Antimicrobial Activity and Probable Mechanisms of Action of Medicinal Plants of Kenya: Withania somnifera,Warbugia ugandensis,Prunus africana and Plectrunthus barbatus

Withania somnifera, Warbugia ugandensis, Prunus africana and Plectrunthus barbatus are used traditionally in Kenya for treatment of microbial infections and cancer. Information on their use is available, but scientific data on their bioactivity, safety and mechanisms of action is still scanty. A study was conducted on the effect of organic extracts of these plants on both bacterial and fungal strains, and their mechanisms of action. Extracts were evaluated through the disc diffusion assay. Bacteria and yeast test strains were cultured on Mueller-Hinton agar and on Sabouraud dextrose agar for the filamentous fungi. A 0.5 McFarland standard suspension was prepared. Sterile paper discs 6 mm in diameter impregnated with 10 µl of the test extract (100 mg/ml) were aseptically placed onto the surface of the inoculated media. Chloramphenicol (30 µg) and fluconazole (25 µg) were used as standards. Discs impregnated with dissolution medium were used as controls. Activity of the extracts was expressed according to zone of inhibition diameter. MIC was determined at 0.78–100 mg/ml. Safety studies were carried using Cell Counting Kit 8 cell proliferation assay protocol. To evaluate extracts mechanisms of action, IEC-6 cells and RT-PCR technique was employed in vitro to evaluate Interleukin 7 cytokine. Investigated plants extracts have both bactericidal and fungicidal activity. W. ugandensis is cytotoxic at IC₅₀<50 µg/ml with MIC values of less than 0.78 mg/ml. Prunus africana shuts down expression of IL 7 mRNA at 50 µg/ml. W. somnifera has the best antimicrobial (1.5625 mg/ml), immunopotentiation (2 times IL 7 mRNA expression) and safety level (IC₅₀>200 µg/ml). Fractions from W. ugandensis and W. somnifera too demonstrated antimicrobial activity. Mechanisms of action can largely be attributed to cytotoxicity, Gene silencing and immunopotentiation. Use of medicinal plants in traditional medicine has been justified and possible mechanisms of action demonstrated. Studies to isolate and characterize the bioactive constituents continue.     

Mechanism of Action of Recombinant Acc-Royalisin from Royal Jelly of Asian Honeybee against Gram-Positive Bacteria

The antibacterial activity of royalisin, an antimicrobial peptide from the royal jelly produced by honeybees, has been addressed extensively. However, its mechanism of action remains unclear. In this study, a recombinant royalisin, RAcc-royalisin from the royal jelly of Asian honeybee Apis cerana cerana, was expressed by fusing with glutathione S-transferase (GST) in Escherichia coli BL21, isolated and purified. The agar dilution assays with inhibition zone showed that RAcc-royalisin, similar to nisin, inhibits the growth of Gram-positive bacteria. The antibacterial activity of RAcc-royalisin was associated with its concentration, and was weakened by heat treatment ranging from 55°C to 85°C for 15 min. Both RAcc-royalisin and nisin exhibited the minimum inhibitory concentrations (MIC) of 62.5 µg/ml, 125 µg/ml, and 250 µg/ml against Gram-positive bacterial strains, Bacillus subtilis and Micrococcus flavus and Staphyloccocus aureus in the microplate assay, respectively. However, RAcc-royalisin did not show antimicrobial activity against tested Gram-negative bacterial and fungal strains. The antibacterial activity of RAcc-royalisin agrees well with the decrease in bacterial cell hydrophobicity, the leakage of 260-nm absorbing materials, and the observation by transmission electron microscopy, all indicating that RAcc-royalisin induced the disruption and dysfunction of cell walls and membranes. This is the first report detailing the antibacterial mechanism of royalisin against Gram-positive bacteria, and provides insight into the application of recombinant royalisin in food and pharmaceutical industries as an antimicrobial agent.     

Photo-mediated optimized synthesis of silver nanoparticles using the extracts of outer shell fibre of Cocos nucifera L. fruit and detection of its antioxidant,cytotoxicity and antibacterial potential

Presently, photo-mediated optimized synthesis of SNPs (CS-AgNPs) was carried out with the help of aqueous extracts of coconut (Cocos nucifera) outer shell fibre. Green synthesis of CS-AgNPs was undertaken under laboratory light conditions and characterized by several standard techniques such as UV–visible spectrophotometer (UV–Vis), X-ray diffraction pattern (XRD), Fourier transform infrared (FT-IR), and scanning electron microscopy (SEM) images and energy dispersive spectroscopy (EDX). UV–Vis spectra displayed a surface plasmon resonance peak at 468 nm equivalent to CS-AgNPs, and the FT-IR spectra confirmed the association of biological molecules from the extract in the synthesis process. The SEM image data confirmed the round and circular nature of CS-AgNPs. The EDX data presented the elemental configuration with a solid peak at 3 KeV that matched with the Ag. The synthesized CS-AgNPs exhibited substantial cytotoxicity potential against the HepG2 cells with (effective concentration (IC₅₀) value of 15.28 µg/ml along with robust antioxidant potential, with respect to its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging (IC₅₀ of 96.39 µg/ml) and reducing assay (IC_0.5 of 209.96 µg/ml). The CS-AgNPs demonstrated encouraging antimicrobial potential against four different pathogenic bacteria and one Candida sp. with inhibition zone diameter ranged between 8.87 and 13.07 mm. Overall, the existing investigation suggested that CS-AgNPs can be an attractive, cost-effective, and environment-friendly candidate for its possible uses in the food, cosmetics, and therapeutic fields.     

Phytochemical,antioxidant and mineral composition of hydroalcoholic extract of chicory (Cichorium intybus L.) leaves

The phytochemical, antioxidant and mineral composition of hydroalcoholic extract of leaves of Cichorium intybus L., was determined. The leaves were found to possess comparatively higher values of total flavonoids, total phenolic acids. The phytochemical screening confirmed the presence of tannins, saponins, flavonoids, in the leaves of the plant. The leaf extract was found to show comparatively low value of IC₅₀ for 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition. The IC₅₀ value of chicory leaves extract was found to be 67.2 ± 2.6 μg/ml. The extracts were found to contain high amount of mineral elements especially Mg and Zn. Due to good phytochemical and antioxidant composition, C. intybus L., leaves would be an important candidate in pharmaceutical formulations and play an important role in improving the human health by participating in the antioxidant defense system against free radical generation.     

Effects of pH and temperature on the wild-type and a mutant form of Neurospora glutamate dehydrogenase

1. We confirm the observation of Bürk (1965) that Neurospora crassa NADP-linked glutamate dehydrogenase normally exists in an inactive form below pH7·0 and in a fully active form above pH8·0 in either tris or orthophosphate buffer. At pH7·4 the enzyme is about half activated at 25°. 2. The variety of the enzyme produced by the mutant am^2l shows a similar behaviour except that the transition is shifted about one pH unit in the alkaline direction. 3. The am^2l enzyme has previously been reported to be activated by brief warming to 30° in phosphate buffer at pH8·0. The wild-type enzyme shows a similar effect at pH7·0. In tris buffer this effect is much less pronounced. 4. The am^2l enzyme is extremely unstable at 47° at pH7·0; its stability is somewhat greater at lower pH, and is markedly increased by increasing the pH in the range 7·0–8·7. The wild-type enzyme also shows an indication of a stability minimum at pH7·0, but a temperature of 60° is needed for a measurable rate of inactivation. 5. The inactive form of the enzyme is much more subject to thermal irreversible denaturation than is the active form.     

Analysis of anti-bacterial and anti oxidative activity of Azadirachta indica bark using various solvents extracts

Herbal medications have been used for relief of symptoms of disease. Regardless of the great advances observed in current medicine in recent decades, plants still make a significant contribution to health care. An alarming increase in bacterial strains resistant to a number of antimicrobial agents demands that a renewed effort be made to seek antibacterial agents effective against pathogenic bacteria resistant to or less sensitive to current antibiotics. Anti-bacterial activity of Azadirachta indica stem bark was tested against pathogenic Salmonella paratyphi and Salmonella typhi using various solvent extracts. The in vitro anti-bacterial activity was performed by agar well diffusion method and the results were expressed as the average diameter of zone of inhibition of bacterial growth around the well. The ethanol and methanol extracts showed better anti-bacterial activity with zone of inhibition (20–25 mm) when compared with other tested extracts and standard antibiotic Erythromycin (15 mcg) with zone of inhibition (13–14 mm). Using Fisher’s exact test of significance difference was found between two Salmonella strains sensitivity patterns against tested extracts (P ⩽ 0.035). Extracts of A. indica stem bark also exhibited significant antioxidant activity, thus establishing the extracts as an antioxidant. The results obtained in this study give some scientific support to the A. indica stem bark for further investigation of compounds and in future could be used as drug.     

Experimental and theoretical analyses of nano-silver for antibacterial activity based on differential crystal growth temperatures

The modulation of antimicrobial properties of nanomaterials can be achieved through various physical and chemical processes, which ultimately affect subsequent properties. In this study, the antibacterial potential of nano-silver was investigated at 0.5, 1.0, 2.0, and 3.0 g/L, and its differential temperature synthesis was achieved at 20, 50, and 70 °C using the solvent evaporation method. Nano-silver particles exhibited FCC (octahedral) crystalline structure with crystallite sizes ranging between 28 and 39 nm calculated using XRD analysis. Moreover, irregular and non-uniform surface morphology was evident from SEM micrographs. The UV–Vis absorbance spectrum of nano-silver exhibited wave maxima at 433 nm, while the FTIR analysis depicted different modes of vibration indicating the CH, OH, C≡C, C-Cl, and CH₂ functional groups attached to the surface. Lastly, nano-silver caused prominent inhibition (12.5 mm) in the Escherichia coli growth, particularly at 70 °C synthesis temperature and 3.0 g/L dose. It is concluded that both the nano-silver crystal growth temperature and dose contributed substantially to bacterial growth inhibition linked with subsequent size, shape-dependent properties.     

Nanocapsular Dispersion of Thymol for Enhanced Dispersibility and Increased Antimicrobial Effectiveness against Escherichia coli O157:H7 and Listeria monocytogenes in Model Food Systems

Essential oils are marginally soluble in water, making it challenging to evenly disperse them in foods and resulting in an increased tendency to bind with food lipids and proteins, resulting in lowered antimicrobial efficacy. In the current study, free and nano-dispersed (ND) thymol were compared in terms of their antimicrobial efficacies against Escherichia coli O157:H7 ATCC 43889 and 43894 and Listeria monocytogenes strains Scott A and 101 in apple cider and 2% reduced-fat milk. Apple cider was adjusted to pHs 5.5 and 3.5, and antimicrobial tests were performed at 0.3-, 0.5-, 0.75-, and 1.0-g/liter thymol concentrations at 35, 32, 25, and 4°C. Overall, 0.5 and 1.0 g/liter thymol in nano-dispersion and along with free thymol were inhibitory and bactericidal, respectively, against bacterial strains under all treatment conditions. At pH 5.5, 0.5 g/liter ND thymol was bacteriostatic against L. monocytogenes and E. coli for up to 48 h. At pH 3.5, L. monocytogenes controls did not survive beyond 12 h but E. coli survived and was inhibited by 0.5 g/liter ND thymol after 12 and 48 h in apple cider. E. coli strains were significantly sensitive to 4°C and pH 3.5 (P < 0.05). When bacteria were tested in 2% reduced-fat milk at 35 or 32°C, ND and free thymol demonstrated inhibition at 4.5 g/liter. Thus, the current technology seems to be promising and novel, enabling thymol-containing nano-dispersions that are not only transparent but also effective against pathogens in food applications, especially in clear beverages.     

Evaluation of in-vitro antioxidant and antibacterial properties of Commelina nudiflora L. extracts prepared by different polar solvents

The study explored on the commonly available weed plant Commelina nudiflora which has potential in-vitro antioxidant and antimicrobial activity. The different polar solvents such as ethanol, chloroform, dichloromethane, hexane and aqueous were used for the soxhlet extraction. The extracts were identified pharmacologically as important bioactive compounds and their potential free radical scavenging activities, and antimicrobial properties were studied. C. nudiflora extracts were monitored on their in-vitro antioxidant ability by DPPH and ABTS radical scavenging assay. Aqueous extract shows significant free radical scavenging activity of 63.4 mg/GAE and 49.10 mg/g in DPPH and ABTS respectively. Furthermore, the aqueous crude extract was used in antibacterial studies, which shows the highest inhibitory activity against Pseudomonas aeruginosa, Escherichia coli and Salmonella typhi. Among all the extracts, aqueous extract of C. nudiflora has significant control over free radical scavenging activity and inhibition of the growth of food pathogenic bacteria. Also, the aqueous extract contains abundance of phenolics and flavonoids higher than other extracts. This study explored weed plant C. nudiflora as a potential source of antioxidant and antibacterial efficacy and identified various therapeutic value bioactive compounds from GC–MS analysis.Abbreviations: ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid), DPPH, 2,2-diphenyl-1-picrylhydrazyl, GAE, gallic acid equivalent, GC–MS, gas chromatography and mass spectrometry     

Plant growth promoting bacteria induce anti-quorum-sensing substances in chickpea legume seedling bioassay

Microorganisms and their hosts communicate through an array of signals. Many physiological processes regulated in quorum sensing (QS) are dependent on auto-inducers, like N-acyl-homoserine lactones (AHLs) as in numerous groups of both gram-positive and gram-negative bacteria. In vitro grown seven-day old chickpea seedlings treated with plant growth promoting bacteria (PGPRs) were used to screen the AHL mimicking and for phytochemical substances like phytohormones and secondary metabolites such as phenolics and flavonoids. Potential anti-quorum sensing (anti-QS) activity surrounding the roots on semi-solid agar lawn of Chromobacterium violaceum (ATCC12742) was observed. Crude protein (4.46–8.30 μg/mL) and methanolic extracts (100 μg/mL) of seedling gave moderate anti-QS activity against CV12742 anti QS bioassay, respectively. Crude protein and methanolic extract of Bacillus amyloliquefaciens (34.00 ± 2.23; 34.00 ± 4.33 mm) and B. subtilis A (27.00 ± 2.10; 3.29 ± 2.16 mm) treated samples showed higher zone of inhibition due to anti-QS activity. Phytohormone analysis using LC–MS for zeatin, auxin and methyl jasmonate (MeJA) indicated that phytohormones were significantly upregulated by 1909.80 ng/g FW, 669.67 ng/g FW and 244.55 ng/g FW, respectively in Pseudomonas brassicacearum treated seedlings compared to control. UHPLC of PGPR treated seedlings showed overly expressed gallic acid, protocatechuic acid, catechin, p-hydroxybenzoic acid, caffeic acid, catechol, vanillin, and ferulic acid in B. amyloliquefaciens treated seedlings compared to others. Enrichment analysis identified significant pathways related to metabolism, biosynthesis of secondary metabolites. The present study indicates that chickpea neutralizes an extensive range of functional responses to AHLs that may play important role in legume host-microbe interactions.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01034-x.     

Efficacy of leaves extract of Calotropis procera Ait. (Asclepiadaceae) in controlling Anopheles arabiensis and Culex quinquefasciatus mosquitoes

The present study aimed to investigate, the larvicidal, adult emergence inhibition and oviposition deterrent activity of aqueous leaves extract of Calotropis procera against Anopheles arabiensis and Culex quinquefasciatus as natural mosquito larvicide. The larvicidal activity was monitored against 2nd, 3rd and 4th instar larvae of each mosquito species 24 h post-treatment. Adult emergence inhibition activity was tested by exposing 3rd instar larvae of each mosquito species to different concentrations of extracts (200, 400, 600, 800 and 1000 ppm for An. arabiensis and 100, 200, 300, 400, 500 and 600 ppm for Cx. quinquefasciatus). Probit analysis was used to analyze data from bioassay experiments. The oviposition deterrent activity was tested by using three different concentrations of extracts (1000, 500 and 200 for An. arabiensis, and 1000, 500 and 100 for Cx. quinquefasciatus) that caused high, moderate and low larval mortality in the larvicidal experiment against 3rd instar larvae. It was found that, LC50–LC90 values calculated were 273.53–783.43, 366.44–1018.59 and 454.99–1224.62 ppm for 2nd, 3rd and 4th larval instars, respectively, of An. arabiensis and 187.93–433.51, 218.27–538.27 and 264.85–769.13 ppm for 2nd, 3rd and 4th larval instars, respectively, of Cx. quinquefasciatus. Fifty percent of adult emergence inhibition (EI50) was shown at 277.90 and 183.65 ppm for An. arabiensis and Cx. quinquefasciatus, respectively. The pupal stage was not affected till a concentration of 5000 ppm. The extract showed oviposition deterrence and effective repellence against both mosquito species at different concentrations, with the observation on that maximal eggs were laid in low concentration of extract. These results suggest that the leaves extract of C. procera possess remarkable larvicidal, adult emergence inhibitor, repellent and oviposition deterrent effect against both An. arabiensis and Cx. quinquefasciatus, and might be used as natural biocides for mosquito control.     

The Effect of an Adding Histidine on Biological Activity and Stability of Pc-pis from Pseudosciaena crocea

Pc-pis is a novel piscidin-like antimicrobial polypeptide that was identified in Pseudosciaena crocea. Although active against most bacteria tested, Pc-pis was inactive against Aeromonas hydrophila and Pseudomonas aeruginosa. The Pc-pis analogue Pc-pis-His was designed by adding a histidine residue at the carboxyl terminal. Pc-pis-His demonstrated a more broad-spectrum and stronger antimicrobial activity against a representative set of microorganisms and more potent antiparasitic activity against Cryptocaryon irritans trophonts than Pc-pis. The stability assay revealed that Pc-pis-His was active against Staphylococcus aureus not only in acidic (pH 5.5–7.3) and relatively low concentration monovalent cation (0–160 mM NaCl) environments but also in alkaline (pH 7.5–9.5), divalent cation (1.25–160 mM MgCl₂ and 1.25–40 mM CaCl₂) and high concentration monovalent cation (320–2560 mM NaCl) environments, which indicates that the added histidine residue conferred better salt-, acid- and alkali-tolerance to Pc-pis-His. Pc-pis-His also possessed the desired heat-tolerance, which was reflected by the antimicrobial activity of the peptide after being boiled for 10–60 minutes. Hemolytic activity analysis revealed that Pc-pis-His at concentrations up to 6 µM exhibited no hemolysis against human erythrocytes, with 6 µM being a concentration that is highly active against most of the microorganisms tested, although the hemolytic activity of Pc-pis-His was enhanced compared to Pc-pis. These results provide a unique, reasonable basis for designing novel piscidins with potent, broad-spectrum and stable antimicrobial activity and new insight into the future development of piscidins as potential therapeutic agents against microbial and external protozoan parasite infections.     

In vivo antipyretic,antiemetic, in vitro membrane stabilization,antimicrobial, and cytotoxic activities of different extracts from Spilanthes paniculata leaves

Background

The study was conducted to evaluate the in vitro antimicrobial activity, cytotoxic, and membrane stabilization activities, and in vivo antiemetic and antipyretic potentials of ethanolic extract, n-hexane and ethyl acetate soluble fractions of Spilanthes paniculata leaves for the first time widely used in the traditional treatments in Bangladesh.

Results

In antipyretic activity assay, a significant reduction (P < 0.05) was observed in the temperature in the mice tested. At dose 400 mg/kg-body weight, the n-hexane soluble fraction showed the effect (36.7 ± 0.63°C ) as like as the standard (dose 150 mg/kg-body weight) after 5 h of administration. Extracts showed significant (P < 0.001) potential when tested for the antiemetic activity compared to the standard, metoclopramide. At dose 50 mg/kg-body weight, the standard showed 67.23% inhibition, whereas n-hexane and ethyl acetate soluble fractions showed 37.53% and 24.93% inhibition of emesis respectively at dose 400 mg/kg-body weight. In antimicrobial activity assay, the n-hexane soluble fraction (400 μg/disc) showed salient activity against the tested organisms. It exerts highest activity against Salmonella typhi (16.9 mm zone of inhibition); besides, crude, and ethyl acetate extracts showed resistance to Bacillus cereus and Bacillus subtilis, and Vibrio cholera respectively. All the extracts were tested for lysis of the erythrocytes. At the concentration of 1mg/ml, ethanol extract, and n-hexane and ethyl acetate soluble fractions significantly inhibited hypotonic solution induced lysis of the human red blood cell (HRBC) (27.406 ± 3.57, 46.034 ± 3.251, and 30.72 ± 5.679% respectively); where standard drug acetylsalicylic acid (concentration 0.1 mg/ml) showed 77.276 ± 0.321% inhibition. In case of heat induced HRBC hemolysis, the plant extracts also showed significant activity (34.21 ± 4.72, 21.81 ± 3.08, and 27.62 ± 8.79% inhibition respectively). In the brine shrimp lethality bioassay, the n-hexane fraction showed potent (LC₅₀ value 48.978 μg/ml) activity, whereas ethyl acetate fraction showed mild (LC₅₀ value 216.77 μg/ml) cytotoxic activity.

Conclusions

Our results showed that the n-hexane extract has better effects than the other in all trials. In the context, it can be said that the leaves of S. paniculata possess remarkable pharmacological effects, and justify its folkloric use as antimicrobial, antipyretic, anti-inflammatory, and antiemetic agent. Therefore, further research may be suggested to find possible mode of action of the plant part.     

Effect of Licochalcone A on Growth and Properties of Streptococcus suis

Streptococcus suis (S.suis) is an important emerging worldwide pig pathogen and zoonotic agent with rapid evolution of virulence and drug resistance. In this study, we wanted to investigate the effect of licochalcone A on growth and properties of Streptococcus suis. The antimicrobial activity of licochalcone A was tested by growth inhibition assay and the minimal inhibitory concentrations (MICs) also were determined. The effect of licochalcone A on S.suis biofilm formation was characterized by crystal violet staining. The effect of licochalcone A on suilysin secretion was evaluated by titration of hemolytic activity. To understand the antimicrobial effect, gene expression profile of S.suis treated by licochalcone A was analyzed by DNA microarray. Our results demonstrated that licochalcone A showed antimicrobial activity on S.suis with MICs of 4 µg/ml for S.suis serotype 2 strains and 8 µg/ml for S.suis serotype 7 strains. Biofilm formation was inhibited by 30–40% in the presence of licochalcone A (3 µg/ml) and suilysin secretion was also significantly inhibited in the presence of licochalcone A (1.5 µg/ml). The gene expression profile of S.suis in the presence of licochalcone A showed that 132 genes were differentially regulated, and we analyzed the regulated genes in the aspect of the bacterial cell cycle control. Among the deregulated genes, the genes responsible for the mass doubling was increased expression, but the genes responsible for DNA replication and cell division were inhibited the expression. So, we think the regulation of the cell cycle genes might provide a mechanistic understanding of licochalcone A mediated antimicrobial effect against S.suis.     

Fermentative Production of Pyranone Derivate I from Marine Vibrio sp. SKMARSP9: Isolation,Characterization and Bioactivity Evaluation

Pyranone derivative I was isolated from fermented broth of isolated marine bacterial strain Vibrio sp. SKMARSP9. The compound I was characterized, and evaluated for its antimicrobial properties. The isolated strain was identified based on 16S rRNA based phylogenetic analysis. The molecular analysis data suggested that this strain is closely related to Vibrio ruber, Vibrio sp. MSSRF10 and Vibrio rhizosphaerae. The best fermentative growth of this isolate was achieved under halophilic conditions and grew efficiently at 30 °C in the presence of 12 % NaCl. The compound I production by this strain is associated with growth. The unpurified extract is hydrophobic in nature, and released only during late growth phase. The extract was purified and characterized by spectral data using NMR, DEPT, and ESI–MS. The purity of I was 97 % which was confirmed by HPLC. The pyranone derivative I exhibited >50 % antioxidant activity and broad spectrum antimicrobial properties against gram negative and gram positive strains. Molecular docking analysis revealed that this pyranone derivative I may be a potential candidate at pharmaceutical sector.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0521-0) contains supplementary material, which is available to authorized users.     

Purification, properties and mechanism of action of a staphylolytic enzyme produced by Aeromonas hydrophila

1. An enzyme produced by Aeromonas hydrophila and capable of lysing Staphylococcus aureus cells was purified 180-fold by gel filtration and chromatography on columns of AG-50 W resin. 2. Physical measurements on the purified enzyme suggest that it is a small basic protein with an isoelectric point between pH9·0 and pH9·5. 3. Maximum lytic activity was obtained in 20mm-tris–glycine buffer, pH8·5, at 45°, with no detectable activity in the absence of a nitrogenous base. 4. The enzyme is active in the above buffer containing 1·5m-sucrose, and is useful for the preparation of protoplasts of Staphylococcus aureus. 5. Purified cell wall peptidoglycans of two strains of Staphylococcus aureus, differing in amino acid composition, were hydrolysed by the enzyme with the liberation of glycine oligopeptides, principally diglycine and triglycine. 6. Synthetic glycine oligopeptides larger than triglycine, but not polyglycine, were hydrolysed, as were a number of leucine-containing dipeptides and tripeptides, but no proteolytic activity could be demonstrated. 7. It is concluded that the enzyme is lytic towards Staphylococcus aureus because it splits the pentaglycine cross-links of the cell-wall peptidoglycan.     

Optimization of nutritional and environmental conditions for pyocyanin production by urine isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is a highly pathogenic bacteria involved in numerous diseases among which, are urinary tract infections (UTIs). The pyocyanin secreted as a virulence factor by this bacterium has many beneficial applications but its high cost remains an obstacle for its widespread use. In this study, a total of fifty urine isolates were identified as P. aeruginosa. All strains produced pyocyanin pigment with a range of 1.3–31 µg/ml. The highest producer clinical strain P21 and the standard strain PA14 were used in optimization of pyocyanin production. Among tested media, king’s A fluid medium resulted in the highest yield of pyocyanin pigment followed by nutrient broth. Growth at 37 °C was superior in pyocyanin production than growth at 30 °C. Both shaking and longer incubation periods (3–4 days) improved pyocyanin production. The pyocyanin yield was indifferent upon growth of P21 at both pH 7 and pH 8. In conclusion, the optimum conditions for pyocyanin production are to use King’s A fluid medium of pH 7 and incubate the inoculated medium at 37 °C with shaking at 200 rpm for a period of three to four days.     

SGK1 negatively regulates inflammatory immune responses and protects against alveolar bone loss through modulation of TRAF3 activity

Serum- and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine kinase that plays important roles in the cellular stress response. While SGK1 has been reported to restrain inflammatory immune responses, the molecular mechanisms involved remain elusive, especially in oral bacteria-induced inflammatory milieu. Here, we found that SGK1 curtails Porphyromonas gingivalis–induced inflammatory responses through maintaining levels of tumor necrosis factor receptor-associated factor (TRAF) 3, thereby suppressing NF-κB signaling. Specifically, SGK1 inhibition significantly enhances production of proinflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-6, IL-1β, and IL-8 in P. gingivalis–stimulated innate immune cells. The results were confirmed with siRNA and LysM-Cre–mediated SGK1 KO mice. Moreover, SGK1 deletion robustly increased NF-κB activity and c-Jun expression but failed to alter the activation of mitogen-activated protein kinase signaling pathways. Further mechanistic data revealed that SGK1 deletion elevates TRAF2 phosphorylation, leading to TRAF3 degradation in a proteasome-dependent manner. Importantly, siRNA-mediated traf3 silencing or c-Jun overexpression mimics the effect of SGK1 inhibition on P. gingivalis–induced inflammatory cytokines and NF-κB activation. In addition, using a P. gingivalis infection–induced periodontal bone loss model, we found that SGK1 inhibition modulates TRAF3 and c-Jun expression, aggravates inflammatory responses in gingival tissues, and exacerbates alveolar bone loss. Altogether, we demonstrated for the first time that SGK1 acts as a rheostat to limit P. gingivalis–induced inflammatory immune responses and mapped out a novel SGK1–TRAF2/3–c-Jun–NF-κB signaling axis. These findings provide novel insights into the anti-inflammatory molecular mechanisms of SGK1 and suggest novel interventional targets to inflammatory diseases relevant beyond the oral cavity.     

Effects of Temperature on Bacterial Communities and Metabolites during Fermentation of Myeolchi-Aekjeot,a Traditional Korean Fermented Anchovy Sauce

Myeolchi-aekjeot (MA) in Korea is produced outdoors without temperature controls, which is a major obstacle to produce commercial MA products with uniform quality. To investigate the effects of temperature on MA fermentation, pH, bacterial abundance and community, and metabolites were monitored during fermentation at 15°C, 20°C, 25°C, and 30°C. Initial pH values were approximately 6.0, and pH values increased after approximately 42 days, with faster increases at higher temperatures. Bacterial abundances increased rapidly in all MA samples after quick initial decreases during early fermentation and then they again steadily decreased after reaching their maxima, which were significantly greater at higher temperatures. Bacterial community analysis revealed that Proteobacteria and Tenericutes were predominant in all initial MA samples, but they were rapidly displaced by Firmicutes as fermentation progressed. Photobacterium and Mycoplasma belonging to Proteobacteria and Tenericutes, respectively, which may include potentially pathogenic strains, were dominant in initial MA, but decreased with the growth of Chromohalobacter, which occurred faster at higher temperatures––they were dominant until 273 and 100 days at 15°C and 20°C, respectively, but not detected after 30 days at 25°C and 30°C. Chromohalobacter also decreased with the appearance of subsequent genera belonging to Firmicutes in all MA samples. Tetragenococcus, halophilic lactic acid bacteria, appeared predominantly at 20°C, 25°C, and 30°C; they were most abundant at 30°C, but not detected at 15°C. Alkalibacillus and Lentibacillus appeared as dominant genera with the decrease of Tetragenococcus at 25°C and 30°C, but only Lentibacillus was dominant at 15°C and 20°C. Metabolite analysis showed that amino acids related to tastes were major metabolites and their concentrations were relatively higher at high temperatures. This study suggests that high temperatures (approximately 30°C) may be appropriate in MA fermentation, in the light of faster disappearance of potentially pathogenic genera, higher amino acids, growth of Tetragenococcus, and faster fermentation.     

Antimicrobial and Antimycobacterial Activities of Methyl Caffeate Isolated from Solanum torvum Swartz. Fruit

Abstract

Solanum torvum Swartz. (Solanaceae) fruit is traditionally used for the treatment of bacterial and fungal infections. The methanolic extract was subjected to activity guided fractionation by column chromatography over silica gel. The structure of the compound was elucidated using physical and spectroscopic data. The antimicrobial activity was screened using five Gram-positive bacteria, six Gram-negative bacteria, seven clinical isolates and four fungi. Antimycobacterial activity was screened against two Mycobacterium strains. The zone of inhibition by methyl caffeate ranged from 0 to 22 mm. The lowest minimum inhibitory concentration (MIC) values of methyl caffeate were: 50 μg/ml against P. vulgaris, 25 μg/ml against K. pneumoniae (ESBL-3971), 8 μg/ml against M. tuberculosis (H³⁷Rv) and 8 μg/ml against M. tuberculosis (Rif^R). Methyl caffeate showed moderate antimicrobial and prominent antimycobacterial activities. Methyl caffeate can be evaluated further for drug development.