Vibriosis

Microb Pathog, 2004 Jul, 37(1), 1 - 9
Effect of W07-toxin on gut physiological response in mice; Bhattacharyya S et al.; A number of unknown secretogenic factor(s) from Vibrio cholerae have been implicated to play a role in inducing cholera-like symptoms observed in patients . The present study has been carried out on the novel W07-toxin (pI 5.2) from V . cholerae W07, an epidemic cholera strain devoid of the ctx gene . The toxin showed maximum binding to GM(1) and interacted with a 20 kDa glycoprotein present on the cell membrane of mice enterocytes in a GM(1) specific manner . The analysis of biochemical parameters in enterocytes triggered with this toxin revealed a significant increase in intracellular calcium concentration and a massive secretion of Cl(-) . However, no absorption of Na(+) was observed under the same condition . This toxin also elevated the level of cyclic adenosine 3',5'-monophosphate (cAMP) as well as protein kinase A (PKA) . Thus, the novel toxin, although distinct from cholera-toxin, showed some functional homology to it and may be one of the key players inducing electrolyte imbalance within intestinal cells in the cholera-like symptoms associated with V . cholerae W07.

Vaccine, 2004 Jun 23, 22(19), 2457 - 69
Biosafety aspects of the recombinant live oral Vibrio cholerae vaccine strain CVD 103-HgR; Viret JF et al.; The development of live attenuated vaccines, allowing for the safe and effective immunisation at mucosal surfaces, is a strategy of great interest for vaccinologists . The main advantage of this approach over conventional parenteral vaccines is the induction of strong mucosal immune responses, allowing targeting of the pathogen at the initial point of contact with the host . Further advantages include the ease of administration, high acceptance by vaccines, and relatively low production costs . Finally, well-characterised, safe and immunogenic vaccine strains are well suited as vectors for the mucosal delivery of foreign vaccine antigens and of DNA vaccines . However, such vaccines, when based on or containing genetically modified organisms (GMOs), are facing new and specific regulatory hurdles, particularly regarding the potential risks for humans and the environment . In this contribution we address selected aspects of the risk assessment of live attenuated bacterial vaccines covered in the course of the registration of vaccine strain CVD 103-HgR as a recombinant live oral vaccine against cholera.

J Fish Dis, 2004 Jun, 27(6), 319 - 26
Competition for attachment of aquaculture candidate probiotic and pathogenic bacteria on fish intestinal mucus; Vine NG et al.; Probiotics for aquaculture are generally only selected by their ability to produce antimicrobial metabolites; however, attachment to intestinal mucus is important in order to remain within the gut of its host . Five candidate probiotics (AP1-AP5), isolated from the clownfish, Amphiprion percula (Lacepede), were examined for their ability to attach to fish intestinal mucus and compete with two pathogens, Aeromonas hydrophila and Vibrio alginolyticus . Two different radioactive isotopes were used to quantify competition between pathogens and probionts . Attachment of the pathogens was enhanced by the presence of the candidate probiotics . However, the addition of the candidate probiotics after the pathogens resulted in reduced pathogen attachment . Only AP5 caused lower attachment success of V . alginolyticus when added before the pathogen . When AP5 was added first, the average attachment change was 41% compared with 72% when added after V . alginolyticus, suggesting that the probiotic is displaced but that enhanced attachment of the pathogen does not occur . Conversely, when V . alginolyticus was added first, followed by AP5, attachment change was 37% while AP5 had 92% attachment change when added second . This implies that the pathogen was displaced by the candidate probiotic and therefore it appeared that, based on the ability of probiont AP5 to attach to mucus, the growth of the pathogen in the digestive tract might be suppressed by the candidate probiont's presence.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Mar-Apr, (2), 11 - 4
{Triacylglycerol lipase activity of hemolytic Vibrio cholerae}; Telesmanich NR; The new method for the determination of the lipase activity of purified preparations of hemolysins and live V . cholerae cell has been developed . On the basis of the determination of triacylglycerollipase (lipase) activity the test for the differentiation of hemolytic and nonhemolytic V . cholerae has been proposed.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Mar-Apr, (2), 7 - 10
{Transition of Vibrio cholerae into the non-culturable state under the conditions of low temperature, different mineralization and illumination of artificial culture medium}; Nikoleishvili LR et al.; The importance of the combined influence of temperature, mineralization and illumination of the medium on the time of the transition of V . cholerae into the uncultivable state has been shown . The reversion of 5- to 60-day variants of uncultivable forms after the elevation of temperature to 20-22 degrees C has been obtained.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Mar-Apr, (2), 3 - 7
{Characterization of Vibrio cholerae O1 cultures isolated from environmental objects on the territory of the Russian Federation in 2002}; Onishchenko GG et al.; The biological properties of 46 V . cholerae O1 eltor cultures isolated in 2002 from water environment on the territory of Russia are presented . All isolated vibrios proved to be typical in their cultural, morphological, biochemical and serological properties . The atypical character of some of them was mainly linked with their phage resistance . The appearance of vibrios, sensitive to bacteriophage ctx+ and containing gene tcp in the absence gene ctx, was noted . Multilocus VNTR typing made it possible to group the cultures under study in 34 genotypes . The presence of toxin coregulated pili was found to be directly related to locus VcB . The necessity of the systematic study of the pheno- and genotypes of the isolated cultures with the aim of epidemiological surveillance of this infection is emphasized.

Proteomics, 2004 May, 4(5), 1491 - 504
A proteome reference map for Vibrio cholerae El Tor; Coelho A et al.; A proteome reference map has been constructed for Vibrio cholerae El Tor, in the pI range of 4.0 to 7.0 . The map is based on two-dimensional gels (2-D) and the identification, by peptide mass fingerprint, of proteins in 94 spots, corresponding to 80 abundant proteins . Two strains are compared, strain N16961 and a Latin American El Tor strain C3294 . The consensus map contains 340 spots consistently seen with both strains grown in Luria-Bertani broth (LB) or minimal M9 medium . The results were obtained from nine gels run with 18 cm immobilized pH gradient strips and precast gels . The 2-D gels were anchored to real N16961 proteins identified by mass spectrometry . Various energy metabolism components and periplasmic ATP-binding cassette (ABC) transporter proteins were identified among the abundant proteins . Two isoforms of OmpU were found . Five operons are proposed and seven hypothetical proteins were experimentally confirmed . Comparisons are made with protein 2-D gels for a classical strain and to microarray analysis available for the N16961 El Tor strain . New results were obtained from the proteome analysis, indicating an abundance of periplasmic ABC transporter proteins not found in microarray studies.

Mol Microbiol, 2004 Jun, 52(6), 1677 - 89
A distinctive dual-channel quorum-sensing system operates in Vibrio anguillarum; Croxatto A et al.; Many bacterial cells communicate using diffusible signal molecules to monitor cell population density via a process termed quorum sensing . In marine Vibrio species, the Vibrio harveyi-type LuxR protein is a key player in a quorum-sensing phosphorelay cascade, which controls the expression of virulence, symbiotic and survival genes . Previously, we characterized Vibrio anguillarum homologues of LuxR (VanT) and LuxMN (VanMN) and, in this study, we have identified homologues of LuxPQ (VanPQ) and LuxOU (VanOU) . In contrast to other Vibrio species, vanT was expressed at low cell density and showed no significant induction as the cell number increased . In addition, although the loss of VanO increased vanT expression, the loss of VanU, unexpectedly, decreased it . Both VanN and VanQ were required for repression of vanT even in a vanU mutant, suggesting an alternative route for VanNQ signal transduction other than via VanU . VanT negatively regulated its own expression by binding and repressing the vanT promoter and by binding and activating the vanOU promoter . The signal relay results in a cellular response as expression of the metalloprotease, empA, was altered similar to that of vanT in all the mutants . Consequently, the V . anguillarum quorum-sensing phosphorelay systems work differently from those of V . harveyi and may be used to limit rather than induce vanT expression.

Environ Microbiol, 2004 Jul, 6(7), 760 - 3
Polylysogeny and prophage induction by secondary infection in Vibrio cholerae; Espeland EM et al.; Strains of Vibrio cholerae O1, biotypes El Tor and classical, were infected with a known temperate phage (PhiP15) and monitored over a 15-day period for prophage induction . Over the course of the experiment two morphologically and three genomically distinct virus-like particles were observed from the phage-infected El Tor strain by transmission electron microscopy and field inversion gel electrophoresis, respectively, whereas only one phage, PhiP15, was observed from the infected classical strain . In the uninfected El Tor culture one prophage was spontaneously induced after 6 days . No induction in either strain was observed after treatment with mitomycin C . Data indicate that El Tor biotypes of V . cholerae may be polylysogenic and that secondary infection can promote multiple prophage induction . These traits may be important in the transfer of genetic material among V . cholerae by providing an environmentally relevant route for multiple prophage propagation and transmission.

Environ Microbiol, 2004 Jul, 6(7), 699 - 706
Occurrence and distribution of Vibrio cholerae in the coastal environment of Peru; Gil AI et al.; The occurrence and distribution of Vibrio cholerae in sea water and plankton along the coast of Peru were studied from October 1997 to June 2000, and included the 1997-98 El Nino event . Samples were collected at four sites in coastal waters off Peru at monthly intervals . Of 178 samples collected and tested, V . cholerae O1 was cultured from 10 (5.6%) samples, and V . cholerae O1 was detected by direct fluorescent antibody assay in 26 out of 159 samples tested (16.4%) . Based on the number of cholera cases reported in Peru from 1997 to 2000, a significant correlation was observed between cholera incidence and elevated sea surface temperature (SST) along the coast of Peru (P < 0.001) . From the results of this study, coastal sea water and zooplankton are concluded to be a reservoir for V . cholerae in Peru . The climate-cholera relationship observed for the 1997-98 El Nino year suggests that an early warning system for cholera risk can be established for Peru and neighbouring Latin American countries.

Microbiology, 2004 Jun, 150(Pt 6), 1769 - 77
Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms; Gomez-Gil B et al.; Fifty strains belonging to Vibrio harveyi, Vibrio campbellii, and the recently described Vibrio rotiferianus, were analysed using phenotypic and genomic techniques with the aim of analysing the usefulness of the different techniques for the identification of V . harveyi-related species . The species V . harveyi and V . campbellii were phenotypically indistinguishable by more than 100 phenotypic features . Thirty-nine experimental strains were phenotypically identified as V . harveyi, but FAFLP, REP-PCR, IGS-PCR and DNA-DNA hybridization proved that they in fact belong to the species V . campbellii . Similar groupings were found among all fingerprinting methodologies (except IGS-PCR) . Thirty-two experimental strains clustered with the V . campbellii type and one reference strain; seven strains clustered with the V . harveyi type and three reference strains; and the type and four reference strains of V . rotiferianus grouped together . The correlations between DNA-DNA hybridization and the genomic fingerprinting by FAFLP and (GTG)(5)-PCR were found to be above 0.68 and statistically significant, suggesting the value of the latter techniques for the reliable identification of V . harveyi-related species . The results presented indicate that strains phenotypically identified as V . harveyi are in fact V . campbellii; these findings position V . campbellii as an important species involved in diseases of reared aquatic organisms.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 243 - 8
A plasmid-encoded class 1 integron carrying sat, a putative phosphoserine phosphatase gene and aadA2 from enterotoxigenic Escherichia coli O159 isolated in Japan; Ahmed AM et al.; A class 1 integron was detected in a single multidrug-resistant strain of enterotoxigenice Escherichia coli (ETEC) O159 after examination of 23 clinical E . coli isolates . This isolate was resistant to streptomycin, kanamycin, gentamicin, chloramphenicol and ampicillin . Sequencing of the class 1 integron identified three-gene cassettes . The first is the streptothricin acetyltransferase gene, sat, which confers resistance to streptothricin . The second is an ORF whose product is a putative phosphoserine phosphatase (PSP), and the last is an aminoglycoside adenyltransferase gene, aadA2, which confers resistance to streptomycin and spectinomycin . The putative PSP gene product was found to be 39%, 38%, 28%, and 27% identical to PSP gene products of Vibrio vulnificus CMCP6, V . vulnificus YJ016, Pseudomonas syringae, and P . aeruginosa, respectively . Southern-blot hybridization showed that this integron is located on a 90 kb plasmid . This is the first report identifying a putative PSP gene in an integron.

Zhonghua Yu Fang Yi Xue Za Zhi, 2004 May, 38(3), 200 - 3
{Characteristics of virulence gene in Vibrio parahaemolyticus strains isolated from clinical patients and environment in Hangzhou, China}; Zhang W et al.; OBJECTIVES: To investigate the characteristics of virulence gene in Vibrio parahaemolyticus strains isolated from clinical patients and environment in Hangzhou, China . METHODS: Thermostable direct hemolysin gene (tdh) and thermostable direct hemolysin-related hemolysin gene (trh) were determined in a total of 174 strains of V . parahaemolyticus isolated from patients and environment (seafood) in Hangzhou area by PCR . RESULTS: The tdh was found in 92 out of 94 V . parahaemolyticus strains from food poisoning patients and in 33 out of 34 strains from sporadic diarrhea patients, and trh was not detected in all above clinical strains . Meanwhile the tdh was negative in all V . parahaemolyticus strains from environment, and the trh was also negative except one strain with urease activity . All strains with trh negative had no the activity of urease . CONCLUSIONS: The V . parahaemolyticus strains from food poisoning patients and sporadic diarrhea patients are tdh positive and trh negative . The V . parahaemolyticus strains with tdh negative and almost trh positive in environment might be a potential pathogen in Hangzhou.

J Infect Dis, 2004 Jun 15, 189(12), 2318 - 22 Epub 2004 May 25.
Incomplete correlation of serum vibriocidal antibody titer with protection from Vibrio cholerae infection in urban Bangladesh; Saha D et al.; The serum vibriocidal antibody is the only recognized predictor of protection from cholera, but no seroepidemiological data have been gathered since the emergence of Vibrio cholerae O139 . We assessed the association between the vibriocidal antibody titer and protection from cholera in an endemic setting . Although a higher baseline vibriocidal titer correlated with protection from V . cholerae O1, infection still developed in some contacts with very high titers . No association between baseline vibriocidal titer and protection from V . cholerae O139 infection was found . Our findings suggest that the vibriocidal antibody is an incomplete predictor of protection from V . cholerae infection.

Protein Expr Purif, 2004 Jul, 36(1), 115 - 23
Expression, purification, and sequence analysis of catalase-1 from the soil bacterium Comamonas terrigena N3H; Zamocky M et al.; Catalases are essential components of the cellular equipment to cope with oxidative stress . We have purified and characterize herein the most abundant heme-containing catalase-1 from the soil bacterium Comamonas terrigena N3H . This oxidative stress-induced enzyme was isolated from exponential phase cells grown in the presence of peroxyacetic acid . We have used consecutive steps of hydrophobic, molecular sieve, and ion exchange chromatography to achieve a high state of purity for this metalloenzyme . The purified sample of catalase exhibited a specific catalytic activity of 55,900 U/mg, allosteric behavior in peroxidic reaction, a broad pH optimum, and a rather atypical electronic spectrum . The sample of highest purity was subjected to mass spectrometry analysis . The molecular weight of the subunit of this homodimeric protein was determined as 55,417 Da . The Qq-TOF mass analysis method allowed us to sequence short tryptic fragments of this catalase . Five such fragments with a total length of 57 amino acids together with several enzymatic properties allowed the classification of this hydroperoxidase as belonging to clade III of monofunctional catalases . The highest sequence similarity is with the catalase from Vibrio fischeri . The presented results imply the significance of this inducible enzyme in the prevention of toxic effects of oxidative stress for bacterial cells.

J Bacteriol, 2004 Jun, 186(12), 4014 - 8
Cross-regulation in Vibrio parahaemolyticus: compensatory activation of polar flagellar genes by the lateral flagellar regulator LafK; Kim YK et al.; Gene organization and hierarchical regulation of the polar flagellar genes of Vibrio parahaemolyticus, Vibrio cholerae, and Pseudomonas aeruginosa appear highly similar, with one puzzling difference . Two sigma(54)-dependent regulators are required to direct different classes of intermediate flagellar gene expression in V . cholerae and P . aeruginosa, whereas the V . parahaemolyticus homolog of one of these regulators, FlaK, appears dispensable . Here we demonstrate that there is compensatory activation of polar flagellar genes by the lateral flagellar regulator LafK.

J Bacteriol, 2004 Jun, 186(12), 3873 - 81
Vibrio fischeri LuxS and AinS: comparative study of two signal synthases; Lupp C et al.; Vibrio fischeri possesses two acyl-homoserine lactone quorum-sensing systems, ain and lux, both of which are involved in the regulation of luminescence gene expression and are required for persistent colonization of the squid host, Euprymna scolopes . We have previously demonstrated that the ain system induces luminescence at cell densities that precede lux system activation . Our data suggested that the ain system both relieves repression and initially induces the lux system, thereby achieving sequential induction of gene expression by these two systems . Analysis of the V . fischeri genome revealed the presence of a putative third system based on the enzyme LuxS, which catalyzes the synthesis of the Vibrio harveyi autoinducer 2 (AI-2) . In this study, we investigated the impact of V . fischeri LuxS on luminescence and colonization competence in comparison to that of the ain system . Similar to the ain system, inactivation of the AI-2 system decreased light production in culture, but not in the squid host . However, while an ainS mutant produces no detectable light in culture, a luxS mutant expressed approximately 70% of wild-type luminescence levels . A mutation in luxS alone did not compromise symbiotic competence of V . fischeri; however, levels of colonization of an ainS luxS double mutant were reduced to 50% of the already diminished level of ainS mutant colonization, suggesting that these two systems regulate colonization gene expression synergistically through a common pathway . Introduction of a luxO mutation into the luxS and ainS luxS background could relieve both luminescence and colonization defects, consistent with a model in which LuxS, like AinS, regulates gene expression through LuxO . Furthermore, while luxS transcription appeared to be constitutive and the AI-2 signal concentration did not change dramatically, our data suggest that ainS transcription is autoregulated, resulting in an over 2,000-fold increase in signal concentration as culture density increased . Taken together, these data indicate that V . fischeri LuxS affects both luminescence regulation and colonization competence; however, its quantitative contribution is small when compared to that of the AinS signal.

J Bacteriol, 2004 Jun, 186(12), 3794 - 805
Quorum sensing regulates type III secretion in Vibrio harveyi and Vibrio parahaemolyticus; Henke JM et al.; In a process known as quorum sensing, bacteria communicate with one another by producing, releasing, detecting, and responding to signal molecules called autoinducers . Vibrio harveyi, a marine pathogen, uses two parallel quorum-sensing circuits, each consisting of an autoinducer-sensor pair, to control the expression of genes required for bioluminescence and a number of other target genes . Genetic screens designed to discover autoinducer-regulated targets in V . harveyi have revealed genes encoding components of a putative type III secretion (TTS) system . Using transcriptional reporter fusions and TTS protein localization studies, we show that the TTS system is indeed functional in V . harveyi and that expression of the genes encoding the secretion machinery requires an intact quorum-sensing signal transduction cascade . The newly completed genome of the closely related marine bacterium Vibrio parahaemolyticus, which is a human pathogen, shows that it possesses the genes encoding both of the V . harveyi-like quorum-sensing signaling circuits and that it also has a TTS system similar to that of V . harveyi . We show that quorum sensing regulates TTS in V . parahaemolyticus . Previous reports connecting quorum sensing to TTS in enterohemorrhagic and enteropathogenic Escherichia coli show that quorum sensing activates TTS at high cell density . Surprisingly, we find that at high cell density (in the presence of autoinducers), quorum sensing represses TTS in V . harveyi and V . parahaemolyticus.

Genetika, 2004 Apr, 40(4), 445 - 53
{Role of moderate bacteriophage 139 in change in production of cholera toxin in a classic strain of Vibrio cholerae}; Eroshenko GA et al.; New data were obtained concerning cell sensitivity of pathogenic strains of cholera vibrions, which belong to the serogroup O1 of classical biovar, to the temperate bacteriophage K139, the native host of which is Vibrio cholerae O139 . Molecular-genetic and biochemical studies showed that phage 139 integrated into the chromosome of strains V . cholerae O1 can change their toxigenic properties . A change in the production of cholera toxin (CT) in lysogens is associated both with an increase in the activity of the toxR regulatory gene and with a distortion of the structure of a chromosomal DNA region that contains a copy of the operon ctxAB encoding CT biosynthesis.

Mar Pollut Bull, 2004 Jun, 48(11-12), 1096 - 101
Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water; Aridgides LJ et al.; There is enormous potential for global transfer of microorganisms, including pathogens, in ships' ballast water . We contend that a major advancement in the study of ballast-water microorganisms in particular, and of aquatic pathogens in general, will be expedited sample analysis, such as provided by the elegant technology of DNA microarrays . In order to use DNA microarrays, however, one must establish the appropriate conditions to bind target sequences in samples to multiple probes on the microarrays . We conducted proof-of-concept experiments to optimize simultaneous detection of multiple microorganisms using polymerase chain reaction (PCR) and Southern hybridization . We chose three target organisms, all potentially found in ballast water: a calicivirus, the bacterium Vibrio cholerae, and the photosynthetic protist Aureococcus anophagefferens . Here, we show simultaneous detection of multiple pathogens is possible, a result supporting the promising future use of microarrays for simultaneous detection of pathogens in ballast water.

J Mol Microbiol Biotechnol, 2004, 7(1-2), 18 - 29
Dual flagellar systems enable motility under different circumstances; McCarter LL; Flagella are extremely effective organelles of locomotion used by a variety of bacteria and archaea . Some bacteria, including Aeromonas, Azospirillum, Rhodospirillum, and Vibrio species, possess dual flagellar systems that are suited for movement under different circumstances . Swimming in liquid is promoted by a single polar flagellum . Swarming over surfaces or in viscous environments is enabled by the production of numerous peritrichous, or lateral, flagella . The polar flagellum is produced continuously, while the lateral flagella are produced under conditions that disable polar flagellar function . Thus at times, two types of flagellar organelles are assembled simultaneously . This review focuses on the polar and lateral flagellar systems of Vibrio parahaemolyticus . Approximately 50 polar and 40 lateral flagellar genes have been identified encoding distinct structural, motor, export/assembly, and regulatory elements . The sodium motive force drives polar flagellar rotation, and the proton motive force powers lateral translocation . Polar genes are found exclusively on the large chromosome, and lateral genes reside entirely on the small chromosome of the organism . The timing of gene expression corresponds to the temporal demand for components during assembly of the organelle: RpoN and lateral- and polar-specific sigma(54)-dependent transcription factors control early/intermediate gene transcription; lateral- and polar-specific sigma(28) factors direct late flagellar gene expression . Although a different gene set encodes each flagellar system, the constituents of a central navigation system (i.e., chemotaxis signal transduction) are shared .

Shokuhin Eiseigaku Zasshi, 2004 Feb, 45(1), 35 - 7
Growth kinetics of Vibrio parahaemolyticus O3:K6 under varying conditions of pH, NaCl concentration and temperature; Nishina T et al.; The growth responses of Vibrio parahamolyticus to pH, NaCl concentration and temperature changes were studied using serotype O3:K6 and other strains . Growth curves were obtained for 27 different sets of conditions, comprised of three levels of NaCl concentration, pH and temperature . The temperature, pH and NaCl concentrations most favorable for growth were in the order of 25 degrees C, 20 degrees C and 15 degrees C, pH 8, 7 and 5.8, and 1%, 3% and 7%, respectively . The bacteria grew most rapidly at 25 degrees C, at a pH of 7 or 8 in the presence of 1% or 3% NaCl, with the population (initial, ca . 2.5 log CFU/mL) reaching a level log 7 CFU/mL at 12 h . A growth predictive model using the Gompertz equation was generated from the experimental data for any combination of NaCl concentration, pH and temperature within the range used in this study.

Mol Gen Mikrobiol Virusol, 2004, (2), 11 - 6
{A comparative analysis of genomes of virulent and avirulent strains of Vibrio cholerae O139}; Eroshenko GA et al.; A comparative analysis of the genome of V . cholerae O139 strains isolated in Russia's territory from patients with cholera and from the environment showed essential differences in their structures . The genome of clinical strains possessed all tested genes associated with virulence (ctxAB, zot, ace, rstC, rtxA, hap, toxR and toxT) and the at-tRS site for the CTXp phage DNA integration . As for the O139 V . cholerae chromosome strains isolated from water, 70% of the studied genes (ctxAB, zot, ace, rstC, tcpA, and toxT) and the attRS sequence were not detected in them . A lack of the key virulence genes in O139-serogroup "water" vibrios, including genes of toxin-coregulated adhesion pili . (that are receptors for the CTXp phage), and of the attachment site of the above phage are indicative of that the O139 V . cholerae strains isolated from open water sources located in different Russia's regions are epidemically negligible.

Microbiol Res, 2004, 159(1), 19 - 28
Yeast DNA sequences initiating gene expression in Escherichia coli; Lewin A et al.; DNA transfer between pro- and eukaryotes occurs either during natural horizontal gene transfer or as a result of the employment of gene technology . We analysed the capacity of DNA sequences from a eukaryotic donor organism (Saccharomyces cerevisiae) to serve as promoter region in a prokaryotic recipient (Escherichia coli) by creating fusions between promoterless luxAB genes from Vibrio harveyi and random DNA sequences from S . cerevisiae and measuring the luminescence of transformed E . coli . Fifty-four out of 100 randomly analysed S . cerevisiae DNA sequences caused considerable gene expression in E . coli . Determination of transcription start sites within six selected yeast sequences in E . coli confirmed the existence of bacterial -10 and -35 consensus sequences at appropriate distances upstream from transcription initiation sites . Our results demonstrate that the probability of transcription of transferred eukaryotic DNA in bacteria is extremely high and does not require the insertion of the transferred DNA behind a promoter of the recipient genome.

Microbes Infect, 2004 Jun, 6(7), 676 - 85
Association of adherence and motility in interleukin 8 induction in human intestinal epithelial cells by Vibrio cholerae; Sarkar M et al.; Interleukin 8 (IL-8) mRNA expression in Vibrio cholerae-infected human intestinal epithelial cells Int407 was determined by quantitative real-time RT-PCR and secretion measured by ELISA . Incubation of Int407 with V . cholerae O395 resulted in increased IL-8 mRNA expression as early as within 2 h of infection . Kinetics of IL-8 secretion reached a peak at about 8 h (780 pg/ml) and decreased thereafter . Induction of IL-8 was significantly high among various toxin-producing strains of V . cholerae belonging to serovar O1, O139 and non-O1 compared to non-toxinogenic strains . Induction of IL-8 was maximum in V . cholerae O395, required live cells and was dependent on de novo protein synthesis . The bacterial culture supernatant and crude cell envelope showed IL-8 stimulating activity . Infection of the monolayer with V . cholerae O395 cheY4 null mutant (O395YN), defective in adherence and motility, resulted in highly reduced levels of IL-8 expression, while hyperadherent and hypermotile mutant (O395Y) with the cheY4 gene duplicated also showed very high IL-8 expression . Another hyperadherent icmF insertion mutant (O395F) with reduced motility showed almost half the amount of IL-8 expression compared to O395Y . These results clearly indicate that both motility and adherence to intestinal epithelial cells are possible triggering factors contributing to IL-8 mRNA expression by V . cholerae.

Biochem Biophys Res Commun, 2004 Jun 18, 319(1), 207 - 13
Bright fluorescence of a novel protein from Vibrio vulnificus depends on NADPH and the expression of this protein is regulated by a LysR-type regulatory gene; Chang CC et al.; A blue fluorescent protein, BfgV, belonging to the short-chain dehydrogenase/reductase superfamily, was found in a non-bioluminescent pathogen Vibrio vulnificus CKM-1 . This protein has fluorescence spectra with two excitation peaks at 283 and 352 nm, and one emission peak at 456 nm . BfgV fluoresces through effectively augmenting the intrinsic fluorescence of NADPH bound to it . Escherichia coli transformants expressing this protein can emit eye-detectable fluorescence . A LysR-type transcriptional regulator gene bfgR was found at the vicinal upstream region of bfgV in CKM-1 genome . The clues that products of bfgR can specifically bind to bfgR-bfgV intergenic promoter region and the deletion of bfgR significantly decreases the expression of bfgV reveal bfgR is a repressor gene of bfgV in V . vulnificus CKM-1.

FEMS Microbiol Lett, 2004 Jun 1, 235(1), 199 - 207
Transcriptional analysis and operon structure of the tagA-orf2-orf3-mop-tagD region on the Vibrio pathogenicity island in epidemic V . cholerae; Zhang D et al.; The Vibrio pathogenicity island (VPI) in epidemic Vibrio cholerae is an essential virulence gene cluster . The VPI can excise from the chromosome and form extrachromosomal circular excision products . The VPI is 41.2-kb in size and encodes 29 potential proteins, several of which have no known function and whose regulation is not well understood . To determine the transcriptional organization of the tagA-orf2-orf3-mop-tagD region located at the 5'-(left) end of the VPI, we used reverse-transcriptase-PCR (RT-PCR), Northern blot analysis and DNA sequencing . RT-PCR primers were designed to transcribe and amplify regions spanning two or more open reading frames so as to establish the transcriptional organization . RT-PCR and Northern blot results demonstrated that the tagA-tagD region is transcribed as a polycistronic message and organized into several potential operons including tagA-orf2, orf3-mop, orf3-mop-tagD and tagD alone . Transcriptional lacZ fusions supported the existence of a promoter upstream of orf3 that was toxT-dependent . Interestingly, our data suggests that the orf3 promoter can drive the expression of either a long transcript (orf3-mop-tagD) or a short transcript (orf3-mop) without tagD . Our data also suggests that tagD can be expressed from two different promoters and that tagD is either transcribed alone or co-expressed with orf3-mop under certain conditions . These studies provide new insight into the genetic structure, transcriptional organization and regulation of a cluster of virulence genes on the VPI of epidemic V . cholerae.

J Food Prot, 2004 May, 67(5), 1005 - 8
Real-time PCR detection of the thermostable direct hemolysin and thermolabile hemolysin genes in a Vibrio parahaemolyticus cultured from mussels and mussel homogenate associated with a foodborne outbreak; Davis CR et al.; Molecular methods have become vital epidemiological tools in the detection and characterization of bacteria associated with a foodborne outbreak . We used both culture and real-time PCR to detect a Vibrio parahaemolyticus isolate associated with a foodborne outbreak . The outbreak occurred in July 2002 in Polk County, Florida, and originated at a Chinese buffet, with one person being hospitalized . The hospital isolated V . parahaemolyticus from the patient but destroyed the sample after diagnosis . From an onsite visit of the restaurant, food samples that possibly contributed to the outbreak were collected and sent to the Florida Department of Health, Tampa Branch Laboratory . Crab legs, crabsticks, and mussel samples were homogenized and incubated according to the Food and Drug Administration Bacteriological Analytical Manual culture protocol . Three sets of primers and a TaqMan probe were designed to target the tdh, trh, and tlh genes and used for real-time PCR . This study was successful in isolating V . parahaemolyticus from a mussel sample and detecting two of its genes (tdh and tlh) in food and pure culture by real-time PCR.

J Bacteriol, 2004 Jun, 186(11), 3304 - 12
Isolation and characterization of rpoS from a pathogenic bacterium, Vibrio vulnificus: role of sigmaS in survival of exponential-phase cells under oxidative stress; Park KJ et al.; A gene homologous to rpoS was cloned from a fatal human pathogen, Vibrio vulnificus . The functional role of rpoS in V . vulnificus was accessed by using an rpoS knockout mutant strain . This mutant was impaired in terms of the ability to survive under oxidative stress, nutrient starvation, UV irradiation, or acidic conditions . The increased susceptibility of the V . vulnificus mutant in the exponential phase to H2O2 was attributed to the reduced activity of hydroperoxidase I (HPI) . Although sigmaS synthesis was induced and HPI activity reached the maximal level in the stationary phase, the mutant in the stationary phase showed the same susceptibility to H2O2 as the wild-type strain in the stationary phase . In addition, HPII activity, which is known to be controlled by sigmaS in Escherichia coli, was not detectable in V . vulnificus strains under the conditions tested . The mutant in the exponential phase complemented with multiple copies of either the rpoS or katG gene of V . vulnificus recovered both resistance to H2O2 and HPI activity compared with the control strain . Expression of the katG gene encoding HPI in V . vulnificus was monitored by using a katG::luxAB transcriptional fusion . The expression of this gene was significantly reduced by deletion of sigmaS in both the early exponential and late stationary phases . Thus, sigmaS is necessary for increased synthesis and activity of HPI, and sigmaS is required for exponentially growing V . vulnificus to develop the ability to survive in the presence of H2O2.

Vaccine, 2004 Jun 2, 22(17-18), 2137 - 45
Experimental immunisation and protection of guinea pigs with Vibrio cholerae toxoid and mucinases, neuraminidase and proteinase; Stewart-Tull DE et al.; As measured by fluid accumulation in ileal loops, Vibrio cholerae mucinase complex, with or without toxoid, protected guinea pigs from challenges with V . cholerae live organisms and enterotoxin . The neuraminidase and proteinases of the complex were combined in modified oil emulsion or aluminum hydroxide adjuvants and the resultant vaccines given by the parenteral or oral routes . There was little difference between the two types of adjuvant . Control of stomach acidity improved oral vaccination . Animals injected intramuscularly (i.m.) with toxoid-containing vaccines were protected from challenge with cholera toxin (CT) whereas those given oral doses were not . Toxoid plus killed V . cholerae cells elicited a more effective protection against toxin challenge than killed V . cholerae cells alone . Vaccines containing mucinases, with or without toxoid, protected the animals from a live V . cholerae challenge . The anti-mucinase immune response may prevent adhesion of the V . cholerae cells and hence reduce delivery of toxin to receptors . These mucinases, neuraminidase and proteinases, may be useful components of acellular, toxoided cholera vaccines for human immunisation.

Genes Cells, 2004 May, 9(5), 479 - 95
Identification of cryptochrome DASH from vertebrates; Daiyasu H et al.; A new type of cryptochrome, CRY-DASH, has been recently identified . The CRY-DASH proteins constitute the fifth subfamily of the photolyase/cryptochrome family . CRY-DASHs have been identified from Synechocystis sp . PCC 6803, Vibrio cholerae, and Arabidopsis thaliana . The Synechocystis CRY-DASH was the first cryptochrome identified from bacteria, and its biochemical features and tertiary structure have been extensively investigated . To determine how broadly the subfamily is distributed within living organisms, we searched for new CRY-DASH candidates within several databases . We found five sequences as new CRY-DASH candidates, which are derived from four marine bacteria and Neurospora crassa . We also found many CRY-DASH candidates from the EST databases, which included sequences from fish and amphibians . We cloned and sequenced the cDNAs of the zebrafish and Xenopus laevis candidates, based on the EST sequences . The proteins encoded by the two genes were purified and characterized . Both proteins contained folate and flavin cofactors, and have a weak DNA photolyase activity . A phylogenetic analysis revealed that the seven candidates actually belong to the new type of cryptochrome subfamily . This is the first report of the CRY-DASH members from vertebrates and fungi.

FEMS Immunol Med Microbiol, 2004 Jun 1, 41(2), 169 - 76
Regulation of proinflammatory mediator production in RAW264.7 macrophage by Vibrio vulnificus luxS and smcR; Shin NR et al.; Vibrio vulnificus causes fatal septicemia in human hosts, which is the consequence of raw shellfish consumption . The mortality following septicemia is dependent on the in vivo production of inflammatory mediators, including tumor necrosis factor-alpha (TNFalpha) . The present study was set up to investigate the association of quorum sensing in V . vulnificus with the host immune response . The effect of quorum sensing on cytotoxicity and the production of proinflammatory mediators was examined using the murine macrophage cell-line RAW264.7 . Cytotoxicity was determined by measuring lactate dehydrogenase release in the culture medium . Extracellular products from luxS- and smcR-deficient mutants exhibited weak cytotoxic effects on RAW264.7 cells . The production of the proinflammatory cytokines TNFalpha, IL-1beta and IL-6 was measured with real-time PCR and ELISA, and production was measured with Griess reagents . Mutation of both luxS and smcR delayed the transcription of TNFalpha, IL-1beta and IL-6 genes . Also, levels of both TNFalpha and nitric oxide induced by luxS- and smcR-deficient mutants were significantly lower than those induced by parent strains . These results suggest that quorum sensing could be involved in the modulation of TNFalpha and nitric oxide produced from host cells by regulating virulence factors, and that V . vulnificus facilitates its host's mortality and bacterial survival by enhancing virulence on host cells.

Fish Shellfish Immunol, 2004 Jul, 17(1), 41 - 51
The immune stimulatory effect of sodium alginate on the white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus; Cheng W et al.; The total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase activity, respiratory burst (release of superoxide anion), superoxide dismutase activity, phagocytic activity and clearance efficiency to the pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (9.4-11.3 g) were injected individually with sodium alginate at 10, 20 or 50 microg g(-1) . No significant differences in THC, DHC and superoxide dismutase activity were observed among the shrimp injected with saline and those injected with sodium alginate at 10, 20 or 50 microg g(-1) . However, L . vannamei injected with sodium alginate at 20 microg g(-1)increased its phenoloxidase activity and respiratory burst after 2 days and one day, respectively . L . vannamei injected with sodium alginate at 50 microg g(-1)maintained a higher phagocytic activity and clearance efficiency to V . alginolyticus after 4 days . In another experiment, L . vannamei which had been injected with sodium alginate, were challenged with V . alginolyticus at 2x10(5)colony-forming units (CFU) shrimp(-1)and then placed in seawater of 34 per thousand . The survival of shrimp that received sodium alginate at either dose was significantly higher than that of control shrimp at the termination of the experiment (6 days after the challenge) . It is therefore concluded that L . vannamei received sodium alginate at 10 microg g(-1)or more and increased its immune ability and resistance from V . alginolyticus infection.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Apr, 21(2), 255 - 8
{Cloning and expression of immunoadjuvant molecule--CTB gene}; Wang T et al.; We cloned cholera toxin subunit B gene from 569B and M045 strain of Vibrio cholerae with polymerase chain reaction, constructed recombinant plasmid pCTB, and transformed pCTB into the prokaryotic cell strain JM109 . The indentification was made by means of restriction enzyme analysis, polymerase chain reaction, DNA sequencing, SDS-polyacrylamine gel electrophoresis analysis and Western blot . The results indicate that we have amplified cholera toxin subunit B gene of 376 bp from Vibrio cholerae and hve constructed the recombinant plasmid pCTB, and we have affained the object amied at successful expression of 12 KD in the prokaryotic cell strain.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 951 - 4
Propionispora hippei sp . nov., a novel Gram-negative, spore-forming anaerobe that produces propionic acid; Abou-Zeid DM et al.; A Gram-negative, spore-forming anaerobe, KS(T), was isolated from an enrichment culture that was set up for anaerobic degradation of the aliphatic polyester poly(propylene adipate) . The strain had the cellular organization of Sporomusa, vibrio-shaped cells and terminal round spores, and fermented sugars and sugar alcohols to propionic and acetic acid . Based on the morphological and physiological features as well as on a 16S rRNA gene similarity of 98 %, it was grouped with Propionispora vibrioides . A relatively low DNA-DNA hybridization value with the type strain of this species (47 %), and differences in substrate utilization and spore morphology, suggested that the strain should be classified in a separate species, Propionispora hippei sp . nov., with KS(T) as the type strain (=DSM 15287(T)=ATCC BAA-665(T)).

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 843 - 6
Vibrio gallicus sp . nov., isolated from the gut of the French abalone Haliotis tuberculata; Sawabe T et al.; Five alginolytic, facultatively anaerobic, non-motile bacteria were isolated from the gut of the abalone Haliotis tuberculata . Phylogenetic analyses based on 16S rDNA data indicated that these strains are related to Vibrio wodanis, Vibrio salmonicida, Vibrio logei and Vibrio fischeri (but with <97 % 16S rRNA gene sequence similarity) . DNA-DNA hybridization and fluorescence amplified fragment length polymorphism fingerprinting demonstrated that the five strains constituted a single species that was different from all currently known vibrios . The name Vibrio gallicus sp . nov . (type strain, CIP 107863(T)=LMG 21878(T)=HT2-1(T); DNA G+C content, 43.6-44.3 mol%) is proposed for this novel taxon . Several phenotypic features were disclosed that discriminated V . gallicus from other Vibrio species: V . gallicus can be differentiated from Vibrio halioticoli on the basis of four traits (beta-galactosidase test and assimilation of three carbon compounds) and from Vibrio superstes by 16 traits.

Biosens Bioelectron, 2004 Jul 15, 19(12), 1711 - 22
On line biomonitors used as a tool for toxicity reduction evaluation of in situ groundwater remediation techniques; Kuster E et al.; Success of groundwater remediation is typically controlled via snapshot analysis of selected chemical substances or physical parameters . Biological parameters, i.e . ecotoxicological assays, are rarely employed . Hence the aim of the study was to develop a bioassay tool, which allows an on line monitoring of contaminated groundwater, as well as a toxicity reduction evaluation (TRE) of different remediation techniques in parallel and may furthermore be used as an additional tool for process control to supervise remediation techniques in a real time mode . Parallel testing of groundwater remediation techniques was accomplished for short and long time periods, by using the energy dependent luminescence of the bacterium Vibrio fischeri as biological monitoring parameter . One data point every hour for each remediation technique was generated by an automated biomonitor . The bacteria proved to be highly sensitive to the contaminated groundwater and the biomonitor showed a long standing time despite the highly corrosive groundwater present in Bitterfeld, Germany . The bacterial biomonitor is demonstrated to be a valuable tool for remediation success evaluation . Dose response relationships were generated for the six quantitatively dominant groundwater contaminants (2-chlortoluene, 1,2- and 1,4-dichlorobenzene, monochlorobenzene, ethylenbenzene and benzene) . The concentrations of individual volatile organic chemicals (VOCs) could not explain the observed effects in the bacteria . An expected mixture toxicity was calculated for the six components using the concept of concentration addition . The calculated EC(50) for the mixture was still one order of magnitude lower than the observed EC(50) of the actual groundwater . The results pointed out that chemical analysis of the six most quantitative substances alone was not able to explain the effects observed with the bacteria . Thus chemical analysis alone may not be an adequate tool for remediation success evaluation in terms of toxicity reduction.

Microbiology, 2004 May, 150(Pt 5), 1283 - 90
Influences of temperature, salinity and starvation on the motility and chemotactic response of Vibrio anguillarum; Larsen MH et al.; The role of growth factors for the motility and chemotaxis of the fish pathogen Vibrio anguillarum was determined . Cells of V . anguillarum were chemotactic to serine in the temperature range 5-25 degrees C and in 0.8-2.7 % NaCl . The chemotactic response was significantly higher at 25 degrees C than at 5 or 15 degrees C . Growth in medium with 1.5 % NaCl gave a higher response than growth with 3 % NaCl; when the salinity of the chemotaxis buffer was raised, the chemotactic response was reduced . The role of starvation was also studied; V . anguillarum showed a high chemotactic response after starvation for 2 and 8 days . Motility and chemotaxis are important virulence factors for this bacterium . Not only was the ability to perform chemotactic motility maintained after starvation, but also it was shown that starvation does not interfere with the ability of the organism to cause infection in rainbow trout after a bath challenge . The swimming speed was reduced at lower temperatures . Within the range of salinity and starvation studied, the motile cells swam with the same velocity, indicating that V . anguillarum under all the examined conditions has a functional flagellum and rotates it with constant speed . Phenamil, a specific inhibitor of Na(+)-driven flagella, reduced the motility of both starved and non-starved cells of V . anguillarum indicating that, in both cases, a Na(+) motive force drives the flagellum.

Pediatr Infect Dis J, 2004 May, 23(5), 475 - 6
Otitis media associated with Vibrio alginolyticus in a child with pressure-equalizing tubes; Feingold MH et al.; Vibrio alginolyticus is an unusual cause of otitis media . Infection usually occurs in the presence of a chronically perforated eardrum or patent pressure-equalizing tube . Infection with V . alginolyticus can occur after even mild, brief exposure to seawater, and the interval between exposure to seawater and onset of clinical infection can be prolonged.

Fish Shellfish Immunol, 2004 Feb, 16(2), 151 - 61
The immune response of the white shrimp Litopenaeus vannamei and its susceptibility to Vibrio infection in relation with the moult cycle; Liu CH et al.; The white shrimp Litopenaeus vannamei (8.0-14.4 g) was examined for haemocyte count, phenoloxidase activity, respiratory burst (release of superoxide anion), phagocytic activity, and clearance efficiency to the pathogen Vibrio alginolyticus in relation with moult cycle (postmoult, A, B; intermoult, C; premoult, D(0)/D(1)D(2)/D(3)) . Granular cells were the highest at C and D(0)/D(1)stage, and the lowest at A stage . Hyaline cells and THC (total haemocyte count) were higher at C stage, but lower at postmoult stages . Phenoloxidase activity was the highest at C stage, and the lowest at A stage . Respiratory burst was lower at A stage . Phagocytic activity of shrimps against V . alginolyticus decreased significantly at postmoult and premoult stages . Additionally, the clearance efficiency of shrimps to V . alginolyticus was significantly lower for shrimps at A stage than those at C stage . In another experiment, L . vannamei at different moult stages were injected with tryptic soy broth (TSB)-grown V . alginolyticus (1x10(5)cfu shrimp(-1)) and then held in 34% seawater . After 10 h, the mortality of V . alginolyticus-injected shrimps was significantly higher for shrimps at postmoult stage than those at intermoult stage . Over 48-120 h, the mortality of V . alginolyticus-injected shrimps was 50.0%, 33.3% and 40.0% at postmoult, intermoult and premoult stage, respectively . It is concluded that L . vannamei showed a decrease in resistance at A stage through a reduction of its haemocyte count, phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency against V . alginolyticus.

Fish Shellfish Immunol, 2004 Feb, 16(2), 93 - 105
Efficacy of a bivalent vaccine against eel diseases caused by Vibrio vulnificus after its administration by four different routes; Esteve-Gassent MD et al.; Vulnivaccine, a vaccine against vibriosis caused by Vibrio vulnificus serovar E (formerly biotype 2), confers acceptable levels of protection to eels after its administration by prolonged immersion in three doses . Recently, a new pathogenic serovar, named serovar A, has been isolated from vaccinated eels in a Spanish freshwater eel farm . The main objective of this work was to design a bivalent vaccine, and to study its effectiveness against the two pathogenic serovars . With this aim, eels weighing around 20 g were immunised with the bivalent vaccine by oral and anal intubation, intraperitoneal injection (i.p.) and prolonged immersion . The overall results indicated that: (i) the new vaccine delivered by oral and anal intubation induced protection levels higher than 80%, to that achieved after i.p . vaccination; (ii) oral and anal vaccination induced a significant systemic and mucosal immune response; (iii) the protection after vaccination by whichever routes was related to antibody titres in plasma; (iv) mucosal and systemic compartments showed different kinetics of antibody production; (v) evidence for passive transfer of antibodies from plasma to gut mucus were found after i.p . and anal vaccination, and finally, (vi) vaccination did not enhance the production of lysozyme, in plasma or mucus . In conclusion, this new vaccine is effective in protecting eels against vibriosis caused by the two eel-pathogenic serovars of V . vulnificus, the oral delivery system is a promising way which may be used in intensive culture facilities during the whole growth period of eels.

Fish Shellfish Immunol, 2004 Mar, 16(3), 407 - 14
Oral vaccination of African catfish with Vibrio anguillarum O2: effect on antigen uptake and immune response by absorption enhancers in lag time coated pellets; Vervarcke S et al.; The impact on antigen uptake and antibody response by the addition of absorption enhancers to Vibrio anguillarum O2 antigen was studied in oral vaccination trials of African catfish (Clarias gariepinus) . Oral vaccination was achieved by feeding lag time coated pellets . The lag time coat prevents premature release of the encapsulated vaccine in the tank, before ingestion of the pellets by the fish . To monitor the antigen uptake, a competitive ELISA was used . The antibody response was measured using an indirect ELISA . Feeding of bacterin-layered pellets without absorption enhancers resulted in a rather low antigen uptake and antibody levels . The addition of absorption enhancers such as sodium salicylate, sodium caprate and vitamin E TPGS increased the serum antigen levels and specific antibody levels in the systemic circulation . Skin mucus antibody levels were higher after oral vaccination compared to the IP and control group . The addition of absorption enhancers in the oral groups further increased the antibody levels obtained in the skin mucus.

Fish Shellfish Immunol, 2004 Mar, 16(3), 321 - 34
Effect of ammonia on the immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus; Liu CH et al.; Growth of Vibrio alginolyticus was not affected by TSB medium containing ammonia-N concentration in the range of 0-20 mg l(-1) . White shrimp Litopenaeus vannamei (7-12 g in the intermolt stage) were challenged with V . alginolyticus, which had been incubated for 24 h in the TSB medium containing different concentrations of ammonia-N (0, 1, 5 . 10 and 20 mg l(-1)) . There was no significant difference in cumulative mortality for shrimp incubated in the TSB medium containing 0, 1, 5, 10 and 20 mg l(-1)ammonia-N after 120 h of challenge . The shrimps were challenged with V . alginolyticus previously incubated in the TSB medium for 24 h, then placed in water containing concentrations of ammonia-N at 0.01 mg l(-1)(control), 1.10, 5.24, 11.10 and 21.60 mg l(-1) . Mortality of shrimp in 5.24, 11.10 and 21.60 mg l(-1)was significantly higher than those in the control solution (0.01 mg l(-1)) after 48-168 h . Shrimps which had been exposed to control, 1.10, 5.24, 11.10 and 21.60 mg l(-1)ammonia-N for 7 days were examined for THC (total haemocyte count), granular cells, hyaline cells, phenoloxidase activity, release of superoxide anion, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to V . alginolyticus . No significant difference in THC, hyaline cells and granular cells were observed among shrimps at different ammonia-N concentrations . Phenoloxidase activity however, decreased when the shrimps were exposed to 5.24 mg l(-1)ammonia-N and greater after 7 days . The release of superoxide anion increased significantly, whereas SOD activity decreased significantly at 21.60 mg l(-1)ammonia-N . With shrimps exposed to 11.21 and 21.22 mg l(-1)ammonia-N for 7 days, phagocytic activity and clearance efficiency to V . alginolyticus significantly decreased . It is therefore suggested that ammonia in water caused a depression in the immune response and an increase in mortality of L . vannamei from the V . alginolyticus infection.

Fish Shellfish Immunol, 2004 Mar, 16(3), 295 - 306
The immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus at different salinity levels; Cheng W et al.; Addition of NaCl at 2.5% to 3.5% to tryptic soy broth (TSB) significantly increased the growth of Vibrio parahaemolyticus . Taiwan abalone Haliotis diversicolor supertexta held in 30 per thousand seawater were injected with V . parahaemolyticus grown in TSB containing NaCl at 0.5, 1.5, 2.5, 3.5 and 4.5% at a dose of 1.6 x 10(5)colony-forming units (cfu) abalone(-1) . After 48 h, the cumulative mortality was significantly higher for the abalone challenged with V . parahaemolyticus grown in 2.5% than those grown in 0.5 and 1.5% NaCl . In other experiments, abalones held in 30 per thousand seawater were injected with TSB-grown V . parahaemolyticus (1.6 x 10(5)cfu abalone(-1)), and then transferred to 20, 25, 30 and 35 per thousand seawater . All abalones held in 20 per thousand were killed in 48 h . The mortality of V . parahaemolyticus-injected abalone held in 30 per thousand was significantly lower over 24-120 h . Abalone held in 30 per thousand seawater and then transferred to 20, 25, 30 and 35 per thousand were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V . parahemolyticus after 24 and 72 h . The THC increased directly related with salinity levels . Phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V . parahaemolyticus decreased significantly for the abalone in 20, 25 and 35 per thousand . It is concluded that the abalone transferred from 30 per thousand to 20, 25 and 35 per thousand had reduced immune ability and decreased resistance against V . parahaemolyticus infection.

J Antimicrob Chemother, 2004 Jun, 53(6), 947 - 51 Epub 2004 Apr 29.
New aminoglycoside acetyltransferase gene, aac(3)-Id, in a class 1 integron from a multiresistant strain of Vibrio fluvialis isolated from an infant aged 6 months; Ahmed AM et al.; OBJECTIVES: To characterize the molecular basis of antibiotic resistance in a multidrug-resistant clinical isolate of Vibrio fluvialis H-08942 . PATIENT AND METHODS: V . fluvialis H-08942 was isolated from a hospitalized infant aged 6 months suffering from cholera-like diarrhoea in India in 2002 . The broth microdilution method was used to determine the MICs of a range of antibiotics for this strain . PCR, DNA sequencing, Southern hybridization, cloning and expression were used to characterize the molecular basis of antibiotic resistances . RESULTS: V . fluvialis H-08942 showed resistance to chloramphenicol, streptomycin, spectinomycin, co-trimoxazole, ampicillin, furazolidone, nalidixic acid and gentamicin . A class 1 integron that contains a novel aminoglycoside acetyltransferase gene, aac(3)-Id, and aminoglycoside adenyltransferase gene, aadA7, was characterized . The aac(3)-Id gene product was found to share 50%, 45% and 44% identity to AAC(3)-Ic, AAC(3)-Ia, and AAC(3)-Ib, respectively . Both aac(3)-Id and aadA7 genes were cloned and expressed in Escherichia coli . Phylogenetic analysis suggested that the aac(3)-Id represents a fourth evolutionary lineage in the aminoglycoside acetyltransferase genes . Southern hybridization showed that this integron is located in the chromosome . CONCLUSIONS: In this study we identified a new type of aminoglycoside acetyltransferase gene, aac(3)-Id . In addition, this is the first report of identification of antibiotic resistance genes and a class 1 integron in V . fluvialis.

Bioessays, 2004 May, 26(5), 463 - 8
Phylogeny of gamma-proteobacteria: resolution of one branch of the universal tree?
Brown JR, Volker C.
The reconstruction of bacterial evolutionary relationships has proven to be a daunting task because variable mutation rates and horizontal gene transfer (HGT) among species can cause grave incongruities between phylogenetic trees based on single genes . Recently, a highly robust phylogenetic tree was constructed for 13 gamma-proteobacteria using the combined alignments of 205 conserved orthologous proteins.1 Only two proteins had incongruent tree topologies, which were attributed to HGT between Pseudomonas species and Vibrio cholerae or enterics . While the evolutionary relationships among these species appears to be resolved, further analysis suggests that HGT events with other bacterial partners likely occurred; this alters the implicit assumption of gamma-proteobacteria monophyly . Thus, any thorough reconstruction of bacterial evolution must not only choose a suitable set of molecular markers but also strive to reduce potential bias in the selection of species .

Int J Food Microbiol, 2004 Apr 15, 92(2), 207 - 15
Susceptibility of Vibrio parahaemolyticus to various environmental stresses after cold shock treatment; Lin C et al.; Vibrio parahaemolyticus was subjected to cold shock treatment at 20 or 15 degrees C for 2 or 4 h . The effect of cold shock on the survival of V . parahaemolyticus subjected to subsequent low temperature (5 and -18 degrees C) and other adverse conditions (47 degrees C, 6 ppm crystal violet, 1000 ppm H(2)O(2), 25 mM acetic acid and 25 mM lactic acid) was investigated . Regardless of the cold shock treatment, survival of V . parahaemolyticus increased when stored at 5 or -18 degrees C, while no increase in survival was noted for cells cold shocked in the presence of chloramphenicol . Cold shock treatment under the conditions tested, in general, enabled V . parahaemolyticus cells to survive better following subsequent challenge by crystal violet, while the cold-shocked organism was more susceptible to high temperature (47 degrees C), H(2)O(2) and organic acids (lactic and acetic acid) than the non-shocked cells . Furthermore, the temperature and time of the cold shock treatment affected the cold shock response of V . parahaemolyticus.

FEMS Microbiol Lett, 2004 May 1, 234(1), 163 - 7
Significance of Na+ in the fish pathogen, Vibrio anguillarum, under energy depleted condition; Fujiwara-Nagata E et al.; Vibrio anguillarum kills various kinds of fish over salinities ranging from seawater to freshwater . In this study, we investigated the role of Na(+) in V . anguillarum, especially under energy-depleted conditions such as in natural seawater . V . angustum S14, which is a typical marine vibrio, was used for comparison . V . anguillarum only required Na(+) for starvation-survival, but in contrast, V . angustum S14 always required Na(+) for both growth and starvation-survival . In marine vibrios, Na(+) is used in the Na(+)-dependent respiratory chain that produces the sodium motive force (SMF) across the cell membrane . It has been considered that marine vibrios always need a SMF produced by Na(+), however in the case of V . anguillarum, the SMF is not required for growth, but becomes more important for starvation-survival.

Dis Aquat Organ, 2004 Mar 10, 58(2-3), 223 - 30
Pathogenicity testing of shellfish hatchery bacterial isolates on Pacific oyster Crassostrea gigas larvae; Estes RM et al.; Bacterial diseases are a major cause of larval mortality in shellfish hatcheries . Even with proper sanitation measures, bacterial pathogens cannot be eliminated in all cases . The pathogenicity of bacteria isolated from Pacific Northwest shellfish hatcheries to Pacific oyster Crassostrea gigas larvae was investigated . We found 3 highly pathogenic strains and 1 mildly pathogenic strain among 33 isolates tested . These strains appear to be members of the genus Vibrio . Although there have been many studies of bivalve bacterial pathogens, a standard method to assess bacterial pathogenicity in bivalve larvae is needed . Thus, we developed 2 methods using either 15 ml conical tubes or tissue culture plates that were employed for rapidly screening bacterial strains for pathogenicity to Pacific oyster larvae . The tissue culture plates worked well for screening both mildly pathogenic strains and LD50 (lethal dose) assays . This method allowed for non-intrusive and non-destructive observation of the oyster larvae with a dissecting microscope . The LD50 for the 3 highly pathogenic strains ranged between 1.6 and 3.6 x 10(4) colony forming units (CFU) ml(-1) after 24 h and between 3.2 x 102 and 1.9 x 10(3) CFU ml(-1) after 48 h.

Dis Aquat Organ, 2004 Mar 10, 58(2-3), 143 - 50
Phylogenetic study and identification of Vibrio splendidus-related strains based on gyrB gene sequences; Le Roux F et al.; Different strains related to Vibrio splendidus have been associated with infection of aquatic animals . An epidemiological study of V . splendidus strains associated with Crassostrea gigas mortalities demonstrated genetic diversity within this group and suggested its polyphyletic nature . Recently 4 species, V . lentus, V . chagasii, V . pomeroyi and V . kanaloae, phenotypically related to V . splendidus, have been described, although biochemical methods do not clearly discriminate species within this group . Here, we propose a polyphasic approach to investigate their taxonomic relationships . Phylogenetic analysis of V . splendidus-related strains was carried out using the nucleotide sequences of 16S ribosomal DNA (16S rDNA) and gyrase B subunit (gyrB) genes . Species delineation based on 16S rDNA-sequencing is limited because of divergence between cistrons, roughly equivalent to divergence between strains . Despite a high level of sequence similarity, strains were separated into 2 clades . In the phylogenetic tree constructed on the basis of gyrB gene sequences, strains were separated into 5 independent clusters containing V . splendidus, V . lentus, V . chagasii-type strains and a putative new genomic species . This phylogenetic grouping was almost congruent with that based on DNA-DNA hybridisation analysis . V . pomeroyi, V . kanaloae and V . tasmaniensis-type strains clustered together in a fifth clade . The gyrB gene-sequencing approach is discussed as an alternative for investigating the taxonomy of Vibrio species.

Microbiol Immunol, 2004, 48(4), 319 - 27
Emergence and serovar transition of Vibrio parahaemolyticus pandemic strains isolated during a diarrhea outbreak in Vietnam between 1997 and 1999; Chowdhury A et al.; We characterized 523 Vibrio parahaemolyticus strains isolated during a survey of diarrhea patients in Khanh Hoa province, Vietnam between 1997 and 1999 . Forty-nine percent of the strains were judged to belong to the pandemic strains that emerged around 1996 and spread to many countries . These strains were positive in the GS-PCR assay and carried the tdh gene . The ORF8 of the f237 phage genome, a possible marker of the pandemic clone, was absent in 10% of these strains . Eleven O: K serovars were detected among the pandemic strains and the strains representing all 11 serovars of pandemic strains were shown to be closely related regardless of the ORF8 genotype using arbitrarily primed PCR and pulsed field gel electrophoresis analyses . It was clear that a transition of major serovars occurred among the pandemic strains represented by the emergence of O3: K6 in 1997, O4: K68 in 1998, and O1: K25 in 1998 and 1999.

Microbiol Immunol, 2004, 48(4), 313 - 8
Cytotoxicity and enterotoxicity of the thermostable direct hemolysin-deletion mutants of Vibrio parahaemolyticus; Park KS et al.; The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of Vibrio parahaemolyticus . We have recently completed the genome sequence of a TDH-producing V . parahaemolyticus strain, RIMD2210633 . In this study, we constructed tdh-deletion mutants from the sequenced strain by homologous recombination and analyzed their phenotypes . Although the deletion of both copies of tdh completely abolished the hemolytic activity of the wild-type strain, the deletion did not affect the cytotoxicity to HeLa cells . Enterotoxicity, assayed by the rabbit ileal loop test, was lowered by tdh deletion, but the mutant still showed partial fluid accumulation in rabbit intestine . These results indicate that the cytotoxicity and enterotoxicity of TDH-producing V . parahaemolyticus are not explained by TDH alone, and suggest that an unknown virulence factor(s) could be involved in these pathogenic activities.

Microbiol Immunol, 2004, 48(4), 243 - 50
A major Li(+) extrusion system NhaB of Pseudomonas aeruginosa : comparison with the major Na(+) extrusion system NhaP; Kuroda T et al.; A gene encoding a Li(+) extrusion system was cloned from the chromosomal DNA of Pseudomonas aeruginosa and expressed in Escherichia coli cells . The gene enabled growth of E . coli KNabc cells, which were unable to grow in the presence of 10 mM LiCl or 0.1 M NaCl because of the lack of major Na(+) (Li(+))/H(+) antiporters . We detected Li(+)/H(+) and Na(+)/H(+) antiport activities in membrane vesicles prepared from E . coli KNabc cells that harbored a plasmid carrying the cloned gene . Activity of this antiporter was pH-dependent with an optimal pH activity between pH 7.5 and 8.5 . These properties indicate that this antiporter is different from NhaP, an Na(+)/H(+) antiporter from P . aeruginosa that we reported previously, and that is rather specific to Na(+) but it cannot extrude Li(+) effectively . The gene was sequenced and an open reading frame (ORF) was identified . The amino acid sequence deduced from the ORF showed homology (about 60% identity and 90% similarity) with that of the NhaB Na(+)/H(+) antiporters of E . coli and Vibrio parahaemolyticus . Thus, we designated the antiporter as NhaB of P . aeruginosa . E . coli KNabc carrying the nhaB gene from P . aeruginosa was able to grow in the presence of 10 to 50 mM LiCl, although KNabc carrying nhaP was unable to grow in these conditions . The antiport activity of NhaB from P . aeruginosa was produced in E . coli and showed apparent Km values for Li(+) and Na(+) of 2.0 mM and 1.3 mM, respectively . The antiport activity was inhibited by amiloride with a Ki value for Li(+) and Na(+) of 0.03 mM and 0.04 mM, respectively.

Microbiol Immunol, 2004, 48(4), 229 - 35
Virulence properties of rough and smooth strains of Vibrio cholerae O1; Islam MS et al.; A comparative study was carried out to see the differences in pathogenicity of rough and smooth strains . A total of 10 strains including 5 each of rough and smooth strains of Vibrio cholerae O1 were tested and found positive for toxin production by enzyme-linked immunosorbent assay (ELISA) in Richardson's and AKI media . All the smooth and rough strains, except one, showed a titre of 1: 10 and 1: 100 in Richardson's and AKI media, respectively . Both types of strains produced enterotoxin in rabbit ileal loop (RIL) . The differences in multiplication abilities of smooth and rough strains in RIL were statistically significant (P <0.05) . However, these differences in multiplying abilities did not influence the adherence potential or enterotoxin production as there was no significant difference (P >0.05) between these properties . This study demonstrated that the rough strains are equally pathogenic and as important as smooth strains.

Arch Environ Contam Toxicol, 2004 Feb, 46(2), 176 - 82
Effect of pH on arsenate and arsenite toxicity to luminescent bacteria (Vibrio fischeri); Fulladosa E et al.; Arsenic is an abundant metalloid and a dangerous pollutant when in solution under the arsenate or arsenite forms-As(V) and As(III), respectively . Since its biological effects are expected to depend on the oxidation state and on speciation, effect of pH on either As(V) or As(III) speciation and resulting toxicity was investigated using the Microtox bioassay based on change in light emission by the luminescent bacteria Vibrio fischeri . Within a 5.0-8.0 pH range, EC50 values for As(V) were found to decrease as pH became basic, reflecting an increase in toxicity; whereas in the case of As(III), EC50 values were almost unchanged within a 6.0-8.0 pH range and lowered only at pH 9.0 . HAsO42- and H2AsO3-were found to be the most toxic species . A statistical approach based on testing the null hypothesis of additive toxicity revealed an antagonistic effect between the arsenate chemical species . At low concentrations, As(V) was regularly found to be more toxic than As(III), independent of the pH value . Conversely, at high concentrations, the toxicity of both As(III) and As(V) was found to chiefly depend on pH, as a consequence of the strong influence of this parameter on the chemical speciation.

Kansenshogaku Zasshi, 2004 Feb, 78(2), 83 - 9
{A basic study of Vibrio vulnificus infection: serotyping and drug sensitivity test of environment-derived strains and human clinical isolates}; Oonaka K et al.; In attempt to elucidate the route and source of Vibrio vulnificus infection . serotyping and drug sensitivity tests of environment-derived strains and human clinical isolates were performed . 1) Serotyping of isolates from the two types of source were determined . Of environment-derived strains, 72.5% were classified into 18 types, and O7 was the most frequent type, accounting for 73.1%, and the second frequent type was O4, accounting for 6.1% . Of human clinical isolates, 87.1% were classified into eight types, and O4 was the most frequent, accounting for 73.5%, and O7 was the secondly most frequent, accounting for 12.9% . 2) Serotypes were investigated by regions . In eastern Japan, 69.2% were classified into 18 types, and O7 and O4 accounting for 44.6% and 5.7%, respectively . In western Japan, 64.8% were classified into eight types, and O7 was the most frequent, accounting for 20.4%, and secondly frequent type was O4, accounting for 11.1% . 3) Regarding the relationship between biotypes and serotypes, environment-derived biotype-I strains were widely distributed in the serotypes, but most biotype-I human clinical isolates were distributed in serotypes O1-O7, showing a difference between the two types of sources . However, many biotype-II strains from the two types of sources included in the serotype O7 group . 4) Drug sensitivity was compared based on MIC90 between strains from the two types of sources . Environment-derived strains were sensitive to ABPC, PIPC, CPZ, CTX, LMOX, MEPM, GM, EM, TC, DOXY, MINO, CP, NA and CPFX, but some strains were resistant to CER, CET, CTX, CMZ, KM and LCM . Human clinical isolates were sensitive to EM, TC, DOXY, MINO, CP, NA and CPFX, but some strains were resistant to ABPC, PIPC, CER, CET, CPZ, CTX, CMZ, LMOX, MEPM, KM, GM, AMK and LCM.

Environ Toxicol, 2004 Jun, 19(3), 191 - 7
Anatoxin-a toxin in the cyanobacterium Planktothrix rubescens from a fishing pond in northern Italy; Viaggiu E et al.; A heavy algal bloom occurring in a fishing pond in northern Italy full of Salmo trutta was examined for algae taxonomy and toxic production . The dominant algal species (98%) was identified as the cyanobacterium Planktothrix rubescens (D.C . ex GOMONT) Komarek Anagnostidis, based on morphological examination, and it was revealed to be toxic in mouse and Vibrio fischeri bioassays . The toxin was identified as anatoxin-a using high-performance liquid chromatography and confirmed using liquid chromatography-mass spectrometry (LC-MS) . The mouse bioassay gave signs of poisoning, as previously reported for anatoxin-a . The LC-MS confirmed the presence of an anatoxin-a peak at m/z 166 (M+H+) . The content of toxin in the field population was estimated at 12.13 microg/g of fresh cells . The bloom was sustained by the very high N/P ratio in the water . This is the first report in Italy of an anatoxin-a-producing Planktothrix rubescens population .

Clin Infect Dis, 2004 Apr 15, 38 Suppl 3, S203 - 11
Survey of physician diagnostic practices for patients with acute diarrhea: clinical and public health implications; Hennessy TW et al.; To understand physician practices regarding the diagnosis of acute diarrheal diseases, we conducted a survey, in 1996, of 2839 physicians in Connecticut, Georgia, Minnesota, Oregon, and California . Bacterial stool culture was requested for samples from the last patient seen for acute diarrhea by 784 (44%; 95% confidence interval, 42%-46%) of 1783 physicians . Physicians were more likely to request a culture for persons with acquired immune deficiency syndrome, bloody stools, travel to a developing country, diarrhea for >3 days, intravenous rehydration, or fever . Substantial geographic and specialty differences in culture-request practices were observed . Twenty-eight percent of physicians did not know whether stool culture included testing for Escherichia coli O157:H7; 40% did not know whether Yersinia or Vibrio species were included . These variabilities suggest a need for clinical diagnostic guidelines for diarrhea . Many physicians could benefit from education to improve their knowledge about tests included in routine stool examinations.

Cell Mol Life Sci, 2004 Apr, 61(7-8), 961 - 72
Involvement of penaeidins in defense reactions of the shrimp Litopenaeus stylirostris to a pathogenic vibrio; Munoz M et al.; The present study reports for the first time the involvement of an antimicrobial peptide in the defense reactions of a shrimp infected by a pathogenic Vibrio, Vibrio penaeicida . New members of the penaeidin family were characterized in the shrimp Litopenaeus stylirostris by RT-PCR and RACE-PCR from hemocyte total RNAs, and by mass spectrometry detection and immunolocalization of mature peptides in shrimp hemocytes . In infected shrimps, bacteria and penaeidin distribution colocalized in the gills and the lymphoid organ that represented the main infected sites . Moreover, the shrimp immune response to infection involved massive hemocyte recruitment to infection sites where released penaeidin may participate in the isolation and elimination of the bacteria, We show that the ability of the shrimps to circumvent shrimp infections is closely related to a recovery phase based on the hematopoietic process.

Toxicol Appl Pharmacol, 2004 May 1, 196(3), 319 - 26
Lysophospholipase inhibition by organophosphorus toxicants; Quistad GB et al.; Lysophospholipases (LysoPLAs) are a large family of enzymes for removing lysophospholipids from cell membranes . Potent inhibitors are needed to define the importance of LysoPLAs as targets for toxicants and potential therapeutics . This study considers organophosphorus (OP) inhibitors with emphasis on mouse brain total LysoPLA activity relative to the mipafox-sensitive neuropathy target esterase (NTE)-LysoPLA recently established as 17% of the total activity and important in the action of OP delayed toxicants . The most potent inhibitors of total LysoPLA in mouse brain are isopropyl dodecylphosphonofluoridate (also for LysoPLA of Vibrio bacteria), ethyl octylphosphonofluoridate (EOPF), and two alkyl-benzodioxaphosphorin 2-oxides (BDPOs){(S)-octyl and dodecyl} (IC50 2-8 nM) . OP inhibitors acting in vitro and in vivo differentiate a more sensitive portion but not a distinct NTE-LysoPLA compared with total LysoPLA activity . For 10 active inhibitors, NTE-LysoPLA is 17-fold more sensitive than total LysoPLA, but structure-activity comparisons give a good correlation (r(2) = 0.94) of IC50 values, suggesting active site structural similarity or identity . In mice 4 h after intraperitoneal treatment with discriminating doses, EOPF, tribufos (a plant defoliant), and dodecanesulfonyl fluoride inhibit 41-57% of the total brain LysoPLA and 85-99% of the NTE-LysoPLA activity . Total LysoPLA as well as NTE-LysoPLA is decreased in activity in Nte(+/-)-haploinsufficient mice compared to their Nte(+/+) littermates . The lysolecithin level of spinal cord but not brain is elevated significantly following EOPF treatment (3 mg/kg), thereby focusing attention on localized rather than general alterations in lysophospholipid metabolism in OP-induced hyperactivity and toxicity.

Biochim Biophys Acta, 2004 Apr 16, 1678(1), 7 - 13
Cloning and characterization of the lipase and lipase activator protein from Vibrio vulnificus CKM-1; Su JH et al.; The gene (lipA) encoding the extracellular lipase and its downstream gene (lipB) from Vibrio vulnificus CKM-1 were cloned and sequenced . Nucleotide sequence analysis and alignments of amino acid sequences suggest that Lip Ais a member of bacterial lipase family I.1 and that LipB is a lipase activator of LipA . The active LipA was produced in recombinant Escherichia coli cells only in the presence of the lipB . In the hydrolysis of p-nitrophenyl esters and triacylglycerols, using the reactivated LipA, the optimum chain lengths for the acyl moiety on the substrate were C(14) for ester hydrolysis and C(10) to C(12) for triacylglycerol hydrolysis.

Environ Pollut, 1992, 76(3), 245 - 9
Effects of temperature and pH on conjugal transfer of zinc-resistant plasmids residing in Gram-negative bacteria isolated from industrial effluents; Hasnain S et al.; Two zinc (Zn)-resistant strains, AnZn-1 and AnZn-2, which were resistant to ZnSO(4) up to 12.5 mg ml(-1) were isolated from industrial effluents . Both were Gram-negative with motile cells . They exhibited tolerance to Ba(2+), Ni(+), Co(2+) Mn(2+), Cu(2+), Fe(2+), Ni(2+), Cd(2+), kanamycin, chloramphenicol, ampillicin and tetracycline, but were sensitive to Hg(2+) and streptomycin . For AnZn-1 and AnZn-2, the optimum pH for growth was 7 . Both were facultative anaerobes and had cytochrome oxidase and urease enzymes, while catalase was present only in AnZn-2 . Both strains had the ability to hydrolyse gelatin, reduce nitrate, and yield acid from arabinose and rhamnose . The two strains shared maximum characters with Vibrionaceae . Each strain carries a single Zn-resistant conjugative plasmid . The plasmid residing in AnZn-1 (pSH1211) displayed a lower level of resistance than the plasmid of AnZn-2 (pSH1212) . Both required a minimum of 24 h for mating and showed highest transfer frequency at 25 degrees C . pSH1211 preferred pH 7 and pSH1212 pH 8.5 for their transfer . Both plasmids, when allowed to mate with Escherichia coli at 25 degrees C, alkaline pH values of 8-8.5 (pSH1211) of pH 7.5 (pSH1212), showed increased transfer frequency.

Blood Coagul Fibrinolysis, 2004 Mar, 15(2), 169 - 78
A secreted metallo protease from Aeromonas hydrophila exhibits prothrombin activator activity; Keller T et al.; Detection, purification, and partial characterization of a protease from Aeromonas hydrophila capable of cleaving prothrombin into active thrombin is described . The protease has been characterized with respect to enzymatic characteristics such as optimum reaction conditions for prothrombin activation, usage of additional substrates, as well as sensitivity against inhibitors . The protease activity can reversibly be inhibited by Me2+ chelating agents like ethylenediamine tetraacetic acid . The enzyme exhibits a pI value of 4.4 and can withstand temperatures up to 55 degrees C without loss of activity . With respect to prothrombin the enzyme exhibits a K(M) value of 1.47 micromol/l and a vmax value of 1.66 mol/min per mol enzyme . Amino terminal sequence analysis as well as mass spectrometry of fragments obtained by trypsin digest showed identity to a recently described elastase type protease from the same organism and homology to known proteases from other procaryotes (e.g . Aeromonas caviae, Vibrio proteolytica, Pseudomonas aeruginosa).

J Bacteriol, 2004 May, 186(9), 2906 - 8
Correlation between osmolarity and luminescence of symbiotic Vibrio fischeri strain ES114; Stabb EV et al.; Vibrio fischeri isolates from Euprymna scolopes are dim in culture but bright in the host . We found the luminescence of V . fischeri to be correlated with external osmolarity both in culture and in this symbiosis . Luminescence enhancement by osmolarity was independent of the lux promoter and unaffected by autoinducers or the level of lux expression, but the addition of an aldehyde substrate for luciferase raised the luminescence of cells grown at high and low osmolarities to the same high level . V . fischeri culture media have lower osmolarities than are typical in seawater or in cephalopods, partially accounting for the bacterium's low light output in culture.

J Bacteriol, 2004 May, 186(9), 2636 - 45
Formation of SXT tandem arrays and SXT-R391 hybrids; Burrus V et al.; SXT is an integrative and conjugative element (ICE) isolated from Vibrio cholerae . This approximately 100-kb ICE encodes resistance to multiple antibiotics and integrates site specifically into the chromosome . SXT excises from the chromosome to form a circular but nonreplicative extrachromosomal molecule that is required for its transfer . Here we found that a significant fraction of freshly isolated SXT exconjugants contained tandem SXT arrays . There was heterogeneity in the size of the SXT arrays detected in single exconjugant colonies . Some arrays consisted of more than five SXTs arranged in tandem . These extended arrays were unstable and did not persist during serial passages . The mechanism accounting for the generation of SXT arrays is unknown; however, array formation was not dependent upon recA and appeared to depend on conjugative transfer . While such arrays did not alter the transfer frequency of wild-type SXT, they partially complemented the transfer deficiency of a Deltaxis SXT mutant, which is ordinarily unable to generate the extrachromosomal intermediate required for SXT transfer . Exconjugants derived from donor strains that harbored tandem arrays of SXT and R391, an SXT-related element, contained functional hybrid elements that arose from recA-independent recombination between the two ICEs . Thus, arrays of SXT-related elements promote the creation of novel ICEs.

J Bacteriol, 2004 May, 186(9), 2558 - 66
The bchU gene of Chlorobium tepidum encodes the c-20 methyltransferase in bacteriochlorophyll c biosynthesis; Maresca JA et al.; Bacteriochlorophylls (BChls) c and d, two of the major light-harvesting pigments in photosynthetic green sulfur bacteria, differ only by the presence of a methyl group at the C-20 methine bridge position in BChl c . A gene potentially encoding the C-20 methyltransferase, bchU, was identified by comparative analysis of the Chlorobium tepidum and Chloroflexus aurantiacus genome sequences . Homologs of this gene were amplified and sequenced from Chlorobium phaeobacteroides strain 1549, Chlorobium vibrioforme strain 8327d, and C . vibrioforme strain 8327c, which produce BChls e, d, and c, respectively . A single nucleotide insertion in the bchU gene of C . vibrioforme strain 8327d was found to cause a premature, in-frame stop codon and thus the formation of a truncated, nonfunctional gene product . The spontaneous mutant of this strain that produces BChl c (strain 8327c) has a second frameshift mutation that restores the correct reading frame in bchU . The bchU gene was inactivated in C . tepidum, a BChl c-producing species, and the resulting mutant produced only BChl d . Growth rate measurements showed that BChl c- and d-producing strains of the same organism (C . tepidum or C . vibrioforme) have similar growth rates at high and intermediate light intensities but that strains producing BChl c grow faster than those with BChl d at low light intensities . Thus, the bchU gene encodes the C-20 methyltransferase for BChl c biosynthesis in Chlorobium species, and methylation at the C-20 position to produce BChl c rather than BChl d confers a significant competitive advantage to green sulfur bacteria living at limiting red and near-infrared light intensities.

Chem Res Toxicol, 2004 Apr, 17(4), 545 - 54
Assessment and modeling of the toxicity of organic chemicals to Chlorella vulgaris: development of a novel database; Cronin MT et al.; This study reports a database of toxicity values for 91 compounds assessed in a novel, rapid, and economical 15 min algal toxicity test . The toxicity data were measured using the unicellular green alga Chlorella vulgaris in an assay that determined the disappearance of fluorescein diacetate . The chemicals tested covered a wide range of physicochemical properties and mechanisms of action . Quantitative activity-activity relationships with the toxicity of the chemicals to other species (Tetrahymena pyriformis, Vibrio fischeri, and Pimephales promelas) showed strong relationships, although some differences resulting from different protocols were established . Quantitative structure-activity relationships (QSARs) were determined using linear {multiple linear regression (MLR)} and nonlinear {k-nearest neighbors (KNN)} methods . Three descriptors, accounting for hydrophobicity, electrophilicity, and a function of molecular size corrected for the presence of heteroatoms, were found to be important to model toxicity . The predictivity of MLR was compared to KNN using leave-one-out cross-validation and the simulation of an external test set . MLR demonstrated greater stability in validation . The results of this study showed that method selection in QSAR is task-dependent and it is inappropriate to resort to more complicated but less transparent methods, unless there are clear indications (e.g., inability of MLR to deal with the data set) for the need of such methods.

Bacteriol Virusol Parazitol Epidemiol, 2002 Jul-Dec, 47(3-4), 119 - 24
{Factors influencing the capacity of cellular substrate adherence of vibrio cholerae O1 and non O1 strains}; Israil AM et al.; Bacterial adherence to eukariotic cells represents an important step of tissue colonization and is mediated by specific molecules called adhesins . Bacterial adherence to cellular substrate is a very complex process consisting in specific interactions between the surface of host cell and bacterial cell surface respectively . Adherence to cellular substrate confers selective advantages to bacterial cells, as: rapid growth rate by shorter lag period and protection against antibodies and lysozime . Adherence and colonization of small bowel represent the early steps of cholera infection (1, 2) . The purposes of this study were to characterize the adherence ability of 46 Vibrio cholerae O1 and non O1 strains with different sources of isolation (acute diarrhea, water sources) to HEp-2 cell; to determine the influence of different factors (culture media, bacterial culture growth phase, proteolytic enzymes, carbohydrates and polyvalent agglutinant anti V . cholerae O1 serum) on the bacterial adherence capacity . Adherence capacity was assayed using the qualitative Cravioto's method . The adherence ability was appreciated by semi quantitative ("+", "++" and "+++") and quantitative assays . The adherence pattern of the tested strains was predominantly a diffuse one . The agar medium proved to be the most appropriate for the early and maximal expression of adhesion molecules, by comparison with nutritive broth and alkaline peptone water . Manose in different concentrations (1% and 3%) inhibited the adherence ability, demonstrating the role of manose-sensitive haemagglutinating fimbriae (MSHA) in mediating the adherence of V . cholerae strains to cellular substrate . Trypsine has no notable effect on the adherence ability, suggesting that the major V . cholerae adhesion molecules are not essentially of protein nature, so that the afimbrial adhesins could also play an important role in bacterial adhesion to eukariotic cells . The agglutinant polyvalent anti-V . cholerae O1 serum had the most significant inhibitory effect on the adherence ability, which was completely abolished in the presence of sub-agglutinant dilutions of serum titer (1/60-1/120) and partially reduced at titers ranging from 1/240 to 1/920 . This inhibitory effect could be explained by bacterial agglutination, but also by the specific blocking of some surface structure implicated in the adherence process (i.e . lipopolysaccharides, as demonstrated by the inhibitory effect of sub-agglutinant serum titers) . The inhibitory effect of polyvalent anti-V . cholerae O1 serum was limited to O1, but was not evident for the non O1 serogroups, demonstrating that the serum antibodies are acting on serogroup specific antigenic fractions.

J Mol Biol, 2004 Apr 30, 338(3), 585 - 96
The crystal structure of the periplasmic domain of the type II secretion system protein EpsM from Vibrio cholerae: the simplest version of the ferredoxin fold; Abendroth J et al.; The terminal branch of the general secretion pathway (Gsp or type II secretion system) is used by several pathogenic bacteria for the secretion of their virulence factors across the outer membrane . In these secretion systems, a complex of 12-15 Gsp proteins spans from the pore in the outer membrane via several associated signal or energy-transducing proteins in the inner membrane to a regulating ATPase in the cytosol . The human pathogen Vibrio cholerae uses such a system, called the Eps system, for the export of the cholera toxin and other virulence factors from its periplasm into the lumen of the gastrointestinal tract of the host . Here, we report the atomic structure of the periplasmic domain of the EpsM protein from V.cholerae, which is a part of the interface between the regulating part and the rest of the Eps system . The crystal structure was determined by Se-Met MAD phasing and the model was refined to 1.7A resolution . The monomer consists of two alphabetabeta-subdomains forming a sandwich of two alpha-helices and a four-stranded antiparallel beta-sheet . In the dimer, a deep cleft with a polar rim and a hydrophobic bottom made by conserved residues is located between the monomers . This cleft contains an extra electron density suggesting that this region might serve as a binding site of an unknown ligand or part of a protein partner . Unexpectedly, the fold of the periplasmic domain of EpsM is an undescribed circular permutation of the ferredoxin fold.

J Virol Methods, 2004 Jun 15, 118(2), 95 - 100
The use of degenerate-primed random amplification of polymorphic DNA (DP-RAPD) for strain-typing and inferring the genetic similarity among closely related viruses; Comeau AM et al.; Often it is necessary to distinguish among strains of closely related viruses, as well as infer the genetic relatedness within large groups of viruses . Current methods for strain-typing viruses are time-consuming, require significant quantities of extracted DNA, and/or may require a priori genetic information . In this study we modified random amplification of polymorphic DNA (RAPD) by using a degenerate primer to produce unique and reproducible banding patterns from viral genomes . In the degenerate-primed RAPD analysis (DP-RAPD), a selection of algal virus and bacteriophage strains were profiled that encompassed an array of genome sizes and virus families . Closely related viruses (e.g . strains infecting Micromonas pusilla) generated similar, yet unique DP-RAPD patterns that could be readily distinguished from viruses within the same family (Phycodnaviridae) infecting a Chlorella-like alga . As well, marine vibriophage from the families Myoviridae, Siphoviridae, and Podoviridae showed high diversity and were distinct from coliphage and cyanophage . Contamination of host DNA, even at levels above those that would normally be encountered, did not interfere with the viral patterns . These findings describe a rapid, PCR-based tool for strain-typing viral isolates that allows inferences to be made on genetic relatedness within groups of closely related viruses.

Water Sci Technol, 2004, 49(4), 273 - 7
Effect of advanced oxidation processes on the toxicity of municipal landfill leachates; Slomczynska B et al.; The aim of the present study was to assess the effect of advanced oxidation processes (AOPs) (oxidation ozone and peroxide/ozone) on the toxicity of leachates from municipal landfill for Warsaw, Poland, using a battery of tests . AOPs used to pre-treat leachates were carried out in laboratory conditions after their coagulation with the use of FeCl3 . The effects of the pre-treatment of leachates using the method of coagulation with FeCl3 depended on the concentration of organic compounds and with optimal conditions of the process ranged from 40 to 70% . Further pre-treatment of the leachates after coagulation, involving the use of oxidation with 03 and H2O2/O3, did not cause significant decrease of leachate toxicity . The data of this study demonstrated the usefulness of the battery of tests using Daphnia magna, Artemia franciscana, Scenedesmus quadricauda and Vibrio fischeri for the toxicity evaluation of raw and pre-treated leachates.

Mikrobiologiia, 2004 Jan-Feb, 73(1), 80 - 8
{Ectothiorhodosinus mongolicum gen . nov., sp . nov.,--a new purple sulfur bacterium from soda lake in Mongolia}; Gorlenko VM et al.; A new purple sulfur bacterium (strain M9) was isolated from the steppe soda Lake Dzun Uldziin Nur (pH 9.4; mineralization, 3.3%) situated in southeastern Mongolia . Individual cells appear as vibrios 0.3-0.5 x 0.7-1 micron in size . The dividing cells often do not separate from each other, forming an almost closed ring . The internal photosynthetic membranes are represented by concentric lamellae lining the cell wall . Photosynthetic pigments are bacteriochlorophyll a and carotenoids of the spirilloxanthin series . The main carotenoid (> 96%) is spirilloxanthin . Two typical light-harvesting complexes (LH1 and LH2) are present in the membranes in a 1:1 ratio . The bacterium is an anaerobe and facultative photoorganoheterotroph . Photolithoautotrophic growth on sulfide is scarce . Thiosulfate is utilized as an electron donor only in the presence of organic matter . Globules of elemental sulfur are formed as an intermediary product of sulfide and thiosulfate oxidation and are deposited outside the cells . The end product of oxidation is sulfate . In the presence of sulfide and carbonates, acetate, lactate, malate, pyruvate, propionate, succinate, and fumarate are used as the additional sources of carbon in anoxygenic photosynthesis . Vitamin are not required . The bacterium is an alkaliphile the pH optimum is at 8.3-9.1, the pH range is 7.6-10.1 . The optimum NaCl concentration in the medium is 1 to 7%; the range is 0.5 to 0.9% . The optimum carbonate content in the medium is 2%; the range is 1 to 10% . The best growth occurs at 30-35 degrees C . The DNA G + C content is 57.5 mol% . According to the results of analysis of the 16S rRNA gene sequences, the new isolate M9 belongs to the phylogenetic cluster containing representatives of the family Ectothiorhodospiraceae within the class "Gammaproteobacteria." In this class, the new isolate forms a new branch, which occupies an intermediate position between the representatives of the genera Ectothiorhodospira and Thiorhodospira . Based on the phenotypic and genetic characteristics, the new purple sulfurbacterium was assigned to a new species of a new genus of the family Ectothiorhodospiraceae, Ectothiorhodosinus mongolicum gen . nov., sp . nov.

Microbiology, 2004 Apr, 150(Pt 4), 911 - 20
Isolation of Vibrio alginolyticus sodium-driven flagellar motor complex composed of PomA and PomB solubilized by sucrose monocaprate; Yakushi T et al.; The polar flagella of Vibrio alginolyticus have sodium-driven motors, and four membrane proteins, PomA, PomB, MotX and MotY, are essential for torque generation of the motor . PomA and PomB are believed to form a sodium-conducting channel . This paper reports the purification of the motor complex by using sucrose monocaprate, a non-ionic detergent, to solubilize the complex . Plasmid pKJ301, which encodes intact PomA, and PomB tagged with a C-terminal hexahistidine that does not interfere with PomB function, was constructed . The membrane fraction of cells transformed with pKJ301 was solubilized with sucrose monocaprate, and the solubilized materials were applied to a Ni-NTA column . The imidazole eluate contained both PomA and PomB, which were further purified by anion-exchange chromatography . Gel-filtration chromatography was used to investigate the apparent molecular size of the complex; the PomA/PomB complex was eluted as approx . 900 kDa and PomB alone was eluted as approx . 260 kDa . These findings suggest that the motor complex may have a larger structure than previously assumed.

Commun Dis Intell, 2004, 28(1), 6 - 68
Australia's notifiable diseases status, 2002: Annual report of the National Notifiable Diseases Surveillance System; Yohannes K et al.; There were 57 infectious diseases notifiable at the national level in Australia in 2002 . States and territories reported 100,278 cases of infectious diseases to the National Notifiable Diseases Surveillance System (NNDSS), a fall of 4 per cent compared to the number of notifications in 2001 . In 2002, the most frequently notified diseases were, sexually transmitted infections (31,929 reports, 32% of total notifications), gastrointestinal infections (26,708 reports, 27% of total notifications) and bloodborne infections (23,741, 24%) . There were 11,711 (12% of total) cases of vaccine preventable diseases, 3,052 (3% of total) cases of vectorborne diseases, 1,155 (1% of total) cases of zoonotic infections, two cases of quarantinable diseases (Vibrio cholerae O1) and 1,980 cases of other bacterial diseases, notified to NNDSS . Compared to 2001, notifications of sexually transmitted infections increased by 16 per cent and gastrointestinal infections by 2 per cent while bloodborne infections fell by 18 per cent . The number of notifications of chlamydial infection and Q fever were the highest since 1991 and 1995 respectively . By contrast, the number of notification for hepatitis A and measles were the lowest since 1991 . For other notifiable diseases, the number of notifications was within the range of the five years between 1997 and 2002 (range = five-year mean plus or minus two standard deviations) . This report also includes 2002 summary data on communicable diseases from other surveillance systems including the Laboratory Virology and Serology Reporting Scheme and sentinel general practitioner schemes.

J Clin Microbiol, 2004 Apr, 42(4), 1657 - 65
Identification of a protein biomarker unique to the pandemic O3:K6 clone of Vibrio parahaemolyticus; Williams TL et al.; The present method of characterizing Vibrio parahaemolyticus strains involves serotyping or detection methods based on assessment of the presence or absence of genes thought to be markers of an organism's pathogenicity . It is unclear whether these assays detect all pathogenic V . parahaemolyticus strains since a clear correlation between the presence of a particular gene and the organism's pathogenicity has not yet been observed . We have described a proteomics-based method to distinguish individual V . parahaemolyticus strains on the basis of their protein profiles and identified a specific protein that is characteristic of the pandemic O3:K6 strain and its clonal derivatives . In the pandemic clone of V . parahaemolyticus, a histone-like DNA-binding protein, HU-alpha, has a C-terminal amino acid sequence different from those of other strains of V . parahaemolyticus . Upon further study, it was discovered that the gene encoding this protein has a 16-kbp insert at the 3' terminus of the open reading frame for this protein . By using the protein sequence of the unique biomarker for the pandemic clone of V . parahaemolyticus, it was possible to rationally design specific PCR-based probes and assays that permit the rapid and precise identification of pandemic strains of V . parahaemolyticus.

Anal Bioanal Chem, 1996 Mar, 354(5-6), 676 - 80
Assessing toxicity and mobilisation of impregnation salts at a contaminated site; Andersen S et al.; Severe soil contamination is often encountered at wood-impregnation plants due to spills, dripping and deposition of sludge associated with dissolved salts of copper, chromium and arsenic (CCA) . Soil samples from a CCA-plant in southern Norway were analysed via a factorial extraction design to investigate mobilisation of contaminated soils . Various concentrations of organic acids, sea-salts, and pH showed that contaminants were not stable, and could be mobilised to the aqueous phase . To further investigate mobilisation of impregnation salts, soil solution collectors were installed at various depths at the site . Concentrations varied considerably . Hydrological changes revealed elevated levels of dissolved salts, which agree with the factorial experiment . Soil chemical processes (not total solid-phase concentrations) dominated the mobilisation and subsequent leaching . Soil solutions were tested for changes in toxicity by chemical analysis and degree of inhibition of luminescence in Vibrio fisheri (Microtox) . Changes in toxicity corresponded to changes in soil solution.

J Biol Chem, 2004 Jun 11, 279(24), 25143 - 8 Epub 2004 Apr 05.
A cellular metalloproteinase activates Vibrio cholerae pro-cytolysin; Valeva A et al.; Many strains of Vibrio cholerae produce a cytolysin (VCC) that forms oligomeric transmembrane pores in animal cells . The molecule is secreted as a procytolysin (pro-VCC) of 79 kDa that must be cleaved at the N terminus to generate the active 65-kDa toxin . Processing can occur in solution, and previous studies have described the action of mature VCC thus generated . However, little is known about the properties of pro-VCC itself . In this study, it is shown that pro-VCC exist as a monomer in solution and binds as a monomer to eukaryotic cells . Bound pro-VCC can then be activated either by exogenous, extracellular, or by endogenous, cell-bound proteases . In both cases, cleavage generates the 65-kDa VCC that oligomerizes to form transmembrane pores . A wide variety of exogenous proteinases can mediate activation . In contrast, the activating cellular protease is selectively inhibited by the hydroxamate inhibitor TAPI, and thus probable candidates are members of the ADAM-metalloproteinase family . Furin, MMP-2, MMP-9, and serine proteinases were excluded . Cells over-expressing ADAM-17, also known as tumor necrosis factor alpha converting enzyme, displayed increased activation of VCC, and knockout cells lacking ADAM-17 had a markedly decreased capacity to cleave the protoxin . The possibility is raised that pro-VCC is targeted to membrane sites that selectively contain or are accessible to cellular ADAM-metalloproteinases . Although many microbial toxins are activated by furin, this is the first evidence for processing by a cellular metalloproteinase . We identified ADAM-17 as a potent activator of pro-VCC.

Appl Environ Microbiol, 2004 Apr, 70(4), 1964 - 72
Phenotypic and genotypic characteristics and epidemiological significance of ctx+ strains of Vibrio cholerae isolated from seafood in Malaysia; Chen CH et al.; Of 97 strains of Vibrio cholerae isolated from various seafoods in Malaysia in 1998 and 1999, 20 strains carried the ctx gene and produced cholera toxin . Fourteen, one, and five of these toxigenic strains belonged to the O139, O1 Ogawa, and rough serotypes, respectively . The rough strains had the rfb gene of the O1 serotype . The toxigenic strains varied in their biochemical characteristics, the amount of cholera toxin produced, their antibiograms, and the presence or absence of the pTLC plasmid sequence . DNA fingerprinting analysis by arbitrarily primed PCR, ribotyping, and a pulsed-field gel electrophoresis method classified the toxigenic strains into 3, 7, and 10 types, respectively . The relatedness of these toxigenic strains to clinical strains isolated in other countries and from international travelers was examined by using a dendrogram constructed from the pulsed-field gel electrophoresis profiles . The results of the examination of the antibiogram and the possession of the toxin-linked cryptic plasmid were consistent with the dendrogram-based relatedness: the O139 strains isolated from Malaysian seafoods could be separated into two groups that appear to have been introduced from the Bengal area independently . The rough strains of Malaysian seafood origin formed one group and belonged to a cluster unique to the Thailand-Malaysia-Laos region, and this group may have persisted in this area for a long period . The single O1 Ogawa strain detected in Malaysian seafood appears to have an origin and route of introduction different from those of the O139 and the rough strains.

Pathol Biol (Paris), 2004 Apr, 52(3), 127 - 30
{Microbial polysaccharides of marine origin and their potential in human therapeutics}; Colliec-Jouault S et al.; Bacterial polysaccharides offer fascinating potential applications for the pharmaceutical industry . Although many known marine bacteria produce exopolysaccharides (EPS), continuation in looking for new polysaccharide-producing micro-organisms is promising . Marine bacteria, isolated from deep-sea hydrothermal vents, have demonstrated their ability to produce in aerobic conditions, unusual EPS . With the aim of discovering biological activities, EPS presenting different structural features were studied . An EPS secreted by Vibrio diabolicus was evaluated on the restoration of bone integrity in experimental model and was demonstrated to be a strong bone-healing material . Another EPS produced by Alteromonas infernus was modified in order to obtain new heparin-like compounds . Unlike the native EPS, the resulting EPS presented anticoagulant properties as heparin . These EPS could provide biochemical entities with suitable functions for obtaining new drugs . They present original structural feature that can be modified to design compounds and improve their specificity.

FEBS Lett, 2004 Apr 9, 563(1-3), 207 - 12
The origin of the sodium-dependent NADH oxidation by the respiratory chain of Klebsiella pneumoniae; Bertsova YV et al.; Properties of Klebsiella pneumoniae respiratory chain enzymes catalyzing NADH oxidation have been studied . Using constructed K . pneumoniae mutant strains, it was shown that three enzymes belonging to different families of NADH:quinone oxidoreductases operate in this bacterium . The NDH-2-type enzyme is not coupled with energy conservation, the NDH-1-type enzyme is a primary proton pump, and the NQR-type enzyme is homologous to the sodium-motive NADH dehydrogenase of Vibrio and is shown to be a primary Na(+) pump . It is concluded that the NQR-type enzyme, not the NDH-1-type enzyme, catalyzes sodium-dependent NADH oxidation in K . pneumoniae.

J Biotechnol, 2004 Apr 8, 109(1-2), 123 - 30
Vibrio cholerae periplasmic superoxide dismutase: isolation of the gene and overexpression of the protein; Gabbianelli R et al.; Superoxide dismutases are ubiquitous enzymes which play an important role in protecting cells against oxidative damage and which have also been shown to contribute to the pathogenicity of many bacterial species . Here we demonstrate that Vibrio cholerae, the causative agent of cholerae, expresses an active periplasmic Cu,Zn superoxide dismutase . Moreover, we have set up an expression system yielding large amounts of V . cholerae recombinant Cu,Zn superoxide dismutase in the periplasm of Escherichia coli and a procedure to obtain the enzyme in a highly purified form . Unlike the bovine enzyme, V . cholerae Cu,Zn superoxide dismutase has been proved to be highly resistant to inactivation by hydrogen peroxide . This property, which appears to be common to other bacterial enzymes of this class, might improve the ability of Cu,Zn superoxide dismutase to protect bacteria against the reactive oxygen species produced by phagocytes.

FEMS Microbiol Lett, 2004 Apr 15, 233(2), 269 - 75
Resuscitation of viable but non-culturable Vibrio parahaemolyticus in a minimum salt medium; Wong HC et al.; Vibrio parahaemolyticus is food-borne pathogen prevalent in Asian countries . This work analyzes factors that influence the resuscitation of the viable but nonculturable (VBNC) state in this bacterium . The MMS-0.5% NaCl medium alone limited cell multiplication, and in this medium, resuscitation was successful when the temperature was upshifted to 25 degrees C but not 37 degrees C . Chloramphenicol inhibition experiments revealed that protein synthesis in the first 24 h of temperature upshift was critical in determining the success of the three-day resuscitation period . The VBNC state induction period and the age of the VBNC cells for successful resuscitation were strain-dependent . Results of this work facilitate further physiological and pathological study of the VBNC state in this pathogen.

Curr Microbiol, 2004 Feb, 48(2), 113 - 7
Predation pattern and phylogenetic analysis of Bdellovibrionaceae from the Great Salt Lake, Utah; Pineiro SA et al.; The Bdellovibrionaceae are predatory, intraperiplasmic bacteria that prey upon a variety of Gram-negative bacteria . The prey susceptibility pattern is frequently used to characterize new isolates . The objective in this study was to isolate and characterize predators from the Great Salt Lake (GSL) by prey susceptibility testing . To recover the predators, water samples were inoculated into an enrichment medium with Vibrio parahaemolyticus as prey . After several days of incubation, the predators were isolated, pure DNA was extracted, and partial 16S rDNA gene was sequenced . Water samples were also plated for isolation of heterotrophic bacteria . The susceptibility of bacterial isolates from the lake and other sources to each predator isolate was determined . The results revealed that there are predators in the GSL, and they preferentially prey on bacteria from the lake . This is the first report of the isolation of Bdellovibrionaceae from GSL and the predators showing preferences for bacteria from the same habitat.

J Biol Chem, 2004 Jun 18, 279(25), 26358 - 69 Epub 2004 Mar 31.
Characterization of polynucleotide kinase/phosphatase enzymes from Mycobacteriophages omega and Cjw1 and vibriophage KVP40; Zhu H et al.; Coliphage T4 Pnkp is a bifunctional polynucleotide 5'-kinase/3'-phosphatase that catalyzes the end-healing steps of a RNA repair pathway . Here we show that mycobacteriophages Omega and Cjw1 and vibriophage KVP40 also encode bifunctional Pnkp enzymes consisting of a proximal 5'-kinase module with an essential P-loop motif, GXGK(S/T), and a distal 3'-phosphatase module with an essential acyl-phosphatase motif, DX- DGT . Biochemical characterization of the viral Pnkp proteins reveals several shared features, including an alkaline pH optimum for the kinase component, an intrinsic RNA kinase activity, and a homotetrameric or homodimeric quaternary structure, that distinguish them from the monomeric DNA-specific phosphatase/kinase enzymes found in mammals and fission yeast . Whereas the phage 5'-kinases differ from each other in their preferences for phosphorylation of 5' overhangs, blunt ends, or recessed ends, none of them displays the preference for recessed ends reported for mammalian DNA kinase . We hypothesize that Pnkp provides phages that have it with a means to evade an RNA-damaging antiviral host response . Genetic complementation of the essential end-healing steps of yeast tRNA splicing by the Omega and Cjw1 Pnkp enzymes establishes their capacity to perform RNA repair reactions in vivo . A supportive correlation is that Omega and Cjw1, which are distinguished from other mycobacteriophages by their possession of a Pnkp enzyme, are also unique among the mycobacteriophages in their specification of putative RNA ligases.

Can J Microbiol, 2004 Feb, 50(2), 127 - 31
Role of cyanobacteria in the persistence of Vibrio cholerae O139 in saline microcosms; Islam MS et al.; Recently, a new strain of cholera, Vibrio cholerae O139, has emerged as an epidemic strain, but there is little information about its environmental reservoir . The present investigation was aimed to determine the role of cyanobacteria in the persistence of V . cholerae O139 in microcosms . An environmental isolate of V . cholerae O139 and three cyanobacteria (Anabaena sp., Nostoc sp., and Hapalosiphon sp.) were used in this study . Survival of culturable V . cholerae O139 in microcosms was monitored using taurocholate-tellurite gelatin agar medium . Viable but nonculturable V . cholerae O139 were detected using a fluorescent antibody technique . Vibrio cholerae O139 could be isolated for up to 12 days in a culturable form in association with cyanobacteria but could not be isolated in the culturable form after 2 days from control water without cyanobacteria . The viable but nonculturable V . cholerae O139 could be detected in association with cyanobacteria for up to 15 months . These results, therefore, suggest that cyanobacteria can act as a long-term reservoir of V . cholerae O139 in an aquatic environment.

Biotechnol Lett, 2004 Feb, 26(3), 197 - 201
Decoloration of a carpet dye effluent using Trametes versicolor; Ramsay JA et al.; Although a non-sterile, undiluted carpet dye effluent (containing two anthraquinone dyes) did not support growth of Trametes versicolor, the pre-grown fungus removed 95% of its color in shake-flasks after 10 h of incubation . After decoloration, the COD of the cell-free supernatant increased and the toxicity was unchanged as determined by the Microtox assay using Vibrio fischeri . Decoloration rates decreased when either glucose alone or Mn2+ and glucose were added . T . versicolor, immobilized on jute twine in a rotating biological contacting reactor, also decolorized four successive batches of the effluent . There was no decoloration in any of the uninoculated, non-sterile controls.

Cell Tissue Res, 2004 May, 316(2), 189 - 95 Epub 2004 Mar 25.
Acidophilic granulocytes of the marine fish gilthead seabream (Sparus aurata L.) produce interleukin-1beta following infection with Vibrio anguillarum; Chaves-Pozo E et al.; The fish immune response to Gram-negative bacteria is poorly understood . In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1beta (IL-1beta) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1beta . We found that gilthead seabream head-kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1beta intracellularly when challenged in vitro with V . anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1beta following infection . Importantly, the blood leukocytes from infected animals that accumulated proIL-1beta were shown to be the acidophilic granulocytes . A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V . anguillarum was also observed .

Analyst, 2004 Apr, 129(4), 309 - 14 Epub 2004 Mar 15.
Functional lipid microstructures immobilized on a gold electrode for voltammetric biosensing of cholera toxin; Cheng Q et al.; Redox functionalized microstructures of diacetylene lipids containing cell surface ligand GM1 have been prepared for the construction of an electrochemical biosensor for cholera toxin from Vibrio cholerae . Incorporation of lipid molecules with disulfide functionality into the microstructures allows for firm attachment of the microstructures on a gold surface to form a sensing interface . The observed morphology of the microstructures is platelet, with size around 240 nm as determined by dynamic light scattering and transmission electron microscopy . The electrochemical response stems from electron transfer between the electrode and the redox sites on the microstructures, and the Faradaic current is influenced by the binding events of protein toxins to the ligands displayed on the crystalline surface . Electrochemical characterization indicates that electron transfer of surface ferrocene on the gold electrode is facile . Differential pulse voltammetry was used to measure the current magnitude as a function of toxin concentration, and a working range expanding from 1.0 x 10(-8) to 5.0 x 10(-7) M was obtained . Bovine serum albumin (BSA) was used as a control agent with which no interference to Faradaic response was found in the same concentration range . Atomic force microscopy (AFM) was used to characterize the morphology and distribution of microstructures on the gold surface . The effectiveness of the design for bypassing surface fouling of proteins in electrochemical detection has been demonstrated, and a binding regulated electron hopping mechanism for the observed electrochemical behavior has been proposed.

Infect Immun, 2004 Apr, 72(4), 2405 - 7
Spatiotemporal analysis of acid adaptation-mediated Vibrio cholerae hyperinfectivity; Angelichio MJ et al.; Acid adaptation has previously been shown to increase the infectivity of Vibrio cholerae in the infant mouse model . To better understand this phenomenon, we monitored the spatial distribution and temporal changes in the ratios of acid-adapted cells to unadapted V . cholerae cells in the small intestine, as well as the timing of virulence factor expression . We found that the competitive advantage afforded by acid adaptation does not become manifest until greater than 3 h postinfection; thus, acid adaptation does not increase V . cholerae passage through the gastric acid barrier . Additionally, acid-adapted and unadapted V . cholerae cells colonize the same sections of the small intestine and show similar kinetics of transcriptional induction of the virulence genes tcpA and ctxA . These studies suggest that the increased infectivity of acid-adapted V . cholerae is due to a more rapid onset of multiplication and/or to an increased multiplication rate within the infant mouse intestine.

J Health Popul Nutr, 2003 Dec, 21(4), 325 - 31
Rice-ORS versus glucose-ORS in management of severe cholera due to Vibrio cholerae O139 Bengal: a randomized, controlled clinical trial; Hossain MS et al.; This study examined the comparative efficacies of rice-based oral rehydration solution (R-ORS) and glucose-based oral rehydration solution (G-ORS) in the management of severe cholera due to Vibrio cholerae O139 Bengal that causes epidemic cholera in many developing countries . Stool culture-proved adult male patients with severe cholera due to V . cholerae O139 Bengal were randomly assigned in a 1:1 ratio to receive either R-ORS or G-ORS after their initial rehydration with intravenous (i.v.) fluid and subsequently four hours of observation . They also received the usual hospital diet and tetracycline capsules (500 mg 6 hourly for three days) immediately after their enrollment in the study . The primary outcomes for observation were stool output during the first 24 hours after intervention and treatment failure as measured by the incidence of re-institution of i.v . fluid after initiation of trial therapy and duration of diarrhoea . Of 113 patients finally included in the study, 57 received R-ORS and 56 G-ORS . The admission characteristics of the two treatment groups were comparable . No significant differences in the first 24 hours of median (inter-quartile range) stool output {179 (67-206) g/kg in R-ORS group vs 193 (80-237) g/kg in G-ORS group; p = 0.52}, incidences of unscheduled i.v . fluid requirement {21% (12/57) in R-ORS group vs 25% (14/56) in G-ORS group; p = 0.78}, and median (inter-quartile range) duration of diarrhoea {32 (24-48) hours in R-ORS group vs 32 (24-56) hours in G-ORS group; p = 0.64} were observed . It is concluded that rice-based ORS is effective but not superior to standard glucose-based ORS in the management of adult males with severe cholera due to V . cholerae O139 Bengal.

Shokuhin Eiseigaku Zasshi, 2003 Dec, 44(6), 289 - 93
Evaluation of MPN method combined with PCR procedure for detection and enumeration of Vibrio parahaemolyticus in seafood; Miwa N et al.; Vibrio parahaemolyticus densities in spiked and naturally contaminated seafood samples were enumerated by the MPN method combined with a PCR procedure (MPN-PCR method) targeting the species-specific thermolabile hemolysin gene (tlh), and by the MPN method using subcultivation of alkaline-peptone-water (APW) enrichment culture on thiosulfate-citrate-bile-sucrose (TCBS) agar (MPN-TCBS method) . In the samples spiked with both V . parahaemolyticus and V . alginolyticus, the numbers of V . parahaemolyticus enumerated by the MPN-PCR method were similar to, or higher than the numbers of spiked cells, whereas those enumerated by the MPN-TCBS method were below the numbers of spiked cells . In naturally contaminated seafood samples, the numbers of V . parahaemolyticus enumerated by the MPN-PCR method were higher than those by the MPN-TCBS method . In the case of the MPN-TCBS method, isolation of V . parahaemolyticus from some APW cultures was difficult because of the overgrowth of many colonies other than V . parahaemolyticus (e.g., V . alginolyticus) on TCBS agar . In contrast, the PCR technique could detect tlh from APW culture without isolation of V . parahaemolyticus, so the possibility of failing to obtain a positive result in APW culture by the MPN-PCR method was considered to be lower than that by the MPN-TCBS method . Furthermore, utilization of the PCR technique reduces the time and labor required for the biochemical identification tests used in the MPN-TCBS method . For the detection and enumeration of V . parahaemolyticus in seafood, especially for samples that show many colonies other than V . parahaemolyticus on TCBS agar, the MPN-PCR method may be more convenient and reliable than the MPN-TCBS method.

Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 5018 - 23 Epub 2004 Mar 22.
Both chemotaxis and net motility greatly influence the infectivity of Vibrio cholerae; Butler SM et al.; The role of chemotaxis in the virulence of gastrointestinal pathogens is ill defined . Counterintuitively, nonchemotactic mutants of the polarly flagellated pathogen Vibrio cholerae greatly out-compete the wild-type strain during infection of the small intestine . We show that the out-competition phenotype is dependent on the direction of flagellar rotation and independent of Toxin Co-regulated Pilus function . Specifically, the out-competition associated with the loss of chemotaxis required the presence of counterclockwise-biased flagellar rotation and smooth straight runs by the bacteria . In contrast, a nonchemotactic strain with clockwise-biased flagellar rotation was confined to small-scale net movement and was attenuated for infection . The significance of the out-competition phenotype was examined and was shown to correlate with a true increase in infectivity . Counterclockwise-biased mutants are aberrantly distributed throughout the infant mouse small intestine and we find that the expression of virulence factors occurs normally in all segments . Thus, alteration of the chemotactic properties of V . cholerae allows it to exploit additional niches in the host intestine.

Biochemistry, 2004 Mar 30, 43(12), 3620 - 7
Local conformational changes in the Vibrio Na+/galactose cotransporter; Veenstra M et al.; Na(+) and sugar transport by cotransporters (symporters) is thought to occur as a series of ordered ligand-induced conformational changes . To localize these conformational changes in a bacterial Na(+)/galactose cotransporter, we have employed a combination of cysteine-scanning and fluorescence techniques . Single or pairs of cysteine residues were introduced into the external face of a cysteine-less Vibrio parahaemolyticus sodium/glucose cotransporter for expression in Escherichia coli, and each transporter was purified using affinity chromatography . All the mutant proteins retained transport activity in bacteria and proteoliposomes . Each mutant was exposed to two different fluorescence reagents, ThioGlo3 or pyrene maleimide, that are essentially nonfluorescent until they react with a thiol . Fluorescence was recorded as a function of time and ligand concentrations . The reagents specifically labeled six of the seven cysteine mutants, but only in Cysteine 423 was the fluorescence affected by ligands . The rate of labeling of Cys423 by ThioGlo3 or pyrene maleimide was reduced by D-galactose in Na(+) buffer . Furthermore, the fluorescence of Thioglo3-labeled Cys423 was quenched by D-galactose, but only in the presence of Na(+) . This quench was not accompanied by a Stokes shift and was not produced by nontransported sugars, e.g., L-glucose . Reducing the sodium concentration from 200 to 10 mM decreased the apparent affinity for d-galactose without altering the maximum quench with saturating D-galactose . Reducing the galactose concentration from 20 to 0.5 mM reduced both the apparent affinity for Na(+) and the maximum quench at saturating Na(+) . These results suggest an ordered reaction scheme with Na(+) binding first . The fluorescence results with ThioGlo3-labeled Cys423 indicate that conformational changes underlying Na(+)/galactose cotransport occur at or near the extracellular domain between transmembrane helices 10 and 11.

J Vet Med Sci, 2004 Feb, 66(2), 205 - 8
Binding of Vibrio anguillarum to neutral glycosphingolipids from intestinal mucosa of rainbow trout (Oncorhynchus mykiss); Irie T et al.; To test whether glycosphingolipids (GSLs) on the intestinal mucosa of rainbow trout (Oncorhynchus mykiss) serve as a binding receptor for Vibrio anguillarum, we analyzed neutral GSLs from rainbow trout intestinal mucosa and investigated the binding of bacteria to neutral GSLs . Two kinds of neutral GSLs, designated N-1 and N-2, were identified on high-performance thin-layer chromatography (TLC) plates . In TLC immunostaining tests, V . anguillarum bound only to galactosylceramide (GalCer), lactosylceramide and N-1 having the same TLC mobility as GalCer, but neither to glucosylceramide nor to N-2 . These results suggest that N-1 is GalCer (Gal beta 1-1Cer) and also that N-1 (GalCer) on rainbow trout intestinal mucosa act as a receptor for V . anguillarum.

Indian J Pathol Microbiol, 2003 Jan, 46(1), 142 - 4
Incidence of Vibrio cholerae serogroup O139 infection with low virulence in Hubli, Karnataka (India); Krishna BV et al.; 384 stool samples from patients with acute gastroenteritis were processed by standard culture techniques and antibiogram of V . cholerae was performed . Stool samples from 93 (24.22%) patients yielded V . cholerae, 58 (62.37%) of which were V . cholerae, El Tor O1 Ogawa, 31 (33.33%) V . cholerae O139 and 4 (4.30%) V . cholerae non O1 non O139 . Of the culture proven cholera cases watery diarrhoea was observed in 79 (84.95%), vomiting in 57(61.29%), muscle cramps in 21 (22.58%) and sweating in 18 (19.35%) . Majority of these patients presented with moderate dehydration 57 (61.29%) . Mild dehydration was found in 19 (20.43%) and severe dehydration in 17 (18.28%) . While majority of patients with O139 infection had mild to moderate dehydration 25 (80.65%), severe dehydration was more common with O1 infection 11 (64.71%) . This study reflects the importance of monitoring the V . cholerae by serogrouping, antibiogram typing, which keep on varying constantly.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Jan-Feb, (1), 29 - 34
{Variable nucleotide tandem repeats (VNTR analysis) in Vibrio cholerae 0139 isolated from humans and from water of surface reservoirs in Russia}; Mishan'kin BN et al.; The comparative study of variable tandem repeats (VNTR analysis) in genomes of V . cholerae 0139 isolated from humans and from water samples taken from surface reservoirs was carried out . The results of the study of the allele state of 5 loci of tandem repeats in 50 strains of vibrios, carried out in the double-primer polymerase chain reaction (PCR), as well as the earlier comparison of the same isolates in the single-primer PCR, showed essential differences and the absence of clonality in the cultures of the clinical and aqueous origin . The suggestion was made that vibrios with individual VNTR genotypes and having no genes ctx and tcpA, isolated from water samples, were epidemic unimportant representatives of the autochthonous microflora of water reservoirs.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Jan-Feb, (1), 23 - 9
{The genome polymorphism of Vibrio cholerae ctxAB(-) strains, containing the proximal part of the CTX element}; Monakhova EV et al.; The comparative analysis of the hybridization patterns of DNA restricts for 20 V . cholerae, groups 01 and non-01 (non-0139), containing the incomplete CTX element (ctxAB-) was carried out with the use of probes, complementary to the genes of the proximal part of the virulence cassettle and flanking its RS1 sequences . This group was found to be heterogeneous both in the number of copies of "truncated" CTX prophage and their localizations in the genome, as well as in the position of the sites of restriction endonucleases HindlII and BglII . Among 17 clinically noncholerigenic isolates, 5 etiologically dangerous clones were found, each of them characterized by the definite time and place of isolation . At least one of them proved to be the causative agent of the local outbreak of diarrheal diseases in Uzbekistan.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Jan-Feb, (1), 13 - 8
{Experimental non-culturable Vibrio cholerae eltor and its biological properties}; Tafel'shtein EE et al.; In experiments with the cultivation of V . cholerae eltor under the conditions of high salt concentration, as well as low temperature and deficiency in nutrient substances, uncultivable forms (UF) of toxigenic and nontoxigenic vibrios were obtained . The absence of growth of seeded vibrios after the filtration of samples (with a filter of 0.22 micron), the preservation of specific antigenic determinants and the initial set of genes, changes in the morphology of cells (small size, coccoid form with the flagella retained) confirm the transition of V . cholerae eltor under study into the uncultivable state which, under unfavorable conditions, more rapidly develops in toxigenic vibrios than in nontoxigenic ones . The analysis of the INT-reductase activity of UF disintegrates revealed that they had endogenic respiration whose activity increased (4.5- to 6.5-fold) in the presence of the exogenic intermediates of the Krebs cycle . The uncultivable forms of the vibrios retain genes responsible for pathogenicity, as well as their antigenic determinants.

Biochemistry, 2004 Mar 23, 43(11), 3183 - 94
Changes in the kinetics and emission spectrum on mutation of the chromophore-binding platform in Vibrio harveyi luciferase; Lin LY et al.; The recently proposed model for the bacteria luciferase-flavin mononucleotide complex identifies a number of critical intermolecular interactions that define a binding platform for the isoalloxazine ring of flavin {Lin, L . Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E . O., and Meighen, E . A . (2001) Protein Sci . 10, 1563-1571} . A key interaction involving van der Waals contact between the isopropyl side chain of alphaVal173 and the 7,8-dimethyl benzene plane of the isoalloxazine chromophore represents an important target to test the validity of the proposed model . Here, structure-function analysis of luciferase variants carrying single point mutations at position alpha173 have verified the functional layout of the active site architecture and implicated this site directly in flavin binding . Moreover, a decrease in the stability of the enzyme-bound C4a-hydroperoxyflavin intermediate in the mutants could account for changes in saturation with the fatty aldehyde substrate . A predicted red-shift on mutation of position alpha173 to increase its polarity confirmed that alphaVal173 was an integral component of the chromophore-binding microenvironment . Introduction of mutations in residues that contact the pyrimidine plane of the isoalloxazine chromophore (alphaA75G/C106V) into the alphaV173A, alphaV173C, alphaV173T, and alphaV173S mutants led to the retention of high levels of enzyme activity (10-40% of wild type) and further red-shifted the emission spectra in the triple mutants . The additivity of the mutation-induced red-shifts in the emission wavelength spectrum provides the basis toward engineering luciferase variants that emit different light colors with the proposed flavin-luciferase model complex as a design reference.

Indian J Pathol Microbiol, 2003 Apr, 46(2), 259 - 60
Role of cell surface antigens of Vibrio cholerae 01 and non 01 serovars in intestinal adhesion; Vijayashree S et al.; Decrease in adherence of Vibrio cholerae to rabbit small intestine was observed following treatment with antisera against outer membrane (OM), lipopolysaccharide (LPS) and flagella . Anti LPS antibodies were more efficient than the other two antibodies in inducing adherence inhibition and promoting in vivo protection.

Klin Lab Diagn, 2004 Jan, (1), 48 - 50
{Analysis of virulent and avirulent strains of Vibrio cholerae by the nest polymerase chain reaction}; Glukhov AI et al.; The detection of cholera enterotoxin in environmental objects and isolation of patients are considered to be the most reliable indices of that the cholera agent is present . Described in the case study is a method of nested polymerase chain reaction (PCR) based on amplifying the ctxA gene fragment coding subunit A of enterotoxin . A possibility was shown to use the above method to confirm the virulence of strains Vibrio cholerae isolated from different sources . The method was tested with 18 virulent and avirulent strains V . cholerae as well as (for the sake of verifying the analysis specificity) with DNAs of other human-pathogenic microorganisms and with the human genome DNA . The results showed a high efficiency of nested PCR in detecting the pathogenicity of cholera-agent strains.

FEMS Microbiol Lett, 2004 Mar 12, 232(1), 23 - 30
Molecular characterisation of rough strains of Vibrio cholerae isolated from diarrhoeal cases in India and their comparison to smooth strains; De K et al.; Sixteen of the 18 Vibrio cholerae rough strains isolated from hospitalised diarrhoea patients were found to contain O1 serotype-specific (wbe) genes and all currently known virulence genes . Expression of the regulatory element ToxR was evident in these strains . Cholera toxin production ability of the rough strains was found to be higher (c . three- to five-fold) as compared to the smooth counterparts and this was transcriptionally regulated . Strains exhibiting the rough phenotype were more amenable to the uptake of CTXphi, which led us to consider that the rough phenotype could play a role in the generation of genetic diversity among V . cholerae strains.

Annu Rev Phytopathol, 1998, 36, 207 - 25
Homoserine lactone-mediated gene regulation in plant-associated bacteria; Pierson LS 3rd et al.; Many plant-associated bacteria produce and utilize diffusible N-acyl-homoserine lactones (AHLs) to regulate the expression of specific bacterial genes and operons . AHL-mediated regulation utilizes two genes that encode proteins similar to the LuxI/LuxR system originally studied in the marine symbiont Vibrio fischeri . The LuxI-type proteins are AHL synthases that assemble the diffusible AHL signal . The LuxR-type proteins are AHL-responsive transcriptional regulatory proteins . LuxR proteins control the transcription of specific bacterial genes in response to the levels of AHL signal . To date, AHL-mediated gene regulation has been identified in a broad range of gram-negative bacteria, most of which are host-associated . However, it seems unlikely that such a widely conserved regulatory mechanism would be limited only to host-microbe interactions . These signals probably play central roles in ecological interactions among organisms in microbial communities by affecting communication among bacterial populations as well as between bacterial populations and their eukaryotic hosts.

J Biol Chem, 2004 May 14, 279(20), 21349 - 55 Epub 2004 Mar 09.
NADH oxidation by the Na+-translocating NADH:quinone oxidoreductase from Vibrio cholerae: functional role of the NqrF subunit; Turk K et al.; The Na(+)-translocating NADH:quinone oxidoreductase from Vibrio cholerae is a six subunit enzyme containing four flavins and a single motif for the binding of a Fe-S cluster on its NqrF subunit . This study reports the production of a soluble variant of NqrF (NqrF') and its individual flavin and Fe-S-carrying domains using V . cholerae or Escherichia coli as expression hosts . NqrF' and the flavin domain each contain 1 mol of FAD/mol of enzyme and exhibit high NADH oxidation activity (20,000 micromol min(-1) mg(-1)) . EPR, visible absorption, and circular dichroism spectroscopy indicate that the Fe-S cluster in NqrF' and its Fe-S domain is related to 2Fe ferredoxins of the vertebrate-type . The addition of NADH to NqrF' results in the formation of a neutral flavosemiquinone and a partial reduction of the Fe-S cluster . The NqrF subunit harbors the active site of NADH oxidation and acts as a converter between the hydride donor NADH and subsequent one-electron reaction steps in the Na(+)-translocating NADH:quinone oxidoreductase complex . The observed electron transfer NADH --> FAD --> {2Fe-2S} in NqrF requires positioning of the FAD and the Fe-S cluster in close proximity in accordance with a structural model of the subunit.

Cell Microbiol, 2004 Apr, 6(4), 391 - 400
Effect of Vibrio parahaemolyticus haemolysin on human erythrocytes; Lang PA et al.; Haemolysin Kanagawa, a toxin from Vibrio parahaemolyticus, is known to trigger haemolysis . Flux studies indicated that haemolysin forms a cation channel . In the present study, channel properties were elucidated by patch clamp and functional significance of ion fluxes by fluorescence-activated cell sorting (FACS) analysis . Treatment of human erythrocytes with 1 U ml-1 haemolysin within minutes induces a non-selective cation permeability . Moreover, haemolysin activates clotrimazole-sensitive K+ channels, pointing to stimulation of Ca2+-sensitive Gardos channels . Haemolysin (1 U ml-1) leads within 5 min to slight cell shrinkage, which is reversed in Ca2+-free saline . Erythrocytes treated with haemolysin (0.1 U ml-1) do not undergo significant haemolysis within the first 60 min . Replacement of extracellular Na+ with NMDG+ leads to slight cell shrinkage, which is potentiated by 0.1 U ml-1 haemolysin . According to annexin binding, treatment of erythrocytes with 0.1 U ml-1 haemolysin leads within 30 min to breakdown of phosphatidylserine asymmetry of the cell membrane, a typical feature of erythrocyte apoptosis . The annexin binding is significantly blunted at increased extracellular K+ concentrations and by K+ channel blocker clotrimazole . In conclusion, haemolysin Kanagawa induces cation permeability and activates endogenous Gardos K+ channels . Consequences include breakdown of phosphatidylserine asymmetry, which depends at least partially on cellular loss of K+.

Environ Microbiol, 2004 Apr, 6(4), 364 - 76
Influence of aquatic microbiota on the survival in water of the human and eel pathogen Vibrio vulnificus serovar E; Marco-Noales E et al.; The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years . The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples . A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition . The bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors . Competitors included V . vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V . vulnificus isolation from water samples . Evidences of bacterial competition that was detrimental for VSE recovery were recorded . Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input . These results suggest that an overgrowth of more efficient competitor bacteria in conventional media used for isolation of V . vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.

Appl Environ Microbiol, 2004 Mar, 70(3), 1833 - 5
Inactivation of Vibrio parahaemolyticus in effluent seawater by alternating-current treatment; Park JC et al.; Vibrio parahaemolyticus, the cause of gastroenteritis in humans, was inactivated by alternating low-amperage electricity . In this study, the application of alternating low-amperage electric treatment to effluent seawater was investigated for the large-scale disinfection of seawater . This method was able to overcome the problem of chlorine generation that results from treatment with continuous direct current . In conclusion, our results showed that alternating-current treatment inactivates V . parahaemolyticus in effluent seawater while minimizing the generation of chlorine and that this alternating-current treatment is therefore suitable for practical industrial applications.

Appl Environ Microbiol, 2004 Mar, 70(3), 1434 - 41
Symbiont-induced changes in host actin during the onset of a beneficial animal-bacterial association; Kimbell JR et al.; The influence of bacteria on the cytoskeleton of animal cells has been studied extensively only in pathogenic associations . We characterized changes in host cytoskeletal actin induced by the bacterial partner during the onset of a cooperative animal-bacteria association using the squid-vibrio model . Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that Vibrio fischeri induced a dramatic increase in actin protein abundance in the bacteria-associated host tissues during the onset of the symbiosis . Immunocytochemistry revealed that this change in actin abundance correlated with a two- to threefold increase in actin in the apical cell surface of the epithelium-lined ducts, the route of entry of symbionts into host tissues . Real-time reverse transcriptase PCR and in situ hybridization did not detect corresponding changes in actin mRNA . Temporally correlated with the bacteria-induced changes in actin levels was a two- to threefold decrease in duct circumference, a 20% loss in the average number of cells interfacing with the duct lumina, and dramatic changes in duct cell shape . When considered with previous studies of the biomechanical and biochemical characteristics of the duct, these findings suggest that the bacterial symbionts, upon colonizing the host organ, induce modifications that physically and chemically limit the opportunity for subsequent colonizers to pass through the ducts . Continued study of the squid-vibrio system will allow further comparisons of the mechanisms by which pathogenic and cooperative bacteria influence cytoskeleton dynamics in host cells.

J Clin Microbiol, 2004 Mar, 42(3), 1280 - 2
Assessment of evolution of pandemic Vibrio parahaemolyticus by multilocus sequence typing; Chowdhury NR et al.; The genetic relatedness of 81 isolates of Vibrio parahaemolyticus was assessed by multilocus sequence typing . The strain with serotype O3:K6 emerged as a pandemic pathogen in 1996, with subsequent expansion to include strains having serotypes O1:KUT, O4:K68, and O1:K25 . Sequence data from gyrB, recA, dnaE, and gnd revealed that 16 distinct serogroups isolated prior to the pandemic were highly variable and only isolates of serogroup O3:K6 shared two alleles with the pandemic strains . The pandemic strains regardless of serotype were clonal, with 51 of 54 isolates having the identical allelic profile (AP) . Serotype alone did not adequately define a pandemic strain: among O1:KUT strains tested, seven strains with the identical pandemic AP carried previously described pandemic markers, while five nonpandemic strains had five distinct APs . Our sequence data provide strong molecular support for the clonal origin of pandemic V . parahaemolyticus O3:K6 and suggest that strains within such a clonal group may acquire previously identified serotypes.

J Biochem (Tokyo), 2004 Jan, 135(1), 43 - 51
Multimeric structure of the PomA/PomB channel complex in the Na+-driven flagellar motor of Vibrio alginolyticus; Yorimitsu T et al.; It is known that PomA and PomB form a complex that functions as a Na(+) channel and generates the torque of the Na(+)-driven flagellar motor of Vibrio alginolyticus . It has been suggested that PomA works as a dimer and that the PomA/PomB complex is composed of four PomA and two PomB molecules . PomA does not have any Cys residues and PomB has three Cys residues . Therefore, a mutant PomB (PomB(cl)) whose three Cys residues were replaced by Ala was constructed and found to be motile as well . We carried out gel filtration analysis and examined the effect of cross-linking between the Cys residues of PomB on the formation of the PomA/PomB complex . In the presence of dithiothreitol (DTT), the elution profile of the PomA/PomB complex was shifted to a lower apparent molecular mass fraction similar to that of the complex of the wild-type PomA and PomB(cl) mutant . Next, to analyze the arrangement of PomA molecules in the complex, we introduced the mutation P172C, which has been shown to cross-link PomA molecules, into tandem PomA dimers (PomA approximately PomA) . These mutant dimers showed a dominant-negative effect . DTT could restore the function of PomA approximately P172C and P172C approximately P172C, but not P172C approximately PomA . Interdimer and intradimer cross-linked products were observed; the interdimer cross-linked products could be assembled with PomB . The formation of the interdimer cross-link suggests that the channel complex of the Na(+)-driven flagellar motor is composed of two units of a complex consisting of two PomA and one PomB, and that they might interact with each other via not only PomA but also PomB.

Exp Parasitol, 2003 Nov-Dec, 105(3-4), 232 - 40
The protistan parasite Perkinsus marinus is resistant to selected reactive oxygen species; Schott EJ et al.; The parasite Perkinsus marinus has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of North America . When viable P . marinus trophozoites are engulfed by oyster hemocytes, the typical accumulation of reactive oxygen species (ROS) normally associated with phagocyte activity is not observed . One hypothesis to explain this is that the parasite rapidly removes ROS . A manifestation of efficient ROS removal should be a high level of resistance to exogenous ROS . We investigated the in vitro susceptibility of P . marinus to ROS as compared to the estuarine bacterium Vibrio splendidus . We find that P . marinus is markedly less susceptible than V . splendidus to superoxide and hydrogen peroxide (H(2)O(2)), but equally sensitive to hypochlorite . Viable P . marinus trophozoites degrade H(2)O(2) in vitro, but lack detectable catalase activity . However, extracts contain an ascorbate dependent peroxidase activity that may contribute to H(2)O(2) removal in vitro and in vivo.

Bioorg Chem, 2004 Apr, 32(2), 63 - 75
Kinetic mechanism of asparagine synthetase from Vibrio cholerae; Fresquet V et al.; Asparagine synthetase B (AsnB) catalyzes the formation of asparagine in an ATP-dependent reaction using glutamine or ammonia as a nitrogen source . To obtain a better understanding of the catalytic mechanism of this enzyme, we report the cloning, expression, and kinetic analysis of the glutamine- and ammonia-dependent activities of AsnB from Vibrio cholerae . Initial velocity, product inhibition, and dead-end inhibition studies were utilized in the construction of a model for the kinetic mechanism of the ammonia- and glutamine-dependent activities . The reaction sequence begins with the ordered addition of ATP and aspartate . Pyrophosphate is released, followed by the addition of ammonia and the release of asparagine and AMP . Glutamine is simultaneously hydrolyzed at a second site and the ammonia intermediate diffuses through an interdomain protein tunnel from the site of production to the site of utilization . The data were also consistent with the dead-end binding of asparagine to the glutamine binding site and PP(i) with free enzyme . The rate of hydrolysis of glutamine is largely independent of the activation of aspartate and thus the reaction rates at the two active sites are essentially uncoupled from one another.

Zhonghua Yu Fang Yi Xue Za Zhi, 2004 Jan, 38(1), 47 - 9
{Study on biology and epidemiological characteristics of vibrio cholerae non-O1 strains isolated from environmental waters in Foshan city}; Chen AZ et al.; OBJECTIVE: To study the distribution of serotype and the positive rate of toxins among vibrio cholerae non-O(1) isolated from environmental waters in Foshan city . METHODS: Water specimens were collected from river and cultured for vibrio cholerae non-O(1) . The PCR method was used to detect cholerae enterotoxin (CT) gene; the ELISA method was used to detect heat-stable toxin (ST) and heat-labile toxin (LT) . RESULTS: 478 vibrio cholerae non-O(1) strains were isolated from 1 644 water specimens, with a positive rate of 29.07% . Serological assay showed that the main serotype of vibrio cholerae non-O(1) in Foshan city is VBO(7) . Positive rate of CT, ST and LT were 1.91%, 13.14% and 12.17%, respectively . CONCLUSIONS: A few non-O(1) strains were found to have several virulent factors simultaneously, and the results suggest that vibrio cholerae non-O(1) in environmental waters is potentially pathogenic and may affect people's health . It is necessary to pay attention to the prevention of diarrhoea caused by vibrio cholerae.

Sci Total Environ, 2004 Mar 5, 320(1), 37 - 50
Toxicity of the 13 priority pollutant metals to Vibrio fisheri in the Microtox chronic toxicity test; Hsieh CY et al.; The Microtox Acute Toxicity Test has been successfully used to measure the toxicity of metals and other pollutants at high concentrations (ppm) in selected environmental samples . However, metals and other toxicants are often found in much lower concentrations (ppb) in many municipal wastewaters and receiving waters . In order to assess the toxicity of these pollutants in these samples, a more sensitive toxicity assay is needed . The Microtox chronic toxicity test has been developed to measure the sublethal effect of toxicants over multiple generations of the test species, Vibrio fisheri . In this study, the toxicity of the 13 priority pollutant metals {i.e . As, Se, Cd, Cr (III and VI), Cu, Pb, Sb, Ag, Tl, Zn, Be, Hg and Ni} to V . fisheri was evaluated using the Microtox chronic toxicity test . In this test, the inhibitory concentration (IC), lowest observable effect concentration (LOEC), and no observable effect concentration (NOEC) were obtained after 22-h of incubation at 27+/-1 degrees C, by comparing the light output of the control to that of the test sample . Among the 13 priority pollutant metals, beryllium (Be) was found to be the most toxic in the test (LOEC=0.742-1.49 microg/l) while thallium (Tl) was the least toxic (LOEC=3840-15300 microg/l) . The LOECs for copper (as Cu) and lead (Pb) in reagent (ASTM Type I) water were 6.78-13.6 microg/l and 626-1251 microg/l, respectively . The toxicity of copper sulfate (as Cu) in reagent water was shown and significantly reduced with the addition of natural organic matter (fulvic acid) or EDTA to the sample . The LOEC values for the 13 priority pollutant metals in this test were comparable to or lower than those reported for commonly used aquatic toxicity tests, such as the Ceriodaphnia dubia assay.

Eur J Med Chem, 2004 Feb, 39(2), 195 - 203
Synthesis, anti-mycobacterial, anti-trichomonas and anti-candida in vitro activities of 2-substituted-6,7-difluoro-3-methylquinoxaline 1,4-dioxides; Carta A et al.; A new series of 23 6,7-difluoro-3-methyl-2-phenylthio/phenylsulfonyl/phenylsulfinyl/benzylamino/phenylamino-quinoxaline 1,4-dioxides variously substituted in the phenyl moiety, was synthesized and submitted to in vitro evaluation for anti-mycobacterial, anti-trichomonas, anti-candida, anti-mycoplasma and antibacterial activities . In anti-mycobacterial assays, several compounds resulted active (MIC90 = 2.0-4.0 microg/ml) against Mycobacterium tuberculosis H37Rv . Anti-trichomonas screening showed a generally good activity of all compounds (MBC = 0.39-25.0 microg/ml) versus Trichomonas vaginalis, in particular the derivatives 5a,d, 7a, 9 and 11c ranged 0.39-0.78 microg/ml (metronidazole MBC = 12.5 microg/ml) . Results of anti-candida assays showed that derivatives 7a, 8a,d and 9 were active against several species of Candida (C . albicans, C . krusei, C . parapsilosis and C . glabrata), having MIC50 between 3.9 and 31.25 microg/ml . The latter compounds were also submitted to anti-mycoplasma assay against Mycoplasma hominis, the results obtained showed that 7a, 8a,d and 9 inhibited the growth of the mycoplasma at the concentration of 0.1 mg/ml . In antibacterial tests only a few compounds showed an MIC50 lower than 62.5 microg/ml against representative strains of Gram-positive and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, Vibrio alginolyticus and Pseudomonas aeruginosa).

Int J Food Microbiol, 2004 Mar 15, 91(3), 319 - 25
Occurrence of the tdh and trh genes in Vibrio parahaemolyticus isolates from waters and raw shellfish collected in two French coastal areas and from seafood imported into France; Robert-Pillot A et al.; The occurrence of the hemolysin genes, tdh and trh, in Vibrio parahaemolyticus strains isolated from environmental samples collected in two French coastal areas, clinical samples, and seafood products imported into France was studied . Polymerase chain reaction (PCR) with two sets of primers was used to detect the hemolysin genes . Most of the clinical isolates (91%) and 1.5% of the isolates from seafood possessed the hemolysin genes . Three and fifteen percent, respectively, of the two groups of environmental strains carried the hemolysin genes depending on the geographic site . The tdh and trh genes play important roles in virulence . Thus, our results indicate that pathogenic V . parahaemolyticus isolates are present in French coastal areas and in seafood imported into France . Furthermore, they may also be present in French seafood products.

Proc Natl Acad Sci U S A, 2004 Feb 24, 101(8), 2524 - 9
The Vibrio cholerae chitin utilization program; Meibom KL et al.; Chitin, an insoluble polymer of GlcNAc, is an abundant source of carbon, nitrogen, and energy for marine microorganisms . Microarray expression profiling and mutational studies of Vibrio cholerae growing on a natural chitin surface, or with the soluble chitin oligosaccharides (GlcNAc)(2-6), GlcNAc, or the glucosamine dimer (GlcN)2 identified three sets of differentially regulated genes . We show that (i) ChiS, a sensor histidine kinase, regulates expression of the (GlcNAc)(2-6) gene set, including a (GlcNAc)2 catabolic operon, two extracellular chitinases, a chitoporin, and a PilA-containing type IV pilus, designated ChiRP (chitin-regulated pilus) that confers a significant growth advantage to V . cholerae on a chitin surface; (ii) GlcNAc causes the coordinate expression of genes involved with chitin chemotaxis and adherence and with the transport and assimilation of GlcNAc; (iii) (GlcN)2 induces genes required for the transport and catabolism of nonacetylated chitin residues; and (iv) the constitutively expressed MSHA pilus facilitates adhesion to the chitin surface independent of surface chemistry . Collectively, these results provide a global portrait of a complex, multistage V . cholerae program for the efficient utilization of chitin.

Environ Toxicol Chem, 2004 Feb, 23(2), 271 - 82
Sediment quality in the Atlantic coast of Spain; Riba I et al.; Sediments from the Atlantic coast of Spain have been studied to evaluate environmental quality by using an integrated approach including chemical and toxicological data . Sediment samples were collected in four littoral ecosystems located in Spain, Bay of Cadiz, Guadalquivir River estuary, Ria of Huelva, and Ria of Coruna . To characterize the sediments, organic carbon, granulometric content, total sulfide, eight trace metals (Hg, Cd, Pb, Cu, Zn, As, Ni, and Cr), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) were measured . The toxicity of sediments was assessed with the amphipod Ampelisca brevicornis, the clam Ruditapes philippinarum, juveniles of the fish Solea senegalensis, populations of the estuarine rotifer Brachionus plicatilis, and populations of the bacterium Vibrio fischeri (Microtox) . The results obtained show that in general, stations located in the Ria of Huelva were associated with heavy metal contamination and with the highest toxicity . Only chronic toxicity tests were capable of identifying the effects associated with PCB concentrations . The sediment quality guidelines calculated by means of a multivariate analysis approach for contaminants not associated with biological effects (mg/kg) are Hg, 0.54; Cd, 0.51; Pb, 260; Cu, 209; Zn, 513; As, 27.4; and total PCBs, 0.05.

South Med J, 2004 Feb, 97(2), 205 - 7
A "fishy remedy": an unusual transmission of Vibrio vulnificus infection; Tal S et al.; This case report describes a unique transmission of Vibrio vulnificus infection . A 38-year-old woman with recurrent cellulitis and chronic ulcer on her leg developed necrotizing cellulitis and sepsis caused by V . vulnificus . Meticulous history investigation revealed the link to contaminated fish blood that had been applied on the ulcer by a traditional healer . Through this case, it may be stressed that a traditional remedy can sometimes be harmful and life-threatening.

Biosci Biotechnol Biochem, 2004 Feb, 68(2), 277 - 85
Cloning and sequencing of the ura3 and ura5 genes, and isolation and characterization of uracil auxotrophs of the fungus Mortierella alpina 1S-4; Takeno S et al.; The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain . In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact . The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced . The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote . Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.

Water Sci Technol, 2004, 49(1), 39 - 46
The relationship bettween composition and toxicity of tannery wastewater; Cotman M et al.; Key toxic components have been identified in pre-treated tannery wastewater with fractionation of samples through chemical and physical means (filtration, air stripping, adsorption on activated carbon,...) . The goal of each fractionation step was to reduce the toxicity due to a specific group of chemicals and compare the results to the toxicity present in the unaltered sample . Toxicity short-term tests with the invertebrate Daphnia magna and thebacterial luminescence inhibition test with Vibrio fischeri were used in combination with chemical analyses . During the toxicity identification and evaluation fractionation, a portion of the sample was pressure filtered . Treated samples contained less organic pollution and metals and were less toxic especially to Daphnia magna . For the removal of ammonia the second portion of sample was air-stripped at different pH levels . We removed 84% of ammonia at pH 11; the toxicity to both organisms decreased but ammonia did not have a deciding effect on the toxicity of tannery wastewater when the organic load was still present . The most successful procedure for toxicity removal was adsorption on powdered activated carbon . We removed organic pollution detected as COD, organic nitrogen compounds and part of the metals . Zeolite treatment was a little less successful for removing ammonia than air-stripping.

Infect Immun, 2004 Mar, 72(3), 1824 - 7
Detection of antibodies to toxin-coregulated pili in sera from cholera patients; Attridge SR et al.; Monoclonal antibodies (MAbs) were prepared against toxin-coregulated pili (TCP) isolated from Vibrio cholerae O1 El Tor . Despite their limited bactericidal potential, two MAbs were able to mediate biotype-specific protection against experimental cholera in infant mice . These MAbs were used in immunoblotting studies to assess seroconversion to El Tor TCP following cholera . Clear anti-pilus responses were observed in five of nine patients.

Photochem Photobiol, 2004 Jan, 79(1), 120 - 5
Oxygen triggering reversible modulation of Vibrio fischeri strain Y1 bioluminescence in vivo; Karatani H et al.; Yellow-emitting Vibrio fischeri Y1 modulates its bioluminescence (BL) depending on the dissolved O2 concentration . On supplying O2 to the cells under anaerobiosis, the cells begin to emit striking yellow BL peaking around 535 nm . The enhanced yellow emission reverts reversibly to the original level after O2 is consumed . Moreover, the reversible rise and fall of the yellow emission occurs repeatedly in accord with the repeating cycles of aeration on and off . This indicates that an increase in the cellular amount of yellow fluorescent protein (YFP) is not an immediate cause of the yellow emission enhancement . One suggested explanation is that the activity of YFP originating from its highly fluorescent property is altered by redox interaction with the respiratory components, including the soluble cytochrome c . Under the O2-limited conditions, the cellular YFP molecules, in part, seem to lose the fluorescent property possibly because of being reduced via redox interaction with some respiratory components in reduced form . On stimulating aerobic respiration with O2 supply, the reduced YFP seems to retrieve its fluorescent property via oxidation possibly with both O2, diffused across the cell membrane, and ferricytochrome c, generated during the respiratory turnover . The suggested redox interactions seem primarily to cause the reversible BL modulation.

J Bacteriol, 2004 Mar, 186(5), 1355 - 61
Expression of cholera toxin under non-AKI conditions in Vibrio cholerae El Tor induced by increasing the exposed surface of cultures; Sanchez J et al.; The regulatory systems controlling expression of the ctxAB genes encoding cholera toxin (CT) in the classical and El Tor biotypes of pathogenic Vibrio cholerae have been characterized and found to be almost identical . Notwithstanding this, special in vitro conditions, called AKI conditions, are required for El Tor bacteria to produce CT . The AKI conditions involve biphasic cultures . In phase 1 the organism is grown in a still tube for 4 h . In phase 2 the medium is poured into a flask to continue growth with shaking . Virtually no expression of CT occurs if this protocol is not followed . Here we demonstrated that CT expression takes place in single-phase still cultures if the volume-to-surface-area ratio is decreased, both under air and under an inert atmosphere . The expression of key genes involved in the regulation of CT production was analyzed, and we found that the expression pattern closely resembles the in vivo expression pattern.

Virology, 2004 Feb 5, 319(1), 141 - 51
Characterization of bacteriophage KVP40 and T4 RNA ligase 2; Yin S et al.; Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a subfamily of RNA strand-joining enzymes that includes the trypanosome RNA editing ligases . A homolog of T4 Rnl2 is encoded in the 244-kbp DNA genome of vibriophage KVP40 . We show that the 335-amino acid KVP40 Rnl2 is a monomeric protein that catalyzes RNA end-joining through ligase-adenylate and RNA-adenylate (AppRNA) intermediates . In the absence of ATP, pre-adenylated KVP40 Rnl2 reacts with an 18-mer 5'-PO(4) single-strand RNA (pRNA) to form an 18-mer RNA circle . In the presence of ATP, Rnl2 generates predominantly AppRNA . Isolated AppRNA can be circularized by KVP40 Rnl2 in the absence of ATP . The reactivity of phage Rnl2 and the distribution of the products are affected by the length of the pRNA substrate . Whereas 18-mer and 15-mer pRNAs undergo intramolecular sealing by T4 Rnl2 to form monomer circles, a 12-mer pRNA is ligated intermolecularly to form dimers, and a 9-mer pRNA is unreactive . In the presence of ATP, the 15-mer and 12-mer pRNAs are converted to AppRNAs, but the 9-mer pRNA is not . A single 5' deoxynucleotide substitution of an 18-mer pRNA substrate has no apparent effect on the 5' adenylation or circularization reactions of T4 Rnl2 . In contrast, a single deoxyribonucleoside at the 3' terminus strongly and selectively suppresses the sealing step, thereby resulting in accumulation of high levels of AppRNA in the absence of ATP . The ATP-dependent "capping" of RNA with AMP by Rnl2 is reminiscent of the capping of eukaryotic mRNA with GMP by GTP:RNA guanylyltransferase and suggests an evolutionary connection between bacteriophage Rnl2 and eukaryotic RNA capping enzymes.

J Environ Qual, 2004 Jan-Feb, 33(1), 80 - 8
Sequential supercritical fluid extraction (SSFE) for estimating the availability of high molecular weight polycyclic aromatic hydrocarbons in historically polluted soils; Szolar OH et al.; Sequential supercritical fluid (CO2) extraction (SSFE) was applied to eight historically contaminated soils from diverse sources with the aim to elucidate the sorption-desorption behavior of high molecular weight polycyclic aromatic hydrocarbons (PAHs) . The method involved five extraction phases applying successively harsher conditions by increasing fluid temperature and density mobilizing target compounds from different soil particle sites . Two groups of soils were identified based on readily desorbing (available) PAH fractions obtained under mildest extraction conditions (e.g., readily desorbing fractions of fluoranthene and pyrene significantly varied between the soils ranging from <10 to >90%) . Moreover, extraction behavior strongly correlated with molecular weight revealing decreasing available PAH fractions with increasing weight . Physicochemical soil parameters such as particle size distribution and organic dry mass were found to have no distinct effect on the sorption-desorption behavior of PAHs in the different soils . However, PAH profiles significantly correlated with readily available pollutant fractions; soils with relatively less mobile PAHs had higher proportions of five- and six-ring PAHs and vice versa . Eventually, biodegradability corresponded well with PAH recoveries under the two mildest extraction phases . However, a quantitative relationship was only established for soils with biodegradable PAHs . Out of eight soils, five showed no biodegradation including the four soils with the lowest fraction of readily desorbing PAHs . Only one soil (which was found to be highly toxic to Vibrio fischeri) did not match the overall pattern showing no PAH biodegradability but large fractions of highly mobile PAHs, concluding that mass transfer limitations may only be one of many factors governing biodegradability of PAHs.

J Appl Microbiol, 2004, 96(3), 447 - 54
Evaluation of a RAPD-based typing scheme in a molecular epidemiology study of Vibrio cholerae O1, Brazil; Leal NC et al.; AIMS: To evaluate the utility of random amplification of polymorphic DNA (RAPD) technique for routine practice in public health laboratories for epidemiological studies of Vibrio cholerae O1 isolates . MATERIALS AND RESULTS: Seventy-nine strains were examined by PCR for the toxin genes (ctx A, zot and ace), virulence-associated genes (tcp A and tox T) and RAPD sequences . Except for one strain (no . 1123) from the Amazonas State, all the strains analysed carried the genes ctx A, zot, ace, tcp A and tox T . RAPD fingerprinting revealed variability but no correlation with serotype, biotype or geographical origin of the isolates was found . CONCLUSION: A standardized RAPD method does not enable the establishment of a pattern data bank for the identification of V . cholerae O1 strains . SIGNIFICANCE AND IMPACT OF THE STUDY: The simplicity and discriminative capacity of this technique make it useful for detecting genetic diversity among micro-organisms from a defined group or for outbreak investigation.

Mar Biotechnol (NY), 2001 Jul, 3(4), 346 - 54
IbpA/B small heat-shock protein of marine bacterium Vibrio harveyi binds to proteins aggregated in a cell during heat shock; Klein G et al.; The IbpA and IbpB are 16-kDa Escherichia coli proteins belonging to a family of small heat-shock proteins (sHsps) . According to the present model, based on the in vitro experiments, sHsps are molecular chaperones that bind and prevent aggregation of nonnative proteins during heat shock . Previously, we have shown that IbpA and IbpB bind to endogenous E . coli proteins aggregated intracellularly by heat shock, which can be separated from soluble proteins and membranes in sucrose density gradients (fraction S) . In this work we have found that marine bacterium Vibrio harveyi contains a single sHsp which is strongly induced by heat shock and reacts with the anti-IbpA/B serum . The 26 amino-terminal amino acids of this sHsp bear high homology to E . coli IbpA and IbpB proteins (73% and 54% identity, respectively) . Fraction S was prepared from heat-shocked cells of V . harveyi, it contained high amounts of the IbpA/B protein . This result indicates that the IbpA/B protein of V . harveyi binds to the proteins that aggregate in V . harveyi cells during heat shock.

Mar Biotechnol (NY), 2001 Jul, 3(4), 336 - 45
Construction and use of a broad-host-range plasmid expressing the lamB gene for utilization of bacteriophage lambda vectors in the marine bacterium Vibrio harveyi; Jasiecki J et al.; The remarkable success of Escherichia coli as a model organism in molecular genetics was dependent, among other things, on its susceptibility to genetic manipulation . Many versatile and sophisticated genetic tools for molecular biology studies are derived from bacteriophage lambda . However, this bacteriophage is specific for E . coli, and thus lambda-based techniques have been restricted to this bacterium . Plasmids expressing the E . coli gene coding for bacteriophage lambda receptor were reported previously, and introduction of such plasmids into cells of some other bacteria made them sensitive to phage lambda infection . However, we found that these systems were not efficient for Vibrio harveyi, one of the most frequently investigated species of marine bacteria . Here we describe construction of a broad-host-range plasmid expressing the lamB gene . Introduction of this plasmid to V . harveyi cells and expression of lamB made this strain susceptible to bacteriophage lambda adsorption and lambda DNA injection . Foreign genetic material could be introduced into cells of this strain using a cosmid vector.

Mar Biotechnol (NY), 2002 Jun, 4(3), 267 - 77
Pathogenesis of gastroenteritis caused by Vibrio carchariae in cultured marine fish; Lee KK et al.; Serious mortality among the cultured grouper Epinephelus coioides, characterized by a swollen intestine containing yellow fluid (gastroenteritis), occurred in 1993 in Taiwan . A bacterium isolated from the intestinal fluid and head kidney of moribund groupers was identified as Vibrio carchariae . Since then, the same Vibrio species has also been isolated from moribund black sea bream Acanthopagrus schlegeli, yellowfin sea bream A . latus, Japanese sea bass Lateolabrax japonicus, and red drum Sciaenops ocellatus suffering from the same syndrome . Each isolate was virulent to the respective fish . Recently, a similar syndrome, flounder infectious necrotizing enteritis, also caused by V . carchariae in summer flounder Paralichthys dentatus, was reported in Rhode Island . The extracellular products (ECPs) of V . carchariae strains EmI82KL (from grouper), Rd (from red drum), and SfUSA (from summer flounder, U.S.A.) were virulent to the grouper or red drum . A 33-kDa serine protease partially purified from the ECP of strain EmI82KL was lethal to the fish . All the moribund or killed fish exhibited gastroenteritis except those killed within 12 hours . This report is the first to show that intraperitoneal injection of the ECP or protease in the fish is virulent and can reproduce gastroenteritis . The serine protease was suggested as a major toxin in the grouper or red drum secreted by V . carchariae.

Dis Aquat Organ, 2003 Dec 29, 57(3), 265 - 70
Effect of methyl parathion on the susceptibility of shrimp Litopenaeus vannamei to experimental vibriosis; Labrie L et al.; Following increasing calls for environmental safety over the past 2 decades, persistent pesticides are being replaced by more rapidly degradable products . However, even these pesticides can affect non-target species, and may be associated with slow growth and increased susceptibility to viral and bacterial infections . In this study, juvenile white shrimp Litopenaeus vannamei (also named Penaeus vannamei) were challenged by intramuscular injection with Vibrio parahaemolyticus after 4 d prior exposure to methyl parathion in feed pellets at 0.080 microg g(-1) . The bacterial injection control group consisted of shrimp fed pellets containing the methyl parathion-carrier solvent acetonitrile . Three additional control groups comprised 2 sterile saline-injection groups fed pellets containing methyl parathion or acetonitrile prior to injection, and 1 uninjected group fed normal pellets . Cumulative mortalities were recorded on the 4th and 8th days, and the presence of histological lesions was recorded on the 8th day . Cumulative mortalities were significantly higher in the group exposed to methyl parathion and bacteria on Day 8 . Histological lesions, typical of vibriosis, were significantly associated with the injection of V . parahaemolyticus . The study provides strong experimental evidence that prior exposure to methyl parathion can increase the severity of Vibrio infections.

Environ Microbiol, 2004 Mar, 6(3), 209 - 17
Determination of Vibrio scophthalmi and its phenotypic diversity in turbot larvae; Cerda-Cuellar M et al.; The association of Vibrio scophthalmi with turbot larvae was assessed, by molecular methods with a species-specific probe, in the rearing stages of turbot (Scophthalmus maximus) larvae using a routine batch of production at a fish farm . The phenotypic diversity of this bacterial species was also studied to identify predominant phenotypes at successive stages of larval development . Vibrio scophthalmi was detected in all turbot larvae samples except in the sample from day 0 after hatching . The percentage of V . scophthalmi in the intestinal microbiota increased throughout larval development . Vibrio scophthalmi was also detected in live food (brine shrimps) and water from the tanks, but not in the sediment . All turbot larvae, 15-57 day old, showed several V . scophthalmi phenotypes, and a pattern of successive waves of phenotypes was observed during successive larval stages . This indicates that certain strains may colonize the intestine more efficiently and thus maintain their population for longer than other strains . Vibrio scophthalmi populations from turbots of different origin were very similar, suggesting that irrespective of geographical area, turbot populations share similar V . scophthalmi strains . Vibrio scophthalmi strain was not isolated from other cultured fish, only turbot larvae, at the same hatchery receiving water from the same supply.

FEMS Microbiol Lett, 2004 Feb 9, 231(1), 145 - 52
In vitro growth characteristics of five candidate aquaculture probiotics and two fish pathogens grown in fish intestinal mucus; Vine NG et al.; The selection of probiotics for aquaculture is usually based on their antagonism towards pathogens . However, other criteria such as growth, attachment to intestinal mucus and production of beneficial compounds should also be considered . We suggest a protocol for the isolation and selection of potential probiotic bacteria based on their in vitro growth characteristics and propose a ranking index (RI) to screen potential aquaculture probionts . We suggest that the lag period and doubling time are the most important criteria for the comparison of growth curves, hence the RI is based on the doubling time (t(d)) and lag period (lambda) obtained from the growth profile of each bacterium . Bacteria were isolated from the gut of the common clownfish, Amphiprion percula, and screened for antagonistic activity towards seven aquatic pathogens . All five candidate probiotics showed antagonism to various aquatic pathogens . When grown in intestinal fish mucus no probiotic had a RI higher than the two tested pathogens (Aeromonas hydrophila and Vibrio alginolyticus) . However, candidate probiont AP1 had a faster specific growth rate (micro) (0.05) than the pathogens (0.049 and 0.047 respectively), while AP5 grown in marine broth had a shorter lag period than the pathogens . Strategies to increase probiotic concentration include the inoculation of high concentrations and the preconditioning of these bacteria to reduce the lag period . It should be tested whether or not such strategies will allow the probiotic bacteria to dominate initially and thereby gain a competitive advantage . This could become an important aspect under in vivo conditions where both attachment and nutrient supply differ from that found in in vitro studies.

Environ Toxicol Chem, 2004 Jan, 23(1), 32 - 40
Toxicological profiling of sediments using in vitro bioassays, with emphasis on endocrine disruption; Houtman CJ et al.; In vitro bioassays are valuable tools for screening environmental samples for the presence of bioactive (e.g., endocrine-disrupting) compounds . They can be used to direct chemical analysis of active compounds in toxicity identification and evaluation (TIE) approaches . In the present study, five in vitro bioassays were used to profile toxic potencies in sediments, with emphasis on endocrine disruption . Nonpolar total and acid-treated stable extracts of sediments from 15 locations in the Rhine Meuse estuary area in The Netherlands were assessed . Dioxin-like and estrogenic activities (using dioxin-responsive chemical-activated luciferase gene expression {DR-CALUX} and estrogen-responsive chemical-activated luciferase gene expression {ER-CALUX} assays) as well as genotoxicity (UMU test) and nonspecific toxic potency (Vibrio fischeri assay) were observed in sediment extracts . For the first time, to our knowledge, in vitro displacement of thyroid hormone thyroxine (T4) from the thyroid hormone transport protein thransthyretin by sediment extracts was observed, indicating the presence of compounds potentially able to disrupt T4 plasma transport processes . Antiestrogenic activity was also observed in sediment . The present study showed the occurrence of endocrine-disrupting potencies in sediments from the Dutch delta and the suitability of the ER- and DR-CALUX bioassays to direct endocrine-disruption TIE studies.

Kansenshogaku Zasshi, 2003 Dec, 77(12), 1015 - 23
{Epidemiological study of outbreaks and sporadic cases due to Vibrio parahaemolyticus--serotype O3:K6 in Aichi Prefecture, Japan, during 1988 and 2001}; Yamazaki M et al.; Percentage of the outbreaks by O3:K6 Vibrio parahaemolyticus (V . p) in Aichi Prefecture Japan increased from 3% (3/86) for 1988-95 to 75% (33/44) for 1996-2001 . The percentage of the sporadic diarrhea cases caused by O3:K6 V . p in a general hospital in Aichi Prefecture also increased from 0% (0/253) to 61% (135/221) during the same periods . Thermostable direct hemolysin (TDH)-positive O3:K6 were isolated from 95% (19/20) of the outbreak incidents and 100% (135/135) of the sporadic cases . Only one TRH (TDH-related hemolysin)-positive O3:K6 was isolated from one outbreak incident . Percentage of the outbreaks by O3:K6 V . p associated with the consumption of boiled shellfishes increased from 5% (6/117) for 1988-95 to 25% (15/59) for 1996-2001, in particular, boiled crabs and squillas associated outbreaks increased from 2% (2/117) to 17% (10/59) and from 2% (2/117) to 10% (6/59), respectively . From 1,548 raw sea foods sampled in the Nagoya Central Wholesale Market in Aichi Prefecture in 1995-99, one TDH-positive O3:K6 was isolated from one live squilla (1/30) . Increase in the percentage of outbreaks associated with TDH-positive O3:K6 V . p after 1996 in Aichi Prefecture was revealed to correlate with the increase in the outbreaks associated with consumption of boiled sea foods, especially boiled crabs as well as squillas . Accordingly, it becomes clear that sanitary handling of these boiled foods is important to prevent outbreaks and sporadic cases of diarrhea caused by O3:K6 V . p infection.

J Basic Microbiol, 2004, 44(1), 23 - 8
Isolation and characterization of pathogenic Vibrio alginolyticus from diseased cobia Rachycentron canadum; Liu PC et al.; Outbreaks of serious mortality among cultured juvenile cobia Rachycentron canadum L . (weighing 8-10 g) characterized by lethargy, dark skin and ascites in the peritoneal cavity while some fish possessing damaged eyes occurred in July and August of 2001 in Taiwan . Fifteen motile bacterial strains were isolated from head kidney and/or the ascites on tryptic soy agar supplemented with 1% NaCl (TSA1) and/or thiosulphate citrate bile salt (TCBS) sucrose agar plates during the two outbreaks . All the isolates were characterized and identified as Vibrio alginolyticus on the basis of biochemical characteristics, and comparisons with those of the reference strain V . alginolyticus ATCC 17749 . The strain C3c01 (a representative of the 15 similar field isolates), was virulent to the cobia with an LD50 value of 3.28 x 10(4) colony forming units/g fish body weight . All the moribund/dead fish exhibited lethargy, dark skin and ascites in the peritoneal cavity as that observed in natural outbreaks . The same bacteria could be reisolated from kidney and the ascites of fish after bacterial challenge using TSA1 and TCBS plates . The results reveal that V . alginolyticus is an infectious agent of vibriosis in the cobia.

Proc Natl Acad Sci U S A, 2004 Feb 17, 101(7), 2123 - 8 Epub 2004 Feb 06.
Genetic diversity and virulence potential of environmental Vibrio cholerae population in a cholera-endemic area; Faruque SM et al.; To understand the evolutionary events and possible selection mechanisms involved in the emergence of pathogenic Vibrio cholerae, we analyzed diverse strains of V . cholerae isolated from environmental waters in Bangladesh by direct enrichment in the intestines of adult rabbits and by conventional laboratory culture . Strains isolated by conventional culture were mostly (99.2%) negative for the major virulence gene clusters encoding toxin-coregulated pilus (TCP) and cholera toxin (CT) and were nonpathogenic in animal models . In contrast, all strains selected in rabbits were competent for colonizing infant mice, and 56.8% of these strains carried genes encoding TCP alone or both TCP and CT . Ribotypes of toxigenic O1 and O139 strains from the environment were similar to pandemic strains, whereas ribotypes of non-O1 non-O139 strains and TCP(-) nontoxigenic O1 strains diverged widely from the seventh pandemic O1 and the O139 strains . Results of this study suggest that (i) the environmental V . cholerae population in a cholera-endemic area is highly heterogeneous, (ii) selection in the mammalian intestine can cause enrichment of environmental strains with virulence potential, (iii) pathogenicity of V . cholerae involves more virulence genes than currently appreciated, and (iv) most environmental V . cholerae strains are unlikely to attain a pandemic potential by acquisition of TCP and CT genes alone . Because most of the recorded cholera pandemics originated in the Ganges Delta region, this ecological setting presumably favors extensive genetic exchange among V . cholerae strains and thus promotes the rare, multiple-gene transfer events needed to assemble the critical combination of genes required for pandemic spread.

Microbiology, 2004 Feb, 150(Pt 2), 383 - 90
Characterization of cyst cell formation in the purple photosynthetic bacterium Rhodospirillum centenum; Berleman JE et al.; Rhodospirillum centenum is an anoxygenic photosynthetic bacterium that is capable of differentiating into several cell types . When grown phototrophically in liquid, cells exhibit a vibrioid shape and have a single polar flagellum . When grown on a solid surface, R . centenum will differentiate into rod-shaped swarm cells that display numerous lateral flagella . Upon starvation for nutrients, R . centenum also forms desiccation-resistant cysts . In this study, it was determined that R . centenum has heat- and desiccation-resistance properties similar to other cyst-forming species . In addition, microscopic analyses of the morphological changes that occur during cyst cell development were performed . It was observed that R . centenum typically forms multi-celled clusters of cysts that contain from four to more than 10 cells per cluster . It was also determined that cell density has a minor effect on the percentage of cyst cells formed, with cell densities of 10(5)-10(7) cells per 5 micro l spot yielding the highest percentage of cyst cells . The striking similarities between the life cycle of R . centenum and the life cycle exhibited by Azospirillum spp . are discussed.

Appl Environ Microbiol, 2004 Feb, 70(2), 855 - 64
Lead precipitation by Vibrio harveyi: evidence for novel quorum-sensing interactions; Mire CE et al.; Three pleiotropic, quorum sensing-defective Vibrio harveyi mutants were observed to precipitate soluble Pb2+ as an insoluble compound . The compound was purified and subjected to X-ray diffraction and elemental analyses . These assays identified the precipitated compound as Pb9(PO4)6, an unusual and complex lead phosphate salt that is produced synthetically at temperatures of ca . 200 degrees C . Regulation of the precipitation phenotype was also examined . Introduction of a luxO::kan allele into one of the mutants abolished lead precipitation, indicating that the well-characterized autoinducer 1 (AI1)-AI2 quorum-sensing system can block lead precipitation in dense cell populations . Interestingly, the V . harveyi D1 mutant, a strain defective for secretion of both AI1 and AI2, was shown to be an effective trans inhibitor of lead precipitation . This suggests that a previously undescribed V . harveyi autoinducer, referred to as AI3, can also negatively regulate lead precipitation . Experiments with heterologous bacterial populations demonstrated that many different species are capable of trans regulating the V . harveyi lead precipitation phenotype . Moreover, one of the V . harveyi mutants in this study exhibited little or no response to intercellular signals from other V . harveyi inocula but was quite responsive to some of the heterologous bacteria . Based on these observations, we propose that V . harveyi carries at least one quorum sensor that is specifically dedicated to receiving cross-species communication.

Cad Saude Publica, 1994 Sep, 10(3), 379 - 86 Epub 2004 Jan 27.
{Occurrence of Vibrio parahaemolyticus in mussels (Perna perna, Linnaeus, 1758) from a natural coastal bed in the municipality of Palhoça, Santa Catarina, Brazil}; Archer RM et al.; A study was carried out on the occurrence of Vibrio parahaemolyticus in forty samples of mussels (Perna perna, Linnaeus, 1758) from a natural bed at Pinheira Beach, Municipality of Palhoca, Santa Catarina (Brazil), during a three-month period . The most probable number technique was used for isolation methodology with prior enrichment of samples in alkaline peptone water and subsequent planting on thiosulfate citrate bile salts sucrose agar . Vibrio parahaemolyticus was found in 52.5% of samples of mussels with a most probable number range of <3 to 93 organisms/g . Serotyping was performed on 61 isolates and our findings indicate that 36.1% of these isolates were serologically non-typable; 54.1% of isolates displayed only flagellate antigenic structures and 8.2% had both antigenic structures . None of the isolates were Kanagawa positive.

Chemosphere, 2004 Apr, 55(2), 207 - 14
Treatment and detoxification of a sanitary landfill leachate; Silva AC et al.; The leachate from an old sanitary landfill (Gramacho Metropolitan Landfill, Rio de Janeiro) was characterized and submitted to coagulation and flocculation treatment followed by ozonation and ammonia stripping . The performance of the treatment was assessed by monitoring the removal of organic matter (COD and TOC), ammonium nitrogen and metals . Detoxification was assessed by determining acute toxicity, using the following organisms: Vibrio fisheri, Daphnia similis, Artemia salina and Brachydanio rerio . Membrane fractionation was employed to infer the range of molecular masses of the pollutants found in the effluent, as well as the toxicity associated to these fractions . Of the techniques under investigation, coagulation and flocculation followed by ammonia stripping were the most effective for toxicity and ammonium nitrogen removal . Membrane fractionation was effective for COD removal; however, acute toxicity was almost the same in all the fractionated samples . Ozonation was moderately effective for COD removal, but significant toxicity removal was only attained when high ozone doses were used.

Int J Med Microbiol, 2003 Dec, 293(6), 413 - 20
Translocation of protein kinase-C with IP3-induced calcium mobilization by heat-stable enterotoxin of Vibrio cholerae non-O1 in isolated rat enterocytes; Hoque KM et al.; The activity of the calcium- and phospholipid-dependent enzyme protein kinase C (PKC) in response to heat-stable enterotoxin (NAG-ST) of Vibrio cholerae non-O1 was examined in isolated rat enterocytes . Optimal stimulation of the membrane-bound PKC activity (about 4.3-fold) was observed after 1 min of incubation of cells with 10 ng/ml toxin; and the effects were dose dependent . Following NAG-ST treatment an increase in PKC activity in the membrane fraction was found with a concomitant decrease in the cytosolic fraction suggesting the redistribution of the enzyme . The pronounced enzyme activity in presence of a classical pseudosubstrate and its complete inhibition by Go 6976 suggested the involvement of a calcium-dependent isoform of PKC (PKC-alpha) . A time course study employing an immunoblot assay provided evidence that NAG-ST led to almost complete translocation of PKC-alpha to the membrane . A 65% inhibition of enzyme activity in the membrane fraction and inhibition of its translocation to some extent by dantrolene treatment further suggested that the enzyme was translocated with the rise of intracellular calcium ({Ca2+}i) . The phosphorylation of three membrane proteins by toxin-induced PKC in vitro and abolition of this phosphorylation by Go 6976 demonstrated that phosphorylation of these membrane proteins was PKC-alpha mediated and might be involved in the alteration of membrane functions.

Environ Toxicol, 2004 Feb, 19(1), 45 - 50
Aquatic toxicity of four alkylphenols (3-tert-butylphenol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol) and their binary mixtures to microbes, invertebrates, and fish; Choi K et al.; The acute and chronic toxicity of four simple alkylphenols with butyl and propyl substitutions was evaluated with aquatic microbes, invertebrates, and fish . These alkylphenols-3-tert-butylphenol, 2-isopropylphenol, 3-isopropylphenol, and 4-isopropylphenol-have been detected in various environmental media, but their impact on aquatic fauna has seldom been evaluated . Relative susceptibility to each phenolic varied by test species . The marine bacterium Vibrio fischeri was the most susceptible to the alkylphenols, up to 3 orders of magnitude more sensitive than species of higher trophic levels . For 4-isopropylphenol, the 5-min Microtox EC(50) value was 0.01 mg/L, whereas the EC(50) for Ceriodaphnia after a 48-h exposure was 10.1 mg/L . Notable differences in sensitivity to the alkylphenols was also observed with the Microtox assay: 4-isopropylphenol was > 200 times more toxic to V . fischeri than was 2-isopropylphenol (EC(50) = 2.72 mg/L) . For V . fischeri, the mixture toxicity of the alkylphenols was additive in nature and was predicted by a concentration addition model . The energy of the lowest unoccupied molecular orbital (ELUMO) explained the observed toxicity of the individual alkylphenols to V . fischeri (r(2) = 0.92, p < 0.05) . These results suggest that the mode of action of polar narcotic alkylphenols to V . fischeri is different than that of other test organisms, possibly because of the differences in the cell structure of the prokaryotic V . fischeri .

Arch Microbiol, 2004 Apr, 181(4), 287 - 93 Epub 2004 Feb 03.
Cloning, identification and expression of an entE homologue angE from Vibrio anguillarum serotype O1; Liu Q et al.; Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, is synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulence plasmid pJM1 . angE, as one of the NRPS genes, is responsible for selecting and activating 2,3-dihydroxybenzoic acid (2,3-DHBA), an important precursor in anguibactin synthesis, into 2,3-DHBA-AMP by adenylylation in the presence of ATP . In this work, an entE homologue, angE, was identified on pEIB1 (a pJM1-like plasmid) from virulent V . anguillarum serotype O1 strain MVM425 . A recombinant clone carrying the complete angE was able to complement an Escherichia coli entE mutant . The angE-encoded protein was overexpressed in E . coli and purified by a three-step procedure . Purified AngE was then used to establish an in vitro enzymatic reaction in which its enzymatic activity of 1-(5'-monophosphate adenyl) 2,3-dihydroxybenzoic acid ligase (2,3-DHBA-AMP ligase) was proved using HPLC to detect AMP formation in the reaction mixture . Moreover, evidence at the level of both transcription and translation confirmed that angE was actively expressed in vivo in V . anguillarum MVM425, and interestingly, unlike many other iron-uptake-system-related genes, its expression is not induced by a low iron concentration in the surrounding environment.

J Environ Monit, 2004 Feb, 6(2), 97 - 102 Epub 2004 Jan 20.
Water toxicity monitoring using Vibrio fischeri: a method free of interferences from colour and turbidity; Faria EC et al.; In this paper the kinetic method for the determination of toxicity using Vibrio fischeri is described and suggested as a potential method for the continuous screening of wastewater toxicity . The kinetic method was demonstrated to be free from interferences due to colour and turbidity normally observed when testing wastewater samples with this organism . This is of great importance for the application of the method to remote toxicity screening of wastewaters . The effect of colour, investigated using 50 ppm Zn(2+) solutions containing the food-dye tropaeolin O, and the effect of turbidity, investigated using 50 ppm Zn(2+) solutions containing white optically reflective and coloured optically absorbing polystyrene beads, is reported . It was also found that the design of the light detection system of the instrument ensures efficient collection of the light scattered by particles in the sample, which enables a greater range of turbid samples to be tested . In addition the natural light decay was found to be negligible during the duration of a 10 min test and thus one channel would be enough to carry out the tests . This would mean halving the quantity of bacterial reagent used and reducing the cost of the tests.

Mar Environ Res, 2004 May, 57(4), 311 - 27
Crude oil bioremediation in sub-Antarctic intertidal sediments: chemistry and toxicity of oiled residues; Pelletier E et al.; The effectiveness of fertilizers for crude oil bioremediation in sub-Antarctic intertidal sediments was tested over a one-year period in a series of ten (10) experimental enclosures . Chemical, microbial and toxicological parameters demonstrated the effectiveness of various fertilizers in a pristine environment where hydrocarbon degrading bacteria (HDB) had not been stimulated by previous accidental spills or human activities . The low temperature of seawater (3-4 degrees C) had no obvious effects on the HDB community and the bioremediation process . Over 90% of n-alkanes were degraded in the first six months and most light aromatics (2-3 rings) disappeared during the first year of observation . The toxicity of oiled residues (Microtox(R) SP) was significantly reduced in the first 6 months of the process, but it increased again in the last months of the experiment . One of the fertilizers containing fishbone compost enriched with urea, inorganic phosphorus and a lipidic surfactant reduced significantly the toxicity of oil residues in the last 3 months of the experiment . Interstitial waters collected below the oil slicks during the remediation showed no toxicity, and even stimulated Vibrio fischeri . When comparing all fertilizers to the control plots, a good correlation (r(2)=0.82) was found between the growth rate of HDB and the degradation rate of n-alkanes in the first 90 days of the experiment only indicating that fertilizers were efficient for at least 3 months but their beneficial effects were lost after 6 months.

Infect Immun, 2004 Feb, 72(2), 629 - 36
A K+ yptake protein, TrkA, is required for serum, protamine, and polymyxin B resistance in Vibrio vulnificus; Chen YC et al.; Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of both serious wound infections and fatal septicemia in many areas of the world . To identify the genes required for resistance to human serum, we constructed a library of transposon mutants of V . vulnificus and screened them for hypersensitivity to human serum . Here we report that one of the isolated serum-susceptible mutants had a mutation in an open reading frame identified as trkA, a gene encoding an amino acid sequence showing high identity to that of TrkA of Vibrio alginolyticus, a protein required for the uptake of potassium . A trkA isogenic mutant was constructed via insertional inactivation, and it was significantly more easily killed by human serum, protamine, or polymyxin B than was the wild type . At K+ concentrations of 1 to 20 mM, this isogenic mutant showed attenuated growth compared to the wild-type strain . In addition, infection experiments demonstrated virulence attenuation when this mutant was administered intraperitoneally or subcutaneously to both normal and iron-treated mice, indicating that TrkA may modulate the transport of potassium and resistance to host innate defenses and that it is important for virulence in mice.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 261 - 5
Vibrio hispanicus sp . nov., isolated from Artemia sp . and sea water in Spain; Gomez-Gil B et al.; Three Gram-negative, small, motile, rod-shaped bacteria were isolated from Artemia sp . and sea water in Barcelona, Spain, during 1990 and 1991 . They were fermentative, oxidase-positive, sensitive to vibriostatic agent O/129, arginine dihydrolase-positive, lysine and ornithine decarboxylase-negative and grew in the absence of NaCl . They differed from phenotypically related species by their ability to grow at 4 degrees C and utilize L-rhamnose . Cloning of the 16S rRNA gene of the type strain produced two different 16S rRNA gene sequences, which differed by 15 bases (0.99%); comparison of these sequences with those deposited in GenBank showed close relationships with Vibrio proteolyticus (97.6% similarity), Vibrio diazotrophicus (97.9%), Vibrio campbellii (96.8%) and Vibrio alginolyticus (96.8%), among others . DNA-DNA hybridization levels with the closest phylogenetically related Vibrio species were <26.4% . Sufficient evidence is provided to support the identity of the three strains analysed as members of a novel species of the genus Vibrio, for which the name Vibrio hispanicus sp . nov . is proposed, with the type strain LMG 13240T (=CAIM 525T=VIB 213T).

Biochem Biophys Res Commun, 2004 Feb 13, 314(3), 838 - 43
Identification and characteristic analysis of the ampC gene encoding beta-lactamase from Vibrio fischeri; Weng SF et al.; Vibrio fischeri ATCC 7744 is an ampicillin resistant (Amp(r)) marine luminous bacterium . The MIC test indicates that V . fischeri is highly resistant to penicillins, and susceptible to cephalosporins . V . fischeri ampC gene was cloned and identified . Nucleotide sequence of an unidentified ufo gene and the ampC, ppiB genes (GenBank Accession No . AY438037) has been determined; whereas the ampC gene encodes the beta-lactamase (AmpC) and the ppiB gene encodes the peptidyl-prolyl cis-trans isomerase B . Alignment and comparison show that V . fischeri beta-lactamase is homologous to the related species' . The specific amino acid residues STFK (62nd to 65th), SDN (122nd to 124th), and D (155th) located 34 residues downstream from the SDN loop of the class A beta-lactamases are highly conserved, but the KTG is not found . V . fischeri ampC gene encoding beta-lactamase has a calculated M(r) 31,181 and comprises 283 amino acid residues (pI 5.35) . There is a signal peptide of 18 amino acid residues MKIKPFLFGLIVLANNAI in the pro-beta-lactamase, which functioned for secretion; thus, the matured protein only has M(r) 29,197 and comprises 265 amino acid residues (pI 4.95) . SDS-PAGE and the beta-lactamase functional assays elicit that the M(r) of the beta-lactamases are close to 29kDa . IEF and the beta-lactamase functional assays show that the beta-lactamases' pI are close to 4.8 as predicted . The results elucidate that V . fischeri ampC gene and the cloned ampC gene in Escherichia coli are the same one . The gene order of the ampC and the related genes is -ufo-(P*-intern)-ampC-ppiB--> (P*-intern: intern promoter for sub-regulation), whereas the P*-intern promoter displays the function to lead the ampC gene's expression for stress response.

Biochem Biophys Res Commun, 2004 Feb 13, 314(3), 681 - 7
A single WAP domain-containing protein from Litopenaeus vannamei hemocytes; Jimenez-Vega F et al.; A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei . The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein . Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes . The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas . mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response . Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.

Lancet, 2004 Jan 17, 363(9404), 223 - 33
Cholera; Sack DA et al.; Intestinal infection with Vibrio cholerae results in the loss of large volumes of watery stool, leading to severe and rapidly progressing dehydration and shock . Without adequate and appropriate rehydration therapy, severe cholera kills about half of affected individuals . Cholera toxin, a potent stimulator of adenylate cyclase, causes the intestine to secrete watery fluid rich in sodium, bicarbonate, and potassium, in volumes far exceeding the intestinal absorptive capacity . Cholera has spread from the Indian subcontinent where it is endemic to involve nearly the whole world seven times during the past 185 years . V cholerae serogroup O1, biotype El Tor, has moved from Asia to cause pandemic disease in Africa and South America during the past 35 years . A new serogroup, O139, appeared in south Asia in 1992, has become endemic there, and threatens to start the next pandemic . Research on case management of cholera led to the development of rehydration therapy for dehydrating diarrhoea in general, including the proper use of intravenous and oral rehydration solutions . Appropriate case management has reduced deaths from diarrhoeal disease by an estimated 3 million per year compared with 20 years ago . Vaccination was thought to have no role for cholera, but new oral vaccines are showing great promise.

Dis Aquat Organ, 2003 Dec 3, 57(1-2), 147 - 50
Vibrio alginolyticus associated with white spot disease of Penaeus monodon; Selvin J et al.; In February 2000, white spot disease outbreaks occurred among cultured Penaeus monodon in extensive shrimp farms on the southwest coast of India . Bacteria were isolated from infected shrimp that showed reddish body coloration and white spots in the cuticle . The isolates were screened on thiosulfate citrate bile salt sucrose (TCBS) agar plates for the selection of Vibrio species . The primary isolate (QS7) was characterized as V . alginolyticus based on morphological, biochemical and physiological characteristics . Antibiotic sensitivity tests of QS7 indicated that the isolate was highly sensitive to chloramphenicol, ciprofloxacin, nalidixic acid and streptomycin . Pathogenicity tests confirmed that the isolate was virulent for P . monodon . Based on the lethal dose (LD50) value (5 x 10(6) cfu per shrimp), it was inferred that shrimp weakened by white spot syndrome virus would succumb to secondary infection by QS7.

Dis Aquat Organ, 2003 Dec 3, 57(1-2), 109 - 16
Reduction of adhesion properties of Ruditapes philippinarum hemocytes exposed to Vibrio tapetis; Choquet G et al.; Vibrio tapetis is the causative agent of brown ring disease (BRD), which affects a species of clam, Ruditapes philippinarum . After incubation with V . tapetis, hemocytes lose filopods and become rounded, indicating cytotoxic activity of the bacterium . To rapidly quantify this cytotoxicity, a flow-cytometry test was developed based on the capacity of V . tapetis to inhibit adhesion of clam hemocytes to plastic . Several bacteria:hemocyte ratios, the cytotoxicity of other Vibrio spp . pathogenic to bivalves, and that of various V . tapetis isolates were tested . Inhibition of adherence is detectable with as few as 5 bacteria per hemocyte . The greater cytotoxic activity of V . tapetis compared to that of V . splendidus and V . pectenicida suggests a specific pathogenicity of V . tapetis to R . philippinarum hemocytes . Although all V . tapetis isolates inhibited adhesion, significant variations in cytotoxicity among isolates was demonstrated.

Mar Biotechnol (NY), 2003 Sep-Oct, 5(5), 480 - 92
Potential of thraustochytrids to partially replace fish oil in Atlantic salmon feeds; Carter CG et al.; The replacement of fish oil with a dried product made from thraustochytrid culture, a marine microorganism, in canola-oil-based diets for Atlantic salmon was investigated . Salmon (37 g) were fed for 51 days on diets containing only canola oil, canola oil and fish oil, or canola oil and the thraustochytrid . There were no significant differences in final weight (106.1 +/- 1.1 g), weight gain (69.6 +/- 1.1 g), feed consumption (16.5 +/- 0.2 mg dry matter g(-1) d(-1)), feed efficiency ratio (1.15 +/- 0.03 g (g-1)), or productive protein value (51.2% +/- 1.7%) between the diets . Nor were there any significant differences in whole-body chemical composition, organ somatic indices, or measures of immune function . However, following transfer to seawater and 2 challenges with Vibrio anguillarum, cumulative mortality was significantly lower in fish fed some fish oil than in those fed the 2 diets containing no fish oil . In conclusion, the thraustochytrid had no detrimental effects on the performance of salmon but, at the current inclusion of 10%, failed to confer the same effect as fish oil under challenging conditions.

J Bacteriol, 2004 Feb, 186(3), 631 - 7
Reversible acyl-homoserine lactone binding to purified Vibrio fischeri LuxR protein; Urbanowski ML et al.; The Vibrio fischeri LuxR protein is the founding member of a family of acyl-homoserine lactone-responsive quorum-sensing transcription factors . Previous genetic evidence indicates that in the presence of its quorum-sensing signal, N-(3-oxohexanoyl) homoserine lactone (3OC6-HSL), LuxR binds to lux box DNA within the promoter region of the luxI gene and activates transcription of the luxICDABEG luminescence operon . We have purified LuxR from recombinant Escherichia coli . Purified LuxR binds specifically and with high affinity to DNA containing a lux box . This binding requires addition of 3OC6-HSL to the assay reactions, presumably forming a LuxR-3OC6-HSL complex . When bound to the lux box at the luxI promoter in vitro, LuxR-3OC6-HSL enables E . coli RNA polymerase to initiate transcription from the luxI promoter . Unlike the well-characterized LuxR homolog TraR in complex with its signal (3-oxo-octanoyl-HSL), the LuxR-30C6-HSL complex can be reversibly inactivated by dilution, suggesting that 3OC6-HSL in the complex is not tightly bound and is in equilibrium with the bulk solvent . Thus, although LuxR and TraR both bind 3-oxoacyl-HSLs, the binding is qualitatively different . The differences have implications for the ways in which these proteins respond to decreases in signal concentrations or rapid drops in population density.

Biochim Biophys Acta, 2004 Jan 5, 1670(1), 69 - 80
Role of the W07-toxin on Vibrio cholerae-induced diarrhoea; Bhattacharyya S et al.; Vibrio cholerae W07 strain isolated from a cholera epidemic in South India, lacked the ctx gene but could still secrete a novel toxin, the W07-toxin that could cause fluid accumulation in ligated rabbit ileal loop . The important intracellular messengers implicated in this study were Ca(2+), cyclic AMP, inositol triphosphate and protein kinase C (PKC) . A number of inhibitors/channel blockers have further shown the major role of {Ca(2+)}(i) in modulation of the toxin-induced cellular response . An increase in the level of reactive oxygen species (ROS) in the W07-toxin-stimulated enterocytes correlated with the decrease in the levels of antioxidant enzymes, catalase and superoxide dismutase (SOD) . The reactive nitrogen intermediates (RNI) detected by measuring the levels of nitrite and citrulline, were found to be high in the enterocytes triggered with the W07-toxin, thereby indicating their role in toxin-mediated change in mucosal permeability . The precise role of the toxin has also been authenticated by conducting the experiments with W07-toxin preincubated in the presence of IgG(WT) (IgG isolated from antitoxin sera) or GM(1) . Thus, a significant increase in the levels of second messengers and a decrease in antioxidant defenses appear to be important in mediating the fluid secretion caused by this novel toxin from V . cholerae W07.

Trop Med Int Health, 2004 Jan, 9(1), 133 - 40
In situ measured elimination of Vibrio cholerae from brackish water; Perez ME et al.; In situ elimination of fluorescently labelled Vibrio cholerae (FLB) was measured in two saline water bodies in Mexico: in a brackish water lagoon, Mecoacan (Gulf of Mexico; State of Tabasco) and an athalassohaline lake, Alchichica (State of Puebla) . Disappearance rates of fluorescently labelled V . cholera O1 showed that they were eliminated from the environment at an average rate of 32% and 63%/day, respectively (based on the bacterial standing stocks) . The indirect immunofluorescence method confirmed the presence of V . cholerae O1 in the lagoon . However, the elimination of FLB was not directly related either to the presence or absence of the bacterium in the water body or to the phytoplankton concentration.

Microb Pathog, 2004 Mar, 36(3), 131 - 9
Molecular-genetic peculiarities of classical biotype Vibrio cholerae, the etiological agent of the last outbreak Asiatic cholera in Russia; Smirnova NI et al.; Molecular-genetic properties of classical biotype Vibrio cholerae strains that caused the Asiatic cholera outbreak in 1942 in Russia have been investigated for the first time . Being characterized by high-level production of cholera toxin and toxin-coregulated adhesion pili both of which are the major virulence factors, all the strains studied, in contrast to the typical cholera pathogens, were autographic requiring purine and/or amino acids added to the minimal medium for their growth . Moreover, these strains containing the structural gene hapA, as shown by the polymerase chain reaction, produced no soluble hemagglutinin/protease, which enables the vibrios to get disseminated in the environment . The peculiarities of the natural V . cholerae strains elucidated in the work are likely to be responsible for the unusual infectious and epidemic processes observed during that cholera outbreak.

Microb Pathog, 2004 Mar, 36(3), 117 - 23
High growing ability of Vibrio vulnificus biotype 1 is essential for production of a toxic metalloprotease causing systemic diseases in humans; Watanabe H et al.; Vibrio vulnificus biotype 1, a causative agent of fatal septicemia or wound infection in humans, is known to produce a toxic metalloprotease as an important virulence determinant . V . vulnificus biotype 2 (serovar E), a primary eel pathogen, was found to elaborate an extracellular metalloprotease that was indistinguishable from that of biotype 1 . The potential of V . vulnificus biotype 1 for production of the metalloprotease was compared with biotype 2 and other human non-pathogenic Vibrio species (Vibrio anguillarum and Vibrio proteolyticus) . When cultivated at 25 degrees C in tryptone-yeast extract broth supplemented with 0.9% NaCl, all bacteria multiplied sufficiently and secreted significant amounts of the metalloprotease . However, at 37 degrees C with 0.9% NaCl, V . anguillarum neither grew nor produced the metalloprotease . In human serum, only V . vulnificus biotype 1 revealed a steady multiplication accompanied with production of the extracellular metalloprotease . This prominent ability of biotype 1 in growth and protease production may contribute to cause serious systemic diseases in humans.

J Microencapsul, 2004 Feb, 21(1), 91 - 106
Inactive Vibrio cholerae whole-cell vaccine-loaded biodegradable microparticles: in vitro release and oral vaccination; Yeh M et al.; An approach is proposed using Vibrio cholerae (VC)-loaded microparticles as oral vaccine delivery systems for improved vaccine bioavailability and increased therapeutic efficacy . The VC-loaded microparticles were prepared with 50:50 poly(DL-lactide-co-glycolide) (PLG), 75:25 poly(DL-lactide-co-glycolide) and poly(lactide acid) (PLA)/PEG blend copolymers by the solvent evaporation method . VC was successfully entrapped in three types of microparticles with loading efficiencies and loading levels as follows: 50:50 PLG systems: 97.8% and 55.4 +/- 6.9 micro g/mg; 75:25 PLG systems: 89.2% and 46.5 +/- 4.4 micro g/mg; PLA/PEG-blended systems: 82.6% and 53.7 +/- 5.8 micro g/mg . The different distributions of VC in the core region and on the surface were as follows: 50:50 PLG systems 25.7 +/- 1.9 and 6.2 +/- 0.9 micro g/mg; 75:25 PLG systems: 25.8 +/- 2.2 and 3.6 +/- 0.4 micro g/mg; PLA/PEG-blended systems: 32.4 +/- 2.1 and 5.2 +/- 1.0 micro g/mg, respectively . In vitro active release of VC was affected mainly by matrix type and VC-loaded location in microparticles . The therapeutic immunogenic potential of VC loaded with 50:50 PLG, 75:25 PLG and PLA/PEG-blended microparticles was evaluated in adult mice by oral immunization . Significantly higher antibody responses and serum immunoglobin Ig G, IgA and IgM responses were obtained when sera from both VC-loaded 75:25 PLG and PLA/PEG-blended microparticles immunized mice were titrated against VC . The most immunogenicity in evoking serum IgG, IgA and IgM responses was immunized by VC-loaded PLA/PEG-blended microparticles, and with VC challenge in mice, the survival rate (91.7%).

Microbiol Res, 2003, 158(4), 299 - 308
Abundance of potentially pathogenic micro-organisms in Penaeus monodon larvae rearing systems in India; Vaseeharan B et al.; Monodon baculovirls (MBV), external fouling organisms (EFO) and bacteria (especially Vibrio species) were monitored during 1996-1997 at nine different Penaeus monodon rearing hatcheries in India . Total cultivable heterotrophic bacteria, Vibrio-like-bacteria, presumptive Vibrio harveyi, Vibrio anguillarum, Vibrio vulnificus counts were determined from shrimp eggs, post larvae, rearing tank water, source sea water, feed (Artemia nauplii and microencapsulated feed) . The MBV infected post larvae and their environment showed higher Vibrio-like-bacteria than uninfected post larvae . An overwhelming predominance of presumptive Vibrio harveyi and Vibrio anguillarum was observed in post larval rearing tank water, MBV infected and uninfected post larvae . Vibrio-like-bacteria in Artemia nauplii clearly showed the possible source of these pathogenic bacteria in the hatchery environments . Quantitative analysis of Vibrio-like-bacteria in hatcheries revealed that when the Vibrio-like-bacteria increases to 2 x 10(2) CFU mortality of the post larvae occurs . Abundance of these micro-organisms in hatchery samples indicated that they are opportunistic pathogens which can invade the shrimp tissue, subsequently cause disease when the post larvae were under stressful conditions.

An Sist Sanit Navar, 2003 Sep-Dec, 26(3), 429 - 31
{Spontaneous bacterial peritonitis and bacteraemia due to Aeromonas hydrophila}; Garcia-Irure JJ et al.; Bacterial peritonitis is a frequent complication in cirrhotic patients . Amongst the aetiological germs, Aeromonas is very infrequent . We present the clinical case of a cirrhotic patient with peritonitis and bacteraemia due to Aeromonas hydrophila . This micro-organism is a Gram-negative bacillus of the Vibrionaceae family . It very frequently produces gastroenteritis in the human being . Exceptionally it can give rise to extraintestinal infections, especially in immunodepressed patients . Peritonitis due to Aeromonas has been described in association with spontaneous bacterial peritonitis in cirrhotic patients, in patients on peritoneal dialysis and in cases of peritonitis secondary to intestinal perforation.

Environ Toxicol Chem, 2003 Dec, 22(12), 3037 - 43
Toxicity of mono- and diesters of o-phthalic esters to a crustacean, a green alga, and a bacterium; Jonsson S et al.; The degradation of phthalic acid diesters may lead to formation of o-phthalic acid and phthalic acid monoesters . The ecotoxic properties of the monoesters have never been systematically investigated, and concern has been raised that these degradation products may be more toxic than the diesters . Therefore, the aquatic toxicity of phthalic acid, six monoesters, and five diesters of o-phthalic acid was tested in three standardized toxicity tests using the bacteria Vibrio fischeri, the green algae Pseudokirchneriella subcapitata, and the crustacean Daphnia magna . The monoesters tested were monomethyl, monoethyl, monobutyl, monobenzyl, mono(2-ethylhexyl), and monodecyl phthalate, while the diesters tested were dimethyl, diethyl, dibutyl, butylbentyl, and di(2-ethylhexyl)phthalate, which were assumed to be below their water solubility . The median effective concentration (EC50) values for the three organisms ranged from 103 mg/L to >4.710 mg/L for phthalic acid, and corresponding values for the monoesters ranged from 2.3 mg/L (monodecyl phthalate in bacteria test) to 4,130 mg/L (monomethyl phthalate in bacteria test) . Dimethyl and diethyl phthalate were found to be the least toxic of the diesters (EC50 26.2-377 mg/L), and the toxicity of the other diesters (butylbenzyl and dibutyl phthalate) ranged from 0.96 to 7.74 mg/L . In general, the phthalate monoesters (degradation products) were less toxic than the corresponding diesters (mother compounds).

Appl Environ Microbiol, 2004 Jan, 70(1), 498 - 507
Rapid detection of Vibrio vulnificus in shellfish and Gulf of Mexico water by real-time PCR; Panicker G et al.; In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water) . Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87 degrees C for 84 clinical and environmental strains of V . vulnificus . No amplification was observed with other vibrios or nonvibrio strains with these primers . The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 10(2) V . vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water . It was possible to improve the level of detection to one V . vulnificus cell in samples that were enriched for 5 h . The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples . Detection of a single cell of V . vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines . The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay . Rapid and sensitive detection of V . vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.

Proc Natl Acad Sci U S A, 2004 Jan 13, 101(2), 627 - 31 Epub 2003 Dec 29.
The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase; Li X et al.; Chitin, a highly insoluble polymer of GlcNAc, is produced in massive quantities in the marine environment . Fortunately for survival of aquatic ecosystems, chitin is rapidly catabolized by marine bacteria . Here we describe a bacterial two-component hybrid sensor/kinase (of the ArcB type) that rigorously controls expression of approximately 50 genes, many involved in chitin degradation . The sensor gene, chiS, was identified in Vibrio furnissii and Vibrio cholerae (predicted amino acid sequences, full-length: 84% identical, 93% similar) . Mutants of chiS grew normally on GlcNAc but did not express extracellular chitinase, a specific chitoporin, or beta-hexosaminidases, nor did they exhibit chemotaxis, transport, or growth on chitin oligosaccharides such as (GlcNAc)(2) . Expression of these systems requires three components: wild-type chiS; a periplasmic high-affinity chitin oligosaccharide, (GlcNAc)(n) (n > 1), binding protein (CBP); and the environmental signal, (GlcNAc)(n) . Our data are consistent with the following model . In the uninduced state, CBP binds to the periplasmic domain of ChiS and "locks" it into the minus conformation . The environmental signal, (GlcNAc)(n), dissociates the complex by binding to CBP, releasing ChiS, yielding the plus phenotype (expression of chitinolytic genes) . In V . cholerae, a cluster of 10 contiguous genes (VC0620-VC0611) apparently comprise a (GlcNAc)(2) catabolic operon . CBP is encoded by the first, VC0620, whereas VC0619-VC0616 encode a (GlcNAc)(2) ABC-type permease . Regulation of chiS requires expression of CBP but not (GlcNAc)(2) transport . (GlcNAc)(n) is suggested to be essential for signaling these cells that chitin is in the microenvironment.

An Med Interna, 2003 Dec, 20(12), 630 - 2
{Septicemia caused by Vibrio cholerae non-01 in immunocompromised patient}; Calduch Broseta JV et al.; We describe the remarkable case of a patient with septicemia caused by Non 0-1 Vibrio Cholerae associated with skin lesion of the lower and upper extremities . This patient suffered from chronic liver disease and a cervix carcinoma in IIIB stage, she had been admitted to the hospital the day before for dicompensated ascites . She received intravenous cefotaxime and had a satisfactory recovery and was completely free of signs and symptoms . We report its epidemiological discovery in inland freshwater and this is the first announced case in Spain with this confirmed environmental isolation and a rare report case in the literature.

Anal Sci, 2003 Dec, 19(12), 1575 - 9
Presence of exclusively bacteriochlorophyll-c containing substrain in the culture of green sulfur photosynthetic bacterium Chlorobium vibrioforme strain NCIB 8327 producing bacteriochlorophyll-d; Saga Y et al.; The light-dependent composition change of light harvesting bacteriochlorophyll(BChl)s in the present culture of a green sulfur photosynthetic bacterium Chlorobium (Chl.) vibrioforme f . sp . thiosulfatophilum strain NCIB 8327 was investigated by visible absorption spectroscopy and HPLC analyses . When the culture was repeatedly grown in liquid media under a low light condition, both the Soret and Qy absorption bands of the in vivo spectrum were shifted to longer wavelengths . Analysis of the extracted pigments by HPLC revealed that the ratio of the amount of BChl-c to that of BChl-d molecules gradually increased during repeated cultivation . In contrast, when the culture grown under a low light intensity was transferred to a high light condition and continued to be grown, the absorption bands were shifted to shorter wavelengths and the ratio of BChls-c/d decreased finally to the almost original value . Colonies were prepared on solid agar media from the liquid culture containing both BChls-c and d, which was grown under a low light intensity . Each colony obtained was found to contain either BChl-c or d, but not both of them . Two types of cells isolated in this study were derived from the same clone, judged from their genetic analyses . The variation of pigment composition in our liquid culture observed here could be ascribed to the difference of growth rates between two substrains containing BChl-c and BChl-d, respectively, depending on light conditions.

Nature, 2004 Jan 1, 427(6969), 72 - 4 Epub 2003 Dec 21.
SOS response promotes horizontal dissemination of antibiotic resistance genes; Beaber JW et al.; Mobile genetic elements have a crucial role in spreading antibiotic resistance genes among bacterial populations . Environmental and genetic factors that regulate conjugative transfer of antibiotic resistance genes in bacterial populations are largely unknown . Integrating conjugative elements (ICEs) are a diverse group of mobile elements that are transferred by means of cell-cell contact and integrate into the chromosome of the new host . SXT is a approximately 100-kilobase ICE derived from Vibrio cholerae that encodes genes that confer resistance to chloramphenicol, sulphamethoxazole, trimethoprim and streptomycin . SXT-related elements were not detected in V . cholerae before 1993 but are now present in almost all clinical V . cholerae isolates from Asia . ICEs related to SXT are also present in several other bacterial species and encode a variety of antibiotic and heavy metal resistance genes . Here we show that SetR, an SXT encoded repressor, represses the expression of activators of SXT transfer . The 'SOS response' to DNA damage alleviates this repression, increasing the expression of genes necessary for SXT transfer and hence the frequency of transfer . SOS is induced by a variety of environmental factors and antibiotics, for example ciprofloxacin, and we show that ciprofloxacin induces SXT transfer as well . Thus, we present a mechanism by which therapeutic agents can promote the spread of antibiotic resistance genes.

Infect Immun, 2004 Jan, 72(1), 389 - 97
Induction of interleukin-8 in T84 cells by Vibrio cholerae; Zhou X et al.; The induction of interleukin-8 (IL-8) in vitro has been suggested to correlate with the reactogenicity of Vibrio cholerae vaccine candidates . V . cholerae vaccine candidate 638, a hemagglutinin protease/hap-defective strain, was recently reported to be well tolerated in human volunteers, suggesting a role for Hap in reactogenicity . We examined the role of hap in the induction of IL-8 in intestinal epithelial T84 cells . Wild-type V . cholerae strains 3038 and C7258 and a vaccine candidate strain, JBK70, induced levels of IL-8 similar to those of their isogenic hap mutants . Supernatant containing Hap did not stimulate IL-8 production at a variety of concentrations tested, suggesting that Hap itself does not induce IL-8 production . Furthermore, supernatant from CVD115, which had deletions of hap and rtxA (encoding repeats in toxin) and was derived from a reactogenic strain, CVD110, induced IL-8 production in T84 cells in a dose-dependent manner . The IL-8-stimulating activity of CVD115 culture supernatants was growth phase dependent and was strongest in stationary phase cultures . This IL-8 stimulator(s) was resistant to heat treatment but sensitive to proteinase . Protease activity in vitro did not correlate with the reactogenicity of V . cholerae vaccine candidates . Our data suggest that Hap is not an IL-8 inducer in T84 cells and that the IL-8 stimulator in the supernatant of V . cholerae culture may play a role in reactogenicity.

Lett Appl Microbiol, 2004, 38(1), 60 - 5
In vitro inhibition of microbial flora of fish by nisin and lactoperoxidase system; Elotmani F et al.; AIMS: To test the antimicrobial effects of nisin and lactoperoxidase system (LP system) against sardines flora . This study is part of a programme designed to investigate the preservability of fish using these inhibitors as potential biopreservatives . METHODS AND RESULTS: Antimicrobial effects of nisin and LP system alone or in combination were tested by the agar diffusion method against bacterial strains isolated from sardines (Sardina pilchardus) . Nisin inhibited only Gram-positive bacteria, whereas LP system inhibited all strains studied . The combination of nisin (100 IU ml-1) and LP system (10 level) was significantly more effective than LP system or nisin alone against all strains, excepting Aeromonas salmonicida subsp . salmonicida and Vibrio alginolyticus . CONCLUSION: These results clearly demonstrated the efficiency of LP System-nisin combination for inhibiting spoilage flora of fish . SIGNIFICANCE AND IMPACT OF THE STUDY: Because LP system has a broad activity spectrum, it may be an interesting additional hurdle to improve the safety of food preservation by nisin . Combination of nisin and LP system could be of great interest as biopreservatives for fish and fish products.

Gut, 2004 Jan, 53(1), 62 - 9
Acute dehydrating disease caused by Vibrio cholerae serogroups O1 and O139 induce increases in innate cells and inflammatory mediators at the mucosal surface of the gut; Qadri F et al.; BACKGROUND AND AIMS: The general concept is that as Vibrio cholerae is not invasive, it mediates a non-inflammatory type of infection . This is being re-evaluated based on available data that natural cholera infection or cholera toxin induces a Th2-type of immune profile and stimulates the humoral immune response, innate cells, and mediators in the host . METHODS: To perform a comprehensive analyses of the inflammatory components, we studied mucosal biopsies from patients, both adults and children with acute watery diarrhoea caused by V cholerae O1 and O139 . Patients with cholera, adults (n = 30) and children (n = 18), as well as healthy controls (n = 24) were studied . Histochemical, immunohistochemical, and ultrastructural studies were carried out to elucidate the contribution of the different factors using paraffin and frozen duodenal and/or rectal sections as appropriate . Samples were collected during the acute stage and during early and/or late convalescence . RESULTS: Following natural cholera infection, patients responded with increases in neutrophil polymorphs during the acute stage (p<0.001) compared with healthy controls whereas mucosal mast cells (MMC) (p = 0.008) and eosinophils (p = 0.034) increased in the gut during convalescence . Electron microscopic analyses of duodenal biopsies from adult patients showed increased piecemeal degranulation in both MMC and eosinophils and accumulation of lipid bodies in MMC . Duodenal biopsies from V cholerae O1 infected patients showed upregulation of myeloperoxidase, lactoferrin, PGHS-1, SCF, tryptase, tumour necrosis factor alpha, alpha-defensin, and eotaxin during the acute stage and chymase, interleukin 3 and major basic protein during convalescence . CONCLUSION: We have shown that innate cells and their mediators are upregulated in acute watery diarrhoea . These cells and factors of the innate arm may be important in the host's defence against cholera . Such effects may need to be simulated in a vaccine to achieve long lasting protection from cholera.

Microb Ecol, 2004 May, 47(4), 341 - 9
Chironomid egg masses as a natural reservoir of Vibrio cholerae non-O1 and non-O139 in freshwater habitats; Halpern M et al.; Cholera is a diarrheal disease caused by the gram-negative bacterium Vibrio cholerae, and an estimated 120,000 deaths from cholera occur globally every year . The natural reservoir of the bacterium is environmental . A recent report indicated an association between V . cholerae and chironomid egg masses . Chironomids, the "non-biting midges" (Diptera; Chironomidae), are the most widely distributed and frequently the most abundant insects in freshwater . Females attach egg masses, each containing hundreds of eggs encased in a layer of gelatin, to the water's edge where bacteria are abundant and may encounter the nutrient-rich substrate . Here we report the isolation of non-O1 and non-O139 V . cholerae from chironomid egg masses from different freshwater bodies in Israel, India, and Africa . In a yearly survey in Israel, chironomid populations were found to peak biannually, and it seemed that those peaks were followed by subsequent bacterial growth and disappearance during the winter in the Mediterranean region . The bacterial population rose as water temperature surpassed 25 degrees C . Thirty-five different serogroups of V . cholerae were identified among the bacteria isolated from chironomids, demonstrating population heterogeneity . Two strains of V . cholerae O37 and O201 that were isolated from chironomid egg masses in Zanzibar Island were NAG-ST positive . Our findings support the hypothesis that the association found between chironomids and the cholera bacteria is not a rare coincidence, indicating that chironomid egg masses may serve as yet another potential reservoir for V . cholerae.

FEMS Microbiol Lett, 2003 Dec 12, 229(2), 223 - 9
Cloning and molecular characterization of a unique hemolysin gene of Vibrio pommerensis sp . nov.: development of a DNA probe for the detection of the hemolysin gene and its use in identification of related Vibrio spp . from the Baltic Sea; Jores J et al.; A group of hemolytic Vibrio strains was isolated from surface water of the Baltic Sea in 1995 . A typical representative strain, CH-291, was found to lyse washed human and animal erythrocytes . Hemolysis was found to be calcium-dependent and occurred over a temperature range from 25 to 37 degrees C . The hemolysin-encoding genes were identified by screening a genomic library of total DNA from strain CH-291 . A cloned chromosomal DNA fragment of 15.6 kb conferred to Escherichia coli DH5alpha a hemolytic phenotype . Hybridization and sequence analysis showed the cloned sequence to be unique to these Baltic Sea Vibrio isolates and therefore provides a useful marker for their identification . Moreover, the cloned 15.6-kb DNA fragment possessed structural features typical for genetic islands, including a decreased GC content and a flanking cryptic insertion sequence element.

Clin Infect Dis, 2004 Jan 1, 38(1), 1 - 9 Epub 2003 Dec 08.
An Outbreak of Vibrio cholerae O1 infections on Ebeye Island, Republic of the Marshall Islands, associated with use of an adequately chlorinated water source; Beatty ME et al.; In December 2000, physicians in the Republic of the Marshall Islands reported the first known outbreak of Vibrio cholerae O1 infection (biotype El Tor, serotype Ogawa) from this country . In a matched case-control study on Ebeye Island, patients with cholera (n=53) had greater odds than persons without cholera (n=104) to have drunk adequately chlorinated water collected from a US military installation on neighboring Kwajalein Island and transported back to Ebeye (matched odds ratio {MOR}, 8.0; P=.01) . Transporting or storing drinking water in a water cooler with a spout and a tight-fitting lid was associated with reduced odds of illness (MOR, 0.24; P<.01), as was drinking bottled water (MOR, 0.08; P<.01), boiled water (MOR, 0.47; P=.02), or water flavored with powdered drink mixes (MOR, 0.18; P<.01) . No cases of cholera were reported among Kwajalein residents . This outbreak highlights the critical importance of handling and storing drinking water safely, especially during outbreaks of gastrointestinal illness.

Hybrid Hybridomics, 2003 Oct, 22(5), 315 - 20
Production and characterization of monoclonal antibodies to E1 Tor toxin co-regulated pilus of Vibrio cholerae; Falero G et al.; Murine monoclonal antibodies (MAbs) against Vibrio cholerae toxin co-regulated pilus (TCP) were generated using conventional hybridoma procedures . Four hybridomas were obtained and two characterized . Hybridomas 10E10E1 and 4D6F9 secreted antibodies of the IgG2a and IgG1 isotypes, respectively, that reacted with a 24-kDa antigen corresponding to the product of the El Tor tcpA gene fused to a six Histidine tail . Additionally, MAbs produced by 4D6F9 selectively recognized the major pilin subunit (TcpA) of El Tor and O139 vibrios in western immunoblot, while MAbs from 10E10E1 also cross-reacted with classical TcpA . Furthermore, vibrios expressing TCP on their surface selectively inhibited binding of the antibodies secreted by both hybridomas to TcpA-coated microtiter plates . Thus, the MAbs reported in this work detected the structural subunit of the pilus either denatured or assembled on the bacterial surface.

Chemosphere, 2004 Mar, 54(11), 1619 - 24
Screening organic micropollutants in surface waters by SPE extraction and ecotoxicological testing; Galassi S et al.; Complex mixtures of toxic substances occurring in surface waters are difficult to characterise by chemical analyses because each compound occurs at a very low concentration and requires a specific analytical method to be identified . Ecotoxicological tests on water extracts can be used as a screening tool to evaluate quickly and simply the overall quality of a water body with regard to micropollutant contamination . In this work, a pre-concentration procedure based on solid-phase extraction (SPE), suitable for both biological testing and analytical determination, is proposed . The extraction procedure is an improved version of a methodology used to evaluate the toxicity of organic micropollutants occurring in surface waters . It offers the advantage of using disposable commercial cartridges, which are easier to manage than the columns prepared with macromolecular resins . Water extracts from two representative Italian rivers, characterised by a different gradient of potential contamination and prepared according to the new concentration techniques, are used . The acute toxicity of the water extracts is tested on Daphnia magna and the bioluminescence inhibition in Vibrio fischeri . Criteria based on the concentration factor (CF) are proposed for assessing the hazard to aquatic life due to the exposure to toxic substances in surface waters . The aim of hazard ranking is to focus analytical efforts towards those samples that show the highest toxic potential.

FEBS Lett, 2003 Dec 18, 555(3), 638 - 42
The intestinal zonula occludens toxin (ZOT) receptor recognises non-native ZOT conformers and localises to the intercellular contacts; Lee A et al.; A preliminary structural analysis of Vibrio cholerae zonula occludens toxin (ZOT) was made by equilibrium denaturation and circular dichroism . ZOT is a structurally unstable protein in aqueous solution (DeltaG((H2O)) 3.82 kcal/mol), the putative intra- and extracellular domains unfold co-operatively, with complete denaturation via observed conformational intermediates . Refolding of denatured ZOT is not dependent on disulphide bridge formation . Partial refolding of a maltose binding protein-ZOT fusion did not prevent its specific binding to the ZOT receptor on Caco-2 cells . Immuno-gold labelling showed that the ZOT receptor localises to the intercellular contacts between cells in a confluent monolayer.

Water Res, 2004 Jan, 38(2), 289 - 300
Toxicity to Daphnia magna and Vibrio fischeri of Kraft bleach plant effluents treated by catalytic wet-air oxidation; Pintar A et al.; Two Kraft-pulp bleaching effluents from a sequence of treatments which include chlorine dioxide and caustic soda were treated by catalytic wet-air oxidation (CWAO) at T=463 K in trickle-bed and batch-recycle reactors packed with either TiO2 extrudates or Ru(3 wt%)/TiO2 catalyst . Chemical analyses (TOC removal, color, HPLC) and bioassays (48-h and 30-min acute toxicity tests using Daphnia magna and Vibrio fischeri, respectively) were used to get information about the toxicity impact of the starting effluents and of the treated solutions . Under the operating conditions, complex organic compounds are mostly oxidized into carbon dioxide and water, along with short-chain carboxylic acids . Bioassays were found as a complement to chemical analyses for ensuring the toxicological impact on the ecosystem . In spite of a large decrease of TOC, the solutions of end products were all more toxic to Daphnia magna than the starting effluents by factors ranging from 2 to 33 . This observation is attributed to the synergistic effects of acetic acid and salts present in the solutions . On the other hand, toxicity reduction with respect to Vibrio fischeri was achieved: detoxification factors greater than unity were measured for end-product solutions treated in the presence of the Ru(3 wt%)/TiO2 catalyst, suggesting the absence of cumulative effect for this bacteria, or a lower sensitivity to the organic acids and salts . Bleach plant effluents treated by the CWAO process over the Ru/TiO2 catalyst were completely biodegradable.

Cell, 2003 Dec 12, 115(6), 705 - 13
The bacterial cytoskeleton: an intermediate filament-like function in cell shape; Ausmees N et al.; Various cell shapes are encountered in the prokaryotic world, but how they are achieved is poorly understood . Intermediate filaments (IFs) of the eukaryotic cytoskeleton play an important role in cell shape in higher organisms . No such filaments have been found in prokaryotes . Here, we describe a bacterial equivalent to IF proteins, named crescentin, whose cytoskeletal function is required for the vibrioid and helical shapes of Caulobacter crescentus . Without crescentin, the cells adopt a straight-rod morphology . Crescentin has characteristic features of IF proteins including the ability to assemble into filaments in vitro without energy or cofactor requirements . In vivo, crescentin forms a helical structure that colocalizes with the inner cell curvatures beneath the cytoplasmic membrane . We propose that IF-like filaments of crescentin assemble into a helical structure, which by applying its geometry to the cell, generates a vibrioid or helical cell shape depending on the length of the cell.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Dec, 35(12), 1145 - 8
{Purification of a big defensin from Ruditapes philippinesis and its antibacterial activity}; Wei YX et al.; A novel big defensin was isolated and characterized from the plasma of Ruditapes philippinesis . It was purified to homogeneity by means of precipitation with (NH(4))(2)SO(4), gel-exclusion chromatography, two kinds of cation-exchange chromatography and named RPD-1 . Its relative molecular mass was 24.8 kD by means of SDS-PAGE . By means of ABI437 amino acid sequence analyser, its 11 NH(2)-terminal amino acid sequence is AVPDVAFNAYG . Two databank systems (NCBI and EBI/EMBL) was indexed, no sequence with homology to RPD-1 was found . Furthermore, it exhibited strong inhibition on the growth of Gram-negative and -positive bacteria . Minimal inhibitory concentration (MIC) of RPD-1 against Staphylococcus aureus, Bacillus subtilis, Micrococcus tetragenus, Escherichia coli, Vibrio parahaemolyticus, Vibrio anguillarum was 9.6 mg/L, 76.8 mg/L, 38.4 mg/L, 76.8 mg/L, 19.2 mg/L, 19.2 mg/L respectively.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Dec, 35(12), 1111 - 6
{Cloning and expression of tumor necrosis factor (TNFalpha) cDNA from red seabream pagrus major}; Cai ZH et al.; A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences . The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence . This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide . In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal . The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile . The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence . The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments . Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta . Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta . RT-PCR was used to study TNFalpha transcript expression . 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen . Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

J Food Prot, 2003 Dec, 66(12), 2283 - 8
Relationship between the effects of stress induced by human bile juice and acid treatment in Vibrio cholerae; Alvarez G et al.; The effects of low pH and human bile juice on Vibrio cholerae were investigated . A mild stress condition (exposure to acid shock at pH 5.5 or exposure to 3 mg of bile per ml for 20 min) slightly decreased (by < or = 1 log unit) V . cholerae cell viability . However, these treatments induced tolerance to subsequent exposures to more severe stress . In the O1 strain, four proteins were induced in response to acid shock (ca . 101, 94, 90, and 75 kDa), whereas only one protein (ca . 101 kDa) was induced in response to acid shock in the O139 strain . Eleven proteins were induced in response to bile shock in the O1 strain (ca . 106, 103, 101, 96, 88, 86, 84, 80, 66, 56, and 46 kDa), whereas only one protein was induced in response to bile shock in the O139 strain (ca . 88 kDa) . V . cholerae O1 and O139 cells that had been preexposed to mild acid shock were twofold more resistant to pH 4.5 (with times required to inactivate 90% of the cell population {D-values} of 59 to 73 min) than were control cells (with D-values of 24 to 27 min) . Likewise, cells that were preexposed to mild bile shock (3 mg/ml) were almost twofold more tolerant of severe bile shock (30 mg/ml; D-values, 68 to 87 min) than were control cells (with D-values of 37 to 43 min) . These protective effects persisted for at least 1 h after the initial shock but were abolished when chloramphenicol was added to the culture during the shock . Cells preexposed to acid shock exhibited cross-protection against subsequent bile shock . However, cells preexposed to bile shock exhibited no changes in acid tolerance . Bile shock induced a modest reduction (0 to 20%) in enterotoxin production in V . cholerae, whereas acid shock had no effect on enterotoxin levels . Adaptation to acid and bile juice and protection against bile shock in response to preexposure to acid shock would be predicted to enhance the survival of V . cholerae in hosts and in foods . Thus, these adaptations may play an important role in the development of cholera disease.

J Food Prot, 2003 Dec, 66(12), 2276 - 82
Sensitivity of Vibrio species in phosphate-buffered saline and in oysters to high-pressure processing; Cook DW; Multiple strains of Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio cholerae non-O1 were tested in phosphate-buffered saline for their sensitivity to high-pressure processing (HPP) . Variability in sensitivity among strains was observed for all species; this variability decreased at higher pressures . V . vulnificus was the species that was most sensitive to treatment at 200 MPa (decimal reduction time {D} = 26 s), and V . cholerae was the species that was most resistant to treatment at 200 MPa (D = 149 s) . The O3:K6 serotype of V . parahaemolyticus was more resistant to pressure than other serotypes of V . parahaemolyticus were . The results of studies involving V . vulnificus naturally occurring in oysters revealed that a pressure treatment of 250 MPa for 120 s achieved a > 5-log reduction in the levels of this bacterium . V . parahaemolyticus serotype O3:K6 in oysters required a pressure of 300 MPa for 180 s for a comparable 5-log reduction . When properly applied, HPP can be effective in improving the safety of shellfish with respect to Vibrio spp.

Plant Physiol, 2004 Jan, 134(1), 137 - 46 Epub 2003 Dec 11.
Chlamydomonas reinhardtii secretes compounds that mimic bacterial signals and interfere with quorum sensing regulation in bacteria; Teplitski M et al.; The unicellular soil-freshwater alga Chlamydomonas reinhardtii was found to secrete substances that mimic the activity of the N-acyl-L-homoserine lactone (AHL) signal molecules used by many bacteria for quorum sensing regulation of gene expression . More than a dozen chemically separable but unidentified substances capable of specifically stimulating the LasR or CepR but not the LuxR, AhyR, or CviR AHL bacterial quorum sensing reporter strains were detected in ethyl acetate extracts of C . reinhardtii culture filtrates . Colonies of C . reinhardtii and Chlorella spp . stimulated quorum sensing-dependent luminescence in Vibrio harveyi, indicating that these algae may produce compounds that affect the AI-2 furanosyl borate diester-mediated quorum sensing system of Vibrio spp . Treatment of the soil bacterium Sinorhizobium meliloti with a partially purified LasR mimic from C . reinhardtii affected the accumulation of 16 of the 25 proteins that were altered in response to the bacterium's own AHL signals, providing evidence that the algal mimic affected quorum sensing-regulated functions in this wild-type bacterium . Peptide mass fingerprinting identified 32 proteins affected by the bacterium's AHLs or the purified algal mimic, including GroEL chaperonins, the nitrogen regulatory protein PII, and a GTP-binding protein . The algal mimic was able to cancel the stimulatory effects of bacterial AHLs on the accumulation of seven of these proteins, providing evidence that the secretion of AHL mimics by the alga could be effective in disruption of quorum sensing in naturally encountered bacteria.

Carbohydr Res, 2003 Nov 14, 338(23), 2711 - 9
Structural characterization of the carbohydrate backbone of the lipopolysaccharide of Vibrio parahaemolyticus O-untypeable strain KX-V212 isolated from a patient; Hashii N et al.; Vibrio parahaemolyticus strain KX-V212 of a novel serotype, which does not belong to any of the known 13 O-serotypes of this vibrio, was isolated from a patient . Its O-antigen harbors a unique strain-specific O-antigenic factor(s), in addition to that shared by the O-antigen of V . parahaemolyticus serotype O2 . A carbohydrate backbone nonasaccharide was isolated from the lipopolysaccharide (LPS) of strain KX-V212 by dephosphorylation, reduction and deacylation and found to consist of one residue each of D-glucose, D-galactose, D-GlcN, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and 5-acetamido-7-(N-acetyl-D-alanyl)amino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (Non5Ac7Ala), and two residues each of D-GlcA and L-glycero-D-manno-heptose (LD-Hep) . Analysis of the isolated and deacylated lipid A showed that this oligosaccharide was an artifact resulting from a loss of one GlcN residue from the lipid A backbone . Therefore, the carbohydrate backbone of the LPS is a decasaccharide having the structure shown below . The initial LPS contains also D-GalA and phosphoethanolamine at unknown positions . Both similarity and differences are observed between the LPS of V . parahaemolyticus serotype O2 and strain KX-V212 . {carbohydrate structure: see text}

Carbohydr Res, 2003 Nov 14, 338(23), 2591 - 603
One-pot preparation of a series of glycoconjugates with predetermined antigen-carrier ratio from oligosaccharides that mimic the O-PS of Vibrio cholerae O:1, serotype Ogawa; Saksena R et al.; Di-through the pentasaccharide that mimic the upstream terminus of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Ogawa were synthesized in the form of 5-methoxycarbonylpentyl glycosides and linked to BSA using squaric acid diester chemistry . The conjugation reactions were monitored by surface-enhanced laser-desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF MS), which allowed conducting the conjugation of the synthetic oligosaccharides in a controlled way and termination of the reaction when the desired molar hapten-BSA ratio had been reached . This made it possible to prepare, from one hapten in a one-pot reaction, a series of neoglycoconjugates having different, predetermined carbohydrate-carrier ratios . The accuracy of molecular mass determination in SELDI-TOF MS analysis could be increased by using the carrier protein as the internal standard.

Vaccine, 2004 Jan 2, 22(3-4), 416 - 21
Suppressive effect of zinc on antibody response to cholera toxin in children given the killed, B subunit-whole cell, oral cholera vaccine; Qadri F et al.; In a previous study, children aged 2-5 years old in Bangladesh were supplemented orally with a single dose of Vitamin A (200,000 IU) and a placebo for zinc (zinc equivalent to 20 mg of elemental zinc) everyday for 42 days (group A), zinc and a placebo for Vitamin A (group Z), zinc and Vitamin A (group AZ) or both placebos (group P) . All children were orally immunised with two doses of the killed cholera vaccine containing whole cells and a recombinant B subunit of cholera toxin (CT) . The number of children who responded with > or = 4-fold vibriocidal antibody (a proxy indicator of protection against cholera) was significantly greater among the zinc-supplemented groups than among the non-zinc-supplemented groups, while Vitamin A supplementation did not appear to have any effect . The sera from these children were assayed for antibody to CT . Antibody to CT is known to exert a synergistic protective effect against cholera in animal studies, and offer significantly higher short-term protection against cholera and significant short-term protection against enterotoxigenic Escherichia coli diarrhoea in humans on oral immunisation with the cholera vaccine . Children who received zinc had significantly reduced levels of serum antibodies to CT than children who received placebos only . Factorial analysis showed a trend for zinc showing a reduction in the number of children responding with CT-antibody, while Vitamin A did not appear to have any effect . Thus, zinc enhanced vibriocidal antibody response, but suppressed CT-antibody response, suggesting that zinc supplementation has different modulating effects on vibriocidal antibody response and CT-antibody response.

Curr Microbiol, 2003 Nov, 47(5), 379 - 82
Experimental evidence for the physiological role of bacterial luciferase in the protection of cells against oxidative stress; Szpilewska H et al.; The origin and function of bioluminescence was considered a problematic question of the Charles Darwin theory . Early evolution of bacterial luminescence and its current physiological importance seem to be especially mysterious . Recently, it was proposed that stimulation of DNA repair may be a physiological role for production of light by bacterial cells . On the other hand, it was also proposed that primary role of luminescent systems could be detoxification of the deleterious oxygen derivatives . Although some previous results might suggest that this hypothesis can be correct, until now experimental evidence for such a mechanism operating in bacterial cells and having physiological importance was generally lacking . Here we demonstrate that in the presence of various oxidants (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, and ferrous ions) at certain concentrations in the culture medium, growth of Vibrio harveyi mutants luxA and luxB, but not of the mutant luxD, is severely impaired relative to wild-type bacteria . This deleterious effect of oxidants on the mutants luxA and luxB could be significantly reduced by addition of the antioxidants A-TEMPO or 40H-TEMPO . We conclude that bacterial luciferase may indeed play a physiological role in the protection of cells against oxidative stress.

Dis Aquat Organ, 2003 Oct 24, 56(3), 249 - 58
Salinity effects on immune parameters of Ruditapes philippinarum challenged with Vibrio tapetis; Reid HI et al.; The occurrence of brown ring disease (BRD) in farmed Manila clams Ruditapes philippinarum is seasonal . Development of the disease is believed to require the presence of the infective agent Vibrio tapetis and particular environmental conditions . This paper studies the effect of salinity (20 to 40 per thousand) on measurable immune parameters of Manila clams, and the progression of BRD in experimentally infected individuals . At 20 per thousand salinity, the total haemocyte count was reduced and disease prevalence was highest . At 40 per thousand salinity significantly fewer clams presented signs of BRD, and this was correlated with increases in the total haemocyte count, hyalinocyte count, phenoloxidase levels and phagocytic activity of haemocytes . Inoculation of clams with V . tapetis did not have a significant effect on the immune parameters measured . Thus, this laboratory-based study relates environmental stress to disease development.

J Biol Chem, 2004 Feb 27, 279(9), 8070 - 5 Epub 2003 Dec 04.
Lipopolysaccharide 3-deoxy-D-manno-octulosonic acid (Kdo) core determines bacterial association of secreted toxins; Horstman AL et al.; In contrast to cholera toxin (CT), which is secreted solubly by Vibrio cholerae across the outer membrane, heat-labile enterotoxin (LT) is retained on the surface of enterotoxigenic Escherichia coli (ETEC) via an interaction with lipopolysaccharide (LPS) . We examined the nature of the association between LT and LPS . Soluble LT binds to the surface of LPS deep-rough biosynthesis mutants but not to lipid A, indicating that only the Kdo (3-deoxy-d-manno-octulosonic acid) core is required for binding . Although capable of binding truncated LPS and Kdo, LT has a higher affinity for longer, more complete LPS species . A putative LPS binding pocket is proposed based on the crystal structure of the toxin . The ability to bind LPS and remain associated with the bacterial surface is not unique to LT, as CT also binds to E . coli LPS . However, neither LT nor CT is capable of binding to the surface of Vibrio . The core structures of Vibrio and E . coli LPS differ in that Vibrio contains a phosphorylated single Kdo-lipid A, and E . coli LPS contains unphosphorylated Kdo2-lipid A . We determined that the phosphate group on the Kdo core of Vibrio LPS prevents CT from binding, resulting in the secretion of soluble toxin . Because LT binds E . coli LPS, it remains associated with the extracellular bacterial surface and is released in association with outer membrane vesicles . We propose that difference in the extracellular fates of LT and CT contribute to the differences in disease caused by ETEC and Vibrio cholerae.

Appl Environ Microbiol, 2003 Dec, 69(12), 7541 - 4
Prevalence of cholera toxin genes (ctxA and zot) among non-O1/O139 Vibrio cholerae strains from Newport Bay, California; Jiang S et al.; The examination of 137 non-O1/O139 Vibrio cholerae isolates from Newport Bay, California, indicated the presence of diverse genotypes and a temporal succession . Unexpectedly, the cholera toxin gene (ctxA) was found in 17% of the strains, of which one-third were also positive for the zot gene . This suggests that ctxA is prevalent in the region of nonepidemicity and is likely to have an environmental origin.

Appl Environ Microbiol, 2003 Dec, 69(12), 7527 - 30
Chemoattraction of Vibrio fischeri to serine, nucleosides, and N-acetylneuraminic acid, a component of squid light-organ mucus; DeLoney-Marino CR et al.; Newly hatched juveniles of the Hawaiian squid Euprymna scolopes rapidly become colonized by the bioluminescent marine bacterium Vibrio fischeri . Motility is required to establish the symbiotic colonization, but the role of chemotaxis is unknown . In this study we analyzed chemotaxis of V . fischeri to a number of potential attractants . The bacterium migrated toward serine and most sugars tested . V . fischeri also exhibited the unusual ability to migrate to nucleosides and nucleotides as well as to N-acetylneuraminic acid, a component of squid mucus.

Appl Environ Microbiol, 2003 Dec, 69(12), 7435 - 46
Characterization of Vibrio fluvialis-like strains implicated in limp lobster disease; Tall BD et al.; Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism . Nineteen isolates were obtained from eight of nine lobsters sampled . Biochemically, the isolates resembled V . fluvialis, and the isolates grew optimally at 20 degrees C; none could grow at temperatures above 23 degrees C . The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37 degrees C . All of the isolates required at least 1% NaCl for growth . Collectively, the data suggest that these isolates may embody a new biotype . Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups . Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease . Several isolates possessed capsules . The isolates were highly susceptible to a variety of antibiotics tested . However, six isolates were resistant to erythromycin . Seventeen isolates harbored plasmids . Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters . Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain . Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph . Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin . Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice . Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process . Further, these results support the thermal reduction data at 37 degrees C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids . In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V . fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.

Appl Environ Microbiol, 2003 Dec, 69(12), 7371 - 6
Effects of saxitoxin (STX) and veratridine on bacterial Na+ -K+ fluxes: a prokaryote-based STX bioassay; Pomati F et al.; Saxitoxin (STX) is a potent natural sodium channel blocker and represents a significant health concern worldwide . We describe here the antagonistic effects of STX and veratridine (VTD), an Na+ channel activator, on three gram-negative bacteria and their application to an STX bioassay . STX reduced the total cellular levels of both Na+ and K+, as measured by flame photometry, whereas VTD increased the cellular concentrations relative to control ion fluxes in the cyanobacterium Cylindrospermopsis raciborskii AWT205 . Endogenous STX production in toxic cyanobacterial strains of C . raciborskii and Anabaena circinalis prevented cell lysis induced by VTD stress . Microscopic cell counts showed that non-STX producing cyanobacteria displayed complete cell lysis and trichome fragmentation 5 to 8 h after addition of VTD and vanadate (VAN), an inhibitor of sodium pumps . The addition of STX, or its analogue neoSTX, prior to treatment with VTD plus VAN prevented complete lysis in non-STX-producing cyanobacteria . VTD also affected cyanobacterial metabolism, and the presence of exogenous STX in the sample also ameliorated this decrease in metabolic activity, as measured by the cellular conversion of tetrazolium into formazan . Reduced primary metabolism was also recorded as a decrease in the light emissions of Vibrio fischeri exposed to VTD . Addition of STX prior to VTD resulted in a rapid and dose-dependent response to the presence of the channel blocker, with samples exhibiting resistance to the VTD effect . Our findings demonstrate that STX and VTD influence bacterial Na+ and K+ fluxes in opposite ways, and these principles can be applied to the development of a prokaryote-based STX bioassay.

Appl Environ Microbiol, 2003 Dec, 69(12), 7137 - 44
Real-time PCR analysis of Vibrio vulnificus from oysters; Campbell MS et al.; Vibrio vulnificus is an opportunistic human pathogen commonly found in estuarine environments . Infections are associated with raw oyster consumption and can produce rapidly fatal septicemia in susceptible individuals . Standard enumeration of this organism in shellfish or seawater is laborious and inaccurate; therefore, more efficient assays are needed . An oligonucleotide probe derived from the cytolysin gene, vvhA, was previously used for colony hybridizations to enumerate V . vulnificus . However, this method requires overnight growth, and vibrios may lack culturability under certain conditions . In the present study, we targeted the same locus for development of a TaqMan real-time PCR assay . Probe specificity was confirmed by amplification of 28 V . vulnificus templates and by the lack of a PCR product with 22 non-V . vulnificus strains . Detection of V . vulnificus in pure cultures was observed over a 6-log-unit linear range of concentration (10(2) to 10(8) CFU ml(-1)), with a lower limit of 72 fg of genomic DNA micro l of PCR mixture(-1) or the equivalent of six cells . Similar sensitivity was observed in DNA extracted from mixtures of V . vulnificus and V . parahaemolyticus cells . Real-time PCR enumeration of artificially inoculated oyster homogenates correlated well with colony hybridization counts (r(2) = 0.97) . Numbers of indigenous V . vulnificus cells in oysters by real-time PCR showed no significant differences from numbers from plate counts with probe (t test; P = 0.43) . Viable but nonculturable cells were also enumerated by real-time PCR and confirmed by the BacLight viability assay . These data indicate that real-time PCR can provide sensitive species-specific detection and enumeration of V . vulnificus in seafood.

FEMS Microbiol Lett, 2003 Dec 5, 229(1), 1 - 7
The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism; Heipieper HJ et al.; Isomerization of cis to trans unsaturated fatty acids is a mechanism enabling Gram-negative bacteria belonging to the genera Pseudomonas and Vibrio to adapt to several forms of environmental stress . The extent of the isomerization apparently correlates with the fluidity effects caused, i.e . by an increase in temperature or the accumulation of membrane-toxic organic compounds . Trans fatty acids are generated by direct isomerization of the respective cis configuration of the double bond without a shift of its position . The conversion of cis unsaturated fatty acids to trans is apparently instrumental in the adaptation of membrane fluidity to changing chemical or physical parameters of the cellular environment . Such an adaptive mechanism appears to be an alternative way to regulate membrane fluidity when growth is inhibited, e.g . by high concentrations of toxic substances . The cis-trans isomerase (Cti) activity is constitutively present and is located in the periplasma, it requires neither ATP nor any other cofactor such as NAD(P)H or glutathione, and it operates in the absence of de novo synthesis of lipids . Its independence from ATP is in agreement with the negative free energy of the reaction . cti encodes a polypeptide with an N-terminal hydrophobic signal sequence, which is cleaved off during or shortly after the enzyme is transported across the cytoplasmic membrane to the periplasmic space . A functional heme-binding site of the cytochrome c-type was identified in the predicted Cti polypeptide and very recently, direct evidence was obtained that isomerization does not include a transient saturation of the double bond.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 2019 - 26
Evolution of symbiosis in the Vibrionaceae: a combined approach using molecules and physiology; Nishiguchi MK et al.; The family Vibrionaceae is considered to be one of the most diverse and well-studied groups of bacteria . Here, evolution is assessed within the Vibrionaceae to determine whether multiple origins of eukaryotic associations have occurred within this diverse group of bacteria . Analyses were based on a large molecular dataset, along with a matrix that consisted of 100 biochemical and restriction digest characters . By using direct optimization methods to analyse both datasets individually and in combination, a total-evidence cladogram has been produced, which supports the hypothesis that several important symbionts (both mutualistic and pathogenic) within the Vibrionaceae are not monophyletic . This leads us to consider that symbiosis (and subsequently, associations with Eukarya) has evolved multiple times within the Vibrionaceae lineage.

Int J Syst Evol Microbiol, 2003 Nov, 53(Pt 6), 1813 - 7
Vibrio superstes sp . nov., isolated from the gut of Australian abalones Haliotis laevigata and Haliotis rubra; Hayashi K et al.; Five alginolytic, facultatively anaerobic, non-motile bacteria were isolated from the gut of abalones Haliotis laevigata and Haliotis rubra . Phylogenetic analyses based on 16S rDNA data indicated that these strains are related closely to Vibrio halioticoli (98 % 16S rDNA sequence similarity) . DNA-DNA hybridization and fluorescent amplified fragment length polymorphism fingerprinting demonstrated that the five strains constituted a single species that was different from all currently known vibrios . The name Vibrio superstes sp . nov . (type strain, LMG 21323(T)=IAM 15009(T)=G3-29(T); DNA G+C content, 48.0-48.9 mol%) is proposed to encompass this novel taxon . Several phenotypic features were disclosed that discriminate V . superstes from other Vibrio species: V . superstes sp . nov . and V . halioticoli can be differentiated on the basis of 17 traits (indole production, beta-galactosidase test and assimilation of 15 carbon compounds).

Eur J Clin Microbiol Infect Dis, 2004 Jan, 23(1), 49 - 52 Epub 2003 Dec 04.
Sepsis with bullous necrotizing skin lesions due to vibrio vulnificus acquired through recreational activities in the Baltic Sea; Kuhnt-Lenz K et al.; This report describes the case of a 59-year-old woman with a history of non-Hodgkin's lymphoma who developed bacteremia with Vibrio vulnificus . The patient had been swimming in the unusually warm Baltic Sea in the summer of 2002 . She presented with symptoms of septicemia and severe bullous necrotizing skin lesions of the extremities . Blood culture revealed Vibrio vulnificus as the pathogenic organism . Under treatment with cefotaxime and gentamicin, she recovered slowly without further complications . Vibrio vulnificus is a marine bacterium that is present in aquatic ecosystems worldwide, especially when water temperatures exceed 20 degrees C . Infections with Vibrio vulnificus are uncommon in Europe, and most cases are reported from subtropical or tropical regions.

FEMS Yeast Res, 2003 Dec, 4(3), 305 - 13
Expression of the Photorhabdus luminescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae; Gupta RK et al.; The luxA, B, C, D, and E genes from Photorhabdus luminescens were cloned and functionally expressed in Saccharomyces cerevisiae to construct a bacterial lux-based yeast bioreporter capable of autonomous bioluminescence emission . The bioreporter was engineered using a series of pBEVY yeast expression vectors that allowed for bi-directional constitutive or inducible expression of the individual luxA, B, C, and E genes . The luxD gene, encoding the acyl-ACP transferase that ultimately supplies the requisite aldehyde substrate for the bioluminescent reaction, was fused to a yeast internal ribosomal entry site (IRES) sequence to ensure high bi-cistronic expression . Although self-generation of bioluminescence was achieved by the bioreporter, the signal was relatively weak and decayed rapidly . To overcome this instability, a flavin oxidoreductase gene (frp) from Vibrio harveyi was co-expressed to provide sufficient concentrations of the FMNH(2) co-factor required for the bioluminescent reaction . Expression of frp with the lux genes not only stabilized but also enhanced bioluminescence to levels approaching 9.0x10(5) times above background.

J Bacteriol, 2003 Dec, 185(24), 7231 - 40
Novel type of specialized transduction for CTX phi or its satellite phage RS1 mediated by filamentous phage VGJ phi in Vibrio cholerae; Campos J et al.; The main virulence factor of Vibrio cholerae, the cholera toxin, is encoded by the ctxAB operon, which is contained in the genome of the lysogenic filamentous phage CTX phi . This phage transmits ctxAB genes between V . cholerae bacterial populations that express toxin-coregulated pilus (TCP), the CTX phi receptor . In investigating new forms of ctxAB transmission, we found that V . cholerae filamentous phage VGJ phi, which uses the mannose-sensitive hemagglutinin (MSHA) pilus as a receptor, transmits CTX phi or its satellite phage RS1 by an efficient and highly specific TCP-independent mechanism . This is a novel type of specialized transduction consisting in the site-specific cointegration of VGJ phi and CTX phi (or RS1) replicative forms to produce a single hybrid molecule, which generates a single-stranded DNA hybrid genome that is packaged into hybrid viral particles designated HybP phi (for the VGJ phi/CTX phi hybrid) and HybRS phi (for the VGJ phi/RS1 hybrid) . The hybrid phages replicate by using the VGJ phi replicating functions and use the VGJ phi capsid, retaining the ability to infect via MSHA . The hybrid phages infect most tested strains more efficiently than CTX phi, even under in vitro optimal conditions for TCP expression . Infection and lysogenization with HybP phi revert the V . cholerae live attenuated vaccine strain 1333 to virulence . Our results reinforce that TCP is not indispensable for the acquisition of CTX phi . Thus, we discuss an alternative to the current accepted evolutionary model for the emergence of new toxigenic strains of V . cholerae and the importance of our findings for the development of an environmentally safer live attenuated cholera vaccine.

J Bacteriol, 2003 Dec, 185(24), 7202 - 12
GacA regulates symbiotic colonization traits of Vibrio fischeri and facilitates a beneficial association with an animal host; Whistler CA et al.; The GacS/GacA two-component system regulates the expression of bacterial traits during host association . Although the importance of GacS/GacA as a regulator of virulence is well established, its role in benign associations is not clear, as mutations in either the gacS or gacA gene have little impact on the success of colonization in nonpathogenic associations studied thus far . Using as a model the symbiotic association of the bioluminescent marine bacterium Vibrio fischeri with its animal host, the Hawaiian bobtail squid, Euprymna scolopes, we investigated the role of GacA in this beneficial animal-microbe interaction . When grown in culture, gacA mutants were defective in several traits important for symbiosis, including luminescence, growth in defined media, growth yield, siderophore activity, and motility . However, gacA mutants were not deficient in production of acylated homoserine lactone signals or catalase activity . The ability of the gacA mutants to initiate squid colonization was impaired but not abolished, and they reached lower-than-wild-type population densities within the host light organ . In contrast to their dark phenotype in culture, gacA mutants that reached population densities above the luminescence detection limit had normal levels of luminescence per bacterial cell in squid light organs, indicating that GacA is not required for light production within the host . The gacA mutants were impaired at competitive colonization and could only successfully cocolonize squid light organs when present in the seawater at higher inoculum densities than wild-type bacteria . Although severely impaired during colonization initiation, gacA mutants were not displaced by the wild-type strain in light organs that were colonized with both strains . This study establishes the role of GacA as a regulator of a beneficial animal-microbe association and indicates that GacA regulates utilization of growth substrates as well as other colonization traits.

J Nat Prod, 2003 Nov, 66(11), 1520 - 3
New indole alkaloids from the North Sea bacterium Vibrio parahaemolyticus Bio249; Veluri R et al.; Several bis- and tris-indole derivatives were isolated from a North Sea bacterium that was closely related to Vibrio parahaemolyticus (98% homology) . 1,1,3-Tris(3-indolyl)butane (3) is a new compound, and 3,3-bis(3-indolyl)butane-2-one (1a), arundine (1b), and 1,1,1-tris(3-indolyl)methane (2a) were isolated from a microorganism for the first time here . Additionally, many other known compounds were obtained from the ethyl acetate extract of the culture . Their structures were established on the basis of various spectral data, and their origin is discussed . All compounds were inactive against a range of bacteria and fungi.

FEMS Microbiol Lett, 2003 Nov 21, 228(2), 193 - 201
Involvement of region 4 of the sigma70 subunit of RNA polymerase in transcriptional activation of the lux operon during quorum sensing; Johnson DC et al.; Quorum sensing-dependent activation of the luminescence (lux) genes of Vibrio fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl)-L-homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR . In its active conformation, LuxR binds to a site known as the lux box centered at position -42.5 relative to the luxI transcriptional start site and is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP) . The specific role of region 4 of the Escherichia coli sigma70 subunit of RNAP in LuxR-dependent activation of the luxI promoter has been investigated . Single-round transcription assays were performed in the presence of purified LuxRDeltaN, the autoinducer-independent C-terminal domain of LuxR, and a variant RNAP which contained a C-terminally truncated sigma70 subunit devoid of region 4 . Results indicated that region 4 is essential for LuxRDeltaN-dependent luxI transcription, therefore 16 single and two triple alanine substitutions in region 4.2 of sigma70 between amino acid residues 590 and 613 were examined for their effects on LuxR- and LuxRDeltaN-dependent transcription at the luxI promoter . Taken together, the analyses performed on these variants of RpoD suggest that some individual residues in region 4.2 are important to the mechanism of activator-dependent transcription initiation under investigation.

J Appl Microbiol, 2003, 95(5), 1106 - 16
Intraspecific characterization of Vibrio alginolyticus isolates recovered from cultured fish in Spain; Zorrilla I et al.; AIMS: Intraspecific differentiation and characterization of Vibrio alginolyticus strains isolated from cultured fish in Spain . MATERIALS AND RESULTS: Thirty-four Vibrio alginolyticus strains isolated from cultured fish were intraspecifically characterized on the basis of biochemical and exoenzymatic patterns, outer membrane protein (OMP) profiles, ribotyping and plasmid analyses . The typing methods used did not allow to group V . alginolyticus isolates on the basis of their sources of collection . A higher homogeneity was observed in OMP profiles . A high percentage of isolates were plasmidless . Ribotyping was the highest discriminatory typing method, as all the isolates tested presented 23 profiles using the HindIII restriction enzyme . On the basis of the ribotyping pattern, a similarity matrix and a dendrogram were constructed . CONCLUSIONS: The results obtained indicate that V . alginolyticus strains isolated from southwestern Spain belong to different clonal lineages . SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown differences with other similar studies carried out in other areas of Europe with strains of V . alginolyticus with respect to the clonal lineages of the strains isolated in southwestern Spain.

J Appl Microbiol, 2003, 95(6), 1293 - 303
The use of PCR to aid in the rapid identification of Vibrio harveyi isolates; Oakey HJ et al.; AIMS: Vibrio harveyi is an important pathogen, causing potential devastation to marine aquaculture . This organism, however, is extremely difficult to identify because it is phenotypically diverse . Biochemical identification can involve many tests and take weeks to perform . The aim of this work is to develop a PCR that can reduce the number of biochemical tests, and the time taken, to get a definitive identification of this organism . METHODS AND RESULTS: The PCR was developed using 16S rDNA sequences from a number of V . harveyi strains, and other vibrios . The described test gave positive results for all strains of V . harveyi tested . However, some strains of V . alginolyticus also gave positive results and a small number of biochemical tests were required to differentiate between these two species . This indicated that preisolation of the bacteria was needed and therefore the test was not applicable to the testing of mixed populations directly . CONCLUSION, SIGNIFICANCE AND IMPACT OF THE STUDY: The duration of identification of this species was significantly reduced from a number of weeks to a few days . Hence, diagnosis of affected animals will be faster and earlier treatment can be administered which may increase the survival rate from vibriosis.

J Appl Microbiol, 2003, 95(6), 1277 - 84
A biochemical protocol for the isolation and identification of current species of Vibrio in seafood; Ottaviani D et al.; AIMS: We report a biochemical method for the isolation and identification of the current species of vibrios using just one operative protocol . METHODS AND RESULTS: The method involves an enrichment phase with incubation at 30 degrees C for 8-24 h in alkaline peptone water and an isolation phase on thiosulphate-citrate-salt sucrose agar plates incubating at 30 degrees C for 24 h . Four biochemical tests and Alsina's scheme were performed for genus and species identification, respectively . All biochemical tests were optimized as regards conditions of temperature, time of incubation and media composition . The whole standardized protocol was always able to give a correct identification when applied to 25 reference strains of Vibrio and 134 field isolates . CONCLUSIONS: The data demonstrated that the assay method allows an efficient recovery, isolation and identification of current species of Vibrio in seafood obtaining results within 2-7 days . SIGNIFICANCE AND IMPACT OF THE STUDY: This method based on biochemical tests could be applicable even in basic microbiology laboratories, and can be used simultaneously to isolate and discriminate all clinically relevant species of Vibrio.

Farmaco, 2003 Dec, 58(12), 1251 - 5
Quinoxalin-2-ones . Part 5 . Synthesis and antimicrobial evaluation of 3-alkyl-, 3-halomethyl- and 3-carboxyethylquinoxaline-2-ones variously substituted on the benzo-moiety; Carta A et al.; A new series of 3-alkyl-, 3-trifluoromethyl-, 3-carboxyethyl- and 3-bromomethylquinoxaline-2-ones and 2,3-bis(bromomethyl)quinoxalines bearing Cl, CF3, morpholine on the benzo-moiety, were synthesised and submitted to a preliminary in vitro evaluation for antibacterial and anticandida activities . Results of the screening showed that compounds 9b, 14b and 19b (MIC=62.5 microg/ml) and 10b (MIC=15.6 microg/ml) were the most active against Vibrio alginolyticus.

J Mol Biol, 2003 Nov 28, 334(3), 567 - 83
The conserved charged residues of the C-terminal region of FliG, a rotor component of the Na+-driven flagellar motor; Yorimitsu T et al.; FliG is an essential component of the flagellar motor and functions in flagellar assembly, torque generation and regulation of the direction of flagellar rotation . The five charged residues important for the rotation of the flagellar motor were identified in Escherichiacoli FliG (FliG(E)) . These residues are clustered in the C terminus and are all conserved in FliG(V) of the Na(+)-driven motor of Vibrioalginolyticus (Lys284, Arg301, Asp308, Asp309 and Arg317) . To investigate the roles of these charged residues in the Na(+)-driven motor, we cloned the VibriofliG gene and introduced single or multiple substitutions into the corresponding positions in FliG(V) . FliG(V) with double Ala replacements in all possible combinations at these five conserved positions still retained significant motile ability, although some of the mutations completely eliminated the function of FliG(E) . All of the triple mutants constructed in this study also remained motile . These results suggest that the important charged residues may be located in different places and the conserved charged residues are not so important for the Na(+)-driven flagellar motor of Vibrio . The chimeric FliG protein (FliG(VE)), composed of the N-terminal domain from V.alginolyticus and the C-terminal domain from E.coli, functions in Vibrio cells . The mutations of the charge residues of the C-terminal region in FliG(VE) affected swarming ability as in E.coli . Both the FliG(V) and the FliG(VE) proteins with the triple mutation were more susceptible to proteolysis than proteins without the mutation, suggesting that their conformations were altered.

Microbes Infect, 2003 Nov, 5(13), 1241 - 7
Activation and suppression of the proinflammatory immune response by Vibrio cholerae toxins; Satchell KJ; Vibrio cholerae induces either non-inflammatory diarrhea or inflammatory gastroenteritis, depending on the presence of cholera toxin, a fluid secretion inducer and a modulator of host immunity . In the absence of cholera toxin, other toxins induce inflammation, resulting in gastroenteritis . Thus, multiple toxins likely affect the safety of live attenuated vaccines.

J Bacteriol, 2003 Dec, 185(23), 6938 - 49
Identification and characterization of genes required for biosynthesis and transport of the siderophore vibrioferrin in Vibrio parahaemolyticus; Tanabe T et al.; In response to low iron availability, Vibrio parahaemolyticus synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, L-alanine, ethanolamine, and citric acid . We have previously reported the cloning and characterization of the pvuA gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin . In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the pvuA gene . The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis . Five of the genes, named pvsABCDE, constitute an operon that is expressed under iron-limiting conditions . Homology searches of their predicted protein products suggested that the four genes pvsABDE are implicated in the biosynthesis of the siderophore . Another gene in the same operon, pvsC, encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps . The remaining four genes, named pvuBCDE, encode proteins strongly homologous to Escherichia coli FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate . Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, psuA and pvuA . Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.

J Bacteriol, 2003 Dec, 185(23), 6893 - 901
The vibrio pathogenicity island of epidemic Vibrio cholerae forms precise extrachromosomal circular excision products; Rajanna C et al.; The Vibrio pathogenicity island (VPI) in epidemic Vibrio cholerae is an essential virulence gene cluster . Like many pathogenicity islands, the VPI has at its termini a phage-like integrase gene (int), a transposase-like gene (vpiT), and phage-like attachment (att) sites, and is inserted at a tRNA-like locus (ssrA) . We report that the VPI precisely excises from the chromosome and that its left and right ends join to form an extrachromosomal circular excision product (pVPI) . Two-stage nested PCR analysis and DNA sequencing confirmed the int-att-vpiT junction and that the core attP of pVPI is identical to the chromosomal VPI attR site . Excision was independent of toxR and toxT . Excision was independent of recA, suggesting that it is mediated by site-specific recombination . Interestingly, while excision was detected in int and vpiT mutants, excision was abolished in a double (int vpiT) mutant and was restored by plasmids containing genes for either recombinase . Excision results in deletion of A361 in the ssrA locus, which flanks the right junction of the VPI . Since A361 encodes U70 in the critical G . U base pair in the acceptor stem of the ssrA RNA that is the determinant for aminoacylation with alanine, this deletion might have deleterious effects on ssrA function . Also, vpiT may have undergone interchromosomal translocation or may represent an independent integration event, as it was found downstream of hutA in some isolates . Our results provide new insight into the molecular biology of the VPI, and we propose that the process of excision and circularization is important in the emergence, pathogenesis, and persistence of epidemic V . cholerae.

J Biol Chem, 2004 Jan 23, 279(4), 2640 - 7 Epub 2003 Nov 10.
The CTXphi repressor RstR binds DNA cooperatively to form tetrameric repressor-operator complexes; Kimsey HH et al.; CTX is a filamentous bacteriophage that encodes cholera toxin and integrates into the Vibrio cholerae genome to form stable lysogens . In CTX lysogens, gene expression originating from the rstA phage promoter is repressed by the phage-encoded repressor RstR . The N-terminal region of RstR contains a helix-turn-helix DNA-binding element similar to the helix-turn-helix of the cI/Cro family of phage repressors, whereas the short C-terminal region is unrelated to the oligomerization domain of cI repressor . Purified His-tagged RstR bound to three extended 50-bp operator sites in the rstA promoter region . Each of the RstR footprints exhibited a characteristic staggered pattern of DNase I-accessible regions that suggested RstR binds DNA as a dimer-of-dimers . In gel permeation chromatography and cross-linking experiments, RstR oligomerized to form dimers and tetramers . RstR was shown to be tetrameric when bound to operator DNA by performing mobility shift experiments with mixtures of RstR and a lengthened active variant of RstR . Binding of RstR to the high affinity O1 site could be fit to a cooperative model of operator binding in which two RstR dimers associate to form tetrameric RstR-operator complexes . The binding of RstR dimers to the left or right halves of O1 operator DNA was not observed in mobility shift assays . These observations support a model in which protein-protein contacts between neighboring RstR dimers contribute to strong operator binding.

Can J Microbiol, 2003 Aug, 49(8), 525 - 9
Isolation and characterization of a zinc-containing metalloprotease expressed by Vibrio tubiashii; Delston RB et al.; A Vibrio tubiashii hemagglutinin, a protease, was purified by ammonium sulfate precipitation, gel filtration, and hydrophobic interaction chromatography . It agglutinates sheep, chicken, bovine, rabbit, guinea pig, and human erythrocytes . It has a molecular mass of 35 kDa, isoelectric points of 3.5 and 3.7, and is inhibited by ortho-phenanthro line, phosphoramidon, and Zincov . The N-terminal amino acid sequence (Ala-Gln-Ala-Thr-Gly-Thr-Gly- Pro-Gly-Gly-Asn-Gln-Lys-Thr-Gly-Gln- Tyr-Asn-Phe-Gly) has strong homology to other Vibrio proteases.

J Microbiol Methods, 2003 Dec, 55(3), 745 - 53
A simple and convenient microtiter plate assay for the detection of bactericidal antibodies to Vibrio cholerae O1 and Vibrio cholerae O139; Boutonnier A et al.; It is believed that the correlate of protection for cholera can be determined by the serum vibriocidal assay . The currently available vibriocidal assays, based on the conventional agar plating technique, are labor intensive . We developed a simple and convenient microtiter plate assay for the detection of vibriocidal antibodies that is equally as efficient for Vibrio cholerae O1 and for V . cholerae O139 . The addition of succinate and neotetrazolium made it possible to measure the growth of surviving bacterial target cells by monitoring a color change . We evaluated assay parameters (target strains, growth of target cells, complement source and concentration) that may affect the reproducibility of the method for V . cholerae O139 . The results obtained with the microtiter plate assay were uniformly similar to those obtained with the conventional agar plating assay, when testing both the Inaba and Ogawa serotypes of V . cholerae O1 . The microtiter plate assay was also convenient for measuring the activity of animal sera and mouse monoclonal antibodies.

J Microbiol Methods, 2003 Dec, 55(3), 667 - 77
Development of bespoke bioluminescent reporters with the potential for in situ deployment within a phenolic-remediating wastewater treatment system; Wiles S et al.; A suite of ecologically relevant, site-specific bioreporters was constructed by transposon mutagenesis of microorganisms isolated from a polluted phenolic-remediating wastewater treatment system . Four Pseudomonad species were engineered to carry a stable chromosomal copy of the lux operon (luxCDABE) derived from Photorhabdus luminescens . These recombinant reporter microorganisms were tested for bioluminescence response to relevant phenol concentrations in the laboratory and to phenolic-containing effluents generated by an industrial wastewater treatment plant . The reporters displayed proportional responses of bioluminescence decay with increasing phenol concentrations up to 800 mg l(-1) of phenol . When deployed against samples from the treatment system, they showed superior operational range and sensing capabilities to that observed for industry standard microorganisms such as Vibrio fischeri . Specifically, the engineered strains accurately predicted toxicity shifts in all the treatment compartments under study (with phenolic concentrations ranging from approximately 10 to 600 mg l(-1)) with a low coefficient of variation of replicate determinations (between 1.16% and 8.32%) . This work highlights the utility of genetic modification of native microorganisms from sites of interest to provide robust and ecologically relevant organism-based reagents for toxicity monitoring with the potential for in situ deployment.

J Mol Biol, 2003 Nov 21, 334(2), 179 - 85
Crystal structure of the N-terminal dimerisation domain of VicH, the H-NS-like protein of Vibrio cholerae; Cerdan R et al.; The histone-like nucleoid structuring (H-NS) protein is a global modulator of gene expression in Gram-negative bacteria . VicH, the H-NS protein of Vibrio cholerae, regulates the expression of certain major virulence determinants implicated in the pathogenesis of cholera . We present here the 2.5A crystal structure of the N-terminal oligomerisation domain of VicH (VicH_Nt) . VicH_Nt adopts the same fold and dimeric assembly as the NMR structure of Escherichia coli H-NS_Nt, thus validating this fold against conflicting data . The structural similarity of V.cholerae VicH_Nt and E.coli H-NS_Nt, despite differences in origin, system of expression, experimental conditions and techniques used, indicates that the fold determined in our studies is robust to experimental conditions . Structural analysis and homology modelling were carried out to further elucidate the molecular basis of the functional polyvalence of the N-terminal domain . Our analysis of members of the H-NS superfamily supports the suggestion that the oligomerisation function of H-NS_Nt is conserved even in more distantly related proteins.

Trends Microbiol, 2003 Nov, 11(11), 505 - 10
Pathogenicity islands and phages in Vibrio cholerae evolution; Faruque SM et al.; The identification of accessory genetic elements (plasmids, phages and chromosomal 'pathogenicity islands') encoding virulence-associated genes has facilitated our efforts to understand the origination of pathogenic microorganisms . Toxigenic Vibrio cholerae, the etiologic agent of cholera, represents a paradigm for this process in that this organism evolved from environmental nonpathogenic V . cholerae by acquisition of virulence genes . The major virulence genes in V . cholerae, which are clustered in several chromosomal regions, appear to have been recently acquired from phages or through undefined horizontal gene transfer events . Evidence is accumulating that the interactions of phages with each other can also influence the emergence of pathogenic clones of V . cholerae . Therefore, to track the evolution of pathogens from their nonpathogenic progenitors, it is also crucial to identify and characterize secondary genetic elements that mediate lateral transfer of virulence genes in trans . Understanding the evolutionary events that lead to the emergence of pathogenic clones might provide new approaches to the control of cholera and other infectious diseases.

Nucleic Acids Res . 2003 Nov 15;31(22):e136.
Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR; Giglio S et al.; SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye and is included in many commercially available kits at undisclosed concentrations . Binding of SG to double-stranded DNA is non-specific and additional testing, such as DNA melting curve analysis, is required to confirm the generation of a specific amplicon . The use of melt curve analysis eliminates the necessity for agarose gel electrophoresis because the melting temperature (T(m)) of the specific amplicon is analogous to the detection of an electrophoretic band . When using SG for real-time PCR multiplex reactions, discrimination of amplicons should be possible, provided the T(m) values are sufficiently different . Real-time multiplex assays for Vibrio cholerae and Legionella pneumophila using commercially available kits and in-house SG mastermixes have highlighted variability in performance characteristics, in particular the detection of only a single product as assessed by T(m) analysis but multiple products as assessed by agarose gel electrophoresis . The detected T(m) corresponds to the amplicon with the higher G+C% and larger size, suggesting preferential binding of SG during PCR and resulting in the failure to detect multiple amplicons in multiplex reactions when the amount of SG present is limiting . This has implications for the design and routine application of diagnostic real-time PCR assays employing SG.

Appl Environ Microbiol, 2003 Nov, 69(11), 6923 - 31
Growth of Vibrio cholerae O1 in red tide waters off California; Mourino-Perez RR et al.; Vibrio cholerae serotype O1 is autochthonous to estuarine and coastal waters . However, its population dynamics in such environments are not well understood . We tested the proliferation of V . cholerae N16961 during a Lingulodinium polyedrum bloom, as well as other seawater conditions . Microcosms containing 100-kDa-filtered seawater were inoculated with V . cholerae or the 0.6- micro m-pore-size filterable fraction of seawater assemblages . These cultures were diluted 10-fold with fresh 100-kDa-filtered seawater every 48 h for four cycles . Growth rates ranged from 0.3 to 14.3 day(-1) (4.2 day(-1) +/- 3.9) for V . cholerae and 0.1 to 9.7 day(-1) (2.2 +/- 2.8 day(-1)) for bacterial assemblage . Our results suggest that dissolved organic matter during intense phytoplankton blooms has the potential to support explosive growth of V . cholerae in seawater . Under the conditions tested, free-living V . cholerae was able to reach concentrations per milliliter that were up to 3 orders of magnitude higher than the known minimum infectious dose (10(4) cell ml(-1)) and remained viable under many conditions . If applicable to the complex conditions in marine ecosystems, our results suggest an important role of the growth of free-living V . cholerae in disease propagation and prevention during phytoplankton blooms.

Appl Environ Microbiol, 2003 Nov, 69(11), 6361 - 9
Cold shock response and major cold shock proteins of Vibrio cholerae; Datta PP et al.; When exponentially growing Vibrio cholerae cells were shifted from 37 degrees C to various lower temperatures, it was found that the organism could adapt and grow at temperatures down to 15 degrees C, below which the growth was completely arrested . There was no difference between the patterns of the cold shock responses in toxinogenic and nontoxinogenic strains of V . cholerae . Gel electrophoretic analyses of proteins of cold-exposed cells revealed significant induction of two major cold shock proteins (Csps), whose molecular masses were 7.7 kDa (CspA(VC)) and 7.5 kDa (CspV), and six other Csps, most of which were much larger . We cloned, sequenced, and analyzed the cspV gene encoding the CspV protein of V . cholerae O139 strain SG24 . Although CspA(VC) and CspV have similar kinetics of synthesis and down-regulation, the corresponding genes, cspA and cspV, which are located in the small chromosome, are not located in the same operon . A comparative analysis of the kinetics of synthesis revealed that the CspV protein was synthesized de novo only during cold shock . Although both CspA(VC) and CspV were stable for several hours in the cold, the CspV protein was degraded rapidly when the culture was shifted back to 37 degrees C, suggesting that this protein is probably necessary for adaptation at lower temperatures . Northern blot analysis confirmed that the cspV gene is cold shock inducible and is regulated tightly at the level of transcription . Interestingly, the cspV gene has a cold shock-inducible promoter which is only 12 nucleotides from the translational start site, and therefore, it appears that no unusually long 5' untranslated region is present in its mRNA transcript . Thus, this promoter is an exception compared to other promoters of cold shock-inducible genes of different organisms, including Escherichia coli . Our results suggest that V . cholerae may use an alternative pathway for regulation of gene expression during cold shock.

Chemosphere, 2004 Feb, 54(5), 679 - 87
Effects of two diluents in the Microtox toxicity bioassay with marine sediments; Onorati F et al.; This paper compares the use of two different diluents, EPA synthetic seawater (salinity 31 per thousand ) and NaCl standard diluent (salinity 35 per thousand ), in the Microtox toxicity bioassay performed on elutriate and solid phase derived from marine sediments . The study was performed comparing three series of data obtained by the use of the two diluents.In the first series the intensity of the natural light output of Vibrio fischeri was considered; in the second series pH value, sulphite and ammonia present in the control and in the diluent after the treatment with the sediment samples; in the last series, the measured toxicity in marine sediments was considered . The light output intensity measured with respect to time, gives information about the bacterial activity due to the different osmotic conditions . pH values joint with ammonia and sulphite content, give information about the effect of the bacterial metabolic activities and of the different interaction between each diluent with the sediment sample . At last, the comparison of the two diluents on real samples show how the different osmotic and hydrogenionic conditions determine different toxicity responses . The results show that the EPA diluent allows more suitable environment for the metabolic activities of bacteria depending on lower stressing conditions than those present when the standard diluent is used . Moreover, the use of EPA diluent reduces the risk of false positive response in the execution of the toxicity bioassay.

Toxicol In Vitro, 2003 Oct-Dec, 17(5-6), 525 - 32
Ecotoxicological evaluation of carbamazepine using six different model systems with eighteen endpoints; Jos A et al.; The occurrence of pharmaceutically active compounds in the aquatic environment has been recognized as one of the emerging issues in environmental chemistry . However, the ecotoxicological effects of pharmaceuticals have still not been researched adequately . Carbamazepine, an anticonvulsant commonly present in surface and groundwater, was studied, using six ecotoxicological model systems with eighteen endpoints evaluated at different exposure time periods . The battery included the immobilization of Daphnia magna, bioluminescence inhibition in the bacterium Vibrio fischeri, growth inhibition of the alga Chlorella vulgaris, and micronuclei induction and root growth inhibition in the plant Allium cepa . Cell morphology, neutral red uptake, total protein content, MTS metabolization, lactate dehydrogenase leakage and activity and glucose-6-phosphate dehydrogenase activity were studied in the salmonid fish cell line RTG-2 . The total protein content, LDH activity, neutral red uptake and MTT metabolization in Vero monkey kidney cells were also investigated . The most sensitive system to carbamazepine was the Vero cell line, followed by Chlorella vulgaris, Vibrio fischeri, Daphnia magna, Allium cepa, and RTG-2 cells . EC50 values from 19 microM in Vero cells at 72 h to more than 1200 microM in other systems, were obtained . Comparing the concentrations in water and the toxicity quantified in our assay systems, carbamazepine is not expected to produce acute toxic effects in the aquatic biota under these circumstances, but chronic and synergistic effects with other chemicals cannot be excluded.

Dis Aquat Organ, 2003 Sep 24, 56(2), 127 - 33
Pharmacokinetics of florfenicol in cod Gadus morhua and in vitro antibacterial activity against Vibrio anguillarum; Samuelsen OB et al.; The pharmacokinetic profile of the antibacterial agent florfenicol was studied in plasma after intravenous (i.v.) injection and in plasma, muscle and liver following oral (p.o.) administration to cod Gadus morhua, held in seawater at 8 degrees C and weighing 100 to 200 g . Following i.v . injection, the plasma drug concentration-time profile showed 2 distinct phases . The plasma distribution half-life (t1/2alpha) was estimated to be 1.6 h, the elimination half-life (t1/2beta) to be 43 h, the total body clearance (ClT) to be 0.015 1 kg(-1) h(-1) and mean residence time (MRT) to be 74 h . The volume of distribution at steady state, Vd(ss), was calculated to be 1.1 l kg(-1) . Following p.o . administration, the bioavailability was estimated to be 91%, the peak plasma concentrations (Cmax) to be 10.8 microg ml(-1) and the time to peak plasma concentrations (Tmax) to be 7 h . Corresponding Cmax and Tmax values were 13.0 microg g(-1) and 9 h, respectively, in muscle and 12.1 microg g(-1) and 9 h, respectively, in liver . The in vitro minimum inhibitory concentration (MIC) values of florfenicol against 3 Vibrio anguillarum strains isolated from diseased cod (A-21, HI-610, HI-618) were 0.5 microg ml(-1) for all 3 strains.

Curr Biol, 2003 Oct 28, 13(21), 1916 - 20
Actin-like proteins MreB and Mbl from Bacillus subtilis are required for bipolar positioning of replication origins; Soufo HJ et al.; Actin-like proteins MreB and Mbl are required for proper cell shape and for viability in B . subtilis and form dynamic helical filaments underneath the cell membrane . We have found that depletion of MreB and Mbl proteins leads to a rapid defect in chromosome segregation before a defect in cell shape becomes detectable . Under these conditions, the SMC chromosome segregation complex that is essential for proper chromosome arrangement and segregation loses its specific subcellular localization, and replication origins fail to localize in a regular bipolar manner as in wild type cells . Time-lapse microscopy showed that during depletion of MreB, origin regions can move towards the same cell pole, showing that bipolar orientation of origin separation is lost . Contrarily, depletion of three other cell shape determinants, MreC, MreD, or MreBH (the third B . subtilis actin homolog) had no effect on chromosome segregation but varying effects on cell morphology . Depletion of MreC and MreD resulted in formation of round cells, while depletion of MreBH led to formation of vibrio-shaped cells . The data show that actin proteins Mbl and MreB are required for proper chromosome segregation and that Mre proteins affect different aspects in cell shape.

Vaccine, 2003 Dec 1, 21(32), 4715 - 21
Cholera vaccine candidate 638: intranasal immunogenicity and expression of a foreign antigen from the pulmonary pathogen Coccidioides immitis; Silva AJ et al.; Vibrio cholerae strain 638 is a live genetically attenuated candidate cholera vaccine in which the CTXPhi prophage encoding cholera toxin has been deleted and hapA, encoding an extracellular Zn-dependent metalloprotease, was insertionally inactivated . Strain 638 was highly immunogenic when inoculated to adult Swiss mice by the intranasal route as judged by the induction of a strong serum vibriocidal antibody response . A side-by-side comparison of strain 638 with its isogenic hapA(+) precursor (strain 81) in the above model indicated that inactivation of hapA does not affect immunogenicity . The spherule-associated antigen 2/proline-rich antigen (Ag2/PRA) of Coccidioides immitis has been shown to protect mice against coccidioidomycosis to an extent dependent on the modes of antigen presentation and challenge with C . immitis arthrospores . In this work, we demonstrate the use of a live genetically attenuated V . cholerae strain to deliver Ag2/PRA . Ag2/PRA was expressed in 638 as a fusion protein with the Escherichia coli heat labile toxin B subunit leader peptide using the strong Tac promoter . The recombinant Ag2/PRA was efficiently expressed, processed and secreted to the periplasmic space . Intranasal immunizations of adult mice with strain 638 expressing Ag2/PRA induced serum vibriocidal antibody response to the vector strain and serum total IgG response to Ag2/PRA . Strain 638 expressing PRA could be recovered from trachea and lung up to 20h after immunization but was effectively cleared 72h post-inoculation.

OMICS, 2003 Fall, 7(3), 317 - 34
Analysis of noise in quorum sensing; Cox CD et al.; Noise may play a pivotal role in gene circuit functionality, as demonstrated for the genetic switch in the bacterial phage lambda . Like the lambda switch, bacterial quorum sensing (QS) systems operate within a population and contain a bistable switching element, making it likely that noise plays a functional role in QS circuit operation . Therefore, a detailed analysis of the noise behavior of QS systems is needed . We have developed a set of tools generally applicable to the analysis of gene circuits, with an emphasis on investigations in the frequency domain (FD), that we apply here to the QS system in the marine bacterium Vibrio fischeri . We demonstrate that a tight coupling between exact stochastic simulation and FD analysis provides insights into the structure/function relationships in the QS circuit . Furthermore, we argue that a noise analysis is incomplete without consideration of the power spectral densities (PSDs) of the important molecular output signals . As an example we consider reversible reactions in the QS circuit, and show through analysis and exact stochastic simulation that these circuits make significant and dynamic modifications to the noise spectra . In particular, we demonstrate a "whitening" effect, which occurs as the noise is processed through these reversible reactions.

J Nat Prod, 2003 Oct, 66(10), 1299 - 301
dd-diketopiperazines: antibiotics active against Vibrio anguillarum isolated from marine bacteria associated with cultures of Pecten maximus; Fdhila F et al.; Bacterial strains CF-20 (CECT5719) and C-148 (CECT5718), isolated from cultures of larvae of molluscs, are shown to produce substances 1-5 with strong antibiotic activity against Vibrio anguillarum (MIC: 0.03-0.07 mug/mL) and identified as the dd-diketopiperazines cyclo(d)-Pro-(d)-Phe (1), cyclo(d)-Pro-(d)-Leu (2), cyclo(d)-Pro-(d)-Val (3), cyclo(d)-Pro-(d)-Ile (4), and cyclo-trans-4-OH-(d)-Pro-(d)-Phe (5) . Comparison with other stereoisomers indicates that inhibition of V . anguillarum is associated with the presence of at least one d-amino acid in the diketopiperazine system . This is the first time a series of dd-diketopiperazines has been isolated from a single natural source and their inhibitory activity against V . anguillarum described.

FEBS Lett, 2003 Oct 23, 553(3), 229 - 31
Differential interaction of the two cholesterol-dependent, membrane-damaging toxins, streptolysin O and Vibrio cholerae cytolysin, with enantiomeric cholesterol; Zitzer A et al.; Membrane cholesterol is essential to the activity of at least two structurally unrelated families of bacterial pore-forming toxins, represented by streptolysin O (SLO) and Vibrio cholerae cytolysin (VCC), respectively . Here, we report that SLO and VCC differ sharply in their interaction with liposome membranes containing enantiomeric cholesterol (ent-cholesterol) . VCC had very low activity with ent-cholesterol, which is in line with a stereospecific mode of interaction of this toxin with cholesterol . In contrast, SLO was only slightly less active with ent-cholesterol than with cholesterol, suggesting a rather limited degree of structural specificity in the toxin-cholesterol interaction.

Appl Microbiol Biotechnol, 2004 Feb, 63(6), 722 - 7 Epub 2003 Oct 21.
Molecular identification of pathogenic and nonpathogenic strains of Vibrio harveyi using PCR and RAPD; Hernandez G et al.; Fifteen environmental samples of Vibrio spp . isolated from healthy and diseased shrimps were tested for pathogenicity to juvenile shrimps . Two isolates, strains Z2 and Z3, were observed to be pathogenic, causing 100% mortality of the target host compared to the control strain Vibrio harveyi ATCC 14126 . Environmental and type strains were subjected to molecular characterization by restriction fragment length polymorphism (RFLP) and PCR using primers targeted to different virulence, transcriptional regulator, or quorum sensing genes from V . harveyi . Primers designed for luxN were specific and identified all the environmental strains as V . harveyi . The random amplified polymorphic DNA (RAPD) method was used to differentiate between pathogenic and nonpathogenic strains of V . harveyi . These methodologies allowed us to detect and distinguish strains virulent and avirulent to juvenile shrimp.

Biosens Bioelectron, 2003 Nov 15, 19(2), 115 - 21
A rapid BOD sensing system using luminescent recombinants of Escherichia coli; Sakaguchi T et al.; A biochemical oxygen demand (BOD) sensing system based on bacterial luminescence from recombinant Escherichia coli containing lux A-E genes from Vibrio fischeri has been developed . It was possible to use frozen cells of luminescent recombinants of E . coli as the bacterial reagents for measurement . Steady bioluminescence was observed during the incubation time between 90 and 150 min in the presence of a sole carbon source such as glucose, acetate, L-glutamate and BOD standard solution (GGA solution) . This disposable bacterial reagent was applied to measure and detect organic pollution due to biodegradable substances in various wastewaters . The obtained values of this study showed a similar correlation with that of the conventional method for BOD determination (BOD5) . Bacterial luminescence that was visualized with an imaging system using a charge coupled device (CCD) camera and a photomulti-counter demonstrated that this method could also be used for multi-sample detection of organic pollution due to biodegradable substances by using a microtiter plate . These results suggested for successful achievement of high-though-put detection of BOD in practical.

FEMS Microbiol Lett, 2003 Oct 10, 227(1), 65 - 71
Ion motive force dependence of protease secretion and phage transduction in Vibrio cholerae and Pseudomonas aeruginosa; Hase CC; Vibrio cholerae is known to secrete a large number of proteins into the extracellular milieu, including the important virulence factor cholera toxin (CT) . However, one of the most abundant proteins found in V . cholerae supernatants is the zinc-metalloprotease HA/protease (HAP) . Whereas efficient protein secretion in Escherichia coli requires ATP hydrolysis and the proton motive force (pmf), little is known about the energy requirements for protein secretion in V . cholerae . To analyze some of the energy requirements for protein secretion in V . cholerae, HAP accumulation in culture supernatants following growth in the presence of various ionophores was assayed . Extracellular production of HAP was strongly reduced in the presence of monensin, an artificial Na(+)/H(+) antiporter that collapses the DeltapNa(+) across the membrane without affecting Deltapsi, whereas the protonophore CCCP had no significant effect on the extracellular accumulation of HAP . In contrast, extracellular protease production in Pseudomonas aeruginosa was affected by CCCP, but not monensin . Furthermore, extracellular protease production of V . cholerae, but not P . aeruginosa, was increased in increasing amounts of NaCl in the culture medium . Together these results indicate that the V . cholerae HAP requires an intact sodium motive force (smf) for its efficient translocation across the membranes, whereas extracellular protease production by P . aeruginosa requires only pmf . As the entry of some bacteriophage genomes has been reported to require pmf, the effects of ionophores on the efficiency of tranduction of V . cholerae by the CTXPhi phage were analyzed . CTXPhi transduction was strongly affected by CCCP, but not monensin, suggesting that phage entry requires pmf but not smf . Understanding the energy requirements for these potentially important virulence aspects of pathogens might lead to novel intervention strategies.

J Bacteriol, 2003 Nov, 185(21), 6434 - 47
Genome sequences of two closely related Vibrio parahaemolyticus phages, VP16T and VP16C; Seguritan V et al.; Two bacteriophages of an environmental isolate of Vibrio parahaemolyticus were isolated and sequenced . The VP16T and VP16C phages were separated from a mixed lysate based on plaque morphology and exhibit 73 to 88% sequence identity over about 80% of their genomes . Only about 25% of their predicted open reading frames are similar to genes with known functions in the GenBank database . Both phages have cos sites and open reading frames encoding proteins closely related to coliphage lambda's terminase protein (the large subunit) . Like in coliphage lambda and other siphophages, a large operon in each phage appears to encode proteins involved in DNA packaging and capsid assembly and presumably in host lysis; we refer to this as the structural operon . In addition, both phages have open reading frames closely related to genes encoding DNA polymerase and helicase proteins . Both phages also encode several putative transcription regulators, an apparent polypeptide deformylase, and a protein related to a virulence-associated protein, VapE, of Dichelobacter nodosus . Despite the similarity of the proteins and genome organization, each of the phages also encodes a few proteins not encoded by the other . We did not identify genes closely related to genes encoding integrase proteins belonging to either the tyrosine or serine recombinase family, and we have no evidence so far that these phages can lysogenize the V . parahaemolyticus strain 16 host . Surprisingly for active lytic viruses, the two phages have a codon usage that is very different than that of the host, suggesting the possibility that they may be relative newcomers to growth in V . parahaemolyticus . The DNA sequences should allow us to characterize the lifestyles of VP16T and VP16C and the interactions between these phages and their host at the molecular level, as well as their relationships to other marine and nonmarine phages.

Gene, 2003 Oct 16, 316, 187 - 95
Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species; Hikima S et al.; Lysozyme cDNA was isolated from a kuruma shrimp, Marsupenaeus japonicus, hemocyte cDNA library . The cDNA consists of 1055 base pairs (bp) and encodes a chicken-type (c-type) lysozyme with a deduced amino acid sequence of 156 residues . The kuruma shrimp lysozyme has a high identity (79.7%) with pacific white shrimp lysozyme, and low to moderate identities (33.3-43.0%) with lysozymes of insects and vertebrates . Comparisons with other c-type lysozymes from invertebrates and vertebrates showed that the two catalytic residues (Glu58 and Asp75) and the eight cysteine residue motif were completely conserved . Two novel insertion sequences were also observed in the kuruma and pacific white shrimp lysozyme amino acid sequences . Interestingly, phylogenetic analysis revealed that the kuruma shrimp lysozyme was more closely related to vertebrate c-type lysozymes . Expression of the cDNA in insect cells, using a baculovirus expression system, yielded a recombinant lysozyme with optimum activity at pH 7.5 and 50 degrees C, as evaluated by a lysoplate assay . The kuruma shrimp lysozyme displayed lytic activities against several Vibrio species and fish pathogens, including Vibrio penaeicida (a pathogenic bacteria to the kuruma shrimp) and suggested that shrimp lysozyme affects a greater variety of pathogens.

Mutat Res, 2003 Sep 29, 530(1-2), 47 - 57
Alleviation of mutagenic effects of polycyclic aromatic agents (quinacrine mustard, ICR-191 and ICR-170) by caffeine and pentoxifylline; Piosik J et al.; Previous studies performed by others indicated that apart from its other biological effects, caffeine (CAF) may have a role in protection of organisms against cancer . However, biological mechanism of this phenomenon remained unknown . Recent studies suggested that caffeine can form stacking (pi-pi) complexes with polycyclic aromatic chemicals . Therefore, one might speculate that effective concentrations of polycyclic aromatic mutagens could be reduced in the presence of caffeine . Here we demonstrate that caffeine and another xanthine, pentoxifylline (PTX), effectively alleviate mutagenic action of polycyclic aromatic agents (exemplified by quinacrine mustard (QM), 2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (ICR-191) and 1,3,7-propanediamine-N-(2-chloroethyl)-N'-(6-chloro-2-methoxy-9-acridinyl)-N-ethyl.2HCl (ICR-170)), but not of aliphatic mutagens (exemplified by mechlorethamine), in the recently developed mutagenicity test based on bacterium Vibrio harveyi . Biophysical studies indicated that caffeine and pentoxifylline can form stacking complexes with the aromatic agents mentioned above . Molecular modeling also confirmed a possibility of stacking interactions between examined molecules.

Rev Environ Contam Toxicol, 2004, 180, 93 - 131
Health effects of Acanthamoeba spp . and its potential for waterborne transmission; Nwachuku N et al.; Risk from Acanthamoeba keratitis is complex, depending upon the virulence of the particular strain, exposure, trauma, or other stress to the eye, and host immune response . Bacterial endosymbionts may also play a factor in the pathogenicity of Acanthamoeba . Which factor(s) may be the most important is not clear . The ability of the host to produce IgA antibodies in tears may be a significant factor . The immune response of the host is a significant risk factor for GAE infection . If so, then a certain subpopulation with an inability to produce IgA in the tears may be at greatest risk . There was no sufficient data on the occurrence or types of Acanthamoeba in tapwater in the U.S . Published work on amoebal presence in tapwater does not provide information on the type of treatment the water received or the level of residual chlorine . Assessment of the pathogenicity by cell culture and molecular methods of Acanthamoeba in tapwater would also be useful in the risk assessment process for drinking water . The possibility that Acanthamoeba spp . might serve as vectors for bacterial infections from water sources also should be explored . The bacterial endosymbionts include an interesting array of pathogens such as Vibrio cholerae and Legionella pneumophila, both of which are well recognized waterborne/water-based pathogens . Work is needed to determine if control of Acanthamoeba spp . is needed to control water-based pathogens in water supplies.

Biochim Biophys Acta, 2003 Oct 15, 1639(2), 65 - 79
Lipopolysaccharides of Vibrio cholerae . I . Physical and chemical characterization; Chatterjee SN et al.; Vibrio cholerae is the causative organism of the disease cholera . The lipopolysaccharide (LPS) of V . cholerae plays an important role in eliciting the antibacterial immune response of the host and in classifying the vibrios into some 200 or more serogroups . This review presents an account of our up-to-date knowledge of the physical and chemical characteristics of the three constituents, lipid-A, core-polysaccharide (core-PS) and O-antigen polysaccharide (O-PS), of the LPS of V . cholerae of different serogroups including the disease-causing ones, O1 and O139 . The structure and occurrence of the capsular polysaccharide (CPS) on V . cholerae O139 have been discussed as a relevant topic . Similarity and dissimilarity between the structures of LPS of different serogroups, and particularly between O22 and O139, have been analysed with a view to learning their role in the causation of the epidemic form of the disease by avoiding the host defence mechanism and in the evolution of the newer pathogenic strains in future . An idea of the emerging trends of research involving the use of immunogens prepared from synthetic oligosaccharides that mimic terminal epitopes of the O-PS of V . cholerae O1 in the development of a conjugate anti cholera vaccine is also discussed.

J Pak Med Assoc, 2003 Aug, 53(8), 335 - 8
Re-emergence of Vibrio cholerae O139 in Pakistan: report from a tertiary care hospital; Jabeen K et al.; OBJECTIVE: This study reports re-emergence of Vibrio cholerae O139 in Pakistan in 2000-2001 from a tertiary care hospital in Karachi, Pakistan . METHODS: This descriptive study was conducted from 2000-2001 . Stool samples were taken from inpatients or those referred to the laboratory from other hospitals, clinics and general practitioners . Samples were processed and Vibrio cholerae isolates were identified according to standard protocols . Tellurite Taurocholate Gelatin agar was used as a selective medium for Vibrio cholerae . Serogroups were identified by slide agglutination with polyvalent antisera . Antimicrobial sensitivities were performed by Kirby Bauer technique . Data was entered and analyzed using SPSS, p values were calculated using t test and two independent samples test . RESULTS: During the study period, 144 samples were found to be infected with Vibrio cholerae O139 in comparison with 545 Vibrio cholerae O1 . Infection with O139 was characteristically observed in the older population (mean age = 40 years) in contrast with Vibrio cholerae O1 strains (mean age = 23 years) (p . value = <0.001) . Sensitivity pattern of 2000-2001 Vibrio cholerae isolates was markedly different to that of 1993-1994 . The earlier isolates were resistant to Cotrimoxazole (99%) and Chloramphenicol (35%) whereas the recent isolates are almost 100% sensitive . CONCLUSION: In conclusion this re-emergent strain seen 6 years after previous episode infected large number of people especially older population suggesting that prior infection with O1 does not provide immunity against O139 and therefore Vibrio cholerae O139 has a potential to cause a major epidemic in an immunologically naive population.

Immunol Lett, 2003 Oct 31, 89(2-3), 143 - 7
Macrophage distinguishes Vibrio cholerae hemolysin from its protease insensitive oligomer by time dependent and selective expression of CD80-CD86; Ray A et al.; The monomeric and oligomeric forms of Vibrio cholerae hemolysin (HlyA), a membrane damaging toxin that forms transmembrane pentameric diffusion channels in target eukaryotic membrane, show a pronounced difference in protease susceptibility, presumably due to masking of sensitive peptide bonds during oligomerization . In this work, we examined if resistance of a protein to proteolytic processing affects the expression of costimulatory molecules, CD80 and CD86, on macrophage exposed to the same antigen . The murine peritoneal cavity macrophages expressed both CD80 and CD86 after 24 h of incubation with HlyA monomer but failed to express the costimulatory molecules when treated with the HlyA oligomer . The expression of CD80 molecule on macrophage after 48 h by the HlyA oligomer that failed to express the costimulatory molecules after 24 h indicates that proteolytic processing plays a decisive role in expression of CD80 and CD86 on cell surface.

J Mol Biol, 2003 Oct 24, 333(3), 657 - 74
Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae; Robien MA et al.; Type II secretion systems consist of an assembly of 12-15 Gsp proteins responsible for transporting a variety of virulence factors across the outer membrane in several pathogenic bacteria . In Vibrio cholerae, the major virulence factor cholera toxin is secreted by the Eps Type II secretion apparatus consisting of 14 Eps proteins . One of these, EpsE, is a cytoplasmic putative NTPase essential for the functioning of the Eps system and member of the GspE subfamily of Type II secretion ATPases . The crystal structure of a truncated form of EpsE in nucleotide-liganded and unliganded state has been determined, and reveals a two-domain architecture with the four characteristic sequence "boxes" of the GspE subfamily clustering around the nucleotide-binding site of the C-domain . This domain contains two C-terminal subdomains not reported before in this superfamily of NTPases . One of these subdomains contains a four-cysteine motif that appears to be involved in metal binding as revealed by anomalous difference density . The EpsE subunits form a right-handed helical arrangement in the crystal with extensive and conserved contacts between the C and N domains of neighboring subunits . Combining the most conserved interface with the quaternary structure of the C domain in a distant homolog, a hexameric model for EpsE is proposed which may reflect the assembly of this critical protein in the Type II secretion system . The nucleotide ligand contacts both domains in this model . The N2-domain-containing surface of the hexamer appears to be highly conserved in the GspE family and most likely faces the inner membrane interacting with other members of the Eps system.

FEMS Microbiol Lett, 2003 Sep 26, 226(2), 281 - 4
Identification of tdh-positive Vibrio parahaemolyticus from an outbreak associated with raw oyster consumption in Spain; Lozano-Leon A et al.; Between August and September 1999, a total of 64 cases of illness were identified in three episodes of acute gastroenteritis associated with the consumption of live oysters from a typical outdoor street market in Galicia (northwest Spain) . Nine case patients were hospitalized and analysis of their stool samples revealed the presence of Vibrio parahaemolyticus . The strains isolated from two stool samples were studied for antibiotic susceptibility, biochemical characteristics and presence of virulence factors . Both isolates were Kanagawa phenomenon positive and produced thermostable direct hemolysin, which is related to pathogenicity in humans . These results show the presence of pathogenic V . parahaemolyticus in mollusks harvested in Europe and reveal the risk of illness associated with their consumption, suggesting the revision of V . parahaemolyticus risk assessment associated with consumption of raw live shellfish.

Environ Toxicol Chem, 2003 Oct, 22(10), 2238 - 42
Bacterial detection of the toxicity of dioxins, polychlorinated diphenyls, and polybrominated diphenyl ethers; Min J et al.; Polychlorinated dibenzo-p-dioxins (PCDDs), biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs) were found to induce several specific stresses within bacterial cells . Four different recombinant Escherichia coli with specific stress promoters (i.e., the recA responsive to DNA damage, fabA responsive to membrane damage, katG responsive to oxidative damage, and grpE responsive to protein damage) that were fused to the lux operon from Vibrio fischeri showed very unique specificities in terms of their stress responses in the presence of PCDD and PCBs . In addition, a recombinant bacterium with the lac promoter fused to the lux operon from Xenorhabdus luminescence also showed dose-dependent responses via a loss of bioluminescence because of the addition of the PCDDs and PCBs . Brominated diphenyl ethers (BDE) -47, -99, and -153 congeners, however, were not found to induce any stress within the bacterial cells, indicating that these chemicals do not stimulate any cellular stresses related to those tested . These three congeners, however, did result in different levels of general cellular toxicity, which was found to be dependent on the position of the bromine . Finally, the cellular toxicity within the bacteria was found to increase when exposed to mixtures of dioxins, PCBs, and PBDEs, possibly from synergistic effects.

Environ Toxicol Chem, 2003 Oct, 22(10), 2228 - 37
Identification of toxic products of anthracene photomodification in simulated sunlight; Brack W et al.; Currently, the evidence of a rapid photomodification of anthracene under sunlight resulting in enhanced toxicity exists; however, the chemical causes of toxicity are still unknown . The present study aimed at filling this gap by irradiation of an anthracene suspension with simulated sunlight and subsequent effect-directed fractionation and analysis of toxic products with respect to the inhibition of bacterial energy metabolism of Vibrio fischeri, reproduction of the green algae Scenedesmus vacuolatus, and genotoxicity in the umuC test . Algal toxicity of anthracene was hardly modified by irradiation prior to testing and distributed over all fractions with emphasis on the fractions containing anthracene-9,10-dione and a photometabolite suggested to be 10-hydroxyanthrone . Bacterial toxicity and genotoxicity in contrast emerged only when anthracene was irradiated . Anthracene-1,4-dione, a so-far-unknown trace photometabolite, was identified as a very potent toxicant dominating the toxicity of photomodified anthracene to V . fischeri . In genotoxic fractions, 1-hydroxyanthracene-9,10-dione and 1,4-dihydroxyanthracene-9,10-dione were identified and confirmed as genotoxicants . The results stress the potential of effect-directed analysis approaches in contrast to mere chemical analysis in studies aiming at toxicologically relevant photomodified substances.

Vet Res Commun, 2003 Sep, 27 Suppl 1, 491 - 6
Application of risk management to the production chain of intensively reared fish; Giuffrida A; Principles of risk management, as a part of risk analysis, are described with respect to their application to the production chain of intensively reared fish . According to the outcomes of the FAO/WHO Expert consultation on the Application of Risk Management (Rome, 1997), there are four steps: risk evaluation, risk management option assessment, implementation of management decisions, and monitoring and review . There is a lack of information on risk assessment for farmed fish though, regarding bacteriological hazards, the data on farmed fish pathology and microbiology suggest a potential prevalence of Vibrio spp . and Aeromonas spp . Other bacterial contaminants could be introduced on handling during selection and packaging of products . On the basis of the above qualitative risk evaluation, risk management options have been individuated . These concern the monitoring of the health status of fish during the fattening period, harvest parameters, hygiene of selection and packaging, and storage characteristics.

Oncol Rep, 2003 Nov-Dec, 10(6), 1759 - 64
Alpha-2,6-sialylation of L-PHA reactive oligosaccharides and expression of N-acetylglucosaminyltransferase V in human diffuse large B cell lymphoma; Suzuki O et al.; Cell surface sialylation and beta1-6 branching of L-PHA reactive oligosaccharides play an important role in metastatic capacities of various tumor cell lines . We analyzed the expression and sialylation of L-PHA reactive oligosaccharides in human diffuse large B cell lymphoma (DLBCL) . DLBCL was grouped into three types; i) . Group A, non-reactive type with no expression of L-PHA reactive oligosaccharides, ii) . Group B, sialylated type with expression of sialylated L-PHA reactive oligosaccharides and iii) . Group C, non-sialylated type with expression of non-sialylated L-PHA reactive oligosaccharides . To clarify the linkage of sialic acid residues in L-PHA reactive oligosaccharides of Group B cases, L-PHA lectin histochemistry after treatment with two different neuraminidases was performed . In all Group B cases, L-PHA binding reactivity was found after treatment with Vibrio cholerae neuraminidase . But not after treatment with Newcastle disease virus neuraminidase . These data indicate that alpha2,6-linked sialic acid residues were predominantly involved in sialylation of L-PHA reactive oligosaccharides of Group B . To clarify the relationship between expression of N-acetylglucosaminyltransferase V (GnT-V), which catalyzes beta1-6 branching of L-PHA reactive oligosaccharides, and L-PHA reactivities in DLBCL, we investigated the expression of GnT-V using immunohistochemical methods . Most of the Group B and C cases expressed GnT-V while 33% of Group A cases showed no expression of GnT-V . These data suggest that expression of GnT-V is not always correlated with the expression of L-PHA reactive glycoconjugates . Furthermore, survival of patients in Group A which showed no expression of GnT-V was significantly shorter than that of patients in Group C which expressed GnT-V . Therefore, loss of non-sialylated L-PHA reactive oligosaccharides due to lack of expression of GnT-V in lymphoma cells may be associated with aggressiveness of DLBCL.

J Med Microbiol, 2003 Nov, 52(Pt 11), 933 - 9
Mutation in tcpR gene (Vc0832) of Vibrio cholerae O1 causes loss of tolerance to high osmolarity and affects colonization and virulence in infant mice; Mishra A et al.; Vibrio cholerae, the agent of cholera, multiplies and colonizes human intestinal tract where it survives high osmolarity due to bile and other sodium salts . In this work, by TnphoA mutagenesis, a mutant of V . cholerae O1 which could not grow and form colonies on LB agar containing 400 mM NaCl has been characterized . The mutant, designated CD83, adhered normally to freshly isolated rabbit intestinal discs, colonized poorly the gut of infant mice and was avirulent in the same model, whereas the parent strain CD81 would colonize the gut and cause death of mice . Attenuation of virulence of CD83 was not attributable to its inability to produce cholera toxin, as no difference was found in the cholera toxin produced in vitro by the mutant and parent strains . Molecular cloning and sequencing of the mutated gene revealed that insertion of transposon occurred in tcpR gene (Vc0832) of V . cholerae . Complementation of the mutant with wild-type tcpR gene resulted in restoration of the ability to survive at high salt concentration (400 mM NaCl), and to colonize the gut and restore virulence . The results suggest that tcpR plays a role in survival of V . cholerae in the small intestine of host as the osmolarity in the intestinal lumen is thought to be equivalent to 300 mM NaCl or higher.

J Clin Microbiol, 2003 Oct, 41(10), 4676 - 82
Genotypic analyses of Vibrio parahaemolyticus and development of a pandemic group-specific multiplex PCR assay; Okura M et al.; A total of 54 Vibrio parahaemolyticus strains including pandemic O3:K6 strains and newly emerged O4:K68, O1:K25, O1:K26, and O1:K untypeable strains (collectively referred to as the "pandemic group") were examined for their pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) profiles and for the presence or absence of genetic marker DNA sequences, toxRS/new or orf8, that had been reported elsewhere to be specific for the pandemic group . Both PFGE and AP-PCR analyses indicated that all strains of the pandemic group formed a distinct genotypic cluster, suggesting that they originated from the same clone . In addition to the pandemic group, four O3:K6 strains that did not possess the thermostable direct hemolysin (tdh) gene also belonged to this cluster and possessed the toxRS/new sequence . However, three O3:K6 strains that clearly belonged to the pandemic group by PFGE and AP-PCR did not possess the orf8 sequence . The evidence suggests that neither the toxRS/new nor the orf8 sequence is a reliable gene marker for definite identification of the pandemic group . We therefore developed a novel multiplex PCR assay specific for the pandemic group . The assay successfully distinguished pandemic group strains from other V . parahaemolyticus strains by yielding two distinct PCR products for tdh (263 bp) and the toxRS/new sequence (651 bp).

J Clin Microbiol, 2003 Oct, 41(10), 4502 - 11
Molecular subtyping of Vibrio cholerae O1 and O139 by pulsed-field gel electrophoresis in Hong Kong: correlation with epidemiological events from 1994 to 2002; Kam KM et al.; Two hundred twenty isolates of Vibrio cholerae O1 and O139 collected from 1994 to 2002 in Hong Kong were analyzed by pulsed-field gel electrophoresis (PFGE) . Chromosomal DNAs from all V . cholerae isolates in agarose plugs were digested with the restriction enzyme NotI, resulting in 20 to 27 bands . Sixty distinctive PFGE patterns in the range of 10 to 300 kb were noted among 213 isolates typeable by PFGE . By comparing the common PFGE patterns obtained from four well-defined outbreaks of V . cholerae O1 and O139 with those obtained from other, epidemiologically unrelated isolates during the study period, indistinguishable and similar PFGE patterns were identified, indicating their close relatedness, in agreement with the results of epidemiological investigations . Heterogeneous PFGE patterns (with four to six banding differences), however, were identified among strains that were imported from other parts of Asia, including Indonesia, India, and Pakistan . Correlations with epidemiological information further support the usefulness of PFGE as an epidemiological tool in laboratory investigations of suspected outbreaks . Standardization of PFGE methodology will allow international comparison of fingerprint patterns and will form the basis of a laboratory network for tracking V . cholerae.

Appl Environ Microbiol, 2003 Oct, 69(10), 6114 - 20
RpoS-dependent stress response and exoenzyme production in Vibrio vulnificus; Hulsmann A et al.; Vibrio vulnificus is an estuarine bacterium capable of causing rapidly fatal infections through both ingestion and wound infection . Like other opportunistic pathogens, V . vulnificus must adapt to potentially stressful environmental changes while living freely in seawater, upon colonization of the oyster gut, and upon infection of such diverse hosts as humans and eels . In order to begin to understand the ability of V . vulnificus to respond to such stresses, we examined the role of the alternate sigma factor RpoS, which is important in stress response and virulence in many pathogens . An rpoS mutant of V . vulnificus strain C7184o was constructed by homologous recombination . The mutant strain exhibited a decreased ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and acidic conditions . The most striking difference was a high sensitivity of the mutant to hydrogen peroxide . Albuminase, caseinase, and elastase activity were detected in the wild type but not in the mutant strain, and an additional two hydrolytic activities (collagenase and gelatinase) were reduced in the mutant strain compared to the wild type . Additionally, the motility of the rpoS mutant was severely diminished . Overall, these studies suggest that rpoS in V . vulnificus is important for adaptation to environmental changes and may have a role in virulence.

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