Search for a command to run...
<b>Background/Objectives</b>: The increasing prevalence of antimicrobial-resistant bacteria highlights the need for improved methodologies to evaluate antimicrobial activity beyond conventional minimum inhibitory concentration testing. While resazurin-based assays are widely used for minimum inhibitory concentration determination due to their simplicity and sensitivity, minimum bactericidal concentration assessment still relies on labor-intensive colony-forming unit counting. The objective of this study was to develop and validate a resazurin-based microwell assay capable of determining both the minimum inhibitory concentration and the minimum bactericidal concentration without routine plate counting, thereby simplifying bactericidal evaluation. <b>Methods</b>: A two-step resazurin-based fluorescence assay was designed and performed in microplates. After determining the minimum inhibitory concentration using resazurin as a metabolic indicator, well-showing inhibited bacterial growths were subjected to a regrowth phase by transferring aliquots into fresh antimicrobial-free medium containing resazurin. This additional step allowed discrimination between reversible metabolic inhibition and irreversible bacterial death. The method was evaluated using ciprofloxacin and chloramphenicol against four bacterial species: <i>Staphylococcus aureus</i>, <i>Enterococcus faecalis</i>, <i>Escherichia coli</i>, and <i>Pseudomonas aeruginosa</i>. Minimum bactericidal concentration values obtained using this assay were compared with those obtained through conventional colony counting on agar plates. <b>Results</b>: Minimum bactericidal concentration values obtained using the two-step fluorescence assay were fully concordant with the conventional colony-forming unit counting method for all tested antibiotics and bacterial species. <b>Conclusions</b>: The proposed two-step resazurin-based microwell assay represents a rapid, reliable, and less labor-intensive alternative for the determination of both the minimum inhibitory concentration and the minimum bactericidal concentration, with potential applications in clinical and industrial microbiology laboratories.