Evaluation and optimization of detection methods for Rhodococcus coprophilus and sorbitol-fermenting Bifidobacteria as source-specific indicator organisms for drinking water sources

Surface drinking water sources are threatened with non-point fecal contamination usually originated from runoff that carries fecal material from wild and farm animals, and from leaking septic and sewerage systems. Identification of the specific source(s) of contamination would allow for more effective management and control programs in a watershed.; Sorbitol-fermenting Bifidobacteria and Rhodococcus coprophilus have been suggested as bacterial indicators of fecal contamination from human and animal origin, respectively. The ecology of the bacteria was investigated. Detection and enumeration methods for the two indicators were evaluated and optimized. The effect of environmental and experimental factors in the recovery, detection levels, and false-positive/false-negative incidences were investigated. Membrane filtration, incubation on Human Bifid Sorbitol Agar, followed by confirmation tests is the recommended method for sorbitol-fermenting Bifidobacteria. Filtration-resuspension, spread-plating and incubation on MM3 agar is recommended for R. coprophilus . A survey of animal and human fecal samples confirmed the potential of sorbitol-fermenting Bifidobacteria as a human-fecal indicator and of R. coprophilus as a grazing (farm) animal fecal indicator. The detection/absence of the indicators in the water samples collected during a one-year survey around the Wachusett Reservoir and its watershed in Central Massachusetts agreed with the potential sources suggested by land use in the sampling areas.; The usefulness of a typing procedure to characterize strains of R. coprophilus originated from different animals was investigated. A total of 45 strains of R. coprophilus were isolated from the fecal samples and subjected to a set of biochemical and antibiotic sensitivity tests. The resulting data was analyzed statistically in an attempt to develop a biotyping method that allows for the classification of the isolates according to their animal of origin. The method included carbohydrate utilization tests, growth on sole carbon source tests and antibiotic resistance tests. This method still needs further development, but could be used for the identification of the source(s) of R. coprophilus detected and isolated from water samples.