Characterization of Vibrio cholerae cultures isolated in foci of cholera in the city of Kazan

During the period of the registered outbreak of cholera in 2001 in Kazan 171 V. cholerae cultures were isolated in the focus of the infection (from patients, carriers and 7 environmental objects). The use of the basic and additional tests, including the polymerase chain reaction, made it possible to establish the circulation of V. cholerae, phagovar 15, in the focus of the infection. The strain isolated from the water reservoir Azino-1 in Kazan was identical in its properties to the epidemically dangerous strains isolated from patients. On the whole, the data obtained in the identification of the strains showed that the cultures isolated from patients, vibrio-carriers and environmental objects were identical.     

Cholera caused by Vibrio cholerae O1 ctxAB- tcpA+

Results of analysis of cholera outbreak during which V. cholerae O1 biovar El-Tor ctxAB- tcpA+ was isolated from 2 patients and 30 carriers are presented. Epidemic was caused by contamination of water source and water route of transmission. Strains identical to ones detected in humans were isolated from water of surface well in zone of water intake. Genome and VNTR-analysis of ctxAB- tcpA+ vibrios that caused outbreak in Rostov region in 2005 showed that they differed from ctxAB- tcpA- and ctxAB- tcpA+ vibrios isolated previously during and beyond of outbreaks from patients, carriers and environment and formed separate group with certain genotype. These results confirms conclusions of epidemiological analysis about imported cause of recent outbreak.     

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.     

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.     

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.     

Hemolytic Vibrio cholerae O1 that is sensitive to Mukerjee's cholera phage IV and the phage produced by the hemolytic vibrio lysogenized with the infection of Mukerjee's cholera phage IV

Strains of hemolytic Vibrio cholerae O1 (El Tor vibrio) which are sensitive to Mukerjee's cholera phage group IV were isolated from cholera patients in North-East Thailand in 1986. Plaques of the phage on these hemolytic V. cholerae O1 were usually translucent but almost transparent on some strains, just like the plaques on non-hemolytic V. cholerae O1 (classical vibrio). These hemolytic V. cholerae O1 were lysogenized with the infection of cholera phage IV, and the lysogenized strains produced phage different from cholera phage IV. These hemolytic strains were classified into Cured type in prophage typing of V. cholerae O1, El Tor, because they were also lysogenized with Kappa phage and were hemolytic. When Cured-type V. cholerae O1, El Tor previously isolated in various countries were examined for the sensitivity to cholera phage IV, some of the isolates were sensitive.     

Method of DNA extraction and application of multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae O1 and O139 from aquatic ecosystems

Vibrio cholerae is a free-living bacterium found in water and in association with plankton. V. cholerae non-O1/non-O139 strains are frequently isolated from aquatic ecosystems worldwide. Less frequently isolated are V. cholerae O1 and V. cholerae O139, the aetiological agents of cholera. These strains have two main virulence-associated factors, cholera toxin (CT) and toxin co-regulated pilus (TCP). By extracting total DNA from aquatic samples, the presence of pathogenic strains can be determined quickly and used to improve a microbiological risk assessment for cholera in coastal areas. Some methods suggested for DNA extraction from water samples are not applicable to all water types. We describe here a method for DNA extraction from coastal water and a multiplex polymerase chain reaction (PCR) for O1 and O139 serogroups. DNA extraction was successfully accomplished from 117 sea water samples collected from coastal areas of Perú, Brazil and the USA. DNA concentration in all samples varied from 20 ng to 480 micro g micro l-1. The sensitivity of the DNA extraction method was 100 V. cholerae cells in 250 ml of water. The specificity of multiplex O1/O139 PCR was investigated by analysing 120 strains of V. cholerae, Vibrio and other Bacteria species. All V. cholerae O1 and O139 tested were positive. For cholera surveillance of aquatic environments and ballast water, total DNA extraction, followed by V. cholerae PCR, and O1/O139 serogroup and tcpA/ctxA genes by multiplex PCR offers an efficient system, permitting risk analysis for cholera in coastal areas.     

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.     

Cholera in the Far East of Russia. Communication 1. Epidemiological characteristics of the outbreak of cholera El tor in Vladivostok

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.     

Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1

A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V. cholerae O1. Enterotoxin-producing V. cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene. Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hlyA gene in the same reaction. The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V. cholerae strains. Enrichment of V. cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food.     

The use of the gene hybridization method for the identification of epidemiologically dangerous strains of cholera vibrios

Using the labeled DNA fragments containing the genes for cholera toxin the strains of cholera vibrios were studied for the presence of cholera toxin genes. Vibrio cholerae strains isolated from natural water reservoirs under the favourable epidemic situation do not contain the cholera toxin genes. The DNA hybridization method was compared with other methods used in research and practical work for estimation of epidemic importance of cholera vibrios.     

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.     

Cholera toxin gene-positive Vibrio cholerae O1 Ogawa and Inaba strains produce the new cholera toxin

Abstract Two strains of cholera toxin (CT) gene-positive Vibrio cholerae O1, Ogawa, isolated from patients with diarrhoea and the hypertoxigenic V. cholerae O1, Inaba (569B), were found to produce the new cholera toxin that has earlier been demonstrated to be elaborated by CT gene-negative human and environmental isolates of V. cholerae O1. The CT gene-positive strains produce the new cholera toxin simultaneously with CT, indicating that they contain the gene coding for the new cholera toxin in addition to that of CT.     

Characterization of a Vibrio cholerae phage isolated from the coastal water of Peru

A Vibrio cholerae bacteriophage, family Myoviridae, was isolated from seawater collected from the coastal water of Lima, Peru. Genome size was estimated to be 29 kbp. The temperate phage was specific to V. cholerae and infected 12/13 V. cholerae O1 strains and half of the four non-O1/non-O139 strains tested in this study. Vibrio cholerae O139 strains were resistant to infection and highest infection rates were obtained in low nutrient media amended with NaCl or prepared using seawater as diluent.     

Glycogen contributes to the environmental persistence and transmission of Vibrio cholerae

Pathogenic Vibrio cholerae cycle between the nutrient-rich human intestinal tract and nutrient-poor aquatic environments and currently few bacterial factors are known that aid in the transition between these disparate environments. We hypothesized that the ability to store carbon as glycogen would facilitate both bacterial fitness in the aquatic environment and transmission of V. cholerae to new hosts. To investigate the role of glycogen in V. cholerae transmission, we constructed mutants that cannot store or degrade glycogen. Here, we provide the first report of glycogen metabolism in V. cholerae and demonstrate that glycogen prolongs survival in nutrient-poor environments that are known ecological niches of V. cholerae , including pond water and rice-water stool. Additionally, glycogen contributes to the pathogenesis of V. cholerae in a transmission model of cholera. A role for glycogen in the transmission of V. cholerae is further supported by the presence of glycogen granules in rice-water stool vibrios from cholera patients, indicating that glycogen is stored during human infection. Collectively, our findings indicate that glycogen metabolism is critical for V. cholerae to transition between host and aquatic environments.     

多重PCR方法检测霍乱弧菌的研究

霍乱弧菌是霍乱的病原体,可以分为O1群、O139群和非O1/非O139群。O1群和O139群霍乱弧菌产生的霍乱肠毒素(也称霍乱毒素)是产生霍乱的主要原因,也只有O1群和O139群霍乱弧菌可引起霍乱。其他群的霍乱弧菌毒性不高,但在食品中也不允许被检出。实验以霍乱胶原酶基因和霍乱毒素基因为目的基因,试图建立一种PCR方法对霍乱弧菌进行检测研究,结果表明此方法可以用于食品中的霍乱弧菌检测。     

霍乱弧菌检测方法的研究进展

烈性肠道传染病霍乱能引起大范围乃至世界性大流行,在我国被列为甲类传染病.霍乱弧菌是导致感染者严重腹泻、引起霍乱的病原菌.霍乱弧菌的快速、准确检测是霍乱预防、控制的重要依据.目前,国内、外针对霍乱弧菌建立了许多有效的检测方法,尤其是分子生物学相关技术的应用,为霍乱弧菌的检测提供了新的手段.本文综述了近年来霍乱弧菌检测方法...     

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.     

Ecology of Vibrio species, including Vibrio cholerae, in natural waters of Kent, England

The distribution of Vibrio species in water from two sites in Kent was studied between 1978 and 1980. They were counted by a most probable number technique using alkaline peptone water for enrichment followed by plating onto thiosulphate citrate bile salt sucrose agar, or by direct plating of water onto the same agar. In a freshwater stream both upstream and downstream from a human sewage works outfall V. metschnikovii was the predominant Vibrio. Vibrio anguillarum was isolated sporadically. Non-O1 serovars of V. cholerae occurred only twice. At the other site in a ditch containing static, brackish water, non-O1 V. cholerae (highest number 400 cfu/ml) was observed as present only from May to November. Vibrio anguillarum was isolated throughout the sampling period. The presence of non-O1 V. cholerae in both sites was not dependent on the input of human sewage. The hypothesis that non-toxigenic V. cholerae can survive and multiply in water was tested in the ditch by the use of submersible chambers constructed of polycarbonate membranes and Plexiglass. The seasonal incidence of non-O1 V. cholerae in the brackish water site could be explained by the multiplication of the organism when the water temperature exceeded 9°C. It was concluded that strains of V. cholerae that are unable to produce cholera toxin are indigenous to static brackish water environments and the possibility that this applies to toxigenic strains as well should be investigated.