New Insight into the Chemical Composition,Antimicrobial and Synergistic Effects of the Moroccan Endemic Thymus atlanticus (Ball) Roussine Essential Oil in Combination with Conventional Antibiotics

This study reported the volatile profile, the antimicrobial activity and the synergistic potential of essential oil (EO) from the Moroccan endemic Thymus atlanticus (Ball) Roussine, in combination with the antibiotics ciprofloxacin and fluconazole for the first time, to the best of our knowledge. The EO chemical composition was determined by gas chromatography coupled to mass spectrometry (GC-MS) analysis and the antimicrobial activity assessed by the disc diffusion method against three Gram positive (Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus) and three Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli and one clinical isolate, Klebsiella pneumonia). The antifungal activity was evaluated in four pathogenic yeasts (Candida albicans, C. glabrata, C. krusei and C. parapsilosis). The minimum inhibition concentration (MIC) and the synergistic effect with ciprofloxacin and fluconazole were determined by the two-fold dilution technique and checkerboard test, respectively. Twenty-one constituents were identified by GC-MS in the EO, including carvacrol (21.62%) and borneol (21.13%) as the major components. The EO exhibited a significant antimicrobial activity with inhibition zones ranging from 0.7 mm to 22 mm for P. aeruginosa and B. subtilis, respectively, and MIC values varying from 0.56 mg/mL to 4.47 mg/mL. The fractional inhibitory concentration index (FICI) values ranged from 0.25 to 0.50 for bacteria and from 0.25 to 0.28 for yeasts. The maximum synergistic effect was observed for K. pneumonia with a 256-fold gain of antibiotic MIC. Our results have suggested that EO from T. atlanticus may be used alone or in association with antibiotics as a new potential alternative to prevent and control the emergence of resistant microbial strains both in the medical field and in the food industry.     

Theoretical analysis of bacterial efflux pumps inhibitors: Strategies in-search of competent molecules and develop next

Multi-drug resistance (MDR) bacteria pose a significant threat to our ability to effectively treat infections due to the development of several antibiotic resistant mechanisms. A major component in the development of the MDR phenotype in MDR bacteria is over expression of different-type of efflux pumps, which actively pump out antibacterial agents and biocides from the periplasm to the outside of the cell. Consequently, bacterial efflux pumps are an important target for developing novel antibacterial treatments. Potent efflux pump inhibitors (EPIs) could be used as adjunctive therapies that would increase the potency of existing antibiotics and decrease the emergence of MDR bacteria. Several potent inhibitors of efflux pumps have been reported which has been summarized here. All the natural and synthetic EPIs were optimized with Gaussian and Avogadro software. The optimized structures were docked with each class of efflux pumps and their bonding parameters were computed. The theoretical analyses were performed with density functional theory (DFT). Overall, computational study revealed a good trend of electrophilicity and ionization potential of the EPIs, the obtained average values are within in the range of 0.001414 AU ± 0.00032 and 0.208821 AU ± 0.015545, respectively. Interestingly, cathinone interacts with most of the efflux pumps among the tested inhibitors. The electrophilicity and ionization potential of cathinone are 0.00198 and 0.2388 AU, respectively. The study opens a new road for designing future-generation target-specific efflux pump inhibitors, as well as one molecule with multiple inhibition abilities.     

Pharmacophore generation of 2-substituted benzothiazoles as AdeABC efflux pump inhibitors in A. baumannii

RND family efflux pumps are important for multidrug resistance in Gram-negative bacteria. To date no efflux pump inhibitors for clinical use have been found, so developing the specific inhibitors of this pump system will be beneficial for the treatment of infections caused by these multidrug-resistant pathogens. A set of BSN-coded 2-substituted benzothiazoles were tested alone and in combination with ciprofloxacin (CIP) against the RND family efflux pump AdeABC overexpressor Acinetobacter baumannii SbMox-2 strain. The results indicated that the BSN compounds did not have antimicrobial activity when tested alone. However, if they were applied in combination with CIP, it was observed that the antibiotic had antimicrobial activity against the tested pathogen, possessing a minimum inhibitory concentration value that could be utilized in clinical treatment. A 3D-common features pharmacophore model was applied by using the HipHop method and the generated pharmacophore hypothesis revealed that the hydrogen bond acceptor property of nitrogen in the thiazole ring and the oxygen of the amide substituted at the second position of the benzothiazole ring system were significant for binding to the target protein. Moreover, three hydrophobic aromatic features were found to be essential for inhibitory activity.     

Quinazoline Derivatives Designed as Efflux Pump Inhibitors: Molecular Modeling and Spectroscopic Studies

Multidrug resistance of bacteria is a worrying concern in the therapeutic field and an alternative method to combat it is designing new efflux pump inhibitors (EPIs). This article presents a molecular study of two quinazoline derivatives, labelled BG1189 and BG1190, proposed as EPIs. In silico approach investigates the pharmacodynamic and pharmacokinetic profile of BG1189 and BG1190 quinazolines. Molecular docking and predicted ADMET features suggest that BG1189 and BG1190 may represent attractive candidates as antimicrobial drugs. UV-Vis absorption spectroscopy was employed to study the time stability of quinazoline solutions in water or in dimethyl sulfoxide (DMSO), in constant environmental conditions, and to determine the influence of usual storage temperature, normal room lighting and laser radiation (photostability) on samples stability. The effects of irradiation on BG1189 and BG1190 molecules were also assessed through Fourier-transform infrared (FTIR) spectroscopy. FTIR spectra showed that laser radiation breaks some chemical bonds affecting the substituents and the quinazoline radical of the compounds.     

Computational Insights and In Vitro Validation of Antibacterial Potential of Shikimate Pathway-Derived Phenolic Acids as NorA Efflux Pump Inhibitors

The expression of the efflux pump systems is the most important mechanism of antibiotic resistance in bacteria, as it contributes to reduced concentration and the subsequent inactivity of administered antibiotics. NorA is one of the most studied antibacterial targets used as a model for efflux-mediated resistance. The present study evaluated shikimate pathway-derived phenolic acids against NorA (PDB ID: 1PW4) as a druggable target in antibacterial therapy using in silico modelling and in vitro methods. Of the 22 compounds evaluated, sinapic acid (−9.0 kcal/mol) and p-coumaric acid (−6.3 kcal/mol) had the best and most prominent affinity for NorA relative to ciprofloxacin, a reference standard (−4.9 kcal/mol). A further probe into the structural stability and flexibility of the resulting NorA-phenolic acids complexes through molecular dynamic simulations over a 100 ns period revealed p-coumaric acid as the best inhibitor of NorA relative to the reference standard. In addition, both phenolic acids formed H-bonds with TYR 76, a crucial residue implicated in NorA efflux pump inhibition. Furthermore, the phenolic acids demonstrated favourable drug likeliness and conformed to Lipinski’s rule of five for ADME properties. For the in vitro evaluation, the phenolic acids had MIC values in the range 31.2 to 62.5 μg/mL against S. aureus, and E. coli, and there was an overall reduction in MIC following their combination with ciprofloxacin. Taken together, the findings from both the in silico and in vitro evaluations in this study have demonstrated high affinity of p-coumaric acid towards NorA and could be suggestive of its exploration as a novel NorA efflux pump inhibitor.     

Photodamage to Multidrug-resistant Gram-positive and Gram-negative Bacteria by 870 nm/930 nm Light Potentiates Erythromycin,Tetracycline and Ciprofloxacin

We have previously shown that 870 nm/930 nm wavelengths cause photodamage at physiologic temperatures in methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli via generation of endogenous radical oxygen species (ROS) and decreased plasma membrane potentials (ΔΨp). We tested MRSA (Strain HSJ216) in vitro with sublethal 870 nm/930 nm laser energy and subinhibitory concentrations of erythromycin, tetracycline, penicillin, rifampin and trimethoprim to surmise whether photodamage could potentiate these antimicrobials. We also tested patient isolates of fluoroquinolone-resistant MRSA and E. coli with subinhibitory concentrations of ciprofloxacin. In MRSA (Strain HSJ216) we observed 97% potentiation (a 1.5 log₁₀ CFU decrease) with erythromycin and tetracycline. In patient isolates of E. coli, we observed 100% potentiation (>3 log₁₀ CFU decrease) in all irradiated samples with ciprofloxacin. To assess whether staphyloxanthin pigment conferred protection against the generated ROS, we created an isogenic carotenoid-deficient mutant of S. aureus that was significantly less tolerant of 870 nm/930 nm exposure than the wild type strain (P < 0.0001). We suggest that antibiotic potentiation results from a photobiological attenuation of ATP-dependent macromolecular synthetic pathways, similar to that observed with daptomycin, via disruption of ΔΨp and endogenous generation of ROS. With erythromycin, tetracycline and ciprofloxacin, attenuation of energy-dependent efflux systems is also a possibility.     

Chemical Characterization and Antibacterial Activity of Essential Oil of Medicinal Plants from Eastern Serbia

The objective of this study was to evaluate wild growing Satureja kitaibelii, Thymus serpyllum, Origanum vulgare, Achillea millefolium and Achillea clypeolata with respect to their essential oil (EO) content, composition and antimicrobial activity. The five species were collected at Mt. Rtanj and the village of Sesalac, Eastern Serbia. The main EO constituents of Lamiaceae plants were p-cymene (24.4%), geraniol (63.4%) and germacrene D (21.5%) in Satureja kitaibelii, Thymus serpyllum and Origanum vulgare ssp. vulgare, respectively. A. millefolium EO had multiple constituents with major ones being camphor (9.8%), caryophyllene oxide (6.5%), terpinen-4-ol (6.3%) and 1,8-cineole (5.6%), while the main EO constituents of A. clypeolata were 1,8-cineole (45.1%) and camphor (18.2%). Antimicrobial testing of the EO showed that Staphylococcus aureus (Gram-positive) was more sensitive to all of the tested EOs than Escherichia coli (Gram-negative). S. kitaibelii EO showed the highest antimicrobial activity against both tested bacterial strains. This is the first study to characterize the EO composition and antimicrobial activity of these five medicinal species from Eastern Serbia in comparison with comprehensive literature data. The results can be utilized by the perfumery, cosmetics, food and pharmaceutical industries, but also for healing purposes in self-medication.     

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. ZIKV^MC-MR766NIID (ZIKV^GFP) 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.     

Farnesol, a potential efflux pump inhibitor in Mycobacterium smegmatis

The active multidrug efflux pump (EP) has been described as one of the mechanisms involved in the natural drug resistance of bacteria, such as mycobacteria. As a result, the development of efflux pumps inhibitors (EPIs) is an important topic. In this study, a checkerboard synergy assay indicated that farnesol both decreased the minimum inhibitory concentration (MIC) of ethidium bromide (EtBr) 8-fold against Mycobacterium smegmatis (M. smegmatis) mc2155 ATCC 700084 when incorporated at a concentration of 32 μg/mL (FICI = 0.625) and decreased MIC 4-fold at 16 μg/mL (FICI = 0.375). Farnesol also showed synergism when combined with rifampicin. A real-time 96-well plate fluorometric method was used to assess the ability of farnesol to inhibit EPs in comparison with four positive EPIs: chlorpromazine, reserpine, verapamil, and carbonyl cyanide m-chlorophenylhydrazone (CCCP). Farnesol significantly enhanced the accumulation of EtBr and decreased the efflux of EtBr in M. smegmatis; these results suggest that farnesol acts as an inhibitor of mycobacterial efflux pumps.     

Nutmeg filler as a natural compound for the production of polyurethane composite foams with antibacterial and anti-aging properties

Polyurethane (PU) composite foams were successfully reinforced with different concentrations (1 wt%, 2 wt%, 5 wt%) of nutmeg filler. The effect of nutmeg filler concentration on mechanical, thermal, antimicrobial and anti-aging properties of PU composite foams was investigated. PU foams were examined by rheological behavior, processing parameters, cellular structure (Scanning Electron Microscopy analysis), mechanical properties (compression test, impact test, three-point bending test, impact strength), thermal properties (Thermogravimetric Analysis), viscoelastic behavior (Dynamic Mechanical Analysis) as well as selected application properties (thermal conductivity, flammability, apparent density, dimensional stability, surface hydrophobicity, water absorption, color characteristic). In order to Disc Diffusion Method, all PU composites were tested against selected bacteria (Escherichia coli and Staphylococcus aureus). Based on the results, it can be concluded that the addition of 1 wt% of nutmeg filler leads to PU composite foams with improved compression strength (e.g. improvement by ~19%), higher flexural strength (e.g. increase of ~11%), improved impact strength (e.g. increase of ~32%) and comparable thermal conductivity (0.023–0.034 W m⁻¹ K⁻¹). Moreover, the incorporation of nutmeg filler has a positive effect on the fire resistance of PU materials. For example, the results from the cone calorimeter test showed that the incorporation of 5 wt% of nutmeg filler significantly reduced the peak of heat release rate (pHRR) by ca. 60% compared with that of unmodified PU foam. It has been also proved that nutmeg filler may act as a natural anti-aging compound of PU foams. The incorporation of nutmeg filler in each amount successfully improved the stabilization of PU composite foams. Based on the antibacterial results, it has been shown that the addition of nutmeg filler significantly improved the antibacterial properties of PU composite foams against both Gram-positive and Gram-negative bacteria.     

Pharmacological and Therapeutic Potential of Myristicin: A Literature Review

Natural products have been used by humanity for many centuries to treat various illnesses and with the advancement of technology, it became possible to isolate the substances responsible for the beneficial effects of these products, as well as to understand their mechanisms. In this context, myristicin, a substance of natural origin, has shown several promising activities in a large number of in vitro and in vivo studies carried out. This molecule is found in plants such as nutmeg, parsley, carrots, peppers, and several species endemic to the Asian continent. The purpose of this review article is to discuss data published in the last 10 years at Pubmed, Lilacs and Scielo databases, reporting beneficial effects, toxicity and promising data of myristicin for its future use in medicine. From 94 articles found in the literature, 68 were included. Exclusion criteria took into account articles whose tested extracts did not have myristicin as one of the major compounds.     

Chemical Composition,Antipathogenic and Cytotoxic Activity of the Essential Oil Extracted from Amorpha fruticosa Fruits

The purpose of this paper was to characterize and investigate the antimicrobial potential of Amorpha fruticosa fruits essential oil (EO). The EO was extracted by hydrodistillation, analyzed by GC-MS, and then evaluated for its interaction with microbial and mammalian cells. The antimicrobial activity was assessed against bacterial and fungal strains, in a planktonic and adherent growth state, using qualitative and quantitative assays. The main components identified in A. fruticosa fruits EO were δ-cadinene, γ-muurolene, and α-muurolene. The Gram-positive strains proved to be more susceptible than Gram-negative bacteria and fungal strains. The EO exhibited good antibiofilm activity, inhibiting the microbial adherence to the inert (96-well plates and Foley catheter section) and cellular substrata. The flow cytometry analysis revealed as one of the possible mechanisms of antimicrobial action the alteration of cell membrane hydrophobicity. The cytotoxicity on the L929 cell line occurred at concentrations higher than 0.3 mg/mL. Taken together, our results demonstrate that A. fruticosa fruits EO contains active compounds with selective inhibitory effect on different microbial strains in planktonic and biofilm growth state, explained at least partially by the interference with microbial membranes due to their hydrophobic character.     

Antimicrobial Activity and Synergy Investigation of Hypericum scabrum Essential Oil with Antifungal Drugs

The chemical composition of Lebanese Hypericum scabrum essential oil (EO) was analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GG-MS). Its antimicrobial activity was evaluated by determining its minimal inhibitory concentrations (MICs) against a Gram-negative and a Gram-positive bacterium, one yeast, and five dermatophytes. H. scabrum EO was most active on filamentous fungi (MIC values of 32–64 µg/mL). Synergy within the oil was investigated by testing each of the following major components on Trichophyton rubrum: α-pinene, limonene, myrcene, β-pinene and nonane, as well as a reconstructed EO. The antifungal activity of the natural oil could not be reached, meaning that its activity might be due, in part, to minor constituent(s). The interactions between H. scabrum EO and commercially available antifungals were assessed by the checkerboard test. A synergistic effect was revealed in the combination of the EO with amphotericin B.     

Assessing HDL Metabolism in Subjects with Elevated Levels of HDL Cholesterol and Coronary Artery Disease

High-density lipoprotein cholesterol (HDL-C) is thought to be atheroprotective yet some patients with elevated HDL-C levels develop cardiovascular disease, possibly due to the presence of dysfunctional HDL. We aimed to assess the metabolic fate of circulating HDL particles in patients with high HDL-C with and without coronary artery disease (CAD) using in vivo dual labeling of its cholesterol and protein moieties. We measured HDL apolipoprotein (apo) A-I, apoA-II, free cholesterol (FC), and cholesteryl ester (CE) kinetics using stable isotope-labeled tracers (D₃-leucine and ¹³C₂-acetate) as well as ex vivo cholesterol efflux to HDL in subjects with (n = 6) and without (n = 6) CAD that had HDL-C levels >90th percentile. Healthy controls with HDL-C within the normal range (n = 6) who underwent the same procedures were used as the reference. Subjects with high HDL-C with and without CAD had similar plasma lipid levels and similar apoA-I, apoA-II, HDL FC, and CE pool sizes with no significant differences in fractional clearance rates (FCRs) or production rates (PRs) of these components between groups. Subjects with high HDL-C with and without CAD also had similar basal and cAMP-stimulated ex vivo cholesterol efflux to HDL. When all subjects were considered (n = 18), unstimulated non-ABCA1-mediated efflux (but not ABCA1-specific efflux) was correlated positively with apoA-I production (r = 0.552, p = 0.017) and HDL FC and CE pool sizes, and negatively with the fractional clearance rate of FC (r = −0.759, p = 4.1 × 10⁻⁴) and CE (r = −0.652, p = 4.57 × 10⁻³). Our data are consistent with the concept that ex vivo non-ABCA1 efflux capacity may correlate with slower in vivo turnover of HDL cholesterol moieties. The use of a dual labeling protocol provided for the first time the opportunity to assess the association of ex vivo cholesterol efflux capacity with in vivo HDL cholesterol metabolic parameters.     

Viability study of clinical bacterial strains by capillary electrophoresis and flow cytometry approaches

One of the challenges medicine faces is the constantly growing resistance of pathogens to various classes of antibiotics. In this study, we investigated the use of capillary electrophoresis (CE) to characterize and assess the physiological states of three clinical bacterial strains—methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA), and Escherichia coli extended-spectrum β-lactamases (ESβL)—exposed to different antibiotics. All chosen bacteria are the leading causes of healthcare-associated and hospital-acquired invasive infections in adults. In the first part of the research, it was determined the optimal incubation time of the tested strains with antibiotics, represented as an optimal time of 24 h. In the second part, we have compared two approaches: flow cytometry (FC) as a standard method and CE as a proposed alternative approach. The viability of clinical strains treated with different class antibiotics calculated in CE measurements was strongly correlated (>0.83 for MSSA, >0.92 for ESβL and MRSA) with the viability obtained on the basis of FC measurements. As a result, CE has a chance to become a modern diagnostic method used in clinical practice. The CE cutoff was found to be 50%; above this value, the strain shows resistance to the action of the antibiotic.     

Antibacterial effect,efflux pump inhibitory (NorA,TetK and MepA) of Staphylococcus aureus and in silico prediction of α, β and δ-damascone compounds

ObjectiveThe present study aimed to evaluate the antibacterial effect and inhibitory capacity against NorA, TetK and MepA efflux pump of Staphylococcus aureus multiresistant by in vitro and in silico approach of α, β and δ-damascone compounds.ResultsThe compounds α, β and δ-damascone showed a clinically relevant effect against S. aureus ATCC 6538 standard strain. A modulating effect was also observed in association with antibiotics against MDR strains. Regarding the inhibition of the efflux pump, the compounds showed a modulating effect with antibiotics, against SA-1199, SA-1199B, SA IS-58 and K2068. Only δ-damascone demonstrated an efflux pump inhibitory effect. Regarding ADME properties, α, β and δ-damascone showed similar physicochemical properties with good pharmacokinetic characteristics, such as lipophilicity, oral bioavailability and low toxicity. In addition, they exhibited the binding energy and Ligand Efficiency (LE) ?81.17 (5.4), ?77.48(-5.4) and ?64.55 (-5.1), which shows good interactions with the critical residues of the MepA receptor.ConclusionsFrom the results it is concluded that the compounds α, β and δ-damascone do not have antibacterial activity, but show a modulating effect in association with antibiotics, as well as not showing direct activity on the efflux pump, but they do have a modulating effect with antibiotics and with EtBr (α and β-damascone).     

Phytochemical prospection,evaluation of antibacterial activity and toxicity of extracts of Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz

Libidibia ferrea (Mart. ex Tul.) L.P. Queiroz is a arboreal species found in the Caatinga from Northeast of Brazil that has been used in popular medicine as an anti-inflammatory, healing, analgesic and for the treatment of respiratory system disorders. Therefore, the objective of this work was to evaluate the composition of ethanol extracts from the leaves and inner bark of Libidibia ferrea, as well as to verify its antibacterial activity and as a potential inhibitor of the TetK efflux pump in Staphylococcus aureus strains, in addition to investigating the toxicity of the extracts in a Drosophila melanogaster model. The analysis and quantification of the extracts markers was performed by High Performance Liquid Chromatography (HPLC). To determine the Minimum Inhibitory Concentration (MIC) broth microdilution tests were carried out. The evaluation of efflux pump inhibition was performed by modifying the MIC of antibiotics and ethidium bromide. Mortality and negative geotaxis tests were used to verify the toxicity of extracts on D. melanogaster. Hydrolysable tannins (gallic acid and ellagic acid) and flavonoids were found in HPLC analysis. The extracts did not show antibacterial activity, demonstrating a MIC ≥ 1024 µg/mL, however the ethanolic extract of the leaves decreased the MIC of the antibiotic from 64 µg/mL to 16 µg/mL, but this effect is not associated with the inhibition of the efflux pump. The extracts did not show toxicity in a D. melanogaster model. This is the first study to evaluate the antibacterial activity of L. ferrea extracts on the IS-58 strain of S. aureus, as well as the first to investigate its toxicity using D. melanogaster. From the results, further studies are needed to determine the mechanisms of action of the extract with other antibiotics.     

Evaluation of a Microbiological Multi-Residue System on the detection of antibacterial substances in ewe milk

To protect both, public health and the dairy industry, from the presence of antibiotic residues in milk, control programmes have been established, which include the needed screening tests. This work focuses on the application of a Microbiological Multi-Residue System in ewe milk, a method based on the use of six different plates, each seeded with one of the following bacteria: Geobacillus stearothermophilus var. calidolactis (beta-lactams), Bacillus subtilis at pH 8.0 (aminoglycosides), Kocuria rhizophila (macrolides), Escherichia coli (quinolones), B. cereus (tetracyclines) and B. subtilis at pH 7.0 (sulphonamides), respectively. Twenty-three antimicrobial substances were analysed and a logistic regression was established for each substance assayed to relate the antibiotic concentration and the zone of microbial growth inhibition. Great linearity in the response was observed (regression coefficients of over 0.97). This fact suggests the possibility of establishing a decision level of antibiotic concentrations near to the Maximum Residue Limits (MRL). Zones of inhibition were suggested as proposed action levels for the different antimicrobial groups (diameters of inhibition of 18 mm for the aminoglycoside, beta-lactam and sulphonamide plates; 19 mm for the tetracycline plate, 21 mm for the macrolide plate, and 24 mm for the quinolone plate). Specificity and cross-reactivity were also assayed.     

The Untargeted Phytochemical Profile of Three Meliaceae Species Related to In Vitro Cytotoxicity and Anti-Virulence Activity against MRSA Isolates

Background: A high mortality rate is associated with about 80% of all infections worldwide, mainly due to antimicrobial resistance. Various antimicrobial and cytotoxic activities have been proposed for Meliaceae species. This study aimed to evaluate the in vitro anti-virulence and cytotoxic effect of the leaf extracts of Aphanamixis polystachya, Toona ciliata and Melia azedarach against five MRSA strains and on three cancer cell lines, followed by biological correlation to their encompassed phytoconstituents. Material and Methods: We explored three plants of this family against a panel of Methicillin-resistant Staphylococcus aureus (MRSA) strains and several cancer cell lines to select the most promising candidates for further in vivo and preclinical studies. The phytochemical composition was evaluated by UHPLC–QTOF–MS untargeted profiling. Cell viability was assessed by SRB assay. Minimum Inhibitory Concentration was carried out by using the agar micro-dilution technique. Inhibition of biofilm formation and preformed biofilm disruption were assessed spectrophotomertically, according to the Sultan and Nabil method (2019). Results: A total of 279 compounds were putatively annotated to include different phytochemical classes, such as flavonoids (108), limonoids/terpenoids (59), phenolic acids (49) and lower-molecular-weight phenolics (39). A. polystachya extract showed the most potent cytotoxic activity against Huh-7, DU-145 and MCF-7 cell lines (IC₅₀ = 3, 3.5 and 13.4 µg mL⁻¹, respectively), followed by M. azedarach, with no effect recorded for T. ciliata extract. Furthermore, both A. polystachya and M. azedarach extracts showed promising anti-virulence and antimicrobial activities, with A. polystachya being particularly active against MRSA. These two latter extracts could inhibit and disrupt the biofilm, formed by MRSA, at sub-lethal concentrations. Interestingly, the extracts inhibited hemolysin-α enzyme, thus protecting rabbit RBCs from lysis. A. polystachya extract reduced the pigmentation and catalase enzyme activity of tested pigmented strains better than M. azedarach at both tested sub-MICs. Consequently, susceptibility of the extract-treated cells to oxidant killing by 200 mM H₂O₂ increased, leading to faster killing of the cells within 120 min as compared to the extract-non-treated cells, likely due to the lower antioxidant-scavenging activity of cells exhibiting less staphyloxanthin production. Conclusion: These findings suggested that both A. polystachya and M. azedarach natural extracts are rich in bioactive compounds, mainly limonoids, phenolics and oxygenated triterpenoids, which can combat MRSA biofilm infections and could be considered as promising sources of therapeutic cytotoxic, antibiofilm and anti-virulence agents.