Inhibition of Staphylococcus aureus Efflux Pump by O-Eugenol and Its Toxicity in Drosophila melanogaster Animal Model.

Background: Efflux pumps are transmembrane proteins that expel drugs out of a bacterial cell contributing to microorganism drug resistance. Several studies addressing the use of natural products with medicinal properties have intensified given the above. Thus, the aim of the present study was to investigate the antibacterial activity and the O-eugenol potential in Staphylococcus aureus resistance reversal by efflux pump inhibition, as well as to evaluate its toxicity in the Drosophila melanogaster arthropod model. The broth microdilution method was used to determine the minimum inhibitory concentration (MIC) and the O-eugenol efflux pump inhibition. For the D. melanogaster toxicity assays, mortality and locomotor system damage were performed using the fumigation method. Results: O-eugenol presented a MIC of 1024 µg/mL against S. aureus. The association of this compound with the antibiotic tetracycline demonstrated a synergistic effect (p < 0.0001), this also being observed when the antibiotic was associated with ethidium bromide (p < 0.0001); thus, these results may be attributable to an efflux pump inhibition. The D. melanogaster mortality and geotaxis assays revealed the compound is toxic, with an EC50 of 18 µg/mL within 48 hours of exposure. Conclusions: While we can conclude that the tested product has an efflux pump inhibitory effect, further studies are needed to elucidate its mechanisms of action, in addition to assays using other strains to verify whether the substance has the same inhibitory effect.

Effect of Carvacrol and Thymol on NorA efflux pump inhibition in multidrug-resistant (MDR) Staphylococcus aureus strains.

Undue exposure to antimicrobials has led to the acquisition and development of sophisticated bacterial resistance mechanisms, such as efflux pumps, which are able to expel or reduce the intracellular concentration of various antibiotics, making them ineffective. Therefore, inhibiting this mechanism is a promising way to minimize the phenomenon of resistance in bacteria. In this sense, the present study sought to evaluate the activity of the Carvacrol (CAR) and Thymol (THY) terpenes as possible Efflux Pump Inhibitors (EPIs), by determining the Minimum Inhibitory Concentration (MIC) and the association of these compounds in subinhibitory concentrations with the antibiotic Norfloxacin and with Ethidium Bromide (EtBr) against strains SA-1199 (wild-type) and SA-1199B (overexpresses NorA) of Staphylococcus aureus. In order to verify the interaction of the terpenes with the NorA efflux protein, an in silico molecular modeling study was carried out. The assays used to obtain the MIC of CAR and THY were performed by broth microdilution, while the Efflux Pump inhibitory test was performed by the MIC modification method of the antibiotic Norfloxacin and EtBr. docking was performed using the Molegro Virtual Docker (MVD) program. The results of the study revealed that CAR and THY have moderate bacterial activity and are capable of reducing the MIC of Norfloxacin antibiotic and EtBr in strains of S. aureus carrying the NorA efflux pump. The docking results showed that these terpenes act as possible competitive NorA inhibitors and can be investigated as adjuvants in combined therapies aimed at reducing antibiotic resistance.

Effect of estragole over the RN4220 Staphylococcus aureus strain and its toxicity in Drosophila melanogaster.

Among the bacterial resistance mechanisms, efflux pumps are responsible for expelling xenobiotics, including bacterial cell antibiotics. Given this problem, studies are investigating new alternatives for inhibiting bacterial growth or enhancing the antibiotic activity of drugs already on the market. With this in mind, this study aimed to evaluate the antibacterial activity of Estragole against the RN4220 Staphylococcus aureus strain, which carries the MsrA efflux pump, as well as Estragole's toxicity in the Drosophila melanogaster arthropod model. The broth microdilution method was used to perform the Minimum Inhibitory Concentration (MIC) tests. Estragole was used at a Sub-Inhibitory Concentration (MIC/8) in association with erythromycin and ethidium bromide to assess its combined effect. As for Estragole's toxicity evaluation over D. melanogaster, the fumigation bioassay and negative geotaxis methods were used. The results were expressed as an average of sextuplicate replicates. A Two-way ANOVA followed by Bonferroni's post hoc test was used. The present study demonstrated that Estragole did not show a direct antibacterial activity over the RN4220 S. aureus strain, since it obtained a MIC ≥1024 µg/mL. The association of estragole with erythromycin demonstrated a potentiation of the antibiotic effect, reducing the MIC from 512 to 256 µg/mL. On the other hand, when estragole was associated with ethidium bromide (EtBr), an antagonism was observed, increasing the MIC of EtBr from 32 to 50.7968 µg/mL, demonstrating that estragole did not inhibited directly the MsrA efflux pump mechanism. We conclude that estragole has no relevant direct effect over bacterial growth, however, when associated with erythromycin, this reduced its MIC, potentiating the effect of the antibiotic.

Evaluation of the Antibacterial Activity and Efflux Pump Reversal of Thymol and Carvacrol against Staphylococcus aureus and Their Toxicity in Drosophila melanogaster.

The antibacterial activity and efflux pump reversal of thymol and carvacrol were investigated against the Staphylococcus aureus IS-58 strain in this study, as well as their toxicity against Drosophila melanogaster. The minimum inhibitory concentration (MIC) was determined using the broth microdilution method, while efflux pump inhibition was assessed by reduction of the antibiotic and ethidium bromide (EtBr) MICs. D. melanogaster toxicity was tested using the fumigation method. Both thymol and carvacrol presented antibacterial activities with MICs of 72 and 256 µg/mL, respectively. The association between thymol and tetracycline demonstrated synergism, while the association between carvacrol and tetracycline presented antagonism. The compound and EtBr combinations did not differ from controls. Thymol and carvacrol toxicity against D. melanogaster were evidenced with EC50 values of 17.96 and 16.97 µg/mL, respectively, with 48 h of exposure. In conclusion, the compounds presented promising antibacterial activity against the tested strain, although no efficacy was observed in terms of efflux pump inhibition.