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The NorA efflux pump of Staphylococcus aureus is known to play a major role in the development of resistance against quinolone drugs by reducing their concentration inside target pathogens. The objective of this study was to evaluate the ability of tannic acid to inhibit the gene expression of the NorA efflux pump in Staphylococcus aureus and to evaluate the in silico effect on the pump. Efflux pump inhibition was evaluated by fluorimetry. The checkerboard method evaluates the effect of the test substance in combination with an antimicrobial at different concentrations. To gene expression evaluation NorA the assay was performed using: a sub-inhibitory concentration preparation (MIC/4) of the antibiotic; a sub-inhibitory concentration preparation (MIC/4) of the antibiotic associated with tannic acid at a sub-inhibitory concentration (MIC/4). In this study, docking simulations were performed by the SWISSDOCK webserver. The ability of tannic acid to inhibit the NorA efflux pump can be related to both the ability to inhibit the gene expression of this protein, acting on signaling pathways involving the ArlRS membrane sensor. As well as acting directly through direct interaction with the NorA protein, as seen in the approach and in silico and in vitro per checkerboard method and fluorimetry of bromide accumulated in the cell.

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Khellin and visnagin are natural furanochromones that photoreact with DNA. Khellin has been used in the treatment of vitiligo and psoriasis, as well as in the treatment of angina pectoris and asthma due to its potent action as a coronary vasodilator and antispasmodic agent. The present study aimed to investigate whether the compounds khellin and visnagin act as inhibitors of NorA protein, an efflux pump overproduced by the strain of Staphylococcus aureus SA-1199B that confers resistance to the fluoroquinolones, such as norfloxacin and ciprofloxacin. These substances alone did not show antibacterial activity against the strain tested. On the other hand, when these compounds were added to the culture medium at subinhibitory concentration, they were able to reduce the minimum inhibitory concentration (MIC) of norfloxacin, ethidium bromide, as well as berberine, suggesting that these compounds are modulating agents of norfloxacin resistance, possibly due to NorA inhibition. Molecular docking analysis showed that both khellin and visnagin form hydrogen bonds with Arg310, an important residue in the interaction between NorA and its substrates, supporting the hypothesis that these compounds are NorA inhibitors. These results suggest a possible application of khellin and visnagin as adjuvants to norfloxacin in the treatment of infections caused by strains of S. aureus that overproduce NorA.

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The objective of this work was to evaluate the inhibitory effect of quercetin on S. aureus Efflux Pumps. The MIC of Quercetin was evaluated through the broth microdilution method, as well as the Efflux Pump inhibition assay through the method of reducing the antibiotic minimum inhibitory concentration as well as that of ethidium bromide. The in silico approach through bioinformatics was performed to demonstrate the molecular mechanism of interaction of the substrate and the binding cavity. The Quercetin inhibition concentration was not clinically relevant. With respect to the reversal of bacterial resistance effect by efflux pump inhibition, this effect was observed with the strains carrying the TetK and NorA pumps. Regarding the interaction between the Quercetin complex and the NorA pump, the extra stability was provided by hydrogen bonds produced by the hydroxyl group.

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INTRODUCTION: Infectious diseases have been responsible for an increasing number of deaths worldwide. Staphylococcus aureus has been recognized as one of the most notable causative agents of severe infections, while efflux pump (EP) expression is one of the main mechanisms associated with S. aureus resistance to antibiotics. OBJECTIVE: This study aimed to investigate the potential of α-pinene as an efflux pump inhibitor in species of S. aureus carrying the TetK and MrsA proteins. METHODS: The minimum inhibitory concentrations (MIC) of α-pinene and other efflux pump inhibitors were assessed using serial dilutions of each compound at an initial concentration above 1024 µg/mL. Solutions containing culture medium and bacterial inoculums were prepared in test tubes and subsequently transferred to 96-well microdilution plates. The modulation of ethidium bromide (EtBr) and antibiotics (tetracycline and erythromycin) was investigated through analysis of the modification in their MICs in the presence of a subinhibitory concentration of α-pinene (MIC/8). Wells containing only culture medium and bacterial inoculums were used as negative control. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control. RESULTS: The MIC of ethidium bromide against S. aureus strains RN-4220 and IS-58 was reduced by association with α-pinene. This monoterpene potentiated the effect of tetracycline against the IS-58 strain but failed in modulating the antibacterial effect of erythromycin against RN-4220, suggesting a selective inhibitory effect on the TetK EP by α- pinene. CONCLUSION: In conclusion, α-pinene has promising effects against S.aureus strains, which should be useful in the combat of antibacterial resistance associated with EP expression. Nevertheless, further research is required to fully characterize its molecular mechanism of action as an EP inhibitor.

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BACKGROUND: Microbial resistance to antibiotics is a global public health problem, which requires urgent attention. Platonia insignis is a native species from the eastern Brazilian Amazon, used in the treatment of burns and wounds. OBJECTIVES: To evaluate the antimicrobial activity of the hydroalcoholic extract of P. insignis (PIHA), the ethyl acetate fraction (PIAE), and its subfraction containing a mixture of biflavonoids (BF). Moreover, the effect of these natural products on the antibiotic activity against S. aureus strains overexpressing efflux pump genes was also evaluated. METHODS: Minimal inhibitory concentrations were determined against different species of microorganisms. To evaluate the modulatory effect on the Norfloxacin-resistance, the MIC of this antibiotic was determined in the absence and presence of the natural products at subinhibitory concentrations. Inhibition of the EtBr efflux assays were conducted in the absence or presence of natural products. RESULTS: PIHA showed a microbicidal effect against S. aureus and C. albicans, while PIAE was bacteriostatic for S. aureus. PIAE and BF at subinhibitory concentrations were able to reduce the MIC of Norfloxacin acting as modulating agents. BF was able to inhibit the efflux of EtBr efflux in S. aureus strains overexpressing specific efflux pump genes. CONCLUSION: P. inignisis, a source of efflux pump inhibitors, including volkensiflavone and morelloflavone, which were able to potentiate the Norfloxacin activity by NorA inhibition, being also able to inhibit QacA/B, TetK and MsrA. Volkensiflavone and morelloflavone could be used as an adjuvant in the antibiotic therapy of multidrug resistant S. aureus strains overexpressing efflux pumps.

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Searching for new alternatives to antibiotic treatments is crucial to surmount the multidrug-resistant bacteria. In this work, the antimicrobial activity of synthetic imidazolidines was evaluated as well as their modulating effect on the resistance to fluoroquinolones in a S. aureus strain (SA-1199B), which overexpresses the norA gene that encodes the NorA efflux pump. Results showed weak antimicrobial activity (512 µg mL-1) for two fluorobenzylidene derivatives against this bacterial strain, while the other benzylidene derivatives were inactive. Despite this fact, both fluorinated compounds were able to enhance the activity of norfloxacin and ciprofloxacin against SA-1199B up to 6.4- and 3.2-fold, respectively. In addition, both derivatives potentiated the action of ethidium bromide against this strain, suggesting that the modulating effect probably involves the inhibition of the NorA efflux pump, which is in concordance with the fluorimetic assays and molecular docking analyses performed in this work.

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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.

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2-aminothiophene derivatives (2AT) in which the thiophene ring is fused with a cycloalkyl or a N-acylated piperidine ring by positions 5 and 6 and carrying a 3-carbethoxy group were synthesized and their bacterial growth and enzyme inhibitory effects against efflux proteins of Staphylococcus aureus leading to resistance to fluoroquinolones and erythromycin (ERY) were investigated. Compounds that most effectively decreases the minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) were assayed for their dose and time effects on the accumulation and efflux of ethidium bromide (EtBr) in the SA-1 strain. None of the compounds displayed antibacterial activity however, three derivatives carrying 2-amino, 2-aminoacetyl and 2-aminotrifluoroacetyl group enhanced the activity of CIP and ERY by 8- and 16-fold, respectively, and were able to restore the sensitivity of resistant strains, acting as typical efflux pump inhibitors (EPIs). The 2-aminoacetyl and 2-aminotrifluoroacetyl derivatives and two other piperidinyl 2-aminotrifluoroacetyl derivatives increased EtBr accumulation in a dose- and time-dependent manner, and one of them was also able to inhibit the EtBr efflux. Taken together, these results represent an important advance in the development of new EPIs, and demonstrate that 2AT represent a good scaffold for developing new antibiotic adjuvants.

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This study was carried out to test the essential oil from C. ambrosioides leaves and its main constituent, α-Terpinene, in an antibacterial activity assay. As well, it was evaluated ability reduce resistance to norfloxacin and ethidium bromide was compared the Staphylococcus aureus 1199B whith 1199 wild type strain. The MIC of the C. ambrosioides essential oil and α-Terpinene were determined by microdilution method. The MIC of the essential oil and α-Terpinene presented a value ≥ 1024 µg/mL. However, when associated with antibacterials, the essential oil from C. ambrosioides leaves significantly reduced the MIC of antibiotics and ethidium bromide, characterizing an efflux pump inhibition. The C. ambrosioides essential oil, despite having no direct antibacterial activity against the S. aureus 1199B strain, showed a potentiating action when associated with antibacterial agents, this being attributed to an inhibition of efflux pumps.

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The widespread use of antibiotics created selective pressure for the emergence of strains that would persist despite antibiotic toxicity. The bacterial resistance mechanisms are several, with efflux pumps being one of the main ones. These pumps are membrane proteins with the function of removing antibiotics from the cell cytoplasm. Due to this importance, the aim of this work was to evaluate the inhibitory effect of tannic acid against efflux pumps expressed by the Staphylococcus aureus RN4220 and IS-58 strains. The efflux pump inhibition was assayed using a sub-inhibitory concentration of efflux pump standard inhibitors and tannic acid (MIC/8), observing their capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due the possible inhibitory effect of these substances. The MICs of EtBr and antibiotics were significantly different in the presence of tannic acid, indicating the inhibitory effect of this product against efflux pumps of both strains. These results indicate the possible usage of tannic acid asan inhibitor and an adjuvant in the antibiotic therapy against multidrug resistant bacteria (MDR).

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