a The Second Department of Internal Medicine, Nagasaki University School of Medicine, 1-7-1 Sakamoto Nagasaki 852, Japan; b Anti-Infectives Research Departments, Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium; c Institut de Microbiologie, Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland
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Abstract |
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Introduction |
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Many studies have investigated the mechanisms of azole resistance in Candida spp. isolates.7,8,9 The results of these studies indicate that resistance to azole antifungal agents may be a consequence of one or more mechanisms. One of the major mechanisms imparting resistance to azole antifungal agents can be drug efflux, similar to the multidrug- resistant (MDR) pumps in tumour cells. Among the MDR genes, CDR1 has been cloned inC. albicans and the level of mRNA of the CDR1 gene was found to be significantly higher in fluconazole-resistant strains than in sensitive strains. 10 The product of another C. albicans gene, CaMDR1, formerly known as BENr resembles proteins of the major facilitator superfamily (MFS) class of MDR proteins.11
In the present study, we used a fluorescent dye, rhodamine 6G (R6G), a substrate of MDR protein in Saccharomyces cerevisiae, to identify MDR activity in azole-resistant isolates of C. albicans.
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Materials and methods |
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Two isolates of C. albicans (B2630 and B67081) held at the Janssen Research Foundation (Beerse, Belgium) were used. Another 10 strains of C. albicans that were originally isolated from AIDS patients with OPC, were obtained from the Institute of Microbiology (Centre Hospitalier Universitaire, Vaudois, Switzerland). The species were identified using standard procedures. All strains were maintained at -80°C. Strains of C. albicans were grown in a CYG medium containing 0.5% casein hydrolysate (Merck, Darmstadt, Germany), 0.5% yeast extract (Difco, Detroit, MI, USA) and 0.5% glucose (Difco). Fluconazole was obtained from Pfizer (Sandwich, UK), and ketoconazole and itraconazole from the Janssen Research Foundation (Beerse, Belgium). Azole antifungal agents were dissolved in dimethyl sulphoxide (DMSO) at 1.5 g/L and used as stock solution.
MIC determination for C. albicans isolates
The stock solution was diluted 100-fold with susceptibility-testing culture medium and a series of 10 two-fold dilutions was prepared. These solutions were pipetted in 100 µL volumes into rows of wells of flat-bottomed microdilution plates (Falcon 3072; Becton Dickinson, Lincoln Park, NJ, USA). The final concentrations of fluconazole ranged from 100 to 0.13 mg/L, and those of ketoconazole and itraconazole from 8 to 0.02 mg/L, in two-fold serial dilutions.
The MIC of antifungal agents was determined by the microdilution method using the 96 flat-bottomed microdilution plate modified from the NCCLS macrodilution method. From deep frozen stock culture, the cells of C. albicans were inoculated into 5 mL of CYG broth and incubated at 37°C for 1824 h with shaking. The final inoculum size was adjusted to 103 cfu/mL. The plates were sealed with plastic stickers and incubated for 48 h at 35°C. MICs were determined as the minimum concentration of the antifungal agents yielding at least 80% inhibition of the growth compared with the growth of control.
Measurement of R6G uptake and glucose-induced efflux
Yeast cells were grown in 100 mL of CYG broth at 37°C for 14 h. 1 x 108 yeast cells/mL were transferred to 100 mL of fresh CYG broth and incubated at 37°C for 4 h. The cells were harvested in 50 mL Falcon tubes (Becton Dickinson) and centrifuged at 5000g for 5 min. The pellets were washed twice with 20 mL of phosphate buffered saline (PBS; Life Technologies, Paisley, Scotland). They were then suspended in a glucose-free PBS buffer, at a concentration of 1 x 108 cells/mL and incubated at 37°C for 1 h in a reciprocating shaker. A stock solution of R6G (Sigma Chemical Co., St Louis, MO, USA) was prepared by dissolving the dye in DMSO at a concentration of 10 mM. A final concentration of 10 µM of R6G was added to the cell suspension and incubated at 37°C in a reciprocating shaker. After incubation for 5, 10, 15, 20, 25 min, 1 mL samples were withdrawn and centrifuged at 9000g for 2 min. The supernatants (750 µL) were collected and absorption was measured at 527 nm. To examine the effect of glucose, the cell suspension was centrifuged after 25 min incubation, at 5000g for 5 min and pellets were resuspended in PBS containing 1 mole of glucose and incubated at 37°C. Samples of 1 mL volume were withdrawn at 5 min intervals and centrifuged at 9000g for 2 min. Then 750 µL of supernatant was collected and absorption was measured at 527 nm. The concentration of R6G was calculated using a standard concentration curve of R6G.
Intracellular concentration of R6G in growing isolates of C. albicans
Yeast cells were grown in 100 mL of CYG medium at 37°C for 14 h in a reciprocating shaker. A total of 1 x 108 cells/mL was transferred to 100 mL of fresh CYG medium and the cells were harvested after 4 h incubation by centrifugation at 5000g for 5 min, and then washed with PBS. Cells were resuspended in CYG medium to a cell concentration of 2.5 x 108 cells/mL. One millilitre of the cell suspension was incubated in a 10 mL glass tube with R6G (final concentration 10 µM) at 37°C for 1 h, under continuous shaking at 300g in an orbital shaker. Cells were collected by centrifugation at 9000g for 2 min and were resuspended with 750 µL of PBS buffer and absorption was measured at 527 nm.
Measurement of CDR1 and CaMDR (BENr) mRNA expression
The measurement of CDR1 and CaMDR (BENr) mRNA expression was reported in our previous study.10 Yeast cells were grown to logarithmic phase in 100 mL of Yeast Nitrogen Base (YNB) medium at 30°C with shaking. The cells were harvested and were ground to a fine powder under liquid nitrogen. The powder was immediately dissolved in a denaturing solution provided by the RNAeasy kit (QIAGEN Inc., Chatsworth, CA, USA). For the Northern analysis, RNA was first denatured in a loading buffer (50% formamide, 100 mM MOPS pH 7.0, 6.4% formaldehyde, 5% glycerol, 5% of a water solution saturated with bromophenol blue) at 85°C for 5 min and then subjected to electrophoresis in 1% agarose. The agarose was melted in a buffer containing 0.1 M MOPS, 0.6 M formaldehyde. The electrophoresis buffer was 0.1 M MOPS pH 7.0.
Northern transfer was performed overnight on GeneScreen Plus (DuPont NEN, Boston, MA, USA) with 10 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) as a transfer buffer. RNA was fixed on the membrane by baking at 80°C under vacuum. Membranes were prehybridized at 42°C with a buffer consisting of 50% formamide, 1% sodium dodecyl sulphate (SDS), 4 x SSC, 10% dextran sulphate and salmon sperm DNA 100 mg/L. 32P-labelled DNA probes were generated by random priming and were added to the hybridization solution overnight. After the washing step, membranes were exposed to X-OMAT AR film (Eastman Kodak Company, Rochester, NY, USA) for documentation. For quantitative analysis, the signals were subsequently analysed by a Phosphor Imager (Molecular Dynamics, Sunnyvale, CA, USA).
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Results |
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To investigate the efflux mechanisms of azole antifungal agents, we measured the uptake and efflux of R6G by C. albicans cells. For this purpose, we compared R6G uptake and efflux in azole-resistant (B67081) and -sensitive (B2630) strains. Intracellular R6G uptake increased immediately when both types of cells were incubated in glucose-free PBS, as was evident from a sharp drop in the measured extracellular concentrations of R6G (Figure 1). However, the uptake reached equilibrium 25 min after incubation. No significant difference in R6G uptake was observed between B67081 (8.86 ± 0.03 nmol/109 cells, n = 10) and B2630 (9.02 ± 0.02 nmol/109 cells, n = 10) 25 min after incubation. In the next step, the cells were resuspended in PBS and 1 mol of glucose was added 30 min after incubation. Azole-resistant C. albicans (B67081) pumped out higher concentrations of R6G (2.00 ± 0.21 nmol/109 cells, n = 10) into the extracellular fluid than azole-sensitive C. albicans (B2630) (0.23 ± 0.14 nmol/109 cells, n = 10). However, no R6G efflux occurred when both strains were maintained for another 35 min in the absence of glucose (Figure).
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We compared the intracellular accumulation of R6G in growing C. albicanscells, both fluconazole-sensitive and -resistant strains (Table II). R6G accumulation measured by the photometric method in the azole-resistant strain (B67081) was markedly lower than in the sensitive strain (B2630). In contrast, intracellular R6G concentration in four of five fluconazole-sensitive strains (MIC of fluconazole was <6.3 mg/L for C27, C33, C23 and C37) was more than 4.0 nmol/mL. However, the concentration in one azole-sensitive strain, C32 (MIC of fluconazole was 3.2 mg/L) was lower than in the other four sensitive strains. Intracellular R6G concentration in three fluconazole-resistant strains (C82, C26 and C39) was <3.0 nmol/mL. The highest accumulation of R6G was noted in azole-resistant strains C34 and C40 (MIC of fluconazole was 25 and 100 mg/L, respectively). We correlated the concentration of intracellular R6G in growing C. albicans strains with the level of CDR1 mRNA (Table II). Six strains with a low expression of CDR1 mRNA showed a high accumulation of intracellular R6G whereas three other azole-resistant strains with a high level of CDR1 expression showed very low intracellular concentration of R6G. These results indicated that accumulation of R6G in the cell was inversely related to the expression level of CDR1 mRNA.
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Discussion |
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The resistance of C40 to fluconazole was as high as that of strain C26. The latter strain also showed cross-resistance to ketoconazole and itraconazole, but strain C40 was less resistant to ketoconazole and itraconazole.10 In the present study, R6G uptake was not significantly different between the two resistant strains. However, we observed R6G efflux only in C26, since the level of CDR1 mRNA expression of C40 was similar to that of the sensitive strain; R6G efflux in C40 was not induced by glucose. The differences in R6G efflux between strains C26 and C40 suggest that R6G is a substrate of the MDR protein produced by CDR1 mRNA.
Intracellular concentrations of R6G were high in fluconazole-sensitive growing C. albicans cells but low in resistant strains. However, one of the sensitive strains, C32, had a low concentration of R6G in growing cells. Comparison between C32 and C37 strains showed that the susceptibility of both strains to fluconazole was similar, but the level of expression of CDR1 mRNA in C37 was almost twice that in the C32 strain. Thus, the differences in expression of CDR1 mRNA between these two strains may explain the difference in intracellular accumulation of R6G. Among azole-resistant strains, C34 and C40 showed a higher accumulation of R6G than other resistant strains. These two strains also showed a low level of expression of CDR1 mRNA. The C40 strain was the only resistant strain included in this study that expressed CaMDR mRNA. The methods used to measure accumulation of R6G could not confirm whether the mechanism of azole resistance was due to the CaMDR gene. The differences in R6G accumulation between these two strains suggest that the efflux mechanism of resistance was induced by a phenotype different from that of the CDR1 gene, or perhaps was caused by an entirely different mechanism. Future studies should compare the accumulation of cytochrome P450 or the binding affinity of azole compounds in these strains.
This method may be simple and convenient for measuring the level of CDR1 mRNA expression in azole-resistant strains of C. albicans and could be useful in determining the basis of azole resistance among clinically isolated strains. The method is based on the molecular mechanism of C. albicans resistance to azoles and can be used for comparing azole-resistant strains of C. albicans defined using other procedures. We suggest that measurement of intracellular accumulation of R6G could be used clinically to identify resistant strains of C. albicans isolated from patients during azole antifungal therapy and to help in selecting a suitable antifungal therapy.
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Acknowledgments |
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Notes |
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References |
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Received 11 October 1998; returned 1 January 1999; revised 18 January 1999; accepted 14 February 1999