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.

Keywords: 16S rRNA; EBC; colistimethate sodium; colistin; lung microbiota; respiratory microbiota; sheep.

FIG 1

Heatmap showing samples grouped by batch based on the time DNA was extracted from the samples. Bacterial OTUs were included where they had an abundance of ≥5% in at least one sample. OTUs which were ≥5% abundant in a DNA extraction kit reagent control are indicated by color (batch 1, green; batch 2, pink; batch 3, blue; and batch 4, yellow). DNA extraction kit reagent controls are labeled as Extraction Kit Batch 1 to 4. EBC samples from conscious and from anesthetized sheep are labeled EBC (cons) and EBC (anaes), respectively. OTUs which were >5% abundant in an extraction kit control do not consistently appear in all samples in the same batch.

FIG 2

Boxplot showing the log 16S rRNA gene concentrations found in sheep respiratory samples (EBC samples from conscious and anesthetized animals and PSB samples) and controls (protected specimen brushes, DNA extraction kit reagents and qPCR reagents). Outliers were defined by SPSS as either “out” values (circles) or “extreme” values (stars). PSB samples contained significantly more bacterial DNA (P < 0.005) than any other respiratory sample type or control.

FIG 3

Heatmap showing EBC samples grouped by sheep and time point. DNA extraction kit reagent controls are labeled as Extraction Kit Batch 1 to 4. EBC samples from conscious and anesthetized sheep are labeled EBC (cons) and EBC (anaes), respectively. Bacterial OTUs were included where they had an abundance of ≥5% in at least one sample. As can be observed, EBC samples taken from the same sheep when it was conscious and when it was anesthetized did not necessarily contain the same bacterial OTUs.

FIG 4

PCoA graph showing the significantly separate clustering of EBC (anaes) and PSB samples from sheep (P = 0.011 by AMOVA), which may be due to the difference in variation between the two sample types (P = 0.026 by HOMOVA). The OTUs which most contributed to samples moving in a positive or negative direction along either axis and which had P values of < 0.00058 (defined by Bonferroni's correction as 0.5 divided by the total number of OTUs), according to the corr.axes command within mothur, are listed. As this graph is only representative of 20.3% of the total variability present between samples, caution should be taken when interpreting how clustered the sample groups appear.

FIG 5

PCoA graph showing the significantly separate clustering of PSB samples from sheep before and after CMS treatment (P = 0.014 by AMOVA). The OTUs which most contributed to samples moving in a positive or negative direction along either axis and which had P values of < 0.00058 (defined by Bonferroni's correction as 0.5 divided by the total number of OTUs), according to the corr.axes command within mothur, are listed. As this graph is only representative of 24% of the total variability present between samples, caution should be taken when interpreting how clustered the sample groups appear.

FIG 6

(A) EBC was collected from conscious animals while they were restrained in a yoke head-restraint holding crate. A face mask was attached and sheep inhaled through a short tube with an inlet valve and exhaled through an RTubeVENT. (B) EBC was collected from anesthetized mechanically ventilated animals by placing the RTubeVENT in-line with the expiratory limb of the ventilator, near the sheep's head.

FIG 7

Diagram of the sheep lung. PSB samples were taken before and after colistimethate sodium treatment from the right ventral diaphragmatic 1 (RVD1), left ventral diaphragmatic 1 (LVD1), right caudal diaphragmatic (RCD), and left caudal diaphragmatic (LCD) lung segments. RC, right cardiac; RA, right apical; LC, left cardiac; LVD2, left ventral diaphragmatic 2; RVD2, right ventral diaphragmatic 2. Adapted from reference .