Tehran University of Medical Sciences Iranian Journal of Microbiology 2008-3289 18 5 2026 10 05 828 840 Seyedeh Samaneh Mostaan Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Bahar Nayeri Fasaei Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Aram Sharifi Department of Animal Science, Faculty of Agriculture, University of Kurdistan, Kurdistan, Iran Aghil Sharifzadeh Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Iradj Ashrafi Tamai Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran Alireza Salmani Ghahyazi Department of Microbiology, Faculty of Veterinary Medicine, Islamic Azad University, Karaj Branch, Karaj, Iran 2026 07 11 2026 09 02 Background and Objectives: Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen responsible for severe infections and increasing therapeutic difficulties due to multidrug resistance and virulence traits such as motility and biofilm formation. This study aimed to evaluate the in vitro antibacterial, antibiotic-synergistic, and anti-motility effects of eugenol in combination with cefixime and gentamicin against nine clinical P. aeruginosa isolates obtained from hospitalized animals. Materials and Methods: This preliminary in vitro laboratory investigation was conducted on a convenience sample of nine independent non-duplicate clinical P. aeruginosa isolates obtained from wound swabs and urinary tract samples of hospitalized animals at the Small Animal Hospital, Faculty of Veterinary Medicine, University of Tehran. Minimum inhibitory concentrations (MICs) of eugenol alone were determined across all nine isolates using the broth microdilution method in accordance with CLSI guidelines (M07-A11). Synergistic interactions between eugenol and the antibiotics cefixime and gentamicin were evaluated using the checkerboard assay and calculation of the fractional inhibitory concentration index (FICI) on a subset of three selected representative isolates (P3, P6, and P9). Furthermore, the effects of sub-inhibitory concentrations of eugenol ("MIC/2" ) on bacterial motility were assessed using standardized media containing 0.3%, 0.7%, and 1.5% agar to evaluate swimming, swarming, and twitching motilities, respectively, across this same subset of isolates (P3, P6, and P9) in biological triplicate (n=3). Results: Antimicrobial analysis across all nine isolates demonstrated intrinsic antibacterial activity of eugenol, with MIC values ranging from 2.5 to 10 µg/mL. Checkerboard synergy testing on the selected representative subset (P3, P6, and P9) revealed synergistic interactions with cefixime ("FICI"=0.5 for isolate P6, P3 and P9) as well as interactions with gentamicin yielding FICI values of 0.256 (for isolates P6 and P9) and 0.384 (for isolate P3), classifying these combinations within synergistic interaction ranges ("FICI"≤0.5). Additionally, sub-inhibitory eugenol treatment of the tested subset significantly reduced bacterial motility compared to untreated controls, decreasing swimming by 44-57%, swarming by 43-46%, and twitching by 39-64% (P<0.05). Conclusion: Eugenol demonstrated notable in vitro antibacterial and anti-motility activities against tested clinical P. aeruginosa isolates from veterinary sources, alongside favorable FICI interactions with gentamicin and cefixime. Because these findings are strictly restricted to limited in vitro laboratory observations without dosage optimization, toxicity, or in vivo efficacy evaluations, further comprehensive investigations are required before establishing any potential clinical relevance. https://ijm.tums.ac.ir/index.php/ijm/article/view/6321 https://ijm.tums.ac.ir/index.php/ijm/article/download/6321/1943