cyp51A Promoter Research Articles

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

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

Exposure to resistant fungi across working environments and time

Antifungal resistance has emerged as a significant health concern with increasing reports of resistant variants in previously susceptible species. At present, little is known about occupational exposure to antifungal-resistant fungi. This study aimed to investigate Danish workers' occupational exposure to airborne fungi resistant to first-line treatment drugs. A retrospective study was performed on a unique collection of personal exposure samples gathered over a twenty-year period from Danish working environments, in sectors including agriculture, animal handling, waste management, and healthcare. A total of 669 samples were cultivated at 37 °C and fungal colonies were identified using MALDI-TOF MS. Subsequently, identification was confirmed by amplicon sequencing the genes of calmodulin and beta-tubulin to unveil potential cryptic species.Infectious fungi (495 isolates from 23 species) were tested for resistance against Itraconazole, Voriconazole, Posaconazole, and Amphotericin B. Working environments were highly variable in the overall fungal exposure, and showed vastly different species compositions. Resistance was found in 30 isolates of the species Aspergillus fumigatus (4 of 251 isolates), A. nidulans (2 of 13), A. niger complex (19 of 131), A. versicolor (3 of 18), and A. lentulus (2 of 2). Sequence analysis revealed several cryptic species within the A. niger complex including A. tubingensis, A. luchuensis, and A. phoenicis. Among the resistant A. fumigatus isolates, two contained the well-described TR34/L98H mutation in the cyp51A gene and promoter region, while the remainder harbored silent mutations.The results indicate that the working environment significantly contributes to exposure to resistant fungi, with particularly biofuel plant workers experiencing high exposure. Differences in the prevalence of resistance across working environments may be linked to the underlying species composition.

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

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

Workflows for detecting fungicide resistance in net form and spot form net blotch pathogens.

Fungicide resistance in Pyrenophora teres f. maculata and P. teres f. teres has become an important disease management issue. Control of the associated barley foliar diseases, spot form and net form net blotch, respectively, relies on three major groups of fungicides, demethylation inhibitors (DMIs), succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs). However, resistance has been reported for the DMI and SDHI fungicides in Australia. To enhance detection of different resistance levels, phenotyping and genotyping workflows were designed. The phenotyping workflow generated cultures directly from lesions and compared growth on discriminatory doses of tebuconazole (DMI) and fluxapyroxad (SDHI). Genotyping real-time polymerase chain reaction (PCR) assays were based on alleles associated with sensitivity or resistance to the DMI and SDHI fungicides. These workflows were applied to spot form and net form net blotch collections from 2019 consisting predominantly of P. teres f. teres from South Australia and P. teres f. maculata from Western Australia. For South Australia the Cyp51A L489-3 and SdhC-R134 alleles, associated with resistance to tebuconazole and fluxapyroxad, respectively, were the most prevalent. These alleles were frequently found in single isolates with dual resistance. This study also reports the first detection of a 134 base pair insertion located at position-66 (PtTi-6) in the Cyp51A promoter of P. teres f. maculata from South Australia. For Western Australia, the PtTi-1 insertion was the most common allele associated with resistance to tebuconazole. The workflow and PCR assays designed in this study have been demonstrated to efficiently screen P. teres collections for both phenotypic and genetic resistance to DMI and SDHI fungicides. The distribution of reduced sensitivity and resistance to DMI and SDHI fungicides varied between regions in south-western Australia, suggesting the emergence of resistance was impacted by both local pathogen populations and disease management programmes. The knowledge of fungicide resistance in regional P. teres collections will be important for informing appropriate management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Isolation of triazole-resistant Aspergillus fumigatus harbouring cyp51A mutations from five patients with invasive pulmonary aspergillosis in Yunnan, China

ABSTRACT Invasive aspergillosis (IA) is the most severe type of Aspergillus infection. Yunnan has developed agriculture, and the proportion of triazole-resistant A. fumigatus induced by triazole fungicides is much higher than that in other regions of China. Inhalation of triazole-resistant A. fumigatus is one of the main factors inducing IA. We gathered five strains of A. fumigatus from the sputum or bronchoalveolar lavage fluid (BALF) of patients with IA in Yunnan. Subsequent testing showed that all of these strains were resistant to triazoles and harboured mutations in the tandem repeat sequence of the cyp51A promoter region, suggesting that they may be triazole-resistant A. fumigatus present in the environment.

Differential Functions of Individual Transcription Factor Binding Sites in the Tandem Repeats Found in Clinically Relevant cyp51A Promoters in Aspergillus fumigatus.

ABSTRACTAspergillus fumigatus is the major filamentous fungal pathogen in humans. The gold standard treatment of A. fumigatus is based on azole drug use, but the appearance of azole-resistant isolates is increasing at an alarming rate. The cyp51A gene encodes the enzymatic target of azole drugs, and azole-resistant alleles of cyp51A often have an unusual genetic structure containing a duplication of a 34- or 46-bp region in the promoter causing enhanced gene transcription. These tandem repeats are called TR34 and TR46 and produce duplicated binding sites for the SrbA and AtrR transcription factors. Using site-directed mutagenesis, we demonstrate that both the SrbA (sterol response element [SRE]) and AtrR binding sites (AtrR response element [ATRE]) are required for normal cyp51A gene expression. Loss of either the SRE or ATRE from the distal 34-bp repeat of the TR34 promoter (further 5′ from the transcription start site) caused loss of expression of cyp51A and decreased voriconazole resistance. Surprisingly, loss of these same binding sites from the proximal 34- or 46-bp repeat led to increased cyp51A expression and voriconazole resistance. These data indicate that these duplicated regions in the cyp51A promoter function differently. Our findings suggest that the proximal 34- or 46-bp repeat in cyp51A recruits a corepressor that requires multiple factors to act while the distal repeat is free of this repression and provides the elevated cyp51A expression caused by these promoter duplications.

An expanded agar-based screening method for azole-resistant Aspergillus fumigatus.

Antifungal susceptibility testing is an essential tool for guiding antifungal therapy. Reference methods are complex and usually only available in specialised laboratories. We have designed an expanded agar-based screening method for the detection of azole-resistant Aspergillus fumigatus isolates. Normally, identification of resistance mechanisms is obtained only after sequencing the cyp51A gene and promoter. However, our screening method provides azole resistance detection and presumptive resistance mechanisms identification. A previous agar-based method consisting of four wells containing voriconazole, itraconazole, posaconazole and a growth control, detected azole resistance to clinical azoles. Here, we have modified the concentrations of voriconazole and posaconazole to adapt to the updated EUCAST breakpoints against A.fumigatus. We have also expanded the method to include environmental azoles to assess azole resistance and the azole resistance mechanism involved. We used a collection of A.fumigatus including 54 azole-resistant isolates with Cyp51A modifications (G54, M220, G448S, TR53 , TR34 /L98H, TR46 /Y121F/T289A, TR34 /L98H/S297T/F495I), and 50 azole susceptible isolates with wild-type Cyp51A. The screening method detects azole-resistant A.fumigatus isolates when there is growth in any of the azole-containing wells after 48h. The growth pattern in the seven azoles tested helps determine the underlying azole resistance mechanism. This approach is designed for surveillance screening of A.fumigatus azole-resistant isolates and can be useful for the clinical management of patients prior to antifungal susceptibility testing confirmation.

Azole resistance in Aspergillus fumigatus isolates from respiratory specimens in Lyon University Hospitals, France: prevalence and mechanisms involved

Resistance of Aspergillus fumigatus to triazoles has been reported increasingly in Europe. As few data are available from Southern France, the objectives of this study were to assess the burden of A. fumigatus isolates with azole resistance from clinical specimens in Lyon, and explore the resistance mechanisms involved. In this retrospective cross-sectional study, 221 consecutive A. fumigatus isolates from respiratory samples were identified from an 8-month period from 195 patients attending the Pulmonary Medicine Departments of Lyon University Hospitals. Morphological identification was confirmed by sequence analysis of the β-tubulin gene. All samples were tested for susceptibilities to itraconazole, voriconazole, posaconazole and isavuconazole using concentration gradient strips, and the results were confirmed using the EUCAST broth microdilution method. Resistance mechanisms were investigated by sequencing the cyp51A gene and its promoter, and by expression analysis of cyp51 and genes encoding several efflux transporters. Four isolates exhibited azole resistance. Three isolates presented with polymorphisms in an intronic region of cyp51A, and one isolate had F46Y, M172V and E427K polymorphisms. No mutations were identified in the cyp51A promoter, but significant induction of cyp51A and cyp51B gene expression was observed for all four and three isolates, respectively. Significant induction of atrF and cdr1B gene expression was observed for two and three isolates, respectively. No significant induction of MDR1/2/3/4, MFS56 and M85 gene expression was observed. To conclude, the observed prevalence of azole resistance was 2.1%. Significant induction of expression of the cyp51 genes and two genes encoding efflux transporters was evidenced, underlying the diversity of resistance mechanisms to be explored.

Hazard of agricultural triazole fungicide: Does cyproconazole induce voriconazole resistance in Aspergillus fumigatus isolates?

Background and Purpose: The present study aimed to evaluate the effect of cyproconazole, the most used fungicide in Iranian wheat farms, on the induction of voriconazole resistance in Aspergillus fumigatus isolates.Materials and Methods:A collection of 20 clinical and environmental isolates were selected for investigation of the in vitro activity of fungicides. The minimum inhibitory concentrations (MICs) were determined by the documented broth microdilution method M38-A2 (CLSI, 2008). Induction experiments were performed and the possibly induced isolate(s) were subjected to antifungal susceptibility testing, sequencing of the CYP51A promoter, and full coding gene. Furthermore, CYP51-protein homology modeling and docking modes were evaluated using SWISS-MODEL (https://swissmodel.expasy.org/) and SEESAR software (version 9.1)Results: Among 10 susceptible isolates, only one strain showed a high MIC value against voriconazole (MIC=4µg/ml) after 25 passages. Nevertheless, sequencing of the CYP51A promoter and full coding gene did not reveal any mutations. Cyproconazole, which has three nitrogen atoms in the aromatic ring, coordinated to the iron atom of heme through a hydrogen bond contact to residue Lys147 present in the active site of the A. fumigates Cyp51 homology model. Conclusion: Cyproconazole is being applied extensively in wheat farms in Iran. According to the results, cyproconazole may not play a key role in the induction of azole resistance in the isolates through the environmental route. However, the potential ability of the fungicide to induce medically triazole-resistant strains over a long period of application should not be neglected.

A 23 bp cyp51A Promoter Deletion Associated With Voriconazole Resistance in Clinical and Environmental Isolates of Neocosmospora keratoplastica.

In the fungal pathogen Aspergillus fumigatus, resistance to azole antifungals is often linked to mutations in CYP51A, a gene that encodes the azole antifungal drug target lanosterol 14α-demethylase. The aim of this study was to investigate whether similar changes could be associated with azole resistance in a Malaysian Fusarium solani species complex (FSSC) isolate collection. Most (11 of 15) clinical FSSC isolates were Neocosmospora keratoplastica and the majority (6 of 10) of environmental isolates were Neocosmospora suttoniana strains. All 25 FSSC isolates had high minimum inhibitory concentrations (MICs) for itraconazole and posaconazole, low MICs for amphotericin B, and various (1 to >32 mg/l) voriconazole susceptibilities. There was a tight association between a 23 bp CYP51A promoter deletion and high (>32 mg/l) voriconazole MICs; of 19 FSSC strains sequenced, nine isolates had voriconazole MICs > 32 mg/l, and they all contained the 23 bp CYP51A promoter deletion, although it was absent in the ten remaining isolates with low (≤12 mg/l) voriconazole MICs. Surprisingly, this association between voriconazole resistance and the 23 bp CYP51A promoter deletion held true across species boundaries. It was randomly distributed within and across species boundaries and both types of FSSC isolates were found among environmental and clinical isolates. Three randomly selected N. keratoplastica isolates with low (≤8 mg/l) voriconazole MICs had significantly lower (1.3–7.5 times) CYP51A mRNA expression levels than three randomly selected N. keratoplastica isolates with high (>32 mg/l) voriconazole MICs. CYP51A expression levels, however, were equally strongly induced (~6,500-fold) by voriconazole in two representative strains reaching levels, after 80 min of induction, that were comparable to those of CYP51B. Our results suggest that FSSC isolates with high voriconazole MICs have a 23 bp CYP51A promoter deletion that provides a potentially useful marker for voriconazole resistance in FSSC isolates. Early detection of possible voriconazole resistance is critical for choosing the correct treatment option for patients with invasive fusariosis.

Development of multiplex real-time PCR for rapid identification and quantitative analysis of Aspergillus species.

The identification of Aspergillus species and azole resistance is highly important for the treatment of invasive aspergillosis (IA), which requires improvements in current fungal diagnostic methods. We aimed to develop multiplex real-time PCR to identify major Aspergillus section and azole resistance. BenA and cyp51A genes were used to design primers, probes, and control DNA for multiplex PCR. Qualitative and quantitative analysis was conducted for 71 Aspergillus and 47 non-Aspergillus isolates. Further, the limit of detection (LOD) and limit of quantitation (LOQ) from hyphae or conidia were determined according to the culture time. Newly developed real-time PCR showed 100% specificity to each Aspergillus section (Fumigati, Nigri, Flavi, and Terrei), without cross-reaction between different sections. In quantitative analysis of sensitivity measurements, LOD and LOQ were 40 fg and 400 fg, respectively. Melting temperature analysis of the cyp51A promoter to identify azole resistance showed temperatures of 83.0 ± 0.3°C and 85.6 ± 0.6°C for susceptible A. fumigatus and resistant isolates with TR34 mutation, respectively. The minimum culture time and fungal colony size required for successful detection were 24 h and 0.4 cm in diameter, respectively. The developed multiplex real-time PCR can identify common Aspergillus sections quantitatively and detect presence of the TR34 mutation. Further, this method shows high sensitivity and specificity, allowing successful detection of early-stage fungal colonies within a day of incubation. These results can provide a template for rapid and accurate diagnosis of IA.

Prospective survey of Aspergillus species isolated from clinical specimens and their antifungal susceptibility: A five-year single-center study in Japan

Aspergillus fumigatus is the most prevalent species that causes aspergillosis. A. fumigatus strains with tandem repeats in the cyp51A promoter have emerged in the environment. Aspergillus species other than A. fumigatus have also been recognized as causative agents of aspergillosis; however, they show lower susceptibility to antifungals compared with A. fumigatus. Therefore, it is important to precisely identify Aspergillus species and determine their antifungal susceptibility. Herein, we collected 119 mold strains isolated from clinical specimens collected at a hospital between November 2013 and December 2018. The collected strains were identified by sequencing several regions, including internal transcribed spacers, and determined their susceptibility to the antifungals itraconazole, voriconazole, and amphotericin B. Of 119 strains, 107 were Aspergillus species, which were identified as A. fumigatus (67), Aspergillus section Nigri (21), A. flavus (7), A. terreus (6), and A. nidulans (6). In Aspergillus section Nigri, the number of A. niger was less than the number of A. welwitschiae and A. tubingensis. Two azole-resistant A. fumigatus samples were included among the isolates. Four of the eight A. tubingensis isolates showed less susceptibility to voriconazole; however, all isolates of A. niger and A. welwitschiae were susceptible to itraconazole and voriconazole. Because of lack of susceptibility data for non-fumigatus Aspergillus and an increasing frequency of antifungal resistance among A. fumigatus, our data along with further surveillance may contribute to determining the frequency and susceptibility of Aspergillus spp. clinical isolates in Japan.

Prospective multicenter surveillance of clinically isolated Aspergillus species revealed azole-resistant Aspergillus fumigatus isolates with TR34/L98H mutation in the Kyoto and Shiga regions of Japan.

The prevalence of azole-resistant Aspergillus fumigatus (ARAF) in Japan is unclear. We aimed to investigate the epidemiology of clinically isolated Aspergillus species and the frequency of azole resistance in Aspergillus species, particularly Aspergillus fumigatus, in the Kyoto and Shiga regions of Japan. Strains of clinically isolated Aspergillus species were prospectively collected from nine acute care hospitals. Species identification was performed by DNA sequence analysis, and all strains were subjected to antifungal susceptibility testing. Sequencing of the Aspergillus cyp51A gene and promoter region and genotyping by short tandem repeats were performed for ARAF isolates. A total of 149 strains were collected, and 130 strains were included for the subsequent analysis after the exclusion of duplicate isolates. The most commonly isolated species was Aspergillus fumigatus, accounting for 43.1% (56 isolates) overall, and seven (12.7%) of 55 strains of A. fumigatus were azole-resistant. Azole-resistance of other Aspergillus species were also found that two (22.2%) of nine strains of A. tubingensis and two (28.6%) of seven strains of A. flavus were azole-resistant. DNA sequence analysis of the ARAF strains revealed that two carried the cyp51A TR34/L98H mutation, one carried G448S, one carried M220I, and three had no relevant mutations (wild type). Genotyping and phylogenetic analyses showed that the TR34/L98H strains were clustered with the strains from the Netherlands and France. These data suggest the emergence of ARAF with TR34/L98H in Japan, and continuous surveillance will be important to identify trends in resistance.

Surveillance for azole resistance in clinical and environmental isolates of Aspergillus fumigatus in Australia and cyp51A homology modelling of azole-resistant isolates.

The prevalence of azole resistance in Aspergillus fumigatus is uncertain in Australia. Azole exposure may select for resistance. We investigated the frequency of azole resistance in a large number of clinical and environmental isolates. A. fumigatus isolates [148 human, 21 animal and 185 environmental strains from air (n = 6) and azole-exposed (n = 64) or azole-naive (n = 115) environments] were screened for azole resistance using the VIPcheck™ system. MICs were determined using the Sensititre™ YeastOne YO10 assay. Sequencing of the Aspergillus cyp51A gene and promoter region was performed for azole-resistant isolates, and cyp51A homology protein modelling undertaken. Non-WT MICs/MICs at the epidemiological cut-off value of one or more azoles were observed for 3/148 (2%) human isolates but not amongst animal, or environmental, isolates. All three isolates grew on at least one azole-supplemented well based on VIPcheck™ screening. For isolates 9 and 32, the itraconazole and posaconazole MICs were 1 mg/L (voriconazole MICs 0.12 mg/L); isolate 129 had itraconazole, posaconazole and voriconazole MICs of >16, 1 and 8 mg/L, respectively. Soil isolates from azole-exposed and azole-naive environments had similar geometric mean MICs of itraconazole, posaconazole and voriconazole (P > 0.05). A G54R mutation was identified in the isolates exhibiting itraconazole and posaconazole resistance, and the TR34/L98H mutation in the pan-azole-resistant isolate. cyp51A modelling predicted that the G54R mutation would prevent binding of itraconazole and posaconazole to the haem complex. Azole resistance is uncommon in Australian clinical and environmental A. fumigatus isolates; further surveillance is indicated.

Insight into the Significance of Aspergillus fumigatus cyp51A Polymorphisms.

Triazole antifungal compounds are the first treatment choice for invasive aspergillosis. However, in the last decade the rate of azole resistance among Aspergillus fumigatus strains has increased notoriously. The main resistance mechanisms are well defined and mostly related to point mutations of the azole target, 14-α sterol demethylase (cyp51A), with or without tandem repeat integrations in the cyp51A promoter. Furthermore, different combinations of five Cyp51A mutations (F46Y, M172V, N248T, D255E, and E427K) have been reported worldwide in about 10% of all A. fumigatus isolates tested. The azole susceptibility profile of these strains shows elevated azole MICs, although on the basis of the azole susceptibility breakpoints, these strains are not considered azole resistant. The purpose of the study was to determine whether these cyp51A polymorphisms (single nucleotide polymorphisms [SNPs]) are responsible for the azole susceptibility profile and whether they are reflected in a poorer azole treatment response in vivo that could compromise patient treatment and outcome. A mutant with a cyp51A deletion was generated and became fully susceptible to all azoles tested. Also, three cyp51A gene constructions with different combinations of SNPs were generated and reintroduced into an azole-susceptible wild-type (WT) strain (the ΔakuBKU80 strain). The alternative model host Galleria mellonella was used to compare the virulence and voriconazole response of G. mellonella larvae infected with A. fumigatus strains with WT cyp51A or cyp51A with SNPs. All strains were pathogenic in G. mellonella larvae, although they did not respond similarly to voriconazole therapeutic doses. Finally, the full genomes of these strains were sequenced and analyzed in comparison with those of A. fumigatus WT strains, revealing that they belong to different strain clusters or lineages.

A simple method to detect the tandem repeat of the cyp51A promoter in azole-resistant strains of Aspergillus fumigatus.

We designed primers and cycling probes to detect the tandem repeat (TR) of cyp51A promoter in Aspergillus fumigatus. A control-probe was designed to anneal to the outside of the TR region, whereas a TR-probe was designed to anneal to the inside of the TR region. For amplification and probe-hydrolysis detection, the CycleavePCR system was used. Although the difference between Ct values of the wild-type genome for the control-probe and the TR-probe was around -0.1, the difference between Ct values of TR-harboring strains was around 0.7. These data indicate that this is a simple method to detect TR in azole-resistant A. fumigatus.

Azole-resistant Aspergillus fumigatus Containing a 34-bp Tandem Repeat in cyp51A Promoter is Isolated from the Environment in Japan.

Azole-resistant strains of Aspergillus fumigatus containing a tandem repeat in the cyp51A promoter and amino acid substitution(s) have been isolated in the environment worldwide; however, this type of resistant strain had never been isolated from the environment in Japan. Our previous study indicated that an azole-resistant A. fumigatus strain OKH50 containing a 34-bp tandem repeat in cyp51A promoter with L98H substitution in Cyp51A was isolated from a patient in Obihiro of Hokkaido, Japan. In this study, we collected azole-resistant Aspergillus spp. by air sampling from the environment in Japan. One Aspergillus-like colony was isolated from one of 10 sampling sites surveyed. The strain Env1 was confirmed as A. fumigatus by nucleotide sequencing and possessed a 34-bp tandem repeat in the promoter region of cyp51A with L98H substitution in Cyp51A. A. fumigatus Env1 has the identical short tandem repeat pattern with the OKH50 strain, indicating that these strains are closely related with each other. Additionally, the short tandem repeat pattern is closely related to Danish and Iranian environmental isolates, suggesting that azole-resistant strains have crossed transnational boundaries and are now present in Japan, and therefore, further analysis throughout Japan is required to determine the distribution of this type of azole-resistant A. fumigatus.

Azole susceptibility in clinical and environmental isolates of Aspergillus fumigatus from eastern Hokkaido, Japan.

Azole antifungals are used not only clinically for fungal infections but also used as agricultural fungicides. Recently, azole-resistant Aspergillus fumigatus containing a tandem repeat in the promoter region of cyp51A combined with amino acid substitution(s) appears in the environment in Eurasia, especially in several European countries. Although azole fungicides have been used in Japan, especially in Hokkaido, surveillance and characterization of A.fumigatus in Hokkaido have not been reported. In this study, we collected soil samples from farms that used an azole fungicide in the Tokachi area of eastern Hokkaido, isolated 91 A.fumigatus strains, and determined the minimal inhibitory concentrations of medical azoles required for these strains. Moreover, because causative agent A.fumigatus is ubiquitous in the air and acquired from the environment, we collected 22 clinical isolates of A.fumigatus to measure their susceptibility to medical azoles in a hospital in the Tokachi area. Our data show that almost all A.fumigatus isolates retained susceptibility to medical azoles. Clinical isolates OKH34 and OKH6 showed 8 and 2μg/mL of voriconazole, respectively, as the minimal inhibitory concentration. Both isolates did not contain tandem repeat in cyp51A promoter region. An isolate contained G448S mutation in cyp51A conferring voriconazole resistance, which is the first report from Japan. Our data shows the existence of azole-resistant and low azole-susceptible clinical isolates and highlight the necessity for continuous surveillance in Japan because resistant A.fumigatus strains can arise through clinical or environmental selection or could be introduced from overseas.

Aspergillus oerlinghausenensis, a new mould species closely related toA. fumigatus

Two isolates belonging to Aspergillus section Fumigati were recovered from German soil on itraconazole containing agar media. Phylogenetic analysis and phenotypic characterization of both isolates show that they represent a novel species named Aspergillus oerlinghausenensis (holotype CBS H-22119(HT), ex-type CBS 139183(T) = IBT 33878 = DTO 316-A3). The species is phylogenetically related to A. fischeri and A. fumigatus. Aspergillus oerlinghausenensis can be differentiated from A. fischeri by its higher growth rate at 50°C. Furthermore, A. oerlinghausenensis is protoheterothallic as only the MAT1-1 idiomorph was detected, while A. fischeri is homothallic. The species differs from A. fumigatus by a weak sporulation on malt extract agar at 25°C, a floccose colony texture on Czapek yeast extract agar and malt extract agar and subglobose instead of subclavate vesicles. The cyp51A promoter region of A. oerlinghausenensis deviates from the previously reported cyp51A promoter regions in A. fumigatus and potentially presents a novel azole resistance conferring modification. Due to the close relationship of A. oerlinghausenensis with A. fischeri and A. fumigatus, this species is placed in a good position for comparative studies involving these species.

Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter.

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.