[Gut microbiota and systemic inflammation in patients with chronic heart failure].

Aim To study the interrelationship between intensity of chronic systemic inflammation (CSI) with severity of the condition and intestinal microbiocenosis parameters in patients with chronic heart failure (CHF).Material and methods 47 hospitalized patients with symptomatic CHF were evaluated. The following parameters were determined: clinical condition; N-terminal pro-B-type natriuretic peptide (NT-proBNP). C-reactive protein (CRP); serum interleukins (IL) 6 and 10; and intestinal microbiocenosis composition by mass-spectrometry of microbial markers in whole blood. Microbiocenosis indexes were compared in the main group and in 38 outpatient patients with arterial hypertension and ischemic heart disease without CHF.Results Direct, medium-power correlations were found between CRP and IL-6 concentrations and severity of clinical condition (NT-proBNP, ХСН stage, and edema severity) in patients with CHF. Most patients with CHF had lower numbers of bifido-, lacto-, propionic-, and eubacteria, and Clostridium (С.) ramosum and higher numbers of aspergillus. Among CHF patients, the highest indexes of endotoxemia, gram (-) bacteria, cocci, actinomycetes, and microfungi were observed in the group with NT-proBNP from 400 to 2000 pg/ml. Direct correlations were observed for amounts of C. hystolyticum, Pseudonocardia spp., and Aspergillus spp. with IL-6 and IL-10 and unidirectional inverse correlation were observed for these cytokines with Propionibacterium acnes and jensenii, Streptomyces spp., and Nocardia asteroides. In addition, IL-6 concentration was negatively correlated with contents of Staphylococcus aureus, C. difficile, C. ramosum, Eggerthella lenta, and Corynebacterium spp. and was positively correlated with C. propionicum, Moraxella spp. and Flavobacterium spp. Concentration of IL-6 directly correlated with the number of Eubacterium spp. and inversely correlated with numbers of Ruminicoccus spp. and Streptomyces farmamarensis. The amount of Streptomyces farmamarensis negatively correlated with CRP concentrations.Conclusion The study results evidence the significance of intestinal microbial-tissue complex in the pathogenesis of CSI in CHF and allow suggesting this complex as a promising target for therapy.

Effect of Prebiotic Complex on Gut Microbiota and Endotoxemia in Female Rats with Modeled Heart Failure.

We studied the levels endotoxin and microbial markers in the blood of female rats with experimental heart failure and the effects of preliminary treatment with a prebiotic complex based on fermented wheat bran and inactivated Saccharomyces cerevisiae culture on these parameters. The concentrations of endotoxin, markers of lactobacilli, and opportunistic microorganisms were found to increase in rats with experimental heart failure and significantly decreased against the background of treatment with prebiotic complex. The dynamics of markers of bifidobacteria, eubacteria, and propionibacteria were reciprocal. The observed effect of the prebiotic complex effect on gut microbiota in rats with experimental heart failure suggests that this complex can be used for the correction of intestinal dysbiosis and endotoxemia in this clinical condition.

[Identification of Hydrocarbon-Oxidizing Dietzia Bacteria from Petroleum Reservoirs Based on Phenotypic Properties and Analysis of the 16S rRNA and gyrB Genes].

The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol%, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002(T), respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classiffication of strain 32d as a D. maris strain and of strain 263, as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

[THE CHROMATOGRAPHY-MASS SPECTROMETRY ANALYSIS OF MICROBIAL FATTY ACIDS IN HUMAN BIOLOGICAL FLUIDS AND THEIR CLINICAL SIGNIFICANCE].

The technique of mass-spectrometric microbial markers is known for almost 20 years. The technique is described in a number of research publications, dissertations and methodological literature. It passed the registration in Roszdravnadzor and is permitted for implementation as a new medical technology in medical institutions on the territory of the Russian Federation ("The evaluation of microecological human status using technique of mass-spectrometry" license FS No 2010/038 of 24.02.2010). The technique of mass-spectrometric microbial markers began to be developed as instrument of clinical routine analysis and monitoring of microecological status, infection and disbiosises in clinical and out-patient practice. The description of technology of mass- spectrometric microbial markers in this aspect requires different than before approach to introduction of clinical laboratory assistants and physicians into technique application. The substantiation given concerning species specificity of composition of fatty acids and (fatty) aldehydes of cellular wall of microorganisms as a basis of their species differentiation in pure culture. The choice is explained concerning molecular markers for their detection in blood and other clinical material with the purpose of further reconstruction of composition of human microbial cenosis (microecology) on blood or calculation of composition of mixed infection in organs om samples of inflammation focus--urine, liquor, phlegm, exudate, drainage, and similar samples containing chemical information about microbes.

[Desulfovibrio hontreensis sp. nov., a Sulfate-Reducing Bacterium Isolated from Marine Biofoulings at the South Vietnam Coastal Area].

A Desulfovibrio strain physiologically similar to and phylogeneticall related to "D. caledoniensis" SEBR 7250, D. portus MSL79, and D. dechloracetivorans ATCC 700912 (96.9, 95.9, and 95.8% similarity of the 16S rRNA gen sequences, respectively) was isolated from marine biofouling in the coastal zone of the South China Sae (Nha Trang, South Vietnam). The cells of strain ME were gram-negative motile vibrios (0.4-0.6 x 1.3-2 µm) with a single flagellum. The strain grew at 20 to 39 degrees C (growth optimum at 34-37 degrees C), pH 5.8 to 8.5 (pH optimum at 6.8-7.5), and salinity from 0.08 to 1.1 M Na+ (optimum at 0.2-0.3 M Na+). In the presence of sulfate, the strain grew autotrophically with hydrogen or on lactate, formate, pyruvate, fumarate, and malate. Weak growth occurred on succinate, glycerol, and fructose. In the absence of sulfate, the strain was able to ferment pyruvate, malate (weakly), but not lactate. Sulfate, sulfite, thiosulfate, elemental sulfur, and dimethyl sulfoxide were used as electron acceptors. Vitamins and yeast extract were not required for growth. The G+C content was 52.4 mol %. Predominant fatty acids were C18:0 (13.9%), C16:0 (9.6%), iso-C16:0 (9.5%), C18: 1w7 (8.8%), anteiso-C15:0 (8.1%), and iso-C 17:1 (7.2%). The fatty acid composition was close to that of D. dechloracetivorans BO and has some similarity to that of D. portus. Based on its genotypic and phenotypic characteristics, strain ME maybe considered as a new species, for which the name Desulfovibrio hontrensis sp. nov. is proposed.

[Fatty acid composition in the carotid artery tissue in the atheromatosis and lipid spot areas. Universal pathogenesis of the atherosclerosis syndrome and its symptom of intimal atheromatosis].

Gas chromatography with mass spectroscopy detection have shown that the major components of atheromatous masses in formed atherosclerotic plaques (surgery material) are the derivatives of essential polyenic fatty acids (ES poly-FA) and of the alcohol cholesterol. They undergo nonphysiological catabolism (hydrolysis) in the lysosomes of resident macrophages as components of protein macromolecules, low-density lipoproteins (LDL). In excess of palmitic triglycer- ides apoB-100 of LDL does not form ligand, and ligand-free LDL become biological in the circulation. They are uptaken via scavenger receptors by resident macrophages in the intima. When ligand LDL are physiologically uptaken via apoB-receptors, they are catabolized (oxidized) in peroxisomes but not in lysosomes. Lipid spots result from functional cellular lipoidosis which fulfills the biological reaction of inflammation and initiate the lipoidosis--vector protein for FA transport--C-reactive protein pathway. Formation of ligand-free LDL lays the basis for intracellular ES poly-FA deficiency and clinical manifestations of the atherosclerosis syndrome. Intima is the spot where large-molecular-weight (> 70 kD) biological rubbish is collected and utilized from a local intravascular pool of the intercellular medium.

[The express mode of identification of agents of bacteriemias using the technique of gas chromatography-mass spectrometry].

The comparative evaluation was carried out concerning the effectiveness of generic identification of hemocultures using the technique of gas chromatography-mass spectrometry by comparison with data of common cultural method. The content of vials with positive hemoculture was analyzed using both the common microbiologic methods and the technique of gas chromatography-mass spectrometry with detection of markers of the most widespread agents of nosocomial bacteriemias: microorganisms of genus Staphylococcus, Enterococcus, Klebsiella, Escherichia, Serratia, Pseudomonas, Acinetobacter, Stenotrophomonas, Candida. The possibility of applying the technique of gas chromatography-mass spectrometry for generic express-identification of agents of bacteriemias was established. The full concurrence of results obtained by gas chromatography-mass spectrometry with the results of common bacteriologic method was revealed. The time saving of analysis during generic identification of hemocultures using gas chromatography-mass spectrometry up to three and less hours against 1.5-2 days in case of common approach. The established information can input into earlier start of etiotropic therapy under severe infections.

Study of human microecology by mass spectrometry of microbial markers.

This review shows that mass spectrometry of microbial markers (MSMM) permits simultaneous in situ determination of more than one hundred microbial fatty acids in clinical, biotechnological or environmental samples, without precultivation and use of biochemical test materials and primers. Unprecedented information about the quantity of anaerobes and uncultivated aerobes, as well as actinobacteria, yeasts, viruses and microscopic fungi in one sample has provided a full understanding of microbial etiology in clinical conditions of patients. The study of intestine dysbiosis has confirmed the hypothesis about the nosological specificity of changes in the intestinal microbiota. It has been proven that infectious processes are polymicrobial. Measurements have shown that anaerobes dominate in number and functional activities in inflammation. The division of microbes into pathogenic and non- pathogenic is artificial. All microbes living in a human body simultaneously stay in both forms. Lactobacilli and bifidobacteria appear as agents of septic conditions and endocarditis. МSММ data confirm that anaerobes of Clostridium, Eubacterium, Propionibacterium, as well as actinobacteria of Streptomyces, Nocardia, Rhodococcus are mixed infection dominants. The data testify translocation of these microbes in inflammation loci from the intestine. Quantitative comparison of concentration of markers in the inflamed organ and blood proves reproduction of microorganisms in this locus. The current hypothesis is confirmed that the goal of translocation is not only infection, but also a biofilm formation similar to intestines, which stimulate local immunity, protection from local pathogens and restoration of the damaged tissues. Quantification using GC-MS revealed that the influence of antibiotics on the normal intestine's microbiota are not as dramatic as believed. Growth-promoting effects are the most important benefits of probiotic applications. The probiotic essence is not the microbial biomass itself, but growth factors, alarm molecules, and other factors of intestinal microbes. There are new possibilities in improving probiotics by using microbial 'consortia', modelling real gut microbiota.

[Spontaneous bacterial peritonitis: scientific and practical achievements and prospects].

We presented the results of our research in comparison with the literature on the etiology, pathogenesis and diagnosis of spontaneous bacterial peritonitis (SBP) at liver cirrhosis (LC), complicated by ascites. Based on these data, was proposed a classification of SBP. Were identified criteria for early diagnosis of SBP on the basis of clinical manifestations of bacterial peritonitis, systemic inflammatory response, results of studies of ascitic fluid (AF) using gas chromatography-mass spectrometry (GC-MS). In accordance with the proposed criteria was proposed a scheme for the diagnosis, treatment and prevention of STDs. Presents data on the effectiveness of therapy and prevention of SBP. Denote the potential major long-term trends in the treatment of SBP in cirrhosis.

[Spontaneous bacterial peritonitis and systemic inflammatory reaction in hepatic cirrhosis patients].

AIM: To develop criteria of early diagnosis of spontaneous bacterial peritonitis (SBP) in hepatic cirrhosis (HC). MATERIAL AND METHODS: Clinical symptoms of SBP including systemic inflammatory reaction were analysed in 286 patients. The count of polymorphonuclear neutrophils (PNL) in ascitic liquid (AL), microbiological study of the blood and AL were made. Ultrasound investigation assessed acoustic homogeneity of AL. Blood serum (BS) and AL were tested for cytokines content: interleukine-1beta, TNF-alpha, interleukine-4, transforming growth factor beta, C-reactive protein. Gas chromatography--mass spectrometry (GC-MC) estimated quantity and quality of chemical components--markers of potential infectious agents of AL. RESULTS: Three groups of patients were identified: group A (a classic SBP) consisted of 23 patients with clinical symptoms of SBP and PNL content > 0.25 x 10(9)/l; group B (control) consisted of 19 patients free of SBP symptoms and PNL content < 0.25 x 10(9)/l; group C (patients at risk)--18 patients with SBP symptoms and PNL content < 0.25 x 10(9)/l. Cultural test of AL was negative in all the groups. Symptoms of SIR occurred equally often in groups A and C. CRP in AL was higher (23.8 +/- 4.3 g/l) in group A than in group B (p < 0.05). CRP concentration (8.6 +/- 2.1 g/l) was higher in group C than in group B (p < 0.05). Changes in cytokine composition were similar in groups A and C. The number of chemical bacterial markers was higher in groups A and C (p < 0.005). CONCLUSION: Patients of group C with SIR, elevated CRP, changed cytokine status, high content of chemical markers of AL infection by GC-MC are at high risk of SBP. Such patients need adequate and early antibacterial therapy.

[Halomonas mongoliensis sp. nov. and Halomonas kenyensis sp. nov., new haloalkaliphilic denitrifiers capable of reducing N2O, isolated from soda lakes].

In the course of the search for N2O-utilizing microorganisms, two novel strains of haloalkaliphilic denitrifying bacteria, Z-7009 and AIR-2, were isolated from soda lakes of Mongolia and Kenya. These microorganisms are true alkaliphiles and grow in the pH ranges of 8.0-10.5 and 7.5-10.6, respectively. They are facultative anaerobes with an oxidative type of metabolism, able to utilize a wide range of organic substrates and reduce nitrate, nitrous oxide, and, to a lesser extent, nitrite to gaseous nitrogen. They can oxidize sulfide in the presence of acetate as the carbon source and nitrous oxide (strain Z-7009) or nitrate (strain AIR-2) as the electron acceptor. The strains require Na+ ions. They grow at medium mineralization levels of 0.16-2.2 M Na+ (Z-7009) and 0.04-2.2 M Na+ (AIR-2). The G+C contents of the DNA of strains Z-7009 and AIR-2 are 67.9 and 65.5 mol %, respectively. According to the results of 16S rRNA gene sequencing and DNA-DNA hybridization, as well as on the basis of physiological properties, the strains were classified as new species of the genus Halomonas: Halomonas mongoliensis, with the type strain Z-7009T (=DSM 17332, =VKM B2353), and Halomonas kenyensis, with the type strain AIR-2T (=DSM 17331, =VKM B2354).

[Thiobacillus sajanensis sp. nov., a new obligately autotrophic sulfur-oxidizing bacterium isolated from Khoito-Gol hydrogen-sulfide springs, Buryatia].

Four strains of rod-shaped gram-negative sulfur-oxidizing bacteria were isolated from Khoito-Gol hydrogen-sulfide springs in the eastern Sayan Mountains (Buryatia). The cells of the new isolates were motile by means of a single polar flagellum. The strains were obligately chemolithoautotrophic aerobes that oxidized thiosulfate (with the production of sulfur and sulfates) and hydrogen sulfide. They grew in a pH range of 6.8-9.5, with an optimum at pH 9.3 and in a temperature range of 5-39 degrees C, with an optimum at 28-32 degrees C. The cells contained ubiquinone Q-8. The DNA G+C content of the new strains was 62.3-64.2 mol %. According to the results of analysis of their 16S rRNA genes, the isolates belong to the genus Thiobacillus within the subclass Betaproteobacteria. However, the similarity level of nucleotide sequences of the 16S rRNA genes was insufficient to assign the isolates to known species of this genus. The affiliation to the genus Thiobacillus was confirmed by DNA-DNA hybridization of the isolates with the type strain of the type species of the genus Thiobacillus, T. thioparus DSM 505T (= ATCC 8158T). Despite the phenotypic similarity, the hybridization level was as low as 21-29%. In addition, considerable differences were revealed in the structure of the genes encoding RuBPC, the key enzyme of autotrophic CO2 assimilation, between the known Thiobacillus species and the new isolates. Based on molecular-biological features and certain phenotypic distinctions, the new isolates were assigned to a new Thiobacillus species, T. sajanensis sp. nov., with the type strain 4HGT (= VKM B-2365T).

[Description of Desulfotomaculum nigrificans subsp. salinus as a new species, Desulfotomaculum salinum sp. nov].

This study focused on the physiological, chemotaxonomic, and genotypic characteristics of two thermophilic spore-forming sulfate-reducing bacterial strains, 435T and 781, of which the former has previously been assigned to the subspecies Desulfotomaculum nigrificans subsp. salinus. Both strains reduced sulfate with the resulting production of H2S on media supplemented with H2 + CO2, formate, lactate, pyruvate, malate, fumarate, succinate, methanol, ethanol, propanol, butanol, butyrate, valerate, or palmitate. Lactate oxidation resulted in acetate accumulation; butyrate was oxidized completely, with acetate as an intermediate product. Growth on acetate was slow and weak. Sulfate, sulfite, thiosulfate, and elemental sulfur, but not nitrate, served as electron acceptors for growth with lactate. The bacteria performed dismutation of thiosulfate to sulfate and hydrogen sulfide. In the absence of sulfate, pyruvate but not lactate was fermented. Cytochromes of b and c types were present. The temperature and pH optima for both strains were 60-65 degrees C and pH 7.0. Bacteria grew at 0 to 4.5-6.0% NaCl in the medium, with the optimum being at 0.5-1.0%. Phylogenetic analysis based on a comparison of incomplete 16S rRNA sequences revealed that both strains belonged to the C cluster of the genus Desulfotomaculum, exhibiting 95.5-98.3% homology with the previously described species. The level of DNA-DNA hybridization of strains 435T and 781 with each other was 97%, while that with closely related species D. kuznetsovii 17T was 51-52%. Based on the phenotypic and genotypic properties of strains 435T and 781, it is suggested that they be assigned to a new species: Desulfotomaculum salinum sp. nov., comb. nov. (type strain 435T = VKM B 1492T).

Novel psychrophilic anaerobic spore-forming bacterium from the overcooled water brine in permafrost: description Clostridium algoriphilum sp. nov.

A gram-positive, motile, strict anaerobic spore-forming bacterium was isolated from the over-cooled brine in the permafrost. The optimal temperature for isolate growth was 5-6 degrees C at pH 6.8-7.2. The bacterium was growing on the medium rich in saccharides and disaccharides. Out of polysaccharides tested, only xylan sustained the growth. Fermentation of the hexoses led to the formation of acetate, butyrate, lactate, H2,CO2 and some formate and ethanol. Cell wall peptidoglycan contained meso-diaminopimelic acid. The major fatty acids of the cell wall were C(14:0) and C(16:1c9). The content of G-C pairs in DNA was 31.4 mol%. As phylogenetic analysis has shown, it is closely linked to the members of cluster 1 of Clostridium. It differs from the other species of the genus by the substrates necessary for the growth, products forming as a result of the fermentation and content of the fatty acids in the cell wall. Thus, it was suggested to describe this strain as a new species named Clostridium algoriphilum. Type strain 14D1 was deposited into the Russian Collection of the Microorganisms VKM B-2271T and German Collection of the Microorganisms DSM 16153T .

[New thermophilic methanotrophs of the genus Methylocaldum]. / Novye termofil'nye metanotrofy roda Methylocaldum.

Two pure cultures of obligate methanotrophs, strains H-11 and 0-12, growing in the temperature range from 30 to 61 degrees C with an optimum at 55 degrees C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes, membrane-bound methane monooxygenase, and phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulose phosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55 degrees C that in cells grown at 37 degrees C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH4+ assimilation occurred through reductive amination of alpha-ketoglutarate and via the glutamate cycle. The relationship between the physiological-biochemical peculiarities of the isolates and their thermophilic nature is discussed.

[Detection of bacterial molecular markers in the tissue of cardiac valves in normal and pathological states by gas chromatography and mass spectrometry]. / Detektsiia molekuliarnykh markerov bakterii v tkani klapanov serdtsa v norme i pri patologii s primeneniem metoda gazovoi khromatografii i mass-spektrometrii.

Samples from cardiac valves of 31 patients were analyzed by gas chromatography and mass spectrometry. The algorithm of mass spectrometric parameters was developed, which permitted the determination of about 200 known microbial fatty acids, aldehydes and sterols, sufficient for the detection and quantitative determination of more that 170 taxons of clinically significant microorganisms on the genus or species levels. The quantitative and qualitative differences in the composition of microbial markers of endocardial valves in normal and pathological states, particularly in cases of infectious endocarditis, were detected. The participation of 37 microbial taxons in the process was confirmed. The level of endocardium colonization in infectious endocarditis reached from 2 to 7 x 10(9) microbial cells/g of valvular tissue (which exceeded twofold the equivalent concentrations of the marker in the normal state). In terms of quantity, the leading role was played by Cardiobacterium hominis.