Efflux Pump (QacA, QacB, and QacC) and ß-Lactamase Inhibitors? An Evaluation of 1,8-Naphthyridines against Staphylococcus aureus Strains.

The bacterial species Staphylococcus aureus presents a variety of resistance mechanisms, among which the expression of ß-lactamases and efflux pumps stand out for providing a significant degree of resistance to clinically relevant antibiotics. The 1,8-naphthyridines are nitrogen heterocycles with a broad spectrum of biological activities and, as such, are promising research targets. However, the potential roles of these compounds on bacterial resistance management remain to be better investigated. Therefore, the present study evaluated the antibacterial activity of 1,8-naphthyridine sulfonamides, addressing their ability to act as inhibitors of ß-lactamases and efflux pump (QacA/B and QacC) against the strains SA-K4414 and SA-K4100 of S. aureus. All substances were prepared at an initial concentration of 1024 µg/mL, and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. Subsequently, their effects on ß-lactamase- and efflux pump-mediated antibiotic resistance was evaluated from the reduction of the MIC of ethidium bromide (EtBr) and ß-lactam antibiotics, respectively. The 1,8-naphthyridines did not present direct antibacterial activity against the strains SA-K4414 and SA-K4100 of S. aureus. On the other hand, when associated with antibiotics against both strains, the compounds reduced the MIC of EtBr and ß-lactam antibiotics, suggesting that they may act by inhibiting ß-lactamases and efflux pumps such as QacC and QacA/B. However, further research is required to elucidate the molecular mechanisms underlying these observed effects.

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.

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.

Aminophenyl chalcones potentiating antibiotic activity and inhibiting bacterial efflux pump.

Chalcones and their derivatives are substances of great interest for medicinal chemistry due to their antibacterial activities. As the bacterial resistance to clinically available antibiotics has become a worldwide public health problem, it is essential to search for compounds capable of reverting the bacterial resistance. As a possibility, the chalcone class could be an interesting answer to this problem. The chalcones (2E)-1-(4'-aminophenyl)-3-(phenyl)­prop-2-en-1-one (APCHAL), and (2E)-1-(4'-aminophenyl)-3-(4-chlorophenyl)­prop-2-en-1-one (ACLOPHENYL) were synthesized by the Claisen-Schmidt condensation and characterized by 1H and 13C nuclear magnetic resonance (NMR), Fourier-transform infrared (FT-IR), and mass spectrometry (MS), In addition, microbiological tests were performed to investigate the antibacterial activity, modulatory potential, and efflux pump inhibition against Staphylococcus aureus (S. aureus) multi-resistant strains. Regarding the S. aureus Gram-positive model, the APCHAL presented synergism with gentamicin and antagonism with penicillin. APCHAL reduced the Minimum inhibitory concentration (MIC) of gentamicin by almost 70%. When comparing the effects of the antibiotic modifying activity of ACLOPHENYL and APCHAL, a loss of synergism is noted with gentamicin due to the addition of a chlorine to the substance structure. For Escherichia coli (E. coli) a total lack of effect, synergistic or antagonistic, was observed between ACLOPHENYL and the antibiotics. In the evaluation of inhibition of the efflux pump, both chalcones presented a synergistic effect with norfloxacin and ciprofloxacin against S. aureus, although the effect is much less pronounced with ACLOPHENYL. The effect of APCHAL is particularly notable against the K2068 (MepA overexpresser) strain, with synergistic effects with both ciprofloxacin and ethidium bromide. The docking results also show that both compounds bind to roughly the same region of the binding site of 1199B (NorA overexpresser), and that this region overlaps with the preferred binding region of norfloxacin. The APCHAL chalcone may contribute to the prevention or treatment of infectious diseases caused by multidrug-resistant S. aureus.

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.

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.