Bacteriophages: modern aspects of application, prospects for the future. The use of phages in medicine and microbiology

In the middle of the last century, biological science took a revolutionary step forward by establishing the molecular basis for the functioning of living systems. A huge role in successful research that led to the determination of the chemical nature of hereditary molecules, decoding genetic code and the creation of gene manipulation technologies, bacteriophages, discovered at the beginning of the last century, played. To date, these bacterial viruses have mastered many “professions” useful for humans: they are used not only as safe antibacterial drugs, but also as disinfectants, and even as the basis for creating electronic nanodevices.

When in the 1930s a group of scientists took up the problems of the functioning of living systems, then in search of the simplest models they paid attention to bacteriophages- bacterial viruses. After all, among biological objects there is nothing simpler than bacteriophages, besides, they can be easily and quickly grown and analyzed, and viral genetic programs are small.

A phage is a minimally sized natural structure containing a densely packed genetic program (DNA or RNA), in which there is nothing superfluous. This program is enclosed in a protein shell, equipped with a minimum set of devices for its delivery inside the bacterial cell. Bacteriophages cannot reproduce by themselves, and in this sense they cannot be considered full-fledged living objects. Their genes begin to work only in bacteria, using the biosynthetic systems available in the bacterial cell and the reserves of molecules necessary for synthesis. However, the genetic programs of these viruses do not fundamentally differ from those of more complex organisms Therefore, experiments with bacteriophages made it possible to establish the fundamental principles of the structure and operation of the genome.

Subsequently, this knowledge and the methods developed during the research became the foundation for the development of biological and medical science, as well as a wide range of biotechnological applications.

Fighters against pathogens

The first attempts to use bacteriophages for treatment infectious diseases were undertaken almost immediately after their discovery, but the lack of knowledge and imperfect biotechnologies of that time did not allow them to achieve complete success. Nevertheless, further clinical practice showed the fundamental possibility of the successful use of bacteriophages in infectious diseases. gastrointestinal tract, genitourinary system, in acute purulent-septic conditions of patients, for the treatment of surgical infections, etc.

Compared with antibiotics, bacteriophages have a number of advantages: they do not cause side effects, moreover, they are strictly specific for certain types of bacteria, therefore, when they are used, the normal human microbiome is not disturbed. However, such high selectivity also creates problems: in order to successfully treat a patient, it is necessary to know exactly the infectious agent and select the bacteriophage individually.

Phages can also be used prophylactically. For example, the Moscow Research Institute of Epidemiology and Microbiology. G. N. Gabrichevsky developed the prophylactic product "FOODFAG" based on a cocktail of bacteriophages, which reduces the risk of infection with acute intestinal infections. Clinical studies have shown that a weekly intake of the drug allows you to get rid of hemolyzing Escherichia coli and other pathogenic and opportunistic bacteria, causing dysbacteriosis intestines.

Bacteriophages treat infectious diseases not only of people, but also of domestic and farm animals: mastitis in cows, colibacillosis and escherichiosis in calves and pigs, salmonellosis in chickens ... It is especially convenient to use phage preparations in the case of aquaculture - for the treatment of industrially grown fish and shrimp, because they stay in water for a long time. Bacteriophages also help to protect plants, although the use of phage technologies in this case is difficult due to the influence of natural factors, such as sunlight and rain, that are detrimental to viruses.

Phages can play a big role in maintaining the microbiological safety of food, since the use of antibiotics and chemical agents in the food industry does not solve this problem, while reducing the level of environmental friendliness of products. The seriousness of the problem itself is evidenced by statistics: for example, in the United States and Russia, up to 40 thousand cases of salmonellosis are registered annually, of which 1% die. The spread of this infection is largely associated with the rearing, processing and consumption of various types of poultry, and attempts to use bacteriophages to combat it have shown promising results.

Yes, an American company Intralytix manufactures phage preparations to combat listeriosis, salmonellosis and bacterial contamination by Escherichia coli. They are approved for use as additives to prevent the growth of bacteria on food - they are sprayed on meat and poultry products, as well as vegetables and fruits. Experiments have shown that a cocktail of bacteriophages can be successfully used in the transportation and sale of live pond fish to reduce bacterial contamination not only of water, but also of the fish itself.

An obvious application of bacteriophages is disinfection, that is, the destruction of bacteria in places where they should not be: in hospitals, food industries, etc. For this purpose, the British company Fixed Phage developed a method for fixing phage preparations on surfaces, which ensures the preservation of biological activity phages up to three years.

Bacteriophages - "Drosophila" of molecular biology

In 1946, at the 11th symposium in the famous American laboratory at Cold Spring Harbor, the theory of "one gene - one enzyme" was proclaimed. Bacteriologist A. Hershey and "former" physicist, molecular biologist M. Delbrück reported on the exchange of genetic traits between different phages while simultaneously infecting Escherichia coli cells. This discovery, made at a time when the physical carrier of the gene was not yet known, testified that the phenomenon of "recombination" - the mixing of genetic traits, is characteristic not only of higher organisms, but also of viruses. The discovery of this phenomenon subsequently made it possible to study in detail the molecular mechanisms of replication. Later, experiments with bacteriophages made it possible to establish the principles of the structure and operation of genetic programs.

In 1952, A. Hershey and M. Chase experimentally proved that the hereditary information of bacteriophage T2 is encoded not in proteins, as many scientists believed, but in DNA molecules (Hershey & Chase, 1952). The researchers followed the reproduction process in two groups of bacteriophages, one carrying radiolabeled proteins and the other carrying DNA molecules. After infection of bacteria with such phages, it turned out that only viral DNA is transmitted into the infected cell, which served as evidence of its role in the storage and transmission of hereditary information.

In the same year, American geneticists D. Lederberg and N. Zindler, in an experiment involving two strains of Salmonella and the bacteriophage P22, found that the bacteriophage is capable of incorporating DNA fragments of the host bacterium during reproduction and transmitting them to other bacteria upon infection (Zinder & Lederberg , 1952). This phenomenon of gene transfer from a donor bacterium to a recipient bacterium has been termed "transduction". The results of the experiment became another confirmation of the role of DNA in the transmission of hereditary information.

In 1969, A. Hershey, M. Delbrück and their colleague S. Luria became Nobel laureates "for their discoveries concerning the mechanism of replication and the genetic structure of viruses."

In 1972, while studying the process of replication (copying cellular information) of E. coli DNA, R. Bird and colleagues used bacteriophages as probes capable of integrating into the bacterial cell genome and found that the replication process proceeds in two directions along the chromosome (Stent, 1974 ).

Seven Days of Creation

Modern methods of synthetic biology make it possible not only to make various modifications to phage genomes, but also to create completely artificial active phages. Technologically, this is not difficult, you just need to synthesize the phage genome and introduce it into a bacterial cell, and there it will start all the processes necessary for the synthesis of proteins and the assembly of new phage particles. In modern laboratories, this work will take only a few days.

Genetic modifications are used to change the specificity of phages and increase their efficiency. therapeutic effect. To do this, the most aggressive phages are provided with recognition structures that bind them to the target bacteria. Also, genes encoding toxic proteins for bacteria that disrupt metabolism are additionally inserted into viral genomes - such phages are more deadly for bacteria.

Bacteria have several defense mechanisms against antibiotics and bacteriophages, one of which is the destruction of viral genomes. restriction enzymes acting on specific nucleotide sequences. To increase the therapeutic activity of phages, due to the degeneracy of the genetic code, the sequences of their genes can be “reformatted” in such a way as to minimize the number of nucleotide sequences that are “sensitive” to enzymes, while simultaneously preserving their coding properties.

A universal way to protect bacteria from all external influences- so called biofilms, films of DNA, polysaccharides, and proteins that bacteria create together and where neither antibiotics nor therapeutic proteins penetrate. Such biofilms are headache doctors, as they contribute to the destruction of tooth enamel, are formed on the surface of implants, catheters, artificial joints, as well as in respiratory tract, on the surface of the skin, etc. To combat biofilms, special bacteriophages were constructed containing a gene encoding a special lytic enzyme that destroys bacterial polymers.

Enzymes "from bacteriophage"

A large number of enzymes that are widely used today in molecular biology and genetic engineering were discovered as a result of research on bacteriophages.

One such example is the restriction enzymes, a group of bacterial nucleases that cleave DNA. Back in the early 1950s. It was found that bacteriophages isolated from cells of one strain of bacteria often reproduce poorly in a closely related strain. The discovery of this phenomenon meant that bacteria have a system for suppressing the reproduction of viruses (Luria & Human, 1952). As a result, an enzymatic restriction-modification system was discovered, with the help of which bacteria destroyed foreign DNA that had entered the cell. The isolation of restriction enzymes (restriction endonucleases) gave molecular biologists an invaluable tool to manipulate DNA: insert one sequence into another or cut out the necessary chain fragments, which ultimately led to the development of recombinant DNA technology.

Another enzyme widely used in molecular biology is bacteriophage T4 DNA ligase, which “crosslinks” the “sticky” and “blunt” ends of double-stranded DNA and RNA molecules. And recently, genetically modified variants of this enzyme with greater activity have appeared.

Most of the RNA ligases used in laboratory practice, which "sew" single-stranded RNA and DNA molecules, also originate from bacteriophages. In nature, they mainly serve to repair broken RNA molecules. Researchers most commonly use bacteriophage T4 RNA ligase, which can “sew” single-stranded polynucleotides onto RNA molecules to label them. This technique is used to analyze the structure of RNA, search for RNA-protein binding sites, oligonucleotide synthesis, etc. Recently, thermostable RNA ligases isolated from bacteriophages rm378 and TS2126 have appeared among routinely used enzymes (Nordberg Karlsson, et al., 2010; Hjorleifsdottir , 2014).

From bacteriophages, some of another group of extremely important enzymes, polymerases, were also obtained. For example, the very "precise" bacteriophage T7 DNA polymerase, which has found application in various fields molecular biology, such as site-directed mutagenesis, but is mainly used to determine the primary structure of DNA.

A chemically modified T7 phage DNA polymerase has been proposed as perfect tool for DNA sequencing as early as 1987 (Tabor & Richardson, 1987). The modification of this polymerase has increased its efficiency by several times: the rate of DNA polymerization in this case reaches more than 300 nucleotides per second, so it can be used to amplify large DNA fragments. This enzyme became the precursor of sequenase, a genetically engineered enzyme optimized for DNA sequencing in the Sanger reaction. Sequenase is characterized by high efficiency and the ability to incorporate nucleotide analogs into the DNA sequence, which are used to improve sequencing results.

The main RNA polymerases used in molecular biology (DNA-dependent RNA polymerases) - enzymes that catalyze the transcription process (reading RNA copies from the DNA template) - also originate from bacteriophages. These include SP6, T7, and T3 RNA polymerases, named after the respective bacteriophages SP6, T7, and T3. All these enzymes are used for in vitro synthesis of antisense RNA transcripts, labeled RNA probes, etc.

The first fully sequenced DNA genome was the φ174 phage genome, over 5000 nucleotides long (Sanger et al., 1977). This decoding was carried out by a group of English biochemist F. Sanger, the creator of the famous DNA sequencing method of the same name.

Polynucleotide kinases catalyze the transfer of a phosphate group from an ATP molecule to the 5' end of a nucleic acid molecule, the exchange of 5' phosphate groups, or the phosphorylation of the 3' ends of mononucleotides. In laboratory practice, bacteriophage T4 polynucleotide kinase is most widely used. It is commonly used in experiments to label DNA with a radioactive isotope of phosphorus. Polynucleotide kinase is also used to search for restriction sites, DNA and RNA fingerprinting, the synthesis of substrates for DNA or RNA ligases.

In molecular biological experiments, bacteriophage enzymes such as T4 phage polynucleotide kinase, commonly used for labeling DNA with a radioactive isotope of phosphorus, DNA and RNA fingerprinting, etc., as well as enzymes that cleave DNA, which are used to obtain single-stranded DNA templates, are also widely used in molecular biological experiments. for sequencing and analysis of nucleotide polymorphism.

Using the methods of synthetic biology, it was also possible to develop bacteriophages armed with the most sophisticated weapons that bacteria use against the phages themselves. It's about about bacterial CRISPR-Cas systems, which are a complex of the nuclease enzyme that cleaves DNA and the RNA sequence that directs the action of this enzyme on a specific fragment of the viral genome. A piece of phage DNA serves as a “pointer”, which the bacterium stores “for memory” in a special gene. When a similar fragment is found inside a bacterium, this protein-nucleotide complex destroys it.

Having figured out the mechanism of operation of CRISPR-Cas systems, the researchers tried to equip the phages themselves with a similar “weapon”, for which a complex of genes encoding a nuclease and addressing RNA sequences complementary to specific regions of the bacterial genome was introduced into their genome. The "target" can be the genes responsible for multiple drug resistance. The experiments were crowned with complete success - such phages with great efficiency affected the bacteria to which they were "tuned".

Phage antibiotics

For therapeutic purposes, phages do not have to be used directly. Over millions of years of evolution, bacteriophages have developed an arsenal of specific proteins - tools for recognizing target microorganisms and manipulating the biopolymers of the victim, on the basis of which antibacterial drugs can be created. The most promising proteins of this type are the endolysin enzymes, which phages use to destroy the cell wall upon exiting the bacterium. By themselves, these substances are powerful antibacterial agents, non-toxic to humans. The efficiency and direction of their action can be increased by changing the addressing structures in them - proteins that specifically bind to certain bacteria.

Most bacteria are divided according to the structure of the cell wall into gram-positive, the membrane of which is covered with a very thick layer of peptidoglycans, and gram-negative, in which the peptidoglycan layer is located between two membranes. The use of natural endolysins is especially effective in the case of gram-positive bacteria (staphylococci, streptococci, etc.), since their peptidoglycan layer is located outside. Gram-negative bacteria (Pseudomonas aeruginosa, Salmonella, coli etc.) are a less accessible target, since the enzyme needs to penetrate the outer bacterial membrane in order to reach the inner peptidoglycan layer.

To overcome this problem, the so-called artilysins were created - modified versions of natural endolysins containing polycationic or amphipathic peptides that destabilize the outer membrane and ensure the delivery of endolysin directly to the peptidoglycan layer. Artilysins have a high bactericidal activity and have already been shown to be effective in the treatment of otitis media in dogs (Briers et al., 2014).

An example of a modified endolysin that selectively acts on certain bacteria is the drug P128 of the Canadian company Ganga Gen Inc.. It is a biologically active fragment of endolysin connected to lysostaphin, a targeting protein molecule that binds to the surface of staphylococcal cells. The resulting chimeric protein has high activity against various strains of staphylococcus, including those with multidrug resistance.

"Counters" of bacteria

Bacteriophages serve not only as a versatile therapeutic and "disinfectant" agent, but also as a convenient and accurate analytical tool for a microbiologist. For example, due to their high specificity, they are natural analytical reagents for the detection of bacteria of a certain type and strain.

In the simplest version of such a study, various diagnostic bacteriophages are added dropwise to a Petri dish with a nutrient medium inoculated with a bacterial culture. If the bacterium turns out to be sensitive to the phage, then at this place of the bacterial "lawn" a "plaque" is formed - a transparent area with killed and lysed bacterial cells.

By analyzing the multiplication of phages in the presence of target bacteria, one can quantify the abundance of the latter. Since the number of phage particles in the solution will increase in proportion to the number of bacterial cells contained in it, it is sufficient to determine the titer of the bacteriophage to estimate the number of bacteria.

The specificity and sensitivity of such an analytical reaction is quite high, and the procedures themselves are simple to perform and do not require sophisticated equipment. It is important that diagnostic systems based on bacteriophages signal the presence of a living pathogen, while other methods, such as PCR and immunoanalytical methods, only indicate the presence of biopolymers belonging to this bacterium. This type of diagnostic methods are particularly suitable for use in environmental studies, as well as in the food industry and agriculture.

Now, special methods are used to identify and quantify different strains of microorganisms. reference species phages. Very fast, almost real-time analytical systems can be created on the basis of genetically modified bacteriophages, which, when they enter a bacterial cell, trigger the synthesis of reporter fluorescent (or capable of luminescence) proteins, such as luciferase. When the necessary substrates are added to such a medium, a luminescent signal will appear in it, the value of which corresponds to the content of bacteria in the sample. Such "light-labeled" phages have been developed to detect dangerous pathogens - the causative agents of plague, anthrax, tuberculosis, and plant infections.

Probably, with the help of modified phages, it will also be possible to solve a long-standing problem of global importance - to develop cheap and fast methods for detecting tuberculosis pathogens at an early stage of the disease. This task is very difficult, since the mycobacteria that cause tuberculosis are characterized by extremely slow growth when cultivated in laboratory conditions. Therefore, the diagnosis of the disease by traditional methods can be delayed for up to several weeks.

Phage technology makes this task easier. Its essence is that bacteriophage D29 is added to the samples of the analyzed blood, capable of infecting wide range mycobacteria. The bacteriophages are then separated, and the sample is mixed with a rapidly growing non-pathogenic culture of mycobacteria, also sensitive to this bacteriophage. If initially there were mycobacteria in the blood that were infected with phages, then the production of bacteriophage will also be observed in the new culture. In this way, single cells of mycobacteria can be detected, and the diagnostic process itself is reduced from 2–3 weeks to 2–5 days (Swift & Rees, 2016).

Phage display

Today, bacteriophages are widely used as simple systems for the production of proteins with desired properties. This is the one developed in the 1980s. extremely effective molecular selection technique - phage display. This term was proposed by the American J. Smith, who proved that on the basis of Escherichia coli bacteriophages, it is possible to create a viable modified virus that carries a foreign protein on its surface. To do this, the corresponding gene is introduced into the phage genome, which merges with the gene encoding one of the surface viral proteins. Such modified bacteriophages can be isolated from a mixture with wild-type phages due to the ability of a “foreign” protein to bind to specific antibodies (Smith, 1985).

Two important conclusions followed from Smith's experiments: first, using recombinant DNA technology, it is possible to create huge populations of 10 6–10 14 phage particles, each of which carries different protein variants on its surface. Such populations are called combinatorial phage libraries. Secondly, by isolating a specific phage from a population (for example, having the ability to bind to a certain protein or organic molecule), this phage can be propagated in bacterial cells and obtain an unlimited number of descendants with desired properties.

Phage display today produces proteins that can selectively bind to therapeutic targets, such as those exposed on the surface of the M13 phage that can recognize and interact with tumor cells. The role of these proteins in the phage particle is to “package” the nucleic acid; therefore, they are well suited for creating gene therapy drugs, only in this case they form a particle already with a therapeutic nucleic acid.

Today, there are two main areas of application of phage display. Peptide-based technology is being used to explore receptors and map antibody binding sites, design immunogens and nanovaccines, and map substrate binding sites for enzyme proteins. Technology based on proteins and protein domains - for the selection of antibodies with desired properties, the study of protein-ligand interactions, screening of expressed complementary DNA fragments and targeted modifications of proteins.

Using phage display, it is possible to introduce recognition groups into all types of surface viral proteins, as well as into the main protein that forms the bacteriophage body. By introducing peptides with desired properties into surface proteins, a whole range of valuable biotechnological products can be obtained. For example, if this peptide mimics the protein of a dangerous virus or bacterium recognized by the immune system, then such a modified bacteriophage is a vaccine that can be easily, quickly and safely produced.

If the terminal surface protein of the bacteriophage is “addressed” to cancer cells, and attach reporter groups (for example, fluorescent or magnetic) to another surface protein, then you get a tool for detecting tumors. And if a cytotoxic drug is also added to the particle (and modern bioorganic chemistry makes it easy to do this), then we get a drug that targets cancer cells.

One of the important applications of protein phage display is the creation of phage libraries of recombinant antibodies, where antigen-binding fragments of immunoglobulins are located on the surface of fd or M13 phage particles. Human antibody libraries are of particular interest because such antibodies can be used in therapy without limitation. AT last years the US pharmaceutical market alone sells about a dozen therapeutic antibodies constructed using this method.

"Industrial" phages

The phage display methodology has also found quite unexpected applications. After all, bacteriophages are primarily nanosized particles of a certain structure, on the surface of which proteins are located, which, using a phage display, can be “provided” with the properties to specifically bind to the desired molecules. Such nanoparticles open the widest possibilities to create materials with a given architecture and "smart" molecular nanodevices, while their production technologies will be environmentally friendly.

Since the virus is a fairly rigid structure with a certain ratio of dimensions, this circumstance makes it possible to use it to obtain porous nanostructures with a known surface area and a desired distribution of pores in the structure. As is known, the catalyst surface area is the critical parameter determining its efficiency. And the existing technologies for the formation of the thinnest layer of metals and their oxides on the surface of bacteriophages make it possible to obtain catalysts with an extremely developed regular surface of a given dimension. (Lee et al., 2012).

MIT researcher A. Belcher used bacteriophage M13 as a template for the growth of rhodium and nickel nanoparticles and nanowires on the surface of cerium oxide. The resulting catalyst nanoparticles facilitate the conversion of ethanol to hydrogen; thus, this catalyst can be very useful for upgrading existing and creating new hydrogen fuel cells. A catalyst grown on a virus template differs from a “conventional” catalyst of similar composition in higher stability, it is less prone to aging and surface deactivation (Nam et al. . , 2012).

By coating filamentous phages with gold and indium dioxide, electrochromic materials were obtained - porous nanofilms that change color when the electric field changes, capable of responding to a change in the electric field one and a half times faster than known analogues. Such materials are promising for creating energy-saving ultra-thin screen devices (Nam et al., 2012).

In Massachusetts Institute of Technology bacteriophages became the basis for the production of very powerful and extremely compact electric batteries. For this, live, genetically modified M13 phages were used, which are harmless to humans and capable of attaching various metal ions to the surface. As a result of the self-assembly of these viruses, structures of a given configuration were obtained, which, when coated with a metal, formed rather long nanowires, which became the basis of the anode and cathode. When self-forming the anode material, a virus capable of attaching gold and cobalt oxide was used, for the cathode - capable of attaching iron phosphate and silver. The latter phage also possessed the ability to "pick up" the ends of a carbon nanotube due to molecular recognition, which is necessary to ensure efficient electron transfer.

Materials for solar cells have also been created based on complexes of the bacteriophage M13, titanium dioxide, and single-walled carbon nanotubes (Dang et al., 2011).

Recent years have been marked by extensive research on bacteriophages, which are finding new applications not only in therapy, but also in bio- and nanotechnologies. Their obvious practical result should be the emergence of a new powerful direction of personalized medicine, as well as the creation of a whole range of technologies in the food industry, veterinary medicine, agriculture and in the production of modern materials. We expect that the second century of bacteriophage research will bring fewer discoveries than the first.

Literature
1. Bacteriophages: biology and applications / Ed.: E. Cutter, A. Sulakvelidze. M.: Scientific world. 2012.
2. Stent G., Kalindar R. Molecular genetics. M.: Mir. 1974. 614 p.
3. Tikunova N. V., Morozova V. V. Phage display based on filamentous bacteriophages: application for the selection of recombinant antibodies // Acta Naturae. 2009. No. 3. C. 6–15.
4. Mc Grath S., van Sinderen D. Bacteriophage: Genetics and Molecular Biology. Horizon Scientific Press, 2007.

№ 10-2013

Photo taken with electron microscope,
shows the process of fixing bacteriophages (coliphages T1) on the surface of the bacterium E. coli.

At the end of the 20th century, it became clear that bacteria undoubtedly dominate the Earth's biosphere, accounting for more than 90% of its biomass. Each species has many specialized types of viruses. According to preliminary estimates, the number of bacteriophage species is about 10 15 . To understand the scale of this figure, we can say that if every person on Earth discovers one new bacteriophage every day, then it will take 30 years to describe all of them.

Thus, bacteriophages are the least studied creatures in our biosphere. Most of the bacteriophages known today belong to the order Caudovirales - tailed viruses. Their particles have a size of 50 to 200 nm. The tail of different lengths and shapes ensures the attachment of the virus to the surface of the host bacterium, the head (capsid) serves as a repository for the genome. Genomic DNA encodes the structural proteins that form the "body" of the bacteriophage and the proteins that ensure the multiplication of the phage inside the cell during infection.

We can say that a bacteriophage is a natural high-tech nanoobject. For example, phage tails are a "molecular syringe" that pierces the wall of a bacterium and injects its DNA into the cell as it contracts. From this point on, the infectious cycle begins. Its further stages consist of switching the bacterial life mechanisms to serving the bacteriophage, multiplying its genome, building multiple copies of viral envelopes, packaging virus DNA in them, and, finally, destruction (lysis) of the host cell.

A bacteriophage is not a living being, but a molecular nanomechanism created by nature.
The tail of a bacteriophage is a syringe that pierces the wall of a bacterium and injects viral DNA,
which is stored in the head (capsid), inside the cell.

In addition to the constant evolutionary competition of defense mechanisms in bacteria and attack in viruses, the reason for the current balance can be considered as the fact that bacteriophages specialized in their infectious action. If available large colony bacteria, where the next generations of phages will find their victims, then the destruction of bacteria by lytic (killing, literally - dissolving) phages proceeds quickly and continuously.

If there are not enough potential victims or the external conditions are not very suitable for efficient reproduction of phages, then phages with a lysogenic development cycle gain an advantage. In this case, after the introduction of the phage DNA into the bacterium, it does not immediately trigger the mechanism of infection, but for the time being it exists inside the cell in a passive state, often invading the bacterial genome.

In this state of the prophage, the virus can exist for a long time, going through cell division cycles together with the bacterium's chromosome. And only when the bacterium enters an environment favorable for reproduction, the lytic cycle of infection is activated. At the same time, when phage DNA is released from the bacterial chromosome, neighboring regions of the bacterial genome are often captured, and their contents can later be transferred to the next bacterium, which the bacteriophage infects. This process (gene transduction) is considered the most important means transfer of information between prokaryotes - organisms without cell nuclei.

How bacteriophage works

All these molecular subtleties were not known in the second decade of the twentieth century, when "invisible infectious agents that destroy bacteria" were discovered. But even without the electron microscope, which was used for the first time in the late 1940s to obtain images of bacteriophages, it was clear that they are capable of destroying bacteria, including pathogens. This property was immediately demanded by medicine.

The first attempts to treat dysentery, wound infections, cholera, typhoid and even plague with phages were carried out quite carefully, and the success looked quite convincing. But after the start of mass production and the use of phage preparations, euphoria turned into disappointment. Very little was known about what bacteriophages are, how to produce, purify and use their dosage forms. Suffice it to say that, according to the results of a test undertaken in the United States in the late 1920s, bacteriophages proper were not found in many industrial phage preparations.

The problem with antibiotics

The second half of the twentieth century in medicine can be called the “era of antibiotics”. However, Alexander Fleming, the discoverer of penicillin, warned in his Nobel lecture that microbial resistance to penicillin arises rather quickly. For the time being, antibiotic resistance has been offset by the development of new types of antimicrobial drugs. But since the 1990s, it has become clear that humanity is losing the “arms race” against microbes.

First of all, the uncontrolled use of antibiotics is to blame, not only in medical, but also in preventive purposes, and not only in medicine, but also in agriculture, the food industry and everyday life. As a result, resistance to these drugs began to develop not only in pathogenic bacteria, but also in the most common microorganisms living in soil and water, making them “conditional pathogens”.

These bacteria thrive in medical institutions, populating plumbing, furniture, medical equipment, sometimes even disinfectant solutions. In people with weakened immune systems, which are the majority in hospitals, they cause severe complications.

No wonder the medical community is sounding the alarm. In 2012, WHO Director-General Margaret Chan issued a statement predicting the end of the era of antibiotics and humanity's defenselessness against infectious diseases. However, practical possibilities combinatorial chemistry - the foundations of pharmacological science - are far from being exhausted. Another thing is that the development antimicrobial agents- a very expensive process that does not bring such profits as many other drugs. So the horror stories about “superbugs” are more of a warning that encourages people to look for alternative solutions.

Bacteriophages and immunity

Since there are a myriad of bacteriophages in nature and they constantly enter the human body with water, air and food, the immune system simply ignores them. There is even a hypothesis about the symbiosis of bacteriophages in the intestine, which regulates the intestinal microflora. It is possible to achieve some kind of immune reaction only with prolonged administration into the body. large doses phages.

But in this way, you can achieve an allergy to almost any substance. Finally, it is very important that bacteriophages are inexpensive. The development and production of a drug consisting of precisely selected bacteriophages with fully decoded genomes, cultivated according to modern biotechnological standards on certain bacterial strains in chemically pure media and highly purified, is orders of magnitude cheaper than modern complex antibiotics.

This makes it possible to quickly adapt phage therapeutic preparations to changing sets of pathogenic bacteria and to use bacteriophages in veterinary medicine, where expensive medicines not economically justified.

In the medical service

It seems logical that there is a resurgence of interest in using bacteriophages, the natural enemies of bacteria, to treat infections. Indeed, during the decades of the “era of antibiotics”, bacteriophages actively served science, not medicine, but fundamental molecular biology. Suffice it to mention the decoding of the "triplets" of the genetic code and the process of DNA recombination. Enough is now known about bacteriophages to reasonably select phages suitable for therapeutic purposes.

Bacteriophages have many advantages as potential drugs. First of all, there are a myriad of them. Although changing the genetic apparatus of a bacteriophage is also much easier than that of a bacterium, and even more so in higher organisms, That is unnecessary. You can always find something suitable in nature. It is more about selection, fixing the desired properties and reproduction of the necessary bacteriophages.

This can be compared with the breeding of dog breeds - sledding, guard, hunting, hounds, fighting, decorative ... All of them remain dogs, but are optimized for a certain type of action, necessary to a person. Secondly, bacteriophages are strictly specific, that is, they destroy only a certain type of microbes without inhibiting normal microflora person.

Thirdly, when a bacteriophage finds a bacterium that it must destroy, it begins to multiply during its life cycle. Thus, the question of dosage becomes not so acute. Fourth, bacteriophages do not cause side effects. All cases of allergic reactions when using therapeutic bacteriophages were caused either by impurities, from which the drug was not sufficiently purified, or by toxins released during the mass death of bacteria. The last phenomenon, the "Herxheimer effect", is often observed with the use of antibiotics.

Two sides of the coin

Unfortunately, medical bacteriophages also have many shortcomings. The most important problem stems from the advantage of the high specificity of phages. Each bacteriophage infects a strictly defined type of bacteria, not even a taxonomic species, but a number of narrower varieties, strains. Relatively speaking, as if guard dog she began to bark only at two-meter-tall thugs dressed in black raincoats, and did not react at all to a teenager in shorts climbing into the house.

Therefore, cases of ineffective use are not uncommon for current phage preparations. A drug made against a certain set of strains and perfectly treating streptococcal tonsillitis in Smolensk may be powerless against all signs of the same tonsillitis in Kemerovo. The disease is the same, caused by the same microbe, and streptococcus strains in different regions are different.

For the most effective use of bacteriophage, accurate diagnostics are necessary. pathogenic microbe, up to strain. The most common diagnostic method now - culture inoculation - takes a lot of time and does not provide the required accuracy. Rapid methods - typing using polymerase chain reaction or mass spectrometry - are slowly introduced due to the high cost of equipment and higher requirements for the qualifications of laboratory assistants. Ideally, the selection of phage components medicinal product could be done against the infection of each individual patient, but this is expensive and unacceptable in practice.

Another important disadvantage of phages is their biological nature. In addition to the fact that bacteriophages require special conditions storage and transportation, such a method of treatment opens up a lot of speculation on the topic of "foreign DNA in a person." And although it is known that a bacteriophage, in principle, cannot infect a human cell and introduce its DNA into it, it is not easy to change public opinion.

From the biological nature and rather large, in comparison with low-molecular drugs (the same antibiotics), the size follows the third limitation - the problem of delivering the bacteriophage into the body. If a microbial infection develops where a bacteriophage can be applied directly in the form of drops, spray or enema - on the skin, open wounds, burns, mucous membranes of the nasopharynx, ears, eyes, large intestine - then there are no problems.

But if the infection occurs in the internal organs, the situation is more complicated. Cases of successful treatment of infections of the kidneys or spleen with the usual oral administration of the bacteriophage preparation are known. However, the mechanism of penetration of relatively large (100 nm) phage particles from the stomach into the bloodstream and into internal organs is poorly understood and varies greatly from patient to patient. Bacteriophages are also powerless against those microbes that develop inside cells, such as tuberculosis and leprosy. Through the wall human cell bacteriophage can't get through.

It should be noted that to oppose the use of bacteriophages and antibiotics in medical purposes it does not follow. With their joint action, a mutual strengthening of the antibacterial effect is observed. This allows, for example, to reduce the doses of antibiotics to values ​​that do not cause pronounced side effects. Accordingly, the mechanism for the development of resistance in bacteria to both components of the combined drug is almost impossible.

The expansion of the arsenal of antimicrobial drugs gives more degrees of freedom in the choice of treatment methods. Thus, the scientifically substantiated development of the concept of using bacteriophages in antimicrobial therapy is a promising direction. Bacteriophages serve not so much as an alternative, but as a complement and enhancement in the fight against infections.

CM. ZAKHARENKO, Candidate of Medical Sciences, Associate Professor, Military medical Academy them. CM. Kirov, St. Petersburg

Bacteriophages are unique microorganisms, on the basis of which a special group of therapeutic and prophylactic preparations has been created in terms of their properties and characteristics. The natural physiological mechanisms of interaction between phages and bacteria underlying their action make it possible to predict an infinite variety of both bacteriophages themselves and possible ways of using them. As bacteriophage collections expand, new target pathogens will undoubtedly appear, and the range of diseases in which phages can be used both as monotherapy and as part of complex treatment regimens will expand.

Thus, the use of the polyvalent pyobacteriophage Sextaphage in the treatment of infected pancreatic necrosis (Perm State Medical Academy named after Academician E.A. Wagner) made it possible to quickly restore the main parameters of homeostasis and the functions of organs and systems in patients. There has also been a significant decrease in the number postoperative complications and deaths: in the group of patients treated with standard therapy, mortality was 100%, while in the troupe treated with BF - 16.6%.

Due to the harmlessness and reactogenicity of BF preparations, it is possible to use them in pediatric practice, including in newborns. The experience of the Nizhny Novgorod Children's Regional Clinical Hospital is interesting, where during the period of complication of the epidemiological situation, along with the usual anti-epidemic measures, BP - Intesti-bacteriophage and BP Pseucfomonas aeruginosa were used. An 11-fold decrease in the incidence of nosocomial infection of Pseudomonas aeruginosa showed the high efficiency of BP use. BF preparations can be prescribed both for the treatment of dysbacteriosis and disorders of the digestive system, and for the prevention of colonization of the mucous membranes of the gastrointestinal tract by opportunistic bacteria. Multicomponent preparations of BF are ideal for immediate relief of the first signs of gastrointestinal upset.

To date, the enterprise has outlined a number of priority areas for the development and production of therapeutic and prophylactic bacteriophages, which correlate with the newly emerging global trends. New preparations are being created and introduced: BF against serrations and enterobacteria have been developed, work is underway to create a phage preparation against Helicobacter pylori.

Only one manufacturer of these drugs - NPO Microgen, according to the report of the deputy head of the department of science and innovative development Alla Lobastova, produces more than 2 million packages annually. Unfortunately, the ideas of many doctors about bacteriophages are far from being objective. Not many people know that bacteriophages active against the same pathogen can belong to different families, have different life cycles, etc. For example, P. aeruginosa bacteriophages belong to the families Myoviridae, Podoviridae, Siphoviridae, life cycle or moderate. Different strains of the same pathogen may have different susceptibility to bacteriophages. Most experts know (heard, someone used) about the existence of liquid and tablet dosage form therapeutic and prophylactic preparations of bacteriophages. However, their range is much wider, which can be attributed to unconditional advantages, especially in combination with a variety of routes of administration (oral administration, enemas, applications, irrigation of wounds and mucous membranes, introduction into wound cavities, etc.). The obvious advantages of bacteriophages traditionally include a specific effect on a rather limited population of bacteria, a limited time existence (until the target population of microorganisms disappears), the absence of such side effects as toxic and allergic reactions, dysbiotic reactions, etc. These drugs can be used in a variety of age groups and during pregnancy. Bacteriophages themselves are not significant allergens. Cases of intolerance to bacteriophage preparations are mostly associated with a reaction to the components of the nutrient medium. All major manufacturers of this group of drugs strive for the maximum quality of the components used, which reduces the likelihood of such reactions. In the context of growing antibiotic resistance, some authors suggest considering bacteriophages as the best alternative to antibiotics. Therapeutic and prophylactic preparations of bacteriophages are a cocktail of specially selected combinations (a complex of polyclonal highly virulent bacterial viruses specially selected against the most common groups of pathogens of bacterial infections) based on the manufacturer's phage collections. Branches of Federal State Unitary Enterprise NPO Microgen in Ufa, Perm and Nizhny Novgorod are modern centers for the production of such drugs. Ability to create customized pathogenic microorganisms therapeutic and prophylactic preparations of bacteriophages is another major advantage of this group of preparations. The growth of bacterial resistance to antimicrobial drugs and the often occurring polyetiology of modern infectious diseases require combined antibiotic therapy (two, three, and sometimes more antimicrobials). To select an effective antibiotic therapy regimen, in addition to the actual sensitivity of the bacteria to the drug, it is necessary to take into account a fairly large number of factors. Phage therapy also has certain advantages in this respect. On the one hand, the use of a combination of bacteriophages is not accompanied by their interaction with each other and does not lead to a change in the schemes of their application. Within the existing set of therapeutic bacteriophages, there are a number of well-proven combinations - bacteriophage coliproteus, pyobacteriophage polyvalent, intesti-bacteriophage. On the other hand, bacteria do not have common mechanisms of resistance to antibiotics and phages; therefore, they can be used both when the pathogen is resistant to one of the drugs, and in the combination "antibiotic + bacteriophage". This combination is especially effective for destroying microbial biofilms. The experiment convincingly showed that the combined use of iron antagonists and a bacteriophage can disrupt the formation of Klebsiella pneumoniae biofilms. At the same time, both a significant decrease in the number of microbial populations and a decrease in the number of "young" cells are noted. One more important feature action of bacteriophages is such a phenomenon as the induction of apoptosis. Some strains of E. coli have genes that cause cell death after the introduction of the T4 bacteriophage into it. Thus, in response to the expression of the late genes of the T4 phage, the lit gene (encodes a protease that destroys the EF-Tu elongation factor necessary for protein synthesis) blocks the synthesis of all cellular proteins. The prrC gene encodes a nuclease that cleaves lysine tRNA. The nuclease is activated by the product of the T4 phage stp gene. In T4 phage-infected cells, rex genes (belonging to the phage genome and expressed in lysogenic cells) cause the formation of ion channels, leading to the loss of vital ions by the cells and, subsequently, to death. The T4 phage itself can prevent cell death by closing the channels with its proteins, products of the rII genes. In the case of the formation of bacterial resistance to an antibiotic, one has to look for new options for modifying the active molecule or fundamentally new substances. Unfortunately, in recent years, the pace of introduction of new antibiotics has slowed down significantly. The situation with bacteriophages is fundamentally different. The collections of major manufacturers include dozens of ready-made bacteriophage strains and are constantly replenished with new active phages. Thanks to constant monitoring of the sensitivity of isolated pathogens to bacteriophages, manufacturers adjust the phage compositions supplied to the regions. Thanks to adapted bacteriophages, it is possible to eliminate outbreaks of nosocomial infections caused by antibiotic-resistant strains.

When taken orally, bacteriophages quickly reach the foci of infection localization: when taken orally by patients with purulent-inflammatory diseases, phages enter the blood after an hour, after 1–1.5 hours they are detected from bronchopulmonary exudate and from the surface of burn wounds, after 2 hours - from the urine , as well as from the cerebrospinal fluid of patients with craniocerebral injuries.

Thus, bacteriophages are unique microorganisms, on the basis of which a special group of therapeutic and prophylactic preparations has been created in terms of their properties and characteristics. The natural physiological mechanisms of interaction between phages and bacteria underlying their action make it possible to predict an infinite variety of both bacteriophages themselves and possible ways of using them. As bacteriophage collections expand, new target pathogens will undoubtedly appear, and the range of diseases in which phages can be used both as monotherapy and as part of complex treatment regimens will expand. A modern view of the future fate of phage therapy should be based both on the high specificity of their action and on the need to strictly observe all the rules of phage therapy. Contrasting bacteriophages with any means of etiotropic therapy is erroneous.

For the first time, the assumption that bacteriophages are viruses was made. D. Errel. In the future, viruses of fungi, etc., were discovered, they began to call them phages.

Phage morphology.

Sizes - 20 - 200nm. Most phages are shaped like tadpoles. The most complex phages consist of a polyhedral head containing nucleic acid, a neck, and processes. At the end of the process is the basal plate, with filaments and teeth extending from it. These threads and teeth serve to attach the phage to the bacterial shell. The most complexly organized phages in the distal part of the process contain an enzyme - lysozyme. This enzyme contributes to the dissolution of the bacterial membrane upon penetration of the 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 contracting sheath
2. Phages with a long process but not a contractile sheath
3. Phages with a short tail
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 - capsomeres.

The most complexly organized phages in the distal part of the process contain an enzyme - lysozyme. This enzyme contributes to the dissolution of the bacterial membrane upon penetration of the phage NK into the cytoplasm.

The phages tolerate freezing, heating up 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 bacterial species

Monophages - species 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 row. The interaction of phages with bacteria can proceed through productive, aproductive and integrative type.

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

With a productive- 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 type of interaction, there are virulent and temperate phages.

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

This is followed by the synthesis of the structural components of phages and the replication of the nucleic acid. 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. Further lysis of the bacterium and the release of a new generation of phages into the external environment.

temperate bacteriophages interact either productively or integratively. The productive cycle goes the same way. 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 replicates synchronously with bacterial DNA, and these structures are transmitted daughter cells. Such built-in phage DNA - prophage, and a bacterium containing a prophage is called lysogenic, and the phenomenon is called lysogeny.

Spontaneously, or under the influence of a series external factors a prophage can be excised from the chromosome, i.e. move 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.

Bacterial lysogenesis underlies phage (lysogenic) conversion. This is understood as a change in traits or properties in lysogenic bacteria, compared with non-lysogenic bacteria of the same species. Different properties can change - morphological, antigenic, etc.

Temperate phages can be defective - unable to form phage progeny outside of natural conditions and in induction.

Virion - a complete 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 phage lizability reaction, as one of the criteria for identifying a bacterial culture, typical phages are used for phage typing, for intraspecific differentiation of bacteria. Conducted for epidemiological purposes, to establish the source of infection and ways to eliminate
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-replication - replicons. Replicons are bacterial chromosomes and plasmids. The bacterial chromosome forms a nucleoid that is not associated with proteins in a closed ring and carries a haploid set of genes.

Plasmids are also a closed ring of the DNA molecule, but much smaller than the chromosome. The presence of plasmids in the cytoplasm of bacteria is not necessary, but they confer 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 tens. Some plasmids are able to reversibly integrate into the bacterial chromosome in a certain region and function as a single replicon. Such plasmids are called integrative. Some plasmids are able to be transferred from one bacterium to another by direct contact - conjugative plasmids. They contain genes responsible for the formation of F-pills, which form a conjugative bridge for the transfer of genetic material.

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, a drug-resistant factor is formed.

Plasmid tox - determining factors of pathogenicity -

Ent - plasmid - contains the gene for the production of enterotoxins.

Hly - destroy the erythrocyte.

mobile genetic elements. These include inserts - 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 of biological substances, such as toxins. Transposable genetic elements can cause gene inactivation, damage to genetic material, replicon fusion, and gene proliferation in 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-heritable changes in traits or properties. Modifications do not affect the genotype, and therefore are not inherited. They are adaptive responses to changes in some specific environmental conditions. As a rule, they are lost in the first generation, after the termination of the factor.

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

Mutations- persistent, inherited changes in the characteristics or properties of the organism. The basis of mutations is qualitative or quantitative change 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 existence of the organism, and indexed arise as a result of the directed action of the mutagenic factor. According to 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 capture one nucleotide. This type of mutation may result from the substitution of one nucleotide for another, the loss of a nucleotide, or the insertion of an extra one.

Chromosomal- can affect several chromosomes.

There may be a deletion - the loss of a chromosome segment, a duplication - doubling of a chromosome segment. A 180 degree rotation of a chromosome segment 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. To chemical mutagens - analogues nitrogenous bases, nitrous acid itself, and even some drugs, cytostatics. To biological - some viruses and transphazones

Recombination- exchange of parts of chromosomes

Transduction – transfer of genetic material by a bacteriophage

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

There are several types of reparation

1. Photoreactivation - this process is provided by a special enzyme that is activated in the presence of visible light. This enzyme moves along the DNA chain and repairs damage. Combines thymers, which 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 synthesizes a patch on the matrix of the remaining complementary chain, and ligases sew the patch into the damaged area.

Gene mutations undergo repair, but chromosomal mutations, as a rule, do not.

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

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

Three types of transmission -

1. Transformation- the process by which a fragment of isolated donor DNA is transferred. Depends on the competence of the recipient and the state of the donor DNA. Competence- the ability to absorb DNA. It depends on the presence of specific proteins in the cell membrane of the recipient and is formed in certain periods bacterial growth. Donor DNA must be double-stranded and not very large in size. Donor DNA penetrates the bacterial membrane, 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 virulent factors. During the assembly of particle phages, 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 moderate phages. When cutting out, cutting it out is strictly carried out along the border. They are inserted between certain genes and transfer them.

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

Gene diagnostics

A set of methods 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 principle of complementarity. This method makes it possible to detect the presence of marker DNA fragments of the pathogen in the genetic material using molecular probes. Molecular probes are short strands of DNA that are complementary to a marker site. A label is introduced into the probe - fluorochrome, a radioactive isotope, an enzyme. The test material is subjected to a special treatment that allows destroying microorganisms, releasing DNA and dividing it into single-stranded fragments. After that, the material is fixed. Then the label activity is detected. This method is not highly sensitive. It is possible to identify the pathogen only with a sufficiently large number of it. 10 to 4 microorganisms. It is rather complicated technically and requires a large number of probes. It has not been widely used in practice. Was designed 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 that is a marker for a given microorganism. Since the process takes place at sufficiently high temperatures of 70-90, the method became possible after the isolation of thermostable DNA polymerase from thermophilic bacteria. The mechanism of amplification 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 that are complementary to the end sequences of the copied fragment of the desired DNA, and the primers not only initiate amplification, but also limit. Now there are several options for PCR, 3 stages are characteristic -

1. DNA denaturation (separation into 1 strand fragments)
2. Primer attachment.
3. Complimentary extension of DNA strands to 2 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 strips. It is held in special device, which is called an amplifier.

Advantages of PCR

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

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

Restriction analysis- with the help of enzymes, the DNA molecule is divided according to certain sequences of nucleoids and the fragments are analyzed according to their composition. This way you can find unique sites.

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 biological objects themselves, for the industrial production of products necessary for humans, for the reproduction of bioeffects that do not manifest themselves in unnatural conditions. As biological objects, unicellular microorganisms, as well as cells, animals and plants are most often used. The cells reproduce very quickly, which makes it possible to increase the biomass of the producer in a short time. At present, biosynthesis complex substances, such as proteins, antibiotics, are more economical and technologically more 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 is reduced to several typical stages - the choice or creation of a productive headquarters. Selection of the optimal nutrient medium, cultivation. Isolation of the target product, its purification, standardization, dosage form. Genetic engineering is reduced to the creation of a target product necessary for a person. The resulting target gene is fused with a vector, and the vector can be a plasmid and inserted into the recipient's cell. Recipient - bacteria - Escherichia coli, yeast. Target products synthesized by recombinants are isolated, purified and used in practice.

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

Bacteriophage gi 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. As a rule, a bacteriophage consists of a protein shell and the genetic material of a single-stranded 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 are actively involved in the cycle chemical substances and energy, have a marked effect on the evolution of microbes and bacteria The structure of a typical bacteriophage myovirus.

The structure of bacteriophages 1 - head, 2 - tail, 3 - nucleic acid, 4 - capsid, 5 - "collar", 6 - tail protein cover, 7 - tail fibril, 8 - spikes, 9 - basal plate

Bacteriophages differ in chemical structure, type of nucleic acid, morphology, and interaction with bacteria. Bacterial viruses are 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 transcriptase enzyme in an inactive state, surrounded by a protein or lipoprotein shell - a capsid that preserves the genome outside the cell. 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. Usually the corners are made up of pentamers of the protein, and the support of each side is made up of hexamers of the same or a similar protein. Moreover, phages can be spherical, lemon-shaped, or pleomorphic in shape. The tail, or process, 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.

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

Interaction of a bacteriophage with bacterial cells According to 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 of a virulent bacteriophage with 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 exit of bacteriophages from the cell. Initially, bacteriophages attach to phage-specific receptors on the surface of the bacterial cell. The tail of the phage, 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 metabolism: the synthesis of bacterial DNA, RNA and proteins stops. The bacteriophage DNA begins to be transcribed using its own transcriptase enzyme, which, after entering the bacterial cell, is activated. Synthesized first early, and then late and. RNA that enters the ribosomes of the host cell, where early (DNA polymerases, nucleases) and late (capsid and tail proteins, lysozyme, ATPase and transcriptase enzymes) 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 occurs - the maturation of phage particles or the combination of phage DNA with an 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. Bacteriophage adsorption on phage-specific cell receptors. Injection of a phage nucleic acid into a host cell. Co-replication of phage and bacterial nucleic acids. Cell division. Further, the bacteriophage can develop according to two models: lysogenic or lytic way. Temperate bacteriophages after division are in a state of prophase (lysogenic pathway). Virulent bacteriophages develop according to a political model: Nucleic acid of the phage directs the synthesis of phage enzymes, using the protein-synthesizing apparatus of the bacterium for this. The phage in one way or another inactivates the host's DNA and RNA, and the phage enzymes completely cleave it; Phage RNA "subdues" the cellular machinery of 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 (capsid) around the phage nucleic acid; under the control of phage RNA, lysozyme is synthesized. 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 fields of application of bacteriophages is antibiotic therapy alternative to taking antibiotics. For example, bacteriophages are used: streptococcal, staphylococcal, klebsiella, dysentery and irrigation alent, pyobacteriophage, coli, proteus and coliproteus and others. 13 registered and applied in Russia medical preparations based on phages. Currently, they are used to treat bacterial infections that are not sensitive to traditional antibiotic treatment, especially in the Republic of Georgia. Usually, the use of bacteriophages is more successful than antibiotics where there are biological membranes coated with polysaccharides, through which antibiotics usually do not penetrate. Currently therapeutic use bacteriophages have not gained acceptance in the West, although phages are used to kill bacteria that cause food poisoning, such as listeria. In many years of experience in the volume of a large city and countryside the unusually high therapeutic and prophylactic efficacy of the dysenteric bacteriophage has been proven (P. M. Lerner, 2010). In Russia, therapeutic phage preparations have been made for a long time; phages were treated even before antibiotics. In recent years, phages have been 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 segments; natural transfer of genes between bacteria by means of certain phages (transduction) is also possible. Phage vectors are usually created on the basis of a temperate bacteriophage λ containing a double-stranded linear DNA molecule. The left and right arms of the phage have all the genes necessary for the lytic cycle (replication, reproduction). middle part bacteriophage genome λ (contains genes that control lysogeny, that is, its integration into the DNA of a bacterial cell) is not essential for its reproduction and is approximately 25 thousand base pairs. This part can be replaced by a foreign DNA fragment. Such modified phages go through the lytic cycle, but lysogeny does not occur. Bacteriophage λ-based vectors are used to clone eukaryotic DNA fragments (i.e., larger genes) up to 23 kb in size. Moreover, phages without inserts are less than 38 kbp. or, on the contrary, with too large inserts - more than 52 kb. do not develop and do not infect bacteria. Since bacteriophage reproduction is possible only 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 the determination of the viability of the cultures used. Using the method of electro-optical analysis of cell suspensions, it was shown that it is possible to study the stages of interaction between a phage-microbial cell

And also in veterinary medicine for: prevention and treatment bacterial diseases birds and animals; treatment of purulent-inflammatory diseases of the mucous membranes of the eyes, oral cavity; prevention of purulent-inflammatory complications in burns, wounds, surgical interventions; in genetic engineering: for transduction - the natural transfer of genes between bacteria; as vectors that transfer sections of DNA; using phages, it is possible to construct directed changes in the genome of the host DNA; in the food industry: in large quantities, phage-containing agents are already processing ready-to-eat meat and poultry products; bacteriophages are used in the production of food products from meat, poultry, cheeses, plant products, etc.;

in agriculture: spraying phage preparations to protect plants and crops from decay and bacterial diseases; to protect livestock and poultry from infections and bacterial diseases; for environmental safety: antibacterial treatment of seeds and plants; cleaning of premises of food processing enterprises; sanitization of the working space and equipment; prevention of hospital premises; carrying out environmental activities

Thus, today bacteriophages are very popular in human and animal life. A number of priority areas for the development and production of therapeutic and prophylactic bacteriophages have been outlined at the enterprises, which correlate with the newly emerging global trends. New drugs are being created and introduced to treat many diseases. Bacteriologists, virologists, biochemists, geneticists, biophysicists, molecular biologists, experimental oncologists, specialists in genetic engineering and biotechnology are engaged in the study and use of bacteriophages.