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Microbiomes have been the focus of a substantial research effort in the last decades. The composition of microbial populations is normally determined by comparing DNA sequences sampled from those populations with the sequences stored in genomic databases. Therefore, the amount of information available in databanks should be expected to constrain the accuracy of microbiome analyses. Albeit normally ignored in microbiome studies, this constraint could severely compromise the reliability of microbiome data. To test this hypothesis, we generated virtual bacterial populations that exhibit the ecological structure of real-world microbiomes. Confronting the analyses of virtual microbiomes with their original composition revealed critical issues in the current approach to characterizing microbiomes, issues that were empirically confirmed by analyzing the microbiome of Galleria mellonella larvae. To reduce the uncertainty of microbiome data, the effort in the field must be channeled towards significantly increasing the amount of available genomic information and optimizing the use of this information., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Serrano-Antón 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.)

Background: Few studies have examined the stability of the pulmonary mycobiome. We report longitudinal changes in the oral and pulmonary mycobiome of participants with and without COPD in a large-scale bronchoscopy study (MicroCOPD)., Methods: Repeated sampling was performed in 30 participants with and 21 without COPD. We collected an oral wash (OW) and a bronchoalveolar lavage (BAL) sample from each participant at two time points. The internal transcribed spacer 1 region of the ribosomal RNA gene cluster was PCR amplified and sequenced on an Illumina HiSeq sequencer. Differences in taxonomy, alpha diversity, and beta diversity between the two time points were compared, and we examined the effect of intercurrent antibiotic use., Results: Sample pairs were dominated by Candida. We observed less stability in the pulmonary taxonomy compared to the oral taxonomy, additionally emphasised by a higher Yue-Clayton measure in BAL compared to OW (0.69 vs 0.22). No apparent effect was visually seen on taxonomy from intercurrent antibiotic use or participant category. We found no systematic variation in alpha diversity by time either in BAL (p-value 0.16) or in OW (p-value 0.97), and no obvious clusters on bronchoscopy number in PCoA plots. Pairwise distance analyses showed that OW samples from repeated sampling appeared more stable compared to BAL samples using the Bray-Curtis distance metric (p-value 0.0012), but not for Jaccard., Conclusion: Results from the current study propose that the pulmonary mycobiome is less stable than the oral mycobiome, and neither COPD diagnosis nor intercurrent antibiotic use seemed to influence the stability., 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, Gunnar Husebø, and Solveig Tangedal declare no conflict of interest. Walter Sanseverino and Andreu Paytuví-Gallart are employed at Sequentia Biotech SL. Tomas M. L. Eagan reports lecture fees from Boehringer and AstraZeneca, and grants from GlaxoSmithKline outside the submitted work. Harald G. Wiker reports being head of the educational programme for medicine at the University of Bergen. Kristel S. Knudsen reports lecture fees from Boehringer Ingelheim and Roche. Rune Nielsen reports grants from Boehringer Ingelheim, GlaxoSmithKline, AstraZeneca, and the Timber Merchant Delphins Endowment, in addition to lecture fees from GlaxoSmithKline and AstraZeneca. Rune Nielsen also reports being unpaid member of The Norwegian Respiratory Society, unpaid GOLD national delegate, unpaid ERS national delegate, and paid member of the Reference group for new Norwegian guidelines for COPD (The Norwegian Directorate of Health). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: 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., 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 Paytuví-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.