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Effect of certain nutrients on the production of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

The effects of certain nutrients on dextransucrase (sucrose: 1,6-alpha-d-glucan 6-alpha-d-glucosyltransferase EC 2.4.1.5) production from Leuconostoc mesenteroides NRRL B-512F were studied. An increase in concentration of sucrose to 4% in the enzyme production medium resulted in the increase of activity of dextransucrase. Higher enzyme yields were obtained at low yeast extract and high phosphate concentrations. The presence of peptone and beef extract in the medium in addition to 2% yeast extract resulted in an enhanced production of dextransucrase. The enzyme activity increased by 30% by both peptone and beef extract. Addition of Tween 80 to the medium enhanced the production of enzyme and the activity was increased by 25%. Magnesium ions stimulated the activity marginally. Sodium fluoride enhanced the activity of dextransucrase by 25%.

Culture Media↗

An evaluation of the antibacterial activities of combinations of sulfonamides, trimethoprim, dibromopropamidine, and silver nitrate compared with their uptakes by selected bacteria.

Modifications of antibacterial activity have been demonstrated using combinations of two antibacterials from trimethoprim, sulfonamides (sulfadiazine, sulfamerazine, and silver sulfadiazine), silver nitrate, and dibromopropamidine isethionate, either formulated in a cream base or dissolved in peptone water. The creams were evaluated using the agar cup diffusion method in isosensitest agar. The peptone water solutions provided fractional inhibitory concentrations for combinations of the antibacterial substances. The test organisms were Pseudomonas aeruginosa, Enterobacter cloacae, and Staphylococcus aureus. Bacterial uptakes of antibacterial combinations, determined by either an HPLC assay method or an atomic absorption method, combined with dry cell weight determinations, indicated that enhancement of activity of the antibacterial combinations against P. aeruginosa (two strains) and E. cloacae were related to marked increases in the bacterial uptake of the chemical agents. Decreases in activity were related to decreased uptake of either dibromopropamidine and/or silver ions. The effect of the trimethoprim and the sulfonamides was shown to depend on their effect on bacterial folate synthesis. It is suggested that partial blockade of the folate synthetic pathway leads to an effect on cell permeability which results in increased uptake of antibacterials. Dibromopropamidine isethionate also has an effect on cell permeability which produces an increased bacterial uptake of a second antibacterial present in the medium. These findings provide further explanation of how subinhibitory concentrations of trimethoprim and sulfonamide combinations are synergistic against a wide range of bacteria even when certain bacteria are resistant to either member of the combination.

Bacteria↗

Structure of microbial communities in activated sludge: potential implications for assessing the biodegradability of chemicals.

Various methods used to assess the biodegradability of chemicals often employ activated sludge as an inoculum since chemicals that ultimately enter the environment are often discharged through wastewater. Differences in the structure and function of activated sludge microbial communities that may complicate interpretation of biodegradation tests could arise from differences in wastewater composition, wastewater treatment plant (WWTP) operation, or manipulations done after collection of the activated sludge. In this study, various methods were used to characterize the structure of microbial communities found in freshly collected activated sludge from WWTPs in Japan, Europe, and the United States, as well as sludge that had been continuously fed either sewage or a glucose-peptone mixture for several weeks after collection. Comparisons of biomass levels, whole-community substrate utilization (determined using Biolog GN and GP plates), and phospholipid fatty acid (PLFA) profiles indicated there were both geographical and temporal differences among freshly collected activated sludge samples. Moreover, marked shifts in the structure of activated sludge microbial communities occurred upon continuous cultivation in the laboratory for 5 weeks using a glucose-peptone feed. These shifts were evident from whole-community substrate utilization and PLFA profiles as well as differences in the profiles of 16S rDNA genes from numerically dominant populations obtained by denaturing gradient gel electrophoresis and terminal restriction fragment analyses. Further studies are needed to better define the variability within and between activated sludge from wastewater treatment plants and laboratory reactors and to assess the impact of such differences on the outcome of biodegradability tests.

Bacteria↗

Non-dystrophic 129 REJ mice are susceptible to i.p. infection with Listeria monocytogenes despite an ability to recruit inflammatory neutrophils to the peritoneal cavity.

In this study we compared the host response to Listeria monocytogenes in 129 REJ mice with listeria-resistant (C57Bl/6j) and susceptible (Balb/c) mouse strains. In all experiments mice were inoculated by the i.p. route. 129 REJ mice and Balb/c mice were sensitive to listeriosis whilst C57Bl/6j mice were relatively resistant to i.p. infection. Relatively large numbers of viable bacteria could be detected in the spleens of 129 REJ mice as early as 6 h following i.p. inoculation suggesting that dissemination of listeria from the peritoneal cavity is rapid in this mouse strain. This contrasted with Balb/c mice which exhibited an early lag phase during which only low numbers of bacteria could be isolated from the spleens of infected animals. In response to both proteose peptone and live listeria, 129 REJ mice demonstrated a greater capacity to recruit neutrophils to the peritoneal cavity than Balb/c and C57Bl/6j mice. In addition, inflammatory phagocytes from 129 REJ mice were as bactericidal in vitro as phagocytes from the Balb/c and C57Bl/6j strains. However, in vivo, inflammatory neutrophils elicited by proteose peptone prior to i.p. infection with L. monocytogenes were not protective in the three mouse strains tested. Despite the apparent inadequacy of peritoneal neutrophils in controlling early bacterial proliferation, depletion of neutrophils in 129 REJ mice severely exacerbated i.p. infection with L. monocytogenes. The results indicate that neutrophils provide an inefficient but essential means of controlling early outgrowth of listeria in the peritoneal cavity of 129 REJ mice. The excessive inflammatory response seen in 129 REJ mice may facilitate the early dissemination of L. monocytogenes from the peritoneal cavity to peripheral sites.

Animals↗

In vitro and in vivo release of cytostatic factors from Lactobacillus casei-elicited peritoneal macrophages after stimulation with tumor cells and immunostimulants.

The effect of tumor cells and immunostimulants on the release of cytostatic factors (CF) from Lactobacillus casei YIT 9018 (LC)-, Corynebacterium parvum (CP)- or peptone-elicited peritoneal macrophages (PM) was investigated in vitro and in vivo. Significant release of CF into the culture medium from PM elicited with LC was induced by seven of eight mitomycin C-pretreated tumor cell lines and not by normal spleen cells, while no CF was released extracellularly from peptone-elicited PM given the same stimulus. CF were released from LC-elicited PM (LCEPM) after stimulation with LC, bacille Calmette-Guérin, streptococcal preparation OK-432, fucoidan or lipopolysaccharide, and LC but not CP induced CF production in the peritoneal cavities of LC- or CP-primed mice. The release of CF from LCEPM after stimulation with mitomycin C-pretreated 3T12-3 cells was inhibited by D-mannose and not by L-fucose. L-Rhamnose and mannose 6-phosphate, but not D-mannose or L-fucose, caused the release of CF from the PM. It was suggested that the release of CF from activated PM is caused by stimulation by some tumor cells, sugars, or bacterial immunostimulants, D-Mannose and L-rhamnose on the surface of tumor cells or bacteria, respectively, may plan an important role in the release of CF from activated macrophages.

Adjuvants, Immunologic↗

Rapid distinction between micrococci and staphylococci with furazolidone agars.

Furazolidone agar proved to be a suitable medium for separating strains of the genera Micrococcus and Staphylococcus. 720 isolates (including 24 type strains) of gram- and catalase-positive cocci were tested for growth on tryptone soya and peptone agar with the addition of 50 microgram/ml furazolidone. The results were compared with the classification obtained by the standard-O/F-test and by the test system of Schleifer and Kloos. For routine identification and separation of staphylococci from micrococci a peptone agar with 20 microgram furazolidone/ml is recommended.

Bacteriological Techniques↗

Reisolation and growth conditions of bacillus agar-exedens.

Several agarolytic Bacillus strains have been isolated. The properties agree with those described by Wieringa (1941) for Bacillus agar-exedens. These strains are the first reisolates since the original cultures were lost. A second group of isolates is related to the agarolytic B. palustris var. gelaticus of Sickles and Shaw (1934). B. agar-exedens requires carbohydrates for growth. In mineral-glucose media growth is inhibited by peptone at pH values of about 7 or less. Under alkaline conditions no inhibition by peptone is observed. A method for the enrichment of B. agar-exedens is described.

Agar↗

Efficacy of seed-based media for the mould-yeast conversion of Blastomyces dermatitidis.

The efficacy of 20 seed-based media is reported for the in vitro mould-yeast conversion of Blastomyces dermatitidis, employing pharmamedia agar, peptone glucose agar, glucose agar and water agar as controls. The mould-yeast conversion varied significantly according to the culture medium, fungal strains and incubation period (p less than 0.01). Garden-pea, chick-pea, cow-pea, soybean, peanut, green gram, French bean, lentil, okra and cottonseed converted all of the 7 B. dermatitidis test strains after 5 days of incubation at 37 degrees C. Although the efficacy of many of these seed media was found to be at par with pharmamedia agar - a commercial cottonseed embryo-derived protein, garden-pea seed agar is adopted because of the wider availability and low fat content of this seed. The recommended composition of the medium comprises 2% aqueous seed extract, 2% glucose and pH 6-7. Only nigerseed and sunflower seeds failed to support the conversion of B. dermatitidis. Of the control media, peptone glucose agar, glucose agar and water agar did not support the conversion of 2 of the B. dermatitidis test strains. The mechanism underlying variable mould-yeast conversion of B. dermatitidis on seed-based media is not clearly understood. However, most of the seeds supporting excellent mould-yeast conversion are known for their high protein content. The conversion was apparently not affected by the fat content of the seeds or by incorporation of glucose in the medium.

Blastomyces↗

Microbial assimilation of alkyl nitro compounds and formation of nitrite.

66 representative strains of bacteria, yeasts and fungi were tested for their ability to grow in a semidefined medium containing 0.5% nitroethane as a nitrogen source. About half of them were found capable of growing in the medium. Hansenula beijerinckii, Candida utilis, and Penicillium chrysogenum were most active in assimilating nitroethane. 2-Nitropropane inhibited growth of most of the microorganisms tested in a medium containing 0.2% peptone and 0.2% glycerol. Hansenula mrakii was found to grow rapidly in the nitroethane-peptone medium after a lag phase. Nitrite was accumulated in the culture fluid after the phase of logarithmic multiplication, and increased with increase of the growth, followed by a decline after the maximum growth. The alkyl nitro compounds were oxidatively denitrified to form nitrite by the crude enzyme from Hansenula mrakii. Nitroethane was generally a poor substrate, but was the best inducer to produce the nitro compounds oxidizing enzyme. 2-Nitro-propane and nitroethane were enzymatically oxidized to nitrite, and acetone and acetaldehyde, respectively, which were isolated as 2,4-dinitrophenylhydrazones and identified. Nitrite formed was found to be reduced into ammonia by the intact cells and also the crude enzyme.

Acetaldehyde↗

Effect of environmental conditions on the production of two extracellular proteolytic enzymes by Vibrio SA1.

The production of two extracellular proteases, an endopeptidase and an aminopeptidase, by the marine bacterium Vibrio SA1 was studied in batch cultures. The production of the proteases was induced during growth of the organism in peptone media and by several amino acids during growth in minimal media. It was repressed by easily metabolisable carbon compounds such as glucose, lactate and succinate during growth in peptone media. During growth in a lactate basal medium, phenylalanine was one of the best inducers and this amino acid was therefore used in further experiments. That lactate did not repress the synthesis of the proteases during growth in the lactate basal medium supplement with 2mM phenylalanine as an inducer, appeared to be a consequence of the low iron content of this medium. Growth curves of Vibrio SA1 on such media showed a period of linear growth during which protease production was observed. When the iron concentration was made sufficiently high to prevent linear growth, the synthesis of the proteases remained repressed. Apparently by imposing an iron limitation on the organism, catabolite repression by lactate was relieved. Similarly, when growth was limited by very low values of the dissolved oxygen tension in the medium, a high rate of protease synthesis was found which was immediately repressed when the oxygen limitation was released. The results indicate that the growth rate and/or a factor associated with the energy metabolism play a role in the regulation of the synthesis of the enzymes.

Amino Acids↗

Chemiluminescence and superoxide generation by leukocytes stimulated by polyelectrolyte-opsonized bacteria. Role of histones, polyarginine, polylysine, polyhistidine, cytochalasins, and inflammatory exudates as modulators of oxygen burst.

Human blood leukocytes generate intense luminol-dependent chemiluminescence (LDCL) following stimulation by streptococci and by Gram negative rods which had been preopsonized by cationic polyelectrolytes (histone, poly L-arginine-PARG, poly L-histidine-PHSTD). Streptococci but not Gram negative rods or hyaluronic acid-rich streptococci (group C) also induced intense LDCL following opsonization with the anionic polyelectrolytes-dextran sulfate or polyanethole sulfonate (liquoid) suggesting that the outer surfaces of different bacteria bound anionic polyelectrolytes to different extents. Both normal and immune serum, synovial fluids and pooled human saliva inhibited the LDCL responses induced by streptococci preopsonized with poly cations. On the other hand, bacteria which had been first preopsonized by the various body fluids and then subjected to a second opsonization by cationic ligands ("sandwiches"), induced a very intense LDCL response in leukocytes. Streptococci which had been preopsonized by PARG, histone or by PHSTD also triggered superoxide generation by blood leukocytes, which was markedly enhanced by a series of cytochalasins. PHSTD alone induced the formation of very large amounts of superoxide. Paradoxically, the same concentrations of cytochalasins B or C which markedly boosted the generation of superoxide following stimulation of leukocytes with soluble or particulate ligands, had a strong inhibitory effect on the generation of LDCL. On the other hand cycochalasins failed to inhibit LDCL which had been induced by phorbol myristate acetate (PMA). Peritoneal macrophages which had been harvested from C. parvum-stimulated mice, generated more LDCL and superoxide following stimulation by PARG than macrophages obtained from proteose peptone-stimulated mice. Macrophages which had been activated either by proteose peptone or by C. parvum and cultivated for 2 hours on teflon surfaces, generated much more LDCL than macrophages which had been cultivated for 24 hours on teflon surfaces. Both cationic and anionic polyelectrolytes mimic the effects of antibodies as activators of the oxygen burst in blood leukocytes and in macrophages. Such polyelectrolytes can serve as models to further study leukocyte-bacteria interactions in infectious and inflammatory sites.

Adult↗

Gastric acid, plasma gastrin, and somatostatin responses to feeding and exogenous gastrin alone and in combination in conscious cats.

Meat in the stomach or duodenum potentiates pentagastrin-induced acid secretion in cats, presumably by a humoral mechanism. In the present study on cats, a meat meal significantly augmented the maximal acid response from a Heidenhain pouch (HP) to pentagastrin or to human synthetic gastrin I by 31 and 30%, respectively. The maximal HP acid response to pentagastrin was augmented also by peptone instilled into the stomach through a gastric fistula. Intravenous infusion of amino acids stimulated acid secretion but did not augment the maximal acid response to pentagastrin. The plasma concentrations of gastrin and somatostatin increased during infusion of pentagastrin and gastrin I and were not further altered by simultaneous feeding. The present results indicate that the mechanism for potentiation of gastrin-induced acid secretion is of physiological significance, since feeding augmented also the acid response to heptadecapeptide gastrin, the only gastrin secreted from the antrum and duodenum in cats. The potentiation of acid secretion is not dependent on the vagal excitation induced by oral feeding, since potentiation was demonstrated also by intragastric peptone instillation. The mechanism for the potentiation is not due to absorbed amino acids or a decrease of plasma somatostatin.

Animals↗

Rapid gastric emptying in duodenal ulcer patients.

Isosmotic liquid peptone meals adjusted to pH 7, 3, and 1.5 were instilled on separate days into the stomachs of 8 duodenal ulcer patients and 7 healthy controls. Using a marker-dilution method, duodenal acid load (DAL) was measured as the amount of unbuffered hydrogen ions delivered to the duodenum per unit time. Gastric emptying was measured as the total volume of gastric contents, including meal plus gastric secretion, passing through the pylorus per unit time (VPP). Mean pentagastrin-stimulated acid output was significantly different between the two groups. However, after all three tests meals, mean DAL was significantly greater in duodenal ulcer than in normal subjects in both hours of the test, and VPP was significantly greater in ulcer than in normal subjects in the first 40 min. In both groups, following peptone meals of pH 7 and 3, the volume of gastric contents delivered through the pylorus decreased as the amount of free hydrogen ions entering the duodenum increased, but a given load of acid was less effective in slowing emptying duodenal ulcer patients than in controls. These studies indicate that duodenal ulcer patients empty liquid meals more rapidly than do normal subjects, independent of the initial pH of the meals, and that, in addition, acid inhibition of gastric emptying is defective in duodenal ulcer.

Adult↗

The production of recombinant beta-galactosidase in Escherichia coli in yeast extract enriched medium.

The productivity of Escherichia coli biomass and recombinant beta-galactosidase was increased in Luria broth (LB) enriched with yeast extract. In flask culture under conditions of LB limitation, yeast extract supplementation gave the highest biomass (strain HB101/pRW756) stimulation per unit of component added compared with supplementation by various amounts of amino acids, vitamins, minerals, purines/pyrimidines, tryptone, casamino acids, casein peptone or gelatin peptone. The biomass production of E. coli HB101/pRW756, XL-1 Blue/puc118, XL-1 Blue FF/puc118 and TB-1/p1034 cells was stimulated in fermentor-scale experiments with additional yeast extract in LB. Total beta-galactosidase production from plasmid genes in fermentor-scale experiments was increased 105.4% in XL-1 blue/puc118 cells, 365.5% in XL-1 blue FF/puc118 cells and 421.4% in TB-1/p1034 cells by 0.5%, 1% and 1% weight per volume of additional yeast extract in LB, respectively. Depending on different strains, the increase of the enzyme production was obtained either by increased biomass, or the combination of enhanced gene expression and increased biomass. Neither the biomass nor beta-galactosidase production was stimulated in N4830/p1034 cells by the increase in yeast extract concentration in the medium.

Culture Media↗

In vitro stimulation of Bacteroides fragilis growth by Escherichia coli.

The interaction between Bacteroides fragilis and Escherichia coli was investigated by following their growth kinetics in an in vitro mixed culture system. In Bacto-peptone or in a semisynthetic medium (SSM-M) Bacteroides fragilis could initiate growth only when a small number of viable Escherichia coli initially coexisted for a short period of time. Both Bacto-peptone and SSM-M previously spent by Escherichia coli also allowed Bacteroides fragilis growth to occur but the supplementation of heat killed Escherichia coli cells or viable cell homogenates to fresh media failed to stimulate Bacteroides fragilis growth. The in vitro stimulation of Bacteroides fragilis growth by Escherichia coli observed in this study may be best explained by the combination of (a) removal of oxygen and other toxic substances and (b) de novo production of Bacteroides fragilis growth stimulating factor(s) by coexistent Escherichia coli.

Abdomen↗

Formation of sulphate, sulphite and S-sulphocysteine by the fungus Microsporum gypseum during growth on cystine.

The dermatophyte Microsporum gypseum was cultivated in media containing 0.5% cystine in suspension, and 0.05% peptone or 1% glucose and 0.05% peptone. During growth on cystine the excess sulphur was oxidized and excreted into the medium not only in the form of sulphate but also in the form of sulphite. Sulphite was produced especially during first phases of growth, where its quantity was higher than that of sulphate and its maximum concentration exceeded 1 mg/ml. S-sulphocysteine, detected chromatographically and determined quantitatively, originated in large quantities by the reaction of sulphite with cystine in the medium. Both sulphite and S-sulphocysteine were further oxidized to sulphate. After exhaustion of cystine 90-93% of the sulphur present was converted to sulphate in the cultivation medium.

Cysteine↗

Optimisation of cellobiose dehydrogenase production by the fungus Sclerotium (Athelia) rolfsii.

The phytopathogenic fungus Sclerotium (Athelia) rolfsii CBS 191.62 is a very efficient producer of the hemoflavoprotein, cellobiose dehydrogenase (CDH), forming up to 225 mg l(-1) (15,000 units cytochrome c activity l(-1)) of this protein, which is of biotechnological interest for sensors, biocatalysis and bioremediation. Both cellulose as inducing substrate and the use of a rich medium containing increased concentrations of peptone from meat or suitable amino acids are important for attaining high CDH yields. CDH, containing a protease-sensitive linker region, can be cleaved by endogenous proteases into a catalytically active flavin fragment and an inactive heme domain. By using increased concentrations of peptone, or certain amino acids such as valine or leucine, or by adding exogenous protease inhibitors, this cleavage can be almost completely inhibited, so that more than 95% intact CDH is obtained under optimised culture conditions. When using non-inhibitory amino acids, e.g. glutamine or lysine, in the medium, more than 80% of the total cellobiose-oxidising activity can be attributed to the flavin fragment.

Amino Acids↗

Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost.

Hydrogen production by thermophilic anaerobic microflora enriched from sludge compost was studied by using an artificial medium containing cellulose powder. Hydrogen gas was evolved with the formation of acetate, ethanol, and butyrate by decomposition of the cellulose powder. The hydrogen production yield was 2.0 mol/mol-hexose by either batch or chemostat cultivation. A medium that did not contain peptone demonstrated a lower hydrogen production yield of 1.0 mol/mol-hexose with less formation of butyrate. The microbial community in the microflora was investigated through isolation of the microorganisms by both plating and denaturing gradient gel electrophoresis (DGGE) of the' PCR-amplified V3 region of 16S rDNA. Sixty-eight microorganisms were isolated from the microflora and classified into nine distinct groups by genetic fingerprinting of the PCR-DGGE or by a random amplified polymorphic DNA analysis and determination of the partial sequence of 16S rDNA. Most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria. Product formation by most of the isolated strains corresponded to that produced by the microflora. Thermoanaerobacterium thermosaccharolyticium was isolated in the enrichment culture with or without added peptone. and was detected with strong intensity by PCR-DGGE. Two other thermophilic cellulolytic microorganisms, Clostridium thermocellum and Clostridium cellulosi, were also detected by PCR-DGGE, although they could not be isolated. These findings imply that hydrogen production from cellulose by microflora is performed by a consortium of several species of microorganisms.

Anaerobiosis↗