Bacteria in permafrost their nature and implications in medicine
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- Fifth Russian Conference on Geocryology Yan-gao Man
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Bacteria in permafrost – their nature and implications in medicine Bacteria in permafrost as well as in other severe environment are distinguished by special properties, including presumably extreme resistance and longevity, and what could be their applications? 
Co-jointed conference of the Department of Geocryology of Lomonosov Moscow State University & International Union for Difficult-to-treat Diseases Within Fifth Russian Conference on Geocryology Yan-gao Man – Co-chairman, Chairman and General Secretary of IUDD, Editor-in-Chief of Journal of Cancer, USA Anatoli Brouchkov – Co-chairman, Head of the Department of Geocryology, Faculty of Geology MSU, Moscow, Russia Moscow June 15th, 12-00 MSU Main building, third floor, room 301
Anatoly Brouchkov1,4, Sergey Naumenko2, Grigoriy Edidin1, Maria Logacheva2, Eugene Brenner3, Alexander Kurilshikov3, Georgii Bazykin3, Alexey Kondrashov2, Valentin Vlassov3 1Lomonosov Moscow State University, Geology Faculty 1Lomonosov Moscow State University, Bioengineering Faculty 3Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 4Tyumen State University There are distinct evidences that permafrost of Mammoth Mountain never thawed for at least last several million years, however we observe a considerable discordance between age of permafrost sampled and the approximate divergence time calculated for the sequenced genomes. It is worth to note that such discordance is tending to be usual when investigating the bacteria isolated from a more or less ancient environment. Although some cases might be explained by possible contamination, others raise questions of great importance. Do these bacteria stay in dormant state while being encaged in permafrost or still divide? What are the energy sources for actively dividing bacteria in permafrost? Are there the genetic determinants for adaptations to live in such environments, as it is stated elsewhere, or such ability is a universal trait of soil bacteria as it can be concluded from regular genomic sequences? We provide some evidences that Bacteria sampled from permafrost sediments exhibit extremely high level of adaptation to the cold, saline and variable osmotic environments. It is shown that particular genes and pathways are important for these adaptations. Combining this knowledge with the fact of recent divergence of permafrost samples makes it possible to study short term positive selection on genome scale level.
Kaprelyants A.1, Greenblatt C.2, Shleeva M1, Nikitushkin V.1, Salina E.1 1Bach Institute of Biochemistry, Research Center for Fundamentals of Biotechnology, RAS, Moscow 2The Hebrew University, Jerusalem Nonsporulating bacteria are able to transit to dormant state in response to stressful conditions. These forms can survive for long time without division. Due to low metabolic activity of dormant bacteria their susceptibility to known drugs is very poor. Latent tuberculosis is a long-term, asymptomatic infection caused by the persistence of dormant Mycobacterium tuberculosis (MTB) within human body. After growth of M.tuberculosis and M.smegmatis cells under sub-optimal conditions bacteria adopted a stable “non-culturable”(NC) state after prolonged incubation in stationary phase. The viability of NC cells could be restored ( resuscitated) by incubation of cells in liquid medium supplemented by Rpf (resuscitation promoting factor- a protein secreted by Microccus luteus). These NC cells could be considered as dormant as their metabolic activity was very low. Microscopic examination revealed morphologically distinct ovoid forms in the population of mycobacterial NC cells. Ovoid forms are characterized by elevated resistance to antibiotics and to heat treatment. Transcriptomic and proteomic analysis suggest that transition of viable bacteria to dormancy and vice versa are programmed processes which include changes on transcriptional and translational levels. Upon transition, dormant mycobacteria develop mechanisms to protect cells against distraction during long storage ( accumulation of pigments ( porhyrins) and tregalose as protective compounds). Synthesis of DNA/RNA binding proteins during transition to dormancy provides protective stability of nucleic acids (DNA and RNA).
Siberian Permafrost Sediment Rosa Margesin1 and Anatoli Brouchkov 2 1Institute of Microbiology, University of Innsbruck 2 Faculty of Geology, Lomonosov Moscow State University We isolated and characterized viable microbial strains from ancient (Neogene) permafrost sediment that was permanently frozen for 3.5 million years. The sampling site was located at Mammoth Mountain in the Aldan river valley in Central Yakutia in Eastern Siberia. Our data demonstrated the presence of viable microorganisms. Analysis of phospolipid fatty acids (PLFA) demonstrated the dominance of bacteria over fungi; the abundance of Gram-negative bacteria was approximately twofold higher than that of Gram-positive bacteria. Viable heterotrophic bacteria were found at 0°C, 10°C and 25°C, but not at 37°C. Viable fungi were only detected at 0°C and 10°C. The low diversity of viable microorganisms was attributed to a number of factors, such as strong selection pressure due to harsh conditions, nutrient deficiency, presence of inorganic and organic inhibitors, permanently freezing conditions for 3.5 million years combined with the lack of contamination by percolating water from surface, groundwater, lakes and rivers. Representatives of the genera Arthrobacters, Subtercola and Glaciimonas were characterized with regard to their growth temperature range, their ability to grow on different media, to produce enzymes, to grow in the presence of NaCl, antibiotics and heavy metals and to degrade hydrocarbons. All strains could grow at 5°C, the upper temperature limit for growth in liquid culture was 25°C or 30°C. Some strains were halotolerant. Sensitivity to rich media, antibiotics, heavy metals and salt increased when temperature decreased (20°C > 10°C > 1°C), which could be a reaction to an increased stress situation at low temperatures. Combined data of phenotypic, phylogenetic and DNA-DNA relatedness studies demonstrated that one of the strains represented a novel species of the genus Glaciimonas, which was recently described as Glaciimonas frigoris sp. nov.
Morgan Zhang, Min Wang Elixiria Biotech Co. Inc A strain of Bacillus sp. isolated from the permafrost by A. V. Brouchkov showed anti-ageing effects in fruit flies and mice. The mechanism of this effect is unknown. Insights into the evolution of longevity from the bowhead whale genome indicate DNA repair, especially, Nucleotide Excision Repair (NER), may be involved in this effect. It was therefore hypothesised that this bacterial strain extends the lifespan, at least in part, by promoting NER. In the presence or absence of the lysis of this stain culture, DNA damage of human Hela cells were induced by ultraviolet radiation at doses of 10 J m-2. These DNAs were repaired for 3, 6, 15, 30 min, and then were analysed by the alkaline comet assay and electrophoresis. Our data indicate the repair kinetics of the DNA in the presence of the lysis have no significant difference from that in the absence of the lysis. This result suggests the NER may not be modulated by the bacterial lysis, and thus may not be involved in its anti-ageing effects, or the Hela cells may be not a good cell line for this anti-ageing experiment.
Oksa Fursova1 , Vasily Potapov2, Anatoli Brouchkov3,5, Gennady Pogorelko1, Gennady Griva4, Nadejda Fursova2, Sergei Ignatov2 1Iowa State University 2State Research Center for Applied Microbiology and Biotechnology (SRCAMB) 3 Faculty of Geology, Lomonosov Moscow State University 4 “Geoengineering” 5Tyumen State University Bacillus cereus strain F, collected from relict permafrost located in Siberia, was analyzed for probiotic activity in the mouse Salmonella enterica model. Viable bacterial cells were found in frozen soils taken at Mammoth Mountain in Yakutia from a depth below the level of seasonal thawing. Geological data indicated the absence of a thawing within millions of years of deposited soils, which helped to ensure the ancient origin of our sample. According to DNA analysis, bacterial cells collected from the relict permafrost appeared to be B. cereus strain F. The morphology of these bacteria was analyzed using atomic force microscopy. B. cereus strain F was assessed as a nonpathogenic bacterium by evaluation of its pathogenicity. A S. enterica model is described in mice after per oral inoculation and serves as a model for the human carrier state. Using this model, probiotic activity by the bacterial strain isolated from the ancient permafrost has been shown against Salmonella infection in mice.
Kalyonova L.F.1,2,3, Melnikov V.P. 1,2,3 1Tyumen State University, 2Tyumen Industrial University, 3Tyumen Scientific Center of SB RAS Incubation temperature of Bacillus F. isolated from late Neogene permafrost modulates biological effects of secondary metabolites. The changes include: 1) spectrum of enzymes secreted by the bacteria; 2) balance of synthesized pro- and anti-inflammatory cytokines by immunocompetent cells of human peripheral blood in vitro; 3) the rate of repair of skin wound and activity of immune system in vivo.
Sergei Ignatov1, Egor Denisenko1, Nina Schiskova1, Anatoly Brouchkov2 1State Research Center for Applied Microbiology and Biotechnology (SRCAMB) 2Lomonosov Moscow State University, Geology Faculty A number of studies claim that it is possible to find alive bacterial cells in special geological samples after a million years of storage. The risk of bacterial contamination exists at all stages of investigation (sample obtaining, transport, and investigation). Ethanol treatment with next burning during 5 min has been proposed for killing contaminated microflora on surfaces of frozen samples from permafrost. On the other hand, the possibility to find ancient pathogens is still probable. Some approaches have been proposed for pathogen indications in permafrost samples before the main analysis.
Akimov V.N.1, Andersen D.2, Galchenko V.F.3 1Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia; 2Carl Sagan Center for the Study of Life in the Universe, SETI Institute, Mountain View, California, United States; 3Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Winogradsky Institute of Microbiology, Moscow, Russia Lake Untersee is the largest surface freshwater lake in East Antarctica, with surface area of 11,4 km2 and maximum depth is 169 m. The lake is permanently covered with 3-4 m of ice and is partly bounded by the Anuchin Glacier. Studies in the 1980s and 90s found the high oxygen supersaturation in most of the lake but significant stratification in the southeastern part of the lake where the water column was anoxic below 80 m. Other important features of the lake Untersee are: pH between 9.8 and 12.1; low primary production in the water column; high proportion of methane in the sediment at the lake bottom. New period in the study of the lake Untersee has begun since 2008. Nearly each year the international interdisciplinary research team “Tawani Expedition” has been carrying out its complex studies on the lake. Dale Andersen and Ian Hawes discovered conical stromatolites growing in the lake Untersee, the largest living ones known to date. Valery Galchenko and Vladimir Akimov investigated the biogeochemical activities of microorganisms in the water column. Dozens of aerobic heterotrophic bacterial strains have been isolated from water column of the lake as well as from permafrost around the lake. Several aerobic phototrophic bacteria have been isolated from the water column and cyanobacterial mat. Nikolay Ravin with his colleagues have been studying metagenomics of cyanobacterial mat and Svetlana Filippova has been studying the diversity of viruses in the lake. Now we know that there are a lot of different organisms in the lake Untersee: aerobic and anaerobic phototrophs, aerobic and anaerobic heterotrophs, methanogens, methanotrophs, homoacetogens, sulfate reducers. It is only a few organisms among them are psychrophiles and alkaliphyles as might be expected, but most of organisms are psychrotolerant and neutrophiles. Complete genome sequences for several isolates from the lake Untersee were obtained recently in hope to find some mechanisms of adaptation to cold and alkaline ecosystem. Also, bacterial isolates from the lake Untersee attract our attention as a source for biomolecules with biotechnological potential.
Robert Khramov Institute of Theoretical and Experimental Biophysics Russian Academy of Sciences, Pushchino, Russia
Gennady Griva1, Anatoli Brouchkov2,4, Ludmila Derimedved3 1 “Geoengineering” 2 Faculty of Geology, Lomonosov Moscow State University 3 Kharkov Pharmaceutical University 4Tyumen State University Full scale pre-clinical studies of ancient bacteria have been performed on the base of National Pharmaceutical University and State Research Center of Drugs (Ukraine). All studies carried out according to standard pharmaceutical procedures and methods accepted for evaluating the efficacy of new drugs. The main biomedical features of the most studied Bacillus F strain are: a super strong geroprotective, cardioprotective, hepatoprotective, cytoprotective and immunomodulatory activity; high level of antioxidant action; anti-inflammatory and antimicrobial effect; probiotic features and radioprotective properties. It also prevents the development of insulin resistance, supports endocrine system, improves psychomotor and locomotor activity, regulates steroid hormone synthesis, reduces allergic reactions, helps to increase muscular strength and physical endurance and improves the functional status of the central nervous system. Strain itself and its derivatives are harmless substances, belong to Class VI toxicity, have no virulent and pathogenic features. A wide range of effective new drugs, probiotics and nutritional supplements, based on relict bacteria from permafrost could be obtained.
Glacial Complex (Central Yakutiya) Svetlana Filippova1, Natalia Surgucheva1, Valentina Sorokina1, Maria Cherbunina2, Elina Karnysheva2, Anatoli Brouchkov2,3 and Valery Galchenko1 1 Winogradsky Institute of Microbiology, Research Center of Biotechnology RAS 2 Lomonosov Moscow State University, Geology Faculty 3Tyumen State University Electron microscopic investigation of four samples of ancient ice wedge from the Pleistocene glacial complex of Mamontova Gora (Yakutiya, Russia) revealed high diversity of bacteriomorphic particles. Their structural features included the presence of electron transparent zones, presumably inclusions containing storage compounds, and microenvironments (capsules or external sheaths). These features may be a result of adaptive strategies providing for microbial survival under permafrost conditions. Predominance of rod-shaped forms morphologically resembling coryneform actinobacteria was found. X-ray microanalysis revealed organic origin of bacteriomorphic particles. Some particles were characterized by incomplete spectra of the major biogenic elements, resulting probably from lowtemperature damage to the cellular structures. Total numbers of aerobic heterotrophic bacteria determined by plating on nutrient media were comparable to the values obtained for permafrost soils and Arctic ice. Predominance of coryneform actinobacteria was observed. Abundance of these evolutionarily early groups of actinobacteria may indicate the ancient origin of the microflora of the relic frozen soils.
Elina Karnysheva1, Anatoli Brouchkov1,4, Maria Cherbunina1, Gennady Griva3, Svetlana Filippova2, Dmitry Skladnev2, Valery Galchenko2 1 Winogradsky Institute of Microbiology, Research Center of Biotechnology RAS 2 Lomonosov Moscow State University, Geology Faculty 3 “Geoengineering” 4Tyumen State University The study of the viral component of ancient microbial communities from permafrost is important for the understanding evolution of microbial communities, possibility of their variations due to climate change, changes in the physical-chemical state of permafrost and practical questions of biosafety. For the first time the virus particles in native samples of ancient ice wedges of the Mammoth Mountain in Siberia have been discovered. Defined morphological diversity of viruses that can be attributed to five main types: miovirus, sifovirus, podovirus, spherical and filamentous. Specific characteristic of these viruses are small size and fever genome.
Gennady Griva1, Anatoli Brouchkov2,5, Natalie Lakstigal3, Erik Terehovs4 1 “Geoengineering” 2 Faculty of Geology, Lomonosov Moscow State University 3 Nakhabino Medical Clinic, Moscow District 4Academy of Tibetian Medicine, Riga 5Tyumen State University Preliminary observations of testing of lizate of Bacillus F. with limited statistics has shown certain changes in blood parameters – concentration of lymphocytes, red blood cells, bilirubin, ALT and hormones etc. No negative effects were detected, but positive results have been received.
Ivanova T.I.1, Sobakin I.P.1, Kuzmina N.P.1, Chevychelov A.P. 1 and V. F. Galchenko2 1 Institute of biological problems of cryolithozone of the Siberian Branch of the Russian Academy of Science 2 Institute of microbiology of S. N. Vinogradsky, FITs "Fundamental Bases of Biotechnology" of the Russian Academy of Sciences, Mikroflora of permafrost soils is adapted to the Arctic conditions possessing a specific physiology. Biological and biogeochemical activity of soil microorganisms of Yakutia are studied insufficiently. Nevertheless, the northern ecosystems which were considered until recently "virgin" are subject to large-scale anthropogenous influence now. One of the most notable forms of technogenesis is local and global radio nuclide pollution. Research of microbic communities in zones of radioactive pollution and estimation of a role of microorganisms in evolution of such pollution was of particular importance after the Chernobyl accident. However influence of radioactive pollution on prokariotny microorganisms is still poorly studied. The taxonomical structure, and also bioindicator and destructive potential of microorganisms on object of underground nuclear explosion "Craton-3" and in a zone of uranium fields of the Central Aldan aren't studied at all. We have obtained the data characterizing levels of maintenance and distribution natural (238U, 226Ra, 222Rn, 210Pb and 232Th) and artificial (137Cs, 90Sr and 238-240Pu) radionuclides in components the permafrost ecosystems; influence of radioactive pollution on microorganisms is estimated; optimum ways of monitoring and rehabilitation of the polluted territories in a zone of distribution of permafrost are revealed. In the soil of the radioactive polluted section an increase in number of all studied groups of microorganisms was observed only (!) with reduction of content of uranium. With increase in content of uranium in the soil a significant decrease in number of microorganisms is observed, or a total disappearance of representatives of the main physiological groups of microorganisms was found. Unlike the polluted section in a "pure" section the number of microorganisms remained rather high on all soil profile. Thus, the assessment of correlation of number of the main groups of microorganisms with content of uranium, temperature and moisture of the soil has shown that in the radioactive polluted alluvial soil there is a considerable negative correlation with the content of uranium. Any correlation with temperature is much less. In the "pure" soil positive correlation of number of microorganisms with temperature is found.
Anatoly Brouchkov1,4, Gennady Griva2, Eugene Brenner3 1 Lomonosov Moscow State University, Geology Faculty 2 ”Geoingeneering” 3 Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 4Tyumen State University The isolation of microorganisms from ancient deposits, including permafrost, and the question do they are as old as the deposits are still areas of controversy. We isolated and characterized bacterial strains from ancient (Neogene) permafrost sediment that was permanently frozen for 3.5 million years (62°56'N, 133°59'E). According to the DNA analysis of bacterial cells collected from the relict permafrost appeared to be Bacillus sp. strains which are similar to modern bacteria at molecular level. Genomic features of microorganisms from old deposits, including permafrost, are still areas of controversy since many representatives of the ancient bacteria are close to modern bacteria at molecular level. Our isolate of the Siberian permafrost was not an exception. The microorganisms in permafrost, if they are really old, apparently have special mechanisms of repair or preservation of genome, and their mutation rates can be low. It seems the ability to keep genome stable might be not only permafrost microorganisms feature, but possibly an attribute of other ancient isolates.
Anatoly Brouchkov1,5, Andrei Mulukin2, Marsel Kabilov3, Svetlana Filippova2, Valery Galchenko2, Viktor Rogov4 1 Lomonosov Moscow State University, Geology Faculty 2 Winogradsky Institute of Microbiology, Research Center of Biotechnology RAS 3 Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk 4 Lomonosov Moscow State University, Geography Faculty 5Tyumen State University Permanently frozen (3 – 3.5 Ma) alluvial Neogene sediments exposed in the Aldan river valley near the Mammoth Mountain (Eastern Siberia) are unique, ancient and poorly studied permafrost environments. So far, the structure of indigenous bacterial community has remained unknown. The 16S metagenomic analysis with total DNA isolation using DNA Spin Kit for Soil (MO-Bio) and QIAamp DNA Stool Mini Kit (Qiagen) revealed the major and minor bacterial lineages in the permafrost alluvium sediments. Totally, 61 OTUs with 31,239 reads (Qiagen kit) and 15,404 reads (Mo-Bio kit) could be assigned to the known taxa; other 26 OTUs remained unclassified. Only three phyla, Bacteroidetes, Proteobacteria and Firmicutes, comprised more than 5% OTUs abundance and accounted for 99% of the total reads. OTUs pertaining to the top 11 families (especially Chitinophagaceae, Caulobacteraceae, Sphingomonadaceae, Bradyrhizobiaceae, Halomonadaceae, in a descending order) held more than 90% of reads. The abundance of Actinobacteria was less (0.7%), whereas members of other phyla (Deinococcus-Thermus, Cyanobacteria/Chloroplast, Fusobacteria, and Acidobacteria) constituted a minor fraction. The bacterial community in the studied ancient alluvium differs from other Canadian and Siberian permafrost sediments, mainly by predominance of Bacteroidetes (more than 52%). Results of this study contributes to a better understanding of a conserved biological component to be subjected to thawing and spreading with water flows.
Taiki Katayama1, Koso Asano1, Anatoli Brouchkov2,4, Masami Fukuda3, Michiko Tanaka1, and Fusao Tomita1 1 Laboratory of Applied Microbiology, Graduate School of Agriculture of Hokkaido University 2 Lomonosov Moscow State University, Geology Faculty 3 Institute of Low Temperature Science of Hokkaido University 4Tyumen State University Living microorganisms were isolated and identified for the first time from ice wedge in permafrost deposits of Alaska, which was never thawed for about 25,000 years. We obtained 248 isolates and 62 different representatives based on a partial 16S rRNA gene analysis. They were mostly Gram-positive related to different groups. Eleven isolates possibly represent new species. Nine isolates are able to grow at -5ºC. These results show that phylogenetically diverse aerobic microorganisms have remained viable within the ice wedge at about -4°C, and the cold-adapted strains are capable of long-term survival in conditions of underground permafrost.
Aleksandra Choporova1, Anatoli Brouchkov2 1Kharkov National Medical University, Phthisiology and Pulmonology Department 2 Lomonosov Moscow State University, Geology Faculty Multidrug-resistant tuberculosis (MDR-TB) has emerged as a global epidemic, with ∼ 425,000 new cases estimated to occur annually. The global human immunodeficiency virus (HIV) infection epidemic has caused explosive increases in TB incidence and may be contributing to increases in MDR-TB prevalence. The risk of adverse reactions to TB treatment is higher in HIV-infected individuals than in HIV-uninfected individuals, occurring in approximately 25% and 13%, respectively. Simultaneous antiretroviral and antimycobacterial treatment requires 6 -10 different drugs. Hepatotoxicity is common in the treatment of MDR-TB in HIV-infected patients, and may be exacerbated by overlapping toxicities with antiretroviral (ARV), by coinfection with viral hepatitis, or by preexisting liver disease. Seaching for optimal method for preventing of hepatotoxic reactions in patients with HIV/MR-TB is extremely important. Based on the results of experimental research, unique features of relic microorganisms from ancient permafrost of Central Yakutia can be useful in complex therapy of the HIV/MDR-TB patient due to wide range of beneficial properties in higher organisms. According to DNA analysis, bacterial cells collected from the relict permafrost appeared to be B. cereus strain F. Studies of hepatoprotective properties of Bacillus F strain lysate with acute tetrahlormetan hepatitis have shown that biologically active substances have the ability to normalize the process of free radical oxidation and to stabilize the hepatocytes antioxidant system. Lysates of relic microorganisms exhibit anti-inflammatory properties. Immunomodulatory activity of B. cereus strain F is based on ability to induce the synthesis and secretion of inflammatory cytokines by cells of the monocyte-macrophage series which promote to expression of adhesion molecules, neutrophil exit into inflammatory focus, activation of NF-kB factor and NK-cells, enhancing phagocytosis and superoxide production, acceleration of lymphocytes proliferation, as well as an increasing levels of interferon-gamma (IFN-γ) by natural killer and reduction of tumor necrosis factor (TNF-α). The positive influence of nonpathogenic bacterium Bacillus F strain on regenerative processes of soft tissue injuries as well as their approved probiotic activity could promote increasing of treatment quality of HIV/MDR-TB patients. Yüklə 2,75 Mb. Dostları ilə paylaş:
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