Dependence of the expression of the biological features of Vibrio cholerae on the conditions of their cultivation

The biological activity of toxigenic and non-toxigenic V. cholerae supernatants was found to depend on the cultivation medium. The use of iron-free tryptone medium made it possible to obtain supernatants of toxigenic V. cholerae with haemolytic activity and destructive action on passaged cell cultures. In the experimental infection of suckling rabbits the influence of the cultivation conditions of V. cholerae on the character and expression of their pathogenic properties was determined. The dissemination of V. cholerae into the internal organs of rabbits after their infection with both toxigenic and non-toxigenic strains correlated neither with the cultivation conditions of these strains, nor with the character of changes in the intestine of the infected animals.     

Effect of changes in the osmolarity of the growth medium on Vibrio cholerae cells.

The rate and extent of lysis of Vibrio cholerae cells under nongrowing conditions were dependent on the osmolarity of the growth medium. Gross alterations in cellular morphology were observed when V. cholerae cells were grown in media of high and low osmolarity. The rate of lysis of V. cholerae cells under nongrowing conditions increased after treatment with chloramphenicol. Chloramphenicol-treated V. cholerae 569B cells showed formation of sphaeroplast-like bodies in medium of high osmolarity, but not in low osmolarity. Changes in the osmolarity of the growth medium also regulated the expression of the outer membrane proteins. This regulation was abolished if V. cholerae cells were grown in Pi-depleted medium. Analysis of the lytic behavior and composition of outer membrane proteins of an osmotically fragile mutant strain revealed a similar dependence on the osmolarity of the growth medium.     

Polyphosphate stores enhance the ability of Vibrio cholerae to overcome environmental stresses in a low-phosphate environment

Vibrio cholerae, the causative agent of Asiatic cholera, has been reported to make large quantities of polyphosphate. Inorganic polyphosphate is a ubiquitous molecule with a variety of functions in prokaryotic and eukaryotic cells. We constructed a V. cholerae mutant with a deletion in the polyphosphate kinase (ppk) gene. The mutant was defective in polyphosphate biosynthesis. Deletion of ppk had no significant effect on production of cholera toxin, hemagglutinin/protease, motility, biofilm formation, and colonization of the suckling mouse intestine. The wild type and mutant had similar growth rates in rich and minimal medium and exhibited similar phosphate uptake and alkaline phosphatase induction. In contrast to ppk mutants from other gram-negative bacteria, the V. cholerae mutant survived prolonged starvation in LB medium and artificial seawater basal salts. The ppk mutant was significantly more sensitive to low pH, high salinity, and oxidative stress when it was cultured in low-phosphate minimal medium. The ppk mutant failed to induce catalase when it was downshifted to phosphorus-limiting conditions. Furthermore, the increased sensitivity of the ppk mutant to environmental stressors in phosphate-limited medium correlated with a diminished capacity to synthesize ATP from intracellular reservoirs. We concluded that polyphosphate protects V. cholerae from environmental stresses under phosphate limitation conditions. It has been proposed that toxigenic V. cholerae can survive in estuaries and brackish waters in which phosphorus and/or nitrogen can be a limiting nutrient. Thus, synthesis of large polyphosphate stores could enhance the ability of V. cholerae to survive in the aquatic environment.     

Evaluation of the multitest medium for rapid presumptive identification of Vibrio cholerae from environmental sources

The multitest V. cholerae medium (VC medium) for rapid presumptive identification of Vibrio cholerae was evaluated. On the basis of reactions in the VC medium, 379 strains recovered during a yearlong ecological study in Calcutta were presumptively identified as V. cholerae. Further phenotypic characterization of these strains revealed that the reactions of 371 (97.9%) isolates were consistent with that of V. cholerae. False-positive reactions were exhibited by eight (2.1%) strains, three of which were identified as Vibrio fluvialis biotype 1. By slightly varying the basic formulation of the VC medium, we could eliminate some false-positive reactions. On the basis of the present evaluation, we recommend the routine use of the VC medium.     

Evaluation of the multitest medium for rapid presumptive identification of Vibrio cholerae from environmental sources.

The multitest V. cholerae medium (VC medium) for rapid presumptive identification of Vibrio cholerae was evaluated. On the basis of reactions in the VC medium, 379 strains recovered during a yearlong ecological study in Calcutta were presumptively identified as V. cholerae. Further phenotypic characterization of these strains revealed that the reactions of 371 (97.9%) isolates were consistent with that of V. cholerae. False-positive reactions were exhibited by eight (2.1%) strains, three of which were identified as Vibrio fluvialis biotype 1. By slightly varying the basic formulation of the VC medium, we could eliminate some false-positive reactions. On the basis of the present evaluation, we recommend the routine use of the VC medium.     

Role of ectoine in Vibrio cholerae osmoadaptation

Vibrio cholerae is both an intestinal pathogen and a microbe in the estuarine community. To persist in the estuarine environment, V. cholerae must adjust to changes in ionic composition and osmolarity. These changes in the aquatic environment have been correlated with cholera epidemics. In this work, we study the response of V. cholerae to increases in environmental osmolarity. Optimal growth of V. cholerae in minimal medium requires supplementation with 200 mM NaCl and KCl. However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed. During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth. We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay. We also demonstrate that high-osmolarity-adapted V. cholerae displays a growth advantage when competed against unadapted cells in high-osmolarity medium. In contrast, low-osmolarity-adapted V. cholerae displays no growth advantage when competed against high-osmolarity-adapted cells in low-osmolarity medium. These results may have implications for V. cholerae population dynamics when seawater and freshwater and their attendant microbes mix.     

Role of cyanobacteria in the persistence of Vibrio cholerae O139 in saline microcosms

Recently, a new strain of cholera, Vibrio cholerae O139, has emerged as an epidemic strain, but there is little information about its environmental reservoir. The present investigation was aimed to determine the role of cyanobacteria in the persistence of V. cholerae O139 in microcosms. An environmental isolate of V. cholerae O139 and three cyanobacteria (Anabaena sp., Nostoc sp., and Hapalosiphon sp.) were used in this study. Survival of culturable V. cholerae O139 in microcosms was monitored using taurocholate-tellurite gelatin agar medium. Viable but nonculturable V. cholerae O139 were detected using a fluorescent antibody technique. Vibrio cholerae O139 could be isolated for up to 12 days in a culturable form in association with cyanobacteria but could not be isolated in the culturable form after 2 days from control water without cyanobacteria. The viable but nonculturable V. cholerae O139 could be detected in association with cyanobacteria for up to 15 months. These results, therefore, suggest that cyanobacteria can act as a long-term reservoir of V. cholerae O139 in an aquatic environment.     

Testing the biological activity of Vibrio cholerae on cell subcultures

Testing the supernatants of ctx(+) strains of V. cholerae eltor and V. cholerae O139 on cell subcultures confirmed the possibility of the synthesis of hemolysin by V. cholerae under the condition of growing them in tripton medium lacking FeCl3. At the same time ctx(+) strains of V. cholerae of both serogroups retained, simultaneously with hemolysin production, their capacity for the synthesis of cholera toxin.     

A new selective, differential agar medium for isolation of Vibrio cholerae O1: PMT (polymyxin-mannose-tellurite) agar

A new selective and differential agar medium, polymyxin-mannose-tellurite (PMT) agar was devised to differentiate easily colonies of Vibrio cholerae O1 from those of V. cholerae non-O1. The differentiation between colonies of the two vibrios is based on mannose-fermentation. Colonies of V. cholerae O1 on the agar are agglutinated with O1 antiserum of V. cholerae much more easily than those on thiosulfate-citrate-bile salts-sucrose (TCBS) agar.     

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.     

霍乱cT-B和LPs-o双价抗原工程菌苗株的构建

作者将霍乱弧菌O抗原及毒素B亚单位基因片段,经DNA体外重组技术,得到了能表达双价抗原的工程菌株1046(pMG305)。经GM1-ELISA分析表明该菌株能够表达特异的霍乱CT-B抗原,且能分泌到胞外,通过菌体凝集,全细胞O抗原酶联分析和血凝抑制试验表明在1046(pMG305)菌体表面表达了霍乱的O抗原,它的脂多糖O抗原通过SDS-PAGE电泳分析,显示它表达了霍乱LPS的特征区带。小鼠腹腔免疫后用霍乱弧菌毒株攻击表明,有良好的保护作用,因此1046(pMG305)可望成为霍乱活疫苗的候选株。     

Inorganic polyphosphate in Vibrio cholerae: genetic, biochemical, and physiologic features

Vibrio cholerae O1, biotype El Tor, accumulates inorganic polyphosphate (poly P) principally as large clusters of granules. Poly P kinase (PPK), the enzyme that synthesizes poly P from ATP, is encoded by the ppk gene, which has been cloned from V. cholerae, overexpressed, and knocked out by insertion-deletion mutagenesis. The predicted amino acid sequence of PPK is 701 residues (81.6 kDa), with 64% identity to that of Escherichia coli, which it resembles biochemically. As in E. coli, ppk is part of an operon with ppx, the gene that encodes exopolyphosphatase (PPX). However, unlike in E. coli, PPX activity was not detected in cell extracts of wild-type V. cholerae. The ppk null mutant of V. cholerae has diminished adaptation to high concentrations of calcium in the medium as well as motility and abiotic surface attachment.     

Cytochrome c maturation and the physiological role of c-type cytochromes in Vibrio cholerae

Vibrio cholerae lives in different habitats, varying from aquatic ecosystems to the human intestinal tract. The organism has acquired a set of electron transport pathways for aerobic and anaerobic respiration that enable adaptation to the various environmental conditions. We have inactivated the V. cholerae ccmE gene, which is required for cytochrome c biogenesis. The resulting strain is deficient of all c-type cytochromes and allows us to characterize the physiological role of these proteins. Under aerobic conditions in rich medium, V. cholerae produces at least six c-type cytochromes, none of which is required for growth. Wild-type V. cholerae produces active fumarate reductase, trimethylamine N-oxide reductase, cbb3 oxidase, and nitrate reductase, of which only the fumarate reductase does not require maturation of c-type cytochromes. The reduction of nitrate in the medium resulted in the accumulation of nitrite, which is toxic for the cells. This suggests that V. cholerae is able to scavenge nitrate from the environment only in the presence of other nitrite-reducing organisms. The phenotypes of cytochrome c-deficient V. cholerae were used in a transposon mutagenesis screening to search for additional genes required for cytochrome c maturation. Over 55,000 mutants were analyzed for nitrate reductase and cbb3 oxidase activity. No transposon insertions other than those within the ccm genes for cytochrome c maturation and the dsbD gene, which encodes a disulphide bond reductase, were found. In addition, the role of a novel CcdA-like protein in cbb3 oxidase assembly is discussed.     

Role for glycine betaine transport in Vibrio cholerae osmoadaptation and biofilm formation within microbial communities

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.     

New selective and differential medium for Vibrio cholerae and Vibrio vulnificus.

Thiosulfate-citrate-bile salts-sucrose agar has been routinely used for the isolation of pathogenic vibrios, although its selectivity for Vibrio cholerae and Vibrio vulnificus is inadequate. Therefore, a new plating medium, cellobiose-polymyxin B-colistin agar, was developed for the isolation of these two species. Cellobiose-polymyxin B-colistin agar demonstrated a significant advantage over other media designed for the isolation or differentiation of vibrios: of both the 136 strains representing 19 Vibrio species and the marine isolates of the genera Pseudomonas, Flavobacterium, and Photobacterium, only V. vulnificus and V. cholerae were able to grow. Furthermore, the fermentation of cellobiose by V. vulnificus allowed for the easy differentiation of these two species. This medium offers significant potential as a selective and differential medium for these two pathogenic vibrios.     

New selective and differential medium for Vibrio cholerae and Vibrio vulnificus

Thiosulfate-citrate-bile salts-sucrose agar has been routinely used for the isolation of pathogenic vibrios, although its selectivity for Vibrio cholerae and Vibrio vulnificus is inadequate. Therefore, a new plating medium, cellobiose-polymyxin B-colistin agar, was developed for the isolation of these two species. Cellobiose-polymyxin B-colistin agar demonstrated a significant advantage over other media designed for the isolation or differentiation of vibrios: of both the 136 strains representing 19 Vibrio species and the marine isolates of the genera Pseudomonas, Flavobacterium, and Photobacterium, only V. vulnificus and V. cholerae were able to grow. Furthermore, the fermentation of cellobiose by V. vulnificus allowed for the easy differentiation of these two species. This medium offers significant potential as a selective and differential medium for these two pathogenic vibrios.     

Induction of melanin biosynthesis in Vibrio cholerae.

Vibrio cholerae synthesized the pigment melanin in response to specific physiological conditions that were stressful to the bacterium. Pigmentation was induced when V. cholerae was subjected to hyperosmotic stress in conjunction with elevated growth temperatures (above 30 degrees C). The salt concentration tolerated by V. cholerae was lowered by additional abiotic factors such as acidic starting pH of the growth medium and limitation of organic nutrients. Although the amount of toxin detected in the culture supernatant decreased significantly in response to stressful culture conditions, no correlation between the physiological conditions that induced melanogenesis and expression of OmpU or cholera toxin was detected. Since conditions that induce melanin production in V. cholerae occur in both the aquatic environment and the human host, it is possible that melanogenesis has a specific function with respect to the survival of the bacterium in these habitats.     

Induction of melanin biosynthesis in Vibrio cholerae.

Vibrio cholerae synthesized the pigment melanin in response to specific physiological conditions that were stressful to the bacterium. Pigmentation was induced when V. cholerae was subjected to hyperosmotic stress in conjunction with elevated growth temperatures (above 30 degrees C). The salt concentration tolerated by V. cholerae was lowered by additional abiotic factors such as acidic starting pH of the growth medium and limitation of organic nutrients. Although the amount of toxin detected in the culture supernatant decreased significantly in response to stressful culture conditions, no correlation between the physiological conditions that induced melanogenesis and expression of OmpU or cholera toxin was detected. Since conditions that induce melanin production in V. cholerae occur in both the aquatic environment and the human host, it is possible that melanogenesis has a specific function with respect to the survival of the bacterium in these habitats.     

The capacity to produce cholera exotoxin in natural strains of Vibrio cholerae

The study of 27 V. cholerae strains, isolated from cholera patients and found to be hemolytically inactive, with a view to establish their capacity for the production of cholera toxin has revealed that 4 strains (V. cholerae cholerae Dacca 35, V. cholerae cholerae Dacca 3, V. cholerae cholerae B1307, V. cholerae cholerae J89) produce this protein. The quantitative determination of enterotoxin has been made with the use of GM1 ELISA technique. Strain Dacca 35 has been found to be highly toxigenic and, as regards the amount of exotoxin it produces, no different from V. cholerae cholerae strain 569B, a well-known producer of cholera toxin. In strain Dacca 35 correlation between the capacity of the cells for toxin production and the morphology of colonies has been established. The study has revealed that the chromosome of strain Dacca 35 contains two copies of gene vctAB responsible for the synthesis of cholera toxin.     

Potentials and conditions for the reversion of pathogenic properties of the causative agent of cholera in an experiment

The passage of V. cholerae noncholerigenic strains and their mutants, both in vitro and in vivo, has demonstrated that strains in which one of such properties as mobility, viability, adhesive, lecithinase and neuraminidase activities, is sharply decreased or lost, are still capable of reversion to cholerigenic forms. V. cholerae strains which have lost two or more of these properties, as well as strains having stable hemolytic activity determined by Greig's test, seem to be incapable of such reversion.