Potential selection and maintenance of manure-originated multi-drug resistant plasmids at sub-clinical concentrations for tetracycline family antibiotics.

The goal of this study was to (a) determine the minimum selection concentrations of tetracycline family antibiotics necessary to maintain plasmids carrying tetracycline-resistant genes and (b) correlate these results to environmental hotspot concentrations reported in previous studies. This study used two plasmids (pT295A and pT413A) originating from dairy manure in a surrogate host CV601. The minimum selection concentrations of antibiotics tested in nutrient-rich medium were determined as follows: 0.1 mg/L for oxytetracycline, 0.45 mg/L for chlortetracycline, and 0.13-0.25 mg/L for tetracycline. Mixing oxytetracycline and chlortetracycline had minimum selection concentration values increased 2-fold compared to those in single antibiotic tests. Minimum selection concentrations found in this study were lower than reported environmental hotspot concentrations, suggesting that tetracycline family antibiotics were likely to be the driver for the selection and maintenance of these plasmids. Relatively high plasmid loss rates (>90%) were observed when culturing a strain carrying a tetracycline-resistant plasmid in antibiotic-free nutrient-rich and nutrient-defined media. Overall, results suggested that these plasmids can be maintained at concentrations environmentally relevant in wastewater treatment plants, sewage, manure, and manured soil; however, they are unstable and easily lost in the absence of antibiotics.