Kretsu Anna PFUR
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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.