ABSTRACTThis study was conducted to determine the effect of testing parameters on thein vitroactivity of gepotidacin, a new triazaacenaphthylene antibacterial agent for the treatment of conventional and biothreat pathogens. CLSI methods, and variations of those methods, were used to test 10Staphylococcus aureus, 10Streptococcus pneumoniae, 10Haemophilus influenzae, and 5Escherichia coliisolates by MIC and 30S. aureus, 15S. pneumoniae, and 15S. pyogenesisolates by disk diffusion (DD) methods. Levofloxacin and linezolid were tested as comparator agents for MIC and DD methods, respectively. Broth microdilution (BMD), macrodilution (MD), and agar dilution (AD) methods were compared. Variations in media, temperature, incubation time, CO2level, and inoculum concentration were tested by all methods, and variations in pH, calcium, magnesium, zinc, potassium, thymidine, and polysorbate 80 levels were tested by BMD and DD. The addition of albumin, serum, and lung surfactant was studied by BMD. The variables that impacted the results the most were high inoculum and pH 5.5 (no growth ofH. influenzaeandS. pneumoniaeby BMD). Gepotidacin AD MIC levels were increased and disk zone diameters were decreased for all species in 10% CO2incubation. The following variables had a minimal effect on gepotidacin results: pH, agar method, atmospheric condition, temperature, and addition of serum and albumin for broth methods. There were also some slight differences in gepotidacin disk results between disk manufacturers and some agar types and also with potassium and thymidine forS. pneumoniae. For all other variations, gepotidacin MIC and disk results were considered comparable to reference results.