Centre de Recherche de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec, Quebec City, Quebec, Canada.
Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Quebec City, Quebec, Canada.
Appl Environ Microbiol. 2019 Apr 18;85(9). doi: 10.1128/AEM.02941-18. Print 2019 May 1.
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 and 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. 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.
由于所使用的方法等原因，在确定真菌暴露与健康影响之间的直接联系方面存在局限性。培养方法忽略了空气中真菌的非活性/不可培养部分。分子方法可以更好地了解微生物群落对环境健康的影响。然而，将这些技术应用于生物气溶胶，特别是真菌细胞时，存在挑战。本研究表明，使用湿采样器从空气中回收样品时，真菌细胞会丢失，本研究旨在创建和测试一种改进的过滤浓缩法，以在 DNA 提取前浓缩霉菌孢子。使用新技术获得的结果表明，使用定量 PCR 从样品中回收的真菌 DNA 多达 3 个数量级。MiSeq 测序方法显示，根据所使用的方法，多样性图谱不同。具体来说，在所测试的 19 个真菌科中，有 8 个真菌科在离心和过滤的样品中丰度不同。在实验室环境中进行的一项实验表明，对于 和 菌株，离心过程中也会发生相同的孢子丢失。我们认为，这项工作有助于确定和解决在处理空气样本过程中（包括离心步骤）真菌细胞丢失的问题，并提出了一种替代方法，以更准确地评估真菌暴露和多样性。这项工作揭示了一个重要问题，即在使用液体培养基和离心来浓缩空气颗粒以在 DNA 提取之前从空气样本中回收真菌孢子时，会发生真菌孢子丢失。我们提供的证据表明，这种损失会影响气溶胶中真菌的总体多样性，并且一些分类群比其他分类群受到的影响更大。此外，实验室实验证实了在野外采样过程中获得的环境结果。本工作中描述的过滤方案提供了对气溶胶中真菌多样性的更好描述，应在真菌气溶胶研究中使用。
