by Viktor KRYLOV, Dr. Sc. (Biol.), Head of Laboratory,

State Scientific Research Institute of Genetics and Selection of Industrial Microorganisms

The current situation with uncontrolled uses of medicinal preparations, pollution of nature with toxic wastes and other adverse phenomena of this kind have produced what experts call another spiral in the evolution of bacteria - the development of their multiple-resistant strains. As often as not, many expensive antibiotics of the last generation (vancomycin, imipenem, etc.) turn out to be powerless in combating ailments caused by these bacteria. And the way medical experts see it - the way out of this situation-consists in pioneering some alternative therapies. The most promising of these is believed to be phagotherapy - the use of specific bacteria viruses (bacteriophages, or phages).

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ON THE NATURE OF BACTERIOPHAGES

By contaminating bacteria in the focus of infection, phages destroy them. This principle lies at the roots of phagotherapy whose idea was suggested in 1917 by one of the pioneer discoverers of phages, Canadian scientist Felix d'Herelle (1873-1949). But the advent of penicillins in 1929, which ushered in the era of antibiotics in medicine, pushed phagotherapy into the background, so to speak.

But research on bacteriophages went on. Thanks to the choice of the colon bacillus (and a very lucky one at that!) as the model for the studies of biological process as in a group of phages, a transition was accomplished from classical to molecular biology which paved the way for genetic engineering, genetic therapy, comparative genomics, etc.

Studies of phages have also been associated with some of the major breakthroughs of the 20th-century science as determining the role of nucleic acids in the transmission of hereditary characters, unraveling the nature of the genetic code, the ways of transmitting its data into protein derivatives, identification of the mechanisms of mutagenesis, etc. The nature of phages was investigated yielding new data which had been lacking at the first stages of these studies. It was established, for example, that these were, in fact, living organisms whose development (same as of any virus) practically fully depends on the host cell. It was also established that there exist many phages, capable of diverse inherited modifications, which makes it possible for them to develop in some related but non-identical bacteria.

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Bacteriophages - which can be classified into species, genera and families - include particles of different morphology. But even despite these external differences, each of them contains inside its membrane (made up mainly of proteins) a genome which determines the program of their development in keeping with the universal principles which apply to all living matter. From this point of view a phage particle can be regarded as a "temporary storage" of genome for its subsequent introduction into bacteria.

Outside of the cell it is biochemically inert, and a living bacteriophage is a bacterium infested with a phage within which a program of development is implemented as "recorded" in its genome. In a simplified way this process can be described as follows: after the infection of a bacterium with a phage there begins what we call a transcription (reading off) with the enzyme RNA- polymerase. This is accompanied by the formation of intermediate data carriers - short molecules of matrix-RNA. These enter the ribosomes ("proteins factories"), where they are aligned into a sequence of the aminoacids in the protein. The subsequent stages include the formation of the structural components of a phage (heads and tails), the "filling up" with nucleic acid and the final "assembly" of phage particles. The successive action of two proteins, which destroy first the inner cytoplasmic membrane, and then the outer strong layer of the cell wall, provides for its rupture and the release of particles of the phage "progeny" capable of infestation of new bacteria. Such is the cycle of development of phages which are best adopted to phagotherapy (these are also known as the truly virulent ones).

Two other types - what we call temperate ones and phages with an uninterrupted development cycle - differ by the nature of infection. The former can "choose" between two models of development-lythic (similar to the development of truly virulent phages) and a moderate one. In the latter case a phage suppresses its genome, synthesizing a special represser protein. This links up with the operator section of the phage genome, which physically obstructs the attachment of RNA-polymerase to the promoters - sections of the beginning of transcription located next to the operator segments.

An inactive genome of a phage is known as prophage, and its carrier bacterium - lysogen. This cell is resistant to the effect of its own phage, and, sometimes, also of other phages. But as soon as the level of represser in the lysogen drops below a critical level, there immediately starts a transcription of the genome of the phage and its lythic development.

By means oftransduction (capture in one bacterium and transfer into another cell of a section of bacterial chromosome), phages can deliver "pathogenic islands" from the bacterial genome of one host into the cells of other bacteria, turning the formerly quite harmless bacteria of common microflora into pathogens (this is known as phage conversion). Ailments caused by phage conversion include diphtheria, scarlatina, whooping cough, some forms of dysentery, cholera and, probably, meningitis. Therefore even moderate phages are not used for therapeutic purposes, even in the form of virulent mutants, because of the possibility of reverse mutations.

Phages with an uninterrupted cycle of development (such as the filiform mp13) are being constantly synthesized in a cell: having infected a bacterium once, they are being constantly reproduced within it and, being clad into a protein coat, are released through a specific pore. And the bacterium itself continues to live and multiply.

WHEN ANTIBIOTICS FAIL

Phages, being intracellular parasites of bacteria, depend on the host cells. Say, practically every stage of development of the former requires the participation of bacterial enzymes, special structures, etc. Therefore the development of a phage can be rather easily arrested by bacterial mutations, which makes the host-cell hereditary resistant to a concrete phage. In order to rule out, or at least substantially reduce this probability, specialists prepare and use mixtures of phages "dwelling" in bacteria of different strains of one kind (or different species). In this case mutants, possessing resistance to one phage, are infected and killed by others. This substantially reduces the rate of survival of phage- resistant bacteria and prolongs the length of using such therapeutic mixtures in the treatment of a concrete patient.

The effectiveness of this therapy can be assessed with the help of the total number of phage types which are active for a certain type of bacteria and their biodiversity. For example, what we call express-classification of a group of samples of virulent phages of the blue pus bacillus Pseudomonas aeruginosa* conducted by us has proved that a considerable segment of phage species is represented by a single phage. And that means that many more phages, active upon these bacteria, have not been identified so far. On the other hand, some phages of one and the same variety occur again and again. And that means that what remains to be identified are exactly the varieties which are missing for phagotherapy; and the introduction into the mixture of even slightly different, but closely related phages may prove to be quite senseless.

Thus the search for new varieties of phages becomes a priority objective for specialists. And when these varieties, with optimal therapeutic properties are picked up, it would be necessary to establish the natural boundaries of variability of their different attributes - something which will make it possible to produce optimal therapeutic mixtures. Incidentally, it order to maintain a high level of effectiveness of preparations consisting of combined phages one often has to replace some phages with different ones. That is why drugstores may offer their customers under one and the same label preparations containing sets of quite different bacteriophages. That depends on what were the initial strains of bacteria used for the selection of the most active phages (as a rule, the former are obtained in clinics located closest to the pharmaceutical plant). As we have established, there can occur paradoxical situations when a preparation which contains, according to a label attached, no less than 10 different spe-

* See: V. Krylov, "These Puzzling Pseudomonade Transposon Phages", Science in Russia, No. 1, 1998. - Ed.

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Morphological types of different phages (on the example of virulent phages Ps. AERUGINOSA).

cific phages against the colon bacillus and assuring a proper level of effectiveness in one area, turns out to be quite useless in another. But still and all, using mixtures of live phages in therapy offers indisputable advantages thanks to the rapid response, relative low costs and simplicity of manufacture. Given a proper, and individually chosen for every patient, selection of phage mixtures, the effectiveness of phagotherapy of, let us say purulent infections, can exceed 90 percent (according to data from our own and Polish researchers). What is more, the use of live phages makes it possible to administer them to the patient in minimal amounts - they will proliferate as long as there remain bacteria which are sensitive to them.

The drawbacks of the suggested method include: highly specific composition of mixtures, calling for constant monitoring and the search for new specific phages; rapid appearance of resistant mutants; working with unstudied material - phages which contain genes controlling the synthesis of products harmful to man.

Thus therapy with mixtures of phages calls for the development of a vast collection of classified phages of different kinds and a painstaking selection of individual mixtures for each and every patient, constant monitoring and timely replacement of phages when they become ineffective.

However, this logically substantiated and time-tested scheme comes into contradiction with an approach which is being envisaged in North America. According to the principles of the Food and Drug Administration (FDA) - the US federal agency which controls the introduction of new drugs - the "uncertainty" of the composition of any new preparation is inadmissible. Therefore there will most likely prevail the use of the same natural phages, possessing a broad spectrum of host bacteria or the only ones for every variety of pathogens of genetic-engineered phages. The basic ideas of this kind of approach are: broaden considerably the spectrum of hosts of some very definite and well- studied phage through the modification of the apparatus of its adsorption and boost "the kill" of the same phage by introducing into its genome some additional genes with different mechanisms of destruction of bacterial cells. As is expected, this will not only limit the number of the phages used, but will make their replacement unnecessary.

The ideas of development of genetically engineered phages rest on the principles of modular theory of evolution. The general principles belong to two Stanford University researchers - Prof. A. Campbell and Prof. D. Bot-

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Classification of bacteriophages on the example of phages of Ps. AERUGINOSA. As one can see, 8 out of 14 virulent phages are embraced by a single representative, which makes it necessary to search for new species with therapeutic properties.

stein. And the idea is that many genes, their sets, or even segments of genes represent what are called modules (mobile and in a certain sense independent parts of a genome). The latter, if transferred into a different genetic environment, will continue to perform their own function (synthesis of a certain section of a protein, structural or enzymat-ically active proteins).

GENETIC ENGINEEMNG APPROACHES

Several different approaches of this kind are mapped out now in genetic engineering research.

One of them is linked with the development of live genetically engineered (GE) phages capable of breeding in a cell and simultaneously possessing several mechanisms of destruction of bacteria thanks to the fusion into their gene of several modules with different "killer" genes. This method, however, has several considerable flaws. First, since a phage is an intracellular parasite, which depends practically at all stages of its development on the main biochemical systems of bacteria, one can introduce into its genome only genes which do not interfere with the operation of these systems - otherwise the development of the phage itself will be stopped. Second, even the introduction into a phage genome of several mechanisms of bacteria destruction offers no guarantee that some new resistant organisms will not appear with time. What we call the absorption- resistant mutants, without any special provisions will be picked up rather quickly, making useless the GE phages and especially phages with a broad spectrum of hosts. Finally, the possibility of transduction of virulent foci from chromosomes or plasmids, even when virulent phages are used, is not ruled out as with the use of live native phages.

To be able to overcome certain drawbacks of this kind, it is proposed to develop GE systems whose cells will be infected not only by the live (proliferating) phage, but by its phantom carrying the plasmide stuffed with modules with several killer genes. In that case the theoretical probability of survival of the resistant bacteria will become negligibly small. But a seemingly attractive solution of this kind can be achieved at the expense of losing the main advantage of a live phage - its ability to multiply. And what would that lead us to? It will be necessary to administer at one and the same time a vast amount of pseudo-phages, producing an overstrain of the immune system. And since the carrier of the killer-plasmid will be a concrete phage, the spectrum of activity of the system will be rather narrow. But the main objection to using this method is the ability of plasmids to enter human cells - something that could have some unpredictable consequences for the patients' health.

The main task of all such genetic engineering approaches is to reduce the specific impact of a system of any type, at least down to the level of a specific

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type of bacteria. According to the logic of its proponents, this approach should do away with the need for individual selection of phage mixtures relative to certain strains of bacteria. But in reality this seems to be highly improbable to achieve. If using antibiotics had led to the development of resistant strains of bacteria, how can one guarantee that no bacteria will appear resistant to all of the killer-modules? And since the search for new killer-proteins will require large collections of classified bacteriophages of different kinds of bacteria, would it not be simpler, on the basis of such collections, to keep up what has been working so well - therapy with phage mixtures?

UNITY OF OPPOSITES

As has been said before, interest in phagotherapy has been aroused, above all, by the advent of vast amounts of bacteria resistant to antibiotics. But why an introduction of every new antibiotic should be followed by the appearance of new resistant strains of bacteria? This is linked, among other things, with human activities and our confidence of being able to change the world around us, including living nature, and use it for our own benefits. This self-confidence has been promoted by the progress of modem science. What is more, it turns out that changing the living world, especially the micro-world, is a relatively simple matter. Unfortunately, as often as not this precipitates some irreversible consequences and, because of the interdependence and interconnection of the macro- and microworlds, this poses a very real threat to mankind.

Bioworlds - identical in their basis - have been progressing, changing and adapting to one another over millions of years. This being so, it is impossible to break off links between them in an abrupt manner without precipitating serious consequences. Now, let's stop and think: the way we see it, bacterial infections are a deplorable eventuality which can and should be done away with in general. As for pathogenic bacteria, their interaction with microorganisms is the only route of evolution, of preserving a certain gene pool which is indispensable for their survival. What is more, for maintaining an effective evolution of microorganisms (and also man) it is necessary to maintain a certain progress of bacteria. And there are reasons to believe that pathogenic bacteria had their own positive role to play in the appearance of Homo sapiens and they did so by reducing the number of our potential enemies and stimulating the development of immunity. And when the Homo sapiens species was formed, it itself provoked the advent of new strains of pathogenic bacteria, specific for the humans only.

Building an absolutely sterile world - without bacteria and infections - means making this world lifeless. Thus, mankind is doomed to an endless struggle with the pathogenic part of the micro-world as a result of which there will appear new types of pathogenic bacteria. And a significant role in their advent can belong to the natural activity of phages, the insufficiently tested methods of their modifications and making use of such modified systems.

One can hardly think of some measures which could help prevent the appearance of bacteria strains with multiple resistance to antibiotics. They are not simply mutants of clinical pathogenes produced by therapeutic errors (low doses of antibiotics, use of just one antibiotic). Though it could well be that

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it were these clinical mutants that had triggered off the mechanism of genetic engineering in nature which has led to the development of multiple-resistant bacteria. These strains are really something special. Some researchers believe they had originated in natural conditions, and the use of antibiotics simply made it possible for them to occupy a new ecological niche - clinics, hospitals and maternity homes. These strains carry specific elements - integrons, containing sets of genes producing resistance not only to various antibiotics, but also to ions of heavy metals, antiseptics, UV radiation and other factors harmful for bacteria. Often integrons are incorporated into plasmids capable of migration between different types of bacteria - something that promotes their rapid propagation.

Some specialists suggest that the "wholesale" application of phages or other antibacterial preparation instead of the classical antibiotics will cause a rapid loss by the multiple-resistant bacteria of plasmids with "sets" or cassettes of genes, because it would be energy-wasteful for cells to sustain the no longer needed plasmides, and it would be possible to revert to the classical antibiotics. This view is supported by occasional replacements of antibiotic-resistant strains with their sensitive versions after phagotherapy. We ourselves saw this happen once even when using phages for a patient with cystic fibrosis (mucoviscidosis), a case in which the use of phages is still believed to be rather unpromising. And although this is possible in theory, there is no confidence as yet about a massive scale of the loss of plasmides. With this aim in view it would be not enough to ban therapies using antibiotics. Since the appearance of cassettes with stability genes by way of natural genetic engineering is not just a reaction to antibiotics, but the consequence of significant changes of the environment (accumulation of toxic compounds, heavy metals, an increased UV radiation background), we have no reasons to expect these cassette structures to disappear. And that means that with repeated uses of the "old" antibiotics genes resistant to them will rapidly spread out again. That being so, the appearance of such multiple- resistant pathogenic bacteria rather attests to the irreversible nature of this process and signals an end to the era of antibiotics as a means of combating and prevention of bacterial infections.

And now one more subject for discussion - the genetic safety of phagotherapy The currently available experience in Russia, Poland and Georgia proves that no undesirable consequences of genetic nature have been traced as yet. But, bearing in mind our limited experience and the fact that with introducing phagotherapy on a mass scale billions of live phage particles will come into contact (not immediate one) with man (they are capable of rapid evolution, including capture and transport into his microflora of undesirable genes) one cannot rule out in advance the genetic possibility of direct contacts of phages with the genetic material of an individual. And this is a very serious problem which calls not only for further studies and observations, but also for extreme caution.

A FEW WORDS IN CONCLUSION

In all probability, phagotherapy, at this stage should be resorted to only if and when therapy with antibiotics fails to cope with a problem. Thus we know of several examples of its successful application in advanced cases of sepsis. But, as clinicists point out, this should be done only with due consideration of what we call the vital factors and bearing in mind the possibility of a shock and allergic reactions.

Deserving of special consideration is peroral administration which can be successfully applied in the treatment of intestinal infections. In recent time it has been almost generally recognized that phages, administered perorally (after neutralization of stomach acidity with alkaline mineral water), rapidly penetrate into the blood and other body fluids. Some even believe that this method can replace the intravenous administration of phages in cases of blood poisoning. But statistical data to that effect are still insufficient and it appears that not all phages can get into the blood through peroral administrations. According to our own observations, that cover, for example, bigger phages related to the phage phiKZ and active on Ps. aeruginosa (blue pus bacillus), which is probably due to their large size.

Another advantage of phagotherapy is its high selectivity. But in the conditions of ordinary clinics, "bristling" with different strains of pathogenic bacteria, the picture is far from being so simple. As often as not, a lesion, rapidly disinfected with specific phages, can become quickly reinfected with other bacteria present in this clinic.

In Russia, it seems to be most appropriate to develop what we call habitual, or customary phagotherapy with mixtures of natural phages, especially broadening their range of species. And it may well be that for many European countries this method will also prevail at least until we are convinced of an obvious superiority of GE techniques (including the cost factor). The way we see it now, the introduction of what we call relatively simple organizational measures into the application of phage mixtures should boost considerably their effectiveness. This includes, above all, using in clinical practice mixtures specially prepared for every individual patient (produced by trained specialists and from factory-made component phages). What is more, this approach should also help reduce the cost of phagotherapy.

Summing it up, time has come for active introduction and perfection of the traditionally accepted methods of phagotherapy in Russia. This is necessary so as not to find ourselves unarmed against infections caused by bacteria resistant to antibiotics and in order not to be dependent on what are often untested (or tested only on cell cultures or mice) "High-tech" methods - often prompted by current market conditions and potentially dangerous, which are now present in such an abundance on the market of medical services.

The author wishes to express his profound gratitude to Prof. G. Ackerman (Canada) for providing the first two illustrations.