Mechanism Of Fluconazole Resistance Research Articles

Analysis of clinical Candida parapsilosis isolates reveals copy number variation in key fluconazole resistance genes.

We used whole-genome sequencing to analyze a collection of 35 fluconazole-resistant and 7 susceptible Candida parapsilosis isolates together with coverage analysis and GWAS techniques to identify new mechanisms of fluconazole resistance. Phylogenetic analysis shows that although the collection is diverse, two persistent clinical lineages were identified. We identified copy number variation (CNV) of two genes, ERG11 and CDR1B, in resistant isolates. Two strains have a CNV at the ERG11 locus; the entire ORF is amplified in one, and only the promoter region is amplified in the other. We show that the annotated telomeric gene CDR1B is actually an artifactual in silico fusion of two highly similar neighboring CDR genes due to an assembly error in the C. parapsilosis CDC317 reference genome. We report highly variable copy numbers of the CDR1B region across the collection. Several strains have increased the expansion of the two genes into a tandem array of new chimeric genes. Other strains have experienced a deletion between the two genes creating a single gene with a reciprocal chimerism. We find translocations, duplications, and gene conversion across the CDR gene family in the C. parapsilosis species complex, showing that it is a highly dynamic family.

Fluconazole-resistant Candida parapsilosis complex candidemia and analysis of mutations in the ERG11 gene from Pakistan.

Recent reports of the emergence of fluconazole resistance in Candida parapsilosis species complex poses a challenge, more specifically in settings where echinocandin-based treatment regime is not feasible. This study reported emergence of fluconazole resistance in C. parapsilosis species complex strains isolated from blood cultures. This retrospective observational study was conducted from 2018 to 2020 at a tertiary care laboratory from Pakistan. Fluconazole-resistant C. parapsilosis species complex fungemia cases were identified from laboratory database and clinical details were collected. Identification of C. parapsilosis species complex was done using API 20C AUX and Cornmeal Tween80 agar morphology. Minimum inhibitory concentrations (MICs) were determined using Sensititre YeastONE and interpretation was done with CLSI M60 ED1:2017. ERG11 gene region was amplified and sequenced by Sanger sequencing and analysed by MEGA 11 Software. A total of 13 (8.5%) fluconazole-resistant isolates were identified from 152 C. parapsilosis species complex candidemia cases. Fluconazole MICs of resistant isolates ranged between 8 and 256 μg/mL. Analysis of ERG11 gene revealed nonsynonymous mutations at position Y132F in 86% of the fluconazole-resistant isolates. Diabetes and hospitalization were important risk factors for candidemia with fluconazole-resistant C. parapsilosis complex. This is the first report of the emergence and molecular mechanisms of fluconazole resistance in C. parapsilosis species complex from Pakistan. Y132F mutation in the ERG11 gene was the most common mutation in fluconazole-resistant strains. These findings are concerning and necessitate better diagnostics, newer antifungals, ongoing surveillance and further insights on resistance mechanisms in the country.

Azole Resistance in Candida albicans Isolates from Oropharyngeal Candidiasis is Associated with ERG11 Mutation and Efflux Overexpression

Background: Azole resistance rates are rising in Candida species. Fluconazole is one of the most important antifungal drugs used in candidiasis treatment. Objectives: We identified the molecular mechanisms of fluconazole resistance of Candida albicans oropharyngeal candidiasis (OPC) isolates obtained from head and neck cancer patients, a study carried out between 2018 and 2020. Methods: One hundred and twenty-five C. albicans clinical isolates were collected. Antifungal susceptibilities were determined by the CLSI- M27-A3 method. The ERG11 gene was amplified and sequenced to discover SNP mutation. Moreover, real-time PCR was carried out to measure the mRNA levels of ERG11, CDR1, CDR2, and MDR1. Results: Resistance to fluconazole was found in 15 C. albicans isolates. Amino acid substitutions E266D and D116E were observed in resistant, sensitive dose-dependent (SDD), and susceptible C. albicans isolates. K128T, G465S, A114S, Y257H and V488I were in relation to fluconazole resistance. D504A, P375A, W520C, G59S, and V51L were novel substitutions detected in the isolates; except for D504A, other mutations were observed only in resistance isolates. The expression levels of CDR2, CDR1, MDR1, and ERG11 were increased compared to susceptible isolates, respectively. Conclusions: ERG11 mutation was the principal mechanism for fluconazole resistance in C. albicans isolated from oropharyngeal candidiasis patients, and caspofungin can be used as the effective antifungal substance in fluconazole resistance situation for C. albicans infection.

S4.3d Mechanism of fluconazole resistance in Candida vulturna, a member of the Candida haemulonii complex of multidrug - resistant yeasts

Emergent species of the Candida genus, September 22, 2022, 10:30 AM - 12:00 PM Objetives Candida vulturna is an emerging yeast firstly isolated in 2016 from flowers and from a blood sample in Malaysia. It is a member of the Candida haemulonii species complex. This complex has 9 species within which C. haemulonii (including the vulnera variety), C. pseudohemulonii, C. duobushaemulonii, C. auris, and now C. vulturna are considered human pathogens. Like the other members of the haemulonii complex, the identification of C. vulturna is difficult. Due to the scarcity of strains to study, the susceptibility pattern of this species is unknown. The objectives of this work were to study C. vulturna strains isolated from clinical samples from different cities of Colombia, to study their susceptibility patterns, and to establish their mechanisms of azole resistance.MethodsFive C. vulturna strains isolated from urine (n = 1) and blood (n = 4) in three different cities in Colombia were studied. Strains were identified as C. duobushaemulonii (n = 4) and C. haemulonii (n = 1) by means of VITEK2 (software version 9.02) and as C. pseudohaemulonii (n = 5) by using Bruker's MALDI-TOF in Colombia. Upon arrival in Argentina, isolates were molecularly identified by sequencing the ITS regions (rDNA). Antifungal susceptibility testing was performed following the CLSI´s M27 4th ed document. Amphotericin B, anidulafungin, caspofungin, 5-flucytosine, fluconazole, isavuconazole, itraconazole, posaconazole, and voriconazole were tested. In addition, ERG11 genes of the five strains were amplified and sequenced.ResultsThe sequences of the ITS regions were analyzed and the 5 strains were identified as C. vulturna (100% homology). All the strains presented low echinocandins MICs (0.06-0.12 μg/ml), 5-flucytosine (0.06-0.12 μg/ml) and triazoles with the exception of fluconazole (isavuconazole: <0.015 -0.12 μg/ml, itraconazole: 0.06-0.50 μg/ml, posaconazole: <0.015-0.015 μg/ml and voriconazole: 0.03-0.50 μg/ml). In the case of amphotericin B and fluconazole, 4 of the 5 strains presented high MICs for both antifungals (>8 μg/ml and >64 μg/ml), while the remaining one presented MIC values = 1 μg/ml for the polyene and for the azole (cross amphotericin B and fluconazole high MICs). The strain with low fluconazole and amphotericina B MICs was the one isolated from urine. ERG11 sequences analysis showed that the 4 strains with high fluconazole MICs (blood isolates) harbor five amino acid substitutions: S119A, P135S, D180E, K266S, N326K (using C. albicans numbering). Only the P135 residue is highly conserved among ERG11 from different species (Fig. 1, box shows the substitution). In addition, this substitution is present in the Erg11p of fluconazole-resistant Debaryomyces hansenii and the P135 residue is located near the Erg11p´s Heme group where azole drugs bind (Fig. 2: Saccharomyces cereviciae Erg11p complexed with voriconazole 3D structure. In yellow the region between Y131 and P135 residues. In grey voriconazole binding the Heme group).ConclusionThis emerging multi-resistant pathogen may be underdiagnosed by the methods commonly used in clinical laboratories. The only method capable of identifying this species is DNA sequencing. The strains studied presented very high MIC values for amphotericin B and fluconazole. The P135S substitution could be responsible for the high fluconazole MIC values observed in 4 of the 5 strains of C. vulturna.

Molecular mechanism of fluconazole resistance and pathogenicity attributes of Lebanese Candida albicans hospital isolates

Hospital infections caused by the opportunistic fungus Candida albicans are increasingly common and life threatening. The first line of defense consists of administering antifungal drugs such as azoles including fluconazole that prevent ergosterol biosynthesis. C. albicans is rapidly developing resistance towards antifungal drugs through various mechanisms including mutations in ERG11 which is a gene involved in the ergosterol biosynthesis pathway. These mutations prevent the binding of the drug and inactivate ergosterol synthesis. Alternatively, upregulation of cell membrane ergosterol content generates resistance by countering the effect of the drug. In this study we sequenced the ERG11 gene in 6 fluconazole sensitive and 8 fluconazole resistant C. albicans isolates recovered from clinical settings in Lebanon and quantified the ergosterol content of their plasma membranes to identify mechanisms linked to fluconazole resistance. A number of pathogenicity attributes were also analyzed to determine any correlation with fluconazole resistance. Our results revealed an increase in ergosterol content in the fluconazole resistant isolates. In addition, we identified both novel and previously reported amino acid substitutions in ERG11 as well as frameshift mutations that might contribute to resistance. The fluconazole resistant isolates did not exhibit an increased virulence potential in a mouse model of systemic infection and showed decreased in vitro potential to form biofilms. No discrepancy between drug resistant and sensitive isolates to cell surface disrupting agents was observed. This approach is the first of its kind to be carried out in Lebanon to identify possible mechanisms and phenotypes of drug resistant C. albicans isolates.

Computational insights into fluconazole resistance by the suspected mutations in lanosterol 14α-demethylase (Erg11p) of Candida albicans.

Mutations in the ergosterol biosynthesis gene 11 (ERG11) of Candida albicans have been frequently reported in fluconazole-resistant clinical isolates. Exploring the mutations and their effect could provide new insights into the underlying mechanism of fluconazole resistance. Erg11p_Threonine285Alanine (Erg11p_THR285ALA), Erg11p_Leucine321Phenylalanine (Erg11p_LEU321PHE) and Erg11p_Serine457Proline (Erg11p_SER457PRO) are three fluconazole-resistant suspected mutations reported in clinical isolates of C. albicans. Therefore, our study aims to investigate the role of these suspected mutations in fluconazole resistance using in-silico methods. Molecular dynamics simulation (MDS) analysis of apo-protein for 25ns (nanosecond) showed that suspected mutant proteins underwent slight conformational changes in the tertiary structure. Molecular docking with fluconazole followed by binding free energy analysis showed reduced non-bonded interactions with loss of heme interaction and the least binding affinity for Erg11p_SER457PRO mutation. MDS of suspected mutant proteins-fluconazole complexes for 50ns revealed that Erg11p_SER457PRO and Erg11p_LEU321PHE have clear differences in the interaction pattern and loss or reduced heme interaction compared to wild type Erg11p-fluconazole complex. MDS and binding free energy analysis of Erg11p_SER457PRO-fluconazole complex showed the least binding similar to verified mutation Erg11p_TYR447HIS-fluconazole complex. Taken together, our study concludes that suspected mutation Erg11p_THR285ALA may not have any role whereas Erg11p_LEU321PHE could have a moderate role. However, Erg11p_SER457PRO mutation has a strong possibility to play an active role in fluconazole resistance of C. albicans.

Antifungal Activity of Thai Cajuput Oil and Its Effect on Efflux-Pump Gene Expression in Fluconazole-Resistant Candida albicans Clinical Isolates.

Candidiasis caused by the fluconazole-resistant opportunistic pathogen Candida albicans is an intractable clinical problem that threatens immunocompromised or normal individuals. The most common mechanism of fluconazole resistance in C. albicans is the failure of cells to accumulate the drug due to increased expression of the efflux proteins encoded by the CDR1, CDR2, and MDR1 genes. Because the number of current antifungal drugs is limited, it is necessary to develop new therapeutic strategies. This study aimed to evaluate the antifungal activity of Thai Cajuput oil, its synergism with fluconazole, and its effect on efflux-pump gene expression in fluconazole-resistant C. albicans clinical isolates. Thus, we first detected the efflux-pump genes in fourteen resistant strains by PCR. The frequencies of the CDR1, CDR2, and MDR1 genes were 68.75%, 62.5%, and 87.5%, respectively, and these efflux-pump genes were distributed in three distinct patterns. Subsequently, the antifungal activity of Thai Cajuput oil was assessed by broth macrodilution and its synergism with fluconazole was evaluated by the checkerboard assay. The changes in the expression levels of CDR1, CDR2, and MDR1 after treatment with Thai Cajuput oil were analyzed by qRT-PCR. The MICs and MFCs of Thai Cajuput oil ranged from 0.31 to 1.25 μl/ml and 0.63 to 1.25 μl/ml, respectively, and its activity was defined as fungicidal activity. The MICs of the combination of Thai Cajuput oil and fluconazole were much lower than the MICs of the individual drugs. Interestingly, sub-MICs of Thai Cajuput oil significantly reduced the MDR1 expression level in resistant strains (P < 0.05). Our study suggests that Thai Cajuput oil can be used to create new potential combination therapies to combat the antifungal resistance of C. albicans.

Experimental Evolution Identifies Adaptive Aneuploidy as a Mechanism of Fluconazole Resistance in Candida auris.

Candida auris is a newly emerging fungal pathogen of humans and has attracted considerable attention from both the clinical and basic research communities. Clinical isolates of C. auris are often resistant to one or more antifungal agents. To explore how antifungal resistance develops, we performed experimental evolution assays using a fluconazole-susceptible isolate of C. auris (BJCA001). After a series of passages through medium containing increasing concentrations of fluconazole, fungal cells acquired resistance. By sequencing and comparing the genomes of the parental fluconazole-susceptible strain and 26 experimentally evolved strains of C. auris, we found that a portion of fluconazole-resistant strains carried one extra copy of chromosome V. In the absence of fluconazole, C. auris cells rapidly became susceptible and lost the extra copy of chromosome V. Genomic and transcriptome sequencing (RNA-Seq) analyses indicate that this chromosome carries a number of drug resistance-related genes, which were transcriptionally upregulated in the resistant, aneuploid strains. Moreover, missense mutations were identified in the genes TAC1B, RRP6, and SFT2 in all experimentally evolved strains. Our findings suggest that the gain of an extra copy of chromosome V is associated with the rapid acquisition of fluconazole resistance and may represent an important evolutionary mechanism of antifungal resistance in C. auris.

Fluconazole-resistant Candida parapsilosis strains with a Y132F substitution in the ERG11 gene causing invasive infections in a neonatal unit, South Africa.

The prevalence of azole resistance in C parapsilosis is very low in most parts of the world. However, South Africa has reported an exceptionally high prevalence of azole resistance in C parapsilosis strains isolated from candidaemia cases. We aimed to determine the possible molecular mechanisms of fluconazole resistance in C parapsilosis isolates obtained through surveillance at a large neonatal unit at a South African academic hospital. We sequenced the ERG11 and MRR1 genes of C parapsilosis isolates recovered from cases of neonatal candidemia, followed by microsatellite genotyping. A total of 73 isolates with antifungal susceptibility results were analysed. Of these, 57 (78%) were resistant, 11 (15%) susceptible dose-dependent and 5 (7%) susceptible. The most commonly identified amino acid substitution within the ERG11 gene was Y132F in 68% (39/57) of fluconazole-resistant isolates and none in susceptible isolates. Three amino acid substitutions (R405K, G583R and A619V) and 1 nucleotide deletion at position 1331 were identified within MRR1 gene in 19 (26%) isolates. Microsatellite genotyping grouped isolates into four clusters (50 isolates). Cluster 1 accounted for 23% (17/73) of all cases, cluster 2 for 22% (16/73), cluster 3 for 14% (10/73) and cluster 4 for 10% (7/73). We found an association between cluster type and fluconazole resistance (P-value=.004). Isolates harbouring the Y132F substitution were more likely to belong to a cluster than non-Y132F isolates. Fluconazole resistance in C parapsilosis strains from a single South African neonatal unit was associated with cluster type and predominantly driven by Y123F amino acid substitutions in the ERG11 gene.

Gene Expression Analysis of Key Players Associated with Fluconazole Resistance in Candida albicans

Background: Fluconazole resistance in Candida albicans has become a serious public health problem. Most previous studies have focused on deciphering the relationship between fluconazole resistance and amino acid substitutions in ERG11, which encodes cytochrome P450 lanosterol 14α-demethylase whose enzymatic activity is inhibited by azoles. However, azole resistance in C. albicans is a multifactorial phenomenon and several lines of evidence indicate that other genes and mechanisms may contribute to the development of fluconazole resistance. Objectives: The present study aimed to investigate the underlying role of six genes in fluconazole-resistant clinical strains of C. albicans, including ERG11, RTA2, and the efflux pump genes CDR1, CDR2, MDR1, and FLU1. Methods: We collected 40 fluconazole-resistant isolates and 40 susceptible isolates from patients with Candida infections in the First Affiliated Hospital of Nanchang University in China from 2005 to 2008. The susceptibility of the isolates to antifungal agents was tested by the M27-A3 broth microdilution method following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Then, the gene expression levels of several key players in azole resistance were quantified. Results: Most fluconazole-resistant strains analyzed in this study were found to be cross-resistant to ketoconazole, itraconazole, and clotrimazole. We observed that the FLU1 gene expression significantly increased (P < 0.05) and exhibited major changes in most of the fluconazole-resistant isolates (75.0%). In addition, the expression of a novel gene, RTA2, was remarkably upregulated (P < 0.05). Interestingly, we found that 10% of the fluconazole-resistant isolates were simultaneously associated with ERG11 mutation and overexpression of RTA2, CDR1, and FLU1 genes. Unlike other studies, we did not find any difference in the expression of CDR2, MDR1, and ERG11 genes between the fluconazole-susceptible and resistant isolates. Conclusions: Our findings suggested that the overexpression of FLU1 and RTA2 genes may cause azole resistance; this finding had not been reported previously in clinical isolates of C. albicans. The upregulation of FLU1 and RTA2 genes was the predominant mechanism of fluconazole resistance in C. albicans in China.

Emergence of clonal fluconazole-resistant Candida parapsilosis clinical isolates in a multicentre laboratory-based surveillance study in India.

The emergence of fluconazole resistance in Candida parapsilosis healthcare-associated infections has recently been increasingly reported. Antifungal susceptibility profiles and mechanisms of fluconazole resistance in C. parapsilosis (n = 199) from nine hospitals in India collected over a period of 3 years were studied. Further, clonal transmission of fluconazole-resistant isolates in different hospitals was investigated. Antifungal susceptibility testing of five azoles, amphotericin B and 5-flucytosine was performed by the CLSI microbroth dilution method. The azole target ERG11 gene was sequenced, and the significance of a novel ERG11 mutation in C. parapsilosis was determined using a gap-repair cloning approach in Saccharomyces cerevisiae. In addition, microsatellite analysis was performed to determine the clonal lineage of C. parapsilosis-resistant strains circulating among different hospitals. A total of 64 (32%) C. parapsilosis isolates were non-susceptible to fluconazole, which included resistant (n = 55; MIC >4 mg/L) and susceptible dose-dependent (n = 9) isolates. Of these 64 non-susceptible isolates, a novel K143R amino acid substitution was noted in 92%, and the remaining five isolates had the Y132F substitution. Elevated azole MICs (≥16-fold) were detected in S. cerevisiae upon expression of C. parapsilosis ERG11 alleles carrying Y132F or K143R substitutions. Two major clusters of non-susceptible isolates were circulating in seven Indian hospitals. We report a novel K143R amino acid substitution in ERG11p causing fluconazole resistance in C. parapsilosis. Fluconazole-non-susceptible C. parapsilosis isolates carrying the novel K143R amino acid substitution should be identified in clinical microbiology laboratories to prevent further clonal transmission.

ERG11 gene polymorphisms and susceptibility to fluconazole in Candida isolates from diabetic and kidney transplant patients

Candidiasis is the most frequent opportunistic mycosis in humans and can cause mortality, particularly in immunodeficient patients. One major concern is the increasing number of infections caused by drug-resistant Candidas trains, as these cannot be efficiently treated with standard therapeutics. The most common mechanism of fluconazole resistance in Candida is mutation of ERG11, a gene involved in the biosynthesis of ergosterol, a compound essential for cell integrity and membrane function. Based on this knowledge, we investigated polymorphisms in the ERG11 gene of 3 Candida species isolated from immunocompromised and immunocompetent patients. In addition, we correlated the genetic data with the fluconazole susceptibility profile of the Candida isolates. A total of 80 Candida albicans, 8 Candida tropicalis and 6 Candida glabrata isolates were obtained from the saliva of diabetic, kidney transplant and immunocompetent patients. Isolates were considered susceptible to fluconazole if the minimum inhibitory concentration was lower than 8 μg/mL. The amino acid mutations F105L, D116E, K119N, S137L, and K128T were observed in C. albicans isolates, and T224C and G263A were found in C. tropicalis isolates. Despite the high number of polymorphisms observed, the mutations occurred in regions that are not predicted to interfere with ergosterol synthesis, and therefore are not related to fluconazole resistance.

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.

Fluconazole resistance in Candida species: a current perspective.

Candida albicans and the emerging non-albicans Candida spp. have significant clinical relevance among many patient populations. Current treatment guidelines include fluconazole as a primary therapeutic option for the treatment of these infections, but it is only fungistatic against Candida spp. and both inherent and acquired resistance to fluconazole have been reported. Such mechanisms of resistance include increased drug efflux, alteration or increase in the drug target, and development of compensatory pathways for producing the target sterol, ergosterol. While many mechanisms of resistance observed in C. albicans are also found in the non-albicans species, there are also important and unexpected differences between species. Furthermore, mechanisms of fluconazole resistance in emerging Candida spp., including the global health threat Candida auris, are largely unknown. In order to preserve the utility of one of our fundamental antifungal drugs, fluconazole, it is essential that we fully appreciate the manner by which Candida spp. manifest resistance to it.

Quantitation of ergosterol content and gene expression profile of ERG11 gene in fluconazole-resistant Candida albicans.

Background and Purpose:The frequency of opportunistic fungal infections in immunocompromised patients, especially by Candida species, has sharply increased in the last few decades. The objective of this study was to analyse the ergosterol content and gene expression profiling of clinical isolates of fluconazole-resistant Candida albicans. Materials and Methods:Sixty clinical samples were identified and collected from immunocompromised patients, namely recurrent oral, vaginal, and cutaneous candidiasis, during 2015-16. Antifungal susceptibility testing of fluconazole against clinical Candida species was performed according to Clinical and Laboratory Standards Institute guidelines. Ergosterol content and gene expression profiling of sterol 14α-demethylase (ERG11) gene in fluconazole-susceptible and –resistant C. albicans were investigated. Results: The specimens consisted of C. albicans (46.67%), Candida krusei (41.67%), and Candida tropicalis (11.67%). All the isolates were resistant to fluconazole. No significant reduction was noted in total cellular ergosterol content in comparison with untreated controls in terms of fluconazole-resistant C. albicans. The expressionlevel of ERG11 gene was down-regulated in fluconazole-susceptible C. albicans. Eventually, the expression pattern of ERG11 gene revealed no significant changes in fluconazole-resistant isolates compared to untreated controls. The results revealed no significant differences between fluconazole-susceptible and –resistant C. albicans sequences by comparison with ERG11 reference sequence. Conclusion:Our findings provide an insight into the mechanism of fluconazole resistance in C. albicans. The mechanisms proposed for clinical isolates of fluconazole-resistant C. albicans are alteration in sterol biosynthesis, analysis of expression level of ERG11 gene, and analysis of gene sequences. Nonetheless, further studies are imperative to find molecular mechanisms that could be targeted to control fluconazole resistance.

Expression Patterns of ABC Transporter Genes in Fluconazole-Resistant Candida glabrata.

Clinical management of fungal diseases is compromised by the emergence of antifungal drug resistance in fungi, which leads to elimination of available drug classes as treatment options. An understanding of antifungal resistance at molecular level is, therefore, essential for the development of strategies to combat the resistance. This study presents the assessment of molecular mechanisms associated with fluconazole resistance in clinical Candida glabrata isolates originated from Iran. Taking seven distinct fluconazole-resistant C. glabrata isolates, real-time PCRs were performed to evaluate the alternations in the regulation of the genes involved in drug efflux including CgCDR1, CgCDR2, CgSNQ2, and CgERG11. Gain-of-function (GOF) mutations in CgPDR1 alleles were determined by DNA sequencing. Cross-resistance to fluconazole, itraconazole, and voriconazole was observed in 2.5% of the isolates. In the present study, six amino acid substitutions were identified in CgPdr1, among which W297R, T588A, and F575L were previously reported, whereas D243N, H576Y, and P915R are novel. CgCDR1 overexpression was observed in 57.1% of resistant isolates. However, CgCDR2 was not co-expressed with CgCDR1. CgSNQ2 was upregulated in 71.4% of the cases. CgERG11 overexpression does not seem to be associated with azole resistance, except for isolates that exhibited azole cross-resistance. The pattern of efflux pump gene upregulation was associated with GOF mutations observed in CgPDR1. These results showed that drug efflux mediated by adenosine-5-triphosphate (ATP)-binding cassette transporters, especially CgSNQ2 and CgCDR1, is the predominant mechanism of fluconazole resistance in Iranian isolates of C. glabrata. Since some novel GOF mutations were found here, this study also calls for research aimed at investigating other new GOF mutations to reveal the comprehensive understanding about efflux-mediated resistance to azole antifungal agents.

Molecular identification, antifungal resistance and virulence of Cryptococcus neoformans and Cryptococcus deneoformans isolated in Seville, Spain.

Cryptococcal meningitis is one of the leading causes of death in HIV/AIDS patients. Our aim was to in order to characterise the epidemiology, antifungal susceptibility pattern and virulence of 28 Cyptococcus sp. strains recovered from 12 AIDS patients during two years in a Spanish single institution. Antifungal susceptibility testing was performed according to the CLSI protocols. Clinical strains were molecularly characterised by serotyping, mating type, PCR fingerprinting (M13 and GACA4 microsatellites) and analysis of two rDNA regions (IGS1 and ITS). Sequencing of the ERG11 gene was used to explore mechanisms of fluconazole resistance. Differences in virulence between species were studied in a Galleria mellonella infection model. Cryptococcus deneoformans and C.deneoformans x Cryptococcus neoformans hybrids were the most frequent variety (65%) followed by C.neoformans (35%). Strains were categorised according to 13 microsatellite genotypes and mixed infections could be detected in three patients. Twenty-nine per cent of the strains were fluconazole resistant. In one of the patients, the fluconazole resistance phenotype was associated with a point mutation in the ERG11 gene responsible for the amino acid substitution G470R. C.neoformans strains were able to kill G.mellonella larvae more efficiently than C.deneoformans and hybrids between both species. Precisely molecular characterisation of C.neoformans species is important for an accurate patient's management.

Molecular mechanisms associated with Fluconazole resistance in clinical Candida albicans isolates from India.

Resistance to azole antifungals is a significant problem in Candida albicans. An understanding of resistance at molecular level is essential for the development of strategies to tackle resistance and rationale design of newer antifungals and target-based molecular approaches. This study presents the first evaluation of molecular mechanisms associated with fluconazole resistance in clinical C.albicans isolates from India. Target site (ERG11) alterations were determined by DNA sequencing, whereas real-time PCRs were performed to quantify target and efflux pump genes (CDR1, CDR2, MDR1) in 87 [Fluconazole susceptible (n = 30), susceptible-dose dependent (n = 30) and resistant (n = 27)] C.albicans isolates. Cross-resistance to fluconazole, ketoconazole and itraconazole was observed in 74.1% isolates. Six amino acid substitutions were identified, including 4 (E116D, F145L, E226D, I437V) previously reported ones and 2 (P406L, Q474H) new ones. CDR1 over-expression was seen in 77.7% resistant isolates. CDR2 was exclusively expressed with CDR1 and their concomitant over-expression was associated with azole cross-resistance. MDR1 and ERG11 over-expression did not seem to be associated with resistance. Our results show that drug efflux mediated by Adenosine-5'-triphosphate (ATP)-binding cassette transporters, especially CDR1 is the predominant mechanism of fluconazole resistance and azole cross-resistance in C. albicans and indicate the need for research directed towards developing strategies to tackle efflux mediated resistance to salvage azoles.

Fluconazole Susceptibility in Cryptococcus gattii Is Dependent on the ABC Transporter Pdr11

Cryptococcus gattii isolates from the Pacific Northwest have exhibited higher fluconazole MICs than isolates from other sites. The mechanism of fluconazole resistance in C. gattii is unknown. We sought to determine the role of the efflux pumps Mdr1 and Pdr11 in fluconazole susceptibility. Using biolistic transformation of the parent isolate, we created a strain lacking Mdr1 (mdr1Δ) and another strain lacking Pdr11 (pdr11Δ). Phenotypic virulence factors were assessed by standard methods (capsule size, melanin production, growth at 30 and 37 °C). Survival was assessed in an intranasal murine model of cryptococcosis. Antifungal MICs were determined by the M27-A3 methodology. No differences in key virulence phenotypic components were identified. Fluconazole susceptibility was unchanged in the Mdr1 knockout or reconstituted isolates. However, fluconazole MICs decreased from 32 μg/ml for the wild-type isolate to <0.03 μg/ml for the pdr11Δ strain and reverted to 32 μg/ml for the reconstituted strain. In murine models, no difference in virulence was observed between wild-type, knockout, or reconstituted isolates. We conclude that Pdr11 plays an essential role in fluconazole susceptibility in C. gattii. Genomic and expression differences between resistant and susceptible C. gattii clinical isolates should be assessed further in order to identify other potential mechanisms of resistance.

Overexpression and mutation as a genetic mechanism of fluconazole resistance in Candida albicans isolated from human immunodeficiency virus patients in Indonesia.

Fluconazole is the standard treatment for oropharyngeal candidiasis, which is the third most common opportunistic infection in human immunodeficiency virus (HIV)/AIDS patients in Indonesia. Overuse of this drug could lead to the emergence of resistance. The objective of this study was to analyse the role of ERG11, CDR1, CDR2 and MDR1 gene overexpression and mutations in the ERG11 gene as a genetic mechanism of fluconazole resistance in Candida albicans isolated from HIV patients in Indonesia. Overexpression of ERG11, CDR1, CDR2 and MDR1 was analysed by real-time reverse transcription PCR, while ERG11 gene mutation analysis was performed using sequencing methods. Seventeen isolates out of 92 strains of C. albicans isolated from 108 HIV patients were found to be resistant to azole antifungals. The highest gene overexpression of ERG11 was found in C. albicans resistant to single fluconazole, while the highest gene overexpression of CDR2 was detected in all isolates of C. albicans resistant to multiple azoles. Amino acid substitutions were observed at six positions, i.e. D116E, D153E, I261V, E266D, V437I and V488I. The amino acid substitution I261V was identified in this study and was probably associated with fluconazole resistance. The combination of overexpression of CDR2 and ERG11 and mutation in the ERG11 gene was found to be a genetic mechanism of fluconazole resistance in C. albicans isolated from HIV patients in Indonesia.