In vitro efficacy of nisin Z against Candida albicans adhesion and transition following contact with normal human gingival cells

Aim:  To investigate the nisin Z innocuity using normal human gingival fibroblast and epithelial cell cultures, and its synergistic effect with these gingival cells against Candida albicans adhesion and transition from blastospore to hyphal form.
Methods and Results:  Cells were cultured to 80% confluence and infected with C. albicans in the absence or presence of various concentrations of nisin Z. Our results indicate that only high concentrations of nisin Z promoted gingival cell detachment and differentiation. Determination of the LD₅₀ showed that the fibroblasts were able to tolerate up to 80  μ g ml⁻¹ for 24 h, dropping thereafter to 62  μ g ml⁻¹ after 72 h of contact, compared to 160  μ g ml⁻¹ after 24 h, and 80  μ g ml⁻¹ after 72 h recorded by the gingival epithelial cells which displayed a greater resistance to nisin Z. The use of nisin Z even at low concentration (25  μ g ml⁻¹) at appropriate concentrations with gingival cells significantly reduced C. albicans adhesion to gingival monolayer cultures and inhibited the yeast's transition.
Conclusion:  These findings show that when used at non-toxic levels for human cells, nisin Z can be effective against C. albicans adhesion and transition and may synergistically interact with gingival cells for an efficient resistance against C. albicans .
Significance and Impact of the Study:  This study suggests the potential usefulness of nisin Z as an antifungal agent, when used in an appropriate range.     

Excretion of butyraldehyde, isobutyraldehyde and valeraldehyde by Streptomyces cinnamonensis

Abstract Streptomyces cinnamonensis produces butyraldehyde, isobutyraldehyde and valeraldehyde in concentrations of 9–39 μg ml⁻¹, with the average molar ratio being 22:33:45. These aldehydes, excreted into fermentation broth or agar, were derivatised with 2,4-dinitrophenylhydrazine, isolated, and their structures were determined by ¹H, ¹³C NMR and mass spectrometry. The highest production of aldehydes was observed in surface cultivation on agar.     

Constitutive mineralization of low concentrations of the herbicide linuron by a Variovorax sp. strain

The mineralization of the herbicide linuron at concentrations of μg and mg L⁻¹ was studied in liquid batch experiments with Variovorax sp. strain SRS16. The strain was highly efficient at mineralizing a range of linuron concentrations (0.002–10 mg L⁻¹) with 20–60% of the added ¹⁴C-ring-labeled linuron metabolized to ¹⁴CO₂ within hours to days depending on the initial linuron concentration and incubation period. At mg L⁻¹ linuron concentrations the mineralization activity by SRS16 was inducible and a shift to constitutive mineralization activity was apparent with a reduction in the linuron concentration to μg L⁻¹ levels. This study revealed that strain SRS16 is a promising candidate for bioaugmentation of water or soil resources contaminated with low linuron concentrations.     

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

Inhibitory effects of sucrose fatty acid esters on survival of Yersinia enterocolitica on eggshell surface and use of blue lake as indicator of bacterial penetration into eggs

Aims:  To determine the effectiveness of sucrose monolaurate (SML) and sucrose monocaprate (SMC), alone and in combination with ethylenediaminetetraacetic acid (EDTA), propionic acid (PA) or citric acid (CA) in reducing mesophilic aerobic bacteria (MAB) and Yersinia enterocolitica O:9 populations on eggshells and their damage potential on the microstructure of shell cuticle.
Methods and Results:  Uninoculated eggs and eggs submerged in a solution of Y. enterocolitica were immersed in solutions of the various treatments. MAB and Y. enterocolitica counts on the surface of the eggs were carried out before and after treatment. MAB counts decreased less than 2 logs on uninoculated eggshells irrespective of treatment and reductions of 3·2 and 3·0 logs of Y. enterocolitica were obtained with 1000 μg ml⁻¹ SML plus 0·1% CA or 1000 μg ml⁻¹ SML plus 600 μg ml⁻¹ EDTA solutions, respectively. Y. enterocolitica 2/O:9 was recovered from natural microflora. Use of blue lake staining revealed minimal damage to the shells from the washing treatments.
Conclusions:  SML and SMC at 1000 μg ml⁻¹ combined with CA or EDTA could be effective in reducing Y. enterocolitica on eggshells with a minimal risk of later bacterial recontamination.
Significance and Impact of the Study:  Eggs are a recognized vehicle for transmission of Y enterocolitica although a prevalence of only 2·7% was detected in this study. Washing eggs in solutions containing SML or SMC could eliminate Y. enterocolitica contamination of egg shells.     

Trifluoperazine inhibits the incorporation of labelled precursors into lipids, proteins and DNA of Mycobacterium tuberculosis H37Rv

Abstract We have recently demonstrated that the calmodulin antagonist trifluoperazine has antitubercular activity in vitro against Mycobacterium tuberculosis H₃₇R_v susceptible and resistant to isoniazid. It is shown that trifluoperazine at a concentration of 50 μ g ml⁻¹ when added to the cells along with the labelled precursors inhibited the incorporation of [¹⁴C]acetate into lipids (63%) and uptake of [¹⁴C]glycine (74%) and [^3H]thymidine (52%) bu whole cells of M. tuberculosis H₃₇R_v by 6 h of exposure. After 48 h, the inhibition was 87%, 97% and 74%, respectively. However, when the drug was added to cells taking up and metabolizing the labelled precursors at a later point (3 h for [¹⁴C]acetate and [³H]thymidine and 12 h for [¹⁴C]glycine) it inhibited completely the uptake of all the precursors, at least up to 24 h. The onset of inhibitory action was very rapid, i.e. 3 h. It is suggested that trifluoperazine has multiple sites of action and acts probably by affecting the synthesis of lipids, proteins and DNA.     

Response of Listeria monocytogenes to liquid smoke

Aims:  To investigate the effect of liquid smoke on growth, survival, proteomic pattern and haemolytic potential of Listeria monocytogenes.
Methods and Results:  Growth and survival curves were recorded in brain–heart infusion broth supplemented with three concentrations of liquid smoke. L. monocytogenes growth was inhibited in the presence of 15 μg ml⁻¹ phenol while a rapid decrease in cell viability occurred in the presence of 30 μg ml⁻¹ phenol. The proteome of L. monocytogenes cytosoluble proteins was slightly modified after 2-h incubation with 30 μg ml⁻¹ phenol but no protein already characterized in response to other known stresses was induced, except the protease ClpP. Liquid smoke inhibited the haemolytic potential without affecting hly gene expression, showing a potential inhibition of protein activity or stability.
Conclusions:  The presence of liquid smoke in a rich medium strongly affected growth and survival of L. monocytogenes . Brief smoke stress affected the metabolic pathways and inhibited the haemolytic activity of L. monocytogenes .
Significance and Impact of Study:  This study is a first step in the investigation of the influence of a smoked product on L. monocytogenes strains.     

Biodegradation of 2,4-dichlorophenol in the presence of volatile organic compounds in soils under different vegetation types

It has been suggested that monoterpenes emitted within the soil profile, either by roots or by decaying biomass, may enhance the biodegradation of organic pollutants. The aim of this study was to evaluate the effect of biogenic volatile organic compounds (VOCs) on the catabolism of 2,4-dichlorophenol in soils. Soils were collected from areas surrounding monoterpene (woodland) and nonmonoterpene (grassland)-emitting vegetation types. Soils were spiked with [UL-¹⁴C] 2,4-dichlorophenol at 10 mg kg⁻¹ and amended with α-pinene, p -cymene or a mix of monoterpenes (α-pinene, limonene and p -cymene in 1 : 1 : 1 ratio). The effects of monoterpene addition on the catabolism of [UL-¹⁴C] 2,4-dichlorophenol to ¹⁴CO₂ by indigenous soil microbial communities were assessed in freshly spiked and 4-week-aged soils. It was found that aged woodland soils exhibited a higher level of [UL-¹⁴C] 2,4-dichlorophenol degradation, which was subsequently enhanced by the addition of monoterpenes ( P <0.001), with the VOC mix and α-pinene amendments showing increased [UL-¹⁴C] 2,4-dichlorophenol catabolism. This study supports claims that the addition of biogenic VOCs to soils enhances the degradation of xenobiotic contaminants.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

Investigation of the anti-fungal activity of coptisine on Candida albicans growth by microcalorimetry combined with principal component analysis

Aims:  This study investigated the anti-fungal activity of coptisine on Candida albicans growth.
Methods and Results:  The metabolic power-time curves of Candida albicans growth at 37°C affected by coptisine were measured by microcalorimetry using an LKB-2277 Bioactivity Monitor with stop-flow mode. Then, the diameter of inhibitory zones in the agar layer was observed using agar cup method, and the minimal inhibitory concentration (MIC) of coptisine on Candida albicans growth was determined by serial dilution method. From the principal component analysis on nine quantitative parameters obtained from the power-time curves, we could easily evaluate the anti-fungal activity of coptisine by analysing the change of values of the main two parameters, growth rate constant k and maximum power output in the log phase P _{m, log}. The results showed that coptisine had strong anti-fungal activity: at a low concentration (45  μ g ml⁻¹) began to inhibit the growth of Candida albicans and at a high concentration (500  μ g ml⁻¹) completely inhibited Candida albicans growth. Coptisine gave big inhibitory zones with diameters between 11 and 43 mm within test range, and the MIC of it was 1000  μ g ml⁻¹.
Conclusions:  Coptisine had strong anti-fungal activity on Candida albicans growth. The method of microcalorimetry applied for the assay of anti-fungal activity of coptisine was quantitative, sensitive and simple.
Significance and Impact of the Study:  This work will provide useful information for the development of chemical biology policy in the use of anti-microbials in food and drug production.     

Robust experimental design for optimizing the microbial inhibitor test for penicillin detection in milk

Aims:  To use experimental design techniques and a multiple logistic regression model to optimize a microbiological inhibition test with dichotomous response for the detection of Penicillin G in milk.
Methods and Results:  A 2³ × 2² robust experimental design with two replications was used. The effects of three control factors (V: culture medium volume, S: spore concentration of Geobacillus stearothermophilus , I: indicator concentration), two noise factors (Dt: diffusion time, Ip: incubation period) and their interactions were studied. The V, S, Dt, Ip factors and V × S, V × Ip, S × Ip interactions showed significant effects.
Conclusions:  The use of 100  μ l culture medium volume, 2 × 10⁵ spores ml⁻¹, 60 min diffusion time and 3 h incubation period is recommended. In these elaboration conditions, the penicillin detection limit was of 3·9  μ g l⁻¹, similar to the maximum residue limit (MRL). Of the two noise factors studied, the incubation period can be controlled by means of the culture medium volume and spore concentration.
Significance and Impact of the Study:  We were able to optimize bioassays of dichotomous response using an experimental design and logistic regression model for the detection of residues at the level of MRL, aiding in the avoidance of health problems in the consumer.     

Symbiotic effectiveness, rate of respiration and glutamine synthetase activity of sodium azide-resistant strains of Rhizobium leguminosarum biovar trifolii

Nitrogen fixing efficiency of sodium azide-resistant strains of Rhizobium leguminosarum bv. trifolii was studied in symbiosis with berseem clover plants in chillum jars. Rate of respiration and glutamine synthetase activity were tested in cultured cells and nodules, respectively. It was observed that shoot dry weight and percentage shoot nitrogen were maximum in plants inoculated with strains resistant to 15 μg ml⁻¹ sodium azide. Rate of respiration in cultured cells was lowest in strains resistant to 15 μg ml⁻¹ sodium azide and highest in strains resistant to 5 μg ml⁻¹ sodium azide. A negative correlation was observed between rate of respiration (in cultured cells) and shoot dry weight of host plants. Glutamine synthetase activity was maximum in nodule extracts of host plants inoculated with strains resistant to 5 and 10 μg ml⁻¹ sodium azide, whereas it was minimum for strains resistant to 15 μg ml⁻¹ sodium azide. Hence, resistance to low doses (15 μg ml⁻¹) of sodium azide, together with lower respiratory and glutamine synthetase activities, could be used as a potential method for isolating the symbiotically effective strains of Rh. leguminosarum bv. trifolii.     

Seasonal heterotrophic flagellate and bacterial plankton dynamics in a large oligotrophic lake– Loch Ness, Scotland

1. Bacterial production in the 0–30 m water column of Loch Ness was measured using a dual labelling procedure with [³H] thymidine and [¹⁴C] leucine between May 1993 and June 1994. In most cases the uptake of the two labels did not covary, suggesting unbalanced growth. Rates of bacterial production varied from undetectable to 46.2 μg C l^–1 day^–1. Highest production coincided with the period of highest primary production, but carbon derived from this source was insufficient to meet the bacterial carbon demand, which was met by allochthonous humic inputs to the system.
2. Heterotrophic flagellate (HNAN) grazing rates, measured using fluorescently labelled bacteria, ranged between 10.3 and 24.5 bacteria cell^–1 day^–1 at temperatures between 5 and 15 °C. They removed up to 27% of the bacterial production per day.
3. Heterotrophic flagellate specific growth rates ranged from 0.043 to 0.093 h^–1 between 5 and 15 °C, giving generation times of 7.4–16.1 h.
4. bacterial and HNAN abundances were not coupled, but the highest HNAN grazing impact related to a time of high bacterial productivity.     

The effect of cryptolepine on the morphology and survival of Escherichia coli, Candida albicans and Saccharomyces cerevisiae

The antimicrobial activity of the indoloquinoline alkaloid, cryptolepine, isolated from Cryptolepis sanguinolenta (Fam. Periplocaceae) was determined against selected micro-organisms. The minimum inhibitory concentration (MIC) ranges obtained, expressed as μg ml⁻¹, were: 5–10 for Saccharomyces cerevisiae NCPF 3139; 10–20 for S. cerevisiae NCPF 3178; 20–40 for Escherichia coli NCTC 10418; 40–80 for E. coli NCTC 11560, Candida albicans ATCC 10231 and C. tropicalis NCPF; and 80–160 for C. albicans NCPF 3242 and NCPF 3262.
Biocidal effects were noted at concentrations 2–4 times those of the MIC of the alkaloid following challenge with 10⁶ cfu ml⁻¹ of micro-organisms. Time-kill studies showed a reduction in viable count from 10⁶ to < 10 cfu ml⁻¹ in 4 h in C. albicans ATCC 10231 exposed to 320 μg ml⁻¹ of the agent; 3 log cycle reductions were recorded for the 6 h counts of E. coli NCTC 10418 and S. cerevisiae NCPF 3139 exposed to 40μg ml⁻¹ and 160 μg ml⁻¹ of the alkaloid respectively.
These results were consistent with findings using scanning electron microscopy. Exposure of cells to biocidal concentrations of cryptolepine produced filamentation prior to lysis in E. coli NCTC 10418 and extreme disturbance of surface structure, including partial and total collapse, followed by lysis in C. albicans ATCC 10231 and S. cerevisiae NCPF 3139.     

Microcystin-producing blooms of Anabaenopsis arnoldi in a potable mountain lake in Saudi Arabia

This study reports for the first time the presence of Anabaenopsis arnoldi blooms in Saudi freshwaters. This species has been investigated with high cell densities (3.8 × 10³–264 × 10³ cells mL⁻¹) during June–November 2007 in Tendaha Lake, one of the major freshwater sources in Saudi Arabia. High temperature and conductivity, and a high concentration of phosphate, and low nitrate concentrations may have contributed to the formation of these blooms. The blooms were found to produce microcystins (MCYSTs) at concentrations up to 364 μg g⁻¹ dry weight as detected by an enzyme-linked immunosorbent assay. MCYSTs were also detected in the raw and treated water of the lake at concentrations (1.6–8.3 and 0.33–1.6 μg L⁻¹, respectively) exceeding the World Health Organization guideline level of 1 μg L⁻¹ for these toxins. HPLC analysis revealed that the extracts of A. arnoldi blooms contained MCYST-RR, -YR and two unidentified MCYSTs, but a pure culture of A. arnoldi isolated from Tendaha Lake during the present study produced MCYST-RR and –YR only. This is the first study to report MCYST production by A. arnoldi . Therefore, this cyanobacterium should be taken into consideration during monitoring of toxic cyanobacterial blooms in drinking and recreational water sources in the world, particularly arid and semi-arid countries including Saudi Arabia.     

Evaluation of in vitro endocytosis and antibody synthesis by rainbow trout head kidney cells treated with bovine lactoferrin

Bovine lactoferrin (LF) was evaluated for its capacity to modulate the in vitro endocytosis (phagocytosis and pinocytosis) and antibody synthesis by head kidney cells of rainbow trout Oncorhynchus mykiss . Phagocytic activity and phagocytic index of head kidney macrophages, determined by measurement of ingested yeast, were influenced by bovine LF starting from the LF concentration of 1 and 0·1 μg ml⁻¹, respectively. Endocytosis, determined by the evaluation of droplet uptake of neutral red dye solution, was significantly enhanced by 10 μg ml⁻¹ of LF. In contrast, antibody synthesis by head kidney cells, evaluated by immunoenzymatic assay, from fish immunized against human‐γ‐globulins (HGG) in vivo was not affected by bovine LF. Although these results showed that bovine LF had no effect on specific immunoglobulin production in vitro , an enhancement of the acquired immune response may be assumed in LF‐treated fish in vivo , as observed in higher vertebrates.     

Electrotransformation of whole cells of Brevibacterium sp. R312 a nitrile hydratase producing strain: Construction of a cloning vector

Abstract: A rapid and effective method is described for electroporation of Brevibacterium sp. R312, a coryneform strain producing nitrile hydratase and amidase. The transformation efficiency of the method is 10⁸ transformants per μg of plasmid under optimal conditions. Parameters optimised included field strength (11.8 kV cm⁻¹), pulse length (2.4 ms), plasmid DNA concentration (0.25 μg ml⁻¹ and cell density (10¹⁰ cells ml⁻¹). Surprisingly, the transformation efficiency did not vary with the growth stage, in contrast to results in the literature. A shuttle vector was constructed containing several unique cloning sites down-stream of the SP6 RNA polymerase promoter.     

Cyanopeptolin S, a sulfate-containing depsipeptide from a water bloom of Microcystis sp.

Abstract A new sulfated, cyclic depsipeptide, called cyanopeptolin S, from Microcystis sp. was isolated from a water bloom in the Auensee/Leipzig (Germany). The depsipeptide had a relative molecular mass of 925 and contained l-arginine, l-threonine, l-isoleucine, N-methyl-l-phenylalanine, a l-glutamic acid-δ-aldehyde ring system and a sulfated d-configurated glyceric acid as a side chain. The structure was elucidated by means of two-dimensional ¹H and ¹³C nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectroscopy, Fourier transformed infrared spectroscopy and combined gas-liquid chromatography/mass spectrometry. Cyanopeptolin S inhibited trypsin with an IC₅₀≤ 0.2 μg ml⁻¹.     

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by ¹⁴CO₂ release in CO₂-free air and by ¹⁴CO₂ release from the oxidation of [1–¹⁴C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO₂ compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I⁻¹1 at −1.08 MPa to 130 μl 1⁻¹ for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non- stomatal inhibition of CO₂ assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.     

Kinetics of photoautotrophic growth of Marchantia polymorpha cells in suspension culture

Chlorophyllous, cultured cells of Marchantia polymorpha L. (HYA-2 cell line) grow actively under photoautotrophic (lithotrophic) conditions. The maximum specific growth rate (μ_cell) was 0.64 day⁻¹ and the doubling time was 1.08 days under optimum conditions (165 μmol m⁻² s⁻¹, 1% carbon dioxide enriched atmosphere, 25^°C). The photosynthetic activity was 1.30 μmol CO₂-fixed (10⁶ cells)⁻¹ h⁻¹ [66 μmol (mg chlorophyll)⁻¹ h⁻¹] in the exponential phase. The growth course has two distinct phases, an exponential and a linear one. The exponential phase is observed as long as the population density is sufficiently low (less than 7.9 × 10⁶ cells ml⁻¹), so that practically all individual cells directly receive the full incident light. The effect of light on the specific growth rate is a linear function of photon flux density. Linear growth occurs after the population density is so high that the incident light is almost completely absorbed by the cell suspension. The growth rate is a logarithmic function of photon flux density, in contrast to the specific growth rate, and saturates at high photon flux densities. The conditions of maximum growth, however, are not wellbalanced between cell mass production and cell division. Therefore, the maximum growth does not continue for a long time.