A workflow for the detection of Vibrio spp. in a tropical estuarine environment via enrichment dependent high throughput sequencing and qPCR methods
Citation:
Cuebas Irizarry, M., B. Parker, J. Hanlon, S. Brown, I. Cappielo Cosme, AND M. Molina. A workflow for the detection of Vibrio spp. in a tropical estuarine environment via enrichment dependent high throughput sequencing and qPCR methods. 2024 American Society for Microbiology Annual Meeting, Atlanta, GA, June 13 - 17, 2024.
Impact/Purpose:
Despite the application of PCR techniques for environmental pathogen detection, only limited information is usually available due to low target detection limits. A combination of enrichment samples and high throughput sequencing can be used to increase the chances of detection. Here, we show a workflow for detection of Vibrio species in an estuarine environment in Puerto Rico (San Juan Bay Estuary (SJBE)), where enrichment-dependent and -independent 16S rRNA amplicon sequencing were combined with detection of pathogens via PCR and qPCR
Description:
Identification of potential pathogens that present risks to human health in recreational waters is necessary to advance in-depth water quality assessments. Despite the application of PCR techniques for environmental pathogen detection, only limited information is usually available due to low target detection limits. A combination of enrichment samples and high throughput sequencing can be used to increase the chances of detection. Here, we show a workflow for detection of Vibrio species in an estuarine environment in Puerto Rico (San Juan Bay Estuary (SJBE)), where enrichment-dependent and -independent 16S rRNA amplicon sequencing were combined with detection of pathogens via PCR and qPCR. SJBE is actively utilized for recreational purposes and frequently experiences flooding of areas with compromised sanitary sewage infrastructure. Water samples were collected from May 2021 thru December 2023 from 16 sites across the estuary and prepared for DNA analysis. Results indicate the dominance of Vibrio spp. across the estuary, including V. cholerae. With the purpose of increasing the detection of species in low abundance, samples from 2023 were enriched using peptone broth for 24 hours at 37°C and a parallel unfiltered sample was stored for further analysis. High throughput 16S rRNA amplicon sequencing from the enriched samples revealed the presence of V. fluvialis, V. mediterranei, V. ponticus, V. tubiashii, V. vulnificus, V. aestuarianus, V. alfacsensis, and V. cidicii. Preliminary qPCR analysis of the unenriched samples indicated that Vibrio cholerae ranged from 2 to 27 gene copies/ml and was mostly found in highly urbanized areas where stormwater was impacted by constant breaches in the sanitary system. 16S rRNA amplicon sequencing and the detection of pathogenic Vibrio via PCR is underway utilizing the unenriched samples. This workflow allows us to increase the pathogen detection, specifically for Vibrio spp., in this estuary system for better monitoring. Methods for quantification and identification are helpful to develop and establish mitigation strategies to reduce contamination and decrease public health risk.