У статті наведені та обговорені результати мікробіологічного дослідження мікрофлори кишечнику пацієнтів із хворобою Альцгеймера. Висвітлені результати in vitro тестів по вивченню біологічних властивостей індигенної та умовно-патогенної мікрофлори кишечнику пацієнтів із хворобою Альцгеймера та різним ступенем дисбіозу.В статье приведены и обсуждены результаты микробиологического исследования микрофлоры кишечника пациентов с болезнью Альцгеймера. Освещены результаты in vitro тестов по изучению биологических свойств идигенной и условно-патогенной микрофлоры кишечника пациентов с болезнью Альцгеймера и разной степенью дисбиоза.The article presents and discusses the results of microbiological study of the intestinal microflora of patients with Alzheimer's disease. The results of in vitro tests to study the biological properties of the indigenic and opportunistic intestinal microflora of patients with Alzheimer's disease and varying degrees of dysbiosis are highlighted

Dovgan, R.

Ivakhniuk, Tetiana Vasylivna

Ivakhniuk, Yurii Petrovych

Makarenko, O.

Molozhava, O.

Ивахнюк, Татьяна Васильевна

Ивахнюк, Юрий Петрович

Івахнюк, Тетяна Василівна

Івахнюк, Юрій Петрович

Electronic Sumy State University Institutional Repository

Modem Science — Modemi véda 2019 Ne 4 IN VITRO TEST-RESEARCH OF THE INDIGENOUS INTESTINAL MICROFLORA OF THE PATIENTS WITH ALZHEIMER'S DISEASE Tatyana Ivakhniuk, Fura Ivakhatuk, Sumy State University, Olha! Taras Shevchenko National University of Kyiv, Oleksandr Makarenka, Interregional Academy of Personne! Management, Bogomolets National Medical University treatment qf disbietic disorders af the intestines, especially for the patients with Alzheimer = disease (4D). in our opinion, remains the personalised therapy an the basis of anes own microflora (Lactobacillus spp. and Byfidumbacteium zpp.) of the intestines of the patient: with.1D became the objective of our study. During the microbiological irepection of 2] patient: with AD it had been found out that at 100% of the examined patients showed the qualitative and quartitative dvshiotic charges of the intestines of different ecterts and qvmptome: in 39.1 = 0.04% af cares dvsbacteriosis of the I degree was registered: in 23.6 = 0.0320 — the IT degree and in 33.3 = 0.03% — IT degrees. After cultivation of the “intestinal” strains of the indigenous micrgfiora on skint neil within 43 hows areliable imerease in the adhesive activity of the all studied bacteria in comparison with the control has been revealed fp < 0.05). gastrointestinal act, Lactobacillus. Introduction. The gutmicrobiotaisa “blackbox™,drawingattention of heresearchers for “interpretation” and understanding of the pathogenesis of many human diseases today. More than 70%. of species of microorganisms resist microbiological cultivation, Le. they cannot be isolated in a pure culture in artificial nutrient media and are identified by means of classical methods of microbiology. The technological breakthrough allowed taking a new step from a simple detection of microorganisms to understanding of their fimctions and a role m organism homeostasis. New methodological approaches allow analyzing all the set of the composite processes, happening at the cellular level or m the whole live organism — genomics, transcriptomics, metagenomic sequencing, proteomics and metabolomucs, which completely transformed our ideas of the structure and function of the “mvisible body” [1]. The gut microbiota actively affects the transformation processes of the molecules of proteins, fats and carbohydrates, synthesis of vitamins m the GIT, mtestinal regulation, takes part in the processes of detoxication and regulates permeability of 136Modem Science — Modermi vada 2019 Ne4  some substances through the mucous membrane of the gui. Studying of the interrelation between the gut and the bram, that is the so-called gastrobrain axis (gut-braim-axis), by means of which the brain has an impact of the function of the gastrointestinal tract and the last one — vice-versa, 1s modem and relevant (2, 3]. At the same time, the main basic components of the microbiota-gut-brain axis is the central nervous system, neuroendocrinal and the neurommoume systems, the autonomic nervous system, and the system of the nerve ganglia of the mtestines and the gut microbiota. These components form a complex multiple-factor network, by means of which the signals from the bram can affect not only the motor, sensory and secretory activity of the intestines, but also its microbiota. And on the contrary, visceral signals from the microbiota-mediated area of intestines, significantly influence brain functions [4]. It is important to emphasize that the probiotics bacteria, applied at certam human diseases and morbid conditions colonize the intestines, can affect the central nervous system through the products of a number of neurotransmutters and biological substances: and acetylcholine. A part of these substances is capable to take effect not only on the mesenterial, but, first of all, on central nervous system [5]. Today more than 60% of the fimctional foodstuffs are used for the purpose of the influence on the digestive system. Prebiotics and probiotics are the drugs, widely used in the world [6]. Despite various formulations of the concept of “probiotics”, most of researchers call them the medicines contammng certain strains of microflora of a healthy human body ma quality of the active Ingredient. It is known that normally bacteria, living im the mucous membrane have their antagonistic effect, concemmng the pathogenic and opportunistic microfiora and provide a vitamim-providing and enzymatic fimection [7] When making pro-biotic drugs the strains of microorganisms, which fulfill certain have to be chosen. They are as follows: 1) safety of the strains, intended for their mtroduction to the structure of probiotics; 2) the existence of antagonistic properties to the pathogenic and opportunistic microfiora; 3) stability to lytic enzymes of saliva (lysozyme), digestive enzymes (pepsm, lipase) and to bile 4) resistance tothe action of gastric juice 5) the adhesive activity and colonial resistance; 6) resistance to antibiotics; 7) the higher specific growth rate of pro-biotic cultures m comparison with a comensalna microflora that allows them to master nutritious substrate faster and consequently, to increase the productivity of cells of pro-biotic strains; 8) the stram has to be technological by production (stable during cultivation and other stages of the technology process); 9) mmune and modulatory and munogenous probiotic action [7]. That is, probiotic strains of microorganisms have to be resistant to antibiotics, due to the synthesis of antibiotic-lke substances as well, so a probiotic and an antibiotic have to work in common, that is the additive effect has to be implemented. However, the widespread introduction of gene-modified strains of microorganisms in the medical practice is limited to a potential unpredictable impact of such microorganisms on the host’s organism (a person or an animal) and also on ecosystems. 137Modem Science — Modemi veda 2019 Ne 4 Some researchers consider that it can be connected with the emergence of the new properties im introduced species, enhancing their competitiveness and also disturbance of the balance of ecosystems. Besides, the possibility of mcontrollable transfering of a recombinant DNA to new hosts is actively discussed. At the same time many researchers ; confirmed the environmental safety of the recombinant microorganisms used as the effective bacteriotherapeutic drugs [8]. There are still a lot of tasks to be solved. Proceedmg from the stated above, at the present stage of development the pnority direction of prevention and treatment of dysbiotic disorders of the intestines, especially for the patients with Alzheimer’s disease, in our opinion, remains the personalized therapy on the basis of one’s own indigenous microflora. Studying of the adhesive properties of the representatives of the mdigenous microflora (Lactobacillus spp. and Bifidumbacteium spp.) of the intestines of the patients with AD became the objective of our study. Materials and methods. Dunmg the research the condition of the gut microfiora was specified accordmg to the methodical structions [9]. For the purpose of obtaming of Lactobacillus spp. and Bifidumbactemm spp. isolates we have conducted a microbiological research of the gut microfiora of the patients with Alzheimer’s disease (AD) (mn = 21). Studying of the adhesive activity of Lactobacillus spp. and Bifidumbacteium spp. isolates was camed out accordmg to the techmique of VI. Bnlis and coauthors. [10]. The statistical processmg of the obtamed results was cared out by means of the Statistica 6.1 software package with the use of the parametrical Student's t-test. Results and their discussion. Analyzing the results of the microbiclogical research of the excrements of the patients with AD we had found out that 100% of the exammed patients showed the qualitative and quantitative dysbiotic changes of the imfestines of different extents of manifestations: I dysbactertosis degree was repistered in 38.1> 0.04% 1% of cases: I degree — in 28.6= 0.03%; I] degree — im 33.3 = 0.03%. Analyzing the qualitatrve and quantitative structure of the mdigenous microbiota of the patients with AD, it had been established that the most evident qualitative and quantitative disturbances of the content of Lactobacillus spp. and Bifidumbacterum spp. ‘rere revealed in the patients with the TT and III degree of eut dysbiosis. The quantity of the indigenous microfiora authentically decreased (p <0,05) in companison with the indices of the patients with dysbiosis degree I; emtically low quantitative mdices of Lactobacillus spp. were registered in the patients with AD and the dysbiosis degree IIT (lg 4.482 0.15 CFU), and Bifidumbacterum spp. — the patients with dysbiosis degree II (lg 3.7 20.2 CFU). Such qualitative and quantitate changes in the gut mucrofiora of the patients with AD reduce colonial resistance and can be comnected also with a decrease in the adhesive potential of the representatives of the mdigenous microflora. The value of the adhesive characteristics of the bacteria for a macroorganism can be considered from two aspects. On the one hand, the adhesive potential of the indigenous 138Modem Science — Modermi véda 2019 Ne4  microfiora is one of the factors of realization of colonial resistance of the mucous membrane of the imtestmes and an obstacle of joming to the receptors of the mucous membrane of the pathogenic microorganisms. On the other hand, in case of development of dysbiotic disorders the adhesive properties of the opportumistic microfiora are considered as a pathog nicity factor as they allow mucrobes to be fimed on the surface of the skin, mucous membrane and to colonize this biotope, reaching a certain population level [11-12]. In the course of studying of the indicators of Lactobacillus spp. (n = 41) and Bifidumbactermm spp. (n= 33) adhesion to erythrocytes of blood 0 (1) of the blood type we have detemnined that among the isolated strains from the patients with AD there was no one with a high adhesion degree (fig. 1). @ Lactobacillus spp. OD Bifidumbacteium spp. Fig. 1. The quantity of the lactobacteria and bifidobacteria, isclated from the intestines of the patients with AD on the adhesion index (1 —the high Al, 2 — the medium AL, 5 — the low AT).        okeee®   1 2 3 In the analysis of the indicators of the adhesion index of microorganisms (AIM) we have established the distinctions: among the “intestmal” Lactobacillus spp. isolates, obtained from the patients with Alzhemer’s disease, the average degree of adhesion (the total value of AIM 3.612 0.05 bact. cell /er) showed the strains, isolated from the patients with I and IT degree the dysbiotic changes of the mtestines. All the patients wath the III intestinal dysbiosis degree Lactobacillus spp. isolates had the low adhesive activity (the total value of AIM 1.93+ 0.03 bact. cell / er). According to the obtamed results on studying of the adhesive properties of Bifidumbactemm spp. isolates it has been determmed that according to AIM these strams were characterized as low (42.4%) and medium (65.9%) on the level of adhesion (fig. 1), and all climiecal Bifidumbacteium spp. isolates, which had them low adhestve activity (AIM values = 2.1220,03 bact. red cell), were isolated from the patients with the I and I] degree of dysbiotic intestine changes. In the second series of the experiment with the isolates of indigenous intestinal microflora, we cared out cultivation of the clinical strains of Lactobacillus spp. and Bifidumbacteimm spp., obtamed from the patients with AD on slam milk within 48 hours: 1 ml of Lactobacillus spp. suspension or Bifidumbacteum spp. in the number of 139Modem Science — Modemi véda 2019 Ne 4 0.5 units, according to the McFarland standard were added to 10 ml of sterile slam milk. After culitvation of these strams ther adhesive activity were studied. As aresult of the analysis of the obtamed results an increase in the adhesive activity of the all studied bacteria in comparison with control is considered to be reliable (p= 0.05) (the first senes of the expermment — pnmary isolation from he patients): Lactobacillus spp. and Bifidumbactemm spp. were characterized by them medium adhesion activity. So, een ee nts SPP. (n= 41) mm the sampling made 3, 860.07 bact. /red cell (from 2.91 to 6.9 bact. /red cell): 12.2% of lactobacteria had the high adhesive activity, 24.4% — the low adhesive activity and 63.4%. — the medium adhesive activity. Among bifidobacteria isolates (n = 33) after cultivation in the sterile skim milk the AIM of Bifidumbacteium spp. in the sampling made 3, $620.07 bact./red cell. (The AIM indices fluctuated from 2.91 to 6.9 bactured cell_): 21.1% were considered as high- adhesive strains, whereas most of them showed the medium (66.7%) adhesion ability and only 12.2% of bifidobacteria strains had thei low adhesive potential. When studying the sensitivity of the isolated microorganisms (Lactobacillus spp. or Bifidumbactertum spp.), which was camied out by disco-diffusion method with the use of paper disks by LLC Pharmaktv production (Kiev, Ukrame.) and Himedia (India), it has been established that 29.27% of Lactobacillus spp. strains showed their resistance to gentamyem; 24.39% — to chloramphenicol and 9.73% p — to erythromycm. When studying the results of the antibiotic record of Bifidumbacterium spp. tt has been revealed that the preatest number of strams (24.2472) was resistant to erythromycm. Tt has been considered that antibiotic-resistance is a useful property of pro-bictic strains of Bifidobacterium and Lactobacillus bacteria that provides the high performance of probiotics for the complex therapy of infectious diseases (joint reception of probiotics and ant biotics). However, m recent years the situation cardmalhy changed, it is connected, first of all, with the global distnbution among antibictic-resistance phenomenon microorganisms that can lead to a number of consequences, adverse for the person. It is connected with the fact that such strams of pro-biotic bacteria can bear the “silent” genes of antibiotic resistance or have their own mechanisms of resistance to For this reason, creation of the personalized bank of indigenous intestinal microflora (bacteria of Bifidobactertum and Lactobacillus type) which can form the further basis for is televant and perspective direction for the future of the Conclusions. Dunng the microbiological mspect on of 21 patients with AD it had been found out that at 100% of the examined patients showed the qualitative and dysbiotic changes of the intestmes of different extents and symptoms: m 348.1 = 0.04% of cases dysbacteriosis of the I degree was registered; in 28.62 0.03% — the II degree and m 33.3 + 0.03% — III degree. The erttically low (p <0,03) quantitative indices of Lactobacillus spp. were registered im the patients with AD and the III degree of dysbiosis (lg 4.48 = 0.15 CFU), and Bifidumbacterum spp. — the patients with he II degree have dysbioais (Ig 3.7 = 0.2 CFU). 140Modem Science — Modemi véda 2019 Ne4  34.1% of strains of Lactobacillus spp. obtamed from the pahents with AD showed the medium adhesion degree (ATM values = 3.6120.05 bact. ‘red cell), obtaimed from the patients with I and I degree of dysbiotic changes of intestines; in 33.3% of the patients with the II] degree of the mtestme dysbiosis Lactobacteria spp. isolates had ther low adhesive activity (AIM values = 1.9520.03 bact. / red cell). Bifidumbacterum spp. isolates were charact rized by the low (42.4%) and medium (65.9%) adhesion level. and all the clinical isolates. which had their low adhesive activity (AIM values = 2.1240.03 bact. ‘red cell.), were isolated from the patients with ] and I extent of dysbiotic changes of intestines. After cultivation of the “intesti al” strains of the indigenous microfiora on skim milk within 48 hours a reliable mcrease im the adhesive activity of the all studied bacteria in comparison with the control has been revealed (p< 0.05) (the first series of the experiment — primary isolation from the patients): Lactobacillus spp. and Bifidumbacterum spp. The prospects of further researches in this direction. It is expedient to carry out restoration of the colomial resistance of the intestines in the patients with Alzheimer’s disease on the basis of using of high-adhesive strams of bifidobactena and lactobacilli. One ofthe ways of the long-time storage of microorganisms is drying that allows keepmg their maim properties. However, tt1s reached not m case of all the ways of drying therefore studying of the various ways of freeze drying of Lactobacillus spp. and Bifidumbacteium spp. will be the followmeg step of our researches that will allow keeping their maim biological properties (adhesive properties and the antagomistic activity, concemming the opportunistic intestinal microfiora). References: 1. Chemevskaia E. A. Mikrobiota kishechnika pri kriticheskih sostoiantyah (obzor) /E. A. Chemevskaia, N. V. Beloboredova // General reanimatology. — Ne 14 (3).- 2018. —. 96-118. (in Russian) 2. Bondarenko VML Znacheme nervnoy sistemyi pr vospalitelnyih zabolevaniiah loshechnika / VM. Bondarenko, E.V. Ryabichenko () Zhum. nukrobiol. — 2011. - Ne 1. —F 92-100. (In Russian) 3. Parfenov AL Chto nam dal vekovoy opyt pomanrya simbiont- noy kishechnoy mukroflory / AT. Parfenov, VM. Bondarenko /! 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In vitro test-research of the indigenous intestinal microflora of the patients with Alzheimer's disease

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In vitro test-research of the indigenous intestinal microflora of the patients with Alzheimer's disease

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