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. SNP analysis of 53 strains circulating at different times in the territory of the continent, as well as isolated in South-Eastern Asia, has been carried out. Phylogenetic relations between the majority of the African and Asian strains have been established. In addition, strains were shown to exist that are, apparently, endemic to the African region. Identified genetic diversity of the strains with varying virulence and drug resistance points out the necessity of continuous molecular monitoring of the cholera agent in Africa.

[Genome structure and origin of nontoxigenic strains of Vibrio cholerae of El Tor biovar with different epidemiological significance].

Intraspecies genetic differentiation of nontoxigenic strains of Vibrio cholerae of El Tor biovar containing one of the key pathogenicity genes, tcpA, is studied along with the phylogenetic relationships between these strains and toxigenic isolates. Comparative analysis of the whole genome nucleotide sequences demonstrates for the first time that ctxA ­ tcpA + strains vary considerably and can be clustered into two separate groups, the CTXφ­RS1φ +VPI+VSP+/CTXφ­RS1φ­VPI+VSP+ isolates and the CTXφ­RS1φ­VPI+VSP­ isolates, differing in their epidemiological significance. In the course of model experiments, it is established that nontoxigenic potentially epidemic CTXφ­RS1φ +VPI+VSP+/CTXφ­RS1φ­VPI+VSP+ isolates are derivatives of toxigenic strains. The results of whole genome SNP analysis of 35 Vibrio cholerae strains confirm these data and indicate genetic remoteness of nontoxigenic CTXφ­RS1φ­VPI+VSP­ strains both from the potentially epidemic strains and from the toxigenic isolates. It is found that the genomes of the CTXφ­RS1φ­VPI+VSP­ strains contain unique SNPs which are characteristic of them alone. The new data on the structure of the genome of nontoxigenic strains with different epidemiological significance may be further used for their genetic differentiation.

[ACTUAL PROBLEMS OF EPIDEMIOLOGIC CONTROL, LABORATORY DIAGNOSTICS AND PROPHYLAXIS OF CHOLERA IN RUSSIAN FEDERATION].

Main problems of system of epidemiologic control for cholera active in Russian Federation, as well as laboratory diagnostics and vaccine prophylaxis of this especially dangerous infection, that had emerged in the contemporary period of the ongoing 7th pandemic of cholera, are discussed. Features of the genome of natural strains of Vibrio cholerae of El Tor biovar, that possess a poten- tial epidemic threat, as well as problems, that have emerged during isolation of these strains from samples of water of surface water bodies during their monitoring, are also examined. The main direction of enhancement of the system of epidemiologic control for cholera consist in develop- ment of a new algorithm of differentiation of administrative territories of Russian Federation by types of epidemic manifestations, as well as optimization of monitoring of environment objects. Integration of modern highly informative technologies into practice, as well as development of new generation diagnostic preparations based on DNA-chips and immunechips is necessary to increase effectiveness of the conducted operative and retrospective diagnostics in the contemporary period. Creation of national cholera vaccine, ensuring simultaneous protection from cholera causative agents of both O1 and O139 serogroups, is also required.

[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. cholerae O1 and O139 serogroup strains. Sensitivity of cultures to antibiotics was determined by disc-diffusion method. DNA isolation was carried out in the presence of 6M guanidine thiocyanate. PCR was carried out in multi-channel amplificator Tercyc. RESULTS: A multiplex PCR was constructed, that includes 5 primer pairs for the detection of O1 and O139 serogroup resistance genes of vibrios to sulfame- thoxazolum, streptomycin B, trimethoprim, the presence of SXT element, an amplification program was developed. Using the developed PCR, V. cholerae O1 serogroup El Tor biovar strains with multiple drug resistance were established to be imported into Russia in 1993. The presence of SXT elements with genes of resistance to 4 antibiotics simultaneously was detected precisely in these strains, that belong to toxigenic genovariants of V. cholerae El Tor biovar. All the El Tor vibrio strains imported in the subsequent years were shown to stably preserve SXT element, this indicates its important role in biology of cholera vibrios. O139 serogroup strains with intact SXT element and having a deletion of the gene coding trimethoprim resistance were isolated. CONCLUSION: The data obtained may be used to establish molecular-genetic mechanisms of emergence of antibiotics resistant strains of cholera vibrio, construction of novel gene diagnostic test-systems and carrying out passportization of strains that are stored in the State collection of pathogenic bacteria.

[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. Protein composition was determined in polyacrylamide gel electrophoresis by Laemmli U.K. Stress resistance of the strains was studied by adding H2O2 to 20 mM concentration and NaCl to 3 mM concentration to the cultural suspension. RESULTS: Loss of cholera toxin genes was shown to be accompanied by changes in the level of expression of 17 proteins including those that take part in energy metabolism, glucose transport, chemotaxis and purine bases. Moreover, non-toxigenic strains were established to be 5-15 times more resistant to saline and oxidative stress compared with toxigenic strains. CONCLUSION: Non-toxigenic V. cholerae mutants adopt better to stress factors, therefore the loss of cholera toxin gene in water environment could be one of the methods of adaptation of pathogenic bacteria to changes in the environment.

[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.

[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. cholerae classical biovar in the chromosome. It is shown that the prophage structure alterations lead to the increase in the toxigenicity and virulence in the genovariants compared to the typical strain-recipient. Moreover, as regards proteomics, changes in the expression of 26 proteins that perform various functions in the cell, such as metabolism, energy exchange, transportation, etc., were demonstrated. The data are indicative of the impact that a new DNA region in the genome of the genovariants has on the expression level of different house-keeping genes. The results obtained testify to the fact that one of the mechanisms of the genovariant emergence in the natural populations of the agent can be horizontal gene transfer.

[Microevolution of cholera agent in the modern period].

AIM: To carry out comparative molecular genetic analysis of highly pathogenic atypical Vibrio cholerae strains biovar El Tor, isolated in the territory of RF, in order to determine micro-evolutionary alterations of cholera agent in the modern period. MATERIALS AND METHODS: 38 clinical strains have been examined by means of polymerase chain reaction, sequencing and MLVA-analysis. The selected strains were isolated at different periods of time during cholera epidemic complications and differed between each other in virulence. RESULTS: It is demonstrated that new variants have emerged in the course of short-term microevolution. Their genome structure and function differ from those of all previously known strains. The genome alterations have been caused by point mutations in ctxB u tcpA genes associated with virulence and located in CTXΦ prophage and pathogenicity island VPI-1 respectively, as well as by the extended deletion in pandemicity island VSP-II. Presented is the dynamics of genome structure and function alterations in modern strains. CONCLUSION: The discovered genomic alterations in the new variants of the agent evolved in the process of microevolution are indicative of their epidemic potential enhancement and probability of virulence potentiation.

[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. cholerae biovar El Tor strains isolated in 1970 - 2011 were studied, 8 of those were typical isolates and 16--genetically altered variants. Strain survival was studied in 0.9% sodium chloride solution and autoclaved river water at various temperature modes (5, 25, 37 and 42 degrees C). Protein composition and exopolysaccharide production were determined by electrophoresis method by U.K. Laemmli. RESULTS: Genovariants as well as typical strains were shown to be able to exist for a long time (up to 5 months) in the absence of nutrients at the temperature of 25 degrees C. However, unlike typical eltor vibrios, genovariants were more resistant to temperature stress. As a result of adaptation to high temperature (42 degrees C) biosynthesis of porin proteins of outer membrane OmpU and/or OmpT is increased in genovariant cells, and at lower temperatures (5 degrees C)--exopolysaccharide. CONCLUSION: V. cholerae biovar El Tor genovariants are able to adapt to temperature change better, that may facilitate their higher survival in the environment.

[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. RESULTS: An algorithm of toxigenic genetically altered V. cholerae biovar El Tor strain identification was developed that includes 4 stages: determination of serogroup, serovar and biovar based on phenotypic properties, confirmation of serogroup and biovar based on molecular-genetic properties determination of strains as genetically altered, differentiation of genetically altered strains by their epidemic potential and detection of ctxB and tcpA key pathogenicity gene polymorphism. The algorithm is based on the use of traditional microbiological methods, PCR and sequencing of gene fragments. CONCLUSION: The use of the developed algorithm will increase the effectiveness of detection of genetically altered variants of the cholera El Tor causative agent, their differentiation by epidemic potential and will ensure establishment of polymorphism of genes that code key pathogenicity factors for determination of origins of the strains and possible routes of introduction of the infection.

[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. An analysis of the VPI-1 structure showed that tcpA gene that encodes the basic protein of the toxin-coregulated pilli (TCP) was represented in the genovariants by four alleles. The variability of the VSP-II was also revealed. It was manifested in the occurence of the deletions of varying length (2069 or 13 105 bp).

[Genetic characterization of Vibrio cholerae strains emerging in Russian Federation during 7th cholera pandemic].

AIM: Comparative molecular-genetic analysis of clinical Vibrio cholerae eltor biovariant strains isolated in Russia during various years. MATERIALS AND METHODS: Microbiological and biochemical methods were used for studies of 25 clinical strains of classic and eltor biovariant cholera, PCR testing and sequencing of various genes was also performed. RESULTS: Phenotypic and genetic analysis of clinical V. cholerae strains isolated in Russia during 7th cholera pandemic has confirmed that they belong to biovariant eltor. PCR testing of 21 isolates obtained from patients in 1970 - 2010 has shown that epidemic complications in Russia from 1993 were caused by altered V. cholerae biovariant eltor. Presence of classic cholera biovariant ctxB coding gene in cholera toxin coding CTX prophage is the genetic alteration of these variants, ctxB sequencing in altered variants has confirmed PCR data and shown 2 ctxB gene alleles (ctxB1 and ctxB7). Altered variants produced significantly more cholera toxin than typical strains. CONCLUSION: In 1970 - 2010 67.6% of clinical isolates were altered V. cholera biovariant eltor variants. These new variants were genetically diverse. Alteration of cholera eltor biovariant genome caused toxigenicity increase.

[Variation in the genome of CTXphi prophage of Vibrio cholerae biovar El Tor caused by Tn5-Mob transposon].

A key pathogenicity factor of the cholera etiologic agent is cholera toxin (CT) whose synthesis is encoded by the ctxAB operon forming apart of the CTXphi ptophage. Alterations in the virulent properties of the cholera vibrios are based on the variability of the CTXphi prophage containing the genes for ctxAB, zot, ace, cep, orfU, and psh in its core region. At the same time, the mechanism of the porophage genome reorganization needs further and more profound analysis. The goal of this work was to demonstrate that transposon Tn5-Mob (Kmr), when introduced into the chromosome of the V. cholera model strain MAK757 El Tor biovar containing two copies of the CTXphi prophage provoked a reorganization in the CTXphi prophage consisting in the deletion of zot, ace, cep, orfU genes. The level of the CT biosynthesis in the insertion mutants MAK757 chr::Tn5-Mob still retaining only the ctxAB operon, increased more than 2000 times as compared to that of the original strain. The enhanced CT production was shown to be associated with the altered structure of the chromosomal DNA region containing one copy of the ctxAB operon encoding this protein biosynthesis. The mutation in the CTXphi genome induced by Tn5-Mob was unstable. Among 600 isolated colonies obtained after dissemination of the MAK757 chr::Tn5-Mob transposant capable of CT overproduction in the full medium with no antibiotics, 5.8% gave clones that in parallel to the loss of Kmr marker, appeared to be deprived of the ctxAB operon thus becoming non-toxinogenic. The observed formation of the V. cholerae insertion mutants both capable of CT overproduction and non-toxinogenic ones, may be indicative of an important role played in the evolution of the cholera pathogen by the CTXphi genome variability induced by Tn elements. The plasmidless V. cholerae El Tor strain characterized by type II CT hyperproduction thus obtained in our experiments could be used for the production of this protein routinely applied to construct efficient cholera diagnostic and prophylactic preparations.

[A study of the prevalence of regulatory genes controlling virulence gene expression among Vibrio choleraeeltor biovariant strains varying in their pandemic potential].

The evolution of the genome of the pathogenic agent of the seventh cholera pandemia Vibrio cholerae eltor biovariant was thought to occur by acquiring not only structural genes of virulence but also regulatory systems as a result of horizontal transfer events. The polymerase chain reaction revealed the presence of the following regulatory genes that control the virulence gene expression in the chromosome of pre-pandemic and pandemic strains of cholera vibrios eltor: toxR, toxT, tcpP, tcpH, luxS, luxO, crp, vicH, pepA. The avirulent V. cholerae strain ATCC14033 isolated in 1910 (hypothetical predecessor of the cholera eltor agent) was shown to be lacking the regulatory genes toxT, tcpP, tcpHlocalized in the pathogenicity island VPI-1, and to be capable of realizing positive control over the expression of the virulence genes involved in the ToxR regulon. The virulent strains isolated from cholera patients during the local cholera outbreak in Indonesia in 1937 did not differ from the strains that caused cholera eltor pandemic in 1961. The strains had identical content of the regulatory genes tested. Only one strain of the four isolates studied contained no tcpPgene. Two key regulatory genes, toxR and toxT, were sequenced in all the isolates. The toxR nucleotide sequence of three pre-pandemic strains was shown to be indistinguishable from that of the pandemic isolates. On the other hand, the clinical strain MAK757 isolated prior to the emergence of the epidemic demonstrated an altered nucleotide sequence in its toxR gene. Experiments with the intra-intestinal challenge of suckling rabbits were indicative of similar virulence levels for the pre-pandemic and pandemic clinical strains. These results may serve as the evidence of the in vivo activity of the pre-pandemic strains of the toxT, tcpH, and tcpP positive regulatory genes that acquired in V. cholerae during the evolutionary process.

[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.

[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(+). Vibrios that had lost all tested genes were also revealed. Genomic rearrangements occurring in water environment in virulent V. cholerae strains, which acquired foreign pathogenicity genes necessary for their existence in human organism, were proposed as one of the mechanisms of formation of clones with an incomplete or no prophage. Infection process in model animals challenged with wild and isogenic strains of V. cholerae differing in the set of the phage genes (ctxA, zot, and ace) was comparatively analyzed. It was shown that variability of CTXphi prophage genome was an important factor of modification of cholera vibrios virulent characteristics. Obtained data point to usefulness of ctxA, zot, and ace phage genes detection in wild V. cholerae isolates as it could permit evaluation of their virulent potential determining the severity of the infection.

[A comparative analysis of molecular-genetic peculiarities of the genomes of cholera, plague and anthrax agents and their evolutional transformations].

Cholera, plague, and anthrax, the diseases that have accounted for millions of human victims, still endanger the entire mankind by possible development of epidemic outbreaks due to their spread or application as bioterrorist agents. Generalized results of research into the genomic features of the Vibrio cholerae, Yersinia pestis, and Bacillus anthracis are discussed. Despite different frequencies of evolutional transformations occurring in their genomes, that are likely to be associated with diverse life cycles of the pathogens, clones with altered diagnostic, and virulence characteristics were shown to have a fair probability of formation. Also presented in the review, are literature data concerning the main evolutional stages for any of these pathogens, determination of new genetic variants, consideration of the mechanisms facilitating maintenance of the microbial agents during the interepidemic periods.

[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. It was shown, that bile serves as a signal from an environment for switching of gene expression of the genes, coding production of factors as virulence, and carrying out protective function of bacterial cell.

[Comparative genomic analysis of vibrio cholerae El Tor preseventh and seventh pandemic strains isolated in various periods].

Genetic organization of 52 Vibrio cholerae El Tor biotype preseventh and seventh pandemic strains isolated in various periods was studied by PCR assay and DNA-DNA hybridization. It was established that the genome of most ancient of analyzed strains isolated from a diarrhea patient in 1910 was devoid of CTX and RS1 prophages, vibrio pathogenicity islands (VPI and VPI-2), and pandemic islands (VSP-1 and VSP-2) that contain key virulence genes. The appearance of pathogenic properties in cholera vibrios for the first time causing a local outbreak of cholera in 1937 is connected with the acquisition of VPI and CTX that carried genes tcpA and ctx-AB, respectively, which are responsible for the colonization of small intestine and encode the production of cholera toxin. The appearance of seventh pandemic agent for cholera was shown to correlate with the acquisition by its precursor of two additional blocks of genes VSP-1 and VSP-2. This finding strongly supports the involvement of these genes in formation of the pandemic potential in strains. Molecular typing methods allowed elucidation of differences in the genetic organization between prepandemic and pandemic strains. The detected variability of the genome of contemporary virulent strains may be a reason for the occurrence of etiological agent for cholera with new properties.

[Genomic variability of vibrio cholerae El Tor biovariant strains].

The authors performed comparative analysis of the genomes of 145 clinical and environmental isolates of Vibrio cholerae El Tor biovariants using single locus and multiplex PCR. The study found that clinical strains isolated from patients with cholera formed a genetically homogenous group, where bacterial chromosome contained all the tested virulence genes, situated on mobile genetic elements that had been acquired by the pathogen at various stages of its evolution. Strains isolated from water ecosystems during interepidemic period were heterogeneous and formed three groups: a small number of virulent strains; non-toxigenic vibrio strains that, in the process of reductional variation in their new econiche, had only managed to maintain individual virulence genes; non-pathogenic "water" vibrios, whose chromosome contained only the genes from its core part, mobile genetic elements being optionally represented only by the persistence island. Molecular typing established genetic relations among V. cholerae strains under study.