Assessing the contributions of DNA gyrase and topoisomerase IV to fluoroquinolone persistence

Abstract

Persisters are dormant variants of regular bacterial cells that are highly tolerant to antibiotic treatments, and they have been clinically implicated in the recalcitrance of chronic bacterial infections to antibiotics. This study focuses on persistence to fluoroquinolones (FQs), a class of potent, broad-spectrum antibiotics commonly used to treat urinary tract infections caused by uropathogenic Escherichia coli. FQs act by binding and inhibiting DNA gyrase and topoisomerase IV. The purpose of this study was to investigate the extent to which DNA gyrase and topoisomerase IV poisoning can affect FQ persistence in E. coli. To answer this question, several strains containing mutations in GyrA (a gyrase subunit), ParC (a topoisomerase IV subunit), and both enzymes were isolated through λ Red recombineering, and subsequent P1 phage transduction, of mutations raised through adaptive laboratory evolution. CRISPR/Cas9-optimized λ Red recombineering was also attempted as a method to isolate strains from synthetic oligonucleotides with clinically relevant mutations; however, this method failed due to a low efficiency for selection of the mutations. Following strain isolation, minimum inhibitory concentration (MIC) analyses and persister assays of the mutants revealed the development of resistance and persistence levels attributable to DNA gyrase and topoisomerase IV poisoning. Finally, a mathematical model was developed to quantitatively assess the contributions of GyrA and ParC mutations to persistence. Comparisons of MIC values confirmed previous findings that GyrA single mutations were sufficient in generating low levels of resistance, while additional ParC mutations were necessary for the development of high levels of FQ resistance. The most striking finding was that the single mutation in topoisomerase IV (G78D in ParC) did not confer resistance on its own, but it did result in high levels of FQ tolerance. This result suggests that the single mutation may develop high persister (hip) mutants and should be utilized in the future as a potential target for the development of FQ tolerance.