Characterization of enteropathogenic Vibrio cholerae non O1/O139 isolated in Uzbekistan

The results of the serotyping of 244 V. cholerae non O1/O139 cultures isolated from patients in Uzbekistan in 2000 and 2001 are presented. All isolates were studied by the method of molecular probing and in the polymerase chain reaction for the presence of virulence genes and for sensitivity to phages ctx+, ctx- and hemolytic activity. The use of monoreceptor O-sera O2-O83 made it possible to determine vibrios of 32 serogroups with the dominating role in the etiology of acute enteric diseases belonging to serogroups O18, O62, O82, O37. Genes ctx AB were detected in none of the isolates, 5 of them contained gene tcp A. A group of cultures, sensitive to phage ctx+ and belonging mainly to enteropathogenic serogroups, was detected.     

Characterization of Vibrio cholerae O1 cultures isolated from environmental objects on the territory of the Russian Federation in 2002

The biological properties of 46 V. cholerae O1 eltor cultures isolated in 2002 from water environment on the territory of Russia are presented. All isolated vibrios proved to be typical in their cultural, morphological, biochemical and serological properties. The atypical character of some of them was mainly linked with their phage resistance. The appearance of vibrios, sensitive to bacteriophage ctx+ and containing gene tcp in the absence gene ctx, was noted. Multilocus VNTR typing made it possible to group the cultures under study in 34 genotypes. The presence of toxin coregulated pili was found to be directly related to locus VcB. The necessity of the systematic study of the pheno- and genotypes of the isolated cultures with the aim of epidemiological surveillance of this infection is emphasized.     

Hemolytic activity and toxigenicity of Vibrio cholerae of different serogroups

Experimental data on the comparative evaluation of the hemolytic activity of ctx+ Hly- and ctx- Hly+ V. cholerae, serogroups O1 and O139, in the process of their cultivation in different nutrient media are presented. The capacity of ctx+ V. cholerae of both serogroups cultivated under the conditions of iron deficiency, for the production of hemolysin capable of lyzing sheep red blood cells was shown. Hemolysin produced by ctx- strains of V. cholerae was synthesized under any conditions. The study of hemolysin preparations obtained from ctx- and ctx+ strains of V. cholerae, serogroups O1 and O139, revealed that they were biologically and immunologically similar.     

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.     

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.     

Selective activation of human intestinal mast cells by Escherichia coli hemolysin

Mast cells (MCs) are recognized to play an important role in bacterial host defense in the murine system. In this study, we studied the interaction of human MCs, isolated from the intestine and purified to homogeneity, with different Escherichia coli and Shigella flexneri strains. We show that alpha-hemolysin (Hly)-producing E. coli strains induce the release of histamine, leukotrienes, and proinflammatory cytokines in intestinal MCs. In contrast, MCs were virtually unresponsive to S. flexneri and several Hly-negative E. coli strains, including the isogenic Hly-deficient mutants of Hly(+) strains. Hly(+) E. coli but not Hly(-) E. coli caused an increase in intracellular Ca(2+) levels. Blocking of extracellular Ca(2+) and of the calmodulin/calcineurin pathway by cyclosporin A inhibited the response to Hly(+) E. coli. Furthermore, inhibition of MAPKs p38 and ERK reduces activation of MCs by Hly(+) E. coli. In addition, using an ex vivo system, we directly record the histamine release by MCs located in the lamina propria after infection with Hly(+) E. coli. Our data indicate that human intestinal mast cells interact with selected Gram-negative bacteria, establish E. coli Hly as a factor regulating MC effector functions, and argue further for a role of human MCs in innate immunity.     

Evaluation of a RAPD-based typing scheme in a molecular epidemiology study of Vibrio cholerae O1, Brazil

AIMS: To evaluate the utility of random amplification of polymorphic DNA (RAPD) technique for routine practice in public health laboratories for epidemiological studies of Vibrio cholerae O1 isolates. MATERIALS AND RESULTS: Seventy-nine strains were examined by PCR for the toxin genes (ctx A, zot and ace), virulence-associated genes (tcp A and tox T) and RAPD sequences. Except for one strain (no. 1123) from the Amazonas State, all the strains analysed carried the genes ctx A, zot, ace, tcp A and tox T. RAPD fingerprinting revealed variability but no correlation with serotype, biotype or geographical origin of the isolates was found. CONCLUSION: A standardized RAPD method does not enable the establishment of a pattern data bank for the identification of V. cholerae O1 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The simplicity and discriminative capacity of this technique make it useful for detecting genetic diversity among micro-organisms from a defined group or for outbreak investigation.     

Phenotypic and genetic features of cultural-morphologic variants of Bacillus anthracis

Comparative analysis of MVLA-genotypes of 6 Bacillus anthracis strains and 40 their variants differing on capsule- and toxin synthesis, hemolytic, proteolytic and lecitinase activity, nutritional requirements, susceptibility to anthrax bacteriophages, virulence, immunogenicity, and presence of genes for capsule and toxin synthesis was performed. Results of phylogenetic analysis of 5 chromosome locuses and plasmid locus pXO1aat which are variable for this sample of B. anthracis cultures showed that all strains divided on 2 main clusters - A and B. Cluster A consisted of 5 genotypes whereas cluster B - of 1 genotype. All highly virulent original strains and variants with characteristic phenotype Cap(CO2)(+)(O2)(-)Tox(+)ProtA(+)Hly(+) Lec(-)Trp(+) had identical genotype in 4 groups and in 5th group differences were present only in vrrA locus. All original strains and variants with the most atypical complex of phenotypic characteristics Cap (CO2)(+)(O2)(+)Tox(-)ProtA(-)Hly(-)Lec(-)Trp(-) also had the same genotype belonging to cluster B and diverged on characteristic of 5 chromosomal VNTR locuses and pXO1aat locus from typical strains. Absence of toxin production in vitro was not related to loss of genetic determinants of toxin components. Cultures with typical characteristics, one of which was ability to produce toxin in vitro, had larger sizes of amplicons of pXO1aat locus (135 and 132 nbp), whereas atoxigenic original strains and variants with complex of atypical characteristics and identical chromosome genotype had the smallest sizes (123 bnp). All original cultures were isolated in Russia, their genotypes are described for the first time.     

PCR-detection of markers for epidemiologically-significant strains of Vibrio cholerae 0139, isolated in Siberia]

Fourteen V. cholerae 0139 strains were isolated in 1996-1999 in Siberia from the Ob river (Novosibirsk) and bogs and lakes (Irkutsk). The strains were tested in PCR for the key virulence determinants (ctx AB, tcp, acf). The genomes lacked these elements, and therefore the strains were acknowledged avirulent. The results correlate completely with the data of phenotypical analysis, characterizing the pathogenic characteristics of isolated strains.     

Effect of pH on the binding of Vicia graminea lectin to erythrocytes. Dependence on the chemical character of red-cell receptors

Binding of the radioactive Vicia graminea lectin to human blood-group M and N erythrocytes and to horse erythrocytes was studied at pH 6-10. Binding of the lectin to untreated human erythrocytes and to those treated with Vibrio cholerae neuraminidase increased severalfold from pH 6 to pH 8 and was maintained at the maximal level up to pH 9/9.5. On the other hand, interaction of V. graminea lectin with native or desialylated horse erythrocytes was not significantly affected by pH and small differences in the binding were opposite to those found with human erythrocytes: the binding decreased when pH increased from 6 to 9.5. Binding of the lectin to all erythrocytes tested at pH 10 was lowered to about 80% of the maximal values. The differences in pH dependence of V. graminea lectin binding to human and horse erythrocytes most probably resulted from the presence of amino groups in human red-cell receptors and their absence from receptors of horse erythrocytes. The earlier data on the enhancing effect of amino group modification on the interaction of human red-cell glycopeptides with V. graminea lectin support the conclusion that an increase in the lectin binding to human erythrocytes at pH 6-8 is confined to the decreased protonization of the receptor amino groups. V. graminea lectin was irreversibly inactivated at pH 3 and was inactivated by EDTA at pH 7.4 and reactivated by Ca2+ or Mn2+. This suggested that the lectin is a metaloprotein, requiring bivalent cations for the full binding activity. Some quantitative differences between the binding properties of V. graminea lectin, prepared from different batches of seeds, are reported.     

The binding of fluorescein-labelled stapbylococcal alpha toxoid to erythrocytes

Erythrocytes of different animal species have variable hemolytic sensitivity to staphylococcal alpha toxin. Specific and non-specific binding of toxin was measured using fluorescein-labelled toxoid. These studies indicate that toxoid binding to erythrocytes increases with concentration for all species tested. Scatchard plot analyses of 35 animals representing seven species indicate that rabbit, pig, cow, and chicken erythrocytes possess 125 980, 103 920, 82 500, and 41 200 receptors per cell, respectively. The number of receptors remains constant over a period of at least 10 days. No detectable receptors were found for human, rat, and guinea pig erythrocytes. A correlation coefficient of 0.992 exists between receptor number and hemolytic sensitivity for those species having receptors. Variation in hemolytic sensitivity is governed by receptor number and not by variation in the dissociation constant. A threshold sensitivity of 37 000 receptors per cell has been calculated. Since species lacking detectable receptors have considerable sensitivity to hemolysis, it is proposed that two binding mechanisms, specific and non-specific, exist which prepare erythrocytes for destruction.     

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.     

Isolation and characterization of an N-acetylgalactosamine specific lectin from Salvia sclarea seeds

Crude extracts from Salvia sclarea seeds were known to contain a lectin which specifically agglutinates Tn erythrocytes (Bird, G. W. G., and Wingham, G. (1974) Vox Sang. 26, 163-166). We have purified the lectin to homogeneity by ion-exchange chromatography and affinity chromatography. The agglutinin was found to be a glycoprotein of Mr = 50,000, composed of two identical subunits of Mr = 35,000 linked together by disulfide bonds. The purified lectin agglutinates specifically Tn erythrocytes and, at higher concentrations, also Cad erythrocytes. Native A, B, or O red blood cells are not agglutinated by the lectin and, even after treatment with sialidase or papain, these cells are not recognized. Tn red cells present 1.45 X 10(6) accessible sites to the lectin which binds to these erythrocytes with an association constant of 1.8 X 10(6) M-1. On Cad red cells, 1.73 X 10(6) sites are accessible to the lectin which binds with an association constant of 1.0 X 10(6) M-1. The carbohydrate specificity of the S. sclarea lectin has been determined in detail, using well defined monosaccharide, oligosaccharide, and glycopeptide structures. The lectin was found to be specific for terminal N-acetylgalactosamine (GalNAc) residues. It binds preferentially alpha GalNAc determinants either linked to Ser or Thr (as in Tn structures) or linked in 1-3 to a beta GalNAc or to an unsubstituted beta Gal. Although more weakly, the lectin binds beta GalNAc residues linked in 1-4 to a beta Gal (as in Cad structures). It does not recognize beta GalNAc determinants linked in 1-3 to a Gal (as in globoside) or the alpha GalNAc residues of blood group A structures.     

alpha-Hemolysin of E. coli]

The activity of alpha-hemolysin increased at the log growth phase in the culture of E. coli P678 Hly+ hemolytic strain; this activity diminished with the change into the stationary phase, and then fell sharply. Replacement of the culture medium in the stationary growth phase by fresh one led to restoration of the hemolytic activity of the culture. The culture fluid separated from the cells at the stationary growth phase produced an inhibitory action on the alpha-hemolysin Ca ions activated and stabilized the alpha-hemolysin. Sodium citrate and sucrose served as hemolysis inhibitors. The action of alpha-hemolysin was maximal against human erythrocytes at pH 6.5. Hemolytic activity was characterized in time by a distinct lag-phase and the phase of the greatest rate of reaction. The duration of the lag-phase and also the rate of hemolysis depended on the concentration of alpha-hemolysin (with the increase of the hemolysin concentration lag-phase was shortened and the reaction was accelerated). There proved to be a linear relationship between the amount of erythrocytes taken into the reaction and the rate of hemoglobin release, and also there was noted a temperature activation of the hemolytic reaction.     

Hemolysis determinant common to Escherichia coli hemolytic plasmids of different incompatibility groups

By using cloned deoxyribonucleic acid fragments from the hemolysis determinant of the hemolytic plasmid pHly152 as hybridization probes, a deoxyribonucleic acid segment of about 3.8 megadaltons was identified as a common sequence in several hemolytic (Hly) plasmids of Escherichia coli belonging in four different incompatibility groups. This segment contained the genetic information for the synthesis and secretion of the extracellular toxin alpha-hemolysin of E. coli. With the exception of pSU5, representing a composite plasmid, one part of which seems to be very similar to pHly152, the overall sequence homology of these Hly plasmids with pHly152 seems to be rather restricted. However, the Hly plasmid pSU316 showed sequence homology with pHly152 that did not extend beyond the hemolysis determinant. The two other plasmids, pSU233 and pSU105, also shared homology with pHly152 in the hemolysis determinant as well as in various other parts of this plasmid which did not seem to be directly linked to the hemolysis determinant. This suggests that the hemolysis determinant has spread to presumably unrelated plasmids of E. coli.     

Variable nucleotide tandem repeats (VNTR analysis) in Vibrio cholerae 0139 isolated from humans and from water of surface reservoirs in Russia

The comparative study of variable tandem repeats (VNTR analysis) in genomes of V. cholerae 0139 isolated from humans and from water samples taken from surface reservoirs was carried out. The results of the study of the allele state of 5 loci of tandem repeats in 50 strains of vibrios, carried out in the double-primer polymerase chain reaction (PCR), as well as the earlier comparison of the same isolates in the single-primer PCR, showed essential differences and the absence of clonality in the cultures of the clinical and aqueous origin. The suggestion was made that vibrios with individual VNTR genotypes and having no genes ctx and tcpA, isolated from water samples, were epidemic unimportant representatives of the autochthonous microflora of water reservoirs.     

Vibrio cholerae hemolytic activity

Mechanisms of realization of Vibrio cholerae hemolytic activitywere analyzed using summarized own results and data from the literature. It has been shown that lectin receptor, which coded by hlyA gene, participates in lysis of sheep erythrocytes, but not of rabbit erythrocytes, as well as interact with D-galactose with selectivity to 3 anomers. Lectin nature of HlyA can determine formation of its complexes with lypopolysaccharides (LPS) and enzymes, which promote realization of hemolysis (by lipase, lecitinase, neuraminidase). It has been determined that lipase activity correlates with hemolytic activity of nonepidemic variants of V. cholerae. Lipase is considered as the enzyme marker of sheep erythrocytes hemolysis. It is assumed that LPS and lipase play shaperon-like role during interaction of HlyA with lipids, which promote denaturation of hemolytic active monomer in hemagglutinating oligomer.     

Primary structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata and its cDNA: structural similarity to the B-chain from plant lectin, ricin

CEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc) specific lectin purified from a marine invertebrate Cucumaria echinata has a strong hemolytic activity especially toward human and rabbit erythrocytes. We determined the primary structure of the CEL-III by examining the amino acid sequences of the protein and the nucleotide sequence of the cDNA. The cDNA encoding CEL-III has 1823 nucleotides and an open reading frame of 1296 nucleotides. CEL-III is composed of 432 amino acid residues with a M(r) of 47? omitted?457 and has six internal tandem repeats, each with of 40-50 amino acids, comprising the N-terminal two-thirds of the molecule. Similar repeats are found in the B-chains of cytotoxic plant lectins, such as ricin and abrin, where six repetitive sequences extend throughout the molecules. A hydropathy plot predicts hydrophobic segments in the C-terminal region of CEL-III. These findings suggest that the N-terminal region of CEL-III plays an important role in binding to carbohydrate receptors on the target cell membranes, an event which triggers an intermolecular hydrophobic interaction of the C-terminal region, the result being oligomerization of CEL-III to lead to pore-formation in erythrocyte membrane.     

Active and inactive forms of hemolysin (HlyA) from Escherichia coli

The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W. & Hedgpeth, J. (1982) J. Bacteriol. 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle. However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase. External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication. The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished. On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA. Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes. Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein. An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product. This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase.