Metagenomics reveals a core macrolide resistome related to microbiota in chronic respiratory disease

This is the author accepted manuscript. The final version is available from the publisher via the DOI in this recordRationale: 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 Objective: To evaluate airway resistomes, and, relate them to host and environmental microbiomes using ultra-deep metagenomic shotgun sequencing Methods: Airway specimens from n=85 individuals with and without chronic respiratory disease (severe asthma, COPD and bronchiectasis) were subjected to metagenomic sequencing to an average depth exceeding twenty million reads. Respiratory and device-associated microbiomes were evaluated based on taxonomical classification and functional annotation including the Comprehensive Antibiotic Resistance Database (CARD) 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. 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 β-lactam, fluoroquinolone and tetracycline resistance genes exist, 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. Conclusion: Metag

Sex Steroids Induce Membrane Stress Responses and Virulence Properties in Pseudomonas aeruginosa.

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection

Microbial Dysregulation of the Gut-Lung Axis in Bronchiectasis

This is the author accepted manuscript. The final version is available from the American Thoracic Society via the DOI in this recordIntroduction: Emerging data supports 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 ITS) 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 (wSNF), 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 significant ‘gut-lung’ interaction. Multi-biome 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 associates with increased exacerbations, greater radiological and overall bronchiectasis severity while 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 (PAO1) infection. This interaction was abrogated following antibiotic (imipenem) pre-treatment 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. Conclusion: A dysregulated ‘gut-lung’ axis, driven by lung Pseudomonas, associates with poorer clinical outcomes in bronchiectasis.Engineering and Physical Sciences Research Council (EPSRC)National Medical Research Council, Singapore Ministry of HealthFondazione IRCCS Cà Grand

Sex steroids induce membrane stress responses and virulence properties in pseudomonas aeruginosa

© 2020 Vidaillac et al. Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa. This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains. IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection

Sensitisation to recombinant Aspergillus fumigatus allergens and clinical outcomes in COPD.

This is the author accepted manuscript. The final version is available from the European Respiratory Society via the DOI in this recordBACKGROUND: Variable clinical outcomes are reported with fungal sensitisation in COPD, and it remains unclear which fungi and what allergens associate with poorest outcomes. The use of recombinant as opposed to crude allergens for such assessment is unknown. METHODS: A prospective multicenter assessment of stable COPD (n=614) was undertaken in five hospitals across three countries: Singapore, Malaysia, and Hong Kong. Clinical and serological assessment was performed against a panel of 35 fungal allergens including crude and recombinant Aspergillus and non-Aspergillus allergens. Unsupervised clustering and Topological Data Analysis (TDA) approaches were employed using the measured sensitisation responses to elucidate if sensitisation sub-groups exist and their related clinical outcomes. RESULTS: Aspergillus fumigatus sensitisation associates with increased exacerbations in COPD. Unsupervised cluster analyses reveal two "fungal sensitisation" groups, one characterized by Aspergillus sensitisation and increased exacerbations, poorer lung function and worse prognosis. Polysensitisation in this group confers even poorer outcome. The second group, characterized by Cladosporium sensitisation is more symptomatic. Significant numbers of individuals demonstrate sensitisation responses to only recombinant (as opposed to crude) Aspergillus fumigatus allergens 1, 3, 5, and 6, and exhibit higher exacerbations, poorer lung function and an overall worse prognosis. TDA validated these findings and additionally identified a sub-group within "Aspergillus sensitised COPD" enriched for frequent exacerbators. CONCLUSION: Aspergillus sensitisation is a treatable trait in COPD. Measuring sensitisation responses to recombinant Aspergillus allergens identifies an important patient subgroup with poor COPD outcomes that remain overlooked by assessment of only crude Aspergillus allergens.Engineering and Physical Sciences Research Council (EPSRC)Singapore General Hospital Research GrantSingapore Ministry of Health's National Medical Research CouncilSingapore Ministry of Health's National Medical Research CouncilNational University of SingaporeSingapore Ministry of Education Academic Research FundSingapore Ministry of Education Academic Research FundSingapore Ministry of Education Academic Research FundSingapore Ministry of Education Academic Research FundSingapore Ministry of Education Academic Research FundSingapore Ministry of Education Academic Research FundSingapore Ministry of Education Academic Research FundSingapore Ministry of Education Academic Research FundSingapore Ministry of Education Academic Research FundSingapore Ministry of Education Academic Research FundBiomedical Research Council (BMRC) (Singapore)Biomedical Research Council (BMRC) (Singapore)Biomedical Research Council (BMRC) (Singapore)Singapore Immunology NetworkSingapore Immunology NetworkNational Medical Research Council (NMRC) (Singapore)Agency for Science Technology and Research (A*STAR) (Singapore)Agency for Science Technology and Research (A*STAR) (Singapore

Neisseria species as pathobionts in bronchiectasis.

Neisseria species are frequently identified in the bronchiectasis microbiome, but they are regarded as respiratory commensals. Using a combination of human cohorts, next-generation sequencing, systems biology, and animal models, we show that bronchiectasis bacteriomes defined by the presence of Neisseria spp. associate with poor clinical outcomes, including exacerbations. Neisseria subflava cultivated from bronchiectasis patients promotes the loss of epithelial integrity and inflammation in primary epithelial cells. In vivo animal models of Neisseria subflava infection and metabolipidome analysis highlight immunoinflammatory functional gene clusters and provide evidence for pulmonary inflammation. The murine metabolipidomic data were validated with human Neisseria-dominant bronchiectasis samples and compared with disease in which Pseudomonas-, an established bronchiectasis pathogen, is dominant. Metagenomic surveillance of Neisseria across various respiratory disorders reveals broader importance, and the assessment of the home environment in bronchiectasis implies potential environmental sources of exposure. Thus, we identify Neisseria species as pathobionts in bronchiectasis, allowing for improved risk stratification in this high-risk group