In this study, CF patient airway colonization was examined using a culture-independent phylogenetic microarray and samples from a cohort of patients defined as clinically stable (no change in pulmonary function for ≥2 months prior to sample collection), aged between 9 months and 72 years (Table S4
). For the purpose of this study it was important to consider only clinically stable time points to determine the longer-term evolution of the CF airway microbiota and avoid the pronounced short-term impact of pulmonary exacerbation and antimicrobial therapy. Deep throat swabs were used to sample pediatric patients in this study, this was to ensure inclusion of neonatal and younger pediatric patients who do not produce sputum. Expectorated sputum samples (following routine spirometry) were collected for adult patients. Despite differences in sampling between the age groups, the mode of sampling did not seem to drive the bacterial community composition. One caveat of airway studies is the necessity to collect the samples through the oral cavity and hence the potential for sample contamination. Both deep throat swab and sputum samples are routinely used for clinical laboratory culture to guide antimicrobial treatment regimes for CF patients. In addition, Rogers and colleagues have previously demonstrated that distinct bacterial communities exists in oral and sputum CF samples 
, also supporting the hypothesis that oral contamination minimally impacts the assessment of airway samples from CF patients.
A total of 158 bacterial families were detected in these samples, the majority of taxa detected in these families have not been previously associated with cystic fibrosis. There are several potential explanations for this including that the PhyloChip is a highly sensitive molecular assay, capable of detecting organisms that are present at levels of only 0.01% of the total community. Validation by sequence analysis of Veillonella parvula
confirms its reported presence in this niche by the array, suggesting that multiple organisms may co-habit this niche. Indeed, other culture-independent T-RFLP-based studies (estimated by the authors as having a sensitivity of 1% of the population), clone library sequencing and temporal temperature gradient gel electrophoresis (TTGE) have also detected a number of species that had not previously been associated with CF airways 
. Even with the limitations of culture-based methods, previous studies have also demonstrated the presence of uncharacteristic species in CF airways using this approach 
. More recently a sequence-based study of a CF patient airway sample revealed the presence of multiple unusual species not previously reported in this niche such as Dolosigranulosum pigrum, Kocuria rosea, Granulicatella
spp. and Bergeyella
spp. amongst others 
. All of the reported species detected in this sequence-based study were also detected by PhyloChip in several of the patients in our study. This in addition to our sequence-based validation, collectively suggest that a multitude of bacterial species do indeed exist in CF airway samples and underscore the need to perform truly deep sequencing to overcome the issue of the dominant species present and detect the “rare biosphere” present.
The impact of sequence depth on interpretation of results was recently demonstrated by Qin et al 
, in a large-scale sequence-based human microbiome study of 124 European individuals. Increasing the sequence depth coverage for samples from two individuals (from ~4 Gb to >8.5 Gb) resulted in an increase in the number of strains common to these two individuals by 25% 
. This demonstrates that human host microbiota are intrinsically diverse and that our current view of these assemblages is only curtailed by the limited depth of sequencing that has been used to interrogate them. Though we recognize that the array-based technology used in this study is potentially subject to cross-hybridization at the taxon-level, it nonetheless provides a standardized tool for high-resolution profiling of samples, permitting detection of low abundance species in dominated communities and relative changes in community composition that can be related to clinical measurements and features of the disease.
In natural systems, colonizing populations are observed to develop progressively, typically leading to increased biomass, productivity and diversity 
. This has recently been exemplified in the human gastrointestinal tract; initial colonization by pioneer aerobic species is followed by facultative anaerobes and finally strict anaerobes, concomitant with an increase in bacterial biomass and productivity characteristic of the stable adult microbiota 
. Though this is a cross-sectional study, data presented here provides insights into the progression of CF airway colonization. Evidence for initial diversification in younger patients, decreases in diversity in older patients and the presence of specialized communities of phylogenetically related species () associated with poor pulmonary function in these older patient suggest that CF airway microbiota may also follow the rules of community assembly previously reported at other host niches. This form of colonization involving an initial rapid rise in species diversity as successive invasions occur, followed by species replacement as the community develops is common, and has previously been reported for other ecosystems and at higher trophic levels 
. Once established, severe antimicrobial-based perturbation of microbial communities has been shown to lead to long-term changes in community composition and a loss of diversity in animal models 
. Subsets of older CF patients exhibit diminished improvement in lung function in response to antimicrobial administration compared with younger CF patients 
, who also typically exhibit better pulmonary function. This suggests that the more phylogenetically-related microbial assemblages detected in older CF patients may be more resistant to these antimicrobials and contribute to poorer airway function, while the more diverse pediatric microbial assemblage is more sensitive to antimicrobial perturbation and linked to improved pulmonary function. More recently a study has demonstrated a “like begets like” phenomenon, in that colonization of a specific niche by particular keystone species results in “invasion” of that community by other phylogenetically related species 
. It appears from our data that a similar scenario exists in older CF patients whose airway microbiota exhibits phylogenetically related members and is largely composed of Pseudomonadaceae. That multiple members of the Pseudomonadaceae may co-exist in CF airways is not unprecedented, Harris and colleagues have previously demonstrated with relatively shallow sequencing depth, the presence of other Pseudomonas
species other than P. aeruginosa,
in 50% of their CF patient airways 
, suggesting that multiple members of the Pseudomonadaecaeae may co-exist in this niche.
Bacterial community structure and composition associated with CF patient age.
It seems counter-intuitive that a greater diversity of organisms (in younger CF patients) would be associated with better lung function, however several studies have demonstrated that dramatic changes in community structure through loss of diversity as reported here, are increasingly being associated with chronic inflammatory diseases, pathogen outgrowth and poor clinical outcome 
. It appears therefore, that bacterial community structure and composition represents an important factor in defining the functionality of the microbial assemblage and host health status. However analysis of CF cohorts is difficult and confounded by the fact that a number of the older patients in this study have less severe disease, which may be associated with their microbial community composition or different treatment regimens. In addition, there is also the possibility that the findings reported here are due to other factors that track with age such as antibiotic use, chest physical therapy, adherence, nutrition or other factors. Nonetheless, this study demonstrates that distinct microbial assemblages are associated with CF patient age and that community composition is correlated with CF genotype and aspects of pulmonary disease in this patient population.
This study also provided information on the relationship between CFTR mutation and the airway microbiota. It has previously been reported that CFTR mutation changes the airway microenvironment 
, which would presumably influence the microbial community that establishes in this niche. A strong relationship between ΔF508 CFTR mutation and absence of multiple members of the Mycobacteriaceae, Staphylococcaceae, Enterobacteriaceae amongst others with a concomitant rise in abundance of members of the Pseudomonadaceae was identified in older CF patients with this genotype. This suggests that mutations rendering the CFTR non-functional are associated with a loss of airway bacterial (including pathogen) diversity and outgrowth of a small group of phylogenetically-related species as these patients age. Whether this is directly related to the severity of the mutation and the creation of a distinct niche due to lack of functional CFTR or due to the treatments necessary to manage these patients (or a combination of both) is unclear. However, patients with heterozygous ΔF508 or non-ΔF508 mutations also exhibited distinct pathogen profiles. Older heterozygous ΔF508CF patient airways were associated with Moraxellaceaea and Sphingobacteriaceae, two bacterial families that have recently been associated with chronic obstructive pulmonary disease 
and invariant Natural Killer T cell induction in asthmatic mice 
. While non-ΔF508 patients were associated with a relatively less severe pathogen profile. This study, albeit small, demonstrates that homozygous-ΔF508 mutation is associated with the most substantial change in airway community structure and phylogeny in older patients. Furthermore, particular CFTR mutations, which are known to influence the airway environment 
, are associated with distinct pathogen profiles, a finding that may explain the range of severity in pulmonary symptoms commonly observed with various CFTR genotypes, and has implications for patient-tailored care 
It is important to note that detailed functional analyses of longitudinal CF airway samples are necessary to comprehensively determine the impact of treatment on the airway microbiota. Given our data, it appears that older CF patients possess a stable core of pathogenic organisms which presumably are selected for over time due to repeated antimicrobial pressure. With the increasing lifespan of CF patients due to successful disease management, understanding the long-term impact of antimicrobials on the CF microbiota may lead to further improvements in treatment strategies and life expectancy. Novel approaches that involve manipulation of microbial consortia, rather than destruction of the community structure may offer an alternative therapeutic approach for patient-tailored management of chronic airway disease. This concept is gaining increasing support 
and there is evidence for its efficacy, although the mechanism underlying these benefits remains unclear. A recent pilot study of pediatric CF patients supplemented with a probiotic Lactobacillus
species or placebo demonstrated that in addition to reduced gastrointestinal inflammation, patients who received the probiotic species exhibited a significant reduction in hospitalizations for pulmonary exacerbation 
. Certainly with the advent of newborn screening programs and sophisticated culture-independent tools to comprehensively monitor patient airways, the opportunity to intervene at a very early age and alter the course of airway microbial colonization to improve patient outcome is unprecedented.
The CF microbiota is a complex community, but the use of molecular ecological approaches permits the progression of CF airway disease to be comprehensively explored. This work provides the foundation for an improved understanding of polymicrobial CF airway colonization, the relationship between community structure, composition and lung function in CF patients and the influence of CFTR mutation on the airway microbiota. Antibiotic administration, the acquisition of organisms from the internal and external environments, and changes associated with patient age (increased lung surface area, hormonal changes) represent key influences on this ecosystem. Understanding the mechanistic relationship between community dynamics, pathogen abundance and behavior, and the host immune response is crucial to further extending the lifespan of this patient population.