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Fungal and Bacterial Diversity of Airway Microbiota in Adults with Cystic Fibrosis: Concordance Between Conventional Methods and Ultra-Deep Sequencing, and Their Practical use in the Clinical Laboratory

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Abstract

Given the complexity of the airway microbiota in the respiratory tract of cystic fibrosis (CF) patients, it seems crucial to compile the most exhaustive and exact list of the microbial communities inhabiting CF airways. The aim of the present study was to compare the bacterial and fungal diversity of sputa from adult CF patients during non-exacerbation period by culture-based and molecular methods, and ultra-deep-sequencing (UDS). Sputum samples from four CF patients were cultured and analysed by DNA extractions followed by terminal restriction fragment length polymorphism analysis through resolution of bacterial ribosomal gene (rDNA) fragments, and cloning plus sequencing of part of fungal rRNA genes. These approaches were compared with UDS method targeting 16S rDNA gene and the internal transcribed spacer (ITS) 2 region of rDNA. A total of 27 bacterial and 18 fungal genera were detected from the four patients. Five (18%) and 3 (16%) genera were detected by culture for bacteria and fungi, respectively, 9 (33%) and 3 (16%) by first generation sequencing (FGS) methods, and 26 (96%) and 18 (100%) by UDS. The mean number of genera detected by UDS per patient was statistically higher than by culture or FGS methods. Patients with severe airway disease as assessed by standard spirometry exhibited a reduced fungal and bacterial diversity. UDS approach evaluates more extensively the diversity of fungal and bacterial flora compared with cultures. However, it currently remains difficult to routinely use UDS mainly because of the lack of standardization, and the current cost of this method.

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Acknowledgements

We would like to thank Damian Rivett and Jean-Philippe Barnier for their technical assistance, and Genoscreen Society for their critical and technical support.

Funding

This work was supported in part by the Assistance Publique-Hôpitaux de Paris (public sector, bourse de mobilité), and Pfizer France Pharmaceutical Division. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. L. Delhaes has received research grants from the French Ministry of Health and Research (PHRC N°2006/1902), Centre Hospitalier Régional Universitaire de Lille, the association “Vaincre la Mucoviscidose” (MucoFong and Mucofong-ATF N8 2006/351), and Pfizer France Pharmaceutical Division (Nu 2006/158).

Author information

Author notes

  1. Franziska A. Stressmann

    Present address: Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Chemical Analytics and Biogeochemistry, Müggelseedamm 310, 12587, Berlin, Germany

  2. Laurence Delhaes

    Present address: Laboratoire de Parasitologie – Mycologie, CHU de Bordeaux, Bordeaux, France

Authors and Affiliations

  1. Unité de Parasitologie-Mycologie, Département de Virologie, Bactériologie-Hygiène, Parasitologie-Mycologie, DHU VIC, CHU Henri Mondor, AP-HP, 51, Avenue du Maréchal De Lattre de TassignyCréteil, 94010, Créteil, France

    Françoise Botterel & Cécile Angebault

  2. Faculté de Médecine de Créteil, EA Dynamyc 7380 UPEC, ENVA, Créteil, France

    Françoise Botterel, Cécile Angebault & Jean-Marc Costa

  3. Département de Biopathologie, Service de Génétique, Institut Gustave Roussy, Villejuif, France

    Odile Cabaret

  4. Laboratoire Cerba, Saint-Ouen-l’Aumône, France

    Jean-Marc Costa

  5. Département de Microbiologie, CHRU de Lille, Lille, France

    Frédéric Wallet

  6. Service de Pneumologie, CHRU de Lille, Lille, France

    Benoit Wallaert & Laurence Delhaes

  7. Molecular Microbiology, King’s College of London, London, UK

    Kenneth Bruce

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Contributions

Authors’ Contributions

FW, BW and LD collected sputum samples and clinical data. FB, OC, JMC, FS and LD prepared sputum samples for molecular analysis including UDS. CA performed the statistical analyses including that of UDS. FB and LD contributed to study design. FB was in charge of all the molecular approaches. FB and CA wrote the manuscript with significant contributions of co-authors, especially KB and LD. All authors read and gave approval on the final manuscript.

Corresponding author

Correspondence to Françoise Botterel.

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Conflict of interest

FB received grants from Astellas, Pfizer, and payment for lectures from Merck and Gilead.

Ethical Approval

All procedures performed in this study involving human participants were in compliance with the ethical standards of the institutional research committee (Comité de Protection des Personnes of Lille University Hospital, CPP 06/84) and with the 1964 Helsinki Declaration and its latest amendments.

Informed Consent

Informed consent forms were obtained from all individual participants included in the study.

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Botterel, F., Angebault, C., Cabaret, O. et al. Fungal and Bacterial Diversity of Airway Microbiota in Adults with Cystic Fibrosis: Concordance Between Conventional Methods and Ultra-Deep Sequencing, and Their Practical use in the Clinical Laboratory. Mycopathologia 183, 171–183 (2018). https://doi.org/10.1007/s11046-017-0185-x

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  • DOI: https://doi.org/10.1007/s11046-017-0185-x

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