by Viktor BONDARENKO, Dr. Sc. (Medicine), Head of the Laboratory of Bacterial Virulence Genetics, N.F. Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences (RAMS)

The total gene pool of the microflora (now known as "microbiome") present in our organism numbers 400,000 genes, or twelve times the size of the human genome. A large population like this predetermines the colossal functional activity of microorganisms implicated in many physiological and immune responses protecting the organism against diseases, infections including.

USEFUL MICROBES

Microbes have always been part and parcel of the human environment. They were the primordial forms of life on earth that have subsequently survived in all stages of biological evolution. Their fitness and fast adaptation to unfavorable environmental conditions is due in many ways to their vast numbers, the diversity and high dynamics of their populations, their high fertility and genetic information transmission rates as well as selection efficiency. But the fitness of microorganism is not infinite at all. The ongoing global pollution of the environment may result in the death of the symbiotic microflora in bodies of water, air and soil. In some of this country's ecologically endangered regions this factor is having an adverse effect on people's health*-fust and foremost, it jeopardizes the normal microflora and immune defenses of the human organism. All that compels scientists to attach greater attention in studying

* See: L. Leontiev, "Ecological Problem of Norilsk: Ways of Solution", Science in Russia, No. 5, 2006. - Ed.

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related dysfunctions and pathologies and in mapping out prevention and correction strategies.

Now what is the normal microflora? From a biological standpoint it involves a totality of biocenoses - in our case microbial communities populating the biotopes* of the open cavities of the host organism. Microorganisms prefer best of all the oral cavity (mouth), the nasopharynx, the esophagus, the stomach, the small and the large intestine, the urethra and other organs. Combined with a biocenosis, a biotope forms an ecosystem of the respiratory, gastrointestinal and urogenital tracts.

By the apt definition of Acad. Anatoly Vorobyev (1923 - 2006), the normal microflora is a qualitative and quantitative correlation of diverse microbial colonies inhabiting individual organs and systems, one that sustains a vital biochemical, metabolic and immunological equilibrium of the host organism. This is an essential condition for its sound health.

In fact all microorganisms inhabiting a biotope are involved in complex symbiotic interrelations and linked by trophic (food) chains. For instance, the ecosystems of the mucous membranes of open cavities and skin are formed at childbirths and change with the further growth and development of the organism. Succession, i.e. the successive change of biocenoses colonizing a definite site of the habitation medium engenders a viable and stable microbial community as a rule.

It was the Russian biologist and pathologist Elie Metchnikoff (Honorary Member of the St. Petersburg Academy of Sciences, Nobel Prize, 1908) who was the first to undertake studies of the normal microflora in man and preparations obtained from lactobacilli (the acidophilic bacilli of sour milk); his concept of the simultaneous usefulness and harm of the symbiotic microflora of the organism has gained worldwide recognition. Today a great contribution to the subject has been made by Russian scientists, namely Drs Valentina Petrovskaya, Otar Chakhava (RAMS N.F Gamaleya Institute of Epidemiology and Microbiology), Inna Kuvayeva (RAMS Institute of Nutrition), Nadezhda Lizko (RAS Institute of Medicobiological Problems), Galina Goncharova, Veronika Pospelova, Stanislav Afanasyev, Boris Shenderov (G.N. Gabrichevsky Institute of Epidemiology and Microbiology), Kira Sokolova (I.N. Blokhina Institute of Epidemiology and Microbiology in Nizhni Novgorod), and their Estonia colleague Akivo Lentsner (Tartu Medical University).

The normal microflora of man is subdivided into resident (obligate) making up as much as 90 percent of the microbes present in the organism; facultative, accounting for less than 9.5 percent; and transitory (transitional, random) - not above 0.5 percent. About 20 percent of the microorganisms colonize the mouth (over 200 species);

* Biotope ("locus of life") - relative to microccology it is a site of the mucous membrane, skin and organ of the host organism (macroorganism) providing adequate conditions for a colony of microorganisms.

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18 to 20 percent are found on the skin; 15 to 16 percent- in the gullet (throat); from 2 to 4 percent populate the urogenital tract in males, and about 10 percent-the vaginal biotope in females. The largest number of microorganisms (up to 40 percent) reside in the gastrointenstinal tract. This will be the subject of our discussion.

These microorganisms are distributed both "vertically"-from the oral (mouth) cavity down to the lower (distal) parts of the large intestine-and "horizontally"-from the lumen to the different layers of the mucous membrane. We distinguish between the parietal (mucosal) and the lumenal microflora. The bulk of the microbial population in the mucous membrane of the small and the large intestine is composed of 15 to 20 associations of the dominant anaerobic bacteria (making do without atmospheric oxygen), facultatively anaerobic ones (capable of growing in the presence of 02, too) and aerobic bacteria (that can exist only in the presence of oxygen); such bacteria include representatives of the genera Bifidobacterium, Bactericides, Fusobacterium, Eubacterium, Clostidium, Lactobacillus, Peptococcus, Peptostreptococcus, Escherichia, Streptococcus, Enterococcus, Staphylococcus, among others. Communities of symbiotic microorganisms on the surface of the mucous membranes of open cavities are found in the form of biofilms which, according to Elie Metchnikoff, cover the mucous membrane like a "glove".

In adult man the total mass of the intestinal microflora exceeds 2.5 kg (about 5 pounds) and its population is as high as 1014. Earlier it was thought to comprise 17 families, 45 genera and about 500 species. These data, however, should be revised in the light of the latest findings obtained by Dr. Paul Eckburg of the United States with the use of molecular genetics methods, and Dr. Georgi Osipov of Russia (A.N. Bakulev Research Center of Cardiovascular Surgery, RAMS), who used the method of gas-liquid chromatography and mass spectroscopy.

Dr. Eckburg and coworkers have shown that the parietal and luminal microflorae include 395 phylogenetically isolated groups (phylotypes) of microorganisms, of which 244 (or 62 percent) were not known before. As many as 195 of the newly discovered microorganisms do not grow on usual nutrient substrates, which means that relevant data will come within our reach only after their food needs and cultivation conditions have been clarified. Most of the new phylotypes belong to the genera Firmicutes and Bacteroides. The sum total of the previously known species (ca. 500) and the newly detected ones is close to fifteen hundred; this figure, however, needs further elucidation.

The basic functions of the normal intestinal microflora are tightly bound up with the immune status and condition of the alimentary and hormonal systems. In particular, these are the provisions for colonization resistance, i.e. mechanisms preventing colonization of the host organism by pathogens and opportunistic pathogens, and stimulation of the physiological and digestive activity of the gastrointestinal tract. Other essential functions include detoxification, stimulation of the synthesis of biologically active substances and renovation of surface cells of the intestinal mucous membrane (that occurs every 48 hours); furthermore, sustaining high levels of the complement (a set of immune proteins), lysozyme (enzyme lysing, or destroying bacterial cell walls), secretory immunoglobulins, interferon and sundry cytokines (signal molecules) important for the realization of natural immunity.

The bacterial activity of the normal microflora in the struggle against pathogens depends on the output of lysozyme, hydrogen perodixe, lactic, acetic, propionic, butyric and other organic acids, and metabolites decreasing the acidity of the medium. In our view, antibiotics-type substances like bacteriocins and microcins play a leading part in this competition.

For instance, bacteriocins secreted by bifidobacteria and lactobacilli suppress the pathogens of dysentery, cholera and abdominal typhoid. They are found to be also active in inhibiting bacteria belonging to the genera

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Destruction of SHIGELLA FLEXNERI (a) by bacteriocin-producing Lactobacillus acidophilus cells (bj (X 60,000). Photo, O. Rybalchenko.

Salmonella, Listeria, Streptococcus, Staphylococcus as well as numerous opportunistic pathogens of the Entero-bacteriaceae family (like Klebsiella, Enterobacter, Hafnia, Serratia, Proteus, Citrobacter, Providencia etc.), fungi of the Candida genus, and so on. Dr. Oksana Rybalchenko (Department of Medicine of St. Petersburg State University) has obtained new data on the change of the cell ultrastructure of the pathogenic and conditionally pathogenic enterobacteria, including the genera Shigella, Klebsiella, Proteus, Citrobacter and fungi of the Candida genus, when acted upon by the bacteriocin-secreting Lactobacillus acidophilus cells. Destructive processes affect not only the cell wall of enterobacteria, but also the cytoplasm and the cell nucleus; the percentage of lysed (destroyed) forms increases at the populational level. Bacteriocins and bacteriocin-like substances (microcins), in contrast to antibiotics, exert a milder antimicrobial action on the indigenous microflora and as such, should be put among a new generation of effective therapeutic remedies.

The normal intestinal microflora is actively involved in the metabolism of substrates of plant, animal and microbial origin. This applies above all to the fermentation of glucose, lactose, starch, cellulose and other substances. It plays an important part in the metabolism of proteins, and of nitrogen- and carbon-containing compounds, and in the recirculation of bile acids. As proved by experiments in recent years, the intestinal microflora - in particular, bifidobacteria, lactobacilli, and enterococci-can bring down the concentration of cholesterol in blood, of lipids in blood serum, thus helping prevent atherosclerosis. The binding of nitrogen by microbial metabolites prevents portal-systemic encephalopathy, while the binding of phosphates lowers the risk of renal insufficiency. Hydrolysis of oxalic acid derivatives (oxalates) inhibits the formation of nephroliths (stones) resulting in the disease known as nephrolithiasis. Lactic-acid bacteria are capable of inactivating histamine, thus easing allergy symptoms. Hypertonic cases partaking of yogurt fermented by Lactobacillus and Saccharomyces show a drop in arterial pressure.

The normal intestinal microflora produces biologically active compounds-volatile or short-chained fatty acids regulating the absorption of sodium, potassium, chlorine and water ions, and ions of calcium, magnesium and zinc, sustaining thereby a water, electrolytic and acid-alkaline balance in the organism. Bacteria synthesize vitamins K, B-thiamine, B2-riboflavin, B3-nicotinic acid, B6-pyridoxine, B12-cyanocobalamine as well as pantothenic and folic acids.

The normal intestinal microflora is likewise involved in providing nonspecific protection realized by activation of macrophages; it stimulates lymphoid tissue and acts upon immunocompetent cells. Immunoglobulins produced by this flora come to be implicated in an intricate mechanism controlling the population of obligate parasites, pathogens including, by blocking their adhesion (attachment) to the epithelium of the mucous membrane and neutralizing their agglutination (sticking together and subsequent precipitation). There are also other bacteriocidal mechanisms at work. Secretory immunoglobulins are all-important for intestinal immunity.

Bifidobacteria, bacteroides, lactobacilli and escherichia work to improve native (congenital) immunity. It has been

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demonstrated that one peptide of the cell wall of bifidobacteria and lactobacilli activates a lympho-proliferative response to T- and B-cell mitogens (substances responsible for cell division, mitosis in particular), thus inducing generation of cytotoxic T-lymphocytes and production of immunoglobulins; this peptide stimulates the cytotoxic function of natural killers and the digestive activity of macrophages; it also stimulates the synthesis of cytokine. An immune response occurs as a consequence of cooperative interactions (synergy) of T- and B-lymphocytes, and macrophages associated with the activation, proliferation and differentiation of immunocompetent cells. Their interaction with bifidobacteria, bacteroides, escherichia, enterococci and lactobacilli is playing an important role in sustaining local immunity, in the stimulation of phagocytosis, and in the synthesis of immunoglobulins, interferons and cytokins (both pro-and anti-inflammatory).

The immune system of the human organism is remarkable for high mobility, its constant reactive preparedness and sensitivity to various exogenic and endogenic effects. This determines the variability of the quantitative composition and correlation of the immune system components at any particular moment of time. Such characteristics constitute a groundwork of the immunological mobility concept advanced by Acad. Rem Petrov. The gist of this concept boils down to the following: there is a definite duality of state (condition) proper to the system of immune defenses. On the one hand, its quantitative composition and correlation of components are in a constant flux under the action of antigen stimuli penetrating the organism or originating within it. On the other hand, the organism seeks a definite equilibrium of the immune system components to enable an adequate response in any concrete situation. This makes the immune system ever-changeful, persisting in perpetual motion.

The immune system is a specific indicator of the impact of unfavorable ecological factors on the human organism. The same is true of the response of the normal microflora to the effect of many damaging factors.

Thus, the symbiotic normal microflora carries out these functions: together with the mucous membrane of the intestine it operates as a barrier preventing microbes and toxins from getting into the organism; it produces biologically active substances responsible for the high antagonistic activity of the resident microflora versus pathogens (opportunistic pathogens, too); it participates in the utilization of food substrates and xenobiotics; it synthesizes amino acids, proteins and vitamins; intensifies intestinal absorption of Ca and Fe ions; and it controls humoral and cellular immunity.

DYSBACTERIOSIS: MICROECOLOGY IN TROUBLE

Should the effect of adverse exogenic and endogenic factors exceed a certain threshold value (maximum), the equilibrium of microbiocenoses gets upset, and this causes microecological and immune dysfunctions. As a result, potentially dangerous (opportunistic) microorganisms gain ascendancy in a biotope; genetic exchange is stepped up to give rise to clones with genomic "islands" of pathogenicity; besides, microorganisms develop drug resistance. This process may lead to grave disorders, such as dysbiosis or dysbacteriosis. The former applies to the biocenoses of bacteria, viruses, protozoa and fungi. The latter (dysbacteriosis) holds for bacteria and microscopic fungi only. We know of the bacterioses of the skin, mouth cavity, intestine and of the urogenital system.

Back in the 1970s Acad. Alexander Bilibin (USSR AMS) pointed to the following trend: while the interest in the normal microflora had slackened somewhat with the onset of the antibiotics era, it experienced a revival when clinicists working up infection cases came up against the side-effects of antibiotics therapy. It was demonstrated that the open-ended use of antibiotics and chemotherapeutic

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drugs of a broad spectrum of action provokes intestinal dysbacteriosis.* Today it is viewed as a clinical and laboratory syndrome characterized by a change in the qualitative and/or quantitative composition of the normal microflora, by metabolic and immunological dysfunctions, and by the translocation (transfer) of microfloral species to foreign biotopes and their excessive growth.

Some pathogenic and opportunistically pathogenic bacteria thrive in overly large colonies (-10⁶) regulated by a system of collective behavior now known as QS (Quorum Sensing). This phenomenon was first described relative to the mechanism of luminescence in sea bacteria. It came out that regulatory cell-to-cell signals activate cells toward cooperative synergic interaction by turning what looks like chaotic cell communities into a kind of multicellular organism (symbiosis) composed of millions of copies. Concerning this process, Acad. Alexander Ginzburg (director of our research institute) elaborated: that QS type behavior is implicated in the regulation of a broad range of physiological processes, including bioluminescence, formation of biofilms, synthesis of the secreted factors of pathogenicity and antibiotics, transfer of conjugative plasmids and even replication. The formation of biofilms by conditionally pathogenic bacteria and yeast-like fungi of the genus Candida cultivated on a solid nutrient substrate was demonstrated by Dr. Oksana Rybalchenko of St. Petersburg.

In the event of intestinal dysbacteriosis bacteria of the transitory microflora may infiltrate the cytoplasm of enterocytes, a process viewed as a symptom of endocytosis, when a cell of a macroorganism captures particulates, liquids and macromolecules. Incidentally, conditionally pathogenic bacteria (like Staphylococcus aureus, hemolytic Escherichia coli, Klebsiella) and fungi of the genus Candida include strains capable of synthesizing substances that destroy lactobacilli; such strains have been detected at the St. Petersburg State Research Center of Highly Pure Biopreparations and at our N.F. Gamaleya Institute of Epidemiology and Microbiology.

The clinical picture of intestinal dysbacteriosis is characterized by certain specific and rather deceptive symptoms. The most frequent ones are expressed in the diminished colonization resistance, and in the suppression of the immune system as well as in the increased sensitivity to infections. Many clinicians in this country justly regard such pathologies as crucial factor behind the ever growing incidence of grave maladies of the alimentary, respiratory and urogenital tracts, both in the acute and in the chronic form.

It has been established that the above condition of a macroorganism is due not only to a significant drop in the population of the normal microflora: the spectrum and level of its antagonist activity with respect to pathogens is likewise down. This downtrend results in the appearance of microbial associations characterized by increased virulence and capable of provoking self-inflicted infections of the organism. These associations form what we term "hospital strains" which, if spreading, cause indoor infections in clinic. Dysbacteriosis may be responsible for an increase in the level of histamine in organs and tissues and for sensitization (enhanced sensitivity) of the organism and allergies.

* See: L. Vorobyeva, "Bacterial Kingdom", Science in Russia, No. 5, 2005. - Ed.

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PROBIOTIC PREPARATIONS

A number of preparations are used for the prevention and treatment of dysbacteriosis. These are probiotics, prebiotics and synbiotics. The probiotics are live microorganisms and substances of microbial origin; administered in the natural, noninvasive mode they have positive effects on the physiological, biochemical and immune reactions of the host organism. These are medicines containing strains of obligate (Bifidobacterium, Lactobacillus, Escherichia, Enterococcus) or transitory (Saccharomyces and aerobic apathogenic sporoforming Bacillus spp.) microorganisms. There are monocomponent (bifidum-bacterin, lactobacterin, colibacterin, sporobacterin, bactisubtil), polycomponent (bificol, bifiform, acylakt, linex), sorptioned (bifidumbacterin forte, florin forte) and metabolitic (hylak forte) probiotics. All of them normalize microbiocenosis and, therefore, exert a remedial effect on the digestive, metabolic and protective function of the intestine and organism as a whole.

Now what concerns prebiotics: different in their mechanism of action, they belong to different pharma-cotherapeutic groups as well. But they cooperate in stimulating the growth and further development of the normal intestinal microflora. This group includes lactulose, oligosaccharides, calcium pantothenate, lysozyme, ambene, and other preparations.

And last, the synbiotics-preparations obtained through rational combination of pro- and prebiotics. Here in Russia we use the polycomponent biocomplexes Normoflorin-B and Normoflorin-L containing B.bifidum and B.longum, or L.acidophilus respectively; biovestin-lacto, supplemented with the biomass B.bifidum, B.adolescentis and L.plantarum; multidophilus containing maltodextrin and the biomass of B.bifidum, L.acidophilus and L.bulgaricus; bifidobac including fructooligosaccharides derived from the topinambour (top-inambo) plant, bifidobacteria and lactobacilli; bifidum-bacterinmulti enriched with different Bifidobacterium species (B.bifidum, B.longum and B.adolscentis).

Probiotics should be prescribed depending on the extent of microbiological disorders and intestinal pathology and with due account of the main disease. Some cases can make do with prevention, but others need treatment. It has been shown that these preparations contribute to a significant increase in the relative and absolute number of B-lymphocytes accompanied by a decrease in the relative and absolute number of T-lymphocytes. Due to its universal immunomodulating properties (including immunostimulation and immunosuppression alike), the microflora is playing an important role in immune response formation. Thus, bacterial liposaccharides and muramylpeptide present within the cell wall of normofloral species have an immunoregulating effect. Therefore probiotics are good both in the acute form of the disease and after (because of their ability to suppress pathogens).

May I stress it once again: preparations based on normal microflora species are in our arsenal against pathogens and opportunistic pathogens and, in addition, they help restore a normal biocenotic balance and upgrade the immune status of the human organism at large.