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Fungicide effects on human fungal pathogens: Cross-resistance to medical drugs and beyond

['Rafael W. Bastos', 'Faculdade De Ciências Farmacêuticas De Ribeirão Preto', 'Universidade De São Paulo', 'Ribeirão Preto-Sp', 'Luana Rossato', 'Federal University Of Grande Dourados', 'Dourados-Ms', 'Gustavo H. Goldman', 'Daniel A. Santos', 'Laboratory Of Mycology']

Date: 2022-01 

Two main hypotheses have been raised to explain this phenomenon: (i) person-to-person transmission of resistant strains; or (ii) infection by an isolate that acquired the resistance mechanism in the environment [ 26 , 30 ]. The first hypothesis has little scientific support because person-to-person or person-to-environment transmissibility has been considered rare or inexistent. In the past, it was thought that transmission happens only through direct donor-to-recipient contact and infected wounds, as most of the transmission happens via aerosolized spores [ 30 ]. However, Engel and colleagues proved that A. fumigatus could be recovered from cough aerosols from CF patients [ 72 ], thus opening the possibility of patient-to-patient and patient-to-environment transmission. Further experiments, however, are still necessary to better detail the transmission of A. fumigatus by coughing. Nevertheless, aerosolized A. fumigatus conidia from patients could not explain all the resistance found in azole-naive patients due to its frequency, and the aerosolized conidia from environmental sources seem to represent a vaster and more constant source of infection [ 72 ].

Although many studies have proved that azole therapy can drive inpatient resistance to emerge in Aspergillus spp. clones [ 57 – 66 ], this route does not explain all cases observed in the genus. Actually, it is estimated that only one-third of the resistant strains arise from in-host adaptation, remarkably those suffering from aspergilloma, allergic or chronic aspergillosis, and predisposing conditions as lung cavities or cystic fibrosis (CF) [ 11 , 64 ]. The main evidence indicating another route is the azole-resistant A. fumigatus isolated from azole-naive patients, which accounts for 64% to 71% of the multiresistant A. fumigatus isolates [ 16 , 67 , 68 ]. Mellado and colleagues recovered 13 multiple triazole-resistant A. fumigatus strains from patients at different hospitals in the Netherlands—4 of them from individuals with no history of azole treatment [ 16 ]. In those cases, the isolates were not only resistant to itraconazole but also had high MIC values of voriconazole, posaconazole, and ravuconazole [ 16 , 69 ]. Subsequently, many studies in different countries have also identified azole-resistant isolates from patients not previously treated with these drugs [ 46 , 68 , 70 , 71 ].

Triazoles are not mutagenic compounds, which means that resistance occurs when genetic changes in the progeny of A. fumigatus are selected during reproduction. In A. fumigatus, 3 modes of reproduction can happen: asexual, sexual, and parasexual. Through asexual sporulation, common in nature, A. fumigatus produces an abundant number of spores (conidia). Even though the progeny from asexual reproduction is clonal, many conidia may harbor spontaneous mutations, ensuring genetic diversity. If one or more mutations give the conidia a better ability to survive and grow under certain stresses (for example, triazole exposure), the mutant will proliferate and might surpass the growth of the wild-type spore. This selective pressure can happen in any environment containing azoles [ 55 , 56 ].

The incidence of clinical A. fumigatus triazole resistance varies according to the country and the patient from which it is isolated. In European countries, clinical resistance ranges from 0.6% to 30%, having reached the highest rate (>20%) in the Netherlands, United Kingdom, and Germany [ 43 , 44 ]. Outside Europe, azole resistance has been detected in China (5.5%), India (1.7%), Iran (3.5%), Japan (12.7%), Thailand (3.2%), Australia (2.6%), and the United States (0.6% to 11.8%) [ 15 , 32 , 43 , 45 – 48 ]. In South America, Brazil, Peru, Mexico, and Argentina have also reported triazole-resistant isolates [ 24 , 49 – 53 ]. The clinical implications of an infection caused by an antifungal-resistant strain are not totally revealed and not always related to therapeutic failure [ 43 ]. Nonetheless, some studies have shown that resistance may ultimately lead to a poor outcome [ 9 – 11 , 54 ].

Aspergillus fumigatus is a saprophytic fungus found in soil, crops, seeds, air, leaves, flowers, and indoor environments [ 15 , 17 , 19 – 21 , 26 , 32 – 36 ]. It also causes a wide range of chronic and life-threatening infections, such as allergic bronchopulmonary aspergillosis (ABPA), chronic pulmonary aspergillosis (CPA), and invasive pulmonary aspergillosis (IPA) [ 37 ]. Such diseases are treated with a restricted arsenal of antifungals from 3 classes: azoles, polyenes, and echinocandins [ 37 – 39 ]. Specifically, the triazoles (voriconazole, itraconazole, posaconazole, and isavulconazole) are the most indicated as the first-line therapy [ 38 , 40 ] and liposomal amphotericin B (polyene) and echinocandins as second-line choices [ 38 , 40 , 41 ]. Unlike echinocandins and polyenes, resistance to azoles is relatively common and has been increasing since the first A. fumigatus azole-resistant strains were reported in 1997 [ 42 ].

2.2 Fungicide-driven resistance: Epidemiological, experimental, and field data

Many epidemiological and experimental data corroborate the theory that the DMIs used in the wood and textile industries, and especially those employed in agriculture, may select azole resistance in A. fumigatus in the environment [29,33,46,73,74] (Fig 1A). These studies presenting data supporting fungicide-driven resistance can be categorized into 4 groups: (i) those in which resistant strains were found in both patients and environment [19–24,32–34,49,68,73–81]; (ii) studies attesting cross-resistance between environmental and medical azoles in isolates from both sources [20,22,30,33,46,75,82]; (iii) investigations demonstrating that susceptible isolates could become resistant when exposed to environmental azoles [29,74,83–85]; and (iv) those proving that more resistant strains could be recovered from places or periods at which the fungicides were applied [20,86].

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TIFF original image Download: Fig 1. Fungicide exposure effects on Aspergillus fumigatus. (a) Azole-susceptible and azole-resistant A. fumigatus can be identified in both fungicide-free and fungicide-containing soils and plant-based materials. There is an enrichment, however, of azole-resistant A. fumigatus in niches containing fungicides. (b) Azole-resistant A. fumigatus isolated from places holding fungicides may present some alterations compared to susceptible isolates that confer them cross-resistance with medical azoles, such as overexpression of efflux pumps and the azole-target enzyme, CYP51A, and CYP51A with a reduced azole affinity. The last 2 physiological changes are due to mutations in the gene cyp51A. The most common mutations are a pair of 34-bp sequence (in tandem) in the gene promoter (TR 34 ), which lead to overexpression of cyp51A, together with a mutation that results in leucine replacement by histidine at position 98 (L98H) in the enzyme CYP51A, reducing the affinity of the enzyme to the azole drugs. (c) Other tandem repeat mutations combined or not with point mutations in the gene cyp51A conferring cross-resistance between environmental and medical azoles also can be detected in azole-resistant A. fumigatus isolated from fungicides-containing places. It is important to notice that the alterations represented correspond to amino acids and not in the DNA and that other tandem repeat mutations have already been observed in the clinical sets, but only TR 34 , TR 46 , and TR 53 have been describing in environmental strains. https://doi.org/10.1371/journal.ppat.1010073.g001

Classically, the studies in the Netherlands started to shed light on how environmental azole exposure could lead to cross-resistance to medical azoles [26,30]. First, they demonstrated that itraconazole-resistant A. fumigatus could be isolated from indoor environments (including patient rooms at hospitals), as well as from cultivable soils, seeds, leaves, and compost—but never from azole-naive soils. These resistant strains also posed high resistance to 2 fungicides, metconazole and tebuconazole, thus demonstrating cross-resistance between medical and environmental azoles [26]. Interestingly, 13 out of the 15 resistant strains isolated from the environment had the same mutation in the gene that encodes the azole-target enzyme (cyp51A) [26], which was identical to the isolate identified in the clinical isolates [14]. Such mutations led to a leucine replaced by histidine at position 98 (L98H) in the enzyme CYP51A, along with a pair of 34-base pair (bp) sequence (in tandem) in the gene promoter region (TR 34 ) (TR 34 /L98H) [16]. The 34-bp sequence in tandem in the cyp51A promoter induces overexpression of cyp51A (about 8-fold) [16], and the point mutation hinders the interaction between the drug and the target enzyme [30] (Fig 1B). This combination of mechanisms results in a consistent itraconazole resistance and variable voriconazole, posaconazole, and isavuconazole susceptibility [30,34,68]. Frequently, TR 34 /L98H also confers a pan-azole resistance, both to medical and environmental azoles [26,30]. Coincidentally, the first resistant clinical isolate carrying TR 34 /L98H was reported infecting a patient in 1998 [14,30], just a few years after triazole fungicides had been introduced into the Netherlands [30], which suggests that this mutant could had emerged after azole fungicide contact in the field. Eventually, the TR 34 /L98H mutation was identified in many other European countries, and also in Asia, North and South America, Australia, and Africa [25,43].

The tandem repeat mutation was also identified in DMI-resistant phytopathogens [82,87], strongly suggesting that this is a common resistance mechanism among molds exposed to these fungicides. Penicillium digitatum, for example, contains tandem repeat mutations varying from 126 bp to 199 bp, which have been associated with DMI resistance [88,89]. However, other resistant isolates of plant pathogens, such as Pyrenopeziza brassicae, Monilinia fructicola, and Venturia inaequalis, have fragments inserted in cyp51A promotor (fragments from 65 bp to 553 bp) [90–92] instead of tandem repeat alteration. In general, both genetic variations result in overexpression of cyp51A as in A. fumigatus [82,87].

If DMIs are really the stressors leading to selection of these mutations in the environment, they should probably share similar molecular structures to clinical azoles and dock similarly to them at the azole-target enzyme in A. fumigatus. In order to address these questions, Snelders and colleagues carried out molecule alignment and docking studies using homology modeling of cyp51A. They identified 5 DMIs, propiconazole, bromuconazole, tebuconazole, epoxiconazole, and difenoconazole, which share structural molecular characteristics to medical triazoles, suggesting that they could select cross-resistance in A. fumigatus. These DMIs also assume a similar configuration when docking to the target enzyme and act against wild-type but not against multi-triazole-resistant A. fumigatus [30], further supporting the idea of DMI as a selection pressure.

Other resistance mechanisms involving promotor duplications, either combined or not with single nucleotide polymorphisms (SNPs), have been described in clinical and environmental strains (Fig 1C). TR 53 (2 copies of a 53-bp sequence in tandem in cyp51A) was the second mechanism discovered [30] and thought to be restricted to clinical isolates until it was identified in resistant A. fumigatus strains isolated from flower fields in Colombia [49]. TR 46 /Y121F/T289A (with 2 copies of a 46-bp sequence in tandem in cyp51A, combined with 2 SNPs) (Fig 1C) was also identified in both clinical and environmental isolates [20,48,49,51,71,73,74,93,94]. This mutation provides resistance especially to voriconazole and in some cases to other medical azoles and environmental fungicides [73]. TR 46 /Y121F/T289A was first reported in the Netherlands [93] and subsequently in Belgium [95], India [73], Denmark [71], Germany [96], Colombia [24,49], and China [86]. The spreading of TR 46 /Y121F/T289A is worrisome, as it can cause high resistance to voriconazole, which is recommended as the first-line therapy for many aspergillosis [97].

Recently, another promoter-repeat mutation (a triple 46-bp promoter repeat), combined with 4 SNPs (TR463/Y121F/M172I/T289A/G448S), which leads to a pan-triazole resistance, was discovered (Fig 1C) [20]. The isolates harboring these mutations came from compost heaps containing azole fungicides and A. fumigatus clinical isolates from the Netherlands [20]. Moreover, additional tandem repeats in cyp51A gene, either combined or not with SNPs, were reported in environmental azole-resistant strains, such as TR464/Y121F/M172I/T289A/G448S [20], TR34/L98H/S297T/F495I [22,86], TR46/Y121F/M172I/T289A/G448S [19], TR92/Y121F/M172I/ T289A/G448S [19], and point mutations without tandem repeat alterations, for example, P216L [33], A284T, G448S, P222Q [74], G54R [34], G138S, Y433N, and N248K [85].

In the environment, azole-resistant isolates harboring the aforementioned genetic modifications have been isolated from several places and materials, including leaves, plant seeds, soil samples, flowerbeds, compost, hospital surroundings, and air samples [19,20,22,24,26,34,49,93,98]. In this way, some researchers have been reporting potential hotspot to isolate those mutants (especially TR34/L98H and TR46/Y121F/T289A), including soils from strawberry fields in China [22]; azole-exposed compost [20], flower bulb waste, green waste material, and wood chippings in the Netherlands [19]. These environments contain several characteristics that may facilitate not only the emergence of azole-resistant strains, but also their maintenance, and spread [19,20]. Such chacharacteristic are beyond the scopus of this review and has been recentely well discussed by Burks and colleagues [98]. Besides the fact that not all the soil or culture seems to be favorable for the emergence of resistant strains, it appears that are some DMIs more prone to select mutations in A. fumigatus and cause cross-resistance with medical azoles, such as propiconazole, bromuconazole, tebuconazole, epoxiconazole, difenoconazole, prothioconazole, and azaconazole [19,30].

Mutations in the cyp51A promoter and its open reading frame (ORF) causing overexpression and/or significant changes in the conformation of lanosterol 14α-demethylase are the primary azole resistance mechanisms in clinical and environmental A. fumigatus isolates. However, azole-resistant strains with wild-type cyp51A have been found, suggesting other resistance means unrelated to cyp51A modifications [29,81,86]. Cui and colleagues exposed azole-susceptible strains to liquid culture medium and soil treated with tebuconazole and then recovered 12 resistant isolates without any alteration in the cyp51A gene [29]. The mRNA quantitative analysis showed that some of these isolates overexpressed the genes encoding a transcription factor involved in resistance (AtrF), 2 efflux pumps (AfuMDR1, AfuMDR2), and paralogue genes for the azole-target enzyme (cyp51A and cyp51B) [29]. Another study also demonstrated that the fungicide propiconazole could select resistance by causing overexpression of cyp51A and the efflux pump genes AfuMDR3 and AfuMDR4 [85]. Overall, these data show how diverse the mechanism behind azole resistance in A. fumigatus is (Fig 1B) and that researchers should also look for alterations beyond the cyp51A gene.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010073

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