[O3:K6 serogroup vibrio parahaemolyticus - the causative agent of food toxic infection outbreaks in Primorsky region of Russian federation].

AIM: Comparative evaluation of biological properties of parahemolytic vibrios that had determined outbreaks and sporadic cases of food toxic infection in Primorsky Region in 2012 and previous years. Materials AND METHODS: 40 clinical strains of Vibrio parahaemolyticus isolated in 2012 were studied in comparison with 62 strains from this region that had been characterized by us previously. Virulence was evaluated by a complex method: hemolytic activitywas determined in Kanagawa test (KT), urease - in Kristensen medium. Serotyping was carried out by a commercial kit of O/K sera. PCR-genotyping was carried out by marker genes of 7 pathogenicity "islands" (VPaI-1-7). RESULTS: All the strains isolated from patients in 2012 had KT-positive and urease-negative phenotype, belonged to O3:K6 serogroup and contained marker genes of 7 VPal that allowed to consider them members of a "pandemic" clone as the other clinical strains from this region. However among 2012 strains an increase of number of antibiotic-resistant variants was established compared with 1997 isolates. CONCLUSION: The data obtained give evidence on the risk of spread of a "pandemic" clone of V. parahaemolyticus in the Far-Eastern region of Russia, a dangerous tendency of antibiotic-resistant variant formation and a necessity to monitor morbidity and the environment with mandatory PCR-detection of genes associated with virulence including integrated into pathogenicity "islands".

[Proteome mass-spectrometric analysis and typing of Vibrio cholerae strains isolated in the territory of Russian Federation in 2010 - 2012].

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 application of mass spectrometry technique MALDI-TOF for inter-specific differentiation of closely-related vibrio].

The study was targeted to apply mass spectrometry analysis for inter-specific differentiation of collection strains of representatives of genus vibrio and identification of comma bacilli extracted from samples of boat ballast waters. The samples consisted of 207 museum strains of cultures and 347 microorganisms from samples of boat ballast waters. The identification of microorganisms was implemented using MALDI-TOF mass spectrometers. The application of MALDI biotyping made it possible to detect inaccuracies in specific and generic characteristics of collection strains V. alginolyticus, V. parahaemolyticus, Shewanella, V. mimicus and to enhance characteristic and quality of collections of microorganisms. The effective application of MALDI to monitor boat ballast waters is demonstrated. This technique allows detecting of complete vibrio landscape of pathogenic and non pathogenic genus. The possibility to detect strains of comma bacillus and cholera agents is demonstrated. The mode of orientation on V. albensis at the stage of selection of suspicious colonies is proposed.

[Collection of Vibrio parahaemolyticus species members: phenotypic and genotypic characteristics].

AIM: Formation of Vibrio parahaemolyticus collection according to modern methodical opportunities and understanding of causative agent biology. MATERIALS AND METHODS: Traditional biochemical tests and PCR-testing of species-specific genes were used to confirm species membership. Catalase, DNAse, proteolytic and tweenase activity was determined by common methods. Virulence was evaluated by a complex method: hemolytic activity was determined in Kanagawa test (KT), urease--in Christensen medium, PRC-testing of tdh and-trh genes. Serotyping was carried out with a commercial O/K-sera kit. PCR-genotyping was carried out by marker genes of 7 pathogenicity islands (VPaI-1-7). RESULTS: Species membership was confirmed for the studied strains. Serologic typing allowed to detect members of 18 serologic groups among the collection strains. All the collection cultures were divided into 4 groups based on KT-Ure-tdh-trh features recombination. A number of genetic variants were detected, strains belonging to a pandemic group and O3:K6 serogroup were determined. CONCLUSION: A collection of V. parahaemolyticus cultures was formed and characterized by a large set of pheno- and genotypic features. A database was developed including information on strain origins, pheno- and genetic features, with genetic variants given, for ease of use of the collection.

[The differentiation of specimiens of Vibrio parahaemolyticua and Vobrio alginolyticus].

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.

[The development of complex technique of evaluation of virulence of parahemolytic vibrio].

The article deals with results of studying parahemolytic vibrio separatedfrom different sources according their phenotype and genotype attributes associated with virulence. In certain cases the mismatch of results of Kanagava tests and polymerase chain reaction test of gene tdh was established. The need in virulence complex evaluation is substantiated. This complex has to include detection of hemolytic activity in Kanagava test and urease activity on the Kristensen medium and polymerase chain reaction detection of genes tdh and trh. The developed complex technique is described. The formula of pathogenic strains is established Three alternatives of virulent parahemolytic vibrio are given. The test-strains Vibrio parahaemolyticus are proposed as control in testing phenotype and genotype strains according virulence signs.

[Use of various methods to isolate exopolysaccharide of eltor cholera Vibrios and its immunochemical characteristics].

AIM: Isolation of Vibrio eltor exopolysaccharide and study of its immunochemical properties. MATERIALS AND METHODS: Rugose variants of strains V. eltor 18895 and V. eltor 18843 obtained by us by selection in M9 medium were used in the study. Exopolysaccharides (EPS) were isolated by K. Kierek (2003), S.P. Zadnova (2004), N.P. Elinova (1984) methods and analyzed for carbohydrate, protein, nucleic acid content and lipopolysaccharide impurity. EPS, LPS, R-LPS structure was compared by high-pressure chromatography. Neutral sugars and amino sugars were identified by thin layer chromatography. Polyclonal antibodies were produced against EPS preparation isolated by N.P. Elinova (1984) method. Specific activity of obtained mice sera was tested by DIA method. RESULTS: EPS isolated by N.P. Elinova method (1984) was shown not to contain extraneous impurities. V. eltor EPS structure differs from LPS and R-LPS. Monosaccharide composition of EPS from ctx+ V. eltor 18895 strain is presented by a wider specter of carbohydrates including glucose, mannose, rhamnose, galacturonic acid. Use in DIA of specific sera produced against EPS from toxigenic strain did not reveal general epitopes with capsule polysaccharides of V. cholerae O139, V. parahaemolyticus and V. vulnificus. CONCLUSION: Use of EPS as an immunogen promoted production of sera that are specific against EPS and rugose variants of Vibrio cholerae eltor that can be used for their detection or characterization.

[GM1-dot-EIA for the detection of toxin-producing Vibrio cholerae strains].

A new variant of enzyme immunoassay (EIA) has been developed on the basis of GM1 gangliosides to detect the toxin-producing Vibrio cholerae strains--GM1-dot-EIA. Experiments were run using a nitrocellulose membrane to bind GM1 gangliosides and polyclonal antitoxic serum to detect cholerogen. GM1-dot-EIA testing identified cholera toxin in 11 of 13 supernatants of V. cholerae eltor ctx(+) strains isolated from man and in 3 of 7 supernatants of V. cholerae eltor ctx(+) strains isolated from water. These data agree with those obtained in CM1-EIA. There was no reaction with the supernatants of other microorganisms. The sensitivity of the technique was 10 ng/ml. Thus, the simple and specific GM1-dot-EIA may be recommended to detect toxin-producing V cholerae strains isolated from man and water.

[Evaluation of polysaccharide antigens of Vibrio cholerae O139 of different origin by monoclonal antibodies]. / Otsenka polisakharidnykh antigenov Vibrio cholerae O139 razlichnogo proiskhozhdeniia s pomoshch'iu monoklonal'nykh antitel.

The epitope composition of O-polysaccharides in the lipopolysaccharide (LPS) of V. cholerae, serogroup O139, isolated from clinical material and water of surface reservoirs was analyzed with the use of monoclonal antibodies. The analysis demonstrated that these O-polysaccharides were similar in their structure and chemical composition. In LPS of V. cholerae O139 clinical strains O-polysaccharide determinants occurred more often. Among V. cholerae isolated from water strains on whose surface individual epitopes of O-polysaccharide occurred less frequently or were absent appeared to be more numerous. A decrease in the concentration of microbial cells in the process of their testing by immunological methods led to increased percent of negative reactions with specific antibodies. Some V. cholerae O139 strains isolated from water were similar in the epitope composition of their O-polysaccharide and binding activity to cultures isolated from humans. As indicated by the results of these studies, cholera vibrios Bengal and vibrios isolated from river water on the territory of Russia had quantitative differences due to a higher level of the production of O-polysaccharide determinants and their occurrence in V. cholerae of serogroup O139.

[Obtaining diagnostic fluorescent monoclonal immunoglobulins against the V. cholerae 0139-serovar]. / Poluchenie diagnosticheskikh fliuorestsiruiushchikh monoklonal'nykh immuhoglobulinov k V. cholerae 0139-serovara.

Below is given a procedure of the obtaining diagnostic fluorescent monoclonal immunoglobulin to detect cholera vibrios of O139 serovar. While obtaining preparations it was managed to determine optimal FTTS-MKA ratio, duration of their conjugation, series of fluorochrome. Test specimens of fluorescent monoclonal immunoglobulin provides intensive glow of V cholerae O139 cells in the working dilution 1:16-1:32. Tests of diagnostic FTTS-MKA on the great number of homologic and heterologic strains showed their strict specificity and high sensibility as to cholera vibrios of O139 serogroup.