Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus

ABSTRACT Clavulanic acid is a β-lactam compound with potent inhibitory activity against β-lactamases. Studies have shown that certain amino acids play essential roles in CA biosynthesis. However, quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production. Here, we report a study of the nutritional requirements of Streptomyces clavuligerus for CA production. Firstly, the influence of the primary nitrogen source and the salts composition was investigated. Subsequently, soybean protein isolate was supplemented with arginine (0.0-3.20 g L-1), threonine (0.0-1.44 g L-1), ornithine (0.0-4.08 g L-1), and glutamate (0.0-8.16 g L-1), according to a two-level central composite rotatable design. A medium containing ferrous sulfate yielded CA production of 437 mg L-1, while a formulation without this salt produced only 41 mg L-1 of CA. This substantial difference suggested that Fe2+ is important for CA biosynthesis. The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence. The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis, so that supplementation was unnecessary. Screening of medium components, together with experimental design tools, could be a valuable way of enhancing CA titers and reducing the process costs.

Explorative investigation of the anti-glycative effect of a rapeseed by-product extract.

The formation of advanced glycation end-products (AGEs) in biological systems is increased during hyperglycaemia due to higher levels of circulating glucose and carbonyl reactive species. AGEs are causative factors of common chronic diseases. Since synthetic AGE-inhibitors exert unwanted side effects and polyphenols act as potent antiglycative agents, vegetables (fruits, seeds and related by-products) are good candidates when searching for natural inhibitors. The aim of this research is to explore the suitability of a polyphenol-rich rapeseed cake extract (RCext) to decrease the formation of AGEs in an in vitro model. Different phenols, amino acids, carbohydrates, organic acids and fatty acids were identified in the RCext by GC-MS. The results confirm a high concentration of polyphenols (73.85 ± 0.64 and 86.85 ± 2.08 mg of gallic acid equivalents per g of RCext spray dried and freeze dried, respectively) which is correlated with the antioxidant capacity and anti-glycative activity in a dose dependent manner. Rapeseed cake extract (3.7 mg mL-1) significantly reduced the formation of free fluorescent AGEs and pentosidine up to 34.85%. The anti-glycative activity of the extract is likely to be due to the high concentration of sinapinic acid (0.108 ± 0.0043 mg g-1) in its metabolic profile, and the mechanism of action is mediated by methylglyoxal trapping. The results show promising potential for using rapeseed cake extract as a food supplement to ameliorate the formation of AGEs. Rapeseed cake extract should therefore be considered a potential candidate for the prevention of glycation-associated complications of age-related pathologies.

Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus.

Clavulanic acid is a ß-lactam compound with potent inhibitory activity against ß-lactamases. Studies have shown that certain amino acids play essential roles in CA biosynthesis. However, quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production. Here, we report a study of the nutritional requirements of Streptomyces clavuligerus for CA production. Firstly, the influence of the primary nitrogen source and the salts composition was investigated. Subsequently, soybean protein isolate was supplemented with arginine (0.0-3.20gL-1), threonine (0.0-1.44gL-1), ornithine (0.0-4.08gL-1), and glutamate (0.0-8.16gL-1), according to a two-level central composite rotatable design. A medium containing ferrous sulfate yielded CA production of 437mgL-1, while a formulation without this salt produced only 41mgL-1 of CA. This substantial difference suggested that Fe2+ is important for CA biosynthesis. The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence. The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis, so that supplementation was unnecessary. Screening of medium components, together with experimental design tools, could be a valuable way of enhancing CA titers and reducing the process costs.

Mesorhizobium soli sp. nov., a novel species isolated from the rhizosphere of Robinia pseudoacacia L. in South Korea by using a modified culture method.

Strain NHI-8(T) was isolated from a forest soil sample, collected in South Korea, by using a modified culture method. Comparative analysis of its nearly full-length 16S rRNA gene sequence showed that strain NHI-8(T) belongs to the genus Mesorhizobium and to be closely related to Mesorhizobium chacoense PR5(T) (97.32 %). The levels of DNA-DNA relatedness between strain NHI-8(T) and reference type strains of the genus Mesorhizobium were 32.28-53.65 %. SDS-PAGE of total soluble proteins and the sequences of the housekeeping genes recA, glnII, and atpD were also used to support the clade grouping in rhizobia. The new strain contained summed feature 8 (57.0 %), cyclo-C19:0ω8c (17.3 %), and C18:0 (11.0 %) as the major fatty acids, as in genus Mesorhizobium. The strain contained cardiolipin, phosphatidylglycerol, ornithine-containing lipid, phosphatidylethanolamine, phosphatidyl-N-dimethylethanolamine, and phosphatidylcholine. Morphological and physiological analyses were performed to compare the characteristics of our strain with those of the reference type strains. Based on the results, strain NHI-8(T) was determined to represent a novel member of the genus Mesorhizobium, and the name Mesorhizobium soli is proposed. The type strain is NHI-8(T) (=KEMB 9005-153(T) = KACC 17916(T) = JCM 19897(T)).

Phenotypes of Escherichia coli isolated from urine: Differences between extended-spectrum ß-lactamase producers and sensitive strains.

BACKGROUND: Escherichia coli is a frequent causative agent of urinary tract infections, and increasing resistance of E. coli to antimicrobials presents a growing challenge. METHODS: Here we compare phenotypes of extended-spectrum ß-lactamase (ESBL) producers (n = 220) with a control group of sensitive strains (non-ESBL producers; n = 150). For each strain, we assessed the presence of O25 antigen, hemolysis, biofilm production, sensitivity to antibiotics, and biochemical profile. RESULTS: Compared to the control group, ESBL producers were more frequently O25 positive (6.0% vs. 42.3%) and less frequently hemolytic (34.7% vs. 6.4%). Comparison of biofilm production in brain-heart infusion (BHI) and in BHI with 4% glucose supplementation showed that ESBL-positive strains produced biofilm in BHI with glucose less intensely than the control group (p < 0.05). Most ESBL producers were ciprofloxacin-resistant (91.8%). Biochemical analyses revealed that ESBL producers more frequently utilized inositol, ornithine, sorbitol, melibiose, and saccharose, whereas the control group more frequently used esculin, lysine, arginine, and dulcitol. The control group strains with O25 antigen were more commonly resistant to ciprofloxacin (p < 0.05). Pulsed-field gel electrophoresis results showed higher variability among the control group of sensitive strains. CONCLUSION: These findings suggest a potential to detect ESBL strains based on virulence factors and biochemical properties, which could be useful in shaping proper empiric antimicrobial therapy, and for initiating such therapy as soon as possible.

Fast derivatization of the non-protein amino acid ornithine with FITC using an ultrasound probe prior to enantiomeric determination in food supplements by EKC.

An EKC method for the determination of ornithine (Orn) enantiomers has been developed after a fast pre-capillary derivatization with FITC. The derivatization step was needed to provide a chemical moiety to the Orn molecule, enabling a sensitive UV detection and the interaction with the CDs used as chiral selectors. To accelerate the derivatization reaction, an ultrasound probe was used. For the development of the chiral method, the influence of different experimental conditions (type and concentration of the chiral selector, temperature, and separation voltage) was investigated. Due to the anionic nature of the analyte (FITC-Orn), five neutral CDs were employed as chiral selectors. The native gamma-CD showed the highest chiral separation power, observing that a low concentration of this CD (1 mM), using a working temperature of 25 degrees C and a separation voltage of 20 kV, enabled to obtain the highest enantioresolution for Orn and its separation from other amino acids usually present in food supplements. After optimizing the method for the preconditioning of the capillary, the analytical characteristics of the chiral method were established. Linearity, LOD and LOQ, precision, and accuracy were evaluated previously to the determination of Orn enantiomers contained in ten commercial food supplements. No interferences from other amino acids present in these samples were observed.

Development of an in-capillary derivatization method by CE for the determination of chiral amino acids in dietary supplements and wines.

A fast in-capillary derivatization method by CE with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate was developed for the first time for the determination of amino acid enantiomers (arginine, lysine, and ornithine) in dietary supplements and wines. Because of the initial current problems due to the formation of precipitates into the capillary during the derivatization reaction, a washing step with an organic solvent as DMSO between injections was necessary. Different approaches were also investigated to enhance the sensitivity of detection. A derivatization procedure, where plugs of ACN, derivatizing agent (10 mM 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate), and sample in borate (1:1 v/v) were injected in tandem (2, 3, and 6 s, respectively, at 50 mbar), was selected because it enabled to obtain the most sensitive and reproducible results. Appropriate analytical characteristics (linearity, LOD and LOQ, precision, absence of matrix interferences, and accuracy) were obtained for this method. Finally, the optimized method was successfully applied to the determination of the enantiomers of arginine, lysine, and ornithine in food samples of different complexities (dietary supplements and wines).

Citrulline modulates muscle protein metabolism in old malnourished rats.

Protein energy malnutrition is common in the elderly, especially in hospitalized patients. The development of strategies designed to correct such malnutrition is essential. Our working hypothesis was that poor response to nutrition with advancing age might be related to splanchnic sequestration of amino acids, which implies that fewer amino acids reach the systemic circulation. Administration of citrulline, which is not taken up by the liver, can offer a means of increasing whole body nitrogen availability and, hence, improve nutritional status. Thirty old (19 mo) rats were submitted to dietary restriction (50% of food intake) for 12 wk. They were randomized into three groups: 10 rats (R group) were killed and 20 others refed (90% of food intake) for 1 wk with a standard diet (NEAA group) or a citrulline-supplemented diet (Cit group). Before being killed, the rats were injected with [(13)C]valine, and the absolute protein synthesis rate (ASR) was measured in the tibialis using the flooding-dose method. When the rats were killed, the tibialis was removed for protein content analysis. Blood was sampled for amino acid and insulin analysis. The standard diet did not have any effect on protein synthesis or on the protein content in the muscle. Citrulline supplementation led to higher protein synthesis and protein content in muscle (117 +/- 9, 120 +/- 14, and 163 +/- 4 mg/organ for protein content in R, NEAA, and Cit groups, P < 0.05). The ASR were 0.30 +/- 0.04, 0.31 +/- 0.04, and 0.56 +/- 0.10 mg/h in the three groups, respectively (R and NEAA vs. Cit, P < 0.05). Insulinemia was significantly higher in the Cit group. For the first time, a realistic therapeutic approach is proposed to improve muscle protein content in muscle in frail state related to malnutrition in aging.

Influence of low-temperature processing of the brackish-water bivalve, Corbicula japonica, on the ornithine content of its extract.

The ornithine content of an extract of the brackish-water bivalve, Corbicula japonica, increased when the bivalve was frozen. It was not influenced by the period of freezing. This phenomenon was not apparent in the scallop, little-neck clam, or hard clam. We applied various low-temperature conditions for processing the bivalve from 4 degrees C to -10 degrees C and measured the ornithine content of each extract. The ornithine content was maximized by processing at - 4 degrees C. The increase in this ornithine content was reduced when the bivalve was stored at 5 degrees C or 15 degrees C after processing at - 4 degrees C, this decrease being reversed when the bivalve was again processed at - 4 degrees C after warming. Low-temperature processing of the brackish-water bivalve therefore increased the ornithine content of the extract.

Aberrations of ammonia metabolism in ornithine carbamoyltransferase-deficient spf-ash mice and their prevention by treatment with urea cycle intermediate amino acids and an ornithine aminotransferase inactivator.

Sparse fur with abnormal skin and hair (spf-ash) mice are deficient in ornithine carbamoyltransferase (OCT) activity, but their OCT protein is kinetically normal. We administered ammonium chloride to spf-ash mice, in order to analyze ammonia metabolism and to find a rationale for the therapy of OCT deficiency. Ammonia concentration in the liver of spf-ash mice increased to a level much higher than in the control. Ammonium chloride injection caused an increase in ornithine (Orn) 5 min after injection and an increase in the sum of Orn, citrulline (Cit) and arginine (Arg) for at least 15 min in the liver of control mice, but no increase in Orn, Cit and Arg in the liver of spf-ash mice. Treatment of spf-ash mice with Arg 5-20 min prior to the injection of ammonium chloride kept the hepatic ammonia concentration at a level comparable to that without the load. A significant reciprocal relationship between ammonia and Orn concentrations in the liver of spf-ash mice 5 min after an ammonium chloride load with or without Arg strongly suggests that ammonia disposal is dependent on the supply of Orn. In spf-ash mice loaded with tryptone as a nitrogen source, Arg supplementation showed a dramatic decrease in urinary orotic acid excretion in a dose-dependent manner. Similar effects were observed with Cit and Orn at the same dose, and a long-lasting effect with an ornithine aminotransferase inactivator, 5-(fluoromethyl)ornithine, at a much lower dose. The rate of urea formation in liver perfused with ammonium chloride was lower in spf-ash mice than in controls, but with the addition of Orn to the medium it increased to a similar level in control and spf-ash mice. These results indicate that OCT is not saturated with Orn in vivo under physiological conditions and that the administration or enrichment of the urea cycle intermediate amino acids enhances the OCT reaction so that the ammonia metabolism of OCT-deficient spf-ash mice is at least partially normalized.

Phenotypic variation of lipid composition in Burkholderia cepacia: a response to increased growth temperature is a greater content of 2-hydroxy acids in phosphatidylethanolamine and ornithine amide lipid.

Burkholderia cepacia produces an unusual range of polar lipids, which includes two forms each of phosphatidylethanolamine (PE) and ornithine amide lipid (OL), differing in the presence or absence of 2-hydroxy fatty acids. By using chemostat cultures in chemically defined media, variations in the lipid content and the proportions of individual lipids have been studied as a function of (a) growth temperature, (b) growth rate and (c) growth-limiting nutrient (carbon, magnesium, phosphorus or oxygen). Total cellular lipid in carbon-limited cultures was lowest at high growth temperatures and low growth rates. Increases in growth temperature over the range 25-40 degrees C led to increases in the proportions of molecular species of PE and OL containing 2-hydroxy acids, without changing the PE:OL ratio. Growth temperature did not alter the balance between neutral and acidic lipids, but the contribution of phosphatidylglycerol to the latter increased with rising growth temperature and growth rate. Pigmentation of cells and the presence of flagella were also temperature-dependent. Change in growth rate also affected the PE:OL ratio and the extent to which monoenoic acids were replaced by their cyclopropane derivatives. Whereas similar lipid profiles were found for carbon-, magnesium- and oxygen-limited cultures, ornithine amides were the only polar lipids detected in phosphorus-limited cells.

Nutritional efficacy of a spermidine supplemented diet.

Polyamines (PA) are ubiquitous cell components essential for growth. Dietary PAs are directed preferentially to tissues and organs that have been stimulated to grow by metabolic signals. Nutritional efficacy and growth potential of an oral PA supplement, spermidine (SD), was examined in growing rats. A group of 24-male Sprague-Dawley rats (200-220 g) was adapted to our vivarium conditions for 3 d, then fed ad libitum continuously for 14 d. During feeding they received either a basal diet (n = 8) or a test diet containing the basal diet with 0.05% SD (test diet 1, n = 8) or 0.10% SD (test diet 2, n = 8). This dose of SD corresponds to an intake of 54 and 108 mumol of SD per rat per day. At the end of 14 d of feeding, the animals were sacrificed and plasma, cerebral spinal fluid (CSF) and tissues (muscle, brain, and liver) were harvested for amino acid analysis. Voluntary food intake, body weight gain, and nitrogen excretion and balance were significantly decreased in test diet 2 fed rats compared to test diet 1. The opposing trends in the accumulation/depletion of free amino acids (AA) in muscle and plasma suggests that the exogenous supply of SD blocks the transport of amino acids, as well as PAs from the cells, since AA and PA share the same transport systems. A trend toward decreased weight gain and feeding efficiency was observed when high concentrations of SD were fed. It was concluded that feeding of SD at moderate intake is not toxic and does not retard growth. Oral administration of a smaller dose (<0.05%) of SD may promote further growth. The optimal level of SD dietary supplementation has thus yet to be established.

Ornithine-alpha-ketoglutarate (OKG) supplementation is more effective than its component salts in traumatized rats.

Addition of an anabolic stimulus during nutritional support seems to be a reasonable adjunct to augment protein synthesis. Ornithine-alpha-ketoglutarate (OKG) has been used for this purpose in many pathological situations, but the mechanism of action is poorly understood. We have evaluated the relative metabolic efficacy of four isonitrogenous diets with or without the addition of alpha-ketoglutarate (alpha KG) or ornithine (ORN), in a rat trauma (bilateral femur fracture) model. Both control and traumatized rats were starved for 2 d. Then for 4 d, the control rats were pair-fed to the traumatized rats, one of the four isonitrogenous diets: the basal diet was a casein-based liquid diet; the ORN and OKG diets were the basal diet in which 10% of the dietary nitrogen was replaced by ORN- or OKG-nitrogen, respectively; the alpha KG diet contained equivalent amounts of alpha KG as were present in the OKG diet. Body weight gain per gram of nitrogen intake was similar in all four diet groups of both control and traumatized rats. The fraction of nitrogen intake that was retained in the body was significantly higher in OKG-fed traumatized rats (23%) than in the corresponding basal diet-fed rats. Plasma and muscle free amino acid concentrations were comparable in OKG- and ORN-fed rats but not in OKG- and alpha KG-fed rats. Our data suggest that the mechanism of OKG action may be associated with increases in growth hormone and insulin, as well as the production of metabolites of ORN and alpha KG. OKG has better metabolic benefits than its two components given separately in the nutritional support of injured rats.

Toxicity and growth-promoting potential of spermine when fed to chicks.

Previous studies have shown that the feeding of putrescine, a biogenic amine and the precursor of the mammalian polyamines, can promote whole-body growth of chicks. The current study was undertaken to determine the effect of spermine, also a biogenic amine and the most cationic of the polyamines, under similar conditions. In Exp. 1, 120 week-old chicks were fed purified crystalline amino acid-based diets containing 0, .2, .4, .6, .8, or 1.0% spermine for 14 d. Spermine proved highly toxic and growth rates were reduced compared with controls when even .2% was fed. In Exp. 2, chicks were fed 0, .0375, .0750, or .1000% spermine. These concentrations proved less toxic than those used in Exp. 1. Supplemental dietary cysteine was then provided at 0, .3, .6, and .9% together with 0, .025, .050, or .400% spermine (Exp. 3) because depletion of cellular glutathione has been suggested as contributing to spermine's toxicity. Even high levels of cysteine supplementation did not overcome spermine's toxicity. Subsequent dietary provision of L-2-oxothiazolidine-4-carboxylic acid (OTC, Exp. 4), a cysteine prodrug, showed that depletion of cellular glutathione was not likely a cause of spermine toxicosis. A trend toward increased weight gain and feed efficiency was observed when low concentrations of spermine were fed. It was concluded, however, that dietary spermine was more toxic to chicks than was previously seen for putrescine, that any growth-promoting effects of dietary spermine are small, and that supplements of dietary cysteine or OTC are unlikely to increase these effects by overcoming spermine toxicosis.

Influence of casein and soy flour proteins on aminoacid content in the liver of experimental animals.

We have observed a significantly increased content of fats and decreased content of proteins in the liver of experimental rats fed a diet supplemented with 25% casein proteins in comparison with the application of de-fatted soy flour. Casein proteins have a higher content of methionine in relation to cystine than baked soy flour. But the soy diet in contrast to the casein diet has a high content of free aminoacids which are not present in casein at all: aspartic acid, asparagine, alpha-aminoadipic acid, methionine, norleucine, lysine, phenylalanine, beta-alanine, ethanolamine, histidine, proline, gamma-aminobutyric acid, taurine. Differences in free valine, alanine, arginine, glycine, ornithine and cysteic acid are also significant. The content of free aminoacids in the liver of experimental animals fed a soy diet is high in the content of cystine, cystathionine, ornithine, beta-aminoisobutyric acid, beta-alanine, gamma-aminobutyric acid, leucine. We have also found accumulation of methionine, glycine, alpha-aminobutyric acid, taurine and citrulline in free aminoacids from the liver of animals fed a casein diet. Citrulline and glycine in free aminoacids from the liver of animals fed a soy protein supplement were not recorded. Our investigations have shown that the application of a soy diet enriched with cystine acts protectively on methionine and that methionine is preferentially utilized for protein synthesis. The catabolic pathway of methionine prevails in animals on a casein diet.

Increased outer membrane ornithine-containing lipid and lysozyme penetrability of Paracoccus denitrificans grown in a complex medium deficient in divalent cations.

Paracoccus denitrificans grown in a complex medium was highly susceptible to lysozyme, in contrast to cells grown in a complex medium supplemented with Mg2+ and Ca2+ or in a succinate-salts medium. The complex medium was deficient in divalent cations needed for optimum outer membrane stability. The major change in molecular compositions of outer membranes isolated from cells grown under the different conditions was a higher ratio of ornithine-containing lipid to phospholipid in complex-medium-grown cells (0.63) than in cells grown in complex medium with Mg2+ and Ca2+ (0.22) or in succinate-salts medium (0.14). We suggest that the dipolarionic ornithine-containing lipid is less dependent than acidic phospholipids on divalent cations for its incorporation into the outer membrane.