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

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

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

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

Approaches for identifying and measuring heteroresistance in azole-susceptible Candida isolates.

Heteroresistance to antifungal agents poses a significant challenge in the treatment of fungal infections. Currently, the absence of established methods for detecting and measuring heteroresistance impedes progress in understanding this phenomenon in fungal pathogens. In response to this gap, we present a comprehensive set of new and optimized methods designed to detect and quantify azole heteroresistance in Candida albicans. Here, we define two primary assays for measuring heteroresistance: population analysis profiling, based on growth on solid medium, and single-cell assays, based on growth in liquid culture. We observe good correlations between the measurements obtained with liquid and solid assays, validating their utility for studying azole heteroresistance. We also highlight that disk diffusion assays could serve as an additional tool for the rapid detection of heteroresistance. These methods collectively provide a versatile toolkit for researchers seeking to assess heteroresistance in C. albicans. They also serve as a critical step forward in the characterization of antifungal heteroresistance, providing a framework for investigating this phenomenon in diverse fungal species and in the context of other antifungal agents. Ultimately, these advancements will enhance our ability to effectively measure antifungal drug responses and combat fungal infections.IMPORTANCEHeteroresistance involves varying antimicrobial susceptibility within a clonal population. This phenomenon allows the survival of rare resistant subpopulations during drug treatment, significantly complicating the effective management of infections. However, the absence of established detection methods hampers progress in understanding this phenomenon in human fungal pathogens. We propose a comprehensive toolkit to address this gap in the yeast Candida albicans, encompassing population analysis profiling, single-cell assays, and disk diffusion assays. By providing robust and correlated measurements through both solid and liquid assays, this work will provide a framework for broader applications across clinically relevant Candida species. These methods will enhance our ability to understand this phenomenon and the failure of antifungal therapy.

Divergent mitochondrial responses and metabolic signal pathways secure the azole resistance in Crabtree-positive and negative Candida species.

Azole drugs are the main therapeutic drugs for invasive fungal infections. However, azole-resistant strains appear repeatedly in the environment, posing a major threat to human health. Several reports have shown that mitochondria are associated with the virulence of pathogenic fungi. However, there are few studies on the mechanisms of mitochondria-mediated azoles resistance. Here, we first performed mitochondrial proteomic analysis on multiple Candida species (Candida albicans, Nakaseomyces glabrata, Pichia kudriavzevii, and Candida auris) and analyzed the differentially expressed mitochondrial proteins (DEMPs) between azole-sensitive and azole-resistant Candida species. Subsequently, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene ontology analysis, and protein-protein interaction network analysis of DEMPs. Our results showed that a total of 417, 165, and 25 DEMPs were identified in resistant C. albicans, N. glabrata, and C. auris, respectively. These DEMPs were enriched in ribosomal biogenesis at cytosol and mitochondria, tricarboxylic acid cycle, glycolysis, transporters, ergosterol, and cell wall mannan biosynthesis. The high activations of these cellular activities, found in C. albicans and C. auris (at low scale), were mostly opposite to those observed in two fermenter species-N. glabrata and P. kudriavzevii. Several transcription factors including Rtg3 were highly produced in resistant C. albicans that experienced a complex I activation of mitochondrial electron transport chain (ETC). The reduction of mitochondrial-related activities and complex IV/V of ETC in N. glabrata and P. kudriavzevii was companying with the reduced proteins of Tor1, Hog1, and Snf1/Snf4.IMPORTANCECandida spp. are common organisms that cause a variety of invasive diseases. However, Candida spp. are resistant to azoles, which hinders antifungal therapy. Exploring the drug-resistance mechanism of pathogenic Candida spp. will help improve the prevention and control strategy and discover new targets. Mitochondria, as an important organelle in eukaryotic cells, are closely related to a variety of cellular activities. However, the role of mitochondrial proteins in mediating azole resistance in Candida spp. has not been elucidated. Here, we analyzed the mitochondrial proteins and signaling pathways that mediate azole resistance in Candida spp. to provide ideas and references for solving the problem of azole resistance. Our work may offer new insights into the connection between mitochondria and azoles resistance in pathogenic fungi and highlight the potential clinical value of mitochondrial proteins in the treatment of invasive fungal infections.

Evolutionary trends in antifungal resistance: a meta-analysis.

The present paper includes a meta-analysis of literature data on 318 species of fungi belonging to 34 orders in their response to 8 antifungal agents (amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole, posaconazole, terbinafine, and voriconazole). Main trends of MIC results at the ordinal level were visualized. European Committee on Antimicrobial Susceptibility Testing and Clinical & Laboratory Standards Institute (CLSI) clinical breakpoints were used as the staff gauge to evaluate MIC values ranging from resistance to susceptibility, which were subsequently compared with a phylogenetic tree of the fungal kingdom. Several orders (Hypocreales, Microascales, and Mucorales) invariably showed resistance. Also the basidiomycetous orders Agaricales, Polyporales, Sporidiales, Tremellales, and Trichosporonales showed relatively high degrees of azole multi-resistance, while elsewhere in the fungal kingdom, including orders with numerous pathogenic and opportunistic species, that is, Onygenales, Chaetothyiales, Sordariales, and Malasseziales, in general were susceptible to azoles. In most cases, resistance vs susceptibility was consistently associated with phylogenetic distance, members of the same order showing similar behavior. IMPORTANCE: A kingdom-wide the largest set of published wild-type antifungal data comparison were analyzed. Trends in resistance in taxonomic groups (monophyletic clades) can be compared with the phylogeny of the fungal kingdom, eventual relationships between fungus-drug interaction and evolution can be described.

Genomic diversity of the pathogenic fungus Aspergillus fumigatus in Japan reveals the complex genomic basis of azole resistance.

Aspergillus fumigatus is a pathogenic fungus with a global distribution. The emergence of azole-resistant A. fumigatus (ARAf) other than the TR-mutants is a problem in Japan. Additionally, the genetic diversity of A. fumigatus strains in Japan remains relatively unknown. Here we show the diversity in the A. fumigatus strains isolated in Japan as well as the complexity in the global distribution of the pathogenic strains. First, we analyzed the genome sequences of 171 strains from Japan as well as the antifungal susceptibility of these strains. Next, we conducted a population analysis of 876 strains by combining the available genomic data for strains isolated worldwide, which were grouped in six clusters. Finally, a genome-wide association study identified the genomic loci associated with ARAf strains, but not the TR-mutants. These results highlight the complexity of the genomic mechanism underlying the emergence of ARAf strains other than the TR-mutants.

cyp51A mutations, protein modeling, and efflux pump gene expression reveals multifactorial complexity towards understanding Aspergillus section Nigri azole resistance mechanism.

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

Triazole resistance in Aspergillus fumigatus isolated from a tomato production environment exposed to propiconazole.

The emergence of azole-resistant Aspergillus fumigatus (ARAf) across the world is an important public health concern. We sought to determine if propiconazole, a demethylase inhibitor (DMI) fungicide, exerted a selective pressure for ARAf in a tomato production environment following multiple exposures to the fungicide. A tomato field trial was established in 2019 and propiconazole was applied weekly until harvest. Soil, leaf, and fruit (when present) samples were collected at baseline and after each propiconazole application. A. fumigatus isolates (n, 178) were recovered and 173 were tested for susceptibility to itraconazole, posaconazole, voriconazole, and propiconazole in accordance with CLSI M38 guidelines. All the isolates were susceptible to medical triazoles and the propiconazole MIC ranged from 0.25 to 8 mg/L. A linear regression model was fitted that showed no longitudinal increment in the log2-fold azole MIC of the isolates collected after each propiconazole exposure compared to the baseline isolates. AsperGenius real-time multiplex assay ruled out TR34/L98H and TR46/Y121F/T289A cyp51A resistance markers in these isolates. Sequencing of a subset of isolates (n, 46) demonstrated widespread presence of F46Y/M172V/E427K and F46Y/M172V/N248T/D255E/E427K cyp51A mutations previously associated with reduced susceptibility to triazoles. IMPORTANCE: The agricultural use of azole fungicides to control plant diseases has been implicated as a major contributor to ARAf infections in humans. Our study did not reveal imposition of selection pressure for ARAf in a vegetable production system. However, more surveillance studies for ARAf in food crop production and other environments are warranted in understanding this public and One Health issue.

Fluconazole-resistant Candida parapsilosis genotypes from hospitals located in five Spanish cities and one in Italy: Description of azole-resistance profiles associated with the Y132F ERG11p substitution.

BACKGROUND: Fluconazole-resistant Candida parapsilosis is a matter of concern. OBJECTIVES: To describe fluconazole-resistant C. parapsilosis genotypes circulating across hospitals in Spain and Rome and to study their azole-resistance profile associated with ERG11p substitutions. PATIENTS/METHODS: We selected fluconazole-resistant C. parapsilosis isolates (n = 528 from 2019 to 2023; MIC ≥8 mg/L according to EUCAST) from patients admitted to 13 hospitals located in five Spanish cities and Rome. Additionally, we tested voriconazole, posaconazole, isavuconazole, amphotericin B, micafungin, anidulafungin and ibrexafungerp susceptibility. RESULTS: Of the 53 genotypes found, 49 harboured the Y132F substitution, five of which were dominating city-specific genotypes involving almost half the isolates. Another genotype involved isolates harbouring the G458S substitution. Finally, we found two genotypes with the wild-type ERG11 gene sequence and one with the R398I substitution. All isolates were fully susceptible/wild-type to amphotericin B, anidulafungin, micafungin and ibrexafungerp. The azole-resistance patterns found were: voriconazole-resistant (74.1%) or voriconazole-intermediate (25.2%), posaconazole-resistant (10%) and isavuconazole non-wild-type (47.5%). Fluconazole-resistant and voriconazole non-wild-type isolates were likely to harbour substitution Y132F if posaconazole was wild type; however, if posaconazole was non-wild type, substitution G458S was indicated if isavuconazole MIC was >0.125 mg/L or substitution Y132F if isavuconazole MIC was ≤0.125 mg/L. CONCLUSIONS: We detected a recent clonal spread of fluconazole-resistant C. parapsilosis across some cities in Spain, mostly driven by dominating city-specific genotypes, which involved a large number of isolates harbouring the Y132F ERG11p substitution. Isolates harbouring substitution Y132F can be suspected because they are non-susceptible to voriconazole and rarely posaconazole-resistant.

Unleashing the potential of vanillic acid: A new twist on nature's recipe to fight inflammation and circumvent azole-resistant fungal infections.

Vanillic acid (VA) - a naturally occurring phenolic compound in plants - is not only used as a flavoring agent but also a prominent metabolite post tea consumption. VA and its associated compounds are believed to play a significant role in preventing diseases, underscoring the need for a systematic investigation. Herein, we report a 4-step synthesis employing the classical organic reactions, such as Willamson's alkylation, Fischer-Spier reaction, and Steglich esterification, complemented with a protection-deprotection strategy to prepare 46 VA derivatives across the five series (1a-1i, 2a-2i, 3, 3a-3i, 4a-4i, 5a-5i) in high yields. The synthesized compounds were investigated for their antifungal, anti-inflammatory, and toxic effects. Notably, compound 1a demonstrated remarkable ROS inhibition with an IC50 value of 5.1 ± 0.7 µg/mL, which is more than twice as effective as the standard ibuprofen drug. A subset of the synthesized derivatives (2b, 2c, 2e, 3b-3d, 4a-4c, 5a, and 5e) manifested their antifungal effect against drug-resistant Candida strains. Compound 5g, in particular, revealed synergism with the established antifungal drugs amphotericin B (AMB) and fluconazole (FLZ), doubling FLZ's potency against azole resistant Candida albican ATCC 36082. Furthermore, 5g improved the potency of these antifungals against FLZ-sensitive strains, including C. glabrata ATCC 2001 and C. parapsilosis ATCC 22019, as well as various multidrug-resistant (MDR) Candida strains, namely C. albicans ATCC 14053, C. albicans CL1, and C. krusei SH2L OM341600. Additionally, pharmacodynamics of compound 5g was examined using time-kill assay, and a benign safety profile was observed with no hemolytic activity in whole blood, and no cytotoxicity towards the normal BJ human cell line. The synergistic potential of 5g was further investigated through both experimental methods and docking simulations.These findings highlight the therapeutic potential of VA derivatives, particularly in addressing inflammation and circumventing FLZ resistance in Candida albicans.

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

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

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

BACKGROUND: Meyerozyma guilliermondii is a yeast species responsible for invasive fungal infections. It has high minimum inhibitory concentrations (MICs) to echinocandins, the first-line treatment of candidemia. In this context, azole antifungal agents are frequently used. However, in recent years, a number of azole-resistant strains have been described. Their mechanisms of resistance are currently poorly studied. OBJECTIVE: The aim of this study was consequently to understand the mechanisms of azole resistance in several clinical isolates of M. guilliermondii. METHODS: Ten isolates of M. guilliermondii and the ATCC 6260 reference strain were studied. MICs of azoles were determined first. Whole genome sequencing of the isolates was then carried out and the mutations identified in ERG11 were expressed in a CTG clade yeast model (C. lusitaniae). RNA expression of ERG11, MDR1 and CDR1 was evaluated by quantitative PCR. A phylogenic analysis was developed and performed on M. guilliermondii isolates. Lastly, in vitro experiments on fitness cost and virulence were carried out. RESULTS: Of the ten isolates tested, three showed pan-azole resistance. A combination of F126L and L505F mutations in Erg11 was highlighted in these three isolates. Interestingly, a combination of these two mutations was necessary to confer azole resistance. An overexpression of the Cdr1 efflux pump was also evidenced in one strain. Moreover, the three pan-azole-resistant isolates were shown to be genetically related and not associated with a fitness cost or a lower virulence, suggesting a possible clonal transmission. CONCLUSION: In conclusion, this study identified an original combination of ERG11 mutations responsible for pan-azole-resistance in M. guilliermondii. Moreover, we proposed a new MLST analysis for M. guilliermondii that identified possible clonal transmission of pan-azole-resistant strains. Future studies are needed to investigate the distribution of this clone in hospital environment and should lead to the reconsideration of the treatment for this species.

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

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

The anti-staphylococcal fusidic acid as an efflux pump inhibitor combined with fluconazole against vaginal candidiasis in mouse model.

BACKGROUND: Candida albicans is the most common fungus that causes vaginal candidiasis in immunocompetent women and catastrophic infections in immunocompromised patients. The treatment of such infections is hindered due to the increasing emergence of resistance to azoles in C. albicans. New treatment approaches are needed to combat candidiasis especially in the dwindled supply of new effective and safe antifungals. The resistance to azoles is mainly attributed to export of azoles outside the cells by means of the efflux pump that confers cross resistance to all azoles including fluconazole (FLC). OBJECTIVES: This study aimed to investigate the possible efflux pump inhibiting activity of fusidic acid (FA) in C. albicans resistant isolates and the potential use of Fusidic acid in combination with fluconazole to potentiate the antifungal activity of fluconazole to restore its activity in the resistant C. albicans isolates. METHODS: The resistance of C. albicans isolates was assessed by determination of minimum inhibitory concentration. The effect of Fusidic acid at sub-inhibitory concentration on efflux activity was assayed by rhodamine 6G efflux assay and intracellular accumulation. Mice model studies were conducted to evaluate the anti-efflux activity of Fusidic acid and its synergistic effects in combination with fluconazole. Impact of Fusidic acid on ergosterol biosynthesis was quantified. The synergy of fluconazole when combined with Fusidic acid was investigated by determination of minimum inhibitory concentration. The cytotoxicity of Fusidic acid was tested against erythrocytes. The effect of Fusidic acid on efflux pumps was tested at the molecular level by real-time PCR and in silico study. In vivo vulvovaginitis mice model was used to confirm the activity of the combination in treating vulvovaginal candidiasis. RESULTS: Fusidic acid showed efflux inhibiting activity as it increased the accumulation of rhodamine 6G, a substrate for ABC-efflux transporter, and decreased its efflux in C. albicans cells. The antifungal activity of fluconazole was synergized when combined with Fusidic acid. Fusidic acid exerted only minimal cytotoxicity on human erythrocytes indicating its safety. The FA efflux inhibitory activity could be owed to its ability to interfere with efflux protein transporters as revealed by docking studies and downregulation of the efflux-encoding genes of both ABC transporters and MFS superfamily. Moreover, in vivo mice model showed that using fluconazole-fusidic acid combination by vaginal route enhanced fluconazole antifungal activity as shown by lowered fungal burden and a negligible histopathological change in vaginal tissue. CONCLUSION: The current findings highlight FA's potential as a potential adjuvant to FLC in the treatment of vulvovaginal candidiasis.

Variability in competitive fitness among environmental and clinical azole-resistant Aspergillus fumigatus isolates.

Azoles are the primary antifungal drugs used to treat infections caused by Aspergillus fumigatus. However, the emergence of azole resistance in A. fumigatus has become a global health concern despite the low proportion of resistant isolates in natural populations. In bacteria, antibiotic resistance incurs a fitness cost that renders strains less competitive in the absence of antibiotics. Consequently, fitness cost is a key determinant of the spread of resistant mutations. However, the cost of azole resistance and its underlying causes in A. fumigatus remain poorly understood. In this observation, we revealed that the 10 out of 15 screened azole-resistant isolates, which possessed the most common azole-targeted cyp51A mutations, particularly the presence of tandem repeats in the promoter region, exhibit fitness cost when competing with the susceptible isolates in azole-free environments. These results suggest that fitness cost may significantly influence the dynamics of azole resistance, which ultimately contributes to the low prevalence of azole-resistant A. fumigatus isolates in the environment and clinic. By constructing in situ cyp51A mutations in a parental azole-susceptible strain and reintroducing the wild-type cyp51A gene into the azole-resistant strains, we demonstrated that fitness cost is not directly dependent on cyp51A mutations but is instead associated with the evolution of variable mutations related to conidial germination or other unknown development-related processes. Importantly, our observations unexpectedly revealed that some azole-resistant isolates showed no detectable fitness cost, and some even exhibited significantly increased competitive fitness in azole-free environments, highlighting the potential risk associated with the prevalence of these isolates. IMPORTANCE: Azole resistance in the human fungal pathogen Aspergillus fumigatus presents a global public health challenge. Understanding the epidemic trends and evolutionary patterns of azole resistance is critical to prevent and control the spread of azole-resistant isolates. The primary cause is the mutation of the drug target 14α-sterol-demethylase Cyp51A, yet its impact on competitive ability remains uncertain. Our competition assays revealed a diverse range of fitness outcomes for environmental and clinical cyp51A-mutated isolates. We have shown that this fitness cost is not reliant on cyp51A mutations but might be linked to unknown mutations induced by stress conditions. Among these isolates, the majority displayed fitness costs, while a few displayed enhanced competitive ability, which may have a potential risk of spread and the need to closely monitor these isolates. Our observation reveals the variation in fitness costs among azole-resistant isolates of A. fumigatus, highlighting the significant role of fitness cost in the spread of resistant strains.

Most Azole-Susceptible Isolates of Aspergillus fumigatus in Hokkaido, Japan are Clustered with Worldwide Strains That Do Not Have Tandem Repeats in Cyp51A Promoter.

In this study, we analyzed Aspergillus fumigatus short tandem repeat patterns of 106 strains isolated from the outdoor air, clinical specimens, and king penguins (Aptenodytes patagonicus) with aspergillosis in Japan, and compared them with those of 668 strains from AfumID (including six isolates from Japan). The results showed that the isolates were classified into three major groups. Group II contained most of the azole-resistant strains with 34- and 46-bp tandem repeats in cyp51A promoter. As in our previous study, OKH50 and Env1 strains were classified in Group II. Most of the azole-susceptible strains obtained in Japan were classified in Group III.

In vitro interactions of proton pump inhibitors and azoles against pathogenic fungi.

Introduction: Azole resistance has been increasingly reported and become an issue for clinical managements of invasive mycoses. New strategy with combination therapy arises as a valuable and promising alternative option. The aim of the present study is to investigate the in vitro combinational effect of proton pump inhibitors (PPIs) and azoles against pathogenic fungi. Methods: In vitro interactions of PPIs including omeprazole (OME), lansoprazole (LAN), pantoprazole (PAN), and rabeprazole (RAB), and commonly used azoles including itraconazole (ITC), posaconazole (POS), voriconazole (VRC) and fluconazole (FLC), were investigated via broth microdilution chequerboard procedure adapted from the CLSI M27-A3 and M38-A2. A total of 67 clinically isolated strains, namely 27 strains of Aspergillus spp., 16 strains of Candida spp., and 24 strains of dematiaceous fungi, were studied. C. parapsilosis (ATCC 22019) and A. flavus (ATCC 204304) was included to ensure quality control. Results: PPIs individually did not exert any significant antifungal activity. The combination of OME with ITC, POS, or VRC showed synergism against 77.6%, 86.6%, and 4% strains of tested pathogenic fungi, respectively, while synergism of OME/FLC was observed in 50% strains of Candida spp. Synergism between PAN and ITC, POS, or VRC was observed against 47.8%, 77.6% and 1.5% strains of tested fungi, respectively, while synergism of PNA/FLC was observed in 50% strains of Candida spp. Synergism of LAN with ITC, POS, or VRC was observed against 86.6%, 86.6%, and 3% of tested strains, respectively, while synergism of LAN/FLC was observed in 31.3% strains of Candida spp. Synergy of the combination of RAB with ITC, POS, or VRC was observed against 25.4%, 64.2%, and 4.5% of tested strains, respectively, while synergism of RAB/FLC was observed in 12.5% of Candida spp.. Among PPIs, synergism was least observed between RAB and triazoles, while among triazoles, synergism was least observed between VRC and PPIs. Among species, synergy was much more frequently observed in Aspergillus spp. and dematiaceous fungi as compared to Candida spp. Antagonism between PPIs with ITC or VRC was occasionally observed in Aspergillus spp. and dematiaceous fungi. It is notable that PPIs combined with azoles showed synergy against azole resistant A. fumigatus, and resulted in category change of susceptibility of ITC and POS against Candida spp. Discussion: The results suggested that PPIs combined with azoles has the potential to enhance the susceptibilities of azoles against multiple pathogenic fungi and could be a promising strategy to overcome azole resistance issues. However, further investigations are warranted to study the combinational efficacy in more isolates and more species, to investigate the underlying mechanism of interaction and to evaluate the potential for concomitant use of these agents in human.

Antifungal chemicals promising function in disease prevention, method of action and mechanism.

The increasing use of antimicrobial drugs has been linked to the rise of drug-resistant fungus in recent years. Antimicrobial resistance is being studied from a variety of perspectives due to the important clinical implication of resistance. The processes underlying this resistance, enhanced methods for identifying resistance when it emerges, alternate treatment options for infections caused by resistant organisms, and so on are reviewed, along with strategies to prevent and regulate the formation and spread of resistance. This overview will focus on the action mechanism of antifungals and the resistance mechanisms against them. The link between antibacterial and antifungal resistance is also briefly discussed. Based on their mechanism action, antifungals are divided into three distinct categories: azoles, which target the ergosterol synthesis; 5-fluorocytosine, which targets macromolecular synthesis and polyenes, which interact physiochemically with fungal membrane sterols. Antifungal resistance can arise through a wide variety of ways. Overexpression of the target of the antifungal drug, changes to the drug target, changes to sterol biosynthesis, decreased intercellular concentration of the target enzyme, and other processes. A correlation exists between the mechanisms of resistance to antibacterial and antifungals, despite the fact that the comparison between the two is inevitably constrained by various parameters mentioned in the review. Drug extrusion via membrane pumps has been thoroughly documented in both prokaryotic and eukaryotic cells, and development of new antifungal compounds and strategies has also been well characterized.

Trends in the activity of mold-active azole agents against Aspergillus fumigatus clinical isolates with and without cyp51 alterations from Europe and North America (2017-2021).

Azole resistance in Aspergillus fumigatus (AFM) is increasing and often associated with cyp51 alterations. We evaluated the activity of isavuconazole and other mold-active azoles against 731 AFM isolates causing invasive aspergillosis collected in Europe (EU; n = 449) and North America (NA; n = 282). Isolates were submitted to CLSI susceptibility testing and epidemiological cutoff value (ECV) criteria. A posaconazole ECV of 0.5 mg/L was used as no CLSI ECV was determined. Azole non-wild-type (NWT) isolates were submitted for cyp51 sequencing by whole genome sequencing. Overall, isavuconazole activity (92.7%/94.0% WT in EU/NA) was comparable to other azoles (WT rate range, 90.9%-96.4%/91.8%-98.6%, respectively), regardless of the region. A total of 79 (10.8%) azole NWT isolates were detected, and similar rates of these isolates were noted in EU (10.7%) and NA (11.0%). Although most AFM were WT to azoles, increasing azole NWT rates were observed in NA (from 6.0% in 2017 to 29.3% in 2021). Azole NWT rates varied from 4.9% (2019) to 20.6% (2018) in EU without an observed trend. cyp51 alterations occurred in 56.3%/54.8% of azole NWT from EU/NA, respectively. The cyp51A TR34/L98H alteration was observed only in EU isolates (72.0% of EU isolates), while cyp51A I242V occurred only in NA isolates (58.3%). Isavuconazole remained active (MIC, ≤1 mg/L) against 18.5/47.1% of azole NWT AFM exhibiting cyp51 alterations in EU/NA, along with voriconazole (29.6/82.4%; MIC, ≤1 mg/L) and posaconazole (48.1/88.2%; MIC, ≤0.5 mg/L). Fourteen different cyp51 alterations were detected in 44 of 79 NWT isolates. The in vitro activity of the azoles varied in AFM that displayed cyp51 alterations. IMPORTANCE A few microbiology laboratories perform antifungal susceptibility testing locally for systemically active antifungal agents. The identification of emerging azole-resistant Aspergillus fumigatus is worrisome. As such, there is a critical role for antifungal surveillance in tracking emerging resistance among both common and uncommon opportunistic fungi. Differences in the regional prevalence and antifungal resistance of these fungi render local epidemiological knowledge essential for the care of patients with a suspected invasive fungal infection.

Four-Arm δ-Ornithine-Based Polypeptoids Resensitize Voriconazole against Azole-Resistant C. albicans.

Worldwide Candida albicans infections cause a huge burden in healthcare and the efficacy of traditional antifungals is diminished because of the rapid development of antifungal resistance. It is necessary to develop new antifungals or new strategies to make multidrug-resistant (MDR) C. albicans to resensitize to existing antifungal drugs. In this work, a series of 4-arm polypeptoids (FAPs) were synthesized through grafting linear ε-l-lysine or δ-ornithine-based oligopeptides to a trimeric lysine core. The most potent 4R-O7 exhibited excellent activities toward three sensitive and two MDR C. albicans strains with MIC values as low as 24-48 µg/mL (vs 375 µg/mL for ε-polylysine, ε-PL). The mechanism studies revealed that 4R-O7 penetrated the cell membrane and generated ROS to kill cells. 4R-O7 exhibited a synergistic effect (FICI < 0.5) with voriconazole (VOR) and also assisted VOR to restore its efficacy to MDR C. albicans. In addition, the combined use of 4R-O7 and VOR significantly improved the elimination efficacy of mature C. albicans biofilms and enhanced the potency in a mouse subcutaneous C. albicans infection model.