Research Project
10215254
Summary Infections that contain biofilms are exceptionally difficult to treat. These infections often respond to antibiotic therapy but quickly relapse, resulting in chronic and recurrent infections. The recalcitrance of biofilm infections is thought to arise from the presence of bacterial persisters. Persisters are antibiotic-tolerant cells that are genetically identical to the overall population that succumbs to antibiotics, but occupy a favorable phenotypic niche at the time of treatment. In general, these survivors are thought to be in a state of dormancy, where the activities of antibiotic primary targets are reduced and the extent of antibiotic-induced damage is severely limited. The current model of a biofilm infection cycle includes clearance of normal cells by antibiotics and immunity, both within the film and shed from it, and clearance of persisters that are shed from the film by immunity. Immune cells are hindered from accessing persisters within the biofilm, and when the antibiotic levels drop, persisters proceed to repopulate the film, causing a relapse infection. With every relapse, the chance for an antibiotic-resistant mutation to occur increases, and since persisters are thought to suffer little to no injury from antibiotic treatment, the rate at which resistant mutants should arise from persister-spawned cultures has been assumed to be the same as that of normal bacterial populations. Recently, we discovered that persisters to fluoroquinolones (FQ) in growth-inhibited populations experience FQ-induced DNA damage that is equivalent to damage in bacteria that die from treatment. These unexpected results suggested that those persisters might be mutagenized by FQ and that populations grown up from persisters, such as those of relapse infections, would be genetically diverse and produce antibiotic-resistant mutants at high rates, which we found to be true. These data suggest that there is a highway between persistence and antibiotic resistance whose entrance ramp is treatment with a commonly prescribed class of antibiotics, FQs. We hypothesize that increased understanding of FQ damage in persisters and how they survive that damage will illuminate strategies to reduce relapse infections and hinder antibiotic resistance development. To test our hypothesis, we will tailor a method that quantifies DNA double-strand breaks (DSBs) at the genome-scale to FQ-induced DSBs and employ it to study strains with different persister levels; use time-lapse microscopy to interrogate the roles of the SOS response, DNA repair, elongation, and septation to the recovery of persisters following FQ treatment; and use genetic mutants, lineage tracking, and whole-genome sequencing to determine whether FQ-induced mutagenesis contributes to heightened antibiotic resistance in populations derived from FQ persisters. Collectively, these experiments and the statistical methods we will use to analyze the resulting data will provide mechanistic knowledge of FQ persisters and how they enhance the incidence of antibiotic resistance, which could illuminate new avenues of therapeutic intervention for recalcitrant infections.
