Tetracycline Resistance Gene Maintenance under Varying Bacterial Growth Rate, Substrate and Oxygen Availability, and Tetracycline Concentration

Abstract

Neither amplification nor attenuation of antibiotic resistance genes (ARG) in the environment are well understood processes. Here, we report on continuous culture and batch experiments to determine how tetracycline (TC), aerobic vs anaerobic conditions, bacterial growth rate, and medium richness affect the maintenance of plasmid-borne TC resistance (Tet^R) genes. The response of <i>E. coli</i> (a model resistant strain excreted by farm animals) versus <i>Pseudomonas aeruginosa</i> (a model bacterium that could serve as a reservoir for ARGs in the environment) were compared to gain insight into response variability. Complete loss of the Tet^R RP1 plasmid (56 kb) occurred for <i>P. aeruginosa</i> in the absence of TC, and faster loss was observed in continuous culture at higher growth rates. In contrast, <i>E. coli</i> retained its smaller pSC101 plasmid (9.3 kb) after 500 generations without TC (albeit at lower levels, with ratios of resistance to 16S rDNA genes decreasing by about 2-fold). A higher rate of ARG loss was observed in <i>P. aeruginosa</i> when grown in minimal growth medium (M9) than in richer Luria broth. Faster ARG loss occurred in <i>E. coli</i> under anaerobic (fermentative) conditions than under aerobic conditions. Thus, in these two model strains it was observed that conditions that ease the metabolic burden of plasmid reproduction (e.g., higher substrate and O₂ availability) enhanced resistance plasmid maintenance; such conditions (in the presence of residual antibiotics) may be conducive to the establishment and preservation of ARG reservoirs in the environment. These results underscore the need to consider antibiotic concentrations, redox conditions, and substrate availability in efforts to evaluate ARG propagation and natural attenuation