Discussion
Here, we report for the first time the development of the airway microbiota in children with PCD, and compare it to that in a similar but more severe disease, CF.
Our study shows that the airway microbiota is highly individual but differs between groups of children with PCD and CF, a novel finding. Several previous studies in CF have also demonstrated high interpatient variability in the airway microbiota,6 18 strengthening arguments for personalised medicine. There was greater diversity in PCD than CF with a significant difference in both alpha and beta diversity. Since most patients with PCD studied had genotypes associated with a milder phenotype, this could suggest increased diversity of the microbiota is associated with milder disease. The significance of this is unclear, however, given that the median FEV1% and hence one marker of disease severity was similar between both groups.
Interpretation of diversity with disease severity is complex. Changes in diversity reflect changes in the relative abundance of organisms. Samples with an even spread of different organisms have greater diversity whereas those dominated by an individual organism have lower diversity. Therefore, changes in diversity may reflect presence or absence of a dominant pathogen, which drives disease severity.
There was no significant relationship between diversity (alpha or beta) and spirometry for either disease. One interpretation is that diversity of the microbiota does not influence disease severity. Disease severity in both groups was however relatively mild, reducing the ability to detect such a relationship. Furthermore, there was no significant difference in FEV1 between PCD and CF with the median FEV1 80% predicted in both groups. This is slightly lower than the median for our centre (RBH) from the UK CF Registry data for that year (FEV1 86.5% predicted in 2013) and a selection bias cannot be excluded. Thus, whether differences in the microbiota between PCD and CF relate to differences in disease prognosis could not be fully determined. FEV1, however, is not a sensitive marker for tracking particularly early lung disease in children36 and may be difficult under 5 years of age. In future studies, relating more sensitive tests of distal airway disease such as lung clearance index, forced oscillation or imaging to airway microbiota diversity could potentially be more revealing.
An inverse relationship was seen between Streptococcus and Haemophilus with age only in PCD. This trend parallels that seen in CF NBS infants in the first 2 years of life.16 Notably the microbiota in PCD and CF NBS appears to have greater similarities than with older children with CF. We speculate the microbiota in PCD is like that of the early microbiota in CF. Whether this is of direct pathological significance, or a marker of another underlying disease process is unclear.
Previous cross-sectional studies in CF identified an increase in diversity during childhood, at least until school age,4 and a decrease in adulthood.6 7 In our study, with the exception of species richness (the number of different organisms), there was no significant difference with age in either alpha or beta diversity in either disease. Many of the children in this study were on prophylactic antibiotics (81% in CF and 58% in PCD). While a treatment effect of prophylactic antibiotics was not seen in either group, given the high proportion of patients receiving prophylaxis, particularly in CF, it is possible that this suppressed changes in the microbiota over time. There were significant differences, however, at genera level: Streptococcus was the most common genus in both groups (31% PCD and 55% CF); Haemophilus was sixfold more abundant in PCD than CF (21% vs 3.2%), whereas Pseudomonas was the second most common genus (8.1%) in CF but not PCD. Several anaerobes were prevalent in PCD as also reported for CF here and elsewhere.
There was reduced diversity with growth of H. influenzae in PCD and P. aeruginosa in CF, suggesting these organisms have important influences on community structure. Previous studies in PCD have demonstrated that while H. influenzae is the most common organism cultured in children, P. aeruginosa becomes the dominant pathogen with age.37 This increase in P. aeruginosa occurs later in PCD than in CF, predominantly after 30 years of age, when transition to a mucoid phenotype also occurs.38 A cross-sectional study of non-CF bronchiectasis found a competitive effect between Haemophilus and Pseudomonas; when Haemophilus were dominant, Pseudomonas were either absent or present in very low abundance and vice versa.39 Possibly the high abundance of Haemophilus in the early CF microbiota and PCD leads to a milder phenotype, with replacement of Haemophilus by Pseudomonas in CF resulting in a more severe phenotype. Interventions aimed at preventing this switch, focussing on the mechanisms whereby Haemophilus is associated with improved prognosis in CF, are needed to further explore this potential relationship and hopefully delineate new therapeutic options.
This study has several strengths. It is the first study of the airway microbiota in children with PCD followed up for over a year. The children with PCD and CF in this study were well matched both by age and disease severity and were sampled frequently (median interval 61 and 42 days in PCD and CF, respectively). This has allowed meaningful comparisons and a detailed investigation of the microbiota to be made. The longitudinal design is important to account for the high interpatient variability in the microbiota seen both in this study and previously.
One limitation is that different sampling methods (TS and sputum) were used at different timepoints. Ideally the same sample type would have been collected at all times, but this proved unfeasible. Comparison of paired TS and sputum (n=16) did not demonstrate a difference in the microbiota and repeating the longitudinal analyses with sputum samples only did not change any conclusions. The relative abundance of Pseudomonas was lower in TS than in sputum samples as in previous studies.40 41 Pseudomonas, however, remained the second most abundant organism whether analysing sputum alone or both sputum and TS combined and the overall trends in community composition and diversity remained unchanged.
While TS are an attractive candidate as a surrogate for lower airway sampling, their accuracy in representing the lower airway microbiota remains debatable. A study of 16 children with CF demonstrated that for many participants, communities were similar between oropharyngeal samples and sputum. In those samples that diverged, there was a higher relative abundance of Pseudomonas, Staphylococcus, Enterobacteriaceae and Haemophilus in sputum samples.41 Similarly, a study of 20 children with CF found high concordance between TS and sputum samples in patients who were Pseudomonas negative.40 In the current study, 94% of patients with PCD and 61% of patients with CF were either ‘free’ or had never grown Pseudomonas at baseline. This may explain why similarity was seen when comparing paired sputum and TS from the children in this study. Previously we have shown that TS correlate with the lower airway microbiota. Although differences exist precluding their use for individual decision making, TS can distinguish between disease groups and are useful for longitudinal study of the microbiota between groups of patients with CSLD.23
More children with CF had gastro-oesophageal reflux disease (GORD) requiring treatment with proton pump inhibitors (PPIs) than children with PCD. GORD and specifically treatment with PPIs may affect the lung microbiota.42 Thus, it is possible that the differences between CF and PCD in this study could relate to GORD. That said we detected no effect of treatment with PPIs.
One limitation of 16S rRNA gene sequencing is that it is often unable to discern species level changes and cannot assess functional changes. It is plausible that while overall community composition may not have changed, functional changes occurred in gene expression, metabolite production or interactions between different species with disease status.43 To this end, analysing longitudinal differences in CSLD with disease status using metagenomics or other functional approaches, as well as including digital droplet PCR to distinguish between absolute and relative abundance changes, would be an important area for future work.
In conclusion, we have demonstrated that the PCD microbiota differs from that of CF. The airway microbiota is highly individual in these diseases, further strengthening arguments for personalised approaches to patient management. Diversity was higher in PCD and community composition differed. There is a greater similarity between PCD and early CF diagnosed on NBS than with later established CF. Pseudomonas is more prevalent in CF contrasting with Haemophilus in PCD. An inverse relationship was seen between Streptococcus and Haemophilus with age in PCD but not CF. A similar relationship has been seen previously in NBS infants with CF.16 This could suggest a switch from a less pathogenic to a more pathogenic community composition later in childhood in CF, potentially influenced by a competitive relationship between Haemophilus and Pseudomonas. Further study to better understand this relationship may lead to new therapeutic approaches.