Antibiotic Potentiating Activity Research Articles

Quantum-chemical calculation, antibiotic potentiating activity, evaluation of efflux pump inhibition against S. aureus multi-resistant strains and molecular docking of the drugs isoniazid and hydralazine

In recent years, due to the uncontrolled use of antibiotics and natural evolution, many pathogenic microorganisms have acquired resistance to currently available drugs. Consequently, the number of infections caused by these microorganisms, considered multi-resistant to antimicrobial drugs, has increased, becoming a serious global public health problem. In the search for alternative therapies, drug repositioning represents a promising way to introduce new medicines. Hydralazine exhibits antihypertensive and peripheral vasodilatory action and isoniazid is a prodrug used as the first choice for the chemotherapy treatment of tuberculosis. In this work, microbiological tests were performed to investigate the modulator potential and efflux pump inhibition on S. aureus multi-resistant strains of the drugs isoniazid and hydralazine. In addition, the Density Functional Theory was used to determine the electronic and reactivity properties. Association of Ciprofloxacin with hydralazine on the K2068 strain proved to be better than the association of Norfloxacin with this antihypertensive on the 1199B strain of S. aureus. Association of isoniazid with ciprofloxacin in the K2068 strain was irrelevant and may have generated antagonism. The opposite effect was observed when associated with Norfloxacin in strain 1199B, where a significant reduction in the MIC of Norfloxacin was observed when associated with isoniazid. Regarding the association of hydralazine and isoniazid with EtBr, the results of this study showed that only hydralazine was able to positively modify the MIC of bromide on strains 1199B and K2068, presenting better results with this last strain, while in tests with to isoniazid. Molecular docking was used to verify the feasibility of forming receptor-ligand complexes and the replicability of the results. The molecular docking study against MepA indicated a possible synergistic effect between hydralazine and the antibiotic ciprofloxacin. Regarding NorA, the data indicated that hydralazine could act through competitive inhibition with the control norfloxacin, since it interacts in the same region of the antibiotic binding site through interactions in common with residues Glu-222, Tyr-225 and Val -302.

3-O-Substituted Quercetin: an Antibiotic-Potentiating Agent against Multidrug-Resistant Gram-Negative Enterobacteriaceae through Simultaneous Inhibition of Efflux Pump and Broad-Spectrum Carbapenemases.

The discovery of safe and efficient inhibitors against efflux pumps as well as metallo-β-lactamases (MBL) is one of the main challenges in the development of multidrug-resistant (MDR) reversal agents which can be utilized in the treatment of carbapenem-resistant Gram-negative bacteria. In this study, we have identified that introduction of an ethylene-linked sterically demanding group at the 3-OH position of the previously reported MDR reversal agent di-F-Q endows the resulting compounds with hereto unknown multitarget inhibitory activity against both efflux pumps and broad-spectrum β-lactamases including difficult-to-inhibit MBLs. A molecular docking study of the multitarget inhibitors against efflux pump, as well as various classes of β-lactamases, revealed that the 3-O-alkyl substituents occupy the novel binding sites in efflux pumps as well as carbapenemases. Not surprisingly, the multitarget inhibitors rescued the antibiotic activity of a carbapenem antibiotic, meropenem (MEM), in NDM-1 (New Delhi Metallo-β-lactamase-1)-producing carbapenem-resistant Enterobacteriaceae (CRE), and they reduced MICs of MEM more than four-fold (synergistic effect) in 8-9 out of 14 clinical strains. The antibiotic-potentiating activity of the multitarget inhibitors was also demonstrated in CRE-infected mouse model. Taken together, these results suggest that combining inhibitory activity against two critical targets in MDR Gram-negative bacteria, efflux pumps, and β-lactamases, in one molecule is possible, and the multitarget inhibitors may provide new avenues for the discovery of safe and efficient MDR reversal agents.

Phytochemical Evaluation of Terminalia canescens DC. Radlk. Extracts with Antibacterial and Antibiotic Potentiation Activities against Selected β-Lactam Drug-Resistant Bacteria.

Terminalia canescens DC. Radlk. (family: Combretaceae) is native to northern Australia. Species of the genus Terminalia are widely used as traditional medicines to treat diverse ailments, including bacterial infections. However, we were unable to find any studies that had examined the antimicrobial activity of T. canescens. In this study, T. canescens was screened against a panel of bacterial pathogens, including multi-antibiotic-resistant strains. Solvents with different polarities were used to extract different complements of phytochemicals from T. canescens leaves. Methanolic and aqueous extracts exhibited substantial antimicrobial activity against various pathogens, including those that are multidrug-resistant strains. When combined with some selected clinical antibiotics, some extracts potentiated the antibacterial inhibitory activity. This study identified two synergistic, eleven additive, eleven non-interactive and eight antagonistic interactions. The toxicities of the plant extracts were examined in the Artemia franciscana nauplii assay and were found to be non-toxic, except the aqueous extract, which showed toxicity. Metabolomic liquid chromatography-mass spectrometry (LC-MS) analyses highlighted and identified several flavonoids, including vitexin, quercetin, orientin and kaempferol, as well as the tannins ellagic acid and pyrogallol, which may contribute to the antibacterial activities observed herein. The possible mechanism of action of these extracts was further explored in this study.

Antibacterial activity and anxiolytic-like effect of Ziziphus joazeiro Mart. leaves in adult zebrafish (Danio rerio)

Ziziphus joazeiro Mart. is an endemic plant of the Caatinga that presents a great socioeconomic importance for the Northeast and Semiarid Region of Brazil. In view of this, this study aimed to evaluate the antibacterial activity and anxiolytic-like effects of Ziziphus joazeiro Mart leaves in adult zebrafish (Danio rerio). The characterization of the main classes of metabolites was performed through chemical reactions. The antibacterial and antibiotic potentiating activity was evaluated by broth microdilution assays. The 96 h acute toxicity, open field test and anxiety models test was evaluated in vivo on adult zebrafish. The results obtained in the phytochemical prospection evidenced the presence of flobabenic tannins, leucoanthocyanidins, flavonois, flavonones, catechins, alkaloids, steroids, and triterpenoids. EEFZJ did not show antibacterial activity for all microorganism tested (MIC ≥ 1024 µg/mL), but reduced the concentration required for bacterial growth inhibition in combination with gentamicin and norfloxacin against multidrug-resistant strains of S. aureus (SA10) and E. coli (EC06), exhibiting synergistic effect with these antibiotics (p<0.0001). In the tests in vivo, EEFZJ was found to be nontoxic, performing reduced locomotor activity and demonstrated an anxiolytic-like effect in adult zebrafish via GABAergic and Serotoninergic systems (5-HT1, 5-HT2A/2C and 5-HT3A/3B).

Antibiotic-Potentiating Activity of the Schinus terebinthifolius Raddi Essential Oil against MDR Bacterial Strains.

Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus are the primary bacteria that cause clinical infections, such as urinary and intestinal infections, pneumonia, endocarditis, and sepsis. Bacterial resistance is an innate natural occurrence in microorganisms, resulting from mutations or the lateral exchange of genetic material. This serves as evidence for the association between drug consumption and pathogen resistance. Evidence has demonstrated that the association between conventional antibiotics and natural products is a promising pharmacological strategy to overcome resistance mechanisms. Considering the large body of research demonstrating the significant antimicrobial activities of Schinus terebinthifolius Raddi, the present study aimed to evaluate the chemical composition and antibiotic-enhancing effects of Schinus terebinthifolius Raddi essential oil (STEO) against the standard and multidrug-resistant strains of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The STEO was extracted by hydrodistillation using a Clevenger-type vacuum rotary evaporator. The Minimum Inhibitory Concentration (MIC) of the STEO was assessed by the microdilution method to evaluate the antibacterial activity. The antibiotic-enhancing activity of the essential oil was assessed by determining the MIC of antibiotics in the presence of a sub-inhibitory concentration (MIC/8) of the natural product. The GC-MS analysis revealed alpha-pinene (24.3%), gamma-muurolene (16.6%), and myrcene (13.7%) as major constituents of the STEO. The STEO potentiated the enhanced antibacterial activity of norfloxacin and gentamicin against all the strains and increased the action of penicillin against the Gram-negative strains. Therefore, it is concluded that although the STEO does not exhibit clinically effective antibacterial activity, its association with conventional antibiotics results in enhanced antibiotic activity.

Anti-Klebsiella and antibiotic-potentiation activities of the methanol extracts of seven Cameroonian dietary plants against multidrug-resistant phenotypes over-expressing AcrAB-TolC efflux pumps

Background: Bacterial infections continue to wreak havoc around the world with high death rates. African medicinal plants and the phytoconstituents showed high efficiency in impeding the growth of the resistance phenotypes of bacteria The present work was designed to determine the antibacterial activity of seven Cameroonian dietary plants against clinical MDR isolated of Klebsiella sp. These plants included Persea americana Miller (Lauraceae), Psidium guajava Linn. (Myrtaceae), Mangifera indica Linn. (Anacardiaceae), Citrus sinensis Linn. (Rutaceae), Passiflora edulis Sims (Passifloraceae), Garcinia kola Heckel (Guttiferae), and Artocarpus heterophylus Lam. (Moraceae). Methods: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations on the used bacterial strains alone, in the presence of an efflux pump inhibitor, phenylalanine arginine beta naphthylamide (PAβN), and in combination with antibiotics, were performed by the microbroth dilution method using a rapid colorimetric para-Iodonitrotetrazolium chloride (INT) assay. Results: The tested botanicals had different extend of antibacterial activities, with MIC ranges of between 256 μg/mL and 2048 μg/mL. Botanicals from Persea americana, Psidium guajava, Mangifera indica, Artocarpus heterophyllus, and Garcinia kola bark had detected MIC values on all 15 tested Klebsiella strains. PAβN potentiated the activity of the botanicals on all tested bacteria, with the increase of activity ranging from 4 to more than 128-fold. The most significant increase of 4 to more than 128-fold was observed with botanicals from leaves and bark of Psidium guajava and Mangifera indica. The botanicals from the leaves of Mangifera indica potentiated the activity of eight out of ten tested antibiotics (Ceftriaxone, Chloramphenicol, Levofloxacin, Ampicillin, Tetracycline, Imipenem, Doxycycline, and Levofloxacin) against 100% of the tested bacteria. Conclusion: In the present study, it was demonstrated that botanicals from Persea Americana, Psidium guajava, Mangifera indica, Artocarpus heterophyllus, and Garcinia kola had the highest spectrum of activity, and can be used to combat the resistance of Klebsiella species

Anti-inflammatory effect, antibiotic potentiating activity against multidrug-resistant strains of Escherichia coli and Staphylococcus aureus, and evaluation of antibiotic resistance mechanisms by the ibuprofen derivative methyl 2-(-4-isobutylphenyl)propanoate.

The prevalence of multidrug-resistant (MDR) bacteria and the limited efficacy of current available antibiotics cause every year approximately 700000 deaths per year. This study aimed to evaluate the anti-inflammatory effect and antibacterial potential of the ibuprofen derivative Methyl 2-(-4-isobutylphenyl)propanoate (MET-IBU). The molecular structure of MET-IBU was confirmed by Nuclear Magnetic Resonance (NMR) and, Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR-FTIR) spectroscopy. Our in vivo study using adult zebrafish model demonstrated that the ibuprofen derivative MET-IBU also possesses anti-inflammatory effect, and in vitro antibacterial activity assays showed that in the association of ampicillin, norfloxacin, and gentamicin with MET-IBU occurred reduction in the minimum inhibitory concentration (MIC) for MDR bacterial strains of Escherichia coli 06 and Staphylococcus aureus 10, indicating a potentiating in the growth inhibition of these pathogenic bacteria. Regarding the strain of Staphylococcus aureus K2068 (overexpressing mepA gene), a potentiation of ethidium bromide was found in the association with MET-IBU, indicating the action of this compound on the efflux pump mechanism present in this strains. This result corroborates the molecular docking study that indicated a high affinity of the MET-IBU with the MepA efflux pump. It was also noticed an antibiotic potentiating activity in the association MET-IBU with norfloxacin against strains of Staphylococcus aureus 1199B (overexpressing norA gene) when compared to the norfloxacin control. This enhanced antibiotic effect of MET-IBU is associated with a second resistance mechanism, which is due to the modification in the topoisomerase enzyme. These results bring attention to the ibuprofen derivative MET-IBU as possible candidate for the development of new options for the treatment of bacterial infections with protective anti-inflammatory action.

Antibacterial and antibiotic-potentiation activities of the hydro-ethanolic extract and protoberberine alkaloids from the stem bark of Enantia chlorantha against multidrug-resistant bacteria expressing active efflux pumps

Ethnopharmacological relevanceEnantia chlorantha is traditionally used to treat various ailments including rickettsia fever, cough and wounds, typhoid fever, infective hepatitis, jaundice, and urinary tract infections. Aim of the studyTo isolate the antibacterial constituents of the hydro-ethanolic extract of the stem bark of E. chlorantha (ECB) and to evaluate the antibacterial and antibiotic-modifying activities of ECB and its constituents against the multidrug-resistant (MDR) phenotypes. Materials and methodsChromatographic methods were used to isolate the constituents of ECB and Spectroscopic methods were used to elucidate the chemical structures of the isolated compounds. The antibacterial activity of samples was determined by the broth microdilution method while spectrophotometric methods were used to evaluate the effects of ECB and its most active constituent on bacterial growth. Their effects on bacterial proton-ATPase pumps was assessed through the acidification of the bacterial culture medium. ResultsSix protoberberine alkaloids were isolated and identified as columbamine (1), pseudocolumbamine (2), jathrorrhizine (3), palmitine (4), 4,13-dihydroxy-3,9,10-trimethoxyprotoberberine (5), and 13-hydroxy-2,3,9,10-tetramethoxyprotoberberine (6). The crude extract (ECB) inhibited the growth of all the tested MDR bacteria, with the minimal inhibitory concentration (MIC) values below 100 μg/mL obtained against Escherichia coli ATCC 10536, AG 102, Enterobacter aerogenes EA 27, Klebsiella pneumoniae ATCC 11296 and KP 55, Providencia stuartii NEA 16, and Staphylococcus aureus MRSA3 and MRSA6. Compound 1 had the best antibacterial effects with MIC values ranging from 16 to 64 μg/mL. The efflux pump inhibitor (EPI), phenylalanine-arginine-β naphthylamide (PAβN) significantly improved the activity of compounds 1–6. Compounds 1–3 significantly potentiated the antibacterial activity of antibiotics such norfloxacin (NOR), ciprofloxacin (CIP), and doxycycline (DOX) against the tested MDR bacteria. ConclusionThe crude extract (ECB) and its isolated compounds 1–6 are potential antibacterial products from Enantia chlorantha. They could be explored more to develop the antibacterial agents that could be used alone or in combination with antibiotics to overcome MDR phenotypes.

Isolation and Characterization of Werneria Chromene and Dihydroxyacidissimol from Burkillanthus malaccensis (Ridl.) Swingle

The secondary metabolites of endemic plants from the Rutaceae family, such as Burkillanthus malaccensis (Ridl.) Swingle from the rainforest of Malaysia, has not been studied. Burkillanthus malaccensis (Ridl.) Swingle may produce antibacterial and antibiotic-potentiating secondary metabolites. Hexane, chloroform, and methanol extracts of leaves, bark, wood, pericarps, and endocarps were tested against bacteria by broth microdilution assay and their antibiotic-potentiating activities. Chromatographic separations of hexane extracts of seeds were conducted to investigate effective phytochemicals and their antibacterial activities. Molecular docking studies of werneria chromene and dihydroxyacidissiminol against SARS-CoV-2 virus infection were conducted using AutoDock Vina. The methanol extract of bark inhibited the growth of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa with the minimum inhibitory concentration of 250, 500, and 250 µg/mL, respectively. The chloroform extract of endocarps potentiated the activity of imipenem against imipenem-resistant Acinetobacter baumannii. The hexane extract of seeds increased the sensitivity of P. aeruginosa against ciprofloxacin and levofloxacin. The hexane extract of seeds and chloroform extract of endocarps were chromatographed, yielding werneria chromene and dihydroxyacidissiminol. Werneria chromene was bacteriostatic for P. aeruginosa and P. putida, with MIC/MBC values of 1000 > 1000 µg/mL. Dihydroxyacidissiminol showed the predicted binding energies of −8.1, −7.6, −7.0, and −7.5 kcal/mol with cathepsin L, nsp13 helicase, SARS-CoV-2 main protease, and SARS-CoV-2 spike protein receptor-binding domain S-RBD. Burkillanthus malaccensis (Ridl.) Swingle can be a potential source of natural products with antibiotic-potentiating activity and that are anti-SARS-CoV-2.

Modulating antibacterial activity against multidrugresistant Escherichia coli and Staphylococcus aureus of the flavonoid pectolinarin isolated from Lantana camara leaves

The antibiotic potentiating activity against standard and multidrug-resistant Escherichia coli and Staphylococcus aureus bacterial strains of the natural compound 5”-O-α-L-raminopyranosyl-1’-β-D-glycopyranosyl-4’,6-dimethoxyflavone (pectolinarin) isolated from L. camara leaves was evaluated. Tests for antibacterial activity of the pure natural substance and analysis of the potentiation of antibacterial activity of pectolinarin associated with antibiotics were carried out against standard and multiresistant bacterial strains of Escherichia coli and Staphylococcus aureus by microdilution. Pectolinarin, when combined with the antibiotic gentamicin, showed synergism, potentiating growth inhibition against Gram-positive S. aureus strains. The pectolinarin flavonoid when combined with the gentamicin antibiotic potentiated its action Gram-positive S. aureus bacteria. Moreover, an antagonistic effect was observed when the pectolinarin was combined with the penicillin antibiotic against the multiresistant S. aureus 358 strain. This research suggests that pectolinarin is a compound with potential application as an antibacterial drug.

Bruguiera gymnorhiza (L.) Lam. at the Forefront of Pharma to Confront Zika Virus and Microbial Infections-An In Vitro and In Silico Perspective.

The recent emergence of Zika virus (ZIKV) in Brazil and the increasing resistance developed by pathogenic bacteria to nearly all existing antibiotics should be taken as a wakeup call for the international authority as this represents a risk for global public health. The lack of antiviral drugs and effective antibiotics on the market triggers the need to search for safe therapeutics from medicinal plants to fight viral and microbial infections. In the present study, we investigated whether a mangrove plant, Bruguiera gymnorhiza (L.) Lam. (B. gymnorhiza) collected in Mauritius, possesses antimicrobial and antibiotic potentiating abilities and exerts anti-ZIKV activity at non-cytotoxic doses. Microorganisms Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70603, methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), Salmonella enteritidis ATCC 13076, Sarcina lutea ATCC 9341, Proteus mirabilis ATCC 25933, Bacillus cereus ATCC 11778 and Candida albicans ATCC 26555 were used to evaluate the antimicrobial properties. Ciprofloxacin, chloramphenicol and streptomycin antibiotics were used for assessing antibiotic potentiating activity. ZIKVMC-MR766NIID (ZIKVGFP) was used for assessing anti-ZIKV activity. In silico docking (Autodock 4) and ADME (SwissADME) analyses were performed on collected data. Antimicrobial results revealed that Bruguiera twig ethyl acetate (BTE) was the most potent extract inhibiting the growth of all nine microbes tested, with minimum inhibitory concentrations ranging from 0.19–0.39 mg/mL. BTE showed partial synergy effects against MRSA and Pseudomonas aeruginosa when applied in combination with streptomycin and ciprofloxacin, respectively. By using a recombinant ZIKV-expressing reporter GFP protein, we identified both Bruguiera root aqueous and Bruguiera fruit aqueous extracts as potent inhibitors of ZIKV infection in human epithelial A549 cells. The mechanisms by which such extracts prevented ZIKV infection are linked to the inability of the virus to bind to the host cell surface. In silico docking showed that ZIKV E protein, which is involved in cell receptor binding, could be a target for cryptochlorogenic acid, a chemical compound identified in B. gymnorhiza. From ADME results, cryptochlorogenic acid is predicted to be not orally bioavailable because it is too polar. Scientific data collected in this present work can open a new avenue for the development of potential inhibitors from B. gymnorhiza to fight ZIKV and microbial infections in the future.

Phytochemical characterization and antibiotic potentiating effects of the essential oil of Aloysia gratissima (Gillies & Hook.) and beta-caryophyllene

The traditional knowledge associated with the use of medicinal plants has significantly contributed to drug development research. In this context, Aloysia gratissima, commonly known as bee-brush or white-brush, has been widely used in Brazilian folk medicine as an analgesic and antimicrobial species. Phytochemical analysis of A. gratissima essential oil has identified sesquiterpene β-caryophyllene (17.3%) as a major component with significant antibacterial activity. Thus, this study aimed to characterize the antibacterial effects of the essential oil obtained from A. gratissima (EOAG) and its major compound β-caryophyllene, as well as to evaluate their potential to act as efflux pumps inhibitors in association with conventional antibiotics. The phytochemical characterization of EOAG was performed by gas chromatography coupled to mass spectrometry (GC-MS), while the antibacterial activity against the strains Pseudomonas aeruginosa 24, Staphylococcus aureus 10, and Escherichia coli 06 was assessed using the broth microdilution method. The antibiotic potentiating activity was investigated by analyzing the ability of the natural products to decrease the minimum inhibitory concentration (MIC) of norfloxacin and ciprofloxacin against S. aureus strains 1199B and K2068. A reduction in the MIC of ethidium bromide in the presence of essential oil components was interpreted as efflux pump inhibition. The GC-MS analysis identified 30 compounds, including β-caryophyllene as the major component. The antibacterial activity analysis demonstrated that both EOAG and β-caryophyllene both EOAG and β-caryophyllene presented antibacterial effects against S. aureus 10 (MIC = 32 μg/mL), in addition to potentiating the activity of norfloxacin against [S. aureus 10], [P. aeruginosa 24], and [E. coli 06]. The isolated compound also reversed the degree of antibiotic resistance observed in strains carrying efflux pumps. In conclusion, the essential oil of A. gratissima and β-caryophyllene have the potential to increase the effectiveness of antibiotics and as such can be used in the development of new therapies against bacterial resistance.

Antibiotic-potentiation activities of three animal species extracts, Bitis arietans, Helix aspersa, and Aristaeomorpha foliacea and mode of action against MDR Gram-negative bacteria phenotypes

Abstract Background: In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world. As antimicrobial activities of most medicinal plants and antibiotics have been already explored, it is more important to make investigations on animal species mainly invertebrates which could constitute an efficient source of antimicrobial molecules. This work was aimed at contributing to the fight against microbial resistance through the study of antibacterial potential of three animal species (Helix aspersa, Bitis arietans, Aristaeomorpha foliacea) on several multidrug-resistant (MDR) Gram-negative strains overexpressing efflux pumps including Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa. Methods: The microdilution technique was used to evaluate the antibacterial activities of the tested samples by determining their minimal inhibitory concentrations (MICs), as well as the effect of their combination with antibiotics. Studies on the mechanisms of action of the most active sample, dried Bitis arietans extract, was carried out using standard methods for evaluating the effects of this extract on bacterial H+-ATPases-mediated proton pumps and on bacterial growth kinetics. In this latter case, the optical density was read spectrophotometrically. Results: Zoochemical screening indicated the presence of protein constituents and alkaloids and the absence of other metabolites in all tested extracts. Dried B. arietans showed the best antibacterial activity by inhibiting the growth of 90% of studied bacterial strains with MICs ranging from 128 to 2048 μg/ml. Moreover, this extract presented a significant activity (100≤MIC≤512 µg/ml) against 35% of bacteria that are E. coli (ATCC8739, AG100ATet, MC4100), E. aerogenes EA27, K. pneumoniae ATCC11296, P. aeruginosa (PA01, PA124) and a moderate activity (512&lt;MIC≤2048 µg/ml) against 55% of studied bacteria. It was followed by fresh B. arietans which inhibited the growth of 65% of bacteria with significant activity on three bacteria (E. coli ATCC8739, E. aerogenes ATCC13048 and K. pneumoniae ATCC11296. These two extracts showed bactericidal effects on many strains. The other extracts samples selectively exhibited an antibacterial activity against less than 40% of strains. All samples potentiated the activity of at least 56% of used antibiotics against at least 70% of studied bacterial strains. B. arietans extracts at MIC/2 and MIC/4 mostly improved the activities of more than 78% of antibiotics on at least 70% of bacteria with improvement activity factors (IAF) ranging from 2 – 128 suggesting that this animal contains bioactive compounds which could act as efflux pumps inhibitors. Bacterial growth kinetic study showed that when treated with dried B. arietans extract (the most active sample) at different concentrations MIC/2, MIC and 2xMIC, the growth of tested bacteria (E. coli ATCC8739) decreased respectively when the concentrations increased. Furthermore, this extract inhibited the H+-ATPase-mediated proton pumps of this bacterium increasing the pH values. Conclusion: Results obtained in the present work provide interesting data for the use of dried B. arietans extract and invertebrates in general in the traditional therapy for the treatment of bacterial infections involving multidrug-resistant phenotypes. Keywords: Gram-negative bacteria; multidrug resistance; efflux pumps; infectious diseases; animal species; secondary metabolites.

In vitro antibacterial and antibiotic-potentiation activities of five edible plant extracts and mode of action against several MDR Gram-negative phenotypes

Abstract Background: Bacterial infections involving the multidrug resistant (MDR) strains are among the top leading causes of death throughout the world. Healthcare system across the globe has been suffering from an extra-ordinary burden in terms of looking for the new and more potent antimicrobial compounds. The aim of the present study was to determine the antibacterial activity of some Cameroonian edible plants (Garcinia lucida bark, Phoenix dactylifera pericarps, Theobroma cacao pod, Solanum macrocarpon leaves and Termitomyces titanicus whole plant) and their antibiotics-potentiation effects against some MDR Gram-negative bacteria phenotypes expressing efflux pumps (Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Providencia stuartii strains). Methods: The antibacterial activities of plant extract alone and in combination with usual antibiotics were carried out using the micro-dilution method. The effects of the most active plant extract (Garcinia lucida bark) on H+-ATPase-mediated proton pumps and on bacterial growth kinetic were performed using experimental protocols, while qualitative reference methods were used to highligh the major groups of secondary metabolites present in the extracts. Results: Qualitative phytochemical screening of plant extracts indicated that all analysed secondary metabolites were present in Theobroma cacao and Termitomyces titanicus while one (saponins) of them was absent in Garcinia lucida and Solanum macrocarpon. Only three of them (polyphenols, flavonoids and saponins) were detected in Phoenix dactylifera. Antibacterial essays showed that G. lucida was the most active plant as it inhibited the growth of all studied bacteria with strong activity (MIC&lt;100 µg/mL) against E. coli ATCC8739, significant activity (100≤MIC≤512 µg/mL) against 80% of bacteria and moderate activity (512&lt;MIC≤2048 µg/mL) against E. coli AG100A and E. aerogenes (EA289 and CM64). It was followed by T. cacao and S. macrocarpon extracts which exhibited an antibacterial potential against 95% and 80% of bacterial strains, respectively. These three extracts exhibited a bactericidal effect on a few bacteria. Extracts from T. titanicus and P. dactylifera were less active as they moderately (512&lt;MIC≤2048 µg/mL) inhibited the growth of 35% and 10% of bacteria. All extracts selectively potentiated the activities of all antibiotics with improvement activity factors (IAF) ranging from 2 to 256. G. lucida, T. cacao and S. macrocarpon potentiated the activities of 100%, 89% and 67% of antibiotics respectively against more than 70%, suggesting that they contain bioactive compounds which could be considered as efflux pumps inhibitors. Whereas T. titanicus and P. dactylifera improved the activities of almost 40% and 20% of antibiotics, respectively. This increase of activities also characterizes synergistic effects between antibiotics and these bioactive compounds. G. lucida extract at all tested concentrations, strongly inhibited the growth of bacterial strain E. coli ATCC8739 and exhibited an inhibitory effect on this bacterial H+-ATPase-mediated proton pumps increasing the pH of the medium. Conclusion: The overall results indicated that food plants among which G. lucida, T. cacao and S. macrocarpon could have a benefit interest in combatting resistant types of bacteria. Keywords: Food plants; infectious diseases; MDR bacteria; efflux pumps; antibiotics; secondary metabolites.

Structural characterization, DFT calculations, ADMET studies, antibiotic potentiating activity, evaluation of efflux pump inhibition and molecular docking of chalcone (E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one

Chalcones are open-chain flavonoids characterized by two aromatic rings joined by a three-carbon α,β-unsaturated carbonyl system. Over the last several years, chalcones have instigated the interest of chemical and pharmacological researchers due to their simple chemical structure and varied biological activities. Here, we performed the electronic properties, of the chalcone, (E)-1-(2-hydroxy-3,4,6-trimethoxyphenyl)-3-(4-methoxyphenyl) prop-2-en-1-one synthesized by Claisen-Schmidt condensation reaction. The density functional theory method was used with the B3LYP/6-311++G(d,p) level of theory to compute the structural, electronic, and reactivity properties of the chalcone. In addition, microbiological tests were performed to investigate the modulator potential and efflux pump inhibition on Staphylococcus aureus multi-resistant strains. The spectroscopic data analyses allowed drawing the molecular structure of the chalcone synthetized with subsequent confirmation using the quantum chemical calculations. The addition of chalcone to the growth medium caused a synergic effect by reduction of the minimum inhibitory concentration (MIC) values for ciprofloxacin strain. Docking results showed that the chalcone docks in almost the same way as the antibiotic against a MepA model. Druglikeness criteria based on the rules of Lipinski and Veber evaluated that the chalcone has the ideal physicochemical and pharmacokinetic properties to be a good candidate for drug orally. The results confirm the structure of the synthesized chalcone and revealing that the compound can be used as a possible inhibitor of the Mep A efflux pump.

Seasonality Effects on Antibacterial and Antibiotic Potentiating Activityagainst Multidrug-Resistant Strains of Escherichia coli and Staphylococcus aureus and ATR-FTIR Spectra of Essential Oils from Vitex gardneriana Leaves.

Plants are natural sources of several bioactive substances, which have been found in extracts, secondary metabolites, and essential oils. Several biological activities have been attributed to essential oils as antiviral, insecticidal, antiparasitic, antioxidant, and antimicrobial. The indiscriminate use of antibiotics has increased the development of resistance mechanisms of microorganisms. Thus, search for efficient natural compounds with antimicrobial activity and low toxicity has increased, so essential oils have been a promising alternative for combating microbial infections. This study was carried out to investigate the seasonality effects on the infrared absorbance spectra, antibacterial activity, and antibiotic potentiating activity of essential oils from Vitex gardneriana leaves. Essential oils were extracted from V. gardneriana Schauer leaves the seasonal period from January to December 2016 and characterized by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The antibacterial effect of these oils and antibiotic potentiating activity, both determined by the minimum inhibitory concentration, were assessed using microtiter plates. For the first time, we present the use of infrared absorbance spectra of these essential oils and show the influence of seasonality on them. Synergistic effects were observed for the essential oils associated with the antibiotics tested (gentamicin, ampicillin, and ofloxacin). The main influence of seasonality on the infrared absorbance spectra of the essential oils of the V. gardneriana occurred in the June month (last month of the rainy season). In regard to antibacterial activity test, the essential oils of the V. gardneriana leaves did not show a direct effect on the strains tested. However, the essential oils when associated with the antibiotics showed variations in the minimum inhibitory concentration with the months of the seasonal period, indicating synergistic effects against Escherichia coli and Staphylococcus aureus bacterial resistance.

Strain-specific anti-biofilm and antibiotic-potentiating activity of 3\u2032,4\u2032-difluoroquercetin

Antibacterial properties of 3′,4′-difluoroquercetin (di-F-Q), a fluorine-substituted stable quercetin derivative, were investigated. Even though di-F-Q itself did not show interesting antibacterial activity, treatment of the Staphylococcus aureus strains with di-F-Q resulted in a dose-dependent reduction in biofilm formation with IC50 values of 1.8 ~ 5.3 mg/L. Also, the antibacterial activity of ceftazidime (CAZ) against carbapenem-resistant Pseudomonas aeruginosa (CRPA) showed eightfold decrease upon combination with di-F-Q. Assessment of the antimicrobial activity of CAZ in combination with di-F-Q against 50 clinical isolates of P. aeruginosa confirmed 15.7% increase in the percentages of susceptible P. aeruginosa isolates upon addition of di-F-Q to CAZ. Further mechanistic studies revealed that di-F-Q affected the antibiotics efflux system in CRPA but not the β-lactamase activity. Thus, di-F-Q was almost equally effective as carbonyl cyanide m-chlorophenyl hydrazine in inhibiting antibiotic efflux by P. aeruginosa. In vivo evaluation of the therapeutic efficacy of CAZ-(di-F-Q) combination against P. aeruginosa showed 20% of the mice treated with CAZ-(di-F-Q) survived after 7 days in IMP carbapenemase-producing multidrug-resistant P. aeruginosa infection group while no mice treated with CAZ alone survived after 2 days. Taken together, di-F-Q demonstrated unique strain-specific antimicrobial properties including anti-biofilm and antibiotic-potentiating activity against S. aureus and P. aeruginosa, respectively.

Synergism of sophoraflavanone G with norfloxacin against effluxing antibiotic-resistant Staphylococcus aureus

Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), remains a challenge in hospital and community settings. The design and discovery of new compounds to deal with resistant bacteria has become one of the most important areas of anti-infective research today. The aim of this study was to address the problem of MRSA by searching for synergistic natural antibacterial products from traditional Chinese herbs that are not substrates for the efflux mechanisms of MRSA and that overcome bacterial drug resistance by other, as yet undescribed, mechanisms. In vitro synergistic activity was determined using the standard chequerboard method, and mechanistic studies were performed by an ethidium bromide efflux assay. Using in vivo experiments, the efficacies of different concentrations of the combinations were compared in a murine model of pyaemia. The natural product sophoraflavanone G showed specific synergistic antibacterial effects both in vitro and in vivo and may serve as a template for agents with antibiotic-potentiating activity for use against infections caused by S. aureus, including MRSA.

Bioautographic identification of alder gray Alnus incana (L.) Moench. fruits and geranium meadow Geranium pratense L. rhizomes extracts active components exhibiting synergism of antimicrobial action with macrolides

Annotation. The increase in the number of isolated antibiotic-resistant strains of microorganisms stimulates the search for alternative sources of biologically active substances that have antibiotic-potentiating action and are able to block the mechanisms of bacterial resistance to antibiotics. The aim of the study was to carry out bioautographic identification of the active components of gray alder fruits, geranium meadow rhizomes extracts and preparation “Altan” (extractant 90% aqueous-ethanolic), which showed synergism of antimicrobial action with erythromycin against skin isolates of staphylococci with different MLS phenotypes. Paper chromatography, thin-layer chromatography and bioautography were performed. Statistical processing of results: UTHSCSA ImageTool 2.0, Microsoft Office Excel 2011 On bioautograms of gray alder fruits extract and Altan with subinhibitory concentrations of erythromycin inoculated with S. epidermidis culture (inductive MLS-resistance phenotype), near the start line, areas with clear edges of growth inhibition were detected indicating the presence of hydrolysable tannins. Bioautograms of geranium meadow rhizomes extract showed the presence of compounds with antibiotic-potentiating activity with pronounced hydrophobic properties, indicating the presence of essential oils. In the extracts of gray alder fruits and geranium meadow rhizomes, the active components with antibiotic-potentiating properties, namely hydrolysable tannins and essential oils, have been identified. The fruits of gray alder and geranium meadow rhizomes are recommended for their preclinical and in-depth pharmacological study.

Constituents of Two Dioscorea Species That Potentiate Antibiotic Activity against MRSA.

The isolation of two diarylnonanoids from Dioscorea cotinifolia possessing antibiotic-potentiating activity against resistant strains of S.aureus are reported. The diarylnonanoids are a class of natural products similar in structure to the diarylheptanoids, which have a wide spectrum of reported biological activities. One of the diarylnonanoids (1) isolated possesses a chiral center, and to deduce its configuration, the modified Mosher ester method was used. Using both 1D and 2D NMR data, as many protons as possible were assigned to both the R- and S-MTPA esters, and the configuration of the chiral center in 1 was determined to be R. Both the chiral and achiral diarylnonanoid (2) exhibited potent antibiotic-potentiating activity with the chiral natural product showing a greater tetracycline-potentiating activity than 2. Interestingly, 2 gave a higher norfloxacin-potentiating activity with a resultant higher efflux pump inhibitory activity. Manipulation of the structure of the diarylnonanoids through synthesis could lead to improved biological activity.