The Lung Microbiome in Young Children with Cystic Fibrosis: A Prospective Cohort Study

Abstract

The cystic fibrosis (CF) lung harbours a diverse microbiome and reduced diversity in the CF lung has been associated with advancing age, increased inflammation and poorer lung function. Data suggest that the window for intervention is early in CF, yet there is a paucity of studies on the lung microbiome in children with CF. The objective of this study was to thoroughly characterise the lower airway microbiome in pre-school children with CF. Bronchoalveolar lavage (BAL) samples were collected annually from children attending the three clinical centres. Clinical and demographic data were collated on all subjects alongside BAL inflammatory markers. 16S rRNA gene sequencing was performed on the Illumina MiSeq platform. Bioinformatics and data analysis were performed using Qiime and R project software. Data on 292 sequenced BALs from 101 children with CF and 51 without CF show the CF lung microbiome, while broadly similar to that in non-CF children, is distinct. Alpha diversity between the two cohorts was indistinguishable at this early age. The CF diagnosis explained only 1.1% of the variation between the cohort microbiomes. However, several key genera were significantly differentially abundant between the groups. While the non-CF lung microbiome diversity increased with age, diversity reduced in CF with age. Pseudomonas and Staphylococcus were more abundant with age, while genera such as Streptococcus, Porphyromonas and Veillonella were less abundant with age. There was a negative correlation between alpha diversity and interleukin-8 and neutrophil elastase in the CF population. Neither current flucloxacillin or azithromycin prophylaxis, nor previous oral or IV antibiotic exposure, was correlated with microbiome diversity. Consecutive annual BAL samples over 5 years from a subgroup of children demonstrated diverse patterns of development in the first years of life.

Keywords: bronchoalveolar lavage; bronchoscopy; children; cystic fibrosis; infection; inflammation; lung; microbiome; microbiota; paediatrics.

Figure 1

Heatmap of all genera present at ≥1% relative abundance in ≥10% of all samples.

Figure 2

Comparison of the CF and control lung microbiome. (A) Violin plots of alpha diversity measures. (B) The percentage relative abundance of genera which were significantly differentially abundant between cohorts (i.e., LDA > 4) as determined by LEfSe (error bars show standard error).

Figure 3

Cross sectional analysis demonstrating differences, with age, in the average phylum-level composition of the lower airway microbiota in CF and control BAL from 1 to 6 years of age.

Figure 4

Age-associated changes in the microbiome of children with CF and without CF (control). (A) alpha diversity displayed as violin plots and (B) beta diversity displayed as unweighted UniFrac PCoA plots (ellipses illustrate 80% confidence intervals) of the lower airway microbiota across three age groups: 0 to 2.5 years old, 2.5 to 5 years old and over 5 years old. (C) Genera which were significantly differentially abundant (i.e., LDA >4.0) across these same groups, as determined using LEfSe.

Figure 5

Longitudinal development of the CF airway microbiome over 5 years in six patients. (A) Relative phyla abundance and (B) alpha diversity in the CF lung microbiome.

Figure 6

Comparison of relative abundance of Staphylococcus genus and Pseudomonas genus with flucloxacillin prophylaxis, and long-term azithromycin treatment, on the day of the bronchoscopy (Kruskal–Wallis test).