Synergistic Activity of Eugenol, Cinnamaldehyde, and Carvacrol in Combination with Different Antibacterial Agents Against Multidrug-Resistant Gram-Negative Clinical Isolates

: Background/Objectives: The WHO has identified carbapenem-resistant Acinetobacter baumannii (CRAb) and carbapenem-producing Enterobacterales (CPE) as the "critical priority" group of multidrug-resistant (MDR) organisms for which new therapeutic strategies are urgently needed. Here, we evaluated the in vitro synergistic activity of eugenol, cinnamaldehyde, and carvacrol in combination with β-lactams, gentamicin, or colistin against MDR Gram-negative bacteria (GNB). Methods: We selected seven MDR-GNB clinical isolates including CRAb, ESBL-producing and CPE clinical isolates displaying different antimicrobial susceptibility profiles. The genomes of clinical isolates were characterized by whole-genome sequencing and synergy testing was performed with checkerboard assay. Results: Our results demonstrate that eugenol, cinnamaldehyde, and carvacrol in combination with colistin exhibited synergistic activity (FICI < 0.5) against MDR-GNB clinical isolates ranging from 37.5 to 50%, while the effect was almost indifferent in combination with different β-lactam molecules or gentamicin against 87.5-100% of MDR-GNB strains. The synergistic interaction of eugenol, cinnamaldehyde, and carvacrol with colistin induced a statistically significant reduction (p < 0.05) in the MIC values compared with the molecules tested alone. Conclusions: Our data demonstrate that this synergistic interaction was not affected by different antimicrobial resistance genes and/or different antimicrobial susceptibility profiles. In conclusion, our results suggest that eugenol, cinnamaldehyde, and carvacrol in combination with colistin represent a potential strategy for the treatment of MDR-GNB pathogens and limit their diffusion.