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Hemolytic Vibrio cholerae biotype El Tor strains were isolated in the United States in 1973 and 1978 after they had supposedly disappeared worldwide during the 1960s and 1970s. We decided to examine the change in prevalence of hemolytic El Tor strains since the beginning of the seventh pandemic and evaluate the usefulness of hemolytic activity as an epidemiological marker. A total of 48 isolates of V. cholerae biotype El Tor isolated in the Eastern Hemisphere between 1960 and 1979, along with 1 Texas (1973) and 38 Louisiana (1978) isolates, were tested for hemolytic activity by each of four methods. One method (utilizing heart infusion broth with 1% glycerol) was slightly superior for detecting hemolytic activity. Titers obtained with this method ranged from less than 2 to 1,024. Of 13 (76.9%) strains from the earliest part of the current pandemic, 10 were hemolytic, compared with 1 of 26 (3.8%) strains isolated in the period from 1966 to 1979 in the Eastern Hemisphere, indicating that nonhemolytic El Tor strains have replaced the hemolytic variety there. In contrast, all 38 Louisiana isolates and the Texas isolate were strongly hemolytic. Hemolytic activity was concluded to be a useful epidemiological marker.

The adsorption of laterally and polarly flagellated bacteria to chitin was measured, and from the data obtained, a modified Langmuir adsorption isotherm was derived. Results indicated that the adsorption of laterally flagellated Vibrio parahaemolyticus follows the Langmuir adsorption isotherm, a type of adsorption referred to as surface saturation kinetics, when conditions are favorable for the production of lateral flagella. When conditions were not favorable for the production of lateral flagella, bacterial adsorption did not follow the Langmuir adsorption isotherm; instead, proportional adsorption kinetics were observed. The adsorption of some polarly flagellated bacteria exhibited surface saturation kinetics. However, the binding index (the product of the number of binding sites and bacterial affinity to the surface) of polarly flagellated bacteria differed significantly from that of laterally flagellated bacteria, suggesting that polarly flagellated bacteria adsorb to chitin by a different mechanism from that used by the laterally flagellated bacteria. From the results of dual-label adsorption competition experiments, in which polarly flagellated V. cholerae competed with increasing concentrations of laterally flagellated V. parahaemolyticus, it was observed that laterally flagellated bacteria inhibited the adsorption of polarly flagellated bacteria. In contrast, polarly flagellated bacteria enhanced the adsorption of V. cholerae. In competition experiments, where V. parahaemolyticus competed against increasing concentrations of other bacteria, polarly flagellated bacteria enhanced V. parahaemolyticus adsorption significantly, whereas laterally flagellated bacteria only slightly enhanced the process. The direct correlation observed between surface saturation kinetics, the production of lateral flagella, and the ability of laterally flagellated bacteria to inhibit the adsorption of polarly flagellated bacteria suggests that lateral flagella represent a component of bacterial structure that is important in the adsorption of laterally flagellated bacteria to surfaces. A model for adsorption events of laterally flagellated bacteria is proposed, based on the evidence presented.

The feces of five patients admitted to a hospital during an outbreak of cholera in Melbourne, Australia, in November 1972, were examined for the presence of tetracycline-resistant coliforms and tetracycline-resistant strains of Vibrio cholerae. Despite the abundance of tetracycline-resistant coliforms able to transfer this resistance to other strains of Escherichia coli, no tetracycline-resistant strains of V. cholerae were detected. In vitro transfer experiments using the V. cholerae strain responsible for the outbreak as recipient revealed that it was a particularly poor host for most R plasmids.

Influenza viruses have been shown to decrease the ability of polymorphonuclear leukocytes (PMN) to respond to a variety of stimuli. This study was done to determine if viral neuraminidase was responsible for decreased PMN function. Treatment of human PMN with purified neuraminidases from influenza virus, Vibrio cholerae, or Clostridium perfringens did not significantly affect the ability of human PMN to respond to stimulation. Occasional virus preparations that lacked the ability to depress PMN function did not differ in neuraminidase activity from viruses capable of causing depression. These results demonstrate that neuraminidase activity is not the cause of influenza virus-induced PMN dysfunction.

Infant mice 8 days of age were infected orally with virulent, motile, classical or El Tor strains of Vibrio cholerae and with nonmotile mutants of low virulence derived from the same strains. At intervals of 8 and 12 h postinfection, frozen thin sections of the ileum were prepared, stained with fluorescein isothiocyanate-labeled rabbit anti-vibrio antibody, and examined with the fluorescence microscope. The motile organisms were present in larger numbers, especially at 12h, and had penetrated the intervillous spaces and crypts of Lieberkuhn more completely than nonmotile vibrios. Dilution counts were made on various regions of the intestines of infant mice challenged orally 12 h previously with either motile or nonmotile strains of V. cholerae. Greater numbers of organisms were found, especially in the upper intestinal regions, when motile organisms were used. Low numbers of vibrios, limited mostly to the lumen, were seen in the ileum of infant mice infected with motile organisms when the infants were the offspring of mothers that had been immunized with crude flagellar vaccine or a vesicular preparation derived from the vibrio cell surface. The distribution of vibrios in this case was similar to that found in infected infants of unvaccinated mothers challenged with nonmotile organisms. Motility appears to enable the bacteria to better populate the upper regions of the intestinal tract and to avoid the washing effects of secretions and peristalsis. Antibacterial immunity may function, at least in part, by making it impossible for motile vibrios to accomplish this widespread distribution within the ileum.

Monoclonal antibodies against the group and type specific antigens of Vibrio cholerae O:1 lipopolysaccharide were used for the rapid detection of Vibrio cholerae strains by motility inhibition and immunofluorescence. Motility inhibition of live Vibrio cholerae O:1 was obtained with group specific monoclonal antibodies. Monoclonal antibodies against the type specific antigens B (Ogawa) and C (Inaba) inhibited motility of strains of homologous serotypes only. Indirect immunofluorescence of heatfixed bacteria with monoclonal antibodies and fluorescein-isothiocyanate conjugated rabbit anti-mouse immunoglobulin was also shown to be suitable for the rapid detection of Vibrio cholerae O:1. Both tests were highly specific and no cross-reactions were observed with strains of non-O:1 vibrios, Escherichia coli or Salmonella spp. tested. However, a weak fluorescence of some Ogawa strains was observed when high concentrations of Inaba specific monoclonal antibodies were used.

The aim of the present studies was to clarify Vibrio cholerae adherence by elucidating the basis for the apparently variable nature of in vitro attachment. An examination of the purported specificity of vibrio adherence did not support the concept of host receptor structures; organisms selected for their capacity to adhere to inert substrates simultaneously acquired the capacity to bind to intestinal tissue in vitro. Further investigation of the bacterial factors involved in adherence confirmed the in vitro significance of the slime agglutinin, although this moiety appeared to have no pathogenic relevance. The conflicting descriptions of V. cholerae attachment are discussed in terms of the nature of the factor responsible for binding in each system.

1) Chemotaxis of cholera vibrios facilitates the association of these bacteria with the mucosal surface. 2) Mucosal extracts can block the chemotactic receptors on the bacterial surface and thereby retard the association of chemotactic bacteria with the mucosal surface. 3) Chemotactic cholera vibrios grow more efficiently than non-chemotactic mutants in germfree mice and in isolated intestinal loops of rabbits. Conversely, non-chemotactic mutants grow more efficiently in infant mice. 4) Chemotaxis is therefore of considerable importance to the in vivo growth of cholera vibrios. The various mechanisms, including those of local immunity, by which bacterial chemotaxis can be exploited for the benefit of the host deserve further exploration.

Attachment of Vibrio cholerae to the mucosal surface of the intestine is considered to be an important virulence characteristic. Vibrio cholerae, an autochthonous member of brackish water and estuarine bacterial communities, also attaches to crustacea, a significant factor in multiplication and survival of V. cholerae in nature. The ability of V. cholerae to attach to the gut wall of the blue crab (Callinectes sapidus) was examined, and attachment was observed only in the hindgut and not the midgut of crabs, confirming a requirement for chitin in the attachment of V. cholerae to invertebrate and zooplankton surfaces. The new finding of attachment of V. cholerae to the hindgut of crabs may be correlated with the epidemiology and transmission of cholera in the aquatic environment. The crab model may also prove useful in elucidating the mechanism(s) of ion transport in crustacea.

The relationship between 433 V. cholerae strains and 571 enterobacterial strains, isolated from different ecosystems, have been studied by the method of "delayed antagonism". The direct correlation between the virulence of V. cholerae and the width of the spectra of their antagonistic activity towards indicator bacteria was detected. The expediency of studies on the role of the vibriocinogenic factor in the survival of V. cholerae in different ecological systems is substantiated.

A number of isolates of Vibrio cholerae were examined with respect to their (i) surface hydrophobicity as measured by hydrophobic interaction chromatography, (ii) capacity to agglutinate erythrocytes, and (iii) ability to bind to an ion-exchange matrix. V. cholerae isolates, cultured under a variety of growth conditions, were conspicuously hydrophobic. The hydrophobicity was accentuated when these strains were (i) cultivated in a chemically defined synthetic medium, (ii) harvested at the exponential phase of growth, and (iii) cultured at a lower temperature. Rough strains were more hydrophobic than smooth strains. Of the various surface components examined, the outer membrane proteins were conspicuously hydrophobic. The cell-bound hemagglutinating activity of V. cholerae strains was increased when these strains were cultured in synthetic medium and harvested at the stationary phase of growth. This property was unaffected by the growth temperature. Only D-mannose, at a high concentration, inhibited hemagglutination of 80% of the isolates examined. L-Fucose did not inhibit the hemagglutinating activity. V. cholerae strains adhered strongly to the anion-exchange matrix DEAE-cellulose. The surface charge density was accentuated when these strains were grown in synthetic medium. These results suggest that the V. cholerae surface contains both specific (hemagglutinating) and nonspecific (hydrophobic and ionic) factors which may influence its eventual adherence to the host cell surface.

Various Sephadex G-15 fractions of pepsin-digested mucosal extract inhibited the in vitro association of cholera vibrios with mucosal slices. Inhibitory activity paralleled the taxin activity of the fractions for these bacteria. This supports the theory that inhibition of mucosal association by pepsin-digested mucosal scrapings was due to the blocking of taxin receptors on the bacterial surface. Nonchemotactic mutants were significantly less efficient than the chemotactic parent or revertant strains in associating with mucosal slices in vitro. Control experiments in which filter paper disks replaced the mucosal slices showed a comparable extent of association of chemotactic and nonchemotactic vibrios with this material. Histological studies indicated that vibrios associated predominantly with the mucus gel of the intestinal slices rather than with the mucosal epithelium or the serosal surface. Intestinal slices attracted chemotactic vibrios even after prolonged washing, suggesting continuous production of the taxin by the tissue. Inert polystyrene particles 1.1 micrometers in diameter penetrated the mucus gel of intestinal slices very poorly, but nevertheless could be detected in low numbers in the deep intervillous spaces within 15 min. In contrast, chemotactic vibrios reached the deep intervillous spaces in significantly higher numbers, whereas motile, non-chemotactic vibrios reacted like the inert particles. It is concluded that mucus gel represents a partial barrier to the penetration of particles of bacterial size and that this barrier can be invaded efficiently by motile bacteria, but only when their locomotion is guided by chemotactic stimuli.

Cells of Vibrio cholerae underwent rapid autolysis when suspended in media of low osmolarity under non-growing conditions. Chaotropes like urea and guanidine. HCl which are potent protein denaturants caused complete and immediate lysis of whole cells. This unique sensitivity of V. cholerae to protein denaturants led to the development of a rapid method for the selective isolation of the outer membrane upon treatment of whole cells with urea. The composition of the outer membrane isolated from both whole cells and crude envelopes by treatment with urea was comparable with that of the outer membrane isolated by other conventional methods.

The prophylactic significance of the nonlipopolysaccharide (non-LPS) antigens of Vibrio cholerae was investigated further with use of the infant mouse cholera model. Of 16 strains examined to date, 12-including eight recent field isolates of both biotypes and both common serotypes-express common non-LPS protective antigens. The exceptional strains are four old isolates of El Tor biotype, the outer membrane proteins of which are not atypical when analyzed by immunoblotting. The protective activities of antibodies to the shared non-LPS components correlated with their capacities to inhibit the in vitro attachment of Vibrio organisms to isolated murine enterocytes.

Isolated rabbit brush borders were used to investigate the adhesive properties of clinical and environmental isolates of non-O1 Vibrio cholerae and clinical isolates of Vibrio parahaemolyticus and Aeromonas hydrophila. A minority of the isolates were found to adhere to brush borders. The adhesion of these isolates was affected by a number of factors, including the bacteria:brush border ratio, incubation time, culture medium and growth temperature. Adhesion was inhibited by L-fucose but was independent on calcium ion concentration.

Human milk and colostrum samples were divided into an immunoglobulin and a nonimmunoglobulin fraction by immunosorbent chromatography. The ability of these fractions to inhibit bacterial cell adhesion and enterotoxin receptor binding of Vibrio cholerae and various Escherichia coli isolates was then tested by in vitro assays. The strongest effect was generally seen with the nonimmunoglobulin fractions, which were shown to significantly inhibit E. coli cell adhesion (hemagglutination) mediated by CFA/I, CFA/II, or K88 fimbriae (but not type 1 pili) and V. cholerae hemagglutination, as well as the binding of cholera toxin and E. coli heat-labile enterotoxin to GM1 ganglioside. Also, the immunoglobulin fractions had significant inhibitory activity in some of these systems. The results are interpreted to suggest that human milk and colostrum may contain secreted structure analogs of the cell receptors for some bacterial adhesions and enterotoxins; this might contribute to the protective effect of milk against enteric infections.

The authors formed a collection of typing phages in order to differentiate cholera vibrios of both biotypes. The collection consists of 7 phages proposed by Mukerjee for typing vibrios of the classical biotype and 3 El Tor phages isolated at the period of the 7th pandemy of cholera. In forming the typing collection the authors observed the following principles: 1) to crate a single set of typing phages and a single scheme of phage type differentiation of classical cholera and El Tor vibrios; 2) to use virulent phages; 3) to form a collection of serologically different phage types. The proposed collection allows to reveal a larger number of phage types as compared with phages proposed by Mukerjee phage typing a larger number of strains enhancing thus the epidemiological significance of the method of phage typing cholera vibrios.

Two strains of boric acid tolerant Vibrio cholerae have been studied in order to identify the characteristics which distinguish them from the 'wild-type' virulent strains. Strain Ib5 requires either amino acid cysteine or methionine while strain Ib5S requires deoxyribonucleic acid (DNA) for growth. Although the strains did not grow on desoxycholate citrate agar (DCA) they grew well on chocolate agar. Morphologically filamentous, bacillary and comma-shaped forms were seen, depending on the media and incubation period. The strains failed to cause accumulation of fluid in gut ligation and permeability factor tests. With the single radial immunodiffusion test, strain Ib5S gave positive reaction in 83% of the tests when DNA was added to the growth medium. On the other hand only 25% of the tests were positive when no DNA was added to the growth medium, even though the antigen was concentrated ten-fold. Strain Ib5 only gave 50% positive tests when the antigen was concentrated ten-fold. It is therefore suggested that Ib5S strain is a cholerangenoid producer in the presence of DNA, and may be useful as an effective vaccine against cholera.

The influence of parenteral immunization with live virulent vibrios and lipopolysaccharide-containing choleragen toxoid and oral immunization with liver avirulent vibrios on the interaction of V. eltor and the mucous membrane was studied on the ligated loops of the small intestine in 164 rabbits. Protection from enterotoxigenicity (the accumulation of fluid) was compared with the suppressive effect on the adhesion and colonization of vibrios. The coefficient of immunization efficacy was determined microbiologically, as the ratio of the adhesion index (i. e. the percentage of the number of vibrios in the media inoculated with the homogenized matter of the washed intestinal wall from the sum of this number and the amount of vibrios in the contents) in the control rabbits to the adhesion index in the immunized ones, and histologically, as the ratio of the average number of vibrios attached to the epithelial number of vibrios attached to the epithelial cells of one intestinal villus in the control rabbits to the corresponding number in the immunized animals. Protection from the enterotoxigenicity of vibrios resulted from the suppression of their adhesion and colonization, lipopolysaccharide being, probably, the factor of adhesiveness. Effective protection was accompanied by an increase in the number of lymphocytes (especially "granular" lymphocytes with lysosome-like granules) between intestinal villi and the number of lymphoid and plasmic cells in the lamina propria of the villi, which indicates the role of lymphocytes in cholera-induced immunity.

Most probable numbers of Vibrio cholerae and related vibrios were determined in Albufera Lake, Valencia, Spain, and in coastal waters under the influence of the lake discharges over the course of an annual cycle. The influence of temperature, kind of water, and characteristics of the different sampling sites on the numbers of vibrios recovered was evaluated. Maximum recovery of vibrios reached 10(3)/ml in both types of waters analyzed. V. cholerae numbers reached 10(3)/ml in the lake and 10(2) in one of the coastal sites. Frequently during the warm season, all vibrios isolated were identified as V. cholerae. Occasionally, no V. cholerae was recovered. The recovery of vibrios was significantly influenced by the temperature of the water and the type of water analyzed. Most of the V. cholerae isolates were included in Heiberg groups I and II, and nearly 50% of the strains used chitin as sole carbon source. Indole was not produced by 100% of the strains. All strains tested were non-O1 serovars.

The efficacy of the 2 antimicrobial compounds, erythromycin and trimethoprim-sulphamethoxazole, were compared in the treatment of the clinical symptoms of cholera and the eradication of Vibrio cholerae O1 organisms from the stools of patients infected with tetracycline-resistant strains. 47 patients with a clinical diagnosis of cholera, without prior antibiotic therapy, were included in the trial and received either erythromycin, trimethoprim-sulphamethoxazole, or a placebo twice daily. The mean number of vibrios per gram of stool decreased from 5.2 x 10(8) +/- 0.3 per ml to 0 within 36 h of admission following either erythromycin or trimethoprim-sulphamethoxazole therapy, while organisms persisted in placebo-treated controls for more than 7 d. Clinically there was a significant reduction in the number of diarrhoeal stools per day and duration of diarrhoea in the erythromycin-treated group compared with the placebo-treated controls. During the study an isolate resistant to trimethoprim-sulphamethoxazole but sensitive to erythromycin was obtained. As an adjunct to oral rehydration therapy, erythromycin may serve as an effective alternative treatment for cholera, especially in areas where trimethoprim-sulphamethoxazole resistance may be emerging.

The existing methods for phagocytosis evaluation are inadequate for assessing all the real events occurring during phagocytosis, or for continuously following the kinetics of the process. Our purpose is to establish the use of luminous bacteria as an object for phagocytosis. The bioluminescence test offers an easy and simple method to determine the kinetics of phagocytosis by following the luminescence of the bacteria. The terrestrial luminous bacteria Vibrio cholerae var. albensis are readily phagocytosed by polymorphonuclear (PMN) cells. The correlation coefficient between the decrease in luminescence and the decrease in viable count is 0.999. The rate of decrease in luminescence and the residual level of luminescence after 60 min of phagocytosis are proportional to the rate of increase in phagocytosis induced chemiluminescence, and to its maximal level, respectively. Opsonization requirements are comparable in both tests. Different inhibitors of the phagocytosis process caused similar changes in the rates of the bio- and chemiluminescence (correlation coefficient 0.974), and in the luminescence maximal level (correlation coefficient 0.804). The validity of the bioluminescence assay being proved, it is suggested as an alternative assay for phagocytosis assessment.

Cell-free hemolymph (serum) of the eastern oyster, Crassostrea virginica, agglutinated Vibrio cholerae, including all O1 serovars and biovars. Seventy-nine other strains of bacteria, including 14 genera and 26 species, were not agglutinated. The A, B, and C factors of O1 antigen were not involved in agglutination. Bacterial agglutinating (BA) activity was demonstrated for oysters inhabiting different environments of the U.S. Atlantic and Gulf coasts. Oyster serum BA titers showed high individual variation. The serum component(s) involved in BA was inhibited by 80 degrees C heat, pronase, EDTA, mucin, and fetuin treatments. N-Acetylneuraminic acid (10 mg/ml) weakly inhibited BA activity. Ligands of V. cholerae were sensitive to neuraminidase and resistant to 80 degrees C and pronase. High salinities (24 and 30%) enhanced BA. Cross-adsorption tests with V. cholerae and human O+ erythrocytes indicated that BA and hemagglutinating activities may involve different serum components. These results imply that the ecology of V. cholerae in C. virginica is influenced by agglutinating activity of oyster serum.

Mutants of Vibrio cholerae was isolated on the basis of reduced ability to induce diarrhea in orally challenged infant mice. Nitrosoguanidine-treated clones were screened for low fluid accumulation ratios in individual mice, and presumptive mutants were confirmed in additional mouse tests. Mutants were examined for alterations in phage type, motility, toxin production, proteolytic activity, neuraminidase production, amylase production, morphology, growth requirements, carbohydrate fermentations, in vitro growth patterns, and cell surface alterations. The types of mutants found included several with previously recognized virulence-associated markers (rough, nonmotile, toxin deficient, protease deficient); several types with pleiotropic alterations (cell morphology, decreased extracellular products); and several with no previously recognized virulence-deficient phenotype (purine requiring, cell surface altered, rapid death in vitro, no defect found). Dose-response kinetics showed that most mutants could provoke diarrhea if given in 100-fold greater numbers than the dose used for screening. Recovery of viable organisms from the gut late in infection showed reduction of survival and/or multiplication capacity for the mutants, with variation in the degree of reduction for the different classes.

A motile, chemotactic, Ogawa strain of Vibrio cholerae was attracted by all 20 L-amino acids tested, in contrast to Escherichia coli AW 405, which did not react to several of these. The maximum number of vibrios entering a capillary was much lower when the capillary contained carbohydrates rather than amino acids, but the minimum effective concentrations of the carbohydrates and amino acids tested were of the same order of magnitude. L-Fucose, a sugar known to inhibit the adhesion of this vibrio strain to brush border membranes, had no attraction (taxin activity) for it. A pepsin digest of rabbit mucosal scrapings or tryptone attracted vibrios as strongly as the most active amino acids. Several nonchemotactic and one nonmotile mutant were selected from the parent vibrio. The nonchemotactic mutants were indistinguishable from the parent in their ability to attach in vitro to isolated intestinal brush border membranes, whereas the nonmotile mutant had lost this ability. Parent and nonchemotactic mutants had equal growth rates in stirred and still continuous flow cultures that were maintained in an anaerobic environment.

In cross-streak tests on nutrient agar containing lactate, acetate, or citrate, the growth of certain enteric bacteria was inhibited by themselves or by Vibrio cholerae, or by both. This mimicked the activity of bacteriocins, but the phenomenon was attributable to the accumulation of carbonate and increased alkalinity of the media.

The role of motility in the pathogenesis of cholera was evaluated in ligated ileal loops of adult rabbits. Four strains of Vibrio cholerae (including both Inaba and Ogawa serotypes of both classical and El Tor biotypes) were compared with their aflagellated, but fully toxigenic and prototrophic, isogenic derivatives as to their ability to produce fluid accumulation in the rabbit gut. The nonmotile mutants required an at least 100-fold-higher dose than their respective wild-type strains to produce comparable fluid accumulation responses. The decreased ability of nonmotile strains to produce a fluid response was not due to their failure to multiply in vivo, since they increased in numbers in the rabbit ileum at the same rate as the wild-type strains, but probably was related to their inability to associate with the intestinal mucosa. After 3 h of incubation, 45 to 53% of motile, [35S]-labeled cells adsorbed to the intestinal wall, whereas only 3 to 15% (depending upon the strain) of the nonmotile bacteria were associated.

Bacteriophage phi 149 which was propagated in Vibrio cholerae (classical) OGAWA 154 strain, killed Vibrio cholerae (El Tor) strain MAK 757 without phage propagation. El Tor vibrios underwent a small degree of lysis only when infected by the phage phi 149 at a high multiplicity of infection and lost their viability at a rate-dependent multiplicity of phage infection. Evidence was obtained with 32P-labelled bacteriophage phi 149 for penetration of phage DNA into both bacterial strains. In host strain (OGAWA 154) phage particle synthesis occurred normally. In El Tor strain MAK 757 the phage DNA was not degraded but its expression was blocked. The killing effect of phi 149 on El Tor strain MAK 757 is supposed to be due to damage of the cytoplasmic membrane, which could not be repaired under the influence of phage information. This was indicated by a blockage of cellular respiration and RNA and protein synthesis.

The effect of UV-irradiation on Vibrio cholerae cells and its changes mediated by the plasmid R245 have been studied. Vibrio cholerae strains 569B and RV31 have been shown to be considerably more sensitive to lethal effect of UV-irradiation as compared with Escherichia coli and Salmonella typhimurium cells. Highly toxigenic strain 569B and practically atoxigenic strain RV31 have the same UV-sensitivity. Lethal effect of UV-irradiation on Vibrio cholerae cells is increased when the irradiated cells are plated on enriched media. UV-induction of mutations was not registered in plasmidless strains of Vibrio cholerae. Plasmid R245 increases UV-resistance of vibrio cells and makes them UV-mutable.

Earlier reports from this laboratory have shown that chemotaxis is an important mechanism that expedites the in vitro association of cholera vibrios with intestinal slices and that affects the in vivo colonization and virulence of these bacteria to a significant degree. The data reported in the present communication indicate that there appears to be a chemotatic gradient attracting cholera vibrios not only to the surface of the mucus gel, but that this gradient continues for at least a considerable distance toward the base of the villi. It is shown further that a strain of Vibrio cholerae was attracted by all 20 amino acids tested, in contrast to Escherichia coli AW405 which is repelled by several of these. Finally, experiments are described that show that superior in vivo colonization of chemotatic vibrios (compared to nonchemotactic mutants) was correlated with a significantly higher degree of mucosal association. Such increased mucosal association of chemotatic vibrios has previously been shown only with mucosal slices in vitro.

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.

Vibrio cholerae strain Htx-3, a hypertoxinogenic mutant of V. cholerae 569B Inaba, produces a dark brown pigment under certain growth conditions, whereas strain 569B does not. We investigated the biochemical basis for this pigment production and the possible relationships between pigmentation and other phenotypic properties in V. cholerae. After mutagenesis of V. cholerae 569B with N-methyl-N'-nitro-N-nitrosoguanidine, 28 independently derived pigment-forming (mel) mutants were isolated and characterized. The mel mutants frequently differed from wild type in toxinogenicity or motility and occasionally differed in other phenotypic traits. Individual mel mutants differed from wild type both in the amount of toxin produced and in the growth conditions optimal for toxin production. It has not yet been established whether multiple phenotypic changes in individual mel mutants represent pleiotropic effects of single mutations or induction of multiple mutations by N-methyl-N'-nitro-N-nitrosoguanidine or both. Production of pigment by mel mutants occurred at temperatures from 22 to 40 degrees C, was inhibited by anaerobiosis, and was stimulated by supplementation of growth media with the amino acid precursors of melanin (l-phenylalanine, l-tyrosine, or l-tyrosine plus l-cysteine). The pigment possessed several other properties reported for microbial melanins. We conclude that a biochemical pathway for melanin production is present in V. cholerae, that this pathway cannot be fully expressed in wild-type strain 569B, and that mutations in the gene(s) which we have designated mel can permit hyperproduction of melanin under appropriate conditions.

Adherence of some wild-type and mutant strains of Vibrio cholerae was studied and the relation between adherence, motility, multiplication, and virulence was examined. Vibrios adhered readily to freshly isolated tissue segments of rabbit intestine at 37 degrees C. The number of vibrios adhering was dependent on the concentration of bacteria to which the tissue was exposed. Adherence was markedly reduced by pretreatment with chloramphenicol and also when intestinal tissue was taken from parenterally or orally immunized rabbits. Non-motile or feebly motile strains were unable to adhere. One motile strain was also found to adhere poorly. The adherence of non-motile or poorly adhering strains was not affected by chloramphenicol. Non-motile, poorly adhering, or slow-growing strains behaved as attenuated strains, suggesting the role of motility, adherence, and multiplication in the virulence of V. cholerae.

The role of temperate phage beta in determining the serology and eltor-lytic phage sensitivity in Vibrio cholerae was investigated. The only serological change found in six host strains was a change to roughness. This was accompanied by failure to adsorb several of the lytic phages. Various phage-sensitivity changes were induced by phage beta in two hosts at the post-adsorption level. In strain HP47, three types of progeny were obtained of which one was universally resistant to lytic phages. These untypable lysogens were culturally stable but gave rise to segregants of the rare phage-type 6 on single colony selection.

Hemagglutination and intestinal adherence properties of non-O1 Vibrio cholerae were studied in vitro. No definite correlation between the cell-associated hemagglutinin titers and the intestinal adhesion indices was noted. Sugar- and glycoprotein-mediated inhibition data also indicated differences between the hemagglutination and adherence processes in respect to the receptor structures. Intestinal adherence of most V. cholerae strains could be inhibited to various extents by N-acetyl D-glucosamine. This observation provides a likely explanation for the ecological behavior of these organisms, which are known to associate themselves with chitinous (chitin:homopolymer of N-acetyl D-glucosamine) surfaces of zooplankton. The absence of any significant difference between the intestinal adherence indices of clinical and environmental isolates suggests that intestinal adhesion may be an essential but not sufficient prerequisite for colonization by and subsequent expression of pathogenicity of these microorganisms.

Experimental studies on survival of Vibrio cholerae in untreated sea-water reveal that they remain alive at least one week. A somewhat longer survival (up to 3 weeks) could be observed if these bacteria were kept in sea-water sterilized by filtration. But if sea-water is autoclaved, cholera vibrios may keep their vitality for periods at least 6 months. Our observations lead to the conclusion that untreated sea-water may contains some filtrable thermolabile factor killing cholera vibrios. Yet these facts should not create false feelings of security. It is known, from recent epidemiological data that sea-medium as a whole may be the origin of cholera outbreak.