This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Antibiotic resistance is one of the world's most pressing public health problems. Widespread use of antibiotics for human and animal health has led to increased levels of antibiotics in wastewater and freshwater ecosystems. Studies indicate that increased levels of antibiotics in water contribute to an increased incidence of antibiotic resistance. It is our hypothesis that wastewater treatment plant (WWTP) effluent is a source of antibiotics and antibiotic resistant bacteria in the environment and contributes to the establishment of environmental reservoirs of antibiotic resistance. Therefore, we propose to characterize bacteria in a freshwater ecosystem that receives effluent from a WWTP. Coliforms will be studied as they are common pollutants in freshwater ecosystems, a well-studied group of organisms, and some strains cause disease in humans. Aeromonads will also be studied as they are ubiquitous in freshwater ecosystems and some strains are human pathogens also. Resistance of bacteria to three antibiotics from three different classes of antibiotics (beta-lactams, tetracyclines, fluoroquinolones) will be studied. Incidence of antibiotic resistance, levels of antibiotic resistance, and genetic determinants of antibiotic resistance will be examined. Samples will be taken at three sites along Tahlequah Creek (one upstream and one downstream of the WWTP and effluent from the WWTP). Enumeration of total coliforms and E. coli levels in water samples will be performed using an EPA-approved most probable number (MPN) method. Enumeration of antibiotic resistant bacteria will be performed using the same method by adding antibiotics to the water samples. Antibiotic resistant bacteria will be isolated from effluent and sediment samples using the MPN kits and differential media. Isolates will be identified based on their 16S rDNA sequences then tested for resistance to various antibiotics. The molecular basis for resistance will be determined using published methods (PCR, IEF). Data and isolates will be collected at regular intervals and used to assess the stability and persistence of antibiotic resistance and resistance genes in this ecosystem. Future plans include examination of horizontal gene transfer of antibiotic resistance genes within and between bacterial species and the mechanisms involved in gene transfer.