Tracking Antibiotic Resistance Genes in the South Platte River Basin Using Molecular Signatures of Urban, Agricultural, And Pristine Sources

A novel approach utilizing antibiotic-resistance-gene (ARG) molecular signatures was applied to track the sources of ARGs at sites along the Cache la Poudre (Poudre) and South Platte Rivers in Colorado. Two lines of evidence were employed: (1) detection frequencies of 2 sulfonamide and 11 tetracycline ARGs and (2) tet(W) phylotype and phylogenetic analysis. A GIS database indicating the locations of wastewater treatment plants (WWTPs) and animal feeding operations (AFOs) in the watershed was also constructed to assess congruence of the surrounding landscape with the putative sources identified by ARG molecular signatures. Discriminant analysis was performed on detection frequencies of tetARG groups that were previously identified to be associated with either WWTPs or AFOs. All but one (South Platte River-3, just downstream from the confluence with the Poudre River) of the eight sites were classified as primarily WWTP-influenced based on discriminant analysis of ARG detection frequencies. tet(W) phylotype analysis also aligned South Platte River-3 with putative AFO sources, while phylogenetic analysis indicated that it was not significantly different from the AFOs or WWTPs investigated. South Platte River-3 is situated in an intense agricultural area, but the upstream portion of the South Platte River receives substantial loading from metropolitan Denver. By contrast, tet(W) phylotype and phylogenetics of site Poudre River-4, located 4 km downstream of a WWTP, was also characterized and found to be significantly different from the AFO lagoons (p < 0.05), as expected. In general, a good correspondence was found between classification of the impacted river sites and the surrounding landscape. While the overall approach could be extended to other watersheds, the general findings indicate that transport of ARGs from specific sources is likely the dominant mechanism for ARG proliferation in this riverine environment relative to selection of ARGs among native bacteria by antibiotics and other pollutants.