Genetic determinants of gene amplifications alter frequency and evolutionary trajectory of antibiotic resistance in Staphylococcus aureus

Gene amplifications are thought to be common in bacterial populations, providing a rapid reversible mode of adaptation to diverse stresses, including the acquisition of antibiotic resistance. We previously showed that the opportunistic pathogen Staphylococcus aureus evolves resistance to the dual-targeting fluoroquinolone delafloxacin (DLX) that inhibits both the DNA gyrase and DNA topoisomerase IV via gene amplifications of an efflux pump encoding gene sdrM. However, the pathways that control the formation or selection of gene amplifications, and consequently adaptive trajectories, remain understudied, especially in gram-positive bacteria like S. aureus. Here, we show that specific DNA repair and chromosomal separation pathways alter the frequency of formation and selection of gene amplifications in S. aureus. Through a screen of 36 mutants deficient in various DNA processes, we found that while sdrM amplification was still the almost universal path to DLX resistance, other mutations that increased sdrM expression reduced the selection frequency of sdrM amplifications, demonstrating the critical role of sdrM in DLX resistance. We found that similar to other bacteria, the formation and loss of sdrM amplifications required a functional RecA recombinase, but multiple other mutants in pathways required for amplifications in other species still exhibited frequent sdrM amplifications, suggesting that S. aureus may have alternate routes of amplification formation. Finally, mutants in the tyrosine recombinase XerC that is involved in chromosomal separation were deficient for sdrM amplifications, indicating that XerC is a novel modulator of amplification formation, maintenance, or selection. Thus, our work sheds light on genetic factors that alter gene amplification-mediated evolutionary trajectories to antibiotic resistance in S. aureus and can potentially unlock mechanisms by which such evolution of resistance can be inhibited.