Project Summary/Abstract It is well known that higher proportions of youth who smoke cigars, e-cigs, and hookahs (waterpipes [WPs]) use flavored tobacco products over unflavored. WP tobacco is unique in that large amounts of sweet humidifying compounds (humectants) are added to provide the user with a sweet, smooth-tasting smoke. Aroma flavor compounds are also added. Some evidence suggests that tobacco flavor additives that are antimicrobial, such as menthol, impact microbiota in cigarettes by reducing bacterial populations while increasing bacterial species resistant to harsh environments. Current understanding is lacking in how additives like humectants and flavors alter microorganisms in WP tobacco. With WP smoking becoming more popular in the U.S. particularly among college students, users may be highly susceptible to the transfer of pathogens when sharing devices, but also to native microorganisms in WP tobacco that could pose significant adverse health effects if inhaled as bioaerosols. It is known that certain bacteria resistant to harsh conditions can survive high temperatures and potentially transfer to the smoke from cigarettes. WP tobacco temperatures are much lower compared to cigarettes, perhaps providing a more suitable environment for pathogenic microorganism to persist. Thus, it is important to characterize microbial species and the impact that humectants and flavors have on microbial growth in WP tobacco, to better understand what harmful microorganisms might be transferred from the tobacco to the WP smoker and elicit adverse respiratory health effects from toxic bioaerosol exposures. The proposed study seeks to fill this knowledge gap by assessing the role of humectants and flavor additives on microbial growth in WP tobacco by apply an innovative alternative toxicology approach. We will add humectants and a select flavor to a plain WP tobacco prepared several ways to isolate the effects of these additives on microbial growth and identify and quantify a broad view of microbial community composition using whole genome sequencing analysis. Shotgun proteomic analysis will also be performed to characterize proteins expressed by organisms colonizing tobacco to explain metabolic pathways involved in tobacco degradation/stability. Additionally, bacteria capable of converting nitrate to nitrite can react with tobacco alkaloids to form carcinogenic tobacco-specific N-nitrosamines (TSNAs). Therefore, we will assess the relationship between TSNA levels and microbes in WP tobacco. This research has important public health regulatory implications in that it will inform the Food and Drug Administration Center for Tobacco Products (FDA CTP) regarding the roles of sweeteners and flavors in newly deemed tobacco products, such as WP. Data from the proposed study can be used to inform whether these compounds influence toxic exposures and, if so, the FDA should ban or limit the use of sweeteners/flavors in WP tobacco. The microorganisms identified will form the foundation of future studies to characterize microbial content in a variety of WP tobacco products and in WP smoke inhaled by the user, providing further needed data for consideration in regulating WP tobacco.