Initial acquisition and succession of the cystic fibrosis lung microbiome is associated with disease progression in infants and preschool children

Abstract

The cystic fibrosis (CF) lung microbiome has been studied in children and adults; however, little is known about its relationship to early disease progression. To better understand the relationship between the lung microbiome and early respiratory disease, we characterized the lower airways microbiome using bronchoalveolar lavage (BAL) samples obtained from clinically stable CF infants and preschoolers who underwent bronchoscopy and chest computed tomography (CT). Cross-sectional samples suggested a progression of the lower airways microbiome with age, beginning with relatively sterile airways in infancy. By age two, bacterial sequences typically associated with the oral cavity dominated lower airways samples in many CF subjects. The presence of an oral-like lower airways microbiome correlated with a significant increase in bacterial density and inflammation. These early changes occurred in many patients, despite the use of antibiotic prophylaxis in our cohort during the first two years of life. The majority of CF subjects older than four harbored a pathogen dominated airway microbiome, which was associated with a further increase in inflammation and the onset of structural lung disease, despite a negligible increase in bacterial density compared to younger patients with an oral-like airway microbiome. Our findings suggest that changes within the CF lower airways microbiome occur during the first years of life and that distinct microbial signatures are associated with the progression of early CF lung disease.

Fig 1. Taxonomic profile of replicate BAL samples.

Relative abundance of bacterial taxa present in replicate BAL samples for each patient at the (A) phylum and (B) genus or lowest identifiable taxonomic level. Secondary axis (B) indicates the Pearson product-moment correlation coefficient (PPMCC) when comparing the proportion of sequences assigned to each taxon between respective replicates.

Fig 2. Merged sequence data for replicate BAL aliquots shows distinct sample groupings.

Scaled Bray-Curtis distance NMDS performed on OTUs in merged replicate BAL samples (Stress: 0.043, non-metric fit R² = 0.998). The average background community (AvgBG, white circle) was treated as an additional sample and included for comparison. Patient age at the time of sampling is denoted and groups are labeled and indicated by a dashed line.

Fig 3. Principle component analysis (PCA) of the 23 most abundant taxa delineates three distinct bacterial associations or cluster types.

Scaled PCA of the 23 most abundant taxa (representing ≥0.5% of the average relative abundance), for all RML samples and the average background community (white circle). Vector length and direction indicate the relative contribution of each taxon. Three associative groups of taxa are apparent. These clusters types can be summarized as background signal (C1, green), oral-like (C2, blue), or pathogen (C3, red) communities.

Fig 4. Relative abundance of each cluster type in BAL samples shows an age-associated trend from C1 to C3.

Relative abundance of each cluster type suggests acquisition of an oral-like airway microbiome between one and two years of age. A gradual transition to a pathogen dominated community type begins around age four. Bacterial density is presented as the log of 16S rRNA gene copies/ml BAL for each sample on the secondary axis; samples with insufficient template for qPCR analysis are displayed as gray filled.

Fig 5. Cluster type is associated with bacterial density and clinical measurements of disease.

(A) Age (years), (B) bacterial density (16S rRNA gene copies/ml BAL; average background signal denoted by dashed line), (C) total immune cell counts (TCC)/ml BAL, (D) total neutrophils/ml BAL, (E) IL-8 (picograms)/ml BAL, (F) bronchial wall thickening, and (G) bronchiectasis scores show significant changes based on cluster type. Significance between groups (C1: green, C2: blue, C3: red) were determined through Tukey’s HSD where single, double, and triple asterisks denote significance below 0.05, 0.005, and 0.0005, respectively. Outliers are defined as values above or below 1.5 times the difference in interquartile range above and below the quartiles within each group. Additional statistical analyses on these data can be found in S1 Table.