CLARITA LEFTHAND
Identification of the source of fecal contamination in Tulalip Bay with Bacteroides 16S rRNA gene and F+ specific coliphage markers
Environmental Health, MS
Preceptor: John Scott Meschke, PhD
Previous work conducted in the Tulalip Bay (Tulalip, WA) has shown high Total Coliform counts resulting in beach closures and shellfish harvesting limitations. TB supports subsistence fishing for many Tulalip Tribal members; therefore, water quality is a significant issue for this area. This project used two genotypic Microbial Source Tracking (MST) methods to determine the sources of fecal contamination in Tulalip Bay (TB). Bacteroides 16S ribosomal RNA gene and F+ RNA coliphage markers were used to differentiate between the sources of fecal contamination. Water samples were collected from TB at 16 sites. Genotyping of F+ RNA coliphage and the 16S ribosomal RNA genes of Bacteroides was utilized to differentiate fecal sources into human and non-human feces and human, ruminate and dog feces, respectively. EPA's 1601 and 1602 protocols were used for the isolation of F+ RNA coliphage. After isolation, RT-PCR was used to amplify F+ RNA coliphage sequences using levivirus- and allolevivirus- specific primer sets. Genogroup specific oligonucleotide probes were used to genotype F+ RNA coliphage in to one of four groups in a hybridization assay. Bacteroides spp. Were collected on membrane filters. DNA was extracted directly from filters and characterized using five host-specific PCR reactions. Our 1601 F+ coliphage data suggest the presence of fecal waste in 15/16 sampling sites in TB. Out of all these sites, four areas appear to be positive during at least 50% of the sampling events. Data from the 1602 procedure show 9/16 positives results for the presence of F+ coliphage. Four sites are positive for 50% or more sampling events. Primers specific for leviviruses and alloleviviruses confirmed the presence of fecal contamination for 10 sites. Our preliminary results show that fecal contamination is present in TB; however, additional work will need to be conducted in order differentiate between sources.
