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 cell