Deepening understanding of persistence to ofloxacin in non-growing bacterial populations

Abstract

Bacterial persisters—cells that can tolerate lethal doses of bactericidal antibiotics and replicate after antibiotic removal—comprise a phenotypic subset of bacterial populations, and are thought to be responsible for the relapse of chronic bacterial infections. Persister frequencies reach their highest in non-growing populations, and non-growing cells are amongst the hardest to kill due to the dependence of many antibiotics on growth-associated processes. Very little is known about factors that distinguish persisters from their antibiotic-sensitive kin in non-growing populations, which suggests that the development of new treatments would be aided by a greater understanding of persister physiology. This thesis works to deepen understanding of persisters in growth-inhibited cultures by using genetic tools to elucidate the physiology of persisters recovering from ofloxacin treatment and by developing a method to assay persister physiology at the systems-level.

In one approach, we used time-lapse microscopy to monitor recovery of Escherichia coli persisters to ofloxacin, and found that persisters extensively filament and induce the SOS response before division, indicating DNA damage. Given these data, we hypothesized that, if damage was repaired incorrectly, antibiotic-resistant mutants would be enhanced in populations derived from persisters compared to untreated controls. We found this to be true. Focusing on rifampicin-resistant mutants, we confirmed that resistance arises from the repair of DNA damage in persisters, and this occurs after a single round of treatment.

Secondly, we recognized that one of the greatest impediments to the study of persisters is their inability to be isolated, which renders most high-throughput methods inapplicable to the study of persisters. To fill this technology gap, we massively parallelized a pre-exisiting method using fluorescence-activated cell sorting, and applied it to interrogate persister physiology in non-growing E. coli populations using a library of promoter reporters. Application of our method uncovered that persistence to ofloxacin in non-growing populations is negatively correlated with ability to synthesize protein.

Collectively, this dissertation deepens understanding of persisters in difficult-to-treat, non-growing populations and provides a high-throughput method to interrogate persister physiology that is applicable to study persistence to any antibiotic in any strain of bacteria that can harbor a fluorescent reporter in any environment.