Date of Award

Summer 8-2013

Degree Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Biological Sciences

Committee Chair

Shiao Wang

Committee Chair Department

Biological Sciences

Committee Member 2

Glenmore Shearer

Committee Member 2 Department

Biological Sciences

Committee Member 3

Rudolph Ellender

Committee Member 3 Department

Biological Sciences

Abstract

Enterococci are microbiological indicators of marine recreation water quality.
Their reliability as fecal indicators is questioned as they are shown to persist in the environment. Multiple laboratory studies on their persistence have been done but few under natural environmental conditions. The purpose of this study was to investigate how long sewage enterococci and enterococcal DNA persist in beach water and to determine whether there is a difference in the genetic diversity and hardiness of sewage vs. environmental isolates. To study persistence, sewage was diluted with beach water, placed in microcosms, and deployed at a beach site in Longbeach and Pass Christian, Mississippi. Samples were analyzed for eight days using membrane filtration to enumerate enterococci, and Quantitative Polymerase Chain Reaction ( qPCR) to quantify enterococcal DNA. To assess genetic diversity, BOX-PCR fingerprints of sewage and environmental isolates were compared using UPGMA cluster analysis and Simpson's diversity index. To study hardiness, growth of sewage and environmental isolates in the presence of 2.5 mM hydrogen peroxide in Enterococcosel broth were monitored for 36 hours. Results showed that after eight days, viable counts decreased on average 3.8 logs and target sequence decreased >87% in surface waters. Conversely, enterococci grew >25 folds at the bottom of the water column after four days. The diversity of sewage and environmental isolates was similar during summer months. However, the diversity of sewage isolates declined in cooler months while that of environmental isolates remained high. The hardiness of sewage vs. environmental isolates differed as a greater portion of sewage isolates grew in the presence of hydrogen peroxide than environmental isolates. Results suggested that enterococci survival in marine water is dependent on their location in the water. Varying nutrient availability of the surface vs. bottom of the water column may be responsible for varying survival based on location. While high counts of brief duration may indicate sewage pollution, persistent high counts with no known sewage leaks may be due to re-growth or resuspension of environmental isolates. Also, consistently high genetic diversity of environmental isolates suggests an accumulation of hardier isolates over time. Their lowered resistant to hydrogen peroxide, however, indicated that oxidative damage is not their main selective agent.

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