Evaluation of isoeugenol in inhibition of Staphylococcus aureus efflux pumps and their toxicity using Drosophila melanogaster model.

The Staphylococcus aureus bacteria is a pathogen considered opportunistic and that has been acquiring resistance to several classes of antibiotics, mainly due to the synthesis of efflux pumps, which are proteins that expel these drugs intracellularly, reducing their effectiveness. The objective of this study was to evaluate the ability of isoeugenol to inhibit S. aureus efflux pumps and to determine its toxicity against a eukaryotic model (Drosophila melanogaster). IS-58, K2068 and K4414 S. aureus strains were used in the study. Isoeugenol minimum inhibitory concentration (MIC) and antibiotic modulation were evaluated in efflux pump inhibitory tests as well as in ethidium bromide (EtBr) assays. Toxicity tests against D. melanogaster assessed mortality and negative geotaxis. Isoeugenol obtained a relevant MIC result and a synergism was observed when isoeugenol was associated with the antibiotics, mainly with ciprofloxacin. Isoeugenol was able to affect all three efflux pumps tested, especially in strain K4414. The mortality of D. melanogaster caused by isoeugenol administration started after 12 h of exposure, being volume dependent and having an LC50 of 81.69 µL/L. In the negative geotaxis test, a statistical difference was observed after 24h of exposure compared to the control, demonstrating that damage to the locomotor apparatus had occurred. Based on the results, isoeugenol is a putative efflux pump inhibitor, becoming an alternative in blocking these proteins, and demonstrated acute toxicity against D. melanogaster.

Evaluation of Benzaldehyde as an Antibiotic Modulator and Its Toxic Effect against Drosophila melanogaster.

Products of natural origin remain important in the discovery of new bioactive molecules and are less damaging to the environment. Benzaldehyde is a product of the metabolism of plants, and similarly to oxygenated terpenes, it can have antibacterial activity against Staphylococcus aureus and toxic action against Drosophila melanogaster; we aimed to verify these activities. The broth microdilution tests determined the minimum inhibitory concentration (MIC) of benzaldehyde alone and in association with antibiotics and ethidium bromide (EtBr). Toxicity against Drosophila melanogaster was determined by fumigation tests that measured lethality and damage to the locomotor system. The results indicated that there was an association of norfloxacin and ciprofloxacin with benzaldehyde, from 64 µg/mL to 32 µg/mL of ciprofloxacin in the strain K6028 and from 256 µg/mL to 128 µg/mL of norfloxacin in the strain 1199B; however, the associations were not able to interfere with the functioning of the tested efflux pumps. In addition, benzaldehyde had a toxic effect on flies. Thus, the results proved the ability of benzaldehyde to modulate quinolone antibiotics and its toxic effects on fruit flies, thus enabling further studies in this area.

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.