Prevalence of azole-resistant Aspergillus fumigatus and other aspergilli in the environment from Argentina.

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

This study intended to inform about the prevalence of Aspergillus species showing triazole resistance in the Argentinian environment. Since azole fungicides are used for crop protection, it was expected that azole resistance in this species with cross-resistance to medical azoles could occur.

Azole resistance in a clinical isolate of Aspergillus fumigatus from Chile.

BACKGROUND: Aspergillus fumigatus is a ubiquitous opportunistic pathogen. This fungus can acquire resistance to azole antifungals due to different mutations in the cyp51A gene. Azole resistance has been observed in several continents and appears to be a globally distributed phenomenon. Specific mutations in cyp51A that lead to azole resistance, such as the TR34/L98H modification, have been reported. AIMS: To evaluate the azole resistance in clinically isolated A. fumigatus strains. METHODS: As a result of our passive surveillance strategy, a total of 23 A. fumigatus isolates from clinical origins were identified through a phylogenetic analysis using the ITS region and ß-tubulin gene fragments, and typed with the CSP microsatellite. Azole susceptibility profiles were performed by disk diffusion and microdilution broth methodologies according to CLSI guidelines. RESULTS: Here we describe, for the first time, the detection of azole-resistant A. fumigatus isolates from clinical origins in Chile with mutations in the cyp51A gene. In addition to the TR34/L98H mutation, one isolate exhibited an F46Y/M172V/E427K-type mutation. Furthermore, microsatellite typing based on cell surface protein (CSP) was performed, showing the t02 (TR34/L98H), t15 (F46Y/M172V/E427K) and t01 (susceptible clinical isolates) genotypes. CONCLUSIONS: Our study demonstrates the presence of mutations related to azole resistance in A. fumigatus strains isolated from clinical samples in Chile. In order to obtain information that may help to tackle the spread of antifungal resistance among A. fumigatus populations, and to ensure the efficacy of future treatments against aspergillosis, a further research is necessary.

Aspergillus fumigatus conidial surface-associated proteome reveals factors for fungal evasion and host immunity modulation.

Aspergillus fumigatus causes aspergillosis and relies on asexual spores (conidia) for initiating host infection. There is scarce information about A. fumigatus proteins involved in fungal evasion and host immunity modulation. Here we analysed the conidial surface proteome of A. fumigatus, two closely related non-pathogenic species, Aspergillus fischeri and Aspergillus oerlinghausenensis, as well as pathogenic Aspergillus lentulus, to identify such proteins. After identifying 62 proteins exclusively detected on the A. fumigatus conidial surface, we assessed null mutants for 42 genes encoding these proteins. Deletion of 33 of these genes altered susceptibility to macrophage, epithelial cells and cytokine production. Notably, a gene that encodes a putative glycosylasparaginase, modulating levels of the host proinflammatory cytokine IL-1ß, is important for infection in an immunocompetent murine model of fungal disease. These results suggest that A. fumigatus conidial surface proteins are important for evasion and modulation of the immune response at the onset of fungal infection.

Brilacidin, a host defense peptide mimetic, potentiates ibrexafungerp antifungal activity against the human pathogenic fungus Aspergillus fumigatus.

Aspergillus fumigatus is the primary etiological agent of aspergillosis. Here, we show that the host defense peptide mimetic brilacidin (BRI) can potentiate ibrexafungerp (IBX) against clinical isolates of A. fumigatus. BRI + IBX can inhibit the growth of A. fumigatus voriconazole- and caspofungin-resistant clinical isolates. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against viruses, bacteria, and fungi. In vitro, combination of BRI + IBX plays a fungicidal role, increases the fungal cell permeability, decreases the fungal survival in the presence of A549 epithelial cells, and appears as a promising antifungal therapeutic alternative against A. fumigatus. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Aspergillus fumigatus causes a series of distinct invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. A. fumigatus causes a spectrum of distinct clinical entities named aspergillosis, which the most severe form is the invasive pulmonary aspergillosis. There are few therapeutic options for treating aspergillosis and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a synergizer o fibrexafungerp (IBX) against A. fumigatus. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. We propose the combination of BRI and IBX as a new antifungal combinatorial treatment against aspergillosis.

Comparison of galactomannan lateral flow assay and enzyme immunoassay to identify Aspergillus spp. in bronchoalveolar lavage fluid.

Aspergillus species can colonize and infect immunocompetent and immunocompromised hosts. Conventional fungal identification depends on microscopic analysis and microorganism medium growth. Other diagnostic methods, non-growth dependent, to invasive fungal infections, are the biomarkers that detect circulating polysaccharides, for example, 1-3-ß-d-Glucan and galactomannan. Both are polysaccharides present on the external layer of fungi cell wall and can be detected in clinical samples during the growth of the fungus in the patient. This study aimed to compare the galactomannan detection of Lateral Flow Assay and Enzyme Immunoassay methods in Bronchoalveolar Lavage Fluid. The galactomannan antigen in Bronchoalveolar Lavage Fluid was measured using Enzyme Immunoassay according to the manufacturer's instructions (PLATELIA ASPERGILLUS™ BioRad) and, using a Lateral Flow Assay according to the manufacturer's instructions (Galactomannan LFA IMMY©). The 71 samples were Bronchoalveolar Lavage Fluid of patients hospitalized at Unicamp Clinical Hospital between 2019 and 2021; of these samples 12/71 (16.9 %) resulted in positive Galactomannan-Lateral Flow Assay. In contrast, Galactomannan-Enzyme Immunoassay resulted as positive in 9/71 (12.6 %) samples, a difference that showed not significant statistically (p-value = 0.36) Comparing both assays' results identified 8 divergences between them, about 11 % of the total sample. The Sensitivity (73.3 %), Specificity (92.35 %), Positive Predictive Value (62.85 %) and Negative Predictive Value (95.15 %) of Lateral Flow Assay were calculated using the Galactomannan Enzyme Immunoassay as standard. The Lateral Flow Assay demonstrated good results when compared with the Enzyme Immunoassay.

Sirtuin E deacetylase is required for full virulence of Aspergillus fumigatus.

Aspergillus fumigatus represents a public health problem due to the high mortality rate in immunosuppressed patients and the emergence of antifungal-resistant isolates. Protein acetylation is a crucial post-translational modification that controls gene expression and biological processes. The strategic manipulation of enzymes involved in protein acetylation has emerged as a promising therapeutic approach for addressing fungal infections. Sirtuins, NAD+-dependent lysine deacetylases, regulate protein acetylation and gene expression in eukaryotes. However, their role in the human pathogenic fungus A. fumigatus remains unclear. This study constructs six single knockout strains of A. fumigatus and a strain lacking all predicted sirtuins (SIRTKO). The mutant strains are viable under laboratory conditions, indicating that sirtuins are not essential genes. Phenotypic assays suggest sirtuins' involvement in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. Deletion of sirE attenuates virulence in murine and Galleria mellonella infection models. The absence of SirE alters the acetylation status of proteins, including histones and non-histones, and triggers significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

Pathogenic potential of an environmental Aspergillus fumigatus strain recovered from soil of Pygoscelis papua (Gentoo penguins) colony in Antarctica.

Aspergillus fumigatus is a common opportunistic pathogen in different animals, including birds such as penguins. For the first time, a fungal strain identified as A. fumigatus was isolated from soil in the nests of gentoo penguins, Pygoscelis papua, on Livingston Island, South Shetland Islands (maritime Antarctica). This isolate (A. fumigatus UFMGCB 11829) displayed a series of potentially pathogenic characteristics in vitro. We evaluated its detailed molecular taxonomy and submitted the A. fumigatus UFMGCB 11829 Antarctic strain to in vivo pathogenic modelling. The isolate was confirmed to represent A. fumigatus morphological and phylogenetic analysis showed that it was closely related to A. fumigatus sequences reported from animals, immunosuppressed humans, storage grains, plants and soils. The strain displayed the best mycelial growth and conidia production at 37 ºC; however, it was also able to grow and produce conidia at 15º, demonstrating its capability to survive and colonize penguin nest at least in the summer season in maritime Antarctica. In pathogenicity tests, healthy mice did not showed symptoms of infection; however, 50% lethality was observed in immunosuppressed mice that were inoculated with 106 and 107 spores. Lethality increased to 100% when inoculated with 108 spores. Our data highlight the potential pathogenicity of opportunistic A. fumigatus that may be present in the Antarctic, and the risks of both their further transfer within Antarctica and outwards to other continents, risks which may be exacerbated due global climatic changes.

Antifungal and Antivirulence Activity of Vanillin and Tannic Acid Against Aspergillus fumigatus and Fusarium solani.

Aspergillus fumigatus and Fusarium solani infections have become severe health threat; both pathogens are considered a priority due to the increasing emergence of antifungal-resistant strains and high mortality rates. Therefore, the discovery of new therapeutic strategies has become crucial. In this study, we evaluated the antifungal and antivirulence effects of vanillin and tannic acid against Aspergillus fumigatus and Fusarium solani. The minimum inhibitory concentrations of the compounds were determined by the microdilution method in RPMI broth in 96-well microplates according to CLSI. Conidial germination, protease production, biofilm formation, and in vivo therapeutic efficacy assays were performed. The results demonstrated that vanillin and tannic acid had antifungal activity against Aspergillus fumigatus, while tannic acid only exhibited antifungal activity against Fusarium solani. We found that vanillin and tannic acid inhibited conidial germination and secreted protease production and biofilm formation of the fungal pathogens using sub-inhibitory concentrations. Besides, vanillin and tannic acid altered the fungal membrane permeability, and both compounds showed therapeutic effect against aspergillosis and fusariosis in an infection model in Galleria mellonella larvae. Our results highlight the antivirulence effect of vanillin and tannic acid against priority pathogenic fungi as a possible therapeutic alternative for human fungal infections.

Estimated burden of fungal infections in Panama.

Data published on Panamanian fungal disease are scarce, mostly case reports. To date, there is no paper that compiles the burden of fungal disease Here we estimate for the first time the incidence and prevalence of fungal diseases in Panama. Data on fungal disease were obtained from different search engines: PubMed, Google Scholar, Scielo and Lilacs. For population and at risk diseases, we used statistics from worldometer, UNAIDS, and WHO. Incidence, prevalence, and absolute numbers were calculated based on the population at risk. Panamanian population in 2022 was 4,429,739. We estimated that 85,530 (1.93 %) people suffer from fungal diseases. The most frequent fungal infection was recurrent Candida vaginitis (3285/100,000). There are 31,000 HIV-infected people in Panama and based on the number of cases not receiving anti-retroviral therapy (14,570), and previous reports of prevalence of opportunistic infections, we estimated annual incidences of 4.0/100,000 for cryptococcal meningitis, 29.5/100,000 for oral candidiasis, 23.1/100,000 for esophageal candidiasis, 29.5/100,000 for Pneumocystis pneumonia, 15.1/100,000, and for histoplasmosis. For chronic pulmonary aspergillosis (CPA) and fungal asthma we used data from Guatemala and Colombia to estimate COPD and asthma prevalence and WHO report for tuberculosis. We estimated annual incidences of 6.1/100,000 for invasive aspergillosis and prevalence of 31.5/100,000 for CPA, 60.2/100,000 for allergic bronchopulmonary aspergillosis, and 79.5/100,000 for severe asthma with fungal sensitisation. Other incidence estimates were 5.0/100,000 for candidaemia, 0.20/100,000 for mucormycosis, and 4.97/100,000 for fungal keratitis. Even though this report on burden of fungal disease is a forward step, more epidemiological studies to validate these estimates are needed.

Aspergillus fumigatus mitogen-activated protein kinase MpkA is involved in gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. The Mitogen-Activated Protein kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.

Evaluation of antifungal activity, mechanisms of action and toxicological profile of the synthetic amide 2-chloro-N-phenylacetamide.

Aspergillus niger causes infections such as otitis and pulmonary aspergillosis in immunocompromised individuals. Treatment involves voriconazole or amphotericin B, and due to the increase in fungal resistance, the search for new compounds with antifungal activity has intensified. In the development of new drugs, cytotoxicity and genotoxicity assays are important, as they allow predicting possible damage that a molecule can cause, and in silico studies predict the pharmacokinetic properties. The aim of this study was to verify the antifungal activity and the mechanism of action of the synthetic amide 2-chloro-N-phenylacetamide against Aspergillus niger strains and toxicity. 2-Chloro-N-phenylacetamide showed antifungal activity against different strains of Aspergillus niger with minimum inhibitory concentrations between 32 and 256 µg/mL and minimum fungicides between 64 and 1024 µg/mL. The minimum inhibitory concentration of 2-chloro-N-phenylacetamide also inhibited conidia germination. When associated with amphotericin B or voriconazole, 2-chloro-N-phenylacetamide had antagonistic effects. Interaction with ergosterol in the plasma membrane is the probable mechanism of action.2-Chloro-N-phenylacetamide has favorable physicochemical parameters, good oral bioavailability and absorption in the gastrointestinal tract, crosses the blood-brain barrier and inhibits CYP1A2. At concentrations of 50 to 500 µg/mL, it has little hemolytic effect and a protective effect for type A and O red blood cells, and in the cells of the oral mucosa it promotes little genotoxic change. It is concluded that 2-chloro-N-phenylacetamide has promising antifungal potential, favorable pharmacokinetic profile for oral administration and low cytotoxic and genotoxic potential, being a promising candidate for in vivo toxicity studies.

Clinical outcomes of aspergillosis among paediatric and adult inpatients: A multicentre study in a Brazilian metropolitan area.

BACKGROUND: Invasive Aspergillosis (IA) is a disease of significant clinical relevance, especially among immunosuppressed patients, and is associated with high mortality rates. In this study, we evaluated the epidemiological features and clinical outcomes in children and adults with IA. METHODS: This was an observational, multicentre, prospective surveillance study of inpatients with IA at two different hospitals in Campinas, Brazil, between 2018 and 2021. RESULTS: A total of 44 patients were identified (54.5% males), with a median age of 42 years (interquartile range (IQR):19.25-59 years, varying between 1 and 89 years). The following baseline conditions were identified: 61.4% were oncohaematological patients and 20.5% were solid organ transplant recipients. Among oncohaematological patients, 77.8% exhibited severe or persistent neutropenia. The median time between the onset of neutropenia and the diagnosis of fungal infection was 20 days (IQR: 10.5-26 days; range, 0-68 days). The interval between neutropenia onset and fungal infection was longer in paediatric than in general hospital (average, 29 vs. 13.4 days; median 26 vs 11 days; p=0.010). After the diagnosis of IA, the survival rates were 44.2% and 30.0% at 180 and 360 days, respectively. Survival was greater in patients aged ≤ 21 years (p = 0.040; log-rank test). They observed no difference in IA mortality related to COVID-19 pandemic. CONCLUSION: High mortality associated with IA was observed in both hospitals. Individuals over the age of 21 have a lower survival rate than younger patients.

A host defense peptide mimetic, brilacidin, potentiates caspofungin antifungal activity against human pathogenic fungi.

Fungal infections cause more than 1.5 million deaths a year. Due to emerging antifungal drug resistance, novel strategies are urgently needed to combat life-threatening fungal diseases. Here, we identify the host defense peptide mimetic, brilacidin (BRI) as a synergizer with caspofungin (CAS) against CAS-sensitive and CAS-resistant isolates of Aspergillus fumigatus, Candida albicans, C. auris, and CAS-intrinsically resistant Cryptococcus neoformans. BRI also potentiates azoles against A. fumigatus and several Mucorales fungi. BRI acts in A. fumigatus by affecting cell wall integrity pathway and cell membrane potential. BRI combined with CAS significantly clears A. fumigatus lung infection in an immunosuppressed murine model of invasive pulmonary aspergillosis. BRI alone also decreases A. fumigatus fungal burden and ablates disease development in a murine model of fungal keratitis. Our results indicate that combinations of BRI and antifungal drugs in clinical use are likely to improve the treatment outcome of aspergillosis and other fungal infections.

Molecular epidemiology and antifungal susceptibilities of Aspergillus species isolated from patients with invasive aspergillosis.

OBJECTIVE: The aim of this study was to evaluate the demographic data, molecular epidemiology, and in vitro antifungal susceptibility results of patients with Aspergillus isolated from various clinical specimens. METHODS: A total of 44 Aspergillus strains were studied. The definition of invasive aspergillosis in patients was made according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) criteria. Strains were phenotypically and molecularly identified. Demographic characteristics of patients and genotypes of strains were evaluated. Phylogenetic analysis was done by the The Unweighted Pair-Group Method with Arithmetic Mean (UPGMA). Antifungal susceptibility of strains was determined according to The Clinical and Laboratory Standards Institute (CLSI)-M61-Ed2 and The European Committee on Antimicrobial Susceptibility Testing (EUCAST). RESULTS: A total of 11 patients were classified as proven and 33 as probable invasive aspergillosis. There was a statistically significant difference in age groups, subdisease, neutropenic, and receiving chemotherapy between groups. A total of 23 strains were identified as Aspergillus fumigatus, 12 as Aspergillus niger, 6 as Aspergillus flavus, and 3 as Aspergillus terreus. Phylogenetic analysis revealed five different genotypes. No statistical difference was found in the comparisons between patients groups and genotype groups. There was a statistically significant difference between genotype groups and voriconazole, posaconazole, and itraconazole Minimum Inhibition Concentration (MIC). CONCLUSION: Accurate identification of strains and antifungal susceptibility studies should be performed due to azole and amphotericin B resistance. Genotyping studies are important in infection control due to identifying sources of infection and transmission routes.

Examination of Genome-Wide Ortholog Variation in Clinical and Environmental Isolates of the Fungal Pathogen Aspergillus fumigatus.

Aspergillus fumigatus is both an environmental saprobe and an opportunistic human fungal pathogen. Knowledge of genomic variation across A. fumigatus isolates is essential for understanding the evolution of pathogenicity, virulence, and resistance to antifungal drugs. Here, we investigated 206 A. fumigatus isolates (133 clinical and 73 environmental isolates), aiming to identify genes with variable presence across isolates and test whether this variation was related to the clinical or environmental origin of isolates. The PanOrtho genome of A. fumigatus consists of 13,085 ortholog groups, of which 7,773 (59.4%) are shared by all isolates (core groups) and 5,312 (40.6%) vary in their gene presence across isolates (accessory groups plus singletons). Despite differences in the distribution of orthologs across all isolates, no significant differences were observed among clinical versus environmental isolates when phylogeny was accounted for. Orthologs that differ in their distribution across isolates tend to occur at low frequency and/or be restricted to specific isolates; thus, the degree of genomic conservation between orthologs of A. fumigatus is high. These results suggest that differences in the distribution of orthologs within A. fumigatus cannot be associated with the clinical or environmental origin of isolates. IMPORTANCE Aspergillus fumigatus is a cosmopolitan species of fungus responsible for thousands of cases of invasive disease annually. Clinical and environmental isolates of A. fumigatus exhibit extensive phenotypic differences, including differences related to virulence and antifungal drug resistance. A comprehensive survey of the genomic diversity present in A. fumigatus and its relationship to the clinical or environmental origin of isolates can contribute to the prediction of the mechanisms of evolution and infection of the species. Our results suggest that there is no significant variation in ortholog distribution between clinical and environmental isolates when accounting for evolutionary history. The work supports the hypothesis that environmental and clinical isolates of A. fumigatus do not differ in their gene contents.

Lethal destructive sinusopathy due to amphotericin B-resistant Aspergillus flavus: A case report.

BACKGROUND: The prevalence of pulmonary aspergillosis and the importance of its early diagnosis are recognized. However, non-pulmonary involvement, including the sinuses region, is not frequently reported, and an infection in this area can affect all paranasal sinuses (pansinusopathy), being a rare pathology that affects immunocompromised hosts. Recent studies have highlighted the occurrence of Aspergillus flavus resistant to antifungal therapy. Therefore, a nasal sinus infection by resistant Aspergillus strains in immunocompromised patients may be linked to a high risk of lethality. CASE REPORT: We are reporting a resistant A. flavus infection in an allogeneic hematopoietic stem cell transplant recipient with episodes of febrile neutropenia, and prolonged use of various antibacterial drugs and antifungal prophylaxis. The patient underwent brain magnetic resonance, which showed the presence of pansinusopathy, and presented necrosis in the left nasal region. Direct microscopic examination of a sample taken from the nasal mucosa revealed the presence of septate hyphae and conidiophores resembling those of A. flavus, that species being the identification achieved with MALDI-TOF MS. Antifungigram was performed by microdilution in broth (EUCAST-E.DEF. 9.3.2) and E-test, and resistance to amphotericin B was shown in both tests. The patient died after septic shock and hemorrhage. CONCLUSIONS: Invasive fungal infections due to amphotericin-B resistant A. flavus may lead to the death of the patient due to an ineffective therapeutic management. Therefore, antifungal susceptibility testing are of utmost importance for administering the proper treatment.

Screening of Chemical Libraries for New Antifungal Drugs against Aspergillus fumigatus Reveals Sphingolipids Are Involved in the Mechanism of Action of Miltefosine.

Aspergillus fumigatus is an important fungal pathogen and the main etiological agent of aspergillosis, a disease characterized by a noninvasive process that can evolve to a more severe clinical manifestation, called invasive pulmonary aspergillosis (IPA), in immunocompromised patients. The antifungal arsenal to threat aspergillosis is very restricted. Azoles are the main therapeutic approach to control IPA, but the emergence of azole-resistant A. fumigatus isolates has significantly increased over recent decades. Therefore, new strategies are necessary to combat aspergillosis, and drug repurposing has emerged as an efficient and alternative approach for identifying new antifungal drugs. Here, we used a screening approach to analyze A. fumigatus in vitro susceptibility to 1,127 compounds. A. fumigatus was susceptible to 10 compounds, including miltefosine, a drug that displayed fungicidal activity against A. fumigatus. By screening an A. fumigatus transcription factor null library, we identified a single mutant, which has the smiA (sensitive to miltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and ΔsmiA strains and chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-Seq) of an SmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipid pathway that are directly or indirectly regulated by SmiA. Sphingolipid analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of SmiA represents the first genetic element described and characterized that plays a direct role in miltefosine response in fungi. IMPORTANCE The filamentous fungus Aspergillus fumigatus causes a group of diseases named aspergillosis, and their development occurs after the inhalation of conidia dispersed in the environment. Very few classes of antifungal drugs are available for aspergillosis treatment, e.g., azoles, but the emergence of global resistance to azoles in A. fumigatus clinical isolates has increased over recent decades. Repositioning or repurposing drugs already available on the market is an interesting and faster opportunity for the identification of novel antifungal agents. By using a repurposing strategy, we identified 10 different compounds that impact A. fumigatus survival. One of these compounds, miltefosine, demonstrated fungicidal activity against A. fumigatus. The mechanism of action of miltefosine is unknown, and, aiming to get more insights about it, we identified a transcription factor, SmiA (sensitive to miltefosine), important for miltefosine resistance. Our results suggest that miltefosine displays antifungal activity against A. fumigatus, interfering in sphingolipid biosynthesis.

Molecular epidemiology of aspergillosis in Magellanic penguins and susceptibility patterns of clinical isolates.

Aspergillus section Fumigati is reported in up to 99% of aspergillosis cases in penguins. So far, no data regarding molecular epidemiology and azole resistance are available for A. fumigatus isolates collected from Magellanic penguins. The aim of this work was to perform molecular identification of Aspergillus section Fumigati at species level, to genotype those isolates using microsatellite markers, to evaluate the in vitro susceptibility patterns of A. fumigatus sensu stricto, and to characterize the cyp51A gene in clinical A. fumigatus strains isolated from Magellanic penguins with proven aspergillosis. All 34 isolates included in the study were identified as A. fumigatus sensu stricto. Analyzing the genetic diversity of the isolates of A. fumigatus sensu stricto, we identified two possible outbreaks in the rehabilitation center and we also observed the maintenance of clonal strains through the years. One A. fumigatus sensu stricto isolate was resistant to posaconazole, but the mutations found in the cyp51A gene of this isolate have not been described as conferring phenotypic resistance, suggesting that other mechanisms of resistance could be involved in the resistance of this isolate. With this study, we were able to understand the molecular diversity of Aspergillus fumigatus isolates collected from Magellanic penguins, to characterize them and to associate them with the described global population of Aspergillus fumigatus.

A. fumigatus sensu stricto is of great importance in penguins' aspergillosis. We could identify two outbreaks in the rehabilitation center and the maintenance of clonal strains through the years. Regarding antifungal prophylaxis, it may proceed, but preferably with surveillance for azole resistance.