Airway microbiota and bronchial hyperresponsiveness in patients with suboptimally controlled asthma.

BACKGROUND: Improvement in lung function after macrolide antibiotic therapy has been attributed to reduction in bronchial infection by specific bacteria. However, the airway might be populated by a more diverse microbiota, and clinical features of asthma might be associated with characteristics of the airway microbiota present. OBJECTIVE: We sought to determine whether relationships exist between the composition of the airway bacterial microbiota and clinical features of asthma using culture-independent tools capable of detecting the presence and relative abundance of most known bacteria. METHODS: In this pilot study bronchial epithelial brushings were collected from 65 adults with suboptimally controlled asthma participating in a multicenter study of the effects of clarithromycin on asthma control and 10 healthy control subjects. A combination of high-density 16S ribosomal RNA microarray and parallel clone library-sequencing analysis was used to profile the microbiota and examine relationships with clinical measurements. RESULTS: Compared with control subjects, 16S ribosomal RNA amplicon concentrations (a proxy for bacterial burden) and bacterial diversity were significantly higher among asthmatic patients. In multivariate analyses airway microbiota composition and diversity were significantly correlated with bronchial hyperresponsiveness. Specifically, the relative abundance of particular phylotypes, including members of the Comamonadaceae, Sphingomonadaceae, Oxalobacteraceae, and other bacterial families were highly correlated with the degree of bronchial hyperresponsiveness. CONCLUSION: The composition of bronchial airway microbiota is associated with the degree of bronchial hyperresponsiveness among patients with suboptimally controlled asthma. These findings support the need for further functional studies to examine the potential contribution of members of the airway microbiota in asthma pathogenesis.

Polymorphisms in the Pseudomonas aeruginosa type III secretion protein, PcrV - implications for anti-PcrV immunotherapy.

The type III secretion system of Pseudomonas aeruginosa, responsible for acute infection, is composed of over twenty proteins that facilitate cytotoxin injection directly into host cells. Integral to this process is production and secretion of PcrV. Administration of a recently developed, anti-PcrV immunoglobulin, either as a therapeutic or prophylactic has previously demonstrated efficacy against laboratory strains of P. aeruginosa in a murine model. To determine if this therapy is universally applicable to a variety of P. aeruginosa clinical isolates, genetic heterogeneity of pcrV was analyzed among strains collected from three geographically distinct regions; United States, France and Japan. Sequence analysis of PcrV demonstrated limited variation among the clinical isolates examined. Strains were grouped according to the presence of non-synonymous single nucleotide polymorphisms. Representative isolates from each mutant group were examined for the ability of anti-PcrV to bind the protein secreted by these strains. The protective effect of anti-PcrV IgG against each strain was determined using an epithelial cell line cytotoxicity assay. The majority of strains tested demonstrated reduced cytotoxicity in the presence of anti-PcrV IgG. This study provides insights into the natural sequence variability of PcrV and an initial indication of the amino acid residues that appear to be conserved across strains. It also demonstrates the protective effect of anti-PcrV immunotherapy against a multitude of P. aeruginosa strains from diverse global regions with a variety of mutations in PcrV.

Airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients.

Bacterial communities in the airways of cystic fibrosis (CF) patients are, as in other ecological niches, influenced by autogenic and allogenic factors. However, our understanding of microbial colonization in younger versus older CF airways and the association with pulmonary function is rudimentary at best. Using a phylogenetic microarray, we examine the airway microbiota in age stratified CF patients ranging from neonates (9 months) to adults (72 years). From a cohort of clinically stable patients, we demonstrate that older CF patients who exhibit poorer pulmonary function possess more uneven, phylogenetically-clustered airway communities, compared to younger patients. Using longitudinal samples collected form a subset of these patients a pattern of initial bacterial community diversification was observed in younger patients compared with a progressive loss of diversity over time in older patients. We describe in detail the distinct bacterial community profiles associated with young and old CF patients with a particular focus on the differences between respective "early" and "late" colonizing organisms. Finally we assess the influence of Cystic Fibrosis Transmembrane Regulator (CFTR) mutation on bacterial abundance and identify genotype-specific communities involving members of the Pseudomonadaceae, Xanthomonadaceae, Moraxellaceae and Enterobacteriaceae amongst others. Data presented here provides insights into the CF airway microbiota, including initial diversification events in younger patients and establishment of specialized communities of pathogens associated with poor pulmonary function in older patient populations.

Increased plasminogen activator inhibitor-1 concentrations in bronchoalveolar lavage fluids are associated with increased mortality in a cohort of patients with Pseudomonas aeruginosa.

BACKGROUND: Increased plasminogen activator inhibitor-1 (PAI-1) concentrations are found in bronchoalveolar lavage (BAL) fluids from patients with ventilator-associated pneumonia or acute respiratory distress syndrome. The authors hypothesized that PAI-1 concentrations were associated with increased mortality in patients with either Pseudomonas aeruginosa-induced ventilator-associated pneumonia or tracheobronchial colonization. METHODS: In a prospective cohort study, daily aspirates from intubated patients were cultured for P. aeruginosa. Positive patients had blind BAL (bBAL) that was processed for biomarker concentrations. Secretion of type III secretion cytotoxins were also analyzed from the P. aeruginosa strains. RESULTS: Thirty-three patients were enrolled. Ten of the 33 patients died. bBAL PAI-1 concentrations were significantly increased in nonsurvivors compared with survivors (31.7 vs. 3.4 ng/ml, P = 0.001 for hospital mortality; 35.9 vs. 4.7 ng/ml, P = 0.02 for 28-day mortality). Even when acute respiratory distress syndrome patients were excluded, there was a significant difference between the survivors and nonsurvivors for bBAL PAI-1 concentrations (P = 0.005). Eighty-three percent of P. aeruginosa strains isolated from patients with high concentrations of bBAL PAI-1 also had strains that secreted cytotoxins. CONCLUSIONS: PAI-1 concentrations in bBALs correlated with mortality in ventilated patients with positive cultures for P. aeruginosa. Elevated bBAL PAI-1 concentrations also correlated with the secretion of type III exotoxins by P. aeruginosa.