MOLECULAR-GENETIC PROPERTIES OF VIBRIO CHOLERAE EL TOR STRAINS CIRCULATING IN AFRICA.

The review contains some brief information on cholera epidemics in Africa. Based on the results of the whole genome sequencing of 30 clinical strains isolated in Africa in different periods of the 7th cholera pandemic (1985-2012), extensive genetic diversity has been revealed. It is demonstrated that at present cholera epidemics in Africa are caused by new variants of the agent, which emerged in South- Eastern Asia in consequence of not only new genes acquisition, but also genome alterations of pandemicity and pathogenicity islands.

[EVALUATION OF FUNCTIONAL FEATURES AND STRESS RESISTANCE OF ISOGENIC TOXIGENIC AND NON-TOXIGENIC BIOVAR EL TOR VIBRIO CHOLERAE STRAINS].

Aim: Comparative evaluation of functional features of toxigenic biovar El Tor Vibrio cholerae strains and their spontaneous non-toxigenic mutants and study of their resistance to saline and oxidative stress.

Materials And Methods: 8 biovar El Tor V. cholerae strains were studied: 4 clinical strains isolated in 1970 from patients in Astrakhan and 4 spontaneous non-toxigenic mutants of these strains that have lost cholera toxin genes as a result of residence in river water at the temperature of 25°C.

[ISOLATION OF ANTIBIOTICS RESISTANCE GENES IN VIBRIO CHOLERAE O1 AND O139 SEROGROUP STRAINS].

Aim: Determination of sensitivity of V. cholerae O1 serogroup El Tor biovar and O139 serogroup strains to antibiotics and determination of the presence of antibiotics resistance genes in their genome.

Materials And Methods: The studies were carried out in 75 V.

[Algorithm of toxigenic genetically altered Vibrio cholerae El Tor biovar strain identification].

Aim: Development of an algorithm of genetically altered Vibrio cholerae biovar El Tor strai identification that ensures determination of serogroup, serovar and biovar of the studied isolate based on pheno- and genotypic properties, detection of genetically altered cholera El Tor causative agents, their differentiation by epidemic potential as well as evaluation of variability of key pathogenicity genes.

Materials And Methods: Complex analysis of 28 natural V. cholerae strains was carried out by using traditional microbiological methods, PCR and fragmentary sequencing.

[Comparative resistance of typical and genetically altered Vibrio cholerae biovar El Tor strains to the effect of unfavorable environmental factors].

Aim: Carry out comparative analysis of survival of typical strains and genovariants of V. cholerae biovar El Tor imported in different years to the territory of Russian Federation, in the absence of nutrients and under the conditions of temperature stress.

Materials And Methods: 24 V.

[Genovariants of the cholera agent biovar El Tor: construction, molecular-genetic, and proteomic analysis].

Experimental modeling of origination of the virulent Vibrio cholerae El Tor genovariants is presented. It was demonstrated that the genovariants obtained did not differ from the natural genetically modified strains emerged in a natural population of the agent, either in phenotypical or genotypic properties. Using the PCR assay and sequencing techniques it was proved that the constructed genovariants carried a CTX(Class phi) prophage genome region with ctxBl gene of the V.

[Comparative molecular-genetic analysis of mobile elements in natural strains of cholera agent].

The molecular-genetic peculiarities of the prophage CTXφ genome, the VPI-1 pathogenicity island, and the VSP-II pandemicity island in recently emerged, genetically altered Vibrio cholerae El Tor strains have been studied. The genome of the prophage CTXφ, which contains the ctxAB operon, which codes cholera toxin (CT), was shown to be unstable. A comparative analysis of the nucleotide sequences of the two phage genome regions (the ctxB gene and ctxAB operon promoter region) among 23 genovariant strains allowed us to reveal the presence of distinct ctxB gene alleles (ctxB1 or ctxB7) that differed in single-nucleotide substitutions and the polymorphism of the ctxAB operon promoter region.

[Prophage CTXphi genome variability and its role in alteration of Vibrio cholerae El Tor virulence characteristics].

Comparative analysis of CTXphi prophage genome of 366 V. cholerae El Tor strains isolated from infected people and water was carried out using the polymerase chain reaction. Four groups of vibrios, which carry different combinations of ctxA, zot, and ace genes from core region of CTXphi prophage coding key (cholera enterotoxin) and accessory (Zot and Ace toxins) pathogenicity factors, were determined: ctxA(+) zot(-) ace(+), ctxA(-) zot(+) ace(+), ctxA(-) zot(+) ace(-), ctxA(-) zot(-) ace(+).

[An avirulent vibrio cholerae strain--producer of the cholera toxin B subunit: obtaining and molecular genetic analysis].

The conjugative recombinant plasmid pIEM3 (KmR TcR) was constructed in order to introduce the cloned ctxB gene encoding the cholera toxin B subunit into the Vibrio cholerae cells. The plasmid was obtained as a result of co-integration of two plasmids: a conjugative plasmid, pIEM1(KmR), carrying mini-kan transposon and IS1 element, as well as the pCTdelta27(TcR) plasmid that is a derivative of the pBR322 which carries the cloned ctxB gene. The avirulent Vibrio cholerae strain eltor biovar deprived (according to the PCR analysis) of the key structural and regulatory pathogenicity genes and carrying a mutation in a single gene of the O1 antigen was chosen as the pIEM3 plasmid carrier strain.

[Proteomic analysis of two isogenic Vibrio cholerae of the classical biovar with the alternative expression of virulence genes].

At carrying out the proteomic analysis of two isogenic Vibrio cholerae Dakka35 of the classical biovar itwas revealed, that toxigenic (1 type) and nontoxigenic (2 type) clones differ from each other not only the expression ofgenes of exopolysaccharide, motility, and soluble haemagglutinin/protease, but also change of activity about other 60 genes. Among 11 identified proteins 5 are the enzymes participating in a metabolism cells. Besides it is revealed, that clones 2 types of Dakka35 strain synthesize in a more level of OmpU and TolC proteins, which provide their more significant stability to action of bile in comparison with clones of 1 type.

[Effects of the recombinant plasmid carrying the genes of cholera prophages CTX and RS1 on the expression of virulence and immunogenicity genes in the cholera pathogen].

Using toxin-coregulated adhesion pili (TCP), the etiologic agent of cholera is able to colonize human small intestine, where this pathogen proceeds with the production of the secreted cholera toxin (CT), inducing the development of severe diarrhea. At the same time, TCP and CT are not only the major factors of pathogenicity but also form a part of the group of key protective antigens. Immunoenzyme, immunoblotting, self-agglutination investigations, electron-microscopic studies, and electrophoretic assay of the outer membrane proteins showed that the recombinant plasmid carrying a number of cloned genes of two prophages, CTX and RS1, introduced into model Vibrio cholerae strains classical biovariant, resulted in the formation of strains with an enhanced rate of synthesis of three protective antigens: CT, TCP, and an outer membrane protein, OmpU.

[Comparative analysis of the major protective antigens production in Vibrio cholerae recombinant and producer strains of the classical biovar].

The comparative study of 4 constructed protective antigen producing strains of the classical biovar and V. cholerae strains 569 B Inaba and M41 Ogawa, used in manufacturing the cholera chemical vaccine "cholerogen-toxoid", was carried out. The study revealed that V.

[Phenotypical analysis of heterogenic Vibrio cholerae strain Dacca 35 Ogawa].

The comparative analysis of the production of the main pathogenicity factors by toxigenic and non-toxigenic clones of V. cholerae natural classical strain Dacca 35 Ogawa has been carried out. The data obtained in this analysis indicate that the appearance of turbid colonies, not synthesing cholera toxin, is linked with the production of an exopolysaccharide layer on the outer surface of the cells, which determines their morphology.

Molecular-genetic peculiarities of classical biotype Vibrio cholerae, the etiological agent of the last outbreak Asiatic cholera in Russia.

Molecular-genetic properties of classical biotype Vibrio cholerae strains that caused the Asiatic cholera outbreak in 1942 in Russia have been investigated for the first time. Being characterized by high-level production of cholera toxin and toxin-coregulated adhesion pili both of which are the major virulence factors, all the strains studied, in contrast to the typical cholera pathogens, were autographic requiring purine and/or amino acids added to the minimal medium for their growth. Moreover, these strains containing the structural gene hapA, as shown by the polymerase chain reaction, produced no soluble hemagglutinin/protease, which enables the vibrios to get disseminated in the environment.

[Lectin of Yersinia pestis vaccine strain EV and its immunological properties].

As the result of the chromatographic separation of Y. pestis EV membrane proteins, a protein fraction with hemagglutinating activity was obtained. The isolated preparation was glycoprotein with a molecular weight of 22 kD, contained 16% of carbohydrates and exhibited thermolabile properties.

[Molecular genetic features of Vibrio cholerae classica strains, that caused the Asiatic cholera epidemic in Russian in 1942].

Molecular genetic features of Vibrio cholerae classical strains which caused an epidemic of Asian cholera in Russia in 1942 have been studied for the first time. These strains had a high level of choleric toxin production and toxin-coregulated adhesion piles, the main virulence factors; all the strains were auxotrophs and needed purine and/or amino acids for growth in minimal medium. Moreover, having hapA structural gene in the chromosome (according to polymerase chain reaction), they did not produce soluble hemagglutinin protease promoting propagation of vibrios in the environment.

[Isolation and phenotyping of Vibrio cholerae strains with the enhanced production of main protective antigens].

Two V. cholerae strains of classical biovar, 2414 (serovar Ogawa) and 2415 (serovar Inaba), with of increased production of main protective antigens--cholera toxin, toxin-coregulated pili of adhesion (TCP), outer membrane protein OmpU, as well as phospholipases and proteases, have been detected among natural and recombinant strains under study. A simultaneous increase in the production of the above-mentioned main immunogenicity factors in strains 2414 and 2415 is seemingly linked with the presence of recombinant plasmid pCT105 with cloned genes of cholera toxin in these microbial cells.

[The role of the protein with molecular weight of 22 kD in the phagocytosis of vaccine strain of Yersinia pestis EV].

The comparative study of different stages of the phagocytosis of vaccine strain Y. pestis EV and its achromogenic variants (AV), has been carried out with the use of peritoneal macrophages as an in vitro experimental model. As revealed in this study, AV whose outer membrane contains no protein with a molecular weight of 22 kD exhibit lower capacity for adherence and for being ingested by phagocytes than the initial strain.

[The properties of the cellular surface of Yersinia pestis strain EV and its achromogenic variants].

The comparative study of the properties of the surface of vaccine strain Y. pestis EV and its achromogenic variants (AV) differing from the initial strain by decreased immunogenicity and by the morphology of colonies, has been made. The achromogenicity of Y.