Semisynthetic and synthetic antibiotics

Production phase - growth is practically ceased, dry weight of microorganism is constant, the antibiotic is intensively synthesized.

Antibiotic producers mostly belong to filamentous microorganisms, which means that, in their culture, cells of various age and at different stages of development are present. The microorganisms grow in pellets, inside which the cultivation conditions differ from those on the pellet surface (nutrient concentrations, oxygen concentration, etc.). An increase in dry weight does not have to always mean the biomass growth since, in streptomycetes, often a thickening of the cell wall or glycocalyx formation occur that increase the dry weight value without rising the cell number . The individual cells can thus be at different stages of development, i.e. in different physiological states. Therefore, we speak about a physiological state of the whole culture that represents an average of physiological states of the individual cells.

Regulation by nutrients. In order to reach a high production of an antibiotic, a sufficient biomass yield is necessary, that is accomplished within a short time, if possible. Thus a danger of contamination is diminished and the economic parameters of the fermentation device are kept at its optimum. For this purpose, readily utilizable carbon, nitrogen and phosphorus sources are used. When they are present in the medium, however, an overproduction of the antibiotic does not take place. The culture medium should be designed in such a way that, after the biomass increased sufficiently, at least one of the basic nutrient sources would become depleted and the culture growth would be consequently limited. However, this limitation is not well understood.

Regulation by nitrogen source.Readily utilizable nitrogen sources present in the culture medium inhibit the production of antibiotics. Mainly ammonium ions decrease the antibiotic synthesis and, therefore, their concentration in the production media is limited to be exhausted at the end of growth phase. Soy flour, peanut flour and other complex substances are used as nitrogen sources in the production phase of antibiotic fermentations. These nitrogen sources are not easily utilizable and are similar to those used by the microorganisms producing antibiotics in nature. Readily utilizable nitrogen sources repress enzymes of secondary metabolism in Cephalosporium acremonium during the biosynthesis of cephalosporin and in Streptomyces clavuligerus producing cephamycin. Similarly, the inhibition of biosyntheses of leucomycin, tylosin , and erythromycin are explained by the repression of enzymes of secondary metabolism. Ammonium salts also inhibit the activity of anhydrotetracycline oxygenase isolated from S. Aureofaciens.

Regulation by phosphate[19]. Phosphate is used as main regulator of overproduction of antibiotics in factories. Inorganic phosphate is carefully added in doses to the medium so as to accomplish an optimal ratio between the biomass production and the capability of antibiotic biosynthesis. Bound to organic compounds normally added to the medium (soy flour, etc.), phosphate does not affect the antibiotic production. The antibiotic biosynthesis is started on the condition the concentration of phosphate in the medium decreased below a certain level.

The influence of inorganic phosphate is explained by repression of the synthesis of enzymes of secondary metabolism . After the inorganic phosphate was depleted from the medium, a significant decrease of the rate of proteosynthesis was observed during the tetracycline biosynthesis and the synthesis of enzymes of secondary metabolism was commenced. If phosphate was kept above the threshold concentration, the significant decrease of the rate of protein synthesis did not occur and enzymes of secondary metabolism were not synthesized. An addition of phosphate to the medium at the beginning of the production phase, after the phosphorus source was depleted and the enzymes of secondary metabolism synthesis initiated, resulted in a decrease of the enzyme levels in the culture and an acceleration of proteosynthesis. The synthesis of secondary metabolism was resumed after the phosphate was depleted again from the medium. Production of oxytetracycline by Streptomyces rimosus is controlled, at least in part, at the level of transcription from promoters overlapped by tandem repeats similar to those of the DNA-binding sites of the OmpR family [20]. The phosphate was found to be consumed at a higher rate than expected, with respect to the actual rate of protein synthesis, and was probably deposited in the cells in the form of polyphosphates.

1.2.2 Influence of low molecular compounds

The antibiotic production can be regulated by different low molecular compounds. The mechanism of their action is not understood. Tryptophan exhibited a stimulatory effect on the production of antibiotics, e.g. mucidin in the basidiomycete Oudemansiella mucida and actinomycin in Streptomyces parvulus. Methionine was found to promote the synthesis of cephalosporin C. Neither tryptophan nor methionine were used as the building units. When enzymes of secondary metabolism were measured, higher levels were detected in the cells of the producing strain.Benzyl thiocyanate increases the production of chlortetracycline and tetracycline in S. aureofaciens. In contrast, it does not influence the production of oxytetracycline in S. rimosus. The effect on the metabolism of S. aureofaciens is multiple, including a number of enzymes but the basic influence of benzylthiocyanate at production is the higher expresion of enzymes of secondary metabolism. This is the reason why benzyl thiocyanate is able to raise the antibiotic production only if it is added in the lag phase, growth phase or at the beginning of the production phase. Its effect is more pronounced in low production strains, where the enzymes of secondary metabolism level and the antibiotic production are increased 10 to 20-fold, as compared to high production strains where the increase is only twofold.

In the streptomycete antibiotic producers, low-molecular, diffusible compounds have been discovered that regulate the metabolism of the producer, where they are present at very low concentrations, and thus affect both the biochemical and morphological differentiation. The most famous of them is factor A, gamma-butyrolactone, that was discovered in Streptomyces griseus producing streptomycin . A non-producing strain started the synthesis of streptomycin after factor A was added to the culture and, in parallel, the formation of aerial mycelium was taking place. Factor A is synthesized by many streptomycetes but the regulatory effect was observed only in Streptomyces griseus, Streptomyces bikiniensis and Streptomyces actuosus. As the result of the addition of factor A to blocked mutants of Streptomyces griseus JA 5142, the synthesis of anthracyclines and leukaemomycin (anthracycline type antibiotic) was resumed. The resistance to streptomycin linked with an enzymatic phosphorylation of the antibiotic is also induced by factor A.

Analogues of factor A have also been found, all of them being gamma-butyrolactones. Virginiae butanolides were detected in Streptomyces virginiae. Factor I was isolated from Streptomyces sp. FR1-5 and its effective concentration was 0.6 ng/ml culture. Most of the factor A analogues, however, were not biologically active.

Factor B was isolated from the yeast Saccharomyces cerevisiae. This substance was capable of eliciting the production of rifamycin in a blocked mutant of Nocardia sp. This substance was effective at a concentration of 10^-8 M, when one molecule elicited a synthesis of about 1500 molecules of the antibiotic. The structure of factor B is similar to cAMP but none of the derivatives of known nucleotides exhibited a comparable effect. Chemically prepared derivatives of factor B have also been tested. The effect was observed with those that had a C₂ -C₁₂ acyl moiety; octylester was the most effective of them, exhibiting the effect at as low a concentration as 10^-10 M. A substitution of guanosine for adenine did not result in a loss of the biological activity of factor B.

Factor C was isolated from the fermentation medium of Streptomyces griseus. This compound causes cytodifferentiation of non-differentiating mutants . Factor C is a protein having a molecular weight of about 34 500 D, whose molecule is rich in hydrophobic amino acids. The effect of autoregulators is easily observable, if they elicit morphological changes, such as the formation of aerial mycelium. Carbazomycinal and 6-methoxcarbazomycinal, isolated from Streptoverticillium species, were capable of inhibition of the aerial mycelium formation at a concentration of 0.5 to 1 microgram per ml. Autoregulators affecting sporulation were found in Streptomyces venezuelae, Streptomyces avermitilis), and Streptomyces viridochromogenes NRRL B-1551. From the same strain of Streptomyces viridochromogenes, germicidin was isolated by Petersen and co-workers [21]. The compound had an inhibitory effect on the germination of arthrospores of Streptomyces viridochromogenes at a concentration as low as 40 picograms per ml. Germicidin (6-(2-butyl)-3-ethyl-4-hydroxy-2-pyrone) is the first known autoregulative inhibitor of spore germination in the genus Streptomyces and was isolated from the supernatant of germinated spores, but also from the supernatant of a submerged culture.Mutants of Streptomyces cinnamonensis resistant to high concentrations of butyrate and isobutyrate produce an anti-isobutyrate factor, that is excreted into the culture medium . On plates, anti-isobutyrate factor efficiently counteracted toxic concentrations of isobutyrate, acetate, propionate, butyrate, 2-methylbutyrate, valerate, and isovalerate in Streptomyces cinnamonensis and other Streptomyces species.General control mechanisms have been looked for that operate in the antibiotic biosynthesis. The energetic state of the cell is thought to be such a general control mechanism. The intracellular ATP level reflects the content of free energy in the cell. In some cases, the start of the antibiotic synthesis is linked with a decrease of the intracellular ATP level. Such a relationship was observed in Streptomyces aureofaciens and Streptomyces fradiae during the production of tetracycline and tylosin , respectively.

Even though the regulatory role of ATP cannot be strictly excluded, the results seem to support a hypothesis that a higher ATP level is accompanying the active primary metabolism. A slow down of growth and of the whole primary metabolism would logically be accompanied by a decrease of the ATP level.

As in the case of ATP, the role of cAMP in the metabolism of antibiotic producers was also studied, especially in connection with the glucose regulation. Hitherto, no indication has been obtained suggesting a significant role of cAMP in the regulation of antibiotic production .

antibiotic medicine microorganisms

1.2.3 Reception of signals from environment

The way of reception of signals from the environment, so that they would be available to the genetic material of the cell to result in the initiation of the antibiotic synthesis, is known quite well. It does not significantly differ from the trasduction of signals for other metabolic processes. Catabolite repression signals or those signalling the depletion of nitrogen or phosphate or the initiation of sporulation are transducted via two-component, signal proteins [22]. In spite of some structural varieties, these proteins are characterized by general mechanistic features and conserved amino acid sequences. The two-component system consists of a cytoplasmic membrane-linked, sensor-transmitter protein and a response-regulator protein, located in the cytoplasm. The sensor-transmitter is composed of a sensor domain located near its N-end the N-end is found outside the cytoplasm. A specific effector is capable of binding directly to this N-end. The transmitter domain is located in the cytoplasm to be linked to the sensor domain via a hydrophobic, amino acid sequence stretching across the membrane. The sensor-transmitter proteins are normal histidine-protein kinases, capable of autophosphorylation at its C-end on receiving a proper signal.

Transcription initiation of structural genes.Regulatory proteins, having been bound to specific DNA sequences and having interacted with RNA polymerase, start the transcription. Regulatory proteins that activate the transcription of structural genes are probably synthesized already during the lag phase. Their binding to DNA and a subsequent biosynthesis of the antibiotic depend mainly on the composition of the growth medium. Provided inorganic phosphate is present in the medium, the activator becomes phosphorylated and thus incapable of binding to DNA. In contrast, for example, the activator of the synthesis of glutamine synthetase, a key enzyme of the assimilation of ammonium salts from the medium and, consequently, of utmost importance for proteosynthesis, is able to bind to DNA only in a phosphorylated form.

One can hypothesize that a depletion of inorganic phosphate from the medium does not stop proteosynthesis as a result of a lack of phosphate in the cell for the biosynthesis of cellular structures, as the phosphate limitation is normally explained, but rather the presence or absence of phosphate in the medium causes respective activation or repression of the activators of the enzyme syntheses in primary or secondary metabolisms.

This idea is also supported by the fact that enzymes of secondary metabolism were synthesized and the antibiotics produced immediately after the phosphate, that had been added at the beginning of the production phase, was depleted from the medium and deposited in the cell [23].

1.3 Technology of antibiotic production

Some antibiotics are commercially produced on a ton scale. The fermentation process during which microorganisms produce antibiotics is carried out in fermentors having a volume of several tens of cubic meters. As in any fermentation process, a conserved strain is used, that is first propagated in the laboratory and then in a plant fermentor. The cells are then used to inoculate production fermentors. The inoculum is most often put into a 10 to 20-fold volume of the fresh medium.

Isolation of a producing microorganism from one cell.The spores are transferred from an agar slope into a volume of 10 ml of sterile H₂O and, after homogenization, the suspension is diluted to contain 30-50 spores in 1 ml. A volume of 0.25 ml of this suspension is transferred on the surface of a suitable agar medium on a Petri dish and spread with a sterile glass stick. Colonies, each of which originates from one cell, grow on the agar. The individual colonies are re-inoculated to agar slopes and their antibiotic production is tested.

1.3.1 Conservation of microorganisms

If cultures are conserved for a long time on agar slopes, being repeatedly transferred from one slope to another, they can degenerate and lose valuable technological properties. Two types of conservation are recommended for long term storage of strains: lyophilization (microbial cells or spores are conserved by quick removal of water by sublimation at a low temperature) or conservation by keeping cultures at a very low temperature (-70^oC) in liquid nitrogen. In both cases cultures keep their properties for at least 10 years.

Laboratory cultivation.Cultivation in the laboratory, irrespective of the fact whether the microorganism will finally be used for inoculation of a production fermentor or in laboratory experiments, is carried out in test tubes or in 200-1000-ml bottles and flasks. The volume of the culture medium mostly represents about one tenth of the total volume of the flask. The flasks are sealed with stoppers allowing diffusion of the air into the flasks to ensure aerobic conditions for growth. At the same time, the stoppers prevent microorganisms from the environment to penetrate into the flasks (cotton-wool stoppers, etc.). Producers of antibiotics require a proper aeration, that is important for both the growth and production of the antibiotic. Therefore, the flask contents is well mixed by agitation on rotary or reciprocal shakers placed in thermostated rooms or boxes. Strictly sterile conditions have to be ensured for the cultivation of antibiotic producers since, in the case of contamination, the producing culture can be suppressed by more rapidly growing microorganisms.

Cultivation in fermentors.Microbial producers of antibiotics are cultivated in fermentors of various size. The lower limit of size of laboratory fermentors is about 1 litre. Owing to the use of complex media, foam is often formed during cultivation and, therefore, the fermentors are filled with the medium up to one half or two thirds of their maximal capacity. When the process of antibiotic production is scaled up from the laboratory conditions to those of true production, basic parameters can be established using several-litre, laboratory fermentors. However, they should be verified in pilot plant fermentors having a size of several cubic meters. The basic equipment of both laboratory and pilot plant fermentors is practically the same. They are made of inert materials such as glass and stainless steel, or their walls are at least lined with an inert material. The fermentors are equipped with a device keeping the cultivation temperature constant (mainly cooling device is important in large fermentors) and with an efficient aeration system, since antibiotic producers require a sufficient oxygen supply for both the growth and synthesis of the antibiotic. The aeration systems based on intensive stirring are not suitable for cultivation of antibiotic producers since a majority of them are filamentous microorganisms that can suffer damage when intensively stirred. The air flowing into the fermentor has to be sterile. It is sterilized by filtration most often glass wool or mineral wool filters are used.

Most antibiotics are produced in a fed batch system, i.e. a certain amount of the culture medium is inoculated with the producing microorganism and, after a time interval, another dose of nutrients is added to the fermentor. Thus a prolonged cultivation can be accomplished that enables us to increase the yield of the antibiotic. The inflow of nutrients makes possible keep their optimal levels. In cultivations whose course is well known, the nutrient inflow is programmed in advance.

Solid-state fermentation.Solid-state, or substrate, fermentation is characterized by a fermentation process on a solid support, which has a low moistre content and occurs in a non-septic and natural state [24]. The use of solid-state technology for the production of antibiotics has some advantages. Due to the lack of free water, smaller fermentors are required and the mycelial microorganisms, used predominantely for antibiotic production are well suited to grow on a solid support. On the contrary, a liquid fermentation process allows greater control and monitoring of parameters, such pH, heat, nutrient condition etc [25].

1.3.2 Isolation, separation and purification of antibiotics

Isolation of an antibiotic from the fermentation medium depends on the fact whether the antibiotic is secreted into the medium or remains in the biomass, inside the cell or bound to the cell wall. If the antibiotic is bound to the biomass or, in contrast, present in the broth supernatant, the two phases are separated by filtration or centrifugation and extracted separately. If the antibiotic is present in both phases the whole broth is used for extraction. Another isolation step usually includes an extraction with solvents of different polarities, followed by evaporation of the extracts to dryness. If the antibiotic is extractable by nonpolar solvents, the extraction is preceded by dehydration, most often using methanol or acetone. By the extraction with nonpolar solvents, a most part of water soluble compounds present in the medium is eliminated. The crude isolate obtained is used as a material for further separation processes.

The antibiotic producers often synthesize a number of compounds or derivatives of the desired compound that have to be separated from the antibiotic produced. The separation is carried out using standard operations such as an extraction into another solvent, chromatography techniques and, in the end, precipitation or crystallization.

2. Experemental part

Streptomyces violatus showed the highest antimicrobial activity in static cultures after 7 days incubation at 30°C. The antibacterial substance was more active against Bacillus subtilis and Staphyllococcus aureus than Escherichia coli or Sarcina lutea. Growth of S. violatus and production of antibiotic in a starch-nitrate medium were monitored over a period of 14 days. The organism produced a blue pigment associated with the antibiotic appearance in the cultures. Optimization of antibiotic production in batch cultures has been carried out. Substitution of starch by glycerol at a concentration of 12.5 g/l showed 1.32-fold increase of antibiotic production. Cultures containing sodium nitrate (2.5g/l) showed the highest antibiotic production followed by peptone, alanine, monosodium glutamate or phenylalanine. A mixture (w/w) of K2HPO4 and KH2PO4 (1g/l) yielded 1.9-fold and 6.1-fold increase in antibiotic production compared to cultures individually supplied with K2HPO4 or KH2PO4, respectively. The presence of ferrous sulphate and manganese chloride improved the production of the antibiotic. An inoculum size of 4x106 spores/ml and initial pH 7.0 at 30°C were optimum for a maximum antibiotic production of 268µg/ml in the culture filtrates of S. violatus [26].

2.1 Abstract to Streptomyces violatus

Streptomycetes are the source of several useful antibiotics that are used not only in the treatment of various human and animal diseases but also in agriculture and biochemistry as metabolic poisons .At least 70 of the approximately 100 marketed antibitics used for the treatment of infections in humans are derived from substances produced by Streptomyces spp., for example Streptomyces aureofaciens is an important industrial microorganism as a producer of chlortetracycline and tetracycline. Discovery of new antibiotics produced by streptomycetes still continues, such as noboritomicins A and B produced by S. Noboritoensis [27], actinomycins X2 produced by S. nasri (El-Naggar et al. 1998), tetrodecamycin produced by S. nashvillensis MJ885-mF8, demethyltetracycline produced by S. aureofaciens and pyrroindomycins produced by S. rugosporus (Abbanat et al. 1999). The ability of streptomycete cultures to form antibiotics is not a fixed property but can be greatly increased or completely lost under different conditions of nutrition and cultivation (Waksman 1961). Therefore, the medium constitution together with the metabolic capacity of the producing organism greatly affects antibiotic biosynthesis. Changes in the nature and type of carbon, nitrogen or phosphate sources and trace elements have been reported to affect antibiotic biosynthesis in streptomycetes . In addition, antibiotic productivity tendes to decrease when metal ion deficient media are used and when the inocula are incubated for long periods and at high temperatures. The present study describes the production of an antimicrobial substance MSW2000 produced by a local isolate of Streptomyces violatus. Improvement of antibiotic production was acheived by optimization of the cultural conditions and by developing of a defined medium for the biosynthesis of the antibiotic.

2.1.1 Producer of experiment

Streptomyces orientalis, S. violatus, S.craterifer and S. astreogriseus were isolated from garden soil, Faculty of Science, Alexandria, Egypt. Soil samples were collected at a depth of 5-10 cm. These strains were identified according to the International Streptomyces Project (ISP) [28].

Target organisms: The following test organisms were used for the bioassay of the antibiotic during the screening experiment: Staphylococcus aureus (209 P FDA), Sarcina leutea (NCIB 495), Bacillus subtilis (ATCC 6051), Escherichia coli (NCIB 1186) and Klepsiella pneumonia (Local isolate). S. aureus was used as a target organism in all other experiments.

Cultivation of Streptomyces violatus for antibiotic production: For studies of antibiotic production, starch-nitrate medium was used as a basal medium. It was composed of (g/l): Starch, 10.0, NaNO3 , 2.5, K2HPO4, 1.0, KH2PO4,1.0 , MgSO4.7H2O, 0.5, KCl, 0.5, trace salt solution 1.0 ml (CuSO4.5H2O (0.64 g/l), FeSO4.7H2O (0.11 g/l), MnCl2.4H2O (0.79 g/l) and ZnSO4.7H2O (0.15 g/l), distilled water,1.0 litre. Medium pH was adjusted to 7.0 before autoclaving using 0.1N NaOH or 0.1 N HCl solution. Fifty-ml aliquots of this medium were dispensed in 250 ml Erlenmeyer flasks. The medium was adjusted to pH 7.0 and sterilised at 121o C for 20 min. Each flask was inoculated with 1.0 ml S. violatus spore suspension obtained from a 6-day-old slant culture. The flasks were then incubated under static conditions at 30o C for 7 days. The antibiotic bioassay was carried out at the end of the incubation period.

Determination of dry weight: The cells were separated from the culture filtrate by

centrifugation at 5,000 rpm for 15 minutes, washed twice with distilled water and then dried at 70o C until reaching a constant weight. Preparation of the crude antibiotic: Following 7 day incubation period, S. Violatus cells were separated from the culture by centrifugation at 5,000 rpm for 15 minutes in a cooling centrifuge at 4o C (Chilspin centrifuge MSE Fisons). The blue-coloured clear supernatant was then tested for its antibiotic activity.

Antibiotic bioassay: This was carried out using the paper-disc diffusion method,

Mueller-Hinton agar as an assay medium and S. aureus as a test organism. The Mueller-Hinton agar (45o C) was poured into sterile Petri-dishes (9 cm diameter) and allowed to solidify. 0.1 ml bacterial suspension (3 x 106 cells) of the test organism was inoculated into the agar surface. Sterile paper discs (6.0 mm diameter Whatman antibiotic assay discs) were placed on the dried surface of the medium using alcohol-flame-sterilised forceps. Each disc received 20 µl of the culture filtrate. Petri-dishes were kept in a refrigerator for 2 hours to allow for the diffusion of the antibiotic. Petri-dishes were then incubated inverted for 18-24 hours at 37o C. The inhibition zone diameter was measured in mm (Amade et al. 1994). The antibiotic concentration (µg/ml) was determined using a standard calibration curve using the purified antimicrobial substance (MSW2000) produced by S. violatus (Said 2001).

Pigment estimation: The blue pigment concentration in the culture broth was estimated colorimetrically at 566 nm. This wave length was selected since it showed a maximum absorption of the coloured supernatant measured in UV VIS 4B Spectrophotometer. Each experiment in this work was repeated three times and the average of the three replicates was taken.

2.2 Results and discusion

Survey of some locally isolated actinomycetes for the production of antibiotic(s). A survey of four locally isolated Streptomyces strains for antibiotic production was carried out in static and shaken cultures (Table 1). It was generally observed that the growth and antibacterial activity obtained in static cultutres were higher than shaken cultures. Streptomyces astreogriseus showed the longest incubation time (12 days) needed to obtain maximum antibacterial activity, while Streptomyces violatus showed a relatively short time (7-days) and produced the highest activity among the tested strains. Streptomyces violatus was also characterised by its broader antibacterial activity, because it affected the growth of all the tested bacteria, showing a stronger activity on S. aureus and B. subtilis. Accordingly, S. violatus was selected for further investigation.

Table 1 - Screening for the antibacterial activity of Streptomyces strains in static (St) and shaken (Sh) cultures.

The growth of S. violatus and the production of antibiotic in a starch-nitrate medium were monitored over a period of 14 days (Fig. 1). The antibiotic production by S. violatus occurred in a growth-phase dependent manner and the highest antibiotic yield was obtained in the late exponential phase and the stationary phase, indicating that it is mainly a product of secondary metabolism. Similar results were observed for streptomycin production in batch cultures of S. griseus [29] when grown in a mineral medium and for the production of candicidin in liquid grown cultures of S. Griseus.The results also showed that S. violatus produced a blue pigment associated with the antibiotic appearance in the culture. It was noticed that a direct tight relationship occurred between the antibiotic production and the intensity of the blue colour formed in the culture (r=0.95). These results may suggest the production of a pigmented antibiotic in S. violatus cultures. The production of the blue-pigmented antibiotic actinorhodin and its physiology are known in S. coelicolor cultures.

Figure 1. Effect of different incubation periods on the production of antibiotic by Streptomyces violatus.

2.2.1 Influence of some cultivation factors on the production of antibiotic

Optimisation of antibiotic production in batch cultures of S. violatus was carried out. This strain was able to grow in all the tested carbon sources (Table 2). However, maximum antibiotic production was obtained in cultures supplemented with glycerol as a sole carbon source followed by cultures containing starch. Cultures containing fructose, maltose, xylose or cellulose did not yield any detectable amounts of the antibiotic. The results also showed that the increase of glycerol level in the culture from 10g/l to 12.5 g/l led to 1.32-fold increase in antibiotic production (Fig 2). The utilisation of glycerol and starch by S. violatus for growth and production of the antibiotic indicates the presence of an active uptake system for these substrates. Glycerol was also found to be used as a sole carbon source by other Streptomyces species.

Figure 2. Effect of glycerol concentration on the production of antibiotic byStreptomyces violatus at different incubation periods: a) 4 days, b) 7 days and c) 10 days.

Table 2 - Effect of different carbon sources on the production of antibiotic by S. violatus.

2.2.2 Influence of nitrogen source

The results revealed that the level of antibiotic production may be greatly influenced by the nature, type and concentration of the nitrogen source supplied in the culture medoium (Table 3). Similar observations have been reported by many investigators. The highest antibiotic production was obtained in cultures of S. Violatus containing sodium nitrate or potassium nitrate as a nitrogen source, followed by cultures containing peptone, alanine, monosodium glutamate or phenylalanine. However, cultures containing asparagine or ammonium citrate did not yield any antibiotic activity and showed lowest growth. The results also showed that the concentration of NaNO3 (Fig. 3) greatly influenced the production of the antibiotic by S. violatus cultures, while the maximum antibiotic yield was obtained in cultures suplemented with 2.5 g/l NaNO3. These results are in partial agreement with those of other investigators. A negative effect of asparagine on the production of cephamycin C was also observed on cultures of S. cattleya, S. latamdurans and Cephalosporium acremonium.

Figure 3. Effect of sodium nitrate (NaNO 3) concentration on the production of antibiotic by Streptomyces violatus at different incubation periods: a) 4 days, b) 7 days and c) 10 days.

Table 3 - Effect of different nitrogen sources on the production of antibiotic by S. violatus.

2.2.3 Influence of potassium phosphate and magnesium sulphate salts

Phosphate is a major factor in the synthesis of a wide range of antibiotics. However, an excessive amount of inorganic phosphate suppresses the production of antibiotics such as tetracycline, actinomycin and candicidin (Kishimoto et al. 1996). The results of the present work (Fig 4) showed that KH2PO4 was not favourable for the production of antibiotic by S. violatus, while K2HPO4 at a concentration of 1g/l yieldes an inhibition zone of 22 mm, equivalent to an antibiotic concentration of 128 µg/ml. It was also observed that addition of a mixture of both phosphate salts (KH2PO4 and K2HPO4) showed the most positive effect on the production of antibiotic by S. violatus. The antibiotic concentration reached its maximum value (245µg/ml) when using a phosphate salt mixture of 1g/l, showing a 1.9-fold and 6.1-fold increase when compared to the highest values obtained when K2HPO4 and KH2PO4 were individually supplied to the medium, respectively. These results are in agreement with those reported by other investigators. The results also showed that addition of 0.5g/l magnesium sulphate to the culture medium was optimal for the production of a maximum yield of antibiotic by S. violatus (Fig 5). At this MgSO4.7H2O concentration, the antibiotic yield was 4.2-fold than that in cultures devoid of magnesium sulphate. The importance of magnesium sulphate for antibiotic production by other Streptomyces species has been reported by several investigators . The effects of magnesium availability are presumably due to requirements of this cation for protein synthesis, and its depletion may restrict enzyme synthesis and activity.

Figure 4. Effect of different (a) KH2PO4 and (b) K2HPO4 concentrations on the production of antibiotic by Streptomyces violatus.

The results also showed that addition of 0.5g/l magnesium sulphate to the culture medium was optimal for the production of a maximum yield of antibiotic by S. violatus (Fig 5). At this MgSO4.7H2O concentration, the antibiotic yield was 4.2-fold than that in cultures devoid of magnesium sulphate. The importance of magnesium sulphate for antibiotic production by other Streptomyces species has been reported by several investigators. The effects of magnesium availability are presumably due to requirements of this cation for protein synthesis, and its depletion may restrict enzyme synthesis and activity (Aasen et al. 1992; mNatsume et al. 1994).

Figure 5. Effect of (MgSO4 .7H2O) concentration on the production of antibiotic by Streptomyces violatus.

2.2.4 Influence of trace elements

The results given in Table 4 showed that iron and manganese could play an important role in the promotion of antibiotic production, the highest dry weight (3.8 mg/ml) was also recorded for manganese. A slight increase in the antibiotic concentration was recorded for Cu, whereas Zn addition lowered the antibiotic concentration compared to the control. The highest antibiotic concentration was achieved in the presence of all elements in the culture medium, yielding a 2.1-fold increase compared to the control reported on the importance of ferrous ions for the growth and antibiotic production by Streptoverticillium rimofaciens. Mansour et al.[30]showed that manganese ions enhanced growth and granaticin production in S. violaceolatus.

Table 4 - The role of trace elements on the production of antibiotic by S. violatus.

3. Protection of workers and life safety

In modern conditions of development of production of a problem in the field of industrial and ecological safety tend to an aggravation. Relevance of a problem of safety of the person and environment is especially sharply shown directly at the enterprises when carrying out technological processes. On trebitel of medicines are interested in receiving qualitative and safe preparations. The workers who are carrying out technological process have to have optimum working conditions.

The main gas emissions in the atmosphere of the enterprises for production of antibiotics containing harmful substances include, except air emissions of all-exchange and local ventilation, technological air emissions at biosynthesis of antibiotics, emissions of boiler and some other auxiliary productions. Various ways of cleaning provide catching about 60% of the harmful substances departing from all sources of pollution.

Gaseous harmful substances consist generally of carbon monoxide (77,4%), sulphurous gas (15,2%) and nitrogen oxides (7,4%).

Vapours of organic solvents making 24,3% of total amount of the thrown-out substances (tab. 3) belong to liquid and gaseous products, specific to production of antibiotics.Besides, at air emissions there is a number of impurity of vapors of various substances making 0,4% of total amount of the liquid and gaseous products released into the atmosphere. Among them chloride hydrogen, vapors of hydrochloric acid, formaldehyde and prevails.

Strong substances, nonspecific for production of antibiotics, in emissions are caught by gas-and-dust cleaning installations for 90%, gaseous emissions of boiler rooms dissipate by means of high pipes. Specific to production of antibiotics firm substances from air emissions for 92,5%, organic solvents - for 10%, 5,4% of the volume of air emissions at biosynthesis of antibiotics are neutralized.

In rooms of storage of finished goods, collecting condensate, preliminary processing of barrels, pump station of reverse water supply, the foreman, the supervising foreman the all-exchange supply and exhaust ventilation is provided. Supply of stitched air and removal of the exhaust is carried out from the top zone, for rooms of packaging of ointment -- from the lower zone. In the stitched P-1 installation external air is cleared of dust in the filter 3 classes, warmed up in the superficial heat exchanger and moistened during the cold period of year, during the warm period -- is only cleared of dust.

Thus, this system of ventilation of air is effective since provides necessary parameters of air for technological process, favorable microclimatic conditions, deletes harmful substances from air of a working zone.

4. Ecological conservation

Ecological factors influencing the effects of antibiotic production were explored experimentally and theoretically. A spatially structured model was used to model the dynamics of antibiotic-producing and nonproducing bacteria in which growth of the nonproducers was reduced by neighbouring antibiotic producers. Various factors affecting spatial interactions between the bacteria were examined for their impact on antibiotic producers. Spatial clustering had a positive impact on the effect of antibiotic production, as measured by the decline in growth of the nonproducing strain, while increasing the initial density of the nonproducing strain had a negative impact. Experiments examined the growth of antibiotic-producing Streptomyces species and a nonproducing, antibiotic-sensitive strain of Bacillus subtilis that were coinoculated on surface media. There was an effect of the Streptomyces on Bacillus growth in some experiments but not in others. In light of the predictions from the model, unintentional clustering of cells is a more likely explanation for this finding than different initial Bacillus densities. The importance of spatial structure seen in this study is consistent with a terrestrial rather than an aquatic distribution of antibiotic-producing bacteria, and may have implications in the search for novel antibiotics.

Over the last 40 years, there has been a steady supply of novel, useful antibiotics produced by microbes isolated from soil and other natural environments. The increased efficiency of screening procedures in the last decade has played a major part in maintaining this supply. However, the selection and sampling of natural environments are still essentially random processes. The main reasons for this are an almost total lack of knowledge of the significance of antibiotics in nature, deficiencies in the taxonomy of antibiotic-producing microbes and its application, and lack of information about the distribution and ecology of known or potential antibiotic producers. The origins of these problems are discussed and some possible solutions are suggested.

A new perspective on the topic of antibiotic resistance is beginning to emerge based on a broader evolutionary and ecological understanding rather than from the traditional boundaries of clinical research of antibiotic-resistant bacterial pathogens. Phylogenetic insights into the evolution and diversity of several antibiotic resistance genes suggest that at least some of these genes have a long evolutionary history of diversification that began well before the `antibiotic era'. Besides, there is no indication that lateral gene transfer from antibiotic-producing bacteria has played any significant role in shaping the pool of antibiotic resistance genes in clinically relevant and commensal bacteria. Most likely, the primary antibiotic resistance gene pool originated and diversified within the environmental bacterial communities, from which the genes were mobilized and penetrated into taxonomically and ecologically distant bacterial populations, including pathogens. Dissemination and penetration of antibiotic resistance genes from antibiotic producers were less significant and essentially limited to other high G+C bacteria.

Conclusion

Antibiotics are biotechnological products that inhibit bacterial growth or kill bacteria. They are naturally produced by microorganisms, such as fungi, to attain an advantage over bacterial populations. Antibiotics are produced on a large scale by cultivating and manipulating fungal cells. Many antibacterial compounds are classified on the basis of their chemical or biosynthetic origin into natural, semisynthetic, and synthetic. Another classification system is based on biological activity. In this classification, antibiotics are divided into two broad groups according to their biological effect on microorganisms: bactericidal agents kill bacteria, and bacteriostatic agents slow down or stall bacterial growth.

Survey of some locally isolated actinomycetes for the production of antibiotic(s). A survey of four locally isolated Streptomyces strains for antibiotic production was carried out in static and shaken cultures. It was generally observed that the growth and antibacterial activity obtained in static cultutres were higher than shaken cultures. Streptomyces astreogriseus showed the longest incubation time (12 days) needed to obtain maximum antibacterial activity, while Streptomyces violatus showed a relatively short time (7-days) and produced the highest activity among the tested strains. Streptomyces violatus [30] was also characterised by its broader antibacterial activity, because it affected the growth of all the tested bacteria, showing a stronger activity on S. aureus and B. subtilis. Accordingly, S. violatus was selected for further investigation.Antibiotics are produced industrially by a process of fermentation, where the source microorganism is grown in large containers (100,000-150,000 liters or more) containing a liquid growth medium. Oxygen concentration, temperature, pHand nutrient levels must be optimal, and are closely monitored and adjusted if necessary.

The effectiveness of individual antibiotics varies with the location of the infection, the ability of the antibiotic to reach the site of infection, and the ability of the microbe to inactivate or excrete the antibiotic. Some anti-bacterial antibiotics destroy bacteria (bactericidal), whereas others prevent bacteria from multiplying (bacteriostatic).Oral antibiotics are simply ingested, while intravenous antibiotics are used in more serious cases, such as deep-seated systemic infections. Antibiotics may also sometimes be administered topically, as with eye drops or ointments.

In the last few years, three new classes of antibiotics have been brought into clinical use. This follows a 40-year hiatus in discovering new classes of antibiotic compounds. These new antibiotics are of the following three classes: cyclic lipopeptides (daptomycin), glycylcyclines (tigecycline), and oxazolidinones (linezolid). Tigecycline is a broad-spectrum antibiotic, while the two others are used for gram-positive infections. These developments show promise as a means to counteract the growing bacterial resistance to existing antibiotics.

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