Effect of the aeration and agitation parameters on the oxytetracycline biosynthesis process under microkinetic conditions

The effect of the aeration levels in flasks on the rate of oxytetracycline biosynthesis and other kinetic characteristics was studied. It was shown that changes in the medium volume in flasks, dilution of the fermentation broth with water or its filtrate and the use of oxygen for aeration had a significant effect on the characteristics studied. Special experiments with low concentrations of the biomass were performed for investigation of the effect of dissolved carbon dioxide on the kinetics of the process.     

Determination of oxytetracycline by flow injection with chemiluminescence detection.

A flow-injection chemiluminescent method for the determination of oxytetracycline was developed. The method is based on an enhancement by oxytetracycline of the chemiluminescence light emission of tris(2,2'-bipyridine) ruthenium (II), generated by the continuous oxidation of tris(2,2'-bipyridine) ruthenium (II) by cerium (IV) sulphate in sulphuric acid. Under the optimum conditions, the calibration curve was linear over the range 1.0 x 10(-7)-1.0 x 10(-5) g/mL for oxytetracycline with the linear equation: DeltaINT = 148.77 x C + 0.6637 (R2 = 0.9994). The detection limit was 4.52 x 10(-8) g/mL. The proposed method was also successfully used to determine oxytetracycline in pharmaceutical formulations. The mean recovery of determination of oxytetracycline was 92.73%. A mechanism for the chemiluminescence enhancement by oxytetracycline of tris(2,2'-bipyridine)-ruthenium (II) and cerium (IV) sulphate system is also proposed.     

Effect of the aeration conditions on the biosynthesis of oxytetracycline and the formation of organic acids by a culture of Actinomyces rimosus]

The effect of the aeration conditions on oxytetracycline biosynthesis and production of organic acids by Act. rimosus was studied. Intensive biosynthesis of oxytetracycline in shaken flasks with concentrated complex media was observed at the rate of oxygen dissolution in the liquid ranging from 14 to 25 mg/1/min. Lower rates of the oxygen dissolution up to 7 mg/1/min resulted in decreased rates of the culture growth and the medium component consumption, decreased antibiotic levels, production of significant amounts of pyruvic and acetic acids.     

Fully automated high-performance liquid chromatographic analysis of whole blood and plasma samples using on-line dialysis as sample preparation: Determination of oxytetracycline in bovine and salmon whole blood and plasma

A fully automated technique for high-performance liquid chromatographic analysis of whole blood and plasma is described. Samples are automatically injected into a dialyser where proteins and blood cells are removed. The dialysates are concentrated on a small column prior to analysis. This technique is used for the determination of oxytetracycline in whole blood and plasma. After dialysis oxytetracycline and the internal standard, tetracycline, are retained on a polystyrene enrichment column and subsequently separated on a polystyrene analytical column by ion-pair chromatography. Using ultraviolet detection 50 ng/ml can be detected. Validation showed good within-day and between-day accuracy and precision. Different oxytetracycline concentrations were found in plasma and whole blood. This difference varied between the species.     

Application of scale-down experiments in the study of kinetics of oxytetracycline biosynthesis

Scale-down experiments in antibiotic biosynthesis were performed by transferring the corresponding amounts of fermentation broth from industrial to laboratory and pilot-plant fermentors where the cultivation process was continued at different cultivation conditions. A previously proposed mathematical model was used to explain the experimental results. The effects of temperature, agitation-aeration intensity, and medium addition during the process were investigated. Computer simulation data were fitted to the experimental data, and good agreement was found. As a consequence of increasing temperature up to 37 degrees C, increases in the specific growth and autolysis rates as well as the specific rates of antibiotic synthesis and carbohydrate utilization were in evidence. Temperature increases of up to 40 degrees C caused a lower oxytetracycline yield. The effect of increased oxygen transfer rate on oxytetracycline biosynthesis was more pronounced at higher temperatures than at lower cultivation temperatures. Culture differentiation (strain segregation) was also studied; it was found that the increased cultivation temperature could be favorable for the growth of biomass active in oxytetracycline biosynthesis. Results of experiments at the pilot-plant scale showed that fed batch and repeated fed batch cultures could be successfully applied and the period of intensive antibiotic synthesis could be prolonged significantly.     

Optimisation of synthetic medium composition for levorin biosynthesis by Streptomyces levoris 99/23 and investigation of its accumulation dynamics using mathematical modelling methods

The composition of a synthetic culture medium for levorin biosynthesis by Streptomyces levoris 99/23 was optimised using mathematical modelling methods. The optimal concentrations of the medium components were established by means of an optimum composition design at three factor variation levels. An adequate regression model was obtained. Levorin biosynthesis by Streptomyces levoris 99/23 in the optimised synthetic medium was over 38% higher than in the initial medium. The antibiotic biosynthesis dynamics in the optimised culture medium was studied by means of a non-linear differential equation system. The resultant model was valid.     

Standardization of the nutrient medium for determining the biological activity of a number of antibiotics by the diffusion-in-agar method

The possibility of using 2 variants of a unified nutrient medium containing no Hottinger's broth for determination of the biological activity of a large number of antibacterial antibiotics was shown. The medium provided satisfactory reproducibility of the results. It contained yeast extract as a source of nitrogen. The medium was selected with the method of the fractional factor experiment. Clear inhibition growth zones and the slope of the dose-response curve were used as the criteria for estimation of the optimal medium. The described nutrient medium allowed a 2-time increase in the sensitivity of the method used for determination of the biological activity of neomycin, monomycin, streptomycin, oxytetracycline, chlortetracycline and erythromycin.     

Recombination between the linear plasmid pPZG101 and the linear chromosome of Streptomyces rimosus can lead to exchange of ends

The 387 kb linear plasmid pPZG101 of Streptomyces rimosus R6 can integrate into the chromosome or form a prime plasmid carrying the oxytetracycline biosynthesis cluster. The integration of plasmid pPZG101 into the linear chromosome of S. rimosus R6-501 in mutant MV25 was shown to be due to a single cross-over at a 4 bp common sequence. pPZG101 had integrated into a 250 kb DNA sequence that was reiterated at a low level. This sequence includes the oxytetracycline biosynthesis cluster, so that homologous recombination generated a mixed population carrying different copy numbers of the region. The 1 Mb linear plasmid pPZG103 in mutant MV17 had also arisen from a cross-over between pPZG101 and the chromosome, so that one end of pPZG103 consists of c . 850 kb of chromosomal sequence including the oxytetracycline biosynthesis cluster. The plasmid pPZG101 was shown to consist of a unique central region of about 30 kb flanked by terminal inverted repeats of about 180 kb. Analysis of a presumed ancestor plasmid pPZG102 suggested that the long terminal repeats had arisen by a recombination event during the strain development programme.     

Study of the effect of certain factors on the process of tetracycline antibiotic crystallization]

Since such factors as temperature and time of the process had a significant effect on the residual activity of the mother solution during crystallization of tetracyclines, it was necessary to study the effect of the above factors on the process for determination of the crystallization optimal conditions. The experimental data provided creation of the regression equations and the curves for similar levels of the main parameters for tetracycline and oxytetracycline were calculated. The lowest values of the mother solution activity within the ranges tested were observed at the temperature of 2 degrees and the time of 3 hours.     

Study of restrictase Sal GI biosynthesis and optimum conditions for its isolation

The effect of the growing phase of Streptomyces albus G. and the components of the culture medium on the biosynthesis of restriction endonuclease Sal GI was studied. The conditions of DNA sedimentation with streptomycin sulfate and polyethylenimine and separation of proteins with ammonium sulfate were investigated as well. A maximal quantity of the enzyme was observed in the cells in 18-20 h of cultivation in flasks on a shaker. The optimal concentrations of the medium components (g/l) were found by mathematical methods of the experiment planning: glucose--19,0; pepton--5.2; K2HPO4 X 3H2O - 5.0. The optimal concentrations for DNA sedimentation with streptomycin sulfate and polyethylenimine were found to be 1.8-2.0% and 0.2-0.3%, respectively. The optimal concentration of ammonium sulfate for protein separation is 70% of saturation.     

Simplified liquid nutrient media for controlling antibiotic activity, the spectrum of their antibacterial action and the sensitivity of microorganisms]

Beaf-peptone broth and some of its modifications, one of which is a simple and in expensive one to a leser extent binding to antibiotics, such as penicillin, oxytetracycline and streptomycin and providing sufficient growth of the test microbes were used to determine the antibiotic activity with the methods of serial dilutions. The simple modification was recommended for practical use. The MIC of the antibiotics in the above simple medium was less than that in the control. The results of the antibiotic activity determination on both media coincided.     

The effect of aeration and agitation on tetracycline biosynthesis

The results of the study on relation between tetracycline biosynthesis and the specific power input for agitation in pilot plant apparatus was studied. No correlation was observed between the levels of tetracycline biosynthesis and changes in the specific power input within a range of 0.6 to 2.3 kW/m3 at the expense of changes in the mixer diameter and the agitation rate, when the aeration rate was constant. It was shown that the aeration conditions were most significant for tetracycline biosynthesis. The study provided determination of the optimal aeration conditions for biosynthesis of tetracycline.     

土霉素暴露对小麦根际抗生素抗性细菌及土壤酶活性的影响

通过培养的方法研究了土霉素暴露和小麦根际抗性细菌的数量、种类、分布特征及土壤酶活性之间的剂量效应关系。结果表明,土霉素暴露下小麦根际单一抗生素抗性细菌数量和抗土霉素—链霉素双重抗性细菌数都明显增加,且与暴露剂量呈正效应关系;同时,土壤磷酸酶、脱氢酶活性下降,但与土霉素的剂量效应关系不明显。从土霉素暴露的土壤中分离到50株抗性细菌,经形态观察、RFLP分组和16S rDNA序列测定与分析,将它们聚集在Actinobacteria、Bacilli、Alphaproteobacteria、Gammaproteobacteria 和Sphingobacteria类群。其中放线菌最多（15株）,占抗性菌总数的30 %;其次是Bacillus属细菌（9株）和Pseudomonas属细菌（8株）,分别占18 %和16 %。同时,具有抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌在土霉素暴露的样品中均被分离到,分别占抗性菌株总数的16 %、8 %和4 %。值得注意的是,随着土霉素暴露剂量的增加,小麦根际优势促生菌Bacillus属细菌的抗性检出率逐步降低;但具有抗生素抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌的检出率却明显增加,提示可能会进一步增大其机会致病性。     

Gas chromatography of bile acids

Bile acids, the end products of cholesterol metabolism in the liver, are of vital importance in the tissue distribution of cholesterol. Abnormalities in cholesterol biosynthesis or metabolism are often reflected in the proportions, concentrations and conjugation of bile acids in various tissues and determination of bile acids in these tissues is important in the diagnosis of hepatobiliary diseases. Several methods for quantitative determination of bile acids in biological fluids are known and have been reviewed. In this review, we have discussed the gas-chromatographic method for determination of bile acids with special reference to bile acid quantitation in plasma, bile, urine and stool.     

龟裂链霉菌zwf2基因阻断提高土霉素生物合成

葡萄糖-6-磷酸脱氢酶(G6PDH)是链霉菌磷酸戊糖途径中第一个酶("看家"酶),也是形成NADPH的关键酶,由zwf1和zwf2基因编码.以温敏型质粒pKC1139为基础构建了用于阻断龟裂链霉菌zwf2的重组质粒pKC1139-zwf2',通过大肠杆菌GM2929去甲基化pKC1139-zwf2'后电转至原始龟裂链霉菌M4018感受态细胞,筛选得到转化子.转化子进一步通过PCR鉴定和点杂交印迹分析鉴定,证明是zwf2基因阻断的阳性突变子命名为M4018-△zwf2.以原始菌株为对照,突变子摇瓶发酵结果表明:突变子的葡萄糖-6-磷酸脱氢酶酶活是原始菌的50%左右,但土霉素生物合成水平则提高了27%;在细胞生长方面,二者均在第4d进入生长稳定期而开始大量合成土霉素,发酵结束时细胞菌体浓度基本相同,但突变子的单位菌丝体土霉素生物合成能力则提高了31%.因此,zwf2的阻断有利于土霉素的生物合成,而对细胞生长没有明显影响.     

Enhancement of oxytetracycline production in a Streptomyces rimosus strain after treatment with acriflavine

Summary A strain ofStreptomyces rimosus was treated with acriflavine as curing agent; morphological and physiologically altered variants were isolated, and changes in sporulation, aerial and substrate mycelia, pigment formation, oxytetracycline (OTC) resistance and biosynthesis are discussed.     

Simultaneous determination of selected veterinary antibiotics in gilthead seabream (Sparus Aurata) by liquid chromatography-mass spectrometry

A method was optimised and validated for simultaneous monitoring of several drugs of different classes of antibiotics such as quinolones (oxilinic acid and flumequine), tetracyclines (oxytetracycline), sulfonamides (sulfadiazine) and trimethoprim in fish muscle and skin. The method is based on solid-liquid extraction without further sample clean up followed by liquid chromatography-mass spectrometry (LC-MS) determination with electrospray ion source (ESI) in positive mode. The limits of quantification (LOQs) were lower than 20 microg/kg for all compounds and repeatability, expressed as relative standard deviations (RSD), were lower than 15%. Therefore, the LC-MS method was successfully applied for the quantitative determination of antibiotics in gilthead sea bream muscle and skin and oxytetracycline in medicated fishes.     

Application of flow cytometry for the determination of minimal inhibitory concentration of several antibacterial agents on Mycoplasma hyopneumoniae

Aim:  In this study, flow cytometry was evaluated for the determination of the minimal inhibitory concentrations (MICs) of nine antibacterial agents (enrofloxacin, ciprofloxacin, oxytetracycline, chloramphenicol, tylosin, lincomycin, gentamycin, spectinomycin and streptomycin) against M. hyopneumoniae .
Methods and Results:  Flow cytometry was able to detect Mycoplasma hyopneumoniae inhibition at 12 h postincubation, whereas the results obtained by the traditional method were only obtained at 48 h, when a visible change in the medium had occurred. At 48 h, both methods gave the same result for eight antibacterial agents, whereas flow cytometry gave slightly higher MIC values for one antibacterial agent (tylosin). This was attributed to the fact that the M. hyopneumoniae growth that had occurred in those tubes was not enough to visibly change the colour of the medium. A good relationship was found between the flow cytometry and the traditional method.
Conclusion:  Flow cytometry was found to be a good method for the determination of antimicrobial MICs in M. hyopneumoniae .
Significance and Impact of the Study:  The flow cytometric method allows the determination of the response of M. hyopneumoniae to each of the antibacterial agents in near real time, and has potential for the identification and study of resistant subpopulations.     

Liquid chromatographic determination and depletion profile of oxytetracycline in milk after repeated intramuscular administration in sheep

A simple, rapid and specific ion-pair liquid chromatographic method for the routine determination of the marker residue of oxytetracycline in sheep milk, at levels as low as 20 microg/kg, has been developed. Milk samples were acidified and extracted with acetonitrile. The extracts were purified by treatment with ammonium sulphate and concentrated into diluted phosphoric acid. Separation was carried out isocratically on a Nucleosil C(18) column using a mobile phase that contained both positively and negatively charged pairing ions. The in-house validated method gave overall recoveries and overall relative standard deviations better than 86% and 4.6%, respectively. The method was successfully applied to study the depletion of oxytetracycline in sheep milk and to estimate the withdrawal period after intramuscular administration of a commercial oxytetracycline formulation.     

Study of oxytetracycline solubility in an aqueous medium]

Solubility of oxytetracycline dihydrate in aqueous media was studied. It was shown that solubility of the drug in bidistillate at a temperature of 20 degrees was 195 gamma/ml, which was much lower than the requirements of some pharmacopoeia with respect to the drug solubility. Dependence of oxytetracycline dihydrate solubility in the aqueous medium on the values of pH and temperature was found. Indigency of the procedure described in the literature for determination of antibiotic solubility according to the dry weight of the filtrate on addition of large excesses of the solid phase to the system was shown.