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.

Keywords: FSMS; MPC; MPC-D; MPC-F; MSW; MSW-D; MSW-F; antibiotic resistance; antimicrobial activity; antimicrobial agents.

FIG 1

Scheme presenting the MSW₁₀¹⁰ ranges. Agents’ concentrations expressed in mg/L (A) or as x-fold MIC (B). CIP, ciprofloxacin; LIN, linezolid; No37, novel benzosiloxaborole compound No37; MSW, mutant selection window; MSW-D, dominant mutant selection window; MSW-F, inferior mutant selection window; AM, agar-dilution method; BM, broth-dilution method; pink color, MSWs of CIP; blue color, MSWs of LIN; green color, MSWs of No37; dotted pattern, MSWs determined by the agar-dilution method; striped pattern, MSWs determined by the broth-dilution method; arrows indicate the MSW’s upper boundary was above the highest concentration tested.

FIG 2

Preparation of the high-density inoculum. SvC, starting volume of bacterial culture; CP, cell pellet; FCP, final cell pellet; BHI, Brain Heart Infusion; 4,000 RPM = 2,061 × g.

FIG 3

Comparison of the agar-dilution method and the broth-dilution method. MPC, mutant prevention concentration; FSMS, frequency of spontaneous mutant selection; MPC-D, dominant mutant prevention concentration; MPC-F, inferior mutant prevention concentration.