Asthma-associated differences in microbial composition of induced sputum

Abstract

Background: It is increasingly evident that microbial colonization of the respiratory tract might have a role in the pathogenesis of asthma.

Objective: We sought to characterize and compare the microbiome of induced sputum in asthmatic and nonasthmatic adults.

Methods: Induced sputum samples were obtained from 10 nonasthmatic subjects and 10 patients with mild active asthma (8/10 were not using inhaled corticosteroids). Total DNA was extracted from sputum supernatants and amplified by using primers specific for the V6 hypervariable region of bacterial 16s rRNA. Samples were barcoded, and equimolar concentrations of 20 samples were pooled and sequenced with the 454 GS FLX sequencer. Sequences were assigned to bacterial taxa by comparing them with 16s rRNA sequences in the Ribosomal Database Project.

Results: All sputum samples contained 5 major bacterial phyla: Firmicutes, Proteobacteria, Actinobacteria, Fusobacterium, and Bacteroidetes, with the first 3 phyla accounting for more than 90% of the total sequences. Proteobacteria were present in higher proportions in asthmatic patients (37% vs 15%, P < .001). In contrast, Firmicutes (47% vs 63%, P = .17) and Actinobacteria (10% vs 14%, P = .36) were found more frequently in samples from nonasthmatic subjects, although this was not statistically significant. Hierarchical clustering produced 2 significant clusters: one contained primarily asthmatic samples and the second contained primarily nonasthmatic samples. In addition, samples from asthmatic patients had greater bacterial diversity compared with samples from nonasthmatic subjects.

Conclusion: Patients with mild asthma have an altered microbial composition in the respiratory tract that is similar to that observed in patients with more severe asthma.

Conflict of interest statement

Disclosure of potential conflict of interest: The rest of the authors declare that they have no relevant conflicts of interest.

Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Figures

FIG 1

Median percentage values of bacterial phyla in samples from asthmatic and nonasthmatic subjects found by using the RDP Classifier, version 2.1. Pie charts show the distribution of organisms for asthmatic and nonasthmatic subjects.

FIG 2

Hierarchical clustering of samples from asthmatic and nonasthmatic subjects. The green and blue boxes indicate the asthmatic and nonasthmatic clusters, respectively. A1–A10, Samples from asthmatic patients; C1–C10, samples from nonasthmatic subjects. The numbers on the branches indicate the bootstrap support for the cluster.

FIG 3

Relative abundance of 15 bacterial families in samples from asthmatic and nonasthmatic subjects. The value of zero indicates a similar percentage contribution of the bacterial family in both asthmatic and nonasthmatic individuals. A positive value for a bacterial family indicates its higher percentage abundance in asthmatics and a negative value indicates the higher abundance in nonasthmatics. For example, Enterobacteriaceae is ~4% more abundant in asthmatics.

Figure 4

Diversity of samples from asthmatic and nonasthmatic subjects based on bacterial genera. The Shannon Index is a mathematic measure commonly used to characterize the species diversity in a community, and it accounts for both the diversity and evenness of the bacterial species present in samples from asthmatic and nonasthmatic subjects.