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.