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

Abstract

ABSTRACT 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 in silico 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 in silico 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. IMPORTANCE 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 in silico. 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.

Keywords

• microbiome
• airway infection
• microbial community dynamics