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The article presents the results of clinical economical analysis, based on "cost-effectiveness" technology, of MALDI-TOF MS, innovative medical technology of express identification of microorganisms, calculation of incremental indicator and application of notion "willingness-to-pay-threshold". Due to the extension of sanctions against the Russian Federation, this medical equipment for national laboratories becomes difficult to access and expensive, that conditions necessity to scientifically substantiate economical effectiveness of implementation of expensive innovative MALDI-TOF MS technology as instrument to contain global antibiotic resistance increase. The understanding of importance of express identification of microorganisms as well as other positive effects that can be achieved by using modern medical equipment on the basis of mass spectrometry results in improving medical care quality, increasing reputation level of medical institution, greater commitment of physicians and patients to microbiological analysis with purpose of prescription of rational antibiotic therapy and improving population health.

Current infectious endocarditis (IE) is characterized by changes in its etiological and epidemiological profiles associated with increased incidence of IE of undetermined etiology. This requires a search for ways to enhance the effectivity of diagnosis. Microbiologistics along with high-tech methods becomes decisively important for identifying the pathogen by studying cultures of blood and tissues from the affected heart valve. This determines timely diagnosis and treatment to be introduced to medical practice as a component of personalized medicine. The article focuses on the validity and features of microbiological (cultural), immunochemical, and molecular biological [MALDI-TOF MS (matrix-activated laser desorption/ionization with time-of-flight mass spectrometry), polymerase chain reaction, sequencing] studies.

A total 267 strains of Neisseria gonorrhoeae obtained in 2016 from 16 regions of the Russian Federation in six federal districts: Southern, Central, Northwestern, Volga, Ural and Siberian were investigated. All microorganisms were identified by biochemical profile on the Vitek 2 Compact analyzer. Matrix-assisted laser desorption ionization-time of flight mass spectrometry(MALDI-ToF MS) was used as an alternative method of identification. Biochemical typing revealed an atypical indistinctive enzymatic profile of N. gonorrhoeae(loss of D-glucose fermentation abilityand reducing of specific enzymes: ProA, TyrA, APPA in 49.1% of studies (131 strains), resulting in 39 strains (14.6%) were assigned to other types of microorganisms. Additional biochemical typing reduced the percentage of error by almost five times (from 14,6 to 3), but 100% confirmation of N. gonorrhoeae was not received.However, verification by mass spectrometer study showed 100% affiliation of the microorganism to N. gonorrhoeae. Biochemical atypia of N. gonorrhoeae represented by the loss of a number of taxonomically significant characters determines the need for an integrated approach to its identification which includes proteomic (massspectrometry) and/or genomic (PCR) studiesalong with biochemical typing., Competing Interests: The authors declare no conflict of interest.

Diagnosing of bloodstream infection (BSI) in outpatients is essential. A large blood volume is required to obtain blood culture (CLSI): 2 sets, 40ml of blood for diagnosing in 95% cases of bacteremia. Molecular-genetic methods can not replace blood culture method, but they accelerate the identification of any pathogen. Culturomics gives a combination of different conditions for isolating microorganisms from a sample and along with their genetic identification. We used the patent method for direct inoculation of buffy-coat from 4,5ml of a venous blood sample and MALDI-ToF identification method. In 382 outpatients examined there were received 183 blood cultures (48,0%), more often among women (65,6%) and young people (74,9%). The causative agents of community-acquired bloodstream infection were aerobes (73,4%), anaerobes (24,2%), fungi (2,4%). The gram-positive cocci were prevailing (51,4%) and the gram-negative rods were isolated rather seldom (9,6%). BSI was monomicrobial (66,5%) and polymicrobial (33,5%). Polymicrobial blood cultures had 2, 3, 4 agents in one blood sample (75,4%, 18,8%, 5,8%, respectively). There were also found combinations of different species of aerobes (47,8%), aerobes with anaerobes (42%). BSI caused complications of the primary disease of the respiratory system, urogenital system and in 100% of cases after plastic surgery. A small blood volume is required for buffy-coat inoculation, the direct agar culture reduces the response time to 2 days, so it makes genetic identification possible on the 2nd day from the moment of blood collection., Competing Interests: The authors declare no conflict of interest.

Corynebacteria non-diphtheria and, in particular, C. pseudodiphtheriticum species that are closely related to C. propinquum and C. striatum form a group of new respiratory pathogens leading to the development of bronchitis, tracheitis, exacerbation of chronic obstructive pulmonary diseases, nosocomial pneumonia and other pathology. The goal is to analyze the frequency of the release of Сorynebacteria non-diphtheria from the upper respiratory tract of patients with various inflammatory diseases of the respiratory tract. Strains of Сorynebacteria non-diphtheria (C. pseudodiphtheriticum, C. propinquum, C. accolens, et al.), isolated from patients with inflammatory diseases of the respiratory tract (60 pcs.) and practically healthy individuals (31 pcs.) were studied. Identification of Сorynebacteria was performed using the method of mass spectrometry (MALDI-ToFMS). Сorynebacteria nondiphtheria in the amount of 105 and higher were more frequently detected with the development of chronic tonsillitis (60.0%) and nasopharyngitis (30%). The strains of C. pseudodiphtheriticum (40.0±6.4%) and the closely related species C. propinquum (21.7±5.3%) were mainly found; much less often - C. accolens (8.3±3.6%), C. afermentans (6.7±3.3%), et al. In 86.7% of cases, Corynebacteria non-diphtheria were isolated from children. In chronic tonsillitis, C. pseudodiphtheriticum and the closely related species of C. propinquum were isolated more often; in nasopharyngitis and bronchitis - С. pseudodiphtheriticum. Isolation of Corynebacteria non-diphtheria and, especially, C. pseudodiphtheriticum, C. propinquum, C. accolens species from patients with inflammatory diseases of the respiratory tract in the amount of 105 and above, if there are no other pathogenic microorganisms in the role of microbial associates, of clinical importance., Competing Interests: The authors declare no conflict of interest.

Identification mass-spectrometry of marker proteins of toxicity of V. cholerae for development the express of diagnostics of the causative agent of cholera on the basis of the computer analysis in the MALDI-ToF format of electronic profiles became the purpose of our research. Subjected to the computer analysis mass and spectrometer electronic passports 140 of strains of V. cholerae with obviously known characteristic of strains which were divided into 2 basic groups in the parameter of existence or lack of a gene of cholera toxin (ctx-and ctx+). We for the first time revealed a taxon - the specific marker protein ceous peak having a molecular mass of 3202 Da characteristic of strains of V.cholerae ctx + and unrepresentative for V.cholerae ctx-., Competing Interests: The authors declare no conflict of interest.

The in-hospital infections are one of the most serious problems of medicine, especially if patients have a background immunosuppression of various genesis conditioned by both disease itself and corresponding therapy. The detection of presence of infection and identification of agent and detection of its resistance are needed for choosing adequate therapy. At that, high heterogeneity of strains and multiple resistance of nosocomial infections to antibiotics and antimicrobial pharmaceuticals and standardization of antibacterial prevention and number of other causes becomes an obstacle for both determination of medicinal sensitivity of bacterium and for identification of pathogen itself in patient. One of the most complicated in antibacterial therapy pathogens causing pyo-septic diseases, are bacteria Stenotrophomonas spp., the only significant species out of them - Stenotrophomonas maltophilia has primary multiple antibiotic resistance. The significance of early identification of S.maltophilia is obvious. The application of MALDI-ToF mass-spectrometry requires shortage of of time of species identification of primary bacterial culture up to 1-2 hours including sampling preparation and analysis of obtained specters. The sequencing of 16S rRNA requires shortage of of total time od species identification of pathogen from clinical sample (blood) up to 1-12 hours, including sampling preparation ans comparison with successions presented in international data base. The given technique permits to exclude out of analysis prolonged period of obtaining a pure hemoculture of agent. The application of sequencing of 16S rRNA and MALDI-ToF mass-spectrometry as an alternative high-precision techniques shorten time of identification of bacteria, including detection of agent directly in blood of patient. Hence, occurs optimization of complex treatment and shortage of time of selection of adequate therapy that is especially important in case of oncological patients because sensitivity of cultural methods can be diminished due to preventive antibiotics' therapy.

The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation playing important functions in various biological processes. By using MALDI-ICR-mass-spectrometry and Western-blot analysis, we have shown the presence of shortened isoform of a-tubulin in complex with the affinity-purified proteasomes from stable cell lines K562 and HEK293.

Aim: An attempt to use MALDI-TOF mass-spectrometry method for identification of lep- tospiral isolates on the serovar level., Materials and Methods: 8 reference Leptospira spp. and 11 leptospira strains isolated from leptospiral patients and infected animals in the North-Western region of Russia were included into the study. Mass-spectra of all the studied strains were obtained by direct profiling of cell extracts. The created main spectral profiles (MSP) of reference strains were used for identification of isolates. Evaluation of identification was carried out by calculating coefficients of matching rate of separate spectra of each isolate with MSP of all the reference strains., Results: Results of identification have shown the similarity of spectra of isolates belonging to Pomona, Icterohaemorrhagiae and Canicola serogroups, with MSP of saprophyte strain L. biflexa Patoc I. It is assumed that spectra of the studied strains contained peaks of polysaccha- ride Ο-antigens. Wherein maximum mean values of matching rate coefficients between spectra of isolates and MSP of pathogenic reference strains of leptospira correctly matched serovar type of the isolate., Conclusion: Further extended studies may form the base of development of a simple and rapid method oftyping of leptospirosis causative agents on the level of serovars using MALDITOF mass-spectrometry.

With the use of whole genome sequencing, the taxonomic status of Lactobacillus fermentum 90 TC-4 strain from Russian collections were studied. Complex analysis of phenotypical and genetic properties was conducted using phenotypic and molecular genetic methods. The main characteristics of the genome and biochemical activity profile of the strain were determined. A comparative analysis of the mass spectrum of ribosomal proteins of the strain, its biochemical properties, a fragment of 16S rRNA gene sequencing, and the entire genome revealed that the present strain belongs to the species L. fermentum, confirming its taxonomic status in accordance with modern taxonomy.

The article presents the results of application of developed methodological approach to identifying Burkholderia pseudomallei and Burkholderia mallei using direct mass spectrometry profiling of cellular proteins. The protocol of sampling preparation of cultures of melioidosis and glanders was optimized with taking in account characteristics of observation of requirements of biological safety for operations with pathogenic biological agents of pathogenicity group II. The dependence of quality of mass spectrums (number of individual peaks and their intensity) from medium of fermentation of microorganisms was evaluated. The characteristic mass spectrums of collection strains B.pseudomallei (5) and B.mallei (5) were obtained. The set of reference mass-spectrums was generated for identification data base S.A.R.A.M.I.S.TM (Anagnostec Gmbh.). The mentioned data base was used for identification of 43 strains of pathogenic Burkholderia. The opportunity of reliable identification of taxonomic belonging of examined microorganisms up to species’ level. The cluster analysis of obtained mass-spectrums of common cellular proteins of collection strains of pathogenic Burkholderia demonstrated grouping of examined strains according to their species’ belonging. The supplemented data base of mass-spectral characteristics hereinafter will permit applying express-identification of isolates suspicious for belonging to agents of melioidosis and glanders. The updated data base will become a basis for developing schemes of hemotyping of strains of Burkholderia using mass spectrometry technique.

The data base “Protein profiles of mass-specters of representatives of species of Vibrio cholerae for program MALDI Biotyper” was used to implement typing of strains of comma bacillus isolated at the territory of the Russian Federation in 2010-2012. Also, analysis of degree of similarity and differences among constant ribosomal proteins was implemented. According the results of MALDI-TOF mass-spectrometry strains of V.cholerae were grouped in two distinct clusters. The first cluster included all epidemically dangerous strains isolated from people arrived in Moscow from India 2010-2012. The second cluster included atoxigenic vibrio with no relation to serogroups O1/O139 isolated from residents of Taganrog in 2011. The analysis of main specters of all collection permitted to identify taxon - specific components distinguishing strains of non-O1/non-O139 from strains of V.cholerae El Tor. Hence, the developed data base of proteom portraits of V.cholerae permits identifying, studying and to typing of agents of cholera and other representatives of V.cholerae species detecting their phylogenetic affinity that is ultimately useful for establishing origin of strains isolated from objects of environment and epidemiological decoding of episodes of disease.

The time-of-flight mass spectrometer analysis of freshly separated human colibacillus populations for identification of markers of proteom specific for hemolytic and non-hemolytic strains, Escherichia-associant of opportunistic enterobacteria. The material consisted of 20 strains of mono-population of hemolytic Escherichia; 100 strains of mono-population of non-hemolytic Escherichia; 15 non-hemolytic strains isolated from association with opportunistic enterobacteria. The protein profiling was implemented on mass spectrometer MALDI-TOF MS Autoflex “Bruker Daltonik”. The value within type of peak mass-charge with 100% intensity equal to 9000 Da can be used as a differentiating indication of detection of hemolytic Escherichia at mass-spectrometer biotyping. The proteom characteristics of strains of various populations within Escherichia coli species are given. The taxon-specific markers for proteom differentiation of population of Escherichia are given too.

Human genetic markers linked with the X chromosome (X-linked) are used in the field of population and medical genetics, as well as for DNA identification of individuals in forensic science and forensic medicine. We proposed an XSNPid panel that consists of 66 unlinked single nucleotide X chromosome markers and developed a protocol for their multiplex genotyping using multilocus PCR and MALDI-TOF mass spectrometry. The XSNPid panel is genotyped within two multiplexes (36 and 30 markers). The developed protocol provides an efficient genotype reading; the fraction of determined genotypes is 98.29%. The high level of gene diversity (0.461) for the X-linked SNPs included in the panel is characteristic of the Russian population. A total of 63 out of 66 markers that provide a high efficiency of genotyping and independent inheritance are suitable for DNA identification purposes. The XSNPid panel is characterized by a very high discriminating ability when studying the Russian population. The probability of genotype coincidence in two unrelated individuals is 9 × 10^(-27) for women and 2 × 10^(-18) for men. Also, the XSNPid panel has a greater multiplex capacity in addition to a higher discriminating ability compared to the other closest analogues of the X chromosome SNP sets, which makes it more cost effective and less time consuming. The XSNPid panel is a convenient tool, not only for individual DNA identification, but also for population genetic studies.

The proper identification of Corynebacterium non diphtheriae is complicated by their wide species variety, variable biochemical activity, large spectrum of diseases at which they are isolated and also by their presence in normal human micro-flora. The bacteriological method is a traditional basis for identification of corynebacteria though long (7-14 days), produces ambiguous results at cultivation of lipophilic and biochemically variable species. For final identification of nondescript species of Corynebacterium non diphtheriae it is recommended to carry out molecular genetic study using golden standard - sequencing on 16S pRNA (DNA), genes rpoB and PLD.In case of receiving of ambiguous responses of sequencing on 16S pRNA precise identification is achieved by sequencing of secondary gene rpoB that permits discovering unique differences in sequences of genomes in different species of corynebacteria (presence of genes of virulence; absence of cluster of genes responsible for production of number of saccharolytic enzymes; presence of genes coding synthesis of particular pigments, etc.). The mass-spectrometric analysis (MALDI-ToF-MS) applied for screening identification of Corynebacterium, is simple in implementation, though requires further development for more accurate differentiation of closely-related species. The poly-phase approach to identification of Corynebacterium non diphtheriae is needed which is to include chemotaxonomic, phenotypic, genotypic information required for reliable description of new clinically significant species of corynebacteria.

Aim: Study mechanisms of interaction of diagnostic bacteriophage El Tor with sensitive strain Vibrio cholerae El Tor 18507 using direct protein profiling, identification of constant and variable proteins, taking part in interaction of the phage and cell, as well as carbohydrate-specific phage receptors., Materials and Methods: . A commercial preparation of cholera diagnostic bacteriophage El Tor, strain V. cholerae El Tor 18507 were used. Effect of carbohydrates on bacteriophage activity was determined in experiments with phage by a classic and modified by us method. Protein profiles of the studied objects were studied using MSP-analysis method., Results: Sucrose was shown to inhibit lytic activity of bacteriophage. Proteome profiles of El Tor bacteriophage and sensitive indicator strains were studied, identification of constant and variable proteins of the studied objects by MSP Peak-list program was carried out., Conclusion: Analysis of changes of profiles of phage and microbial cell during interaction with sucrose gave a basis for assuming, that sucrose in the mixture of culture-phage enters interaction namely with phage protein receptors, blocking receptors specific for cholera vibrio, that subsequently manifests in a sharp decrease of phage activity against the sensitive strain.

Aim: Confirmation of taxonomic position of Lactobacillus fermentum 90 TC-4 strain using phenotypic (classic microbiological, MALDI TOF mass-spectrometry) and genetic (16S rRNA gene segment sequencing and full genome sequencing) methods., Materials and Methods: Object of the study--Lactobacillus fermentum 90 TC-4 strains from various collections. Mass-spectrometric analysis was carried out using Autoflex MALDI TOF mass-spectrometer (Bruker Daltonics, Germany), study of biochemical properties of the strain was carried out using API 50 CHL strips (Biomerueux, France), "DNA-sorb B" kitwas used for isolation ofgenome DNA (CRIE, Moscow). Sequencing of the accumulated fragments of 16S rRNA gene was carried out using GenomeLab GeXP sequencing (Beckman Coulter, USA), full genome sequencing was carried out in MiSeq platform (Illumina). Assembly of genome and bioinformation analysis was carried out using BLAST program (www.blast.ncbi.nlm.nih.gov/blast.cgi), "CLC Bio Assembly" and genome server RAST (rast.nmpdr.org)., Results: L. fermentum 90 TC-4 strain was established to be contaminated by L. plantarum culture in a series of cases. As a result of identification of a pure culture of L. fermentum 90 TC-4 strain using a specter of high-technology methods, membership of the strain in L. fer- mentum species has been proven., Conclusion: Taxonomic status of L. fermentum 90 TC-4 strain was confirmed.

Aim: Study transformation of skin microflora during development of atopic dermatitis., Materials and Methods: 45 patients with various forms of atopic dermatitis (AtD) were examined. Control group consisted of 26 healthy individuals. The strains were cultivated on elective nutrient media. Identification ofthe isolated strains was carried out by MALDI-TOF mass-spectrometry method., Results: A low frequency of occurrence of taxon Staphylococcus epidermidis on face skin and high frequency of occurrence of Staphylococcus aureus on upper and lower limb skin was established for AtD patients compared with healthy individuals. The frequency of occurrence of proteolyti-cally active isolates of S. aureus in AtD patients was 3 times higher than in healthy carriers of this taxon. Taxons of microorganisms not inherent to healthy individuals such as Bacillus mycoides, Pseudomonas putida, Pseudomonas radiobacter were isolated on lower limb and neck skin of AtD patients. A high frequency of occurrence of Cryptococcus satoi, Candida albicans, Malassezia globosa fungi was noted., Conclusion: A decrease of barrier functions of skin during AtD facilitates contamination of patients' skin with rare bacterial taxons and fungi. One of the possible mechanisms of suppression of immune competent cell functions could be proteolytic enzymes of S. aureus.

Aim: Carry out comparative analysis using time-of-flight mass-spectrometry with matrix laser desorption/ionization (MALDI-TOF MS) of protein profiles of brucellosis causative agents (Brucella melitensis Rev-1 and Brucella abortus 19BA), cultivated in various nutrient media: Albimi agar, brucellagar and erythrit-agar., Materials and Methods: Vaccine,strains: Brucella melitensis Rev-1 and Brucella abortus 19BA. Protein profiling in linear mode on Microflex "Bruker Daltonics" MALDI-TOF mass-spectrometer., Results: A number of characteristic features of brucella mass-spectra was detected: in particular, preservation of the total qualitative composition of protein profiles of cultures and significant differences in the intensity of separate peaks depending on the nutrient medium used., Conclusion: Based on the analysis of the data obtained, use of Albimi agar as the nutrient medium for preparation of brucella culture samples for mass-spectrometric analysis was shown to be optimal.

Aim: Comparative evaluation of species identification of microorganisms by MALDI-TOF mass-spectrometry and automatic biochemical analyzer VITEK2 Compact 30., Materials and Methods: Species identification of 18,400 isolates of microorganisms (staphylococci, streptococci, enterococci, enterobacteria, nonfermenting gram-negative bacteria, lactobacilli, anaerobes, yeast fungi, neisseriae), isolated from vagina of pregnant and non-pregnant women and from newborns, was carried out. Identification of the isolated microorganisms was carried out by automatic bacteriologic analyzer VITEK2 Compact30 (BioMerieuX, France) and MALDI-TOF-MS analysis method on AutofleXIII (Bruker Daltonics, Germany) mass-spectrometer., Results: Comparative identification of 2005 isolates of microorganisms was carried out. Sequencing of ribosomal RNA was used as a reference method. Authenticity of species identification my MALDI-TOF-MS analysis method was: for staphylococci (95.8%), enterococci (97.5%), enterobacteria (98.4%), nonfermenting gram-negative bacteria (93.6%), β-hemolytiC staphylococci (93.8%), lactobacilli (92.8%), yeast fungi (99.9%)., Conclusion: Introduction of MALDI-TOF-MS analysis technology into practical work of microbiological laboratories exceeds previously used methods of microbiological testing in terms of speed; cost and authenticity of identification of a wide spectrum of microorganisms.

Purpose: Study the characteristics of proteomic profile of esophageal mucosa in different forms and gradations of gastro-esophageal reflux disease (GERD)., Materials and Methods: The study included 68 patients with verified GERD. Studied the proteomic profile of a gullet a method of tandem time-of-flight MALDI mass spectrometry using bioinformation algorithms., Results: The identified proteins are involved in the regulation of inflammation, apoptosis, adhesion and transport of ions and other important biological processes. The data obtained allow to expand the understanding of the molecular mechanisms of this pathology development. Identified proteins in the esophagus can be used as informative markers of the complicated course of GERD.

The comparative analysis was carried out concerning effectiveness of three techniques of identification of Corynebacterium non diphtheriae: bacteriological, molecular genetic (sequenation on 16SpRNA) andmass-spectrometric (MALDI-ToFMS). The analysis covered 49 strains of Corynebacterium non diphtheriae (C.pseudodiphheriticum, C.amycolatum, C.propinquum, C.falsenii) and 2 strains of Corynebacterium diphtheriae isolated under various pathology form urogenital tract and upper respiratory ways. The corinbacteria were identified using bacteriologic technique, sequenation on 16SpRNA and mass-spectrometric technique (MALDIToF MS). The full concordance of results of species' identification was marked in 26 (51%) of strains of Corynebacterium non diphtheriae at using three analysis techniques; in 43 (84.3%) strains--at comparison of bacteriologic technique with sequenation on 16S pRNA and in 29 (57%)--at mass-spectrometric analysis and sequenation on 16S pRNA. The bacteriologic technique is effective for identification of Corynebacterium diphtheriae. The precise establishment of species belonging of corynebacteria with variable biochemical characteristics the molecular genetic technique of analysis is to be applied. The mass-spectrometric technique (MALDI-ToF MS) requires further renewal of data bases for identifying larger spectrum of representatives of genus Corynebacterium.

Aim: Creation of a classification model of Leptospira spp. serovar model using ClinProTools 3.0 software and evaluation of use of MALDI-TOF MS as a method of quality control of reference strains of leptospira., Materials and Methods: 10 reference strains of Leptospira spp. were used in the study according to microscopic agglutination reaction from the collection of Pasteur RIEM. All the strains were cultivated for 10 days in Terskikh medium at 28 degrees C. Cell extracts were obtained by ethanol/formic acid method. α-cyano-4-hydroxycinnamic acid solution was used as a matrix. Mass-spectra were obtained in Microflex mass-spectrometer (Bruker Daltonics, Germany). External validation of the test-model was carried out using novel spectra of every reference strain during their repeated reseeding., Results: Values of cross-validation and confirmatory ability of the optimal model, built on a genetic algorithm, was 99.14 and 100%, respectively. This model contained 11 biomarker peaks (m/z 2959, 3447, 3548, 3764, 3895, 5221, 5917, 6173, 6701, 7013, 8364) for serovar classification. Results of the external validation have shown a 100% correct classification in serovar classesin Sejroe, Ballum, Tarassovi; Copenhageni, Mozdoc, Grippotyphosa and Patoc, that indicates a high prognostic ability of the model in these classes. However, data from verification matrix have shown, that 50%.of the spectra from Canicola and Pomona serovars were classified as Patoc class, that could be associated with cross serological activity of Patoc serovar L. biflexa with pathogenic leptospirae., Conclusion: MALDI-TOF mass-spectrometry method combined with building and using the classification model could be a useful instrument for intra-laboratory control of leptospira reseeding.

The diagnostic of infections of blood flow using technique of hemofermentation (blood inoculation) is one of the most significant functions of laboratory of clinical microbiology. The effectiveness of the given technique depends on many factors, including criteria of evaluation of clinical significance of episode of bacteriemia and isolated microorganism applied by physician-microbiologist. The intelligent analysis of received results is needed. The physician-microbiologist has to determine if microorganism isolated from given blood sample, is a genuine agent of infections of bloodflow or it is only effect of contamination of analyzed sample at certain stage. The article presents data concerning taxonomic structure of microorganisms isolated under episodes of bacteriemia of adult oncologic hematologic patients during 2005-2013. The criteria of evaluation of clinical significance of episode of bacteriemia and isolated microorganism are described. The given criteria are developed in the N.N. Blokhin Russian oncological research center and are applied since 1977. The cases of contamination and genuine bacteriemia are established. The comparative analysis of international data and results of one's own study are carried out.

Aim: Isolate bacteriocins from Enterococcus faecium metabolites and characterize their effect on cells of Gram positive (Listeria monocytogenes) and Gram negative (Escherichia coli) bacteria., Materials and Methods: Methods of solid-phase extraction, ion-exchange and reversed phase chromatography were applied for isolation of bacteriocins from cultural medium of bacteria MALDI time-of-flight mass-spectrometry was used for characterization of the obtained preparations. The mechanism of biological effect of peptides was evaluated using DNA-tropic dyes (SYTO 9 and PI) with subsequent registration of fluorescence spectra: Atomic-force microscopy (AFM) was used for characterization of morpho-functional reaction of target cells., Results: Peptide fractions with mass of 1.0 - 3.0 kDa were isolated from enterococci metabolites, that inhibit the growth of indicator microorganisms. E. faecium strain exoproducts were shown to increase membrane permeability during interaction with L. monocytogenes, that results in subsequent detectable disturbance of normal cell morphology of listeria. Alterations of E. coli surface during the effect of purified peptide fraction was detected using AFM., Conclusion: The studies carried out have revealed the effect of bacteriocins of enterococci on microorganisms with various types of cell wall composition and have confirmed the importance of bacterial barrier structure permeability disturbance in the mechanism of antimicrobial effect of enterocins.

Species classification of a strain of the bacterium oil-destructor Acinetobacter sp. I B DT-5.1/l is established by means of gas chromatography-mass spectrometry fatty acid methyl esters in the cell wall and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of proteins of a cell.

A phospholipid drug delivery nanosystem with particle size up to 30 nm elaborated at the Institute of Biomedical Chemistry has been used earlier for incorporation of doxorubicin (Doxolip). This system demonstrated higher antitumor effect in vivo as compared with free doxorubicin. In this study the effect of this nanosystem containing doxorubicin on HepG2 cell proteome has been investigated. Cells were incubated in a medium containing phospholipid nanoparticles (0.5 mg/ml doxorubicin, 10 mg/mL phosphatidylcholine). After incubation for 48 h their survival represented 10% as compared with untreated cells. Cell proteins were analyzed by quantitative two-dimensional gel electrophoresis followed by identification of differentially expressed proteins with MALDI-TOF mass spectrometry. The phospholipid transport nanosystem itself insignificantly influenced the cell proteome thus confirming previous data on its safety. Doxorubicin, as both free substance and Doxolip (i.e. included into phospholipid nanoparticles) induced changes in expression of 28 proteins. Among these proteins only four of them demonstrated different in response to the effect of the free drug substance and Doxolip. Doxolip exhibited a more pronounced effect on expression of certain proteins; the latter indirectly implies increased penetration of the drug substance (included into nanoparticles) into the tumor cells. Increased antitumor activity of doxorubicin included into phospholipid nanoparticles may be associated with more active increase of specific protein expression.

The proteome profile of Danio rerio embryos grown in the medium containing doxorubicin, included in the phospholipid transport nanosystem (doxolip) has been investigated using combination of 1D-electrophoresis with subsequent MALDI-TOF-PMF mass spectrometry. Cultivation of growing of D. rerio embryos in the medium with doxolip caused a substantial increase in expression of the cytoskeletal proteins, a decrease in the number of nuclear proteins involved in DNA and RNA synthesis and disappearance of vitellogenin 2 in comparison with control (the cultivation medium containing the phospholipid transport nanosystem). Analysis of the proteomic profiles of doxolip-treated embryos suggests lower toxicity of doxorubicin incorporated in the phospholipid nanosystem.

Aim: Analysis of differences in protein spectra of various bifidobacteria strains of intestine microsymbiocenosis using identification results from MALDI-TOF mass-spectrometer., Materials and Methods: Results of mass-spectrometry ("Bruker Daltonics", Germany) for 57 intestine isolates' of Bifidobacterium spp. are provided. 500,laser impulses were used for obtaining every mass-spectrum; parameters of mass-spectrometer were optimized for the 1000-18000 m/z (mass to charge) range., Results: Comparative analysis of mass-spectrometry biomarkers for Bifidobacterium genus members has detected variations in the quantity of peaks (4 to 56) among both various species and within bifidobacteria species, that reflects uniqueness of the protein profile of separate strains. Along with biomarkers, specific for most cultures, significant differences of the examined peaks were detected; including among microorganisms, that belong to the same species. As such, for B. bifidum species strains--only in 67 ± 7.5% of cultures the presence of common peaks in'the 9282-9901 m/z was detected, whereas protein spectra in other ranges differed by both quantity and molecular mass., Conclusion: Differences in protein profile of Bifidobacterium genus microorganisms reflect uniqueness of protein spectra (proteome) of every separate strain; determining their functional activity, features of interaction, with associative microsymbionts and host organism in human associative symbiosis.

Aim: Characteristics of quantitative and qualitative composition of cultured microorganisms isolated from axilla skin of practically healthy individuals., Materials and Methods: 77 practically healthy individuals aged 18 to 40 years were examined. Species identification of microorganisms was carried out byculture-morphologic, tinctorial and biochemical properties using time-of-flight mass spectrometer and rpoB gene amplification with subsequent direct sequencing., Results: Quantitative evaluation of microbial composition of axilla skin microbiota in most of the practically healthy individuals varied in the 4-5 lg CFU/ml interval, whereas seeding of skin by this microbiota at the level of 8 lg CFU/ml was not detected. 158 strains of 24 microorganism species were identified in this biotope. Most of these strains (68.9%) belonged to Corynebacterium genus, 21.6% of strains--to Staphylococcus genus, 7.6% of strains--to Micrococcus genus and 1.9% of strains--Candida albicans. 16 species of corynebacteria were isolated with predomination of C. tuberculostearicum (40.3%), C. amycolatum (18.4%) and C. ureicelerivorans (14.8%) strains. The microbial landscape in most of the examined individuals (77.9%) was presented by microorganism association., Conclusion: Quantitative and qualitative species composition of cultured microorganisms isolated from axilla skin biotope of practically healthy individuals was characterized for the first time.

The technique MALDI-TOF mass spectrometry was applied using device Microflex with database MALDI Biotyper (Bruckeer Daltonics Inc.) to identify with high level of reliability 8 strains P. fulva from collection of pseudo monads isolated from clinical material in St. Petersburg. When analyzing the same strains applying technique MALDI TOF mass spectrometry using device Vitek MS (bioMerieux) these starins were wrongly identifies as P. putida. The complex of tests of common analysis was approved and proposed for control differentiation of P. fulva and P. putida. The medical significance of P. fulva was approved.

It has been shown that a producer strain of reactivating factor (RF) is identical to a typical strain of Luteococcus japonicus DSM 10546 from the Propionibacteriaceae family according to the physiological and biochemical properties and the sequencing of 16S rRNA fragments. A number of phenotypical differences from the model strain allowed the producer strain to be considered a subspecies of Luteococcus japonicus, and it was named Luteococcus japonicus subsp. casei. At cultivation of the producer, RF is secreted into the medium and plays the role of a signaling molecule. RF antioxidant activities towards various organic radicals may be a possible mechanism of its protective and reactivating effects. Metabolites secreted by the L. casei producer strain into the culture medium were separated by a combination of liquid chromatographies. Four components possessing biological activities were found. The most active one was studied by MALDI-TOF mass spectrometry, which revealed that it is a polypeptide. Primary identification of some amino acid residues was performed. Sugar residues were found in the structure.

Vibrio parahaemolyticua and Vobrio alginolyticus are phylogenetically closely-related species. They have common ecological niches, same cultural features and similar biochemical characteristics. The phenotype variability and taxonomy similarity of strains of these species impedes differentiation of Vibrio parahaemolyticua and Vobrio alginolyticus according biochemical characteristics. To obtain reliable results of diagnostic application of additional methods of differentiation and identification these two species of bacteria are needed. The study was organized to comparatively evaluate effectiveness of biochemical testing, polymerase chain reaction analysis and mass-spectrometry technique in differentiation of species of Vibrio parahaemolyticua and Vibrio alginolyticus. The study implemented analysis of methods of differentiation of species of Vibrio parahaemolyticua and Vibrio alginolyticus using model of collection including atypical strains of these species. To substantiate species belonging of strains of Vibrio parahaemolyticua and Vobrio alginolyticus such techniques are to be additionally applied to biochemical methods of identification as polymerase chain reaction analysis with species-specific primers of genes of metalloproteinase (collagenase) vppC and vapC. The MALDI-TOFF method of mass-spectrometry can be used as additional effective method of identification and inter-species differentiation of species of Vibrio parahaemolyticua and Vibrio alginolyticus isolated from various sources.

Homology of the amino acid sequence of the mitochondrial potassium-transporting protein (MW 57kDa), having the properties of a channel subunit of the mitochondrial ATP-dependent potassium channel, and calreticulin (MW 55kDa) was detected by MALDI-TOF-TOF analysis method. Inhibitory analysis of ATP-dependent potassium transport in mitochondria with polyclonal antibodies to calreticulin was carried out. A dose-dependent inhibition of potassium transport in mitochondria by these antibodies was shown. The maximum value of inhibition was 55-60%. Based on these data it is hypothesized that at least two types of ATP-sensitive potassium channels are localized in mitochdndrial membrane. It is expected that the type of mitochondrial ATP-dependent potassium channel, which includes homologous calreticulin protein is localized mainly at mitochondrial and reticulum membrane contact sites.

Gastric cancer, one of the most widespread malignant tumors, still lacks reliable serum/plasma biomarkers of its early detection. In this study we have developed, unified, and tested a new methodology for search of gastric cancer biomarkers based on profiling of low molecular weight proteome (LMWP) (1-17 kDa). This approach included three main components: sample pre-fractionation, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS), data analysis by a bioinformatics software package. Applicability and perspectives of the developed approach for detection of potential gastric cancer markers during LMWP analysis have been demonstrated using 69 plasma samples from patients with gastric cancer (stages I-IV) and 238 control samples. The study revealed peptides/polypeptides, which may be potentially used for detection of this pathology.

Aim: Conduction of a comparative proteomic mass-spectrometric (MS) analysis using a personal database of V. cholerae protein mass-spectra and genetic VNTR-typing of cholera causative agent strains., Materials and Methods: V. cholerae O1 El Tor strains - 7, V. cholerae non O1/non O139 - 2. Protein profiling and VNTR-genotyping of strains was carried out on MALDI TOF-MS Autoflex (Bruker Daltonics) mass-spectrometer and SIS Cholera-strains-VNTR., Results: The established community of a proteomic profile of epidemic cholera vibrio strains isolated in 2010 - 2012 in Moscow allowed to determine Indian origin of a toxigenic strain isolated in Taganrog in 2011. M/z proteins distinguishing V. cholerae O1 and non O1/non O139 strains were identified. Proteomic analysis confirms the results of VNTR-genotyping., Conclusion: Study and typing of V. cholerae members with determination of their origin and phylogenetic relationship is possible using a collection of V. cholerae mass-spectra.

The aim of the research was the search of the mechanism of Cytoflavin action in patients with isolated systolic arterial hypertension (ISAH) as the aging model. The following research methods were applied: the assessment of complaints, 24-hour arterial pressure monitoring, ultrasound diagnostic, volume sphygmography, lipid profiles and coagulogram, molecular phenotyping by MALDI-TOF-TOF-MS. The combination of Cytoflavin with standard therapy of ISAH led to the most expressed return development of clinical symptoms, the restoration of the haemodynamics, structural and geometrical parameters of cardiovascular system, indicators lipid profiles and coagulogram in the comparison with patients with ISAH, who accepted standard therapy and those of the control groups. Molecular mechanisms of Cytoflavin action, including regulation of the activity of cell signaling pathways through intermolecular interactions were found. We recommend using Cytoflavin together with the standard therapy in treatment of ISAH, which provides the action of geroprotectors for cardiovascular system.

The reaction of high temperature solid state catalytic isotope exchange in peptides and proteins under the action of catalyst-activated spillover hydrogen was studied. The reaction of human gene-engineered insulin with deuterium and tritium was conducted at 120-140° C to produce insulin samples containing 2-6 hydrogen isotope atoms. To determine the distribution of the isotope label over tritium-labeled insulin's amino acid residues, oxidation of the S-S bonds of insulin by performic acid was performed and polypeptide chains isolated; then their acid hydrolysis, amino acid analysis and liquid scintillation counts of tritium in the amino acids were conducted. The isotope label was shown to be incorporated in all amino acids of the protein, with the peptide fragment FVNQHLCGSHLVE of the insulin β-chain showing the largest incorporation. About 45% of the total protein isotope label was incorporated in His5 and His10 of this fragment. For the analysis of isotope label distribution in labeled insulin's peptide fragments, the recovery of the S-S bonds by mercaptoethanol, the enzymatic hydrolysis by glutamyl endopeptidase from Bacillus intermedius and HPLC division of the resulting peptides were carried out. Attribution of the peptide fragments formed due to hydrolysis at the Glu-X bond in the β-chain was accomplished by mass spectrometry. Mass spectrometry analysis data of the deuterium-labeled insulin samples' isotopomeric composition showed that the studied solid state isotope exchange reaction equally involved all the protein molecules. Biological studying of tritium-labeled insulin showed its physiological activity to be completely retained.

The earliest eight clinical strains of Pseudomonas fulva were identified in the culture collection of pseudomonads isolated in St. Petersburg in 1995-2005, that confirmed the medical importance of the species. A high level of the species identification of all the strains of P. fulva by MALDI-TOF mass-spectrometry with the use of Microflex device with database MALDI Biotyper (Bruker Daltonics Inc.) was shown. Tests for routine studies providing identification of P. fulva without the use of genetic methods were approved. The profile of the antibiotic susceptibility of the clinical strains of P. fulva was described. Acquired resistance of two P. fulva isolates to the 3rd generation cephalosporins and chloramphenicol was detected.

The molecular pharmacological effects of cytoflavin in patients with hypertensive encephalopathy (HE) and isolated systolic arterial hypertension (ISAH) have been investigated using the following methods: assessment of complaints, 24-hour arterial pressure monitoring, ultrasound diagnostics including echocardiography, measurement of lipid profiles and coagulograms, and molecular phenotyping by MALDI-TOF/TOF-MS. A combination of cytoflavin administration with standard therapy of HE and ISAH led to the most expressed return development of clinical symptoms, restoration of the hemodynamic, structural, and geometrical parameters of cardiovascular system, and normalization of the indices of lipid profiles and coagulograms in comparison to patients with HE and ISAH, which accepted only standard therapy. Molecular mechanisms of cytoflavin action have been revealed, which include control of the activity of cellular signaling pathways by means of intermolecular interactions. The optimized therapy of HE and ISAH is recommended for clinical application, which assumes a combined use of standard therapy and cytoflavin and provides a geroprotective action upon the cardiovascular system.

The goal of this work was to develop methodological approaches to identification of the Vibrio genus representatives using the MALDI-TOF mass-spectrometric analysis technologies. The aspects of the biological safety in sample preparations for mass-spectrometric analysis were studied, reference spectra of six typical V. cholerae strains were developed. Identification of 55 strains, representatives of the Vibrio genus, including 45 V. cholerae strains with different epidemic importance, was performed using the MALDI Biotyper 3.0 basis comprising V. cholerae reference spectra. The possibility of reliable definition of the tested strain taxonomic belonging to the species level was demonstrated. Thus, the results completely corresponded to the data of classical microbiological identification. Stability and reproducibility of the offered research method was experimentally shown. The results allow identification of the Vibrio genus representatives to be implemented with the use of the mass-spectrometric analysis as an effective method that defines a species belonging of the basic Vibrio genus representatives in the shortest-terms.

Phycocyanin, a high value pigment was purified from diazotrophic cyanobacteria Anabaena variabilis CCC421 using a strategy involving ammonium sulfate precipitation, dialysis and anion exchange chromatography using DEAE-cellulose column. 36% phycocyanin with a purity of 2.75 was recovered finally after anion exchange chromatography. Purified phycocyanin was found to contain 2 subunits of 17 and 18 kDa which were identified as a-and (3 subunits by SDS-PAGE and MALDI-TOE HPLC method using a C5 column coupled with fluorescence or photodiode-based detection was also developed to separate and detect the A. variabilis CCC421 phycocyanin subunits. The fluorescence method was more sensitive than photodiode one. The purified phycocyanin from A. variabilis CCC421 as well as its subunits was characterized with respect to absorption and IR spectra. Spectral characterization of the subunits revealed that alpha and beta subunits contained one and two phycocyanobilin groups as chromophores, respectively.

Protein fraction able to induce K(+)-selective transport across bilayer lipid membrane was isolated from human blood plasma with the use of the detergent and proteolytic enzyme-free method developed at our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel forming activity of fraction was observed only in the presence of K+. Using a threefold gradient of KCl in the presence of studied proteins the potassium-selective potential balanced by voltage of -29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to data obtained with MS-MALDI-TOF/TOF and database NCBI three protein components were identified in isolated researched sample.

Extracellular beta-D-glucosidase was isolated in a homogeneous state from the Penicillium canescens marine fungus. According to SDS-electrophoresis, the molecular weight of the enzyme was 64 kDa and the maximal activity was observed at pH 5.2 and 70 degrees C. Glucosidase catalyzed the hydrolysis of beta-glycosidic bonds both in glycosides and in glucose disaccharides and had transglycosylation activity. The enzyme can be used for the deglycosylation of natural glycosides and in enzymatic synthesis of new carbon-containing compounds.

In the present work the studies ofbacteriolytic factors from sheep blood plasma have been performed. Three novel enzymes have been identified and characterized. Two of them have a molecular weight 15 +/- 2 kDa and able to lyse the gram-negative Escherichia coli bacteria. The third enzyme has a molecular weight 34 +/- 4 kDa and is able to lyse both gram-negative Escherichia coli and gram-positive Micrococcus luteus bacteria. The bacteriolytic reactions have been studied for all three enzymes; particularly, pH-optima have been identified with respect to the substrate. To identify the enzymes trypsinolysis and consequent MALDI-TOF mass spectrometry studies were performed. The results were compared to data from publicly available databases, such as Swiss-Prot, NCBI, MSDB.

We identified individual proteins characterizing starvation-induced metabolic stress. Sustained changes at the level of proteome caused by metabolic stress were demonstrated. Intensified synthesis of S6 kinase ribosomal protein, immunoglobulin lambda light chains, and inter-alpha-trypsin inhibitor was observed in the experimental groups.

Point mutations associated with isoniazid resistance in Mycobacterium tuberculosis (MTB) have been analyzed in codon 315 of the katG gene by conventional polymerase chain reaction (PCR) using primers containing locked nucleic acid (LNA) modified nucleotides. Purity and structure of primers containing 5 LNA monomers of 17 nucleotides in length were characterized by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and a 17-mer duplex formed by two complementary oligonucleotides was characterized by the method of thermal denaturation. The duplex containing five LNA monomers per each strand was characterized by a higher melting temperature than it was expected using extrapolation of theoretical calculation for nucleotide modification of one strand of the duplex. Detection of any of six possible mutations in katG codon 315 (i.e. discrimination between sensitive and resistant MTB) requires just one PCR employing a set of two primers with one LNA-modified primer; this is an important advantage of oligonucleotides containing LNA over unmodified nucleotides: employment of multiplex PCR would require up to 12 primers. Problems of control of oligonucleotide modification by LNA monomers are discussed.

There was studied an influence of intragastric administration of titanium dioxide (anatase form) nanoparticles (NP) on protein expression profiles in rat's liver microsomes by methods of proteomics. Animals received water suspension of NP in doses from 0,1 to 10 mg per kg body weight intragastrically daily during 28 days. Microsomes were isolated from liver by means of preparative ultracentrifugation. Proteins composition was studied by 2D-electrophoresis in acrylamide gel. Protein spots were identified by MALDI-TOF analysis. The results demonstrated appearance of 53 new protein spots and disappearance of 19 spots in animals subjected to NP irrespective of their dose. In addition 25 new spots appeared and 3 disappeared in higher doses of NP when compared to low dose group and control animals. Mass-spectrum analysis showed presence of few polypeptides registered in international database among proteins expressed under influence of NP. One of this dominant expressed proteins corresponded to enzyme glutathione transpherase Mu 2 isoform (M=41,55 kD, pI=8,0). The conclusion was made of well advances of proteomic analysis in artificial NP influences on biosynthetic processes estimation.

Exosomes are 20-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro and in vivo. Since exosomes contain both RNA (mRNA and microRNA) and proteins, which can be transferred to another cell, and be functional in that new environment, these vesicles may be involved in the communication between cells. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo suggest their participation in pathological situations. Our purpose here is to describe methods for the production, purification, and proteomic characterization of exosomes derived from human cancer cells in vitro. Based on exosomes' unique lipidic composition, we have developed the new approach to increase production of exosomes by cells in vitro. Secondly, we have developed quality control by laser correlation spectroscopy for exosomal assays based on the amount of MHC class I and CD63 molecules on their surface. At last, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was used after 2D electrophoresis for the proteomic analysis of exosomes derived from cancer cell lines. This study describes the protein composition of brain tumor cell-derived exosomes in more detail.

In the present study, a proteomic technology combining one-dimensional gel electrophoresis (1DE) with subsequent mass spectrometry (MALDI-TOF-PMF) has been successfully applied for revelation of changes in the protein profile of zebrafish (Danio rerio) 52 hpf embryos. Prior to 1DE separation of zebrafish embryonic proteins, the procedure for obtaining embryos homogenate was optimized by ultrasonic treatment. A total of 84 proteins, including 15 vitellogenins, were identified. It was shown that growing ofzebrafish embryos in the medium with doxorubicin (DOX) stimulated Caspase-3 induction and promoted the disappearance of cardiac troponins, both these findings being consistent with literature data on doxorubicin-induced cardiotoxicity. The 1DE-based proteomic mapping approach proposed herein enabled not only to identify proteins but also to register those changes in embryos' proteomic profile that were caused by doxorubicin.