Phages can be used for practical purposes. The use of bacteriophages in medicine and beyond

The achievements of modern medicine and pharmaceuticals are great, but pathogenic microorganisms are also constantly improving and adapting to the action of those drugs that were deadly to them just a couple of years ago. Where antibiotics are powerless, bacteriophages will help fight pathogenic microorganisms.

What are bacteriophages

Literally translated from ancient Greek, bacteriophages are bacteria eaters. This biological term refers to viruses that selectively infect bacterial cells.

Bacteriophages are present wherever bacteria live, so their habitat can be air, water, soil, the human body, food, and clothing.

Structural features of a bacteriophage: this virus does not have cellular structure, there is only genetic material covered with a protein coat on top. Therefore, they have to look for suitable cellular microorganisms to reproduce.

The phage begins its destructive activity for the bacterium by injecting its own genetic information into its body, and then begins active reproduction. When a bacterial cell is destroyed, from 100 to 200 new bacteriophages emerge through its fragments, which immediately begin to infect nearby bacteria.

Kinds

The most famous bacteriophages:

• dysenteric;
• staphylococcal;
• streptococcal;
• potassium;
• pseudomonas;
• Pseudomonas aeruginosa.

Advantages

Some scientists argue that soon the use of drugs based on bacteriophages will compete with the use of antibiotics in the treatment of a variety of diseases.

The basis for this bold assumption is provided by the following advantages of using phages:

• absence of addiction and contraindications to the use of the drug;
• no inhibitory effect on the immune system;
• selective action (beneficial bacterial flora remains intact);
• harmonious combination with other methods of treatment, including therapy with antibiotics (according to research results, phages even enhance their effect);
• a pronounced effect in the treatment of sluggish painful conditions caused by bacterial agents that are insensitive to antibiotics.

This allows the bacteriophage to be successfully used for children, the elderly, pregnant women, and debilitated patients.

Indications

Indications for including bacteriophages in the treatment regimen are the following infections:

• surgical (abscess, panaritium, paraproctitis, osteomyelitis, boils, burns, phlegmon, carbuncles, purulent wounds);
• urogenital (cystitis, pyelonephritis, colpitis, urethritis, endometritis, salpingoophoritis);
• enteral (cholecystitis, gastroenterocolitis, intestinal dysbiosis);
• blood poisoning;
• diseases of the ENT organs (tonsillitis, sinusitis, otitis media);
• diseases respiratory tract and lungs (tracheitis, pleurisy, laryngitis, bronchitis, pneumonia).

Methods of application

The method by which the bacteriophage should be used directly depends on the nature and location of the source of inflammation. IN different situations The following methods of application would be appropriate:

• orally ( medicinal product taken orally);
• rectally (bacteriophage enema);
• locally (in the form of washing, lotions, irrigation, instillation, rinsing, administration of turundas soaked in the drug).

The bacteriophage acts more effectively if the treatment combines different methods of application. There are certain clinical indications, according to which bacteriophage is taken orally in tablets, and local action provides liquid bacteriophage in the form of a lotion.

Preparations based on bacteriophages, produced in the form of solutions, aerosols, tablets, suppositories and gels, are gaining popularity. Pharmacy forms drugs are provided with detailed instructions on how to take the bacteriophage.

Contraindications

Most people with a certain degree of mistrust consider the possibility of treatment with bacteriophages, although the effectiveness and, most importantly, safety of such therapy has already been proven.

The only one possible contraindication May be increased sensitivity to bacteriophages, although cases allergic reaction bacteriophages are not typical.

Bacteriophage preparations

The pharmaceutical industry offers many drugs whose principle of action is based on the antimicrobial activity of bacteriophages.

• Intesti-bacteriophage (Intestiphage)

  

  Liquid immunobiological antimicrobial preparation. It inhibits the activity of microorganisms, causing diseases gastrointestinal tract(bacterial dysentery, typhoid fever, enterocolitis, paratyphoid fever, dysbacteriosis, salmonellosis). It is used internally and as an enema. Contraindications: hypersensitivity to the drug. Side effects: In newborns, skin rashes and regurgitation are possible in the first 2 days of use.

• Pyobacteriophage polyvalent (Sextaphage)

  

  Copes successfully with purulent-septic diseases newborns and infants, purulent-inflammatory diseases of the ENT organs, enteral infections. Used to treat newly infected wounds. There are no contraindications or side effects.

• Bacteriophage Klebsiella pneumoniae (Klebsifag)

  

  It affects bacteria that cause pneumonia, ozena, and rhinoscleroma. Also helps with generalized septic conditions, to prevent contamination nosocomial strains Klebsiella There are no side effects. Contraindication: hypersensitivity to the components.

• Salmonella bacteriophage

  

  Destroys Salmonella cells and microorganisms similar in antigenic structure. Suitable for the treatment of salmonellosis in children and adults. There are no contraindications or side effects.

• Pseudomonas aeruginosa bacteriophage (Pseudomonas aeruginosa)

  

  Used for treatment of lesions various organs Pseudomonas aeruginosa. Side effects not identified. Contraindication: hypersensitivity to the drug.

• Bacteriophage streptococcal (Streptophage)

  

  Kills streptococcal bacteria, which makes drugs based on it indispensable in the treatment of sore throat, tonsillitis, sinusitis, panaritium, festering wounds and many other ailments. To treat sinusitis, it is recommended to instill this bacteriophage into the nose. There are no side effects. Contraindication: hypersensitivity to the drug.

• Bacteriophage coli

  

  Has a specific antibacterial effect, directed exclusively against pathogenic strains of Escherichia coli. Prescribed for lesions of the gastrointestinal tract, suppuration of wounds, sepsis of newborns, conjunctivitis, urogenital infections. Contraindication: hypersensitivity to the drug. No side effects were identified.

• Bacteriophage Klebsiella polyvalent

  

  Effective in the treatment of peritonitis, pleurisy, purulent-inflammatory diseases in gynecology. It is also used in the treatment of stomatitis, periodontitis and inflammation of the sinuses. There are no side effects. Contraindication: hypersensitivity to the components of the drug.

• Coliproteus bacteriophage

  

  IN liquid form in demand for the prevention and treatment of colpitis and enterocolitis. In tablet form, it is more often used for advanced forms of pyelonephritis and cystitis, inflammatory processes in the pelvic organs. Contraindication: allergy to any of its components of the drug. There are no side effects.

• Dysenteric bacteriophage

  

  Used for the treatment and prevention of dysentery. No side effects were identified. Contraindications: hypersensitivity to the components, and for the tablet form of the drug - the patient’s age is less than 1 year, pregnancy and breastfeeding.

Do not exaggerate the danger of viruses included in similar drugs and bacteriophage analogues. They are only deadly to bacteria that cause disease. If the doctor considers it appropriate to include bacteriophages in the treatment regimen, you should trust and be prepared for a speedy recovery.

The use of bacteriophages is carried out exclusively for the intended purpose and under the supervision of the attending physician.

Bacteriophages or phages (from other Greek φᾰγω “I devour”) are viruses that selectively infect bacterial cells. Most often, bacteriophages multiply inside bacteria and cause their lysis. Typically, a bacteriophage consists of a protein shell and genetic material of single- or double-stranded nucleic acid (DNA or, less commonly, RNA). The total number of bacteriophages in nature is approximately equal to the total number of bacteria (1030 – 1032 particles). Bacteriophages actively participate in the circulation chemical substances and energy, have a noticeable effect on the evolution of microbes and bacteria. The structure of a typical bacteriophage myovirus.

Structure of bacteriophages 1 - head, 2 - tail, 3 - nucleic acid, 4 - capsid, 5 - “collar”, 6 - protein sheath of the tail, 7 - tail fibril, 8 - spines, 9 - basal plate

Bacteriophages differ in chemical structure, type of nucleic acid, morphology and nature of interaction with bacteria. To size bacterial viruses hundreds and thousands of times smaller than microbial cells. A typical phage particle (virion) consists of a head and a tail. The length of the tail is usually 2-4 times the diameter of the head. The head contains genetic material - single-stranded or double-stranded RNA or DNA with the enzyme transcriptase in an inactive state, surrounded by a protein or lipoprotein shell - the capsid, which stores the genome outside the cell. The nucleic acid and capsid together make up the nucleocapsid. Bacteriophages may have an icosahedral capsid assembled from multiple copies of one or two specific proteins. Typically, the corners are made of pentamers of a protein, and the support of each side is made of hexamers of the same or similar protein. Moreover, phages can be spherical, lemon-shaped or pleomorphic in shape. The tail, or appendage, is a protein tube - a continuation of the protein shell of the head; at the base of the tail there is an ATPase that regenerates energy for the injection of genetic material. There are also bacteriophages with a short process, without a process and filamentous.

Taxonomy of bacteriophages The large number of isolated and studied bacteriophages determines the need for their systematization. This is done by the International Committee on Taxonomy of Viruses (ICTV). Currently, according to International classification and the nomenclature of viruses, bacteriophages are divided depending on the type of nucleic acid and morphology. On this moment Nineteen families are distinguished. Of these, only two are RNA-containing and only five families are enveloped. Of the families of DNA viruses, only two families have single-stranded genomes. Nine DNA-containing families have a circular DNA genome, while the other nine have linear DNA. Nine families are specific only to bacteria, the remaining nine are only specific to archaea, and (Tectiviridae) infects both bacteria and archaea

Interaction of a bacteriophage with bacterial cells Based on the nature of the interaction of a bacteriophage with a bacterial cell, virulent and temperate phages are distinguished. Virulent phages can only increase in number through the lytic cycle. The process of interaction between a virulent bacteriophage and a cell consists of several stages: adsorption of the bacteriophage on the cell, penetration into the cell, biosynthesis of phage components and their assembly, and release of bacteriophages from the cell. Initially, bacteriophages attach to phage-specific receptors on the surface of the bacterial cell. The phage tail, with the help of enzymes located at its end (mainly lysozyme), locally dissolves the cell membrane, contracts, and the DNA contained in the head is injected into the cell, while the protein shell of the bacteriophage remains outside. Injected DNA causes a complete restructuring of the cell's metabolism: the synthesis of bacterial DNA, RNA and proteins stops. The bacteriophage's DNA begins to be transcribed using its own transcriptase enzyme, which is activated after entering the bacterial cell. The early ones are synthesized first, and then the late ones. RNAs that enter the ribosomes of the host cell, where early (DNA polymerases, nucleases) and late (capsid and tail proteins, enzymes lysozyme, ATPase and transcriptase) bacteriophage proteins are synthesized. Bacteriophage DNA replication occurs according to a semi-conservative mechanism and is carried out with the participation of its own DNA polymerases. After the synthesis of late proteins and the completion of DNA replication, the final process begins - the maturation of phage particles or the combination of phage DNA with the envelope protein and the formation of mature infectious phage particles

Life cycle Moderate and virulent bacteriophages at the initial stages of interaction with a bacterial cell have the same cycle. Adsorption of bacteriophage on phage-specific cell receptors. Injection of phage nucleic acid into a host cell. Co-replication of phage and bacterial nucleic acid. Cell division. Further, the bacteriophage can develop according to two models: lysogenic or lytic path. Temperate bacteriophages after division are in a state of prophase (lysogenic pathway). Virulent bacteriophages develop according to the lytic model: The nucleic acid of the phage directs the synthesis of phage enzymes, using the protein synthesizing apparatus of the bacterium. The phage in one way or another inactivates the host DNA and RNA, and the phage enzymes completely break it down; The RNA of the phage “subordinates” the cellular apparatus for protein synthesis. The phage nucleic acid replicates and directs the synthesis of new envelope proteins. New phage particles are formed as a result of spontaneous self-assembly of the protein shell (capsids) around the phage nucleic acid; Lysozyme is synthesized under the control of phage RNA. Cell lysis: the cell bursts under the influence of lysozyme; about 200-1000 new phages are released; phages infect other bacteria.

Application In medicine One of the areas of use of bacteriophages is antibacterial therapy, an alternative to taking antibiotics. For example, bacteriophages are used: streptococcal, staphylococcal, klebsiella, dysentery and polyalent, pyobacteriophage, coli, proteus and coliproteus and others. 13 are registered and used in Russia medical supplies based on phages. Currently they are used to treat bacterial infections, which are not sensitive to traditional treatment antibiotics, especially in the Republic of Georgia. Typically, the use of bacteriophages is accompanied by greater success than antibiotics where they are present. biological membranes, coated with polysaccharides, through which antibiotics usually do not penetrate. Currently, the therapeutic use of bacteriophages has not received approval in the West, although phages are used to kill bacteria that cause food poisoning, such as Listeria. With many years of experience in the amount of large city And rural areas the unusually high therapeutic and prophylactic effectiveness of the dysentery bacteriophage has been proven (P. M. Lerner, 2010). In Russia, therapeutic phage preparations have been made for a long time; they were treated with phages even before antibiotics. IN last years phages were widely used after the floods in Krymsk and Khabarovsk to prevent dysentery.

In biology, bacteriophages are used in genetic engineering As vectors that transfer DNA sections, natural gene transfer between bacteria through certain phages (transduction) is also possible. Phage vectors are usually created on the basis of the temperate bacteriophage λ, containing a double-stranded linear DNA molecule. Left and right shoulders phage have all the genes necessary for the lytic cycle (replication, reproduction). middle part The genome of the bacteriophage λ (contains genes that control lysogeny, that is, its integration into the DNA of the bacterial cell) is not essential for its reproduction and is approximately 25 thousand nucleotide pairs. This part can be replaced with a foreign DNA fragment. Such modified phages undergo a lytic cycle, but lysogeny does not occur. Bacteriophage λ vectors are used to clone eukaryotic DNA fragments (that is, larger genes) up to 23 thousand nucleotide pairs (kb). Moreover, phages without inserts are less than 38 kb. or, on the contrary, with too large inserts - more than 52 kb. do not develop or infect bacteria. Since bacteriophage reproduction is only possible in living cells, bacteriophages can be used to determine the viability of bacteria. This direction has great prospects, since one of the main issues in various biotechnological processes is determining the viability of the crops used. Using the method of electro-optical analysis of cell suspensions, the possibility of studying the stages of phage-microbial cell interaction was shown

And also in veterinary medicine for: prevention and treatment of bacterial diseases of birds and animals; treatment of purulent-inflammatory diseases of the mucous membranes of the eyes and oral cavity; prevention of purulent-inflammatory complications in burns, wounds, surgical interventions; in genetic engineering: for transduction - natural transmission genes between bacteria; as vectors transferring DNA sections; using phages, it is possible to engineer targeted changes in the host DNA genome; V Food Industry: ready-to-eat meat and poultry products are already being processed en masse with phage-containing agents; bacteriophages are used in the production of food products from meat, poultry, cheese, plant products, etc.;

V agriculture: spraying phage preparations to protect plants and crops from rotting and bacterial diseases; to protect livestock and poultry from infections and bacterial diseases; For environmental safety: antibacterial treatment of seeds and plants; cleaning premises of food processing enterprises; sanitization of workspace and equipment; prevention of hospital premises; carrying out environmental activities

Thus, today bacteriophages are very popular in the lives of humans and animals. At the enterprises it is planned whole line priority areas for the development and production of therapeutic and prophylactic bacteriophages, which correlate with newly emerging global trends. New drugs are being created and introduced to treat many diseases. The study and use of bacteriophages is carried out by bacteriologists, virologists, biochemists, geneticists, biophysicists, molecular biologists, experimental oncologists, specialists in genetic engineering and biotechnology

Bacteriophages in medical practice used in diagnosis, treatment and prevention infectious diseases.

A. In diagnostics, bacteriophage is used when carrying out a cultural research method to determine the type of isolated pure culture, also for its typing. The method described below for using a bacteriophage to indicate the presence of a certain type of bacteria in a pathological material without isolating it in a pure culture has not become widespread.

1. The reaction of increasing phage titer is based on the ability of a specific bacteriophage to replicate only in the cells of bacteria of “its own” species. It is carried out according to the following principle. A certain amount of a specific bacteriophage is added to the pathological material, it is incubated in a thermostat, and then the amount of phage is again determined. If it has increased, it means that the bacteriophage has “found” a cell of “its” species to replicate, therefore, bacteria of the desired species are present in the pathological material.

2. In the process of identifying a pure culture, species and type bacteriophages are used.
A. Species-specific bacteriophages are used for phage indication. The isolated pure culture is inoculated onto a plate agar and a drop of a specific bacteriophage is dropped onto it. If the culture belongs to the desired species, then there will be no growth at the site where the drop is applied, otherwise phage will be observed at the site where the drop is applied. bacterial growth. Sometimes, after applying the bacteriophage, the Petri dish containing the agar plate is tilted, allowing the droplet to drain into the edge of the dish (which is why this method is called “drip dripping”).

b. Typical bacteriophages are used for phage typing. The principle of the method is as follows.
1. The strain to be typed is inoculated onto plate agar.
2. Then drops of typical bacteriophages are dropped onto the inoculated surface (each into its own square, marked in advance, for example, with a glass graph at the bottom of a Petri dish).
3. The inoculated dish is incubated in a thermostat.
4. Take into account the experience by recording “sterile spots” or “plaques” - places of lack of growth at the site of application of a drop of the bacteriophage to which it is sensitive this option bacteria.
5. Phagovar (phagotype) is designated by listing the typical phages that lyse a given variant.
B. The use of bacteriophages (usually species) for treatment is referred to as phage therapy. For the purpose of treatment, bacteriophages are used topically (in the form of irrigation of the affected surface, injection into a local focus pathological process etc.), since their administration by the parenteral route leads to the development of an immune response to the foreign phage protein. If a therapeutic bacteriophage is administered orally (to treat intestinal infections), then it is best to use a tablet form of the drug, coated with an acid-resistant coating that dissolves in the alkaline environment of the intestine - bacteriophages are very sensitive to low pH and are quickly inactivated in the acidic environment of the stomach.
B. Phage prophylaxis is the use of a bacteriophage (also, as a rule, a specific one) to prevent the development of a bacterial infection. Currently used for emergency prophylaxis typhoid fever and dysentery (under emergency prevention refers to a set of measures to prevent the development of the disease after the act of infection has occurred, i.e. entry of the pathogen into the patient's body).

For the first time, the assumption that bacteriophages are viruses was made. D. Errel. Subsequently, fungal viruses, etc., were discovered and became known as phages.

Phage morphology.

Dimensions - 20 - 200nm. Most phages are tadpole-shaped. The most complex phages consist of a multifaceted head, in which the nucleic acid is located, a neck and processes. At the end of the process there is a basal plate, with threads and teeth extending from it. These threads and teeth serve to attach the phage to the bacterial membrane. In the most complexly organized phages, the distal part of the process contains the enzyme - lysozyme. This enzyme promotes the dissolution of the bacterial membrane during the penetration of phage NK into the cytoplasm. In many phages, the process is surrounded by a sheath, which in some phages can contract.

There are 5 morphological groups

1. Bacteriophages with a long process and a contractile sheath
2. Phages with a long process but no contractile sheath
3. Short branch phages
4. Phages with a process analogue
5. Filamentous phages

Chemical composition.

Phages are composed of nucleic acid and proteins. Most of them contain 2-stranded DNA, closed in a ring. Some phages contain a single strand of DNA or RNA.

Phage shell - capsid, consists of ordered protein subunits - capsomers.

In the most complexly organized phages, the distal part of the process contains the enzyme - lysozyme. This enzyme promotes the dissolution of the bacterial membrane during the penetration of phage NK into the cytoplasm.

Phages tolerate freezing, heating to 70, and drying well. Sensitive to acids, UV and boiling. Phages infect strictly defined bacteria by interacting with specific cell receptors.

According to the specificity of the interaction -

Polyphages - interacting with several related species of bacteria

Monophages - species-specific phages - interact with one type of bacteria

Type phages - interact with individual variants of bacteria within a species.

According to the action of typical phages, the species can be divided into phage series. The interaction of phages with bacteria can occur through productive, aproductive and integrative type.

Productive type- phage progeny is formed, and the cell is lysed

With aproductive- the cell continues to exist, the interaction process is interrupted at the initial stage

Integrative type- the phage genome integrates into the bacterial chromosome and coexists with it.

Depending on the types of interaction, they distinguish virulent and temperate phages.

Virulent interact with bacteria in a productive manner. First, the absorption of the phage on the bacterial membrane occurs due to the interaction of specific receptors. There is penetration or penetration of viral nucleic acid into the cytoplasm of bacteria. Under the influence of Lysozyme, a small hole is formed in the bacterial shell, the sheath of the phage contracts and NK is injected. The phage shell outside the bacterium. Next, the synthesis of early proteins occurs. They ensure the synthesis of phage structural proteins, replication of phage nucleic acid and repression of the activity of bacterial chromosomes.

After this, synthesis occurs structural components phages and nucleic acid replication. From these elements, a new generation of phage particles is assembled. The assembly is called morphogenesis, new particles, of which 10-100 can be formed in one bacterium. Next is the lysis of the bacteria and the release of a new generation of phages into the external environment.

Temperate bacteriophages interact either in a productive or integrative manner. The productive cycle proceeds similarly. With integrative interaction, the DNA of a temperate phage, after entering the cytoplasm, is integrated into the chromosome in a certain area, and during cell division it is replicated synchronously with bacterial DNA and these structures are transmitted to daughter cells. Such built-in phage DNA - prophage, and a bacterium containing a prophage is called lysogenic, and the phenomenon is lysogeny.

Spontaneously, or under the influence of a number of external factors, the prophage can be cut out from the chromosome, i.e. pass into a free state, exhibit the properties of a virulent phage, which will lead to the formation of a new generation of bacterial bodies - prophage induction.

Lysogenesis of bacteria underlies phage (lysogenic) conversion. This is understood as a change in characteristics or properties in lysogenic bacteria compared to non-lysogenic bacteria of the same species. Subject to change different properties- morphological, antigenic, etc.

Temperate phages may be defective - unable to form phage progeny not in natural conditions and in induction.

Virion is a full-fledged viral particle consisting of NK and a protein shell

Practical application of phages -

1. Application in diagnostics. In relation to a number of bacterial species, monophages are used in the phagolysability reaction as one of the criteria for identifying a bacterial culture; typical phages are used for phagotyping and for intraspecific differentiation of bacteria. Conducted for epidemiological purposes, to establish the source of infection and ways to eliminate it
2. For the treatment and prevention of a number of bacterial infections - abdominal type, staphylococcal and streptococcal infections (acid-resistant tablets)
3. Temperate bacteriophages are used in genetic engineering as a vector capable of introducing genetic material into a living cell.

Genetics of bacteria

The bacterial genome consists of genetic elements capable of self-reproduction - replicons. Replicons are bacterial chromosomes and plasmids. The bacterial chromosome forms a nucleoid, a closed ring not associated with proteins and carries a haploid set of genes.

A plasmid is also a closed ring of a DNA molecule, but much smaller in size than a chromosome. The presence of plasmids in the cytoplasm of bacteria is not necessary, but they provide an advantage in environment. Large plasmids are reduced with the chromosome and their number in the cell is small. And the number of small plasmids can reach several dozen. Some plasmids are capable of reversibly integrating into the bacterial chromosome in a certain region and functioning as a single replicon. Such plasmids are called integrative. Some plasmids are capable of being transmitted from one bacterium to another through direct contact - conjugative plasmids. They contain genes responsible for the formation of F-piles, which form a conjugative bridge for the transfer of genetic materials.

The main types of plasmids are

F - integrative congative plasmid. The sex factor determines the ability of bacteria to be donors during conjugation

R - plasmids. Resistant. Contains genes that determine the synthesis of factors that destroy antibacterial drugs. Bacteria possessing such plasmids are not sensitive to many drugs. Therefore, drug-resistant factors are formed.

Plasmid tox - determining factors of pathogenicity -

Ent - plasmids - contains a gene for the production of enterotoxins.

Hly - destroy red blood cells.

Mobile genetic elements. These include insertion - insertion elements. The generally accepted designation is Is. These are sections of DNA that can move both within the replicon and between them. They contain only the genes necessary for their own movement.

Transposons- larger structures that have the same properties as Is, but in addition they contain structural genes that determine the synthesis biological substances, such as toxins. Mobile genetic elements can cause gene inactivation, damage to genetic material, replicon fusion, and the spread of genes throughout a bacterial population.

Variability in bacteria.

All types of variability are divided into 2 groups - non-hereditary (phenotypic, modification) and hereditary (genotypic).

Modifications- phenotypic non-inherited changes in traits or properties. Modifications do not affect the genotype and therefore are not inherited. They are adaptive reactions to changes in specific conditions external environment. As a rule, they are lost in the first generation, after the factor ceases to act.

Genotypic variability affects the genotype of the organism, and therefore can be transmitted to descendants. Genotypic variability is divided into mutations and recombinations.

Mutations- persistent, heritable changes in the characteristics or properties of an organism. The basis of mutations is a qualitative or quantitative change in the sequence of nucleotides in a DNA molecule. Mutations can change almost any property.

By origin, mutations are spontaneous and induced.

Spontaneous mutations occurs in the natural conditions of the organism’s existence, and induced arise as a result of the directed action of a mutagenic factor. Based on the nature of changes in the primary structure of DNA in bacteria, gene or point mutations and chromosomal aberrations are distinguished.

Gene mutations occur within a single gene and minimally involve one nucleotide. This type of mutation can be the result of the replacement of one nucleotide with another, the loss of a nucleotide, or the insertion of an extra one.

Chromosomal- may affect several chromosomes.

There may be a deletion - the loss of a section of a chromosome, or a duplication - the doubling of a section of a chromosome. Rotating a section of a chromosome by 180 degrees is an inversion.

Any mutation occurs under the influence of a certain mutagenic factor. By their nature, mutagens are physical, chemical and biological. Ionizing radiation, X-rays, UV rays. Chemical mutagens include analogues of nitrogenous bases, nitrous acid itself, and even some medicines, cytostatics. Biological - some viruses and transphasons

Recombination- exchange of chromosome sections

Transduction - transfer of genetic material using a bacteriophage

Reparation of genetic material - restoration of damage resulting from mutations.

There are several types of reparation

1. Photoreactivation - this process is ensured by a special enzyme that is activated in the presence of visible light. This enzyme moves along the DNA strand and repairs damage. Combines timers that are formed under the action of UV. The results of dark reparation are more significant. It does not depend on light and is provided by several enzymes - first, nucleases cut out the damaged section of the DNA chain, then DNA polymerase, on the matrix of the complementary chain preserved, synthesizes a patch, and ligases sew the patch into the damaged area.

Reparations are subject to gene mutations, but chromosomal ones are usually not

1. Genetic recombination in bacteria. They are characterized by the penetration of genetic material from the donor bacterium into the recipient bacterium with the formation of a daughter genome containing the genes of both original individuals.

Incorporation of a donor DNA fragment into the recipient occurs by crossing over

Three types of transmission -

1. Transformation- a process in which a fragment of isolated donor DNA is transferred. Depends on the competence of the recipient and the condition of the donor DNA. Competence- ability to absorb DNA. It depends on the presence of special proteins in the recipient’s cell membrane and is formed during certain periods of bacterial growth. Donor DNA must be double-stranded and not very large in size. Donor DNA penetrates the bacterial membrane, and one of the chains is destroyed, the other is integrated into the recipient's DNA.
2. Transduction- carried out with the help of bacteriophages. General transduction and specific transduction.

General - occurs with the participation of virulence factors. During the assembly of phage particles, the phage head may mistakenly include not phage DNA, but a piece of the bacterial chromosome. Such phages are defective phages.

Specific- it is carried out by temperate phages. When cutting, cutting it is strictly carried out along the border. They are built between certain genes and transfer them.

1. Conjugation- transfer of genetic material from the donor bacterium to the recipient, upon their direct contact. A necessary condition- presence of a congative plasmid in the donor cell. During conjugation, a conjugation bridge is formed due to pili, through which the genetic material is transferred from the donor to the patient.

Gene diagnostics

A set of methods that make it possible to identify the genome of a microorganism or its fragment in the material under study. The method of NC hybridization was the first to be proposed. Based on the use of the principle of complementarity. This method allows you to detect the presence of marker DNA fragments of the pathogen in the genetic material using molecular probes. Molecular probes are short DNA chains complementary to the marker region. A fluorescent label is added to the probe, radioactive isotope, enzyme. The material being studied is subjected to special treatment, which allows it to destroy microorganisms, release DNA and divide it into single-stranded fragments. After this, the material is fixed. The activity of the tag is then detected. This method is not highly sensitive. It is possible to identify the pathogen only if its quantity is sufficiently large. 10 to 4 microorganisms. It is quite technically complex and requires large quantities probes. Widespread in practice he did not find it. Was developed new method - polymerase chain reaction- PCR.

This method is based on the ability of DNA and viral RNA to replicate, i.e. to self-reproduction. The essence of the patient is repeated copying - in vitro amplification of a DNA fragment, which is a marker for a given microorganism. Since the process takes place at sufficiently high temperatures 70-90, the method became possible after the isolation of thermostable DNA polymerase from thermophilic bacteria. The amplification mechanism is such that copying of DNA chains does not begin at any point, but only at certain starting blocks, for the creation of which so-called primers are used. Primers are polynucleotide sequences complementary to the terminal sequences of the copied fragment of the desired DNA, and primers not only initiate amplification, but also limit it. Now there are several PCR options, characterized by 3 stages -

1. Denaturation of DNA (division into 1 chain fragments)
2. Attaching the primer.
3. Complementary addition of DNA strands to double strands

This cycle lasts 1.5-2 minutes. As a result, the number of DNA molecules doubles 20-40 times. The result is 10 to the 8th power of copies. After amplification, electrophoresis is performed and isolated in the form of stripes. It is carried out in a special device called an amplifier.

Advantages of PCR

1. Gives direct indications of the presence of a pathogen in the test material, without isolating a pure culture.
2. Very high sensitivity. Theoretically, one can detect the 1st.
3. The material for research can be immediately disinfected after collection.
4. 100% specificity
5. Fast results. Full analysis- 4-5 hours. Express method.

It is widely used for the diagnosis of infectious diseases, the causative agents of which are unculturable or difficult to cultivate organisms. Chlamydia, mycoplasma, many viruses - hepatitis, herpes. Test systems have been developed to determine anthrax, tuberculosis.

Restriction analysis- with the help of enzymes, the DNA molecule is separated according to certain nucleoid sequences and the fragments are analyzed based on their composition. This way you can find unique areas.

Biotechnology and genetic engineering

Biotechnology is a science that, based on the study of the vital processes of living organisms, uses these bioprocesses, as well as the biological objects themselves, for the industrial production of products necessary for humans, to reproduce bioeffects that do not manifest themselves in unnatural conditions. As biological objects Most often, single-celled microorganisms are used, as well as cells from animals and plants. Cells reproduce very quickly, which allows for a short time increase the biomass of the producer. Currently, the biosynthesis of complex substances, such as proteins, antibiotics, is more economical and technologically accessible than other types of raw materials.

Biotechnology uses the cells themselves as a source of the target product, as well as large molecules synthesized by the cell, enzymes, toxins, antibodies and primary and secondary metabolites - amino acids, vitamins, hormones. The technology for obtaining products of microbial and cellular synthesis comes down to several typical stages - selection or creation of a productive headquarters. Selection of optimal nutrient medium, cultivation. Isolation of the target product, its purification, standardization, giving dosage form. Genetic engineering comes down to the creation of target products necessary for humans. The resulting target gene is fused to a vector, and the vector can be a plasmid, and it is inserted into the recipient cell. Recipient - bacteria - E. coli, yeast. The target products synthesized by recombinants are isolated, purified and used in practice.

The first to be created were insulin and human interferon. Erythropoietin, growth hormone, monoclonal antibodies. Hepatitis B vaccine.

Practical application of phages. Bacteriophages are used in laboratory diagnostics of infections for intraspecific identification of bacteria, i.e., determination of the phagovar (phagotype). For this purpose the method is used phage typing, based on the strict specificity of the action of phages: drops of various diagnostic type-specific phages are applied to a plate with a dense nutrient medium seeded with a “lawn” of a pure culture of the pathogen. The phage of a bacterium is determined by the type of phage that caused its lysis (formation of a sterile spot, “plaque”, or “negative colony”, phage). The phage typing technique is used to identify the source and routes of spread of infection (epidemiological marking). Isolation of bacteria of the same phagovar from different patients indicates a common source of their infection.

Phages are also used for treatment and prevention a number of bacterial infections. They produce typhoid, salmonella, dysentery, pseudomonas, staphylococcal, streptococcal phages and combined preparations (coliproteus, pyobacteriophages, etc.). Bacteriophages are prescribed according to indications orally, parenterally or topically in the form of liquid, tablet forms, suppositories or aerosols.

Bacteriophages are widely used in genetic engineering and biotechnology as vectors for producing recombinant DNA.

Causative agents of escherichiosis. Taxonomy and characteristics. The role of Escherichia coli in normal and pathological conditions. Microbiological diagnosis of enteral escherichiosis. Principles of treatment and prevention.

Escherichiosis- infectious diseases, the causative agent of which is Escherichia coli.

There are enteral (intestinal) and parenteral escherichiosis. Enteral escherichiosis is an acute infectious disease characterized by primary damage to the gastrointestinal tract. They occur in the form of outbreaks; the causative agents are diarrheagenic strains of E. coli. Parenteral escherichiosis is a disease caused by opportunistic strains of E. coli - representatives of the normal microflora of the colon. With these diseases, damage to any organs is possible.

Taxonomic position. The causative agent - Escherichia coli - is the main representative of the genus Escherichia, family Enterobacteriaceae, belonging to the department Gracilicutes.

Morphological and tinctorial properties. E.coli are small gram-negative rods with rounded ends. In smears they are arranged randomly, do not form spores, peritrichous. Some strains have a microcapsule, pili.

Cultural properties. Escherichia coli is a facultative anaerobe, optimal. pace. for height - 37C. E.coli not demanding nutrient media and grows well on simple media, giving diffuse turbidity on liquid media and forming colonies on dense media. To diagnose escherichiosis, differential diagnostic media with lactose are used - Endo, Levin.

Enzyme activity. E.coli has a large set of different enzymes. Most hallmark E.coli is its ability to ferment lactose.

Antigenic structure. Escherichia coli has somatic ABOUT-, flagellar H and surface K antigens. O-antigen has more than 170 variants, K-antigen - more than 100, H-antigen - more than 50. The structure of O-antigen determines its serogroup. Strains E. coli having their own set of antigens (antigenic formula) are called serological variants (serovars).

According to antigenic, toxigenic properties, two are distinguished: biological variant E. coli:

1) opportunistic coli;

2) “certainly” pathogenic, diarrheagenic.

Pathogenicity factors. Forms endotoxin, which has enterotropic, neurotropic and pyrogenic effects. Diarrheagenic Escherichia produces an exotoxin that causes a significant disruption of water-salt metabolism. In addition, some strains, like the causative agents of dysentery, contain an invasive factor that promotes the penetration of bacteria into cells. The pathogenicity of diarrheagenic Escherichia is in the occurrence of hemorrhage and nephrotoxic effect. To pathogenicity factors of all strains E.coli include pili and outer membrane proteins that promote adhesion, as well as a microcapsule that prevents phagocytosis.

Resistance. E.coli has a higher resistance to action various factors external environment; it is sensitive to disinfectants and quickly dies when boiled.

RoleE.coli. Escherichia coli is a representative of the normal microflora of the colon. It is an antagonist of pathogenic intestinal bacteria, putrefactive bacteria and fungi of the genus Candida. In addition, it is involved in the synthesis of vitamins B, E And TO, partially breaks down fiber.

Strains that live in the large intestine and are opportunistic can get beyond the gastrointestinal tract and, with a decrease in immunity and their accumulation, become the cause of various nonspecific purulent-inflammatory diseases (cystitis, cholecystitis) - parenteral escherichiosis.

Epidemiology. The source of enteric escherichiosis is sick people. Mechanism of infection - fecal-oral, transmission routes - alimentary, contact and household.

Pathogenesis. Oral cavity. Gets into small intestine, is adsorbed in epithelial cells with the help of pili and outer membrane proteins. Bacteria multiply and die, releasing endotoxin, which increases intestinal motility, causes diarrhea, fever and other symptoms of general intoxication. Produces exotoxin - severe diarrhea, vomiting and significant disruption of water-salt metabolism.

Clinic. Incubation period is 4 days. The disease begins acutely, with fever, abdominal pain, diarrhea, and vomiting. There are disturbances in sleep and appetite, and headache. In the hemorrhagic form, blood is found in the stool.

Immunity. After past illness immunity is fragile and short-lived.

Microbiological diagnostics . Basic method - bacteriological. The type of pure culture is determined (gram-negative bacilli, oxidase-negative, fermenting glucose and lactose to acid and gas, forming indole, not forming hydrogen sulfide) and belonging to the serogroup, which makes it possible to distinguish opportunistic E. coli from diarrheagenic ones. Intraspecific identification, which has epidemiological significance, consists of determining the serovar using diagnostic adsorbed immune sera.

83. Structure and functions of the immune system.