Characterization of Aspergillus fumigatus Isolates from Air and Surfaces of the International Space Station

Abstract

One mission of the Microbial Observatory Experiments on the International Space Station (ISS) is to examine the traits and diversity of fungal isolates to gain a better understanding of how fungi may adapt to microgravity environments and how this may affect interactions with humans in a closed habitat. Here, we report an initial characterization of two isolates, ISSFT-021 and IF1SW-F4, of Aspergillus fumigatus collected from the ISS and a comparison to the experimentally established clinical isolates Af293 and CEA10. Whole-genome sequencing of ISSFT-021 and IF1SW-F4 showed 54,960 and 52,129 single nucleotide polymorphisms, respectively, compared to Af293, which is consistent with observed genetic heterogeneity among sequenced A. fumigatus isolates from diverse clinical and environmental sources. Assessment of in vitro growth characteristics, secondary metabolite production, and susceptibility to chemical stresses revealed no outstanding differences between ISS and clinical strains that would suggest special adaptation to life aboard the ISS. Virulence assessment in a neutrophil-deficient larval zebrafish model of invasive aspergillosis revealed that both ISSFT-021 and IF1SW-F4 were significantly more lethal than Af293 and CEA10. Taken together, these genomic, in vitro, and in vivo analyses of two A. fumigatus strains isolated from the ISS provide a benchmark for future investigations of these strains and for continuing research on specific microbial isolates from manned space environments. IMPORTANCE As durations of manned space missions increase, it is imperative to understand the long-term consequence of microbial exposure on human health in a closed human habitat. To date, studies aimed at bacterial and fungal contamination of space vessels have highlighted species compositions biased toward hardy, persistent organisms capable of withstanding harsh conditions. In the current study, we assessed traits of two independent Aspergillus fumigatus strains isolated from the International Space Station. Ubiquitously found in terrestrial soil and atmospheric environments, A. fumigatus is a significant opportunistic fungal threat to human health, particularly among the immunocompromised. Using two well-known clinical isolates of A. fumigatus as comparators, we found that both ISS isolates exhibited normal in vitro growth and chemical stress tolerance yet caused higher lethality in a vertebrate model of invasive disease. These findings substantiate the need for additional studies of physical traits and biological activities of microbes adapted to microgravity and other extreme extraterrestrial conditions.

Keywords: Aspergillus fumigatus; International Space Station; SNP analysis; secondary metabolites; virulence.

FIG 1

Isolation and phylogenetic characterization of ISS strains. (A) IF1SW-F4 was isolated from the wall area outlined in blue adjacent to the cupola window aboard the ISS. ISSFT-021 was independently isolated from a HEPA filter (not shown). (B) Frequency distribution of total SNPs found in 94 sequenced clinical and environmental isolates of A. fumigatus in comparison to a reference genome (Af293). Colored arrows designate the bin groups to which each strain included in this study belongs. (C) Phylogenetic tree of 95 sequenced isolates of A. fumigatus, showing mating type (MAT1-1 or MAT1-2), clinical or environmental origin, and geographical sampling location. Strains of interest used in this study are highlighted in yellow.

FIG 2

In vitro growth of ISS isolates compared to growth of the clinical isolates Af293 and CEA10. (A) Growth on GMM at 37°C, showing colony morphology and color. (B) Radial growth at 37°C on GMM. Statistical analyses were performed by one-way ANOVA. (C) Germination rates in liquid GMM at 37°C, 250 rpm. Spores were considered germinated after germ tube lengths were observed to be greater than or equal to the swollen spore base.

FIG 3

ISS isolates showed no enhanced resistance to chemical stresses in vitro. (A) Colony appearance after point inoculations of 10 µl containing 1 × 10⁴ conidia on solid GMM supplemented with the following stressors: 1.0 M NaCl, 0.02% MMS, and 25 mg/ml Congo red (CR). (B) Quantification of growth inhibition was measured by colony diameters after a 72-h incubation at 37°C. Data shown are the radial growth versus that in controls. (C) Hydrogen peroxide sensitivity was assayed by diffusion assay with 1-cm holes filled with 100 µl of 4% H₂O₂ in plates containing 5 × 10⁷ spores suspended in top agar. (D) Zones of inhibition were measured as diameters after 48 h of growth, and significance determined via one-way analysis of variance.

FIG 4

Secondary metabolite production of ISS strains. (A) Secondary metabolite profiles of ISSFT-021, IF1SW-F4, CEA10, and Af293 when grown on GMM. Individual metabolite production is reported as either increased, decreased, or no difference. compared to that of Af293. (B) Metabolite quantification, showing the percent change for each metabolite in relation to Af293; significance was determined using a one-way ANOVA.

FIG 5

Virulence assessment in a larval zebrafish model of invasive aspergillosis. (A) Survival outcome through 7 days postinfection (dpi) of neutrophil-deficient mpx:mCherry-2A-Rac2D57N larvae, in which neutrophils specifically are unable to reach the site of infection. (B) Survival outcome of the second ISS isolate, IF1SW-F4, compared to that of CEA10 and ISSFT-021 in mpx:mCherry-2A-Rac2D57N larvae. Shown are data pooled from three (A) or four (B) independent experimental replicates. Statistical analyses were performed using the Cox proportional hazard regression analysis.