Ultrastructural Changes in the Intestine of Suckling Rabbits Infected with Cholerogenic and Non-Cholerogenic nonO1/nonO139 Vibrio cholerae Strains.

We performed an electron microscopic study of the small intestine of suckling rabbits infected with cholerogenic and non-cholerogenic strains nonO1/nonO139 Vibrio cholerae. Cholerogenic strain induced mostly hydropic degeneration of the epithelium typical of cholera toxin effect, while non-cholerogenic strain induced the formation of lacunae along the borders of adjacent epithelial cells typical of hemagglutinin/protease effect. In both cases, reduction of microvilli, destruction of intracellular organelles, two types of mitochondrial reaction (condensation and swelling with destruction of cristae), appearance of myelin figures, defects in the capillary walls, and activation of pinocytosis were observed. These data confirm our previous assumption on interchangeability of different pathogenic factors of Vibrio cholerae, including nonO1/nonO139 strains.

[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".

[VNTR-genotyping of Vibrio cholerae strains isolated from objects in the territory of Russian Federation in 2012].

AIM: VNTR-typing of Vibrio cholerae strains isolated in the territory of Russian Federation in 2012. MATERIALS AND METHODS: 71 Vibrio cholerae O3 and 3 V cholerae O1/O139 strains were used in the study. Genotyping was performed by using PCR for 5 VNTR-loci. RESULTS: Multilocus VNTR-typing allowed to group the strains into 31 VNTR-genotypes. Genotypes were divided among 10 discrete clusters by results of a cluster analysis. The presence of tcpA gene is clearly linked with the presence of VcB locus. Each geographic region was characterized by their own VNTR-genotypes. CONCLUSION: In the course of the carried out VNTR-genotyping of V. cholerae isolated in 2012, 2 types of vibrio population formation were detected. A geographic attachment to specific regions was characteristic for most of the genotypes.

[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.

[Study of possibility of formation of Vibrio parahaemolyticus pandemic clones based on retrospective PCR-screening of clinical strains].

AIM: PCR-genotyping of Vibrio parahaemolyticus strains that had caused sporadic diseases in Novorossiysk from 1973 to 1976. MATERIALS AND METHODS: 24 clinical strains of V. parahaemolyticus isolated in Novorossiysk, most of which belonged to serogroups O4:K12 and O4:K8; 10 O3:K6 strains--causative agents of gastroenteritis outbreak in Vladivostok (1997) and 3 from Japan (1971) were used. PCR genotyping was performed by a set of marker genes of 7 pathogenicity islands (VPaI-1 - VPaI-7) and a number of other pathogenicity factors. RESULTS: All the strains isolated in 1970s differed significantly by sets of VPaI marker genes. In contrast to causative agents of outbreak in Vladivostok that contain all 7 VPaI genes (that is, members of the pandemic group that had spread globally since 1996) none of the O4:K12 and O4:K8 Novorossiysk strains contained the full set of all the VPaI genes. However this set was distributed among the members of the group. CONCLUSION: Taking into account that O4:K12 and O4:K8 serogroups are considered by a number of authors as O3:K6 serovariants, PCR-screening data obtained by us allows to assume that horizontal transfer of mobile elements (VPaI) between strains circulating in the region could have led to the formation of pandemic clones already in the 1970s. This implies that in several coastal regions in certain periods of time conditions that favor these process may form, and risk of infection with pandemic clones is associated not only with import of seafood.

[Characteristics of Vibrio cholerae Cef (CHO cell elongating factor): bioinformatics analysis and experimental data].

Bioinformatics analysis of the primary and secondary structure of the Vibrio cholerae Cef (CHO cell elongating factor) protein was conducted. Similarity with triacylglycerol lipases and cytotonic toxins of other bacterial species was observed. Cef was predicted to be a heat-tolerant serine lipase with the Kunitz domain and leucine zipper. These data were confirmed experimentally. The Cefs of the two biotypes of V. cholerae O1, as well as O139 and nonO1/nonO139 serogroups, were purified from the recombinant Escherichia coli strains carrying corresponding cloned genes, and their physicochemical properties, biochemical and biological activities in vitro and in vivo were characterized. Biological activity against the cultured cells was not associated with estherase activity. Evidently, Cef is a bifunctional protein contributing both to pathogenicity of the cholera agent and to its competitive ability in different ecological niches.

Ultrastructural changes in cultured cells under the effect of Vibrio Cholerae hemagglutinin/protease.

Electron microscopic study of changes in cultured cells caused by Vibrio cholerae recombinant hemagglutinin/protease (HA/P) showed significant structural changes, most pronounced in HeLa and L-929 cells not forming a compact monolayer with tight junctions between the cells: formation of numerous vesicles on the cell surface and clasmatosis, vacuolation of the cytoplasm, swelling of mitochondria, clarification of their matrix and crist distortions, and increase in the number of lysosomes. Cytoplasm vacuolation predominated in MDCK culture, while clasmatosis was less intense. Addition of HA/P to CaCo2 cells forming a differentiated polarized monolayer, led to extension of cell-cell spaces not impairing tight junctions, swelling of mitochondria, cytoplasm vacuolation, and clasmatosis on the apical surface. These changes virtually completely coincided with those caused by the so-called NMDCY factor (non-membrane-damaging cytotoxin), described as new Vibrio cholerae toxin. These findings confirm our previous hypothesis about the identity of these factors.

[Point mutation of the Virbrio cholerae O139 cef (CHO cell elongating factor) gene alters the substrate specificity of its product].

Sequencing of the cef (CHO cell elongating factor) of Vibrio cholerae serogroup O139 revealed one nucleotide substitution (C for T in position 2015) in comparison with classical V. cholerae O1 and two substitutions (AC for GT in positions 2014-2015) in comparison with V. cholerae O1 E1 Tor. A comparative bioinformatic analysis showed that the substitution determines a threonine residue in position 672 of the Cefprotein, while the position is occupied by an isoleucine residue in the classical strains and a valine residue in the El Tor group. The last two amino acids are hydrophobic, while threonine is hydrophilic, having a polar R group. The non- synonymous substitution affects the predicted secondary and, probably, tertiary structures of the Cef-O139 protein and explained our previous finding that the protein fails to degrade tributyrin, while retaining the tweenase activity spectrum and all other characteristics. It cannot be excluded that the inability of Cef-O139 to cleave triglycerides, along with other genetic specifics, contribute to the fact that the O139 serogroup has been displaced from a dominating position in etiology of cholera by the El Tor genotype. The nucleotide sequence of the V. cholerae O139 cefgene and the deduced amino acid sequence of its product are reported for the first time and were deposited in GenBank under accession nos. JF499787 and AEC04822.1, respectively.

[Effectiveness of expression of tdh gene of Vibrio parahaemolyticus depends on two point mutations in promoter region].

A molecular-biological study of the clinical strains of Vibrio parahaemolyticus that contain genes of thermostable direct hemolysin Tdh) and Tdh-related hemolysin (Trh). Using Southern blot hybridization, it is shown that genomes of strains that carry determinants of both hemolysins (tdh(+)-trh+) represent a single copy, whereas in tdh2+RH+ strains, there are two copies (tdh1 and tdh2). All of the examined tdh+trh+ and some of the tdh+trh strains either did not express the tdh gene or did not express the tdh gene (Kanagawa negative or KP-) or expressed it weakly and not often (Kanagawa intermediate, KP+), unlike several Kanagawa positive tdh+trh- strains. To establish the reasons for KP -/+ phenotypes, tdh, tdh11, and tdh2 genes of 13 strains isolated in Russia and neighboring foreign countries were sequenced, followed by the biotransformation analysis of the obtained sequences, as well as a comparison with those of a number of strains presented in GenBank. The results revealed that the weak expression of the tdh gene depends, not only on one point mutation in the promoter region (substitution of A for G in the -35 region), as was thought previously, but also on the second substitution (G for A in the -3 position relative to the -10 sequence), which is quite sufficient when the former is absent. Therefore, the reversion of KP -/+ strains that contain one of these substitutions can take place as a result of a single reverse point mutation, and they should be considered potentially dangerous. Strains that contain both substitutions may revert with lesser probability because, in this case, both mutations are necessary.

[Characteristics of Vibrio cholerae nonO1/nonO139 serogroup strains that caused diseases in population of Rostov region].

AIM: Genotype characteristic and determination of serological properties of Vibrio cholerae nonO1/nonO139 strains that caused diseases in population of Rostov region from 2000 to 2009. MATERIALS AND METHODS: 15 clinical strains of V. cholerae nonO1/nonO139 were studied. Serotyping was performed by using a kit of monospecific typing sera against serogroup 02-084 cholera vibrios obtained from Rostov Research Institute for Plague Control, PCR and VNTR-genotyping--by using specific primers described in scientific publications and constructed by us. RESULTS: Serologic features of strains are very diverse and strains contain various combination of pathogenicity factor genes that seem to be interchangeable. Similar pattern was observed for VNTR-genotyping. Distribution of the examined strains by VNTR-genotyping did not correlate with either PCR-genotyping data or serotyping, or place and time of isolation. CONCLUSION: The data obtained indicates a lack of general source of human infection even in the same location and time period. On the other hand, serological and genotypic features of V. cholerae nonO1/nonO139 may undergo changes in the process of staying in the macro organism or environment due to high plasticity of their genome.

[Study of expression of contact-dependent secretion systems in Vibrio cholerae on the model of Dictyostelium discoideum].

AIM: To detect T3SS and T6SS genes in Vibrio cholerae genomes and assessment of resistance of strains with different genotype characteristics to ingestion by Dictyostelium discoideum amoeba. MATERIALS AND METHODS: One hundred thirty-five toxigenic and non-toxigenic strains of Echolerae O1, O139, and non-O1/non-O139 were studied by PCR and on Dictyostelium discoideum model. RESULTS: T3SS cluster of genes was detected in several non-toxigenic representatives of 01 and non-O1/non-O139 serogroups. All toxigenic vibrios except one strain belonging to non-O1/ non-O139 serogroup lack this cluster. T6SS genes were presented in all studied strains although far from allofthem contained sequence coding ACD-domain of vgrG1 gene product--key effector of T6SS. Susceptibility of vibrios to ingestion by amoeba did not depend from presence of T3SS genes. Only strains with genetic determinants ACD-vgrG1 together with pathogenicity locus VPI were resistant to ingestion, all others were susceptible. CONCLUSION: D. discoideum model is adequate for study of expression of T6SS but not T3SS in V. cholerae. The latter is rather needed for colonization of intestine than for antagonistic activity against protozoa in external environment. Expression of T6SS is characteristic for many non-toxigenic strains of V. cholerae, whereas use of contact-dependent secretion is unlikely necessary for majority of toxigenic strains.

[Serotyping and genotypic characteristic of Vibrio cholerae non-O1/non-O139 serogroups isolated from water of surface basins and sewages of Rostov-on-Don city in 2003 - 2008].

AIM: Determination of serogroup and PCR-genotyping of Vibrio cholerae non-O1/non-O139 strains isolated from surface basins and sewages of Rostov-on-Don city in 2003 - 2008. MATERIALS AND METHODS: Seven hundred strains of V. cholerae non-O1/non-O139 serogroups were studied in reaction of slide-agglutination with array of 80 diagnostic sera for non-O1/non-O139 serogroups. Selective screening of strains representing dominating serogroups was performed for extended number of genetic determinants of pathogenicity factors. RESULTS: It was established that V. cholerae belonging to serogroups O53, O67, O75, and O76 are dominating in water ecosystems of Rostov-on-Don city at this time. All studied strains were characterized by lack of cholera toxin genes and toxin-coregulated pili but had different combinations of genes of additional virulence factors. There was no correlation between genotypic characteristics and serogroup. CONCLUSION: The study showed that change of serologic landscape of V. cholerae non-O1/non-O139 occurred in water objects in studied area during last decades. Necessity of dynamic surveillance for circulation of V. cholerae non-O1/non-O139 in aquatic environment with widening of studied spectrum of their biological features was demonstrated.

Ultrastructural changes in the small intestine of suckling mice, caused by vibrio cholerae hemagglutinin/protease.

Suckling mice aged 4-5 days were injected with Vibrio cholerae hemagglutinin/protease and ultrastructural changes in their small intestine were studied after 5 h. The preparation caused a statistically significant accumulation of fluid in the intestine, appearance of large gaps along cell-cell spaces in the villi and crypts, intense production and secretion of the mucus by goblet cells. The formation of interepithelial cavities was paralleled by vascular changes, supplemented by extravasal disorders caused by mast cell reaction. The role of enterochromaffin cells and lipofibroblasts, modulating the secretion in the intestine, is confirmed.

[Virulence of pre-CTXphi-carrying Vibrio cholerae: genotypic and phenotypic characteristics].

AIM: Complex assessment of virulence of cholera vibrios carrying the truncated CTX element (pre-CTXphi prophage). MATERIALS AND METHODS: Twenty-two strainsof Vibriocholerae O1 and non-O1/non-O139 were studied by PCR and laboratory models. RESULTS: Genomes of all strains, besides pre-CTXphi genes, contained genes hapA (hemagglutinin/proteases), cef (CHO cell elongating factor), rtxA (high-molecular cytotoxin), and rtxC (its activator). Nucleotide sequences of rtxA and vgrG genes from ACD domains, genes VPI and VPI-2 from islands of pathogenicity, mshA (mannose-sensitive pili) gene were presented in different combinations. None strains contained shiga-like toxin (slt1) aswell as thermostable direct (tdh) and thermostable direct-related (trh) hemolysin genes of V. parahaemoliticus. On the model of infant rabbits almost all strains caused a significant enteropathogenic effect sometimes resembling cholera effect and in a number of cases dissemination of bacteria into various organs and tissues took place. Cultural supernatants of the majority of strains stipulated cell rounding in CHO cultures (one of them caused cell destruction) and disconnection of cells in McCoy and L-929 dense monolayers as well as increase of skin permeability in Craig's test. Conclusion. Apparently, diarrhea of different severity observed in patients from whom these strains were isolated as well as signs of virulence revealed in the laboratory models were determined by the expression of genes of accessory pathogenicity factors including those detected in the present study.

[Cholera caused by Vibrio cholerae O1 ctxAB- tcpA+].

Results of analysis of cholera outbreak during which V. cholerae O1 biovar El-Tor ctxAB- tcpA+ was isolated from 2 patients and 30 carriers are presented. Epidemic was caused by contamination of water source and water route of transmission. Strains identical to ones detected in humans were isolated from water of surface well in zone of water intake. Genome and VNTR-analysis of ctxAB- tcpA+ vibrios that caused outbreak in Rostov region in 2005 showed that they differed from ctxAB- tcpA- and ctxAB- tcpA+ vibrios isolated previously during and beyond of outbreaks from patients, carriers and environment and formed separate group with certain genotype. These results confirms conclusions of epidemiological analysis about imported cause of recent outbreak.

[Characterization of enteropathogenic Vibrio cholerae non O1/O139 isolated in Uzbekistan].

The results of the serotyping of 244 V. cholerae non O1/O139 cultures isolated from patients in Uzbekistan in 2000 and 2001 are presented. All isolates were studied by the method of molecular probing and in the polymerase chain reaction for the presence of virulence genes and for sensitivity to phages ctx+, ctx- and hemolytic activity. The use of monoreceptor O-sera O2-O83 made it possible to determine vibrios of 32 serogroups with the dominating role in the etiology of acute enteric diseases belonging to serogroups O18, O62, O82, O37. Genes ctx AB were detected in none of the isolates, 5 of them contained gene tcp A. A group of cultures, sensitive to phage ctx+ and belonging mainly to enteropathogenic serogroups, was detected.

[Toxins of Vibrio cholerae].

Surveyed in the paper are published data on properties, biological activity, genetic determinants and action mechanisms of recently known toxins produced by different strains of Vibrio cholerae irrespectively of their capacity for the synthesis of choleric toxin--the main virulence factor. Their possible importance both for the general clinical pattern of cholera provoked by cholerogenic agents and as independent virulence factors causing diarrhea without cholera is elucidated. The sets and levels of expression of additional toxins can differ for different pathogenic clones and they can correspondingly condition degrees of their epidemic and etiological safety.

[Cloning and expression of Vibrio cholerae zonula occludens toxin (zot) gene in Escherichia coli].

Two recombinant plasmids containing the cloned PCR-amplifled Vibrio cholerae zonula occludens toxin (zot) gene was constructed in orientation providing its transcription from lac-promoter. One of them contained also its own zot promoter. The third plasmid was obtained by subcloning a Vibrio cholerae DNA fragment including intact zot and ace (accessory cholera enterotoxin) genes. The expression levels of the cloned genes in Escherichia coli varied depending on a promoter type, host strain and culture conditions. The human intestinal cell line CaCo2 appeared to be a suitable model for assessing the biological activity of toxin preparations. The product of zot gene possessed a marked activity in respect to CaCo2 in spite of the lack of the molecule cleavage and transport of its toxic C-terminal part from alien host cells into the culture media. The constructed recombinant plasmids can be used as a source of molecular hybridization probes; and E.col transformants carrying those plasmids can serve in zot purification both for the scientific and practical purposes.