Microtiter Broth Dilution Method Research Articles

First Report on Salvia Sahendica Boiss & Buhs roots biocomponents characterization by GC-MS and HPLC and Antibacterial Potency

Treatment of bacterial infections with antibiotic resistance is complicated. For this reason, new therapeutic methods are needed to control bacterial infections. Therefore, it is necessary to discover new antibiotics from medicinal plants that can destroy drug-resistant bacteria. Essential oils (EO) of some medicinal plants have antimicrobial effects and can be used as antimicrobial agents in the treatment of infections and food industries. The aim of this study was to determine the EO composition and antimicrobial effects of EO and polyphenols profile of Salvia Sahendica roots for the first time. Chemical compounds and antibacterial activity of EOs against Staphylococcus aureus and Bacillus cereus as gram-positive and Escherichia coli and Pseudomonas aeruginosa as gram- negative bacteria were evaluated using microtiter broth dilution method. Studied organs of S. sahendica showed significant diversity in terms of type and percentage of essential oil and polyphenol compounds. Overall, 44, 46, 42, and 45 compounds were identified in the essential oil of leaves, flowers, stems, and roots of this plant, which constituted 99.6, 99.3, 98.2, and 99.4% of total oil composition, respectively. The current study collectively demonstrated inhibitory effects ranged from strong (S. aureus) to low inhibition (P. aeruginosa). According to the HPLC result, the amounts of coumaric acid, chlorogenic acid, and gallic acid compounds in the root are higher than in other plant parts, which indicate that the root can be a suitable source for the production of these valuable polyphenol compounds. Due to the results, it is possible to hope for the application of EO from S. sahendica as natural antibacterial in the pharmaceutical and food industries.

Can flow cytometric measurements of reactive oxygen species levels determine minimal inhibitory concentrations and antibiotic susceptibility testing for Acinetobacter baumannii?

Current antimicrobial susceptibility testing (AST) requires 16-24 hours, delaying initiation of appropriate antibiotics. Hence, there is a need for rapid AST. This study aims to develop and evaluate the feasibility of a rapid flow cytometric AST assay to determine minimum inhibitory concentration (MIC) for carbapenem-resistant Acinetobacter baumannii (CRAB). Antibiotic exposure causes increased intracellular reactive oxygen species (ROS) in bacteria. We hypothesized that ROS can be used as a marker to determine MIC. We assessed three CRAB clinical isolates across fifteen antibiotics at various concentrations in a customized 96-well microtiter plate. The antibiotics assessed include amikacin, beta-lactams (ampicillin/sulbactam, aztreonam, cefepime, ceftolozane/tazobactam, doripenem, imipenem, meropenem, and piperacillin/tazobactam), levofloxacin, polymyxin B, rifampicin, trimethoprim/sulfamethoxazole, and tetracyclines (tigecycline and minocycline). These clinical CRAB isolates were assessed for ROS after antibiotic treatment. Increased ROS levels indicated by increased RedoxSensorTM Green (RSG) fluorescence intensity was assessed using flow cytometry (FCM). MIC was set as the lowest antibiotic concentration that gives a ≥1.5-fold increase in mode RSG fluorescence intensity (MICRSG). Accuracy of MICRSG was determined by comparing against microtiter broth dilution method performed under CLSI guidelines. ROS was deemed accurate in determining the MICs for β-lactams (83.3% accuracy) and trimethoprim/sulfamethoxazole (100% accuracy). In contrast, ROS is less accurate in determining MICs for levofloxacin (33.3% accuracy), rifampicin (0% accuracy), amikacin (33.3% accuracy), and tetracyclines (33.3% accuracy). Collectively, this study described an FCM-AST assay to determine antibiotic susceptibility of CRAB isolates within 5 hours, reducing turnaround time up to 19 hours.

Evaluating antibacterial efficacy of extracts from Ludwigia hyssopifolia against pathogenic bacteria

Ludwigia hyssopifolia extracts have historically been consumed as a healthful drink and rich in biomass. Therefore, the aims of this study were to determine the most effective fractionated extracts of L. hyssopifolia against pathogens. The whole plants of L. hyssopifolia were extracted various fractions with methanol, hexane, chloroform, ethyl acetate, and aqueous. The total phenolics and flavonoids was screened using quantitative approaches. The extracts tested for antibacterial activity against pathogens, including Bacillus cereus, Staphylococcus aureus, Escherichia coli, Salmonella Typhi, and Helicobacter pylori through the agar diffusion technique and the microtiter broth dilution method. The largest yield was obtained from the aqueous fractional extract. The phytochemical composition discovered in ethyl acetate was more abundant in phenolics and flavonoid compounds. Fractionation enhanced the antibacterial effect of the extract. The extracts showed a broad spectrum of antibacterial activity against all the bacteria tested, particularly H. pylori. In parallel, ethyl acetate extract showed antibacterial activity. The current results could provide rational support for the traditional use of L. hyssopifolia to treat infectious diseases and provide data for developing natural products.

Polymeric micelle gel with luliconazole: in vivo efficacy against cutaneous candidiasis in Wistar rats.

The objective of this research was focused on the design and development of luliconazole-loaded polymeric micelle hydrogel (LUL-PM-CHG) using quality by design (QbD) principle to improve the penetration and retention of LUL in the skin. The optimization of the formulation involved the utilization of a Box-Behnken design with three factors and three levels. The impact of specific formulation variables, namely the ratio of poloxamer P123 and F127, sonication time, and the quantity of drug, was investigated in terms of particle size, micellar incorporation efficiency, and polydispersity index. The LUL-loaded P123/F127 mixed micelles involved the thin film hydration method for thin preparation. The characteristics of optimized formulation include a particle size of 226 ± 8.52nm, a polydispersity index (PDI) of 0.153 ± 0.002, a zeta potential (ZP) of 30.15 ± 2.32mV, and a micellar incorporation efficiency (MIE) of 88.38 ± 3.84%. In vitro release studies indicated a sustained release of LUL-PM-CHG for a duration of up to 8h. The MIC, GI50, and GI90 of different formulations on Candida albicans were determined using both the microtiter broth dilution method and the plate method and showed that LUL-PM-CHG exhibited the highest antifungal activity compared to the other formulations, with MIC values of 3.25 ± 0.19ng/mL, GI50 values of 37.11 ± 2.89, and GI90 values of 94.98 ± 3.41 The study also measured the % of inhibition activity and the generation of intracellular reactive oxygen species (ROS) using flow cytometry. LUL-PM-CHG showed the highest percentage of inhibition (75.5%) and ROS production (MFI-140951), indicating its enhanced activity compared to LUL-CHG and LUL. Fungal infection was induced in Wistar rats using immunosuppressant's treatment followed by exposure to C. albicans. Finally, in vivo fungal scaling and histopathological studies indicated a reduction in fungal infection in Wistar rat skin after treatment. The obtained results suggested that LUL-PM can serve as a promising formulation to enhance luliconazole antifungal activity and increase patient compliance.

Evaluation of Antisickling Activity and Antibacterial Potential of Methanolic Root Extract of Ximenia americana Linn. (Olacacaea)

Aims: This research work was done in order to evaluate invitro antisickling activity, and antibacterial potentials of the methanolic root extract of Ximenia americana, Linn. Olacaceae. Study Design: It is an experimental research work. Place and Duration of Study: Department of Microbiology and Biotechnology, National Institute of Pharmaceutical Research and Development (NIPRD), Idu, Abuja, Nigeria. The study was carried out between December, 2019 to June, 2020. Methodology: Evaluation of the antisickling activity of the methanolic root extract of Ximenia americana was carried out in two-fold; viz. the ability of the plant extract to inhibit or prevent sickling (antisickling effect) of erythrocytes from blood samples obtained from sickle cell disease (SCD) volunteers on one hand, and the ability of the extract to reversed pre-sickled erythrocytes using standard protocols. The antibacterial assay was done using microtiter broth dilution method to determine the minimum inhibitory concentration (MIC) on some selected encapsulated bacterial (E. coli, Salmonella typhi, Staphylococcus aureus, Pseudomonas mirabilis, Pseudomonas aeruginosa, Bacillus subtilis, and Klebsiella pneumonia) that have been reported to caused complications in SCD. Results: The methanolic root extract of Ximenia americana showed a concentration dependent antisickling activity across concentration gradient. The percent sickling inhibition was dose-dependent (0.05 mg = 60 %, 0.5 mg = 62 %, 1 mg = 88 %, 2 mg = 87 %, and 4 mg = 90 %), this result was statistically significant (p < 0.05 vs control) when compared to the untreated group (25 %). Similarly, the result of the sickle red cell reversal by the test extract demonstrated a percent dose-dependent reversibility (0.05 mg = 36 %, 0.5 mg = 40 %, 1 mg = 48 %, 2 mg = 58 %, and 4 mg = 63 %), a result that was significantly (p < 0.5 vs control) compared to the control (19 %). The antimicrobial assay result showed that, the sensitivity of the selected microorganisms to the plant extract was concentration dependent. Staphylococcus aureus and Bacillus subtilis showed better sensitivity to the extract with higher zones of inhibition (8 mm and 7 mm) at 80 mg/mL concentration. Conclusion: Methanolic root extract of Ximenia americana L demonstrated a significant dose-dependence antisickling and sickle cell reversal activities, with p < 0.05 vs control. Also, the extract showed sensitivity against some selected bacteria isolates as a result possess antibacterial activity against susceptible microorganism.

In Vitro Antimicrobial Activity of Plant Species against the Phytopathogens Ralstonia solanacearum, Phytophthora infestans, and Neopestalotiopsis javaensis

Plants are prone to be attacked by various pathogenic microorganisms, leading to significant crop yield losses. Pesticides are used to control agricultural pathogens; however, frequently, they are toxic synthetic products. This work evaluated the antibacterial and antifungal activity of Pernettya prostrata, and Rubus roseus plant extracts against three phytopathogens Ralstonia solanacearum, Phytophthora infestans, and Neopestalotiopsis javaensis responsible for causing banana bacterial wilt, late blight y scab diseases, respectively. The extracts were obtained in methanol. The phytopathogens were isolated from diseased plants grown in Ecuador, R. solanacearum was isolated from samples of Musa paradiasiaca, P. infestans from samples of Solanum lycopersicum and N. javaensis from diseased samples of Persea americana—morphological and molecular methods identified the isolated pathogens. The antibacterial activity was determined by the microtiter broth dilution method; six serial concentrations of the extracts were tested. The antifungal activity was determined based on the effects of the plant extracts on the inhibition of radial growth of fungi; five serial concentrations of the extracts were tested. The extracts of P. prostrata and R. roseus showed activity against R. solanacearum and P. infestans with a MIC of 22.5 and 31.25 mg/mL, respectively.

Antibacterial Activities and Chemical Compounds of Plantago lanceolata (Ribwort Plantain) and Plantago major (Broadleaf Plantain) Leaf Extracts

Background: Plantago lanceolata L. (ribwort plantain) and Plantago major L. (broadleaf plantain) are widely used in ethnobotanical studies and for treating various diseases. This study aims to investigate the antimicrobial activity and chemical compounds of these plants. Methods: The leaf extracts of P. lanceolata and P. major were fractioned using different solvents. The phytochemical screening was carried out by the gas chromatography-mass spectrometry (GC-MS) method. The antibacterial activity of extracts was assessed using the disc diffusion method, and the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) were measured by microtiter-broth dilution method. Results: The dichloromethane leaf extract of P. lanceolata and P. major showed the highest antibacterial activity against Salmonella paratyphi (diameter of the inhibition zone: 18.83 and 20.00 mm, respectively) at 100 mg/mL concentration. The lowest MIC was related to dichloromethane extracts of both plants against S. paratyphi (500 µg/mL). The lowest MBC (1000 µg/mL) was related to the dichloromethane extract of P. major against S. paratyphi. The main compounds of P. lanceolata leaf extracts were bis(2-ethylhexyl) phthalate (41.96%), 1-methoxy-3-(2-hydroxyethyl)nonane (32.69%), bicyclo[3.1.1]heptane, 2,6,6-trimethyl- (1.alpha.,2.beta.,5.alpha.)- (10.45%), and cycloheptasiloxane tetradecamethyl- (27.96% and 31.33%). The main compounds of P. major leaf extracts were eicosane (23.62%), cyclohexasiloxane dodecamethyl- (18.21%), 1-methyl-3-n-propyl-2-pyrazolin-5-one (18.08%), cycloheptasiloxane tetradecamethyl- (33.85%), and 1,2-benzisothiazole-3-acetic acid, methyl ester (34.26%). Conclusion: Fractionation of the methanolic leaf extract of P. lanceolata and P. major can help better isolate active components from these plants. The antibacterial properties of the extracts of two plants may be due to the presence of antibacterial compounds detected in GC-MS.

Antibacterial activity and phytochemical components of leaf extract of Calpurnia aurea

Local Ethiopians use Calpurnia aurea to treat skin infections. However, there is no adequate scientific confirmation. The aim of this study was to evaluate the antibacterial activities of the crude and the fractionated extracts of C. aurea leaves against different bacterial strains. The crude extract was made by maceration. The Soxhlet extraction method was used to obtain fractional extracts. The antibacterial activity against gram positive and gram negative American Type Culture Collection (ATCC) strains was performed using the agar diffusion technique. The minimum inhibitory concentration was determined through the microtiter broth dilution method. Preliminary phytochemical screening was done using standard techniques. The largest yield was obtained from ethanol fractional extract. Except for chloroform, which provided a relatively low yield when compared to petroleum ether, increasing the polarity of the extracting solvent improved the yield. The crude extract, solvent fractions, and the positive control showed inhibitory zone diameter, while the negative control did not. When used at a concentration of 75 mg/ml, the crude extract had similar antibacterial effects as gentamicin (0.1 mg/ml) and the ethanol fraction. The 2.5 mg/ml crude ethanol extract of C. aurea suppressed the growth of Pseudomonas aeruginosa, Streptococcus pneumoniae, and Staphylococcus aureus, according to the MIC values. The extract of C. aurea was more effective in inhibiting P. aeruginosa than the other gram-negative bacteria. Fractionation enhanced the antibacterial effect of the extract. All fractionated extracts showed the highest inhibition zone diameter against S. aureus. Petroleum ether extract had the greatest inhibition zone diameter against all bacterial strains. The non-polar components were more active compared to the more polar fractions. The phytochemical components discovered in the leaves of C. aurea included alkaloids, flavonoids, saponins, and tannins. Among these, the tannin content was remarkably high. The current results could provide a rational support for the traditional use of C. aurea to treat skin infections.

Effect of alkynyloxy derivatives of lawsone as an antifungal spray for acrylic denture base: An in vitro study

ObjectiveThe purpose of this study was to (i) synthesize and develop an alkynyloxy derivative of lawsone as an antifungal spray and (ii) assess the antifungal spray’s effectiveness in reducing the viability of Candida albicans (C. albicans) on polymethylmethacrylate (PMMA) specimens. MethodsLawsone methyl ether (LME) and its derivative, 2-(prop-2-ynyloxy)naphthalene-1,4-dione (compound 1) were synthesized and characterized. The synthetic compounds were screened for antimicrobial activities against C. albicans using the microtiter broth dilution method to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Compound 1 was further formulated as an antifungal spray in three concentrations (100, 200, and 400 μg/mL). C. albicans biofilms were developed for 48 h on PMMA specimens. The efficacy of using an antifungal spray for 1 and 3 min to remove biofilm was assessed using colony counting and scanning electron microscopy (SEM). Chlorhexidine gluconate (CHX), polident®, and distilled water were used as positive and negative control cleansing solutions, respectively. ResultsLME and compound 1 showed comparable inhibition against C. albicans with a MIC of 25 μg/mL and MFC of 50 μg/mL. For immediate treatment, C. albicans was not detected on PMMA specimens when expose to 2% CHX and compound 1 (100, 200, and 400 μg/mL) antifungal spray for 3 min. However, after recolonization, a small number of viable cells were observed in denture soaked in compound 1 antifungal spray for 3 min group. Following recolonization, polident® and distilled water had comparable viable cell counts of C. albicans to the no treatment group. Scanning electron microscope (SEM) images revealed that CHX, polident®, and compound 1 caused cell damage in various forms. ConclusionDenture spray containing synthetic alkynyloxy derivative of lawsone is a promising antifungal agent for C. albicans biofilm removal from the PMMA surface.

Investigation of the effects of antimicrobial and anti-biofilm peptide IDR1018 and chitosan nanoparticles on ciprofloxacin-resistant Escherichia coli.

Peptide IDR1018 and chitosan nanoparticles (CNs) showed antimicrobial and anti-biofilm activity against bacteria. In this study, the antimicrobial effects of peptide IDR1018 and CNs were evaluated on 50 clinical isolates of uropathogenic Escherichia coli (UPEC) resistant to ciprofloxacin. Ion gelation method was applied for CNs synthesis. Scanning electron microscope (SEM) and dynamic light scattering (DLS) were utilized to evaluate the nanoparticles. Antimicrobial and synergistic activity of peptide IDR1018 and CNs with ciprofloxacin were evaluated by microtiter broth dilution method. The checkerboard test was used to investigate the antimicrobial effects of IDR1018 and CNS in combination with ciprofloxacin. Anti-biofilm effect of ciprofloxacin, peptide IDR1018, and CNs was evaluated using crystal violet method. Fourteen (28%), 21 (42%), and 15 (30%) of clinical isolates produced strong, moderate, and weak biofilm, respectively. The CNs were spherical and uniform under electron microscopy with an average diameter of 246 nm. The minimum inhibitory concentration (MIC) values were 16-128, 20-40, and 375-750 (µg/ml) for ciprofloxacin, peptide IDR1018, and CNs, respectively. Fractional inhibitory concentration (FIC) analysis indicated a synergistic effect of ciprofloxacin in combination with peptide IDR1018, but in combination with CNs, this antibiotic showed an additive effect. Our results revealed that peptide IDR1018 and CNs have antimicrobial properties on UPEC isolates. Biofilm inhibition and biofilm eradication of clinical isolate were shown by peptide IDR1018 and CNs in a concentration-dependent manner. The antimicrobial agents alone and in combination decreased the number of viable bacteria in the biofilms. Therefore, these components seem to be a treating approach against biofilm-forming UPEC isolates.

Antibacterial, Antiradical and Antiproliferative Potential of Green, Roasted, and Spent Coffee Extracts

The phytochemical compositions of green coffee beans (GB), roasted coffee (RC), and the solid residue known as spent coffee grounds (SCG) have been associated with beneficial physiological effects. The objective of this study was to analyze the total phenolic compounds, antiradical scavenging ability, antibacterial activity, and antiproliferative activity on cancer cells of aqueous and ethanolic extracts of GB, RC, and SCG samples. The total phenolic content was quantified by Folin–Ciocalteu assay, while the antiradical activity was evaluated by ABTS●+ and DPPH radical assays, antibacterial activity was determined using the microtiter broth dilution method, and antiproliferative activity was evaluated by MTT assay in lung carcinoma cells (A549) and cervical cancer cells (C33A); furthermore, apoptosis and cell cycle arrest were evaluated by flow cytometry. Ethanolic extracts of RC and SCG showed the highest content of total phenols. The SCG ethanolic extract exhibited the lowest inhibitory capacity 50 (IC50) values for free radicals. The SCG extracts also had the lowest MIC values in bacteria. In antiproliferative assays, SCG extracts exhibited a significant decrease in viability in both cell lines, as well as increased apoptotic cells and promoted cell cycle arrest. The higher content of total phenols and antiradical activity of SCG ethanolic extracts was related to their antiproliferative activity in cancer cells, as well as their antibacterial activity against clinical isolates; therefore, the utilization of SCG adds value to an abundant and inexpensive residue.

Inhibitory effects of Lactobacillus casei Shirota against both Candida auris and Candida spp. isolates that cause vulvovaginal candidiasis and are resistant to antifungals

BackgroundVulvovaginal candidiasis (VVC), the second leading cause of genital infection in women of reproductive age, is caused by yeasts of the genus Candida. Treatment is usually empirical and performed with azoles, which have shown increasing ineffectiveness due to resistance from these species. This therapeutic challenge has led to the search for new treatment strategies. Lactobacillus spp. produce several components with microbicidal effects, such as lactic acid. These species are the main components of a healthy vaginal microbiota and have been used as probiotics. The aim of this work was to investigate the in vitro inhibitory effects of Lactobacillus casei Shirota on both the Candida spp. that cause VVC and on C. auris.MethodsThe microbicidal effects of L. casei Shirota on the main VVC-causing species, C. albicans, C. tropicalis, C. norvegensis and C. parapsilosis, in addition to C. auris were investigated by counting the Colony-forming Units (CFUs) after cocultivation. The antifungal activity of lactic acid against these Candida strains was assessed using the microtiter broth dilution method to determine the minimum inhibitory concentrations (MICs). The effects of L. casei Shirota on hyphal and early biofilm formation was measured by optical microscopy.ResultsL. casei Shirota showed inhibitory action against all tested Candida spp., ranging from 66.9 to 95.6% inhibition depending on the species. This inhibition is possibly related to the production of lactic acid, since lactic acid has shown microbicidal action against these same Candida spp. at a concentration of 5 mg/mL, which corresponds to half of the normal physiological concentration. In addition, L. casei Shirota was able to reduce the formation of C. albicans hyphae and early biofilms, showing strong anti-Candida effects.ConclusionsThese results suggest that L. casei Shirota has antifungal activity against the Candida species that cause VVC. L. casei also has microbicidal action against C. auris.

Physicochemical and nutritional profile of leaves, flowers, and fruits of the edible halophyte chorão-da-praia (Carpobrotus edulis) on Portuguese west shores

Leaves, flowers and fruits from the edible halophyte chorão-da-praia (Carpobrotus edulis), harvested in the Portuguese western sea shores, were characterized regarding their proximate composition, fatty acid profile, pigments, phenolic compounds, antioxidant and antimicrobial activities. Fruits had the highest amount of carbohydrates (70.2 ± 1.6%) and the lowest of protein (4.67 ± 0.22%) and ash (22.0 ± 1.0%) contents. No significant differences were observed in the lipid fraction. Leaves were mainly constituted by saturated (52.6 ± 1.3%) fatty acids (FA), whereas fruits showed the highest amount of polyunsaturated FA (48.6 ± 3.1%). Chlorophylls and carotenoids were more abundant in leaves. Aqueous-ethanolic (1:1, v/v) extract of flowers exhibited the highest phenolic content (299 ± 8 μg/mg of gallic acid equivalents), by the Folin–Ciocalteu methodology, as well as the strongest antioxidant activity (IC50 = 11.83 ± 1.0 μg/mL and 7.82 ± 0.24 μmol/mg of ascorbic acid equivalents by the DPPH and FRAP methods, respectively). Also, flowers extract showed the most pronounced antimicrobial activity by the microtiter broth dilution method. Mass and tandem mass spectra revealed that the most abundant species in all extracts were glycosylated and/or acetylated O-Methyl flavonols, mainly derived from syringetin and laricitrin. Nuclear magnetic resonance was also employed for complementary confirmation of the syringetin moieties. Regarding the profile in polyphenols, when comparing leaves, flowers and fruits, one concluded that there were clearly differences in the relative amount of these polyphenols, being flowers richer in the syringetin derivatives. In conclusion, this edible plant, classified as an invasive species of the Portuguese coast, highlights for its richness in bioactive compounds with potential application in the food industry.

Evaluation of Antibacterial Activity of Aqueous, Ethanolic and Methanolic Extracts of Areca Nut Fruit on Selected Bacteria.

Today, the tendency to use of natural preservatives to increase food security has expanded. In the present study, antibacterial effects of Areca Nut fruit extracts were evaluated against Staphylococcus aureus, Escherichia coli, Salmonella enterica, and Enterobacter aerogenes bacteria using agar disc diffusion technique. Methanol, ethanol, and water were used as solvents for extraction by maceration method, and extracts were analyzed by GC-MS. The antibacterial activity was evaluated using microtiter broth dilution method to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results revealed that all ATCC strains were significantly inhibited by ethanolic and methanolic extracts. Escherichia coli produced a significantly larger zone of inhibition for Gentamicin (35 ± 0.65 mm) and Penicillin (25 mm ± 0.56), while Enterobacter aerogenes produced smaller zone of inhibition for Gentamicin (20 ± 0.87 mm) and Penicillin (15 ± 0.87 mm). Also, methanolic extract had considerable antibacterial activity with MIC value of 1.56 mg/mL against Escherichia coli. All of extracts were used to evaluate antibacterial effects in prepared cake, and as a result, all pathogenies were the most sensitive by methanolic extract in 100 mg/L of concentration except Escherichia coli that were more sensitive by ethanolic extract. In conclusion, the Areca Nut fruit extracts may be used as a natural preservative in food industries. Future studies should focus on the effect of Areca Nut fruit extracts in bakery and drinking industries.

Antibiofilm Activity of Nystatin, Aspirin and EDTA Against Candida albicans Isolated from Iraqi Women with Vulvovaginitis

The vulvovaginitis candidiasis is often associated with biofilm formation by Candida albicans and using ofantifungal agents against C. albicans biofilms is urgently needed. Microtiter plate assay using crystal violetwas used for detection the ability of Candida albicans to form biofilm and the microtiter broth dilutionmethod was used for determination the minimum inhibitory concentrations (MICs). Out of 42 Candidaalbicans isolated from vulvovaginitis, 37 (88%) can produce biofilm at the varying degrees. Twenty-eight(75.7%) isolates could form a strong biofilm. The results of minimum inhibitory concentrations (MICs) ofNystatin, Aspirin and EDTA (Ethylenediaminetetraacetic acid) against 28 C. albicans isolates which formedthe strong biofilm, revealed that range of concentrations of Nystatin were (6.25-100 µg/ml), while the MICsof aspirin and EDTA were more than 1000 µg/ml. It was obvious that the Nystatin had the inhibitory activityat the concentrations 6.25 and 12.5 µg/ml. The highest antibiofilm activity by Nystatin were demonstratedat the subinhibitory concentration 50 ?g/ml with biofilm eradication percent (75.80%), while the lowestantifungal effect (2.86-10.70%) was at very low concentrations (3.125-6.25 ?g/ml). Also, there was anobvious biofilm eradication of Aspirin and EDTA at the concentrations 500 and 1000 ?g/ml but the effectof aspirin at the concentration 1000 ?g/ml (70.51%) is more than EDTA (60.12%) in contrast with theconcentration 500 ?g/ml, it was found that the effect of EDTA (51.29%) is more than aspirin (34.25%). Inconclusion, the present study highlights the role of Aspirin and EDTA as antibiofilm agents when used withNystatin which have the ability to inhibit the growth of C. albicans in patients with vulvovaginitis.

Characterizing Antimicrobial Resistance in Chicken Pathogens: A Step towards Improved Antimicrobial Stewardship in Poultry Production in Vietnam.

In the Mekong Delta of Vietnam, farmers use large quantities of antimicrobials to raise small-scale chicken flocks, often including active ingredients regarded of “critical importance’” by the World Health Organization. Due to limitations in laboratory capacity, the choice of antimicrobials normally does not follow any empirical criteria of effectiveness. The aim of this study was to highlight non-critically important antimicrobials against which chicken pathogens are likely to be susceptible as a basis for treatment guidelines. Microtiter broth dilution method was performed to determine the minimal inhibitory concentration (MIC) of 12 commonly used antimicrobials for 58 isolates, including Ornithobacterium rhinotracheale (ORT) (n = 22), Gallibacterium anatis (n = 19), and Avibacterium endocarditidis (n = 17). Unfortunately, internationally accepted breakpoints for resistance in these organisms do not exist. We drew tentative epidemiological cut-offs (TECOFFs) for those antimicrobial-pathogen combinations where MIC distributions suggested the presence of a distinct non-wild-type population. Based on the observed results, doxycycline would be the drug of choice for A. endocarditidis (11.8% presumptive non-wild type) and G. anatis infections (5.3% presumptive non-wild type). A total of 13.6% ORT isolates were non-wild type with regards to oxytetracycline, making it the drug of choice against this pathogen. This study illustrates the challenges in interpreting susceptibility testing results and the need to establish internationally accepted breakpoints for veterinary pathogens.

Antibacterial activities of the extracts, fractions and isolated compounds from Canarium patentinervium Miq. against bacterial clinical isolates

BackgroundCanarium patentinervium leaves are used by the local indigenous people of Malaysia for wound healing. The current study is undertaken to screen the comprehensive antibacterial activity of the leaves and barks extracts, fractions and isolated compounds from this plant. Bioassay guided fractionation was also undertaken to deeply evaluate the antibacterial activity of the water fraction of the leaves extract. This is to provide preliminary scientific evidence to the ethnopharmacology usage of this plant by investigating antibacterial properties of the plant and its isolated constituents.MethodsBio-assay guided fractionation and subsequent isolation of compounds using open column chromatography. The antibacterial activity against gram positive and gram negative ATCC strain and resistant clinical strains were evaluated using microtiter broth dilution method to determine minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill assay. The chemical structure of the isolated compounds from the water fraction of the ethanol extract of leaves was elucidated using Nuclear Magnetic Resonance (NMR).ResultsThe ethanol extract of the leaves and barks showed antimicrobial activity against all four ATCC and eight clinical isolates. The ethanol extract of the leaves and the corresponding water fraction had good activity against MRSA S. aureus. (MIC: 250 μg/ml) and had bactericidal effect on eight of the clinical strains (MSSA,MRSA, oxacillin-resistant CONS, oxacillin-sensitive CONS, Enterococcus faecalis, Klebsiela species, Kleb pneumoniae ESBL and Candida parapsilosis). Further phytochemical investigation of the water fraction of the crude ethanol extract of leaves afforded compound 7 (hyperin) and compound 8 (cynaroside) that had bactericidal activity against tested bacterial species (MIC 50 μg/ml and 100 μg/ml). The two compounds were isolated from this genus for the first time.ConclusionsThese results may provide a rational support for the traditional use of Canarium patentinervium Miq. in infections and wound healing, since the antimicrobial compounds isolated were also present in the leaves extract.

Determination of Antimicrobial Properties of Crude Aqueous Leaves Extracts of Selected Medicinal Plants Using Resazurin-Based Microtiter Broth Dilution Method

The Philippines has a wide range of medicinal plants species that need to be tested for safety and efficacy for the possibility of serving as basis in drug development in the future. Five medicinal plants commonly used in the province of Cavite, Philippines were selected for crude aqueous leaf extraction. These were Annona muricata L., Cymbopogon citratus DC., Graptophyllum pictum (L.) Griff, Jatropha curcas L. and Piper betle L. Extracts were tested for antimicrobial activities using resazurin-based microtiter broth dilution method. P. betle showed the maximum antimicrobial activity with minimum inhibitory concentrations (MIC) of 4.69 mg/ ml against Pseudomonas aeruginosa and Staphylococcus aureus and 37.50 mg/ ml against Candida albicans . This then validated the efficiency of P. betle in treating skin diseases and other infections as reported in the locality.

Starvation- and antibiotics-induced formation of persister cells in Pseudomonas aeruginosa.

Planktonic stationary and exponential cultures of Pseudomonas aeruginosa are highly resistant to killing by bactericidal antimicrobials because of the presence of persisters, cells that are multidrug tolerant and play a key role in the recalcitrance of biofilm infections. The aim of this study was to investigate the formation of persister cells in P. aeruginosa stationary vs. exponential cultures using different class antimicrobials. The susceptibilities of P. aeruginosa PAO1 wild-type and mutant strains to antimicrobials were determined by standard microtiter broth dilution method. In order to determine persister formation, dose- and time-dependent killing experiments were performed with antibiotics. Ceftazidime (Cephalosporin) showed little efficacy against either culture. Stationary-phase cells were more tolerant to imipenem (Carbapenem) than exponential cells, leaving a small fraction of persisters at high imipenem concentration in both populations. Polymyxin B (Polymyxin) appeared to be ineffective at low concentrations against both cell populations. Very high polymyxin B concentration completely eradicated exponential cells and regrowth was seen in a stationary population. Stationary cells were more tolerant to tobramycin (Aminoglycoside) than exponential cells but a higher concentration of tobramycin completely eliminated survivors. Ciprofloxacin (Fluoroquinolone) at a low concentration resulted in killing of both cultures of P. aeruginosa, producing persisters that were invulnerable to killing. Stationary cells appear to be somewhat more tolerant than exponential cells in all of these assays. We also showed that nutrient deprivation (serine starvation) regulated by stringent and general stress response, contribute to the increased tolerance of P. aeruginosa exponential and stationary planktonic cells via production of persisters.

Screening for antibacterial and antibiofilm activities in Astragalus angulosus.

In a search for finding novel therapeutic agents, extracts from an endemic Lebanese plant, Astragalus angulosus, were evaluated for their potential in-vitro antibacterial and antibiofilm activities against three Gram-positive bacterial strains; Staphylococcus epidermidis (CIP444), Staphylococcus aureus (ATCC25923), and Enterococcus faecalis (ATCC29212); in addition to two Gram-negative strains, Escherichia coli (ATCC35218) and Pseudomonas aeruginosa (ATCC27853). The plant was collected in April of 2013 and divided into several different portions, then its extracts were obtained by maceration using two different solvents. Extract analysis followed directly where microtiter broth dilution method was employed to assess antibacterial activity, while antibiofilm potential was tested using colorimetric method. Whole plant ethanolic extract showed the highest bacteriostatic effect at a concentration of 12.78 mg/ml and also was the most versatile exerting its effect against 3 different strains. Other extracts also exhibited an effect but at higher concentrations and each against a single strain. Regarding antibiofilm activity, the majority of the extracts were able to eradicate >50% of S. epidermidis preformed biofilm, where the highest activity was obtained with flower fraction extracted in water, achieving 67.7% biofilm eradication at 0.2 mg/ml. This plant possesses a promising potential in regard to eradicating bacteria and their biofilms and it is the first contributing step of establishing a library for the endemic Lebanese plants in this domain.