Date Published: July 13, 2017
Publisher: Public Library of Science
Author(s): Marion Engel, David Endesfelder, Brigitte Schloter-Hai, Susanne Kublik, Michael S. Granitsiotis, Piera Boschetto, Mariarita Stendardo, Imre Barta, Balazs Dome, Jean-François Deleuze, Anne Boland, Joachim Müller-Quernheim, Antje Prasse, Tobias Welte, Jens Hohlfeld, Deepak Subramanian, David Parr, Ivo Glynne Gut, Timm Greulich, Andreas Rembert Koczulla, Adam Nowinski, Dorota Gorecka, Dave Singh, Sumit Gupta, Christopher E. Brightling, Harald Hoffmann, Marion Frankenberger, Thomas P. Hofer, Dorothe Burggraf, Marion Heiss-Neumann, Loems Ziegler-Heitbrock, Michael Schloter, Wolfgang zu Castell, Brenda A. Wilson.
Changes in microbial community composition in the lung of patients suffering from moderate to severe COPD have been well documented. However, knowledge about specific microbiome structures in the human lung associated with CT defined abnormalities is limited.
Bacterial community composition derived from brush samples from lungs of 16 patients suffering from different CT defined subtypes of COPD and 9 healthy subjects was analyzed using a cultivation independent barcoding approach applying 454-pyrosequencing of 16S rRNA gene fragment amplicons.
We could show that bacterial community composition in patients with changes in CT (either airway or emphysema type changes, designated as severe subtypes) was different from community composition in lungs of patients without visible changes in CT as well as from healthy subjects (designated as mild COPD subtype and control group) (PC1, Padj = 0.002). Higher abundance of Prevotella in samples from patients with mild COPD subtype and from controls and of Streptococcus in the severe subtype cases mainly contributed to the separation of bacterial communities of subjects. No significant effects of treatment with inhaled glucocorticoids on bacterial community composition were detected within COPD cases with and without abnormalities in CT in PCoA. Co-occurrence analysis suggests the presence of networks of co-occurring bacteria. Four communities of positively correlated bacteria were revealed. The microbial communities can clearly be distinguished by their associations with the CT defined disease phenotype.
Our findings indicate that CT detectable structural changes in the lung of COPD patients, which we termed severe subtypes, are associated with alterations in bacterial communities, which may induce further changes in the interaction between microbes and host cells. This might result in a changed interplay with the host immune system.
Chronic Obstructive Pulmonary Disease (COPD) is characterized by chronic cough, increased sputum production and dyspnoea. More than 3 million people died of COPD in 2012, approximately 6% of all deaths globally . Whereas in high-income countries COPD is primarily caused by tobacco smoking, in low- and middle-income countries both indoor and outdoor air pollution play an important role in disease etiology .
After direct extraction of the DNA derived from bronchial brushings from patients suffering from COPD stages 1 and 2 according to the GOLD classification, and from healthy subjects, bacterial community composition was determined by 454 pyro-sequencing of 16S rRNA gene amplification products. Sequencing resulted in 77515 chimera free, high-quality reads. After removal of 16S rRNA sequences derived from mitochondria of epithelium cells of the lung and of reads identified as potential PCR contaminations according to , numbers of reads varied between 722 to 4789 reads per sample. To account for differences in the number of sequencing reads, all libraries were subsampled to 722 reads and reads were clustered on 95% sequence similarity level. This number of reads was sufficient to cover the majority of OTUs in all samples as indicated by analysis of the individual rarefaction curves (S2 Fig). Samples with less than 700 reads in total were discarded for the analysis. In total, 25 samples (9 samples from healthy subjects and 16 from cases) were used for further analysis.
The current microbiota analysis was performed on samples obtained from the EvA study . This study defined subtypes of COPD using CT image analysis of lung density for determination of the degree of emphysema and airway wall thickness as a measure of bronchitis. We were interested in early changes in bacterial communities during disease development in COPD, with special emphasis on interrelation of COPD subtypes with and without abnormalities in CT and changes in microbial community composition.
This study revealed changes in lung microbial community composition in GOLD stage 1 and 2 COPD cases with abnormalities in CT. We could show that lung community composition in COPD patients without abnormalities in CT resembles that of the control group, whereas, communities of patients with abnormalities in CT were significantly different. Our approach using community detection in association networks of bacteria hints to the presence of different communities of associated bacteria in the lung.