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Candida glabrata exhibits innate resistance to azole antifungal drugs but also has the propensity to rapidly develop clinical drug resistance. Azole drugs, which target Erg11, is one of the major classes of antifungals used to treat Candida infections. Despite their widespread use, the mechanism controlling azole-induced ERG gene expression and drug resistance in C. glabrata has primarily revolved around Upc2 and/or Pdr1. Phylogenetic and syntenic analyses revealed that C. glabrata , following a whole genome duplication event, maintained HAP1A and HAP1B , whereas Saccharomyces cerevisiae only retained the HAP1A ortholog, HAP1 . In this study, we determined the function of two zinc cluster transcription factors, Hap1A and Hap1B, as direct regulators of ERG genes. In S. cerevisiae, Hap1, an ortholog of Hap1A, is a known transcription factor controlling ERG gene expression under aerobic and hypoxic conditions. Interestingly, deleting HAP1 or HAP1B in either S. cerevisiae or C. glabrata, respectively, showed altered susceptibility to azoles. In contrast, the strain deleted for HAP1A did not exhibit azole susceptibility. We also determined that the increased azole susceptibility in a hap1B Δ strain is attributed to decreased azole-induced expression of ERG genes, resulting in decreased levels of total ergosterol. Surprisingly, Hap1A protein expression is barely detected under aerobic conditions but is specifically induced under hypoxic conditions, where Hap1A is required for the repression of ERG genes. However, in the absence of Hap1A, Hap1B can compensate as a transcriptional repressor. Our study shows that Hap1A and Hap1B is utilized by C. glabrata to adapt to specific host and environmental conditions., Importance: Invasive and drug-resistant fungal infections pose a significant public health concern. Candida glabrata , a human fungal pathogen, is often difficult to treat due to its intrinsic resistance to azole antifungal drugs and its capacity to develop clinical drug resistance. Therefore, understanding the pathways that facilitate fungal growth and environmental adaptation may lead to novel drug targets and/or more efficacious antifungal therapies. While the mechanisms of azole resistance in Candida species have been extensively studied, the roles of zinc cluster transcription factors, such as Hap1A and Hap1B, in C. glabrata have remained largely unexplored until now. Our research shows that these factors play distinct yet crucial roles in regulating ergosterol homeostasis under azole drug treatment and oxygen-limiting growth conditions. These findings offer new insights into how this pathogen adapts to different environmental conditions and enhances our understanding of factors that alter drug susceptibility and/or resistance., Competing Interests: The authors declare no conflict of interest.

Background: Candida parapsilosis is a pathogenic yeast that has reduced susceptibility to echinocandins and ranks as the second or third leading cause of candidaemia, depending on the geographical region. This yeast often causes nosocomial infections, which are frequently detected as outbreaks. In recent years, resistance to azoles in C. parapsilosis has increased globally, primarily due to the accumulation of mutations in the ERG11 gene., Objectives: In this study, we have developed an assay based on real-time PCR and high-resolution melting (HRM) curve analysis to detect two of the most prevalent mutations at ERG11 that confer resistance to fluconazole (Y132F and G458S)., Methods: We designed allele-specific oligonucleotides that selectively bind to either the wild type or mutated sequences and optimised the conditions to ensure amplification of the specific allele, followed by detection via high-resolution melting (HRM) analysis., Results: The designed oligonucleotides to detect the Erg11 Y132F and Erg11 G458S mutations produced specific amplification of either WT or mutated alleles. We conducted a duplex real-time PCR combining oligonucleotides for the wild-type sequences in one mix, and oligonucleotides for the mutated alleles in another. Following this, we performed an analysis of the HRM curve to identify the amplified allele in each case. This technique was blindly evaluated on a set of 114 C. parapsilosis isolates, all of which were unequivocally identified using our approach., Conclusion: This technique offers a new method for the early detection of azole resistance mechanism in C. parapsilosis., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Background: Meyerozyma guilliermondii is a yeast species responsible for invasive fungal infections. It has high minimum inhibitory concentrations (MICs) to echinocandins, the first-line treatment of candidemia. In this context, azole antifungal agents are frequently used. However, in recent years, a number of azole-resistant strains have been described. Their mechanisms of resistance are currently poorly studied., Objective: The aim of this study was consequently to understand the mechanisms of azole resistance in several clinical isolates of M. guilliermondii., Methods: Ten isolates of M. guilliermondii and the ATCC 6260 reference strain were studied. MICs of azoles were determined first. Whole genome sequencing of the isolates was then carried out and the mutations identified in ERG11 were expressed in a CTG clade yeast model (C. lusitaniae). RNA expression of ERG11, MDR1 and CDR1 was evaluated by quantitative PCR. A phylogenic analysis was developed and performed on M. guilliermondii isolates. Lastly, in vitro experiments on fitness cost and virulence were carried out., Results: Of the ten isolates tested, three showed pan-azole resistance. A combination of F126L and L505F mutations in Erg11 was highlighted in these three isolates. Interestingly, a combination of these two mutations was necessary to confer azole resistance. An overexpression of the Cdr1 efflux pump was also evidenced in one strain. Moreover, the three pan-azole-resistant isolates were shown to be genetically related and not associated with a fitness cost or a lower virulence, suggesting a possible clonal transmission., Conclusion: In conclusion, this study identified an original combination of ERG11 mutations responsible for pan-azole-resistance in M. guilliermondii. Moreover, we proposed a new MLST analysis for M. guilliermondii that identified possible clonal transmission of pan-azole-resistant strains. Future studies are needed to investigate the distribution of this clone in hospital environment and should lead to the reconsideration of the treatment for this species., (© 2024 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Objectives: Infections caused by azole-resistant Candida tropicalis strains are increasing in clinical settings. The reason for this epidemical change and the mechanisms of C. tropicalis azole resistance are not fully understood., Methods: In this study, we performed biological and genomic analyses of 239 C. tropicalis strains, including 115 environmental and 124 human commensal isolates., Results: Most (99.2%) of the isolates had a baseline diploid genome. The strains from both environmental and human niches exhibit similar abilities to survive under stressful conditions and produce secreted aspartic proteases. However, the human commensal isolates exhibited a stronger ability to filament than the environmental strains. We found that 19 environmental isolates (16.5%) and 24 human commensal isolates (19.4%) were resistant to fluconazole. Of the fluconazole-resistant strains, 37 isolates (86.0%) also exhibited cross-resistance to voriconazole. Whole-genome sequencing and phylogenetic analyses revealed that both environmental and commensal isolates were widely distributed in a number of genetic clusters, but the two populations exhibited a close genetic association. The majority of fluconazole-resistant isolates were clustered within a single clade (X)., Conclusions: The combination of hotspot mutations (Y132F and S154F) and genomic expansion of ERG11, which encodes the azole target lanosterol 14-α-demethylase and represents a major target of azole drugs, was a major mechanism for the development of azole resistance. The isolates carrying both hotspot mutations and genomic expansion of ERG11 exhibited cross-resistance to fluconazole and voriconazole. Moreover, the azole-resistant isolates from both the environmental and human commensal niches showed similar genotypes., (Copyright © 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Methods: Over a four-year period, 123 Candida bloodstream isolates were collected at a quaternary care hospital. The isolates were identified by MALDI-TOF MS and their fluconazole (FLC) susceptibility patterns were assessed according to CLSI guidelines. Subsequently, sequencing of ERG11, TAC1 or MRR1, and efflux pump activity were performed for resistant isolates., Results: Out of 123 clinical strains,C. albicans accounted for 37.4%, followed by C. tropicalis 26.8%, C. parapsilosis 19.5%, C. auris 8.1%, C. glabrata 4.1%, C. krusei 2.4% and C. lusitaniae 1.6%. Resistance to FLC reached 18%; in addition, a high proportion of isolates were cross-resistant to voriconazole. Erg11 amino acid substitutions associated with FLC-resistance (Y132F, K143R, or T220L) were found in 11/19 (58%) of FLCresistant isolates. Furthermore, novel mutations were found in all genes evaluated. Regarding efflux pumps, 8/19 (42%) of FLC-resistant Candida spp strains showed significant efflux activity. Finally, 6/19 (31%) of FLC-resistant isolates neither harbored resistance-associated mutations nor showed efflux pump activity. Among FLC-resistant species, C. auris 7/10 (70%) and C. parapsilosis 6/24 (25%) displayed the highest percentages of resistance (C. albicans 6/46, 13%)., Discussion: Overall, 68% of FLC-resistant isolates exhibited a mechanism that could explain their phenotype (e.g. mutations, efflux pump activity, or both). We provide evidence that isolates from patients admitted to a Colombian hospital harbor amino acid substitutions related to resistance to one of the most commonly used molecules in the hospital setting, with Y132F being the most frequently detected., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ceballos-Garzon, Peñuela, Valderrama-Beltrán, Vargas-Casanova, Ariza and Parra-Giraldo.)

We analyzed a cohort of Trichosporon asahii strains with different MICs of fluconazole and voriconazole and evaluated the presence of ERG11 mutations. ERG11 mutation conferring an amino acid change was found and its resistance potential was evaluated by cloning into Saccharomyces cerevisiae susceptible host strain. Transformants were not resistant to either fluconazole nor voriconazole. Our results suggest that ERG11 variants exist among T. asahii isolates, but are not responsible for resistance phenotypes.

Fungal infections are a major health concern because of limited antifungal drugs and development of drug resistance. Candida can develop azole drug resistance by overexpression of drug efflux pumps or mutating ERG11 , the target of azoles. However, the role of epigenetic histone modifications in azole-induced gene expression and drug resistance is poorly understood in Candida glabrata. In this study, we show that Set1 mediates histone H3K4 methylation in C. glabrata. In addition, loss of SET1 and histone H3K4 methylation increases azole susceptibility in both C. glabrata and S. cerevisiae. This increase in azole susceptibility in S. cerevisiae and C. glabrata strains lacking SET1 is due to distinct mechanisms. For S. cerevisiae, loss of SET1 decreased the expression and function of the efflux pump Pdr5, but not ERG11 expression under azole treatment. In contrast, loss of SET1 in C. glabrata does not alter expression or function of efflux pumps. However, RNA sequencing revealed that C. glabrata Set1 is necessary for azole-induced expression of all 12 genes in the late ergosterol biosynthesis pathway, including ERG11 and ERG3. Furthermore, chromatin immunoprecipitation analysis shows histone H3K4 trimethylation increases upon azole-induced ERG gene expression. In addition, high performance liquid chromatography analysis indicated Set1 is necessary for maintaining proper ergosterol levels under azole treatment. Clinical isolates lacking SET1 were also hypersusceptible to azoles which is attributed to reduced ERG11 expression but not defects in drug efflux. Overall, Set1 contributes to azole susceptibility in a species-specific manner by altering the expression and consequently disrupting pathways known for mediating drug resistance.

Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-14 demethylase enzyme, which is referred to as Erg11. Inhibition of Erg11 ultimately leads to a reduction in ergosterol production, an essential fungal membrane sterol. Many Candida species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. In C. glabrata, these azole resistant mutations typically cause hyperactivity of the Pdr1 transcription factor and rarely lie within the ERG11 gene. Here, we generated C. glabrata ERG11 mutations that were analogous to azole resistance alleles from C. albicans ERG11 . Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. Resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. Crucially, the presence of the DM ERG11 allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in ERG11 activity can involve cellular effects beyond an alteration in this key azole target enzyme. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata.

Azole resistance constitutes a serious clinical problem in the management of infections caused by Candida albicans. This study aimed to explore azole-resistant mechanisms in clinical C. albicans isolates collected in Jinan, Shandong, China. In total, 22 samples were collected and analyzed. Among these, four isolates (28A, 28D, 28I, and 28J) exhibited high level of pan-azole-resistance that was Hsp90 dependent. Gene sequencing revealed that the four Hsp90-dependent strains contained different ERG3 mutations that led to four novel amino acid substitutions (S265Y, N322D, N324S, and E355D) in Erg3. The role of these substitutions in azole resistance development was determined by constructing one copy of the mutated ERG3 from the 28A, 28D, and 28I strains into C. albicans CAI4, respectively. The minimum inhibitory concentration value of fluconazole (FLC) against C. albicans CAI4- ERG3 28I increased fourfold compared with the wild-type C. albicans strain, suggesting that the novel combination of substitutions S265Y, N322D, and N324S played an important role in mediating azole resistance in 28I. Besides, we identified several different mechanisms in other three isolates. Strains 28A and 28D displayed increased efflux ability and overexpression of MDR1 . Strain 28J showed high level of ERG11 expression, but no mutation in its regulator Upc2 was observed. Our study revealed that multiple factors confer azole resistance in clinical C. albicans isolates and combination therapy should be conducted clinically.

An increasing number of outbreaks due to resistant non-albicans Candida species have been reported worldwide. Between 2014 and 2016, Candida isolates causing invasive candidiasis were recovered in a Mexican hospital. Isolates were identified to species level and antifungal susceptibility was determined. In the time period studied, 74 invasive candidiasis cases were identified, with 38% (28/74) caused by Candida parapsilosis, out of which 54% (15/28) were fluconazole resistant. The ERG11 gene was sequenced for 12 recoverable fluconazole-resistant C. parapsilosis isolates and SNPs identified. The 12 isolates had one common silent point mutation in ERG11 (T591C) and seven isolates had an additional (A395T) mutation, which corresponded to Y132F. Four of the isolates carrying this mutation were closely related within the same cluster by microsatellite typing. This is the first report of an invasive candidiasis outbreak in Mexico due to azole-resistant C. parapsilosis associated with the Y132F substitution., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Background: Candida tropicalis (C. tropicalis) is an important opportunistic pathogenic Candida species that can cause nosocomial infection. In this study, we analyzed the distribution and drug susceptibility of C. tropicalis and the relationship between ERG11 and UPC2 expression and resistance to azole antifungal agents., Methods: C. tropicalis was cultured and identified by Sabouraud Agar Medium, CHROM Agar Candida and ATB tests (Bio-Mérieux, France). Total RNA was extracted from the collected strains, and the ERG11 and UPC2 mRNA expression levels were analyzed by quantitative real-time PCR., Results: In total, 2872 clinical isolates of Candida, including 319 strains of C. tropicalis, were analyzed herein; they were mainly obtained from the Departments of Respiratory Medicine and ICU. The strains were predominantly isolated from airway secretion samples, and the detection trend in four years was mainly related to the type of department and specimens. The resistance rates of C. tropicalis to fluconazole, itraconazole and voriconazole had been increasing year by year. The mRNA expression levels of ERG11 and UPC2 in the fluconazole-resistant group were significantly higher than they were in the susceptible group. In addition, there was a significant positive linear correlation between these two genes in the fluconazole-resistant group., Conclusions: Overexpression of the ERG11 and UPC2 genes in C. tropicalis could increase resistance to azole antifungal drugs. The routine testing for ERG11 and UPC2 in high-risk patients in key departments would provide a theoretical basis for the rational application of azole antifungal drugs.

Objective: We investigated molecular mechanisms responsible for azole resistance in Candida tropicalis isolates., Methods: We studied 507 C. tropicalis isolates causing invasive candidiasis from ten hospitals over 5 years. Antifungal susceptibility was determined by broth microdilution methods. Point mutations in the C. tropicalis ERG11 gene that may confer azole resistance were explored and verified. The expression levels of ERG11, CYTb, MDR1 and CDR1 genes were compared in 20 fluconazole-susceptible and 20 fluconazole-resistant isolates., Results: Fluconazole-susceptible, -susceptible dose-dependent and -resistant strains accounted for 76.7% (389/507), 10.5% (53/507) and 12.8% (65/507) of C. tropicalis isolates, respectively. The ERG11 mutation A395T/W occurred in 10.7% (54/507) of isolates, all of which were resistant to fluconazole. The nucleotide mutation C461T/Y was the second most common (50/507 isolates, 9.9%), and all isolates carrying C461T/Y also had the mutation A395T/W. However, the presence of C461T did not contribute to the azole-resistant phenotype. Substitutions V125A, Y257H and G464S (<2% of isolates), which were reported for the first time in C. tropicalis, also conferred fluconazole non-susceptible phenotypes. Compared with fluconazole susceptible isolates, fluconazole-resistant isolates had higher ERG11 (fold expression level 1.42 versus 0.79, p < 0.01) but lower CYTb (fold expression level 1.26 versus 2.67, p < 0.01) gene expression levels. Three azole-resistant isolates carrying the wild-type ERG11 gene had higher levels of CDR1 and MDR1 expression., Conclusions: ERG11 missense mutations were the major mechanism responsible for azole resistance in C. tropicalis isolates, but overexpression of ERG11, CDR1 and MDR1, as well as reduced expression of CYTb, also contributed to resistance., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

In the case presented here, we describe the isolation of an azole-resistant strain of M. pachydermatis from a canine Malassezia dermatitis. The isolate (NUBS18001) from this case exhibited a minimum inhibitory concentration (MIC) of 320 μg/ml to itraconazole (ITZ) by broth microdilution (BM) assay, >32 μg/ml to ITZ by E-test, and >32 μg/ml to KTZ by E-test. Synergistic effects between FK506 and ITZ in the azole-resistant strain was evaluated using the microdilution checker-board method. The ITZ-resistant strain exhibited MICs of 320 μg/mL of ITZ alone and 5 μg/ml of FK506 alone; the addition of FK506 attenuated the ITZ MIC to 2.5 μg/ml, yielding an ITZ FICI value of 0.507. This result suggested that the combination of ITZ and FK506 exerted an additive effect against the ITZ-resistant strain. To understand the other mechanism inferred to be present in our multi-azole-resistant strain, we sequenced the ERG11 gene from this isolate, and detected missense mutations (A412G and C905T) in the sequence of the ERG11 open reading frame (ORF). To the best of our knowledge, this work is the first report that a multi-azole-resistant M. pacydermatis strain contains mutations in ERG11., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Multiple Erg11 amino acid substitutions were identified in clinical isolates of Candida auris originating from India and Colombia. Elevated azole MICs were detected in Saccharomyces cerevisiae upon heterologous expression of C. auris ERG11 alleles that encoded for Y132F or K143R substitutions; however, expression of alleles encoding I466M, Y501H, or other clade-defined amino acid differences yielded susceptible MICs. Similar to other Candida species, specific C. auris ERG11 mutations resulted directly in reduced azole susceptibility., (Copyright © 2018 Healey et al.)

This study was undertaken to characterize the mechanism(s) of azole resistance in clinical isolates of Candida albicans collected in Shanghai, China, focusing on the role of efflux pumps, target enzymes of fluconazole (Erg11), respiratory status and the ergosterol biosynthetic pathway. Clinical isolates of C. albicans (n = 30) were collected from 30 different non-HIV-infected patients in four hospitals in Shanghai. All 30 C. albicans isolates were susceptible to amphotericin B and 5-fluorocytosine. Twelve C. albicans isolates showed resistance to at least one type of triazole antifungal. Flow cytometry analysis of rhodamine 6G efflux showed that azole-resistant isolates had greater efflux pump activity, which was consistent with elevated levels of CDR1 and CDR2 genes that code for ABC efflux pumps. However, we did not observe increased expression of ERG11 and MDR1 or respiratory deficiency. Several mutations of ERG11 and TAC1 genes were detected. The F964Y mutation in the TAC1 gene was identified for the first time. Two main sterols, ergosterol and lanosterol, were identified by GC-MS chromatogram, and no missense mutations were found in ERG3. Furthermore, seven amino acid substitutions in ERG11, A114S, Y132H, Y132F, K143Q, K143R, Y257H and G448E were found, by Type II spectral quantitative analysis, to contribute to low affinity binding between Erg11 and fluconazole., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Fungal resistance is a public health concern due to the limited availability of antifungal resources and the complexities associated with treating persistent fungal infections. Azoles are thus far the primary line of defense against fungi. Specifically, azoles inhibit the conversion of lanosterol to ergosterol, producing defective sterols and impairing fluidity in fungal plasmatic membranes. Studies on azole resistance have emphasized specific point mutations in CYP51/ERG11 proteins linked to resistance. Although very insightful, the traditional approach to studying azole resistance is time-consuming and prone to errors during meticulous alignment evaluation. It relies on a reference-based method using a specific protein sequence obtained from a wild-type (WT) phenotype. Therefore, this study introduces a machine learning (ML)-based approach utilizing molecular descriptors representing the physiochemical attributes of CYP51/ERG11 protein isoforms. This approach aims to unravel hidden patterns associated with azole resistance. The results highlight that descriptors related to amino acid composition and their combination of hydrophobicity and hydrophilicity effectively explain the slight differences between the resistant non-wild-type (NWT) and WT (nonresistant) protein sequences. This study underscores the potential of ML to unravel nuanced patterns in CYP51/ERG11 sequences, providing valuable molecular signatures that could inform future endeavors in drug development and computational screening of resistant and nonresistant fungal lineages. [ABSTRACT FROM AUTHOR]

Fungal resistance is a public health concern due to the limited availability of antifungal resources and the complexities associated with treating persistent fungal infections. Azoles are thus far the primary line of defense against fungi. Specifically, azoles inhibit the conversion of lanosterol to ergosterol, producing defective sterols and impairing fluidity in fungal plasmatic membranes. Studies on azole resistance have emphasized specific point mutations in CYP51/ERG11 proteins linked to resistance. Although very insightful, the traditional approach to studying azole resistance is time-consuming and prone to errors during meticulous alignment evaluation. It relies on a reference-based method using a specific protein sequence obtained from a wild-type (WT) phenotype. Therefore, this study introduces a machine learning (ML)-based approach utilizing molecular descriptors representing the physiochemical attributes of CYP51/ERG11 protein isoforms. This approach aims to unravel hidden patterns associated with azole resistance. The results highlight that descriptors related to amino acid composition and their combination of hydrophobicity and hydrophilicity effectively explain the slight differences between the resistant non-wild-type (NWT) and WT (nonresistant) protein sequences. This study underscores the potential of ML to unravel nuanced patterns in CYP51/ERG11 sequences, providing valuable molecular signatures that could inform future endeavors in drug development and computational screening of resistant and nonresistant fungal lineages.

Acquired fluconazole resistance (FR) in bloodstream infection (BSI) isolates of Candida albicans is rare. We investigated the FR mechanisms and clinical features of 14 fluconazole non-susceptible (FNS; FR and fluconazole-susceptible dose-dependent) BSI isolates of C. albicans recovered from Korean multicenter surveillance studies during 2006- 2021. Mutations causing amino acid substitutions (AASs) in the drug-target gene ERG11 and the FR-associated transcription factor genes TAC1, MRR1, and UPC2 of the 14 FNS isolates were compared with those of 12 fluconazole-susceptible isolates. Of the 14 FNS isolates, eight and seven had Erg11p (K143R, F145L, or G464S) and Tac1p (T225A, R673L, A736T, or A736V) AASs, respectively, which were previously described in FR isolates. Novel Erg11p, Tac1p, and Mrr1p AASs were observed in two, four, and one FNS isolates, respectively. Combined Erg11p and Tac1p AASs were observed in seven FNS isolates. None of the FR-associated Upc2p AASs were detected. Of the 14 patients, only one had previous azole exposure, and the 30-day mortality rate was 57.1% (8/14). Our data show that Erg11p and Tac1p AASs are likely to contribute to FR in C. albicans BSI isolates in Korea and that most FNS C. albicans BSIs develop without azole exposure. [ABSTRACT FROM AUTHOR]

Background: Candida parapsilosis complex consists of three species, the prevalence and geographical distribution of which might vary. Increasing rates of fluconazole resistance among C. parapsilosis complex were reported from various centres. Objectives: Aim of this study was to identify invasive C. parapsilosis complex strains up to species level, explore rates and molecular mechanisms of azole resistance and analyse temporal changes at a single centre. Methods: Isolates from blood cultures from 1997 to 2017 were included. Species were identified using RFLP of the SADH gene and confirmed with ITS sequencing when needed. In vitro susceptibility to fluconazole, voriconazole and posaconazole was tested and evaluated using EUCAST guidelines. Sequences of ERG11 and MRR1 genes were analysed for fluconazole non‐susceptible isolates. Results: A total of 283 isolates from 181 patients were tested for azole susceptibility. All were C. parapsilosis sensu stricto, except one C. orthopsilosis. All three azoles were effective against 213 of the isolates from 135 patients, including one C. orthopsilosis. Fluconazole resistance was 13.3% (24/181 patients). While the first fluconazole‐resistant isolates were detected in 2004, increase was evident after 2011. In ERG11, Y132F mutation was the most common among fluconazole non‐susceptible isolates (71.7%), followed by G458S (10.9%) and D421N (4.3%). In MRR1, R405K (56.5%) and G927C (8.7%) were detected. However, association of these mutations to azole resistance is yet to be investigated. Conclusions: Rising azole resistance rates in C. parapsilosis sensu stricto isolates particularly after 2011 were of concern. The well‐known Y132F mutation was the predominant mechanism of azole resistance while accompanied with other genetic mutations. [ABSTRACT FROM AUTHOR]

Candida auris is a remarkable emerging pathogen. It has emerged separately, yet simultaneously in different parts of the world, establishing four phylogenetic and geographic distinct clades with a potential fifth clade that was recently reported. C. auris is often perceived as a pathogen in disguise, due to its frequent misidentification and its immune evasion. On the other hand, many of the recovered isolates are multidrug-resistant. In fact, some of these isolates are resistant to the three main antifungal classes: echinocandins, azoles, and polyenes. Moreover, C. auris has the ability to persist and survive on different objects for a long time, aided by different adhering mechanisms including aggregation and biofilm formation, thereby causing outbreaks of invasive infections in hospital settings. However, C. auris ability to maintain its pathogenicity at high temperatures remains among its most unique properties. This is why C. auris represents a challenging threat, and more studies are needed to meet this challenge. This review highlights different characteristics of this emerging yeast with emphasis on its antifungal resistance, its ability to persistent on different surfaces, and its immune evasion capability. [ABSTRACT FROM AUTHOR]

Background: Candida vulvovaginitis is an important cause of morbidity among women. Fluconazole and other azoles are among the commonest antifungal agents used for the treatment of this condition. Azole resistance among Candida species is an increasing problem, and mutations in the ERG11 gene is the commonest cause of fluconazole resistance in Candida. The objectives of this study are to determine antifungal susceptibility of vaginal Candida isolates and detect carriage of mutant ERG11 gene by them. Methods: High vaginal swabs obtained from 260 participants were cultured on Saboraud's Dextrose agar (SDA) for isolation of Candida, and identified by growth on CHROMagar Candida, germ tube and carbohydrate fermentation tests. Antifungal susceptibility to fluconazole, voriconazole, nystatin and flucytosine was determined by the Kirby Bauer disc diffusion method on supplemented Mueller Hinton agar. ERG11 gene was detected by conventional singleplex polymerase chain reaction (PCR) assay. Results: Candida was isolated from 126 of 260 (48.5%) participants, and the identified species were Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilopsis and Candida famata. There were 112 (88.9%) isolates susceptible to fluconazole, 122 (96.8%) to voriconazole, 111 (88.1%) to nystatin, and 16 (6.6%) to flucytosine. The mutant ERG11 gene was detected in all four fluconazole-resistant isolates but not from any of five randomly selected fluconazole susceptible dose dependent (SDD) isolates. Conclusion: Azole resistance among Candida in this environment is associated with mutant ERG11 gene expression. [ABSTRACT FROM AUTHOR]

Objective: We aimed to investigate whether mutations and/or overexpressions of ERG4 and ERG11 genes were involved in drug resistance to azoles in Candida albicans. Methods: Totally, 34 clinical isolates of C. albicans were included in this study. Antimicrobial susceptibility tests, including 5-fluorocytosine (5-FC), amphotericin B (AMB), fluconazole (FCA), itraconazole (ITR), and voriconazole (VRC), were performed by broth microdilution method. Mutations in the ERG4 and ERG11 genes sequence were detected. The messenger RNA (mRNA) levels of ERG4 and ERG11 were measured by quantitative real-time polymerase chain reaction. The correlation of the expression levels of ERG4 with ERG11 genes in susceptible isolates and resistant isolates was analyzed by Pearson's correlation analysis. Results: Among 34 C. albicans isolates, 52.94%, 70.59%, and 50.00% isolates were resistant to FCA, ITR, and VRC, respectively. Sequencing results revealed that only 2 silent mutations were found in ERG4 gene, while 10 amino acid substitutions, including 6 reported previously and 4 new identified, were frequently found in ERG11 gene. The mRNA levels of ERG4 and ERG11 genes were significantly elevated in resistant compared with susceptible C. albicans isolates. Furthermore, the mRNA level of ERG4 was positively correlated with ERG11 in susceptible but not resistant C. albicans isolates. Conclusions: The resistance to azoles may be associated with the mutations in ERG11 but not ERG4 gene in C. albicans isolates. In addition, overexpressed ERG4 and ERG11 genes are found in resistant C. albicans isolates, and the mRNA levels of ERG4 may be irrelevant to ERG11 in resistant C. albicans isolates. [ABSTRACT FROM AUTHOR]

The mechanisms of azole resistance in Trichosporon asahii have not yet been fully clarified. We previously showed that T. asahii has the ERG11 gene, coding lanosterol 14-α-demethylase（Erg11 protein; Erg11p）, which is the primary target of azoles. A single amino acid substitution at G453R in Erg11p was found to induce changes in the affinity of this enzyme for azoles, especially fluconazole, in vitro. In the present study, we investigated the DNA sequences of the ERG11 gene using six different strains of clinically isolated T. asahii that were highly resistant to multi-azoles, including fluconazole, itraconazole, and voriconazole. All of the T. asahii strains had a point mutation（G448A）that caused a single amino acid substitution at G150S in Erg11p. This amino acid is highly conserved among major fungal pathogens. We identified a new point mutation in the ERG11 gene that is common to clinically isolated azole-resistant T. asahii strains, suggesting that this mutation is associated with the multi-azole resistance of T. asahii. [ABSTRACT FROM AUTHOR]

Objectives To explore the mechanisms underlying azole resistance in clinical isolates of Candida tropicalis collected in China by focusing on their efflux pumps, respiratory status and azole antifungal target enzyme. Methods Fifty-two clinical isolates of C. tropicalis were collected from five hospitals in four provinces of China and antifungal susceptibility tests were performed. Rhodamine 6G and rhodamine 123 were used to investigate the efflux pumps and respiratory status, respectively. Transporter-related genes CDR1 and MDR1, mitochondrial gene CYTb, as well as ERG11, were quantified by real-time RT–PCR. Meanwhile, ergosterol content was analysed using liquid chromatography-mass spectrometry/mass spectrometry. An ERG11-deficient (erg11Δ) Saccharomyces cerevisiae strain was generated to study the function of mutations in ERG11. Results MICs showed that 31 isolates were resistant to at least one type of azole antifungal. Flow cytometry using rhodamine 123 revealed increased respiration for the azole-resistant isolates, but CYTb was not overexpressed. No significant difference in the efflux of rhodamine 6G was found, which was consistent with the comparable expression levels of CDR1 and MDR1. In contrast, the azole-resistant isolates overexpressed ERG11 and showed increased ergosterol content. Moreover, the isolates resistant to three azole antifungals expressed higher levels of ERG11 mRNA than those resistant to only fluconazole or itraconazole. Two ERG11 mutations, Y132F and S154F, were found in azole-resistant isolates and could be shown to mediate azole resistance by expression in S. cerevisiae. Conclusions The up-regulation and mutations of ERG11 mediate azole resistance of C. tropicalis. [ABSTRACT FROM AUTHOR]

Lanosterol 14-α-demethylase ( Erg11 protein; Erg11p), encoded by the ERG11 gene, is the primary target of azoles. Recently, a change in affinity of this enzyme for azoles has been reported as a resistance mechanism in several fungal species. Trichosporon asahii ( T. asahii) is susceptible to fluconazole ( FLC). This report identified the ERG11 gene of T. asahii ( NCBI accession; ). The Erg11p of T. asahii, presumed from the DNA sequence, was closely related to the Erg11p of Cryptococcus neoformans. Furthermore, a FLC-susceptible strain was cultured in medium containing FLC at concentrations from 4.0 to 16 μg mL−1 in order to analyze the development of FLC resistance in T. asahii. The degree of resistance was related to the FLC concentration of the growth medium. One highly resistant strain that was cultured in the medium containing 16 μg mL−1 FLC contained 1 point mutation ( G1357 C) that caused a single amino acid substitution at G453 R. This amino acid is highly conserved among major fungal pathogens, and it is in a region very close to the heme-binding domain, which is characteristic of the cytochrome P450 superfamily, the primary target for the azole class of antifungal agents. This amino acid substitution may have caused the high resistance to azoles in T. asahii. [ABSTRACT FROM AUTHOR]

Azoles have been applied widely to combat pathogenic fungi in medicine and agriculture and, consequently, loss of efficacy has occurred in populations of some species. Often, but not always, resistance was found to result from amino acid substitutions in the molecular target of azoles, 14α-sterol demethylase (CYP51 syn. ERG11). This review summarizes CYP51 function, evolution, and structure. Furthermore, we compare the occurrence and contribution of CYP51 substitutions to azole resistance in clinical and field isolates of important fungal pathogens. Although no crystal structure is available yet for any fungal CYP51, homology modeling using structures from other origins as template allowed deducing models for fungal orthologs. These models served to map amino acid changes known from clinical and field isolates. We conclude with describing the potential consequences of these changes on the topology of the protein to explain CYP51-based azole resistance. Knowledge gained from molecular modeling and resistance research will help to develop novel azole structures. [ABSTRACT FROM AUTHOR]

Azole resistance in Aspergillus fumigatus isolates is increasingly reported in different nosologic contexts with variable prevalence in different countries. Mutations in the target of triazoles are widely described in azole-resistant clinical isolates. The recovery of mutated/resistant isolates is described either in patients undergoing long-term azole treatment or after inhalation of environmentally acquired resistant isolates. Acquisition in patients during azole therapy highlights the capacity of this fungus to adapt to its environment, but it has a low impact in terms of public health, as interhuman transmission of A. fumigatus is uncommon. Environmentally acquired resistant isolates may propagate and affect populations at risk. The use of triazoles as first-line therapy or prophylaxis could lead to selection of resistant isolates in patients, because most isolates harbor azole cross-resistance. Although mold-active triazoles have provided major progress in the prophylaxis and treatment of Aspergillus infection, the increase of azole resistance could question their use in humans. [ABSTRACT FROM AUTHOR]

Abstract: For several years, azole antifungal drugs have been a treatment option for potentially life-threatening Candida infections. However, azole resistance can occur through various mechanisms such as alterations in ERG11, encoding lanosterol 14α-demethylase (CYP51). In this study, we investigated the antifungal susceptibility to fluconazole, itraconazole, and voriconazole of 73 clinical isolates of Candida albicans. Screening for amino acid substitutions in Erg11 was performed on each of the 73 isolates. Twenty isolates displayed a marked decrease in azole susceptibility. Amino acid substitutions were detected in more than two-thirds of the strains. In all, 23 distinct substitutions were identified. Four have not been described previously, among which N136Y and Y447H are suspected to be involved in azole resistance. We suggest that the high genetic polymorphism of ERG11 must be considered in the rationale design of new azole compounds targeting lanosterol 14α-demethylase. A review of all Erg11 amino acid polymorphisms described to date is given. [Copyright &y& Elsevier]

Aims: To clarify the underlying synergistic antifungal mechanisms of retigeric acid B (RAB) in combination with azoles against Candida albicans. Methods and Results: Increased accumulation of rhodamine 123 in C. albicans was measured by both spectrophotometric method and flow cytometry. The inhibitory properties to the drug efflux of C. albicans were determined spectrophotometrically. The decreased cellular ergosterol synthesis was measured using its unique spectrophotometric absorbance profile, and the downregulation expression levels of CDR1 and ERG11 were detected by real-time reverse transcription polymerase chain reaction. Transmission electron microscopy investigation found the wrinkled cell membrane and the impaired cell wall. Conclusions: RAB synergizes the antifungal effect of azoles against C. albicans by inhibiting efflux pump activity, targeting the ergosterol biosynthesis pathway and increasing the fluidity for the resulted ergosterol depletion. Significance and Impact of the Study:  Investigating the mechanism of the synergy between RAB and azoles against C. albicans will help us to uncover the antifungal roles of this lichen-derived triterpene acid and find its possible clinical applications in overcoming fungal resistance. [ABSTRACT FROM AUTHOR]

Abstract: Candida glabrata is an important human pathogen that is naturally less susceptible to antimycotics compared with Candida albicans. Ten unmatched C. glabrata clinical isolates were selected from a collection of isolates exhibiting decreased susceptibilities to azole antifungals. Overexpression of the CgPDR1 gene, encoding the main multidrug resistance transcription factor, and its target genes CgCDR1 and CgCDR2, coding for drug efflux transporters, was observed in six fluconazole-resistant isolates. Sequence analysis of the polymerase chain reaction (PCR)-amplified DNA fragments of each isolate''s CgPDR1 gene was used to identify two novel L347F and H576Y mutations in CgPdr1p. These proved to be responsible for fluconazole resistance in transformants of the C. glabrata pdr1Δ mutant strain. Five isolates harbouring the H576Y mutation also contained the mutation E502V in CgErg11p 14C-lanosterol-demethylase. Heterologous expression of the CgERG11 mutant allele did not provide evidence for its involvement in azole resistance. In four fluconazole-sensitive isolates that were itraconazole-resistant, slightly enhanced CgCDR2 expression was observed. No upregulation of the CgERG11 gene was observed in any of the ten isolates. The results demonstrate that decreased susceptibilities of C. glabrata clinical isolates to azole antifungals mainly results from gain-of-function mutations in the gene encoding the CgPdr1p transcription factor. [Copyright &y& Elsevier]

Background: One of the mechanisms for resistance to azole antifungal drugs used in C. albicans therapy is the contribution of overexpression and point mutations in ERG11 gene that codes 14mα-demethylase (14DM) which is the primary target for the azole class of antifungals. These mutations can cause amino acids substitutions in the enzyme and alteration of 14DM sequence may lead to a decreased affinity of the enzyme for azoles. Aim: The purpose of this study was to detect genetic differences present in collection of C. albicans isolates demonstrating resistance and sensitivity to azoles. Material and methods: C. albicans strains used in this study were isolated from clinical samples obtained from hospitalized patients. To identify such alterations ERG11 gene was investigated by MSSCP technique (Multitemperature Single Strand Conformation Polymorphism). Results and conclusions: MSSCP analysis revealed the presence of genetic differences in ERG11 gene in analysed C. albicans strains. [ABSTRACT FROM AUTHOR]