Assessment of Airway Microbiota and Inflammation in Cystic Fibrosis Using Multiple Sampling Methods

Abstract

Rationale: Oropharyngeal (OP) swabs and induced sputum (IS) are used for airway bacteria surveillance in nonexpectorating children with cystic fibrosis (CF). Molecular analyses of these airway samples detect complex microbial communities. However, the optimal noninvasive sampling approach for microbiota analyses and the clinical relevance of microbiota, particularly its relationship to airway inflammation, is not well characterized.Objectives: The goals of this study were to compare molecular analyses of concurrently collected saliva, OP swabs, IS, and expectorated sputum (ES) from children with CF and to determine the association between microbiota, lung function, and airway inflammation.Methods: Saliva, OP swabs, IS, and ES were collected from 16 children with CF. Spirometry was performed.Measurements and Main Results: Respiratory and saliva samples (n = 61) were sequenced for bacterial microbial communities, and total and CF-specific bacterial quantitative PCR assays were performed. Airway samples underwent conventional culture for CF-specific pathogens. Neutrophil elastase, IL-1b, IL-1ra, IL-6, Il-8, TNF-a, and vascular endothelial growth factor were measured in ES and IS. Sequencing results from individual subjects were similar across samples, with greater between-subject than within-subject variation. However, Pseudomonas and Staphylococcus were detected in higher relative abundance from lower airways (ES and IS) compared with paired upper airway samples (OP and saliva). Pseudomonas, Staphylococcus, and Enterobacteriaceae correlated with increased airway inflammation. Divergence between microbiota in upper airway compared with lower airway samples, indicating greater differences between communities, was associated with increased sputum neutrophil elastase.Conclusions: Bacteria detected in IS samples resemble ES samples, whereas OP samples may underrepresent bacteria associated with airway inflammation. Divergence of lower airway communities from upper airway was associated with airway inflammation and may portend disease progression.

Evidence and role for bacterial mucin degradation in cystic fibrosis airway disease

“…The near ubiquitous presence of mucin-fermenting anaerobes in sputum [5,27,28, this study], coupled with numerous studies demonstrating that the respiratory mucins MUC5B and MUC5AC are degraded in CF patients compared to healthy controls [18,39] supports the idea that bacterial mucin degradation is commonplace within the CF airways. Streptococcus sp., for example, which were consistently abundant in mucin-enriched sputum cultures, have been extensively characterized for their ability to degrade salivary mucins [21].…”

“…Notably, obligately anaerobic taxa have gained recent attention for their abundance in expectorated sputum, bronchoalveolar lavage fluid and explanted lungs [5,27,28,[32][33][34]. A role for these organisms in CF disease has not yet been established; however, some have been characterized for their ability to degrade and ferment salivary mucins in the oral cavity [21,23,35].…”

“…Traditionally, culture-based studies have focused on a small number of taxa associated with CF lung disease (e.g. Pseudomonas aeruginosa, Staphylococcus aureus), however, culture-independent surveys of the CF lung microbiome have revealed a far more complex bacterial community than previously appreciated [3][4][5]. While the temporal dynamics of these communities and their association with disease states have been studied in detail, the in vivo host environment, and microbial metabolism therein, is relatively understudied.…”

Different next generation sequencing platforms produce different microbial profiles and diversity in cystic fibrosis sputum

“…Since sample collection and handling have been shown to influence sequencing results [7,23,24], the extracted DNA used for sequencing was taken from the same aliquot to eliminate any potential upstream variations. Likewise, the same computational pipeline and 16S reference database to analyze sequencing results were used to limit any variations in analysis.…”

“…In much of the CF literature, the mainstay of taxonomic identification has been next-generation 454 pyrosequencing of different variable regions within the 16S rRNA gene [4–7]. Since Roche will be discontinuing the 454 platform [8], two commercially available state-of-the-art next-generation sequencing (NGS) platforms have been proposed to characterize the CF lung microbiome: MiSeq (Illumina, San Diego CA) and PacBio RSII (Pacific Biosciences, Menlo Park CA).…”