Vibriosis

Vet Immunol Immunopathol, 1994 Dec, 44(1), 85 - 95
Genetic variation in the humoral immune response against Vibrio salmonicida and in antibody titre against Vibrio anguillarum and total IgM in Atlantic salmon (Salmo salar); Stromsheim A et al.; Total IgM level and antibody titre to Vibrio anguillarum O-antigen after bath-vaccination, and specific antibody response to V . salmonicida O-antigen at three different samplings were analysed in family material of Atlantic salmon (Salmo salar), consisting of 791 fish belonging to 34 maternal full-sib groups within 12 paternal half-sib groups . The fish were immunized twice, and blood samples collected three times . After the third blood sampling, the fish were challenged with V . anguillarum . Medium to low genetic variation was recorded in total IgM and in the antibody titres against V . anguillarum O-antigen and V . salmonicida O-antigen, with heritability estimates of 0.12, 0.18 and from 0.03 to 0.12, respectively . Moderate to high genetic and phenotypic correlations were found between the V . salmonicida O-antigen titres at different samplings . Genetic and phenotypic correlations between the initial titres were moderate to low . The effect of different immune traits, including Aeromonas salmonicida A-layer titres (previously described), on the ability to survive the challenge was examined . The likelihood of surviving the challenge was affected positively by the A . salmonicida A-layer titre at the second sampling, and almost significantly affected by the initial V . anguillarum O-antigen titre . Production traits, such as mean slaughter weight and mean proportion of survivors in a corresponding full-sib material, were obtained in the sea-rearing period . No significant full-sib correlation between immune parameters and production traits was detected.

Int J Epidemiol, 1994 Dec, 23(6), 1292 - 9
Epidemic cholera during refugee resettlement in Malawi; Hatch DL et al.; BACKGROUND . In June 1988 a cholera epidemic occurred in a Mozambican refugee population resettling in southern Malawi . METHODS . A case-control study was conducted to determine possible risk factors for disease . The characteristics of 48 refugee households with any member(s) hospitalized for suspected cholera were compared to 441 randomly sampled refugee households without hospitalizations . RESULTS . Vibrio cholerae 01 was isolated from 50% (5/10) of case-patient stool cultures . Having any water containers with > or = 10 T capacity was associated with a significantly lower odds of suspected cholera in households (adjusted odds ratio {aOR} = 0.02, 95% confidence interval {CI} : 0.003-0.12), as was having metal cooking pots (aOR = 0.3, 95% CI : 0.12-0.7), after adjusting for length of residence and socioeconomic status (logistic regression model) . Households with two or more children < 5 years old were at markedly increased odds of suspected cholera (P < 0.0001) . These results suggest that water containers and cooking pots served important preventive functions during this cholera outbreak . Young children may have contributed to cholera transmission, but the reason(s) remains undetermined.

Chem Pharm Bull (Tokyo), 1994 Dec, 42(12), 2449 - 51
Marine natural products . XXXIV . Trisindoline, a new antibiotic indole trimer, produced by a bacterium of Vibrio sp . separated from the marine sponge Hyrtios altum; Kobayashi M et al.; A new antibiotic indole trimer named trisindoline (1) was isolated, together with a known dioxopiperazine brevianamide F (2), from the culture of a bacterium of Vibrio sp., which was separated from the Okinawan marine sponge Hyrtios altum . The structure of trisindoline (1) has been determined on the bases of physicochemical evidence and chemical synthesis.

Cell Biol Toxicol, 1994 Dec, 10(5-6), 345 - 51
Biochemiluminescence and biomedical applications; Champiat D et al.; Although used for analytical purposes for more than 40 years it is only recently that biochemiluminescence (BCL) has found widespread acceptance . Methods employing BCL reactions now play an important role in biomedical research and laboratory medicine . The main attractions for the assay technology include exquisite sensitivity (attomole-zeptomole), high selectivity, speed and simplicity . In biomedical research, the most important applications of BCL are: (1) to estimate microbial numbers and to assess cellular states (e.g., after exposure to antibiotic or cytotoxic agents) and in reporter gene studies (firefly luciferase gene); (2) NAD(P)H involved in redox/dehydrogenase studies using Vibrio luciferase complex; (3) BCL labels and CL detection of enzyme labels in immunoassays are the most widespread routine application for this technology . BCL enzyme immunoassays represent the most active area of development, e.g., enhanced BCL method for peroxidase and BCL assays for alkaline phosphatase labels using adamantyl 1,2-dioxetane.

Glycoconj J, 1994 Dec, 11(6), 518 - 26
Chitovibrin: a chitin-binding lectin from Vibrio parahemolyticus; Gildemeister OS et al.; A novel 134 kDa, calcium-independent chitin-binding lectin, 'chitovibrin', is secreted by the marine bacterium Vibrio parahemolyticus, inducible with chitin or chitin-oligomers . Chitovibrin shows no apparent enzymatic activity but exhibits a strong affinity for chitin and chito-oligomers > dp9 . The protein has an isoelectric pH of 3.6, shows thermal tolerance, binds chitin with an optimum at pH 6 and is active in 0-4 M NaCl . Chitovibrin appears to be completely different from other reported Vibrio lectins and may function to bind V . parahemolyticus to chitin substrates, or to capture or sequester chito-oligomers . It may be a member of a large group of recently described proteins in Vibrios related to a complex chitinoclastic (chitinivorous) system.

Asian Pac J Allergy Immunol, 1994 Dec, 12(2), 155 - 9
A monoclonal antibody-based dot-blot ELISA diagnostic kit for the detection of Vibrio cholerae 01 in stools of diarrheic patients and household contacts; Supawat K et al.; A "cholera diagnostic kit" was developed for sensitive, specific, rapid, and inexpensive detection of Vibrio cholerae 01 . The monoclonal antibody specific to antigen A of Vibrio cholerae 01 was used as an antigen detection reagent and the principle of dot-blot ELISA was adopted . The kits were used in seven Regional Medical Sciences Centres, Ministry of Public Health, located at various regions of Thailand where diarrhea occurs frequently . Diagnostic efficiency of the kits in the detection of Vibrio cholerae 01 from rectal swabs of the diarrheic patients and their household contacts was evaluated in comparison with the conventional culture method . The two methods were found to have excellent degree of agreement (kappa values > 95%) . The dot-blot ELISA has several advantages over the culture methods, ie rapid (dot-blot ELISA takes 1-2 hours while the culture method takes at least two days) and inexpensive . It requires no sophisticated equipment . The procedure is not complicated thus it is easy to train personnel . The diagnostic kits are recommended for use in the detection of severe diarrhea caused by V . cholerae 01 not only in hospitals and health centres where adequate treatment of the patients is required as a life-saving measure but also for early recognition of cholera cases and their contacts so that other action, ie prevention and control of outbreaks and surveillance can be promptly implemented.

Rev Biol Trop, 1994 Dec, 42(3), 487 - 92
Extinction of Vibrio cholerae in acidic substrata: contaminated cabbage and lettuce treated with lime juice; Mata L et al.; Lime juice killed millions of Vibrio cholerae O1, El Tor, Inaba, present on cabbage and lettuce contaminated in the laboratory . The lethal effect was evident within 5 min of exposure to lime juice . No vibrios could be recovered at dilution 1:10 using alkaline peptone water (APW) and thiosulfate-citrate-bile salts-saccharose agar (TCBS) . More than 99.9% of the initial inoculum was effectively destroyed . The number of vibrios killed by lime juice was 2 to 6 logarithms greater than the maximum infecting dose, and 4 to 8 logs greater than the minimum infecting dose for cholera El Tor . The time interval needed for killing was smaller than the usual waiting time for serving food in homes and restaurants . The addition of lime juice to non-acidic foods, beverages and water, is strongly recommended to prevent infection with cholera vibrios and other acid-sensitive microorganisms . This measure is particularly important for rural and slum populations in the tropics and subtropics.

Rev Biol Trop, 1994 Dec, 42(3), 479 - 85
Extinction of Vibrio cholerae in acidic substrata: contaminated fish marinated with lime juice (ceviche); Mata L et al.; Millions of Vibrio cholerae O1 El Tor were rapidly eliminated when added to commercial ceviche prepared by marination of mahi-mahi fish in lime juice . Likewise, large masses of viable vibrios present in laboratory contaminated fish, were readily eliminated after immersion in lime juice, during the preparation of ceviche . The killing effect was evident within 5 min of exposure of vibrios to lime juice, with reductions of more than 99.9% of the initial bacterial mass . After 2 h of marination of fish with lime juice (the minimum recommended), no vibrios were detected in the lowest working dilutions (1:10, 1:100) . The Vibrio mass eliminated by lime juice was 2 to 6 logarithms greater than the maximum infectious dose, and 4 to 8 logs greater than the minimum infectious dose to induce cholera El Tor . Also, the killing time was shorter than the elapsing time between preparing and serving food in homes or restaurants . The traditional marination of fish with lime juice or its addition to seafood and meals immediately before consumption, should be protected and promoted to prevent infection with cholera vibrios . In the face of an epidemic of cholera, consumption of ceviche prepared with lime juice would be one of the safest ways to avoid infection with V . cholerae.

Biochem Biophys Res Commun, 1994 Nov 30, 205(1), 275 - 81
Stereospecificity of hydride transfer and substrate specificity for FMN-containing NAD(P)H-flavin oxidoreductase from the luminescent bacterium, Vibrio fischeri ATCC 7744; Inouye S et al.; The stereospecificity of the hydride transfer in NAD(P)H-flavin reductase reaction of V . fischeri ATCC 7744 was determined by 1H-NMR spectroscopy using stereospecifically labeled reduced beta-nicotinamide adenine dinucleotide (beta-NADH) . The recombinant flavoenzyme, purified from E . coli cells, selectively transferred the pro-R hydrogen at the C-4 position of the nicotinamide ring to flavin and is therefore classified as an A-side specific enzyme . Lumiflavin was used for the reductase reaction, but lumichrome and alpha-NADH were not utilized as electron acceptor and donor, respectively.

Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11388 - 92
The Vibrio cholerae O139 serogroup antigen includes an O-antigen capsule and lipopolysaccharide virulence determinants; Waldor MK et al.; Vibrio cholerae serogroup O139 emerged on the Indian subcontinent in October 1992 to become the first non-O1 V . cholerae serogroup documented to cause epidemic cholera . Although related to V . cholerae El Tor O1 strains, O139 strains have unique surface structures that include a capsular surface layer and lipopolysaccharide (LPS) . Immunoblot analysis of either whole-cell lysates or LPS preparations revealed three electrophoretic forms of the O139 antigen: two slowly migrating forms and one rapidly migrating form that appeared identical to O139 LPS . All three forms of the antigen shared an epitope defined by an O139-specific monoclonal antibody . A serum-sensitive nonencapsulated mutant was isolated that lacks only the slow migrating forms . The slow migrating forms did not stain with silver whereas the rapidly migrating form did, suggesting that the former might constitute highly polymerized O-antigen side-chain molecules that were not covalently bound to core polysaccharide and lipid A (an "O-antigen capsule") . A single transposon insertion resulted in the loss of immunoreactivity of both the LPS and the O-antigen capsule, implying that there are genes common to the biosynthesis of both these macromolecules . The O139 LPS and O-antigen capsule were both important for colonization of the small intestine of the newborn mouse and for serum resistance, demonstrating that both of these forms of the O139 serogroup antigen are virulence factors.

Biochim Biophys Acta, 1994 Nov 22, 1219(3), 701 - 5
Cloning and sequencing of a K+ transport gene (trk A) from the marine bacterium Vibrio alginolyticus; Nakamura T et al.; A gene has been cloned from the marine bacterium Vibrio alginolyticus that functionally complements a mutant strain of Escherichia coli, TK420, defective in K+ transport genes (kdpABC, trkD, trkA) . The cloned Vibrio gene allowed TK420 to grow in a synthetic medium containing less than 10 mM K+ and concomitantly led to an increase in K+ uptake activity . The nucleotide sequence of the cloned fragment revealed an open reading frame, which encodes a protein with a predicted 458 amino acid sequence and molecular mass of 50,122 Da . This gene has 71% homology to trkA gene at the DNA level from E . coli and the deduced amino acid sequence is 79% identical with E . coli TrkA, implying that V . alginolyticus has a trkA-like gene as a component of K+ transport systems.

Gene, 1994 Nov 18, 149(2), 211 - 7
Insertion of a HIV-1-neutralizing epitope in a surface-exposed internal region of the cholera toxin B-subunit; Backstrom M et al.; The non-toxic B-subunit of cholera toxin (CTB) is a powerful immunogen and has been investigated as a carrier for foreign peptide epitopes, with peptides genetically fused to either the N- or C terminus of CTB . In the present study, we have constructed a plasmid encoding a novel intrachain CTB fusion protein with a peptide epitope inserted into an internal region of CTB: eight amino acids (aa) in CTB (56-63) were substituted with a 10-aa peptide from the third variable (V3) loop of the HIV-1 envelope protein gp120 . The resulting chimeric protein retained important functional characteristics of the native CTB including pentamerization and GM1 ganglioside receptor binding . The internal hybrid protein was also shown to be resistant to proteolytic degradation during production in Vibrio cholerae, whereas a terminal hybrid protein, where the same gp120-epitope was fused to the N terminus of CTB, was rapidly cleaved during culture . The inserted epitope, which is known to give rise to HIV-1 neutralizing Ab, could be detected with a V3 loop-specific monoclonal Ab when the chimeric protein was analyzed in ELISA and immunoblot, indicating that the epitope inserted at this site is presented on the surface of the protein . Consistent with these observations, immunization of mice with the CTB::HIV hybrid protein elicited a high titered serum Ab response to the CTB moiety and also, in some but not all animals, a detectable response to the inserted gp120 epitope.

Virology, 1994 Nov 15, 205(1), 7 - 16
Identification of a 40- to 42-kDa attachment polypeptide for canine parvovirus in A72 cells; Basak S et al.; The attachment of canine parvovirus (CPV) to different cell lines was quantitated by a fluorescence-activated cell sorter assay . The viral attachment was observed to both permissive A72 and nonpermissive ST cells but not to nonpermissive MDBK cells . The binding of and infectivity for CPV to A72 cells was reduced upon prior treatment of cells with Vibrio cholerae neuraminidase or lectins, specific for sialic acid . Similarly, treatment of cells with any of several proteases reduced virus binding; however, phospholipase treatment had no effect indicating that one or more membrane glycoproteins were involved in virus binding . These proteins were characterized with a virus overlay protein blot assay . Virus bound to a protein with a molecular mass of 40 to 42 kDa in membranes prepared from A72 and ST cells and not from MDBK cells . The binding to this polypeptide was specific since increasing amounts of unlabeled virions competitively inhibited binding of radiolabeled virions in a dose-dependent manner . A polypeptide of similar molecular mass was immunoprecipitated from radiolabeled octyl glucoside (OG) extract of A72 cells using purified virions, virion-specific antiserum, and protein A . The binding to this polypeptide was decreased but not abolished upon prior treatment of the membrane with V . cholerae neuraminidase . CPV preferentially recognized a polypeptide of similar molecular size in the OG extract prepared from the biotinylated basolateral surface of polarized MDCK monolayer . Hence, we propose that the 40- to 42-kDa glycoprotein represents a specific attachment molecule for CPV in A72 cells.

Lancet, 1994 Nov 5, 344(8932), 1273 - 6
Protective efficacy of oral whole-cell/recombinant-B-subunit cholera vaccine in Peruvian military recruits; Sanchez JL et al.; The cholera epidemic in South America has reinforced the need for safe and effective oral vaccines . In a randomised, double-blind, placebo-controlled efficacy trial among 1563 Peruvian military recruits we have investigated the protective efficacy of an oral inactivated whole-cell/recombinant-B-subunit (WC/rBS) cholera vaccine . Participants were given two oral doses of cholera vaccine or Escherichia coli K12 placebo, with an interval of 7-14 days . 1426 (91%) subjects received the two prescribed doses and were followed up for a mean of 18 weeks (median 21 weeks) . After vaccination, Vibrio cholerae O1 El Tor Ogawa was isolated from 17 subjects with diarrhoea . 16 of the cholera cases occurred 2 weeks or longer after the second dose of vaccine (14 placebo recipients, 2 vaccinees) . We also detected 14 symptomless infections (11 {7 placebo recipients, 4 vaccinees}) 2 weeks or longer after the second dose . The vaccine had significant protective efficacy against cholera (86% {95% CI 37-97}, p < 0.01) but not against symptomless infection (42% {-96 to 85}) . All cholera cases were in people of blood group O, who made up 76% of the study population (p < 0.01) . Two doses of WC/rBS vaccine, given 1 to 2 weeks apart, provide rapid, short-term protection against symptomatic cholera in adult South Americans, who are predominantly of blood group O . Long-term efficacy studies in Peruvian adults and children are under way.

J Bacteriol, 1994 Nov, 176(22), 6986 - 91
Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri; Graf J et al.; Vibrio fischeri is found both as a free-living bacterium in seawater and as the specific, mutualistic light organ symbiont of several fish and squid species . To identify those characteristics of symbiosis-competent strains that are required for successful colonization of the nascent light organ of juvenile Euprymna scolopes squids, we generated a mutant pool by using the transposon Mu dI 1681 and screened this pool for strains that were no longer motile . Eighteen independently isolated nonmotile mutants that were either flagellated or nonflagellated were obtained . In contrast to the parent strain, none of these nonmotile mutants was able to colonize the juvenile squid light organ . The flagellated nonmotile mutant strain NM200 possessed a bundle of sheathed polar flagella indistinguishable from that of the wild-type strain, indicating that the presence of flagella alone is not sufficient for colonization and that it is motility itself that is required for successful light organ colonization . This study identifies motility as the first required symbiotic phenotype of V . fischeri.

J Bacteriol, 1994 Nov, 176(22), 6885 - 91
A plasmid-encoded prepilin peptidase gene from enteropathogenic Escherichia coli; Zhang HZ et al.; Enteropathogenic Escherichia coli, a leading agent of infantile diarrhea worldwide, adheres to tissue culture cells in a pattern called "localized adherence." Localized adherence is associated with bundle-forming pili encoded by the plasmid bfpA gene, the product of which is homologous with the major structural subunit proteins of type IV fimbriae in other bacteria . Several of these proteins have been shown to be processed from a precursor by a specific prepilin peptidase . We cloned restriction fragments downstream of the bfpA gene into an E . coli-Pseudomonas aeruginosa shuttle vector and mobilized them into a P . aeruginosa prepilin peptidase (pilD) mutant . A plasmid containing a 1.3-kb PstI-BamHI fragment was able to complement the pilD mutation, as demonstrated by restoration of sensitivity to the pilus-specific bacteriophage PO4 . The DNA sequence of this fragment revealed an open reading frame, designated bfpP, the predicted product of which is homologous to other prepilin peptidases, including TcpJ of Vibrio cholerae (30% identical amino acids), PulO of Klebsiella oxytoca (29%), and PilD of P . aeruginosa (28%) . A bfpA::TnphoA mutant complemented with a bfpA-containing DNA fragment only partially processes the BfpA protein . When complemented with a larger fragment containing bfpP as well as bfpA, the mutant expresses the fully processed BfpA protein . P . aeruginosa PAK, but not a pilD mutant of PAK, expresses mature BfpA protein when the bfpA gene is mobilized into this strain . Thus, as in other type IV fimbria systems, enteropathogenic E . coli utilizes a specific prepilin peptidase to process the major subunit of the bundle-forming pilus . This prepilin petidase contains sequence and reciprocal functional homologies with the PilD protein of P . aeruginosa.

J Bacteriol, 1994 Nov, 176(22), 6812 - 8
Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp . strain JT0107; Sugano Y et al.; A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose}, agarotriose {O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose}, agarobiose {O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose}, 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp . strain JT0107 and characterized . This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography . The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric . Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases . The optimum temperature and pH were 30 degrees C and 7.7, respectively . The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.

Biochim Biophys Acta, 1994 Nov 1, 1188(1-2), 69 - 74
F0F1-ATPase of Vibrio parahaemolyticus: purification using new detergents and characterization; Ogawa W et al.; Previous attempts to isolate a stable F0F1-ATPase complex (H(+)-translocating ATPase) from Vibrio parahaemolyticus have been unsuccessful . Using new non-ionic detergents (alkyl thiomaltosides), a stable F0F1 complex with a high specific activity (15-25 units/mg protein) was purified and characterized . The purified F0F1-ATPase consists of eight subunits (alpha, beta, gamma, delta, epsilon, a, b and c) . The new detergents, in combination with sucrose (or glycerol), lipid, dithiothreitol and phenylmethylsulfonyl fluoride, effectively stabilized the F0F1 complex . The ATPase activity of the F0F1 complex was greatly increased by anions, such as SO4(2-) and SO3(2-) . Sodium ion increased the activity by about 2-fold . Dicyclohexylcarbodiimide, Zn2+, 4-acetamido-4'-isothiocyanostilben-2,2'disulfonate and tetrachlorosalicylanilide inhibited F0F1-ATPase activity . Ethanol, which stimulated F1-ATPase activity, inhibited F0F1-ATPase activity . Methanol, Na3VO4 and bafilomycin A1 did not have any significant effect on F0F1-ATPase activity, although methanol, like ethanol, stimulated F1-ATPase activity.

Biochim Biophys Acta, 1994 Nov 1, 1188(1-2), 101 - 7
The effect of pH on the growth and motility of Rhodobacter sphaeroides WS8 and the nature of the driving force of the flagellar motor; Packer HL et al.; Rhodobacter sphaeroides WS8 grew, and swam vigorously, over the pH range 6 to 9 . Sustained motility was, however, observed in populations of cells resuspended at pH values between 4.9 and 10.4, although the mean run speed was reduced at the extremes of pH . The ability of R . sphaeroides to swim in strong alkaline conditions prompted the question of whether motility at alkaline pH was powered by a sodium motive force, as has been found in the facultative alkalophilic Bacillus and Vibrio species, particularly as motility was found to be sensitive to the sodium channel inhibitor amiloride . The nature of the driving force of the flagellar motor was therefore investigated . It was found that R . sphaeroides was motile over the same pH range in the absence and presence of sodium ions . The protonophore CCCP was found to inhibit motility under all conditions, whereas monensin, an inhibitor of sodium pumps, had no effect upon motility in the presence or absence of sodium . It was concluded that the delta p is the driving force for the flagellar motor in R . sphaeroides at all values of pH . Amiloride, a specific inhibitor of the sodium-driven flagellar motor in alkalophilic Bacillus and Vibrio was shown to act non-specifically on the proton driven motor of R . sphaeroides, reducing the swimming speed of this organism in media with and without sodium to the same extent and over the complete pH range . Measurement of the delta p by using the electrochromic absorbance change of the carotenoid pigments to measure delta psi and 31P-NMR to measure delta pH showed that the maximum delta p was about -215 mV . At pH 10 the cells swam more slowly and the delta p was about -90 mV . These data suggest that the flagellar motor of R . sphaeroides is proton-driven under all conditions with a threshold for motor rotation below -90 mV and saturation at above -90 mV and below -215 mV.

Infect Immun, 1994 Nov, 62(11), 5120 - 5
Vibrio cholerae iron transport systems: roles of heme and siderophore iron transport in virulence and identification of a gene associated with multiple iron transport systems; Henderson DP et al.; Vibrio cholerae iron transport mutants were tested for their ability to cause disease in an infant mouse model . The mice were challenged with either the wild-type strain, a vibriobactin synthesis mutant, a heme utilization mutant, or double mutants containing both the vibriobactin synthesis defect and the heme utilization defect . When mice were challenged with 10(7) bacteria, the ability of the double mutant to survive in the intestines was greatly reduced and that of the heme utilization mutant was slightly reduced compared with that of the wild type or the vibriobactin synthesis mutant . When the inoculum size was reduced 10-fold, all of the iron transport mutants failed to colonize the intestines and failed to cause diarrhea in the mice, whereas the wild-type strain was not cleared and elicited a diarrheal response . These data indicate that disruption of either the heme utilization or the vibriobactin uptake system reduces the ability of V . cholerae to cause disease . One of the heme utilization mutants, DHH1, was found to be defective also in utilization of vibriobactin and ferrichrome, mimicking the Escherichia coli TonB- phenotype . This mutant was the least virulent of the iron transport mutants tested . Transformation of DHH1 with the recombinant plasmid pHUT4 restored the abilities to use hemin, vibriobactin, and ferrichrome as iron sources, suggesting that pHUT4 encodes a gene(s) involved globally in the iron transport systems . Hybridization of Vibrio DNA with the V . cholerae heme utilization genes demonstrated the presence of DNA homologous to the genes encoding the outer membrane protein HutA and the inner membrane protein HutB in all the V . cholerae strains tested . The probe containing hutA, but not that containing hutB, also hybridized to DNA from Vibrio parahaemolyticus.

Infect Immun, 1994 Nov, 62(11), 4781 - 8
The maltose regulon of Vibrio cholerae affects production and secretion of virulence factors; Lang H et al.; The effects of maltose on production and secretion of virulence factors of Vibrio cholerae in strain X28214, classical biotype, and in maltose-defective transposon mutants constructed from this strain were characterized . Maltose was found to inhibit secretion of cholera toxin and to reduce production of the mannose-sensitive hemagglutinin and the soluble hemagglutinin-protease . In contrast, the amount of toxin-coregulated pilus was increased in the presence of maltose . The maltose effect was apparently mediated by genes of the maltose regulon, since inactivation of the malQ or malF gene of V . cholerae by transposon insertion was found to affect production and secretion of the same virulence factors that were responsive to maltose . The malQ and malF mutants showed, in addition, reduced virulence in an infant-mouse model . These results suggest that maltose may have a significant regulatory role in the production of virulence factors and that an intact maltose regulon is needed for full virulence of V . cholerae.

Rev Invest Clin, 1994 Nov-Dec, 46(6), 495 - 8
{Vibrio vulnificus in Mexico: a case report and review of the literature}; Porras-Cortes G et al.; Vibrio vulnificus is an etiologic agent of septicemia and skin lesions . Patients with chronic diseases, and more frequently chronic liver disease, are specially susceptible to this infection . The main risk factor is shellfish ingestion . In this report we present a patient with chronic liver disease who suffered fulminant sepsis and necro-hemorrhagic bullaes secondary to a V . vulnificus infection . The patient had ingested shrimps two days before . A review of the recent literature on the subject is also presented.

Vaccine, 1994 Nov, 12(15), 1384 - 8
Comparative efficacy of biodegradable liposomes and microspheres as carriers for delivery of Vibrio cholerae antigens in the intestine; Chandrasekhar U et al.; The effect of the encapsulated antigens of Vibrio cholerae and their route of administration in induction of immune response was studied in experimental cholera . The antigenic proteins of V . cholerae El Tor strain KB207 were obtained by fractionation of cell-free lysate by high-performance liquid chromatography . The antigenic proteins were pooled and encapsulated in biodegradable liposomes and poly(D,L) lactic co-glycolic acid microspheres . Rabbits were immunized with free as well as encapsulated antigens by different routes . Liposome-encapsulated antigens delivered intraintestinally offered maximum protection . Orally or intraintestinally delivered antigens in microspheres failed to elicit a significant immune response, although as a carrier microspheres were comparable to liposomes when judged by the subcutaneous route . The results suggested that liposomes and microspheres could be used as carriers of protective antigens of V . cholerae for effective immunization.

Toxicon, 1994 Nov, 32(11), 1397 - 412
Aspects of the haemolytic reaction induced by Kanagawa haemolysin of Vibrio parahaemolyticus; Huntley JS et al.; Vibrio parahaemolyticus, an important enteric pathogen, produces toxin (Kanagawa haemolysin, KH), the presence of which correlates well with pathogenicity . KH induced lysis of human red blood cells (HRBC); the kinetics were strongly dependent on KH concentration (0-1 HU/ml) and rather independent of target cell concentration {0.5 < or = haematocrit (%) < or = 6} and the ratio KH:HRBC . The suggestion that KH-induced haemolysis is due to colloid osmosis is supported by results indicating: (1) osmotic protection (by suspension in iso-osmotic choline chloride, D-sorbitol or L-valine, or MOPS-buffered saline with added sucrose), (2) a cell volume increase prior to lysis, and (3) an increase in HRBC cation (86Rb+) influx after KH addition, indicating raised passive cation permeation . The effect of temperature on KH-induced haemolysis indicates the importance of processes other than the action of a simple water-filled pore, because of the high activation energy {53.30 +/- 2.79 kJ (mol.)-1} involved . Although haemolytic rate was attenuated by washout after 5 min KH exposure, the KH-induced lesion itself was not susceptible to washout by either extracellular volume expansion (at constant osmolarity) or centrifugation/resuspension . This suggests that HRBC binding of KH from aqueous solution still continues after 5 min exposure at 37 degrees C . Pre-vortexing KH with dibutyl phthalate (DBP) dramatically reduced the haemolytic activity of the aqueous toxin preparation, suggesting a protein-lipid interaction, which may support the contention that KH can move from a hydrophilic to a hydrophobic environment . Two features were identified that are characteristic of highly purified TDH preparations: (1) thermostability of haemolysin, and (2) monovalent cation selectivity series of lesion: Cs+ > Li+ > K+ > Rb+ > Na+, confirming that TDH is the important leak-inducing agent of KH.

Mol Biol (Mosk), 1994 Nov-Dec, 28(6), 1299 - 307
{A kinetic method of determining the frequency of homologous recombination of plasmids in Escherichia coli cells}; Zavil'gel'skii GB et al.; To test the frequency of recombination by the RecF pathway the hybrid plasmids have been constructed which allow the bioluminescence recombination assay in the transformed E . coli cells . pF2(+) and pF6(+) are derivatives of pUC18 and pACYC184 respectively, with the luxA and luxB genes of Vibrio fischeri cloned downstream of the lac promoter . The luxA genes in pF2(+) and pF6(+) were mutated at the XhoI site and the HindIII site, accordingly, pF8 is a pACYC184 derivative with two copies luxA and luxB genes . The one copy of luxA gene was mutated at the XhoI site and another copy of the luxA gene was mutated at the HindIII site . A kinetic analysis of the population of the replicating and recombinating plasmids has been carried out . Experimental values of the frequency of recombination per one generation (P) were determined for the different E . coli strains.

Vaccine, 1994 Nov, 12(14), 1330 - 4
Immunogenicity of Vibrio cholerae ghosts following intraperitoneal immunization of mice; Eko FO et al.; The immunogenic potential of Vibrio cholerae ghosts (VCG) in comparison with heat-killed whole-cell vibrios (WCV) was evaluated after intraperitoneal immunization of adult mice . Swiss white mice received four doses of VCG or WCV intraperitoneally, consisting of 500 micrograms of lyophilized material in 200 microliters of phosphate-buffered saline (PBS), pH 7.4 . The control group received 200 microliters of PBS . Serum samples were collected from all mice on the day of immunization and on days 14, 24, 35 and 62 postimmunization . Sera were examined for vibriocidal antibodies by the microtitre and tube-dilution methods and Vibrio-specific serum IgG antibodies were assessed by ELISA . IgG antibodies to intact WCV were detected in sera from all animals immunized with VCG or WCV . The response was specific and of high magnitude . Significantly higher antibody responses were obtained when sera from both VCG- and WCV-immunized mice were titrated against VCG . The immunogenicity of VCG in evoking serum IgG responses was higher than that of WCV . However, the immunogenicity of the two antigen preparations was comparable in terms of seroconversion for vibriocidal antibodies . These results demonstrate that VCG administered intraperitoneally evoke Vibrio-specific serum IgG responses as well as vibriocidal antibody activity in mice.

J Nat Prod, 1994 Nov, 57(11), 1587 - 90
Vibrindole A, a metabolite of the marine bacterium, Vibrio parahaemolyticus, isolated from the toxic mucus of the boxfish Ostracion cubicus; Bell R et al.; The EtOAc extract of the whole culture medium of Vibrio parahaemolyticus, which inhabits the toxic mucus of the box fish Ostracion cubicus, afforded a new indole-derived natural product, vibrindole A {1}, along with some known cyclic dipeptides and indoles . The structure of 1 was determined by analysis of its physicochemical characteristics.

J Clin Microbiol, 1994 Nov, 32(11), 2775 - 9
Characterization of phenotypic, serological, and toxigenic traits of Vibrio cholerae O139 bengal; Nair GB et al.; Biochemical and physiological traits of a collection of strains of Vibrio cholerae O139 Bengal isolated from India, Bangladesh, and Thailand showed that these strains formed a phenotypically homogeneous group with identical characteristics that were essentially similar to those of the O1 serogroup . Resistance to 150 micrograms of the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine) and Mukherjee's El Tor phage 5 and classical phage IV and the nonagglutinability of the strains with O1 antiserum were the only discernible differences between the O139 and O1 serogroups . Extensive serological characterization further revealed the O139 serogroup to be distinct from the existing 138 serogroups of V . cholerae . Antiserum raised against the O139 serogroup required absorption with the R reference strain CA385 and with the reference strain representing serogroup O22 to remove cross-reacting agglutinins . All of the 223 representative strains of V . cholerae O139 examined hybridized with DNA probes specific for the cholera toxin (CT) gene, zonula occludens toxin gene, and El Tor hemolysin gene but not with the probe specific for the heat-stable enterotoxin gene . The amount of CT present in stool samples of patients infected with the O139 serogroup was higher than that found in stools of patients infected with O1 El Tor, and this echoed findings that the amount of CT produced by O139 strains in vitro was higher than that produced by the O1 El Tor strains . The nucleotide sequences of the genes encoding the A and B subunits of CT of the O139 serogroup were identical to the sequences reported for the CT gene of O1 El Tor . The CT gene of O139 strains could be amplified by using primers developed for detection of the CT gene of the O1 serogroup by a PCR assay, which could also be used to detect the CT gene in stool samples of patients infected with strains of the O139 serogroup.

APMIS, 1994 Nov, 102(11), 874 - 6
The first fatal case of Vibrio vulnificus infection in Denmark; Bock T et al.; Vibrio vulnificus can cause severe infections in humans and persons with preexisting liver disorders are especially at risk . In this paper we report what is to our knowledge the first fatal case of V . vulnificus infection in Denmark . The patient was a 68-year-old man with a history of chronic lymphatic leukemia and hepatic cirrhosis . Physicians should be aware of the clinical manifestations of this disease and should be especially attentive to patients at risk of acquiring the infection if there has been possible exposure to V . vulnificus by contact with seawater or contaminated material such as eels.

Indian J Med Res, 1994 Nov, 100, 217 - 8
Outbreak of cholera due to Vibrio cholerae 01 in Orissa state; Niyogi SK et al.; During May-June 1993, an outbreak of acute diarrhoea resulting in deaths primarily in adults was reported in two districts of Orissa state . Epidemiological and microbiological investigations revealed that this outbreak was caused by V . cholerae 01 biotype EITor . V . cholerae 01 strains were uniformly resistant to furazolidone.

Indian J Med Res, 1994 Nov, 100, 213 - 6
Epidemic of Vibrio cholerae 0139 in Calcutta; Bhattacharya SK et al.; As one of large outbreaks of cholera-like illness in the Indian subcontinent, Calcutta and its neighbouring areas experienced an unprecedented epidemic due to a new strain of V . cholerae non-01, designated as V . cholerae 0139 Bengal, since January 1993 . This epidemic predominantly affected the adult population of Calcutta as evidenced by the hospitalization of more adults at the Infectious Disease Hospital, Calcutta (IDH), which bore the main brunt of the epidemic in and around Calcutta . During the peak of the epidemic about 180 to 300 diarrhoea patients were admitted daily at the IDH . Of the 807 patients screened, 407 were positive for V . cholerae 0139 and majority (82.8%) of the cases were > 10 yr of age . Severe dehydration was recorded in 85.5 per cent of the cases.

J AOAC Int, 1994 Nov-Dec, 77(6), 1492 - 9
Identification of Vibrio vulnificus by cellular fatty acid composition using the Hewlett-Packard 5898A Microbial Identification System: collaborative study; Landry WL; A gas chromatographic method using a capillary column for rapid identification of Vibrio vulnificus was examined in a collaborative study . Identifications were performed by analysis of cellular fatty acid profiles which were automatically searched against reference profiles stored in a computer-generated library . Each of the 13 collaborators was sent 15 unknown isolates, which included 10 V . vulnificus isolates and 5 negative control isolates . Each collaborator was furnished with a computer-generated library, developed by the Dallas U.S . Food and Drug Administration laboratory, which contained entries for V . vulnificus, V . cholerae, V . fluvialis, V . parahaemolyticus, V . mimicus, and Aeromonas hydrophila . Of the 195 isolates sent to the collaborators, results for 190 isolates were received . The other 5 isolates were nonviable before analyses began . Of the 126 V . vulnificus isolates analyzed, 118 (93.7%) were correctly identified . Of the 65 negative control isolates sent, one was nonviable, one was misidentified as V . vulnificus, and 2 were misidentified as V . parahaemolyticus . Of the 64 negative controls analyzed, 95.3% were correctly identified . Statistical analysis shows a sensitivity rate of 0.872, specificity rate of 0.982, false positive rate of 0.010, and false negative rate of 0.206 . The gas chromatographic method for identification of Vibrio vulnificus by microbial fatty acid profile has been adopted first action by AOAC INTERNATIONAL.

Microbiology, 1994 Nov, 140 ( Pt 11), 3117 - 24
Cloning and nucleotide sequence of the carboxynorspermidine decarboxylase gene from Vibrio alginolyticus; Yamamoto S et al.; The gene (nspC) encoding carboxynorspermidine decarboxylase (CANS DC), the last enzyme in norspermidine biosynthesis, in Vibrio alginolyticus was isolated by immuno-screening and its complete nucleotide sequence was determined . Sequence analysis of the subcloned fragment (2.0 kb) revealed an ORF of 1131 bp encoding a protein of 377 amino acids with a calculated molecular mass of 42,008 Da . The sequence of 20 N-terminal amino acids of purified CANS DC was found to be identical to that predicted from the nspC gene . A putative ribosome binding sequence was observed 8 bp upstream from the translation start site (ATG), and promoter- and terminator-like sequences were detected upstream and downstream of the ORF, respectively . Database searches identified no similar proteins, but the deduced amino acid sequence contained a putative pyridoxal 5'-phosphate binding region similar to those of the bacterial meso-2,6-diaminopimelate decarboxylases and eukaryotic ornithine decarboxylases . Another full ORF was found on the opposite strand downstream from the nspC gene . It encoded a protein of 69 amino acids with a calculated molecular mass of 7441 Da, which exhibited some weak similarity to ScrR, a repressor protein of V . alginolyticus, in the helix-turn-helix DNA binding domain, but did not appear to be expressed in the host cells.

Zentralbl Bakteriol, 1994 Nov, 281(4), 475 - 80
Antigenicity and antigenic cross-reactivity of outer membrane proteins of Vibrio parahaemolyticus; Biswas T et al.; Antigenicity of outer membrane proteins was studied and compared between Kanagawa positive (clinical) and negative (environmental) strains of Vibrio parahaemolyticus . Murine antibodies recognized a wide range of outer membrane proteins of Kanagawa negative strains as antigens with molecular masses ranging between 102 kDa to 14 kDa . However, only a few of the total outer membrane proteins of clinical isolates were antigenic and comprised a 55kDa protein as the major antigen . Although a marked difference in antigenicity was found, molecular masses of outer membrane proteins of Kanagawa positive and negative strains migrated similarly in SDS-PAGE . Several outer membrane proteins of Kanagawa-positive strains were found to be antigenic and ubiquitous in a cross-reactivity study indicating that the ubiquitous proteins which could not be recognized by anti-KP antibodies were buried in the outer membrane.

Microb Pathog, 1994 Nov, 17(5), 339 - 46
Expression and mutagenesis of recombinant cholera toxin A subunit; Vadheim KL et al.; ADP-ribosylating protein exotoxins from Vibrio cholerae (CT) and Escherichia coli (LT-I) share two short regions of sequence similarity with Bordetella pertussis toxin (PT) . Previous studies have indicated that substitution of arginine for lysine 7 within the first region of CT drastically decreases ADP ribosyltransferase activity . We have more closely defined the role of other amino acids in this region by generating modified proteins in which arginine 7 was replaced with lysine (R7K), aspartate 9 was replaced with arginine (D9R), glycine was substituted for proline 12 (P12G), amino acids 6 to 13 were deleted (delta 613) or the C-terminal KDEL sequence was changed to NEDL . The modified proteins R7K, D9R and delta 613 exhibited undetectable ADP ribosyltransferase activity . Comparison of the tryptic digest of R7K with native CT suggested that changes in protein conformation may be responsible for the loss of ADP-ribosylation activity.

FEMS Microbiol Lett, 1994 Nov 1, 123(3), 289 - 98
Monoclonal antibodies against Vibrio anguillarum O2 and Vibrio ordalii identify antigenic differences in lipopolysaccharide O-antigens; Mutharia LM et al.; Monoclonal antibodies (mAbs) that recognize distinct species-specific antigenic epitopes in O-antigens from Vibrio anguillarum O2, O2a and certain O2b strains (mAb 7B4) and from Vibrio ordalii strains (mAbs A16 and 7D11) were generated . Western immunoblot analysis using these mAbs revealed that vibrio strains grown in the presence of fresh rainbow trout blood expressed lipopolysaccharide (LPS) with longer (high molecular mass) O-antigens and extracellular capsular layers when compared to strains grown without rainbow trout blood . We also generated mAbs that react with O-antigens from V . anguillarum serotype O1 (mAbs 7B8, 7B5 and 1C3) and serotype O3 (mAbs 13A1 and 14C5) strains . These mAbs provide rapid and accurate diagnostic reagents for serological differentiation of V . ordalii from serotype O2 strains of V . anguillarum, and for serotyping of these pathogenic vibrios.

Eur J Biochem, 1994 Nov 1, 225(3), 1029 - 39
Isolation and structural analysis of oligosaccharide phosphates containing the complete carbohydrate chain of the lipopolysaccharide from Vibrio cholerae strain H11 (non-O1); Bock K et al.; For the first time, an oligosaccharide has been prepared comprising the lipid A backbone, the core oligosaccharide and one repeating unit of the O-specific polysaccharide (O-chain) of a lipopolysaccharide . Lipopolysaccharide from Vibrio cholerae strain H11 (non-O1) was deacylated and the products were separated by high-performance anion-exchange chromatography . Major fractions were a hexadecasaccharide trisphosphate 1, representing the core-lipid A oligosaccharide substituted by one modified repeating unit of the O-antigenic polysaccharide, a dodecasaccharide trisphosphate 2 and an undecasaccharide trisphosphate 3, representing the core-lipid A region . Oligosaccharide 1 originated from beta-elimination upon alkaline hydrolysis of alpha-galacturonic acid of the O-chain; oligosaccharides 2 and 3 were most likely obtained from naturally occurring lipopolysaccharide species carrying no O-chain . The structures of these compounds were elucidated on the basis of monosaccharide composition, and NMR investigations comprising correlation spectroscopy, total correlation spectroscopy and nuclear Overhauser enhancement spectroscopy experiments, as well as heteronuclear 13C, 1H correlation spectroscopy . The structures are as follows: {formula: see text} where R is beta-L-threo-hex-4-enuronopyranosyl-(1-4)-alpha-Neu-(2-3)-beta-Gal A-(1-3)- beta-QuiN-(1-4)-beta-Sedf-(2- in 1, beta-Sedf-(2- in 2, and H in 3 . Where not stated otherwise, sugars are pyranoses of the D-series . Hep is L-glycero-D-manno-heptose, QuiN is 2-amino-2,6-dideoxy-glucose, Kdo is 3-deoxy-D-manno-2-octulosonic acid, Sed is D-altro-heptulose and GalA is galacturonic acid.

JAMA, 1994 Oct 19, 272(15), 1203 - 5
Diagnosis and treatment of cholera in the United States . Are we prepared?
Besser RE, Feikin DR, Eberhart-Phillips JE, Mascola L, Griffin PM.
OBJECTIVE--To assess cholera recognition and treatment by US health care workers in the largest cholera outbreak in the United States this century . DESIGN--We reviewed the medical records of passengers from a flight on which a cholera outbreak occurred . To determine the availability of oral rehydration solutions, we surveyed treatment facilities and referral pharmacies . SETTING--On February 14, 1992, more than 100 passengers on a flight from South America to Los Angeles, Calif, were infected with toxigenic Vibrio cholerae O1 . SUBJECTS--Fifty-four of 67 passengers who sought care in California and Nevada . RESULTS--We reviewed the records of 54 passengers, including 39 with diarrhea and 15 without symptoms . All 17 persons who sought treatment before the outbreak was widely reported by the media had diarrhea . For 12 of these persons, recent travel to South America was noted, but only those four whose records listed cholera as a possible diagnosis were immediately hospitalized . Seven sought care again within 3 days; three were dehydrated, two of these three were hospitalized, and one of these two died . None of the 26 patients suspected to have cholera received appropriate fluids; severely dehydrated patients did not receive Ringer's lactate solution and those not severely dehydrated did not receive an oral rehydration solution . None of the facilities and pharmacies involved stocked World Health Organization oral rehydration salts solution, the preferred solution for treating cholera and other diarrheal diseases . CONCLUSIONS--Treatment of cholera in the United States was suboptimal . Oral fluids appropriate for the treatment of cholera and other diarrheal diseases were generally unavailable . Widespread cholera in the developing world means that US physicians should be prepared to treat "imported" cases . Physicians evaluating patients with diarrhea should obtain a travel history, should consider cholera in patients returning from countries with endemic or epidemic cholera, and should instruct patients in appropriate use of World Health Organization oral rehydration salts solution or other oral rehydration solutions containing 75 to 90 mmol/L of sodium . Pharmacies and medical facilities should stock these solutions.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 185 - 91
Cloning and sequencing of the gene encoding Vibrio cholerae O1 fimbrial subunit (fimbrillin); Ehara M et al.; The gene encoding an 18 kDa fimbrial subunit of Vibrio cholerae O1 was identified in a fimbriate strain Bgd17 . Mixed oligoprimers were prepared based on the amino acid sequence of the N-terminus and that from a cyanogen bromide-cleaved fragment of the fimbrillin . A PCR-amplified 185 bp DNA fragment was sequenced . This 185 bp fragment was further extended to 540 bp to 3' and 5' termini by RNA-PCR using a primer containing a random hexamer at its 3' end . This fragment did not contain the stop codons . It was further extended by a gene walking method using Eco RI cassette and its primers . Finally a 660 bp fragment was obtained and sequenced . This fragment contained the complete open reading frame of the structural subunit of the fimbriae, composed of 169 amino acids with a molecular mass of 17435.65 and a leader sequence of 6 or 9 amino acids . The deduced amino acid sequence of the polypeptide encoded by the gene, designated fimA, displayed a highly conserved sequence of MKXXXGFTLI EL of type 4 fimbriae.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 179 - 84
Morphology of the viable but nonculturable Vibrio cholerae as determined by the freeze fixation technique; Kondo K et al.; The morphology of the nonculturable Vibrio cholerae strain TSI-4 was examined by the freeze fixation technique of electron microscopy and subsequently four unique structures were found in the fine structure s of this bacterium . The size of the cell was about 2/3 of the growing cell . Although the cell was observed to have an outer membrane as well as the cell membrane and cytoplasm, the outer membrane was undulated and had a surface layer of fine fibers . The peptidoglycan layer was thick and more electron dense than that of normal cells.

Biochemistry, 1994 Oct 11, 33(40), 12194 - 201
Probing the Vibrio harveyi luciferase beta subunit functionality and the intersubunit domain by site-directed mutagenesis; Xin X et al.; While the critical role of the bacterial luciferase alpha subunit in catalysis has been amply documented, the beta subunit was only known to be involved in thermal stability and substrate binding . Two conserved histidyl residues at position 81 and 82 of the beta subunit of Vibrio harveyi luciferase were each mutated to an alanine, aspartate, or lysine to probe further the beta functionality . These mutations resulted in higher Km values for reduced riboflavin 5'-phosphate, less efficient oxidations of the aldehyde substrate, and decreased light-emitting activities . beta His82 appears to be significantly more critical than beta His81 . For the beta His82-mutated luciferases, the maximal light intensities and total light outputs were reduced to 19-4% of that for the wild-type enzyme, and the values of Vmax/Km,flavin were decreased by 2-3 orders of magnitude . The reduced light emission activities for these mutated luciferases can be correlated to lower yields of the flavin 4a-hydroperoxide intermediate, reduced productions of the excited flavin emitter, and/or enhanced quenching of the emitter . The beta subunit and the conserved beta His82 in particular have thus been shown to be critical not only to flavin binding but also to catalytic characteristics of luciferase . The dimeric structure of luciferase is essential to its high catalytic efficiency . To characterize the intersubunit domain, three sets of single/double mutants were constructed, and the additivities of mutational effects were tested to screen for residues that could interact across the subunit interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1994 Oct 11, 148(1), 91 - 5
Identification of a ToxR-activated gene, tagE, that lies within the accessory colonization factor gene cluster of Vibrio cholerae O395; Kovach ME et al.; The nucleotide (nt) sequence has been determined for a Vibrio cholerae ToxR-activated gene designated tagE that is located within a cluster of genes required for efficient intestinal colonization . The tagE gene encompasses 909 nt and is predicted to encode a 303-amino-acid (aa) protein with an estimated molecular mass of 34,468 Da . Computer-assisted similarity searches revealed that TagE possesses aa sequence similarity with Escherichia coli OrfU and Staphylococcus simulans lysostaphin, two proteins that are involved in cell-wall biosynthesis and peptidoglycan degradation, respectively . The role, if any, that TagE plays in the accessory colonization factor phenotype is currently under investigation.

Arthritis Rheum, 1994 Oct, 37(10), 1553 - 4
Systemic lupus erythematosus presenting as a non-O:1 Vibrio cholerae abscess; Nedunchezian D et al.; The usual presentations and manifestations of systemic lupus erythematosus (SLE) are well known . We describe a patient with SLE that was discovered in the course of evaluation of an abscess, found to be associated with non-O:1 Vibrio cholerae.

J Trop Med Hyg, 1994 Oct, 97(5), 317 - 20
Vibrio cholerae 0139 'Bengal' in Singapore; Tay L et al.; Vibrio cholerae 0139 was isolated from five patients with cholera-like illness . All were imported cases . Laboratory investigations found our five isolates in show similar morphological, biochemical and serological characteristics to the V . cholerae 0139 strains causing epidemics in Bangladesh and India . Our isolates were toxin producers resistant to streptomycin and co-trimoxazole . No local transmission was known to have occurred following introduction of these imported cases.

J Bacteriol, 1994 Oct, 176(20), 6199 - 206
Molecular evolution of the seventh-pandemic clone of Vibrio cholerae and its relationship to other pandemic and epidemic V . cholerae isolates; Karaolis DK et al.; Genetic variation and molecular evolution within the seventh-pandemic clone of Vibrio cholerae O1 and its relationship to other V . cholerae isolates were examined by studying 58 clinical isolates that were epidemiologically unassociated and isolated from patients in different countries over 62 years (1931 to 1993) . The sample consisted of 45 isolates from the seventh cholera pandemic (1961 to the present), 3 from the sixth pandemic, 3 from sporadic El Tor outbreaks prior to the seventh pandemic, 2 from the U.S . Gulf Coast, and 5 O139 Bengal isolates . Ribotyping detected 11 polymorphic restriction sites within the seventh-pandemic isolates and showed major differences in ribotypes in comparison with sixth- and pre-seventh-pandemic isolates . O139 isolates were very similar to isolates from the start of the seventh pandemic, differing at only two sites . The majority of seventh-pandemic isolates fall into two groups, the first present from 1961 to the present and found only in Asia and the second arising in 1966 and spreading worldwide . Both groups underwent change over time, allowing a provisional estimate for the nucleotide substitution rate within the seventh pandemic clone.

J Bacteriol, 1994 Oct, 176(19), 5988 - 98
MotX, the channel component of the sodium-type flagellar motor; McCarter LL; Thrust for propulsion of flagellated bacteria is generated by rotation of a propeller, the flagellum . The power to drive the polar flagellar rotary motor of Vibrio parahaemolyticus is derived from the transmembrane potential of sodium ions . Force is generated by the motor on coupling of the movement of ions across the membrane to rotation of the flagellum . A gene, motX, encoding one component of the torque generator has been cloned and sequenced . The deduced protein sequence is 212 amino acids in length . MotX was localized to the membrane and shown to interact with MotY, which is the presumed stationary component of the motor . Overproduction of MotX, but not that of a nonfunctional mutant MotX, was lethal to Escherichia coli . The rate of lysis caused by induction of motX was proportional to the sodium ion concentration . Li+ and K+ substituted for Na+ to promote lysis, while Ca2+ did not enhance lysis . Protection from the lethal effects of induction of motX was afforded by the sodium channel blocker amiloride . The data suggest that MotX forms a sodium channel . The deduced protein sequence for MotX shows no homology to its ion-conducting counterpart in the proton-driven motor; however, in possessing only one hydrophobic domain, it resembles other channels formed by small proteins with single membrane-spanning domains.

J Bacteriol, 1994 Oct, 176(19), 5949 - 57
Stringent control during carbon starvation of marine Vibrio sp . strain S14: molecular cloning, nucleotide sequence, and deletion of the relA gene; Flardh K et al.; In order to evaluate the role of the stringent response in starvation adaptations of the marine Vibrio sp . strain S14, we have cloned the relA gene and generated relaxed mutants of this organism . The Vibrio relA gene was selected from a chromosomal DNA library by complementation of an Escherichia coli delta relA strain . The nucleotide sequence contains a 743-codon open reading frame that encodes a polypeptide that is identical in length and highly homologous to the E . coli RelA protein . The amino acid sequences are 64% identical, and they share some completely conserved regions . A delta relA::kan allele was generated by replacing 53% of the open reading frame with a kanamycin resistance gene . The Vibrio relA mutants displayed a relaxed control of RNA synthesis and failed to accumulate ppGpp during amino acid limitation . During carbon and energy starvation, a relA-dependent burst of ppGpp synthesis concomitant with carbon source depletion and growth arrest was observed . Also, in the absence of the relA gene, there was an accumulation of ppGpp during carbon starvation, but this was slower and smaller than that which occurred in the stringent strains, and it was preceded by a marked decrease in the {ATP}/{ADP} ratio . In both the wild-type and the relaxed strains, carbon source depletion caused an immediate decrease in the size of the GTP pool and a block of net RNA accumulation . The relA mutation did not affect long-term survival or the development of resistance against heat, ethanol, and oxidative stress during carbon starvation of Vibrio sp . strain S14.

J Bacteriol, 1994 Oct, 176(19), 5897 - 903
Glucose upshift of carbon-starved marine Vibrio sp . strain S14 causes amino acid starvation and induction of the stringent response; Flardh K et al.; The physiological status of carbon-starved cells of the marine Vibrio sp . strain S14 has been investigated by the analysis of their immediate response to carbon and energy sources . During the first minute after glucose addition to 48-h-starved cells, the pools of ATP and GTP increased rapidly, and the {ATP}/{ADP} ratio reached the level typical for growing cells within 4 min . The total rates of RNA and protein synthesis increased initially but were inhibited 4 to 5 min after glucose addition by the induction of the stringent response . A mutation in the relA gene abolished stringent control during the recovery and significantly prolonged the lag phase, before the starved cells regrew, after the addition of a single source of carbon . However, both the wild-type and the relA cells regrew without a significant lag phase when given glucose supplemented with amino acids . On the basis of these results, it is suggested that carbon-starved cells are deficient in amino acid biosynthesis and that ppGpp and the stringent response are involved in overcoming this deficiency, presumably by depressing the synthesis of amino acid biosynthetic enzymes . Furthermore, the data suggest that the starved cells primarily are starved for energy, and evidence is presented that the step-up in the rate of protein synthesis after refeeding is partially dependent on de novo RNA synthesis.

Infect Immun, 1994 Oct, 62(10), 4176 - 85
Importance of ADP-ribosylation in the morphological changes of PC12 cells induced by cholera toxin; Glineur C et al.; Cholera toxin (CTX) is composed of two subunits, subunit A, which possesses ADP-ribosyltransferase activity, and subunit B, which is responsible for receptor binding . It has previously been shown that agents that increase cyclic AMP (cAMP) levels in cells induce differentiation of PC12 cells into neurite-like cells . In this report, we show that as little as 100 pg of CTX per ml induces such changes . CTX was found to ADP-ribosylate at least four membrane proteins of PC12 cells in vitro and in vivo and to increase intracellular cAMP levels . We have developed an inducible ctx gene expression system in Vibrio cholerae by using the tac promoter . The culture medium of the CTX-producing bacteria was able to induce the morphological changes and the ADP-ribosylation of the PC12 cell membrane proteins . We have constructed two CTX-cross-reactive mutant proteins (CTX-CRM) by site-directed mutagenesis . The choice of glutamic acid 29 as the target amino acid was based on sequence similarities with other bacterial toxins . CTX-CRM-E29 delta, in which the Glu-29 of the A subunit was deleted, showed strongly reduced ADP-ribosyltransferase activity and did not induce significant morphological changes of PC12 cells . In contrast, CTX-CRM-E29D, in which the Glu-29 was replaced by an aspartic acid, was as active as the wild-type protein . We conclude that the ADP-ribosylation activity of CTX is important for the toxin-induced differentiation of PC12 cells . Pertussis toxin, which had no visible effect on PC12 cell morphology, was also able to ADP-ribosylate a membrane-bound protein(s) in vitro and in vivo . Pertussis toxin alone did not significantly increase cAMP levels in PC12 cells, but it acted synergistically with CTX.

Protein Sci, 1994 Oct, 3(10), 1670 - 86
Protein crystallography and infectious diseases; Verlinde CL et al.; The current rapid growth in the number of known 3-dimensional protein structures is producing a database of structures that is increasingly useful as a starting point for the development of new medically relevant molecules such as drugs, therapeutic proteins, and vaccines . This development is beautifully illustrated in the recent book, Protein structure: New approaches to disease and therapy (Perutz, 1992) . There is a great and growing promise for the design of molecules for the treatment or prevention of a wide variety of diseases, an endeavor made possible by the insights derived from the structure and function of crucial proteins from pathogenic organisms and from man . We present here 2 illustrations of structure-based drug design . The first is the prospect of developing antitrypanosomal drugs based on crystallographic, ligand-binding, and molecular modeling studies of glycolytic glycosomal enzymes from Trypanosomatidae . These unicellular organisms are responsible for several tropical diseases, including African and American trypanosomiases, as well as various forms of leishmaniasis . Because the target enzymes are also present in the human host, this project is a pioneering study in selective design . The second illustrative case is the prospect of designing anti-cholera drugs based on detailed analysis of the structure of cholera toxin and the closely related Escherichia coli heat-labile enterotoxin . Such potential drugs can be targeted either at inhibiting the toxin's receptor binding site or at blocking the toxin's intracellular catalytic activity . Study of the Vibrio cholerae and E . coli toxins serves at the same time as an example of a general approach to structure-based vaccine design . These toxins exhibit a remarkable ability to stimulate the mucosal immune system, and early results have suggested that this property can be maintained by engineered fusion proteins based on the native toxin structure . The challenge is thus to incorporate selected epitopes from foreign pathogens into the native framework of the toxin such that crucial features of both the epitope and the toxin are maintained . That is, the modified toxin must continue to evoke a strong mucosal immune response, and this response must be directed against an epitope conformation characteristic of the original pathogen.

Immunology, 1994 Oct, 83(2), 288 - 94
Effect of injected yeast glucan on the activity of macrophages in Atlantic salmon, Salmo salar L., as evaluated by in vitro hydrogen peroxide production and phagocytic capacity; Brattgjerd S et al.; A prepared polysaccharide from the cell wall of yeast, M-Glucan, has previously been demonstrated to have immunostimulatory effects in salmonids as observed by enhanced in vivo non-specific disease resistance in Atlantic salmon, Salmo salar L., and increased in vitro bactericidal activity of rainbow trout, Oncorhynchus mykiss (Walbaum), macrophages . In the present study M-Glucan was injected intraperitoneally into Atlantic salmon and the effect on core components in the non-specific part of the immune system was observed . The hydrogen peroxide (H2O2) production of isolated head kidney macrophages from glucan-injected fish was measured 3 and 6 weeks after M-Glucan treatment and was increased at both time-points upon phorbol myristate acetate-(PMA) triggering . Without PMA triggering the difference was only significant 3 weeks after glucan injection when compared to a control group injected with saline . In a phagocytic assay with macrophages and Vibrio salmonicida the initial uptake of bacteria was elevated at both 3 and 6 weeks after glucan treatment . There was no significant difference when uptake of another fish pathogenic bacteria, Renibacterium salmoninarum, was studied . Treatment of Atlantic salmon with M-Glucan also resulted in enhanced serum lysozyme activity in week 3 of the experimental period . The results indicate that M-Glucan elevates the activity of the non-specific part of the immune system and the use of M-Glucan as an immunostimulant is discussed.

Mol Microbiol, 1994 Oct, 14(2), 255 - 62
Proximal and distal sites bind LuxR independently and activate expression of the Vibrio harveyi lux operon; Miyamoto CM et al.; The LuxR regulatory protein of Vibrio harveyi as well as the autoinducer molecule, N-(3-hydroxybutanoyl) homoserine lactone, are known to be required for expression of luminescence . Although LuxR has been implicated in the activation of the promoter of the lux operon of V . harveyi, and can bind to two distinct sites upstream of the transcription initiation start site, its mode of action is unknown . In the present experiments, mobility shift assays were used to demonstrate that LuxR bound to the distal and proximal sites in an independent rather than co-operative interaction with a much tighter binding to the distal site . Deletion mutation analyses of DNA upstream of the lux promoter followed by transconjugation into V . harveyi in trans using the chloramphenicol acetyltransferase (cat) gene as a reporter demonstrated, however, that the proximal site for LuxR was absolutely critical for promoter activation while the distal LuxR site was only necessary for maximum activation . This result was confirmed by mutation of the proximal site which blocked activation of the lux promoter and binding of LuxR to this site, but did not prevent LuxR binding to the distal site.

Mol Microbiol, 1994 Oct, 14(1), 17 - 29
Transcriptional control of toxT, a regulatory gene in the ToxR regulon of Vibrio cholerae; Higgins DE et al.; Co-ordinate expression of many virulence genes in Vibrio cholerae is under the control of the ToxR and ToxT proteins . These proteins function in a regulatory cascade in which ToxR is required to activate toxT, and ToxT activates virulence genes . The precise mechanism for ToxR activation of toxT is unknown, but data presented in this report suggest a direct involvement of ToxR . Primer extension and gene fusion analyses identified a ToxR-regulated promoter directly upstream of toxT, immediately following a region of inverted repeats capable of terminating transcription . Gel mobility shift studies indicate that ToxR binds DNA within the inverted repeat region, yet preliminary evidence suggests that ToxR binding alone is not sufficient for activation of toxT . Possible mechanisms of ToxR-dependent toxT expression are discussed.

J Fla Med Assoc, 1994 Oct, 81(10), 676 - 8
Non 0-1 Vibrio cholerae septicemia and culture negative neutrocytic ascites in a patient with chronic liver disease; Poulos JE et al.; Non 0-1 Vibrio cholerae infection is often associated with ingestion of contaminated seafood and its common presentation is gastroenteritis . Septicemia may be found in immunocompromised hosts resulting in mortality approaching 50% . A case is reported of non 0-1 Vibrio cholerae infection presenting with septicemia in a patient with neutrocytic ascites suggestive of spontaneous bacterial peritonitis.

Rev Latinoam Microbiol, 1994 Oct-Dec, 36(4), 295 - 306
{Polymerase chain reaction (PCR) for the identification of toxigenic Vibrio cholerae O1 in oysters}; Rodriguez-Angeles MG et al.; PCR was made with ctx2 (CGG GCA GAT TCT AGA CCT CCT G) y ctx3 (CGA TGA TCT TGG AGC ATT CCC AC) primers for subunit A of cholera toxin, 30 cycles of temperature on samples of 50 g of oysters added in 450 ml of peptone alcaline water that were inoculated with 15 x 10(6), 0.75 x 10(6) and 0.15 x 10(6) CFU/ml of toxigenic 6707 V . cholerae O1 reference strain . The samples were tested by three microbiological methods: INDRE's method uses 1 x 10(-1) dilution of sample, two fold pass to peptone alcaline water pH 9 incubated 18 h and 6 h at 37 degrees C, the Food and Drugs Administration (FDA) method uses 10(-1) to 10(-6) dilutions of sample, 6 h incubation and reincubation for 18 h at 37 and 42 degrees C and the Mexican laboratories (LMD) with 10(-4) to 10(-3) dilutions, the samples were incubated for 6 h and then reincubated for 18 h at two temperatures 37 and 42 degrees C . The PCR by INDRE's method was positive with 3 x 10(2) CFU/ml/g oyster . In the FDA's method the PCR detected DNA in 10(-4) dilution with 3 x 10(1) CFU/ml/g oyster and in LMD's method the PCR was positive in 10(-3) with 3 CFU/ml/g oyster . The results of the PCR were obtained between 5-6 h, and later V . cholerae O1 was isolated by three microbiological methods . The PCR reproducibility was better on DNA sample diluted 1:4 and 10 microliters of sample increased from 1:1000 to 1:10000 the sensitivity of PCR.

Rev Latinoam Microbiol, 1994 Oct-Dec, 36(4), 283 - 93
{Identification of Vibrio cholerae O1 by flow cytometry}; Alvarado-Aleman FJ et al.; A total of 72 peptonated water samples suspected of carrying Vibrio cholerae were assessed by laser flow cytometry (LFC) and compared with positive culture . We used a direct fluorescence technique using polyclonal (PolAb) and monoclonal antibodies (MoAb) conjugated to fluorescein . The PolAb were able to detect 33 positive samples . A clear difference among the 20 positive samples was found with only three V . cholerae O1 false negatives when MoAb were used whereas all 13 V . cholerae Non O1 samples were detected . The correlation index comparing control autofluorescence with peptonated water samples show a R = 0.69, versus 0.96 with pure V . cholerae O1 strains . Our data suggest that the LFC technique is able to recognize V . cholerae O1 from a mixture of microorganisms with high sensitivity and specificity in a few hours.

Rev Latinoam Microbiol, 1994 Oct-Dec, 36(4), 277 - 81
{Cytotoxic effect of Vibrio cholerae non-O1 on Vero cells}; Figueroa-Arredondo P et al.; At the present time there is still in Mexico a diarrhoeal outbreak due to Vibrio cholerae O1 . In INDRE we have isolated from the same outbreak last year (jan-apr), 70 strains of Vibrio cholerae Non-O1 . These were isolated from patients with a diarrhoeal illness different from cholera . Patients were of different ages and sex, and from various geographic areas . The isolated strains were confirmed by serological agglutination test with polyclonal antisera, and they neither belong to O1 serogroup or O139 . We assayed all the 70 strains in Vero cells, searching for cytotoxic effect, probably attributed to cholera toxin, or any other toxin . The strains were screened by PCR for cholera toxin gene detection, and negative results were obtained . We have found only one CT-producer strain, but it was a rough one so, we are not able to affirm that is not a V . cholerae O1 serotype . Vibrio cholerae Non-O1 strains, tested in Vero cells assay, produced cytotoxic effect within 24 h . It was found that 48/70 strains (66.6%), had cytotoxic activity, showing rounding and then lysis of cells . From our results we concluded that this cytotoxic effect, is not cholera toxin related, instead we propose it could be due to an unknown virulence factor, probably a different toxin in mexican Vibrio cholerae Non-O1 strains.

Rev Latinoam Microbiol, 1994 Oct-Dec, 36(4), 273 - 6
{Evaluation of the ELISA method for cholera toxin determination in Vibrio cholerae cultures}; Gonzalez-Bonilla C et al.; ELISA test was evaluated in 503 cultures of Vibrio cholerae O1 y 303 Non-O1 . The cultures were isolated from sewage from different states of Mexico between june 1991 and october 1992 . The sensitivity was 100% and specificity was 96% . Only 12 strains of V . cholerae Non-O1 were positive for CT toxin . When these cultures were confirmed by polymerase chain reaction (PCR) for cholera toxin, the results were negative . ELISA test is a good alternative to be used for toxin production in cultures of V . cholerae, it needs confirmation only with O1 negative and Non-O1 positive reactions.

Rev Latinoam Microbiol, 1994 Oct-Dec, 36(4), 263 - 71
{Cytotonic and cytotoxic effect of cholera toxin on Vero cells and its relation to PCR}; Rodriguez-Angeles MG et al.; We studied 40 Vibrio cholerae strains: 16 from stool, 16 from sewage and 8 from food . The serotypes were Inaba in 21 strains, 8 Ogawa strains and 11 V . cholerae non-O1 . PCR was made with ctx2 and ctx3 primers with 25 cycles of temperature: 1 min at 94 degrees C, 1 min at 60 degrees C and 1 min at 72 degrees C . 24 V . cholerae strains were positive: 18/24 Inaba y 6/24 Ogawa . PCR was negative for 16 strains: 3 Inaba serotype, 2 Ogawa y 11 V . cholerae non-O1 . In Vero culture cells 18 strains were cytotonic, 21 cytotoxic and 1 strain was negative . ELISA was positive for 11 strains with PCR positive . The PCR sensitivity was 95.83% compared with culture cells . V . cholerae O1 produced cytotoxic effect on Vero culture cells, maybe related to ACE factor . Colony blot was made with a specific probe labeled with digoxigenin and it could detect 4 Vibrio cholerae toxigenic strains with PCR negative . All V . cholerae Non O1 strains were PCR negative.

Rev Latinoam Microbiol, 1994 Oct-Dec, 36(4), 253 - 6
{Seroepidemiology of cholera in Mexico}; Gonzalez-Bonilla C et al.; Antibodies against Vibrio cholerae were determined in 2352 serum samples obtained from patients with clinical diagnosis of cholera . Samples from their contacts and from healthy people living in the same communities were also analyzed . Vibriocidal antibodies with titers 1:160 or higher were observed in 25% of the samples . An increase of vibriocidal and antitoxin antibody titers were observed in 56 to 60% of the patients in which paired samples were available, one obtained in the acute phase of the disease and the other in the convalescence, confirming the diagnosis of cholera . Differences in the antibody titers were noticed when comparing the serotype according to the geographic area and the season of the year.

Rev Latinoam Microbiol, 1994 Oct-Dec, 36(4), 243 - 51
{Phenotypic and genotypic characterization of Vibrio cholerae O1}; Giono-Cerezo S et al.; We made 52180 tests for isolation and identification of toxigenic V . cholerae O1 from rectal swabs and reference strains . We isolated 17.6% V . cholerae O1 strains in 1991, 43.5% in 1992 and 38.9% in 1993 . The main serovar in 1991 was Inaba, whereas in 1993 a similar percentage was serovar Ogawa . The phenotype of V . cholerae strains was determined by hemolysis test, Voges-Proskauer test, polymyxin B resistance and phages 4 and 5 resistance . All of the mexican strains were El Tor . There were 2.9-0.75% hemolytic strains from 1991 to 1993, but they were negative when the test was made in tube with human erythrocytes . The resistotypes were performed in 24526 selected strains by Kirby-Bauer method and MIC tests . All of the strains were sensitive, except more than 100 strains isolated in Veracruz that were resistant to tetracycline and doxycycline . Detection of cholera toxin was made by ELISA and on culture of Vero and CHO cells . All the V . cholerae O1 strains were toxigenic . The genotype was determined by PCR and ribotyping . The PCR amplified one 564 pb fragment on V . cholerae O1 . The ribotypes of mexican strains were 5 and 6a.

Vaccine, 1994 Oct, 12(13), 1231 - 7
Production of Vibrio cholerae ghosts (VCG) by expression of a cloned phage lysis gene: potential for vaccine development; Eko FO et al.; The protein E-specific lysis mechanism of the Escherichia coli-specific bacteriophage PhiX174 was employed to produce Vibrio cholerae ghosts (VCG) . VCG consist of both rounded and collapsed cells that have lost their cytoplasmic contents through an E-specific hole in the cell envelope . These ghosts are proposed as non-living material for immunization against cholera . A specific membrane anchor sequence was used to insert the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) fusion protein into the cell envelope of V . cholerae . The identity of the expression products was confirmed by Western blot analysis employing an RT-specific monoclonal antibody . HIV-1 RT was chosen as a model for the purpose of evaluating heterologous gene expression in V . cholerae and the carrier potential of VCG . Intraperitoneal immunization of mice was used to evaluate the immunogenic potential of VCG . Preliminary results showed significant seroconversions to intact whole-cell vibrio antigens in mice immunized with VCG or a heat-killed whole-cell vibrio preparation.

J Biotechnol, 1994 Sep 15, 37(1), 33 - 7
A model system for the continuous production of a heterologous protein using a novel secretion promoting factor which operates in Escherichia coli; Tokugawa K et al.; The 'PAS factor' whose gene has been cloned from a species of Vibrio, is a novel protein secretion factor which is functional in Escherichia coli cells . To demonstrate that practical use of the PAS factor gene is possible, we have constructed a model secretion vector 'pAS23' . Using this system, beta-lactamase was produced and secreted into the medium of a continuous culture system, after optimization of culture conditions.

J Bacteriol, 1994 Sep, 176(18), 5631 - 8
Identification, cloning, and sequencing of a gene required for ferric vibriobactin utilization by Vibrio cholerae; Butterton JR et al.; Chromosomal DNA downstream of the Vibrio cholerae ferric vibriobactin receptor gene, viuA, was cloned and sequenced, revealing an 813-bp open reading frame encoding a deduced protein of 271 amino acids . In vitro transcription-translation of this DNA confirmed expression of a protein of the expected size . A deletion mutation of this gene, viuB, was created in the classical V . cholerae strain O395 by in vivo marker exchange . By cross-feeding studies, this mutant was unable to utilize exogenous ferric vibriobactin but synthesized the siderophore normally; synthesis of siderophore by the mutant was also confirmed by the Arnow assay . Complementation of the mutant with a plasmid encoding only viuB restored ferric vibriobactin utilization to normal . Unexpectedly, hydropathicity analysis of ViuB did not reveal a signal sequence or transmembrane domain, suggesting that ViuB is not a periplasmic or membrane protein but may be a cytoplasmic protein involved in ferric vibriobactin uptake and processing, perhaps analogous to the Escherichia coli protein Fes . ViuB was not, however, homologous to Fes or to other proteins in the database . Complementation studies revealed that the cloned V . cholerae viuB gene could complement an E . coli fes mutant but that the cloned E . coli fes gene could not complement a V . cholerae viuB mutant . Northern (RNA) blot analysis of RNA from wild-type V . cholerae grown in high- and low-iron media revealed a monocistronic viuB message that was negatively regulated by iron at the transcriptional level . The promoter of viuB was located by primer extension and contained a nucleotide sequence highly homologous to the E . coli Fur binding consensus sequence, suggesting that expression of viuB is under the control of the V . cholerae fur gene.

J Infect Dis, 1994 Sep, 170(3), 701 - 4
The novel epidemic strain O139 is closely related to the pandemic strain O1 of Vibrio cholerae; Berche P et al.; A new Vibrio cholerae serogroup O139 strain of unknown origin recently emerged in India and Bangladesh, causing a major outbreak of cholera . The genetic relationship between this epidemic strain and the O1 strain responsible for the 7th pandemic of cholera was studied by analyzing the DNA polymorphism of V . cholerae by pulsed-field gel electrophoresis and arbitrarily primed polymerase chain reaction . The restriction patterns of the reference strain O139 Bengal and 10 wild O139 strains isolated early in the Indian outbreak strikingly resemble that of the pandemic O1 strain of V . cholerae El Tor, thus suggesting a close genetic relationship among these strains . This similarity contrasts with the genetic heterogeneity of sporadic non-O1 strains isolated in various parts of the world . Study results strongly suggest that the new epidemic O139 strain is closely related to and might be derived from the pandemic O1 strain of V . cholerae.

J Bacteriol, 1994 Sep, 176(17), 5450 - 8
Identification of VCR, a repeated sequence associated with a locus encoding a hemagglutinin in Vibrio cholerae O1; Barker A et al.; We have determined the nucleotide sequence of a 6.3-kb BamHI fragment of the chromosome of Vibrio cholerae 569B that includes the sequence of the mannose-fucose-resistant hemagglutinin reported previously (V.L . Franzon, A . Barker, and P . A . Manning, Infect . Immun . 61:3032-3037, 1993) . This region contains nine copies of a 124-bp direct repeat, here named VCR, of imperfect dyad symmetry, that are shown by Southern hybridization to occur at least 60 to 100 times in the V . cholerae O1 chromosome . Large-scale chromosomal mapping suggests that the repeats are confined to about 10% of the chromosome . Related sequences are also found in non-O1 V . cholerae but not in other members of the family Vibrionaceae . However, VCR is unrelated to other previously described repetitive sequences.

Infect Immun, 1994 Sep, 62(9), 3859 - 63
Vibrio cholerae non-O1 serogroup associated with cholera gravis genetically and physiologically resembles O1 E1 Tor cholera strains; Hall RH et al.; Until recently, only Vibrio cholerae strains of the O1 serogroup have been associated with epidemic cholera . In December 1992, an outbreak of cholera gravis in Vellore, India, was attributed to a new serogroup of V . cholerae recently designated O139 . Serogroup O139 cholera has since spread to 13 countries and has reached pandemic proportions . Serogroup O139 cholera evades immunity to O1 cholera and is not detected by the standard O1 antigen test . Understanding the origins of O139 cholera and determining the relatedness of O139 to O1 cholera are necessary to device strategies for detecting, reporting, and controlling this new pandemic . In order to determine the origins of this novel cholera serogroup, O139 was analyzed for virulence genes, for virulence proteins and their regulation, and for its genomic background . We found that O139 and O1 V . cholera strains of the E1 Tor biotype possess highly homologous virulence genes encoding cholera toxin and toxin-coregulated pili and that the regulation of virulence protein expression likewise was indistinguishable between O139 and O1 . Pulsed-field gel electrophoresis (PFGE) revealed the restriction digest pattern of O139 strains to be closely related to that of O1 serogroup E1 Tor biotype cholera strains from the Indian subcontinent . However, PFGE showed minor differences among individual O139 cholera isolates, suggesting that O139 V . cholerae is evolving.

Vaccine, 1994 Sep, 12(12), 1078 - 82
Immunological memory after immunization with oral cholera B subunit--whole-cell vaccine in Swedish volunteers; Jertborn M et al.; The capacity of peroral immunization with either two or three doses of B subunit-whole cell (B-WC) cholera vaccine to induce immunological memory was examined in Swedish volunteers by testing the immune responses to a single dose of B-WC vaccine given 10 months after the initial immunization . Antibody responses in serum and antibody-secreting cell (ASC) responses in peripheral blood were studied, since these responses seem to reflect the gut mucosal IgA immune responses after oral immunization with B-WC vaccine . Previously immunized volunteers responded to a single dose of B-WC vaccine more frequently and with higher levels of IgA and IgG antitoxin antibodies as well as vibriocidal antibodies in serum than did previously unvaccinated controls . The IgA-ASC responses to cholera toxin B subunit were also higher in primed volunteers than in controls . Two doses of B-WC vaccine were as effective as three doses in inducing immunological memory for cholera immunity . A new B-WC cholera vaccine based on recombinant B subunit had the same capacity as the first generation of B-WC vaccine to induce immunological memory for cholera antitoxin immunity.

Kansenshogaku Zasshi, 1994 Sep, 68(9), 1068 - 74
{Serotypes of urease producing Vibrio parahaemolyticus and their relation to possession of tdh and trh genes}; Suzuki N et al.; We analysed 467 isolates of Vibrio parahaemolyticus for possession of tdh/trh gene in comparison with urease production and serotypes . Strains possessing tdh+/trh-, tdh+/trh+, tdh-/trh-, and tdh-/trh- show positive urease production 2.1, 100, 65.7, 100%, respectively . Serotypes of O1:K69, O3:K6, O3:K72, O6:K18, O6:K46 and O1:KUT were frequently positive (100% except 91.7% of the latest one) in urease production . All isolates of O3:K6 prossessed trh, whereas all isolates of certain serotypes including O1:K69 and O3:K72 possessed both trh and trh and tdh genes . Among these, most of O1:K69 and O3:K72 were urease producer . From these results, we speculate that urease production is closely related to the presence of the trh gene or/and lesser production of TDH . We also found the new combination of serovar, O3:K25, O4:K37 and O13:K72 in Kanagawa phenomenon-positive strains.

Indian J Med Res, 1994 Sep, 100, 95 - 7
Endemicity of cholera among rural areas of Loni, Ahmednagar district of Maharashtra; Jain RC et al.; A total of 130 Vibrio cholerae strains isolated during November 1989 to December 1992 from the rural population of Loni areas--Ahmednagar district of Maharashtra were characterised . Of these isolates, 124 were El tor vibrios serotype Ogawa, and 6 were El tor vibrios serotype Inaba . One hundred twenty two strains belonging to T4 phage, while 8 strains of El tor vibrio serotype Ogawa were untypable . All the strains isolated, showing haemolytic and non-haemolytic colony variants of El tor V . cholerae, and had resistance of one or more antibiotics . Maximum incidence was observed in November-December, the illness had a mild onset and no fatality was reported.

Indian J Med Res, 1994 Sep, 100, 93 - 4
Changing bacteriological profile of cholera in Nagpur, 1991-93; Agarwal V et al.; In Nagpur (Maharashtra) during the period 1991-93, Vibrio cholerae serogroup 01 predominated in 1991 (94.7%) and 1992 (86.4%) but significantly declined in 1993 (10.7%) . Serogroups 02-0138 were infrequently encountered . A new strain V . cholerae serogroup 0139 emerged in 1993 and accounted for 89.3 per cent of the total vibrios isolated in the year . Replacement of the endemic 01 strain by the new 0139 strain was observed.

Appl Environ Microbiol, 1994 Sep, 60(9), 3483 - 4
Effect of time and temperature on multiplication of Vibrio vulnificus in postharvest Gulf Coast shellstock oysters; Cook DW; After harvest, shellstock oysters stored under controlled temperatures of 10, 13, and 18 degrees C and at ambient outside air temperature (23 to 34 degrees C) were sampled after 12 and 30 h for Vibrio vulnificus . At 13 degrees C and below, V . vulnificus failed to multiply in the oysters . In oysters held at 18 degrees C for 30 h and under ambient conditions for 12 and 30 h, V . vulnificus numbers were statistically greater (P < 0.05) than those in oysters at harvest . These data indicate that endogenous V . vulnificus can multiply in unchilled shellstock oysters.

Zh Mikrobiol Epidemiol Immunobiol, 1994 Sep-Oct, (5), 37 - 41
{A prognostic model of a cholera epidemic}; Boev BV et al.; A new model for the prognostication of cholera epidemic on the territory of a large city is proposed . This model reflects the characteristic feature of contacting infection by sensitive individuals due to the preservation of Vibrio cholerae in their water habitat . The mathematical model of the epidemic quantitatively reflects the processes of the spread of infection by kinetic equations describing the interaction of the streams of infected persons, the causative agents and susceptible persons . The functions and parameters of the model are linked with the distribution of individuals according to the duration of the incubation period and infectious process, as well as the period of asymptomatic carrier state . The computer realization of the model by means of IBM PC/AT made it possible to study the cholera epidemic which took place in Mexico in 1833 . The verified model of the cholera epidemic was used for the prognostication of the possible spread of this infection in Guadalajara, taking into account changes in the epidemiological situation and the size of the population, as well as improvements in sanitary and hygienic conditions, in the city.

J Diarrhoeal Dis Res, 1994 Sep, 12(3), 222 - 4
Distribution of Zonula occludens toxin (zot) gene among clinical isolates of Vibrio cholerae O1 from Bangladesh and Africa; Faruque SM et al.; Seventy-two clinical isolates of Vibrio cholerae O1 from Bangladesh, and 12 and 9 isolates respectively from Tanzania and Nigeria were screened for sequences homologous to zonula occludens toxin (zot) and cholera toxin (ctx) genes . As observed previously, all isolates in the present study also possessed sequences for both toxins which suggested that zot does not occur independent of ctx . It appears that along with the virulence genes located in the "virulence cassette" region of the bacterial chromosome, zot may play a role in the pathogenesis of cholera.

J Diarrhoeal Dis Res, 1994 Sep, 12(3), 214 - 8
Severity of cholera during concurrent infections with other enteric pathogens; Faruque AS et al.; In a clinic-based case-control study in Bangladesh we evaluated whether children with diarrhoea due to V . cholerae O1 in association with other enteric pathogen(s) are likely to manifest more severe disease as indicated by development of moderate or severe dehydration . Children with moderate or severe dehydration were defined as cases and those with no dehydration were controls; both cases and controls had acute diarrhoea . A systematic sample of 268 dehydrated cases and 699 nondehydrated controls aged 1-35 months with acute watery diarrhoea of 6 days or less was included . In a multivariate analysis it has been shown that infection with Vibrio cholerae O1 in association with another diarrhoea pathogen (odds ratio = 7.07) was strongly correlated with status of dehydration than those with the V . cholerae O1 infection as a single pathogen (odds ratio = 3.63) . Either group was associated with significant risk of dehydration . The results of the study suggest that more than one enteropathogen may be simultaneously involved in causing severe diarrhoea, and appropriate public health measures to reduce environmental contamination should be beneficia

Mol Microbiol, 1994 Sep, 13(6), 1013 - 20
TcpA pilin sequences and colonization requirements for O1 and O139 vibrio cholerae; Rhine JA et al.; The distribution, characterization and function of the tcpA gene was investigated in Vibrio cholerae O1 strains of the El Tor biotype and in a newly emergent non-O1 strain classified as serogroup O139 . The V . cholerae tcpA gene from the classical biotype strain O395 was used as a probe to identify a clone carrying the tcpA gene from the El Tor biotype strain E7946 . The sequence of the E7946 tcpA gene revealed that the mature El Tor TcpA pilin has the same number of residues as, and is 82% identical to, TcpA of classical biotype strain O395 . The majority of differences in primary structure are either conservative or clustered in a manner such that compensatory changes retain regional amino acid size, polarity and charge . In a functional analysis, the cloned gene was used to construct an El Tor mutant strain containing an insertion in tcpA . This strain exhibited a colonization defect in the infant mouse cholera model similar in magnitude to that previously described for classical biotype tcpA mutants, thus establishing an equivalent role for TCP in intestinal colonization by El Tor biotype strains . The tcpA analysis was further extended to both a prototype El Tor strain from the Peru epidemic and to the first non-O1 strain known to cause epidemic cholera, an O139 V . cholerae isolate from the current widespread Asian epidemic . These strains were shown to carry tcpA with a sequence identical to E7946 . These results provide further evidence that the newly emergent non-O1 serogroup O139 strain represents a derivative of an El Tor biotype strain and, despite its different LPS structure, shares common TCP-associated antigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Food Addit Contam, 1994 Sep-Oct, 11(5), 549 - 58
Antimicrobial action of some GRAS compounds against Vibrio vulnificus; Sun Y et al.; Vibrio vulnificus is a bacterium indigenous to estuarine waters and is known to be a significant human pathogen . Infections are generally associated with the consumption of raw oyster . In an attempt to identify possible antimicrobial agents against this organism that might be used in foods, ten compounds that are generally recognized as safe (GRAS) by the FDA were tested against both the opaque and translucent morphotypes of V . vulnificus . Eight of those compounds had a lethal effect for both morphotypes of this bacterium . Diacetyl had the lowest lethal concentration (50 ppm) of the GRAS compounds tested within 24 h . Lactic acid and butylated hydroxyanisole possessed lethal activities at 300 ppm and 400 ppm, respectively, within 3 h . The mode of action of lactic acid against V . vulnificus appears to be an effect primarily of pH, while the antimicrobial activities of diacetyl and BHA appeared not to be affected by pH . No significant differences were found for opaque to translucent, or from translucent to opaque switching, in examining the possible effects of the GRAS compounds on colonial morphology.

Immunol Lett, 1994 Sep, 42(1-2), 67 - 73
Binding of serum autoantibodies to sialidase-treated tracheal epithelial cells . Determination of autoantibodies isotypes in normal and influenza virus infected guinea pig sera; Nahori MA et al.; Cultured epithelial cells isolated from guinea pig trachea were treated with Vibrio cholerae sialidase . The treatment was not cytotoxic and resulted in membrane desialylation as assessed by measurement of sialic acids released, along with an increased fixation of the galactose-specific lectin peanut agglutinin . After incubation in serum from normal guinea pigs, membrane-bound immunoglobulins were detected using peroxidase-labelled antibodies . Sialidase-treated cells bound significantly more IgM than controls (P < 0.0005), whereas binding of IgG was not significantly different between treated and untreated cells (0.1 < P < 0.375); IgA were never detected . In influenza-infected guinea-pigs, as assessed by reactivity with peanut agglutinin, the tracheal and lung epithelium, as well as alveolar cells were hyposialylated . In these animals, the level of serum IgG autoantibodies capable to bind sialidase treated cultured cells increased, while the level of IgM autoantibodies did not change . These autoantibodies may participate in cellular dysfunctions and modified bronchoreactivity that occur during infection of the respiratory tract by sialidase-producing microorganisms, either through activation of the complement system, or subsequently to their reaction with cells expressing membrane complement and/or Fc receptors.

Rev Med Chil, 1994 Sep, 122(9), 986 - 92
{Characterization of a multiresistant strain of Vibrio cholerae O1, isolated from a case of cholera in Chile}; Castillo L et al.; This report characterizes a multiresistant Vibrio Cholerae O1 strain, isolated from a patient with cholera, and investigates the mechanism of resistance . The analyzed strain was resistant to tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole . The resistance was mediated by a 101 megadalton plasmid that was transferred to the resultant of a conjugation assay between the multiresistant V . Cholerae strain and E . coli C-600 used as receptor strain, that acquired the triple resistance of the parental strain . The resistant V . cholerae strain had a Ogawa serotype, El Tor biotype and toxigenic capacity, demonstrated by ELISA and latex agglutination techniques . The biochemical features of the strain were identical to those of susceptible strains, except for the resistance to 10 and 150 ug o 129 vibriostatic factor . The emergence of plasmid mediated resistance to drugs of choice in the treatment of cholera must alert Chilean and Latin American health authorities, considering the cholera will continue affecting the region.

FEMS Microbiol Lett, 1994 Sep 1, 121(3), 321 - 5
Vibrio vulnificus may produce a metalloprotease causing an edematous skin lesion in vivo; Miyoshi S et al.; Vibrio vulnificus, an opportunistic human pathogen, secretes a metalloprotease which has been suspected of being the causative factor for edematous skin lesions . The antibody against alpha-macroglobulin, the sole plasma inactivator of V . vulnificus metalloprotease, delayed clearance of the protease administered into dorsal skin, and increased the edema-forming ability of living bacterial cells . The derivative of the protease, which is resistant to the inactivating action of alpha-macroglobulin, was not excluded from the dorsal skin . Furthermore, the vibrio inoculated into the mammalian serum was found to produce the protease in adequate amounts . These results suggest that V . vulnificus secretes a metalloprotease into the interstitial-tissue space, resulting in the development of an edematous skin lesion, and that the protease is immediately inactivated by alpha-macroglobulin and subsequently excluded.

Biochim Biophys Acta, 1994 Aug 24, 1194(1), 166 - 70
Inhibitory mechanism of Ca2+ on the hemolysis caused by Vibrio vulnificus cytolysin; Park JW et al.; Calcium in millimolar concentrations protected mouse erythrocytes from hemolysis caused by Vibrio vulnificus cytolysin without affecting the release of intracellular K+ from the cells . This effect was maximal at 25 mM CaCl2 . The protection was not absolute and could be partially overcome by increased concentrations of cytolysin . Calcium failed to block both the binding and oligomer formation of cytolysins on the erythrocyte membrane . After pore formation, the continued presence of calcium is required for the prevention of hemolysis . There was hardly any inflow of calcium into the erythrocytes through pores as measured by 45Ca2+ uptake . The presence of calcium after the abolition of Ca2+ gradient by ionomycin cannot inhibit the hemolysis caused by cytolysin . These results suggest that calcium exerts its major inhibitory effect on V . vulnificus cytolysin-induced hemolysis as an osmotic protectant, and that cytolysin may become an useful tool for permeabilizing cells selectively for small ions such as potassium or sodium while preventing the Ca2+ flow.

Biochemistry, 1994 Aug 16, 33(32), 9382 - 8
Structure of a myristoyl-ACP-specific thioesterase from Vibrio harveyi; Lawson DM et al.; The crystal structure of a myristoyl acyl carrier protein specific thioesterase (C14ACP-TE) from a bioluminescent bacterium, Vibrio harveyi, was solved by multiple isomorphous replacement methods and refined to an R factor of 22% at 2.1-A resolution . This is the first elucidation of a three-dimensional structure of a thioesterase . The overall tertiary architecture of the enzyme resembles closely the consensus fold of the rapidly expanding superfamily of alpha/beta hydrolases, although there is no detectable homology with any of its members at the amino acid sequence level . Particularly striking similarity exists between the C14ACP-TE structure and that of haloalkane dehalogenase from Xanthobacter autotrophicus . Contrary to the conclusions of earlier studies {Ferri, S . R., & Meighen, E . A . (1991) J . Biol . Chem . 266, 12852-12857} which implicated Ser77 in catalysis, the crystal structure of C14ACP-TE reveals a lipase-like catalytic triad made up of Ser114, His241, and Asp211 . Surprisingly, the gamma-turn with Ser114 in a strained secondary conformation (phi = 53 degrees, psi = -127 degrees), characteristic of the so-called nucleophilic elbow, does not conform to the frequently invoked lipase/esterase consensus sequence (Gly-X-Ser-X-Gly), as the positions of both glycines are occupied by larger amino acids . Site-directed mutagenesis and radioactive labeling support the catalytic function of Ser114 . Crystallographic analysis of the Ser77-->Gly mutant at 2.5-A resolution revealed no structural changes; in both cases the loop containing the residue in position 77 is disordered.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1994 Aug 15, 121(2), 181 - 8
Role of iron in the pathogenicity of Vibrio damsela for fish and mammals; Fouz B et al.; The ability to obtain iron of 14 isolates of Vibrio damsela with different degrees of virulence for mice and turbot (Scophthalmus maximus) has been evaluated in artificial and natural iron-restricted environments . All strains were capable of utilizing haemoglobin (Hb) and ferric ammonium citrate (FAC) as the sole iron sources in vitro . However, only virulent V . damsela strains were able to resist the bacteriostatic and bactericidal effects of human and turbot sera, their growth being enhanced by the addition of Hb and FAC . The inhibitory effect of these sera on the growth of the non-pathogenic strain (ATCC 35083), however, was reversed by heat treatment (56 degrees C for 60 min) . The role of iron-availability on the virulence was investigated in iron-overloaded animals . The iron-treatment before the infection resulted in a significant reduction in the LD50 of virulent strains . This fact demonstrates a positive correlation between iron availability in host fluids and degree of virulence in the species Vibrio damsela.

J Mol Biol, 1994 Aug 12, 241(2), 283 - 7
Crystallization and preliminary crystallographic analysis of NADPH:FMN oxidoreductase from Vibrio harveyi; Tanner J et al.; Crystals of NADPH:FMN oxidoreductase from Vibrio harveyi have been obtained and characterized by X-ray diffraction . This enzyme plays a role in the generation of light in luminescent bacteria by providing reduced FMN to luciferase . Large, high quality crystals were grown using polyethylene glycol 6000 at pH 7.0 . They crystallize in the monoclinic space group P2(1) with cell dimensions a = 51.2 A, b = 85.9 A, c = 58.1 A, beta = 109.3 degrees, and diffract to 1.8 A . We expect two molecules per asymmetric unit . High resolution data sets have been recorded and a search is under way for heavy-atom derivatives.

J Biol Chem, 1994 Aug 12, 269(32), 20785 - 90
Biosynthesis of poly-3-hydroxybutyrate in the luminescent bacterium, Vibrio harveyi, and regulation by the lux autoinducer, N-(3-hydroxybutanoyl)homoserine lactone; Sun W et al.; Poly-3-hydroxybutyrate (PHB), a biopolymer of important commercial applications, is found in a wide range of Gram-negative and Gram-positive bacteria and cyanobacteria . The present study has resulted in the identification of PHB in the luminescent marine bacteria, Vibrio harveyi, in spite of it being previously classified as PHB-negative . PHB granules with distinct membranes were detected by electron microscopy after fixation and staining of V . harveyi cells with malachite green . Analyses by gas chromatography, nuclear magnetic resonance, infrared, and ultraviolet spectroscopy clearly established the presence of PHB . The synthesis of PHB in V . harveyi was found to be under cell density regulation with the levels increasing from 0 (< 0.2) to 26 mg of PHB/g of dry cell weight during growth in a manner analogous to the induction of luminescence in this bacteria . Moreover, synthesis of PHB in V . harveyi was shown to be controlled by the lux autoinducer, N-(3-hydroxybutanoyl)homoserine lactone, providing not only a potential link between luminescence and PHB production but also showing that the lux autoinducer acts as a general signal transductant . These results have also extended the role of homoserine lactones in metabolic regulation to include the control of synthesis of potential energy reserves.

Mol Gen Genet, 1994 Aug 2, 244(3), 295 - 302
Gene sequence of recA+ and construction of recA mutants of Vibrio cholerae; Stroeher UH et al.; The recA+ gene of Vibrio cholerae O1 has been cloned, its nucleotide sequence determined and the product characterized . A deletion mutation was constructed in the recA gene and mutants showed the typical sensitivity to UV and to DNA-damaging agents, as well as an inability to mediate homologous DNA recombination . The chromosomal recA deletion mutants in V . cholerae do not show altered virulence in the infant mouse cholera model and are thus ideal strains for use in complementation studies.

Appl Environ Microbiol, 1994 Aug, 60(8), 3020 - 2
Urea hydrolysis can predict the potential pathogenicity of Vibrio parahaemolyticus strains isolated in the Pacific Northwest; Kaysner CA et al.; The ability of some strains of Vibrio parahaemolyticus to hydrolyze urea (uh+) can be used as a marker to predict which strains isolated from molluscan shellfish harvested in the Pacific Northwest are potentially pathogenic . The thermostable direct hemolysin-producing (TDH+) characteristic is a marker that is correlated with potential pathogenicity, and all of the TDH+ strains that we have isolated have been found to be uh+ . Most of the uh+ strains belong to somatic antigen groups O3, O4 and O5 . TDH+ strains are usually members of groups O4 and O5 . The strains most often associated with human illness are members of the uh+, O4 group . The test for urease production is a simple screening test that can be helpful in predicting which strains are potentially pathogenic.

J Bacteriol, 1994 Aug, 176(16), 5116 - 22
Structural and functional analyses of mutant Fur proteins with impaired regulatory function; Wertheimer AM et al.; Vibrio anguillarum Fur mutants, 775met9 and 775met11, were characterized . V . anguillarum 775met9 had a change of D to G at position 104 located in the carboxy terminus resulting in impaired Fur activity.Computer analysis predicts perturbation of an alpha-helix in the carboxy terminus which may interfere with Fur protein conformation . Strain 775met11 had a change in the start codon resulting in no protein synthesis . The mutants are unstable, and reversion to the wild type occurs frequently.

J Bacteriol, 1994 Aug, 176(16), 5108 - 15
Vibrio cholerae fur mutations associated with loss of repressor activity: implications for the structural-functional relationships of fur; Lam MS et al.; We used the Vibrio cholerae Fur protein as a model of iron-sensitive repressor proteins in gram-negative bacteria . Utilizing manganese mutagenesis, we isolated twelve independent mutations in V . cholerae fur that resulted in partial or complete loss of Fur repressor function . The mutant fur genes were recovered by PCR and sequenced; 11 of the 12 contained point mutations (two of which were identical), and one contained a 7-bp insertion that resulted in premature truncation of Fur . All of the mutants, except that containing the prematurely truncated Fur, produced protein by Western blot (immunoblot) analysis, although several had substantially smaller amounts of Fur and two made an immunoreactive protein that migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Nine of the 11 point mutations altered amino acids that are identical in all of the fur genes sequenced so far, suggesting that these amino acids may play important structural or functional roles in Fur activity . Eight of the point mutations occurred in the amino-terminal half of Fur, which is thought to mediate DNA binding; most of these mutations occurred in conserved amino acids that have been previously suggested to play a role in the interaction between adjacent alpha-helices of the protein . Three of the point mutations occurred in the carboxy-terminal half of Fur, which is thought to bind iron . One mutation at histidine-90 was associated with complete loss of Fur function; this amino acid is within a motif previously suggested as being involved in iron binding by Fur . The fur allele mutant at histidine-90 interfered with iron regulation by wild-type fur in the same cell when the mutant allele was present at higher copy number; wild-type fur was dominant over all other fur mutant alleles studied . These results are analyzed with respect to previous models of the structure and function of Fur as an iron-sensitive repressor.

J Bacteriol, 1994 Aug, 176(15), 4757 - 60
Construction and characterization of an isogenic mutant of Vibrio parahaemolyticus having a deletion in the thermostable direct hemolysin-related hemolysin gene (trh)
Xu M, Yamamoto K, Honda T, Ming X {corrected to Xu M.
A mutant, Vp-MX02, having a deletion in the gene (trh) for the thermostable direct hemolysin-related hemolysin (TRH) was constructed by double-crossover gene conversion from a TRH-producing Vibrio parahaemolyticus strain, TH3996 . The deleted region was the upstream half of trh . Hemolysis was completely lost, and TRH antigen was undetectable in the mutant . Administration of the mutant into ligated rabbit small intestines elicited partial, but apparent, fluid accumulation . These results suggest that the enterotoxicity of TRH-producing V . parahaemolyticus TH3996 may be attributed to the remaining C-terminal of TRH or, more likely, to an unknown virulence factor(s) in addition to TRH.

Infect Immun, 1994 Aug, 62(8), 3299 - 304
A mutant toxin of Vibrio parahaemolyticus thermostable direct hemolysin which has lost hemolytic activity but retains ability to bind to erythrocytes; Tang GQ et al.; A mutant toxin, R7, of thermostable direct hemolysin (TDH) with a single amino acid substitution at glycine 62 was analyzed . The hemolytic activity of R7 decreased to less than 1/1,000 of that of wild-type TDH, and its mouse lethality was undetectable . This mutant toxin, however, showed a marked inhibitory effect on hemolysis by wild-type TDH . Enzyme immunoassay and flow cytometric analysis demonstrated that R7 retained approximately 50% of the ability to bind to erythrocytes compared with that of wild-type TDH, suggesting that its inhibition of hemolysis by wild-type TDH might be due to blocking the binding sites on the erythrocyte membrane . Wild-type TDH affected the erythrocyte membrane by causing an influx of calcium and propidium iodide, while R7 showed no detectable effects of these kinds . These results suggest that hemolysis by TDH consists of at least two steps, binding and postbinding, and that R7 is likely to be a postbinding activity-deficient mutant toxin of TDH.

Infect Immun, 1994 Aug, 62(8), 3289 - 98
The Vibrio cholerae acfB colonization determinant encodes an inner membrane protein that is related to a family of signal-transducing proteins; Everiss KD et al.; Vibrio cholerae accessory colonization factor genes (acfA, B, C, and D) are required for efficient intestinal colonization . Expression of acf genes is under the control of a regulatory cascade that also directs the synthesis of cholera toxin and proteins involved in the biogenesis of the toxin-coregulated pilus . The gene for acfB was cloned by using an acfB::TnphoA fusion junction to probe a V . cholerae O395 bacteriophage lambda library . DNA sequence analysis revealed that acfB is predicted to encode a 626-amino-acid protein related to the V . cholerae HlyB and TcpI proteins . These three Vibrio proteins have amino acid sequence similarity in a region highly conserved among bacterial methyl-accepting chemotaxis proteins . Analysis of the predicted AcfB amino acid sequence suggests that this colonization determinant possesses a membrane topology and domain organization similar to those of methyl-accepting chemotaxis proteins . Heterologous expression of acfB in Escherichia coli generates four polypeptide species with apparent molecular masses of 34, 35, 74, and 75 kDa . The 74- and 75-kDa proteins appear to represent modified forms of the full-length AcfB protein . The 34- and 35-kDa polypeptide species most likely correspond to a C-terminal 274-amino-acid polypeptide that results from internal translation initiation of acfB mRNA . Localization studies with AcfB-PhoA hybrid proteins indicate that AcfB resides in the V . cholerae inner membrane . V . cholerae acfB::TnphoA mutants display an altered motility phenotype in semisolid agar . The relationship between AcfB and Vibrio motility and the amino acid similarities between AcfB and chemotaxis signal-transducing proteins suggest that AcfB may interact with the V . cholerae chemotaxis machinery . The data presented in this report provide preliminary evidence that acfB encodes an environmental sensor/signal-transducing protein involved in V . cholerae colonization.

Infect Immun, 1994 Aug, 62(8), 3051 - 7
Construction and characterization of recombinant Vibrio cholerae strains producing inactive cholera toxin analogs; Hase CC et al.; The catalytic A subunit of cholera toxin (CT-A) is capable of ADP-ribosylating the guanine nucleotide-binding protein, which regulates cell adenylyl cyclase, leading to the life-threatening diarrhea of cholera . Amino acids involved in the enzymatic activity of CT-A have previously been identified . By means of site-directed mutagenesis, an analog of the CT-A subunit gene was created with codon substitutions for both Arg-7 and Glu-112, each of which has been shown to produce subunits lacking ADP-ribosyltransferase activity . The mutated gene fragment was exchanged for the wild-type copy in the previously cloned ctxAB operon from El Tor biotype, Ogawa serotype Vibrio cholerae strain 3083, which produces CT-2 . Further, the zonula occludens toxin gene, zot, was inactivated by an insertional mutation to create the new plasmid construct pCT-2* . Additionally, a DNA fragment encoding the B subunit of CT-1 (CT produced by classical biotype, Inaba serotype V . cholerae strain 569B) was exchanged for the homologous part in pCT-2*, resulting in the creation of pCT-1* . These plasmid constructs were introduced into the CT-negative V . cholerae mutant strain JBK70 (E1 Tor biotype, Inaba serotype); CT-A-B+ derivatives CVD101 and CVD103 of classical biotype Ogawa and Inaba serotype strains 395 and 569B, respectively; El Tor biotype Inaba and Ogawa serotype strains C6706 and C7258, respectively, recently isolated in Peru; and O139 (synonym Bengal) strain SG25-1 from the current epidemic in India . Recombinant toxins (CT-1* and CT-2*), partially purified from culture supernatants of transformed JBK70, were shown to be inactive on mouse Y1 adrenal tumor cells and in an in vitro ADP-ribosyltransferase assay . CT-1* and CT-2* reacted with polyclonal and monoclonal antibodies against both A and B subunits of CT . The toxin analogs reacted with antibodies against CT-A and CT-B on cellulose acetate strips and in a GM1 enzyme-linked immunosorbent assay; they reacted appropriately with B-subunit epitype-specific monoclonal antibodies in checkerboard immunoblots, and they formed precipitin bands with GM1-ganglioside in Ouchterlony tests . However, the reactions of the modified proteins with anti-A-subunit monoclonal antibodies were weaker than the reactions with wild-type holotoxins . V, cholerae strains carrying ctxA*, with either ctxB-1 or ctxB-2, and inactivated zot genes were created by homologous recombination . The recombinant strains and the purified toxin analogs were inactive in the infant rabbit animal model.(ABSTRACT TRUNCATED AT 400 WORDS)

J Infect Dis, 1994 Aug, 170(2), 468 - 72
Severe life-threatening cholera associated with blood group O in Peru: implications for the Latin American epidemic; Swerdlow DL et al.; A household survey in 1991, at the onset of the Latin American cholera epidemic, investigated high attack rates in Trujillo, Peru, and determined the association between blood group O and severe cholera . Of 463 persons in 69 households, 173 (37%) reported diarrhea, 21% required rehydration therapy, and 4% were hospitalized; these treatment requirements greatly exceeded estimates based on other populations . Elevated vibriocidal or antitoxic antibody titers were present in 52% of 321 from whom serum was obtained; 73% were blood group O . Blood group O was strongly associated with severe cholera: Infected persons had more diarrheal stools per day than persons of other blood groups, were more likely to report vomiting and muscle cramps, and were almost eight times more likely to require hospital treatment . Since prevalence of blood group O in Latin America may be the world's highest, estimates of treatment requirements should be increased to prevent unnecessary deaths.

J Infect Dis, 1994 Aug, 170(2), 278 - 83
Emergence of a new cholera pandemic: molecular analysis of virulence determinants in Vibrio cholerae O139 and development of a live vaccine prototype; Waldor MK et al.; In October 1992, a non-O1 strain of Vibrio cholerae emerged as a cause of epidemic cholera in India and Bangladesh . This antigenically novel clone has been designated serogroup O139 synonym Bengal . Since its emergence, V . cholerae O139 has caused a massive cholera epidemic throughout and beyond the Indian subcontinent . Molecular analysis of virulence determinants in clinical isolates suggests that O139 strains are highly related to El Tor O1 strains . Unlike other non-O1 strains, O139 strains carry multiple copies of the cholera toxin genetic element and also genes for the toxin-coregulated pilus . These results guided construction of a live V . cholerae O139 vaccine prototype through deletion of genes for at least four specific virulence determinants (ctxA, ace, zot, and cep) as well as other factors involved in site-specific and homologous recombination (RS1, attRS1, and recA) . It is hoped that this attenuated live vaccine will help control the pandemic spread of V . cholerae O139.

Mol Mar Biol Biotechnol, 1994 Aug, 3(4), 200 - 5
M13 DNA fingerprinting in differentiation of marine Vibrio species; Chikarmane H et al.; The identification and differentiation of bacterial strains and species are frequently carried out by the use of diagnostic biochemical profiles, serology, and the detection of restriction fragment length polymorphisms in genomic DNA . We show here that DNA restriction fragment length polymorphisms detected using a probe derived from bacteriophage M13 can discriminate between several marine Vibrio species . We have also demonstrated that individual isolates of Vibrio species can be differentiated using the M13 probe.

Mol Microbiol, 1994 Aug, 13(3), 485 - 94
Analysis of membrane protein interaction: ToxR can dimerize the amino terminus of phage lambda repressor; Dziejman M et al.; The ToxR protein of Vibrio cholerae is an integral membrane protein that co-ordinately regulates virulence determinant expression . ToxR directly activates the cholera toxin operon, but maximal activation is achieved in the presence of ToxS, an integral membrane protein thought to interact with ToxR periplasmic sequences . Studies that substitute alkaline phosphatase sequences for the periplasmic domain of ToxR have led to a model for ToxR activation based on dimerization and ToxS interaction . We constructed lambda-ToxR chimeric proteins using the DNA-binding domain of the phage lambda repressor, which cannot effectively dimerize by itself, to assess the ability of ToxR to form dimers in Escherichia coli . The results suggest that ToxR sequences can propagate dimerization, and that ToxS can influence the ability to dimerize.

Vaccine, 1994 Aug, 12(11), 1000 - 3
Kinetics of the vibriocidal antibody response to live oral cholera vaccines; Wasserman SS et al.; The best correlate of protection against cholera is the level of serum vibriocidal antibodies, which are primarily directed against the O antigen of Vibrio cholerae O1 and lyse V . cholerae in the presence of complement . We established the timing of peak vibriocidal antibody response using sera from safety/immunogenicity studies of live oral cholera vaccines CVD 103-HgR, CVD 103-HgR2 and CVD 110 among immunologically naive North Americans and Colombians . The serum reciprocal vibriocidal antibody titre was consistently higher 10 days postimmunization than on either day 7 or day 14 . This study suggests that recent phase 2 studies of CVD 103-HgR may have underestimated the peak vibriocidal titre by collecting serum on days 7-8 rather than on day 10; future studies of live oral cholera vaccines should take these results into account to obtain the best measurement of peak immunological responses . Because of the rapid drop in vibriocidal antibody titres about 2 weeks after immunization, care must be exercised in comparing immunogenicity of different vaccine candidates, formulations, dosage levels and immunization schedules.

Arch Dis Child, 1994 Aug, 71(2), 161 - 2
Vibrio cholerae O139 in Calcutta; Bhattacharya SK et al.; Vibrio cholerae O139 was recovered from 28 of 79 children with acute watery diarrhoea . Clinically, they presented with watery diarrhoea (100%), vomiting (79%), abdominal cramps (61%), anorexia (61%), dehydration (100%), and absence of fever . Both clinical and blood biochemical parameters of these cases were similar to the illness caused by the new strain in adults . Hypoglycaemia was seen in 40% of those screened.

FEMS Microbiol Rev, 1994 Aug, 14(4), 369 - 74
Identification and characterization of genetically programmed responses to toxic metal exposure in Escherichia coli; Guzzo A et al.; In order to identify chromosomal genetically programmed responses to toxic metal exposure, a library of 3000 Escherichia coli clones was created that contained the promoterless luxAB genes of Vibrio harveyi inserted at single and random chromosomal loci . Changes in gene expression, as measured by a change in luminescence, were monitored after exposure of the clones to various metals . In this manner, we have identified two clones that showed an increase in luminescence in the presence of aluminum, one clone in the presence of nickel, and two clones in the presence of selenite . Identification of the metal-induced gene(s), and characterization of their biochemical function, will provide important clues about the effects of these metals at the molecular level.

FEMS Microbiol Lett, 1994 Aug 1, 121(1), 47 - 54
Biotype-specific tcpA genes in Vibrio cholerae; Iredell JR et al.; The tcpA gene, encoding the structural subunit of the toxin-coregulated pilus, has been isolated from a variety of clinical isolates of Vibrio cholerae, and the nucleotide sequence determined . Strict biotype-specific conservation within both the coding and putative regulatory regions was observed, with important differences between the El Tor and classical biotypes . V . cholerae O139 Bengal strains appear to have El Tor-type tcpA genes . Environmental O1 and non-O1 isolates have sequences that bind an El Tor-specific tcpA DNA probe and that are weakly and variably amplified by tcpA-specific polymerase chain reaction primers, under conditions of reduced stringency . The data presented allow the selection of primer pairs to help distinguish between clinical and environmental isolates, and to distinguish El Tor (and Bengal) biotypes from classical biotypes of V . cholerae . While the role of TcpA in cholera vaccine preparations remains unclear, the data strongly suggest that TcpA-containing vaccines directed at O1 strains need include only the two forms of TcpA, and that such vaccines directed at (O139) Bengal strains should include the TcpA of El Tor biotype.

Microb Pathog, 1994 Aug, 17(2), 69 - 78
A 53 kDa protein of Vibrio cholerae classical strain 0395 involved in intestinal colonization; Singh SN et al.; Mutants of Vibrio cholerae 01 strain 0395 (classical) mutated in genes encoding secretory or cell surface proteins were induced by TnphoA mutagenesis and were selected as blue colonies on L-agar plates containing 5-bromo-4-chloro-3-indolyl phosphate . Southern analysis of the total DNA from blue colonies showed that all mutants had TnphoA insertion in genomic DNA . These mutants were analysed for adherence, colonization and protein profile . Adherence to freshly isolated rabbit intestinal discs was affected in some mutants . The less adhesive mutants were examined for colonization of the intestine of infant mice . One mutant, designated T-87, was extremely poor at colonization and less diarrhaegenic than the parent strain . Analysis of T-87 by SDS-PAGE revealed that two proteins of 53 and 38 kDa were lacking . The 38 kDa protein was identified as OmpU . The 53 kDa protein was extracellular and cells treated with anti-53-kDa antibodies could not colonize the gut of infant mice . The expression of the 53 and 38 kDa proteins in T-87 was dependent of the growth medium . The data suggest that T-87 is mutated in a regulatory gene which regulates the expression of proteins involved in intestinal colonization.

Eur J Epidemiol, 1994 Aug, 10(4), 393 - 8
Construction of plasmids useful for production of the B subunit of cholera toxin from Vibrio cholerae or a heat-labile enterotoxin from enterotoxigenic Escherichia coli; Tsuji T et al.; A simple method to construct the plasmids producing the B subunit of porcine or human heatlabile enterotoxin or cholera toxin was developed, and the B subunits produced by the resulting plasmids were purified . The gene of LTp from pEWD 299 was ligated to pHSG 396 or pBluescript SK(+)-1 and the vector carrying one Xbal and EcoR1 site in the LTp-A gene was constructed . The Xbal-EcoR1 fragment of LTp-A gene was exchanged for the multicloning site of pHSG 396 containing Xbal, BamH1, Cla 1, Kpn1, Sac1 and EcoR1 sites . This plasmid (pTSU28) produced the LTp-B subunit . Moreover, the fragment of the LTp-B gene of pTSU 28 was exchanged by the EcoR1-HindIII fragment of LTh-B from E . coli H10407 strain (pTSU 35) or by the Cla 1-Hind III fragment of CT-B gene amplified by the PCR procedure with the chromosomal DNA of V . cholerae 86KT25 (pTSU 32) . The DNA sequence of the CT-B subunit amplified by PCR procedure was compared and found identical to that cited in the literature {11} . After these plasmids were transformed into E . coli MV 1184 strain, the toxins produced by them were purified using a Bio-Gel A 5m affinity column for both LT-Bs and an immunobilized D-galactose affinity column for CT-B . Though both columns absorbed only the B subunit, the eluates contained a single protein corresponding to the B subunit, suggesting that each mutant produces only the B subunit.

Glycobiology, 1994 Aug, 4(4), 429 - 35
Carbohydrate receptor-mediated gene transfer to human T leukaemic cells; Thurnher M et al.; The mucin-type carbohydrate Tn cryptantigen (GalNAc alpha 1-O-Ser/Thr, where GalNAc is N-acetyl-D-galactosamine) is expressed in many carcinomas, in haemopoietic disorders including the Tn syndrome, and on human immunodeficiency virus (HIV) coat glycoproteins, but is not expressed on normal, differentiated cells because of the expression of a Tn-processing galactosyltransferase . Using Jurkat T leukaemic cells which express high levels of Tn antigen due to deficient Tn galactosylation, we have established the Tn antigen-mediated gene transfer and demonstrate the considerable efficiency of this approach . We used poly(L-lysine) conjugates of the monoclonal antibody 1E3 directed against the Tn antigen to deliver the luciferase and beta-galactosidase reporter genes to Jurkat cells by receptor-mediated endocytosis . Addition of unconjugated 1E3 reduced transfection efficiency in a concentration-dependent manner and incubation with free GalNAc abolished DNA transfer completely, indicating that gene delivery is indeed mediated by the Tn antigen . Pre-treatment of Jurkat cells with Vibrio cholerae sialidase, which uncovers additional Tn antigens, resulted in an improvement of gene transfection . Both human and chicken adenovirus particles attached to the DNA/polylysine complex strongly augmented transgene expression . When the beta-galactosidase (lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis, up to 60% of the cells were positive in the cytochemical stain using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a chromogenic substrate . The efficiency of the transferrin receptor-mediated DNA uptake into Jurkat cells was comparatively low, although these cells were shown to express considerable amounts of transferrin receptor . We show here that a mucin-type carbohydrate antigen mediates highly efficient DNA uptake by endocytosis into Jurkat T cells . This method represents a 50-fold improvement of Jurkat cell transfection efficiency over other physical gene transfer techniques . Specific gene delivery to primary cancer cells exhibiting Tn epitopes may especially be desirable in immunotherapy protocols.

Carbohydr Res, 1994 Jul 16, 260(2), 203 - 18
Improved synthesis and the crystal structure of methyl 4,6-dideoxy-4-(3-deoxy-L-glycero-tetronamido)-alpha-D-mannopyrano side, the methyl alpha-glycoside of the intracatenary repeating unit of the O-polysaccharide of Vibrio cholerae O:1; Gotoh M et al.; The crude product of deamination of the commercially available L-homoserine was acetylated and the 2-O-acetyl-3-deoxy-L-glycero-tetronolactone (18) formed was used to N-acylate methyl perosaminide (methyl 4-amino-4,6-dideoxy-alpha-D-mannopyranoside, 12) and its 2,3-O-isopropylidene derivative . The major product isolated from the reaction was the crystalline methyl 4-(4-O-acetyl-3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-alpha-D-+ ++mannopyranoside (1, 70-75%) resulting from acetyl group migration in the initially formed 2'-O-acetyl derivative . O-Deacetylation of 1 gave the title amide 2 . Compound 2, obtained crystalline for the first time, was fully characterized, and its crystal structure was determined . Deoxytetronamido derivatives diastereomeric with 1 and 2, respectively, were obtained by the acylation of 12 with 2-O-acetyl-3-deoxy-D-glycero-tetronolactone (prepared from D-homoserine), and subsequent deacetylation . Structures of several byproducts of the reaction of 12 with 18 have been deduced from their spectral characteristics . Since these byproducts were various O-acetyl derivatives of 2, the title compound could be obtained in approximately 90% yield by deacetylating (Zemplen) the crude mixture of N-acylation products, followed by chromatography.

Wkly Epidemiol Rec, 1994 Jul 15, 69(28), 205 - 12
Cholera in 1993 . Part I; International dissemination of epidemic Vibrio cholerae by cargo ship ballast and other nonpotable waters; Gulf Coast Seafood Laboratory, Food and Drug Administration, Dauphin Island, Alabama 36528In 1991 and 1992, toxigenic Vibrio cholerae O1, serotype Inaba, biotype El Tor, was recovered from nonpotable (ballast, bilge, and sewage) water from five cargo ships docked in ports of the U.S . Gulf of Mexico . Four of these ships had taken on ballast water in cholera-infected countries; the fifth took on ballast in a noninfected country . Isolates examined by pulsed-field gel electrophoresis were indistinguishable from the Latin American epidemic strain, C6707; however, they differed significantly from the endemic Gulf Coast strain (VRL 1984), the sixth-pandemic strain (569-B), and a V . cholerae non-O1 strain isolated from a ship arriving from a foreign port . On the basis of our findings, the Food and Drug Administration recommended that the U.S . Coast Guard issue an advisory to shipping agents and captains requesting that ballast waters be exchanged on the high seas before entry of ships into U.S . ports.

FEMS Microbiol Lett, 1994 Jul 1, 120(1-2), 57 - 61
Isolation of a Vibrio cholerae transposon-mutant with an altered viable but nonculturable response; Ravel J et al.; We have isolated more than 2500 mutants of Vibrio cholerae by using transposon mutagenesis . Mutants were screened under low nutrient conditions in artificial seawater for an altered viable but nonculturable response, compared to the wild-type . Mutant JR09H1 entered the viable but nonculturable state more rapidly than the wild-type at both 25 degrees C and 4 degrees C.

FEMS Microbiol Lett, 1994 Jul 1, 120(1-2), 143 - 8
Cholera DFA: an improved direct fluorescent monoclonal antibody staining kit for rapid detection and enumeration of Vibrio cholerae O1; Hasan JA et al.; An improved fluorescent monoclonal antibody staining kit, Cholera DFA, for direct detection and enumeration of Vibrio cholerae O1 has been developed, employing a highly specific anti-A antigen monoclonal antibody, COLTA, labeled with fluorescein isothiocyanate (FITC) . An optimized quantity of anti-photobleaching agent is used in a glycerol mounting medium to retard the rapid fading of immunofluorescent stained cells during fluorescent microscopy, thus enabling prolonged inspection of individual fields, as well as improved photographic recording of results without loss of fluorescence intensity . When tested for specificity, all 30 strains of V . cholerae O1 reacted with Cholera DFA, whereas 100 heterologous species examined did not, yielding 100% specificity for all strains examined in this study . A field trial was conducted in Bangladesh, employing Cholera DFA and the results were compared with those obtained by conventional culture methods . Of 44 diarrheal stool specimens tested, Cholera DFA was positive for V . cholerae O1 in all culture-positive stool specimens and negative for all culture-negative stool specimens . The procedure is sensitive and highly specific, as well as simple, i.e., less complex than the indirect fluorescent assay, requiring only one reagent and less than 30 min to complete the staining process, while retarding rapid fading that often occurs with fluorescent microscopy.

FEMS Microbiol Lett, 1994 Jul 1, 120(1-2), 137 - 42
Colonization of professional divers by toxigenic Vibrio cholerae O1 and V . cholerae non-O1 at dive sites in the United States, Ukraine and Russia; Huq A et al.; Vibrio cholerae, recognized as the causative agent of epidemic cholera, was isolated from healthy professional divers and from water samples collected at dive sites in the United States, Ukraine and Russia . Swabs of nose, ear and throat of divers and their tank regulators, i.e . the divers and their diving gear, were taken before and after routine dives . Blood samples were collected before and 30-60 days after each dive to measure IgG and IgA titers against the whole cell antigen of V . cholerae O1 . Nine strains of V . cholerae O1 and nine strains of V . cholerae non-O1 were isolated during this study . These isolates were identified by conventional biochemical tests and indirect fluorescent antibody staining methods, using fluorescein isothiocyanate-labeled monoclonal antibody, COLTA, prepared against the 'A' antigenic factor of the lipopolysaccharide of V . cholerae O1, and serotyped by slide agglutination . Seven of the nine strains of V . cholerae O1 isolated and successfully cultured during the studies, were toxigenic by enzyme-linked immunosorbent assay and polymerase chain reaction . Analyses of IgG and IgA antibodies of the divers showed that most of the divers had prior exposure to V . cholerae O1 . V . cholerae serotype non-O1 strains isolated during the study were found to be non-toxigenic.

J Bacteriol, 1994 Jul, 176(14), 4219 - 25
MotY, a component of the sodium-type flagellar motor; McCarter LL; Energy to power the rotation of bacterial flagella can be derived from the proton or sodium transmembrane potential . Until now, genes encoding a bacterial sodium-type flagellar motor have not been defined . A gene, motY, encoding one component of the sodium-type flagellar motor of Vibrio parahaemolyticus was cloned by complementation of a Mot- mutant strain . Sequencing revealed an open reading frame of 879 nucleotides in which a transposon conferring a motility defect mapped . Overexpression of motY in Escherichia coli allowed identification of a product 33 kDa in apparent size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . This size correlated well with the predicted molecular mass of 33,385 Da . Unlike mot genes identified in other bacteria, localized transposon mutagenesis suggested that the locus was not an extended region containing multiple genes required for swimming motility . Sequencing upstream and downstream of motY confirmed that the gene maps alone and placed it within a locus homologous to the E . coli rnt locus . Although data bank searches failed to reveal significant similarity to known motility components, the carboxyl terminus of MotY showed extensive homology to a number of outer membrane proteins known to interact with peptidoglycan, including OmpA and peptidoglycan-associated lipoproteins . To a limited extent, this domain could also be identified in the Bacillus subtilis MotB protein . This finding suggests that MotY plays the role of a stator in the sodium flagellar motor, stabilizing the force-generating unit through direct interaction with the cell wall.

Infect Immun, 1994 Jul, 62(7), 2669 - 78
The Vibrio cholerae toxin-coregulated-pilus gene tcpI encodes a homolog of methyl-accepting chemotaxis proteins; Harkey CW et al.; Virulence gene activation in Vibrio cholerae is under the control of the ToxR-ToxT regulatory cascade . The ToxR regulon consists of genes required for toxin-coregulated-pilus (TCP) biogenesis, accessory colonization factor genes, cholera toxin genes, and ToxR-activated genes (tag) of unknown function . The tagB gene was isolated by using a tagB::TnphoA fusion junction to probe a V . cholerae )395 bacteriophage lambda library . Nucleotide sequence analysis revealed that tagB is identical to tcpI, a gene which encodes a protein that negatively regulates the synthesis of the major pilin subunit of TCP (TcpA) . Our results show that the tcpI gene encodes a 620-amino-acid protein that shares extensive sequence similarity with the highly conserved signaling domain in methyl-accepting chemotaxis proteins . Expression of tcpI in Escherichia coli results in the synthesis of a 71-kDa polypeptide that becomes localized to the inner membrane . Similarly, TcpI-PhoA alkaline phosphatase activity is enriched in V . cholerae inner membrane preparations . Colonies of V . cholerae tcpI::TnphoA mutant cells display increased swarming on solid media when compared with those of the parental V . cholerae O395 . Taken together, these observations suggest that TcpI may play a dual role in promoting vibrio colonization of the small bowel . In response to the appropriate environmental signal(s), TcpI permits maximum expression of tcpA while simultaneously reducing vibrio chemotaxis-directed motility . We believe coordinate regulation of colonization and motility determinants, in such a fashion, facilitates efficient V . cholerae microcolony formation.

Mol Microbiol, 1994 Jul, 13(2), 273 - 86
Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway; Bassler BL et al.; Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium . One signal-response system is encoded by the luxL,M,N locus . The luxL and luxM genes are required for the production of an autoinducer (probably beta-hydroxybutyl homoserine lactone), and the luxN gene is required for the response to that autoinducer . Analysis of the phenotypes of LuxL,M and N mutants indicated that an additional signal-response system also controls density sensing . We report here the identification, cloning and analysis of luxP and luxQ, which encode functions required for a second density-sensing system . Mutants with defects in luxP and luxQ are defective in response to a second autoinducer substance . LuxQ, like LuxN, is similar to members of the family of two-component, signal transduction proteins and contains both a histidine protein kinase and a response regulator domain . Analysis of signalling mutant phenotypes indicates that there are at least two separate signal-response pathways which converge to regulate expression of luminescence in V . harveyi.

Mol Microbiol, 1994 Jul, 13(1), 153 - 60
The human gastric pathogen Helicobacter pylori has a gene encoding an enzyme first classified as a mucinase in Vibrio cholerae; Smith AW et al.; The human bacterial pathogen Helicobacter pylori has been suggested to be the causative agent of the most common chronic infection of man . Since its first isolation in 1982, H . pylori has been associated with gastric and duodenal ulcer disease, and more recently, gastric cancer . The proteolytic digestion of gastric mucus by this microorganism has been suggested as an important mechanism by which its pathogenicity is at least partly exerted . Here we report the detection of protease activity in H . pylori total-cell and supernatant extracts . On the basis that zinc metalloproteases are common microbial pathogenicity factors, we identified a single protein in H . pylori protein extracts with antibodies to the Pseudomonas aeruginosa elastase (a secreted zinc metalloprotease) . This same protein was identified by pooled serum from patients infected with H . pylori . We used the functional and immunological relationship between the P . aeruginosa elastase and the Vibrio cholerae haemagglutinin/protease (HAP) to clone the H . pylori hap gene, which was over 99% similar to the V . cholerae hap gene in the coding region . A 4 kb DNA fragment containing the entire cloned gene was highly unstable in Escherichia coli and Bacillus subtilis cloning vectors . We also demonstrated that a hap-like gene sequence is present in all nine Helicobacter species so far discovered . The V . cholerae HAP was first classified on the basis of its mucinase activity.

Lipids, 1994 Jul, 29(7), 527 - 8
Production of docosahexaenoic acid by marine bacteria isolated from deep sea fish; Yano Y et al.; Five bacterial strains isolated from the intestine of deep sea fish were shown to produce docosahexaenoic acid (22:6n-3; DHA) at a level of 6.4 to 11.6% of total fatty acids when incubated in DHA-free medium . In all of the strains examined, other polyunsaturated fatty acids were barely detectable, except for eicosapentaenoic acid (20:5n-3) . A typical strain, such as T3615, produced DHA at a concentration of about 0.8 mg/L within six days of aerobic incubation at 5 degrees C and under atmospheric pressure . The T3615 strain, belonging to the genus Vibrio, is rod-shaped, Gram-negative, motile and facultatively anaerobic.

J Clin Microbiol, 1994 Jul, 32(7), 1805 - 6
Identification of Vibrio hollisae associated with severe gastroenteritis after consumption of raw oysters; Carnahan AM et al.; Vibrio hollisae was recovered from the stool culture of a 40-year-old female hospitalized for severe abdominal cramping, vomiting, fever, and watery diarrhea . She had consumed two dozen raw oysters 5 days prior . There was only a single colony on thiosulfate-citrate-bile salts-sucrose-agar, and definitive identification required conventional test media with 1% NaCl.

J Clin Microbiol, 1994 Jul, 32(7), 1685 - 90
Molecular characterization of Vibrio cholerae O1 strains by pulsed-field gel electrophoresis; Cameron DN et al.; Pulsed-field gel electrophoresis (PFGE) was performed on 180 isolates of Vibrio cholerae serogroup O1 representing 6 different multilocus enzyme electrophoresis (MEE) types and 27 rRNA restriction fragment length polymorphism types (ribotypes) . Isolates were digested with the restriction enzyme NotI and were separated into 63 patterns on the basis of differences in band arrangements . In general, strains which were different by MEE or ribotyping also had different PGFE patterns . PFGE identified individual strains within a single MEE type or ribotype; isolates with one PFGE pattern were less frequently distinguished by ribotyping . All V . cholerae O1 isolates tested from the Latin American epidemic were indistinguishable by their MEE, ribotype, or PFGE patterns . PFGE could further distinguish strains of this same ribotype isolated in Africa, Europe, the South Pacific, or Southeast Asia . Although both MEE and PFGE could identify the strain from the Latin American epidemic, PFGE was more rapid and less labor intensive . PFGE also distinguished nontoxigenic isolates endemic to the U.S . Gulf Coast from unrelated nontoxigenic isolates . In the present study PFGE was more discriminating than other previously described subtyping assays for V . cholerae O1 and appears to be a useful epidemiologic tool.

Development, 1994 Jul, 120(7), 1719 - 29
Bacterial symbionts induce host organ morphogenesis during early postembryonic development of the squid Euprymna scolopes; Montgomery MK et al.; The mutualistic association between the squid Euprymna scolopes and the bacterium Vibrio fischeri is an emerging experimental system for the study of the influence of bacteria on animal development . Taking advantage of the ability to raise both this host and its microbial partner independently under laboratory conditions, we describe the effects of bacterial interactions on morphogenesis of the juvenile host symbiotic organ . Our results show that bacteria are essential for normal postembryonic development of the symbiotic organ, which involves changes in both the surface epithelium and the epithelial tissue within the organ where the bacterial culture will take up residence . Cell death induced by exposure to symbiotic V . fischeri results in the regression of a complex ciliated surface epithelium, a tissue that apparently functions to facilitate inoculation of the juvenile organ with the appropriate specific bacterial species . Regression of this tissue begins within hours of exposure to symbiosis-competent bacteria and progresses over the next 5 days, at which time full regression is complete, resulting in a symbiotic organ whose epithelial surface resembles that of the fully mature organ . Moreover, symbiosis-competent bacteria induce modification of the epithelial cells of the crypts that will house these symbionts; these cells undergo significant changes in shape and size in response to interactions with symbiotic V . fischeri . In contrast, we find that when these tissues are not exposed to the proper bacterial symbionts they remain in a state of arrested morphogenesis, a condition that can be rescued by interactions with symbionts . The results of these studies are the first experimental data demonstrating that a specific bacterial symbiont can play an inductive role in animal development.

FEMS Microbiol Lett, 1994 Jul 1, 120(1-2), 207 - 10
Correlation between cell-associated mannose-sensitive hemagglutination by Vibrio parahaemolyticus and adherence to a human colonic cell line Caco-2; Nagayama K et al.; Cell-associated hemagglutination (cHA) activity with human erythrocytes was examined for 468 clinical and 71 environmental strains of Vibrio parahaemolyticus . Approximately 95% of the strains tested were cHA positive irrespective of source or Kanagawa phenomenon . 75% of clinical strains showed relatively strong mannose-sensitive hemagglutination (MSHA), whereas 88% of the environmental strains showed relatively weak mannose-resistant hemagglutination (MRHA) . Adherence of V . parahaemolyticus to Caco-2 cells was also determined . A clear positive correlation between cell-associated MSHA and adherence to Caco-2 cells was observed.

Infect Immun, 1994 Jul, 62(7), 2901 - 7
Role of antibodies against biotype-specific Vibrio cholerae pili in protection against experimental classical and El Tor cholera; Osek J et al.; Vibrio cholerae O1, which exists as two biotypes, classical and El Tor, expresses fimbrial antigens called toxin-coregulated pili (TCP) and mannose-sensitive hemagglutinin (MSHA) pili, respectively . We have raised rabbit antisera and monoclonal antibodies against these fimbrial antigens and prepared Fab fragments which possess specific antibodies directed against the respective fimbrial antigens from these antisera . The protective effect of these antibody preparations was studied in the infant mouse cholera model . Antibodies against TCP were able to protect baby mice against challenge with V . cholerae O1 of the classical but not of the El Tor biotype . Similar but reverse biotype differences in protection against challenge with classical and El Tor vibrios were observed when antibodies against MSHA pili were used . The protective effect of V . cholerae O1 antilipopolysaccharide (anti-LPS) antibodies, both alone and in combination with antifimbrial antibodies, was also evaluated . We showed that antibodies to the LPS component also prevented infections with V . cholerae O1 . Moreover, our results indicate that antibodies against TCP or MSHA pili and against LPS cooperate at least additively, and possible even synergistically, in protecting baby mice against challenge with group O1 vibrios . These results indicate that TCP and MSHA pili as well as LPS play an important role in the pathogenesis of experimental cholera . We could also demonstrate that antibacterial immunity preventing colonization is biotype specific . Our results might be used for the generation of new oral cholera vaccines including both TCP and MSHA fimbrial antigens.

Indian J Pathol Microbiol, 1994 Jul, 37(3), 289 - 92
Cholera epidemic in Goa; Verenkar M et al.; Two hundred and fifty stool samples were studied during an outbreak of cholera in Goa during the months of July to September, 1988 . 80 strains of Vibrio were isolated with an isolation rate of 32% . 72.5% of those affected were adults . All strains of Vibrio cholerae isolated belonged to Eltor biotype, Fifty three (66.25%) of them being Ogawa serotype while 21 (26.25%) were Inaba . NAG Vibrios accounted for 6 (7.5%) strains . Antimicrobial sensitivity pattern showed high degree of sensitivity to chloramphenicol, gentamicin and naladixic acid.

Microbiology, 1994 Jul, 140 ( Pt 7), 1775 - 9
Unusual in vivo turnover of transfer RNA in Vibrio cholerae; Mukhopadhyay P et al.; Two lines of evidence suggest that, unlike in other organisms, the transfer RNAs of Vibrio cholerae undergo rapid turnover in vivo . Firstly, the tRNA content of V . cholerae cells treated with rifampicin (an inhibitor of initiation of RNA synthesis) decreased rapidly and continuously . Secondly, the newly synthesized tRNAs were rapidly degraded even under normal conditions of growth; the average half life of tRNA was 11.8 min . The degradation is mediated by an enzyme(s), present in V . cholerae cytoplasm, that apparently degrades tRNA completely . Rapid turnover is balanced by an enhanced rate of tRNA biogenesis, which was calculated to be 2.5 times higher than that in Escherichia coli.

Infect Immun, 1994 Jul, 62(7), 2811 - 7
Neutralizing monoclonal antibodies to an extracellular Pseudomonas cepacia protease; Kooi C et al.; Pseudomonas cepacia produces at least two extracellular proteases with apparent molecular masses of 36,000 and 40,000 Da . The 36-kDa protease has high proteolytic activity and the 40-kDa protease has low proteolytic activity with hide powder azure as a substrate . Monoclonal antibodies (MAbs) were raised against the purified 36- and 40-kDa proteases . Several MAbs directed against the 36-kDa protease were found to recognize the 40-kDa protease by Western immunoblot analysis . Similarly, a MAb directed against the 40-kDa protease recognized the 36-kDa protease, suggesting that these two proteases may be immunologically related . A MAb directed against the 36-kDa protease, designated 36-6-8, and a MAb directed against the 40-kDa protease (MAb G-11) cross-reacted with other extracellular proteases, such as Pseudomonas aeruginosa elastase and alkaline protease, Pseudomonas pseudomallei protease, and the Vibrio cholerae hemagglutinin/protease . MAb 36-6-8 neutralized the P . cepacia 36-kDa protease, P . aeruginosa elastase, P . pseudomallei protease, and V . cholerae hemagglutinin/protease but did not affect P . aeruginosa alkaline protease activity . In contrast, MAb G-11 to the 40-kDa protease neutralized only the P . cepacia 36-kDa protease . This evidence suggests that the neutralizing MAb, 36-6-8, recognizes an epitope conserved among some metalloproteases . This epitope may lie at or near the active site of the P . cepacia 36-kDa protease and P . aeruginosa elastase.

FEBS Lett, 1994 Jun 27, 347(2-3), 163 - 8
NAD(P)H-flavin oxidoreductase from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, is a flavoprotein; Inouye S; The NAD(P)H-flavin oxidoreductase gene from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, was expressed in Escherichia coli, and the enzyme purified using Cibacron Blue 3G-A affinity column chromatography from crude extracts in a single step . The purified enzyme had a typical flavoprotein absorption spectrum and flavin mononucleotide (FMN) was identified as a prosthetic group, non-covalently bound in a molar ratio of 1:1 . The enzyme catalyzed the electron transfer from NADH via FMNH2 to various other electron acceptors . Reduced flavin produced by flavin reductase participated non-enzymatically in the following reactions: H2O2-forming NADH oxidase-like, oxygen-insensitive nitroreductase-like, diaphorase (quinone reductase)-like and bacterial luciferase reactions.

FEMS Microbiol Lett, 1994 Jun 15, 119(3), 377 - 80
A gene for the enterotoxin zonula occludens toxin is present in Vibrio mimicus and Vibrio cholerae O139; Chowdhury MA et al.; The presence of the zonula occludens toxin (ZOT) gene, which encodes an enterotoxin produced by serotype O1 strains of the pathogenic bacterium, Vibrio cholerae, in addition to cholera toxin, was investigated in selected strains of V . mimicus and the new pandemic V . cholerae non-O1 serotype O139 . The zot gene was detected by polymerase chain reaction (PCR) amplification, using sets of primers based on the sequence of the V . cholerae O1 zot sequence . PCR amplification of genomic DNAs of both cholera toxin gene (ctx) positive and ctx- strains of V . mimicus detected the presence of zot gene . An AccI-EcoRV V . cholerae zot gene fragment designed to overlap PCR products was used as a probe . Southern hybridization studies confirmed that the PCR fragments from V . mimicus and V . cholerae O139 were strongly homologous to the V . cholerae O1 zot gene . The zot gene was found with 3 of 5 strains of V . mimicus of which only one strain harbored the ctx gene . The presence of a zot gene in ctx- toxigenic V . mimicus indicates a possible role of ZOT in the toxigenicity of this species . We conclude that, in addition to ctx, V . mimicus and V . cholerae O139 have the potential to produce ZOT.

Structure, 1994 Jun 15, 2(6), 535 - 44
Crystal structure of Vibrio cholerae neuraminidase reveals dual lectin-like domains in addition to the catalytic domain; Crennell S et al.; BACKGROUND: Vibrio cholerae neuraminidase is part of a mucinase complex which may function in pathogenesis by degrading the mucin layer of the gastrointestinal tract . The neuraminidase, which has been the target of extensive inhibitor studies, plays a subtle role in the pathology of the bacterium, by processing higher order gangliosides to GM1, the receptor for cholera toxin . RESULTS: We report here the X-ray crystal structure of V . cholerae neuraminidase at 2.3 A resolution . The 83 kDa enzyme folds into three distinct domains . The central catalytic domain has the canonical neuraminidase beta-propeller fold, and is flanked by two domains which possess identical legume lectin-like topologies but without the usual metal-binding loops . The active site has many features in common with other viral and bacterial neuraminidases but, uniquely, has an essential Ca2+ ion which plays a crucial structural role . CONCLUSIONS: The environment of the small intestine requires V . cholerae to secrete several adhesins, and it is known that its neuraminidase can bind to cell surfaces, and remain active . The unexpected lectin-like domains possibly mediate this attachment . These bacterial lectin folds represent additional members of a growing lectin superfamily.

J Biotechnol, 1994 Jun 15, 35(1), 69 - 76
A novel protein secretion factor from a Vibrio species which operates in Escherichia coli; Tokugawa K et al.; A DNA fragment specific to a Vibrio species was found to promote extracellular secretion of proteins, when cloned into Escherichia coli . Cells harboring a plasmid carrying this fragment secreted significant amounts of periplasmic beta-lactamase and alkaline phosphatase into the medium, however most cytoplasmic beta-galactosidase was retained within the cell . The DNA sequence essential for this property was found to be a gene encoding 76 amino acids, which was designated as the 'PAS factor' . Highly expressed PAS factor is harmful to the cell, this may be due to a disruption of the membrane structure and/or function.

J Bacteriol, 1994 Jun, 176(12), 3552 - 8
Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme; Lei B et al.; NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction . By using an oligonucleotide probe based on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme . The DNA sequence of the frp gene was determined; the deduced amino acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312 . The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1 . The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step . The same chromatography material was also highly effective in concentrating diluted flavin reductase P . The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor . Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent . The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined . In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism.

J Bacteriol, 1994 Jun, 176(12), 3544 - 51
Identification of the genes encoding NAD(P)H-flavin oxidoreductases that are similar in sequence to Escherichia coli Fre in four species of luminous bacteria: Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio orientalis; Zenno S et al.; Genes encoding NAD(P)H-flavin oxidoreductases (flavin reductases) similar in both size and sequence to Fre, the most abundant flavin reductase in Escherichia coli, were identified in four species of luminous bacteria, Photorhabdus luminescens (ATCC 29999), Vibrio fischeri (ATCC 7744), Vibrio harveyi (ATCC 33843), and Vibrio orientalis (ATCC 33934) . Nucleotide sequence analysis showed Fre-like flavin reductases in P . luminescens and V . fischeri to consist of 233 and 236 amino acids, respectively . As in E . coli Fre, Fre-like enzymes in luminous bacteria preferably used riboflavin as an electron acceptor when NADPH was used as an electron donor . These enzymes also were good suppliers of reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction . In V . fischeri, the Fre-like enzyme is a minor flavin reductase representing < 10% of the total FMN reductase . That the V . fischeri Fre-like enzyme has no appreciable homology in amino acid sequence to the major flavin reductase in V . fischeri, FRase I, indicates that at least two different types of flavin reductases supply FMNH2 to the luminescence system in V . fischeri . Although Fre-like flavin reductases are highly similar in sequence to luxG gene products (LuxGs), Fre-like flavin reductases and LuxGs appear to constitute two separate groups of flavin-associated proteins.

J Infect Dis, 1994 Jun, 169(6), 1381 - 4
Epidemic cholera in the Amazon: the role of produce in disease risk and prevention; Mujica OJ et al.; Epidemic cholera struck Peru in January 1991 and spread within a month to the Amazon headwaters . A case-control study was done in the Amazonian city of Iquitos, Peru . Cholera-like illness was associated with eating unwashed fruits and vegetables (odds ratio {OR} = 8.0; 95% confidence limits {CL} = 2.2, 28.9) and drinking untreated water (OR = 2.9; 95% CL = 1.3, 6.4) . Consumption of a drink made from toronja, a citrus fruit, was protective against illness (OR = 0.4; 95% CL = 0.2, 0.7) . Illness was inversely associated with the quantity of toronja drink consumed (P < .01) . Produce has not previously been convincingly documented as a risk factor for cholera; this study underscores the importance of washing produce before eating it . Acidic juices, such as toronja drink (pH 4.1), inhibit vibrio growth and may make contaminated water safer . Wild citrus fruits such as toronja are abundant, cheap, and popular in the Amazon region . Promoting the consumption of toronja drink may be a useful cholera prevention strategy in this region.

J Bacteriol, 1994 Jun, 176(11), 3269 - 77
Characterization of the Vibrio cholerae outer membrane heme transport protein HutA: sequence of the gene, regulation of expression, and homology to the family of TonB-dependent proteins; Henderson DP et al.; The regulation of hutA, the Vibrio cholerae gene encoding a 77-kDa iron-regulated outer membrane protein required for heme iron utilization, was characterized, and the DNA sequence of the gene was determined . A hutA::Tn5 lac fusion generated previously (D . P . Henderson and S . M . Payne, Mol . Microbiol . 7:461-469, 1993) was transformed into Fur- and Fur+ strains of Escherichia coli and V . cholerae . The results of beta-galactosidase assays on the transformed strains demonstrated that transcription of hutA is regulated by the Fur repressor protein in E . coli and at least partially regulated by Fur in V . cholerae . Analysis of the DNA sequence of hutA indicated that a sequence homologous to the E . coli consensus Fur box was present in the promoter region of hutA . The amino acid sequence of HutA is homologous to those of several TonB-dependent outer member proteins . However, when the V . cholerae heme utilization system, which requires one or more genes encoded by the recombinant plasmid pHUT10 in addition to hutA carried on a second vector, was transferred to a wild-type strain and an isogenic tonB mutant of E . coli, the tonB mutant could utilize heme iron as efficiently as the wild-type strain . These data indicate that the V . cholerae heme utilization system reconstituted in E . coli does not require a functional TonB protein . The tonB mutant transformed with the heme utilization plasmids could not utilize the siderophore ferrichrome as an iron source, indicating that none of the genes encoded on the heme utilization plasmids complements the tonB defect in E . coli . It is possible that a gene(s) encoded by the recombinant heme utilization plasmids encodes a protein serving a TonB-like function in V . cholerae . A region in the carboxy terminus of HutA is homologous to the horse hemoglobin gamma chain, and the amino acids involved in forming the heme pocket in the gamma chain are conserved in HutA . These data suggest that this region of HutA is involved in heme binding.

J Clin Microbiol, 1994 Jun, 32(6), 1589 - 90
Development and evaluation of rapid monoclonal antibody-based coagglutination test for direct detection of Vibrio cholerae O139 synonym Bengal in stool samples; Qadri F et al.; A monoclonal antibody-based coagglutination test directly detected Vibrio cholerae O139 synonym Bengal in 83 of 120 watery diarrheal stool specimens; on culture, 90 samples were positive . Thus, with 92% sensitivity, 100% specificity, and 100% positive and 95% negative predictive values, the coagglutination test is a useful rapid test for V . cholerae O139.

Kansenshogaku Zasshi, 1994 Jun, 68(6), 744 - 50
{Survival of Vibrio cholerae O139 Synonym Bengal in water from a river}; Okitsu T et al.; Survival of five strains of Vibrio cholerae, including serotypes O139 Synonym Bengal, O1 El Tor, and non-O1 were compared in water from a river . These bacteria were mixed with water from a river and the water filtered through 0.45 micron millipore filters, respectively, to yield a concentration of 10(6) CFU/ml and incubated at 5 degrees C and 20 degrees C for 21 days . The survival curve of V . cholerae O139 was almost the same with V . cholerae O1 and non-O1 . The number of these bacilli decreased to less than 10(2) CFU/ml at 7 days of the incubation at 20 degrees C . When incubated at 5 degrees, however, these bacilli survived much longer and the number decreased to the same value after 14 days . Therefore, these results indicated that the temperature during the incubation greatly affects the survival of V . cholerae . On the other hand, when the water was filtered and used for the experiment, V . cholerae survived longer than in the polluted water . From these observations, if the river is polluted with V . cholerae O139 in the future, it is suggested that the distribution of V . cholerae O139 in the river may be the same as the present condition with V . cholerae O1 and non-O1.

Can J Microbiol, 1994 Jun, 40(6), 446 - 55
Numerical analysis and the application of random amplified polymorphic DNA polymerase chain reaction to the differentiation of Vibrio strains from a seasonally cold ocean; Martin-Kearley J et al.; Eighty regional strains of Vibrio isolated from the seasonally cold waters of coastal Newfoundland, and a number of Vibrio reference cultures, were studied . The regional strains had been isolated from the brown macroalga Alaria esculenta and the giant scallop Placopecten magellanicus and were known to grow at 4 degrees C . The strains were grouped according to their arginine-dihydrolase reactions and examined by numerical analysis . According to phenotypic properties the arginine-dihydrolase positive strains closely resembled Vibrio splendidus biovar I . Most clusters of the arginine-dihydrolase negative strains appeared to be unique but the closest phenotypic resemblance among some strains was with Vibrio ordalii . Some strains were examined using the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique for fingerprinting and it was shown that the regional strains were significantly different from either V . splendidus biovar I or V . ordalii . Generally, the strains from seaweed clustered separately from those that were from scallops . Strains in some clusters, especially those from the seaweed, were able to utilize most of the compounds that were tested as sole sources of carbon and energy.

FEMS Microbiol Lett, 1994 Jun 1, 119(1-2), 229 - 35
Adherence to human small intestines of capsulated Vibrio cholerae O139; Yamamoto T et al.; Capsulated cells of V . cholerae O139 adhered to formalin-fixed or native mucosa of the small intestines from an adult and a child . The primary adherence target was mucus . Capsulated O139 cells adhered better to the antigen sampling cells (M cells) of ileal Peyer's patch than to the absorptive cells . O139 cells on the mucosa appeared as small aggregates . Similar organisms were found on the mucosa of duodenal biopsy samples from patients infected with V . cholerae O139 . The findings indicated that capsulated cells of V . cholerae O139 tend to autoagglutinate and contribute to the effective adherence to the intestinal mucosa.

Appl Environ Microbiol, 1994 Jun, 60(6), 1749 - 53
Regulation of extracellular copper-binding proteins in copper-resistant and copper-sensitive mutants of Vibrio alginolyticus; Harwood VJ et al.; Extracellular proteins of wild-type Vibrio alginolyticus were compared with those of copper-resistant and copper-sensitive mutants . One copper-resistant mutant (Cu40B3) constitutively produced an extracellular protein with the same apparent molecular mass (21 kDa) and chromatographic behavior as copper-binding protein (CuBP), a copper-induced supernatant protein which has been implicated in copper detoxification in wild-type V . alginolyticus . Copper-sensitive V . alginolyticus mutants displayed a range of alterations in supernatant protein profiles . CuBP was not detected in supernatants of one copper-sensitive mutant after cultures had been stressed with 50 microM copper . Increased resistance to copper was not induced by preincubation with subinhibitory levels of copper in the wild type or in the copper-resistant mutant Cu40B3 . Copper-resistant mutants maintained the ability to grow on copper-amended agar after 10 or more subcultures on nonselective agar, demonstrating the stability of the phenotype . A derivative of Cu40B3 with wild-type sensitivity to copper which no longer constitutively expressed CuBP was isolated . The simultaneous loss of both constitutive CuBP production and copper resistance in Cu40B3 indicates that constitutive CuBP production is necessary for copper resistance in this mutant . These data support the hypothesis that the extracellular, ca . 20-kDa protein(s) of V . alginolyticus is an important factor in survival and growth of the organism at elevated copper concentrations . The range of phenotypes observed in copper-resistant and copper-sensitive V . alginolyticus indicate that altered sensitivity to copper was mediated by a variety of physiological changes.

Epidemiol Infect, 1994 Jun, 112(3), 463 - 71
An epidemic of cholera due to Vibrio cholerae O139 in Dhaka, Bangladesh: clinical and epidemiological features; Mahalanabis D et al.; We describe the disease spectrum and socio-demographic and epidemiological features of an epidemic of cholera due to a new pathogen, Vibrio cholerae O139, in patients attending a very large hospital in the metropolitan city of Dhaka, Bangladesh . This hospital treats 70,000-90,000 patients a year with diarrhoeal diseases . A 4% systematic sample of 1854 patients attending from January to April 1993 were studied . Five hundred and two (27%) of the 1854 patients were culture positive for V . cholerae O139 and 63 (3%) were culture positive for V . cholerae O1 biotype El Tor . Patients with V . cholerae O139 were mainly adults with a short history of watery diarrhoea . Eight-three percent of patients had moderate to severe dehydration . All recovered except one 80-year-old man with compromised renal function who died . Seventy-eight percent of patients required initial intravenous rehydration followed by oral rehydration therapy with rice ORS; they also received tetracycline to reduce diarrhoea severity . Most patients were from urban slums with inadequate sanitation facilities and hygiene practices . The newly recognized V . cholerae O139 infection produced an epidemic of severe dehydrating diarrhoea indistinguishable from clinical cholera in a population which experiences two epidemic peaks of cholera in a year due to V . cholerae O1 . Infection with the latter does not appear to confer any cross-protection from V . cholerae O139 . The new pathogen suppressed, albeit temporarily, V . cholerae O1 . Unlike other non-O1 serogroups of V . cholerae this new serogroup appears to have epidemic potential.

Vet Immunol Immunopathol, 1994 Jun, 41(3-4), 353 - 66
Monoclonal antibodies to turbot (Scophthalmus maximus) immunoglobulins: characterization and applicability in immunoassays; Estevez J et al.; Five monoclonal antibodies (mAbs) to immunoglobulins (Igs) of the turbot Scophthalmus maximus were produced and characterized . All the mAbs (denominated UR1, UR3, UR4, UR6 and UR7) are of isotype IgG1/kappa and show good anti-turbot Ig reactivity in enzyme-linked immunosorbent assay (ELISA) and immunoblotting . Results of competitive ELISA and immunoblotting analysis indicate that these five mAbs react with at least three different epitopes on the turbot Ig H chain . Except in the case of UR1, reactivity with periodate-treated purified turbot Ig was much lower than with the untreated Ig, suggesting that carbohydrate residues are involved in epitope recognition . All the mAbs showed reactivity with sera from the closely related species Scophthalmus rhombus but not with sera from species of other flatfish genera . One of these mAbs (UR3) has been successfully applied for the detection of antibodies against Vibrio anguillarum in ELISA.

J Nat Prod, 1994 Jun, 57(6), 858 - 61
Structure and antimicrobial activity of diterpenes from the roots of Plectranthus hereroensis; Batista O et al.; Two abietane-type diterpenoids have been isolated from the roots of Plectranthus hereroensis (Labiatae), one being the already known horminone {1} and the other a new substance, 7 alpha,12-dihydroxy-17(15-->16)-abeo-abieta-8,12,16-triene-11 ,14-dione {2}, whose structure was established by spectroscopic means . Compounds 1 and 2 showed antimicrobial activity against Staphylococcus aureus, Vibrio cholerae, Candida albicans, and Pseudomonas aeruginosa.

Trends Microbiol, 1994 Jun, 2(6), 187 - 92
The toxin-co-regulated pilus of Vibrio cholerae O1: a model for type 4 pilus biogenesis?
Iredell JR, Manning PA.
The toxin-co-regulated pilus (TCP), an important colonization factor of Vibrio cholerae, is similar to the type 4 pilus produced by a variety of pathogenic Gram-negative bacteria . The putative translocation and assembly machinery of TCP has broad similarities with known pilin and nonpilin export mechanisms.

Cent Eur J Public Health, 1994 Jun, 2(1), 37 - 41
Epidemiology and spectrum of vibrio diarrheas in the lower cross river basin of Nigeria; Eko FO et al.; In 1991 a cholera epidemic occurred in Nigeria . The features of this cholera outbreak in a single hospital in Cross River, Nigeria, were examined . Microbiologic techniques included the use of thiosulphate citrate bile-salts sucrose (TCBS) medium for culture of all stool specimens . Vibrio isolates from diarrheic patients included V . cholerae-O1 (75), V . cholerae non-O1 (10) and V . parahaemolyticus (21) . The illnesses were diverse, ranging from mild to severe, and in most instances requiring hospitalization, rehydration as well as antibiotic treatment . Eighty patients were hospitalized and six died mainly from hypovolemic shock and acute renal failure arising from excessive fluid loss . The low vibrio-associated mortality observed in this outbreak may have been influenced by the proximity and easy transit access to the health care facilities offered by the teaching hospital . This contrasts with the high mortality figures reported by Health Centers in the rural areas during the same period . Some features of vibrio diarrheas were comparable with those of other enteric pathogens . Poorly developed water and sewage disposal systems, contact with sea water, consumption of fishery products and leftover foods were the main risk factors identified.

Gene, 1994 May 27, 143(1), 117 - 21
Cloning and sequencing of the Legionella pneumophila fur gene; Hickey EK et al.; Iron is required for the intracellular and extracellular growth of Legionella pneumophila (Lp) . In addition, variations in iron levels may serve as a signal for changes in gene expression . In a number of bacterial pathogens, the regulation of gene expression by iron is usually mediated by the Fur (ferric uptake regulation) repressor protein . Through complementation of an Escherichia coli fur mutation and nucleotide sequence analysis, we have cloned and characterized the Lp fur gene . Lp fur encoded a 15.0-kDa protein whose repressive activity was, as expected, highest in bacteria grown in iron-rich media . Computer analysis determined that Lp Fur had an amino-acid identity of over 54% and a similarity of over 72% to the Fur of E . coli, Yersinia pestis, Vibrio species and Pseudomonas aeruginosa . The promoter region of Lp fur contained sequences homologous to the Fur-binding site, suggesting that fur is autoregulated in Lp . Finally, Southern blot hybridizations demonstrated that fur is conserved among Lp strains and Legionella species.

Biochim Biophys Acta, 1994 May 17, 1218(1), 105 - 8
Sequence analysis of the agaB gene encoding a new beta-agarase from Vibrio sp . strain JT0107; Sugano Y et al.; An agarase gene (agaB) was cloned from genomic DNA of Vibrio sp . strain JT0107 . Analysis of the 3200 nucleotide sequence just before the agarase 0107 gene (agaA) which existed in genomic DNA of Vibrio sp . strain JT0107 revealed a putative single open reading frame coding for 955 amino acids . Comparison of the deduced amino acid sequence of AgaB to that of agarase 0107 revealed the existence of partially highly homologous regions . A part of this gene was expressed in Escherichia coli to yield a protein with agarase activity . This is the first report of evidence by genetic analysis that at least two different kinds of agarases exist in strain JT0107.

FEMS Microbiol Lett, 1994 May 15, 118(3), 265 - 71
Epidemic isolates of Vibrio cholerae 0139 express antigenically distinct types of colonization pili; Sengupta TK et al.; Vibrio cholerae belonging to the recently described serogroup 0139, which are responsible for the current cholera epidemics in India and Bangladesh, were shown to express pilus-like structures partially cross-reacting with the toxin-coregulated pilus of V . cholerae strain (0395) belonging to the 01 serogroup and classical biotype . The 0139 pili were composed of 20 kDa subunit proteins which were antigenically related to the 20 kDa pilus protein of another diarrhoeagenic non-01 V . cholerae strain (serogroup 034) isolated earlier . The pili described in this study were found to be involved in the intestinal colonization process and, therefore, may contribute towards the virulence of the 0139 epidemic isolates.

Am J Trop Med Hyg, 1994 May, 50(5), 566 - 9
Epidemic cholera in Trujillo, Peru 1992: utility of a clinical case definition and shift in Vibrio cholerae O1 serotype; Vugia DJ et al.; Epidemic cholera continues in Peru . Since 1991, cholera surveillance in Peru has been based mainly on clinical recognition . To determine the proportion of reported cholera patients who actually have cholera and to evaluate the clinical case definition used in surveillance, we cultured rectal swabs from patients presenting with acute diarrhea in March 1992 in Trujillo, Peru . Of 197 patients meeting the clinical case definition, 174 (88%) had confirmed Vibrio cholerae O1 infection . In this epidemic setting, watery diarrhea of sudden onset in a person of any age presenting for treatment is highly predictive of cholera . Of note, 90% of the current V . cholerae O1 El Tor isolates were of serotype Ogawa, while a year earlier, all were of serotype Inaba.

J Bacteriol, 1994 May, 176(10), 3085 - 8
The light organ symbiont Vibrio fischeri possesses a homolog of the Vibrio cholerae transmembrane transcriptional activator ToxR; Reich KA et al.; A cross-hybridizing DNA fragment to Vibrio cholerae toxR was cloned from the nonpathogenic light organ symbiont Vibrio fischeri, and three proteins homologous to V . cholerae ToxR, ToxS, and HtpG were deduced from its DNA sequence . V . fischeri ToxR was found to activate a V . cholerae ToxR-regulated promoter, and an antiserum raised against the amino-terminal domain of V . cholerae ToxR cross-reacts V . fischeri ToxR.

J Bacteriol, 1994 May, 176(10), 3076 - 80
Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa; Gray KM et al.; Autoinduction is a conserved mechanism of cell density-dependent gene regulation that occurs in a variety of gram-negative bacteria . Autoinducible luminescence in Vibrio fischeri requires a transcriptional activator, LuxR, while a LuxR homolog, LasR, activates elastase expression in Pseudomonas aeruginosa . Both LuxR and LasR require specific signal molecules, called autoinducers, for activity . We show here the activation in Escherichia coli of the V . fischeri luminescence (lux) operon by LasR and of the P . aeruginosa elastase gene (lasB) by LuxR when each is in the presence of its cognate autoinducer . Neither LuxR nor LasR showed appreciable activity with the heterologous V . fischeri or P . aeruginosa autoinducer . This supports the view that there is a direct interaction of each transcriptional activator with its proper autoinducer and suggests that there are conserved, autoinduction-related elements within the promoter regions of these genes.

J Infect Dis, 1994 May, 169(5), 1029 - 34
Spread of Vibrio cholerae O139 Bengal in India; Nair GB et al.; Vibrio cholerae serogroup O139 Bengal, a novel strain with epidemic potential, completely displaced V . cholerae serogroup 01 in Calcutta in January 1993, which was followed by an epidemic caused by V . cholerae O139 in March-May 1993 . From November 1992 to July 1993, 95.6% of 916 V . cholerae isolates submitted to the National Institute of Cholera and Enteric Diseases from 28 locations in India were confirmed as serogroup O139 . As of July 1993, V . cholerae O139 had been isolated from 13 Indian states and a union territory, and large outbreaks of cholera caused by the O139 serogroup had occurred in several cities . The extent of spread of V . cholerae O139 Bengal in India and its ability to swiftly disseminate leaves little doubt that this is the beginning of the eighth pandemic of cholera.

Infect Immun, 1994 May, 62(5), 2108 - 10
Vibrio cholerae O139 synonym bengal is closely related to Vibrio cholerae El Tor but has important differences; Johnson JA et al.; Although Vibrio cholerae O139 synonym Bengal strains, from the current epidemics in India and Bangladesh, are closely related to seventh-pandemic strains, as shown by multilocus enzyme electrophoresis, Bengal strains are encapsulated and portions of the O1 antigen biosynthetic complex genes found in O1 strains are altered or lacking . Encapsulated Bengal strains showed resistance to killing by normal human serum . The presence of the capsule suggests the potential for bloodstream invasion in susceptible hosts and has profound implications for vaccine development.

Hear Res, 1994 May, 75(1-2), 145 - 50
Human fetal auditory threshold improvement during maternal oxygen respiration; Sohmer H et al.; It has been suggested that the near full-term fetus in-utero has a sensori-neural hearing loss compared to the neonate due to the relative hypoxia resulting from placental oxygenation compared to pulmonary oxygenation . This hypothesis was tested by estimating the threshold of the fetus to vibrio-acoustic stimulation applied to the maternal abdomen while the mother was breathing room air and again when breathing oxygen . Fetal response was assessed by maternal perception of fetal movement and by objective demonstration of movement by ultrasound . It has been shown that the fetal responses are to the acoustic component of the stimulus, that the acoustic stimulus is not overly attenuated or masked, and that maternal oxygen inhalation enhances fetal oxygenation . The results showed that the threshold was lower and/or the response was stronger when the mother was breathing oxygen compared to when she was breathing room air . Thus it is confirmed that in-utero the fetus has an hypoxia-induced sensori-neural hearing loss . At birth, with the shift to more efficient pulmonary oxygenation, there is an improvement in auditory threshold.

Can J Microbiol, 1994 May, 40(5), 408 - 11
Responses of diverse heterotrophic bacteria to elevated copper concentrations; Gordon AS et al.; The influence of copper on the growth of Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis, Vibrio parahaemolyticus, Vibrio alginolyticus (three strains), and an unidentified Vibrio sp . was examined in batch cultures . The effects of copper at micromolar concentrations varied from undetectable to complete growth inhibition . Each strain was able to recover from a growth lag observed after copper addition at a characteristic concentration . Copper concentrations that allowed recovery ranged from 25 to 150 microM . Extracellular proteins in the medium of cultures that had recovered from copper stress were compared with those from control cultures . Protein profiles were analyzed for the presence of proteins similar to extracellular copper-binding proteins (CuBP) previously reported in V . alginolyticus . CuBP-like proteins were found in each Vibrio sp . examined . A protein of similar molecular mass was also detected in copper-stressed cultures of P . aeruginosa and not in control cultures . Escherichia coli and Bacillus spp . did not produce CuBP-like proteins . The data show that CuBP-like proteins are not produced by all bacteria in response to copper stress and indicate that such proteins are common in marine Vibrio spp.

Mol Microbiol, 1994 May, 12(3), 403 - 12
Sequence and function of LuxO, a negative regulator of luminescence in Vibrio harveyi; Bassler BL et al.; Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of extracellular signal molecules (autoinducers) in the culture medium . A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode a function required for the density-dependent response . Transposon Tn5 insertions in the recombinant clone were isolated, and the mutations were transferred to the genome of V . harveyi for examination of mutant phenotypes . Expression of luminescence in V . harveyi strains with transposon insertions in one locus, luxO, was independent of the density of the culture and was similar in intensity to the maximal level observed in wild-type bacteria . Sequence analysis of luxO revealed one open reading frame that encoded a protein, LuxO, similar in amino acid sequence to the response regulator domain of the family of two-component, signal transduction proteins . The constitutive phenotype of LuxO- mutants indicates that LuxO acts negatively to control expression of luminescence, and relief of repression by LuxO in the wild type could result from interactions with other components in the Lux signalling system.

FEMS Immunol Med Microbiol, 1994 May, 8(4), 293 - 8
Production and cross-reactivity patterns of a panel of high affinity monoclonal antibodies to Vibrio cholerae O139 Bengal; Garg S et al.; A series of monoclonal antibodies of different isotypes specific for Vibrio cholerae O139, the new pandemic strain of cholera, was produced . These mAbs reacted only with the reference strain (MO45) representing serovar O139 but did not react with any of the other reference strains representing serovars O1 to O140 . Significantly, the mAbs did not agglutinate the R-cultures of V . cholerae (CA385, 20-93) which demonstrated the exceptional specificity of these mAbs and indicated that the mAbs recognized antigenic determinants unique for the O139 serovar . There was heterogeneity in the intensity of reactivity of the mAbs with strains of V . cholerae O139 isolated from diverse sources . Apart from 4H6, the other mAbs agglutinated all the O139 strains examined . 2D12 and 2F8 were the best mAbs based on the intensity of agglutination with all the O139 strains . Evaluation of 3A10 in comparison with a polyclonal anti-O139 antibody raised in rabbit using the slide agglutination format revealed that 3A10 fared as well as the polyclonal antibody for the laboratory identification of the O139 serovar . The acquisition of these mAbs provide reagents which would be very useful in the development of simple immunodiagnostic assays for the diagnosis of V . cholerae O139 infections.

Plasmid, 1994 May, 31(3), 242 - 50
Localization of the replication region of the pMJ101 plasmid from Vibrio ordalii; Bidinost C et al.; The 30-kb pMJ101 plasmid is found as a high-copy-number pool in all the pathogenic strains of Vibrio ordalii examined so far . The replication functions of pMJ101 were localized within a 2.4-kb EcoRV-HindIII restriction fragment by using different subclones in combination with Bal31 exonuclease deletions and Tn5 insertion mutants . Recombinant clones carrying this fragment were able to replicate in Escherichia coli cells deficient in either DNA Polymerase I (PolA-) or integration host factor functions . However, the viability of recombinant plasmids containing the pMJ101 origin of replication was dependent on the expression of the gene encoding the DnaA protein . Electrophoretic analysis of plasmid-encoded proteins in an in vitro transcription-translation coupled system revealed that the replication region of pMJ101 encodes a 36-kDa protein . The expression of this protein was correlated with the ability of different recombinant plasmids harboring this pMJ101 DNA region to replicate in the PolA- E . coli strain . Replication typing showed that pMJ101 is not related to any of the plasmid incompatibility groups contained in the bank of rep probes described by M . Couturier et al . (Microbiol . Rev . 52, 375-395, 1988).

J Clin Microbiol, 1994 May, 32(5), 1246 - 8
Direct detection of Vibrio cholerae in stool samples; Varela P et al.; A direct method to detect Vibrio cholerae in stool samples was developed by using a PCR procedure that did not require a DNA purification step . Dilution (1/100) of stool samples prevented inhibition of the reaction by contaminants, and two consecutive PCRs, the second one with a nested primer, achieved the desired sensitivity . Comparison of the results obtained from stool swab samples processed by the two-step PCR and by an enzyme-linked immunosorbent assay using GM1 as the capture molecule showed that the former is more sensitive and gave positive results even when V . cholerae was not culturable or dead.

Microbiology, 1994 May, 140 ( Pt 5), 1217 - 22
recA mutations reduce adherence and colonization by classical and El Tor strains of Vibrio cholerae; Kumar KK et al.; Two recA mutants of Vibrio cholerae (classical and El Tor biotypes) were constructed by disruption of the wild-type recA gene with mutated recA sequences of V . cholerae cloned in the suicide vector pGP704 . Mutants defective in the recA gene were compared with their respective RecA+ parent strains with regard to their adherence to isolated rabbit intestine and colonization of intestine of infant mice . The recA mutation in V . cholerae was found to diminish adherence and markedly affected colonization.

Appl Environ Microbiol, 1994 May, 60(5), 1684 - 6
Isolation of Vibrio cholerae O139 synonym Bengal from the aquatic environment in Bangladesh: implications for disease transmission; Islam MS et al.; Currently, Bangladesh is experiencing an epidemic of acute watery diarrhea caused by Vibrio cholerae O139 . Surface waters were collected and cultured for vibrious following enrichment . Twelve percent (11 of 92) of samples yielded V . cholerae O139, and all of them were positive for cholera toxin . The data suggest that V . cholerae O139 is easily culturable from surface water samples.

Appl Environ Microbiol, 1994 May, 60(5), 1681 - 3
A medium for presumptive identification of Vibrio anguillarum; Alsina M et al.; A medium (VAM) for differentiation of Vibrio anguillarum is described . The presence of bile salts, the high pH, and the high NaCl concentration select mainly for Vibrio species . The high salinity and the ampicillin select for a fraction of Vibrio species, and sorbitol fermentation differentiates among those vibrios still able to grow . One hundred ninety-seven of 227 strains of V . anguillarum were identified with this medium . Only 3 of 66 strains of Vibrio that were not V . anguillarum or V . anguillarum-like were recognized with this medium, and any of 7 non-Vibrio strains related to fish diseases or Escherichia coli grew on the medium . It is our contention that the medium described here constitutes an efficient instrument for presumptive detection of V . anguillarum in pathological and environmental samples.

Appl Environ Microbiol, 1994 May, 60(5), 1414 - 20
Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions; Van Dyk TK et al.; Heat shock gene expression is induced by a variety of environmental stresses, including the presence of many chemicals . To address the utility of this response for pollutant detection, two Escherichia coli heat shock promoters, dnaK and grpE, were fused to the lux genes of Vibrio fischeri . Metals, solvents, crop protection chemicals, and other organic molecules rapidly induced light production from E . coli strains containing these plasmid-borne fusions . Introduction of an outer membrane mutation, tolC, enhanced detection of a hydrophobic molecule, pentachlorophenol . The maximal response to pentachlorophenol in the tolC+ strain was at 38 ppm, while the maximal response in an otherwise isogenic tolC mutant was at 1.2 ppm . Stress responses were observed in both batch and chemostat cultures . It is suggested that biosensors constructed in this manner may have potential for environmental monitoring.

Trans R Soc Trop Med Hyg, 1994 May-Jun, 88(3), 296 - 7
The effect of iron on the survival of Vibrio cholerae O1 in dechlorinated tap water; Patel M et al.; Many factors, such as temperature, pH, organic nutrients, types of water storage containers, etc., determine the survival of Vibrio cholerae in water . Since the survival of V . cholerae O1 has been shown to be much longer in metal drums used as household water storage containers than in clay pots and plastic drums, the present study was designed to explore the possible role played by insoluble iron on the survival of V . cholerae O1 in water . The possibility of iron acting as particulate matter for the organisms to adhere to was also examined by using inert glass beads in water . Survival of V . cholerae O1 in dechlorinated tap water, with and without inert glass beads, ranged from < 24 h to 10 d . The number of surviving bacteria was, however, very low . In the presence of impure ferric oxide (Fe2O3), survival in tap water ranged from 4 to 12 d and the numbers of surviving bacteria were very high . Iron was thought to play an important role in the survival of V . cholerae O1 in water . Differences between the numbers of bacteria and the length of survival in surface water and in sediment were unremarkable . The El Tor and classical biotypes gave similar results.

J Biochem (Tokyo), 1994 May, 115(5), 868 - 74
Structure and iron transport activity of vibrioferrin, a new siderophore of Vibrio parahaemolyticus; Yamamoto S et al.; The structure of vibrioferrin, a siderophore from Vibrio parahaemolyticus, was elucidated based on a combination of partial hydrolysis and spectroscopic techniques . HPLC of purified vibrioferrin showed two peaks with an area ratio of approximately 2:1 . However, upon reinjection of each of those isolated compounds, the original chromatographic pattern was obtained, indicating an equilibrium between two compounds in aqueous solution . Consistent with this finding, most of the NMR signals of vibrioferrin were duplicated . The structure was determined as 1-(2-{2-(5-carboxy-5-hydroxy-2-oxo-1-pyrrolidinyl)propionamide}ethyl) citrate, which exists in two epimeric forms resulting from cyclization between an amidic nitrogen of the alanine residue and a keto group of the 2-ketoglutaric acid residue . Transport experiments with 55Fe-labeled vibrioferrin demonstrated the function of vibrioferrin as a siderophore in V . parahaemolyticus . Kinetic studies with mid-log phase cells revealed that the iron uptake system was receptor-mediated, with Km and Vmax values of 67 nM and 54 pmol Fe/mg cell protein/min, respectively . Moreover, iron uptake mediated by vibrioferrin was blocked both by uncouplers and by ATPase inhibitors.

Zh Mikrobiol Epidemiol Immunobiol, 1994 May-Jun, (3), 49 - 51
{The ecological and epidemiological aspects of the spread of Vibrio parahaemolyticus in a freshwater region}; Boiko AV et al.; For the first time Vibrio parahaemolyticus have been isolated in this country in a freshwater region both from patients with the diarrheal syndrome and from environmental objects . In the region of the lower course of the Volga morbidity in acute enteric infections (AEI) caused by these vibrios may reach 9.1% of all AEI cases in some years . The formation of an ecological niche suitable for the circulation of these bacteria has taken place in the Volga delta . All these facts indicate that freshwater reservoirs and their hydrobios present potential epidemic danger and, therefore, the possibility of sporadic AEI morbidity and outbreaks of infections caused by V.parahaemolyticus exists in freshwater regions with natural and climatic conditions similar to those of the region of the lower course of the Volga.

Zh Mikrobiol Epidemiol Immunobiol, 1994 May-Jun, (3), 34 - 9
{The epidemic situation re: cholera in the world: a morbidity analysis and trends}; Onishchenko GG et al.; The analysis of cholera morbidity throughout the world over the period of 1988-1992 indicates the existence of a tendency towards an increase in morbidity due to epidemic outbreaks in the countries of South and Central America and in Africa . Using the data of literature, attempts have been made to elicit the causes of the sudden appearance and spread of cholera in South America . The increase of cholera morbidity in Africa is associated with the activization of cholera in endemic foci and intensified migration caused by military conflicts in the countries of Central and East Africa . Cholera morbidity in Asia appears to be declining; however, large outbreaks of cholera, as well as diarrheal diseases clinically similar to cholera and caused by Vibrio cholerae non 01, serogroup 139, have been recorded . In Europe, including Russia, cholera outbreaks and sporadic cases, mainly imported, have been reported . The prognosis of cholera situation remain unfavorable due to the activization of epidemic processes and the constant risk of the infection being imported to any country of the world.

Gac Sanit, 1994 May-Jun, 8(42), 139 - 45
{Cholera in the world . The cholera epidemic of 1991 in Peru}; Galdos-Tanguis H; The objective of this article was to review the epidemic of cholera disease that affected Peru in 1991, its epidemiologic impact and the natural regions most affected . Also it is reviewed the state of this epidemic two years after . A description of the incidence of natural regions, the main mechanisms of transmission and the control strategy that public health authorities hold on . This review concludes that professionals and public health authorities should be prepared to control this disease, that can suddenly erupt in any country or region, due to the easy way of transmission of Vibrio cholerae.

J Med Assoc Thai, 1994 May, 77(5), 249 - 52
A second outbreak of cholera in the home for mentally handicapped, Nonthaburi; Jiraphongsa C et al.; A second outbreak of cholera, due to the Ogawa strain, occurred in the home for Mentally Handicapped Children in Nonthaburi between July 29 and August 9, 1992 . An outbreak of cholera due to the Inaba strain was reported in the same institution and season tin 1987 . In 1992, the clinical attack rate was 8 per cent of 440 children; there were two deaths . Bath water was contaminated with Vibrio cholerae O1 E1 Tor Ogawa, the same strain as was isolated from the ill children . Chlorination of the water supply, obtained from an underground well, was insufficient . The water supply needs further investigation, and the sanitary conditions in the institutions should be improved.

Mutat Res, 1994 May, 321(3), 127 - 32
Induction of adaptive response by nitrofurantoin against oxidative DNA damage in some bacterial cells; Basak J et al.; Pretreatment with a sublethal dose of nitrofurantoin did not give any protection to Vibrio cholerae OGAWA 154 (wild-type) cells against subsequent treatment with challenging doses of MNNG and vice versa . However, pretreatment with a sublethal dose of nitrofurantoin offered significant protection to the bacterial cells against subsequent treatment with challenging doses of H2O2 and vice versa . Further, sublethal doses of nitrofurantoin or H2O2 produced almost the same degree of protection against challenges by H2O2 or nitrofurantoin . Both the alkylating agent MNNG and the oxidative agent H2O2 induced adaptive responses in Vibrio cholerae OGAWA 154 cells against subsequent challenge by the respective agents . The experiments presented in this communication revealed that nitrofurantoin produced an adaptive response in bacterial cells against oxidative and not alkylating DNA damage.

Biochim Biophys Acta, 1994 Apr 28, 1185(2), 243 - 6
Cloning and nucleotide sequence of the gene for NADH:FMN oxidoreductase from Vibrio harveyi; Izumoto Y et al.; The gene encoding the enzyme NADH:FMN oxidoreductase (EC 1.6.99.3) from Vibrio harveyi has been isolated from a recombinant library of genomic DNA and sequenced . The deduced amino acid sequence, 237 amino acids long, shows 48% identity with E . coli NAD(P)H:flavin oxidoreductase and 40% identity with Vibrio harveyi luxG gene product.

Ned Tijdschr Geneeskd, 1994 Apr 23, 138(17), 873 - 5
{Sepsis caused by non-O1-Vibrio cholerae: a patient in The Netherlands}; de Groot R et al.; In a 84-year-old woman extraintestinal infection by non-OI Vibrio cholerae was diagnosed . She had septicaemia with cholangitis and cholecysto- and choledocholithiasis . Until now 26 patients with non-OI V . cholerae septicaemia have been reported . Most had an underlying disease, usually a chronic liver disease or haematological malignancy . These disorders were not present in our patient . She was treated with co-trimoxazole and afterwards she underwent a cholecystectomy and common bile duct exploration . At the time of operation no non-OI V . cholerae could be isolated from the gallbladder or the bile from the common bile duct . A possible cause of the infection was a herring which the patient had eaten six weeks before hospital admission.

Ned Tijdschr Geneeskd, 1994 Apr 23, 138(17), 871 - 3
{2 Dutch travelers returning from Thailand with cholera}; Smit AA et al.; Cholera is a disease rarely imported in the Netherlands . Recently a 34-year-old woman who had returned from a trip through Thailand was admitted to our hospital with complaints of vomiting, watery stools and moderate dehydration . Vibrio cholerae OI serotype Ogawa biotype El Tor was isolated from the faeces . She recovered after antimicrobial and fluid therapy . Her 29-year-old travelling companion had only mild symptoms of diarrhoea, but the bacterium was isolated from her stool also . Cholera should be considered in travellers with vomiting and diarrhoea coming back from Thailand.

Int J Syst Bacteriol, 1994 Apr, 44(2), 330 - 7
Sequence determination of rRNA genes of pathogenic Vibrio species and whole-cell identification of Vibrio vulnificus with rRNA-targeted oligonucleotide probes; Aznar R et al.; A comparative analysis of seven new 16S rRNA gene sequences of pathogenic Vibrio species with previously published vibrio sequences confirmed that Vibrio vulnificus represents a group that is not closely related to the core organisms of the genus Vibrio . In addition, we found that V . vulnificus, Listonella (Vibrio) anguillarum and Vibrio diazotrophicus branch off separately from the core group . A comparison of the 16S rRNA gene sequences of V . vulnificus strains belonging to biotypes 1 and 2 revealed that the sequences of all but four biotype 1 strains were identical to each other but slightly different (17 bases) from the sequences of the rest of the V . vulnificus strains investigated . In addition, the sequences of variable regions of the 23S rRNA genes of Vibrio fluvialis, Vibrio furnissii, Vibrio harveyi, Vibrio cholerae, and V . vulnificus C7184 and TW1 were determined, aligned, and compared with all available bacterial 23S rRNA sequences in order to search for specific target sites . As a result, four oligonucleotide probes specific for V . vulnificus were synthesized, and the specificities of these probes were evaluated by dot blot hybridization to membrane-bound RNAs from 21 V . vulnificus strains, 13 strains belonging to other Vibrio species, 61 strains belonging to species that are members of the alpha, beta, and gamma subclasses of the Proteobacteria, and 3 eucaryotic microorganisms . Two probes hybridized with all of the V . vulnificus strains tested, and the other two probes distinguished V . vulnificus biotype 1 strains from all other organisms . In situ identification of V . vulnificus by using tetramethylrhodamine- or fluorescein-labelled oligonucleotides is now possible.

FEMS Microbiol Lett, 1994 Apr 1, 117(2), 197 - 202
Structure and arrangement of the cholera toxin genes in Vibrio cholerae O139; Lebens M et al.; The sequence of the ctxB gene encoding the B subunit of cholera toxin has been determined for a strain of Vibrio cholerae of the novel O139 serotype associated with recent outbreaks of severe cholera throughout South-East Asia and found to be identical to the ctxB gene in V . cholerae O1 of the El Tor biotype . Analyses by Southern hybridization and PCR showed that all strains of the O139 serotype V . cholerae tested carried cholera toxin genes and other genes associated with a virulence cassette DNA region at two loci identical or homologous to those identified in the Classical rather than the El Tor biotype of V . cholerae serotype O1 although these loci in O139 could reside on restriction fragments of variable size.

J Am Acad Dermatol, 1994 Apr, 30(4), 626 - 8
Cutaneous manifestations of non-01 Vibrio cholerae septicemia with gastroenteritis and meningitis; Chan HL et al.; A 58-year-old man with diabetes had fever and chills 5 days after ingestion of raw seafood . Nausea, vomiting, watery diarrhea, bilateral calf pain, and neck stiffness subsequently developed . Generalized edema and ecchymotic patches with a vesiculobullous eruption appeared on the extremities . Four blood cultures were positive for Vibrio cholerae non-01 . The patient was successfully treated with antibiotics . This is the first documented case of V . cholerae non-01 septicemia with cutaneous lesions and meningitis in Taiwan.

J Bacteriol, 1994 Apr, 176(8), 2293 - 9
Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli; Gonzalez-Flecha B et al.; Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells . We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi . Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents . Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers . This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome . These cells exhibited increased beta-galactosidase expression as they grew into mid-log phase . This increase was ascribed to luciferase activity because beta-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium . The soxS'::luxAB plasmid transformed superoxide dismutase-deficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions . The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal . Taken collectively, these data point to the generation of significant amounts of intracellular superoxide by bacterial luciferase, the possible mechanism of which is discussed . In addition to providing insights into the role of superoxide in the activation of the SoxR protein, these results suggest caution in the interpretation of experiments using luciferase as a reporter of gene expression.

J Med Microbiol, 1994 Apr, 40(4), 246 - 51
Influence of animal passage on haemolysin and enterotoxin production in Vibrio cholerae O1 biotype El Tor strains; Tikoo A et al.; Of 43 strains of Vibrio cholerae O1 biotype El Tor isolated over a span of almost three decades (1964-1990) from stools of children and adults with diarrhoea (25 isolates) and from sewage (three) and water from the river Ganges (15) examined for production of haemolysin and its correlation with enterotoxin production, 17 isolates showed haemolysis . The majority of isolates (26), including 68% of diarrhoeal and 50% of environmental origin, were non-haemolytic . The titre of haemolysin produced was 4-16 HU/ml, irrespective of the source of isolation . Haemolytic strains caused significantly more fluid accumulation than the non-haemolytic strains in the rabbit ileal loop (RIL) test . Twenty nine (67.4) V . cholerae biotype El Tor isolates--all the haemolytic and most (61.5%) of the non-haemolytic isolates tested--caused fluid accumulation . The remaining non-haemolytic strains that caused little or no accumulation of fluid did so after one to four consecutive passage(s) through RIL without change in haemolytic character; these strains required more consecutive passage through rabbit gut to show haemolysis . All these strains reverted to their original non-haemolytic character on repeated subculture or on storage in the laboratory but continued to show enterotoxic activity . The present study indicated that El Tor haemolysin is not responsible for fluid accumulation in rabbit gut.

Epidemiol Infect, 1994 Apr, 112(2), 285 - 90
Seasonal variations in the occurrence of Vibrio vulnificus along the Dutch coast; Veenstra J et al.; The seasonal variation in the occurrence of V . vulnificus in relation to water temperature and salinity was studied along the Dutch coast . In two consecutive years V . vulnificus strains could be isolated in August when the water temperature was highest . The indole-positive strains isolated from North Sea water samples were identical to most strains isolated from human disease and from the environment . However, strains isolated from four of five patients living in countries around the North Sea were different from the North Sea isolates in that they were indole-negative and have a lower NaCl tolerance.

Biometals, 1994 Apr, 7(2), 109 - 16
Structure of vulnibactin, a new polyamine-containing siderophore from Vibrio vulnificus; Okujo N et al.; A new siderophore named vulnibactin has been isolated from low iron cultures of Vibrio vulnificus, a human pathogen . The structure was established as N-{3-(2,3-dihydroxybenzamido)propyl}-1,3-bis{2-(2-hydroxy-phenyl)- trans-5 - methyl-2-oxazoline-4-carboxamido}propane by a combination of acid hydrolysis, nuclear magnetic resonance spectroscopy and positive fast atom bombardment mass spectrometry . Vulnibactin is characterized as containing one residue of 2,3-dihydroxybenzoic acid as well as two residues of salicylic acid, both of which are involved in the formation of oxazoline rings with L-threonine bound to a norspermidine backbone . In addition, two other compounds with siderophore activity were purified and their structures were also determined . These two compounds provided further support for the structure of vulnibactin.

J Bacteriol, 1994 Apr, 176(7), 1985 - 91
Competition between Vibrio fischeri strains during initiation and maintenance of a light organ symbiosis; Lee KH et al.; Colonization of the light-emitting organ of the Hawaiian squid Euprymna scolopes is initiated when the nascent organ of a newly hatched squid becomes inoculated with Vibrio fischeri cells present in the ambient seawater . Although they are induced for luminescence in the light organ, these symbiotic strains are characteristically non-visibly luminous (NVL) when grown in laboratory culture . The more typical visibly luminous (VL) type of V . fischeri co-occurs in Hawaiian seawater with these NVL strains; thus, two phenotypically distinct groups of this species potentially have access to the symbiotic niche, yet only the NVL ones are found there . In laboratory inoculation experiments, VL strains, when presented in pure culture, showed the same capability for colonizing the light organ as NVL strains . However, in experiments with mixed cultures composed of both VL and NVL strains, the VL ones were unable to compete with the NVL ones and did not persist within the light organ as the symbiosis became established . In addition, NVL strains entered light organs that had already been colonized by VL strains and displaced them . The mechanism underlying the symbiotic competitiveness exhibited by NVL strains remains unknown; however, it does not appear to be due to a higher potential for siderophore activity . While a difference in luminescence phenotype between VL and NVL strains in culture is not likely to be significant in the symbiosis, it has helped identify two distinct groups of V . fischeri that express different colonization capabilities in the squid light organ . This competitive difference provides a useful indication of important traits in light organ colonization.

Infect Immun, 1994 Apr, 62(4), 1480 - 3
Potential for reacquisition of cholera enterotoxin genes by attenuated Vibrio cholerae vaccine strain CVD 103-HgR; Kaper JB et al.; The potential for reacquisition of ctxA genes by attenuated Vibrio cholerae O1 vaccine strain CVD 103-HgR was examined by performing a series of mating experiments under a variety of in vivo and in vitro conditions . We found no evidence that CVD 103-HgR could reacquire ctxA genes from wild-type V . cholerae O1 strains . However, if the donor V . cholerae O1 strains were genetically manipulated to add genes that allow chromosomal gene transfer, then ctxA sequences could be acquired by CVD 103-HgR . The minimal excretion of CVD 103-HgR by vaccinees and the refractoriness to reacquisition of ctxA sequences suggest that this well-tolerated, highly immunogenic live oral cholera vaccine will have a minimal environmental impact.

Eur J Clin Microbiol Infect Dis, 1994 Apr, 13(4), 299 - 303
Molecular subtyping of Vibrio cholerae O1 strains recently isolated from patient, food and environmental samples in Spain; Usera MA et al.; Nineteen Vibrio cholerae O1 strains isolated in Spain from patient, food and environmental samples in the period 1990-1992 were characterized by detection of cholera toxin by enzyme immunoassay, detection of cholera toxin gene by polymerase chain reaction, and by biotyping, ribotyping and pulsed-field gel electrophoresis . Ten isolates were toxigenic and were further characterized by multilocus enzyme electrophoresis . Molecular subtyping methods allowed precise differentiation between isolates, indicating their geographic origin . Isolates associated with the ongoing seventh pandemic were distinguishable from those associated with the present Latin American epidemic . All isolates from the environment and seafood were nontoxigenic, and were genetically different and more diverse than toxigenic isolates . The data suggest that a focus of endemic cholera does not exist in Spain, and that the analyzed nontoxigenic Vibrio cholerae O1 isolates from imported seafood were not a threat to public health.

Ann Trop Med Parasitol, 1994 Apr, 88(2), 109 - 22
Cholera; Shears P; Although it is more than a century since the discovery of the vibrio bacillus, cholera remains one of the great epidemic diseases of the tropical world . The epidemiology of cholera is an interaction between the biological and ecological properties of Vibrio cholerae and the complex patterns of human behaviour in tropical environments . The seventh pandemic has spread through all areas of the tropics, and cholera has become endemic in many new areas . The view that cholera was primarily water borne and that humans were the only long-term reservoir has been challenged by the discovery that V . cholerae can survive, often in a dormant state, in aquatic environments . The recent appearance of V . cholerae 0139, a new serotype that causes a disease clinically and epidemiologically indistinct from cholera, has further complicated our understanding of this ancient disease . Developments in the molecular characterization of V . cholerae are providing new information to explain the genetic and epidemiological variations.

New Microbiol, 1994 Apr, 17(2), 155 - 8
A microbiological assay for determining sarafloxacin and oxolinic acid concentrations in Atlantic salmon plasma; Giles JS et al.; A microbiological assay was developed to quantify the concentrations of the quinolone antibiotics sarafloxacin and oxolinic acid in Atlantic salmon (Salmo salar) plasma . The assay was a modification of the Association of Official Analytical Chemists (AOAC) method for antibiotic concentrations in feeds using disk diffusion and a lawn of Vibrio anguillarum ATCC 19264 as the test organism . With these modifications, sensitivities of 0.04 microgram/mL for sarafloxacin and 0.125 micrograms/mL for oxolinic acid were obtained.

Protein Expr Purif, 1994 Apr, 5(2), 198 - 204
Purification of the B-subunit oligomer of Escherichia coli heat-labile enterotoxin by heterologous expression and secretion in a marine vibrio; Amin T et al.; Heat-labile enterotoxins (Etx) are plasmid-encoded, multimeric proteins produced by certain diarrheagenic strains of Escherichia coli . The nontoxic, receptor-binding B subunit (EtxB) of such toxins may be useful as a component of vaccines against enterotoxigenic E . coli, or as a carrier for the delivery of heterologous epitopes to the mucosal immune system . Here we describe a simple method for the purification of EtxB from a marine vibrio harboring a broad-host range controlled expression vector containing the etxB gene . Induction of EtxB resulted in its specific secretion to the medium, to a concentration of greater than 25 mg/liter of culture . The techniques of ultrafiltration and hydrophobic interaction chromatography were used to purify EtxB to homogeneity from the medium of this organism (with a yield of 60.7%) . EtxB-epitope fusion proteins were also successfully expressed and secreted in this marine vibrio, suggesting that this system may be of general use in the preparation of EtxB-based vaccines.

Indian J Med Res, 1994 Apr, 99, 159 - 61
Validity of new phage typing scheme against Vibrio cholerae 01 biotype ElTor strains; Sarkar BL et al.; A total of 538 strains of V . cholerae 01 biotype ElTor were phage typed by the conventional Basu and Mukerjee and also the new typing scheme developed at the National Institute of Cholera and Enteric Diseases, Calcutta . The strains could be clustered into seven types by the new scheme as against only two by the conventional method . The results provide conclusive evidence on the validity of the new scheme for phage typing of V . cholerae strains.

Mol Microbiol, 1994 Apr, 12(1), 71 - 82
Longus: a long pilus ultrastructure produced by human enterotoxigenic Escherichia coli; Giron JA et al.; Enterotoxigenic Escherichia coli (ETEC) causes an acute cholera-like diarrhoea in both humans and animals . We describe a new pilus termed longus produced by ETEC, which can extend for over 20 microns from the cell surface . Longus is composed of a repeating subunit of 22 kDa and its NH2-terminal amino acid sequence revealed homology with the toxin-coregulated pilus of Vibrio cholerae, the bundle-forming pilus of enteropathogenic E . coli and type IV pilins of some Gram-negative bacterial pathogens . The longus structural gene (lngA) is encoded in a large plasmid and was cloned in a 5 kb fragment, which proved to be sufficient for pilus production and assembly in E . coli K-12 . The presence of lngA was restricted to human ETEC strains . In contrast to other ETEC pili, lngA was widely distributed among ETEC strains independent of their geographical origin, serotype, toxin production, or other pili antigens expressed . Longus is a new member of the type IV pili family, which may represent a highly conserved intestinal colonization factor of ETEC . Common antigenic determinants exist among longus and their pilin subunits, produced by heterologous ETEC . Longus could be significant in the immunoprophylaxis of diarrhoeal disease caused by ETEC, especially against those strains in which no colonization factors have been identified and that produce heat-stable toxin only.

Mem Inst Oswaldo Cruz, 1994 Apr-Jun, 89(2), 221 - 3
Vibrio fluvialis attachs to but does not enter HeLa cell monolayers; Carvalho IT et al.; Considering the possibility that invasiveness could be a neglected factor of virulence in Vibrio fluvialis-linked enteritis, since a dysenteric form of the disease was seen in Bangladesh, we studied 12 Brazilian strains of the organism, six clinical and six environmental, to determine whether they might be able to enter into HeLa cell monolayers or would carry plasmids incidentally involved in invasiveness . Four human and two environmental isolates attached to but did not enter into the cells . Though five strains harbored plasmids, no relationship was found between the carriage of these genetic elements and adhesiveness.

Glycoconj J, 1994 Apr, 11(2), 97 - 104
An improved method for the measurement of total lipid-bound sialic acids after cleavage of alpha 2,8 sialic acid linkage with Vibrio cholerae sialidase in the presence of cholic acid, SDS and Ca2+; Maliakal MA et al.; In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD3 and GD2) are not involved because their alpha 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment . However, the sialic acids from these glycolipids can be recovered completely after cleavage of alpha 2,8 linkages by V . cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium . Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides . Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca2+ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.

Glycoconj J, 1994 Apr, 11(2), 89 - 95
Identification and characterization of the Sda beta 1,4,N-acetylgalactosaminyltransferase from pig large intestine; Malagolini N et al.; The high occurrence in large intestine epithelial cells from pig of a beta-N-acetylgalactosaminyltransferase with a substrate specificity very similar to that of the Sda beta 1,4-N-acetylgalactosaminyltransferase from other tissues is reported . The enzyme strictly recognized the NeuAc alpha 2,3Gal beta terminal sequence of N- and O-linked oligosaccharides bound to glycoproteins . The transferase activity required Mn2+ and an optimum pH of 7.4 . In contrast to the kidney Sda-enzyme from humans and other mammals, the microsomal fraction of pig colonic cells expressed a very high activity even in the absence of Triton X-100 . A rapid procedure is presented for the large scale preparation of GalNAc beta 1,4(NeuAc alpha 2,3)Gal beta 1,4Glc from NeuAc alpha 2,3Gal beta 1,4Glc . The biosynthesized tetrasaccharide was completely resistant to the action of neuraminidase from Vibrio cholerae, whereas about 60% of N-acetylneuramic acid was cleaved by neuraminidase from Newcastle disease virus . HPLC separation of different compounds is reported.

J Clin Microbiol, 1994 Apr, 32(4), 1050 - 3
Molecular analysis of rRNA and cholera toxin genes carried by the new epidemic strain of toxigenic Vibrio cholerae O139 synonym Bengal; Faruque SM et al.; Vibrio cholerae O139 synonym Bengal recently caused large epidemics of cholera-like disease in Bangladesh and India . We compared the restriction fragment length polymorphisms of ctxA and rRNA genes (ribotypes) in 27 isolates of V . cholerae O139 from patients in Bangladesh and India with those of 48 isolates of V . cholerae O1 from patients and 21 V . cholerae isolates from surface waters in Bangladesh, which included 2 O139 and 19 other non-O1 isolates . Ribotyping of the isolates with BglI revealed that all 29 isolates of O139 vibrios belonged to a single ribotype, suggesting a clonal nature of the infection . However, the O139 vibrios comprised two ctxA genotypes and carried three or more copies of the ctxA gene, and the chromosomal locations of these copies were unlike those of the El Tor or classical vibrios . Analysis of the restriction fragment length polymorphisms of the rRNA genes suggested that V . cholerae O139 isolates are more closely related to El Tor strains of V . cholerae O1 than were 19 other non-O1 vibrios and 33 classical V . cholerae O1 isolates that were studied . However, further studies are needed to determine whether V . cholerae O139 originated from mutations and genetic changes in a V . cholerae O1 strain or was due to the acquisition of virulence genes by a previously unknown V . cholerae non-O1 strain.

Infect Immun, 1994 Apr, 62(4), 1504 - 6
Comparison of Vibrio cholerae O139 with V . cholerae O1 classical and El Tor biotypes; Calia KE et al.; Vibrio cholerae O139 is a recently identified non-O1 V . cholerae strain responsible for outbreaks of epidemic cholera in India, Bangladesh, and Thailand in the past 2 years . Other workers have demonstrated the presence of the cholera toxin genetic element in V . cholerae O139, unlike the situation for other non-O1 V . cholerae strains . We sought to compare further this strain with strains of V . cholerae O1, classical and El Tor biotypes, by classic microbiologic methods, Southern blot analysis for restriction fragment length polymorphisms with probes for iron-regulated genes of V . cholerae O1, and comparisons of outer membrane protein profiles . Our results were similar for V . cholerae O139 and the El Tor biotype of V . cholerae O1, with the exception of the constitutive expression in V . cholerae O139 of OmpS, an outer membrane protein that was maltose inducible in comparison strains of V . cholerae O1.

Biochim Biophys Acta, 1994 Mar 23, 1190(2), 465 - 8
Cloning and sequencing of an Na+/H+ antiporter gene from the marine bacterium Vibrio alginolyticus; Nakamura T et al.; A gene has been cloned from a DNA library from the marine bacterium Vibrio alginolyticus that functionally complements a mutant strain of Escherichia coli, NM81, defective in an Na+/H+ antiporter (NhaA) . The cloned Vibrio gene restored NM81 to grow in a medium containing 0.5 M NaCl at pH 7.5 and concomitantly led to an increase in Na+/H+ antiport activity . The nucleotide sequence of the cloned fragment revealed an open reading frame, which encodes a protein with a predicted 383 amino acid sequence and molecular mass of 40,400 Da . The hydropathy profile is characteristic of a membrane protein with 11 membrane spanning regions . The deduced amino acid sequence is 58% identical with E . coli NhaA.

Carbohydr Res, 1994 Mar 18, 256(1), 113 - 28
Identification of a novel sugar, 4-amino-4,6-dideoxy-2-O-methylmannose in the lipopolysaccharide of Vibrio cholerae O1 serotype Ogawa; Ito T et al.; A novel sugar in the lipopolysaccharide of Vibrio cholerae O1 serotype Ogawa has been identified . The sugar was liberated from the lipopolysaccharide when hydrolyzed in 10 M HCl at 90 degrees C for 15 min . The sugar was purified and identified as 4-amino-4,6-dideoxy-2-O-methylmannose (2-O-methylperosamine) . Since it was found only in the lipopolysaccharide of Vibrio cholerae O1 serotype Ogawa, it seems that the sugar is one of the specific constituents determining Ogawa serotype specificity.

FEMS Microbiol Lett, 1994 Mar 15, 117(1), 47 - 51
Efficient extracellular production of hybrid E . coli heat-labile enterotoxin B subunits in a marine Vibrio; Marcello A et al.; Escherichia coli heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides to target cells, particularly for the oral delivery of epitopes to the mucosal immune system . In this study, two extensions to the C-terminus of EtxB were genetically engineered that correspond to a well-characterized neutralising epitope of glycoprotein D from herpes simplex virus (EtxB-gD) and to the C-terminal nine amino acids from the 38 kDa subunit of HSV-encoded ribonucleotide reductase (EtxB-R2) . Here we describe the extracellular secretion of the two hybrid EtxBs from a marine Vibrio harbouring a broad-host range inducible expression vector containing the hybrid genes . Large amounts of intact fusion proteins (15-20 mg per liter of culture) were secreted into the medium upon induction . These hybrid proteins maintained the receptor-binding activity of the native toxin as well as being cross-reactive with anti-EtxB and anti-heterologous peptide monoclonal antibodies.

Biochem J, 1994 Mar 15, 298 Pt 3, 675 - 80
Nucleotide sequence of a novel arylesterase gene from Vibro mimicus and characterization of the enzyme expressed in Escherichia coli; Shaw JF et al.; A gene coding for an arylesterase of Vibrio mimicus was cloned . Sequence determination reveals that the esterase gene has an open reading frame of 600 nucleotides which encodes a protein of M(r) 22,300 . The deduced amino acid sequence contain a pentapeptide GDSLS (residues 27-31), which was also found in the phospholipid-cholesterol acyltransferase from Aeromonas hydrophila . Substitution of Ser-29 by alanine or cysteine in the cloned gene abolished the esterase activity in the tributyrin plate assay . On the other hand, the activity was not lost when Ser-31 was changed to alanine . The cloned gene was expressed in Escherichia coli, and the protein purified by a four-step procedure . The purified protein migrated on SDS/PAGE as a single band with an apparent M(r) of 22,100 . This enzyme favoured the hydrolysis of several arylesters and was classified as an arylesterase (EC 3.1.1.2) . N-Terminal analysis showed that Ser-20 was the first amino acid of the mature secreted protein, suggesting that the N-terminal 19 hydrophobic amino acids served as a signal peptide.

Arch Intern Med, 1994 Mar 14, 154(5), 551 - 6
Cholera in the United States, 1965-1991 . Risks at home and abroad; Weber JT et al.; OBJECTIVE: To assess risks for cholera in the United States . DESIGN: Review of published reports of cholera outbreaks and sporadic cases and Centers for Disease Control and Prevention (CDC) memoranda and laboratory reports . PATIENTS: Persons with symptomatic laboratory-diagnosed cholera treated in the United States and territories . RESULTS: From 1965 through 1991, 136 cases of cholera were reported . Fifty-three percent of the patients were hospitalized and three persons died (case-fatality rate, 0.02) . Ninety-three infections were acquired in the United States and 42 overseas; for one case the source was unknown . Domestically acquired cholera was largely related to the endemic Gulf Coast focus of Vibrio cholerae 01 (56 cases) . The major domestic food vehicle was shellfish, particularly crabs harvested from the Gulf of Mexico or nearby estuaries . In 1991, 14 (54%) of 26 domestically acquired cases were caused by food from Ecuador (n = 11) and Thailand (n = 3) . During 1991, the first cases of cholera in travelers returning from South America were reported . In 1991, the rate of cholera among air travelers returning from South America was estimated as 0.3 per 100,000; among air travelers returning from Ecuador, 2.6 per 100,000 . CONCLUSIONS: Cholera remains a small but persistent risk in the United States and for travelers . An endemic focus on the Gulf Coast, the continuing global pandemic, and the epidemic in South America make this likely to continue for years to come . Physicians should know how to diagnose and treat cholera and should report all suspected cases to their state health departments.

Gene, 1994 Mar 11, 140(1), 67 - 71
The DNA adenine methyltransferase-encoding gene (dam) of Vibrio cholerae; Bandyopadhyay R et al.; The DNA adenine methyltransferase (MTase)-encoding gene (dam) of Vibrio cholerae, an organism belonging to the family Vibrionaceae, has been cloned and the complete nucleotide (nt) sequence determined . V . cholerae dam encodes a 21.5-kDa protein and is directly involved in methyl-directed DNA mismatch repair . It can substitute for the Escherichia coli enzyme and can suppress the phenotypic traits associated with E . coli dam mutants . Overproduction of V . cholerae Dam MTase does not result in hypermutability in either V . cholerae or E . coli cells . Overproduction of V . cholerae Dam in a pUC plasmid, however, fails to suppress the 2-aminopurine (2-AP)-sensitive phenotype of E . coli dam mutants . Homology between the nt and deduced amino acid (aa) sequences of the E . coli and V . cholerae dam genes is only 30-35%.

J Clin Microbiol, 1994 Mar, 32(3), 856 - 7
Rapid detection of Vibrio cholerae O1 in stools of Peruvian cholera patients by using monoclonal immunodiagnostic kits . Loyaza Cholera Working Group in Peru; Carillo L et al.; We compared stool culture with two commercial Vibrio cholerae O1 rapid diagnostic kits which detect antigen in 100 adults with cholera in Peru . Serum vibriocidal-antibody titer was used as an external reference . Both rapid diagnostic kits appeared to detect cholera more frequently than did culture and were highly specific.

Genetika, 1994 Mar, 30(3), 337 - 41
{Lon-protease participates in the regulation of transcription of the Lux-operon of Vibrio fischeri}; Zavil'gel'skii GB et al.; Lon protease of Escherichia coli specifically inhibits the activity of the regulatory protein LuxI responsible for synthesis of the autoinducer N-(3-oxo-hexanoyl)homoserine lactone . As a result, transcription of the right lux operon is initiated in lon+ bacteria much later than in lon- mutants . Lon protease does not affect the activity of LuxAB proteins (luciferase) and LuxC, LuxD, and LuxE proteins involved in synthesis of an aldehyde substrate for luciferase.

Mol Gen Mikrobiol Virusol, 1994 Mar-Apr, (2), 25 - 8
{Mapping a genetic determinant determining the increased synthesis of cholera toxin by the Dakka 35 strain of Vibrio cholerae}; Smirnova NI et al.; A new mutation tox-2 defining the increased level of cholera exotoxin production by the strain Vibrio cholerae Dakka 35 isolated from nature has been mapped by conjugational crosses of donor and recipient strains differing by toxin production and serovar . The mutation has been localized on the chromosomal fragment containing the ilv, pur, ura, rfb genes adjacent to ura-94 locus . The linkage of the tox-2 mutation with the rfb locus coding for the synthesis of somatic Ol-antigen has been also established.

Vaccine, 1994 Mar, 12(4), 359 - 64
Recombinant derivative of a naturally occurring non-toxinogenic Vibrio cholerae 01 expressing the B subunit of cholera toxin: a potential oral vaccine strain; Dasgupta U et al.; A clinical isolate of Vibrio cholerae 01 was identified which did not possess the heat-labile (CT), the heat-stable (ST) or the zonula occludens (Zot) toxin genes . Rabbit ileal loop assays showed that no other CT-like toxin was produced by this strain . The partly deleted cholera toxin gene which carries the intact gene for the B subunit was cloned and the recombinant plasmid, pURD110, was introduced into this non-toxinogenic natural human isolate . The transformed cells (strain URD2) secreted the B subunit gene product which competed with the holotoxin secreted by the hypertoxinogenic strain 569B of V . cholerae for the GM1 ganglioside binding sites in vivo . This strain can colonize the rabbit intestine as detected by the removable intestinal tie adult rabbit diarrhoea (RITARD) model . This construct has an advantage over other live oral attenuated V . cholerae strains used as vaccines in that the latter strains were made non-toxinogenic by only deleting part of the gene coding for the A subunit of cholera toxin while the strain described here is naturally non-toxinogenic.

Appl Environ Microbiol, 1994 Mar, 60(3), 984 - 8
Densities of Vibrio vulnificus in the intestines of fish from the U.S . Gulf Coast; DePaola A et al.; Densities of Vibrio vulnificus in the intestinal contents of various finfish, oysters, and crabs and in sediment and waters of the U.S . Gulf Coast were determined by the most probable number procedure . Species were identified by enzyme immunoassay . During the winter, densities of V . vulnificus were low, and the organism was isolated more frequently from sheepshead fish than from sediment and seawater . From April to October, V . vulnificus densities were considerably higher (2 to 5 logs) in estuarine fish than in surrounding water, sediment, or nearby oysters and crustacea . Highest densities were found in the intestinal contents of certain bottom-feeding fish (10(8)/100 g), particularly those that consume mollusks and crustaceans . Densities of V . vulnificus in fish that feed primarily on plankton and other finfish were similar to those in oysters, sediment, and crabs (10(5)/100 g) . V . vulnificus was found infrequently in offshore fish . The presence of high densities of V . vulnificus in the intestines of common estuarine fish may have both ecological (growth and transport) and public health (food and wound infections) implications.

Appl Environ Microbiol, 1994 Mar, 60(3), 908 - 12
Distribution of tetracycline resistance determinants among gram-negative bacteria isolated from polluted and unpolluted marine sediments; Andersen SR et al.; Tetracycline-resistant gram-negative bacteria were isolated from four different marine sediments in Scandinavia and analyzed with DNA probes for the determinant classes A to E . Colony hybridizations of 429 isolates revealed that class E is the dominating resistance determinant in these marine sediments . Comparison of fecally polluted and unpolluted sediments showed few determinant classes in unpolluted sediment and a complex composition of several determinant classes in polluted sediment . Total DNA extraction and analysis with DNA probes for determinant classes A to E resulted in no hybridization signal, because of the low number of gram-negative tetracycline-resistant bacteria . Identification of class E isolates revealed that this determinant is present not only in Aeromonas hydrophila, Escherichia coli, and Vibrio salmonicida but also in additional strains.

J Med Microbiol, 1994 Mar, 40(3), 194 - 6
Vibrio mimicus with multiple toxin types isolated from human and environmental sources; Ramamurthy T et al.; A collection of 13 strains of Vibrio mimicus, including both clinical and environmental isolates from different geographic regions, was examined for various toxins . One strain of environmental origin produced cholera-like toxin (CT) which was completely absorbed with anti-CT immunoglobulin G, five strains produced a haemolysin that cross-reacted with the thermostable direct haemolysin of V . parahaemolyticus and DNA from two strains hybridised with a DNA probe specific for the heat-stable enterotoxin of V . cholerae non-O1 . Culture supernates of all strains produced a factor that was cytotoxic to Vero and Chinese hamster ovary cells . In this study, we were able to identify strains of V . mimicus that produced, or had the genetic potential to produce, several toxin types simultaneously . The role of these strains as genetic reservoirs is discussed.

Infect Immun, 1994 Mar, 62(3), 887 - 91
Oral administration of polymeric immunoglobulin A prevents colonization with Vibrio cholerae in neonatal mice; Lee CK et al.; A simple animal model was used to demonstrate passive protection by immunoglobulin A (IgA) against a mucosal pathogen, Vibrio cholerae . Oral administration of a monoclonal IgA directed against a lipopolysaccharide component of the vibrio protected neonatal mice against oral challenge, as measured by reduced intestinal colonization . A single dose of 0.1 microgram of polymeric monoclonal IgA given 1 h prior to challenge reduced the number of recoverable vibrios by at least 100-fold . An additional dose 3 h before challenge or 1 h after challenge did not enhance protection . A 10-fold-higher concentration of monomeric IgA was required to achieve the same level of protection as that conferred by polymeric IgA . Polymeric IgA digested with trypsin or human duodenal aspirates to lower-molecular-weight fragments retained most of its ability to protect mice against challenge.

Microb Pathog, 1994 Mar, 16(3), 235 - 41
Vibrio cholerae O139 Bengal possesses a capsular polysaccharide which may confer increased virulence; Weintraub A et al.; A newly described Vibrio cholerae serogroup--O139 Bengal, the causative agent of the recent large epidemics of cholera-like disease in the Indian subcontinent and neighbouring countries--possesses a high molecular weight capsular polysaccharide (CPS) that can be visualized by electron microscopy and in composition differs from the lipopolysaccharide (LPS) . The CPS and LPS can be separated from each other by a two-step extraction procedure, a phenol-water extraction in order to extract all polysaccharides from the bacterial suspension followed by a phenol-chloroform-petroleum ether (PCP) extraction . The CPS is mainly composed of 3,6-dideoxyhexose (abequose or colitose), quinovosamine and glucosamine . The LPS of the O139 Bengal strain appears to possess a short polysaccharide which contains glucose, galactose, glucosamine and heptose . Both the LPS and CPS are immunogenic . They react in an enzyme immunoassay with rabbit antibodies generated against whole heat-killed bacteria . By analogy with other capsulated bacteria, the possession of a capsule may confer increased virulence of O139 Bengal.

Indian J Med Res, 1994 Mar, 99, 97 - 100
The appearance & spread of Vibrio cholerae 0139 in India; Jesudason MV et al.; A new clone of non-01 V . cholerae designated as serogroup 0139, which produces cholera toxin, was detected first in south India in September 1992 and has spread to many parts of India since then . It was identified in Bangladesh in December 1992 and in Thailand in April 1993 . By May 1993 it was found in Haryana and Punjab . Its clinical manifestations are typical of cholera, occurring in outbreaks . This clone has largely replaced the previously prevalent 01 V . cholerae in several cholera endemic areas indicating that a new cholera pandemic has begun.

Indian J Med Res, 1994 Mar, 99, 107 - 8
Outbreak of gastroenteritis due to a new strain of non O group 1 Vibrio cholerae, in Ludhiana in May-August 1993; Prabhakar H et al.; The isolation of the new serotype 0139 of non 01 V . cholerae from an outbreak of gastroenteritis is reported . The study of 35 such isolates revealed their similarity with the El tor vibrios biochemically and by other characters . All were strongly haemolytic and 97.1 per cent of the strains showed a positive haemagglutination.

Indian J Med Res, 1994 Mar, 99, 105 - 6
Emergence of Vibrio cholerae serogroup 0139 in Haryana in May-June 1993; Sabherwal U et al.; In an outbreak of acute watery diarrhoea, 11 strains of V . cholerae were isolated in May-June 1993 at Medical College, Rohtak . Eight of these belonged to serogroup Ogawa and three were identified as V . cholerae serogroup 0139 . This is the first report of isolation of this novel serotype from this region.

Indian J Med Res, 1994 Mar, 99, 103 - 4
Characteristics of Vibrio cholera 0139 strains isolated in Sevagram (Maharashtra) during April-August 1993; Narang P et al.; A total of 44 strains of Vibrio 0139 serotype isolated between April and August 1993 at Sevagram (Wardha) were examined for expression of a number of biochemical and physiological characteristics . All strains fermented lactose within 24 h and belonged to Heiberg group III . Salt tolerance to 8 per cent NaCl was seen in 22.72 per cent strains . Haemolysis of sheep RBCs and haemagglutination of human 'O', chicken and rabbit RBCs was consistently positive . All the strains were sensitive to tetracycline and resistant to polymyxin B and cotrimoxazole.

Indian J Med Res, 1994 Mar, 99, 101 - 2
Cholera outbreak due to Vibrio cholerae serogroup 0139 in Yavatmal (Maharashtra) in March-July, 1993; Jalgaonkar SV et al.; A total of 34 strains of V . cholerae were isolated during March to July, 1993 . Of the 34 V . cholerae isolated 26 strains were non 01 and remaining eight were 01 El tor vibrio . Non-01 strains were identified as novel epidemic strains designated as 0139 . The shift in the relative and absolute prevalence of V . cholera serotype 01 and non 01 was noted.

West Indian Med J, 1994 Mar, 43(1), 7 - 8
The occurrence of non-01 Vibrio cholerae in non-potable water samples in Barbados; Moore R et al.; Fourteen freshwater or brackish-water samples taken from different sites were examined for the presence of Vibrio cholerae . Standard enrichment techniques, using pre-incubation in alkaline peptone water and plating on thiosulfate citrate bile sucrose agar (TCBS) followed by biochemical, physiological and morphological characterization of the isolates, revealed the presence of Vibrio cholerae at nine of the sites examined . Serotyping for type 01 only was performed . All the strains isolated were non-01 Vibrio cholerae.

Bull Pan Am Health Organ, 1994 Mar, 28(1), 76 - 9
Food protection activities of the Pan American Health Organization; The DnaK homologue of the marine Vibrio sp . strain S14 binds to the unprocessed form of a carbon starvation-specific periplasmic protein; Department of General and Marine Microbiology, University of Goteborg, SwedenThe Escherichia coli DnaK homologue in Vibrio sp . strain S14 was shown to possess chaperone function for translocation during carbon starvation . This was demonstrated by using the method of co-immunoprecipitation . DnaK co-precipitated with the carbon starvation-specific periplasmic space protein Csp5 three hours after the onset of carbon starvation . Pulse-chasing of the protein with radiolabelled methionine followed by the addition of an excess of unlabelled methionine demonstrated that the Csp5 protein was translocated across the inner membrane . Only the cytoplasmic unprocessed precursor form of Csp5 co-precipitated with DnaK . The non-covalent binding between the two proteins was found to be ATP-dependent, as the addition of ATP released the interaction between DnaK and the precursor form of Csp5, as was shown on silver-stained SDS-polyacrylamide gels and by Western blot analysis . We suggest that DnaK maintains the carbon starvation-inducible protein Csp5 in a translocation-competent form in the cytoplasm.

J Biol Chem, 1994 Feb 25, 269(8), 5612 - 8
Mechanism of aldehyde inhibition of Vibrio harveyi luciferase . Identification of two aldehyde sites and relationship between aldehyde and flavin binding; Lei B et al.; Vibrio harveyi luciferase is sensitive to aldehyde substrate inhibition, and two kinetic schemes have been previously postulated to account for such an inhibition . One scheme depicts a sequential binding of 2 aldehyde molecules, yielding an active enzyme-aldehyde binary complex and subsequently an inactive enzyme-(aldehyde)2 ternary complex (Holzman, T . F., and Baldwin, T . O . (1983) Biochemistry 22, 2838-2846) . This two-aldehyde model was later withdrawn, and recently, a different scheme was proposed, following which the prior binding of one aldehyde to the native luciferase forms an inactive dead-end complex (Abu-Soud, H . M., Clark, A . C., Francisco, W . A., Baldwin, T . O., and Raushel, F . M . (1993) J . Biol . Chem . 268, 7699-7706) . In this work, kinetic and equilibrium studies were carried out to elucidate further the mechanism of aldehyde inhibition . Two, presumably independent, aldehyde-binding sites were detected, with a higher affinity site for the aldehyde substrate and a weaker affinity site for the aldehyde inhibitor . Binding to and dissociation from the inhibitor site by decanal were revealed by chemical relaxation analysis to be slow processes . Furthermore, whereas the binding of the decanal substrate enhances the affinity of the reduced riboflavin 5'-phosphate (FMNH2) site, the binding of decanal to the inhibitor site competes against FMNH2 binding, thus resulting in inhibition of luciferase activity . These findings are not compatible with either of the two earlier schemes mentioned above . A new kinetic model is formulated for the mechanism of aldehyde inhibition . Theoretical kinetic behaviors predicted on the basis of this model are in excellent agreement with experimental observations . A particularly reactive cysteine (residue 106) on the alpha subunit has been previously demonstrated to be at or near an aldehyde site (Fried, A., and Tu, S.-C . (1984) J . Biol . Chem . 259, 10754-10759) . Evidence is presented to indicate that this residue is at or near the aldehyde inhibitor site . Relative locations of this residue and binding sites for FMNH2, the aldehyde substrate, and the aldehyde inhibitor are proposed.

FEMS Microbiol Lett, 1994 Feb 15, 116(2), 215 - 9
Mineralization of monofluorobenzoate by a diculture under sulfate-reducing conditions; Drzyzga O et al.; A mesophilic, dehalogenating, sulfate-reducing diculture was isolated from an anaerobic lake sediment . One strain of the diculture is proposed to be an endospore-forming Desulfotomaculum species, the second strain was a vibrioid, motile and non-sporeforming species which is tentatively assigned to the genus Desulfovibrio . The diculture was able to mineralize 4- and 2-fluorobenzoate both isomers being incompletely oxidized with the release of acetate, which was subsequently used by both sulfate-reducing strains . Other electron donors used for growth included benzoate, 3- and 4-hydroxybenzoate, protocatechuate, catechol, phenol, 2,5-dimethoxyphenol, fatty acids up to C8, malate and pyruvate . The culture obtained from a freshwater habitat grew optimally at NaCl concentrations of 0.3-0.5 g l-1, 33-37 degrees C, and pH 7.4 . Our experiments showed that certain fluorinated aromatic hydrocarbons could serve as sole sources of carbon and energy for sulfate-reducing bacteria.

Infect Immun, 1994 Feb, 62(2), 759 - 63
Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice; Amaro C et al.; The virulence mechanisms of Vibrio vulnificus biotype 2 have been studied and compared with those of biotype 1 in mice as the experimental animals . Biotype 2 isolates from European eels were as virulent for mice as biotype 1 strains (50% lethal dose, about 10(5) CFU per mouse); a septicemic infection developed in less than 24 h . These strains had several properties in common with biotype 1 organisms including capsule expression, uptake of various iron sources, and production of exoproteins, whose role in mouse virulence has been demonstrated . We also discuss the implication of biotype 2 strains in human infections.

J Cell Biochem, 1994 Feb, 54(2), 161 - 73
Expression of PNA-binding sites on specific glycoproteins by human melanoma cells is associated with a high metastatic potential; Zebda N et al.; Lectin-binding patterns of seven human melanoma clones and variants selected from the same parental cell line and differing in their spontaneous metastatic potential in an animal model were compared by flow cytometry and Scatchard analysis . Human melanoma clones and variants with high and low metastatic potential could be distinguished by their peanut agglutinin (PNA)-binding patterns, but not by their wheat germ agglutinin (WGA)-, Ulex europaeus agglutinin I (UEA I)-, and soybean agglutinin (SBA)-binding patterns . Low metastatic clones and variants proved to be made up of single poorly peanut agglutinin-binding cell population (2.20-3.52 x 10(6) sites/cell, Ka = 2.48-2.75 x 10(6) M-1) . By contrast, highly metastatic variants were found to be constituted by two cellular subpopulations, exhibiting respectively a moderate 2.62-3.72 x 10(6) sites/cell) and a high peanut agglutinin staining (17.68-18.76 x 10(6) sites/cell) . One highly metastatic clone was found to be homogeneously constituted by a single population of cells strongly binding this lectin (18.86 x 10(6) sites/cell) with an association constant of 4.06 +/- 10(6) M-1 . Using an EPICS V cytometer, these two subpopulations were sorted from a highly metastatic variant and tested for their metastatic abilities: cells with high PNA binding generated a higher frequency of metastases than did moderately PNA-binding cells . Following treatment with Vibrio cholerae neuraminidase, all cells from all variants and clones were brightly labeled by PNA, collecting in a single peak with similar fluorescence intensities . Electrophoresis of total cellular proteins and subsequent detection with labeled PNA om Western blots show two major PNA-reactive glycoproteins with apparent molecular weights of 140 and 110 kDa (MAGP1 and MAGP2), expressed only in highly metastatic cells, but which can be strongly labeled by PNA in slightly metastatic cells following a treatment with neuraminidase . These results provide evidence that the expression of terminal galactose (beta 1-3)N-acetyl galactosamine structure, positioned on MAGP1 and MAGP2 glycoproteins, is associated with the metastatic potential of human melanoma cells.

Res Microbiol, 1994 Feb, 145(2), 151 - 6
An analysis of the V1 and V2 regions of Vibrio cholerae and Vibrio mimicus 16S rRNA; Coelho A et al.; The V1 and V2 variable regions of the 16S rRNA gene of three strains of V . cholerae and one strain of V . mimicus were amplified by PCR . Fragments containing both regions were cloned into M13mp18 using Smal and sequenced by the dideoxy method . The 263-bp sequence from a strain isolated during the 1991 cholera outbreak in Brazil was deposited in Genbank under the accession number L05178 . Except for an extra G in one of the strains, the three V . cholerae sequences were identical . The V . mimicus sequence was very similar, with only two substitutions . We compared these sequences with the Vibrio 16S rRNA sequences described by Dorsch et al . in 1992 . It was noted that the V1 region, including helix 6 and its associated loop, comprised two different sizes and sequences in the various Vibrio species . While V . cholerae, V . mimicus, V . vulnificus, V . anguillarum and V . diazotrophicus had a 46-nucleotide V1, other species such as V . parahaemolyticus, V . proteolyticus, V . alginolyticus, V . campbellii and V . hollisae had longer 54- or 55-nucleotide regions, with a different consensus sequence . The phylogeny of Vibrio was analysed using the sequenced region and its equivalent in other species, by means of the "Phylip" software package . Species with a short helix 6 were grouped together, as were species with a long helix . Dorsh et al.'s analysis is discussed in relation to this "helix 6 split".

Mol Cell Probes, 1994 Feb, 8(1), 39 - 44
Detection of the Vibrio cholerae heat-stable enterotoxin gene by polymerase chain reaction; Guglielmetti P et al.; A polymerase chain reaction assay was developed for detection of the Vibrio cholerae heat-stable enterotoxin gene (sto) . The assay is based on two oligonucleotide primers suitable for amplification of the entire sto open reading frame . Reaction conditions were defined to obtain optimal results in terms of specificity and sensitivity . Under these conditions the assay was highly sensitive and enabled good results to be obtained using, as a template, crude DNA preparations from single bacterial colonies . A rapid protocol for direct sequence analysis of the amplification product, which may be used in combination with the PCR assay to confirm the product identity and analyse sequence polymorphisms within the sto gene, was also developed . Twenty-two V . cholerae non-O1 isolates from sporadic cases of V . cholerae non-O1-associated gastroenteritis from Cuba were analysed by this PCR assay . Four strains (18.2%) were found to carry the sto gene . The prevalence of V . cholerae non-O1 strains carrying the sto gene among clinical isolates is higher than that reported for other geographical areas, except in the case of epidemics.

Comp Immunol Microbiol Infect Dis, 1994 Feb, 17(1), 63 - 70
Detection of potential virulence markers of Vibrio vulnificus strains isolated from fish in Sweden; Krovacek K et al.; A variety of potential virulence markers such as the production of cytotoxin, haemolysin, exoenzymes, bactericidal action of sera, presence of capsule and adhesion to human intestinal cells were investigated on Vibrio vulnificus strains isolated from eels in Sweden . The strains had the capacity of producing all or some of the above-mentioned virulence markers, to varying degrees though none of the strains produced any capsule . The strains also bound specifically to human intestinal cells in vitro with maximum adhesion levels of 30 bacteria/cell . The results on binding of V . vulnificus cytotoxin to HeLa cells, showed that a very short exposure time (30 min) was required for inducing the cytotoxic effects . V . vulnificus is a relatively new addition to the list of bacteria pathogenic for humans, and since there are increasing reports on its isolation from aquatic environments and seafood (e.g . raw oysters, crabs and shellfish), the results on virulence profiles of V . vulnificus strains presented above emphasize the importance of these organisms in public health and epidemiological studies.

Protein Sci, 1994 Feb, 3(2), 166 - 75
Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide; Merritt EA et al.; Cholera toxin (CT) is an AB5 hexameric protein responsible for the symptoms produced by Vibrio cholerae infection . In the first step of cell intoxication, the B-pentamer of the toxin binds specifically to the branched pentasaccharide moiety of ganglioside GM1 on the surface of target human intestinal epithelial cells . We present here the crystal structure of the cholera toxin B-pentamer complexed with the GM1 pentasaccharide . Each receptor binding site on the toxin is found to lie primarily within a single B-subunit, with a single solvent-mediated hydrogen bond from residue Gly 33 of an adjacent subunit . The large majority of interactions between the receptor and the toxin involve the 2 terminal sugars of GM1, galactose and sialic acid, with a smaller contribution from the N-acetyl galactosamine residue . The binding of GM1 to cholera toxin thus resembles a 2-fingered grip: the Gal(beta 1-3)GalNAc moiety representing the "forefinger" and the sialic acid representing the "thumb." The residues forming the binding site are conserved between cholera toxin and the homologous heat-labile enterotoxin from Escherichia coli, with the sole exception of His 13 . Some reported differences in the binding affinity of the 2 toxins for gangliosides other than GM1 may be rationalized by sequence differences at this residue . The CTB5:GM1 pentasaccharide complex described here provides a detailed view of a protein:ganglioside specific binding interaction, and as such is of interest not only for understanding cholera pathogenesis and for the design of drugs and development of vaccines but also for modeling other protein:ganglioside interactions such as those involved in GM1-mediated signal transduction.

Appl Environ Microbiol, 1994 Feb, 60(2), 732 - 7
Rapid detection and identification of Vibrio anguillarum by using a specific oligonucleotide probe complementary to 16S rRNA; Martinez-Picado J et al.; Partial 16S rDNA from Vibrio collection type strains and recent isolates of Vibrio-related strains were sequenced and compared with previously published sequences . A 24-base DNA oligonucleotide (VaV3) was designed and used as a specific probe for detection and identification of Vibrio anguillarum . Its specificity was tested against collection type strains and environmental isolates and no cross-reaction was found . The probe detected 8 of the 10 V . anguillarum serovars . It was applied to screen different Vibrio-related strains isolated from marine hatcheries and fish farms . The detection limit in DNA-DNA slot blot hybridization was 150 pg.

FEMS Microbiol Lett, 1994 Jan 15, 115(2-3), 265 - 71
The effect on enterotoxicity of protease purified from Vibrio cholerae O1; Ichinose Y et al.; The effect on enterotoxicity of protease purified from Vibrio cholerae O1 was investigated by the inoculation of live vibrio cells into protease-treated loops of the ileal loop model . Fluid accumulation ratios in the protease-treated loops were elevated in a dose-dependent manner by challenge with live vibrio cells but not by that with toxin . An enhancement effect of protease on enterotoxicity was observed in both serotypes of V . cholerae O1 and V . cholerae non-O1 . It is suggested, therefore, that the enterotoxicity was enhanced by treatment with protease when live vibrio cells were inoculated into the ileal loops of rabbits.

FEMS Microbiol Lett, 1994 Jan 15, 115(2-3), 329 - 34
Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes; Choudhury SR et al.; The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases . The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb . The NotI and SfiI digestion patterns of the genomes of several V . cholerae strains belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP) . RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V . cholerae.

FEMS Microbiol Lett, 1994 Jan 15, 115(2-3), 247 - 52
Purification and characterization of vibrio cholerae O139 fimbriae; Yamashiro T et al.; A Vibrio cholerae O139 (strain Al-1841) isolated from a patient with a cholera-like disease in Bangladesh predominantly produced new curved, wavy fimbriae (Al-1841 fimbriae) and small numbers of previously reported V . cholerae non-O1 S7-like pili . The former was purified and characterized . The molecular mass of the Al-1841 fimbrial subunit was less than 2.5 kDa, and it was immunologically different from that of V . cholerae non-O1 S7 pili . This novel fimbrial antigen was detected in all 182 Gram-negative strains from five genera tested but was absent from the Gram-positive bacteria tested . The purified Al-1841 fimbriae did not agglutinate human or rabbit erythrocytes.

J Med Microbiol, 1994 Jan, 40(1), 31 - 6
The use of gene probes, immunoassays and tissue culture for the detection of toxin in Vibrio cholerae non-O1; Said B et al.; Vibrio cholerae non-O1 strains were screened for the presence of cholera enterotoxin (CT) genes by means of digoxigenin-labelled polynucleotide CTA and CTB probes . In-vitro production of CT was investigated by the Y1 mouse adrenal cell assay, enzyme-linked immunosorbent assay (ELISA) and a commercial, reversed passive latex agglutination (RPLA) kit . Only two (0.25%) of 790 strains tested gave positive results with the CTA and CTB probes . The production of other bacterial cytotoxin(s) made it impossible to use the characteristic cell-rounding effect on Y1 cells for the detection of CT . CT production by the probe-positive strains was confirmed by the immunoassays . Two hundred and fifty-two of the 788 probe-negative strains were tested by both cell assay and immunoassays . Of these, 90% produced cytotoxin(s) in the cell assay . In addition, 37% gave positive results in CT-ELISA, but negative results with LT-ELISA and VET-RPLA . These results indicate the presumed presence of a toxin in V . cholerae non-O1 that is able to bind GM1 and react with antisera to CT, but which is not identical to CT.

J Bacteriol, 1994 Jan, 176(1), 77 - 83
Exogenous myristic acid can be partially degraded prior to activation to form acyl-acyl carrier protein intermediates and lipid A in Vibrio harveyi; Shen Z et al.; To study the involvement of acyl carrier protein (ACP) in the metabolism of exogenous fatty acids in Vibrio harveyi, cultures were incubated in minimal medium with {9,10-3H}myristic acid, and labeled proteins were analyzed by gel electrophoresis . Labeled acyl-ACP was positively identified by immunoprecipitation with anti-V . harveyi ACP serum and comigration with acyl-ACP standards and {3H}beta-alanine-labeled bands on both sodium dodecyl sulfate- and urea-polyacrylamide gels . Surprisingly, most of the acyl-ACP label corresponded to fatty acid chain lengths of less than 14 carbons: C14, C12, C10, and C8 represented 33, 40, 14, and 8% of total {3H}14:0-derived acyl-ACPs, respectively, in a dark mutant (M17) of V . harveyi which lacks myristoyl-ACP esterase activity; however, labeled 14:0-ACP was absent in the wild-type strain . 14:0- and 12:0-ACP were also the predominant species labeled in complex medium . In contrast, short-chain acyl-ACPs (< or = C6) were the major labeled derivatives when V . harveyi was incubated with {3H}acetate, indicating that acyl-ACP labeling with {3H}14:0 in vivo is not due to the total degradation of {3H}14:0 to {3H}acetyl coenzyme A followed by resynthesis . Cerulenin increased the mass of medium- to long-chain acyl-ACPs (> or = C8) labeled with {3H}beta-alanine fivefold, while total incorporation of {3H}14:0 was not affected, although a shift to shorter chain lengths was noted . Additional bands which comigrated with acyl-ACP on sodium dodecyl sulfate gels were identified as lipopolysaccharide by acid hydrolysis and thin-layer chromatography . The levels of incorporation of {3H} 14:0 into acyl-ACP and lipopolysaccharide were 2 and 15%, respectively, of that into phospholipid by 10 min . Our results indicate that in contrast to the situation in Escherichia coli, exogenous fatty acids can be activated to acyl-ACP intermediates after partial degradation in V . harveyi and can effectively label products (i.e., lipid A) that require ACP as an acyl donor.

J Bacteriol, 1994 Jan, 176(1), 240 - 8
Analysis of the complexity of gene regulation by fur in Vibrio cholerae; Litwin CM et al.; Iron concentration influences the expression of a number of genes involved in iron uptake and virulence in bacteria . In Escherichia coli, coordinate regulation of these genes by iron depends on the product of the fur gene, which acts as an iron-responsive, DNA-binding repressor protein . Several genes in Vibrio cholerae are also repressed by iron; and a fur gene, homologous to E . coli fur, has been previously cloned from this organism . The present study was undertaken to define the roles of Fur and iron in regulating gene expression in V . cholerae . V . cholerae strains with a mutation in fur by virtue of suicide plasmid integration into this gene showed derepressed expression of two previously characterized, iron-regulated genes, irgA and viuA, in high concentrations of iron; even in the fur mutants, however, residual two- to threefold regulation by iron persisted . The fur mutant strains constructed by suicide plasmid integration required antibiotic selection to maintain the mutation . To analyze further the effect of Fur and iron on gene regulation in V . cholerae without the need for antibiotic selection, we used in vivo marker exchange to construct a nonrevertible V . cholerae fur mutant . This V . cholerae fur mutant grew significantly less well in Luria-Bertani medium than the wild-type parent but grew slightly better than the wild type under iron-restricted conditions . The V . cholerae fur mutant was unable to utilize a number of carbon sources including glycerol, acetate, succinate, lactate, and fumarate, that supported growth of the wild-type strain on minimal media . We utilized two-dimensional gel electrophoresis of whole-cell protein extracts from the fur mutant and wild-type strains following growth in conditions of either low or high concentrations of iron to identify proteins regulated by iron and/or Fur . Twenty-two proteins were negatively regulated by iron in the wild-type strain but constitutively expressed in the fur mutant, consistent with the model of Fur as an iron-dependent repressor . However, many other proteins were regulated in a different manner by iron and/or Fur . Seventeen proteins were negatively regulated by iron but independent of Fur, suggesting the presence of an additional iron-dependent repressor(s) . Six proteins were strongly iron regulated in the fur mutant but hardly expressed at all in the wild-type strain regardless of the iron concentration, suggesting an interaction between Fur and another iron regulatory mechanism . There were 11 proteins that were induced rather than repressed by iron, in four different regulatory classes . Gene regulation in V . cholerae by Fur and iron is much more complex than previously thought and is reminiscent of the Lrp regulon in E.coli.

J Bacteriol, 1994 Jan, 176(1), 213 - 20
Characterization of the Vibrio anguillarum fur gene: role in regulation of expression of the FatA outer membrane protein and catechols; Tolmasky ME et al.; The chromosomally encoded Vibrio anguillarum fur gene was characterized . The amino acid sequence of the Fur protein showed a very high degree of homology with those of V . cholerae and V . vulnificus . The degree of homology was lower, although still high, with the Escherichia coli and Yersinia pestis Fur amino acid sequences, while the lowest degree of homology was found with the Pseudomonas aeruginosa Fur protein . The C-terminal portion of Fur is the least conserved region among these Fur proteins . Within this portion, two regions spanning amino acids 105 to 121 and 132 to the end are the least conserved . A certain degree of variation is also present in the N termini spanning amino acids 28 to 46 . Regulation of expression of the V . anguillarum fur gene by iron was not detected by immunoblot analysis . Mutations in the cloned fur gene were generated either by site-directed mutagenesis (the Lys-77 was changed to a Gly to generate the derivative FurG77) or by insertion of a DNA fragment harboring the aph gene in the same position . FurG77 was impaired in its ability to regulate a reporter gene with the Fur box in its promoter, while the insertion mutant was completely inactive . V . anguillarum fur mutants were obtained by isolating manganese-resistant derivatives . In one of these mutants, which encoded a Fur protein with an apparent lower molecular weight, the regulation of the production of catechols and synthesis of the outer membrane protein FatA were partially lost . In the case of another mutant, no protein was detected by anti-Fur serum . This derivative showed a total lack of regulation of biosynthesis of catechols and FatA protein by iron.

Infect Immun, 1994 Jan, 62(1), 166 - 71
Demonstration and characterization of simultaneous production of a thermostable direct hemolysin (TDH/I) and a TDH-related hemolysin (TRHx) by a clinically isolated Vibrio parahaemolyticus strain, TH3766; Xu M et al.; Simultaneous production of a thermostable direct hemolysin (TDH)-like toxin (TDHx) and a TDH-related hemolysin (TRH)-like toxin (TRHx) by a clinical isolate (strain TH3766) of Kanagawa phenomenon-positive Vibrio parahaemolyticus was demonstrated and characterized . The two hemolysins were differentially purified by column chromatography on hydroxyapatite and immunoaffinity columns . The molecular weight of the two hemolysins were estimated to be 23,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) . The purified TDHx was indistinguishable from the previously reported TDH/I (from strain TH012) but was different from the authentic TDH of a Kanagawa phenomenon-positive strain (T4750) physicochemically . The mobility of TRHx in nondenaturing PAGE differed from all the known TDHs and TRHs . The genes (tdhX and trhX) coding for TDHx and TRHx were cloned and sequenced . Homologies of nucleotide sequences of the coding regions between tdhX and tdhA (a gene for the authentic TDH) and between trhX and trh (a gene for the authentic TRH) were 98.1 and 99.1%, respectively, and homology between tdhX and trhX was 68.1% . At the amino acid level, TdhX was completely identical to TDH/I, although two base differences were found in the nucleotide sequences between tdhX and tdh/I . Two amino acid differences were observed between TrhX and Trh . Thus, these findings suggest that the TH3766 strain produces two types of hemolysins simultaneously . This is the first evidence that a strain of V . parahaemolyticus produces two types of toxins of the TDH-TRH family at the same time.

Placenta, 1994 Jan, 15(1), 1 - 11
GTP-binding proteins associated with the human placental syncytiotrophoblast plasma membrane; Kenton P et al.; The nature of GTP-binding components associated with isolated human term placental syncytiotrophoblast microvillous plasma membrane vesicles (SPMV) was determined; these are relevant to elucidation of intracellular signal transduction mechanisms . Four proteins were identified, with molecular weights of 29, 27, 23 and 21 kDa, which specifically bound {alpha-32P}GTP in the presence of Mg2+ . Studies employing anti-p21c-ras monoclonal antibodies indicated these four GTP-binding components were ras-related and one, the 21 kDa component, may be p21c-ras . In addition, SPMV were also found to express the alpha subunits of three separate G proteins . A 45 kDa SPMV GTP-binding protein was identified as a substrate for Vibrio cholera toxin and was recognized by a rabbit antibody to the alpha subunit of the adenylate cyclase stimulating G protein, Gs . A 41 kDa SPMV GTP-binding protein substrate of Bordetella pertussis toxin was also recognized by rabbit antibodies to the alpha subunits of the adenylate cyclase inhibiting G proteins, Gi-1 and Gi-3 . No evidence was found to support the presence of the 21 kDa Gp, a G protein previously associated with membranes prepared from whole placental tissue homogenates.

Microbios, 1994, 77(312), 191 - 8
Seawater effects on various Vibrio species; Munro PM et al.; This study compared the effects of sea water on Vibrio cholerae and six other Vibrio spp . Survival in seawater microcosms as well as uptake of a carbonated substrate in marine or non-marine conditions were investigated . Except for V . vulnificus becoming non-culturable, all the other selected species survived in sea water for at least 15 days at 20 degrees C . Depending on the species tested, the substrate was better transported in a high salt medium (V . cholerae, V . fluvialis and V . metschnikovii), than in a low salt medium (V . fluvialis, V . furnissii, V . parahaemolyticus and V . vulnificus) . In terms of the response of the species to marine conditions, no correlation was found between survival in sea water and substrate uptake.

Trop Geogr Med, 1994, 46(1), 42 - 3
The origin of non-outbreak Vibrio parahaemolyticus infections on Guam; Haddock RL et al.; A study of Vibrio parahaemolyticus infected patients not associated with known outbreaks and controls matched for sex, ethnicity and age (+/- 5 years) was conducted on Guam . Cases were asked if they had eaten seafood within the 24-hour period preceding onset of illness and controls were asked if they had eaten seafood within the 24-hour period preceding their interview . Cases were more likely than controls to have eaten seafood (OR = 37.59, CL {8.30-220.24}).

J Appl Bacteriol, 1994 Jan, 76(1), 79 - 85
A set of keys for biochemical identification of environmental Vibrio species; Alsina M et al.; A set of biochemical keys which provide fast and presumptive identification for Vibrio spp . is presented . They have been specially designed for environmental isolates, and can be used for strains that are Gram-negative, give a positive oxidase test, grow on TCBS medium and are facultative anaerobes . The keys are constituted by 28 tests and a maximum of 10 tests are needed for the most complicated identification . They have been designed for routine purposes, especially for studies with a high number of isolates . Some tests are included in enzyme-activity based kits that could be used with these keys through certain results, principally for environmental isolates, should be confirmed by standard methods.

Kansenshogaku Zasshi, 1994 Jan, 68(1), 8 - 12
{The first report of traveler's diarrhea associated with a newly described toxigenic Vibrio cholerae O139 strain in Japan}; Kurazono T et al.; A newly described Vibrio cholerae O139 was isolated from a patient who had traveled in India on April 1993 . The patient experienced 5 to 6 watery diarrhea per day after he returned to Japan . The isolated strain registered as K111 did not agglutinate with O1-O138 antiserum and agglutinated with O139 antiserum . This strain resembled V . cholerae O1 strain in biochemical characters and had ctx and zot, although was resistant to the vibrio static agent O/129 . This is the first report of cholera-like illness by the newly described toxigenic V . cholerae O139 strain in Japan.

Kansenshogaku Zasshi, 1994 Jan, 68(1), 163 - 7
{A case of cardiogenic shock caused by Vibrio parahaemolyticus}; Tsujimoto M et al.; A case of a 53 year old healthy female complaining of diarrhea and abdominal pain after taking raw fish is presented . She immediately went into shock and unconsciousness . Central venous pressure was 8 cmH2O and her ECG showed a first-degree AV block and ST-T changes in almost all leads . After mechanical ventilation and administration of dopamine, dobutamine, cefotiam, ciprofloxacin, she became alert and recovered from her critical condition . V . parahaemolyticus which produces thermostable direct hemolysin (TDH) was cultured from the feces on admission . Kanagawa phenomenon was positive . Arterial blood culture was negative and the titer of serum endotoxin was low . The diagnosis of cardiogenic shock due to exotoxin produced by V . parahaemolyticus was made . Serological examination by ELISA showed elevation of IgG class antibody against TDH and TRH (TDH related hemolysin) . And antibody against TDH was normalized after 180 days . By review of literature, there are some case reports of cardiogenic shock complicated with V . parahaemolyticus infection, but few showed elevation of antibody against TDH and TRH in the serum of the survived patient.

J Clin Microbiol, 1994 Jan, 32(1), 249 - 52
A novel kit for rapid detection of Vibrio cholerae O1; Hasan JA et al.; We report on the development and testing of a novel, rapid, colorimetric immunodiagnostic kit, Cholera SMART, for direct detection of the presence of Vibrio cholerae O1 in clinical specimens . Unlike conventional culture methods requiring several days to complete, the Cholera SMART kit can be used directly in the field by untrained or minimally skilled personnel to detect V . cholerae O1 in less than 15 min, without cumbersome laboratory equipment . A total of 120 clinical and environmental bacterial strains, including both O1 and non-O1 serotypes of V . cholerae isolated from samples collected from a variety of geographical regions, were tested, and positive reactions were observed only with V . cholerae O1 . Also, results of a field trial in Bangladesh, employing Cholera SMART, showed 100% specificity and 96% sensitivity compared with conventional culture methods . Another field trial, in Mexico, showed that Cholera SMART was 100% in agreement with a recently described coagglutination test when 108 stool specimens were tested.

Zhonghua Yu Fang Yi Xue Za Zhi, 1994 Jan, 28(1), 16 - 9
{Protection of Vibrio cholerae from heating and chlorination by chitin}; Liu SG et al.; El Tor Vibrio cholerae and non-01 V . cholerae absorbed onto chitin particles could not only multiply in vitro, but also could partly survive with heating at 65-75 degrees C for 60-160 sec (7/40) or with 3.8-15 ppm of effective chlorine for 10 min (24/29) . None of those unabsorbed onto chitin could survive at the above temperature, and only very few of those could survive with the above concentration of effective chlorine . These findings are conducive to the control of cholera transmission caused by the crustacean contaminated with chitin.

Microbios, 1994, 79(318), 7 - 17
Antigens of Vibrio vulnificus; Bradley JL et al.; Rabbit antisera raised against a formalinized culture of Vibrio vulnificus were examined for the development of IgM and IgG antibody by ELISA analysis of serum samples harvested weekly for 5 weeks following the initial injection . V . vulnificus antigens recognized by the sera in three fractions of the culture were identified by immunoblot analysis . The major species recognized in the culture supernatant solution by both IgM and IgG had a molar mass of 58 kD . In fractions derived from the bacterial pellet, IgM recognized a species of 56 kD, while IgG reacted strongly with 29.5, 56, 59, 68, 87, and 186 kD species.

Indian J Exp Biol, 1994 Jan, 32(1), 44 - 8
Effect of rehydrating fluid 'Electral' on Vibrio cholerae cells; Bhattacharya R et al.; V . cholerae OGAWA 154 cells underwent rapid loss of colony forming capacity during the first few minutes' incubation in the Electral medium at 37 degrees C, the turbidity of the suspension however increasing with time of incubation and leading to a plateau from 5 min onward . The vibrio suspension in the Electral medium released small amounts of 280 nm and much higher amounts of 260 nm absorbing materials . On withdrawal of the Electral medium, the cells underwent significant liquid holding recovery in the phosphate buffered saline, pH 7 . Majority of the cells underwent no significant ultrastructural change but grew into long filamentous forms . The mode of action of the Electral medium on the vibrios is discussed.

Arch Microbiol, 1994, 161(5), 439 - 41
Restriction endonucleases from Selenomonas ruminantium which recognize and cleave 5'-AT/TAAT-3'; Pristas P et al.; Two natural isolates from fallow-deer rumen identified as Selenomonas ruminantium were found to produce a restriction endonuclease which we called Sru4DI . This enzyme was isolated from cell extracts by phosphocellulose chromatography . Analysis of the Sru4DI recognition site showed that Sru4DI recognizes the hexanucleotide sequence 5'-AT/TAAT-3' generating 5'dinucleotide protruding ends upon cleavage and thus is a true isoschizomer of vspI, a restriction enzyme isolated from Vibrio sp.

Microbios, 1994, 78(314), 7 - 16
Anti-bacillus substance in the marine sponge, Hyatella species, produced by an associated Vibrio species bacterium; Oclarit JM et al.; The bacterial isolate, M22-1, belonging to the genus Vibrio was obtained from an homogenate of the sponge, Hyatella sp . The bacterium was cultured in marine agar and was found to produce an anti-Bacillus compound . The substance was chemically identified as a peptide antibiotic, an andrimid . The same substance was found in the sponge extract, suggesting that the active component was synthesized by the associated microorganism.

Rev Gastroenterol Peru, 1994 Jan-Apr, 14(1), 27 - 31
{The in vitro action of plants on Vibrio cholerae}; Guevara JM et al.; Natural products of several plants, according to the geographic location, are used by Peruvian people in the popular treatment of diarrhea, with good success . When cholerae cases appeared in Peru, we were interested to know the "in vitro" effect against Vibrio cholerae 01, of these useful plants to treat diarrhea . The following plants were tested: Cichorium intybus, Althaea officinalis, Psorela glandulosa, Geranium maculatum, Punica granatum, Malus sativa, Cydonia oblonga, Chenopodium ambrosoides, Krameria triandria, Tea chinensis, Daucus carota, Persea gratissima, Psidium guayaba and Lippia dulcis . Decoction or infusion of the plants were used in the "in vitro" experiments . The following plants showed no "in vitro" effect against V . cholerae: Cichorium intybus, Althaea officinalis, Psorela glandulosa, Geranium maculatum, Chenopodium ambrosoides, Krameria triandria, Psidium guayaba, Lippia dulcis and Daucus carota . Decoction of Malus sativa and Cydenia oblonga showed bactericidal effect for their acidity and stone avocado (Persea gratissima) a late bactericidal effect . Tea infusion and the decoction of Punica granatum peel, showed the best bactericidal effect and we suggest to use them as to stop cholera spreading.

Zh Mikrobiol Epidemiol Immunobiol, 1994 Jan-Feb, (2), 31 - 6
{Genetic determination and the spectrum of the hemolytic activity of Vibrio cholerae: their significance in the complex of biovar-specific traits}; Ushakova IE et al.; A collection of 363 V . cholerae strains isolated from different sources were studied by the spectrum of their hemolytic activity in combination with biovar-associated properties . The strains were analyzed for the presence of the cholera toxin (CT) gene (vct) and the hemolysin gene (hly) with the use of the CT probe and a previously cloned 6.56 kb fragment of V . eltor DNA coding the synthesis of hemolysin . The study revealed that all V . cholerae strains had the hly gene irrespective of the spectrum of their hemolytic activity, biovar, 01 agglutinability and the presence of the vct gene in their genome, while other species of the genus Vibrio and related groups contained no hly gene sequence . The results of the comparative study of the hemolytic activity of V . cholerae and V . eltor are discussed.

Zh Mikrobiol Epidemiol Immunobiol, 1994 Jan-Feb, (2), 11 - 5
{The characteristic properties of Vibrio cholerae eltor isolated from environmental objects on the territory of the former USSR during the 7th cholera pandemic}; Lomov IuM et al.; The properties of 22,382 V . eltor strains isolated from environmental objects on the territory of different climatic and geographical zones during the period of 1970-1988 were studied . The study was made on the morphology of their colonies, the agglutinability of the strains by cholera O serum, type-specific serum and RO serum, their capacity for being lyzed by V . eltor bacteriophage, their hemolytic activity and virulence . Differences in the occurrence of strains with any of the above-mentioned properties, depending on the object from which they were isolate, the climatic and geographical zone and the intensity of the epidemiological situation with regard to cholera.

Bull Soc Pathol Exot, 1994, 87(1), 38 - 40
{In vitro sensitivity of Vibrio cholerae serotype 0:139 to an intestinal antiseptic tiliquinol-tilbroquinol combination}; Bougoudogo F et al.; O:139 is a new serotype of Vibrio cholerae that is not agglutinated by an O:1 antiserum but causes epidemics of cholera . Strains of O:139 serotype are resistant to O/129 compound and many antibiotics but are sensitive to tetracyclines and tiliquinol-tilbroquinol (Intetrix) . The clinical management of the patients infected with serotype O:139 is identical to that of usual choleric patients . However, the immunological difference with O:1 serotype must lead to reconsider both the diagnosis and the vaccinating strategies of cholera.

Bull Soc Pathol Exot, 1994, 87(1), 33 - 7
{Value of vibriocidal antibody research in endemic areas of Vibrio cholerae 0:1}; Bendib A et al.; We made vibriocidal antibody titration in the serum of some populations in Algeria and in Mali either during or between cholera epidemics . The seropositivity rate was 43.3% in healthy contacts in Alger in 1990 during an epidemic of cholera . For 12/16 healthy contacts examined two times in a 25-day interval, the seropositivity rate increased during the epidemic and the mean of antibody titres rose 8.88 folds . In Constantine, 53% of 195 blood donors had significant titres of vibriocidal antibodies in 1992, 6 years after an epidemic of cholera . The seropositivity rate in population seemed decreasing during this year . In Bamako, 46% of selected patients had significant vibriocidal antibody titres 8 years after the last epidemic of cholera in Mali . Seven of 10 children born after the epidemic had vibriocidal antibodies . These data confirm the persistence of vibriocidal antibodies in population during many years . The importance of the seropositivity rate in healthy contacts and in children born during a non epidemic period shows that asymptomatic infection is frequent and that Vibrio cholerae O:1 may be circulating in population between epidemics . As part of surveillance of cholera outbreaks in endemic areas, it might be of interest to study on a regular basis the vibriocidal antibody seropositivity rate in populations.

Biol Chem Hoppe Seyler, 1994 Jan, 375(1), 61 - 70
Dimerization of Bence Jones proteins: linking the rate of transcription from an Escherichia coli promoter to the association constant of REIV; Kolmar H et al.; Homodimers of immunoglobulin VL domains are minimal models of antibodies in that they display an ensemble of six hypervariable loops . Bence Jones protein REI is a mixture of a complete kappa light chain and the corresponding variable domain (REIV) . The known three-dimensional structure of the REIV dimer (Epp et al., 1975, Biochemistry 14, 4943-4952) provides a basis for studying dimer stabilization by protein engineering . Mutant REIV-L94H was constructed and shown to have an equilibrium constant of dimerization about one order of magnitude higher than wildtype REIV . By fusing REIV and variants to the aminoterminal part of the Vibrio cholerae ToxR regulator protein (Miller et al., 1987, Cell 48, 271-279), a transcriptional signal in E . coli can be derived from REIV homodimer formation constant . The system senses dimerization of the immunoglobulin part of the fusion protein, located in the periplasmatic space, and transduces the signal as transcriptional activation to a ctx::lacZ gene construct integrated into the E . coli chromosome . There is positive correlation between the propensities of homodimer formation and the rate of transcriptional initiation at the ctx promoter . Since beta-galactosidase levels can easily be measured colorimetrically in crude cell lysates of a large number of clones using an ELISA reader, this procedure constitutes all elements required for a genetic screen in E . coli for immunoglobulin variants with altered association constants.

Scand J Infect Dis, 1994, 26(4), 493 - 4
Mixed bacteremia with Vibrio metschnikovii in an 83-year-old female patient; Hardardottir H et al.; An 83-year-old woman suddenly fell ill and was admitted to the hospital on suspicion of a heart attack . After admission she developed high fever, chills and malaise . Vibrio metshnikovii and Staphylococcus hominis were isolated from 2 separately obtained blood cultures . One of the cultures also yielded Escherichia coli . The patient's condition improved rapidly after treatment with ampicillin intravenously . To our knowledge, this is the fourth reported case of V . metschnikovii bacteremia in humans.

Microbiol Immunol, 1994, 38(6), 467 - 70
Existence of a novel hemagglutinin having no protease activity in Vibrio mimicus; Alam M et al.; The protease elaborated by Vibrio mimicus is known to possess hemagglutinating ability to chicken erythrocytes, the well-known HA/protease . A non-protease hemagglutinin (HA) with strong agglutinating ability towards rabbit erythrocytes was obtained from 32 hr culture supernatant of a pathogenic environmental strain of V . mimicus . This HA (V . mimicus HA: VMHA) appeared stable at relatively higher temperature and agglutinated the erythrocytes from rabbit, guinea pig and mouse but not the erythrocytes from chicken, bovine, horse and sheep . Simple sugars, metal ions and chelating agents failed to inhibit the activity of VMHA . The activity of VMHA was found to be sensitive to digestion by proteolytic enzymes including HA/protease . These results provide evidence for the existence of novel HA other than HA/protease in V . mimicus.

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