Vibriosis

Infect Immun, 2001 Oct, 69(10), 6091 - 101
The toxR gene of Vibrio (Listonella) anguillarum controls expression of the major outer membrane proteins but not virulence in a natural host model; Okuda J et al.; To examine the hypothesis that the ancestral role of the toxR gene in the family Vibrionaceae is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathogen . The toxR gene of V . angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed . The toxR sequence was 63 to 51% identical to those reported for other species of the family Vibrionaceae . Distribution of the Va-toxR gene sequence in V . anguillarum strains of various serotypes was confirmed by using DNA probe and PCR methods . An isogenic toxR mutant of V . anguillarum PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method . The wild-type strain and the toxR mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods . A 35-kDa major OMP was not produced by the toxR mutant . However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the toxR mutant . For the toxR mutant, the MICs of two beta-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain . Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the toxR-regulated major OMPs of the family Vibrionaceae . The results indicate that the toxR gene is not involved in virulence expression in V . anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae . These results support the hypothesis that the ancestral role of the toxR gene is regulation of OMP gene expression and that only in some Vibrio species has ToxR been appropriated for the regulation of a virulence gene(s).

Infect Immun, 2001 Oct, 69(10), 6084 - 90
Diminished diarrheal response to Vibrio cholerae strains carrying the replicative form of the CTX(Phi) genome instead of CTX(Phi) lysogens in adult rabbits; Faruque SM et al.; Toxigenic Vibrio cholerae strains are lysogens of CTX(Phi), a filamentous bacteriophage which encodes cholera toxin (CT) . Following infection of recipient V . cholerae cells by CTX(Phi), the phage genome either integrates into the host chromosome at a specific attachment site (attRS) or exists as a replicative-form (RF) plasmid . We infected naturally occurring attRS-negative nontoxigenic V . cholerae or attenuated (CTX(-) attRS negative) derivatives of wild-type toxigenic strains with CTX(Phi) and examined the diarrheagenic potential of the strains carrying the RF of the CTX(Phi) genome using the adult rabbit diarrhea model . Under laboratory conditions, strains carrying the RF of CTX(Phi) produced more CT than corresponding lysogens as assayed by a G(M1)-based enzyme-linked immunosorbent assay and by fluid accumulation in ligated ileal loops of rabbits . However, when tested for diarrhea in rabbits, the attRS-negative strains (which carried the CTX(Phi) genome as the RF) were either negative or produced mild diarrhea, whereas the attRS-positive strains with integrated CTX(Phi) produced severe fatal diarrhea . Analysis of the strains after intestinal passage showed that the attRS-negative strains lost the phage genome at approximately a fivefold higher frequency than under in vitro conditions, and 75 to 90% of cells recovered from challenged rabbits after 24 h were CT negative . These results suggested that strains carrying the RF of CTX(Phi) are unable to cause severe disease due to rapid loss of the phage in vivo, and the gastrointestinal environment thus provides selection of toxigenic strains with an integrated CTX(Phi) genome . These results may have implications for the development of live V . cholerae vaccine candidates impaired in chromosomal integration of CTX(Phi) . These findings may also contribute to understanding of the etiology of diarrhea occasionally associated with nontoxigenic V . cholerae strains.

Zh Mikrobiol Epidemiol Immunobiol, 2001 May-Jun, (3), 69 - 72
{Comparative study of the diagnostic value of new cholera eltor bacteriophages ctx+, ctx-, and KhDF-3, 4, and 5}; Saiapina LV et al.; The comparative evaluation of the diagnostic value of new cholera eltor bacteriophages ctx+ and ctx-, as well as monophages X{symbol: see text}-3, 4, 5, demonstrated their high activity and specificity . Using of these bacteriophages epidemic potential of 95% Vibrio cholerae eltor strains ctx+ and 84.5% of V . cholerae eltor stains ctx- was determined . Commercial monophages X{symbol: see text}-3, 4, 5 were inferior to bacteriophages ctx+ and ctx- in their diagnostic value: only 55% of strains having gene ctxAB were found to be epidemically dangerous, i.e . 45% of strains capable of causing the disease were not detected . On the basis of the results obtained in this investigation cholera eltor bacteriophages ctx+ and ctx- were recommended for introduction into practical use, while further production of cholera diagnostic monophages X{symbol: see text}-3, 4, 5 was recommended to be stopped.

Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 448 - 52
Isolation and characterization of a bacterium that produces hydrocarbons extracellularly which are equivalent to light oil; Park MO et al.; A halotorelant bacterial strain that produces a significant amount of lipids from short-chain fatty acids was isolated from the sludge of a sewage disposal plant . This strain displayed a significant extracellular accumulation of lipids . The yield of lipids including hydrocarbons was highest (120% of cell dry weight) at the end of the linear growth phase . Fractionation of the lipids using thin-layer chromatography and subsequent gas chromatography showed that hydrocarbons were also obtained following an increase in total lipids . Their yield was the highest (50% of cell dry weight) in the linear growth phase . Additional analysis using infrared absorption spectrum and gas chromatography-mass spectrometry showed that the hydrocarbon fraction was composed of alkanes, such as C15H32, C18H38, C21H44, C22H46 and C24H50 . Homology analysis of the 16s rDNA sequence as well as studies of the morphological and physiological characteristics indicated that the bacterium is a strain of Vibrio furnissii.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Mar-Apr, (2), 6 - 9
{Comparative pathomorphological assessment of the action of Vibrio cholerae serogroups 01 and 0139}; Isupov IV et al.; The pathomorphological picture of experimental infection caused by the infective agent of cholera was shown to have some specific features observed in infections caused by vibrios belonging to the serogroups under study . Infection caused by V . cholerae of serogroup O139 induced some morphological changes in the gastrointestinal tract which were quite characteristic of this disease, but inflammatory changes with the prevalence of proliferative infiltrative processes came to the foreground simultaneously with less developed processes of edema and dystrophic lesions of enterocytes . These specific morphological features in animals infected with V . cholerae of serogroup O139 appeared to be probably due to the production of new surface structures by these strains.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Mar-Apr, (2), 3 - 6
{Isolation and phenotyping of Vibrio cholerae strains with the enhanced production of main protective antigens}; Zadnova SP et al.; Two V . cholerae strains of classical biovar, 2414 (serovar Ogawa) and 2415 (serovar Inaba), with of increased production of main protective antigens--cholera toxin, toxin-coregulated pili of adhesion (TCP), outer membrane protein OmpU, as well as phospholipases and proteases, have been detected among natural and recombinant strains under study . A simultaneous increase in the production of the above-mentioned main immunogenicity factors in strains 2414 and 2415 is seemingly linked with the presence of recombinant plasmid pCT105 with cloned genes of cholera toxin in these microbial cells . As the result of plasmid-chromosomal relationships, this plasmid probably ensures the effective expression of global regulating gene toxR . The strains capable of the hyperproduction of cholera toxin, TCP and protein OmpU may be used for the manufacture of more effective chemical vaccine (choleragen-toxoid).

Zh Mikrobiol Epidemiol Immunobiol, 2001 Mar-Apr, (2), 25 - 9
{Analysis of toxin genetic determinants of Vibrio cholerae virulence cassette and neuraminidase with DNA probes}; Monakhova EV et al.; V . cholerae strains of different origin have been studied for the presence of cholera toxin genes (vct), the proximal part of the virulence cassette including genes zot, ace and orfU, as well as neuraminidase genes (neu), in their genomes with the use of molecular DNA probes . The possibility, in principle, for some strains to lose only a part of their virulence cassette (gene vct), while retaining its proximal part has been shown . In most cases such strains are isolated from patients with diarrhea of different severity and may probably play some etiological role, provided that the expression of the genes of additional toxins of the virulence cassette occurs . The gene expressing neuraminidase which facilitates the penetration of cholera toxin into the epithelial cells of the intestine is always present in vct+ strains and may be absent in vct- strains . The absence of genetic relationship between neuraminidases in V . cholerae O139 and V . cholerae O1 and non-O1 (non-O139) has been confirmed . The problems in connection with the integration and deletion of genetic determinants of V . cholerae virulence factors are discussed.

J Bacteriol, 2001 Oct, 183(19), 5762 - 7
Residue aspartate-147 from the third transmembrane region of Na(+)/H(+) antiporter NhaB of Vibrio alginolyticus plays a role in its activity; Nakamura T et al.; NhaB is a bacterial Na(+)/H(+) antiporter with unique topology . The pH dependence of NhaB from Vibrio alginolyticus differs from that of the Escherichia coli NhaB homolog . Replacement of Asp-147 with Glu made high H(+) concentrations a requirement for the NhaB activity . Replacement of Asp-147 with neutral amino acids inactivated NhaB.

J Bacteriol, 2001 Oct, 183(19), 5751 - 5
Identification, characterization, and functional analysis of a gene encoding the ferric uptake regulation protein in Bartonella species; Park SY et al.; Environmental iron concentrations coordinately regulate transcription of genes involved in iron acquisition and virulence via the ferric uptake regulation (fur) system . We identified and sequenced the fur gene and flanking regions of three Bartonella species . The most notable difference between Bartonella Fur and other Fur proteins was a substantially higher predicted isoelectric point . No promoter activity or Fur autoregulation was detected using a gfp reporter gene fused to the 204 nucleotides immediately upstream of the Bartonella fur gene . Bartonella henselae fur gene expression complemented a Vibrio cholerae fur mutant.

Syst Appl Microbiol, 1997 Jan, 20(1), 133 - 6
A low molecular weight artificial RNA of unique size with multiple probe target regions; Pitulle C et al.; Artificial RNAs (aRNAs) containing novel sequence segments embedded in a deletion mutant of Vibrio proteolyticus 5S rRNA have previously been shown to be expressed from a plasmid borne growth rate regulated promoter in E . coli . These aRNAs accumulate to high levels and their detection is a promising tool for studies in molecular microbial ecology and in environmental monitoring . Herein a new construct is described which illustrates the versatility of detection that is possible with aRNAs . This 3xPen aRNA construct carries a 72 nucleotide insert with three copies of a unique 17 base probe target sequence . This aRNA is 160 nucleotides in length and again accumulates to high levels in the E . coli cytoplasm without incorporating into ribosomes . The 3xPen aRNA illustrates two improvements in detection . First, by appropriate selection of insert size, we obtained an aRNA which provides a unique and hence, easily quantifiable peak, on a high resolution gel profile of low molecular weight RNAs . Second, the existence of multiple probe targets results in a nearly commensurate increase in signal when detection is by hybridization . These aRNAs are naturally amplified and carry sequence segments that are not found in known rRNA sequences . It thus may be possible to detect them directly . An experimental step involving RT-PCR or PCR amplification of the gene could therefore be avoided.

Syst Appl Microbiol, 1993, 16, 280 - 6
A prototype stable RNA identification cassette for monitoring plasmids of genetically engineered microorganisms; Hedenstierna KO et al.; A prototype stable RNA identification cassette for monitoring genetically engineered plasmids carried by strains of Escherichia coli has been developed . The cassette consists of a Vibrio proteolyticus 5S ribosomal RNA (rRNA) gene surrounded by promoters and terminators from the rrnB operon of Escherischia coli . The identifier RNA is expressed and successfully processed so that approximately 30% of the 5S rRNA isolated from either whole cells or 70S ribosomes is of the V . proteolyticus type . Cells carrying the identifier are readily detectable by hybridization . Accurate measurements show that the identification cassette has little effect on fitness compared to a strain containing an analogous plasmid carrying wild type E . coli 5S rRNA, and the V . proteolyticus 5S rRNA gene is not inactivated after prolonged growth . These results demonstrate the feasibility of developing small standardized identification cassettes that can utilize already existing highly sensitive rRNA detection methods . Cassettes of this type could in principle be incorporated into either the engineered regions of recombinant plasmids or their hosts.

Appl Environ Microbiol, 1988 Jan, 54(1), 250 - 6
34S/32S fractionation in sulfur cycles catalyzed by anaerobic bacteria; Fry B et al.; Stable isotopic distributions in the sulfur cycle were studied with pure and mixed cultures of the anaerobic bacteria, Chlorobium vibrioforme and Desulfovibrio vulgaris . D . vulgaris and C . vibrioforme can catalyze three reactions constituting a complete anaerobic sulfur cycle: reduction of sulfate to sulfide (D . vulgaris), oxidation of sulfide to elemental sulfur (C . vibrioforme), and oxidation of sulfur to sulfate (C . vibrioforme) . In all experiments, the first and last reactions favored concentration of the light 32S isotope in products (isotopic fractionation factor epsilon = -7.2 and -1.7%, respectively), whereas oxidation of sulfide favored concentration of the heavy 34S isotope in products (epsilon = +1.7%) . Experimental results and model calculations suggest that elemental sulfur enriched in 34S versus sulfide may be a biogeochemical marker for the presence of sulfide-oxidizing bacteria in modern and ancient environments.

J Photochem Photobiol B, 1992 Aug 14, 15(1-2), 171 - 9
Förster energy transfer in chlorosomes of green photosynthetic bacteria; Causgrove TP et al.; Energy transfer properties of whole cells and chlorosome antenna complexes isolated from the green sulfur bacteria Chlorobium limicola (containing bacteriochlorophyll c), Chlorobium vibrioforme (containing bacteriochlorophyll d) and Pelodictyon phaeoclathratiforme (containing bacteriochlorophyll e) were measured . The spectral overlap of the major chlorosome pigment (bacteriochlorophyll c, d or, e) with the bacteriochlorophyll a B795 chlorosome baseplate pigment is greatest for bacteriochlorophyll c and smallest for bacteriochlorophyll e . The absorbance and fluorescence spectra of isolated chlorosomes were measured, fitted to gaussian curves and the overlap factors with B795 calculated . Energy transfer times from the bacteriochlorophyll c, d or e to B795 were measured in whole cells and the results interpreted in terms of the Forster theory of energy transfer.

Biochim Biophys Acta, 1990 Feb 22, 1015(3), 457 - 63
Effects of oxidants and reductants on the efficiency of excitation transfer in green photosynthetic bacteria; Wang J et al.; The efficiency of energy transfer in chlorosome antennas in the green sulfur bacteria Chlorobium vibrioforme and Chlorobium limicola was found to be highly sensitive to the redox potential of the suspension . Energy transfer efficiencies were measured by comparing the absorption spectrum of the bacteriochlorophyll c or d pigments in the chlorosome to the excitation spectrum for fluorescence arising from the chlorosome baseplate and membrane-bound antenna complexes . The efficiency of energy transfer approaches 100% at low redox potentials induced by addition of sodium dithionite or other strong reductants, and is lowered to 10-20% under aerobic conditions or after addition of a variety of membrane-permeable oxidizing agents . The redox effect on energy transfer is observed in whole cells, isolated membranes and purified chlorosomes, indicating that the modulation of energy transfer efficiency arises within the antenna complexes and is not directly mediated by the redox state of the reaction center . It is proposed that chlorosomes contain a component that acts as a highly quenching center in its oxidized state, but is an inefficient quencher when reduced by endogenous or exogenous reductants . This effect may be a control mechanism that prevents cellular damage resulting from reaction of oxygen with reduced low-potential electron acceptors found in the green sulfur bacteria . The redox modulation effect is not observed in the green gliding bacterium Chloroflexus aurantiacus, which contains chlorosomes but does not contain low-potential electron acceptors.

Mol Gen Mikrobiol Virusol, 2001, (3), 23 - 8
{Prevalence of major virulence genes among various Vibrio cholerae El-TOR strains for evaluating their epidemiological significance}; Smirnova NI et al.; Specific oligonucleotide primers were chosen for identifying the fragments of the four major virulence genes of V . cholerae eltor (ctxA, tcpA, toxR, and hap) using the polymerase chain reaction (PCR) . In order to estimate the efficiency of complex PCR testing of V . cholerae for evaluation of their epidemiological significance, a collection of 80 V . cholerae eltor strains with known virulence was selected, whose most important specific features had been studied previously . The hap was appropriate species-specific gene making it possible to detect V . cholerae strains regardless of their virulence . The most complete and objective data for evaluating the epidemic significance can be obtained by detecting the presence of three virulence genes (ctxA, tcpA, and toxR) in their chromosome . The prevalence of the above four genes in various V . cholerae strains isolated from the environment during epidemic and non-epidemic periods was studied.

J Biol Chem, 2001 Nov 23, 276(47), 43645 - 52 Epub 2001 Aug 31.
A secreted aminopeptidase of Pseudomonas aeruginosa . Identification, primary structure, and relationship to other aminopeptidases; Cahan R et al.; Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains . The aminopeptidase was partially purified by DEAE-cellulose chromatography and found to be heat stable . The apparent molecular mass of the enzyme was approximately 56 kDa; hence, it was designated AP(56) . Heating (70 degrees C) of the partially purified aminopeptidase preparations led to the conversion of AP(56) to a approximately 28-kDa protein (AP(28)) that retained enzyme activity, a reaction that depended on elastase (LasB) . The pH optimum for Leu-pNA hydrolysis by AP(28) was 8.5 . This activity was inhibited by Zn chelators but not by inhibitors of serine- or thiol-proteases, suggesting that AP(28) is a Zn-dependent enzyme . Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate . The sequences of the first 20 residues of AP(56) and AP(28) were determined . A search of the P . aeruginosa genomic data base revealed a perfect match of these sequences with positions 39-58 and 273-291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase . A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus aminopeptidase, approximately 35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29-32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases . The residues potentially involved in zinc coordination were conserved in all these proteins . Thus, P . aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases.

J Mol Biol, 2001 Aug 31, 311(5), 1001 - 9
Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts; Daniell H et al.; The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens . Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome) . However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen . PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome . Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB . Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts . In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts . Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses . Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible . Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants . Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts .

Microbiol Immunol, 2001, 45(7), 543 - 7
Identification of a H+/glucose and galactose symporter gene glt from Xanthomonas oryzae pv . oryzae; Tsuge S et al.; We identified a glucose and galactose transporter gene from the plant-pathogenic bacterium Xanthomonas oryzae pv . oryzae . Sequence analysis indicated that the gene, named glt, encoded a polypeptide of 592 amino acid residues and the product was significantly homologous with members of the Na+/glucose cotransporter (SGLT) family from mammalian and bacterial origin, especially with vSGLT from Vibrio parahaemolyticus (50% identity) . GLT functioned as a glucose and galactose transporter in an Escherichia coli mutant deficient in glucose and galactose transport activity . A protonophore inhibited the transport activity, suggesting that GLT is a H+-coupled glucose/galactose symporter.

Microbiol Mol Biol Rev, 2001 Sep, 65(3), 445 - 62, table of contents
Polar flagellar motility of the Vibrionaceae; McCarter LL; Polar flagella of Vibrio species can rotate at speeds as high as 100,000 rpm and effectively propel the bacteria in liquid as fast as 60 microm/s . The sodium motive force powers rotation of the filament, which acts as a propeller . The filament is complex, composed of multiple subunits, and sheathed by an extension of the cell outer membrane . The regulatory circuitry controlling expression of the polar flagellar genes of members of the Vibrionaceae is different from the peritrichous system of enteric bacteria or the polar system of Caulobacter crescentus . The scheme of gene control is also pertinent to other members of the gamma purple bacteria, in particular to Pseudomonas species . This review uses the framework of the polar flagellar system of Vibrio parahaemolyticus to provide a synthesis of what is known about polar motility systems of the Vibrionaceae . In addition to its propulsive role, the single polar flagellum of V . parahaemolyticus is believed to act as a tactile sensor controlling surface-induced gene expression . Under conditions that impede rotation of the polar flagellum, an alternate, lateral flagellar motility system is induced that enables movement through viscous environments and over surfaces . Although the dual flagellar systems possess no shared structural components and although distinct type III secretion systems direct the simultaneous placement and assembly of polar and lateral organelles, movement is coordinated by shared chemotaxis machinery.

J Clin Microbiol, 2001 Sep, 39(9), 3241 - 6
Concomitant infection of enterotoxigenic Escherichia coli in an outbreak of cholera caused by Vibrio cholerae O1 and O139 in Ahmedabad, India; Chakraborty S et al.; In Ahmedabad, a major city in the state of Gujarat, India, an outbreak of acute secretory diarrhea caused by Vibrio cholerae O1 Ogawa El Tor, V . cholerae O139, and multiple serotypes of enterotoxigenic Escherichia coli (ETEC) occurred in January 2000 . All of the representative V . cholerae O1 and O139 isolates examined harbored the ctxA gene (encoding the A subunit of cholera toxin) and the El Tor variant of the tcpA gene (encoding toxin-coregulated pilus) . ETEC isolates of different serotypes were positive for the elt gene, encoding heat-labile enterotoxin . To further understand the molecular characteristics of the pathogens, representative isolates were examined by ribotyping and pulsed-field gel electrophoresis (PFGE) . Ribotyping showed that the isolates of V . cholerae O1 Ogawa exhibited a pattern identical to that of the prevailing clone of O1 in areas where cholera is endemic in India, and all of the O139 isolates were identical to the BII clone of V . cholerae O139 . PFGE of the representative O1 Ogawa isolates exhibited an identical pattern, comparable to the H pattern of the new clone of O1 reported in Calcutta, India . PFGE analysis of the V . cholerae O139 isolates showed identical patterns, but these differed from the PFGE patterns of O139 isolates reported during 1992 to 1997 in Calcutta . ETEC isolates showed genetic heterogeneity among isolates belonging to the same serotype, although the identical PFGE pattern was also observed among ETEC isolates of different serotypes . Antibiograms of the isolates were unusual, because all of the O139 isolates were resistant to nalidixic acid . Likewise, all of the E . coli isolates showed resistance to ciprofloxacin, norfloxacin, and nalidixic acid . This is a unique outbreak, and we believe that it is the first in which V . cholerae and ETEC were concomitantly involved.

Biochemistry, 2001 Sep 4, 40(35), 10655 - 63
Heterocycle formation in vibriobactin biosynthesis: alternative substrate utilization and identification of a condensed intermediate; Marshall CG et al.; The iron-chelating peptide vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit nonribosomal peptide synthetase complex, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two 2,3-dihydroxyphenyl- (DHP-) methyloxazolinyl groups in amide linkage on a norspermidine scaffold . We have tested the ability of the six-domain VibF subunit (Cy-Cy-A-C-PCP-C) to utilize various L-threonine analogues and found the beta-functionalized amino acids serine and cysteine can function as alternate substrates in aminoacyl-AMP formation (adenylation or A domain), aminoacyl-S-enzyme formation (A domain), acylation by 2,3-dihydrobenzoyl- (DHB-) S-VibB (heterocyclization or Cy domain), heterocyclization to DHP-oxazolinyl- and DHP-thiazolinyl-S-enzyme forms of VibF (Cy domain) as well as transfer to DHB-norspermidine at both N(5) and N(9) positions (condensation or C domain) to make the bis(oxazolinyl) and bis(thiazolinyl) analogues of vibriobactin . When L-threonyl-S-pantetheine or L-threonyl-S-(N-acetyl)cysteamine was used as a small-molecule thioester analogue of the threonyl-S-VibF acyl enzyme intermediate, the Cy domain(s) of a CyCyA fragment of VibF generated DHB-threonyl-thioester products of the condensation step but not the methyloxazolinyl thioesters of the heterocyclization step . This clean separation of condensation from cyclization validates a two-stage mechanism for threonyl, seryl, and cysteinyl heterocyclization domains in siderophore and antibiotic synthetases . Full heterocyclization activity could be restored by providing CyCyA with the substrate L-threonyl-S-peptidyl carrier protein (PCP)-C2, suggesting an important role for the protein scaffold component of the heterocyclization acceptor substrate . We also examined heterocyclization donor substrate specificity at the level of acyl group and protein scaffold and observed intolerance for substitution at either position.

Eur J Pharm Biopharm, 2001 Sep, 52(2), 145 - 50
Enhancing the permeation of marker compounds and enaminone anticonvulsants across Caco-2 monolayers by modulating tight junctions using zonula occludens toxin; Cox DS et al.; Zonula occludens toxin (Zot), a protein elaborated from Vibrio cholerae, has been shown to be capable of reversibly opening tight junctions between intestinal cells The objective of this study was to examine the effect of Zot on the flux of various molecules across Caco-2 cell monolayers . In addition, the transport of a series of anticonvulsants, the enaminones was also evaluated in the presence of Zot . The flux of {(14)C}mannitol, {(14)C}inulin and various enaminones across Caco-2 cell monolayers (n=6) was examined after pre-incubation for 1h with Zot (0 or 4000ng/ml) or phosphate-buffered saline (PBS) . At the end of the incubation period, the flux of radiolabeled compounds or enaminones (1x10(-4)M) was assessed over a 2-h period . In addition, dose-response studies with Zot (0, 1000, 2000 or 4000ng/ml) were performed using mannitol . The flux of both mannitol and inulin significantly increased (P<0.05) in the presence of Zot . The transport of the enaminones with Zot ranged from 9.42 to 26.83x10(-5)cm/s vs . 4.68 to 13.83x10(-5)cm/s without Zot . Zot significantly increased the transport of all agents tested . This suggests that the co-administration of drugs with Zot may be a useful delivery strategy to increase the intestinal permeability and hence oral absorption of poorly bioavailable agents.

J Infect Dis, 2001 Sep 15, 184(6), 799 - 802 Epub 2001 Aug 07.
Cholera in the United States, 1995-2000: trends at the end of the twentieth century; Steinberg EB et al.; To evaluate recent trends in cholera in the United States, surveillance data from all cases of laboratory-confirmed toxigenic Vibrio cholerae O1 and O139 infection reported to the Centers for Disease Control and Prevention between 1995 and 2000 were reviewed . Sixty-one cases of cholera, all caused by V . cholerae O1, were reported . There was 1 death, and 35 (57%) of the patients were hospitalized . Thirty-seven (61%) infections were acquired outside the United States; 14 (23%) were acquired through undercooked seafood consumed in the United States, 2 (3%) were acquired through sliced cantaloupe contaminated by an asymptomatically infected food handler, and no source was identified for 8 (13%) infections . The proportion of travel-associated infections resistant to trimethoprim-sulfamethoxazole, sulfisoxazole, streptomycin, and furazolidone increased from 7 (8%) of 88 in 1990-1994 to 11 (31%) of 35 in 1995-2000 . Foreign travel and undercooked seafood continue to account for most US cholera cases . Antimicrobial resistance has increased among V . cholerae O1 strains isolated from ill travelers.

Carbohydr Res, 2001 Aug 30, 334(3), 195 - 205
Synthetic explorations towards 3-deoxy-3-fluoro derivatives of D-perosamine; Poirot E et al.; Based on a literature precedent, preparation of methyl 4-azido-3,4,6-trideoxy-3-fluoro-alpha-D-mannopyranoside (18) was attempted via fluorination of methyl 4-azido-2-O-benzyl-4,6-dideoxy-alpha-D-altropyranoside with diethylaminosulfur trifluoride (DAST) . Contrary to expectations, the reaction took place with retention of configuration at the site of the fluorination yielding methyl 4-azido-2-O-benzyl-3,4,6-trideoxy-3-fluoro-alpha-D-altropyranoside . Treatment with DAST of methyl 4-azido-2-O-benzyl-4,6-dideoxy-alpha-D-allopyranoside (8), or its 2-(p-methoxybenzyl) analog 9 resulted in fluorination with inversion of configuration at position 3, to give the corresponding 3-deoxy-3-fluoro glucopyranosides 10 and 11, respectively . Accordingly, compound 18 was prepared from 11, by de-p-methoxybenzylation at O-2, followed by inversion of configuration at C-2 in the resulting methyl 4-azido-3,4,6-trideoxy-3-fluoro-alpha-D-glucopyranoside . The 2-O-methyl analog of 18 (19) was prepared by methylation of 18 . Compounds 18 and 19 were converted, conventionally, into the 3-fluoro analogs of the terminal determinants of the O-PS of Vibrio cholerae O:1, serotype Inaba and Ogawa, respectively.

J Food Prot, 2001 Aug, 64(8), 1172 - 7
Heterogeneity of environmental, retail, and clinical isolates of Vibrio vulnificus as determined by lipopolysaccharide-specific monoclonal antibodies; Zuppardo AB et al.; The opportunistic pathogen Vibrio vulnificus expresses lipopolysaccharide (LPS) antigens on its outer membrane surface . A serological typing system was developed for these antigens, utilizing the discriminatory recognition of monoclonal antibodies (MAb) by ELISA . MAb were used to recognize five unique types of LPS-associated antigens for examination of clinical . environmental, and retail isolates of V . vulnificus . The overall serotype profile of the clinical isolates was significantly different (P < 0.05) from that of the environmental and retail isolates . A higher percentage of clinical isolates were typable (61%) compared to the environmental isolates (41%) and retail isolates (44%) . In particular, the percentage of serotype 1/5 among clinical isolates (33%), compared to that of environmental (9%) and retail (4%), was highly significant (P < 0.0001) . Among the environmental Gulf Coast isolates, there were differences in the prevalence of serotypes 2 and 3 (P < 0.05), depending on whether isolates were obtained from Louisiana or Alabama harvest sites . There were no statistically significant differences between the serotype profiles of Gulf and Atlantic Coast retail isolates despite the absence of serotype 1/5 from the Atlantic Coast . While some serotype diversity was detected in V . vulnificus isolated during different seasons, from different geographic locations, and at retail versus at harvest, there was no apparent concordance between any of the serotype distributions obtained from oysters versus that isolated clinically . The heterogeneity of environmental isolates and relative homogeneity among clinical isolates suggest that human risk may not be predicted on quantitative exposure alone.

FEMS Microbiol Lett, 2001 Aug 7, 202(1), 79 - 83
Presence of high-affinity iron uptake systems in fish-isolated and environmental strains of Vibrio anguillarum serotype O3; Muino L et al.; This work describes the presence of high-affinity iron uptake systems in Vibrio anguillarum serotype O3 strains (subgroups A and B), isolated from diseased fish and environmental samples, as well as the presumptive effect of iron on their virulence . All strains demonstrated an ability to grow under iron-limiting conditions, production of catechol-type siderophores and synthesis of iron-regulated outer membrane proteins . However, clear differences were found depending on the isolation source, suggesting a more efficient iron uptake system in fish-isolated strains . Comparing the iron-regulated outer membrane protein profiles with those described for O1 and O2 serotypes, we found a protein which is specific for serotype O3, and another one that allows the differentiation of serotype O3 fish isolates from environmental strains . Moreover, only the strains showing this protein increased their virulence when iron was added to the inoculum in pathogenicity assays.

Med J Malaysia, 2001 Mar, 56(1), 4 - 9
Detection of Vibrio cholerae 01 from aquatic environment in Sarawak; Norazah A et al.; The detection of Vibrio cholerae 01 from the aquatic environment of Daro and Bintulu in Sarawak was carried out following an outbreak of cholera . Conventional culture methods and detection of ctx gene by polymerase chain reaction technique were carried out on 80 water samples . Only one sample was positive by culture methods while 8 were positive by PCR . DNA finger printing by pulsed-field gel electrophoresis showed that the clinical isolates in Daro and Bintulu were genetically identical while the environmental isolate was closely related . Recovery of Vibrio cholerae by culture method is poor and newer methods of detection should be developed.

Infect Immun, 2001 Sep, 69(9), 5943 - 8
Isolation and characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin; Fan JJ et al.; We isolated a Vibrio vulnificus mutant that was deficient in both metalloprotease and cytolysin by allelic exchange . The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties . The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice.

Infect Immun, 2001 Sep, 69(9), 5864 - 73
Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli; Taniguchi T et al.; The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems . CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane . In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP . Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E . coli . The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E . coli genome (50%) . The introduction of a recombinant plasmid containing the cof gene cluster into the E . coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2 . This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection.

Microbiol Immunol, 2001, 45(6), 417 - 24
Gene analysis of Vibrio cholerae NAGV14 pilus and its distribution; Kuroki H et al.; Adhesive pilus of Vibrio cholerae 034, strain NAGV14, was genetically analyzed . The deduced amino acid (aa) sequence of the major pilin structural gene (VcfA) was 67% homologous to the MshA pilin in the N-terminal region, but no homology was found in the C-terminal region which contained the antigenic epitopes . Upstream and downstream flanking regions examined were highly homologous to mshB and mshC of the MSHA (mannose-sensitive hemagglutinin) gene locus . A short leader sequence and a pair of cysteines near the C-terminus which are the characteristics of type 4a pilus family were found . The major pilin structural gene of NAGV14 was compared to that of a strain V10 producing non-adhesive pili . The deduced aa sequences showed 60% homology, and the distance between two cysteines in the C-terminal region was different . A total of 177 V . cholerae strains were investigated for the presence of a type 4 pilus gene locus by PCR, and 95% were positive . The major pilin gene of NAGV14 was detected in 4 of 93 V . cholerae non-O1, non-0139 strains tested, but none of the V . cholerae O1 and O139 (72 and 12 strains, respectively) . Our result suggested that a type 4 pilus gene locus similar to the MSHA gene locus is widely distributed among V . cholerae strains . We proposed naming this type 4 pilus gene locus the VCF (for V . cholerae flexible pili) gene locus.

New Microbiol, 2001 Jul, 24(3), 273 - 80
Virulence factors in Vibrios and Aeromonads isolated from seafood; Scoglio ME et al.; Thirty-one isolates from seafood, identified as Aeromonas hydrophila (7), Aeromonas caviae (11), Vibrio parahaemolyticus (3), Vibrio fluvialis (5), Vibrio alginolytictus (3), Vibrio metschnikovii (1) and Vibrio damsela (1), were tested for possible virulence factors including extracellular hydrolytic enzymes, haemolysins, cytotoxins (VERO and HEp-2 cells) and adherence ability (HEp-2 cells) . All the A . hydrophila strains were beta-haemolytic and produced cytotoxins as well as one strain of V . fluvialis . A . hydrophila and A . caviae strains, frequently adhesive, showed both aggregative and diffusive patterns, while five Vibrio strains only (three V . fluvialis, one V . parahaemolyticus and one V . alginolyticus) were adhesive with an aggregative pattern.

Microbiology, 2001 Aug, 147(Pt 8), 2089 - 101
Endogenous isolation of replicon probes for assessing plasmid ecology of marine sediment microbial communities; Cook MA et al.; Six functional replication origins (repGA14, repGA33, repGA70, repSD41, repSD164 and repSD172), obtained from endogenously isolated, broad-host-range (BHR) marine plasmids ranging in size from 5 to 60 kb, were used to determine plasmid occurrence in three coastal marine sediment sites (in California, Georgia and South Carolina, USA) . The plasmid-specific replicons were isolated from plasmid-bearing marine sediment bacteria belonging to the alpha and gamma subclasses of the Proteobacteria . The plasmid sources of the endogenous replicons were considered to be cryptic due to a lack of identifiable phenotypic traits . The putative Rep proteins from a number of these replicons showed similarity to replicons of two recognized families: RCR group III (repSD164) and the FIA family of theta group A (repSD41, repSD121, repGA33 and repGA14) . Plasmids isolated from marine bacteria belonging to the genera Pseudoalteromonas, Shewanella and Vibrio cultivated from geographically different coastal sites exhibited homology to two of the marine plasmid replicons, repSD41 and repGA70, obtained from a Vibrio sp . The repGA33 plasmid origin, obtained from a Shewanella sp . isolated from coastal Georgia, was detected in 7% of the Georgia marine sediment Shewanella sp . isolates . Microbial community DNA extracted from marine sediments was also screened for the presence of the plasmid replication sequences . Community DNA samples amplified by PCR yielded a positive signal for the repSD172 and repGA14 replication sequences . The replication origin of BHR plasmid RK2 (IncP) was also detected in marine Vibrio sp . and microbial community DNA extracted from the three coastal sites . These findings provide molecular evidence that marine sediment bacteria harbour an untapped population of BHR plasmids.

Kansenshogaku Zasshi, 2001 Jun, 75(6), 485 - 9
{The trends of Vibrio parahaemolyticus foodborne outbreaks in Tokyo: 1989-2000}; Obata H et al.; The total number of foodborne outbreaks due to Vibrio parahaemolyticus in Tokyo during the last 12 years between 1989 and 2000 were 710 . The number of outbreaks in a year was 55 in 1989, 75 in 1990, and there was a gradual decrease to 24 outbreaks in 1993 which was the smallest number during those 12 years . After 1994, the number of outbreaks increased dramatically year by year until 1998 (107 outbreaks) . Then they had decreased slightly to 74 in 1999, 65 in 2000 . The monthly incidence of V . parahaemolyticus foodborne outbreaks showed a peak in August (44.2%) each year . In the last 12 years, 88.7% of V . parahaemolyticus foodborne outbreaks occurred during the 3 months between July and September, while 99.9% occurred between June and October . The most prevalent serotype of V . parahaemolyticus also changed, the most prevalent was O4:K4 in 1989, O4:K8 in both 1990 and 1991, O1:K56 in 1992, and O4:K8 from 1993 through 1995 . Serotype O3:K6 became the most prevalent in 1996 and has remained so to date . In addition, the new serotype O4:K68 had also appeared in 1998 . The number of outbreaks due to serotype O4:K68 followed that of O3:K6 . Thus, the trends of V . parahaemolyticus foodborne outbreaks during the last 12 years in Tokyo showed various characteristics and dramatic changes in causal organisms.

J Infect Dis, 2001 Sep 1, 184(5), 643 - 7 Epub 2001 Aug 09.
Local production of anti-vibrio cholerae mucosal antibody in reproductive tract tissues after cholera; Ryan ET et al.; To investigate whether intestinal presentation of an antigen by Vibrio cholerae, a noninvasive organism, could induce an anatomically distant mucosal immune response in reproductive tract tissues, the endocervical immune responses of women in Bangladesh were evaluated after cholera . Endocervical secretions were analyzed for secretory IgA (sIgA) antibody against the B subunit of cholera toxin (CtxB) in 9 women with cholera and 8 women with diarrhea caused by neither V . cholerae nor heat labile enterotoxin-producing Escherichia coli . Women infected with V . cholerae developed significant sIgA anti-CtxB responses in endocervical samples (P< or =.02) . Antibody subtype analysis of endocervical IgA was consistent with local mucosal production (P< or =.001) . Women with cholera did not develop sIgA anti-CtxB responses in serum . The ability to generate specific mucosal immune responses in reproductive tract tissues after intestinal presentation of antigen could facilitate development of vaccines effective against reproductive tract pathogens.

Environ Toxicol Chem, 2001 Aug, 20(8), 1733 - 9
Freeze concentration of ambient waters for toxicity testing; deBruyn AM et al.; We have developed a method to concentrate aqueous samples for toxicity testing . This method relies on the phenomenon of freezing exclusion, whereby solutes are rejected from the interstices of a growing ice crystal . Tenfold freeze concentration gave excellent recoveries of inorganic and organic analytes, phenol and ZnSO4 toxicity from spiked natural waters, and toxicity of both pre- and postdischarge municipal wastewater . Simultaneous 10-fold concentration of strong mineral or humic ambient matrices did not substantially modify the expressed toxicity of phenol or ZnSO4, and it did not seem to generate spurious toxicity to the marine bioassay organism used (Vibrio fischeri) . Hundredfold freeze concentration permitted the quantification of low levels of ambient toxicity in a wide variety of natural waters using a rapid, inexpensive microbioassay . Precipitation of matrix elements may limit the degree of concentration that can be achieved with highly mineralized or strongly humic waters . This approach is well suited to ambient toxicity testing, because it is nonspecific and has low potential for solvent contamination . Furthermore, the low temperatures involved minimize volatilization and degradation of organic contaminants.

Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1383 - 8
Vibrio shiloi sp . nov., the causative agent of bleaching of the coral Oculina patagonica; Kushmaro A et al.; The aetiological agent of bleaching of the coral Oculina patagonica was characterized as a new Vibrio species on the basis of 16S rDNA sequence, DNA-DNA hybridization data and phenotypic properties, including the cellular fatty acid profile . Based on its 16S rDNA and DNA-DNA hybridization, the new Vibrio species is closely related to Vibrio mediterranei . The name Vibrio shiloi sp . nov . is proposed for the new coral-bleaching species, the type strain being AK1T (= ATCC BAA-91T = DSM 13774T).

Mol Microbiol, 2001 Jul, 41(2), 393 - 407
Overlapping binding sites for the virulence gene regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter; Kovacikova G et al.; The expression of the Vibrio cholerae virulence factors, toxin-co-regulated pilus (TCP) and cholera toxin (CT), are dependent on the ability of the LysR regulator AphB to co-operate with a second protein, AphA, to activate the expression of the membrane-bound transcription factors TcpP and TcpH . To gain insights into the mechanism by which AphA and AphB co-operate to activate the expression of tcpPH, we have purified these two proteins to near homogeneity and show that they are each capable of interacting with the classical tcpPH promoter at distinct binding sites . As shown by tcpP-lacZ promoter deletion experiments, gel shift and DNase I footprinting, AphA binds to and activates from a region of the promoter between -101 and -71 from the start of transcription . AphB binds to and activates from a partially overlapping downstream site between -78 and -43, and these functions are dependent upon a region of partial dyad symmetry that resembles the well-characterized LysR-binding motif . A single basepair difference in this region of dyad symmetry has been shown previously to play a critical role in the expression of virulence genes between the two disease-causing biotypes of V . cholerae, classical and El Tor . We also show here that the tcpPH promoter is negatively influenced by the global regulator cAMP-CRP . Purified CRP binds to a near-consensus sequence in the tcpPH promoter in a cAMP-dependent manner and protects from DNase I digestion a region that is completely within the region protected by AphA and AphB . These findings raise the possibility that the negative effect of cAMP-CRP on virulence gene expression is the result of its ability to influence AphA- and AphB-dependent transcriptional activation of tcpPH under various conditions.

Mol Microbiol, 2001 Jul, 41(2), 311 - 23
Evidence for a rolling-circle mechanism of phage DNA synthesis from both replicative and integrated forms of CTXphi; Moyer KE et al.; The genes encoding cholera toxin, the principal virulence factor of Vibrio cholerae, are part of the circular single-stranded DNA genome of CTXphi . In toxigenic V . cholerae strains, the CTXphi genome is typically found in integrated arrays of tandemly arranged CTX prophages . Infected cells that lack a chromosomal integration site harbour the CTXphi genome as a plasmid (pCTX) . We studied the replication of pCTX and found several indications that this plasmid replicates via a rolling-circle (RC) mechanism . The initiation and termination sites for pCTX plus-strand DNA synthesis were mapped to a 22 bp sequence that contains inverted repeats and a nonanucleotide motif found in the plus-strand origins of several RC replicons . Furthermore, similar to other RC replicons, replication of plasmids containing duplicated pCTX origins resulted in the deletion of sequences between the two origins and the formation of a single chimeric origin . Our previous work revealed that CTX prophage arrays give rise to hybrid CTX virions that contain sequences derived from two adjacent prophages . We now report that the boundaries between the sequences contributed to virions by the upstream and the downstream prophages in an array correspond to the site at which synthesis of plus-strand pCTX DNA is initiated and terminated . These data support the model that plus-strand CTXphi DNA is generated from chromosomal prophages via a novel process analogous to RC replication.

Eur J Biochem, 2001 Aug, 268(15), 4261 - 8
Identification of protein-coding genes in the genome of Vibrio cholerae with more than 98% accuracy using occurrence frequencies of single nucleotides; Wang J et al.; The published sequence of the Vibrio cholerae genome indicates that, in addition to the genes that encode proteins of known and unknown function, there are 1577 ORFs identified as conserved hypothetical or hypothetical gene candidates . Because the annotation is not 100% accurate, it is not known which of the 1577 ORFs are true protein-coding genes . In this paper, an algorithm based on the Z curve method, with sensitivity, specificity and accuracy greater than 98%, is used to solve this problem . Twenty-fold cross-validation tests show that the accuracy of the algorithm is 98.8% . A detailed discussion of the mechanism of the algorithm is also presented . It was found that 172 of the 1577 ORFs are unlikely to be protein-coding genes . The number of protein-coding genes in the V . cholerae genome was re-estimated and found to be approximately 3716 . This result should be of use in microarray analysis of gene expression in the genome, because the cost of preparing chips may be somewhat decreased . A computer program was written to calculate a coding score called VCZ for gene identification in the genome . Coding/noncoding is simply determined by VCZ > 0/VCZ < 0 . The program is freely available on request for academic use.

Emerg Infect Dis, 2001, 7(3 Suppl), 583 - 7
Cholera outbreak in southern Tanzania: risk factors and patterns of transmission; Acosta CJ et al.; To identify risk factors and describe the pattern of spread of the 1997 cholera epidemic in a rural area (Ifakara) in southern Tanzania, we conducted a prospective hospital-based, matched case- control study, with analysis based on the first 180 cases and 360 matched controls . Bathing in the river, long distance to water source, and eating dried fish were significantly associated with risk for cholera . Toxigenic Vibrio cholerae O1, biotype El Tor, serotype Ogawa, was isolated in samples from Ifakara's main water source and patients' stools . DNA molecular analyses showed identical patterns for all isolates.

Southeast Asian J Trop Med Public Health, 2001 Mar, 32(1), 95 - 9
Transition of drug susceptibilities of Vibrio cholerae O1 in Lao People's Democratic Republic; Phantouamath B et al.; The changes of drug susceptibilities of Vibrio cholerae O1 isolated during the past 7 years (1993-1999) in Lao PDR were investigated . The most noteworthy finding was the appearance of polymyxin B sensitive El Tor vibrios . Until 1996, the susceptibilities were almost as expected and cholera disappeared in 1997 . When a cholera outbreak resurfaced in 1998, the susceptibilities of isolated V . cholerae O1 against tetracycline, sulfamethoxazol-trimethoprim, chloramphenicol and polymyxin B were quite different from those of previously isolated organisms . Minimum inhibitory concentrations (MICs) of tetracycline and chloramphenicol against the isolates in 1998 were about 16 times higher than those against the previous isolates, and the MICs of sulfamethoxazol-trimethoprim were about 256 times higher than those against the previous isolates, (trimethoprim 32 microg/ml: sulfamethoxazol 608 microg/ml) . Eleven percent of the isolates (11/99) were as sensitive to polymyxin B as the classic cholera vibrios (MIC < 2 microg/ml) . In 1999, the susceptibility pattern was almost the same as that in 1998 except for polymyxin B to which 58% of the isolates (21/36) became sensitive.

Southeast Asian J Trop Med Public Health, 2001 Mar, 32(1), 100 - 4
Detection and molecular characterization of the zot gene in Vibrio cholerae and V . alginolyticus isolates; Raja N et al.; A total of 11 Vibrio cholerae isolates from 1996-1998 outbreaks in Malaysia and 4 V . alginolyticus were analyzed . Isolates were characterized by polymerase chain reaction (PCR) and Southern hybridization for the presence of the gene encoding zonula occludens toxin (zot) . Screening of zot gene by PCR revealed the presence of this gene in V . cholerae and V . alginolyticus . The zot gene from one V . cholerae Ogawa isolate that was cloned in a pCR 2.1 TOPO vector was sequenced . The sequences obtained were 99% homologous to the zot gene sequence from the Gene Bank.

Glycobiology, 2001 Aug, 11(8), 655 - 61
Expression and identification of the RfbE protein from Vibrio cholerae O1 and its use for the enzymatic synthesis of GDP-D-perosamine; Albermann C et al.; The 4-amino-6-deoxy-monosaccharide D-perosamine is an important element in the glycosylation of interesting cell products, such as antibiotics and lipopolysaccharides (LPS) of Gram-positive and Gram-negative bacteria . The biosynthetic pathway of the precursor molecule, GDP-D-perosamine, in Vibrio cholerae O1 starts with an isomerisation of fructose-6-phosphate catalyzed by the bifunctional enzyme phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase (RfbA; E.C . 2.7.7.22) creating the intermediate mannose-6-phosphate, which is subsequently converted by the phosphomanno-mutase (RfbB; E.C . 5.4.2.8) and further by RfbA to GDP-D-mannose, to GDP-4 keto-6-deoxymannose by a 4,6-dehydratase (RfbD; E.C . 4.2.1.47) and finally to GDP-D-perosamine by an aminotransferase (RfbE; E.C . not yet classified) . We cloned the rfbD and the rfbE genes of V . cholerae O1 in Escherichia coli expression vectors . Both biosynthetic enzymes were overproduced in E . coli BL21 (DE3) and their activities were analyzed . The enzymatic conversion from GDP-D-mannose to GDP-D-perosamine was optimized and the final product, GDP-D-perosamine, was purified and identified by nuclear magnetic resonance, mass spectrometry, and chromatography . The catalytically active form of the GDP-4-keto-6-deoxy-D-mannose-4-aminotransferase seems to be a tetramer of 170 kDa . The His-tag RfbE fusion protein has a Km of 0.06 mM and a Vmax value of 38 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose . The Km and Vmax values for the cosubstrate L-glutamate were 0.1 mM and 42 nkat/mg protein, respectively . The intention of this work is to establish a basis for both the in vitro production of GDP-D-perosamine and for an in vivo perosaminylation system in a suitable bacterial host, preferably E . coli.

Fish Shellfish Immunol, 2001 Jul, 11(5), 415 - 31
In vitro interactions between rainbow trout (Oncorhynchus mykiss) macrophages and Vibrio anguillarum serogroup O2a; Boesen HT et al.; The sensitivity of Vibrio anguillarum serogroup O2a to killing by rainbow trout macrophages in the presence or absence of specific antibodies and complement components was evaluated using an in vitro assay . Fluorescence microscopy revealed that V . anguillarum serogroup O2a was phagocytosed by rainbow trout macrophages . In the absence of specific antibodies and complement components the bacteria were killed to a limited extent by the macrophages and there was no increased killing if the bacteria were opsonised with either antibodies or antibodies and complement . Furthermore, activated macrophages did not show enhanced ability to kill the bacteria . Vibrio anguillarum serogroup O2a were susceptible to both cell-free superoxide anion (O2-) and hydrogen peroxide (H2O2), which might be generated during the macrophage respiratory burst and the bacteria did not quench cell-free O2- . However, the production of O2- by macrophages was undetectable during the first 30 min following infection and no respiratory burst was inducible by phorbol myristate acetate (PMA) 4 h after infection with V . anguillarum . This suggests that the bacteria were able to inhibit the production of O2- by the infected macrophages . Naive fish were protected when passively immunised with anti-V . anguillarum serogroup O2a antiserum . However, previous results suggest that antibodies are unlikely to provide the fish with protective immunity directly through activation of the complement system and lysis of the bacterial cells . The present in vitro findings suggest that the protective mechanisms of antibody against V . anguillarum serogroup O2a may not involve the opsonising effect of antibodies for enhanced killing by macrophages . However, the possibility exists that such antibodies may prevent the attachment of the pathogen to the host's tissues.

Fish Shellfish Immunol, 2001 Jul, 11(5), 399 - 413
Immunoglobulin genes and antibody responses in the spotted wolffish (Anarhichas minor Olafsen); Espelid S et al.; The spotted wolffish Anarhichas minor Olafsen is a promising new species in aquaculture in the cold waters of northern Norway . In this paper, some basic immunological studies of this marine species are reported . Of comparative interest are the cDNA sequences of the immunoglobulin transcript and the antibody responses to model antigens . Of more practical importance are the humoral immune responses and antibody specificities to potentially pathogenic bacteria . Full length cDNA clones encoding the immunoglobulin heavy and light chains in the spotted wolffish were sequenced demonstrating variable degrees of similarity to other teleost fish species . Also in the spotted wolffish the CH4 domain was deleted in the transmembrane form of the immunoglobulin heavy chain (IgH) as a receptor on B cells, with the transmembrane exon spliced directly to the CH3 domain . The antibody responses to various antigens like hapten-carrier molecules, protein antigens and bacterial pathogens were relatively high, but with some interesting exceptions . Anti-hapten responses to NIP and FITC were high while anti-DNS responses were low, but more surprisingly, there was hardly any B-cell response to the carrier molecule LPH . On the other hand, protein antigens like CGG and BSA were highly immunogenic in the spotted wolffish as were the bacterial antigens Vibrio anguillarum, V . salmonicida and Aeromonas salmonicida.

J Appl Microbiol, 2001 Aug, 91(2), 322 - 7
Comparison of selective media for the detection of Vibrio vulnificus in environmental samples; Cerda-Cuellar M et al.; AIMS: To compare two selective agars, cellobiose-colistin (CC) agar and a modification of the Vibrio vulnificus medium (VVMc agar), for the isolation of Vibrio vulnificus from environmental samples . METHODS AND RESULTS: The efficiencies of recovery of V . vulnificus collection strains on CC, VVM, VVMc and on thiosulphate-citrate-bile salts-sucrose (TCBS) agar were compared and similar efficiencies were obtained . A slightly higher recovery was observed on VVMc agar . The detection of V . vulnificus in environmental samples (eels and water) was performed by combining culture-based methods (CC and VVMc agars) with DNA-based methods using species-specific probes based on the cytolysin-haemolysin and the 16S rDNA genes . A lower accompanying microbiota was found on CC agar than on VVMc agar . CONCLUSION: The comparison between CC and VVMc agars confirms that both are useful for the detection of V . vulnificus in environmental samples . However, the use of any of these media should be combined with a species-specific probe . SIGNIFICANCE AND IMPACT OF THE STUDY: The combined use of a selective medium and a specific probe provides a feasible method for the detection of V . vulnificus for epidemiological and ecological studies.

Arch Pathol Lab Med, 2001 Aug, 125(8), 1107 - 9
Vibrio vulnificus septicemia in a patient with the hemochromatosis HFE C282Y mutation; Gerhard GS et al.; Vibrio vulnificus is an extremely invasive gram-negative bacillus found in marine waters that causes overwhelming bacteremia and shock that is associated with high mortality . Impaired iron metabolism has been implicated in the susceptibility to V vulnificus bacterial infections . We report a case of fatal V vulnificus sepsis in a 56-year-old man who died within 1 to 3 days after consuming raw seafood . At autopsy, he was found to have micronodular cirrhosis and iron overload . Postmortem genetic analysis revealed the presence of the hemochromatosis gene (HFE) C282Y mutation . To our knowledge, this is this first documented fatal case of V vulnificus infection in a patient proven to carry the HFE C282Y mutation . Because this patient was heterozygous for the major hereditary hemochromatosis mutation and was not previously diagnosed with clinical iron overload, the spectrum of clinical susceptibilities to V vulnificus infection may include carriers of the C282Y mutation.

Appl Environ Microbiol, 2001 Aug, 67(8), 3707 - 11
Purification and characterization of a vulnificolysin-like cytolysin produced by Vibrio tubiashii; Kothary MH et al.; An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200 . This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3 . In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture . Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH . Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.

Lett Appl Microbiol, 2001 Aug, 33(2), 137 - 43
Diversity of Vibrio spp . populations in several exhibition aquaria with a shared water supply; Blanch AR et al.; AIMS: Abiotic factors may influence the settlement of bacterial populations in similar marine environments . Exhibition aquaria are a model for the study of the settlement of bacteria in different environment . Vibrio populations in the seawater reservoir, the Mediterranean tank and the Tropical tank from an exhibition aquarium on the western coast of the Mediterranean were compared and the effect of abiotic factors on the structure of these populations was considered . METHODS AND RESULTS: High diversity indexes and similar Vibrio populations were found in the water of the reservoir and of the Mediterranean tank, whereas a lower diversity and different main populations were found in the water of the Tropical tank . The antibiotic resistance profiles of the most representative strains, presented a number of differences depending on the origin of the sample . CONCLUSION: Abiotic conditions, mainly temperature, may determine the structure and composition of Vibrio populations in exhibition aquaria . SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial monitoring of water could be useful for health management of aquatic environments.

Microbiol Immunol, 2001, 45(5), 393 - 7
Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other vibrios; Alam MJ et al.; We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V . parahaemolyticus using both selective and non-selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment . Culture temperatures (20 or 37 C) did not affect the recovery of V . parahaemolyticus but did for other vibrios . We observed similar seasonality of V parahaemolyticus as in most other environmental studies . V . parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile-salt sucrose) agar counts . Even with the replica plating method, however, vibrios number goes down to a minimum level and V . parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CFU on nutrient agar (with 2% NaCl) did not vary so much during the study period.

Arch Biochem Biophys, 2001 Aug 1, 392(1), 110 - 6
Flavin specificity and subunit interaction of Vibrio fischeri general NAD(P)H-flavin oxidoreductase FRG/FRase I; Tang CK et al.; Apoenzyme of the major NAD(P)H-utilizing flavin reductase FRG/FRase I from Vibrio fischeri was prepared . The apoenzyme bound one FMN cofactor per enzyme monomer to yield fully active holoenzyme . The FMN cofactor binding resulted in substantial quenching of both the flavin and the protein fluorescence intensities without any significant shifts in the emission peaks . In addition to FMN binding (K(d) 0.5 microM at 23 degrees C), the apoenzyme also bound 2-thioFMN, FAD and riboflavin as a cofactor with K(d) values of 1, 12, and 37 microM, respectively, at 23 degrees C . The 2-thioFMN containing holoenzyme was about 40% active in specific activity as compared to the FMN-containing holoenzyme . The FAD- and riboflavin-reconstituted holoenzymes were also catalytically active but their specific activities were not determined . FRG/FRase I followed a ping-pong kinetic mechanism . It is proposed that the enzyme-bound FMN cofactor shuttles between the oxidized and the reduced form during catalysis . For both the FMN- and 2-thioFMN-containing holoenzymes, 2-thioFMN was about 30% active as compared to FMN as a substrate . FAD and riboflavin were also active substrates . FRG/FRase I was shown by ultracentrifugation at 4 degrees C to undergo a monomer-dimer equilibrium, with K(d) values of 18.0 and 13.4 microM for the apo- and holoenzymes, respectively . All the spectral, ligand equilibrium binding, and kinetic properties described above are most likely associated with the monomeric species of FRG/FRase I . Many aspects of these properties are compared with a structurally and functionally related Vibrio harveyi NADPH-specific flavin reductase FRP .

Protein Sci, 2001 Aug, 10(8), 1563 - 71
Modeling of the bacterial luciferase-flavin mononucleotide complex combining flexible docking with structure-activity data; Lin LY et al.; Although the crystal structure of Vibrio harveyi luciferase has been elucidated, the binding sites for the flavin mononucleotide and fatty aldehyde substrates are still unknown . The determined location of the phosphate-binding site close to Arg 107 on the alpha subunit of luciferase is supported here by point mutagenesis . This information, together with previous structure-activity data for the length of the linker connecting the phosphate group to the isoalloxazine ring represent important characteristics of the luciferase-bound conformation of the flavin mononucleotide . A model of the luciferase-flavin complex is developed here using flexible docking supplemented by these structural constraints . The location of the phosphate moiety was used as the anchor in a flexible docking procedure performed by conformation search by using the Monte Carlo minimization approach . The resulting databases of energy-ranked feasible conformations of the luciferase complexes with flavin mononucleotide, omega-phosphopentylflavin, omega-phosphobutylflavin, and omega-phosphopropylflavin were filtered according to the structure-activity profile of these analogs . A unique model was sought not only on energetic criteria but also on the geometric requirement that the isoalloxazine ring of the active flavin analogs must assume a common orientation in the luciferase-binding site, an orientation that is also inaccessible to the inactive flavin analog . The resulting model of the bacterial luciferase-flavin mononucleotide complex is consistent with the experimental data available in the literature . Specifically, the isoalloxazine ring of the flavin mononucleotide interacts with the Ala 74-Ala 75 cis-peptide bond as well as with the Cys 106 side chain in the alpha subunit of luciferase . The model of the binary complex reveals a distinct cavity suitable for aldehyde binding adjacent to the isoalloxazine ring and flanked by other key residues (His 44 and Trp 250) implicated in the active site.

J Bacteriol, 2001 Aug, 183(16), 4737 - 46
Characterization of VPI pathogenicity island and CTXphi prophage in environmental strains of Vibrio cholerae; Mukhopadhyay AK et al.; Environmental isolates of Vibrio cholerae of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into V . cholerae evolution . We found that each isolate contained a variant form of the VPI pathogenicity island . Distinguishing features included (i) four new alleles of tcpF (which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical tcpF; (ii) a new allele of toxT (virulence regulatory gene), 36% divergent (at the protein level) in its 5' half and nearly identical in its 3' half to canonical toxT; (iii) a new tcpA (pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of toxT . Also found were transpositions of an IS903-related element and function-unknown genes to sites in VPI . Cholera toxin (ctx) genes were found in isolates of two RAPD types, in each case embedded in CTXphi-like prophages . Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types . New possible prophage repressor and replication genes were also identified . Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic V . cholerae strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species.

Mar Environ Res, 2000 Jul-Dec, 50(1-5), 163 - 8
Immune function, hepatic CYP1A, and reproductive biomarker responses in the gulf killifish, Fundulus grandis, during dietary exposures to endocrine disrupters; Rice CD et al.; The gulf killifish, Fundulus grandis, was used to determine the influence of biological rhythms on three biomarker responses . We first developed monoclonal antibodies against the model's immunoglobulins and vitellogenin in order to measure antibody responses and vitellogenesis, respectively . We then treated adults with 10, 1, 1, and 10 ppm of Aroclor 1254, tribuyltin, 3-methylcholanthrene, and nonyl-phenol, respectively, in mixtures over a 16-week period . The study followed Vibrio anguillarum-specific antibody responses, hepatic CYP1A, and plasma vitellogenin levels in the morning and again in the evening at 2-week intervals . The contaminated diet suppressed secondary antibody responses, but only in the morning . The contaminated diet also altered CYP1A, but not vitellogenesis . In addition, fish in the control group exhibited daily and seasonal differences in specific antibody levels and CYP1A induction . Moreover, circulating vitellogenin levels in control males sampled in the morning increased throughout the exposure, but remained below those of females . This study underscores the need to consider normal physiological rhythms when employing biomarkers in toxicology.

Carbohydr Res, 2001 Jul 19, 333(4), 263 - 9
Depolymerization of the capsular polysaccharide from Vibrio cholerae O139 by a lyase associated with the bacteriophage JA1; Linnerborg M et al.; We have studied the interaction between the Vibrio cholerae O139 specific phage JA1, belonging to the Podoviridae family, and the capsular polysaccharide (CPS) of the parent strain from which the phage was isolated . Upon incubation of the JA1 phage with the CPS, oligosaccharides were isolated and purified . The oligosaccharides derived from one (shown below) and two repeating units of the CPS were characterized using NMR spectroscopy, mass spectrometry and sugar analysis (structure: see text) . The cleavage was found to occur by beta-elimination at the 4-substituted alpha-linked galacturonic acid, which results in a 4-deoxy-beta-L-threo-hex-4-enopyranosyl uronic acid group (Sug) . The enzyme associated with the JA1 phage responsible for the depolymerization of the V . cholerae O139 CPS is thus a lyase.

Environ Sci Technol, 2001 Jul 1, 35(13), 2773 - 7
Rhizosphere effects on microbial community structure and dissipation and toxicity of polycyclic aromatic hydrocarbons (PAHs) in spiked soil; Joner EJ et al.; Phytoremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs) has so far neglected the possible role of the ubiquitous symbiotic associations between plant roots and fungi known as arbuscular mycorrhizas . A time course laboratory experiment with clover and ryegrass grown on spiked {500 + 500 + 50 mg kg-1 of anthracene, chrysene and dibenz(a,h)anthracene} soil demonstrated for the first time that dissipation of condensed PAHs may be enhanced in the presence of arbuscular mycorrhiza {66 and 42% reductions in chrysene and dibenz(a,h)anthracene, respectively, versus 56 and 20% reductions in nonmycorrhizal controls} . Addition of a surfactant accelerated initial PAH dissipation but did not attain final PAH concentrations below those obtained with nonmycorrhizal plants . Toxicity tests (earthworm survival and bioluminescence inhibition in Vibrio fischeri) indicated that mycorrhiza reduced the toxicity of PAHs and/or their metabolites and counteracted a temporally enhanced toxicity mediated by surfactant addition . Phospholipid fatty acid profiles demonstrated that the imposed treatments altered the microbial community structure and indicated that the mycorrhiza-associated microflora was responsible for the observed reductions in PAH concentrations in the presence of mycorrhiza.

Mol Gen Mikrobiol Virusol, 2001, (2), 24 - 6
{PCR-detection of markers for epidemiologically-significant strains of Vibrio cholerae 0139, isolated in Siberia}; Balakhonov SV et al.; Fourteen V . cholerae 0139 strains were isolated in 1996-1999 in Siberia from the Ob river (Novosibirsk) and bogs and lakes (Irkutsk) . The strains were tested in PCR for the key virulence determinants (ctx AB, tcp, acf) . The genomes lacked these elements, and therefore the strains were acknowledged avirulent . The results correlate completely with the data of phenotypical analysis, characterizing the pathogenic characteristics of isolated strains.

J Biomol Struct Dyn, 2001 Jun, 18(6), 872 - 80
Analysis of the codon usage pattern in the Vibrio cholerae genome; Wang J et al.; The codon usage in the Vibrio cholerae genome is analyzed in this paper . Although there are much more genes on the chromosome 1 than on chromosome 2, the codon usage patterns of genes on the two chromosomes are quite similar, indicating that the two chromosomes may have coexisted in the same cell for a very long history . Unlike the base frequency pattern observed in other genomes, the G+C content at the third codon position of the V . cholerae genome varies in a rather small interval . The most notable feature of codon usage of V . cholerae genome is that there is a fraction of genes show significant bias in base choice at the second codon position . The 2,006 known genes can be classified into two clusters according to the base frequencies at this position . The smaller cluster contains 227 genes, most of which code for proteins involved in transport and binding functions . The encoding products of these genes have significant bias in amino acids composition as compared with other genes . The codon usage patterns for the 1,836 function unknown ORFs are also analyzed, which is useful to study their functions.

Prikl Biokhim Mikrobiol, 2001 May-Jun, 37(3), 359 - 63
{Lipids of microorganisms from the family Vibrionaceae causing diseases in fishes}; Bogdan VV et al.; Lipid fractional and fatty acid compositions of microorganisms from the genera Aeromonas, Pseudomonas, and Vibrio (the family Vibrionaceae), causing diseases of different fish species, were studied . Motile aeromonads and vibrios displayed higher relative contents of membrane lipids and oleic acid and lower relative contents of storage lipids compared with immotile aeromonads and pseudomonads, which is connected with the activities of their movements . Immotile aeromonads and vibrios exhibited higher levels of polyunsaturated fatty acids and higher absolute phospholipid contents compared to motile aeromonads and pseudomonads . This is likely to be related to host specificity of these bacteria and reflects the specific patterns of fatty acid compositions of the infected fish (salmonid and cyprinid) tissues.

Arch Environ Contam Toxicol, 2001 Apr, 40(3), 386 - 91
The effects of tributyltin (TBT) and 3,3',4,4',5-pentachlorobiphenyl (PCB-126) mixtures on antibody responses and phagocyte oxidative burst activity in channel catfish, Ictalurus punctatus; Regala RP et al.; The organotin tributyltin (TBT) is an antifouling biocide used in marine paints and is a common pollutant in harbor estuaries . We previously demonstrated that the immune system of channel catfish, Ictalurus punctatus, is a sensitive target organ of TBT . Exposure strongly suppresses humoral immune responses . Harbor estuaries often contain polychlorinated biphenyls (PCBs) due to their ubuquitous distribution . The coplanar congener 3,3',4,4'5'-polychlorinated biphenyl (PCB-126) is also immunotoxic to channel catfish, but it suppresses only the innate immune responses and only at high doses . In this study we exposed channel catfish to TBT, PCB-126, or both in mixtures, with canola oil (CO) serving as the carrier control . Antibody responses to Vibrio anguillarum and phagocyte oxidative burst activity were measured after (1) a single dose of 0.01 or 1 mg/kg of each or both in combination, and (2) six injections of 1.7 or 170 microg/kg of each (or in combination) given every 3 days over a 16-day period to yield a cumulative dose of 0.01 or 1 mg/kg, respectively . We measured antibody responses to V . anguillarum 21 days after immunization and oxidative burst activities 14 and 21 days after the final treatment . The highest dose of TBT suppressed antibody responses after a single exposure . The high dose of PCB-126 also suppressed antibody responses . The addition of PCB-126 to TBT doses did not alter the antibody responses beyond the effects of TBT alone . In the repeated exposure group, only the high dose of TBT suppressed antibody responses . In animals exposed to mixtures, high levels of PCB-126 enhanced suppression associated with low levels of TBT, whereas PCB-126 protected against suppression associated with high levels of TBT . Single exposures to TBT or PCB-126 suppressed phagocyte oxidative burst activity . In animals exposed to mixtures, as a single exposure, the addition of a low dose PCB-126 protected against low dose TBT-related oxidative burst activity suppression . In the repeated exposure groups TBT suppressed oxidative burst activity, but only at the highest dose on day 21, while high doses of PCB-126 suppressed activity on day 14 . Furthermore, low levels of PCB-126 reversed the suppressed oxidative burst activity associated with high levels of TBT on day 21 . Overall, this study demonstrates moderate additivity in terms of the immunotoxicity of TBT and PCB-126 mixtures using these two endpoints of immune function in the channel catfish model.

J Biol Chem, 2001 Sep 21, 276(38), 35934 - 9 Epub 2001 Jul 06.
Site-directed mutagenesis of acyl carrier protein (ACP) reveals amino acid residues involved in ACP structure and acyl-ACP synthetase activity; Flaman AS et al.; Acyl carrier protein (ACP) interacts with many different enzymes during the synthesis of fatty acids, phospholipids, and other specialized products in bacteria . To examine the structural and functional roles of amino acids previously implicated in interactions between the ACP polypeptide and fatty acids attached to the phosphopantetheine prosthetic group, recombinant Vibrio harveyi ACP and mutant derivatives of conserved residues Phe-50, Ile-54, Ala-59, and Tyr-71 were prepared from glutathione S-transferase fusion proteins . Circular dichroism revealed that, unlike Escherichia coli ACP, V . harveyi-derived ACPs are unfolded at neutral pH in the absence of divalent cations; all except F50A and I54A recovered native conformation upon addition of MgCl(2) . Mutant I54A was not processed to the holo form by ACP synthase . Some mutations significantly decreased catalytic efficiency of ACP fatty acylation by V . harveyi acyl-ACP synthetase relative to recombinant ACP, e.g . F50A (4%), I54L (20%), and I54V (31%), whereas others (V12G, Y71A, and A59G) had less effect . By contrast, all myristoylated ACPs examined were effective substrates for the luminescence-specific V . harveyi myristoyl-ACP thioesterase . Conformationally sensitive gel electrophoresis at pH 9 indicated that fatty acid attachment stabilizes mutant ACPs in a chain length-dependent manner, although stabilization was decreased for mutants F50A and A59G . Our results indicate that (i) residues Ile-54 and Phe-50 are important in maintaining native ACP conformation, (ii) residue Ala-59 may be directly involved in stabilization of ACP structure by acyl chain binding, and (iii) acyl-ACP synthetase requires native ACP conformation and involves interaction with fatty acid binding pocket residues, whereas myristoyl-ACP thioesterase is insensitive to acyl donor structure.

Ecotoxicol Environ Saf, 2001 Jul, 49(3), 293 - 301
Use of the ciliated protozoan Tetrahymena pyriformis for the assessment of toxicity and quantitative structure--activity relationships of xenobiotics: comparison with the Microtox test; Bogaerts P et al.; Cytotoxicity and quantitative structure-activity relationships of 13 inorganic and 21 organic substances were determined using three bioassays performed on the ciliated protozoan Tetrahymena pyriformis and the luminescent bacterium Vibrio fischeri . The best concordance of toxicity results was observed between the T . pyriformis FDA--esterase activity and population growth inhibition tests for the organic compounds . The sensitivity of these two assays is compared with that of the Microtox test . The T . pyriformis FDA test showed a high sensitivity is most cases . The aim of the current research was to determine whether the relative toxicity of metal ions and organic molecules, with these three bioassays, was predictable using three ion characteristics and hydrophobicity, respectively . For metal ions, the variable that best modeled the toxicity data obtained with the two T . pyriformis tests was the softness index {sigma(p), i.e., (coordinate bond energy of the metal fluoride--coordinate bond energy of the metal iodide)/(coordinate bond energy of the metal fluoride)} . No correlation was found with the Microtox test . For organic compounds, a significant correlation was observed between the hydrophobicity coefficient and the toxicity data . This correlation is closer with the two tests using Tetrahymena .

J Microbiol Methods, 2001 Sep, 46(3), 217 - 25
Isolation and characterization of a temperature-sensitive generalized transducing bacteriophage for Vibrio cholerae; Hava DL et al.; CP-T1 is the only described generalized transducing bacteriophage for the intestinal pathogen Vibrio cholerae, yet many of its basic biological parameters remain unknown . Due to low frequencies of transduction and pseudolysogen formation, CP-T1 has not been widely used as a genetic tool . To overcome these limitations, we have isolated a conditional mutant of CP-T1 that exhibits temperature-sensitive plaque formation . Several biological properties of CP-T1ts were determined, including its restrictive temperature, adsorbance profile to host cells, burst time, and burst size . Based on these properties, an optimized transduction protocol was designed which resulted in several fold higher transduction frequencies for a variety of genetic markers from a number of chromosomal loci . Generalized transduction was also demonstrated between classical and E1 Tor biotype strains of V . cholerae.

Int J Med Microbiol, 2001 May, 291(2), 81 - 8
Regulation of virulence in Vibrio cholerae; Klose KE; Vibrio cholerae causes the diarrheal disease cholera primarily because it expresses a colonization factor (toxin-coregulated pilus; TCP) and a potent toxin (cholera toxin; CT) within the human intestine . While the true environmental signals that induce CT and TCP expression within the intestine remain unknown, much progress has been made identifying the regulatory factors that modulate their expression . Transcriptional regulation of the genes encoding TCP and CT involves a cascade consisting of a number of regulatory factors located on recently acquired mobile genetic elements as well as others residing within the ancestral Vibrio genome . In vivo studies have revealed interesting differences between the regulation of TCP and CT expression in the laboratory and within the intestine.

Environ Toxicol Chem, 2001 Jul, 20(7), 1438 - 49
The use of acute and chronic bioassays to determine the ecological risk and bioremediation efficiency of oil-polluted soils; van Gestel CA et al.; To compare the effectiveness of acute and chronic bioassays for the ecological risk assessment of polluted soils, soil samples from a site with an historical mineral oil contamination (< 50-3,300 mg oil/kg dry soil) at the Petroleum Harbour in Amsterdam, The Netherlands, were screened for ecological effects using acute and chronic bioassays . A two-step 0.001 M Ca(NO3)2 extraction at a final solution-to-soil ratio of 1:1 was used to prepare extracts for the acute bioassays . Acute bioassays (< or = 5 d) applied to the 0.001 M Ca(NO3)2 extracts from the polluted and reference soils included growth tests with bacteria (Bacillus sp.), algae (Raphidocelis subcapitata), and plants (Lactuca sativa), immobility tests with nematodes (Plectus acuminatus), springtails (Folsomia candida), and cladocerans (Daphnia magna), and the Microtox test (Vibrio fischeri) . Chronic bioassays (four weeks) performed on the same soil samples included tests with L . sativa, F . candida, and earthworms (Eisenia fetida) and the bait-lamina test (substrate consumption) . The acute bioassays on Microtox showed a response that corresponded with the level of oil pollution . All other acute bioassays did not show such a consistent response, probably because pollutant levels were too low to cause acute effects . All chronic bioassays showed sublethal responses according to the contaminant levels (oil and in some soils also metals) . This shows that chronic bioassays on soil samples are more sensitive in assessing the toxicity of mineral oil contamination in soil than acute bioassays on soil extracts . A pilot scale bioremediation study on soils taken from the two most polluted sites and a control site showed a rapid decline of oil concentrations to reach a stable level within eight weeks . Acute bioassays applied to the soils, using Microtox, algae, and D . magna, and chronic bioassays, using plants, Collembola, earthworms, and bait-lamina consumption, in all cases showed a rapid reduction of toxicity, which could be attributed to the degradation of light oil fractions.

FEMS Microbiol Lett, 2001 May 1, 198(2), 141 - 6
Structures of ribonuclease P RNAs of Vibrio core species; Maeda T et al.; The structures of an RNA component of ribonuclease P (RNase P RNA) were examined for Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio natriegens, Vibrio campbellii, Vibrio proteolyticus, Vibrio pelagius and Vibrio harveyi to clearly determine their genetic differences . The RNase P RNAs ranged from 382 to 454 nucleotides (nt) in size, and were remarkably different from each other in the structure of two helices, P3 and P12 . The P3 helices were comprised of tandem repeats of a palindromic sequence (24 nt), resulting in the longitudinal repetition of a stem structure . The number of repetitions ranged from four in V . harveyi, to one in both V . alginolyticus and V . proteolyticus . The genes for the RNase P RNAs of all species were located between two open reading frames, the amino acid sequences of which were similar to the hypothetical proteins located at 70.92 and 1.94 min in the Escherichia coli chromosome.

FEMS Microbiol Lett, 2001 May 1, 198(2), 123 - 4
Helical growth and the curved shape of Vibrio cholerae; Cooper S; The curved, comma, or bent shape of Vibrio cholerae is attributed to, and explained by, the normal helical growth of the cell . The comma-like shape of V . cholerae is not due to an asymmetrical positioning of peptidoglycan such that some chains of peptidoglycan are placed so they are more spread out on one side of the cell and squeezed together on the other side.

J Zoo Wildl Med, 2000 Dec, 31(4), 523 - 31
Common cuttlefish (Sepia officinalis) mortality at the National Zoological Park: implications for clinical management; Sherrill J et al.; Six out of seven cuttlefish acquired by the Smithsonian National Zoological Park in July 1998 died before 1 November 1998 . Postmortem examinations showed mantle ulcers, secondary bacterial infections, inanition, and cuttlebone fractures . The surviving cuttlefish developed a progressive focal mantle ulcer, was treated with oral chloramphenicol intermittently for 9 wk, and maintained a normal appetite and growth rate until death at 7 mo of age . The National Zoological Park pathology database showed signalments, histories, and causes of mortality of 186 common cuttlefish, each 1-14 mo old, that received gross and histologic examinations; for example, the largest group of cuttlefish of known sex, age, and body weight at postmortem were 7-9 mo old and weighed an average of 376.2 g (males, n = 18) and 299.0 g (females, n = 15) . Many cuttlefish had multiple pathologic diagnoses . Significant diseases included inflammation and secondary bacterial infections, especially gastrointestinal, cardiovascular, respiratory, reproductive, and ophthalmic, and septicemia due to Vibrio spp . or other gram-negative bacteria . Mantle lesions, including ulceration/dermatitis, abscess/granuloma, necrosis/fibrosis/cellulitis, and laceration/abrasion/erosion, were also identified, along with inanition, cuttlebone lesions, and trauma . Mantle lesions were associated with secondary bacterial infections and death . On the basis of this information, if captive cuttlefish behavior creates risk for development of mantle lesions, administration of antibiotics effective against gram-negative bacteria may delay or halt disease progression . Cuttlefish exhibits require proper design, husbandry, economic resources, and staffing to minimize disease syndromes and mortality.

J Clin Microbiol, 2001 Jul, 39(7), 2594 - 7
Detection of RTX toxin gene in Vibrio cholerae by PCR; Chow KH et al.; A PCR that amplifies a recently discovered Vibrio cholerae RTX (repeat in toxin) toxin gene was developed . Among 166 clinical and environmental isolates of V . cholerae causing epidemics and sporadic cases of cholera in various parts of the world, all were found to be toxigenic by both PCR and HEp-2 cell cytotoxicity assay . Standard strains of the classical biotype containing a deletion within the gene cluster exhibited negative results by both assays . This is the first rapid genotyping method for differentiation of V . cholerae O1 classical biotype strains from El Tor biotype strains as well as strains of other non-O1 serogroups including serogroup O139 . The PCR assay that was developed also specifically detects RTX toxin genes in V . cholerae, as clinical isolates of Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Aeromonas species, and Plesiomonas species were all negative by the RTX toxin-specific PCR as well as the HEp-2 cytotoxicity assay . These findings highlight the characteristics of the RTX toxins in V . cholerae . Their role in the pathogenicity of the bacterium requires further investigation.

Appl Environ Microbiol, 2001 Jul, 67(7), 3220 - 5
The mannose-sensitive hemagglutinin of Vibrio cholerae promotes adherence to zooplankton; Chiavelli DA et al.; The bacterium Vibrio cholerae, the etiological agent of cholera, is often found attached to plankton, a property that is thought to contribute to its environmental persistence in aquatic habitats . The V . cholerae O1 El Tor biotype and V . cholerae O139 strains produce a surface pilus termed the mannose-sensitive hemagglutinin (MSHA), whereas V . cholerae O1 classical biotype strains do not . Although V . cholerae O1 classical does not elaborate MSHA, the gene is present and expressed at a level comparable to that of the other strains . Since V . cholerae O1 El Tor and V . cholerae O139 have displaced V . cholerae O1 classical as the major epidemic strains over the last fifteen years, we investigated the potential role of MSHA in mediating adherence to plankton . We found that mutation of mshA in V . cholerae O1 El Tor significantly diminished, but did not eliminate, adherence to exoskeletons of the planktonic crustacean Daphnia pulex . The effect of the mutation was more pronounced for V . cholerae O139, essentially eliminating adherence . Adherence of the V . cholerae O1 classical mshA mutant was unaffected . The results suggest that MSHA is a factor contributing to the ability of V . cholerae to adhere to plankton . The results also showed that both biotypes of V . cholerae O1 utilize factors in addition to MSHA for zooplankton adherence . The expression of MSHA and these additional, yet to be defined, adherence factors differ in a serogroup- and biotype-specific manner.

Appl Environ Microbiol, 2001 Jul, 67(7), 3161 - 7
Duplication of hemolysin genes in a virulent isolate of Vibrio harveyi; Zhang XH et al.; Vibrio harveyi VIB 645, which is very pathogenic towards salmonids and produces extracellular product with a high titer of hemolytic activity towards fish erythrocytes, was found to contain two closely related hemolysin genes (designated vhhA and vhhB), whereas the majority of strains examined (11 of 13) carried only a single hemolysin gene . Both genes from VIB 645 were cloned and sequenced . The open reading frames (ORFs) of vhhA and vhhB shared a high level of identity (98.8%) and were predicted to encode identical polypeptides comprising 418 amino acid residues . The VHH protein shows homology to the lecithinase of V . mimicus and V . cholerae . Transformants of Escherichia coli containing the ORF of either vhhA or vhhB displayed weak hemolytic activity in rainbow trout blood agar . The hemolytic activity was very high when the ORF of vhhB was cloned in E . coli together with the native promoter . Surprisingly, the level of vhh-specific RNA transcript produced by VIB 645 was found to be very low . We conclude that the hemolytic phenotype of VIB 645 is not due to increased expression of one or both copies of the vhh gene.

Appl Environ Microbiol, 2001 Jul, 67(7), 2895 - 902
Antacid increases survival of Vibrio vulnificus and Vibrio vulnificus phage in a gastrointestinal model; Koo J et al.; Viable counts of three strains of Vibrio vulnificus and its phage were determined during exposure to a mechanical gastrointestinal model with or without antacid for 9 h at 37 degrees C . V . vulnificus was eliminated (>4-log reduction) within 30 min in the gastric compartment (pH decline from 5.0 to 3.5) . Viable V . vulnificus cells delivered from the gastric compartment during the first 30 min of exposure reached 10(6) to 10(8) CFU/ml in the intestinal compartment after 9 h (pH 7.0) . Phages were eliminated within 45 min in the gastric compartment (pH decline from 5.1 to 2.5) . Less than a 2-log reduction of phage was observed in the intestinal compartment after 9 h (pH 7.0) . When the gastric compartment contained antacid V . vulnificus counts decreased slightly (<2 log) during 2 h of exposure (pH decline from 7.7 to 6.0), while counts in the intestinal compartment (pH 7.5) reached 10(7) to 10(9) CFU/ml . Phage numbers decreased 1 log after 2 h in the gastric compartment (pH decline from 7.7 to 5.7) containing antacid and decreased 1 log in the intestinal compartment (pH 7.6) after 9 h . Presence of antacid in the gastric compartment of the model greatly increased the ability of both V . vulnificus and its phage to survive simulated gastrointestinal transit and may be a factor involved with oyster-associated illness.

Biophys J, 2001 Jul, 81(1), 382 - 93
Photoinduced transient absorbance spectra of P840/P840(+) and the FMO protein in reaction centers of Chlorobium vibrioforme; Vassiliev IR et al.; The kinetics of photoinduced absorbance changes in the 400-ns to 100-ms time range were studied between 770 and 1025 nm in reaction center core (RCC) complexes isolated from the green sulfur bacterium Chlorobium vibrioforme . A global, multiple stretched-exponential analysis shows the presence of two distinct but strongly overlapping spectra . The spectrum of the 70-micros component consists of a broad bleaching with two minima at 810 and 825 nm and a broad positive band at wavelengths greater than 865 nm and is assigned to the decay of (3)Bchl a of the Fenna-Matthews-Olson (FMO) protein . The contribution of the 70-micros component correlates with the amount of FMO protein in the isolated RCC complex . The spectrum of the 1.6-micros component has a sharp bleaching at 835 nm, a maximum at 805 nm, a broad positive band at wavelengths higher than 865 nm, and a broad negative band at wavelengths higher than 960 nm . When the RCC is incubated with inorganic iron and sulfur, the 1.6-micros component is replaced by a component with a lifetime of approximately 40 micros, consistent with the reconstruction of the F(X) cluster . We propose that the 1.6-micros component results from charge recombination between P840(+) and an intermediate electron acceptor operating between A(0) and F(X) . Our studies in Chlorobium RCCs show that approaches that employ a single wavelength in the measurement of absorption changes have inherent limitations and that a global kinetic analysis at multiple wavelengths in the near-infrared is required to reliably separate absorption changes due to P840/P840(+) from the decay of (3)Bchl a in the FMO protein.

Environ Toxicol, 2001 Jun, 16(3), 277 - 86
Effects of a pre-incubation period on the photoinduced toxicity of polycyclic aromatic hydrocarbons to the luminescent bacterium Vibrio fischeri; el-Alawi YS et al.; Irradiation of polycyclic aromatic hydrocarbons (PAHs) in aqueous solution with simulated solar radiation (SSR; a light source with a visible light: UV-A:UV-B ratio similar to that of sunlight) can greatly enhance their toxicity . Two microbial toxicity tests with Vibrio fischeri were used to investigate the effect of composition of the growth medium and pre-incubation on the photoinduced toxicity of PAHs . The assays were a short-term test (15 min) and long-term test (18 h) . Both assays were carried out in SSR and darkness to examine for photoinduced toxicity of PAHs . For the short-term toxicity assay, inhibition of bacterial luminescence was measured . For the long-term toxicity assay, both inhibition of bacterial luminescence and inhibition of growth were recorded . To broaden this test, V . fischeri cells were pre-incubated with PAHs in medium without a carbon source (minimal medium) for 8 h to facilitate assimilation and photooxidation of the contaminants, and to prevent bacterial growth at the outset of the assay . V . fischeri was more sensitive in minimal medium than in complex medium in both the short- and long-term toxicity assays . Moreover, in the long-term assay, SSR greatly increased toxicity, especially if there was a pre-incubation period in minimal medium . This indicates that both assimilation and photooxidation of PAHs are important to their toxicity to V . fischeri.

J Appl Microbiol, 2001 Jun, 90(6), 919 - 27
Characterization of Aeromonas and Vibrio species isolated from a drinking water reservoir; Ivanova EP et al.; AIMS: To study the phenotypic and chemotaxonomic (i.e . phospholipid and cellular fatty acid composition) characteristics of environmental Aeromonas spp . and Vibrio spp . isolated from a drinking water reservoir near Vladivostok City, and the application of some chemotaxonomic markers for discrimination of the two genera and species . METHODS AND RESULTS: Presumptive Aeromonas species were dominant in surface water samples (up to 25% of the total number of bacteria recovered) . These strains were consistent with respect to the cultural and biochemical properties used to define the species Aeromonas sobria (seven strains) and Aer . popoffii (three strains) . Vibrio mimicus (two strains) and Vibrio metschnikovii (one strain) were identified according to phenotypic features and cellular fatty acid composition . CONCLUSION: Environmental Aer . sobria isolates were atypical in their ability to grow at 42 degrees C, and were haemolytic, proteolytic and cytotoxic . Although it was present in a high proportion in the water samples, atypical Aer . sobria is not an indicator of polluted water . SIGNIFICANCE AND IMPACT OF THE STUDY: The incidence of Aeromonas in the drinking water reservoirs in the Far East of Russia is reported for the first time.

Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1035 - 44
Isolation of Desulfomicrobium orale sp . nov . and Desulfovibrio strain NY682, oral sulfate-reducing bacteria involved in human periodontal disease; Langendijk PS et al.; The species of sulfate-reducing bacteria that prevail in sites affected by periodontal disease may be different from those commonly occurring in the digestive tracts of healthy individuals . Ten strains of mesophilic sulfate-reducing bacteria (SRB) were isolated from subgingival plaque in periodontal lesions of ten patients with periodontitis . Characterization on the basis of morphological, physiological and phylogenetic properties demonstrated two distinct types of oral SRB . One strain was a curved rod with high motility . For dissimilatory sulfate reduction, lactate or pyruvate was oxidized incompletely to equimolar amounts of acetate . Desulfoviridin and cytochrome c3 were present in this mesophilic vibrio and the cellular lipid profile was similar to that from members of the genus Desulfovibrio . The 16S rDNA sequence was similar to that of the proposed 'Desulfovibrio fairfieldensis' . Cells of the nine other strains were straight, rod-shaped, exhibited a low growth rate and oxidized substrates incompletely to acetate . These SRB, like members of the genus Desulfomicrobium, lacked desulfoviridin . Analysis of the 16S rDNA sequences of seven of the nine isolates showed a high degree of similarity among these oral strains, forming a distinct lineage within the genus Desulfomicrobium . The cellular lipid profile of a representative oral strain, NY678T, was in accordance with that of other Desulfomicrobium species, but also showed dissimilar features . The phenotypic and phylogenetic analyses indicate that these rod-shaped SRB from the oral cavity could be regarded as a new species, for which the designation Desulfomicrobium orale sp . nov . is proposed.

FEMS Microbiol Lett, 2001 Jun 12, 200(1), 111 - 6
Unusual adaptive, cross protection responses and growth phase resistance against peroxide killing in a bacterial shrimp pathogen, Vibrio harveyi; Vattanaviboon P et al.; Oxidant induced protection against peroxide killing was investigated in a prawn bacterial pathogen, Vibrio harveyi . Exposure to 250 microM H(2)O(2) induced adaptive protection against subsequent exposure to killing concentrations of H(2)O(2) . In addition, 200 microM t-butyl hydroperoxide (tBOOH) induced cross protection to H(2)O(2) killing . On the other hand, peroxide pretreatment did not induce protection against tBOOH killing . Peroxide induced adaptive and cross protection responses required new protein synthesis and were abolished by addition of a protein synthesis inhibitor . Pretreatments of V . harveyi with 250 microM H(2)O(2) and 200 microM tBOOH induced an increase in peroxide scavenging enzymes, catalase and alkyl hydroperoxide reductase subunit C . In addition, stationary phase cells of V . harveyi were more resistant to H(2)O(2) and iodoacetamide killing but highly susceptible to tBOOH killing compared to exponential phase cells . Many aspects of the oxidative stress response of V . harveyi are different from those of other bacteria and these factors may be important for bacterial survival in the environment and during interactions with host shrimp.

J Commun Dis, 2000 Sep, 32(3), 207 - 11
An outbreak of Eltor cholera in Aizwal town of Mizoram, India; Sengupta PG et al.; During the months of May, June and through early part of July 1994, an unusual occurrence of severe dehydrating watery diarrhoea cases and deaths were reported from Aizwal town, the capital of Mizoram, a North-Eastern state of India . Vibrio cholerae 01 biotype Eltor, the causative agent responsible for this outbreak, was isolated from 50.0% of hospitalised cases . The disease affected older children and adults more (52.9%) than younger children below five years of age . Vibrio cholerae 01 strains isolated were uniformly resistant to furazolidone and co-trimoxazole, which are commonly advocated in the treatment of cholera specially in children of developing countries . Emergence of such resistant strain is alarming and is of great public health importance.

Infect Immun, 2001 Jul, 69(7), 4681 - 5
Vibrio cholerae tolC is required for bile resistance and colonization; Bina JE et al.; TolC and its homologues are outer membrane proteins that are essential for the transport of many molecules across the cell envelope . In this study we characterized the gene encoding Vibrio cholerae TolC . V . cholerae tolC mutants failed to secrete the RTX cytotoxin, were hypersensitive to antimicrobial agents, and were deficient in intestinal colonization.

Biochemistry, 2001 Jun 19, 40(24), 7318 - 23
Sodium-dependent steps in the redox reactions of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi; Bogachev AV et al.; The Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi was purified and studied by EPR and visible spectroscopy . Two EPR signals in the NADH-reduced enzyme were detected: one, a radical signal, and the other a line around g = 1.94, which is typical for a {2Fe-2S} cluster . An E(m) of -267 mV was found for the Fe-S cluster (n = 1), independent of sodium concentration . The spin concentration of the radical in the enzyme was approximately the same under a variety of redox conditions . The time course of Na+-NQR reduction by NADH indicated the presence of at least two different flavin species . Reduction of the first species (most likely, a FAD near the NADH dehydrogenase site) was very rapid in both the presence and absence of sodium . Reduction of the second flavin species (presumably, covalently bound FMN) was slower and strongly dependent on sodium concentration, with an apparent activation constant for Na+ of approximately 3.4 mM . This is very similar to the Km for Na+ in the steady-state quinone reductase reaction catalyzed by this enzyme . These data led us to conclude that the sodium-dependent step within the Na+-NQR is located between the noncovalently bound FAD and the covalently bound FMN.

Comp Biochem Physiol B Biochem Mol Biol, 2001 Jun, 129(2-3), 687 - 93
Hormonal status and phagocytic activity in sea bream infected with vibriosis; Deane EE et al.; Serum taken from female, sexually mature, silver sea bream (Sparus sarba) displaying either no symptoms of vibriosis, mild infection, severe infection or moribundity were assayed for a number of key hormones . Serum cortisol levels were not significantly different among symptomless, mildly- and severely-infected groups, whereas moribund fish displayed hypercortisolemia with a 14-fold increase in serum cortisol in comparison to symptomless fish . Serum estradiol levels were significantly reduced 19-fold in mildly-infected fish and remained at a low level as infection progressed, whereas serum testosterone increased gradually during vibriosis with a 1.8-fold increase in moribund groups in comparison to symptomless groups . Both serum thyroxine (T(4)) and triiodothyronine (T(3)) gradually decreased during vibriosis being 26- and 2.8-fold lower, respectively, in moribund fish in comparison to symptomless fish . The non-specific immune response, as determined by phagocytic activity, was also assessed using macrophages isolated from the pronephros and spleen of infected fish . Phagocytic indices significantly increased in mildly- and severely-infected fish and then decreased from these stimulated levels in moribund fish.

J Bacteriol, 2001 Jul, 183(13), 4024 - 32
Biological and biochemical characterization of variant A subunits of cholera toxin constructed by site-directed mutagenesis; Jobling MG et al.; Cholera toxin (CT) is the prototype for the Vibrio cholerae-Escherichia coli family of heat-labile enterotoxins having an AB5 structure . By substituting amino acids in the enzymatic A subunit that are highly conserved in all members of this family, we constructed 23 variants of CT that exhibited decreased or undetectable toxicity and we characterized their biological and biochemical properties . Many variants exhibited previously undescribed temperature-sensitive assembly of holotoxin and/or increased sensitivity to proteolysis, which in all cases correlated with exposure of epitopes of CT-A that are normally hidden in native CT holotoxin . Substitutions within and deletion of the entire active-site-occluding loop demonstrated a prominent role for His-44 and this loop in the structure and activity of CT . Several novel variants with wild-type assembly and stability showed significantly decreased toxicity and enzymatic activity (e.g., variants at positions R11, I16, R25, E29, and S68+V72) . In most variants the reduction in toxicity was proportional to the decrease in enzymatic activity . For substitutions or insertions at E29 and Y30 the decrease in toxicity was 10- and 5-fold more than the reduction in enzymatic activity, but for variants with R25G, E110D, or E112D substitutions the decrease in enzymatic activity was 12- to 50-fold more than the reduction in toxicity . These variants may be useful as tools for additional studies on the cell biology of toxin action and/or as attenuated toxins for adjuvant or vaccine use.

J Bacteriol, 2001 Jul, 183(13), 3903 - 9
Signaling system in Porphyromonas gingivalis based on a LuxS protein; Chung WO et al.; The luxS gene of quorum-sensing Vibrio harveyi is required for type 2 autoinducer production . We identified a Porphyromonas gingivalis open reading frame encoding a predicted peptide of 161 aa that shares 29% identity with the amino acid sequence of the LuxS protein of V . harveyi . Conditioned medium from a late-log-phase P . gingivalis culture induced the luciferase operon of V . harveyi, but that from a luxS insertional mutant did not . In P . gingivalis, the expression of luxS mRNA was environmentally controlled and varied according to the cell density and the osmolarity of the culture medium . In addition, differential display PCR showed that the inactivation of P . gingivalis luxS resulted in up-regulation of a hemin acquisition protein and an arginine-specific protease and reduced expression of a hemin-regulated protein, a TonB homologue, and an excinuclease . The data suggest that the luxS gene in P . gingivalis may function to control the expression of genes involved in the acquisition of hemin.

Vaccine, 2001 Jun 14, 19(27), 3720 - 5
Cost of immunization with a locally produced, oral cholera vaccine in Viet Nam; Naficy AB et al.; Policy decisions regarding whether to incorporate new vaccines into routine public health practice in developing countries will depend in part on the costs of vaccine purchase and of vaccine delivery . In March, 1997, a large-scale effectiveness trial of a locally produced, orally administered bivalent vaccine against Vibrio cholerae 01 and 0139 began in Viet Nam . Empirical data obtained from the trial was used to determine the costs of the immunization campaign from the government perspective . The study population, including the children less than one year of age and pregnant women who were ineligible for immunization, was 353926 . A total of 289041 persons received two doses of vaccine, and 13340 persons received one dose of vaccine . Two-dose vaccine coverage was 83.4% . The total cost of vaccine delivery during the immunization campaign was $66527 . The cost of each dose of vaccine was $0.31 . Therefore, the total cost of the immunization campaign was $0.44 per dose administered, and $0.91 per fully immunized person . Attempts to reduce the cost per dose of vaccine (e.g . the use of a monovalent vaccine against serogroup 01) are likely to have a large impact on the cost of future similar immunization campaigns.

J Health Popul Nutr, 2001 Mar, 19(1), 39 - 42
Vibrio cholerae resistant to 2,4-diamino-6,7-diisopropylpteridine (O/129) isolated from patients with enteritis in Ceará, Brazil; Hofer E et al.; This paper reports the characterization of clinical Vibrio cholerae resistant to vibriostatic agent O/129, using classical and plasmid analysis . In a study conducted during December 1991-September 1993, two of 7,058 V . cholerae strains, obtained from patients suspected to have cholera in the State of Ceara, northeast Brazil, were resistant to 150 micrograms of the vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine) . One strain was identified as V . cholerae O1 El Tor Inaba and the other one as serogroup O22 . Only one O1 strain harboured a plasmid of 147 kb transferable to Escherichia coli K12, and five strains of V . cholerae O1 and non-O1 were sensitive to O/129 and plasmid-negative at a frequency between 8 x 10(-2) and 3.6 x 10(-5) . Additionally, O/129-resistant strains of V . cholerae O1 and O22 were resistant to trimethoprim/sulphamethoxazole.

J Med Microbiol, 2001 Jun, 50(6), 499 - 508
Immunochemical characterisation of Vibrio cholerae O139 O antigens and production of a diagnostic antiserum without absorption; Feodorova VA et al.; Rabbits and mice immunised with chemically extracted O-antigens (O-Ags) of Vibrio cholerae O139 (O-AgB and O-AgD) developed antibodies (Abs) which appeared to be highly specific in ELISA for the relevant antigens and V . cholerae O139 strains without absorption, in contrast to the Abs against the heated O-Ag (O-AgH) . An ELISA test based on the use of these Abs was shown to detect V . cholerae O139 strains down to concentrations of (9.4 x 10(4))-(7.5 x 10(5)) vibrios/ml and demonstrated no cross-reaction with other vibrios including representatives of serogroup O22 . Native and proteinase K-treated O-AgB, O-AgD, O-AgH, as well as whole-cell lysates of V . cholerae O139 strains of different origin were used in immunoblotting with these Abs . Clear differences in the patterns of zones of specific reaction between chemically extracted and heated O-Ags and between lipopolysaccharide profiles of the V . cholerae O139 strains of different origin were observed . Serogroup-specific protein bands in the native O-AgB and O-AgD preparations were defined . The approach described for obtaining serogroup-specific Abs against vibrios and other bacteria seems to be promising for the development of specific diagnostic tests and further investigation of bacterial antigenic structure.

J Med Microbiol, 2001 Jun, 50(6), 489 - 98
Immunogenicity and protective role of an IgA reactive 31-kDa antigen of Vibrio cholerae O139; Kumar S et al.; Immunoglobulin A (IgA) is important in protective immunity against infection by Vibrio cholerae . In this study, the immune response to and protective role of a 31-kDa antigen of V . cholerae O139, reacting with IgA antibodies present in the sera of cholera patients and common to V . cholerae strains O139 and O1 was evaluated in BALB/c mice . From the various antigens of V . cholerae O139 and V . cholerae O1 which reacted with IgA antibodies in sera of a cholera patient, a 31-kDa common antigen was selected and purified by DEAE-Sepharose CL 6B column chromatography . Oral administration of live V . cholerae O139 in BALB/c mice elicited an IgA response to the 31-kDa antigen in serum and intestinal fluid, and a proliferation of the splenic lymphocytes on stimulation with the same antigen . The cytokine profile of these splenic lymphocytes revealed a shift from a mixed Th1 and Th2 response--interleukin-10 (IL-10) and interferon-gamma--in the first week after infection to a Th2 type of response--IL-10--in the third week . In passive protection studies, hyperimmune serum to the 31-kDa antigen was able to protect infant mice against challenge with O139 and O1 strains . These results demonstrate the ability of the 31-kDa antigen of V . cholerae O139 to induce humoral and cellular immune responses in mice, and its immunoprotective nature.

Microb Ecol, 2001 Apr, 41(3), 264 - 271
Grazing Characteristics and Growth Efficiencies at Two Different Temperatures for Three Nanoflagellates Fed with Vibrio Bacteria at Three Different Concentrations; Ishigaki T et al.; Small inocula of one of the flagellates Paraphysomonas imperforata, Pteridomonas danica, and Cafeteria roenbergensis were added to suspensions of the bacterium Vibrio natriegens at each of three concentrations between 107 and 108 cells ml-1 and incubated at each of the temperatures 10 degrees C and 25 degrees C . Samples were taken at intervals for counting the flagellates and bacteria to determine the timing of the maximum of flagellate numbers and the concentrations at that time . Measurements of the protein concentration of the suspensions during incubation were used to determine the gross growth efficiency (GGE) or yield of flagellate grazing in each experiment . The most effective grazer was Pteridomonas, followed by Paraphysomonas, with Cafeteria being least effective, as judged by the threshold bacterial concentrations at which flagellate multiplication ceased, which were about 2 x 105, 2 x 106, and 2 x 107, respectively, and by the finding that Pteridomonas consumed 99%, Paraphysomonas about 95%, and Cafeteria only 60-70% of the available bacteria in the experiments . Peak concentrations of flagellates were reached later at the lower temperature, but the numbers of flagellates produced and of bacteria eaten were of a similar order at the two temperatures and the GGE was only slightly higher at the lower temperature . The time taken to reach peak flagellate numbers changed little with a threefold increase in bacterial concentrations, but the GGE increased and the numbers of bacteria eaten to produce one flagellate decreased when the bacterial concentration was increased . The three flagellates show clear evidence of niche specialization in differences in thresholds of bacterial prey concentration.

Proc Natl Acad Sci U S A, 2001 Jun 5, 98(12), 6889 - 94
Selection for in vivo regulators of bacterial virulence; Lee SH et al.; We devised a noninvasive genetic selection strategy to identify positive regulators of bacterial virulence genes during actual infection of an intact animal host . This strategy combines random mutagenesis with a switch-like reporter of transcription that confers antibiotic resistance in the off state and sensitivity in the on state . Application of this technology to the human intestinal pathogen Vibrio cholerae identified several regulators of cholera toxin and a central virulence gene regulator that are operative during infection . These regulators function in chemotaxis, signaling pathways, transport across the cell envelope, biosynthesis, and adherence . We show that phenotypes that appear genetically independent in cell culture become interrelated in the host milieu.

Emerg Infect Dis, 2001 May-Jun, 7(3), 477 - 8
Filamentous phage associated with recent pandemic strains of Vibrio parahaemolyticus; Iida T et al.; A group of pandemic strains of Vibrio parahaemolyticus has recently appeared in Asia and North America . We demonstrate that a filamentous phage is specifically associated with the pandemic V . parahaemolyticus strains . An open reading frame unique to the phage is a useful genetic marker to identify these strains.

Prim, Care Update Ob Gyns . 2001 May, 8(3), 106 - 109
Cholera; Parsi VK; Cholera, an infectious disease caused by Vibrio cholerae, is primarily transmitted by ingestion of contaminated food or water . In severe cases, cholera may lead to severe dehydration, metabolic acidosis, and ultimately, hypovolemic shock and death . The diagnosis is confirmed by identification of V . cholerae in a stool specimen . Treatment should be started immediately by rapid replacement of fluid and electrolytes . Antibiotics such as tetracycline and doxycycline shorten the duration of illness but do not significantly affect overall mortality . Cholera can be prevented by limiting spread, survival, and growth of the organism . The current parenteral cholera vaccine is not recommended by the Public Health Service or the World Health Organization because of its limited protection . A number of oral vaccines are currently being tested in clinical trials.

FEMS Microbiol Lett, 2001 May 30, 199(2), 229 - 33
Description and application of a rapid method for genomic DNA direct sequencing; Krin E et al.; We describe a rapid method for determining nucleotide sequences directly from total genomic DNA . This technique was used to determine genomic DNA sequences in various prokaryotic and eukaryotic microorganisms with a G+C content between 40 and 50%, e.g . Escherichia coli, Vibrio cholerae, Bacillus subtilis and Saccharomyces cerevisiae . Furthermore, the method was applied to accurately sequence up to 300 DNA base pairs in Photorhabdus luminescens, whose genome sequencing is currently under way . Taken together, these results provide evidence that our technique can be widely used to easily and efficiently determine genomic DNA sequences.

Carbohydr Res, 2001 Jun 4, 332(3), 279 - 84
Structure of the O-specific polysaccharide of Vibrio cholerae O9 containing 2-acetamido-2-deoxy-D-galacturonic acid; Kocharova NA et al.; The O-specific polysaccharide (OPS) was isolated by mild-acid degradation of the lipopolysaccharide of Vibrio cholerae O9 and studied by carboxyl reduction, sugar and methylation analyses, Smith degradation, and two-dimensional NMR spectroscopy, including COSY, TOCSY, NOESY, and H-detected 1H,(13)C HMQC experiments . The following structure of the pentasaccharide-repeating unit of the OPS was established:

Appl Environ Microbiol, 2001 Jun, 67(6), 2421 - 9
Genotypes associated with virulence in environmental isolates of Vibrio cholerae; Rivera IN et al.; Vibrio cholerae is an autochthonous inhabitant of riverine and estuarine environments and also is a facultative pathogen for humans . Genotyping can be useful in assessing the risk of contracting cholera, intestinal, or extraintestinal infections via drinking water and/or seafood . In this study, environmental isolates of V . cholerae were examined for the presence of ctxA, hlyA, ompU, stn/sto, tcpA, tcpI, toxR, and zot genes, using multiplex PCR . Based on tcpA and hlyA gene comparisons, the strains could be grouped into Classical and El Tor biotypes . The toxR, hlyA, and ompU genes were present in 100, 98.6, and 87.0% of the V . cholerae isolates, respectively . The CTX genetic element and toxin-coregulated pilus El Tor (tcpA ET) gene were present in all toxigenic V . cholerae O1 and V . cholerae O139 strains examined in this study . Three of four nontoxigenic V . cholerae O1 strains contained tcpA ET . Interestingly, among the isolates of V . cholerae non-O1/non-O139, two had tcpA Classical, nine contained tcpA El Tor, three showed homology with both biotype genes, and four carried the ctxA gene . The stn/sto genes were present in 28.2% of the non-O1/non-O139 strains, in 10.5% of the toxigenic V . cholerae O1, and in 14.3% of the O139 serogroups . Except for stn/sto genes, all of the other genes studied occurred with high frequency in toxigenic V . cholerae O1 and O139 strains . Based on results of this study, surveillance of non-O1/non-O139 V . cholerae in the aquatic environment, combined with genotype monitoring using ctxA, stn/sto, and tcpA ET genes, could be valuable in human health risk assessment.

Zhonghua Yu Fang Yi Xue Za Zhi, 2000 Sep, 34(5), 278 - 80
{Simultaneous detection and differentiation of O1 classical E1 Tor, O139 and nonO1 nonO139 strains of Vibrio cholerae by using multiplex PCR}; Rui Y et al.; OBJECTIVE: To explore the rapid and sensitive procedures for detection and differentiation of O1 classical(CVC), El Tor(EVC), O139 and nonO1 nonO139 strains of Vibrio cholerae . METHODS: Four pairs of primers were designed from toxin sub-unit A(ctxA) gene, O139 special gene, Haemolytic subunit A gene and Toxin coregulated pilus subunit A gene . Multiplex PCR was established to simultaneously detect those four genes . CVC, EVC, O139 and nonO1 nonO139 strains can be detected and differentiated by the size and numbers of Multiplex PCR bands via electrophoresis . Two pairs of inside primers were designed from ctx A and O139 special gene, and a nested PCR was established . RESULTS: 60 EVC, 2 CVC, 15 O139 and 30 nonO1 nonO139 strains isolated from patients were detected with multiplex PCR and nested PCR, and the results were correct . 10 EVC and 15 O139 strains isolated from environment samples were detected, and the results meant that EVC were toxic and O139 were non-toxic . The sensitivity of multiplex PCR reached 100 cfu, and that of nested PCR reached 1-10 cfu . CONCLUSION: This method is rapid, specific, and sensitive, and possess great value for practical application.

J Bacteriol, 2001 Jun, 183(12), 3784 - 90
Flagellum-independent surface migration of Vibrio cholerae and Escherichia coli; Brown II et al.; Surface translocation has been described in a large variety of microorganisms, including some gram-negative enteric bacteria . Here, we describe the novel observation of the flagellum-independent migration of Vibrio cholerae and Escherichia coli on semisolid surfaces with remarkable speeds . Important aspects of this motility are the form of inoculation, the medium composition, and the use of agarose rather than agar . Mutations in several known regulatory or surface structure proteins, such as ToxR, ToxT, TCP, and PilA, did not affect migration, whereas a defect in lipopolysaccharide biosynthesis prevented translocation . We propose that the observed surface migration is an active process, since heat, protease, or chloramphenicol treatments of the cells have strong negative effects on this phenotype . Furthermore, several V . cholerae strains strongly expressing the hemagglutinin/protease but not their isogenic hap-negative mutants, lacked the ability of surface motility, and the treatment of migrating strains with culture supernatants from hap strains but not hap-null strains prevented surface translocation.

J Bacteriol, 2001 Jun, 183(12), 3652 - 62
Characterization of the role of the ToxR-modulated outer membrane porins OmpU and OmpT in Vibrio cholerae virulence; Provenzano D et al.; ToxR, the transmembrane regulatory protein required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae, directly activates and represses the transcription of two outer membrane porins, OmpU and OmpT, respectively . In an attempt to dissect the role of the OmpU and OmpT porins in viability and virulence factor expression, in-frame chromosomal deletions were constructed in the coding sequences of ompU and ompT of V . cholerae . Two separate deletions were introduced into ompU; the first (small) deletion, Delta ompU1, removed the coding sequence for 84 internal amino acids (aa), while the second (large) deletion, Delta ompU2, removed the coding sequence for the entire amino-terminal 274 aa . The Delta ompU1 strain had a growth defect that could not be complemented by episomal expression of full-length ompU . In contrast, a strain with Delta ompU2 displayed wild-type growth kinetics in rich media, suggesting that this is the true phenotype of a strain lacking OmpU and that the truncated OmpU protein, rather than the absence of OmpU, may be the cause for the Delta ompU1 phenotype . A large deletion removing the coding sequence for the entire N-terminal 273 aa of OmpT (Delta ompT) was also constructed in wild-type as well as Delta toxR and Delta ompU2 strains, and these strains displayed wild-type growth kinetics in rich media . However, the Delta ompU2 strain was deficient for growth in deoxycholate compared to wild-type, Delta ompT, and Delta ompU2 Delta ompT strains, reinforcing a positive role for the OmpU porin and a negative role for the OmpT porin in V . cholerae resistance to anionic detergents . The Delta ompU2, Delta ompT, and Delta ompU2 Delta ompT strains exhibited wild-type levels of in vitro virulence factor expression and resistance to polymyxin B and serum and in vivo colonization levels similar to a wild-type strain in the infant mouse intestine . Our results demonstrate that (i) OmpU and OmpT are not essential proteins, as was previously thought; (ii) these porins contribute to V . cholerae resistance to anionic detergents; and (iii) OmpU and OmpT are not essential for virulence factor expression in vitro or intestinal colonization in vivo.

J Bacteriol, 2001 Jun, 183(12), 3537 - 47
The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone; Milton DL et al.; Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL) . However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V . anguillarum . In this study, we have characterized this second system . Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL) . Quantification of each AHL present in stationary-phase V . anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively . Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi . However, resequencing of the V . harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM) . Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V . harveyi . A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL . In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI . However, a vanN mutant displayed a wild-type AHL profile . Neither mutation affected either the production of proteases or virulence in a fish infection model . These data indicate that V . anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V . fischeri (the LuxRI homologues VanRI) and V . harveyi (the LuxMN homologues, VanMN).

Pediatr Infect Dis J, 2001 May, 20(5), 550 - 1
Vibrio cholerae non-O1 facial cellulitis in a North Queensland, Australian child; Norton R et al.; Vibrio cholerae is an uncommon cause of cellulitis in Australia . Most reported cases worldwide have involved marine or brackish water contact . A recognized risk factor for acquiring this infection is chronic liver disease secondary to hepatitis B . We describe a case of extensive facial cellulitis caused by Vibrio cholerae non-O1, non-0139, in an 11-year-old indigenous girl from North Queensland, Australia, who was hepatitis B surface antigen-negative . Treatment consisted of extensive debridement, antibiotics, hyperbaric oxygen and facial reconstructive surgery . Early microbiologic diagnosis and a combined therapeutic approach are important in the management of this condition.

AIDS Treat News . 1995 Aug 18;(no 229):6.
FDA warning on raw oysters . Food and Drug Administration.
{Identification of Vibrio vulnificus from patients with otitis media and septicemia by PCR method}
Sato S, Miura T, Saitoh M, Tsukiashi S, Hongo T, Toba T.

Department of Medical Technology, School of Health Sciences, Hirosaki UniversitySucrose-negative colonies were isolated on TCBS agar plate from otorrhea specimens from otitis media patient in the Adult Diseases Examination Center, Hirosaki Medical Association . The isolate was identified as Vibrio vulnificus by nested PCR method which amplify V . vulnificus-specific sequences directed against 23S rRNA genes . The PCR method was applied to identify 6 strains originally isolated from septicemia patients in Kurashiki Central Hospital and formerly identified as V . vulnificus by phenotypic characteristics . When examined by the API20E system, the above PCR confirmed-V . vulnificus isolates were correctly identified as V . vulnificus with % ID 99.8, though this gave 3 different profiles . Cytotoxin-hemolysin gene was detected in otorrhea strain as well as septicemia strains by PCR method . The above results suggest that PCR method targeted cytotoxin-hemolysin gene is suitable for rapid and accurate identification of the isolate, because the result is obtained in less than 4 h . To our knowledge this is the first report on the V . vulnificus infection in Aomori Prefecture and the isolation from otorrhea.

FEMS Microbiol Lett, 2001 May 15, 199(1), 33 - 7
Role of endosymbiotic zooxanthellae and coral mucus in the adhesion of the coral-bleaching pathogen Vibrio shiloi to its host; Banin E et al.; Vibrio shiloi, the causative agent of bleaching the coral Oculina patagonica in the Mediterranean Sea, adheres to its coral host by a beta-D-galactopyranoside-containing receptor on the coral surface . The receptor is present in the coral mucus, since V . shiloi adhered avidly to mucus-coated ELISA plates . Adhesion was inhibited by methyl-beta-D-galactopyranoside . Removal of the mucus from O . patagonica resulted in a delay in adhesion of V . shiloi to the coral, corresponding to regeneration of the mucus . DCMU inhibited the recovery of adhesion of the bacteria to the mucus-depleted corals, indicating that active photosynthesis by the endosymbiotic zooxanthellae was necessary for the synthesis or secretion of the receptor . Further evidence of the role of the zooxanthellae in producing the receptor came from a study of adhesion of V . shiloi to different species of corals . The bacteria failed to adhere to bleached corals and white (azooxanthellate) O . patagonica cave corals, both of which lacked the algae . In addition, V . shiloi adhered to two Mediterranean corals (Madracis and Cladocora) that contained zooxanthellae and did not adhere to two azooxanthellate Mediterranean corals (Phyllangia and Polycyathus) . V . shiloi demonstrated positive chemotaxis towards the mucus of O . patagonica . The data demonstrate that endosymbiotic zooxanthellae contribute to the production of coral mucus and that V . shiloi infects only mucus-containing, zooxanthellate corals.

Dev Comp Immunol, 2001 Jun-Jul, 25(5-6), 365 - 75
Haemocyte parameters associated with resistance to brown ring disease in Ruditapes spp . clams; Allam B et al.; Brown ring disease (BRD) is a shell disease caused by Vibrio tapetis . This pathogen disturbs the periostracal lamina causing the appearance of a brown conchiolin deposit on the inner face of the shell, within the extrapallial space . Although differences in resistance to BRD have been documented, their relationship to possible defense functions has never been investigated . In this study, flow cytometry was used to analyze cellular parameters in asymptomatic and experimentally infected Ruditapes philippinarum from France and the west coast of the USA . Parallel analyses were made on Ruditapes decussatus, the native European clam, which is highly resistant to BRD . In the haemolymph and extrapallial fluid of animals without BRD, total haemocyte counts, the percentage of granulocytes, and the phagocytic activity against latex beads or V . tapetis by the haemocytes were significantly higher in American R . philippinarum than in French R . philippinarum . In most cases, levels in R . decussatus were the highest of all three groups . Four weeks following challenge with V . tapetis, BRD prevalence reached 52 in American clams and 100% in French specimens, but only 37% in R . decussatus . In symptomatic animals, phagocytosis of V . tapetis increased significantly in the resistant species of clam, R . decussatus, was unchanged in US clams, and decreased significantly in FR specimens when compared to asymptomatic individuals from each population . Ingestion of V . tapetis by haemocytes in the extrapallial fluid, which is in contact with the periostracal lamina, could be the main defense mechanism used to counter the pathogen . Our results suggest that resistance to BRD may well be related to the concentration of granular haemocytes and the phagocytic activity of haemocytes.

J Invertebr Pathol, 2001 Apr, 77(3), 165 - 72
Toxicity to bivalve hemocytes of pathogenic vibrio cytoplasmic extract; Lambert C et al.; Using a chemiluminescence (CL) test, it had been previously demonstrated that Vibrio pectenicida, which is pathogenic to Pecten maximus larvae, was able to inhibit completely the CL activity of P . maximus hemocytes and partially inhibit those of Crassostrea gigas . Conversely, a Vibrio sp . strain, S322, pathogenic to C . gigas larvae was more active in reducing the CL activity of oyster hemocytes than of scallop hemocytes . Using this same CL biotest, V . pectenicida and S322 cytoplasmic extracts were shown to reproduce CL inhibition while the cytoplasmic extract of a nonpathogenic strain (U1, Pseudoalteromonas) was without effect . Moreover, cytoplasmic extract as well as live V . pectenicida cells provoked, within a few hours, death of P . maximus hemocytes adhering to a glass slide . After partial purification, it was shown that toxic activities of V . pectenicida cytoplasmic extract was due to a toxin, named VHKT (for vibrio hemocyte-killer toxin), which is heat stable, acid and protease resistant, and less than 3 kDa in molecular weight . Attempts to purify VHKT by reverse-phase (C18) HPLC separated activity into the fraction eluted by water at a retention time of 4.02 min .

Extremophiles, 2001 Apr, 5(2), 119 - 26
Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp . strain Fi:7; Bendt A et al.; A marine psychrotolerant bacterium from the Antarctic Ocean showing high chitinolytic activity on chitin agar at 5 degrees C was isolated . The sequencing of the 16S rRNA indicates taxonomic affiliation of the isolate Fi:7 to the genus Vibrio . By chitinase activity screening of a genomic DNA library of Vibrio sp . strain Fi:7 in Escherichia coli, three chitinolytic clones could be isolated . Sequencing revealed, for two of these clones, the same open reading frame of 2,189 nt corresponding to a protein of 79.4 kDa . The deduced amino acid sequence of the open reading frame showed homology of 82% to the chitinase ChiA from Vibrio harveyi . The chitinase of isolate Fi:7 contains a signal peptide of 26 amino acids . Sequence alignment with known chitinases showed that the enzyme has a chitin-binding domain and a catalytic domain typical of other bacterial chitinases . The chitinase ChiA of isolate Fi:7 was overexpressed in E . coli BL21(DE3) and purified by anion-exchange and hydrophobic interaction chromatography . Maximal enzymatic activity was observed at a temperature of 35 degrees C and pH 8 . Activity of the chitinase at 5 degrees C was 40% of that observed at 35 degrees C . Among the main cations contained in seawater, i.e., Na+, K+, Ca2+, and Mg2+, the enzymatic activity of ChiA could be enhanced twofold by the addition of Ca2+.

Indian J Exp Biol, 2001 Jan, 39(1), 85 - 6
Antifungal and cytotoxic effects of methanol extracts of three marine molluscs; Rajaganapathi J et al.; Methanol extracts of whole body (Melampus ceylonicus), blood (Anadara rhombea) and hepatopancreas (Sepiella inermis) were screened for antifungal and cytotoxic activity . Blood of A . rhombea and hepatopancreas of S . inermis strongly inhibited Candida albicans, Mucor racemosus and Penicillium expansum . A . rhombea appear to exert strong cytotoxic activity against vertebrate erythrocytes . The bacterial pathogens (Escherichia coli and Vibrio cholarae) were not inhibited by any of the three tested methanol extracts.

J Food Prot, 2001 May, 64(5), 682 - 6
Populations of Vibrio parahaemolyticus in retail oysters from Florida using two methods; Ellison RK et al.; Vibrio parahaemolyticus is a naturally occurring estuarine bacterium that is often associated with gastroenteritis in humans following consumption of raw molluscan shellfish . A number of studies have investigated the environmental distribution of V . parahaemolyticus, but little is known about the levels of this organism during distribution of oysters or at the point of consumption . Duplicate samples of shellstock oysters were collected monthly (September 1997 to May 1998) from the same four restaurants and three wholesale seafood markets in the Gainesville, Fla . area and analyzed for total V . parahaemolyticus densities using two methods: a standard MPN method (BAM-MPN) and a new direct plating procedure (direct-VPAP) . Both methods employed an alkaline phosphatase-labeled DNA probe (VPAP) targeting the species-specific thermolabile hemolysin (tlh) gene to confirm suspect colonies as V . parahaemolyticus . The highest monthly geometric mean V . parahaemolyticus density was observed in October of 1997 (approximately 3,000/g) with similarly high values during September and November of 1997 . From December 1997 to May 1998 mean densities were generally less than 100/g, falling to approximately 10/g in February and March . A strong correlation (r = 0.78) between the direct-VPAP and BAM-MPN methods for determining V . parahaemolyticus densities in market-level oysters was observed . The direct-VPAP method was more rapid and precise while the BAM-MPN was more sensitive and may better recover stressed cells . The utilization of the VPAP probe for identification of V . parahaemolyticus sharply reduced the labor for either method compared to biochemical identification techniques used in earlier V . parahaemolyticus surveys.

Presse Med, 2001 Apr 7, 30(13), 631 - 3
{Isolation of Vibrio strains in French coastal waters and infection with Vibrio cholerae non-O1/non-O139}; Aubert G et al.; OBJECTIVE: Although the incidence in France of V . cholerae non-O1/non-O139 infection in man has increased since 1996, it remains low (7 cases in 1999) . After the death in 1994 of an immunodepressed patient presenting a skin lesion showing superinfection by a strain of non-O1/non-O139 V . cholerae following exposure to seawater, we examined 22 samples of sea-water collected from 20 French coastal areas (Mediterranean coast) . METHODS: The sea-water samples were filtered and enriched with alkaline peptone water (APW), and the strains of Vibrio were isolated on TCBS, SS and BCP media and identified using the API 20 E system (bioMerieux, France) . RESULTS: We isolated 6 strains belonging to 3 species of Vibrio: 2 V . cholerae (non-O1/non-O139), 3 V . parahaemolyticus and 1 V . alginolyticus . One of the V . cholerae strains was isolated from sea-water sampled at the coastal town in which the patient had been staying . The seawater strains exhibited high sensitivity (MIC determined by agar dilution) to the following antibiotics: aminoglycosides, tetracyclines, azithromycin, cotrimoxazole, rifampicin and fluoroquinolones . The beta-lactams were very active against strains of V . cholerae isolated from seawater, while the strain isolated from this patient presented a new carbenicillinase (CARB-6) recently described . CONCLUSION: The presence of Vibrio in seawater along the French coast-line constitutes a risk for immunocompromised patients, and the severity of Vibrio infections warrants improved monitoring both of these organisms and of the marine environment . In addition, awareness on the part of doctors would allow patients at risk to be warned against these dangers.

Environ Toxicol Chem, 2001 Apr, 20(4), 839 - 45
Toxicity-based criteria for the evaluation of textile wastewater treatment efficiency; Cordova Rosa EV et al.; Brazilian textile mills import wastewater treatment technologies, performances of which are generally evaluated only on a physicochemical basis . Thus, a battery of bioassays was used to evaluate the performance of an ozonation system to treat textile effluents . Comparative toxicological profiles for bacteria (Vibrio fischeri), algae (Scenedesmus subspicatus), daphnia (Daphnia magna), fish (Poecilia reticulata), and plants (soybean--Glycine max, rice--Oryza sativa, and wheat--Triticum aestivum), as well as genotoxic effects (Vicia faba micronucleus assay), are presented for both raw and ozonated textile effluents . The relative sensitivity of bioassays (or end points) to textile effluents found in this study in decreasing order was plant enzymes > bacteria > algae daphnids approximately = plant biomass approximately = germination rate > fish . No significant genotoxic effect was found . We have concluded that ozonation was relatively effective in reducing toxicity of textile effluents . Bioassays used in this study proved to be sensitive and reliable tools for determining the toxicity of industrial effluents, and thus they can be used to evaluate emerging technology efficiency.

J Mol Evol, 2001 Apr, 52(4), 321 - 32
The evolution of bioluminescent oxygen consumption as an ancient oxygen detoxification mechanism; Timmins GS et al.; Endogenous reductants such as hydrogen sulfide and alkylthiols provided free radical scavenging systems during the early evolution of life . The development of oxygenic photosynthesis spectacularly increased oxygen levels, and ancient life forms were obliged to develop additional antioxidative systems . We develop here the hypothesis of how "prototypical" bioluminescent reactions had a plausible role as an ancient defense against oxygen toxicity through their "futile" consumption of oxygen . As oxygen concentrations increased, sufficient light would have been emitted from such systems for detection by primitive photosensors, and evolutionary pressures could then act upon the light emitting characteristics of such systems independently of their use as futile consumers of oxygen . Finally, an example of survival of this ancient mechanism in present-day bioluminescent bacteria (in the Euprymna scolopes-Vibrio fischeri mutualism) is discussed . Once increasing ambient oxygen levels reached sufficiently high levels, the use of "futile" oxygen consumption became too bioenergetically costly, so that from this time the evolution of bioluminescence via this role was made impossible, and other mechanisms must be developed to account for the evolution of bioluminescence by a wide range of organisms that patently occurred after this (e.g., by insects).

Environ Toxicol, 2001, 16(2), 136 - 41
Toxicity evaluation of metal plating wastewater employing the Microtox assay: a comparison with cladocerans and fish; Choi K et al.; The relative sensitivity of the Microtox assay is closely related to the type of toxicant, and hence its utility in biomonitoring effluents is better evaluated on a case-by-case basis . The Microtox assay, employing the marine bacterium Vibrio fischeri, was evaluated for its applicability in monitoring metal plating wastewater for toxicity . The results of the Microtox assay after 5, 15, and 30 min of exposure, were compared with data obtained from conventional whole effluent toxicity testing (WET) methods that employed Daphnia magna, Ceriodaphnia dubia, and the fathead minnow (Pimephales promelas) . The Microtox assay produced notably comparable EC50 values to the LC50 values of the acute fathead minnow toxicity test (< 0.5 order of difference) . The Spearman's rank correlation analyses showed that the bacterial assay, regardless of exposure duration, correlated better with the acute fish than the daphnid results (p < 0.05) . These observations were consistent to other studies conducted with inorganic contaminants . The relative sensitivity of the 30-min Microtox assay was within the range of the two frequently used acute daphnid/fish toxicity tests . In conclusion, the Microtox assay correlated well with the acute fathead minnow data and is well suited for toxicity monitoring for these types of industrial wastes.

Int J Antimicrob Agents, 2001 May, 17(5), 407 - 9
In vitro susceptibility of Vibrio spp . isolated from the environment; Zanetti S et al.; Bacteria of the genus Vibrio include harmless aquatic strains as well as strains capable of causing epidemics of cholera and human intestinal diseases . Some of these species may show resistance to different antibiotics including cefotaxime, tetracycline and chloramphenicol . The susceptibility to different antibiotics was tested using 40 Vibrio alginolyticus, eight V . parahaemolyticus and six V . vulnificus strains isolated in the coastal waters of Northern Sardinia (Italy) . The frequency of resistance to beta-lactams was unexpectedly high . More than 80% of Vibrio isolates were resistant to ampicillin and 2.5% of V . alginolyticus were resistant to ceftazidime and cefotetan . Forty percent of V . alginolyticus and three V . vulnificus isolates gave a positive nitrocefin test . PCR was also performed using selected primers chosen for having common sequences of bla(TEM) and bla(SHV) genes.

Int J Antimicrob Agents, 2001 May, 17(5), 383 - 7
In vitro susceptibility to 15 antibiotics of vibrios isolated from penaeid shrimps in Northwestern Mexico; Roque A et al.; The sensitivity of 144 isolates of Vibrio spp isolated from shrimp was compared using common antibiotics and those used in the shrimp industry . The in vitro susceptibility of the isolates was studied using amikacin, ampicillin, carbenicillin, cephalothin, cefotaxime, ceftriaxone, chloramphenicol, gentamicin, netilmicin, nitrofurantoin, pefloxacin, trimethoprim-sulphamethoxazole, enrofloxacin, oxytetracycline and florfenicol . The relationship between the minimum inhibitory concentration and the disk inhibition zone was also studied for some antibiotics.

Biochim Biophys Acta, 2001 May 2, 1512(1), 53 - 63
Lumen geometry of ion channels formed by Vibrio cholerae EL Tor cytolysin elucidated by nonelectrolyte exclusion; Yuldasheva LN et al.; Vibrio cholerae EL Tor cytolysin, a water-soluble protein with a molecular mass of 63 kDa, forms small pores in target cell membranes . In this communication, planar lipid bilayers under voltage clamp conditions were used to investigate the geometric properties of the pores . It was established that all cytolysin channels were inserted into membranes with the same orientation . Sharp asymmetry in the I-V curve of fully open cytolysin channels persisting at high electrolyte concentrations indicated asymmetry in the geometry of the channel lumen . Using the nonelectrolyte exclusion method, evidence was obtained that the cis opening of the channel had a larger diameter (< or = 1.9 nm) than the trans opening (< or = 1.6 nm) . The channel lumen appeared constricted, with a diameter of < or = 1.2 nm . Cup-shaped lumen geometry was deduced for both channel openings, which appeared to be connected to each other via a central narrow part . The latter contributed significantly to the total electrical resistance and determined the discontinuous character of channel filling with nonelectrolytes . Comparisons of the properties of pores formed by cytolysins of two V . cholerae biotypes (EL Tor and non-O1) indicated that the two ion channels possessed a similar geometry.

J Protein Chem, 2001 Jan, 20(1), 25 - 32
Synthesis of esculentin-1 antibacterial peptide fragments on 1,4-butanediol dimethacrylate cross-linked polystyrene support; Roice M et al.; Peptide segments corresponding to antibacterial esculentin-1 (1-15), (3344), (9-27), and their modified forms were synthesized on 1,4-butanediol dimethacrylate cross-linked polystyrene (PS-BDODMA) support . Hydroxymethyl and aminomethyl 2% PS-BDODMA supports were used for the synthesis . The HMPB linker was appended to the aminomethyl resin using HBTU in presence of HOBt and the first amino acid was incorporated using MSNT . The conventional Fmoc synthetic protocol was used for the synthesis of peptides . The peptides were cleaved from the support using TFA . The peptides were purified by HPLC, and characterized by amino acid analysis and MALDI TOF MS . The secondary structures of the peptides were revealed by CD measurements . The synthesis of these peptides illustrates the utility of the new support for the synthesis of long-chain bioactive peptides . The synthetic peptides were tested for antimicrobial activity against Escherichia coli Mos blue, E . coli 2, Bacillus brevis, B . megaterium, Pseudomonas HTL, and Vibrio mimicus . The antibacterial activity of the peptides was explained on the basis of the helicity and charged nature of the sequences.

Biochemistry, 2001 Feb 13, 40(6), 1749 - 54
Differential transfers of reduced flavin cofactor and product by bacterial flavin reductase to luciferase; Jeffers CE et al.; It is believed that the reduced FMN substrate required by luciferase from luminous bacteria is provided in vivo by NAD(P)H-FMN oxidoreductases (flavin reductases) . Our earlier kinetic study indicates a direct flavin cofactor transfer from Vibrio harveyi NADPH-preferring flavin reductase P (FRP(H)) to the luciferase (L(H)) from the same bacterium in the in vitro coupled luminescence reaction . Kinetic studies were carried out in this work to characterize coupled luminescence reactions using FRP(H) and the Vibrio fischeri NAD(P)H-utilizing flavin reductase G (FRG(F)) in combination with L(H) or luciferase from V . fischeri (L(F)) . Comparisons of K(m) values of reductases for flavin and pyridine nucleotide substrates in single-enzyme and luciferase-coupled assays indicate a direct transfer of reduced flavin, in contrast to free diffusion, from reductase to luciferase by all enzyme couples tested . Kinetic mechanisms were determined for the FRG(F)-L(F) and FRP(H)-L(F) coupled reactions . For these two and the FRG(F)-L(H) coupled reactions, patterns of FMN inhibition and effects of replacement of the FMN cofactor of FRP(H) and FRG(F) by 2-thioFMN were also characterized . Similar to the FRP(H)-L(H) couple, direct cofactor transfer was detected for FRG(F)-L(F) and FRP(H)-L(F) . In contrast, despite the structural similarities between FRG(F) and FRP(H) and between L(F) and L(H), direct flavin product transfer was observed for the FRG(F)-L(H) couple . The mechanism of reduced flavin transfer appears to be delicately controlled by both flavin reductase and luciferase in the couple rather than unilaterally by either enzyme species.

Appl Environ Microbiol, 2001 May, 67(5), 2360 - 4
Differentiation of environmental and clinical isolates of Vibrio mimicus from Vibrio cholerae by multilocus enzyme electrophoresis; Vieira VV et al.; In this study, we demonstrated that analyzed strains of Vibrio mimicus and Vibrio cholerae could be separated in two groups by using multilocus enzyme electrophoresis (MEE) data from 14 loci . We also showed that the combination of four enzymatic loci enables us to differentiate these two species . Our results showed that the ribosomal intergenic spacer regions PCR-mediated identification system failed, in some cases, to differentiate between V . mimicus and V . cholerae . On the other hand, MEE proved to be a powerful molecular tool for the discrimination of these two species even when atypical strains were analyzed.

Appl Environ Microbiol, 2001 May, 67(5), 2304 - 9
Stress and stress-induced neuroendocrine changes increase the susceptibility of juvenile oysters (Crassostrea gigas) to Vibrio splendidus; Lacoste A et al.; Oysters are permanently exposed to various microbes, and their defense system is continuously solicited to prevent accumulation of invading and pathogenic organisms . Therefore, impairment of the animal's defense system usually results in mass mortalities in cultured oyster stocks or increased bacterial loads in food products intended for human consumption . In the present study, experiments were conducted to examine the effects of stress on the juvenile oyster's resistance to the oyster pathogen Vibrio splendidus . Oysters (Crassostrea gigas) were challenged with a low dose of a pathogenic V . splendidus strain and subjected to a mechanical stress 3 days later . Both mortality and V . splendidus loads increased in stressed oysters, whereas they remained low in unstressed animals . Injection of noradrenaline or adrenocorticotropic hormone, two key components of the oyster neuroendocrine stress response system, also caused higher mortality and increased accumulation of V . splendidus in challenged oysters . These results suggest that the physiological changes imposed by stress, or stress hormones, influenced host-pathogen interactions in oysters and increased juvenile C . gigas vulnerability to Vibrio splendidus.

Water Res, 2001 Apr, 35(6), 1415 - 24
Assessment of toxicity reduction after metal removal in bioleached sewage sludge; Renoux AY et al.; Sewage sludge can be applied to land to supply and recycle organic matter and nutrients . Trace elements in sludge, however, may accumulate in the soil with repeated sludge applications . Reducing metal content may therefore reduce the adverse effects of sludge application . The objective of this study was to evaluate the efficiency of bioleaching technology in reducing metal content and toxicity as measured by a battery of terrestrial and liquid-phase bioassays . Sludge-soil mixtures simulating the application of sludge to land were tested by means of terrestrial bioassays, barley (Hordeum vulgare L.) seed germination (5 d) and sprout growth (14 d), lettuce (Lactuca sativa) seed germination (5 d), and worm (Eisenia andrei) mortality (14 d) . Liquid-phase bioassays, Microtox (Vibrio fischeri, 15 min), lettuce root elongation (L . sativa, 5 d), cladoceran mortality (Daphnia magna, 48 h), and SOS Chromotest (Escherichia coli) were used after elutriation of the sludge . Comparison of the bioassay results (except for D . magna) before and after treatment demonstrated that this bioleaching process reduced both sludge toxicity and metal content . In addition, lower Cu and Zn concentrations found in barley sprouts following treatment supported the assumption that the bioleaching process, by decreasing metal content and bioavailability, reduced sewage sludge toxicity . This study also emphasized the interest of using ecotoxicological bioassays for testing biosolids . In particular, the terrestrial bioassays after simulation of land application and the Microtox test after sludge elutriation proved to be the most appropriate procedures.

Microb Pathog, 2001 Apr, 30(4), 237 - 46
Isolation and characterization of bacteriophage-resistant mutants of Vibrio cholerae O139; Attridge SR et al.; Vibrio cholerae O139 strains produce a capsule which is associated with complement resistance and is used as a receptor by bacteriophage JA1 . Spontaneous JA1-resistant mutants were found to have several phenotypes, with loss of capsule and/or O-antigen from the cell surface . Determination of the residual complement resistance and infant mouse colonization potential of each mutant suggested that production of O-antigen is of much greater significance than the presence of capsular material for both of these properties . Two different in vitro assays of complement resistance were compared and the results of one shown to closely reflect the comparative recoveries of bacteria from the colonization experiments . Preliminary complementation studies implicated two rfb region genes, wzz and wbfP, as being essential for the biosynthesis of capsule but not O-antigen .

FEMS Microbiol Ecol, 2001 May, 35(3), 305 - 312
Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola; Hentschel U et al.; The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola . The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes . The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas) . One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains . Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria . The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans . A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates . Antimicrobial activities were also found against clinical isolates, i.e . multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients . The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.

Chemotherapy, 2001 May-Jun, 47(3), 184 - 93
Effect of Sub-MICs of antibiotics on the hydrophobicity and production of acidic polysaccharide by Vibrio vulnificus; Farzam F et al.; BACKGROUND: Subinhibitory concentrations of antibiotics, which can occur in vivo, have been demonstrated to alter the production of bacterial virulence factors, including the capsule, or the interaction between microorganism and phagocyte by affecting surface hydrophobicity . METHODS: Using a microtiter assay system, the effect of subinhibitory concentrations of amikacin, gentamicin, cephalothin and doxycycline on the surface hydrophobicity and production of acidic polysaccharide by Vibrio vulnificus (8 human isolates, 8 environmental isolates) was determined . RESULTS: All four drugs, in a dose-dependent manner, caused alterations in adherence to polystyrene, a measure of surface hydrophobicity, and the production of acidic polysaccharides, as determined by Alcian blue staining . CONCLUSION: The changes in capsule production and surface hydrophobicity measured in response to sub-MICs of antibiotics appear to be independent variables .

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 29 - 38
Differences in nucleotide specificity and catalytic mechanism between Vibrio harveyi aldehyde dehydrogenase and other members of the aldehyde dehydrogenase superfamily; Zhang L et al.; The fatty aldehyde dehydrogenase (Vh-ALDH) isolated from the luminescent bacterium, Vibrio harveyi, differs from other aldehyde dehydrogenases in its high affinity for NADP(+) . The binding of NADP(+) appears to arise from the interaction of the 2'-phosphate of the adenosine moiety of NADP(+) with a threonine (T175) in the nucleotide recognition site just after the beta(B) strand as well as with an arginine (R210) that pi stacks over the adenosine moiety . The active site of Vh-ALDH contains the usual suspects of a cysteine (C289), two glutamates (E253 and E377) and an asparagine (N147) involved in the aldehyde dehydrogenase mechanism . However, Vh-ALDH has one polar residue in the active site that distinguishes it from other ALDHs; a histidine (H450) is in close contact with the cysteine nucleophile . As a glutamate has been implicated in promoting the nucleophilicity of the active site cysteine residue in ALDHs, the close contact of a histidine with the cysteine nucleophile in Vh-ALDH raises the possibility of alternate routes to increase the reactivity of the cysteine nucleophile . The effects of mutation of these residues on the different functions catalyzed by Vh-ALDH including acylation, (thio)esterase, reductase and dehydrogenase activities should help define the specific roles of the residues in the active site of ALDHs.

J Biol Chem, 2001 Apr 27, 276(17), 13875 - 80 Epub 2001 Jan 31.
Differential expression of Vibrio vulnificus elastase gene in a growth phase-dependent manner by two different types of promoters; Jeong HS et al.; Elastase activity of Vibrio vulnificus was highly dependent on growth phase, reached a maximum during the stationary phase, and was regulated at the level of transcription . The stationary phase production of elastase in crp or rpoS mutants, which were constructed by allelic exchanges, decreased about 3- and 10-fold, respectively . However, the promoter activity of vvpE encoding elastase was unaffected by those mutations in the log phase when analyzed using a vvpE-lux fusion . A primer extension analysis revealed that the transcription of vvpE begins at two different sites, consisting of putative promoter L (PL) and promoter S (PS) . The PL activity was constitutive through the log and stationary phases, lower than the PS activity, and unaffected by the crp or rpoS mutations . The transcription of PS, induced only in the stationary phase, was dependent on RpoS . The mutation in crp reduced the activity of PS; however, the additional inactivation of crp did not influence the PS activity in the rpoS mutant, indicating that CRP exerted its effects through PS requiring RpoS . These results demonstrate that vvpE expression is differentially directed by PL and PS depending on the growth phase and elevated by RpoS and CRP in the stationary phase.

Microbios, 2001, 104(408), 71 - 7
Pathogenicity of Vibrio alginolyticus isolated from diseased small abalone Haliotis diversicolor supertexta; Liu PC et al.; Outbreaks of mass mortality among cultured small abalone Haliotis diversicolor supertexta with abscess/ulcers in the mantle occurred in 1998 at Kao-Hsiung, Taiwan . A swarming bacterium, strain H-11 was isolated from the haemolymph of the moribund small abalone using tryptic soy agar supplemented with 3% NaCl and/or thiosulphate citrate bile salt sucrose agar . This strain was characterized and identified as Vibrio alginolyticus on the basis of various biochemical tests . The H-11 strain and its extracellular products were virulent to small abalones with LD50 values of 3.6 x 10(5) colony forming units and 2.96 microg protein/g body weight, respectively.

Glycoconj J, 2000 Jun, 17(6), 425 - 33
Evaluation of synthetic schemes to prepare immunogenic conjugates of Vibrio cholerae O139 capsular polysaccharide with chicken serum albumin; Kossaczka Z et al.; Vibrio cholerae serotype O139 is a new etiologic agent of epidemic cholera . There is no vaccine available against cholera caused by this serotype . V . cholerae O139 is an encapsulated bacterium, and its polysaccharide capsule is an essential virulent factor and likely protective antigen . This study evaluated several synthetic schemes for preparation of conjugates of V . cholerae O139 capsular polysaccharide (CPS) with chicken serum albumin as the carrier protein (CSA) using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as activating agents . Four conjugates described here as representative of many experiments were synthesized in 2 steps: 1) preparation of adipic acid hydrazide derivative of CPS (CPS(AH)) or of CSA (CSA(AH)), and 2) binding of CPS(AH) to CSA or of CPS to CSA(AH) . Although all conjugates induced CPS antibodies, the conjugate prepared by EDC-mediated binding of CPS and CSA(AH) (EDC:CPS-CSA(AH)) was statistically significantly less immunogenic than the other three conjugates . Representative sera from mice injected with these three conjugates contained antibodies that mediated the lysis of V . cholerae O139 inoculum . Evaluation of the different synthetic schemes and reaction conditions in relation to the immunogenicity of the resultant conjugates provided the basis for the preparation of a V . cholerae O139 conjugate vaccine with a medically useful carrier protein such as diphtheria toxin mutant.

Microbiol Immunol, 2001, 45(2), 135 - 42
Identification and characterization of the sodA genes encoding manganese superoxide dismutases in Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus; Kimoto R et al.; Sequencing of Fur titration assay-positive clones obtained from genomic DNA libraries of Vibrio parahaemolyticus, V . mimicus and V . vulnificus revealed open reading frames encoding proteins of 202, 205 and 202 amino acid residues, respectively . Each open reading frame was preceded by a predicted Fur box which overlaps a likely promoter with similarity to the -10 and -35 consensus sequence of Escherichia coli . The deduced amino acid sequences shared considerable homology with bacterial Mn-containing superoxide dismutases (MnSODs) . Consistent with this, these Vibrio strains produced proteins with SOD activity resistant to inhibition by H2O2 and KCN only when grown under iron-limiting conditions . Primer extension analysis of the total RNA from these vibrios revealed iron-repressible expression of the genes . Furthermore, when grown under iron-limiting conditions, E . coli carrying a plasmid with each cloned gene overexpressed protein with the same electrophoretic mobility and insensitivity of SOD activity to H2O2 and KCN . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing revealed that proteins (MnSODs) having N-terminal amino acid sequences consistent with those deduced from the corresponding genes were present in cell lysates of the vibrios grown under these iron-limited conditions . These results demonstrate that the genes cloned in this study are sodA homologs encoding MnSODs, whose expression is regulated by the iron status of the growth medium . PCR using a primer set based on the V . parahaemolyticus sodA sequence revealed the presence of homologous genes in certain other Vibrio species.

J Bacteriol, 2001 May, 183(9), 2918 - 28
Mapping stress-induced changes in autoinducer AI-2 production in chemostat-cultivated Escherichia coli K-12; DeLisa MP et al.; Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density . Recently, this phenomenon has been discovered in Escherichia coli, and while pathogenic E . coli utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic E . coli is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins . The production of autoinducer AI-2, a signaling molecule employed by E . coli for intercellular communication, was studied in E . coli W3110 chemostat cultures using a Vibrio harveyi AI-2 reporter assay (M . G . Surrette and B . L . Bassler, Proc . Natl . Acad . Sci . USA 95:7046-7050, 1998) . Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli . Results demonstrated that AI-2 levels increased with the steady-state culture growth rate . In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-beta-D-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2) . In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress . Because of our interest in the expression of heterologous proteins in E . coli, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2 . Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes.

J Bacteriol, 2001 May, 183(9), 2746 - 54
The ToxR-mediated organic acid tolerance response of Vibrio cholerae requires OmpU; Merrell DS et al.; It was previously demonstrated that the intestinal pathogen Vibrio cholerae could undergo an adaptive stress response known as the acid tolerance response (ATR) . The ATR is subdivided into two branches, inorganic ATR and organic ATR . The transcriptional regulator ToxR, while not involved in inorganic ATR, is required for organic ATR in a ToxT-independent manner . Herein, we investigate the effect of organic acid stress on global protein synthesis in V . cholerae and show by two-dimensional gel electrophoresis that the stress response alters the expression of more than 100 polypeptide species . The expression of more than 20 polypeptide species is altered in a toxR strain compared to the wild type . Despite this, ectopic expression of the porin OmpU from an inducible promoter is shown to be sufficient to bypass the toxR organic ATR defect . Characterization of the effect of organic acid stress on ompU and ompT transcription reveals that while ompU transcription remains virtually unaffected, ompT transcription is repressed in a ToxR-independent manner . These transcript levels are similarly reflected in the extent of accumulation of OmpU and OmpT . Possible roles for OmpU in organic acid resistance are discussed.

Infect Immun, 2001 May, 69(5), 3488 - 93
Preparation, immunogenicity, and protective efficacy, in a murine model, of a conjugate vaccine composed of the polysaccharide moiety of the lipopolysaccharide of Vibrio cholerae O139 bound to tetanus toxoid; Boutonnier A et al.; The epidemic and pandemic potential of Vibrio cholerae O139 is such that a vaccine against this newly emerged serogroup of V . cholerae is required . A conjugate made of the polysaccharide moiety (O-specific polysaccharide plus core) of the lipopolysaccharide (LPS) of V . cholerae O139 (pmLPS) was prepared by derivatization of the pmLPS with adipic acid dihydrazide and coupling to tetanus toxoid (TT) by carbodiimide-mediated condensation . The immunologic properties of the conjugate were tested using BALB/c mice injected subcutaneously three times at 2 weeks interval and then a fourth time 4 weeks later . Mice were bled 7 days after each injection and then once each month for the following 6 months . LPS and TT antibody levels were determined by enzyme-linked immunosorbent assay using immunoplates coated with either O139 LPS or TT . Both pmLPS and pmLPS-TT conjugate elicited low levels of immunoglobulin M (IgM), peaking 5 weeks after the first immunization . The conjugate elicited high levels of IgG antibodies, peaking 3 months after the first immunization and declining slowly during the following 5 months . TT alone, or as a component of conjugate, induced mostly IgG antibodies . Antibodies elicited by the conjugate recognized both capsular polysaccharide and LPS from V . cholerae O139 and were vibriocidal . They were also protective in the neonatal mouse model of cholera infection . The conjugation of the O139 pmLPS, therefore, enhanced its immunogenicity and conferred T-dependent properties to this polysaccharide.

Infect Immun, 2001 May, 69(5), 3431 - 4
Periodontal pathogens produce quorum sensing signal molecules; Frias J et al.; Different species of bacteria important in the composition of dental plaque were tested for production of extracellular autoinducer-like activities that stimulate the expression of the luminescence genes in Vibrio harveyi . Several strains of Prevotella intermedia, Fusobacterium nucleatum, and Porphyromonas gingivalis were found to produce such activities . Interestingly, these bacteria belong to the same phylogenetic group, and they are periodontal pathogens important in the development of periodontal disease . They specifically produce extracellular signaling molecule related autoinducer-2 from V . haveyi . Nevertheless, they seem to be unable to produce homologues of acyl-homoserine lactones . Furthermore, Escherichia coli DH5alpha can be complemented by the introduction of a P . gingivalis gene with high homology to the luxS gene, which has been called luxS(P.g.).

Am J Physiol Gastrointest Liver Physiol, 2001 May, 280(5), G781 - 6
Microbes and microbial Toxins: paradigms for microbial-mucosal toxins . V . Cholera: invasion of the intestinal epithelial barrier by a stably folded protein toxin; Lencer WI; Cholera toxin (CT) produced by Vibrio cholerae is the virulence factor responsible for the massive secretory diarrhea seen in Asiatic cholera . To cause disease, CT enters the intestinal epithelial cell as a stably folded protein by co-opting a lipid-based membrane receptor, ganglioside G(M1) . G(M1) sorts the toxin into lipid rafts and a retrograde trafficking pathway to the endoplasmic reticulum, where the toxin unfolds and transfers its enzymatic subunit to the cytosol, probably by dislocation through the translocon sec61p . The molecular determinants that drive entry of CT into this pathway are encoded entirely within the structure of the protein toxin itself.

Mol Cell Probes, 2001 Apr, 15(2), 61 - 9
DNA packaging and developmental intermediates of a broad host range Vibrio vulnificus bacteriophage 71A-6; Khan SA et al.; The structural intermediates in the capsid assembly and DNA packaging pathway of Vibrio vulnificus bacteriophage 71A-6, a rod-shaped double-stranded DNA podovirus, were isolated by ultracentrifugation and studied by electron microscopy, SDS-PAGE and pulsed-field gel electrophoretic analysis . Bacteriophage 71A-6 synthesized rod-shaped capsids (mean length=200+/-8 nm; mean width=47+/-3 nm n=50) during its development . Several host proteins that probably help in the assembly and maturation of the capsids were attached to these capsids as spherical structures . A capsid-DNA or DNA packaging complex that consisted of the mature capsids, DNA and a 42.5-kDa protein was also isolated . The size of the capsids increased in length and decreased in width (mean length=220+/-8 nm; mean width=45+/-3 nm n=50) either during or after the DNA packaging . The capsid fractions contained about 12 phage structural proteins and eight host proteins . At least three proteins were tentatively identified: a 38.5-kDa major capsid protein, a 35.2-kDa tail protein and 42.5-kDa packaging initiator or terminator protein . The size of the bacteriophage 71A-6 genome was determined to be 143.0-kb by pulsed-field gel electrophoresis . The total mass of all the mature phage proteins corresponded to only 14.0% of the coding capacity of phage genome.

Cell, 2001 Mar 23, 104(6), 937 - 48
Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin; Tsai B et al.; Cholera toxin is assembled from two subunits in the periplasm of Vibrio cholerae and disassembled in the analogous compartment of target cells, the lumen of the endoplasmic reticulum (ER), before a fragment of it, the A1 chain, is transported into the cytosol . We show that protein disulfide isomerase (PDI) in the ER lumen functions to disassemble and unfold the toxin once its A chain has been cleaved . PDI acts as a redox-driven chaperone; in the reduced state, it binds to the A chain and in the oxidized state it releases it . Our results explain the pathway of cholera toxin, suggest a role for PDI in retrograde protein transport into the cytosol, and indicate that PDI can act as a novel type of chaperone, whose binding and release of substrates is regulated by a redox, rather than an ATPase, cycle.

Microbiology, 2001 Apr, 147(Pt 4), 831 - 7
Analysis of the role of flagellar activity in virulence gene expression in Vibrio cholerae; Hase CC; Expression of virulence factors and motility of VIBRIO: cholerae are intimately linked by an as yet uncharacterized mechanism . Several lines of evidence indicate that the activity of the flagellum of V . cholerae might have an impact on virulence gene regulation, as alterations of the motility phenotype, either by mutation or by inhibitory drugs, result in varied levels of virulence factor production . The Na(+)-driven polar flagella of some VIBRIO: species are proposed to act as mechanosensors, sensing media viscosity . It has been suggested that the V . cholerae flagellum might act as a 'voltmeter', responding to changes in membrane potential, or might sense some environmental conditions that lead to the repression of virulence factors in V . cholerae . To test these hypotheses, beta-galactosidase levels of several types of non-motile mutant derivatives of a V . cholerae toxT::lacZ reporter strain were analysed following changes in media viscosity, membrane potential and other environmental conditions . Like the parental strain, the non-motile strain showed increased toxT::lacZ expression in high-viscosity media, suggesting that the sensing of media viscosity does not occur via the flagella . Other molecules that might be able to detect changes in media viscosity could include mechanosensitive (MS) ion channels found in the bacterial membrane . However, a V . cholerae derivative strain mutated in two putative MS channels still showed increased toxT::lacZ expression in high-viscosity media, indicating that these putative ion channels of V . cholerae are not involved in the viscosity effect and suggesting an as yet uncharacterized mechanism for sensing of media viscosity . The flagellum does not appear to act as a voltmeter, as beta-galactosidase activities of the non-flagellate derivative strain were found to be similar to those of the parental strain after artificially changing the sodium membrane bioenergetics . Similarly, several environmental conditions known to reduce toxin expression were equally effective in reducing toxT::lacZ expression in the motile or non-motile strains . In conclusion, the flagellum of V . cholerae does not act as a mechanosensor, voltmeter or signal transducer for environmental conditions . Thus, alternative mechanisms for the detection of these conditions must exist that likely do not involve the ToxR molecule, as the sensing of all of the tested parameters occurred when the TcpP/H proteins alone activated the toxT::lacZ reporter gene.

Appl Environ Microbiol, 2001 Apr, 67(4), 1710 - 7
Effect of exogenous siderophores on iron uptake activity of marine bacteria under iron-limited conditions; Guan LL et al.; More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM . The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore, N,N'-bis (2,3-dihydroxybenzoyl)-O-serylserine from a Vibrio sp . Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria . Radioisotope studies using (59)Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores . Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols . An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores . This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores . Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.

Appl Environ Microbiol, 2001 Apr, 67(4), 1536 - 41
Proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of Zooxanthellae; Banin E et al.; The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae) . Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29 degrees C . Glycerol inhibited the production of toxin P . Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54) . The structure of toxin P was confirmed by chemical synthesis . In the presence of 12.5 mM NH(4)Cl, pure natural or synthetic toxin P (10 microM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min . The inhibition was proportional to the toxin P concentration . Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH(4)Cl resulted in rapid inhibition of photosynthesis . When zooxanthellae were incubated in the presence of NH(4)Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH(3) into the cell . It is known that uptake of NH(3) into cells can destroy the pH gradient and block photosynthesis . This mode of action of toxin P can help explain the mechanism of coral bleaching by V . shiloi.

J Biol Chem, 2001 May 4, 276(18), 14628 - 33 Epub 2001 Feb 02.
Coupling of cholesterol and cone-shaped lipids in bilayers augments membrane permeabilization by the cholesterol-specific toxins streptolysin O and Vibrio cholerae cytolysin; Zitzer A et al.; Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol . Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide . Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers . A wide variety of ceramide analogs were as effective as D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC . Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin . In contrast, the activity of staphylococcal alpha-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape . The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically . Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.

J Biol Chem, 2001 Jun 1, 276(22), 19160 - 5 Epub 2001 Feb 14.
Zonula occludens toxin structure-function analysis . Identification of the fragment biologically active on tight junctions and of the zonulin receptor binding domain; Di Pierro M et al.; Zonula occludens toxin (Zot) is an enterotoxin elaborated by Vibrio cholerae that increases intestinal permeability by interacting with a mammalian cell receptor with subsequent activation of intracellular signaling leading to the disassembly of the intercellular tight junctions . Zot localizes in the bacterial outer membrane of V . cholerae with subsequent cleavage and secretion of a carboxyl-terminal fragment in the host intestinal milieu . To identify the Zot domain(s) directly involved in the protein permeating effect, several zot gene deletion mutants were constructed and tested for their biological activity in the Ussing chamber assay and their ability to bind to the target receptor on intestinal epithelial cell cultures . The Zot biologically active domain was localized toward the carboxyl terminus of the protein and coincided with the predicted cleavage product generated by V . cholerae . This domain shared a putative receptor-binding motif with zonulin, the Zot mammalian analogue involved in tight junction modulation . Amino acid comparison between the Zot active fragment and zonulin, combined with site-directed mutagenesis experiments, confirmed the presence of an octapeptide receptor-binding domain toward the amino terminus of the processed Zot.

J Assoc Physicians India, 2000 May, 48(5), 505 - 6
Clinical profile of non-O1 strain-O139 of Vibrio cholerae in the region of Ambajogai, Maharashtra; Kamble TK et al.; OBJECTIVES: To study clinical profile of the newly emerged novel strain non-O1, O139 of Vibrio cholerae, in the region of Ambajogai, District Beed of Maharashtra . METHODS: Out of 208 patients of acute gastroenteritis, 41 revealed to be positive for Vibrio cholerae by recommended method of stool examination . All the strains were sent to National Institute of Cholera and Infectious Diseases, Calcutta for confirmation . RESULTS: Out of 41 cases, 12 were of Vibrio cholerae O1, 29 Non-O1, of which nine found to be O139 strain . All patients were from 2-80 years of age with low-socioeconomic status and maximum incidence was in August (64.70%), presented with severe rice watery loose motions . Vomiting was observed in 26 (63.41%), more so in patients of O139 infection (88.88%) than four (33.33%) of O1 infection . Sweating was observed in three patients (33.33%) of O139 infection, cramps in gastrocnemis muscles in three patients (33.33%) of O139 infection and two (16.66%) of O1 infection . Signs of dehydration were mild to moderate in four patients (33.33%) of O1 infection; severe dehydration in six (66.66%), moderate in two (22.22%) and mild in one patient (11.11%) of O139 infection . While dehydration was severe in four (20%), moderate in one (5%) and mild in three patients (15%) of Non-O1 infection (excluding O139 cases) . Clinical features were more severe in patients of serotype O139 than the patients of O1 and Non-O1 (excluding O139 cases) . However all patients responded to intravenous fluids, oral rehydration and antibiotics (tetracycline) within 24-48 hours without any mortality . CONCLUSIONS: This study reflects the first emergence of Non-O1, strain O139 during the year 1997 with severe and critical clinical features in Ambajogai region causing high morbidity in the form of severe dehydration and peripheral circulatory collapse which requires early and correct diagnosis and prompt treatment.

Fish Shellfish Immunol, 2001 Jan, 11(1), 39 - 52
Antibodies against Vibrio salmonicida lipopolysaccharide (LPS) and whole bacteria in sera from Atlantic salmon (Salmo salar L.) vaccinated during the smolting and early post-smolt period; Steine NO et al.; The antibody response to Vibrio salmonicida LPS was studied by ELISA and immunoblot after vaccination of salmon with an aqueous vaccine containing the bacterium . The vaccination of the groups took place from February to July . Antibodies to LPS were present in all sera . Sera from unvaccinated fish and samples collected a short time after vaccination contained low antibody levels . The antibody levels in vaccinated groups increased with time after vaccination except for fish vaccinated during the smolting period . For these fish groups decreasing levels were found in the autumn . Vaccination provided high levels of antibodies to LPS at least 6 months later, even with low water temperatures at primary and secondary vaccination . Fish vaccinated during smolting at higher water temperatures reached high antibody levels a short time after vaccination, but for these groups a decrease took place resulting in the lowest antibody levels of the vaccinated groups in September . Immunoblots showed that antibody reacted with low molecular weight components corresponding to the O-side chain . Immunoblots using whole bacteria as antigen showed a few immunoreactive bands and individual reaction patterns with respect to location of the bands and immunodominance.

Fish Shellfish Immunol, 2001 Jan, 11(1), 15 - 22
Uptake and processing of a Vibrio anguillarum bacterin in Artemia franciscana measured by ELISA and immunohistochemistry; Bergh O et al.; Nauplii of Artemia franciscana were incubated in two different concentrations (undiluted and 1:9 in autoclaved sea water) of a divalent bacterin composed of two different serovars of Vibrio anguillarum . In order to investigate uptake and further processing of a bacterin in the live feed organism A . franciscana, immunohistochemistry was applied, visualising the presence of whole bacterial cells and antigens from the bacterin in individual nauplii . By using ELISA, it was shown that approximately 1.5-2-5 x 10(5) cells were incorporated into each Artemia under the conditions used . Maximum incorporation of cells was measured after 30 min, whereas after 60 min there was a decline to levels of 0.9-1.6 x 10(5) cells per Artemia . Immediately after incubation in the bacterin solution, the nauplii were transferred to a culture of the alga Isochrysis galbana, in order to simulate transfer of the nauplii to rearing tanks for fish larvae . From the ELISA, it could be concluded that the incorporated bacterial cells were excreted from the Artemia nauplii rapidly, however a large variation among different nauplii could be visualised by immunohistochemistry.

Carbohydr Res, 2001 Feb 15, 330(3), 347 - 56
Characterization of cytosolic sialidase from Chinese hamster ovary cells: part II . Substrate specificity for gangliosides; Muthing J et al.; Cytosolic Chinese hamster ovary (CHO) cell sialidase has been cloned as a soluble glutathione S-transferase (GST)-sialidase fusion protein with an apparent molecular weight of 69 kD in Escherichia coli . The enzyme has then been produced in mg quantities at 25-L bioreactor scale and purified by one-step affinity chromatography on glutathione sepharose (Burg, M.; Muthing, J . Carbohydr . Res . 2001, 330, 335-346) . The cloned sialidase was probed for desialylation of a wide spectrum of different types of gangliosides using a thin-layer chromatography (TLC) overlay kinetic assay . Different gangliosides were separated on silica gel precoated TLC plates, incubated with increasing concentrations of sialidase (50 degreesU/mL up to 1.6 mU/mL) without detergents, and desialylated gangliosides were detected with specific anti-asialoganglioside antibodies . The enzyme exhibited almost identical hydrolysis activity in degradation of GM3(Neu5Ac) and GM3(Neu5Gc) . A slightly enhanced activity, compared with reference Vibrio cholerae sialidase, was detected towards terminally alpha(2-3)-sialylated neolacto-series gangliosides IV3-alpha-Neu5Ac-nLc4Cer and VI3-alpha-Neu5Ac-nLc6Cer . The ganglio-series gangliosides G(D1a), G(D1b), and G(T1b), the preferential substrates of V . cholerae sialidase for generating cleavage-resistant G(M1), were less suitable targets for the CHO cell sialidase . The increasing evidence on colocalization of gangliosides and sialidase in the cytosol strongly suggests the involvement of the cytosolic sialidase in ganglioside metabolism on intracellular level by yet unknown mechanisms.

Acta Med Croatica, 2000, 54(3), 107 - 11
Improvements for multipurpose bacteriological identification tables to suit the diagnosis of Vibrio cholerae; Muic V et al.; The aim of the study was to reduce to key tests the 4 extensive polyvalent diagnostic biochemical tables most widely used in Croatia and to adapt them for the demonstration of Vibrio cholerae and its differentiation from the 3 Vibrios (V . alvinolyticus, V . mentschikovii, V . fluvialis) important in differential diagnosis . The fourth table has now been adapted to differentiate among all 12 Vibrio species known to be human pathogens (V . mimicus, V . cincinatiensis, V . holisae, V . damsela, V . furnisi, V . parahaemolyticus, V . vulnificus, V . carchariae) . Using the inductive Learning by Logic Minimization Method (ILLM), we analyzed 2 tables (i.e . identification matrices) that were a part of bioMerieux's commercial packaged polyvalent identification systems widely used in Croatia (API 20E and ATB 32E), as well as 2 compilation tables by M . T . Kelly et al . The tables contained 27, 32, 59 and 8 tests, respectively . Cutting these solely to the key tests involved rationalizing them from 59 to the 5 necessary to differentiate Vibrio cholerae from 3 related Vibrios . Further rationalizations were from 32 to 2 and from 27 to the 3 necessary to differentiate Vibrio cholerae from 2 related Vibrios . By reducing the table of 8 tests to 7, and adding 4 new ones to these we achieved an optimization permitting mutual differentiation of all 12 known human Vibrio pathogens . Use of the selective TCBS plating medium was the only precondition for making these tables effective.

FEMS Microbiol Lett, 2001 Mar 15, 196(2), 99 - 105
Monodansylcadaverine inhibits cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin on cultured rat embryonic fibroblast cells; Naim R et al.; The mechanism of action of Vibrio parahaemolyticus thermostable direct hemolysin (TDH) on cultured cells still remains unclear . We show that addition of osmotic stabilizers, such as polyethylene glycol and dextran, could not protect cultured rat embryonic fibroblast cells (Rat-1) against cytotoxicity induced by TDH, unlike their protection against the hemolytic activity of TDH . By contrast, 100 microM monodansylcadaverine, as well as the presence of 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) in medium, protected the cells against cytotoxicity of TDH . Binding of TDH to Rat-1 cells and intracellular localization of TDH were affected by monodansylcadaverine and EGTA as analyzed by flow cytometry and confocal microscopy . On the hemolytic activity of TDH, monodansylcadaverine and EGTA had no effect . These results suggest that the mechanism of cytotoxicity of TDH on Rat-1 cells was different from that of hemolytic activity of TDH on red blood cells.

Clin Infect Dis, 2001 Apr 1, 32(7), E117 - 9 Epub 2001 Mar 20.
Non-serogroup O:1 Vibrio cholerae bacteremia and cerebritis; Suankratay C et al.; We describe a case of non-serogroup O:1 Vibrio cholerae bacteremia and cerebritis in a 41-year-old Thai man with alcoholism who presented with fever and cellulitis of the right ankle . He was successfully treated with parenteral cefotaxime and then was switched to treatment with oral ciprofloxacin.

Med Sci Monit, 2001 Mar-Apr, 7(2), 242 - 5
Detection of etiological agent for cholera by PCR protocol; Kapley A et al.; BACKGROUND: PCR protocol for Vibrio cholerae, the causative agent of the diarrheal disease cholera has been described in this report . We report the detection of Vibrio species in drinking water samples in a duplex PCR reaction . The target loci used in the study were ctxA and tcpA . The sensitivity and efficiency of detection of this protocol can be applied in epidemic conditions, wherein monitoring of target organisms is very crucial . MATERIAL AND METHODS: Single step thermocycling programs have been reported for amplification of specific target loci . We have demonstrated that a gradient multi-step thermocycling program is more efficient in improving the sensitivity of detection by PCR . The method for preparation of template DNA from environmental sample uses filtration of water followed by harvesting the possible bacterial residue . This was further treated with proteinase K and heat to yield DNA compatible for PCR . The protocol was optimized for amplification of target loci from standard strains as well as from environmental water samples . RESULTS: The method can simultaneously detect two different loci of Vibrio cholerae in a single reaction . The sensitivity of detection achieved for the pathogen was 100 cells per reaction . The specificity of the primers was demonstrated by spiking the reaction with non-specific template . The developed protocol was successfully extended to environmental samples . CONCLUSIONS: The developed duplex PCR protocol allows the simultaneous detection of two genetic loci of the target pathogen from water samples . This enhances the efficiency of detection and provides a sensitive tool for the rapid detection of the pathogen that can be useful in epidemic conditions where the time factor involved in the identification of target pathogen is very crucial.

Biotechnol Bioeng, 2001 May 5, 73(3), 179 - 87
Kinetic resolution of chiral amines with omega-transaminase using an enzyme-membrane reactor; Shin JS et al.; A kinetic resolution process for the production of chiral amines was developed using an enzyme-membrane reactor (EMR) and a hollow-fiber membrane contactor with (S)-specific omega-transaminases (omega-TA) from Vibrio fluvialis JS17 and Bacillus thuringiensis JS64 . The substrate solution containing racemic amine and pyruvate was recirculated through the EMR and inhibitory ketone product was selectively extracted by the membrane contactor until enantiomeric excess of (R)-amine exceeded 95% . Using the reactor set-up with flat membrane reactor (10-mL working volume), kinetic resolutions of alpha-methylbenzylamine (alpha-MBA) and 1-aminotetralin (200 mM, 50 mL) were carried out . During the operation, concentration of ketone product, i.e., acetophenone or alpha-tetralone, in a substrate reservoir was maintained below 0.1 mM, suggesting efficient removal of the inhibitory ketone by the membrane contactor . After 47 and 32.5 h of operation using 5 U/mL of enzyme, 98.0 and 95.5% ee of (R)-alpha-MBA and (R)-1-aminotetralin were obtained at 49.5 and 48.8% of conversion, respectively . A hollow-fiber membrane reactor (39-mL working volume) was used for a preparative-scale kinetic resolution of 1-aminotetralin (200 mM, 1 L) . After 133 h of operation, enantiomeric excess reached 95.6% and 14.3 g of (R)-1-aminotetralin was recovered (97.4% of yield) . Mathematical modeling of the EMR process including the membrane contactor was performed to evaluate the effect of residence time . The simulation results suggest that residence time should be short to maintain the concentration of the ketone product in EMR sufficiently low so as to decrease conversion per cycle and, in turn, reduce the inhibition of the omega-TA activity .

FEMS Microbiol Lett, 2001 Mar 1, 196(1), 45 - 50
Evidence of calcium influx across the plasma membrane depends upon the initial rise of cytosolic calcium with activation of IP(3) in rat enterocytes by heat-stable enterotoxin of Vibrio cholerae non-O1; Hoque KM et al.; In response to heat-stable enterotoxin of Vibrio cholerae non-O1, the initial rise of cytosolic Ca(2+) occurred with activation of IP(3) . Chelation of extracellular Ca(2+) with EGTA and suspension of cells in Ca(2+) free buffer both demonstrated the involvement of internal stores in the rise of {Ca(2+)}i . Cells pretreated with dantrolene resulted in decrease of {Ca(2+)}i response which suggested that the rise of intracellular level of Ca(2+) was mostly due to the mobilization from IP(3) sensitive stores . When the cytosolic Ca(2+) was chelated by loading the cells with BAPTA, NAG-ST could not induce Ca(2+) entry to the cell as assessed by Mn(2+) quenching of fura-2 fluorescence which suggested that calcium influx across the plasma membrane depends upon initial rise of this bivalent cation that maintained the sustained phase of {Ca(2+)}i response . Addition of toxin to the fura-2-loaded cells, preincubated with lanthanum chloride, resulted in reduction of {Ca(2+)}i level with a short duration of irregular sustained phase further suggesting that the influx of Ca(2+) across the plasma membrane might be through the calcium channel.

Rev Invest Clin, 2000 Nov-Dec, 52(6), 632 - 7
{Fulminant sepsis caused by Vibrio vulnificus . A case series}; Cornejo-Juarez P et al.; BACKGROUND: Vibrio vulnificus is a marine bacteria associated with the ingestion of raw shellfish or contact with seawater . It can produce wound infection, diarrhea and sepsis . The main risk factor for infection is the presence of chronic liver disease . Prior studies have shown mortality from 40% to 63% . OBJECTIVE: Report of 8 cases of disseminated infection with V . vulnificus causing fulminant sepsis . DESIGN: Series of cases . METHODS: We reviewed the database of the laboratory of clinical microbiology from 1990 to 1999 . A computer-based review of the worldwide medical literature was also accomplished . RESULTS: There were 8 cases of V . vulnificus infection . All patients had chronic liver disease, 3 also had diabetes mellitus and 1 received immunosuppressive agents . Five patients were known to have ingested raw shellfish . The mean duration of illness before death was 4 days . All patients presented with sepsis, seven had cutaneous lesions . Five patients received early antimicrobial treatment during the first 24 hours and all of them in the first 48 hours . Regardless of susceptibility to the antimicrobial agents used, the mortality was of 87.5% . Disk-diffusion test showed 100% susceptibility to imipenem, ceftazidime and tetracycline; 83% to cefepime, ticarcillin and cotrimoxazole and 50% to quinolones . CONCLUSION: The V . vulnificus infection appears in patients with chronic liver disease and it is associated with high mortality . This infection has to be suspected in high-risk patients who have eaten raw shellfish and therapy must be initiated as soon as possible.

Syst Appl Microbiol, 2000 Dec, 23(4), 523 - 7
Identification and characterization of carnobacteria associated with the gills of Atlantic salmon (Salmo salar L.); Ringo E et al.; Using the surface plate technique, the population level of aerobic bacteria, occurring in the gills of Atlantic salmon (Salmo salar L.), was determined to be approximately 3 x 10(4) g(-1) . Of 100 isolates investigated, 58 were gram-negative . Out of the 42 gram-positive isolates, 26 belonged to the carnobacteria, of which ten were further identified on the basis of 16S rDNA sequence analysis and AFLP fingerprinting . All were identified as Carnobacterium piscicola-like . These carnobacteria strains were also screened for their ability to produce growth inhibitory compounds active against the fish pathogens Aeromonas salmonicida subsp . salmonicida, Vibrio anguillarum and Vibrio salmonicida . Nine out of the ten C . piscicola isolates tested strongly inhibited growth of the three pathogens.

Diagn Microbiol Infect Dis, 2001 Feb, 39(2), 71 - 5
Vibrio parahaemolyticus associated with cholera-like diarrhea among patients in North Jakarta, Indonesia; Lesmana M et al.; A diarrhea study was conducted in North Jakarta, Indonesia from December 1996 through December 1997 . Vibrio parahaemolyticus was isolated from 333 (6.1%) of 5442 rectal swab samples collected from patients with cholera-like diarrhea . Vibrio cholerae O1 was isolated from 545 (10.0%) and V . cholerae non-O1 from 183 samples (3.4%), respectively . Patients positive for V . parahaemolyticus were mostly adults between 20 and 40 years of age, with males constituting 62% . A majority (65%) of these patients demonstrated watery diarrhea with a frequency of fewer than 10 episodes per 24 hour . A large number of the patients had abdominal pain (83%) and vomiting (76%) and were non-febrile (90%) . The highest isolation rate (9.6%) of V . parahaemolyticus was found during the dry season (June, July) and the lowest (4.5%) in the rainy season (December, January, February) . All of the V . parahaemolyticus isolates were hemolytic on human blood agar (positive Kanagawa) but none was urease positive . Disk diffusion antibiotic susceptibility tests performed on the isolates demonstrated resistance to ampicillin (98%), cephalothin (24%), kanamycin (15%), colistin (97%), neomycin (2%) and ceftriaxone (0.3%) . All isolates (100%) were sensitive to chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, gentamicin, and ciprofloxacin.

FEBS Lett, 2001 Mar 9, 492(1-2), 45 - 9
Expression and mutagenesis of the NqrC subunit of the NQR respiratory Na(+) pump from Vibrio cholerae with covalently attached FMN; Barquera B et al.; The Na(+)-translocating NADH:quinone oxidoreductase (Na(+)-NQR) is present in the membranes of a number of marine bacteria and pathogenic bacteria . Two of the six subunits of the Na(+)-NQR, NqrB and NqrC, have been previously shown to contain covalently bound flavin adenine mononucleotide (FMN) . In the current work, the cloning of nqrC from Vibrio cholerae is reported . The gene has been expressed in V . cholerae and shown to contain one equivalent of covalently bound FMN . In contrast, no covalent flavin was detected when threonine-225 was replaced by leucine . The data show that the FMN attachment does not require assembly of the enzyme and are consistent with the unusual threonine attachment site.

Biochim Biophys Acta, 2001 May 1, 1505(1), 82 - 93
Na(+)-driven flagellar motor of Vibrio; Yorimitsu T et al.; Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane . Bacteria move using rotating helical flagellar filaments . The flagellar motor is located at the base of the filament and is buried in the cytoplasmic membrane . Flagellar motors are classified into two types according to the coupling ion: namely the H(+)-driven motor and the Na(+)-driven motor . Analysis of the flagellar motor at the molecular level is far more advanced in the H(+)-driven motor than in the Na(+)-driven motor . Recently, the genes of the Na(+)-driven motor have been cloned from a marine bacterium of Vibrio sp . and some of the motor proteins have been purified and characterized . In this review, we summarize recent studies of the Na(+)-driven flagellar motor.

Biochim Biophys Acta, 2001 May 1, 1505(1), 45 - 56
Na(+) translocation by bacterial NADH:quinone oxidoreductases: an extension to the complex-I family of primary redox pumps; Steuber J; The current knowledge on the Na(+)-translocating NADH:ubiquinone oxidoreductase of the Na(+)-NQR type from Vibrio alginolyticus, and on Na(+) transport by the electrogenic NADH:Q oxidoreductases from Escherichia coli and Klebsiella pneumoniae (complex I, or NDH-I) is summarized . A general mode of redox-linked Na(+) transport by NADH:Q oxidoreductases is proposed that is based on the electrostatic attraction of a positively charged Na(+) towards a negatively charged, enzyme-bound ubisemiquinone anion in a medium of low dielectricity . A structural model of the {2Fe-2S}- and FAD-carrying NqrF subunit of the Na(+)-NQR from V . alginolyticus based on ferredoxin and ferredoxin:NADP(+) oxidoreductase suggests that a direct participation of the Fe/S center in Na(+) transport is rather unlikely . A ubisemiquinone-dependent mechanism of Na(+) translocation is proposed that results in the transport of two Na(+) ions per two electrons transferred . Whereas this stoichiometry of the pump is in accordance with in vivo determinations of Na(+) transport by the respiratory chain of V . alginolyticus, higher (Na(+) or H(+)) transport stoichiometries are expected for complex I, suggesting the presence of a second coupling site.

Zh Mikrobiol Epidemiol Immunobiol, 2001 Jan-Feb, (1), 5 - 9
{Biological properties of Vibrio cholerae as a part of epidemiologic surveillance of cholera}; Khatovich AB; Systematic dynamic surveillance of the complex of biological properties of V . cholerae makes it possible to find out specific features of this infective agent, to improve diagnostics and to use the data thus obtained for epidemiological surveillance on cholera . The study of the complex of biological properties of V . cholerae O1, its ecological relationships and interactions give evidence to assert that microbiological aspects as one of the primary tasks in monitoring water ecosystems, as well as the necessity of surveillance on strains isolated from humans . Different properties of V . cholerae should be determined irrespective of the object, time and territory of their isolation in the process of epidemiological surveillance on cholera.

Ann Ig, 2000 Nov-Dec, 12(6), 487 - 91
Virulence factors in Vibrio alginolyticus strains isolated from aquatic environments; Zanetti S et al.; In a microbiological monitoring carried out in various aquatic environment of Sardinia Island (Italy) Vibrio alginolyticus with different virulence phenotypes appeared widely spread . Hemolysis, hemoagglutination and protease production might be together particularly in strains isolated from polluted environments . Adherence capacities to two epithelial cells (Hep-2 and Caco-2) available in laboratory were widely spread in the examined bacterial strains . The adhesion degree was influenced by the utilized cellular clone . The lack of a correspondence between adhesion capacity and more traditional virulence tests do not permit its replacement at screening level.

J Med Microbiol, 2001 Mar, 50(3), 268 - 76
Molecular characterisation of rough variants of Vibrio cholerae isolated from hospitalised patients with diarrhoea; Mitra RK et al.; Seven rough isolates of Vibrio cholerae isolated as the sole infecting agent from patients with cholera-like diarrhoea were examined for the presence of the regulatory element toxR and certain virulence-associated genes of the CTX genetic element and V . cholerae pathogenicity island (VPI) . Multiplex PCR analysis with wb-specific genes of either O1 or O139 origin showed that six of the seven isolates produced an O1 wb-specific amplicon and the remaining isolate produced an O139-specific amplicon . Analysis of lipopolysaccharide profiles of smooth variants of V . cholerae revealed the presence of long repeated units of 'O' polysaccharide side chains but all the rough variants appeared to be devoid of the latter and possessed only core oligosaccharide . PCR amplification with primers specific to the ctxA, ctxB, tcpA, tagA, int, aldA, toxT, LJ, RJ and toxR genes revealed that six of the seven rough isolates were positive for these genes . One isolate was found to be negative for tagA and RJ, indicating the presence of an altered VPI . Each of these isolates showed media-dependent expression of cholera toxin (CT) and produced more toxin than the reference V . cholerae O1 El Tor strain VC20 or O139 strain SG24 under comparable conditions . Studies on the clonality of these isolates by the analysis of rRNA genes indicated their relatedness to strains of V . cholerae O1 El Tor or O139, isolated during the same time period.

Int J Infect Dis, 2000, 4(4), 198 - 202
Seasonal, nontoxigenic Vibrio cholerae O1 Ogawa infections in the Eastern Region of Saudi Arabia; Bubshait SA et al.; BACKGROUND: Surveillance for Vibrio cholerae in the Eastern Region of Saudi Arabia has been ongoing since 1985 to detect and prevent local proliferation of imported cholera . In 1996 and 1997 the authors performed additional microbiologic and epidemiologic assessment of V . cholerae surveillance to better characterize a recurrent summertime pattern of V . cholerae infections in the Eastern Region of Saudi Arabia . METHODS: All health facilities routinely submitted stool or rectal swab specimens for isolation of V . cholerae from patients with gastroenteritis . In addition, specimens were taken from expatriate workers and household contacts of persons with confirmed V . cholerae infection . Forty-two isolates were evaluated for cholera enterotoxin by enzyme-linked immunosorbent assay, cholera toxin polymerase chain reaction, and Y1 adrenal cell assay; 12 isolates also were characterized by pulsed-field gel electrophoresis (PFGE) . Interviews about potential exposures were done for all V . cholerae infections . RESULTS: Vibrio cholerae O1 serotype Ogawa biotype El Tor was identified in 113 gastroenteritis patients (6.0 per 100,000 population per year), 28 asymptomatic expatriate workers, and 16 of 982 household contacts of index patients . All symptomatic infected persons had mild illness that was not typical of cholera, and all 42 isolates evaluated were nontoxigenic . All 12 isolates evaluated by PFGE had an indistinguishable pattern (pattern 81) . Infections appeared in late May, decreased in mid-July through August, increased again in September, and disappeared from December through April . Infections had a uniform geographic distribution and affected all ages . No linkage was identified between affected households, or between community cases and food-handlers or domestic servants . DISCUSSION: Surveillance in the Eastern Region of Saudi Arabia has identified a novel strain of nontoxigenic V . cholerae O1 Ogawa . This strain probably has a local environmental reservoir . Since cholera toxin is the primary virulence factor involved in the cause of cholera, assays for cholera toxin should be included in cholera surveillance.

Appl Environ Microbiol, 2001 Mar, 67(3), 1292 - 9
Specific growth rate plays a critical role in hydrogen peroxide resistance of the marine oligotrophic ultramicrobacterium sphingomonas alaskensis strain RB2256; Ostrowski M et al.; The marine oligotrophic ultramicrobacterium Sphingomonas alaskensis RB2256 has a physiology that is distinctly different from that of typical copiotrophic marine bacteria, such as Vibrio angustum S14 . This includes a high level of inherent stress resistance and the absence of starvation-induced stress resistance to hydrogen peroxide . In addition to periods of starvation in the ocean, slow, nutrient-limited growth is likely to be encountered by oligotrophic bacteria for substantial periods of time . In this study we examined the effects of growth rate on the resistance of S . alaskensis RB2256 to hydrogen peroxide under carbon or nitrogen limitation conditions in nutrient-limited chemostats . Glucose-limited cultures of S . alaskensis RB2256 at a specific growth rate of 0.02 to 0.13 h(-1) exhibited 10,000-fold-greater viability following 60 min of exposure to 25 mM hydrogen peroxide than cells growing at a rate of 0.14 h(-1) or higher . Growth rate control of stress resistance was found to be specific to carbon and energy limitation in this organism . In contrast, V . angustum S14 did not exhibit growth rate-dependent stress resistance . The dramatic switch in stress resistance that was observed under carbon and energy limitation conditions has not been described previously in bacteria and thus may be a characteristic of the oligotrophic ultramicrobacterium . Catalase activity varied marginally and did not correlate with the growth rate, indicating that hydrogen peroxide breakdown was not the primary mechanism of resistance . More than 1,000 spots were resolved on silver-stained protein gels for cultures growing at rates of 0.026, 0.076, and 0.18 h(-1) . Twelve protein spots had intensities that varied by more than twofold between growth rates and hence are likely to be important for growth rate-dependent stress resistance . These studies demonstrated the crucial role that nutrient limitation plays in the physiology of S . alaskensis RB2256, especially under oxidative stress conditions.

Water Res, 2001 Feb, 35(2), 557 - 60
Toxicity of benzotriazole and benzotriazole derivatives to three aquatic species; Pillard DA et al.; Benzotriazole and its derivatives comprise an important class of corrosion inhibitors, typically used as trace additives in industrial chemical mixtures such as coolants, deicers, surface coatings, cutting fluids, and hydraulic fluids . Recent studies have shown that benzotriazole derivatives are a major component of aircraft deicing fluids (ADFs) responsible for toxicity to bacteria (Microtox) . Our current research compared the toxicity of benzotriazole (BT), two methylbenzotriazole (MeBT) isomers, and butylbenzotriazole (BBT) . Acute toxicity assays were used to model the response of three common test organisms: Microtox bacteria (Vibrio fischeri), fathead minnow (Pimephales promelas) and water flea (Ceriodaphnia dubia) . The response of all the three organisms varied over two orders of magnitude among all compounds . Vibrio fischeri was more sensitive than either C . dubia or P . promelas to all the test materials, while C . dubia was less sensitive than P . promelas . The response of test organisms to unmethylated benzotriazole and 4-methylbenzotriazole was similar, whereas 5-methylbenzotriazole was more toxic than either of these two compounds . BBT was the most toxic benzotriazole derivative tested, inducing acute toxicity at a concentration of < or = 3.3 mg/l to all organisms.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Sep-Oct, (5), 87 - 91
{Genetic markers of epidemic strains of Vibrio cholerae}; Smirnova NI et al.; In this review new data on the key pathogenicity genes of V . cholerae are presented . As shown on the basis of the analysis of the latest information on the structure of the genomes of different V . cholerae strains, structural genes ctxAB coding cholera toxin may not serve as the only marker of epidemically dangerous strains . More complete and reliable information for the evaluation of the epidemic potential of V . cholerae isolated from the environment may be obtained by the simultaneous detection of 4 genetic markers: genes ctxAB, tcpA and hap coding, respectively, cholera toxin, toxin-corregulated adhesion pili and soluble hemagglutinin/protease, as well as regulatory virulence gene toxR.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Sep-Oct, (5), 84 - 6
{Bactericidal properties of the Vibrio cholerae non O1 bacteriolysin}; Telesmanich NR et al.; The influence of the preparation of hemocytolysin, obtained from V . cholerae non O1 (strain P-11702), on the growth of V . cholerae cells was studied . The study revealed that hemocytolysin is capable of inducing partial or complete bacterial lysis on the place of its application, depending on the protein load of the substance and the inoculation dose of microbes . Two electrophoretic fractions with molecular weights of 14 and 14.5 kD induced the cytolysis of sheep, rabbit, guinea-pig red blood cells and showed a bactericidal effect . The different sensitivity of Vct+ and Vct- strains of V . cholerae to different doses of hemocytolysin was studied.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Sep-Oct, (5), 75 - 8
{The use if Francisella tularensis lipopolysaccharide in the dot solid phase enzyme immunoassay}; Aronova NV et al.; To determine antitularemia antibodies in the sera of humans and animale, the possibility of using dot immunoassay with the use of F . tularensis lipopolysaccharide (LPS) as antigen-containing preparation was ascertained . Experiments demonstrated that this method made it possible to determine specific antitularemia antibodies in the sera of sick and immunized humans and animals . Investigetions carried out with the use of heterologous antisera to F . novicida, F . novicida-like and F . philomiragia, as well as Brucellf abortus, Vibrio cholerae and Yersinia enterocolitica, revealed that F . tularensis S-LPS was highly specific . The results obtained in this investigation are indicative of good prospects of using F . tularensis LPS in dot blotting for the laboratory diagnostics of tularemia in humans.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Sep-Oct, (5), 22 - 5
{Microbiological monitoring of causative agents of sapronoses in the water of the Bogatinskoe water reservoir}; Ivanova EP et al.; The results of the microbiological monitoring of potential causative agents of sapronoses in the water of the Bogatinskoye reservoir revealed that in the summer period of 1998 the mass accumulation of virulent Aeromonas sobria (up to 25% of the total number of heterotrophic bacteria) took place . The autumn period was characterized by a decrease in the number of A . sobria and the detection of bacteria of the genus Vibrio (up to 22%) with V . mimicus and V . metschnikovii identified among them in the water ecosystems of the southern regions of the Maritime Territory.

Am J Trop Med Hyg, 2000 Apr, 62(4), 513 - 7
New insights on the emergence of cholera in Latin America during 1991: the Peruvian experience; Seas C et al.; After a century of absence, in late January 1991, Vibrio cholerae invaded the Western Hemisphere by way of Peru . Although a number of theories have been proposed, it is still not understood how that invasion took place . We reviewed the clinical records of persons attending hospital emergency departments in the major coastal cities of Peru from September through January of 1989/1990 and 1990/1991 . We identified seven adults suffering from severe, watery diarrhea compatible with a clinical diagnosis of cholera during the four months preceding the cholera outbreak, but none during the previous year . The patients were scattered among five coastal cities along a 1,000 km coastline . We postulate that cholera vibrios, autochthonous to the aquatic environment, were present in multiple coastal locations, and resulted from environmental conditions that existed during an El Nino phenomenon . Once introduced into the coastal communities in concentrations large enough for human infection to occur, cholera spread by the well-known means of contaminated water and food.

Microbiol Immunol, 2000, 44(12), 963 - 70
Characterization of Vibrio parahaemolyticus manganese-resistant mutants in reference to the function of the ferric uptake regulatory protein; Funahashi T et al.; In many bacteria, the ferric uptake regulatory protein (Fur) has a central role in the negative regulation of genes affected by iron limitation . In this study, Vibrio parahaemolyticus strains carrying mutations in the fur gene encoding Fur were isolated by the manganese selection method to assess the function of Fur in connection with alternations in the coordinate expression of the siderophore vibrioferrin (VF) and iron-repressible outer membrane proteins (IROMPs) . Ten out of 25 manganese-resistant mutants constitutively produced VF and expressed at least two IROMPs irrespective of the iron concentration in the medium . PCR-direct DNA sequencing of the fur genes in these mutants identified four different point mutations causing amino acid changes . Moreover, a fur overexpressing plasmid was constructed to prepare antiserum against V . parahaemolyticus Fur . Western blotting with this antiserum revealed that the intracellular abundance of the wild-type Fur was not significantly affected by the iron concentrations in the growth medium, and that the Fur proteins of the mutant strains occurred at substantially smaller amounts and/or migrated more rapidly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the wild-type Fur . These data afford an additional insight into the structure-function relationship of Fur and imply its involvement in the iron acquisition systems of V . parahaemolyticus, although it is yet unknown whether its action on the target genes is direct or indirect.

Carbohydr Res, 2001 Jan 15, 330(1), 83 - 92
Structural studies of the O-specific polysaccharide of Vibrio cholerae O8 using solvolysis with triflic acid; Kocharova NA et al.; The O-specific polysaccharide (OPS) of Vibrio cholerae 08 was isolated by mild acid degradation of the lipopolysaccharide and studied by two-dimensional NMR spectroscopy, including NOESY and heteronuclear multiple-bond correlation (HMBC) experiments . The OPS was found to have a tetrasaccharide repeating unit with the following structure: --> 4)-beta-D-Glcp NAc3NAcylAN-(1 --> 4)-beta-D-Manp NAc3NAcAN-(1 --> 4)-alpha-L-Gulp NAc3NAcA-(1 --> 3) -beta-D-QuipNAc4NAc-(1 --> where QuiNAc4NAc is 2,4-diacetamido-2,4,6-trideoxyglucose, GlcNAc3NAcylAN is 2-acetamido-3-(N-formyl-L-alanyl)amino-2,3-dideoxyglucuronamide, ManNAc3NAcAN is 2,3-diacetamido-2,3-dideoxymannuronamide, and GulNAc3NAcA is 2,3-diacetamido-2,3-dideoxyguluronic acid . The OPS was stable towards acid hydrolysis and solvolysis with anhydrous hydrogen fluoride, but could be cleaved selectively with trifluoromethanesulfonic (triflic) acid by the glycosidic linkages of beta-QuiNAc4NAc and alpha-GulNAc3NAcA . The structures of the oligosaccharides obtained that were elucidated by electrospray ionization (ESI) MS and NMR spectroscopy, confirmed the OPS structure.

Carbohydr Res, 2001 Jan 15, 330(1), 7 - 20
Syntheses of the L-manno and some other analogs of the terminal determinants of the O-PS of Vibrio cholerae O:1; Poirot E et al.; Analogs of the methyl alpha-glycosides of the terminal residues of the O-specific polysaccharides (O-PS) of Vibrio cholerae O:1, serotype Inaba and Ogawa, have been prepared as probes to study their interaction with anti V . cholerae O:1 antibodies . They differ from the termini of the respective O-PSs in anomeric or absolute configuration of perosamine, position of the O-methyl group in D-perosamine, and nature of the N-acyl side chain.

J Med Assoc Thai, 2000 Nov, 83(11), 1289 - 95
Food poisoning outbreak from contaminated fish-balls; Tangkanakul W et al.; On February 9th, 1998, a food poisoning outbreak occurred at a boarding school for underprivileged students . An unmatched case-control study was done . An environmental survey, laboratory study of rectal swab culture, fish-balls, water and the cooking utensils were also performed . There were 132 suspect cases, of which the attack rate in teachers was 9.8 per cent (4/41), 16.7 per cent (1/6) in the food handlers and 15.7 per cent (127/810) in the students . The median incubation period was 18 hours . Analysis of food consumption revealed those who ate lunch noodles had the highest risk (OR 3.8, 95% CI 0.6-5.9) . In details of food components, those who ate fish-balls in curry had the only significant risk (OR 3.5, 95% CI 1.2-0.8) of becoming ill when compared to those who did not . Fish-balls in noodles and curry had a dose response relationship . Bacterial culture from 25 grams of fish-balls was positive for Vibrio parahaemolyticus . The fish-balls in noodles and curry were identified as the implicated food . The modes of contamination were uncooked food, cooking utensils and the food handlers . The manufacturer, which had no license to operate and had poor standards of sanitation, was closed by the Food and Drug Administration.

Int J Syst Evol Microbiol, 2001 Jan, 51(Pt 1), 61 - 6
Vibrio cyclotrophicus sp . nov., a polycyclic aromatic hydrocarbon (PAH)-degrading marine bacterium; Hedlund BP et al.; Strain P-2P44T was isolated from creosote-contaminated marine sediments by using a most-probable number procedure in which phenanthrene was the sole carbon and energy source . Growth experiments showed that P-2P44T utilized several two- and three-ring polycyclic aromatic hydrocarbons (PAHs) as substrates, including naphthalene, 2-methylnaphthalene and phenanthrene . Additionally, gas-chromatography experiments showed that P-2P44T degraded several other PAHs, though it was unable to use them as sole sources of carbon and energy . Phylogenetic analyses confirmed that strain P-2P44T is a member of the genus Vibrio, most closely related to Vibrio splendidus . However, strain P-2P44T shared only 98.3% 16S rDNA identity and 35% DNA-DNA reassociation with the type strain of V . splendidus . Strain P-2P44T differed phenotypically from V . splendidus . Together, these differences indicated that strain P-2P44T represents a novel species in the genus Vibrio, for which the name Vibrio cyclotrophicus sp . nov . is proposed; the type strain is P-2P44T (= ATCC 700982T = PICC 106644T).

Zh Mikrobiol Epidemiol Immunobiol, 2000 Nov-Dec, (6), 99 - 104
{Factors contributing to preservation of Vibrio cholerae in water reservoirs}; Mikhailova AE et al.; The summarized data of literature concerning the survival of V . cholerae in the environment and the influence of abiotic and biotic factors on this process are presented . These data make it possible to regard cholera as sapronosis and to form an idea of the role of factors contributing to the survival of V . cholerae in the environment and to its spread among human population.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Nov-Dec, (6), 72 - 4
{Synthesis of protective antigens during submerged cultivation of Vibrio cholerae}; Fedorova VA et al.; The effectiveness of dot immunoanalysis for evaluating the dynamics of the synthesis of O-antigen, cholera toxin, neuraminidase, adhesin CFA1 in the process of the reactor cultivation of V . cholerae used for the production of oral chemical cholera vaccine is shown . The established regularities of the synthesis of the protective antigens of V . cholerae in the process of scaled-up cultivation are discussed.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Nov-Dec, (6), 28 - 30
{Vibrio cholerae O139 bacteriophages}; Kudriakova TA et al.; Cholera bacteriophages have been isolated from 27 lysogenic cultures of V . cholerae O139 . As shown the pages under study belong to two morphological groups A1 and F1 and serological types II and XII . The use of prophage typing and the sensitivity test to specific phage made it possible to differentiate V . cholerae strains, serogroup O139.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Nov-Dec, (6), 104 - 8
{Pathogenicity factors of various vibrios and aeromonads}; Boiko AV; A review of literature on the pathogenicity factors of non-cholera vibrios and Aeromonas is presented . A detailed analysis of such pathogenicity factors as hemolysins, lecithinases, cytotoxins, adhesins, etc . is given . Information on some mechanisms of the pathogenetic action of these factors on warm-blooded animals is presented . The necessity of more extensive research on pathogenicity and persistence factors in opportunistic bacteria causing sapronotic infections is emphasized.

Dis Aquat Organ, 2000 Dec 21, 43(3), 159 - 73
Diseases of the eye of farmed shrimp Penaeus monodon; Smith PT; Lesions were found in the eyes of cultured shrimp Penaeus monodon that displayed non-specific signs of disease, including lethargy, dark pigmentation, brown gills, empty midgut, anorexia, white tail muscle, necrosis of uropods and fouled cuticle . Eye lesions were associated with sexual development in moribund shrimp in at least 1 disease event . Suppurative inflammation, granuloma and malacia were observed in histological examination of the eye and the causative agents of lesions appear to be Vibrio spp . and a rod-shaped virus (similar to Lymphoid Organ Virus, Gill-Associated Virus {GAV} and Yellow-Head Virus) . Suppurative inflammation was characterised by edema, infiltration of haemocytes and local sites of abscesses . Eyes with granuloma usually appeared white in pond-side examinations, and histology showed that fibrous tissue replaced ommatidia, ganglia and internal structures of the eye . Malacia of the eye was characterised by necrosis of nervous tissue, vacuolation and vascular proliferation in the medulla ganglia . Levels of presumptive Vibrionaceace were high in moribund specimens and Gram-negative rods were observed in some specimens as free particles in the interstitial fluid and haemolymph in the eye . Transmission electron microscopy showed that nerve cells in the fasciculated zone (near the basement membrane) contained cytoplasmic vesicles (1 to 3 microm in diameter) with particles (15 to 26 nm in diameter) and rod-shaped nucleocapsids . The rods were similar to those of GAV and were 130 to 260 nm long, 10 to 16 nm in diameter and had helical symmetry with a screw-like thread (2.4 to 3.5 nm pitch) . Also, unidentified enveloped virions, averaging 74 nm in diameter, were observed in cytoplasmic vesicles in the fasciculated zone . In conclusion, it is suggested that bacterial and viral infections of the eye could result in impaired neuroendocrine functions, which may cause a range of clinical signs of disease.

J Basic Microbiol, 2000, 40(5-6), 377 - 84
A highly homologous 68 kbp plasmid found in Vibrio vulnificus strains virulent for eels; Lewin A et al.; Vibrio vulnificus serovar E (biotype 2) strains are virulent for eels and have also been reported to cause illness in humans . Studies on the plasmid content of serovar E strains revealed the existence of a plasmid of approximately 70 kbp present in most of these strains . In this study we characterized the 70 kbp plasmids of seven biotype 2 strains isolated from seawater, diseased eels and wound infections in humans . We determined the exact size of the high molecular weight plasmids to be 68 kbp . A comparison of the plasmids of the seven strains by restriction length polymorphism and hybridization analysis showed them to be almost identical.

Sheng Wu Gong Cheng Xue Bao, 2000 Sep, 16(5), 570 - 3
{Cloning of the zot gene of Vibrio cholerae and its expression in Escherichia coli}; He ZY et al.; The zot gene encoding Zonula occludens toxin was amplified from classic Vibrio cholerae genomic DNA by PCR . The result of sequencing indicated that zot gene encodes 399 amino acid residues . The sequence of zot gene was a little bit different from that of reported including 14 nucleotides and four amino acid residues . The expression plasmid pET-ZOT was constructed by inserting zot gene into plasmid pET-28a(+) containing the T7 promoter . The expression plasmid was induced into E . coli BL21 (DE3) and expression strain BLZOT was selected . SDS-PAGE analysis revealed that the ZOT protein was expressed and accumulated up to above 15% of bacterial soluble protein after induced by IPTG . A protein of 47 kD was expressed as including body . Western blot analysis revealed that the expressed protein was ZOT.

Yakugaku Zasshi, 2000 Nov, 120(11), 1149 - 57
{Effects of Vibrio vulnificus metalloprotease on the capillaries: pathological actions and inactivation by alpha-macroglobulin}; Miyoshi S; Vibrio vulnificus is an opportunistic human pathogen causing wound infection and septicemia, characterized by hemorrhagic and edematous damage to the skin of limbs . When injected into the dorsal skin, an extracellular metalloprotease from this vibrio (V . vulnificus protease: VVP) enhanced the vascular permeability through activation of the Hageman factor-plasma kallikrein-kinin cascade and/or stimulation of exocytotic histamine release . Additionally, VVP caused the hemorrhagic skin lesion through disorganization of the vascular basement membrane layer due to specific degradation of type IV collagen, which is known to form the backbone structure of the basement membrane . However, injected VVP was quickly inactivated by a plasma glycoprotein, alpha-macroglobulin, at a molar ratio of 1:1 . This glycoprotein was leaked from the capillaries by the actions of VVP, which resulted in in situ inactivation by physical entrapment . When VVP (45,000 Da) was incubated at 37 degrees C, a 35,000 Da fragment was generated by the autocatalytic removal of a 10,000 Da C-terminal polypeptide . This N-terminal fragment showed significant proteolytic activity, however, because of a markedly decreased affinity to the protein substrates, its permeability-enhancing and hemorrhagic activity was reduced to less than 50% . These findings indicate that the C-terminal polypeptide is not essential for but promotes skin reactions caused by VVP.

Wei Sheng Wu Xue Bao, 1997 Dec, 37(6), 449 - 54
{Comparison of outer membrane protein profiles of Vibrio sp.}; Zhang X et al.; The outer membrane protein (OMP) profiles of 32 Vibrio type strains have been compared . The major OMP profiles of different Vibrio species had a considerable heterogenecity . Most of the strains had 3-7 major OMPs, with molecular masses ranging between 91,000 and 14,000 . Many strains had common major OMPs such as 54,000, 43,000 and 27,000 . However, no common major OMPs in all Vibrio type strains had been found.

Proc Natl Acad Sci U S A, 2001 Jan 16, 98(2), 652 - 7
The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons; Rowe-Magnus DA et al.; Integrons are genetic elements that acquire and exchange exogenous DNA, known as gene cassettes, by a site-specific recombination mechanism . Characterized gene cassettes consist of a target recombination sequence (attC site) usually associated with a single open reading frame coding for an antibiotic resistance determinant . The affiliation of multiresistant integrons (MRIs), which contain various combinations of antibiotic resistance gene cassettes, with transferable elements underlies the rapid evolution of multidrug resistance among diverse Gram-negative bacteria . Yet the origin of MRIs remains unknown . Recently, a chromosomal super-integron (SI) harboring hundreds of cassettes was identified in the Vibrio cholerae genome . Here, we demonstrate that the activity of its associated integrase is identical to that of the MRI integrase, IntI1 . We have also identified equivalent integron superstructures in nine distinct genera throughout the gamma-proteobacterial radiation . Phylogenetic analysis revealed that the evolutionary history of the system paralleled that of the radiation, indicating that integrons are ancient structures . The attC sites of the 63 antibiotic-resistance gene cassettes identified thus far in MRIs are highly variable . Strikingly, one-fifth of these were virtually identical to the highly related yet species-specific attC sites of the SIs described here . Furthermore, antimicrobial resistance homologues were identified among the thousands of genes entrapped by these SIs . Because the gene cassettes of SIs are substrates for MRIs, these data identify SIs as the source of contemporary MRIs and their cassettes . However, our demonstration of the metabolic functions, beyond antibiotic resistance and virulence, of three distinct SI gene cassettes indicates that integrons function as a general gene-capture system for bacterial innovation.

J Bacteriol, 2001 Feb, 183(3), 835 - 42
Two-component sensor required for normal symbiotic colonization of euprymna scolopes by Vibrio fischeri; Visick KL et al.; The light organ of the squid Euprymna scolopes is specifically colonized to a high density by the marine bacterium Vibrio fischeri . To date, only a few factors contributing to the specificity of this symbiosis have been identified . Using a genetic screen for random transposon mutants defective in initiating the symbiotic association or in colonizing the light organ to high density, we identified a mutant of V . fischeri that exhibited an apparent defect in symbiosis initiation . This mutant was not defective in motility, luminescence, or growth in minimal medium, suggesting that it lacks an essential, previously unidentified symbiotic function . By sequence analysis, we showed that the locus inactivated in this mutant encodes a predicted 927-amino-acid protein with a high degree of similarity to the sensor component of hybrid two-component regulatory systems . We have therefore designated this locus rscS, for regulator of symbiotic colonization-sensor . Sequence analysis revealed two hydrophobic regions which may result in the formation of a periplasmic loop involved in signal recognition; PhoA fusion data supported this proposed membrane topology . We have investigated the start site of rscS transcription by primer extension and identified a putative promoter region . We hypothesize that RscS recognizes a signal associated with the light organ environment and responds by stimulating a putative response regulator that controls protein function or gene expression to coordinate early colonization events . Further studies on RscS, its cognate response regulator, and the signaling conditions will provide important insight into the interaction between V . fischeri and E . scolopes.

Am J Physiol Regul Integr Comp Physiol, 2001 Feb, 280(2), R301 - 12
Evolution of the Na-P(i) cotransport systems; Werner A et al.; Membrane transport systems for P(i) transport are key elements in maintaining homeostasis of P(i) in organisms as diverse as bacteria and human . Two Na-P(i) cotransporter families with well-described functional properties in vertebrates, namely NaPi-II and NaPi-III, show conserved structural features with prokaryotic origin . A clear vertical relationship can be established among the mammalian protein family NaPi-III, a homologous system in C . elegans, the yeast system Pho89, and the bacterial P(i) transporter Pit . An alternative lineage connects the mammalian NaPi-II-related transporters with homologous proteins from Caenorhabditis elegans and Vibrio cholerae . The present review focuses on the molecular evolution of the NaPi-II protein family . Preliminary results indicate that the NaPi-II homologue cloned from V . cholerae is indeed a functional P(i) transporter when expressed in Xenopus oocytes . The closely related NaPi-II isoforms NaPi-IIa and NaPi-IIb are responsible for regulated epithelial Na-dependent P(i) transport in all vertebrates . Most species express two different NaPi-II proteins with the exception of the flounder and Xenopus laevis, which rely on only a single isoform . Using an RT-PCR-based approach with degenerate primers, we were able to identify NaPi-II-related mRNAs in a variety of vertebrates from different families . We hypothesize that the original NaPi-IIb-related gene was duplicated early in vertebrate development . The appearance of NaPi-IIa correlates with the development of the mammalian nephron.

Environ Microbiol, 1999 Jun, 1(3), 223 - 9
Inhibition of photosynthesis and bleaching of zooxanthellae by the coral pathogen Vibrio shiloi; Ben-Haim Y et al.; Vibrio shiloi is the causative agent of bleaching (loss of endosymbiotic zooxanthellae) of the coral Oculina patagonica in the Mediterranean Sea . To obtain information on the mechanism of bleaching, we examined the effect of secreted material (AK1-S) produced by V . shiloi on zooxanthellae isolated from corals . AK1-S caused a rapid inhibition of photosynthesis of the algae, as measured with a Mini-PAM fluorometer . The inhibition of photosynthesis was caused by (i) ammonia produced during the growth of V . shiloi on protein-containing media and (ii) a non-dialysable heat-resistant factor . This latter material did not inhibit photosynthesis of the algae by itself but, when added to different concentrations of NH4Cl, enhanced the inhibition approximately two- to threefold . Ammonia and the enhancer were effective to different degrees on zooxanthellae isolated from four species of coral examined . In addition to the rapid inhibition of photosynthesis, AK1-S caused bleaching (loss of pigmentation) and lysis of zooxanthellae . Bleaching was more rapid than lysis, reaching a peak (25% bleached algae) after 6 h . The factors in AK1-S responsible for bleaching and lysis were different from those responsible for the inhibition of photosynthesis, because they were heat sensitive, non-dialysable and active in the dark . Thus, the coral pathogen V . shiloi produces an array of extracellular materials that can inhibit photosynthesis, bleach and lyse zooxanthellae.

Cell Microbiol, 2000 Feb, 2(1), 11 - 7
Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction-associated proteins occludin and ZO-1; Wu Z et al.; Vibrio cholerae produces a little-studied cytotoxin, haemagglutinin/protease (HA/P), in addition to several better-characterized enterotoxins, i.e . cholera toxin (CT), zonula occludens toxin (ZOT) and accessory cholera enterotoxin (Ace) . We have found recently that HA/P perturbs the barrier function of Mardin-Darby canine kidney epithelial cell line I (MDCK-I) by affecting the intercellular tight junctions (TJs) and the F-actin cytoskeleton . In the present study we have assessed more specifically how TJs are affected by HA/P by investigating the cellular localization and biochemical integrity of two well-characterized TJ-associated proteins, occludin and ZO-1 . Western blot analysis showed that occludin bands of 66-85 kDa were digested by HA/P to two predominant bands of around 50 kDa and 35 kDa, and that this degradation was greatly attenuated when the specific bacterial metalloproteinase inhibitor Zincov was co-administered . Trypsin, on the other hand, did not degrade occludin when it was applied in the same way, suggesting that the degradation of occludin by HA/P is an early and specific event . The other TJ-associated protein ZO-1 was not degraded by HA/P in parallel experiments, suggesting the selectivity of HA/P-associated protein degradation . Moreover, immunofluorescence labelling and confocal microscopy showed that ZO-1, but not occludin, around cell-cell boundaries was rearranged by HA/P treatment . Since ZO-1 is located on the inside of the plasma membrane and is directly associated with occludin, the results indicate that breakdown of occludin may send signals to ZO-1 that affect its organization and the structure of the F-actin cytoskeleton . Our finding that the zinc-containing metalloprotease of V . cholerae specifically degraded occludin suggests that specific degradation of important host proteins by bacterial zinc-containing metalloproteases may be an important mechanism in microbial pathogenesis.

Genome Biol . 2000;1(3):REVIEWS1016 . Epub 2000 Sep 01.
The complete genome sequence of Vibrio cholerae: a tale of two chromosomes and of two lifestyles; Schoolnik GK et al.; Vibrio cholerae O1 has figured prominently in the history of infectious diseases as a cause of periodic global epidemics, an affliction of refugees in areas of social strife and as the disease first subjected to modern epidemiological analysis during the classic investigations of John Snow in mid-19th century London {1} . Thus, publication of the entire genome sequence of V . cholerae O1 (biotype El Tor) in Nature {2} by a consortium of investigators from The Institute for Genomic Research, the University of Maryland and Harvard Medical School is properly regarded as an historic event that will trigger a paradigm shift in the study of this organism.

Luminescence, 2001 Jan-Feb, 16(1), 29 - 32
Inhibition of light emission from the bioluminescent bacterium Vibrio fischeri after exposure to triclosan and related hygiene care products; Martin EB et al.; The affect of the anti-microbial agent triclosan (alternative names Microban and Irgasan DP300) on the light emission by the bioluminescent bacterium Vibrio fischeri was determined . Triclosan at concentrations greater than 0.2% (w/v) caused cell lysis and immediate (< 5 s) loss of light emission . Exposure to triclosan at lower concentrations caused a decrease in light output over time . The rate of the decrease in light output followed a cuboid relationship, of which the initial rate (first 60 s) of light loss was proportional to the concentration of triclosan . The effect on light output by two commercially available hygiene products containing triclosan also caused a similar response in light loss .

J Travel Med, 2000 Nov-Dec, 7(6), 304 - 8
Serum antibody response induced in mice after oral administration of three different antigens of enterotoxigenic Escherichia coli in enteric coated microparticles; Adachi JA et al.; BACKGROUND: Gastric digestion of these antigens plays an important role, decreasing the ability to deliver antigens to the gut-associated lymphoid tissue . To overcome this obstacle, microencapsulated antigens from enterotoxigenic Escherichia coli (ETEC) were evaluated for oral immunization of mice . METHODS: Four groups of 10 each received 3 series of 3 doses each of (1) B subunit of cholera toxin (CTB), similar to heat-labile toxin of ETEC, (2) formalin-killed whole cell ETEC H10407 (FK-ETEC), (3) crude preparation of colonization factor antigen I (CFA/I), or (4) placebo . Serum antibody was measured on day 0 and 60 by ELISA . RESULTS: In group 1 a CTB antibody response was induced in all mice, 3 with 1:105 titer and 7 with 1:106 . These antibodies neutralized cholera toxin-induced steriodogenesis of Y-1 adrenal cells . In group 2, 8 mice developed a whole H10407 bacteria antibody titer of 1:100, one 1:200 and one showed no immune response . In the same group, an anti-CFA/I response was observed in 6 mice and anti-LPS in 4 mice as determined by Western blot . All mice in group 3 showed > 1:104 anti-CFA/I antibody titer . Group 4 mice did not develop an immune response to any ETEC antigens . CONCLUSIONS: Microencapsulation appears to be a suitable approach for oral vaccination against ETEC and Vibrio cholerae.

FEMS Microbiol Lett, 2001 Feb 20, 195(2), 237 - 44
Vibrio parahaemolyticus thermostable direct hemolysin can induce an apoptotic cell death in Rat-1 cells from inside and outside of the cells; Naim R et al.; Rat-1 cells exposed to Vibrio parahaemolyticus thermostable direct hemolysin (TDH) developed morphological changes including shrinkage of the cells and reduction in the size of nuclei . Cells either microinjected with TDH or transfected with the tdh gene also showed morphological changes similar to those induced by externally added toxin . Furthermore, TDH-exposed or tdh-transfected cells both showed chromatin condensation and DNA fragmentation which suggest cells undergoing apoptosis . In contrast, expression of a TDH mutant (R7) did not reveal any cytotoxic effects . We demonstrate that expressed TDH was distributed in the cytoplasm . The interleukin-1beta-converting enzyme-related protease inhibitor ZVAD-FMK did not inhibit TDH cytotoxicity . Our results suggest that TDH can induce its cytotoxicity both from outside and from inside the cells and killed the cells through apoptosis.

Infect Immun, 2001 Mar, 69(3), 1947 - 52
Comparison of Vibrio cholerae pathogenicity islands in sixth and seventh pandemic strains; Karaolis DK et al.; Epidemic Vibrio cholerae strains possess a large cluster of essential virulence genes on the chromosome called the Vibrio pathogenicity island (VPI) . The VPI contains the tcp gene cluster encoding the type IV pilus toxin-coregulated pilus colonization factor which can act as the cholera toxin bacteriophage (CTXPhi) receptor . The VPI also contains genes that regulate virulence factor expression . We have fully sequenced and compared the VPI of the seventh-pandemic (El Tor biotype) strain N16961 and the sixth-pandemic (classical biotype) strain 395 and found that the N16961 VPI is 41,272 bp and encodes 29 predicted proteins, whereas the 395 VPI is 41,290 bp . In addition to various nucleotide and amino acid polymorphisms, there were several proteins whose predicted size differed greatly between the strains as a result of frameshift mutations . We hypothesize that these VPI sequence differences provide preliminary evidence to help explain the differences in virulence factor expression between epidemic strains (i.e., the biotypes) of V . cholerae.

Infect Immun, 2001 Mar, 69(3), 1613 - 24
Cell vacuolation caused by Vibrio cholerae hemolysin; Figueroa-Arredondo P et al.; Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood . Here we report that at least one-third of diarrhea-associated nonepidemic V . cholerae strains from Mexico cause vacuolation of cultured Vero cells . Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of Aeromonas; it involved primarily the endoplasmic reticulum at early times (approximately 1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (approximately 16 h) . In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V . cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases . It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization . The gene for V . cholerae vacuolating activity was cloned and was found to correspond to hlyA, the structural gene for hemolysin . HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis . Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin . We propose that this vacuolation, in itself, contributes to the virulence of V . cholerae strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses.

Infect Immun, 2001 Mar, 69(3), 1528 - 35
Differential biological and adjuvant activities of cholera toxin and Escherichia coli heat-labile enterotoxin hybrids; Bowman CC et al.; Two bacterial products that have been demonstrated to function as mucosal adjuvants are cholera toxin (CT), produced by various strains of Vibrio cholerae, and the heat-labile enterotoxin (LT) produced by some enterotoxigenic strains of Escherichia coli . Although LT and CT have many features in common, they are clearly distinct molecules with biochemical and immunologic differences which make them unique . The goal of this study was to determine the basis for these biological differences by constructing and characterizing chimeric CT-LT molecules . Toxin gene fragments were subcloned to create two constructs, each expressing the enzymatically active A subunit of one toxin and the receptor binding B subunit of the other toxin . These hybrid toxins were purified, and the composition and assembly of CT A subunit (CT-A)-LT B subunit (LT-B) and LT A subunit (LT-A)-CT B subunit (CT-B) were confirmed . Hybrids were evaluated for enzymatic activity, as measured by the accumulation of cyclic AMP in Caco-2 cells, and the enterotoxicity of each toxin was assessed in a patent-mouse assay . The results demonstrated that LT-A-CT-B induces the accumulation of lower levels of cyclic AMP and has less enterotoxicity than either wild-type toxin or the other hybrid . Nonetheless, this hybrid retains adjuvant activity equivalent to or greater than that of either wild-type toxin or the other hybrid when used in conjunction with tetanus toxoid for intranasal immunization of BALB/c mice . Importantly, the ability of LT to induce a type 1 cytokine response was found to be a function of LT-A . Specifically, LT-A-CT-B was able to augment the levels of antigen-specific gamma interferon (IFN-gamma) and interleukin 5 to levels comparable to those achieved with native LT, while CT-A-LT-B and native CT both produced lower levels of antigen-specific IFN-gamma . Thus, these toxin hybrids possess unique biological characteristics and provide information about the basis for differences in the biological activities observed for CT and LT.

ALTEX, 1998, 15(3), 115 - 122
{Microbial test combination with Vibrio fischeri as a screening toxicity test}; Schicktanz S et al.; Two different microbial toxicity tests were investigated concerning the possibility to reduce animal experiments in product testing . In the acute bioluminescence test the inhibition of the bacterial luminescence was measured . In the chronic DMS test the inhibition of the microbial metabolism of DMSO to DMS was recorded . The combination of both tests with the same organism Vibrio fischeri enhances the power as a toxicological indicator . We used well known alcohols and tensides . For 15 substances, the correlation between the experimental results and the Draize eye irritation test (rs = 0,943) and the intravenous toxicity (rs = 0,958) was statistically significant . If both tests gave EC50-values >10000mg/L, the substance could be categorised as non or mild toxic . If the EC50-values were <5mg/L, an extremely toxic substance could be expected . Neither false positive nor false negative results have been found, therefore the application of the method as a screening test or as a part in a test battery is conceivable.

Eur J Immunol, 2001 Jan, 31(1), 64 - 71
Effects of cholera toxin on macrophage production of co-stimulatory cytokines; Cong Y et al.; Cholera toxin (CT), the enterotoxin of Vibrio cholerae, is a potent mucosal and systemic immunogen and adjuvant . The precise mechanism of the adjuvanticity of CT is poorly understood . Our previous work has showed that CT up-regulates B7.2, but not B7.1 expression on macrophages, and thus increases their co-stimulatory activity . In the current study, the effects of CT on macrophage co-stimulatory cytokine production were investigated . Bone marrow macrophages were generated by culturing bone marrow cells with macrophage colony-stimulating factor . CT treatment increased endotoxin-stimulated macrophage IL-10, IL-6, and IL-1beta production, whereas it decreased IL-12, TNF-alpha and nitric oxide production . Antibody blocking experiments showed that CT inhibition of IL-12 and TNF-alpha production was mediated by increased IL-10 production, in that addition of anti-IL-10 monoclonal antibody abrogated CT inhibition . The decrease in nitric oxide production was in turn secondary to inhibition of TNF-alpha production . Taken together, our study demonstrated that CT has differential effects on various macrophage co-stimulatory cytokines, effects that are likely to contribute to its adjuvanticity.

Mol Microbiol, 2001 Feb, 39(3), 801 - 12
The two TonB systems of Vibrio cholerae: redundant and specific functions; Seliger SS et al.; The two TonB systems in Vibrio cholerae were found to have unique as well as common functions . Both systems can mediate transport of haemin and the siderophores vibriobactin and ferrichrome . However, TonB1 specifically mediates utilization of the siderophore schizokinen, whereas TonB2 is required for utilization of enterobactin by V . cholerae . Although either TonB system was sufficient for the use of haemin as an iron source, in vitro competition between TonB1 and TonB2 system mutants indicates a preferential role for TonB1 in haemin utilization . This was most pronounced in conditions of high osmolarity, in which TonB1 system mutants were unable to grow with haemin as the sole iron source . Sequence analysis predicted that the two TonB proteins differ in both amino acid sequence and protein size . An internal deletion in TonB1 was constructed in order to generate a protein of approximately the same size as TonB2 . A strain expressing the TonB1 deletion protein, and no other TonB, used haemin as the iron source in low-osmolarity medium, but could not use haemin in high osmolarity . This is the same phenotype as a strain expressing only TonB2 and suggests that TonB1, but not TonB2, can span the increased periplasmic space in high osmolarity and thus mediate haemin transport . Mouse colonization assays indicated a role for both TonB systems, and mutations in either system resulted in reduced ability to compete with the wild type in vivo.

Dev Comp Immunol, 2001 Apr, 25(3), 205 - 17
Immune-relevant (including acute phase) genes identified in the livers of rainbow trout, Oncorhynchus mykiss, by means of suppression subtractive hybridization; Bayne CJ et al.; To develop tools for analysis of the acute phase response, we used suppression subtractive hybridization of cDNAs from the livers of trout in an unchallenged state and in the course of a response to injection with a Vibrio bacterin emulsified in Freund's Incomplete Adjuvant . The resulting cDNA library contains 300-600bp long fragments of 25 or more immune-relevant genes . Fifteen were previously unreported for salmonids, and 12 were not known from any fish species . Known acute phase proteins include serum amyloid A, transferrin and precerebellin-like protein; trout C-polysaccharide-binding protein 1 is probably also an acute phase protein . Components of both the complement system (n=5) and the clotting system (n=3), as well as lectins, various binding proteins, a putative antibacterial peptide, a chemotaxin, an anti-oxidant enzyme, as well as some likely cell-surface receptors and metabolic and lysosomal enzymes are represented in the library . One clone closely resembles a group of Toll-like receptors, including the human IL-1 receptor . Three cDNAs appear to represent complete open reading frames.

FEBS Lett, 2001 Jan 12, 488(1-2), 5 - 8
FMN is covalently attached to a threonine residue in the NqrB and NqrC subunits of Na(+)-translocating NADH-quinone reductase from Vibrio alginolyticus; Hayashi M et al.; The Na(+)-translocating NADH-quinone reductase (NQR) from Vibrio alginolyticus is composed of six subunits (NqrA to NqrF) . We previously demonstrated that both NqrB and NqrC subunits contain a flavin cofactor covalently attached to a threonine residue . Fluorescent peptide fragments derived from the NqrB and NqrC subunits were applied to a matrix-assisted laser desorption ionization time-of-flight mass spectrometer, and covalently attached flavin was identified as FMN in both subunits . From post-source decay fragmentation analysis, it was concluded that FMN is attached by a phosphate group to Thr-235 in the NqrB subunit and to Thr-223 in the NqrC subunit . The phosphoester binding of FMN to a threonine residue reported here is a new type of flavin attachment to a polypeptide.

Ecotoxicol Environ Saf, 2001 Feb, 48(2), 161 - 9
Automated biomonitoring using real time movement analysis of Euglena gracilis; Tahedl H et al.; An automated biomonitoring system for early warning of pollutants in aquatic environments is described and characterized . The system uses sublethal changes in the movement behavior of the flagellate Euglena gracilis as biological endpoints . The movement is determined by real time image analysis . All parameters describing motility, velocity, orientation, and form of the cells are calculated during measurement, and changes of these parameters are interpreted as effect . By automatic dilution of the water sample, dose-effect relationships can be recorded automatically . A total measurement procedure, including control and sample measurement and filling and rinsing of the system, typically requires 8 min . Measurements with different organic and inorganic toxic compounds were performed and the calculated EC(50) values compared with literature data for the bioluminescence test with Vibrio fischeri . Also, measurements with waste water samples from different industrial plants were performed . The fast response time, the small size, the reliable image analysis system, the calculation of several endpoints, and the automatic measuring procedure are major advantages compared to other biological test systems .

Microbiology, 2001 Jan, 147(Pt 1), 183 - 91
A Vibrio harveyi insertional mutant in the cgtA (obg, yhbZ) gene, whose homologues are present in diverse organisms ranging from bacteria to humans and are essential genes in many bacterial species; Czyz A et al.; The cgtA gene product is a member of the subfamily of small GTP-binding proteins that have been identified in diverse organisms ranging from bacteria to humans . In bacteria that sporulate or display another special developmental programme, this gene (referred to as cgtA, obg or yhbZ) appears to be involved in the regulation of these processes . However, this gene has also been found to be essential in all bacterial species investigated to date, although its role in bacteria that do not sporulate and do not undergo a specific development remains unknown . Here the authors characterize a Vibrio harveyi mutant bearing a transposon insertion into the cgtA gene . This mutant reveals a multiple phenotype: it grows more slowly than the wild-type strain in a rich medium; its growth is completely inhibited in minimal media; its survival in 3% NaCl is dramatically reduced; it is very sensitive to UV irradiation; it is more susceptible to mutation upon treatment with different mutagens; its luminescence is decreased; its quorum-sensing regulation is less effective than in the wild-type strain; and the elongated shape of the mutant cells may suggest problems with the regulation of cell division and/or DNA replication . These defects in diverse cellular processes found in the insertional cgtA mutant of V . harveyi indicate that in a bacterium that does not sporulate and does not display other special development programmes, the CgtA protein is involved in the regulation of many crucial biochemical reactions, possibly at the stage of signal transduction.

J Bacteriol, 2001 Mar, 183(5), 1830 - 4
VibD and VibH are required for late steps in vibriobactin biosynthesis in Vibrio cholerae; Wyckoff EE et al.; Vibrio cholerae synthesizes the catechol siderophore vibriobactin . In this report, we present the complete map of a vibriobactin gene region containing two previously unreported vibriobactin biosynthetic genes . vibD encodes a phosphopantetheinyl transferase, and vibH encodes a novel nonribosomal peptide synthase . Both VibD and VibH are required for vibriobactin biosynthesis.

Microbiology, 2001 Feb, 147(Pt 2), 499 - 506
Frankia sequences exhibiting RNA polymerase promoter activity; Bock JV et al.; Frankia are Gram-positive, filamentous bacteria capable of fixing atmospheric dinitrogen either in the free-living state or in symbiosis with a variety of woody plants . Only a few Frankia genes have been sequenced and gene expression is not well characterized . To isolate a segment of Frankia DNA that functions as an RNA polymerase promoter, fragments of Frankia strain ArI5 genomic DNA were cloned upstream of a promoterless, Vibrio harveyi luxAB cassette . Constructs were screened for luminescence in E . coli and positive clones assayed for in vitro transcription activity with a partially purified Frankia RNA polymerase extract . Primer extension analysis of in vitro transcripts produced from one clone, GLO7, identified two major transcription start sites, TSP-1 and TSP-2, 52 bp apart . Deletion analysis then localized sequences essential for promoter activity . The upstream promoter region, GLO7p1, contains sequences resembling the -35 element of a Streptomyces promoter and the -35 and -10 elements of the canonical E . coli promoter . Also within this region are two pentamers identical to sequences near the 5' end of the Frankia strain CpI1 glutamine synthetase gene . The second promoter, GLO7p2, contains a putative NtrC binding site at -145 and a possible sigma(N)-RNA polymerase recognition sequence at -14 suggesting that GLO7p2 may be a nitrogen-regulated promoter . An in vivo transcript representing an ORF of 498 aa starting 64 bp downstream of the distal transcription start, TSP-1, was detected by RT-PCR . This supports the conclusion that this DNA fragment has promoter activity in vivo as well as in vitro.

J Bacteriol, 2001 Feb, 183(4), 1369 - 75
Regulation of metalloprotease gene expression in Vibrio vulnificus by a Vibrio harveyi LuxR homologue; Shao CP et al.; Expression of the Vibrio vulnificus metalloprotease gene, vvp, was turned up rapidly when bacterial growth reached the late log phase . A similar pattern of expression has been found in the metalloprotease gene of Vibrio cholerae, and this has been shown to be regulated by a Vibrio harveyi LuxR-like transcriptional activator . To find out whether a LuxR homologue exists in V . vulnificus, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various luxR-like genes of vibrios . A gene containing a 618-bp open reading frame was identified and found to be identical to the smcR gene of V . vulnificus reported previously . An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique . This mutant exhibited an extremely low level of vvp transcription compared with that of the parent strain . On the other hand, the cytolysin gene, vvhA, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth . These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene . Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for V . vulnificus virulence in mice.

Appl Environ Microbiol, 2001 Feb, 67(2), 721 - 4
Evaluation of two direct plating methods using nonradioactive probes for enumeration of Vibrio parahaemolyticus in oysters; Gooch JA et al.; Oysters (Crassostrea virginica) were collected monthly from May 1998 to April 1999 from Mobile Bay, Ala., and analyzed to determine Vibrio parahaemolyticus densities at zero time and after 5, 10, and 24 h of postharvest storage at 26 degrees C . After 24 h of storage at 26 degrees C, oysters were transferred to a refrigerator at 3 degrees C and then analyzed 14 to 17 days later . The V . parahaemolyticus numbers were determined by the most-probable-number procedure using alkaline phosphatase-labeled DNA probe VPAP, which targets the species-specific thermolabile hemolysin gene (tlh), to identify suspect isolates (MPN-VPAP procedure) . Two direct plating methods, one using a VPAP probe (Direct-VPAP) and one using a digoxigenin-labeled probe (Direct-VPDig) to identify suspect colonies, were compared to the MPN-VPAP procedure . The results of the Direct-VPAP and Direct-VPDig techniques were highly correlated (r = 0.91), as were the results of the Direct-VPAP and MPN-VPAP procedures (r = 0.91) . The correlation between the Direct-VPDig and MPN-VPAP results was 0.85 . The two direct plating methods in which nonradioactive DNA probes were used were equivalent to the MPN-VPAP procedure for identification of total V . parahaemolyticus, and they were more rapid and less labor-intensive.

Appl Environ Microbiol, 2001 Feb, 67(2), 575 - 85
gfp-based N-acyl homoserine-lactone sensor systems for detection of bacterial communication; Andersen JB et al.; In order to perform single-cell analysis and online studies of N-acyl homoserine lactone (AHL)-mediated communication among bacteria, components of the Vibrio fischeri quorum sensor encoded by luxR-P(luxI) have been fused to modified versions of gfpmut3* genes encoding unstable green fluorescent proteins . Bacterial strains harboring this green fluorescent sensor detected a broad spectrum of AHL molecules and were capable of sensing the presence of 5 nM N-3-oxohexanoyl-L-homoserine lactone in the surroundings . In combination with epifluorescent microscopy, the sensitivity of the sensor enabled AHL detection at the single-cell level and allowed for real-time measurements of fluctuations in AHL concentrations . This green fluorescent AHL sensor provides a state-of-the-art tool for studies of communication between the individuals present in mixed bacterial communities.

Trends Microbiol, 2000 Apr, 8(4), 189 - 91
The suckling mouse model of cholera; Klose KE; Vibrio cholerae colonization of the suckling mouse intestine is a commonly used animal model for the human diarrheal disease cholera . This model has a number of advantages as well as disadvantages, and has been extremely useful in the identification and characterization of proven and putative virulence factors involved in human cholera.

APMIS, 2000 Mar, 108(3), 178 - 86
Mucosal and systemic antibody responses after peroral or intranasal immunization: effects of conjugation to enterotoxin B subunits and/or of co-administration with free toxin as adjuvant; Rask C et al.; The mucosa-binding molecules cholera toxin (CT) from Vibrio cholerae and heat-labile enterotoxin (LT) from Escherichia coli have previously been used as mucosal adjuvants and carriers for many types of antigen . However, since these molecules are toxic and cannot be used in human vaccines, it is important to study whether their non-toxic mucosa-binding B subunits, CTB and LTB, can be used as alternative safe mucosal adjuvants and/or carrier molecules . We have as a model protein antigen used human gammaglobulin (HGG) for admixture with or chemical conjugation to recombinantly produced CTB and LTB, respectively, and measured antigen-specific local secretory IgA antibodies in saponin extracts from intestine and lung tissue by ELISA following intra-nasal (i.n.) or per-oral (p.o.) immunization . The results show that local antibody formation against HGG was increased after immunization with conjugated as compared to free HGG . However, while the conjugates alone gave rise to significant immune responses in the lung and also, to a lesser degree, in the intestine after i.n . immunization, co-administration of a small amount of free CT/LT as adjuvant was needed to induce a significant immune response in the intestine after p.o . immunization . We also found that following i.n . immunization, the addition of CTB to HGG, without coupling, increased the mucosal immune response to some extent, indicating that CTB by itself can work as an adjuvant by the i.n . route of immunization . A striking finding was that, as a carrier, CTB was superior to LTB when the conjugates were used by the oral but not by the i.n . route of immunization . In conclusion, conjugation of an antigen to mucosa-binding molecules such as CTB and/or LTB can dramatically increase their mucosal immunogenicity . This approach may thus be useful in the preparation of mucosal vaccines.

FEMS Microbiol Lett, 2001 Jan 1, 194(1), 1 - 5
Purification and preliminary characterization of the zonula occludens toxin receptor from human (CaCo2) and murine (IEC6) intestinal cell lines; Uzzau S et al.; In the present study, we report the preliminary characterization of the epithelial cell receptor for Vibrio cholerae zonula occludens toxin (Zot) . Zot receptor was purified by ligand-affinity chromatography . Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis revealed a protein of ca . 66 kDa . Partial N-terminal sequence obtained from purified murine and human Zot receptor revealed homology between the two proteins and with human alpha-1-chimaerin . Zot protein domain(s) involved in receptor binding were also analyzed by constructing several in frame deletion derivatives of a recombinant fusion Zot protein tagged with maltose binding protein . Our results suggest that Zot binding to its cellular membrane receptor requires a sequence that spans between amino acids 118 and 299.

Dis Aquat Organ, 2000 Nov 14, 43(2), 91 - 101
Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla; Collado R et al.; Vibriosis due to Vibrio vulnificus serovar E (biotype 2) is one of the main causes of mortality in European eels cultured in Europe . The main objective of this study was to develop a vaccine and a vaccination procedure against this pathogen . With this aim, we tested several vaccine formulations (inactivated whole-cells with and without toxoids--inactivated extracellular products--from capsulated and uncapsulated strains, attenuated live vaccines and purified lipopolysaccharide {LPS}) on eels maintained under controlled laboratory conditions using different delivery routes (injection and immersion) . To study the immune response we estimated antibody titers and bactericidal/bacteriostatic activity in mucus and serum . To evaluate protection, we calculated the relative percent survival (RPS) after intraperitoneal (i.p.) injection and bath challenge of the pathogen . The overall results indicate that: (1) capsular antigens seem to be essential for protective immunization; (2) vaccines confer the highest protection when administered by i.p . injection; (3) booster is needed to achieve good protection by immersion; (4) enriching the vaccine with toxoids enhances protection to optimal levels (RPS values around 70 to 100%, depending on the delivery route); and (5) the protective effect in serum and mucus depends on the route of administration and seems to be related to the production of specific antibodies.

Dis Aquat Organ, 2000 Nov 14, 43(2), 127 - 37
Luminous vibriosis in rock lobster Jasus verreauxi (Decapoda: Palinuridae) phyllosoma larvae associated with infection by Vibrio harveyi; Diggles BK et al.; Studies were conducted to determine the cause of outbreaks of luminous vibriosis in phyllosoma larvae of the packhorse rock lobster Jasus verreauxi reared in an experimental culture facility . On 2 separate occasions mortalities of up to 75% over a period of 4 wk were observed in 4th to 5th and 8th to 10th instar phyllosomas at water temperatures of 20 and 23 degrees C, respectively . Affected larvae became opaque, exhibited small red spots throughout the body and pereiopods, and were faintly luminous when viewed in the dark . Histopathology showed that the gut and hepatopancreas tubules of moribund phyllosomas contained massive bacterial plaques . The hepatopancreas tubules of moribund larvae were atrophic and some contained necrotic cells sloughed into the lumen . Dense, pure cultures of a bacterium identified as Vibrio harveyi were isolated from moribund larvae . The disease syndrome was reproduced by in vivo challenge and V . harveyi was successfully reisolated from diseased larvae after apparently healthy larvae were exposed by immersion to baths of more than 10(4) V . harveyi ml(-1) at 24 degrees C . Injured larvae were more susceptible to infection than were healthy larvae . Survival of larvae experimentally and naturally exposed to V . harveyi was improved when antibiotics were administered via bath exposures.

Microbiol Immunol, 2000, 44(11), 953 - 6
Characterization of a novel Vibrio parahaemolyticus phage, KVP241, and its relatives frequently isolated from seawater; Matsuzaki S et al.; A vibriophage, KVP241, and six of its relatives were isolated independently from seawater using Vibrio parahaemolyticus as the host . All of the phages had the same morphology (a hexagonal head and a tail with a contractile sheath) and the same host range (specific for some V . parahaemolyticus strains) . DNA-DNA hybridization experiments elucidated that their genomes are highly homologous to each other . Analyses of amino acid sequences of putative major capsid proteins indicated that KVP241 may be weakly related to T4-type phages having a more elongated head.

Microbiol Immunol, 2000, 44(11), 941 - 4
The O-polysaccharide of lipopolysaccharide isolated from Vibrio fluvialis O19 is identical to that of Vibrio bioserogroup 1875 variant; Kondo S et al.; A structural analysis has been carried out on the O-polysaccharide of lipopolysaccharide (LPS) isolated from Vibrio fluvialis 181-86 (Kobe) serotype O19 (O19) which has the Inaba antigen factor C of O1 V . cholerae and factors D and E in common with Vibrio bioserogroup 1875 . The O-polysaccharide of O19 was characterized as an alpha (1-->2)-linked homopolymer of N-3-hydroxypropionyl-D-perosamine (4-amino-4,6-dideoxy-D-mannopyranose), which was identical to that of Vibrio bioserogroup 1875 Variant . Passive hemolysis and passive hemolysis inhibition analysis performed using anti-factor D, E and anti-factor E antisera, demonstrated that the LPS from O19 harbored O-antigenic factors identical to those of the LPS from Vibrio bioserogroup 1875 Variant.

Microbiol Immunol, 2000, 44(11), 931 - 40
Spectrum of gut immunologic reactions: selective induction of distinct responses to Vibrio cholerae WO7 and its toxin; Walia K et al.; Past studies with Vibrio cholerae have shown that cholera toxin (CT) is mainly responsible for inducing T helper type 2 (Th2) responses with systemic IgG1, IgE and mucosal secretory IgA (sIgA) antibodies . In this study, V . cholerae WO7, which produces novel toxin unrelated to CT, was given orally to mice in order to determine whether the strain V . cholerae WO7 differs from V . cholerae 569B, which produces CT, in the nature of responses generated at the gut and splenic level . The analysis of immune responses evoked by V . cholerae WO7 in the gut of mice revealed striking differences as compared to those elicited by V . cholerae 569B infection . To assess the T helper cell type responses, lymphocytes from Peyer's patches and the spleen were stimulated in vitro for studying the cytokine patterns . PP and SP lymphoid cells from V . cholerae WO7 infected animals elaborated significant amounts of IL-2, IFN-gamma and IL-12 by 7 days p.i., suggesting a Th1 type of response . However by 15 days p.i., the PP and SP lymphoid cells secreted only IL-6 and IL-10 with traces of IFN-gamma . On the other hand, infection with V . cholerae 569B yielded mainly Th2 type responses at Peyer's patches as well as the splenic level . Infection with both V . cholerae WO7 and 569B induced toxin-specific IgA secreting cells at the gut and splenic level along with IgG1 secreting cells, indicating that both V . cholerae WO7 and 569B evoke an antigen-specific Th2 type of response in the gut as well as spleen . The persistence of IgA along with Th1-type cytokines indicates an alternate induction mechanism since mucosal IgA responses are usually associated with Th2-type responses . These observations are suggestive of a common mechanism employed by the host to clear different strains of V . cholerae infection (569B and WO7 in this case), while the nature of toxins elaborated failed to modulate the net outcome of the infection caused by V . cholerae.

Microbiol Immunol, 2000, 44(11), 871 - 8
Mechanism of high susceptibility of iron-overloaded mouse to Vibrio vulnificus infection; Hor LI et al.; Vibrio vulnificus produces fulminant septicemia in humans with underlying conditions, particularly those with diseases that elevate the iron level . The effect of a high iron level on the virulence of V . vulnificus was therefore investigated in mice treated with iron dextran . The mice loaded with iron became highly susceptible to V . vulnificus infection, the LD50 (50% lethal dose) decreased five logs when infected per peritoneum . However, when infected via the oral route, the LD50 was affected little unless the mouse was treated with an additional drug such as cyclophosphamide or D-galactosamine . Mice with or without iron-overloading died when the bacterial concentration in the blood reached 10(5) cfu/ml or above . Iron increased the growth rate of the bacteria, both inside and outside of the animal, quickly reaching a lethal concentration in the iron-overloaded mouse . V . vulnificus, grown with or without the addition of iron, showed strong cytotoxicity on the isolated cells or within the animal at high bacterial concentration . Iron overload stimulated the production of tumor necrosis factor alpha (TNF-alpha), a major factor of septic shock, in mice upon infection with the bacteria, probably caused by the endotoxin; however, the neutrophils, whose migration is effected by TNF-alpha, appeared to be less active . Taken together, the major virulence factor of V . vulnificus appeared to be the accelerated growth of bacteria to quickly reach the lethal level and the lower activity of immune cells including neutrophil as a result of iron-overloading . These two effects manifest other virulence factors, the host's as well as bacterial . Such factors, other than TNF-alpha stimulated by the endotoxin, enhanced cytotoxicity, which kills the host cells including the host's immune cells.

J Indian Med Assoc, 2000 Jul, 98(7), 389 - 90
Outbreak of cholera caused by Vibrio cholerae 01 intermediately resistant to norfloxacin at Malda, West Bengal; Bhattacharya MK et al.; During the end of September 1997, an unusual outbreak of severe dehydrating watery diarrhoea cases and deaths were reported from Malda town . Vibrio cholerae 01 El tor, the causative agent responsible for this episode was isolated from 56.5% of cases sampled . Three of the five drinking water samples were also positive for V cholerae 01 . Majority of cases were adults . Isolated strains were uniformly resistant to furazolidone and intermediately to norfloxacin . Indiscriminate use of antibiotic should be discouraged for development of multidrug resistant strains.

J Indian Med Assoc, 2000 Jul, 98(7), 371 - 6
Cholera in its present day scenario; Sanyal SC; Cholera existed in many parts of the world since olden days . Gangetic delta is considered as the home of the disease . Since 1970 there has been a significant development of the disease with its ecologic and epidemiologic aspects . Vibrio cholerae non-01 strain in taxonomically separated from V cholerae 01 strain . Though 01 strain causes epidemic outbreaks, still non-01 has been implicated to cause cholera like illness . While humans are long considered to be the only reservoir of V cholerae 01 strain, but the organism appears to have a free-living cycle in the natural environment . The organism survives more rapidly in the brackish water than fresh water . It has been demonstrated that V cholerae undergoes conversion to a viable but non-culturable state, whereby the cells are reduced in size, become ovoid, but in contrast to starved cells, do not grow at all on standard laboratory media . Seasonality coupled with starvation response and dormancy phenomenon, reflects the origin of V cholerae as an autochthonous estuary dweller . All biotypes of this organism can grow on media containing chitin as the sole carbon source . Outbreaks of the disease are related to plankton blooms associated with warmer sea-surface temperature . In 1997 V cholerae 01 biotype El tor continued to occur in all regions of the world . In 1982 a new classical variant initially displaced entrenched El tor in Bangladesh and coexisted with it for almost a decade . V cholerae 0139 Bangal has arisen along the Bay of Bengal and has spread in Asia.

Ann Ig, 2000 Jul-Aug, 12(4), 297 - 305
{Isolation of Listeria spp., Aeromonas spp., and Vibrio spp . from seafood products}; Scoglio ME et al.; Forty-one strains of Listeria, Aeromonas and Vibrio have been isolated in 71 samples of seafood, both raw and ready to eat and frozen . L . monocytogenes, detected by PCR also, is found in the smoked salmon only . Aeromonas spp . and Vibrio spp . are isolated in the raw products (shrimps and shellfish) . No relationship is found between the presence of such microrganisms and the common indicator bacteria . Finally, the health hazard related to strong contamination and the need to diversify the food safety assurance programmes, for the various products, are underlined.

J Bacteriol, 2001 Jan, 183(2), 758 - 62
SmcR-dependent regulation of adaptive phenotypes in Vibrio vulnificus; McDougald D et al.; Vibrio vulnificus contains homologues of the V . harveyi luxR and luxS genes . A null mutation in smcR (luxR) resulted in a defect in starvation survival, inhibition of starvation-induced maintenance of culturability that occurs when V . vulnificus is starved prior to low-temperature incubation, and increased expression of stationary-phase phenotypes.

Appl Environ Microbiol, 2001 Jan, 67(1), 82 - 8
Cloning and characterization of a periplasmic nuclease of Vibrio vulnificus and its role in preventing uptake of foreign DNA; Wu SI et al.; We have cloned a nuclease gene, vvn, from Vibrio vulnificus, an estuarine bacterium that causes wound infections and septicemia in humans and eels . The gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa . The deduced amino acid sequence of the mature nuclease predicted a molecular mass of 25 kDa, which was confirmed by vital stain, and a pI of 8.6 . Vvn was produced in the periplasm of either V . vulnificus or recombinant Escherichia coli strains and was active in the oxidized (but not the reduced) form . This nuclease was able to digest DNA and RNA, with differential thermostability in DNase and RNase activities . Expression of Vvn in E . coli DH5alpha reduced the frequencies of transformation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively . In addition, the transformation frequency of a Vvn-deficient V . vulnificus mutant (ND) was 10-fold higher than that of the parent strain . These data suggested that Vvn may be involved in preventing uptake of foreign DNA by transformation . However, Vvn expressed in the recipients had little effect on the conjugation frequency in either E . coli or V . vulnificus . Some other DNase(s) may be present in the periplasm and responsible for a residual DNase activity, which was about one-fourth of that of the parent strain, detected in the ND mutant . We also demonstrated that Vvn was not required for the virulence of V . vulnificus mice.

J Med Microbiol, 2000 Dec, 49(12), 1085 - 90
Molecular epidemiological study of Vibrio cholerae isolates from infected patients in Teheran, Iran; Pourshafie MR et al.; A total of 110 clinical isolates of Vibrio cholerae O1 biotype El Tor serotype Ogawa isolated in a recent outbreak from different districts of Teheran, Iran, was subjected to 99 carbon source utilisation tests, ribotyping and toxinogenotyping . PCR showed that the genes encoding cholera toxin (ctxA), toxin co-regulated pilus (tcp), accessory cholera enterotoxin (ace) and zonula occludens toxin (zot) were present in 100%, 100%, 97.3% and 99.1% of the isolates, respectively . Restriction fragment length polymorphism (RFLP) study of the BglI-digested DNA probed with five oligonucleotides targeting the conserved regions of 16S and 23S rRNA genes revealed a similar ribotype pattern for 109 isolates . All but one isolate showed ribotype pattern B21a, containing seven bands with molecular sizes ranging from 11 to 3.9 kb . The toxin gene restriction pattern (toxinogenotype) showed that the isolates carried either three or two copies of the toxin genes (ace, zot, ctx) which were recognised as TB41a and TB69 patterns, respectively . Overall, the ribotyping data showed that, despite biochemical differences in 12 of the 99 carbon sources, most of the isolates studied belonged to a single clone.

Southeast Asian J Trop Med Public Health, 2000 Jun, 31(2), 360 - 5
Characterization of the pili isolated from Vibrio parahaemolyticus O3:K6; Nakasone N et al.; Pilus of Vibrio parahaemolyticus O3:K6 strain LVP9 belonging to the newly identified clone was purified and characterized . The molecular mass of the pilin was estimated to be about 18 kDa by SDS-PAGE, and the isoelectric point of the pilin was 5.0 +/- 0.2 . The LVP9 pili were antigenically different from the other V . parahaemolyticus Na2 pili and Ha7 pili as previously reported, nevertheless all three had indistinguishable morphology and shared a high degree of homology in their N-terminal amino acid sequences . Strain LVP9 and its purified pili did not agglutinate human and rabbit erythrocytes . The LVP9 organisms and the purified pili were adhesive to the rabbit intestine . The adhesion was inhibited by pretreatment of the rabbit intestine with the purified pili or by pretreatment of the organisms with the Fab fractions of anti-pilus antibody . These results indicate that the LVP9 pilus is an adherent factor to the rabbit intestine.

Lett Appl Microbiol, 2000 Dec, 31(6), 433 - 7
Virulence of Vibrio parahaemolyticus isolated from cultured small abalone, Haliotis diversicolor supertexta, with withering syndrome; Liu PC et al.; Outbreaks of mass mortality among cultured small abalone Haliotis diversicolor supertexta with withering syndrome occurred in May and September 1998 in Kao-Hsiung, Taiwan . Bacterial strains CH-1 and B4 were isolated from the haemolymph of the moribund small abalone using tryptic soy agar supplemented with 3% NaCl and/or thiosulphate citrate bile salt sucrose agar . These two strains were characterized and identified as Vibrio parahaemolyticus on the basis of various biochemical tests . The B4 strain and its extracellular products were virulent to small abalone with LD(50) values of 1.6 x 10(5) colony-forming units and 7.58 microg protein g-1 body weight, respectively.

Curr Opin Microbiol, 2000 Dec, 3(6), 603 - 7
Developmental biology in marine invertebrate symbioses; McFall-Ngai MJ et al.; Associations between marine invertebrates and their cooperative bacterial symbionts offer access to an understanding of the roots of host-microbe interaction; for example, several symbioses like the squid-vibrio light organ association serve as models for investigating how each partner affects the developmental biology of the other . Previous results have identified a program of specific developmental events that unfolds as the association is initiated . In the past year, published studies have focused primarily on describing the mechanisms underlying the signaling processes that occur between the juvenile squid and the luminous bacteria that colonize it.

Biochim Biophys Acta, 2000 Dec 1, 1494(3), 226 - 35
Cloning and functional studies of a luxO regulator LuxT from Vibrio harveyi; Lin YH et al.; LuxO is the central regulator integrating the quorum sensing signals controlling autoinduction of luminescence in Vibrio harveyi . We have previously purified to homogeneity a new lux regulator, LuxT, that binds to the luxO promoter . Based on the sequence of the tryptic peptides of LuxT, degenerate oligonucleotides were designed for PCR of the genomic DNA . A 273 bp PCR DNA fragment containing sequences encoding the tryptic peptides was extended by inverse PCR to obtain the complete gene (luxT) encoding a protein of 153 amino acids which shares homology with the AcrR/TetR family of transcriptional regulators . The recombinant and native LuxT gave the same footprint binding between 117 and 149 bp upstream from the luxO initiation codon . Gene disruption of luxT in V . harveyi increased luxO expression and affected the cell density dependent induction of luminescence showing that LuxT was a repressor of luxO . As LuxT also affected the survival of the V . harveyi cells at high salt concentration and homologous proteins are present in other bacterial species, including the pathogen, Vibrio cholerae, the LuxT regulatory protein appears to be a general rather than a lux-specific regulator.

Infect Immun, 2001 Jan, 69(1), 613 - 6
Differential interleukin-8 response of intestinal epithelial cell line to reactogenic and nonreactogenic candidate vaccine strains of Vibrio cholerae; Rodriguez BL et al.; In this study, we analyzed whether attachment of Vibrio cholerae vaccine strains to human intestinal epithelial cells can induce an interleukin-8 (IL-8) response . The IL-8 transcripts were detected by PCR amplification of reverse-transcribed mRNA, and the gene product secretion was measured by an enzyme-linked immunosorbent assay . Infection of monolayers of the undifferentiated HT29-18N2 cell line with reactogenic (JBK70 and 81) and nonreactogenic (CVD103HgR and 638) vaccine strains of V . cholerae resulted in markedly higher IL-8 expression by epithelial cells exposed to reactogenic strains than by cells exposed to the nonreactogenic strains . Additionally, epithelial cells produced IL-8 transcripts following stimulation with cholera vaccine strains in a concentration-dependent manner . These results represent a new insight into the inflammatory component of reactogenicity and could be used as a predictive marker of vaccine reactogenicity prior to human testing.

Yeast, 2000 Dec, 17(4), 307 - 13
Featured organism: pathogen special: Vibrio cholerae, Pseudomonas aeruginosa and Xylella fastidiosa; Wixon J; One could almost say that it is the latest fashion to sequence a bacterial genome . However, this would belittle the efforts of those working on these important organisms, whose data will greatly help those working on the prevention of disease in the fields of medicine and agriculture . In this feature we present a guided tour of the latest additions to the 'sequenced microbes' club . Vibrio cholerae is the causative agent of cholera, which is still a threat in countries with poor sanitation and unsafe drinking water . Pseudomonas aeruginosa is responsible for a large proportion of opportunistic human infections, typically infecting those with compromised immune systems, particularly cystic fibrosis patients, those patients on respirators and burn victims . Xylella fastidiosa is a plant pathogen that attacks citrus fruits by blocking the xylem, resulting in juiceless fruits of no commercial value .

J Appl Microbiol, 2000 Nov, 89(5), 735 - 43
Feather keratin hydrolysis by a Vibrio sp . strain kr2; Sangali S et al.; The aim of the study was to characterize feather-degrading bacteria isolated from poultry industry waste . A Vibrio sp . strain kr2 producing a high keratinolytic activity when cultured on native feather-containing broth was isolated . The bacterium grew with an optimum at pH 6.0 and 30 degrees C, where maximum featherdegrading activity was also observed . Keratinase production was similar at both 25 and 30 degrees C, while the maximum concentration of soluble protein was reached at 30 degrees C . Reduction of disulphide bridges was also observed, increasing with cultivation time . The keratinase of strain kr2 was active on azokeratin, azocasein, benzoyl-arginine-p-nitroanilide and Ala-Ala-p-nitroanilide as substrates . The amino acid composition of the feather hydrolysate was determined, presenting similarities with that reported for feather lysate, feather meal and raw feathers . A novel feather-degrading bacterium was isolated and characterized, showing high keratinolytic activity . Complete feather degradation was achieved during cultivation . Strain kr2 shows potential for use for biotechnological processes involving keratin hydrolysis.

Biochim Biophys Acta, 2000 Dec 20, 1509(1-2), 264 - 74
Vibrio cholerae cytolysin: assembly and membrane insertion of the oligomeric pore are tightly linked and are not detectably restricted by membrane fluidity; Zitzer A et al.; Hemolytic strains of Vibrio cholerae secrete a cytolysin that, upon binding as a monomer, forms pentameric pores in animal cell membranes . Pore formation is inhibited at low temperature and in the absence of cholesterol . We here posed the following questions: firstly, can oligomerization be observed in the absence of pore formation? Secondly, is membrane fluidity responsible for the effect of temperature or of cholesterol upon pore formation? The first issue was approached by chemical cross-linking, by electrophoretic heteromer analysis, and by electron microscopy . None of these methods yielded any evidence of a non-lytic pre-pore oligomer . The second question was addressed by the use of two susceptible liposome models, consisting of cholesterol admixed to bovine brain lipids and to asolectin, respectively . The two liposome species clearly differed in membrane fluidity as judged by diphenylhexatriene fluorescence polarization . Nevertheless, their permeabilization by the cytolysin decreased with temperature in a closely parallel fashion, virtually vanishing at 5 degrees C . Omission of cholesterol from the liposomes uniformly led to an increase in membrane fluidity but prevented permeabilization by the cytolysin . The effects of temperature and of cholesterol upon cytolysin activity are thus not mediated by fluidization of the target membrane . The findings of our study distinguish V . cholerae cytolysin from several previously characterized pore-forming toxins.

J Bacteriol, 2001 Jan, 183(1), 387 - 92
Amino acid residues in LuxR critical for its mechanism of transcriptional activation during quorum sensing in Vibrio fischeri; Trott AE et al.; PCR-based site-directed mutagenesis has been used to generate 38 alanine-substitution mutations in the C-terminal 41 amino acid residues of LuxR . This region plays a critical role in the mechanism of LuxR-dependent transcriptional activation of the Vibrio fischeri lux operon during quorum sensing . The ability of the variant forms of LuxR to activate transcription of the lux operon was examined by using in vivo assays in recombinant Escherichia coli . Eight recombinant strains produced luciferase at levels less than 50% of that of a strain expressing wild-type LuxR . Western immunoblotting analysis verified that the altered forms of LuxR were expressed at levels equivalent to those of the wild type . An in vivo DNA binding-repression assay in recombinant E . coli was subsequently used to measure the ability of the variant forms of LuxR to bind to the lux box, the binding site of LuxR at the lux operon promoter . All eight LuxR variants found to affect cellular luciferase levels were unable to bind to the lux box . An additional 11 constructs that had no effect on cellular luciferase levels were also found to exhibit a defect in DNA binding . None of the alanine substitutions in LuxR affected activation of transcription of the lux operon without also affecting DNA binding . These results support the conclusion that the C-terminal 41 amino acids of LuxR are important for DNA recognition and binding of the lux box rather than positive control of the process of transcription initiation.

J Bacteriol, 2001 Jan, 183(1), 382 - 6
Quorum sensing in Vibrio fischeri: analysis of the LuxR DNA binding region by alanine-scanning mutagenesis; Egland KA et al.; LuxR is the transcriptional activator for quorum-sensing control of luminescence in Vibrio fischeri . A series of alanine-scanning mutants spanning a predicted helix-turn-helix region in the DNA binding domain of LuxR was constructed, and the activity of each of the LuxR mutant proteins in recombinant Escherichia coli was investigated . The region covered by the mutagenesis spanned residues 190 to 224 . About half of the alanine-scanning mutants showed activities similar to that of the wild-type LuxR: at least two were positive-control mutants, four appeared to be defective in DNA binding, and several others were characterized as DNA binding affinity mutants . This analysis, taken together with information about other bacterial transcription factors, provides insights into amino acid residues in LuxR that are involved in DNA binding and transcriptional activation.

J Bacteriol, 2001 Jan, 183(1), 309 - 17
Vibrio fischeri genes hvnA and hvnB encode secreted NAD(+)-glycohydrolases; Stabb EV et al.; HvnA and HvnB are proteins secreted by Vibrio fischeri ES114, an extracellular light organ symbiont of the squid Euprymna scolopes, that catalyze the transfer of ADP-ribose from NAD(+) to polyarginine . Based on this activity, HvnA and HvnB were presumptively designated mono-ADP-ribosyltransferases (ARTases), and it was hypothesized that they mediate bacterium-host signaling . We have cloned hvnA and hvnB from strain ES114 . hvnA appears to be expressed as part of a four-gene operon, whereas hvnB is monocistronic . The predicted HvnA and HvnB amino acid sequences are 46% identical to one another and share 44% and 34% identity, respectively, with an open reading frame present in the Pseudomonas aeruginosa genome . Four lines of evidence indicate that HvnA and HvnB mediate polyarginine ADP-ribosylation not by ARTase activity, but indirectly through an NAD(+)-glycohydrolase (NADase) activity that releases free, reactive, ADP-ribose: (i) like other NADases, and in contrast to the ARTase cholera toxin, HvnA and HvnB catalyzed ribosylation of not only polyarginine but also polylysine and polyhistidine, and ribosylation was inhibited by hydroxylamine; (ii) HvnA and HvnB cleaved 1, N(6)-etheno-NAD(+) and NAD(+); (iii) incubation of HvnA and HvnB with {(32)P}NAD(+) resulted in the production of ADP-ribose; and (iv) purified HvnA displayed an NADase V(max) of 400 mol min(-1) mol(-1), which is within the range reported for other NADases and 10(2)- to 10(4)-fold higher than the minor NADase activity reported in bacterial ARTase toxins . Construction and analysis of an hvnA hvnB mutant revealed no other NADase activity in culture supernatants of V . fischeri, and this mutant initiated the light organ symbiosis and triggered regression of the light organ ciliated epithelium in a manner similar to that for the wild type.

J Bacteriol, 2001 Jan, 183(1), 270 - 9
Transcriptional organization and dynamic expression of the hbpCAD genes, which encode the first three enzymes for 2-hydroxybiphenyl degradation in Pseudomonas azelaica HBP1; Jaspers MC et al.; Pseudomonas azelaica HBP1 degrades the toxic substance 2-hydroxybiphenyl (2-HBP) by means of three enzymes that are encoded by structural genes hbpC, hbpA, and hbpD . These three genes form a small noncontiguous cluster . Their expression is activated by the product of regulatory gene hbpR, which is located directly upstream of the hbpCAD genes . The HbpR protein is a transcription activator and belongs to the so-called XylR/DmpR subclass within the NtrC family of transcriptional activators . Transcriptional fusions between the different hbp intergenic regions and the luxAB genes of Vibrio harveyi in P . azelaica and in Escherichia coli revealed the existence of two HbpR-regulated promoters; one is located in front of hbpC, and the other one is located in front of hbpD . Northern analysis confirmed that the hbpC and hbpA genes are cotranscribed, whereas the hbpD gene is transcribed separately . No transcripts comprising the entire hbpCAD cluster were detected, indicating that transcription from P(hbpC) is terminated after the hbpA gene . E . coli mutant strains lacking the structural genes for the RNA polymerase sigma(54) subunit or for the integration host factor failed to express bioluminescence from P(hbpC)- and P(hbpD)-luxAB fusions when a functional hbpR gene was provided in trans . This pointed to the active role of sigma(54) and integration host factor in transcriptional activation from these promoters . Primer extension analysis revealed that both P(hbpC) and P(hbpD) contain the typical motifs at position -24 (GG) and -12 (GC) found in sigma(54)-dependent promoters . Analysis of changes in the synthesis of the hbp mRNAs, in activities of the 2-HBP pathway enzymes, and in concentrations of 2-HBP intermediates during the first 4 h after induction of continuously grown P . azelaica cells with 2-HBP demonstrated that the specific transcriptional organization of the hbp genes ensured smooth pathway expression.

J Bacteriol, 2001 Jan, 183(1), 178 - 88
pepA, a gene mediating pH regulation of virulence genes in Vibrio cholerae; Behari J et al.; ToxT, a member of the AraC family of transcriptional regulators, controls the expression of several virulence factors in Vibrio cholerae . In the classical biotype of V . cholerae, expression of toxT is regulated by the same environmental conditions that control expression of the virulence determinants cholera toxin and the toxin coregulated pilus . Several genes that activate toxT expression have been identified . To identify genes that repress toxT expression in nonpermissive environmental conditions, a genetic screen was used to isolate mutations which alter the expression of a toxT-gusA transcriptional fusion . Several mutants were isolated, and the mutants could be divided into two classes . One class of mutants exhibited higher expression levels of toxT-gusA at both the nonpermissive pH and temperature, while the second class showed elevated toxT-gusA expression only at the nonpermissive pH . One mutant from the second class was chosen for further characterization . This mutant was found to carry a TnphoA insertion in a homolog of the Escherichia coli pepA gene . Disruption of pepA in V . cholerae resulted in elevated levels of expression of cholera toxin, tcpA, toxT, and tcpP at the noninducing pH but not at the noninducing temperature . Elevated levels of expression of toxT and tcpP at the nonpermissive pH in the pepA mutant were abolished in tcpP toxR mutant and aphB mutant backgrounds, respectively . A putative binding site for PepA was identified in the tcpPH-tcpI intergenic region, suggesting that PepA may act at the level of tcpPH transcription . Disruption of pepA caused only partial deregulation at the noninducing pH, suggesting the involvement of additional factors in the pH regulation of virulence genes in V . cholerae.

Biochemistry, 2000 Dec 19, 39(50), 15522 - 30
Reconstitution and characterization of the Vibrio cholerae vibriobactin synthetase from VibB, VibE, VibF, and VibH; Keating TA et al.; Vibriobactin {N(1)-(2,3-dihydroxybenzoyl)-N(5),N(9)-bis{2-(2, 3-dihydroxyphenyl)-5-methyloxazolinyl-4-carboxamido}norspermidine} , is an iron chelator from the cholera-causing bacterium Vibrio cholerae . The six-domain, 270 kDa nonribosomal peptide synthetase (NRPS) VibF, a component of vibriobactin synthetase, has been heterologously expressed in Escherichia coli and purified . VibF has an unusual NRPS domain organization: cyclization-cyclization-adenylation-condensation-peptidyl carrier protein-condensation (Cy(1)-Cy(2)-A-C(1)-PCP-C(2)) . VibF activates and covalently loads its PCP with L-threonine, and together with vibriobactin synthetase proteins VibE (adenylation) and VibB (aryl carrier protein) condenses and heterocyclizes 2, 3-dihydroxybenzoyl-VibB with L-Thr to 2-dihydroxyphenyl-5-methyloxazolinyl-4-carboxy-VibF in the first demonstration of oxazoline formation by an NRPS cyclization domain . This enzyme-bound aryl oxazoline can be transferred by VibF to various amine acceptors but most efficiently to N(1)-(2, 3-dihydroxybenzoyl)norspermidine (k(cat) = 122 min(-1), K(m) = 1.7 microM), the product of 2,3-dihydroxybenzoyl-VibB, norspermidine, and VibH . This diacylated product undergoes a second aryl oxazoline acylation on its remaining secondary amine, also catalyzed by VibF, to yield vibriobactin . Vibriobactin biosynthesis in vitro has thus been accomplished from four proteins, VibE, VibB, VibF, and VibH, with the substrates 2,3-dihydroxybenzoic acid, L-Thr, norspermidine, and ATP . Vibriobactin synthetase is an unusual NRPS in that all intermediates are not covalently tethered as PCP thioesters and in that it represents an NRPS pathway with two branch points.

Biochemistry, 2000 Dec 19, 39(50), 15513 - 21
Vibriobactin biosynthesis in Vibrio cholerae: VibH is an amide synthase homologous to nonribosomal peptide synthetase condensation domains; Keating TA et al.; The Vibrio cholerae siderophore vibriobactin is biosynthesized from three molecules of 2,3-dihydroxybenzoate (DHB), two molecules of L-threonine, and one of norspermidine . Of the four genes positively implicated in vibriobactin biosynthesis, we have here expressed, purified, and assayed the products of three: vibE, vibB, and vibH . All three are homologous to nonribosomal peptide synthetase (NRPS) domains: VibE is a 2,3-dihydroxybenzoate-adenosyl monophosphate ligase, VibB is a bifunctional isochorismate lyase-aryl carrier protein (ArCP), and VibH is a novel amide synthase that represents a free-standing condensation (C) domain . VibE and VibB are homologous to EntE and EntB from Escherichia coli enterobactin synthetase; VibE activates DHB as the acyl adenylate and then transfers it to the free thiol of the phosphopantetheine arm of VibB's ArCP domain . VibH then condenses this DHB thioester (the donor) with the small molecule norspermidine (the acceptor), forming N(1)-(2, 3-dihydroxybenzoyl)norspermidine (DHB-NSPD) with a k(cat) of 600 min(-1) and a K(m) for acyl-VibB of 0.88 microM and for norspermidine of 1.5 mM . Exclusive monoacylation of a primary amine of norspermidine was observed . VibH also tolerates DHB-acylated EntB and 1,7-diaminoheptane, octylamine, and hexylamine as substrates, albeit at lowered catalytic efficiencies . DHB-NSPD possesses one of three acylations required for mature vibriobactin, and its formation confirms VibH's role in vibriobactin biosynthesis . VibH is a unique NRPS condensation domain that acts upon an upstream carrier-protein-bound donor and a downstream amine, turning over a soluble amide product, in contrast to an archetypal NRPS-embedded C domain that condenses two carrier protein thioesters.

Int J Med Microbiol, 2000 Oct, 290(4-5), 345 - 50
Characterization of the multimeric Eps complex required for cholera toxin secretion; Sandkvist M et al.; Vibrio cholerae causes diarrheal disease through colonization of the small intestine . A critical aspect of V . cholerae pathogenesis is its ability to actively secrete cholera toxin to the extracellular environment . This occurs via the type II secretion pathway, where the toxin subunits are first transported to the periplasm through the Sec pathway . Following folding and assembly the toxin is then translocated across the outer membrane by a specialized Extracellular Protein Secretion (Eps) machinery encoded by at least 13 genes . Although the Eps proteins are believed to form a secretion apparatus that spans both membranes, cholera toxin is thought to engage this complex first in the periplasm . In order to determine the organization of the Eps apparatus and to understand the mechanism of secretion, the Eps apparatus has been dissected and three of the components, EpsE, EpsL and EpsM, have been purified and characterized . They were shown to form a stable, multiprotein complex spanning the cytoplasmic membrane.

Int J Hyg Environ Health, 2000 Oct, 203(2), 169 - 75
Isolation of Vibrio alginolyticus from seawater aquaria; Hormansdorfer S et al.; The seawater bacterium Vibrio alginolyticus was detected in 5 of 20 water samples from seawater aquaria (from 3 of 5 units) and also from the surface of diseased stony corals . A total of 45 isolates were differentiated biochemically, of which 13 isolates (29%) proved to be V . alginolyticus . All those strains produced the virulence factors caseinase and lipase, 11 strains amylase and gelatinase . 7 strains showed lecithinase activity and 2 strains produced hemolysins . All examined strains showed a marked toxicity to vero cells proven by the MTT-bioassay, but no toxicity to plant cells with the saline alga Asteromonas gracilis as model . The isolates were mostly resistant to beta-lactam antibiotics, macrolides and lincomycin . However, they proved to be susceptible to aminoglycoside- and polypeptide-antibiotics as well as to tetracyclines, chloramphenicol, florfenicol, enrofloxacin and sulfamethoxazol-trimethoprim . The possible participation of this bacterium in the bleaching and dying of stony corals is mentioned as well as its role as human pathogen.

Int Microbiol, 2000 Mar, 3(1), 51 - 3
Identification of Vibrio spp . (other than V . vulnificus) recovered on CPC agar from marine natural samples; Macian MC et al.; Two hundred and eighty four presumptive but not confirmed Vibrio vulnificus isolates grown on cellobiose-polymixin B-colistin agar (CPC) at 40 degrees C, recovered from sea water samples from Valencia, Spain, during a microbiological survey for V . vulnificus, were phenotypically identified . Most of the isolates (91%) corresponded to Vibrio species . V . harveyi (24%) and V . splendidus(19%) were the most abundant species identified, followed by V . navarrensis (13%), V . alginolyticus (8%) and V . parahaemolyticus (5%) . The ability to grow on CPC agar and ferment cellobiose of several V . vulnificus strains from different origins and serovars, including reference strains, was tested . Most serovar E isolates and 25% of non-serovar E isolates could not grow on CPC agar.

Syst Appl Microbiol, 2000 Oct, 23(3), 409 - 17
Ribotyping of vibrio populations associated with cultured oysters (Ostrea edulis); Macian MC et al.; The intraspecific variability of Vibrio splendidus, V . harveyi and V . tubiashii recovered from oysters (Ostrea edulis) collected at the Mediterranean coast near Valencia, Spain, was analyzed by ribotyping . The two former species represented the most abundant ones, and the third one was the only species described as pathogenic for oysters . A total of 115 environmental strains were studied, 84 of V . splendidus, 23 of V . harveyi and 8 of V . tubiashii . Chromosomal DNA was digested with KpnI and hybridized with an oligonucleotide probe complementary to a highly conserved sequence in the 23S rRNA gene . Ribotyping among natural populations of the three species rendered 5 to 9 bands, and showed a high genetic diversity, with a ratio no . of strains/no . of ribotypes between 1.1 and 1.5 . Cluster analysis of V . splendidus ribotypes suggests a seasonal pattern of incidence, with those ribotypes corresponding to winter and spring samples being maintained in the oysters over the year.

Syst Appl Microbiol, 2000 Oct, 23(3), 373 - 5
Vibrio pelagius: differences of the type strain deposited at various culture collections; Macian MC et al.; A critical evaluation of published and own taxonomic and phylogenetic studies on Vibrio pelagius showed substantial diversity of strains received as type strains from various Culture Collections . The comparison of data based upon 16S rRNA sequence analyses, earlier genomic DNA-DNA similarity studies as well as physiological investigations and the original description indicate that Vibrio pelagius strains CECT 4202T and ATCC 25916T really represent the originally described type species whereas strains NCIMB 1900T and CIP 102762T highly likely are representatives of Vibrio natriegens.

Rev Cubana Med Trop, 2000 May-Aug, 52(2), 106 - 9
{Toxigenic Vibrio cholerae non-01}; Bravo L et al.; The antimicrobial susceptibility and the presence of a heat-stable toxin were researched into 100 non-01 Vibrio cholerae strains sent by 7 different health centers to the National Reference Laboratory of Acute Diarrheal Diseases in "Pedro Kouri" Tropical Medicine Institute . The presence of 20% toxigenic non-01 Vibrio cholerae was detected, a figure substantially higher than that reported in other geographic areas, except for endemic areas . This result will make it possible to set epidemiological alert in Cuba because these strains can be infected by CTX phages (element transporting genes that encode for choleric toxin) which will give such strains an epidemic potential similar to that of the etiologic agent of cholera . The identified strains could be studied as possible cholera vaccine candidates.

Med Dosw Mikrobiol, 2000, 52(2), 139 - 50
{Evaluation of cytotoxic activity of Vibrio cholerae non-01 culture filtrate on established cell lines and human diploid cells}; Pancer K et al.; The cell-destroying effect of cell free filtrates of 90 V . cholerae non-01 cultures was measured by titration method in 3 established cell lines: CHO, HeLa and Vero and in 3 human diploid cells cultures: MRC-5, WI-38 and PZ . The vibrio strains differed in the titre of toxic effect . Most sensitive was CHO cell line, least sensitive were human diploid cell cultures . It was found that bacterial strains produced different substances toxic for various cell lines . Among them NAG-ST toxin produced by 41% of examined strains was identified and hemolysins/cytolysins activity was evaluated . Both may play a role in the pathogenicity of those strains for humans.

J Clin Microbiol, 2000 Dec, 38(12), 4621 - 5
Vibrio parahaemolyticus serovar O3:K6 as cause of unusually high incidence of food-borne disease outbreaks in Taiwan from 1996 to 1999; Chiou CS et al.; The occurrence of food-borne disease outbreaks in Taiwan increased dramatically in 1996, and the incidence has since remained elevated . This increase in outbreaks is correlated with a high rate of isolation of Vibrio parahaemolyticus, which caused between 61 and 71% of the total outbreaks for the period 1996 to 1999 . By serotyping, 40 serovars were identified from 3743 V . parahaemolyticus isolates, of which O3:K6 was the most frequently detected . The O3:K6 serovar could have emerged in Taiwan as early as October 1995 and at that time accounted for only 0.6% of the V . parahaemolyticus infections . This level increased suddenly to 50.1% in 1996 and reached a peak (83.8%) in 1997 . Comparison of the outbreak profiles for the etiology groups indicates that the high incidence of food-borne disease outbreaks during 1996 to 1999 can be attributed to the extraordinarily high O3:K6 infections . In 1999, the O3:K6 serovar was still prevalent, and accounted for 61.3% of all V . parahaemolyticus infections . Due to its extraordinarily high infection frequency and its capability to spread globally, this organism needs to be intensively monitored internationally.

Braz J Infect Dis, 1999 Apr, 3(2), 31 - 49
Tourism and Emerging and Re-emerging Infectious Diseases in the Americas: What Physicians Must Remember for Patient Diagnosis and Care; Schmunis GA et al.; Emerging diseases are those which have shown an increased in humans over the last 20 years . Re-emerging diseases are those which have reappeared after a period of significant decrease in incidence . The etiological agents of these diseases in the Western Hemisphere are viruses (HIV, dengue, oroupuche, sabia, guanarito, or hanta), bacteria (Vibrio cholera, Borrellia burgdorferi, Legionella pneumofila, Eseherichia coli 0157:H7, or other bacteria with a new pattern of antibiotic resistance), or parasites (Cryptosporidia, Cyclosporidia or drug resistant Plasmodium falciparum) . Due to the widespread geographical distribution of these infectious diseases in the Americas, and an increasing number of travellers (more than 87 million persons within the region in 1997), there are many opportunities to contract an infection when travelling in developed or undeveloped countries . The infection may present with symptoms during the trip, or following the traveler s return to his or her place of origin . However, too often practicing physicians do not inquire about the travel history of their patients and, when they do, they often lack the information about diseases relevant to travelers . From the regional perspective, the emerging or reemerging agents that pose a higher risk to tourists or travelers are: 1) those that cause enteric infections; 2) sexually transmitted diseases; and 3) vector-borne diseases, including those present in ecotourism areas . Emerging and re-emerging diseases that physicians may encounter in their clinical practice while caring for travelers returning from different countries of the Western Hemisphere are briefly described (Lyme disease, legionellosis, dengue, yellow fever, P . falciparum malaria, cyclosporidiosis and cryptosporidiosis) . This report attempts to draw attention to the fact that new clinical and etiological entities are present in several geographical areas of the Americas; to place each of these entities into an epidemiological context; and to end the misconception that only travel to poor countries carries a risk of acquiring an infection . By knowing which infectious agents occur in each area and the incubation period of each disease, the treating physician can often treat patients successfully . Health care professionals must be aware of the organisms circulating in the region so that they have them in mind during their clinical practice.

Indian J Med Res, 2000 Sep, 112, 78 - 85
Cluster-analysis & patterns of dissemination of multidrug resistance among clinical strains of Vibrio cholerae in Calcutta, India; Ramamurthy T et al.; BACKGROUND & OBJECTIVES: Antimicrobial resistance among Vibrio cholerae has been monitored for several years in Calcutta . To investigate the changing trends in multidrug resistance (MDR) among different serogroups of V . cholerae and to perform software assisted cluster analysis the current study was undertaken . METHODS: Strains isolated from patients with cholera and "cholera-like" diarrhoea admitted in the Infectious Diseases Hospital, Calcutta were analysed . Eight hundred and forty V . cholerae strains isolated from 1992 through 1997 were tested for susceptibility to 11 antibiotics . Cluster analysis was done using SPSS software . RESULTS: Most of the strains exhibited MDR with fluctuating trends as the resistance profile diverged each year . A total of 119 different resistance profiles exhibited by V . cholerae O1, O139 and non-O1, non-O139 serogroups were analysed by cluster combination method . During 1993 and 1994, 53 per cent of V . cholerae O139 and 82 per cent of V . cholerae O1 serogroups, respectively, exhibited maximal number of new resistance patterns . The frequency of new resistance patterns among V . cholerae non-O1, non-O139 was constantly high (33-47%) during 1995 to 1997 . INTERPRETATION & CONCLUSIONS: With a few exceptions, preponderance of the resistance profiles was generally not confined to any serogroup . The cluster analysis depicted dissemination of some of the resistance patterns commonly found among V . cholerae non-O1, non-O139 belonging to different serogroups to the O139 serogroup in the succeeding years . In this study we have shown that the V . cholerae strains are resistant to several antibiotics with constant change in the MDR profiles . It is imperative to define the susceptibility pattern of the strains to determine the effective drug of choice for the treatment of cholera.

Int J Cancer, 2000 Dec 15, 88(6), 866 - 72
Monoclonal antibody DS6 detects a tumor-associated sialoglycotope expressed on human serous ovarian carcinomas; Kearse KP et al.; A newly developed murine monoclonal antibody, DS6, immunohistochemically reacts with an antigen, CA6, that is expressed by human serous ovarian carcinomas but not by normal ovarian surface epithelium or mesothelium . CA6 has a limited distribution in normal adult tissues and is most characteristically detected in fallopian tube epithelium, inner urothelium and type 2 pneumocytes . Pre-treatment of tissue sections with either periodic acid or neuraminidase from Vibrio cholerae abolishes immunoreactivity with DS6, indicating that CA6 is a neuraminidase-sensitive and periodic acid-sensitive sialic acid glycoconjugate ("sialoglycotope") . SDS-PAGE of OVCAR5 cell lysates has revealed that the CA6 epitope is expressed on an 80 kDa non-disulfide-linked glycoprotein containing N-linked oligosaccharides . Two-dimensional non-equilibrium pH gradient gel electrophoresis indicates an isoelectric point of approximately 6.2 to 6.5 . Comparison of the immunohistochemical distribution of CA6 in human serous ovarian adenocarcinomas has revealed similarities to that of CA125; however, distinct differences and some complementarity of antigen expression were revealed by double-label, 2-color immunohistochemical studies . The DS6-detected CA6 antigen appears to be distinct from other well-characterized tumor-associated antigens, including MUC1, CA125 and the histo-blood group-related antigens sLea, sLex and sTn .

J Bacteriol, 2000 Dec, 182(24), 6992 - 8
CTX prophages in classical biotype Vibrio cholerae: functional phage genes but dysfunctional phage genomes; Davis BM et al.; CTXphi is a filamentous, lysogenic bacteriophage whose genome encodes cholera toxin, the primary virulence factor produced by Vibrio cholerae . CTX prophages in O1 El Tor and O139 strains of V . cholerae are found within arrays of genetically related elements integrated at a single locus within the V . cholerae large chromosome . The prophages of O1 El Tor and O139 strains generally yield infectious CTXphi . In contrast, O1 classical strains of V . cholerae do not produce CTXphi, although they produce cholera toxin and they contain CTX prophages integrated at two sites . We have identified the second site of CTX prophage integration in O1 classical strains and characterized the classical prophage arrays genetically and functionally . The genes of classical prophages encode functional forms of all of the proteins needed for production of CTXphi . Classical CTX prophages are present either as solitary prophages or as arrays of two truncated, fused prophages . RS1, a genetic element that is closely related to CTXphi and is often interspersed with CTX prophages in El Tor strains, was not detected in classical V . cholerae . Our model for CTXphi production predicts that the CTX prophage arrangements in classical strains will not yield extrachromosomal CTX DNA and thus will not yield virions, and our experimental results confirm this prediction . Thus, failure of O1 classical strains of V . cholerae to produce CTXphi is due to overall deficiencies in the structures of the arrays of classical prophages, rather than to mutations affecting individual CTX prophage genes.

J Bacteriol, 2000 Dec, 182(24), 6964 - 74
Evidence for a role of rpoE in stressed and unstressed cells of marine Vibrio angustum strain S14; Hild E et al.; We report the cloning, sequencing, and characterization of the rpoE homolog in Vibrio angustum S14 . The rpoE gene encodes a protein with a predicted molecular mass of 19.4 kDa and has been demonstrated to be present as a single-copy gene by Southern blot analysis . The deduced amino acid sequence of RpoE is most similar to that of the RpoE homolog of Sphingomonas aromaticivorans, sigma(24), displaying sequence similarity and identity of 63 and 43%, respectively . Northern blot analysis demonstrated the induction of rpoE 6, 12, and 40 min after a temperature shift to 40 degrees C . An rpoE mutant was constructed by gene disruption . There was no difference in viability during logarithmic growth, stationary phase, or carbon starvation between the wild type and the rpoE mutant strain . In contrast, survival of the mutant was impaired following heat shock during exponential growth, as well as after oxidative stress at 24 h of carbon starvation . The mutant exhibited microcolony formation during optimal growth temperatures (22 to 30 degrees C), and cell area measurements revealed an increase in cell volume of the mutant during growth at 30 degrees C, compared to the wild-type strain . Moreover, outer membrane and periplasmic space protein analysis demonstrated many alterations in the protein profiles for the mutant during growth and carbon starvation, as well as following oxidative stress, in comparison with the wild-type strain . It is thereby concluded that RpoE has an extracytoplasmic function and mediates a range of specific responses in stressed as well as unstressed cells of V . angustum S14.

Microbiol Immunol, 2000, 44(9), 787 - 98
Production of serine protease of Aeromonas sobria is controlled by the protein encoded by the gene lying adjacent to the 3' end of the protease gene; Okamoto K et al.; We cloned a protease gene of Aeromonas sobria and determined its nucleotide sequence . The protease is composed of 624 amino acid residues and its calculated molecular weight is 66,737.7 . The amino acid sequence showed the characteristic features of a bacterial serine protease . We expressed the protease gene in Vibrio parahaemolyticus from which the synthesized protease is secreted into the culture medium as the mature form, and purified the mature protease by successive column chromatographies . The size of the mature protease is 65,000 daltons and the amino acid sequence analysis revealed that a 24-amino acid peptide at the amino terminal of the precursor is removed from the mature protease . This peptide might function as a signal peptide in translocation across the inner membrane . Subsequently, we found that the protein, designated ORF2 protein, encoded by the gene lying adjacent to the 3' end of the protease gene plays an important role in production of the protease . Mutation of the ORF2 gene did not affect transcription of the protease gene, but resulted in degradation of the protease in the cell . This shows that ORF2 protein is required for the successful production of the serine protease by cell.

Food Addit Contam, 2000 Sep, 17(9), 787 - 91
Low temperature pasteurization to reduce the risk of vibrio infections from raw shell-stock oysters; Andrews LS et al.; Vibrio vulnificus and V . parahaemolyticus are natural inhabitants of estuarine environments and may be transmitted to humans by ingestion of raw oysters . This study focused on the use of low temperature pasteurization, to reduce these Vibrio spp . to nondetectable levels, thus reducing the risk of infection associated with raw oyster consumption . Artificially-inoculated V . vulnificus and V . parahaemolyticus and naturally-contaminated V . vulnificus in live oysters were pasteurized at 50 degrees C for up to 15 min . Samples of processed and unprocessed oysters were enumerated for V . vulnificus, V . parahaemolyticus, and aerobic spoilage bacteria for 0-14 days . Low temperature pasteurization was effective in reducing these pathogens from > 100,000 to non-detectable levels in less than 10 min of processing . Spoilage bacteria were reduced by 2-3 logs, thus increasing the shelf-life for up to 7 days beyond live unprocessed oysters . Vibrio vulnificus in control oysters was reduced by 10(2) during ice storage alone . Following pasteurization and during a temperature storage abuse study (24 h at 22 degrees C), V . vulnificus was not recovered . During this storage period spoilage bacteria exceeded 1 million/g oyster meat.

Biochemistry, 2000 Nov 28, 39(47), 14409 - 18
A histidine residue in the catalytic mechanism distinguishes Vibrio harveyi aldehyde dehydrogenase from other members of the aldehyde dehydrogenase superfamily; Zhang L et al.; Aldehyde dehydrogenases (ALDHs) catalyze the transfer to NAD(P) of a hydride ion from a thiohemiacetal derivative of the aldehyde coupled with a cysteine residue in the active site . In Vibrio harveyi aldehyde dehydrogenase (Vh-ALDH), a histidine residue (H450) is in proximity (3.8 A) to the cysteine nucleophile (C289) and is thus capable of increasing its reactivity in sharp contrast to other ALDHs in which more distantly located glutamic acid residues are proposed to act as the general base . Mutation of H450 in Vh-ALDH to Gln and Asn resulted in loss of dehydrogenase, (thio)esterase, and acyl-CoA reductase activities; the residual activity of H450Q was higher than that of the H450N mutant in agreement with the capability of Gln but not Asn to partially replace the epsilon-imino group of H450 . Coupled with a change in the rate-limiting step, these results indicate that H450 increases the reactivity of C289 . Moreover, for the first time, the acylated enzyme intermediate could be directly monitored after reaction with {(3)H}tetradecanoyl-CoA showing that the H450Q mutant was acylated more rapidly than the H450N mutant . Inactivation of the wild-type enzyme with N-ethylmaleimide was much more rapid than the H450Q mutant which in turn was faster than the H450N mutant, demonstrating directly that the nucleophilicity of C289 was affected by H450 . As the glutamic acid residue implicated as the general base in promoting cysteine nucleophilicity in other ALDHs is conserved in Vh-ALDH, elucidation of why a histidine residue has evolved to assist in this function in Vh-ALDH will be important to understand the mechanism of ALDHs in general, as well as help delineate the specific roles of the active site glutamic acid residues.

Infect Immun, 2000 Dec, 68(12), 7180 - 5
Phylogeny of Vibrio cholerae based on recA sequence; Stine OC et al.; We sequenced a 705-bp fragment of the recA gene from 113 Vibrio cholerae strains and closely related species . One hundred eighty-seven nucleotides were phylogenetically informative, 55 were phylogenetically uninformative, and 463 were invariant . Not unexpectedly, Vibrio parahaemolyticus and Vibrio vulnificus strains formed out-groups; we also identified isolates which resembled V . cholerae biochemically but which did not cluster with V . cholerae . In many instances, V . cholerae serogroup designations did not correlate with phylogeny, as reflected by recA sequence divergence . This observation is consistent with the idea that there is horizontal transfer of O-antigen biosynthesis genes among V . cholerae strains.

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