Vibriosis

J Clin Microbiol, 1996 Jul, 34(7), 1843 - 5
Phage specific for Vibrio cholerae O139 Bengal; Albert MJ et al.; From the stool of a Vibrio cholerae O139 Bengal-infected patient, a phage that specifically lysed capsulated V . cholerae O139 strains only was isolated . The phage is useful for the confirmatory diagnosis of V . cholerae O139 infection and for the differentiation of variants that lack the capsule.

J Clin Microbiol, 1996 Jul, 34(7), 1760 - 4
Probable new species of Desulfovibrio isolated from a pyogenic liver abscess; Tee W et al.; A fastidious, slowly growing, spiral gram-negative bacterium was isolated from the liver abscess of an 82-year-old man with a 3-week history of febrile illness . The organism was an obligate anaerobe that grew at 37 and 42 degrees C but not at 25 degrees C . Its vibrioid or spiral morphology on Gram staining, rapid progressive motility, electron micrograph features, and biochemical tests were all consistent with the organism belonging to the genus Desulfovibrio . 16S rRNA gene sequencing of this organism demonstrated a 97% similarity to Desulfovibrio desulfuricans with 45 nucleotide differences, suggesting that it is a new species of Desulfovibrio.

Indian J Med Res, 1996 Jul, 104, 148 - 51
Association of metal tolerance with multiple antibiotic resistance of enteropathogenic organisms isolated from coastal region of deltaic Sunderbans; Choudhury P et al.; A survey of microorganisms was conducted in four coastal regions of the deltaic Sunderbans . Among ten different isolates, three were enteropathogenic . They were Vibrio cholerae non-O1 (CT+), enterotoxinogenic Escherichia coli (ETEC) and Pseudomonas aeruginosa . These enteropathogens were able to grow in the presence of zinc (Zn++), cadmium (Cd++), lead (Pb++), cobalt (Co++), copper (Cu++), nickel (Ni++) and silver (Ag+) up to 10 mM concentration . They also showed resistance against 5 to 10 antibiotics . Metal tolerance and drug resistance were not determined by plasmid(s) . Synthesis of outer membrane protein among the marine isolates was higher in presence of metal . Enteropathogens isolated from the deltaic Sunderbans were well adapted for growth of the saline environment with higher concentrations of toxic metals.

Indian J Med Res, 1996 Jul, 104, 139 - 41
Biological activity & interaction of Vibrio cholerae bacteriophages in rabbit ileal loop; Sarkar BL et al.; A set of ten V . cholerae EITor phages is in routine use for phage typing of V . cholerae O1 biotype EITor strains . These phages were used in rabbit ileal loop experiment to investigate whether these phages have any prophylactic value as regards their lytic capability on V . cholerae strains . The phages were found to have no prophylactic use as they were unable to lyse the standard bacterial strain V . cholerae MAK 757.

Indian J Med Res, 1996 Jul, 104, 134 - 8
Electron microscopic studies on Vibrio cholerae O139; Garg S et al.; 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.

Indian J Med Res, 1996 Jul, 104, 129 - 33
Comparative analysis of factors promoting optimal production of cholera toxin by Vibrio cholerae O1 (classical & E1Tor biotypes) & O139; Mukhopadhyay AK et al.; Various culture media {AKI, Brain heart infusion broth (BHI), Casamino acid-yeast extract broth (CAYE), Casamino acid-yeast extract broth supplemented with 90 micrograms/ml of lincomycin (CAYE-L), Tryptic soy broth (TSB) and Yeast extract peptone (YEP)}, cultural conditions (stationary and shaking) and incubation temperatures (30 degrees C and 37 degrees C) were evaluated to determine optimal conditions for production of cholera toxin (CT) by different biotypes (classical and E1Tor) and serogroups (O1 and O139) of V . cholerae . It was found that V . cholerae O1 E1Tor grown in CAYE-L and incubated at 30 degrees C with constant shaking was optimal for production of CT, while for the classical biotype and for the O139 serogroup, CT was maximally produced when grown in YEP and incubated at 30 degrees C in a shaker . Temperature appeared to be a prominent factor affecting the production of CT by the O1 E1Tor biotype when the media used were AKI, CAYE-L and YEP and also for the classical biotype when the media used were the AKI, BHI, CAYE and YEP . In the case of the O1 E1Tor biotype, CAYE-L was the best medium for CT production whereas for the classical biotype, CAYE-L was a poor medium as far as CT production was concerned . Irrespective of the media used, 30 degrees C shake culture condition seemed to be more favourable for supporting CT production except in CAYE medium for the O1 E1Tor biotype where incubation at 37 degrees C in a shaker was as good as incubation at 30 degrees C.

Indian J Med Res, 1996 Jul, 104, 125 - 8
Comparison of the sensitivity & specificity of a polyclonal versus monoclonal capture antibody based Bead ELISA for direct detection of cholera toxin from stool specimens; Ramamurthy T et al.; This study was conducted to examine whether substitution of polyclonal anti-CT IgG (PAb) coated beads with monoclonal anti-CT IgG, (MAb) coated beads would enhance the sensitivity and specificity of the Bead ELISA for direct detection of CT in stool samples of cholera patients . Sensitivity of MAb Bead ELISA was found to be more efficient (92.7%) than the PAb Bead ELISA (88.2%) in comparison to the 'gold standard' method of conventional culture method (CM) . However, the MAb based Bead ELISA had lower specificity (45%) than that of PAb Bead ELISA (51.8%) . Further, the positive predictive value was also lower in MAb Bead ELISA (69.9%) as compared to PAb Bead ELISA (82.1%) . Generally, all the statistical evaluation demonstrated better agreement between PAb (77.9%) and MAb (72.6%) Bead ELISAs in direct detection of free CT and culture method in confirming the causative organism i.e., Vibrio cholerae in stool specimens . When the two assay systems, viz., PAb and MAb Bead ELISAs were compared, the superiority of the PAb Bead ELISA over MAb Bead ELISA was clearly evident as it had 82.4 per cent of agreement value.

Indian J Med Res, 1996 Jul, 104, 60 - 75
Strategies for production of a potential candidate vaccine for cholera; Ghosh A et al.; First attempt at cholera vaccination was made by Jaime Ferran in 1884 . Since then, a variety of strategies and methods have been evolved to create a safe, efficacious vaccine against cholera . For the first few years emphasis was on the development of parenteral vaccines . However, as a result of accumulation of a tremendous amount of knowledge, not only on Vibrio cholerae-the causative agent, but also on its interaction with the host, emphasis has shifted towards the development of oral vaccines . Two such vaccines, one killed, a whole cell/B subunit combination vaccine and the other a live attenuated one, have shown promise . The combination vaccine in its present state of development confers only a transient protection in young children, while the live attenuated one produces adverse reaction . To combat these, various strategies are being evolved . In one attempt, a potential candidate vaccine strain has been constructed from a non-reactogenic clinical isolate of V . cholerae, which is devoid of all known major virulence genes and is also a good colonizer . In animal studies this construct has shown considerable promise . This review discusses the various strategies that have been employed so far in the quest for an ideal cholera vaccine.

Indian J Med Res, 1996 Jul, 104, 38 - 51
Adherence & colonization properties of Vibrio cholerae & diarrhoeagenic Escherichia coli; Ghose AC; Bacterial adherence to host cells is the initial key step towards colonization and establishment of infection within the host . The adherence process requires the participation of two components: an 'adhesin' (adherence or colonization factor) of bacteria and a 'receptor' on the host (eucaryotic) cell surface . Many bacteria express several distinct and alternative mechanisms of cell adherence depending on the environmental conditions and nature of the adhesins as well as receptors . Bacteria causing gastrointestinal infection need to penetrate the mucous layer before attaching themselves to epithelial and other absorptive cells in the intestine . This attachment is usually mediated by fimbriae or pilus structures although other cell surface components of bacteria may also take part in the process . Adherent bacteria colonize intestinal epithelium by multiplication and initiation of a series of biochemical reactions inside the target cell through signal transduction mechanisms (with or without the help of toxins) . Alternatively, adherent bacteria induce extensive rearrangement of the cytoskeletal structure of the epithelial cell thereby making more intimate contact with the cell or even forcing their entry into it . This is followed by bacterial multiplication and intercellular spread leading to eventual death of the target cell . Available information on the adherence and colonization properties of V . cholerae and E . coli, the two important causative agents of gastrointestinal illness in man, is discussed and summarized in this article.

Indian J Med Res, 1996 Jul, 104, 14 - 27
Epidemiology & molecular biology of Vibrio cholerae O139 Bengal; Albert MJ; The emergence of Vibrio cholerae O139 Bengal as the second aetiologic agent of epidemic cholera in October 1992 in the south Indian coastal city of Madras has shattered the long-held notion that only V . cholerae belonging to serogroup O1 are capable of causing epidemic (and pandemic) cholera . Within months of its appearance in Madras, V . cholerae O139 engulfed the entire Indian subcontinent in a series of outbreaks of cholera . It also spread to several neighbouring countries in Asia . Several western countries also reported imported cholera cases due to this organism . In the regions of the Indian subcontinent where cholera due to V . cholerae O1 is endemic, children are mostly susceptible because adults would have acquired at least some immunity due to earlier exposure . However, when V . cholerae O139 struck people in these areas, even though all age groups were affected, the disease was more prevalent in adults, which suggested that the disease is new in this population . As with O1 cholera, water and food seemed to be the vehicles of infection . Many family contracts of index cases of O139 cholera were found to be infected with V . cholerae O139, and in many of them, the infection was asymptomatic which is reminiscent of O1 EITor infection . Again as with O1 EITor infection, individuals of blood group O were more susceptible to O139 infection than those with other blood groups . In its molecular aspects, O139 vibrio resembles O1 EITor vibrio . The virulence genes encoding cholera toxin, zonula occludens toxin, accessory cholera enterotoxin and core-encoded pilin are present in a 4.5 kb 'virulence cassette' region of the chromosome as in EITor vibrios and the expression of these virulence factors, toxin coregulated pilus (TCP) and several outer membrane proteins are found to be under the control of the master regulator ToxR as in EITor vibrios . The iron-regulated genes involved in virulence are also found in the same locus as in EITor vibrios . However, the genes involved in the somatic antigen synthesis in O1 vibrios are found to be deleted in O139 vibrios and are replaced by a new region of chromosome which encodes the new surface antigen synthesis in O139 vibrios . When V . cholerae O139 emerged and caused outbreaks, the prevailing O1 EITor vibrios virtually disappeared from most of the areas . The disappearance of EITor vibrios, the rapid spread of O139 vibrios and the resemblance of O139 vibrios to EITor vibrios seemed to suggest that O139 vibrios might be the causative agent of the 'eighth' pandemic of cholera . However, after a year of its appearance, O139 vibrios are on the wane and O1 EITor vibrios have re-emerged as the predominant organism, in the Indian subcontinent . Thus, the immediate threat of a new cholera pandemic posed by V . cholerae O139 may not be as large as it first seemed . However, whether it will follow the pattern of EITor vibrio which took approximately 60 yr since its first isolation before emerging as the seventh pandemic strain of cholera, is not clear . The factor(s) contributing to the diminished isolation of O139 vibrios and the re-emergence of O1 EITor vibrios are not understood . The vibrios might have undergone changes that would have affected their ability to survive and compete in the environment.

Indian J Med Res, 1996 Jul, 104, 5 - 13
Molecular insights into the evolution & epidemiology of Vibrio cholerae; Nair GB; The past few years have witnessed a resurgence in the global incidence of cholera . The increasing application of procedures employing concepts and techniques assimilated from molecular biology has provided new means of discriminating V . cholerae . Such studies are providing a wealth of critical information that has assisted the epidemiologist in tracing the spread of epidemics and has provided new insights into the evolution and origin of newer variants of V . cholerae . In this article, an effort is made to collect all the recent information and present the impact this new information has made on our understanding of V . cholerae.

Appl Environ Microbiol, 1996 Jul, 62(7), 2331 - 7
Toxic and enzymatic activities of Vibrio vulnificus biotype 2 with respect to host specificity; Biosca EG et al.; In this work, the enzymatic activities of selected strains of biotypes 1 and 2 of Vabrio vulnificus were analyzed by using conventional methods and the API ZYM system . The toxic activities of extracellular products (ECPs) were further evaluated by in vitro and in vivo experiments . The ECPs of both biotypes (i) showed high-level hydrolytic activities, (ii) displayed cytotoxicity for fish cell lines, and (iii) were lethal for eels . Exotoxins seem to be proteinaceous since heat treatment of ECP samples destroyed their toxicity . Only biotype 2 strains were virulent for cels, suggesting that host specificity must be related to differences in cell surface properties . Infectivity trials with other fish species also revealed that only biotype 2 strains were virulent.

Appl Environ Microbiol, 1996 Jul, 62(7), 2252 - 6
Oxidative stress detection with Escherichia coli harboring a katG'::lux fusion; Belkin S et al.; A plasmid containing a transcriptional fusion of the Escherichia coli katG promoter to a truncated Vibrio fischeri lux operon (luxCDABE) was constructed . An E . coli strain bearing this plasmid (strain DPD2511) exhibited low basal levels of luminescence, which increased up to 1,000-fold in the presence of hydrogen peroxide, organic peroxides, redox-cycling agents (methyl viologen and menadione), a hydrogen peroxide-producing enzyme system (xanthine and xanthine oxidase), and cigarette smoke . An oxyR deletion abolished hydrogen peroxide-dependent induction, confirming that oxyR controlled katG'::lux luminescence . Light emission was also induced by ethanol by an unexplained mechanism . A marked synergistic response was observed when cells were exposed to both ethanol and hydrogen peroxide; the level of luminescence measured in the presence of both inducers was much higher than the sum of the level of luminescence observed with ethanol and the level of luminescence observed with hydrogen peroxide . It is suggested that this construction or similar constructions may be used as a tool for assaying oxidant and antioxidant properties of chemicals, as a biosensor for environmental monitoring and as a tool for studying cellular responses to oxidative hazards.

J Struct Biol, 1996 Jul-Aug, 117(1), 70 - 2
Crystallization and preliminary crystallographic analysis of the major NAD(P)H: FMN oxidoreductase of Vibrio fischeri ATCC 7744; Koike H et al.; The major flavin reductase from Vibrio fischeri, FRase I, has been crystallized in the presence of FMN by the vapor diffusion method using polyethylene glycol 4000 as a precipitant . The crystals belonged to the monoclinic space group C2 with unit cell dimensions, a = 101.6 A, b = 63.2 A, c = 74.4 A, and beta = 100.0 degrees . The crystals are expected to contain two FRase I molecules per asymmetric unit . The crystals diffracted X-rays to at least 2.2 A resolution and are appropriate for structural analysis at high resolution.

Can J Microbiol, 1996 Jul, 42(7), 662 - 71
Expression of the Escherichia coli chromosomal ars operon; Cai J et al.; A chromosomally located operon (ars) of Escherichia coli has been previously shown to be functional in arsenic detoxification . DNA sequencing revealed three open reading frames homologous to the arsR, arsB, and arsC open reading frames of plasmid-based arsenic resistance operons isolated from both E . coli and staphylococcal species . To examine the outline of transcriptional regulation of the chromosomal ars operon, several transcriptional fusions, using the luciferase-encoding luxAB genes of Vibrio harveyi, were constructed . Measurement of the expression of these gene fusions demonstrated that the operon was rapidly induced by sodium arsenite and negatively regulated by the trans-acting arsR gene product . Northern blotting and primer extension analyses revealed that the chromosomal ars operon is most likely transcribed as a single mRNA of approximately 2100 nucleotides in length and processed into two smaller mRNA products in a manner similar to that found in the E . coli R773 plasmid-borne ars operon . However, transcription was found to initiate at a position that is relatively further upstream of the initiation codon of the arsR coding sequence than that determined for the E . coli R773 plasmid's ars operon.

FEMS Microbiol Lett, 1996 Jul 1, 140(2-3), 287 - 94
Construction and use of a new vector/transposon, pLBT::mini-Tn10:lac:kan, to identify environmentally responsive genes in a marine bacterium; Albertson NH et al.; The previously described pLOFKm transposon delivery plasmid (J.Bacteriol . (1990) 172, 6557-6567) was engineered such that a promotorless lacZ gene was cloned within the transposon cassette, generating the vector pLBT . Using pLBT, stable insertion mutations were generated at high frequencies in Vibrio sp . S141 and Pseudomonas sp . S91, and the interrupted genes could be monitored for their pattern of regulation . Genetic screens isolated mutants defective in a variety of activities . We describe the construction and use of pLBT as a tool for reporter gene mutant analysis in bacteria other than well-characterized laboratory strains.

J Bacteriol, 1996 Jul, 178(14), 4182 - 8
Characterization of a porin from the outer membrane of Vibrio anguillarum; Simon M et al.; The outer membranes of the 10 serovars of Vibrio anguillarum showed a common major protein with a size of around 40 kDa . Antibodies against the major outer membrane protein (MOMP) of V . anguillarum AO18 (serovar O1) cross-reacted with the MOMPs of all the other serovars but not with the outer membrane proteins of Escherichia coli . The MOMP of V . anguillarum serovar O1 was isolated, reconstituted to two-dimensional crystals, and structurally characterized by electron microscopy and image processing . The unit cell structure of the crystalline MOMP, as well as the secondary structure composition of the protein with a high amount of beta-structure, is strongly reminiscent of that of bacterial porins . The functional properties of the pores were investigated by conductance measurements with the MOMP reconstituted in planar lipid membranes . The V . anguillarum MOMP is characterized by a relatively weak cation selectivity and a moderate surface charge, and it shows voltage-dependent conductance effects . The MOMP is functionally similar to OmpF from E . coli, and it can be classified as a general diffusion porin.

J Bacteriol, 1996 Jul, 178(14), 4157 - 65
A new type of conjugative transposon encodes resistance to sulfamethoxazole, trimethoprim, and streptomycin in Vibrio cholerae O139; Waldor MK et al.; Vibrio cholerae O139 is the first non-O1 serogroup of V . cholerae to give rise to epidemic cholera . Apparently, this new serogroup arose from an El Tor O1 strain of V cholerae, but V . cholerae O139 is distinguishable from V . cholerae El Tor O1 by virtue of its novel antigenic structure and also its characteristic pattern of resistances to the antibiotics sulfamethoxazole, trimethoprim, streptomycin, and furazolidone . We found that the first three of these antibiotic resistances are carried on an approximately 62-kb self-transmissible, chromosomally integrating genetic element which we have termed the SXT element . This novel conjugative transposon-like element could be conjugally transferred from V . cholerae O139 to V cholerae O1 and Escherichia coli strains, where it integrated into the recipient chromosomes in a site-specific manner independent of recA . To study the potential virulence properties of the SXT element as well as to improve upon the live attenuated O139 vaccine strain Bengal-2, a large internal deletion in the SXT element was crossed on to the Bengal-2 chromosome . The resulting strain, Bengal-2.SXT(s), is sensitive to sulfamethoxazole and trimethoprim and colonizes the intestines of suckling mice as well as wild-type strains do, suggesting that the SXT element does not encode a colonization factor . Derivatives of Bengal-2.SXT(s) are predicted to be safe, antibiotic-sensitive, live attenuated vaccines for cholera due to the O139 serogroup.

J Am Osteopath Assoc, 1996 Jul, 96(7), 432 - 3
Acute appendicitis secondary to non-0 group I Vibrio cholerae; Cook MA et al.; Acute appendicitis is the most common abdominal surgical condition and is usually associated with colonic flora . The patient described had acute appendicitis associated with an uncommon microorganism . This report underscores the importance of obtaining an adequate occupational, travel, and dietary history.

Microbiology, 1996 Jul, 142 ( Pt 7), 1675 - 84
Analysis of starvation conditions that allow for prolonged culturability of Vibrio vulnificus at low temperature; Paludan-Muller C et al.; The response of the estuarine human pathogen Vibrio vulnificus to starvation for carbon, nitrogen or phosphorus, or all three nutrients simultaneously (multiple-nutrient), was examined with respect to the maintenance of culturability during incubation at low temperature . V . vulnificus showed similar survival patterns during starvation for the individual nutrients when kept at 24 degrees C . On the other hand, cultures prestarved at 24 degrees C and then shifted to 5 degrees C maintained culturability at low temperature in a starvation-condition-dependent manner . Carbon and multiple-nutrient starvation were indistinguishable in their ability to mediate maintenance of culturability in the cold . Prolonged starvation for phosphorus had a similar effect, but nitrogen starvation did not allow for maintenance of culturability . Extracellular factors produced during starvation were not observed to have an effect on the culturability of cells incubated at low temperature . Protein synthesis during starvation for individual nutrients was analysed by two-dimensional PAGE of pulse-labelled proteins . Carbon and multiple-nutrient starvation gave nearly identical protein induction patterns involving at least 34 proteins, indicating that carbon starvation determines both responses . Nitrogen starvation for 1 h induced 24 proteins, while phosphorus starvation induced a set of 10 proteins after 1 h and about 40 proteins after 18 h . It is suggested that starvation for carbon or phosphorus induces maintenance of culturability of V . vulnificus incubated at low temperature via the synthesis of distinct sets of starvation-specific proteins.

Infect Immun, 1996 Jul, 64(7), 2873 - 6
Pulmonary damage by Vibrio vulnificus cytolysin; Park JW et al.; Vibrio vulnificus is an estuarine bacterium that causes septicemia and serious wound infection . Cytolysin produced by V . vulnificus has been incriminated as one of the important virulence determinants of bacterial infection . Cytolysin (8 hemolytic units) given intravenously to mice via their tail veins caused severe hemoconcentration and lethality . Cytolysin treatment greatly increased pulmonary wet weight and vascular permeability as measured by (125)I-labeled albumin leakage without affecting those factors of other organs significantly . Blood neutrophils were markedly decreased in number after cytolysin injection, with a concomitant increase in the level of pulmonary myeloperoxidase activity, indicating that cytolysin-induced neutropenia might be due to pulmonary sequestration of neutrophils . By microscopic examination, severe perivascular edema and neutrophil infiltration were evident in lung tissues . These results suggest that increased vascular permeability and neutrophil sequestration in the lungs are important factors in lethal activity by cytolysin.

Infect Immun, 1996 Jul, 64(7), 2853 - 6
Toxin-coregulated pilus, but not mannose-sensitive hemagglutinin, is required for colonization by Vibrio cholerae O1 El Tor biotype and O139 strains; Thelin KH et al.; The relative contributions of toxin-coregulated pilus (TCP) and cell-associated mannose-sensitive hemagglutinin (MSHA) to the colonization ability of Vibrio cholerae O1 El Tor biotype strains and O139 Bengal strains was determined by using isogenic parental and in-frame deletion mutant pairs in the infant mouse cholera model . Both the El Tor and O139 tcpA mutant strains showed a dramatic defect in colonization as indicated by their competitive indices, whereas deletion of mshA had a negligible effect on colonization in either background.

Infect Immun, 1996 Jul, 64(7), 2834 - 8
Role of catechol siderophore synthesis in Vibrio vulnificus virulence; Litwin CM et al.; We isolated a Vibrio vulnificus TnphoA mutant that was unable to produce catechol siderophores or to acquire iron from transferrin . This mutant showed reduced virulence in an infant mouse model . The TnphoA insertion was in an open reading frame designated venB . The venB gene cloned on a plasmid restored catechol production to the mutant . The deduced amino acid sequence of venB is 41% identical to the enzyme isochorismatase of Escherichia coli (EntB), an enzyme involved in the biosynthesis of the catechol siderophore enterobactin.

J Med Microbiol, 1996 Jul, 45(1), 35 - 9
Haemolysin produced by Vibrio cholerae non-O1 is not enterotoxic; Singh DV et al.; Of 28 isolates of Vibrio cholerae non-O1 (10 from diarrhoeal patients and 18 from environmental sources) examined for haemolytic activity and its correlation, if any, with enterotoxic activity, 24 showed haemolysis . The four non-haemolytic isolates showed haemolysis after consecutive passages through rabbit ileal loops (RILs) . The titres of haemolytic activity were 4-64 HU/ml irrespective of their source . Eight (28.5%) of the non-O1 isolates caused fluid accumulation; six (25%) were haemolytic and two (50%) non-haemolytic . The remaining isolates showed enterotoxic activity after one-to-three consecutive passages through RILs irrespective of their haemolytic character and source . Environmental isolates caused significantly more fluid accumulation than the diarrhoeal isolates . All these isolates reverted to their original non-toxigenic character on repeated subculture or on storage in the laboratory, but continued to show haemolytic activity . The results of the present study indicate that V . cholerae non-O1 strains are potentially enterotoxigenic independent of their haemolytic character and source, and enterotoxin, not haemolysin, is the factor most likely to be responsible for their enterotoxic activity.

J Med Microbiol, 1996 Jul, 45(1), 31 - 4
Production of the new cholera toxin by environmental isolates of Vibrio cholerae non-O1; Singh DV et al.; One of five strains of Vibrio cholerae non-O1 isolated from environmental sources caused fluid accumulation in an initial rabbit ileal loop (RIL) test . The four strains that caused little or no accumulation of fluid gave a positive response after one-to-three consecutive passages through RILs . The amount of fluid produced increased after each passage . Filtrates of cultures of all five environmental isolates caused fluid accumulation similar to that produced by live cells . The enterotoxin showed a precipitin band with new cholera antitoxin and was neutralised completely by new cholera antitoxin diluted 1 in 32, indicating its close immunobiological relationship to the new cholera toxin . The present study indicates that V . cholerae non-O1 strains produce an enterotoxin that is similar to the new cholera toxin.

Science, 1996 Jun 28, 272(5270), 1910 - 4
Lysogenic conversion by a filamentous phage encoding cholera toxin; Waldor MK et al.; Vibrio cholerae, the causative agent of cholera, requires two coordinately regulated factors for full virulence: cholera toxin (CT), a potent enterotoxin, and toxin-coregulated pili (TCP), surface organelles required for intestinal colonization . The structural genes for CT are shown here to be encoded by a filamentous bacteriophage (designated CTXphi), which is related to coliphage M13 . The CTXphi genome chromosomally integrated or replicated as a plasmid . CTXphi used TCP as its receptor and infected V . cholerae cells within the gastrointestinal tracts of mice more efficiently than under laboratory conditions . Thus, the emergence of toxigenic V . cholerae involves horizontal gene transfer that may depend on in vivo gene expression.

Carbohydr Res, 1996 Jun 21, 287(2), 225 - 45
Structural studies of the Vibrio salmonicida lipopolysaccharide; Edebrink P et al.; The oligosaccharide part of the Vibrio salmonicida (strain NCMB 2262) lipopolysaccharide was isolated by mild acid hydrolysis followed by gel-permeation chromatography . The structure was established mainly by methylation analysis, mass spectrometry, and NMR spectroscopy . It is concluded that the oligosaccharide has the following structure, in which L-alpha-D-Hep p is L-glycero-alpha-D-manno-heptopyranose, D-alpha-D-Hepp is D-glycero-alpha-D-manno-heptopyranose, alpha-D-Fuc p4N is 4-amino-4,6-dideoxy-alpha-D-galactopyranose, alpha-NonA is 5-acetamidino-7-acetamido-3,5,7, 9-tetradeoxy-L-glycero-alpha-D-galacto-nonulosonic acid, BA is (R)-3-hydroxybutanoyl, and PEA is phosphoethanolamine . The substitution pattern of the branching heptosyl residue was deduced from 1H NMR chemical shifts and conformations of the branching region, obtained by molecular modelling . The absolute configuration for NonA was determined by NMR spectroscopy from NOE correlations to the neighbouring sugar and 13C NMR chemical shift data . It could also be shown that assignments of nonulosonic acids with the D-glycero-L-galacto configuration, reported by previous investigators, are erroneous and should be changed to L-glycero-D-galacto . The oligosaccharide is assumed to be linked to the 5-position of a Kdo residue, phosphorylated in the 4-position as observed for other lipopolysaccharides from Vibrionaceae . {formula: see text}

J Mol Biol, 1996 Jun 21, 259(4), 687 - 95
Rotational fluctuation of the sodium-driven flagellar motor of Vibrio alginolyticus induced by binding of inhibitors; Muramoto K et al.; Rotation of the Na(+)-driven flagellar motor of Vibrio alginolyticus was investigated under the influence of inhibitors specific to the motor, amiloride and phenamil . The rotation rate of a single flagellum on a cell stuck to a glass slide was examined using laser dark-field microscopy . In the presence of 50 mM NaCl, the average rotation rate (omega) was about 600 r.p.s . with a standard deviation (sigma omega) of 9% of omega . When omega was decreased to about 200 r.p.s . by the presence of 1.5 mM amiloride, sigma omega increased to 15% of omega . On the other hand, when omega was decreased to about 200 r.p.s . by the addition of 0.6 microM phenamil, a large increase in sigma omega up to 50% of omega, was observed . Similarly large fluctuations were observed at other concentrations of phenamil . These observations suggest that dissociation of phenamil from the motor was much slower than that of amiloride . A very low concentration of phenamil caused a transient but substantial reduction in rotation rate . This might suggest that binding of only a single molecule of phenamil strongly inhibits the torque generation in the flagellar motor.

Structure, 1996 Jun 15, 4(6), 665 - 78
Crystal structure of a new heat-labile enterotoxin, LT-IIb; van den Akker F et al.; BACKGROUND: Cholera toxin from Vibrio cholerae and the type I heat-labile enterotoxins (LT-Is) from Escherichia coli are oligomeric proteins with AB5 structures . The type II heat-labile enterotoxins (LT-IIs) from E . coli are structurally similar to, but antigenically distinct from, the type I enterotoxins . The A subunits of type I and type II enterotoxins are homologous and activate adenylate cyclase by ADP-ribosylation of a G protein subunit, G8 alpha . However, the B subunits of type I and type II enterotoxins differ dramatically in amino acid sequence and ganglioside-binding specificity . The structure of LT-IIb was determined both as a prototype for other LT-IIs and to provide additional insights into structure/function relationships among members of the heat-labile enterotoxin family and the superfamily of ADP-ribosylating protein toxins . RESULTS: The 2.25 A crystal structure of the LT-IIb holotoxin has been determined . The structure reveals striking similarities with LT-I in both the catalytic A subunit and the ganglioside-binding B subunits . The latter form a pentamer which has a central pore with a diameter of 10-18 A . Despite their similarities, the relative orientation between the A polypeptide and the B pentamer differs by 24 degrees in LT-I and LT-IIb . A common hydrophobic ring was observed at the A-B5 interface which may be important in the cholera toxin family for assembly of the AB5 heterohexamer . A cluster of arginine residues at the surface of the A subunit of LT-I and cholera toxin, possibly involved in assembly, is also present in LT-IIb . The ganglioside receptor binding sites are localized, as suggested by mutagenesis, and are in a position roughly similar to the sites where LT-I binds its receptor . CONCLUSIONS: The structure of LT-IIb provides insight into the sequence diversity and structural similarity of the AB5 toxin family . New knowledge has been gained regarding the assembly of AB5 toxins and their active-site architecture.

Biochim Biophys Acta, 1996 Jun 11, 1281(2), 220 - 6
Nature of the cation leak induced in erythrocyte membranes by Kanagawa haemolysin of Vibrio parahaemolyticus; Huntley JS et al.; Vibrio parahaemolyticus is an important enteric pathogen that produces an exotoxin prepared as Kanagawa haemolysin (KH) . Isotope flux techniques were used to analyse toxin action on the basal permeability of human erythrocytes . KH induced a cation leak that was (i) rapid in onset (lag phase < 1 min), (ii) 'pore-like' in terms of kinetic characteristics, and (iii) of high magnitude initially (first 10 min) and then subsequently lower (but still raised with reference to control cells) . The susceptibilities of the induced flux pathway to washout in initial and later periods suggested a protracted binding time course for toxin action . Neuraminidase treatment of erythrocytes enhanced both haemolysis and flux induced by KH, suggesting that the affinity of the toxin for the membrane had increased, possibly as a result of additional toxin receptors being unmasked by this enzyme . These results show that KH elevates the basal permeability of human erythrocytes in a complex manner, a process that probably underlies the deleterious effects of this toxin on cellular function.

FEBS Lett, 1996 Jun 3, 387(2-3), 167 - 70
Efficient expression, processing and secretion of a biologically active mammalian protein by Vibrio cholerae; Ghorpade A et al.; The use of Vibrio cholerae as a secretory expression system for the expression of a mammalian protein, namely human growth hormone, under the control of the heat labile enterotoxin chain B signal sequence is reported . The protein is efficiently expressed and processed . The mature protein is exported to the periplasm after which it is secreted to the extracellular milieu . The expressed and secreted hGH actively binds to its receptor as established by its receptor binding activity . The biological activity of the protein is demonstrated in vitro in a Nb2 proliferation assay.

Antibiot Khimioter, 1996 Jun, 41(6), 29 - 33
{Dynamics of changes in antibiotic sensitivity of Vibrio cholerae 01, isolated from environmental objects}; Khaitovich AB et al.; The analysis of the dynamics of the antibiotic susceptibility of 442 strains of V . cholerae 01 isolated within 1986-1994 from the environment showed that the susceptibility level was different . Strains of V . cholerae 01 with high susceptibility to tetracyclines, erythromycin, gentamicin, rifampicin and cefazolin and with moderate susceptibility to monomycin, kanamycin, ampicillin, carbenicillin and levomycetin (chloramphenicol) were detected as well as the strains resistant to streptomycin and polymyxin B . The susceptibility of the V . cholerae 01 strains to certain antibiotics (tetracyclines, levomycetin, streptomycin) changed in regard to the isolation time and object and its geographical location . In 1991-1994 a tendency was observed towards an increase in the number of the strains resistant to the drugs . The resistance of the isolates from the objects connected with the vital activity of humans (sewage, washings from the cholera foci, fish from polluted water reservoirs) was higher than that of the isolates from open water reservoirs . There were definite difficulties in the detection of the cultures with antibiotic susceptibility characteristic of various regions because of their different origin . The results of the study were indicative of a necessity of monitoring of the biological properties of V . cholerae 01 isolates from the environment important for cholera control.

Antibiot Khimioter, 1996 Jun, 41(6), 25 - 8
{Antibiotic sensitivity of Vibrio cholerae 01, isolated in the Ukraine in 1994}; Khaitovich AB et al.; One thousand and four hundred strains of V . cholerae 01 isolated in 1994 in the Ukraine were studied with respect to their antibiotic susceptibility and resistance . The study showed that it was possible not only to estimate the present tendencies in and the regularities of the change in their character but also to presuppose the probable circulation and incidence of the microbe based on the differences in the susceptibility, frequency and resistance pattern of the strains of V . cholerae 01 isolated from the environment and humans before and during the cholera outbreak . Unlike the strains of V . cholerae 01 isolated from the environment before the outbreak, the strains isolated during the outbreak from the environment and humans were characterized by resistance to levomycetin (chloramphenicol) and streptomycin . The results suggested that the cholera outbreak in 1994 was incidental . The data are useful for cholera epidemic surveillance . However, the final conclusion is possible after investigation of the gene type pattern in the circulating V . cholerae strains.

Genetika, 1996 Jun, 32(6), 744 - 9
{Emergence of toxigenic Vibrio cholerae strains on non-O1 serotype as a result of the exchange of genetic information}; Smirnova NI et al.; To study the possibilities of genetic exchange between Vibrio cholerae of O1 and non-O1 serogroups, donor and recipient strains were developed . It was shown that toxicogenic strains of V . cholerae non-O1 appeared in vitro and in vivo as the result of conjugative transfer of rfb-NAG genes from avirulent V . cholerae non-O1 strains to toxicogenic strains belonging to V . cholerae O1 classical and eltor biovars . These genes are responsible for synthesis of O antigen of non-O1 serotype . It was established that foreign rfb-NAG genes have no effect on virulence properties of a causative agent of cholera . Apparently, pathogenic V . cholerae non-O1 strains with cholera toxin genes are generated due to transfer of rfb-NAG genes under natural conditions.

J Diarrhoeal Dis Res, 1996 Jun, 14(2), 113 - 6
Factors affecting production of haemolysin by strains of Vibrio fluvialis; Rahim Z et al.; The in vitro production of haemolysin by Vibrio fluvialis was studied using sheep erythrocyte . The effect of the composition of various media and different concentrations of sodium chloride on the production of haemolysin and heat-stability was investigated . Comparatively higher titre of haemolysin production was noted in brain heart infusion (BHI) broth . Adding 0.5% NaCl to BHI broth reduced the production of haemolysin; adding 5.0% NaCl to the medium totally inhibited it . The highest titre of haemolysin was produced at 30 degrees C and 37 degrees C, whereas no haemolysin was produced at 50 degrees C . Haemolytic activity was totally destroyed when heated at 56 degrees C for 30 minutes . Haemolysin could be assayed easily following this method.

J Diarrhoeal Dis Res, 1996 Jun, 14(2), 107 - 9
Unusual occurrence of cholera in Delhi during January 1994: epidemiological investigations; Singh J et al.; Hundreds of laboratory-confirmed cholera cases occur every year in Delhi . However from 1965 through 1993, no cases of cholera nor carriers of Vibrio cholerae have been detected in the months January and February of all these years . Nevertheless, two cases occurred in January 1994 . Both were children who acquired their infection locally . Six hundred fifty-eight rectal swabs collected from possible contacts were negative for V . cholerae . The next isolations could be made only in April, which is the usual beginning of the cholera season . The study suggests that cholera transmission can occur during the winter months in Delhi, but that it is not sustained.

FEMS Immunol Med Microbiol, 1996 Jun, 14(2-3), 67 - 72
Development of an improved synthetic medium for a better production of the new cholera toxin and its immunological relationship with the toxin produced by Vibrio cholerae O139 strains; Tikoo A et al.; An improved synthetic medium (M4) comprising syncase medium supplemented with sodium chloride (1%) and sucrose (0.5%) pH adjusted to 7.4 was developed for a better production of the new cholera toxin (NCT) . The culture filtrates prepared in the M4 medium caused significantly (P > 0.05) more fluid accumulation than that in syncase medium . Crude toxin, prepared in the M4 medium with V . cholerae O1 strains (X-392 and 2740-80) caused a reaction similar to that of the same amount of NCT (32 micrograms) prepared in the syncase medium . The neutralization of the optimal loop reacting dose of the NCT prepared in the M4 medium by anti-NCT raised against syncase prepared toxin indicates the release of the same kind of toxin in both media . These observations indicate that the modified M4 medium may be used for NCT preparation and further characterization . All the strains of Vibro cholerae O139 used in this study produced a toxin antigenically similar to NCT.

Infect Control Hosp Epidemiol, 1996 Jun, 17(6), 371 - 2
Nosocomial infection due to Vibrio cholerae in two referral hospitals in Guatemala; Hernandez JE et al.; We report nosocomial infection with Vibrio cholerae 01, in four seriously ill individuals and one infant in Guatemala . Nosocomial cholera occurs in developing countries in Latin America and should be suspected in hospitalized patients with diarrhea, especially during community outbreaks, in order to institute appropriate diagnostic, therapeutic, and control measures.

Ecotoxicol Environ Saf, 1996 Jun, 34(1), 76 - 84
A mixture toxicity study employing combinations of tributyltin chloride, dibutyltin dichloride, and tin chloride using the marine bacterium Vibrio harveyi as the test organism; Thomulka KW et al.; Mixture toxicity studies in dual combinations for three metals, tributyltin chloride, dibutyltin dichloride, and tin chloride, and one experiment using all chemicals were performed using the bioluminescent marine bacterium Vibrio harveyi as the test organism in a direct toxicity test procedure . Combination toxicity was evaluated using an additive index equation method and two isopleth procedures, isobole plot and isobologram . Additive index values were determined at both estimated effective median concentration (EC50) and one-third EC50 values for each dual combination and all three chemicals for one-third EC50 values . Isopleths employed chemical combinations at 20% intervals of the EC50 concentrations . Additive index values for various mixtures were either additive or antagonistic (less than additive) . Isobolograms for all mixtures were descriptively additive or synergistic (greater than additive) . Isobole plots were also descriptively additive or synergistic, although a few measurements were statistically different for synergism . Statistical evaluation between mixtures and single values, using the z test, were in some cases different at the 5% level . Bioluminescent counts were determined to be normally distributed using a statistical test for small sample numbers at the 1% level . Evaluation for outliers, using the Dixon test, was also performed and found one mixture to have an outlier . This single outlier had no influence on the combined toxicity results . The use of low-cost and rapid bioluminescent microbial toxicity tests for mixture studies is discussed.

Glycoconj J, 1996 Jun, 13(3), 377 - 84
Gangliosides protect human melanoma cells from ionizing radiation-induced clonogenic cell death; Thomas CP et al.; With an experimental model of spontaneous lung metastases of melanoma developed in this laboratory, a range of sublines (variants and clones) with different metastatic potential and ganglioside expression was established from a single human melanoma cell line M4Be . Using an in vitro clonogenic assay and provided that cells were cultured for no more than five passages, variations in cellular radioresistance of M4Be and seven sublines derived from M4Be were detected . This study shows a positive correlation between the cell intrinsic radioresistance of M4Be and its seven sublines and their total ganglioside content . More precisely, the proportion of radioresistant cells in M4Be and the seven sublines correlated with the number of cells determined by flow cytometry that were positively labelled with a monoclonal antibody directed to GD3 disialoganglioside . Blocking the cellular biosynthesis of gangliosides with the inhibitor Fumonisin B1 or cleaving with Vibrio cholerae neuraminidase the cell surface ganglioside-bound sialic acid in a radioresistant poorly metastatic subline increased its radiosensitivity in vitro . In contrast, enrichment of a radiosensitive metastatic subline with exogenous bovine brain GM1 increased its radioresistance in vitro . These results suggest that, in the radiation dose range important for radioprotection (0-1 Gy), membrane gangliosides radioprotect human melanoma cells in vitro.

Drugs, 1996 Jun, 51(6), 966 - 73
Practical guidelines for the treatment of cholera; Seas C et al.; Cholera is a dramatic clinical illness that requires rapid diagnosis and aggressive therapy . Clinical signs and symptoms of mild, moderate and severe dehydration must be determined, before beginning fluid therapy . Fluid therapy has 2 phases: rehydration (first 3 to 4 hours to correct deficits) and maintenance (to match continuing losses) . The route and speed of fluid administration will depend on the degree of dehydration . Patients with severe dehydration should be treated intravenously, as should those patients who do not tolerate oral rehydration solution (ORS) . Ringer's lactate is the preferred intravenous solution, although normal saline may be used along with ORS . For most patients with cholera, an ORS using one of the higher sodium-containing solutions and plain water optimally provide the fluid and salt needed . Close monitoring of intake, outputs and hydration status should be performed for all patients . Antimicrobial therapy should be given to moderately and severely ill patients in order to decrease the volume of fluids lost and to shorten the period of excretion of vibrios.

J Clin Microbiol, 1996 Jun, 34(6), 1535 - 9
Subspecies typing of Vibrio parahaemolyticus by pulsed-field gel electrophoresis; Wong HC et al.; Vibrio parahaemolyticus is one of the most important food-borne pathogens in Taiwan, Japan, and other costal regions . We report on the development of a pulsed-field gel electrophoresis (PFGE) method for the molecular typing of this pathogen . Genomic DNA was digested with SfiI, and the fragments were resolved on 1% agarose with a contour-clamped homogeneous electric field apparatus set at 190 V and a pulse time of 3 to 80 s . A total of 130 selected isolates obtained from outbreaks during 1993 and 1994 on Taiwan were also characterized by this PFGE method . These isolates were grouped into 14 PFGE types which consisted on one to six patterns, and a total of 39 patterns were identified . Most of these domestic clinical isolates could be clustered into several major types (types A, B, C, and G) . These major types showed relatively low degrees of similarity to several foreign strains and other domestic but environmental strains . Strain CCRC12863, which originated from Japan, was close to the group consisting of F, G, and H PFGE types, suggesting a clonal relationship between this Japanese strain and other domestic isolates.

J Clin Microbiol, 1996 Jun, 34(6), 1453 - 61
DNA fingerprinting of Vibrio cholerae strains with a novel insertion sequence element: a tool to identify epidemic strains; Bik EM et al.; A novel Vibrio cholerae insertion sequence element, designated IS1004, was characterized and used for DNA fingerprinting of Vibrio spp . IS1004 comprises 628 bp and contains an open reading frame whose product shows a large degree of sequence identity with the IS200-encoded transposase . IS1004 was present in one to eight copies in most of the V . cholerae strains analyzed . The IS1004-generated fingerprints of epidemic V . cholerae strains with serotype O1 were closely related, although it was possible to distinguish between the two biotypes, classical and El Tor . Non-O1 serotype strains generally showed heterogeneous patterns unrelated to those of the epidemic O1 strains . Several strains were observed with identical or related fingerprint patterns but expressed different serotypes . Conversely, strains with different fingerprint patterns but identical serotypes were also found . These observations indicate that the gene clusters coding for distinct O antigens may be transferred horizontally between V . cholerae strains . Two examples of non-O1 strains with a fingerprint resembling that of epidemic O1 strains were found; they were the O139 Bengal strain and an O37 strain . The O139 Bengal strain is closely related to the El Tor biotype . The O37 strain was responsible for a large cholera outbreak in Sudan in 1968 and was classified as a noncholera vibrio . Our study, however, shows that the O37 Sudan strain is genetically closely related to classical O1 strains . Similar to O139 Bengal, O37 Sudan lacked most of the O1 antigen cluster but did contain flanking genes . Thus, O37 Sudan represents a second example of an epidemic V . cholerae strain carrying non-O1 antigens . This study underlines the importance of genotypic methods for the differentiation of V . cholerae strains and for recognition of strains with epidemic potential.

Microbiology, 1996 Jun, 142 ( Pt 6), 1499 - 504
An immunochemical study of serological cross-reaction between lipopolysaccharides from Vibrio cholerae O22 and O139; Isshiki Y et al.; A comparative chemical and serological study of the LPS of Vibrio cholerae O139 and O22 was performed . Chemical analysis revealed that the sugar composition of the LPS of strain O22 was quite similar to that of O139 LPS . Each contained D-glucose, L-glycero-D-manno-heptose, colitose (3,6-dideoxy-L-galactose), D-fructose, D-glucosamine, D-quinovosamine and D-galacturonic acid . The O-antigenic relationship between the two strains was analysed by passive haemolysis (PH) and passive haemolysis inhibition (PHI) tests with the respective LPS being used as antigens to sensitize sheep red blood cells (SRBC) and, in the latter case, as inhibitors in a PH system that consisted of LPS-sensitized SRBC, guinea-pig complement and anti-O139 or anti-O22 antiserum, both unabsorbed and absorbed with the heterologous antigen . In the PH experiment, unabsorbed anti-O139 antiserum had haemolytic titres of 66,000 and 22,000 against O139 LPS- and O22 LPS-sensitized SRBC, respectively; unabsorbed anti-O22 antiserum had haemolytic titres of 900 and 13,000, respectively . Thus, the anti-O139 antiserum contained an antibody that reacted with a heterologous O22 antigen at a high titre (22,000) and this antibody was completely removed from anti-O139 antiserum with the O22 antigen . The anti-O22 antiserum contained an antibody that reacted with the heterologous O139 antigen at a low titre (900) and this antibody was completely removed from anti-O22 antiserum with the O139 antigen . In PHI tests O139 LPS and O22 LPS each strongly inhibited (the ID50 of LPS ranged from 0.03 to 0.14 microgram ml-1) the heterologous haemolytic systems of both O139 LPS-sensitized SRBC/anti-O22 antiserum and O22 LPS-sensitized SRBC/anti-O139 antiserm, which are substantially equivalent to the common antigen factor in the O139 LPS-sensitized SRBC/anti-O22 antiserum system and the common antigen factor in the O22 LPS-sensitized SRBC/anti-O139 antiserum system, respectively . The results indicated that the O antigen of O139 is closely related to that of O22 in an a,b-a,c type of relationship where a is common antigenic factor, b is an O139-specific antigenic factor and c is an O22-specific antigenic factor.

Infect Immun, 1996 Jun, 64(6), 2362 - 4
New evidence for an inflammatory component in diarrhea caused by selected new, live attenuated cholera vaccines and by El Tor and Q139 Vibrio cholerae; Silva TM et al.; Using a lactoferrin latex agglutination assay, we have compared the inflammatory responses to a cholera vaccine candidate, CVD 110, in which all known toxin genes have been deleted or mutated yet still produced significant diarrhea, with a less reactive vaccine strain and wild-type El Tor and 0139 Vibrio cholerae strains . Data suggest that diarrhea due to attenuated and wild-type El Tor V . cholerae, and to a lesser extent 0139 V . cholerae, involves an inflammatory response . Further study is required to further elucidate the mechanism of the process(es) involved.

Infect Immun, 1996 Jun, 64(6), 2246 - 55
Alterations in Vibrio cholerae motility phenotypes correlate with changes in virulence factor expression; Gardel CL et al.; Motility is thought to contribute to the virulence of Vibrio cholerae, but the role it plays in pathogenesis is not completely understood . To investigate the influence of motility on virulence gene expression and intestinal colonization, we have isolated mutants with altered swarming abilities in soft agar medium . Both spontaneous hyperswarmer (exhibiting faster swarm rates) and spontaneous or transposon-induced nonmotile mutants of strain 0395 were obtained . Surprisingly, we found that two of three classes of hyperswarmer mutants were defective in autoagglutination, a phenotype associated with expression of toxin-coregulated pili (TCP), an essential ToxR-regulated colonization factor of V . cholerae . In contrast, nonmotile mutants exhibited autoagglutination under growth conditions that normally repress this phenotype . Further characterization of mutant strains revealed differences in the expression of other virulence determinants . Class I hyperswarmer mutants were defective in production of TCP, cholera toxin, and a cell-associated hemolysin but showed increased levels of protease and fucose-sensitive hemagglutinin . All nonmotile mutants examined, including those with insertions in a sequence homologous to motB, exhibited increased expression of TCP pilin, cholera toxin, and cell-associated hemolysin but dramatically decreased levels of fucose-sensitive hemagglutinin and HEp-2 adhesins . In general, nonmotile mutants displayed few or no defects in intestinal colonization, while class I hypermotile mutants were highly defective in colonization . These results suggest that the motility phenotype of V . cholerae is tightly coupled to the expression of multiple ToxR-regulated and non-ToxR-regulated virulence determinants.

Infect Immun, 1996 Jun, 64(6), 2220 - 4
Preclinical immunoprophylactic and immunotherapeutic efficacy of antisera to capsular polysaccharide-tetanus toxoid conjugate vaccines of Vibrio vulnificus; Devi SJ et al.; Vibrio vulnificus is an oyster-associated bacterial pathogen that causes life-threatening fulminating septicemia and necrotizing wound infections in humans . The capsular polysaccharide of V . vulnificus (VvPS) is critical for virulence . Previously we showed that active immunization of mice with a VvPS-tetanus toxoid (VvPS-TTa) conjugate vaccine conferred significantly higher protection against subsequent lethal challenge than immunization with VvPS alone . In the current study, we examined the utility of immunoprophylaxis or immunotherapy with hyperimmune antisera elicited by VvPS-TTa and VvPS-TTb conjugate vaccines prepared by different synthetic schemes . First we demonstrated that the Ribi adjuvant significantly enhanced the murine antibody response (P < or = 0.02) to both conjugates . Subsequently, high-titered polyclonal antisera were raised to VvPS-TTa and VvPS-TTb conjugate vaccines by using Ribi adjuvant or Freund's adjuvants . Antisera were observed to have protective effects when administered before and after acute lethal infection . All animals receiving prophylactic antisera intraperitoneally 24 h before lethal challenge with homologous carbotype 1 were protected, while 73 to 100% of control mice succumbed . Immunotherapy was also effective, with survival rates of 60 to 73% seen among mice when antisera were administered 2 h after bacterial challenge, at a time when symptoms of infection were already apparent . The protective effect of capsular antiserum appeared to be serotype specific . Antisera to the, carbotype 1 VvPS-TTa vaccine did not confer cross-protection against lethal challenge with carbotype 2 V . vulnificus despite partial structural similarity and a weak serological cross-reaction between the two carbotypes . Immune globulins induced by a potential multivalent VvPS conjugate vaccine composed of clinically prevalent carbotypes may have utility in the management of V . vulnificus infections and deserve further evaluation.

Infect Immun, 1996 Jun, 64(6), 2144 - 50
Synthesis of hybrid molecules between heat-labile enterotoxin and cholera toxin B subunits: potential for use in a broad-spectrum vaccine; Lebens M et al.; Three variants of the cholera toxin B subunit (CTB) were generated by site-specific mutagenesis in which regions of the mature protein were altered to the composition found at the corresponding positions of the closely related B subunit of the heat-labile enterotoxin of enterotoxigenic Escherichia coli (LTB) . The mutant proteins were expressed in Vibrio cholerae and purified from the growth medium . In the first of the mutant proteins, the first 25 amino acids corresponded to the sequence found in LTB, and in the second, changes were made at positions 94 and 95 of the mature protein . The third mutant protein combined the changes made in the first two . Analysis of the immunological properties of these novel proteins by using monoclonal antibodies and absorbed polyclonal antiserum demonstrated that they had acquired LTB-specific epitopes . Immunizations with the mutant proteins resulted in antisera containing LTB-specific as well as CTB-specific and cross-reactive antibodies . The sera were also found to be more strongly cross-reactive in the in vitro neutralization of both cholera toxin and heat-labile enterotoxin than were antisera raised against either CTB or LTB . The results suggest that such hybrid CTB-LTB proteins may be useful in a broad-spectrum vaccine against enterotoxin-induced diarrhea.

Trop Med Int Health, 1996 Jun, 1(3), 393 - 8
Vibrio cholerae O139: how great is the threat of a pandemic?
Siddique AK, Akram K, Zaman K, Mutsuddy P, Eusof A, Sack RB.
The emergence of the new strain Vibrio cholerae O139 and its rapid spread in Bangladesh and India together with its detection in several other countries, have raised the question whether this constitutes the beginning of the eighth pandemic of cholera, and if so, how large a threat it poses . In an attempt to answer this question, epidemic spread patterns of Vibrio cholerae O139 strain in Bangladesh were studied . Initially the epidemic moved quickly and affected the entire coastal and estuarine tidal plains of southern Bangladesh . In the flood plains of the northern regions it affected mostly the north-eastern and north-central areas, at a slower pace than in the southern areas . In the beginning the new strain totally displaced both biotypes (classic and El Tor) of Vibrio cholerae O1 . Nearly 2 years after its initial detection, striking differences in the distribution of V . cholerae O139 and O1 were observed . In most northern areas, the new strain was replaced by V . cholerae O1, whereas in the southern coastal regions, the O139 strain continues to dominate epidemics . The study suggests that the O139 strain may become endemic in the coastal ecosystem . The threat of a pandemic, therefore, may not be as large as it first seemed.

Epidemiol Infect, 1996 Jun, 116(3), 275 - 8
Changing epidemiology of cholera due to Vibrio cholerae O1 and O139 Bengal in Dhaka, Bangladesh; Faruque AS et al.; At the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) Dhaka we studied the trends in cholera for the period January 1992 to May 1995 . Vibrio cholerae O139 Bengal emerged as a second aetiologic agent of cholera in Dhaka in January 1993 . In 1993, the majority of cholera cases was due to V . cholerae O139, with V . cholerae O1 accounting for a small proportion of cases . During the latter part of the study period (Jan 1994-May 1995), V . cholerae O1 re-emerged as the predominant cholera strain . The predominant age group affected in endemic cholera due to V . cholerae O1 was children 2-9 years old, and the organism was isolated from more females than from males at all ages . In contrast, cholera due to V . cholerae O139 caused disease mostly in adults 15 years and older, which indicated that this organism was new in this population . As with V . cholerae O1, V . cholerae O139 was isolated from more females than males . The initial rapid emergence and predominance of V . cholerae O139 was considered possibly to herald the start of the eighth pandemic of cholera . However, just after a year, the prevalence of V . cholerae O139 decreased dramatically with V . cholerae O1 resuming the role of the dominant cholera strain . The factor(s) contributing to the dramatic decline in prevalence of V . cholerae O139 is not well understood.

Arch Microbiol, 1996 Jun, 165(6), 370 - 6
Geovibrio ferrireducens, a phylogenetically distinct dissimilatory Fe(III)-reducing bacterium; Caccavo F Jr et al.; A new, phylogenetically distinct, dissimilatory, Fe(III)-reducing bacterium was isolated from surface sediment of a hydrocarbon-contaminated ditch . The isolate, designated strain PAL-1, was an obligately anaerobic, non-fermentative, motile, gram-negative vibrio . PAL-1 grew in a defined medium with acetate as electron donor and ferric pyrophosphate, ferric oxyhydroxide, ferric citrate, Co(III)-EDTA, or elemental sulfur as sole electron acceptor . PAL-1 also used proline, hydrogen, lactate, propionate, succinate, fumarate, pyruvate, or yeast extract as electron donors for Fe(III) reduction . It is the first bacterium known to couple the oxidation of an amino acid to Fe(III) reduction . PAl-1 did not reduce oxygen, Mn(IV), U(VI), Cr(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor . Cell suspensions of PAL-1 exhibited dithionite-reduced minus air-oxidized difference spectra that were characteristic of c-type cytochromes . Analysis of the 16S rRNA gene sequence of PAL-1 showed that the strain is not related to any of the described metal-reducing bacteria in the Proteobacteria and, together with Flexistipes sinusarabici, forms a separate line of descent within the Bacteria . Phenotypically and phylogenetically, strain PAl-1 differs from all other described bacteria, and represents the type strain of a new genus and species, Geovibrio ferrireducens.

Biochem Biophys Res Commun, 1996 May 24, 222(3), 774 - 9
Cloning and sequencing of the gene for Na+/H+ antiporter of Vibrio parahaemolyticus; Nozaki K et al.; A gene encoding an Na+/H+ antiporter was cloned from vibrio parahaemolyticus into the plasmid pBR322 and expressed in Escherichia coli cells . The gene enabled mutant E . coli cells to grow in the presence of 0.2 M NaCl (or 10 mM LiCl) . These cells were originally unable to grow under such conditions because of the lack of major Na+(Li+)/H+ antiporters . We detected Na+/H+ antiporter activity due to the gene in membrane vesicles . The gene was sequenced and the deduced amino acid sequence was found to be 72% identical to the NhaB Na+/H+ antiporter of E . coli.

Arch Microbiol, 1996 May 22, 165(5), 306 - 10
Influence of growth conditions on fatty acid composition of a polyunsaturated-fatty-acid-producing Vibrio species
Jostensen JP, Landfald B.
The influence on fatty acid composition of growth medium composition and phase of growth during batch culture and of dilution rate and growth temperature during continuous culture was studied in the eicosapentaenoic-acid (20:5 n-3)-producing Vibrio CCUG 35308 . In glucose-mineral medium, even-numbered normal fatty acyl residues, primarily 16:0, 16:1, 18:1, and 20:5, strongly dominated (ca . 90%), and the fatty acid profile remained practically unchanged throughout a batch-growth cycle . In nutrient broth, the contribution by "uncommon" fatty acids, mainly i-13:0, 15:0, i-15:0, and 17:1 was generally higher, and increased from 15.4% of total fatty acids in early exponential growth phase to 33.2% in the stationary phase . Reduction of the dilution rate in a chemostat from 0.27 to 0.065 h-1 also led to an almost threefold increase in the proportion of odd-numbered residues at the expense of the even-numbered normal ones . Contrary to this plasticity in the overall fatty acid profile influenced by variations in nutrient composition and availability, the level of eicosapentaenoic acid seemed exclusively dictated by growth temperature . The synthesis of this polyunsaturated fatty acid may be a key regulatory process in maintaining membrane fluidity.

Biochim Biophys Acta, 1996 May 22, 1281(1), 1 - 4
Sequence of a Na+/glucose symporter gene and its flanking regions of Vibrio parahaemolyticus; Sarker RI et al.; The nucleotide sequence of an approximately 6 kbp segment of chromosomal DNA of Vibrio parahaemolyticus was determined . The nucleotide sequence revealed four open reading frames (ORFs) in this region . Hydropathy profiles of the deduced amino acid sequence of the ORFs indicate that ORF1 encodes a hydrophobic polypeptide with typical characteristics of a membrane transport protein . All other ORFs encode hydrophilic polypeptides . ORF1 showed significant amino acid sequence similarity to proteins of the SGLT (Na+/glucose symporter) family, and the amino acid sequence of ORF4 showed very high similarity to several bacterial transcriptional repressor proteins (GalR-LacI family) . We observed elevated glucose transport activity in cells harboring a plasmid carrying the DNA region corresponding to ORF1, and the glucose transport was greatly stimulated by Na+ . Thus, we believe that ORF1 encodes a Na+/glucose symporter.

Biochemistry, 1996 May 21, 35(20), 6233 - 42
NADH:ubiquinone oxidoreductase of Vibrio alginolyticus: purification, properties, and reconstitution of the Na+ pump; Pfenninger-Li XD et al.; The Na+-activated NADH:ubiquinone oxidoreductase of Vibrio alginolyticus was extracted from the membranes with lauryldimethylamine-N-oxide and purified by two successive anion exchange columns . This preparation, yielding four major and several minor stained bands after SDS-PAGE, retained the NADH-dehydrogenase activity (with menadione as an artificial electron acceptor) and ubiquinone-1 (Q) reductase activity . On further fractionation of the enzyme, the Q-reductase activity essentially disappeared . Chemical analyses revealed the presence of FAD but not FMN, of non-heme iron and of acid-labile sulfur and tightly-bound ubiquinone-8 in the purified Q-reductase preparation . The participation of an iron-sulfur cluster of the {2Fe-2S} type in the electron translocation was demonstrated by the appearance of a typical EPR signal for this prosthetic group after the reduction of Q-reductase with NADH . A strong EPR signal typical for a radical observed upon reduction of the enzyme might arise from the formation of quinone radicals . In the absence of Na+, the path of the electrons apparently ends with the reduction of ubiquinone-1 to the semiquinone derivative which in the presence of O2 becomes reoxidized with concomitant formation of superoxide radicals . In the presence of Na+, these oxygen radicals are not formed and the semiquinone is further reduced to the quinol derivative . These results indicate that the Na+-dependent step in the electron transfer catalyzed by NADH:ubiquinone oxidoreductase is the reduction of ubisemiquinone to ubiquinol . After reconstitution of the purified Q-reductase into proteoliposomes, NADH oxidation by ubiquinone-1 was coupled to Na+ transport with an apparent stoichiometry of 0.5 Na+ per NADH oxidized . The transport was stimulated by valinomycin (+ K+) or by the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) . The transport of Na+ is therefore a primary event and does not involve the intermediate formation of a proton gradient.

Eur J Biochem, 1996 May 15, 238(1), 160 - 5
Structure of the O-specific polysaccharide of an Aeromonas trota strain cross-reactive with Vibrio cholerae O139 Bengal; Knirel YA et al.; The O-specific polysaccharide of an Aeromonas trota strain was isolated by hydrolysis of the lipopolysaccharide at pH 4.5 followed by gel-permeation chromatography and found to consist of hexasaccharide repeating units containing D-galactose, L-rhamnose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-galactose in the ratios 1:1:2:1:1 . Partial hydrolysis of the polysaccharide with 48% hydrofluoric acid resulted in selective removal of colitose to give a modified polysaccharide containing the other four sugar constituents . On the basis of methylation analysis and NMR spectroscopic studies of the initial and modified, colitose-free polysaccharide, it was concluded that the repeating unit of the O-specific polysaccharide has the following structure {sequence: see text} The known cross-reactivity between the strain studied and Vibrio cholerae O139 Bengal is substantiated by the presence of a common colitose-containing epitope shared by the O-specific polysaccharide of A . trota and the capsular polysaccharide of V . cholerae, which is thought to carry determinants of O-specificity.

Ann N Y Acad Sci, 1996 May 15, 782, 252 - 63
Protein expression in the stressed Vibrio strains; Toth D et al.; In a conjunction process using Escherichia coli SM10 (pLOF) KmR APR as donor and Vibrio S141 SmR as recipient, several mutants were constructed: Vibrio PH 101, V . PH 106, and V . PH 109 with lowered ability to synthesize poly-beta-hydroxybutyrate . The survival and metabolic activities of parent and mutant strains were estimated when they were subjected to stress conditions (starvation of carbon and energy sources and/or cadmium treatment) . Using two-dimensional electrophoresis, the synthesis of stress proteins was demonstrated . Vibrio cultures consecutively exposed to CdCl2 and then to starvation or vice versa responded similarly metabolically . These results show increased proteosynthetic activity of the stressed Vibrio cells, indicating that the primary cadmium treatment induced the expression and synthesis of the protective proteins, enabling the cells to cope with the secondary stress.

FEMS Microbiol Lett, 1996 May 1, 138(2-3), 227 - 32
Phenotypic expression of a mannose-sensitive hemagglutinin by a Vibrio cholerae O1 E1Tor strain and evaluation of its role in intestinal adherence and colonization; Mukhopadhyay S et al.; A Vibrio cholerae O1 strain (1150) of the EIT or biotype and Ogawa serotype with haemagglutination (HA) activity was subjected to TnphoA mutagenesis . Out of several mutants isolated, one HA- and another HA+ mutant were further characterised . The HA- mutant showed about 50% reduction in its intestinal adherence capacity in vitro and about 9-fold decrease of its colonisation ability in vivo, as compared to the wild-type strain . Subsequent studies showed that the HA activity of strain 1150 was mediated by a mannose-sensitive haemagglutinin (MSHA) . Thus, the phenotypic expression of MSHA appears to be partly responsible for the intestinal adherence and colonisation properties of strain 1150.

J Invertebr Pathol, 1996 May, 67(3), 213 - 8
Association of Aeromonas hydrophila and Vibrio alginolyticus with Larval Mortalities of Scallop (Argopecten purpuratus)
Riquelme C, Toranzo AE, Barja JL, Vergara N, Araya R.
A bacteriological study was carried out in a hatchery of Argopecten purpuratus located in northern Chile which had been affected by severe larval mortalities . The phenotypic characterization of the bacterial strains revealed that Vibrio alginolyticus was the predominant species isolated in the majority of samples taken from the different units of the hatchery (microalgae, swimming larvae, seawater of larval culture tanks, and a reservoir tank of 50-mum filtered seawater) . However, the bacterial population of dying larvae was composed of only Aeromonas hydrophila strains which proved to be resistant to most of the chemotherapeutic agents tested . The bioassays conducted to evaluate the effect of these bacteria on larval survival showed that all of the isolated Vibrio and Aeromonas strains possessed a high degree of pathogenicity, since they produced dying larvae on concentrations ranging from 5.5 x 10(4) to 5.5 x 10(2) cells/ml . The possible virulence mechanism of these bacteria is discussed, as well as the potential use of drugs to prevent larval mortalities.

J Foot Ankle Surg, 1996 May-Jun, 35(3), 222 - 4
Necrotizing fasciitis of the foot caused by an unusual organism, Vibrio vulnificus; Yip KM et al.; In modern medicine, there are very few infectious disease processes occurring in the foot that can cause death within 48 hr . and have an overall mortality rate of 50% despite appropriate antibiotic and surgical treatment . Such a condition must be regarded as being potentially deadly . The authors report a case of necrotizing fasciitis of the left foot resulting from an unusual organism of Vibrio vulnificus.

Mol Microbiol, 1996 May, 20(4), 799 - 811
Genetic organization and functional analysis of the otn DNA essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139; Bik EM et al.; In 1992 a new Vibrio cholerae strain, designated V . cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh . Previously, we have shown that this strain arose from a V . cholerae O1 strain by the acquisition of novel DNA . Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains . The mosaic structure of rfaDVCO139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA . However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1-acceptor DNA is probably located downstream of rfbDVCO139 . The DNA region between rfaDVCO139 and rfbQRSVCO139, designated otn, contained seven open reading frames (ORFs) . Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis . Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis . The otn DNA is also found in V . cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain . However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains . No antigenic relationship was found between the different V . cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA . The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species . Furthermore, a repeat motif was observed in extragenic regions . A number of observations suggest that these sequences may facilitate gene flow between V . cholerae strains and the assembly of clusters of functionally related genes.

Mol Microbiol, 1996 May, 20(4), 693 - 9
The sodium-driven polar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar expression; Kawagishi I et al.; Certain marine Vibrio species swim in sea water, propelled by a polar flagellum, and swarm over surfaces using numerous lateral flagella . The polar and the lateral flagellar motors are powered by sodium- and proton-motive forces, respectively . The lateral flagella are produced in media of high viscosity, and the relevant viscosity sensor is the polar flagellum . The cell might monitor either the rotation rate of the flagellar motor or the mechanical force applied against the flagellum . To test these possibilities, we examined the effects of amiloride and its derivatives, which inhibit the rotation of the sodium-driven motor, on lateral flagellar gene (laf) expression in Vibrio parahaemolyticus . Phenamil, an amiloride analogue that inhibits swimming at micromolar concentrations, induced laf transcription in media devoid of viscous agents in a dose-dependent manner . The relationship between the average swimming speed and laf induction in the presence of various concentrations of phenamil was very similar to that observed when viscosity was changed . These results indicate that marine Vibrio sense a decrease in the rotation rate of (or the sodium influx through) the polar flagellar motor as a trigger for laf induction . Alternative mechanisms for laf induction are also discussed.

Eur J Obstet Gynecol Reprod Biol, 1996 May, 66(1), 57 - 64
First line immunochemotherapy with cisplatin-based protocol by intraperitoneal and intravenous routes in ovarian cancer: technique and results of 82 cases; Zylberberg B et al.; OBJECTIVES: The aim of this study, initiated in 1982, was to determine the feasibility and the interest of a first-line immunochemotherapy delivered by intraperitoneal (i.p.) and intravenous (i.v.) routes combined in ovarian cancer . STUDY DESIGN: Eighty-two naive patients with a common epithelial cancer entered the study from January 1982 to December 1990 (median follow up > 70 months) . For i.p . infusion, we used a simple lumbar puncture needle left in situ for < 2 h . The first 18 patients received monthly by i.p . route: Adriamycin (DXR) 40 mg/m2, Fluorouracil 1000 mg/m2, Cisplatin (CDDP) 90 mg/m2, Bleomycin 30 mg -DGZ (extract of vibrio cholerae) 60 mg/m2 . For the remaining 64 patients Aracytin 500 mg/m2 replaced DXR and the dose of CDDP was more than doubled (200 mg/m2) thanks to the use of sodium thiosulfate . All 82 patients received Ifosfamid 1300 mg/m2 intravenously . RESULTS: Local toxicity consisted in one subcutaneous abscess and one bacterial peritonitis out of 1508 abdominal punctures . Stage III turned out to be the most interesting group to evaluate the efficacy of a first-line protocol . In this group 34 out of 47 patients (72.3%) who underwent an initial incomplete surgery were in complete remission (CR) at second-look . Nevertheless, 21 out of the 34 patients in CR relapsed (61.7%) and 14 died (43.2%) . CONCLUSION: These results show the efficacy of our regimen administered i.p., and the safety of the delivery by a simple needle which avoids the complications of the implantable systems . Nevertheless, the usefulness of a systematic second-line chemotherapy (Paclitaxel?), despite a prognosis situation as favourable as a CR at second-look, must be taken into consideration.

J Clin Microbiol, 1996 May, 34(5), 1293 - 5
Restriction fragment length polymorphism of the tdh and trh genes in clinical Vibrio parahaemolyticus strains; Suthienkul O et al.; The restriction fragment length polymorphism of the genes encoding thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (trh) was analyzed for 137 strains of Vibrio parahaemolyticus isolated from specimens from diarrheal patients in Thailand . The HindIII restriction fragment patterns of tdh and trh were grouped into five and four types, respectively . A strong association between the restriction fragment patterns of tdh and trh was observed with V . parahaemolyticus strains.

J Clin Microbiol, 1996 May, 34(5), 1189 - 92
Molecular characterization of Vibrio cholerae O1 strains isolated during cholera outbreaks in Guinea-Bissau; Dalsgaard A et al.; In the present study, 19 strains of Vibrio cholerae O1 biotype El Tor isolated during outbreaks of cholera in Guinea-Bissau in 1987, 1994, and 1995 were characterized to investigate a possible epidemiological relationship among the isolates . On the basis of ribotyping with the restriction enzyme BglI, 5 strains isolated in 1987 showed two closely related ribotypes, while 14 strains isolated in 1994 and 1995 showed the same ribotype that was distinct from the ribotypes of strains isolated in 1987 . Southern blot hybridization of BglI-digested genomic DNA with a cholera toxin probe demonstrated that the strains isolated in 1987 showed an identical cholera toxin genotype, whereas O1 strains isolated in 1994 and 1995 showed the same genotype that was distinct from the genotype of strains isolated in 1987 . These results were supported by the results of antibiotic susceptibility testing, in which strains isolated in 1987 showed resistance to polymyxin B only, while each of the strains from 1994 and 1995 showed resistance to polymyxin B, trimethoprim-sulfamethoxazole, and the vibriostatic agent O/129 . Although our results are based on a limited number of V . cholerae O1 strains, they suggest that the epidemic in Guinea-Bissau in 1994 and 1995 was due to the introduction of a new strain to the country.

J Clin Microbiol, 1996 May, 34(5), 1114 - 7
Nontoxigenic Vibrio cholerae 01 serotype Inaba biotype El Tor associated with a cluster of cases of cholera in southern India; Saha PK et al.; Thirteen strains of Vibrio cholerae 01 belonging to the Inaba serotype El Tor biotype isolated from patients during an outbreak of cholera in the town of Warangal in southern India were found to be nontoxigenic (NT), since they did not produce cholera toxin or hybridize with DNA probes specific for cholera toxin, Zot, or Ace . The unheated and heated culture supernatants of the NT V . cholerae 01 evoked a rapid cell-rounding effect when introduced on confluent layers of CHO and HeLa cells which could not be inhibited by antiserum against known toxins . Culture supernatants of two representative NT V . cholerae 01 strains caused an increase in short-circuit current in rabbit ileal tissue mounted on an Ussing chamber, and the pattern of increase in short-circuit current was consistent with the presence of a quickly acting toxin like stable toxin . None of the strains of NT V . cholerae 01 hybridized with a DNA probe specific for the heat-stable enterotoxin of V . cholerae non-01, nor did the factor produced by NT V . cholerae 01 resemble the recently described heat-stable enterotoxin produced by enteroaggregative Escherichia coli as determine by a PCR assay . To our knowledge, this is the first report of NT V . cholerae 01 being associated with a cluster of cases of cholera, and it appears that a clone of NT V . cholerae 01 has the potential to cause localized outbreaks of cholera.

Pediatr Infect Dis J, 1996 May, 15(5), 415 - 8
Incidence of Vibrio cholerae O1 diarrhea in children at the onset of cholera epidemic in periurban Lima, Peru; Gil AI et al.; OBJECTIVE . To determine the incidence of Vibrio cholerae O1-associated diarrhea in children during the onset of the 1991 cholera epidemic in Peru . METHODS . Stool cultures were obtained from children (mean age, 26 months) participating in a prospective community-based study of diarrhea in a periurban community in Lima between February and May, 1991 . RESULTS . Of the 409 diarrheal episodes cultured V . cholerae O1 was isolated in 14 (3.4%) episodes . This represented an incidence of 0.11 episode per child year, higher than previously reported rates in children from endemic cholera areas . Most cases were mild; only 1 case required hospitalization . CONCLUSIONS . Our study indicates that from the beginning of this epidemic, V . cholerae O1 caused diarrhea in children as well as adults and should therefore be considered as one of the possible pathogens when children from a cholera-affected area develop diarrhea.

Kansenshogaku Zasshi, 1996 May, 70(5), 456 - 62
{Clinical study on bacteremia in patients with liver cirrhosis}; Mizuno R et al.; Infections and fever are frequent in patients with liver cirrhosis . Study on bacteremia in cirrhotic patients has not been reported in Japan . In a 16 year period from 1979 to 1994, we collected 39 cases with 40 episodes and 44 microorganisms of bacteremia for this study . The incidence of bacteremia in cirrhotic hospital admissions was 4.8% (39/808) . Gram negative bacteria were the predominant microorganisms of bacteremia (66%, 29/44) . Among them, Escherichia coli, Klebsiella pneumoniae, Vibrio sp . were the three most commonly detected microorganisms . Pseudomonas aeruginosa bacteremia has not been detected . Laboratory data of cirrhotic patients showed that positive blood culture patients had significantly lower serum albumin, prothrombin time and hepaplastin test than negative patients . Focal infection could be diagnosed in only 45% (20/44) . The mortality rate was 28% (11/39), but the bacteremia related death (by septic shock) were only 2 cases . The other causes of death were hepatic failure in 9 cases . In conclusion, bacteremia is a important complication of liver cirrhosis . Blood culture is necessary in cirrhotic patients with fever.

Biotechnol Prog, 1996 May-Jun, 12(3), 387 - 92
Characterization of the stress response of a bioluminescent biological sensor in batch and continuous cultures; Rupani SP et al.; The effects of temperature, growth stage, and inducer (ethanol) concentration on the kinetics and magnitude of the stress response were investigated by using an Escherichia coli strain with the grpE heat shock promoter fused to the Vibrio fischeri lux genes . When stressed, the cells responded by changing the level of specific light emission, which was measured both on- and off-line . These measurements were used to characterize and optimize the sensitivity of the construct by determining the conditions at which the culture exhibited maximum specific bioluminescence and minimum response time to ethanol induction in batch cultivation . The results of the batch study were then applied to continuous cultivation, and the effect of dilution rate was determined . These results are of considerable interest in the development of an on-line biological sensor system for the detection and toxicity assessment of chemical pollutants.

Biochem J, 1996 May 1, 315 ( Pt 3), 1001 - 5
pH-dependence of the dithiol-oxidizing activity of DsbA (a periplasmic protein thiol:disulphide oxidoreductase) and protein disulphide-isomerase: studies with a novel simple peptide substrate; Ruddock LW et al.; A decapeptide containing two cysteine residues at positions 3 and 8 has been designed for use in monitoring the disulphide bond-forming activity of thiol:disulphide oxidoreductases . The peptide contains a tryptophan residue adjacent to one of the cysteine residues and an arginine residue adjacent to the other . Oxidation of this dithiol peptide to the disulphide state is accompanied by a significant change in tryptophan fluorescence emission intensity . This fluorescence quenching was used as the basis for monitoring the disulphide bond-forming activity of the enzymes protein disulphide-isomerase (PDI) and DsbA (a periplasmic protein thiol:disulphide oxidoreductase) in the pH range 4.0-7.5, where the rates of spontaneous or chemical oxidation are low . Reaction rates were found to be directly proportional to enzyme concentration, and more detailed analysis indicated that the rate-determining step in the overall process was the reoxidation of the reduced form of the enzyme by GSSG . The pH-dependence of the enzyme-catalysed reaction reflected primarily the pKa of the reactive cysteine residue at the active site of each enzyme . The data indicate a pKapp of 5.6 for bovine PDI and of 5.1 for Vibrio cholerae DsbA.

J Bacteriol, 1996 May, 178(10), 2897 - 901
Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactions with autoinducer analogs; Schaefer AL et al.; The Vibrio fischeri luminescence genes are activated by the transcription factor LuxR in combination with a diffusible signal compound, N-(3-oxohexanoyl) homoserine lactone, termed the autoinducer . We have synthesized a set of autoinducer analogs . Many analogs with alterations in the acyl side chain showed evidence of binding to LuxR . Some appeared to bind with an affinity similar to that of the autoinducer, but none showed a higher affinity, and many did not bind as tightly as the autoinducer . For the most part, compounds with substitutions in the homoserine lactone ring did not show evidence of binding to LuxR . The exceptions were compounds with a homocysteine thiolactone ring in place of the homoserine lactone ring . Many but not all of the analogs showing evidence of LuxR binding had some ability to activate the luminescence genes . None were as active as the autoinducer . While most showed little ability to induce luminescence, a few analogs with rather conservative substitutions had appreciable activity . Under the conditions we employed, some of the analogs showing little or no ability to induce luminescence were inhibitors of the autoinducer.

J Infect Dis, 1996 May, 173(5), 1176 - 83
The epidemiology of Vibrio infections in Florida, 1981-1993; Hlady WG et al.; The epidemiology of 690 Vibrio infections reported in Florida during 1981-1993 is described . Most infections resulted in one of three clinical syndromes: gastroenteritis (51%), wound infections (24%), or primary septicemia (17%) . Case-fatality rates were 1% for gastroenteritis, 5% for wound infections, and 44% for primary septicemia . While gastroenteritis had little seasonal variation, 91% of primary septicemias and 86% of wound infections occurred from April through October, mostly due to the seasonality of Vibrio vulnificus and Vibrio parahaemolyticus infections . Infected wounds were largely a result of occupational activities around seawater . Some 68% of gastroenteritis cases and 83% of the primary septicemias were associated with raw oyster consumption . Preexisting liver disease was present in 48% of patients with primary septicemia and was associated with a fatal outcome in both wound infections (relative risk {RR}, 28.3; 95% confidence interval {CI}, 6.3-127.5; P < .0001) and primary septicemia (RR, 1.9; 95% CI, 1.2-3.1; P < .01).

Infect Immun, 1996 May, 64(5), 1756 - 61
Relative importance of three iron-regulated outer membrane proteins for in vivo growth of Vibrio cholerae; Tashima KT et al.; Iron is an essential nutrient to support the growth of most bacterial species . However, iron is not easily available to microorganisms infecting mammalian hosts, because it is largely sequestered by iron-binding proteins, such as transferrin or lactoferrin, or complexed to heme . In response to environmental iron stress, Vibrio cholerae produces the siderophore vibriobactin as well as a number of iron-induced outer membrane proteins . Previous data on the role of iron acquisition systems for the intraintestinal growth of mucosal pathogens such as V . cholerae are conflicting . In this report, we isolated mutants of V . cholerae with TnphoA fusions in each of viuA, hutA, and irgA, as well as strains mutant in each pair of these genes and all three simultaneously, to analyze the role of these iron-induced outer membrane protein receptors for in vivo growth of V . cholerae . The fusion between hutA and TnphoA in a single copy on the chromosome allowed the study of in vitro regulation of hutA in response to iron, fur, and irgB; transcription of hutA was tightly iron regulated (70-fold) and dependent on a functional Fur but did not require IrgB . To investigate the effects of mutations in these iron-induced outer membrane proteins on in vivo growth, we inoculated ileal loops in a rabbit model of infection . This avoids exposure of organisms to the potential killing effects of gastric acid, allows several logarithmic increases in growth in the in vivo environment, and facilitates direct comparison of multiple strains in the same animal to avoid any differences between animals . We grew each mutant to be tested in competition with the wild-type strain in the same loop, to provide an internal control . We confirmed that the inocula for these experiments were grown under conditions of iron stress prior to in vivo inoculation, by measuring the alkaline phosphatase activity of the iron-regulated fusion in each strain . The results confirmed that mutation of irgA produced a much more substantial in vivo growth defect than mutation of either hutA or viuA alone . Double mutants of irgA with either viuA or hutA, or the strain mutant in all three genes, showed an in vivo growth defect comparable to the strain mutant in irgA only, suggesting that mutation of irgA was the most relevant for in vivo growth . The strain mutant in both hutA and viuA was also markedly impaired for in vivo growth, suggesting that mutation of both of these iron uptake systems simultaneously can also produce a substantial in vivo growth defect.

Gene, 1996 Apr 17, 170(1), 9 - 16
Comparison of the promoter proximal regions of the toxin-co-regulated tcp gene cluster in classical and El Tor strains of Vibrio cholerae O1; Ogierman MA et al.; A physical map has been constructed of the 5-kb XbaI fragment encoding the promoter proximal of region the tcp gene cluster encoding the toxin-coregulated pilus (TCP) of Vibrio cholerae . This fragment contains the major regulatory regions for TCP . Comparison of the nucleotide (nt) sequences from strains of the classical and El Tor biotypes demonstrates that the regions are essentially identical, with several notable exceptions . The intergenic regions, between tcpI and tcpP, and between tcpH and tcpA, show significant sequence divergence which may account for the biotype-related differences in TCP, since this is the location of the major promoter sequences . The C-terminal coding regions of the major pilin subunit, TcpA, also differ . Southern hybridization analyses suggest that the tcpA nt sequence is conserved within a biotype, and Western blot analysis suggests that the two forms of TcpA are antigenically different, but related . Besides tcpA, tcpB, tcpH and tcpI, the genes encoding two additional proteins, TcpP and TcpQ, but not previously defined, were also identified . TcpH and TcpI have been previously suggested to be regulatory proteins but homology data imply that TcpI is a methyl-accepting chemotaxis protein (MCP), as recently reported {Harkey et al., Infect . Immun . 62 (1994) 2669-2678}, and TcpH is predicted to be a periplasmic or exported protein . TcpP is thought to be a trans-cytoplasmic membrane (CM) protein which may have a regulatory role.

Biochem Biophys Res Commun, 1996 Apr 16, 221(2), 477 - 83
Site-directed mutagenesis of a novel serine arylesterase from Vibrio mimicus identifies residues essential for catalysis; Chang RC et al.; Site-directed mutagenesis (SDM) of an arylesterase (the arylesterase) from Vibrio mimicus revealed that residues S29, H153, and D96 constituted a catalytic triad . The use of a serine residue for ester hydrolysis by the arylesterase proves that the enzyme is a novel serine arylesterase . SDM also showed that D28 was necessary for the esterase activity; to our knowledge it is the first time that a residue immediately preceding the active-site serine in esterases was shown biochemically to possess such a property . The results further suggest that D28 plays a role in substrate-binding . Residue 31 was firmly shown to participate in the binding of N-acetyl-D, L-phenylalanine beta-naphthyl ester (NAPNE), an artificial substrate for chymotrypsin . The S31G enzyme showed a 4 fold decrease in the Km for NAPNE over that of wild type enzyme, proving residue 31 is important for substrate-specificity . A mechanism for binding and catalysis of esters by the arylesterase is proposed, which includes the unique role of S31 for aromatic (hydrophobic) acyl-binding . The biochemical properties of the arylesterase suggest that the enzyme stands out as a member of a distinct subfamily within a recently proposed, lipolytic enzyme family.

J Biol Chem, 1996 Apr 5, 271(14), 8176 - 82
Structure and expression of the Chlorobium vibrioforme hemB gene and characterization of its encoded enzyme, porphobilinogen synthase; Rhie G et al.; Plasmids containing DNA from the green photosynthetic bacterium Chlorobium vibrioforme complement a heme-requiring Escherichia coli hemB mutant that is deficient in porphobilinogen (PBG) synthase activity . PBG synthase activity was detected in extract of complemented cells but not in that of cells transformed with control plasmid . The sequence of the C . vibrioforme hemB gene predicts a HemB protein that contains 328 amino acids, has a molecular weight of 36,407, and is 53% identical to the homologous proteins of Synechocystis sp . PCC 6301 and Rhodobacter capsulatus . The response of C . vibrioforme PBG synthase to divalent metals is unlike that of any previously described PBG synthase; Mg2+ stimulates but is not required for activity, and Zn2+ neither stimulates nor is required . This response correlates with predicted sequences of two putative variable metal binding regions of C . vibrioforme HemB . The C . vibrioforme hemB open reading frame begins 1585 bases downstream from the end of the hemD open reading frame and is transcribed in the same direction as hemA, hemC, and hemD . However, hemB is not part of the same transcription unit as these genes, and the hemB transcript is approximately the same size as the hemB gene alone . Between hemD and hemB there is an intervening open reading frame that is oriented in the opposite direction and encodes a protein with a predicted amino acid sequence significantly similar to that of inositol monophosphatase, an enzyme that is not involved in tetrapyrrole biosynthesis . The gene order within hem gene clusters is highly conserved in phylogenetically diverse prokaryotic organisms . This conservation suggests that there are functional constraints on the relative order of the hem genes.

FEMS Microbiol Lett, 1996 Apr 1, 137(2-3), 247 - 52
Characterization of malate dehydrogenase from deep-sea psychrophilic Vibrio sp . strain no . 5710 and cloning of its gene; Ohkuma M et al.; A metabolic key enzyme malate dehydrogenase (MDH) was purified from a deep-sea psychrophilic bacterium, Vibrio sp . strain no . 5710 . The enzyme displayed an optimal activity shifted toward lower temperature and a pronounced heat lability . A gene encoding this enzyme was isolated and cloned . Recombinant Escherichia coli cells harboring the isolated clone expressed MDH activity with temperature stability identical to that of the parental psychrophile . Nucleotide sequencing of the gene revealed that its primary sequence was similar to that of a mesophile E . coli MDH (78% amino acid identity), for which the three-dimensional structure is known . The enzyme is thus suitable for the analysis of molecular adaptations to low temperatures.

Microbiology, 1996 Apr, 142 ( Pt 4), 845 - 53
Stress resistance and recovery potential of culturable and viable but nonculturable cells of Vibrio vulnificus; Weichart D et al.; The estuarine, human-pathogenic bacterium Vibrio vulnificus responds to low temperature by the formation of viable but nonculturable (VBNC) cells, while starvation at moderate temperatures allows for maintenance of culturability of this organism . Recovery of cold-incubated populations of V . vulnificus was restricted to the culturable fraction in slide cultures and most probable number assays . These populations, however, gave between 1.1- and 8-fold higher c.f.u . counts on soft agar plates than on ordinary agar plates, indicating that a small and variable fraction of the cell population was injured rather than nonculturable . Thus, the population of cold-incubated cells is composed of culturable, injured and nonculturable cells, with the numbers of the culturable and injured cells rapidly decreasing during cold incubation . Recovery of nonculturable cells of the organism, however, could not be obtained by any combination of temperature and nutrient shifts in any of the assays . VBNC cells of the organism were assessed with regard to their persistence and stress resistance in comparison to growing and starved cells . The sonication resistance of VBNC cells was initially similar to that of growing cells, but increased during prolonged cold incubation . The final resistance of cold-incubated VBNC cells was equal to the markedly increased resistance of starving cells, which also displayed increased resistance against exposure to ethanol and mechanical stress . Our results indicate that in spite of the apparent absence of recovery under a wide range of laboratory conditions, VBNC cells of V . vulnificus undergo changes at low temperature which potentially allow them to persist for extended periods.

Mol Gen Mikrobiol Virusol, 1996 Apr-Jun, (2), 14 - 8
{Creating a system of conjugated chromosome transfer and genetic mapping of Vibrio cholerae serogroup O139 chromosomes}; Smirnova NI et al.; A conjugational gene transfer system consisting of donor and recipient strains has been developed for genetic analysis of Vibrio cholerae 0139 serogroup, a new cholera agent . Donor strains constructed using the Tn5-Mob carrying the origin of transfer (ori T) of plasmid RP4 and helper plasmid pRP4-4 were able to perform a directed transfer of chromosomal markers . Recipient strains carried mutations in auxotrophic genes as well as in virulence genes . Based on this gene transfer system, a genetic map of V . cholerae chromosome has been created showing the order of 17 gene markers . Relationship between the genetic structure of V . cholerae 0139 and 01 is discussed.

Appl Environ Microbiol, 1996 Apr, 62(4), 1454 - 7
Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans; Amaro C et al.; We report that the eel pathogen Vibrio vulnificus biotype 2 is also an opportunistic pathogen for humans . Results from a detailed comparative study using reference strains of both biotypes revealed that the clinical strain ATCC 33817, originally isolated from a human leg wound and classified as V . vulnificus (no reference on its biotype is noted), belongs to biotype 2 of the species . As a biotype 2 strain, it is negative for indole and pathogenic for eels and mice, harbors two plasmids of high MrS, and belongs to serogroup E, recently proposed as characteristic of biotype 2 strains . In consequence, appropriate measures must be taken by consumers, particularly by those running a health risk, and by fish farmers, above all when manipulating eels during epizootic outbreaks.

Appl Environ Microbiol, 1996 Apr, 62(4), 1378 - 82
Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification; Coleman SS et al.; DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed . In addition, PCR amplification of V . vulnificus from oysters seeded with biotype 1 cells was demonstrated . By the methods described, V . vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells . It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters . V . vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin . The protocol used for detection of V . vulnificus cells in eels required less than 5 h to complete . Optimum MgCl2 concentrations for the PCR of V . vulnificus from oysters and eels were different, although the same primer pair was used for both . This is the first report on the detection of cells of V . vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen.

Appl Environ Microbiol, 1996 Apr, 62(4), 1141 - 4
Vibrio mimicus diarrhea following ingestion of raw turtle eggs; Campos E et al.; Clinical and epidemiological characteristics of diarrhea associated with Vibrio mimicus were identified in 33 hospitalized patients referred to the Costa Rican National Diagnostic Laboratory Network between 1991 and 1994 . The relevant symptoms presented by patients included abundant watery diarrhea, vomiting, and severe dehydration that required intravenous Dhaka solution in 83% of patients but not fever . Seroconversion against V . mimicus was demonstrated in four patients, from whom acute- and convalescent-phase sera were obtained . Those sera did not show cross-reaction when tested against Vibrio cholerae O1 strain VC-12 . All the V . mimicus isolates from these cases produced cholera toxin (CT) and were susceptible to commonly used antibiotics . Attempts to isolate this bacterium from stool samples of 127 healthy persons were not successful . Consumption of raw turtle eggs was recalled by 11 of the 19 (58%) individuals interviewed . All but two V . mimicus diarrheal cases were sporadic . These two had a history of a common source of turtle (Lepidochelys olivacea) eggs for consumption, and V . mimicus was isolated from eggs from the same source (a local market) . Among the strains, variations in the antimicrobial susceptibility pattern were observed . None of the strains recovered from market turtle eggs nor the four isolates from river water showed CT production . Further efforts to demonstrate the presence of CT-producing V . mimicus strains in turtle eggs were made . Successful results were obtained when nest eggs were tested . In this case, it was possible to isolate CT- and non-CT-producing strains, even from the same egg . For CT detection we used PCR, enzyme-linked immunosorbent assay (ELISA), and Y-1 cell assay, obtaining a 100% correlation between ELISA and PCR results . Primers Col-1 and Col-2, originally described as specific for the V . cholerae O1 ctxA gene, also amplified a 302-bp segment with an identical restriction map from V . mimicus . These results have important implications for epidemiological surveillance in tropical countries where turtle eggs are used for human consumption, serving as potential sources of cholera-like diarrhea.

Appl Environ Microbiol, 1996 Apr, 62(4), 1133 - 40
Construction of luciferase reporter bacteriophage A511::luxAB for rapid and sensitive detection of viable Listeria cells; Loessner MJ et al.; Specific transfer and expression of bacterial luciferase genes via bacteriophages provides an efficient way to detect and assay viable host cells . Listeria bacteriophage A511 is a genus-specific, virulent myovirus which infects 95% of Listeria monocytogenes serovar 1/2 and 4 cells . We constructed recombinant derivative A511::luxAB, which carries the gene for a fused Vibrio harveyi LuxAB protein inserted immediately downstream of the major capsid protein gene (cps) . Efficient transcription is initiated by the powerful cps promoter at 15 to 20 min postinfection . Site-specific introduction of the luciferase gene into the phage genome was achieved by homologous recombination in infected cells between a plasmid carrying A511 DNA flanking luxAB and phage DNA . Recombinants occurred in the lysate at a frequency of 5 x 10(-4) and were readily identified by the bioluminescent phenotype conferred on newly infected host cells . A511::luxAB can be used to directly detect Listeria cells . Following infection and a 2-h incubation period, numbers as low as 5 x 10(2) to 10(3) cells per ml were detected by using a single-tube luminometer . Extreme sensitivity was achieved by including an enrichment step prior to the lux phage assay; under these conditions less than 1 cell of L . monocytogenes Scott A per g of artificially contaminated salad was clearly identified . The assay is simple, rapid, inexpensive, and easy to perform . Our findings indicate that A511::luxAB is useful for routine screening of foods and environmental samples for Listeria cells.

Emerg Infect Dis, 1996 Apr-Jun, 2(2), 93 - 102
The evolution and maintenance of virulence in microparasites; Levin BR; In recent years, population and evolutionary biologists have questioned the traditional view that parasite-mediated morbidity and mortality inverted question markvirulence inverted question markis a primitive character and an artifact of recent associations between parasites and their hosts . A number of hypotheses have been proposed that favor virulence and suggest that it will be maintained by natural selection . According to some of these hypotheses, the pathogenicity of HIV, Vibrio cholerae, Mycobacterium tuberculosis,theShigella,as well as Plasmodium falciparum,and many other microparasites, are not only maintained by natural selection, but their virulence increases or decreases as an evolutionary response to changes in environmental conditions or the density and/or behavior of the human population . Other hypotheses propose that the virulence of microparasites is not directly favored by natural selection; rather, microparasite-mediated morbidity and mortality are either coincidental to parasite-expressed characters (virulence determinants that evolved for other functions) or the product of short-sighted evolution in infected hosts . These hypotheses for the evolution and maintenance of microparasite virulence are critically reviewed, and suggestions are made for testing them experimentally.

Curr Microbiol, 1996 Apr, 32(4), 229 - 31
Virulence of Vibrio alginolyticus isolated from diseased tiger prawn, Penaeus monodon; Lee KK et al.; Outbreaks of mass mortality among cultured tiger prawns (Penaeus monodon) with white spotted syndrome (WSS) in the carapace occurred in the summer of 1994 in I-Lan, Taiwan . A swarming strain Val was isolated from hemolymph of the moribund prawns with tryptic soy agar (TSA, supplemented with 1% NaCl, Oxoid) and/or thiosulfate citrate bile salt sucrose (TCBS, Difco) agar . This strain was characterized and identified to be Vibrio alginolyticus . The strain was susceptible to antibiotics such as chloramphenicol, ciprofloxacin, doxycycline hydrochloride, nalidixic acid, oxolic acid, and oxytetracycline while resistant to ampicillin, novobiocin, penicillin G, sulfisoxazole, and sulfonamide . The bacteria and their extracellular products (ECP) were lethal to both tiger prawns (P . monodon) and kuruma prawns (P . japonicus) with LD50 values of 1.13 x 10(5), 2.46 x 10(5) CFU/g, and 0.23, 0.63 micrograms protein/g prawn body weight, respectively.

Mol Microbiol, 1996 Apr, 20(1), 213 - 22
Genetic footprint on the ToxR-binding site in the promoter for cholera toxin; Pfau JD et al.; The transmembrane DNA-binding protein, ToxR, of Vibrio cholerae is a global transcriptional regulator of virulence gene expression . ToxR has been shown to interact with promoter regions upstream of both the ctxAB operon encoding cholera toxin, and the regulatory gene toxT . Deletion analysis has shown that a repeated sequence, TTTTGAT, is required for ToxR binding and activation of the ctxAB promoter . However, this sequence is not found upstream of the toxT promoter . Genetic selections using P22 challenge phages were used to define sites within the promoter for ctxAB which are critical for ToxR-DNA interactions . Single-base-pair changes and deletion mutations that impair ToxR binding cluster within two regions: -57 to -69 within two of three tandem TTTTGAT sequences; and from -39 to -47, between the repeat sequences; and the -35 region of the promoter . ToxR does not bind to a synthetic target that has three tandem repeats which lack a flanking upstream and downstream sequence . These results suggest that the ToxR-binding site lies immediately upstream of the - 35 position of the ctx promoter, and that the affinity of ToxR binding to this site is influenced by the repeat sequences.

Mol Microbiol, 1996 Apr, 20(1), 137 - 49
Vibrio parahaemolyticus FlaJ, a homologue of FliS, is required for production of a flagellin; Stewart BJ et al.; The flaA locus of Vibrio parahaemolyticus encodes one of the four polar flagellin genes, the flagellum-capping protein HAP2, and three additional flagellar genes . Sequence analysis downstream of the gene encoding HAP2 revealed the region to be similar to the fliD (HAP2) locus of Pseudomonas aeruginosa . The deduced protein sequences for the newly identified genes suggest that one protein belongs to the family of transcriptional regulatory proteins known to interact with sigma (54), one may be a rod component of the flagellum, and one resembles the FliS protein . fliS is an essential flagellar gene in many bacteria; however its function is not clear . The V . parahaemolyticus polar flaC flagellin gene was poorly expressed in Escherichia coli Production of FlaC was stimulated by provision of the flaA locus in trans . Dissection of this locus revealed that the fliS-like gene, flaJ, was required for increased expression of flaC . Stimulation by FlaJ occurred in E.coli mutants defective in either the master flagellar-controlling operon or the gene encoding the flagellar sigma (28) . Therefore the effect of FlaJ was not mediated through flagellar proteins . Nor was it mediated through sigma (54) for enhanced FlaC production was observed in mutants with defects in the gene encoding sigma (54).

J Clin Microbiol, 1996 Apr, 34(4), 897 - 900
Vitek system antimicrobial susceptibility testing of O1, O139, and non-O1 Vibrio cholerae; Sciortino CV et al.; Vibrio cholerae causes epidemic diarrhea throughout the world . Fluid replacement is the primary therapy for cholera; however, high mortality rates often necessitate the use of antibiotics . V . cholerae, like most bacteria, has developed resistance to some antibiotics . In the early 1990s a new serotype strain, Bengal 0139, began a new wave of cholera epidemics . Bengal isolates showed unique trends in antimicrobial resistance . Many clinical laboratories use automated antibiotic susceptibility testing for V . cholerae . It is important to know if automated susceptibility test results for V . cholerae coincide with reported trends in antibiotic susceptibility . In the present study, we used the Vitek automated susceptibility system to determine the susceptibilities of 79 V . cholerae O1 isolates, 100 O139 isolates, and 112 non-O1 isolates . Vitek susceptibilities for V . cholerae showed a good correlation with preestablished epidemiological data . Although the new O139 serogroup showed a trend of increased resistance to trimethoprim-sulfamethoxazole and nitrofurantoin, it was more susceptible to ampicillin than previous serogroup O1 and non-O1 strains . Regardless of serogroup, > or = 98% of the V . cholerae isolates tested were susceptible to most antibiotics tested by us . It is important to continue susceptibility testing of all new isolates of V . cholerae because of emerging resistant strains . However, V . cholerae remains susceptible to most of the available antibiotics.

J Clin Microbiol, 1996 Apr, 34(4), 1038 - 40
Simple differentiation of Vibrio cholerae O139 from V . cholerae O1 and non-O1, non-O139 by modified CAMP test; Lesmana M et al.; Strong positive CAMP reactions were demonstrated by 121 Vibrio cholerae O139 and 504 El Tor isolates, and weak positive CAMP reactions were shown by 235 non-O1, non O139 isolates when these isolates were tested by a modified CAMP technique . Thirty-five classical biotype V . cholerae O1 isolates included in the tests were all CAMP negative.

Int J Food Microbiol, 1996 Apr, 29(2-3), 311 - 9
Rapid detection of Vibrio parahaemolyticus in oysters by immunofluorescence microscopy; Chen HC et al.; A method of indirect immunofluorescence microscopy was developed for the rapid detection of Vibrio parahaemolyticus in oysters . Affinity-purified antibodies against two outer membrane proteins (molecular mass 36 and 34 kDa, respectively) of V . parahaemolyticus were used as the primary antibodies to label the vibrio, and fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G was used to reveal the antigen-antibody reaction . Of 85 strains of V . parahaemolyticus tested, all produced strong fluorescence under the fluorescence microscope . However, for 63 strains (including 27 vibrios) of other bacteria tested, 6 produced weak to moderate fluorescence . The sensitivity and specificity of the immunostaining technique were 100 and 90.5%, respectively . In the inoculation experiments, as low as 1.7 CFU/g of V . parahaemolyticus spiked in oysters could be detected by the immunostaining method after 18-h enrichment in alkaline peptone water containing 3% NaCl . The immunofluorescence microscopy is recommended for rapid screening of V . parahaemolyticus in oysters; a presumptive positive result could be obtained within 24 h.

Vaccine, 1996 Apr, 14(6), 526 - 31
Further molecular characterization and stability of the live oral attenuated cholera vaccine strain CVD103-HgR; Favre D et al.; Vibrio cholerae CVD103-HgR, the first live attenuated vaccine licensed for human use produced by recombinant DNA technology, was genetically compared to its parent strains 569B and CVD103 . The genetic stability for both lyophilized vaccine in final container form and for viable organisms shed from vaccinees was determined . Results obtained lead us to conclude: (i) the genetic composition of the examined genes in CVD103-HgR is identical to that of the parent strains except for the alterations induced; (ii) the level of mercury resistance depends on the orientation of the mer operon within hlyA, with the highest level being observed for the orientation found in CVD103-HgR; (iii) no DNA sequences from plasmids used in construction remain in the genome; (iv) the strain is genetically stable; and (v) both CVD103-HgR and its parent strains contain defective lysogenic prophages . We have further confirmed that a certain amount of restriction fragment length polymorphism (RFLP) exists around the chromosomal ctx locus within V . cholerae strains of the classical biotype (detectable on chromosomal DNA restricted by either HindIII or EcoRI, but not PstI).

Biometals, 1996 Apr, 9(2), 157 - 67
Physical and structural characterization of yersiniophore, a siderophore produced by clinical isolates of Yersinia enterocolitica; Chambers CE et al.; Clinical isolates of Yersinia enterocolitca, which belong to mouse-lethal serotypes, produce the siderophore yersiniophore . Siderophore production was shown to be iron regulated and to reach maximum production in late log phase . Yersiniophore is a fluorescent siderophore with maximum excitation at 270 nm and a major emission peak at 428 nm . Absorption maxima were seen at 210 and 250 nm with a low broad peak from 280 to 320 nm . Purification of unchelated yersiniophore for structural analysis was made difficult by low yields (1-2 mg mg-1), and susceptibility to acid hydrolysis, oxidation and possibly polymerization . Yersinophore was therefore purified as an Al3+ chelate, which was found to be stable in solution for several weeks . To purify Al(3+)-yersinophore, unchelated yersiniophore was first extracted from culture supernatants with dichloromethane, concentrated by rotary evaporation and adsorbed to a DEAE-sephacel column . Al(3+)-yersiniophore was eluted with 0.01 M AlCl3 and further purified by HPLC . The structure was established by a combination of elemental analysis, high resolution mass spectrometry and two-dimensional NMR experiments . Yersiniophore is a phenolate-thiazole siderophore with the formula C21H24N3O4S3Al and a molecular weight of 505.07404 when chelated to Al3+ . The structure of yersiniophore was determined to be closely related to the structures of pyochelin, produced by Pseudomonas aeruginosa, and anguibactin, produced by Vibrio anguillarum.

Mol Microbiol, 1996 Apr, 20(2), 415 - 25
Autoregulation of luxR: the Vibrio harveyi lux-operon activator functions as a repressor; Chatterjee J et al.; Mobility-shift assays have been used to demonstrate that the activator of the Vibrio harveyi lux operon, LuxR, binds independently, and with similar affinity, to two sites upstream of its own open reading frame . One site was located between 52 and 107 bp upstream of, and the other site in a region 25 bp downstream of, the transcriptional start site . The luxR promoter, in a transcriptional fusion with the chloramphenicol acetyl transferase (cat) gene, could readily be expressed in Escherichia coli as well as V . harveyi in the absence of LuxR . In both species, the presence of the luxR gene product resulted in repression of luxR promotion . These results show that LuxR directly regulates its own expression by functioning as an autorepressor . A mechanism for this repression is suggested by evidence showing that LuxR has a negative effect on RNA polymerase binding to the luxR promoter . In light of the fact that LuxR is also part of a regulatory family of repressors, the mechanism by which LuxR functions as a transcriptional activator of the lux operon has been re-examined.

New Microbiol, 1996 Apr, 19(2), 155 - 66
Distribution and numerical taxonomy of Vibrionaceae in the waters of the Straits of Messina; Caruso G et al.; The results of a study carried out by numerical analysis on Vibrio strains isolated from the waters of the Straits of Messina are reported . The quantitative data showed the presence of low bacterial densities (ranging from 9 to 99 CFU/100 ml of water) due to the intense currents which characterize this area; also the highest bacterial counts were generally found during the "montante" current . With regard to the qualitative results, there was a predominance of vibrios belonging to V . mediterranei, V . splendidus II and V . pelagius II species, which represented respectively 25%, 19% and 13% of the total bacterial population . The species distribution did not seem related to sampling stations or depth . The taxonomic structure, obtained using the simple matching coefficient and unweighted average linkage clustering, revealed the presence of 7 main clusters (S = 80-95%), which included strains of various origin because of the particular turbulence of the waters of the Straits.

J Bacteriol, 1996 Apr, 178(8), 2409 - 15
Cloning and characterization of motY, a gene coding for a component of the sodium-driven flagellar motor in Vibrio alginolyticus; Okunishi I et al.; The bacterial flagellar motor is a molecular machine that couples proton or sodium influx to force generation for driving rotation of the helical flagellar filament . In this study, we cloned a gene (motY) encoding a component of the sodium-driven polar flagellar motor in Vibrio alginolyticus . Nucleotide sequence analysis revealed that the gene encodes a 293-amino-acid polypeptide with a single putative transmembrane segment that is very similar (94.5% identity) to the recently described MotY of V . parahaemolyticus . Their C-terminal domains were similar to the C-terminal domains of many peptidoglycan-interacting proteins, e.g., Escherichia coli MotB and OmpA, suggesting that MotY may interact with peptidoglycan for anchoring the motor . By using the lac promoter-repressor system, motY expression was controlled in V . alginolyticus cells . Swimming ability increased with increasing concentrations of the inducer isopropyl-beta-D-thiogalactopyranoside, and the swimming fraction increased after induction . These results are consistent with the notion that MotY is a component of the force-generating unit . V . alginolyticus motY complemented the motY mutation of V . parahaemolyticus . However, motY appeared to lack a region corresponding to the proposed motY promoter of V . parahaemolyticus . Instead, sequences similar to the sigma54 consensus were found in the upstream regions of both species . We propose that they are transcribed from the sigma54 -specific promoters.

Ann Surg, 1996 Apr, 223(4), 428 - 33
A role for tumor necrosis factor-alpha in the increased mortality associated with Vibrio vulnificus infection in the presence of hepatic dysfunction; Espat NJ et al.; OBJECTIVE: The present study was designed to evaluate whether pre-existing hepatic dysfunction (cirrhosis) leads to increased morbidity and mortality, in part through an inappropriate in vivo tumor necrosis factor-alpha response . SUMMARY BACKGROUND DATA: Vibrio vulnificus is the most commonly isolated member of the noncholera Vibrio sp., responsible for fulminant and frequently fatal septicemia . A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from Vibrio sp . infection . However, the underlying mechanism behind this association has not been fully delineated . METHODS: Cirrhosis was induced in C57BL/6 (15 to 20 g) mice using thrice-weekly injections of carbon tetrachloride (CCl4) for 7 weeks . Either a 7.0 to 9.5 X 10(7) (low dose) or a 0.8 to 1.2 X 10(9) colony-forming unit (high dose) of V . vulnificus was administered through a mini-laparotomy incision via transgastric puncture into both cirrhotic and control animals . RESULTS: Mortality in cirrhotic mice to low- and high-dose Vibrio infection was 88% (7/8) and 100% (8/8), respectively, whereas mortality in control animals was 0% (0/8) and 12% (1/8), respectively (p<0.01) . Tumor necrosis factor-alpha mRNA could be detected by reverse transcriptase polymerase chain reaction in livers and lungs from infected animals 2 and 4 hours after Vibrio administration in both control and cirrhotic animals . Lung and liver tumor necrosis factor-alpha bioactivity, however, was significantly lower in cirrhotic animals infected with Vibrio when compared with controls . Serum tumor necrosis factor-alpha was only sporadically detected in both groups of Vibrio-infected animals . When cirrhotic mice challenged with a low dose of Vibrio sp . were pretreated with 1.0 mg/kg body weight of a novel tumor necrosis factor-alpha receptor immunoadhesin, the increased mortality was completely prevented . CONCLUSIONS: Cirrhotic mice show increased mortality to Vibrio infection, and this increased mortality is dependent on an in vivo tumor necrosis factor-alpha response.

Epidemiol Infect, 1996 Apr, 116(2), 121 - 6
Epidemic cholera in Guatemala, 1993: transmission of a newly introduced epidemic strain by street vendors; Koo D et al.; Epidemic cholera reached Guatemala in July 1991 . By mid-1993, Guatemala ranked third in the hemisphere in reported cases of cholera . We conducted a case-control study with two age-, sex-, and neighbourhood-matched controls per patient in periurban Guatemala City . Twenty-six patients hospitalized for cholera and 52 controls were enrolled . Seven (47%) of 15 stool cultures obtained after admission yielded toxigenic Vibrio cholerae O1 . All seven were resistant to furazolidone, sulfisoxazole, and streptomycin, and differed substantially by pulsed-field gel electrophoresis from the Latin American epidemic strain dominant in the hemisphere since 1991 . In univariate analysis, illness was associated with consumption of left-over rice (odds ratio {OR} = 7.0, 95% confidence interval {CI} = 1.4-36), flavored ices (-helados') (OR = 3.6, CI = 1.1 - 12), and street-vended non-carbonated beverages (OR = 3.8, CI = 1.2-12) and food items (OR = 11.0, CI = 2.3-54) . Street-vended food items remained significantly associated with illness in multivariate analysis (OR = 6.5, CI = 1.4-31) . Illness was not associated with drinking municipal tap water . Maintaining water safety is important, but slowing the epidemic in Guatemala City and elsewhere may also require improvement in street vendor food handling and hygiene.

J Infect Dis, 1996 Apr, 173(4), 1019 - 22
Enteroaggregative Escherichia coli heat-stable enterotoxin is not restricted to enteroaggregative E . coli; Savarino SJ et al.; Enteroaggregative Escherichia coli (EAggEC) have been implicated as diarrheal pathogens in several settings . Some EAggEC produce a distinct heat-stable enterotoxin named EAST1 . The distribution and prevalence of the EAST1 gene in selected groups of bacterial enteropathogens were determined by colony hybridization . One hundred percent of 75 O157:H7 enterohemorrhagic E . coli (EHEC), 41% of 227 EAggEC, 41% of 149 enterotoxigenic E . coli, 22% of 65 enteropathogenic E . coli (EPEC), and 38% of 47 E . coli stool isolates from asymptomatic children hybridized with an EAST1 DNA probe . None of 55 enteroinvasive E . coli, 12 Yersinia enterocolitica, or 20 Vibrio cholerae non-O1 strains were EAST1 probe-positive . Concordance between EAST1 genotype and enterotoxicity was shown in examined strains of EAggEC, EHEC, and EPEC . The gene encoding EAST1 is more broadly distributed among diarrheogenic E . coli than previously known and may represent an additional determinant in the pathogenesis of E . coli diarrhea.

Biochim Biophys Acta, 1996 Mar 29, 1300(1), 1 - 4
Genetic analysis of the chromosomal region encoding lysophospholipase L2 of Vibrio cholerae O1; Whayeb SA et al.; From the Tn5-inserted mutant library of Vibrio cholerae O1, we found a mutant, NF404, which lost the production of both hemolysin and cholera toxin (CT) even though the Tn5-insertion site was out side from the structural genes for hemolysin and CT . Cloning and sequencing analysis of the homologous region from the wild-type strain, revealed that the sequence spanning the coding region of an ORF1 nominated as lypA, encoding a 39.5 kDa protein . Deduced amino acid sequence of the lypA gene had 37.6% identity to the lysophospholipase L2 (EC 3.1.1.5) of Escherichia coli . In the downstream of lypA, a second open reading frame (ORF2) encoding an unknown protein with molecular weight of 19.9 kDa was found . Assaying the lysophospholipase L2 activity in the cell extract of E.coli harboring lypA in an expression vector showed clear increase in the enzymatic activity.

Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 509 - 14
On the nature of the adaptive response induced by mitomycin C in Vibrio cholerae OGAWA 154 cells; Basak J; The induction of an adaptive response in Vibrio cholerae OGAWA 154 cells was obtained using an alkylating agent, mitomycin C, as both stimulating and challenging agent . Cross-adaptive response was observed in V . cholerae cells when pretreated with a sublethal dose of another alkylating agent, N-methyl-N'nitro-N-nitrosoguanidine, followed by challenging treatment with mitomycin C . The dose of mitomycin C for 50% survival (D50) became almost double for mitomycin C pretreated cells and 1.5 times for N-methyl-N-'nitro-N-nitrosoguanidine pretreated cells, compared to nonpretreated cells . It was also shown that pretreatment with a sublethal dose of oxidative DNA damaging agents, viz, hydrogen peroxide or nitrofurantoin, did not show any cross-adaptive response against subsequent challenge by mitomycin C.

Carbohydr Res, 1996 Mar 22, 283, 111 - 27
Structural studies of the lipopolysaccharide O-antigen and capsular polysaccharide of Vibrio anguillarum serotype O:2; Sadovskaya I et al.; Vibriosis caused by Vibrio anguillarum affects salmonid and marine fish species worldwide and is considered to be one of the most serious threats to the success of commercial fish farming . In the course of this study, it was found that V . anguillarum serotype O:2 strains produce an acidic capsular polysaccharide having the identical structure to that of the O-chain polysaccharide . One-dimensional and two-dimensional nuclear magnetic resonance techniques, together with partial hydrolysis and various specific modifications, were used to determine the structure of these polysaccharides . It is proposed that both O-chain and capsular polysaccharide of V . anguillarum serotype O:2 are composed of linear tetrasaccharide repeating units having the following structure, in which Glc2NAc3NAN represents 2-acetamido-3-amino-2,3-dideoxy-D-glucuronamide, Man2NAc3AmA is 3-acetamidino-2-acetamido-2,3-dideoxy-D-mannuronic acid . Am represents an acetamidino group, Gal(NAc)2A is 2,3-diacetamido-2,3-dideoxy-L-galacturonic acid, Bac(NAc)2 is 2,4-diacetamido-2,4,6-trideoxy-D-glucose (N,N'-diacetylbacillosamine) and Fo is formyl.

Biochim Biophys Acta, 1996 Mar 13, 1279(2), 149 - 56
Properties of a Na+/galactose (glucose) symport system in Vibrio parahaemolyticus; Sarker RI et al.; We have investigated galactose transport in a mutant strain of Vibrio parahaemolyticus that lacks a glucose-PTS (phosphoenolpyruvate:carbohydrate phosphotransferase system) and a trehalose-PTS . Cells of the V . parahaemolyticus actively transported D-galactose and Na+ greatly stimulated the transport . Maximum stimulation of D-galactose transport activity was observed at 10mM NaCl, and Na+ could be replaced with Li+ . Addition of galactose to the cell suspension under anaerobic conditions elicited Na+ uptake . Therefore, we conclude that this organism accomplishes galactose transport by a Na+/solute symport mechanism . Judging from inhibition results, D-galactose, D-glucose and to a lesser extent alpha-D-fucose are substrates of this transport system . The Na+/galactose symport system exhibited a high affinity for D-galactose (Km: 40 microM) and showed a relatively lower affinity for D-glucose (Km: 420 microM), but the maximum velocities for galactose and glucose transport were almost same (about 52 nmol/min per mg protein) . The Na+/D-galactose symport system was induced by either D-galactose or alpha-D-fucose, and repressed by D-glucose.

J Antimicrob Chemother, 1996 Mar, 37(3), 575 - 81
Efficacy of norfloxacin and doxycycline for treatment of vibrio cholerae 0139 infection; Dutta D et al.; An open randomised controlled clinical trial with 160 adults with acute watery diarrhoea and severe dehydration compared the efficacy of varying regimens of norfloxacin and doxycycline for the treatment of cholera caused by Vibrio cholerae 0139 Bengal . Data were analysed for the 111 patients who were faeces culture positive for V . cholerae 0139 . In addition to rehydration therapy, 28 patients received 300 mg of doxycycline as a single dose on admission, 26 patients received norfloxacin 400 mg bd for three days, 28 patients received a single dose of 800 mg of norfloxacin and 29 patients received no antibiotic (control group) . Patients in the three treatment groups and control group had comparable characteristics on admission . All three treatment groups had reduced stool output, duration of diarrhoea and fluid intake compared with the control group . Multidose norfloxacin treatment significantly reduced stool output, duration of diarrhoea and fluid requirement compared with the other regimens.

Southeast Asian J Trop Med Public Health, 1996 Mar, 27(1), 126 - 31
Hemolysins and plasmid profiles of Vibrio parahaemolyticus; Vadivelu J et al.; Forty clinical isolates of Vibrio parahaemolyticus were studied for the production of the thermostable direct hemolysin (TDH), and the TDH-related hemolysin (TRH) including the respective encoding genes, tdh and trh . The presence of TDH and its encoding genes were found amongst 95% of the strains, whereas the TRH was absent amongst these isolates . Thirty-two isolates were found to be plasmid-free, whereas eight isolates possessed plasmids with sizes ranging from 2.4 > or = 23 kb . Using a DNA probe coding for the homologous region of the tdh and trh, it was found that the tdh genes were present on the chromosomal DNA.

Appl Environ Microbiol, 1996 Mar, 62(3), 928 - 35
Siderophore-mediated iron acquisition mechanisms in Vibrio vulnificus biotype 2; Biosca EG et al.; Vibrio vulnificus biotype 2 is a primary pathogen for eels and, as has recently been suggested, an opportunistic pathogen for humans . In this study we have investigated the ability of V . vulnificus biotype 2 to obtain iron by siderophore-mediated mechanisms and evaluated the importance of free iron in vibriosis . The virulence degree for eels was dependent on iron availability from host fluids, as was revealed by a reduction in the 50% lethal dose for iron-overloaded eels . This biotype produced both phenolate- and hydroxamate-type siderophores of an unknown nature and two new outer membrane proteins of around 84 and 72 kDa in response to iron starvation . No alterations in lipopolysaccharide patterns were detected in response to iron stress . Finally, our data suggest that V . vulnificus biotype 2 uses the hydroxamate-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein.

Appl Environ Microbiol, 1996 Mar, 62(3), 918 - 27
Phenotypic characterization of Vibrio vulnificus biotype 2, a lipopolysaccharide-based homogeneous O serogroup within Vibrio vulnificus; Biosca EG et al.; In this study, we have reevaluated the taxonomic position of biotype 2 of Vibrio vulnificus . For this purpose, we have biochemically and serologically characterized 83 biotype 2 strains from diseased eels, comparing them with 17 biotype 1 strains from different sources . Selected strains were also molecularly analyzed and tested for eel and mouse pathogenicity . Results have shown that biotype 2 (i) is biochemically homogeneous, indole production being the main trait that distinguishes it from biotype 1, (ii) presents small variations in DNA restriction profiles and outer membrane protein patterns, some proteins being immunologically related to outer membrane proteins from biotype 1, (iii) expresses a common lipopolysaccharide (LPS) profile, which is immunologically identical among strains and distinct from that of LPS of tested biotype 1 strains, and (iv) contains at least two high-Mr plasmids . Regarding host range, we have confirmed that both biotypes are pathogenic for mice but only biotype 2 is pathogenic for eels . On the basis of these data, we propose that biotype 2 of V . vulnificus constitutes an LPS-based O serogroup which is phenotypically homogeneous and pathogenic for eels . In this article, the serogroup is designated serogroup E (for eels).

Microb Pathog, 1996 Mar, 20(3), 141 - 53
The toxin-coregulated pilus is a colonization factor and protective antigen of Vibrio cholerae El Tor; Voss E et al.; We have previously shown that insertional inactivation of tcpA, the gene encoding the major pilin subunit of the toxin-coregulated pilus (TCP), renders Vibrio cholerae O1 strains of El Tor biotype virtually avirulent in the infant mouse cholera model (IMCM) . We now report that more refined mutants, bearing an in-frame deletion in tcpA, show a similar dramatic attenuation in vivo . In mixed-infection competition experiments the ratio of wild-type:mutant vibrios increased c . 10(3)-10(5) fold during a period of in vivo growth . An attempt to complement the delta tcpA mutants by providing a functional El Tor tcpA gene in trans was only partially successful . Sera raised against El Tor TcpA were able to passively protect infant mice against challenge with TCP-positive strains of homologous biotype and were also protective against isolates of the novel O139 serovar . These sera failed to protect against challenge with a strain of classical biotype, nor could antibodies to classical TCP confer immunity to El Tor challenge . We conclude that TCP is a critical colonization factor of V . cholerae O1 El Tor and that antibodies to TCP are sufficient to confer protection against such strains in the IMCM . Our data suggest that the biotype-specific epitopes carried by TcpA are of greater vaccine significance than those epitopes common to both proteins.

Bull Pan Am Health Organ, 1996 Mar, 30(1), 36 - 42
Comparative effectiveness of co-trimoxazole and tetracycline in the treatment of Cholera; Grados P et al.; The purpose of the study reported here was to compare the bactericidal effectiveness of tetracycline and co-trimoxazole (a combination of sulfamethoxazole and trimethoprim) in treating cholera . The study, an open-ended random trial using adult patients with cholera cases confirmed by stool culture, was carried out in March 1993 at the Cholera Treatment Unit (CTU) of the Hospital de Apoyo Departmental Maria Auxiliadora in Lima, Peru . A total of 107 subjects were divided into two groups (A and B) . The 50 in Group A received 500 mg of tetracycline orally every 6 hours for 3 days; the 57 in Group B received co-trimoxazole (160 mg of trimethoprim and 800 mg of sulfamethoxazole) orally every 12 hours for 3 days . The two groups were comparable in terms of age, sex, duration of symptoms prior to hospital admission, time at which antibiotic treatment was initiated, and clinical evolution . Control stool cultures of specimens obtained after treatment showed Vibrio cholerae O-1 present in 2% of the Group A and 12.3% of the Group B patients, and also showed V . cholerae non-O-1 present in 2% of the Group A patients and 3.5% of the Group B patients . Overall, it was concluded that both therapeutic treatment regimens were effective and that the strains of V . cholerae observed in the southern sector of the city of Lima were still susceptible to both antibiotics.

Biosci Biotechnol Biochem, 1996 Mar, 60(3), 463 - 7
A novel alcohol resistant metalloproteinase, vimelysin, from vibrio sp . T1800: purification and characterization; Oda K et al.; We found a novel metalloproteinase, which has high activity at low temperatures and in the presence of organic solvents, in the culture supernatant of a marine bacterium, Vibrio sp . T1800 . The metalloproteinase, named vimelysin, was purified from the culture supernatant by three column chromatographies . About 150 mg of purified vimelysin was obtained from 3.3 liters of the culture supernatant with a high yield of 57% . The purified vimelysin showed a single protein band on SDS-PAGE with molecular weight of 38,000 . The isoelectric point of vimelysin was 4.3 by isoelectric focusing . The optimum pH of vimelysin was pH 8.0 or pH 6.5 using casein or furylacryloyl-glycyl-leucine amide (FAGLA) as substrates, respectively . The optimum temperature of vimelysin was 50 degrees C when casein was used as a substrate, but it was 15 degrees C when FAGLA was used as a substrate . Interestingly, vimelysin activity was completely retained after 48 h of incubation at 25 degrees C in the presence of 50% ethanol . Moreover, vimelysin showed 40% activity of the control even in the presence of 10% ethanol, while thermolysin showed only 5% activity under the same conditions.

Microbiol Res, 1996 Mar, 151(1), 43 - 8
Isolation and partial characterization of polymyxin B-resistant isolates of Vibrio parahaemolyticus; Koga T et al.; Two polymyxin-resistant isolates V2PXR and P1R4PXR of Vibrio parahaemolyticus were spontaneously isolated and compared with the polymyxin-sensitive strains as to their chemical compositions of cell envelopes and their outer membrane protein compositions . When grown in the presence or absence of polymyxin, the cell envelopes of polymyxin-resistant isolates showed a significantly increased content of protein as compared with those of respective polymyxin-sensitive strains . Both polymyxin-resistant isolates produced the major outer membrane protein b' after growth in the presence or absence of polymyxin . A new major outer membrane protein with an apparent molecular weight of 26,000 (26 K protein) was produced by polymyxin-resistant isolates grown in the presence of polymyxin . The production of 26 K protein was also observed in the polymyxin-resistant isolates grown in the medium containing low concentrations of NaCl (0.2% and 0.5%).

J Biochem (Tokyo), 1996 Mar, 119(3), 456 - 62
The CDw65 monoclonal antibodies VIM-8 and VIM-11 bind to the neutral glycolipid V3FucnLc8Cer; Kniep B et al.; At the IVth and Vth Workshop on Human Leukocyte Differentiation Antigens a group of monoclonal antibodies recognizing myeloid cells was found to bind to the ganglioside X3-NeuAcVII3FucnLc10Cer (VIM-2 dodecasaccharide) . These antibodies were given the provisional cluster of differentiation designation CDw65 . Three antibodies of this cluster (VIM-2, VIM-8, and VIM-11) have now been studied in detail at the molecular and the cellular level . Binding of VIM-2 is abolished after treatment of cells with Vibrio cholerae neuraminidase, whereas VIM-8 and VIM-11 show enhanced binding to neuraminidase-treated cells . We investigated binding of the three mAbs to glycolipid antigens with shorter carbohydrate chains . Distinct differences were observed in the binding of CDw65 antibodies to VIII3-NeuAcV3FucnLc8Cer (VIM-2 decasaccharide) . VIM-2 strongly bound to this antigen, whereas no binding was observed with the other two mAbs . Conversely, the asialoganglioside of the VIM-2 decasaccharide, V3FucnLc8Cer, was not recognized by VIM-2, but this antigen bound strongly VIM-8 and VIM-11 . Thus, VIM-2 and the other CDw65 antibodies represented two different antigen specificities.

Comp Biochem Physiol B Biochem Mol Biol, 1996 Mar, 113(3), 639 - 44
Antibacterial activity in the coelomocytes of the sea urchin Paracentrotus lividus; Stabili L et al.; Naturally present antibacterial activity directed against Vibrio alginolyticus was demonstrated in coelomocytes lysate (CL) and cell-free coelomic fluid (CF) of the marine echinoderm Paracentrotus lividus . Kinetic analysis revealed that 5 min of contact was enough to induce significant bactericidal effect . Maximum activity required 30 min of contact . Nonsensitive to the effect of trypsin, the activity was almost completely suppressed by incubation with subtilisin . Purified from CL by three successive steps of chromatography (gel filtration, anion exchange, reverse phase), active antibacterial protein appeared as a single polypeptide chain of approximate molecular weight of 60 kDa.

Eur J Clin Microbiol Infect Dis, 1996 Mar, 15(3), 227 - 32
Clinical manifestations and molecular epidemiology of Vibrio vulnificus infections in Denmark; Dalsgaard A et al.; The clinical manifestations of and epidemiological data from 11 patients infected with Vibrio vulnificus admitted to Danish hospitals during the unusually warm summer of 1994 are reported . All patients contracted the disease after exposure to seawater; however, none had consumed seafood . Four patients developed bacteremia, one of whom subsequently died; nine patients, including the four with bacteremia, exhibited skin manifestations . Four patients contracted the disease while fishing; in at least one case the patient had handled eels . All Vibrio vulnificus strains were highly susceptible to 11 antimicrobial agents tested . Plasmid analysis revealed that 8 of 11 strains carried plasmids . Ribotyping using the enzyme HindIII on the 11 strains showed five different types, two of which comprised four strains each . The present study provides the first clinical and epidemiological data about a series of human Vibrio vulnificus infections from a temperate zone.

Diagn Microbiol Infect Dis, 1996 Mar, 24(3), 165 - 7
Antibodies that react with the capsular polysaccharide of Vibrio vulnificus are detectable in infected patients, and in persons without known exposure to the organism; Fiore A et al.; In serious infections with Vibrio vulnificus, IgG antibodies to the capsular polysaccharide of the infecting strain were demonstrable in patient serum . It was not possible to show that persons with probable increased exposure to V . vulnificus (shellfish industry workers) had increased levels of antibodies to any one of three capsular types tested when compared with persons who would be expected to have had minimal exposure to the organism (Seventh Day Adventists) . Antibodies that reacted with the capsular polysaccharides were demonstrable in persons without a history of V . vulnificus infection, suggesting that cross-reacting antibodies are present in the general population.

J Diarrhoeal Dis Res, 1996 Mar, 14(1), 16 - 26
Protection against mortality due to Vibrio cholerae infection in infant rabbits caused by immunization of mothers with cholera protective antigen; Sciortino CV; Vaccination of female rabbits with cholera protective antigen (CPA) protected their F1 progeny from lethal challenge with Vibrio cholerae . Protection was determined by the choleragenic score and survival rates . Serum and milk IgG, IgM, IgA titres to CPA, cholera toxin, and LPS were determined . At 8 and 20 weeks post-immunization, mothers' milk, sera, and infants' sera showed elevated CPA-specific IgG and IgA, and infants were protected . Mothers' serum and milk antibody remained elevated for 36 weeks . At 26 weeks, mothers were re-bred, but their progeny were swapped and cross-fed . Infants born to the placebo-vaccinated mothers and nursed by CPA-immune nannies were partially protected from challenge . Infants born to CPA-immune mothers and cross-fed by the placebo-vaccinated nannies were less protected . CPA stimulated both transplacental and milk antibody, but passive immunity was primarily milk-derived . A 36-week booster vaccine stimulated an anamnestic serological response that did not provide protection equivalent to the original vaccine . CPA provided partial protective immunity to the milk-fed infant rabbits that suggests that CPA may be important in the development of a cholera vaccine.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Mar-Apr, (2), 52 - 5
{The use of a new strain of Vibrio cholerae O139 as a producer of an enteric chemical vaccine}; Dzhaparidze MN et al.; Conditions for the submerged cultivation of a strain of V . cholerae O139, were worked out . These conditions ensured a high yield of biomass, soluble O-antigen and exoenzymes (proteinase, phospholipase A) into the culture medium, which exceeded their production by strains of serovar O1, respectively, 2, 3, 4 and 8 times . The preparation, isolated from the culture fluid and lyophilized, contained up to 50% of O-antigen and exoenzymes . In experiments on white mice the preparation exhibited low toxicity (LD50 was equal, on the average, to 1.2 mg) and immunogenicity (ED50 was equal to 3-5 micrograms) with respect to V . cholerae O139, which corresponded to the protective potency of commercial vaccine against V . cholerae O1 infection.

Vet Med (Praha), 1996 Mar, 41(3), 77 - 81
Vibriosis in rainbow trout cultured in the Krka estuary, Croatia: occurrence and comments; Malnar L et al.; The frequency of vibriosis in cultured rainbow trout, maintained under different rearing conditions in the Krka estuary, was examined over a 6 yr period . Annual studies commenced regularly in October and ended in June of the following year . Every month during study periods, 37-75 trouts were randomly taken from each of 4 farm sites for routine examination . Twenty fish from these samples were subsequently employed for bacteriological analyses . Based on morphological and biochemical properties, the bacterial isolates were identified as Vibrio anguillarum, (biotype I) . The findings demonstrate high occurrence of vibriosis in trout cultivated in the Krka estuary . Furthermore, there was a direct relationship between water quality parameters and the severity of vibriosis epizootics . Moreover, the causative agent has been isolated from free-living species, fish, notably eel and mullet, which are abundant to the Krka aquatorium . The findings from these long-term studies will be considered with reference to the developing salmon farming industry of Croatia.

Infect Immun, 1996 Mar, 64(3), 1081 - 3
A classical strain of Vibrio cholerae with diminished ability to process the proteolytically sensitive site in the A subunit of cholera toxin; Ichinose Y et al.; Vibrio cholerae O1, No . 31, a strain isolated from a patient with mild diarrhea, produced mainly the unnicked cholera toxin . The amount of toxin that had accumulated in the cells was approximately 200 times lower than that secreted into the culture medium . When the unnicked toxin was purified by three successive column chromatographies and then extracted from the polyacrylamide gel, the unnicked toxin showed two bands corresponding to the A and B subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the A1 fragment was detected by trypsinization . Biological and enzymatic activities of the purified toxin with trypsinization were identical to those of cholera toxin from V . cholerae 569B as seen in the rabbit skin permeability test and the NAD:agmatine ADP-ribosyltransferase assay . DNA sequences of the A and B subunits were identical to those of the A- and B-subunit genes from the El Tor 2125 and classical 0395 strains, respectively . These data suggest that the wild V . cholerae strain, No . 31, produces a toxin identical to toxins previously reported in the literature and secretes it without accumulation in the cell, as is the case with other strains . However, strain No . 31's ability to nick the toxin is diminished compared with such abilities of other strains.

J Bacteriol, 1996 Mar, 178(5), 1310 - 9
Flagellin A is essential for the virulence of Vibrio anguillarum; Milton DL et al.; A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized . The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit . A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made . Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy . Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene . Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa . N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA . Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence . In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection . In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host.

Gene, 1996 Feb 22, 169(1), 47 - 52
Positive selection vectors for allelic exchange; Skorupski K et al.; We describe here the development and use of two new allelic exchange vectors, pKAS32 and pKAS46 . These vectors can be used for allelic exchange in a wide variety of bacterial species because their R6K origin of replication functions only in bacteria engineered to produce the replication protein pi . In addition, these vectors express the Escherichia coli rpsL gene, encoding ribosomal protein S12, which provides a positive selection for bacteria that have exchanged cloned plasmid sequences with the corresponding chromosomal sequences . In this report, we show that these vectors can be used to efficiently introduce point mutations and deletions into the chromosome of Vibrio cholerae.

FEMS Microbiol Lett, 1996 Feb 15, 136(2), 187 - 91
Resuscitation of Vibrio cholerae O1 strain TSI-4 from a viable but nonculturable state by heat shock; Wai SN et al.; Vibrio cholerae strain TSI-4 was incubated in an M9 salt solution at 15 degrees C for more than 100 days . The plate counts showed no viable cells on day 30, but a broth culture from that day showed the growth of bacteria . However, after 35 days the bacteria entered the nonculturable state, based on the assessment of both the plate counts and broth culture . A portion of the culture was heated at 45 degrees C for 1 min in a water bath and subsequently plated onto a nutrient agar plate . More than 1000 colonies were recovered after this heat-shock treatment . The recovered cells showed the same chromosomal DNA pattern in the restriction map and the same outer membrane protein pattern in SDS-PAGE . Recovery of viable cells by heat-shock was achieved in cultures grown on M9 salt but not from cultures grown in phosphate-buffered saline . This suggests that the presence of NH4Cl in the M9 salt solution may support the growth of the bacteria in a low nutrient medium, while also playing an important role in resuscitation.

FEMS Microbiol Lett, 1996 Feb 15, 136(2), 175 - 80
Virulence-associated characteristics and phage lysogenicity of two morphologically distinct colonies of Vibrio cholerae O139 serogroup; Smirnova NI et al.; The presence of a temperate phage was demonstrated in a strain of Vibrio cholerae O139 isolated from a patient . Spontaneous variants with translucent colonies had lost this phage . The loss of the phage was associated with increased hydrophobicity, indicating the loss of the capsule . These clones were sensitive to serum bactericidal activity, showed decreased expression of such presumed virulence factors as proteases, motility and mannose-sensitive pili . Furthermore, excision of the phage made the strain dependent on purines for growth.

Carbohydr Res, 1996 Feb 7, 281(1), 47 - 60
Synthesis of the methyl alpha-glycosides of a di-, tri-, and a tetra-saccharide fragment mimicking the terminus of the O-polysaccharide of Vibrio cholerae O:1, serotype Ogawa; Lei P et al.; Methyl 4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-alpha-D- mannopyranoside was acetylated, and the fully protected methyl glycoside was treated with dichloromethyl methyl ether-ZnCl2 (DCMME-ZnCl2) reagent to give 3-O-acetyl-4-(2,4-di-O-acetyl-3- deoxy-L-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-alpha-D-mannop yranosyl chloride (3) . Condensation of 3 with methyl 3-O-acetyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6- dideoxy-alpha-D-mannopyranoside (4) gave the fully acetylated disaccharide 5, which was deacetylated yielding the methyl alpha-glycoside of title disaccharide . The disaccharide glycosyl donor required for the blockwise synthesis of the title tri- and the tetra-saccharide, 3-O-acetyl-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-d ideoxy-2-O- methyl-alpha-D-mannopyranosyl-(1-->2)-3-O-acetyl-4- (2,4-di-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-alpha- D- mannopyranosyl chloride (12), was obtained by condensation of 3 with the 1-O-acetyl analog of 4, followed by treatment of the disaccharide formed with DCMME-ZnCl2 . The synthesis of the methyl alpha-glycoside of the title trisaccharide involved a condensation of 12 with 4, followed by deacetylation . Similarly, the condensation of 12 with 15, the latter being the analog of 5 having a free HO-2, followed by deacetylation, gave the methyl alpha-glycoside of the title tetrasaccharide . All glycosylation reactions were mediated by silver trifluoromethanesulfonate in the presence of 2,4,6-trimethylpyridine . 4-(3-Deoxy-L-glycero-tetronamido)-4,6-dideoxy-2-O-methyl-alpha,bet a-D- mannopyranose was prepared for the first time . It was characterized by NMR spectroscopy, and via its crystalline per-O-acetyl derivative . It is the saccharide whose alpha-form constitutes the terminal, non-reducing end-group of the O-PS of V . cholerea O:1, serotype Ogawa.

Lett Appl Microbiol, 1996 Feb, 22(2), 184 - 8
Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus; Dalsgaard A et al.; Methods for the identification and isolation of environmental isolates of Vibrio vulnificus were evaluated . Alkaline peptone water supplemented with polymyxin B and colistin-polymyxin B-cellobiose agar were employed for the isolation of suspected V . vulnificus from water, sediment and shellfish samples . When comparing the identification of putative V . vulnificus obtained with the API 20E assay and an oligonucleotide probe, 29 API 20E profiles were obtained with only four profiles (representing 20 isolates) reaching the identification threshold of V . vulnificus among a total of 66 isolates hybridizing with the probe . The results indicated that, compared with colony hybridization, the API 20E assay was not adequate for the identification of environmental isolates of V . vulnificus.

Lett Appl Microbiol, 1996 Feb, 22(2), 111 - 4
Isolation and characterization of Vibrio alginolyticus isolated from diseased kuruma prawn, Penaeus japonicus; Lee KK et al.; Outbreaks of serious mortality among cultured kuruma prawns (Penaeus japonicus) with white spotted syndrome in the carapace occurred in the summer of 1993 in I-Lan, Taiwan . A swarming bacterium, strain Swy, was isolated from the hepatopancreas of the moribund prawns using tryptic soy agar supplemented with 1% NaCl and/or thiosulphate citrate bile salt sucrose agar . This strain was characterized and identified as Vibrio alginolyticus on the basis of a number of biochemical tests . The Swy strain was virulent to both kuruma prawns (P . japonicus) and tiger prawns (P . monodon) with LD50 values of 4.43 x 10(4) and 1.57 x 10(5) cfu g body weight-1, respectively.

Microbiology, 1996 Feb, 142 ( Pt 2), 309 - 13
Aeromonas trota strains, which agglutinate with Vibrio cholerae O139 Bengal antiserum, possess a serologically distinct fimbrial colonization factor; Nakasone N et al.; Pili of Aeromonas trota strain 1220, which agglutinates with Vibrio cholerae O139 Bengal antiserum, were purified and characterized . The molecular mass of the subunit protein was estimated to be 20 kDa and the pl was 5 center dot 4 . The pili were immunologically unrelated to the other Aeromonas pili reported so far . However, the N-terminal amino acid sequence of the subunit pilin was similar to those of the pilins from other Aeromonas pili reported previously . Neither A . trota cells nor pili purified from strain 1220 agglutinated human and rabbit erythrocytes, but both adhered to the rabbit intestine . Bacterial cells pretreated with antipilus antibody (Fab portion) failed to adhere to the rabbit intestine . Moreover, bacteria did not adhere to the rabbit intestine pretreated with the purified pili . This pilus antigen was not detected in V . cholerae O139 Bengal and other Aeromonas spp . These findings suggest that the pilus of the A . trota strain is a novel colonization factor of Aeromonas spp.

FEMS Microbiol Lett, 1996 Feb 1, 136(1), 39 - 44
Identification and characterization of an extracellular protease activity produced by the marine Vibrio sp . 60; Marcello A et al.; The marine fish pathogen Vibrio sp . 60 has been used as a host for heterologous expression of the Escherichia coli heat-labile enterotoxin B-subunit and derivatives carrying a C-terminal extension . In this study, a chimeric enterotoxin B-subunit with an extension corresponding to the carboxy-terminal nine amino acids -Tyr-Ala-Gly-Ala-Val-Val-Asn-Asp-Leu-COOH from the small subunit of herpes simplex virus type 1-encoded ribonucleotide reductase, is shown to be proteolytically cleaved in the extracellular medium by a single virus type 1-encoded ribonucleotide reductase, is shown to be proteolytically cleaved in the extracellular medium by a single protease that is secreted by the host strain . Such protease behaves as a typical metalloprotease, being inhibited by EDTA but not by a serine protease inhibitor . Purification and amino acid composition analysis of the two proteolysis products revealed a specific cleavage of the peptide bond between amino acids glycine and alanine of the nine amino acid extension with loss of activity . The above observation is relevant for the biotechnological exploitation of Vibrio sp . 60.

Appl Microbiol Biotechnol, 1996 Feb, 44(6), 795 - 800
Bioremediation of 2,4,6-trinitrotoluene-contaminated soils by two different aerated compost systems; Breitung J et al.; Two composting systems were compared on a laboratory scale as a bioremediation technology for degradation or immobilization of 2,4,6-trinitrotoluene (TNT) in contaminated soils . The first compost was aerated from the beginning whereas the second compost was only aerated after an anaerobic prephase of 65 days . In the first compost system the TNT concentration declined rapidly by 92% but, at the end, TNT could be partially recovered . During the anaerobic prephase of the second compost system, TNT was almost completely converted to aminodinitrotoluenes, which during the subsequent aeration almost entirely disappeared . In addition, the second compost generated less toxic material than the first one as confirmed by inhibition of bioluminescence of Vibrio fischeri . These data show that microbiological TNT-degradation systems can be successfully designed which are prerequisite for an efficient bioremediation of contaminated soils.

Mol Biotechnol, 1996 Feb, 5(1), 1 - 10
Detection of viable Vibrio cholerae by reverse-transcriptase polymerase chain reaction (RT-PCR); Bej AK et al.; The use of conventional PCR can amplify target DNA from both viable and nonviable cells of Vibrio cholera . Detection of only viable microbial pathogens in biological samples, especially clinical and food samples, is usually desired to ensure positive test results are associated with active agents, and not the remains of dead cells . Positive identifications caused by nonliving causative agents may lead to misguided decisions concerning the effectiveness of treatment, and whether patient treatment should be continued or whether the food should be discarded . Consequently, this work was directed toward development of a reverse-transcriptase polymerase chain reaction (RT-PCR)-based in vitro DNA amplification method, which specifically detects only viable cells . Total RNA from both viable and nonviable cells was purified by using a FastPrep Cell Disrupter ({symbol: see text}Bio 101/Savant) and FastRNA extraction reagents ({symbol: see text}Bio 101) . The purified RNA was treated with DNase I (RNase-free) to avoid any amplification from the contaminating target DNA . An RT-PCR approach using this rapid and effective method for RNA purification showed amplification of the target mRNA only from the viable cells . The sensitivity of detection of viable cells of V . cholerae was > or = 10(3), which is well within the minimum number of cells (10(5)-10(6)) required for infection . The use of a reliable prokaryotic RNA extraction method followed by RT-PCR amplification of the target mRNA can be used for specific detection of viable microbial pathogen, such as V . cholerae.

Kansenshogaku Zasshi, 1996 Feb, 70(2), 175 - 9
{Isolation of Vibrio cholerae in imported frozen seafood and their cholera-enterotoxin production}; Shiraishi S et al.; A survey study for Vibrio cholerae in imported seafood was conducted during January 1991 to December 1994 . A total of 7,439 specimens (approximately 20% of all imported food) were randomly picked up and examined for contamination of V . cholerae . Among these, V . cholerae O1 were isolated from 9 specimens, but they were all cholerae enterotoxin (CT)-negative . In terms of V . cholerae non-O1, a total of 2,803 specimens (37.4%) were contaminated with this vibrio . Shrimp, especially the ones still in their shells and imported from Asian countries such as India and Indonesia, were highly contaminated with V . cholerae . Although no strains of V . cholerae O1 isolated in this study produced CT, 2 strains of V . cholerae non-O1 were proved to be CT-producers . Taking together the high contamination of V . cholerae in imported seafood and a part of those strains producing CT, we believe that careful survey for the possible contamination of V . choleare in imported seafood is necessary.

Mol Microbiol, 1996 Feb, 19(3), 625 - 37
Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum; O'Toole R et al.; The role of the flagellum and motility in the virulence of the marine fish pathogen Vibrio anguillarum was examined . Non-motile mutants were generated by transposon mutagenesis . Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500-fold decrease in virulence when fish were inoculated by immersion in bacteria-containing water . However, the flagellar filament and motility were not required for pathogenicity following intraperitoneal injection of fish . The transposon-insertion site for six mutants was determined by cloning and sequencing of the Vibrio DNA flanking the transposon . V . anguillarum genes whose products showed strong homology to proteins with an established role in flagellum biosynthesis were identified . One of the aflagellate mutants had a transposon insertion in the rpoN gene of V . anguillarum . This rpoN mutant failed to grow at low concentrations of available iron and was avirulent by both the immersion and intraperitoneal modes of inoculation . A chemotaxis gene, cheR, was located upstream of one transposon insertion and an in-frame deletion was constructed in the coding region of this gene . The resulting non-chemotactic mutant exhibited wild-type pathogenicity when injected intra-peritoneally into fish but showed a decrease in virulence similar to that seen for the non-motile aflagellate mutants following immersion infection . Hence, chemotactic motility is a required function of the flagellum for the virulence of V . anguillarum.

Mol Microbiol, 1996 Feb, 19(4), 815 - 26
Cloning and sequence of a region encoding a surface polysaccharide of Vibrio cholerae O139 and characterization of the insertion site in the chromosome of Vibrio cholerae O1; Comstock LE et al.; Vibrio cholerae serogroup O139 Bengal is the first documented serogroup other than O1 to cause epidemic cholera . The O139 Bengal strains are very similar to V . cholerae serogroup O1 biotype El Tor strains . The major differences between the two serogroups are that O139 Bengal contains a distinct O antigen and produces a polysaccharide capsule . We previously described three TnphoA mutants of O139 strain AI1837 which abolish both O antigen and capsule production . These TnphoA insertions were mapped to a 21.5 kb EcoRI fragment of the O139 chromosome . We describe here the cloning and mapping of this 21.5 kb EcoRI fragment and it was shown to complement each of the mutants in trans to produce O antigen and capsule . The EcoRI fragment contains 13 kb of DNA that is specific to O139 and 8.5 kb of DNA that is common to O1 and O139 . Sequence analysis of the 13 kb of O139-specific DNA revealed that it contains 11 open reading frames all of which are transcribed in the same direction . Eight of the 11 open reading frames are homologous to sugar biosynthesis genes from other organisms . Using extended polymerase chain reactions, we show that the extent of the DNA region in O139 that is not present in O1 is approximately 35 kb . The site of insertion of this O139-specific DNA in the O1 chromosome was mapped to the rfbO1 region . We also demonstrate that O139 Bengal strain AI1837 contains a deletion of 22 kb that in serogroup O1 strains contains the rfb region . Therefore, O139 Bengal probably arose from an O1 strain that had undergone genetic rearrangements including deletion of the O1 rfb region and acquisition of a 35 kb region of DNA which encodes O139 surface polysaccharide.

Mol Microbiol, 1996 Feb, 19(4), 767 - 75
The role of lux autoinducer in regulating luminescence in Vibrio harveyi; control of luxR expression; Miyamoto CM et al.; Analysis of Vibrio harveyi dark autoinducer mutants has demonstrated that the level of LuxR was much lower than that observed in wild-type cells . Complementation with luxR fully restored luminescence suggesting that the lux autoinducer may control expression of the luxR regulatory gene . By primer extension, the transcriptional start site of luxR was located 78 bp from the initiation codon . The level of the primer-extended product was enhanced upon addition of the lux autoinducer to the autoinducer mutants, which was confirmed by hybridization of lux mRNA with specific probes . By using chloramphenicol acetyltransferase as a reporter gene in a transcriptional fusion with luxR, the stimulatory effect of autoinducer on luxR expression was shown to occur at the level of the luxR promoter . The results provide evidence that the autoinducer signal in V . harveyi can be transduced through luxR, resulting in stimulation of luminescence.

Indian J Biochem Biophys, 1996 Feb, 33(1), 35 - 8
Estimation of single-strand breaks induced in the dried film of DNA by high energy alpha particle from a cyclotron; Basak J; DNA extracted and purified from Vibrio cholerae OGAWA 154 cells and prepared in the form of a dry thin film was exposed in air to a beam of alpha particles obtained from a Variable Energy Cyclotron . The number of single-strand breaks per DNA unit exhibited a linear dose-effect relationship indicating the occurrence of single-hit kinetics . The efficiency and yield of alpha-induced single-strand breaks were approximately 72 eV/break and 1.39 respectively.

FEMS Immunol Med Microbiol, 1996 Feb, 13(2), 101 - 5
Role of cell-associated N-acetyl-D-glucosamine specific haemagglutinin in the adhesion of Vibrio cholerae O1 to rabbit intestinal epithelial cells in vitro; Sasmal D et al.; Previously a N-acetyl-D-glucosamine specific cell-associated haemagglutinin (HA) had been purified from a Vibrio cholerae O1 strain . This study documents the role of this purified HA as an adhesin of V . cholerae O1 . A significant inhibition in the adhesion of V . cholerae O1 bacterial cells to isolated rabbit intestinal brush borders (RIBB) was observed when the latter were pretreated with purified HA in ELISA . Antibody raised against purified HA and Fab (IgG) fragment of this serum inhibited adhesion of the bacteria to isolated rabbit intestinal epithelial cells (RIEC) . V . cholerae O1 (both Ogawa and Inaba serovars) showed less adherence to isolated RIEC of animals immunised with the purified HA . Patients convalescing from V . cholerae O1 infection showed high ELISA titres against the purified HA indicating that it is expressed in the host during the disease process.

Burns, 1996 Feb, 22(1), 44 - 7
Vibrio cholerae non-O1 primary septicaemia following a large thermal burn; Magnusson MR et al.; A case of primary septicaemia with a Vibrio cholerae not agglutinable with O group 1 sera is reported in a burn patient . This appears to be the first reported case of this organism causing infection in a burn patient . The case is discussed, highlighting the difficulties encountered in treating this unexpected organism and the course of the infection in this patient . It is probable that the organism was obtained during first aid for the burn wound.

Indian J Med Res, 1996 Feb, 103, 71 - 3
Cluster of cases of clinical cholera due to Vibrio cholerae 010 in east Delhi; Rudra S et al.; A total of 514 samples of acute diarrhoeal stools received over a period of four months yielded 315 isolates morphologically and biochemically resembling V . cholerae . Out of 315 isolates, 223 (70.8%) were identified as V . cholerae 01, 20 (6.4%) as 0139 and 42 (13.3%) as 010 . Thirty (9.5%) isolates did not agglutinate with any of the available antisera . All V . cholerae 010 isolates showed complete homogeneity in their biochemical and physiological properties . This strain appears to be closely related to El Tor biotype of V . cholerae 01, since it was positive for some of the tests used for identification of El Tor . The ability of strain 010 to grow in the presence of 6 per cent salt provides it the status of an important environmental pathogen . Acquisition of some virulence genes from El Tor vibrios by this strain 010 appears to be one of the mechanisms involved in the emergence of this serogroup.

J Photochem Photobiol B, 1996 Feb, 32(3), 153 - 7
Meso-substituted cationic porphyrins as efficient photosensitizers of gram-positive and gram-negative bacteria; Merchat M et al.; Previous studies on the photosensitization of bacterial cells with different neutral or negatively charged porphyrins and phthalocyanines have demonstrated that, although Gram-positive bacteria are efficiently photoinactivated, Gram-negative bacteria become photosensitive only after modification of the permeability of their outer membrane . The results described in this paper show that two meso-substituted cationic porphyrins, namely tetra(4N-methyl-pyridyl)porphine tetraiodide and tetra(4N,N,N-trimethyl-anilinium)porphine, efficiently photosensitize the inactivation of Gram-negative bacteria, such as Vibrio anguillarum and Escherichia coli . A negatively charged meso-substituted porphyrin, tetra(4-sulphonatophenyl)porphine, has no appreciable photosensitizing activity towards Gram-negative bacteria, although all three porphyrins exhibit a similar subcellular distribution pattern, being mainly localized in the protoplasts or spheroplasts . Moreover, the three porphyrins show similar efficiency in the photoinactivation of the Gram-positive bacterium Entorecoccus seriolicida.

Am J Gastroenterol, 1996 Feb, 91(2), 336 - 40
Non-O1 Vibrio cholerae bacteremia in patients with cirrhosis: 5-yr experience from a single medical center; Lin CJ et al.; OBJECTIVES: To assess the clinical features and susceptibility of cirrhotic patients to non-O1 Vibrio cholerae bacteremia and to provide our therapeutic experiences in this rare and high lethal infection . METHODS: Twenty-eight blood culture isolates of non-O1 V . cholerae were identified by our clinical microbiology laboratory between July 1989 and June 1994 . Patients with underlying cirrhosis and the aforementioned bacteremia were retrospectively reviewed . RESULTS: Twenty-one cirrhotic patients (16 male, five female; mean age, 50.9 yr; range 28-67 yr) were identified and classified as Child B (6 cases) and Child C (15 cases) . Bacteremic episodes occurred most often from March to September . Seafood ingestion (seven cases) and seawater exposure (two cases) were risk factors, but nosocomial infections were also noted in six cases . Presenting symptoms and signs included ascites (95.2%), fever (81%), abdominal pain (52.4%), diarrhea (33.3%), and cellulitis with bullae formation (19%) . Concurrent spontaneous bacterial peritonitis was determined in 10 cases, seven with positive ascites cultures . Antibiotic therapy (either cephalothin with gentamicin or ceftriaxone alone) cured most of the bacteremic episodes . The overall case-fatality rate was 23.8%, but 75% of the deaths were observed in patients with skin manifestation . CONCLUSIONS: Patients with decompensated cirrhosis are susceptible to non-O1 V . cholerae bacteremia and should not ingest raw seafood or expose skin wounds to salt water . A high index of suspicion and early administration of antibiotics may lower the mortality rate.

Pflugers Arch, 1996 Feb, 431(4), 668 - 70
Changes to erythrocyte membrane cation permeability induced by a bacterial toxin; Huntley JS et al.; Vibrio parahaemolyticus secretes an exotoxin prepared as Kanagawa haemolysin (KH) which causes marked alterations to the function of epithelial, cardiac and other cell types, but whose cellular mode of action is poorly understood . Using human red cells as a model system with radiotracer flux techniques, we have shown that KH (1) markedly elevated the basal leak to K+, (2) raised Ca2+ influx, and as a consequence of this, (3) stimulated the Ca2+-activated K+ channel . These results suggest that an important deleterious effect of this toxin is to elevate cation permeability, which will have both direct and indirect actions on the behaviour of a variety of cell types in vivo.

Appl Environ Microbiol, 1996 Feb, 62(2), 725 - 7
Occurrence of urease-positive Vibrio parahaemolyticus in Kanagawa, Japan, with specific reference to presence of thermostable direct hemolysin (TDH) and the TDH-related-hemolysin genes; Osawa R et al.; A total of 132 strains of V . parahaemolyticus isolated from patients and from the suspected causal food items of past food poisoning cases occurring in Kanagawa Prefecture, Japan, were examined for the ability to hydrolyze urea, with specific reference to the presence of the thermostable direct hemolysin gene (tdh) and the gene for thermostable direct hemolysin-related hemolysin (trh) . Ten strains belonging to five different O-antigen serotypes were positive for urea hydrolysis (UH+), and four of these strains did not carry tdh . A total of 106 strains carried tdh, but less than 6% of them were UH+, whereas all trh-carrying strains were UH+ . The evidence suggests that urea hydrolysis is not a reliable marker for identifying tdh-carrying V . parahaemolyticus strains in Japan (the Pacific Northeast) but may be a marker for trh-carrying strains.

Appl Environ Microbiol, 1996 Feb, 62(2), 717 - 24
Distribution of Vibrio vulnificus in the Chesapeake Bay; Wright AC et al.; Vibrio vulnificus is a potentially lethal human pathogen capable of producing septicemia in susceptible persons . Disease is almost always associated with consumption of seafood, particularly raw oysters, or with exposure of wounds to seawater . An oligonucleotide DNA probe (V . vulnificus alkaline phosphatase-labeled DNA probe {VVAP}), previously shown to be highly specific for V . vulnificus, was used to enumerate this species in environmental samples collected from the Chesapeake Bay between April 1991 and December 1992 . Total aerobic, heterotrophic, culturable bacteria were enumerated by plate counts on nonselective medium . The number of V . vulnificus organisms was determined by colony lifts of spread plates for subsequent hybridization with VVAP . V . vulnificus was not detected in any samples collected during February and March (water temperature of < 8 degrees C) but was found in 80% of the water samples collected during May, July, September, and December (water temperature of > 8 degrees C), with concentrations ranging from 3.0 x 10(1) to 2.1 x 10(2)/ml (ca . 8% of the total culturable heterotrophic bacteria) . In a multiple regression analysis, increased V . vulnificus concentrations were correlated with lower salinities and with isolation from samples collected closer to the bottom . Isolation from oysters was demonstrable when water temperatures were 7.6 degrees C, with concentrations ranging from 1.0 x 10(3) to 4.7 x 10(4)/g (ca . 12% of total culturable bacteria) . In samples collected in May and July, V . vulnificus was identified in seven of seven plankton samples and four of nine sediment samples . Our data demonstrate that V . vulnificus is a widespread and important component of the bacterial population of the Chesapeake Bay, with counts that are comparable to those reported from the Gulf of Mexico.

Appl Environ Microbiol, 1996 Feb, 62(2), 450 - 5
Effect of low temperature on starvation-survival of the eel pathogen Vibrio vulnificus biotype 2; Biosca EG et al.; At present, no reports exist on the isolation of the eel pathogen Vibrio vulnificus biotype 2 from water samples . Nevertheless, it has recently been demonstrated that this biotype can use water as a route of infection . In the present study, the survival of this pathogen in artificial seawater (ASW) microcosms at different temperatures (25 and 5 degrees C) was investigated during a 50-day period, with biotype 1 as a control, V . vulnificus biotype 2 was able to survive in the culturable state in ASW at 25 degrees C in the free-living form, at least for 50 days, entering into the nonculturable state when exposed to low temperature . In this state, this microorganism survived with reduced rates of activity, showing marked changes in size and morphology . The rate at which cells became nonculturable was dependent on their physiological age . The capsule seems not to be necessary for the survival of biotype 2 in aquatic environments as a free-living organism . Culturability remained the highest on modified salt water yeast extract agar, which is closer in salt and nutrient composition to ASW than heart infusion agar . Biotype 2 cells recovered culturability on solid media after an increase of incubation temperature from 5 to 25 degrees C . Culturable cells of this bacterium maintained infectivity for either eel or mice, while dormant cells seemed to lose their virulence . The former finding suggests that the aquatic environment is a reservoir and vehicle of transmission of this pathogen.

J Bacteriol, 1996 Feb, 178(4), 971 - 6
Modulation of luminescence operon expression by N-octanoyl-L-homoserine lactone in ainS mutants of Vibrio fischeri; Kuo A et al.; Population density-dependent expression of luminescence in Vibrio fischeri is controlled by the autoinducer N-3-oxohexanoyl-L-homoserine lactone (autoinducer 1 {AI-1}), which via LuxR activates transcription of the lux operon (luxICDABEG, encoding the putative autoinducer synthase {LuxI} and the luminescence enzymes) . We recently identified a novel V . fischeri locus, ainS, necessary for the synthesis of a second autoinducer, N-octanoyl-L-homoserine lactone (AI-2), which via LuxR can activate lux operon transcription in the absence of AI-1 . To define the regulatory role of AI-2, a luxI ainS double mutant was constructed; in contrast to the parental strain and a luxI mutant, the luxI ainS mutant exhibited no induction of luminescence and produced no detectable luminescence autoinducer, demonstrating that V . fischeri makes no luminescence autoinducers other than those whose synthesis is directed by luxI and ainS . A mutant defective only in ainS exhibited accelerated luminescence induction compared with that of the parental strain, indicating that AI-2 functions in V . fischeri to delay luminescence induction . Consistent with that observation, the exogenous addition of AI-2 inhibited induction in a dose-dependent manner in V . fischeri and Escherichia coli carrying the lux genes . AI-2 did not mediate luxR negative autoregulation, alone or in the presence of AI-1, and inhibited luminescence induction in E . coli regardless of whether luxR was under the control of its native promoter or a foreign one . Increasing amounts of AI-1 overcame the inhibitory effect of AI-2, and equal activation of luminescence required 25- to 45-fold-more AI-2 than AI-1 . We conclude that AI-2 inhibits lux operon transcription . The data are consistent with a model in which AI-2 competitively inhibits the association of AI-1 with LuxR, forming a complex with LuxR which has a markedly lower lux operon-inducing specific activity than that of AI-1-LuxR . AI-2 apparently functions in V . fischeri to suppress or delay induction at low and intermediate population densities.

J Bacteriol, 1996 Feb, 178(4), 1134 - 40
Analysis of the Pseudomonas aeruginosa elastase (lasB) regulatory region; Rust L et al.; The enzyme elastase is an important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa . Previous studies have shown that expression of the P . aeruginosa elastase gene (lasB) requires both an activator protein, LasR, and an N-acylhomoserine lactone compound termed Pseudomonas autoinducer (PAI) . In this study, we analyzed the lasB promoter region to learn more about lasB activation by LasR and PAI . We report that the lasB transcriptional start is located 141 nucleotides upstream from the lasB translational start . It was also discovered that the lasB promoter region contains two putative operator sequences (OP1 and OP2) that are similar to each other and the Vibrio fischeri lux operator . OP1 is located directly upstream from, and may overlap with, the lasB promoter region, and OP2 is centered 102 nucleotides upstream from the lasB transcriptional start site . To study the effects of these putative operators and other sequences upstream from the lasB transcriptional start site on lasB activation, a series of transcriptional lasBp-lacZ gene fusions was constructed . Data from these fusions indicate that both putative operators are involved in LasR- and PAI-mediated lasB activation, with OP1 being more important than OP2.

J Bacteriol, 1996 Feb, 178(4), 1105 - 12
Physical map of the genome of Vibrio cholerae 569B and localization of genetic markers; Majumder R et al.; A combined physical and genetic map of the genome of the classical O1 hypertoxinogenic strain 569B of Vibrio cholerae has been constructed . The enzymes NotI, SfiI and CeuI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis . The digests produced 37, 22, and 7 fragments, respectively . The CeuI maps of the genomes of strains 569B and O395, constructed by partial restriction digestion, were identical, and the data are consistent with the concept of circular chromosomes . The genome size of each of the strains was estimated to be about 3.2 Mb . The NotI and SfiI digestion profiles of the genomic DNAs of strains 569B and O395 exhibited distinct restriction fragment length polymorphism . The linkages between the 37 NotI fragments of the genome of strain 569B were determined by combining three approaches: isolation of linking clones, analysis of partial digestion fragments, and identification of NotI fragments in isolated CeuI and SfiI fragments . To align linked fragments precisely, NotI-digested genomic DNA was end labeled and separated in the same gel with the NotI-digested DNA to be probed with linking clones . This also allowed the identification of smaller restriction fragments that are not visible in ethidium bromide-stained gels . The presence of repetitive DNA sequences in the V . cholerae 569B genome has been demonstrated . Twenty cloned homologous and heterologous genes and seven rrn operons have been positioned on the physical map . The two copies of the Ctx genetic element in the genome of strain 569B are located about 1,000 kb apart.

J Bacteriol, 1996 Feb, 178(4), 1080 - 7
Genetic analysis of the interaction between Vibrio cholerae transcription activator ToxR and toxT promoter DNA; Higgins DE et al.; Expression of many virulence genes in Vibrio cholerae is under the control of the ToxT protein . These include genes whose products are required for the biogenesis of the toxin-coregulated pilus, accessory colonization factor, and cholera toxin . ToxT is a member of the AraC family of transcriptional activators and is part of the ToxR regulatory cascade . ToxR is a transmembrane DNA-binding protein that is required for transcription of toxT and also can directly activate transcription of the cholera toxin operon (ctxAB) . The sequences upstream of ctxAB and toxT to which ToxR binds show no obvious similarity, which implies that ToxR may be recognizing a degenerate sequence or, alternatively, a common structural motif within both binding sites . Data presented in this report demonstrate that nucleotides within the upstream half-site of an inverted repeat element in the toxT promoter are critical for ToxR-regulated activation of transcription in V . cholerae . In addition, gene fusion and DNA-binding studies with mutant ToxR proteins indicate that residues of ToxR required for binding to the ctx promoter are also required for binding to the toxT promoter . These data suggest that ToxR is not recognizing an inverted repeat sequence per se in the activation of toxT but, rather, some motif composed in part of sequences within the upstream half-site of the inverted repeat and that ToxR recognizes similar motifs within the ctxAB and toxT promoters.

Am J Epidemiol, 1996 Feb 1, 143(3), 263 - 8
Epidemiologic study of Vibrio cholerae O1 and O139 in Thailand: at the advancing edge of the eighth pandemic; Hoge CW et al.; Vibrio cholerae O139 Bengal emerged on the Indian subcontinent in late 1992 and was first recognized in Thailand in 1993 . To characterize the epidemiology of this disease, a hospital-based case-control study was conducted in Samutsakorn, a port city 30 km southwest of Bangkok . Between November 15, 1993, and June 3, 1994, 366 patients were confirmed to have cholera by culture, including 165 (45%) with O139 Bengal, 191 (52%) with O1 Ogawa, and 10 (3%) with both serogroups . During the same time period the previous year, 319 culture-confirmed cholera cases occurred, all serogroup O1 . Questionnaires were obtained from 105 patients with O139 Bengal and 103 with O1 infections; for each case patient, two asymptomatic age- and sex-matched control persons were selected . Of the patients with O139 Bengal infections, 93% were adults (> or = 15 years) compared with 92% of patients with O1 infections . Risk factors for cholera identified by case-control comparisons were similar for the two serogroups and included consumption of untreated water, uncooked seafood, and food served at group gatherings . V . cholerae O139 Bengal has emerged in Thailand as a cause of endemic cholera, with epidemiologic features and incidence similar to those of the preexisting O1 strainPIP: Vibrio cholera 0139 Bengal emerged on the Indian subcontinent in late 1992 and was first recognized in Thailand in 1993 . To characterize the epidemiology of this disease, a hospital-based case-control study was conducted in Samutsakorn, a port city 30 km southwest of Bangkok . Between November 15, 1993, and June 3, 1994, cultures confirmed that 366 patients had cholera, including 165 (45%) with O139 Bengal, 191 (52%) with O1 Ogawa, and 10 (3%) with both serogroups . During the same time period in the previous year, 319 culture-confirmed cholera cases occurred, all serogroup O1 . Questionnaires were completed for 217 (59%) of the 366 patients . 105 patients were infected with 0139 Bengal, 103 with V . cholera O1, and 9 with both serogroups . For each case patient, two asymptomatic age- and sex-matched control persons were selected . Of the 105 case patients with 0139 Bengal infections, 98 (93%) were adults (age 15 or older) compared with 95 (92%) of 103 patients with 01 infections . Patient infected with 0139 Bengal were more often male than patients with O1 (58% vs . 42%, p = .018) . Cholera cases and matched controls were similar with regard to matching criteria of age and sex . Risk factors for cholera identified by case-control comparisons were similar for the two serogroups and included consumption of untreated water, uncooked seafood, and food served at group gatherings . Raw seafood exhibited a strong trend toward an association with O1 infections, and this variable was a significant risk factor among all cholera cases (matched odds ratio = 2.54) . V . cholera 0139 Bengal rapidly displaced existing strains of V . cholera O1 and accounted for over 95% of V . cholera isolates in India and Bangladesh during the first year of its appearance . It has emerged in Thailand as a cause of endemic cholera with epidemiologic features and incidence similar to those of the preexisting O1 strain .

J Bacteriol, 1996 Feb, 178(3), 817 - 22
Synthesis of immediate upshift (Iup) proteins during recovery of marine Vibrio sp . strain S14 subjected to long-term carbon starvation; Marouga R et al.; Proteins induced during the initial phase of recovery after long-term carbon starvation in the marine Vibrio sp . strain S14 were identified by two-dimensional gel electrophoresis analysis . Nutritional upshift experiments with pulse-labeled cells were performed after addition of glucose to cells starved for 48 h . Eighteen proteins synthesized during the first 3 min after substrate addition were identified and designated immediate upshift proteins (Iup proteins) . They were induced at least 10-fold compared with the rate of synthesis during starvation . Of the Iup proteins, five are not found in exponentially growing cells . Subsequent to the first 3 min of glucose addition, a complex pattern of sequential synthesis of proteins made during a transient phase as well as proteins made during 60 min of the outgrowth response was monitored . To resolve whether the Iup proteins were synthesized from stable transcripts, the initiation of transcription was inhibited by rifampin (Rif) . Addition of Rif 5 min prior to glucose promoted upshift resulted in the synthesis of 12 Iup proteins . Furthermore, three Iup proteins were still induced by cells that were Rif treated 20 min prior to the upshift . These results suggest that stable but silent transcripts exist during starvation and that the translation of these mRNA species is initiated by substrate addition . This regulatory mechanism may be essential for an immediate initiation of the recovery program by the nongrowing cell.

Infect Immun, 1996 Feb, 64(2), 460 - 5
Physical linkage of the Vibrio cholerae mannose-sensitive hemagglutinin secretory and structural subunit gene loci: identification of the mshG coding sequence; Marsh JW et al.; Vibrio cholerae O1 expresses a variety of cell surface factors which mediate bacterial adherence and colonization at the intestinal epithelium . The mannose-sensitive hemagglutinin (MSHA), a type IV pilus, is a potential attachment factor of the V . cholerae El Tor biotype . We describe a TnphoA mutant that is defective in its ability to hemagglutinate mouse erythrocytes . The TnphoA insertion maps to a recently identified genetic locus that encodes products that are predicted to be essential for assembly and export of the MSHA pilus . Insertional disruption at this locus in a mshA-phoA reporter strain provides evidence for a role of this locus in the latter stages of pilus assembly and/or export . These constructs have provided physical markers by which we have established close physical linkage of this secretion locus to a set of genes that includes the mshA structural gene . Sequence analysis of the intervening region between these two loci has revealed the presence of an open reading frame with homology to pilus biogenesis genes of several gram-negative bacteria . This genetic organization suggests an entire operon encoding the MSHA pilus and the components necessary for its assembly and secretion to the bacterial cell surface . The nomenclature of the MSHA structural and secretory locus has been redefined accordingly.

Rev Prat, 1996 Jan 15, 46(2), 197 - 205
{Cholera}; Geffray L; Cholera remains a great epidemic disease . The spread of the current pandemic due to Vibrio cholerae O1, the discovery of a permanent aquatic reservoir due to dormant state of vibrio, the occurrence in 1992 of the new pathogenic serotype O139 in India, and increasing antibiotic resistance are clear demonstration that this old disease is far from conquered . Clinical manifestations are due to the stimulation of enterocytic cAMP by vibrio's enterotoxin . Treatment by appropriate early rehydration is highly effective . During major outbreaks it requires "military logistics" . Hygiene and chlorine remain the best prophylaxis; WHO's recommendations on the use of the new oral vaccines, effective against O1-strains, will probably evolve as the results of ongoing trials will become available.

FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 265 - 70
Identification of heme-binding proteins in the cell membranes of Vibrio anguillarum; Mazoy R et al.; Two strains of Vibrio anguillarum belonging to O1 and O2 serotypes were examined for their ability to bind hemin and hemoglobin . Whole cells as well as membrane extracts from both strains could clearly bind hemin and hemoglobin constitutively . Hemoglobin binding was completely inhibited by a 100-fold excess of unlabelled hemoglobin and also by hemin, suggesting the existence of specific receptors for heme groups in the cell membranes . Several hemin-binding and hemoglobin-binding bands with similar molecular sizes were detected in polyacrylamide gels as well as in Western blots . Only two of these protein bands in both strains were iron-regulated while the others were independent of the cell iron status . We conclude that both serotypes of V . anguillarum possess heme-binding abilities by means of membrane proteins.

Microbiol Immunol, 1996, 40(11), 791 - 8
Utilization of iron sources and its possible roles in the pathogenesis of Vibrio parahaemolyticus; Wong HC et al.; Vibrio parahaemolyticus is an important enteropathogen in Japan, Taiwan and other coastal regions . The influence of the regulation of iron on the pathogenesis of this pathogen has not been well characterized . The growth of pathogenic and non-pathogenic strains of V . parahaemolyticus on iron-limited agar plates was stimulated by ferritin, lactoferrin and transferrin at 30 microM, and also by hemin, hemoglobin and ferric ammonium citrate at 100 microM . Spontaneous iron-utilizing mutant strains (mutants) were derived from a clinical strain, ST550 . Compared with the parent strain, lowered virulence was demonstrated for these mutants, as assayed by adult mouse and suckling mouse models . The in vivo growth and enterotoxigenicity of these mutants were also lower in the suckling mice . Adherence of the mutants to excised mouse intestine was lower as demonstrated by scanning electron microscopy . The iron-regulated outer membrane protein profile also changed in selected mutants . These results indicate that iron-regulated outer membrane proteins and other unknown factors associated with iron utilization may have profound influences, besides iron acquisition, on the pathogenesis of V . parahaemolyticus.

Morfologiia, 1996, 109(3), 67 - 71
{Ultrastructural changes in the interstitial cells of the kidney medullary substance in suckling rabbits with experimental cholera}; Kharlanova NG et al.; A culture of virulent selection of cholera vibrios L-top 5879 was introduced through the probe to suckling rabbits-pups 10 to 12 days old . Ultrastructural changes of interstitial cells and capillaries of kidney medulla were studied . During vibrio adhesion (4 hrs after the inaction) interstitial cells acquire dystrophic changes, lipid granules content reduces, while vascular permeability grows higher which suggests the presence of prostaglandin precursors elimination into blood flow . Cholera development (1-2 days later) is accompanied with progressing of signs of prostaglandin synthesis activation and their precursors passage into the vascular bed.

Microbiol Immunol, 1996, 40(10), 735 - 41
An N-{(R)-(-)-2-hydroxypropionyl}-alpha-L-perosamine homopolymer constitutes the O polysaccharide chain of the lipopolysaccharide from Vibrio cholerae O144 which has antigenic factor(s) in common with V . cholerae O76; Sano Y et al.; Chemical and serological studies were performed with the lipopolysaccharide (LPS) from Vibrio cholerae O144 (O144) . The LPS of O144 contained D-glucose, D-galactose, L-glycero-D-manno-heptose, D-fructose, D-quinovosamine (2-amino-2,6-dideoxy-D-gluco-pyranose) and L-perosamine (4-amino-4,6-dideoxy-L-manno-pyranose) . The perosamine, a major component sugar of the LPS from O144, was in an L-configuration, as is also the case in the LPS from V . cholerae O76 (O76), in contrast to the D-configuration of the perosamine in the LPS of V . cholerae O1 . A structural analysis revealed that the O polysaccharide chain of the LPS from O144 is an alpha(1-->2)-linked homopolymer of (R)-(-)-2-hydroxypropionyl-L-perosamine . The serological cross-reactivity between O144 and O76 was clearly revealed by cross-agglutination and cross-agglutinin absorption tests with whole cells, as well as by passive hemolysis tests with sheep red-blood cells that had been sensitized with the LPS from O144 and O76 . In contrast, in passive hemolysis tests, the LPS of O144 did not cross-react serologically with the LPSs from other strains such as V . cholerae O1 (Ogawa and Inaba), V . cholerae O140, Vibrio bio-serogroup 1875 (Original and Variant) and Yersinia enterocolitica O9 . The LPSs from these strains consist of O polysaccharide chains composed of alpha(1-->2)-linked homopolymers of D-perosamine with various N-acyl groups, and they share the Inaba antigen factor C of V . cholerae O1 in common . The results obtained in this study demonstrate that the absolute configuration of the perosamine residue in homopolymers plays a very important role in the expression of the serological specificity of the Inaba antigen factor C of V . cholerae O1.

Indian J Med Res, 1996 Jan, 103, 55 - 7
CAMP test for the identification of Vibrio cholerae 0139; George V et al.; The confirmation of the identity of Vibrio cholerae serogroup 01 and serogroup 0139 is usually done by slide agglutination tests using specific antisera . Antiserum to V . cholerae 01 is freely available but not antiserum to V . cholerae 0139, thus making specific identification of the latter more difficult . A modified CAMP (Christie Atkins and Muench - Paterson) test has been described as a possibility in the identification of V . cholerae 0139 and we have evaluated this on 197 strains of organisms including 48 V . cholerae 0139, 50 V . cholerae 01, 49 non-01, non-139 V . cholerae and 50 Aeromonas species . The test had a sensitivity of 98.9 per cent and a specificity of 92.9 per cent in the identification of these strains, when compared with faeces culture as the gold standard.

Microbiol Immunol, 1996, 40(9), 621 - 6
Lipopolysaccharides of Escherichia coli K12 strains that express cloned genes for the Ogawa and Inaba antigens of Vibrio cholerae O1: identification of O-antigenic factors; Hisatsune K et al.; Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No . 30 and No . 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively . The two recombinant strains, No . 30 (Ogawa) and No . 64 (Inaba), produced LPS that included, in common, the O-polysaccharide chain composed of an alpha (1-->2)-linked N-(3-deoxy-L-glycero-tetronyl)-D-perosamine (4-amino-4,6-dideoxy-D-manno-pyranose) homopolymer attached to the core oligosaccharide of the LPS of E . coli K12 . Structural analysis revealed the presence of N-(3-deoxy-L-glycero-tetronyl)-2-O-methyl-D-perosamine at the non-reducing terminus of the O-polysaccharide chain of LPS from No . 30 (Ogawa) but not from No . 64 (Inaba) . Serological analysis revealed that No . 30 (Ogawa) and No . 64 (Inaba) LPS were found to share the group antigen factor A of V . cholerae O1 . They were distinguished by presence of the Ogawa antigen factor B {co-existing with relatively small amounts of the Inaba antigen factor (c)} in the former LPS and the Inaba antigen factor C in the latter LPS . It appears, therefore, that No . 30 (Ogawa) and No . 64 (Inaba) have O-antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O-forms of V . cholerae O1, respectively . Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2-O-methyl group at the non-reducing terminus of the alpha (1-->2)-linked N-(3-deoxy-L-glycero-tetronyl)-D-perosamine homopolymer that forms the O-polysaccharide chain of LPS of V . cholerae O1 (Ogawa).

Annu Rev Microbiol, 1996, 50, 591 - 624
Lessons from a cooperative, bacterial-animal association: the Vibrio fischeri-Euprymna scolopes light organ symbiosis; Ruby EG; Although the study of microbe-host interactions has been traditionally dominated by an interest in pathogenic associations, there is an increasing awareness of the importance of cooperative symbiotic interactions in the biology of many bacteria and their animal and plant hosts . This review examines a model system for the study of such symbioses, the light organ association between the bobtail squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri . Specifically, the initiation, establishment, and persistence of the benign bacterial infection of the juvenile host light organ are described, as are efforts to understand the mechanisms underlying this specific colonization program . Using molecular genetic techniques, mutant strains of V . fischeri have been constructed that are defective at specific stages of the development of the association . Some of the lessons that these mutants have begun to teach us about the complex and long-term nature of this cooperative venture are summarized.

Microbiol Immunol, 1996, 40(8), 595 - 8
Involvement of vulnibactin and exocellular protease in utilization of transferrin- and lactoferrin-bound iron by Vibrio vulnificus; Okujo N et al.; In vitro growth experiments were conducted to evaluate the ability of vulnibactin, a siderophore produced by Vibrio vulnificus, to sequester transferrin- or lactoferrin bound iron for growth . Comparative studies with the strain producing vulnibactin and its exocellular protease-deficient mutant revealed the involvement of the protease in addition to vulnibactin in effective utilization of iron ion (Fe3+) bound to transferrin and lactoferrin . It appears that the protease causes cleavage of these proteins, thereby making bound iron more accessible to vulnibactin.

Epidemiol Rev, 1996, 18(1), 29 - 51
Emerging foodborne pathogens: Escherichia coli O157:H7 as a model of entry of a new pathogen into the food supply of the developed world; Armstrong GL et al.; There would appear to be little argument that the large outbreaks of E . coli O157:H7 which have occurred since the early 1980s represent a distinct, new phenomenon . The number of reported cases have increased dramatically, starting from zero in 1981; however, it is also clear that this increase in reported cases is in part an artifact of improved surveillance and reporting . Available data suggest that E . coli O157:H7 infections were present prior to 1982, although numbers appear to have been small . At a molecular level, the organism shows evidence of clonal origin, but there is not the striking clonality, with virtually identical pulsed-field gel electrophoresis and ribotyping patterns, which has been seen in situations such as the emergence of Vibrio cholerae O139 Bengal in the Indian subcontinent in 1992 or the introduction of V . cholerae O1 into naive populations in South America in 1991 (127-129) . Findings are more consistent with the image of an organism which arose from a common ancestor, but which has had time to become distributed geographically and to show some evidence of genetic divergence . While this is an "emerging" infection, at least in terms of its distribution and public recognition, it is unlikely that it will be possible to identify the "first" O157:H7 case or to track the clonal spread of the organism through cattle or human populations.

Microbiol Immunol, 1996, 40(1), 59 - 65
The thermostable direct hemolysin gene (tdh) of Vibrio hollisae is dissimilar in prevalence to and phylogenetically distant from the tdh genes of other vibrios: implications in the horizontal transfer of the tdh gene; Nishibuchi M et al.; Vibrio hollisae strains isolated recently from patients in various locations were examined for the presence of the thermostable direct hemolysin gene (tdh) using nucleic acid hybridization and polymerase chain reaction assays . The results were consistent with the previous finding that all strains of V . hollisae carry the tdh gene . In contrast, the tdh gene has been detected in a minority of strains for other Vibrio species (V . parahaemolyticus, V . cholerae non-O1, and V . mimicus) . Detailed phylogenetic analysis showed that the tdh genes of the non-V . hollisae species were very closely related to each other and that the tdh gene of V . hollisae was distantly related to the tdh genes of the non-V . hollisae species . These results and the proposed insertion sequence-mediated tdh transfer mechanism suggest that the tdh gene may have been maintained stably in V . hollisae and that the tdh genes of the non-V . hollisae species may have been involved in recent horizontal transfer.

Arch Med Res, 1996 Autumn, 27(3), 275 - 83
Genetic manipulation of Vibrio cholerae for vaccine development: construction of live attenuated El Tor candidate vaccine strains; Benitez JA et al.; The recent spread of El Tor cholera to America augments the need for an effective, safe and economical vaccine . In the present paper we describe the construction of live attenuated V . Cholerae strains by specifically deleting the genes encoding cholera toxin and other putative toxins from the bacterial chromosome . To maximize the likelihood of exposing protective antigens relevant to currently circulating vibrios we selected for genetic manipulation recent epidemic V . cholerae isolates from Peru . The mutant strains did not produce cholera toxin in vitro and in vivo . Deletion of the virulence cassette was accompanied by marked attenuation in the infant mouse cholera model . A selected El Tor Ogawa candidate vaccine strain was refractory to acquisition of foreign genes by conjugation with toxigenic vibrios.

Vet Pathol, 1996 Jan, 33(1), 55 - 65
A sequential light microscopic and ultrastructural study on the uptake and handling of Vibrio salmonicida in phagocytes of the head kidney in experimentally infected Atlantic salmon (Salmo salar L.); Brattgjerd S et al.; The uptake and handling of Vibrio salmonicida in phagocytes of the head kidney of Atlantic salmon (Salmo salar L.) were evaluated by light and electron microscopy, including in situ identification of the bacterium by immunolabeling at the light microscopical and the ultrastructural level . Fish were injected with live bacteria, and 4, 24, 48, and 72 hours after inoculation, samples were collected after perfusion fixation . Morphologically, the most prominent change in the course of the experiment was an increasing number of intrasinusoidal, endothelial cell-adherent phagocytes and the elevated number of interstitial melanomacrophages . Immunohistochemically, bacterial antigens were initially identified in intrasinusoidal phagocytes, and at 24 hours postinfection in endothelium-adherent phagocytes and intrasinusoidal melanomacrophages . Later, (48 and 72 hours postinfection), the interstitial melanomacrophages were also found to harbor bacterial antigen . Ultrastructurally, bacteria were identified in phagosomes in intrasinusoidal phagocytes, and morphological findings also indicated an increased cellular degradation, including autophagocytosis . Immunoelectron microscopy indicated that bacterial antigens were associated with melanomacrophages, specifically in their electron-dense cytoplasmic granules . These findings indicate that intrasinusoidal phagocytes and melanomacrophages participate in the rapid and active clearance of particulate material from the circulation, i.e., pathogenic microorganisms, and in the scavenging of cellular degradation products . The process of formation of melanomacrophages and their possible function is discussed.

Rev Argent Microbiol, 1996 Jan-Mar, 28(1), 23 - 30
{Survival of different strains of Vibrio cholerae on fresh lettuce}; Wachsman MB et al.; Temperature effect on the survival of Vibrio cholerae in fresh lettuce was studied . Remaining infectivity in leaves experimentally contaminated with different strains of Vibrio cholerae was determined at 4 degrees C and 25 degrees C . The number of colonies in TCBS agar (Vibrio cholerae) and in nutrient agar (Vibrio cholerae and secondary microorganisms) was determined at to (initial time) and at ti (different post-contamination times) . The ratio ti/to was calculated in both cases . Different survival features were found among the Vibrio cholerae strains . The CDC-185 was the most sensitive one in the experimental conditions used . At 25 degrees C, Vibrio cholerae grew faster than the secondary microorganisms and perhaps in this way it would survive in nature . At 4 degrees C lettuce leaves contaminated with the CDC-185 and the Peruano strains showed an important overgrowth relative to the secondary microorganisms at the 7th day post-contamination, whereas No O1 and 425 strains were able to inhibit this multiplication . Lettuce leaves were treated with acetic acid 0,05% to determine the inactivation of Vibrio cholerae at low pH . This decontamination treatment was effective, since a reduction of 2 log was obtained in all cases.

Biol Signals, 1996 Jan-Feb, 5(1), 22 - 9
Effects of dibutyryl cAMP and bacterial toxins on indoleamine-induced encystment of dinoflagellates; Tsim ST et al.; Dinoflagellates are the causative agents of red tides with worldwide occurrence and can be induced to encyst by in doleamines such as melatonin and 5-methoxytryptamine (5-MOT) . This biological response may be mediated via indoleamine-binding proteins or receptors . Here we report the initial characterization of the signal transduction mechanisms by which indoleamines induce encystment of dinoflagellates . In particular, we explored the possible involvement of G proteins and cAMP in cyst formation . Both melatonin and 5-MOT promoted the encystment of Gonyaulax tamarensis and Crypthecodinium cohnii . Exposure of dinoflagellates to dibutyryl cAMP, which directly activates cAMP-dependent pathways, did not affect the ability of indoleamines to promote encystment . However, dibutyryl cAMP dose-dependently diminished the indoleamine-induced suppression of cell growth . Exposure of dinoflagellates to the bacterial toxins from Vibrio cholerae and Bordetella pertussis had no effect on the indoleamine-induced encystment response, indicating the lack of involvement of Gs or Gi-like proteins . Moreover, {32P}ADP ribosylation of dinoflagellate membranes by either toxin failed to identify substrate proteins . These results suggest that although the indoleamine-induced encystment of dinoflagellates may involve a G-protein-coupled signal transduction pathway, the identity of the G protein concerned may be distinct from those that regulate adenylyl cyclases in mammalian cells.

Microbiol Immunol, 1996, 40(4), 303 - 5
Analysis of Vibrio cholerae O139 Bengal isolated from different geographical areas using macrorestriction DNA analysis; Kurazono H et al.; Vibrio cholerae O139 isolated from different countries, as well as from different locations within a country, were examined using macrorestriction DNA analysis to determine the clonality of the O139 strains . NotI digests of genomic DNA of representative strains from Nepal, India, Bangladesh, China, Thailand, and Malaysia revealed very similar but not identical patterns . Examinations of the banding patterns generated by pulsed-field gel electrophoresis of strains isolated within countries revealed complete homogeneity . These results further reiterate the spread of an identical clone of V . cholerae O139 although it appears that genetic polymorphism among the O139 strains is becoming apparent.

Life Sci, 1996, 59(3), PL41 - 7
Vibrio vulnificus hemolysin dilates rat thoracic aorta by activating guanylate cyclase; Kook H et al.; Hemolysin produced by Vibrio vulnificus caused hypotension and tachycardia in rats and dilated rat thoracic aorta . Hemolysin-induced vasodilatation of the aorta was not affected by N omega-nitro-L-arginine methyl ester and aminoguanidine, NO synthase inhibitors, whereas the vasodilatation was inhibited by LY 83,583, a guanylate cyclase inhibitor . Hemolysin elevated cGMP levels, and the elevation was abolished by LY 83,583 . These results suggest that V . vulnificus hemolysin activates guanylate cyclase independently of NO synthase, and the subsequent increase in cGMP levels results in vasodilatation.

FEMS Microbiol Lett, 1996 Jan 1, 135(1), 143 - 7
Effect of Vibrio cholerae non-O1 protease on lysozyme, lactoferrin and secretory immunoglobulin A; Toma C et al.; The effect of Vibrio cholerae non-O1 protease on host defense proteins (lysozyme, secretory immunoglobulin A and lactoferrin) was studied in relation to its virulence mechanism . The proteins treated with the protease were analysed by SDS-PAGE . There was no influence of the protease on lysozyme . The protease cleaved lactoferrin into two fragments of 50 kDa and 34 kDa . N-terminal amino acid sequencing of these fragments revealed that the cleavage site was near the hinge region, between serine 420 and serine 421 . This cleavage could affect the transition from open to closed configuration which is involved in iron binding and release . The anti-bacterial activity of lactoferrin was not affected by protease treatment . Secretory immunoglobulin A yielded a 42-kDa protein as the cleavage product . The susceptibility of secretory immunoglobulin A to V . cholerae non-O1 protease suggests a mechanism by which bacteria might evade the effect of this immunoglobulin.

Can J Microbiol, 1996 Jan, 42(1), 87 - 93
Serogroup conversion of Vibrio cholerae non-O1 to Vibrio cholerae O1: effect of growth state of cells, temperature, and salinity; Montilla R et al.; Recently, we reported the occurrence of seroconversion from Vibrio cholerae non-O1 to V . cholerae O1, but little is known about the environmental and physiological factors influencing seroconversion . We investigated effects of temperature (4, 25, and 35 degrees C) and salinity ( < 0.05 and 10%0.), as well as the stage of growth of cells, on serogroup conversion . Seroconversion of V . cholerae occurred under various environmental conditions . However, the rate of seroconversion in natural water ( < 0.5% salinity) and synthetic seawater microcosms (10%0 salinity), employing cells harvested from stationary phase culture, was approximately 2 logs higher than cells harvested from cultures in the logarithmic phase (i.e., 10(5) versus 10(3) per 10(10) cells . Thus, the physiological state of the cells, and to a lesser degree, temperature and salinity, is an important factor in the conversion of V . cholerae from non-O1 to O1 serogroup.

Infect Immun, 1996 Jan, 64(1), 355 - 7
Protective immunity to Shiga-like toxin I following oral immunization with Shiga-like toxin I B-subunit-producing Vibrio cholerae CVD 103-HgR; Acheson DW et al.; This study addresses a mechanism for inducing systemic immunity to Shiga-like toxins by oral administration of a Shiga-like toxin I B-subunit-expressing Vibrio cholerae vaccine strain {CVD 103-HgR(pDA60)} . Two sets of three rabbits were given either CVD 103-HgR or CVD 103-HgR(pDA60) orally . All rabbits immunized with CVD 103-HgR(pDA60) developed neutralizing serum antibodies to Shiga-like toxin I . None of the controls developed such antibodies.

Infect Immun, 1996 Jan, 64(1), 346 - 8
A nontoxic cholera enterotoxin (CT) analog is chimeric with regard to both epitypes of CT-B subunits, CT-B-1 and CT-B-2; Boesman-Finkelstein M et al.; The gene encoding a nontoxic analog, CT-2*, of cholera enterotoxin (CT) with attenuating codon substitutions in the A subunit was introduced into the attenuated Vibrio cholerae classical biotype mutant candidate vaccine strain CVD103, which produces the B subunit (but not the A subunit) of CT-1 . The recombinant strain produces a chimeric nontoxic analog holotoxin containing both CT-B-1 and CT-B-2 subunits . This offers potential advantages over CVD103 in the induction of immunity against E1 Tor biotype and V . cholerae O139 strains which produce CT-B-2 . The recombinant protein may also be useful in polysaccharide-protein conjugate vaccines against both O1 and O139 serovars of V . cholerae.

Infect Immun, 1996 Jan, 64(1), 343 - 5
Antibody against the capsule of Vibrio cholerae O139 protects against experimental challenge; Sengupta DK et al.; Antiserum to the capsular polysaccharide of an opaque variant of Vibrio cholerae O139 strain MDO-12 recognizes capsular antigen in three different colonial variants of the strain, although the amount of recognition varies with the extent of opacity . The anti-capsular-polysaccharide serum, at subagglutinating doses, protected suckling mice against challenge with both the most opaque variant and the most translucent variant . Further studies indicated that the protection was associated with inhibition of intestinal colonization by the vibrios . These results thus highlight the potential importance of the capsule in immunoprophylaxis against cholera caused by V . cholerae O139.

Infect Immun, 1996 Jan, 64(1), 283 - 9
Vibrio cholerae Hcp, a secreted protein coregulated with HlyA; Williams SG et al.; Hcp is a 28-kDa secreted protein of Vibrio cholerae regulated coordinately with the hemolysin, HlyA . Both proteins show a dependence on HlyU for expression, suggesting that Hcp may be secreted by V . cholerae in vivo . We have identified and sequenced two genes for Hcp, designated hcpA and hcpB (hemolysin-coregulated protein) . The genes encode identical amino acid sequences . Both express a 28-kDa protein, despite open reading frames with only a 19-kDa capacity, suggesting that the Hcp protein runs aberrantly on polyacrylamide gel electrophoresis . There is no cleavage involved in secretion of Hcp from the cell, suggesting a novel mechanism of secretion . An hcp null mutant was constructed, and this strain displayed no deficiency in virulence or colonization in the infant mouse cholera model . From sequence data and primer extension analysis, we predict that the hcp promoter is the sigma 54 type, with a candidate integration host factor binding site upstream . Although hcp and hlyA are coregulated by HlyU, there are no obvious similarities between their promoters . We predict that an intermediate regulator may be involved in the activation of hcp by HlyU . This raises the possibility that HlyU is part of a regulatory cascade.

Infect Immun, 1996 Jan, 64(1), 10 - 5
Factors influencing secondary vibriocidal immune responses: relevance for understanding immunity to cholera; Losonsky GA et al.; Although serum vibriocidal activity is used extensively as a marker of immunity to O1 Vibrio cholerae, there are limitations in this assay to detect instances of reexposure . We define the conditions operative in producing secondary vibriocidal responses in North American volunteers primed with either wild-type V . cholerae 1, 4, or 6 months later . Secondary serum vibriocidal responses occurred under two distinct secondary challenge conditions . The first occurred when secondary challenge produced a breakthrough in clinical protection . Following secondary exposure, 14 of 22 (64%) and 1 of 29 (3%) subjects with and without vibrio stool excretion, respectively, had secondary responses (P < 0.001); 5 of 6 (83%) and 10 of 45 (22%) subjects with or without diarrhea, respectively, mounted a secondary response (P = 0.006) . The second condition occurred in the presence of full clinical protection but was dependent on the time interval between exposure . No subject (0 to 17) vaccinated with CVD 103-HgR and given homologous wild-type challenge within 4 months mounted a secondary vibriocidal response (P = 0.0009) . The majority of the serum vibriocidal activity was of the immunoglobulin M (IgM) isotype, seen in 96 and 73% of subjects following primary and secondary exposure, respectively . Vibriocidal activity in the IgG fraction following primary and secondary exposures occurred with < or = 50% of volunteers; lipopolysaccharide (LPS)-specific IgG1 and IgG3 subclass responses supported the vibriocidal isotype data . However, following primary exposure, IgG4 LPS responses predominated, occurring in 81% of responding volunteers . These data suggest that, under certain conditions of secondary exposure to V . cholerae O1 antigens, when there is sufficient active local immunity present, there is a block of vibrio antigen resampling at the M cell level . We discuss the implications of and possible explanations for these findings.

J Bacteriol, 1996 Jan, 178(2), 571 - 3
Isolation of Vibrio harveyi acyl carrier protein and the fabG, acpP, and fabF genes involved in fatty acid biosynthesis; Shen Z et al.; We report the isolation of Vibrio harveyi acyl carrier protein (ACP) and cloning of a 3,973-bp region containing the fabG (encoding 3-ketoacyl-ACP reductase, 25.5 kDa), acpP (encoding ACP, 8.7 kDa), fabF (encoding 3-ketoacyl-ACP synthase II, 43.1 kDa), and pabC (encoding aminodeoxychorismate lyase, 29.9 kDa) genes . Predicted amino acid sequences were, respectively, 78, 86, 76, and 35% identical to those of the corresponding Escherichia coli proteins . Five of the 11 sequence differences between V . harveyi and E . coli ACP were nonconservative amino acid differences concentrated in a loop region between helices I and II.

J Bacteriol, 1996 Jan, 178(2), 524 - 30
Porins of Vibrio cholerae: purification and characterization of OmpU; Chakrabarti SR et al.; Three outer membrane proteins with molecular masses of 40, 38, and 27 kDa of the hypertoxinogenic strain 569B of Vibrio cholerae have been purified to homogeneity . The synthesis of all the three proteins is regulated by the osmolarity of the growth medium . The pore-forming ability of the 40-kDa protein, OmpT, and the 38-kDa protein, OmpU, has been demonstrated by using liposomes, in which these proteins were embedded . The 27-kDa protein, OmpX, though osmoregulated, is not a porin . OmpU constitutes 30% of the total outer membrane protein when grown in the presence of 1.0% NaCl in the growth medium and 60% in the absence of NaCl . OmpU is an acidic protein and is a homotrimer of 38-kDa monomeric units . Its secondary structure contains predominantly a beta-sheet, and three to four Ca2+ ions are associated with each monomeric unit . Removal of Ca2+ irreversibly disrupts the structure and pore-forming ability of the protein . The pore size of OmpU is 1.6 nm, and the specific activity of the OmpU channel is two- to threefold higher than that of Escherichia coli porin OmpF, synthesis of which resembles that of OmpU with respect to the osmolarity of the growth medium . The pore size of OmpT, which is analogous to OmpC of E . coli, is smaller than that of OmpU . Southern blot hybridization of V . cholerae genomic DNA digested with several restriction endonucleases with nick-translated E . coli ompF as the probe revealed no nucleotide sequence homology between the ompU and ompF genes . OmpU is also not antigenically related to OmpF . Anti-OmpF antiserum, however, cross-reacted with the 45-kDa V . cholerae outer membrane protein, OmpS, the synthesis of which is regulated by the presence of maltose in the growth medium . OmpU hemagglutinated with rabbit and human blood . This toxR-regulated protein is one of the possible virulence determinants in V . cholerae (V . L . Miller and J . J . Mekalanos, J . Bacteriol . 170:2575-2583, 1988).

J Bacteriol, 1996 Jan, 178(1), 209 - 15
Halovibrin, secreted from the light organ symbiont Vibrio fischeri, is a member of a new class of ADP-ribosyltransferases; Reich KA et al.; The purification, cloning, and deduced amino acid sequence of an ADP-ribosyltransferase secreted from the marine bacterium Vibrio fischeri (V . fischeri ADP-r) is described . This enzyme was purified from culture supernatant, and partial amino acid sequence obtained from the purified protein was used to design a degenerate oligonucleotide probe that was used to clone a cross-hybridizing DNA fragment from V . fischeri genomic DNA . Recombinant Escherichia coli clones harboring this fragment possessed ADP-ribosyltransferase activity . The DNA fragment was sequenced, and deletion analysis localized the ADP-ribosyltransferase activity to one of the three possible open reading frames in the fragment; the deduced amino acid sequence from this open reading frame matched the amino acid sequence obtained from the purified protein . V . fischeri ADP-r has no significant homology (DNA or amino acid) with other known ADP-ribosyltransferases . This enzyme appears to require neither proteolytic cleavage nor a reducing agent for enzymatic activity . The cloned gene is expressed but not secreted in E . coli; however, it is secreted from a heterologous marine Vibrio species . We have named this enzyme halovibrin.

J Bacteriol, 1996 Jan, 178(1), 156 - 62
The ToxR protein of Vibrio cholerae forms homodimers and heterodimers; Ottemann KM et al.; The ToxR protein of Vibrio cholerae regulates the expression of several virulence factors that play important roles in the pathogenesis of cholera . Previous experiments with ToxR-alkaline phosphatase (ToxR-PhoA) fusion proteins suggested a model for gene regulation in which the inactive form of ToxR was a monomer and the active form of ToxR was a dimer (V . L . Miller, R . K . Taylor, and J . J . Mekalanos, Cell 48:271-279, 1987) . In order to examine whether ToxR exists in a dimeric form in vivo, biochemical cross-linking analyses were carried out . Different dimeric cross-linked species were detected depending on the expression level of ToxR: when overexpressed, ToxR+ToxR homodimers and ToxR+ToxS heterodimers were detected, and when ToxR was expressed at normal levels, exclusively ToxR+ToxS heterodimers were detected . The amount of overexpression was quantitated by using ToxR-PhoA fusion proteins and was found to correspond to 2.7-fold the normal level of ToxR . The formation of both homodimeric ToxR species and heterodimeric ToxR+ToxS species is consistent with previously reported genetic data that suggested that both types of ToxR oligomeric interactions occur . However, variation in the amount of either the homodimeric or heterodimeric form detectable by this cross-linking analysis was not observed to correlate with laboratory culture conditions known to modulate ToxR activity . Thus, genetic and biochemical data indicate that ToxR is able to interact with both itself and ToxS but that these interactions may not explain mechanistically the observed changes in ToxR activity that occur in response to environmental conditions.

Carbohydr Res, 1995 Dec 27, 279, 117 - 31
Synthesis of the methyl alpha-glycoside of a trisaccharide mimicking the terminus of the O antigen of Vibrio cholerae O:1, serotype Inaba; Lei PS et al.; Coupling of methyl 4-amino-4,6-dideoxy-2-O-4-methoxybenzyl-alpha-D-mannopyranoside, obtained from the corresponding 4-azido derivative by treatment with H2S, with 3-deoxy-L-glycero-tetronolactone gave the crystalline methyl 4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-2-O-4-methoxybenzyl- alpha-D- mannopyranoside (7) . Subsequent acetylation of 7, followed by O-demethoxybenzylation of the 8 formed gave the crystalline methyl 3-O-acetyl-4,6-dideoxy-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetronami do)-alpha- D-mannopyranoside (9), which was used as the key intermediate in the construction of the title trisaccharide . To make a glycosyl donor allowing the extension of the oligosaccharide chain at O-2, compound 9 was converted, via conventional transformations, into 3-O-acetyl-2-O-bromoacetyl-4,6-dideoxy-4-(2,4-di-O-acetyl-3-deoxy-L-glyc ero- tetronamido)-alpha-D-mannopyranosyl chloride (12) . Condensation of 12 with 9 afforded the disaccharide 20 having a selectively removable protecting group at O-2(2) . The latter was O-debromoacetylated, and the disaccharide nucleophile thus obtained was treated with 2,3-di-O-acetyl-4,6-dideoxy-4-(2,4-di-O-acetyl-3-deoxy-L-glycero-tetr onamido)- alpha-D-mannopyranosyl chloride to give, after O-deacetylation, the target, title trisaccharide . The constituent monosaccharide of the O-specific polysaccharide antigen of Vibrio cholerae serotype Inaba, 4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-D-mannopyranose (18), was obtained from the peracetate of its methyl alpha-glycoside by acetolysis, followed by O-deacetylation . The amorphous compound 18 was characterized by 1H and 13C NMR spectroscopy and through its crystalline alpha-per-O-acetyl derivative.

Biochemistry, 1995 Dec 26, 34(51), 16725 - 32
Involvement of cysteine 289 in the catalytic activity of an NADP(+)-specific fatty aldehyde dehydrogenase from Vibrio harveyi; Vedadi M et al.; Fatty aldehyde dehydrogenase (Vh.ALDH) from the luminescent bacterium, Vibrio harveyi, may be implicated in controlling luminescence as it catalyzes the oxidation of the fatty aldehyde substrate for the light-emitting reaction . On the basis of the amino-terminal sequence of Vh.ALDH, a degenerate probe was used to screen a genomic library of V harveyi in pBR322, a positive clone was selected containing the Vh.ALDH gene and expressed in Escherichia coli, and the enzyme was purified to homogeneity . Although the sequence of the V . harveyi ALDH significantly diverged from other aldehyde dehydrogenases, mutation of a conserved cysteine implicated in catalysis completely inactivated the enzyme without loss of its ability to bind nucleotides, consistent with a catalytic role for this residue . Using absorption and fluorescence assays for NAD(P)H, it was shown that NAD+ and NADP+ bound to the same site and that saturation of Vh.ALDH with NADP+ occurred with a Michaelis constant (Km = 1.4 microM) over 40 times lower than that reported for other aldehyde dehydrogenases . Although V . harveyi aldehyde dehydrogenase is unique in terms of its high specificity for NADP+, the identification of a catalytic conserved cysteine in Vh.ALDH clearly indicates that a highly related mechanism and structure have been retained among even the most diverged aldehyde dehydrogenases.

Biochemistry, 1995 Dec 19, 34(50), 16519 - 23
High-ionic strength interference of ribosomal inhibition produced by aminoglycoside antibiotics; Marin I et al.; A protein synthesis cell-free system capable of performing with similar efficiencies in different ionic conditions has been developed for the halotolerant marine bacterium Vibrio costicola . The system has been used to test the effect of ionic strength on the interference produced by thirty translation inhibitors with different structural, functional, and domain specificities . In general, at high ionic strengths, the inhibition of protein synthesis produced by polycationic antibiotics like the aminoglycosides is much less pronounced than the inhibition obtained at low ionic strengths, while non-aminoglycosidic antibiotics show similar inhibitory activities at both high and low ionic conditions . These results strongly suggest that competition between polycationic antibiotics and cations at high concentrations in the media is responsible for the lack of inhibition by aminoglycoside antibiotics at high ionic strengths, rather than a lack of binding sites.

Harefuah, 1995 Dec 15, 129(12), 552 - 5, 615
{Acute renal failure as a complication of cholera}; Knobel B et al.; We present a 72-year-old man who had episodes of severe, acute renal failure during severe attacks of diarrhea caused by Vibrio cholerae . Patterns of acute tubular necrosis and tubulointerstitial nephritis developed following hypotension and decrease in renal blood flow, causing secondary renal ischemia . There was severe dehydration with profound hypovolemia and infection . The clinical picture included fever, weakness, arthralgia, pedal edema, mild bilateral pleural effusions, anemia, leukocytosis, azotemia with a maximum of 330 mg/dl of urea, creatine to a maximum of 9.8 mg/dl, hypoproteinemia, severe metabolic acidosis, marked increase in lactate dehydrogenase (LDH) and creatine phosphokinase (CPK), microscopic hematuria, sterile leukocyturia, normoglycemic glucosuria and phosphaturia with diminished tubular reabsorption of phosphorus . A short oliguric phase was followed by a polyuric phase lasting about 10 days, and glomerular and tubular function became normal after about 3 weeks . Treatment was by intensive infusions of fluids, electrolytes, sodium bicarbonate, salt-free albumin and antibiotics . To the best of our knowledge, this renal complication of cholera has not yet been described in Israel.

FEMS Microbiol Lett, 1995 Dec 15, 134(2-3), 245 - 9
Cloning and sequence analysis of Vibrio parahaemolyticus ompK gene encoding a 26-kDa outer membrane protein, OmpK, that serves as receptor for a broad-host-range vibriophage, KVP40; Inoue T et al.; The ompK gene of Vibrio parahaemolyticus 1010 (RIMD 2210001) encoding an outer membrane protein (OMP), OmpK, which serves as the receptor for a broad-host-range vibriophage, KVP40, was cloned and sequenced . The gene consisted of 789 nucleotides encoding 263 amino acids . Since the first 20 amino acids most likely constitute the signal peptide, mature OmpK would consist of 243 amino acids with a calculated molecular mass of 27458 Da . Sequence comparisons indicate that OmpK is unique among Vibrio OMPs so far sequenced, but may be distantly related to Tsx of enteric bacteria and is homologous to an Aeromonas hydrophila OMP, protein IV.

FEMS Microbiol Lett, 1995 Dec 15, 134(2-3), 233 - 8
Ca(2+)-independent cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin (TDH) on Intestine 407, a cell line derived from human embryonic intestine; Tang GQ et al.; The effects of Vibrio parahaemolyticus thermostable direct hemolysis on Intestine 407, a cell line derived from the intestine of human embryos, were investigated . The hemolysin was shown to be cytotoxic to Intestine 407 . This cytotoxicity is accompanied by the damage of plasma membrane and lysosomes, as well as cellular degeneration in the form of large transparent blebs . Although an increase in cytosolic free Ca2+ due t the influx of extracellular Ca2+ was observed in cells treated with thermostable direct hemolysin, it was found to be irrelevant to any of the above effects . These results suggest that the effects of thermostable direct hemolysin observed in this study on Intestine 407 are not mediated by Ca(2+)-dependent pathways.

J Diarrhoeal Dis Res, 1995 Dec, 13(4), 229 - 31
Prescribing pattern by doctors for acute diarrhoea in children in Delhi, India; Singh J et al.; Parents (mostly mothers) of 264 children aged less than 5 years with acute watery diarrhoea were interviewed about their treatment profile before hospitalization in Delhi, India in 1993 . Only 22% of the cases were given prescriptions for oral rehydration solutions (ORS), whereas a very high proportion (64%) of them were given drugs, including antibiotics and antidiarrhoeals and 40% were given intravenous fluids . The differences among the treatment groups were highly significant . The government and private doctors were equally responsible for the low rate of prescription of ORS . The confidence of health professionals in ORS appears to be very low . The findings suggest the need of a high-profile continuing education programme to encourage ORS prescription in DelhiPIP: During June-September 1993 two investigators visited the Infectious Disease Hospital 2-3 times a week to interview parents (mostly mothers) of 264 children aged under 5 years with acute watery diarrhea about their children's age, clinical symptoms, and the type of treatment received before hospitalization in Delhi, India . 107 and 46 were found positive for Vibrio cholera 01 and 0139, respectively . The remaining 111 had non-cholera watery diarrhea . Among the 46 patients with V . cholerae 0139, 17 received ORS, 34 received drugs, and 22 received iv fluids; among the 107 patients with V . cholerae 01, the respective figures were 20, 66, and 45, while among the 111 patients with non-cholera diarrhea the figures were 22, 68, and 39 . The difference in administration of ORS to 0139 cholera patients vs . 01 cholera patients was significant (p = 0.01) . 49% of the 59 cases who received ORS were treated by private physicians, as compared to 46% of the patients who were treated by government physicians . Significantly more cases were prescribed drugs by private doctors (57%) than by government doctors (41%) . Only 22% of the cases were given prescriptions for oral rehydration solutions (ORS), whereas 64% of them were given drugs, including antibiotics and antidiarrheals, and 40% were given intravenous fluids . The difference among the treatment groups was highly significant . The government and private doctors were equally responsible for the low rate of prescription of ORS . The confidence of health professionals in ORS appears to be very low, as they preferred to prescribe antibiotics and antidiarrheals rater than ORS for the treatment of acute watery diarrhea . This phenomenon indicates the need for a high-profile continuing education program for both types of physicians to encourage ORS prescription in Delhi .

J Diarrhoeal Dis Res, 1995 Dec, 13(4), 219 - 23
Effect of iron on the recovery of viable bacteria from the cardiac blood in an experimental mouse model and on the serum resistance and macrophage phagocytosis of Vibrio parahaemolyticus; Wong HC et al.; The role of iron in the pathogenesis of Vibrio parahaemolyticus, an intestinal pathogen, is not clearly understood . In an intraperitoneally infected mouse model, viable bacteria of a Kanagawa-negative food isolate CCRC12958 and a Kanagawa-positive clinical isolate ST550 of V . parahaemolyticus were rapidly recovered from the cardiac blood before the death of these animals . Iron-limitation during the preparation of bacterial culture decreased the appearance of CCRC12958 strain in the cardiac blood, and this effect was compensated for by the addition of complementary iron during infection . This phenomenon may be related to the survival and multiplication of this pathogen in 50% murine serum and in phagocytosis by murine peritoneal macrophages . However, the effect of iron-limitation on the serum survival of this pathogen was strain-dependent, and the resistance to phagocytosis was not significant.

Biomed Environ Sci, 1995 Dec, 8(4), 350 - 8
A natural vaccine candidate strain against cholera; Liu YQ et al.; E1 Tor Vibrio cholerae (EVC) strains may be classified into two kinds-epidemigenic (EEVC) strains and non-epidemigenic (NEEVC) strains-based on a phage-biotyping system . A large number of EEVC strains have been screened for toxigenic and putative colonization attributes . One such naturally occurring strains (designated IEM101) has been found which is devoid of genes encoding cholera toxin (CT), accessory cholera enterotoxin (ACE), zonula occludens toxin (ZOT), but possesses RS1 sequences and toxin-coregulated pilus A gene (icpA) although icpA is poorly expressed . It expresses type B pili but does not possess type C pili . It is an E1 Tor Ogawa strain and does not cause fluid accumulation in rabbit ilcal loop tests . Active immunization of rabbits with strain IEM101 elicited good protection against challenge with virulent strains of V . cholerae O1 . Oral administration caused no side effects in 15 human volunteers, colonized the gut for four to ten days and elicited good immune responses.

Berl Munch Tierarztl Wochenschr, 1995 Dec, 108(12), 462 - 5
{Preliminary studies of the immunization of shrimp (Penaeus monodon) against vibrio infections}; Bechteler C et al.; There is a great demand for an applicable vaccine against bacterial infections of prawns, especially Vibriosis . The results of the tests that had been carried out can be evaluated as promising and indicate that the vaccination of prawns against bacterial diseases is possible . Nevertheless it is still necessary to increase the scale of research on this subject, above all, the basics of the immuno-system of prawns . Adult prawns should be vaccinated to check if they are able to pass their immuno-protection to their progeny . If that is the case only a few breeding animals have to be vaccinated, instead of all the larvae . Actually the prophylactic application of antibiotics is the only method to prevent infections with Vibriosis . 100-150 mg of Oxytetracycline per kg of prawns are fed during one production period and these antibiotics are also used in humans . Assuming that the average amount of harvested prawns per production unit is 8-10 metric tons/pond (1 ha) . 800-1500 g of antibiotics are used . Since different pathogenic strains have developed resistance to Oxytetracycline, also other kinds of antibiotics (for example oxolinic acid) are given today . Antibiotics are often fed until harvesting, however, there are laws which prohibit use to antibiotics during the last thirty days before harvesting, to prevent residues in the prawn body . A vaccine against bacterial diseases could decrease the production costs and reduce the amount of the applied antibiotics.

Bull Pan Am Health Organ, 1995 Dec, 29(4), 312 - 21
Safety and immunogenicity of oral killed whole cell recombinant B subunit cholera vaccine in Barranquilla, Colombia; Concha A et al.; In January and February 1992, an assessment was conducted of the safety and immunogenicity of two doses of a new oral cholera vaccine prepared from the recombinant B subunit of the toxin and from killed whole cells (rBS/WC) in 1,165 individuals between the ages of 12 months and 64 years in Barranquilla, Colombia . This was a randomized, double-blind placebo-controlled study . Participants received two doses of either the vaccine or a placebo (killed Escherichia coli K12) over a two-week interval . Few symptoms were detected during the three days following administration of the initial dose and even fewer following the second . Sera obtained upon administration of the first dose and two weeks after administration of the second were tested for Vibrio cholerae 01 Inaba vibriocidal antibodies and antitoxins . Geometric mean titers (GMT) of vibriocidal antibodies were found to increase two-fold in subjects receiving the vaccine . In the paired samples taken from vaccinated subjects, two-fold or greater increases were observed in 44% and four-fold or greater increases were observed in 34%, as compared to similar increases in 9.2% and 2.2% of the sera taken from those receiving the placebo (P < 0.05) . The GMTs of IgG and IgA antitoxins, as determined by ELISA, increased by factors of 4 and 3.2, respectively, in those receiving the vaccine, as compared to factors of 1.1 and 1.1 in those given the placebo (P < 0.001 for IgG, P < 0.01 for IgA) . Approximately 80% of the paired samples from the vaccinated group showed an increase of both IgG and IgA antitoxins > or = 1.5, as compared to only about 20% of those in the placebo group (P < 0.000001) . Belonging to the O blood group did not significantly affect the immune response . Children under age four tended to show a weaker vibriocidal antibody response and a stronger antitoxin response than older subjects . The two doses of oral vaccine were found to be safe and without attributable side-effects . The vibriocidal antibody and antitoxin responses were similar to those obtained previously with the conventional oral killed whole cell B subunit cholera vaccinePIP: In a randomized, double-blind, placebo-controlled study in January and February 1992, the safety and immunogenicity of two doses of a new oral cholera vaccine was assessed . The vaccine was prepared from the recombinant B subunit of the toxin and from killed whole cells (rBS/WC) in 1165 individuals between the ages of 12 months and 64 years in Barranquilla, Colombia . Participants received two doses of either the vaccine or a placebo (killed Escherichia coli K12) over a 2-week interval . Few symptoms were detected during the 3 days following administration of the initial dose and even fewer following the second one . Sera obtained upon administration of the first dose and 2 weeks after administration of the second dose were tested for Vibrio cholera 01 Inaba vibriocidal antibodies and antitoxins . Geometric mean titers (GMTs) of vibriocidal antibodies were found to increase two-fold in subjects receiving the vaccine . In the paired samples taken from vaccinated subjects, two-fold or greater increases were observed in 44% and four-fold or greater increases were observed in 34% . In comparison, similar increases were found only in 9.2% and 2.2% of the sera taken from those receiving placebo (p .05) . The GMTs of IgG and IgA antitoxins, as determined by ELISA, increased by factors of 4 and 3.2, respectively, in those receiving the vaccine as compared with factors of 1.1 and 1.1, respectively, in those given the placebo (p .001 for IgG and p .01 for IgA) . Approximately 80% of the paired samples from the vaccinated group showed an increase of both IgG and IgA antitoxins or= 1.5 as compared with only about 20% of those in the placebo group (p .000001) . Belonging to the O blood group did not significantly affect the immune response . Children under the age of 4 years tended to show a weaker vibriocidal antibody response and stronger antitoxin response than did older subjects . The two doses of oral vaccine were found to be safe and without attributable side effects .

Infusionsther Transfusionsmed, 1995 Dec, 22(6), 344 - 9
{Flow cytometry detection of erythrocyte antigens and antibodies . Technical aspects and new clinical applications}; Fischer K et al.; BACKGROUND: Although hemagglutination techniques have proved worthwhile since many years in immunohematology, they also have several disadvantages . They are manual and subjective visual methods, which make it difficult to quantitate red cell antibodies or surface antigens . Flow cytometric analysis overcomes these limitations because of its ability to analyze individual populations of cells by sensitive, reproducible, and objective methods . MATERIALS AND METHODS: Washed red cells from regular blood donors and patients were analyzed natively and after treatment with enzymes (sialidase, protease) or pneumococcal polysaccharides, using monoclonal Rh antibodies, human 7s-immunoglobulin, and FITC-labeled anti-human IgG or FITC anti-T lectin . The fluorescence intensity of single red cells was determined in the Ortho Cytoron Absolute flow cytometer . RESULTS: We determined the optimal test conditions and normal values by investigation of 50 blood donors . The fluorescence intensity of untreated red cells proved to be constant and therefore was used to adjust the instrument . Furthermore, the data of experimental adsorptions of red cells with pneumococcal antigens, sialidase (Vibrio cholerae) and protease (papain) as well as data from patients suffering from chronic HBV infection and autoimmune hemolytic anemia and acute pancreatitis are presented . A special software program was developed for statistical analysis and graphical presentation of the raw data . The computer program permits to analyze results from different experiments or from different dates and depicts them comparatively in overlay histograms, which may be useful in a serial study of changes of the antibody concentration or antigen expression . CONCLUSION: The flow cytometric analysis of red cells proves to be a simple, rapid, reproducible, and objective method for antigen and antibody quantitation . Furthermore, this technique may be a useful new tool for the investigation of acute, infection-associated hemolytic anemia.

Microbiologia, 1995 Dec, 11(4), 439 - 46
Role of extracellular products in the pathogenicity of Vibrio strains on cultured gilt-head seabream (Sparus aurata); Balebona MC et al.; The enzymatic activities of extracellular products (ECP) from different pathogenic Vibrio strains and their virulence for fish (cultured gilt-head seabream, Sparus aurata L.) were tested . ECP were obtained by growing the bacteria on cellophane overlays . Cytotoxic effects of ECP on fish cell lines were rapid and strong, provoking mortality of fish after 24 to 72 h of the ECP inoculation . A close relationship between proteolytic activities (gelatinase and caseinase) of the ECP and the presence of lytic effects in the muscular tissue at the injection site was observed . Therefore, these activities may be considered responsible for the fish tissue damage.

J Clin Microbiol, 1995 Dec, 33(12), 3119 - 23
Characterization of Aeromonas trota strains that cross-react with Vibrio cholerae O139 Bengal; Albert MJ et al.; It has previously been shown that Vibrio cholerae O139 Bengal shares antigens with V . cholerae serogroups O22 and O155 . We detected six surface water isolates of Aeromonas trota that agglutinated in polyclonal antisera to V . cholerae O139 and V . cholerae O22 but not in antiserum to V . cholerae O155 . On the basis of agglutinin-absorption studies, the antigenic relationship between the cross-reacting bacteria were found to be in an a,b-a,c fashion, where a is the common antigenic epitope and b and c are unique epitopes . The antigen sharing between A . trota strains and V . cholerae O139 was confirmed in immunoblot studies . However, A . trota strains did not react with two monoclonal antibodies specific for V . cholerae O139 and, consequently, tested negative in the Bengal SMART rapid diagnostic test for V . cholerae O139 which uses one of the monoclonal antibodies . A polyclonal antiserum to a cross-reacting A . trota strain cross-protected infant mice against cholera on challenge with virulent V . cholerae O139 . All A . trota strains were cytotoxic for HeLa cells, positive for adherence to HEp-2 cells, and weakly invasive for HEp-2 cells; one strain was heat-stable toxin positive in the suckling mouse assay; however, all strains were negative for cholera toxin-like enterotoxin . Studies on bacteria that share somatic antigen with V . cholerae O139 may shed further light on the genesis of V . cholerae O139.

Epidemiol Infect, 1995 Dec, 115(3), 435 - 46
An epidemiological study of Vibrio cholerae O1 in the Australian environment based on rRNA gene polymorphisms; Desmarchelier PM et al.; Since 1977, Vibrio cholerae O1 has been isolated from the Australian aquatic environment and periodically cholera cases have occurred following exposure to these environments . To study the relationships between clinical isolates and environmental isolates from rivers and aquatic life, widely distributed throughout the country, a wide range of molecular typing methods were employed . In this paper we report the analysis of the 180 Australian isolates (10 clinical and 170 environmental) using ribotyping . Seven ribotype patterns were observed among the Australian inaba isolates, 2 of which included all clinical inaba isolates and 84% environmental inaba isolates collected from 9 rivers and creeks in eastern Australia during an 8-year period . Isolates from epidemiologically related clinical cases, asymptomatic household contacts and sewage were indistinguishable . The ogawa isolates were more diverse, with 9 ribotypes observed among 24 isolates from 8 rivers during the same period . Ribotype patterns were not shared between the serotypes with the exception of one ogawa isolate which could be distinguished using PFGE . Ribotyping has been useful in confirming an association between epidemiologically related clinical isolates and the aquatic environment and the persistence of several clones of the O1 serovar in the Australian environment during an 8-year period.

Epidemiol Infect, 1995 Dec, 115(3), 427 - 34
Biotype traits and antibiotic susceptibility of Vibrio cholerae serogroup O1 before, during and after the emergence of the O139 serogroup; Mukhopadhyay AK et al.; Sixty-nine strains of Vibrio cholerae O1 isolated at different times were analysed to investigate if there were any differences among the O1 strains isolated before, during and after the advent of the O139 serogroup . Of the 69 O1 strains examined, 68 belonged to the Ogawa serotype while one belonged to the Inaba serotype . With the exception of one strain all other strains of V . cholerae O1 belonged to the eltor biotype . A single O1 strain isolated before the emergence of the O139 serogroup could not be classified as either eltor or classical biotype because it was resistant to both classical and eltor specific bacteriophages . Marked variations in the susceptibility to antibiotics of V . cholerae O1 isolated during the different periods were observed . In addition, strains of V . cholerae isolated after the epidemic of serogroup O139 in Calcutta showed an expanding R-type with resistance to a variety of drugs as compared to the O1 strains isolated before the advent of the O139 serogroup . From this study, it is clear that there is a substantial mobility in genetic elements of V . cholerae O1 which necessitates a continuous monitoring to keep abreast of the changing traits of the etiologic agent of cholera.

J Trop Med Hyg, 1995 Dec, 98(6), 419 - 27
Epidemic cholera in Latin America: spread and routes of transmission; Guthmann JP; PIP: Latin America's first cholera epidemic this century struck along the Peruvian coast in January 1991 . Rapid and intense surveillance could not stop it from crossing Peru's borders . Public health interventions did keep the case fatality rate low (0.92%), however . Cholera first spread to Ecuador, then Colombia . By the end of 1993, all countries of Latin America except Uruguay and the Caribbean reported cholera cases . The greatest proportion of cholera cases and the highest incidence rate were in Peru (63.7% and 26.9/1000, respectively) . Most cholera cases were reported in 1991 and were concentrated in Peru (82.3%) . 45.5% of all cholera deaths occurred in 1991 . Central America had the highest case fatality rates . The routes of transmission of Vibrio cholerae in this Latin America cholera epidemic included unwashed fruit and vegetables, contaminated food and ice from street vendors, contaminated drinking water, and contaminated crab meat transported in luggage . The source of the epidemic in Peru has not been identified . Peru had in place an extensive oral rehydration therapy program, an epidemic field investigation service, and laboratory resources . Most Latin American countries, particularly in rural areas and the outskirts of big cities, lack water supply and basic sanitation . Untreated waste water is discharged into rivers and the ocean . Inadequate sanitation facilities and the use of inadequately treated water are likely responsible for the spread of the cholera epidemic in Latin America .

Mol Mar Biol Biotechnol, 1995 Dec, 4(4), 365 - 8
Rapid detection of Vibrio cholerae contamination of seafood by polymerase chain reaction; Karunasagar I et al.; The possibility of detecting Vibrio cholerae contamination of seafood using a technique based on polymerase chain reaction (PCR) was studied . Direct PCR on lysate prepared from fish homogenates containing 10(3) V . cholerae/ml gave a positive reaction . When combined with alkaline peptone water (APW) enrichment, homogenates containing 1.4 cells/ml gave amplification signal . The technique could also detect V . cholerae O139, the recent epidemic serotype in the Indian subcontinent . An environmental isolate of non-O1 V . cholerae that produced cholera toxin was also positive in this assay . The results suggest that PCR-based techniques have great potential in quick detection of toxigenic V . cholerae in seafoods.

Appl Environ Microbiol, 1995 Dec, 61(12), 4454 - 8
Purification and characterization of an extracellular beta-1,4-mannanase from a marine bacterium, Vibrio sp . strain MA-138; Tamaru Y et al.; A beta-mannanase (EC 3.2.1.78) from Vibrio sp . strain MA-138 was purified by ammonium sulfate precipitation and several chromatographic procedures including gel filtration, adsorption, and ion-exchange chromatographies . The final ion-exchange chromatography Mono Q yielded one major active fraction and three minor active fractions . The major active fraction was purified to homogeneity on the basis of native polyacrylamide gel electrophoresis (PAGE) . This purified enzyme was identified as a glycoprotein by periodic acid-Schiff staining and a monomeric protein with a molecular mass of 49 kDa by sodium dodecyl sulfate-PAGE . The pI of the enzyme was 3.8 . The purified enzyme exhibited maximal activity at pH 6.5 and 40 degrees C and hydrolyzed at random the internal beta-1,4-mannosidic linkages in beta-mannan to give various sizes of oligosaccharides . The first 20 N-terminal amino acid sequence of the purified enzyme showed high homology with the N-terminal region of beta-mannanase from Streptomyces lividans 66.

Gene, 1995 Dec 1, 166(1), 43 - 8
Regulation of tcp genes in classical and El Tor strains of Vibrio cholerae O1; Thomas S et al.; Expression of genes encoding the toxin-co-regulated pilus (TCP) varies between the two biotypes of Vibrio cholerae O1 . Sequence analysis of the tcp locus from the classical and El Tor strains has revealed differences in the intergenic regions between tcpI and tcpP, and tcpH and tcpA, which may be involved in regulation . To investigate this possibility, transcription of tcpA, and the predicted upstream promoters for tcpI and tcpP, has been analysed in the classical and El Tor strains using promoter-cat (chloramphenicol acetyltransferase) fusions . Together with primer extension analyses, these studies indicate that the tcpA and tcpP promoters are toxR-dependent and suggest that TcpP may be involved in activation of both the tcpI and tcpP promoters . We conclude that differences in the level of tcpA expression in a classical and an El Tor strain are likely to be due to the effect of sequence variation on the ability of control factors to act on these regulatory regions.

Gene, 1995 Dec 1, 166(1), 33 - 42
A putative pathway for perosamine biosynthesis is the first function encoded within the rfb region of Vibrio cholerae O1; Stroeher UH et al.; The first four genes (rfbA,B,D,E) of the rfb region of Vibrio cholerae O1 are predicted to encode the enzymes required for the biosynthesis of perosamine, which constitutes the backbone structure of the O-antigen of the lipopolysaccharide . Based on homology to known proteins/protein families, the following functions are predicted: RfbA, phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase; RfbB, phosphomanno-mutase; RfbD, oxido reductase and RfbE, perosamine synthetase (amino-transferase) . Thus, perosamine is synthesized from fructose 6-phosphate via the intermediates mannose 6-phosphate by RfbA, to mannose 1-phosphate by RfbB, to GDP-mannose by RfbA, to GDP-4-keto-6-dideoxymannose by RfbD and to GDP-perosamine by RfbE . This final product would then serve as the substrate for the addition of the tetronate, which could then be polymerized into the O-antigen for transfer to the lipid A plus core oligosaccharide and export to the cell surface . The organization of these genes are such that one would expect them to be translationally coupled as part of the rfb operon . However, the absence of readily detectable promoter sequences suggests low levels of transcription, in line with other studies . The nucleotide sequence of these genes is absolutely conserved in the two isolates 569B (classical, Inaba) and O17 (El Tor, Ogawa) which were expected to show maximal sequence variation . This suggests very tight constraints on the micro-evolution within these sequences.

Gene, 1995 Dec 1, 166(1), 19 - 31
A putative pathway for biosynthesis of the O-antigen component, 3-deoxy-L-glycero-tetronic acid, based on the sequence of the Vibrio cholerae O1 rfb region; Morona R et al.; The nucleotide sequence of a region of the rfb genes, encoding biosynthesis of the Vibrio cholerae (Vc) O1 O-antigen, was determined . Analysis of the open reading frames (ORFs) within this region has revealed similarities with a number of different classes of biosynthetic proteins and enzymes . The ORFs have been designated RfbK, RfbL, RfbM, RfbN and RfbO . RfbK is a small, acidic protein which has similarity to the family of proteins known as acyl-carrier proteins (ACP) . The RfbL protein has similarity to a super-family of enzymes which adenylate their substrates as a part of their reaction mechanism . Included in these are several acetyl-CoA ligases . Alignment of RfbL with these proteins reveals a highly conserved domain containing the motif GlyXaaXaaGlyXaaPro . This resembles the ATP-binding site motif and may represent a variant of the usual motif, except that Pro replaces Gly . The VcRfbM protein has similarity with a family of long-chain, iron-containing alcohol dehydrogenases, of which the Escherichia coli K-12 fucO and adhE gene products are also members . The RfbN protein has sequence homology with LuxE and LuxC of Vibrio harveyi (Vh) and other bioluminescent bacterial species . The latter are two components of the enzyme complex which synthesizes the long-chain aldehyde used in the V . harveyi bioluminescence system . Finally, the VcRfbO protein has sequence similarity with acetyl-CoA transferases . We were able to identify a number of the gene products using a T7 expression system, confirming several of the allocated ORFs . A biosynthetic pathway for the Vc O-antigen component 3-deoxy-L-glycero-tetronic acid, based on the enzymatic functions predicted for the RfbK, RfbL, RfbM, RfbN and RfbO proteins, is presented.

Biochem J, 1995 Dec 1, 312 ( Pt 2), 439 - 44
The lux autoinducer-receptor interaction in Vibrio harveyi: binding parameters and structural requirements for the autoinducer; Cao JG et al.; To assess the binding parameters and the structure-function relationship of the Vibrio harveyi lux autoinducer, N-(D-3-hydroxybutanoyl)homoserine lactone (D-HBHL), to light emission, a series of acylhomoserine lactone analogues were synthesized and their effects on the stimulation of luminescence of an autoinducer-deficient mutant of V . harveyi, D1, examined . Of the analogues with 3-hydroxyacyl chains, only N-(3-hydroxyvaleryl)homoserine lactone (HVHL) could act as an inducer, with about 85% of the potency of D-HBHL in stimulation of luminescence; the apparent Kd of the putative receptor for HVHL was 3.8 microM, close to that for the natural autoinducer (1.4 microM) . Analogues with longer 3-hydroxyacyl chains, N-(3-hydroxyhexanoyl)homoserine lactone and N-(3-hydroxyheptanoyl)homoserine lactone, acted as competitive inhibitors of HBHL with apparent KI values of 77 and 53 microM respectively . Replacement of the 3-hydroxybutanoyl moiety with a 3-methylbutanoyl or 3-methoxybutanoyl group created weak competitive inhibitors, N-(isovaleryl)- and N-(3-methoxybutanoyl)- homoserine lactones, with apparent KI values of 150 and 360 microM respectively . Two other analogues, N-(2-hydroxybutanoyl)- and N-(4-hydroxybutanoyl)-homoserine lactone, could neither stimulate nor inhibit luminescence . The approach used in these studies to demonstrate binding of autoinducer analogues at the same site, as well as measurement of the relative dissociation constant, may be of value in analysing analogues activating or inhibiting luminescence and other processes that are under control of acylhomoserine lactone autoregulators.

J Bacteriol, 1995 Dec, 177(24), 7155 - 63
Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under the control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family; Brint JM et al.; Mutants of Pseudomonas aeruginosa PAO1 that were deficient in the ability to produce proteases that degrade casein were detected among the survivors of chemical mutagenesis . One such mutant (PDO31) showed reduced production of elastolytic activity, beta-hemolytic activity, and pyocyanin . A 4.3-kb EcoRI fragment from a gene bank of PAO1 that complemented defects in PDO31 was found . Transposon mutagenesis and deletion derivatives of the clone were used in conjunction with complementation tests to determine the physical location of the gene of interest . Nucleotide sequence analysis revealed an open reading frame (rhlR) encoding a putative 27.6-kDa protein (RhlR) with homology to autoinducer-responsive regulators of quorum sensing systems such as LuxR of Vibrio fischeri and LasR of P . aeruginosa . Further sequence analysis downstream of rhlR revealed an independently transcribed gene (rhlI) that encodes a putative 22.2-kDa protein with homology to members of the family of autoinducer synthetases, such as LuxI of V . fischeri and LasI of P . aeruginosa . The rhlRI sequences were also recently reported by others (U.A . Ochsner and J . Reiser, Proc . Natl . Acad . Sci . USA 92: 6424-6428, 1995) as an autoinducer-mediated regulation mechanism for rhamnolipid biosurfactant synthesis in P . aeruginosa PG201 . Mutants with defects in rhlR or rhlI were constructed in PAO1 by gene replacement, using clones modified by Tn501 insertion . Compared with the wild type, the rhlR and rhlI mutants both showed defects in the production of elastase, LasA protease, rhamnolipid, and pyocyanin . Transcription from the gene for elastase, as measured with a lasB-cat fusion, demonstrated that production of elastase was subject to cell density-dependent gene activation in PAO1 . However, transcription of lasB-cat in the rhlI mutant, which had lost the presumptive autoinducer synthetase (predicted to activate RhlR), showed low basal activity and had lost all cell density-dependent transcription of lasB . Thus, RhlR-RhlI represent the second autoinducer-responsive regulatory mechanism found in P . aeruginosa that controls expression of multiple virulence factor exoproducts, including elastase.

J Bacteriol, 1995 Dec, 177(23), 6978 - 82
Isolation of a carbon starvation regulatory mutant in a marine Vibrio strain; Ostling J et al.; A carbon starvation-responding lac fusion of the marine Vibrio sp . strain S14 was used as a reporter strain in order to identify genes critical in the regulation of the carbon starvation response . Interestingly, sequence data together with an altered phenotype with respect to the accumulation of guanosine 3',5'-bispyrophosphate (ppGpp) imply that one of the genes (csrS) identified by this approach is an Escherichia coli spoT equivalent . Complementary data suggest that the function encoded by the csrS gene is essential for the successful development of starvation and stress resistance.

J Bacteriol, 1995 Dec, 177(23), 6946 - 51
AinS and a new family of autoinducer synthesis proteins; Gilson L et al.; In Vibrio fischeri, the autoinducer N-3-oxohexanoyl-L-homoserine lactone (AI-1) governs the cell density-dependent induction of the luminescence operon via the LuxR transcriptional activator . The synthesis of AI-1 from bacterial metabolic intermediates is dependent on luxI . Recently, we found a second V . fischeri autoinducer molecule, N-octanoyl-L-homoserine lactone (AI-2), that in E . coli also activates the luminescence operon via LuxR . A locus independent of luxI was identified as being required for AI-2 synthesis . This 2.7-kb ain (autoinducer) locus was characterized by transposon insertion mutagenesis, deletion and complementation analysis, and DNA sequencing . A single 1,185-bp gene, ainS, was found to be the sole exogenous gene necessary for the synthesis of AI-2 in Escherichia coli . In addition, a V . fischeri ainS mutant produced AI-1 but not AI-2, confirming that in its native species ainS is specific for the synthesis of AI-2 . ainS is predicted to encode a 45,580-Da protein which exhibits no similarity to LuxI or to any of the LuxI homologs responsible for the synthesis of N-acyl-L-homoserine lactones in a variety of other bacteria . The existence of two different and unrelated autoinducer synthesis genes suggests the occurrence of convergent evolution in the synthesis of homoserine lactone signaling molecules . The C-terminal half of AinS shows homology to a putative protein in Vibrio harveyi, LuxM, which is required for the synthesis of a V . harveyi bioluminescence autoinducer . Together, AinS and LuxM define a new family of autoinducer synthesis proteins . Furthermore, the predicted product of another gene, ainR, encoded immediately downstream of ainS, shows homology to LuxN, which is similarly encoded downstream of luxM in V . harveyi and proposed to have sensor/regulator functions in the bioluminescence response to the V . harveyi auto inducer . This similarity presents the possibility that AI-2, besides interacting with LuxR, also interacts with AinR under presently unknown conditions.

Infect Immun, 1995 Dec, 63(12), 4959 - 63
Adherence to and invasion of tissue culture cells by Vibrio hollisae; Miliotis MD et al.; The adherence to and invasion of cultured epithelial cells by Vibrio hollisae were examined by quantitative studies and by light, fluorescent, and electron microscopy . Condensed actin was observed around clustered adherent and intracellular bacteria . Bacteria multiplied intracellularly . Inhibitor studies indicated that internalization occurred by an integrated pleiotropic process involving eukaryotic and prokaryotic protein syntheses, microfilaments, microtubules, and receptor-mediated endocytosis.

J Biotechnol, 1995 Nov 21, 43(1), 45 - 51
Studies on plasmid stability and LTB production by recombinant Vibrio cholerae in batch and chemostat cultures: a lesson for optimizing conditions for chemical induction; Ramesh A et al.; Plasmid content, its stability and the expression of B-subunit of Escherichia coli heat-labile enterotoxin (LTB) in Vibrio cholerae/r-pMMB68 system have been studied in batch as well as in chemostat cultures . Upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), cultures secreted LTB into the extracellular milieu . Highest specific LTB production rate of 7.3 mg mg-1h-1 was achieved in batch culture induced at the late exponential growth phase . The plasmid pMMB68 was fairly stable up to 20 generations, even in the absence of selection pressure . Instability of the plasmid was accelerated in the presence of IPTG and at higher dilution rates . Maximum productivity of 2.1 mg l-1 h-1 was achieved in continuous culture, which remained constant at a range of dilution rates from 0.20 to 0.35 h-1.

Biochemistry, 1995 Nov 21, 34(46), 15084 - 90
Tryptophan 250 on the alpha subunit plays an important role in flavin and aldehyde binding to bacterial luciferase . Effects of W-->Y mutations on catalytic function; Li Z et al.; Bacterial luciferase is a heterodimer (alpha beta) that catalyzes the oxidation of FMNH2 and a fatty aldehyde, resulting in light emission . To explore the nature of the flavin binding site with respect to the role of tryptophan residues, the catalytic and binding properties of single-point mutants of Xenorhabdus luminescens luciferase with one of the eight tryptophans converted to a tyrosine residue were investigated by luminescence and fluorescence measurements . Conversion of tryptophans 194 and 250 on the alpha subunit to tyrosine had relatively large effects on the properties of luciferase with only minor changes in the properties on mutation of the other four tryptophans on alpha and the two on the beta subunit . Mutation of alpha W250 decreased the binding to FMNH2, FMN, aldehyde, and fatty acid, causing major changes in luminescence emission and decay . The results are consistent with alpha W250 interacting with flavin which in turn affects aldehyde binding . Mutation of alpha W194 did not affect the interaction with flavin or aldehyde but did change the relative rate of decay of light emission with aldehydes of different chain lengths as well as the activation energy for this process . Moreover, these results provide evidence for alpha W250, and to a lesser extent alpha W194, being in contact with the isoalloxazine ring of flavin, a proposal that has been recently made based on a model with flavin bound to the alpha subunit and anchored at a binding site for the phosphate moiety of FMN(H2) identified in the crystal structure of Vibrio harveyi luciferase {Fisher, A . J . Raushel, F . M., Baldwin, T . O., & Rayment, I . (1995) Biochemistry 34, 6581-6586}.

J Immunol Methods, 1995 Nov 16, 187(1), 121 - 5
Quartz crystal microbalance detection of Vibrio cholerae O139 serotype; Carter RM et al.; A piezoelectric (PZ) quartz crystal microbalance (QCM) biosensor for the rapid detection of Vibrio cholerae serotype O139 has been developed . The antibody to this serotype was immobilized on the gold transducer surface of a 10 MHz AT cut PZ crystal . Solutions containing known antigen concentrations were then incubated for 1 h on the antibody-bound transducer . The biosensor was able to detect 10(5) cells per ml of O139 versus a background of O1 (Ogawa) serotype.

Structure, 1995 Nov 15, 3(11), 1197 - 205
The three domains of a bacterial sialidase: a beta-propeller, an immunoglobulin module and a galactose-binding jelly-roll; Gaskell A et al.; BACKGROUND: Sialidases, or neuraminidases, have been implicated in the pathogenesis of many diseases, but are also produced by many non-pathogenic bacteria . Bacterial sialidases are very variable in size, often possessing domains in addition to the catalytic domain . The sialidase from the non-pathogenic soil bacterium Micromonospora viridifaciens is secreted in two forms with molecular weights of 41 kDa or 68 kDa, depending on the nature of the carbohydrate used to induce expression . RESULTS: We report here the X-ray crystal structures of the 41 kDa and 68 kDa forms of the sialidase from M . viridifaciens at 1.8 A and 2.5 A resolution respectively . In addition, we report a complex of the 41 kDa form with an inhibitor at 2.0 A resolution, and a complex of the 68 kDa form with galactose at 2.5 A . The 41 kDa form shows the canonical sialidase beta-propeller fold . The 68 kDa form possesses two additional domains, one with an immunoglobulin-like fold that serves as a linker to the second, which is homologous to the galactose-binding domain of a fungal galactose oxidase . CONCLUSIONS: The presence of the additional carbohydrate-binding domain in the 68 kDa form of the bacterial sialidase reported here is a further example of a combination of carbohydrate binding and cleaving domains which we observed in the sialidase from Vibrio cholerae . This dual function may be common, but only to other bacterial and parasitic sialidases, but also to other secreted glycosidases involved in pathogenesis . The bacterium may have acquired both the immunoglobulin module and the galactose-binding module from eukaryotes, as the enzyme shows a remarkable similarity to a fungal galactose oxidase which possesses similar domains performing different functions and assembled in a different order.

FEMS Microbiol Lett, 1995 Nov 15, 133(3), 203 - 8
The viable but non-culturable state in the human pathogen Vibrio vulnificus; Oliver JD; Vibrio vulnificus is a serious human pathogen, accounting for 95% of all seafood-related deaths in the United States . During the winter months, when coastal water temperatures drop below 10 degrees C, investigators have repeatedly reported their inability to isolate this estuarine bacterium from the environment . We now realize that this apparent 'die-off' is actually due to entry of the cells into a 'viable but non-culturable' state, a survival response to the low temperature stress . Cells in this state appear dormant, and cannot be cultured in or on routine bacteriological media, but are capable of returning to the actively metabolizing state when the environmental stress is removed . This review describes this non-culturable state in V . vulnificus, and its role in the ecology, physiology, and epidemiology of this pathogen.

FEBS Lett, 1995 Nov 13, 375(1-2), 5 - 10
Predicted structure and possible ionmotive mechanism of the sodium-linked NADH-ubiquinone oxidoreductase of Vibrio alginolyticus; Rich PR et al.; Two groups have now published sequences of the six genes contained in the operon coding for the sodium-linked NADH-ubiquinone oxidoreductase of Vibrio alginolyticus . Sequence analyses indicate that this enzyme is unrelated to other known respiratory NADH dehydrogenases . A search for cofactor motifs suggests that the enzyme contains only one FAD, a ferredoxin-type iron sulphur centre, and the NADH-binding site . These are all located on NqrF, a subunit that can be recognized as a new member of a large diverse family of NAD(P)H-oxidizing flavoenzymes . A possible model of ion-coupling is presented, based upon this new information.

Mol Microbiol, 1995 Nov, 18(4), 685 - 701
Characterization of Vibrio cholerae bacteriophage K139 and use of a novel mini-transposon to identify a phage-encoded virulence factor; Reidl J et al.; Temperate bacteriophage K139 was isolated from a Vibrio cholerae O139 isolate and characterized in this study . The phage genome consists of a 35 kbp, double-stranded, linear DNA molecule that circularizes and integrates into the chromosome in a site-specific manner . DNA sequences that cross-hybridize with K139 phage DNA are present in all strains of V . cholerae serogroup O1 of the classical biotype examined and in some strains of the El Tor biotype . Phage K139 produces plaques on El Tor O1 strains that do not carry the K139-related sequences but does not plaque on O139 strains that lack detectable phage DNA . This results suggests that O139 strains arose in part by horizontal gene transfer of the O139 antigen genes into an El Tor O1 strain that harboured a K139 prophage . Consistent with this interpretation, the morphology of K139 phage particles is identical to that displayed by the widely distributed family of O1 phages referred to as 'kappa' . In order to test whether K139 phage is involved in lysogenic conversion of V . cholerae, we constructed a novel mini-transposon, Tn10d-bla, which was designed to produce beta-lactamase fusions to phage-encoded, exported proteins . All Tn10d-bla insertions obtained were closely linked to one location on the K139 phage genome . DNA sequence determination of the fusion joints revealed an open reading frame (ORF1), encoding a gene product of 137 amino acids with a typical N-terminal hydrophobic signal sequence . ORF1 was designated the glo gene (G protein-like ORF) because its amino acid sequence shows similarity to eukaryotic Gs(alpha) protein (34.5% identity over an 81-amino-acid overlap) and its C-terminus displays the consensus motif (CAAX) which is found in many small eukaryotic GTP-binding proteins . LD50 assays with isogenic Glo+ and Glo- K139 lysogens suggest that glo encodes a secreted virulence determinant of V . cholerae.

Mol Microbiol, 1995 Nov, 18(4), 671 - 83
Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection; Camilli A et al.; A complete understanding of host-parasite interactions must necessarily include the identification and characterization of gene products expressed by both parties during the infectious process . We have developed a new screen to identify bacterial genes that are transcriptionally induced during infection of a host animal . The method is based on pre-selection of strains carrying tnpR operon fusions (encoding resolvase, a site-specific DNA recombinase) which are not expressed in vitro, followed by screening for a subset of these strains that subsequently express resolvase within the host environment . The latter subset was recognized as recombinants that had deleted a resolvase-specific reporter construct . Thirteen transcription units of Vibrio cholerae were identified that were induced during infection in an infant mouse model of cholera . Five of these were predicted to encode polypeptides with diverse functions in metabolism, biosynthesis and motility; one encoded a secreted lipase; two appear to be antisense to genes involved in motility; and five are predicted to encode polypeptides of unknown function . Three of the transcripts were shown to be required for full virulence in infant mice, as assessed by competition experiments.

Arkh Patol, 1995 Nov-Dec, 57(6), 58 - 63
{Ultrastructural changes in atria dna small intestines of suckling rabbits in experimental cholera}; Lomov IuM et al.; 10-12-day old suckling rabbits received intragastrically culture of V . eltor 5879 and the resultant ultrastructural changes in the small intestine, atrial cardiomyocytes and capillaries were studied . In the period of cholera vibrio adhesion (4 hours after the culture administration) epithelial cells underwent hydropic degeneration . Degenerative changes due to cell vacuolization and contractile alterations were noted in cardiomyocytes of both atria . The atrial Na-uretic factor secretion prevailed over its synthesis in line with increased vascular permeability . The development of experimental cholera (on day 1-2) was characterized by balloon degeneration in the small intestinal epithelium . Numerous secretory granules were formed in the atrial cardiomyocytes without releasing peptides into the circulation . These changes are of compensatory nature and serve for liquid retention in the body . Microcirculatory disturbances take place at three levels: intravascular, endothelial and perivascular.

Clin Infect Dis, 1995 Nov, 21(5), 1330 - 3
Primary septicemia caused by Vibrio cholerae non-O1 acquired on Cape Cod, Massachusetts; Kontoyiannis DP et al.; We describe a patient with non-O1, non-O139 Vibrio cholerae septicemia associated with hemorrhagic bullous skin lesions of the lower extremities . The patient had underlying liver disease, and he probably acquired the organism through ingestion of raw clams . Although his condition rapidly improved during appropriate therapy, the patient's cellulitis and skin lesions persisted and he developed a fluid collection of the lower extremity that required drainage . Molecular methods were used to examine the non-O1 V . cholerae isolate for several known virulence factors of V . cholerae O1 . The isolate failed to express cholera toxin and toxin-coregulated pilus (Tcp) and was negative in Southern hybridizations for ctxB, tcpA, toxR, and toxT . The vast majority of vibrio infections in the United States are clustered in the Gulf Coast area . This patient acquired the infection on Cape Cod . To our knowledge, this is the first case of non-O1 V . cholerae septicemia reported to have occurred in Massachusetts . Given the high fatality rate of this infection, it is important for physicians to consider this diagnosis in patients who have underlying risk factors and appropriate epidemiologic exposures, even when they reside as far north as the New England states.

Biophys J, 1995 Nov, 69(5), 2154 - 62
Simultaneous measurement of bacterial flagellar rotation rate and swimming speed; Magariyama Y et al.; Swimming speeds and flagellar rotation rates of individual free-swimming Vibrio alginolyticus cells were measured simultaneously by laser dark-field microscopy at 25, 30, and 35 degrees C . A roughly linear relation between swimming speed and flagellar rotation rate was observed . The ratio of swimming speed to flagellar rotation rate was 0.113 microns, which indicated that a cell progressed by 7% of pitch of flagellar helix during one flagellar rotation . At each temperature, however, swimming speed had a tendency to saturate at high flagellar rotation rate . That is, the cell with a faster-rotating flagellum did not always swim faster . To analyze the bacterial motion, we proposed a model in which the torque characteristics of the flagellar motor were considered . The model could be analytically solved, and it qualitatively explained the experimental results . The discrepancy between the experimental and the calculated ratios of swimming speed to flagellar rotation rate was about 20% . The apparent saturation in swimming speed was considered to be caused by shorter flagella that rotated faster but produced less propelling force.

J Clin Microbiol, 1995 Nov, 33(11), 2935 - 9
Development and testing of monoclonal antibody-based rapid immunodiagnostic test kits for direct detection of Vibrio cholerae O139 synonym Bengal; Hasan JA et al.; We report on the development and testing of two monoclonal antibody-based rapid immunodiagnostic test kits, BengalScreen, a coagglutination test, and Bengal DFA, a direct fluorescent-antibody test, for direct detection of Vibrio cholerae O139 synonym Bengal in clinical and environmental specimens . The BengalScreen test requires less than 5 min to complete and can be used in the field . Bengal DFA, being more sensitive than BengalScreen, requires only one reagent and less than 20 min for detection and enumeration of V . cholerae O139 synonym Bengal . In tests for specificity, all 40 strains of V . cholerae O139 reacted with both test kits, whereas 157 strains of heterologous species examined did not, yielding 100% specificity in this study . A field trial was conducted in with both BengalScreen and Bengal DFA, and the results were compared with those obtained by conventional culture methods . BengalScreen demonstrated a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94% . Results obtained by Bengal DFA, on the other hand, were 100% sensitive and 100% specific and yielded 100% positive and negative predictive values compared with culture methods . In a second evaluation, 93 stool specimens from Mexico that were negative for V . cholerae O139 by culture were also tested with both the BengalScreen and Bengal DFA kits . None of the 93 specimens were positive for V . cholerae O139 by both tests . A concentration method was optimized for screening of environmental water samples for V . cholerae O139 synonym Bengal with rapid test kits . BengalScreen results were unequivocally positive when water samples contained at least 2.0 x 10(3) CFU/ml, whereas Bengal DFA demonstrated an unequivocally positive reaction when the water sample contained at least 1.5 x 10(2) CFU/ml . When Bengal DFA was compared with conventional culture methods for enumeration of V . cholerae O139 synonym Bengal organisms, no difference was observed.

J Clin Microbiol, 1995 Nov, 33(11), 2833 - 8
Molecular epidemiology of toxigenic Vibrio cholerae in Bangladesh studied by numerical analysis of rRNA gene restriction patterns; Faruque SM et al.; Cholera is endemic in Bangladesh, and a regular seasonal pattern of cholera epidemics occurs . We examined the clonal relationships among 103 clinical and environmental Vibrio cholerae isolates belonging to O1, O139, or non-O1 non-O139 serogroups isolated during epidemic and interepidemic periods in Bangladesh and compared them with those of 51 V . cholerae isolates from four countries in Asia and Africa . These studies were done by a computer-assisted numerical analysis of the restriction endonuclease cleavage patterns of rRNA genes (ribotypes) . Unweighed pair-group cluster analysis of BglI- and HindIII-generated band patterns revealed 16 clusters . Ribotypes were defined as clusters of strains possessing > 98% similarity . The results showed that 154 isolates could be differentiated into 15 different ribotypes, and strains belonging to 3 of these ribotypes (ribotypes I, V, and VIIIA and VIIIB) were isolated more frequently during the epidemic periods than during interepidemic periods in Bangladesh . Classical vibrios belonged to six different ribotypes (ribotypes I to VI), with a mean similarity coefficient of 0.84, and the El Tor vibrios belonged to five different ribotypes (ribotypes VIIIA and IX to XII), with a mean similarity coefficient of 0.82 . A single clone of El Tor vibrios (ribotype XII) was resident in Tanzania, whereas Nigeria, Syria, and India shared toxigenic El Tor strains with Bangladesh . Cholera toxin (CT)-positive O139 vibrios isolated from Bangladesh and India belonged to a single ribotype (ribotype VIIIB) and were > 98% similar to one of the ribotypes of El Tor vibrios (ribotype VIIIA), but a CT-negative O139 vibrio from Argentina (ribotype XIII) was < 75% similar to the same cluster of El Tor vibrios, thus suggesting more than one possible origin for O139 vibrios . Strains belonging to the same ribotypes (ribotypes VIII to X) were isolated from both patients and surface water in Bangladesh, indicating possible transmission through surface water . A clone of a CT-positive environmental isolate of non-O1 V . cholerae (ribotype VII) was found to be closely related (76.3% similarity) to a clone of classical vibrios (ribotype I) and was only between 27.2 and 56.1% similar to clusters of El Tor, O139, and two other non-O1 nontoxigenic clones.

Ecotoxicol Environ Saf, 1995 Nov, 32(2), 201 - 4
Use of the bioluminescent bacterium Vibrio harveyi to detect biohazardous chemicals in soil and water extractions with and without acid; Thomulka KW et al.; An investigation was undertaken using the bioluminescence-reduction bioassay of Vibrio harveyi to study the toxicity of 31 chemicals in a soil and water extraction that was treated with and without hydrochloric acid . Soil had the chemical added and was washed with 0.2 N HC1 and afterward this acid-treated soil containing the chemical was evaluated . Each chemical was tested independently . Endpoints determined were either an estimated effective concentration at 50 or 20%, which was based on bacterial-luminescence remaining after treatment . This investigation suggests that metals are more effectively bound in treatments without acid than those with acid, while no difference in toxicity was observed for organics . In comparison to a previous study of toxicity using these chemicals in water, soil was effective in reducing toxicity as determined by this assay . However, each chemical appears to have its own unique toxicity characteristic when evaluated under different conditions.

Clin Diagn Lab Immunol, 1995 Nov, 2(6), 685 - 8
Comparison of the vibriocidal antibody response in cholera due to Vibrio cholerae O139 Bengal with the response in cholera due to Vibrio cholerae O1; Qadri F et al.; Vibrio cholerae serogroup O139, now considered to be the second organism capable of causing epidemic severe dehydrating cholera, contains a capsular polysaccharide which makes it difficult for it to be used in the conventional vibriocidal antibody assay optimized for V . cholerae O1 . After modification of the procedure, which involved the use of specific bacterial strains, a lower bacterial inoculum, and increased amounts of complement, the vibriocidal antibody responses to V . cholerae O139 were measured in acute- and convalescent-phase sera from 33 V . cholerae O139-infected and 18 V . cholerae O1-infected patients and in single serum samples from 20 healthy control subjects . The responses in these individuals to V . cholerae O1 strains were also determined . Significant elevations in the homologous antibody response were found only in the convalescent-phase sera from both groups of patients with cholera . These findings may explain the basis for the lack of heterologous protection between the two serogroups of V . cholerae . Healthy controls had higher background levels of vibriocidal antibody to V . cholerae O1 than to V . cholerae O139.

FEMS Microbiol Lett, 1995 Nov 1, 133(1-2), 151 - 4
Aberrant gene for E1 Tor hemolysin from Vibrio cholerae non-O1, N037; Honma Y et al.; Vibrio cholerae non-O1 strain N037 produced a hemolysin (NO37-Hly) which was antigenically similar to E1 Tor hemolysin (E1 Tor-Hly) but different in molecular size, hemolytic activity, and glucose binding capacity . In the gene encoding NO37-Hly, a 4-bp insertion into the structural gene for E1 Tor-Hly (hlyA) was found . The insertion in a shift of codon frames generating a new stop codon in the downstream region . NO37-Hly was a truncated product of E1 Tor-Hly sharing 90% of the N terminal region . This suggested that the 10% C-terminal region of E1 Tor-Hly is needed for the maximal hemolytic activity and glucose binding capacity.

Biosci Biotechnol Biochem, 1995 Nov, 59(11), 2068 - 73
Molecular cloning and sequence analysis of the gene encoding the collagenase from Cytophaga sp . L43-1 strain; Sasagawa Y et al.; Cytophaga sp . strain L43-1 secretes a collagenase {Y . Sasagawa et al., Biosci . Biotech . Biochem., 57, 1894-1898 (1993)} . A cog gene encoding the collagenase from this strain was cloned, and the nucleotides were sequenced . The structural gene of cog consisted of 3846 base pairs, which encoded a polypeptide consisting of 1282 amino acid residues with a predicted molecular mass of 130 kDa which was synthesized as a pre-matured enzyme . The deduced N-terminal 14 amino acids sequence, molecular mass of 120 kDa, and pI of 4.96 of the predicted matured enzyme were consistent with those previously found for the collagenase purified from the strain . The cog gene was expressed in Escherichia coli using the lac promoter and ribosomal binding sequence in plasmid vector pUC119 or pKK223-3, but not its own putative promoter and Shine-Dalgarno sequence . The consensus amino acid sequence (His-Glu-Xaa-Xaa-His) of an active site of the metal proteases including the collagenase from Vibrio arginolyticus and a series of human MMPs was found in the Cog protein of the strain.

J Infect Dis, 1995 Nov, 172(5), 1405 - 8
Urease production correlates with possession of the trh gene in Vibrio parahaemolyticus strains isolated in Thailand; Suthienkul O et al.; A total of 489 Vibrio parahaemolyticus isolates from patients in Thailand with diarrhea was examined for the presence of thermostable direct hemolysin (TDH) and TDH-related hemolysin genes (tdh and trh, respectively), their serovars, TDH production, and urease activity . Of the strains, 81% were positive only for the tdh gene, 6% for both trh and tdh genes, and 2% for the trh gene only . Thirty-seven (8%) of the 489 isolates were positive for urease production . Of special interest, all urease-positive strains possessed the trh gene, and conversely, urease-negative strains lacked the gene, indicating that urease production by V . parahaemolyticus strains strongly correlates with the possession of the trh gene . Thus, the urease-positive phenotype of V . parahaemolyticus can be considered an indication of virulent (trh-possessing) V . parahaemolyticus strains in clinical diagnosis.

J Infect Dis, 1995 Nov, 172(5), 1401 - 4
Vibrio cholerae O139 Bengal infections among tourists to Southeast Asia: an intercontinental foodborne outbreak; Boyce TG et al.; To determine the source and extent of an outbreak of Vibrio cholerae O139 Bengal infections among 630 cruise ship passengers to Southeast Asia, a retrospective cohort study was done . Questionnaires were sent to all passengers from the United States, Canada, and the United Kingdom, and serum samples were requested from all passengers reporting diarrhea . A case was defined as diarrheal illness with onset between 8 and 28 February 1994 and a cholera antitoxic antibody titer > or = 800 . Six passengers, including 1 with bacteremia, met the case definition . Illness was associated with eating yellow rice at a buffet restaurant in Bangkok on 10 February (relative risk undefined, P = .005) . This international outbreak demonstrates foodborne transmission of Vibrio cholerae O139 Bengal, an emerging cause of epidemic cholera in Asia, to tourists from Western countries . Physicians should suspect infection with either V . cholerae O1 or O139 in any patient with severe watery diarrhea after travel to the developing world.

J Cell Biol, 1995 Nov, 131(4), 951 - 62
Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL; Lencer WI et al.; Vibrio cholerae and Escherichia coli heat labile toxins (CT and LT) elicit a secretory response from intestinal epithelia by binding apical receptors (ganglioside GM1) and subsequently activating basolateral effectors (adenylate cyclase) . We have recently proposed that signal transduction in polarized cells may require transcytosis of toxin-containing membranes (Lencer, W . I., G . Strohmeier, S . Moe, S . L . Carlson, C . T . Constable, and J . L . Madara . 1995 . Proc . Natl . Acad . Sci . USA . 92:10094-10098) . Targeting of CT into this pathway depends initially on binding of toxin B subunits to GM1 at the cell surface . The anatomical compartments in which subsequent steps of CT processing occur are less clearly defined . However, the enzymatically active A subunit of CT contains the ER retention signal KDEL (RDEL in LT) . Thus if the KDEL motif were required for normal CT trafficking, movement of CT from the Golgi to ER would be implied . To test this idea, recombinant wild-type (wt) and mutant CT and LT were prepared . The COOH-terminal KDEL sequence in CT was replaced by seven unrelated amino acids: LEDERAS . In LT, a single point mutation replacing leucine with valine in RDEL was made . Wt and mutant toxins displayed similar enzymatic activities and binding affinities to GM1 immobilized on plastic . Biologic activity of recombinant toxins was assessed as a Cl- secretory response elicited from the polarized human epithelial cell line T84 using standard electrophysiologic techniques . Mutations in K(R)DEL of both CT and LT delayed the time course of toxin-induced Cl- secretion . At T1/2, dose dependencies for K(R)DEL-mutant toxins were increased > or = 10-fold . KDEL-mutants displayed differentially greater temperature sensitivity . In direct concordance with a slower rate of signal transduction . KDEL-mutants were trafficked to the basolateral membrane more slowly than wt CT (assessed by selective cell surface biotinylation as transcytosis of B subunit) . Mutation in K(R)DEL had no effect on the rate of toxin endocytosis . These data provide evidence that CT and LT interact directly with endogenous KDEL-receptors and imply that both toxins may require retrograde movement through Golgi cisternae and ER for efficient and maximal biologic activity.

Proc Natl Acad Sci U S A, 1995 Oct 24, 92(22), 10374 - 8
Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139; Stroeher UH et al.; The recent emergence of a pathogenic new non-O1 serotype (O139) of Vibrio cholerae has led to numerous studies in an attempt to identify the origins of this new strain . Our studies indicate that O139 strains have clear differences in the surface polysaccharides when compared with O1 strains: the lipopolysaccharide can be described as semi-rough . Southern hybridization with the O1 rfb region demonstrates that O139 strains no longer contain any of the rfb genes required for the synthesis of the O1 O-antigen or its modification and also lack at least 6 kb of additional contiguous DNA . However, O139 strains have retained rfaD and have a single open reading frame closely related to three small open reading frames of the O1 rfb region . This region is closely related to the H-repeat of Escherichia coli and to the transposases of a number of insertion sequence elements and has all the features of an insertion sequence element that has been designated VcIS1 . Transposon insertion mutants defective in O139 O-antigen (and capsule) biosynthesis map to the same fragment as VcIS1 . Preliminary sequence data of complementing clones indicate that this DNA encodes a galactosyl-transferase and other enzymes for the utilization of galactose in polysaccharide biosynthesis . We propose a mechanism by which both the Ogawa serotype of O1 strains and the O139 serotype strains may have evolved.

Rev Argent Microbiol, 1995 Oct-Dec, 27(4), 185 - 90
{Determination of bactericidal antibodies directed against vibrio cholerae in an adult population in Montevideo}; Varela G et al.; Uruguay is the only Latinoamerican country that remains free of cholera . Thus, to obtain a baseline for future diagnosis vibriocidal antibodies were investigated in 100 sera from blood donors serogroup "O" . The reaction was carried out in microplates with live antigen Ogawa VC 12 . In 95% of sera, titers were below 160, suggesting a high degree of susceptibility to V . cholerae in the population.

Mol Gen Mikrobiol Virusol, 1995 Oct-Dec, (4), 23 - 6
{Genetic analysis of Vibrio cholerae chromosomal regions containing the tox-2 mutation, affecting production of cholera toxin}; Smirnova NI et al.; Conjugational-mating experiments showed mrh genes coding for the synthesis of mannose and fucose resistant hemagglutinin/adhesin and vibrio motility gene mot to be localized in the chromosome of V . cholerae Dacca strain 35 close to tox-2 mutation providing a high level of toxin production and rfb locus which controls 01 antigen synthesis . The results indicate the presence of a block of virulence genes within ura-pur chromosomal region of V . cholerae.

J Wildl Dis, 1995 Oct, 31(4), 562 - 5
Fusarium solani fungal infection of the lateral line canal system in captive scalloped hammerhead sharks (Sphyrna lewini) in Hawaii; Crow GL et al.; Two of five scalloped hammerhead sharks (Sphyrna lewini) captured May 1987 in Hawaii (USA) developed granulomatous exudative mycotic dermatitis localized in the lateral line canal system . The lesion initially was noted in the cephalic canals, but over a period of months extended into the lateral canal . Fusarium solani and Vibrio spp . were isolated from the canal exudate of both sharks . Bacterial colonies were not observed in the canal walls or surrounding tissues . Fusarium solani infection resulted in a chronic physical and behavioral deterioration of the two sharks; one shark was euthanized in September 1988 and the other in July 1989 . This is the first report of Fusarium solani infection in the lateral line canal system and the third account in hammerhead sharks.

Can J Microbiol, 1995 Oct, 41(10), 946 - 50
Serogroup conversion of Vibrio cholerae; Colwell RR et al.; Vibrio cholerae serogroup O1 can be detected in the environment in a viable but nonculturable form, whereas V . cholerae non-O1 cells can be readily cultured during interepidemic periods in geographical regions where cholera is endemic . In the present study, pure cultures of V . cholerae non-O1 cells contained O1 cells when examined by immune-fluorescence microscopy . Laboratory microcosms were used to examine the outgrowth of the O1 cells in cultures of non-O1 V . cholerae . One O1 cell per 10(6) non-O1 cells could be detected by direct fluorescent-monoclonal antibody staining but only after incubation of the non-O1 culture for 48 h . Individual O1 cells were not detected in cultures incubated less than 48 h . Hybridization study, using a polymerase chain reaction (PCR) amplified fragment of the O-antigen of V . cholerae O1 as a probe, revealed the existence of a homologous gene in a microcosm sample of V . cholerae non-O1 containing serogroup-converted cells . The mechanism by which O1 cells can occur in cultures of non-O1 V . cholerae most likely resulted from spontaneous mutation of gene(s) encoding the O-somatic properties and (or) chemical, physical, or biological changes in the environment inducing expression or repression of the controlling gene(s) . These findings have important implications for the epidemiology of cholera and the environmental source(s) of toxin producing V . cholerae O1.

FEMS Immunol Med Microbiol, 1995 Oct, 12(2), 113 - 20
Attachment of non-culturable toxigenic Vibrio cholerae O1 and non-O1 and Aeromonas spp . to the aquatic arthropod Gerris spinolae and plants in the River Ganga, Varanasi; Shukla BN et al.; Non-cultivable, pathogenic O1 and non-O1 Vibrio cholerae and Aeromonas spp . were resuscitated from aquatic arthropods and plant homogenate respectively, by rabbit ileal loop (RIL) assay . These organisms adhered to the aquatic arthropod Gerris spinolae and various species of phytoplankton in the River Ganga, but failed to grow after direct inoculation on artificial media except for only 10 homogenates of the arthropod . The number of non-O1 V . cholerae and Aeromonas recovered on direct inoculation of G . spinolae homogenates were in the order of 10(5)-10(6) whereas those of the Ganga water were 10(2)-10(3) ml-1 . A total of 119 strains of O1 and non-O1 V . cholerae and Aeromonas spp . (69 isolates from G . spinolae and 50 from aquatic plants) were recovered from the loop contents . The results indicate that production of the enzyme chitinase by O1 and non-O1 V . cholerae and Aeromonas spp . might facilitate their adsorption and multiplication on different species of zoo- and phyto-plankton . Most of the isolates were enterotoxic, haemolytic and resistant to different antibiotics . This study suggests that species of zoo- and phyto-planktons, until now not reported to be associated with O1 and non-O1 V . cholerae, may act as reservoirs of these organisms as well as different species of Aeromonas in a fresh-water riverine ecosystem.

Res Microbiol, 1995 Oct, 146(8), 671 - 83
The distinction of pathogenic Vibrio cholerae groups using arbitrarily primed PCR fingerprints; Coelho A et al.; Pathogenic Vibrio cholerae strains were compared by fingerprinting with arbitrarily primed polymerase chain reaction (AP-PCR) . They were O1 classical and El Tor strains and recent non-O1 Bengal strains . Ten oligonucleotides from a total of fifty-two tested gave distinctive patterns, and these strains were separated into four groups . A second technique, amplification of 16S/23S rRNA spacers with a pair of oligonucleotides, was also used . Various bands were obtained, and the result can be treated as an additional fingerprint with a different pattern for each of the groups . The method of AP-PCR fingerprinting is fast and sensitive . A test of the stability of the El Tor patterns was done with a set of strains isolated during the present Brazilian epidemics . Examples of AP-PCRs with non-O1 strains are given . A typing scheme is proposed in which oligo 1 is first used, and depending on the fingerprint obtained, additional oligonucleotides are used to confirm the classification of the strain . It is proposed that the AP-PCR technique be used for epidemiological studies, analysing strains reaching new locations or environmental isolates suspected of being pathogenic . It will be particularly helpful in cases in which traditional methods cannot clearly classify the strain.

J Clin Microbiol, 1995 Oct, 33(10), 2715 - 22
Characterization of Vibrio cgolerae non-O1 serogroups obtained from an outbreak of diarrhea in Lima, Peru; Dalsgaard A et al.; In February 1994, an outbreak of diarrhea caused by non-O1 Vibrio cholerae occurred among volunteers in a vaccine trial study area in Lima, Peru . Clinically, 95% of the patients presented with liquid diarrhea with either no or mild dehydration . Serogrouping of 58 isolates recovered from diarrheal patients affected in the outbreak revealed seven different serogroups, with serogroups O10 (21%) and O12 (65%) being predominant . Most of these isolates were susceptible to a variety of antimicrobial agents . None of the 58 isolates hybridized with a DNA probe previously used to detect the gene encoding the heat-stable enterotoxin NAG-ST or produced cholera toxin as assessed by GM1 ganglioside enzyme-linked immunosorbent assay . Ribotyping exhibited 10 different BglI ribotype patterns among the 58 V . cholera non-O1 strains studied . However, ribotyping showed that all isolates belonging to serogroup O12 exhibited identical ribotypes and that 83% of the serogroup O10 isolates belonged to another identical ribotype, thus showing excellent correlation between ribotypes and serogroups . Among a group of O10 and O12 isolates selected for virulence studies, none produced enterotoxin whereas the majority produced a cytotoxin, as assessed in Y1 and HeLa cells . These isolates were also negative for the gene encoding zonula occludens toxin (Zot) as assessed by a PCR assay . The isolates tested showed strong adherence and some degree of invasion in the HEp-2 cell assay, whereas none of the isolates was positive in the PCR assay for the gene encoding the toxin coregulated pilus subunit A antigen (tcpA) . In the removable intestinal tie adult rabbit diarrhea model, O10 and O12 serogroup isolates produced severe diarrhea and occasionally death when rabbits were challenged with 10(10) bacterial cells . Fluid accumulation was shown in the rabbit intestinal loop test when whole cultures were injected . No significant difference in virulence was shown between serogroup O10 and O12 isolates . This study provides further evidence that V . chlorae non-O1 non-O139 strains have diarrhegenic potential for humans through a yet-undefined mechanism(s) and that such strains can cause outbreaks.

J Appl Bacteriol, 1995 Oct, 79(4), 384 - 92
Ribotyping and plasmid profiling of Vibrio anguillarum serovar O2 and Vibrio ordalii; Tiainen T et al.; One hundred and twenty-nine strains of Vibrio anguillarum serovar O2 and 14 strains of Vibrio ordalii were ribotyped and examined for plasmid contents . A total of 35 different ribotypes were detected . The V . anguillarum serovar O2 strains were divided into 32 different ribotypes . The V . ordalii strains showed three different ribotypes, clearly distinct from those of the V . anguillarum strains . Ribotypes were separated into seven clusters, of which one comprised the V . ordalii strains . Clustering of the strains indicated a genetic difference between North European and South European V . anguillarum O2 strains . Sero-subgroups O2a and O2b shared ribotypes; however, three of the clusters did not include O2a strains . All V . ordalii strains had a plasmid of 32 kb . This plasmid was not detected in any of the V . anguillarum strains . Seventeen different plasmid profiles with 17 different sized plasmids were detected among the V . anguillarum strains . Most of the plasmids were small (< 6 kb) and found in several strains . Except for one South European strain, plasmids were detected only in the North European strains of V . anguillarum O2.

Eur J Biochem, 1995 Oct 1, 233(1), 152 - 8
The structure of the lipid A-core region of the lipopolysaccharides from Vibrio cholerae O1 smooth strain 569B (Inaba) and rough mutant strain 95R (Ogawa); Vinogradov EV et al.; The lipopolysaccharides (LPS) from Vibrio cholerae 95R, a rough mutant strain of O1 V . cholerae 162 (Ogawa), and from smooth O1 V . cholerae 569B (Inaba) were de-O-acylated . In each case, one part of the products was treated with 48% aqueous HF which removed the phosphoryl and fructose residues, then reduced, de-N-acylated, and N-acetylated . Another part was de-N-acylated by treatment with hot KOH . The products of both degradation pathways were separated by high-performance anion-exchange chromatography . The major dephosphorylated and defructosylated product 1 was obtained in pure form, whereas the minor products 2 and 3 were eluted as a mixture, as were, from the second degradation, the phosphorylated oligosaccharides 4 (major product) and 5 (minor product) . No phosphorylated component corresponding to oligosaccharide 3 could be identified by NMR spectroscopy in the latter mixture . The following structures of oligosaccharides 1-5 were established on the basis of monosaccharide and methylation analyses, Smith degradation, and 1H- and 13C-NMR investigations (correlated, total correlated, NOE and heteronuclear correlation spectroscopy; all sugars are present as alpha-D-pyranoses except where indicated otherwise; Hep, L-glycero-D-manno-heptose; Kdo, 3-deoxy-D-manno-2-octulosonic acid) . {formula: see text} In the untreated lipopolysaccharide, the amino group of the non-reducing terminal glucosamine residue is not substituted.

Microbiology, 1995 Oct, 141 ( Pt 10), 2569 - 76
Molecular analysis of an extracellular protease gene from Vibrio parahaemolyticus; Lee CY et al.; The structural gene prtVp encoding the extracellular protease of Vibrio parahaemolyticus strain 93 was cloned in Escherichia coli and sequenced . The cloned DNA fragment contained a 1761 bp ORF encoding a 587 amino acid protein . The deduced polypeptide is composed of a 25 amino acid signal peptide and a 562 amino acid extracellular polypeptide with a calculated molecular mass of 63,156 Da . Protease analysis using a gelatin-containing SDS-polyacrylamide gel detected the presence of a 62 kDa protease that was present in the culture supernatant fractions of the wild-type V . parahaemolyticus strain and of E . coli bearing a pUC119 recombinant with the prtVp DNA insert . The protease activity was inhibited by zinc- and metal-specific inhibitors such as EDTA and 1,10-phenanthroline, which suggested that it is a metalloprotease . The deduced amino acid sequence of PrtVp has 32% identity with that of the collagenase of Vibrio alginolyticus, but has no identity with those of the bacterial proteases . A conserved zinc-binding domain was also found in PrtVp from homology comparison with other metalloproteases . This PrtVp can cause weak haemolysis on blood agar.

Am J Epidemiol, 1995 Oct 1, 142(7), 759 - 64
Impaired immune response to natural infection as a correlate of vaccine failure in a field trial of killed oral cholera vaccines; Clemens J et al.; In a field trial carried out in 1985 in Matlab, Bangladesh, the authors evaluated whether subjects who developed Vibrio cholerae 01 infections during the first year after earlier receipt of B subunit-killed whole cell (BS-WC) or killed whole cell-only (WC) oral cholera vaccines exhibited deficient serum vibriocidal immune responses to these infections . After severe V . cholerae 01 infections (n = 70) in subjects > 5 years of age, the age group in which both vaccines were efficacious, a 6.5 geometric mean-fold rise of serum vibriocidal antibodies was observed among vaccinees, compared with an 18.6 geometric mean-fold rise in placebo-recipients (p < 0.01) . Depressions of serum vibriocidal responses among vaccinees were even more marked after asymptomatic infections (n = 30): a 1.1 geometric mean-fold rise in vaccinees versus a 5.9 geometric mean-fold rise in placebo-recipients (p < 0.01) . The authors conclude that subjects who failed to be protected by BS-WC and WC, despite being in the age group for which these vaccines were protective, exhibited poor immune responses even to the vigorous stimulus of natural infection . These findings raise the possibility that immune hyporesponsiveness may limit the potential efficacy attainable by cholera vaccines in populations with endemic choleraPIP: Natural infections by Vibrio cholerae 01 are known to confer substantial protection against recurrent infections in populations where cholera is endemic . This suggests that it may one day be possible to develop a highly effective oral vaccine against cholera . It is, however, curious that cholera continues to occur into adulthood in populations which have endemic cholera . This phenomenon could be the result of an inability among some individuals in endemic populations to mount suitable immune responses to natural infections . If such immune hyporesponsiveness is truly at work, it may be an important barrier against the development and use of an effective oral cholera vaccine . The authors evaluated whether deficient immune responses to natural infection were associated with the risk of vaccine failure among recipients of killed oral cholera vaccines in a field trial in Bangladesh during 1985 . Their findings support the hypothesis that immune hyporesponsiveness, even after the vigorous stimulus of natural infection, may have limited the protection conferred by the vaccines studied in the trial .

J Infect Dis, 1995 Oct, 172(4), 1126 - 9
Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1; Kenner JR et al.; Cholera vaccine candidate Peru-15 was derived from a Vibrio cholerae O1 El Tor Inaba strain by deleting the cholera toxin genetic element, introducing the gene encoding cholera toxin B subunit into recA, and screening for nonmotility . In a controlled study, Peru-15 (2 x 10(8) cfu) was administered to 11 volunteers . No vaccinee developed diarrhea, and 10 of 11 had > 4-fold rises in vibriocidal antibody titers . One month later, 5 vaccinees and 5 control volunteers were challenged with wild type V . cholerae O1 . Four of 5 controls developed diarrhea (mean, 1.9 L) . Two Peru-15 vaccinees developed diarrhea, 1 with < 0.3 L and 1 with approximately 1.0 L; this latter volunteer had not developed a significant vibriocidal immune response to vaccination . Peru-15 shows promise as a single-dose, oral cholera vaccine that is safe, immunogenic, and protective.

J Bacteriol, 1995 Oct, 177(19), 5606 - 11
Circadian expression of the dnaK gene in the cyanobacterium Synechocystis sp . strain PCC 6803; Aoki S et al.; The expression of the dnaK gene in the cyanobacterium Synechocystis sp . strain PCC 6803 was continuously monitored as bioluminescence by an automated monitoring system, using the bacterial luciferase genes (luxAB) of Vibrio harveyi as a reporter of promoter activity . A dnaK-reporting bioluminescent Synechocystis strain was constructed by fusing a promoterless segment of the luxAB gene set downstream of the promoter region of the Synechocystis dnaK gene and introduction of this gene fusion into a BglII site downstream of the ndhB gene in the Synechocystis chromosome . Bioluminescence from this strain was continuously monitored and oscillated with a period of about 22 h for at least 5 days in continuous light . The phase of the rhythm was reset by the timing of the 12-h dark period administered prior to the continuous light . The period of the rhythm was temperature compensated between 25 and 35 degrees C . Thus, the bioluminescence rhythm satisfied the three criteria of circadian rhythms . Furthermore, the abundance of dnaK mRNA also oscillated with a period of about 1 day for at least 2 days in continuous light conditions, indicating circadian control of dnaK gene expression in Synechocystis sp . strain PCC 6803.

Infect Immun, 1995 Oct, 63(10), 4091 - 8
Initial studies of the structural signal for extracellular transport of cholera toxin and other proteins recognized by Vibrio cholerae; Connell TD et al.; The specificity of the pathway used by Vibrio cholerae for extracellular transport of cholera toxin (CT) and other proteins was examined in several different ways . First, V . cholerae was tested for the ability to secrete the B polypeptides of the type II heat-labile enterotoxins of Escherichia coli . Genes encoding the B polypeptide of LT-IIb in pBluescriptKS- phagemids were introduced into V . cholerae by electroporation . Culture supernatants and periplasmic extracts were collected from cultures of the V . cholerae transformants, and the enterotoxin B subunits were measured by an enzyme-linked immunosorbent assay . Results confirmed that the B polypeptides of both LT-IIa and LT-IIb were secreted by V . cholerae with efficiencies comparable to that measured for secretion of CT . Second, the plasmid clones were introduced into strain M14, an epsE mutant of V . cholerae . M14 failed to transport the B polypeptides of LT-IIa and LT-IIb to the extracellular medium, demonstrating that secretion of type II enterotoxins by V . cholerae proceeds by the same pathway used for extracellular transport of CT . These data suggest that an extracellular transport signal recognized by the secretory machinery of V . cholerae is present in LT-IIa and LT-IIb . Furthermore, since the B polypeptide of CT has little, if any, primary amino acid sequence homology with the B polypeptide of LT-IIa or LT-IIb, the transport signal is likely to be a conformation-dependent motif . Third, a mutant of the B subunit of CT (CT-B) with lysine substituted for glutamate at amino acid position 11 was shown to be secreted poorly by V . cholerae, although it exhibited immunoreactivity and ganglioside GM1-binding activity comparable to that of wild-type CT-B . These findings suggest that Glu-11 may be within or near the extracellular transport motif of CT-B . Finally, the genetic lesion in the epsE allele of V . cholerae M14 was determined by nucleotide sequence analysis.

Appl Environ Microbiol, 1995 Oct, 61(10), 3661 - 6
A novel approach for monitoring genetically engineered microorganisms by using artificial, stable RNAs; Pitulle C et al.; Further improvements in technology for efficient monitoring of genetically engineered microorganisms (GEMs) in the environment are needed . Technology for monitoring rRNA is well established but has not generally been applicable to GEMs because of the lack of unique rRNA target sequences . In the work described herein, it is demonstrated that a deletion mutant of a plasmid-borne Vibrio proteolyticus 5S rRNA gene continues to accumulate to high levels in Escherichia coli although it is no longer incorporated into 70S ribosomes . This deletion construct was subsequently modified by mutagenesis to create a unique recognition site for the restriction endonuclease BstEII, into which new sequences could be readily inserted . Finally, a novel 17-nucleotide identifier sequence from Pennisetum purpureum was embedded into the construct to create an RNA identification cassette . The artificial identifier RNA, expressed from this cassette in vivo, accumulated in E . coli to levels comparable to those of wild-type 5S rRNA without being seriously detrimental to cell survival in laboratory experiments and without entering the ribosomes . These results demonstrate that artificial, stable RNAs containing sequence segments remarkably different from those present in any known rRNA can be designed and that neither the deleted sequence segment nor ribosome incorporation is essential for accumulation of an RNA product.

Am J Trop Med Hyg, 1995 Oct, 53(4), 351 - 9
Vibrio cholerae in the horn of Africa: epidemiology, plasmids, tetracycline resistance gene amplification, and comparison between O1 and non-O1 strains; Coppo A et al.; The prevalence of Vibrio cholerae O1 and non-O1 has been investigated in numerous Somali regions of the Horn of Africa from 1983 to 1990 . From January 1983 to January 1985 and between December 1986 and December 1990, no strains of V . cholerae O1 and 226 strains (5.3%) of V . cholerae non-O1 were isolated from 4,295 diarrhea cases . During a cholera epidemic in 1985 and 1986, the overall case-fatality rate was 13% and the attack rate was 3-3.5 per 1,000 population . Matched case-control studies identified a waterborne route of transmission . A drug-susceptible Ogawa strain from Ethiopia caused the introduction of the disease into northern Somalia . There were two major resistant derivatives of the original strain, and the one resistant to ampicillin, kanamycin, streptomycin, sulfonamide, and tetracycline (TC) predominated in the spreading disease . In 1986, susceptible Ogawa strains quickly displaced this resistant strain . The two incompatibility group C plasmids responsible for the resistance patterns had complex and scattered differences in their structures . Physical analysis of the plasmid DNA region coding for TC resistance demonstrated its genetic amplification in highly resistant variants of Ogawa strains.

Carbohydr Res, 1995 Sep 15, 275(1), 117 - 29
Synthesis and crystal structure of methyl 4-6-dideoxy-4-(3-deoxy-L- glycero-tetronamido)-2-O-methyl-alpha-D-mannopyranoside, the methyl alpha-glycoside of the terminal unit, and presumed antigenic determinant, of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Ogawa; Lei PS et al.; Methyl 4-azido-4,6-dideoxy-3-O-benzyl-alpha-D-mannopyranoside and its analogous 3-O-(4-methoxybenzyl) derivative were methylated and the 2-O-methyl derivatives formed were converted into methyl 4-amino-4,6-dideoxy-2-O-methyl-alpha-D- mannopyranoside {sequence: see text} . Reaction of the latter with 3-deoxy-L-glycero-tetronolactone gave the methyl glycoside of 4,6-dideoxy-4-(3-deoxy-L-glycero- tetronamido)-2-methyl-alpha-D-mannopyranose {sequence: see text}, the monosaccharide that is reported to be the terminal moiety of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Ogawa . The unit cell packing of the compound, which crystallized as a monohydrate, differs from that of the previously described crystalline compound lacking the 2-O-methyl group . The unmethylated sugar is the terminal moiety of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Inaba . The crystal structure of methyl 4,6-dideoxy-2-O- methyl-4-trifluoroacetamido-alpha-D-mannopyranoside {sequence: see text} is also described.

Tijdschr Diergeneeskd, 1995 Sep 15, 120(18), 531 - 4
{A study of the cause of massive mortality among marine-cultured rainbow trout (Oncorrhynchus mykiss, Walbaum) in a fish farm in the southwestern Netherlands}; Oorschot RW et al.; The present study investigated the mass mortality of marine cultured young rainbow trout (Oncorhynchus mykiss, Walbaum) in the Netherlands . The course of the disease, the clinical symptoms, and bacteriological and virological investigations lead to the diagnosis: 'primary infectious pancreatic necrosis virus (IPNV) infection followed by secondary vibriosis' . Treatment with flumequine seemed to be effective . The trout were possible infected with IPNV at the trout hatchery and nursery from which they originated.

Virology, 1995 Sep 10, 212(1), 77 - 83
Neuraminidase is essential for fowl plague virus hemagglutinin to show hemagglutinating activity; Ohuchi M et al.; When hemagglutinin (HA) of fowl plague virus (FPV) was expressed in CV-1 cells by a simian virus 40 vector, hemadsorption was barely detectable, although HA was exposed at the cell surface . However, treatment of HA-expressing cells with Vibrio cholerae neuraminidase (VCNA) resulted in extensive hemadsorption . VCNA treatment enhanced the electrophoretic mobility of the HA1 subunit of HA, indicating the removal of sialic acid . When two oligosaccharides in the vicinity of the receptor binding site of FPV HA were deleted by site-specific mutagenesis, VCNA treatment was not required for hemadsorption . Mutants which retained one of these oligosaccharides and mutants in which oligosaccharides not adjacent to the receptor binding site were deleted needed VCNA treatment to show hemadsorption . VCNA treatment also enhanced hemadsorption of vector-expressed HA of the WSN strain, which had a complex-type oligosaccharide in the vicinity of the receptor binding site, but had no effect on hemadsorption of Hong Kong type HA, which has a high-mannose type oligosaccharide adjacent to the receptor binding site . These results indicate that sialic acid on oligosaccharides near the receptor binding site interferes with hemadsorption . Thus, the neuraminidase is essential for FPV HA to show hemagglutinating activity.

Biol Pharm Bull, 1995 Sep, 18(9), 1189 - 93
N-terminal quarter part of tetracycline transporter from pACYC184 complements K+ uptake activity in K+ uptake-deficient mutants of Escherichia coli and Vibrio alginolyticus; Nakamura T et al.; In an attempt to clone a gene encoding the K+ uptake system from Vibrio alginolyticus, two plasmids, pKT2 and pKT4, were derived from pACYC184 . These plasmids allowed the growth of K+ uptake-deficient mutant strains of Escherichia coli TK420 and V . alginolyticus FS181 in a low K+ medium . The pKT2 and pKT4 had an insertion about 7 and 6 kb, respectively, from the genome of V . alginolyticus . We prepared deletion plasmids from both plasmids and found that the site of genes inserted in the two was not identical and that the active locus corresponded to the structural gene encoding the N-terminal quarter part of tetA(C) gene . The N-terminal region of tetA(C) gene was ligated in another vector plasmid pHG165 to produce pHGK23 . pHGK23 complemented the growth of TK420 in the low K+ medium . It contained only 62 bp from the genome of V . alginolyticus, and the open reading frame was composed of 98 amino acid residues from the N-terminal quarter part of tetA(C) and 5 amino acid residues attached by gene fusion . Using the Na+ -loaded cells of TK420, pHGK23 was found to increase the activity of K+ uptake . These results show that the N-terminal side tetA(C) gene product functions as a K+ uptake system.

Mol Microbiol, 1995 Sep, 17(5), 801 - 12
Transcriptional regulation of bioluminesence genes from Vibrio fischeri; Sitnikov DM et al.; The phenomenon of cell-density-dependent control of gene expression, called autoinduction, has long been a subject of interest and investigation in bioluminescent marine bacteria . It is now becoming clear that many other bacteria, including animal and plant pathogens, use an autoinduction mechanism to regulate a variety of functions . Cell-density-dependent gene expression provides an excellent example of multicellular behaviour in the prokaryotic kingdom where a single cell is able to communicate and sense when a minimal population unit, a 'quorum' of bacteria, is achieved in order for certain behaviour of the population to be performed efficiently . Regulation of bacterial bioluminescence has been studied for many years and represents the best model system for understanding the mechanism of cell-density-dependent gene expression . This review will focus on transcriptional regulation of the Vibrio fischeri luminescence genes emphasizing the role of the transcriptional activator LuxR and possible autoinduction mechanisms that occur in E . coli . Alternative views and opinions regarding the molecular details of the autoinduction mechanism will be discussed.

Mol Microbiol, 1995 Sep, 17(6), 1035 - 44
Vibrio spp . secrete proaerolysin as a folded dimer without the need for disulphide bond formation; Hardie KR et al.; Proaerolysin is an extracellular dimeric protein that is secreted across the inner and outer membranes of Aeromonas spp . in separate steps . To investigate the role of protein folding in the second step, one or more cysteine residues were introduced and the mutant proaerolysins were expressed in Aeromonas hydrophila and Aeromonas salmonicida, as well as Vibrio cholerae . Replacing Met-41 with Cys resulted in expression of a protein that could form a dimer in which the monomers were linked together by a disulphide bridge . A double mutant was also made, in which Gly-202 and Ile-445 were replaced with cysteine in order to allow the formation of an intrachain disulphide bridge when the molecule was correctly folded . The M41C covalent dimer and G202C/I445C proaerolysin with the new intrachain bridge were both easily detected inside the bacteria, and they later appeared in the culture supernatants . Small amounts of incorrectly folded proaerolysin were also observed in the cells, but they were not secreted . We observed in the cells, but they were not secreted . We conclude that proaerolysin folds and dimerizes before being released from the cell, and that correct folding is a requirement for secretion to occur . The proton ionophore CCCP reduced release of the folded proteins . Unoxidized protein was secreted by cells grown in beta-mercaptoethanol and by a dsbA mutant of V . cholerae, indicating that disulphide bond formation may not be essential for release.

J Diarrhoeal Dis Res, 1995 Sep, 13(3), 176 - 9
Vibrio cholerae non-O1 as a causal pathogen in cholera patients in Yangon, Myanmar; Oo KN et al.; Vibrio cholerae non-O1 was studied in patients with rice watery diarrhoea admitted to the Infectious Diseases Hospital, Yangon . The study was conducted during 1993-1994 to determine the association of the pathogen with the disease . Altogether 771 rectal swabs were collected and examined . V . cholerae were isolated by the standard methods . The seasonal, age and sex distribution, serotyping and susceptibility of these isolates to antibiotics were investigated, V . cholerae were isolated from 233 (3O.2%) samples . Among them, V . cholerae O1 were isolated from 117 (5O%) samples and V . cholerae non-O1 from 116 (5O%) samples . The seasonal, age and sex distribution was identical in both V . cholerae O1 and V . cholerae non-O1 groups . V . cholerae O139 was isolated during February 1994 . Thus V . cholerae non-O1 was also one of the causal pathogens of cholera, like V . cholerae O1 in this community.

What Is Genome?, What Is Molecular Biology?, What Is Rhizobia?, What Is Genetic Engineering?, What Is Environmental Microbiology?, c, Microbes, c, Bacterium, a, Microorganisms, r, Bacteriology, e, Microbiology, o, Photobacteria, i, Escherichia coli, n, Escherichia coli, r, Microbial, i, Bactericidal, i, Culture medium, a, Growth media, o, Staphylococcus, n, Staphylococcus, a, S. cerevisiae, r, Microorganism, e, Bacillus, e, Cell cultures, o, S. cerevisiae, a, Botulin, r, Salmonella, r, Bacillus, s, Brevibacteria, o, Shigella, o, Antibiotic resistance, i, Escherichia coli

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005