Rate of osmotic pressure change in drying saliva microdroplets drives inactivation of model respiratory bacteria

ABSTRACT Transmission of respiratory pathogens depends on their ability to remain viable in drying respiratory droplets, yet the physicochemical drivers of bacterial inactivation during droplet evaporation remain poorly quantified. This study combines controlled droplet experiments with physicochemical modeling to investigate how osmotic pressure dynamics influence bacterial survival. Using Escherichia coli and Staphylococcus epidermidis as gram-negative and gram-positive surrogates, respectively, we measured viability loss in artificial saliva droplets dried at multiple relative humidities, and reconstructed the time-resolved osmotic pressure using the respiratory aerosol model. Both organisms remained stable while droplets were liquid and osmotic pressure was low but lost viability as decreasing water activity during drying produced rapid increases in osmotic pressure. For droplets that effloresced, these changes were most pronounced around efflorescence. The extent of inactivation scaled log-linearly with the rate of osmotic pressure change: E. coli decayed faster than S. epidermidis , and relationships derived in artificial saliva predicted survival in independent phosphate-buffered saline experiments. A lower relative humidity produced a more rapid increase in osmotic pressure and greater inactivation. These results identify the rate of osmotic pressure change during respiratory droplet drying as a quantitative predictor of bacterial survival across different solution matrices and drying conditions. IMPORTANCE The risk of infection by respiratory pathogens depends on how long microorganisms remain viable in respiratory particles after exhalation, yet the physical mechanisms controlling bacterial survival during droplet drying are not well defined. Evaporation of respiratory droplets concentrates salts and can impose sudden and extreme osmotic stress on microbes, but this process has been difficult to quantify because osmotic pressure cannot be measured directly inside microscopic droplets. Integration of droplet experiments with a physicochemical aerosol model shows that bacterial inactivation is governed primarily by the rate of osmotic pressure increase during droplet drying rather than by static values of humidity or solute concentration alone. This mechanism explains why rapid drying produces strong inactivation.