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The San Juan Bay Estuary (SJBE) is a mixed watershed highly impacted by urbanization in San Juan, Puerto Rico. Currently, communities around SJBE are affected by failures in wastewater infrastructure and/or flooding events that may represent a public health risk. To develop tools to support quantitative microbial assessments, we characterized bacterial communities across the system; identified factors impacting microbial communities based on seasonality; and identified potential emergent or opportunistic pathogens. Water samples were collected from 16 sites across the estuary during two seasons: wet (June-November) and dry (December-May) from May 2021 thru December 2023. DNA was extracted from filters for 16S rRNA amplicon sequencing. Among the top 20 taxonomic groups across the estuary, members of Arcobacter spp. and Aeromonas spp. which include pathogenic species were detected. To increase the chance of identifying potential pathogens in low concentrations, a subset of filters was enriched with peptone broth for 24 hours at 37°C. DNA was extracted for 16S rRNA amplicon sequencing and data was compared in parallel to unenriched filters. Permutation tests indicated significant differences (p-value=0.001) between microbial composition based on seasonality for 2022 and 2023 only. Another permutation test indicated that the microbial composition in enriched and unenriched samples is significantly different with members of Photobacterium spp., and Vibrio spp. dominating enriched samples. Additionally, potential pathogens such as V. vulnificus, and V. cholerae were not identified in unenriched samples. NMDS analyses indicated that location in the estuary influenced the composition of the bacterial communities, with samples collected at the sites forming distinct clusters. Other analyses are underway to elucidate the effect of environmental factors, including land use on bacterial community composition and the abundance of pathogens across the estuary . Our findings attempt to provide a comprehensive understanding of bacterial diversity and potential pathogen in urban recreational water by applying NGS. |