Fluconazole-resistant Candida albicans, a cause of recurrent oropharyngeal candidiasis in patients with human immunodeficiency virus infection, has recently emerged as a cause of candidiasis in patients receiving cancer chemotherapy and marrow transplantation (MT). In this study, we performed detailed molecular analyses of a series of C. albicans isolates from an MT patient who developed disseminated candidiasis caused by an azole-resistant strain 2 weeks after initiation of fluconazole prophylaxis (K. A. Marr, T. C. White, J. A. H. vanBurik, and R. A. Bowden, Clin. Infect. Dis. 25:908–910, 1997). DNA sequence analysis of the gene (ERG11) for the azole target enzyme, lanosterol demethylase, revealed no difference between sensitive and resistant isolates. A sterol biosynthesis assay revealed no difference in sterol intermediates between the sensitive and resistant isolates. Northern blotting, performed to quantify mRNA levels of genes encoding enzymes in the ergosterol biosynthesis pathway (ERG7, ERG9, and ERG11) and genes encoding efflux pumps (MDR1, ABC1, YCF, and CDR), revealed that azole resistance in this series is associated with increased mRNA levels for members of the ATP binding cassette (ABC) transporter superfamily, CDR genes. Serial growth of resistant isolates in azole-free media resulted in an increased susceptibility to azole drugs and corresponding decreased mRNA levels for the CDR genes. These results suggest that C. albicans can become transiently resistant to azole drugs rapidly after exposure to fluconazole, in association with increased expression of ABC transporter efflux pumps.
Some Candida albicans isolates from AIDS patients with oropharyngeal candidiasis are becoming resistant to the azole antifungal agent fluconazole after prolonged treatment with this compound. Most of the C. albicans isolates resistant to fluconazole fail to accumulate this antifungal agent, and this has been considered a cause of resistance. This phenomenon was shown to be linked to an increase in the amounts of mRNA of a C. albicans ABC (ATP-binding cassette) transporter gene called CDR1 and of a gene conferring benomyl resistance (BENr), the product of which belongs to the class of major facilitator multidrug efflux transporters (D. Sanglard, K. Kuchler, F. Ischer, J. L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378-2386, 1995). To analyze the roles of these multidrug transporters in the efflux of azole antifungal agents, we constructed C. albicans mutants with single and double deletion mutations of the corresponding genes. The mutants were tested for their susceptibilities to these antifungal agents. Our results indicated that the delta cdr1 C. albicans mutant was hypersusceptible to the azole derivatives fluconazole, itraconazole, and ketoconazole, thus showing that the ABC transporter Cdr1 can use these compounds as substrates. The delta cdr1 mutant was also hypersusceptible to other antifungal agents (terbinafine and amorolfine) and to different metabolic inhibitors (cycloheximide, brefeldin A, and fluphenazine). The same mutant was slightly more susceptible than the wild type to nocodazole, cerulenin, and crystal violet but not to amphotericin B, nikkomycin Z, flucytosine, or pradimicin. In contrast, the delta ben mutant was rendered more susceptible only to the mutagen 4-nitroquinoline-N-oxide. However, this mutation increased the susceptibilities of the cells to cycloheximide and cerulenin when the mutation was constructed in a delta cdr1 background. The assay used in the present study could be implemented with new antifungal agents and is a powerful tool for assigning these substances as putative substrates of multidrug transporters.
Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753–2765, 1999). Deletion of CgCDR1 in C. glabrata rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type C. glabrata exposed to fluconazole in a medium containing the drug at 50 μg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 × 10−4 to 4 × 10−4). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation of CgCDR1. The CgCDR1 deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 μg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to CgCDR1, called CgCDR2, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both CgCDR1 and CgCDR2 suppressed the development of HFAR in a medium containing fluconazole at 5 μg/ml, showing that both genes are important mediators of resistance to azole derivatives in C. glabrata. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in C. glabrata. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both CgCDR1 and CgCDR2.
Candida albicans is an opportunistic human fungal pathogen and a causative agent of oropharyngeal candidiasis (OPC), the most frequent opportunistic infection among patients with AIDS. Fluconazole and other azole antifungal agents have proven effective in the management of OPC; however, with increased use of these agents treatment failures have occurred. Such failures have been associated with the emergence of azole-resistant strains of C. albicans. In the present study we examined changes in the genome-wide gene expression profile of a series of C. albicans clinical isolates representing the stepwise acquisition of azole resistance. In addition to genes previously associated with azole resistance, we identified many genes whose differential expression was for the first time associated with this phenotype. Furthermore, the expression of these genes was correlated with that of the known resistance genes CDR1, CDR2, and CaMDR1. Genes coordinately regulated with the up-regulation of CDR1 and CDR2 included the up-regulation of GPX1 and RTA3 and the down-regulation of EBP1. Genes coordinately regulated with the up-regulation of CaMDR1 included the up-regulation of IFD1, IFD4, IFD5, IFD7, GRP2, DPP1, CRD2, and INO1 and the down-regulation of FET34, OPI3, and IPF1222. Several of these appeared to be coordinately regulated with both the CDR genes and CaMDR1. Many of these genes are involved in the oxidative stress response, suggesting that reduced susceptibility to oxidative damage may contribute to azole resistance. Further evaluation of the role these genes and their respective gene products play in azole antifungal resistance is warranted.
Molecular mechanisms of azole resistance in Candida albicans, including alterations in the target enzyme and increased efflux of drug, have been described, but the epidemiology of the resistance mechanisms has not been established. We have investigated the molecular mechanisms of resistance to azoles in C. albicans strains displaying high-level fluconazole resistance (MICs, ≥64 μg/ml) isolated from human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis. The levels of expression of genes encoding lanosterol 14α-demethylase (ERG11) and efflux transporters (MDR1 and CDR) implicated in azole resistance were monitored in matched sets of susceptible and resistant isolates. In addition, ERG11 genes were amplified by PCR, and their nucleotide sequences were determined in order to detect point mutations with a possible effect in the affinity for azoles. The analysis confirmed the multifactorial nature of azole resistance and the prevalence of these mechanisms of resistance in C. albicans clinical isolates exhibiting frank fluconazole resistance, with a predominance of overexpression of genes encoding efflux pumps, detected in 85% of all resistant isolates, being found. Alterations in the target enzyme, including functional amino acid substitutions and overexpression of the gene that encodes the enzyme, were detected in 65 and 35% of the isolates, respectively. Overall, multiple mechanisms of resistance were combined in 75% of the isolates displaying high-level fluconazole resistance. These results may help in the development of new strategies to overcome the problem of resistance as well as new treatments for this condition.
The opportunistic fungal pathogen Candida albicans is the major causative agent of oropharyngeal candidiasis (OPC) in AIDS. The development of azoles, such as fluconazole, for the treatment of OPC has proven effective except in cases where C. albicans develops resistance to fluconazole during the course of treatment. In the present study, we used microarray technology to examine differences in gene expression from a fluconazole-susceptible and a fluconazole-resistant well-characterized, clinically obtained matched set of C. albicans isolates to identify genes which are differentially expressed in association with azole resistance. Among genes found to be differentially expressed were those involved in amino acid and carbohydrate metabolism; cell stress, cell wall maintenance; lipid, fatty acid, and sterol metabolism; and small molecule transport. In addition to CDR1, which has previously been demonstrated to be associated with azole resistance, the drug resistance gene RTA3, the ergosterol biosynthesis gene ERG2, and the cell stress genes CRD2, GPX1, and IFD5 were found to be upregulated. Several genes, such as the mitochondrial aldehyde dehydrogenase gene ALD5, the glycosylphosphatidylinositol synthesis gene GPI1, and the iron transport genes FET34 and FTR2 were found to be downregulated. Further study of these differentially regulated genes is warranted to evaluate how they may be involved in azole resistance. In addition to these novel findings, we demonstrate the utility of microarray analysis for studying the molecular mechanisms of drug resistance in pathogenic organisms.
Oral infections with the pathogenic yeast Candida albicans are one of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients. The widespread use of azole antifungal drugs has led to the development of drug-resistant isolates. Several molecular mechanisms that contribute to drug resistance have been identified, including increased mRNA levels for two types of efflux pump genes: the ATP binding cassette transporter CDRs (CDR1 and CDR2) and the major facilitator MDR1. Using Northern blot analyses, the expression patterns of these genes have been determined during logarithmic and stationary phases of cell growth and during growth in different carbon sources in a set of matched susceptible and fluconazole-resistant isolates that have been characterized previously. MDR1, CDR1, and CDR2 are expressed early during logarithmic growth, CDR4 is expressed late during logarithmic growth, and CDR1 is preferentially expressed in stationary-phase cells. There is a small decrease in expression of these genes when the cells are grown in carbon sources other than glucose. While increased mRNA levels of efflux pump genes are commonly associated with azole resistance, the causes of increased mRNA levels have not yet been resolved. Southern blot analysis demonstrates that the increased mRNA levels in these isolates are not the result of gene amplification. Nuclear run-on assays show that MDR1 and CDR mRNAs are transcriptionally overexpressed in the resistant isolate, suggesting that the antifungal drug resistance in this series is associated with the promoter and trans-acting factors of the CDR1, CDR2, and MDR1 genes.
Infections with fluconazole-resistant Candida albicans isolate have rarely been described in clinical settings other than oropharyngeal candidiasis in patients with late-stage AIDS. We report on two patients with leukemia who developed fungemia caused by fluconazole-resistant C. albicans after receiving fluconazole prophylaxis (400 mg/day) and empiric amphotericin B therapy (0.5 mg/kg of body weight per day). The fluconazole MICs for the isolates were > or = 64 micrograms/ml, and the isolates were resistant to other azoles and had membrane sterol changes consistent with a mutation in the delta 5,6-sterol desaturase gene. The lack of ergosterol in the cytoplasmic membrane of the fluconazole-resistant strains also imparted resistance to amphotericin B. Both patients were successfully treated with high-dose amphotericin B (1 to 1.25 mg/kg/day) and flucytosine (150 mg/kg/day).
Candida albicans biofilm infections are usually treated with azole antifungals such as fluconazole. However, the development of resistance to this drug in C. albicans biofilms is very common, especially in immunocompromised individuals. The upregulation of the sterol biosynthetic pathway gene ERG and the efflux pump genes CDR and MDR may contribute to this azole tolerance in Candida species. We hypothesize that farnesol, an endogenous quorum sensing molecule with possible antimicrobial properties which is also the precursor of ergosterols in C. albicans, may interfere with the development of fluconazole resistance in C. albicans biofilms. To test this hypothesis, MICs were compared and morphology changes were observed by confocal laser scanning microscopy (CLSM) for farnesol-treated and -untreated and fluconazole-resistant groups. The expression of possible target genes (ERG11, ERG25, ERG6, ERG5, ERG3, ERG1, MDR1, CDR1, and CDR2) in biofilms was analyzed by reverse transcription-PCR (RT-PCR) and quantitative PCR (qPCR) to investigate the molecular mechanisms of the inhibitory effects of farnesol. The results showed a decreased MIC of fluconazole and thinner biofilms for the farnesol-treated group, indicating that farnesol inhibited the development of fluconazole resistance. The sterol biosynthetic pathway may contribute to the inhibitory effects of farnesol, as the transcription levels of the ERG11, ERG25, ERG6, ERG3, and ERG1 genes decreased in the farnesol-treated group.
A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1- and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [3H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1-regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1-, CDR2-, or MDR1-overexpressing cells accumulated much more [3H]fluconazole than did PGVs from empty-vector controls. The Kms (expressed in micromolar concentrations) and Vmaxs (expressed in picomoles per milligram of protein per minute), respectively, for [3H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [3H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [3H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [3H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC50s; expressed as proportions of the [3H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi.
The present study tracks the development of low-level azole resistance in in vitro fluconazole-adapted strains of Candida albicans, which were obtained by serially passaging a fluconazole-susceptible dose-dependent strain, YO1-16 (fluconazole MIC, 16 μg ml−1) in increasing concentrations of fluconazole, resulting in strains YO1-32 (fluconazole MIC, 32 μg ml−1) and YO1-64 (MIC, 64 μg ml−1). We show that acquired resistance to fluconazole in this series of isolates is not a random process but is a gradually evolved complex phenomenon that involves multiple changes, which included the overexpression of ABC transporter genes, e.g., CDR1 and CDR2, and the azole target enzyme, ERG11. The sequential rise in fluconazole MICs in these isolates was also accompanied by cross-resistance to other azoles and terbinafine. Interestingly, fluorescent polarization measurements performed by using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene revealed that there was a gradual increase in membrane fluidity of adapted strains. The increase in fluidity was reflected by observed change in membrane order, which was considerably decreased (decrease in fluorescence polarization values, P value) in the adapted strain (P value of 0.1 in YO1-64, compared to 0.19 in the YO1-16 strain). The phospholipid composition of the adapted strain was not significantly altered; however, ergosterol content was reduced in YO1-64 from that in the YO1-16 strain. The asymmetrical distribution of phosphatidylethanolamine (PE) between two monolayers of plasma membrane was also changed, with PE becoming more exposed to the outer monolayer in the YO1-64 strain. The results of the present study suggest for the first time that changes in the status of membrane lipid phase and asymmetry could contribute to azole resistance in C. albicans.
Resistance to azole antifungals continues to be a significant problem in the common fungal pathogen Candida albicans. Many of the molecular mechanisms of resistance have been defined with matched sets of susceptible and resistant clinical isolates from the same strain. Mechanisms that have been identified include alterations in the gene encoding the target enzyme ERG11 or overexpression of efflux pump genes including CDR1, CDR2, and MDR1. In the present study, a collection of unmatched clinical isolates of C. albicans was analyzed for the known molecular mechanisms of resistance by standard methods. The collection was assembled so that approximately half of the isolates were resistant to azole drugs. Extensive cross-resistance was observed for fluconazole, clotrimazole, itraconazole, and ketoconazole. Northern blotting analyses indicated that overexpression of CDR1 and CDR2 correlates with resistance, suggesting that the two genes may be coregulated. MDR1 overexpression was observed infrequently in some resistant isolates. Overexpression of FLU1, an efflux pump gene related to MDR1, did not correlate with resistance, nor did overexpression of ERG11. Limited analysis of the ERG11 gene sequence identified several point mutations in resistant isolates; these mutations have been described previously. Two of the most common point mutations in ERG11 associated with resistance, D116E and E266D, were tested by restriction fragment length polymorphism analysis of the isolates from this collection. The results indicated that the two mutations occur frequently in different isolates of C. albicans and are not reliably associated with resistance. These analyses emphasize the diversity of mechanisms that result in a phenotype of azole resistance. They suggest that the resistance mechanisms identified in matched sets of susceptible and resistant isolates are not sufficient to explain resistance in a collection of unmatched clinical isolates and that additional mechanisms have yet to be discovered.
Fluconazole (FLC) remains the antifungal drug of choice for non-life-threatening Candida infections, but drug-resistant strains have been isolated during long-term therapy with azoles. Drug efflux, mediated by plasma membrane transporters, is a major resistance mechanism, and clinically significant resistance in Candida albicans is accompanied by increased transcription of the genes CDR1 and CDR2, encoding plasma membrane ABC-type transporters Cdr1p and Cdr2p. The relative importance of each transporter protein for efflux-mediated resistance in C. albicans, however, is unknown; neither the relative amounts of each polypeptide in resistant isolates nor their contributions to efflux function have been determined. We have exploited the pump-specific properties of two antibody preparations, and specific pump inhibitors, to determine the relative expression and functions of Cdr1p and Cdr2p in 18 clinical C. albicans isolates. The antibodies and inhibitors were standardized using recombinant Saccharomyces cerevisiae strains that hyper-express either protein in a host strain with a reduced endogenous pump background. In all 18 C. albicans strains, including 13 strains with reduced FLC susceptibilities, Cdr1p was present in greater amounts (2- to 20-fold) than Cdr2p. Compounds that inhibited Cdr1p-mediated function, but had no effect on Cdr2p efflux activity, significantly decreased the resistance to FLC of seven representative C. albicans isolates, whereas three other compounds that inhibited both pumps did not cause increased chemosensitization of these strains to FLC. We conclude that Cdr1p expression makes a greater functional contribution than does Cdr2p to FLC resistance in C. albicans.
Resistance to antifungal drugs, specifically azoles such as fluconazole, in the opportunistic yeast Candida albicans has become an increasing problem in human immunodeficiency virus (HIV)-infected individuals. The molecular mechanisms responsible for this resistance have only recently become apparent and can include alterations in the target enzyme of the azole drugs (lanosterol 14alpha demethylase [14DM]), or in various efflux pumps from both the ABC transporter and major facilitator gene families. To determine which of these possible mechanisms was associated with the development of drug resistance in a particular case, mRNA levels have been studied in a series of 17 clinical isolates taken from a single HIV-infected patient over 2 years, during which time the levels of fluconazole resistance of the strain increased over 200-fold. Using Northern blot analysis of steady-state levels of total RNA from these isolates, we observed increased mRNA levels of ERG16 (the 14DM-encoding gene), CDR1 (an ABC transporter), and MDR1 (a major facilitator) in this series. The timing of the increase in mRNA levels of each of these genes correlated with increases in fluconazole resistance of the isolates. Increased mRNA levels were not observed for three other ABC transporters, two other genes in the ergosterol biosynthetic pathway, or the NADPH-cytochrome P-450 oxidoreductase gene that transfers electrons from NADPH to 14DM. Increases in mRNA levels of ERG16 and CDR1 correlated with increased cross-resistance to ketoconazole and itraconazole but not to amphotericin B. A compilation of the genetic alterations identified in this series suggests that resistance develops gradually and is the sum of several different changes, all of which contribute to the final resistant phenotype.
Candida albicans frequently develops resistance to treatment with azole drugs due to the acquisition of gain-of-function mutations in the transcription factor Tac1p. Tac1p hyperactivation in azole-resistant isolates results in the constitutive overexpression of several genes, including CDR1 and CDR2, which encode two homologous transporters of the ATP-binding cassette family. Functional studies of Cdr1p and Cdr2p have been carried out so far by heterologous expression in the budding yeast Saccharomyces cerevisiae and by gene deletion or overexpression in azole-sensitive C. albicans strains in which CDR1 expression is low and CDR2 expression is undetectable. Thus, the direct demonstration that CDR1 and CDR2 overexpression causes azole resistance in clinical strains is still lacking, as is our knowledge of the relative contribution of each transporter to clinical azole resistance. In the present study, we used the SAT1 flipper system to delete the CDR1 and CDR2 genes from clinical isolate 5674. This strain is resistant to several azole derivatives due to a strong hyperactive mutation in Tac1p and expresses high levels of Cdr1p and Cdr2p. We found that deleting CDR1 had a major effect, reducing resistance to fluconazole (FLC), ketoconazole (KTC), and itraconazole (ITC) by 6-, 4-, and 8-fold, respectively. Deleting CDR2 had a much weaker effect, reducing FLC or KTC resistance by 1.5-fold, and had no effect on ITC resistance. These results demonstrate that Cdr1p is a major determinant of azole resistance in strain 5674 and potentially in other clinical strains overexpressing Cdr1p and Cdr2p, while Cdr2p plays a more minor role.
The inactivation of ERG3, a gene encoding sterol Δ5,6-desaturase (essential for ergosterol biosynthesis), is a known mechanism of in vitro resistance to azole antifungal drugs in the human pathogen Candida albicans. ERG3 inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified a C. albicans clinical isolate (VSY2) with high-level resistance to azole drugs in vitro and an absence of ergosterol but normal filamentation. Sequencing of ERG3 in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (erg3-1) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming that ERG3 inactivation was the mechanism of azole resistance. Additionally, the replacement of both ERG3 alleles by erg3-1 in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinical ERG3 mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole both in vitro and in vivo, the ERG3-derived mutant of SC5314 was resistant only in vitro and was less virulent than the wild type. This suggests that VSY2 compensated for the in vivo fitness defect of ERG3 inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation of ERG3 does not necessarily affect filamentation and virulence.
Clinically relevant azole resistance in the fungal pathogen Candida albicans is most often associated with the increased expression of plasma membrane efflux pumps, specifically the ATP-binding cassette (ABC) transporters CaCdr1p and CaCdr2p and the major facilitator superfamily (MFS) transporter, CaMdr1p. Development of potent pump inhibitors that chemosensitize cells to azoles is a promising approach to overcome antifungal resistance. Here we identify Nile Red as a new fluorescent substrate for CaCdr1p, CaCdr2p and CaMdr1p. Nile Red was effluxed efficiently from Saccharomyces cerevisiae cells heterologously expressing these transporters. Enniatin selectively inhibited the efflux of Nile Red from S. cerevisiae cells expressing CaCdr1p or CaMdr1p but not from cells expressing CaCdr2p. This indicates that Nile Red can be used for the identification of inhibitors specific for particular transporters mediating antifungal resistance in pathogenic yeast.
MDR; yeast; fluconazole; efflux pumps; fluorescent substrate; Candida albicans
The micafungin and caspofungin susceptibilities of Candida albicans laboratory and clinical isolates and of Saccharomyces cerevisiae strains stably hyperexpressing fungal ATP-binding cassette (ABC) or major facilitator superfamily (MFS) transporters involved in azole resistance were determined using three separate methods. Yeast strains hyperexpressing individual alleles of ABC transporters or an MFS transporter from C. albicans gave the expected resistance profiles for the azoles fluconazole, itraconazole, and voriconazole. The strains hyperexpressing CDR2 showed slightly decreased susceptibility to caspofungin in agar plate drug resistance assays, as previously reported, but increased susceptibility to micafungin compared with either the strains hyperexpressing CDR1 or the null parent deleted of seven ABC transporters. The strains hyperexpressing CDR1 showed slightly decreased susceptibility to micafungin in these assays. A C. albicans clinical isolate overexpressing both Cdr1p and Cdr2p relative to its azole-sensitive isogenic progenitor acquired resistance to azole drugs and showed reduced susceptibility to caspofungin and slightly increased susceptibility to micafungin in agar plate drug resistance assays. None of the strains showed significant resistance to micafungin or caspofungin in liquid microdilution susceptibility assays. The antifungal activities of micafungin and caspofungin were similar in agarose diffusion assays, although the shape and size of the caspofungin inhibitory zones were affected by medium composition. The assessment of micafungin and caspofungin potency is therefore assay dependent; the differences seen with agar plate drug resistance assays occur over narrow ranges of echinocandin concentrations and are not of clinical significance.
Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.
The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.
The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.
Despite considerable evidence of azole resistance in oral candidiasis due to Candida species, little is known about the azole susceptibilities of the genital tract isolates responsible for vaginitis. The fluconazole susceptibilities of vaginal isolates obtained during a multicenter study of 556 women with complicated Candida vaginitis were determined by evaluating two fluconazole treatment regimens. Of 393 baseline isolates of Candida albicans, 377 (96%) were highly susceptible to fluconazole (MICs, <8 μg/ml) and 14 (3.6%) were resistant (MICs, ≥64 μg/ml). Following fluconazole therapy, one case of in vitro resistance developed during 6 weeks of monitoring. In accordance with the NCCLS definition, in vitro fluconazole resistance correlated poorly with the clinical response, although a trend of a higher mycological failure rate was found (41 versus 19.6% on day 14). By using an alternative breakpoint of 1 μg/ml, based upon the concentrations of fluconazole achievable in vaginal tissue, no significant differences in the clinical and mycological responses were observed when isolates (n = 250) for which MICs were ≤1 μg/ml were compared with isolates (n = 30) for which MICs were >1 μg/ml, although a trend toward an improved clinical outcome was noted on day 14 (odds ratio, >2.7; 95% confidence interval, 0.91, 8.30). Although clinical failure was uncommon, symptomatic recurrence or mycological relapse almost invariably occurred with highly sensitive strains (MICs, <1.0 μg/ml). In vitro fluconazole resistance developed in 2 of 18 initially susceptible C. glabrata isolates following fluconazole exposure. Susceptibility testing for women with complicated Candida vaginitis appears to be unjustified.
The cytochrome P-450 lanosterol 14α-demethylase (CYP51A1) of yeasts is involved in an important step in the biosynthesis of ergosterol. Since CYP51A1 is the target of azole antifungal agents, this enzyme is potentially prone to alterations leading to resistance to these agents. Among them, a decrease in the affinity of CYP51A1 for these agents is possible. We showed in a group of Candida albicans isolates from AIDS patients that multidrug efflux transporters were playing an important role in the resistance of C. albicans to azole antifungal agents, but without excluding the involvement of other factors (D. Sanglard, K. Kuchler, F. Ischer, J.-L. Pagani, M. Monod, and J. Bille, Antimicrob. Agents Chemother. 39:2378–2386, 1995). We therefore analyzed in closer detail changes in the affinity of CYP51A1 for azole antifungal agents. A strategy consisting of functional expression in Saccharomyces cerevisiae of the C. albicans CYP51A1 genes of sequential clinical isolates from patients was designed. This selection, which was coupled with a test of susceptibility to the azole derivatives fluconazole, ketoconazole, and itraconazole, enabled the detection of mutations in different cloned CYP51A1 genes, whose products are potentially affected in their affinity for azole derivatives. This selection enabled the detection of five different mutations in the cloned CYP51A1 genes which correlated with the occurrence of azole resistance in clinical C. albicans isolates. These mutations were as follows: replacement of the glycine at position 129 with alanine (G129A), Y132H, S405F, G464S, and R467K. While the S405F mutation was found as a single amino acid substitution in a CYP51A1 gene from an azole-resistant yeast, other mutations were found simultaneously in individual CYP51A1 genes, i.e., R467K with G464S, S405F with Y132H, G129A with G464S, and R467K with G464S and Y132H. Site-directed mutagenesis of a wild-type CYP51A1 gene was performed to estimate the effect of each of these mutations on resistance to azole derivatives. Each single mutation, with the exception of G129A, had a measurable effect on the affinity of the target enzyme for specific azole derivatives. We speculate that these specific mutations could combine with the effect of multidrug efflux transporters in the clinical isolates and contribute to different patterns and stepwise increases in resistance to azole derivatives.
In Candida albicans, caspofungin (CSP) susceptibilities, determined by broth microdilution methods, have not been found to be related to azole susceptibilities or resistance. In contrast, it has been observed that azole-resistant clinical isolates that overexpress the efflux pump gene CDR2 are less susceptible to CSP when tested using an agar dilution method commonly used with Saccharomyces cerevisiae. The goal of this study is to further understand the effects of azole resistance mechanisms on CSP susceptibility testing. A collection of 69 isolates exhibiting known mechanisms of azole resistance and resistance-associated phenotypes were analyzed by broth microdilution methods to determine standard minimum inhibitory concentrations (MIC) for CSP. The same isolates were then analyzed for MIC by Etest strips, an agar based method that has been shown generally to be comparable to broth methods. The MIC as analyzed by both methods were not significantly different. However, a collection of strains overexpressing the efflux pump CDR2 does exhibit a spectrum of CSP susceptibilities when examined by agar dilution susceptibility tests, ranging from standard to reduced susceptibilities. This work demonstrates that a change in CSP susceptibility with CDR2 overexpressing cells in agar dilution studies is a variable phenotype and it is not the result of growth conditions (broth versus agar).
Candida albicans; Caspofungin; drug resistance; Etest
Study of in vivo antifungal activity of the hydroalcoholic extract (HE) and n-BuOH extract (BUTE) of Sapindus saponaria against azole-susceptible and -resistant human vaginal Candida spp.
The in vitro antifungal activity of HE, BUTE, fluconazole (FLU), and itraconazole (ITRA) was determined by the broth microdilution method. We obtained values of minimal inhibitory concentration (MIC) and minimum fungicide concentration (MFC) for 46 strains of C. albicans and 10 of C. glabrata isolated from patients with vulvovaginal candidiasis (VVC). VVC was induced in hyperestrogenic Wistar rats with azole-susceptible C. albicans (SCA), azole-resistant C. albicans (RCA), and azole-resistant C. glabrata (RCG). The rats were treated intravaginally with 0.1 mL of HE or BUTE at concentrations of 1%, 2.5% and 5%; 100 μg/mL of FLU (treatment positive control); or distilled water (negative control) at 1, 24, and 48 h after induction of the infection, and the progress of VVC was monitored by culturing and scanning electron microscopy (SEM). The toxicity was evaluated in cervical cells of the HeLa cell line.
The extracts showed in vitro inhibitory and fungicidal activity against all the isolates, and the MIC and MFC values for the C. glabrata isolates were slightly higher. In vivo, the SCA, RCA, and RCG infections were eliminated by 21 days post-infection, with up to 5% HE and BUTE, comparable to the activity of FLU. No cytotoxic action was observed for either extract.
Our results demonstrated that HE and BUTE from S. saponaria show inhibitory and fungicidal activity in vitro, in addition to in vivo activity against azole-resistant vaginal isolates of C. glabrata and azole-susceptible and resistant isolates of C. albicans. Also considering the lack of cytotoxicity and the low concentrations of the extracts necessary to eliminate the infection in vivo, HE and BUTE show promise for continued studies with purified antifungal substances in VVC yeast isolates.
Sapindus saponaria; vaginal yeasts; antifungal activity; in vivo
Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains.
A major mechanism of azole resistance in Candida albicans is overexpression of the genes encoding the ATP binding cassette transporters Cdr1p and Cdr2p due to gain-of-function mutations in Tac1p, a transcription factor of the zinc cluster family. To identify the Tac1p regulon, we analyzed four matched sets of clinical isolates representing the development of CDR1- and CDR2-mediated azole resistance by using gene expression profiling. We identified 31 genes that were consistently up-regulated with CDR1 and CDR2, including TAC1 itself, and 12 consistently down-regulated genes. When a resistant strain deleted for TAC1 was examined similarly, expression of almost all of these genes returned to levels similar to those in the matched azole-susceptible isolate. Using genome-wide location (ChIP-chip) analysis (a procedure combining chromatin immunoprecipitation with hybridization to DNA intergenic microarrays), we found 37 genes whose promoters were bound by Tac1p in vivo, including CDR1 and CDR2. Sequence analysis identified nine new genes whose promoters contain the previously reported Tac1p drug-responsive element (CGGN4CGG), including TAC1. In total, there were eight genes whose expression was modulated in the four azole-resistant clinical isolates in a TAC1-dependent manner and whose promoters were bound by Tac1p, qualifying them as direct Tac1p targets: CDR1, CDR2, GPX1 (putative glutathione peroxidase), LCB4 (putative sphingosine kinase), RTA3 (putative phospholipid flippase), and orf19.1887 (putative lipase), as well as IFU5 and orf19.4898 of unknown function. Our results show that Tac1p binds under nonactivating conditions to the promoters of its targets, including to its own promoter. They also suggest roles for Tac1p in regulating lipid metabolism (mobilization and trafficking) and oxidative stress response in C. albicans.