Feasibility of low-biomass exhaled breath microbiome sequencing using a PDC-sampler in febrile and healthy individuals.

Exhaled breath is a noninvasive and repeatable biological matrix offering new opportunities for respiratory microbiome analysis, yet its extremely low microbial biomass limits current high-throughput applications. Building on our previously developed phase-change drywall cyclone sampler (PDC-sampler), which integrates condensational growth with dry-wall cyclone separation, we established a validated workflow for efficient aerosol collection and multi-Omics sequencing of exhaled breath. Using this platform, exhaled breath from 15 febrile patients and 6 healthy volunteers was analyzed via shotgun metagenomic and 16 S rRNA sequencing to assess microbial composition, diversity, and functional features. The PDC-sampler significantly increased microbial DNA yield, enabling stable detection of bacterial taxa dominated byPseudomonadota, Bacillota, Bacteroidota, andActinomycetota. Functional annotations and diversity metrics revealed distinct microbial and metabolic patterns between individuals, confirming the platform's analytical sensitivity and biological representativeness. This work experimentally validates the feasibility of exhaled breath microbiome sequencing using the PDC-sampler, providing a practical and generalizable framework for noninvasive respiratory microecology studies and future diagnostic applications.