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Abstract

Resistance to antibiotics has made diseases that previously healed easily become more difficult to treat. Staphylococcus aureus is an important cause of hospital-acquired infections and multi-drug resistant. NorA efflux pump, present in bacteria S. aureus, is synthesized by the expression of the norA gene. Menadione, also known as vitamin K3, is one of the synthetic forms of vitamin K. Therefore, the aim of this study is to verify the menadione effect on efflux inhibition through NorA pump gene expression inhibition and assess the effects of menadione in bacterial membrane. The effect of menadione as an efflux pump inhibitor (EPI) was evaluated by the microdilution method, fluorimetry, electron microscopy, and by RT-qPCR to evaluate gene expression. In the molecular docking, association with menadione induces increased fluorescence intensity. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA. The results showed the norA gene had its expression significantly diminished in the presence of menadione. The simulation showed that several menadione molecules were able to go through the bilayer and allow the entry of water molecules into the hydrophobic regions of the bilayer. When present within membranes, menadione may have caused membrane structural changes resulting in a decline of the signaling pathways involved in norA expression. Menadione demonstrated to be an efflux pump inhibitor with dual mechanism: affecting the efflux pump by direct interaction with protein NorA and indirectly inhibiting the norA gene expression, possibly by affecting regulators present in the membrane altered by menadione.

2.1. Bacterial Strain and Culture

The SA-1199B strain of Staphylococcus aureus was kindly donated by Prof. S. Gibbons of the University of London. Before all microbiological tests, the strain was maintained in the culture medium blood agar base (Laboratorios Difco Ltd., São Paulo, Brazil). For use in testing, the cells were grown overnight at 37 °C in the culture medium: Heart Infusion Agar (HIA, Difco). The strain used has the generic characteristic of presenting the norA resistance gene responsible for expressing the efflux pump (NorA) for antibiotics in the group of fluoroquinolones.

2.7. Sample Preparation for the Real-Time PCR Assay

The samples were prepared using the minimum inhibitory concentration (MIC) as obtained in Tintino et al. []. The assays were performed as described in the . The preparations were cultured in Brain Heart Infusion (BHI) culture medium for 24 h at 37 °C and then centrifuged at 3000 rpm (Fanem Centrífuga Excelsa® 3280) for pellet formation, procedures performed prior to RNA extraction.

3.1. Efflux Pump Inhibition by MIC Reduction of Ethidium Bromide (EtBr)

In the evaluation of inhibition by MIC reduction of ethidium bromide, the results in , menadione was shown to significantly reduce the ethidium bromide MIC, which indicates NorA inhibition. In the inhibition of the efflux pump, it is known that decreasing the MIC of bromide, of ethidium or antibiotic, by up to three-fold, in relation to the control (substrate of efflux pump) alone, is indicative of efflux pump inhibition.

3.2. Evaluation of Pump Inhibition by Fluorescence Emission of Ethidium Bromide

In the evaluation of inhibition by fluorescence emission of ethidium bromide, according to the results in , the association with menadione induces increased fluorescence intensity when compared to the bacterium associated only with bromide, indicating inhibition of efflux pump. Regarding the Carbonyl Cyanide m-Chlorophenylhydrazine (CCCP) pump inhibitor, the same showed low fluorescence intensity when compared to the others.

3.3. Homology Modeling and Molecular Docking

The molecular docking method was used to investigate the binding affinity of menadione in the channel formed by the transmembrane alpha-helices of the NorA protein. Menadione was observed (100% of the clusters) interacting with residues ILE12, ILE15, PHE16, ILE19, PHE47, GLN51, ALA105, and MET109 from NorA (). This protein region has also been observed in other docking studies in the literature with other binders such as boeravinone B [], riparin B [], salvin, thioxanthene, and levofloxacin [].