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3BIOLOGY AND MEDICINE
INTESTINAL MICROBIOTA PATTERNS
OF ELDERLY PEOPLE WITH VARIOUS PSYCHOPHYSICAL STATUS
Abstract
Based on specific chemical markers of microorganisms more than 50 childbirth/species of microorganisms of a small intestine of the person was defined in blood of donors and elderly people. The analysis of patterns of microbiota of small intestine revealed existence of both big interindividual variations in specific structure of microorganisms, and significant intergroup distinctions in structure and quantity of microorganisms between groups of young and elderly individuals, and between groups of people of advanced age with the different psychophysical status. For elderly people with the expressed psychophysical insufficiency microbic patterns of small intestine with the high maintenance of "patobiont", such as Enterococcus spp and Staphylococcus aureus are characteristic in comparison with people of advanced age with moderate cognitive frustration and insignificant physical violations.
Keywords
microbiota of small intestine, elderly people, psychophysical status
Introduction. Commensal microorganisms live in a condition of harmonious symbiosis with each other and with the owner. But violation of balance among an intestines microbiota, as it is considered now, is associated and even can be a cause of cardiovascular diseases, inflammatory diseases of intestines, oncological disease, defeat of the central nervous system (CNS), which are demonstrated by shifts in the psycho-emotional sphere (behavior, mood, a depression, etc.) and violations of cognitive functions of a brain from light to heavy clinical phenotypes - dementia and Alzheimer's disease (Forsythe, Sudo, Dinan, et al, 2010). Exact mechanisms, by means of which the brain and intestines/microbiota carry out conversion, are not absolutely clear for today, but communication through autonomous nervous system, intestinal nervous system, immune system and hypothalamus-hyporphysical-adrenal axis (Collins, Surette, Bercik, 2012) are well documented. Accumulation in the last decades of experimental data (generally on models of animals) initiates search of proofs for confirmation of a hypothesis about existence of difficult communicative network interactions of commensal intestinal microbiota with a brain and its participation in development and function of a brain.
Purpose: to create patterns of microbiota of small intestine of elderly people with various psychophysical status.
Methodological base. The research included 31 persons of advanced age, living in the Chelyabinsk gerontological center". Average age of the surveyed was 79 ± 5 years. Doctors-clinical physicians of the center carried out monitoring of the psychophysical status of the elderly people. For assessment of the cognitive violations there was a classification of stages of distsircular encephalopathy of N. N. Yakhno with coauthors (2003), physical state - Barthel index (Barthel Index of Activities of Daily Living, 1993). By results of assessment of psychophysical status of surveyed were divided into 2 groups: 16 people with moderate cognitive frustration and insignificant physical violations - group 'Active longevity' and 15 people, with the expressed
Aleksandra Burmistrova
PhD in Medicine, Professor Head of Immune Microbiology and General Biology Department Dean of Biology Faculty Chelyabinsk State University Chelyabinsk, Russia burmal@csu. ru
Authors
Yulia Filippova
PhD in Biology Associate Professor Immune Microbiology and General Biology Department Chelyabinsk State University Chelyabinsk, Russia julse@rambler. ru
Arina Timofeeva
Postgraduate student Immune Microbiology and General Biology Department Chelyabinsk State University Chelyabinsk, Russia timofeew.sn@yandex. ru
cognitive insufficiency, which is shown in vascular dementia, and physical insolvency - group 'Mercy'. Elderly people of all groups had identical age-associated diseases: cardiovascular, bronchial-lungs, diseases of digestive tract and musculoskeletal device. Thus, individuals with obesity, oncological diseases, Parkinson and Alzheimer's illness, congenital violations of the central nervous system were excluded from the research. Group of comparison is randomly selected 19 regular blood donors of "Chelyabinsk Regional Station of Blood Transfusion" (group 'Donors') with average age - 39 ± 4 years.
Applied a method of a gas chromatography to studying of qualitative and quantitative structure of microbiota of small intestine - the mass of spectrometry with use of the gas chromatograph of the Maestro of GH 7820 (GH-MS, Russia) with the mass and selective detector Agilent Technologies of 5975 (USA). Samples of whole blood of donors and elderly people dried up at methanol addition, and subjected to a sour methanolysis in 1 M of HCl in methanol (1 hour, 80zs). At this stage, there was a release of fatty acids and aldehydes from difficult lipids of microorganisms and other cages and liquids in the form of methyl air and dimethylated. These components were twice extracted by hexane, dried up and processed within 15 min. at 80 °C of N, the O-bis (trimetilsilit) - tripleftoracetamid for receiving trimethylsilyl air of hydroxyacids and stirols. The received mix of air was entered into an injector of GH-MS system. Regular programs of the device were used for management and data processing. Chromatographic division of test was carried out on a capillary column with the methyl-silicone imparted phase HP-5 ms Agilent Technologies (USA). As a result of the conducted researches in tests of blood of donors and people of advanced age identified specific chemical markers (carbonic acids, aldehydes, sterols, sterins), which were correlated to the corresponding type and quantity of microorganisms, by means of the program developed by G. A. Osipov (Osipov, Parfyonov, et. al.).
During statistical data processing, serial statisticians and criteria were used: for an assessment of the central tendency - calculation of medians and 25-75 quartiles, for an assessment of the importance of intergroup distinctions - Kraskel-Wallice criterion with aposterior pair comparisons on Konover-Inman. Distinctions considered statistically significant at p<0,05.
Results. In blood of donors and elderly people on the basis of specific chemical markers of microorganisms we defined more than 50 childbirth/species of microorganisms of small intestine of the person (data are presented in the table 1).
Table 1. Microbic markers in blood of donors and elderly people with varios psychophysical status
Name of a microorganism Group 'Donors' (n=19) Group 'Active longevity' (n=16) Group 'Mercy' (n=15)
Streptococcus spp. 6 (0-26,0) 23,5 (11,0-39,0) * 17 (10,0-31,0)
Eggerthella lenta 164 (100,0-262,0) 144,5 (121,5-182,5) 139 (78,0-162,0)
Bacillus cereus 0 (0-15,0) 29 (24,0-39,5) * 27 (4,0-35,0) *
Peptostreptococcus anaerobius 18623 0 (0-0) 0 (0-0) 0 (0-0)
Cl. hystolyticum/Str. pneumonia 0 (0-0) 0 (0-0) 0 (0-0)
Nocardia spp. 210 (116,0-442,0) 389 (306,0-4993,0) 393 (256,0-600,0)
Peptostreptococcus anaerobius 17642 0 (0-0) 0 (0-0) 0 (0-0)
Moraxella
spp./Acinetobacter spp. 0 (0-2,0) 1,5 (0-8,5) 0 (0-0) **
Pseudomonas aeruginosa 0 (0-0) 0 (0-0) 0 (0-0)
Propionibacterium spp. 0 (0-0) 0 (0-0) 0 (0-0)
Bacillus megaterium 0 (0-0) 0 (0-0) 0 (0-0)
Clostridium propionicum 0 (0-0) 0 (0-0) 0 (0-0)
Steno trophomonas maltophilia 0 (0-0) 0 (0-0) 0 (0-0)
Bacteroides hypermegas 0 (0-0) 0 (0-0) 0 (0-0)
Actinomyces spp. 16 (13,0-18,0) 14 (12,5-17,5) 16 (14,0-18,0)
Pseudonocardia spp. 8 (6-11) 14 (9,5-15,5) * 13 (8,0-15,0) *
Streptomyces spp. 117 (60,0-214,0) 174,5 (121,0-204,0) 157 (111,0-214,0)
Clostridium ramosum 3129 (2338,0-3794,0) 3905,5 (3220,0-4323) * 3239 (2607,0-3595,0) **
Fusobacterium
spp. /Haemophilus spp. 0 (0-0) 2 (2,0-3,0) * 1 (1,0-2,0) */**
Alcaligenes spp. 14 (8,0-19,0) 22 (16,0-31,0) * 22 (12,0-28,0) *
Str. farmamarensis 0 (0-0) 0 (0-0) 0 (0-0)
Flavobacterium spp. 0 (0-0) 0 (0-0) 0 (0-0)
Rhodococcus spp. 32 (19,0-40,0) 37 (24,0-83,0) 43 (26,0-80,0)
Staphylococcus epidermidis 76 (54,0-101,0) 105 (46,0-141,0) 93 (72,0-128,0)
Porphyromonas spp. 0 (0-0) 0 (0-0) 0 (0-0)
Corynebacterium spp. 123 (61,0-181,0) 157 (113,5-187,5) 158 (149,0-202,0)
Lactobacillus spp. 3915 (3538,0-4945,0) 4673 (3660,5-4878,0) 4534 (3770,0-5441,0)
Campilobacter mucosalis 3 (0-5,0) 5,5 (3,0-7,5) * 5 (4,0-6,0) *
Candida spp. 255 (172,0-292,0) 399,5 (260,5-525,5) * 338 (294,0-473,0) *
Enterobacteriaceae spp. 0 (0-0) 0 (0-0) 0 (0-0)
Clostridium spp (C. tetani group) 70 (12,0-179,0) 136 (64,0-221,0) 98 (47,0-164,0)
Clostridium difficile 129 (90,0-155,0) 162,5 (120,5-193,0) 188 (137,0-224,0) *
Aspergillus spp. 11 (7,0-16,0) 21 (14,5-26,5) * 19 (12,0-23,0) *
Prevotella spp. 26 (19,0-29,0) 45 (39,5-55,5) * 43 (28,0-48,0) *
Eubacterium spp. 11778 (10388,0-12749,0) 7386,5 (5635,5-8575,0) * 7474 (5176,0-8876,0) *
Bacteroides fragilis 0 (0-0) 0 (0-0) 0 (0-0)
Staphylococcus aureus 210 (149,0-360,0) 225,5 (184,0-325,0) 356 (238,0-482,0) */**
Bifidobacterium spp. 3103 (1900,0-4499,0) 325 (105,5-672,0) * 276 (44,0-821,0) *
Helicobacter pylori 0 (0-0) 0 (0-0) 0 (0-0)
Clostridium perfringens 17 (12,0-19,0) 28 (21,5-37,0) * 29 (22,0-62,0) *
Enterococcus spp. 46 (31,0-55,0) 22,5 (9,0-34,0) * 32 (19,0-59,0) **
Chlamydia trachomatis 0 (0-0) 0 (0-0) 0 (0-0)
Propionibacterium freudenreichii 1755 (1270,0-2165,0) 2109 (1670,0-2445,5) 1996 (1721,0-2178,0)
Streptococcus mutans 324 (198,0-387,0) 297 (255,0-381,0) 280 (199,0-339,0)
Herpes spp. 27 (18,0-49,0) 61 (1,0-142,5) 28 (2,0-142,0)
Microprobe, campesterol 332 (279,0-385,0) 147,5 (113,0-182,5) * 131 (98,0-273,0) *
Nocardia asteroides 468 (441,0-721,0) 767,5 (497,0-885,5) 445 (371,0-672,0)
Epstein-Barre virus 15 (8,0-52,0) 163 (57,5-795,5) * 111 (71,0-295,0) *
Mikr mushrooms,
sitosterol 153 (98,0-343,0) 104 (80,5-355,5) 130 (76,0-238,0)
Propionibacterium acnes 80 (53,0-95,0) 72,5 (52,5-86,5) 55 (21,0-76,0)
Ruminicoccus spp. 880 (684,0-928,0) 789 (588,5-1010,0) 961 (705,0-1030,0)
Mycobacterium spp. 0 (0-0) 0 (0-0) 0 (0-0)
Blautia coccoides 0 (0-13) 27 (3,0-36,0) * 20 (0-68,0) *
Kingella spp. 12 (4,0-22,0) 17 (10,0-21,0) 17 (12,0-27,0)
Actinomyces viscosus 704 (630,0-751,0) 629 (488,5-787,5) 704 (605,0-766,0)
Propionibacterium jensenii 75 (26,0-142,0) 65 (36,0-86,5) 28 (0-56,0) */**
Cytomegalovirus 419 (275,0-570,0) 349,5 (278,5-415,5) 314 (190,0-411,0)
Endotoksin (sum) 0,25 (0,17-0,30) 0,47 (0,41-0,53) * 0,43 (0,26-0,46) *
Note: concentration of microorganisms x105, WHICH/g (concentration is calculated on chemical markers of microorganisms in blood).
Data are presented in the form of a median and 25-75 quartiles; * - distinctions between groups of 'donors' and elderly people (r<0,05); ** - distinctions between groups 'active longevity' and 'mercy' (r<0,05).
The analysis of patterns of microbiota of small intestine revealed existence of big interindividual variations in specific structure of microorganisms, and significant intergroup distinctions in structure and quantity of microorganisms between groups of young and elderly individuals, and also between groups of people of advanced age with the different psychophysical status (see table 1).
The prevailing groups of microorganisms in a small intestine of young conditionally healthy people are bacteria Firmicutes (the sorts Eubacterium spp, Bifidobacterium spp., Lactobacillus spp., types of Clostridium ramosum), microorganisms Actinobacteria (Propionibacterium freudenreichi) and mushrooms Actinomyces viscosus (see table 1). Elderly people, not depending on the psychophysical status, in small intestine have the same prevailing microorganisms, as young donors, however the quantity of eubacteria in 2, and bifidobacteria is reduced by 10 times, in comparison with similar indicators at young age. Eu-and bifidobacteria in a small intestine of elderly people are replaced with other representatives like Firmicutes (types of Clostridium perfringens, Bacillus cereus, Blautia coccoides) and microorganisms of other types: Bacteroidetes (sort Prevotella spp.), Actinobacteria (sort Pseudonocardia spp.) Proteobacteria (sort Alcaligenes spp., type of Campilobacter mucosalis), Fusobacteria, and mushrooms of the sorts Candida spp. and Aspergillus spp., which quantity is significantly above among elderly people, than young donors. Besides, in blood of advanced age people, it is not dependent on the psychophysical status, levels of markers of viruses of Epstein-Barre and the general endotoxicosis, in comparison with individuals of young age are authentically increased (see table 1).
In groups of elderly people with various psychophysical status: 'Active longevity' and 'Mercy', structure of community of bacteria of a small intestine was identical, however we found features in quantitative structure of microorganisms. So, at individuals with considerable violations of the psychophysical status (miloserdiye group) the number of bacteria of the sort Enterococcus spp and Staphylococcus aureus was authentically raised; the quantity of microorganisms Moraxella spp./Acinetobacter spp., Fusobacterium spp./Haemophilus spp., Clostridium ramosum and Propionibacterium jensenii was reduced, in comparison with similar indicators of elderly people of group 'Active longevity' (people with moderate cognitive frustration and insignificant physical violations) (see table 1).
Discussion. It is known that in intestinal microflora of the person there are five bacterial types (Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia) and one Archean type (Euryarchaeota) (Biagi, Candela, Turroni, et al., 2013). In the conducted research, we obtained data on considerable decrease of bacteria Firmicutes (especially bacteria Eubacterium spp. and Bifidobacterium spp.) among elderly people, in comparison with young donors. Microorganisms Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria and mushrooms, occupy the released niche. Our data agree the results of a number of authors (Mueller, Saunier, Hanisch, et al., 2006; Woodmansey, McMurdo, Macfarlane, Macfarlane, 2004) about change of a ratio of Bacteroidetes/Firmicutes and decrease in quantity of bifidobacteria among people of advanced age.
When comparing groups of people of advanced age, significant increase at individuals with the expressed violations of the psychophysical status attracts attention (group 'Mercy') of facultative anaerobe bacterias, including, enterococci and Staphylococcus aureus. Stafilokokki, enterokokk streptococci, and enterobakteriya often classify as "patobionts", being, as we know, initiators of the low-graduated inflammatory process prolonged system chronic among elderly people (Pedron, Sansonetti, 2008), which affects the level of metabolic homeostasis of an organism and functional activity of CNS.
Conclusion. Thus, for elderly people with the expressed psychophysical insufficiency microbic patterns of small intestine with the high maintenance of "patobionts", such as Enterococcus spp and Staphylococcus aureus are characteristic. In comparison with people of advanced age with moderate cognitive frustration and insignificant physical violations.
REFERENCES
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TLR10-TLR1 -TLR6 HAPLOTYPES IN CHELYABINSK REGION POPULATIONS
Abstract
This paper is devoted to assessing the prevalence of different haplotypes in toll-like receptor genes in Chelyabinsk Region populations. Here is examined gene cluster TLR10-TLR1-TLR6, containing single nucleotide polymorphisms, effected by natural selection during development of human world populations. Differences in the frequency of certain three-locus haplotypes in populations of Russians, Bashkirs and Nagaybaks of Chelyabinsk Region are demonstrated.
Keywords
toll-like receptors, TLR10-TLR1-TLR6 cluster, Bashkirs, Nagaybaks, Russians, single nucleotide polymorphisms
Authors
Aleksandr Evdokimov
Postgraduate, Teaching Assistant Immune Microbiology and General Biology Department Chelyabinsk State University Chelyabinsk, Russia avdax@yandex. ru
Aleksandra Burmistrova
PhD in Medicine, Professor Head of Immune Microbiology and General Biology Department Dean of Biology Faculty Chelyabinsk State University Chelyabinsk, Russia burmal@csu. ru
Dariya Stashkevich
PhD in Biology, Associate Professor Immune Microbiology and General Biology Department Chelyabinsk State University
Chelyabinsk, Russia stashkevich_dary@mail. ru
Introduction. The evolutionary development of animals and plants were always in close interaction with various microorganisms, symbionts and pathogens. This interaction implies the existence of structures, involved in the recognition of microorganisms. The first level of recognition of microorganisms is associated with innate immune system, providing recognition of "images" of alien and dangerous microorganisms through its receptors. Such receptors are called pathogen- or pattern-recognition receptors (pathogen/pattern recognition receptors, PRRs), since they act as microbial sensors, that recognize molecular motifs (patterns), conservative for specific groups of microorganisms - pathogen-associated molecular patterns (pathogen-associated molecular patterns, PAMPs). As a result of binding PPRs with PAMPs the inflammatory response activates (Barreiro, Ben-Ali, Quach, et al., 2009; Casanova, Abel, Quintana-Murci, 2011).
The most studied PRRs are toll-like receptors (toll-like receptors, TLRs), located on the membranes of innate immune cells. In humans, the family of TLRs include 12 receptors (TLR1 -
