Submicronic fungal bioaerosols: high-resolution microscopic characterization and quantification

Abstract

Submicronic particles released from fungal cultures have been suggested to be additional sources of personal exposure in mold-contaminated buildings. In vitro generation of these particles has been studied with particle counters, eventually supplemented by autofluorescence, that recognize fragments by size and discriminate biotic from abiotic particles. However, the fungal origin of submicronic particles remains unclear. In this study, submicronic fungal particles derived from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum cultures grown on agar and gypsum board were aerosolized and enumerated using field emission scanning electron microscopy (FESEM). A novel bioaerosol generator and a fungal spores source strength tester were compared at 12 and 20 liters min(-1) airflow. The overall median numbers of aerosolized submicronic particles were 2 × 10(5) cm(-2), 2.6 × 10(3) cm(-2), and 0.9 × 10(3) cm(-2) for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. A. fumigatus released significantly (P < 0.001) more particles than A. versicolor and P. chrysogenum. The ratios of submicronic fragments to larger particles, regardless of media type, were 1:3, 5:1, and 1:2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Spore fragments identified by the presence of rodlets amounted to 13%, 2%, and 0% of the submicronic particles released from A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Submicronic particles with and without rodlets were also aerosolized from cultures grown on cellophane-covered media, indirectly confirming their fungal origin. Both hyphae and conidia could fragment into submicronic particles and aerosolize in vitro. These findings further highlight the potential contribution of fungal fragments to personal fungal exposure.

FIG 1

Instruments used for aerosolization and collection of particles from fungal cultures. (A) Stami particle generator (SPG). (B) Fungal spores source strength tester (FSSST).

FIG 2

Experimental setup. The generator (SPG) with mounted cassette is connected to the system setup. Flowmeters 1 and 2 measure the airflows at the inlet and outlet of the generator, respectively. HEPA 1 and HEPA 2 filter the airflows from the outlet and to the inlet, respectively. The dryer (in-line tube containing silica gel) maintains a constant relative humidity in the air at the inlet, and the in-line P2021 ionizer reduces electrostatic charges.

FIG 3

Micrographs of aerosolized particles. (A) Warted to spiny spores from A. fumigatus (8-week-old cultures grown on GB and aerosolized by the use of the FSSST at 12 liters min⁻¹). (B) Spiny spores from A. versicolor (2-week-old cultures grown on MEAC and aerosolized by the use of the SPG at 12 liters min⁻¹). (C) Rugose spores from P. chrysogenum (2-week-old cultures grown on MEAC and aerosolized by the use of the SPG at 12 liters min⁻¹). (D) Shattered spores of A. fumigatus (8-week-old cultures grown on GB and aerosolized by the use of the FSSST at 12 liters min⁻¹). (E) Spore with rodlet structure from A. versicolor (2-week-old cultures grown on MEAC and aerosolized by the use of the SPG at 12 liters min⁻¹). Arrows shows submicronic (a) and larger (b) particles on the filter membrane. SE, secondary electron.

FIG 4

(A to E) Surface structures of submicronic fragments with rodlets from A. versicolor (2-week-old cultures grown on MEA and aerosolized by the use of the SPG at 12 liters min⁻¹) (A) and A. fumigatus (2-week-old cultures grown on MEA and aerosolized by the use of the SPG at 20 liters min⁻¹) (B) and surface structures of submicronic fragments without rodlets from A. versicolor (2-week-old cultures grown on MEAC and aerosolized by the use of the SPG at 12 liters min⁻¹) (C), A. fumigatus (2-week-old cultures grown on MEA and aerosolized by the use of the SPG at 12 liters min⁻¹) (D), and P. chrysogenum (2-week-old cultures grown on MEAC and aerosolized by the use of the SPG at 12 liters min⁻¹) (E). (F to H) Control micrographs of freeze-dried hyphal fragments without rodlets from A. versicolor (F), A. fumigatus (G), and P. chrysogenum (H).