Identifying Sources of Fecal Pollution in the Roanoke River, Roanoke County, Virginia

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. INTRODUCTION To date 3,486 km of the 78,000 km of streams and rivers in Virginia are listed as impaired, with only one third being adequately monitored (FOR VA). The Roanoke River, used as a source of drinking water and recreation, originates in the mountains of Montgomery County, runs eastward through the highly populated areas of Roanoke County, Salem City, and Roanoke City, continues into North Carolina and empties into Albemarle Sound, North Carolina. In the Roanoke area, land usage is both agricultural, on which horses, cattle and other agriculturally important animals are present, as well as urban from which human indicator bacteria may originate. Large numbers of resident geese and ducks as well as other wildlife are also present. Of the 803 stream segments in Virginia that are listed as impaired waters, fecal indicator bacteria are the leading cause of the impairment (DEQ). In the next 10 years, Virginia must develop TMDLs (Total Maximum Daily Load) for 600 impaired segments (DEQ). Public watersheds can be restricted from human recreational use if they exceed the Environmental Protection Agency (EPA) standard of 126 Escherichia coli or 33 Enterococcus colony forming units (cfu) per 100 mL (geometric mean) in fresh water (EPA). Diseases caused by enteric pathogens potentially transmitted through contaminated water include cholera (Vibrio cholerae), gasteroenteritis (Escherichia coli) , giardiasis (Giardia) , salmonellosis and typhoid fever (Salmonella sp.), shigellosis (dysentery, Shigella sp.), and viruses, such as hepatitis A and Norwalk group viruses (Parveen et al., 1999; United States Environmental Protection Agency, 2001). 158 VIRGINIA JOURNAL OF SCIENCE With current water testing procedures, the presence of fecal indicator organisms indicate the presence of fecal material but not the source of the contamination. Transforming a non-point source into a point source is valuable in order to improve water quality, reduce the nutrient load leaving the watershed, and prevent possible transmission of disease (Hagedorn et al, 1999). Several methodologies have been implemented to determine human and non-human sources of contamination. While many methods exist, several have been used extensively and successf\illy, or show promise. These methods include antibiotic resistance analysis (ARA; Wiggins; 1996; Hagedorn et al, 1999; Wiggins et al., 1999; Bowman et al., 2000; Harwood et al., 2000; Bower, 2001), ribotyping (Parveen et al, 1999; Hartel etal., 2000; Carson et al., 2001), pulsed-field gel electrophoresis (PFGE; Simmons, 2000), and utilization of specific carbon sources (Hagedorn, et al., in review). In the work presented here, Enterococci were used as a fecal indicator for our library of known-source isolates. While fecal coliforms are the standard indicator in Virginia, ARA using the enterococci has been highly successful (Hagedorn et al., 1999; Wiggins et al., 1999; Bower, 2000; Bowman et al., 2000; Harwood et al., 2000). Enterococci are an appropriate indicator in brackish and salt water primarily because they are more apt to survive in marine environments than fecal coliforrns because they can tolerate high (6 .5%) salt concentrations (Hagedorn et al, 1999). Enterococci also have a higher survival rate through wastewater treatment processes than fecal coli forms making them an attractive target in fresh water (Harwood et al., 2000). This provides the basis for using antibiotic resistance patterns (ARPs) as a "phenotypic fingerprint" to compare and categorize unknown Enterococcus spp. isolates. Although antibiotics are primarily used in humans and livestock, we find antibiotic resistance is widespread and common, even in wildlife such as Canada goose, white-tailed deer, muskrat, and raccoon. We report here that ARA of fecal enterococci from known fecal sources, used in conjunction with discriminate analysis (DA), effectively predicted the sources of isolates taken from four Roanoke river sample sites over three sample dates. We have reinforced the bacterial source tracking method and we report both the extent of fecal contamination in the Roanoke River on these dates and the source of fecal contamination. MATERIALS AND METHODS Bacterial Library Fresh fecal material, ranging from a swab to several grams, from known sources (horse, human, raccoon, sheep, chicken, cow, white-tailed deer, Canada goose, and muskrat), was diluted in sterile distilled water. Samples from humans are presumed to be a mixture of isolates from several individuals from a portable toilet or pump out truck. Multiple manure samples were collected and mixed, while goose isolates were from single individuals. Several horses were swabbed to obtain fecal material. One hundred L fecal suspension was pipetted onto each mEnterococcus agar (Difeo) plate and spread with a sterile glass hockey stick. Plates were inverted and incubated at 37C for 48h . Burgundy and pink colonies were picked off with sterile toothpicks and placed into 200μL enterococcosel broth (BBL) in sterile 96-well plates. Black wells (positive for Enterococcus spp.) were noted. A 48 prong replicaplater was used to transfer isolates onto Trypticase Soy Agar with lecithin and polysorbate 80 (BBL) Antibiotic Plates (Table 1). Antibiotic plates were inverted and incubated for 48h at 37C, and SOURCE TABLE 1. Antibiotics and Final Plate