Triazole Fungicides Can Induce Cross-Resistance to Medical Triazoles in Aspergillus fumigatus

Contains fulltext : 103858.pdf (publisher's version ) (Open Access)BACKGROUND: Azoles play an important role in the management of Aspergillus diseases. Azole resistance is an emerging global problem in Aspergillus fumigatus, and may develop through patient therapy. In addition, an environmental route of resistance development has been suggested through exposure to 14alpha-demethylase inhibitors (DMIs). The main resistance mechanism associated with this putative fungicide-driven route is a combination of alterations in the Cyp51A-gene (TR(34)/L98H). We investigated if TR(34)/L98H could have developed through exposure to DMIs. METHODS AND FINDINGS: Thirty-one compounds that have been authorized for use as fungicides, herbicides, herbicide safeners and plant growth regulators in The Netherlands between 1970 and 2005, were investigated for cross-resistance to medical triazoles. Furthermore, CYP51-protein homology modeling and molecule alignment studies were performed to identify similarity in molecule structure and docking modes. Five triazole DMIs, propiconazole, bromuconazole, tebuconazole, epoxiconazole and difenoconazole, showed very similar molecule structures to the medical triazoles and adopted similar poses while docking the protein. These DMIs also showed the greatest cross-resistance and, importantly, were authorized for use between 1990 and 1996, directly preceding the recovery of the first clinical TR(34)/L98H isolate in 1998. Through microsatellite genotyping of TR(34)/L98H isolates we were able to calculate that the first isolate would have arisen in 1997, confirming the results of the abovementioned experiments. Finally, we performed induction experiments to investigate if TR(34)/L98H could be induced under laboratory conditions. One isolate evolved from two copies of the tandem repeat to three, indicating that fungicide pressure can indeed result in these genomic changes. CONCLUSIONS: Our findings support a fungicide-driven route of TR(34)/L98H development in A. fumigatus. Similar molecule structure characteristics of five triazole DMIs and the three medical triazoles appear the underlying mechanism of cross resistance development. Our findings have major implications for the assessment of health risks associated with the use of triazole DMIs

Emergence of Azole Resistance in Aspergillus fumigatus and Spread of a Single Resistance Mechanism

Paul Verweij and colleagues show that azole resistance has emerged inAspergillus fumigatus in The Netherlands and that a dominant resistance mechanism is present in clinical isolates

Emergence of a Pathogenic Fungus Resistant to Triazole Antifungal Drugs

International audienc

Azole resistance in Aspergillus fumigatus

Invasive aspergillosis remains an important complication of immunosuppressive treatment regimens in humans. Triazoles are increasingly used in the prevention and management of invasive aspergillosis. Voriconazole is first choice for the primary therapy of invasive aspergillosis, and posaconazole was shown to be highly effective in preventing invasive aspergillus disease when given to high-risk groups prophylactically. Resistance in moulds is uncommon in clinical medicine, but might occur in specific patient groups. It appears that azole resistance might evolve in patients that harbor a high number of reproducing Aspergillus and are treated with azoles for long period of time. These conditions are present in patients with aspergilloma or other cavitary lung diseases caused by Aspergillus species. Azole resistance has been described to emerge in this patient group. Recently azole resistance was also reported in patients with acute invasive aspergillosis, where the conditions for resistance development appear to be less that optimal: fungal proliferation through hyphal elongation and relative short treatment episodes. This might suggest that azole resistance is caused by exposure outside the patient, for instance in the environment. Molecular studies have shown that triazole resistance in A. fumigatus is associated with amino acid substitutions in the cyp51A protein. Alterations might result in different patterns of azole resistance, primarily of itraconazole, with varying reduced activity of other triazoles such as voriconazole and posaconazole. Azole resistance in A. fumigatus has been associated with treatment failure. At present clinical microbiology laboratories do not routinely perform in vitro susceptibility testing, but it appears to be appropriate to test A. fumigatus isolates at least in those isolates recovered from patients that fail to azole therapy

Next-Generation Sequencing in the Mycology Lab

New state-of-the-art techniques in sequencing offer valuable tools in both detection of mycobiota and in understanding of the molecular mechanisms of resistance against antifungal compounds and virulence. Introduction of new sequencing platform with enhanced capacity and a reduction in costs for sequence analysis provides a potential powerful tool in mycological diagnosis and research. In this review, we summarize the applications of next-generation sequencing techniques in mycology

Azole-resistance development; how the Aspergillus fumigatus lifecycle defines the potential for adaptation

In order to successfully infect or colonize human hosts or survive changing environments, Aspergillus fumigatus needs to adapt through genetic changes or phenotypic plasticity. The genomic changes are based on the capacity of the fungus to produce genetic variation, followed by selection of the genotypes that are most fit to the new environment. Much scientific work has focused on the metabolic plasticity, biofilm formation or the particular genetic changes themselves leading to ad-aptation, such as antifungal resistance in the host. Recent scientific work has shown advances made in understanding the natural relevance of parasex and how both the asexual and sexual reproduction can lead to tandem repeat elongation in the target gene of the azoles: the cyp51A gene. In this review, we will explain how the fungus can generate genetic variation that can lead to adaptation. We will discuss recent advances that have been made in the understanding of the lifecycle of A. fumigatus to explain the differences observed in speed and type of mutations that are generated under different environments and how this can facilitate adaptation, such as azole-resistance selec-tion

Flower Bulb Waste Material is a Natural Niche for the Sexual Cycle in Aspergillus fumigatus

With population genetic evidence of recombination ongoing in the natural Aspergillus fumigatus population and a sexual cycle demonstrated in the laboratory the question remained what the natural niche for A. fumigatus sex is. Composting plant-waste material is a known substrate of A. fumigatus to thrive and withstand temperatures even up to 70°C. Previous studies have shown indirect evidence for sexual reproduction in these heaps but never directly demonstrated the sexual structures due to technical limitations. Here, we show that flower bulb waste material from stockpiles undergoing composting can provide the conditions for sexual reproduction. Direct detection of ascospore structures was shown in agricultural flower bulb waste material by using a grid-based detection assay. Furthermore, we demonstrate that ascospores can germinate after exposure to 70°C for up to several days in contrast to asexual conidia that are unable to survive a two-hour heat shock. This indicates a sufficient time frame for ascospores to survive and escape composting stockpiles. Finally, sexual crosses with cleistothecium and viable ascospore formation could successfully be performed on flower bulb waste material. Recombination of A. fumigatus can now be explained by active sexual reproduction in nature as we show in this study that flower bulb waste material provides an environmental niche for sex

Azole Resistance Profile of Amino Acid Changes in Aspergillus fumigatus CYP51A Based on Protein Homology Modeling▿

Molecular studies have shown that the majority of azole resistance in Aspergillus fumigatus is associated with amino acid substitutions in the cyp51A gene. To obtain insight into azole resistance mutations, the cyp51A gene of 130 resistant and 76 susceptible A. fumigatus isolates was sequenced. Out of 130 azole-resistant isolates, 105 contained a tandem repeat of 34 bp in the promoter region and a leucine-to-histidine substitution in codon 98 (designated TR/L98H). Additionally, in 12 of these TR/L98H resistant isolates, the mutations S297T and F495I were found, and in 1 isolate, the mutation F495I was found. In eight azole-resistant isolates, known azole resistance mutations were detected in codon G54, G138, or M220. In three azole-susceptible isolates, the mutation E130D, L252L, or S400I was found and in 13 azole-susceptible isolates but also in 1 azole-resistant isolate, the mutations F46Y, G98G, M172V, N248T, D255E, L358L, E427K, and C454C were found. All of the nonsynonymous mutations, apart from the mutations in codons G54, G138, and M220 and L98H, were located at the periphery of the protein, as determined by a structural model of the A. fumigatus Cyp51A protein, and were predicted neither to interact with azole compounds nor to affect structural integrity. Therefore, this wide diversity of mutations in the cyp51A gene in azole-susceptible A. fumigatus isolates is not correlated with azole resistance. Based on the Cyp51A protein homology model, the potential correlation of a mutation to azole resistance can be predicted

Generation of Affibody® ligands binding interleukin-2 receptor α/CD25

Affibody® molecules specific for human IL-2Rα, the IL-2 (interleukin-2) receptor α subunit, also known as CD25, were selected by phage-display technology from a combinatorial protein library based on the 58-residue Protein A-derived Z domain. The IL-2R system plays a major role in T-cell activation and the regulation of cellular immune responses. Moreover, CD25 has been found to be overexpressed in organ rejections, a number of autoimmune diseases and T-cell malignancies. The phage-display selection using Fc-fused target protein generated 16 unique Affibody® molecules targeting CD25. The two most promising binders were characterized in more detail using biosensor analysis and demonstrated strong and selective binding to CD25. Kinetic biosensor analysis revealed that the two monomeric Affibody® molecules bound to CD25 with apparent affinities of 130 and 240 nM respectively. The Affibody® molecules were, on biosensor analysis, found to compete for the same binding site as the natural ligand IL-2 and the IL-2 blocking monoclonal antibody 2A3. Hence the Affibody® molecules were assumed to have an overlapping binding site with IL-2 and antibodies targeting the IL-2 blocking Tac epitope (for example, the monoclonal antibodies Daclizumab and Basiliximab, both of which have been approved for therapeutic use). Furthermore, immunofluorescence microscopy and flow-cytometric analysis of CD25-expressing cells demonstrated that the selected Affibody® molecules bound to CD4 + CD25+ PMBCs (peripheral-blood mononuclear cells), the IL-2-dependent cell line NK92 and phytohaemagglutinin-activated PMBCs. The potential use of the CD25-binding Affibody® molecules as targeting agents for medical imaging and for therapeutic applications is discussed