Mutant Prevention Concentration, Frequency of Spontaneous Mutant Selection, and Mutant Selection Window-a New Approach to the In Vitro Determination of the Antimicrobial Potency of Compounds.

Abstract

The analysis of antimicrobial activity is usually MIC- and minimal bactericidal concentration (MBC)-focused, though also crucial are resistance-related parameters, e.g., the frequency of spontaneous mutant selection (FSMS), the mutant prevention concentration (MPC), and the mutant selection window (MSW). In vitro-determined MPCs, however, are sometimes variable, poorly repeatable, and not always reproducible in vivo. We propose a new approach to the in vitro determination of MSWs, along with novel parameters: MPC-D, MSW-D (for dominant mutants, i.e., selected with a high frequency, without a fitness loss), and MPC-F, MSW-F (for inferior mutants, i.e., with an impaired fitness). We also propose a new method for preparing the high-density inoculum (>1011 CFU/mL). In this study, the MPC and MPC-D (limited by FSMS of <10-10) of ciprofloxacin, linezolid, and novel benzosiloxaborole (No37) were determined for Staphylococcus aureus ATCC 29213 using the standard agar method, while the MPC-D and MPC-F were determined by the novel broth method. Regardless of the method, MSWs1010 of linezolid and No37 were the same. However, MSWs1010 of ciprofloxacin in the broth method was narrower than in the agar method. In the broth method, the 24-h incubation of ~1010 CFU in a drug-containing broth differentiates the mutants that can dominate the cell population from those that can only be selected under exposure. We consider MPC-Ds in the agar method to be less variable and more repeatable than MPCs. Meanwhile, the broth method may decrease discrepancies between in vitro and in vivo MSWs. The proposed approaches may help establish MPC-D-related resistance-restricting therapies.