Mechanisms Of Azole Resistance Research Articles

High-Resolution Melting Assay to Detect the Mutations That Cause the Y132F and G458S Substitutions at the ERG11 Gene Involved in Azole Resistance in Candida parapsilosis.

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. 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). 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. The designed oligonucleotides to detect the Erg11Y132F and Erg11G458S 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. This technique offers a new method for the early detection of azole resistance mechanism in C. parapsilosis.

Exploration of novel mechanisms of azole resistance in Candida auris.

Candida auris is a pathogenic yeast of particular concern because of its ability to cause nosocomial outbreaks of invasive candidiasis (IC) and to develop resistance to all current antifungal drug classes. Most C. auris clinical isolates are resistant to fluconazole, an azole drug that is used for the treatment of IC. Azole resistance may arise from diverse mechanisms, such as mutations of the target gene (ERG11) or upregulation of efflux pumps via gain of function mutations of the transcription factors TAC1 and/or MRR1. To explore novel mechanisms of azole resistance in C. auris, we applied an in vitro evolutionary protocol to induce azole resistance in a TAC1A/TAC1B/MRR1 triple-deletion strain. Azole-resistant isolates without ERG11 mutations were further analyzed. In addition to a whole chromosome aneuploidy of chromosome 5, amino acid substitutions were recovered in the transcription factor Upc2 (N592S, L499F), the ubiquitin ligase complex consisting of Ubr2 (P708T, H1275P) and Mub1 (Y765*), and the mitochondrial protein Mrs7 (D293H). Genetic introduction of these mutations in an azole-susceptible wild-type C. auris isolate of clade IV resulted in significantly decreased azole susceptibility. Real-time reverse transcription PCR analyses were performed to assess the impact of these mutations on the expression of genes involved in azole resistance, such as ERG11, the efflux pumps CDR1 and MDR1 or the transcription factor RPN4. In conclusion, this work provides further insights in the complex and multiple pathways of azole resistance of C. auris. Further analyses would be warranted to assess their respective role in azole resistance of clinical isolates.

Candida glabrata maintains two HAP1 ohnologs, HAP1A and HAP1B, for distinct roles in ergosterol gene regulation to mediate sterol homeostasis under azole and hypoxic conditions.

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.

A ubiquitin-mediated post-translational degradation of Cyp51A contributes to a novel azole resistance mode in Aspergillus fumigatus

The airborne fungus Aspergillus fumigatus is a major pathogen that poses a serious health threat to humans by causing aspergillosis. Azole antifungals inhibit sterol 14-demethylase (encoded by cyp51A), an enzyme crucial for fungal cell survival. However, the most common mechanism of azole resistance in A. fumigatus is associated with the mutations in cyp51A and tandem repeats in its promoter, leading to reduced drug-enzyme interaction and overexpression of cyp51A. It remains unknown whether post-translational modifications of Cyp51A contribute to azole resistance. In this study, we report that the Cyp51A expression is highly induced upon exposure to itraconazole, while its ubiquitination level is significantly reduced by itraconazole. Loss of the ubiquitin-conjugating enzyme Ubc7 confers resistance to multiple azole antifungals but hinders hyphal growth, conidiation, and virulence. Western blot and immunoprecipitation assays show that deletion of ubc7 reduces Cyp51A degradation by impairing its ubiquitination, thereby leading to drug resistance. Most importantly, the overexpression of ubc7 in common environmental and clinical azole-resistant cyp51A isolates partially restores azole sensitivity. Our findings demonstrate a non-cyp51A mutation-based resistance mechanism and uncover a novel role of post-translational modification in contributing to azole resistance in A. fumigatus.

Mechanism of azole resistance in Candida vulturna, an emerging multidrug resistant pathogen related with Candida haeumulonii and Candida auris.

Candida vulturna is an emerging pathogen belonging to the Metshnikowiaceae family together with Candida auris and Candida haemulonii species complex. Some strains of this species were reported to be resistant to several antifungal agents. This study aims to address identification difficulties, evaluate antiungal susceptibilities and explore the molecular mechanisms of azole resistance of Candida vulturna. We studied five C. vulturna clinical strains isolated in three Colombian cities. Identification was performed by phenotypical, proteomic and molecular methods. Antifungal susceptibility testing was performed following CLSI protocol. Its ERG11 genes were sequenced and a substitution was encountered in azole resistant isolates. To confirm the role of this substitution in the resistance phenotype, Saccharomyces cerevisiae strains with a chimeric ERG11 gene were created. Discrepancies in identification methods are highlighted. Sequencing confirmed the identification as C. vulturna. Antifungal susceptibility varied among strains, with four strains exhibiting reduced susceptibility to azoles and amphotericin B. ERG11 sequencing showed a point mutation (producing a P135S substitution) that was associated with the azole-resistant phenotype. This study contributes to the understanding of C. vulturna's identification challenges, its susceptibility patterns, and sheds light on its molecular mechanisms of azole resistance.

Adhesive and biofilm-forming Candida glabrata Lebanese hospital isolates harbour mutations in subtelomeric silencers and adhesins.

The prevalence of Candida glabrata healthcare-associated infections is on the rise worldwide and in Lebanon, Candida glabrata infections are difficult to treat as a result of their resistance to azole antifungals and their ability to form biofilms. The first objective of this study was to quantify biofilm biomass in the most virulent C. glabrata isolates detected in a Lebanese hospital. In addition, other pathogenicity attributes were evaluated. The second objective was to identify the mechanisms of azole resistance in those isolates. A mouse model of disseminated systemic infection was developed to evaluate the degree of virulence of 41 azole-resistant C. glabrata collected from a Lebanese hospital. The most virulent isolates were further evaluated alongside an isolate having attenuated virulence and a reference strain for comparative purposes. A DNA-sequencing approach was adopted to detect single nucleotide polymorphisms (SNPs) leading to amino acid changes in proteins involved in azole resistance and biofilm formation. This genomic approach was supported by several phenotypic assays. All chosen virulent isolates exhibited increased adhesion and biofilm biomass compared to the isolate having attenuated virulence. The amino acid substitutions D679E and I739N detected in the subtelomeric silencer Sir3 are potentially involved- in increased adhesion. In all isolates, amino acid substitutions were detected in the ATP-binding cassette transporters Cdr1 and Pdh1 and their transcriptional regulator Pdr1. In summary, increased adhesion led to stable biofilm formation since mutated Sir3 could de-repress adhesins, while decreased azole susceptibility could result from mutations in Cdr1, Pdh1 and Pdr1.

The TAC1 Gene in Candida albicans: Structure, Function, and Role in Azole Resistance: A Mini-Review.

Candidiasis is a common fungal infection caused by Candida species, with Candida albicans being the most prevalent. Resistance to azole drugs, commonly used to treat Candida infections, poses a significant challenge. Transcriptional activator candidate 1 (TAC1) gene has emerged as a key player in regulating drug resistance in C. albicans. This review explores the structure and function of the TAC1 gene and its role in azole resistance. This gene encodes a transcription factor that controls the expression of genes involved in drug resistance, such as efflux pump genes (CDR1, CDR2, and MDR1) and ERG11. Mutations in TAC1 can increase these genes' expression and confer resistance to azoles. Various TAC1 gene mutations, mostly gain-of-function mutations, have been identified, which upregulate CDR1 and CDR2 expression, resulting in azole resistance. Understanding the mechanisms of azole resistance mediated by the TAC1 gene is crucial for the strategies in the effective antifungal development pipeline.

Investigation of Azole Resistance Involving cyp51A and cyp51B Genes in Clinical Aspergillus flavus Isolates.

This study aimed to investigate azole resistance mechanisms in Aspergillus flavus, which involve cyp51A and cyp51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of cyp51A and cyp51B genes for 34 A. flavus isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of cyp51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in cyp51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in cyp51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the cyp51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the cyp51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the cyp51A and cyp51B genes is the primary mechanism responsible for resistance in A. flavus collection. Nevertheless, other resistance mechanisms can be involved.

Azole resistance mechanisms and population structure of the human pathogen Aspergillus fumigatus on retail plant products.

Aspergillus fumigatus is a ubiquitous saprotroph and human-pathogenic fungus that is life-threatening to the immunocompromised. Triazole-resistant A. fumigatus was found in patients without prior treatment with azoles, leading researchers to conclude that resistance had developed in agricultural environments where azoles are used against plant pathogens. Previous studies have documented azole-resistant A. fumigatus across agricultural environments, but few have looked at retail plant products. Our objectives were to determine if azole-resistant A. fumigatus is prevalent in retail plant products produced in the United States (U.S.), as well as to identify the resistance mechanism(s) and population genetic structure of these isolates. Five hundred twenty-five isolates were collected from retail plant products and screened for azole resistance. Twenty-four isolates collected from compost, soil, flower bulbs, and raw peanuts were pan-azole resistant. These isolates had the TR34/L98H, TR46/Y121F/T289A, G448S, and H147Y cyp51A alleles, all known to underly pan-azole resistance, as well as WT alleles, suggesting that non-cyp51A mechanisms contribute to pan-azole resistance in these isolates. Minimum spanning networks showed two lineages containing isolates with TR alleles or the F46Y/M172V/E427K allele, and discriminant analysis of principle components identified three primary clusters. This is consistent with previous studies detecting three clades of A. fumigatus and identifying pan-azole-resistant isolates with TR alleles in a single clade. We found pan-azole resistance in U.S. retail plant products, particularly compost and flower bulbs, which indicates a risk of exposure to these products for susceptible populations and that highly resistant isolates are likely distributed worldwide on these products.IMPORTANCEAspergillus fumigatus has recently been designated as a critical fungal pathogen by the World Health Organization. It is most deadly to people with compromised immune systems, and with the emergence of antifungal resistance to multiple azole drugs, this disease carries a nearly 100% fatality rate without treatment or if isolates are resistant to the drugs used to treat the disease. It is important to determine the relatedness and origins of resistant A. fumigatus isolates in the environment, including plant-based retail products, so that factors promoting the development and propagation of resistant isolates can be identified.

Pan-azole- and multi-fungicide-resistant Aspergillus fumigatus is widespread in the United States.

Aspergillus fumigatus is an important global fungal pathogen of humans. Azole drugs are among the most effective treatments for A. fumigatus infection. Azoles are also widely used in agriculture as fungicides against fungal pathogens of crops. Azole-resistant A. fumigatus has been increasing in Europe and Asia for two decades where clinical resistance is thought to be driven by agricultural use of azole fungicides. The most prevalent mechanisms of azole resistance in A. fumigatus are tandem repeats (TR) in the cyp51A promoter coupled with mutations in the coding region which result in resistance to multiple azole drugs (pan-azole resistance). Azole-resistant A. fumigatus has been isolated from patients in the United States (U.S.), but little is known about its environmental distribution. To better understand the distribution of azole-resistant A. fumigatus in the U.S., we collected isolates from agricultural sites in eight states and tested 202 isolates for sensitivity to azoles. We found azole-resistant A. fumigatus in agricultural environments in seven states showing that it is widespread in the U.S. We sequenced environmental isolates representing the range of U.S. sample sites and compared them with publicly available environmental worldwide isolates in phylogenetic, principal component, and ADMIXTURE analyses. We found worldwide isolates fell into three clades, and TR-based pan-azole resistance was largely in a single clade that was strongly associated with resistance to multiple agricultural fungicides. We also found high levels of gene flow indicating recombination between clades highlighting the potential for azole-resistance to continue spreading in the U.S.IMPORTANCEAspergillus fumigatus is a fungal pathogen of humans that causes over 250,000 invasive infections each year. It is found in soils, plant debris, and compost. Azoles are the first line of defense antifungal drugs against A. fumigatus. Azoles are also used as agricultural fungicides to combat other fungi that attack plants. Azole-resistant A. fumigatus has been a problem in Europe and Asia for 20 years and has recently been reported in patients in the United States (U.S.). Until this study, we did not know much about azole-resistant A. fumigatus in agricultural settings in the U.S. In this study, we isolated azole-resistant A. fumigatus from multiple states and compared it to isolates from around the world. We show that A. fumigatus which is resistant to azoles and to other strictly agricultural fungicides is widespread in the U.S.

Distribution of Aspergillus species and prevalence of azole resistance in clinical and environmental samples from a Spanish hospital during a three-year study period.

Surveillance studies are crucial for updating trends in Aspergillus species and antifungal susceptibility information. Determine the Aspergillus species distribution and azole resistance prevalence during this 3-year prospective surveillance study in a Spanish hospital. Three hundred thirty-five Aspergillus spp. clinical and environmental isolates were collected during a 3-year study. All isolates were screened for azole resistance using an agar-based screening method and resistance was confirmed by EUCAST antifungal susceptibility testing. The azole resistance mechanism was confirmed by sequencing the cyp51A gene and its promoter. All Aspergillus fumigatus strains were genotyped using TRESPERG analysis. Aspergillus fumigatus was the predominant species recovered with a total of 174 strains (51.94%). The rest of Aspergillus spp. were less frequent: Aspergillus niger (14.93%), Aspergillus terreus (9.55%), Aspergillus flavus (8.36%), Aspergillus nidulans (5.37%) and Aspergillus lentulus (3.28%), among other Aspergillus species (6.57%). TRESPERG analysis showed 99 different genotypes, with 72.73% of the strains being represented as a single genotype. Some genotypes were common among clinical and environmental A. fumigatus azole-susceptible strains, even when isolated months apart. We describe the occurrence of two azole-resistant A. fumigatus strains, one clinical and another environmental, that were genotypically different and did not share genotypes with any of the azole-susceptible strains. Aspergillus fumigatus strains showed a very diverse population although several genotypes were shared among clinical and environmental strains. The isolation of azole-resistant strains from both settings suggest that an efficient analysis of clinical and environmental sources must be done to detect azole resistance in A. fumigatus.

Cyp51A mutations, protein modeling, and efflux pump gene expression reveals multifactorial complexity towards understanding Aspergillus section Nigri azole resistance mechanism

Black Aspergillus species are the most common etiological agents of otomycosis, and pulmonary aspergillosis. However, limited data is available on their antifungal susceptibility profiles and associated resistance mechanisms. Here, we determined the azole susceptibility profiles of black Aspergillus species isolated from the Indian environment and explored the potential resistance mechanisms through cyp51A gene sequencing, protein homology modeling, and expression analysis of selected genes cyp51A, cyp51B, mdr1, and mfs based on their role in imparting resistance against antifungal drugs. In this study, we have isolated a total of 161 black aspergilli isolates from 174 agricultural soil samples. Isolates had variable resistance towards medical azoles; approximately 11.80%, 3.10%, and 1.24% of isolates were resistant to itraconazole (ITC), posaconazole (POS), and voriconazole (VRC), respectively. Further, cyp51A sequence analysis showed that non-synonymous mutations were present in 20 azole-resistant Aspergillus section Nigri and 10 susceptible isolates. However, Cyp51A homology modeling indicated insignificant protein structural variations because of these mutations. Most of the isolates showed the overexpression of mdr1, and mfs genes. Hence, the study concluded that azole-resistance in section Nigri cannot be attributed exclusively to the cyp51A gene mutation or its overexpression. However, overexpression of mdr1 and mfs genes may have a potential role in drug resistance.

Importance of the Aspergillus fumigatus Mismatch Repair Protein Msh6 in Antifungal Resistance Development.

One of the systems responsible for the recognition and repair of mistakes occurring during cell replication is the DNA mismatch repair (MMR) system. Two major protein complexes constitute the MMR pathway: MutS and MutL. Here, we investigated the possible relation of four A. fumigatus MMR genes (msh2, msh6, pms1, and mlh1) with the development of azole resistance related to the phenomenon of multi-drug resistance. We examined the MMR gene variations in 163 Aspergillus fumigatus genomes. Our analysis showed that genes msh2, pms1, and mlh1 have low genetic variability and do not seem to correlate with drug resistance. In contrast, there is a nonsynonymous mutation (G240A) in the msh6 gene that is harbored by 42% of the strains, most of them also harboring the TR34/L98H azole resistance mechanism in cyp51A. The msh6 gene was deleted in the akuBKU80A. fumigatus strain, and the ∆msh6 isolates were analyzed for fitness, azole susceptibility, and virulence capacity, showing no differences compared with the akuBKU80 parental strain. Wild-type msh6 and Δmsh6 strains were grown on high concentrations of azole and other non-azole fungicides used in crop protection. A 10- and 2-fold higher mutation frequency in genes that confer resistance to boscalid and benomyl, respectively, were observed in Δmsh6 strains compared to the wild-type. This study suggests a link between Msh6 and fungicide resistance acquisition.

Pan-azole-resistant Meyerozyma guilliermondii clonal isolates harbouring a double F126L and L505F mutation in Erg11.

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. The aim of this study was consequently to understand the mechanisms of azole resistance in several clinical isolates of M. guilliermondii. 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, invitro experiments on fitness cost and virulence were carried out. 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. 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.

Bimodal distribution of azole susceptibility in Sporothrix brasiliensis isolates in Brazil.

Sporothrix brasiliensis is an emerging zoonotic fungal pathogen that can be difficult to treat. Antifungal susceptibility testing was performed on the mold phase of a convenience sample of 61 Sporothrix spp. isolates from human and cat sporotrichosis cases in Brazil using the Clinical and Laboratory Standards Institute standard M38. A bimodal distribution of azole susceptibility was observed with 50% (28/56) of S. brasiliensis isolates showing elevated itraconazole minimum inhibitory concentrations ≥16 µg/mL. Phylogenetic analysis found the in vitro resistant isolates were not clonal and were distributed across three different S. brasiliensis clades. Single nucleotide polymorphism (SNP) analysis was performed to identify potential mechanisms of in vitro resistance. Two of the 28 resistant isolates (MIC ≥16 mg/L) had a polymorphism in the cytochrome P450 gene, cyp51, corresponding to the well-known G448S substitution inducing azole resistance in Aspergillus fumigatus. SNPs corresponding to other known mechanisms of azole resistance were not identified in the remaining 26 in vitro resistant isolates.

Upc2-mediated mechanisms of azole resistance in Candida auris.

Candida auris is an emerging yeast pathogen of major concern because of its ability to cause hospital outbreaks of invasive candidiasis and to develop resistance to antifungal drugs. A majority of C. auris isolates are resistant to fluconazole, an azole drug used for the treatment of invasive candidiasis. Mechanisms of azole resistance are multiple, including mutations in the target gene ERG11 and activation of the transcription factors Tac1b and Mrr1, which control the drug transporters Cdr1 and Mdr1, respectively. We investigated the role of the transcription factor Upc2, which is known to regulate the ergosterol biosynthesis pathway and azole resistance in other Candida spp. Genetic deletion and hyperactivation of Upc2 by epitope tagging in C. auris resulted in drastic increases and decreases in susceptibility to azoles, respectively. This effect was conserved in strains with genetic hyperactivation of Tac1b or Mrr1. Reverse transcription PCR analyses showed that Upc2 regulates ERG11 expression and also activates the Mrr1/Mdr1 pathway. We showed that upregulation of MDR1 by Upc2 could occur independently from Mrr1. The impact of UPC2 deletion on MDR1 expression and azole susceptibility in a hyperactive Mrr1 background was stronger than that of MRR1 deletion in a hyperactive Upc2 background. While Upc2 hyperactivation resulted in a significant increase in the expression of TAC1b, CDR1 expression remained unchanged. Taken together, our results showed that Upc2 is crucial for azole resistance in C. auris, via regulation of the ergosterol biosynthesis pathway and activation of the Mrr1/Mdr1 pathway. Notably, Upc2 is a very potent and direct activator of Mdr1.IMPORTANCECandida auris is a yeast of major medical importance causing nosocomial outbreaks of invasive candidiasis. Its ability to develop resistance to antifungal drugs, in particular to azoles (e.g., fluconazole), is concerning. Understanding the mechanisms of azole resistance in C. auris is important and may help in identifying novel antifungal targets. This study shows the key role of the transcription factor Upc2 in azole resistance of C. auris and shows that this effect is mediated via different pathways, including the regulation of ergosterol biosynthesis and also the direct upregulation of the drug transporter Mdr1.

A live-cell ergosterol reporter for visualization of the effects of fluconazole on the human fungal pathogen Candida albicans.

Ergosterol is a critical membrane lipid in fungi. In Candida albicans, this essential plasma membrane amphipathic lipid is important for interactions with host cells, in particular, host immune responses. Here, we use a live-cell reporter for specifically visualizing ergosterol and show that apical enrichment of this sterol is not critical for budding and filamentous growth in this human fungal pathogen. Our results highlight that this live-cell reporter is likely to be a useful tool in the analyses of azole resistance and tolerance mechanisms, including alterations in drug targets and upregulation of efflux activities.

Emergence of multiple fluconazole-resistant Candida parapsilosis sensu stricto clones with persistence and transmission in China.

We explored the epidemiological and molecular characteristics of Candida parapsilosis sensu stricto isolates in China, and their mechanisms of azole resistance. Azole susceptibilities of 2318 non-duplicate isolates were determined using CLSI broth microdilution. Isolates were genotyped by a microsatellite typing method. Molecular resistance mechanisms were also studied and functionally validated by CRISPR/Cas9-based genetic alterations. Fluconazole resistance occurred in 2.4% (n = 56) of isolates, and these isolates showed a higher frequency of distribution in ICU inpatients compared with susceptible isolates (48.2%, n = 27/56 versus 27.8%, 613/2208; P = 0.019). Microsatellite-genotyping analysis yielded 29 genotypes among 56 fluconazole-resistant isolates, of which 10 genotypes, including 37 isolates, belonged to clusters, persisting and transmitting in Chinese hospitals for 1-29 months. Clusters harbouring Erg11Y132F (5/10; 50%) were predominant in China. Among these, the second most dominant cluster MT07, including seven isolates, characteristically harbouring Erg11Y132F and Mrr1Q625K, lent its carriage to being one of the strongest associations with cross-resistance and high MICs of fluconazole (>256 mg/L) and voriconazole (2-8 mg/L), causing transmission across two hospitals. Among mutations tested, Mrr1Q625K led to the highest-level increase of fluconazole MIC (32-fold), while mutations located within or near the predicted transcription factor domain of Tac1 (D440Y, T492M and L518F) conferred cross-resistance to azoles. This study is the first Chinese report of persistence and transmissions of multiple fluconazole-resistant C. parapsilosis sensu stricto clones harbouring Erg11Y132F, and the first demonstration of the mutations Erg11G307A, Mrr1Q625K, Tac1L263S, Tac1D440Y and Tac1T492M as conferring resistance to azoles.

Comparative analysis of the biological characteristics and mechanisms of azole resistance of clinical Aspergillus fumigatus strains.

Aspergillus fumigatus is a common causative pathogen of aspergillosis. At present, triazole resistance of A. fumigatus poses an important challenge to human health globally. In this study, the biological characteristics and mechanisms of azole resistance of five A. fumigatus strains (AF1, AF2, AF4, AF5, and AF8) were explored. There were notable differences in the sporulation and biofilm formation abilities of the five test strains as compared to the standard strain AF293. The ability of strain AF1 to avoid phagocytosis by MH-S cells was significantly decreased as compared to strain AF293, while that of strains AF2, AF4, and AF5 were significantly increased. Fungal burden analysis with Galleria mellonella larvae revealed differences in pathogenicity among the five strains. Moreover, the broth microdilution and E-test assays confirmed that strains AF1 and AF2 were resistant to itraconazole and isaconazole, while strains AF4, AF5, and AF8 were resistant to voriconazole and isaconazole. Strains AF1 and AF2 carried the cyp51A mutations TR34/L98H/V242I/S297T/F495I combined with the hmg1 mutation S541G, whereas strains AF4 and AF8 carried the cyp51A mutation TR46/Y121F/V242I/T289A, while strain AF5 had no cyp51A mutation. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis revealed differences in the expression levels of genes associated with ergosterol synthesis and efflux pumps among the five strains. In addition, transcriptomics, RT-qPCR, and the NAD+/NADH ratio demonstrated that the mechanism of voriconazole resistance of strain AF5 was related to overexpression of genes associated with energy production and efflux pumps. These findings will help to further elucidate the triazole resistance mechanism in A. fumigatus.

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.