Metagenomic whole genome shotgun analysis of the airway microbiome in laryngotracheal stenosis: a pilot study.

The airway microbiome has been implicated in the pathogenesis of laryngotracheal stenosis (LTS), yet prior studies using 16 S rRNA sequencing have limited sub-genus level resolution. Metagenomic whole genome shotgun sequencing (mWGS) allows for strain-level taxonomic and functional genomic analysis, providing detailed insights into specific organisms and pathways. A pilot study was conducted to explore the advantages and challenges of mWGS in investigating the airway metagenome in LTS. mWGS was conducted on 12 intraoperative swab samples from 8 LTS patients, divided into tracheostomy-dependent (n = 3) and non-tracheostomy (n = 5) groups, and 4 controls. Patient comorbidities, antibiotic use, and medications were documented. Biobakery workflows were used for taxonomic and functional profiling. Species-specific reference databases were constructed for 6 abundant species for strain-level analyses. LTS samples had decreased taxonomic diversity and were dominated by species with previously described roles in other chronic inflammatory processes such as Staphylococcus aureus, Streptococcus parasanguinis, Streptococcus mitis, and Corynebacterium pseudogenitalium. LTS samples were enriched for pathways involved in fatty acid biosynthesis and formaldehyde metabolism. Our results identified tracheostomy as an important potential confounder in airway metagenomics but show mWGS techniques are promising in uncovering microbiota correlates in LTS that could reveal disease-specific biomarkers, comorbidity links, and therapeutic targets.