Evolutionary perspective on the origin of Haitian cholera outbreak strain

Cholera epidemic has not been reported in Haiti for at least 100?years, although cholera has been present in Latin America since 1991. Surprisingly, the recent cholera epidemic in Haiti (October 2010) recorded more than 250,000 cases and 4000 deaths in the first 6?months and became one of the most explosive and deadly cholera outbreak in recent history. In the present study, we conducted genomic analyses of pathogenicity islands of three Haitian Vibrio cholerae strains and compared them with nine different V. cholerae O1 El Tor genomes. Although CIRS101 is evolutionarily most similar to the Haitian strains, our study also provides some important differences in the genetic organization of pathogenicity islands of Haitian strains with CIRS101. Evolutionary analysis suggests that unusual functional constraints have been imposed on the Haitian strains and we hypothesize that amino acid substitution is more deleterious in Haitian strains than in nonHaitian strains.     

High depth, whole-genome sequencing of cholera isolates from Haiti and the Dominican Republic

ABSTRACT: BACKGROUND: Whole-genome sequencing is an important tool for understanding microbial evolution and identifying the emergence of functionally important variants over the course of epidemics. In October 2010, a severe cholera epidemic began in Haiti, with additional cases identified in the neighboring Dominican Republic. We used whole-genome approaches to sequence four Vibrio cholerae isolates from Haiti and the Dominican Republic and three additional V. cholerae isolates to a high depth of coverage (>2000x); four of the seven isolates were previously sequenced. RESULTS: Using these sequence data, we examined the effect of depth of coverage and sequencing platform on genome assembly and identification of sequence variants. We found that 50x coverage is sufficient to construct a whole-genome assembly and to accurately call most variants from 100 base pair paired-end sequencing reads. Phylogenetic analysis between the newly sequenced and thirty-three previously sequenced V. cholerae isolates indicates that the Haitian and Dominican Republic isolates are closest to strains from South Asia. The Haitian and Dominican Republic isolates form a tight cluster, with only four variants unique to individual isolates. These variants are located in the CTX region, the SXT region, and the core genome. Of the 126 mutations identified that separate the Haiti-Dominican Republic cluster from the V. cholerae reference strain (N16961), 73 are non-synonymous changes, and a number of these changes cluster in specific genes and pathways. CONCLUSIONS: Sequence variant analyses of V. cholerae isolates, including multiple isolates from the Haitian outbreak, identify coverage-specific and technology-specific effects on variant detection, and provide insight into genomic change and functional evolution during an epidemic.     

Mobilization of the Vibrio pathogenicity island between Vibrio cholerae isolates mediated by CP-T1 generalized transduction

Pathogenicity islands are large chromosomal regions encoding virulence genes that were acquired by horizontal gene transfer and are found in a wide range of pathogenic bacteria. In toxigenic Vibrio cholerae isolates the receptor for the cholera toxin encoding filamentous phage CTXphi, the toxin-coregulated pilus, is part of the Vibrio pathogenicity island (VPI). In this paper, we show that the VPI can be transferred between O1 serogroup strains, the predominant cause of epidemic cholera, via a generalized transducing phage CP-T1.     

High-Frequency Rugose Exopolysaccharide Production by Vibrio cholerae Strains Isolated in Haiti

In October, 2010, epidemic cholera was reported for the first time in Haiti in over 100 years. Establishment of cholera endemicity in Haiti will be dependent in large part on the continued presence of toxigenic V. cholerae O1 in aquatic reservoirs. The rugose phenotype of V. cholerae, characterized by exopolysaccharide production that confers resistance to environmental stress, is a potential contributor to environmental persistence. Using a microbiologic medium promoting high-frequency conversion of smooth to rugose (S–R) phenotype, 80 (46.5%) of 172 V. cholerae strains isolated from clinical and environmental sources in Haiti were able to convert to a rugose phenotype. Toxigenic V. cholerae O1 strains isolated at the beginning of the epidemic (2010) were significantly less likely to shift to a rugose phenotype than clinical strains isolated in 2012/2013, or environmental strains. Frequency of rugose conversion was influenced by incubation temperature and time. Appearance of the biofilm produced by a Haitian clinical rugose strain (altered biotype El Tor HC16R) differed from that of a typical El Tor rugose strain (N16961R) by confocal microscopy. On whole-genome SNP analysis, there was no phylogenetic clustering of strains showing an ability to shift to a rugose phenotype. Our data confirm the ability of Haitian clinical (and environmental) strains to shift to a protective rugose phenotype, and suggest that factors such as temperature influence the frequency of transition to this phenotype.     

Comparative genomic analyses of the vibrio pathogenicity island and cholera toxin prophage regions in nonepidemic serogroup strains of Vibrio cholerae

Two major virulence factors are associated with epidemic strains (O1 and O139 serogroups) of Vibrio cholerae: cholera toxin encoded by the ctxAB genes and toxin-coregulated pilus encoded by the tcpA gene. The ctx genes reside in the genome of a filamentous phage (CTXphi), and the tcpA gene resides in a vibrio pathogenicity island (VPI) which has also been proposed to be a filamentous phage designated VPIphi. In order to determine the prevalence of horizontal transfer of VPI and CTXphi among nonepidemic (non-O1 and non-O139 serogroups) V. cholerae, 300 strains of both clinical and environmental origin were screened for the presence of tcpA and ctxAB. In this paper, we present the comparative genetic analyses of 11 nonepidemic serogroup strains which carry the VPI cluster. Seven of the 11 VPI(+) strains have also acquired the CTXphi. Multilocus sequence typing and restriction fragment length polymorphism analyses of the VPI and CTXphi prophage regions revealed that the non-O1 and non-O139 strains were genetically diverse and clustered in lineages distinct from that of the epidemic strains. The left end of the VPI in the non-O1 and non-O139 strains exhibited extensive DNA rearrangements. In addition, several CTXphi prophage types characterized by novel repressor (rstR) and ctxAB genes and VPIs with novel tcpA genes were found in these strains. These data suggest that the potentially pathogenic, nonepidemic, non-O1 and non-O139 strains identified in our study most likely evolved by sequential horizontal acquisition of the VPI and CTXphi independently rather than by exchange of O-antigen biosynthesis regions in an existing epidemic strain.     

Genetics of stress adaptation and virulence in toxigenic Vibrio cholerae

Vibrio cholerae, a Gram-negative bacterium belonging to the gamma-subdivision of the family Proteobacteriaceae is the etiologic agent of cholera, a devastating diarrheal disease which occurs frequently as epidemics. Any bacterial species encountering a broad spectrum of environments during the course of its life cycle is likely to develop complex regulatory systems and stress adaptation mechanisms to best survive in each environment encountered. Toxigenic V. cholerae, which has evolved from environmental nonpathogenic V. cholerae by acquisition of virulence genes, represents a paradigm for this process in that this organism naturally exists in an aquatic environment but infects human beings and cause cholera. The V. cholerae genome, which is comprised of two independent circular mega-replicons, carries the genetic determinants for the bacterium to survive both in an aquatic environment as well as in the human intestinal environment. Pathogenesis of V. cholerae involves coordinated expression of different sets of virulence associated genes, and the synergistic action of their gene products. Although the acquisition of major virulence genes and association between V. cholerae and its human host appears to be recent, and reflects a simple pathogenic strategy, the establishment of a productive infection involves the expression of many more genes that are crucial for survival and adaptation of the bacterium in the host, as well as for its onward transmission and epidemic spread. While a few of the virulence gene clusters involved directly with cholera pathogenesis have been characterized, the potential exists for identification of yet new genes which may influence the stress adaptation, pathogenesis, and epidemiological characteristics of V. cholerae. Coevolution of bacteria and mobile genetic elements (plasmids, transposons, pathogenicity islands, and phages) can determine environmental survival and pathogenic interactions between bacteria and their hosts. Besides horizontal gene transfer mediated by genetic elements and phages, the evolution of pathogenic V. cholerae involves a combination of selection mechanisms both in the host and in the environment. The occurrence of periodic epidemics of cholera in endemic areas appear to enhance this process.     

Genetic markers of epidemic strains of Vibrio cholerae

In this review new data on the key pathogenicity genes of V. cholerae are presented. As shown on the basis of the analysis of the latest information on the structure of the genomes of different V. cholerae strains, structural genes ctxAB coding cholera toxin may not serve as the only marker of epidemically dangerous strains. More complete and reliable information for the evaluation of the epidemic potential of V. cholerae isolated from the environment may be obtained by the simultaneous detection of 4 genetic markers: genes ctxAB, tcpA and hap coding, respectively, cholera toxin, toxin-corregulated adhesion pili and soluble hemagglutinin/protease, as well as regulatory virulence gene toxR.     

An outbreak of cholera (El Tor) in Samarkand Province, Uzbekistan in 1990]

A group outbreak of cholera caused by Vibrio eltor in the Samarkand Province++ in 1990 is analyzed. The retrospective analysis of the isolation of V. cholerae from water of surface reservoirs has been made. The study points out that since the cholera epidemic of 1985 V. cholerae avirulent strains, serogroup O1, have been periodically isolated in the Samarkand Province. The conclusion has been made that the isolation of even avirulent strains only from environmental objects is indicative of the unfavorable epidemiological situation in this area and of the necessity to take measures for improving epidemiological surveillance.     

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.     

Genetic Traits of Vibrio cholerae O1 Haitian Isolates That Are Absent in Contemporary Strains from Kolkata,India

The world''s worst cholera epidemic in Haiti (2010) coerced to trace the origin and dissemination of the causative agent Vibrio cholerae O1 for proper management of cholera. Sequence analysis of the Haitian strain showed several variations in the genes encoding cholera toxin B subunit (ctxB); toxin-co-regulated pilus (tcpA), repeat in toxins (rtxA), quinolone resistance-determining region (QRDR) of gyrase A (gyrA), rstB of RS element along with the change in the number of repeat sequences at the promoter region of ctxAB. Our earlier studies showed that variant tcpA (tcpA CIRS) and ctxB (ctxB7) first appeared in Kolkata during 2003 and 2006, respectively. The present study revealed that a variant rtxA was first isolated in Kolkata during 2004 and probably formed the genetic background for the emergence of the ctxB7 allele as we were unable to detect a single strain with the combination of El Tor rtxA and ctxB7. The variant gyrA was first time detected in Kolkata during 1994. The Kolkata strains contained four heptad repeats (TTTTGAT) in their CT promoter regions whereas Haitian strains carried 5 heptad repeats. Haitian strains had 3 nucleotide deletions at the rstB gene, which is a unique feature of the classical biotype strains. But the Kolkata strains did not have such deletion mutations in the rstB. Our study demonstrated the existence of some Haitian genetic traits in Kolkata isolates along with the dissimilarities in genomic content with respect to rstB and ctxAB promoter region. Finally, we conclude that Haitian variant strain may be evolved due to sequential event in the Indian subcontinent strain with some cryptic modification in the genome.     

Genome Sequence of Hybrid Vibrio cholerae O1 MJ-1236, B-33, and CIRS101 and Comparative Genomics with V. cholerae

The genomes of Vibrio cholerae O1 Matlab variant MJ-1236, Mozambique O1 El Tor variant B33, and altered O1 El Tor CIRS101 were sequenced. All three strains were found to belong to the phylocore group 1 clade of V. cholerae, which includes the 7th-pandemic O1 El Tor and serogroup O139 isolates, despite displaying certain characteristics of the classical biotype. All three strains were found to harbor a hybrid variant of CTXΦ and an integrative conjugative element (ICE), leading to their establishment as successful clinical clones and the displacement of prototypical O1 El Tor. The absence of strain- and group-specific genomic islands, some of which appear to be prophages and phage-like elements, seems to be the most likely factor in the recent establishment of dominance of V. cholerae CIRS101 over the other two hybrid strains.Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a life-threatening disease that causes severe, watery diarrhea. Cholera bacteria are serogrouped based on their somatic O antigens, with more than 200 serogroups identified to date (6). Only toxigenic strains of serogroups O1 and O139 have been identified as agents of cholera epidemics and pandemics; serogroups other than O1 and O139 have the potential to cause mild gastroenteritis or, rarely, local outbreaks. Genes coding for cholera toxin (CTX), ctxAB, and other virulence factors have been shown to reside in bacteriophages and various mobile genetic elements. In addition, V. cholerae serogroup O1 is differentiated into two biotypes, classical and El Tor, by a combination of biochemical traits, by sensitivity to biotype-specific bacteriophages, and more recently by nucleotide sequencing of specific genes and by molecular typing (5, 17, 19).There have been seven pandemics of cholera recorded throughout human history. The seventh and current pandemic began in 1961 in the Indonesian island of Sulawesi and subsequently spread to Asia, Africa, and Latin America; the six previous pandemics are believed to have originated in the Indian subcontinent. Isolates of the sixth pandemic were almost exclusively of the O1 classical biotype, whereas the current (seventh) pandemic is dominated by the V. cholerae O1 El Tor biotype as the causative agent, a transition occurring between 1923 and 1961. Today, the disease continues to remain a scourge in developing countries, confounded by the fact that V. cholerae is native to estuaries and river systems throughout the world (8).Over the past 20 years, several new epidemic lineages of V. cholerae O1 El Tor have emerged (or reemerged). For example, in 1992, a new serogroup, namely, O139 of V. cholerae, was identified as the cause of epidemic cholera in India and Bangladesh (25). The initial concern was that a new pandemic was beginning; however, the geographic range of V. cholerae O139 is currently restricted to Asia. Additionally, V. cholerae O1 hybrids and altered El Tor variants have been isolated repeatedly in Bangladesh (Matlab) (23, 24) and Mozambique (1). Altered V. cholerae O1 El Tor isolates produce cholera toxin of the classical biotype but can be biotyped as El Tor by conventional phenotypic assays, whereas V. cholerae O1 hybrid variants cannot be biotyped based on phenotypic tests and can produce cholera toxin of either biotype. These new variants have subsequently replaced the prototype seventh-pandemic V. cholerae O1 El Tor strains in Asia and Africa, with respect to frequency of isolation from clinical cases of cholera (27).Here, we report the genome sequence of three V. cholerae O1 variants, MJ-1236, a Matlab type I hybrid variant from Bangladesh that cannot be biotyped by conventional methods, CIRS101, an altered O1 El Tor isolate from Bangladesh which harbors ctxB of classical origin, and B33, an altered O1 El Tor isolate from Mozambique which harbors classical CTXΦ, and we compare their genomes with prototype El Tor and classical genomes. From an epidemiological viewpoint, among the three variants characterized in this study, V. cholerae CIRS101 is currently the most “successful” in that strains belonging to this type have virtually replaced the prototype El Tor in Asia and many parts of Africa, notably East Africa. This study, therefore, gives us a unique opportunity to understand why V. cholerae CIRS101 is currently the most successful El Tor variant.     

Multiple antibiotic resistance of Vibrio cholerae serogroup O139 in China from 1993 to 2009

Regarded as an emerging diarrheal micropathogen, Vibrio cholerae serogroup O139 was first identified in 1992 and has become an important cause of cholera epidemics over the last two decades. O139 strains have been continually isolated since O139 cholera appeared in China in 1993, from sporadic cases and dispersed foodborne outbreaks, which are the common epidemic types of O139 cholera in China. Antibiotic resistance profiles of these epidemic strains are required for development of clinical treatments, epidemiological studies and disease control. In this study, a comprehensive investigation of the antibiotic resistance of V. cholerae O139 strains isolated in China from 1993 to 2009 was conducted. The initial O139 isolates were resistant to streptomycin, trimethoprim-sulfamethoxazole and polymyxin B only, while multidrug resistance increased suddenly and became common in strains isolated after 1998. Different resistance profiles were observed in the isolates from different years. In contrast, most V. cholerae O1 strains isolated in the same period were much less resistant to these antibiotics and no obvious multidrug resistance patterns were detected. Most of the non-toxigenic strains isolated from the environment and seafood were resistant to four antibiotics or fewer, although a few multidrug resistant strains were also identified. These toxigenic O139 strains exhibited a high prevalence of the class I integron and the SXT element, which were rare in the non-toxigenic strains. Molecular subtyping of O139 strains showed highly diverse pulsed-field gel electrophoresis patterns, which may correspond to the epidemic state of sporadic cases and small-scale outbreaks and complex resistance patterns. Severe multidrug resistance, even resistance transfers based on mobile antibiotic resistance elements, increases the probability of O139 cholera as a threat to public health. Therefore, continual epidemiological and antibiotic sensitivity surveillance should focus on the occurrence of multidrug resistance and frequent microbial population shifts in O139 strains.     

Molecular analysis of the rstR and orfU genes of the CTX prophages integrated in the small chromosomes of environmental Vibrio cholerae non-O1, non-O139 strains

The ctxAB genes encoding cholera toxin, reside in the genome of a filamentous bacteriophage CTXphi. The presence of CTX prophage in non-epidemic environmental Vibrio cholerae strains is rare. The CTX prophage, the lysogenic form of CTXphi in V. cholerae, is comprised of the 'RS2' and the 'Core'. Analysis of the rstR gene present in the RS2 region of the CTX prophage revealed the presence of new alleles of the prophages in four environmental non-O1, non-O139 strains VCE22 (O36), VCE228 (O27), VCE232 (O4) and VCE233 (O27), and the CTX prophages are located in the small chromosomes. Phylogenetic analysis based on the nucleotide sequences of the rstR and orfU (present in the core) genes of these prophages placed them in a single unique cluster, which is distally located compared with that of epidemic V. cholerae O1 strains. Further analysis indicated that the genome of the prophage present in the strain VCE22 is devoid of the ctxAB genes, called pre-CTX prophage and the strain also possess the toxin-coregulated pilus protein coding gene tcpA of classical type, another important pathogenicity determining locus of the epidemic V. cholerae strains. Comparative analysis of the nucleotide sequences of the rstR and orfU genes indicated that the pre-CTX prophage of VCE22 might be the progenitor of new alleles of the CTX prophages present in these environmental strains.     

Critical factors influencing the occurrence of Vibrio cholerae in the environment of Bangladesh

The occurrence of outbreaks of cholera in Africa in 1970 and in Latin America in 1991, mainly in coastal communities, and the appearance of the new serotype Vibrio cholerae O139 in India and subsequently in Bangladesh have stimulated efforts to understand environmental factors influencing the growth and geographic distribution of epidemic Vibrio cholerae serotypes. Because of the severity of recent epidemics, cholera is now being considered by some infectious disease investigators as a "reemerging" disease, prompting new work on the ecology of vibrios. Epidemiological and ecological surveillance for cholera has been under way in four rural, geographically separated locations in Bangladesh for the past 4 years, during which both clinical and environmental samples were collected at biweekly intervals. The clinical epidemiology portion of the research has been published (Sack et al., J. Infect. Dis. 187:96-101, 2003). The results of environmental sampling and analysis of the environmental and clinical data have revealed significant correlations of water temperature, water depth, rainfall, conductivity, and copepod counts with the occurrence of cholera toxin-producing bacteria (presumably V. cholerae). The lag periods between increases or decreases in units of factors, such as temperature and salinity, and occurrence of cholera correlate with biological parameters, e.g., plankton population blooms. The new information on the ecology of V. cholerae is proving useful in developing environmental models for the prediction of cholera epidemics.     

Molecular genetic features of Vibrio cholerae classica strains, that caused the Asiatic cholera epidemic in Russian in 1942

Molecular genetic features of Vibrio cholerae classical strains which caused an epidemic of Asian cholera in Russia in 1942 have been studied for the first time. These strains had a high level of choleric toxin production and toxin-coregulated adhesion piles, the main virulence factors; all the strains were auxotrophs and needed purine and/or amino acids for growth in minimal medium. Moreover, having hapA structural gene in the chromosome (according to polymerase chain reaction), they did not produce soluble hemagglutinin protease promoting propagation of vibrios in the environment. These features of natural V. cholerae classical strains are apparently responsible for the peculiar infectious and epidemic processes in the cholera epidemic.     

Comparative analysis of the major protective antigens production in Vibrio cholerae recombinant and producer strains of the classical biovar

The comparative study of 4 constructed protective antigen producing strains of the classical biovar and V. cholerae strains 569 B Inaba and M41 Ogawa, used in manufacturing the cholera chemical vaccine "cholerogen-toxoid", was carried out. The study revealed that V. cholerae plasmid strains 2414 Ogawa, 2415 Inaba and nonplasmid strains 2416 Ogawa, 2417 Inaba had a higher level of production of the main protective antrigens in comparison with producer strains. They also synthesized much more (4-5 fold) cholera toxin, toxin co-regulated adhesion pili, contained protein OmpU in their outer membrane, exceeded 2- to 3-fold in the synthesis of pathogenicity enzymes (proteases, phospholipases) and synthesized the same amounts of 01 antigen, serovars Inaba and Ogawa. The use of the newly created protective-antigen producing strains in vaccine manufacturing could facilitate the preparation of a more effective cholera chemical vaccine "cholerogen-toxoid".     

Incidence and toxigenicity of Vibrio cholerae in a freshwater lake during the epidemic of cholera caused by serogroup O139 Bengal in Calcutta, India

Abstract The extent of contamination of a freshwater lake with Vibrio cholerae 0139 Bengal and the toxigenicity of all the V. cholerae isolates recovered during the period of the study were examined during and after an explosive outbreak of 0139 cholera in Calcutta. Strains biochemically characterized as V. cholerae could be isolated throughout the period of study examined from the freshwater lake samples. Most probable number of V. cholerae belonging to the 0139 serogroup in surface waters was 3 to 4 per 100 ml during major part of the study but isolation of this serogroup from sediment and plankton samples was infrequent. Of the total of 150 strains recovered, 23 (15.3%) agglutinated with the 0139 antiserum while the remaining belonged to the non-O1 non-O139 serogroups. None of the strains agglutinated with the O1 antiserum. All the 23 strains of V. cholerae O139 produced cholera toxin while 7.9% of the 127 non-O1 non-O139 strains also produced cholera toxin. Resistance to ampilicillin, furazolidone and streptomycin was encountered among strains belonging to both V. cholerae O139 and V. cholerae non-O1 non-O139 strains, but the percentage of resistant strains in the former was much higher than in the latter. During this cholera epidemic, possibly due to the introduction of large numbers of toxigenic V. cholerae such as the O139 serogroup, there was an increase in the number of toxigenic vibrios among the innocuous aquatic residents. This presumably occured through genetic exchange and, if substantiated, could play an important role in the re-emergence of epidemics.     

O1群和O139群霍乱弧菌主要抗原研究进展

霍乱弧菌是引起人和动物烈性肠道传染病霍乱的病原体。在霍乱弧菌的200多个血清群中,只有O1群和O139群霍乱弧菌能引起霍乱。快速准确检测O1群和O139群霍乱弧菌是霍乱防治的关键。表面抗原在O1群和O139群霍乱弧菌检测中发挥着重要作用。简要综述了O1群和O139群霍乱弧菌的脂多糖、霍乱肠毒素、外膜蛋白W、毒素共调菌毛和甘露糖敏感血凝素等5种主要抗原的研究进展。     

Characterization of Vibrio cholerae eltor isolates according to their epidemic potential using new diagnostic cholera bacteriophages eltor ctx+ and ctx- and by the polymerase chain reaction

The epidemic potential of 113 V. cholerae eltor strains of different origin was determined with new diagnostic cholera bacteriophages eltor ctx+ and ctx-, as well as the test for hemolytic activity. Of these strains 50 were epidemically safe and 51 were epidemically dangerous, while the epidemic potential of 12 other strains could not be detected. Determination of genes ctxA, tcpA and toxR in the strains under study by means of the polymerase chain reaction (PCR) revealed that epidemically dangerous strains carried the whole set of the above genes in 92.2% of cases. 98.0% of epidemically safe cultures were lacking either gene ctxA, or genes ctxA and tcpA, or genes ctxA, tcpA and toxR, which confirmed their incapacity to cause cholera. The results of the differentiation of the cultures with new diagnostic cholera phages coincided with the results of PCR in 90% of cases. The most complete and reliable evaluation of the epidemic potential of individual vibrio isolates may be obtained using the two compared methods. The amplification test system gives more information when isolates with unclear epidemic potential are analyzed.