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. 2020 Oct 1;6(4):199. doi: 10.3390/jof6040199

Clonal Expansion of Environmental Triazole Resistant Aspergillus fumigatus in Iran

Fatemeh Ahangarkani 1,2, Hamid Badali 3,4, Kiana Abbasi 5, Mojtaba Nabili 6, Sadegh Khodavaisy 7, Theun de Groot 1, Jacques F Meis 1,8,9,*
PMCID: PMC7712205  PMID: 33019714

Abstract

Azole-resistance in Aspergillus fumigatus is a worldwide medical concern complicating the management of aspergillosis (IA). Herein, we report the clonal spread of environmental triazole resistant A. fumigatus isolates in Iran. In this study, 63 A. fumigatus isolates were collected from 300 compost samples plated on Sabouraud dextrose agar supplemented with itraconazole (ITR) and voriconazole (VOR). Forty-four isolates had the TR34/L98H mutation and three isolates a TR46/Y121F/T289A resistance mechanism, while two isolates harbored a M172V substitution in cyp51A. Fourteen azole resistant isolates had no mutations in cyp51A. We found that 41 out of 44 A. fumigatus strains with the TR34/L98H mutation, isolated from compost in 13 different Iranian cities, shared the same allele across all nine examined microsatellite loci. Clonal expansion of triazole resistant A. fumigatus in this study emphasizes the importance of establishing antifungal resistance surveillance studies to monitor clinical Aspergillus isolates in Iran, as well as screening for azole resistance in environmental A. fumigatus isolates.

Keywords: Aspergillus fumigatus, azole resistance, compost, TR34/L98H, TR46/Y121F/T289A

1. Introduction

Aspergillus fumigatus is the most common agent of various forms of aspergillosis, including allergic bronchopulmonary aspergillosis (ABPA), chronic pulmonary aspergillosis (CPA), aspergilloma, and invasive aspergillosis (IA) [1]. Voriconazole (VOR) is the recommended primary and most effective therapy in the management of aspergillosis [2]. However, azole resistant A. fumigatus isolates are increasingly found worldwide with major epidemiological and clinical implications [3,4]. Therapeutic failure caused by azole-resistant A. fumigatus is becoming a significant concern to clinicians who are caring for patients at high risk for IA [1,4,5,6,7,8]. Azole resistance in A. fumigatus is mainly linked to cyp51A-mediated resistance mechanism, such as a 34-basepair (bp) sequence tandem repeat (TR34) in the promoter region of the cyp51A gene, in combination with a L98H substitution and a 46 bp tandem repeat (TR46) in the cyp51A promoter in combination with two amino acid changes (Y121F and T289A) in the CYP51A protein (TR46/Y121F/T289A) [9]. Isolates carrying these mutations exhibit a pan-azole resistant phenotype that can develop through long-term treatment with azole antifungals in the clinical setting or extensive exposure of the fungus to azole compounds in the environment [10,11]. Azole-resistant A. fumigatus with the TR34/L98H mutation isolated from environmental and clinical samples have been reported earlier in Iran, and we recently reported the occurrence of TR46/Y121F/T289A mutations in the cyp51A gene in A. fumigatus isolates from compost [12,13,14,15,16,17,18]. High concentrations of azole-resistant A. fumigatus spores are released during incomplete composting processes, especially when azole residues from agricultural waste are present [19]. Agricultural use of fungicides has driven the emergence and spread of azole-resistant A. fumigatus. The existence of an environmental route of azole resistance development involves serious risks for patients, as well, as they can become infected with azole resistant A. fumigatus strains before starting their treatment [12,13,14,15,16,17,18,19,20,21,22,23,24]. Notably, genetic exploration of azole resistant A. fumigatus strains indicates that isolates with the TR34/L98H allele are less genetically variable than susceptible isolates [12,23]. For instance, analysis of azole resistant A. fumigatus isolates in the Netherlands showed five distinct genotype groups in this country, while all the azole resistant isolates with the TR34/L98H mutation belonged to one group [5]. On the other hand, all clinical and environmental azole resistant A. fumigatus strains carrying TR34/L98H obtained from India were genetically identical [14]. These studies illustrate that A. fumigatus carrying this azole resistance mutation may preferentially spread clonal within a population. Major data gaps remain regarding the genotype distribution of azole resistance A. fumigatus in Iran. As ongoing reports indicate an expansion in the frequency of azole resistant A. fumigatus isolates worldwide, understanding the genetic structure of this potentially lethal fungus is critical. In this study, the genetic characterization of azole resistant A. fumigatus isolated from compost samples in Iran was explored.

2. Materials and Methods

2.1. Isolate Collection

According to a previously described protocol, commercial and home-made compost samples from different region of Iran (located about 300 km apart) were collected. To recover A. fumigatus strains, 1 cm2 of compost was dissolved in 5 mL sterile saline solution containing Tween 40 (0.05%), vortexed, and allowed to settle. For primary screening of azole-resistant A. fumigatus strains, 100 μL supernatant was plated on a Sabouraud dextrose agar plate (SDA; Difco, Franklin Lakes, NJ, USA), supplemented with 4 and 1 mg/L itraconazole and voriconazole, respectively, and incubated at 45 °C for 72 h in the dark [17]. Molecular identification of all A. fumigatus isolates that grew on the supplemented plate was performed with sequencing of the partial beta-tubulin gene as previously described [16].

2.2. In Vitro Antifungal Susceptibility Testing

Minimum inhibitory concentrations (MICs) were determined by broth microdilution susceptibility testing according to the methods in the Clinical and Laboratory Standards Institute (CLSI) reference standard (M38) [25]. For the preparation of the microdilution trays, itraconazole (Janssen, Beerse, Belgium) and voriconazole (Pfizer, Sandwich, UK) were obtained from the respective manufacturers as reagent-grade powders. All drugs were dissolved in 1% dimethyl sulfoxide (DMSO; Sigma, Zwijndrecht, the Netherlands) and were prepared at a final concentration of 0.031–16 mg/L. Paecilomyces variotii (ATCC 22319) and Candida parapsilosis (ATCC 22019) were used as quality controls [25].

2.3. Detection of Cyp51a Gene Mutations

All A. fumigatus isolates were subjected to a mixed-format real-time PCR assay specific for TR34/L98H and TR46/Y121F/T289A mutations of cyp51A gene leading to triazole resistance in A. fumigatus as described previously [26]. Those isolates with negative or inconclusive results in the real-time PCR assay, were further evaluated by sequencing the cyp51A gene as described previously [27].

2.4. Microsatellite Genotyping

Genotyping of all A. fumigatus isolates was performed with a panel of nine short tandem repeats (STRs) loci (namely short tandem repeats Aspergillus fumigatus (STRAf) 2A, 2B, 2C, 3A, 3B, 3C, 4A, 4B, and 4C), as previously described [28]. Genotypes were considered identical when they showed the same alleles for all nine loci [29,30]. Finally, the genetic relatedness between Iranian isolates from compost and 633 resistant A. fumigatus strains with clinical or environmental sources collected during 2001–2019 from different countries (The Netherlands, India, United Kingdom, Tanzania, France, Colombia, Romania, Ireland, China, Kuwait, Germany, and Japan) and previous Iranian isolates in the database at the Center of Expertise in Mycology, Radboudumc/Canisius-Wilhelmina Ziekenhuis (CWZ), in Nijmegen, The Netherlands, already barcoded using a panel of nine short tandem repeat loci, were analysed using BioNumerics software v7.6.1 (Applied Maths, Saint-Martens-Latem, Belgium).

3. Results

3.1. Triazole Resistant A. fumigatus with Mutation in cyp51A Gene

A total of 63 A. fumigatus colonies from 300 compost samples were obtained from SDA supplemented with itraconazole and voriconazole. Of these, 55 A. fumigatus isolates had high MICs of itraconazole (≥8 mg/L) and voriconazole (≥2 mg/L) by in vitro antifungal susceptibility testing. Exploring the mechanisms of resistance in these isolates by sequencing cyp51A and its promoter region showed that 44 isolates harbored the TR34/L98H mutation, three isolates the TR46/Y121F/T289A mutation and two isolates a M172V mutation. No mutations were found in 14 resistant isolates. Data of resistant isolates are summarized in Table 1. Details of isolates with the TR46/Y121F/T289A mutation have been previously described [17].

Table 1.

Description of all A. fumigatus isolates from compost.

Strain Longitude and Latitude of Sampling MIC (mg/L) 3 STRAf
1 ITR 2 VOR Mutation in cyp51A 2A 2B 2C 3A 3B 3C 4A 4B 4C
mn224 35.9548° N, 52.1100° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn225 36.6717° N, 52.4439° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn226 36.6717° N, 52.4439° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn229 36.6717° N, 52.4439° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn227 36.7049° N, 52.6547° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn228 36.6329° N, 52.2667° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn231 36.4684° N, 52.8634° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn235 36.4684° N, 52.8634° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn232 36.4684° N, 52.8634° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn241 36.4684° N, 52.8634° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn233 36.6858° N, 52.5265° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn234 36.6858° N, 52.5265° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn236 36.4676° N, 52.3507° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn246 36.4676° N, 52.3507° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn247 36.5971° N, 52.6654° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn250 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn251 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn252 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn253 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn254 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn255 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn256 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn257 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn258 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn260 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn261 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn263 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn265 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn266 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn267 35.6892° N, 51.3890° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn268 35.6892° N, 51.3890° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn269 35.6892° N, 51.3890° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn270 35.6892° N, 51.3890° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn271 35.6892° N, 51.3890° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn272 35.6892° N, 51.3890° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn273 35.6892° N, 51.3890° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn274 35.6892° N, 51.3890° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn277 36.5659° N, 53.0586° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn279 36.9268° N, 50.6431° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn280 36.7284° N, 53.8102° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
mn281 36.7284° N, 53.8102° E 16 2 TR34/L98H 22 10 9 9 9 23 8 10 8
IFRC: 1854 35.6892° N, 51.3890° E 16 2 TR34/L98H 14 10 8 9 10 5 8 10 27
IFRC: 1858 35.6892° N, 51.3890° E 8 1 TR34/L98H 13 21 8 32 9 6 8 10 10
IFRC: 1866 35.6892° N, 51.3890° E 16 8 TR34/L98H 14 24 14 31 9 31 10 9 5
mn248 36.6329° N, 52.2667° E 16 16 M172V 11 15 16 19 29 4 7 5 8
IFRC: 1867 35.6892° N, 51.3890° E 16 16 M172V 11 16 9 19 20 5 7 5 5
IFRC: 1860 35.6892° N, 51.3890° E 16 0.125 Wild type 27 18 16 7 12 28 27 5 8
IFRC: 1868 35.6892° N, 51.3890° E 16 16 Wild type 27 18 16 7 12 28 27 5 8
IFRC: 1862 35.6892° N, 51.3890° E 16 0.5 Wild type 27 20 13 8 14 35 10 11 10
IFRC: 1864 35.6892° N, 51.3890° E 16 0.25 Wild type 20 10 8 37 9 6 10 9 15
IFRC: 1859 35.6892° N, 51.3890° E 16 0.25 Wild type 21 20 14 30 21 5 11 6 5
mn245 36.7049° N, 52.6547° E 16 1 Wild type 13 19 8 34 29 7 10 9 8
mn276 36.5659° N, 53.0586° E 16 2 Wild type 18 22 15 43 13 27 13 8 10
mn278 36.5659° N, 53.0586° E 16 2 Wild type 24 10 10 28 11 6 8 7 15
mn249 36.5659° N, 53.0586° E 0.125 0.5 Wild type 23 22 14 44 12 27 13 8 7
mn223 36.5659° N, 53.0586° E 0.125 0.5 Wild type 22 23 11 9 10 6 11 7 6
mn240 36.6329° N, 52.2667° E 0.5 1 Wild type 11 15 16 19 29 4 7 5 5
mn242 36.6329° N, 52.2667° E 0.125 0.5 Wild type 24 22 18 24 13 17 9 8 10
mn230 36.6717° N, 52.4439° E 0.5 0.5 Wild type 24 20 18 22 10 6 9 12 9
IFRC: 1863 35.6892° N, 51.3890° E 4 0.125 Wild type 24 18 15 94 10 6 14 11 10
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1 ITR: itraconazole; 2 VOR: voriconazole; 3 STRAf: Short tandem repeats Aspergillus fumigatus.

3.2. Microsatellite Typing Results and Evidence for Clonal Spread of a Single Triazole-Resistant A. fumigatus Genotype

Genotypic analysis identified that 41 A. fumigatus isolates with TR34/L98H shared the same allele across all nine examined microsatellite loci. These isolates came from compost in 13 different cities. The three remaining isolates with TR34/L98H exhibited three different genotypes. The two isolates with M172V differed by five microsatellite loci (2B, 2C, 3B, 3C, 4C). From the 14 azole resistant isolates with wild type cyp51A, which originated from 5 different cities, two isolates shared the same alleles across all nine microsatellite loci, while the 12 other isolates were genetically very diverse. A minimum spanning tree (MST) based on azole-resistant strains from various countries showed that the 41 Iranian A. fumigatus isolates with TR34/L98H formed a separate cluster (Figure 1).

Figure 1.

Figure 1

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Minimum-spanning tree showing the genetic relationship of resistant Aspergillus fumigatus genotypes. Iranian clonal complex is illustrated by the arrow. Solid thick and thin branches demonstrate 1 or 2 microsatellite markers difference, respectively; dashed branches indicate 3 microsatellite markers difference between two genotypes; 4 or more microsatellite markers difference between genotypes are demonstrated with dotted branches.

4. Discussion

In this study, about 70% A. fumigatus isolates from compost samples grew on SDA supplemented with azoles and had the TR34/L98H mutation in the cyp51A gene. Indeed, the high rate of resistance to azole drugs due to the TR34/L98H mutation in A. fumigatus in Iran outperforms previous studies done during 2013–2016. The prevalence of clinical or environmental azole-resistant A. fumigatus isolates harboring this mutation was much lower in a previous episode and has been estimated between 3.2–6.6% [16,18,31]. Concurrent genetic studies of worldwide A. fumigatus isolates harboring the TR34/L98H resistance mechanism also suggested clonal expansion from a common resistant ancestor [32,33]. In the current study the azole resistant A. fumigatus population with TR34/L98H was grouped into four microsatellite genotypes, in which the genotype with STRAf profile: 2A:22, 2B:10, 2C:9, 3A:9, 3B:9, 3C:23, 4A:8, 4B:10, 4C:8 included 41 (93%) identical isolates, showing clonal expansion across different geographic locations. Furthermore, MST showed Iranian A. fumigatus isolates harboring TR34/L98H were apart from isolates of other countries and previously recovered Iranian isolates. Similar to our finding, Chowdhary et al. described a clonal spread and emergence of environmental azole resistant A. fumigatus strains carrying the TR34/L98H mutation from different parts of India. All Indian azole resistant isolates shared the same multilocus microsatellite genotype not found in any other analyzed samples within India or from other Asian or European countries [14]. In agreement with our findings, there is strong evidence that azole-susceptible or cyp51A single point mutation resistance strains have a greater genetic diversity than isolates harboring TR34/L98H and TR46/Y121F/T289A mutations, since the expansion of latter strains at a local level is predominantly clonal [14,34,35,36]. The dispersal of A. fumigatus with the TR34/L98H genotype supports the hypothesis that these strains have robust fitness in natural environments, with comparable or even higher fitness than that of wild-type strains [11]. Clonal spread of a single genotype in our study reinforced the hypothesis that geographic distances are not a barrier for the global spread from its centers of origin and their ability to cover thousands of miles by producing a large number of airborne spores or by anthropogenic means [14,31,37,38,39]. The widespread application of azole fungicides in Iran could have contributed to the spread of azole resistant A. fumigatus in environment niches, such as compost. To mitigate spread of azole resistant A. fumigatus in environment, changing of practices to prevent fungal diseases in plants on the fields is necessary. Procedures, such as prudent and restricted use of fungicides, controlling doses, and periods of fungicide application could be helpful. In cases where resistance to fungicides is observed, either the dosage can be increased or alternative fungicides can be used. In addition, environmental surveillance studies aimed to collect precise information of azole resistance monitoring to investigate the size and impact of this emerging problem is necessary [40].

Interestingly, we found that a sizable number of isolates (8 out of 54 resistant isolates) with azole MICs ≥16 mg/L exhibited no mutations in cyp51A. Other mechanisms of resistance, such as increased production of drug target Cyp51A protein, multidrug efflux pumps, or other proposed but not yet fully characterized mechanisms of resistance, such as amino acid substitutions in 3-hydroxy-3-methylglutaryl-CoA, stress response, and biofilm formation, can contribute to azole resistance in these isolates [32]. The limitation of our study was the absence of STRAf profiles of TR34/L98H A. fumigatus from neighbor countries of Iran, such as Pakistan or Turkey, for comparison with Iranian isolates [41,42]. In addition, the absence of clinical A. fumigatus was another drawback of our study. As most clinical microbiology laboratories in Iran do not routinely perform antifungal susceptibility testing of Aspergillus, the prevalence of azole resistance and mechanism of resistance in clinical A. fumigatus isolates in Iran is unknown [17].

5. Conclusions

Clonal spread of triazole resistant A. fumigatus isolated from compost, which is used widely in gardens and indoor plants in Iran, is concerning. This study highlights the importance of antifungal resistance surveillance studies of clinical and environmental Aspergillus isolates in Iran.

Acknowledgments

F.A. is a recipient of an ESCMID observership grant to visit ESCMID observership center 58 (CWZ, Nijmegen, The Netherlands).

Author Contributions

Conceptualization, F.A., H.B. and J.F.M.; Data curation, F.A., K.A., M.N. and S.K.; Formal analysis, F.A. and T.d.G.; Funding acquisition, J.F.M.; Investigation, K.A. and T.d.G.; Methodology, T.d.G.; Supervision, J.F.M.; Writing—original draft, F.A., H.B., and J.F.M.; Writing—review and editing, K.A., M.N., S.K. and T.d.G. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by National Institutes for Medical Research Development (NIMAD), Grant/Award Number: 982677 and Mazandaran University of Medical Sciences, Sari, Iran, Grant/Award Number: 1352.

Conflicts of Interest

J.F.M. received grants from Pulmozyme and F2G. He has been a consultant to Scynexis and received speaker’s fees from United Medical, TEVA, and Gilead Sciences. The other authors report no conflicts of interest.

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