Repeated bronchoscopy examination of the airway microbiome

Background: Airway microbiota potentially plays a role in obstructive lung diseases. The stability of the lower airways is still unknown. Methods: 21 healthy controls and 41 patients with obstructive lung disease completed two volunteer bronchoscopies. Median days between the two bronchoscopies were 140 in the disease group and 150 in the controls. We sampled oral wash (OW) before protected bronchoalveolar lavage in two fractions (PBAL1 and PBAL2), and protected specimen brushes (PSB). After DNA extraction and PCR for the V3V4 region of the 16S rRNA gene, samples were sequenced on an Illumina MiSeq. Up-stream bioinformatics were carried out with QIIME-2, identifying amplicon sequence variants (ASVs). Contaminating ASVs were identified using the decontam package in R. Results: A final table of 551 ASVs consisted of 19x10^6 sequences. Alpha diversity was lower in the second exam for OW samples, and borderline lower for PBAL1. Permutational tests of beta diversity indicated that within-individual comparison was lower than what could be expected by chance (Figure). A non-parametric trend test showed that beta-diversity between the two procedures followed a pattern of PSB>PBAL2>PBAL1>OW. Conclusion: The airways microbiota diversity varied between examinations, and more so in more protected, low-biomass samples. However, the overall composition (beta-diversity) shows stability within a person, beyond that of chance.