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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.

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.

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.

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.

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.This study aimed to investigate the potential ofamp;#945;-pinene as an efflux pump inhibitor in species of S. aureus carrying the TetK and MrsA proteins.The minimum inhibitory concentrations (MIC) ofamp;#945;-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 ofamp;#945;-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.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 byamp;#945;- pinene.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.

Staphylococcus aureus has been reported as one of the most difficult to treat. In the search for new treatment alternatives, isolated plant substances such as phenolic compounds, have demonstrated the ability to reverse bacterial resistance. The present study aims to evaluate the inhibitory action of caffeic acid and gallic acid on efflux pumps from S. aureus resistant strains. The broth microdilution assay was carried out to obtain the MICs of caffeic acid and gallic acid while the efflux pump inhibition test was assessed through the reduction of the minimum inhibitory concentration of the antibiotic and ethidium bromide. In addition, in silico theoretical parameters were analyzed to determine the theoretical efficacy of the compound and its free energy of interaction. In the results, the inhibition concentration of the two compounds did not certify clinical relevance with 1024 μg/mL for all strains. In the efflux pump inhibition effect, caffeic acid inhibited the MrsA pumps of the strain RN-4220 and NorA of the strain 1199B. Caffeic acid showed greater efficacy in the docking model, in agreement with the demonstrated experimental efficacy. Isolated compounds can be indicated as efficient options in the inhibition of resistance mechanisms.

Compounds capable of inhibiting the efflux pump mechanism are a promising alternative against bacterial resistance because, when combined with antibiotics, they can increase the effectiveness of these drugs by inhibiting active efflux. Elaiophylin, derived from Streptomyces hygroscopicus, is a natural antibiotic that exhibits a variety of biological activities, including antibacterial activity. However, its potential as an inhibitor of the bacterial efflux mechanism has not been investigated. This study evaluated the ability of Elaiophylin to inhibit the NorA efflux pump in Staphylococcus aureus strains. Therefore, tests were performed to obtain the Minimum Inhibitory Concentration (MIC) and to verify the ability of Elaiophylin to potentiate the MIC of the antibiotic Norfloxacin and Ethidium Bromide (EtBr), known substrates of NorA efflux. Real-time PCR and molecular docking assays were also performed to assess the potential of Elaiophylin against NorA. The strains SA-1199 (wild type) and SA-1199B (NorA over-expressed) of S. aureus were used for this study. The results showed that Elaiophylin significantly decreased the MIC of Norfloxacin and EtBr, increasing the activity of these substrates against S. aureus, which carries the efflux protein NorA. However, Elaiophylin provided a non-significant reduction in norA gene expression, however, molecular docking demonstrated a high binding affinity between Elaiophylin and NorA efflux protein, indicating that Elaiophylin can act as a potential NorA in S. aureus.

Pilosocereus pachycladus F. Ritter, Cactaceae, popularly known as “facheiro”, is used as food and traditional medicine in Brazilian caatinga ecoregion. The plant is used to treat prostate inflammation and urinary infection. The present work reports the first secondary metabolites isolated from P. pachycladus. Therefore, the isolated compound 4-hydroxy-3,5-dimethoxy benzaldehyde (syringaldehyde) was evaluated as modulator of Staphylococcus aureus pump efflux-mediated antibiotic resistance. The isolation of compounds was performed using chromatographic techniques and the structural elucidation was carried out by spectroscopic methods. In order to evaluate syringaldehyde ability to modulate S. aureus antibiotic resistance, its minimum inhibitory concentrations (μg/ml) was first determinate, then, the tested antibiotics minimum inhibitory concentrations were determined in the presence of the syringaldehyde in a sub-inhibitory concentration. The chromatographic procedures led to isolation of twelve compounds from P. pachycladus including fatty acids, steroids, chlorophyll derivatives, phenolics and a lignan. The syringaldehyde did not show any antibacterial activity at 256 μg/ml against S. aureus. On the other hand the compound was able to reduce the antibiotic concentration (tetracycline, norfloxacin, ethidium bromide) required to inhibit the growth of drug-resistant bacteria, showing the ability of syringaldehyde of inhibiting the efflux pump on these bacteria. Keywords: Forage cactus, Caatinga ecoregion, Cactaceae, Phytochemical study, Antibacterial activity, Resistant Staphylococcus aureus

The first occurrences and dissemination of resistant microorganisms led to the inefficacy of many antibiotics, available in the market nowadays, therefore, the search for new substances with antimicrobial activity from natural sources has gained a great importance. The purpose of this work is to evaluate the antibacterial activity and modulation of drug resistance in Staphylococcus aureus by coumarins such as bergapten, xantotoxin, isopimpinellin and imperatorin obtained from two Rutaceae species (Metrodorea mollis and Pilocarpus spicatus). The antimicrobial activity was assessed based on the minimum inhibitory concentration (MIC), using the microdilution method. The MIC was >256 g/mL for all coumarins tested. Regarding the modulation of drug resistance assay, the isopimpinellin reducted the MIC of erytromicin by 4 times, whereas imperatorin exhibited the best result, reducing the MIC of tetracycline (2 times), erytomicin (4 times) and norfloxacin (4 times). By reducing the MIC of ethidium bromide, the imperatorin is consider in fact, as a putative efflux pump inhibitor of bacteria.

The aim of this study is to evaluate the effect of ergosterol on steroids and cholesterol efflux pumps in multidrug resistant strains of S. aureus. Were used RN4220 harboring plasmid pUL5054, which carries the gene encoding the MsrA macrolide efflux protein; and IS-58, which possesses the TetK tetracycline efflux protein; 1199B resists hydrophilic fluoroquinolones via a NorA-mediated mechanism and wild strain 1199B. The Minimal Inhibitory Concentration (MIC) was determined and the evaluation of possible inhibition of efflux pumps by reduction of MIC. Some of the detrimental effects on bacterial cells can be attributed to the detergent properties of cholesterol and ergosterol on account of their amphipathic structure. Besides the cholesterol did not affect directly the pump structure, a synergism was observed, maybe due the interaction with the cell membrane and interference in the lipid bilayer.

Bacterial resistance to antibiotics has become a public health issue around the world. The present study aimed to evaluate the antibacterial activity of chalcones isolated from flowers of Arrabidaea brachypoda, and their potential as efflux pump inhibitors of Staphylococcus aureus efflux pumps. Microdilution assays were performed with natural products from A. brachypoda. Chalcones 1, 3, 4, and 5 did not show intrinsic antimicrobial activity against all S. aureus strains tested, but they were able to potentiate the Norfloxacin action against the SA1199-B (norA) strain, with a better modulating action for the 4 trimethoxylated chalcone. All chalcones were also able to potentiate the action of EtBr against SA1199-B strain, suggesting a potential NorA inhibition. Moreover, chalcone 4 was able to interfere in the activity of MepA, and interfered weakly in the QacA/B activity. Molecular docking analyzes showed that tested chalcones are capable of binding in the hydrophobic cavity of NorA and MepA, in the same Norfloxacin binding site, indicating that chalcone 4 compete with the antibiotic for the same NorA and MepA binding sites. Association of chalcone 4 with Norfloxacin could be an alternative against multidrug resistant S. aureus over-productive of NorA or MepA. This study was funded by the Fundação de Amparo à Pesquisa do Estado do Piauí (grant number: 050/2019) and Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq (grant number: 050/2019), Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (grant number: 00849/18-Universal) and CAPES (grant number: 88887.472618/2019-00-PROCAD-AM).

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.

This study investigated whether different fruit flavonoids are capable of modulating norfloxacin (Nor) resistance in Staphylococcus aureus SA-1119B, an S. aureus strain that overexpresses norA. The minimum inhibitory concentration (MIC) of flavonoids and norfloxacin (Nor) were determined using broth microdilution tests. To assess the antibiotic resistance modulatory effect, the MIC of Nor was measured in growth media with or without flavonoids at a sub-MIC level. The lowest MIC (256 μg/mL) against S. aureus SA-1199B was observed for myricetin; all other tested flavonoids produced MICs >256 μg/mL. When tested at sub-MIC levels in combination with Nor, hesperetin reduced the MIC of Nor from 128 μg/mL to 8 μg/mL (16-fold), whereas phloretin, diosmetin, and myricitrin reduced the MIC of Nor from 128 μg/mL to 32 μg/mL (4-fold), and quercitrin reduced the MIC of Nor from 128 μg/mL to 64 μg/mL (2-fold). No modulatory effects were observed for flavonoids with 7-O-glycoside residue or for non-glycosylated forms of 3-O-glycoside flavonoids. These results suggest that fruit flavonoids in aglycone forms or 3-O-glycoside flavonoids are capable of modulating Nor-resistance in S. aureus that overexpresses NorA.

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.

The aim of this study was to evaluate the antimicrobial activity of ethanoic extract of P. amarus (PAEE) and its compound Phyllanthin, as well as, investigate if these natural products could modulate the fluoroquinolone-resistance in S. aureus SA1199-B by way of overexpression of the NorA efflux pump. Microdilution tests were carried out to determine the minimal inhibitory concentration (MIC) of the PAEE or Phyllanthin against several bacterial and yeast strains. To evaluate if PAEE or Phyllanthin were able to act as modulators of the fluoroquinolone-resistance, MICs for Norfloxacin and ethidium bromide were determined in the presence or absence of PAEE or Phyllanthin against S. aureus SA1199-B. PAEE showed antimicrobial activity against Gram-negative strains, meanwhile Phyllanthin was inactive against all strains tested. Addition of PAEE or Phyllanthin, to the growth media at sub-inhibitory concentrations enhanced the activity of the Norfloxacin as well as, Ethidium Bromide, against S. aureus SA1199-B. These results indicate that Phyllanthin is able to modulate the fluoroquinolone-resistance possibly by inhibition of NorA. This hypothesis was supported by in silico docking analysis which confirmed that Phyllantin is a NorA ligand. Thus, this compound could be used as a potentiating agent of the Norfloxacin activity in the treatment of infections caused by fluoroquinolone-resistant S. aureus.

Nine new compounds derived from selenoglycolic acid were synthesized, and their structures were fully characterized by elemental analysis, infrared (IR), 1H and 13C nuclear magnetic resonance (NMR). The compounds were evaluated in an in silico study and showed strong to moderate antibacterial activity against several strains of Staphylococcus aureus. In particular, three compounds exhibited excellent antibacterial activity, with minimum inhibitory concentrations (MICs) between 16 and 64 µg mL-1. Furthermore, two of the nine compounds showed antifungal activity, with MIC of 1024 and 512 µg mL-1. In silico studies of the parameters of Lipinski's rule of five indicate that these compounds have potential to be new drug candidates.

Efflux pumps are integral parts of the bacterial plasma membrane that are responsible for many cases of antibiotic resistance. Modulators of drug resistance are regarded as the most suitable new antibacterial therapies. We evaluated the extracts of Sargassum polyceratium and the isolated compound pheophytin (Sp-1) for antibiotic modifying activity in strains of Staphylococcus aureus with efflux pump. The minimum inhibitory concentrations (MICs) for norfloxacin, tetracycline and erythromycin were determined by the microdilution broth method, in the absence and presence of the extract at a sub-inhibitory concentration (MIC/4). The extracts and isolated compounds showed no significant antimicrobial activity, but they changed the antibiotic activity, decreasing bacterial resistance by 2 to 4x. Using a checkerboard method, it was also possible to observe the synergistic effect (ΣFIC ≤ 0.5) between Sp-1 and the antibiotics erythromycin and norfloxacin. The results indicate that the seaweed Sargassum polyceratium and pheophytin are potential sources of an antibiotic adjuvant that modulates bacterial resistance, acting as a putative efflux pump inhibitor.

Arrabidaea brachypoda is a native shrub of the Brazilian Cerrado widely used in the folk medicine for treatment of renal diseases and articular pains. This study aimed to, first, evaluate the antimicrobial activity of both extracts and isolated molecules Brachydins BR-A and BR-B obtained from the flowers of A. brachypoda against Staphylococcus aureus, Escherchia coli and Candida albicans species. A second objective was to investigate if these natural products were able to potentiate the Norfloxacin activity against the strain Staphylococcus aureus SA1199-B that overexpress the norA gene encoding the NorA efflux pump. Extracts and isolated compounds were analyzed by HPLC-PDA and LC-ESI-MS respectively. Minimal inhibitory concentrations of Norfloxacin or Ethidium Bromide (EtBr) were determined in the presence or absence of ethanolic extract, dichloromethane fraction, as well as BR-A or BR-B by microdilution method. Only BR-B showed activity against Candida albicans. Addition of ethanolic extract, dichloromethane fraction or BR-B to the growth media at sub-inhibitory concentrations enhanced the activity of both Norfloxacin and EtBr against S. aureus SA1199-B, indicating that these natural products and its isolated compound BR-B were able to modulate the fluoroquinolone-resistance possibly by inhibition of NorA. Moreover, BR-B inhibited the EtBr efflux in the SA1199-B strain confirming that it is a NorA inhibitor. Isolated BR-B was able to inhibit an important mechanism of multidrug-resistance very prevalent in S. aureus strains, thus its use in combination with Norfloxacin could be considered as an alternative for the treatment of infections caused by S. aureus strains overexpressing norA.

Background: Tannins have shown inhibitory effects against pathogenic bacteria, and these properties make tannins potential modifying agents in bacterial resistance. Methods: The minimum inhibitory concentration (MIC) of tannic acid (TA), gallic acid (GA) and norfloxacin (Nor) against Staphylococcus aureus SA-1119 (NorA-effluxing strain) was determined using broth microdilution tests. To assess the modulation of antibiotic resistance, the MIC of Nor was determined in growth media with or without TA or GA at a subinhibitory concentration (1/4 MIC). The checkerboard method was performed to obtain the fractional inhibitory concentration index (FICI) for the combined application of TA and Nor. Results: TA displayed a weak inhibitory effect (MIC 512 μg/ml) against S. aureus SA-1119, while no inhibitory effect was displayed by GA (MIC >512 μg/ml). However, when TA was tested at a subinhibitory concentration in combination with Nor, the MIC of Nor against S. aureus SA-1119 decreased from 128 to 4 μg/ml (32-fold); this effect was not observed for GA. In the checkerboard assay, the MIC of TA and Nor decreased from 512 to 128 μg/ml (4-fold) and from 128 to 8 μg/ml (16-fold), respectively. The combination of TA and Nor presented an FICI as low as 0.31, which indicates a synergistic interaction. Conclusions: TA is a potential agent for increasing the clinical efficacy of Nor to control resistant S. aureus.

Ultraviolet radiation B (UVB) represents 5% of all solar UV radiation and chronic exposure can induce harmful biological responses, including skin cancer. Prospection of new drugs with photoprotective properties and less toxic effects is constant and natural products have been the main options in this field. Coumarins are a group of natural phenolic compounds that shows several pharmacological activities. The aim of present work was to investigate the effect of coumarin and six derivatives in sea urchin gametes and zygotes exposed to UVB. Embryonic development assay was used to monitor UVB embryotoxicity. Firstly, we demonstrated that coumarin inhibited first embryonic cell division from 5 μM (EC50 = 52.9 μM) and its derivatives showed an embryotoxic effect ten times higher. Then, gametes or zygotes were treated with coumarin compounds before or after UVB exposure (UVB doses ranged from 0.056 to 0.9 kJ m−2). Pretreatment of gametes or zygotes with coumarin or 3-hydroxycoumarin (1 μM, both) decreased UVB embryotoxic effect. Protective effect of the compounds was observed only when cells were treated previous to UVB exposure. Coumarin derivatives 4-hydroxycoumarin, 6-hydroxycoumarin, 7-hydroxycoumarin, 6,7-dihydroxycoumarin and 6-methoxy-7-hydroxycoumarin did not exhibit photoprotective activity. Our data provides evidences that coumarin and 3-hydroxycoumarin can be a promising class of photoprotective drugs.

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.

The present study is pioneer in analyzing the chemical composition of the essential oil from aerial parts of Turnera subulata Sm. and evaluates their antibacterial activity against a panel of drug-resistant strains of Staphylococcus aureus. The aerial parts were kiln-dried and then powdered in mechanical mill, and was subjected to hydrodistillation in a Clevenger-type apparatus. The components were analyzed using a mass spectrometrycoupled gas chromatography (GC-MS), and the identification of substances was performed by comparison of the mass spectra obtained with the mass spectra of the database of the GC-MS and retention indices . The essential oil was evaluated using the method of broth dilution at concentrations ranging from 3.125µg/mL to 3200µg/mL. It was possible to identify 45 substances (92.1%) of the essential oils, and the major components were trans-caryophyllene (6.7%), citronellol (5.6%), sphatulenol (5.3%), α-cadinol (4.3%), n-tricosano (4.3%), geraniol (4.1%) and trans-geranilacetone (3.7%), n-pentacosano (3.5%), globulol (3.4%), caryophyllene oxide (3.2%). The essential oil of T. subulata Sm. showed effective antibacterial activity for the various strains of S. aureus tested, with the MIC values between 25 µg/mL and 1600 µg/mL. The study of the essential oil of Turnera subulata Sm. showed that it consists of a complex mixture of several classes of compounds having the sesquiterpenes as major constituents followed by monoterpenes, and showed an antibacterial activity significant front the strains tested. O presente estudo é pioneiro em analisar a composição química dos óleos essenciais das partes aéreas de Turnera subulata Sm. e sua atividade antibacteriana frente à Staphylococcus aureus resistentes a antibióticos. As partes aéreas da planta foram secas em estufa, pulverizadas em moinho mecânico e submetidas à hidrodestilaçao em aparato tipo Clevenger. A composição dos óleos essenciais foi analisada por cromatografia gasosa acoplada a espectrometria de massas (CG-EM), e a identificação realizada por comparação dos espectros de massas com a biblioteca do CG-EM e índices de retenção. A atividade antimicrobiana do óleo essencial foi avaliada usando o método de diluição em caldo, em concentrações que variaram de 3.125µg/mL to 3200µg/mL. Foi possível identificar 45 substâncias (92,1%) do óleo essencial, sendo os componentes majoritários: trans-cariofileno (6,7%), citronelol (5,6%), espatulenol (5,3%), α-cadinol (4,3%), n-tricosano (4,3%), geraniol (4,1%) e trans-geranilacetona (3,7%), n-pentacosano (3,5%), globulol (3,4%) e óxido de cariofileno (3,2%). O óleo essencial de T. subulata Sm. mostrou atividade antibacteriana eficaz para as várias cepas de S. aureus testadas, com valores de CIM entre 25 µg/mL e 1600 µg/mL. O estudo do óleo essencial de Turnera subulata Sm. evidenciou sua complexa mistura, contendo várias classes de substâncias, tendo os sesquiterpenos como constituintes majoritários seguido dos monoterpenos, e mostrou significativa atividade antibacteriana frente as cepas testadas.

Background: In an ongoing project to evaluate essential oils as modulators of antibiotic resistance, the essential oil from Origanum vulgare L. (OVEO), as well as its individual constituents carvacrol (CAR) and thymol (THY), were investigated using Staphylococcus aureus strains possessing efflux mechanisms of resistance to norfloxacin, erythromycin and tetracycline. Methods: The minimum inhibitory concentration (MIC) values of the antibiotics were determined by agar dilution method, in the absence and in the presence of subinhibitory concentrations of OVEO, CAR or THY. Results: Along with relevant antistaphylococcal activity, OVEO, CAR and THY modulated the activity of tetracycline, i.e. in combination with antibiotics a reduction in the MIC was observed (up to fourfold). Conclusions: The results presented here represent, as far as we know, the first report of OVEO, CAR and THY as putative efflux pump inhibitors. Broadly, these findings indicate that essential oils could serve as potential sources of compounds capable of modulating drug resistance.

SUMMARY Embryos of marine organisms whose development occurs externally are particularly sensitive to ultraviolet (UV) light (bands A and B, respectively, UVA and UVB). ATP-binding cassette (ABC) transporters are the first line of cellular defense against chemical or physical stress. The present work investigated the involvement of ABC transporters on UVA or UVB effects on eggs, spermatozoa, and embryonic cells of the sea urchin Echinometra lucunter. Gametes or embryos were exposed to UVA (3.6–14.4 kJ m−2) or UVB (0.112–14.4 kJ m−2), and embryonic development was monitored by optical microscopy at different developmental stages in the presence or absence of the ABC-transporter blockers reversin205 (ABCB1 blocker) or MK571 (ABCC1 blocker). E. lucunter eggs, spermatozoa and embryos were resistant to UVA exposure. Resistance to the harmful effects of UVB was strongly associated to ABC transporter activity (embryos > eggs > spermatozoa). ABCB1 or ABCC1 blockage promoted the injurious effects of UVA on spermatozoa. ABCC1 transporter blockage increased UVB-dependent damage in eggs while ABCB1 transporter inhibition increased harmful effects of UVB in embryonic cells. ABC-transporter activity was not, however, affected by UVB exposure. In conclusion, the present study is the first report on the protective role of ABC transporters against harmful effects of UVA and UVB on sea urchin eggs and embryonic cells. Mol. Reprod. Dev. 81: 66–83, 2014. © 2013 Wiley Periodicals, Inc.

The essential oil from leaves of Croton grewioides Baill was obtained by hydrodistillation using Clevenger apparatus, and its chemical composition was analyzed by GC-MS, where 18 compounds were identified, mostly as monoterpenes (55.56%) and sesquiterpenes (44.44%), in which the major constituent was the α-pinene (47.43%). The essential oil of Croton grewioides (EOCg) and its major compound (α-pinene) were evaluated as modulators of antibiotic resistance in strain SA-1199B and IS-58 of Staphylococcus aureus that overexpresses efflux protein. The minimum inhibitory concentrations (MICs) of the antibiotics were determined by the microdilution assay in the absence and in the presence of sub-inhibitory concentration of EOCg and α-pinene. Although the EOCg and α-pinene did not indicate relevant antibacterial activity in vitro, they acted as antibiotic resistance modulators, i.e., EOCg in combination with norfloxacin, reducted its MIC, by 64× whereas in combination with tetracycline it was observed a reduction of 4×. Additionally, it was observed a MIC reduction of tetracycline by 32×, when combined with α-pinene. The results suggest that EOCg and α-pinene modulate or even reverse bacterial resistance as a putative efflux pump inhibitor.

The use of natural products is crucial to suppress the development of these micro-organisms and to reduce the concentration necessary to inhibit these microrganisms, reducing the toxicity risks also. In this study, the essential oil from Chenopodium ambrosioides Leaves and its main constituent α-Terpinene were used in the antibacterial and potentiating activity of antibiotics and ethidium bromide assays, against the bacterial strains Staphylococcus aureus IS-58, carriers of efflux pumps. The Minimum Inhibitory Concentration (MIC) was determined using a microdilution method. The capacity of the aforementioned was also tested in combination with tetracycline and ethidium bromide, with the aim of improving the activity of the antibacterials. The MIC of the C. ambrosioides L. essential oil and of α-Terpinene were above 1024 μg/mL, comprising a clinically irrelevant value. However, when associated with the antibiotics, the C. ambrosioides L. essential oil, significantly decreased the MIC of tetracycline and ethidium bromide. The efflux pump is the only mechanism the bacteria possesses to reduce the toxicity of ethidium bromide, and thus this reduction in the MIC demonstrates that the C. ambrosioides L. essential oil is an effective option in the inhibition of the efflux pump present in these micro-organisms.

This study evaluated the efficacy of glycone (myricitrin, hesperidin and phloridzin) and aglycone flavonoids (myricetin, hesperetin and phloretin) in inhibiting biofilm formation by Staphylococcus aureus RN4220 and S. aureus SA1199B that overexpress the msrA and norA efflux protein genes, respectively. The minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC50 - defined as the lowest concentration that resulted in ≥50% inhibition of biofilm formation) of flavonoids were determined using microdilution in broth procedures. The flavonoids showed MIC >1024 μg/mL against S. aureus RN4220 and S. aureus SA1199B; however, these compounds at lower concentrations (1-256 μg/mL) showed inhibitory effects on biofilm formation by these strains. Aglycone flavonoids showed lower MBIC50 values than their respective glycone forms. The lowest MBIC50 values (1 and 4 μg/mL) were observed against S. aureus RN4220. Myricetin, hesperetin and phloretin exhibited biofilm formation inhibition >70% for S. aureus RN4220, and lower biofilm formation inhibition against S. aureus SA1199B. These results indicate that sub-MICs of the tested flavonoids inhibit biofilm formation by S. aureus strains that overexpress efflux protein genes. These effects are more strongly established by aglycone flavonoids.

During the early periods of antibiotic usage, bacterial infections were considered tamed. However, widespread antibiotic use has promoted the emergence of antibiotic-resistant pathogens, including multidrug resistant strains. Active efflux is a mechanism for bacterial resistance to inhibitory substances, known simply as drug efflux pumps. The bacterium Staphylococcus aureus is an important pathogenic bacterium responsible for an array of infections. The NorA efflux pump has been shown to be responsible for moderate fluoroquinolone resistance of S. aureus. The inhibition of the efflux pump was assayed using a sub-inhibitory concentration of standard efflux pump inhibitors and tannic acid (MIC/8), where its capacity to decrease the MIC of Ethidium bromide (EtBr) and antibiotics due to the possible inhibitory effect of these substances was observed. The MICs of EtBr and antibiotics were significantly reduced in the presence of tannic acid, indicating the inhibitory effect of this agent against the efflux pumps of both strains causing a three-fold reduction of the MIC when compared with the control. These results indicate the possible usage of tannic acid as an adjuvant in antibiotic therapy against multidrug resistant bacteria (MDR).

This study aimed to assess the adhesion, detachment kinetic and biofilm formation of Staphylococcus aureus isolates from food services surfaces on stainless steel and polypropylene surfaces when cultivated in a vegetable-based broth at 7 and 28 °C, and the efficacy of peracetic acid (30 mg/L) and sodium hypochlorite (250 mg/L) in removing the bacterial cells from the matrix of the preformed biofilm. The isolates adhered over 4 Log cfu/cm 2 regardless the surface kind and incubation temperature. Cell detachment was around 3 Log cfu/cm 2 over the first six contacts with agar characterizing a high persistence of cells on the tested surfaces. Number of cells (5–7 Log cfu/cm 2 ) needed for biofilm formation was noted at all experimental systems already after 3 days of incubation. A range of 2.0–3.3 and 1.5 to 2.1 Log cfu/cm 2 was observed in the reduction of cells in biofilm matrix caused by peracetic acid and sodium hypochlorite, respectively. The isolates of S. aureus revealed high capability to adhere and form biofilm on the tested surfaces in both assayed incubation temperature.

Solanum paludosum extracts obtained by maceration (SP-EtOH CRUDE and SP-EtOH MARC ) and supercritical fluid extraction ( SP-SFE CO 2 ) were evaluated. Three extracts with distinct chromatographic profiles were obtained: SP-EtOH CRUDE , with a complex phenolic composition; SP-SFE CO 2 , containing methoxylated flavonoids; and SP-EtOH MARC , which bears mainly polar polyphenols. All extracts were strong antioxidants, as measured using the diphenylpicrylhydrazyl (DPPH ) method, xanthine/xanthine oxidase/luminol and iron-induced lipid peroxidation systems. Extracts were also evaluated as modifiers of antibiotic activity in strains of Staphylococcus aureus expressing fluoroquinolone, macrolide and tetracycline efflux proteins. Despite their lack of antibacterial activity, all extracts were able to potentiate antibiotics activities by as much as eight fold. Both the maceration and supercritical fluid extraction processes were adequate to obtain extracts with different biological activities according to their patterns of phenolic compounds. The data suggest that S. paludosum could be a valuable source of antioxidants and putative efflux pumps inhibitors.

Introduction: Some species of Staphylococcus are often recognized as etiological agents of many animal and human opportunistic infections. This study is the first test of change in resistance ofantibiotic activity by Croton campestris A. and Ocimum gratissimum L. against multiresistant strains of Staphylococcus aureus.Objective: In this study, the hexane and methanol extract of Croton campestris A. and Ocimum gratissimum L. was tested for antibacterial activity alone and in combination with norfloxacin against the strain SA1199B.Materials and methods: The minimum inhibitory concentration (MIC) and the modulatory effect of extracts was assayed using microtitre assay.Results: By the fact of the MIC observed was not clinically relevant (MIC= 512 to ≥1.024 μg/ml), the antibiotic activity of norfloxacin was enhanced when this antibiotic was combined with sub-inhibitory concentrations of extracts, mainly the hexane extracts.Conclusions: These results indicate that the assayed extracts present compounds that can be used as a putative efflux pump inhibitor, indicating that Croton campestris A. and Ocimum gratissimum L. can be a source of plant derived products with antibiotic modifier activity.Introducción. Algunas especies de Staphylococcus suelen ser reconocidas como agentes etiológicos de muchas infecciones oportunistas en animales y em humanos. Este estudio es la primera prueba delcambio en la resistencia de la actividad antibiótica por Croton campestris A. y Ocimum gratissimum L. contra cepas multirresistentes de Staphylococcus aureus.Objetivo. Ensayar la actividad antibacteriana de los extractos hexánicos y metanólicos de Croton campestris A. y Ocimum gratissimum L. sola y en combinación con norfloxacina sobre la cepa SA1199B.Materiales y métodos. Se analizó la concentración inhibitoria mínima (CIM) y el efecto modulador de los extractos usando el ensayo de microtitulación.Resultados. Por el hecho de que la CIM observada no era clínicamente relevante (CIM: 512 ≥ 1.024 mg/ml), la actividad antibiótica de norfloxacina fue potenciada cuando se combinó este antibiótico conconcentraciones subinhibitorias de los extractos, principalmente con los extractos hexánicos. Conclusiones. Estos resultados indican que los extractos ensayados presentan compuestos que pueden ser utilizados como inhibidores putativos de la bomba de eflujo, lo que indica que Croton campestris A. y Ocimum gratissimum L. pueden ser una fuente de productos derivados de plantas con actividad modificadora de antibióticos.

This is the first report of antibiotic modifying activity by the volatile compounds from essential oils of Lippia microphylla Cham. (Verbenaceae). The leaves of Lippia microphylla Cham. (Verbenaceae) were extracted by hydrodistillation, and the essential oil obtained was examined with respect to its effects on the antibiotic activity of gentamicin, tetracycline and norfloxacin by minimal inhibitory dose method and gaseous contact. The gaseous component of the oil inhibited the bacterial growth of the strains of Staphylococcus aureus with a MID of 0.5 mg/L air. The activity of gentamicin against Pseudomonas aerugionosa and norfloxacin against Staphylococcus aureus 1199B was increased by 47% and 225%, whereas no effect or an increased resistance to tetracycline was observed with an inhibition zone 14.3% smaller after contact with the gaseous component of the essential oil. These results show that the volatile compounds of this oil can enhance antibiotic activity, which may lead to the development o...

In an ongoing project to evaluate natural compounds isolated from by-products or wastes from vegetables and fruits (edible plants) as modulators of antibiotic resistance, ethanol extract from mango peel was investigated using Staphylococcus aureus strains possessing efflux mechanisms of resistance to norfloxacin, erythromycin and tetracycline. The minimum inhibitory concentrations (MIC) of the antibiotics were determined by the micro dilution assay in the absence and in the presence of sub-inhibitory mango peel extract concentration. Although the extract did not display relevant antibacterial activity (MIC>2048 µg/mL), it modulated the activity of antibiotics, i.e. in combination with antibiotics (at 512 µg/mL), a four-fold reduction in the MIC values for tetracycline and erythromycin was observed. The results presented here indicates that mango peel could serve as a source of potential adjuvant of antibiotics which add value to this mango by-product.

Two salts of the mesoionic compounds 1,4-diphenyl-5-(5-nitro-2-furanyl)-1,3,4-thiadiazolium-2-thiol chloride (MC-1) and 4-phenyl-5-(5-nitro-2-furanyl)-1,3,4-thiadiazolium-2-phenylamine chloride (MC-2) were synthesized utilizing 1,4-diphenyl-thiosemicarbazide and 5-nitro-2-furoyl chloride as starting materials. Their structures were characterized by IR, 1H-NMR, 13C-NMR and elemental analysis. These compounds were analyzed for their influence on the effectiveness of norfloxacin, tetracycline, and erythromycin (standard antibiotics) against resistant strains of Staphylococcus aureus. MC-1 and MC-2, at sub-inhibitory concentrations of 16 μg/mL, favourably modulated the antibiotic activity of tetracycline by 16- and 32-fold, respectively (MIC), and that of erythromycin by 4-fold.

From the aerial parts of Sidastrum micranthum (A. St.-Hil.) Fryxell (Malvaceae) were isolated m-methoxy-p-hydroxy-benzaldehyde, o-hydroxy-benzoic acid, acacetin, quercetin, 7,4′-Di-O-methylisoscutellarein, genkwanin and tiliroside. These compounds were identified by data analyses of spectroscopic methods. Although acacetin and 7,4′-Di-O-methylisoscutellarein did not display relevant antibacterial activity (MIC = 256 µg/mL), they modulated the activity of antibiotics, i.e. in combination with antibiotics at 64 µg/mL (¼ MIC), a two-fold reduction in the MIC was observed for norfloxacin and ethidium bromide; regarding tetracycline and erythromycin a two-fold reduction in the MIC was observed only with 7,4′-Di-O-methylisoscutellarein.

The antibacterial activity of ethanol extracts of Eugenia jambolana L. and E. uniflora L. (Myrtaceae) were evaluated by agar-well diffusion method and MICs by microdillution method. E. jambolana showed antibacterial activity against MRSA strains (MIC 1024 μg/mL). It is therefore suggested that extracts from E. jambolana could be used as an anti– Staphylococcus agent. When compared with Methicillin and Gentamicin, the extract was more effective against, being a promising antibacterial agent. Based on the author's knowledge, this is the first report about the antibacterial activity of leave ethanol extract from Eugenia jambolana.

Bacterial infectious agents represent a risk to populations, where they are responsible for the high morbidity and mortality. In combating these pathogens, our main line of defense is the use of antibiotics. However, the indiscriminate use of these drugs select resistant strains to these same drugs.In this study the ethanol extract of Hyptis martiusii Benth. (EEHM) (Lamiaceae) was tested for its antimicrobial activity against aminoglycoside multi-resistant Staphylococcus aureus (MRSA).In this study, the ethanol extract of H. martiusii was prepared and tested with chlorpromazine for its antimicrobial activity using the microdilution method. Chlorpromazine and the ethanol extract were used alone and also in combination with aminoglycosides against a MRSA strain resistant to these antibiotics to determine the participation of efflux systems in resistance mechanisms. The FIC index was calculated and evaluated by the checkerboard method.A potentiating effect between this extract and aminoglycosides was demonstrated. Similarly, a potentiating effect of chlorpromazine with kanamycin was detected, indicating the involvement of an efflux system in the resistance to this aminoglycoside. The checkerboard method with combinations of aminoglycosides and EEHM demonstrated additive effect with kanamycin and gentamicin. It is therefore suggested that extracts from H. martiusii could be used as a source of plant-derived natural products with resistance- modifying activity.This is the first report about the modifying antibiotic activity of Hyptis martiusii, constituting a new approach against bacterial resistance to antibiotics as aminoglycosides.

Staphylococcus genus is widely spread in nature being part of the indigenous microbiota of skin and mucosa of animal and birds. Some Staphylococcus species are frequently recognized as etiological agents of many animal and human opportunistic infections. This is the first report of efflux pump inhibition by the volatile compounds from essential oil of Zanthoxylum articulatum. The aim of this study was evaluated the synergism between the essential oil of Zanthoxylum articulatum and aminoglycosides. The leaves of Zanthoxylum articulatum Engler (Rutaceae) were subjected to hydrodistillation, and the essential oil extracted was examined with respect to inhibitory activity of efflux pump by gaseous contact. The gaseous component of the oil enhanced the antibiotic activity of gentamicin, possibly by affecting the efflux system and enhanced the inhibition zone of norfloxacin in 70.9 %. This result shows that this oil influences the antibiotic activity and may be used as an adjuvant in the antibiotic the...

In this study, the ethanol extract of Momordica charantia L. (EEMC) (Cucurbitaceae) was tested for its antimicrobial activity against strains of Escherichia coli. The growth of two E. coli strains tested was not inhibited by the extract. The MIC and MBC values were ≥ 1 mg/mL for both strains of E. coli used. A synergistic effect between this extract and aminoglycosides was demonstrated. Similarly, a synergistic effect was observed of chlorpromazine on kanamycin and amikacin, indicating the involvement of an efflux system in the resistance to these aminoglycosides. The checkerboard method with combinations of aminoglycosides and EEMC demonstrated synergism with kanamycin and an additive effect with amikacin and neomycin. It is therefore suggested that extracts from M. charantia could be used as a source of plant-derived natural products with resistance-modifying activity. This is the first report about the modifying antibiotic activity of Momordica charantia, constituting a new weapon against bacterial res...

In an ongoing project to evaluate natural compounds isolated from plants from the Brazilian biodiversity as modulators of antibiotic resistance, kaempferol-3-O-beta-d-(6''-E-p-coumaroyl) glucopyranoside (tiliroside), isolated from Herissantia tiubae (Malvaceae) was investigated using the strain SA-1199B of Staphylococcus aureus, which overexpresses the norA gene encoding the NorA efflux protein which extrudes hydrophilic fluorquinolones and some biocides, such as benzalkonium chloride, cetrimide, acriflavine and ethidium bromide. The minimum inhibitory concentrations (MICs) of the antibiotics and biocides were determined by the microdilution assay in the absence and in the presence of sub-inhibitory concentration of tiliroside. Although tiliroside did not display relevant antibacterial activity (MIC = 256 microg/mL), it modulated the activity of antibiotics, i.e. in combination with antibiotics a reduction in the MIC was observed for norfloxacin (16-fold), ciprofloxacin (16-fold), lomefloxacin (four-fold) and ofloxacin (two-fold), and an impressive reduction in the MICs for the biocides (up to 128-fold). The results presented here represent the first report of a kaempferol glycoside as a putative efflux pump inhibitor in bacteria. The present finding indicates that H. tiubae (and broadly Malvaceae) could serve as a source of plant-derived natural products that modulate bacterial resistance, i.e. a source of potential adjuvants of antibiotics.

In our continuous study of the Malvaceae family, we describe here the isolation of four triterpenes (frideline, lupeol, cycloartenol, and cycloeucalenol), a steroid (β-sitosterol), and four phenolic compounds from the aerial parts of Herissantia tiubae (K. Schum.) Brizicky (i.e., a benzoic acid derivative, a coumarin and two flavonoids, kaempferol 7-O-α-l-rhamnopyranoside and 4′,5-dihydroxy-3,6,7,8,3′-pentamethoxyflavone). The structural identification of the compounds was made by comparison with literature data and spectral analysis, including two-dimensional NMR techniques. These compounds are reported here for the first time in genus Herissantia. The pentamethoxyflavone was assayed against strains of Staphylococcus aureus possessing efflux mechanisms of resistance to norfloxacin, tetracycline, and erythromycin. Although the compound did not display relevant antibacterial activity (MIC ≥ 512 μg/mL), it modulated the activity of antibiotics, i.e., in combination with antibiotics (at 128 μg/mL), a two-fol...

Semisynthetic and commercial coumarins were investigated for their antibacterial and adjuvant properties with antibiotic agents against norfloxacin, erythromycin, and tetracycline resistantStaphylococcus aureusas based on efflux mechanisms. The coumarins and certain commercial antibiotics had their Minimum Inhibitory Concentrations determined by broth microdilution assay against resistantS. aureusstrains which overexpress efflux pump proteins. For evaluation of the modulatory activity, the antibiotics MICs were determined in the presence of the coumarin derivatives at subinhibitory concentration. Although the coumarins did not display relevant antibacterial activity (MIC ≥ 128 µg/mL), they did modulate the antibiotics activities. Various coumarins, especially the alkylated derivatives in combination with antibiotics at subinhibitory concentrations, modulated antibiotic activity, reducing the MIC for tetracycline and norfloxacin by 2 to 8 times. Polar Surface Area (PSA) studies were performed and the fact that the presence of apolar groups is an important factor for the modulatory activity of coumarins was corroborated. Docking on the Penicillin-Binding Protein from MRSA identified that18is a potential ligand presenting lowEbinding. The results indicate that coumarin derivatives modulated antibiotic resistance and may be used as potential antibiotic adjuvants, acting by bacterial efflux pump inhibition inS. aureus.

Tannins have shown inhibitory effects against pathogenic bacteria, and these properties make tannins potential modifying agents in bacterial resistance.The minimum inhibitory concentration (MIC) of tannic acid (TA), gallic acid (GA) and norfloxacin (Nor) against Staphylococcus aureus SA-1119 (NorA-effluxing strain) was determined using broth microdilution tests. To assess the modulation of antibiotic resistance, the MIC of Nor was determined in growth media with or without TA or GA at a subinhibitory concentration (1/4 MIC). The checkerboard method was performed to obtain the fractional inhibitory concentration index (FICI) for the combined application of TA and Nor.TA displayed a weak inhibitory effect (MIC 512 μg/ml) against S. aureus SA-1119, while no inhibitory effect was displayed by GA (MIC512 μg/ml). However, when TA was tested at a subinhibitory concentration in combination with Nor, the MIC of Nor against S. aureus SA-1119 decreased from 128 to 4 μg/ml (32-fold); this effect was not observed for GA. In the checkerboard assay, the MIC of TA and Nor decreased from 512 to 128 μg/ml (4-fold) and from 128 to 8 μg/ml (16-fold), respectively. The combination of TA and Nor presented an FICI as low as 0.31, which indicates a synergistic interaction.TA is a potential agent for increasing the clinical efficacy of Nor to control resistant S. aureus.

Cepas de Staphylococcus aureus de origem bovina foram submetidas ao tratamento com quatro fluoquinolonas na concentração subinibitória (1/2 x CMI), para avaliar a influência desses agentes sobre plasmídios. A ciprofloxacina mostrou ser a fluorquinolona mais eficiente, eliminando marcas de resistência para estreptomicina, tetraciclina, penicilina e cádmio. A norfloxacina e a pefloxacina eliminaram resistência para penicilina e tetraciclina, respectivamente; no entanto, não foi evidenciada a eliminação de plasmídio com ofloxacina. Os resultados confirmam a eficácia das fluor-quinolonas em eliminar plasmídios de resistência mostrando a importância desses estudos como contribuição para o entendimento da prevenção de linhagens multiresistentes, uma vez que as quinolonas em concentrações subinibitórias podem aumentar a sensibilidade das linhagens a outros agentes antimicrobianos.Bovine strains of Staphylococcus aureus were submitted to treatment with four fluoro-quinolones in subinhibitory concentrations (1/2 x MICs) to evaluate their influence on the curing of plasmids. Ciprofloxacin showed to be the most efficient by eliminating resistance to streptomycin, tetracyclin, penicillin, and cadmium nitrate. Norfloxacin and pefloxacin eliminated penicillin- and tetracyclin-resistance respectively. Otherwise, plasmids elimination by ofloxacin was not evidenced. The results obtained in this study confirm the potential of fluoroquinolones to eliminate antibiotic-resistant plasmids, and showed to be a valuable contribution for the prevention of multi-resistant strains, and may even enhance their sensitivity to other chemotherapeutic agents.

An ethanol extract and different partition fractions obtained from Lippia origanoides H.B.K. were assayed for light-mediated activity against strains of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Identical assays were conducted with and without exposure to UV-A (400-315 nm) light to test for light enhanced activity. The ethanol extract and dichloromethane fraction showed light-mediated activity against the S. aureus strain, but not against the E. coli strain. The dichloromethane fraction was more active than the ethanol extract. Naringenin did not display light-mediated activity against the tested bacteria, indicating that the light-mediated antimicrobial activity of the dichloromethane fraction is not due to its major component. The results represent the first report of light-mediated antimicrobial activity of Lippia origanoides and show that its phytochemicals could be used as light-mediated antimicrobial agents.

In an ongoing project to evaluate essential oils as modulators of antibiotic resistance, the essential oil from Origanum vulgare L. (OVEO), as well as its individual constituents carvacrol (CAR) and thymol (THY), were investigated using Staphylococcus aureus strains possessing efflux mechanisms of resistance to norfloxacin, erythromycin and tetracycline.The minimum inhibitory concentration (MIC) values of the antibiotics were determined by agar dilution method, in the absence and in the presence of subinhibitory concentrations of OVEO, CAR or THY.Along with relevant antistaphylococcal activity, OVEO, CAR and THY modulated the activity of tetracycline, i.e. in combination with antibiotics a reduction in the MIC was observed (up to fourfold).The results presented here represent, as far as we know, the first report of OVEO, CAR and THY as putative efflux pump inhibitors. Broadly, these findings indicate that essential oils could serve as potential sources of compounds capable of modulating drug resistance.