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.     

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.     

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.     

Alteration of cholera toxin biosynthesis in Vibrio cholerae 01 as a result of temperate phage 139 integration into bacterial chromosome

Infection of V. cholerae 01 (classical and eltor biovars) cells with the temperate cholera phage 139 derived from V. cholerae serogroup 0139 followed by integration of the phage genome into the bacterial chromosome significantly increased the production of cholera toxin, the main virulence factor. The level of toxin biosynthesis in the lysogenic V. cholerae classical strain increased 3-fold and that in V. eltor thirty times in comparison with the parental strains. Increased production of cholera toxin was not associated with an increase in the number of copies of genes involved in its biosynthesis but seemed to be due to changes in toxinogenesis regulation.     

A 14-kilodalton inner membrane protein of Vibrio cholerae biotype e1 tor confers resistance to group IV choleraphage infection to classical vibrios.

Choleraphage phi 149 differentiates the two biotypes, classical and el tor, of Vibrio cholerae. This phage cannot replicate in V. cholerae biotype el tor cells because the concatemeric DNA intermediates produced are unstable and cannot be chased to mature phage DNA. A V. cholerae biotype el tor gene coding for a 14,000-Da inner membrane protein which destabilizes the concatemeric DNA intermediates by hindering their binding to the cell membrane has been identified. Presumably, a 22,000-Da V. cholerae biotype el tor protein might also have a role in conferring phage phi 149 resistance to cells belonging to the biotype el tor. A nucleotide sequence homologous to the 1.2-kb V. cholerae biotype el tor DNA coding for both the 14,000- and 22,000-Da proteins is present in all strains of classical vibrios but is not transcribed. The nucleotide sequence of the gene coding for the 14,000-Da protein has been determined.     

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.     

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.     

A comparative analysis of genomes of virulent and avirulent strains of Vibrio cholerae O139

A comparative analysis of the genome of V. cholerae O139 strains isolated in Russia's territory from patients with cholera and from the environment showed essential differences in their structures. The genome of clinical strains possessed all tested genes associated with virulence (ctxAB, zot, ace, rstC, rtxA, hap, toxR and toxT) and the at-tRS site for the CTXp phage DNA integration. As for the O139 V. cholerae chromosome strains isolated from water, 70% of the studied genes (ctxAB, zot, ace, rstC, tcpA, and toxT) and the attRS sequence were not detected in them. A lack of the key virulence genes in O139-serogroup "water" vibrios, including genes of toxin-coregulated adhesion pili. (that are receptors for the CTXp phage), and of the attachment site of the above phage are indicative of that the O139 V. cholerae strains isolated from open water sources located in different Russia's regions are epidemically negligible.     

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.     

Phage typing of cholera vibrios with different sets of phages]

A comparative study was made of two sets of Mukerjee and Drozhevkina-Arutyunova's bacteriophages in typing 514 strains of the El Tor vibrios and 45 strains of clasic biotype. It was shown that the Mukerjee or Drozhevkina-Arutyunova's phages could be used for the typing of cholera vibrios. The phages of the latter set prove to detect more phage types (18 against 11); they determine both the phage type and the biotype at the same time. The typing of cholera vibrios of both biotypes is possible, and the percentage of nontyping strains left is comparatively low (5.2 against 23.5 after Mukerjee). A table of the phage correspondence was made; it permits to obtain comparable data in using any set of the typing phages.     

Determination of the degree of similarity of cholera and NAG vibrios

The numeric taxonomy taking into account 80 signs has demonstrated the similarity of NAG and cholera vibrios, the average similarity coefficient exceeding 80 %. Among NAG vibrios, 53 % of the strains have been found to deviate from the central strain of V. cholerae mainly with regard to their utilization of the sources of carbon. The use of the citrate sign for the study of the biological properties of NAG vibrios is proposed.     

Lipopolysaccharides of Vibrio cholerae: III. Biological functions

This review presents the salient features of the biological functions including the (i) endotoxic activities, (ii) antigenic properties, (iii) immunological responses to and (iv) phage receptor activities of the Vibrio cholerae lipopolysaccharides (LPS). The biological functions of the capsular polysaccharide (CPS) of V. cholerae have also been discussed briefly as a relevant topic. The roles of LPS and other extracellular polysaccharides in the (i) intestinal adherence and virulence of the vibrios and (ii) the biofilm formation by the organisms have been analysed on the basis of the available data. Every effort has been made to bring out, wherever applicable, the lacunae in our knowledge. The need for the continuous serogroup surveillance and monitoring of the environmental waters and the role of LPS in the designing of newer cholera vaccines has been discussed briefly in conclusion.     

Cholera and NAG vibrio utilization of different C-containing substrates in Hiss media and a medium with a limited mineral content

A total of 55 V. cholerae strains and 175 NAG vibrio strains were studied with a view to establish their capacity for utilizing citrate in Simmons citrate agar or for growing in it in the absence of the source of carbon. The strains were divided into 3 groups, each containing approximately an equal number of cholera and NAG vibrios irrespective of their origin or serovars. None of 50 signs used in this investigation permitted the reliable differentiation of the cholera and NAG vibrio groups due to considerable differences between the strains within each group. The use of Hiss medium with starch instead of Kodam medium is proposed for the determination of the diastatic activity of cholera and NAG vibrios.     

Vibrio cholerae temperate phage O139: characteristics and role in changing expression of chromosomal virulence genes

Restriction analysis of temperate cholera phage 139 isolated from Vibrio cholerae P16064, serogroup 0139, showed its DNA to be double-stranded linear with cohesive terminals. DNA-DNA hybridization on nylon membranes revealed that many V. cholerae strains of serogroup 0139 isolated in different regions contained a temperate cholera phage 139 in their genomes. Southern blot hybridization of chromosomal DNA PST-fragments with phage probe showed that the temperate phage 139 was identical to the temperate phage of serogroup II V. eltor. The phage integrated in the chromosome near genes encoding motility (mot) and production of the capsule (cap) and purine (pur). Phage genome is apparently responsible for instability of cap, pur, and mot genes whose products are important for the development of an infectious process in cholera.     

Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor

A method that stimulates cholera toxin (CT) production by Vibrio cholerae O1 biotype El Tor (El Tor vibrios) to the level of several micrograms per ml in the culture fluid was established. Such a large amount of CT was obtained by the following method: El Tor vibrios were cultured in AKI medium (1.5% Bacto peptone, 0.4% yeast extract-Difco, 0.5% NaCl, 0.3% NaHCO3) at 37 C for 4 hr in a stationary test tube and then for 16 hr in a shaken flask, with inoculum sizes of 10(5) to 10(7)/ml. With this method, 35 strains out of 60 examined produced 2 to 16 micrograms/ml of CT as determined by the reversed passive latex agglutination test (RPLA). Thirty-three randomly selected strains out of the 60 produced reasonable amounts of rabbit skin vascular permeability factor, reflecting the amount of CT titrated with RPLA.     

Role of moderate bacteriophage 139 in change in production of cholera toxin in a classic strain of Vibrio cholerae

New data were obtained concerning cell sensitivity of pathogenic strains of cholera vibrions, which belong to the serogroup O1 of classical biovar, to the temperate bacteriophage K139, the native host of which is Vibrio cholerae O139. Molecular-genetic and biochemical studies showed that phage 139 integrated into the chromosome of strains V. cholerae O1 can change their toxigenic properties. A change in the production of cholera toxin (CT) in lysogens is associated both with an increase in the activity of the toxR regulatory gene and with a distortion of the structure of a chromosomal DNA region that contains a copy of the operon ctxAB encoding CT biosynthesis.     

Prevalence of type III secretion system genes in cholera vibrios from different serogroups

Prevalence of vcs genes coding the type III secretion system (T3SS) in cholera vibrios of different serogroups isolated in Russia and neighboring countries was studied for the first time. Virulent strains of O1 and O139 serogroups as well as toxigenic Vibrio cholerae strains of other serogroups contained no T3SS genes. Unlike mentioned strains, 29.2% of atoxigenic non O1/non O139 cholera vibrios isolated from patients in Russia and neighboring countries contained the T3SS genes cluster, which might contribute to the pathogenic properties of these strains.     

Antibacterial susceptibility/resistance of Vibrio cholerae eltor clinical strains isolated in the Caucasus during the seventh cholera pandemic

The data on antibacterial susceptibility and resistance of Vibrio cholerae eltor phenotypes with different sets of the susceptibility or resistance markers conditioning the outbreaks and sporadic cases of cholera in the Caucasus within 1970-1998 are presented. An increase of the number of the Vibrio cholerae phenotypes resistant to tetracycline and chloramphenicol usually used in the treatment of cholera was recorded in 1990-1994 vs. 1970-1989. The El Tor cholera vibrios stored on synthetic media lost some of their resistance markers, therefore the retrospective investigation of the antibioticograms was only of approximate prognostic value in the choice of the drugs for the etiotropic treatment of cholera in view of possible outbreak of the disease.     

Use of a new method for restoring the type properties of atypical Vibrio cholerae

Subcultures with a number of signs characteristic of epidemically significant strains have been isolated from cholera vibrios, nonpathogenic and atypical in a number of properties, by a new in vitro method developed by the authors. This method makes it possible to increase the virulence of poorly agglutinating cultures of V. cholerae O1 and their agglutinability with cholera antisera.