European Respiratory Society
The Lung Microbiome

Studying the lung microbiome requires a specialist approach to sampling, laboratory techniques and statistical analysis. This Monograph introduces the techniques used and discusses how respiratory sampling, 16S rRNA gene sequencing, metagenomics and the application of ecological theory can be used to examine the respiratory microbiome. It examines the different components of the respiratory microbiome: viruses and fungi, in addition to the more frequently studied bacteria. It also considers a range of contexts from the paediatric microbiome and how this develops to disease of all ages including asthma and chronic obstructive pulmonary disease, chronic suppurative lung diseases, interstitial lung diseases, acquired pneumonias, transplantation, cancer and HIV, and the interaction of the respiratory microbiome and the environment.

  • ERS Monograph
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    Robert P. Dickson, Dept of Internal Medicine, University of Michigan, 6220 MSRB III/SPC 5642, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5642, USA. E-mail:

    The human respiratory tract is an enormous and spatially heterogeneous ecosystem, comprising hundreds of kilometres of airways and a surface area 30 times greater than that of the skin. It extends from the microbe-dense pharynx to the microbe-sparse alveoli, and harbours a dynamic balance of microbial immigration and elimination. Thus, sampling of the lung microbiota requires special considerations: anatomical, physiological, microbiological and procedural. In this chapter, we discuss commonly used techniques for sampling the lung microbiota, review the rationale and evidence behind common approaches, and provide recommendations regarding the crucial issue of pharyngeal and sequencing contamination.

    Cite as: Dickson RP, Cox MJ. Sampling. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 1–17 [].

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    Gisli G. Einarsson, Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, UK. E-mail:

    The resident airway microbiota plays an important role in both health and complex respiratory disorders, including CF, bronchiectasis, COPD and asthma which are characterised by recurrent lung infections and an alteration in innate and adaptive immunity. Recurrent cycles of infection and bronchial inflammation progressively lead to lung injury and long-term damage, and it is therefore imperative to better understand the aetiology of the infective micro-organisms to allow appropriate treatment steps to be implemented. The most prevalent pathogenic bacteria detected in the airways of people with chronic respiratory disease are Haemophilus influenzae, Pseudomonas aeruginosa, Burkholderia cepacia complex, Streptococcus pneumoniae, Staphylococcus aureus and Moraxella catarrhalis, although a number of other potentially pathogenic micro-organisms (PPM) have been proposed to play a role during infective episodes. Variations in the sampling techniques and detection methods used can influence the isolation rates of PPM; it is therefore important to assess the most informative approaches used to identify all clinically relevant micro-organisms. This chapter describes various culture-dependent and -independent methodologies that are currently being employed in an effort to detect and identify the resident microbiota in clinical specimens. It also considers how these methodologies may affect clinical decision-making in the future.

    Cite as: Einarsson GG, Boutin S. Techniques: culture, identification and 16S rRNA gene sequencing. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 18–34 [].

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    Themoula Charalampous, Bob Champion Research and Educational Building, University of East Anglia, Norwich Research Park, Colney Lane, Norwich NR4 7UQ, UK. E-mail:

    Metagenomics is, by definition, the direct identification and characterisation of all genomes within a sample. When metagenomics is used to characterise pathogens directly from clinical samples, it is then referred to as clinical metagenomics. Clinical metagenomics has the potential to replace conventional methodologies, such as culture, for the diagnosis of infection due to its unbiased approach and the potential for a rapid turnaround time to results. An efficient metagenomics-based pipeline needs to be comprehensive, rapid and cost-effective, and will include: 1) sample preparation, including pathogen DNA enrichment and/or host DNA depletion strategies, 2) library preparation and sequencing, and 3) rapid data analysis. Each of these steps will be discussed (focusing on bacterial infections), with the aim of producing high-quality data while reducing cost and turnaround time. We review the literature on clinical metagenomics for diagnostic and epidemiological applications, and discuss the challenges in applying clinical metagenomics methodologies.

    Cite as: Charalampous T, Kay GL, O'Grady J. Applying clinical metagenomics for the detection and characterisation of respiratory infections. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 35–49 [].

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    Bärbel U. Foesel, Research Unit Comparative Microbiome Analysis, Helmholtz Center Munich, Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany. E-mail:

    The persisting dogma that the healthy human lung is sterile led to neglect of the lung microbiome for a long time and it is only recently that it has been acknowledged as an issue. Culture-independent methods have shown that a diverse microbial community is present in the lung of healthy individuals and that it harbours important functional traits. However, as in the whole field of human microbiome research, empirical work is lagging far behind the overwhelming amounts of data produced as a result of advances in NGS techniques. Adaptations of classical models and theories from ecology and evolution might help to close this gap and provide the basis for directed, theory-driven (lung) microbiome research in health and disease. In this chapter, we will provide some ecological theories that are widely applied in microbial ecology and discuss their relevance for future lines of research in lung microbiome research.

    Cite as: Foesel BU, Pfeiffer S, Raj ACD, et al. Applying ecological theories in research: lessons learned from microbial ecology and evolution? In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 50–66 [].

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    Debby Bogaert, Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK. E-mail:

    Microbial colonisation of mucosal surfaces starts at birth and diversifies within the first months of life. This process is driven mainly by niche specificity but also by early environmental exposures, ultimately shaping the composition of the microbiome. The early-life microbiota probably performs important functions, including respiratory tract morphogenesis, pathogen resistance and immune-system development. Microbial dysbiosis or imbalance, instigated by altered exposure to lifestyle factors including antimicrobial treatment, is coupled to a dysregulated immune response, possibly leading to microbial overgrowth, infection and inflammation. Shifts in microbial communities have been associated with the early stages of (respiratory) diseases including acute infection, chronic wheeze and asthma, causing a paradigm shift in our current understanding of disease pathogenesis. Mechanistic insights obtained from animal, in vitro and computational models are slowly starting to highlight key host–microbiome–environment interactions contributing to disease. In the future, a systems science approach integrating microbiome data with host and environment characteristics may contribute to novel interventions to better prevent, diagnose and treat respiratory diseases.

    Cite as: de Koff EM, Pattaroni C, Marsland BJ, et al. The early-life microbiome: the key to respiratory health? In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 67–87 [].

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    Laurence Delhaes, Laboratory of Parasitology–Mycology, Centre Hospitalier Universitaire de Bordeaux, INSERM, U1045, 33000 Bordeaux, France. E-mail:

    The Fungi Kingdom includes about 5 million species of yeasts, moulds and the more familiar mushrooms, all producing spores that are present in the human environment. A large proportion of respiratory and endemic fungal infections start with spore inhalation. Inhaled fungi affect human health related to the host immune status and any underlying pulmonary disorder such as primary infections, progressive opportunistic infections, chronic infections and allergic disease. Usually, inhalation of fungi results in transient colonisation of the respiratory tract, as they are cleared by an intact respiratory tract and immune system. However, when the patient has a chronic respiratory disorder (CRD) or is immunocompromised, fungal colonisation, notably with Aspergillus spp., may lead to allergic disease or infection. Whatever persists defines the respiratory mycobiome. This chapter focuses on the mycobiome in the context of CRDs and reviews the patient populations at risk, advances in the laboratory tools used to diagnose fungal respiratory disease ranging from conventional methods (culturomics) to NGS approaches (targeted metagenomics), and the concept of the “fungal exposome”.

    Cite as: Vandenborght L-E, Enaud R, Coron N, et al. From culturomics to metagenomics: the mycobiome in chronic respiratory diseases. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 88–118 [].

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    William G. Flight, Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK. E-mail:

    A wide variety of viruses make up the virome, the compartment of the microbiome that includes bacteriophages and commensal eukaryotic viruses, as well as common pathogenic viruses such as influenza virus, rhinovirus and RSV. Susceptibility to viruses varies by age, immunocompetence and the presence of underlying respiratory disorders. Viral RTIs may cause dysbiosis of the lung microbiome, and conversely the make-up of the lung microbiome may affect susceptibility to and severity of a viral infection. Bacteriophages play a key role in modulating the bacterial microbiota of the lung and are a potential avenue of therapy for lung infection, especially in the context of antimicrobial resistance. The study of the lung virome and virus–bacteria interactions at the community level remains in its infancy but is an exciting area of future research with the potential for therapeutic benefits in respiratory disease.

    Cite as: Flight WG, Turkington CJR, Clokie MRJ. Viruses. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 119–139 [].

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    K.F. Chung, National Heart & Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, UK. E-mail:

    With the advent of 16S rRNA gene sequencing, a respiratory microbiome has been described in healthy lungs. The microbiota can be modulated by various environmental factors, including diet, antibiotics and early-life microbial and viral exposures that could influence both the inception and progress of allergies and of chronic obstructive lung diseases. The lung microbiota is dysregulated in asthma and COPD, with a reduced diversity and community composition that has been linked to disease severity and inflammatory phenotypes. In COPD, the microbiota has been associated with recurrent exacerbations and airflow obstruction. There is a relative abundance of Haemophilus or Moraxella spp. at the onset of a COPD exacerbation, while specific bacteria have been associated with the inflammatory phenotype of the exacerbation. The airway microbiome is likely to influence the host immune response and contribute to disease pathogenesis in chronic airways disease by mechanisms that need to be better understood. Targeted therapies addressing the microbiome could then be devised.

    Cite as: Chung KF, Huffnagle GB, Huang YJ. Obstructive airways disease: potential pathogenetic roles. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 140–157 [].

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    Yvonne J. Huang, 6301 MSRB3/SPC5642, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-5642, USA. E-mail:

    Study of the lung microbiome in disease began in CF, the cardinal condition of impaired mucociliary airway clearance due to mutations in a transmembrane ion channel protein regulating epithelial fluid transport. Thick mucus serving as a nidus for progressive airway infection is the hallmark complication in bronchiectasis, which, untreated, causes progressive lung function decline. Remarkable progress has been made in understanding the microbiology of airway infection in CF, furthered by the recent application of advanced molecular and cultivation tools. Findings have highlighted the complex interactions among the airway microbiota, from birth through adulthood, that shape bronchiectasis development, progression and clinical outcomes in CF. Such approaches are now being adopted to understand the lung microbiome in non-CF aetiologies of bronchiectasis and similarly complex interactions among the microbiota, the host and treatment effects that together shape clinical outcomes. Applying an ecological perspective to these areas of study promises greater insights that will allow for more precise therapeutic strategies in the future.

    Cite as: Rogers G, Huang YJ. Chronic suppurative lung disease: cystic fibrosis and non-cystic fibrosis bronchiectasis. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 158–172 [].

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    Philip L. Molyneaux, Fibrosis Research Group, National Heart and Lung Institute, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK. E-mail:

    Interstitial lung diseases (ILDs) occur in response to a variety of triggers, both environmental and immune, and result in an exaggerated or unregulated inflammatory or fibrotic response within the lung interstitium. While there may be a clear trigger identified in some cases, for the majority this remains elusive. In this chapter, we discuss the growing evidence that the bacterial communities of the lower airways may play a role in the pathogenesis and progression of ILDs. In idiopathic pulmonary fibrosis, dysbiosis and bacterial burden may act as persistent stimuli driving repetitive alveolar injury, while in sarcoidosis there is intriguing evidence of a direct role for microbes in the immunopathogenesis of the disease. In other ILDs, including hypersensitivity pneumonitis and those related to an underlying connective tissue disease, the microbiome may act in synergy with a dysregulated immune system. We discuss the observational and associative work in humans and the emerging studies in pre-clinical models, which have begun to elucidate a relationship between the lung microbiota and alveolar inflammation and fibrogenesis.

    Cite as: O'Dwyer DN, Moore BB, Molyneaux PL. Interstitial lung disease. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 173–187 [].

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    Lieuwe Bos, Dept of Respiratory Medicine, Intensive Care, Amsterdam UMC Location AMC, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail:

    Pneumonia has long been regarded as a disease with a relatively simple pathogenesis: a pathogen enters the LRT and starts growing in an otherwise sterile environment. With new insights into the pulmonary microbiome, pneumonia could be defined in ecological terms as “the acute loss of biodiversity due to the overgrowth of a single or several pathogenic micro-organisms causing lung inflammation and damage”. Understanding the ecological perspective is important when dealing with pneumonia patients with pre-existing problems; underlying colonisation can frequently be detected by traditional microbiological methods, and more subtle aberrations in the microbiome may be important, especially in immunocompromised patients and in those with chronic pulmonary diseases. Intubation and mechanical ventilation result in a rapid change in the pulmonary microbiome, and these changes are associated with ventilator-associated pneumonia.

    Cite as: Bos LDJ, Rylance J, Gordon SB. The lung bacterial microbiome in community-acquired and nosocomial pneumonia. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 188–194 [].

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    Laurent P. Nicod, Division of Pulmonology, Lausanne University Hospital, Rue du Bugnon 46, BH-10.664, CH 1011 Lausanne, Switzerland. E-mail:

    Lung microbial communities and host immunity have evolved in a coordinated manner. A major challenge for current research is to characterise the clinical implications of a breakdown of this homeostatic crosstalk. In this chapter, we sequentially discuss host–microbiota interactions in lung transplantation, cancer and HIV infection, considering expected commonalities in the effects of underlying active immunosuppression, chemotherapy, radiotherapy and virus-induced immunodeficiency. Exploring the interplay between microbes and host immunity in immunocompromised conditions is intrinsically important for these groups of patients but is also very likely to help increase our understanding of corresponding interactions in an immunocompetent context, such as in major respiratory diseases.

    Cite as: Bernasconi E, Aubert J-D, Koutsokera A, et al. Compromised immunity: transplantation, cancer and HIV. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 195–215 [].

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    Pirkka V. Kirjavainen, Environmental Health Unit, National Institute for Health and Welfare, PO Box 95, FI-70701 Kuopio, Finland. E-mail:

    From ancient times, we have adapted to survive among the millions of microbes that surround us and that we unavoidably inhale, ingest and touch. In the modern, urbanised environment with increasing time spent indoors, we encounter less biodiversity and consequently fewer different microbes than our immune systems have evolved to encounter. This may result in insufficient signals needed to train the immune system away from hypersensitivity, which may explain the rise in asthma and allergy prevalence. While a causal relationship between environmental microbial exposures and asthma and allergies remains to be proven, it is strongly supported by experimental studies. Indoors, the abundance of microbes sourced from the human microbiome and potential human pathogens are characteristically enriched and some, such as moulds growing in damp indoor structures, are associated with an increased risk of asthma. In contrast, asthma- and allergy-protective environments such as farms with livestock and homes with children or pets are associated with a more diverse microbiome. With the exception of a few asthma-predisposing potential respiratory pathogens, specific microbial taxa or properties explaining the protective or predisposing associations are poorly understood. To date, high richness is the most consistent determinant of the protective microbial exposure.

    Cite as: Kirjavainen PV, Hyytiäinen H, Täubel M. The environmental microbiota and asthma. In: Cox MJ, Ege MJ, von Mutius E, eds. The Lung Microbiome (ERS Monograph). Sheffield, European Respiratory Society, 2019; pp. 216–239 [].

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