Compounds from Vernonia arborea Buch.-Ham. Inhibit Microbes that Impair Wound Healing

Aims: To identify the antimicrobial potency of the leaf fractions of Vernonia arborea against selected wound microbes viz., Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Stenotrophomonas maltophilia. Background: Wound healing is often delayed due to the presence of polymicrobial load, that have to be abolished to facilitate the healing process. A major class of antimicrobial phytocompound reported to occur in Vernonia arborea species include sesquiterpenes. Reports on the wound healing potency of V. arborea in wound models of Wistar rats however did not report antimicrobial activity of the aqueous or methanolic extracts. Methodology: The column fractions of the hexane leaf extract were tested against the selected strains by agar well diffusion assay and the zone of inhibition confirmed with TLC bioautography at specific Rf. The minimum inhibitory concentration (MIC) of the bioactive fractions was identified using resazurin microtiter assay (REMA) and the minimum bactericidal concentration (MBC) was determined. HPTLC quantification was also performed. Results: Out of the 30 pooled fractions, six showed antimicrobial potency against all the five tested wound microbes. The minimum inhibitory concentrations of these fractions were determined, ranging from 15.62 µg/mL to 500 µg/mL for the different microbes. Quantitative High-Performance Thin Layer Chromatography (HPTLC) revealed two compounds (a and b) in the bioactive fraction10 with yields of 633 mg (63%) and 97 mg (9.7%) per gram of the extract. Conclusion: The findings suggest the potential use of the bioactive compound in chronic infectious wound management therapy.

Antibiotic Susceptibility of Environmental Legionella pneumophila Strains Isolated in Northern Italy

Legionella pneumophila is ubiquitous in aquatic environments and responsible for severe pneumonia in humans through inhalation of aerosol containing Legionella spp. Macrolides and fluoroquinolones are frequently used antimicrobials, but treatment failures are increasingly being reported. As susceptibility testing is not routinely performed, this study aimed to determine the minimum inhibitory concentrations (MICs) on 58 environmental Legionella pneumophila strains (24 of serogroup 1 and 34 of non-serogroup 1) isolated in Northern Italy. MICs of azithromycin, erythromycin, ciprofloxacin, levofloxacin, and rifampicin were determined by the microdilution method using buffered yeast extract broth supplemented with α-ketoglutarate (BYEα). Seventy-five percent of Legionella pneumophila isolates showed MIC values below the tentative highest MICs indicated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); rifampicin was the most active agent with MIC90 values below 0.008 mg/L. Interestingly, one isolate was tested and found to be PCR-positive for the azithromycin LpeAB active efflux system, further confirmed by the reserpine/resazurin microtiter assay. In conclusion, this study has provided additional susceptibility data for environmental Legionella pneumophila isolates from Northern Italy demonstrating, in general, low MICs values for the tested antimicrobials, although one strain tested was shown to possess the LpeAB resistance determinant, indicating that future surveillance studies are warranted.

Antibacterial activity of secondary metabolites isolated from Ficus aurata (Miq.) fruits

This research aimed to isolate chemical constituents from ethyl acetate and n-hexane fractions of Ficus aurata (Miq.) fruits and investigated their antibacterial activities. Chemical constituents were separated by using vacuum column chromatography, gravity column chromatography, and thin layer chromatography. The structures of the isolated compounds 3,4-dihydroxybenzoic acid (1) and β-sitosterol (2) were elucidated using various spectroscopic methods, including UV, IR, and NMR (1H-NMR, 13C-NMR, and DEPT). While, antibacterial activity was done by assay using Resazurin Microtiter Assay method. These 2 compounds reported as new constituents from F. aurata fruit. In addition, analysis of the bioassay showed that compound 1 had a notable antibacterial activity against gram negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATTC 27853), and gram-positive bacteria (Staphylococcus aureusATCC 23235) with the Minimum Inhibitory Concentration (MIC) value of 0.1563 µg/mL, and it indicated the same MIC as cefadroxil (positive control). Compound 2 has weak antibacterial activity compared to cefadroxil (positive control). The results found that Ficus aurata (Miq.) fruits metabolites have antibacterial activity and show potency as antibacterial agent. Different kinds of bacteria and assay could be needed to investigate and support its antibacterial activity in the future.

An Antisense yycF RNA Modulates Biofilm Organization of Methicillin-Resistant Staphylococcus aureus and Pathogenicity in a Rat Model of Osteomyelitis

Staphylococcus aureus (S. aureus) is one of most common opportunistic pathogens and is attributed to several human infections. The increasing incidence of methicillin-resistant S. aureus (MRSA) is a serious clinical threat for osteomyelitis crisis. The YycFG two-component system of S. aureus regulates genes associated with biofilm formation. To investigate the potential role of an antisense yycF RNA in the regulation of transcription levels of yycF and associated effects on biofilm formation and pathogenicity, antisense yycF (ASyycF) RNA was detected by RT-PCR and 5′ RACE assays. ASyycF overexpression mutants were constructed, and the biofilm biomass was determined by crystal violet microtiter assay and scanning electron microscopy (SEM). Quantitative RT-PCR and Western blotting analyses were used to detect whether ASyycF overexpression inhibited the transcription and translation of biofilm-related genes. Then, a rat tibial infective model was used to evaluate the pathogenicity of ASyycF overexpression in vivo. ASyycF transcription led to reductions in YycF production and biofilm formation. Overexpression of ASyycF inhibited the transcription and translation of biofilm-related genes. The sensitivity to vancomycin was improved in ASyycF-overexpressing MRSA. Furthermore, ASyycF inhibited MRSA invasion in a rat tibial infection model. From this study, the expression of the YycF protein was found to be inversely correlated with different levels of ASyycF transcription. The biofilm biomass and pathogenicity decreased in the ASyycF-overexpressing mutant. Thus, the current evidence may support ASyycF as a supplementary strategy for managing S. aureus and MRSA infections.

Antisense RNA Enhanced Sensitivity of Methicillin-resistant Staphylococcus Aureus to Hydrogen Peroxide

Background: Staphylococcus aureus (S. aureus) is the leading cause of various infective diseases including topical soft tissue infections. Due to an increasing prevalence of the methicillin-resistant Staphylococcus aureus (MRSA), S. aureus infection become more challenging to being treated over recent years. The goals of this study were to investigate the roles of antisense yycF/G RNAs in the regulation of the biofilm formation and pathogenicity. Methods: MRSA ASyycF/G overexpression mutants were constructed. The biofilm biomass was determined by crystal violet microtiter assay and scanning electron mi-ceroscopy (SEM). Quantitative RT-PCR and western blotting analysis were used to detect the transcripts and translations of biofilm-related genes. The effects of the antisense RNAs (ASyycF/G) strategy on the susceptibility of biofilm-producing S. aureus to hydrogen peroxide were also investigated. Result: The ASyycF/G transcript leaded to reduction in the biofilm formation. Overexpression of ASyycF/G inhibited the transcripts and translations of biofilm-related genes. The sensibility to vancomycin was improved in ASyycF/G overexpression MRSA. Conclusions: The biofilm biomass and the transcripts of the pathogenicity associated genes decreased in the ASyycF/G overexpression mutant. Thus, the current evidence may provide a supplementary strategy for managing MRSA infections.

Outlining In Vitro and In Silico Cholinesterase Inhibitory Activity of Twenty-Four Natural Products of Various Chemical Classes: Smilagenin, Kokusaginine, and Methyl Rosmarinate as Emboldening Inhibitors

Cholinesterase (ChE) inhibition is an important treatment strategy for Alzheimer’s disease (AD) as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are involved in the pathology of AD. In the current work, ChE inhibitory potential of twenty-four natural products from different chemical classes (i.e., diosgenin, hecogenin, rockogenin, smilagenin, tigogenin, astrasieversianins II and X, astragalosides I, IV, and VI, cyclocanthosides E and G, macrophyllosaponins A-D, kokusaginin, lamiide, forsythoside B, verbascoside, alyssonoside, ipolamide, methyl rosmarinate, and luteolin-7-O-glucuronide) was examined using ELISA microtiter assay. Among them, only smilagenin and kokusaginine displayed inhibitory action against AChE (IC50 = 43.29 ± 1.38 and 70.24 ± 2.87 µg/mL, respectively). BChE was inhibited by only methyl rosmarinate and kokusaginine (IC50 = 41.46 ± 2.83 and 61.40 ± 3.67 µg/mL, respectively). IC50 values for galantamine as the reference drug were 1.33 ± 0.11 µg/mL for AChE and 52.31 ± 3.04 µg/mL for BChE. Molecular docking experiments showed that the orientation of smilagenin and kokusaginine was mainly driven by the interactions with the peripheral anionic site (PAS) comprising residues of hAChE, while kokusaginine and methyl rosmarinate were able to access deeper into the active gorge in hBChE. Our data indicate that similagenin, kokusaginine, and methyl rosmarinate could be hit compounds for designing novel anti-Alzheimer agents.

Phytochemical Profiling of Methanolic Fruit Extract of Gardenia latifolia Ait. by LC-MS/MS Analysis and Evaluation of Its Antioxidant and Antimicrobial Activity

Gardenia latifolia Ait. (Rubiaceae) is also known as Indian Boxwood is a small deciduous tree often growing in southern states of India. In the present study, phytochemical profiling of methanolic extract of G. latifolia fruits were carried out using FTIR and LC-MS/MS analysis. Besides, its antioxidant and antimicrobial potential have been analysed using DPPH activity, differential pulse voltammetry and resazurin microtiter assay, respectively. Phytochemical profiling revealed the presence of 22 major diversified compounds and main were 3-caffeoyl quinic acid (chlorogenic acid), 3,4-Di-O-caffeoyl quinic acid, 6-O-trans-feruloylgenipin gentiobioside, 10-(6-O-trans sinapoyl glucopyranosyl) gardendiol, isoquercitrin, scortechinones, secaubryenol, iridoids and quercetin 3-rutinoside (rutin). The extract showed antioxidant activity (IC50 = 65.82) and powerful antibacterial activity with lowest minimum inhibitory concentration against Gram-positive Staphylococcus aureus (15.62 µg/µL), Bacillus subtilis (31.25 µg/µL) than gram negative Escherichia coli (62.5 µg/µL), Klebsiella pneumoniae (62.5 µg/µL), Pseudomonas aeruginosa (31.25 µg/µL). This study shows that the fruits of G. latifolia have tremendous potential to be used in food industries, phyto-therapeutics and cosmetic industries.

Encapsulation and Characterization of Meropenem in Niosome Nanoparticles and Inhibitory Effects of Antibiofilm and Antibacterial on Methicillin and Vancomycin Resistant Strains of Staphylococcus Aureus

Background: We aim to assess the antibacterial and anti-biofilm properties of niosome-encapsulated meropenem. Methods: After isolating S. aureus isolates and determining their microbial sensitivity, their ability to form biofilms was examined using plate microtiter assay. Various formulations of niosome-encapsulated meropenem were prepared using the thin-film hydration method, Minimum Biofilm Inhibitory Concentration (MBIC) and Minimum Inhibitory Concentration (MIC) were determined, and biofilm genes expression was examined. Drug formulations’ toxicity effect on HDF cells were determined using MTT assay.Results: Out of the 162 separated Staphylococcus aureus, 106 were resistant to methicillin. 87 MRSA isolates were vancomycin-resistant, all of which could form biofilms. The F1 formulation of neoplastic meropenem with a size of 51.3 ± 5.84 and an encapsulation index of 84.86 ± 3.14 was detected, which prevented biofilm growth with a BDI index of 69% and reduced icaD, FnbA, Ebps biofilms’ expression with p ≤0.05 in addition to reducing MBIC and MIC by 4-6 times. Interestingly, F1 formulation of neoplastic meropenem indicated cell viability over 90% at all tested concentrations. Conclusions: Results of the present study indicate that niosome-encapsulated meropenem reduces the resistance of Staphylococcus aureus MRSA to antibiotics in addition to increasing its anti-biofilm and antibiotic activity, and could prove useful as a new strategy for drug delivery.

Fungicide Management of Pasmo of Flax and Sensitivity of Septoria linicola to Pyraclostrobin and Fluxapyroxad

Among the diseases that have the potential to cause damage to flax every year, pasmo, caused by Septoria linicola, is the most important. Fungicide application and a diverse crop rotation are the most important strategies to control this disease because there is little variation in resistance among flax cultivars. However, few fungicide products are available to flax growers. Field studies were conducted at four locations in Western Canada in 2014, 2015 and 2016 to determine the effect of two fungicide active ingredients applied singly and in combination: pyraclostrobin, fluxapyroxad and fluxapyroxad+pyraclostrobin; and two application timings (early-flower, mid-flower and at both stages) on pasmo severity, seed yield and quality of flaxseed. The results indicated that among the three fungicide treatments, both pyraclostrobin and fluxapyroxad+pyraclostrobin controlled pasmo effectively, however, fluxapyroxad+pyraclostrobin was the most beneficial to improve the quality and quantity of the seed at most of the site-years. Disease severity in the fungicide-free control was 70%, application of fluxapyroxad+pyraclostrobin decreased disease severity to 18%, followed by pyraclostrobin (23%) and fluxapyroxad (48%). Application of fluxapyroxad+pyraclostrobin also improved seed yield to 2562 kg ha-1 compared with 1874 kg ha-1 for the fungicide-free control, followed by pyraclostrobin (2391 kg ha-1) and fluxapyroxad (2340 kg ha-1). Fungicide application at early and mid-flowering stage had the same effects on disease severity and seed yield; however, seed quality was improved more when fungicide was applied at mid-flowering stage. Continuous use of the same fungicide may result in the development of fungicide insensitivity in the pathogen population. Thus, sensitivity of S. linicola isolates to pyraclostrobin and fluxapyroxad fungicides were determined by the spore germination and microtiter assay methods. Fungicide insensitivity was not detected among the 73 isolates of S. linicola tested against either of these fungicides.

Synthesis of New 3-Arylaminophthalides and 3-Indolyl-phthalides using Ammonium Chloride, Evaluation of their Anti-Mycobacterial Potential and Docking Study

Objective: The study aims at the derivatization of “Phthalides” and synthesizes 3- arylaminophthalides & 3-indolyl-phthalides compounds, and evaluates their anti-tubercular and antioxidant activities. The study has also intended to employ the in silico methods for the identification of possible drug targets in Mycobacterium and evaluate the binding affinities of synthesized compounds. Methods: This report briefly explains the synthesis of phthalide derivatives using ammonium chloride. The synthesized compounds were characterized using spectral analysis. Resazurin Microtiter Assay (REMA) plate method was used to demonstrate the anti-mycobacterial activity of the synthesized compounds. An in-silico pharmacophore probing approach was used for target identification in Mycobacterium. The structural level interaction between the identified putative drug target and synthesized phthalides was studied using Lamarckian genetic algorithm-based software. Results and Discussion: In the present study, we report an effective, environmentally benign scheme for the synthesis of phthalide derivatives. Compounds 5c and 5d from the current series appear to possess good anti-mycobacterial activity. dCTP: deaminasedUTPase was identified as a putative drug target in Mycobacterium. The docking results clearly showed the interactive involvement of conserved residues of dCTP with the synthesized phthalide compounds. Conclusion: On the eve of evolving anti-TB drug resistance, the data on anti-tubercular and allied activities of the compounds in the present study demonstrates the enormous significance of these newly synthesized derivatives as possible candidate leads in the development of novel anti-tubercular agents. The docking results from the current report provide a structural rationale for the promising anti-tubercular activity demonstrated by 3-arylaminophthalides and 3-indolyl-phthalides compounds.