Treatment of a Clinically Relevant Plant-Pathogenic Fungus with an Agricultural Azole Causes Cross-Resistance to Medical Azoles and Potentiates Caspofungin Efficacy

Abstract

Azoles are extensively applied in agriculture and medicine, and a relationship between the development of azole resistance in agriculture and the development of azole resistance in clinical practice may exist. The maize pathogen Colletotrichum graminicola, causing cutaneous mycosis and keratitis, has been used to investigate the acquisition of resistance to an agricultural azole and the resulting cross-resistance to various medical antifungal agents. Azole-adapted strains were less sensitive to all azoles tested but showed increased sensitivity to caspofungin, amphotericin B, and nystatin. Viability staining and infection assays with excised human skin confirmed these data.Five plant-pathogenic Colletotrichum species, including Colletotrichum graminicola, have been reported to cause cutaneous phaeohyphomycosis and keratitis in humans and are of clinical relevance (2,8,9). Most Colletotrichum crassipes, C. gloeosporioides, C. coccodes, and C. dematium strains isolated from infected tissue displayed a major degree of resistance to all azoles tested (3).In modern agriculture, Colletotrichum species have been implicated in anthracnose plant diseases worldwide, with substantial economical losses (7). Among other antifungal agents, azoles have been used extensively to control Colletotrichum species (12). Azoles differing in structure but exhibiting the same mode of action are used to treat both fungal diseases of plants and medical mycoses. As keratitis caused by Colletotrichum infection occurs primarily after injury by plant material (8,9,19), it is tempting to speculate that azole-resistant strains isolated from human tissues may have developed in fungicidetreated fields (see the report on azole resistance published by the scientific steering committee of the European Commission [http://ec.europa.eu/food/fs/sc/ssc/out278_en.pdf]). As Colletotrichum species are exposed to azoles in agricultural environments and are of clinical relevance, these pathogens are well suited to investigate the acquisition of resistance to agricultural azoles and the resulting cross-resistance to antifungal agents of various different chemical classes and modes of action used in medicine. MATERIALS AND METHODSCultivation of C. graminicola and adaptation to tebuconazole. Wild-type C. graminicola isolate CgM2 was cultivated as described previously (24).To adapt C. graminicola to tebuconazole, 1 ϫ 10 6 conidia were inoculated into 50 ml complete medium (CM) (15) and incubated in darkness at 23°C for 7 days. Cultures were then transferred into fresh liquid CM with increasing tebuconazole concentrations (1 g ml Ϫ1 for 24 h, 5 g ml Ϫ1 for 96 h, and 20 g ml Ϫ1 for 1 h). The mycelium was blended (Ultra Turrax; IKA Labortechnik, Staufen, Germany) at level 5 for 30 s, further cultivated on solidified CM containing 5 g tebuconazole ml Ϫ1 and 8 g tebuconazole ml Ϫ1 for 4 weeks each, and maintained on CM plates containing 30 g tebuconazole ml Ϫ1 . Quantitative determination of drug sensitivity. Radial growth rates of nonadapted and matched, adapted isol...

Possible Environmental Origin of Resistance of Aspergillus fumigatus to Medical Triazoles

“…As A. fumigatus is a thermotolerant fungus and readily grows in compost heaps, resistance to azole fungicides might arise under these conditions. Stepwise increased exposure of the maize pathogen Colletotrichum graminicola, a fungus also capable of causing cutaneous mycosis and keratitis in humans, to the fungicide tebuconazole showed loss of activity by this triazole as well as by ITZ and voriconazole, indicating that fungicides are capable of inducing resistance to medical azoles (17).…”

In Vitro Susceptibility of the Yeast Pathogen Cryptococcus to Fluconazole and Other Azoles Varies with Molecular Genotype

“…However, we cannot completely rule out prior exposure to all azoles, as azole-based formulations are used in many countries, including Australia, to protect crops from pathogenic fungi and as wood preservatives (2,16,28). Studies have shown that some environmental fungi of clinical importance can develop cross-resistance to medical azoles following exposure to agricultural azoles (34,39,46). C. gattii and C. neoformans are environmental fungi, and contact with agricultural azoles may occur.…”

Mechanisms and significance of fungicide resistance

“…It is important to note that some fungicides belonging to classes that do not need to invade the cell to reach their target and to be active, i.g. amphothericin B and nystatin, which form complexes with fungal sterols and induces pore formation, or the β-1,3-glucan synthase inhibitor caspofungin, were able to control azole-adapted strains more efficiently than non-adapted strains (47).…”

“…Fungicide sensitivity assays performed with C. graminicola in vitro showed that adaptation to an azole fungicide used in agricultural environments also resulted in resistance to azoles used in clinical environments. Fungicide sensitivity determined by radial growth assays in vitro correlated well with viability staining of fungal hyphae and infection assays with excised human skin (47). It is important to note that some fungicides belonging to classes that do not need to invade the cell to reach their target and to be active, i.g.…”