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Antibiotic Resistance Patterns (ARPs) of Enterococcus spp. were used as a phenotypic fingerprint to compare and categorize unknown-source isolates in an impaired segment of the Roanoke River, Roanoke County, Virginia. Antibiotic resistance analysis (ARA) of enterococci has been effectively used to differentiate among sources of fecal contamination in many geographic regions in the United States. Enterococcus spp. were used as a fecal indicator in a library consisting of 1,562 known-source isolates. Two-way analysis indicated that approximately 95% of the unknown-source isolates collected were of animal origin. A 3-way analysis indicated that 61% of the unknowns were of livestock origin while 34% were of wildlife origin. Of the isolates determined to be of wildlife origin, almost all were from raccoons and geese while enterococci from deer were present at low percentages. For one sample date, 20% of the isolates at one site were of human origin. This bacterial source tracking (BST) data will prove valuable for the development of TMDLs for this impaired waterway.