Microbiota in Exhaled Breath Condensate and the Lung

Abstract

The lung microbiota is commonly sampled using relatively invasive bronchoscopic procedures. Exhaled breath condensate (EBC) collection potentially offers a less invasive alternative for lung microbiota sampling. We compared lung microbiota samples retrieved by protected specimen brushings (PSB) and exhaled breath condensate collection. We also sought to assess whether aerosolized antibiotic treatment would influence the lung microbiota and whether this change could be detected in EBC. EBC was collected from 6 conscious sheep and then from the same anesthetized sheep during mechanical ventilation. Following the latter EBC collection, PSB samples were collected from separate sites within each sheep lung. On the subsequent day, each sheep was then treated with nebulized colistimethate sodium. Two days after nebulization, EBC and PSB samples were again collected. Bacterial DNA was quantified using 16S rRNA gene quantitative PCR. The V2-V3 region of the 16S rRNA gene was amplified by PCR and sequenced using Illumina MiSeq. Quality control and operational taxonomic unit (OTU) clustering were performed with mothur. The EBC samples contained significantly less bacterial DNA than the PSB samples. The EBC samples from anesthetized animals clustered separately by their bacterial community compositions in comparison to the PSB samples, and 37 bacterial OTUs were identified as differentially abundant between the two sample types. Despite only low concentrations of colistin being detected in bronchoalveolar lavage fluid, PSB samples were found to differ by their bacterial compositions before and after colistimethate sodium treatment. Our findings indicate that microbiota in EBC samples and PSB samples are not equivalent.IMPORTANCE Sampling of the lung microbiota usually necessitates performing bronchoscopic procedures that involve a hospital visit for human participants and the use of trained staff. The inconvenience and perceived discomfort of participating in this kind of research may deter healthy volunteers and may not be a safe option for patients with advanced lung disease. This study set out to evaluate a less invasive method for collecting lung microbiota samples by comparing samples taken via protected specimen brushings (PSB) to those taken via exhaled breath condensate (EBC) collection. We found that there was less bacterial DNA in EBC samples compared with that in PSB samples and that there were differences between the bacterial communities in the two sample types. We conclude that while EBC and PSB samples do not produce equivalent microbiota samples, the study of the EBC microbiota may still be of interest.

Age-related differences in the respiratory microbiota of chickens

“…Quantification of the V3 region of the 16S rRNA gene was performed as described previously [ 22 ] using the primers UniF340 ) and UniR514 ) at a final concentration of 0.4 μM using the LightCycler 480 SYBR green I master mix (Roche Applied Science).…”

The Madness of Microbiome: Attempting To Find Consensus “Best Practice” for 16S Microbiome Studies

“…Second, there are conflicting results in the literature with regard to the variation introduced by different sample collection methodologies. For example, there have been attempts to replace invasive sampling with less invasive methods; however, significant differences have been found in microbial populations in comparisons of swab and biopsy samples from human intestines ( 6 ), breath condensate and lung brushings ( 7 ), and rumen fluid samples obtained via oral stomach tubing and a fistula ( 8 ). However, other works contradict these findings, with two studies showing no statistically significant differences when studying the rumen microbiota in cattle using a variety of sampling methods ( 9 , 10 ).…”

Exhaled Breath Condensate—A Non-Invasive Approach for Diagnostic Methods in Asthma

“…A study by Glendinning et al compared EBC and protected specimen brushings (PSB) in an animal model. EBC samples had significantly less DNA material, which is the main limitation of the method [ 116 ]. The advantage of exhaled breath condensates is non-invasive sample collection.…”