TOWARDS A BETTER UNDERSTANDING OF PERSISTENCE AND TOLERANCE OF STARVING E. COLI POPULATIONS

Abstract

Bacterial persisters - subpopulations of isogenic bacterial cultures that can tolerate lethal doses of antibiotics, constitute a major threat to public health due to their abilities to repopulate after the conclusion of treatments and spawn recurrent infections. Many antibiotics, such as ß -lactams, are ineffective against growth-inhibited populations. In clinical settings, however, microbes can be slowing-growing or starved, especially in biofilms, granulomas, or intracellular bacterial reservoirs. Fluoroquinolones (FQs) and nitrofurans are two classes of antibiotics that are known to be effective against both actively growing and growth-inhibited bacteria, and used in the treatment of urinary tract infections. FQs kill by targeting topoisomerases, such as DNA gyrase, and at saturating concentrations produce lethal DNA double-stranded breaks (DSBs). Previous studies have shown that FQ persisters are not spared from DNA damage; however, little is known about the characteristics of FQ-induced DNA damage that persisters endure. In this dissertation, we developed GCS-seq that can precisely map FQ-stabilized gyrase-DNA cleavage sites (GCSs) at the genome scale. Applying GCS-seq to FQ-treated stationary-phase E. coli, we identified a total over 20000 GCSs. Importantly, we showed that the genome cleavage strength is an extremely strong predictor of persistence. Following that study, we investigated DNA damage repair in FQ persisters, and demonstrated that the DNA repair machinery used depends on the chromosome copy number (ploidy) in individual bacteria. Specifically, homologous recombination (HR)-deficient mutants (∆recN and lexA3) showed a significant decrease in survivability in diploid cells compared to wild-type, and such depression was not observed in monoploids. Further we identified DNA repair mutants that are crucial for monoploid persister survival (∆uvrD and exonuclease VII mutants). Beyond FQs, we studied the efficacies of nitrofurans, which are prodrugs that once activated lead to pleiotropic damage, on growth-inhibited populations. We observed near-complete tolerance to nitrofurans in stationary-phase E. coli, and found that metabolite supplementation could resensitize them without stimulating growth. We demonstrated that the tolerance observed was due to insufficient reducing equivalent supply for nitrofuran activation. Collectively, this work deepens understanding of FQ persistence and nitrofuran tolerance and showed that closer inspection of these phenomena could illuminate strategies to address hard-to-treat infections.