Evaluation of the effects of hybrids of metronidazole and eugenol analogues as inhibitors of the Staphylococcus aureus NorA efflux pump: an in vitro and in silico analysis

Staphylococcus aureus is a Gram-positive coccus-type bacterium responsible for colonizing various sites of the host’s body, thus generating several pathologies such as pneumonia, osteomyelitis, endocarditis, and even cases of sepsis. Eugenol, dihydroeugenol, guaiacol, and phenol are phenolic compounds with antimicrobial activities, as is the drug metronidazole; therefore, eight substances were synthesized by coupling metronidazole with the molecules mentioned above, thus originating the compounds AD1–AD8. The present study aimed to evaluate the antibacterial activity and the capacity of the compounds AD1–AD8 to amplify the action of norfloxacin and ethidium bromide against the S. aureus 1199B strain through the inhibition of the NorA efflux pump. Broth microdilution assays were performed to determine the MIC and to analyze the association of the compounds AD1–AD8 with norfloxacin and ethidium bromide, along with the membrane permeability test and molecular docking. The compounds AD1–AD8 obtained positive results as they exhibited a potentiating effect by reducing the MIC of norfloxacin and ethidium bromide against 1199B, thus evidencing interference with the functioning of the NorA efflux pump, which had already been predicted by the data obtained from the molecular docking assay, in addition to having demonstrated significant effects by acting on the S. aureus membrane. The information obtained from this research is in line with what is reported in the literature regarding the effects observed with eugenol, dihydroeugenol, and guaiacol and their derivatives when combined with other antibiotics against S. aureus strains that possess efflux pumps, with competitive inhibition being observed, as well as the possibility that the reduction in MIC may have occurred due to a reduction in gene expression levels. Further assays are needed to evaluate these possibilities. Based on the presented data, it was found that the compounds AD1–AD8 exhibit promising aspects to act as antibiotic adjuvants.