[EVALUATION OF NUTRIENT MEDIA TO GROW SOME INFECTION DISEASES CAUSATIVE AGENTS].

The detection methods for microbial agents that have eidemiological significance are diversity but cultivation on nutritional media remains the gold standard in microbiological diagnostics. Choice of medium depends on the conditions in which bacteria were early and is present. The nature life determines its physiological peculiarity then a metabolic plasticity promote to survive and to save the virulence. In this review on the example of Yersihia pestis and Vibrio cholerae performed evaluations of the efficient decisions for the bacterial media development. It is declared advantage of baker's yeast hydrolisate as the nutrition media base.

[STUDY OF COMPOSITION OF PLANT EXTRACTS, POSSESSING ANTIMICROBIAL EFFECT AGAINST VIBRIO CHOLERAE EL TOR, USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY].

AIM: Study the composition of plant extracts using high-performance liquid chromatography. (HPLC) and evaluation of their antimicrobial effect against Vibrio cholerae El Tor. MATERIALS AND METHODS: Qualitative and quantitative composition of plant extracts was studied using HPLC. Determination of sensitivity of microorganisms to plant extracts was carried out by diffusion into agar method and serial dilutions method. RESULTS: Antibacterial effect of water, water-alcohol and acetone extracts of roots of Limonium gmelinii L., Berberis vulgaris L. and Glycyrrhiza glabra L. was studied. The most effective methods of extraction of biologically active substances, possessing antimicrobial effect against various strains of V. cholerae El Tor, were determined. CONCLUSION: The use of HPLC allowed to establish the presence of catechines, alkaloids protoberberines and glycyrrhizic acid in xtracts, possessing antimicrobial effect against V. cholera El Tor strains.

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.

[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.

[Serologic study of experimental cholera polymer antigen diagnosticums].

AIM: Evaluation of quality indicators of constructed cholera antigen polymer diagnosticums by using a complex of specific anti-cholera sera. MATERIALS AND METHODS. Cell lysates of cholera vibrio strains Vibrio cholerae cholerae 1395, V. eltor Ogawa 2044, V. eltor Inaba 13020, V. cholerae O139 16064 were sensitins for experimental preparations. 3 sera from cholera patients, normal human sera, cholera O1 (Ogawa, Inaba) commercial horse, cholera O139 commercial rabbit and heterologic sera against shigella, salmonella, escherichia and yersinia as well as experimental cholera rabbit sera against O1 and O139 were used as control. RESULTS: The study established that diagnosticums based on V. cholerae cholerae 1395 and V. cholerae O139 16064 strain sensitins by quality indicators may be used in the future for construction of these diagnosticums. CONCLUSION: Antibody containing preparations--commercial horse O1 sera, rabbit experimental and commercial sera and MCA O139 demonstrating titers not lower than 1/5120-1/10240 may serve as a control of experimental diagnosticums in the absence of human sera from cholera patients.

[The analysis of possibility to apply new preparations in serologic diagnostic of agent of cholera in working activity of specialized anti-epidemic brigades].

The approbation of diagnostic preparations on the substrate of monoclonal antibodies developed in the institute was carried out during tactical specialized exercise with building up of units on the basis of mobile complex of specialized anti-epidemic brigades. It is established that diagnostic agglutinating and fluorescent monoclonal immunoglobulins by their sensitivity are equal to polyclonal commercial preparations and can be used at the stages of laboratory diagnostic of cholera both in conditions of stationary laboratory and mobile complex of specialized anti-epidemic brigades. The method of dot immunoanalysis on the substrate of monoclonal antibodies can, on a par with such common methods as immunofluorescence, slide-agglutination and polymerase chain reaction, be applied in complex of methods of express-diagnostic of cholera.

[Phylogenetic analysis of genomes of Vibrio cholerae strains isolated on the territory of Rostov region].

AIM: Determination of origin of 2 Vibrio cholerae strains isolated on the territory of Rostov region by using full genome sequencing data. MATERIALS AND METHODS: Toxigenic strain 2011 EL- 301 V. cholerae 01 El Tor Inaba No. 301 (ctxAB+, tcpA+) and nontoxigenic strain V. cholerae O1 Ogawa P- 18785 (ctxAB-, tcpA+) were studied. Sequencing was carried out on the MiSeq platform. Phylogenetic analysis of the genomes obtained was carried out based on comparison of conservative part of the studied and 54 previously sequenced genomes. RESULTS: 2011EL-301 strain genome was presented by 164 contigs with an average coverage of 100, N50 parameter was 132 kb, for strain P- 18785 - 159 contigs with a coverage of69, N50 - 83 kb. The contigs obtained for strain 2011 EL-301 were deposited in DDBJ/EMBL/GenBank databases with access code AJFN02000000, for strain P-18785 - ANHS00000000. 716 protein-coding orthologous genes were detected. Based on phylogenetic analysis strain P- 18785 belongs to PG-1 subgroup (a group of predecessor strains of the 7th pandemic). Strain 2011EL-301 belongs to groups of strains of the 7th pandemic and is included into the cluster with later isolates that are associated with cases of cholera in South Africa and cases of import of cholera to the USA from Pakistan. CONCLUSION: The data obtained allows to establish phylogenetic connections with V cholerae strains isolated earlier.

[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.

[Comparative evaluation of protein hydrolysates in designing their based universal culture medium for the diagnosis of plague and cholera].

Various protein hydrolysates made in Russia and foreign countries were comparatively evaluated to use them to design a universal agarized culture medium for the diagnosis of plague and cholera. Pancreatic baker's yeast broth was found to be most effective among the test media.

[New nutrient medium for the cultivation and isolation of the plague microbe ChDS-37 as an element of the mobilization reserve of specialized antiepidemic teams of the Russian Inspectorate for the Protection of Consumer Rights and Human Welfare].

A new nutrient medium has been designed to culture and isolate the plague microbe ChDS-37 on the basis of the pancreatic digest of baker's yeast. The results of laboratory tests of the designed medium, by using 10 plague microbe strains and those of approval during the tactical and special training of a specialized antiepidemic team (SAET), suggest that the medium has some advantage over reference media and creates prerequisites for being incorporated into the mobilization reserve of a SAET.

[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.

[Role of iron ions in the production of hemolysin by toxigenic and nontoxigenic Vibrio cholerae of different serogroups]. / Rol' ionov zheleza v produktsii gemolizina toksigennymi i netoksigennymi kholernymi vibrionami razlichnykh serogrupp.

The hemolytic activity of ctx- and ctx+ V. cholerae, serogroups eltor and O39, in a medium free of FeCl3 was studied. During the cultivation in this medium, the strains of both V. cholerae serogroups proved to be capable of lysing sheep red blood cells in the Graig test, irrespective of the presence of ctx genes. The cultivation of V. cholerae ctx+ strains of both serogroups under such conditions facilitated the production of hemolysin with the same spectrum of lytic activity as hemolysin produced by ctx- strains.

[Dynamics of the isolation and biological properties of Vibrio cholerae cultures, serogroups 01 and 0139, isolated from water reservoirs and sewage water on the territory of the city of Rostov-on-Don]. / Dinamika vydeleniia i biologicheskie svoistva kul'tur kholernykh vibrionov 01 i 0139 serogrupp, vydelennykh iz poverkhnostnykh vodoemov i stochnykh vod na territorii g. Rostov-na-Donu.

The dynamics of the isolation of V. cholerae cultures from various water objects on the territory of Rostov-on-Don during the period of 1994-2001 was analyzed and biological properties of 14 such cultures were studied. In the absence of epidemic complications during the above-mentioned period, a growth in the amount of V. cholerae isolates, serogroups 01 and 0139, including toxigenic V. cholerae 01, was registered. The microbiological and epidemiological aspects of the monitoring of surface reservoirs and sewage were considered and the expediency of the profound and systematic study of its results for epidemiological surveillance on cholera was emphasized.

[Effect of various amino acids and ammonium salts in a synthetic culture media on the cholera enterotoxin production]. / Vliianie razlichnykh aminokislot i solei ammoniia v sostave sinteticheskoi pitatel'noi sredy na produktsiiu kholernogo énterotoksina.

The influence of amino acids and ammonium salts on the production of cholera enterotoxin (CT) by 3 Vibrio cholerae strains of different biovars and serogroups was evaluated. As revealed in this study, toxin formation in each of the strains was quantitatively and qualitatively determined by their individual sets of amino acids. The amino acid compositions ensuring the maximum production of CT by the V. cholerae strains under study were formed. The use of ammonium salts as the only source of nitrogen in the composition of a synthetic nutrient medium for the accumulation of CT was shown to be inexpedient.

[The determination of the optimal conditions for the production of a number of pathogenicity factors in Vibrio cholerae]. / Opredelenie optimal'nykh uslovii produktsii riada faktorov patogennosti kholernykh vibrionov.

Conditions for the cultivation of V. cholerae of different sero- and biovars on tryptone medium, ensuring the maximum production of cholera toxin (CT), dermonecrotic factor (DNF), hemorrhagic factor (HF) and new cholera toxin (NCT) have been determined. The lack of coincidence in the optimum conditions ensuring the maximum production of CT, DNF and HF has been established, which may be indicative of different nature of these toxic substances. NCT, produced by vct- strains, is similar to CN in biological activity as determined in the skin permeability test and in the conditions of accumulation in tryptone medium.

[The characteristics of the antibacterial therapy for cholera in Dagestan]. / Osobennosti antibakterial'noi terapii kholery v Dagestane.

Wide circulation of antibiotic-resistant Vibrio cholerae strains again gives prominence to the problem of etiotropic therapy. The results of the treatment of 428 persons infected with V.cholerae (237 cholera patients and 191 Vibrio carriers) in different regions of Daghestan during the outbreak of epidemic in 1994 are presented. The main criterion of the effectiveness of antibacterial therapy was the determination of the percentage of bacterial relapses. The sensitivity of 118 V.cholerae strains to different antibacterial preparations was studied by the method of serial dilutions. After the clinical use of chloramphenicol 29.7% of bacterial relapses were registered, the in vitro resistance of V. cholerae being 32-64 mkg/ml. After the use of tetracycline 16.5% bacterial relapses were registered with in vitro resistance being the same. The use of the combination of these preparations gave 15% of bacterial relapses. Furazolidone gave 4.3% of bacteria relapses, while after the use of ciprofloxacin 2.8% of bacterial relapses were registered with in vitro sensitivity equal to 0.25-0.5 mkg/ml. Ciprofloxacin was recommended for the treatment of cholera patients and furazolidone, for the treatment of Vibrio carriers.

[The preservation of the causative agent of cholera in the water supplies of the central regions of Dagestan (experimental data)]. / O sokhranenii vozbuditelia kholery v vodnykh istochnikakh tsentral'nykh raionov Dagestana (éksperimental'nye dannye).

The acidic pH of water of surface water reservoirs in Izberbash and two adjoining regions, including sea water, seems to be unfavorable for the prolonged preservation of Vibrio cholerae eltor, but additional ecological investigations are necessary to study the possibility for infection to take root at this territory. Water from the Zam-Zam spring, if contaminated with V. cholerae, may serve as a transmission factor, but the duration of its action is limited by the survival term of V. cholerae. The water route of transmission did not play any essential role in the spread of cholera in the central regions of Daghestan.