Beyond One-Size-Fits-All: Addressing Methodological Constraints in Novel Antimicrobials Discovery.

: Antimicrobial resistance is a growing global health concern that requires multiple strategies to be tackled effectively. While the discovery of new antimicrobial molecules is essential, the repurposing of existing compounds also plays a significant role. Standard methods to evaluate antimicrobial efficacy, regulated by the Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI), such as the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), are available. However, several potential antimicrobics show interference with these standard methods, resulting in underestimated activity and their premature dismissal from further studies. This work compares reference methods in evaluating different compounds with unique physico-chemical characteristics. We aim to demonstrate that combining different susceptibility tests is mandatory for a successful preclinical screening of antimicrobial compounds. : A selection of substances including natural extracts, both free and in the form of nanocomposites with fumed silica, ionic liquids, ozonated oils, commercial and pure antibiotics, was tested using broth microdilution, disk diffusion, and agar dilution. These methods were chosen following EUCAST and CLSI guidelines, and comparisons were made to evaluate their applicability and limitations for non-conventional substances. : The study highlighted significant variability in the outcomes depending on the method used, especially for substances with intrinsic properties such as high viscosity, poor solubility, or specific interactions with the testing medium. In several cases, the use of a single standard method failed to accurately reflect the real antimicrobial activity, leading to potential misinterpretation of effectiveness. : A combined methodological approach is recommended to overcome the limitations of individual techniques. The integration of multiple reference methods offers a more accurate screening strategy for identifying and characterizing new and repurposed antimicrobials.