The fecal loading of aquatic environments by various animal hosts is of concern
for public and environmental health. The microbiological contamination of waterways is
amongst the most commonly listed water quality impairment in the U. S. The federal
Clean Water Act (CWA) requires individual states to provide the U. S. Environmental
Protection Agency (USEPA) with an aggregate quality assessment of its waterways every
biennium, in effort to identify waters that do not meet state and federal quality standards.
Once waterways are deemed impaired, states must conduct a Total Maximum Daily Load
(TMDL) assessment in order to mitigate the impairment and restore the water body to
acceptable quality. Successful bacterial TMDL implementation strategies require the use
of microbial source tracking (MST) technologies that accurately and efficiently
characterize the host-specific source of bacterial loading of waterways, and the relative
quantities of each bacterium. Here I report that suppression subtraction hybridization
(SSH) was found useful to identify gene markers specific to E. coli derived from swine
fecal sources. The ability of this marker gene set to identify 62.3% of E. coli isolated
from swine hosts suggests it may be useful in determining their fecal contribution to
impaired waterways. I also investigated the influence of cattle grazing operations on the
microbiological impairment of a small stream system in Southeastern Minnesota.
Impairment by fecal indicator bacteria (FIB) was assessed by using plate count analyses
and a quantitative PCR (q-PCR) was developed to estimate the presence of a bovinespecific
Bacteroides marker gene in the waterway. The q-PCR data were compared to E.coli plate count data, revealing a lack of correlation between the two methods. Several physical and environmental factors likely influenced the level of E. coli found in the
stream, confirming the hypothesis that other information will be needed to supplement
current efforts to monitor fecal indicator bacteria in order to determine accurate sourcespecific
fecal impacts. Lastly, spatial and temporal variation in the population structure of
a fecal pathogen (Salmonella) in association with an alternate host and habitats, including
the green alga Cladophora found in stream and lake water, aquatic plants, beach sand,
and sediments, was evaluated by use of horizontal fluorophore-enhanced rep-PCR
(HFERP) DNA fingerprinting. It was revealed that Salmonella populations associated
with Cladophora varied both spatially and temporally, suggesting potentially different
input sources of Salmonella over space and time. In addition, differing environmental
stressors may play a role in selecting particular Salmonella genotypes that are best suited
for growth in these environments The use of rapid molecular-based assays to determine
the presence and source-specific loading of fecal indicator bacteria and pathogens has the potential to improve the accuracy and efficacy of TMDL studies, and to expedite
implementation strategies to remediate impaired waterways.
University of Minnesota M.S. thesis. November 2009. Major: Water Resources Science. Advisor: Michael J. Sadowsky. 1 computer file (PDF); xii, 138 pages. Ill. (some col.)
Sawdey, Richard Charles.
Development, validation, and application of molecular microbial source tracking methods to be used in the assessment of environmental waterways..
Retrieved from the University of Minnesota Digital Conservancy,
Content distributed via the University of Minnesota's Digital Conservancy may be subject to additional license and use restrictions applied by the depositor.