Microbial source tracking of fecal contamination in oyster growing areas

Objectives

Our project objectives address the MASGC 2018-21 strategic plan’s focus area “Healthy Coastal Ecosystem” Goals: “(Goal 1) Habitat, ecosystems and the services they provide are protected, enhanced or restored” and (Goal 2) “Land, water and living resources are managed by applying sound science, tools and services to sustain ecosystems”. Our overall project goal is to compare the specificity and performance of DNA-based technologies and fecal indicator gene markers to existing standard EPA methods for quantifying indicators of fecal contamination in oyster growing waters. Assays developed in this study will be done with watershed samples as a first step towards the development and validation of tools and technologies for monitoring fecal pollution in oyster growing areas receiving waters from an Alabama watershed. This project will provide data on sources of fecal pollution (e.g., human, avian, animal) contributing to the measured total maximum daily load (TMDL) of fecal indicators detected in local oyster growing waterbodies. The tools and data generated from this project will; (i) contribute to water quality knowledge and baseline data on sources of fecal coliform in a large Alabama watershed, (ii) validate the use of alternative indicator/methods such as MST tracking and next generation sequencing technologies for water quality monitoring.

The specific objectives of this project are:

1. To identify potential sources of fecal coliform bacteria detected in oyster growing areas.
2.  To test the performance of next generation sequencing tools for the identification of pathogens and sources of fecal coliforms detected in oyster growing areas. 

Methodology

During Year 1, the focus of the project will be to identify potential fecal coliforms sources in environmental samples collected in the West Fowl River Watershed. Fecal coliform levels will be determined by standard fecal indicator culture-based methods for comparison to DNA-based microbial source tracking (MST) methods using: (i) human, (ii) avian and (iii) ruminant and domestic animal fecal indicators gene markers. Either one or both of our null hypotheses will be accepted if we detect statistically significant differences in (i) host sources impacting the Fowl River Bay (West Fowl River Watershed) during regular or low-flow periods and (ii) during heavy precipitation rainfall events that occur in the West Fowl River Watershed.

During Year 2, we will conduct a proof-of-principle study to compare a small subset of Year 1 samples (three to five samples) with either high or low E. coli culturing counts with be shotgun metagenomic sequenced and 16S rRNA gene amplicon sequenced. Specifically, we will determine if there is a correlation between E. coli culturing counts with rRNA gene amplicon sequencing via the (i) Illumina MiSeq platform and shotgun metagenomic sequencing using the (ii) Oxford Nanopore MinION platform. Unlike the MiSeq platform, the MinION system is a portable device which can provide rapid, real-time sequencing and analysis without being limited to laboratory constraints.

Rationale

To ensure the sanitary water quality conditions of shellfish growing areas, total coliform and fecal coliform densities are routinely monitored. Elevated levels of fecal coliforms exceeding the National Shellfish Safety Program (NSSP) 14/31 standard trigger closures of recreational areas, beaches and shellfish harvesting areas as higher fecal coliform levels correlate with increased risk to human health.