A Clinical Drug Library Screen Identifies Tosufloxacin as Being Highly Active against Staphylococcus aureus Persisters

Abstract

To identify effective compounds that are active against Staphylococcus aureus (S. aureus) persisters, we screened a clinical drug library consisting of 1524 compounds and identified six drug candidates that had anti-persister activity: tosufloxacin, clinafloxacin, sarafloxacin, doxycycline, thiostrepton, and chlorosalicylanilide. Among them, tosufloxacin had the highest anti-persister activity, which could completely eradicate S. aureus persisters within 2 days in vitro. Clinafloxacin ranked the second with very few persisters surviving the drug exposure. Interestingly, we found that both tosufloxacin and trovafloxacin that had high activity against persisters contained at the N-1 position the 2,4-difluorophenyl group, which is absent in other less active quinolones and may be associated with the high anti-persister activity. Further studies are needed to evaluate tosufloxacin in animal models and to explain its unique activity against bacterial persisters. Our findings may have implications for improved treatment of persistent bacterial infections.

Keywords: Staphylococcus aureus; clinafloxacin; clinical drug library; persisters; tosufloxacin.

Figure 1

Structures of the six drug candidates with activity against S. aureus persisters.

Figure 2

Colony counts to determine the relative activity of the drug candidates and in comparison to known agents active against S. aureus persisters. The S. aureus stationary phase culture was treated with ofloxacin (10 μg/mL) for four hours to enrich persisters. Then, the surviving persisters from the treatment were subjected to drug exposure with different antibiotics (50 μM) as described in the text. The viability of the bacteria was determined by colony forming unit (CFU) count 1 to 4 days after drug exposure. (A) Tosufloxacin (50 μM) was the most active among the six drugs, and completely eliminated all persisters after 2 days; (B) acyldepsipeptide (ADEP4) + rifampin (RIF) could not completely eradicate S. aureus persisters even after 4 days. The final concentrations of ADEP4 and RIF were 5 μg/mL and 0.4 μg/mL, respectively.

Figure 3

The effect of tosufloxacin against S. aureus persisters that survived from different cidal antibiotic treatments and comparison of the activity of antibiotics in aerobic and anaerobic environments. (A) S. aureus persisters enriched by ofloxacin, gentamycin or vancomycin were similarly killed by tosufloxacin (50 μM). The S. aureus stationary phase culture was treated with ofloxacin (10 μg/mL), gentamycin (10 μg/mL) or vancomycin (10 μg/mL) for four hours to enrich persisters. Then, the surviving persisters from the treatment were subjected to drug exposure with tosufloxacin (50 μM); (B) The activities of antibiotics were comparable in aerobic and anaerobic environments. The S. aureus stationary phase culture was exposed to 20 μM of the respective drugs and incubated for 3 days at 37 °C (aerobically and anaerobically) when the CFU was determined.

Figure 4

Comparison of the structure and activity of tosufloxacin with the other quinolone drugs. (A) The activity of quinolone drugs (10 μM) against S. aureus persisters. S. aureus stationary phase culture was treated with ofloxacin (10 μg/mL) for 4 h to enrich persisters followed by treatment with different quinolone antibiotics. The bacterial viability was determined at different times by CFU count; (B) Structures of tosufloxacin and the other quinolone antibiotics. Tosufloxacin and trovafloxacin that have high activity against persisters both contain the 2,4-difluorophenyl group at the N-1 position, which is highlighted by the red circle and is absent in other less active quinolones.