Long-term survival of E1 Tor cholera vibrios in naturally contaminated sewage]

El Tor cholera vibrios of Ogava serological type were revealed in the sewage of the locomotive shed for 15 months. In experiment with an oil catcher in naturally infected sewage El Tor vibrios survived 36 days, in storage of this sewage at the laboratory--39 days, in the artificially infected sewage of a settlement and of a milk plant--2 and 11 days, respectively, in the oil and disel fuel--14 months. Consequently, El Tor vibrio can survive in the sewage with a high oil product content for a long time.     

Lipopolysaccharides of R mutants isolated from Vibria cholerae.

The chemical and serological properties of lipopolysaccharides isolated from the S form and from the R form of cholera vibrios were compared. It was found that the S-R mutation of cholera vibrios involves total elimination of the two component amino sugars of S-form lipopolysaccharides, i.e. quinovosamine and perosamine. This elimination resulted in the loss of O-specificity of S-form lipopolysaccharides and concomitant appearance of strong serological cross-reactivity, in the passive-haemolysis-inhibition test, among R-form lipopolysaccharides regardless of the serotypes (Inaba and Ogawa) of their S parent strains.     

Phenotypic and genetic characterization of Vibrio cholerae O1 clinical isolates collected through national antimicrobial resistance surveillance network in Nepal

Cholera occurs in sporadic cases and outbreaks in Nepal each year. Vibrio cholerae O1 (n = 522) isolated during 2007-2010 from diarrheal patients at 10 different hospital laboratories in Nepal were characterized. Biochemical and serologic identifications showed that all the isolates belonged to serogroup O1, El Tor biotype. Except 72 isolates of Inaba serotype isolated in the year 2007, all the remaining isolates were of Ogawa serotype. All isolates were resistant to nalidixic acid and furazolidone. Resistance to tetracycline, ciprofloxacin, erythromycin and co-trimoxazole were 21, 4, 16 and 90 % respectively. Seventy-seven of these isolates were selected for further characterization for ctxB gene and MLVA typing. Two different variants of classical type cholera toxin were observed. Ogawa strains from 2007 and 2010-Western Nepal outbreak harbored CTX-3 type cholera toxin, whereas Inaba serotypes in 2007 and the remaining Ogawa serotypes in 2008-2010 harbored CTX 3b-type toxin. MLVA analysis showed circulation of four different groups of altered V. cholerae O1 El Tor strains. Two different profiles were seen among 2007 Inaba (9, 3, 6, x, x) and Ogawa (10, 7, 6, x, x) isolates. The MLVA profile of 2008 and 2009 Ogawa isolates were similar to those of Inaba strains of 2007. Isolates from 2010 also showed three different MLVA profiles; profile 9, 3, 6, x, x in 3 isolates, 11, 7, 6, x, x among 2010 Western Nepal outbreak strains and profile 8, 3, 6, x, x among isolates from Butwal and Kathmandu.     

The isolation of Vibrio fluvialis on the territory of the USSR]

For the first time V. fluvialis strains were detected on the territory of the USSR. The taxonomic position of these vibrios was determined by their nucleotide DNA composition (the content of guanine + cytosine was 49.3-51.0 mole%) and the characteristic features of their phenotype. The individual features of the strains consisted in their capacity for agglutination with cholera antisera, groups 01 and Inaba, in diagnostic dilutions in the presence of differences in genomes and phenotypes with cholera vibrios. Molecular hybridization DNA-DNA also gave no confirmation of their relationship to cholera vibrios (23-26% homology). The comparative study of V. fluvialis strains from the USSR and other countries by a broader set of their phenotypical signs confirmed their identity.     

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.     

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.     

A method for studies of an El Tor-associated antigen of Vibrio cholerae O1

A method for studying the biotype El Tor associated mannose-sensitive haemagglutinin (MSHA) of V. cholerae O1 has been developed. By using crude MSHA adsorbed to chicken erythrocytes as solid phase antigen in an enzyme-linked immunosorbent assay (ELISA), antisera against V. cholerae of the El Tor biotype reacted in high titre with the MSHA-coated cells, whereas antisera against vibrios of the classical biotype did not bind significantly, i.e. in higher titre than pre-immune sera. The binding of anti-MSHA serum, or a monoclonal antibody against MSHA, to the MSHA-coated erythrocytes could be efficiently inhibited by crude MSHA as well as by El Tor vibrios whereas neither V. cholerae lipopolysaccharide nor different strains of classical vibrios had any inhibitory effect. These results support the existence of an El Tor-associated immunogen. They also suggest a possibility of determining antibodies against different haemagglutinins in ELISA without having access to purified antigens.     

Monoclonal antibodies to antigens of cholera vibrios of the Ogava serovar

A set of hybrids is obtained synthesizing monoclonal antibodies to the surface antigenic determinants of the choleric vibrio of the Ogava serovar. The antigenic structure of vibrios of the typical and atypical strains isolated from a man and from environmental objects is studied using a collection of highly specific immunoglobulins. The high-specific diagnostic preparations for identification of the 01-group cholera agent serovar may be created on their base.     

Expression and detection of different biotype-associated cell-bound haemagglutinins of Vibrio cholerae O1

The expression of two cell-bound haemagglutinins, one sensitive to L-fucose (FSHA) and the other to D-mannose (MSHA), on Vibrio cholerae O1 strains of both the classical and the El Tor biotypes was studied by (i) agglutination of chicken and human group O erythrocytes in the presence of L-fucose or D-mannose, (ii) binding of the bacteria to L-fucose- and D-mannose-coated agarose beads, and (iii) agglutination of the bacteria by 'biotype-specific' antisera. All of the 12 classical strains studied that were isolated before 1979 gave FSHA of human O erythrocytes whereas only 6 of 17 classical strains isolated during recent epidemics expressed FSHA; a few of the classical strains expressed MSHA in addition to FSHA. All the El Tor strains gave MSHA of chicken erythrocytes and one strain also expressed FSHA. Both the cell-bound HAs were optimally expressed during the exponential phase of growth; FSHA markedly decreased during the late exponential phase while the MSHA usually persisted into the stationary phase. The expression of FSHA and MSHA correlated very well with the direct binding of vibrios to fucose- and mannose-coated agarose beads, respectively. Antiserum 'specific' for classical vibrios agglutinated classical strains expressing FSHA and also the El Tor strain exhibiting FSHA. Similarly, the anti-El Tor serum agglutinated all El Tor strains and also classical strains expressing MSHA, suggesting that the 'biotype-specific' sera were specific for the biotype-associated cell-bound HAs.     

The composition of a population of cloned cultures of revertant Vibrio cholerae L forms

The process of L-transformation and L-transformed state duration have been studied for their effect on variability of main characters of revertant cultures of choleric vibrions L-forms at the population level with the use of cloned cultures of the choleric vibrions. The study was conducted on two strains of the choleric vibrion of the eltor biovar in different periods of storage in the L-transformed state (1, 3, 6 months). It has been revealed that characters of the species and biovar remained stable despite the influence of L-transforming agents. The characters of clone cultures characterizing virulence (sensitivity to KhDF phages, hemolytic activity, toxin production and virulence for sucking rabbits proved to be subjected to variability to the greatest extent with simultaneous preservation of the toxin-production gene. A resistant change of the serovar (from Inaba to Ogava) is observed only in one revertant-subculture of the virulent strain.     

Electron microscopy of cholera vibrios from the intestinal contents of infected suckling rabbits]

Electron microscopic study of the cells of classic, El Tor and NAG-vibrios showed them to be no different by the structure type from the cells of ofter Gram-negative bacteria. A characteristic peculiarity of the cholera vibrios revealed after their passage through the intestine of nursling rabbits was the presence of microcapsules and protrusions of the areas of the wall membranous apparatus.     

The lysogeny of Vibrio parahaemolyticus of serovar O4:K12]

Lysogeny has been first established in strains of parahemolytic vibrios of serovar O4:K12. Moderate phages belonged to morphological group IV by home A. S. Tikhonenko's classification and were presented by one serological type. No correlation has been revealed between sensitivity to moderate phages of parahemolytic vibrios and specificity of "O"- or "K"-serotypes.     

Effect of sulfide water from natural springs on the properties of Vibrino cholerae

The authors studied the properties of E1 Tor cholera vibrios isolated from sulfide water of the natural sources. There was demonstrated under experimental and natural conditions the influence of ecological conditions of sulfide water on such vibrio properties as cholerogenicity, sensitivity to diagnostic and typing phages, hemolytic activity and the value of the hemolysin-destructive factor. A short-liver stay of cholera vibrios in sulfide water was accompanied by some reduction of their virulence.     

Enteropathogenicity of hemolysing El Tor Vibrios

The authors studied the enteropathogenic properties of 11 strains of hemolysing E1 Tor vibrios, of which 8 in enteric administration to suckling rabbits caused no death of the animals, and 3 caused the animal death with the phenomena of diarrhea, but without any typical cholerogenicity syndrome. In case of administration with mucine the pathogenic properties were revealed in 6 strains more. Use of strains grown on media with starch for the infection led, in individual cases, to the manifestation of enteropathogenic properties. Consequently, the strains of hemolysing E1 for vibrios under study should be regarded as weakly virulent, and some--as avirulent ones.     

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.     

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.     

Isolation and molecular characterization of genetically diverse antagonistic, diazotrophic red-pigmented vibrios from different mangrove rhizospheres

Genetic diversity of red-pigmented vibrios from different mangrove rhizospheres ( Porteresia coarctata, Avicennia marina and Rhizophora mucronata ) collected from Pichavaram mangrove, India was investigated. Twenty red–pink pigmented strains were isolated, 16S rRNA gene analyses indicted that these isolates belong to the genus Vibrio and were phylogenetically closely related to the type strains of Vibrio rhizosphaerae and Vibrio ruber . The rep-PCR analysis using GTG₅ and BOX primers had similar groupings, and segregated these pigmented Vibrio isolates including two type strains into seven unique genotypic groups (REP groups A1–A7). The rhizosphere of P. coarctata harbors highly genetically diverse groups of red-pigmented vibrios compared to other plants. Multilocus sequence analysis using four genetic loci ( pyrH, recA, rpoA , 16S rRNA) clearly supported the hypothesis that strains MSSRF38 (REP group A5) and MSSRF39 (REP group A6) could represent new Vibrio species. Biological functions of these vibrios were also determined and it was found that all these isolates have antagonistic activity against phytopathogens, and isolates belonging to REP groups A5 and A6 were positive for nifH gene by PCR. In conclusion, this study for the first time demonstrates the occurrence of genetically diverse groups of antagonistic, diazotrophic red-pigmented vibrios from different mangrove plants and suggests a new ecological role for vibrios as heterotrophic plant associated rhizobacteria.     

Monoclonal antibodies against Vibrio anguillarum O2 and Vibrio ordalii identify antigenic differences in lipopolysaccharide O-antigens

Abstract Monoclonal antibodies (mAbs) that recognize distinct species-specific antigenic epitopes in O-antigens from Vibrio anguillarum O2, O2a and certain O2b strains (mAb 7B4) and from Vibrio ordalii strains (mAbs A16 and 7D11) were generated. Western immunoblot analysis using these mAbs revealed that vibrio strains grown in the presence of fresh rainbow trout blood expressed lipopolysaccharide (LPS) with longer (high molecular mass) O-antigens and extracellular capsular layers when compared to strains grown without rainbow trout blood. We also generated mAbs that react with O-antigens from V. anguillarum serotype O1 (mAbs 7B8, 7B5 and 1C3) and serotype O3 (mAbs 13A1 and 14C5) strains. These mAbs provide rapid and accurate diagnostic reagents for serological differentiation of V. ordalii from serotype O2 strains of V. anguillarum , and for serotyping of these pathogenic vibrios.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> isolates from cholera outbreaks in north India

Vibrio cholerae isolates recovered from cholera outbreaks in Bhind district of Madhya Pradesh and Delhi, Northern India were characterized. The O1 serogroup isolates from Bhind outbreak were of Inaba serotype whereas both Ogawa and Inaba serotypes were recovered from Delhi. PCR analysis revealed that only O1 serogroup V. cholerae isolates carried the virulence-associated genes like ctxA, tcpA, ace, and zot. Molecular typing by repetitive sequence based ERIC, VCR1, and VC1 PCR’s revealed similar DNA profile for both Inaba and Ogawa serotypes. A discrete VC1-PCR band identified among the El Tor strains had greater similarity (>97%) to the V. cholerae genome sequence and therefore has the potential to be used as a marker for the identification of the V. cholerae strains. Non-O1 strains recovered from Bhind region differed among themselves as well as from that of the O1 isolates. All the O1 serogroup isolates possessed SXT element and were uniformly resistant to the antibiotics nalidixic acid, polymyxin-B, furazolidone, cloxacilin, trimethoprim-sulfamethaxazole, and vibriostatic agent 0129. Inaba strains from both Delhi and Bhind differed from Ogawa strains by their resistance to streptomycin despite sharing similar DNA patterns in all the three rep-PCRs. Though Delhi and Bhind are separate geographical regions in Northern India, Inaba strains from both these places appear to be closely related owing to their similarity in antibiogram and genetic profile.