INCREASING THE DETECTION OF VIBRIO SPP. IN A TROPICAL ESTUARINE ENVIRONMENT VIA ENRICHMENT DEPENDENT HIGH THROUGHPUT SEQUENCING AND QPCR METHODS

Identification of potential pathogens that pose human health risk in recreational waters is necessary to provide in-depth water quality assessments. Despite the advances in NGS techniques, limited information can be obtained for pathogen detection due to low concentrations in the environment. Here, we show a workflow for detection of Vibrio species in a tropical environment: San Juan Bay Estuary (SJBE) in Puerto Rico. SJBE is actively utilized for recreational purposes and frequently experiences flooding of areas with compromised sanitary sewage infrastructure. Water samples were collected from 2021-2023 from 16 sites across the estuary. To increase the detection of species in low abundance, samples from 2023 were enriched using peptone broth for 24 hours at 37°C and a parallel unenriched sample for further comparison in 16S amplicon sequencing. High throughput 16S rRNA amplicon sequencing from the enriched samples revealed the presence of 9 species of Vibrio. qPCR analysis of the unenriched samples indicated that Vibrio cholerae ranged from 2 to 27 gene copies/ml in highly urbanized areas impacted by constant breaches in the sanitary system. 16S rRNA amplicon sequencing of unenriched samples is expected to reveal less detection of Vibrio spp and the detection of pathogenic Vibrio via PCR is underway. This workflow allows us to increase potential pathogen detection, specifically for Vibrio spp. in this estuary system. Quantification and identification methods are helpful to develop mitigation strategies to reduce contamination and decrease public health risk.

Presentation Preference: Poster

Primary Presenter: Mara Cuebas Irizarry, Environmental Protection Agency (cuebasirizarry.mara@epa.gov)

Authors:

Marirosa Molina, U.S. Environmental Protection Agency, Office of Research and Development  (molina.marirosa@epa.gov)

Brandon Parker, U.S. Environmental Protection Agency, Oak Ridge Associated Universities  (parker.brandon@epa.gov)

Sarah Brown, U.S. Environmental Protection Agency, Oak Ridge Institute for Science and Education  (brown.sarah@epa.gov)

Ivelisse Cappielo Cosme, U.S. Environmental Protection Agency, Oak Ridge Institute for Science and Education, U.S.Environmental Protection Agency Region 2 CEPD  (cappielocosme.ivelisse@epa.gov)