Parallel Analysis of Cystic Fibrosis Sputum and Saliva Reveals Overlapping Communities and an Opportunity for Sample Decontamination

Abstract

<jats:title>ABSTRACT</jats:title> <jats:p>Culture-independent studies of the cystic fibrosis (CF) airway microbiome typically rely on expectorated sputum to assess the microbial makeup of lower airways. These studies have revealed rich bacterial communities. There is often considerable overlap between taxa observed in sputum and those observed in saliva, raising questions about the reliability of expectorated sputum as a sample representing lower airway microbiota. These concerns prompted us to compare pairs of sputum and saliva samples from 10 persons with CF. Using 16S rRNA gene sequencing and droplet digital PCR (ddPCR), we analyzed 37 pairs of sputum and saliva samples, each collected from the same person on the same day. We developed an <jats:italic>in silico</jats:italic> postsequencing decontamination procedure to remove from sputum the fraction of DNA reads estimated to have been contributed by saliva during expectoration. We demonstrate that while there was often sizeable overlap in community membership between sample types, expectorated sputum typically contains a higher bacterial load and a less diverse community compared to saliva. The differences in diversity between sputum and saliva were more pronounced in advanced disease stage, owing to increased relative abundance of the dominant taxa in sputum. Our effort to model saliva contamination of sputum <jats:italic>in silico</jats:italic> revealed generally minor effects on community structure after removal of contaminating reads. Despite considerable overlap in taxa observed between expectorated sputum and saliva samples, the impact of saliva contamination on measures of lower airway bacterial community composition in CF using expectorated sputum appears to be minimal.</jats:p> <jats:p><jats:bold>IMPORTANCE</jats:bold> Cystic fibrosis is an inherited disease characterized by chronic respiratory tract infection and progressive lung disease. Studies of cystic fibrosis lung microbiology often rely on expectorated sputum to reflect the microbiota present in the lower airways. Passage of sputum through the oropharynx during collection, however, contributes microbes present in saliva to the sample, which could confound interpretation of results. Using culture-independent DNA sequencing-based analyses, we characterized the bacterial communities in pairs of expectorated sputum and saliva samples to generate a model for “decontaminating” sputum <jats:italic>in silico</jats:italic>. Our results demonstrate that salivary contamination of expectorated sputum does not have a large effect on most sputum samples and that observations of high bacterial diversity likely accurately reflect taxa present in cystic fibrosis lower airways.</jats:p>

Microbiome data enhances predictive models of lung function in people with cystic fibrosis

“…Third, our observation of a consistent retention of specific non-pathogen taxa across multiple models (with and without the addition of potentially confounding EMR features, including age and BMI) points to the potential for a distinct causal pathway that is orthologous to age or BMI. We note that the interpretation that oral bacteria are active players in the lung environment is further buttressed by a recent study on people with established CF disease (Lu et al 2020) that used paired sputum and saliva samples to infer the presence of substantial populations of oral bacteria in the lung.…”

“…Under this contamination model, increasing pathogen load compared to a constant background of oral microbiome contamination generates a spurious link between oral microbes, microbiome diversity, and patient health [27]. While recent paired sputum-saliva sampling analysis indicates that oral sample contamination is not a substantial contributor to sputum microbiome profiles in people with established CF lung disease [29], these conflicting hypotheses highlight the uncertainty in the role specific taxa present in sputum.…”

The sputum microbiome associated with different sub-types of AECOPD in a Chinese cohort

“…Future studies are needed to quantify the total bacterial load by qPCR to determine the differences in absolute bacterial load of total and specific bacteria between groups of different clinical status in COPD. Of note, although sputum has been widely used to study the lung microbiome, considerable overlap between sputum microbiome and saliva microbiome has been observed [19]. As negative correlation between oral hygiene on COPD has been reported [20,37], the spatial dynamics between upper and lower respiratory tract microbiome and the potential roles of these oral microbiome in COPD development in the Chinese population is highly desired.…”

“…Although it is prone to oral microbiome contamination given the topological continuity of the oral cavity and the lower respiratory tract, sputum has been widely used as a surrogate to study the lung microbiome in different respiratory diseases [19,20]. The objectives of this study were to characterize the AECOPD sputum microbiome in a Chinese population and to determine whether specific sputum microbiome biomarkers could be identified in order to differentiate eosinophilic from other subtypes of AECOPD.…”

Antibiotics drive expansion of rare pathogens in a chronic infection microbiome model

“…A number of approaches to address the sputum contamination issue have been taken, including mouth cleaning and sputum rinsing 29 , as well as more invasive sampling techniques (subject to clinical need 28,[30][31][32] ). Most recently, computational analysis of paired sputum and saliva samples from adults with established CF lung disease has demonstrated that saliva contamination during sample collection has a minimal quantitative impact on the community profile 33 .…”