Potential selection and maintenance of manure-originated multi-drug resistant plasmids at sub-clinical antibiotic concentrations

Abstract

The goal of this study was to determine minimum selection concentrations of various antibiotics using four manure-originated multi-drug resistant plasmids in a surrogate Escherichia coli host. These plasmids carried genes conferring resistance phenotypes to several antibiotic classes including beta-lactams, lincosamides, phenicols, macrolides, sulfonamides and tetracyclines. The minimum selection concentrations of antibiotics tested in nutrient-rich medium were determined: 14.1-28.2 mg/L for penicillin G, 0.1 mg/L for oxytetracycline, 0.45 mg/L for chlortetracycline, 2 mg/L for lincomycin, 1 mg/L for florfenicol, 1.3-4 mg/L for azithromycin, 0.13-0.25 mg/L for tetracycline, 0.004-0.01 mg/L for cefotaxime. Penicillin G, oxytetracycline, chlortetracycline, lincomycin and florfenicol had minimum selection concentrations in nutrient-defined medium slightly changed within 3.5-fold range compared to those in nutrient-rich medium. The minimum selection concentrations of antibiotics interfering folic acid synthesis in bacteria were also determined: 63 mg/L for sulfamethoxazole, 11.2 mg/L for sulfisoxazole and 0.06 mg/L for trimethoprim. Mixing two antibiotics changed minimum selection concentrations within 3.7-fold range compared to those in single antibiotic tests. Relatively high plasmid loss rates (> 90%) were observed when culturing plasmid-bearing strains in antibiotic-free nutrient-rich and nutrient-defined media. Overall results suggested that these plasmids can be maintained at concentrations environmentally relevant in waste water treatment plants, sewage, manure and manured soil although they are not stable in antibiotic-free environments.