Aspergillus fumigatus forms biofilms with reduced antifungal drug susceptibility on bronchial epithelial cells

Abstract

Aspergillus fumigatus is a leading cause of death in immunocompromised patients and a frequent colonizer of the respiratory tracts of asthma and cystic fibrosis (CF) patients. Biofilms enable bacteria and yeasts to persist in infections and can contribute to antimicrobial resistance. We investigated the ability of A. fumigatus to form biofilms on polystyrene (PS) and human bronchial epithelial (HBE) and CF bronchial epithelial (CFBE) cells. We developed a novel in vitro coculture model of A. fumigatus biofilm formation on HBE and CFBE cells. Biofilm formation was documented by dry weight, scanning electron microscopy (SEM), and confocal scanning laser microscopy (CSLM). The in vitro antifungal activities of seven antifungal drugs were tested by comparing planktonic and sessile A. fumigatus strains. A. fumigatus formed an extracellular matrix on PS and HBE and CFBE cells as evidenced by increased dry weight, SEM, and CSLM. These biofilms exhibited decreased antifungal drug susceptibility and were adherent to the epithelial cells, with fungi remaining viable throughout 3 days. These observations might have implications for treatment of A. fumigatus colonization in chronic lung diseases and for its potential impact on airway inflammation, damage, and infection.

FIG. 1.

Quantification of A. fumigatus biofilms in coculture (dark-grey bars, 16HBE; light-gray bars, CFBE41o). Each bar represents the mean biomass of three independent experiments with dry-weight measurement, and the error bars indicate the standard errors of the mean. Neg., PBS control containing MEM plus 10% PBS after 2 days of incubation.

FIG. 2.

Densities of A. fumigatus biofilms in coculture (dark-grey bars, 16HBE; light-grey bars, CFBE41o−) were quantified in triplicate by safranin staining. The error bars indicate the standard errors of the mean. Neg., PBS control containing MEM plus 10% PBS after 2 days of incubation.

FIG. 3.

(a) SEM under nonbiofilm conditions at 48 h. (b) SEM of biofilm surface at 48 h. The boxes indicate parallel-packed hyphae. The arrow indicates ECM embedded in the hyphal network.

FIG. 4.

CSLM image of a lateral view of an Aspergillus biofilm on PS.

FIG. 5.

CSLM images. (A to E) Biofilm formation on 16 HBE cells (panel A without infecting conidia). (F to K) Biofilm formation on CFBE41o− cells (panel F without infecting conidia) after 4 h (B and D), 1 day (C and H; the arrows indicate early ECM), and 2 days (D and I; the arrows indicate ECM proliferation as regions of bright-green fluorescence without clear edges) and a 3D reconstruction after 3 days (E and K; the arrows indicate highly active [red-stained] conidia and hyphae, as well as green-stained areas without clear edges between and around the hyphal structures).