Medical Triazoles Research Articles

Greenhouses represent an important evolutionary niche for Alternaria alternata.

Greenhouses have become increasingly important for food production and food security. However, our understanding of how greenhouses may contribute to genetic variations in soil microbial populations is very limited. In this study, we obtained and analyzed soil populations of the cosmopolitan fungal pathogen Alternaria alternata in seven greenhouses in Shijiazhuang, northeast China. Our analyses revealed high proportions of isolates being resistant to agricultural triazole fungicides and medical triazole drugs, including cross-resistance to both groups of triazoles. In addition, we found that greenhouse populations of A. alternata located within a few kilometers showed similar levels of genetic differentiation as those separated by over 2,000 km between northeast and southwest China. Our study suggests that greenhouse populations of this and potentially other fungal pathogens represent an important ecological niche and an emerging threat to food security and human health.

Genetic structure and triazole resistance among Aspergillus fumigatus populations from remote and undeveloped regions in Eastern Himalaya.

Aspergillus fumigatus is a ubiquitous mold and a common human fungal pathogen. Recent molecular population genetic and epidemiological analyses have revealed evidence for long-distance gene flow and high genetic diversity within most local populations of A. fumigatus. However, little is known about the impact of regional landscape factors in shaping the population diversity patterns of this species. Here we sampled extensively and investigated the population structure of A. fumigatus from soils in the Three Parallel Rivers (TPR) region in Eastern Himalaya. This region is remote, undeveloped and sparsely populated, bordered by glaciated peaks more than 6,000 m above sea level, and contained three rivers separated by tall mountains over very short horizontal distances. A total of 358 A. fumigatus strains from 19 sites along the three rivers were isolated and analyzed at nine loci containing short tandem repeats. Our analyses revealed that mountain barriers, elevation differences, and drainage systems all contributed low but statistically significant genetic variations to the total A. fumigatus population in this region. We found abundant novel alleles and genotypes in the TPR population of A. fumigatus and significant genetic differentiation between this population and those from other parts of Yunnan and the globe. Surprisingly, despite limited human presence in this region, about 7% of the A. fumigatus isolates were resistant to at least one of the two medical triazoles commonly used for treating aspergillosis. Our results call for greater surveillance of this and other human fungal pathogens in the environment. IMPORTANCE The extreme habitat fragmentation and substantial environmental heterogeneity in the TPR region have long known to contribute to geographically shaped genetic structure and local adaptation in several plant and animal species. However, there have been limited studies of fungi in this region. Aspergillus fumigatus is a ubiquitous pathogen capable of long-distance dispersal and growth in diverse environments. In this study, using A. fumigatus as a model, we investigated how localized landscape features contribute to genetic variations in fungal populations. Our results revealed that elevation and drainage isolation rather than direct physical distances significantly impacted genetic exchange and diversity among the local A. fumigatus populations. Interestingly, within each local population, we found high allelic and genotypic diversities, and with evidence ~7% of all isolates being resistant to two medical triazoles, itraconazole and voriconazole. Given the high frequency of ARAF found in mostly natural soils of sparsely populated sites in the TPR region, close monitoring of their dynamics in nature and their effects on human health is needed.

Genetic Structure and Triazole Antifungal Susceptibilities of Alternaria alternata from Greenhouses in Kunming, China.

ABSTRACTAlternaria alternata is an opportunistic human fungal pathogen and a ubiquitous phytopathogen capable of causing diseases to >100 agricultural crops and ornamental plants. To control plant diseases caused by A. alternata, triazole fungicides have been widely used both in open crop and vegetable fields and in indoor growth facilities such as greenhouses. At present, the effect of fungicide use on triazole resistance development in A. alternata populations is not known. Here, we isolated 237 A. alternata strains from nine greenhouses around metropolitan Kunming in Yunnan, southwest China, determined their genotypes using 10 short tandem repeat markers, and quantified their susceptibility to four triazoles (difenoconazole, tebuconazole, itraconazole, and voriconazole). Abundant allelic and genotypic diversities were detected among these A. alternata strains. Significantly, over 17% of the strains were resistant to difenoconazole, and both known and new drug-resistance mutations were found in the triazole target gene cyp51. Our findings of high-level genetic variation of A. alternata in greenhouses coupled with high-frequency fungicide resistance call for greater attention to continued monitoring and to developing alternative plant fungal disease management strategies in greenhouses.IMPORTANCE Alternaria alternata is among the most common fungi in our environments, such as indoor facilities, the soil, and outdoor air. It can cause diseases in >100 crop and ornamental plants. Furthermore, it can cause human infections. However, our understanding of its genetic diversity and antifungal susceptibility is very limited. Indeed, the critical threshold values for resistance have not been defined for most antifungal drugs in this species. Greenhouses are known to have heavy applications of agricultural fungicides. In this study, we analyzed strains of A. alternata from nine greenhouses near metropolitan Kunming in southwestern China. Our study revealed very high genetic diversity and identified strains with high MIC values against two agricultural and two medical triazole antifungals within each of the nine greenhouses. Our study calls for greater attention to this emerging threat to food security and human health.

Citizen Science Surveillance of Triazole-Resistant Aspergillus fumigatus in United Kingdom Residential Garden Soils.

Compost is an ecological niche for Aspergillus fumigatus due to its role as a decomposer of organic matter and its ability to survive the high temperatures associated with the composting process. Subsequently, composting facilities are associated with high levels of A. fumigatus spores that are aerosolized from compost and cause respiratory illness in workers. In the UK, gardening is an activity enjoyed by individuals of all ages, and it is likely that they are being exposed to A. fumigatus spores when handling commercial compost or compost they have produced themselves. In the present study, 246 citizen scientists collected 509 soil samples from locations in their gardens in the UK, from which were cultured 5,174 A. fumigatus isolates. Of these isolates, 736 (14%) were resistant to tebuconazole: the third most-sprayed triazole fungicide in the UK, which confers cross-resistance to the medical triazoles used to treat A. fumigatus lung infections in humans. These isolates were found to contain the common resistance mechanisms in the A. fumigatus cyp51A gene TR34/L98H or TR46/Y121F/T289A, as well as the less common resistance mechanisms TR34, TR53, TR46/Y121F/T289A/S363P/I364V/G448S, and (TR46)2/Y121F/M172I/T289A/G448S. Regression analyses found that soil samples containing compost were significantly more likely to grow tebuconazole-susceptible and tebuconazole-resistant A. fumigatus strains than those that did not and that compost samples grew significantly higher numbers of A. fumigatus than other samples. IMPORTANCE The findings presented here highlight compost as a potential health hazard to individuals with predisposing factors to A. fumigatus lung infections and as a potential health hazard to immunocompetent individuals who could be exposed to sufficiently high numbers of spores to develop infection. Furthermore, we found that 14% of A. fumigatus isolates in garden soils were resistant to an agricultural triazole, which confers cross-resistance to medical triazoles used to treat A. fumigatus lung infections. This raises the question of whether compost bags should carry additional health warnings regarding inhalation of A. fumigatus spores, whether individuals should be advised to wear facemasks while handling compost, or whether commercial producers should be responsible for sterilizing compost before shipping. The findings support increasing public awareness of the hazard posed by compost and investigating measures that can be taken to reduce the exposure risk.

Dissection of the Activity of Agricultural Fungicides against Clinical Aspergillus Isolates with and without Environmentally and Medically Induced Azole Resistance.

Azole resistance is an emerging problem in patients with aspergillosis. The role of fungicides for resistance development and occurrence is not fully elucidated. EUCAST reference MICs of 17 fungicides (11 azoles and 6 others), five azole fungicide metabolites and four medical triazoles were examined against two reference and 28 clinical isolates of A. fumigatus, A. flavus and A. terreus with (n = 12) and without (n = 16) resistance mutations. Eight/11 azole fungicides were active against wild-type A. fumigatus, A. flavus and A. terreus, including four (metconazole, prothioconazole-desthio, prochloraz and imazalil) with low MIC50 (≤2 mg/L) against all three species and epoxiconazole, propiconazole, tebuconazole and difenoconazole also against wild-type A. terreus. Mefentrifluconazole, azole metabolites and non-azole fungicides MICs were >16 mg/L against A. fumigatus although partial growth inhibition was found with mefentrifluconazole. Moreover, mefentrifluconazole and axozystrobin were active against wild-type A. terreus. Increased MICs (≥3 dilutions) were found for TR34/L98H, TR34(3)/L98H, TR46/Y121F/T289A and G432S compared to wild-type A. fumigatus for epoxiconazole, propiconazole, tebuconazole, difenoconazole, prochloraz, imazalil and metconazole (except G432S), and for prothioconazole-desthio against TR46/Y121F/T289A, specifically. Increased MICs were found in A. fumigatus harbouring G54R, M220K and M220R alterations for five, one and one azole fungicides, respectively, compared to MICs against wild-type A. fumigatus. Similarly, increased MICs wer found for A. terreus with G51A, M217I and Y491H alterations for five, six and two azole fungicides, respectively. Azole fungicides showed activity against wild-type A. fumigatus, A. terreus and A. flavus, but not against all mutant isolates, suggesting the environmental route of azole resistance may have a role for all three species.

The Medical Triazole Voriconazole Can Select for Tandem Repeat Variations in Azole-Resistant Aspergillus Fumigatus Harboring TR34/L98H Via Asexual Reproduction.

Azole-resistant Aspergillus fumigatus isolates recovered at high frequency from patients, harbor mutations that are associated with variation of promoter length in the cyp51A gene. Following the discovery of the TR34/L98H genotype, new variations in tandem repeat (TR) length and number of repeats were identified, as well as additional single nucleotide polymorphisms (SNPs) in the cyp51A gene, indicating that the diversity of resistance mutations in A. fumigatus is likely to continue to increase. Investigating the development routes of TR variants is critical to be able to design preventive interventions. In this study, we tested the potential effects of azole exposure on the selection of TR variations, while allowing haploid A. fumigatus to undergo asexual reproduction. Through experimental evolution involving voriconazole (VOR) exposure, an isolate harboring TR343/L98H evolved from a clinical TR34/L98H ancestor isolate, confirmed by whole genome sequencing. TR343/L98H was associated with increased cyp51A expression and high VOR and posaconazole MICs, although additional acquired SNPs could also have contributed to the highly azole-resistant phenotype. Exposure to medical azoles was found to select for TR343, thus supporting the possibility of in-host selection of TR34 variants.

Diagnostic-driven management of invasive fungal disease in hematology in the era of prophylaxis and resistance emergence: Dutch courage?

Patients receiving intensive anti-leukemic treatment or recipients of allogeneic hematopoietic stem cell transplantation (HSCT) are prone to develop invasive fungal disease caused by both Aspergillus and non-Aspergillus moulds. Overall mortality following invasive mould disease (IMD) is high; adequate and timely antifungal treatment seems to ameliorate the outcome, yet early diagnosis in the haematological patient remains a challenge for most clinicians. Prophylaxis and the empiric addition of antifungal therapy to neutropaenic patients with fever persisting or recurring during broad-spectrum antibiotic treatment is therefore standard of care in many institutions. However, aside from the potential for overtreatment and important side effects, the emergence of resistance to medical triazoles in Aspergillus fumigatus poses a risk for inadequate initial treatment. Initial voriconazole therapy in patients with azole-resistant invasive aspergillosis was recently shown to be associated with a 23% increased mortality rate compared to the patients with azole-susceptible infection, despite changing to appropriate antifungal therapy once resistance was detected. Moreover, fever is not always present with IMD; therefore, cases may be missed when relying solely on this symptom for starting diagnostic procedures and antifungal treatment. At our institution, a diagnostic-driven treatment approach for IMD was implemented relying on clinical but also laboratory markers to start antifungal treatment. We describe the basis and clinical implementation of our diagnostic-driven approach in this review.

Pesticide behavior in paddy fields and development of azole-resistant Aspergillus fumigatus: Should we be concerned?

Tricyclazole as a common fungicide wildly used to control rice blast disease in the Asian country may induce azole resistance in Aspergillus fumigatus isolates. The main reason of the acquired azole resistance is probably environmental exposure through wide fungicide use in agriculture. The present study was conducted with the aim of investigating the current status of the azole-resistant A. fumigatus obtained from the paddy fields with exposure to tricyclazole. A total of 108 soil samples were collected from four different locations of paddy fields in Mazandaran Province, Iran. Pure fungal colonies were initially identified based on the conventional tools, and then reconfirmed by using DNA sequencing of the partial ß-tubulin gene. In addition, the in vitro antifungal susceptibility was determined using the Clinical and Laboratory Standards Institute document (CLSI) M38-A2. The identification of the mutations in the CYP51A gene was accomplished by the implementation of the polymerase chain reaction amplification assay on the selected isolates. Overall, 31 of 108 (28.7%) isolates were identified as A.fumigatus, four (3.7%) of which were recognized as azole-resistant with MICs of itraconazole ≥8μg/ml and voriconazole ≥4μg/ml. Only two out of the four azole-resistant A. fumigatus isolates harboured TR34/L98H variant and the other two isolates were identified as azole-resistant without any CYP51A gene mutations. However, other point mutations (TR46/Y121F/T289A) were not detected in the CYP51A gene. The high molecular structure similarity between environmental and medical triazoles may result in the selection of resistance mechanisms. Nonetheless, one might conclude that tricyclazole with different molecular structures against medical azoles induces azole-resistance in A.fumigatus isolates. The behavior of such pesticides as tricyclazole in the rice paddy fields would have an effective role in the development of azole-resistance that requires detailed information.

Evolution of cross-resistance to medical triazoles in Aspergillus fumigatus through selection pressure of environmental fungicides.

Resistance to medical triazoles in Aspergillus fumigatus is an emerging problem for patients at risk of aspergillus diseases. There are currently two presumed routes for medical triazole-resistance selection: (i) through selection pressure of medical triazoles when treating patients and (ii) through selection pressure from non-medical sterol-biosynthesis-inhibiting (SI) triazole fungicides which are used in the environment. Previous studies have suggested that SI fungicides can induce cross-resistance to medical triazoles. Therefore, to assess the potential of selection of resistance to medical triazoles in the environment, we assessed cross-resistance to three medical triazoles in lineages of A. fumigatus from previous work where we applied an experimental evolution approach with one of five different SI fungicides to select for resistance. In our evolved lines we found widespread cross-resistance indicating that resistance to medical triazoles rapidly arises through selection pressure of SI fungicides. All evolved lineages showed similar evolutionary dynamics to SI fungicides and medical triazoles, which suggests that the mutations inducing resistance to both SI fungicides and medical triazoles are likely to be the same. Whole-genome sequencing revealed that a variety of mutations were putatively involved in the resistance mechanism, some of which are in known target genes.

Is the emergence of fungal resistance to medical triazoles related to their use in the agroecosystems? A mini review.

Triazole fungicides are used broadly for the control of infectious diseases of both humans and plants. The surge in resistance to triazoles among pathogenic populations is an emergent issue both in agriculture and medicine. The non-rational use of fungicides with site-specific modes of action, such as the triazoles, may increase the risk of antifungal resistance development. In the medical field, the surge of resistant fungal isolates has been related to the intensive and recurrent therapeutic use of a limited number of triazoles for the treatment and prophylaxis of many mycoses. Similarities in the mode of action of triazole fungicides used in these two fields may lead to cross-resistance, thus expanding the spectrum of resistance to multiple fungicides and contributing to the perpetuation of resistant strains in the environment. The emergence of fungicide-resistant isolates of human pathogens has been related to the exposure to fungicides used in agroecosystems. Examples include species of cosmopolitan occurrence, such as Fusarium and Aspergillus, which cause diseases in both plants and humans. This review summarizes the information about the most important triazole fungicides that are largely used in human clinical therapy and agriculture. We aim to discuss the issues related to fungicide resistance and the recommended strategies for preventing the emergence of triazole-resistant fungal populations capable of spreading across environments.

Determination of antifungal susceptibility patterns among the environmental isolates of Aspergillus fumigatus in Iran.

Background:In recent years, triazole-resistant environmental isolates of Aspergillus fumigatus have emerged in Europe and Asia. Azole resistance has been reported in patients who are treated with long-term azole therapy or exposure of the fungus spores to the azole fungicides used in agriculture. To date, a wide range of mutations in A. fumigatus have been described conferring azole-resistance, which commonly involves modifications in the cyp51A gene. We investigated antifungal susceptibility pattern of environmental isolates of A. fumigatus.Materials and Methods:In this study, 170 environmental samples collected from indoors surfaces of three hospitals in Iran. It was used β-tubulin gene to confirm the all of A. fumigatus isolates, which was identified by conventional methods. Furthermore, the antifungal susceptibility of itraconazole, voriconazole, and posaconazole was investigated using broth microdilution test, according to European Committee on Antimicrobial Susceptibility testing reference method.Results:From a total of 158 environmental molds fungi obtained from the hospitals, 58 isolates were identified as A. fumigatus by amplification of expected size of β-tubulin gene (~500 bp). In this study, in vitro antifungal susceptibility testing has shown that there were not high minimum inhibitory concentration values of triazole antifungals in all of the 58 environmental isolates of A. fumigatus.Conclusion:Our findings demonstrated that there was not azole-resistant among environmental isolates of A. fumigatus. Medical triazoles compounds have structural similarity with triazole fungicide compounds in agriculture, therefore, resistance development through exposure to triazole fungicide compounds in the environment is important but it sounds there is not a serious health problem in drug resistance in environmental isolates in Iran.

Triazole Susceptibilities in Thermotolerant Fungal Isolates from Outdoor Air in the Seoul Capital Area in South Korea.

Emerging fungi resistant to triazoles are a concern because of the increased use of medical triazoles and exposure to agricultural triazoles. However, little is known about the levels of triazole susceptibility in outdoor airborne fungi making it difficult to assess the risks of inhalation exposure to airborne, antifungal-resistant fungi. This study examined triazole susceptibilities of the airborne thermotolerant fungi isolated from the ambient air of the Seoul Capital Area of South Korea. We used impactor air sampling with triazole-containing nutrient agar plates as the collection substrates to screen for airborne fungal isolates based on their triazole susceptibilities. This study estimated that 0.17% of all the culturable fungi belong to the pathogenic thermotolerant taxa, among which each isolate of Aspergillus niger and Aspergillus tubingensis showed a minimum inhibitory concentration (MIC) of 2 μg/mL or greater for itraconazole. Their concentration in air was 0.4 CFU/m3. Seven human pathogenic Paecilomyces variotii isolates had MICs of 32 μg/mL or greater and lower than 2 μg/mL for the agricultural fungicide tebuconazole and the medical triazole itraconazole, respectively. Though the concentration was low, our results confirm the presence of airborne fungi with high MICs for itraconazole in ambient air. Inhalation is an important exposure route because people inhale more than 10 m3 of air each day. Vigilance is preferred over monitoring for the emergence of triazole-resistant fungal pathogens in ambient outdoor air.

Triazole-resistant Aspergillus fumigatus harbouring G54 mutation: Is it de novo or environmentally acquired?

Triazole resistance in Aspergillus fumigatus develops in patients with chronic lung diseases receiving long-term azole therapy or by environmental selection of resistant A. fumigatus. Here we report for the first time the isolation of triazole-resistant A. fumigatus (TRAF) harbouring the G54E mutation from environmental samples in India, Romania and Tanzania. This mutation in the cyp51A azole target gene of A. fumigatus is so far considered as de novo occurring in patients due to prolonged exposure to azoles. A total of 81 soil and woody debris samples from India, Romania and Tanzania were processed for detection of TRAF and determination of their susceptibility to medical triazoles and fungicides. cyp51A sequencing and real-time PCR were performed for detection of mutations. The isolates were genotyped by microsatellite typing. Overall, 25% of samples (20/81) from India, Romania and Tanzania harboured TRAF. Of the 20 samples harbouring TRAF, a single resistance mechanism, the G54E mutation, was found in 16 samples from three countries. This mechanism was responsible for 46.4% of resistant isolates from Tanzania, 30.4% from Romania and 20.0% from India. The G54E isolates revealed high MICs of itraconazole and posaconazole and were cross-resistant to agricultural fungicides. The majority of the Romanian and Tanzanian G54E isolates had an identical genotype. The present report describes the genetic heterogeneity of TRAF strains harbouring the G54E mutation in the environment of India, Romania and Tanzania. It may be anticipated that long-term exposure of A. fumigatus to fungicides may induce selection of G54 mutants in the environment.

Environmental azole fungicide, prochloraz, can induce cross-resistance to medical triazoles in Candida glabrata.

Acquisition of azole resistance by clinically relevant yeasts in nature may result in a significant, yet undetermined, impact in human health. The main goal of this study was to assess the development of cross-resistance between agricultural and clinical azoles by Candida spp. An in vitro induction assay was performed, for a period of 90 days, with prochloraz (PCZ) - an agricultural antifungal. Afterward, the induced molecular resistance mechanisms were unveiled. MIC value of PCZ increased significantly in all Candida spp. isolates. However, only C.glabrata developed cross-resistance to fluconazole and posaconazole. The increased MIC values were stable. Candida glabrata azole resistance acquisition triggered by PCZ exposure involved the upregulation of the ATP binding cassette multidrug transporter genes and the transcription factor, PDR1. Single mutation previously implicated in azole resistance was found in PDR1 while ERG11 showed several synonymous single nucleotide polymorphisms. These results might explain why C.glabrata is so commonly less susceptible to clinical azoles, suggesting that its exposure to agricultural azole antifungals may be associated to the emergence of cross-resistance. Such studies forward potential explanations for the worldwide increasing clinical prevalence of C.glabrata and the associated worse prognosis of an infection by this species.

Clonal Expansion and Emergence of Environmental Multiple-Triazole-Resistant Aspergillus fumigatus Strains Carrying the TR34/L98H Mutations in the cyp51A Gene in India

Azole resistance is an emerging problem in Aspergillus which impacts the management of aspergillosis. Here in we report the emergence and clonal spread of resistance to triazoles in environmental Aspergillus fumigatus isolates in India. A total of 44 (7%) A. fumigatus isolates from 24 environmental samples were found to be triazole resistant. The isolation rate of resistant A. fumigatus was highest (33%) from soil of tea gardens followed by soil from flower pots of the hospital garden (20%), soil beneath cotton trees (20%), rice paddy fields (12.3%), air samples of hospital wards (7.6%) and from soil admixed with bird droppings (3.8%). These strains showed cross-resistance to voriconazole, posaconazole, itraconazole and to six triazole fungicides used extensively in agriculture. Our analyses identified that all triazole-resistant strains from India shared the same TR34/L98H mutation in the cyp51 gene. In contrast to the genetic uniformity of azole-resistant strains the azole-susceptible isolates from patients and environments in India were genetically very diverse. All nine loci were highly polymorphic in populations of azole-susceptible isolates from both clinical and environmental samples. Furthermore, all Indian environmental and clinical azole resistant isolates shared the same multilocus microsatellite genotype not found in any other analyzed samples, either from within India or from the Netherlands, France, Germany or China. Our population genetic analyses suggest that the Indian azole-resistant A. fumigatus genotype was likely an extremely adaptive recombinant progeny derived from a cross between an azole-resistant strain migrated from outside of India and a native azole-susceptible strain from within India, followed by mutation and then rapid dispersal through many parts of India. Our results are consistent with the hypothesis that exposure of A. fumigatus to azole fungicides in the environment causes cross-resistance to medical triazoles. The study emphasises the need of continued surveillance of resistance in environmental and clinical A. fumigatus strains.

Triazole fungicides and the selection of resistance to medical triazoles in the opportunistic mould Aspergillus fumigatus

Azole resistance is an emerging problem in the opportunistic mould Aspergillus fumigatus. The triazoles are the most important agents for the management of Aspergillus diseases in humans. Selection for acquired resistance may occur in the hospital setting through exposure to high doses of azoles during azole therapy, but evidence is accumulating that A. fumigatus may become resistant to medical triazoles through environmental exposure to fungicides. The recovery of A. fumigatus isolates resistant to the medical triazoles from azole-naive patients as well as from the environment strongly indicates an environmental route of resistance selection. Molecule alignment studies have identified five fungicides that share a very similar molecule structure with the medical triazoles, and thus may have selected for mechanisms that confer resistance to both groups of compounds. It is important to explore further the presumed fungicide-driven route of resistance selection in order to implement effective preventive measures as the prevalence of azole resistance in A. fumigatus continues to increase and causes major challenges in the management of azole-resistant Aspergillus diseases.

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

BackgroundAzoles 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 14α-demethylase inhibitors (DMIs). The main resistance mechanism associated with this putative fungicide-driven route is a combination of alterations in the Cyp51A-gene (TR34/L98H). We investigated if TR34/L98H could have developed through exposure to DMIs.Methods and FindingsThirty-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 TR34/L98H isolate in 1998. Through microsatellite genotyping of TR34/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 TR34/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.ConclusionsOur findings support a fungicide-driven route of TR34/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.

Azole resistance in Aspergillus fumigatus: a side-effect of environmental fungicide use?

Invasive aspergillosis due to multi-azole-resistant Aspergillus fumigatus has emerged in the Netherlands since 1999, with 6.0-12.8% of patients harbouring resistant isolates. The presence of a single resistance mechanism (denoted by TR/L98H), which consists of a substitution at codon 98 of cyp51A and a 34-bp tandem repeat in the gene-promoter region, was found in over 90% of clinical A fumigatus isolates. This is consistent with a route of resistance development through exposure to azole compounds in the environment. Indeed, TR/L98H A fumigatus isolates were cultured from soil and compost, were shown to be cross-resistant to azole fungicides, and genetically related to clinical resistant isolates. Azoles are abundantly used in the environment and the presence of A fumigatus resistant to medical triazoles is a major challenge because of the possibility of worldwide spread of resistant isolates. Reports of TR/L98H in other European countries indicate that resistance might already be spreading.

Possible Environmental Origin of Resistance of Aspergillus fumigatus to Medical Triazoles

We reported the emergence of resistance to medical triazoles of Aspergillus fumigatus isolates from patients with invasive aspergillosis. A dominant resistance mechanism was found, and we hypothesized that azole resistance might develop through azole exposure in the environment rather than in azole-treated patients. We investigated if A. fumigatus isolates resistant to medical triazoles are present in our environment by sampling the hospital indoor environment and soil from the outdoor environment. Antifungal susceptibility, resistance mechanisms, and genetic relatedness were compared with those of azole-resistant clinical isolates collected in a previous study. Itraconazole-resistant A. fumigatus (five isolates) was cultured from the indoor hospital environment as well as from soil obtained from flower beds in proximity to the hospital (six isolates) but never from natural soil. Additional samples of commercial compost, leaves, and seeds obtained from a garden center and a plant nursery were also positive (four isolates). Cross-resistance was observed for voriconazole, posaconazole, and the azole fungicides metconazole and tebuconazole. Molecular analysis showed the presence of the dominant resistance mechanism, which was identical to that found in clinical isolates, in 13 of 15 environmental isolates, and it showed that environmental and clinical isolates were genetically clustered apart from nonresistant isolates. Patients with azole-resistant aspergillosis might have been colonized with azole-resistant isolates from the environment.