Comparison of In Vitro Methods for Assaying the Antibacterial Activity of a Mix of Natural Essential Oils Against Zoonotic Bacteria

With the increasing occurrence of bacterial resistance, it is now essential to look for new alternatives to protect the curative utilization of antibiotics within the One Health concept. Here, we adapt and optimize a broth microdilution method and compare it against the broth macrodilution method for evaluating the antibacterial activity of a complex essential oils mix (EO mix) against four livestock pathogens: <i>Escherichia coli</i>, <i>Bacillus cereus</i>, <i>Pseudomonas aeruginosa</i>, and <i>Staphylococcus aureus.</i> Microdilution method performance (final volume well: 300 µL; inoculum: 1.0 × 10⁶ CFU/mL) was evaluated following CLSI recommendations, by comparing the MIC of each of the four strains with the MICs obtained with the macrodilution method (final volume tube: 2 mL; inoculum 1.0 × 10⁶ CFU/mL). Microdilution analysis was performed with an automated plate reader (Bioscreen C), and three bacterial growth parameters (OD max, lag phase, and growth rate) were calculated (DMFit curve-fitting software (v2.1; courtesy of the Institute of Food Research, Norwich, UK)). EO mix MICs were determined for <i>E. coli</i>, <i>S. aureus</i>, and <i>B. cereus</i>. Our results emphasize the importance of ensuring the accuracy of MIC results by performing three technical and three biological replicates, and combining OD max, lag phase, and growth rate to assess the impact of an EO mix at sub-MIC levels.