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Background: The increasing detection rate of C. tropicalis and its azole resistance have made clinical treatment difficult. The presence of candiduria seems to correlate with invasive candida infection, especially for patients admitted to ICUs. However, the prevalence and antifungal resistance of C. tropicalis isolates in urine samples has not been well studied., Aim: To retrospectively investigate the clinical features, antifungal resistance, and genetic relatedness of C. tropicalis isolates from urine samples., Methods: A total of 107 clinical C. tropicalis isolates were retrospectively studied, including phenotypes of isolates and characteristics of patients. The genetic profiles of 107 isolates were genotyped using multi-locus sequence typing (MLST). Phylogenetic analysis was inferred using unweighted pair group method with arithmetic averages. MLST clonal clusters (CCs) were analysed by goeBURST., Findings: Of the 107 isolates, 27.1% were resistant to fluconazole, and there was a notable increasing trend of fluconazole resistance from 16.1% in 2019 to 40.0% in 2021. Forty-seven diploid sequence types (DSTs) were assigned to ten major CCs. CC1 was the predominant fluconazole-susceptible group; 24 isolates from CC1 belonged to DST333, an outbreak clone in NICU ward. The azole-resistant CC4 contained 19 isolates, accounting for 65.5% of the azole-resistant isolates in this study. CC4 belongs to a prevalent FNS CC1 globally, of which the putative founder genotype was DST225., Conclusion: This study revealed an outbreak of azole-susceptible C. tropicalis isolates in urine specimens and a high azole resistance rate of C. tropicalis in candiduria, and the MLST type showed clonal aggregation in azole-resistant isolates from urine samples., Competing Interests: Conflict of interest statement None declared., (Copyright © 2024 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

The widespread use of azole antifungals in agriculture and clinical settings has led to serious drug resistance. Overexpression of the azole drug target 14α-demethylase ERG11 (CYP51) is the most common fungal resistance mechanism. However, the presence of additional regulatory proteins in the transcriptional response of erg11 is not yet fully elucidated. In this study, leveraging the identified key promoter region of erg11 that controls its response to azoles in Neurospora crassa , we pinpointed a protein, Crf4-3, which harbors a PWWP domain and exerts a positive regulatory influence on azole resistance, as determined by DNA pulldown assays. The removal of Crf4-3 results in heightened sensitivity to azoles while remaining unaffected by other stressors tested. Additionally, the deletion leads to the abolition of transcriptional responses of genes such as erg11 and erg6 to ketoconazole. Interestingly, the basal expression of erg1 , erg11 , erg25 , and erg3A is also affected by the deletion of crf4-3 , indicating its role in sterol homeostasis. Crf4-3 homologs are broadly distributed across the Pezizomycotina fungi. The gene deletion for its homologous protein in Aspergillus fumigatus also significantly improves sensitivity to azoles such as voriconazole, primarily through the attenuation of the transcriptional response of erg11 . Our data, for the first time, identified Crf4-3 as a novel regulatory protein in the azole stress response of filamentous fungi, offering fresh insights into the mechanisms of azole resistance.IMPORTANCETranscriptional control of pivotal genes, such as erg11 , stands as the primary driver of azole resistance. Although considerable effort has been dedicated to identifying transcription factors involved, our knowledge regarding the use of transcriptional regulation strategies to combat azole resistance is currently limited. In this study, we reveal that a PWWP domain-containing protein Crf4-3, which is conserved in Pezizomycotina fungi, modulates fungal azole sensitivity by transcriptionally regulating sterol biosynthetic genes, including erg11 . These results also broaden the understanding of fungal PWWP domain-containing proteins regarding their roles in regulating resistance against azole antifungals. Considering research on small molecules targeting the PWWP domain in humans, Crf4-3 homolog emerges as a promising target for designing fungal-specific drugs to combat azole resistance., Competing Interests: The authors declare no conflict of interest.

This study aimed to evaluate whether the MIC Test Strip (MTS) quantitative assay for determining the minimum inhibitory concentration (MIC) correlated with the CLSI reference broth microdilution (BMD) method for antifungal susceptibility testing of wild-type and non-wild-type Aspergillus species against antifungal agents known to be usually effective against Aspergillus spp. This study was performed to assist in the decision-making process for possible deployment of the MTS assay for antimicrobial susceptibility testing of Aspergillus species into regional public health laboratories of Mycology due to difficulties in equipping the reference BMD methods in a laboratory routine. For this purpose, a set of 40 phenotypically diverse isolates (27 wild-type, 9 non-wild-type, and 4 species with reduced susceptibility to azoles and amphotericin B [AMB]) collected from clinical samples were tested. MICs were performed by both MTS and reference BMD for AMB and azoles. MTS results for posaconazole correlated well with reference BMD, rendering an almost perfect agreement (kappa value = 1.000) by category interpretation (CI)/category distribution of MICs (CDM) (100%), while voriconazole MTS results yielded a substantial correlation with BMD (kappa value = 0.788) by CI/CDM (97.5%). In contrast, itraconazole and AMB yielded the poorest correlation with BMD, rendering moderate agreement (kappa values of 0.554 and 0.437, respectively) by CI/CDM (87.5% and 85%, respectively). In conclusion, the MTS method represents a valid option for antimicrobial susceptibility testing of Aspergillus species against posaconazole and voriconazole. Itraconazole and AMB MTS results showed some concerning lack of correlation with the corresponding reference BMD results., (© The Author(s) 2025. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Multidrug resistance in the pathogenic fungus Candida glabrata is a growing global threat. Here, we study mechanisms of multidrug resistance in this pathogen. Exposure of C. glabrata cells to micafungin (an echinocandin) leads to the isolation of a mutant exhibiting resistance to echinocandin and azole antifungals. The drug-resistant phenotype is due to a non-synonymous mutation (R70H) in gene IPI1, which is involved in pre-rRNA processing. Azole resistance in the ipi1 R70H mutant depends on the Pdr1 transcription factor, which regulates the expression of multidrug transporters. The C. glabrata Ipi1 protein physically interacts with the ribosome-related chaperones Ssb and Ssz1, both of which bind to Pdr1. The Ipi1-Ssb/Ssz1 complex inhibits Pdr1-mediated gene expression and multidrug resistance in C. glabrata, in contrast to Saccharomyces cerevisiae where Ssz1 acts as a positive regulator of Pdr1. Furthermore, micafungin exposure reduces metabolic activity and cell proliferation in the ipi1 R70H mutant, which may contribute to micafungin tolerance., Competing Interests: Competing interests: T.M. has received a research grant from MSD for this project. T.M. and H.M. have received research grants and lecture honoraria from MSD, Astellas, and Pfizer outside this work. The sponsors and pharmaceutical companies had no role in the study design; collection, analysis, and interpretation of the data; and writing of the manuscript. The other authors have no competing interests., (© 2025. The Author(s).)

Background: Topical antiseptics are crucial for preventing infections and reducing transmission of pathogens. However, commonly used antiseptic agents have been reported to cause cross-resistance to other antimicrobials in bacteria, which has not yet been described in yeasts. This study aims to assess the in vitro efficacy of antiseptics against clinical and reference isolates of Candida albicans and Nakaseomyces glabratus, and whether prolonged exposure to antiseptics promotes the development of antifungal (cross)resistance., Methods: A high-throughput approach for in vitro resistance development was established to simultaneously expose 96 C. albicans and N. glabratus isolates to increasing concentrations of a given antiseptic - chlorhexidine, triclosan or octenidine. Susceptibility testing and whole genome sequencing of yeast isolates pre- and post-exposure were performed., Results: Long-term exposure to antiseptics does not result in the development of stable resistance to the antiseptics themselves. However, 50 N. glabratus isolates acquired resistance to azole antifungals after long-term exposure to triclosan or chlorhexidine, revealing newly acquired mutations in the PDR1 and PMA1 genes., Conclusions: Chlorhexidine as well as triclosan, but not octenidine, were able to introduce selective pressure promoting resistance to azole antifungals. Although we assessed this phenomenon only in vitro, these findings warrant critical monitoring in clinical settings., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Azole and benzimidazole fungicides are widely used agrochemicals to prevent and treat fungal growth and are frequently detected in aquatic environments. Here, we aimed to assess the aquatic ecological risks of ten currently used azole and benzimidazole fungicides, which with the aryl hydrocarbon receptor (AhR) agonistic activity, and their transformation products (TPs). We obtained over 400 types of aerobic TPs for ten fungicides. Some fungicides and their TPs (approximately 26.7%) exhibited the potential AhR agonistic activity and toxicity to different aquatic species. Meanwhile, some compounds with the chlorine element and benzene ring structure exhibited environmental persistence and mobile ability. Several of them were frequently detected in aquatic environments, posing potential risks to aquatic ecosystems. These harmful fungicides and their TPs should be given attention. This study provides important insight into the aquatic ecological risks caused by azole and benzimidazole fungicides, which can provide theoretical guidance for their pollution control.

Candida ( Nakaseomyces ) glabrata , an opportunistic human fungal pathogen, causes mucosal and deep-seated infections in immunocompromised individuals. Recently designated as a high-priority fungal pathogen by the World Health Organization (WHO), C. glabrata exhibits low inherent susceptibility to azole antifungals. In addition, about 10% clinical isolates of C. glabrata display co-resistance to both azole and echinocandin drugs. Molecular mechanisms of antifungal resistance and virulence in C. glabrata are currently being delineated in-depth. This Review provides an overview of the epidemiology, biology, drug resistance, tools and host model systems for C. glabrata . Additionally, we discuss the immune evasion strategies that aid C. glabrata in establishing infections in the host. Overall, this Review aims to contribute to ongoing efforts to raise awareness of human pathogenic fungi, the growing threat of antifungal drug resistance and the unmet need for novel antifungal therapies, with an ultimate goal of improving clinical outcomes of affected individuals.

Background: Scedosporium/Lomentospora species are ranked as the second most frequently isolated filamentous fungi from cystic fibrosis (CF) patients. Previously, we demonstrated that the minimum inhibitory concentration (MIC) for voriconazole and posaconazole increased when performed on a mucin-containing synthetic CF sputum medium (SCFM) compared to the standard medium, RPMI-1640. In this study, we have expanded the MIC comparison to four additional azoles and investigated characteristics linked to azole resistance in Scedosporium apiospermum, Scedosporium minutisporum, Scedosporium aurantiacum and Lomentospora prolificans., Methods: MIC was assayed by CLSI protocol, efflux pump activity was assessed by rhodamine 6G and sterols were analysed by gas chromatography-mass spectrometry (GC-MS)., Results: Overall, MICs for fluconazole, itraconazole, voriconazole, posaconazole, miconazole and ketoconazole increased by least 2-fold when susceptibility tests were performed using SCFM compared to RPMI. The activity of efflux pumps was similar in both media; however, in RPMI, but not in SCFM, the activity was induced by voriconazole and fluconazole. Additionally, MICs for those antifungals decreased more noticeably in SCFM than in RPMI in the presence of the efflux pump inhibitor PaβN. The SCFM-grown cells presented fewer sterols in their composition, and consequently higher membrane fluidity, than RPMI-grown cells. GC-MS analysis demonstrated a remodulation in the sterol profile in SCFM- compared to RPMI-grown cells. Accordingly, when the MIC assay was performed in the presence of the membrane stressor NaCl (3%), the susceptibility to voriconazole and fluconazole increased more in SCFM- than RPMI-grown cells., Conclusions: Scedosporium/Lomentospora species undergo cellular adaptations in SCFM that favours their growth in face of the challenges imposed by azole antifungals., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

The synthesis and spectroscopic characterization of three complexes containing a substituted 2-(2-pyridyl)benzothiazole (PyBTh) group in the ligand frame are reported along with the comparative biological activity. The ligands have been substituted at the 6-position with either a methoxy (Py(OMe)BTh) or a methyl group (Py(Me)BTh). Reaction of Py(OMe)BTh with either CuCl 2 or Cu(NO 3 ) 2 ·2.5 H 2 O yielded the monomeric [Cu(Py(OMe)BTh)) 2 (NO 3 )]NO 3 ·1.5 MeOH, (1·1.5 MeOH) complex or the dimeric [Cu(Py(OMe)BTh)Cl 2 ] 2 (2), respectively, with the nuclearity of the complex dependent on the starting Cu(II) salt. Reaction between the methyl substituted ligand and Cu(NO 3 ) 2 ·2.5 H 2 O resulted in the isolation of Cu(Py(Me)BTh)(NO 3 ) 2 ·0.5 THF (3·0.5 THF). Complexes 1-3 were fully characterized. Cyclic voltammetry measurements were performed on all three complexes as well as on [Cu(PyBTh) 2 (H 2 O)](BF 4 ) 2 (4), a compound previously reported by us which contains the unsubstituted 2-(2-pyridyl)benzothiazole ligand. The biological activity was studied and included concentration dependent DNA binding and cleavage, antibacterial activity, and cancer cell toxicity. All complexes exhibited DNA cleavage activity, however 2 and 4 were found to be the most potent. Mechanistic studies revealed that the nuclease activity is dependent on an oxidative mechanism reliant principally on O 2 - . Antibacterial studies revealed complex 4 was more potent compared to 1-3. Cancer cell toxicity studies were carried out on HeLa, PC-3, and MCF7 cells with 1-4, Cu(QBTh)(NO 3 ) 2 (H 2 O) and Cu(PyBIm) 3 (BF 4 ) 2 . The differences in the observed toxicities suggests the importance of the ligand and its substituents in modulating cell death., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Atherosclerosis is often accompanied by inflammation and oxidative stress. Excessive reactive oxygen species (ROS) can damage the vascular endothelium, leading to endothelial dysfunction and reduced nitric oxide (NO) bioavailability. Further accumulation of ROS contributes to vascular cell damage, lipid peroxidation, and extracellular matrix deposition. Thus, clearing excess ROS and reshaping the oxidative microenvironment is essential for treating atherosclerosis (AS). In this study, Ebselen, which mimics glutathione peroxidase and possesses redox capabilities, was successfully synthesized. Subsequently, a multifunctional coating was designed using a combination of Ebselen and poly (trimethylene carbonate) (PTMC), capable of protecting cells from ROS-induced damage, promoting vascular endothelialization, and exhibiting anti-proliferative properties. The Ebselen-loaded coating effectively scavenges free radicals (with an elimination rate of 89 %), catalytically releases NO (0.96 × 10⁻¹⁰ to 1.26 × 10⁻¹⁰ mol/cm²/min), and sustainably delivers Ebselen to the lesion site through a redox cycle. Notably, this coating shows excellent hemocompatibility and cytocompatibility. Subcutaneous implantation results indicated that the fibrous capsule thickness of PTMC10 was the lowest, at just 47.7 % of that of PTMC. Therefore, the Ebselen-loaded coating presents promising applications in cardiovascular stents., Competing Interests: Declaration of Competing Interest We would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication. All the authors listed have approved the manuscript that is enclosed., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The global epidemiology of aspergillosis varies and is influenced by various factors. To elucidate the disease burden and identify effective control strategies, the epidemiological characteristics of Aspergillus infections have to be investigated. The aim of this study was to assess the epidemiological characteristics of various Aspergillus species, including their morphological features, species identification, and in vitro susceptibility to nine antifungal agents in a large tertiary hospital in northern China., Methods: Ninety-five clinical isolates of Aspergillus were collected from patients. Aspergillus species identification was performed using conventional morphological methods, MALDI-TOF MS, and gene sequencing. In vitro susceptibility to nine antifungal agents was evaluated using the Sensititre YeastOne system. Target genes (cyp51A and cyp51b) of A. tubinazole were sequenced using the Sanger method., Results: Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, Aspergillus tubingensis, and Aspergillus terreus were the most common isolated species. Rare species included Aspergillus tamarii, Aspergillus usamil, Aspergillus versicolor, Aspergillus udagawae, Aspergillus lentulus, Aspergillus sydowii, and Aspergillus quadrilineatus. Pulmonary infections accounted for 86.3 % (82/95) of collected cases, and the in-hospital mortality rate was 22.1 %. The median minimum inhibitory concentration (MIC) range of amphotericin B was 1.5-4 mg/L. The MIC range of triazoles against Aspergillus species, excluding Aspergillus udagawae and Aspergillus lentulus, was 0.12-0.5 mg/L. The median minimum effective concentration range of echinocandins was < 0.008-0.03 mg/L. Non-wild-type resistance to amphotericin B was observed in 29.6 % (16/54) of Aspergillus fumigatus isolates, and non-wild-type resistance to voriconazole was observed in 11.1 % (1/9) of Aspergillus tubingensis isolates. Moreover, CYP51A and CYP51b of Aspergillus tabinensis had 2-29 and 10-13 nucleotide mutations, respectively., Conclusion: Patients with non- Aspergillus fumigatus infection accounted for 43.2 %. The T256A amino acid substitution in CYP51A of Aspergillus tabinensis did not lead to increased azole drug MICs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In recent years, the widespread emergence of drug resistance in yeasts and filamentous fungi to existing antifungal armamentariums has become a severe threat to global health. There is also concern regarding increased rates of azole resistance in Aspergillus fumigatus and Terbinafine resistance in Trichophyton species. To overcome this concern of resistance to regular therapies, new antifungal drugs with novel and effective mechanisms are crucially needed. Herbal remedies may be promising strategies for the treatment of resistant infections. We aimed to investigate the in vitro and silico activity of piperlongumine against clinical azole susceptible/resistant A. fumigatus and terbinafine-susceptible/resistant Trichophyton species. In the current study, piperlongumine demonstrated potent antifungal activity, with minimum inhibitory concentrations (MICs) ranging from 0.016-4 μg/mL against Trichophyton isolates and 0.25-2 μg/mL for A. fumigatus isolates. Additionally, molecular docking studies indicated that piperlongumine has a strong binding affinity to the active sites of squalene epoxidase and sterol 14-alpha demethylase. However, further studies are warranted to correlate these findings with clinical outcomes and provide the basis for further investigations to pave the way for developing novel antifungal agents., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Iman Haghani reports financial support was provided by Mazandaran University of Medical Sciences. This research was financially supported by grant number 17588 from Mazandaran University of Medical Sciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Fluconazole-resistant Candida parapsilosis has emerged as a significant health-care-associated pathogen with a propensity to spread patient to patient and cause nosocomial outbreaks, similar to Candida auris. This study investigates a long-lasting outbreak of fluconazole-resistant C parapsilosis that was initially detected in December, 2018, and January, 2019, and officially declared in November, 2019; lasted multiple years; and involved several health-care centres in Berlin, Germany., Methods: In this retrospective, observational, and genomic epidemiology study, we used whole-genome sequencing (WGS) of isolates sent by German health-care facilities and laboratories to the National Reference Center for Invasive Fungal Infections (Jena, Germany) for antifungal susceptibility testing between Jan 1, 2016, and Dec 31, 2022. We included all potential outbreak samples (ie, isolates originating from Berlin that were resistant to fluconazole and voriconazole but susceptible to posaconazole) and all non-outbreak isolates that originated from outside of Berlin and were resistant to at least one azole. We also included a number of non-outbreak isolates from outside Berlin that were susceptible or resistant to azoles so that the total study dataset included a matching amount of outbreak and non-outbreak samples from Germany. We used admission and discharge records for patients involved in the outbreak and constructed a network of patient transfers in time and space. We used WGS data for included samples, complemented with WGS data for global samples obtained from the National Center for Biotechnology Information Sequence Read Archive, to construct single-nucleotide variant (SNV)-based phylogeny and perform SNV distance-based analyses. Additionally, we used the whole genomic dataset to identify loci with high discriminatory power to establish a multi-locus sequence typing (MLST) strategy for C parapsilosis., Findings: We identified 38 clonal, azole-resistant isolates of C parapsilosis causing 33 cases of invasive infection during a 2018-22 outbreak in multiple hospitals in Berlin. We also sequenced the genomes of 37 non-outbreak isolates. WGS revealed that outbreak strains were separated by a mean of 36 SNVs (SD 20), whereas outbreak strains differed from outgroup samples from Berlin and other regions of Germany by a mean of 2112 SNVs (828). Temporal and genomic reconstruction of the outbreak cases indicated that transfer of patients between health-care facilities was probably responsible for the persistent reimportation of the drug-resistant clone and subsequent person-to-person transmission. German outbreak strains were closely related to strains responsible for an outbreak in Canada and to isolates from Kuwait, Türkiye, and South Korea. Including the outbreak clone, we identified three distinct azole-resistant lineages carrying ERG11 Y132F in Germany. We identified four 750 bp loci in CPAR2&#95;101400, CPAR2&#95;101470, CPAR2&#95;108720, and CPAR2&#95;808110 for inclusion in our MLST strategy. Application of the MLST method to a global collection of 386 isolates identified 62 sequence types, with the outbreak strains all belonging to the same sequence type., Interpretation: This study underscores the emergence of drug-resistant C parapsilosis that can spread patient to patient within a health-care system, but also, possibly, internationally. Our findings highlight the importance of monitoring C parapsilosis epidemiology globally and of continuous surveillance and rigorous infection control measures at the local scale. We also developed a novel MLST scheme for genetic epidemiology and outbreak investigations, which could represent a faster and less expensive alternative to WGS., Funding: German Federal Ministry for Education and Research, German Research Foundation, and German Ministry of Health., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Emergence of azole-resistant Aspergillus fumigatus represents a concern for public health worldwide. Here we applied a spatial analysis to a large number of records on A. fumigatus azole resistance in the Netherlands to evaluate the relationship of a set of environmental factors with the emergence of resistant isolates and to identify the potential risk areas. A total of 1850 aspergillosis cases were included in the analysis: 1559 caused by wild-type (WT) and 291 by azole-resistant (RES) strains. High-resolution maps containing environmental variables (climate, crop cultivations) were used as layers for spatial analysis. QGIS software was used for transformation and calculation of layers, and visualizing maps, whereas MaxEnt software was used to perform spatial analysis. A separate distribution map for WT and RES was generated using each layer, then the ratio between RES and WT suitability was calculated, and a new map was generated showing the areas where suitability of RES was higher than WT (ratio > 1). Layers presenting areas with a ratio > 1 were then used for a further analysis including all these layers together to generate a final risk map, to identify the most contributing environmental variables, and to estimate the number of people potentially exposed to the risk. Results showed that the areas with the highest RES/WT ratio were associated with the following variables: legume cultivations ratio 1.5-2.7; fruit tree plantations, ratio 2.1; dump sites and artificial sites, ratio 2.3-2.7; spring minimum and mean temperatures, ratio 1.6-1.8. Areas with medium, high, and very high risk covered a surface of about 2330 km 2 and people exposed were >319,000, with Zeeland and Noord-Holland being the provinces with the highest risk. All together these results suggest that azole-resistance emergence is a complex phenomenon depending on a multitude of environmental variables that are not yet been fully explored., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Incidences of fluconazole (FLC) resistance among Candida glabrata clinical isolates are a growing issue in clinics. The pleiotropic drug response network in C. glabrata confers azole resistance and is defined primarily by the Zn 2 Cys 6 zinc cluster-containing transcription factor Pdr1 and target genes such as CDR1 , which encodes an ATP-binding cassette transporter protein thought to act as an FLC efflux pump. Mutations in the PDR1 gene that render the transcription factor hyperactive are the most common cause of fluconazole resistance among clinical isolates. The phenothiazine class drug fluphenazine and a molecular derivative, CWHM-974, which both exhibit antifungal properties, have been shown to induce the expression of Cdr1 in Candida spp. We have used a firefly luciferase reporter gene driven by the CDR1 promoter to demonstrate two distinct patterns of CDR1 promoter activation kinetics: gradual promoter activation kinetics that occur in response to ergosterol limitations imposed by exposure to azole and polyene class antifungals and a robust and rapid CDR1 induction occurring in response to the stress imposed by fluphenazines. We can attribute these different patterns of CDR1 induction as proceeding through the promoter region of this gene since this is the only segment of the gene included in the luciferase reporter construct. Genetic analysis indicates that the signaling pathways responsible for phenothiazine and azole induction of CDR1 overlap but are not identical. The short time course of phenothiazine induction suggests that these compounds may act more directly on the Pdr1 protein to stimulate its activity., Importance: Candida glabrata has emerged as the second-leading cause of candidiasis due, in part, to its ability to acquire high-level resistance to azole drugs, a major class of antifungal that acts to block the biosynthesis of the fungal sterol ergosterol. The presence of azole drugs causes the induction of a variety of genes involved in controlling susceptibility to this drug class, including drug transporters and ergosterol biosynthetic genes such as ERG11. We found that the presence of azole drugs leads to an induction of genes encoding drug transporters and ERG11, while exposure of C. glabrata cells to antifungals of the phenothiazine class of drugs caused a much faster and larger induction of drug transporters but not ERG11. Coupled with further genetic analyses of the effects of azole and phenothiazine drugs, our data indicate that these compounds are sensed and responded to differentially in the yeast cell., Competing Interests: The authors declare no conflict of interest.

The environmental use of azole fungicides has led to selective sweeps across multiple loci in the Aspergillus fumigatus genome causing the rapid global expansion of a genetically distinct cluster of resistant genotypes. Isolates within this cluster are also more likely to be resistant to agricultural antifungals with unrelated modes of action. Here we show that this cluster is not only multi-azole resistant but has increased propensity to develop resistance to next generation antifungals because of variants in the DNA mismatch repair system. A variant in msh6-G233A is found almost exclusively within azole resistant isolates harbouring the canonical cyp51A azole resistance allelic variant TR 34 /L98H. Naturally occurring isolates with this msh6 variant display up to 5-times higher rate of mutation, leading to an increased likelihood of evolving resistance to other antifungals. Furthermore, unlike hypermutator strains, the G233A variant conveys no measurable fitness cost and has become globally distributed. Our findings further suggest that resistance to next-generation antifungals is more likely to emerge within organisms that are already multi-azole resistant due to close linkage between TR 34 /L98H and msh6-G233A, posing a major problem due to the prospect of dual use of novel antifungals in clinical and agricultural settings., Competing Interests: Competing interests: M.J.Br. is a former employee of F2G Ltd (to 2008) and has received funding from F2G Ltd for a Ph.D. studentship. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Today, Candida albicans is still the most common cause of both local and life-threatening systemic candidiasis. The spread of resistant fungal strains has resulted in an urgent need to search for new promising antimycotics. Here, we investigated the antifungal action of the tobacco defensin NaD1 against susceptible and resistant to azoles and echinocandins strains of C. albicans . We demonstrated that NaD1 was equally effective and fungicidal against all tested strains. The MIC and MFC values were 6.25 and 12.5 µM, respectively. We showed for the first time that NaD1 could act synergistically not only with caspofungin but also with human host defense antimicrobial peptides cathelicidin LL-37 and β-defensin-2 (HBD2) against susceptible and resistant fungal strains. Using flow cytometry, we demonstrated that NaD1 in combinations with LL-37 or HBD2 can reinforce each other by enhancing membrane disruption. Using the Caco-2 cell monolayer model, we demonstrated that NaD1 impaired the adhesion of C. albicans cells to the human epithelium. Moreover, NaD1 inhibited the formation of fungal biofilms in Sabouraud broth and less markedly in nutrient-rich RPMI-1640 medium, and enhanced the antibiofilm activity of caspofungin. Thus, we hypothesized that NaD1 might affect the development of candidiasis in vivo, including that caused by resistant fungal strains.

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., Competing Interests: F. Lamoth has received research grants from Novartis, MSD, Gilead, and Pfizer outside of the present work and speaker honoraria from MSD, Gilead, Pfizer, Mundipharma, and Becton-Dickinson. All fees were paid to his institution (CHUV).

With the recent clinical success of anti-amyloid-β (Aβ) monoclonal antibodies, there is a renewed interest in agents which target the Aβ peptide of Alzheimer's disease (AD). Metal complexes are particularly well-suited for this development, given their structural versatility and ability to form stabile interactions with soluble Aβ. In this report, a small series of ruthenium-arene complexes were evaluated for their respective ability to modulate both the aggregation and cytotoxicity of Aβ. First, the stability of the complexes was evaluated in a variety of aqueous media where the complexes demonstrated exceptional stability. Next, the ability to coordinate and modulate the Aβ peptide was evaluated using several spectroscopic methods, including thioflavin T (ThT) fluorescence, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Overall, the complex RuBO consistently gave the greatest inhibitory action towards Aβ aggregation, which correlated with its ability to coordinate to Aβ in solution. Furthermore, RuBO also had the lowest affinity for serum albumin, which is a key consideration for a neurotherapeutic, as this protein does not cross the blood brain barrier. Lastly, RuBO also displayed promising neuroprotective properties, as it had the greatest inhibition of Aβ-inducted cytotoxicity.

Aspergillus fumigatus is a widely distributed pathogen responsible for severe infections, particularly in immunocompromised individuals. Triazoles are the primary treatments options for Aspergillus infections; however, the emergence of acquired resistance to this antifungal class is becoming a growing concern. In this study, we investigated the potential of the antiviral drug, lopinavir (LPV) to restore the susceptibility of A. fumigatus strains to a set of azoles, while also reducing the required azole dosage for treatment of susceptible isolates. The combination of LPV with either itraconazole (ITC) or posaconazole (POS) demonstrated potent synergistic interactions against 16 out of 23 (~70%) and 21 out of 23 (~91%) A. fumigatus isolates, respectively. Moreover, the combination showed synergistic activity against other clinically important Aspergillus species, including A. niger, A. flavus, and A. brasiliensis. The fractional inhibitory concentration index (FICI) for the combinations ranged from 0.18 to 0.313 for ITC and 0.091 to 0.313 for POS, indicating strong synergistic effects. Further investigation revealed that efflux pump inhibition contributed to the synergy observed between azole and LPV. Morphological examination of the fungal cells subjected to this combinational therapy at sub-inhibitory doses showed the presence of carbohydrate granules/patches. The identification of LPV as a promising adjunct therapy holds promise for addressing the emerging challenge of azole resistance in Aspergillus species and improving treatment outcomes for patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Burns et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The review examines recent advances in the design and synthesis of 1,3-selenazole derivatives since 2000. Various synthetic approaches to 1,3-selenazoles and reaction conditions are discussed. The beneficial properties of 1,3-selenazoles, especially their biological activity, are emphasized. Compounds with antitumor, antiviral (HIV-1 and HIV-2), antibacterial, antifungal, antiproliferative, anticonvulsant, and antioxidant activity are highlighted., Competing Interests: The authors declare no conflicts of interest.

Candida albicans is the most common pathogen in systemic fungal diseases, exhibits a complex pathogenic mechanism, and is increasingly becoming drug tolerant. Therefore, it is particularly important to study the genes associated with virulence and resistance of C. albicans . Here, we identified a gene ( orf19.1588 ) that encodes a conserved mitochondrial protein known as CaSDH8 , upon deletion of CaSdh8 , the deleted strain ( Casdh8Δ/Δ ) experienced impaired growth, hyphal development, and virulence. Casdh8Δ/Δ displayed a reduced capacity to utilize alternative carbon sources, along with detrimental alterations in reactive oxygen species (ROS), mitochondrial membrane potential (MMP) depolarization, and adenosine triphosphate (ATP) levels. Interestingly, Casdh8Δ/Δ demonstrated resistance to azole drugs, and under the influence of fluconazole, the cell membrane permeability and mitochondrial function of Casdh8Δ/Δ were less compromised than those of the wild type, indicating a reduction in the detrimental effects of fluconazole on Casdh8Δ/Δ . These findings highlight the significance of CaSDH8 as a crucial gene for the maintenance of cellular homoeostasis. Our study is the first to document the effects of the CaSDH8 gene on the virulence and azole resistance of C. albicans at both the molecular and animal levels, providing new clues and directions for the antifungal infection and the discovery of antifungal drug targets.

We aimed to access the genetic diversity of Apergillus fumigatus strains obtained from cystic fibrosis (CF) patients from southern Brazil. A. fumigatus sensu stricto isolates from respiratory clinical specimens were genotyped by microsatellite markers and azole resistance was evaluated by azole-agar screening. Twenty-seven isolates from twenty-seven patients showed a high genetic diversity, with the differentiation of 25 different genotypes (25 unique and one common to two isolates). All isolates were susceptible to the azoles tested. We believe that prospectively monitoring A. fumigatus genetic diversity is essential to identify interpatient transmission and outbreaks, as is the identification of resistant strains., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Candida albicans is an important human pathogenic yeast, that can become resistant to commonly used antifungal agents, such as azoles. One mechanism of drug resistance is efflux via ATP binding cassette transporters, such as Cdr1. Several studies have investigated the structural organization, binding mechanisms, function and regulation of Cdr1. This review summarizes the findings on the structure and function of Cdr1 and highlights important aspects to consider in future research relating to multidrug ABC transporters., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Helminths or worm-like parasites are common infectious pathogens that cause significant diseases and mortality in animals and humans. To be noted, nematodes or soil-transmitted helminths (STH) have been infected about 1.5 billion people according to the recent reports of world health organization (WHO). Helminths-based infections are a significant type of neglected tropical diseases (NTDs) that show higher occurrence in deprived regions of tropical and subtropical areas. Low-level sanitation and limited facilities increase the risk of affliction with these infectious diseases. Helminths infections are known to be associated with non-significant symptoms to heavier ones such as gastrointestinal (GI) manifestations, weakness, impaired growth, disturbed physical development and death on the basis of involved worm population. Despite the implementations of preventive and mass-treatment strategies, incomplete curative effects and drug-resistance potentiality inspires the discovery of new and potent anthelmintic agents. In this context, several efforts have been focused on the synthesis and biological evaluation of various heterocycles, and in particular azoles, as anthelmintic agents. Structural elucidations of the emerged anti-infective agents from the medicinal chemistry perspective is a rational way to tackle the burden of infections throughout development of new anthelmintic medications. In continuation to our previous contributions, privileged azole-based anthelmintic compounds that have been documented in the 2012-2023 period and their structure activity relationship (SAR), are going to be reviewed in this study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: A predominate azole-resistant Candida tropicalis clade 4 genotype causing candidemia has been detected in not only Taiwan but also China, Singapore, and Australia. It can also be detected on fruit surfaces. In addition to determining distribution and drug susceptibilities of pathogenic yeasts in environments of intensive care units of 25 hospitals in Taiwan, we would also like to investigate whether the azole-resistant C. tropicalis exists in Taiwan's hospital environment., Methods: The swabs of hospital environments were collected from August to November in 2020 and were cultured for yeasts. The yeasts were identified by rDNA sequence and the antifungal susceptibilities of those isolates were determined by the broth microdilution method., Results: The average yeast-culture rate of hospitals was 9.4% (217/2299). Sinks had the highest yeast-positive culture rate (32.7%), followed by bedside tables (28.9%), floors (26.0%), water-dispenser buttons (23.8%), and TV controller/touch panels (19.0%). Of 262 identified isolates, Candida parapsilosis was the most common species, accounting for 22.1%, followed by Filobasidium uniguttulatum (18.3%), Candida albicans (9.5%), C. tropicalis (8.0%), Candida glabrata (Nakaseomyces glabratus) (6.9%), and 30 other species (35.1%). Of the 21 C. tropicalis isolates from 11 units in 9 hospitals, 15 diploid sequence types (DSTs) were identified. The two DST506 fluconazole-resistant ones belonged to clade 4., Conclusion: We detected not only various pathogenic yeast species but also the predominant clade 4 genotype of azole-resistant C. tropicalis. Our findings highlight and re-emphasize the importance of regular cleaning and disinfection practices., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ling Lu reports financial support was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The PI3K/Akt pathway is crucial in numerous cellular functions such as cell growth, survival proliferation and movement in both normal and cancer cells. It plays also a key role in epithelial-mesenchymal transitions and angiogenesis during the tumorigenesis processes. Since many transformative events in cancer are driven by increased PI3K/Akt pathway signaling, Akt is considered a valuable target for developing new therapies against various tumor types, including pancreatic cancer. This is because the PI3K/AKT/mTOR pathway is a key downstream effector of RAS, and RAS activation is the most prominent genetic alteration in pancreatic cancer. Herein we report the synthesis and the biological evaluation of a new series of azole urea compounds that exhibited promising antiproliferative and antimigratory activities against pancreatic cancer cells through an Akt inhibition mechanism. These effects were demonstrated using a variety of assays, including Sulforhodamine B, cell-cycle, wound-healing, and kinase activity, apotposis and ELISA assays. Additionally, the anticancer properties of the most active compound in the series were confirmed in the 3D spheroid model of PATU-T cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Ebselen is a neuroprotective drug that protects cellular components from oxidative damage by modulating enzymatic cofactors, metalloproteins, gene expression, antioxidant-anti-inflammatory effects, and immunological systems. Our goal was to compare the efficacy of Ebselen and methylprednisolone on rat facial nerve crush injury., Methods: Thirty healthy male Wistar rats (mean weight of 245 g) were used in this study. The rats were randomly divided into four groups: Group 1 (ebselen group), Group 2 (methylprednisolone group), Group 3 (control group), and Group 4 (sham group (the right side of the control group)). Except for the sham group, all groups had their left facial nerve crushed. Three weeks after surgery, prospective functional, histologic, and electrophysiologic recovery was reported., Results: The ebselen group and methylprednisolone group had similar and more significant recovery at Nerve Excitability Thresholds (NET) at the end of three weeks. These groups also showed similar features in terms of histopathological parameters such as axonal degeneration, vascular congestion, axon diameter, and myelin thickness. Except for the macrovacuolization parameter, both showed statistically better results than the control group. Although there was an earlier improvement in the whiskers and blink tests in the ebselen group compared to the methylprednisolone and control groups, complete recovery was observed in all groups on the 21st day., Conclusion: Ebselen was found to be similarly effective to methylprednisolone in nerve regeneration in a rat model of experimental facial nerve crush. Considering that methylprednisolone has serious systemic side effects, ebselen may be a good alternative.

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

The World Health Organization (WHO) recognizes Candida albicans and Cryptococcus neoformans as the critical priority fungal pathogens for which therapeutic solutions are needed. Azole-based antifungal agents, including triazoles, diazoles, and thiazoles, are widely used in the treatments for fungal infections. In light of past successes in the transformation of antibacterial kanamycin into antifungal derivatives via chemical modifications, a new library of kanamycin-azole hybrids was synthesized and tested against a panel of azole-resistant and susceptible Candida and Cryptococcus strains. Structure activity relationship (SAR) studies revealed pivotal roles for antifungal activity of the azole ring (imidazole vs triazole) and halogen substituents on the benzene ring (F vs Cl). Most notably, hybrids 13, 14 and 15 were active against resistant C. albicans, C. tropicalis and C. neoformans strains and non-toxic towards mammalian cells. Mode of action investigations using fluorogenic dyes, (SYTOX TM ) showed the fungal active compounds could permeabilize fungal membranes even at ¼ MICs. These findings reveal novel azole-based antifungals that could offer new therapeutic options for candidiasis and cryptococcosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chagas disease is caused by the eukaryote parasite Trypanosoma cruzi . Current treatment exhibits limited efficacy and selenium-based compounds emerged as promising candidates for new therapies which is surpassing its bioisoster, sulfur. We designed new thiosemicarbazones, thiazoles, selenosemicarbazones and selenazoles, using isosteric substitution. We synthesized 57 new chalcogen compounds which were evaluated against T. cruzi , C2C12 cells and cruzain, the main target of this parasite. Additionally, human cathepsin L, was tested for selectivity. Three compounds were selected, based on their activity against the intracellular amastigotes (EC 50 < 1 μM, SI > 10) and cruzain (IC 50 < 100 nM, SI > 5.55) which compared favorably with the approved drug, Benznidazole, and the well-established cruzain inhibitor K777. Seleno-compounds demonstrated enhanced activity and selenazoles showed a decrease in selenium-associated toxicity. Compound 4-methyl-2-(2-(1-(3-nitrophenyl)ethylidene)hydrazineyl)-1,3-selenazole ( Se 2h ) emerged as a promising candidate, and its binding to cruzain was investigated. Pharmacokinetic assessment was conducted, showing a favorable profile for subsequent in vivo assays.

We evaluated the combined performance of itraconazole and voriconazole Etest® gradient concentration strips for the detection of Aspergillus fumigatus azole resistance associated with cyp51a mutations confirmed by gene sequencing. Among 118 A. fumigatus clinical isolates collected in a French center, 6 (5%) had azole resistance mutations, 5 of which were probably of environmental origin. Using recent method-dependent epidemiological cutoff values as thresholds, the combination's sensitivity and specificity were 100% [95% confidence interval 61-100] and 99% [95-100]. Our results support itraconazole and voriconazole Etest® combined use as a promising self-sufficient method for simple, efficient and reliable cyp51a-related azole-resistant A. fumigatus detection., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Carbon dioxide (CO 2 ) is an economically viable and abundant carbon source that can be incorporated into compounds such as C2-carboxylated 1,3-azoles relevant to the pharmaceutical, cosmetics, and pesticide industries. Of the 2.4 million commercially available C2-unsubstituted 1,3-azole compounds, less than 1 % are currently purchasable as their C2-carboxylated derivatives, highlighting the substantial gap in compound availability. This availability gap leaves ample opportunities for exploring the synthetic accessibility and use of carboxylated azoles in bioactive compounds. In this study, we analyze and quantify the relevance of C2-carboxylated 1,3-azoles in small-molecule research. An analysis of molecular databases such as ZINC, ChEMBL, COSMOS, and DrugBank identified relevant C2-carboxylated 1,3-azoles as anticoagulant and aroma-giving compounds. Moreover, a pharmacophore analysis highlights promising pharmaceutical potential associated with C2-carboxylated 1,3-azoles, revealing the ATP-sensitive inward rectifier potassium channel 1 (K ATP ) and Kinesin-like protein KIF18 A as targets that can potentially be addressed with C2-carboxylated 1,3-azoles. Moreover, we identified several bioisosteres of C2-carboxylated 1,3-azoles. In conclusion, further exploration of the chemical space of C2-carboxylated 1,3-azoles is recommended to harness their full potential in drug discovery and related fields., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

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

Azole antifungals are the main drugs used to treat fungal infections. Amino acid substitutions in the drug target Erg11 (Cyp51) are a common resistance mechanism in pathogenic yeasts. How many and which mutations confer resistance is, however, largely unknown. Here we measure the impact of nearly 4,000 amino acid variants of Candida albicans Erg11 on the susceptibility to six clinical azoles. This was achieved by deep mutational scanning of CaErg11 expressed in Saccharomyces cerevisiae. We find that a large fraction of mutations lead to resistance (33%), most resistance mutations confer cross-resistance (88%) and only a handful of resistance mutations show a significant fitness cost (9%). Our results reveal that resistance to azoles can arise through a large set of mutations and this will probably lead to azole pan-resistance, with little evolutionary compromise. This resource will help inform treatment choices in clinical settings and guide the development of new drugs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

We compared the minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of azoles in antifungal drug-susceptible, terbinafine-resistant, and lowly itraconazole (ITCZ)-susceptible strains of dermatophytes. To assess the MICs of ITCZ, ravuconazole (RVCZ), efinaconazole (EFCZ), and luliconazole (LUCZ) in the isolates, broth microdilution assays were performed based on the Clinical and Laboratory Standards Institute M38-A2 guidelines with modifications. After the assays for determining the MICs, the inoculum suspensions in wells were resuspended, then 10 μL of the growth solution in each well was inoculated onto potato dextrose agar with the use of a pipette. After 7 days of incubation at 28°C, the MFCs were determined as the lowest concentration of a drug that allowed the growth of colonies on the potato dextrose agar. The MICs in the dermatophytes were <0.03 to >32 mg/L for ITCZ, <0.03 to 4 mg/L for RVCZ, <0.03 to 2 mg/L for EFCZ, and <0.03 mg/L for LUCZ. The MFCs in the dermatophytes were 1 to >32 mg/L for ITCZ, 0.06 to >32 mg/L for RVCZ, <0.03 to 4 mg/L for EFCZ, and <0.03 to 2 mg/L for LUCZ. If the drug susceptibility test shows that the fungi are resistant to the drug, the treatment can be changed to a susceptible drug in advance, or if the fungi are low-susceptible, the treatment can be done with the recognition that it may require a longer treatment period than usual., (© 2024 Japanese Dermatological Association.)

Mortalities due to mycoses have dramatically increased with the emergence of drug-resistant strains and growing immune-compromised populations globally. Azole antifungals have been the first choice against fungal infections of a wide spectrum and several azole derivatives with ester function were reported for their potentially promising and favorable activity against Candida spp. In this study, we designed and synthesized a series of 1-(aryl)-2-(1H-imidazol-1-yl/1H-1,2,4-triazol-1-yl)ethyl esters, and tested them against seven reference Candida strains using EUCAST reference microdilution method. Among the series, 6a, 6d, and 6g proved highly potent in vitro compared to fluconazole; especially against Candida albicans and Candida tropicalis with minimum inhibitor concentration (MIC) values as low as 0.125 and 0.06 mg/L, respectively, although their activities against Candida krusei and Candida glabrata remained limited. The compounds also showed minimal toxicity to murine fibroblasts according to the in vitro cytotoxicity tests. Molecular modeling predicted 6g as an orally available druglike compound according to all parameters and CYP51 inhibition as the likely mechanism for their antifungal effects. The study underpins the promise of azoles with ester functionality as a potential scaffold for small-molecule antifungal drug design., (© 2024 Wiley Periodicals LLC.)

Fluconazole-resistant clade 4 Candida tropicalis causing candidemia in humans has been detected in tropical/subtropical areas, including those in China, Singapore, and Australia. We analyzed 704 individual yeasts isolated from fruits, soil, water, and farmers at 80 orchards in Taiwan. The most common pathogenic yeast species among 251 isolates recovered from farmers were Candida albicans (14.7%) and C. parapsilosis (11.6%). In contrast, C. tropicalis (13.0%), C. palmioleophila (6.6%), and Pichia kudriavzevii (6.0%) were prevalent among 453 environmental isolates. Approximately 18.6% (11/59) of C. tropicalis from the environment were resistant to fluconazole, and 81.8% (9/11) of those belonged to the clade 4 genotype. C. tropicalis susceptibility to fluconazole correlated with susceptibilities to the agricultural azole fungicides, difenoconazole, tebuconazole, and triadimenol. Tandem gene duplications of mutated ERG11 contributed to azole resistance. Agriculture environments are a reservoir for azole-resistant C. tropicalis; discontinuing agricultural use of azoles might reduce emergence of azole-resistant Candida spp. strains in humans.

Visceral leishmaniasis is a life-threatening parasitic disease, but current antileishmanial drugs have severe drawbacks. Antifungal azoles inhibit the activity of cytochrome P450 (CYP) 51 enzymes which are responsible for removing the C14α-methyl group of lanosterol, a key step in ergosterol biosynthesis in Leishmania. However, they exhibit varying degrees of antileishmanial activities in culture, suggesting the existence of unrecognized molecular targets. Our previous study reveals that, in Leishmania, lanosterol undergoes parallel C4- and C14-demethylation to form 4α,14α-dimethylzymosterol and T-MAS, respectively. In the current study, CYP5122A1 is identified as a sterol C4-methyl oxidase that catalyzes the sequential oxidation of lanosterol to form C4-oxidation metabolites. CYP5122A1 is essential for both L. donovani promastigotes in culture and intracellular amastigotes in infected mice. CYP5122A1 overexpression results in growth delay, increased tolerance to stress, and altered expression of lipophosphoglycan and proteophosphoglycan. CYP5122A1 also helps to determine the antileishmanial effect of antifungal azoles in vitro. Dual inhibitors of CYP51 and CYP5122A1 possess superior antileishmanial activity against L. donovani promastigotes whereas CYP51-selective inhibitors have little effect on promastigote growth. Our findings uncover the critical biochemical and biological role of CYP5122A1 in L. donovani and provide an important foundation for developing new antileishmanial drugs by targeting both CYP enzymes., (© 2024. The Author(s).)

The global epidemic of drug-resistant Candida auris continues unabated. The initial report on pan-drug resistant (PDR) C. auris strains in a hospitalized patient in New York was unprecedented. PDR C. auris showed both known and unique mutations in the prominent gene targets of azoles, amphotericin B, echinocandins, and flucytosine. However, the factors that allow C. auris to acquire pan-drug resistance are not known. Therefore, we conducted a genomic, transcriptomic, and phenomic analysis to better understand PDR C. auris . Among 1,570 genetic variants in drug-resistant C. auris , 299 were unique to PDR strains. The whole-genome sequencing results suggested perturbations in genes associated with nucleotide biosynthesis, mRNA processing, and nuclear export of mRNA. Whole transcriptome sequencing of PDR C. auris revealed two genes to be significantly differentially expressed-a DNA repair protein and DNA replication-dependent chromatin assembly factor 1. Of 59 novel transcripts, 12 transcripts had no known homology. We observed no fitness defects among multi-drug resistant (MDR) and PDR C. auris strains grown in nutrient-deficient or -enriched media at different temperatures. Phenotypic profiling revealed wider adaptability to nitrogenous nutrients and increased utilization of substrates critical in upper glycolysis and tricarboxylic acid cycle. Structural modeling of a 33-amino acid deletion in the gene for uracil phosphoribosyl transferase suggested an alternate route in C. auris to generate uracil monophosphate that does not accommodate 5-fluorouracil as a substrate. Overall, we find evidence of metabolic adaptations in MDR and PDR C. auris in response to antifungal drug lethality without deleterious fitness costs., Competing Interests: The authors declare no conflict of interest.

Azole resistance has emerged as a new therapeutic challenge in patients with aspergillosis. Various resistance mutations are attributed to the widespread use of triazole-based fungicides in agriculture. This study explored the prevalence of azole-resistant Aspergillus fumigatus (ARAF) and other aspergilli in the Argentine environment. A collection of A. fumigatus and other aspergilli strains isolated from soil of growing crops, compost, corn, different animal feedstuffs, and soybean and chickpea seeds were screened for azole resistance. No ARAF was detected in any of the environmental samples studied. However, five A. flavus, one A. ostianus, one A. niger and one A. tamarii recovered from soybean and chickpea seeds showed reduced susceptibility to medical azole antifungals (MAA). The susceptibility profiles of five A. flavus isolates, showing reduced susceptibility to demethylase inhibitors (DMIs), were compared with those of 10 isolates that exhibited susceptibility to MAA. Aspergillus flavus isolates that showed reduced MAA susceptibility exhibited different susceptibility profiles to DMIs. Prothioconazole and tebuconazole were the only DMIs significantly less active against isolates with reduced susceptibility to MAA. Although no ARAF isolates were found in the samples analysed, other aspergilli with reduced susceptibility profile to MAA being also important human pathogens causing allergic, chronic and invasive aspergillosis, are present in the environment in Argentina. Although a definitive link between triazole-based fungicide use and isolation of azole-resistant human pathogenic aspergilli from agricultural fields in Argentina remains elusive, this study unequivocally highlights the magnitude of the environmental spread of azole resistance among other Aspergillus species., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

Aspergillus fumigatus is the predominant pathogen responsible for aspergillosis infections, with emerging drug-resistant strains complicating treatment strategies. The role of mitochondrial functionality in fungal resistance to antifungal agents is well-documented yet not fully understood. In this study, the mitochondrial protein Bcs1A, a homolog of yeast Bcs1, was found to regulate colony growth, ion homeostasis, and the response to antifungal drugs in A. fumigatus . Microscopic observations revealed substantial colocalization of Bcs1A-GFP fusion protein fluorescence with mitochondria. Bcs1A deletion compromised colony growth and the utilization of non-fermentable carbon sources, alongside causing abnormal mitochondrial membrane potential and reduced reactive oxygen species production. These findings underscore Bcs1A's vital role in maintaining mitochondrial integrity. Phenotypic analysis and determinations of minimum inhibitory concentrations indicated that the Δ bcs1A mutant was more resistant to various antifungal agents, such as azoles, terbinafine, and simvastatin, compared to wild-type strain. RNA sequencing and RT-qPCR analysis highlighted an upregulation of multiple efflux pumps in the Δ bcs1A mutant. Furthermore, loss of the principal drug efflux pump, mdr1, decreased azole tolerance in the Δbcs1A mutant, suggesting that Bcs1A's modulated of azoles response via efflux pump expression. Collectively, these results establish Bcs1A as essential for growth and antifungal drug responsiveness in A. fumigatus mediated through mitochondrial regulation.IMPORTANCEDrug resistance presents a formidable obstacle in the clinical management of aspergillosis. Mitochondria are integral to various biochemical pathways, including those involved in fungi drug response, making mitochondrial proteins promising therapeutic targets for drug therapy. This study confirms that Bcs1A, a mitochondrial respiratory chain protein, is indispensable for mitochondrial functionality and multidrug tolerance in Aspergillus fumigatus . Mutation of Bcs1A not only leads to a series of drug efflux pumps upregulated but also shows that loss of the primary efflux pump, mdr1 , partial reduction in drug tolerance in the Bcs1A mutant, highlighting that Bcs1A's significant influence on mitochondria-mediated drug resistance., Competing Interests: The authors declare no conflict of interest.

Aims: Aspergillus lentulus is an important newly recorded species in the A. fumigatus complex and its resistance to azole drugs and the high mortality rate of infected individuals have emerged as problems. Comprehensive understanding of the A. lentulus is limited due to lack of genome-wide fine mapping data. The aim of this study was to investigate the A. lentulus signature at the molecular level, analyze the genome-wide profile of this strain, and predict its possible genes that execute azole resistance. Methods: In this study, a whole-genome sequencing of a clinically isolated A. lentulus strain (named A. lentulus PWCAL1) was studied by PacBio Sequel sequencing platform. Azole resistance genes were predicted based on whole-genome sequencing data analysis, gene function annotation, comparative genomic analysis, and BLASTP alignment using the Mycology Antifungal Resistance Database to comprehensively understanding the genome-wide features, pathogenicity, and resistance mechanisms of A. lentulus . Results: The results of whole-genome sequencing demonstrated that the total length of A. lentulus PWCAL1 genome was 31255105 bp, the GC content was 49.24%, and 6883 coding genes were predicted. A total of 4565, 1824, and 6405 genes were annotated in the Gene Ontology, Clusters of Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes databases, respectively. In the Pathogen Host Interactions Database and the Database of Fungal Virulence Factors, 949 and 259 interacting virulence factors were identified, respectively, with the main virulence factor-mutant virulence phenotype, being enriched in reduced virulence. Comparative genomic analysis showed that there were 5456 consensus core genes in this strain and four closely related strains of A. fumigatus complex, which were mainly involved in human diseases, metabolism, organismal systems, etc. Among the three aligned A. lentulus strains, the number of unique genes of this bacterium was the highest with a number of 171, and these genes were mainly associated with carbohydrate metabolism and cell growth and death. Resistance gene prediction demonstrated that the A5653 gene of this bacterium had F46Y/N248T double point mutations on the CYP51A gene, but no tandem repeat mutations in the promoter region were detected. Furthermore, 12 genes belonging to the fungal multidrug resistance ATP-binding cassette (ABC) transporters were identified based on the complete genome sequence and phylogenetic analysis of A. lentulus, which belonged to the ALDp subfamily, the PDR subfamily ( AtrB , CDR1 , and CDR2 ), and the MDR subfamily ( MDR1 ), respectively, and there were four genes that are annotated to the major facilitator superfamily multidrug transporter. Further phylogenetic tree classification of the ABC transporter subfamilies predicted in the nine selected A. fumigatus complex strains showed that these putative ABC proteins were divided into two main clusters, which belonged to the PDR ( CDR1 , CDR2 , AtrB , and AtrF ) and MDR subfamilies ( MDR1 , MDR2 , and MDR3 ). The distribution of these ABC proteins varies among different species of the A. fumigatus complex. Conclusions: The main result obtained from this study for the whole genome of A. lentulus provide new insights into better understanding the biological characteristics, pathogenicity, and resistance mechanisms of this bacterium. In this study, two resistance mechanisms, which include CYP51A gene mutation and multidrug-resistant ABC transporter, were predicted in a single isolate. Based on the predicted CYP51A-F46Y/N248T site mutation combination, we speculate that the CYP51A gene of A. lentulus may be partially responsible for azole resistance. Based on the predicted ABC transporter family genes, we hypothesize that resistance to multiple azoles in A. lentulus is mediated, at least in part, by these ABC transporters with resistance.

Objectives: We aimed to report the emergence of azole-resistant invasive aspergillosis in hematologic patients admitted to a tertiary hospital in Spain during the last 4 months., Methods: Prospective, descriptive study was performed to describe and follow all consecutive proven and probable invasive aspergillosis resistant to azoles from hematological cohort during the last 4 months. All patients had fungal cultures and antifungal susceptibility or real-time PCR detection for Aspergillus species and real-time PCR detection for azole-resistant mutation., Results: Four cases of invasive aspergillosis were diagnosed in 4 months. Three of them had azole-resistant aspergillosis. Microbiological diagnosis was achieved in three cases by means of fungal culture isolation and subsequent antifungal susceptibility whereas one case was diagnosed by PCR-based aspergillus and azole resistance detection. All the azole-resistant aspergillosis presented TR34/L98H mutation. Patients with azole-resistant aspergillosis had different hematologic diseases: multiple myeloma, lymphoblastic acute leukemia, and angioimmunoblastic T lymphoma. Regarding risk factors, one had prolonged neutropenia, two had corticosteroids, and two had viral co-infection. Two of the patients developed aspergillosis under treatment with azoles., Conclusion: We have observed a heightened risk of azole-resistant aspergillosis caused by A. fumigatus harboring the TR 34 /L98H mutation in patients with hematologic malignancies. The emergence of azole-resistant aspergillosis raises concerns for the community, highlighting the urgent need for increased surveillance and the importance of susceptibility testing and new drugs development., (© 2024. The Author(s).)

Antifungal drugs have had a tremendous impact on human health and the yields of crops. However, in recent years, due to usage both in a health setting and in agriculture, there has been a rapid emergence of antifungal drug resistance that has outpaced novel compound discovery. It is now globally recognized that new strategies to tackle fungal infection are urgently needed, with such approaches requiring the cooperation of both sectors and the development of robust antifungal stewardship rationales. In this review, we examine the current antifungal regimes in clinical and agricultural settings, focusing on two pathogens of importance, Candida auris and Aspergillus fumigatus, examining their drivers of antifungal resistance, the impact of dual-use azoles and the impact agricultural practices have on driving the emergence of resistance. Finally, we postulate that a One Health approach could offer a viable alternative to prolonging the efficacy of current antifungal agents.

The global outbreak of the COVID-19 pandemic caused by the SARS-CoV-2 virus had led to profound respiratory health implications. This study focused on designing organoselenium-based inhibitors targeting the SARS-CoV-2 main protease (M pro ). The ligand-binding pathway sampling method based on parallel cascade selection molecular dynamics (LB-PaCS-MD) simulations was employed to elucidate plausible paths and conformations of ebselen, a synthetic organoselenium drug, within the M pro catalytic site. Ebselen effectively engaged the active site, adopting proximity to H41 and interacting through the benzoisoselenazole ring in a π-π T-shaped arrangement, with an additional π-sulfur interaction with C145. In addition, the ligand-based drug design using the QSAR with GFA-MLR, RF, and ANN models were employed for biological activity prediction. The QSAR-ANN model showed robust statistical performance, with an r 2 training exceeding 0.98 and an RMSE test of 0.21, indicating its suitability for predicting biological activities. Integration the ANN model with the LB-PaCS-MD insights enabled the rational design of novel compounds anchored in the ebselen core structure, identifying promising candidates with favorable predicted IC 50 values. The designed compounds exhibited suitable drug-like characteristics and adopted an active conformation similar to ebselen, inhibiting M pro function. These findings represent a synergistic approach merging ligand and structure-based drug design; with the potential to guide experimental synthesis and enzyme assay testing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In Candida albicans, Cdr1 pumps azole drugs out of the cells to reduce intracellular accumulation at detrimental concentrations, leading to azole-drug resistance. Milbemycin oxime, a veterinary anti-parasitic drug, strongly and specifically inhibits Cdr1. However, how Cdr1 recognizes and exports azole drugs, and how milbemycin oxime inhibits Cdr1 remain unclear. Here, we report three cryo-EM structures of Cdr1 in distinct states: the apo state (Cdr1 Apo ), fluconazole-bound state (Cdr1 Flu ), and milbemycin oxime-inhibited state (Cdr1 Mil ). Both the fluconazole substrate and the milbemycin oxime inhibitor are primarily recognized within the central cavity of Cdr1 through hydrophobic interactions. The fluconazole is suggested to be exported from the binding site into the environment through a lateral pathway driven by TM2, TM5, TM8 and TM11. Our findings uncover the inhibitory mechanism of milbemycin oxime, which inhibits Cdr1 through competition, hindering export, and obstructing substrate entry. These discoveries advance our understanding of Cdr1-mediated azole resistance in C. albicans and provide the foundation for the development of innovative antifungal drugs targeting Cdr1 to combat azole-drug resistance., (© 2024. The Author(s).)

We describe for the first time, a high-quality genome for a rare human yeast pathogen Candida mucifera, from a patient with chronic suppurative otitis media. This pathogen exhibited reduced azole susceptibility, similar to its close relatives within the Trichomonascus ciferrii species complex., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Multidrug resistance (MDR) poses a significant problem in cancer treatment, often causing adverse effects during chemotherapy. Ebselen (Ebs), a synthetic organoselenium compound, affects cellular redox status in cancer cells. In the study, we observed that Ebs disrupted cellular redox balance and sensitized drug-resistant cells to doxorubicin (DOX) treatment. The combination of Ebs and DOX led to increased intracellular reactive oxygen species (ROS) levels and lipid peroxidation while decreasing the activity of thioredoxin reductase (TrxR) and cellular antioxidants in drug-resistant cells. Furthermore, this combination treatment demonstrated notable chemosensitizing effects by reducing cell viability and proliferation in MDR cells compared to DOX treatment alone. Additionally, the combination of Ebs and DOX induced DNA fragmentation and exhibited G2/M phase cell cycle arrest. Immunofluorescent analysis revealed that the Ebs and DOX combination upregulated the expression of p53 and p21, which activated the mitochondrial-dependent apoptotic pathway. The combination treatment also enhanced the upregulation of proapoptotic markers such as Bax, Caspase-3, -9, and cytochrome C, while downregulating the expression of the antiapoptotic marker Bcl-2. Therefore, the current discoveries suggest that Ebs could be employed as a drug candidate for reversing MDR in cancer cells by regulating cellular redox homeostasis., (© 2024 John Wiley & Sons Ltd.)