Submicronic Fungal Bioaerosols: High-Resolution Microscopic Characterization and Quantification

Abstract

dSubmicronic particles released from fungal cultures have been suggested to be additional sources of personal exposure in moldcontaminated 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.

Aerosolization of Mycotoxins after Growth of Toxinogenic Fungi on Wallpaper

“…For these two species, an air velocity of 2 m/s, which matches the air speed observed due to mechanical and natural ventilation in tertiary buildings (28), allowed the aerosolization of numerous particles from wallpaper. These particles were distributed in two main categories: one including very small particles (Ͻ0.15 m), and the second including particles ranging from 2 to 6 m. This second group may correspond to spores, groups of spores, or mycelium debris (38,39), in agreement with previous data on aerosolization of these fungal species (40)(41)(42). One can note that for P. brevicompactum, the total number of particles aerosolized from substrate was higher than for A. versicolor.…”

Profile and Morphology of Fungal Aerosols Characterized by Field Emission Scanning Electron Microscopy (FESEM)

“…The characteristics of these fungal isolates and the procedure for inoculum preparation have been previously described elsewhere (Afanou et al 2014). Briefly, spores from 2-week-old cultures grown on malt extract agar (MEA) (Samson et al 2004) were harvested by gently scraping cultures submerged in 20 mL phosphate buffered saline (PBS) containing 0.1% Tween 20 using a sterile inoculation loop.…”

“…10 8 spores) of this spore suspension was used to inoculate gypsum board (GB) (Lafarge Gips Dekoform 6 AK, Oberursel, Germany) commonly used as indoor building material in Norwegian residential environments. The GB was cut into 80 mm diameter circular plates that were prepared following the procedure previously described (Afanou et al 2014). Following 8 weeks of incubation at 25 ± 1°C and 45 ± 5% relative humidity (in the incubator), the GB plates were utilized for aerosolization experiments.…”

“…Following our previous publication on the enumeration and origin of submicronic fungal fragments by high-resolution microscopy (Afanou et al 2014), we describe in this study the morphology of fragments and spore particles, and their distribution in in vitro generated fungal aerosols. The size, shape and origin characteristics of various fungal particle types are described, as well as the profile of the aerosols generated from pure fungal cultures.…”

“…The morphology and size of spores, and hyphal and spore fragments have been assessed by microscopy in attempts to describe the fungal aerosols (Pady and Kramer 1960; Pady and Gregory 1963; Eduard et al 1988, 1990; Heikkila et al 1988; Karlsson and Malmberg 1989; Green et al 2005; Halstensen et al 2007; Afanou et al 2014; Vestlund et al 2014). Quantification and size-characterization of fungal particles have also been performed by measuring the optical or aerodynamic diameter of aerosol particles using automatic particle counters or sizers.…”