Heterogeneity and within-host adaption observed in clinical isolates of Aspergillus fumigatus

Objective: We explored the phenotypical and genetic variability among isolates of the ubiquitous and saprophytic fungus Aspergillus fumigatus which has the remarkable ability to adapt and grow in many different niches. Due to this ability it can also cause invasive and non-invasive infections in humans and animals, for example invasive pulmonary aspergillosis (IPA) in humans and sinonasal aspergillosis (SNA) in dogs. Our main objective is to understand how this fungus adapts to different niches and to find the factors and genetic traits that influences host adaptation and development in the context of fungal infections. To address this question we have compared a set of clinical and environmental isolates at a genetic and phenotypic level. Isolates were derived from sputum or bronchoalveolar lavage from human patients at the intensive care unit who were suspected to developed IPA. In addition we cultured isolates from fungal plaques isolated from the sinus of dogs suffering with SNA using endoscopy or trephination. Methods: We have compared a set of isolates of A. fumigatus from A) humans (29 isolates from a preselected set of 9 patients), B) dogs with SNA (27 isolates form 9 patients) C) environmental isolates (27 isolates) with reference strains. Azole resistance was determined by microdilution assay antifungal susceptibility testing and tandem repeats in the promotor region of the cyp51A gene. Sequencing of calmodulin (CaM), beta-tubulin(benA) and mating type genes (MAT1-1 and 1-2) and microsatellite (STRAf) analysis were performed to detect genetic differences between isolates. Plating on different media was performed to observe differences in macro and micromorphology Results: Genotyping of the different isolates showed that each human patient carried multiple fungal genotypes. In contrast, each dog suffering from SNA appeared to be infected by only one single genotype. Remarkably, different isolates from each dogs, and having the same genotype, showed a large phenotypic variability. In particular “white isolates” with apparent reduced sporulation were frequently isolated (13 out of 27 isolates) from dogs but not in human patients or in environmental isolates. In terms of azole resistance only human isolates and one of the indoor and outdoor environmental isolates were found to be resistant. Principal component analysis using colony diameter as a proxy for growth speed suggests that canine isolates might represent a subgroup of A. fumigatus that are responsible for SNA. Conclusion: Our observations shows thatfumigatus from dogs with SNA are phenotypically very diverse in contrast to their environmental and human counterparts. Phenotypic variability seems to be generated 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. We expect that appearance of the phenotypic “white isolates” in dogs is a result of within-host adaption and is triggered by environmental factors in the sinus which we address in ongoing research.