Fungal rRNA Sequencing of Indoor and Occupational Air Samples

Rationale: Fungal exposure may exacerbate adverse health effects; however, the etiological agents remain uncharacterized due to the limitations of traditional sampling and analysis methods. In this pilot study, rRNA sequencing was utilized to examine the diversity of fungal species present in indoor and occupational environments.

Methods: Air samples were collected using the NIOSH BC212 sampler. Sampling sites included two homes, one green house, and one horse farm. For traditional microscopic analyses, spores were extracted from samples and stained with lactophenol cotton blue. For molecular analyses, DNA was collected and rRNA sequences were amplified with universal fungal primers. Amplicons were cloned, sequenced, and used in BLAST searches against the NCBI nucleotide database to determine the closest fungal relative based on sequence similarity.

Results: PCR amplicons were observed from each of the four samples. The greenhouse samples predominantly yielded the fungal genera Cladosporium , Alternaria , and Sclerotinia , as well as the class Polyporales. In the horse farm, 16/20 isolated clones were 100% identical with Wallemia sebi , a basidiomycete associated with hypersensitivity pneumonitis in European farmers. The clones from one home were dominated by the dry-rot basidiomycete, Antrodia sinuosa . Samples from the second home contained a variety of clones from the genera Epicoccum , Cladosporium , and Phlebia . Traditional microscopic counting showed air samples to be predominantly composed of Aspergillus/Penicillium amerospores in all samples.

Conclusions: Compared to traditional methods of analysis, our data demonstrate a much wider diversity of fungi that may possibly contribute to personal exposure and adverse health effects in home and occupational environments.