In-host microevolution of Aspergillus fumigatus: a phenotypic and genotypic analysis

E Ballard1, WJG Melchers2,3, J Zoll2,3, AJP Brown1, PE Verweij2,3, A Warris1

Author address: 

1Medical Research Council Centre for Medical Mycology, University of Aberdeen, United Kingdom 2Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands 3Center of Expertise in Mycology, Radboud University Medical Centre/CWZ, Nijmegen, The Netherlands

Abstract: 

Purpose: In order to survive, Aspergillus fumigatus must adapt to specific niche environments. Adaptation to the human host includes modifications that enable persistent colonisation and, following antifungal therapy, the development of azole resistance. The aim of this study is to advance understanding of the genetic and physiological adaptation of A. fumigatus occurring in patients during infection and treatment. This study has potential to inform novel therapeutic strategies to prevent and overcome persistent and/or recurrent fungal infection.

Methods: Central to this study are 13 A. fumigatus strains isolated from a single chronic granulomatous disease patient suffering from recurrent invasive aspergillosis. The strains were isolated over a period of 2 years. Initial characterisation of the strains involved measuring antifungal susceptibility, using the EUCAST broth microdilution reference method, and microsatellite genotyping. In detail characterisation of the strains involved phenotypic and genotypic analysis including whole genome sequencing.

Results: All strains had identical microsatellite genotypes and were considered isogenic. The first 2 strains isolated were azole susceptible, whereas later isolates were itraconazole, voriconazole and/or posaconazole resistant. The first 5 isolates produced wild-type green colonies whereas subsequent isolates produced predominantly white, sterile hyphae. Remarkably, the final isolate collected produced green colonies again.

Single nucleotide polymorphism (SNP) based phylogenetic analysis verified the isolates to be isogenic and showed V130-15, the first strain to be isolated, as the precursor the other 12 strains. Whole genome comparisons identified 248 non-synonymous SNPs thought to have developed in-host. Cyp51A SNPs were identified in 9 of the isolates in the series; the SNPs identified were G54R, G54V, P216L and M220R. Growth assays in the presence and absence of various antifungal stressors highlighted minor changes in growth rate and stress resistance, with exception of one isolate (V157-62) showing a significant growth defect (p = 0.003). Interestingly, later isolates produced significantly fewer conidia in comparison to earlier isolates (p = 0.038). In certain drug resistant isolates conidiation was restored in the presence of itraconazole. The series of isolates possessed differences in their virulence as demonstrated in a Galleria mellonella infection model.

Conclusions: This study describes for the first time in detail the genotypic and phenotypic changes that occur in A. fumigatus during the course of infection. We conclude that the microevolution of A. fumigatus in this patient has driven the emergence of both Cyp51A-independent and Cyp51A-dependent azole resistance mechanisms, and additional phenotypes that are likely to have promoted fungal persistence. These wide ranging changes represent adaptation to the human host environment. Understanding this adaptation is vitally important as it gives insight into the type of stresses encountered by the fungus in host. 

2018

Full conference title: 

The 8th Advances Against Aspergillus, Lisbon Conference Center, Lisbon, Portugal
    • AAA 8th (2018)