Johana Rondevaldova

Synergistic interactions of epigallocatechin gallate and oxytetracycline against various drug resistant Staphylococcus aureus strains in vitro.

Epigallocatechin gallate (EGCG), the major catechin contained in tea leaves, is known to possess the synergistic anti-staphylococcal activity in combination with various β-lactam antibiotics and tetracycline. In the present study, we explored the in vitro combinatory effect of EGCG in combination with oxytetracycline against eight standard strains and clinical isolates of Staphylococcus aureus, including erythromycin, methicillin and tetracycline resistant strains. The minimum inhibitory concentrations were determined by the broth microdilution assay and the data were evaluated according to the sum of fractional inhibitory concentrations (∑FIC). Our results showed synergistic and additive interactions against all S. aureus strains tested (∑FIC 0.288-0.631), two of which were multidrug resistant. According to our best knowledge, it is the first report on the EGCG synergy with oxytetracycline. Considering its significant synergistic antimicrobial effect and low toxicity, we suggest EGCG as a promising compound for the development of new anti-staphylococcal formulations.

The relationship between structure and in vitro antibacterial activity of selected isoflavones and their metabolites with special focus on antistaphylococcal effect of demethyltexasin.

In this study, we tested 15 naturally occurring isoflavones and their metabolites for their possible antibacterial properties against nine Gram‐positive and Gram‐negative bacteria. The in vitro antibacterial activity was determined using the broth microdilution method, and the results were expressed as minimum inhibitory concentrations (MICs). 6,7,4′‐trihydroxyisoflavone (demethyltexasin), 7,3′,4′‐trihydroxyisoflavone (hydroxydaidzein), 5,7‐dihydroxy‐4′‐methoxyisoflavone (biochanin A), 7,8,4′‐trihydroxyisoflavone (demethylretusin) and 5,7,4′‐trihydroxyisoflavone (genistein) produced significant antibacterial activity (MICs ≥ 16 μg ml−1). The most effective compound, demethyltexasin, was subsequently tested for its growth‐inhibitory effect against Staphylococcus aureus, and it exhibited significant antistaphylococcal effects against various standard strains and clinical isolates, including methicillin and tetracycline resistant ones with the MICs ranging from 16 to 128 μg ml−1.

Composition and Antimicrobial Activity of Euphrasia rostkoviana Hayne Essential Oil

Eyebright, Euphrasia rostkoviana Hayne (Scrophulariaceae), is a medicinal plant traditionally used in Europe for the treatment of various health disorders, especially as eyewash to treat eye ailments such as conjunctivitis and blepharitis that can be associated with bacterial infections. Some Euphrasia species have been previously reported to contain essential oil. However, the composition and bioactivity of E. rostkoviana oil are unknown. Therefore, in this study, we investigated the chemical composition and antimicrobial activity of the eyebright essential oil against some organisms associated with eye infections: Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, and Candida albicans. GC-MS analysis revealed more than 70 constituents, with n-hexadecanoic acid (18.47%) as the main constituent followed by thymol (7.97%), myristic acid (4.71%), linalool (4.65%), and anethole (4.09%). The essential oil showed antimicrobial effect against all organisms tested with the exception of P. aeruginosa. The best activity was observed against all Gram-positive bacteria tested with the minimum inhibitory concentrations of 512 µg/mL. This is the first report on the chemical composition of E. rostkoviana essential oil and its antimicrobial activity.

Antimicrobial activity of traditional medicinal plants from Ankober District, North Shewa Zone, Amhara Region, Ethiopia

Abstract Context: Traditional medicinal plants have long been used in Ethiopia to treat human and livestock ailments. Despite a well-documented rich tradition of medicinal plant use in the country, their direct antimicrobial effects are still poorly known. Objective: To investigate the antimicrobial activity of 19 medicinal plant species that were selected based on the ethnobotanical information on their traditional use to treat infectious diseases in Ankober District. Methods: About 23 different ethanol extracts of plants obtained by maceration of various parts of 19 medicinal plant species were studied for potential antimicrobial activity using a broth microdilution method against Bacillus cereus, Bacteroides fragilis, Candida albicans, Clostridium perfringens, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella enteritidis, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes. Results: Plant extracts from Embelia schimperi Vatke (Myrsinaceae) showed the strongest antibacterial activity with a minimum inhibitory concentration (MIC) value of 64 µg/ml against B. cereus, L. monocytogenes, and S. pyogenes. Growth inhibitory activities were also observed for extracts of Ocimum lamiifolium Hochst. (Lamiaceae) against S. pyogenes, and those of Rubus steudneri Schweinf. (Rosaceae) against S. epidermidis at an MIC value of 128 µg/ml. Generally, 74% of ethanol extracts (17 extracts) showed antimicrobial activity against one or more of the microbial strains tested at an MIC value of 512 µg/ml or below. Discussion and conclusions: Results confirm the antimicrobial role of traditional medicinal plants of Ankober and warrant further investigations on promising medicinal plant species so as to isolate and characterise chemicals responsible for the observed strong antimicrobial activities.

Thymoquinone vapor significantly affects the results of Staphylococcus aureus sensitivity tests using the standard broth microdilution method

The broth microdilution (BMD) method is widely used for the determination of minimum inhibitory concentrations of antimicrobial agents, including volatile oils and their components. In this series of various experiments, we have demonstrated the influence of thymoquinone (TQ) vapor on the results of the BMD test performed with Staphylococcus aureus as a model organism. The spread of vapor from the TQ containing wells (32-512 μg/mL) caused the complete inhibition of staphylococcal growth in adjoining wells initially containing bacterium-inoculated pure Mueller-Hinton broth only and thus produced false positive results of the test. The ability of TQ to pass into the adjoined wells was subsequently confirmed by gas chromatography-mass spectrometry, whereas TQ at concentrations up to 84 μg/mL was detected in these wells after five hours. Based on these results, we suppose that vapors of TQ as well as of other naturally occurring volatile compounds and their mixtures (for example essential oils and plant extracts) can significantly influence results of the standard BMD assay. These observations, therefore, call for development of new appropriate BMD method suitable for assessment of antimicrobial activity of volatile substances.

Evaluation of antibacterial potential and toxicity of plant volatile compounds using new broth microdilution volatilization method and modified MTT assay

With aim to develop effective proof-of-concept approach which can be used in a development of new preparations for the inhalation therapy, we designed a new screening method for simple and rapid simultaneous determination of antibacterial potential of plant volatiles in the liquid and the vapour phase at different concentrations. In addition, EVA (ethylene vinyl acetate) capmat™ as vapour barrier cover was used as reliable modification of thiazolyl blue tetrazolium bromide (MTT) assay for cytotoxicity testing of volatiles on microtiter plates. Antibacterial activity of carvacrol, cinnamaldehyde, eugenol, 8-hydroxyquinoline, thymol and thymoquinone was determined against Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae using new broth microdilution volatilization method. The cytotoxicity of these compounds was evaluated using MTT test in lung fibroblast cells MRC-5. The most effective antibacterial agents were 8-hydroxyquinoline and thymoquinone with the lowest minimum inhibitory concentrations (MICs) ranging from 2 to 128μg/mL, but they also possessed the highest toxicity in lung cell lines with half maximal inhibitory concentration (IC50) values 0.86-2.95μg/mL. The lowest cytotoxicity effect was identified for eugenol with IC50 295.71μg/mL, however this compound produced only weak antibacterial potency with MICs 512-1024μg/mL. The results demonstrate validity of our novel broth microdilution volatilization method, which allows cost and labour effective high-throughput antimicrobial screening of volatile agents without need of special apparatus. In our opinion, this assay can also potentially be used for development of various medicinal, agricultural, and food applications that are based on volatile antimicrobials.

In vitro Combinatory Antimicrobial Effect of Plumbagin with Oxacillin and Tetracycline against Staphylococcus aureus

Plumbagin (5‐hydroxy‐2‐methyl‐1,4‐naphthoquinone), a bicyclic naphthoquinone naturally distributed among Plumbago species, has been reported to have antimicrobial activity against a wide range of microorganisms. In this study, plumbagin was examined for its combinatory antimicrobial effect with tetracycline or oxacillin against nine strains of Staphylococcus aureus, including its methicillin‐ and multidrug‐resistant strains. Minimum inhibitory concentrations were determined through the broth microdilution method, whereas the combinatory effect was evaluated according to the sum of fractional inhibitory concentration (ΣFIC) indices. Additive interactions were obtained for both combinations against most of the strains tested. Synergy was obtained for combination with oxacillin against two out of seven strains (ΣFIC range 0.273–0.281), both were methicillin resistant. Our results proved plumbagin as a compound suitable for anti‐Staphylococcal combinatory testing. Moreover, to the best of our knowledge, this is the first report of plumbagin synergy with oxacillin against S. aureus strains, including its resistant forms. Copyright

In vitro antistaphylococcal effects of Embelia schimperi extracts and their component embelin with oxacillin and tetracycline

Bacterial infections are in less-developed countries traditionally treated by remedies prepared from medicinal plants. Embelia schimperi (Vatke) is a plant used as a taenicide or disinfectant in Ethiopia, very often taken mixed with another plant species. In the present study, we examined two extracts prepared from seeds and twigs with leaves of E. schimperi and its main present secondary metabolite embelin for their antibacterial combinatory effect with oxacillin and tetracycline against sensitive and resistant Staphylococcus aureus strains. Minimum inhibitory concentrations were determined through the broth microdilution method, whereas the combinatory effect was evaluated through fractional inhibitory concentration sum (ΣFIC) indices. Results show many positive interactions and synergy occurring in embelin and oxacillin combinations against 4 out of 9 strains (ΣFIC 0.203–0.477) and for embelin and tetracycline combination against 3 out of 9 strains (ΣFIC 0.400–0.496). Moreover, the resistance to oxacillin has been overcome in 2 strains and to tetracycline in 3 strains. According to our knowledge, this is the first study showing antimicrobial combinatory effect of E. schimperi as well as of embelin. These findings can be used for the further research targeted on the development of new antistaphylococcal agents.

Screening of in vitro antimicrobial activity of plants used in traditional Indonesian medicine

Abstract Context: In many regions of Indonesia, there are numerous traditional herbal preparations for treatment of infectious diseases. However, their antimicrobial potential has been poorly studied by modern laboratory methods. Objective: This study investigates in vitro antimicrobial activity of 49 ethanol extracts from 37 plant species used in Indonesian traditional medicine for treatment against Candida albicans, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Materials and methods: The plants were collected from the Biopharmaca collection garden, Bogor, Indonesia. The plant material was dried, finely grounded, extracted using ethanol, concentrated, and the dried residue was dissolved in 100% DMSO. Antimicrobial activity was determined in terms of a minimum inhibitory concentration (MIC) using a broth microdilution method in 96-well microplates. Results: The extract of Orthosiphon aristatus (Blume) Miq. (Lamiaceae) leaf produced the strongest antimicrobial effect, inhibiting the growth of C. albicans (MIC 128 μg/mL), S. aureus (MIC 256 μg/mL), E. faecalis (MIC 256 μg/mL) and P. aeruginosa (MIC 256 μg/mL). The leaf extract of Woodfordia floribunda Salisb. (Lythraceae) also exhibited significant effect against C. albicans (MIC 128 μg/mL), S. aureus (MIC 256 μg/mL) and E. faecalis (MIC 256 μg/mL). Rotheca serrata (L.) Steane & Mabb. (Lamiaceae) leaf extract inhibited the growth of S. aureus (MIC 256 µg/mL) and C. albicans (MIC 256 µg/mL). Discussion and conclusions: The leaf extract of O. aristatus and W. floribunda exhibited a significant anti-candidal effect. Therefore, both of these plants can serve as prospective source materials for the development of new anti-candidal agents.

Evaluation of in vitro growth-inhibitory effect of carvacrol and thymol combination against Staphylococcus aureus in liquid and vapour phase using new broth volatilization chequerboard method

Carvacrol and thymol, both plant-derived volatile compounds, have extensively been studied individually as well as in combination with other agents for their antimicrobial activity in liquid phase. However, in contrast to well-established assays for testing of antimicrobial combinatory effects in liquid media, there are no standardized methods for evaluation of interactions between volatile compounds in vapour phase. The objective of this study was to verify new broth volatilization chequerboard method by testing the combination of carvacrol and thymol and to determine in vitro inhibitory effect of these compounds in liquid and vapour phase against twelve Staphylococcus aureus strains. The new method, based on combination of standard microdilution chequerboard and new broth volatilization tests allowing calculation of fractional inhibitory concentrations (FICs), was used. Combination of carvacrol and thymol produced the additive antimicrobial effect against all strains tested. In several cases, they reached ΣFIC values lower than 0.6, which can be considered as a strong additive interaction. The best result was found in vapour phase against one standard strain at combination of 128 μg/mL of carvacrol and 16-256 μg/mL of thymol (ΣFIC = 0.51) and in liquid phase against one clinical isolate at combination of 256 μg/mL of carvacrol and 256 μg/mL of thymol (ΣFIC = 0.53). The study verified that the new technique is suitable for simple and rapid high-throughput combinatory antimicrobial screening of volatile compounds simultaneously in vapour and liquid phase and that it allows determination and comparison of MIC and FIC values in both, liquid and solid media.