Synergy Assessment of Four Antimicrobial Bioactive Compounds for the Combinational Treatment of Bacterial Pathogens.

Antimicrobial resistance (AMR) has become a topic of great concern in recent years, with much effort being committed to developing alternative treatments for resistant bacterial pathogens. Drug combinational therapies have been a major area of research for several years, with modern iterations using combining well-established antibiotics and other antimicrobials with the aim of discovering complementary mechanisms. Previously, we characterised four GRAS antimicrobials that can withstand thermal polymer extrusion processes for novel medical device-based and therapeutic applications. In the present study, four antimicrobial bioactive-silver nitrate, nisin, chitosan and zinc oxide-were assessed for their potential combined use as an alternative synergistic treatment for AMR bacteria via a broth microdilution assay based on a checkerboard format. The bioactives were tested in arrangements of two-, three- and four-drug combinations, and their interactions were determined and expressed in terms of a synergy score. Results have revealed interesting interactions based on treatments against recognised test bacterial strains that cause human and animal infections, namely E. coli, S. aureus and S. epidermidis. Silver nitrate was seen to greatly enhance the efficacy of its paired treatment. Combinations with nisin, which is a lantibiotic, exhibited the most interesting results, as nisin has no effect against Gram-negative bacteria when used alone; however, it demonstrated antimicrobial effects when combined with silver nitrate or chitosan. This study constitutes the first study to both report on practical three- and four-drug combinational assays and utilise these methods for the assessment of established and emerging antimicrobials. The novel methods and results presented in this study show the potential to explore previously unknown drug combination compatibility measures in an ease-of-use- and high-throughput-based format, which can greatly help future research that aims to identify appropriate alternative treatments for AMR, including the screening of potential new bioactives biorefined from various sources.

Chemical composition and antibacterial activity of bee venom against multi-drug resistant pathogens.

Bee venom with an antimicrobial effect is a powerful natural product. One of the most important areas where new antimicrobials are needed is in the prevention and control of multi-drug resistant pathogens. Today, antibacterial products used to treat multi-drug resistant pathogen infections in hospitals and healthcare facilities are insufficient to prevent colonisation and spread, and new products are needed. The aim of the study is to investigate the antibacterial effect of the bee venom (BV), a natural substance, on the species of Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcus faecalis, Carbapenem resistant Escherichia coli, Carbapenem resistant Klebsiella pneumoniae and Carbapenem resistant Acinetobacter baumannii. As a result of this study, it was found that MIC90 and MBC90 values ranged from 6.25 µg/mL - 12.5 µg/mL and numbers of bacteria decreased by 4-6 logs within 1-24 h for multi-drug resistant pathogens. In particular, Vancomycin resistant Enterococcus faecalis isolate decreased 6 log cfu/mL at 50 µg/mL and 100 µg/mL concentrations in the first hour. The effective bacterial inhibition rate of bee venom suggests that it could be a potential antibacterial agent for multi-drug resistant pathogens.Contribution: The treatment options of antibiotic-resistant pathogens are a major problem in both veterinary and human medicine fields. We have detected a high antibacterial effect against these agents in this bee venom study, which is a natural product. Apitherapy is a fashionable treatment method all over the world and is used in many areas of health. Bee venom is also a product that can be used as a drug or disinfectant raw material and can fill the natural product gap that can be used against resistant bacteria.

Antibacterial effect of essential oils and their components against Xanthomonas arboricola pv. pruni revealed by microdilution and direct bioautographic assays.

Bacterial spot of stone fruits caused by Xanthomonas arboricola pv. pruni (Xap) is one of the most significant diseases of several Prunus species. Disease outbreaks can result in severe economic losses while the control options are limited. Antibacterial efficacy of essential oils (EOs) of thyme, cinnamon, clove, rosemary, tea tree, eucalyptus, lemon grass, citronella grass, and lemon balm was assessed against two Hungarian Xap isolates. The minimal inhibitory concentration (MIC) was determined by broth microdilution assay and for the identification of active EOs' components a newly introduced high-performance thin-layer chromatography (HPTLC)-Xap (direct bioautography) method combined with solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS) was applied. All EOs inhibited both bacterium isolates, but cinnamon proved to be the most effective EO with MIC values of 31.25 µg/mL and 62.5 µg/mL, respectively. Compounds in the antibacterial HPTLC zones were identified as thymol in thyme, trans-cinnamaldehyde in cinnamon, eugenol in clove, borneol in rosemary, terpinen-4-ol in tea tree, citral (neral and geranial) in lemon grass and lemon balm, and citronellal and nerol in citronella grass. Regarding active compounds, thymol had the highest efficiency with a MIC value of 50 µg/mL. Antibacterial effects of EOs have already been proven for several Xanthomonas species, but to our knowledge, the studied EOs, except for lemon grass and eucalyptus, were tested for the first time against Xap. Furthermore, in case of Xap, this is the first report demonstrating that direct bioautography is a fast and suitable method for screening anti-Xap components of complex matrices, like EOs.

Antiproliferative assay of suma or Brazilian ginseng (Hebanthe eriantha) methanolic extract on HCT116 and 4T1 cancer cell lines, in vitro toxicity on Artemia salina larvae, and antibacterial activity.

Hebanthe eriantha is a medicinal plant used in folk medicine and a subject of commercial interest. The cytotoxicity effects from H. eriantha root extracts on cancerous and normal cells were assessed by the MTT method, and in vitro toxicity was evaluated on Artemia salina. The inhibition of the proliferation of bacteria and MIC values were examined by the disc diffusion and the broth microdilution method, respectively. Human colon cancer HCT116 and mouse breast tumour model 4T1 cells treated with methanolic extract showed a significant decrease in viability of cells with IC50: 272.6 and 88.5 µg/mL at 72h, respectively. The methanolic extract of H. eriantha showed moderate toxicity against A. salina (LC50: 589.4 µg/mL). In antimicrobial activity, the methanolic extract showed the highest inhibitory function against S. aureus and P. vulgaris (17.5 and 16 mm) with MICs of 500 µg/mL. The results confirmed the potential of plant roots as cytotoxic agents.

Antimicrobial Activity of Eucalyptus globulus, Azadirachta indica, Glycyrrhiza glabra, Rheum palmatum Extracts and Rhein against Porphyromonas gingivalis.

Novel plant-derived antimicrobials are of interest in dentistry, especially in the treatment of periodontitis, since the use of established substances is associated with side effects and concerns of antimicrobial resistance have been raised. Thus, the present study was performed to quantify the antimicrobial efficacy of crude plant extracts against Porphyromonas gingivalis, a pathogen associated with periodontitis. The minimal inhibitory concentrations (MICs) of Eucalyptus globulus leaf, Azadirachta indica leaf, Glycyrrhiza glabra root and Rheum palmatum root extracts were determined by broth microdilution for P. gingivalis ATCC 33277 according to CLSI (Clinical and Laboratory Standards Institute). The MICs for the E. globulus, A. indica and G. glabra extracts ranged from 64 mg/L to 1024 mg/L. The lowest MIC was determined for an ethanolic R. palmatum extract with 4 mg/L. The MIC for the anthraquinone rhein was also measured, as the antimicrobial activity of P. palmatum root extracts can be partially traced back to rhein. Rhein showed a remarkably low MIC of 0.125 mg/L. However, the major compounds of the R. palmatum root extract were not further separated and purified. In conclusion, R. palmatum root extracts should be further studied for the treatment of periodontitis.

Azelaic acid and Melaleuca alternifolia essential oil co-loaded vesicular carrier for combinational therapy of acne.

Aim: Azelaic acid (AzA), a comedolytic, antibacterial, anti-inflammatory anti-melanogenic agent, prescribed against acne vulgaris is safe on skin. Its combination with another widely used anti-acne agent, tea tree oil (EO) whose delivery is limited by volatility, instability and lipophilicity constraints was attempted. Method: Solvent injection was used to prepare AzA-EO integrated ethosomes. Result: Ethosomes were transformed into carbopol hydrogel, which exhibited pseudo-plastic properties with appreciable firmness, work of shear, stickiness and work of adhesion. The hydrogel showed better permeation and retention characteristics vis-a-vis commercial formulation (AzidermTM), when evaluated in Wistar rat skin. Further, ethosome hydrogel composite was better tolerated with no side effects. Conclusion: The findings suggests that the aforementioned strategy could be a potential treatment used for acne management.

Hydrogel composite containing azelaic acid and tea tree essential oil as a therapeutic strategy for Propionibacterium and testosterone-induced acne.

Azelaic acid (AzA) is a USFDA bioactive prescribed against acne vulgaris. It possesses delivery challenges like poor aqueous solubility, low skin-penetrability, and dose-dependent side effects, which could be overcome by its synergistic combination with tea tree oil (TTO) as a microemulsion (ME)-based hydrogel composite. AzA-TTO ME was prepared to employ pseudo-ternary phase diagram construction. The best AzA-TTO ME was of uniform size (polydispersity index < 0.7), nano-range (~357.4 ± 2% nm), transmittance (> 90%), and negative zeta potential (-1.42 ± 0.25% mV) values. ME hydrogel composite with optimum rheological and textural attributes showed better permeation, retention, and skin-compliant characteristics, vis-a-vis marketed formulation (Aziderm™) when evaluated in Wistar rat skin. In vitro antibacterial efficacy in bacterial strains, i.e., Staphylococcus aureus, Propionibacterium acne, and Staphylococcus epidermidis, was evaluated employing agar well plate diffusion and broth dilution assay. ME hydrogel has shown an increase in zone of inhibition by two folds and a decrease in minimum inhibitory concentration (MIC) by eightfold against P. acnes vis-a-vis AzA. Finally, ME hydrogel composite exhibited a better reduction in the papule density (93.75 ± 1.64%) in comparison to Aziderm™ 72.69 ± 4.67%) on acne as developed in rats by inducing testosterone. Thus, the developed AzA-TTO ME hydrogel composite promises an efficacious and comparatively safer drug delivery system for the topical therapy of acne vulgaris.

Validation of a method of broth microdilution for the determination of antibacterial activity of essential oils.

OBJECTIVE: The aim of the present study was to adapt and optimize a broth microdilution method and compare it to the agar dilution method for the evaluation of activity of essential oils from medicinal plants against Gram-negative bacteria. Based on bibliographic research, active and not active oils were selected. The sensitivity and specificity were established as parameters for validation. The comparison between both methods was made using contingency analysis tables, based on the observed frequencies. For both methods, the minimum inhibitory concentration was determined against Escherichia coli strains, in an essential oil concentration range between 0.03 and 0.48% (v/v). RESULTS: A stable emulsion formation was achieved with the addition of Tween 80 and constant agitation, guaranteeing the continuous contact of oil with bacteria (critical step in the microdilution method). The statistical analysis of results obtained with both methods presented a good sensitivity and specificity (100% in both cases), which let us correctly discriminate between active and non-active oils. The values obtained for the minimal inhibitory concentration were independent of the technique used. Finally, the obtained results show that the validated microtechnique allows important diminishment of time and resources for investigations dealing with essential oils or lipophilic extracts evaluation.

Chemical Profile of the Volatile Constituents and Antimicrobial Activity of the Essential Oils from Croton adipatus, Croton thurifer, and Croton collinus.

The aim of this study was to determine the volatile phytochemical constituents and evaluate the antimicrobial activity of the essential oils of the leaves from Croton adipatus, Croton thurifer, and Croton collinus. Essential oils were extracted by hydro-distillation using the Clevenger extractor and the phytochemical analysis was determined by Gas chromatography-Mass Spectrometry (GC-MS). The antimicrobial activity was assessed using the agar diffusion and colorimetric broth microdilution methods against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, and Candida albicans ATCC The essential oils from C. adipatus, C. thurifer, and C collinus had 46, 38, and 35 volatile constituents respectively. The main compounds determined in C. adipatus were ß-myrcene (18.34%), while in C. collinus was ß-caryophyllene (44.7%), and in C. thurifer was an unknown component (C10H16: 22.38%). Essential oil of C. adipatus showed a MIC against B. subtilis (286.4 µg/mL) and C. albicans (572.8 ± 0 µg/mL); C. thurifer against S. aureus (296.1 ± 0 µg/mL) and B. subtilis (148 ± 0 µg/mL); and C. collinus against B. subtilis (72 ± 0 µg/mL) and C. albicans (576.2 ± 0 µg/mL). The three essential oils of Croton species demonstrated in vitro antimicrobial activity against a strain of bacteria or fungi.

Antimicrobial Effects of Plant Extracts against Clostridium perfringens with Respect to Food-Relevant Influencing Factors.

ABSTRACT: The application of plant extracts (PEs) could be a promising option to satisfy consumers' demand for natural additives to inhibit growth of variable pathogenic bacteria. Thus, the aim of this study was to develop a standardized microdilution method to examine the antimicrobial effects of 10 hydrophilic PEs against two strains of Clostridium perfringens facing various food-relevant influencing factors. Because of the high opacity of PEs, resazurin was used as an indicator for bacterial growth instead of pellet formation. The highest value of the MIC of the replications of each PE was defined as effective plant extract concentration (EPC), whereas the next concentration beneath the lowest MIC was defined as the ineffective plant extract concentration (IEPC). The EPCs of seven PEs, allspice, cardamom, cinnamon, clove, coriander, ginger, and mace, were between 0.625 and 10 g/kg, whereas extracts of caraway, nutmeg, and thyme showed no antimicrobial activity up to the maximum concentration tested (10 g/kg) against C. perfringens in vitro. Two intrinsic factors, sodium chloride (NaCl) and sodium nitrite (NaNO2), displayed either synergistic or additive effects or no interaction with most PEs. By combination with PEs at their IEPC (0.08 to 1.25 g/kg), MIC of NaCl and NaNO2 decreased from between 25 and 50 g/kg to between 6 and 25 g/kg and from more than 200 mg/kg to between 0.2 and 100 mg/kg, respectively. In contrast, lipid (sunflower oil) at a low concentration inhibited the antimicrobial effects of all tested PEs. For extrinsic factors, only allspice, ginger, and coriander could maintain their antimicrobial effects after being heated to 78°C for 30 min. The synergistic effect between PEs and pH values (5.0 and 5.5) was also found for all PEs. The established screening method with resazurin and defining EPC and IEPC values allows the verification of antimicrobial effects of PEs under various food-relevant influencing factors in a fast and reproducible way.

Collection, Extraction, and In Vitro Antibacterial Evaluation of Plants Used in Traditional Medicine.

Medicinal plants are used to treat infectious and inflammatory diseases in various traditional medical systems, and thus could represent a promising source of antimicrobials. To establish the scientific basis for the therapeutic actions of traditional plant medicines, we provide a general workflow for evaluating the anti-infective properties of crude extracts from plants. We provide guidance starting from plant collections in the field and the creation of herbarium voucher specimens, moving to the processing of plants by drying, grinding, and extracting the plant parts collected, and finally ending with the antimicrobial investigation of these plant extracts. In this protocol, we provide a description of our workflow for the growth inhibitory evaluation of plant extracts against common human pathogenic bacteria.

Vapors of Volatile Plant-Derived Products Significantly Affect the Results of Antimicrobial, Antioxidative and Cytotoxicity Microplate-Based Assays.

Volatile plant-derived products were observed to exhibit broad spectrum of biological effects. However, due to their volatility, results of conventional microplate-based bioassays can be significantly affected by the vapors. With aim to demonstrate this phenomenon, antimicrobial, antioxidant, and cytotoxic activities of three essential oils (Alpinia elegans, Cinnamomum iners, and Xanthostemon verdugonianus), one supercritical CO2 extract (Nigella sativa), and four plant-derived compounds (capsaicin, caryophyllene oxide, 8-hydroxyquinoline, and thymoquinone) were evaluated in series of experiments including both ethylene vinyl acetate (EVA) Capmat sealed and nonsealed microplates. The results clearly illustrate that vapor transition to adjoining wells causes false-positive results of bioassays performed in nonsealed microtiter plates. The microplate layout and a duration of the assay were demonstrated as the key aspects defining level of the results affection by the vapors of volatile agents. Additionally, we reported biological activities and chemical composition of essential oils from A. elegans seeds and X. verdugonianus leaves, which were, according to our best knowledge, analyzed for the first time. Considering our findings, certain modifications of conventional microplate-based assays are necessary (e.g., using EVA Capmat as vapor barrier) to obtain reliable results when biological properties of volatile agents are evaluated.

Volatilome Analyses and In Vitro Antimicrobial Activity of the Essential Oils from Five South African Helichrysum Species.

Helichrysum genus was used in folk South African medicine to treat various human disorders. As a part of our on-going research addressing the exploitation of South African plants belonging to this genus, five species were investigated for their volatile and antimicrobial activities. The volatile organic compounds (VOCs) and the essential oils (EOs) were analysed by gas chromatography mass spectrometry (GC-MS). Microdilution was the method used for assessing both antimycotic and antibacterial activities, which was also tested by Kirby-Bauer agar disc diffusion. Total monoterpenes (TMs) dominated the VOCs of four species (H. trilineatum (70.6%), H. edwardsii (79.3%), H. cooperi (84.5%), and H. pandurifolium (57.0%)). H. cooperi and H. edwardsii EOs showed the predominance of TMs (68.2% and 84.5%, respectively), while H. pandurifolium and H. trilineatum EOs were characterized by the prevalence of TSs (86.5% and 43.6%, respectively). H. odoratissimum EO evidenced a similar amount of both TMs (49.5%) and TSs (46.4%). Microsporum canis was more sensitive to these EOs. The lowest minimum inhibitory concentration (MIC) was observed with H. pandurifolium and H. edwardsii EOs (0.25%). H. pandurifolium and H. trilineatum had a good effect on Staphylococcus aureus (MIC 5%). These findings open new perspectives for the exploitation of these natural compounds for application in cosmetics and pharmaceutics.

Antibacterial Activity of Four Plant Extracts Extracted from Traditional Chinese Medicinal Plants against Listeria monocytogenes, Escherichia coli, and Salmonella enterica subsp. enterica serovar Enteritidis.

The worldwide ethnobotanical use of four investigated plants indicates antibacterial properties. The aim of this study was to screen and determine significant antibacterial activity of four plant extracts in vitro and in a poultry digest model. Using broth microdilution, the concentrations at which four plant extracts inhibited Listeria monocytogenes, Salmonella enteritidis, and Escherichia coli over 24 hours was determined. Agrimonia pilosa Ledeb, Iris domestica (L.) Goldblatt and Mabb, Anemone chinensis Bunge, and Smilax glabra Roxb all exhibited a minimum inhibitory concentration (MIC) of 62.5mg/L and a minimum bactericidal concentration (MBC) of 500mg/L against one pathogen. A. pilosa Ledeb was the most effective against L. monocytogenes and E. coli with the exception of S. enteritidis, for which A. chinensis Bunge was the most effective. Time-kills of A. pilosa Ledeb and A. chinensis Bunge against L. monocytogenes, E. coli and S. enteritidis incubated in poultry cecum were used to determine bactericidal activity of these plant extracts. A. chinensis Bunge, significantly reduced S. enteritidis by ≥ 99.99% within 6 hours. A. pilosa Ledeb exhibited effective significant bactericidal activity within 4 hours against L. monocytogenes and E. coli. This paper highlights the potential of these plant extracts to control pathogens commonly found in the poultry gastrointestinal tract.

Incidence, Clinical Manifestation, Treatment Outcome, and Drug Susceptibility Pattern of Nontuberculous Mycobacteria in HIV Patients in Tehran, Iran.

BACKGROUND: Nontuberculous mycobacterial (NTM) infections have radically increased worldwide due to the increase in HIV infections. The disease activity increases with progressive immunodeficiency. METHODS: A total of 216 HIV seropositive patients suspected of having mycobacterial infection were recruited for this study. Clinical samples were collected from each patient and cultured on Lowenstein-Jensen media. Detection and species identification were simultaneously done using Reverse Blot Hybridization Assay System. Also, the minimum inhibitory concentrations (MIC) for each isolate were determined in 7H9 broth media for 10 antibiotics. RESULTS: In this study, 4 rapid and 4 slow-growing NTM species were isolated and identified. Mycobacterium fortuitum was the most common NTM species, 3/8 (37.5%), followed by Mycobacterium kansasii, 2/8 (25%). The cases were identified as pulmonary disease, 5/8 (62.5 %), disseminated infection, 2/8 (25%), and skin abscess, 1/8 (12.5%). M. chelonae and Mycobacterium avium were isolated from patients diagnosed with disseminated infection with treatment failure. The skin abscess was caused by infection with M. simiae. The results of the MIC testing were as follows: M. kansasii and M. fortuitum were susceptible to amikacin (AMK); M. avium to clarithromycin (CLA); M. fortuitum 2/3 (67%) to ciprofloxacin (CIP); 1/2 (50%) of M. kansasii isolates to CLA, and M. chelonae to rifampin (RIF), linezolid (LIN), AMK, and CIP at medium and high concentrations. CONCLUSION: AMK showed incredible in vitro activity against M. kansasii and M. fortuitum. Also, M. avium was susceptible to CLA, whereas M. simiae and M. chelonae were resistant to the tested drugs in this study.

Chemical constituents with antimicrobial and antioxidant activity from the aerial parts of Callistemon lanceolatus (Sm.) Sweet.

Callistemon lanceolatus (Sm.) Sweet grows all over the world and used to treat cough and bronchitis. The air-dried powder of the aerial parts was exhaustively extracted with methanol and the concentrated extract was adsorbed on silica gel for preparation of slurry. It was dried and subjected to silica gel column packed in petroleum ether. The column was eluted with organic solvents in order of increasing polarity to isolate 1-triacosanol (1), n-eicosanyl palmitate (2), n-heptadecanyl arachidate (3), n-tricosanyl palmitate, (4), 4-hydroxyphenethyl carbocerate (5), 4-hydroxyphenethyl gheddate (6), urs-12-en-3α-acetoxy-18ß-H-28-oic acid (7) and stigmast-5-en-3ß-ol-3ß-D-glucuronopyranoside (8). Among them, compound 5 and 6 were new fatty acid ester isolated from this plant. Compound 7 showed MIC 32 µg/mL against E. coli which was comparable to amoxicillin having same MIC 32 µg/mL. Compound 5 and 6 showed significant antioxidant activity by inhibiting DPPH due to the presence of phenolic groups.

Antifungal activity study of the monoterpene thymol against Cryptococcus neoformans.

Cryptococcus neoformans is a yeast fungus, which causes cryptococcosis, triggered by basidiospore inhalation and consequent dissemination to the central nervous system. In this study, we analyzed the antifungal action of thymol against 10 clinical strains of C. neoformans and analyzed the interaction of this monoterpene with sterols. The MICs of thymol ranged from 20 to 51 µg/ml, while the MFC values varied between 40 and 101 µg/ml. For the strains ICB-2601 and LM-39, in the presence of ergosterol, the MIC of thymol was 64 µg/ml, and in the presence of cholesterol, its MIC was 32 µg/ml. Based on the results, thymol presents antifungal action and seems to interact with ergosterol, but not with cholesterol. Complementary studies are needed to analyze its full effects.

Surveying the chemical composition and antibacterial activity of essential oils from selected medicinal plants against human pathogens.

BACKGROUND AND OBJECTIVES: Essential oils (EOs) with different biological activities, such as antibacterial properties, are a valuable resource for developing new drugs. MATERIALS AND METHODS: Ingredients of six medicinally important EOs, including Artemisia dracunculus, Anethum graveolens, Citrus limon, Citrus sinensis, Cinnamomum zeylanicum and Zingiber officinale, were identified using GC-MS analysis. Moreover, their five major compounds were also listed. Furthermore, the half-maximal inhibitory concentration (IC50) against four important human bacteria was also investigated using the 96-well plate microdilution. RESULTS: C. sinensis EO with IC50 of 1.0 and 4.7 mg.mL-1 have the most effect on the growth of S. aureus and P. aeruginosa. Moreover, EOs of Cinnamomum zeylanicum (IC50: 1.0 mg. Ml-1) and Artemisia dracunculus (IC50: 1.3 mg.mL-1) significantly showed better inhibitory effect on E. coli and K. pneumoniae. CONCLUSION: These EOs could be used for developing inexpensive, potent, and green antibacterial agents.

Chemical composition and antimicrobial activity of two extract of propolis against isolates of Staphylococcus spp. and multiresistant bacterials / Composição química e atividade antimicrobiana de dois extratos de própolis contra isolados de Staphylococcus spp. e bactérias multirresistentes

There is a growing need to discover and develop alternative therapies for the treatment of mastitis caused by Staphylococcus spp. and multidrug-resistant bacterial infections. This study examined the chemical composition and antimicrobial potential of two propolis extracts (EPA and EPB) against seventy-seven isolates of Staphylococcus spp. obtained from subclinical bovine mastitis; three clinical strains of MRSA and two from clinical strains of S. aureus ATCC, identified as S. aureus ATCC 6538 and S. aureus ATCC 25923. The total phenolic content was determined by the Folin-Ciocalteau method, the total flavonoid content by the Dowd method and the phenolic profile was quantified by HPLC-DAD. The MBC values of the extracts were evaluated by broth microdilution method. The amount of total phenolic and flavonoid compounds was higher in EPA than EPB. Both extracts revealed the presence of caffeic, coumaric, cinnamic, ferulic and 3,4-dihydroxybenzoic acids, with higher concentrations of coumaric and cinnamic acids. Staphylococcus spp. isolates were susceptible to EPA (90.9%), EPB (83.1%) and oxacillin (80.5%). The oxacillin susceptible isolates were also susceptible to EPA (70.1%) and EPB (80.6%), whereas those oxacillin-resistant strains were also susceptible to EPA (40.0%) and to EPB (26.7%). MBC ranged from 34.3 to 68.7µm/mL for EPA and from 68.7 to 137.5µg/mL for EPB. Both extracts inhibited significantly (100%) the clinical strains of MRSA, S. aureus ATCC 6538 and S. aureus ATCC 25923 at the concentration of 68.7µg/mL. It is concluded that both extracts of propolis, whose main constituents are coumaric and cinnamic acids, have high antimicrobial activity against the microorganisms studied, and EPA also against oxacillin-resistant strains. These findings reinforce its potential use for the treatment of bovine mastitis.(AU)

É cada vez mais oportuna a necessidade de descobrir e desenvolver terapias alternativas para tratamento da mastite causada por Staphylococcus spp. e de infecções bacterianas multirresistentes. Este estudo examinou a composição química e o potencial antimicrobiano de dois extratos etanólicos de própolis (EPA e EPB) contra setenta e sete isolados de Staphylococcus spp. obtidos a partir de mastite bovina subclínica; três estirpes clínicas de MRSA e duas de linhagens clínicas de S. aureus ATCC, identificadas como, S. aureus ATCC 6538 e S. aureus ATCC 25923, ambas metacilina resistentes. O teor total de fenólicos foi determinado pelo método de Folin-Ciocalteau, o teor de flavonoides totais pelo método Dowd e o perfil fenólico foi quantificado por HPLC-DAD. CBM dos extratos foi avaliada pelo método de microdiluição em caldo. A quantidade total de compostos fenólicos e flavonoides foi maior no EPA do que no EPB. Ambos os extratos revelaram a presença dos ácidos cafeico, cumárico, cinâmico, ferúlico e 3,4-di-hidroxibenzóico, com maiores concentrações de ácidos cumárico e cinâmico. Os isolados de Staphylococcus spp. foram sensíveis a EPA (90,9%), EPB (83,1%) e oxacilina (80,5%). Os isolados suscetíveis à oxacilina também foram suscetíveis ao EPA (70,1%) e ao EPB (80,6%), enquanto os do resistente à oxacilina foram suscetíveis ao EPA (40,0%) e ao EPB (26,7%). MBC variou de 34,3 a 68,7µm/mL para EPA e de 68,7 a 137,5µg/mL para EPB. Ambos os extratos inibiram significativamente (100%) as linhagens clínicas de MRSA, S. aureus ATCC 6538 e S. aureus ATCC 25923 na concentração de 68,7µg/mL. Conclui-se que os extratos etanólicos da própolis, cujos principais constituintes são os ácidos cumário e cinâmico, possuem atividade antimicrobiana contra os micro-organismos estudados, e o EPA também contra as cepas resistentes à oxacilina. Estes achados reforçam seu potencial uso para o tratamento da mastite bovina.(AU)

Inhibitory Effects on Clinical Isolated Bacteria and Simultaneous HPLC Quantitative Analysis of Flavone Contents in Extracts from Oroxylum indicum.

Oroxylum indicum is a medicinal plant in Thailand, which has been used as a tonic and for the treatment of various diseases. Extracts from various parts of O. indicum were reported as promoting in vitro antioxidant and antibacterial effects. Phytochemical analysis suggested that this plant contained some flavones. O. indicum fruit and seed water and ethanol extracts and their major flavonoids including baicalein, baicalin, and chrysin were tested for in vitro antibacterial activities on four clinical isolated bacteria, namely, Staphylococcus intermedius, Streptococcus suis, Pseudomonas aeruginosa, and ß-Escherichia coli, using a broth micro-dilution assay. The amounts of these three major flavonoids were also quantitatively analyzed using the high-performance liquid chromatographic (HPLC) method. O. indicum fruit ethanol extract from Nakhon Pathom province (OFNE) promoted the strongest antimicrobial activity against four clinical pathogenic bacteria, including S. intermedius (IC50 = 1.30 mg/mL), S. suis (13.59% inhibition at 7.81 mg/mL), P. aeruginosa (IC50 = 39.20 mg/mL), and ß-E. coli (IC50 = 66.85 mg/mL). Baicalin showed high in vitro antibacterial effect to all tested bacteria. From the optimized and validated HPLC method, baicalin, baicalein, and chrysin contents in O. indicum extracts were 0.19 ± 0.00 - 9.45 ± 0.13, 0.14 ± 0.00 - 1.27 ± 0.02, and 0.02 ± 0.00 - 0.96 ± 0.02 g/100 g extract, respectively. Baicalin was found to be the major compound in O. indicum seed extract followed by baicalein, whereas chrysin was found in lower amounts than the amounts of the other two flavonoids in all O. indicum extracts.