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The role of fecal indicator bacteria (FIB) in water quality assessment is to provide a warning of the increased risk of pathogen presence. An effective surrogate for waterborne pathogens would have similar survival characteristics in aquatic environments. Although the effect of abiotic factors such as sunlight and salinity on the survival of FIB and pathogens are becoming better understood, the effect of the indigenous microbiota is not well characterized. The influence of biotic factors on the survival of non-pathogenicEscherichia coli, Enterococcus faecalis, andE. coliO157:H7 were compared in fresh (river) water and sediments over 5 days. Treatments were (i) disinfection (filtration of water and baking of sediments) to remove indigenous protozoa (predators) and bacteria (competitors), and (ii) kanamycin treatment to reduce competition from indigenous bacteria. The disinfection treatment significantly increased survival ofE. coli, E. coliO157:H7 andEnt. faecalisin the water column. In sediments, survival of FIB but not that ofE. coliO157:H7 increased in disinfected treatments, indicating that the pathogen's survival was unaffected by the natural microbiota. Location (water or sediment) influenced bacterial survival more than species/type in the disinfection experiment. In the competition experiments where only the natural bacterial flora was manipulated, the addition of kanamycin did not affect the survival ofEnt. faecalis, but resulted in greater survival ofE. coliin water and sediment. Species/type influenced survival more than the level of competition in this experiment. This study demonstrates the complexity of interactions of FIB and pathogens with indigenous microbiota and location in aquatic habitats, and argues against over-generalizing conclusions derived from experiments restricted to a particular organism or habitat.