Multiple routes to fungicide resistance: Interaction of Cyp51 gene sequences, copy number and expression.

We examined the molecular basis of triazole resistance in Blumeria graminis f. sp. tritici (wheat mildew, Bgt), a model organism among powdery mildews. Four genetic models for responses to triazole fungicides were identified among US and UK isolates, involving multiple genetic mechanisms. Firstly, only two amino acid substitutions in CYP51B lanosterol demethylase, the target of triazoles, were associated with resistance, Y136F and S509T (homologous to Y137F and S524T in the reference fungus Zymoseptoria tritici). As sequence variation did not explain the wide range of resistance, we also investigated Cyp51B copy number and expression, the latter using both reverse transcription-quantitative PCR and RNA-seq. The second model for resistance involved higher copy number and expression in isolates with a resistance allele; thirdly, however, moderate resistance was associated with higher copy number of wild-type Cyp51B in some US isolates. A fourth mechanism was heteroallelism with multiple alleles of Cyp51B. UK isolates, with significantly higher mean resistance than their US counterparts, had higher mean copy number, a high frequency of the S509T substitution, which was absent from the United States, and in the most resistant isolates, heteroallelism involving both sensitivity residues Y136+S509 and resistance residues F136+T509. Some US isolates were heteroallelic for Y136+S509 and F136+S509, but this was not associated with higher resistance. The obligate biotrophy of Bgt may constrain the tertiary structure and thus the sequence of CYP51B, so other variation that increases resistance may have a selective advantage. We describe a process by which heteroallelism may be adaptive when Bgt is intermittently exposed to triazoles.

Harnessing Machine Learning to Uncover Hidden Patterns in Azole-Resistant CYP51/ERG11 Proteins.

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.

Candida glabrata maintains two Hap1 homologs, Zcf27 and Zcf4, for distinct roles in ergosterol gene regulation to mediate sterol homeostasis under azole and hypoxic conditions.

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 three 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. In this study, we determined the function of two zinc cluster transcription factors, Zcf27 and Zcf4, as direct but distinct regulators of ERG genes. Our phylogenetic analysis revealed C. glabrata Zcf27 and Zcf4 as the closest homologs to Saccharomyces cerevisiae Hap1. Hap1 is a known zinc cluster transcription factor in S. cerevisiae in controlling ERG gene expression under aerobic and hypoxic conditions. Interestingly, when we deleted HAP1 or ZCF27 in either S. cerevisiae or C. glabrata, respectively, both deletion strains showed altered susceptibility to azole drugs, whereas the strain deleted for ZCF4 did not exhibit azole susceptibility. We also determined that the increased azole susceptibility in a zcf27Δ strain is attributed to decreased azole-induced expression of ERG genes, resulting in decreased levels of total ergosterol. Surprisingly, Zcf4 protein expression is barely detected under aerobic conditions but is specifically induced under hypoxic conditions. However, under hypoxic conditions, Zcf4 but not Zcf27 was directly required for the repression of ERG genes. This study provides the first demonstration that Zcf27 and Zcf4 have evolved to serve distinct roles allowing C. glabrata to adapt to specific host and environmental conditions.

Synthesis and antifungal evaluation of novel triazole derivatives bearing a pyrazole-methoxyl moiety.

Life-threatening invasive fungal infections pose a serious threat to human health. A series of novel triazole derivatives bearing a pyrazole-methoxyl moiety were designed and synthesized in an effort to obtain antifungals with potent, broad-spectrum activity that are less susceptible to resistance. Most of these compounds exhibited moderate to excellent in vitro antifungal activities against Candida albicans SC5314 and 10,231, Cryptococcus neoformans 32,609, Candida glabrata 537 and Candida parapsilosis 22,019 with minimum inhibitory concentration (MIC) values of ≤0.125 µg/mL to 0.5 µg/mL. Use of recombinant Saccharomyces cerevisiae strains showed compounds 7 and 10 overcame the overexpression and resistant-related mutations in ERG11 of S. cerevisae and several pathogenic Candida spp. Despite being substrates of the C. albicans and Candida auris Cdr1 drug efflux pumps, compounds 7 and 10 showed moderate potency against five fluconazole (FCZ)-resistant fungi with MIC values from 2.0 µg/mL to 16.0 µg/mL. Growth kinetics confirmed compounds 7 and 10 had much stronger fungistatic activity than FCZ. For C. albicans, compounds 7 and 10 inhibited the yeast-to-hyphae transition, biofilm formation and destroyed mature biofilm more effectively than FCZ. Preliminary mechanism of action studies showed compounds 7 and 10 blocked the ergosterol biosynthesis pathway at Erg11, ultimately leading to cell membrane disruption. Further investigation of these novel triazole derivatives is also warranted by their predicted ADMET properties and low cytotoxicity.

Role of ERG11 and MDR1 genes in cycloheximide and multidrug resistance in Candida species.

Candida species are amongst the commensals of the mucosal surfaces of the human body which include the oral cavity, vagina, and intestinal mucosa. Fungal infections are on the rise worldwide. The overall burden of infections due to fungi is difficult to estimate because the majority of them remain undiagnosed. The present study aims to determine the burden of antifungal resistance in low socioeconomic country, Pakistan and the frequency of ERG11 and MDR1 genes involved. A total of 636 Candida isolates were obtained from various tertiary care institutions in Lahore in the form of culture on various culture plates. Sabouraud agar culture plates were used to culture the Candida spp. Antifungal resistance was determined against Fluconazole, Itraconazole, Ketoconazole, and Nystatin via disk diffusion technique. Most resistance was observed against Fluconazole followed by Itraconazole, Ketoconazole, and Nystatin. The Candida isolates recovering from CVP tip and tissue have a high resistance profile. Candida species resistant to at least two antifungals were chosen for further ERG11 and MDR1 detection through real-time PCR. Among 255 Candida isolates, 240 contained ERG11 gene while MDR1 gene is present in 149 Candida isolates. The isolates carrying both genes were tested by the broth microdilution technique for the susceptibility against cycloheximide, all of them were able to grow in cycloheximide. The genetic determinants of antifungal resistance such as ERG11 and MDR1 are as important in the multidrug resistance against a variety of compounds and antifungal drugs.

Molecular and Epidemiological Investigation of Fluconazole-resistant Candida parapsilosis-Georgia, United States, 2021.

Background: Reports of fluconazole-resistant Candida parapsilosis bloodstream infections are increasing. We describe a cluster of fluconazole-resistant C parapsilosis bloodstream infections identified in 2021 on routine surveillance by the Georgia Emerging Infections Program in conjunction with the Centers for Disease Control and Prevention. Methods: Whole-genome sequencing was used to analyze C parapsilosis bloodstream infections isolates. Epidemiological data were obtained from medical records. A social network analysis was conducted using Georgia Hospital Discharge Data. Results: Twenty fluconazole-resistant isolates were identified in 2021, representing the largest proportion (34%) of fluconazole-resistant C parapsilosis bloodstream infections identified in Georgia since surveillance began in 2008. All resistant isolates were closely genetically related and contained the Y132F mutation in the ERG11 gene. Patients with fluconazole-resistant isolates were more likely to have resided at long-term acute care hospitals compared with patients with susceptible isolates (P = .01). There was a trend toward increased mechanical ventilation and prior azole use in patients with fluconazole-resistant isolates. Social network analysis revealed that patients with fluconazole-resistant isolates interfaced with a distinct set of healthcare facilities centered around 2 long-term acute care hospitals compared with patients with susceptible isolates. Conclusions: Whole-genome sequencing results showing that fluconazole-resistant C parapsilosis isolates from Georgia surveillance demonstrated low genetic diversity compared with susceptible isolates and their association with a facility network centered around 2 long-term acute care hospitals suggests clonal spread of fluconazole-resistant C parapsilosis. Further studies are needed to better understand the sudden emergence and transmission of fluconazole-resistant C parapsilosis.

Comparative Genomics of the First Resistant Candida auris Strain Isolated in Mexico: Phylogenomic and Pan-Genomic Analysis and Mutations Associated with Antifungal Resistance.

Candida auris is an emerging multidrug-resistant and opportunistic pathogenic yeast. Whole-genome sequencing analysis has defined five major clades, each from a distinct geographic region. The current study aimed to examine the genome of the C. auris 20-1498 strain, which is the first isolate of this fungus identified in Mexico. Based on whole-genome sequencing, the draft genome was found to contain 70 contigs. It had a total genome size of 12.86 Mbp, an N50 value of 1.6 Mbp, and an average guanine-cytosine (GC) content of 45.5%. Genome annotation revealed a total of 5432 genes encoding 5515 proteins. According to the genomic analysis, the C. auris 20-1498 strain belongs to clade IV (containing strains endemic to South America). Of the two genes (ERG11 and FKS1) associated with drug resistance in C. auris, a mutation was detected in K143R, a gene located in a mutation hotspot of ERG11 (lanosterol 14-α-demethylase), an antifungal drug target. The focus on whole-genome sequencing and the identification of mutations linked to the drug resistance of fungi could lead to the discovery of new therapeutic targets and new antifungal compounds.

Genome-wide analysis of in vivo-evolved Candida auris reveals multidrug-resistance mechanisms.

Candida auris, an emerging and multidrug-resistant fungal pathogen, has led to numerous outbreaks in China. While the resistance mechanisms against azole and amphotericin B have been studied, the development of drug resistance in this pathogen remains poorly understood, particularly in in vivo-generated drug-resistant strains. This study employed pathogen whole-genome sequencing to investigate the epidemiology and drug-resistance mutations of C. auris using 16 strains isolated from two patients. Identification was conducted through Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and antimicrobial susceptibilities were assessed using broth microdilution and Sensititre YeastOne YO10. Whole-genome sequencing revealed that all isolates belonged to the South Asian lineage, displaying genetic heterogeneity. Despite low genetic variability among patient isolates, notable mutations were identified, including Y132F in ERG11 and A585S in TAC1b, likely linked to increased fluconazole resistance. Strains from patient B also carried F214L in TAC1b, resulting in a consistent voriconazole minimum inhibitory concentration of 4 µg/mL across all isolates. Furthermore, a novel frameshift mutation in the SNG1 gene was observed in amphotericin B-resistant isolates compared to susceptible ones. Our findings suggest the potential transmission of C. auris and emphasize the need to explore variations related to antifungal resistance. This involves analyzing genomic mutations and karyotypes, especially in vivo, to compare sensitive and resistant strains. Further monitoring and validation efforts are crucial for a comprehensive understanding of the mechanisms of drug resistance in C. auris.

Precise genome editing underlines the distinct contributions of mutations in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in Candida parapsilosis.

Candida parapsilosis has recently emerged as a major threat due to the worldwide emergence of fluconazole-resistant strains causing clonal outbreaks in hospitals and poses a therapeutic challenge due to the limited antifungal armamentarium. Here, we used precise genome editing using CRISPR-Cas9 to gain further insights into the contribution of mutations in ERG11, ERG3, MRR1, and TAC1 genes and the influence of allelic dosage to antifungal resistance in C. parapsilosis. Seven of the most common amino acid substitutions previously reported in fluconazole-resistant clinical isolates (including Y132F in ERG11) were engineered in two fluconazole-susceptible C. parapsilosis lineages (ATCC 22019 and STZ5). Each mutant was then challenged in vitro against a large array of antifungals, with a focus on azoles. Any possible change in virulence was also assessed in a Galleria mellonella model. We successfully generated a total of 19 different mutants, using CRISPR-Cas9. Except for R398I (ERG11), all remaining amino acid substitutions conferred reduced susceptibility to fluconazole. However, the impact on fluconazole in vitro susceptibility varied greatly according to the engineered mutation, the stronger impact being noted for G583R acting as a gain-of-function mutation in MRR1. Cross-resistance with newer azoles, non-medical azoles, but also non-azole antifungals such as flucytosine, was occasionally noted. Posaconazole and isavuconazole remained the most active in vitro. Except for G583R, no fitness cost was associated with the acquisition of fluconazole resistance. We highlight the distinct contributions of amino acid substitutions in ERG11, ERG3, MRR1, and TAC1 genes to antifungal resistance in C. parapsilosis.

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

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.

COVID-19 associated candidemia: From a shift in fungal epidemiology to a rise in azole drug resistance.

Our understanding of fungal epidemiology and the burden of antifungal drug resistance in COVID-19-associated candidemia (CAC) patients is limited. Therefore, we conducted a retrospective multicenter study in Iran to explore clinical and microbiological profiles of CAC patients. Yeast isolated from blood, were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and subjected to antifungal susceptibility testing (AFST) using the broth microdilution method M27-A3 protocol. A total of 0.6% of the COVID-19 patients acquired CAC (43/6174). Fluconazole was the most widely used antifungal, and 37% of patients were not treated. Contrary to historic candidemia patients, Candida albicans and C. tropicalis were the most common species. In vitro resistance was high and only noted for azoles; 50%, 20%, and 13.6% of patients were infected with azole-non-susceptible (ANS) C. tropicalis, C. parapsilosis, and C. albicans isolates, respectively. ERG11 mutations conferring azole resistance were detected for C. parapsilosis isolates (Y132F), recovered from an azole-naïve patient. Our study revealed an unprecedented rise in ANS Candida isolates, including the first C. parapsilosis isolate carrying Y132F, among CAC patients in Iran, which potentially threatens the efficacy of fluconazole, the most widely used drug in our centers. Considering the high mortality rate and 37% of untreated CAC cases, our study underscores the importance of infection control strategies and antifungal stewardship to minimize the emergence of ANS Candida isolates during COVID-19.

Rapid Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction Protocol for Detection of Y132F Mutation in Fluconazole Resistant Candida parapsilosis.

There is an emerging fluconazole resistance in Candida parapsilosis in recent years. The leading mechanism causing azole resistance in C. parapsilosis is the Y132F codon alteration in the ERG11 gene which encodes the target enzyme of azole drugs. In this study, we evaluated the sensitivity, compatibility, and specificity of a novel tetra-primer amplification refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method for rapid detection of the Y132F mutation in fluconazole nonsusceptible C. parapsilosis. Antifungal susceptibility tests for detection of fluconazole resistance were performed by broth microdilution according to the CLSI guidelines. All susceptible and nonsusceptible C. parapsilosis isolates were analyzed for ERG11 mutations with Sanger sequencing. T-ARMS-PCR was fully concordant with the Sanger sequencing (100% of sensitivity and specificity) for detection of Y132F mutations. T-ARMS-PCR method could be a rapid, simple, accurate, and economical assay in the early detection of the most common cause of fluconazole resistance in C. parapsilosis isolates. In routine laboratories with high C. parapsilosis isolation rates, performing the T-ARMS-PCR for early detection of the most common reason of fluconazole resistance in C. parapsilosis, could be a life-saving approach for directing antifungal therapy before obtaining the definitive antifungal susceptibility tests results.

Prevalence and drug susceptibility of clinical Candida species in nasopharyngeal cancer patients in Vietnam.

In the nature, Candida species are normal inhabitants and can be observed in a wide variety of vertebrates. In humans, especially for cancer patients who fall prey to opportunistic pathogens, this group of susceptible multi-drug resistant and biofilm-forming yeasts, are among the commonest ones. In this study, Candida species in 76 oral lesion samples from Vietnamese nasopharyngeal-cancer patients were isolated, morphologically identified using CHROMagar™, germ tube formation, and chlamydospore formation tests, and molecularly confirmed by PCR-RFLP. The drug susceptibility of these isolates was then tested, and the gene ERG11 was DNA sequenced to investigate the mechanism of resistance. The results showed that Candida albicans remained the most prevalent species (63.16% of the cases), followed by Candida glabrata, Candida tropicalis, and Candida krusei. The rates of resistance of non-albicans Candida for tested drugs were 85.71%, 53.57%, and 57.14% to fluconazole, clotrimazole, and miconazole, respectively. Although the drug-resistance rate of Candida albicans was lower than that of non-albicans Candida, it was higher than expected, suggesting an emerging drug-resistance phenomenon. Furthermore, ERG11 DNA sequencing revealed different mutations (especially K128T), implying the presence of multiple resistance mechanisms. Altogether, the results indicate an alarming drug-resistance situation in Candida species in Vietnamese cancer patients and emphasize the importance of species identification and their drug susceptibility prior to treatment.

Antifungal susceptibility profile and local epidemiological cut-off values of Yarrowia (Candida) lipolytica: an emergent and rare opportunistic yeast.

IMPORTANCE: Yarrowia lipolytica, also known as Candida lipolytica, is an emerging opportunistic "rare pathogenic yeast". Due to the limited data on its antifungal susceptibility, the clinical treatments become challenging. Based on the China Hospital Invasive Fungal Surveillance Network (2009-2022), we conducted a comprehensive multi-method study on clinical isolates from various central hospitals. This study is currently the largest study carried out to assess the antifungal susceptibility of Y. lipolytica. It is also the first to establish local epidemiological cut-off values (L-ECOFFs), identify its ERG11 mutations, and assess the consistency between the three prevalent commercial antifungal susceptibility testing methods and the broth microdilution method. We recommend the Sensititre YeastOne as the best option for antifungal susceptibility testing for Y. lipolytica, followed by the ATB FUNGUS 3. Nevertheless, practitioners should use the MIC test strip with discretion.

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

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

First Canadian report of transmission of fluconazole-resistant Candida parapsilosis within two hospital networks confirmed by genomic analysis.

Candida parapsilosis is a common cause of non-albicans candidemia. It can be transmitted in healthcare settings resulting in serious healthcare-associated infections and can develop drug resistance to commonly used antifungal agents. Following a significant increase in the percentage of fluconazole (FLU)-nonsusceptible isolates from sterile site specimens of patients in two Ontario acute care hospital networks, we used whole genome sequence (WGS) analysis to retrospectively investigate the genetic relatedness of isolates and to assess potential in-hospital spread. Phylogenomic analysis was conducted on all 19 FLU-resistant and seven susceptible-dose dependent (SDD) isolates from the two hospital networks, as well as 13 FLU susceptible C. parapsilosis isolates from the same facilities and 20 isolates from patients not related to the investigation. Twenty-five of 26 FLU-nonsusceptible isolates (resistant or SDD) and two susceptible isolates from the two hospital networks formed a phylogenomic cluster that was highly similar genetically and distinct from other isolates. The results suggest the presence of a persistent strain of FLU-nonsusceptible C. parapsilosis causing infections over a 5.5-year period. Results from WGS were largely comparable to microsatellite typing. Twenty-seven of 28 cluster isolates had a K143R substitution in lanosterol 14-α-demethylase (ERG11) associated with azole resistance. As the first report of a healthcare-associated outbreak of FLU-nonsusceptible C. parapsilosis in Canada, this study underscores the importance of monitoring local antimicrobial resistance trends and demonstrates the value of WGS analysis to detect and characterize clusters and outbreaks. Timely access to genomic epidemiological information can inform targeted infection control measures.

Monoterpenes as potential antifungal molecules against Candida cell membranes: in-vitro and in-silico studies.

Azoles are the frequently used antifungal drugs that target the enzyme lanosterol 14 α-demethylase (erg11p). This enzyme plays a vital role in ergosterol biosynthesis and hence maintainenance of cell membrane fluidity and integrity. The emergence of resistance to azoles and their fungistatic nature against several fungal pathogens is the major challenge to combat invasive candidiasis. Therefore, there is an urgent need to discover new antifungals with better efficacy. This study targets erg11 protein using in silico approach and identifies the monoterpene compounds (α-terpineol, carveol, and terpinene-4-ol) based on docking score and ligand interaction analysis. Further dynamic behavior of best-docked compounds with erg11p was analyzed by various parameters of MD simulation. The binding free energy of selected compounds towards the definitive pocket was also calculated. To further investigate the antifungal activity of selected compounds, in vitro studies were conducted on C. albicans. Studies thus suggest that the proposed the mechanism of antifungal action of test compounds involves targeting the ergosterol biosynthetic pathway. The compounds were explored for their effect on the disruption of membrane integrity by studying ERG11gene expression analysis, scanning electron microscopy, PI uptake (fluorescence microscopy,) and H+-extrusion. The results suggest that the selected monoterpenes are safer natural antifungals that disrupt membrane integrity by inhibiting ergosterol biosynthesis and other membrane associated structures.Communicated by Ramaswamy H. Sarma.

Synthesis of new imidazole-based ionic liquids with antifungal activity against Candida albicans.

Background and Objectives: Candida albicans cause a problematic condition in immunocompromised patients that could not be treated quickly due to the resistant behavior of microorganisms. This study aimed to investigate the effect of a novel ionic liquid (IL) as a new drug on C. albicans strains. Materials and Methods: Seven newly binary ionic liquids mixtures were synthesized, and among them, ([prollinium chloride] [1-methylimidazolium 3-sulfonate] ([pro-HCl][MImS]) was selected and characterized by 1HNMR, 13C NMR, and FT-IR methods. Samples from patients (n=50) with candidiasis were collected and identified through culture media. ERG11 gene overexpression was related to resistance against azole-bearing drugs. The antibiogram, well diffusion assay, MICs, and MFCs tests were operated. PCR and Real-time evaluated the expression of the ERG11 gene, and the rate of cell death was detected using Flow Cytometry. Results: Our data manifested that this novel IL (Ionic Liquid) can inhibit C. albican's growth, reduce the expression of ERG11 and increase dead cells. Conclusion: The newly synthesized IL had an inhibiting effect on the growth of the C. albicans strains and may be used as an alternative candidate for novel drug design.

A New Genotype of Trichophyton quinckeanum with Point Mutations in Erg11A Encoding Sterol 14-α Demethylase Exhibits Increased Itraconazole Resistance.

Trichophyton quinckeanum, the causative agent of mouse favus, has been responsible for several infections of animal owners in recent years and showed an infection peak around 2020 in Jena, Thuringia. The isolated T. quinckeanum strains from Thuringia differ in some positions of the ITS region compared to strains from the IHEM collection as well as to Trichophyton schoenleinii. All T. quinckeanum strains of the new genotype show up to a 100-fold increased itraconazole resistance as measured by microplate laser nephelometry (MLN) assays. Analysis of genes involved in Trichophyton indotineae azole resistance, such as Erg1, which encodes squalene epoxidase, and Erg11B, one of two copies of the sterol 14-α demethylase gene, show a 100% identity between the two T. quinckeanum genotypes. In contrast, Erg11A fragments differ in 15-nucleotide positions between both T. quinckeanum genotypes, resulting in the unique amino acid substitution Ala256Ser in resistant strains. The new T. quinckeanum genotype may have evolved through interspecies mating. Mating type analysis showed a nearly 100% identity of the minus type MAT1-1-1 fragment for all T. quinckeanum isolates. The closely related Trichophyton schoenleinii belongs to the plus mating type and has 100% identical fragments of Erg1 and Erg11B. Erg11A protein sequences of T. schoenleinii and T. quinckeanum showed increased diversity.

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

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.