Severity-related changes of bronchial microbiome in chronic obstructive pulmonary disease

Abstract

Bronchial colonization by potentially pathogenic microorganisms (PPMs) is often demonstrated in chronic obstructive pulmonary disease (COPD), but culture-based techniques identify only a portion of the bacteria in mucosal surfaces. The aim of the study was to determine changes in the bronchial microbiome of COPD associated with the severity of the disease. The bronchial microbiome of COPD patients was analyzed by 16S rRNA gene amplification and pyrosequencing in sputum samples obtained during stable disease. Seventeen COPD patients were studied (forced expiratory volume in the first second expressed as a percentage of the forced vital capacity [FEV1%] median, 35.0%; interquartile range [IQR], 31.5 to 52.0), providing a mean of 4,493 (standard deviation [SD], 2,598) sequences corresponding to 47 operational taxonomic units (OTUs) (SD, 17) at a 97% identity level. Patients were dichotomized according to their lung function as moderate to severe when their FEV1% values were over the median and as advanced when FEV1% values were lower. The most prevalent phyla in sputum were Proteobacteria (44%) and Firmicutes (16%), followed by Actinobacteria (13%). A greater microbial diversity was found in patients with moderate-to-severe disease, and alpha diversity showed a statistically significant decrease in patients with advanced disease when assessed by Shannon (ρ = 0.528; P = 0.029, Spearman correlation coefficient) and Chao1 (ρ = 0.53; P = 0.028, Spearman correlation coefficient) alpha-diversity indexes. The higher severity that characterizes advanced COPD is paralleled by a decrease in the diversity of the bronchial microbiome, with a loss of part of the resident flora that is replaced by a more restricted microbiota that includes PPMs.

FIG 1

Principal coordinate (PC) analysis with Bray-Curtis dissimilarity index. Red and blue dots represent moderate-to-severe and advanced COPD patients, respectively (Adonis test, P = 0.221).

FIG 2

Taxonomic identification at phylum level in each sample from moderate-to-severe (labeled with A) and advanced (labeled with B) chronic obstructive pulmonary disease (COPD) patients.

FIG 3

Scatterplot of Proteobacteria relative abundances and Firmicutes relative abundances (Spearman correlation coefficient, ρ = −0.629 and P = 0.007) in chronic obstructive pulmonary disease (COPD) patients.

FIG 4

Heat map showing clustering of genera communities and the most predominant genera in chronic obstructive pulmonary disease (COPD) patients with moderate-to-severe (A) and advanced (B) disease. Sample numbers are listed horizontally along the bottom. The genera are listed vertically on the right. The relative abundance of each genus is represented by the color key.

FIG 5

Microbiome diversity in chronic obstructive pulmonary disease (COPD) patients according to lung function by the Shannon index (a) and Chao1 index (b) (P = 0.029, ρ = 0.53; P = 0.028, Spearman correlation test).

FIG 6

LEfSe (24) rank plot of differentially abundant taxa (bacterial OTUs) at different taxonomic levels in moderate-to-severe versus advanced COPD patients. Statistical analysis found 10 bacterial OTUs overrepresented in moderate-to-severe COPD patients (A) compared with patients with advanced disease. unid, unidentified.