Employment of a Resazurin Viability-Based Assay for Minimum Inhibitory and Bactericidal Concentration Determination

Area of Science:

• Microbiology
• Biotechnology
• Analytical Chemistry

Background:

• Rising antimicrobial resistance necessitates advanced evaluation methods beyond standard minimum inhibitory concentration (MIC) tests.
• Current minimum bactericidal concentration (MBC) determination relies on time-consuming colony-forming unit (CFU) counting.
• Resazurin assays offer sensitivity for MIC but lack simplicity for MBC.

Purpose of the Study:

• Develop and validate a novel resazurin-based microwell assay.
• Enable simultaneous determination of MIC and MBC without routine plate counting.
• Simplify and expedite bactericidal activity assessment.

Main Methods:

• A two-step resazurin fluorescence assay was designed for microplates.
• MIC determined using resazurin as a metabolic indicator.
• MBC assessed via a regrowth phase in fresh medium with resazurin to distinguish reversible inhibition from bacterial death.

Main Results:

• The two-step assay demonstrated full concordance with conventional CFU counting for MBC determination.
• Results were consistent across tested antibiotics (ciprofloxacin, chloramphenicol) and bacterial species (S. aureus, E. faecalis, E. coli, P. aeruginosa).

Conclusions:

• The proposed two-step resazurin assay is a rapid, reliable, and less labor-intensive method.
• It offers an efficient alternative for determining both MIC and MBC.
• Potential applications exist in clinical and industrial microbiology settings.