Time-kill curve analysis and pharmacodynamic functions for in vitro evaluation of antimicrobials against Neisseria gonorrhoeae

Abstract

Gonorrhea is a sexually transmitted infection caused by the Gram-negative bacterium Neisseria gonorrhoeae. Resistance to first-line empirical monotherapy has emerged, so robust methods are needed to appropriately evaluate the activity of existing and novel antimicrobials against the bacterium. Pharmacodynamic functions, which describe the relationship between the concentration of antimicrobials and the bacterial net growth rate, provide more detailed information than the MIC only. In this study, a novel standardized in vitro time-kill curve assay was developed. The assay was validated using five World Health Organization N. gonorrhoeae reference strains and various concentrations of ciprofloxacin, and then the activity of nine antimicrobials with different target mechanisms were examined against a highly susceptible clinical wild type isolate (cultured in 1964). From the time-kill curves, the bacterial net growth rates at each antimicrobial concentration were estimated. Finally, a pharmacodynamic function was fitted to the data, resulting in four parameters that describe the pharmacodynamic properties of each antimicrobial. Ciprofloxacin resistance determinants shifted the pharmacodynamic MIC (zMIC) and attenuated the bactericidal effect at antimicrobial concentrations above the zMIC. Ciprofloxacin, spectinomycin and gentamicin had the strongest bactericidal effect during the first six hours of the assay. Only tetracycline and chloramphenicol showed a purely bacteriostatic effect. The pharmacodynamic functions differed between antimicrobials, showing that the effect of the drugs at concentrations below and above the MIC vary widely. In conclusion, N. gonorrhoeae time-kill curve experiments analyzed with pharmacodynamic functions have potential for in vitro evaluation of new and existing antimicrobials and dosing strategies to treat gonorrhea.