RESUMEN
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.
Asunto(s)
Aspergilosis , Aspergillus fumigatus , Filogenia , Microbiología del Suelo , Spheniscidae , Animales , Spheniscidae/microbiología , Regiones Antárticas , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/patogenicidad , Ratones , Aspergilosis/microbiología , Aspergilosis/veterinaria , Enfermedades de las Aves/microbiología , VirulenciaRESUMEN
Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.
Asunto(s)
Aspergillus fumigatus/clasificación , Cromosomas Fúngicos/genética , Heterogeneidad Genética , Histonas/metabolismo , Aspergilosis Pulmonar/microbiología , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Cromatina , Elementos Transponibles de ADN , Proteínas Fúngicas/metabolismo , Regulación de la Expresión Génica de las Plantas , Aptitud Genética , Código de Histonas , Humanos , Regiones Promotoras Genéticas , Metabolismo Secundario , VirulenciaRESUMEN
BACKGROUND: Mutations in cyp51A gene are known as main mechanisms of azole resistance in Aspergillus fumigatus, whereas azole-susceptible strains also carry cyp51A mutations (polymorphisms). The polymorphisms found in Europe mainly consist of two combinations of mutations, that is combinations of five single-nucleotide polymorphisms (SNPs) of cyp51A, referred to as cyp51A-5SNPs, and combinations of three SNPs of cyp51A, referred to as cyp51A-3SNPs. Few studies have compared the distributions of cyp51A polymorphisms between different regions. OBJECTIVES: The aim of this study was to investigate the regional differences of cyp51A polymorphisms. METHODS: We compared the proportions of cyp51A polymorphisms in clinical and environmental strains isolated in various countries, and analysed the strains phylogenetically using short tandem repeats (STRs) and whole-genome sequence (WGS). RESULTS: Among the Japanese strains, 15 out of 98 (15.3%) clinical strains and 8 out of 95 (8.4%) environmental strains had cyp51A polymorphisms. A mutation of cyp51AN248K was the most prevalent polymorphism in both clinical (n = 14, 14.3%) and environmental strains (n = 3, 3.2%). Only one environmental strain harboured cyp51A-5SNPs, which was reported to be the most prevalent in Europe. For phylogenetic analyses using STRs and WGS, 183 and 134 strains, respectively, were employed. They showed that most of the strains with cyp51AN248K clustered in the clades different from those of the strains with cyp51A-5SNPs and cyp51A-3SNPs as well as from those with TR34 /L98H mutations. CONCLUSIONS: This study suggests that there are genetic differences between cyp51A polymorphisms of A. fumigatus in Japan and Europe.
Asunto(s)
Aspergillus fumigatus/genética , Sistema Enzimático del Citocromo P-450/genética , Proteínas Fúngicas/genética , Aspergilosis Pulmonar Invasiva/microbiología , Polimorfismo de Nucleótido Simple , Aspergilosis Pulmonar/microbiología , Animales , Antifúngicos/farmacología , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/patogenicidad , Bombyx/microbiología , Enfermedad Crónica , Microbiología Ambiental , Europa (Continente) , Genotipo , Humanos , Japón , Pruebas de Sensibilidad Microbiana , Mutación , Filogenia , Virulencia , Secuenciación Completa del GenomaRESUMEN
The ongoing global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), first described in Wuhan, China. A subset of COVID-19 patients has been reported to have acquired secondary infections by microbial pathogens, such as opportunistic fungal pathogens from the genus Aspergillus. To gain insight into COVID-19-associated pulmonary aspergillosis (CAPA), we analyzed the genomes and characterized the phenotypic profiles of four CAPA isolates of Aspergillus fumigatus obtained from patients treated in the area of North Rhine-Westphalia, Germany. By examining the mutational spectrum of single nucleotide polymorphisms, insertion-deletion polymorphisms, and copy number variants among 206 genes known to modulate A. fumigatus virulence, we found that CAPA isolate genomes do not exhibit significant differences from the genome of the Af293 reference strain. By examining a number of factors, including virulence in an invertebrate moth model, growth in the presence of osmotic, cell wall, and oxidative stressors, secondary metabolite biosynthesis, and the MIC of antifungal drugs, we found that CAPA isolates were generally, but not always, similar to A. fumigatus reference strains Af293 and CEA17. Notably, CAPA isolate D had more putative loss-of-function mutations in genes known to increase virulence when deleted. Moreover, CAPA isolate D was significantly more virulent than the other three CAPA isolates and the A. fumigatus reference strains Af293 and CEA17, but similarly virulent to two other clinical strains of A. fumigatus. These findings expand our understanding of the genomic and phenotypic characteristics of isolates that cause CAPA. IMPORTANCE The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has already killed millions of people. COVID-19 patient outcome can be further complicated by secondary infections, such as COVID-19-associated pulmonary aspergillosis (CAPA). CAPA is caused by Aspergillus fungal pathogens, but there is little information about the genomic and phenotypic characteristics of CAPA isolates. We conducted genome sequencing and extensive phenotyping of four CAPA isolates of Aspergillus fumigatus from Germany. We found that CAPA isolates were often, but not always, similar to other reference strains of A. fumigatus across 206 genetic determinants of infection-relevant phenotypes, including virulence. For example, CAPA isolate D was more virulent than other CAPA isolates and reference strains in an invertebrate model of fungal disease, but similarly virulent to two other clinical strains. These results expand our understanding of COVID-19-associated pulmonary aspergillosis.
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Aspergillus fumigatus/genética , COVID-19/complicaciones , Genómica , Fenotipo , Aspergilosis Pulmonar/complicaciones , Anciano , Antifúngicos , Aspergillus , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/aislamiento & purificación , Femenino , Humanos , Masculino , Metabolómica , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Mutación , Polimorfismo de Nucleótido Simple , SARS-CoV-2 , Metabolismo Secundario/genética , Virulencia/genéticaRESUMEN
The main objective of the study is to characterize two new strains of Aspergillus fumigatus through morphometric, biochemical, molecular methods, and to evaluate their antimicrobial potentiality. The micro-morphotaxonomy, growth, and metabolic behavior of the strains, nHF-01 and PPR-01, were studied in different growth conditions and compared with standard strain. The molecular characterization was done by sequencing the ncrDNA ITS1-5.8S-ITS2 and D1-D2 domains of the nc 28S rDNA region and compared with a secondary structure-based phylogenetic tree. The secretory antimicrobials and pigments were characterized by TLC, UV-Vis, and FT-IR spectroscopy. Both the strains showed distinct growth patterns in different nutritional media and could assimilate a wide range of carbohydrates with distinctive biochemical properties. The molecular characterization revealed the strains, nHF-01 and PPR-01, as Aspergillus fumigatus (GenBank Accession No. MN190286 and MN190284, respectively). It was observed that the strain nHF-01 produces red to brownish pigments having mild antimicrobial activity while the strain PPR-01 does not represent such transformations. The extractable compounds had a significant antimicrobial potentiality against the human pathogenic bacteria. From this analysis, it can be concluded that the nHF-01 and PPR-01 strains are distinct from other A. fumigatus by their unique characters. Large-scale production and detailed molecular elucidation of the antimicrobial compounds may lead to the discovery of new antimicrobial compounds from these strains.
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Antiinfecciosos/metabolismo , Aspergillus fumigatus/metabolismo , Antiinfecciosos/farmacología , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/genética , Aspergillus fumigatus/crecimiento & desarrollo , Medios de Cultivo/química , ADN de Hongos/genética , ADN Ribosómico/genética , Humanos , Filogenia , Pigmentos Biológicos/metabolismo , Pigmentos Biológicos/farmacología , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Aspergillus fumigatus is the main cause of invasive aspergillosis (IA) with a high annual global incidence and mortality rate. Recent studies have indicated an increasing prevalence of azole-resistant A. fumigatus (ARAF) strains, with agricultural use of azole fungicides as a potential contributor. China has an extensive agricultural production system and uses a wide array of fungicides for crop production, including in modern growth facilities such as greenhouses. Soils in greenhouses are among the most intensively cultivated. However, little is known about the occurrence and distribution of ARAF in greenhouse soils. Here, we investigated genetic variation and triazole drug susceptibility in A. fumigatus from greenhouses around metropolitan Kunming in Yunnan, southwest China. Abundant allelic and genotypic variations were found among 233 A. fumigatus strains isolated from nine greenhouses in this region. Significantly, â¼80% of the strains were resistant to at least one medical triazole drug, with >30% showing cross-resistance to both itraconazole and voriconazole. Several previously reported mutations associated with triazole resistance in the triazole target gene cyp51A were also found in our strains, with a strong positive correlation between the frequency of mutations at the cyp51A promoter and that of voriconazole resistance. Phylogenetic analyses of cyp51A gene sequences showed evidence for multiple independent origins of azole-resistant genotypes of A. fumigatus in these greenhouses. Evidence for multiple origins of azole resistance and the widespread distributions of genetically very diverse triazole-resistant strains of A. fumigatus in greenhouses calls for significant attention from public health agencies.IMPORTANCE The origin and prevalence of azole-resistant Aspergillus fumigatus have been attracting increasing attention from biologists, clinicians, and public health agencies. Current evidence suggests agricultural fungicide use as a major cause. In southwest China, greenhouses are used to produce large amounts of fruits, flowers, and vegetables for consumers throughout China as well as those in other countries, primarily in southeast Asia. Here, we found a very high frequency (â¼80%) of triazole-resistant A. fumigatus in our sample, the highest reported so far, with a significant proportion of these strains resistant to both tested agricultural fungicides and medical triazole drugs. In addition, we found novel allelic and genotypic diversities and evidence for multiple independent origins of azole-resistant genotypes of A. fumigatus in greenhouse populations in this region. Our study calls for a systematic evaluation of the effects of azole fungicide usage in greenhouses on human health.
Asunto(s)
Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Azoles/farmacología , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Variación Genética , Agricultura , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/patogenicidad , China , Fungicidas Industriales/efectos adversos , Fungicidas Industriales/farmacología , Genotipo , Humanos , Mutación , Filogenia , Salud Pública , Suelo/química , Microbiología del SueloRESUMEN
The origin of isolates routinely used by the community of Aspergillus fumigatus researchers is periodically a matter of intense discussion at our centre, as the construction of recombinant isolates have sometimes followed convoluted routes, the documentation describing their lineages is fragmented, and the nomenclature is confusing. As an aide memoir, not least for our own benefit, we submit the following account and tabulated list of strains (Table 1) in an effort to collate all of the relevant information in a single, easily accessible document. To maximise the accuracy of this record we have consulted widely amongst the community of Medical Mycologists using these strains. All the strains described are currently available from one of these organisations, namely the Fungal Genetics Stock Centre (FGSC), FungiDB, Ensembl Fungi and The National Collection of Pathogenic Fungi (NCPF) at Public Health England. Display items from this manuscript are also featured on FungiDB. LAY ABSTRACT: We present a concise overview on the definition, origin and unique genetic makeup of the Aspergillus fumigatus isolates routinely in use by the fungal research community, to aid researchers to describe past and new strains and the experimental differences observed more accurately.
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Aspergillus fumigatus/clasificación , Aspergillus fumigatus/genética , Evolución Biológica , Genotipo , Filogenia , Variación Genética , HumanosRESUMEN
Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is both an environmental saprobe and an opportunistic human fungal pathogen, resistance is suggested to arise from fungicide use in agriculture, as the azoles used for plant protection share the same molecular target as the frontline antifungals used clinically. However, limiting azole fungicide use on crop fields to preserve their activity for clinical use could threaten the global food supply via a reduction in yield. In this study, we clarify the link between azole fungicide use on crop fields and resistance in a prototypical human pathogen through systematic soil sampling on farms in Germany and surveying fields before and after fungicide application. We observed a reduction in the abundance of A. fumigatus on fields following fungicide treatment in 2017, a finding that was not observed on an organic control field with only natural plant protection agents applied. However, this finding was less pronounced during our 2018 sampling, indicating that the impact of fungicides on A. fumigatus population size is variable and influenced by additional factors. The overall resistance frequency among agricultural isolates is low, with only 1 to 3% of isolates from 2016 to 2018 displaying resistance to medical azoles. Isolates collected after the growing season and azole exposure show a subtle but consistent decrease in susceptibility to medical and agricultural azoles. Whole-genome sequencing indicates that, despite the alterations in antifungal susceptibility, fungicide application does not significantly affect the population structure and genetic diversity of A. fumigatus in fields. Given the low observed resistance rate among agricultural isolates as well the lack of genomic impact following azole application, we do not find evidence that azole use on crops is significantly driving resistance in A. fumigatus in this context.IMPORTANCE Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is an environmental fungus that also causes life-threatening infections in humans, antimicrobial resistance is suggested to arise from fungicide use in agriculture, as the chemicals used for plant protection are almost identical to the antifungals used clinically. However, removing azole fungicides from crop fields threatens the global food supply via a reduction in yield. In this study, we survey crop fields before and after fungicide application. We find a low overall azole resistance rate among agricultural isolates, as well as a lack of genomic and population impact following fungicide application, leading us to conclude azole use on crops does not significantly contribute to resistance in A. fumigatus.
Asunto(s)
Aspergillus fumigatus/efectos de los fármacos , Farmacorresistencia Fúngica , Fungicidas Industriales/farmacología , Agricultura , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Azoles/química , Azoles/farmacología , Relación Dosis-Respuesta a Droga , Genética de Población , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Filogenia , Dinámica Poblacional , Microbiología del SueloRESUMEN
Aspergillus fumigatus is a pathogenic fungus responsible for invasive aspergillosis (IA). Typically, it can produce abundant conidia to survive and spread. The infection by A. fumigatus usually occurs in immunocompromised patients due to failed clearance of inhaled conidia. However, the incidence of aspergillosis in immunocompetent hosts has been increasing, the pathogenesis of which is still unknown. Our team previously obtained two clinical nonsporulating A. fumigatus isolates from non-immunocompromised patients, which only have the form of hyphae. This present study demonstrated the in vitro and in vivo characteristics of the two nonsporulating A. fumigatus isolates and verified that their conidiation defects are associated to abolished expression of the sporulation-related central regulatory pathway brlA gene. In addition, we confirmed the mutation site of brlA gene (c.657_660delTCCT) contributes to the nonsporulating phenotype in one clinical isolate. Plate assay showed that the two nonsporulating isolates have a similar resistance to antifungal drugs, cell wall disturbing substances, and oxidative stress compared with the wild-type reference Af293. Most important of all, we employed an immunocompetent mouse model to mimic the pathogenesis of pulmonary aspergillosis in non-immunocompromised patients. It revealed that the hyphae of two nonsporulating isolates and Af293 have similar virulence in immunocompetent hosts. Interestingly, the hyphae fragments of Af293 but not conidia are able to induce invasive aspergillosis in immunocompetent mice. In conclusion, our study indicate that the form of hyphae may play a dominant causative role in pulmonary aspergillosis of immunocompetent hosts rather than conidia.
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Aspergillus fumigatus/clasificación , Hifa/crecimiento & desarrollo , Inmunocompetencia , Aspergilosis Pulmonar/microbiología , Aspergilosis Pulmonar/patología , Animales , Aspergillus fumigatus/aislamiento & purificación , Aspergillus fumigatus/patogenicidad , Modelos Animales de Enfermedad , Farmacorresistencia Fúngica/genética , Femenino , Proteínas Fúngicas/genética , Humanos , Hifa/patogenicidad , Ratones , Ratones Endogámicos C57BL , Mutación , Esporas Fúngicas/fisiología , Factores de Transcripción/genética , VirulenciaRESUMEN
Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions.
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Aspergillus niger/enzimología , Aspergillus niger/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Transferasas/genética , Transferasas/metabolismo , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/enzimología , Aspergillus fumigatus/genética , Aspergillus nidulans/clasificación , Aspergillus nidulans/enzimología , Aspergillus nidulans/genética , Aspergillus niger/clasificación , Pared Celular , Eliminación de Gen , Mutación , FilogeniaRESUMEN
Azole antifungal resistance in Aspergillus fumigatus is a worldwide concern. As in most public hospitals in Brazil, antifungal susceptibility tests are not routinely performed for filamentous fungi at our institution. A 4-year retrospective azole antifungal resistance screening revealed two azole-resistant A. fumigatus clinical isolates carrying the CYP51A TR34 (34-bp tandem repeat)/L98H (change of L to H at position 98)/S297T/F495I resistance mechanism mutations, obtained from two unrelated patients. Broth microdilution antifungal susceptibility testing showed high MICs for itraconazole, posaconazole, and miconazole. Short tandem repeat (STR) typing analysis presented high levels of similarity between these two isolates and clinical isolates with the same mutations reported from the Netherlands, Denmark, and China, as well as environmental isolates from Taiwan. Our findings might indicate that active searching for resistant A. fumigatus is necessary. They also represent a concern considering that our hospital provides tertiary care assistance to immunocompromised patients who may be exposed to resistant environmental isolates. We also serve patients who receive prophylactic antifungal therapy or treatment for invasive fungal infections for years. In these two situations, isolates resistant to the antifungal in use may be selected within the patients themselves. We do not know the potential of this azole-resistant A. fumigatus strain to spread throughout our country. In this scenario, the impact on the epidemiology and use of antifungal drugs will significantly alter patient care, as in other parts of the world. In summary, this finding is an important contribution to alert hospital laboratories conducting routine microbiological testing to perform azole resistance surveillance and antifungal susceptibility tests of A. fumigatus isolates causing infection or colonization in patients at high risk for systemic aspergillosis.
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Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Azoles/farmacología , Sistema Enzimático del Citocromo P-450/genética , Proteínas Fúngicas/genética , Aspergillus fumigatus/clasificación , Brasil , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Repeticiones de Microsatélite/genética , Mutación Missense/genética , Estudios Retrospectivos , Secuencias Repetidas en Tándem/genéticaRESUMEN
Aspergillus fumigatus is a globally distributed opportunistic fungal pathogen capable of causing highly lethal invasive aspergillosis in immunocompromised individuals. Recent studies have indicated that the global population consists of multiple, divergent genetic clusters that are geographically broadly distributed. However, most of the analyzed samples have come from continental Eurasia and the Americas where the effects of ancient versus recent factors are difficult to distinguish. Here, we investigated environmental A. fumigatus isolates from Auckland, New Zealand, a geographically isolated population, and compared them with those from other parts of the world to determine the relative roles of historical differentiation and recent gene flow in shaping A. fumigatus populations. Our data suggest that the Auckland A. fumigatus population contains both unique indigenous genetic elements as well as genetic elements that are similar to those from other regions such as Europe, Africa, and North America. Though the hypothesis of random recombination was rejected, we found abundant evidence for phylogenetic incompatibility and recombination within the Auckland A. fumigatus population. Additionally, susceptibility testing identified two triazole-resistant strains, one of which contained the globally distributed mutation TR34/L98H in the cyp51A gene. Our results suggest that contemporary gene flow, likely due to anthropogenic factors, is a major force shaping the New Zealand A. fumigatus population.
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Aspergillus fumigatus/clasificación , Aspergillus fumigatus/genética , Microbiología Ambiental , Evolución Molecular , Flujo Génico , Variación Genética , Alelos , Aspergillus fumigatus/aislamiento & purificación , Farmacorresistencia Fúngica , Genes Fúngicos , Nueva Zelanda , Recombinación GenéticaRESUMEN
Azole resistance is a major concern for treatment of infections with Aspergillus fumigatus. Environmental resistance selection is a main route for Aspergillus spp. to acquire azole resistance. We investigated the presence of environmental hotspots for resistance selection in the Netherlands on the basis of the ability of A. fumigatus to grow and reproduce in the presence of azole fungicide residues. We identified 3 hotspots: flower bulb waste, green waste material, and wood chippings. We recovered azole-resistant A. fumigatus from these sites; all fungi contained cyp51A tandem repeat-mediated resistance mechanisms identical to those found in clinical isolates. Tebuconazole, epoxiconazole, and prothioconazole were the most frequently found fungicide residues. Stockpiles of plant waste contained the highest levels of azole-resistant A. fumigatus, and active aerobic composting reduced Aspergillus colony counts. Preventing plant waste stockpiling or creating unfavorable conditions for A. fumigatus to grow in stockpiles might reduce environmental resistance burden.
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Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Azoles/farmacología , Farmacorresistencia Fúngica , Microbiología Ambiental , Antifúngicos/uso terapéutico , Aspergilosis/tratamiento farmacológico , Aspergilosis/epidemiología , Aspergilosis/microbiología , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Azoles/uso terapéutico , Genes Bacterianos , Humanos , Pruebas de Sensibilidad Microbiana , Países Bajos/epidemiologíaRESUMEN
BACKGROUND: Studies on Aspergillus fumigatus azole resistance in cystic fibrosis patients are scarce despite the fact that it is the most frequently isolated fungus from respiratory samples from these individuals. OBJECTIVES: To evaluate resistance prevalence, investigate mechanisms of resistance and explore the relationship between resistant isolates by genotyping. METHODS: We conducted a prospective 1 year study (from 1 January to 31 December 2015), based on the investigation of up to five colonies per sample from cystic fibrosis patients. RESULTS: Twenty-three (6.5%) isolates among the 355 tested were resistant to at least one triazole drug, using the EUCAST reference method, leading to a prevalence of 6.8% (6/88 patients). Analysis of resistance mechanisms highlighted TR34/L98H (nâ=â10), TR46/Y121F/T289A (nâ=â1), WT cyp51A (nâ=â11) and F46Y/M172V/N248T/D255E/E427K (nâ=â1). No genotype was shared between patients. CONCLUSIONS: This study showed a relatively stable resistance prevalence in comparison with the previous study conducted in 2010-11 (8%), although resistance mechanisms varied between the two studies.
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Antifúngicos/farmacología , Aspergilosis/epidemiología , Aspergillus fumigatus/efectos de los fármacos , Azoles/farmacología , Fibrosis Quística/complicaciones , Farmacorresistencia Fúngica , Adolescente , Adulto , Anciano , Aspergilosis/microbiología , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Niño , Preescolar , Femenino , Francia/epidemiología , Variación Genética , Genotipo , Humanos , Lactante , Masculino , Persona de Mediana Edad , Epidemiología Molecular , Tipificación Molecular , Técnicas de Tipificación Micológica , Prevalencia , Estudios Prospectivos , Adulto JovenRESUMEN
The emergence of azole resistance in the pathogenic fungus Aspergillus fumigatus has continued to increase, with the dominant resistance mechanisms, consisting of a 34-nucleotide tandem repeat (TR34)/L98H and TR46/Y121F/T289A, now showing a structured global distribution. Using hierarchical clustering and multivariate analysis of 4,049 A. fumigatus isolates collected worldwide and genotyped at nine microsatellite loci using analysis of short tandem repeats of A. fumigatus (STRAf), we show that A. fumigatus can be subdivided into two broad clades and that cyp51A alleles TR34/L98H and TR46/Y121F/T289A are unevenly distributed across these two populations. Diversity indices show that azole-resistant isolates are genetically depauperate compared to their wild-type counterparts, compatible with selective sweeps accompanying the selection of beneficial mutations. Strikingly, we found that azole-resistant clones with identical microsatellite profiles were globally distributed and sourced from both clinical and environmental locations, confirming that azole resistance is an international public health concern. Our work provides a framework for the analysis of A. fumigatus isolates based on their microsatellite profile, which we have incorporated into a freely available, user-friendly R Shiny application (AfumID) that provides clinicians and researchers with a method for the fast, automated characterization of A. fumigatus genetic relatedness. Our study highlights the effect that azole drug resistance is having on the genetic diversity of A. fumigatus and emphasizes its global importance upon this medically important pathogenic fungus.IMPORTANCE Azole drug resistance in the human-pathogenic fungus Aspergillus fumigatus continues to emerge, potentially leading to untreatable aspergillosis in immunosuppressed hosts. Two dominant, environmentally associated resistance mechanisms, which are thought to have evolved through selection by the agricultural application of azole fungicides, are now distributed globally. Understanding the effect that azole resistance is having on the genetic diversity and global population of A. fumigatus will help mitigate drug-resistant aspergillosis and maintain the azole class of fungicides for future use in both medicine and crop protection.
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Antifúngicos/farmacología , Aspergilosis/microbiología , Aspergillus fumigatus/efectos de los fármacos , Azoles/farmacología , Farmacorresistencia Fúngica , Microbiología Ambiental , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Análisis por Conglomerados , Variación Genética , Genotipo , Humanos , Repeticiones de Microsatélite , Tipificación Molecular , Técnicas de Tipificación Micológica , Filogenia , Secuencias Repetidas en TándemRESUMEN
More than a decade ago a short tandem repeat-based typing method was developed for the fungus Aspergillus fumigatus. This STRAf assay is based on the analysis of nine short tandem repeat markers. Interpretation of this STRAf assay is complicated when there are only one or two differences in tandem repeat markers between isolates, as the stability of these markers is unknown. To determine the stability of these nine markers, a STRAf assay was performed on 73-100 successive generations of five clonally expanded A. fumigatus isolates. In a total of 473 generations we found five times an increase of one tandem repeat unit. Three changes were found in the trinucleotide repeat marker STRAf 3A, while the other two were found in the trinucleotide repeat marker STRAf 3C. The di- or tetranucleotide repeats were not altered. The altered STRAf markers 3A and 3C demonstrated the highest number of repeat units (≥50) as compared to the other markers (≤26). Altogether, we demonstrated that 7 of 9 STRAf markers remain stable for 473 generations and that the frequency of alterations in tandem repeats is positively correlated with the number of repeats. The potential low level instability of STRAf markers 3A and 3C should be taken into account when interpreting STRAf data during an outbreak.
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Aspergillus fumigatus/clasificación , Aspergillus fumigatus/genética , Inestabilidad Genómica , Técnicas de Genotipaje/métodos , Repeticiones de Microsatélite , Técnicas de Tipificación Micológica/métodos , Secuencias Repetidas en TándemRESUMEN
We collected sputum samples and cough plates from 15 cystic fibrosis patients in the Netherlands who were colonized with Aspergillus fumigatus; we recovered A. fumigatus of the same genotype in cough aerosols and sputum samples from 2 patients. The belief that transmission of A. fumigatus from cystic fibrosis patients does not occur should be reconsidered.
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Aspergilosis/etiología , Aspergilosis/transmisión , Aspergillus fumigatus , Fibrosis Quística/complicaciones , Exposición por Inhalación/efectos adversos , Antifúngicos/farmacología , Antifúngicos/uso terapéutico , Aspergilosis/epidemiología , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/genética , Aspergillus fumigatus/aislamiento & purificación , Fibrosis Quística/epidemiología , Genotipo , Humanos , Tipificación Molecular , Países Bajos/epidemiología , Vigilancia en Salud Pública , Esputo/microbiologíaRESUMEN
Aspergillus fumigatus resistant to azole as first-line therapy has been reported in azole-naïve patients. This worldwide resistance phenomenon has been linked to fungicide-driven alterations in the cyp51A gene and its promoter region (such as TR34/L98H and TR46/Y121F/T289A). Azole-resistant A. fumigatus related to the use of triazole fungicides in flower fields was recently reported In Colombia. The purpose of this study was to investigate the presence of azole-resistant A. fumigatus in soil samples from vegetable crops such as carrots, potatoes, maize, strawberries, and pea, and from prepared farming land surrounding the city of Bogotá. Species identification was based on sequencing of the ß-tubulin and calmodulin genes. All A. fumigatus strains were screened for azole resistance on agar supplemented with itraconazole or voriconazole. Among the 60 soil samples, 34 (56.6%) were positive for A. fumigatus and 15 samples exhibited strains (n = 18) that grew on agar supplemented with itraconazole or voriconazole. Triazole-resistant strains were isolated from soil samples associated with carrot, potato, maize, and pea crops. Sequencing of the cyp51A gene and its promoter region indicated polymorphism, mainly with the presence of TR46/Y121F/T289A (n = 8), TR34/L98H, and TR53. Eight resistant isolates exhibited cyp51A wild type without alterations in the promoter region. Our study showed evidence of dissemination of azole-resistant A. fumigatus, with high genetic diversity, in vegetable crops in Colombia. These data underline the need to determine the prevalence of azole resistance in A. fumigatus in clinical and environmental settings for other regions of Colombia as well as Latin America.
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Aspergillus fumigatus/efectos de los fármacos , Azoles/administración & dosificación , Sistema Enzimático del Citocromo P-450/genética , Farmacorresistencia Fúngica , Proteínas Fúngicas/genética , Fungicidas Industriales/administración & dosificación , Enfermedades de las Plantas/prevención & control , Verduras/microbiología , Aspergillus fumigatus/clasificación , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/aislamiento & purificación , Azoles/farmacología , Calmodulina/genética , Colombia , Fungicidas Industriales/farmacología , Humanos , Polimorfismo Genético , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Microbiología del Suelo , Tubulina (Proteína)/genéticaRESUMEN
AIMS: Aspergillus sp. are ubiquitous saprophytic fungi and their conidia easily inhaled. This is particularly important in immunocompromised patients, more susceptible to developing invasive aspergillosis. In addition to A. fumigatus sensu stricto, cryptic species, many resistant to antifungal drugs, have been associated with invasive infections, making it important to assess their presence and diversity in different environments. Therefore, the aim of this study was to assess the presence, diversity and susceptibility to antifungal drugs of airborne fungi. Moreover, in azole-resistant A. fumigatus sensu stricto isolates the presence of underlying molecular mechanisms of resistance was investigated. METHODS AND RESULTS: Eighty-four Aspergillus isolates were collected from the environment air in hospitals and the Water Treatment Plant. The use of molecular tools allowed to detect 12 different cryptic species, showing a prevalence of 21·4%. The majority of isolates (69·0%) belonged to A. fumigatus complex and 65·4% were A. fumigatus sensu stricto. Among these, 21·8% were resistant to itraconazole (ITZ), 38·2% to posaconazole and 87·3% to isavuconazole; none of them were resistant to voriconazole or amphotericin B. Sequencing of the cyp51A gene on the 12 A. fumigatus sensu stricto ITZ-resistant isolates revealed the presence of mutations. CONCLUSION: Our study reports a large number of environmental-resistant Aspergillus species, including A. fumigatus sensu stricto that display an important role in invasive fungal infections. None of the environmental isolates showed mutations on cyp51A gene related to azole resistance. SIGNIFICANT AND IMPACT OF THE STUDY: This study is the first assessment of molecular resistance mechanisms in A. fumigatus sensu stricto environmental isolates, in Portugal. Since TR34/L98H and TR46/Y121F/T289A cyp51A mutations were already reported in the clinical setting in Portugal (Monteiro et al. J Glob Antimicron Resist 13: 190-191, 2018; Pinto et al. Front Microbiol 9: 1656, 2018), and have been linked to environmental route, it is utmost importance to perform surveillance network for azole-resistant A. fumigatus.