16 research outputs found

    Sputum metagenomics of people with bronchiectasis

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    BACKGROUND: The microbiota in the sputum of people with bronchiectasis has repeatedly been investigated in cohorts of different geographic origin, but so far has not been studied to the species level in comparison to control populations including healthy adults and smokers without lung disease.METHODS: The microbial metagenome from sputa of 101 European Bronchiectasis Registry (EMBARC) study participants was examined by using whole-genome shotgun sequencing.RESULTS: Our analysis of the metagenome of people with bronchiectasis revealed four clusters characterised by a predominance of Haemophilus influenzae, Pseudomonas aeruginosa or polymicrobial communities with varying compositions of nonpathogenic commensals and opportunistic pathogens. The metagenomes of the severely affected patients showed individual profiles characterised by low alpha diversity. Importantly, nearly 50% of patients with severe disease were grouped in a cluster characterised by commensals. Comparisons with the sputum metagenomes of healthy smokers and healthy nonsmokers revealed a gradient of depletion of taxa in bronchiectasis, most often Neisseria subflava, Fusobacterium periodonticum and Eubacterium sulci.CONCLUSION: The gradient of depletion of commensal taxa found in healthy airways is a key feature of bronchiectasis associated with disease severity.</p

    Sputum metagenomics of people with bronchiectasis

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    BACKGROUND: The microbiota in the sputum of people with bronchiectasis has repeatedly been investigated in cohorts of different geographic origin, but so far has not been studied to the species level in comparison to control populations including healthy adults and smokers without lung disease.METHODS: The microbial metagenome from sputa of 101 European Bronchiectasis Registry (EMBARC) study participants was examined by using whole-genome shotgun sequencing.RESULTS: Our analysis of the metagenome of people with bronchiectasis revealed four clusters characterised by a predominance of Haemophilus influenzae, Pseudomonas aeruginosa or polymicrobial communities with varying compositions of nonpathogenic commensals and opportunistic pathogens. The metagenomes of the severely affected patients showed individual profiles characterised by low alpha diversity. Importantly, nearly 50% of patients with severe disease were grouped in a cluster characterised by commensals. Comparisons with the sputum metagenomes of healthy smokers and healthy nonsmokers revealed a gradient of depletion of taxa in bronchiectasis, most often Neisseria subflava, Fusobacterium periodonticum and Eubacterium sulci.CONCLUSION: The gradient of depletion of commensal taxa found in healthy airways is a key feature of bronchiectasis associated with disease severity.</p

    An association study on contrasting cystic fibrosis endophenotypes recognizes KRT8 but not KRT18 as a modifier of cystic fibrosis disease severity and CFTR mediated residual chloride secretion

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    <p>Abstract</p> <p>Background</p> <p>F508del-CFTR, the most frequent disease-causing mutation among Caucasian cystic fibrosis (CF) patients, has been characterised as a mutant defective in protein folding, processing and trafficking. We have investigated the two neighbouring cytokeratin genes <it>KRT8 </it>and <it>KRT18 </it>in a candidate gene approach to ask whether variants in <it>KRT8 </it>and/or <it>KRT18 </it>modify the impaired ion conductance known as the CF basic defect, and whether they are associated with correct trafficking of mutant CFTR and disease severity of CF.</p> <p>Methods</p> <p>We have selected contrasting F508del-<it>CFTR </it>homozygous patient subpopulations stratified for disease severity, comparing 13 concordant mildly affected sib pairs vs. 12 concordant severely affected sib pairs, or manifestation of the CF basic defect in intestinal epithelium, comparing 22 individuals who exhibit CFTR-mediated residual chloride secretion vs. 14 individuals who do not express any chloride secretion, for an association. The <it>KRT8</it>/<it>KRT18 </it>locus was initially interrogated with one informative microsatellite marker. Subsequently, a low density SNP map with four SNPs in KRT8 and two SNPs in KRT18, each selected for high polymorphism content, was used to localize the association signal.</p> <p>Results</p> <p><it>KRT8</it>, but not <it>KRT18</it>, showed an association with CF disease severity (P<sub>best </sub>= 0.00131; P<sub>corr </sub>= 0.0185) and CFTR mediated residual chloride secretion (P<sub>best </sub>= 0.0004; P<sub>corr </sub>= 0.0069). Two major four-marker-haplotypes spanning 13 kb including the entire <it>KRT8 </it>gene accounted for 90% of chromosomes, demonstrating strong linkage disequilibrium at that locus. Absence of chloride secretion was associated with the recessive haplotype 1122 at rs1907671, rs4300473, rs2035878 and rs2035875. The contrasting haplotype 2211 was dominant for the presence of CFTR mediated residual chloride secretion. In consistency, the <it>KRT8 </it>haplotype 2211 was associated with mild CF disease while 1122 was observed as risk haplotype. Analysis of microsatellite allele distributions on the SNP background suggests that the mild <it>KRT8 </it>haplotype 2211 is phylogenetically older than its severe counterpart.</p> <p>Conclusions</p> <p>The two opposing <it>KRT8 </it>alleles which have been identified as a benign and as a risk allele in this work are likely effective in the context of epithelial cell differentiation. As the mild <it>KRT8 </it>allele is associated with CFTR mediated residual chloride secretion among F508del-<it>CFTR </it>homozygotes, the KRT8/KRT18 heterodimeric intermediary filaments of the cytoskeleton apparently are an essential component for the proper targeting of CFTR to the apical membrane in epithelial cells.</p

    Caenorhabditis elegans Semi-Automated Liquid Screen Reveals a Specialized Role for the Chemotaxis Gene cheB2 in Pseudomonas aeruginosa Virulence

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    Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity

    Factors associated with worse lung function in cystic fibrosis patients with persistent staphylococcus aureus

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    Background Staphylococcus aureus is an important pathogen in cystic fibrosis (CF). However, it is not clear which factors are associated with worse lung function in patients with persistent S. aureus airway cultures. Our main hypothesis was that patients with high S. aureus density in their respiratory specimens would more likely experience worsening of their lung disease than patients with low bacterial loads. Methods Therefore, we conducted an observational prospective longitudinal multi-center study and assessed the association between lung function and S. aureus bacterial density in respiratory samples, co-infection with other CF-pathogens, nasal S. aureus carriage, clinical status, antibiotic therapy, IL-6- and IgG-levels against S. aureus virulence factors. Results 195 patients from 17 centers were followed; each patient had an average of 7 visits. Data were analyzed using descriptive statistics and generalized linear mixed models. Our main hypothesis was only supported for patients providing throat specimens indicating that patients with higher density experienced a steeper lung function decline (p<0.001). Patients with exacerbations (n = 60), S. aureus small-colony variants (SCVs, n = 84) and co-infection with Stenotrophomonas maltophilia (n = 44) had worse lung function (p = 0.0068; p = 0.0011; p = 0.0103). Patients with SCVs were older (p = 0.0066) and more often treated with trimethoprim/sulfamethoxazole (p = 0.0078). IL-6 levels positively correlated with decreased lung function (p<0.001), S. aureus density in sputa (p = 0.0016), SCVs (p = 0.0209), exacerbations (p = 0.0041) and co-infections with S. maltophilia (p = 0.0195) or A. fumigatus (p = 0.0496). Conclusions In CF-patients with chronic S. aureus cultures, independent risk factors for worse lung function are high bacterial density in throat cultures, exacerbations, elevated IL-6 levels, presence of S. aureus SCVs and co-infection with S. maltophilia

    Fitness of Isogenic Colony Morphology Variants of Pseudomonas aeruginosa in Murine Airway Infection

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    Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called ‘dissociative behaviour’. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of–function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild

    Differential decay of parent-of-origin-specific genomic sharing in cystic fibrosis-affected sib pairs maps a paternally imprinted locus to 7q34

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    Cystic fibrosis (CF) is a monogenic disease characterized by a high variability of disease severity and outcome that points to the role of environmental factors and modulating genes that shape the course of this multiorgan disease. We genotyped families of cystic fibrosis sib pairs homozygous for F508del-CFTR who represent extreme clinical phenotypes at informative microsatellite markers spanning a 38 Mb region between CFTR and 7qtel. Recombination events on both parental chromosomes were compared between siblings with concordant clinical phenotypes and siblings with discordant clinical phenotypes. Monitoring parent-of-origin-specific decay of genomic sharing delineated a 2.9-Mb segment on 7q34 in which excess of recombination on paternal chromosomes in discordant pairs was observed compared with phenotypically concordant sibs. This 2.9-Mb core candidate region was enriched in imprinting-related elements such as predicted CCCTC-binding factor consensus sites and CpG islands dense in repetitive elements. Moreover, allele frequencies at a microsatellite marker within the core candidate region differed significantly comparing mildly and severely affected cystic fibrosis sib pairs. The identification of this paternally imprinted locus on 7q34 as a modulator of cystic fibrosis disease severity shows that imprinted elements can be identified by straightforward fine mapping of break points in sib pairs with informative contrasting phenotypes
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