G.G. Einarsson

Lung microbiota and bacterial abundance in patients with bronchiectasis when clinically stable and during exacerbation.

RATIONALE Characterization of bacterial populations in infectious respiratory diseases will provide improved understanding of the relationship between the lung microbiota, disease pathogenesis, and treatment outcomes. OBJECTIVES To comprehensively define lung microbiota composition during stable disease and exacerbation in patients with bronchiectasis. METHODS Sputum was collected from patients when clinically stable and before and after completion of antibiotic treatment of exacerbations. Bacterial abundance and community composition were analyzed using anaerobic culture and 16S rDNA pyrosequencing. MEASUREMENTS AND MAIN RESULTS In clinically stable patients, aerobic and anaerobic bacteria were detected in 40 of 40 (100%) and 33 of 40 (83%) sputum samples, respectively. The dominant organisms cultured were Pseudomonas aeruginosa (n = 10 patients), Haemophilus influenzae (n = 12), Prevotella (n = 18), and Veillonella (n = 13). Pyrosequencing generated more than 150,000 sequences, representing 113 distinct microbial taxa; the majority of observed community richness resulted from taxa present in low abundance with similar patterns of phyla distribution in clinically stable patients and patients at the onset of exacerbation. After treatment of exacerbation, there was no change in total (P = 0.925), aerobic (P = 0.917), or anaerobic (P = 0.683) load and only a limited shift in community composition. Agreement for detection of bacteria by culture and pyrosequencing was good for aerobic bacteria such as P. aeruginosa (κ = 0.84) but poorer for other genera including anaerobes. Lack of agreement was largely due to bacteria being detected by pyrosequencing but not by culture. CONCLUSIONS A complex microbiota is present in the lungs of patients with bronchiectasis and remains stable through treatment of exacerbations, suggesting that changes in microbiota composition do not account for exacerbations.

Comparison of microbiomes from different niches of upper and lower airways in children and adolescents with cystic fibrosis.

Changes in the airway microbiome may be important in the pathophysiology of chronic lung disease in patients with cystic fibrosis. However, little is known about the microbiome in early cystic fibrosis lung disease and the relationship between the microbiomes from different niches in the upper and lower airways. Therefore, in this cross-sectional study, we examined the relationship between the microbiome in the upper (nose and throat) and lower (sputum) airways from children with cystic fibrosis using next generation sequencing. Our results demonstrate a significant difference in both α and β-diversity between the nose and the two other sampling sites. The nasal microbiome was characterized by a polymicrobial community while the throat and sputum communities were less diverse and dominated by a few operational taxonomic units. Moreover, sputum and throat microbiomes were closely related especially in patients with clinically stable lung disease. There was a high inter-individual variability in sputum samples primarily due to a decrease in evenness linked to increased abundance of potential respiratory pathogens such as Pseudomonas aeruginosa. Patients with chronic Pseudomonas aeruginosa infection exhibited a less diverse sputum microbiome. A high concordance was found between pediatric and adult sputum microbiomes except that Burkholderia was only observed in the adult cohort. These results indicate that an adult-like lower airways microbiome is established early in life and that throat swabs may be a good surrogate in clinically stable children with cystic fibrosis without chronic Pseudomonas aeruginosa infection in whom sputum sampling is often not feasible.

Community dynamics and the lower airway microbiota in stable chronic obstructive pulmonary disease, smokers and healthy non-smokers

Rationale The role bacteria play in the progression of COPD has increasingly been highlighted in recent years. However, the microbial community complexity in the lower airways of patients with COPD is poorly characterised. Objectives To compare the lower airway microbiota in patients with COPD, smokers and non-smokers. Methods Bronchial wash samples from adults with COPD (n=18), smokers with no airways disease (n=8) and healthy individuals (n=11) were analysed by extended-culture and culture-independent Illumina MiSeq sequencing. We determined aerobic and anaerobic microbiota load and evaluated differences in bacteria associated with the three cohorts. Culture-independent analysis was used to determine differences in microbiota between comparison groups including taxonomic richness, diversity, relative abundance, ‘core’ microbiota and co-occurrence. Measurement and main results Extended-culture showed no difference in total load of aerobic and anaerobic bacteria between the three cohorts. Culture-independent analysis revealed that the prevalence of members of Pseudomonas spp. was greater in the lower airways of patients with COPD; however, the majority of the sequence reads for this taxa were attributed to three patients. Furthermore, members of Bacteroidetes, such as Prevotella spp., were observed to be greater in the ‘healthy’ comparison groups. Community diversity (α and β) was significantly less in COPD compared with healthy groups. Co-occurrence of bacterial taxa and the observation of a putative ‘core’ community within the lower airways were also observed. Conclusions Microbial community composition in the lower airways of patients with COPD is significantly different to that found in smokers and non-smokers, indicating that a component of the disease is associated with changes in microbiological status.

Neutrophil extracellular traps are associated with disease severity and microbiota diversity in patients with chronic obstructive pulmonary disease

&NA; Figure. No caption available. Background: Neutrophil extracellular traps (NETs) have been observed in the airway in patients with chronic obstructive pulmonary disease (COPD), but their clinical and pathophysiologic implications have not been defined. Objective: We sought to determine whether NETs are associated with disease severity in patients with COPD and how they are associated with microbiota composition and airway neutrophil function. Methods: NET protein complexes (DNA‐elastase and histone‐elastase complexes), cell‐free DNA, and neutrophil biomarkers were quantified in soluble sputum and serum from patients with COPD during periods of disease stability and during exacerbations and compared with clinical measures of disease severity and the sputum microbiome. Peripheral blood and airway neutrophil function were evaluated by means of flow cytometry ex vivo and experimentally after stimulation of NET formation. Results: Sputum NET complexes were associated with the severity of COPD evaluated by using the composite Global Initiative for Obstructive Lung Disease scale (P < .0001). This relationship was due to modest correlations between NET complexes and FEV1, symptoms evaluated by using the COPD assessment test, and higher levels of NET complexes in patients with frequent exacerbations (P = .002). Microbiota composition was heterogeneous, but there was a correlation between NET complexes and both microbiota diversity (P = .009) and dominance of Haemophilus species operational taxonomic units (P = .01). Ex vivo airway neutrophil phagocytosis of bacteria was reduced in patients with increased sputum NET complexes. Consistent results were observed regardless of the method of quantifying sputum NETs. Failure of phagocytosis could be induced experimentally by incubating healthy control neutrophils with soluble sputum from patients with COPD. Conclusion: NET formation is increased in patients with severe COPD and associated with more frequent exacerbations and a loss of microbiota diversity.

Reduced bacterial colony count of anaerobic bacteria is associated with a worsening in lung clearance index and inflammation in cystic fibrosis.

Anaerobic bacteria have been identified in abundance in the airways of cystic fibrosis (CF) subjects. The impact their presence and abundance has on lung function and inflammation is unclear. The aim of this study was to investigate the relationship between the colony count of aerobic and anaerobic bacteria, lung clearance index (LCI), spirometry and C-Reactive Protein (CRP) in patients with CF. Sputum and blood were collected from CF patients at a single cross-sectional visit when clinically stable. Community composition and bacterial colony counts were analysed using extended aerobic and anaerobic culture. Patients completed spirometry and a multiple breath washout (MBW) test to obtain LCI. An inverse correlation between colony count of aerobic bacteria (n = 41, r = -0.35; p = 0.02), anaerobic bacteria (n = 41, r = -0.44, p = 0.004) and LCI was observed. There was an inverse correlation between colony count of anaerobic bacteria and CRP (n = 25, r = -0.44, p = 0.03) only. The results of this study demonstrate that a lower colony count of aerobic and anaerobic bacteria correlated with a worse LCI. A lower colony count of anaerobic bacteria also correlated with higher CRP levels. These results indicate that lower abundance of aerobic and anaerobic bacteria may reflect microbiota disruption and disease progression in the CF lung.

CORK Study in Cystic Fibrosis: Sustained Improvements in Ultra-Low-Dose Chest CT Scores After CFTR Modulation With Ivacaftor

Background Ivacaftor produces significant clinical benefit in patients with cystic fibrosis (CF) with the G551D mutation. Prevalence of this mutation at the Cork CF Centre is 23%. This study assessed the impact of cystic fibrosis transmembrane conductance regulator modulation on multiple modalities of patient assessment. Methods Thirty‐three patients with the G551D mutation were assessed at baseline and prospectively every 3 months for 1 year after initiation of ivacaftor. Change in ultra‐low‐dose chest CT scans, blood inflammatory mediators, and the sputum microbiome were assessed. Results Significant improvements in FEV1, BMI, and sweat chloride levels were observed post‐ivacaftor treatment. Improvement in ultra‐low‐dose CT imaging scores were observed after treatment, with significant mean reductions in total Bhalla score (P < .01), peribronchial thickening (P = .035), and extent of mucous plugging (P < .001). Reductions in circulating inflammatory markers, including interleukin (IL)‐1&bgr;, IL‐6, and IL‐8 were demonstrated. There was a 30% reduction in the relative abundance of Pseudomonas species and an increase in the relative abundance of bacteria associated with more stable community structures. Posttreatment community richness increased significantly (P = .03). Conclusions Early and sustained improvements on ultra‐low‐dose CT scores suggest it may be a useful method of evaluating treatment response. It paralleled improvement in symptoms, circulating inflammatory markers, and changes in the lung microbiota.

Genetic mannose binding lectin deficiency is associated with airway microbiota diversity and reduced exacerbation frequency in COPD

Background In cystic fibrosis and bronchiectasis, genetic mannose binding lectin (MBL) deficiency is associated with increased exacerbations and earlier mortality; associations in COPD are less clear. Preclinical data suggest MBL interferes with phagocytosis of Haemophilus influenzae, a key COPD pathogen. We investigated whether MBL deficiency impacted on clinical outcomes or microbiota composition in COPD. Methods Patients with COPD (n=1796) underwent MBL genotyping; linkage to health records identified exacerbations, lung function decline and mortality. A nested subcohort of 141 patients, followed for up to 6 months, was studied to test if MBL deficiency was associated with altered sputum microbiota, through 16S rRNA PCR and sequencing, or airway inflammation during stable and exacerbated COPD. Findings Patients with MBL deficiency with COPD were significantly less likely to have severe exacerbations (incidence rate ratio (IRR) 0.66, 95% CI 0.48 to 0.90, p=0.009), or to have moderate or severe exacerbations (IRR 0.77, 95% CI 0.60 to 0.99, p=0.047). MBL deficiency did not affect rate of FEV1 decline or mortality. In the subcohort, patients with MBL deficiency had a more diverse lung microbiota (p=0.008), and were less likely to be colonised with Haemophilus spp. There were lower levels of airway inflammation in patients with MBL deficiency. Interpretation Patients with MBL deficient genotype with COPD have a lower risk of exacerbations and a more diverse lung microbiota. This is the first study to identify a genetic association with the lung microbiota in COPD.

Chronic lung disease in common variable immune deficiency (CVID): A pathophysiological role for microbial and non-B cell immune factors

Abstract One of the most common and most severe forms of primary antibody deficiency encountered in the clinical setting is a heterogeneous group of syndromes termed common variable immune deficiency (CVID). This disorder is characterized by reduced immunoglobulin production and increased susceptibility to infection, particularly of the respiratory tract. Infection and subsequent immunological/inflammatory processes may contribute to the development of pulmonary complications such as bronchiectasis and interstitial lung disease. Immunoglobulin replacement and/or antibiotic therapy, to prevent infection, are routinely prescribed treatments. However, chronic lung disease, the major cause of morbidity and mortality in this patient cohort, may still progress. This clinical progression suggests that pathogens recalcitrant to currently prescribed treatments and other immunological defects may be contributing to the development of pulmonary disease. This review describes the potential role of microbiological and non-B cell immunological factors, including T-cells, neutrophils, complement, toll like receptors, and antimicrobial peptides, in the pathogenicity of chronic lung disease in patients with CVID.

Anaerobic bacteria cultured from cystic fibrosis airways correlate to milder disease: a multisite study

Anaerobic and aerobic bacteria were quantitated in respiratory samples across three cystic fibrosis (CF) centres using extended culture methods. Subjects aged 1–69 years who were clinically stable provided sputum (n=200) or bronchoalveolar lavage (n=55). 18 anaerobic and 39 aerobic genera were cultured from 59% and 95% of samples, respectively; 16 out of 57 genera had a ≥5% prevalence across centres. Analyses of microbial communities using co-occurrence networks in sputum samples showed groupings of oral, including anaerobic, bacteria, whereas typical CF pathogens formed distinct entities. Pseudomonas was associated with worse nutrition and F508del genotype, whereas anaerobe prevalence was positively associated with pancreatic sufficiency, better nutrition and better lung function. A higher total anaerobe/total aerobe CFU ratio was associated with pancreatic sufficiency and better nutrition. Subjects grouped by factor analysis who had relative dominance of anaerobes over aerobes had milder disease compared with a Pseudomonas-dominated group with similar proportions of subjects that were homozygous for F508del. In summary, anaerobic bacteria occurred at an early age. In sputum-producing subjects anaerobic bacteria were associated with milder disease, suggesting that targeted eradication of anaerobes may not be warranted in sputum-producing CF subjects. Anaerobic bacteria are cultured across all ages, occur as communities and correlate with milder CF disease in adults http://ow.ly/7wQ430khMmE

Bacterial proteases and haemostasis dysregulation in the CF lung

BACKGROUND Pathogenic bacteria which chronically colonise the cystic fibrosis (CF) lung produce a number of virulence determinants, including distinct proteolytic activities. The potential role bacterial proteases play on haemostatic dysregulation within the CF lung is, however, poorly defined, despite haemoptysis being a common complication in CF. METHODS The potential impact of known CF pathogens (Pseudomonas aeruginosa and Burkholderia cepacia complex spp.) on haemostasis was examined for their ability to degrade fibrinogen and dysregulate fibrin clot formation and platelet aggregation. RESULTS Results demonstrate that key CF pathogens growing as a biofilm on mucin exhibit considerable fibrinogenolytic activity, resulting in fibrinogen breakdown, impaired clot formation, and modulation of platelet aggregation. Human neutrophil elastase may also contribute to fibrinogen breakdown and dysregulated clot formation at high concentration. CONCLUSION Bacterial-derived proteases may play an important role in the dysregulation of airway haemostasis, and potentially contribute to episodes of haemoptysis within the CF lung.