Evaluation of the antibacterial and inhibitory activity of the MepA efflux pump of Staphylococcus aureus by riparins I, II, III, and IV.

The efflux pump mechanism contributes to the antibiotic resistance of widely distributed strains of Staphylococcus aureus. Therefore, in the present work, the ability of the riparins N-(4-methoxyphenethyl)benzamide (I), 2-hydroxy-N-[2-(4-methoxyphenyl)ethyl]benzamide (II), 2, 6-dihydroxy-N-[ 2-(4-methoxyphenyl)ethyl]benzamide (III), and 3,4,5-trimethoxy-N-[2-(4-methoxyphenethyl)benzamide (IV) as potential inhibitors of the MepA efflux pump in S. aureus K2068 (fluoroquinolone-resistant). In addition, we performed checkerboard assays to obtain more information about the activity of riparins as potential inhibitors of MepA efflux and also analyzed the ability of riparins to act on the permeability of the bacterial membrane of S. aureus by the fluorescence method with SYTOX Green. A molecular coupling assay was performed to characterize the interaction between riparins and MepA, and ADMET (absorption, distribution, metabolism, and excretion) properties were analyzed. We observed that I-IV riparins did not show direct antibacterial activity against S. aureus. However, combination assays with substrates of MepA, ciprofloxacin, and ethidium bromide (EtBr) revealed a potentiation of the efficacy of these substrates by reducing the minimum inhibitory concentration (MIC). Furthermore, increased EtBr fluorescence emission was observed for all riparins. The checkerboard assay showed synergism between riparins I, II, and III, ciprofloxacin, and EtBr. Furthermore, riparins III and IV exhibited permeability in the S. aureus membrane at a concentration of 200 µg/mL. Molecular docking showed that riparins I, II, and III bound in a different region from the binding site of chlorpromazine (standard pump inhibitor), indicating a possible synergistic effect with the reference inhibitor. In contrast, riparin IV binds in the same region as the chlorpromazine binding site. From the in silico ADMET prediction based on MPO, it could be concluded that the molecules of riparin I-IV present their physicochemical properties within the ideal pharmacological spectrum allowing their preparation as an oral drug. Furthermore, the prediction of cytotoxicity in liver cell lines showed a low cytotoxic effect for riparins I-IV.

NorA, Tet(K), MepA, and MsrA Efflux Pumps in Staphylococcus aureus, their Inhibitors and 1,8-Naphthyridine Sulfonamides.

Antibiotic resistance can be characterized, in biochemical terms, as an antibiotic's inability to reach its bacterial target at a concentration that was previously effective. Microbial resistance to different agents can be intrinsic or acquired. Intrinsic resistance occurs due to inherent functional or structural characteristics of the bacteria, such as antibiotic-inactivating enzymes, nonspecific efflux pumps, and permeability barriers. On the other hand, bacteria can acquire resistance mechanisms via horizontal gene transfer in mobile genetic elements such as plasmids. Acquired resistance mechanisms include another category of efflux pumps with more specific substrates, which are plasmid-encoded. Efflux pumps are considered one of the main mechanisms of bacterial resistance to antibiotics and biocides, presenting themselves as integral membrane transporters. They are essential in both bacterial physiology and defense and are responsible for exporting structurally diverse substrates, falling into the following main families: ATP-binding cassette (ABC), multidrug and toxic compound extrusion (MATE), major facilitator superfamily (MFS), small multidrug resistance (SMR) and resistance-nodulation-cell division (RND). The Efflux pumps NorA and Tet(K) of the MFS family, MepA of the MATE family, and MsrA of the ABC family are some examples of specific efflux pumps that act in the extrusion of antibiotics. In this review, we address bacterial efflux pump inhibitors (EPIs), including 1,8-naphthyridine sulfonamide derivatives, given the pre-existing knowledge about the chemical characteristics that favor their biological activity. The modification and emergence of resistance to new EPIs justify further research on this theme, aiming to develop efficient compounds for clinical use.

Antibacterial activity of eugenol on the IS-58 strain of Staphylococcus aureus resistant to tetracycline and toxicity in Drosophila melanogaster.

The indiscriminate use of antibiotics contributes significantly to the selection of bacteria resistant to several antibiotics. Among the resistance mechanisms are the Efflux Pumps which are responsible for extruding solutes from the cell cytoplasm through proteins in the cell membrane. Because of this, new strategies are needed to control multidrug-resistant pathogenic strains. In this way, the objective of this study was to evaluate the antibacterial activity of eugenol by inhibition of TetK Efflux Pump in strains of Staphylococcus aureus resistant to Tetracycline, in addition to evaluating its toxicity in Drosophila melanogaster. To determine the Minimum Inhibitory Concentration (MIC), the broth microdilution method was used. The modulated effect of antibiotic and Ethidium Bromide associated with eugenol in subinhibitory concentrations (MIC/8) was evaluated. To evaluate the toxic effect of eugenol on D. melanogaster, fumigation tests were used, in which the parameters of mortality and damage to the locomotor system were evaluated. The results showed that eugenol has no direct activity in S. aureus, with an MIC ≥1024 µg/mL. However, it demonstrated that the synergistic potential when associated with Tetracycline, reducing the MIC of the antibiotic, already associated with Ethidium Bromide, had an antagonistic effect. When the toxicity in D. melanogaster was evaluated, eugenol demonstrated a non-toxic profile, since it presented EC50: 2036 µL/mL in 48 h of exposure. In conclusion, eugenol had no relevant direct effect against S. aureus, however, it potentialized the action of the antibiotic by decreasing its MIC.

Inhibition of the MepA efflux pump by limonene demonstrated by in vitro and in silico methods.

Bacterial resistance is a natural process carried out by bacteria, which has been considered a public health problem in recent decades. This process can be triggered through the efflux mechanism, which has been extensively studied, mainly related to the use of natural products to inhibit this mechanism. To carry out the present study, the minimum inhibitory concentration (MIC) tests of the compound limonene were performed, through the microdilution methodology in sterile 96-well plates. Tests were also carried out with the association of the compound with ethidium bromide and ciprofloxacin, in addition to the ethidium bromide fluorimetry, and later the molecular docking. From the tests performed, it was possible to observe that the compound limonene presented significant results when associated with ethidium bromide and the antibiotic used. Through the fluorescence emission, it was observed that when associated with the compound limonene, a greater ethidium bromide fluorescence was emitted. Finally, when analyzing the in silico study, it demonstrated that limonene can efficiently fit into the MepA structure. In this way, it is possible to show that limonene can contribute to cases of bacterial resistance through an efflux pump, so that it is necessary to carry out more studies to prove its effects against bacteria carrying an efflux pump and assess the toxicity of the compound.

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.

In Vitro and In Silico Inhibition of Staphylococcus aureus Efflux Pump NorA by α-Pinene and Limonene.

Since the discovery of the first antibiotics, bacteria have acquired a variety of resistance mechanisms, with efflux pump (EP) being the most prominent mechanism for intracellular targeting drugs. These proteins have become efficient mechanisms of resistance to antibiotics in species such as Staphylococcus aureus and, therefore, have been identified as promising therapeutic targets in antibacterial drug development. Accordingly, evidence suggests that monoterpenes can act as EP inhibitors and can be useful in circumventing bacterial resistance. This study aimed to evaluate the effectiveness of monoterpenes α-pinene and limonene as EP inhibitors against a strain of S. aureus expressing NorA protein. The minimum inhibitory concentration (MIC) against the 1199B strain of S. aureus, which carries genes encoding efflux proteins associated with antibiotic resistance to norfloxacin, was assessed through the broth microdilution method. The results obtained served as a subsidy for the analysis of the NorA pump inhibition with norfloxacin and ethidium bromide. Docking techniques, in silico, were used to evaluate the interaction of monoterpenes with NorA. Both monoterpenes showed no clinically effective antibacterial activity. Nevertheless, these compounds were found to decrease the MICs of ethidium bromide and norfloxacin indicating EP inhibition, which was confirmed by molecular docking analyses. In conclusion, α-pinene and limonene showed promising antibiotic-enhancing properties in S. aureus 1199B strain, indicating that monoterpenes can be used in targeted drug development to combat antibiotic resistance associated with EP expression.

Chemical synthesis, molecular docking and MepA efflux pump inhibitory effect by 1,8-naphthyridines sulfonamides.

This study aimed to evaluate the antibacterial activity and to verify, in silico and in vitro, the inhibition of efflux mechanisms using a series of synthesized 1,8-naphthyridines sulfonamides against Staphylococcus aureus strains carrying MepA efflux pumps. The chemical synthesis occurred through the thermolysis of the Meldrum's acid adduct. The sulfonamide derivatives were obtained by the sulfonylation of 2-amino-5­chloro-1,8-naphthyridine with commercial benzenesulfonyl chloride. Antibacterial activity was assessed by the broth microdilution test. Efflux pump inhibitory capacity was evaluated in silico by molecular docking and in vitro by analyzing synergistic effects on ciprofloxacin and ethidium bromide (EtBr) and by EtBr fluorescence emission assays. The following 1,8-naphthyridines were synthesized: 4-methyl-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10a); 2,5-dichloro-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10b); 4-fluoro-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10c); 2,3,4-trifluoro-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10d); 3-trifluoromethyl-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10e); 4­bromo-2,5-difluoro-N-(5­chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Compound 10f). The 1,8-naphthyridines derivatives associated with sulfonamides did not show antibacterial activity. However, they showed a favorable pharmacokinetic profile with possible MepA efflux pump inhibitory action, demonstrated in molecular docking. In addition to the promising results in reducing the concentration of intracellular EtBr. 1,8-naphthyridines act as putative agents in the inhibitory action of the MepA efflux pump.

The 1,8-naphthyridines sulfonamides are NorA efflux pump inhibitors.

OBJECTIVE: Efflux pumps are transmembrane proteins associated with bacterial resistance mechanisms. Bacteria use these proteins to actively transport antibiotics to the extracellular medium, preventing the pharmacological action of these drugs. This study aimed to evaluate in vitro the antibacterial activity of 1,8-naphthyridines sulfonamides, as well as their ability to inhibit efflux systems of Staphylococcus aureus strains expressing different levels of the NorA efflux pump. METHODS: The broth microdilution test was performed to assess antibacterial activity. Efflux pump inhibition was evaluated in silico by molecular docking and in vitro by fluorometric tests, and the minimum inhibitory concentration (MIC) was determined. The MIC was determined in the association between 1,8-naphthyridine and norfloxacin or ethidium bromide. RESULTS: The 1,8-naphthyridines did not show direct antibacterial activity. However, they effectively reduced the MIC of multidrug-resistant bacteria by associating with norfloxacin and ethidium bromide, in addition to increasing the fluorescence emission. In silico analysis addressing the binding between NorA and 1,8-naphthyridines suggests that hydrogen bonds and hydrophilic interactions represent the interactions with the most favourable binding energy, corroborating the experimental data. CONCLUSION: Our data suggest that 1,8-naphthyridines sulfonamides inhibit bacterial resistance through molecular mechanisms associated with inhibition of the NorA efflux pump in S. aureus strains.

In vitro and in silico inhibitory effects of synthetic and natural eugenol derivatives against the NorA efflux pump in Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium responsible for a number of diseases and has demonstrated resistance to conventional antibiotics. This study aimed to evaluate the antibacterial activity of eugenol and its derivatives allylbenzene, 4-allylanisole, isoeugenol and 4-allyl-2,6-dimethoxyphenol against the S. aureus NorA efflux pump (EP) in association with norfloxacin and ethidium bromide. The antibacterial activity of the compounds was assessed using the broth microdilution method to determine the minimum inhibitory concentration (MIC). A reduction in the MIC of ethidium bromide (a substrate for several efflux pumps) or norfloxacin was used as a parameter of EP inhibition. Molecular modeling studies were used to predict the 3D structure and analyze the interaction of selected compounds with the binding pocket of the NorA efflux pump. Except for 4-allylanisole and allylbenzene, the compounds presented clinically effective antibacterial activity. When associated with norfloxacin against the SA 1199B strain, 4-allyl-2,6-dimethoxyphenol eugenol and isoeugenol caused significant reduction in the MIC of the antibiotic, demonstrating synergistic effects. Similar effects were observed when 4-allyl-2,6-dimethoxyphenol, allylbenzene and isoeugenol were associated with ethidium bromide. Together, these findings indicate a potential inhibition of the NorA pump by eugenol and its derivatives. This in vitro evidence was corroborated by docking results demonstrating favorable interactions between 4-allyl-2,6-dimetoxypheno and the NorA pump mediated by hydrogen bonds and hydrophobic interactions. In conclusion, eugenol derivatives have the potential to be used in antibacterial drug development in strains carrying the NorA efflux pump.

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.

Do 1,8-naphthyridine sulfonamides possess an inhibitory action against Tet(K) and MsrA efflux pumps in multiresistant Staphylococcus aureus strains?

Naphthyridines represent a class of heterocyclic compounds formed by two condensed aromatic rings. This study aimed to evaluate the antibacterial activity and in vitro inhibition of efflux resistance mechanisms of a series of 1,8-naphthyridine sulfonamides against strains carrying Tet(K) and MsrA efflux pumps. The efflux pump inhibitory capacity was evaluated by analyzing synergistic effects between 1,8-naphthyridine sulfonamides and standard antibiotics, as well as ethidium bromide. The following 1,8-naphthyridines were used: 4-methyl-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Naph 1); 2,5-Dichloro-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Naph 2); 2,3,4-trifluoro-N-(5-chloro-1,8-naphthyridin-2-yl)benzenesulfonamide (Naph 7); 3-trifluoromethyl-N-(5-chloro-1,8-naphthyridin-2-yl)-benzenesulfonamide (Naph 9). The 1,8-naphthyridine sulfonamide derivatives possessed a potential Tet(K) and MsrA efflux pump inhibitory action.

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.

Characterization and antibacterial activity of the essential oil obtained from the leaves of Baccharis coridifolia DC against multiresistant strains.

Essential oils are secondary metabolites with immense pharmacological potential.These substances are abundantly produced by plants of the family Asteraceae, such as Baccharis coridifolia. Previous studies have demonstrated that this species has pharmacological properties that make it a promising source of new antibacterial agents. Therefore, the present study aimed to evaluate the antibacterial and antibiotic-modulating activity of Baccharis coridifolia essential oil against multidrug-resistant (MDR) strains. The phytochemical analysis was carried out by gas chromatography coupled to Mass Spectroscopy (GC/MS), and realized the Minimum Inhibitory Concentation (MIC) and antibiotic-modulation from the microdilution method in 96-well plates. It was revealed the presence of germacrene D (23.7%), bicyclogermacrene (17.1%), and (E)-caryophyllene (8.4%) as major components. The minimum inhibitory concentration of essential oil against strains of Pseudomonas aeruginosa (512 µg/mL) and Staphylococcus aureus (128 µg/mL) demonstrated clinically relevant antibacterial activity. In addition, the combination of subinhibitory doses of the oil with conventional antibiotics showed synergism, indicating potentiation of the antibacterial effect. In conclusion, the essential oil of Baccharis coridifolia (EOBc) presented antibacterial and antibiotic-modulating activities that place this species as a source of molecules useful in the fight against bacterial resistance.

GC-MS-FID characterization and antibacterial activity of the Mikania cordifolia essential oil and limonene against MDR strains.

The present study evaluated the effect of the essential oil of Mikania cordifolia (EOMc) and its major constituent limonene alone or associated with antibacterial drugs against Multidrug Resistant Bacteria (MDR). To evaluate the antibacterial activity, the minimum inhibitory concentrations (MIC) of the oil and limonene against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus were determined. The antibiotic-modulating activity was assessed using subinhibitory concentrations (MIC/8) of these substances in combination with conventional antibacterial drugs. Although no relevant antibacterial activity of the natural products was detected, both substances modulated the action of antibiotics against resistant bacteria. The EOMc demonstrated the best modulating effect against P. aeruginosa, presenting synergistic effects when associated with gentamicin and norfloxacin. In addition, the oil reduced the MIC of norfloxacin against E. coli as well as reduced the MIC of gentamicin against S. aureus. On the other hand, the best effect of limonene was obtained against S. aureus. Thus, it is concluded that the essential oil Mikania cordifolia and the isolated compound limonene do not have clinically significant antibacterial effect, but modulate the action of antibiotics against MDR bacteria.

Chemical Profile, Antibacterial Activity and Antibiotic-Modulating Effect of the Hexanic Zea Mays L. Silk Extract (Poaceae).

The present study aimed to determine the chemical profile and to evaluate the antibacterial activity and antibiotic-modulating action of the hexanic Zea mays silk extract in association with aminoglycosides. Standard Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 strains, as well as multi-resistant Escherichia coli 27, Staphylococcus aureus 35, and Pseudomonas aeruginosa 31 strains, were used in this study. Phytochemical prospection revealed the presence of the following secondary metabolites: tannins, flavones, flavonoids, and xanthones, with the main chemical constituents being identified in plant extracts obtained with apolar organic solvents such as hexane. The extract presented a minimum inhibitory concentration (MIC) ≥1024 µg/mL against all the tested strains. The association of the extract with aminoglycoside antibiotics showed significant synergistic effects against Staphylococcus aureus and Pseudomonas aeruginosa, except for amikacin, which was antagonized by the extract against E. coli. These results indicate the Zea mays silk presents bioactive compounds with antibiotic-modulating properties. However, further research is required to characterize the effects of isolated compounds and determine their potential for drug development.

Use of Flavonoids and Cinnamates, the Main Photoprotectors with Natural Origin.

Many pathological problems are initiated by ultraviolet radiation (UVR), such as skin cancer, the most commonly diagnosed cancer worldwide. The UVA (320-400 nm) and UVB (290-320 nm) wavelengths may cause effects such as photoaging, DNA damage, and a series of cellular alterations. The UVA radiation can damage the DNA, oxidize the lipids, and produce dangerous free radicals, which can cause inflammation, modify the gene expression in response to stress, and weaken the skin immune response. With a minor penetration, the UVB radiation is more harmful, being responsible for immediate damage. Ultraviolet radiation light emitted by the sun is considered necessary for the existence of life but cause radiation problems, especially in the skin. The photoprotective activities of plant extracts and isolated composts were evaluated by many reports, as well as the correlation of these compounds with the antioxidant activity. This review presents plant compounds with interest to the cosmetic industry to be used in sunscreens such as flavonoids and cinnamates.