[EFFECT OF OSMOTIC AND OXIDATIVE STRESS ON STRAINS OF GENOVARIANTS OF VIBRIO CHOLERAE EL TOR BIOVAR].

AIM: Study the effect of osmotic and oxidative stress on survivability and changes in phenotypic and genetic properties of strains of genovariants of V. cholerae El Tor biovar. MATERIALS AND METHODS: 22 strains of V. cholerae El Tor biovar were used in the study. Phenotypic properties of strains were studied in LB medium with the addition of the appropriate ingredients. Surface structures of cells were studied using scanning probe microscope "Solver P47-PRO". PCR was carried out using specific primers in "Tercic" amplificator. RESULTS: After 60 minutes of incubation in 3 M solution of NaCl and after 6 minutes in 20 mM solution of hydrogen peroxide, the amount of surviving cells of genovariants was, respectively, 3.0 - 25.0 and 4.3 - 7.6 times higher than for typical strains. One of the mechanisms of increased resistance of genovariants to high concentrations of salt was associated with the production of an extra exopolysaccharide layer on the cell surface at earlier periods than in typical strains. Osmotic stress results in a reversible reduction of mobility in strains of genovariants of V. cholerae El Tor biovar. Osmotic and oxidative stress was revealed to result in a loss of a number of mobile genetic elements in strains of genovariants. CONCLUSION: Genovariants of V. cholerae El Tor biovar, that had caused cholera outbreaks in Russia in 1993 -2001, in contrast to typical strains, isolated in 1970 - 1990, are more resistant to th effect of osmotic and oxidative stress, that, probably, facilitates their higher survivability in both the environment and macroorganism.

[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. The cointegrate uncoupling was shown to take place in 5% the cholera vibrio cells followed by retention of only the multi-copy pCTdelta7 plasmid. This event leads to the formation of the TcRKmS clones characterized by high levels of the cholera toxin secreted B subunit production (10 to 14 microg/ml), one of these (KM93) being selected as a strain-producer of the protein. Molecular-genetic and biochemical assays were used to elucidate peculiar features of inheritance and expression of the cloned ctxAB gene within the KM93 cells. The expression of the cloned ctxB gene was shown to be independent of the presence of the toxR, tcpP, tcpH, toxT regulatory genes suggesting the existence of some other mechanisms that might exert their control over the transcriptional activity of the cholera toxin B subunit gene. Effective production of the cholera toxin B subunit would be also observed if the constructed producer strain was cultured under the conditions of industrial process. This indicates a possibility of its employment as a source of this protein involved in manufacturing cholera immunodiagnostic and prophylactic preparations.