Figures
- FIGURE 1
Flow diagram for BEAT-COPD (Biomarkers to Target Antibiotic and Systemic Corticosteroid Therapy in COPD Exacerbations) subject enrolment.
- FIGURE 2
Microbiome shifts during exacerbations. a) α diversity (Faith's phylogenetic diversity (PD)) and composition of major taxonomic groups at both phylum and genus levels in samples collected across the four visit types: stable, exacerbation (Exac), post-therapy (Post) and recovery (Rec). b) Box and whisker plots showing the relative abundances of Streptococcus, Haemophilus and Moraxella in samples collected across the four visits. c) Heterogeneity in Moraxella changes among individuals. Lines connect paired stable and exacerbation samples collected in the same visit series from the same subjects, and were coloured by increase or decrease of Moraxella during exacerbations. Only paired stable and exacerbation samples were included.
- FIGURE 3
The microbiome discriminates bacterial and eosinophilic exacerbations. a) α diversity (Shannon's H) and composition of major taxonomic groups at both phylum and genus levels in exacerbation samples with different exacerbation phenotypes. The number of samples is indicated for each subgroup in the bar chart. b) Principal coordinate analysis (PCoA) and c) unweighted pair group method with arithmetic mean clustering show distinct clustering of samples in bacterial and eosinophilic subgroups. d) Partial least squares discriminant analysis classification of bacterial and eosinophilic exacerbations using clinical, microbiome and their combined variables at both phylum (L6) and operational taxonomic unit (OTU) levels. The models were evaluated in terms of area under the curve (AUC), R2 and Q2 scores. B: bacterial; V: viral; E: eosinophilic; BE: bacterial and eosinophilic; BV: bacterial and viral; and Pauci: pauci-inflammatory. *: p<0.05.
- FIGURE 4
Microbiome changes due to oral corticosteroids and antibiotics. α diversity (Shannon's H) and composition of major taxonomic groups at both phylum and genus levels in exacerbations (Exac), post-therapy (Post) and recovery (Rec) samples of subjects treated with steroids, antibiotics or a combination of both. Only visit series with a complete cycle of exacerbation, post-therapy and recovery visits were included. The number of visit series is indicated for each subgroup. *: p<0.05 using ANOVA.
- FIGURE 5
Bacterial co-existence and co-exclusion relationships with operational taxonomic units (OTUs) and host factors. Interaction networks of a) microbiome and b) microbiome and clinical factors. Each node represents an OTU or a clinical trait. The OTUs were coloured by the class-level taxonomy. The five co-existence OTUs are highlighted in the dotted ellipse in a). The clinical traits were grouped together at the left of the network in b). Each edge represents a significant correlation coloured by co-existence or co-exclusion relationships. Edge width is proportional to the absolute value of Pearson correlation coefficient. The size of the node is proportional to its degree of connectivity. The degrees are shown in parentheses for highly connected nodes. For the clinical variables, both the total degree and the degree of connectivity to OTUs are shown. MMP: matrix metalloproteinase; CXCL: chemokine (C-X-C motif) ligand; IL: interleukin.
Tables
- TABLE 1
Major clinical characteristics of subjects at baseline
Sex male/female 65/22 Age at baseline 68 (45–87) Age at diagnosis 61 (30–83) BMI kg·m−2 26.40 (16.67–38.19) GOLD stage# 1 1 2 35 3 32 4 19 Smoking status Current smoker 37 Ex-smoker 48 Nonsmoker 2 Smoking history pack-years 50 (6–158) Number of exacerbations 1 46 2 31 3 9 4 1 Treatment¶,+ Antibiotics 21 Steroids 8 Antibiotics and steroids 65 FEV1 L 1.3±0.1 FEV1 % pred 47.4±2.0 FEV1/FVC ratio 46.7±1.4 CRQ score 16.2±0.5 Symptom VAS total mm 159.6±8.5 SGRQ score 52.9±1.9 Data are presented as n, mean (range) or mean±sem. BMI: body mass index; GOLD: Global Initiative for Chronic Obstructive Lung Disease; FEV1: forced expiratory volume in 1s; FVC: forced vital capacity; CRQ: Chronic Respiratory Disease Questionnaire; VAS: visual analogue scale; SGRQ: St George's Respiratory Questionnaire. #: baseline visits prior to the stable visits for sputum collection; ¶: treatments administered for exacerbations; assessments at exacerbation were prior to initiation of therapy; +: numbers represent exacerbation events, thus include subjects with more than one exacerbation.
- TABLE 2
Major clinical characteristics of subjects over four visits
Visits p-value Stable Exacerbation Post-therapy Recovery Visits 106 137 136 97 FEV1 L 1.3±0.1 1.1±0.1 1.2±0.1 1.2±0.1 0.1 FEV1 % pred 49.6±1.8 48.1±1.6 48.5±1.6 49.0±2.0 0.94 FEV1/FVC ratio 48.0±1.3 48.9±1.3 49.2±1.2 48.7±1.4 0.92 CRQ score 16.6±0.5 12.7±0.4 16.0±0.4 16.6±0.5 <0.001 Symptom VAS total mm 174.2±8.5 255.7±6.5 150.2±7.5 151.7±8.5 <0.001 Sputum pathogen detection 47 55 42 32 0.30 Sputum cell count cells×106·g−1 5.8 (4.4–7.3) 14.6 (11.7–17.5) 8.1 (5.7–10.4) 5.1 (3.6–6.6) <0.001# Sputum neutrophil count % 69.1 (64.8–73.5) 79.1 (75.4–82.9) 74.2 (70.6–77.8) 68.7 (63.9–73.5) <0.001 Sputum eosinophil count % 2.8 (1.9–3.7) 4.3 (2.6–6.0) 1.5 (1.0–2.0) 3.8 (2.3–5.3) <0.01# Sputum lymphocyte count % 0.5 (0.4–0.7) 0.5 (0.4–0.6) 0.6 (0.4–0.7) 0.5 (0.4–0.7) 0.91 Blood cell count cells×109·L−1 8.3 (7.7–8.9) 9.5 (8.8–10.2) 10.4 (9.5–11.2) 8.8 (8.1–9.6) <0.01 Blood neutrophils cells×109·L−1 5.5 (5.1–5.9) 6.9 (6.4–7.4) 7.6 (7.1–8.2) 5.9 (5.4–6.4) <0.001 Blood eosinophils cells×109·L−1 0.2 (0.2–0.3) 0.3 (0.2–0.3) 0.2 (0.2–0.3) 0.3 (0.2–0.3) 0.16 Blood lymphocytes cells×109·L−1 2.1 (1.9–2.3) 2.1 (2.0–2.3) 2.3 (2.1–2.5) 2.2 (2.0–2.5) 0.29 Blood basophils cells×109·L−1 0.04 (0.03–0.04) 0.04 (0.04–0.04) 0.04 (0.04–0.04) 0.04 (0.04–0.05) 0.62 Data are presented as n, mean±sem or mean (95% CI). FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity; CRQ: Chronic Respiratory Disease Questionnaire; VAS: visual analogue score. #: these variables were log transformed for statistical analysis.
- TABLE 3
The prevalence (P) and average relative abundance (RA) of predominant operational taxonomic units (OTUs) (average relative abundance >1%) in the lung microbiome
OTU ID Species/subspecies All Stable Exacerbation Post-therapy Recovery P RA P RA P RA P RA P RA Visits n 476 106 137 136 97 4439603 Streptococcus spp. 82.1 13.1 83.0 12.3 78.8 11.6 82.4 13.4 85.6 15.8 4445466 Streptococcus spp. 44.7 10.1 47.2 12.1 45.3 8.9 46.3 10.1 39.2 9.5 509773 Streptococcus spp. 78.4 6.6 85.8 7.3 80.3 6.3 71.3 6.2 77.3 6.9 1059655 Streptococcus spp. 31.5 5.2 30.2 5.2 33.6 5.4 30.9 4.9 30.9 5.6 4462083 Streptococcus infantis 31.7 3.9 29.2 4.3 32.1 4.0 31.6 4.2 34.0 3.0 240755 Haemophilus spp. 69.7 10.7 61.3 9.5 72.3 12.4 71.3 9.5 73.2 11.3 956702 Haemophilus spp. 25.2 5.1 33.0 6.5 24.1 5.1 23.5 5.4 20.6 3.0 4385138 Haemophilus spp. 23.5 1.4 25.5 0.8 27.0 1.9 22.1 0.9 18.6 1.9 861881 Moraxella spp. 46.2 5.6 45.3 5.0 57.7 10.0 41.9 3.4 37.1 3.3 269930 Pseudomonas veronii 33.8 2.4 42.5 3.0 32.8 2.6 32.4 2.2 27.8 1.8 269901 Pseudomonas spp. 12.8 1.6 14.2 1.8 12.4 1.2 14.7 1.4 9.3 2.4 342427 Veillonella dispar 93.3 2.7 91.5 2.4 93.4 2.2 94.9 3.2 92.8 3.0 4326277 Unclassified in Gemellaceae 84.0 2.6 88.7 2.8 81.8 2.3 78.7 2.4 89.7 3.0 4411138 Rothia mucilaginosa 73.1 2.4 75.5 3.0 74.5 2.3 71.3 2.1 71.1 2.5 4396235 Neisseria spp. 58.6 2.2 60.4 2.3 59.9 2.2 55.9 2.4 58.8 1.8 12574 Actinomyces spp. 79.2 1.6 84.0 1.7 79.6 1.3 72.8 1.7 82.5 1.7 257492 Granulicatella spp. 83.8 1.4 86.8 1.4 85.4 1.2 80.9 1.3 82.5 1.7 4307391 Prevotella melaninogenica 39.1 1.1 41.5 1.1 39.4 1.2 40.4 1.2 34.0 0.8 Data are presented as %, unless otherwise stated. The OTUs were firstly grouped by their genera and then ranked by their RAs.
- TABLE 4
List of clinical variables significantly associated with microbial α and β diversity in group I subjects
α diversity β diversity Shannon's H Observed species Faith's phylogenetic diversity Chao1 Positive/negative correlation OTU Genus (L6) Phylum (L2) Sputum CXCL8/IL-8 *** * *** *** Negative # ** ** Baseline SGRQ symptom # *** *** *** Negative BMI * * * # Positive # # #,¶ Sputum CXCL11/ITAC # * * ** Positive Serum TNF-α * * # # Positive Serum SAA-1 # * * # Positive Serum CCL26/eotaxin3 * # # # Negative Serum CSF-2 * # # # Positive # # * Serum IL-10 * # # # Positive Sputum MMP-8 # * # # Negative Blood monocytes # # ** # Positive Blood basophils # # * # Positive Serum MMP-7 ** ** # Sputum neutrophil percentage ** ** ** Exacerbation frequency # # #,¶ OTU: operational taxonomic units; CXCL: chemokine (C-X-C motif) ligand; IL: interleukin; SGRQ: St George's Respiratory Questionnaire; BMI: body mass index; ITAC: interferon inducible T-cell α chemoattractant; TNF: tumour necrosis factor; SAA: serum amyloid A; CCL: CC-chemokine ligand; CSF: colony stimulating factor; MMP: matrix metalloproteinase. #: p≥0.05 and absent in the model; ¶: not significantly associated with L2 β diversity, but present in its model. *: p<0.05; **: p<0.01; ***: p<0.001.
Disclosures
Supplementary material
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- Supplementary material - Supplementary methods, figures S1-S5 and tables S1-S5
- Supplementary table 1
