OBJECTIVES/GOALS: The goal of this project is to apply wastewater-based epidemiology to determine the trends of NTS through analysis of influent wastewater, and to isolate and identify circulating serotypes among samples that are representative of the human population in Gainesville, FL. METHODS/STUDY POPULATION: This study is being conducted in Gainesville, FL, a suburban city in North Central Florida with approximately 133,997 residents served by two wastewater reclamation facilities (WRF). Fifty-three weekly influent composite wastewater samples were collected between September 2020 and September 2021 from each WRF. Wastewater samples were processed using an adsorption-based sample concentration method and invA genetic target for NTS DNA detection and quantification via qPCR. The quantified NTS DNA wastewater concentration from each WRF was summed to determine the overall concentration. Bacterial isolation was conducted on stored wastewater of qPCR positive samples using EPA method 1682. Recovered isolate serotypes were identified through whole-genome sequence analysis. RESULTS/ANTICIPATED RESULTS: Overall, NTS was positive in Gainesville wastewater in 48/53 weeks between September 2020 – September 2021. 83/106 (78%) samples tested positive for NTS from both WRFs. The mean wastewater concentration from each WRF was 3.53 (Main St.) and 3.31 Log10 invA Genetic Copies/L. The overall mean city concentration was 6.85 Log10 invA Genetic Copies/L. There were sustained periods of NTS in the wastewater during the months of December-February and May-August. There was no observed seasonal trend in NTS wastewater concentrations over the study period. Culture-based methods will provide a comprehensive review of circulating serotypes that are responsible for both clinical and subclinical infections within the community. DISCUSSION/SIGNIFICANCE: Florida has an incidence of NTS twice the national average. Clinical surveillance is the current standard for community surveillance of NTS. Only 28% of NTS cases are identified in a clinical setting, leaving a major gap in community surveillance. This project has helped fill this gap and provided a comprehensive community-level assessment of NTS.