Detection of azole-resistant Aspergillus fumigatus in the environment from air, plant debris, compost, and soil

Abstract

Aspergillus fumigatus is a ubiquitous fungus, a saprophyte of plants, and an opportunistic pathogen of humans. Azole fungicides are used in agriculture to control plant pathogens, and azoles are also used as a first line of treatment for aspergillosis. The continued exposure of A. fumigatus to azoles in the environment has likely led to azole resistance in the clinic where infections result in high levels of mortality. Pan-azole resistance in environmental isolates is most often associated with tandem-repeat (TR) mutations containing 34 or 46 nucleotides in the cyp51A gene. Because the rapid detection of resistance is important for public health, PCR-based techniques have been developed to detect TR mutations in clinical samples. We are interested in identifying agricultural environments conducive to resistance development, but environmental surveillance of resistance has focused on labor-intensive isolation of the fungus followed by screening for resistance. Our goal was to develop assays for the rapid detection of pan-azole-resistant A. fumigatus directly from air, plants, compost, and soil samples. To accomplish this, we optimized DNA extractions for air filters, soil, compost, and plant debris and standardized two nested-PCR assays targeting the TR mutations. Sensitivity and specificity of the assays were tested using A. fumigatus DNA from wild type and TR-based resistant isolates and with soil and air filters spiked with conidia of the same isolates. The nested-PCR assays were sensitive to 5 fg and specific to A. fumigatus without cross-reaction with DNA from other soil microorganisms. Environmental samples from agricultural settings in Georgia, USA were sampled and tested. The TR46 allele was recovered from 30% of samples, including air, soil and plant debris samples from compost, hibiscus and hemp. These assays allow rapid surveillance of resistant isolates directly from environmental samples improving our identification of hotspots of azole-resistant A. fumigatus.

Molecular Diagnostics for Invasive Fungal Diseases: Current and Future Approaches

“…fumigatus complex species as well as further delineating the role of membrane transporter protein gene and non-cyp51A mutations such as cholesterol import and HapE mutations [144][145][146]. Targeted sequencing with pre-amplification of tandem repeat regions can be performed, but the utility of these methods is unclear when non-cyp51A mediated triazole-resistant clinical isolates ranges from 15 to 60% [146,147].…”

Detection of pan-azole resistant Aspergillus fumigatus in horticulture and a composting facility in Belgium

“… 9 Since then, it has been demonstrated that the majority of cases of azole-resistant disease arise when an azole-naive patient is infected with an azole-resistant strain from the environment, which is frequently found in air and soil. 8–10 , 16–18 …”

“…Since all azoles target the enzyme lanosterol-14-alpha-demethylase ( cyp 51), which inhibits the biosynthesis of ergosterol, cross-resistance is seen between the medical and agricultural azoles. 8 , 24 , 25 Aspergillus fumigatus has two cyp 51 isoforms, namely c yp 51A and cyp 51B. Most azole-resistant strains display mutations in the cyp 51A gene, although other resistance mechanisms have been described.…”