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.     

Lecithinase activity of Vibrio cholerae and of vibrios not agglutinated by O-cholera serum

The lecithinase activities of 187 V. cholerae strains differing in their virulence and 70 cultures of NAG vibrios of different origin varied in all experimental groups. The level of lecithinase activity in the groups of V. cholerae strains having low virulence, or no virulence at all, was considerably higher than in virulent strains. NAG vibrios isolated from diarrhea patients and from samples of lake and pond water did not differ in the activity of this enzyme.     

The fatty acid composition of the total lipids in Vibrio cholerae]

The fatty acid composition of total cell lipids of V. albensis typing strains, serovars 01-012, isolated from patients and environmental objects, as well as V. eltor and NAG vibrios, has been studied. Fatty acids contain 14-18 carbon atoms and are mainly represented by unsaturated monoacids. Palmitoleic and oleic acids constitute the greater part of unsaturated acids (their total content is 55.2-71.0%). The level of saturated fatty acids in considerably lower (27.0-43.1%), and these acids are mainly represented by palmitic acid, its maximum level being 28.4%. The similarity of the fatty acid profiles of the lipids common to the strains under study may indicate that these strains are phylogenetically related.     

Neuraminidase activity of NAG vibrios and of some representatives of the intestinal group of bacteria

It was studied 203 strains of NAG vibrios and 71 strains of different enterobacteria for the ability to produce neuraminidase. The most frequent neuraminidase producers were found among the strains isolated from humans (99 strain of 131). There was no correlation between neuraminidase production and other properties of the vibrios. The examined strains of the familyEnterobacteriaceae did not produce the enzyme.     

Factors in the spread of diseases caused by nonagglutinating vibrios studied by using the data from strain serotyping

The method of the serotyping of strains was used for the epidemiological evaluation of the role of different factors in the transfer of infective agents in 147 cases of diseases and carrier state, caused by NAG vibrios, in Karakalpakia. Out of 150 NAG vibrio strains, 136 strains were serotyped and classified with 26 serovars. The strains were found to belong mostly to serovars 47, 37, 5 and 6 (42.0%). Most of the infected persons (58.5%) used water from open water bodies for household purposes. The role of water factor in the spread of infection was confirmed by a wide spectrum of serologic variants, a low index of the focal outbreaks (1.02), and sporadic pattern of infection. No group morbidity or toxinfection-type outbreaks were recorded.     

Sensitivity of the vibrios to ultraviolet-radiation.

The ultraviolet-inactivation kinetics of a number of strains of Vibrio cholerae (classical), Vibrio cholerae (el tor), NAG vibrios and Vibrio parahaemolyticus were investigated. Statistical analyses revealed significant differences between any two of the four types of vibrio in respect to their sensitivity to U.V.     

Inferring the evolutionary history of vibrios by means of multilocus sequence analysis

We performed the first broad study aiming at the reconstruction of the evolutionary history of vibrios by means of multilocus sequence analysis of nine genes. Overall, 14 distinct clades were recognized using the SplitsTree decomposition method. Some of these clades may correspond to families, e.g., the clades Salinivibrio and Photobacteria, while other clades, e.g., Splendidus and Harveyi, correspond to genera. The common ancestor of all vibrios was estimated to have been present 600 million years ago. We can define species of vibrios as groups of strains that share >95% gene sequence similarity and >99.4% amino acid identity based on the eight protein-coding housekeeping genes. The gene sequence data were used to refine the standard online electronic taxonomic scheme for vibrios (http://www.taxvibrio.lncc.br).     

NAG infection of grass frog (Rana temporaria) tadpoles]

Rana temporaria tadpoles (2250) were contaminated with three types of NAG vibrio cultures. Clinical, bacteriological and morphological examinations showed the larvae to be suffering from an acute infection during the first 2 days after the contamination. Then the vibrios persisted in the tadpole organism for a long time and were excreted in the feces.     

Immunochemical Studies on Lipopolysaccharide from Agglutinable and Non-Agglutinable Vibrios

Lipopolysaccharide (LPS) prepared from Vibrio cholerae and a non-agglutinable (NAG; not agglutinable with O-group I serum according to Gardner and Venkatraman [13]) vibrio strain, isolated from a patient with cholera-like clinical symptoms, have been compared with respect to their chemical composition and immunological behavior. In addition to a significant difference in the chemical composition between the two lipopolysaccharides, the LPS from V. cholerae, unlike that from the NAG vibrio, requires prior treatment with alkali for it to be an effective antigen in the indirect hemagglutination test with sheep cells. It has been suggested that the alkali acts by removing excess O-acetyl group from LPS of agglutinable vibrios. LPS from the NAG vibrio consistently showed a lower antibody response in rabbits in terms of agglutinin and vibriocidal titer. Also, the class of agglutinin antibody elicited by LPS of the NAG vibrio was predominantly immunoglobin M, and that from V. cholerae was immunoglobulin G under comparable conditions.     

High surface hydrophobicity of hemagglutinatingVibrio cholerae and other vibrios

Surface hydrophobicity of hemagglutinatingVibrio cholerae, Vibrio parahaemolyticus, and NAG vibrios has been investigated. Most strains caused mannose-sensitive hemagglutination of monkey, guinea pig, chicken, and mannose-resistant hemagglutination of human erythrocytes with different degrees of hemagglutinating activity. Hemagglutinating strains adsorbed to a hydrophobic gel (Octyl Sepharose), whereas nonhemagglutinating strains failed to adsorb.Vibrio cholerae and other vibrios investigated seem to have pronounced surface hydrophobicity as estimated by Octyl Sepharose and they correspondingly autoaggregated into visible cell clumps in ammonium sulfate solution at low molarity (0.2–0.4 M). Nonhemagglutinating strains did not aggregate even at high (2 M) ammonium sulfate concentration. The presence of surface hemagglutinins of vibrios is growth-media-dependent. Strains, grown in four different liquid media, produced hemagglutinins and expressed pronounced surface hydrophobicity. Studies with electron microscopy revealed the presence of fimbriae on the vibrio cells. The number of fimbriae on the cells varied from strain to strain. Some strains possessed more than 300 fimbriae/cell whereas others had less than 10 fimbriae/cell. Vibrio hemagglutinins are easily detached from the cell surface by heating or sonication, and their cell surface hydrophobicity decreased simultaneously.     

In Vitro and In Vivo Studies of Streptomycin-Dependent Cholera Vibrios

Streptomycin-dependent cholera vibrio strains were derived from Inaba, Ogawa, and NAG vibrios by the method of Mel. These phenotypes grew more slowly and attacked fermentable substances after a longer period of time than the streptomycin-sensitive parent strains. Rabbits injected with streptomycin-sensitive strains and their streptomycin-dependent forms showed homologous agglutinin production. Patas monkeys fed with 10(9) streptomycin-dependent strains shed them for 1 to 2 days without ill effect, whereas the same number of streptomycin-independent organisms caused disease. The possibility of the application of multiple doses of streptomycin-dependent organisms in oral immunization against cholera was considered.     

The lipids of phosphorescent Vibrio albensis bacteria]

The lipid composition of fluorescent vibrios, V. eltor and nonagglutinating vibrios has been studied. In the fraction of polar lipids phosphatidylethanolamine, phosphatidylinositol and cardiolipin and in the fraction of neutral lipids monoglycerides, free fatty acids, diglycerides, triglycerides, sterol esters have been identified. The fatty acid composition of some classes of neutral lipids have been determined. Both similarity and differences between the strains under study in their lipid and fatty acid composition have been established.     

Effect of normal and specific immune sera on neuraminidase activity]

We have got evidence that there is no antigenic relationship reflecting the structural similarity between neuraminidases synthesized by noncholera vibrios and Arthrobacter nicotianae. The cross-reactions between the enzymes and heterological antisera were not observed. Antibodies against the A. nicotianae neuraminidase inhibited the activity of the enzyme for a glycomacropeptide of milk whey and for components of the blood serum, and had no effect no the neuraminidase from noncholera vibrios. Antibodies against the neuraminidase of noncholera vibrios inhibited only the activity of the homologous enzyme. Upon gel-filtration on Sephadex G-200 the antibodies inhibiting the activity of the enzymes under study were found in the fraction of 7S-gamma-globulins.     

The agglutination reactions of cholera vibrios.

The present study with 11 strains of vibrio using single-dose and hyperimmune antisera confirmed earlier observations on the cross-reactivity of the flagellar (H) agglutinating antigens of cholera and NAG vibrios. The effect of several variables on the agglutinating sensitivity of cell suspensions was determined by measuring the reaction rate in the presence of constant O- or H-antibody. The variables investigated were culture conditions, antigen dilution, reaction temperature, formalin fixation and heat-treatment; all were found to affect cholera and NAG serovars similarly. The optimal conditions for the O- and H-tests were markedly different. Dilute, young living broth cultures were highly sensitive to O- but not H-antibody. Conditions favoring the H-reaction were 48-hr culture on firm dry agar, high suspension opacity, a reaction temperature of 45 C and formalin fixation. The inverse relationship of O- and H-sensitivity under these conditions indicated that the flagellar antigen in the growing vibrio is masked by an O-sensitive layer. The temperature of denaturation of the unfixed H-antigen before or after reaction with antiserum was 64 +/- 0.5 C and could be used as a criterion of the H-reaction.     

Development of antilipase immunoglobulin polymer diagnosticum for the detection of Vibrio cholerae eltor, possessing hemolytic and lipase activity

The possibility of using a heterogeneous, but structurally similar antigen--the commercial preparation of Pseudomonas sp. lipase (Sigma, USA)--for the development of polymer diagnosticum aimed at determination of lipase production in cholera vibrios was shown. The new diagnosticum (antilipase antibodies) on a polymer carrier was used in the serological volume agglomeration test for the detection of hemolytic atoxigenic V. eltor, obtained from environmental, objects, which produced lipase in 80% of cases. The differentiating capacity of the diagnosticum was confirmed on 120 V. eltor cultures isolated from environmental objects. The newly developed diagnosticum makes it possible to determine the lipase activity in cholera vibrios of different biovars and serovars.     

Crystal structures of Urtica dioica agglutinin and its complex with tri-N-acetylchitotriose

Urtica dioica agglutinin is a small plant lectin that binds chitin. We purified the isolectin VI (UDA-VI) and crystal structures of the isolectin and its complex with tri-N-acetylchitotriose (NAG3) were determined by X-ray analysis. The UDA-VI consists of two domains analogous to hevein and the backbone folding of each domain is maintained by four disulfide bridges. The sequence similarity of the two domains is not high (42 %) but their backbone structures are well superimposed except some loop regions. The chitin binding sites are located on the molecular surface at both ends of the dumbbell-shape molecule. The crystal of the NAG3 complex contains two independent molecules forming a protein-sugar 2:2 complex. One NAG3 molecule is sandwiched between two independent UDA-VI molecules and the other sugar molecule is also sandwiched by one UDA-VI molecule and symmetry-related another one. The sugar binding site of N-terminal domain consists of three subsites accommodating NAG3 while two NAG residues are bound to the C-terminal domain. In each sugar-binding site, three aromatic amino acid residues and one serine residue participate to the NAG3 binding. The sugar rings bound to two subsites are stacked to the side-chain groups of tryptophan or histidine and a tyrosine residue is in face-to-face contact with an acetylamino group, to which the hydroxyl group of a serine residue is hydrogen-bonded. The third subsite of the N-terminal domain binds a NAG moiety with hydrogen bonds. The results suggest that the triad of aromatic amino acid residues is intrinsic in sugar binding of hevein-like domains.     

Investigation of populations heterogeneous according to O-antigen in Vibrio cholerae cultures

The O-antigenic composition of 36 cultures of Vibrio cholerae agglutinating simultaneously with 01 cholera sera and 0 sera to NAG vibrios of the Sakazaki collection was investigated. It has been established experimentally that under the effect of medium and environmental conditions such cultures dissociate to subcultures differing in their affiliation to different serological groups according to 0 antigen. The passage of these cultures in the organism of susceptible animals promotes preservation of 01-group clones whereas the passage in peptone water or prolonged storage under unfavourable conditions result in the predominance of clones of different serological affiliation. The proposition has been put forward that the observed vibrio cultures are genotypically capable of producing, besides the 01 group, a number of 0 antigens. Phenotypical manifestation of the antigenic structure in the respective individuals of the population depends on the conditions of the environment.     

Ecological relationship between Vibrio parahaemolyticus and agar-digesting vibrios as evidenced by bacteriophage susceptibility patterns.

Twenty bacteriophages active against Vibrio parahaemolyticus and agar-digesting vibrios, isolated from oysters (Crassostrea gigas) and Dungeness crab (Cancer magister) and by induction of a lysogenic agar digester, were tested as to their host range. These phages were specific for V. parahaemolyticus and various agar-digesting vibrios, and interspecies lysis occurred only between these two groups. V. alginolyticus, V. anguillarum and related species, V. cholerae, and a group of marine psychrophilic and psychrotrophic vibrios were not affected. No correlation was observed between the O and K serotypes of V. parahaemolyticus strains and bacteriophage susceptibility patterns, and 7 of 28 strains of V. parahaemolyticus were not lysed by any of the phages. Only two of the phage isolates were capable of lysing all susceptible V. parahaemolyticus strains. No correlation was observed between the inter-and intraspecies genetic relatedness (DNA homologies) of V. parahaemolyticus and agar-digesting vibrios and susceptibility patterns to different bacteriophages. Some of the phages were capable of plaque formation on V. parahaemolyticus as well as on some strains of agar-digesting vibrios that were separated by 70 to 80% differences in their DNA homologies. The possible ecological significance of these vibrio bacteriophages, particularly those having a wide host range, is discussed.     

DNA base composition and biochemical characters of Campylobacter strains

We studied the relationship between ten authentic Campylobacter fetus and two C. bubulus strains, and seventeen named vibrios of animal and human origin. All organisms fall within the genus Campylobacter, as defined. Their DNA base composition ranges from 29.8 to 35.9% (G+C). On the basis of similarity of differential biochemical tests and % (G+C), they can be divided into four closely similar groups.

1. C. fetus var. intestinalis and var. venerialis with 34.7 to 35.9% (G+C).
2. Vibrio fecalis with 32.0 to 32.8% (G+C).
3. C. bubulus with 30.1 and 30.6% (G+C).
4. The cluster V. jejuni, V. coli, Vibrio sp. from fowl, and related vibrios from man and sheep with 29.8 to 34.0% (G+C).

We propose that all the vibrios used here be included in the genus Campylobacter.     

Rapid Method of Determining Cholera Vibrio Biotype

The characteristic motility of cholera vibrios, as viewed through a dark-field microscope, and the adhesiveness of chicken cell-positive vibrios provide a means for rapidly identifying and biotyping cholera vibrios. Dilute suspensions of vibrios, such as one might find in a fresh rectal swab specimen from a cholera patient, when mixed with a 0.25% suspension of chicken erythrocytes in saline, can be used to biotype the cholera vibrios without prior isolation in pure culture. This is accomplished by using a dark-field microscope through which the chicken cell-positive cholera vibrios are observed to attach to the scattered erythrocytes and to propel them with a characteristic flipping motion.