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Antibiotics LOW-MOLECULAR EFFECTORS OF INITIALLY NATURAL ORIGIN, CAPABLE TO INHIBIT LIVING CELLS GROWTH In the world of antibiotics are produced annually at $ 20 billion. Antibiotics: antimicrobial antineoplastic drugs. The discovery of antibiotics revolutionized the treatment of infectious diseases. Gone idea of incurable bacterial infections (tuberculosis, sepsis, syphilis, and others.). The application of antibiotics: crop, animal husbandry, veterinary medicine, food industry and others. (used more widely than in medicine). Organization of large-scale production of antibiotics has played a crucial role in the development of industrial biotechnology. (Biotechnical period) History 1871 AG Polotebnov Russian dermatologist first described the therapeutic properties of this mold. 1928 A. Flemming - the ability to set a filamentous fungus green mold Penicillium notatum cause the death of microorganisms The amount offered antibiotics is growing. 1940 only 6 known antibiotics, now be described in more 12000 similar compounds including about 200 drugs used in clinic. 97% of the known antibiotics are toxic, however not used in practice. Getting penicillin Alexander Fleming (08.06.1881 – 03.11.1955). Nobel Prise on physiology and medicine with H.Flory and A. Chain in 1945. Since the beginning of the Second World War there was an urgent need for antibacterial agents. In 1941, penicillin was first tested for the treatment of a person suffering from a staph infection. Despite came in the early treatment of short-term improvement, the patient died. The doctors had only 3 grams of penicillin. This treatment was insufficient. For penicillin in an amount sufficient to cure one patient required to process about 1000 l “fungi broth". It was necessary to find more active producer, learn to cultivate it in large quantities and develop a method for isolating penicillin and get it in pure form. This problem was solved in 1943 in the United States: the culture of the fungus was irradiated with X-rays and the best producers were selected. After repeating this procedure more than 20 times a suitable strain producer was obtained . And now continue to select the most promising strains of this fungus. The productivity of the best of them is about 50 g per 1 liter of penicillin nutrient solution. Getting streptomycin Waxman Zelman Abraham (1888 – 1973, Hayenis, USA) Microbiologist. Nobel Prise on Medicine and Physiology at 1952. For some bacteria penicillin harmless. These bacteria true causative agent of tuberculosis Mycobacterium.? However, in 1941, an American microbiologist Z.Waxman isolated from soil microorganisms streptomycetes. These organisms produce a substance streptomycin - extremely active antibiotic acting including the causative agent of tuberculosis. Waxman suggested calling antibiotics all substances produced by microorganisms and to inhibit, damage or kill other microbes. Currently identified about five thousand. Different antibiotics from which the medicine is used in only about 100. Chemical Classification 1. β-lactam (penicillins, cephalosporins) make up more than 50% market share of antibiotics. 2. tetracyclines (tetracycline, morfotsiklin, metatsiklin). 3. Macrolides (erythromycin, oleandomycin). 4. The aminoglycosides (gentamicin, amikacin). 5. Glycopeptides (vancomycin, ristomycin). 6. Amfenikoly (chloramphenicol). 7. lincosamides (lincomycin). 8. Polyene [antifungal (nystatin, levorin)]. 9. Antineoplastic (bleomycin), and others. Other classifications From the spectrum of action: narrow wide action. (tetracyclines, macrolides, aminoglycosides) "+" In the case of unidentified pathogens, "-" Long-term use in patients cause goiter By type of action: 1. bactericidal (lactam, aminoglycosides), causing the death of microorganisms, 2. bacteriostatic (macrolides, tetracyclines, chloramphenicol), impairs the ability of microorganisms to share. By the nature of the impact on the bacterial cell: bacteriostatic (bacteria alive, but unable to replicate) bactericides (bacteria sacrificed but still physically present in the medium), bacteriolytic (bacteria are killed, and bacterial cell walls are destroyed). Mechanisms of action 1) violation of the biosynthesis of peptidoglycan cell wall (penicillins, vancomycin, cephalosporins); 2) violation of the individual stages of the translation process (amphenicols, aminoglycosides, tetracyclines, macrolides, lincosamides); 3) damage to the cytoplasmic membrane (gramicidin, polymyxin); 4) violation biosynthesis of nucleic acids (rifamycins, actinomycin D, antitumor antibiotics); 5) violation of energy metabolism (oligomycin, chlorhexidine). The main stages of microbiological synthesis of antibiotics Cultivation of microorganism-producer Separation of biomass (centrifugation, filtration) Biomass Cell lysis (desintegration) Extraction Separation of extract from disintegrated cells (centrifugation, membrane filtration) Cultural liquid Concentration (ultrafiltration, ion exchange) Purifying of target product (extraction, ion exchange) Dosage form development Manufacturing of antibiotics 1. finding microorganisms (MO) - antagonists in nature and their isolation in pure culture; 2. study of the spectrum of action and determination of antibiotic activity of isolated MO antagonists: 3. Selection of cultivation conditions of antibiotics producers; 4. primary identification of the antibiotic in the early stages of the study: isolation and purification of chemical active principle from the culture medium and cells comparison of the antibiotic in the biological and chemical parameters with known drugs to identify new properties of the substance; 5. study of the mechanism of action and toxicity testing and therapeutic qualities of antibiotics in animals 6. Development of technology for production of the antibiotic in the laboratory and its introduction in industrial production; 7. obtained from the initial strains of new genotypes of MO, with increased activity by mutation and recombination by methods of genetic and cell engineering. Alternative ways of getting new antibiotics 1. chemical or biochemical modification of the antibiotic molecule of the original drug for new properties; 2. directed biochemical modification of the structure obtained by chemical method; 3. chemical synthesis using natural structures as templates; 4. Mutasynthesis: getting of idiotrofs (mutants requiring to form a specific section of its antibiotic molecule (precursor); getting of a mutasinton (analogue of predecessor) by chemical methods; idiotrofs cultivation in a medium containing mutasinton. Thus idiotrof molecule comprises mutasinton antibiotic produced by it. The result is a new mutasynthetic structure; 5. hybridization (producer -genetic hybrid); antibiotic hybrid structure may contain two different metabolites - a product of combination of genes. The main stages of production of hybrid antibiotics: 1. choice of producer, forming a well-known antibiotic; 2. finding a new MO for hybridization; 3. study the biochemical pathways of synthesis of the antibiotic intermediates and enzymes; 4. identification of genes that control the formation of biosynthetic enzymes and regulators; 5. providing a recombinant DNA sequence comprising a gene for biosynthesis favorable; 6. cloning the genetic structure of the new culture of the recipient; 7. chemical, microbiological and pharmacological study of a new antibiotic. Ways to control metabolic processes of a producer The purpose of biotechnology - Based on the understanding of the physiological and genetic properties of the producer to obtain the maximum yield of the end product. changes in the composition of the nutrient medium; changing ambient conditions (temperature, pH, aeration); design bioreactor (fermentor); regulation introducing additional substrate; fixation of the physiological state of the culture using the method of continuous culture; use of genetically modified strains of a producer. Producers of antibiotics Producers called organisms that serve as source of any substances used by man. Sources of antibiotics: actinomycetes, fungi; bacteria, higher plants; tissues of animals and fish. penicillin - some strains of Penicillium notatum and P. chrysogenum, streptomycin - a certain strain of Streptomyces griseus, whereas other strains of the same species did not produce any antibiotics, or produce, but other. Some microorganisms secrete not one, but several antibiotics. Pseudomonas aeruginosa forms piotsianazu, pyocyanin, piolipoevuyu acid and other piosoedineniya; Bacillus brevis produces gramicidin and tirotsidin; P. notatum - penicillin and penatin; Streptomyces rimosus - oxytetracycline and rimotsidin; Streptomyces aureofaciens - chlortetracycline and tetracycline. One and the same antibiotic can be produced by microorganisms of various kinds. Gliotoksin form and Gliocladium species Trichoderma, Aspergillus fumigatus, and others. Different strains or microorganisms may produce different chemical forms of the same antibiotic, for example penicillins or various different forms of streptomycin. Non-spore bacteria Bacillus pyocyaneus or Pseudomonas aeruginosa - pyocyanin, piotsianaza, piolipoevaya acid. Different strains of E. coli (Escherichia coli) - colicin, Spore-forming bacteria. Aerobic spore-forming Gram-positive bacteria (Bacillus) relate to the actual bacteria. Like all bacteria not have a nucleus, the genome is more simple, i.e. contains less than the number of genes in actinomycetes, have mitochondria, the cell wall consists of peptidoglycan component. The life cycle of the bacteria around one and a half days. strains of Bacillus subtilis produce bacitracin, subtilin, etc .; B. brevis -gramitsidin C tirotsidin (tyrothricin); B. polimixa (B. aerosporus) - polymyxin (aerosporin). B. mycoides, B. mesentericus and B. simplex marked variety, still insufficiently studied compounds: batsillin, kolistatin and others. Many of them inhibit the growth of fungi. Actinomycetes are prokaryotes, the genome is not enclosed in the core, and represents a ring chromosome, is not separated from the cytoplasm by a nuclear membrane, do not contain mitochondria, the cell wall of the heteropolymer - peptidoglycan. All this brings actinomycetes bacteria. Unlike "true" bacteria (eubacteria) - multicellular organisms with complex life cycle, usually within 5-6 days. Actinomycetes form sporophores and spores. Mitsetin (Krasil'nikov Korenyako, 1939). streptomycin, tetracycline, erythromycin, novobiocin, neomycin, and others. Aspergillus flavus- penicillin and aspergilovuyu acid; A.fumigatus-fumigatin, spinulozin, fumigatsin (gelvolevuyu acid) and glitoksin; Streptomyces rimosus- oxytetracycline and rimotsidin; S.aurofaciens-chlortetracycline and tetracycline. One and the same A / B can be produced by various kinds of microorganisms. Moulds -mnogokletochnye organisms with complex life cycle, form different kinds of mycelium, sporophores disputes and others. Morphological education. producers betalaktamov: Penicillium chrysogenum - benzylpenicillin P.notatum - penicillin and penatin; Acremonium chrysogenum - cephalosporin C Fusidium coccineum - fusidic acid (an antibiotic steroid structure) most important - griseofulvin, mycophenolic acid, gliotoksin, klavatsin, aspergillovaya acid, and many others The main part of fungal antibiotics have not yet found practical applications mainly because of their high toxicity. Algae. Many algae are capable of producing a substance having antibiotic properties, but so far none of them have found clinical application. Lichens - lihenin and usnic acid. Higher plants - antibacterial agents, similar in its properties to the true antibiotics volatile - allicin, Tomatin and others. Allicin - garlic (Allium sativum), (Kavallito, 1944) - extracted with organic solvents and purified by steam distillation. Garlic contains alliin (odorless garlic and has antibiotic properties) is transformed into allicin under the influence of enzyme allinnazy contained in the juice of garlic. Allicin inhibits the development of gram + and gram bacteria, the tubercle bacillus. Allicin has relatively high toxicity. Lethal dose if it intravenously to mice is 60 mg / kg, sc - 120 mg / kg. The high toxicity of allicin and instability of the drug makes it impossible to use in medicine. Aqueous extracts of the seeds of radish (Raphanus sativum) contain antibiotic rafanin; radish roots and leaves do not contain antibiotic. Rafanin was isolated in 1947, 1 kg of seeds is possible to obtain 3 g of pure antibiotic. Rafanin like allicin, are not found in the seeds as a free compound, and a proantibiotika; converted into rafanin under the action of the enzyme contained in the seeds of radish. Rafanin inhibits Gram + and Gram- bacteria in a concentration of 40-200 ug / ml. Low concentrations of antibiotic delayed germination of seeds of many plants, other than seeds of radish. Pets. Among the products of animal origin, has antibacterial properties, occupies an important place lysozyme. Lysozyme is found in egg white, spleen, heart, liver, lung, in various secretions (tears, nasal mucus: saliva et al.), In the juices of certain plants, microorganisms and bacteriophages. More fully studied lysozyme derived from egg white. Currently crystalline lysozyme prepared from egg white directly by adsorption on bentonite clay. With clay lysozyme eluted with 5% aqueous pyridine at pH 5, and then the enzyme was precipitated with ammonium sulfate, dialyzed and freeze-dried. Hen egg lysozyme protein is active against Gram + bacteria (Bacillus, Micrococcus, Streptococcus, Sarcina). Less sensitive to lysozyme Gram bacteria. The action of lysozyme on microorganisms sensitive to lysis of cells thereto, due primarily to the action of the substance on the cell walls, which under the influence of the antibiotic, and thus break occurs "pouring" of the cytoplasmic contents of the cells. Lysozyme does not exhibit toxic properties in relation to human or animal, on the contrary, he, like bio-stimulants, activates protective properties makroorgannzma. In animals, lysozyme has a protective function against penetration saprophytic and pathogenic microorganisms. Lysozyme studied and continues to be studied as a therapeutic factor used in infectious diseases, dermatology, ophthalmology, surgery, and when applied to malignant tumors. From the tissues of animals and fish (erythrite, ekteritsid). ability to synthesize antibiotics is a useful tool for the species, develop and enforceable in the evolution of organisms. The production of antibiotics - one of the factors that gives certain advantages microorganism-antagonist in the fight for survival in complex natural microbial associations - one of the mechanisms of adaptation. Antagonism may be due to microbes and other substances and adaptive mechanisms that are not connected with the formation of chemical compounds. All this can also contribute to a wide spread of germs that have not identified the ability to synthesize antibiotics. in vitro, in isolated growing actinomycetes (outside the natural ecological community) on artificial media, is not always possible to identify the ability to synthesize an antibiotic. That is inactive in vitro actinomycetes strains are capable of the biosynthesis antibiotic substances produced by microorganisms, are random, depending only on the conditions of cultivation (Z.Vaksman, X.Leshevale) because antibiotics are not formed by all widely spread of germs and antibiotics are rapidly inactivated in the soil. The literature contains numerous data on the availability of antagonism between microbes in the soil due to the release of antibiotics. In the micro wherein the antibiotic is present (in the ground), it certainly affects the microbes in contact with it. Indeed, in the process of destruction of the soil under the influence of antibiotics of various physico-chemical factors (pH, the presence of colloids, etc.), And inactivation of specific enzymes formed by microbes. But this does not prove the absence of action of antibiotics on microbes in the soil. Inactivation of antibiotics by microorganisms - a natural reaction of a living organism to the harmful effects of the environment. In response to antibiotics microorganisms produce different forms of protection. In any case, the final result of this interaction will be determined by: the speed of propagation, the intensity of synthesis of antibiotics and inactivating substances capable of fuller use of the substance of the environment for life, the degree of resistance to adverse physical and chemical environmental conditions, and so on. Isolation and selection methods Isolation producers of antibiotics can be produced from a wide variety of substrates: soil, decaying plant and animal residues, sludge, water, lakes and rivers, air and other sources. However, the most rich in microorganisms that produce antibiotics, soil. Up much of the isolated organisms and antibioticproducing substances. To isolate microorganisms - producers of antibiotics from natural habitat used a large number of different methods: Seeding soil suspension in water on the surface of an agar plate. Certain weighed soil is pounded in a mortar with a small amount of water, quantitatively transferred to a flask with sterile water. The flask contents were shaken for 5 imaginary, and then an aqueous slurry is made of a number of successive dilutions, which were plated onto appropriate agar medium. For further separate pure cultures of colonies after incubation in an incubator at a desired temperature, the tubes were subcultured into nutrient agar with bevelled. Each pure culture of the microorganism replated on different composition of the medium, and after a fairly good development verified its antibiotic properties. Sowing soil nutrient agar previously seeded test organism. The surface of nutrient agar seeded with the required test organism culture and then spread out on an agar plate are small, no more millet grains, lumps of soil or applied to the soil as dusts, spreading it over the entire surface of the plate. The plates were then placed in an incubator and after a certain period of time (24-48 hours and sometimes more) pieces of viewing soil or individual portions thereof, which are formed around the zone of delay of growth of the test organism. From these plots pure cultures of organisms isolated and subjected to further study. The method of soil enrichment. The soil from which will be allocated antagonists, enriches the body of species in relation to which want to get antagonist. To this end, the soil samples placed in turn in glass vessels systematically added The washed slurry relevant microorganisms. Then, at regular intervals, such soil is planted in separate lumps on agar plates in petri dishes previously inoculated with the same organism that has been used to enrich the soil. Centrifugation method of soil suspension To isolate actinomycetes from soil, and especially of the soil in the spring, when it develops a large number of fungi and bacteria, the method of centrifugation soil slurry. The method is based on the difference in sedimentation rate of certain types of microorganisms in a centrifugal field. At 3000 rev / min for 20 min particle size corresponding mold spores or bacteria cell type you. mesentericus, you. mycoides, you. subtilis, deposited on the bottom of the tube. Particles of the same size corresponding disputes actinomycetes are spinning at a given speed of the liquid in the surface layer. Inoculating the supernatant, it is possible in most cases (92%) available on nutrient agar plates only colonies actinomycetes. The method of freezing and thawing of the soil. Selected for the isolation of actinomycetes soil sample is placed in a domestic refrigerator evaporator at a temperature of -8 °. After one hour the sample is removed from the refrigerator and kept at room temperature until completely thawed. Freeze-thaw procedure was repeated twice. Then, a sample of soil was placed in sterile tap water, the suspension is shaken for 15 min on a rotary shaker at 230 rev / min, after which the different dilutions of the suspension were plated on nutrient agar plate in a Petri dish. Method of freezing and thawing of the soil can detect them in 1,23,6 times actinomycetes than in the same samples without freezing. This is apparently due to the increase in desorption from the surface of soil actinomycetes particles. Use of nutrient media containing antibiotics. When planting soil suspension on agar plates created difficulties for the development of rare species of actinomycetes as a result of the rapid development of bacteria and widespread species of actinomycetes in soil. Therefore, for the purposes of directional allocation of certain groups of microorganisms in the environment for seeding soil suspezii add various antibiotics. When adding antibiotics to the culture medium of microorganisms is suppressed normal microflora, the conditions for the development of resistance to these antibiotics forms of microbes; the latter may be new or rare species capable of forming new antibiotics. Actinomycetes used for the isolation medium containing in their structure the antibiotics such as tetracycline, neomycin, nystatin, streptomycin, chloramphenicol, penicillin, and others. In allocating producing new antibiotic substances used medium containing streptomycin at concentrations ranging from 25 to 100 micrograms / ml and rubomycin - from 5 to 20 micrograms / ml. According to some microbiologists, to date, isolated and studied no more than 10 percent of all existing in the nature of microorganisms. It is therefore necessary to explore and develop new methods of isolation of microbes, which could contribute to their maximum detection in nature. The main objective of the first rounds of antibiotic substances - the concentration of biologically active compounds and removal of associated fiber. The main methods of isolation from native antibiotic solution (a culture liquid, freed from the biomass producing) include: antibiotic precipitation, extraction methods antibiotics ogranicheskimi solvents sorption methods using surfactants (activated carbon, activated alumina and others.) Or ion-exchange materials (ion exchange resins). Antibiotic isolated from one of these methods represent only technically pure drug, but more may be used in medical practice. Further purification of the preparation is carried out or by readsorption, recrystallization, dissolution in organic solvents, antibiotics, or other methods. After antibiotic substance using either method is well isolated and purified, its biological activity is checked against a broad range of micro-organisms (antimicrobial spectrum). Furthermore, the antibiotic tested for sterility, toxicity, pyrogenicity, tested in regard to actions of white blood cells and determining other metrics. The aim of clarifying sterility of the finished product - the establishment of its lack of microorganisms and spores, especially pathogenic. For this purpose, if it is possible to inactivate the antibiotic substances, and then seeding it to produce a variety of nutritional composition of the medium (meat-peptone broth, liver broth, blood agar and m. P.). In Gram + cell wall comprises peptidoglycan, polysaccharides, teichoic acid (composed of sugars, sugar alcohols, amino acids and phosphoric acid). Teichoic acid and polysaccharides are connected with the frame walls - murein. Using electronic images of thin slices (lamination) in the walls of gram-positive bacteria are not detected. ? In the cell wall of Gram-more complex, contains significant amounts of lipids (fats), associated with proteins and sugars in complex systems - lipoproteins and lipopolysaccharides. Murein in the bacterial cell wall of Gram generally less than that of Gram + bacteria. Using an electron microscope, it was found that the multilayer walls of the bacteria Penicillin is widely used in the treatment of staphylococcal infections - osteomyelitis, infectious arthritis, pneumonia, bronchitis, empyema, endocarditis, boils, laryngotracheitis, mastitis, meningitis, otitis media, peritonitis, infected wounds and burns, septicemia, sinusitis, tonsillitis, and many other diseases. His successfully used in a variety of infections caused by hemolytic streptococci and anaerobic, pneumococci, gonococci, meningococci, anaerobic clostridia (gas gangrene agents), diphtheria bacillus, anthrax, spirochetes, and many other bacteria. However, in mixed infections caused by gram-negative bacteria, as well as malaria, tuberculosis, viral infections, fungal and other diseases penicillin ineffective. Toxic effects of penicillin appears mainly in the form of allergic reactions (even at the lowest dose) and seizures (administered very large doses). Cephalosporins chemical structure similar to penicillin, but resistant to beta-lactamase. Therefore, highly active against bacteria coliform (rod-shaped bacteria such as gram-negative Escherichia coli) .Currently obtained a large number of cephalosporins, among them - are used in the clinic cephalothin, cefazolin, cephalexin, cefamandole, defoksitin and ceftriaxone. Streptomycin is used in many infections. This is an effective treatment for meningitis, endocarditis, laryngotracheitis, as well as diseases of the urinary tract and the lungs caused by a bacillus Pfeiffer (Hemophilus influenzae). Treatment with streptomycin lend themselves well meningitis, pneumonia and urinary tract infections, if the cause of these diseases are sensitive to it strains of Escherichia coli, Proteus vulgaris, Klebsiella pneumoniae (Friedlander bacillus), Aerobacter aerogenes and Pseudomonas. Chloramphenicol and tetracyclines They are effective when taken orally (p.o.) and are widely used in many infectious diseases caused by bacteria and some large viruses. Such diseases include typhoid fever, various forms of typhus, spotted fever, gonococcal infection, syphilis, brucellosis, urinary tract infection, lymphogranuloma venereum, and many others. These antibiotics are also effective in most diseases for which treatment is shown penicillin, and are often prescribed for penicillin-resistant infections and in those cases where preferred oral therapy.. Erythromycin and novobiocin Erythromycin and other antibiotics (e.g., carbomycin, oleandomycin) having a special (macrolide) the chemical structure and novobiocin have a broad spectrum of action - approximately the same as that of penicillin, but also include some Gram-negative bacteria. Their advantage lies in the possibility of ingestion and low toxicity; they are relatively rarely cause gastrointestinal disturbances.