated increase in mRNA for an efflux pump in the CDR family. In these clinical isolates, phenotypic resistance to these drugs was transient, as the azole antifungal MICs for the resistant isolates decreased after serial growth in the absence of drug (9).

Rapid development of antimicrobial resistance in vivo can result from drug pressure that selects for a resistant clone or from selection for a resistant subset of cells within a heterogeneous population. Alternatively, the presence of the drug can influence transcription of resistance genes within a clonal population of cells. Heterogeneous resistance, or selection for a resistant subpopulation of cells, is a phenomenon that has been well documented for Staphylococcus species (3) and more recently described for Cryptococcus neoformans (11).

Two series of isogenic C. albicans isolates with inducible yet transient resistance to azole drugs have been identified from patients who received BMTs at our center (data presented here and in references 9 and 10). In this study, we show that the molecular mechanism of azole resistance in the second series of isolates is similar to that in the first series, as both series of isolates become resistant with associated increased expression of CDR mRNA. The phenotypic pattern of resistance in both series of isolates suggests that the inducible, transient nature of resistance is associated with heterogeneous resistance, or selection of a resistant subpopulation of cells from a phenotypically heterogeneous population.

	MATERIALS AND METHODS

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Microorganisms. The isolates used in this study are listed in Table 1. The first series of nine isolates of C. albicans, FH1 to FH9 has been previously described (9). This series consists of colonizing and bloodstream isolates of the same strain from a patient who underwent BMT (10). The second series of isolates, TL1 to TL3, consists of two colonizing (mouthwash) isolates (TL1 and -2) and a third isolate (TL3) obtained from the blood of a patient who underwent BMT. Individual yeast colonies were picked from Sabouraud dextrose agar (Difco), identified as C. albicans by germ tube testing and use of API 20C strips, and stored frozen at 70°C in yeast extract-peptone-dextrose (YEPD) (10 g of yeast extract, 20 g of peptone, and 20 g of dextrose per liter) containing 10% glycerol. Azole-susceptible laboratory strains (SC5314 and 3153a) and azole-susceptible (2-76) and -resistant (12-99) clinical isolates described previously (16) were also used as controls.

Drugs, media, and antifungal susceptibility testing. Powder formulations of fluconazole (Roerig-Pfizer, New York, N.Y.), itraconazole (Janssen Pharmaceutica, Beerse, Belgium), and amphotericin B (Sigma, St. Louis, Mo.) were suspended in distilled water, filter sterilized, and stored at 70°C. Media utilized in these studies include yeast nitrogen base (1.7 g/liter) (Difco, Detroit, Mich.) with ammonium sulfate (5 g/liter) and dextrose (10 g/liter) (YAD), YEPD, and RPMI 1640 with 0.165 M MOPS (morpholinepropanesulfonic acid), pH 7.0 (American Bioorganics, Niagra Falls, N.Y.). Antifungal susceptibility testing was performed by the standardized microdilution method (13). E tests were performed as instructed by the manufacturer (AB Biodisk North America Inc., Piscataway, N.J.).

DNA extraction, strain typing, and sequence analysis. Genomic DNA was prepared using glass bead cell shearing (5). Restriction fragment length polymorphism analyses were performed to determine genetic relatedness of isolates TL1 to -3 and to compare susceptible and resistant isolates after serial transfer. Southern blotting was performed (7), and blots were hybridized with the C. albicans strain-specific Ca3 probe (20). The Ca3 probe was labeled with [-³²P]dATP with random priming.

RNA preparation and Northern blotting. Quantities of mRNAs for genes previously reported to be involved in azole resistance (MDR1, CDR, and ERG11), other genes in the ergosterol synthesis pathway (ERG1, ERG7, and ERG9), and the housekeeping gene TEF3 were compared in the susceptible and the resistant isolates. To quantitate mRNAs for multiple genes simultaneously, reverse Northern blotting was performed (6), followed by confirmation with standard Northern blotting. Yeast RNAs were prepared using cell-shearing methods (21). To perform the reverse Northern blotting, 10 µg of total cellular RNA was denatured in 100 µl of 0.05 M sodium carbonate at 55°C for 30 min, precipitated in ethanol, resuspended in water containing RNase inhibitor (Boehringer Mannheim, Indianapolis, Ind.), and 5' end labeled with [-³²P]ATP using T4 polynucleotide kinase (Promega, Madison, Wis.). Labeled RNA was cleaned using a G-25 MicroSpin Column (Amersham Pharmacia Biotech, Piscataway, N.J.). Target DNAs were PCR fragments previously cloned into PCR Script-Amp cloning vectors (Promega). Ten micrograms of each of these fragments was electrophoresed in 1% agarose gels and transferred to membranes using standard techniques for Southern blotting (7). Hybridizations were performed with labeled mRNAs from both the susceptible (TL1) and resistant (TL3) isolates. Signal intensities were quantified after exposure to a phosporimaging screen (Storm 1860; Molecular Dynamics), using the Molecular Dynamics ImageQuant program. To adjust for the quantity of labeled RNA used for hybridization, signal intensities of genes of interest were normalized with TEF3.

Testing for stability and induction. The stability of the fluconazole-resistant phenotype was determined by serial transfer in the absence of drug. A single colony of each isolate was cultured in drug-free YEPD at 30°C and transferred with 1- to 5,000-µl dilutions every 2 or 3 days. To determine if susceptible isolates become resistant in vitro in the presence of the drug (induction), single colonies were cultured in YAD containing fluconazole at a concentration equivalent to two times the original MIC for the isolate. YAD was used instead of yeast extract-based medium (YEPD) to ensure that ergosterol was not supplemented by the medium. MICs for the transferred isolates were determined weekly, and isolates were stored at 70°C in YEPD containing 10% glycerol.

Population analysis. A previously described method that utilizes CHROMagar (CHROMagar Company, Paris, France) containing fluconazole to identify resistant C. albicans isolates (15) was modified for population analyses. A single colony was cultured overnight in YEPD broth at 30°C, cells were counted with a hemocytometer, and 100, 1000, and 10,000 cells were plated onto CHROMagar plates without and with fluconazole (1, 4, 16, and 64 µg/ml). Growth was quantified using an automated colony counter (Eagle Eye II; Stratagene, La Jolla, Calif.) after 2 days of incubation (30°C). The number of colonies that grew in the presence of the drug at each concentration, relative to the number that grew in the absence of the drug, was calculated and plotted.

	RESULTS

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Azole resistance in the TL series of isolates. The first two TL isolates (TL1 and TL2) are susceptible to fluconazole, and the third isolate (TL3) is resistant (Table 1). TL1 and TL2 are also susceptible to itraconazole and ketoconazole (data not shown), while TL3 is resistant (MICs, 16 and 1 µg/ml, respectively). All isolates are susceptible to amphotericin B (MICs, 1 µg/ml).

View larger version (55K): [in this window] [in a new window]   FIG. 1.   (A) Southern blot of TL genomic DNA digested with EcoRI and hybridized with Ca3. (B) Reverse Northern blots to detect mRNA levels of the indicated genes. Blots were probed with mRNA from the azole-susceptible isolate TL1 (left) and the azole-resistant isolate TL3 (right). The resistant isolate contained sevenfold more CDR mRNA (after normalization with the TEF3 gene) than the susceptible isolate.

Resistance mechanism of the FH series. Our previous studies showed that azole drug resistance developed in a colonizing isolate of C. albicans along with an associated increase in mRNA for a member of the CDR efflux pump family. We explored the mechanism of resistance further by performing Northern analyses with probes specific for the CDR1 and CDR2 genes (6). The CDR1 mRNA levels increase approximately 2.3-fold between FH1 and FH8. CDR2 is not detectable in FH1, and the mRNA levels in FH8 are at least 24-fold above background (data not shown).

View larger version (21K): [in this window] [in a new window]   FIG. 2.   (A) Fluconazole MICs (y axis, log scale) for the resistant isolates of the FH (open symbols) and TL (solid symbols) series decrease after serial passage in the absence of drug. Shown are MICs, obtained from E tests, for the resistant isolates FH5 (open squares) and FH8 (open diamonds) in the FH series and the resistant isolate TL3 (solid squares) in the TL series. E tests were used for these experiments in order to appreciate early losses of high levels of resistance (between 64 and 256 µg of fluconazole per ml). Each transfer represents approximately 12 generations of growth. (B) Induction of resistance is shown as fluconazole MICs (y axis) after growth in the presence of drug. Shown are the MICs determined by microdilution methods (13), for the inducible isolates FH1 (squares), and TL1 (circles) and the noninducible isolates 3153a (×), SC5314 (triangles), and 2-76 (diamonds). Results represent the averages from at least two separate experiments.

Inducibility of susceptible C. albicans isolates. To determine if the original, fluconazole-susceptible isolates in both series (FH1 and TL1) become resistant to the drug in vitro, the isolates were serially transferred in the presence of fluconazole. Figure 2B diagrams the increase in fluconazole MIC in both FH1 and TL1. The fluconazole-susceptible laboratory controls (3153a and SC5314) and the fluconazole-susceptible clinical isolate 2-76 (27) did not show increased resistance after growth in the presence of the drug (Fig. 2B). The experiments were repeated twice, with similar results, and genetic similarity was confirmed with Southern analyses using the Ca3 probe (not shown).

View larger version (12K): [in this window] [in a new window]   FIG. 3.   Inducibility (left panel) and transience (right panel) for the susceptible isolate FH1. MICs (y axis, log scale) of fluconazole (circles), ketoconazole (squares), and itraconazole (triangles) were determined by microdilution methods (13). Each transfer in the presence of fluconazole (left) and in the absence of fluconazole (right) represents approximately 12 generations of growth (x axis) (29).

Analysis of cellular populations. To determine if the transient, inducible azole resistance in the FH and TL isolates is associated with selection for a resistant subpopulation or increased expression of CDR in one clone, analysis of resistance within the cellular population was performed. Figure 4 shows the results of at least two population analyses for the susceptible, inducible strains FH1 and TL1 compared to the susceptible, noninducible strains 3153a, 2-76, and SC5314. FH1 has a majority of cells for which the MIC corresponds with the serial dilution MIC (4 µg/ml) but there is also a minority population of cells (approximately 10%) for which the apparent MIC is >64 µg/ml. TL1 has a majority of cells for which the MIC is 1 µg/ml and a minority for which the MIC is between 4 and 64 µg/ml. Alternatively, the noninducible isolates have homogeneous populations, for which the MICs correspond with the serial dilution MICs (1 µg/ml).

View larger version (29K): [in this window] [in a new window]   FIG. 4.   Population analyses of susceptible, inducible isolates FH1 and TL1 and noninducible isolates 3153a, SC5314, and 2-76. The percentage of resistant cells (y axis, log scale) is defined as the ratio of colonies with growth on plates containing the indicated concentration of fluconazole relative to growth in the absence of drug.

View larger version (51K): [in this window] [in a new window]   FIG. 5.   Population analyses of fluconazole-resistant isolates and susceptible transfers. Shown are the resistant isolate FH8, the susceptible isolate FH8-T, and the resistant transfer of the susceptible isolate FH8-T-R. Results are also shown for the resistant transfer of the first susceptible isolate, FH1-R, and its susceptible transfer, FH1-R-T. The percentage of resistant cells (y axis, log scale) is defined as the ratio of colonies with growth on plates containing the indicated concentration of fluconazole relative to growth in the absence of drug. Results represent averages from at least two experiments.

View larger version (134K): [in this window] [in a new window]   FIG. 6.   Fluconazole E-test results for the heterogeneous isolate FH1 (left) and the homogeneous isolate 2-76 (right). E tests were performed using equivalent inocula as directed by the manufacturer.

	DISCUSSION

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We have documented that C. albicans can become resistant to azole antifungals through a mechanism that involves selection of a resistant isolate from a heterogeneous population of cells. These findings highlight the complexity of antimicrobial susceptibility testing and clinical interpretation for both bacteria and yeasts.

Recently, a heterogeneous phenotype in clinical isolates of Cryptococcus neoformans isolated from AIDS patients with persistent meningitis was described (11). The two series of C. albicans isolates described here also acquired clinically significant resistance, as they became resistant to fluconazole in vivo and caused disseminated infection despite prophylactic administration of large doses of the drug in BMT recipients. The prevalence of these organisms and whether they account for clinical failures of fluconazole therapy in other situations are unknown.

Inducible drug resistance associated with a heterogeneous phenotype in C. albicans is consistent with previous reports of heterogeneity in susceptibility phenotypes (22) and resistance mechanisms (4) within clonal populations of Candida spp. Cultures of C. glabrata, C. krusei, and C. albicans contain heterogeneous populations of colonies that vary in susceptibility to fluconazole and itraconazole (22). Also, the rapid development of reversible fluconazole resistance in C. albicans after serial growth in the presence of drug was reported (2), although the mechanisms associated with resistance were not determined. Other investigators found that populations of C. albicans that are experimentally induced to become resistant to fluconazole acquire resistance through multiple mechanisms, despite clonal derivation by single-cell progenitors (4). More recently, in vitro induction of resistance in C. tropicalis was documented, with associated increased expression of CDR1 and MDR1 (1). Our findings complement these observations by documenting that the variability in the susceptibility phenotype within cellular populations is associated with the clinical development of resistance and an inducible phenotype in vitro.

This phenomenon might be similar to the heterogeneous resistance that occurs in staphylococci and enterococci that become resistant to -lactam and glycopeptide antibiotics (3, 23). In staphylococci, heterogeneous resistance appears to involve several genes and control is complex (3). Although the mechanisms remain elusive, factors in the cellular environment (temperature and medium pH, etc.) affect the phenotypic expression of resistance (3). One mechanism by which these organisms become resistant to -lactam and glycopeptide antibiotics appears to be mediated by a change in cell wall components, such as penicillin-binding proteins and peptidoglycan, resulting in a decrease in susceptibility due to altered binding (and possibly cellular entry) of the drug (12, 24). Whether similar mechanisms are associated with heterogeneous azole drug resistance in C. albicans is currently unknown, but it is of interest that all of the antimicrobials that have been associated with heterogeneous resistance (-lactams, glycopeptides, and azoles) are static drugs that directly or indirectly target the cell wall or membrane.

As noted previously (22), another possible cellular mechanism associated with heterogeneity in C. albicans might involve phenotypic switching between susceptibility phenotypes (26). Isolates of Candida lusitaniae undergo a reversible phenotypic switch that is associated with the development of resistance to amphotericin B (30). Since different strains of C. albicans undergo reversible phase switching, this is a potential mechanism for heterogeneity between susceptibility phenotypes (26).

The high frequency at which resistant cellular subpopulations are detected suggests that genetic mutations are not responsible for the generation of resistance, because mutations occur at frequencies approximating 10⁶ to 10⁸ per gene. Although we utilized a DNA fingerprinting method that has the best resolution, our conclusions are limited by the fact that no methods to fully ascertain the genetic distance between strains are available (25). Also, our molecular studies suggest that the two series of isolates both became resistant to the drug in vivo and in vitro along with associated increased mRNA levels for CDR1 and CDR2 (reference 9 and this study). Although we do not know how the cellular and molecular makeups of these isolates otherwise compare, it is possible that the heterogeneous phenotype is specifically associated with CDR expression, as increased expression of this efflux pump might cause a metabolic growth disadvantage that could explain the transient nature of resistance. Studies investigating the cellular phenotypes and molecular mechanisms of heterogeneous isolates are ongoing.

The development of resistance in these strains should be differentiated from the trailing phenotype that is observed in serial dilution susceptibility testing. Isolates that trail exhibit a low azole MIC after 24 h of growth and a high MIC after 48 h (18). The isolates described here do not trail. This is an important distinction, as trailing isolates behave as susceptible strains in vivo (18), while these heterogeneous isolates were clearly the cause of resistant disease in patients on prophylactic fluconazole. Preliminary population analyses on trailing isolates do not show a heterogeneous pattern as described in this study, but instead these isolates have a high degree of variability in colony morphology in the absence of drug (data not shown). Also, the trailing isolates do not become resistant with serial passage in the presence of drug in vitro (data not shown). These findings suggest that the heterogeneous and trailing phenotypes differ in both in vitro and in vivo behavior. While isolates that have the heterogeneous phenotype are a potential cause of clinically resistant infection, the significance of the trailing phenotype appears to be limited to being a cause of falsely elevated MICs in susceptibility testing.

The finding that isolates of C. albicans can become resistant to fluconazole rapidly, despite MICs being within the susceptible range, has important implications for the clinical microbiology laboratory. Previous studies have noted that serial dilution susceptibility testing is less successful in predicting therapeutic success than in predicting failure (19). Although it is likely that host factors are the major explanation, it is possible that isolate heterogeneity contributes to these limitations. Given the use of fluconazole as a single agent for candidemia in immunocompetent hosts (17), the laboratory should be able to differentiate homogeneous, susceptible isolates from heterogeneous, susceptible isolates that can become resistant after drug exposure. Our preliminary results suggest that E tests might be useful, but further studies are necessary to characterize large numbers of heterogeneous isolates and to document the correlation between heterogeneity and clinical failure.

In summary, we have described an important mechanism by which C. albicans can become resistant to azole antifungals in immunosuppressed patients. Clinical microbiologists and clinicians should be aware of this as a potential cause of azole treatment failure. Further studies are required to determine the prevalence of this phenotype and the cellular and molecular factors involved in conferring resistance.