The pulmonary mycobiome—A study of subjects with and without chronic obstructive pulmonary disease

PLOS ONE RESEARCH ARTICLE The pulmonary mycobiome—A study of subjects with and without chronic obstructive pulmonary disease Einar M. H. Martinsen ID1*, Tomas M. L. Eagan1,2, Elise O. Leiten1, Ingvild Haaland1, Gunnar R. Husebø1,2, Kristel S. Knudsen2, Christine Drengenes1,2, Walter Sanseverino3, Andreu Paytuvı́-Gallart3, Rune Nielsen1,2 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Clinical Science, University of Bergen, Bergen, Norway, 2 Department of Thoracic Medicine, Haukeland University Hospital, Bergen, Norway, 3 Sequentia Biotech SL, Barcelona, Spain * Abstract Background OPEN ACCESS Citation: Martinsen EMH, Eagan TML, Leiten EO, Haaland I, Husebø GR, Knudsen KS, et al. (2021) The pulmonary mycobiome—A study of subjects with and without chronic obstructive pulmonary disease. PLoS ONE 16(4): e0248967. https://doi. org/10.1371/journal.pone.0248967 Editor: Sanjay Haresh Chotirmall, Lee Kong Chian School of Medicine, SINGAPORE Received: November 19, 2020 Accepted: March 8, 2021 Published: April 7, 2021 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0248967 Copyright: © 2021 Martinsen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: The dataset and code supporting the conclusions of this article is available in the DRYAD repository. Age and sex are omitted from the metadata due to privacy The fungal part of the pulmonary microbiome (mycobiome) is understudied. We report the composition of the oral and pulmonary mycobiome in participants with COPD compared to controls in a large-scale single-centre bronchoscopy study (MicroCOPD). Methods Oral wash and bronchoalveolar lavage (BAL) was collected from 93 participants with COPD and 100 controls. Fungal DNA was extracted before sequencing of the internal transcribed spacer 1 (ITS1) region of the fungal ribosomal RNA gene cluster. Taxonomic barplots were generated, and we compared taxonomic composition, Shannon index, and beta diversity between study groups, and by use of inhaled steroids. Results The oral and pulmonary mycobiomes from controls and participants with COPD were dominated by Candida, and there were more Candida in oral samples compared to BAL for both study groups. Malassezia and Sarocladium were also frequently found in pulmonary samples. No consistent differences were found between study groups in terms of differential abundance/distribution. Alpha and beta diversity did not differ between study groups in pulmonary samples, but beta diversity varied with sample type. The mycobiomes did not seem to be affected by use of inhaled steroids. Conclusion Oral and pulmonary samples differed in taxonomic composition and diversity, possibly indicating the existence of a pulmonary mycobiome. PLOS ONE | https://doi.org/10.1371/journal.pone.0248967 April 7, 2021 1 / 16 PLOS ONE concerns. Available from: https://doi.org/10.5061/ dryad.w3r2280nz. Funding: The MicroCOPD study was funded by unrestricted grants and fellowships from Helse Vest, GlaxoSmithKline, Bergen Medical Research Foundation, and the Endowment of Timber Merchant A. Delphin and Wife through the Norwegian Medical Association. The MicroCOPD funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Sequentia Biotech SL provided support in the form of salaries for authors Walter Sanseverino and Andreu Paytuvı́-Gallart, but not study design or data collection. All funding of data collection and laboratory analyses were from the MicroCOPD Study. Walter Sanseverino and Andreu Paytuvı́-Gallart both contributed to interpretation of results, and preparation of the manuscript. The specific roles of all authors are articulated in the ‘author contributions’ section. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: Einar M. H. Martinsen, Elise O. Leiten, Ingvild Haaland, Gunnar R. Husebø, Kristel S. Knudsen, and Christine Drengenes declare no conflict of interest. Walter Sanseverino and Andreu Paytuvı́-Gallart are employed at Sequentia Biotech SL. Rune Nielsen reports grants from the Endowment of Timber Merchant A. Delphin and Wife (The Norwegian Medical Association) and grants from GlaxoSmithKline during the conduct of the study, and grants and personal fees from AstraZeneca, grants and personal fees from GlaxoSmithKline, grants and personal fees from Boehringer Ingelheim, and grants from Novartis outside the submitted work. Tomas M. L. Eagan reports grants from Helse Vest (Western Norway Regional Health Authority) during the conduct of the study, and personal fees from Boehringer Ingelheim outside the submitted work. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The pulmonary mycobiome in COPD Introduction Fungi are ubiquitous, and are found in indoor and outdoor environments [1]. Due to its direct communication with surrounding air, the respiratory tract is constantly exposed to fungal spores through inhalation [2]. Healthy airways possess effective removal of such spores through mucociliary clearance and phagocytosis. In contrast, impaired defence mechanisms, use of immunosuppressant, and frequent use of antibiotics probably predispose for increased fungal growth [2], and all factors are quite frequent in chronic obstructive pulmonary disease (COPD). The fungal part of the microbiome, the mycobiome, of the lungs is understudied [3], and only three studies have used next generation sequencing to study the mycobiome of the respiratory tract in COPD particularly [4–6]. Notably, participants in Cui et al.´s study were also HIV infected, and only ten had COPD [4]. The study by Su et al. [5] and Tiew et al. [6] used sputum samples, which are vulnerable to contamination from the high-biomass oral cavity. By contrast, mycobiome studies of other respiratory diseases have evolved rapidly. For instance, a study on asthma patients showed higher fungal burdens in participants receiving corticosteroid therapy [7], while another study has revealed associations between Aspergillus-specific immunisation and bronchiectasis severity [8]. There is clearly a need for large studies of the mycobiome, with a well-characterised COPD disease population and healthy controls. The Bergen COPD Microbiome study (short name “MicroCOPD”) fills this scientific void [9]. Samples were collected from the lower airways of participants with and without COPD using bronchoscopy. The aim of th (...truncated)