Epidemiological Studies of Pan-Azole Resistant Aspergillus fumigatus Populations Sampled during Tulip Cultivation Show Clonal Expansion with Acquisition of Multi-Fungicide Resistance as Potential Driver

Abstract

Pan-azole resistant isolates are found in clinical and environmental Aspergillus fumigatus (Af) populations. Azole resistance can evolve in both settings, with Af directly targeted by antifungals in patients and, in the environment, Af unintendedly exposed to fungicides used for material preservation and plant disease control. Resistance to non-azole fungicides, including methyl benzimidazole carbamates (MBCs), quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs), has recently been reported. These fungicide groups are not used in medicine but can play an important role in the further spread of pan-azole resistant genotypes. We investigated the multi-fungicide resistance status and the genetic diversity of Af populations sampled from tulip field soils, tulip peel waste and flower compost heaps using fungicide sensitivity testing and a range of genotyping tools, including STRAf typing and sequencing of fungicide resistant alleles. Two major clones were present in the tulip bulb population. Comparisons with clinical isolates and literature data revealed that several common clonal lineages of TR₃₄/L98H and TR₄₆/Y121F/T289A strains that have expanded successfully in the environment have also acquired resistance to MBC, QoI and/or SDHI fungicides. Strains carrying multiple fungicide resistant alleles have a competitive advantage in environments where residues of multiple fungicides belonging to different modes of action are present.

Keywords: Aspergillus fumigatus; CYP51A; aspergillosis; azole fungicides; clonal lineages; fungicide resistance; fungicide target proteins.

Figure 1

MIC distribution of A. fumigatus isolates sampled from tulip field soils (n = 180) in the Netherlands. Numbers show amount (sum) of isolates within each MIC range.

Figure 1

MIC distribution of A. fumigatus isolates sampled from tulip field soils (n = 180) in the Netherlands. Numbers show amount (sum) of isolates within each MIC range.

Figure 2

Comparison of fungicide sensitivity levels for strains isolated from tulip field soils (n = 180), tulip peel waste (n = 42), tulip compost (n = 19) and flower bulbs (tulips (n = 128) and daffodils (n = 30)). For voriconazole, tebuconazole and carbendazim out of range values spiral plating MIC values were displayed as the log values of 19.120, 17.349 and 11.464 µg/mL, respectively.

Figure 3

Minimum-spanning networks showing the genetic relationship of Aspergillus fumigatus multilocus genotypes (MLGs) originating from different sources. The relatedness between MLGs is based on Bruvo’s genetic distances, which accounts for the stepwise mutation of microsatellite loci. Each node represents an MLG with one or more individuals. Nodes with multiple isolates (clonal lineages), clusters, are arranged alphabetically. Nodes that are more closely related have darker and thicker edges, whereas nodes that are more distantly related have lighter and thinner edges. Names of a selection of isolates, including all medical strains, are also displayed and all details on strains can be found in Table S1.