Tocopherol,Carotene, Phenolic Contents and Antibacterial Properties of Rose Essential Oil,Hydrosol and Absolute

The antioxidant and antibacterial activities, and total phenolic contents of Rosa damascena Mill. flower extracts (absolute, essential oil and hydrosol) were investigated. The chemical compositions of these extracts were analysed by GC-MS. Phenylethyl alcohol (78.38%) was found to be the main constituent of rose absolute, while citrenellol and geraniol were the major compounds (>55%) of rose essential oil and hydrosol. Tocopherol and carotene levels were determined by high performance liquid chromatography (HPLC) analysis. The levels of beta carotene (422.3±35.6 ppm), alpha tocopherol (2397.1±72.5 ppm) and gamma tocopherol (343.1±28.4 ppm) of rose absolute were found to be higher than that of essential oil and hydrosol. Their total phenolic contents were also evaluated. The total phenolic content of the tested extracts varied from 5.2 to 2134.3 GAE/mg L⁻¹. Rose absolute and essential oil contained high levels of phenolics and demonstrated strong antibacterial activity against Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), Chromobacterium violaceum (ATCC 12472) and Erwinia carotovora (ATCC 39048) strains.     

Functional and ultrastructural changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Cinnamomum verum essential oil

Aims: To study cellular damage induced by Cinnamomum verum essential oil in Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. Methods and Results: The effect of cinnamon bark essential oil on these two strains was evaluated by plate counts, potassium leakage, flow cytometry and transmission electron microscopy (TEM). Exposure to this oil induced alterations in the bacterial membrane of Ps. aeruginosa, which led to the collapse of membrane potential, as demonstrated by bis‐oxonol staining, and loss of membrane‐selective permeability, as indicated by efflux of K⁺ and propidium iodide accumulation. Thus, respiratory activity was inhibited, leading to cell death. In Staph. aureus, cells treated with the oil entered a viable but noncultivable (VNC) state. The oil initially caused a considerable decrease in the metabolic activity and in the replication capacity of these bacterial cells. The loss of membrane integrity appeared later, as indicated by bis‐oxonol and Propidium iodide (PI) staining. Data provided by TEM showed various structural effects in response to cinnamon essential oil. In Ps. aeruginosa cells, coagulated cytoplasmic material was observed, and intracellular material was seen in the surrounding environment, while oil‐treated Staph. aureus showed fibres extending from the cell surface. Conclusions: Cinnamon essential oil damages the cellular membrane of Ps. aeruginosa, which leads to cell death. There is evidence of VNC Staph. aureus after exposure to the oil. Significance and Impact of the Study: Cinnamon essential oil shows effective antimicrobial activity and health benefits and is therefore considered a potential food additive. To use this oil as a natural food preservative, especially in combination with other preservation methods, a thorough understanding of the mechanism through which this oil exerts its antibacterial action is required.     

Chemical Composition and Antibacterial Activity of Iranian Lavandula × hybrida

Lavandin (Lavandula × hybrida) is an evergreen shrub and cultivated worldwide for its essential oil which possesses various biological activities. In this study, the essential oils were isolated from the leaves of ten lavandin populations in western Iran. The hydrodistilled essential oils were analyzed by GC‐FID/MS. Results indicated significant differences (P ≤ 0.05) among the various populations for the main essential oil constituents. The major components from different populations were 1,8‐cineole (31.64 – 47.94%), borneol (17.11 – 26.14%), and camphor (8.41 – 12.68%). In vitro antibacterial activity was evaluated against S. agalactiae, S. aureus, E. coli, and K. pneumoniae. The inhibition zones were in the range of 09.36 mm for S. aureus to 23.30 mm for E. coli. Results indicated that there was a significant correlation between essential oil composition and level of antibacterial efficacy expressed as inhibition zones.     

The Entner–Doudoroff pathway empowers Pseudomonas putida KT2440 with a high tolerance to oxidative stress

Glucose catabolism of Pseudomonas putida is carried out exclusively through the Entner–Doudoroff (ED) pathway due to the absence of 6‐phosphofructokinase. In order to activate the Embden–Meyerhof–Parnas (EMP) route we transferred the pfkA gene from Escherichia coli to a P. putida wild‐type strain as well as to an eda mutant, i.e. lacking 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase. PfkA^{E. coli} failed to redirect the carbon flow from the ED route towards the EMP pathway, suggesting that ED was essential for sugar catabolism. The presence of PfkA^{E. coli} was detrimental for growth, which could be traced to the reduction of ATP and NAD(P)H pools along with alteration of the NAD(P)H/NADP⁺ ratio. Pseudomonas putida cells carrying PfkA^{E. coli} became highly sensitive to diamide and hydrogen peroxide, the response to which is very demanding of NADPH. The inhibitory effect of PfkA^{E. coli} could in part be relieved by methionine, the synthesis of which relies much on NADPH. These results expose the role of the ED pathway for generating the redox currency (NADPH) that is required for counteracting oxidative stress. It is thus likely that environmental bacteria that favour the ED pathway over the EMP pathway do so in order to gear their aerobic metabolism to endure oxidative‐related insults.     

四种植物精油对黑翅土白蚁触杀和驱避作用

为筛选出高效防治黑翅土白蚁的天然植物精油,减少有机合成农药的使用,该文研究了大蒜精油、肉桂油、丁香油和印楝素油四种植物精油对黑翅土白蚁的触杀效果和驱避作用。结果表明:大蒜精油、肉桂油和丁香油的浓度为5和10 mg·m L~(-1)时,处理2 h后,黑翅土白蚁的校正死亡率达100%,而相同浓度的印楝素油和对照处理的黑翅土白蚁校正死亡率低于5%。随着处理时间延长,浓度为1.25和2.5 mg·m L~(-1)的大蒜精油、肉桂油和丁香油处理6 h时,黑翅土白蚁的校正死亡率仍达100%,而此时对应的印楝素油和对照处理的黑翅土白蚁校正死亡率仅为10%,说明大蒜精油、肉桂油和丁香油对黑翅土白蚁具有较强的触杀效果。大蒜精油、丁香油和肉桂油在处理黑翅土白蚁2 h后LC_(50)值(半致死量)分别为1.572、1.05和1.03mg·m L~(-1),说明肉桂油对黑翅土白蚁的毒性相对最大,触杀效果最好。此外,10 mg·m L~(-1)的大蒜精油、肉桂油、丁香油和印楝素油的驱避试验表明,处理4、6、8和12 h后,大蒜精油、肉桂油和丁香油三精油处理区的黑翅土白蚁数均显著低于对照区的,驱避率总体93%,而对应的印楝素油的驱避率总体28.5%,表明大蒜精油、丁香油和肉桂油三种植物精油对黑翅土白蚁均有显著的驱避活性。综上可知,四种植物精油中大蒜精油、肉桂油和丁香油在防治黑翅土白蚁方面应用潜力很好,是开发绿色环保白蚁防治药剂的可选材料。     

Study on the toxic mechanism of La3+ to Escherichia coli

The toxic mechanism of La³⁺ to Escherichia coli is investigated by detecting the concentration change of La³⁺ in E. coli cells growing in La³⁺-containing medium. Stimulatory action and inhibitory effect of La³⁺ in different concentrations can be attributed to the permeability alteration of the cell. Low concentration of La³⁺ increases the nutrition absorbability of the cells from the cultures as a result of increased cell permeability, and high concentration of La³⁺ causes the accumulation of La³⁺ in cells, resulting in the toxic effects on the E. coli cells.     

Essential Oil Compositions and Antibacterial and Antioxidant Activities of Five Lavandula stoechas Cultivars Grown in Thailand

The essential oils of five Lavandula stoechas cultivars grown in Thailand were characterized for their volatile compounds using GC‐FID and GC/MS methods as well as screened for antibacterial and antioxidant activities. Dried aerial parts, including flowers and stems from each cultivar, were subjected to hydrodistillation for 4 h. The essential oil yields were 0.18 %–0.82 % w/w. Of the 95 compounds detected and identified, 1,8‐cineole, fenchone, and camphor were considered the major compounds. Essential oil from each cultivar demonstrated different patterns of antibacterial activity and a variety of antioxidant properties. The highest antibacterial activity, MIC=0.39 mg mL^?1, was observed from the essential oil of L. stoechas ‘major’ (against Klebsiella pneumoniae and Salmonella typhimurium) and the essential oil of L. stoechas ‘white lavender’ (against S. typhimurium). The essential oil of L. stoechas×viridis ‘St. Brelade’ possessed the highest antioxidant capacity, as determined by the DPPH and ABTS assays (IC₅₀ of 67.65 and 89.26 mg mL^?1, respectively). The results indicated that some of these essential oils could be used as key ingredients in lavender oil products in Thailand to increase their therapeutic efficacy, depending on their intended application.     

A novel medium for expression of proteins selectively labeled with 15N-amino acids in Spodoptera frugiperda (Sf9) insect cells

Whereas bacterial expression systems are widely used for production of uniformly or selectively ¹⁵N-labeled proteins the usage of the baculovirus expression system for labeling is limited to very few examples in the literature. Here we present the complete formulations of the two insect media, IML406 and 455, for the high-yield production of selectively ¹⁵N-labeled proteins in insect cells. The quantities of ¹⁵N-amino acids utilized in the production of labeled GST were similar in the case of bacterial and viral expression. For the most studied amino acids essential for insect cells the ¹⁵N-HSQC spectra, recorded with GST labeled in insect cells, showed no cross labeling and provided therefore spectra of better quality compared to NMR spectra of GST expressed in E. coli. Also in the case of amino acids not essential for Sf9 cells we were able to label a defined number of amino acid species. Therefore the selective labeling using the baculovirus expression vector system represents a complement or even an alternative to the bacterial expression system. Based on these findings we can provide a first simple overview of the network of the amino acid metabolism in E. coli and insect cells focused on nitrogen. For some amino acids the expression of labeled proteins in insect cells can replace the cell-free protein expression.     

A new gene controlling the frequency of cell division per round of DNA replication in Escherichia coli

Summary A novel mutant of Escherichia coli, named cfcA1, was isolated from a temperature-sensitive dnaB42 strain, and found to have the following characteristics. Division arrest and lethality induced by inhibition of DNA replication was reduced and delayed in the cfcA1 dnaB42 strain, as compared with the parental dnaB42 strain. Two types of inhibition of division induced by the addition of nalidixic acid or hydroxyurea were suppressed by the cfcA1 mutation. Under permissive conditions for DNA replication, the colony forming ability of cfcA1 cells was significantly reduced as compared with that of cfc ⁺ cells; conversely the division rate of cfcA1 cells was higher than that of cfc ⁺ cells. The cfcA1 mutation partially restored division arrest induced in the thermosensitive ftsZ84 mutant at the restrictive temperature and suppresed the UV sensitivity of the lon mutation. The mutation was mapped at 79.2 min on the E. coli chromosome. Taking these properties into account, it is hypothesized that the cfcA gene is involved in determining the frequency of cell division per round of DNA replication by interacting with the FtsZ protein which is essential for cell division.     

Investigation of enhancement of two processes, sedimentation and conjugation, when bacteria are concentrated in ultrasonic standing waves

Cells aggregate and can be recovered from suspension when exposed to an ultrasonic standing wave field. The acoustic force on individual cells in a standing wave decreases with particle volume. A plane ultrasonic field generated by a transducer driven at 3.3 MHz was used here to investigate the removal of Escherischia coli, cells with dimensions of the order of 1.0 m, from batch suspension by sedimentation over a range of concentrations (10³ to 10¹⁰ cells ml^–1). Cell removal efficiencies greater than 90% were achieved at initial concentrations of 10¹⁰ cells ml^–1. Removal efficiencies decreased gradually to zero, as initial bacterial concentration was reduced to 10⁷ cells ml^–1. It was found that, when low concentrations of E. coli (10³ to 10⁵ cells ml^–1) were added to suspensions of larger particles (i.e. yeast cells) that were of sufficient concentration to form aggregates in the sound field, E. coli could be harvested to an efficiency of 40%. The results imply that the E. coli became trapped and sediment with aggregates of larger particles. Some strains of bacteria are capable of DNA transfer by conjugation. The transfer rate of E. coli RP4 plasmid is order of magnitudes greater when conjugation occurs on solid medium rather than in liquid suspension. We have investigated whether the conjugation rate would also be higher in ultrasonically induced E. coli clumps than in free suspension. The donor strain was mixed with a recipient strain of E. coli, then sonicated in a capillary at 4.6 MHz in a tubular transducer for 5 min. The bacteria aggregated successfully. Results showed a three-fold increase in the rate of conjugation compared to a liquid mating control.     

Development and characterization of a carvacrol nanoemulsion and evaluation of its antimicrobial activity against selected food-related pathogens

Carvacrol has been recognized as an efficient growth inhibitor of food pathogens. However, carvacrol oil is poorly water-soluble and can be oxidized, decomposed or evaporated when exposed to the air, light, or heat. To overcome these limitations, a carvacrol nanoemulsion was developed and its antimicrobial activity against food pathogens evaluated in this study. The nanoemulsion containing 3% carvacrol oil, 9% surfactants (HLB 11) and 88% water, presented good stability over a period of 90 days. In general, the carvacrol nanoemulsion (MIC: 256 µg ml⁻¹ for E. coli and Salmonella spp., 128 µg ml⁻¹ for Staphylococcus aureus and Pseudomonas aeruginosa) exhibited improved antimicrobial activity compared to the free oil. The carvacrol nanoemulsion additionally displayed bactericidal activity against Escherichia coli, P. aeruginosa and Salmonella spp. Therefore, the results of this study indicated that carvacrol oil nanoemulsions can potentially be incorporated into food formulations, wherein their efficacy for the prevention and control of microbial growth could be evaluated.     

Synergistic antibacterial activity of the essential oil of Cuminum cyminum L. seed and nisin in a food model

Aims: To investigate effects of various concentrations of the essential oil of Cuminum cyminum L. seed alone and in combination with nisin on survival of vegetative forms of Bacillus cereus and Bacillus subtilis in a food model (commercial barley soup) and their ultrastructure. Methods and Results: Gas chromatography–mass spectrometry analysis indicated that cumin aldehyde (29·02%) and α‐terpinen‐7‐al (20·70%) constituted the highest amount of the essential oil. The lowest concentration of the essential oil significantly affected the growth of the bacteria at 8°C but not at 25°C. Synergistic effect of the essential oil in combination with the lowest concentration of nisin was observed on the bacteria at 8°C. Evaluation of the sensory properties showed that concentration of 0·15 μl ml^?1 of the essential oil was the most acceptable. Conclusions: The essential oil of C. cyminum L. seed showed the most bactericidal effects on B. cereus at 8°C. Ultrastructural studies of vegetative cells confirmed the synergistic destructive effects of the essential oil and nisin on membrane and cell wall of the bacteria. Significance and Impact of the Study: Utilization of essential oil of C. cyminum L. seed in combination with nisin can inhibit growth of food‐borne pathogens in food.     

Induction of Phage Production in the Lysogenic Escherichia coli by Hydroxyurea

1) Hydroxyurea, a reversible DNA synthesis inhibitor, was used to study the mechanism of prophage λ induction in Escherichia coli K12. Induction of prophage was judged on two criteria: increase of phage-producing cells and loss of colony-forming ability of the cells. 2) Hydroxyurea induced an increase of phage-producing cells only in lysogenic strains known to be inducible with ultraviolet irradiation for prophage development and not in strains such as E. coli K12 (λind^–) or E. coli K12 recA (λ⁺). 3) When protein synthesis was inhibited, hydroxyurea did not increase phage-producing cells of lysogenic strains; it showed a bacteriocidal effect on lysogenic recA⁺ strains, but not on nonlysogenic strains. 4) The sensitivity of E. coli K12 recA to hydroxyurea was independent of whether or not the cells were lysogenic. 5) From the results it is suggested that certain steps leading to loss of colony-forming ability (i.e. prophage induction) do not require de novo protein synthesis but require the presence of the host recA⁺ gene.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR

Aims: The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real‐time PCR for the detection of viable Escherichia  coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results: Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan^® real‐time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan^® probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0·25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false‐negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 μg ml^?1, was demonstrated to effectively bind DNA from 10⁸ CFU ml^?1 dead cells, and the optimized method could detect as low as 10⁴ CFU g^?1 of viable E. coli O157:H7 cells in ground beef without interference from 10⁸ CFU g^?1 of dead cells. Conclusions: EMA real‐time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study: The EMA real‐time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products.     

Comparison of solid‐phase cytometry and the plate count method for the evaluation of the survival of bacteria in pharmaceutical oils

Aim: To compare the survival of four bacterial strains (Escherichia coli, Proteus mirabilis, Staphylococcus aureus, Pseudomonas aeruginosa) in pharmaceutical oils, including jojoba oil/tea tree oil, carbol oil, jojoba oil and sesame oil. Methods and Results: Oils were spiked with the test bacteria in a concentration of 10⁴ CFU ml^?1. Bacteria were extracted from oils with phosphate‐buffered saline containing 0·5% Tween 20. Aliquots of the pooled water layers were analysed by solid‐phase cytometry and plate counting. Plate counts dropped to zero for all test strains exposed for 24 h to three of the four oils. In contrast, significant numbers of viable cells were still detected by SPC, except in the jojoba oil/tea tree oil mixture and partly in sesame oil. Conclusions: Exposure of bacteria for 24 h to the two oils containing an antimicrobial led to a loss of their culturability but not necessarily of their viability. The antibacterial activity of the jojoba oil/tea tree oil mixture supersedes that of carbol oil. Significance and Impact of the Study: These in vitro data suggest that the jojoba oil/tea tree oil mixture more than carbol oil inhibits bacterial proliferation when used for intermittent self‐catherization.     

Plasmids expressing the Herpes simplex virus thymidine kinase gene in mammalian and bacterial cells

Summary Two plasmids containing either the complete thymidine kinase gene of Herpes simplex virus type I (pSK2) or the gene without the remote control sequence (pSK1) just behind the lac promoter and the first codons of the lacZ gene were constructed. Both plasmids efficiently transform mouse Ltk^- cells as well as E. coli tk^- cells to the Tk⁺ phenotype and are well suited for plasmid rescue from transformed mouse cells by direct functional selection for tk expression using a tk ^- mutant of E. coli C600.     

In Vitro Antibacterial Efficacy of Cymbopogon flexuosus Essential Oil against Aeromonas hydrophila of Fish Origin and in Silico Molecular Docking of the Essential Oil Components against DNA Gyrase-B and Their Drug-Likeness

In aquaculture, diseases caused by the Aeromonads with high antibiotic resistance are among the most common and troublesome diseases. Application of herbs is emerging as a tool in controlling these diseases. Plant extracts besides disease control, favor various physiological activities in fish. In this study, essential oil of Cymbopogon flexuosus (Poaceae family) was studied in vitro for its antibacterial efficacy against two oxytetracycline (OTC) resistant and one sensitive strains of Aeromonas hydrophila. The oil was found rich (86.93 %) in oxygenated terpenoids containing 74.15 % of citral. The oil exhibited dose dependent growth inhibition of the bacteria. Mean MIC value of the oil against the sensitive strain was recorded as 2.0 mg mL⁻¹ whereas MBC value was recorded as 4.0 mg mL⁻¹. The oil was found effective against the OTC resistant isolates with the MIC and MBC values ranging from 2.67–3.33 and 4.0–6.67 mg mL⁻¹, respectively. In silico molecular docking of the essential oil components against DNA gyrase-B, a vital macromolecule in bacterial cell, was carried out to computationally asses the efficacy of the oil against the bacteria. Some of the components of the essential oil strongly bonded with the enzyme to inhibit its efficacy. Binding energy of some components of the oil was comparable to that of the conventional antibiotic, OTC. The identified phytochemicals exhibited favorable physicochemical and pharmacokinetic properties and satisfied the rule of five (Ro5).     

Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress

Aims: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. Methods and Results: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55–70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE? system to measure net K⁺ fluxes. No difference in K⁺ fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K⁺ loss from the heat‐treated E. coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K⁺ flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K⁺ flux disruption was not clearly observed until 68°C was reached. Conclusions: No changes in net K⁺ flux from heat‐stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K⁺ fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two‐stage process enabled evaluation of the lethality of heat‐treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. Significance and Impact of the Study: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture‐dependent test offers an alternative system to culture‐based methods.