Analysis of the airway microbiota of healthy individuals and patients with chronic obstructive pulmonary disease by T-RFLP and clone sequencing.

Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways.The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) length polymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease

Levels of cytokines in broncho-alveolar lavage fluid, but not in plasma, are associated with levels of markers of lipid peroxidation in breath of ventilated ICU patients

Alkanes and alkenes in the breath are produced through fatty acid peroxidation, which is initialized by reactive oxygen species. Inflammation is an important cause and effect of reactive oxygen species. We aimed to evaluate the association between fatty acid peroxidation products and inflammation of the alveolar and systemic compartment in ventilated intensive care unit (ICU) patients.Volatile organic compounds were measured by gas chromatography and mass spectrometry in the breath of newly ventilated ICU patients within 24 h after ICU admission. Cytokines were measured in non-directed bronchial lavage fluid (NBL) and plasma by cytometric bead array. Correlation coefficients were calculated and presented in heatmaps.93 patients were included. Peroxidation products in exhaled breath were not associated with markers of inflammation in plasma, but were correlated with those in NBL. IL-6, IL-8, IL-1β and TNF-α concentration in NBL showed inverse correlation coefficients with the peroxidation products of fatty acids. Furthermore, NBL IL-10, IL-13, GM-CSF and IFNγ demonstrated positive associations with breath alkanes and alkenes. Correlation coefficients for NBL cytokines were high regarding peroxidation products of n-6, n-7 and particularly in n-9 fatty acids.Levels of lipid peroxidation products in the breath of ventilated ICU patients are associated with levels of inflammatory markers in NBL, but not in plasma. Alkanes and alkenes in breath seems to be associated with an anti-inflammatory, rather than a pro-inflammatory state in the alveol

Confusion matrix of the classification results using a NaiveBayes classifier and cross validation.

<p>Confusion matrix of the classification results using a NaiveBayes classifier and cross validation.</p

An overview of the bacterial genera detected by sequencing and terminal restriction fragment length polymorphism analysis in COPD patients and healthy individuals.

<p>Due to technical limitation of T-RFLP analysis with one labeled primer, this method can only predict the phylogenetic ID. Identification of a specific genera by both methods is marked by an “X”.</p

T-RFLP profiles revealed highly interindividual variation of detected bands.

<p>Band sizes were correlated with bacterial general, keeping in mind that T-FRLP only allows a prediction but not a determination of the microbial taxa.</p

Sample richness (a) and Shannon diversity index (b) based on fragment lengths.

<p>Terminal restriction fragments shorter than 50 bp were excluded from the analysis. Empty squares correspond to healthy individuals, filled squares to COPD patients.</p