Resolving Airborne Bacterial Viability in Megacity Beijing: Temperature-Linked Human-Associated Potential Pathogens in the Intact Fraction

DNA sequencing has revealed extensive airborne microbial diversity beyond culturable taxa. However, DNA from viable cells and extracellular sources is often conflated, obscuring their distinct health and ecological implications. Here, we partitioned airborne bacterial DNA into three fractions: extracellular DNA (eDNA) recovered from Cetyltrimethylammonium bromide (CTAB)-treated supernatants, cellular DNA (celDNA) from pellets containing intact and compromised cells, and DNA from intact cells (iDNA) obtained after propidium monoazide (PMA) pretreatment. Each fraction was characterized using 16S rRNA gene sequencing. Ambient eDNA harbored significantly higher species richness than iDNA and celDNA, whereas this pattern was absent in an unoccupied indoor environment. The three fractions displayed distinct community structures that varied by season and environment type (ambient vs indoor). dbRDA (distance-based redundancy analysis) identified season and temperature as major drivers. The iDNA fraction was enriched in host-associated bacteria from human, animal, and plant sources. Potentially pathogenic respiratory species, including Haemophilus influenzae, Pseudomonas aeruginosa, and Streptococcus pneumoniae, were significantly enriched in the ambient iDNA fraction during winter, and negatively correlated with ambient temperature. These findings highlight the importance of DNA partitioning in deciphering inhalation risks and ecological impacts of bioaerosols.