Field-testing Enterococcus Faecalis for Bacterial Source Tracking

A waste stabilization pond was suspected of causing fecal contamination to Holt Branch, a tributary of Juniper Creek, near Byron, Georgia. We used ribotyping, a genotypic (DNA-based) bacterial source tracking (BST) method, to match Enterococcus faecalis isolates from Holt Branch and the waste stabilization pond. Fecal enterococci in the effluent from the waste stabilization pond averaged 13,863 colony-forming units (CFU) per 100 mL, and numbers of fecal enterococci from Holt Branch averaged 3,290 CFU per 100 mL. The 17 Ent. faecalis isolates from the Holt Branch yielded 4 ribotypes, and the 41 isolates obtained from the waste stabilization pond yielded 9 ribotypes. All four ribotypes from Holt Branch matched the ribotypes from the waste stabilization pond at a 100% similarity index, which suggested that the waste stabilization pond was the source of fecal contamination to Holt Branch. This is the first use of Ent. faecalis for BST. INTRODUCTION A waste stabilization pond located in Byron, Georgia, was suspected as a source of fecal contamination of Holt Branch, a tributary of Juniper Creek. The Georgia Environmental Protection Agency requested that this suspected contamination be confirmed because Juniper Creek supplied water to a pond used by the Boy Scouts of America for swimming. Because the flow of Holt Branch was solely dependent on the effluent from the waste stabilization pond, the situation provided conditions ideal to field-test Ent. faecalis, a fecal indicator bacterium, for BST. BACKGROUND AND RELATED WORK In most BST methods, environmental isolates of a fecal bacterium are matched to isolates of the same bacterium obtained from various warm-blooded animals either phenotypically (based on characteristics expressed by the bacterium, like antibiotic resistance; e.g., Wiggins et al., 1999) or genotypically (e.g., Parveen et al., 1999). By matching the two, the host origin of the environmental isolates can be determined. One BST method is ribotyping (Farber, 1996). This method is based on identifying highly conserved sections of DNA (not mutated readily) that encode for ribosomal RNA. Ribotyping yields banding patterns (ribotypes) that can be compared to each other for similarity. As a method for BST, it has excellent reproducibility, good discriminatory power, excellent ease of interpretation, and good ease of performance. However, manual ribotyping is tedious and time consuming. It is possible to automate the process with a RiboPrinter (DuPont Qualicon, Wilmington, DE). This instrument increases the accuracy and precision of ribotyping while making the method quicker and simpler. One of the most important objectives of BST is to distinguish between human and other sources of fecal contamination. Water managers are interested in this because human feces are commonly presumed to be the reservoir of many human pathogens. Also, compared to other sources of fecal contamination (e.g., wildlife), human sources are relatively easy to fix and remedy. One bacterium that may be linked to humans and not to many other warm-blooded animals is Ent. faecalis (Pourcher et al., 1991; Wheeler et al., 2002).