Comparative genotyping and phenotyping of Aspergillus fumigatus isolates from humans, dogs and the environment

Abstract

Background: Aspergillus fumigatus is a ubiquitous saprotrophic fungus and an opportunistic pathogen of humans and animals. Humans and animals can inhale hundreds of A. fumigatus spores daily. Normally this is harmless for humans, but in case of immunodeficiency, invasive pulmonary aspergillosis (IPA) can develop with a high mortality rate. A. fumigatus also causes non-invasive mycoses like sino-nasal aspergillosis (SNA) in dogs.

Results: In this study we compared A. fumigatus isolates from humans with suspected IPA, dogs with SNA, and a set of environmental isolates. Phylogenetic inference based on calmodulin (CaM) and beta-tubulin (benA) sequences did not reveal A. fumigatus sub-groups linked to the origin of the isolates. Genotyping and microsatellite analysis showed that each dog was infected by one A. fumigatus genotype, whereas human patients had mixed infections. Azole resistance was determined by antifungal susceptibility testing and sequencing of the cyp51A gene. A total of 12 out of 29 human isolates and 1 out of 27 environmental isolates were azole resistant. Of the azole resistant strains, 11 human isolates showed TR₃₄/L98H (n = 6) or TR46/Y121F/T289A (n = 5). Phenotypically, isolates from dogs were more variable in growth speed and morphology when compared to those isolated from human and the environment.

Conclusions: 1. A. fumigatus from dogs with SNA are phenotypically very diverse in contrast to their environmental and human counterparts. 2. Phenotypic variability can be induced during the chronic infection process in the sinus of the dogs. The basis of this heterogeneity might be due to genomic differences and/or epigenetic variations. 3. Differences in dogs is a could be a result of within-host adaption and might be triggered by environmental factors in the sinus, however this hypothesis still needs to be tested.

Fig. 1

Minimum spanning tree of A. fumigatus isolates. Circles indicate different genotypes, numbers inside the circles indicate the number of isolates belonging to that particular genotype, thickness of the line indicates the relatedness of the connected isolates determined by Bruvo distance; values close to zero indicate identical isolates, while values close to 1 indicate unrelated isolates [35]. Additionally, TR mutations present in the isolates are depicted as color-coded stars. The black star (WT) refers to azole sensitive phenotype

Fig. 2

Schematic overview of the nine human and canine patients. The total number of genotypes and the codes of the A. fumigatus isolates from each patient is indicated. Isolates that are underlined belong to the same genotype in the case of canine patients, an asterisk indicates the same patient (CP8 – CP8.2) being re-infected with a different A. fumigatus isolate

Fig. 3

Morphology of colonies (a) and conidiophores and conidia (B-D) of A. fumigatus isolates from dogs, humans and the environment. Top row of panel A shows the obverse side of the colony grown on MEA of isolates DTO 028-D6, DTO 271-A5, and DTO 303-E8, while the bottom row shows the reverse side of CYA-grown colonies of DTO 028-D6, DTO 326-I9, DTO 326-H2. A conidiophore of DTO 327-D3 with flask-shaped phialides and abundant production of conidia is shown in (b), while (c) shows similar phialides of isolate DTO 326-I9 with sparse production of conidia. Reduced, cylindrical shaped phialides of DTO 303-F2 and abundant production of conidia are shown in (d)

Fig. 4

PCA and K-means clustering of A. fumigatus isolates based on colony grown on several media. The arrow indicates the position of A. fumigatus var. ellipticus