CDR1 Expression Research Articles

MDR1 overexpression combined with ERG11 mutations induce high-level fluconazole resistance in Candida tropicalis clinical isolates

BackgroundMarked increases in fluconazole resistance in Candida tropicalis have been recently reported. In this study, the molecular mechanisms behind fluconazole resistance were investigated.MethodsTwenty-two C. tropicalis clinical isolates, including 12 fluconazole-resistant isolates and 10 fluconazole-susceptible isolates, were collected from a tertiary care teaching hospital in Beijing between 2013 and 2017. Antifungal susceptibility testing, multilocus sequence typing, ERG11 amplification and sequencing, quantitative real-time reverse transcription-polymerase chain reaction (ERG11, UPC2, MDR1, and CDR1), and clinical data collection were performed for all C. tropicalis isolates.ResultsMultilocus sequence typing revealed that the 10 fluconazole-susceptible isolates and 12 fluconazole-resistant isolates were divided into nine and seven diploid sequence types, respectively. Of the 12 patients with fluconazole-resistant isolates, six had been previously exposed to azole and four had a fatal outcome. Y132F and S154F amino acid substitutions in Erg11p were found in all fluconazole-resistant isolates except one. MDR1 gene overexpression was identified in fluconazole-resistant isolates. In particular, seven high-level fluconazole resistant isolates (minimum inhibitory concentration ≥ 128 mg/L) and three pan-azole resistant isolates were identified. CDR1, ERG11, and UPC2 gene expression levels in fluconazole-resistant isolates were not significantly different from the control isolates (P = 0.262, P = 0.598, P = 0.114, respectively).ConclusionsThis study provides evidence that the combination of MDR1 gene overexpression and ERG11 missense mutations is responsible for high-level fluconazole resistance and pan-azole resistance in C. tropicalis clinical isolates. To the best of our knowledge, this is the first study investigating the relationship between MDR1 gene overexpression and increased fluconazole resistance.

Jjj1 Is a Negative Regulator of Pdr1-Mediated Fluconazole Resistance in Candida glabrata.

The high prevalence of fluconazole resistance among clinical isolates of Candida glabrata has greatly hampered the utility of fluconazole for the treatment of invasive candidiasis. Fluconazole resistance in this yeast is almost exclusively due to activating mutations in the transcription factor Pdr1, which result in upregulation of the ABC transporter genes CDR1, PDH1, and SNQ2 and therefore increased fluconazole efflux. However, the regulation of Pdr1 is poorly understood. In order to identify genes that interact with the Pdr1 transcriptional pathway and influence the susceptibility of C.glabrata to fluconazole, we screened a collection of deletion mutants for those exhibiting increased resistance to fluconazole. Deletion of the gene coding for a protein homologous to the Saccharomyces cerevisiae J protein Jjj1 resulted in decreased fluconazole susceptibility. We used the SAT1 flipper method to generate independent deletion mutants for JJJ1 in an SDD clinical isolate. Expression of both CDR1 and PDR1 was increased in the absence of JJJ1. In the absence of CDR1 or PDR1, deletion of JJJ1 has only a modest effect on fluconazole susceptibility. Transcriptional profiling using transcriptome sequencing (RNA-seq) revealed upregulation of genes of the Pdr1 regulon in the absence of JJJ1. Jjj1 appears to be a negative regulator of fluconazole resistance in C.glabrata and acts primarily through upregulation of the ABC transporter gene CDR1 via activation of the Pdr1 transcriptional pathway. IMPORTANCECandida glabrata is the second most common species of Candida recovered from patients with invasive candidiasis. The increasing number of infections due to C.glabrata, combined with its high rates of resistance to the commonly used, well-tolerated azole class of antifungal agents, has limited the use of this antifungal class. This has led to the preferential use of echinocandins as empirical treatment for serious Candida infections. The primary mechanism of resistance found in clinical isolates is the presence of an activating mutation in the gene encoding the transcription factor Pdr1 that results in upregulation of one or more of the efflux pumps Cdr1, Pdh1, and Snq2. By developing a better understanding of this mechanism of resistance to the azoles, it will be possible to develop strategies for reclaiming the utility of the azole antifungals against this important fungal pathogen.

Antimicrobial Activity of Green Silver Nanoparticles against Fluconazole-resistant Candida albicans in Animal Model

FUNGAL infections due to fluconazole-resistant Candida albicans are a serious clinical problem, requiring new efficient antifungal treatment. The present study evaluated the susceptibility of fluconazole-resistant C. albicans to green silver nanoparticles. A total of 40 isolates were examined for the expressions of CDR1, CDR2, and MDR1 gene by quantitative reverse transcription polymerase chain reaction. The antifungal activities of green silver nanoparticles alone and/or in combination with fluconazole (to improve antifungal activity) were assessed by broth microdilution assay and transmission electron microscopy. The effect of fluconazole and/or green silver nanoparticles on the production of C. albicans to protease and phospholipase was also evaluated. And finally, animal model was used to prove the safe and effective use of green silver nanoparticles in the treatment of fluconazole-resistant C. albicans. For all the tested C. albicans strains, the minimum inhibitory concentrations (MICs) of fluconazole and green silver nanoparticles were 64 and 4.84 μg/ml, respectively. Green silver nanoparticles decrease the production of protease and phospholipase enzymes. The expression of both CDR1 and CDR2 were decreased after exposure to green silver nanoparticles, while the expression of CDR 1, CDR 2 and MDR 1 were all decreased when fluconazole and green silver nanoparticles were used. Green silver nanoparticles may be causing suppression of the CDR1, CDR2 and MDR1 expression in fluconazole-resistant C. albicans. The results suggest the use of green silver nanoparticles as a safe and effective treatment against fluconazole-resistant C. albicans.

T cells presenting viral antigens or autoantigens induce cytotoxic T cell anergy.

In the course of modeling the naturally occurring tumor immunity seen in patients with paraneoplastic cerebellar degeneration (PCD), we discovered an unexpectedly high threshold for breaking CD8+ cytotoxic T cell (CTL) tolerance to the PCD autoantigen, CDR2. While CDR2 expression was previously found to be strictly restricted to immune-privileged cells (cerebellum, testes, and tumors), unexpectedly we have found that T cells also express CDR2. This expression underlies inhibition of CTL activation; CTLs that respond to epithelial cells expressing CDR2 fail to respond to T cells expressing CDR2. This was a general phenomenon, as T cells presenting influenza (flu) antigen also fail to activate otherwise potent flu-specific CTLs either in vitro or in vivo. Moreover, transfer of flu peptide-pulsed T cells into flu-infected mice inhibits endogenous flu-specific CTLs. Our finding that T cells serve as a site of immune privilege, inhibiting effector CTL function, uncovers an autorepressive loop with general biologic and clinical relevance.

Monitoring Antifungal Resistance in a Global Collection of Invasive Yeasts and Molds: Application of CLSI Epidemiological Cutoff Values and Whole-Genome Sequencing Analysis for Detection of Azole Resistance in Candida albicans.

The activity of 7 antifungal agents against 3,557 invasive yeasts and molds collected in 29 countries worldwide in 2014 and 2015 was evaluated. Epidemiological cutoff values (ECVs) published in the Clinical and Laboratory Standards Institute (CLSI) M59 document were applied for species with no clinical breakpoints. Echinocandin susceptibility rates were 95.9% to 100.0% for the 5 most common Candida species, except for the rates for Candida parapsilosis to anidulafungin (88.7% susceptible, 100.0% wild type). Rates of fluconazole resistance ranged from 8.0% for Candida glabrata to 0.4% for Candida albicans Seven Candida species displayed 100.0% wild-type amphotericin B MIC results, and Candida dubliniensis and Candida lusitaniae exhibited wild-type echinocandin MIC results. The highest fluconazole, voriconazole, and posaconazole MIC values for Cryptococcus neoformans var. grubii were 8 μg/ml, 0.12 μg/ml, and 0.25 μg/ml, respectively. Aspergillus fumigatus isolates were 100.0% wild type for caspofungin and amphotericin B, but 3 (0.8%) of these isolates were non-wild type to itraconazole (2 isolates) or voriconazole (1 isolate). Mutations in FKS hot spot (HS) regions were detected among 13/20 Candida isolates displaying echinocandin MICs greater than the ECV (16 of these 20 isolates were C. glabrata). Most isolates carrying mutations in FKS HS regions were resistant to 2 or more echinocandins. Five fluconazole-nonsusceptible C. albicans isolates were submitted to whole-genome sequencing analysis. Gain-of-function, Erg11 heterozygous, and Erg3 homozygous mutations were observed in 1 isolate each. One isolate displayed MDR1 promoter allele alterations associated with azole resistance. Elevated levels of expression of MDR1 or CDR2 were observed in 3 isolates and 1 isolate, respectively. Echinocandin and azole resistance is still uncommon among contemporary fungal isolates; however, mechanisms of resistance to antifungals were observed among Candida spp., showing that resistance can emerge and monitoring is warranted.

Expression of Major Efflux Pumps in Fluconazole-Resistant Candida albicans.

Resistance to azole antifungals is considered as a significant problem in Candida albicans infections. Several molecular mechanisms of fluconazole resistance including alterations in the gene encoding the target enzyme ERG11 or overexpression of efflux pump genes including CDR1, CDR2, MDR1, MDR2 and FLU1 have been reported. The aim of this study was to investigate overexpression of efflux pump genes including CDR1, CDR2, MDR1, MDR2 and FLU1 in fluconazole- resistant C. albicans. In this study, a total of 97 clinical isolates of C. albicans were isolated from hospitalized children in Children medical center, an Iranian referral hospital. Fluconazole susceptibility testing of C. albicans was performed using the broth microdilution method according to the CLSI guideline. Expression of CDR1, CDR2, MDR1, MDR2 and FLU1 genes was measured using quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and 18SrRNA gene was used as a housekeeping gene. Among 97 C. albicans isolates, 5 strains were categorized as fluconazoleresistant. Overexpression of CDR1, CDR2 and MDR2 genes was found in all isolates. MDR1 overexpression was observed in four resistant isolates. None of the resistant strains displayed increases in FLU1 transcript levels. Overexpression of the CDR1, CDR2, MDR1 and MDR2 genes might play an important role in fluconazole-resistant C. albicans. No link between expression of FLU1 and fluconazole resistance was found.

Effect of biogenic selenium nanoparticles on ERG11 and CDR1 gene expression in both fluconazole-resistant and -susceptible Candida albicans isolates.

Background and Purpose: Candida albicans is the most common Candida species (sp.) isolated from fungal infections. Azole resistance in Candida species has been considerably increased in the last decades. Given the toxicity of the antimicrobial drugs, resistance to antifungal agents, and drug interactions, the identification of new antifungal agents seems essential. In this study, we assessed the antifungal effects of biogenic selenium nanoparticles on C. albicans and determined the expression of ERG11 and CDR1 genes.Materials and Methods:Selenium nanoparticles were synthesized with Bacillus sp. MSH-1. The ultrastructure of selenium nanoparticles was evaluated with a transmission electron microscope. The antifungal susceptibility test was performed according to the modified Clinical and Laboratory Standards Institute M27-A3 standard protocol. The expression levels of the CDR1 and ERG11 genes were analyzed using the quantitative real-time polymerase chain reaction (PCR) assay.Results:The azole-resistant C. albicans and wild type C. albicans strains were inhibited by 100 and 70 µg/mL of selenium nanoparticle concentrations, respectively. The expression of CDR1 and ERG11 genes was significantly down-regulated in these selenium nanoparticle concentrations.Conclusion:As the findings indicated, selenium nanoparticles had an appropriate antifungal activity against fluconazole-resistant and -susceptible C. albicans strains. Accordingly, these nanoparticles reduced the expression of CDR1 and ERG11 genes associated with azole resistance. Further studies are needed to investigate the synergistic effects of selenium nanoparticles using other antifungal drugs.

Strong synergism of dexamethasone in combination with fluconazole against resistant Candida albicans mediated by inhibiting drug efflux and reducing virulence

Candida albicans is the most commonly isolated Candida spp. in the clinic and its resistance to fluconazole (FLC) has been emerging rapidly. Combination therapy may be a potentially effective approach to combat drug resistance. In this study, the combination antifungal effects of dexamethasone (DXM) and FLC against resistant C. albicans in vitro were assayed using minimum inhibitory concentrations (MICs), sessile MICs and time–kill curves. The in vivo efficacy of this drug combination was evaluated using a Galleria mellonella model by determining survival rate, fungal burden and histological damage. In addition, the impact of DXM on efflux pump activity was investigated using a rhodamine 6G assay. Expression of CDR1, CDR2 and MDR1 was determined by real-time quantitative PCR, and extracellular phospholipase activity was detected by the egg yolk agar method to reveal the potential synergistic mechanism. The results showed that DXM potentiates the antifungal effect of FLC against resistant C. albicans strains both in vitro and in vivo, and the synergistic mechanism is related to inhibiting the efflux of drugs and reducing the virulence of C. albicans.

Azole resistance in Candida albicans from animals: Highlights on efflux pump activity and gene overexpression.

This study investigated potential mechanisms of azole resistance among Candida albicans from animals, including efflux pump activity, ergosterol content and gene expression. For this purpose, 30 azole-resistant C.albicans strains from animals were tested for their antifungal susceptibility, according to document M27-A3, efflux pump activity by rhodamine 6G test, ergosterol content and expression of the genes CDR1, CDR2, MDR1, ERG11 by RT-qPCR. These strains were resistant to at least one azole derivative. Resistance to fluconazole and itraconazole was detected in 23 and 26 strains respectively. Rhodamine 6G tests showed increased activity of efflux pumps in the resistant strains, showing a possible resistance mechanism. There was no difference in ergosterol content between resistant and susceptible strains, even after fluconazole exposure. From 30 strains, 22 (73.3%) resistant animal strains overexpressed one or more genes. From this group, 40.9% (9/22) overexpressed CDR1, 18.2% (4/22) overexpressed CDR2, 59.1% (13/22) overexpressed MDR1 and 54.5% (12/22) overexpressed ERG11. Concerning gene expression, a positive correlation was observed only between CDR1 and CDR2. Thus, azole resistance in C. albicans strains from animals is a multifactorial process that involves increased efflux pump activity and the overexpression of different genes.

Overexpression of MDR-1 and CDR-2 genes in fluconazole resistance of Candida albicans isolated from patients with vulvovaginal candidiasis

Background and Purpose: Candida albicans (C. albicans) is an opportunistic fungus that can colonize women’s mucosal epithelial cell surfaces, causing vulvovaginitis in specific circumstances. The major genes contributing to drug resistance in C. albicans are the candida drug resistance (CDR) and multi drug resistance (MDR) genes. The purpose of this study was to evaluate the CDR-2 and MDR-1 gene expression patterns in C. albicans strains isolated from patients with recurrent vulvovaginal candidiasis. Materials and Methods:In this study, 40 isolates of fluconazole-resistant C. albicans were cultured on Sabouraud dextrose agar. These isolates were collected from women with vulvovaginitis who were referred to a clinic in Tehran, Iran, and transferred to a mycology laboratory. Then, RNA was extracted from the isolates using phenol-chloroform and glass beads, and the complementary DNA (cDNA) was synthetized. To detect the semi-quantitative expression of CDR-2 and MDR-1 genes, the reverse transcriptase-PCR (RT-PCR) technique was performed using specific primers. Results:Our findings indicated that of the 40 C. albicans isolates, 35 (87.5%) strains were positive for mRNA of the CDR-2 gene, 32 (80%) strains expressed mRNA of the MDR-1 gene, and 30 (75%) strains were confirmed to express mRNA of both the CDR-2 and MDR-1 genes simultaneously using the RT-PCR assay. Conclusion:According to the obtained results, the expression rates of CDR-2 and MDR-1 genes were high in fluconazole-resistant C. albicans isolates, which can cause treatments to fail and result in chronic infections. Inhibiting these important genes using novel or natural agents can help with the treatment of chronic and recurrent vaginitis.

Resistance Mechanisms and Clinical Features of Fluconazole-Nonsusceptible Candida tropicalis Isolates Compared with Fluconazole-Less-Susceptible Isolates.

We investigated the azole resistance mechanisms and clinical features of fluconazole-nonsusceptible (FNS) isolates of Candida tropicalis recovered from Korean surveillance cultures in comparison with fluconazole-less-susceptible (FLS) isolates. Thirty-five clinical isolates of C. tropicalis, comprising 9 FNS (fluconazole MIC, 4 to 64 μg/ml), 12 FLS (MIC, 1 to 2 μg/ml), and 14 control (MIC, 0.125 to 0.5 μg/ml) isolates, were assessed. CDR1, MDR1, and ERG11 expression was quantified, and the ERG11 and UPC2 genes were sequenced. Clinical features of 16 patients with FNS or FLS bloodstream isolates were analyzed. Both FNS and FLS isolates had >10-fold higher mean expression levels of CDR1, MDR1, and ERG11 genes than control isolates (P values of <0.02 for all). When FNS and FLS isolates were compared, FNS isolates had 3.4-fold higher mean ERG11 expression levels than FLS isolates (P = 0.004), but there were no differences in those of CDR1 or MDR1 Of all 35 isolates, 4 (2 FNS and 2 FLS) and 28 (8 FNS, 11 FLS, and 9 control) isolates exhibited amino acid substitutions in Erg11p and Upc2p, respectively. Both FNS and FLS bloodstream isolates were associated with azole therapeutic failure (3/4 versus 4/7) or uncleared fungemia (4/6 versus 4/10), but FNS isolates were identified more frequently from patients with previous azole exposure (6/6 versus 3/10; P = 0.011) and immunosuppression (6/6 versus 3/10; P = 0.011). These results reveal that the majority of FNS C. tropicalis isolates show overexpression of CDR1, MDR1, and ERG11 genes, and fungemia develops after azole exposure in patients with immunosuppression.

A New Endogenous Overexpression System of Multidrug Transporters of Candida albicans Suitable for Structural and Functional Studies.

Fungal pathogens have a robust array of multidrug transporters which aid in active expulsion of drugs and xenobiotics to help them evade toxic effects of drugs. Thus, these transporters impose a major impediment to effective chemotherapy. Although the Saccharomyces cerevisiae strain AD1-8u− has catered well to the need of an overexpression system to study drug transport by multidrug transporters of Candida albicans, artifacts associated with a heterologous system could not be excluded. To avoid the issue, we exploited a azole-resistant clinical isolate of C. albicans to develop a new system devoid of three major multidrug transporters (Cdr1p, Cdr2p, and Mdr1p) for the overexpression of multidrug transporters under native hyperactive CDR1 promoter due to gain of function (GOF) mutation in TAC1. The study deals with overexpression and functional characterization of representatives of two major classes of multidrug transporters, Cdr1p and Mdr1p, to prove the functionality of this newly developed endogenous expression system. Expression of native Cdr1 and Mdr1 protein in C. albicans cells was confirmed by confocal microscopy and immunodetection and resulted in increased resistance to the putative substrates as compared to control. The system was further validated by overexpressing a few key mutant variants of Cdr1p and Mdr1p. Together, our data confirms the utility of new endogenous overexpression system which is devoid of artifactual factors as most suited for functional characterization of multidrug transporter proteins of C. albicans.

Antifungal effect of Echinophora platyloba on expression of CDR1 and CDR2 genes in fluconazole-resistant Candida albicans

Background: Several studies examined the effect of the Echinophora platyloba extract in treatment of azole-resistant Candida albicans clinical isolates.Objective: We investigated the effect of E. platyloba extract on expression of CDR1 and CDR2 genes in fluconazole-resistant clinical isolates of C. albicans using real-time PCR.Materials and Methods: The crude extract of E. platyloba was obtained using percolation method. Using serial dilution method, different concentrations of extract were achieved. Two hundred microlitres of fungal suspension (106 CFU/ml) was added to the media and cultured with different concentrations and then incubated at 37 °C for 48 h. The concentration of extract in the first tube, which inhibited the growth of C. albicans, was recorded as the Minimal Inhibitory Concentration (MIC). In order to analyse the expression of CDR1 and CDR2 genes, RNA was extracted from C. albicans isolates before and after treatment with MIC of E. platyloba using glass beads and the denaturing buffer agents in an RNase-free environment and then the cDNA was synthesised and used for real-time PCR assay.Results: Twenty of total of 148 isolates were resistant to fluconazole. The MIC and MFC for the alcoholic extract of E. Platyloba were 64 mg/ml and 128 mg/ml, respectively. Real-time PCR results revealed that the mRNA levels of CDR1 and CDR2 genes significantly declined after incubation with E. Platyloba (both p values < 0.001).Conclusion: E. Platyloba is effective in reducing CDR1 and CDR2 expression which in turn plays an important role in fluconazole resistance in Candida species.

Mechanisms of resistance to fluconazole in Candida albicans clinical isolates from Iranian HIV-infected patients with oropharyngeal candidiasis

The opportunistic pathogen Candida albicans is the major agent of oropharyngeal candidiasis (OPC) in HIV/AIDS patients. The increased use of fluconazole can lead to the emergence of azole-resistant strains and treatment failures in PLWH (people living with HIV) receiving long-term therapy for OPC. The purpose of this study was to evaluate CDR1, CDR2, MDR1, and ERG11 gene expression in C. albicans clinically isolated from HIV-infected patients in Iran. In this study, we evaluated the molecular mechanisms of azole resistance in 20 fluconazole-resistant C. albicans isolates obtained from Iranian HIV-infected patients with oropharyngeal candidiasis by Real-Time polymerase chain reaction. The overexpression of drug efflux pump CDR1 gene was found to be the major resistance mechanism observed in these isolates. The overexpression of the CDR1 gene correlated strongly with increasing resistance to fluconazole (P<0.05). Additionally, an increased level of mRNA in ERG11 was not observed in any of the tested isolates. Our findings suggested that the CDR1 gene expression to fluconazole resistance in C. albicans is greater than other known genes.

The roles of CDR1, CDR2, and MDR1 in kaempferol-induced suppression with fluconazole-resistant Candida albicans

Context: Fungal infections caused by fluconazole-resistant Candida albicans are an intractable clinical problem, calling for new efficient antifungal drugs. Kaempferol, an active flavonoid, has been considered a potential candidate against Candida species. Objective: This work investigates the resistance reversion of kaempferol in fluconazole-resistant C. albicans and the underlying mechanism. Materials and methods: The antifungal activities of fluconazole and/or kaempferol were assessed by a series of standard procedures including broth microdilution method, checkerboard assay and time-kill (T-K) test in nine clinical strains as well as a standard reference isolate of C. albicans. Subsequently, the morphological changes, the efflux of rhodamine 6G, and the expressions of CDR 1, CDR 2, and MDR 1 were analysed by scanning electron microscope (SEM), inverted fluorescence microscope and quantitative reverse transcription polymerase chain reaction (qRT-PCR) in C. albicans z2003. Results: For all the tested C. albicans strains, the minimum inhibitory concentrations (MICs) of fluconazole and kaempferol ranged 0.25–32 and 128–256 μg/mL with a range of fractional inhibitory concentration index of 0.257–0.531. In C. albicans z2003, the expression of both CDR 1 and CDR 2 were decreased after exposure to kaempferol alone with negligible rhodamine 6G accumulation, while the expression of CDR 1, CDR 2 and MDR 1 were all decreased when fluconazole and kaempferol were used concomitantly with notable fluorescence of rhodamine 6G observed. Discussion and conclusion: Kaempferol-induced reversion in fluconazole-resistant C. albicans might be likely due to the suppression of the expression of CDR1, CDR2 and MDR1.

Using PCR to Compare the Expression of CDR1, CDR2, and MDR1 in Candida Albicans Isolates Resistant and Susceptible to Fluconazole

Using PCR to Compare the Expression of CDR1, CDR2, and MDR1 in Candida Albicans Isolates Resistant and Susceptible to Fluconazole

Mutations in transcription factor Mrr2p contribute to fluconazole resistance in clinical isolates of Candida albicans

The Candida albicans zinc cluster proteins are a family of transcription factors (TFs) that play essential roles in the development of antifungal drug resistance. Gain-of-function mutations in several TFs, such as Tac1p, Mrr1p and Upc2p, have been previously well documented in azole-resistant clinical C. albicans isolates. Mrr2p (multidrug resistance regulator 2) is a novel TF controlling expression of the ABC transporter gene CDR1 and mediating fluconazole resistance. In this study, the relationship between naturally occurring mutations in MRR2 and fluconazole resistance in clinical C. albicans isolates was investigated. Among a group of 20 fluconazole-resistant clinical C. albicans and 10 fluconazole-susceptible C. albicans, 12 fluconazole-resistant isolates overexpressed CDR1 by at least two-fold compared with the fluconazole-susceptible isolates. Of these 12 resistant isolates, three (C7, C9, C15) contained 11 identical missense mutations, 6 of which occurred only in the azole-resistant isolates. The contribution of these mutations to CDR1 overexpression and therefore to fluconazole resistance was further verified by generating recombinant strains containing the mutated MRR2 gene. The mutated MRR2 alleles from isolate C9 contributed to an almost six-fold increase in CDR1 expression and an eight-fold increase in fluconazole resistance; the missense mutations S466L and T470N resulted in an increase in CDR1 expression of more than two-fold and a four-fold increase in fluconazole resistance. In contrast, the other four missense mutations conferred only two- to four-fold increases in fluconazole resistance, with no significant increase in CDR1 expression. These findings provide some insight into the mechanism by which MRR2 regulates C. albicans multidrug resistance.

Selected mechanisms of molecular resistance of Candida albicans to azole drugs.

A phenomenon of increasing resistance of Candida spp. to azoles has been observed for several years now. One of the mechanisms of lack of sensitivity to azoles is associated with CDR1, CDR2, MRD1 genes (their products are active transport pumps conditioning drug efflux from pathogen's cell), and ERG11 gene (encoding lanosterol 14α-demethylase). Test material was 120 strains of Candida albicans (60 resistant and 60 susceptible to azole drugs) obtained from clinical samples. The first stage of experiment assessed the expression of CDR1, CDR2, MDR1 and ERG11 genes by Q-PCR. The impact of ERG11 gene's mutations on the expression of this gene was analysed. The final stage of the experiment assessed the level of genome methylation of Candida albicans strains. An increase in the expression of CDR2, MDR1 and ERG11 was observed in azole-resistant strains of Candida albicans in comparison to strains sensitive to this class of drugs. Furthermore, 19 changes in the sequence of ERG11 were detected in tested strains. Four of the discovered mutations: T495A, A530C, G622A and A945C led to the following amino acid substitutions: D116E, K128T, V159I and E266D, respectively. It has also been found that statistically five mutations: T462C, G1309A, C216T, C1257T and A945C affected the expression of ERG11. The applied method of assessing the level of methylation of Candida albicans genome did not confirm its role in the development of resistance to azoles. The results indicate however, that resistance of Candida albicans strains to azole drugs is multifactorial.

Characterization of azole resistance mechanisms in Chilean clinical isolates of Candida albicans

The commensal yeast Candida albicans, can cause superficial or systemic candidiasis in susceptible hosts. In Chile, azole antifungals are the most widely used drugs in the treatment of candidiasis. In a previous study performed at our center, 2.1 and 1.6% of clinical isolates of C. albicans were found to be resistant to fluconazole and voriconazole, respectively. To characterize the resistance mechanisms involved in azoles resistance in Chilean clinical isolates. Eight resistant, nine susceptible-dose dependent (SDD) and 10 susceptible strains (n: 27) were selected according to the Clinical Laboratory Standards Institute (CLSI) M27-S3 criteria, from vaginal and urine samples. Mutations in the 408-488 region of the ERG11 gene were studied by sequencing, and the relative expression of ERG11 gene and efflux pump genes CDR1, CDR2 and MDR1, was evaluated by quantitative real-time PCR (q-PCR). No mutations were detected in the ERG11 gene and its overexpression was found only in 12.5% of the resistant strains (1/8). The most prevalent mechanism of resistance was the over-expression of efflux pumps (62.5%; 5/8). The study of the expression of efflux pumps by q-PCR could be a useful diagnostic tool for early detection of azole resistance in C. albicans.

Vulvovaginal candidiasis: species distribution, fluconazole resistance and drug efflux pump gene overexpression.

The increasing incidence of vulvovaginal candidiasis (VVC) and the emergence of fluconazole resistance are an indisputable fact. However, little information is available regarding the correlation between fluconazole resistance in vaginal Candida albicans and the expression of drug efflux pump genes. In this study, we investigated the species distribution, fluconazole susceptibility profiles and the mechanisms of fluconazole resistance in Candida strains. In total, 785 clinical Candida isolates were collected from patients with VVC. C. albicans was the most frequently isolated species(n = 529) followed by C. glabrata (n = 164) and C. krusei (n = 57). Of all Candida isolates, 4.7% were resistant to fluconazole. We randomly selected 18 fluconazole resistant isolates of C. albicans to evaluate the expression of CDR1, CDR2, MDR1 and FLU1 genes. Compared with fluconazole-susceptible C. albicans isolates, CDR1 gene expression displayed 3.16-fold relative increase, which was statistically significant. CDR2, MDR1 and FLU1 overexpression was observed in several fluconazole-resistant C. albicans isolates, but statistical significance was not achieved. These results demonstrate a high frequency of non-albicans species (32.6%); however, C. albicans is the most common Candida species implicated in vaginitis, and this strain displays considerable fluconazole resistance. Meanwhile, our study further indicates that fluconazole resistance in C. albicans may correlate with CDR1 gene overexpression.