Characterization of Antibacterial Drugs Against Non-Growing Bacteria

Abstract

Non-growing bacterial cells, such as those found in chronic infections, are highly tolerant to antibiotics and contribute to treatment failure and relapse. A high-throughput screen of over 6,000 drugs and drug candidates from repurposing libraries identified 39 with activity against non-growing uropathogenic Escherichia coli. These hit compounds were subsequently tested against non-growing Pseudomonas aeruginosa and Staphylococcus aureus, and the 24 most active candidates were selected for further evaluation. Dose-dependent effects on regrowth delay and bacterial killing were assessed across all three species. Statistical analysis confirmed that the observed regrowth delays were significant and reproducible, with p-values below 0.0001 for the most effective treatments. The impact of treatment duration, testing conditions, and efflux pump activity on drug efficacy was also investigated. Most compounds exhibited rapid activity, with significant effects detected within the first hour of exposure. The composition of the growth medium—particularly phosphate buffering—strongly influenced drug activity, whereas bacterial strain and regrowth conditions had minimal effect. Inhibition of efflux pumps during treatment or regrowth had no clear effect. Finally, delafloxacin—a fluoroquinolone not included in the original libraries—was tested due to its structural similarity to sitafloxacin, one of the most potent hit compounds. Despite its enhanced halogenation, delafloxacin showed inferior activity against non-growing bacteria compared to sitafloxacin.