[Cholera Vibrio biofilm: production, characterization and role in reservation of causative agent in water environment].

AIM: Experimental production, characterization and evaluation of the role of cholera vibrio biofilm. MATERIALS AND METHODS: 33 strains of Vibrio cholerae eltor O1 and V. cholerae O139 of various epidemic significance and origin were studied in a series of experiments by bacteriologic, microscopic (light-optic, luminescent, scanning electron microscopy), molecular genetics, spectrophotometric and statistical methods. RESULTS: Formation of a biofilm involving inter-cellular bonds, pili and extracellular material and variability of the microorganism (RO-phenotype and transition into uncultivable forms) was shown at various temperature and substrate conditions. A more pronounced ability to form biofilms was detected for strains isolated from environmental samples compared with isolated from clinical material regardless of their epidemic significance. Toxigenic strains of eltor biovar (from surface reservoirs during cholera outbreaks) have demonstrated the highest parameters of optical density compared with toxigenic clinical isolates and non-toxigenic O1 and O139 serogroup cultures. The presence of mbaA1 and mbaA2, vpsR, toxR, hapA genes is common for strains that form a biofilm. CONCLUSION: The data obtained confirm the role of biofilm in reservation of cholera vibrio strains of various epidemic significance in saprophytic phase of microorganism existence.

[Membrane-bound proteases of ompT+ and ompT- Vibrio cholerae strains].

AIM: Detection ofproteases in outer membranes (OM) of ompT+ and ompT- Vibrio cholerae strains of O1 and O139 serogroups. MATERIALS AND METHODS: Specific sterile preparations of OM were obtained by lysis of live V. cholerae cells by 4.5 M urea solution with subsequent differential centrifugation and treatment by nucleases. Extraction of OM proteins previously treated by sodium sarcosinate was carried out by Triton X-100 in the presence of EDTA. Protease and polypeptide spectra were studied in substrate and SDS electrophoresis. Sensitivity of proteases to inhibitors was determined in diffusion test in agarose gel containing substrate by using soy trypsin inhibitor (STI) and phenylmethylsulfonyl fluoride (PMSF). The presence of ompT was determined in PCR by using specific primers. RESULTS: According to PCR data 13 Vibrio cholerae O1 strains and 3 V. cholerae O139 strains isolated from clinical material as well as 22 V. cholerae O1 strains isolated from environmental objects contained ompT gene. 2 V. cholerae O1 human isolated strains, 9 V. cholerae O1 strains and 2 V. cholerae O139 strains isolated from the environment did not have ompT gene. By using SDS- and enzyme-electrophoresis in polyacrylamide gel quantitative and qualitative differences in composition of polypeptides and proteases of OM ompT+ and ompT- V. cholerae strains that hydrolyze gelatin, casein and protamine sulfate were detected. Inhibition of OM by STI and PMSF resulted in a decrease of their proteolytic activity. CONCLUSION: In preparations and extracts of ompT+ and ompT- V. cholerae OM up to 3 proteases some of which may be related to ompT-like were detected.

[Molecular-genetic analysis of the epidemical strains of the Vibrio cholerae El Tor isolated from the Siberian and maritime regions of Russia].

The detection of the biotype-specificity, pathogenicity determinants, and sequencing of the ctxB gene and the ctxAB promoter was carried out for analysis of the Vibrio cholerae El Tor strains genome structure. The strains (n = 90) were isolated during cholera epidemic outbreaks in Siberia and the Far East. All toxigenic Vibrio cholerae El Tor strains were divided into two groups: the first group included strains isolated during 1970s: they had the genotype ctxB3+rstREl+rstRCl-rstC+TLC+tbr4. All epidemic dangerous El Tor biotype strains isolated in 1990s belonged to the second group. The strains were characterized as atypical variants because of classical genotype (ctxB1) ctxB gene harboring. The second group fell into three genotypes according to the set of genetic markers (ctxB, rstR, rstC, TLC, tbr). It was suggested that the set of genetic determinants could be used as a marker for epidemiological analysis of spreading of atypical ET strains. The comparative analysis of genome structure enables to suggest possible ways of pathogen evolution.

[Detection of "hybrid" Vibrio cholerae eltor strains during epidemic complications in Syberia and Far East].

AIM: Biotyping of Vibrio cholerae eltor isolated during epidemic complications of cholera in Syberia and Far East by phenotypic and genotypic properties complex. MATERIALS AND METHODS: 45 strains of V. cholerae were studied. Phenotypic analysis was performed by using a complex ofbiovar determining tests. Genotyping was performed by detecting ctxAB, tcpA, toxR, rstRgenes, and ctxB gene structure analysis. RESULTS: All the V. cholerae during epidemiologic complications in Syberia in the 1970s belong to eltor biovar by phenotypic properties and have eltor specific alleles of tcpA and rstR genes, and ctxB of the third genotype in the genome. In the 1990s the strains were phenotypically matching eltor biovar, but had genetical determinants of both eltor(tcpAE1, rstRE1) and classical (ctxB1, rstR(Cl) biovar. CONCLUSION: The cause of epidemic complications of cholera in Syberia in the 1970s was V. cholerae eltor with typical eltor biovarphenotypical and genotypical properties. In the 1990s cases of introduction into the region of "hybrid: V. cholerae eltor strain were ascertained, developing into acute cholera outbreaks in several cases.

[Evolution of the epidemiology of cholera].

The problems of the evolution of cholera at the stages of its pandemic spread are described. A short characterization of endemic zones in the countries of Asia, Africa and Latin America, stipulating the preservation of infection and the appearance of periodic epidemics in the world, is presented. Special attention is paid to the genesis of epidemic outbreaks in endemic and introduced foci of cholera, differing in the specific features of their formation and the accumulation of the epidemic variant of the infective agent in aqueous habitat. The role of the ecosystem of surface water reservoirs in the prolonged survival of cholera vibrios is evaluated. The necessity of the detailed study of the mechanisms and forms of existence of serogroup 0139 vibrio in open water reservoirs is substantiated. In the system of epidemiological surveillance on cholera the microbiological monitoring of environmental objects assumes the leading importance.

[Experimental non-culturable Vibrio cholerae eltor and its biological properties]. / Eksperimental'noe poluchenie nekul'tiviruemykh form Vibrio cholerae eltor i kharakteristika ikh biologicheskikh svoistv.

In experiments with the cultivation of V. cholerae eltor under the conditions of high salt concentration, as well as low temperature and deficiency in nutrient substances, uncultivable forms (UF) of toxigenic and nontoxigenic vibrios were obtained. The absence of growth of seeded vibrios after the filtration of samples (with a filter of 0.22 micron), the preservation of specific antigenic determinants and the initial set of genes, changes in the morphology of cells (small size, coccoid form with the flagella retained) confirm the transition of V. cholerae eltor under study into the uncultivable state which, under unfavorable conditions, more rapidly develops in toxigenic vibrios than in nontoxigenic ones. The analysis of the INT-reductase activity of UF disintegrates revealed that they had endogenic respiration whose activity increased (4.5- to 6.5-fold) in the presence of the exogenic intermediates of the Krebs cycle. The uncultivable forms of the vibrios retain genes responsible for pathogenicity, as well as their antigenic determinants.

[PCR-detection of markers for epidemiologically-significant strains of Vibrio cholerae 0139, isolated in Siberia]. / PtsR-detektsiia markerov épidemicheskoi znachimosti shtammov Vibrio cholerae 0139, izolirovannykh v Sibiri.

Fourteen V. cholerae 0139 strains were isolated in 1996-1999 in Siberia from the Ob river (Novosibirsk) and bogs and lakes (Irkutsk). The strains were tested in PCR for the key virulence determinants (ctx AB, tcp, acf). The genomes lacked these elements, and therefore the strains were acknowledged avirulent. The results correlate completely with the data of phenotypical analysis, characterizing the pathogenic characteristics of isolated strains.

[Eltor cholera in Latin America]. / Kholera El'tor v Latinskoi Amerike.

Cholera epidemics in South and Central America in the period 1991-1999 are characterized. The mechanisms of existence of Vibrio eltor in environmental objects have been substantiated. The causes and conditions of endemic foci formation which determine the infection spread not only within the countries of the subcontinent, but also far beyond its borders are shown. The effectiveness of cholera control interventions is evaluated.

[Cholera in the Far East of Russia. Communication 1. Epidemiological characteristics of the outbreak of cholera El tor in Vladivostok]. / Kholera na Dal'nem Vostoke Rossii. Soopshchenie 1. Epidemiologicheskaia kharakteristika vspyshki kholery él'tor v g. Vladivostoke.

The materials on the investigation of the outbreak cholera eltor in Vladivostok, caused by the import of infection from China. The leading role of the water route of transmission of this infection is shown due to the contamination of water sources with non-decontaminated sewage water. The complex of antiepidemic measures was carried out, which made it possible to arrest the spread of cholera and liquidate its foci.

[Comparative study of various methods for determining Vibrio cholerae toxigenicity]. / Sravnitel'noe izuchenie razlichnykh metodov opredeleniia toksigennosti Vibrio cholerae.

Testing of 138 Vibrio cholerae strains for gene determinants responsible for the production of cholera enterotoxin by the polymerase chain reaction (PCR) and gene probing using molecular CT-probe showed good correlation of the results of different methods and correlation of these data with studies of V. cholerae strain virulence in vivo and in hemolytic activity test. The advantages of PCR in rapid assessment of the toxigenicity and epidemic significance of V. cholerae strains are demonstrated.

[The characteristics of the protectiveness of a preparation of Vibrio cholerae outer membrane based on the data from a study using the ligated intestinal loop]. / Kharakteristika protektivnosti preparata naruzhnoi membrany kholernogo vibriona po dannym issledovaniia pereviazannoi kishechnoi petli.

The study aimed at finding out the antiadhesive capacity of antigenic preparation, earlier obtained from V. cholerae outer membrane and highly effective with respect to cholera infection, was undertaken. The study was made on previously immunized adult rabbits who had been subjected to laparotomy under anesthesia and the ligation of intestinal loops, subsequently inoculated with the broth culture of V. cholerae eltor (P-3122, serovar Ogawa). The intestinal loops were studied histologically and bacteriologically with the calculation of the number of vibrios, the deduction of the adhesion index and the coefficient of the efficacy of immunization. The data thus obtained indicated that the specific immunization of rabbits with their subsequent inoculation with V. cholerae virulent strain suppressed the adhesive activity of the infective agent which was more pronounced in rabbits immunized with the preparation of V. cholerae outer membrane.

[The outer membranes of Vibrio cholerae as a potential component in a chemical vaccine]. / Naruzhnye membrany kholernogo vibriona kak potentsial'nyi komponent khimicheskoi vaktsiny.

The results of the study of the preparation of V. cholerae eltor membrane, obtained by the lysis and inactivation of microbial cells with urea and the subsequent differential centrifugation and nuclease treatment. As revealed in this study, the outer membrane preparation, when introduced parenterally and orally to mice, induced pronounced immunity to experimental cholera infection and the production of vibriocidal antibodies in high titers. The treatment of V. cholerae eltor membranes with trypsin led to further increase of the immunogenic potency of the preparation. The protective action of V. cholerae eltor outer membranes considerably exceeded the protective effect of currently used whole-cell eltor vaccine. This opens prospects for using the above-mentioned preparation for the improvement of chemical vaccine as a component ensuring the formation of antibacterial immunity.

[Comparative antibiotic sensitivity of NAG vibrios]. / Sravnitel'naia chuvstvitel'nost' k antibiotikam NAG-vibrionov.

Six hundred and forty eight NAG vibrio strains isolated at various periods from patients and carriers and from environmental objects such as surface of water reservoirs and sewage were studied with respect to their sensitivity to 14 antibiotics with the method of serial dilutions in solid media. Irrespective of the isolation place, object and time, the NAG vibrios were highly resistant to penicillins and polymyxin M. At the same time they were highly sensitive to gentamicin (MIC 1-2 micrograms/ml), levomycetin (MIC 0.5-1 micrograms/ml) and tetracyclines (MIC 0.25-1 micrograms/ml). Study of the recipient capacity of NAG vibrios with respect to R plasmids showed that they could be recipients of exogenic R plasmids of various incompatibility groups.

[Phage typing of nonagglutinating vibrios isolated in the territory of the USSR]. / Fagotipirovanie neaggliutiniruiushchikhsia vibrionov, vydelennykh na territorii SSSR.

The work presents the results of the phage typing of 2,437 NAG vibrio strains isolated in the USSR from patients and carriers (374 strains), from open water basins (1,675 strains), from sewage (234 strains) and from bottom silt (172 strains). Phage typing was carried out with a set of 5 phages capable of lyzing enteropathogenic NAG vibrios (phages TEPV 1, 2, 3, 4, 5). NAG vibrios isolated from humans were sensitive to phages TEPV in 45% of cases and NAG vibrios isolated from the environment, only in 29.9% of cases. Enteropathogenic NAG vibrios belonging to phagovar 1 were isolated most frequently from samples taken both from humans and from the environment. The NAG vibrios isolated from patients, carriers and the sources of water supply nearest to them belonged to the same phagovars.

[Serological typing of Vibrios, nonagglutinating with O-cholera serum, isolated in USSR]. / Serologicheskoe tipirovanie ne aggliutiniruiuschikhsia O-kholernoi syvorotkoi vibriunov, vedelennykh na territorii SSSR.

The authors carried out serological typing of 2008 strains of NAG vibrios isolated in 1968--1975 in 18 territories of the Soviet Union from humans and from the objects of environment. Serological type was established in 40.5% of the srains isolated from humans and in 16% of the strains obtained from the environmental objects. Among persons with acute gastrointestinal diseases serotype 5 was the one which dominated, and among strains from the environment--serotype 8 Sakazaki. Serological type 15 was revealed for the first time in the USSR at the territory of Siberia and the Far East among the NAG-vibrio strains isolated from the environment.

[Patterns in the body's immune response to administration of El Tor vibrio cholera toxin to experimental animals]. / Nekotorye zakonomernosti immunnoi reaktsii organizma na vvedenie èksperimentalnym zhivvotnym toksina kholernogo vibriona El Tor

A study was made of some regularities attending the mechanisms of antibody production in the organism of experimental animals after the administration of El Tor cholera vibrio toxin. As revealed, the indices of the immune response depended on the chosen model, the method of its determination, the method of administration and the amount of the toxin administered. Dynamics of the primary and secondary responses of the immunocompetent cells of mouse spleen was determined. With the action of the El Tor toxin on the lymphoid tissue of mice there were expressed the general biological regularities of the response -- the optimal amount of the antigen-antibody complexes caused activation of the antibody-producing cells; high doses depressed the most reactive immunocompetent Y-cells. Apparently, there existed a genetic determinination of the immune response of the organism to a definite antigen -- the toxin of the El Tor cholera vibrio. It is supposed that it is necessary to choose groups which mostly require cholera vaccination.