Plant Extracts Containing Saponins Affects the Stability and Biological Activity of Hempseed Oil Emulsion System.

In this study, two saponins-rich plant extracts, viz. Saponaria officinalis and Quillaja saponaria, were used as surfactants in an oil-in-water (O/W) emulsion based on hempseed oil (HSO). This study focused on a low oil phase content of 2% v/v HSO to investigate stable emulsion systems under minimum oil phase conditions. Emulsion stability was characterized by the emulsification index (EI), centrifugation tests, droplet size distribution as well as microscopic imaging. The smallest droplets recorded by dynamic light scattering (droplets size v. number), one day after the preparation of the emulsion, were around 50-120 nm depending the on use of Saponaria and Quillaja as a surfactant and corresponding to critical micelle concentration (CMC) in the range 0-2 g/L. The surface and interfacial tension of the emulsion components were studied as well. The effect of emulsions on environmental bacteria strains was also investigated. It was observed that emulsions with Saponaria officinalis extract exhibited slight toxic activity (the cell metabolic activity reduced to 80%), in contrast to Quillaja emulsion, which induced Pseudomonas fluorescens ATCC 17400 growth. The highest-stability samples were those with doubled CMC concentration. The presented results demonstrate a possible use of oil emulsions based on plant extract rich in saponins for the food industry, biomedical and cosmetics applications, and nanoemulsion preparations.

Allicin, a natural antimicrobial defence substance from garlic, inhibits DNA gyrase activity in bacteria.

Allicin (diallylthiosulfinate) is a potent antimicrobial substance, produced by garlic tissues upon wounding as a defence against pathogens and pests. Allicin is a reactive sulfur species (RSS) that oxidizes accessible cysteines in glutathione and proteins. We used a differential isotopic labelling method (OxICAT) to identify allicin targets in the bacterial proteome. We compared the proteomes of allicin-susceptible Pseudomonas fluorescens Pf0-1 and allicin-tolerant PfAR-1 after a sublethal allicin exposure. Before exposure to allicin, proteins were in a predominantly reduced state, with approximately 77% of proteins showing less than 20% cysteine oxidation. Protein oxidation increased after exposure to allicin, and only 50% of proteins from allicin-susceptible Pf0-1, but 65% from allicin-tolerant PfAR-1, remained less than 20% oxidised. DNA gyrase was identified as an allicin target. Cys433 in DNA gyrase subunit A (GyrA) was approximately 6% oxidized in untreated bacteria. After allicin treatment the degree of Cys433 oxidation increased to 55% in susceptible Pf0-1 but only to 10% in tolerant PfAR-1. Allicin inhibited E. coli DNA gyrase activity in vitro in the same concentration range as nalidixic acid. Purified PfAR-1 DNA gyrase was inhibited to greater extent by allicin in vitro than the Pf0-1 enzyme. Substituting PfAR-1 GyrA into Pf0-1 rendered the exchange mutants more susceptible to allicin than the Pf0-1 wild type. Taken together, these results suggest that GyrA was protected from oxidation in vivo in the allicin-tolerant PfAR-1 background, rather than the PfAR-1 GyrA subunit being intrinsically less susceptible to oxidation by allicin than the Pf0-1 GyrA subunit. DNA gyrase is a target for medicinally important antibiotics; thus, allicin and its analogues may have potential to be developed as gyrase inhibitors, either alone or in conjunction with other therapeutics.

Identification of natural product compounds as quorum sensing inhibitors in Pseudomonas fluorescens P07 through virtual screening.

Pseudomonas fluorescens, a Gram-negative psychrotrophic bacteria, is the main microorganism causing spoilage of chilled raw milk and aquatic products. Quorum sensing (QS) widely exists in bacteria to monitor their population densities and regulate numerous physiological activities, such as the secretion of siderophores, swarming motility and biofilm formation. Thus, searching for quorum sensing inhibitors (QSIs) may be another promising way to control the deterioration of food caused by P. fluorescens. Here, we screened a traditional Chinese medicine (TCM) database to discover potential QSIs with lesser toxicity. The gene sequences of LuxI- and LuxR-type proteins of P. fluorescens P07 were obtained through whole-genome sequencing. In addition, the protein structures built by homology modelling were used as targets to screen for QSIs. Twenty-one compounds with a dock score greater than 6 were purchased and tested by biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136). The results showed that 10 of the compounds were determined as hits (hit rate: 66.67%). Benzyl alcohol, rhodinyl formate and houttuynine were effective QSIs. The impact of the most active compound (benzyl alcohol) on the phenotypes of P. fluorescens P07, including swimming and swarming motility, production of extracellular enzymes and siderophores, N-acylhomoserine lactone (AHLs) content and biofilm formation were determined. The inhibitory mechanism of benzyl alcohol on the QS system of P. fluorescens P07 is further discussed. This study reveals the feasibility of searching for novel QSIs through virtual screening.

Negative effects of artemisinin on phosphorus solubilizing bacteria in vitro.

The anti-malarial drug artemisinin is extracted from the leaves of Artemisia annua L. Due to toxicity to some microorganisms, the release of artemisinin from this medicinal plant in commercial cultivation might produce a potential risk for phosphorus (P) solubilizing bacteria (PSB). Therefore, the growth, P mobilization, and proton and organic acid efflux by two PSB isolates, Bacillus subtilis and Pseudomonas fluorescens, obtained from the soil without growing A. annua L. in history in the region for growing A. annua L., Chongqing, China, were studied through soil and solution incubations with different nominal concentrations of artemisinin (0, 2.5, 5.0, and 10.0 mg/kg or mg/L). Addition of artemisinin into soil and culture solutions decreased significantly the number of PSB except P. fluorescens at a low artemisinin concentration (2.5 mg/L) in culture solution which remained unchanged in comparison with the control (without artemisinin). This suggests high artemisinin inhibited the cell division or led to the death of PSB, and the different species responded differently to artemisinin. Compared with original soil, PSB inoculation significantly increased Olsen P, whilst the addition of artemisinin decreased this P form in soil. There was a positive correlation between the number of PSB and Olsen P content in soils (r2 = 0.824, n = 8), indicating the involvement of PSB in P mobilization of insoluble minerals. Oxalate and acetate were commonly found in the bacterial culture solutions, which accounted for 73.6-84.4% of all organic acids in the culture medium without artemisinin. Malate was detected in the culture solution of B. subtilis, and citrate and succinate in P. fluorescens. The percentage of tricalcium phosphate solubilization (PTPS) positively correlated to the concentrations of protons and all organic acids (r2proton＝0.901, n＝8, P＜0.01; r2organic acids＝0.923, n＝8, P＜0.01). The concentrations of protons, organic acids and soluble inorganic P in culture solutions, and PTPS were decreased simultaneously as nominal artemisinin concentrations increased. For these decreases it implies the metabolic inhibition and the death of PSB caused by artemisinin could be the main reasons for the less efflux of protons and organic acids, presumably resulting in the decreased ability of PSB to mobilize inorganic P. Therefore, artemisinin released from A. annua L. in commercial and continual cultivation could adversely affect the community structure and inorganic P mobilization of PSB in soils.

Research for the lichen Usnea barbata metabolites.

This work presents investigations of biologically active metabolites of Usnea barbata lichen. Extraction conditions for usnic acid and other biologically active phytocomponents using various solvent systems were chosen. Modern analytical techniques were used to study composition of the obtained extracts; usnic acid and phenolic compound contents were estimated. Antioxidant activity and antimicrobial properties of lichen dry extract against bacteria Bacillus subtilis and Pseudomonas fluorescens were studied.

Transcriptomic analysis of the response of Pseudomonas fluorescens to epigallocatechin gallate by RNA-seq.

Epigallocatechin gallate (EGCG) is a main constituent of green tea polyphenols that are widely used as food preservatives and are considered to be safe for consumption. However, the underlying antimicrobial mechanism of EGCG and the bacterial response to EGCG are not clearly understood. In the present study, a genome-wide transcriptional analysis of a typical spoilage bacterium, Pseudomonas fluorescens that responded to EGCG was performed using RNA-seq technology. A total of 26,365,414 and 23,287,092 clean reads were generated from P. fluorescens treated with or without 1 mM EGCG and the clean reads were aligned to the reference genome. Differential expression analysis revealed 291 upregulated genes and 134 downregulated genes and the differentially expressed genes (DEGs) were verified using RT-qPCR. Most of the DGEs involved in iron uptake, antioxidation, DNA repair, efflux system, cell envelope and cell-surface component synthesis were significantly upregulated by EGCG treatment, while most genes associated with energy production were downregulated. These transcriptomic changes are likely to be adaptive responses of P. fluorescens to iron limitation and oxidative stress, as well as DNA and envelope damage caused by EGCG. The expression of specific genes encoding the extra-cytoplasmic function sigma factor (PvdS, RpoE and AlgU) and the two-component sensor histidine kinase (BaeS and RpfG) were markedly changed by EGCG treatment, which may play important roles in regulating the stress responses of P. fluorescens to EGCG. The present data provides important insights into the molecular action of EGCG and the possible cross-resistance mediated by EGCG on P. fluorescens, which may ultimately contribute to the optimal application of green tea polyphenols in food preservation.

Enhancement of antibacterial properties of silver nanoparticles-ceftriaxone conjugate through Mukia maderaspatana leaf extract mediated synthesis.

Green synthesis of nanoparticles with low range of toxicity and conjugation to antibiotics has become an attractive area of research for several biomedical applications. Nanoconjugates exhibited notable increase in biological activity compared to free antibiotic molecules. With this perception, we report the biosynthesis of silver nanoparticles using aqueous extract of leaves of Mukia maderaspatana and subsequent conjugation of the silver nanoparticles to antibiotic ceftriaxone. The leaves of this plant are known to be a rich source of phenolic compounds with high antioxidant activity that are used as reducing agents. The size, morphology, crystallinity, composition of the synthesized silver nanoparticles and conjugation of ceftriaxone to silver nanoparticles were studied using analytical techniques. The activity of the conjugates against Bacillus subtilis (MTCC 1790), Klebsiella pneumoniae (MTCC 3384), Staphylococcus aureus (ATCC 25923), and Salmonella typhi (MTCC 3224) was compared to ceftriaxone and unconjugated nanoparticles using disc diffusion method. The effect of silver nanoparticles on the reduction of biofilms of Pseudomonas fluorescens (MTCC 6732) was determined by micro plate assay method. The antioxidant activities of extract, silver nitrate, silver nanoparticles, ceftriaxone and conjugates of nanoparticles were evaluated by radical scavenging 1, 1- diphenyl-2-picrylhydrazyl test. Ultraviolet visible spectroscopy and Fourier transform infrared spectroscopy confirmed the formation of metallic silver nanoparticles and conjugation to ceftriaxone. Atomic force microscopy, transmission electron microscopy and particle size analysis showed that the formed particles were of spherical morphology with appreciable nanosize and the conjugation was confirmed by slight increase in surface roughness. The results thus showed that the conjugation of ceftriaxone with silver nanoparticles has better antioxidant and antimicrobial effects than ceftriaxone and unconjugated nanoparticles. It can be suggested that M. maderaspatana mediated nanoparticle-ceftriaxone conjugate can be used effectively in the production of potential antioxidant and antimicrobial agents. The present study offers a significant overview to the development of novel antimicrobial nanoparticles.

Multiple antibiotic-resistant bacteria on fluted pumpkin leaves, a herb of therapeutic value.

Fluted pumpkin (Telfairia occidentalis) is a minimally-processed green leafy vegetable traditionally used for its antianaemic properties in the form of leaf juice without a heating or inactivation step before consumption. The aim of the study was to assess the presence of surface microbiota on T. occidentalis leaves and also to determine the antimicrobial susceptibility of isolated organisms. Bacterial contaminants on 50 samples of T. occidentalis leaves were isolated and characterized using standard biochemical methods and the antimicrobial susceptibility of isolated organisms was determined using the antibiotic disc diffusion assay. The results obtained show that the leaves of T. occidentalis is contaminated with organisms which included Enterobacter agglomerans (25.9%), Proteus vulgaris (24.9%), Klebsiella spp. (2.6%), and Serratia liquefaciens (2.1%). Other bacterial isolates recovered in order of frequency included: Staphylococcus spp. (33.7%), Bacillus spp. (8.3%), and Pseudomonas fluorescens (2.6%). Of the 193 bacterial isolates from the leaves of T. occidentalis samples tested for antimicrobial resistance, all (100%) were found to be resistant to ampicillin, cloxacillin, augmentin, erythromycin, and tetracycline while 96% of the isolates were resistant to cephalothin. Resistance to trimethoprim (93%) and gentamicin (83%) was also observed. Approximately, 22% of the isolates were resistant to ciprofloxacin; however, only 11 (5.8%) were resistant to ofloxacin. Thus, uncooked T. occidentalis is a potential source of highly-resistant epiphytic bacteria which could be opportunistic pathogens in consumers.

Synergistic antimicrobial effect against early biofilm formation: micropatterned surface plus antibiotic treatment.

The detrimental effects of biofilms are a cause of great concern in medical, industrial and environmental areas. In this study, we proposed a novel eradication strategy consisting of the combined use of micropatterned surfaces and antibiotics on biofilms to reduce the rate of bacterial colonisation. Pseudomonas fluorescens biofilms were used to perform a comparative evaluation of possible strategies to eradicate these biological layers. First, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration of planktonic cultures were determined. Subsequently, adhesion of bacteria on microstructured gold surfaces (MS) with patterned features that were similar to the bacterial diameter as well as on smooth nanostructured gold (NS) was assessed. As expected, lower bacterial attachment as well as inhibition of bacterial aggregation were observed on MS. The effect of streptomycin treatment (ST) in the concentration range 1-4 mg/L (0.25-1× MIC) on biofilms grown on MS and NS was also evaluated. The combined strategy involving the use of micropatterned surfaces and antibiotic treatment (MS+ST) to eradicate Pseudomonas biofilms was then investigated. Results showed a synergistic effect of MS+ST that yielded a reduction of ≥1000-fold in the number of surviving biofilm bacteria with respect to those obtained with single ST or MS. The combined strategy may be a significant contribution to the eradication of biofilms from different environments. In addition, the important role of early monolayer bacterial aggregates in increasing resistance to antimicrobial agents was demonstrated.

Antagonistic control of microbial pathogens under iron limitations by siderophore producing bacteria in a chemostat setup.

Certain bacteria develop iron chelation mechanisms that allow them to scavenge dissolved iron from the environment and to make it unavailable to competitors. This is achieved by producing siderophores that bind the iron which is later liberated internally in the cell. Under conditions of iron limitation, siderophore producing bacteria have therefore an antagonistic growth advantage over other species. This has been observed in particular in agricultural and aquacultural systems, as well as in food microbiology. We investigate here the possibility of a probiotic biocontrol strategy to eradicate a well established, often pathogenic, non-chelating population by supplementing the system with generally regarded as safe siderophore producing bacteria. Set in a chemostat setup, our modeling and simulation studies suggest that this is indeed possible in a finite time treatment.

Antiseptic activity and phenolic constituents of the aerial parts of Vitex negundo var. cannabifolia.

Four phenolics, salviaplebeiaside (1), γ-tocopherol (2), chrysosplenol-D (4), and isovitexin (5), along with α-tocoquinone (3) and ß-sitosterol (6) were isolated from the aerial parts of Vitex negundo var. cannabifolia. The isolation was performed using bio-assay tracking experiments. The structures of compounds 1-5 were established by spectroscopic means. The antibacterial activities of the compounds were assessed against Escherichia coli, Bacillus subtilis, Micrococcus tetragenus and Pseudomonas fluorescens. Chrysosplenol-D (4) exhibited activities against all the four spoilage microorganisms.

Antimicrobial action of essential oil vapours and negative air ions against Pseudomonas fluorescens.

The aim of this study was to investigate the antibacterial activity of essential oil (in liquid as well as in vapour phase) and negative air ions (NAI) against Pseudomonas fluorescens. The combined effect of NAI with essential oil vapour was also investigated to determine kill time and morphological changes in bacterial cells. The MIC of Cymbopogon citratus (0.567 mg/ml), Mentha arvensis (0.567 mg/ml), Mentha piperita (1.125 mg/ml) and Eucalyptus globulus (2.25 mg/ml) was studied via the agar dilution method. To estimate the antibacterial activity of essential oils in the vapour phase, agar plates inoculated with P. fluorescens were incubated with various concentrations of each essential oil vapour and zone of inhibition was recorded. Further, in order to assess the kill time, P. fluorescens inoculated agar plates were exposed to selected bactericidal essential oil vapour and NAI, separately, in an air-tight chamber. A continuous decrease in bacterial count was observed over time. A significant enhancement in the bactericidal action was observed by exposure to the combination of essential oil vapour and NAI as compared to their individual action. Scanning electron microscopy was used to study the alteration in morphology of P. fluorescens cells after exposure to C. citratus oil vapour, NAI, and combination of C. citratus oil vapour and NAI. Maximum morphological deformation was found due to the combined effect of C. citratus oil vapour and NAI. This study demonstrates that the use of essential oils in the vapour phase is more advantageous than the liquid phase. Further the antibacterial effect of the essential oil vapours can be significantly enhanced by the addition of NAI. The work described here offers a novel and efficient approach for control of bacterial contamination that could be applied for food stabilization practices.

Evaluation of different carbon sources for growth and biosurfactant production by Pseudomonas fluorescens isolated from wastewaters.

The indigenous strain Pseudomonas fluorescens, isolated from industrial wastewater, was able to produce glycolipid biosurfactants from a variety of carbon sources, including hydrophilic compounds, hydrocarbons, mineral oils, and vegetable oils. Hexadecane, mineral oils, vegetable oils, and glycerol were preferred carbon sources for growth and biosurfactant production by the strain. Biosurfactant production was detected by measuring the surface and interfacial tension, rhamnose concentration and emulsifying activity. The surface tension of supernatants varied from 28.4 mN m(-1) with phenanthrene to 49.6 mN m(-1) with naphthalene and heptane as carbon sources. The interfacial tension has changed in a narrow interval between 6.4 and 7.6 mN m(-1). The emulsifying activity was determined to be highest in media with vegetable oils as substrates. The biosurfactant production on insoluble carbon sources contributed to a significant increase of cell hydrophobicity and correlated with an increased growth of the strain on these substrates. Based on these results, a mechanism of biosurfactant-enhanced interfacial uptake of hydrophobic substrates could be proposed as predominant for the strain. With hexadecane as a carbon source, the pH value of 7.0-7.2 and temperature of (28 +/- 2) degrees C were optimum for growth and biosurfactant production by P. fluorescens cells. The increased specific protein and biosurfactant release during growth of the strain on hexadecane in the presence of NaCl at contents up to 2% could be due to increased cell permeability. The capability of P. fluorescens strain HW-6 to adapt its own metabolism to use different nutrients as energy sources and to keep up relatively high biosurfactant levels in the medium during the stationary phase is a promising feature for its possible application in biological treatments.

Cephalosol: an antimicrobial metabolite with an unprecedented skeleton from endophytic Cephalosporium acremonium IFB-E007.

Cephalosol (1), a potent antimicrobial secondary metabolite with a new carbon skeleton, was characterized from the culture of Cephalosporium acremonium IFB-E007 that used to reside as an endophyte in Trachelospermum jasminoides (Apocynaceae). Its structure and absolute configuration were unambiguously determined by spectroscopic and computational approaches.

Study on the synergic effect of natural compounds on the microbial quality decay of packed fish hamburger.

The effectiveness of natural compounds in slowing down the microbial quality decay of refrigerated fish hamburger is addressed in this study. In particular, the control of the microbiological spoilage by combined use of three antimicrobials, and the determination of their optimal composition to extend the fish hamburger Microbiological Stability Limit (MAL) are the main objectives of this work. Thymol, grapefruit seed extract (GFSE) and lemon extract were tested for monitoring the cell growth of the main fish spoilage microorganisms (Pseudomonas fluorescens, Photobacterium phosphoreum and Shewanella putrefaciens), inoculated in fish hamburgers, and the growth of mesophilic and psychrotrophic bacteria. A Central Composite Design (CCD) was developed to highlight a possible synergic effect of the above natural compounds. Results showed an increase in the MAL value for hamburgers mixed with the antimicrobial compounds, compared to the control sample. The optimal antimicrobial compound composition, which corresponds to the maximal MAL value determined in this study, is: 110 mgL(-1) of thymol, 100 mgL(-1) of GFSE and 120 mgL(-1) of lemon extract. The presence of the natural compounds delay the sensorial quality decay without compromising the flavor of the fish hamburgers.

Assessment of toxicity of the untreated and treated olive mill wastewaters and soil irrigated by using microbiotests.

Hazard assessments based on two measures of toxicity were conducted for the untreated olive mill wastewaters (U), untreated olive mill wastewaters organic extract (UOE), treated olive mill wastewaters (T), treated olive mill wastewaters organic extract (TOE) and extracts of soils ferti-irrigated with untreated (SU) and with treated olive mill wastewaters (ST). The measures of toxicity were achieved by the determination of the bioluminescence inhibition percent (I(B)%) of Vibrio fischeri and by the growth inhibition (GI) of Bacillus megaterium, Pseudomonas fluorescens and Escherichia coli. A bioluminescence inhibition of V. fischeri of 100%, 100%, 65%, 47%, 46% and 30% were obtained with U, UOE, T, TOE, SU and ST respectively. Indeed, even diluted 24 times, a significant bioluminescence inhibition of 96% was obtained by U. However, only 30% bioluminescence inhibition was obtained by 24 times diluted T. Whereas, 24 times diluted, SU and ST did not show a bioluminescence inhibition (3% and 1%, respectively). The GI of B. megaterium, P. fluorescens and E. coli were, respectively, 93%, 72% and 100% by U; 100%, 80% and 100% by UOE; 70%, 60% and 89% by T; 63%, 54% and 68% by TOE; 39%, 27% and 43% by SU and 23%, 0% and 34% by ST. The incubation of U or T in the soil during four months reduced their toxicity by 54% and 35%, respectively. As it was expected, the most resistant bacterium to OMW toxicity is P. fluorescens then B. megaterium and E. coli. V. fischeri remained the most sensitive strain to the toxicity of this sewage what proves again its utilisation as standard of measure of the toxicity.

Antimicrobial activity of several herb and spice extracts in culture medium and in vacuum-packaged pork.

Extracts prepared from honeysuckle, Scutellaria, Forsythia suspensa (Thunb), cinnamon, and rosemary with 75% ethanol and from clove oil dissolved in 75% ethanol were applied to inoculated agar media to observe their inhibitory effects on the growth of Escherichia coli, Pseudomonas fluorescens, and Lactobacillus plantarum. All the extracts suppressed the growth of these bacteria; Scutellaria exhibited the strongest effect against E. coli. An orthogonal test revealed that the most effective antimicrobial composite extracts were equal-volume mixtures of 0.125 g/ml Scutellaria + 0.5 g/ml honeysuckle + 0.125 g/ml Forsythia + 0.25 g/ml cinnamon and 0.25 g/ml cinnamon + 0.125 g/ml rosemary + 0.25% clove oil. These mixed extracts also produced strong antimicrobial effects in vacuum-packaged fresh pork, with 1.81- to 2.32-log reductions in microbial counts compared with the control when stored for up to 28 days. The sensory panel detected minimal differences in surface color and off-odors between meat samples treated with herb-spice extracts and the control. These results indicate that combined herb and spice extracts can be used as natural antimicrobials for food preservation.

Susceptibility of pseudomonads to Melaleuca alternifolia (tea tree) oil and components.

OBJECTIVES: Thirty isolates of Pseudomonas aeruginosa, 15 isolates of Pseudomonas putida and 11 isolates of Pseudomonas fluorescens were tested for susceptibility to tea tree oil (TTO), the essential oil of Melaleuca alternifolia, and the components terpinen-4-ol, alpha-terpineol, cineole, gamma-terpinene and rho-cymene. METHODS: MICs were determined by broth microdilution in Mueller-Hinton medium supplemented with 0.002% (v/v) Tween 80. RESULTS: The MIC90 of TTO for all isolates tested was 4% (v/v) or less. Susceptibility to components tested varied between species. CONCLUSIONS: Pseudomonas spp. are susceptible to TTO and some of its components although they are less susceptible than many other bacteria tested previously.