Accelerated Lung Function Decline and Mucus-Microbe Evolution in Chronic Obstructive Pulmonary Disease.

RATIONALE: Progressive lung function loss is recognized in COPD; however, no study concurrently evaluates how accelerated lung function decline relates to mucus properties and the microbiome in COPD. OBJECTIVE: Longitudinal assessment of mucus and microbiome changes accompanying accelerated lung function decline in COPD patients. METHODS: Prospective, longitudinal assessment of the London COPD cohort exhibiting the greatest FEV1 decline (n=30; "accelerated decline"; 156 mL/year FEV1 loss) and with no FEV1 decline (n=28; "non-decline"; 49 mL/year FEV1 gain) over time. Lung microbiomes from "paired" sputum (total 116 specimens) were assessed by shotgun metagenomics and corresponding mucus profiles evaluated for biochemical and biophysical properties. RESULTS: Biochemical and biophysical mucus properties are significantly altered in the accelerated decline group. Unsupervised principal component analysis showed clear separation, with mucus biochemistry associated with accelerated decline, while biophysical mucus characteristics contributed to inter-individual variability. When mucus and microbes are considered together, an accelerated decline mucus-microbiome association emerges, characterized by increased mucin (MUC5AC and MUC5B) concentration and the presence of Achromobacter and Klebsiella. As COPD progresses, mucus-microbiome shifts occur, initially characterized by low mucin concentration and transition from viscous to elastic dominance accompanied by the commensals Veillonella, Gemella, Rothia and Prevotella (GOLD A and B) before transition to increased mucus viscosity, mucins, and DNA concentration along with the emergence of pathogenic microorganisms including Haemophilus, Moraxella and Pseudomonas (GOLD E). CONCLUSION: Mucus-microbiome associations evolve over time with accelerated lung function decline, symptom progression and exacerbations affording fresh therapeutic opportunities for early intervention. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Airway "Resistotypes" and Clinical Outcomes in Bronchiectasis.

Rationale: Chronic infection and inflammation shapes the airway microbiome in bronchiectasis. Utilizing whole-genome shotgun metagenomics to analyze the airway resistome provides insight into interplay between microbes, resistance genes, and clinical outcomes. Objectives: To apply whole-genome shotgun metagenomics to the airway microbiome in bronchiectasis to highlight a diverse pool of antimicrobial resistance genes: the "resistome," the clinical significance of which remains unclear. Methods: Individuals with bronchiectasis were prospectively recruited into cross-sectional and longitudinal cohorts (n = 280), including the international multicenter cross-sectional Cohort of Asian and Matched European Bronchiectasis 2 (CAMEB 2) study (n = 251) and two independent cohorts, one describing patients experiencing acute exacerbation and a further cohort of patients undergoing Pseudomonas aeruginosa eradication treatment. Sputum was subjected to metagenomic sequencing, and the bronchiectasis resistome was evaluated in association with clinical outcomes and underlying host microbiomes. Measurements and Main Results: The bronchiectasis resistome features a unique resistance gene profile and increased counts of aminoglycoside, bicyclomycin, phenicol, triclosan, and multidrug resistance genes. Longitudinally, it exhibits within-patient stability over time and during exacerbations despite between-patient heterogeneity. Proportional differences in baseline resistome profiles, including increased macrolide and multidrug resistance genes, associate with shorter intervals to the next exacerbation, whereas distinct resistome archetypes associate with frequent exacerbations, poorer lung function, geographic origin, and the host microbiome. Unsupervised analysis of resistome profiles identified two clinically relevant "resistotypes," RT1 and RT2, the latter characterized by poor clinical outcomes, increased multidrug resistance, and P. aeruginosa. Successful targeted eradication in P. aeruginosa-colonized individuals mediated reversion from RT2 to RT1, a more clinically favorable resistome profile demonstrating reduced resistance gene diversity. Conclusions: The bronchiectasis resistome associates with clinical outcomes, geographic origin, and the underlying host microbiome. Bronchiectasis resistotypes link to clinical disease and are modifiable through targeted antimicrobial therapy.

Microbial Dysregulation of the Gut-Lung Axis in Bronchiectasis.

Rationale: Emerging data support the existence of a microbial "gut-lung" axis that remains unexplored in bronchiectasis. Methods: Prospective and concurrent sampling of gut (stool) and lung (sputum) was performed in a cohort of n = 57 individuals with bronchiectasis and subjected to bacteriome (16S rRNA) and mycobiome (18S Internal Transcribed Spacer) sequencing (total, 228 microbiomes). Shotgun metagenomics was performed in a subset (n = 15; 30 microbiomes). Data from gut and lung compartments were integrated by weighted similarity network fusion, clustered, and subjected to co-occurrence analysis to evaluate gut-lung networks. Murine experiments were undertaken to validate specific Pseudomonas-driven gut-lung interactions. Results: Microbial communities in stable bronchiectasis demonstrate a significant gut-lung interaction. Multibiome integration followed by unsupervised clustering reveals two patient clusters, differing by gut-lung interactions and with contrasting clinical phenotypes. A high gut-lung interaction cluster, characterized by lung Pseudomonas, gut Bacteroides, and gut Saccharomyces, is associated with increased exacerbations and greater radiological and overall bronchiectasis severity, whereas the low gut-lung interaction cluster demonstrates an overrepresentation of lung commensals, including Prevotella, Fusobacterium, and Porphyromonas with gut Candida. The lung Pseudomonas-gut Bacteroides relationship, observed in the high gut-lung interaction bronchiectasis cluster, was validated in a murine model of lung Pseudomonas aeruginosa infection. This interaction was abrogated after antibiotic (imipenem) pretreatment in mice confirming the relevance and therapeutic potential of targeting the gut microbiome to influence the gut-lung axis. Metagenomics in a subset of individuals with bronchiectasis corroborated our findings from targeted analyses. Conclusions: A dysregulated gut-lung axis, driven by lung Pseudomonas, associates with poorer clinical outcomes in bronchiectasis.

The current understanding and future directions for sputum microbiome profiling in chronic obstructive pulmonary disease.

PURPOSE OF REVIEW: Next-generation sequencing (NGS) has deepened our understanding of the respiratory microbiome in health and disease. The number of microbiome studies employing sputum as an airway surrogate has continued to increase over the past decade to include multiple large multicentre and longitudinal studies of the microbiome in chronic obstructive pulmonary disease (COPD). In this review, we summarize the recent advances to our understanding of the bacteriome, virome and mycobiome in COPD. RECENT FINDINGS: Diverse microbiome profiles are reported in COPD. The neutrophilic Haemophilus-predominant bacteriome remains a prominent COPD phenotype, relatively stable over time and during exacerbations. Studies of the virome remain limited but reveal a potential involvement of viruses and bacteriophages particularly during COPD exacerbations and advancing disease severity. Mycobiome signatures, even in stable COPD are associated with poorer clinical outcomes including mortality. SUMMARY: The sputum microbiome in COPD is being increasingly recognized for its clinical relevance, even in the stable state. Future studies integrating microbial kingdoms holistically (i.e. bacterial, viral and fungal) will provide deeper insight into its functionality including the relevance of microbial interactions and effect of treatment on microbiome-associated clinical outcomes.

Human and Porcine Transmission of Clostridioides difficile Ribotype 078, Europe.

Genomic analysis of a diverse collection of Clostridioides difficile ribotype 078 isolates from Ireland and 9 countries in Europe provided evidence for complex regional and international patterns of dissemination that are not restricted to humans. These isolates are associated with C. difficile colonization and clinical illness in humans and pigs.

A high-risk airway mycobiome is associated with frequent exacerbation and mortality in COPD.

INTRODUCTION: The chronic obstructive pulmonary disease (COPD) bacteriome associates with disease severity, exacerbations and mortality. While COPD patients are susceptible to fungal sensitisation, the role of the fungal mycobiome remains uncertain. METHODS: We report the largest multicentre evaluation of the COPD airway mycobiome to date, including participants from Asia (Singapore and Malaysia) and the UK (Scotland) when stable (n=337) and during exacerbations (n=66) as well as nondiseased (healthy) controls (n=47). Longitudinal mycobiome analysis was performed during and following COPD exacerbations (n=34), and examined in terms of exacerbation frequency, 2-year mortality and occurrence of serum specific IgE (sIgE) against selected fungi. RESULTS: A distinct mycobiome profile is observed in COPD compared with controls as evidenced by increased α-diversity (Shannon index; p<0.001). Significant airway mycobiome differences, including greater interfungal interaction (by co-occurrence), characterise very frequent COPD exacerbators (three or more exacerbations per year) (permutational multivariate ANOVA; adjusted p<0.001). Longitudinal analyses during exacerbations and following treatment with antibiotics and corticosteroids did not reveal any significant change in airway mycobiome profile. Unsupervised clustering resulted in two clinically distinct COPD groups: one with increased symptoms (COPD Assessment Test score) and Saccharomyces dominance, and another with very frequent exacerbations and higher mortality characterised by Aspergillus, Curvularia and Penicillium with a concomitant increase in serum sIgE levels against the same fungi. During acute exacerbations of COPD, lower fungal diversity associates with higher 2-year mortality. CONCLUSION: The airway mycobiome in COPD is characterised by specific fungal genera associated with exacerbations and increased mortality.

Aspergillus-Associated Endophenotypes in Bronchiectasis.

Bronchiectasis is a chronic condition of global relevance resulting in permanent and irreversible structural airway damage. Bacterial infection in bronchiectasis is well studied; however, recent molecular studies identify fungi as important pathogens, either independently or in association with bacteria. Aspergillus species are established fungal pathogens in cystic fibrosis and their role is now increasingly being recognized in noncystic fibrosis bronchiectasis. While the healthy airway is constantly exposed to ubiquitously present Aspergillus conidia in the environment, anatomically damaged airways appear more prone to colonization and subsequent infection by this fungal group. Aspergilli possess diverse immunopathological mechanistic capabilities and when coupled with innate immune defects in a susceptible host, such as that observed in bronchiectasis, it may promote a range of clinical manifestations including sensitization, allergic bronchopulmonary aspergillosis, Aspergillus bronchitis, and/or invasive aspergillosis. How such clinical states influence "endophenotypes" in bronchiectasis is therefore of importance, as each Aspergillus-associated disease state has overlapping features with bronchiectasis itself, and can evolve, depending on underlying host immunity from one type into another. Concurrent Aspergillus infection complicates the clinical course and exacerbations in bronchiectasis and therefore dedicated research to better understand the Aspergillus-host interaction in the bronchiectasis airway is now warranted.

Metagenomics Reveals a Core Macrolide Resistome Related to Microbiota in Chronic Respiratory Disease.

Rationale: Long-term antibiotic use for managing chronic respiratory disease is increasing; however, the role of the airway resistome and its relationship to host microbiomes remains unknown.Objectives: To evaluate airway resistomes and relate them to host and environmental microbiomes using ultradeep metagenomic shotgun sequencing.Methods: Airway specimens from 85 individuals with and without chronic respiratory disease (severe asthma, chronic obstructive pulmonary disease, and bronchiectasis) were subjected to metagenomic sequencing to an average depth exceeding 20 million reads. Respiratory and device-associated microbiomes were evaluated on the basis of taxonomical classification and functional annotation including the Comprehensive Antibiotic Resistance Database to determine airway resistomes. Co-occurrence networks of gene-microbe association were constructed to determine potential microbial sources of the airway resistome. Paired patient-inhaler metagenomes were compared (n = 31) to assess for the presence of airway-environment overlap in microbiomes and/or resistomes.Measurements and Main Results: Airway metagenomes exhibit taxonomic and metabolic diversity and distinct antimicrobial resistance patterns. A "core" airway resistome dominated by macrolide but with high prevalence of ß-lactam, fluoroquinolone, and tetracycline resistance genes exists and is independent of disease status or antibiotic exposure. Streptococcus and Actinomyces are key potential microbial reservoirs of macrolide resistance including the ermX, ermF, and msrD genes. Significant patient-inhaler overlap in airway microbiomes and their resistomes is identified where the latter may be a proxy for airway microbiome assessment in chronic respiratory disease.Conclusions: Metagenomic analysis of the airway reveals a core macrolide resistome harbored by the host microbiome.

Respiratory Mycoses in COPD and Bronchiectasis.

Chronic obstructive pulmonary disease (COPD) and bronchiectasis represent chronic airway diseases associated with significant morbidity and mortality. Bacteria and viruses are commonly implicated in acute exacerbations; however the significance of fungi in these airways remains poorly defined. While COPD and bronchiectasis remain recognized risk factors for the occurrence of Aspergillus-associated disease including chronic and invasive aspergillosis, underlying mechanisms that lead to the progression from colonization to invasive disease remain uncertain. Nonetheless, advances in molecular technologies have improved our detection, identification and understanding of resident fungi characterizing these airways. Mycobiome sequencing has revealed the complex varied and myriad profile of airway fungi in COPD and bronchiectasis, including their association with disease presentation, progression, and mortality. In this review, we outline the emerging evidence for the clinical importance of fungi in COPD and bronchiectasis, available diagnostic modalities, mycobiome sequencing approaches and association with clinical outcomes.

Environmental fungal sensitisation associates with poorer clinical outcomes in COPD.

INTRODUCTION: Allergic sensitisation to fungi such as Aspergillus are associated to poor clinical outcomes in asthma, bronchiectasis and cystic fibrosis; however, clinical relevance in COPD remains unclear. METHODS: Patients with stable COPD (n=446) and nondiseased controls (n=51) were prospectively recruited across three countries (Singapore, Malaysia and Hong Kong) and screened against a comprehensive allergen panel including house dust mites, pollens, cockroach and fungi. For the first time, using a metagenomics approach, we assessed outdoor and indoor environmental allergen exposure in COPD. We identified key fungi in outdoor air and developed specific-IgE assays against the top culturable fungi, linking sensitisation responses to COPD outcomes. Indoor air and surface allergens were prospectively evaluated by metagenomics in the homes of 11 COPD patients and linked to clinical outcome. RESULTS: High frequencies of sensitisation to a broad range of allergens occur in COPD. Fungal sensitisation associates with frequent exacerbations, and unsupervised clustering reveals a "highly sensitised fungal predominant" subgroup demonstrating significant symptomatology, frequent exacerbations and poor lung function. Outdoor and indoor environments serve as important reservoirs of fungal allergen exposure in COPD and promote a sensitisation response to outdoor air fungi. Indoor (home) environments with high fungal allergens associate with greater COPD symptoms and poorer lung function, illustrating the importance of environmental exposures on clinical outcomes in COPD. CONCLUSION: Fungal sensitisation is prevalent in COPD and associates with frequent exacerbations representing a potential treatable trait. Outdoor and indoor (home) environments represent a key source of fungal allergen exposure, amenable to intervention, in "sensitised" COPD.

Distinct "Immunoallertypes" of Disease and High Frequencies of Sensitization in Non-Cystic Fibrosis Bronchiectasis.

RATIONALE: Allergic sensitization is associated with poor clinical outcomes in asthma, chronic obstructive pulmonary disease, and cystic fibrosis; however, its presence, frequency, and clinical significance in non-cystic fibrosis bronchiectasis remain unclear. OBJECTIVES: To determine the frequency and geographic variability that exists in a sensitization pattern to common and specific allergens, including house dust mite and fungi, and to correlate such patterns to airway immune-inflammatory status and clinical outcomes in bronchiectasis. METHODS: Patients with bronchiectasis were recruited in Asia (Singapore and Malaysia) and the United Kingdom (Scotland) (n = 238), forming the Cohort of Asian and Matched European Bronchiectasis, which matched recruited patients on age, sex, and bronchiectasis severity. Specific IgE response against a range of common allergens was determined, combined with airway immune-inflammatory status and correlated to clinical outcomes. Clinically relevant patient clusters, based on sensitization pattern and airway immune profiles ("immunoallertypes"), were determined. MEASUREMENTS AND MAIN RESULTS: A high frequency of sensitization to multiple allergens was detected in bronchiectasis, exceeding that in a comparator cohort with allergic rhinitis (n = 149). Sensitization was associated with poor clinical outcomes, including decreased pulmonary function and more severe disease. "Sensitized bronchiectasis" was classified into two immunoallertypes: one fungal driven and proinflammatory, the other house dust mite driven and chemokine dominant, with the former demonstrating poorer clinical outcome. CONCLUSIONS: Allergic sensitization occurs at high frequency in patients with bronchiectasis recruited from different global centers. Improving endophenotyping of sensitized bronchiectasis, a clinically significant state, and a "treatable trait" permits therapeutic intervention in appropriate patients, and may allow improved stratification in future bronchiectasis research and clinical trials.

The Mycobiome in Health and Disease: Emerging Concepts, Methodologies and Challenges.

Fungal disease is an increasingly recognised global clinical challenge associated with high mortality. Early diagnosis of fungal infection remains problematic due to the poor sensitivity and specificity of current diagnostic modalities. Advances in sequencing technologies hold promise in addressing these shortcomings and for improved fungal detection and identification. To translate such emerging approaches into mainstream clinical care will require refinement of current sequencing and analytical platforms, ensuring standardisation and consistency through robust clinical benchmarking and its validation across a range of patient populations. In this state-of-the-art review, we discuss current diagnostic and therapeutic challenges associated with fungal disease and provide key examples where the application of sequencing technologies has potential diagnostic application in assessing the human 'mycobiome'. We assess how ready access to fungal sequencing may be exploited in broadening our insight into host-fungal interaction, providing scope for clinical diagnostics and the translation of emerging mycobiome research into clinical practice.

Whole-Genome Sequencing of Aspergillus terreus Species Complex.

Aspergillus terreus species complex is an opportunistic fungal pathogen increasingly implicated in invasive infection, as well as chronic respiratory disease. Currently, an understanding of A. terreus pathogenicity is impeded by a limited number of whole-genome sequences of this fungal pathogen. We here describe a high-quality whole-genome assembly of European A. terreus clinical isolate M6925, derived by single-molecule real-time sequencing with short-read polishing.

Bronchiectasis and cough: An old relationship in need of renewed attention.

Bronchiectasis is an increasingly recognised respiratory condition with limited therapeutic options and a complex spectrum of clinical manifestations that invariably includes chronic cough. As the primary presentation of bronchiectasis in most cases, chronic cough and its mechanistic underpinnings are of central importance but remain poorly understood in this setting. Bronchiectasis is also increasingly identified as an underlying cause of chronic cough highlighting the interrelationship between the two conditions that share overlapping clinical features. Several therapeutic approaches have illustrated positive effects on bronchiectasis-associated cough, however, more focused treatment of heterogeneous cough subtypes may yield better outcomes for patients. A current challenge is the identification of bronchiectasis and cough endophenotypes that may allow improved patient stratification and more targeted therapeutic matching of the right treatment to the right patient. Here we discuss the complex disease phenotypes of bronchiectasis and their interrelationship with cough while considering current and emerging treatment options. We discuss some key cough promoters in bronchiectasis including infection, allergy and immune dysfunction.

MycopathologiaGENOMES: The New 'Home' for the Publication of Fungal Genomes.

The wider availability of information on genomes has become essential for future advances in fungal biology, pathogenesis and epidemiology, and for the discovery of new drugs and diagnostics. MycopathologiaGENOMES is designed for the rapid publication of new genomes of human and animal pathogenic fungi using a checklist-based, standardized format.

Optimisation and Benchmarking of Targeted Amplicon Sequencing for Mycobiome Analysis of Respiratory Specimens.

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway.

Immunological corollary of the pulmonary mycobiome in bronchiectasis: the CAMEB study.

Understanding the composition and clinical importance of the fungal mycobiome was recently identified as a key topic in a "research priorities" consensus statement for bronchiectasis.Patients were recruited as part of the CAMEB study: an international multicentre cross-sectional Cohort of Asian and Matched European Bronchiectasis patients. The mycobiome was determined in 238 patients by targeted amplicon shotgun sequencing of the 18S-28S rRNA internally transcribed spacer regions ITS1 and ITS2. Specific quantitative PCR for detection of and conidial quantification for a range of airway Aspergillus species was performed. Sputum galactomannan, Aspergillus specific IgE, IgG and TARC (thymus and activation regulated chemokine) levels were measured systemically and associated to clinical outcomes.The bronchiectasis mycobiome is distinct and characterised by specific fungal genera, including Aspergillus, Cryptococcus and ClavisporaAspergillus fumigatus (in Singapore/Kuala Lumpur) and Aspergillus terreus (in Dundee) dominated profiles, the latter associating with exacerbations. High frequencies of Aspergillus-associated disease including sensitisation and allergic bronchopulmonary aspergillosis were detected. Each revealed distinct mycobiome profiles, and associated with more severe disease, poorer pulmonary function and increased exacerbations.The pulmonary mycobiome is of clinical relevance in bronchiectasis. Screening for Aspergillus-associated disease should be considered even in apparently stable patients.

Inhaled nanomaterials and the respiratory microbiome: clinical, immunological and toxicological perspectives.

Our development and usage of engineered nanomaterials has grown exponentially despite concerns about their unfavourable cardiorespiratory consequence, one that parallels ambient ultrafine particle exposure from vehicle emissions. Most research in the field has so far focused on airway inflammation in response to nanoparticle inhalation, however, little is known about nanoparticle-microbiome interaction in the human airway and the environment. Emerging evidence illustrates that the airway, even in its healthy state, is not sterile. The resident human airway microbiome is further altered in chronic inflammatory respiratory disease however little is known about the impact of nanoparticle inhalation on this airway microbiome. The composition of the airway microbiome, which is involved in the development and progression of respiratory disease is dynamic, adding further complexity to understanding microbiota-host interaction in the lung, particularly in the context of nanoparticle exposure. This article reviews the size-dependent properties of nanomaterials, their body deposition after inhalation and factors that influence their fate. We evaluate what is currently known about nanoparticle-microbiome interactions in the human airway and summarise the known clinical, immunological and toxicological consequences of this relationship. While associations between inhaled ambient ultrafine particles and host immune-inflammatory response are known, the airway and environmental microbiomes likely act as intermediaries and facilitate individual susceptibility to inhaled nanoparticles and toxicants. Characterising the precise interaction between the environment and airway microbiomes, inhaled nanoparticles and the host immune system is therefore critical and will provide insight into mechanisms promoting nanoparticle induced airway damage.