Development Of Azole Resistance Research Articles

UPC2 mutations and development of azole resistance in Candida albicans hospital isolates from Lebanon

ObjectivesThis study evaluated the role of Upc2 in the development of azole resistance in Candida albicans isolates from Lebanese hospitalized patients and determined a correlation between resistance and virulence. MethodsThe UPC2 gene which codes for an ergosterol biosynthesis regulator was sequenced and analysed in two azole-resistant and one azole-susceptible C. albicans isolates. An amino acid substitution screening was carried out on Upc2 with a focus on its ligand binding domain (LBD) known to interact with ergosterol. Then, Upc2 protein secondary structure prediction and homology modelling were conducted, followed by total plasma membrane ergosterol and cell wall chitin quantifications. For virulence, mouse models of systemic infection were generated and an agar adhesion and invasion test was performed. ResultsAzole-resistant isolates harboured novel amino acid substitutions in the LBD of Upc2 and changes in protein secondary structures were observed. In addition, these isolates exhibited a significant increase in plasma membrane ergosterol content. Resistance and virulence were inversely correlated while increased cell wall chitin concentration does not seem to be linked to resistance since even though we observed an increase in chitin concentration, it was not statistically significant. ConclusionsThe azole-resistant C. albicans isolates harboured novel amino acid substitutions in the LBD of Upc2 which are speculated to induce an increase in plasma membrane ergosterol content, preventing the binding of azoles to their target, resulting in resistance.

Azole resistance in Aspergillus fumigatus- comprehensive review.

Aspergillus fumigatus is a ubiquitous filamentous fungus commonly found in the environment. It is also an opportunistic human pathogen known to cause a range of respiratory infections, such as invasive aspergillosis, particularly in immunocompromised individuals. Azole antifungal agents are widely used for the treatment and prophylaxis of Aspergillus infections due to their efficacy and tolerability. However, the emergence of azole resistance in A. fumigatus has become a major concern in recent years due to their association with increased treatment failures and mortality rates. The development of azole resistance in A. fumigatus can occur through both acquired and intrinsic mechanisms. Acquired resistance typically arises from mutations in the target enzyme, lanosterol 14-α-demethylase (Cyp51A), reduces the affinity of azole antifungal agents for the enzyme, rendering them less effective, while intrinsic resistance refers to a natural resistance of certain A. fumigatus isolates to azole antifungals due to inherent genetic characteristics. The current review aims to provide a comprehensive overview of azole antifungal resistance in A. fumigatus, discusses underlying resistance mechanisms, including alterations in the target enzyme, Cyp51A, and the involvement of efflux pumps in drug efflux. Impact of azole fungicide uses in the environment and the spread of resistant strains is also explored.

Hotspot mutations and genomic expansion of ERG11 are major mechanisms of azole resistance in environmental and human commensal isolates of Candida tropicalis

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. In this study, we performed biological and genomic analyses of 239 C. tropicalis strains, including 115 environmental and 124 human commensal isolates. 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). 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.

TRIAZOLE RESISTANCE IN ASPERGILLUS FUMIGATUS-A COMPREHENSIVE REVIEW

Aspergillus fumigatus is a common ubiquitous fungal pathogen present in the environment including soil, decaying leaves, compost, grain crops and household dust. It can turn opportunistic in immunocompromised individuals causing allergic diseases, respiratory problems and invasive blood stream infections. Being saprophytic, it propagates by producing minute grey-green conidia that can become airborne and reach humans. In majority of cases, aspergillosis due to this organism can heal with treatment by antifungals. The azole compounds, one of the most important antifungal drugs, generally target the 14-α sterol demethylase enzyme, encoded by the cyp51A gene of the fungus, causing impaired membrane ergosterol synthesis. However in recent years the situation has turned grave due to development of azole resistant fungal species both in the environment as well as in the clinics. The main mechanism of resistance in A. fumigatus involves incorporation of tandem repeats in the promoter region of cyp51A, along with point mutations in the gene sequence. Resistant isolates tend to arise either due to excessive use of azole fungicides in the agricultural fields, from where they reach human through sporulation; or by prolonged treatment of patients with azole drugs. The early diagnosis of aspergillosis is very crucial for successful treatment of a patient. The article brings forth a comprehensive study of this organism, the mechanism of development and spread of azole resistant fungal isolates, genetic basis of development of azole resistance, detection of resistant strains and finally drugs used to combat the fungal infection.

TRIAZOLE RESISTANCE IN ASPERGILLUS FUMIGATUS-A COMPREHENSIVE REVIEW

Aspergillus fumigatus is a common ubiquitous fungal pathogen present in the environment including soil, decaying leaves, compost, grain crops and household dust. It can turn opportunistic in immunocompromised individuals causing allergic diseases, respiratory problems and invasive blood stream infections. Being saprophytic, it propagates by producing minute grey-green conidia that can become airborne and reach humans. In majority of cases, aspergillosis due to this organism can heal with treatment by antifungals. The azole compounds, one of the most important antifungal drugs, generally target the 14-α sterol demethylase enzyme, encoded by the cyp51A gene of the fungus, causing impaired membrane ergosterol synthesis. However in recent years the situation has turned grave due to development of azole resistant fungal species both in the environment as well as in the clinics. The main mechanism of resistance in A. fumigatus involves incorporation of tandem repeats in the promoter region of cyp51A, along with point mutations in the gene sequence. Resistant isolates tend to arise either due to excessive use of azole fungicides in the agricultural fields, from where they reach human through sporulation; or by prolonged treatment of patients with azole drugs. The early diagnosis of aspergillosis is very crucial for successful treatment of a patient. The article brings forth a comprehensive study of this organism, the mechanism of development and spread of azole resistant fungal isolates, genetic basis of development of azole resistance, detection of resistant strains and finally drugs used to combat the fungal infection.

Azole-Resistance Development; How the Aspergillus fumigatus Lifecycle Defines the Potential for Adaptation.

In order to successfully infect or colonize human hosts or survive changing environments, Aspergillus fumigatus needs to adapt through genetic changes or phenotypic plasticity. The genomic changes are based on the capacity of the fungus to produce genetic variation, followed by selection of the genotypes that are most fit to the new environment. Much scientific work has focused on the metabolic plasticity, biofilm formation or the particular genetic changes themselves leading to adaptation, such as antifungal resistance in the host. Recent scientific work has shown advances made in understanding the natural relevance of parasex and how both the asexual and sexual reproduction can lead to tandem repeat elongation in the target gene of the azoles: the cyp51A gene. In this review, we will explain how the fungus can generate genetic variation that can lead to adaptation. We will discuss recent advances that have been made in the understanding of the lifecycle of A. fumigatus to explain the differences observed in speed and type of mutations that are generated under different environments and how this can facilitate adaptation, such as azole-resistance selection.

Molecular Mechanisms of Azole Resistance in Four Clinical Candida albicans Isolates.

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-ERG328I 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.

The Multi-Fungicide Resistance Status of Aspergillus fumigatus Populations in Arable Soils and the Wider European Environment.

The evolution and spread of pan-azole resistance alleles in clinical and environmental isolates of Aspergillus fumigatus is a global human health concern. The identification of hotspots for azole resistance development in the wider environment can inform optimal measures to counteract further spread by minimizing exposure to azole fungicides and reducing inoculum build-up and pathogen dispersal. We investigated the fungicide sensitivity status of soil populations sampled from arable crops and the wider environment and compared these with urban airborne populations. Low levels of azole resistance were observed for isolates carrying the CYP51A variant F46Y/M172V/E427K, all belonging to a cluster of related cell surface protein (CSP) types which included t07, t08, t13, t15, t19, and t02B, a new allele. High levels of resistance were found in soil isolates carrying CYP51A variants TR34/L98H and TR46/Y121F/T289A, all belonging to CSP types t01, t02, t04B, or t11. TR46/Y121F/M172V/T289A/G448S (CSP t01) and TR46/Y121F/T289A/S363P/I364V/G448S (CSP t01), a new haplotype associated with high levels of resistance, were isolated from Dutch urban air samples, indicating azole resistance evolution is ongoing. Based on low numbers of pan-azole resistant isolates and lack of new genotypes in soils of fungicide-treated commercial and experimental wheat crops, we consider arable crop production as a coldspot for azole resistance development, in contrast to previously reported flower bulb waste heaps. This study also shows that, in addition to azole resistance, several lineages of A. fumigatus carrying TR-based CYP51A variants have also developed acquired resistance to methyl benzimidazole carbamate, quinone outside inhibitor and succinate dehydrogenase (Sdh) inhibitor fungicides through target-site alterations in the corresponding fungicide target proteins; beta-tubulin (F200Y), cytochrome b (G143A), and Sdh subunit B (H270Y and H270R), respectively. Molecular typing showed that several multi-fungicide resistant strains found in agricultural soils in this study were clonal as identical isolates have been found earlier in the environment and/or in patients. Further research on the spread of different fungicide-resistant alleles from the wider environment to patients and vice versa can inform optimal practices to tackle the further spread of antifungal resistance in A. fumigatus populations and to safeguard the efficacy of azoles for future treatment of invasive aspergillosis.

Triazole Evolution of Candida parapsilosis Results in Cross-Resistance to Other Antifungal Drugs, Influences Stress Responses, and Alters Virulence in an Antifungal Drug-Dependent Manner.

The number of invasive infections caused by Candida species is increasing worldwide. The incidence of candidiasis cases caused by non-albicans Candida species, such as Candida parapsilosis, is also increasing, and non-albicans Candida species are currently responsible for more invasive infections than C. albicans Additionally, while the development of azole resistance during invasive disease with C. albicans remains uncommon, azole-resistant C. parapsilosis strains are frequently isolated in the hospital setting. In this study, we applied direct selection to generate azole-adapted and azole-evolved C. parapsilosis strains in order to examine the effect of azole resistance development on fungal viability and pathogenesis progression. Depending on the drug applied, the different evolved strains developed distinct cross-resistance patterns: the fluconazole-evolved (FLUEVO) and voriconazole-evolved (VOREVO) strains gained resistance to fluconazole and voriconazole only, while posaconazole evolution resulted in cross-resistance to all azoles and the posaconazole-evolved (POSEVO) strains showed higher echinocandin MIC values than the FLUEVO and VOREVO strains. Whole-genome sequencing results identified the development of different resistance mechanisms in the evolved strains: the FLUEVO and VOREVO strains harbored amino acid substitutions in Mrr1p (A808T and N394Y, respectively), and the POSEVO strain harbored an amino acid change in Erg3p (D14Y). By revealing increased efflux pump activity in both the FLUEVO and the VOREVO strains, along with the altered sterol composition of the POSEVO strain, we now highlight the impact of the above-mentioned amino acid changes in C. parapsilosis azole resistance development. We further revealed that the virulence of this species was only slightly or partially affected by fluconazole and voriconazole adaptation, while it significantly decreased after posaconazole adaptation. Our results suggest that triazole adaptation can result in azole cross-resistance and that this process may also result in virulence alterations in C. parapsilosis, depending on the applied drug.IMPORTANCECandida parapsilosis causes life-threatening fungal infections. In the last 2 decades, the increasing number of azole-resistant C. parapsilosis clinical isolates has been attributable to the overuse and misuse of fluconazole, the first-line antifungal agent most commonly used in several countries. To date, the range of applicable antifungal drugs is limited. As a consequence, it is essential to understand the possible mechanisms of antifungal resistance development and their effect on virulence in order to optimize antifungal treatment strategies in the clinical setting. Our results revealed that the prolonged exposure to azoles resulted not only in azole resistance but also in cross-resistance development. Our data further indicate that resistance development may occur through different mechanisms that can also alter the virulence of C. parapsilosis These results highlight the consequences of prolonged drug usage and suggest the need for developing alternative antifungal treatment strategies in clinical practice.

Parasexual recombination enablesAspergillus fumigatusto persist in cystic fibrosis

Aspergillus fumigatus is a saprobic fungus that causes a range of pulmonary diseases, some of which are characterised by fungal persistence such as is observed in cystic fibrosis (CF) patients. Creation of genetic variation is critical for A. fumigatus to adapt to the lung environment, but biofilm formation, especially in CF patients, may preclude mutational supply in A. fumigatus due to its confinement to the hyphal morphotype. We tested our hypothesis that genetic variation is created through parasexual recombination in chronic biofilms by phenotypic and genetic analysis of A. fumigatus isolates cultured from different origins.As diploids are the hallmark of parasex, we screened 799 A. fumigatus isolates obtained from patients with CF, chronic pulmonary lung disease and acute invasive aspergillosis, and from the environment for spore size. Benomyl sensitivity, nuclear content measurements through fluorescence-activated cell sorting and scanning electron microscopy were used to confirm the diploid state of large size spores. Whole genome sequencing was used to characterise diploid-associated genetic variation.We identified 11 diploids in isolates recovered from six of 11 (55%) CF patients and from one of 24 (4%) chronic aspergillosis patients, but not in 368 isolates from patients with acute Aspergillus infection and the environment. Diploid formation was associated with accumulation of mutations and variable haploid offspring including a voriconazole-resistant isolate.Parasexual recombination allows A. fumigatus to adapt and persist in CF patients, and plays a role in azole resistance development. Our findings are highly significant for understanding the genetics and biology of A. fumigatus in the human lung.

Dynamics of in vitro development of azole resistance in Candida tropicalis

ObjectivesIncreasing incidence of azole resistance in Candida tropicalis, especially to fluconazole, has been seen in Asian countries including India. Limited knowledge is available on the molecular mechanisms associated with the development of azole resistance in C. tropicalis. The present study examined the dynamics of in vitro azole resistance in C. tropicalis after prolonged treatment with fluconazole. MethodsNine fluconazole-susceptible isolates of C. tropicalis were used in this study. Fluconazole resistance was induced experimentally in C. tropicalis isolates. The stability of induced resistance and cross-resistance to other azoles was examined. The molecular mechanisms of azole resistance were assessed by measuring the expression and mutation analysis of different genes. ResultsVarying degrees of resistance [five with minimum inhibitory concentrations (MICs) ≤32 mg/L and four with MICs ≥128 mg/L] were noticed, and the resistance was developed in 3 months. Of the nine resistant isolates, four induced resistant isolates with MICs ≥128 mg/L presented temporal resistance stability up to 10 subcultures. These four isolates presented cross-resistance to other azoles and also an inducible overexpression of transporters (CDR1, CDR2, CDR3 and MDR1), ergosterol biosynthesis pathway genes (ERG1, ERG2, ERG3 and ERG11), transcription factors (TAC1 and UPC2) and stress-responsive genes (HSP90 and MKC1) was noticed. No mutations were seen in any of the four genes (ERG1, ERG3, ERG11 and UPC2) tested. ConclusionsCandida tropicalis isolates adapt themselves in the presence of continuous drug exposure and switch back to being susceptible in the absence of the drug. The acquisition of resistance in C. tropicalis is mediated by the overexpression of different resistance-related genes without any molecular alterations.

In Vivo Selection of a Unique Tandem Repeat Mediated Azole Resistance Mechanism (TR120) inAspergillus fumigatus cyp51A, Denmark

We report a fatal aspergillosis case in which STRAf typing and whole-genome sequencing substantiated in vivo emergence of an azole-resistant Aspergillus fumigatus with a 120-bp tandem repeat in the promoter region of cyp51A. This event, previously restricted to the environment, challenges current understanding of azole resistance development in A. fumigatus.

Azole-resistant Aspergillus fumigatus in the Italian environment.

Azole resistance in Aspergillus fumigatus environmental and clinical isolates is recognised as an emerging problem worldwide. Development of azole resistance may be environmentally driven because of the massive use of azole fungicides in agriculture. The mechanism of azole resistance is mostly related to mutations in the cyp51A gene. A. fumigatus azole resistance in the environment was previously documented in northern Italy. This study extended the research in the agricultural environment also in central and southern Italy and investigated differences in the Italian geographical areas and in the different types of crops. A total of 177 samples (173 soil samples and 4 Dutch bulbs) collected in the period 2014-20117 in 14 Italian regions were analysed. Itraconazole-resistant A. fumigatus isolates grew in 16.9% of the screened samples. Differences were observed in soil samples from the three Italian geographic areas: 12.5% in the north, 15.2% in the centre and 24.1% in the south. Resistant isolates were from different cultivations, treated or officially not treated with azole fungicides. Sequencing of the cyp51A gene confirmed that resistance was mainly associated with the TR34/L98H mutation (29/30 isolates); 1 isolate showed the G54E mutation. The risk for patients to acquire multi-azole-resistant strains from the environment could have a serious impact on the management of life-threatening invasive infections. The azole resistance rate of 16.9% found in Italy requires suitable monitoring of antifungal susceptibility of clinical isolates.

Negative regulation of Candida glabrata Pdr1 by the deubiquitinase subunit Bre5 occurs in a ubiquitin independent manner

The primary route for development of azole resistance in the fungal pathogen Candida glabrata is acquisition of a point mutation in the PDR1 gene. This locus encodes a transcription factor that upon mutation drives high level expression of a range of genes including the ATP-binding cassette transporter-encoding gene CDR1. Pdr1 activity is also elevated in cells that lack the mitochondrial genome (ρ° cells), with associated high expression of CDR1 driving azole resistance. To gain insight into the mechanisms controlling activity of Pdr1, we expressed a tandem affinity purification (TAP)-tagged form of Pdr1 in both wild-type (ρ+ ) and ρ° cells. Purified proteins were analyzed by multidimensional protein identification technology mass spectrometry identifying a protein called Bre5 as a factor that co-purified with TAP-Pdr1. In Saccharomyces cerevisiae, Bre5 is part of a deubiquitinase complex formed by association with the ubiquitin-specific protease Ubp3. Genetic analyses in C. glabrata revealed that loss of BRE5, but not UBP3, led to an increase in expression of PDR1 and CDR1 at the transcriptional level. These studies support the view that Bre5 acts as a negative regulator of Pdr1 transcriptional activity and behaves as a C. glabrata-specific modulator of azole resistance.

CoERG11 A395T mutation confers azole resistance in Candida orthopsilosis clinical isolates

Candida orthopsilosis is a human fungal pathogen responsible for a wide spectrum of symptomatic infections. Evidence suggests that C. orthopsilosis is mainly susceptible to azoles, the most extensively used antifungals for treatment of these infections. However, fluconazole-resistant clinical isolates are reported. This study evaluated the contribution of a single amino acid substitution in the azole target CoErg11 to the development of azole resistance in C. orthopsilosis. C. orthopsilosis clinical isolates (n = 40) were tested for their susceptibility to azoles and their CoERG11 genes were sequenced. We used a SAT1 flipper-driven transformation to integrate a mutated CoERG11 allele in the genetic background of a fluconazole-susceptible isolate. Susceptibility testing revealed that 16 of 40 C. orthopsilosis clinical isolates were resistant to fluconazole and to at least one other azole. We identified an A395T mutation in the CoERG11 coding sequence of azole-resistant isolates only that resulted in the non-synonymous amino acid substitution Y132F. The SAT1 flipper cassette strategy led to the creation of C. orthopsilosis mutants that carried the A395T mutation in one or both CoERG11 alleles (heterozygous or homozygous mutant, respectively) in an azole-susceptible genetic background. We tested mutant strains for azole susceptibility and for hot-spot locus heterozygosity. Both the heterozygous and the homozygous mutant strains exhibited an azole-resistant phenotype. To the best of our knowledge, these findings provide the first evidence that the CoErg11 Y132F substitution confers multi-azole resistance in C. orthopsilosis.

In-host microevolution of Aspergillus fumigatus: A phenotypic and genotypic analysis

In order to survive, Aspergillus fumigatus must adapt to specific niche environments. Adaptation to the human host includes modifications facilitating persistent colonisation and the development of azole resistance. The aim of this study is to advance understanding of the genetic and physiological adaptation of A. fumigatus in patients during infection and treatment. Thirteen A. fumigatus strains were isolated from a single chronic granulomatous disease patient suffering from persistent and recurrent invasive aspergillosis over a period of 2 years. All strains had identical microsatellite genotypes and were considered isogenic. Whole genome comparisons identified 248 non-synonymous single nucleotide polymorphisms. These non-synonymous mutations have potential to play a role in in-host adaptation. The first 2 strains isolated were azole susceptible, whereas later isolates were itraconazole, voriconazole and/or posaconazole resistant. Growth assays in the presence and absence of various antifungal stressors highlighted minor changes in growth rate and stress resistance, with exception of one isolate showing a significant growth defect. Poor conidiation was observed in later isolates. In certain drug resistant isolates conidiation was restored in the presence of itraconazole. Differences in virulence were observed as demonstrated in a Galleria mellonella infection model. We conclude that the microevolution of A. fumigatus in this patient has driven the emergence of both Cyp51A-independent and Cyp51A-dependent, azole resistance mechanisms, and additional phenotypes that are likely to have promoted fungal persistence.

Mutations in Hotspot Regions of ERG11 Gene in Fluconazole Resistant Isolates of Candida albicans in Guilan Province, Northern Iran

Widespread use of azoles has resulted in rapid development of azole resistance in Candida albicans strains. Mutations in ERG11, a target enzyme of azoles, alter the binding ability of azoles to this enzyme and result in the development of resistant strains. In this study, we evaluated ERG11 mutations in fluconazole resistant isolates of C. albicans. In this study, 60 clinical samples were isolated from Guilan hospitals. Then differential tests were used to identify C. albicans strains. Disc diffusion and MIC tests were used to the analyze fluconazole susceptibility. Then, the resistant isolates were evaluated by PCR and sequencing methods for ERG11 mutations. Of 60 clinical samples, 40 C. albicans strains were identified through specific symptoms. Susceptibility tests showed that four C. albicans strains were resistant to high dose fluconazole (≥512 μg/mL). In all resistant isolates was found missense mutations such as K291N, C470G and Q474R and three isolates had premature nonsense mutation (Y477stop). Our study indicates that the level of fluconazole resistance in C. albicans strains is high in Guilan province and other drugs should be used in resistant infections. It seems that missense mutations in four isolates play role in azole resistance. However in three isolates premature stop codon may be involved in high dose resistance. And it is suggested that in fourth isolates another mechanisms introduce increase of resistance dose in combination with missense mutation in ERG11. Results of this study suggest that in patients by high dose of resistance do not use azole because of mutations that decrease azole effects.

Triazole Resistance in Aspergillus Species: An Emerging Problem

Aspergillus species are ubiquitous fungal saprophytes found in diverse ecological niches worldwide. Among them, Aspergillus fumigatus is the most prevalent and is largely responsible for the increased incidence of invasive aspergillosis with high mortality rates in some immunocompromised hosts. Azoles are the first-line drugs in treating diseases caused by Aspergillus spp. However, increasing reports in A. fumigatus azole resistance, both in the clinical setting and in the environment, are threatening the effectiveness of clinical and agricultural azole drugs. The azole target is the 14-α sterol demethylase encoded by cyp51A gene and the main mechanisms of resistance involve the integration of tandem repeats in its promoter and/or single point mutations in this gene. In A. fumigatus, azole resistance can emerge in two different scenarios: a medical route in which azole resistance is generated during long periods of azole treatment in the clinical setting and a route of resistance derived from environmental origin due to extended use of demethylation inhibitors in agriculture. The understanding of A. fumigatus azole resistance development and its evolution is needed in order to prevent or minimize its impact. In this article, we review the current situation of azole resistance epidemiology and the predominant molecular mechanisms described based on the resistance acquisition routes. In addition, the clinical implications of A. fumigatus azole resistance and future research are discussed.

Amino acid substitutions in Erg11p of azole-resistant Candida glabrata: Possible effective substitutions and homology modelling.

Understanding the mechanisms responsible for fluconazole resistance in Candida glabrata is not only crucial for the development of new antifungals but is also important in choosing appropriate antifungals for patients at the earliest stages. The aim of this study was to determine the Erg11p amino acid substitutions in fluconazole-resistant C. glabrata isolates. Sixty clinical isolates of C. glabrata were investigated. In vitro antifungal activities of fluconazole, itraconazole and voriconazole were determined using the broth microdilution reference method. The ERG11 gene for resistant (n=4) and susceptible (n=1) isolates were sequenced and multi-aligned using MEGA6 software. A homology model of the C. glabrata ERG11 gene was created by SWISS-MODEL software using the crystal structure of Saccharomyces cerevisiae Erg11p as a template, and the predicted binding sites to fluconazole were investigated. Fluconazole and multi-azole resistance were observed in 6.7% and 3.3% of the isolates, respectively. Several amino acid substitutions were identified, among which some were also identified in susceptible isolates. The amino acid substitution G236V was at the binding site, and substitutions H146Q and D234E were near to the binding site of triazoles according to the SWISS-MODEL. According to the homology modelling results, the amino acid substitution G236V is highly likely to play a key role in azole resistance development.

The Rpd3/Hda1 family of histone deacetylases regulates azole resistance in Candida albicans.

The histone deacetylase (HDAC) has recently been linked to the morphogenesis and virulence of yeast. However, the effects of HDAC on antifungal susceptibility are not well understood. We sought to characterize the action of histone deacetylation on azole resistance in Candida albicans and its possible mechanism of action. A total of 40 C. albicans strains were studied. Azole susceptibility with or without trichostatin A (TSA) was determined according to the CLSI microdilution method. The null mutants of HDA1 and RPD3 (genes targeted by TSA) were also investigated using drop-plate assays and a rapid acquisition of adaptation to the azole test. Transcriptional levels of HDAC genes and efflux genes were quantified using RT-PCR for both the basal and fluconazole-induced conditions. The inhibition of HDACs by TSA (0.25 mg/L) markedly reduced the trailing growth and the growth of most C. albicans strains. Trailing growth for C. albicans strains was decreased from 2-fold to 256-fold at 48 h. The deletion of HDA1 or RPD3 increased the susceptibility to azoles compared with the WT strain. The expression of HDA1 and RPD3 was up-regulated to different levels, and returned to the level of the susceptible parental strain when stable resistance had formed during the course of acquired fluconazole resistance both in vitro and in vivo. Efflux genes were poorly expressed in mutant strains compared with those of the WT strain. Our results indicate the important role of the Rpd3/Hda1 family in the development of azole resistance in C. albicans. Histone deacetylation may govern the expression of genes related to the early stages of adaptation to azole stress, such as efflux pump genes.