Synergy could be an effective strategy to potentiate and recover antibiotics nowadays useless in clinical treatments against multi-resistant bacteria. In this study, synergic interactions between antibiotics and grape pomace extract that contains high concentration of phenolic compounds were evaluated by the checkerboard method in clinical isolates of Staphylococcus aureus and Escherichia coli. To define which component of the extract is responsible for the synergic effect, phenolic compounds were identified by RP-HPLC and their relative abundance was determined. Combinations of extract with pure compounds identified there in were also evaluated. Results showed that the grape pomace extract combined with representatives of different classes of antibiotics as β-lactam, quinolone, fluoroquinolone, tetracycline and amphenicol act in synergy in all S. aureus and E. coli strains tested with FICI values varying from 0.031 to 0.155. The minimal inhibitory concentration (MIC) was reduced 4 to 75 times. The most abundant phenolic compounds identified in the extract were quercetin, gallic acid, protocatechuic acid and luteolin with relative abundance of 26.3, 24.4, 16.7 and 11.4%, respectively. All combinations of the extract with the components also showed synergy with FICI values varying from 0.031 to 0.5 and MIC reductions of 4 to 125 times with both bacteria strains. The relative abundance of phenolic compounds has no correlation with the obtained synergic effect, suggesting that the mechanism by which the synergic effect occurs is by a multi-objective action. It was also shown that combinations of grape pomace extract with antibiotics are not toxic for the HeLa cell line at concentrations in which the synergistic effect was observed (47 μg/mL of extract and 0.6–375 μg/mL antibiotics). Therefore, these combinations are good candidates for testing in animal models in order to enhance the effect of antibiotics of different classes and thus restore the currently unused clinical antibiotics due to the phenomenon of resistance. Moreover, the use of grape pomace is a good and low-cost alternative for this purpose being a waste residue of the wine industry.

In this study, we tested eight naturally-occurring flavonoids—three flavanones and five flavones—for their possible antibacterial properties against four Gram-positive and four Gram-negative bacteria. Flavonoids are known for their antimicrobial properties, and due their structural diversity; these plant-derived compounds are a good model to study potential novel antibacterial mechanisms. The lipophilicity and the interaction of antibacterial compounds with the cell membrane define the success or failure to access its target. Therefore, through the determination of partition coefficients in a non-polar/aqueous phase, lipophilicity estimation and the quantification of the antibacterial activity of different flavonoids, flavanones, and flavones, a relationship between these parameters was assessed. Active flavonoids presented diffusion coefficients between 9.4 × 10−10 and 12.3 × 10−10 m2/s and lipophilicity range between 2.0 to 3.3. Active flavonoids against Gram-negative bacteria showed a narrower range of lipophilicity values, compared to active flavonoids against Gram-positive bacteria, which showed a wide range of lipophilicity and cell lysis. Galangin was the most active flavonoid, whose structural features are the presence of two hydroxyl groups located strategically on ring A and the absence of polar groups on ring B. Methylation of one hydroxyl group decreases the activity in 3-O-methylgalangin, and methylation of both hydroxyl groups caused inactivation, as shown for 3,7-O-dimethylgalangin. In conclusion, the amphipathic features of flavonoids play a crucial role in the antibacterial activity. In these compounds, hydrophilic and hydrophobic moieties must be present and could be predicted by lipophilicity analysis.

Microcin E492, a polypeptide antibiotic, has been shown to have an M, of 6,000 by urea-SDS-polyacrylamide gel electrophoresis of the fluorescently labelled compound. It is known that the bactericidal action of microcin involves a loss of the transmembrane potential. In this study we show that microcin forms cation-selective channels in planar phospholipid bilayers. The channels have two main conductance states the current-voltage curves of which rectify. The reversal potentials measured under biionic conditions indicate a permeability sequence of NH,+ > K' = Rb' = Cs+ > Na+ = Li' > Tris+. The results suggest that membrane potential dissipation induced by microcin is a consequence of the formation of pores in the bacterial membrane.

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Microcin E492 is a polypeptide antibiotic that is produced and excreted by Klebsiella pneumoniae RYC492. The genetic determinants for microcin synthesis and immunity were cloned in Escherichia coli VCS257 into the cosmid vector pHC79, starting from total DNA of K. pneumoniae RYC492. The microcin E492 expressed in E. coli had the same properties as that of K. pneumoniae, i.e., the same molecular weight, the ability to form ionic channels in planar phospholipid bilayers, and essentially identical biological properties. Microcin E492 expression in E. coli, like that in K. pneumoniae, was mainly in the exponential phase of growth, declining in the stationary phase. The immunity determinant was subcloned into the same vector, and its expression was found to disappear in the stationary phase. This phenomenon is not dependent on rpoS, the stationary-phase sigma factor.Microcins are a family of low-molecular-weight antibiotics produced by members of the family Enterobacteriaceae. Microcins, unlike colicins, are not induced by DNA-damaging agents, but like for colicins, bacterial strains producing a specific microcin are immune to the same microcin (3).Microcin E492 is a polypeptide with an M r of 6,000 (14) that is produced by Klebsiella pneumoniae RYC492 and is active on strains of Escherichia coli, Klebsiella, Salmonella, Citrobacter, Enterobacter, and Erwinia (6). The mechanism of action is through membrane depolarization (9) induced by the formation of pores in the bacterial membrane (14). Studies on channel-forming bacteriocins produced by gram-negative bacteria have been reported only for colicins, all of them of high molecular weight (reviewed in reference 5). The other described bacteriocin which has the cytoplasmic membrane as target is colicin V. This bacteriocin has been reclassified as a microcin because it has a molecular weight of 6,000 and does not have most of the properties which are associated with colicins (10). Despite the fact that microcin E492 has many feature in common with colicin V, they are not closely related, because ColV ϩ strains that have high-level immunity to colicin V are fully susceptible to microcin E492 (9). Those colicins which act by depolarizing the bacterial energy-transducing membrane in vivo are able to form aqueous channels in both closed liposomes and planar membranes. However, despite the fact that colicin V inhibits the capacity of E. coli to carry out active transport of proline and to generate a membrane potential (26), it has not been possible to find an effect of colicin V on asolectin liposomes, and it remains to be established whether this difference reflects a difference in the in vivo mode of action. Thus, so far the only microcin described to be a channel-forming bacteriocin is microcin E492 (14). Nevertheless, low-molecular-weight pore-forming bacteriocins of gram-positive bacteria have been described, among them the lantibiotic nisin and lactococcins A and B, which are nonlantibiotic heatstable bacteriocins (reviewed in references 1, 13, and 24). The former has...

Kaurenoic acid is a diterpene with selective antibacterial activity against Gram-positive bacteria. The compound is bacteriolytic for Bacillus cereus. This activity was only partially affected by the composition and pH of the culture medium. Loss of the ability to retain the Gram stain and morphological alterations were produced in B. cereus cells exposed to kaurenoic acid. On the other hand, LPS mutants of Salmonella typhi were resistant to the compound, but spheroplasts of Escherichia coli became more sensitive to kaurenoic acid.

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Staphylococcus aureus is a serious human pathogen that is highly adaptive to environmental conditions and rapidly develops antibiotic resistance. The use of efflux pumps to reduce antibiotic concentrations at the intracellular level is one of the main mechanisms by which bacteria develop antibiotic resistance. The management of efflux pumps, specifically NorA, which is expressed by S. aureus strains, is a valuable strategy for restoring susceptibility in strains resistant to antibacterial agents. In recent years, many studies have focused on searching for natural substances that can reverse efflux pump-mediated resistance in S. aureus . Extracts and compounds obtained from plants can be efficient efflux pump inhibitors (EPIs) and represent a potentially patient-friendly strategy for controlling S. aureus . In the present study, we evaluated the ability of essential oils, petroleum ether extracts, dichloromethane extract (DCME) and six compounds isolated from the heartwood of Pilgerodendron uviferum (Cupressaceae) and two synthetic derivatives to inhibit efflux in NorA pumps in the following three S. aureus strains: K2378, which overexpressed the norA gene ( norA ++), K1902 ( norA -deleted, Δ norA ) and the parental strain, NCTC 8325-4. Efflux activity was evaluated using a fluorometric method that measured the accumulation of the universal efflux pump substrate ethidium bromide (EtBr). Only DCME and the compounds 15-copaenol and epi -cubenol inhibited EtBr efflux by K2378. Even the lowest concentration of 15-copaenol exhibited a stronger inhibitory effect than carbonyl cyanide m -chlorophenyl hydrazone on EtBr efflux by K2378. 15-copaenal only showed inhibition of EtBr efflux in K2378 cells at 125 μg/mL, but not superior to the control inhibitor and 15-copaenyl acetate exerted no intrinsic EPI activity against K2378. Fractional inhibitory concentration index (FICI) values obtained in the checkerboard assays, indicated that all combinations between DCME, epi -cubenol and 15-copaenol, and tested antibiotics showed a synergistic effect in wild type, norA ++ and Δ norA strains. Moreover, those were not toxic for the HeLa cell line at concentrations in which the synergistic effect and inhibitory activity of efflux pumps was determined. Other extracts and compounds obtained from P. uviferum did not display EtBr efflux-inhibiting activity against the evaluated S. aureus strains.