** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** WHILE AGRICULTURE PRODUCTION URGES A GROWING DEMAND OF FRESHWATER, WASTEWATER REUSE IS RECOGNIZED AS A PROMISING STRATEGY TO SUPPLY WATER FOR IRRIGATION AND PROMOTE WATER SUSTAINABILITY. HOWEVER, A SERIOUS PUBLIC HEALTH CONCERN ON WASTEWATER REUSE IS THE POTENTIAL TRANSMISSION OF PATHOGENS, ESPECIALLY ANTIBIOTIC-RESISTANT BACTERIA (ARB) FROM REUSED TREATED WASTEWATER TO FOOD. ENVIRONMENTAL BACTERIA MAY ACQUIRE ANTIBIOTIC RESISTANCE THROUGH HORIZONTAL GENE TRANSFER OF ANTIBIOTIC RESISTANCE GENES (ARGS). WASTEWATER CARRIES A LARGE NUMBER OF ARGS, AND THE HIGH BACTERIAL DENSITY AND DIVERSITY ENVIRONMENTS IN BIOLOGICAL WASTEWATER TREATMENT PROCESSES CAN FACILITATE THE TRANSFER OF ARGS AMONG BACTERIA. WHILE THREE MAJOR PATHWAYS (INCLUDING NATURAL TRANSFORMATION, TRANSDUCTION, AND CONJUGATION) WERE BELIEVED TO HORIZONTALLY TRANSFER ARGS AMONG BACTERIA, VESIDUCTION (HORIZONTAL GENE TRANSFER MECHANISM VIA VESICLE-ASSOCIATED ARGS RELEASED FROM BACTERIA) HAS BEEN RECENTLY RECOGNIZED AS AN EMERGING BUT OVERLOOKED ROUTE TO TRANSFER GENES HORIZONTALLY. THE VESICLE-ASSOCIATED ARGS CAN BE EXTENSIVELY RELEASED FROM BACTERIA, STABLE AND PERSISTENT IN ENVIRONMENT, AND ESCAPE FROM OR RESIST TO WATER TREATMENT PROCESSES. HOWEVER, OUR KNOWLEDGE ON TRANSFER AND FATE OF THESE VESICLE-ASSOCIATED ARGS IN WASTEWATER TREATMENT AND REUSE IS NOT SUFFICIENT TO DEVELOP CONTROL STRATEGIES. IN THE PROPOSED RESEARCH, WE AIM TO I) UNDERSTAND THE HORIZONTAL TRANSFER OF ARGS MEDIATED BY MEMBRANE VESICLES IN WATER MATRICES RELEVANT TO AGRICULTURE WATER REUSE, II) ELUCIDATE THE PERSISTENCE AND REMOVAL OF VESICLE-ASSOCIATED ARGS IN KEY STEPS OF WASTEWATER TREATMENT AND RECLAMATION, AND III) DETERMINE THE OCCURRENCE, ABUNDANCE, AND REMOVAL OF VESICLE-ASSOCIATED ARGS IN EACH STEP IN MUNICIPAL WATER RECLAMATION AND RECYCLING FACILITIES. FIRST, WE WILL CONDUCT LAB TESTS TO EVALUATE THE HORIZONTAL TRANSFER ABILITIES OF VESICLE-ASSOCIATED ARGS TO SELECTED BACTERIAL SPECIES THAT ARE ENVIRONMENTALLY AND CLINICALLY RELEVANT, AND WE WILL ALSO EXPLORE THE MECHANISMS/FACTORS CONTROLLING THIS TRANSFER IN WATER MATRICES. NEXT, BENCH-TOP TESTS WILL BE PERFORMED TO QUANTITATIVELY INVESTIGATE THE DAMAGE/DEGRADATION OF VESICLE-ASSOCIATED ARGS THROUGH CHARACTERIZING DNA, PROTEINS, LIPIDS, VESICLE SIZES, AND VESICLE INTEGRITY/MORPHOLOGY UNDER EXPOSURE TO SIMULATED WASTEWATER TREATMENT CONDITIONS. FINALLY, REAL WASTEWATER AND RECLAIMED WATER WILL BE COLLECTED IN WATER RECLAMATION AND RECYCLING FACILITIES, AND THE METAGENOMIC AND QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS WILL BE CONDUCTED TO UNDERSTAND THE OCCURRENCE, ABUNDANCE, AND REMOVAL OF VESICLE-ASSOCIATED ARGS.THIS STUDY WILL ADVANCE OUR KNOWLEDGE OF THE FATE, TRANSFER, AND RISKS OF ARGS IN AGRICULTURAL WATER REUSE, PROVIDE INSIGHTS INTO THE CONTROL OF ANTIBIOTIC-RESISTANT PATHOGENS IN AGRICULTURAL WATER REUSE, AND SHED LIGHT ON THE MANAGEMENT OF WATER REUSE FOR AGRICULTURAL IRRIGATION.