Genome‐wide screen reveals a universal role of ATP in ciprofloxacin tolerance among genetically distinct Escherichia coli persisters

Bacterial persisters show tolerance to bactericidal antibiotics and play essential roles in chronic infections; however, the general mechanisms underlying persister formation and antibiotic tolerance remain insufficiently characterized. In this study, the Escherichia coli Keio library was used to identify genes involved in ciprofloxacin tolerance by culturing each mutant to the late stationary phase (to induce persistence via starvation), followed by dilution into fresh medium for antibiotic exposure. This two-step, genome-wide screening approach enabled the identification of 37 ciprofloxacin-sensitive mutants with diverse biological functions and 11 ciprofloxacin-tolerant mutants related to amino acid and β-nicotinamide adenine dinucleotide (NAD⁺) biosynthesis, with 25 genes being identified as persister-related genes for the first time. Notably, sensitive mutants (ΔatpC, ΔatpF, ΔruvC, and Δrnr) were specifically sensitive to quinolone antibiotics, whereas tolerant mutants (ΔmetR, ΔleuB, and ΔnadB) showed tolerance to ampicillin and gentamicin. Importantly, adenosine triphosphate (ATP) levels were downregulated in ciprofloxacin-tolerant mutants and upregulated in ciprofloxacin-sensitive mutants, implying a negative correlation between ATP levels and ciprofloxacin tolerance among these genetically distinct persisters. This negative correlation was further observed when ATP levels in different mutants were chemically modulated using specific metabolites, nutrients, and respiration inhibitors. In addition, ciprofloxacin persistence across different mutants was found to correlate closely with antibiotic uptake and reactive oxygen species (ROS) levels. Collectively, these findings establish a universal role for ATP in the ciprofloxacin tolerance of genetically diverse persisters under varying resuscitation conditions, conceivably through the modulation of antibiotic uptake and ROS accumulation, and it is implied that the provision of abundant nutrients is potentially beneficial for anti-persister chemotherapy in clinic settings.