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. 2017 Aug 30;3(3):00019-2017.
doi: 10.1183/23120541.00019-2017. eCollection 2017 Jul.

Protected sampling is preferable in bronchoscopic studies of the airway microbiome

Rune Grønseth  1 , Christine Drengenes  1   2 , Harald G Wiker  2   3 , Solveig Tangedal  1   2 , Yaxin Xue  4 , Gunnar Reksten Husebø  1   2 , Øistein Svanes  1   2 , Sverre Lehmann  1   2 , Marit Aardal  1 , Tuyen Hoang  2 , Tharmini Kalananthan  1 , Einar Marius Hjellestad Martinsen  2 , Elise Orvedal Leiten  2 , Marianne Aanerud  1 , Eli Nordeide  1   2 , Ingvild Haaland  1   2 , Inge Jonassen  4 , Per Bakke  2 , Tomas Eagan  1   2
Affiliations

Protected sampling is preferable in bronchoscopic studies of the airway microbiome

Rune Grønseth et al. ERJ Open Res. .

Abstract

The aim was to evaluate susceptibility of oropharyngeal contamination with various bronchoscopic sampling techniques. 67 patients with obstructive lung disease and 58 control subjects underwent bronchoscopy with small-volume lavage (SVL) through the working channel, protected bronchoalveolar lavage (PBAL) and bilateral protected specimen brush (PSB) sampling. Subjects also provided an oral wash (OW) sample, and negative control samples were gathered for each bronchoscopy procedure. DNA encoding bacterial 16S ribosomal RNA was sequenced and bioinformatically processed to cluster into operational taxonomic units (OTU), assign taxonomy and obtain measures of diversity. The proportion of Proteobacteria increased, whereas Firmicutes diminished in the order OW, SVL, PBAL, PSB (p<0.01). The alpha-diversity decreased in the same order (p<0.01). Also, beta-diversity varied by sampling method (p<0.01), and visualisation of principal coordinates analyses indicated that differences in diversity were smaller between OW and SVL and OW and PBAL samples than for OW and the PSB samples. The order of sampling (left versus right first) did not influence alpha- or beta-diversity for PSB samples. Studies of the airway microbiota need to address the potential for oropharyngeal contamination, and protected sampling might represent an acceptable measure to minimise this problem.

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Conflict of interest statement

Conflict of interest: Disclosures can be found alongside this article at openres.ersjournals.com

Figures

FIGURE 1
FIGURE 1
Mean taxonomic distribution at the phylum level, by sampling method, for all 125 individuals (unrarefied). OW: oral wash; SVL: small-volume lavage in the left upper lobe; BAL1: first fraction of protected bronchoalveolar lavage (BAL) from right middle lobe; BAL2: second fraction of protected BAL from right middle lobe; PSB: protected specimen brush from right lower lobe and left upper lobe. No legend for smallest phylae.
FIGURE 2
FIGURE 2
Box-plot of alpha-diversity measured by wholetree phylogenetic differences grouped according to sampling method and chronic obstructive pulmonary disease (COPD) status. Rarefied at 1000 sequences. OW: oral wash sample; SVL: small-volume lavage from left upper lobe; PBAL1: first fraction of protected bronchoalveolar lavage (BAL); PBAL2: second fraction of protected BAL; right PSB: protected specimen brush from right lower lobe; left PSB: protected specimen brush from left upper lobe.
FIGURE 3
FIGURE 3
Principal coordinates analyses on unweighted UniFrac distance matrix comparing sampling methods in the MicroCOPD to oral wash samples. Rarefied at 1000 sequences. LUL: left upper lobe; BAL: bronchoalveolar lavage; RML: right middle lobe; RLL: right lower lobe.
See this image and copyright information in PMC

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