Carbapenem antibiotics are widely used for their broad antibacterial effects, but the emergence of carbapenem-resistant has recently become a global problem. To solve this problem, research is needed to find compounds that increase antibiotic activity. Therefore, this study aimed to validate the antibacterial and anti-biofilm effects, as well as the inhibition of gene expression of thymoquinone, an extract of commonly used as a spice in many dishes. The minimum inhibitory concentration of carbapenem antibiotics and thymoquinone was determined. Phenotypic analysis was performed to confirm the effect of thymoquinone on motility, which is one of the virulence factors of carbapenem-resistant uropathogenic (CR-UPEC). Furthermore, quantitative real-time polymerase chain reaction analysis was used to determine the expression levels of carbapenemase gene ( ), efflux pump genes (, , , ), as well as motility and adhesion genes (, ). In addition, biofilm inhibition and biofilm eradication assays were performed. All strains showed resistance to carbapenem antibiotics, while an antibacterial effect was confirmed at a concentration of 256 ��g/mL of thymoquinone. Phenotypic analysis revealed a nearly 50% suppression in migration distance compared to the control group at 128 ��g/mL of thymoquinone. Subsequent gene expression tests indicated the downregulation of carbapenemase-, efflux pump-, motility-, and adhesion genes by thymoquinone. Furthermore, our findings demonstrated that thymoquinone exhibits both biofilm formation inhibition and eradication effects. These findings suggest that thymoquinone may serve as a potential antibiotic adjuvant for treating CR-UPEC and could be a valuable resource in combating UTIs caused by multidrug-resistant bacteria.
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