Alternate genetic paths of adaptation to spectinomycin in Escherichia coli

Unregulated antibiotic use leads to their accumulation in the environment at sublethal levels, facilitating the evolution of antimicrobial resistance in bacteria. Antibiotics used in farms and veterinary medicine are frequently detected in soil and water, though genetic adaptations to them in human-relevant bacteria are poorly characterized. In this study, we investigated adaptation of Escherichia coli to low concentrations of spectinomycin, an inhibitor of bacterial translation and broad-spectrum antibacterial for domestic animals. Using laboratory evolution, we elucidate 2 distinct strategies of adaptation to low levels of spectinomycin, distinguished by higher fitness in the antibiotic with or without an appreciable change in minimum inhibitory concentration. Both strategies were effective against a natural spectinomycin-producing strain and showed a partially overlapping mutational signature. Increase in drug minimum inhibitory concentration, i.e. canonical resistance, required target site mutations in the ribosomal S5 protein. Adaptation without change in minimum inhibitory concentration, however, was mediated by target-associated as well as nontarget mutations. A novel nontarget locus was the multidrug efflux pump MdfA. Interestingly, loss of MdfA rather than overproduction conferred growth advantage in spectinomycin, contrary to its established function as an efflux channel. We demonstrate that MdfA's role in proton homeostasis contributed to this phenotype. Finally, we show that low drug-adapted bacteria were "primed" for resistance acquisition when challenged with high spectinomycin pressure. Thus, our study identifies alternate genetic strategies of bacterial adaptation to low concentrations of an environmentally relevant antibiotic and establishes an interplay between them.