Recovery of Fungal Cells from Air Samples: a Tale of Loss and Gain

Abstract

There are limitations in establishing a direct link between fungal exposure and health effects due to the methodology used, among other reasons. Culture methods ignore the nonviable/uncultivable fraction of airborne fungi. Molecular methods allow for a better understanding of the environmental health impacts of microbial communities. However, there are challenges when applying these techniques to bioaerosols, particularly to fungal cells. This study reveals that there is a loss of fungal cells when samples are recovered from air using wet samplers and aimed to create and test an improved protocol for concentrating mold spores via filtration prior to DNA extraction. Results obtained using the new technique showed that up to 3 orders of magnitude more fungal DNA was retrieved from the samples using quantitative PCR. A sequencing approach with MiSeq revealed a different diversity profile depending on the methodology used. Specifically, 8 fungal families out of 19 families tested were highlighted to be differentially abundant in centrifuged and filtered samples. An experiment using laboratory settings showed the same spore loss during centrifugation for Aspergillus niger and Penicillium roquefortii strains. We believe that this work helped identify and address fungal cell loss during processing of air samples, including centrifugation steps, and propose an alternative method for a more accurate evaluation of fungal exposure and diversity.IMPORTANCE This work shed light on a significant issue regarding the loss of fungal spores when recovered from air samples using liquid medium and centrifugation to concentrate air particles before DNA extraction. We provide proof that the loss affects the overall fungal diversity of aerosols and that some taxa are differentially more affected than others. Furthermore, a laboratory experiment confirmed the environmental results obtained during field sampling. The filtration protocol described in this work offers a better description of the fungal diversity of aerosols and should be used in fungal aerosol studies.

Keywords: bioaerosols; centrifugation; filtration; fungi; recovery; taxon loss.

FIG 1

Concentration of Aspergillus fumigatus using culture and molecular (qPCR) methods in aerosol samples collected from three different composting facilities. Each point represents a different visit. The bars represent the mean value for each condition. The biological replicates are represented by the site sampled at each visit. Two visits in vegetal composting and one visit in animal composting were negative for A. fumigatus. Therefore, they do not appear on the figure.

FIG 2

Concentrations of Penicillium and Aspergillus (Pen-Asp/m³) using qPCR on filtered and centrifuged samples (left y axis) compared with concentrations of mesophilic molds (CFU/m³) using culture counts (right y axis) from bioaerosol samples collected from two different biomethanization facilities. The points on the graph represent the sites sampled in each facility. The bars represent the mean value for each condition. The biological replicates are represented by the site sampled during the different visits. The bars represent mean concentrations.

FIG 3

Boxplot representing the number of observed fungal OTUs in bioaerosol samples collected with the Coriolis sampler in biomethanization facilities. The samples were categorized into two groups, centrifugation and filtration, according to the concentration protocol used.

FIG 4

Distribution of fungi detected in the centrifuged samples. All 50 fungi listed were present in 100% of the filtered samples. Each pie represents the percentage of detection of the taxa listed under the centrifuged samples. The taxa listed under each pie chart were present in the percentage of centrifuged samples shown above. Numbers in parentheses represent the number of taxa listed above. C, class; O, order; F, family.

FIG 5

Triboelectric effect on spore pelleting of A. niger N402 (A), P. roquefortii FM164 (B), and A. niger ΔpptA strain (C) after centrifugation for 10 min at 1,700 × g in an Eppendorf fixed-angle rotor. Spores in H₂O (lanes 1 and 2) or 150 mM NaCl (lane 3) were added to a 1.5-ml tube either before (lanes 1 and 3) or after (lane 2) the transfer via a charged 15-ml tube.

FIG 6

Diagram of the fungal cell concentration protocols prior to DNA extraction. The diagram shows each step from sampling to DNA extraction. The red circle shows the hypothesis of fungal cell loss during the disposal of the supernatant in the centrifugation protocol. The SASS 3100 dry sampler was used as a control condition (Fig. S1 to S3). After particle collection on the electret filter, a SASS 3010 particle extractor is required to elute the captured particles in a buffer. The particles are trapped in the filter via electric charges, and the use of the buffer changes the charges of the particles, which are collected in the liquid buffer.