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Objectives: Worldwide emergence of clonal outbreaks caused by fluconazole‐resistant (FLCR) and the recent emergence of echinocandin‐ and multidrug‐resistant (ECR and MDR) Candida parapsilosis isolates pose serious threats to modern clinics. Conducting large‐scale epidemiological studies aimed at determining the genetic composition and antifungal resistance rates is necessary to devise antifungal stewardship and infection control strategies at international, national and local levels. Despite being severely hit by outbreaks due to FLCR C. parapsilosis isolates, such knowledge at the national level is lacking in Türkiye. Herein, we conducted a prospective multicentre study involving five major clinical centres in Türkiye to determine antifungal resistance rates, underlying mechanisms and genetic composition of all isolates. Methods: In total, 341 isolates were collected from 265 patients including clinical information. Antifungal susceptibility testing against common antifungals was performed in addition to sequencing of ERG11 and FKS1. Last, isolates were genotyped with short tandem repeat (STR) genotyping to investigate potential nosocomial transmission. Results: The FLCR rate was 26.7% (91/341), out of which 75.8% (69/91) harboured the ERG11Y132F mutation. Patients infected with FLCR isolates had a higher mortality rate compared to their susceptible counterparts (49% for FLCR vs. 42% for susceptible). ECR rate was 2.1% (7/341) and isolates carried FKS1F652L/R658G/W1370R mutations. Concerningly, four ECR isolates were MDR. FLCR isolates grouped in distinct clusters without evidence of inter‐hospital transmission, whereas large clusters containing susceptible isolates from all centres were noted. Conclusion: Overall, the increasing prevalence of FLCR C. parapsilosis at national level and the emergence of ECR and MDR isolates pose serious clinical challenges in Türkiye. Therefore, conducting large‐scale epidemiological studies are critical to determine the trend of antifungal resistance and to tailor pertinent antifungal stewardship and infection control strategies to effectively curb the spread of drug‐resistant C. parapsilosis. [ABSTRACT FROM AUTHOR]

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 Erg11Y132F and Erg11G458S mutations produced specific amplification of either WT or mutated alleles. We conducted a duplex real‐time PCR combining oligonucleotides for the wild‐type sequences in one mix, and oligonucleotides for the mutated alleles in another. Following this, we performed an analysis of the HRM curve to identify the amplified allele in each case. This technique was blindly evaluated on a set of 114 C. parapsilosis isolates, all of which were unequivocally identified using our approach. Conclusion: This technique offers a new method for the early detection of azole resistance mechanism in C. parapsilosis. [ABSTRACT FROM AUTHOR]

The steep increase in acquired drug resistance in Candida isolates has posed a great challenge in the clinical management of candidiasis globally. Information of genes and codon sites that are positively selected during evolution can provide insights into the mechanisms driving antifungal resistance in Candida. This study aimed to create a manually curated list of genes of Candida spp. reported to be associated with antifungal resistance in literature, and further investigate the structure-function implications of positively selected genes and mutation sites. Sequence analysis of antifungal drug resistance associated gene sequences from various species and strains of Candida revealed that ERG11 and MRR1 of C. albicans were positively selected during evolution. Four sites in ERG11 and two sites in MRR1 of C. albicans were positively selected and associated with drug resistance. These four sites (132, 405, 450, and 464) of ERG11 are predictive markers for azole resistance and have evolved over time. A well-characterized crystal structure of sterol-14-α-demethylase (CYP51) encoded by ERG11 is available in PDB. Therefore, the stability of CYP51 in complex with fluconazole was evaluated using MD simulations and molecular docking studies for two mutations (Y132F and Y132H) reported to be associated with azole resistance in literature. These mutations induced high flexibility in functional motifs of CYP51. It was also observed that residues such as I304, G308, and I379 of CYP51 play a critical role in fluconazole binding affinity. The insights gained from this study can further guide drug design strategies addressing antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Trichophyton indotineae is an emerging pathogen causing recalcitrant skin infections and exhibiting multiple resistances to azoles and allylamines. Squalene epoxidase erg1Ala448Thr mutants often show association with azole resistance. RT-PCR gene expression analysis helps to elucidate the connection between ergosterol biosynthesis regulation and efflux control through the activation of multidrug resistance (MDR) and major facilitator superfamily (MFS1) transporters as well as heat shock proteins (HSP). Several T. indotineae isolates demonstrated a heat-dependent increase of Erg11B transcripts combined with downregulation of Erg1, suggesting a protective role for Erg11B. They also showed persistent upregulation of MFS1. The addition of fluconazole or voriconazole induced the expression of Erg11A, MDR3 and, to a lesser extent, Erg11B and Erg1. The azole-resistant erg1Ala448Thr mutant UKJ 476/21 exhibited exceptionally high transcript levels of sterol 14-αdemethylase Erg11B, combined with the inability of HSP60 and HSP90 to respond to increasing growth temperatures. Itraconazole demonstrated similar effects in a few T. indotineae isolates, but terbinafine did not enhance Erg1 transcription at all. Overexpression of Erg11B may explain the multiple azole resistance phenotype, whereas Erg11B point mutations are not associated with resistance to azoles used for medical treatment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fungicide resistance is often conferred through the mutation of genes encoding fungicide targets or proteins that remove fungicides from cells, but mechanisms can vary widely between taxa. Discovering the specific resistance alleles present in pathogen populations is essential for monitoring the evolution and movement of resistant genotypes. In this study, we explored the genomic basis of demethylase inhibitor (DMI) resistance in Leptosphaeria maculans, the main pathogen of the canola crop Brassica napus. Using an international collection of over 200 genome‐sequenced isolates, we assayed in vitro sensitivity to the DMI tebuconazole and conducted a genome‐wide association study on a variant set including single‐nucleotide polymorphisms (SNPs), small indels and structural variants. The main resistance allele identified was a 237 bp remnant transposable element insertion in the promoter of the erg11/CYP51 DMI target gene in a large proportion of isolates from Europe, an allele known to confer DMI resistance in Australia. Several associated loci were identified, none of which are commonly linked to DMI resistance in other phytopathogens. We also found little to no relationship between DMI tolerance and baseline growth rate, suggesting minimal fitness effects of fungicide resistance in these isolates. This study indicates common DMI resistance alleles in L. maculans are shared across continents and erg11/CYP51 coding mutations, which are near‐ubiquitous in other fungal pathogens, may not underpin DMI resistance in this species. Furthermore, that resistance occurs frequently in numerous canola‐growing regions suggests management is essential for growers. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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

ABSTRACT The antifungal susceptibility profile and epidemiological cut-off values (ECOFFs) of Yarrowia lipolytica, a rare opportunistic yeast, remain unclear. We conducted a comprehensive multi-method study on clinical isolates from various central hospitals, based on the China Hospital Invasive Fungal Surveillance Network (2009–2022). Our objective was to evaluate the antifungal susceptibility of Y. lipolytica, establish its local ECOFFs (L-ECOFFs), and compare the performance of the ATB FUNGUS 3 (ATB), Sensititre YeastOne (SYO), and minimum inhibitory concentration (MIC) test strip (MTS) with that of the broth microdilution (BMD) method. L-ECOFFs were established using ECOFFinder, and we examined ERG11 mutations to assess the reliability of the L-ECOFFs. The L-ECOFF for fluconazole was 8 µg/mL. Non-wild-type isolates of antifungal drugs, such as flucytosine and azoles, were exclusively isolated from patients. Additionally, we detected that four strains with the ERG11 A395T mutation (azole MIC >L-ECOFF) may be associated with the exposure to azole drugs. For azoles, ATB showed the highest essential agreement with the BMD (98.18%–100%), followed by SYO (85.45%–100%). However, ATB could not detect susceptibility to echinocandins, while SYO exhibited the highest agreement (98.18%–100%) in detecting echinocandin susceptibility. Our findings indicate that acquired azole cross-resistance has emerged despite Y. lipolytica infections being rare. This research provides crucial antifungal susceptibility data and establishes the initial L-ECOFFs for Y. lipolytica. The SYO is recommended as the optimal laboratory antifungal susceptibility testing method for Y. lipolytica, followed by ATB, whereas the use of MTS requires caution. We hope that this study will facilitate improved clinical management of Y. lipolytica infections. 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.

Treatment options for Candidiasis include polyenes, azoles, echinocandins, nucleoside analogues, and allylamines, but drug resistance is a growing problem. Some species, like C. krusei and C. glabrata, exhibit high resistance to fluconazole and azoles. Accurate identification of Candida species and their susceptibility patterns is crucial for effective treatment. Mutations in the ERG11 gene, responsible for lanosterol 14a-demethylase enzyme production, lead to azole drug resistance, reducing their efficacy. This study focuses on identifying ERG11 gene mutations, offering prognostic and therapeutic significance in preventing drug resistance emergence. The triazole resistant Candida albicans isolates were selected and the DNA is extracted. ERG11 gene is amplified and sequenced to find point mutations and the substituted amino acids by using ABI 3500 XL genetic analyzer. A total of 83 mutations in the ERG11 gene of 10 isolates of C. albicans which were resistant to Fluconazole, Voriconazole and Itraconazole were detected in this study. Of the 83 mutations, 56 mutations resulted in change in amino acid (missense mutations) and 27 were silent mutations where the change in nucleotide sequence did not result in any change in the amino acid. D116E, E266D and G464S mutations occurred 6 times each in the 10 isolates. K128T, I147T mutations were found in 4 isolates. A114S, S405F, T229A, G465S, K143E mutations were found in 3 isolates. R467K, R523G, V488I, W520C mutations were found in 2 isolates. This study further establishes that the ERG11 gene point mutations are one of the major causes of azole antifungal drug resistance in Candida albicans. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

Background 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.

Introduction: Candida palmioleophila is a rare human pathogenic fungus, which has been poorly characterized at the genome level. In this study, we reported the first fatal case of C. palmioleophila infection in China and investigate the microevolution of C. palmioleophila in the human host environment. Methods: A series of C. palmioleophila stains were collected from the patient at different time points for routine microbial and drug sensitivity testing. The first C. palmioleophila isolate 07202534 was identified by de novo whole genome sequencing. The in vitro and in vivo genetic evolutionary characteristics of C. palmioleophila were discussed based on the analysis of bioinformatics data. Results: The six C. palmioleophila isolates displayed dose-dependent sensitivity to fluconazole. The C. palmioleophila genome contained homologous genes such as CDR1 and MDR1, which were recognized to be related to azole resistance. In addition, amino acid variation was detected at F105L and other important sites of ERG11. In addition, the mean divergence time between C. palmioleophila and Scheffersomyces stipites CBS 6054 was 406.04 million years, indicating that C. palmioleophila originated earlier than its closest relative. In addition, the six strains of C. palmioleophila isolated form the patient had higher homology and fewer mutation sites, which indicated the stability in C. palmioleophila genome. We also found that C. palmioleophila had a wide natural niche and may evolve slowly. Discussion: We believe that this study will contribute to improve our understanding of the genetic evolution, pathogenicity, and drug resistance of C. palmioleophila and will aid in the prevention and control of its spread. [ABSTRACT FROM AUTHOR]

Background and Aim: Nowadays, non-albicans Candida are common in human pathogens, and some of these cases were found in milk. Therefore, as well as the lack of accurate estimates of its global prevalence and severity, the present study aims to assess the demographic features of non-albicans Candida (NAC) spp. and determine the species distribution of NAC. It also evaluated the in vitro Azole susceptibility of NAC species and identified the erg11 gene and erg11 expression in fluconazole-resistant isolates of NAC spp., in Iran. Materials and Methods: In the present study, non-albicans Candida, including Candida glabrata, Candia krusei, Candida parapsilosis, and Candida tropicalis, were isolated and identified from 14 farms (raw milk) and human patients using culture methods, Real-Time PCR and sequencing. The resistance and susceptibility of the samples to azole were examined and erg11 expression was evaluated by RT-qPCR. The results were analyzed by REST Software to compare the levels of erg11 gene expression involved in drug resistance of NAC. Results: 74 and 52 NAC strains were isolated in 262 collected milk samples and human samples. Based on ITS sequencing, 0.76% were identified as C. glabrata, 2.29% as C. tropicalis, 4.19% as C. parapsilosis, and 19.8% as C. krusei. The expression of the erg11 gene in the NAC was increased in samples isolated from humans compared to samples isolated from livestock (P>0.05), while no significant difference was found in the case of Candida glabrata isolated from both sources (P<0.05). All NAC isolates were sensitive to flucytosine. Conclusion: non-albicans Candida (NAC) isolates from cows' milk have antifungal resistance genes while they had not taken any antifungal drugs. The resistance gene is transferred from antifungal agents in crop protection medications. Clinical isolates also had increased resistance to antifungal activity. Also, using Azole antibiotics can increase resistance gene level activity. This phenomenon should be considered for treatment program protocols. [ABSTRACT FROM AUTHOR]

Introduction. Interactions between the 2 microbiota components – bacteria and fungi – are of interest as diagnostic and prognostic markers in selection of treatment tactics for oncological patients.Aim. To study microbiota of the oral cavity in patients with primary squamous cell carcinoma of the oropharyngeal area before and after surgical intervention to find biomarkers for rational selection of antifungal drugs.Materials and methods. At the Surgical Department of Head and Neck Tumors of the N. N. Blokhin National Research Center of Oncology, three-component study was performed: investigations of spectrum of Candida spp. isolates, Candida spp. strains’ resistance to antifungals, and oral washes in primary patients before and after surgery. mALDI-Tof microflex LT (Biotyper, Bruker Daltonics, germany) was used for strain identification; Sensititre Yeast ONE, YO10 (Trek Diagnostic System, united kingdom) plates were used for determination of minimal inhibiting concentrations of anti fungals. values of minimal inhibiting concentrations were evaluated based on the European Committee on Antimicrobial Susceptibility Testing (EuCAST) criteria (version 10.0).Results. four-year observation of patients at the surgical department of head and neck tumors of the N. N. Blokhin National Research Center of Oncology showed that the most common species of Candida is C. albicans (73.5 % of cases). Candida spp. resistance to antifungals was detected only for fluconazole (9.3 % of cases) and micafungin (8.0 % of cases), mostly among C. albicans strains. In 31.8 % of primary patients, oral washes prior to surgery showed growth of Candida spp. (probably, tissue colonization). After surgical intervention, Candida spp. growth was detected in 36.4 % of cases, only 1 of which was diagnosed as invasive mycosis. In 54.5 % of cases before and in 72.7 % of cases after surgery, gram-negative rods were detected. After surgical intervention, percentage of enterobacteria and non-fermenters significantly increased: 59.1 % versus 27.3 % (p

Candida fungi are common opportunistic microorganisms capable of causing infections of various localization, as well as life-threatening conditions in immunocompromised patients, such as HIV-infected individuals, oncology patients, subjects undergoing HSCT, which number has been steadily increasing in recent years. In addition, resistance to anti-fungal drugs has been spreading as well. Naturally sensitive to azoles, C. albicans possess a variety of mechanisms of acquired resistance, including efflux transporters and target protein-encoding gene amplification. This study was conducted to assess a prevalence of such mechanisms in the isolates sample obtained from HIV-infected patients in the Moscow region of the Russian Federation, characterize a relationship between these mechanisms and patterns of developing drug resistance. 18 strains of C. albicans resistant to fluconazole and voriconazole were isolated from HIV-infected patients with recurrent oropharyngeal candidiasis in the Moscow region. The expression levels of the ERG11, MDR1, CDR1, CDR2 genes involved in the formation of acquired azole resistance were measured using quantitative PCR, the 2CT method with ACT and PMA genes as control genes and reference values of sensitive isolates. Expression levels exceeding the average values of sensitive isolates by more than 3 standard deviations were considered significantly elevated. In most of the isolates, elevated levels of CDR1 and CDR2 gene expression were found: 89% and 78%, respectively. The expression level of the MDR1 gene was increased only in 28% of cases. ERG11 expression levels were significantly elevated in 78% of the isolates. Expression levels of all resistance genes studied were significantly increased in 4 strains. In this sample of C. albicans isolates, acquired resistance is mainly associated with efflux vectors encoded by the CDR1 and CDR2 genes. Also, in most isolates, an increased expression level for the azole target protein gene ERG11 was detected. The expression level of the efflux transporter gene MDR1 was increased in the smallest number of samples. It is also impossible to exclude a potential role of other mechanisms in developing acquired resistance, such as mutations in the ERG11 gene. It can be assumed that the identified mechanisms of resistance result from long-term, widespread, and sometimes uncontrolled use of azoles, including those in treatment and prevention of candidiasis in HIV-infected patients.

ABSTRACT Cryptococcus neoformans and Cryptococcus gattii cause cryptococcosis, a life-threatening fungal infection affecting mostly immunocompromised patients. In fact, cryptococcal meningitis accounts for about 19% of AIDS-related deaths in the world. Because of long-term azole therapies to treat this mycosis, resistance to fluconazole leading to treatment failure and poor prognosis has long been reported for both fungal species. Among the mechanisms implicated in resistance to azoles, mutations in the ERG11 gene, encoding the azole target enzyme lanosterol 14-α-demethylase, have been described. This study aimed to establish the amino acid composition of ERG11 of Colombian clinical isolates of C. neoformans and C. gattii and to correlate any possible substitution with the in vitro susceptibility profile of the isolates to fluconazole, voriconazole, and itraconazole. Antifungal susceptibility testing results showed that C. gattii isolates are less susceptible to azoles than C. neoformans isolates, which could correlate with differences in the amino acid composition and structure of ERG11 of each species. In addition, in a C. gattii isolate with high MICs for fluconazole (64 μg/mL) and voriconazole (1 μg/mL), a G973T mutation resulting in the substitution R258L, located in substrate recognition site 3 of ERG11, was identified. This finding suggests the association of the newly reported substitution with the azole resistance phenotype in C. gattii. Further investigations are needed to determine the exact role that R258L plays in the decreased susceptibility to fluconazole and voriconazole, as well as to determine the participation of additional mechanisms of resistance to azole drugs. IMPORTANCE The fungal species Cryptococcus neoformans and C. gattii are human pathogens for which drug resistance or other treatment and management challenges exist. Here, we report differential susceptibility to azoles among both species, with some isolates displaying resistant phenotypes. Azoles are among the most commonly used drugs to treat cryptococcal infections. Our findings underscore the necessity of testing antifungal susceptibility in the clinical setting in order to assist patient management and beneficial outcomes. In addition, we report an amino acid change in the sequence of the target protein of azoles, which suggests that this change might be implicated in resistance to these drugs. Identifying and understanding possible mechanisms that affect drug affinity will eventually aid the design of new drugs that overcome the global growing concern of antifungal resistance.

Neuromeningeal cryptococcosis is a life-threatening infection of the central nervous system, caused by encapsulated yeast belonging to the Cryptococcus neoformans and Cryptococcus gattii species complexes. Recent data showed that virulence and antifungal resistance are variable for yeasts belonging to the C. gattii species complex. There is an increase in resistance to fluconazole for yeasts of the C. gattii species complex and the virulence is variable according to the genotype. In the present study, (i) we explored and compared the mechanisms of resistance to fluconazole between C. deuterogattii clinically resistant strains and induced fluconazole-resistant strains by exposure to fluconazole in vitro, and (ii) we studied their virulence in the Galleria mellonella study model. We demonstrated that the fluconazole resistance mechanisms involved were different between clinically resistant strains and induced resistant strains. We also demonstrated that fluconazole-induced resistant strains are less virulent when compared to the original susceptible strains. On the contrary, the clinically resistant strain tested maintains its virulence compared to fluconazole-susceptible strains of the same sequence type. [ABSTRACT FROM AUTHOR]

Arome Solomon Odiba,1,2 Olanrewaju Ayodeji Durojaye,3– 5 Ifeoma Maureen Ezeonu,6 Anthony Christian Mgbeahuruike,7 Bennett Chima Nwanguma1,2 1Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, 410001, Nigeria; 2Department of Molecular Genetics and Biotechnology, University of Nigeria, Nsukka, Enugu State, 410001, Nigeria; 3Department of Chemical Sciences, Coal City University, Emene, Enugu State, Nigeria; 4Department of Molecular and Cell Biology, University of Science and Technology of China, Hefei, Anhui, 230026, People’s Republic of China; 5MOE Key Laboratory of Membraneless Organelle and Cellular Dynamics, Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China; 6Department of Microbiology, Faculty of Biological Sciences, University of Nigeria, Nsukka, Enugu State, 410001, Nigeria; 7Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State, 410001, NigeriaCorrespondence: Bennett Chima Nwanguma; Anthony Christian Mgbeahuruike, Department of Biochemistry, Faculty of Biological Sciences, University of Nigeria, Enugu State, Nsukka, 410001, Nigeria, Tel +234 8063655062, Email bennett.nwanguma@unn.edu.ng; anthony.mgbeahuruike@unn.edu.ngBackground: Resistance to antifungal drugs for treating Candida infections remains a major concern globally despite the range of medications available. Most of these drugs target key proteins essential to the life cycle of the organism. An enzyme essential for fungal cell membrane integrity, lanosterol 14–α demethylase (CYP51), is encoded by the ERG11 gene in Candida species. This enzyme is the target of azole–based drugs. The organism has, however, devised molecular adaptations to evade the activity of these drugs.Materials and Methods: Classical methods were employed to characterize clinical isolates sampled from women and dogs of reproductive age. For fluconazole efficacy studies, CLSI guidelines on drug susceptibility testing were used. To understand the susceptibility pattern, various molecular and structural analytic approaches, including sequencing, in silico site-directed mutagenesis, and protein-ligand profiling, were applied to the ERG11 gene and CYP51 protein sequences. Several platforms, comprising Clustal Omega, Pymol plugin manager, Pymol molecular visualizer, Chimera–curated Dynameomics rotamer library, protein–ligand interaction profiler, Charmm36 force field, GROMACS, Geneious, and Mega7, were employed for this analysis.Results: The following Candida species distribution was obtained: 37.84% C. albicans, 8.12% C. glabrata, 10.81% C. krusei, 5.41% C. tropicalis, and 37.84% of other unidentified Candida species. Two codons in the nucleotide sequence of the wild-type (CTC and CCA) coding for LEU– 370 and PRO– 375, respectively, were mutated to L370S and P375H in the resistant strain. The mutation stabilized the protein at the expense of the heme moiety. We found that the susceptible isolate from dogs (Can–iso– 029/dog) is closely related to the most resistant isolate from humans.Conclusion: Taken together, our results showed new mutations in the heme-binding pocket of caCYP51 that explain the resistance to fluconazole exhibited by the Candida isolates. So far, the L370S and P375H resistance-linked mutations have not been previously reported.Keywords: Candida infection, CYP51, drug resistance, ERG11, fluconazole, mutation

Relevance. The global trend of rapid increase in resistance to antifungal drugs due to multiple factors, dictates the need for continuous monitoring of taxonomic structure and susceptibility of nosocomial pathogens, causing invasive fungal infections, for permanent correction of the optimal prevention and treatment strategies.Purpose: to determine antifungal susceptibility of the main yeast pathogens in candidemia in cancer patients, as well as to determine resistance genes and pathogenic factor genes.Material and Methods. Eighty-two strains of Candida spp. isolated from blood of cancer patients from 2015 to 2021 were analyzed. Minimum inhibitory concentrations of fuconazole, voriconazole, posaconazole, anidulafungin and micafungin were determined by a gradient method (E-test, BioMerieux, France). The EUCAST and CLSI criteria were used for MIC value assessment. The genes, associated with pathogenicity factors, and resistance to antifungal drugs were identifed.Results. Our study results based on EUCAST 2020, v.10.0 criteria showed that triazoles, especially fuconazole, were the least effective drugs in empirical therapy for invasive candidiasis (including candidemia). Resistance of Candida spp. fuconazole was superior to that of voriconazole (47.2 % vs 23.2 %, respectively, p0.05). The ERG11 and FKS1 genes associated with resistance to antifungal drugs were detected in 28.6 % of Candida spp. strains. The ERG11 gene was detected in 8.6 % of cases, exclusively in Candida albicans strains. The FKS1 gene was identifed in 20.0 % of strains (85.7 % of them were C. parapsilosis, 7.1 % each were C. tropicalis and C. glabrata). Pathogenic factor genes were identifed in 78.6 % of C. albicans and in 79.1 % of C. parapsilosis strains.Conclusion. Molecular genetic methods for the detection of Candida spp strains carrying resistance genes to antifungal drugs, and the determination of pathogenicity factors are promising trends in searching for biomarkers. They facilitate interpretation of results of microbiological study to assess the ability of Candida spp. strains to develop invasive mycoses.

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

Introduction: The rapid rise of azole resistance in Candida tropicalis causing invasive infections has become a public health concern; however, the prevalence of resistant isolates in urine samples was not well studied, because the clinical significance of candiduria was not unambiguous due to possible host colonization. Methods: We performed a 12-year laboratory-based surveillance study of C. tropicalis causing either invasive infection or candiduria and studied their susceptibility profiles to common antifungal drugs. The complete coding domain sequence of the ERG11 gene was amplified in all fluconazole resistant isolates, and aligned with the wild-type sequence to detect nucleotide mutations. Results: A total of 519 unique C. tropicalis strains isolates, 69.9% of which were isolated from urine samples and remaining 30.1% were invasive strains. Overall, 16.5% isolates were confirmed to be resistant to fluconazole, of which 91.9% were cross-resistant voriconazole. Of note, at the beginning of surveillance (2010-2011), the fluconazole resistance rates were low in both candiduria and invasive groups (6.8% and 5.9%, respectively). However, the resistant rate in the candiduria group significantly increased to 29.5% since 2012-2013 (p = 0.001) and stayed high since then, whilst the resistance rate in the invasive group only showed a gradually increasing trends till 2021 (p > 0.05). Sequence analysis of ERG11 from fluconazoleresistant strains revealed the prevalence of A395T/W mutations were relatively low (16.7%) in the beginning but reached 87.5-100% after 2014. Moreover, the A395W heterozygous mutation isolates became predominant (>60% of resistant strains) after 2016, and indeed isolates carrying corresponding amino acid substitution (Y132F) was highly resistant to fluconazole with MIC50 exceeded 256 µg/ml. Conclusion: Our study revealed high azole resistant rate in candiduria with its increasing trends observed much earlier than stains causing invasive infections. Given antimicrobial resistance as a critical "One Health" issue, the emergence of antifungal resistance in Candida species that are common commensal colonizers in the human body should be concerned. [ABSTRACT FROM AUTHOR]

Background: Epidemiological surveillance of Candidozyma sp. has become important because many species of this new genus have been reported to be responsible for nosocomial outbreaks and to exhibit elevated minimal inhibitory concentrations (MIC) to one or more classes of antifungal drugs., Objectives: To describe the genetic relationships among Argentinian clinical isolates belonging to the Candidozyma genus and to study the molecular mechanisms associated with antifungal resistance., Methods: We performed whole-genome sequencing of 41 isolates. Identification was based on ribosomal DNA sequencing and susceptibility testing was determined according to the EUCAST document. Phylogenetic analysis, non-synonymous mutations in genes associated with antifungal resistance and the presence of copy number variations (CNVs) were investigated., Results: We identified 12 Candidozyma haemuli, 11 Candidozyma haemuli var. vulneris, 5 Cz. haemuli/ Cz. haemuli var. vulneris ITS hybrids, 8 Candidozyma duobushaemuli and 5 Candidozyma cf. pseudohaemuli. Phylogenetic analysis, together with clinical data, demonstrated nosocomial transmission events. In addition, Cz. haemuli and Cz. haemuli var. vulneris were not separated in the phylogenetic tree; the Cz. cf. pseudohaemuli isolates clustered distantly from the Cz. pseudohaemuli type strain. Most isolates were resistant to amphotericin B, and two Cz. haemuli isolates showed fluconazole resistance and Y132F mutation in ERG11. We did not find CNV in genes associated with antifungal resistance., Conclusions: These findings highlight the need for epidemiological surveillance of these species and the study of molecular mechanisms associated with antifungal resistance. Furthermore, we propose a taxonomic revision for Cz. haemuli var. vulneris and Cz. pseudohaemuli based on genomic data., (© 2025 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

MethodsOver a four-year period, 123 Candida bloodstream isolates were collected at a quaternary care hospital. The isolates were identified by MALDI-TOF MS and their fluconazole (FLC) susceptibility patterns were assessed according to CLSI guidelines. Subsequently, sequencing of ERG11, TAC1 or MRR1, and efflux pump activity were performed for resistant isolates.ResultsOut of 123 clinical strains,C. albicans accounted for 37.4%, followed by C. tropicalis 26.8%, C. parapsilosis 19.5%, C. auris 8.1%, C. glabrata 4.1%, C. krusei 2.4% and C. lusitaniae 1.6%. Resistance to FLC reached 18%; in addition, a high proportion of isolates were cross-resistant to voriconazole. Erg11 amino acid substitutions associated with FLC-resistance (Y132F, K143R, or T220L) were found in 11/19 (58%) of FLCresistant isolates. Furthermore, novel mutations were found in all genes evaluated. Regarding efflux pumps, 8/19 (42%) of FLC-resistant Candida spp strains showed significant efflux activity. Finally, 6/19 (31%) of FLC-resistant isolates neither harbored resistance-associated mutations nor showed efflux pump activity. Among FLC-resistant species, C. auris 7/10 (70%) and C. parapsilosis 6/24 (25%) displayed the highest percentages of resistance (C. albicans 6/46, 13%).DiscussionOverall, 68% of FLC-resistant isolates exhibited a mechanism that could explain their phenotype (e.g. mutations, efflux pump activity, or both). We provide evidence that isolates from patients admitted to a Colombian hospital harbor amino acid substitutions related to resistance to one of the most commonly used molecules in the hospital setting, with Y132F being the most frequently detected.

Candida haemulonii var. vulnera is a rare variant of C. haemulonii, which has been previously reported to cause human infections. Owing to the close kinship between C. haemulonii sensu stricto and C. haemulonii var. vulnera, accurate identification of C. haemulonii var. vulnera relied on DNA sequencing assay targeting, for example, rDNA internal transcribed spacer (ITS) region. In this work, two strains of C. haemulonii var. vulnera were collected from the China Hospital Invasive Fungal Surveillance Net (CHIF-NET). The identification capacity of three matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and VITEK 2 YST ID biochemical methods were evaluated against ITS sequencing. In addition, antifungal susceptibility testing was performed using Sensititre YeastOne. Moreover, we comprehensively screened drug-resistant related genes by whole-genome sequencing. The two strains were not correctly identified to species variant level using MALDI-TOF MS and YST ID cards. Both strains were resistant to amphotericin B (minimum inhibitory concentration [MIC] > 2 µg/ml). Moreover, strain F4564 and F4584 exhibited high MIC to fluconazole (>256 µg/ml) and 5-flucytosine (>64 µg/ml), respectively, which were supposed to result from key amino acid substitutions Y132F and G307A in Erg11p and V58fs and G60K substitutions in Fur1p. The rare species C. haemulonii var. vulnera has emerged in China, and such drug-resistant fungal species that can cause invasive diseases require further close attention. [ABSTRACT FROM AUTHOR]

Candida parapsilosis is a leading cause of invasive candidiasis in southern Europe, Latin America and Asia. C. parapsilosis has been mostly considered susceptible to triazoles, but fluconazole resistance is on the rise in some countries. The main mechanism related to fluconazole resistance is the presence of ERG11p substitutions, dominated by the Y132F amino acid substitution. Isolates harbouring this substitution mimic C. auris given that they may cause hospital outbreaks, become endemic, and emerge simultaneously in distant areas around the world. At the moment, Spain is experiencing a brusque emergence of fluconazole resistance in C. parapsilosis; isolates harbouring the Y132F substitution were detected for the first time in 2019. A recent study on Candida spp isolates from blood cultures collected in 16 hospitals located in the Madrid metropolitan area (2019 to 2021) reported that fluconazole resistance in C. parapsilosis reached as high as 13.6%. Resistance rates rose significantly during those three years: 3.8% in 2019, 5.7% in 2020, and 29.1% in 2021; resistant isolates harboured either the dominant Y132F substitution (a single clone found in four hospitals) or G458S (another clone found in a fifth hospital). The COVID-19 pandemic may have increased the number of candidaemia cases. The reason for such an increase might be a consequence of uncontrolled intra-hospital patient-to-patient transmission in some hospitals, as an increase not only in C. parapsilosis candidaemia episodes but also in the spread of clonal fluconazole-resistant isolates might have occurred in other hospitals during the pandemic period. Patients affected with fluconazole-resistant C. parapsilosis harbouring the Y132F substitution presented a mortality rate ranging from 9% to 78%, were mainly admitted to intensive care wards but did not have differential risk factors compared to those infected by susceptible isolates. With scarce exceptions, few patients (≤20%) infected with fluconazole-resistant isolates had previously received fluconazole, thus supporting the fact that, although fluconazole might have been a key factor to promote resistance, the main driver promoting the spread of fluconazole-resistant isolates was patient-to-patient transmission.

Abstract Background Candida albicans, the main human fungal pathogen, can cause fungal infection and seriously affect people's health and life. This study aimed to investigate the effects of ritonavir (RIT) on C. albicans and the correlation between SAP2 as well as ERG11 and drug resistance. Results Secreted aspartyl proteinases (Saps) activities and pathogenicity of C. albicans with different drug resistance were measured. M27‐A4 broth microdilution method was used to analyze the drug sensitivity of RIT combined with fluconazole (FCA) on C. albicans. After that, SAP2 and ERG11 mutations were examined by polymerase chain reaction (PCR) and sequencing, and quantitative real‐time PCR was utilized to determine the expression of the two genes. By analyzing pz values, the Saps activity of cross‐resistant strains was the highest, followed by voriconazole (VRC)‐resistant strains, FCA‐resistant strains, itraconazole (ITR)‐resistant strains, and sensitive strains. The pathogenicity of C. albicans in descending order was as follows: cross‐resistant strains, VRC‐resistant strains, ITR‐resistant strains, FCA‐resistant strains, and sensitive strains. With the increase of RIT concentrations, the Saps activity was gradually inhibited. Drug sensitivity results showed that there was no synergistic effect between RIT and FCA. Additionally, no gene mutation sites were found in SAP2 sequencing, and 17 synonymous mutations and 6 missense mutations occurred in ERG11 sequencing. Finally, the expression of SAP2 and ERG11 was significantly higher in the resistant strains compared with the sensitive strains, and there was a positive liner correlation between SAP2 and ERG11 messenger RNA expression (r = .6655, p

Histoplasmosis has been previously diagnosed in animals from Europe. The aim of this study is to review the literature on these reports, to analyze cases diagnosed at our laboratory (2000–2022) and to improve molecular typing of Histoplasma capsulatum directly from tissue to study the molecular epidemiology of Histoplasma capsulatum causing animal infections in Europe. Including 15 cases studied in our laboratory, we identified 39 cases of animal histoplasmosis between 1968 and 2022. They were diagnosed mostly in superficial tissue biopsies from cats and badgers from Central Europe. Using phylogenetic analyses of six partial genes, we were able to classify eight of the etiological agents as belonging to a highly supported lineage within the Eurasian clade. This study confirms the occurrence of autochthonous histoplasmosis in animals in Central Europe and proposes the addition of new loci to the MLST scheme to study the molecular epidemiology of histoplasmosis using either formalin-fixed paraffin-embedded tissue and fresh or cadaveric biopsies. [ABSTRACT FROM AUTHOR]

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

Candida auris is an emerging pathogenic fungal species found worldwide. Since April 2016, C. auris colonization/infection cases have been found in a general hospital in Shenyang, China. The genome-based phylogenetic studies of these isolates remain undefined. In the current study, the microbiological characteristics and antifungal susceptibility of these C. auris isolates, which were collected in Shenyang during the three-year period (2016–2018), were investigated. Whole-genome sequencing was applied to investigate the genetic variation and molecular epidemiological characteristics. A total of 93 C. auris isolates, including 92 clinical isolates and 1 environmental screening isolate were identified. Among the investigated wards, the C. auris cases were the most prevalent (97.4%, 37/38) in four intensive care units (ICUs). The Shenyang isolates carrying the VF125AL mutation in the key drug-resistance gene ERG11 were mainly fluconazole resistant and formed a distinct subclade under the South African clade according to the phylogenetic and population structural analyses. In addition, the Shenyang subclade was found to be closely related to the British subclade in the aspect of genetic distance. As a conclusion, this study provides an important clue for revealing the origin of C. auris found in Shenyang and could also contribute to improve the understanding of the epidemiological characteristics of C. auris worldwide.

Fluconazole is the drug of choice for cryptococcal meningitis (CM) monoprophylaxis in resource-limited settings such as Uganda. Emerging fluconazole resistance linked to mutations in the Cryptococcus neoformansERG11 gene (CYP51) has been observed in clinical isolates. Currently, the single nucleotide polymorphisms [SNPs] in the Cryptococcus spp. ERG11 gene that could be responsible for fluconazole resistance are poorly characterized within Ugandan C. neoformans clinical isolates. If available, this information would be useful in the management of cryptococcosis among HIV patients. This cross-sectional study investigates the SNPs present in the coding region of the C. neoformansERG11 gene to determine the relationship between the SNPs identified and fluconazole susceptibility of the clinical isolates. 310 C. neoformans isolates recovered from the Cerebrospinal Fluid (CSF) of patients with HIV and cryptococcal meningitis were examined. The fluconazole half-maximal inhibitory concentrations (IC50 range: 0.25–32 μg/mL) was determined using the microbroth dilution method. A total of 56.1% of the isolates had low IC50 values of <8 μg/mL while 43.9% had high IC50 values ≥ 8 μg/mL. We amplified and sequenced 600 bp of the ERG11 coding sequence from 40 of the clinical isolates. Novel synonymous and 2 missense mutations, S460T and A457V, were identified in the ERG11 gene. The identified SNPs were not associated with differences in fluconazole IC50 values in vitro (p = 0.179). [ABSTRACT FROM AUTHOR]

Objective To screen the genes associated with fluconazole resistance in Trichosporon asahii to facilitate mechanistic studies of fluconazole resistance and the development of new antifungal agents. Methods Three clinical isolates of fluconazole-sensitive Trichosporon asahii were cultured in the presence of increasing concentrations of fluconazole until the drug concentration reached 128 μg/mL. After further culture for 30 generations in the absence of fluconazole, the MIC value of the induced bacteria was measured. The total RNA was extracted from both the sensitive and resistant strains of the bacteria for construction of the stranded RNA-Seq libraries and transcriptome sequencing to identify the differentially expressed genes (DEGs) in the resistant strains. The genomes of the 3 sensitive and resistant strains were sequenced for comparison of the sequence of the differentially expressed ERG11 gene. Results We successfully obtained stable Trichosporon asahii strains with fluconazole resistance. Transcriptome sequencing data showed that the genes associated with drug transport, ergosterol synthesis and antioxidation were up-regulated in the resistant strains, and among them ERG11 gene, the target gene of fluconazole, was up-regulated by 3.7 folds. Genomic sequencing proved no mutations in the sequence of ERG11 gene in the 3 resistant strains. Conclusion The molecular mechanism for drug resistance in Trichosporon asahii is complex. The upregulation of ergosterol synthesis pathway, especially ERG11 gene, along with other genes associated with efflux transfer and oxidative stress, is one of the main mechanisms for drug resistance in Trichosporon asahii.

ABSTRACT Fungal infections contribute to over 1.5 million deaths annually, with Candida albicans representing one of the most concerning human fungal pathogens. While normally commensal in nature, compromise of host immunity can result in C. albicans disseminating into the human bloodstream, causing infections with mortality rates of up to 40%. A contributing factor to this high mortality rate is the limited arsenal of antifungals approved to treat systemic infections. The most widely used antifungal class, the azoles, inhibits ergosterol biosynthesis by targeting Erg11. The rise of drug resistance among C. albicans clinical isolates, particularly against the azoles, has escalated the need to explore novel antifungal strategies. To address this challenge, we screened a 9,600-compound subset of the University of Tokyo Core Chemical Library to identify molecules with novel antifungal activity against C. albicans. The most potent hit molecule was CpdLC-6888, a 2,5-disubstituted pyridine compound, which inhibited growth of C. albicans and closely-related species. Chemical-genetic, biochemical, and modeling analyses suggest that CpdLC-6888 inhibits Erg11 in a manner similar to the azoles despite lacking the canonical five-membered nitrogen-containing azole ring. This work characterizes the antifungal activity of a 2,5-disubstituted pyridine against C. albicans, supporting the mining of existing chemical collections to identify compounds with novel antifungal activity. IMPORTANCE Pathogenic fungi represent a serious but underacknowledged threat to human health. The treatment and management of these infections relies heavily on the use of azole antifungals, a class of molecules that contain a five-membered nitrogen-containing ring and inhibit the biosynthesis of the key membrane sterol ergosterol. By employing a high-throughput chemical screen, we identified a 2,5-disubstituted pyridine, termed CpdLC-6888, as possessing antifungal activity against the prominent human fungal pathogen Candida albicans. Upon further investigation, we determined this molecule exhibits azole-like activity despite being structurally divergent. Specifically, transcriptional repression of the azole target gene ERG11 resulted in hypersensitivity to CpdLC-6888, and treatment of C. albicans with this molecule blocked the production of the key membrane sterol ergosterol. Therefore, this work describes a chemical scaffold with novel antifungal activity against a prevalent and threatening fungal pathogen affecting human health, expanding the repertoire of compounds that can inhibit this useful antifungal drug target.

Neuromeningeal cryptococcosis is a life-threatening infection of the central nervous system, caused by encapsulated yeast belonging to the Cryptococcus neoformans and Cryptococcus gattii species complexes. Recent data showed that virulence and antifungal resistance are variable for yeasts belonging to the C. gattii species complex. There is an increase in resistance to fluconazole for yeasts of the C. gattii species complex and the virulence is variable according to the genotype. In the present study, (i) we explored and compared the mechanisms of resistance to fluconazole between C. deuterogattii clinically resistant strains and induced fluconazole-resistant strains by exposure to fluconazole in vitro, and (ii) we studied their virulence in the Galleria mellonella study model. We demonstrated that the fluconazole resistance mechanisms involved were different between clinically resistant strains and induced resistant strains. We also demonstrated that fluconazole-induced resistant strains are less virulent when compared to the original susceptible strains. On the contrary, the clinically resistant strain tested maintains its virulence compared to fluconazole-susceptible strains of the same sequence type.

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.

A feature of Candida tropicalis is its ability to undergo phenotypic switching that can affect antifungal sensitivity and virulence traits. Here, we investigated the effect of switching on alterations at the cellular structure level of C. tropicalis morphotypes and whether exposure to fluconazole (FLC) in vitro could be associated with these alterations in a morphotype-dependent manner. Candida tropicalis morphotypes included clinical isolate (Parental) and two switch strains (Crepe variant and revertant of Crepe-RC). The minimum inhibitory concentration (MIC50) of fluconazole was determined according to EUCAST. Cell wall porosity, quantification of cell wall components, cell size/complexity, and expression of ERG11 and CDR1 genes in morphotypes pre- and post-exposure to fluconazole were determined. Crepe and RC showed an eightfold higher MIC50 (1 µg/ml) than the Parental (0.125 µg/ml). Exposure to FLC resulted in twofold higher MIC50 for Parental and RC. The Crepe variant exhibited a fourfold higher expression of ERG11, and the RC showed 10-fold higher expression of CDR1 than the clinical isolate. Switch strains showed reduced cell wall porosity compared to Parental, and exposure to FLC resulted in a significant reduction in the porosity of Parental and RC cells. Furthermore, phenotypic switching affected cell wall β-1,3-glucan and chitin contents in a morphotype-dependent manner. Our findings indicate that switching affects cellular structure in C. tropicalis and the occurrence of differential alterations between the clinical isolate and its switched states in response to fluconazole exposure., (© The Author(s) 2024. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

ABSTRACT Resistance to fluconazole is one of clinical characteristics most frequently challenging the treatment of invasive Candida auris infections, and is observed among >90% of all characterized clinical isolates. In this work, the native C. auris ERG11 allele in a previously characterized fluconazole-susceptible clinical isolate was replaced with the ERG11 alleles from three highly fluconazole-resistant clinical isolates (MIC ≥256 mg/L), encoding the amino acid substitutions VF125AL, Y132F, and K143R, using Cas9-ribonucleoprotein (RNP) mediated transformation system. Reciprocally, the ERG11WT allele from the same fluconazole-susceptible clinical isolate, lacking any resistance-associated mutation, was introduced into a previously characterized fluconazole-resistant clinical isolate, replacing the native ERG11K143R allele, using the same methods. The resulting collection of strains was subjected to comprehensive triazole susceptibility testing, and the direct impact each of these clinically-derived ERG11 mutations on triazole MIC was determined. Introduction of each of the three mutant ERG11 alleles was observed to increase fluconazole and voriconazole MIC by 8- to 16-fold. The MIC for the other clinically available triazoles were not significantly impacted by any ERG11 mutation. In the fluconazole-resistant clinical isolate background, correction of the K143R encoding mutation led to a similar 16-fold decrease in fluconazole MIC, and 8-fold decrease in voriconazole MIC, while the MIC of other triazoles were minimally changed. Taken together, these findings demonstrate that mutations in C. auris ERG11 significantly contribute to fluconazole and voriconazole resistance, but alone cannot explain the substantially elevated MIC observed among clinical isolates of C. auris. IMPORTANCE Candida auris is an emerging multidrug-resistant and health care-associated pathogen of urgent clinical concern. The triazoles are the most widely prescribed antifungal agents worldwide and are commonly utilized for the treatment of invasive Candida infections. Greater than 90% of all C. auris clinical isolates are observed to be resistant to fluconazole, and nearly all fluconazole-resistant isolates of C. auris are found to have one of three mutations (encoding VF125AL, Y132F, or K143R) in the gene encoding the target of the triazoles, ERG11. However, the direct contribution of these mutations in ERG11 to fluconazole resistance and the impact these mutations may have the susceptibility of the other triazoles remains unknown. The present study seeks to address this knowledge gap and potentially inform the future application the triazole antifungals for the treatment of infections caused by C. auris.

Diutina catenulata (Candida catenulata) is an ascomycete yeast species widely used in environmental and industrial research and capable of causing infections in humans and animals. At present, there are only a few studies on D. catenulata, and further research is required for its more in-depth characterization and analysis. Eleven strains of D. catenulata collected from China Hospital Invasive Fungal Surveillance Net (CHIF-NET) and the CHIF-NET North China Program were identified using matrix-assisted laser desorption ionization–time of flight mass spectrometry and internal transcribed spacer sequencing. The antifungal susceptibility of the Diutina catenulata strains was tested using the Clinical and Laboratory Standards Institute broth microdilution method and Sensititre YeastOne™. Furthermore, ERG11 and FKS1 were sequenced to determine any mutations related to azole and echinocandin resistance in D. catenulata. All isolates exhibited low minimum inhibitory concentration (MIC) values for itraconazole (0.06–0.12 μg/ml), posaconazole (0.06–0.12 μg/ml), amphotericin B (0.25–1 μg/ml), and 5-flucytosine (range,