IUBMB Life, 1999 Sep, 48(3), 345 - 52 Cloning and characterization of manganese superoxide dismutase gene from Vibrio parahaemolyticus and application to preliminary identification of Vibrio strains; Shyu YC et al.; The sodA gene coding for manganese superoxide dismutase (Mn-SOD) from the marine microorganism Vibrio parahaemolyticus was cloned, sequenced, and overexpressed in Escherichia coli by use of the pET20b (+) expression vector . The full-length gene consisted of a 588-bp open reading frame and encoded a polypeptide of 196 amino acid residues, with a calculated molecular mass of 21,713 Da . The recombinant enzyme was efficiently purified from crude E . coli cell lysate by metal ion affinity chromatography . The recombinant VPMn-SOD resisted thermo-denaturation up to 60 degrees C and was insensitive to such inhibitors as EDTA, NaN3 and diethyldithiocarbamic acid . The specificity of V . parahaemolyticus Mn-SOD gene probe was analyzed by cross-species polymerase chain reaction to provide information for Vibrio strain identification. Infect Immun, 2000 Mar, 68(3), 1700 - 5 Cytotoxic cell vacuolating activity from Vibrio cholerae hemolysin; Coelho A et al.; A Vibrio cholerae cytotoxin, designated VcVac, was found to cause vacuolation in Vero cells . It was originally detected in the pathogenic O1 Amazonia variant of V . cholerae and later shown to be produced in environmental strains and some El Tor strains . Comparison of VcVac production in various strains suggested that hemolysin was responsible for the vacuolating phenotype . Genetic experiments established a firm correlation between vacuolation and hemolysin production . The mammalian cell vacuolating activity of the V . cholerae hemolysin is a new property of this protein and points to a previously unknown type of interaction between V . cholerae and its host. Infect Immun, 2000 Mar, 68(3), 1507 - 13 Infectious CTXPhi and the vibrio pathogenicity island prophage in Vibrio mimicus: evidence for recent horizontal transfer between V . mimicus and V . cholerae; Boyd EF et al.; Vibrio mimicus differs from Vibrio cholerae in a number of genotypic and phenotypic traits but like V . cholerae can give rise to diarrheal disease . We examined clinical isolates of V . mimicus for the presence of CTXPhi, the lysogenic filamentous bacteriophage that carries the cholera toxin genes in epidemic V . cholerae strains . Four V . mimicus isolates were found to contain complete copies of CTXPhi . Southern blot analyses revealed that V . mimicus strain PT5 contains two CTX prophages integrated at different sites within the V . mimicus genome whereas V . mimicus strains PT48, 523-80, and 9583 each contain tandemly arranged copies of CTXPhi . We detected the replicative form of CTXPhi, pCTX, in all four of these V . mimicus isolates . The CTX prophage in strain PT5 was found to produce infectious CTXPhi particles . The nucleotide sequences of CTXPhi genes orfU and zot from V . mimicus strain PT5 and V . cholerae strain N16961 were identical, indicating contemporary horizontal transfer of CTXPhi between these two species . The receptor for CTXPhi, the toxin-coregulated pilus, which is encoded by another lysogenic filamentous bacteriophage, VPIPhi, was also present in the CTXPhi-positive V . mimicus isolates . The nucleotide sequences of VPIPhi genes aldA and toxT from V . mimicus strain PT5 and V . cholerae N16961 were identical, suggesting recent horizontal transfer of this phage between V . mimicus and V . cholerae . In V . mimicus, the vibrio pathogenicity island prophage was integrated in the same chromosomal attachment site as in V . cholerae . These results suggest that V . mimicus may be a significant reservoir for both CTXPhi and VPIPhi and may play an important role in the emergence of new toxigenic V . cholerae isolates. Infect Immun, 2000 Mar, 68(3), 1491 - 7 The virulence regulatory protein ToxR mediates enhanced bile resistance in Vibrio cholerae and other pathogenic Vibrio species; Provenzano D et al.; The transmembrane regulatory protein ToxR is required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae, including cholera toxin (CT) and the toxin coregulated pilus (TCP) . ToxR is necessary for transcription of the gene encoding a second regulatory protein, ToxT, which is the direct transcriptional activator of CT and TCP genes . However, ToxR, independent of ToxT, directly activates and represses transcription of the outer membrane porins OmpU and OmpT, respectively . The genes encoding TCP and CT (and including ToxT) lie on horizontally acquired genetic elements, while the toxR, ompU, and ompT genes are apparently in the ancestral Vibrio chromosome . The contribution of ToxR-dependent modulation of outer membrane porins to cholera pathogenesis has remained unknown . We demonstrate that ToxR mediates enhanced bile resistance in a ToxT-independent manner . In both classical and El Tor biotypes of V . cholerae, a toxR mutant strain has a reduced minimum bactericidal concentration (MBC) of bile, the bile component deoxycholate (DC), and the anionic detergent sodium dodecyl sulfate (SDS) compared to both wild-type and toxT mutant strains . Classical and El Tor toxR mutant strains also exhibit reduced growth rates at subinhibitory concentrations of DC and SDS . Growth of either V . cholerae biotype in subinhibitory concentrations of bile or DC induces increased ToxR-dependent production of a major 38-kDa outer membrane protein, which was confirmed to be OmpU by Western blot . Measurement of transcription of a ompUp-lacZ fusion in both biotypes reveals stimulation (about two- to threefold) of ToxR-dependent ompU transcription by the presence of bile or DC, suggesting that ToxR may respond to the presence of bile . The toxR mutant strains of three additional human intestinal pathogenic Vibrio species, V . mimicus, V . fluvialis, and V . parahaemolyticus, display lower MBCs of bile, DC, and SDS and have altered outer membrane protein profiles compared to the parental wild-type strains . Our results demonstrate a conserved role for ToxR in the modulation of outer membrane proteins and bile resistance of pathogenic Vibrio species and suggest that these ToxR-dependent outer membrane proteins may mediate enhanced resistance to bile . We speculate that ToxR-mediated bile resistance was an early step in the evolution of V . cholerae as an intestinal pathogen. Infect Immun, 2000 Mar, 68(3), 1400 - 7 Iha: a novel Escherichia coli O157:H7 adherence-conferring molecule encoded on a recently acquired chromosomal island of conserved structure; Tarr PI et al.; The mechanisms used by Shiga toxin (Stx)-producing Escherichia coli to adhere to epithelial cells are incompletely understood . Two cosmids from an E . coli O157:H7 DNA library contain an adherence-conferring chromosomal gene encoding a protein similar to iron-regulated gene A (IrgA) of Vibrio cholerae (M . B . Goldberg, S . A . Boyko, J . R . Butterton, J . A . Stoebner, S . M . Payne, and S . B . Calderwood, Mol . Microbiol . 6:2407-2418, 1992) . We have termed the product of this gene the IrgA homologue adhesin (Iha), which is encoded by iha . Iha is 67 kDa in E . coli O157:H7 and 78 kDa in laboratory E . coli and is structurally unlike other known adhesins . DNA adjacent to iha contains tellurite resistance loci and is conserved in structure in distantly related pathogenic E . coli, but it is absent from nontoxigenic E . coli O55:H7, sorbitol-fermenting Stx-producing E . coli O157:H-, and laboratory E . coli . We have termed this region the tellurite resistance- and adherence-conferring island . We conclude that Iha is a novel bacterial adherence-conferring protein and is contained within an E . coli chromosomal island of conserved structure . Pathogenic E . coli O157:H7 has only recently acquired this island. Infect Immun, 2000 Mar, 68(3), 1171 - 5 In vitro and in vivo analyses of constitutive and in vivo-induced promoters in attenuated vaccine and vector strains of Vibrio cholerae; John M et al.; The optimal promoter for in vivo expression of heterologous antigens by live, attenuated vaccine vector strains of Vibrio cholerae is unclear; in vitro analyses of promoter activity may not accurately predict expression of antigens in vivo . We therefore introduced plasmids expressing the B subunit of cholera toxin (CtxB) under the control of a number of promoters into V . cholerae vaccine strain Peru2 . We evaluated the tac promoter, which is constitutively expressed in V . cholerae, as well as the in vivo-induced V . cholerae heat shock htpG promoter and the in vivo-induced V . cholerae iron-regulated irgA promoter . The functionality of all promoters was confirmed in vitro . In vitro antigenic expression was highest in vaccine strains expressing CtxB under the control of the tac promoter (2 to 5 microgram/ml/unit of optical density at 600 nm {OD(600)}) and, under low-iron conditions, in strains containing the irgA promoter (5 microgram/ml/OD(600)) . We orally inoculated mice with the various vaccine strains and used anti-CtxB immune responses as a marker for in vivo expression of CtxB . The vaccine strain expressing CtxB under the control of the tac promoter elicited the most prominent specific anti-CtxB responses in vivo (serum immunoglobulin G {IgG}, P </= 0.05; serum IgA, P </= 0.05; stool IgA, P </= 0.05; bile IgA, P </= 0.05), despite the finding that the tac and irgA promoters expressed equivalent amounts of CtxB in vitro . Vibriocidal antibody titers were equivalent in all groups of animals . Our results indicate that in vitro assessment of antigen expression by vaccine and vector strains of V . cholerae may correlate poorly with immune responses in vivo and that of the promoters examined, the tac promoter may be best suited for expression from plasmids of at least certain heterologous antigens in such strains. Dis Aquat Organ, 1999 Nov 30, 38(3), 201 - 10 Infectious necrotizing enteritis and mortality caused by Vibrio carchariae in summer flounder Paralichthys dentatus during intensive culture; Soffientino B et al.; An epizootic causing mortality among cultured summer flounder Paralichthys dentatus occurred in summer of 1998 at a land-based facility on Narragansett Bay, Rhode Island, USA . The disease, flounder infectious necrotizing enteritis (FINE), was characterized by reddening around the anal area, distended abdomens filled with opaque serosanguineous fluid, enteritis and necrosis of the posterior intestine . In extreme cases of the disease, the posterior intestine was detached from the anus and was observed coming out the vent . The intestine of individuals that recovered from the disease ended in a blind-sac; the abdomens of these fish were distended, due to food and water inside the intestinal blind-sac . A bacterium was isolated from ascites fluid and kidney of moribund flounder and identified as the causative agent in challenge experiments . The pathogen was identified as Vibrio carchariae by morphological and biochemical characteristics and sequence of the 16S rRNA . The LD50 estimate was 5 x 10(5) colony-forming units injected intraperitoneally into 100 to 200 g summer flounder. J Mol Biol, 2000 Mar 3, 296(4), 1127 - 37 Luminescence control in the marine bacterium Vibrio fischeri: An analysis of the dynamics of lux regulation; James S et al.; A mathematical model has been developed based on the fundamental properties of the control system formed by the lux genes and their products in Vibrio fischeri . The model clearly demonstrates how the components of this system work together to create two, stable metabolic states corresponding to the expression of the luminescent and non-luminescent phenotypes . It is demonstrated how the cell can "switch" between these steady states due to changes in parameters describing metabolic processes and the extracellular concentration of the signal molecule N-3-oxohexanoyl-l-homoserine lactone . In addition, it is shown how these parameters influence how sensitive the switch mechanism is to cellular LuxR and N-3-oxohexanoyl-l-homoserine lactone and complex concentration . While these properties could lead to the collective phenomenon known as quorum sensing, the model also predicts that under certain metabolic circumstances, basal expression of the lux genes could cause a cell to luminesce in the absence of extracellular signal molecule . Finally, the model developed in this study provides a basis for analysing the impact of other levels of control upon lux regulation . Ecotoxicol Environ Saf, 2000 Jan, 45(1), 87 - 91 Sensitivity and significance of luminescent bacteria in chronic toxicity testing based on growth and bioluminescence; Gellert G; This study explored the use of luminescent bacteria (Vibrio fischeri) for chronic aquatic toxicity tests . The evaluated inhibition of growth to Cu2+, Cr6+, Zn2+, Hg2+, Cd2+, Pb2+, cetyl-trimethylammonium bromide, 3,4-dichloroaniline, acetone, dimethylsulfoxide, ethanol, nitrobenzene, methanol, and 3,5-dichlorophenol was compared with results from another investigation, where the inhibition was determined by bioluminescence . Growth inhibition was found to indicate more reliably the presence of substances with chronic toxic properties than the loss of bioluminescence . But growth responded weaker to the majority of the analyzed toxicants than bioluminescence . This must be connected with the test parameters and the experimental conditions . But among growth experiments with freshwater bacteria species the sensitivity of the growth inhibition assay with V . fischeri is competitive when a poor medium is employed. J Biol Chem, 2000 Feb 25, 275(8), 5718 - 22 Functional reconstitution of the Na(+)-driven polar flagellar motor component of Vibrio alginolyticus; Sato K et al.; The bacterial flagellar motor is a molecular machine that couples the influx of specific ions to the generation of the force necessary to drive rotation of the flagellar filament . Four integral membrane proteins, PomA, PomB, MotX, and MotY, have been suggested to be directly involved in torque generation of the Na(+)-driven polar flagellar motor of Vibrio alginolyticus . In the present study, we report the isolation of the functional component of the torque-generating unit . The purified protein complex appears to consist of PomA and PomB and contains neither MotX nor MotY . The PomA/B protein, reconstituted into proteoliposomes, catalyzed (22)Na(+) influx in response to a potassium diffusion potential . Sodium uptake was abolished by the presence of Li(+) ions and phenamil, a sodium channel blocker . This is the first demonstration of a purification and functional reconstitution of the bacterial flagellar motor component involved in torque generation . In addition, this study demonstrates that the Na(+)-driven motor component, PomA and PomB, forms the Na(+)-conducting channel. Indian J Med Res, 1999 Nov, 110, 155 - 9 Cytotoxicity of non O1, non O139 Vibrios isolated from fresh water bodies in Vellore, south India; Balaji V et al.; The samples of plankton, soil sediment and water from a pond, a lake and a moat respectively in and around Vellore were studied for environmental vibrios . Vibrios were isolated from all these specimens after enrichment in alkaline peptone water and subculture on selective media . Non O1, non O139 Vibrio cholerae, Aeromonas spp . and Plesiomonas spp . were isolated . There were no isolates of V . cholerae serogroup O1 and O139 . Representative strains of non O1 and non O139 V . cholerae from environmental sources were tested for toxin production in Chinese hamster ovary (CHO) and Vero cell monolayer in microtitre plates . Thirty-three (91.7%) of the 36 strains tested demonstrated cytopathic effect (CPE) in both cell lines indicating their toxigenicity . PCR done on representative strains of non O1 and non O139 V . cholerae showed that none of the strains were positive for ctx A, tcp A-E and tcp C genes . These results indicate that these non agglutinating environmental vibrios produced cytotoxins other than the cholera toxin. Am J Trop Med Hyg, 1999 Dec, 61(6), 869 - 73 Safety, immunogenicity, and lot stability of the whole cell/recombinant B subunit (WC/rCTB) cholera vaccine in Peruvian adults and children; Taylor DN et al.; To assess the safety, immunogenicity, and lot stability of the whole cell/recombinant B subunit cholera vaccine, 2 lots manufactured in June 1991 and February 1992 were tested in January 1995 . Two oral doses of vaccine or placebo given 2 weeks apart were given with buffer to 216 Peruvian adults and children . Symptoms were elicited for 3 days after each dose . Serum and plasma specimens obtained from each volunteer before vaccination and 10-14 days after the second dose were tested for vibriocidal and anti-cholera toxin antibodies . The vaccine was well-tolerated . Nearly half of the 100 vaccinees had pre-vaccination vibriocidal titers > or = 1:40 . Elevated titers were observed in 22% of 37 children 2-5 years of age compared with 66% of 63 vaccinees 6-65 years (P < 0.001) . A > or =2-fold serum vibriocidal response was observed in 55% of 100 vaccinees and 6% of 32 placebo recipients . An elevated pre-vaccination titer (< or =1:40) did not change the proportion of vaccinees who responded with a > or =2-fold increase in vibriocidal titer (51% versus 59%, difference not significant), but did change the proportion responding with a > or =4-fold increase (41% versus 22%; P < 0.05) . The vibriocidal seroconversion rate was lowest in children 2-5 years old despite low pre-vaccination titers . Two-fold or greater serum antitoxic responses in IgA and IgG were observed in >90% of the vaccinees; > or =4-fold responses were seen in 65-70% of the vaccinees with a 6-8-fold increase over baseline . Plasma specimens were as good as sera for determining anti-toxic antibodies by ELISA, but were less satisfactory for determining vibriocidal antibody titers. Res Microbiol, 1999 Nov-Dec, 150(9-10), 641 - 51 Super-integrons; Rowe-Magnus DA et al.; Integrons represent the primary mechanism for antibiotic resistance gene capture and dissemination among gram-negative bacteria . The recent finding of super-integron (SI) structures in the genomes of several bacterial species has expanded their role in genome evolution . The Vibrio cholerae superintegron is gathered in a single chromosomal super-structure harbouring hundreds of gene cassettes . The encoded functions, when identifiable, are linked to adaptations extending beyond antibiotic resistance and pathogenicity . Comparison of the cassette contents of super-integrons from remote Vibrio species suggests that most of their cassettes are species-specific . Many bacterial species belonging to several distinct genera of the gamma- and beta-proteobacteria undoubtedly carry or show strong evidence for the presence of chromosomal SIs . If each bacterial species harbouring a SI has its own cassette pool, the resource in terms of gene cassette availability may be immense. Eur J Biochem, 2000 Feb, 267(4), 979 - 83 Activity and stability of a neutral protease from Vibrio sp . (vimelysin) in a pressure-temperature gradient; Ikeuchi H et al.; The apparent second-order rate constant of hydrolysis of Fua-Gly-LeuNH2 by vimelysin, a neutral protease from Vibrio sp . T1800, was measured in a variable pressure-temperature gradient (0 . 1-400 MPa and 5-40 degrees C) . The apparent maximum rate was observed at approximately 15 degrees C and 150-200 MPa; the pressure-activation ratio (kcat/Km(max)/kcat/Km(0.1 MPa)) was reached about sevenfold . The pressure dependence of the kcat and Km parameters at constant temperature (25 degrees C) revealed that the pressure-activation below 200 MPa was mainly caused by a change in the kcat parameter . The change in the intrinsic fluorescence intensity of vimelysin was also measured in a pressure-temperature plane (0.1-400 MPa and -20 to +60 degrees C) . The fluorescence intensity was found to decrease by increasing pressure and temperature, and the isointensity contours were more or less circular . The tangential lines to the contours at high temperatures and low to medium pressures seem to have slightly positive slopes, which was reflected by the higher residual activities left after incubations at higher temperatures and medium pressure (200 MPa and 50 degrees C) and by the almost intact secondary structure left after 1 h of incubation at 200 MPa and 40 degrees C, as studied by circular dichroism . These results were compared with the corresponding results for thermolysin, a moderately thermostable protease from Bacillus thermoproteolyticus . Apparent differences that might be related to the temperature adaptations of the respective source microbes are also discussed. J Microbiol Methods, 2000 Feb, 39(3), 213 - 24 Comparative study of the abundance of various bacterial morphotypes in an eutrophic freshwater environment determined by AODC and TEM; Fischer UR et al.; Transmission electron microscopy (TEM) and epifluorescence microscopy were used to obtain comparative measurements of total bacterial counts, and to enumerate abundances of various bacterial morphotypes in an eutrophic freshwater habitat . Although particulate matter would have been expected to interfere with counting by obscuring large areas of the electron microscope grids, estimates of total bacterial abundance made by TEM were on average 1.2 times greater than those obtained using the acridine orange direct counting method (AODC) . However, the precision of the AODC method was greater than that for TEM, with a coefficient of variation (C.V.) of 4.0% versus 8.8%, respectively . The total bacterial abundance ranged from 1.1 to 3.2 x 10(6) ml(-1) . As was the case for total bacterial density, the numbers of rod- and vibrio-shaped cells were lower when counted in the epifluorescence microscope, indicating the presence of potential starvation forms or ultramicrobacteria . Greatest variations in counts made by TEM and AODC were found for filamentous and coccoid bacteria . Counts of filamentous bacteria made by AODC were only about half of those detected by TEM . In contrast, cocci were on average 1.5 times greater when counted by AODC compared to TEM estimates . Both counting differences were probably caused by the morphology and low density of filamentous and coccoid bacteria (1.7 and 1.4 x 10(5) ml(-1), respectively), which led to an uneven distribution on polycarbonate filters as well as on electron microscope grids . Besides, cocci might easily be mistaken for large viral particles when counted by AODC . Hence, the study supports the use of TEM over AODC for obtaining accurate estimates of total bacterial abundance and especially bacterial morphotypes in natural waters. Rev Fac Cien Med Univ Nac Cordoba, 1999, 56(1), 85 - 9 {Isolation of Vibrio cholerae non 01 from patients with acute gastroenteritis}; Patrito E et al.; It was assay by biochemical and immunological tests, strains of V . Cholerae non-01 non-0139 isolated from two patients of the province of Cordoba, Argentina . They showed episodes of acute gastroenteritis . Strains non-01 non-0139 were isolated from water samples ingested by patients . We conclude that strains identified from patients would have the source from contaminated environmental water by V . Cholerae. Lancet, 2000 Jan 29, 355(9201), 377 - 8 Helicobacter pylori and epidemic Vibrio cholerae O1 infection in Peru; Shahinian ML et al.; In a cross-sectional study of the 1991 Peruvian cholera epidemic, Vibrio cholerae O1 infection was associated with Helicobacter pylori infection, particularly in young children . These data support the hypothesis that hypochlorhydria induced by H . pylori is important in the pathogenesis of diarrhoeal disease. Yi Chuan Xue Bao, 1999, 26(5), 585 - 90 {Cloning and sequence analysis of ATCase genes from Psychrophilic vibrio}; Zhang YF et al.; The gene encoding for aspartate transcarbamoylase (ATCase) from Psychrophilic vibrio, strain 2693 was cloned and sequenced . The sequence revealed the existence of two gene encoding respectively for a catalytic chain (pyrB) and a regulatory chain (pyr I) . The catalytic and regulatory polypeptide chains of Vibrio 2693 ATCase are encoded by a single pyrBI bicistronic operon, and appear to be transcribed under the control of the same promoter . The 3'-terminus of the catalytic cistron (pyrB) is adjacent to the 5'-terminus of the regulatory cistron, these is only a 4 bp space between the two coding regions. Cent Eur J Public Health, 1999 Nov, 7(4), 216 - 20 Quality of water--quality of life; Sixl W et al.; Especially in developing countries, the problem of adequate drinking water supply is an ever growing one . Public health programmes have been established to improve the population's health conditions, but these programmes require big financial means for guaranteeing adequate supply of potable water and medical therapy for sick people . Too little emphasis is still put on regular testing of drinking water for microorganisms such as Aeromonas sp . and Vibrio sp . In a spot check analysis in various countries, the importance of Aeromonas sp . is shown--not a single sample complied with international norms and guidelines for drinking water. J Clin Microbiol, 2000 Feb, 38(2), 578 - 85 Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analyses; Matsumoto C et al.; Vibrio parahaemolyticus O3:K6 strains responsible for the increase in the number of cases of diarrhea in Calcutta, India, beginning in February 1996 and those isolated from Southeast Asian travelers beginning in 1995 were shown to belong to a unique clone characterized by possession of the tdh gene but not the trh gene and by unique arbitrarily primed PCR (AP-PCR) profiles (J . Okuda, M . Ishibashi, E . Hayakawa, T . Nishino, Y . Takeda, A . K . Mukhopadhyay, S . Garg, S . K . Bhattacharya, G . B . Nair, and M . Nishibuchi, J . Clin . Microbiol . 35:3150-3155, 1997) . Evidence supporting a hypothesis that this clone emerged only recently and is spreading to many countries was obtained in this study . Of 227 strains isolated in a hospital in Bangladesh between 1977 and 1998, only 22 strains isolated between 1996 and 1998 belonged to the new O3:K6 clone (defined by the serovar, the tdh and trh typing, and AP-PCR profiles) . The O3:K6 strains isolated from clinical sources in Taiwan, Laos, Japan, Thailand, Korea, and the United States between 1997 and 1998 were also shown to belong to the new O3:K6 clone . The clonality of the new O3:K6 strains was also confirmed by analysis of the toxRS sequence, which has been shown to be useful for phylogenetic analysis of the members of the genus Vibrio . The toxRS sequences of the representative strains of the new O3:K6 clone differed from those of the O3:K6 strains isolated before 1995 at least at 7 base positions within a 1,346-bp region . A new PCR method targeted to 2 of the base positions unique to the new O3:K6 clone was developed . This PCR method could clearly differentiate all 172 strains belonging to the new O3:K6 clone from other O3:K6 strains isolated earlier . One hundred sixty-six strains belonging to 28 serovars other than O3:K6 were also examined by the new PCR method . The tdh-positive and trh-lacking strains that belonged to the O4:K68 and O1:K untypeable serovars and were isolated in three countries and from international travelers beginning in 1997 gave positive results . The AP-PCR profiles of these strains were nearly identical to those of the new O3:K6 clone, and their toxRS sequences were 100% identical to that of the new O3:K6 clone . The results suggest that these strains may have diverged from the new O3:K6 clone by alteration of the O:K antigens . In conclusion, this study presents strong evidence for the first pandemicity in the history of V . parahaemolyticus and reports a novel toxRS-targeted PCR method that will be useful in epidemiological investigation of the cases associated with the current pandemic spread. J Exp Zool, 2000 Feb 15, 286(3), 280 - 96 Aposymbiotic culture of the sepiolid squid Euprymna scolopes: role of the symbiotic bacterium Vibrio fischeri in host animal growth, development, and light organ morphogenesis; Claes MF et al.; The sepiolid squid Euprymna scolopes forms a bioluminescent mutualism with the luminous bacterium Vibrio fischeri, harboring V . fischeri cells in a complex ventral light organ and using the bacterial light in predator avoidance . To characterize the contribution of V . fischeri to the growth and development of E . scolopes and to define the long-term effects of bacterial colonization on light organ morphogenesis, we developed a mariculture system for the culture of E . scolopes from hatching to adulthood, employing artificial seawater, lighting that mimicked that of the natural environment, and provision of prey sized to match the developmental stage of E . scolopes . Animals colonized by V . fischeri and animals cultured in the absence of V . fischeri (aposymbiotic) grew and survived equally well, developed similarly, and reached sexual maturity at a similar age . Development of the light organ accessory tissues (lens, reflectors, and ink sac) was similar in colonized and aposymbiotic animals with no obvious morphometric or histological differences . Colonization by V . fischeri influenced regression of the ciliated epithelial appendages (CEAs), the long-term growth of the light organ epithelial tubules, and the appearance of the cells composing the ciliated ducts, which exhibit characteristics of secretory tissue . In certain cases, aposymbiotic animals retained the CEAs in a partially regressed state and remained competent to initiate symbiosis with V . fischeri into adulthood . In other cases, the CEAs regressed fully in aposymbiotic animals, and these animals were not colonizable . The results demonstrate that V . fischeri is not required for normal growth and development of the animal or for development of the accessory light organ tissues and that morphogenesis of only those tissues coming in contact with the bacteria (CEAs, ciliated ducts, and light organ epithelium) is altered by bacterial colonization of the light organ . Therefore, V . fischeri apparently makes no major metabolic contribution to E . scolopes beyond light production, and post-embryonic development of the light organ is essentially symbiont independent . J . Exp . Zool . 286:280-296, 2000 . Appl Environ Microbiol, 2000 Feb, 66(2), 855 - 9 A selective medium and a specific probe for detection of Vibrio vulnificus; Cerda-Cuellar M et al.; A selective medium (VVM) and a specific 16S rRNA gene (rDNA) probe (V3VV) for the detection of Vibrio vulnificus were developed . The medium contains D-(+)-cellobiose as the main carbon source and electrolytes (MgCl(2)-6H(2)O and KCl), which stimulate bacterial growth . Polymyxin B, colistin, and moderate alkalinity and salinity provide selectivity properties . V . vulnificus grows on VVM as flat, bright yellow colonies . Other Vibrio species tested either did not grow or showed green-bluish colonies, with the exception of V . campbelli, V . carchariae, and V . navarrensis . There is a higher colony count on VVM agar than on cellobiose-colistin agar or on modified cellobiose-polymyxin B-colistin agar . The specific probe was evaluated by colony hybridization and dot blot hybridization with PCR-amplified 16S rDNA using collection strains and environmental isolates . No strain studied other than V . vulnificus showed positive hybridization with this oligonucleotide . The combined use of VVM agar and the V3VV probe provided the recovery of V . vulnificus from mixed bacterial suspensions and spiked mussels. Appl Environ Microbiol, 2000 Feb, 66(2), 718 - 22 beta-cyanoalanine production by marine bacteria on cyanide-free medium and its specific inhibitory activity toward cyanobacteria; Yoshikawa K et al.; In screening the culture broth of marine bacteria collected at Yap (Micronesia), Palau (Belau), and Okinawa (the southwest islands of Japan) for antimicroalgal activity, 37 out of 2,594 bacterial isolates tested were found to produce anticyanobacterial substances against Oscillatoria amphibia NIES-361 . One strain, C-979, identified as a Vibrio sp., was selected and cultured in 2.4 liters of marine broth 2216 to identify the bioactive compound produced by the strain . The purified very hydrophilic compound (16.4 mg) was determined to be beta-cyano-L-alanine (L-CNAla) by instrumental analyses and the application of the advanced Marfey method . L-CNAla did not inhibit the growth of bacteria, yeast, or eukaryotic microalgae, but some cyanobacteria were found to be sensitive to L-CNAla at a concentration of 0.4 to 25 microg/ml . The effect of L-CNAla on some other environmental organisms, including invertebrates and a macroalgae, is discussed . CNAla production in marine broth was examined by thin-layer chromatography for the 37 bacterial isolates which produced an anticyanobacterial substance . The broth of 36 of these strains contained CNAla, suggesting the wide distribution of CNAla production by marine bacteria . This is the first report on bacteria that produce CNAla without a supply of the cyanide ion in the medium. J Eukaryot Microbiol, 2000 Jan-Feb, 47(1), 62 - 9 Comparison of growth efficiencies of protozoa growing on bacteria deposited on surfaces and in suspension; Zubkov MV et al.; Bacteria were deposited in tubes as compact pellets by centrifuging suspensions of cultured Vibrio at stationary phase . Numbers and protein biomass of flagellates added to these tubes and of the Vibrio, were followed and compared with the growth of the same and other protists on identical, uncentrifuged Vibrio . The flagellates Bodo saliens and Caecitellus parvulus, which could not be seen to multiply in tubes of suspended bacteria, grazed deposited bacteria actively as did the more versatile flagellate Cafeteria roenbergensis . The growth of these flagellates and their consumption of deposited bacteria were very similar to those of the flagellate Pteridomonas danica or the ciliate Uronema marinum fed with suspended bacteria, although deposit-feeders grew more slowly . Gross growth efficiencies (30-60%) of deposit-feeding flagellates were similar to those of the suspension-feeding protists . Caecitellus consumed 55 Vibrio to produce one flagellate, while 4,500 Vibrio were consumed to produce one Uronema . Surface-feeding flagellates are shown to be efficient bacterivores, capable of restricting the numbers of bacteria deposited on surfaces just as other protozoa control numbers of suspended bacteria. J Bacteriol, 2000 Feb, 182(4), 1001 - 7 Cysteine-scanning mutagenesis of the periplasmic loop regions of PomA, a putative channel component of the sodium-driven flagellar motor in Vibrio alginolyticus; Asai Y et al.; The sodium-driven motor consists of the products of at least four genes, pomA, pomB, motX, and motY, in Vibrio alginolyticus . PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ion channel . In PomA, two periplasmic loops were predicted at positions 21 to 36 between membrane segments 1 and 2 (loop(1-2)) and at positions 167 to 180 between membrane segments 3 and 4 (loop(3-4)) . To characterize the two periplasmic loop regions, which may have a role as an ion entrance for the channel, we carried out cysteine-scanning mutagenesis . The T186 residue in the fourth transmembrane segment and the D71, D148, and D202 residues in the predicted cytoplasmic portion of PomA were also replaced with Cys . Only two mutations, M179C and T186C, conferred a nonmotile phenotype . Many mutations in the periplasmic loops and all of the cytoplasmic mutations did not abolish motility, though the five successive substitutions from M169C to K173C of loop(3-4) impaired motility . In some mutants that retained substantial motility, motility was inhibited by the thiol-modifying reagents dithionitrobenzoic acid and N-ethylmaleimide . The profiles of inhibition by the reagents were consistent with the membrane topology predicted from the hydrophobicity profiles . Furthermore, from the profiles of labeling by biotin maleimide, we predicted more directly the membrane topology of loop(3-4) . None of the loop(1-2) residues were labeled, suggesting that the environments around the two loops are very different . A few of the mutations were characterized further . The structure and function of the loop regions are discussed. Indian J Med Res, 1999 Oct, 110, 126 - 7 Outbreak of cholera in arid zone of Bikaner; Gupta A et al.; Bikaner being an arid zone was more or less unaffected by cholera until 1994 when an outbreak of clinical cholera occurred . We isolated 64 Vibrio cholerae strains out of 475 stool samples received (isolation rate 13.47%) . All the Vibrio strains belonged to biotype El Tor serotype Ogawa . Low isolation rate was probably related to the poor transportation and medical facilities available at remote areas and indiscriminate and irrational use of antibiotics . The antimicrobial susceptibility testing of the isolates has not shown significant resistance against commonly used antimicrobials. Infect Immun, 2000 Feb, 68(2), 977 - 81 Optimizing the germfree mouse model for in vivo evaluation of oral Vibrio cholerae vaccine and vector strains; Crean TI et al.; The germfree mouse model of Vibrio cholerae infection can be used to judge immune responses to V . cholerae vaccine and vector strains . In the original model, a single oral inoculation was administered on day 0, a booster oral inoculation was administered on day 14, and immune responses were analyzed with samples collected on day 28 . Unfortunately, immune responses in this model frequently were low level, and interanimal variability occurred . In order to improve this model, we evaluated various primary and booster V . cholerae inoculation schedules . The most prominent systemic and mucosal antibody responses were measured in mice that received a multiple primary inoculation series on days 0, 2, 4, and 6 and booster inoculations on days 28 and 42 . These modifications result in improved preliminary evaluation of V . cholerae vaccine and vector strains in mice. Infect Immun, 2000 Feb, 68(2), 948 - 52 Molecular characterization of a new variant of toxin-coregulated pilus protein (TcpA) in a toxigenic non-O1/Non-O139 strain of Vibrio cholerae; Nandi B et al.; A toxigenic non-O1/non-O139 strain of Vibrio cholerae (10259) was found to contain a new variant of the toxin-coregulated pilus (TCP) protein gene (tcpA) as determined by PCR and Southern hybridization experiments . Nucleotide sequence analysis data of the new tcpA gene in strain 10259 (O53) showed it to be about 74 and 72% identical to those of O1 classical and El Tor biotype strains, respectively . The predicted amino acid sequence of the 10259 TcpA protein shared about 81 and 78% identity with the corresponding sequences of classical and El Tor TcpA strains, respectively . An antiserum raised against the TCP of a classical strain, O395, although it recognized the TcpA protein of strain 10259 in an immunoblotting experiment, exhibited considerably less protection against 10259 challenge compared to that observed against the parent strain . Incidentally, the tcpA sequences of two other toxigenic non-O1/non-O139 strains (V2 and S7, both belonging to the serogroup O37) were determined to be almost identical to that of classical tcpA . Further, tcpA of another toxigenic non-O1/non-O139 strain V315-1 (O nontypeable) was closely related to that of El Tor tcpA . Analysis of these results with those already available in the literature suggests that there are at least four major variants of the tcpA gene in V . cholerae which probably evolved in parallel from a common ancestral gene . Existence of highly conserved as well as hypervariable regions within the sequence of the TcpA protein would also predict that such evolution is under the control of considerable selection pressure. Infect Immun, 2000 Feb, 68(2), 526 - 34 Cloning and characterization of vuuA, a gene encoding the Vibrio vulnificus ferric vulnibactin receptor; Webster AC et al.; The ability of Vibrio vulnificus to acquire iron from the host has been shown to correlate with virulence . Many iron transport genes are regulated by iron, and in V . vulnificus, transcriptional regulation by iron depends on the fur gene . The N-terminal amino acid sequence of a 72-kDa iron-regulated outer membrane protein purified from a V . vulnificus fur mutant had 53% homology with the first 15 amino acids of the mature protein of the Vibrio cholerae vibriobactin receptor, ViuA . In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for VuuA, the vulnibactin receptor of V . vulnificus . Analysis of the DNA sequence of the vuuA promoter region demonstrated a sequence identical to the upstream Fur box of V . cholerae viuA . Northern blot analysis showed that the transcript was strongly regulated by iron . The amino acid sequence of VuuA was 74% identical to the sequence of V . cholerae ViuA and was homologous to those of several TonB-dependent outer membrane receptors . An internal deletion of the V . vulnificus vuuA gene resulted in the loss of expression of the 72-kDa protein and the loss of the ability to use transferrin or vulnibactin as a source of iron . This mutant showed reduced virulence in an infant mouse model . Introduction of a plasmid containing the complete viuA coding sequence and 342 bp of upstream DNA into the mutant restored ferric vulnibactin and ferric transferrin utilization to the mutant. Trends Microbiol, 2000 Jan, 8(1), 39 - 42 An endogenous retrovirus and exogenous scrapie in a mouse model of aging; Carp RI et al.; As we enter the post-genomic era, there is an increasing need for accurate methods of identifying host and pathogen factors that contribute to bacterial, viral and fungal disease . In addition, there is a requirement for fast and precise techniques to evaluate potential therapies for the prevention of infectious diseases . The development of useful and cost-effective model systems will be crucial in advancing our knowledge of all aspects of microbial pathogenesis . In this series, we will learn of animal models used to investigate diseases caused by a wide variety of pathogens, including HIV, Vibrio cholerae and Pseudomonas aeruginosa . A description of a model system specifically designed to study intracellular pathogens will be presented, as will a variety of the techniques currently used to exploit other useful models of infection . Additionally, a description of the mathematical models used to analyse the population biology of human onchocerciasis will be discussed . The series begins with an intriguing look at the possible connections between an endogenous retrovirus, the infectious agent of scrapie and accelerated senescence in a mouse model of early aging. Carbohydr Res, 1999 Dec 12, 322(3-4), 201 - 8 The synthesis and evaluation of novel sialic acid analogues bound to matrices for the purification of sialic acid-recognising proteins; Abo S et al.; A novel N-acetylneuraminic acid analogue, 2-S-(5'-aminopentyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, as well as the thiosialoside 2-S-(2'-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, have been synthesised and successfully coupled to CNBr-activated Sepharose 4B through the terminal amino group . The resultant affinity resins have proved efficient in purifying a number of sialic acid-recognising proteins such as Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Trypanosoma cruzi, and sialyltransferases from rat liver, all in high yield. Biosci Biotechnol Biochem, 1999 Nov, 63(11), 2017 - 9 Purification and characterization of beta-1,3-xylanase from a marine bacterium, Vibrio sp . XY-214; Araki T et al.; beta-1,3-Xylanase was purified to gel electrophoretic homogeneity and 83-fold from a cell-free culture fluid of Vibrio sp . XY-214 by ammonium sulfate precipitation and successive chromatographies . The enzyme had a pl of 3.6 and a molecular mass of 52 kDa . The enzyme had the highest level of activity at pH 7.0 and 37 degrees C . The enzyme activity was completely inhibited by Cu2+, Hg2+, and N-bromosuccinimide . The enzyme hydrolyzed beta-1,3-xylan to produce mainly xylotriose and xylobiose but did not act on xylobiose, p-nitrophenyl-beta-D-xyloside, beta-1,4-xylan, beta-1,3-glucan, or carboxymethyl cellulose. Science, 1982 Nov 19, 218(4574), 791 - 3 Bacterial bioluminescence: isolation and expression of the luciferase genes from Vibrio harveyi; Belas R et al.; Genes for the luciferase enzyme of Vibrio harveyi were isolated in Escherichia coli by a general method in which nonluminous, transposon insertion mutants were used . Conditions necessary for light production in E . coli were examined . Stimulation of transcription of the genes for luciferase (lux A and lux B) was required for efficient synethesis of luciferase . To enhance transcription bacteriophage promoter elements were coupled to the cloned lux gene fragments. Sci Total Environ, 1999 Dec 15, 243-244, 141 - 8 Acute toxicity assessment of Polish (waste) water with a microplate-based Hydra attenuata assay: a comparison with the Microtox test; Pardos M et al.; The use of Hydra attenuata in acute toxicity assessment is a potentially useful tool in (waste) water biomonitoring . The purpose of this study was to compare the sensitivity of H . attenuata with the extensively used Microtox test on 14 (waste) water samples from the Krakow region (South Poland) . To this end, specific morphological changes displayed by the freshwater cnidarian Hydra attenuata (lethal LC50s and sublethal EC50s effects) and bioluminescence of the marine bacteria Vibrio fisheri (Microtox) were compared . Clearly, the Hydra assay was the more sensitive indicator of toxicity . No relationship was found among Hydra toxicological responses and water levels of As, Cd, Co, Cu, Pb and Zn . However, it appeared that toxicity to Hydra might be due to ammonia levels . Additional studies to better circumscribe the tolerance of H . attenuata to 'natural' water characteristics are needed. J Bacteriol, 2000 Feb, 182(3), 805 - 11 Conversion of the Vibrio fischeri transcriptional activator, LuxR, to a repressor; Egland KA et al.; The Vibrio fischeri luminescence (lux) operon is regulated by a quorum-sensing system that involves the transcriptional activator (LuxR) and an acyl-homoserine lactone signal . Transcriptional activation requires the presence of a 20-base inverted repeat termed the lux box at a position centered 42.5 bases upstream of the transcriptional start of the lux operon . LuxR has proven difficult to study in vitro . A truncated form of LuxR has been purified, and together with sigma(70) RNA polymerase it can activate transcription of the lux operon . Both the truncated LuxR and RNA polymerase are required for binding to lux regulatory DNA in vitro . We have constructed an artificial lacZ promoter with the lux box positioned between and partially overlapping the consensus -35 and -10 hexamers of an RNA polymerase binding site . LuxR functioned as an acyl-homoserine lactone-dependent repressor at this promoter in recombinant Escherichia coli . Furthermore, multiple lux boxes on an independent replicon reduced the repressor activity of LuxR . Thus, it appears that LuxR can bind to lux boxes independently of RNA polymerase binding to the promoter region . A variety of LuxR mutant proteins were studied, and with one exception there was a correlation between function as a repressor of the artificial promoter and activation of a native lux operon . The exception was the truncated protein that had been purified and studied in vitro . This protein functioned as an activator but not as a repressor in E . coli . The data indicate that the mutual dependence of purified, truncated LuxR and RNA polymerase on each other for binding to the lux promoter is a feature specific to the truncated LuxR and that full-length LuxR by itself can bind to lux box-containing DNA. J Bacteriol, 2000 Feb, 182(3), 742 - 8 Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae; Sandkvist M et al.; Extracellular secretion of proteins via the type II or general secretion pathway in gram-negative bacteria requires the assistance of at least 12 gene products that are thought to form a complex apparatus through which secreted proteins are translocated . Although this apparatus is specifically required only for the outer membrane translocation step during transport across the bacterial cell envelope, it is believed to span both membranes . The EpsE, EpsL, and EpsM proteins of the type II apparatus in Vibrio cholerae are thought to form a trimolecular complex that is required to either control the opening and closing of the secretion pore or to transduce energy to the site of outer membrane translocation . EpsL is likely to play an important role in this relay by interacting with both the cytoplasmic EpsE protein and the cytoplasmic membrane protein EpsM, which is predominantly exposed on the periplasmic side of the membrane . We have now extended this model and mapped the separate regions within EpsL that contain the EpsE and EpsM binding domains . By taking advantage of the species specificity of the type II pathway, we have used chimeric proteins composed of EpsL and its homologue, ExeL, from Aeromonas hydrophila together with either EpsE or its Aeromonas homologue, ExeE, to complement the secretion defect in both epsL and exeL mutant strains . These studies have mapped the species-specific EpsE binding site to the N-terminal cytoplasmic region between residues 57 and 216 of EpsL . In addition, the species-specific EpsM binding site was mapped to the C-terminal half of EpsL by coimmunoprecipitation of EpsM with different EpsL-ExeL chimeras . This site is present in the region between amino acids 216 and 296, which contains the predicted membrane-spanning segment of EpsL. Mol Microbiol, 2000 Jan, 35(1), 189 - 203 Molecular cloning and transcriptional regulation of ompT, a ToxR-repressed gene in Vibrio cholerae; Li CC et al.; In pathogenic Vibrio cholerae, at least 17 genes are co-ordinately regulated by ToxR . Most of these genes, including those that encode cholera toxin (CT), toxin co-regulated pilus (TCP), accessory colonization factor (ACF) and OmpU, are positively regulated . OmpT is the only identified protein under negative regulation of ToxR . To understand the molecular mechanism by which ToxR represses OmpT expression, we cloned ompT and characterized the ompT promoter and its interaction with ToxR . Sequence analysis revealed that ompT encodes a predicted 35.8 kDa outer membrane porin of V . cholerae . Primer extension analysis identified a transcriptional start site 104 bp upstream of the translational start codon . Both primer extension analysis and promoter fusion studies showed that ToxR represses OmpT expression at the transcriptional level . Promoter fusion studies also suggest that cyclic AMP receptor protein (CRP) is involved in ompT activation . Gel mobility shift assays combined with DNase I footprinting analysis demonstrated that ToxR mediates repression of ompT transcription by directly binding to an A/T-rich region between -95 and -30 of the ompT promoter . To further understand how the interaction of ToxR with different promoters results in its function as an activator or repressor, we have also mapped the regions on the ctxAB and toxT promoters to which ToxR binds . The regions protected by ToxR on each of these promoters are all A/T rich and large in size, although they are positioned differently relative to each transcriptional start site. Mol Microbiol, 2000 Jan, 35(1), 139 - 49 A genetic analysis of the functions of LuxN: a two-component hybrid sensor kinase that regulates quorum sensing in Vibrio harveyi; Freeman JA et al.; The bioluminescent marine bacterium Vibrio harveyi controls light production using two parallel quorum-sensing systems . V . harveyi produces two autoinducers (AI-1 and AI-2), which are recognized by cognate membrane-bound two-component hybrid sensor kinases called LuxN and LuxQ respectively . Under conditions of low cell density, in the absence of autoinducer, the hybrid sensors are kinases, and under conditions of high cell density, in the presence of autoinducer, the sensors are phosphatases . These activities allow LuxN and LuxQ to modulate the level of phosphorylation of the response regulator protein LuxO . LuxO, in turn, controls the transcription of the genes encoding luciferase . The phosphorelay protein LuxU is required for signalling to LuxO . In this report, we present a genetic analysis of the activities of the AI-1 sensor LuxN . Point mutations and in frame deletions were constructed in luxN and recombined onto the chromosome of V . harveyi for in vivo phenotypic analysis . We show that the conserved histidine (H471) in the sensor kinase domain of LuxN is required for kinase activity but not for phosphatase activity . In contrast, the conserved aspartate (D771) in the response regulator domain of LuxN is required for both activities . Furthermore, the LuxN phosphatase activity is localized to the response regulator domain . Our results indicate that the LuxN kinase activity is regulated by the presence of AI-1, whereas the LuxN phosphatase activity is constitutive . We also show that signalling from the two V . harveyi quorum-sensing systems is not equivalent . AI-1 and LuxN have a much greater effect on the level of LuxO phosphate and therefore Lux expression than do AI-2 and LuxQ. Cornea, 2000 Jan, 19(1), 108 - 9 Vibrio vulnificus corneal ulcer: rapid resolution of a virulent pathogen; Massey EL et al.; PURPOSE: To describe a corneal ulcer due to Vibrio vulnificus that resolved rapidly with antibiotic therapy alone . METHOD: Case report and review of literature . RESULTS: This is the third reported case of corneal ulcer due to V . vulnificus . All followed trauma sustained during oyster shucking . Both prior cases required invasive therapy to achieve a cure . Our case responded rapidly to hourly treatment with ciprofloxacin, Neosporin, and fortified vancomycin . CONCLUSION: V . vulnificus is a virulent pathogen that can infect the cornea after shellfish injury to the eye . Clinical suspicion and early therapy with appropriate antibiotics can lead to an excellent outcome. Carbohydr Res, 1999 Nov 23, 322(1-2), 40 - 5 Structure of the exopolysaccharide of Vibrio diabolicus isolated from a deep-sea hydrothermal vent; Rougeaux H et al.; The structure of the exopolysaccharide produced under laboratory conditions by Vibrio diabolicus, a bacterium recovered from a deep-sea hydrothermal vent, has been investigated using sugar and methylation analysis and NMR spectroscopy . The polysaccharide consists of a linear tetrasaccharide repeating unit with the following structure . -->3)-beta-D-Glcp Nac-(1-->4)-beta-D-Glcp A-(1-->4)-beta-D-Glcp A-(1-->4)-alpha-D-Galp NAc-(1--> J Food Prot, 2000 Dec, 63(12), 1665 - 9 Effect of simulated gastric fluid and bile on survival of Vibrio vulnificus and Vibrio vulnificus phage; Koo J et al.; Bacteria and phages may be exposed to acid conditions in the stomach and to bile in the intestine . Survival of three strains of Vibrio vulnificus and three strains of its phages was examined at 37 degrees C after exposure to simulated gastric fluid at pH 3 to 4 or to 0, 1, and 2% bile in broth or buffer . Mean D-values (decimal reduction times) at pH 4 and 3 were 3.3 and 1.3 min for V . vulnificus and 97.8 and 0.7 min for its phages . No V . vulnificus survivors were found at pH 2.0 . There were few survival differences among strains of V . vulnificus or its phages . Numbers of V . vulnificus increased 1 log in tryptic soy broth containing 1 or 2% bile after 3 h . Numbers of V . vulnificus and its phages remained constant in phosphate-buffered saline regardless of bile concentrations up to 3 h . Those V . vulnificus bacteria and phages that survive stomach acidity may proliferate in the small intestine, since they are resistant to bile. J Food Prot, 2000 Dec, 63(12), 1660 - 4 Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of vibrio parahaemolyticus; McCarthy SA et al.; Production of the thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus is associated with pathogenicity of the organism and is encoded by the tdh gene . The timely resolution of seafood-associated outbreaks requires rapid and accurate detection of pathogenic V . parahaemolyticus . The specificity of alkaline phosphatase- and digoxigenin-labeled tdh gene probes was evaluated against 61 strains of V . parahaemolyticus (including isolates from recent outbreaks involving oysters from the Pacific Northwest, Texas, and New York), 85 strains of other vibrios, and 7 strains of non-vibrio species from clinical and environmental sources . The probes were specific for detection of the V . parahaemolyticus tdh gene. Singapore Med J, 1999 Sep, 40(9), 596 - 7 Fulminant necrotising fasciitis caused by Vibrio parahaemolyticus; Lim TK et al.; We report a patient with septicaemia and fulminant necrotising fasciitis caused by Vibrio parahaemolyticus . This organism is strongly associated with seawater exposure and seafood ingestion . The patient recovered due to expedient management, prompt recognition of the organism, appropriate antimicrobial cover and surgical debridement . The lesson to be learned is that this organism should be clinically suspected and recognised from its typical history of injury and fulminant clinical progress as a delay in diagnosis and treatment may result in an increased risk of mortality. Biochemistry, 2000 Jan 11, 39(1), 117 - 26 Mechanism of action and identification of Asp242 as the catalytic nucleophile of Vibrio furnisii N-acetyl-beta-D-glucosaminidase using 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosyl fluoride; Vocadlo DJ et al.; The novel mechanism-based reagent 2-acetamido-2-deoxy-5-fluoro-alpha-L-idopyranosykl fluoride has been synthesized, and the kinetic parameters K(M) = 0.23 mM and K(CAT)= 0.55 min(-1) for its hydrolysis by vibrio furnisi beta-N-acetylglucosaminidase (ExoII) HAVE been determined . Investigation of mixtures of enzyme with this slow substrate by electrospray mass spectrometry revealed a high steady-state population of the 2-acetamido-2-deoxy-5-fluoro-beta-L-idopyranosyl-enzyme, indicating that the hydrolytic mechanism of ExoII involves the formation and rate-determining hydrolysis of a glycosyl-enzyme intermediate . Analysis of a peptic digest of the glycosyl-enzyme by HPLC/ESMS/MS in the netural-loss mode permitted identification of a peptide bearing the 5-fluoro-sugar moiety . Tandem MS sequencing of the labeled peptide, in conjuction with multiple sequence alignmentsS of family 3 members, allowed the identification of ASP242 as the catalytic nucleophile within the sequence IVFSDDLSM. Kansenshogaku Zasshi, 1999 Nov, 73(11), 1159 - 62 {A case of a sudden death from Vibrio vulnificus septicemia in a patient with liver cirrhosis}; Izumikawa K et al.; A 46-year-old male patient with alcoholic cirrhosis of the liver was carried to our out-patient clinic as he had developed shock while under routine follow-up, and died on the way to the hospital . He had been admitted several times since the diagnosis eight years ago, and was finally discharged from the hospital six weeks ago with improved physical condition and laboratory findings . A vesicle and bulla formation with phlegmon on the skin of right leg and sole of foot was noticed . Vibrio vulnificus was detected from the purulent discharge of the skin on culture . We conclude that the patient developed V . vulnificus-septicemia which resulted in sudden death . Since V . vulnificus infection may frequently take a fulminant course in patients with liver cirrhosis, adequate measures should be taken for early diagnosis and treatment to prevent the fatal outcome. Microbios, 1999, 98(390), 95 - 111 Purification and partial characterization of a toxic serine protease produced by pathogenic Vibrio alginolyticus; Chen FR et al.; An extracellular lethal toxin produced by Vibrio alginolyticus strain Swy originally isolated from diseased kuruma prawn (Penaeus japonicus) was purified using the AKTA purifier system with hydrophobic interaction chromatography, anion exchange and gel filtration columns . The toxin is an alkaline serine protease, inhibited by phenyl methylsulphonyl fluoride (PMSF), antipain and shows maximal activity at pH 8 to 11, having a pI of 4.3 and a molecular weight of approximately 33 kD . The toxin was completely inhibited by FeCl2 but partially inhibited by 3,4-dichloroisocoumarin (3,4-DCI), ethylenediamine tetraacetic acid (EDTA), ethylene glycol-bis(beta-amino-ethyl ether) N,N,N',N'-tetraacetic acid (EGTA), CuCl2 and ZnCl2 . The purified protease was lethal for kuruma prawn at an LD50 of 0.29 microgram protein/g body weight . The haemolymph withdrawn from the moribund prawns injected with the toxic protease was unable to clot . The coagulogen in the kuruma prawn plasma showed an increased migration rate after incubation with this serine protease, and a plasma colour change from blue to pink was recorded . The addition of PMSF completely inhibited the lethal toxicity of the purified protease, indicating that this serine protease was a lethal toxin produced by the bacterium . The 33 kD protease was therefore a toxic protease produced by V . alginolyticus strain Swy. FEMS Microbiol Lett, 2000 Jan 15, 182(2), 285 - 9 Construction of mini-Tn10luxABcam/Ptac-ATS and its use for developing a bacteriophage that transduces bioluminescence to Escherichia coli O157:H7; Waddell TE et al.; Mini-Tn10luxABcam/Ptac-ATS was constructed in order to develop a luciferase-transducing bacteriophage for detecting Escherichia coli O157:H7 . The transposon was designed to deliver a 3.6-kb insertion that confers n-decanal-dependent bioluminescence and resistance to chloramphenicol and was constructed using mini-Tn10cam/Ptac-ATS in the plasmid pNK2884 and luxAB from Vibrio harveyi . PhiV10, a temperate bacteriophage infecting common phage types of Escherichia coli O157:H7, was mutagenized as a prophage in E . coli O157:H7 strain R508 . PhiV10::luxABcamA1-23 was rescued from the strain by propagating it on a strain lacking the bacteriophage and the vector containing the transposon . The bacteriophage transduced n-decanal-dependent bioluminescence to E . coli O157:H7 strain R508 that was measurable approximately 1 h post infection. Mol Gen Mikrobiol Virusol, 1999, (4), 3 - 11 {Genetic control of Vibrio cholerae pathogenicity: the temperate filamentous phage CTX, coding for cholera toxin and the "island of pathogenicity"}; Smirnova NI; Reviews modern data on the genetic control of the key factors of Vibrio cholerae pathogenicity: cholera toxin and toxin-coregulated adhesion pili . Pays special attention to the temperate filamentous CTX bacteriophage, whose genome contains structural genes of cholera toxin, and the "pathogenicity island" carrying tcp genes responsible for the most important factor of the human small intestine colonization with V . cholerae . Discusses the mechanism of coordinated regulation of the activity of the main genes of V . cholerae pathogenicity genes. J Clin Microbiol, 2000 Jan, 38(1), 424 - 6 Pulsed-field gel electrophoresis-based molecular comparison of vibrio cholerae O1 isolates from domestic and imported cases of cholera in Japan; Arakawa E et al.; Sixty-seven Vibrio cholerae O1 El Tor isolates (36 domestic and 31 imported) were classified into 19 subtypes by NotI- and SfiI-digested pulsed-field gel electrophoresis . Twenty-five of 36 domestic and 4 imported isolates were assigned to a NotI-A1-SfiI-A1 subtype, suggesting that this pulse type is widely distributed in Asia and Japan. J Clin Microbiol, 2000 Jan, 38(1), 44 - 9 Development and evaluation of a phage typing scheme for Vibrio cholerae O139; Chakrabarti AK et al.; The scenario of cholera that existed previously changed in 1992 and 1993 with the emergence of toxigenic Vibrio cholerae O139 in India . The genesis of the new serogroup formed the impetus to search for O139 phages in and around the country . A total of five newly isolated phages lytic to V . cholerae O139 strains were used for the development of this phage typing scheme . These phages differed from each other and also differed from the existing O1 phages in their lytic patterns, morphologies, restriction endonuclease digestion profiles, and immunological criteria . With this scheme, 500 V . cholerae O139 strains were evaluated for their phage types, and almost all strains were found to be typeable . The strains clustered into 10 different phage types, of which type 1 (38.2%) was the dominant type, followed by type 2 (22.4%) and type 3 (18%) . Additionally, a comparative study of phage types in 1993 and 1994 versus those from 1996 to 1998 for O139 strains showed a higher percentage of phage type 1 (40.5%), followed by type 3 (18.8%) during the period between 1993 and 1994, whereas phage type 2 (32 . 1%) was the next major type during the period from 1996 to 1998 . This scheme comprising five newly isolated phages would be another useful tool in the study of the epidemiology of cholera caused by V . cholerae O139. Appl Environ Microbiol, 2000 Jan, 66(1), 148 - 53 Genetic diversity of clinical and environmental isolates of Vibrio cholerae determined by amplified fragment length polymorphism fingerprinting; Jiang SC et al.; Vibrio cholerae, the causative agent of major epidemics of diarrheal disease in Bangladesh, South America, Southeastern Asia, and Africa, was isolated from clinical samples and from aquatic environments during and between epidemics over the past 20 years . To determine the evolutionary relationships and molecular diversity of these strains, in order to understand sources, origin, and epidemiology, a novel DNA fingerprinting technique, amplified fragment length polymorphism (AFLP), was employed . Two sets of restriction enzyme-primer combinations were tested for fingerprinting of V . cholerae serogroup O1, O139, and non-O1, O139 isolates . Amplification of HindIII- and TaqI-digested genomic DNA produced 30 to 50 bands for each strain . However, this combination, although capable of separating environmental isolates of O1 and non-O1 strains, was unable to distinguish between O1 and O139 clinical strains . This result confirmed that clinical O1 and O139 strains are genetically closely related . On the other hand, AFLP analyses of restriction enzyme ApaI- and TaqI-digested genomic DNA yielded 20 to 30 bands for each strain, but were able to separate O1 from O139 strains . Of the 74 strains examined with the latter combination, 26 serogroup O1 strains showed identical banding patterns and were represented by the O1 El Tor strain of the seventh pandemic . A second group, represented by O139 Bengal, included 12 strains of O139 clinical isolates, with 7 from Thailand, 3 from Bangladesh, and 2 from India . Interestingly, an O1 clinical isolate from Africa also grouped with the O139 clinical isolates . Eight clinical O1 isolates from Mexico grouped separately from the O1 El Tor of the seventh pandemic, suggesting an independent origin of these isolates . Identical fingerprints were observed between an O1 environmental isolate from a river in Chile and an O1 clinical strain from Kenya, both isolated more than 10 years apart . Both strains were distinct from the O1 seventh pandemic strain . Two O139 clinical isolates from Africa clustered with environmental non-O1 isolates, independent of other O139 strains included in the study . These results suggest that although a single clone of pathogenic V . cholerae appears responsible for many cases of cholera in Asia, Africa, and Latin America during the seventh pandemic, other cases of clinical cholera were caused by toxigenic V . cholerae strains that appear to have been derived locally from environmental O1 or non-O1 strains. Appl Environ Microbiol, 2000 Jan, 66(1), 140 - 7 Genetic diversity of Vibrio cholerae in Chesapeake Bay determined by amplified fragment length polymorphism fingerprinting; Jiang SC et al.; Vibrio cholerae is indigenous to the aquatic environment, and serotype non-O1 strains are readily isolated from coastal waters . However, in comparison with intensive studies of the O1 group, relatively little effort has been made to analyze the population structure and molecular evolution of non-O1 V . cholerae . In this study, high-resolution genomic DNA fingerprinting, amplified fragment length polymorphism (AFLP), was used to characterize the temporal and spatial genetic diversity of 67 V . cholerae strains isolated from Chesapeake Bay during April through July 1998, at four different sampling sites . Isolation of V . cholerae during the winter months (January through March) was unsuccessful, as observed in earlier studies (J . H . L . Kaper, R . R . Colwell, and S . W . Joseph, Appl . Environ . Microbiol . 37:91-103, 1979) . AFLP fingerprints subjected to similarity analysis yielded a grouping of isolates into three large clusters, reflecting time of the year when the strains were isolated . April and May isolates were closely related, while July isolates were genetically diverse and did not cluster with the isolates obtained earlier in the year . The results suggest that the population structure of V . cholerae undergoes a shift in genotype that is linked to changes in environmental conditions . From January to July, the water temperature increased from 3 degrees C to 27.5 degrees C, bacterial direct counts increased nearly an order of magnitude, and the chlorophyll a concentration tripled (or even quadrupled at some sites) . No correlation was observed between genetic similarity among isolates and geographical source of isolation, since isolates found at a single sampling site were genetically diverse and genetically identical isolates were found at several of the sampling sites . Thus, V . cholerae populations may be transported by surface currents throughout the entire Bay, or, more likely, similar environmental conditions may be selected for a specific genotype . The dynamic nature of the population structure of this bacterial species in Chesapeake Bay provides new insight into the ecology and molecular evolution of V . cholerae in the natural environment. J Neurochem, 2000 Jan, 74(1), 320 - 6 Affinity purification and partial characterization of the zonulin/zonula occludens toxin (Zot) receptor from human brain; Lu R et al.; The intercellular tight junctions (TJs) of endothelial cells represent the limiting structure for the permeability of the blood-brain barrier (BBB) . Although the BBB has been recognized as being the interface between the bloodstream and the brain, little is known about its regulation . Zonulin and its prokaryotic analogue, zonula occludens toxin (Zot) elaborated by Vibrio cholerae, both modulate intercellular TJs by binding to a specific surface receptor with subsequent activation of an intracellular signaling pathway involving phospholipase C and protein kinase C activation and actin polymerization . Affinity column purification revealed that human brain plasma membrane preparations contain two Zot binding proteins of approximately 55 and approximately 45 kDa . Structural and kinetic studies, including saturation and competitive assays, identified the 55-kDa protein as tubulin, whereas the 45-kDa protein represents the zonulin/Zot receptor . Biochemical characterization provided evidence that this receptor is a glycoprotein containing multiple sialic acid residues . Comparison of the N-terminal sequence of the zonulin/Zot receptor with other protein sequences by BLAST analysis revealed a striking similarity with MRP-8, a 14-kDa member of the S-100 family of calcium binding proteins . The discovery and characterization of this receptor from human brain may significantly contribute to our knowledge on the pathophysiological regulation of the BBB. Carbohydr Res, 1999 Oct 15, 321(3-4), 157 - 67 Studies on vaccines against cholera . Synthesis of neoglycoconjugates from the hexasaccharide determinant of Vibrio cholerae O:1, serotype Ogawa, by single-point attachment or by attachment of the hapten in the form of clusters; Zhang J et al.; The terminal hexasaccharide of the O-antigen of Vibrio cholerae O:1, serotype Ogawa, has been synthesized in the form of a glycoside whose aglycon (linker) allows conjugation to carrier proteins by reductive amination . The conjugate obtained from direct, single-point attachment of the linker-equipped hapten to chicken serum albumin (CSA) contained seven hapten residues/CSA . A neoglycoconjugate containing the carbohydrate antigen in the form of clusters was obtained using, as a hapten subcarrier, an oligopeptide containing 16 amino groups . It was treated with a limited amount of hapten, to give a hapten-carrying subcarrier (HCS) . Subsequent conjugation of HCS to CSA, using squaric acid diethyl ester as a conjugation reagent, gave a cross-linked, glycocluster conjugate containing 51% (w/w) of the carbohydrate. FEMS Microbiol Lett, 2000 Jan 1, 182(1), 35 - 40 Diversity in the arrangement of the CTX prophages in classical strains of Vibrio cholerae O1; Basu A et al.; This study reports the results of a molecular analysis of the CTX prophages in classical biotype strains of Vibrio cholerae O1 of clinical origin isolated between 1970 and 1979 in India . All strains were sensitive to group IV classical phage and polymyxin B but resistant to group 5 El Tor phage . These phenotypic traits are consistent to that exhibited by the classical biotype . PCR studies reconfirmed their biotype assignment and showed the presence of intact CTX prophages and the presence of the recently described toxin linked cryptic plasmid . Restriction fragment length polymorphism of rRNA genes and pulsed-field gel electrophoresis showed clonal diversity among the strains . The most notable observation was the finding that one strain (GP13) has three CTX prophages while another (GP147) has four CTX prophages . This is the first time heterogeneity is reported in the arrangement of the CTX prophages among classical strains of V . cholerae O1. Cell, 1999 Dec 10, 99(6), 625 - 34 Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection; Lee SH et al.; The temporal expression patterns of the critical Vibrio cholerae virulence genes, tcpA and ctxA, were determined during infection using a recombinase reporter . TcpA was induced biphasically in two temporally and spatially separable events in the small intestine, whereas ctxA was induced monophasically only after, and remarkably, dependent upon, tcpA expression; however, this dependence was not observed during in vitro growth . The requirements of the virulence regulators, ToxR, TcpP, and ToxT, for expression of tcpA and ctxA were determined and were found to differ significantly during infection versus during growth in vitro . These results illustrate the importance of examining virulence gene expression in the context of bona fide host-pathogen interactions. Carbohydr Res, 1999 Sep 15, 321(1-2), 88 - 95 Solution conformation and dynamics of the trisaccharide fragments of the O-antigen of Vibrio cholerae O1, serotypes Inaba and Ogawa; Gonzalez L et al.; The conformational behavior of the trisaccharide fragments of the Ogawa and Inaba Vibrio cholera serotypes has been studied using NMR and molecular dynamics (MD) . The obtained results indicate that there are no significant differences in the major conformation and in the extent of motion of the glycosidic torsions of these molecules . The differences in biological activity are probably not due to conformational effects but to van der Waals and/or hydrogen bonding interactions between the antigens and the biological receptor. Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 15196 - 201 Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli; Sperandio V et al.; Enterohemorrhagic Escherichia coli O157:H7 and enteropathogenic E . coli cause a characteristic histopathology in intestinal cells known as attaching and effacing . The attaching and effacing lesion is encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells . Using lacZ reporter gene fusions, we show that expression of the LEE operons encoding the type III secretion system, translocated intimin receptor, and intimin is regulated by quorum sensing in both enterohemorrhagic E . coli and enteropathogenic E . coli . The luxS gene recently shown to be responsible for production of autoinducer in the Vibrio harveyi and E . coli quorum-sensing systems is responsible for regulation of the LEE operons, as shown by the mutation and complementation of the luxS gene . Regulation of intestinal colonization factors by quorum sensing could play an important role in the pathogenesis of disease caused by these organisms . These results suggest that intestinal colonization by E . coli O157:H7, which has an unusually low infectious dose, could be induced by quorum sensing of signals produced by nonpathogenic E . coli of the normal intestinal flora. Electrophoresis, 1999 Nov, 20(17), 3343 - 6 Electrophoretic characterization of a novel cysteine protease produced by Vibrio harveyi; Lee KK et al.; Electrophoretic characterization of a novel cysteine protease produced by pathogenic luminous Vibrio harveyi, originally isolated from diseased tiger prawn Penaeus monodon in Taiwan, is demonstrated in the present study using native polyacrylamide gel electrophoresis (native PAGE), sodium dodecyl sulfate-PAGE (SDS-PAGE), crossed immunoelectrophoresis (CIE) and isoelectric focusing (IEF) gels . The protease has a pI of 6.4 and exhibits a fast-migrating feature in native-PAGE and CIE gels indicating that it is a negatively charged protease . The protease electrophoresed as a 22 kDa protein band in native- and SDS-PAGE (in SDS - buffer with or without the presence of 2-mercaptoethanol) while it electrophoresed as a 38 kDa protein band in SDS-PAGE when the samples were boiled for 10 min prior to electrophoresis . The results reveal that the enzyme is an SDS-resistant monomeric protease and its high negative charge is not influenced by SDS (detergent) without boiling the sample . The present results are useful in determining proteins of similar nature to this unique cysteine protease. Eur J Biochem, 2000 Jan, 267(1), 163 - 70 Characterization of adhesive epitopes with the OmpS display system; Lang H et al.; OmpS is an outer membrane protein of Vibrio cholerae where it forms trimeric pores that function in the uptake of maltose and maltodextrins . Based on sequence similarity to LamB proteins, a model of OmpS folding in the outer membrane has been constructed . According to this model, OmpS contains 18 transmembrane beta-strands and nine surface-accessible loops . Adhesive epitopes can, when inserted into surface-accessible loop 4 (L4) and expressed in Escherichia coli, retain their functional characteristics . We inserted three D-repeats from the Staphylococcus aureus fibronectin-binding protein FnBPA into L4 of OmpS and showed that E . coli cells expressing these hybrids bind fibronectin . DNA fragments covering the N-terminal half of the globoside-binding P-fimbrial adhesin class II PapG of E . coli were cloned into the same surface accessible loop (L4) of OmpS . Fragments of papG encoding 53 or 186 amino acids from the N-terminal end of class II PapG adhesin were found to confer bacterial adhesiveness to globoside . Removal of 23 amino acids from the N-terminus of PapG did not affect receptor binding, but removal of 31 amino acids abolished it . The newly developed night sky image technique was also used to demonstrate the binding properties of membrane vesicles carrying the hybrid proteins . We raised antibodies against the purified hybrid protein containing 53 amino acids from PapG . This antiserum recognized the P-fimbriae on E . coli cells . These data provide evidence that the N-terminal first 53 amino acids of class II PapG contain the receptor-binding domain. FEBS Lett, 1999 Dec 17, 463(3), 336 - 40 N-terminus of mature heat-labile enterotoxin chain B is critical for its extracellular secretion in Vibrio cholerae; Mukhija R et al.; The effects of addition of a few amino acids to the amino- and carboxy-terminal regions of the mature portion of the heat-labile enterotoxin chain B (LTB) of Escherichia coli on protein export, secretion and assembly were investigated . In E . coli, LTB (secretory protein) with or without the extension at the N- or C-terminus accumulated in the periplasmic fraction . For Vibrio cholerae, LTB with the extension at the C-terminus was exported to the periplasm followed by secretion to the extracellular milieu . However, LTB with the N-terminus extension was exported to the periplasm only . Our findings suggest that in the case of V . cholerae, the N-terminus of the mature LTB plays an important role in its secretion to the extracellular milieu. Ann Trop Paediatr, 1999 Mar, 19(1), 15 - 20 Aeromonas-associated diarrhoea in Bangladeshi children: clinical and epidemiological characteristics; Teka T et al.; We studied the clinical and epidemiological features associated with Aeromonas diarrhoea by a hospital survey of 7,398 children under 5 years of age presenting with diarrhoea . The data were actually based upon two cohorts from this survey, the majority of the data being identified from 405 (5.5%) in whom Aeromonas was the sole enteric pathogen . Aeromonas caviae was the most prevalent species, accounting for 32% (129/405) of all isolates . Eighty-three per cent of children with Aeromonas-associated diarrhoea were younger than 3 years . The majority of the children had acute onset of vomiting and watery diarrhoea resulting in mild to moderate dehydration . Fever, non-watery diarrhoea, age less than 3 years, and diarrhoea of 7-14 days duration were found to be significantly associated with Aeromonas diarrhoea compared with Vibrio cholerae O1 infection after adjusting for confounders . Aeromonas-associated diarrhoea was most common from March to May (during the peak of the hot and humid season), and September to October, similar to Vibrio cholerae O1 . Our results indicate that Aeromonas infection is common in young children presenting with diarrhoea in Bangladesh. Acta Vet Scand, 1999, 40(3), 263 - 70 Vibrios associated with mortality in cultured plaice Pleuronectes platessa fry; Pedersen K et al.; Fifty two bacterial strains, identified as Vibrio spp., were isolated from diseased plaice fry . The most numerous group comprised V . anguillarum (26/52), of which 3 isolates belonged to serogroup O2a, 16 corresponded to serogroup O18, and 7 isolates were non-typeable . All serogroup O18 isolates had identical ribotype patterns . Fourteen isolates were identified as V . splendidus biotype I (n = 11) or V . splendidus-like (n = 3) . Seven isolates were V . fluvialis, representing the first isolation of this species in Denmark and the first description of V . fluvialis associated with diseased fish . All V . fluvialis isolates had identical ribotype patterns, indicating the presence of a single clone . The last 5 isolates belonged to 2 different, unidentified Vibrio species (n = 2 and 3, respectively) . Although all isolates were recovered from diseased plaice fry, their exact role as pathogens for the fry is as yet uncertain . Selected isolates were tested for virulence to salmon and turbot . When injected into juvenile salmonid fish, the recorded LD50 values were higher than 10(6), indicating that their virulence was relatively low . However, virulence seemed to deteriorate upon subculturing, and therefore, the strains may have been more virulent upon primary isolation from the plaice fry. Schweiz Med Wochenschr, 1999 Nov 20, 129(46), 1744 - 8 Molecular approaches for safer and stronger vaccines; Del Giudice G et al.; Progress in molecular biology and biotechnology is making possible the development of new vaccines or the improvement of already existing ones . Recombinant DNA technology, genetic attenuation of bacterial and viral pathogens and their use as vectors for heterologous proteins, expression of microbial antigens in transgenic edible plants, and naked nucleic acid technology represent the most popular approaches hitherto adopted . A successful biotechnological approach to the development of new and improved vaccines has been based on genetic detoxification of bacterial toxins, such as the toxin of Bordetella pertussis, the heat-labile enterotoxin of Escherichia coli, and the toxin of Vibrio cholerae . Genetically detoxified Bordetella pertussis is now included in a commercially available acellular vaccine against pertussis . Genetically detoxified Escherichia coli and Vibrio cholerae have been shown to behave as very strong and safe mucosal adjuvants and are now entering the clinical stage . These results demonstrate that a rational molecular approach to the development of safer and stronger vaccines is feasible. J Bacteriol, 1999 Dec, 181(24), 7639 - 42 Evidence that expression of the Vibrio vulnificus hemolysin gene is dependent on cyclic AMP and cyclic AMP receptor protein; Bang YB et al.; Glucose repressed hemolysin production in Vibrio vulnificus . Promoter activity of the hemolysin gene, vvh, assessed with a vvh-luxCDABE transcriptional fusion, required cyclic AMP (cAMP) and cAMP receptor protein (CRP) in Escherichia coli . Hemolysin production in V . vulnificus increased after the addition of cAMP and was undetectable in a putative crp mutant, suggesting that vvh is also regulated by cAMP-CRP in V . vulnificus. J Bacteriol, 1999 Dec, 181(24), 7588 - 96 A multifunctional ATP-binding cassette transporter system from Vibrio cholerae transports vibriobactin and enterobactin; Wyckoff EE et al.; Vibrio cholerae uses the catechol siderophore vibriobactin for iron transport under iron-limiting conditions . We have identified genes for vibriobactin transport and mapped them within the vibriobactin biosynthetic gene cluster . Within this genetic region we have identified four genes, viuP, viuD, viuG and viuC, whose protein products have homology to the periplasmic binding protein, the two integral cytoplasmic membrane proteins, and the ATPase component, respectively, of other iron transport systems . The amino-terminal region of ViuP has homology to a lipoprotein signal sequence, and ViuP could be labeled with {(3)H}palmitic acid . This suggests that ViuP is a membrane lipoprotein . The ViuPDGC system transports both vibriobactin and enterobactin in Escherichia coli . In the same assay, the E . coli enterobactin transport system, FepBDGC, allowed the utilization of enterobactin but not vibriobactin . Although the entire viuPDGC system could complement mutations in fepB, fepD, fepG, or fepC, only viuC was able to independently complement the corresponding fep mutation . This indicates that these proteins usually function as a complex . V . cholerae strains carrying a mutation in viuP or in viuG were constructed by marker exchange . These mutations reduced, but did not completely eliminate, vibriobactin utilization . This suggests that V . cholerae contains genes in addition to viuPDGC that function in the transport of catechol siderophores. Microbiol Immunol, 1999, 43(9), 909 - 12 Siderophore production of Vibrio parahaemolyticus strains from different sources; Yamamoto S et al.; Vibrio parahaemolyticus strains isolated from different sources were assayed for their ability to produce a siderophore, vibrioferrin, under iron-limited growth conditions . The mean value +/- standard error of mean (microM vibrioferrin in spent culture supernatant/optical density at 660 nm) was 832.3 +/- 66.9 for clinical isolates (n=44), which was significantly higher (P<0.01) than those for food isolates (461.0 +/- 66.5; n=37) and coastal isolates (378.8 +/- 37.2; n=26) . This suggests that greater productivity of vibrioferrin by clinical isolates may be associated with a selective advantage for survival and proliferation under conditions of iron-limitation such as in the intestine {corrected}. J Appl Microbiol, 1999 Nov, 87(5), 757 - 63 Pathogenicity of vibrio splendidus strains associated with turbot larvae, scophthalmus maximus Gatesoupe FJ, Lambert C, Nicolas JL. Turbot larvae were challenged with eight strains of Vibrio splendidus isolated from diseased larvae, plus a ninth strain pathogenic to scallop larvae (A515; Nicolas et al . 1996) . Six strains caused heavy mortality but the scallop pathogen and the other two strains did not . All the strains shared a large number of phenotypic traits, and an attempt was made to relate virulence to genotype and phenotype . Five of the six pathogenic strains were very similar, as shown by RAPD fingerprinting and phenotypic characteristics . The relatedness of the other strains was intermediate between the main pathogenic group and V . splendidus ATCC 33125, but the DNA-DNA homology between the pathogenic group and the reference strain was still high (78% of reassociation rate) . The non-pathogenic isolates may be a useful tool for determining the possible virulence factors, as all the isolates differed by few characteristics. J Microbiol Immunol Infect, 1997 Feb, 30(1), 32 - 42 {Characterization of haemolysis of the Vibrio parahaemolyticus no.93}; Su SC et al.; Vibrio parahaemolyticus is a causative bacterium of food poisoning, and the haemolysin produced by this organism has been considered as one of the important virulence factors . In order to understand the pathogenic mechanism of this bacterium, the characteristics of haemolysin from Vibrio parahaemolyticus isolated from Taiwan were studied . One of the clinical strains, V . parahaemolyticus No.93, presents a weak hemolytic zone on 7% NaCl-Wagatsuma medium . The DNA hybridization results show that V . parahemolyticus has neither tdh nor trh gene . V . parahaemolyticus No.93 shows obviously hemolytic zone on 3%-NaCl Wagatsuma medium (human blood) . The crude extracellular protein of V . parahaemolyticus No . 93 was evaluated for its heat tolerance and enzyme activities by media assay . The results show that this crude extracellular protein is thermolabile . The crude extracellular protein of V . parahaemolyticus No.93 was analyzed on 10% SDS-PAGE and an apparent band of 64 kDa protein was observed . Furthermore, the crude extracellular protein was analyzed by running gelatin-SDS-PAGE and hemoglobin-SDS-PAGE, and three clear zones on 62 kDa, 52 kDa and 41 kDa were observed on both SDS-PAGEs . Thus we propose that the crude extracellular protein of the V . parahaemolyticus No.93 can degrade gelatin as well as hemoglobin . Whether these protease being the virulence factors of Vibrio parahaemolyticus No.93 needs to be further studied. Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1996 Nov, 29(4), 210 - 24 {K-serotype analyses of Vibrio parahaemolyticus isolated in northern Taiwan, 1983 through 1993}; Wang TK et al.; From 1983 through 1993, 786 strains of Vibrio parahaemolyticus were collected from food-borne disease outbreaks and sporadic cases of diarrheal illness in northern Taiwan, involving 42 K-serotypes . Five top leading serotypes were K8 (36.8%), K15 (10.8%), K12 (8.7%), K56 (7.9%) and K63 (4.7%) . However, a variation of K-serotypes was found during this study period . From 112 food-borne outbreaks associated with this microorganism, only 54 (48.2%) outbreaks were caused by a single serotype, while 58 (51.8%) were caused by multiple K-serotypes . Numbers of outbreaks caused by two, three and more than three K-serotypes were 29 (26%), 16 (14.2%), and 13 (11.6%), respectively . In a special outbreak, eight K-serotypes was found . Outbreaks caused by party caterers were most frequently associated with multiple K-serotypes. Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1996 Nov, 29(4), 197 - 209 Survey on the distribution of Vibrionaceae at the seaport areas in Taiwan, 1991-1994; Wu HS et al.; A monthly survey on the distribution of human-pathogenic Vibrionaceae of the seawater from five principal harbors in Taiwan was conducted by National Quarantine Service from July, 1991 to February, 1994 . Of the total 1,167 Vibrionaceae isolates, strains of Vibrio alginolyticus (449 strains) were the most frequently isolated, followed by Vibrio parahaemolyticus (262) , Aeromonas hydrophila (153), Vibrio cholerae non-O1 (86), and Vibrio vulnificus (67) . None of Vibrio cholerae O1 was isolated . The pH, salinity, and water temperature in this study ranged from 5.8 to 8.6, 0.1 to 4.2% , and 15 to 34 degrees C, respectively . It is concluded that the family Vibrionaceae exists autochthonously around the coastal waters in Taiwan. Microb Pathog, 1999 Dec, 27(6), 377 - 85 Expression of Vibrio cholerae zonula occludens toxin and analysis of its subcellular localization; Uzzau S et al.; Vibrio cholerae elaborates zonula occludens toxin (Zot), a protein that increases the permeability of small intestinal mucosa by opening intercellular tight junctions . The zot gene is located, together with the genes encoding CT and Ace enterotoxins, within the genome of V . cholerae filamentous phage CTXsmall ef, Cyrillic . Interestingly, Zot appears to be structurally and functionally related to the gene I product of other filamentous phages and it has been shown to be required for CTXsmall ef, Cyrillic morphogenesis . In this study we described the cloning of zot in several expression plasmid systems and we examined the subcellular localization of Zot by using affinity purified anti-Zot antibodies . We found that Zot localizes in the V . cholerae cell envelope with M(r);45 kDa which is consistent with the predicted primary translation product from the first methionine of zot (44.8 kDa) . A second molecule, corresponding to the 33 kDa N-terminal region of Zot, was also detected . Both molecules are exposed at the bacterial cell surface . The production of the 33 kDa Zot, that might represent a processing product, was abolished in mutant ZotG59 . N-terminal tagged 6xHis-Zot fusion protein retained the capability to reach the outer membrane and the 6xHis tag was not cleaved off during the translocation to the periplasm, whereas the presence of the tag partially blocked the formation of the 33 kDa molecule . Zot secretion and anchorage to the bacterial outer membrane was also observed in E . coli strains expressing Zot, suggesting that the toxin may be directed to the outer membrane via the same pathway in E . coli and V . cholerae . Zot cleavage might be due to a V . cholerae specific protease activity, since the 33 kDa protein was not efficiently produced in E . coli . On the basis of these data and Zot amino acid sequence analysis, we suggest that while the N-terminal part of the molecule is involved in the morphogenesis of CTXsmall ef, Cyrillic, the C-terminal region might carry the domain(s) responsible for Zot enterotoxic activity . Biochemistry, 1999 Dec 7, 38(49), 16136 - 45 Folding, stability, and physical properties of the alpha subunit of bacterial luciferase; Noland BW et al.; Bacterial luciferase is a heterodimeric (alphabeta) enzyme composed of homologous subunits . When the Vibrio harveyi luxA gene is expressed in Escherichia coli, the alpha subunit accumulates to high levels . The alpha subunit has a well-defined near-UV circular dichroism spectrum and a higher intrinsic fluorescence than the heterodimer, demonstrating fluorescence quenching in the enzyme which is reduced in the free subunit {Sinclair, J . F., Waddle, J . J., Waddill, W . F., and Baldwin, T . O . (1993) Biochemistry 32, 5036-5044} . Analytical ultracentrifugation of the alpha subunit has revealed a reversible monomer to dimer equilibrium with a dissociation constant of 14.9 +/- 4.0 microM at 18 degrees C in 50 mM phosphate and 100 mM NaCl, pH 7.0 . The alpha subunit unfolded and refolded reversibly in urea-containing buffers by a three-state mechanism . The first transition occurred over the range of 0-2 M urea with an associated free-energy change of 2.24 +/- 0.25 kcal/mol at 18 degrees C in 50 mM phosphate buffer, pH 7.0 . The second, occurring between 2.5 and 3.5 M urea, comprised a cooperative transition with a free-energy change of 6.50 +/- 0.75 kcal/mol . The intermediate species, populated maximally at ca . 2 M urea, has defined near-UV circular dichroism spectral properties distinct from either the native or the denatured states . The intrinsic fluorescence of the intermediate suggested that, although the quantum yield had decreased, the tryptophanyl residues remained largely buried . The far-UV circular dichroism spectrum of the intermediate indicated that it had lost ca . 40% of its native secondary structure . N-Terminal sequencing of the products of limited proteolysis of the intermediate showed that the C-terminal region of the alpha subunit became protease labile over the urea concentration range at which the intermediate was maximally populated . These observations have led us to propose an unfolding model in which the first transition is the unfolding of a C-terminal subdomain and the second transition represents the unfolding of a more stable N-terminal subdomain . Comparison of the structural properties of the unfolding intermediate using spectroscopic probes and limited proteolysis of the alpha subunit with those of the alphabeta heterodimer suggested that the unfolding pathway of the alpha subunit is the same, whether it is in the form of the free subunit or in the heterodimer. Biotechnol Bioeng, 2000 Jan 5, 67(1), 72 - 9 Biodesulfurization of dibenzothiophene in Escherichia coli is enhanced by expression of a Vibrio harveyi oxidoreductase gene; Reichmuth DS et al.; One possible alternative to current fuel hydrodesulfurization methods is the use of microorganisms to remove sulfur compounds . Biodesulfurization requires much milder processing conditions, gives higher specificity, and does not require molecular hydrogen . In the present work we have produced two compatible plasmids: pDSR3, which allows Escherichia coli to convert dibenzothiophene (DBT) to hydroxybiphenyl (HBP), and pDSR2, which produces a Vibrio harveyi flavin oxidoreductase . We show that the flavin oxidoreductase enhances the rate of DBT removal when co-expressed in vivo with the desulfurization enzymes . The plasmids pDSR2 and pDSR3 were co-expressed in growing cultures . The expression of oxidoreductase caused an increase in the rate of DBT removal but a decrease in the rate of HBP production . The maximum rate of DBT removal was 8 mg/h . g dry cell weight . Experiments were also conducted using resting cells with the addition of various carbon sources . It was found that the addition of glucose or glycerol to cultures with oxidoreductase expression produced the highest DBT removal rate (51 mg/h . g dry cell weight) . The culture with acetate and no oxidoreductase expression had the highest level of HBP production . For all carbon sources, the DBT removal rate was faster and the HBP generation rate slower with the expression of the oxidoreductase . Analysis of desulfurization intermediates indicates that the last enzyme in the pathway may be limiting . Epidemiol Infect, 1999 Oct, 123(2), 225 - 32 Molecular characterization of Vibrio cholerae O1 and non-O1 from human and environmental sources in Malaysia; Radu S et al.; A total of 31 strains of Vibrio cholerae O1 (10 from outbreak cases and 7 from surface water) and non-O1 (4 from clinical and 10 from surface water sources) isolated between 1993 and 1997 were examined with respect to presence of cholera enterotoxin (CT) gene by PCR-based assays, resistance to antibiotics, plasmid profiles and random amplified polymorphic DNA (RAPD) analysis . All were resistant to 9 or more of the 17 antibiotics tested . Identical antibiotic resistance patterns of the isolates may indicate that they share a common mode of developing antibiotic resistance . Furthermore, the multiple antibiotic resistance indexing showed that all strains tested originated from high risk contamination . Plasmid profile analysis by agarose gel electrophoresis showed the presence of small plasmids in 12 (7 non-O1 and 5 O1 serotypes) with sizes ranging 1.3-4.6 MDa . The CT gene was detected in all clinical isolates but was present in only 14 (6 O1 serotype and 8 non-O1 serotype) isolates from environmental waters . The genetic relatedness of the clinical and environmental Vibrio cholerae O1 and non-O1 strains was investigated by RAPD fingerprinting with four primers . The four primers generated polymorphisms in all 31 strains of Vibrio cholerae tested, producing bands ranging from < 250 to 4500 bp . The RAPD profiles revealed a wide variability and no correlation with the source of isolation . This study provides evidence that Vibrio cholerae O1 and non-O1 have significant public health implications. Blood Cells Mol Dis, 1999 Jun-Aug, 25(3-4), 180 - 92 Hemostasis of tiger prawn Penaeus monodon affected by Vibrio harveyi, extracellular products, and a toxic cysteine protease; Lee KK et al.; The effects of bacterial cells, extracellular products (ECP) and a purified cysteine protease of Vibrio harveyi on hemostasis and plasma components of tiger prawn (Penaeus monodon) were studied . The clotting ability of the hemolymph withdrawn from moribund prawns pre-injected with the bacteria, ECP, cysteine protease of PBS (control) was observed for 2 h at 25 C . Of these, only the control group was clottable while all the other groups were unclottable . A component of the plasma, previously identified as coagulogen-like protein, was further confirmed to be a coagulogen by the comparison of plasma with serum on non-reduced SDS-PAGE or using rabbit antiserum to the coagulogen-like protein (R alpha coagulogen) to neutralize the clotting ability of normal prawn hemolymph . The coagulogen was reduced in amount in plasma of moribund prawns after injection with the bacteria, ECP or cysteine protease while it apparently disappeared after pre-incubation with the ECP or cysteine protease for 2 h at 25 C compared with normal prawn plasma as observed in crossed immunoelectrophoresis (CIE) gels . The reduction of the amount of coagulogen in plasma of moribund prawns was also evident in CIE gels using R alpha coagulogen . In addition, the apparent disapperance of the coagulogen mentioned above was eventually proven to be due to the change of its migration rate in CIE gels after pre-incubation with ECP or cysteine protease, since the disappeared coagulogen arc (arc 2) (migrated into arc 1) could be visualized by using R alpha coagulogen or by reducing the time for pre-incubation from 2 h to 30 min . Thus, the effects of cysteine protease on plasma coagulogen observed in vitro and in vivo may markedly interfere with hemostasis leading to the occurrence of unclottable hemolymph . These complex events may significantly contribute to the pathogenicity of V . harveyi in the prawn. J Food Prot, 1999 Nov, 62(11), 1266 - 9 Effects of a commercial heat-shock process on Vibrio vulnificus in the American oyster, Crassostrea virginica, harvested from the Gulf Coast; Hesselman DM et al.; Oysters (Crassostrea virginica) harvested from the Gulf Coast, containing 10(2) to 10(4) most probable number (MPN) per gram of Vibrio vulnificus, were subjected to a commercial heat-shock process . After 1 to 4 min at internal oyster meat temperatures exceeding 50 degrees C, shellstock oysters were shucked, chilled, washed, and packed . V . vulnificus and total bacterial levels in Gulf Coast oysters were significantly reduced from 1 to 4 logs in the finished product . Similar reductions were not observed in shellstock oysters that were subject to conventional processing . Under the National Shellfish Sanitation Program, heat shocking is an acceptable process to use to assist in the shucking of shellstock . This research revealed that the heat-shock process may also serve to significantly reduce V . vulnificus in summer Gulf Coast oysters. Infect Immun, 1999 Dec, 67(12), 6496 - 509 Characterization of the angR gene of Vibrio anguillarum: essential role in virulence; Wertheimer AM et al.; The ability to utilize the iron bound by high-affinity iron-binding proteins in the vertebrate host is an important virulence factor for the marine fish pathogen Vibrio anguillarum . Virulence in septicemic infections is due to the presence of a highly efficient plasmid-encoded iron transport system . AngR, a 110-kDa protein component of this system, appears to play a role in both regulation of the expression of the iron transport genes fatDCBA and the production of the siderophore anguibactin . Therefore, study of the expression of the angR gene and the properties of its product, the AngR protein, may contribute to the understanding of the mechanisms of virulence of this pathogen . In this work, we present genetic and molecular evidence from transposition mutagenesis experiments and RNA analysis that angR, which maps immediately downstream of the fatA gene, is part of a polycistronic transcript that also includes the iron transport genes fatDCBA and angT, a gene located downstream of angR which showed domain homology to certain thioesterases involved in nonribosomal peptide synthesis of siderophores and antibiotics . In order to dissect the specific domains of AngR associated with regulation of iron transport gene expression, anguibactin production, and virulence, we also generated a panel of site-directed angR mutants, as well as deletion derivatives . Both virulence and anguibactin production were dramatically affected by each one of the angR modifications . In contrast to the need for an intact AngR molecule for anguibactin production and virulence, the regulation of iron transport gene expression does not require the entire AngR molecule, since truncation of the carboxy terminus carrying the nonribosomal peptide synthetase cores, as well as the site-directed mutations, resulted in derivatives that retained their ability to regulate gene expression which was only abolished after truncation of amino-terminal sequences containing helix-turn-helix and leucine zipper motifs and a specialized heterocyclization and condensation domain found in certain nonribosomal peptide synthetases . The evidence, while not rigorously eliminating the possibility that a separate regulatory polypeptide exists and is encoded somewhere within the 5'-end region of the angR gene, strongly supports the idea that AngR is a bifunctional protein and that it plays an essential role in the virulence mechanisms of V . anguillarum . We also show in this study that the angT gene, found downstream of angR, intervenes in the mechanism of anguibactin production but is not essential for virulence or iron transport gene expression. Infect Immun, 1999 Dec, 67(12), 6346 - 9 Validation and characterization of a human volunteer challenge model for cholera by using frozen bacteria of the new Vibrio cholerae epidemic serotype, O139; Cohen MB et al.; Until recently, all epidemic strains of Vibrio cholerae were of the O1 serotype . Current epidemics have also been caused by a new serotype, Vibrio cholerae O139 . Although the pathogenesis and clinical features of O139 cholera are similar to those of O1 cholera, immunity to serotype O1 does not confer immunity to serotype O139 . Therefore, prior to beginning vaccine efficacy studies, we sought to validate the use of a large standardized frozen inoculum of virulent V . cholerae O139 4260B for use in a human volunteer challenge model . Healthy volunteers (n = 25) were recruited for an Internal Review Board-approved inpatient dose-escalation challenge . Our goal was to identify a dose at which the cholera attack rate and the geometric mean purge were sufficient for determining vaccine efficacy against moderate and severe disease . At a dose of 10(5) CFU, 8 of 10 volunteers experienced purging and had a positive stool culture for V . cholerae . However, at this dose, the geometric mean stool volume of 2,175 g was insufficient by study criteria . At a dose of 10(6) CFU, 14 of 15 volunteers experienced purging, with a geometric mean stool volume of 5,621 g . Disease severity was significantly greater in volunteers with blood group O than those with non-O blood types (10,353 g versus 3,555 g, P < 0.001) . Following challenge, all volunteers demonstrated a significant rise in antitoxin antibodies but the serum vibriocidal titer was attenuated compared to that seen after challenge with an O1 strain . This model provides a reproducible illness of sufficient severity for testing the efficacies of new O139 or combined O1-O139 vaccines. Infect Immun, 1999 Dec, 67(12), 6341 - 5 Randomized, double-blind, placebo-controlled, multicentered trial of the efficacy of a single dose of live oral cholera vaccine CVD 103-HgR in preventing cholera following challenge with Vibrio cholerae O1 El tor inaba three months after vaccination; Tacket CO et al.; CVD 103-HgR is a live oral cholera vaccine strain constructed by deleting 94% of the gene for the enzymatically active A subunit of cholera toxin from classical Inaba Vibrio cholerae O1 569B; the strain also contains a mercury resistance gene as an identifying marker . This vaccine was well tolerat