Outgrowth of the bacterial airway microbiome after rhinovirus exacerbation of chronic obstructive pulmonary disease

Abstract

Rationale: Rhinovirus infection is followed by significantly increased frequencies of positive, potentially pathogenic sputum cultures in chronic obstructive pulmonary disease (COPD). However, it remains unclear whether these represent de novo infections or an increased load of organisms from the complex microbial communities (microbiome) in the lower airways.

Objectives: To investigate the effect of rhinovirus infection on the airway bacterial microbiome.

Methods: Subjects with COPD (n = 14) and healthy control subjects with normal lung function (n = 17) were infected with rhinovirus. Induced sputum was collected at baseline before rhinovirus inoculation and again on Days 5, 15, and 42 after rhinovirus infection and DNA was extracted. The V3-V5 region of the bacterial 16S ribosomal RNA gene was amplified and pyrosequenced, resulting in 370,849 high-quality reads from 112 of the possible 124 time points.

Measurements and main results: At 15 days after rhinovirus infection, there was a sixfold increase in 16S copy number (P = 0.007) and a 16% rise in numbers of proteobacterial sequences, most notably in potentially pathogenic Haemophilus influenzae (P = 2.7 × 10(-20)), from a preexisting community. These changes occurred only in the sputum microbiome of subjects with COPD and were still evident 42 days after infection. This was in contrast to the temporal stability demonstrated in the microbiome of healthy smokers and nonsmokers.

Conclusions: After rhinovirus infection, there is a rise in bacterial burden and a significant outgrowth of Haemophilus influenzae from the existing microbiota of subjects with COPD. This is not observed in healthy individuals. Our findings suggest that rhinovirus infection in COPD alters the respiratory microbiome and may precipitate secondary bacterial infections.

Figure 1.

16S ribosomal RNA (rRNA) gene copy number at baseline and after rhinovirus (RV) infection and its correlation with inflammatory markers. (A) There is no significant difference between the bacterial load before RV infection in the chronic obstructive pulmonary disease (COPD) and control cohorts (P = 0.08). (B and C) After RV infection, there is an increase in bacterial load on Day 15 compared with baseline in (B) the COPD cohort (P = 0.0068) but not in (C) the control cohort (P = 0.072). (D) On Day 15, the bacterial copy number of 16S rRNA correlates with the concentrations of sputum inflammatory cells (P = 0.0001), neutrophils (P = 0.001), and sputum neutrophil elastase levels (P = 0.045) (Spearman’s rank correlation).

Figure 2.

A phylogenetic tree and heat map of bacterial 16S ribosomal RNA (rRNA) sequences derived from sputum at baseline. This depicts the top 100 operational taxonomic units (OTUs) organized phylogenetically by tree with abundance indicated by the color (darker blue, more abundant). The samples are grouped into chronic obstructive pulmonary disease (COPD), smokers, and nonsmoking control subjects (NS). Taxonomy assignments at the phylum level are shown in the inner column and are color-coded.

Figure 3.

Mean (±SEM) Bray–Curtis dissimilarity between communities and their respective baseline. A Bray–Curtis measure of dissimilarity of 0 means the two groups have the same composition (i.e., share all species), and 1 means the two sites do not share any species. There is a significant difference between the bacterial communities on Day 15 in the subjects with chronic obstructive pulmonary disease (COPD) compared with the baseline community. *P < 0.001 (two-tailed t test).

Figure 4.

Distribution of bacterial phyla at each time point after rhinovirus (RV) inoculation. (A) In the control subjects, no significant difference was observed between the percentage compositions at the phylum level between any time points. (B) In subjects with chronic obstructive pulmonary disease (COPD), significant increases in Proteobacteria (dark blue) were observed on Day 15 after RV inoculation compared with baseline (P = 2.2 × 10^–16).

Figure 5.

The distribution of the top four phyla grouped at each time point. (A) In the control subjects, no significant change from baseline was observed in any of the phyla after rhinovirus (RV) infection. (B) The response to RV in the chronic obstructive pulmonary disease (COPD) cohort was not uniform, but there was a significant increase from baseline in Proteobacteria on Day 15 after RV inoculation (P = 2.2 × 10^–16).

Figure 6.

Phylogenetic identification of Haemophilus sp. operational taxonomic unit (OTU). Phylogenetic analysis of the representative sequence of OTU0768 (boldface type) shows there is strong clustering of this bacterium within the Haemophilus genus. Bootstrapping analysis provides a method to judge the strength of confidence for nodes on phylogenetic trees, and a value greater than 95% seen here supports confident assignment of this OTU as Haemophilus influenzae. The tree was rooted with a near neighbor outgroup constructed with sequences from Morganella morganii, Proteus mirabilis, and Providencia stuartii.