High-throughput functional screening reveals low frequency of antibiotic resistance genes in DNA recovered from the Upper Mississippi River

In this study, we determined the frequency of antibiotic resistance genes (ARGs) in the Upper Mississippi River using a high-throughput, functional, metagenomic screening procedure. Fosmid libraries containing ∼10,000 clones were screened for resistance to ampicillin, cephalothin, kanamycin, and tetracycline. We hypothesized that nutrient concentrations, land cover type, and taxonomic community composition may select for ARGs. Resistance to ampicillin, cephalothin, and kanamycin was low (<1.00%), and no resistance to tetracycline was detected. Ammonium and total dissolved solids (TDS) concentrations were correlated with kanamycin and cephalothin resistances (r= 0.617 and -0.449, P = 0.002 and 0.036, respectively). Cephalothin resistance was also positively correlated with the percentage of forested land cover (r = 0.444, P = 0.039). Only the candidate division OD1, among 35 phyla identified, was correlated with ampicillin resistance (r = 0.456, P = 0.033), suggesting that minority members of the community may be responsible for dissemination of ARGs in this ecosystem. Results of this study suggest that ammonium and TDS may be involved in a complex selection process for ARGs. Furthermore, we suggest that minority species, potentially contributed in low numbers from sediment and biofilm reservoirs, may be the primary carriers of ARGs in this riverine system.