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A Molecular Approach to Determine the Origin of Fecal Bacteria in the Catoma Creek Watershed




Wijesinghe, Rasanthi

Type of Degree



Agronomy and Soils


High concentrations of fecal indicator bacteria are the most common cause of surface-water impairment in Alabama. A 37 km segment of Catoma Creek in Montgomery Country has been included on Alabama 303(d) List of impaired water bodies due to elevated concentrations of fecal coliform bacteria and organic enrichment. Fecal contamination can originate from both human and non-human sources, including surface runoff from land application of animal wastes or farm animal feedlots, inadequate septic or sewer systems, improper waste disposal, and wildlife impact. The objectives of this study were to monitor the fecal contamination and identify sources of contamination in the Catoma Creek watershed. Water samples were collected monthly at eight locations in the watershed for a period of one year. E. coli was enumerated using the modified m-TEC media. Data showed that E. coli concentrations varied from 18 to 12,650 CFU/100 ml, with 70% of the samples exceeding the EPA criterion for swimming water. There was a positive correlation between flow rates and E. coli concentrations. Chemical analyses of the water samples showed that the concentration of total phosphorus in all the samples was above the proposed Ecoregion IX nutrient criterion, 78% of samples were above the NO3-N criterion, and 50% of samples were above the total nitrogen criterion, suggesting that there is a serious risk of eutrophication in this watershed. The rep-PCR DNA fingerprint technique was used to identify the sources of fecal contamination in the Catoma Creek watershed. A known source library of DNA fingerprints was developed using 582 E. coli isolates obtained from humans, dogs, cattle, chickens, horses, wild turkeys, waterfowl and deer. DNA fingerprints generated using the BOX A1R primer demonstrated great genetic diversity of E. coli. Cluster analyses of DNA fingerprint patterns were performed with BioNumerics software using a densitometric curve based matching function (Cosine) and unweighted pair group method with arithmetic average. Jackknife analysis was used to determine cluster/group validity, revealing that the average rate of correct classification for the entire library was 88% and that of the decloned library was 74%. The DNA fingerprints (502) obtained from E. coli isolated from the water samples of the Catoma Creek watershed were compared against those in the known source library. Results showed that 18% of the E. coli isolates were from humans, 14% each from dogs and waterfowl, 4% each from deer and wild turkeys, 2% each from cattle and chickens, 0% from horses, and the remaining 41% unidentified. Further research is needed to improve the representativeness of the library by including more source groups and E. coli isolates.