INTESTINAL DYSBIOSIS, HUMAN HEALTH AND FUNCTIONAL NUTRITION

The interest of scientists, physicians, pediatricians, general practitioners to the problem of intestinal dysbiosis is caused by the fact that there are very close relations between the intestinal microbiocenosis disorders and organic and functional pathology not only of the gastrointestinal tract, but also of other organs. For more than 50 years probiotics have been the leading tool for correction of intestinal microbiocenosis. However, the response of intestinal microbiota to exogenous intake of probiotics is variable, or is completely absent. The article presents the analysis of the literature data and results of our own experimental studies on human volunteers, fully confirming previous findings on laboratory animals, according to which the probiotic microorganisms are foreign and do not survive in the gastrointestinal tract of humans and animals, and their interaction with the host organism has the character of «host against probiotic». The results of the comparative study on the effectiveness of 18 modern preparations for correction of intestinal microbiocenosis at antibiotic-associated dysbiosis in experiments on animals are given. The comparative experimental research on the effectiveness of modern preparations that are frequently used in clinical practice as well as recent innovative commercial and experimental preparations (Actoflor-C. Stimbifid plus, Stimex) regarding intestinal microbiota recovery at antibioticassociated dysbiosis in conventional white mice was carried out. IgorYu. Chicherin1, Ivan P. Pogorelsky2, Irina A. Lundovskikh2, IlyaV. Darmov2, Marina R. Shabalina2 1 Scientific society «Microbiota», Sergiev Posad, Moscow Region, Russia 2 Vyatka State University, Kirov, Russia УДК/UDC 621.3 DOI 10.21323/2414–438X-2017–2–4-44-61 Для цитирования: Чичерин и.Ю., Погорельский и.П., лундовских и.а., Дармов и.в., Шабалина М.р. Дисбиоз кишечника, здоровье человека и функциональное питание. Теория и практика переработки мяса. 2017; 2(4):44-61. DOI:10.21323/2414– 438X-2017–2–4–44-61 FOr cItatIOn: chicherin I.Yu., Pogorelsky I.P., Lundovskikh I.a., Darmov I.V., Shabalina M.r. Intestinal dysbiosis, human health and functional nutrition. Theory and practice of meat processing. 2017;2(4):44-61. (In russ.) DOI:10.21323/2414–438X-2017–2–4–44-61 Чичерин И.Ю. 1, Погорельский И.П. 2, Лундовских И.А.2, Дармов И.В.2, Шабалина М.Р.2 1 Научное общество «Микробиота», Сергиев Посад, Московская область, Россия 2 Вятский государственный университет, Киров, Россия

The intestinal microbiota as a discrete organ, which is named in the scientific literature the «paracytological system» [1], plays a significant role in the vital activity of the host body: metabolism, detoxification, formation of innate immunity, development of colonization resistance against pathogenic and conditionally pathogenic microorganisms at the first stage of the infectious process development [2,3,4].The knowledge about the role of the intestinal microbiota is constantly extending and renewing towards an acquisition of new data about its functions as well as properties, which gave grounds to regard the intestinal microbiota as the main biogenic factor determining health or disease development [5].An imbalance in the abovementioned system due to intake of antimicrobials, changes in the habitat and nutrition character, gastroenterological diseases as well as an impact of other factors leads to the development of the clinico-laboratory syndrome -dysbiosis [6,7,8,9,10,11,12,13,14].
A range of tools for prophylaxis and treatment of disbiotic intestinal microbiocenosis disorders is quite wide.For more than 50 years, a special place in it has been given to probiotics -preparations containing live microorganisms that are representatives of the normal colon microbiota [6,7].It is remarkable that only after decades of using probiotics in Russia, the Resolution of the Expert Council of 07.11.2015 on the effectiveness, safety and regulatory aspects of the use of probiotics in the Russian Federation and other countries [15] acknowledged on the basis of scientific publications (although with reservations), the possibility of the appearance of systemic infections (endocarditis, sepsis, meningitis, bacteremia, pneumonia), caused by lactobacilli when using probiotics.
According to Besselink, M.G.H [16], in 2004-2007, 15 clinics of the University Medical Center Utrecht (Netherlands) studied an effect of the multispecies probiotic preparation on reduction of the risk of infectious complications in 296 patients with pancreatitis in the multicenter, randomized, double-blind, placebo-controlled trial.During treatment, 24 patients in the group that received the probiotic died.In the control group of patients, who did not intake the probiotic, the mortality was 2.5 times lower.
The reasons for this are still unknown; however, in October 2015, the similar study carried out in the Czech Republic was terminated on the insistence of the Dutch scientists.
The group of Russian scientists did the experimental research with the aim of revealing a possible cause of lethal cases in patients of the university clinics, which gave evidence about the biologically hazardous potential of probiotic preparations.In the course of the investigations with the use of the laboratory animals, it was found that the potential hazard of probiotics consists in the ability of foreign microorganisms in their composition to initiate the translocation (transfer into the foreign biotope) of the intestinal microbiota into the blood stream and abdominal cavity due to the increased antigenic and allergenic load, which resulted in the death of more than 30 % of the experimental animals associated with the infectious toxic (endotoxic) shock with the development of bacteremia and peritonitis [17,18].
Therefore, the practice of clinical and experimental investigations clearly showed the problem of effectiveness and safety of probiotics.Only in 2015, did the experts acknowledge the fact of the potential hazard of probiotics [15]; however, the results of the experimental studies published in 2005-2006 [19,20], brought the scientists closer to understanding the fact that a single statement about the presence or absence of effectiveness of probiotic therapy or its transitory character is absolutely insufficient.
It was found that in violation of standards, to which probiotics have to correspond [7], their effectiveness in the animal experiments (as required by the United Nations Ethics Committee) was often not proved (Fig. 1).Primarily, there should be correspondence to the standards of microorganisms in the composition of preparations, which, in particular, have to be protected from an impact of the digestive enzymes and bile.Their effectiveness and safety for humans have to be proved experimentally; the genetic stability of microbial properties should be guaranteed; high quality lyophilization of microorganisms has to provide the high survival rate after peroral intake of probiotics.
In fact, immediately after the theoretical substantiation of inclusion of one or another microorganism as promis-ing for creation of a probiotic preparation on its basis, all activity of developers switched over the biotechnology of a new preparation and its promotion onto the market of probiotics.
The second very important stage of pre-clinical experimental substantiation of safety, harmlessness and effectiveness of microorganisms and supplementary ingredients included in the composition of probiotic preparations was excluded (with the tacit agreement) from the logical chain of actions in development and clinical trials of a preparation itself.This led to the following problems in clinical trials associated not only with an increase in dysbiosis in the population despite a significant range of probiotic preparations, but also with their safety.
Over a long period of time, there was no method for determination of bioavailability of probiotic microorganisms (survival during transit through the gastrointestinal tract); the mechanism of action of probiotic microorganisms (substitutive action, that is, the establishment in a biofilm of the intestinal mucosa) was not recognized; the safety of the use of probiotic preparations (biocompatibility of microorganisms in a probiotic composition with indigenous microbiota, the possibility of its translocation through the intestinal walls etc.) was not proved.
This problem was successfully solved by a group of Russian scientists, who developed, first of all, the universal method for differentiation of probiotic and indigenous microorganisms by obtaining labeled derivatives of probiotic bacteria isolated from intestinal contents of laboratory animals that stably inherited a trait of rifampicin resistance and preserved species characteristics [14,17,18,21,22,23,24,25].
Thirdly, in 2012, a universal indicator was introduced into the scientific use for the first time.The indicator characterizes the quantitative changes in the intestinal microbiota in animals in an experiment under an effect of the means of microbiocenosis correction, namely, «the rate of intestinal microbiota recovery» [27,28].The universal-ity of the indicator resides in the fact that it is possible to assess with its help an effect of both positive (the rate of recovery) and negative (the rate of inhibition) factors on total microbial content and the number of individual representatives of the intestinal microbiota.This integrative indicator, which includes all complex of factors affecting the intestinal microbiota, enables obtaining the quantitative characteristics of the process of recovery (inhibition) of the intestinal microbiota (CFU•g -1 •day -1 ) compared with the similar indicator that is determined in the control experiment on animals upon self-recovery of their intestinal microbiocenosis (ratio to control).
The results of the in vitro experiments on the study of bifidobacteria and lactobacilli survival in the gastric juice and duodenal contents of humans [29] show that during incubation of bifidobacteria and lactobacilli in the indicated biological media, the quantity of viable probiotic microorganisms was significantly reduced compared to their initial quantity up to individual cells in case of bifidobacteria.
A significant decrease (by 4-7 orders) in the number of the probiotic microorganisms entering the organism of the experimental animals was established [21].These data confirm the results of the previous studies in vitro [30,31] about extremely low survival of probiotic microorganisms in the aggressive media.
In the context of the above mentioned, it is necessary to note the results of the experiments, which were published in the work [32].According to the presented data, removal of probiotic microorganisms from the commercial biocomplex «Normoflorin-B1» increased the effectiveness of the preparation regarding recovery of normobiota in animals with antibiotic-associated dysbiosis almost by 60 times, and the live bacteria per se, which were removed from the biocomplex, did not have an ability to stimulate recovery of the intestinal microbiota when administered enterally to the animals with antibiotic-associated dysbiosis.Therefore, it was proved experimentally that not probiotic microorganisms but their metabolites made a significant contribution to normalization of the intestinal microbiocenosis disorder [32,33].
It can be stated that in the course of the transit through the digestive tract of the experimental animals, the number of probiotic microorganisms decreases to the value lower than critical, after which population survival, in general, is impossible.This data supported by the results of the study on species, tissue and individual specificity as well as heterology of probiotic bacteria for the organism of a new host [18], their incompatibility with his/her residential microbiota indicate ecological and functional marginality of probiotic microorganisms [34] and impossibility to change with their help inherent microbiota, which was already formed at birth both in healthy individuals [35], and in individuals with disbiotic changes in the intestinal microbiota [33,36].
The published results of the experimental studies on laboratory animals, which demonstrated on the factual The second very important stage of pre-clinical experimental substantiation of safety, harmlessness and effectiveness of microorganisms and supplementary ingredients included in the composition of probiotic preparations was excluded (with the tacit agreement) from the logical chain of actions in development and clinical trials of a preparation itself.This led to the following problems in clinical trials associated not only with anincrease indysbiosis in the population despite a significant range of probiotic preparations, but also with their safety.material the necessity to change the view on the problem of probiotics and probiotic therapy for the sake of patients' health, were not disproved.The opponents expressing their doubts state that the data obtained on laboratory animals should not be extrapolated to humans.Pediatricians are especially skeptical asserting that everything is different in children (children are not mice!) and constantly emphasizing the necessity to carry out studies with participation of volunteers to achieve full clarity in solving this complex problem.Indeed, this problem required solving several theoretical, methodological and organizational problems at the first stage of investigations; in particular, obtaining spontaneous rifampicin resistant (Rif r ) mutants of probiotic lactic acid bacteria L. plantarum 8Р-А3 with stably inherited species characteristics and a trait of antibiotic resistance (R-trait), growing them on the special culture media; adapting to human volunteers the method for initiation of experimentally antibiotic-associated dysbiosis that was developed for mice; detecting the representatives of the intestinal microbiota and marked according to the R-trait lactic acid bacteria L. plantarum 8Р-А3 in faeces of laboratory animals and volunteers using selective solid culture media, their quantification in 1 g of faeces; comparatively assessing the effectiveness of modern methods regarding correction of intestinal microbiota at experimental antibiotic-associated dysbiosis both in laboratory animals and human volunteers.
Before consideration and assessment of our own experimental results, it is necessary to note that developers and producers who have been engaged in probiotic therapy for decades, regarded all microorganisms isolated from healthy people as «good and healthy», and, therefore, promising for the use in the biotechnology of probiotics.
They, as well as physicians and patients, had a priori beliefs about probiotics as preparations with curative and prophylactic activities, which can be included into the treatment regimen for most diseases without experimental trial on animals of their effectiveness and safety.
It has turned out that this belief is far from the reality as it is the questions of effectiveness and safety of probiotic microorganisms as well as the regulatory aspects of their use that were discussed in the special session of the Expert Council in November 2015 in Moscow [15].Based on the results of the session, the experts stated in the Resolution that probiotics are live microorganism, which upon administration in adequate doses have a positive effect on human health.
The Resolution of the Expert Council [15] and the appeal of the experts to the professional medical community with a proposal to discuss the main problems that have arisen over the last years indicate that these questions have accumulated much faster than solutions have been found.
In the light of the results of the experimental studies that point to the involvement of the probiotic microorganisms to initiation of the intestinal microbiota translocation [17,18], the warning of M.D. Ardatsky and O.N. Minushkin [37] expressed as far back as in 2006 is perceived differently.
The authors are sure that it is necessary to take into consideration a potential hazard of the intestinal microbiota and its metabolites, when translocation of microorganisms into the non-residential biotopes and sterile cavities occurs.According to the data of the cited authors, penetration of the intestinal microbiota into the abdominal cavity leads to its infection and development of spontaneous peritonitis.With that, the mortality of patients with liver cirrhosis of B and C classes by Child-Pugh in this case reaches 50 %, while 69 % of patients had a recurrence within a year.
In a series of publications [14,21,26,27,29,30,31,32,34], which contain the results of their own experimental investigations, a group of the Russian scientists also actually appeals to the professional medical community with a proposal to look differently at the problem of probiotics and probiotic therapy.
Recently, the strategy for intestinal microbiota maintenance was reconsidered: the conclusive evidence was obtained with regard to the fact that the use of the traditional preparations (probiotics) based on live microorganisms is not the most effective and safe way for correction of microecological disorders in the intestine.
Low survival of lyophilized microorganisms (in capsules or in flasks) in the composition of probiotic preparations has been established experimentally: when plating suspensions of rehydrated lyophilized probiotic preparations (within the shelf life) on the special solid culture media, survival of microorganisms is 15-25 % of the total number of «live microorganisms» specified in instructions.Only insignificant part of remained probiotic microorganisms does reach colon, which results, among other things, in their low survival during the transit through the intestine -less than ten-thousandths of a percent of the initial quantity entered the stomach in the composition of the preparation.It was also proved that in the intestine, even at dysbiosis, viable indigenous strains (auto-strains) continue to reside and a probiotic that was created on the basis of any biotechnological «universal» strain is heterologous (foreign) and enters the antagonistic relations with auto-strains.
It is also necessary to take into account that probiotic microorganisms entered into the intestine have the lymphocytotoxic activity [38,39], which affects an ability of the patient's immune system to detect, which microorganisms (indigenous or foreign) enter the gastrointestinal tract.The presence of the undesirable for live organism properties in probiotic microorganisms points once more to a necessity of the individual approach to selection of a probiotic, the right choice of the course and single doses of a preparation.
At the first stage of our own research, carried out on animals and volunteers (8 individuals, one of whom was placebo-controlled) without the signs of the intestine microbiocenosis disorder, the transitory character of the presence of labeled (rifampicin resistant -Rif r ) lactobacilli in the gastrointestinal tract of animals and humans as well as low level of bacteria shedding with faeces were revealed (Table 1).
At the same time, an important regularity was revealed: survival of lactic acid bacteria L. plantarum 8Р-А3 (Rif r ) in the body of guinea pigs was lower than in the body of white mice, while survival in the gastrointestinal tract of volunteers was lower than in the body of white mice and guinea pigs.
The reason for the difference in survival of lactic acid bacteria L. plantarum 8Р-А3 (Rif r ) in the gastrointestinal tract of white mice, guinea pigs and volunteers, in our opinion, is the length of the intestine: with an increase in the length of the intestine (white mice -guinea pigsvolunteers) and the time of bacteria transit through the gastrointestinal tract, the numbers of lactobacilli decreased from 0.0000034 % (in the intestine of white mice) to 0.00000016 % (in the intestine of guinea pigs) and to 0.000000008 % in humans.
It is necessary to note another fact: the active phase of lactic acid bacteria L. plantarum 8Р-А3 (Rif r ) shedding from the body of volunteers lasted up to 5 days, while in white mice and guinea pigs it was up to 15 days.
At the second stage, the investigations were carried out on animals and volunteers with antibiotic-associated intestine dysbiosis induced by gentamicin peroral administration.The daily dose of gentamicin peroral administration was calculated for animals on the basis of the daily dose for humans with consideration for conversion coefficient.
In the course of the investigations, the process of recovery of indigenous lactobacilli under an influence of the peroral administration of labeled probiotic lactic acid bacteria L. plantarum 8Р-А3 (Rif r ) and possibility of survival of the latter in the intestine during their transit were studied.Based on the data of the quantification by the bacteriological method of the total microbial count and the number of individual representatives of the intestinal microbiota in the intestine of animals and volunteers, it is possible to suggest the development of the third stage of intestinal dysbiosis by I.K. Maksimov [40] or the third decompensated uncomplicated degree of dysbiosis by O.N. Minushkin et al. [7].
The bacteriological study of faeces from animals and volunteers during the experiment gives grounds to suggest two processes in animals: on one hand, a slow increase in the numbers of indigenous lactobacilli (the recovery rate of the intestinal microbiota on the 12th day of the experiment was 1.3•10 4 CFU•g -1 •day -1 for white mice and 7.2•10 4 CFU•g -1 •day -1 for guinea pigs), and on the other hand, an increase in the number of the probiotic lactic acid bacteria L. plantarum 8Р-А3 (Rif r ) in the intestinal contents in the first two days of their peroral administration, but quite a fast decrease and full elimination by 13-14 days of observation.
Lactic acid bacteria L. plantarum 8Р-А3 (Rif r ) in the quantity of several tens in 1 g of faeces were found in all 7 volunteers from 4 to 8 days (with delay of 2 days compared to animals).From the 9th day, immediately after termination of the per os intake of lactic acid bacteria L. plantarum 8Р-А3 (Rif r ), they were not detected any more in the faeces of three volunteers and, subsequently, the number of probiotic lactic acid bacteria L. plantarum 8Р-А3 (Rif r ) sharply reduced in the faeces of all volunteers; by the 12th day (the 4th day after termination of intake), labeled lactobacilli were not isolated.
The average value of the indicators of the recovery rate of indigenous lactobacilli in the volunteers from the experimental group (1,2•10 5 CFU•g -1 •day -1 ) practically did not differ from the rate of self-recovery of indigenous lactobacilli in the volunteer (biocontrol) (3,8•10 5 CFU•g-1•day -1 ) in the placebo controlled investigation, which suggests the absence of the probiotic effect of the peroral administration of probiotic lactobacilli.
The presented results give grounds to suggest that probiotic lactic acid bacteria L. plantarum 8Р-А3(Rif r ) being in the human digestive tract, do not exert their potential abilities to stimulate recovery, at least, regarding indigenous lactobacilli.It is obvious that in the body of volunteers as well as in the body of white mice and guinea pigs, probiotic lactobacilli remain to be foreign, do not survive in the intestine, do not multiply, do not produce their natural metabolites and do not have a stimulating effect on the indigenous microbiota.
In the conditions of antibiotic-associated dysbiosis, the maximum number of lactic acid bacteria L. plantarum 8Р-А3 (Rif r ) in the faeces of all volunteers was observed on the 4-8th days from the beginning of per os intake; however, already one day after the last intake of lactobacilli, on the background of an increase in the number of the indigenous intestinal microbiota and natural enhancement of its antagonism, they were not found in the faeces of 3 of 7 volunteers, and on the 4th day after termination of intake, lactic acid bacteria L. plantarum 8Р-А3 (Rif r ) were not detected by the bacteriological method in the faecas of all volunteers, similar to what was observed in the experiment in the conditions of intestinal normobiosis.
The results of the study showed that: -the intestinal microbiota effectively counteracted colonization of the intestine by foreign microorganisms.-the behavior of probiotic microorganisms was identical in the body of the experimental animals and humans.We also carried out the comparative experimental study on the effectiveness of modern preparations that are frequently used in clinical practice as well as recent innova-tive commercial and experimental preparations (Actoflor-C, Stimbifid plus, Stimex) regarding intestinal microbiota recovery at antibiotic-associated dysbiosis in conventional white mice.The characteristics of the preparations are given in Table 2.
The effectiveness of the peroral administration of probiotics was assessed by the rate of recovery of the intestinal microbiota in animals of the experimental groups compared to the rate of self-recovery of microbiota: the total microbial count, 1.8•10 5 CFU•g -1 •day -1 ; lactobacilli, 2.9•10 3 CFU•g -1 •day -1 ; bifidobacteria, 1.0•10 4 CFU•g -1 •day -1 ; Esche-  •day -1 in the animals from the control group, which did not receive preparations per os (the rate of self-recovery of the total microbial count and the individual representatives of microbiota was taken as 1).
The summarizing data on the comparative assessment of the effectiveness of the modern means for correction of the intestinal microbiocenosis disorders in conventional white mice with antibiotic-associated dysbiosis are presented in Figure 2.
As follows from Figure 2, there are significant differences in the effectiveness of the studied modern means for correction of the intestinal microbiota disorders regarding intestinal microbiota recovery in conventional white mice with antibiotic-associated dysbiosis.Among 18 preparations, only 3 (Stimbifid, Stimbifid plus and Stimex) were significantly superior by the rate of intestinal microbiota recovery compared to the control animal group (selfrecovery of the intestinal microbiota).
By the rate of intestinal microbiota recovery, 11 preparations were at the level of its self-recovery or slightly higher, and 3 preparations selectively stimulated the growth of lactobacilli.
Therefore, a necessity of the most effective and safe recovery of the autochthonous (indigenous) intestinal microbiota by the use of prebiotics and metabiotics, and their compositions -metaprebiotics was scientifically substantiated and experimentally proved.
Complete information about preparations intended for correction of intestinal microbiocenosis is critical for a physician.And similar to the case of administration of antimicrobials when physicians are ultimately responsible for prescribed antimicrobial therapy, they have to be absolutely sure in the effectiveness and safety of a prescribed preparation for correction of the intestinal microbiocenosis.
It is very important that the results of the theoretical and experimental, fundamental and applied studies find understanding in developers of the modern effective and safe preparations that affect the disturbed pathogenetic element of self-regulation -the intestinal microbiota.The choice of such means is a prerogative of a physician, who takes the final decision about prescription of the correction means, which will effectively stimulate recovery of the patient's autochthonous (indigenous) individual microbiota with highly specific qualitative and quantitative composition.

Conclusions
The microbiota of the digestive tract, which includes more than 5000 species of microorganisms is a very important discrete organ having many functions that are crucial for body health.Disturbance of the intestinal microbiocenosis structure is manifested as dysbiosis leading to symbiontic digestion disorder, which, in turn, is a cause of many systemic and local pathological processes developing in the human body.Treatment measures on correction of dysbiosis envisage the use of the preparations to correct intestinal microbiocenosis.The most numerous group of these preparations consists of prebiotics.The performed investigations proved conclusively that the use of the traditional probiotic preparations based on live microorganisms is not the most active and safe method for correction of microecological disorders in the intestine.
It was shown that after peroral administration to humans, probiotic microorganisms L.plantarum 8Р-А3 (Rif r ) lived their own life being in the antagonistic relations with the indigenous intestinal microbiota both in the conditions of normobiocenosis and dysbiosis of the volunteers' intestine experiencing the effect of bio-incompatibility of the «host against probiotic» type.
The low effectiveness of measures for correction of dysbiotic disorders of the intestinal microbiota by using probiotics gives grounds for revision of the strategy of maintenance and recovery of the individual intestinal microbiota, which was formed after birth.
In this connection, it seems expedient to further develop the evidential basis for detection of the effectiveness and safety of the created means for correction of the intestinal microbita disorders with the obligatory conduction of the experiments on animals and confirmation of the obtained data with the clinical trials.This sequence of action to a large extent will facilitate an improvement in quality and effectiveness of preparations, increase in their safety as well as physicians' and patients' confidence in them.

Fig. 1 .
Fig. 1.Traditionally established sequence of actions in development of probiotic preparations

Figure 2 . 1 richia, 3 . 3 •
Figure 2. Comparative experimental assessment of the effectiveness of modern probiotics, prebiotics, synbiotics and metabiotics in correction of the intestinal microbiocenosis disorders in animals with antibiotic-associated dysbiosis