The inhibitory effects of pyrocarbonic acid diethyl ester on lactobacillus casei and its phage Jl – A novel strategy for phage control in technical fermentation processes

The effects of pyrccarbonic acid diethyl ester (PADE) on Lactobacillus casei Sl and its phage Jl was investigated in relation to the control of phages in the dairy industry and other technica fermentation processes. PADE exhibited a bacteriostatic effect at 0.5 to 8 mM and a bactericidal effect at 10 mM or higher. It inhibited the growth of the phage at its bacteriostatic and bactericidal concentrations. The growth inhibition of the phage was reversible at the bacteriostatic concentrations but complete and irreversible at the bactericidal concentrations. PADE inactivated the free phage within several minutes; 10 and 30 mM of PADE inactivated 90 and 100%, respectively, of the phage. It completely decomposed into ineffective components in several minutes. The bacteria grew almost normally when they were inoculated after the complete decomposition of PADE. These four characteristics of PADE – its bactericidal effect, its inhibitory effect on phage growth, its phage-inactivating effect and its decomposition – suggest a novel strategy for phage control in technical fermentation processes, including the dairy industry.     

Photo-Irradiation of Proanthocyanidin as a New Disinfection Technique via Reactive Oxygen Species Formation

In the present study, the bactericidal effect of photo-irradiated proanthocyanidin was evaluated in relation to reactive oxygen species formation. Staphylococcus aureus suspended in proanthocyanidin aqueous solution was irradiated with light from a laser at 405 nm. The bactericidal effect of photo-irradiated proanthocyanidin depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power. When proanthocyanidin was used at the concentration of 1 mg/mL, the laser irradiation of the bacterial suspension could kill the bacteria with a >5-log reduction of viable cell counts. By contrast, bactericidal effect was not observed when proanthocyanidin was not irradiated. In electron spin resonance analysis, reactive oxygen species, such as hydroxyl radicals, superoxide anion radicals, and hydrogen peroxide, were detected in the photo-irradiated proanthocyanidin aqueous solution. The yields of the reactive oxygen species also depended on the concentration of proanthocyanidin, the laser irradiation time, and the laser output power as is the case with the bactericidal assay. Thus, it is indicated that the bactericidal effect of photo-irradiated proanthocyanidin is exerted via the reactive oxygen species formation. The bactericidal effect as well as the yield of the oxygen radicals increased with the concentration of proanthocyanidin up to 4 mg/mL, and then decreased with the concentration. These findings suggest that the antioxidative activity of proanthocyanidin might prevail against the radical generation potency of photo-irradiated proanthocyanidin resulting in the decreased bactericidal effect when the concentration is over 4 mg/mL. The present study suggests that photo-irradiated proanthocyanidin whenever used in an optimal concentration range can be a new disinfection technique.     

Bactericidal effect of a silica gel-supported porphyrinatoantimony(V) complex under visible light irradiation

In order to develop a bactericidal agent operating under visible light irradiation, a silica gel-supported dihydroxo(tetraphenylporphyrinato)antimony(V) complex (SbTPP/SiO₂) was prepared. The SbTPP/SiO₂ particles irradiated by fluorescent light in a test tube induced remarkable bactericidal activity for Escherichia coli cells. The bactericidal activity of the SbTPP/SiO₂ was affected by both the concentration of the SbTPP/SiO₂ and the light intensity. Under irradiation by visible light, the SbTPP/SiO₂ photocatalyst showed much superior bactericidal activity to the commercially available TiO₂. Moreover, under irradiation by sunlight, bactericidal activity of the SbTPP/SiO₂ was observed, and the bactericidal effect of the SbTPP/SiO₂ particles was effective for continuous treatment on a column photoreactor under fluorescent-light irradiation.     

Bacteria of Porcine Skin, Xenografts, and Treatment with Neomycin Sulfate

Homogenized 4-mm punch biopsies were taken from pigs and bacteriologically evaluated to determine the efficacy of surgical scrub procedures and the subsequent treatment of tissue with 0.5% neomycin sulfate-sodium bisulfite (neomycin-bisulfite) as a decontaminating agent. The majority of the lots of porcine skin taken directly from animals for xenografts in the treatment of burns contained viable bacteria at the time of grafting although scrubbing procedures substantially reduced the skin bacteria. The porcine bacteria consisted primarily of coagulase-negative staphylococci with most strains exhibiting caseinolytic and elastase activity. Staphylococci were the only abundant bacteria found in postscrub biopsies and in saline solutions used to wash the dermatome during its use. After an overnight exposure of grafting tissue soaked in neomycin-bisulfite, the spent neomycin-bisulfite solutions were tested for bacteriostatic and bactericidal activity by comparison to unused neomycin. All solutions tested were equal in bacteriostatic strength, but the bactericidal action of some spent solutions was decreased. Neomycin alone exerted a more lethal effect on sensitive bacteria than the neomycin-bisulfite solution. The desirability of having viable porcine skin for a xenograft necessitated using or discarding the tissue after storage in neomycin-bisulfite at 4 C for a maximum of 72 hr. Certain contaminating microorganisms were unaffected by antibiotic treatment, and the prolonged use of neomycin without bisulfite would have primarily eradicated only the porcine coagulase-negative staphylococci. Neither the presence of this group in grafting tissue nor their proteolytic activity had any observed adverse effect on xenografting success.     

Some further observations on synergism between chloramphenicol and polymyxin B in relation toSalmonella bacteria

Summary The synergism between chloramphenicol and polymyxin B sulphate, earlier noticed using a replica technique, has been reexamined applying quantitative methods. Vital count experiments in suspensions ofSalmonella typhimurium in broth have revealed that addition of bacteriostatic concentrations of chloramphenicol in a range of 5–100 μg/ml to bacteriostatic concentrations of polymyxin (0.1–0.9 μg/ml) produce a strong bactericidal effect (synergism). Combinations of bactericidal concentrations of polymyxin (1 μg/ml and higher) with chloramphenicol also exert a very high degree of bactericidal activity, though no more than with polymyxin alone (no synergism). Chloramphenicol therapy sustained with polymyxin, may be of therapeutic value in the eradication of stubborn enteralSalmonella infections. With the technical assistance of MissM. J. Wisse.     

Targets for hydrogen-peroxide-induced damage to suspension and biofilm cells of Streptococcus mutans

Hydrogen peroxide (H2O2) is considered a major endogenous source of oxidative stress to oral bacteria and also is widely used in oral care products. Our study objectives were to identify specific targets for H2O2-induced damage to cells of Streptococcus mutans in suspensions and monospecies biofilms and to differentiate bacteriostatic and bactericidal actions of the peroxide. Streptococcus mutans was grown in suspension cultures and fed-batch biofilms for assessing relative sensitivities of viability, glycolysis, and protein synthesis to H2O2 damage. Biofilm cells were found to have essentially the same peroxide sensitivity as cells in suspensions. H2O2 at low concentrations of about 16.3 mmol/L was highly inhibitory for glycolysis and mainly bacteriostatic. The most sensitive target detected for glycolytic inhibition was glyceraldehyde-3-phosphate dehydrogenase with IC50 (50% inhibitory concentration) values of ca. 2.2 mmol/L for suspension cells and 2.3 mmol/L for biofilms with 15 min treatments. The phosphoenolpyruvate:glucose phosphotransferase pathway was less sensitive with an IC50 of ca. 10 mmol/L. Aldolase was not inhibited at bacteriostatic concentrations of the peroxide. For suspensions and biofilms, acidification somewhat diminished peroxide sensitivity, while increased temperature enhanced sensitivity. At concentrations above about 30 mmol/L, H2O2 became mainly bactericidal but not mutagenic for S. mutans. A major target for bactericidal damage was protein synthesis, thus rendering cells incapable of repairing or replacing oxidatively damaged proteins.     

The effect of the sugar source on citric acid production by Aspergillus niger

Summary Under otherwise identical fermentation conditions, the sugar source has been shown to have a marked effect on citric acid production by Aspergillus niger. Sucrose was the most favourable source, followed by glucose and fructose and then lactose. No citric acid was produced from galactose. Strong relationships were observed between citric acid production and the activities of certain enzymes in myccelial cell-free extracts prepared from fermentation samples. When sucrose, glucose, or fructose was the sugar source pyruvate carboxylase activity was high, but 2-oxoglutarate dehydrogenase activity was not detected. When galactose was the sugar source pyruvate carboxylase activity was low, but 2-oxoglutarate dehydrogenase activity was high. It is suggested that whereas glucose and fructose repress 2-oxoglutarate dehydrogenase, thereby causing accumulation of citric acid, galactose does not. The activity of aconitase showed a direct relationship to the citric acid production rate. Thus, the activity was highest when sucrose was the sugar source, and lowest when galactose was the source. It is suggested that when large amounts of citric acid are lost from the cell the activity of aconitase increases as a response to the diminished intracellular supply of its substrate.     

Antibacterial Activity of l-Lysine α-Oxidase against rec+ and rec− Strains of Bacillus subtilis

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker’s yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker’s yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Studies on mutagenicity of irradiated sugar solutions in Salmonella typhimurium.

Irradiated sugar solutions are mutagenic towards Salmonella typhimurium, the effect being dose-dependent up to 2.0 Mrad. At all doses, ribose solution exhibited greater mutagenicity than did sucrose solution. The mutagenic effect was observed only in dividing cells and appears to be directly related to the growth rate. A larger proportion of revertants was observed after incubation with irradiated sugar solution for a period of 4 h than for 24 h. Irradiation of the sugar solutions in the frozen conditions was effective in completely preventing the development of mutagenic potential. Post-irradiation storage of the sugar solutions for a prolonged period (25 weeks) also minimized their mutagenic effect. The irradiated sugar solutions gave rise to both missense and frame-shift (additon as well as deletion) types of mutation; ribose was more effective in inducing the latter type. The irradiated sugar solutions failed to show a mutagenic response in the host-mediated assay with mice as the mammalian host.     

Potentiation of Inhibitory Activity of Colistin on Pseudomonas aeruginosa by Sulphamethoxazole and Sulphamethizole

Potentiation of colistin by sulphamethoxazole and sulphamethizole was demonstrated with 19 out of 20 strains of Pseudomonas aeruginosa. This enhancement was bactericidal as well as bacteriostatic. Synergy between trimethoprim and sulphamethoxazole was also demonstrated with four strains of Ps. aeruginosa, but even when the two drugs were combined high concentrations of trimethoprim were still required to produce a bactericidal effect. Combinations of sulphamethoxazole and gentamicin appeared to be synergistic when the bacteriostatic effect was measured, but the combined bactericidal effect was indifference. The bactericidal and bacteriostatic effect of combinations of carbenicillin with sulphamethoxazole was also indifference.     

Influence of fructose and glucose on the production of exopolysaccharides and the activities of enzymes involved in the sugar metabolism and the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772

 The effect of fructose and glucose on the growth, production of exopolysaccharides and the activities of enzymes involved in the synthesis of sugar nucleotides in Lactobacillus delbrueckii subsp. bulgaricus grown in continuous culture was investigated. When grown on fructose, the strain produced 25 mg l^-1 exopolysaccharide composed of glucose and galactose in the ratio 1:2.4. When the carbohydrate source was switched to a mixture of fructose and glucose, the exopolysaccharide production increased to 80 mg l^-1, while the sugar composition of the exopolysaccharide changed to glucose, galactose and rhamnose in a ratio of 1:7.0:0.8. A switch to glucose as the sole carbohydrate source had no further effect. Analysis of the enzymes involved in the synthesis of sugar nucleotides indicates that in cell-free extracts of glucose-grown cells the activity of UDP-glucose pyrophosphorylase was higher than that in cell-free extracts of fructose-grown cells. The activities of dTDP-glucose pyrophosphorylase and the rhamnose synthetic enzyme system were very low in glucose-grown cultures but could not be detected in fructose-grown cultures. Cells grown on a mixture of fructose and glucose showed similar enzyme activities as cells grown on glucose. Analysis of the intracellular level of sugar nucleotides in glucose-grown cultures of L. delbrueckii subsp. bulgaricus showed the presence of UDP-glucose and UDP-galactose in a ratio of 3.3:1, respectively, a similar ratio and slightly lower concentrations were found in fructose-grown cultures. The lower production of exopolysaccharides in cultures grown on fructose may be caused by the more complex pathway involved in the synthesis of sugar nucleotides. The absence of activities of enzymes leading to the synthesis of rhamnose nucleotides in fructose-grown cultures appeared to result in the absence of rhamnose monomer in the exopolysaccharides produced on fructose. Received: 1 February 1996/Received revision: 31 May 1996/Accepted: 2 June 1996     

Enrichment of sugar content in melon fruits by hydrogen peroxide treatment

Since sweetness is one of the most important qualities of many fruits, and since sugars are translocated from leaves to fruits, the present study investigates photosynthetic activity, activity of sugar metabolizing enzymes, sugar content in leaves and fruits and endogenous levels of hydrogen peroxide in leaves of melon plants treated with various dilutions of hydrogen peroxide, a nonspecific signaling molecule in abiotic stress. For this purpose, 4-month-old melon plants were treated with various concentrations (<50mM) of hydrogen peroxide by applying 300mL per day to the soil of potted plants. The treatments resulted in increased fructose, glucose, sucrose and starch in the leaves and fruits. The most effective concentration of hydrogen peroxide was 20mM. During the day, soluble sugars in leaves were highest at 12:00h and starch at 15:00h. Furthermore, the peroxide treatment increased the photosynthetic activity and the activities of chloroplastic and cytosolic fructose-1,6-bisphosphatase, sucrose phosphate synthase and invertases. Thus, our data show that exogenous hydrogen peroxide, applied to the soil, can increase the soluble sugar content of melon fruits.     

Genetic dimorphism in the taste sensitivity to trehalose inDrosophila melanogaster

Summary A quantitative behavioral assay was developed for the measurement of taste responses to sugars inDrosophila. The amount of the intake of a sugar solution was measured colorimetrically after homogenization of flies which had consumed sugar solutions mixed with a food-dye. A two-choice method was utilized to determine the taste sensitivity to sugars. Two kinds of sugar solutions were marked with either blue or red food-dye and placed alternately in the wells of a micro test plate. Flies were allowed to choose between the two sugar solutions. By classifying and counting the coloured flies, the relative taste sensitivity could be determined. Employing these methods, a genetic dimorphism in the taste sensitivity to trehalose was found among some laboratory strains ofDrosophila melanogaster. No difference in the taste sensitivity to glucose, fructose and sucrose was found between the trehalose high-sensitivity (T-1) and the low-sensitivity (Oregon-R) strains. Trehalose concentration equivalent to 2 mmol/1 sucrose, in terms of stimulating activity, was 57 mmol/1 inOregon-R and was 10 mmol/1 inT-1. Genetic analysis showed that theTre gene, whose locus is closely linked tocx (13.6) on theX chromosome, is responsible for the difference in the taste sensitivity to trehalose.     

Synthesis of 2′-O-Photocaged Ribonucleoside Phosphoramidites

The chemical synthesis and incorporation of the phosphoramidite derivatives of 2?′-O-photocaged ribonucleosides (A, C, G and U) with o-nitrobenzyl, α-methyl-o-nitrobenzyl or 4,5-dimethoxy-2-nitrobenzyl group into oligoribonucleotides are described. The efficiency of UV irradiated uncaging of these 2′-O-photocaged oligoribonucleotides was found in the order of α-methyl-o-nitrobenzyl < 4,5-dimethoxy-2-nitrobenzyl < 2′-O-o-nitrobenzyl.     

Purification and Properties of UDP-galactose 4-Epimerase from Bifidobacterium bifidum

UDP-galactose 4-epimerase (EC 5.1.3.2) was purified to a homogeneous state from Bifidobacterium bifidutn grown on a glucose medium. The molecular weight was estimated to be about 90,000. The purified enzyme was very stable and 60 % of its initial activity survived three months of storage at 4°C even at a low protein concentration (0.2 mg/ml). The optimum pH was 9.0, and the Km values for UDP-galactose and UDP-glucose were 5.4 × 10^-4 M and 1.4×10 ^-3 M. UDP was a competitive inhibitor. The enzyme activity was stimulated by various sugar phosphates, but was slightly inhibited by fructose 1,6-diphosphate (FDP). A high concentration of galactose or glucose, which had no effect by itself, inhibited the activity in combination with UMP. The inhibition by FDP was also enhanced by combination with UMP.     

Sugar Concentrations in Guard Cells of Vicia faba Illuminated with Red or Blue Light : Analysis by High Performance Liquid Chromatography

Concentrations of soluble sugars in guard cells in detached, sonicated epidermis from Vicia faba leaves were analyzed quantitatively by high performance liquid chromatography to determine the extent to which sugars could contribute to changes in the osmotic potentials of guard cells during stomatal opening. Stomata were illuminated over a period of 4 hours with saturating levels of red or blue light, or a combination of red and blue light. When stomata were irradiated for 3 hours with red light (50 micromoles per square meter per second) in a solution of 5 millimolar KCl and 0.1 millimolar CaCl₂, stomatal apertures increased a net maximum of 6.7 micrometers and the concentration of total soluble sugar was 289 femtomoles per guard cell (70% sucrose, 30% fructose). In an identical solution, 2.5 hours of irradiation with 25 micromoles per square meter per second of blue light caused a maximum net increase of 7.1 micrometers in stomatal aperture and the total soluble sugar concentration was 550 femtomoles per guard cell (91% sucrose, 9% fructose). Illumination with blue light at 25 micromoles per square meter per second in a solution lacking KCl caused a maximum net increase in stomatal aperture of 3.5 micrometers and the sugar concentration was 382 femtomoles per guard cell (82% sucrose, 18% fructose). In dual beam experiments, stomata irradiated with 50 micromoles per square meter per second of red light opened steadily with a concomitant increase in sugar production. Addition of 25 micromoles per square meter per second of blue light caused a further net gain of 3.7 micrometers in stomatal aperture and, after 2 hours, sugar concentrations had increased by an additional 138 femtomoles per guard cell. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were performed with epidermis illuminated with 50 micromoles per square meter per second of red light or with 25 micromoles per square meter per second of blue light in solutions containing or lacking KCl. DCMU completely inhibited sugar production under red light, had no effect on guard cell sugar production under blue light when KCl was present, and inhibited sugar production by about 50% when guard cells were illuminated with blue light in solutions lacking KCl. We conclude that soluble sugars can contribute significantly to the osmoregulation of guard cells in detached leaf epidermis of V. faba. These results are consistent with the operation of two different sugar-producing pathways in guard cells: a photosynthetic carbon reduction pathway and a pathway of blue light-induced starch degradation.     

Cyanide, Peroxide and Nitric Oxide Formation in Solutions of Hydroxyurea Causes Cellular Toxicity and May Contribute to Its Therapeutic Potency

Hydroxyurea (HU) is a potent remedy against a variety of ailments and an efficient inhibitor of DNA synthesis, yet its pharmacology is unclear. HU acts in Escherichia coli by the same mechanism as it does in eukaryotes, via inhibition of ribonucleotide reductase. When examining a controversy about concentrations of HU that prevent thymineless death in E. coli, we found instability in HU solutions that avoided prior detection due to its peculiar nature. In contrast to freshly dissolved HU, which did not affect respiration and was bacteriostatic, 1-day-old HU solutions inhibited respiration and were immediately bactericidal. Respiration was inhibited by two gases, hydrogen cyanide (HCN) and nitric oxide (NO), whose appearance we detected in “aged” HU stocks by gas chromatography-mass spectrometry; however, neither gas was bactericidal. While determining the cause of toxicity, we found that HU damages DNA directly. We also demonstrated accumulation of peroxides in HU solutions by enzymatic assays, which explains the toxicity, as both NO and HCN are known to kill bacteria when combined with hydrogen peroxide. Remarkably, we found that bactericidal effects of NO + H₂O₂ and HCN + H₂O₂ mixtures were further synergistic. Accumulation of decomposition products in solutions of HU may explain the broad therapeutic effects of this drug.     

Mechanism of bactericidal action of phenethyl alcohol inEscherichia coli

The mechanism of bactericidal action of phenethyl alcohol (PEA) inE. coli, which was previously demonstrated to be dependent on protein synthesis, has been investigated. Mutants resistant to PEA were selected, but the resistance observed was associated with a change in permeation. PEA effects on DNA, RNA, and protein synthesis were studied with bacteriostatic and bactericidal concentrations Similar results (inhibition of DNA synthesis and decrease in RNA synthesis) were obtained with lethal concentrations of PEA in cells pretreated with chloramphenicol, and with bacteriostatic concentrations of PEA in unpretreated cells. The PEA intracellular accumulation reached a maximum within 4 min and was not inhibited by KCN or by 2,4-dinitrophenol. The presence of phenylacetaldehyde was demonstrated in both stationary and exponential growth phase cells exposed to PEA but not in cells pretreated with chloramphenicol. These results suggested that the bactericidal mechanism of action of PEA involves its conversion into the corresponding aldehyde.     

Effect of Inoculum Size on In Vitro Susceptibility Testing with Lincomycin

There is disagreement in the literature as to whether lincomycin is primarily a bacteriostatic or a bactericidal agent against gram-positive cocci and also regarding the levels of activity of this agent against susceptible microorganisms. These questions were examined in a study of the effect of inoculum size on the results of tube dilution susceptibility determinations with lincomycin against 49 clinical isolates of Staphylococcus aureus and 25 strains of streptococci and pneumococci. Lincomycin was both highly active and bactericidal when tested against 40 strains of S. aureus with inocula containing a maximum of 10(4) cells per ml [median minimal inhibitory concentration (MIC), 0.78 mug/ml; median minimal bactericidal concentration (MBC), 1.56 mug/ml]. With inocula of 10(5) cells per ml, lincomycin was primarily bacteriostatic (median MIC, 1.56 mug/ml; median MBC, 12.5 mug/ml). There were further decreases in inhibitory levels and significant losses of bactericidal activity when inocula containing more than 10(7) cells were tested (median MIC, 3.13 mug/ml; median MBC > 100 mug/ml). Similar measurements with streptococci and pneumococci revealed a lesser effect of inoculum size. The mean MBC value for alpha-hemolytic streptococci increased from 0.40 to 1.05 mug/ml with an increase in inocula from 10(4) to 10(6) cells per ml, but without a marked increase in MIC values. Similar results were obtained for beta-hemolytic streptococci and pneumococci.     

Growth rate,sugar consumption,chlortetracycline production and anhydrotetracycline oxygenase activity inStreptomyces aureofaciens

The relationship between the activity of ATC oxygenase, CTC production and growth rate was investigated in a low-producing strain ofStreptomyces aureofaciens, closely related to the wildtype strain, and in a higher-producing variant. Different growth rates were achieved by using glucose, fructose and sucrose as carbon sources. Activity of the enzyme and CTC yield in both strains were inversely proportional to the rate of sugar utilization but in the higherproducing variant sugar utilization was slower than in the low-producing strain. The expression of ATC oxygenase was less sensitive to “catabolite repression” in the higherproduc ing strain. BT, a stimulator of CTC production, markedly inhibited growth of the higher-producing variant in a medium with slowly utilized sugars (fructose and sucrose) but had little effect on growth of the lowproducing strain. It also increased the level of ATC oxygenase in both strains under all experimental conditions. It could be established that there was no obligatory relationship between the increase of antibiotic synthesis and the increase of enzyme activity.