Mechanistic Understanding of Breath-Borne Bacterial Inactivation Kinetics in Oxidant-Rich and Natural Air

Abstract Bioaerosols play a central role in respiratory infection transmission, but how atmospheric oxidants affect their viability, especially in human respiratory fluids, remains unexplored. Here, we investigated the inactivation of five bacterial species with distinct cell envelope architectures (Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis, Staphylococcus aureus, and Mycobacterium smegmatis) suspended in human exhaled breath condensate (EBC) droplets exposed to gas-phase hydroxyl radicals (•OH) produced by ultraviolet irradiation of H2O2. Compared with the other three exposure conditions, the •OH-generating (H2O2+UV) treatment caused the greatest viability loss, with 4-h survival ranging from 24.3% (E. coli) to 63.0% (M. smegmatis). Gram-negative bacteria were sensitive to both H2O2 and •OH, whereas Gram-positive species resisted H2O2 but were inactivated by •OH. M. smegmatis showed the highest tolerance. Inactivation accelerated in smaller droplets, and the size-dependent kinetics were characterized using Weibull and first-order models incorporating surface-area-to-volume ratio. Morphological, biochemical, and metabolomic analyses indicated that the observed inactivation differences were associated with bacterial envelope types and surface characteristics. •OH preferentially degrades outer membrane glycolipids in Gram-negative bacteria and oxidizes cytoplasmic membrane lipids in Gram-positive bacteria, while all species shared a core signature of nucleic acid oxidative damage. These findings provide a molecular understanding of bacterial inactivation kinetics in human EBC droplets and may inform the assessment of bioaerosol transmission risks.