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Cholera toxins: purification and preliminary characterization of ileal loop reactive type 2 toxin.

Details for the preparation and partial purification of culture supernatant fluids of Vibrio cholerae (V. comma) 569B which retain rabbit ileal loop fluid-accumulating activity are presented. These preparations were fractionated on Sephadex G-200 and on diethylaminoethyl-Sephadex. On the latter, two fractions were obtained by elution with a linear sodium chloride gradient. The fraction designated "fraction I" retains the toxic activity as demonstrated in the rabbit ileal loop model. Chemical and immunological properties of this active fraction are described.

Amino Acids↗

LYSIS OF BACTERIAL PROTOPLASTS AND SPHEROPLASTS BY STAPHYLOCOCCAL ALPHA-TOXIN AND STREPTOLYSIN S.

Bernheimer, Alan W. (New York University School of Medicine, New York, N.Y.), and Lois L. Schwartz. Lysis of bacterial protoplasts and spheroplasts by staphylococcal alpha-toxin and streptolysin S. J. Bacteriol. 89:1387-1392. 1965.-Protoplasts of Bacillus megaterium, Sarcina lutea, and Streptococcus pyogenes, and spheroplasts of Escherichia coli were lysed by staphylococcal alpha-toxin, whereas spheroplasts of Vibrio metschnikovii and V. comma were not. In the spectrum of its lytic action, streptolysin S qualitatively resembled staphylococcal alpha-toxin except for failure to lyse S. pyogenes protoplasts. In contrast to the two foregoing agents, streptolysin O did not lyse protoplasts and spheroplasts. The observations are interpreted in relation to similarities and differences in lipid composition of bacterial and mammalian cell membranes.

Animals↗

Complete sequence of virulence plasmid pJM1 from the marine fish pathogen Vibrio anguillarum strain 775.

The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.

Animals↗

[A boy with cholera from India].

A 7-year-old Indian boy travelling from India to the United Kingdom was brought to the Emergency Clinic of Airport Medical Services at Schiphol airport in Amsterdam, the Netherlands. He had had watery diarrhoea in the aircraft and had lost consciousness. In view of the strong indications for cholera and the rice water-like diarrhoea, he was admitted to the paediatric ward of the VU Medical Centre where intravenous rehydration was carried out. He recovered within three days. A large number of comma-shaped, motile, Gram-negative rods were found in the faeces. After two days, the faeces culture revealed Vibrio cholerae O1 El Tor, serotype Inaba. On the day of the flight, the patient had drunk a litre of water from a bottle that later turned out to have been from the New Delhi water supply. Cholera is rare as an import disease in the Netherlands. Due to the severe dehydration, the infection can run a serious course and even be fatal. The infection is not transmitted from person to person. Therefore, no special measures are needed when a patient with cholera is admitted to hospital.

Child↗

Electron microscopic studies on Vibrio cholerae O139.

We conducted studies to investigate the surface architecture of V. cholerae O139 using electron microscopy and compared it with O1 and other serogroups of V. cholerae. The bacterium is comma-shaped and has a single polar flagellum and morphologically resembles the classical and E1Tor biotypes of V. cholerae O1. High power electron microscopy showed a few pili, 5 to 7 nm in diameter, and 2 to 3 in number per bacterium. The presence of a capsule on electron microscopy of ultrathin sections of V. cholerae O139 treated with polycationic ferritin clearly distinguished the O139 serogroup from the O1 serogroup which are not encapsulated. Immunoelectron microscopy further revealed that an anti-O139 monoclonal antibody of the IgG2a isotype bound specifically only to an O139 strain but not to any other serogroup of V. cholerae indicating that O139 has unique epitopes not found in other serogroups of V. cholerae.

Bacterial Capsules↗

Effect of high oxygen tensions on the growth of selected, aerobic, gram-negative, athogenic bacteria.

The in vitro effects of high O(2) tensions (P(O2)) on aerobic, enteric pathogens were examined at pressures of up to 3 atm absolute. Organisms from the genera Salmonella, Shigella, and Vibrio were usually subjected to 24-hr exposures. Tensions of 0.87, 1.87, and 2.87 atm absolute of O(2) (plus traces of CO(2) and N(2)) became progressively inhibitory for Salmonella and Shigella growth, but were bactericidal only for V. comma strains at tensions greater than 0.87 atm absolute of O(2). Growth inhibition of enteric organisms resulted from increased P(O2), rather than pressure per se, and could be mitigated nutritionally; an appropriate carbohydrate source is at least partially involved. Further studies with vibrios indicated that such mitigation was independent of medium pH. In addition, a synergistic relationship existed between O(2) and sulfisoxazole when tensions from 0.87 to 2.87 atm absolute of O(2) were maintained for 3 to 24 hr. Synergism occurred even under nutritional conditions which negated growth inhibition by O(2) alone. Bactericidal concentrations of sulfisoxazole, in the presence of increased P(O2), were reducible up to 4,000-fold. The combined procedure employed in this investigation, by use of an antimicrobial drug of known action, which also synergizes with O(2), plus nutritional studies, suggests a means for establishing a site of O(2) toxicity. These data support the concept that O(2) inhibition of growth represents a metabolic disturbance and that metabolic pathways involving p-aminobenzoic acid may be O(2)-labile. Such an approach could also guide development of antimicrobial agents as O(2) substitutes for promoting synergism.

Carbohydrate Metabolism↗

A Halophilic vibrio isolated from a case of chronic cholecystitis.

We found a halophilic vibrio in B bile from a 75-year-old female patient with chronic cholecystitis, and examined its biochemical characteristics. The organisms are gram-negative short rods or comma shaped, with some ring forms. They have a single polar flagellum, but not capsule. The strains can grow in peptone water with 1.0 to 4.0% NaC1, but not with no NaC1 or 6.0% NaC1. The characteristics of the organisms are positive dextrose fermentation, catalase, oxidase, and ornithine decarboxylase, and negative lysine decarboxylase, arginine dihydrolase, and absence of gas from glucose. They are sensitive to 2,4-diamine-6,7-diisopropyl pteridine (0/129). These characteristics indicate that the isolated strain should be a halophilic vibrio. however, no growth on Salmonella-Shigella (SS), SS with added sucrose and bromcresol purple (SS-SB), MacConkey's or thiosulfate citrate bile salts (TCBS) agar plates was demonstrated. Nitrate reduction, Simmons' citrate agar, indole, omicron-nitrophenol-beta-d-galactopyranoside (ONPG), motility and esculin hydrolysis were positive. Urease, gelatinase, Voges-Proskauer, phenylalanine deaminase and malonate reactions were negative. Acid was produced from amygdalin, arabinose, cellobiose, fructose, galactose, glucose, lactose, maltose, mannitol, salicin, starch, sucrose, trehalose, and xylose, but not from adonitol, dulcitol, inositol, mannose, melibiose, raffinose, rhamnose and sorbitol. From these characteristics the isolate is considered to be not identical with V. parahaemolyticus, V. Cholerae, V. Vulnificus or other vibrios. It can be presumed that this isolate represents another species of halophilic vibrio.

Aged↗

Use of multiparameter analysis for Vibrio alginolyticus viable but nonculturable state determination.

BACKGROUND: Vibrio alginolyticus is known to enter into a viable but nonculturable (VBNC) state in response to environmental conditions unfavorable to the growth. Cells in VBNC condition pose a public health threat because they are potentially pathogenic. METHODS: We constructed a pathway for the identification of the most significant variables and the characterization of those variables able to discriminate the groups under investigation. Different parameters measured by the image processing software were chosen as the most representative of V. alginolyticus cell morphology (length index for dimension) and metabolic activity (density profile indexes). To detect relationships between the groups of treatment performed, we carried out a principal components analysis (PCA). RESULTS: The PCA analysis indicated that increasing coccoid shape transformation was related to both metabolic and dimension variations, delineating a well defined graph profile. Indeed, we discovered that specific morphological variations occur when cells in the culturable state pass into VBNC condition, namely comma-shaped culturable bacteria are converted into coccoid-shaped VBNC cells. The results were also supported by scanning electron microscopy analysis. CONCLUSIONS: This technique allows the analysis of a large number of vibrio samples in a short period of time. The obtained multiparameter information may complement genetic/molecular analyses facilitating, in an automatic fashion, further studies to evaluate the potential risk of this pathogen in the environment. It may also be a useful tool for large-scale cell biology studies and high content screening.

Bacterial Physiological Phenomena↗

Vibrio cholerae non-O1 septicaemia in a patient with liver cirrhosis and Billroth-II-gastrectomy.

This report deals with the first diagnosed case of Vibrio cholerae non-O1 septicaemia in Austria. After a vacation in Tunisia, a 51-year-old male patient with liver cirrhosis and Billroth-II-gastrectomy was admitted to hospital because of abdominal pain, growing ascites, and jaundice. Four days later, the patient developed a single peak of high fever (39.6 degrees C). A blood culture was drawn and treatment with amoxycillin/clavulanic acid commenced. The blood culture yielded Gram-negative comma-like rods which were identified as V. cholerae non-O1.

Abdominal Pain↗

Characteristics of boric acid tolerant Vibrio cholerae.

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.

Amino Acids↗

[Clinical bacteriological analysis of Vibrio vulnificus infection--a report of five case].

Clinical features in Vibrio infection are generally represented by gastrointestinal involvements such as food poisoning, and its prognosis is usually good. However, Vibrio vulnificus infection not uncommonly causes serious problems including sepsis, necrotizing fasciitis of the extremities, and other conditions, sometimes resulting in fatal outcome. In the present study, we analyzed clinical microbiological aspects of five cases with V. vulnificus infection. All the strains of V. vulnificus isolated in five patients are oxidase-positive Gram negative rods presenting comma-like configuration, which were yielded on TCBS agar forming green colonies; they were grayish-white in color and viscous in texture on 5% sheep blood agar, identification of bacteria were done using VITEK AMS (BioMérieux). Piperacillin and third-generation cephalosporins were found to have bactericidal activities against these strains. All five cases we experienced have primary ailments, and three cases out of the five had taken perishable sea-food before showing disease symptoms. V. vulnificus has two infection channels; one is external wound and the other is oral intake. The latter is said that it may become serious. This has a rather short period from the starting the symptoms to death, and there is high death rate. For life-saving, it is inevitably necessary to dose an effective antibacterial medicine in the early stage. If we suspect this bacteria in the test laboratory, it is important to report this to the clinical doctor. In other words, this is one of the bacteria that needs prompt treatment and further microbiology testing.

Adult↗

[Microtest systems in the diagnosis of cholera].

Micro test systems for the biochemical identification of comma bacilli (MTS-V) and for a rapid determination of their groups according Heiberg (MTS-H) were designed and commissioned into medical practice. The micro test systems showed their advantages before the control media of Hiss and Bacterium Incubation Media at studying the museum-kept and freely-obtained vibrio strains as well as at studying the concomitant microflora and clinical materials taken from ill people and vibrio-carriers during the 1994 outbreak of cholera in Daghestan.

Bacterial Typing Techniques↗