Graduation Year

2008

Document Type

Thesis

Degree

M.S.

Degree Granting Department

Biology

Major Professor

Valerie J. Harwood, Ph.D.

Committee Member

Gordon A. Fox, PhD.

Committee Member

Kathleen M. Scott, PhD.

Keywords

Indicator organisms, Water quality, DNA purification, Phylotypes, Flume

Abstract

Indicator organisms (IOs) such as Escherichia coli and Enterococcus spp. are used to predict the presence of pathogens in waters. Determining the relationships between environmental factors, IOs, and pathogens is a key to assessing water quality and ensuring public health, yet certain strains of E. coli and Enterococcus spp. survive for long periods in natural waters. Molecular subtyping, using repetitive extragenic palindromic DNA sequences (BOX-PCR), has been used to discriminate among environmental E. coli and Enterococcus spp. isolates. The reproducibility of BOX-PCR patterns varies with DNA purification methods; therefore, it is important to develop a standardized, rapid, high throughput DNA purification protocol for population biology studies. I have compared the effects of DNA purification methods on the reproducibility, cost, and speed of producing BOX-PCR patterns using three methods: a commercially available Qiagen kit (Qiagen DNeasy tissue), a whole cell method requiring no pre-treatment, and a method developed in-house using the MacConnell Mini-prep 96 (mini-prep) instrument. The whole cell method was the least expensive, but demonstrated the least precision (reproducibility). The Qiagen kit and the Mini-prep 96 showed high reproducibility (90-95%); however, the Mini-prep 96 is less expensive and very rapid, allowing processing of up to 192 isolates/day.

Water and sediment from a Florida river were placed in an outdoor flume that maintained turbulent flow and oxic conditions in the water column (~11 mg/L). The flume was inoculated with seven E. coli strains and nine Enterococcus spp. of distinct BOX-PCR phylotypes. Putative "survivor" strains previously isolated from mesocosms and disinfected wastewater effluent and control laboratory strains were chosen to test the hypothesis of differential survival of strains under hydrodynamically active conditions. IO strains isolated each day were typed by BOX-PCR (n=100 isolates/day), revealing differential survival of certain E. coli and Enterococcus strains. Ultimately, a better understanding of the effect of hydrodynamic regime and phylotype distribution on IO survival in water will allow more accurate modeling of the fate of these organisms in aquatic environments. This will in turn lead to a better understanding of the organisms we use as indicators of pollution. This is necessary to ensure the health and safety of all recreational water users.

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