Investigating the role of dna gyrase and topoisomerase iv mutants in fluoroquinolone persistence

Abstract

Persistence refers to the phenomenon in which a subpopulation of bacterial cells does not demonstrate increased resistance to antibiotics but dies more slowly under drug treatment. Since persister populations are not detectable through an increase in MIC, as resistant strains are, they often go undetected in the clinic and will begin to grow normally again once antibiotic treatment is removed, resulting in recurrent infections. This study examined persistence against fluoroquinolones (FQs), an antibiotic class often used to treat respiratory and urinary-tract infections. Our objective was to construct Escherichia coli strains containing single point mutations observed in the background of FQ-resistant clinical isolates, and to determine their resistance and persistence to FQs. Using CRISPR/Cas9-optimized λ Red recombineering, a technique which combines the recombination capabilities of the λ Red system to introduce point mutations and the cleavage abilities of the CRISPR/Cas9 system to select against non-mutated cells, we developed seven E. coli mutant strains: gyrB S464Y, gyrB K447E, parC S57T, parC A56T, parE I529L, parE I444F, and parE S458T. We found that six of these mutants exhibited increased persistence to the FQ levofloxacin (relative to wild-type E. coli ) without an increase in the level of resistance. These results demonstrate the importance of further investigation into gyrB, parC, and parE mutations in strains that are not initially resistant but are subsequently implicated in recalcitrant infection under FQ treatment — in turn widening the mutational landscape to include genes beyond gyrA in the study of FQ resistance.