The airway microbiota of neonates colonized with asthma-associated pathogenic bacteria

Abstract

Culture techniques have associated colonization with pathogenic bacteria in the airways of neonates with later risk of childhood asthma, whereas more recent studies utilizing sequencing techniques have shown the same phenomenon with specific anaerobic taxa. Here, we analyze nasopharyngeal swabs from 1 month neonates in the COPSAC₂₀₀₀ prospective birth cohort by 16S rRNA gene sequencing of the V3-V4 region in relation to asthma risk throughout childhood. Results are compared with previous culture results from hypopharyngeal aspirates from the same cohort and with hypopharyngeal sequencing data from the later COPSAC₂₀₁₀ cohort. Nasopharyngeal relative abundance values of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are associated with the same species in the hypopharyngeal cultures. A combined pathogen score of these bacteria's abundance values is associated with persistent wheeze/asthma by age 7. No other taxa are associated. Compared to the hypopharyngeal aspirates from the COPSAC₂₀₁₀ cohort, the anaerobes Veillonella and Prevotella, which have previously been implicated in asthma development, are less commonly detected in the COPSAC₂₀₀₀ nasopharyngeal samples, but correlate with the pathogen score, hinting at latent community structures that bridge current and previous results. These findings have implications for future asthma prevention efforts.

Fig. 1. Study overview.

Characteristics and previously published results on airway bacteria and asthma from the two COPSAC cohorts^,. Created with BioRender.com.

Fig. 2. Nasopharyngeal microbiota associates with hypopharyngeal culture and childhood asthma.

a Comparison of results from culturing of hypopharyngeal aspirates with relative abundance values from 16S rRNA gene sequencing of nasopharyngeal swabs, for each of the four species identified in the cultures. Box plots indicate median (middle line), 25th, 75th percentile (box), and 1.5× interquartile range (whiskers). P values are from Wilcoxon tests. b Kaplan–Meier curve showing the risk of asthma by each tertile of summed relative abundance of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis (pathogen score) with 7-year risk estimates per curve. Censoring is marked with +. c Associations between the pathogen score and asthma-related endpoints at 3, 5, and 7 years. All Hazard Ratios (HR, dots) are quantified per standard deviation (SD) increase in the pathogen score with corresponding 95% confidence intervals (error bars), calculated with Cox proportional hazards regression. OCS oral corticosteroid, ICS Inhaled corticosteroid. N = 285. Data from the COPSAC₂₀₀₀ cohort. P values are two-sided and not adjusted for multiple comparisons.

Fig. 3. Individual taxa and childhood asthma risk.

Differential abundance tests of individual bacterial species and asthma by age 7, using two different statistical models; a Cox regression of time-to-asthma quantified as Hazard Ratio per log2 increase in species relative abundance and b DESeq2 analysis comparing abundance of the species between children who later developed asthma vs those who did not. The same set of four species, that are significant in either analysis, are highlighted. FDR, false discovery rate. N = 285. Data from the COPSAC₂₀₀₀ cohort. P values are two-sided and not adjusted for multiple comparisons—instead, the False Discovery Rate (FDR) cutoff is shown with a dotted line.

Fig. 4. Apparent observed cohort differences in the detection of Veillonella and Prevotella.

a, b Comparison of COPSAC₂₀₁₀ hypopharyngeal aspirates and COPSAC₂₀₀₀ nasopharyngeal swabs by genus names. Veillonella and Prevotella are highlighted due to their significant association with asthma in COPSAC₂₀₁₀ (dark gray). Gemella, Streptococcus, and Neisseria also contributed to the bacterial asthma score, but were not individually significant after FDR correction (light gray). Different regions were amplified in the two cohorts (COPSAC₂₀₀₀—V3-V4; COPSAC₂₀₁₀—V4), but the bioinformatics and data processing pipeline was identical (DADA2 + AnnotIEM, see methods). c, d Genus abundance values of Veillonella and Prevotella are not associated with persistent wheeze/asthma by age 7 in COPSAC₂₀₀₀. e Veillonella and/or Prevotella presence is not associated with persistent wheeze/asthma by age 7 in COPSAC₂₀₀₀. Their association estimate attenuated after mutual adjustment with the pathogen score, which in contrast did not attenuate. Dots indicate estimates, error bars indicate 95% confidence intervals (CI). N = 285 (COPSAC₂₀₀₀), N = 641 (COPSAC₂₀₁₀). P values are two-sided and not adjusted for multiple comparisons.

Fig. 5. Correlation structure between Veillonella, Prevotella, and pathogenic genera.

Correlation heatmap between the most common genera in COPSAC₂₀₀₀, arranged by hierarchical clustering, using the Spearman rank correlation metric. We observe that Veillonella and Prevotella form a cluster with two of the three pathogen genera, Streptococcus and Haemophilus, which also includes a few other taxa. Highlighting was performed manually. See also Supplementary Fig. 6. N = 285.