Gepotidacin is a first-in-class, novel triazaacenaphthylene antibiotic that 
inhibits bacterial DNA replication and has in vitro activity against susceptible 
and drug-resistant pathogens. Reference in vitro methods were used to 
investigate the MICs and minimum bactericidal concentrations (MBCs) of 
gepotidacin and comparator agents for Staphylococcus aureus, Streptococcus 
pneumoniae, and Escherichia coli Gepotidacin in vitro activity was also 
evaluated by using time-kill kinetics and broth microdilution checkerboard 
methods for synergy testing and for postantibiotic and subinhibitory effects. 
The MIC90 of gepotidacin for 50 S. aureus (including methicillin-resistant S. 
aureus [MRSA]) and 50 S. pneumoniae (including penicillin-nonsusceptible) 
isolates was 0.5 μg/ml, and for E. coli (n = 25 isolates), it was 4 μg/ml. 
Gepotidacin was bactericidal against S. aureus, S. pneumoniae, and E. coli, with 
MBC/MIC ratios of ≤4 against 98, 98, and 88% of the isolates tested, 
respectively. Time-kill curves indicated that the bactericidal activity of 
gepotidacin was observed at 4× or 10× MIC at 24 h for all of the isolates. S. 
aureus regrowth was observed in the presence of gepotidacin, and the resulting 
gepotidacin MICs were 2- to 128-fold higher than the baseline gepotidacin MICs. 
Checkerboard analysis of gepotidacin combined with other antimicrobials 
demonstrated no occurrences of antagonism with agents from multiple 
antimicrobial classes. The most common interaction when testing gepotidacin was 
indifference (fractional inhibitory concentration index of >0.5 to ≤4; 82.7% for 
Gram-positive isolates and 82.6% for Gram-negative isolates). The postantibiotic 
effect (PAE) of gepotidacin was short when it was tested against S. aureus (≤0.6 
h against MRSA and MSSA), and the PAE-sub-MIC effect (SME) was extended (>8 h; 
three isolates at 0.5× MIC). The PAE of levofloxacin was modest (0.0 to 2.4 h), 
and the PAE-SME observed varied from 1.2 to >9 h at 0.5× MIC. These in vitro 
data indicate that gepotidacin is a bactericidal agent that exhibits a modest 
PAE and an extended PAE-SME against Gram-positive and -negative bacteria and 
merits further study for potential use in treating infections caused by these 
pathogens.