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 antibiotics curative utilization in 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 4 livestock pathogens Escherichia coli (E. coli), Bacillus cereus (B. cereus), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus. Microdilution method performance (final volume well: 300 µL; inoculum: 106 CFU/mL), was evaluated following CLSI recommendations, by comparing each MIC of each 4 strains, with each MIC’s obtained with macrodilution method (final volume tube: 2 mL; inoculum 106 CFU/mL). Microdilution was performed with automated plate reeder (Bioscreen C) and 3 bacterial growth parameters (OD max, lag phase, and growth rate) were calculated (Dmfit software). EO Mix MICs were determined for E. coli, S. aureus and B. cereus. Our results emphasize the importance of ensuring the accuracy of MIC results by performing 3 technical and 3 biological replicates, and combining OD max, lag phase, and growth rate to assess the impact of an EO mix at sub-MIC levels.