The isolation of monoclonal antibodies to the hemolysin of noncholerigenic vibrios]

The results of the work on obtaining monoclonal antibodies (McAb) to hemolysin of noncholerigenic vibrios and the study of hemolysin preparations and Vibrio cholerae strains are presented. As established with the use of McAb, hemolysin preparations proved to be complex compounds containing, in addition to hemolytic protein, some lipopolysaccharide determinants, which had not been detected with the use of traditional biochemical tests. The study revealed that V. cholerae non-01 and V. cholerae eltor hemolysins have common antigenic determinants, which was confirmed by the data of literature on the homogeneity of these substances. McAb obtained in this study interacted with V. cholerae avirulent hemolytic strains, not interacting with V. cholerae classical strains.     

Differences in virulence genes in Vibrio cholerae eltor strains isolated from different sources in Turkmenistan territory

Polymerase chain reaction (PCR) detected the presence of various genes associated with virulence in genome of strains V. cholerae eltor isolated in Turkmenistan territory during epidemic and epidemic-free perios. It was found that a complete set of virulence genes (ctxA+, tcpA+ and toxR+) contained strains isolated from patients, carriers and environment only in cholera epidemics. Strains isolated from the environment in the period free of epidemics did not contain ctxA and tcpA in 78.2% of cases, but 5.2% of the strains carried a complete set of virulence genes. There were also nontoxigenic strains containing genes tcpA and toxR. Such strains were isolated from the environment (16.6%) and vibrion carriers (42.9%). Isolated were also strains V.cholerae eltor carrying bacteriophage CTX phi with incomplete set of virulence genes and having genotype ctxA-, ace+ and zot+. Almost all the strains ctxA-, tcpA+ carry attRS1-site in genome. This shows that such strains may transform into toxigenic as a result of infection with bacteriophage CTX phi.     

Morphology and enzyme activity of nonculturable forms of Vibrio cholerae

The transition of V. cholerae into the uncultivable state under experimental conditions was accompanied by gradual changes in their morphology, motility and metabolic activity. The vibrios took the oval form, lost their flagellum, motility and enzyme activity on diagnostic media. Dehydrogenase activity tested by reduction of triphenyl tetrazolium chloride, increased at the initial stages and dropped to the initial level or even lower by the end of the observation period (10 months). Similar dynamics was noted when the cytoplasmatic marker enzyme--ATPase activity was studied. Glucose catabolism in the uncultivable forms shifted towards glycolysis. During 1-2 months ctx and tcp genes could be detected in these forms by the PCR. The dynamics of the biological properties under study made it possible to find out the existence of 3 functionally different stages in the development of an uncultivable population.     

Cell surface characteristics of environmental and clinical isolates of Vibrio cholerae non-O1.

The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations.     

Cell surface characteristics of environmental and clinical isolates of Vibrio cholerae non-O1.

The cell surfaces of several toxigenic and nontoxigenic environmental and clinical isolates of Vibrio cholerae non-O1 have been examined. The environmental strains, irrespective of toxigenicity, are significantly more resistant to antibiotics and detergents than are V. cholerae O1 strains. The clinical isolates of non-O1 vibrios are as sensitive to a wide variety of chemicals as the O1 vibrios. The environmental non-O1 strains are also less susceptible to lysis when treated with protein denaturants or neutral and anionic detergents than are O1 vibrios and the clinical non-O1 strains. In contrast to O1 vibrios, the environmental non-O1 vibrios do not have exposed phospholipids in their outer membranes. These features of the cell surfaces of environmental non-O1 vibrios might have a role in the better survival of these organisms under environmental fluctuations.     

Detection of lipase in Vibrio cholerae serogroup O1 in reaction of volumetric agglomeration

Study showed that El-Tor strains of V. cholerae isolated from different sources produce lipase for hemolysis after cultivation during 24 h on meat-peptone broth independently from their toxigenic and hemolytic abilities. Study of 3- and 4-hours broth cultures of vibrios revealed possibility to differentiate between hemolytic nontoxigenic strains and toxigenic nonhemolytic ones. Using antilipaze diagnostic kit it was possible to differentiate El-Tor vibrios from vibrios of classic biovar basing on lipase production 24 h after cultivation on meat-peptone broth that was evident in El-Tor vibrios but not in classic biovar strains.     

Genetic diversity of toxigenic and nontoxigenic Vibrio cholerae serogroups O1 and O139 revealed by array-based comparative genomic hybridization

Toxigenic serogroups O1 and O139 of Vibrio cholerae may cause cholera epidemics or pandemics. Nontoxigenic strains within these serogroups also exist in the environment, and also some may cause sporadic cases of disease. Herein, we investigate the genomic diversity among toxigenic and nontoxigenic O1 and O139 strains by comparative genomic microarray hybridization with the genome of El Tor strain N16961 as a base. Conservation of the toxigenic O1 El Tor and O139 strains is found as previously reported, whereas accumulation of genome changes was documented in toxigenic El Tor strains isolated within the 40 years of the seventh pandemic. High phylogenetic diversity in nontoxigenic O1 and O139 strains is observed, and most of the genes absent from nontoxigenic strains are clustered together in the N16961 genome. By comparing these toxigenic and nontoxigenic strains, we observed that the small chromosome of V. cholerae is quite conservative and stable, outside of the superintegron region. In contrast to the general stability of the genome, the superintegron demonstrates pronounced divergence among toxigenic and nontoxigenic strains. Additionally, sequence variation in virulence-related genes is found in nontoxigenic El Tor strains, and we speculate that these intermediate strains may have pathogenic potential should they acquire CTX prophage alleles and other gene clusters. This genome-wide comparison of toxigenic and nontoxigenic V. cholerae strains may promote understanding of clonal differentiation of V. cholerae and contribute to an understanding of the origins and clonal selection of epidemic strains.     

Model of Vibrio cholerae biofilm as a mechanism of its survival in surface water reservoirs

Vibrio cholerae eltor strains with different epidemic importance isolated from river water in the city of Vladivostok during a cholera outbreak (1999) and in the city of Irkutsk during a safe cholera period (2005) are used in the experiment. A biofilm structure consisting of a peripheral part, bundles, polysaccharide matrix, canals, and polymorphic vibrios is presented by light and luminescent microscopy. The metachromatic pink coloring of the matrix (crystal violet and toluidine blue) or fluorescent reddish orange color (acridine orange) are evidence of acid mucopolysaccharide (glucosaminoglycans) content. The biofilm of a toxigenic strain as opposed to a nontoxigenic one is formed much later, while the elements comprising its structure are more apparent. The viability of vibrio cells during the experiment (90 days) preserving the initial pathogenic potential testifies to the highly adaptable properties of the Vibrio cholerae eltor, which promotes its survival and existence in surface water reservoirs under favorable ecological conditions (optimal temperature, existence of chitin-containing substratum, etc.).     

The epidemiological importance of Vibrio cholerae isolated from different ecological systems

The analysis of the data on the isolation of V. cholerae from different ecological systems indicates that V. eltor do not constantly inhibit the rivers and sea at the territory under control. Hemolytically active V. cholerae without the vct gene, found to be faintly virulent and avirulent when studied on suckling rabbits used as a model and when evaluated by the complex method, show no tendency towards epidemic spread in the presence of conditions for the realization of the transmission of vibrios by the water route.     

Effect of environmental factors on expression and activity of chitinase genes of vibrios with special reference to Vibrio cholerae

AIMS: The aim of this study was to investigate the distribution and inducibility of chitinase genes in vibrios and the effect of environmental factors on the expression level and activity of chitinase genes in Vibrio cholerae strains. METHODS AND RESULTS: Chitin agar plate assays showed that V. cholerae strains were more chitinolytic than non-cholerae vibrios. All of the identified or putative chitinase genes were expressed in V. cholerae (four strains) but not in non-cholerae vibrios (seven species/strains) under standard laboratory growth conditions. In non-cholerae vibrios, these genes were induced by chitin, its monomer N-acetyl-d-glucosamine and on exposure to rabbit intestine, while in V. cholerae strains, these genes showed significant variation in expression levels. To study the effects of environmental factors on the expression and activity of chitinase genes in V. cholerae, bacteria were cultured in different pH, temperature, sodium chloride and nutrients. RT-PCR analysis showed that lower temperatures and higher pH, salinity and nutrition favoured expression of these genes, while their activity increased under higher nutrition content and salinity. CONCLUSIONS: Chitinase genes are distributed in all the relatively small number of strains studied here, and biotic and abiotic factors have significant role in the induction, expression level and activity of this gene family in vibrios. SIGNIFICANCE AND IMPACT OF THE STUDY: Chitinases have important applications especially in recycling of chitin. Vibrios can be used as chitinolytic agents, using suitable culture conditions that maximize the expression and activity of these genes.     

Hemolytic activity of Vibrio cholerae eltor and V. cholerae O139 toxigenic and nontoxigenic strains

A total of 20 ctx- and 16 ctx+ V. cholerae eltor strains, 20 ctx- and 22 ctx+ V. cholerae O139 strains were under study. Hemolytic activity was tested in modified Greig test with sheep, guinea pig and rabbit red blood cells. The comparative study of the hemolytic properties of V. cholerae O1 and O139 under different conditions of cultivation demonstrated their capacity of lysing sheep red blood cells (SRBC) irrespective of the presence of toxigenic properties. A wider spectrum of lytic activity of ctx- strains in Greig test with respect to red blood cells of different animals and the capacity of lysing SRBC, most resistant to the action of toxin, may be due to a considerably greater content of Hly+ clones in their population.     

Whole genome PCR scanning reveals the syntenic genome structure of toxigenic Vibrio cholerae strains in the O1/O139 population

Vibrio cholerae is commonly found in estuarine water systems. Toxigenic O1 and O139 V. cholerae strains have caused cholera epidemics and pandemics, whereas the nontoxigenic strains within these serogroups only occasionally lead to disease. To understand the differences in the genome and clonality between the toxigenic and nontoxigenic strains of V. cholerae serogroups O1 and O139, we employed a whole genome PCR scanning (WGPScanning) method, an rrn operon-mediated fragment rearrangement analysis and comparative genomic hybridization (CGH) to analyze the genome structure of different strains. WGPScanning in conjunction with CGH revealed that the genomic contents of the toxigenic strains were conservative, except for a few indels located mainly in mobile elements. Minor nucleotide variation in orthologous genes appeared to be the major difference between the toxigenic strains. rrn operon-mediated rearrangements were infrequent in El Tor toxigenic strains tested using I-CeuI digested pulsed-field gel electrophoresis (PFGE) analysis and PCR analysis based on flanking sequence of rrn operons. Using these methods, we found that the genomic structures of toxigenic El Tor and O139 strains were syntenic. The nontoxigenic strains exhibited more extensive sequence variations, but toxin coregulated pilus positive (TCP+) strains had a similar structure. TCP+ nontoxigenic strains could be subdivided into multiple lineages according to the TCP type, suggesting the existence of complex intermediates in the evolution of toxigenic strains. The data indicate that toxigenic O1 El Tor and O139 strains were derived from a single lineage of intermediates from complex clones in the environment. The nontoxigenic strains with non-El Tor type TCP may yet evolve into new epidemic clones after attaining toxigenic attributes.     

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.     

Role of iron ions in the production of hemolysin by toxigenic and nontoxigenic Vibrio cholerae of different serogroups

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

Incidence of toxigenic vibrios in foods available in Taiwan

A total of 1088 vibrios and related species were isolated from seafood and aquacultured foods available in Taiwan. They were identified as Vibrio alginolyticus, V. cholerae, V. fluvialis I, V. fluvialis II, V. parahaemolyticus, V. mimicus, Aeromonas caviae, A. hydrophila, A. sobria and other species. Incidence of these Vibrio and Aeromonas species in these foods was high. Vibrio parahaemolyticus was frequently found in seawater and in foods of freshwater origin. The Vibrio isolates were examined for enzymatic and toxigenic activities. Most of them showed strong lipase or protease activities. Haemolytic activities of V. cholerae, V. fluvialis I and V. fluvialis II isolates were mostly strong. About 49% showed cytotoxic activity and 5% cytotonic activity in Chinese hamster ovary cell culture assay. Nevertheless, only three non-1 V. cholerae (2.07%) and two V. parahaemolyticus isolates (1.65%) produced cholera toxin and thermostable direct haemolysin activity, respectively. Various toxigenic vibrios may be important food-borne pathogens in this region because of their high incidence in foods.     

Effect of various biophysicochemical conditions on toxigenicity of Vibrio cholerae 01 during survival with a green alga, Rhizoclonium fontanum, in an artificial aquatic environment

Toxigenic and nontoxigenic strains of Vibrio cholerae 01 occur in the natural aquatic environment. It is not clear whether V. cholerae 01 lose toxigenicity and become nontoxigenic during survival in the aquatic environment as a result of the effect of various biophysicochemical conditions (e.g., sunlight, pH, temperature, competition with other bacteria for nutrients, etc.). Five toxigenic strains were exposed to artificial aquatic environments in the presence of a filamentous green alga. Rhizoclonium fontanum, and recovered after different time intervals (0 and 0.5 h, 3, 6, 9, and 15 days). This experimental system was exposed to sunlight and the V. cholerae 01 were in competition for nutrients with resident bacterial flora from R. fontanum. The toxigenicity of Vibrio cholerae 01 that were recovered at different time intervals was assessed by tissue culture assay using Vero cells. The toxigenicity of recovered strains was compared with that of the parent strains. The results demonstrated that toxigenic V. cholerae 01 are unlikely to lose their toxigenicity in aquatic environments as a result of the effects of various biophysicochemical conditions. These results are consistent with the hypothesis of environmental reservoirs of V. cholerae.     

Incidence of toxigenic vibrios in foods available in Taiwan.

A total of 1088 vibrios and related species were isolated from seafood and aquacultured foods available in Taiwan. They were identified as Vibrio alginolyticus, V. cholerae, V. fluvialis I, V. fluvialis II, V. parahaemolyticus, V. mimicus, Aeromonas caviae, A. hydrophila, A. sobria and other species. Incidence of these Vibrio and Aeromonas species in these foods was high. Vibrio parahaemolyticus was frequently found in seawater and in foods of freshwater origin. The Vibrio isolates were examined for enzymatic and toxigenic activities. Most of them showed strong lipase or protease activities. Haemolytic activities of V. cholerae, V. fluvialis I and V. fluvialis II isolates were mostly strong. About 49% showed cytotoxic activity and 5% cytotonic activity in Chinese hamster ovary cell culture assay. Nevertheless, only three non-O1 V. cholerae (2.07%) and two V. parahaemolyticus isolates (1.65%) produced cholera toxin and thermostable direct haemolysin activity, respectively. Various toxigenic vibrios may be important food-borne pathogens in this region because of their high incidence in foods.     

Genotypes associated with virulence in environmental isolates of Vibrio cholerae

Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans. Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood. In this study, environmental isolates of V. cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR. Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes. The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V. cholerae isolates, respectively. The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V. cholerae O1 and V. cholerae O139 strains examined in this study. Three of four nontoxigenic V. cholerae O1 strains contained tcpA ET. Interestingly, among the isolates of V. cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene. The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V. cholerae O1, and in 14.3% of the O139 serogroups. Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V. cholerae O1 and O139 strains. Based on results of this study, surveillance of non-O1/non-O139 V. cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.     

Evaluation of a method for the serological identification of Vibrio cholerae non-01

The development of a method for serological identification of toxigenic and nontoxigenic V. cholerae non-O1, as well as on their role in human pathology, is reviewed. The evaluation on this method when used for establishing the etiology of acute diarrhea cases and analysis of sporadic diseases and group outbreaks of alimentary toxicoinfection type. Different points of view on the formation of toxigenic clones of V. cholerae non-O1 in nature is considered. The necessity for further development of the serological typing method is substantiated and the tasks of its improvement are set.     

Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae O1, O139, and non-O1 strains.

Enterobacterial repetitive intergenic consensus (ERIC) sequence polymorphism was studied in Vibrio Cholerae strains isolated before and after the cholera epidemic in Brazil (in 1991), along with epidemic strains from Peru, Mexico, and India, by PCR. A total of 17 fingerprint patterns (FPs) were detected in the V. cholerae strains examined; 96.7% of the toxigenic V. cholerae O1 strains and 100% of the O139 serogroup strains were found to belong to the same FP group comprising four fragments (FP1). The nontoxigenic V. cholerae O1 also yielded four fragments but constituted a different FP group (FP2). A total of 15 different patterns were observed among the V. cholerae non-O1 strains. Two patterns were observed most frequently for V. cholerae non-01 strains, 25% of which have FP3, with five fragments, and 16.7% of which have FP4, with two fragments. Three fragments, 1.75, 0.79, and 0.5 kb, were found to be common to both toxigenic and nontoxigenic V. cholerae O1 strains as well as to group FP3, containing V. cholerae non-O1 strains. Two fragments of group FP3, 1.3 and 1.0 kb, were present in FP1 and FP2 respectively. The 0.5-kb fragment was common to all strains and serogroups of V. cholerae analyzed. It is concluded from the results of this study, based on DNA FPs of environmental isolates, that it is possible to detect an emerging virulent strain in a cholera-endemic region. ERIC-PCR constitutes a powerful tool for determination of the virulence potential of V. cholerae O1 strains isolated in surveillance programs and for molecular epidemiological investigations.