Graduation Year

2018

Document Type

Thesis

Degree

M.S.

Degree Name

Master of Science (M.S.)

Degree Granting Department

Biology (Integrative Biology)

Major Professor

Valerie Harwood, Ph.D.

Committee Member

David Lewis, Ph.D.

Committee Member

Kathleen Scott, Ph.D.

Keywords

E. coli, Fecal indicator bacteria, H genes, MST

Abstract

Fecal pollution of surface water can lead to human health issues because waterborne transmission of pathogens is a common cause of gastroenteritis. Genetic markers developed for human-associated Escherichia coli (H8, H12, H14, and H24) are promising tools for microbial source tracking (MST) of sewage in environmental waterbodies but are untested in the US. H gene marker performance characteristics (specificity, sensitivity, and prevalence) were assessed, with the goal of developing a quantitative PCR assay for the markers with the best performance. Individual E. coli isolates from reference samples were tested to determine end-point PCR assay performance. Three hundred E. coli strains isolated from sewage and 900 obtained from animal fecal samples were screened for H8, H12, H14, and H24. Specificity values against animal fecal isolates were 92%, 97%, 86%, and 81% for H8, H12, H14, and H24, respectively. Sensitivity values against sewage were 100%, 90%, 100%, and 100%. Prevalence values for the marker genes in E. coli isolates were 16.6%, 7.7%, 16.3%, and 21.3%. Two new TaqMan qPCR assays were developed for the highest performing markers, H8 and H12. Specificity and sensitivity for the qPCR assays were determined against reference fecal samples. Host specificity for the H8 and H12 qPCR assays was 96% and 93%, respectively. The H8 and H12 qPCR assays were positive in all sewage samples. Mean H8 qPCR concentrations were higher in sewage compared to H12, ranging from 3.2x107 to 3.9x107 gene copies/100 mL, while H12 qPCR concentrations varied from 1.5x106 to 1.7x10 6 gene copies/100 mL. Concentrations in sewage were significantly and positively correlated with the human-associated Bacteroides marker HF83.These results indicate that the H8 TaqMan assay developed in this study for quantification of human host-associated E. coli is a promising addition to the MST toolbox due to high specificity for sewage, abundance in untreated and partially-treated wastewater, and correlation with the well-established HF183 marker. While the H12 assays also showed strong performance characteristics, the gene’s concentration was over ten-fold lower than that of H8, making it less likely to be detected in diluted sewage. Host-associated E. coli markers are a tool that can improve water quality management decisions by allowing investigators to determine whether human sources contribute to elevated E. coli densities in surface waters, which can inform total maximum daily load plans and risk assessment.

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