[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.

[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.

[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.

[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.

[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.