Airway microbiota across age and disease spectrum in cystic fibrosis

Abstract

Our objectives were to characterise the microbiota in cystic fibrosis (CF) bronchoalveolar lavage fluid (BALF), and determine its relationship to inflammation and disease status.BALF from paediatric and adult CF patients and paediatric disease controls undergoing clinically indicated bronchoscopy was analysed for total bacterial load and for microbiota by 16S rDNA sequencing.We examined 191 BALF samples (146 CF and 45 disease controls) from 13 CF centres. In CF patients aged <2 years, nontraditional taxa (e.gStreptococcus, Prevotella and Veillonella) constituted ∼50% of the microbiota, whereas in CF patients aged ≥6 years, traditional CF taxa (e.gPseudomonas, Staphylococcus and Stenotrophomonas) predominated. Sequencing detected a dominant taxon not traditionally associated with CF (e.gStreptococcus or Prevotella) in 20% of CF BALF and identified bacteria in 24% of culture-negative BALF. Microbial diversity and relative abundance of Streptococcus, Prevotella and Veillonella were inversely associated with airway inflammation. Microbiota communities were distinct in CF compared with disease controls, but did not differ based on pulmonary exacerbation status in CF.The CF microbiota detected in BALF differs with age. In CF patients aged <2 years, Streptococcus predominates, whereas classic CF pathogens predominate in most older children and adults.

FIGURE 1

a, b) Total bacterial load (TBL) and c, d) Shannon diversity index detected in bronchoalveolar lavage fluid (BALF) samples from cystic fibrosis (CF) and disease control participants. a) TBL for disease control, CF paediatric and CF adult BALF. b) TBL for disease control and CF paediatric BALF based on positive (+) or negative (–) bacterial culture results. c) Shannon diversity index for disease control, CF paediatric and CF adult BALF. d) Shannon diversity index for disease control and CF paediatric BALF based on (+) or negative (–) bacterial culture results. TBL was highest and Shannon diversity index lowest in CF BALF with positive cultures. TBL was measured on all BALF samples, whereas the Shannon diversity index was calculated only for samples with successful amplification and sequencing. Boxes show 25–75th interquartile range (IQR) with whiskers showing 1.5 times the IQR. Median indicated by a solid line in the box. Outliers are shown as individual data points. TBL limit of detection based on reagent control samples is indicated by the dashed line. *: p<0.05; **: p<0.01 for statistically significant differences.

FIGURE 2

a) Prevalence (% of samples with specific taxa detected) and b) relative abundance of the most frequently detected taxa and taxa associated with traditional cystic fibrosis (CF) pathogens from disease control (DC) and paediatric CF bronchoalveolar lavage fluid (BALF). a) Prevalence of bacterial taxa detected from CF and disease control BALF. Most taxa were detected at higher prevalence in CF compared with disease control BALF. b) Median relative abundance for bacterial taxa associated with nontraditional and traditional CF bacteria detected from CF and disease control BALF. Streptococcus, Prevotella, Veillonella, Neisseria and Porphyromonas were detected at higher relative abundance in disease control compared with CF BALF. Examining traditional CF taxa, Staphylococcus was detected at higher relative abundance in CF compared with disease control BALF. Although not statistically significant, taxa associated with Pseudomonas+, Stenotrophomonas, Burkholderia and Achromobacter+ had a wider range of relative abundance in CF compared with disease controls. “Pseudomonas+” contains sequences assigned to the taxa Pseudomonadales and Pseudomonas. “Achromobacter+” contains sequences assigned to the taxa Alcaligenaceae and Achromobacter. Median relative abundance was calculated based on BALF samples positive for a given taxa. Relative abundance is displayed as median (line), boxes indicate 25–75th interquartile range, and whiskers extend from minimum and maximum values. *: p<0.05; **: p<0.01 for statistically significant differences.

FIGURE 3

Heatmap of associations with clinical characteristics, microbiota and airway inflammation in cystic fibrosis patients. Spearman’s correlation coefficients for total bacterial load, diversity and relative abundance of bacterial taxa related to age, lung function (forced expiratory volume in 1 s (FEV₁) % pred) and bronchoalveolar lavage fluid cell counts (total white blood cells, absolute neutrophil count and percentage neutrophils). “Pseudomonas+” contains sequences assigned to the taxa Pseudomonadales and Pseudomonas. “Achromobacter+” contains sequences assigned to the taxa Alcaligenaceae and Achromobacter. Red/brown: positive correlations; blue/black: negative correlations. For example, lower diversity was associated with older age and higher markers of inflammation. Higher relative abundance of Prevotella, Streptococcus and Veillonella was associated with younger age and less inflammation. Positive (up) and negative (down) correlations are also indicated by the direction of the arrowhead symbol in the heatmap. Correlation values >0.5 have p-values <0.05.

FIGURE 4

Cross-sectional results by age group and pulmonary exacerbation status for patients with cystic fibrosis for a) total bacterial load (TBL) by pulmonary exacerbation status, b) Shannon diversity index by pulmonary exacerbation status, c) TBL by age and d) Shannon diversity index by age. Disease controls are shown in panels a) and b). Boxes show 25–75th interquartile range (IQR) with whiskers showing 1.5 times the IQR. Median indicated by a solid line in the box. TBL was measured on all bronchoalveolar lavage fluid samples, whereas the Shannon diversity index was calculated only for samples with successful amplification and sequencing. TBL limit of detection based on reagent control samples is indicated by a dashed line.

FIGURE 5

Cross-sectional plots of major taxa detected by sequencing by age group in cystic fibrosis (CF). a) Median relative abundance, based on samples with detectable taxa, for Pseudomonas+, Stenotrophomonas, Streptococcus, Prevotella, Staphylococcus, Haemophilus, Burkholderia and Veillonella. b) Combined median relative abundance for traditional CF taxa (Pseudomonas+, Staphylococcus, Stenotrophomonas, Haemophilus and Burkholderia) and taxa not traditionally identified from culture (Streptococcus, Neisseria, Porphyromonas, Prevotella and Veillonella). “Pseudomonas+” contains sequences assigned to the taxa Pseudomonadales and Pseudomonas.