Bacteriophages in medicine
Bacteria eaters
Konstantin Miroshnikov, "Popular Mechanics" No. 10-2013
All creatures living on earth have microscopic parasites – viruses. Bacteria also have their own viruses. The reproduction cycle of bacterial viruses inevitably ends with the death of the microbe. To emphasize this feature, one of the discoverers of this effect, Felix D'erel, came up with a special name – "bacteriophages", translated from Greek – "bacteria eaters".
Photo taken with an electron microscope,
shows the process of fixing bacteriophages (T1 coliphages) on the surface of the E. coli bacterium.
At the end of the twentieth century, it became clear that bacteria certainly 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 from 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 structural proteins that form the "body" of the bacteriophage, and proteins that ensure the reproduction of the phage inside the cell during infection.
We can say that a bacteriophage is a natural high–tech nanoobject. For example, the tails of phages are a "molecular syringe" that pierces the bacterial wall and, contracting, injects its DNA into the cell. From this moment the infectious cycle begins. Its further stages consist of switching the mechanisms of the bacterium's vital activity to the maintenance of the bacteriophage, the reproduction of its genome, the construction of many copies of the viral shells, the packaging of the virus DNA in them, and, finally, the 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 bacterial wall and injects viral DNA,
which is stored in the head (capsid), inside the cell.
Each stage has many nuances that have a deep evolutionary and ecological meaning. After all, bacteria and their viral parasites have coexisted for hundreds of millions, if not billions of years. And this struggle for survival did not end with the total destruction of unicellular cells, nor with the acquisition of total resistance to phages and uncontrolled reproduction of bacteria.
In addition to the constant evolutionary competition of defense mechanisms in bacteria and attack in viruses, the reason for the current equilibrium can also be considered the fact that bacteriophages specialized in their infectious action. If there is a large colony of bacteria where the next generations of phages will find their victims, then the destruction of bacteria by lytic (killing, literally – dissolving) phages goes on quickly and continuously.
If there are not enough potential victims or the external conditions are not too suitable for effective 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 exists inside the cell in a passive state, often being introduced into the bacterial genome.
In this state of the prophage, the virus can exist for a long time, passing through cell division cycles together with the bacterial chromosome. And only when the bacterium enters a breeding environment, the lytic cycle of infection is activated. At the same time, when the phage DNA is released from the bacterial chromosome, neighboring sections of the bacterial genome are often captured, and their contents can later be transferred to the next bacterium that the bacteriophage infects. This process (gene transduction) is considered to be the most important means of transferring information between prokaryotes – organisms without cell nuclei.
How the bacteriophage worksAll 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 an electron microscope, with the help of which it was possible to obtain images of bacteriophages for the first time in the late 1940s, it was clear that they were capable of destroying bacteria, including pathogens. This property was immediately demanded by medicine.
The first attempts to treat dysentery, wound infections, cholera, typhus and even plague with phages were carried out quite carefully, and success looked quite convincing. But after the start of mass production and use of phage drugs, euphoria was replaced by disappointment. Very little was known about what bacteriophages are, how to produce, purify and apply their dosage forms. Suffice it to say that according to the results of the inspection undertaken in the USA at the end of the 1920s, there were no bacteriophages in many industrial phage preparations at all.
The problem with antibioticsThe 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 the resistance of microbes to penicillin occurs quite quickly. For the time being, antibiotic resistance was compensated 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 for therapeutic, but also for 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".
Such bacteria comfortably exist in medical institutions, inhabiting plumbing, furniture, medical equipment, sometimes even disinfectant solutions. In people with weakened immune systems, which are the majority in hospitals, they cause severe complications.
Unsurprisingly, the medical community is sounding the alarm. In 2012, WHO Director General Margaret Chan made a statement predicting the end of the era of antibiotics and the vulnerability of humanity to infectious diseases. However, the practical possibilities of combinatorial chemistry – the foundations of pharmacological science – are far from being exhausted. Another thing is that the development of antimicrobials is a very expensive process that does not bring such profits as many other drugs. So horror stories about "superbugs" are rather a warning that encourages people to search for alternative solutions.
Bacteriophages and immunitySince there are countless 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 of large doses of phages into the body.
But in this way you can achieve allergies to almost any substances. And 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, cultured according to modern biotechnological standards on certain strains of bacteria in chemically pure environments and highly purified, is orders of magnitude cheaper than modern complex antibiotics.
This makes it possible to quickly adapt phage therapeutics to changing sets of pathogenic bacteria and use bacteriophages in veterinary medicine, where expensive drugs are not economically justified.
In the medical serviceIt seems quite logical to revive interest in the use of bacteriophages – natural enemies of bacteria – for the treatment of infections.
Indeed, during the decades of the "era of antibiotics" bacteriophages actively served science, but not medicine, but fundamental molecular biology. It is enough to mention the decoding of the "triplets" of the genetic code and the process of DNA recombination. Now enough is known about bacteriophages to reasonably choose phages suitable for therapeutic purposes.
There are many advantages of bacteriophages as potential medicines. First of all, there are countless of them. Although it is also much easier to change the genetic apparatus of a bacteriophage than that of a bacterium, and even more so in higher organisms, there is no need for this. You can always find something suitable in nature. It is more about breeding, securing the required properties and multiplying the necessary bacteriophages.
This can be compared with the breeding of breeds of dogs – sled, guard, hunting, hounds, fighting, decorative… At the same time, they all remain dogs, but they are optimized for a certain type of actions that a person needs. Secondly, bacteriophages are strictly specific, that is, they destroy only a certain type of microbes, without inhibiting the normal human microflora.
Thirdly, when a bacteriophage finds a bacterium that it must destroy, it begins to multiply during its life cycle. Thus, the issue of dosage is 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 insufficiently purified, or by toxins released during the mass death of bacteria. The latter phenomenon, the "Herxheimer effect", is often observed with the use of antibiotics.
Two sides of the coinUnfortunately, there are also many disadvantages of medical bacteriophages.
The most important problem stems from the advantage – 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, it's as if the guard dog started barking 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, for current phage preparations, cases of ineffective use are not uncommon. A drug made against a certain set of strains and perfectly treating streptococcal angina in Smolensk may be powerless against all signs of the same angina in Kemerovo. The disease is the same, caused by the same microbe, and the strains of streptococcus in different regions are different.
For the most effective use of the bacteriophage, an accurate diagnosis of the pathogenic microbe, up to the strain, is necessary. The most common diagnostic method now – culture sowing – takes a lot of time and does not give the required accuracy. Rapid methods – typing by polymerase chain reaction or mass spectrometry - are being implemented slowly due to the high cost of equipment and higher requirements for the qualification of laboratory assistants. Ideally, the selection of phage components of the drug 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 storage and transportation conditions to maintain infectivity, this method of treatment opens up space for a lot of speculation on the topic of "extraneous DNA in a person". And although it is known that a bacteriophage, in principle, cannot infect a human cell and inject its DNA into it, it is not easy to change public opinion.
From the biological nature and rather large, in comparison with low–molecular-weight drugs (the same antibiotics), the third limitation follows - the problem of delivering the bacteriophage into the body. If a microbial infection develops where the 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 cure of kidney or spleen infections with the usual oral administration of the bacteriophage drug are known. But the mechanism of penetration of relatively large (100 nm) phage particles from the stomach into the bloodstream and into the internal organs is poorly understood and varies greatly from patient to patient. Bacteriophages are also powerless against those microbes that develop inside cells, for example, pathogens of tuberculosis and leprosy. A bacteriophage cannot get through the wall of a human cell.
It should be noted that the use of bacteriophages and antibiotics for medical purposes should not be opposed. When they act together, there is a mutual strengthening of the antibacterial effect. This allows, for example, to reduce the doses of antibiotics to values that do not cause pronounced side effects. Accordingly, the mechanism of developing 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 based development of the concept of the use of bacteriophages in antimicrobial therapy is a promising direction. Bacteriophages serve not so much as an alternative, but as a supplement and reinforcement in the fight against infections.
The author is the acting head of the Laboratory of Molecular Bioengineering of the Institute of Bioorganic Chemistry. Shemyakina and Ovchinnikova RAS
Portal "Eternal youth" http://vechnayamolodost.ru01.11.2013