Vibriosis

Biochem Biophys Res Commun, 2003 Feb 28, 302(1), 35 - 40
Glutamate-41 of Vibrio harveyi acyl carrier protein is essential for fatty acid synthase but not acyl-ACP synthetase activity; Gong H et al.; Bacterial acyl carrier protein (ACP) is a small, acidic, and highly conserved protein that supplies acyl groups for biosynthesis of a variety of lipid products . Recent modelling studies predict that residues primarily in helix II of Escherichia coli ACP (Glu-41, Ala-45) are involved in its interaction with the condensing enzyme FabH of fatty acid synthase . Using recombinant Vibrio harveyi ACP as a template for site-directed mutagenesis, we have shown that an acidic residue at position 41 is essential for V . harveyi fatty acid synthase (but not acyl-ACP synthetase) activity . In contrast, various replacements of Ala-45 were tolerated by both enzymes . None of the mutations introduced dramatic structural changes based on circular dichroism and native gel electrophoresis . These results confirm that Glu-41 of ACP is a critical residue for fatty acid synthase, but not for all enzymes that utilize ACP as a substrate.

Dev Comp Immunol, 2003 Mar, 27(3), 197 - 205
Nitric oxide production by carpet shell clam (Ruditapes decussatus) hemocytes; Tafalla C et al.; We have demonstrated that carpet shell clam (Ruditapes decussatus) hemocytes produce nitric oxide (NO) in response to zymosan or bacterial lipopolysaccharide (LPS) . This NO production was partially inhibited by the NO synthase inhibitor, N-omega-nitro-L-arginine (L-NAME) . The capability of clam hemocytes to produce NO in response to the bacterial pathogen Vibrio tapetis was also studied . Incubation with bacteria induced a significant NO production by clam hemocytes, even though exogenous NO only slightly decreased the growth of V . tapetis.The effect of exogenous NO on the capability of clam hemocytes to phagocytose labeled Escherichia coli was studied using two different NO donors S-nitroso-N-acetyl-penicillamine (SNAP), and diethylenetriamine NO adduct (DETA/NO) . Exogenous NO did not increase hemocyte phagocytosis, indicating that NO does not mediate phagocytosis in this species . These results are in accordance to those observed in other mollusk species, in which NO was independent of phagocytosis and constitutes an alternative method of killing invading pathogens.

Biochem Biophys Res Commun, 2003 Feb 21, 301(4), 1093 - 8
General function of N-terminal propeptide on assisting protein folding and inhibiting catalytic activity based on observations with a chimeric thermolysin-like protease; Tang B et al.; Pro-aminopeptidase processing protease (PA protease) is a thermolysin-like metalloprotease produced by Aeromonas caviae T-64 . The N-terminal propeptide acts as an intramolecular chaperone to assist the folding of PA protease and shows inhibitory activity toward its cognate mature enzyme . Moreover, the N-terminal propeptide strongly inhibits the autoprocessing of the C-terminal propeptide by forming a complex with the folded intermediate pro-PA protease containing the C-terminal propeptide (MC) . In order to investigate the structural determinants within the N-terminal propeptide that play a role in the folding, processing, and enzyme inhibition of PA protease, we constructed a chimeric pro-PA protease by replacing the N-terminal propeptide with that of vibriolysin, a homologue of PA protease . Our results indicated that, although the N-terminal propeptide of vibriolysin shares only 36% identity with that of PA protease, it assists the refolding of MC, inhibits the folded MC to process its C-terminal propeptide, and shows a stronger inhibitory activity toward the mature PA protease than that of PA protease . These results suggest that the N-terminal propeptide domains in these thermolysin-like proteases may have similar functions, in spite of their primary sequence diversity . In addition, the conserved regions in the N-terminal propeptides of PA protease and vibriolysin may be essential for the functions of the N-terminal propeptide.

Klin Lab Diagn, 2002 Dec, (12), 50 - 1
{Obtaining diagnostic fluorescent monoclonal immunoglobulins against the V . cholerae 0139-serovar}; Alekseeva LP et al.; Below is given a procedure of the obtaining diagnostic fluorescent monoclonal immunoglobulin to detect cholera vibrios of O139 serovar . While obtaining preparations it was managed to determine optimal FTTS-MKA ratio, duration of their conjugation, series of fluorochrome . Test specimens of fluorescent monoclonal immunoglobulin provides intensive glow of V cholerae O139 cells in the working dilution 1:16-1:32 . Tests of diagnostic FTTS-MKA on the great number of homologic and heterologic strains showed their strict specificity and high sensibility as to cholera vibrios of O139 serogroup.

Syst Appl Microbiol, 2002 Dec, 25(4), 544 - 50
Comparison of ribotyping, randomly amplified polymorphic DNA, and pulsed-field gel electrophoresis for molecular typing of Vibrio tapetis; Romalde JL et al.; Brown ring disease, caused by Vibrio tapetis, is an important pathological problem in different species of cultured clams . In order to evaluate the genetic diversity of the pathogen, twenty-seven isolates of V tapetis with different origin were screened by ribotyping (RT), pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA PCR (RAPD) . Restriction with PvuII, SalI, and SmaI gave 2 RT patterns, differentiating in all cases the strain 0202RD isolated from carpet-shell clams (Ruditapes decussatus) from the other strains tested . The use of NotI generated strain specific PFGE profiles, which could be grouped in two main clusters . Cluster 1 grouped all but one strain and was subdivided into six PFGE subtypes (1a to 1f) which joined at a similarity level of 75.6% . Cluster 2 included again only the isolate 0202RD . RAPD analysis yielded the same results with three different primers, this method being able to differentiate the isolates from R . decussatus from those isolated from other clam species . Of the three techniques evaluated, PFGE was the most discriminating of the three techniques evaluated, followed in discriminating power by RAPD and RT tests . On the basis of the results obtained, we conclude that the RAPD procedure, which is more rapid and easier to perform than the other techniques, shows to be very useful to analyze large amounts of strain collections from an epidemiological monitoring stanpoint . In addition, PFGE is of great utility to evaluate the genetic diversity of strains involved in an outbreak and to study the spreading of a specific clone.

J Invertebr Pathol, 2003 Jan, 82(1), 23 - 33
Response of Penaeus indicus females at two different stages of ovarian development to a lethal infection with Vibrio penaeicida; Avarre JC et al.; An association between vitellogenesis and the immune system was suggested in crustaceans from studies on plasma lipoproteins . The present research studies the effect of an experimentally induced bacterial infection on vitellogenesis in females of the shrimp Penaeus indicus, as a model for penaeid species . Pre-vitellogenic and vitellogenic P . indicus females were experimentally infected with an extremely pathogenic bacterium, Vibrio penaeicida . The peak in mortality occurred earlier in pre-vitellogenic animals than in vitellogenic ones, although the final mortality level ( approximately 64-74%) 52h post-infection was nearly the same for the two groups . Twenty hours after infection, the total number of haemocytes was significantly reduced in vitellogenic females while there was no change in the pre-vitellogenic group . Protein synthesis in ovaries was not significantly affected by infection, at the two stages of ovarian development . No differences were found in mRNA levels of shrimp ovarian peritrophin protein (SOP), but preliminary results showed that mRNA expression of vitellin (VT) was reduced in a heavily infected vitellogenic female . The total amount of lipids in the haemolymph of vitellogenic females was almost twice higher than that of pre-vitellogenic ones . However, there was no change in the total content of lipids, lipid classes and fatty acid distribution in haemolymph or hepatopancreas following infection . Although vitellogenic and pre-vitellogenic females probably respond differently to a lethal bacterial infection, physiological differences may be concealed by the rapid onset of mortality.

Lett Appl Microbiol, 2003, 36(3), 150 - 1
A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments; Pfeffer C et al.; AIMS: The purpose of this study was to compare a recently described medium, thiosulphate-chloride-iodide (TCI), for the isolation of estuarine vibrios with thiosulphate-citrate-bile salts-sucrose (TCBS) . METHODS: A total of 492 colonies which developed on these media from estuarine water samples taken monthly over a 10-month period were examined . RESULTS: A much larger number of colonies developed on TCBS than TCI, and minimal taxonomic criteria indicated that a higher percentage (61%) of TCBS colonies could be identified as Vibrio spp . when compared with TCI (46%) . SIGNIFICANCE: This study suggests that TCBS is a superior medium when compared with TCI for the isolation of Vibrio spp . from estuarine waters . Because of the public health risk presented by V . vulnificus, V . parahaemolyticus, V . cholerae and other vibrios, the selection of the most appropriate medium for their isolation is extremely important.

Microbiology, 2003 Jan, 149(Pt 1), 89 - 97
Genesis of variants of Vibrio cholerae O1 biotype El Tor: role of the CTXphi array and its position in the genome; Nandi S et al.; The gene encoding cholera toxin, the principal virulence factor of Vibrio cholerae, is encoded by a filamentous, lysogenic bacteriophage known as CTXphi . The genome of V . cholerae, the host for CTXphi, consists of two chromosomes, one large and one small . Here, it is shown that localization and array of CTX prophage DNA in either the large or small chromosome of V . cholerae is likely to be one of the reasons for the emergence of O1 biotype El Tor variants isolated just before and after the V . cholerae O139 cholera outbreak in 1992 . Analyses of the organization of the CTX region of the genome of pre-O139 El Tor strains revealed that these strains carry two distinct CTX prophages integrated in the small chromosome in tandem: CTX(ET), the prophage having a conserved NotI site in its repeat sequence segment which seems to be specific for the El Tor strains so far examined, followed by CTX(calc)-like genome, the prophage found in recent O139 clinical isolates from Calcutta . In sharp contrast, in post-O139 El Tor strains only one copy of the CTX(ET) prophage was found to be integrated in the large chromosome . To the authors' knowledge, the presence of CTX prophage in the small chromosome of O1 El Tor strains has not been reported previously . It is also shown that the difference in the CTX copy number and the position of the bacteriophage on the genomes of pre- and post-O139 El Tor strains have an effect on cholera toxin production . While a pre-O139 strain produced maximum cholera toxin in yeast extract/peptone medium at 30 degrees C, a post-O139 El Tor strain showed maximal yield at 37 degrees C, indicating differential regulation of cholera toxin between the strains . It appears from this study that the variation in the integration site of the CTX prophage, its copy number and the presence of diverse phage genomes in V . cholerae O1 biotype El Tor may be strategically important for generating variants with subtle phenotypic modulations of virulence factor production in this longest-ruling seventh pandemic strain.

Biochem J, 2003 May 1, 371(Pt 3), 989 - 95
Biosynthesis of 2-acetamido-2,6-dideoxy-L-hexoses in bacteria follows a pattern distinct from those of the pathways of 6-deoxy-L-hexoses; Kneidinger B et al.; 6-Deoxy-L-hexoses have been shown to be synthesized from dTDP-D-glucose or GDP-D-mannose so that the gluco/galacto-configuration is converted into the manno/talo-configuration, and manno/talo is switched to gluco/galacto . Our laboratory has been investigating the biosynthesis of 2-acetamido-2,6-dideoxy-L-hexoses in both Gram-positive and Gram-negative bacteria, and in a recent paper we described the biosynthesis of the talo (pneumosamine) and galacto (fucosamine) derivatives from UDP-D-N-acetylglucosamine a 2-acetamido sugar {Kneidinger, O'Riordan, Li, Brisson, Lee and Lam (2003) J . Biol . Chem . 278, 3615-3627} . In the present study, we undertake the task to test the hypothesis that UDP-D-N-acetylglucosamine is the common precursor for the production of 2-acetamido-2,6-dideoxy-L-hexoses in the gluco-, galacto-, manno- and talo-configurations . We present data to reveal the steps for the biosynthesis of the gluco (quinovosamine)- and manno (rhamnosamine)-configured compounds . The corresponding enzymes WbvB, WbvR and WbvD from Vibrio cholerae serotype O37 have been overexpressed and purified to near homogeneity . The enzymic reactions have been analysed by capillary electrophoresis and NMR spectroscopy . Our data have revealed a general feature of reaction cascades due to the three enzymes . First, UDP-D-N-acetylglucosamine is catalysed by the multi-functional enzyme WbvB, whereby dehydration occurs at C-4, C-6 and epimerization at C-5, C-3 to produce UDP-2-acetamido-2,6-dideoxy-L-lyxo-4-hexulose . Secondly, this intermediate is converted by the C-4 reductase, WbvR, in a stereospecific reaction to yield UDP-2-acetamido-L-rhamnose . Thirdly, UDP-2-acetamido-L-rhamnose is epimerized at C-2 to UDP-2-acetamido-L-quinovose by WbvD . Interestingly, WbvD is also an orthologue of WbjD, but not vice versa . Incubation of purified WbvD with UDP-2-acetamido-2,6-dideoxy-L-talose and analysing the reaction products by capillary electrophoresis revealed the same product peak as when WbjD was used . This sugar nucleotide is a specific substrate for WbjD and is a C-4 epimer of UDP-2-acetamido-L-rhamnose.

EMBO J, 2003 Feb 17, 22(4), 870 - 81
Vibrio harveyi quorum sensing: a coincidence detector for two autoinducers controls gene expression; Mok KC et al.; In a process called quorum sensing, bacteria communicate with one another by exchanging chemical signals called autoinducers . In the bioluminescent marine bacterium Vibrio harveyi, two different auto inducers (AI-1 and AI-2) regulate light emission . Detection of and response to the V.harveyi autoinducers are accomplished through two two-component sensory relay systems: AI-1 is detected by the sensor LuxN and AI-2 by LuxPQ . Here we further define the V.harveyi quorum-sensing regulon by identifying 10 new quorum-sensing-controlled target genes . Our examination of signal processing and integration in the V.harveyi quorum-sensing circuit suggests that AI-1 and AI-2 act synergistically, and that the V.harveyi quorum-sensing circuit may function exclusively as a 'coincidence detector' that discriminates between conditions in which both autoinducers are present and all other conditions.

Appl Environ Microbiol, 2003 Feb, 69(2), 820 - 6
Contribution of pilA to competitive colonization of the squid Euprymna scolopes by Vibrio fischeri; Stabb EV et al.; Vibrio fischeri colonizes the squid Euprymna scolopes in a mutualistic symbiosis . Hatchling squid lack these bacterial symbionts, and V . fischeri strains must compete to occupy this privileged niche . We cloned a V . fischeri gene, designated pilA, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins . Unlike its closest known relatives, Vibrio cholerae mshA and vcfA, pilA is monocistronic and not clustered with genes associated with pilin export or assembly . Using wild-type strain ES114 as the parent, we generated an in-frame pilA deletion mutant, as well as pilA mutants marked with a kanamycin resistance gene . In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (P < 0.05) but not by an unmarked pilA mutant, for squid colonization . In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing . The competitive defect of pilA mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for V . fischeri populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used . ES114 also outcompeted a pilA mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain . V . fischeri strain ET101, which was isolated from Euprymna tasmanica and is outcompeted by ES114, lacks pilA; however, 11 other diverse V . fischeri isolates apparently possess pilA . The competitive defect of pilA mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria.

J Mol Evol, 2003 Jan, 56(1), 69 - 76
Common 5S rRNA variants are likely to be accepted in many sequence contexts; Zhang Z et al.; Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA . These sequences together comprise the structure space of the RNA . In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not . We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space . One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts . To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position . Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region . All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found . Each variant was evaluated for its ability to function as a valid 5S rRNA in an E . coli cellular context . It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context . In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position . In this case, 86% (6/7) are likely valid . As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space . In this case only two of seven were found to be potentially valid . The results demonstrate that changes that occur multiple times in a local region of RNA sequence space in fact usually will be accepted in any sequence context in that same local region.

Toxicon, 2003 Feb, 41(2), 245 - 9
Studies of the effect of Psi-APONIN from Nannochloris sp . on the Florida red tide organism Karenia brevis; Derby ML et al.; Studies were conducted on the conditions under which the red tide organism, Karenia brevis (a.k.a., Gymnodinium breve), was treated with Nannochloris sp . The latter organism is known to produce cytolytic agents called Apparent Oceanic Naturally Occurring Cytolin (APONINs) . Conventional wisdom might suggest that brevetoxins would be released upon destruction of the single-celled dinoflagellate K . brevis and that efforts to treat red tide outbreaks would lead to release of brevetoxins and enhanced toxicity toward marine species . Earlier studies described conditions by which K . brevis cells were converted to a non-motile form when cultures of K . brevis were treated with an isolate (Psi-APONIN) produced by Nannochloris sp . but when centrifuged only a small amount of the toxin was released . The present study confirms that the toxin is not released when the K . brevis is undisturbed, however, when the culture is stressed (stirred with a magnetic stirring bar for 24, 48, and 72h) toxin was released, and the toxicity could be measured using a Microtox analyzer . In the study, it was found that at as few as eighty cells of K . brevis produced a toxic effect of 20% as measured by the effect on Vibrio fischeri . Nannochloris sp . had no effect on the bacteria used in the Microtox analyzer, nor did interaction of Nannochloris sp . with K . brevis in the short term . This effect is presumed to be due to the production of Psi-APONIN and conversion of K . brevis to a non-motile or resting form.

Trends Microbiol, 2002 Dec, 10(12), 571 - 4
Information overload: assigning genetic functionality in the age of genomics and large-scale screening; Merrell DS et al.; As more and more genome sequences are completed, it is becoming increasingly evident that our understanding of the function of most bacterial gene products is lacking . This is frustrating, particularly in the study of pathogens, where an understanding of the role of individual gene products would probably facilitate the development of novel antimicrobials and vaccines . Recently, we devised a technique known as virulence-attenuated pool (VAP) screening to help assign genetic functionality to gene products that the pathogen Vibrio cholerae requires for colonization . This screen and potential new applications of the VAP technique are discussed here.

Biochemistry, 2003 Feb 11, 42(5), 1334 - 44
Dissection of the EntF condensation domain boundary and active site residues in nonribosomal peptide synthesis; Roche ED et al.; Nonribosomal peptide synthetases (NRPSs) make many natural products of clinical importance, but a deeper understanding of the protein domains that compose NRPS assembly lines is required before these megasynthetases can be effectively engineered to produce novel drugs . The N-terminal amide bond-forming condensation (C) domain of the enterobactin NRPS EntF was excised from the multidomain synthetase using endpoints determined from sequence alignments and secondary structure predictions . The isolated domain was well-folded when compared by circular dichroism to the vibriobactin NRPS VibH, a naturally free-standing C domain . The EntF domain was also fully functional in an assay based on a synthetic small-molecule substrate, seryl N-acetylcysteamine . Active site mutants of the EntF C domain were surprisingly inactive in vitro as compared to their VibH counterparts, yet maintained the overall domain structure . An in vivo assay was developed in the context of the full-length EntF protein to more sensitively probe the activity level of the C domain mutants, and this supported strong effects for the active site mutations . The crucial role of histidine-138 was confirmed by assay of the full-length protein in vitro . These results suggest a strong resemblance of catalysis by the EntF C domain to chloramphenicol acetyltransferase, including an active site organized by an arginine-aspartate salt bridge, a key histidine acting as a general base, and an asparagine instead of a serine stabilizing the proposed tetrahedral intermediate by hydrogen bonding . The precise definition of a functional C domain excised from a NRPS should aid efforts at swapping NRPS domains between assembly lines.

J Bacteriol, 2003 Feb, 185(4), 1289 - 98
KtrAB and KtrCD: two K+ uptake systems in Bacillus subtilis and their role in adaptation to hypertonicity; Holtmann G et al.; Recently, a new type of K+ transporter, Ktr, has been identified in the bacterium Vibrio alginolyticus (T . Nakamura, R . Yuda, T . Unemoto, and E . P . Bakker, J . Bacteriol . 180:3491-3494, 1998) . The Ktr transport system consists of KtrB, an integral membrane subunit, and KtrA, a subunit peripherally bound to the cytoplasmic membrane . The genome sequence of Bacillus subtilis contains two genes for each of these subunits: yuaA (ktrA) and ykqB (ktrC) encode homologues to the V . alginolyticus KtrA protein, and yubG (ktrB) and ykrM (ktrD) encode homologues to the V . alginolyticus KtrB protein . We constructed gene disruption mutations in each of the four B . subtilis ktr genes and used this isogenic set of mutants for K+ uptake experiments . Preliminary K+ transport assays revealed that the KtrAB system has a moderate affinity with a Km value of approximately 1 mM for K+, while KtrCD has a low affinity with a Km value of approximately 10 mM for this ion . A strain defective in both KtrAB and KtrCD exhibited only a residual K+ uptake activity, demonstrating that KtrAB and KtrCD systems are the major K+ transporters of B . subtilis . Northern blot analyses revealed that ktrA and ktrB are cotranscribed as an operon, whereas ktrC and ktrD, which occupy different locations on the B . subtilis chromosome, are expressed as single transcriptional units . The amount of K+ in the environment or the salinity of the growth medium did not influence the amounts of the various ktr transcripts . A strain with a defect in KtrAB is unable to cope with a sudden osmotic upshock, and it exhibits a growth defect at elevated osmolalities which is particularly pronounced when KtrCD is also defective . In the ktrAB strain, the osmotically mediated growth defect was associated with a rapid loss of K+ ions from the cells . Under these conditions, the cells stopped synthesizing proteins but the transcription of the osmotically induced proHJ, opuA, and gsiB genes was not impaired, demonstrating that a high cytoplasmic K+ concentration is not essential for the transcriptional activation of these genes at high osmolarity . Taken together, our data suggest that K+ uptake via KtrAB and KtrCD is an important facet in the cellular defense of B . subtilis against both suddenly imposed and prolonged osmotic stress.

J Bacteriol, 2003 Feb, 185(4), 1236 - 44
Roles of NhaA, NhaB, and NhaD Na+/H+ antiporters in survival of Vibrio cholerae in a saline environment; Herz K et al.; Vibrio cholerae, the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity . In this work, we investigated the role of three Na+/H+ antiporters on the survival of V . cholerae in a saline environment . We have previously cloned the Vc-nhaA gene encoding the V . cholerae homolog of Escherichia coli . Here we identified two additional antiporter genes, designated Vc-nhaB and Vc-nhaD, encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of Vibrio species and E . coli . We showed that both Vc-NhaA and Vc-NhaB confer Na+ resistance and that Vc-NhaA displays an antiport activity in E . coli, which is similar in magnitude, kinetic parameters, and pH regulation to that of E . coli NhaA . To determine the roles of the Na+/H+ antiporters in V . cholerae, we constructed nhaA, nhaB, and nhaD mutants (single, double, and triple mutants) . In contrast to E . coli, the inactivation of the three putative antiporter genes (Vc-nhaABD) in V . cholerae did not alter the bacterial exponential growth in the presence of high Na+ concentrations and had only a slight effect in the stationary phase . In contrast, a pronounced and similar Li+-sensitive phenotype was found with all mutants lacking Vc-nhaA during the exponential phase of growth and also with the triple mutant in the stationary phase of growth . By using 2-n-nonyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the electron-transport-linked Na+ pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na+/H+ antiporter activity becomes essential for the resistance of V . cholerae to Na+ at alkaline pH . Since the ion pump NQR is Na+ specific, we suggest that its activity masks the Na+/H+ but not the Li+/H+ antiporter activities . Our results indicate that the Na+ resistance of the human pathogen V . cholerae requires a complex molecular system involving multiple antiporters and the NQR pump.

J Bacteriol, 2003 Feb, 185(4), 1195 - 207
Analysis of residues determining specificity of Vibrio cholerae TonB1 for its receptors; Mey AR et al.; In gram-negative organisms, high-affinity transport of iron substrates requires energy transduction to specific outer membrane receptors by the TonB-ExbB-ExbD complex . Vibrio cholerae encodes two TonB proteins, one of which, TonB1, recognizes only a subset of V . cholerae TonB-dependent receptors and does not facilitate transport through Escherichia coli receptors . To investigate the receptor specificity exhibited by V . cholerae TonB1, chimeras were created between V . cholerae TonB1 and E . coli TonB . The activities of the chimeric TonB proteins in iron utilization assays demonstrated that the C-terminal one-third of either TonB confers the receptor specificities associated with the full-length TonB . Single-amino-acid substitutions near the C terminus of V . cholerae TonB1 were identified that allowed TonB1 to recognize E . coli receptors and at least one V . cholerae TonB2-dependent receptor . This indicates that the very C-terminal end of V . cholerae TonB1 determines receptor specificity . The regions of the TonB-dependent receptors involved in specificity for a particular TonB protein were investigated in experiments involving domain switching between V . cholerae and E . coli receptors exhibiting different TonB specificities . Switching the conserved TonB box heptapeptides at the N termini of these receptors did not alter their TonB specificities . However, replacing the amino acid immediately preceding the TonB box in E . coli receptors with an aromatic residue allowed these receptors to use V . cholerae TonB1 . Further, site-directed mutagenesis of the TonB box -1 residue in a V . cholerae TonB2-dependent receptor demonstrated that a large hydrophobic amino acid in this position promotes recognition of V . cholerae TonB1 . These data suggest that the TonB box -1 position controls productive interactions with V . cholerae TonB1.

Expert Opin Pharmacother, 2003 Feb, 4(2), 141 - 6
An evaluation of current cholera treatment; Bhattacharya SK; Cholera, caused by Vibrio cholerae O1 and O139, is characterised by profuse purging of watery stools, and vomiting and dehydration . The mainstay of therapy of cholera patients is rehydration with oral rehydration salt solution or intravenous Ringer's lactate depending upon the degree of dehydration . Antibiotics such as tetracycline, doxycycline, norfloxacin, ciprofloxacin and furazolidone may be used as an adjunct to rehydration therapy for severely purging cholera patients to reduce the rate of stool output . This shortens the duration of hospital stay, stops excretion of vibrios in the stool and minimises the requirement of fluids . Resistance to many of these drugs has been observed and is a matter of concern . Other antidiarrhoeals are not recommended . Many antisecretory drugs have been tried as an adjunct therapy, unfortunately, until today, none has been found useful in the treatment of cholera . Feeding during and after cholera is emphasised.

Yi Chuan Xue Bao, 2002 Oct, 29(10), 936 - 40
{Analysis of gene cluster of Tat-dependent protein export system of Vibrio cholerae and its function}; Zhang LJ et al.; The Tat (Twin-arginine translocatin) system is a recently defined protein export pathway that serves to translocate folded proteins . The substrates of the Tat pathway contain specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif-S/T-R-R-x-F-L-X . Here is the report of knocked out the tatA, tatB and tatC genes of the V . cholerae by suicide plasmid homologous recombination technology . Mutant strains showed obvious changes of growth characteristics . The transport of a typical Tat pathway substrate, trimethylamine N-oxide reductase (molybdoenzyme), was completely blocked . The physiochemical reactions of the parent and mutant strains were also analyzed . Four physiochemical reactions using D-galactose, L-asparagine, glyl-L-aspartic acid and D-L-alpha-glycerol phosphate as substrates were negative in mutant strains, which might be affected by the inactivation of the Tat-dependent system.

Sheng Wu Gong Cheng Xue Bao, 2002 Sep, 18(5), 614 - 8
{Biosynthesis and accumulation of poly(3-hydroxybutyrate) in Vibrio natriegens}; Liu SJ; Accumulation of poly(3-hydroxybutyrate) {poly(3HB)} by V . natriegens was studied . Results indicated that V . natriegens used glucose, gluconate, fructose and molasses as carbon sources for poly(3HB) synthesis . When molasses was used, up to 28.4% of poly(3HB) to cellular dry weight was accumulated . The accumulation of poly(3HB) followed, was not simultaneously to, the cell growth . Analysis of the PHA polymerase, beta-ketothiolase, and acetoacetyl-CoA reductase showed that the poly(3HB) accumulation was correlated to the increase of their activities in cells . Poly(3HB) accumulation was also related to the de novo fatty acid synthesis, as revealed by the results that cerulenin, a specific inhibitor to the de novo fatty acid synthesis, significantly reduced accumulation of poly(3HB) . Based on the results from this study, the synthetic pathway of poly(3HB) was proposed.

Vaccine, 2003 Mar 7, 21(11-12), 1282 - 91
Construction and characterisation of O139 cholera vaccine candidates; Ledon T et al.; The hemagglutinin/protease (HA/P) seems to be an attractive locus for the insertion of heterologous tags in live cholera vaccine strains . A deltaCTXphi spontaneous mutant derived from a pathogenic strain of O139 Vibrio cholerae was sequentially manipulated to obtain hapA Colon, two colons celA derivatives which were later improved in their environmental safety by means of a thyA mutation . All the strains here obtained showed similar phenotypes in traits known to be remarkable for live cholera vaccines irrespective of their motility phenotypes, although the hapA mutants had a 10-fold decrease in their colonisation capacity compared with their parental strains in the infant mouse cholera model . However, the subsequent thyA mutation did not affect their colonisation properties in the same model . These preliminary results pave the way for further clinical assays to confirm the possibilities of these vaccine prototypes as safe and effective tools for the prevention of O139 cholera .

Gene, 2003 Jan 16, 303, 147 - 56
Cloning and characterization of a metalloprotease from Vibrio harveyi strain AP6; Teo JW et al.; A metalloprotease gene pap6 was cloned from Vibrio harveyi strain AP6 . Sequence analysis showed that pap6 was 2034 bp in length and predicted to encode a peptide of 677 amino acids with a molecular mass of 75 kDa . SDS-PAGE analysis of the purified Pap6 revealed that it was 35 kDa in size . N-terminal amino acid sequencing established that the mature protein began at Leu-191, suggesting that the preprotein of Pap6 was processed to generate a mature protease . Purified Pap6 was characterized as a zinc metalloprotease as it was inhibited by zinc- and metal-specific inhibitors such as 1, 10-phenanthroline, EGTA and EDTA . The deduced amino acid sequence revealed the presence of a zinc-binding motif HEXXH approximately 19aa approximately E . Substitution of these active site residues by site-directed mutagenesis caused significant losses in enzyme activity, thus demonstrating their involvement in catalysis . Pap6 was shown to digest a range of host proteins, including gelatin, fibronectin, and type IV collagen, indicating a potential role in pathogenesis.

Environ Toxicol Chem, 2003 Feb, 22(2), 285 - 95
Immunocompetence of juvenile Chinook salmon against Listonella anguillarum following dietary exposure to Aroclor 1254; Powell DB et al.; Controlled laboratory challenges with pathogenic Listonella (formerly Vibrio) anguillarum bacteria were used to examine potential effects of dietary exposure to polychlorinated biphenyls (PCBs) on the growth and immunocompetence of juvenile Puget Sound (WA, USA) Chinook salmon (Oncorhynchus tschawytscha) . Salmon were fed four levels of the PCB congener mixture Aroclor 1254 for 28 d to bracket likely exposure to PCBs in the lower Duwamish waterway near Seattle, Washington, USA . Fish were transferred to five replicate tanks per dose, exposed to L . anguillarum, and monitored for 14 d . Half the PCB-dosed fish were vaccinated against L . anguillarum, and specific immunity was allowed to develop in this group for three weeks prior to challenge . All mortalities following challenge were individually sampled for bacteria to identify the cause of death . The data indicate that dietary PCB exposure, even at relatively high levels, did not have a significant effect on growth, innate disease resistance, or acquired immunity to L anguillarum . The controlled laboratory experiments in this study suggest that the immune system of Chinook salmon is not sensitive to orally delivered PCBs at environmentally relevant concentrations.

Environ Toxicol Chem, 2003 Feb, 22(2), 233 - 8
Specific responses of bacterial cells to dioxins; Min J et al.; Five different recombinant bioluminescent strains of Escherichia coli that contain the recA (responsive to DNA damage related stress), fabA (membrane damage), katG (oxidative damage), grpE (protein damage), and lac (constitutive expression, general toxicity) promoters fused to the bacterial lux operon from either Vibrio fischeri or Photorhabdus luminescens were used to describe the different mechanisms of toxicity that several dibenzo-p-dioxins and dibenzofurans have on bacteria, as well as to determine whether bacteria can sensitively detect the presence of these compounds . 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) was found to cause only DNA-related damage to bacterial cells . However, the four stress-responsive strains showed positive responses after addition of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD), whereas 2,3,7,8-tetrachlorodibenzo-p-furan (2,3,7,8-TCDF) caused only DNA . oxidative, and protein damage . However, 2,8-dichlorodibenzo-p-dioxin (2,8-DCDD) was not found to induce any stresses tested for in this study, that is, DNA, membrane, oxidative, and protein damage, indicating that each congener might differentially interact with the cell, stimulating differential stress responses within them . By using the constitutive strain, we found that the level of cellular toxicity experienced due to the addition of these four dioxins decreased in the order of 2,3,7,8-TCDD (the most toxic) . 1,2,3,4-TCDD, 2.8-DCDD, and 2,3,7,8-TCDF . The 20% effective concentration (EC20), defined in this study the concentration of chemical that causes a 20% decrease in the bioluminescence 60 min after induction, was only 0.1 microg/L for 2,3,7,8-TCDD, a value that is lower than that of the other congeners and demonstrates that 2,3,7,8-TCDD was the most toxic compound tested in this study.

Wei Sheng Wu Xue Bao, 2002 Jun, 42(3), 263 - 9
{Classification for one pathogenic Vibrio anguillarum strain isolated from skin-ulcer flounder}; Mo Z et al.; A pathogenic bacterial strain M3 revealed gram negative rod shape, motile and translucent clone was studied . This bacterium was isolated from the skin-ulcer flounder in the fish farm of RongCheng City, Shandong Provice, and could not be identified by BIOLOG ID SYSTEM . Comparative 16S rDNA sequence analyses revealed that strain M3 was most related to the genus of Vibrio . The overall similarity value between strain M3 and Vibrio species were 94% to 98% . Phylogentic analysis showed that M3 strain exhibited the highest level of similarity to Vibrio anguillarum, and the biological features of strain M3 were very similar to that of Vibrio anguillarum . Based on the results obtained, strain M3 was identified as Vibrio anguillarum.

Wei Sheng Wu Xue Bao, 1999 Oct, 39(5), 461 - 8
{Antibiotic resistance and plasmid profiles of Vibrio isolates from cultured Sparus sarba}; Li J et al.; A total of 51 potential pathogenic vibrios were isolated from moribund silver seabream Sparus sarba, which were collected from local fish farms of Hong Kong . All the isolates were classified and identified as 7 species by the API 20 E system and the scheme of Alsina & Blanch . These species were Vibrio alginolyticus (24 strains), Vibrio vulnificus (12 strains), Vibrio parahaemolyticus(7 strains), Vibrio logei(4 strains), Vibrio pelagius II(2 strains), Vibrio fluvialis (1 strains) and Vibrio meditterranei (1 strains) . Among these isolates, the three predominant species (V . alginolyticus, V . vulnificus and V . parahaemolyticus) were confirmed to be virulent to sea bream by experimental challenge . All isolates were also screened for plasmid DNA by agarose gel electrophoresis and tested for susceptibility to 16 antimicrobial agents by the agar dilution method . Of the 51 isolates examined, all strains were sensitive to ceftriaxone, streptomycin, nalidixic acid and rifampicin, and almost all were sensitive to ceftazidime, netilimicin, chloramphenicol and sulfamethoxazole except one or two strains . Most isolates were resistant to ampicillin (60 . 8%), cefuroxime (66.7%), amikacin(55%), kanamycin(58.8%) and trimethoprinm (76.5%) . Fifteen of the 51 isolates harboured 1-4 plasmids, with sizes ranging from 9 to 123 kb . Both the plasmids and the associated antimicrobial resistance (ampicillin, cefuroxime and trimethoprim) of 9 isolates could be transferred to recipient by single-step conjugation, however, the frequencies were very low, ranging from 10(-11) to 10(-9) . The present results indicate that resistance to these antibiotics is chromosomal.

Wei Sheng Wu Xue Bao, 2001 Aug, 41(4), 447 - 51
{Production and detection of monoclonal anti-idiotype antibodies against Vibrio anguillarum}; Xia Y et al.; Vibrio anguillarum is the pathogenic bacteria of Vibriosis, which is an infectious disease found in various fish species . Seven monoclonal anti-idiotype antibodies(mAb2) were raised against mAb1 4A6 . Identification of subgroup showed that 1H5, 1D1, 2B12 and 2F12 belonged to IgG2b, 2H12 and 1H12 to IgG2a and 1E10 to IgG3 . The titers of these mAb2 ascites were 1 x 10(-4)-1 x 10(-6) . The capacity of the mAb2 to inhibit the binding between mAb1 and antigen was investigated with the competitive inhibition ELISA . The results showed that 1D1, 1E10 1H5 and 2H12 mAb2 were able to inhibit this binding . Another experiment demonstrated that four mAb2(1D1, 1E10, 1H5 and 2H12) might induce Balb/c mice to produce Ab3 and these Ab3 competed the same antigen epitopes with Ab1 . These results indicate that mAb2(1D1, 1E10, 1H5 and 2H12) are likely to represent internal image of antigen and belong to Ab2 beta . They might be employed to induce antibodies against pathogenic epitopes of V . anguillarum in vivo so as to gave the safe and effective vaccine.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 1286 - 91 Epub 2003 Jan 27.
Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro; Xu Q et al.; Vibrio cholerae is the etiologic bacterial agent of cholera, a severe diarrheal disease endemic in much of the developing world . The V . cholerae genome contains 3,890 genes distributed between a large and a small chromosome . Although the large chromosome encodes the majority of recognizable gene products and virulence determinants, the small chromosome carries a disproportionate number of hypothetical genes . Thus, little is known about the role of the small chromosome in the biology of this organism or other Vibrio species . We have used the rabbit ileal loop model of V . cholerae infection to obtain in vivo-grown cells under near midexponential conditions in the small-intestinal environment . We compared the global transcriptional pattern of these in vivo-grown cells to those grown to midexponential phase in rich medium under aerobic conditions . Under both conditions, the genes showing the highest levels of expression reside primarily on the large chromosome . However, a shift occurs in vivo that results in many more small chromosomal genes being expressed during growth in the intestine . Our analysis further suggests that nutrient limitation (particularly iron) and anaerobiosis are major stresses experienced by V . cholerae during growth in the rabbit upper intestine . Finally, relative to in vitro growth, the intestinal environment significantly enhanced expression of several virulence genes, including those involved in phenotypes such as motility, chemotaxis, intestinal colonization, and toxin production.

Wei Sheng Wu Xue Bao, 2000 Feb, 40(1), 14 - 20
{Characterization and phylogenic analysis on a new isolate of genus Rhodocista}; Zhang D et al.; A strain of purple non sulfur bacteria was isolated from waste water plant in Beijing . It is phototaxic, and can converted the vibrioid cells into thick-wall cyst . Internal synthetic membrane is present as lamellae . It has a low absorption maxima at 798 nm . and require thiamine and vitamin B12 as growth factors . It can use succinate as carbon and energy source, but glucose can not be used . It can not grow in 3% NaCl and had Q-9 as major quinone . The 16S rRNA gene was amplified, cloned and sequenced . A phylogenic tree was constructed on the basis of the 16S rDNA sequences . It showed that the previously known member of the genus Rhodocista, Rc . centenaria, is the nearest neighbor to strain 3-p . The level of binary sequence similarity between Rc . centenaria and strain 3-p of 95%, and their phenotypic differences and genetic DNA-DNA relatedness of 56%, shows they are different species of Rhodocista . A new species name, Rhodocista pekinense, was proposed for strain 3-p.

Infect Immun, 2003 Feb, 71(2), 1020 - 5
Pathogenic potential of environmental Vibrio cholerae strains carrying genetic variants of the toxin-coregulated pilus pathogenicity island; Faruque SM et al.; The major virulence factors of toxigenic Vibrio cholerae are cholera toxin (CT), which is encoded by a lysogenic bacteriophage (CTXPhi), and toxin-coregulated pilus (TCP), an essential colonization factor which is also the receptor for CTXPhi . The genes for the biosynthesis of TCP are part of a larger genetic element known as the TCP pathogenicity island . To assess their pathogenic potential, we analyzed environmental strains of V . cholerae carrying genetic variants of the TCP pathogenicity island for colonization of infant mice, susceptibility to CTXPhi, and diarrheagenicity in adult rabbits . Analysis of 14 environmental strains, including 3 strains carrying a new allele of the tcpA gene, 9 strains carrying a new allele of the toxT gene, and 2 strains carrying conventional tcpA and toxT genes, showed that all strains colonized infant mice with various efficiencies in competition with a control El Tor biotype strain of V . cholerae O1 . Five of the 14 strains were susceptible to CTXPhi, and these transductants produced CT and caused diarrhea in adult rabbits . These results suggested that the new alleles of the tcpA and toxT genes found in environmental strains of V . cholerae encode biologically active gene products . Detection of functional homologs of the TCP island genes in environmental strains may have implications for understanding the origin and evolution of virulence genes of V . cholerae.

J Food Prot, 2003 Jan, 66(1), 125 - 9
Oyster-to-oyster variability in levels of Vibrio parahaemolyticus; Kaufman GE et al.; This study examined the variability in the levels of total and pathogenic Vibrio parahaemolyticus in individual oysters . Twenty oysters were collected on three occasions (in June, July, and September 2001) from a site near Mobile Bay, Ala . Ten of these oysters were tested immediately, and 10 were tested after 24 h of storage at 26 degrees C . Levels of total and pathogenic V . parahaemolyticus were determined by alkaline phosphatase-labeled DNA probe procedures targeting the thermolabile hemolysin and thermostable direct hemolysin genes, respectively . Similar V . parahaemolyticus levels (200 to 2,000 CFU/g) were found in nearly 90% of the oysters (for all sampling occasions) prior to storage . The log-transformed densities (means +/- standard deviations) of V . parahaemolyticus in oysters immediately after harvest were 2.90 +/- 0.91, 2.88 +/- 0.36, and 2.47 +/- 0.26 log10 CFU/g for June, July, and September, respectively . After storage for 24 h at 26 degrees C, the mean V . parahaemolyticus densities increased approximately 13- to 26-fold . Before storage, pathogenic V . parahaemolyticus was detected in 40% (10 to 20 CFU/g) of the oysters collected in June and July but was not detected in any oysters collected in September . After storage, pathogenic V . parahaemolyticus was detected in some oysters at levels of > 100 CFU/g . These data should aid in the development of sampling protocols for oyster monitoring programs and in the determination of exposure distributions associated with raw oyster consumption.

J Food Prot, 2003 Jan, 66(1), 38 - 43
Use of diacetyl to reduce the load of Vibrio vulnificus in the Eastern oyster, Crassostrea virginica; Birkenhauer JM et al.; Vibrio vulnificus is a highly virulent human pathogen that occurs naturally among the microflora of oysters . This organism has two portals of entry into humans, one of which is ingestion . Oysters containing V . vulnificus consumed in a raw or undercooked state often serve as a vehicle for the transmission of this organism . Previous studies conducted in our laboratory have examined various generally recognized as safe compounds and have determined that diacetyl, a component of butter, is among the most effective of these compounds in reducing loads of V . vulnificus in oysters . The purpose of this study was to further examine the role of diacetyl, along with that of depuration, in reducing loads of V . vulnificus . Shellstock oysters were treated with various concentrations of diacetyl, and we found that many of the oysters ceased pumping when diacetyl was added . The data obtained in this study indicated that treatment with diacetyl is ineffective; however, any reduction in V . vulnificus numbers may be masked when groups of oysters, some of which may not have taken up diacetyl, are sampled . We then investigated the efficacy of diacetyl in lowering levels of V . vulnificus in shucked oysters . Diacetyl was found to significantly reduce the load of V . vulnificus in shucked oysters containing natural populations . Overall, it appears that treatment with diacetyl is ineffective for shellstock oysters, although it has potential for use in reducing loads of V . vulnificus in shucked oysters.

Protein Sci, 2003 Feb, 12(2), 379 - 83
Vibrio cholerae cytolysin is composed of an alpha-hemolysin-like core; Olson R et al.; The enteric pathogen Vibrio cholerae secretes a water-soluble 80-kD cytolysin, Vibrio cholerae cytolysin (VCC) that assembles into pentameric channels following proteolytic activation by exogenous proteases . Until now, VCC has been placed in a unique class of pore-forming toxins, distinct from paradigms such as Staphyloccal alpha-hemolysin . However, as reported here, amino acid sequence analysis and three-dimensional structure modeling indicate that the core component of the VCC toxin is related in sequence and structure to a family of hemolysins from Staphylococcus aureus that include leukocidin F and alpha-hemolysin . Furthermore, our analysis has identified the channel-forming region of VCC and a potential lipid head-group binding site, and suggests a conserved mechanism of assembly and lysis . An additional domain in the VCC toxin is related to plant lectins, conferring additional target cell specificity to the toxin.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 1304 - 9 Epub 2003 Jan 21.
Emergence and evolution of Vibrio cholerae O139; Faruque SM et al.; The emergence of Vibrio cholerae O139 Bengal during 1992-1993 was associated with large epidemics of cholera in India and Bangladesh and, initially, with a total displacement of the existing V . cholerae O1 strains . However, the O1 strains reemerged in 1994 and initiated a series of disappearance and reemergence of either of the two serogroups that was associated with temporal genetic and phenotypic changes sustained by the strains . Since the initial emergence of the O139 vibrios, new variants of the pathogen derived from multiple progenitors have been isolated and characterized . The clinical and epidemiological characteristics of these strains have been studied . Rapid genetic reassortment in O139 strains appears to be a response to the changing epidemiology of V . cholerae O1 and also a strategy for persistence in competition with strains of the O1 serogroup . The emergence of V . cholerae O139 has provided a unique opportunity to witness genetic changes in V . cholerae that may be associated with displacement of an existing serogroup by a newly emerging one and, thus, provide new insights into the epidemiology of cholera . The genetic changes and natural selection involving both environmental and host factors are likely to influence profoundly the genetics, epidemiology, and evolution of toxigenic V . cholerae, not only in the Ganges Delta region of India and Bangladesh, but also in other areas of endemic and epidemic cholera.

Appl Microbiol Biotechnol, 2003 Jan, 60(5), 577 - 80 Epub 2002 Dec 13.
Construction of a sodA::luxCDABE fusion Escherichia coli: comparison with a katG fusion strain through their responses to oxidative stresses; Lee HJ et al.; A recombinant bioluminescent Escherichia coli strain, EBHJ, (sodA::luxCDABE), containing the promoter for the manganese superoxide dismutase ( sodA) gene fused to the Vibrio fischeri luxCDABE operon, was successfully constructed and characterized . Redox-cycling agents, such as paraquat and chromium, strongly induced a sodA- regulated response in dose-dependent manners, resulting in an increase of the bioluminescence . In a comparison with an existing oxidative stress responsive strain, DPD2511 (katG::luxCDABE), which is sensitive to H(2)O(2), the mechanism of chemicals that cause oxidative damage was elucidated via the key transcriptional factors involved in induction of the sodA and katG promoters, i.e . SoxRS and OxyR, respectively . It was found that responses from the katG- and sodA-based strains were significantly different dependent upon the chemicals being tested . Therefore, EBHJ, alone or in parallel with DPD2511, can be used to characterize and monitor chemicals that cause oxidative damage.

Mar Pollut Bull, 2003 Jan, 46(1), 56 - 64
A toxicity identification evaluation of silty marine harbor sediments to characterize persistent and non-persistent constituents; Stronkhorst J et al.; Sediment toxicity in silty marine harbor sediments is frequently dominated by ammonia or sulfide, leaving the adverse effects of persistent toxic substances unnoticed . To investigate the latter, we subjected interstitial water from three contaminated silty sediments to toxicity identification evaluation (TIE) phase I manipulations and tested for toxicity with four bioassays: the amphipod Corophium volutator (survival as an endpoint), the sea urchin Psammechinus miliaris (fertilization, embryo development) and the bacterium Vibrio fischeri (bioluminescence inhibition).The graduated pH manipulations identified the prominent toxicity of ammonia in the amphipod and sea urchin embryo tests, and also sulfide toxicity in the bacterium test . In two of the three samples tested with the amphipods, sea urchin embryos and bacteria, a small but significant reduction in interstitial water toxicity was achieved by removing persistent compounds through C(18) solid phase extraction . EDTA chelation resulted in a slight detoxification of the interstitial water for the amphipods and sea urchin embryos, but this was not related to any measured trace metals . Despite the presence of toxic levels of ammonia and sulfide in the harbor sediments, we established the adverse biological effects of persistent constituents by means of the TIE manipulations and in vivo interstitial water bioassays.

Lett Appl Microbiol, 2003, 36(2), 83 - 7
Control of pathogenic Vibrio spp . by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon; Vaseeharan B et al.; AIMS: The present study evaluated the in vitro and in vivo antagonistic effect of Bacillus against the pathogenic vibrios . METHODS AND RESULTS: Cell-free extracts of Bacillus subtilis BT23 showed greater inhibitory effects against the growth of Vibrio harveyi isolated by agar antagonism assay from Penaeus monodon with black gill disease . The probiotic effect of Bacillus was tested by exposing shrimp to B . subtilis BT23 at a density of 106-108 cfu ml-1 for 6 d before a challenge with V . harveyi at 103-104 cfu ml-1 for 1 h infection . The combined results of long- and short-term probiotic treatment of B . subtilis BT23 showed a 90% reduction in accumulated mortality . CONCLUSIONS: This study reports that pathogenic vibrios were controlled by Bacillus under in vitro and in vivo conditions . SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicated that probiotic treatment offers a promising alternative to the use of antibiotics in shrimp aquaculture.

Biochemistry, 2003 Jan 28, 42(3), 766 - 75
Dimeric structure of the six-domain VibF subunit of vibriobactin synthetase: mutant domain activity regain and ultracentrifugation studies; Hillson NJ et al.; Nonribosomal peptide synthetases (NRPS), fatty acid synthases (FAS), and polyketide sythases (PKS) are multimodular enzymatic assembly lines utilized in natural product biosynthesis . Previous data on FAS and PKS subunits have indicated that they are homodimers and that some of their catalytic functions can work in trans . When NRPS assembly lines have been probed for comparable formation of stable oligomers, no evidence had been forthcoming that species other than monomer forms were active . In this work we focus on the six-domain (Cy1-Cy2-A-C1-PCP-C2) enzyme VibF from the vibriobactin synthetase assembly line, which contains three other proteins, VibB, VibE, and VibH, that--when purified and mixed with VibF and the substrates ATP, threonine, 2,3-dihydroxybenzoate (DHB), and norspermidine--produce the iron chelator vibriobactin . Using a deletion of the Cy1 domain and separate inactivating mutations in the Cy2, A, PCP, and C2 domains of VibF, we report regain of catalytic activity upon mutant protein mixing that argues for heterodimer formation, stable for hundreds to thousands of catalytic cycles, with acyl chain processing and transfer around blocked domains . Ultracentrifugation data likewise confirm a dimeric structure for VibF and establish that domains within NRPS dimeric modules can act on acyl chains in trans . The results described here are the first indication for an NRPS subunit that homodimerization can occur and that there is a continuum of functional oligomerization states between monomers and dimers in nonribosomal peptide synthetases.

Mol Gen Mikrobiol Virusol, 2002, (4), 12 - 8
{Differences in virulence genes in Vibrio cholerae eltor strains isolated from different sources in Turkmenistan territory}; Smirnova NI et al.; Polymerase chain reaction (PCR) detected the presence of various genes associated with virulence in genome of strains V . cholerae eltor isolated in Turkmenistan territory during epidemic and epidemic-free perios . It was found that a complete set of virulence genes (ctxA+, tcpA+ and toxR+) contained strains isolated from patients, carriers and environment only in cholera epidemics . Strains isolated from the environment in the period free of epidemics did not contain ctxA and tcpA in 78.2% of cases, but 5.2% of the strains carried a complete set of virulence genes . There were also nontoxigenic strains containing genes tcpA and toxR . Such strains were isolated from the environment (16.6%) and vibrion carriers (42.9%) . Isolated were also strains V.cholerae eltor carrying bacteriophage CTX phi with incomplete set of virulence genes and having genotype ctxA-, ace+ and zot+ . Almost all the strains ctxA-, tcpA+ carry attRS1-site in genome . This shows that such strains may transform into toxigenic as a result of infection with bacteriophage CTX phi.

J Bacteriol, 2003 Feb, 185(3), 1037 - 44
pIIICTX, a predicted CTXphi minor coat protein, can expand the host range of coliphage fd to include Vibrio cholerae; Heilpern AJ et al.; CTXphi is a filamentous bacteriophage that encodes cholera toxin . CTXphi infection of its host bacterium, Vibrio cholerae, requires the toxin-coregulated pilus (TCP) and the products of the V . cholerae tolQRA genes . Here, we have explored the role of OrfU, a predicted CTXphi minor coat protein, in CTXphi infection . Prior to the discovery that it was part of a prophage, orfU was initially described as an open reading frame of unknown function that lacked similarity to known protein sequences . Based on its size and position in the CTXphi genome, we hypothesized that OrfU may function in a manner similar to that of the coliphage fd protein pIII and mediate CTXphi infection as well as playing a role in CTXphi assembly and release . Deletion of orfU from CTXphi dramatically reduced the number of CTXphi virions detected in supernatants from CTXphi-bearing cells . This defect was complemented by expression of orfU in trans, thereby confirming a role for this gene in CTXphi assembly and/or release . To evaluate the requirement for OrfU in CTXphi infection, we introduced fragments of orfU into gIII in an fd derivative to create OrfU-pIII fusions . While fd is ordinarily unable to infect V . cholerae, an fd phage displaying the N-terminal 274 amino acids of OrfU could infect V . cholerae in a TCP- and TolA-dependent fashion . Since our findings indicate that OrfU functions as the CTXphi pIII, we propose to rename OrfU as pIII(CTX) . Our data also provide new evidence for a conserved pathway for filamentous phage infection.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 1051 - 5 Epub 2003 Jan 14.
Reduction of cholera in Bangladeshi villages by simple filtration; Colwell RR et al.; Based on results of ecological studies demonstrating that Vibrio cholerae, the etiological agent of epidemic cholera, is commensal to zooplankton, notably copepods, a simple filtration procedure was developed whereby zooplankton, most phytoplankton, and particulates >20 microm were removed from water before use . Effective deployment of this filtration procedure, from September 1999 through July 2002 in 65 villages of rural Bangladesh, of which the total population for the entire study comprised approximately 133,000 individuals, yielded a 48% reduction in cholera (P < 0.005) compared with the control.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 1280 - 5 Epub 2003 Jan 14.
CTXphi-independent production of the RS1 satellite phage by Vibrio cholerae; Faruque SM et al.; The cholera toxin genes of Vibrio cholerae are encoded by the filamentous phage, CTXphi . Chromosomal CTXphi prophage DNA is often found flanked by copies of a related genetic element designated RS1, and RS1 DNA can be packaged into filamentous phage particles (designated RS1phi) by using the CTXphi morphogenesis genes . RS1phi is a satellite phage that further controls expression and dissemination of CTXphi . Here we describe a CTXphi-independent mechanism for production of RS1phi . A nontoxigenic environmental V . cholerae strain (55V71) was identified that supports production of RS1phi . However, newly infected CTX-negative strains did not produce RS1phi, indicating that additional 55V71 genes were involved in production of RS1phi . Analysis of nucleic acids from phage preparations of 55V71 revealed a 7.5-kb single-stranded DNA, whose corresponding replicative form was found in plasmid preparations . This DNA likely corresponds to the genome of a new filamentous phage, which we have designated KSF-1phi . The replicative form DNA of KSF-1phi was cloned into pUC18, and the resulting construct pKSF-1.1 supported the production of RS1phi particles by CTX-negative V . cholerae strains . RS1phi particles produced in this way infect recipient V . cholerae strains by a mechanism that is independent of the CTXphi receptor, the toxin-coregulated pilus . Thus, KSF-1phi is capable of facilitating the transfer of the RS1 element to strains that do not express toxin coregulated pilus . Given that RS1phi can enhance coproduction of CTXphi particles, KSF-1phi-mediated dissemination of RS1 may indirectly promote the spread of toxin genes among V . cholerae strains . This study also shows that filamentous phages can package diverse DNA elements and thus may play a role in horizontal transfer of more genes than previously appreciated.

Fish Shellfish Immunol, 2003 Feb, 14(2), 105 - 14
In the spiny lobster (Panulirus interruptus) the prophenoloxidase is located in plasma not in haemocytes; Hernandez-Lopez J et al.; In the spiny lobster (Panulirus interruptus), unlike other crustaceans most of the prophenoloxidase (proPO) was detected in cell-free plasma (86.3%) . In spite of its location, lobster proPO activating system has a similar activation mechanism to other crustacean proPO systems . Haemocyte lysate was able to activate the plasma proPO indicating location of the prophenoloxidase activating enzyme (PPAE) in haemocytes . Lobster haemocyte PPAE was isolated by affinity chromatography and its participation as activating enzyme was demonstrated . This enzyme is a serine-proteinase that transforms the inactive form (proPO) to an active one (phenoloxidase) . The PPAE was also present in the cell-free supernatant of haemocytes previously incubated with Vibrio alginolyticus.

Biochemistry, 2003 Jan 21, 42(2), 529 - 34
Complex formation between Vibrio harveyi luciferase and monomeric NADPH:FMN oxidoreductase; Jeffers CE et al.; A direct transfer of the reduced flavin mononucleotide (FMNH(2)) cofactor of Vibrio harveyi NADPH:FMN oxidoreductase (FRP) to luciferase for the coupled bioluminescence reaction has been indicated by recent kinetic studies {Lei, B., and Tu, S.-C . (1998) Biochemistry 37, 14623-14629; Jeffers, C., and Tu, S.-C . (2001) Biochemistry 40, 1749-1754} . For such a mechanism, a complex formation of luciferase with FRP is essential, but until now, no evidence for such a complex has been reported . In this work, FRP was labeled at 1:1 molar ratio with the fluorophore eosin . The labeled enzyme was about 30% active in either the reductase single-enzyme or the luciferase-coupled assay . The labeled FRP in either the holo- or apoenzyme form was similar to the native FRP in undergoing a monomer-dimer equilibrium . By measuring the steady-state fluorescence anisotropy of eosin-labeled FRP, it was shown that luciferase formed a complex at 1:1 molar ratio with the monomer of either the apoenzyme or the holoenzyme form of FRP with K(d) values of 7 and 11 microM, respectively . Neither the holo- nor the apoenzyme of the labeled FRP in the dimeric form was effective in complexing with luciferase . At maximal in vivo bioluminescence, the V . harveyi cellular contents of luciferase and FRP were estimated to be 172 and 3 microM, respectively . The vast majority of FRP would be trapped in the luciferase/FRP complex . Plausible physiological significance of such a finding is discussed.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Sep-Oct, (5), 15 - 21
{Morphology and enzyme activity of nonculturable forms of Vibrio cholerae}; Sokolenko AV et al.; The transition of V . cholerae into the uncultivable state under experimental conditions was accompanied by gradual changes in their morphology, motility and metabolic activity . The vibrios took the oval form, lost their flagellum, motility and enzyme activity on diagnostic media . Dehydrogenase activity tested by reduction of triphenyl tetrazolium chloride, increased at the initial stages and dropped to the initial level or even lower by the end of the observation period (10 months) . Similar dynamics was noted when the cytoplasmatic marker enzyme--ATPase activity was studied . Glucose catabolism in the uncultivable forms shifted towards glycolysis . During 1-2 months ctx and tcp genes could be detected in these forms by the PCR . The dynamics of the biological properties under study made it possible to find out the existence of 3 functionally different stages in the development of an uncultivable population.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Sep-Oct, (5), 7 - 11
{Hemolytic activity and toxigenicity of Vibrio cholerae of different serogroups}; Men'shikova EA et al.; Experimental data on the comparative evaluation of the hemolytic activity of ctx+ Hly- and ctx- Hly+ V . cholerae, serogroups O1 and O139, in the process of their cultivation in different nutrient media are presented . The capacity of ctx+ V . cholerae of both serogroups cultivated under the conditions of iron deficiency, for the production of hemolysin capable of lyzing sheep red blood cells was shown . Hemolysin produced by ctx- strains of V . cholerae was synthesized under any conditions . The study of hemolysin preparations obtained from ctx- and ctx+ strains of V . cholerae, serogroups O1 and O139, revealed that they were biologically and immunologically similar.

J Biotechnol, 2003 Feb 27, 101(1), 49 - 56
The toxicity of textile reactive azo dyes after hydrolysis and decolourisation; Gottlieb A et al.; The toxicity of C.I . Reactive Black 5 and three Procion dyes, as found in textile effluents, was determined using the bioluminescent bacterium Vibrio fischeri . Hydrolysed Reactive Black had a slightly greater toxicity than the parent form (EC(50) 11.4+/-3.68 and 27.5+/-4.01 mg l(-1), respectively) . A baffled bioreactor with anaerobic and aerobic compartments was used to decolourise hydrolysed Reactive Black 5 in a synthetic effluent . Decolourisation of hydrolysed Reactive Black resulted in an increased toxicity (EC(50) 0.2+/-0.03 mg l(-1)) . Toxicity was not detectable when decolourised Reactive Black 5 was metabolised under aerobic conditions . No genotoxicity was detected after the decolourisation of either the parent or the hydrolysed reactive dyes, either in vitro or in the bioreactor . The toxicity and genotoxicity of decolourised C.I . Acid Orange 7 was due to the production of 1-amino-2-naphthol (EC(50) 0.1+/-0.03 mg l(-1)).

Water Sci Technol, 2002, 46(11-12), 253 - 6
Bio-affecting mercury detection using mercury resistance gene module fused with bioluminescence reporter genes; Yamagata T et al.; Bioluminescence sensor systems were developed for monitoring environmental mercury contamination . The biological mercury measurement sensor systems were constructed by DNA recombination technique . A bacterial mercury-resistant operon (meroperon) from Pseudomonas sp . K-6y4 and a bacterial bioluminescence operon (lux operon) from an ocean bacterium Vibrio fischeri were fused in avector plasmid . The resulting recombinant plasmids were cloned in Escherichia coli cells . The bioluminescence sensor systems responded to mercury chloride of 0.1 nM to 100 nM . The mercury bioluminescence sensor developed in this study can be used for monitoring of the bio-affecting mercury instead of total mercury that is measured by conventional analytical equipment . The fundamental feature of the bioluminescence sensor system is attractive for use as a monitoring system for bio-affecting environmental mercury contamination.

Ecotoxicology, 2002 Dec, 11(6), 435 - 50
Statistical analysis of sediment toxicity by additive monotone regression splines; De Boer WJ et al.; Modeling nonlinearity and thresholds in dose-effect relations is a major challenge, particularly in noisy data sets . Here we show the utility of nonlinear regression with additive monotone regression splines . These splines lead almost automatically to the estimation of thresholds . We applied this novel method to explore the relation between the toxicity of aquatic sediments, as observed in bioassays with Daphnia magna, Chironomus riparius and Vibrio fischeri, and the degree of contamination of the sediments . Despite the low signal-to-noise ratio in the data, some interesting thresholds and (non)linear effects were found . The method has added value compared to the linear multivariate methods applied earlier to these data . Percentages of explained variance remained low, but could be doubled by diminishing the effect of local variability.

J Clin Microbiol, 2003 Jan, 41(1), 442 - 6
Sequence polymorphism of the 16S rRNA gene of Vibrio vulnificus is a possible indicator of strain virulence; Nilsson WB et al.; Vibrio vulnificus exhibits considerable strain-to-strain variation in virulence . Attempts to associate phenotypic or genotypic characteristics with strain virulence have been largely unsuccessful . Based on a 17-nucleotide difference throughout the sequence of the small subunit 16S rRNA gene, there are two major groups of V . vulnificus designated types A and B . In a survey of the 16S rRNA genotype in 67 V . vulnificus human clinical and nonclinical strains, we determined that the majority of nonclinical isolates are type A (31 of 33) and that there is a statistically significant association between the type B genotype and human clinical strains (26 of 34).

J Clin Microbiol, 2003 Jan, 41(1), 124 - 34
Genetic diversity of Vibrio cholerae O1 in Argentina and emergence of a new variant; Pichel M et al.; The genetic diversity of Vibrio cholerae O1 strains from Argentina was estimated by random amplified polymorphic DNA (RAPD) analysis and pulsed-field gel electrophoresis (PFGE) . Twenty-nine isolates carrying the virulence genes ctxA, zot, ace, and tcpA appeared to represent a single clone by both typing methods; while 11 strains lacking these virulence genes exhibited several heterogeneous RAPD and PFGE patterns . Among the last group, a set of isolates from the province Tucuman showed a single RAPD pattern and four closely related PFGE profiles . These strains, isolated from patients with diarrhea, did not produce the major V . cholerae O1 virulence determinants, yet cell supernatants of these isolates caused a heat-labile cytotoxic effect on Vero and Y-1 cells and elicited significant variations on the water flux and short-circuit current in human small intestine mounted in an Ussing chamber . All these effects were completely abolished by incubation with a specific antiserum against El Tor hemolysin, suggesting that this virulence factor was responsible for the toxic activity on both the epithelial cells and the small intestine specimens and may hence be involved in the development of diarrhea . We propose "Tucuman variant" as the designation for this new cluster of cholera toxin-negative V . cholerae O1 strains.

Dis Aquat Organ, 2002 Nov 7, 52(1), 39 - 46
16S ribosomal DNA sequencing confirms the synonymy of Vibrio harveyi and V . carchariae; Gauger EJ et al.; Seventeen bacterial strains previously identified as Vibrio harveyi (Baumann et al . 1981) or V . carchariae (Grimes et al . 1984) and the type strains of V . harveyi, V . carchariae and V . campbellii were analyzed by 16S ribosomal DNA (rDNA) sequencing . Four clusters were identified in a phylogenetic analysis performed by comparing a 746 base pair fragment of the 16S rDNA and previously published sequences of other closely related Vibrio species . The type strains of V . harveyi and V . carchariae and about half of the strains identified as V . harveyi or V . carchariae formed a single, well-supported cluster designed as 'bona fide' V . harveyi/carchariae . A second more heterogeneous cluster included most other strains and the V . campbellii type strain . Two remaining strains are shown to be more closely related to V . rumoiensis and V . mediterranei . 16S rDNA sequencing has confirmed the homogeneity and synonymy of V . harveyi and V . carchariae . Analysis of API20E biochemical profiles revealed that they are insufficient by themselves to differentiate V . harveyi and V . campbellii strains . 16S rDNA sequencing, however, can be used in conjunction with biochemical techniques to provide a reliable method of distinguishing V . harveyi from other closely related species.

J Am Chem Soc, 2003 Jan 8, 125(1), 265 - 75
X- and W-band EPR and Q-band ENDOR studies of the flavin radical in the Na+ -translocating NADH:quinone oxidoreductase from Vibrio cholerae; Barquera B et al.; Na(+)-NQR is the entry point for electrons into the respiratory chain of Vibrio cholerae . It oxidizes NADH, reduces ubiquinone, and uses the free energy of this redox reaction to translocate sodium across the cell membrane . The enzyme is a membrane complex of six subunits that accommodates a 2Fe-2S center and several flavins . Both the oxidized and reduced forms of Na(+)-NQR exhibit a radical EPR signal . Here, we present EPR and ENDOR data that demonstrate that, in both forms of the enzyme, the radical is a flavin semiquinone . In the oxidized enzyme, the radical is a neutral flavin, but in the reduced enzyme the radical is an anionic flavin, where N(5) is deprotonated . By combining results of ENDOR and multifrequency continuous wave EPR, we have made an essentially complete determination of the g-matrix and all major nitrogen and proton hyperfine matrices . From careful analysis of the W-band data, the full g-matrix of a flavin radical has been determined . For the neutral radical, the g-matrix has significant rhombic character, but this is significantly decreased in the anionic radical . The out-of-plane component of the g-matrix and the nitrogen hyperfine matrices are found to be noncoincident as a result of puckering of the pyrazine ring . Two possible assignments of the radical signals are considered . The neutral and anionic forms of the radical may each arise from a different flavin cofactor, one of which is converted from semiquinone to flavohydroquinone, while the other goes from flavoquinone to semiquinone, at almost exactly the same redox potential, during reduction of the enzyme . Alternatively, both forms of the radical signal may arise from a single, extremely stable, flavin semiquinone, which becomes deprotonated upon reduction of the enzyme.

Exp Mol Med, 2002 Sep 30, 34(4), 308 - 12
Vibrio vulnificus cytolysin induces hyperadhesiveness of pulmonary endothelial cells for neutrophils through endothelial P-selectin: a mechanism for pulmonary damage by Vibrio vulnificus cytolysin; Kim BS et al.; Vibrio vulnificus cytolysin forms transmembrane pores that are permeable to calcium ions in pulmonary endothelial cells, and has been suggested as an important virulence factor that sequestrate neutrophils primarily in the lung . To elucidate the mechanism we investigated whether the cytolysin affect the expression of endothelial P-selectin and adhesiveness of pulmonary endothelial cells for neutrophils . The cytolysin increased the adhesiveness of CPAE cell, a pulmonary endothelial cell line, for neutrophils in a concentration- and time-dependent manner . The increase of adhesiveness occurred within several minutes after the cytolysin exposure, persisted up to 90 min, and was not affected by cycloheximide . Furthermore, flow cytometric analyses showed that cytolysin enhanced the level of P-selectin on CPAE cell surface . Therefore, these results suggest that the cytolysin-induced hyperadhesiveness of pulmonary endothelial cells for neutrophils is mediated by the mobilization of endothelial P-selectin to the cell surface.

Appl Environ Microbiol, 2003 Jan, 69(1), 199 - 211
Differential growth response of colony-forming alpha- and gamma-proteobacteria in dilution culture and nutrient addition experiments from Lake Kinneret (Israel), the eastern Mediterranean Sea, and the Gulf of Eilat; Pinhassi J et al.; Even though it is widely accepted that bacterioplankton growth in lakes and marine ecosystems is determined by the trophic status of the systems, knowledge of the relationship between nutrient concentrations and growth of particular bacterial species is almost nonexistent . To address this question, we performed a series of culture experiments with water from Lake Kinneret (Israel), the eastern Mediterranean Sea, and the Gulf of Eilat (northern Red Sea) . In the initial water samples, the proportion of CFU was typically <0.002% of the 4',6'-diamidino-2-phenylindole (DAPI) counts . During incubation until the early stationary phase, the proportion of CFU increased to 20% of the DAPI counts and to 2 to 15% of the DAPI counts in unenriched lake water and seawater dilution cultures, respectively . Sequencing of the 16S ribosomal DNA of colony-forming bacteria in these cultures consistently revealed an abundance of alpha-proteobacteria, but notable phylogenetic differences were found at the genus level . Marine dilution cultures were dominated by bacteria in the Roseobacter clade, while lake dilution cultures were dominated by bacteria affiliated with the genera Sphingomonas and CAULOBACTER: In nutrient (glucose, ammonium, phosphate) addition experiments the CFU comprised 20 to 83% of the newly grown cells . In these incubation experiments fast-growing gamma-proteobacteria dominated; in the marine experiments primarily different Vibrio and Alteromonas species appeared, while in the lake water experiments species of the genera Shewanella, Aeromonas, and Rheinheimera grew . These results suggest that major, but different, gamma-proteobacterial genera in both freshwater and marine environments have a preference for elevated concentrations of nutrients and easily assimilated organic carbon sources but are selectively outcompeted by alpha-proteobacteria in the presence of low nutrient concentrations.

J Bacteriol, 2003 Jan, 185(2), 674 - 8
Experimental verification of a sequence-based prediction: F(1)F(0)-type ATPase of Vibrio cholerae transports protons, not Na(+) ions; Dzioba J et al.; The membrane energetics of the intestinal pathogen Vibrio cholerae involves both H(+) and Na(+) as coupling ions . The sequence of the c subunit of V . cholerae F(0)F(1) ATPase suggested that this enzyme is H(+) specific, in contrast to the results of previous studies on the Na(+)-dependent ATP synthesis in closely related Vibrio spp . Measurements of the pH gradient and membrane potential in membrane vesicles isolated from wild-type and DeltaatpE mutant V . cholerae show that the F(1)F(0) ATPase of V . cholerae is an H(+), not Na(+), pump, confirming the bioinformatics assignments that were based on the Na(+)-binding model of S . Rahlfs and V . Muller (FEBS Lett . 404:269-271, 1999) . Application of this model to the AtpE sequences from other bacteria and archaea indicates that Na(+)-specific F(1)F(0) ATPases are present in a number of important bacterial pathogens.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2015 - 22
Enterovibrio norvegicus gen . nov., sp . nov., isolated from the gut of turbot (Scophthalmus maximus) larvae: a new member of the family Vibrionaceae; Thompson FL et al.; Twenty-two isolates originating from the gut of healthy cultured turbot larvae in Norway were investigated using a polyphasic approach . Amplified fragment length polymorphism fingerprinting analysis showed that the isolates have typical patterns and form two main groups . Phylogenetic analysis revealed that the isolates belong to the gamma-Proteobacteria, with Vibrio hollisae as their closest neighbour . DNA-DNA hybridization, chemotaxonomic and phenotypic analyses further proved that these isolates represent a tight novel taxon that differs from currently described species in the family Vibrionaceae . It is proposed that these novel isolates be accommodated in a new genus, Enterovibrio gen . nov., with Enterovibrio norvegicus sp . nov . as the type species . Isolates were motile by a polar flagellum, positive for oxidase, catalase, arginine dihydrolase and beta-galactosidase, but negative for the Voges-Proskauer reaction . They produced indole, did not reduce nitrate and were resistant to the vibriostatic agent O/129 . The DNA G+C content of E . norvegicus was 47.1-47.9 mol% . The type strain is E . norvegicus LMG 19839(T) (= CAIM 430(T)).

J Infect Dis, 2003 Jan 1, 187(1), 96 - 101 Epub 2002 Dec 13.
A 4-year study of the epidemiology of Vibrio cholerae in four rural areas of Bangladesh; Sack RB et al.; How Vibrio cholerae spreads around the world and what determines its seasonal peaks in endemic areas are not known . These features of cholera have been hypothesized to be primarily the result of environmental factors associated with aquatic habitats that can now be identified . Since 1997, fortnightly surveillance in 4 widely separated geographic locations in Bangladesh has been performed to identify patients with cholera and to collect environmental data . A total of 5670 patients (53% <5 years of age) have been studied; 14.3% had cholera (10.4% due to V . cholerae O1 El Tor, 3.8% due to O139) . Both serogroups were found in all locations; outbreaks were seasonal and often occurred simultaneously . Water-use patterns showed that bathing and washing clothes in tube-well water was significantly protective in two of the sites . These data will be correlated with environmental factors, to develop a model for prediction of cholera outbreaks.

Int J Antimicrob Agents, 2003 Jan, 21(1), 71 - 4
Lysis of methicillin-resistant Staphylococcus aureus by 2,4-diacetylphloroglucinol produced by Pseudomonas sp . AMSN isolated from a marine alga; Kamei Y et al.; Previously 2,4-diacetylphloroglucinol (DAPG) produced by Pseudomonas sp . AMSN isolated from a marine alga, had demonstrated a high level of anti-methicillin-resistant Staphylococcus aureus (MRSA) comparable with that of vancomycin . In this study, this substance had bacteriolytic activity against MRSA at 1 microg/ml as well as similar activity against Vibrio parahaemolyticus at 24 microg/ml that suggests a novel antibacterial mode of action by this substance . Heat and pH stability tests showed it to be stable at temperatures ranging from 35 to 70 degrees C and at pHs ranging from 2-7 . It was not acutely toxic to mice at levels up to 100 mg/kg.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Nov-Dec, (6), 78 - 80
{Characterization of Vibrio cholerae cultures isolated in foci of cholera in the city of Kazan}; Ziatdinov VB; During the period of the registered outbreak of cholera in 2001 in Kazan 171 V . cholerae cultures were isolated in the focus of the infection (from patients, carriers and 7 environmental objects) . The use of the basic and additional tests, including the polymerase chain reaction, made it possible to establish the circulation of V . cholerae, phagovar 15, in the focus of the infection . The strain isolated from the water reservoir Azino-1 in Kazan was identical in its properties to the epidemically dangerous strains isolated from patients . On the whole, the data obtained in the identification of the strains showed that the cultures isolated from patients, vibrio-carriers and environmental objects were identical.

Mar Environ Res, 2003 Mar, 55(2), 161 - 79
Serum antibody levels against select bacterial pathogens in Atlantic bottlenose dolphins, Tursiops truncatus, from Beaufort NC USA and Charleston Harbor, Charleston, SC, USA; Beck BM et al.; Concern over the emergence of zoonotic diseases in marine organisms is growing . In response to this concern, this study set out to measure antibody activities against bacterial pathogens in Atlantic bottlenose dolphins, Tursiops truncatus, from the coastal estuaries of NC and SC, USA . Individuals from Charleston SC harbor, a heavily industrialized shipping harbor estuary, and from Beaufort NC, a non-shipping estuary, were examined . Purified IgG was obtained from pooled sera using ammonium sulfate precipitation steps and protein-G procedures, which was then used to generate a panel of IgG-specific monoclonal antibodies . Two of these antibodies, mAbs BB-10-2 (IgG1) and BB-32-2 (IgG2b), were then used to determine total serum IgG concentrations using a sandwich capture ELISA . Circulating IgG levels were variable between individuals and between the two pods . MAb BB-10-2 was then used in an indirect ELISA to determine serum antibody activities against several common marine bacteria as well as the human pathogens E . coli and E . coli strain 0157:H7, Vibrio parahemolyticus, V . vulnificus, V . cholerae, Mycobacteria marinum, M . fortuitum, and M . chelonae . The highest antibody activities were against mycobacteria, two of which are zoonotic pathogens . Males had the highest antibody activities, thus suggesting low cell-mediated immunity against intracellular pathogens in these individuals . T-cell proliferation in response to Con-A, an indicator of cell-mediated immune function, was then measured in the Beaufort population . Males had the lowest proliferation responses, however a negative correlation between antibody activities and T-cell proliferation in individuals could not be established for either of the Mycobacteria species . Overall, antibody activities against all bacteria, including innocuous species such as V . anguillarum, V . natrigens, and M . xenopi were highly variable between individual dolphins and the two pods, with some animals exhibiting very high activities . These studies suggests that dolphin populations should be monitored by following the health and seroprevalence of pathogens of interest in select individual animals over time.

Microbiol Res, 2002, 157(4), 249 - 55
Alkaline adaptation induces cross-protection against some environmental stresses and morphological change in Vibrio parahaemolyticus; Koga T et al.; The relationship between alkaline adaptation and the resistance against environmental stresses was examined in Vibrio parahaemolyticus . Alkali-adapted cells were found to have increased resistance against various stresses, including heat, crystal violet, deoxycholic acid, and hydrogen peroxide . However, alkali-adapted cells showed no increased resistance against acid stress and heat-adapted cells did not show increased resistance against alkaline stress . Furthermore, alkaline treatment induced cell elongation with heterogenous size of the bacterium.

Hua Xi Yi Ke Da Xue Xue Bao, 2000 Mar, 31(1), 27 - 9
{PCR amplification and cloning of virulence expression regulatory gene toxR of Vibrio cholerae}; Chen J et al.; In order to construct a genomic bivalent oral vaccine of Leishmania donovani and Vibrio cholerae, we amplified a 1.3 kb DNA fragment from 7 strains of Vibrio cholerae with primers P1 and P2 . Restriction endonuclease analysis of PCR amplified products from 9 strains of Vibrio cholerae was performed by digestion with EcoR I . The results revealed an EcoR I site in the central part of toxR gene . The entire toxR gene of Vibrio cholerae Non-CT strain 7743 was amplified by PCR with primers P1 and P2, digested with endonucleases BamH I and Hind III, then was orientationally cloned into plasmid pAT153 . The restriction endonuclease analysis demonstrates that the recombinant plasmid ptR4 contains a 1.3 kb toxR gene fragment.

J Gen Appl Microbiol, 1999 Aug, 45(4), 155 - 161
Acid adaptation induces cross-protection against some environmental stresses in Vibrio parahaemolyticus; Koga T et al.; The relationship of acid adaptation to the resistance of other environmental stresses was examined in Vibrio parahaemolyticus . Acid-adapted cells were found to have increased resistance to various stresses, including heat, crystal violet, bile, and deoxy cholic acid . However, heat-adapted cells showed no increased resistance against acid stress . Adaptation required protein synthesis, since treatment with chloramphenicol during adaptation to pH 5.3 prevented the development of acid resistance . Acid-adapted cells showed an increased amount of outer membrane protein with an apparent molecular weight of 27,000 . These results show that acid-induced cross-protection involved changes in outer membrane protein composition and the known enhancement of intracellular pH homeostasis.

J Gen Appl Microbiol, 1997 Dec, 43(6), 317 - 324
Cellular fatty acid profiles of ninety-five strains of Vibrio vulnificus isolated from clinical specimens in Korea; Shin MG et al.; There have been many reports of primary Vibrio vulnificus septicemia in Korea since 1987 . This study was undertaken to determine the cellular fatty acid (CFA) compositions of 95 clinical strains of V . vulnificus isolated in Korea during 1985-1995 . We compared these results with the CFA profile of V . vulnificus in the Microbial Identification System (MIS) (CLIN library version 3.9; Microbial ID Inc., Newark, DE, U.S.A.), and the grouping of V . vulnificus by CFA analysis was also performed . The relationship between groups and serotypes of V . vulnificus was described . Although most of the CFAs in V . vulnificus strains were similar to the CFA profile of V . vulnificus in the MIS, some distinctive differences were observed between the results of this study and the MIS CFA profile of V . vulnificus . First, the means of 2 major CFAs, 16 : 0 and 16 : 1w7c, were 22.16 and 18.26% in this study but 23.52 and 25.44% in the MIS, respectively . Second, all isolates had 11 : 0iso3OH, which was not present in the MIS . Of 95 strains, 10 strains (10.5%) showed 'NO MATCH' results by the MIS identification . Eighty five strains (89.5%) were identified as V . vulnificus by the MIS (the first choice identification), but they disclosed low SI values of <0.6 (not 'EXCELLENT MATCH') except 2 strains (2.1%) . This showed that V . vulnificus strains isolated in Korea had different characteristics in CFA composition in comparison with the MIS V . vulnificus library . Nine groups comprising all the strains were obtained by cluster analysis and were further characterized by principal-component analysis . There was heterogeneity between the groups by CFA and serotypes of V . vulnificus . The same serovars were distributed into diverse groups.

J Gen Appl Microbiol, 1998 Feb, 44(1), 27 - 33
Differences among marine and hospital strains of Vibrio cholerae during Peruvian epidemic; Carvajal GH et al.; During a period of 18 months of an epidemic of Vibrio cholerae, cultures from 450 samples of fish, shellfish and seawater were isolated . The highest frequencies of occurrence observed were 5.2% in fish from inshore waters, 3.9% in marine snails, and 1.8% in mussels and crabs . No incidents were isolated from cultures of fish in the open seas or cultures from frozen shrimp . Cultures of marine origin were compared with cultures from hospitalized patients, and these revealed marked serological and toxigenic differences . Marine strains were mainly non-O1 V . cholerae, non toxigenic . We presume fishing off-shore not to be the cause of this outbreak . However, marine species from contaminated waters could contain toxigenic V . cholerae remaining viable and potentially pathogenic . Methods used were more sensitive and specific for detecting marine strains . In this paper the need to use more specific methods is discussed.

Mol Biol (Mosk), 2002 Nov-Dec, 36(6), 1074 - 9
{Variable tandem repeat VcA of Vibrio cholerae}; Vodop'ianov SO et al.; Computer analysis revealed seven potential variable-number tandem-repeat (VNTR) loci in the Vibrio cholerae genome . Specific primers were designed to amplify locus VcA located on chromosome 2 and containing a TGCTGT repeat . The locus was found in all tested strains from a V . cholerae strain collection, the repeat number varying 3 to 23 . In total, 14 VcA alleles were observed . The VcA locus was proposed as a marker for the molecular typing of V . cholerae strains.

Infect Immun, 2003 Jan, 71(1), 533 - 5
Vibrio vulnificus induces macrophage apoptosis in vitro and in vivo; Kashimoto T et al.; In this study, we compared the apoptotic activities of clinical and environmental isolates of Vibrio vulnificus toward macrophages in vitro and in vivo . The clinical isolates induced apoptosis in macrophage-like cells in vitro and in macrophages in vivo . This suggests that macrophage apoptosis may be important for the clinical virulence of V . vulnificus.

Infect Immun, 2003 Jan, 71(1), 510 - 5
The vibrio pathogenicity island-encoded mop protein modulates the pathogenesis and reactogenicity of epidemic vibrio cholerae; Zhang D et al.; Epidemic Vibrio cholerae possess the VPI (Vibrio pathogenicity island) essential virulence gene cluster . The VPI is 41.2 kb in size and encodes 29 potential proteins, several of which have no known function . We show that the VPI-encoded Orf4 is a predicted 34-kDa periplasmic protein containing a zinc metalloprotease motif . V . cholerae seventh-pandemic (El Tor) strain N16961 carrying an orf4 mutation showed no obvious difference relative to its parent in the production of cholera toxin and the toxin-coregulated pilus, motility, azocasein digestion, and colonization of infant mice . However, analysis of rabbit ileal loops revealed that the N16961 orf4 mutant is hypervirulent, causing increased serosal hemorrhage and reactogenicity compared to its parent . Histology revealed a widening of submucosa, with an increase in inflammatory cells, diffuse lymphatic vessel dilatation, edema, endothelial cell hypertrophy of blood vessels, blunting of villi, and lacteal dilatation with lymphocytes and polymorphonuclear leukocytes . The mutant could be complemented in vivo with an orf4 gene on a plasmid but not with an orf4 gene containing a site-directed mutation in the putative zinc metalloprotease motif . Although its mechanism of its action is being studied further, our results suggest that the Orf4 protein is a zinc metalloprotease that modulates the pathogenesis and reactogenicity of epidemic V . cholerae . Based on our findings, we name this VPI-encoded protein Mop (for modulation of pathogenesis).

Infect Immun, 2003 Jan, 71(1), 298 - 308
luxS and arcB control aerobic growth of Actinobacillus actinomycetemcomitans under iron limitation; Fong KP et al.; LuxS is responsible for the production of autoinducer 2 (AI-2), which functions in Vibrio harveyi as a quorum-sensing signal that controls the cell density-dependent expression of the lux operon . In nonluminescent organisms, the physiologic role of AI-2 is not clear . We report that inactivation of luxS in Actinobacillus actinomycetemcomitans JP2 results in reduced growth of the mutant, but not the wild-type organism, under aerobic, iron-limited conditions . Stunted cultures of the luxS mutant A . actinomycetemcomitans JP2-12 grew to high cell density when subcultured under iron-replete conditions . In addition, the mutant strain grew to high cell density under iron limitation after transformation with a plasmid containing a functional copy of luxS . Results of real-time PCR showed that A . actinomycetemcomitans JP2-12 exhibited significantly reduced expression of afuA (eightfold), fecBCDE (10-fold), and ftnAB (>50-fold), which encode a periplasmic ferric transport protein, a putative ferric citrate transporter, and ferritin, respectively . The expressions of putative receptors for transferrin, hemoglobin, and hemophore binding protein were also reduced at more modest levels (two- to threefold) . In contrast, expressions of sidD and frpB (encoding putative siderophore receptors) were increased 10- and 3-fold, respectively, in the luxS mutant . To better understand the mechanism of the AI-2 response, the A . actinomycetemcomitans genome was searched for homologs of the V . harveyi signal transduction proteins, LuxP, LuxQ, LuxU, and LuxO . Interestingly, ArcB was found to be most similar to LuxQ sensor/kinase . To determine whether arcB plays a role in the response of A . actinomycetemcomitans to AI-2, an arcB-deficient mutant was constructed . The isogenic arcB mutant grew poorly under anaerobic conditions but grew normally under aerobic iron-replete conditions . However, the arcB mutant failed to grow aerobically under iron limitation, and reverse transcriptase PCR showed that inactivation of arcB resulted in decreased expression of afuA and ftnAB . Thus, isogenic luxS and arcB mutants of A . actinomycetemcomitans exhibit similar phenotypes when cultured aerobically under iron limitation, and both mutants exhibit reduced expression of a common set of genes involved in the transport and storage of iron . These results suggest that LuxS and ArcB may act in concert to control the adaptation of A . actinomycetemcomitans to iron-limiting conditions and its growth under such conditions.

J Food Prot, 2002 Dec, 65(12), 1873 - 80
Density of total and pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and Gulf coast molluscan shellfish at harvest; Cook DW et al.; The densities of total and pathogenic Vibrio parahaemolyticus in 671 samples of molluscan shellfish harvested in 1999 and 2000 from 14 sites in seven Gulf and Atlantic coast states were determined at 2-week intervals over a period of 12 to 16 months in each state . Changes in V . parahaemolyticus densities in shellfish between harvest and sample analysis were minimized with time and temperature controls . Densities were measured by direct plating techniques, and gene probes were used for identification . Total and pathogenic V . parahaemolyticus organisms were identified with probes for the thermolabile direct hemolysin (tlh) gene and the thermostable direct hemolysin (tdh) gene, respectively . An enrichment procedure involving 25 g of shellfish was also used for the recovery of pathogenic V . parahaemolyticus . The densities of V . parahaemolyticus in shellfish from all harvest sites were positively correlated with water temperature . Shellfish from the Gulf Coast typically had higher densities of V . parahaemolyticus than did shellfish harvested from the North Atlantic or mid-Atlantic coast . Vibrio parahaemolyticus counts exceeded 1,000 CFU/g for only 5% of all samples . Pathogenic (tdh+) V . parahaemolyticus was detected in approximately 6% of all samples by both procedures, and 61.5% of populations in the positive samples from the direct plating procedure were at the lower limit of detection (10 CFU/g) . The frequency of detection of pathogenic V . parahaemolyticus was significantly related to water temperature and to the density of total V . parahaemolyticus . The failure to detect pathogenic V . parahaemolyticus in shellfish more frequently was attributed to the low numbers and uneven distribution of the organism.

Protein Sci, 2003 Jan, 12(1), 27 - 33
A structural basis for the mechanism of aspartate-beta-semialdehyde dehydrogenase from Vibrio cholerae; Blanco J et al.; L-Aspartate-beta-semialdehyde dehydrogenase (ASADH) catalyzes the reductive dephosphorylation of beta-aspartyl phosphate to L-aspartate-beta-semialdehyde in the aspartate biosynthetic pathway of plants and micro-organisms . The aspartate pathway produces fully one-quarter of the naturally occurring amino acids, but is not found in humans or other eukaryotic organisms, making ASADH an attractive target for the development of new antibacterial, fungicidal, or herbicidal compounds . We have determined the structure of ASADH from Vibrio cholerae in two states; the apoenzyme and a complex with NADP, and a covalently bound active site inhibitor, S-methyl-L-cysteine sulfoxide . Upon binding the inhibitor undergoes an enzyme-catalyzed reductive demethylation leading to a covalently bound cysteine that is observed in the complex structure . The enzyme is a functional homodimer, with extensive intersubunit contacts and a symmetrical 4-amino acid bridge linking the active site residues in adjacent subunits that could serve as a communication channel . The active site is essentially preformed, with minimal differences in active site conformation in the apoenzyme relative to the ternary inhibitor complex . The conformational changes that do occur result primarily from NADP binding, and are localized to the repositioning of two surface loops located on the rim at opposite sides of the NADP cleft.

Carbohydr Res, 2002 Nov 29, 337(24), 2437 - 42
The binding of synthetic analogs of the upstream, terminal residue of the O-polysaccharides (O-PS) of Vibrio cholerae O:1 serotypes Ogawa and Inaba to two murine monoclonal antibodies (MAbs) specific for the Ogawa lipopolysaccharide (LPS); Liao X et al.; The binding of nineteen analogues of the upstream, terminal, monosaccharide residue of each of the O-polysaccharide (O-PS) of Vibrio cholerae O:1, serotype Ogawa and Inaba, with two murine monoclonal IgG antibodies both specific for the Ogawa LPS were measured using fluorescence spectroscopy . The use of the deoxy and the deoxyfluoro analogs allowed further refinement of the hydrogen-bonding pattern involved in the binding . Based on the binding characteristics observed for some of the ligands in the Inaba series, the binding of the monosaccharide that represents the upstream, terminal unit of the O-PS of V . cholerae O:1 serotype Inaba was redefined . We show for the first time that the upstream, terminal monosaccharide of the Inaba O-PS shows weak binding with these two anti-Ogawa antibodies . The results obtained allow further rationalization of the structural basis for the binding of V . cholerae O:1 antigens to their homologous antibodies.

Diagn Microbiol Infect Dis, 2002 Nov, 44(3), 221 - 5
A DNA probe specific for Aeromonas colonies; Chacon MR et al.; Members of the genus Aeromonas are important enteropathogens . Commercial identification systems are often unable to correctly identify Aeromonas strains and misidentification as Vibrio spp . is common . A digoxigenin-DNA probe based on a 237 bp of the glycerophospholipid-cholesterol acyltransferase gene has been tested in a colony hybridization assay . The probe hybridized with all Aeromonas species tested (n = 16) but not with strains of other enteropathogenic bacteria (n = 20) . The probe allowed the unequivocal identification of Aeromonas in primary isolation media within 36 h.

Can J Microbiol, 2002 Oct, 48(10), 933 - 9
Biochemical evidence against protein-mediated uptake of myristic acid in the bioluminescent marine bacterium Vibrio harveyi; Byers DM et al.; The bioluminescent marine bacterium, Vibrio harveyi, can utilize exogenous myristic acid (14:0) for beta-oxidation, phospholipid and lipid A synthesis, and as an source of myristyl aldehyde for light emission in the V . harveyi dark mutant M17 . A variety of genetic and biochemical strategies were employed in an attempt to isolate V . harveyi mutants defective in myristate uptake and to characterize proteins involved in this process . Although {3H}myristate uptake in a tritium suicide experiment decreased the survival of nitrosoguanidine-treated M17 cells by a factor of 10(5), none of the surviving cells characterized were defective in either incorporation of exogenous myristate into phospholipid or stimulation of light emission . These parameters were also unaffected when intact M17 cells were treated with proteases . Moreover, M17 double mutants selected on the basis of diminished luminescence response to myristate all incorporated {3H}myristate into lipids normally . Finally, no resistant colonies were obtained using the bacteriocidal fatty acid analogue, 11-bromoundecanoate, and experiments with decanoate (10:0) indicated that the V . harveyi cell envelope is very sensitive to physical disruption by fatty acids . Taken together, these results support an unfacilitated uptake of myristic acid in V . harveyi, in contrast with the regulated vectorial transport and activation of long chain fatty acids in Escherichia coli.

Ecotoxicol Environ Saf, 2002 Sep, 53(1), 170 - 7
Correlation of two bioluminescence and one fluorogenic bioassay for the detection of toxic chemicals; Peinado MT et al.; The linear correlation between the EC50 values of 50 substances obtained in luminescence bioassays using Vibrio fischeri and Vibrio harveyi and in a fluorogenic bioassay using Escherichia coli was investigated . As a result, a significant correlation was found between the said values in all three toxicity tests . The bioassay using V . harveyi had a sensitivity similar to that of the fluorogenic bioassay, and the bioassay using V . fischeri was the least sensitive of all . The sensitivity of the three bioassays for each of the tested substances, chiefly heavy metals, organic solvents, orgnochlorated compounds, and pesticides, differed in the majority of the cases . The three bioassays were quantified using the same laboratory apparatus and the data were processed in the same way . The possibility of designing a battery of toxicity tests that can be performed using the same apparatus but different organisms and parameters is discussed.

J Pak Med Assoc, 2002 Aug, 52(8), 347 - 8
Drug sensitivity pattern of cholera in children; Memon IA et al.; OBJECTIVE: To study the drug sensitivity pattern of cholera in children . SETTING: DTU of Civil Hospital, Karachi . PATIENTS AND METHODS: All children age 2 months to 15 years attending Diarrhoea Treatment Unit (DTU) with acute onset of diarrhoea and dehydration were screened for cholera . Stool samples were collected in alkaline peptone and those positive for cholera had their antibiotic sensitivity determined . RESULTS: Of 846 stool specimens, 161 were positive for V . Cholera . All were sensitive to third generation cephalosporins and quinolones, 98-100% to nalidixic acid, 82-86% to chloramphenicol and 67-75% to doxycycline and all were resistant to cotrimaxazole . CONCLUSION: Cholera can be treated with nalidixic acid or chloramphenical in young children while doxycycline for older children . Cotrimoxazole is not effective . Efforts should be done for identification and surveillance of cholera cases, along with change of sensitivity pattern of Vibrio cholera.

Lancet, 2002 Nov 30, 360(9347), 1722 - 7
Comparison of single-dose azithromycin and 12-dose, 3-day erythromycin for childhood cholera: a randomised, double-blind trial; Khan WA et al.; BACKGROUND: Cholera is a major public-health problem, with children most affected . However, effective single-dose antimicrobial regimens have been identified only for adults . Our aim was to compare the efficacy of azithromycin and erythromycin regimens in the treatment of children . METHODS: We did a double-blind, randomised study of 128 severely dehydrated children (age 1-15 years) with cholera, treated at one of two treatment centres in Bangladesh in 1999 . Children were assigned single-dose azithromycin (20 mg/kg bodyweight, maximum individual dose 1 g; n=65) or 12.5 mg/kg erythromycin (maximum dose 500 mg; n=63) every 6 h for 3 days . Patients stayed in hospital for 5 days . We measured fluid balance every 6 h, and obtained a rectal swab or stool sample for culture daily . Our primary outcome measures were clinical success of treatment-ie, cessation of watery diarrhoea within 48 h-and bacteriological success-ie, absence of Vibrio cholerae O1 or O139 from cultures of stool or rectal swab samples after study day 2 . Analysis was per protocol . FINDINGS: Two children in both groups withdrew from the study, and we excluded one child in the erythromycin group . Treatment was clinically successful in 48 (76%) patients who received azithromycin and 39 (65%) who received erythromycin (difference 11%, 95% CI -5 to 27, p=0.244); and bacteriologically successful in 45 (71%) and 49 (82%) patients, respectively (10%, -5 to 25, p=0.261) . Patients treated with azithromycin had a shorter duration of diarrhoea (median 24 h vs 42 h; difference 12 h, 0-18 h, p=0.019) and fewer episodes of vomiting (1 vs 4; difference 1 episode, 0-3 episodes, p=0.023) . INTERPRETATION: Single-dose azithromycin is as effective for treatment of cholera in children as standard erythromycin therapy, but is associated with less vomiting.

Biochim Biophys Acta, 2002 Sep 23, 1599(1-2), 106 - 14
Purification, characterization and molecular cloning of Vibrio fluvialis hemolysin; Han JH et al.; Hemolysin of Vibrio fluvialis (VFH) was purified from culture supernatants by ammonium sulfate precipitation and successive column chromatographies on DEAE-cellulose and Mono-Q . N-terminal amino acid sequences of the purified VFH were determined . The purified protein exhibited hemolytic activity on many mammalian erythrocytes with rabbit erythrocytes being the most sensitive to VFH . Activity of the native VFH was inhibited by the addition of Zn2+, Ni2+, Cd2+ and Cu2+ ions at low concentrations . Pores formed on rabbit erythrocytes were approximately 2.8-3.7 nm in diameter, as demonstrated by osmotic protection assay . Nucleotide sequence analysis of the vfh gene revealed an open reading frame (ORF) consisting of 2200 bp which encodes a protein of 740 amino acids with a molecular weight of 82 kDa . Molecular weight of the purified VFH was estimated to be 79 kDa by SDS-PAGE and N-terminal amino acid sequence revealed that the 82 kDa prehemolysin is synthesized in the cytoplasm and is then secreted into the extracellular environment as the 79 kDa mature hemolysin after cleavage of 25 N-terminal amino acids . Deletion of 70 amino acids from the C-terminus exhibited a smaller hemolytic activity, while deletion of 148 C-terminal amino acids prevented hemolytic activity.

Arch Inst Pasteur Madagascar, 1999, 65(1-2), 71 - 4
{First cases of cholera observed in children at the Befelatanana General Hospital--Antananarivo University Hospital Center (Madagascar)}; Raobijaona H et al.; Ravages caused by cholera among children are well known . The disease invaded Madagascar in 1999 May . This retrospective study reported the first childhood cholera cases . The survey was carried out at the Befelatanana Hospital during the period of cholera outbreak from April 23th to July 31st . The purpose of the study was to specify clinical, epidemiological and bacteriological characteristics of the disease . 5 out of 178 suspected cholera cases were less than 15 years old . 2 young girls out these 5 children, inhabitants of Antananarivo-City were hospitalized for acute diarrhoea with serious dehydratation . Their disease was confirmed by bacteriology . Vibrio cholera O1 strain, serovar Ogawa was identified . Epidemiological investigation allowed to identify the contamination modal in the file no 1 . The authors conclude that cholera is an important problem of public health in developing country like Madagascar . Disease control needs environmental sanitation and good individual hygiene practices.

Arch Inst Pasteur Madagascar, 2001, 67(1-2), 6 - 13
{Vibrio cholerae in Madagascar: study of a multiresistant strain}; Rakoto Alson AO et al.; Madagascar was cholera free until March 1999 . The first case was reported in Mahajanga, a north west coast harbor . Ten months later and despite a massive use of tetracycline as prophylactic drug, cholera had reached every region of the island . All suspected cholera samples were analysed at the Pasteur Institute of Madagascar where susceptibility to tetracycline was systematically performed . On February 2000, a multidrug resistant strain of V . cholerae was isolated . We studied this strain by performing Minimal Inhibitory Concentration (MIC) and by plasmidic and conjugative assay . As the original strain, this multiresistant V . cholerae showed a resistance to cotrimoxazole, to streptomycin and chloramphenicol but, in addition to, appeared strongly resistant to ampicillin and tetracycline . This strain harboured a 26 kb self-transmissible plasmid . Conjugation tests showed the possibility of plasmidic segregates or acquisition of two different plasmids . The weak transfer rate could explain why we have isolated only one multiresistant strain . The emergence of a such multiresistant strain should encourage the medical authorities to reinforce the epidemic survey in every medical Malagasy district and to carry out new antimicrobial surveys to describe the mechanisms of the spread of these resistances.

Arch Microbiol, 2002 Dec, 179(1), 57 - 65 Epub 2002 Nov 09.
The Vibrio fischeri sapABCDF locus is required for normal growth, both in culture and in symbiosis; Lupp C et al.; Inactivation of the sapABCDF genes results in a loss of virulence in several bacterial pathogens of animals and plants . The role of this locus in the growth physiology of Vibrio fischeri, and in the symbiotic colonization of the squid Euprymna scolopes was investigated . In rich medium, a V . fischeri sapA insertion mutant grew at only 85% the rate of its wild-type parent . While a similar effect has been attributed to a potassium-transport defect in sap mutants of enteric bacteria, the V . fischeri mutant grew more slowly regardless of the potassium concentration of the medium . Similarly, the growth-rate defect was independent of the source of either carbon, nitrogen, or phosphorous, indicating that the V . fischeri sap genes do not encode functions required for the transport of a specific form of any of these nutrients . Finally, while a delay in colonizing the nascent light organ of the squid could be accounted for by the lower growth rate of the mutant, a small but statistically significant reduction in its final population size in the host, but not in medium, suggests that the sap genes play another role in the symbiosis . All of these phenotypic defects could be genetically complemented in trans by the sapABCDF genes, but not by the sapA gene alone, indicating that the insertion in sapA is polar to the four downstream genes in the locus . Thus, while the sap locus is important to the normal growth of V . fischeri, it plays different physiological roles in growth and tissue colonization than it does in enteric pathogens.

Ecotoxicology, 2002 Oct, 11(5), 379 - 83
A marine bioassay test set to assess marine water and sediment quality-its need, the approach and first results; Peters C et al.; There is a need for establishing a marine bioassay test set to assess marine water and sediment samples in Germany . The selected marine bioassay test set, two tests for the water phase (with the luminescence bacteria Vibrio fischeri and the algae Phaeodactylum tricornutum Bohlin) and a whole sediment test with the marine amphipod Corophium volutator (Pallas) is described and first results are shown.

Biol Res, 2002, 35(3-4), 433 - 40
Characterization by PCR of Vibrio parahaemolyticus isolates collected during the 1997-1998 Chilean outbreak; Cordova JL et al.; Between November 1997 and April 1998, several human gastroenteritis cases were reported in Antofagasta, a city in the north of Chile . This outbreak was associated with the consumption of shellfish, and the etiologic agent responsible was identified as Vibrio parahaemolyticus . This was the first report of this bacterium causing an epidemic in Chile . V . parahaemolyticus was the only pathogenic bacterium isolated from patient stools and from shellfish samples . These isolates were analyzed by polymerase chain reaction (PCR) amplification of the pR72H gene, a species-specific sequence . Based on the pR72H gene amplification pattern, at least three different isolates of V . parahaemolyticus were found . Two isolates (named amplicons A and C) generated PCR products of approximately 400 bp and 340 bp respectively, while another type of isolate designated B, did not generate a PCR product, regardless of which method of DNA purification was used . Sequence analysis of the amplicons A and C shows that they have an 80 bp and 183 bp conserved region at the 5' end of the gene . However, both isolates have different sequences at their 3' terminus and are also different from the pR72H sequence originally reported . Using this PCR assay we demonstrated that these three isolates were found in clinical samples as well as in shellfish . The warm seawater caused by the climatological phenomena "El Nino" perhaps favored the geographic dispersion of the bacterium (bacterial bloom) occurring in Antofagasta that occurred during that time of year.

Biochim Biophys Acta, 2002 Dec 2, 1556(2-3), 113 - 20
Kinetics of the spectral changes during reduction of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi; Bogachev AV et al.; Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy . The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone . The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone . The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species . The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH2 at the NADH dehydrogenating site . The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMN's . Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH . The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone . One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR.

Plasmid, 2002 Nov, 48(3), 222 - 8
Plasmid-mediated iron uptake and virulence in Vibrio anguillarum; Stork M et al.; The plasmid pJM1 of Vibrio anguillarum harbors genes encoding proteins that enable the bacterial cell to survive under iron limiting conditions . A subset of these proteins are involved in the biosynthesis of the siderophore anguibactin and in the internalization of the ferric-siderophore into the cell cytosol . We have identified several genes encoding non-ribosomal peptide synthetases that catalyze the synthesis of anguibactin, these genes are: angB/G, angM, angN, angR, and angT . In addition, the genes fatA, fatB, fatC, and fatD are involved in the transport of ferric-anguibactin complexes . These transport genes, together with the biosynthesis genes angR and angT, are included in the iron transport biosynthesis operon (ITBO) . Both the biosynthesis and the transport genes are under tight positive as well as negative control . We have identified four regulators; two of them, a chromosomally encoded Fur and a plasmid-mediated antisense RNA, RNAbeta, act in a negative fashion, while positive regulation is facilitated by AngR and TAFr . We also have evidence that the siderophore itself plays a positive role in the regulatory mechanism of the expression of both transport and biosynthesis genes.

FEBS Lett, 2002 Dec 4, 532(1-2), 221 - 6
Involvement of in vivo induced cheY-4 gene of Vibrio cholerae in motility, early adherence to intestinal epithelial cells and regulation of virulence factors; Banerjee R et al.; Using a global transcription profile approach cheY-4 of Vibrio cholerae was identified as an in vivo induced gene . In the present study, duplication of the gene in the chromosome resulted in increased motility, increased chemotactic response towards isolated intestinal mucus layer and stronger adhesion to human intestinal epithelial cell line at an early phase of infection compared to wild type and a null mutant strain . In contrast to the cheY-4 null mutant, duplication of cheY-4 gene resulted in increased expression of ctxAB and tcpA, the two major virulence genes of V . cholerae.

J Microbiol Methods, 2003 Feb, 52(2), 273 - 7
Improved recovery of pathogenic Vibrio parahaemolyticus from oysters using colony hybridization following enrichment; Nordstrom JL et al.; The traditional streak plating and alternative spread-plating methods were compared for detection of pathogenic Vibrio parahaemolyticus (Vp) in oyster enrichments . We found the alternative method to be more efficient: it was quicker (2d vs . 3d) and had a significantly (p < 0.05) greater detection rate than streak plating.

Pharm Res, 2002 Nov, 19(11), 1680 - 8
Enhanced permeability of molecular weight markers and poorly bioavailable compounds across Caco-2 cell monolayers using the absorption enhancer, zonula occludens toxin; Cox DS et al.; PURPOSE: Zonula occludens toxin (Zot), a protein elaborated from Vibrio cholerae, has been shown to be capable of reversibly opening tight junctions . The objective of this work was to determine the stability of Zot and to examine the permeability of a series of molecular weight hydrophilic markers and therapeutic agents in the presence of Zot . METHOD: The transport of molecular weight markers (i.e., PEG 4000, FITC-dextran 10.000 and inulin) and therapeutic agents (i.e., acyclcovir, cyclopsorin, paclitaxel . doxorubicin) was evaluated with Zot (0, 2, and 4 microg/mL) using Caco-2 cell monolayers . RESULTS: Zot was found to be stable over a 10-day period . Significantly higher (p < 0.05) permeability of the molecular weight markers, in lin, and PEG4000 were observed with Zot (4 microg/mL) . The transport of each therapeutic marker was significantly increased with paclitaxel displaying a >3-fold enhancement in Papp values with Zot (4 microg/mL) . A 30% decrease in transepithelial electrical resistance values wa observed, which returned to baseline 30 min after Zot was removed . CONCLUSIONS: Considering the problems of poor oral bioavailability, it is concluded that Zot is a promising drug delivery technology to be used to enhance drug transport across the intestinal mucosa . Future applications are targeted at assessing its usefulness in oral drug delivery using in vivo systems.

J Appl Microbiol, 1997 Mar, 82(3), 365 - 71
Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production; Pedersen K et al.; The virulence of 18 strains of Vibrio anguillarum serogroup O1 was compared to plasmid content, expression of siderophores and outer membrane proteins . All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions . Only strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2 . All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence . However, some strains carrying the virulence plasmid or a derivative of it, produced siderophores as well as OM2 but were non-pathogenic to fish . Likewise, among the virulent strains, considerable variation in LD50 values was recorded . Plasmid profiling and restriction analysis showed that the virulence plasmid existed in various molecular weights from 26 to 80 kbp, with 65-67 kbp being the most common, and that this plasmid displayed various restriction profiles . The presence of other plasmids did not seem to affect the pathogenic properties.

J Appl Microbiol, 2002, 93(6), 1089 - 98
The complete nucleotide sequence of the Vibrio harveyi bacteriophage VHML; Oakey HJ et al.; AIMS: To determine the complete nucleotide sequence of the bacteriophage VHML and establish a hypothesis for the virulence conversion caused by VHML infection of Vibrio harveyi . METHODS AND RESULTS: The complete nucleotide sequence of VHML was determined (43,193 bp) and used to identify putative genes . The translated products of these genes were compared with reported sequences to assign hypothetical functions . All anticipated structural genes and putative genes for lysogeny were identified . In addition, we found a complete N6-adenine methyltransferase (Dam) gene that appeared to have an essential site for ADP-ribosylating toxins at the C-terminal of the translated product . CONCLUSIONS: Virulence conversion of V . harveyi by VHML may be associated with Dam transcriptional regulation . The Dam gene may also encode for a toxin component similar to ADP-ribosylating toxins . SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript lays the foundation for understanding the virulence of toxin-producing V . harveyi . Further research into aspects discussed here will lead to a greater comprehension regarding the invertebrate disease vibriosis and its control in the farming of these animals.

J Appl Microbiol, 1997 Feb, 82(2), 211 - 8
Vibrio anguillarum serogroup O3 and V . anguillarum-like serogroup O3 cross-reactive species--comparison and characterization; Tiainen T et al.; Forty-five Vibrio anguillarum-like isolates reacting with V . anguillarum serogroup O3 antiserum were examined in 30 characters to clarify their phenotypical properties, while their genotype was examined by ribotyping . The strains were isolated from diseased and dead fish or from environmental sources such as water, sediment, plankton, and faeces and gills of healthy fish . Phenotypically, the similarity of all the strains was more than 90% . However, significant differences between the fish-associated and environmental strains were detected . Biochemically, deviations were found in the Voges-Proskauer test and lysine decarboxylase reaction . Clustering analysis of the ribotypes showed two distinct clusters with a similarity of only 32% . Two strains representing each of these groups were used in a LD50 study, which showed some difference also in the pathogenicity between environmental and fish strains . It is suggested that the environmental strains belong to another species than V . anguillarum, but serologically cross-reacting with the V . anguillarum serogroup O3 . The ribotyping as well as biochemical results indicated that the environmental strains possibly belong to Vibrio aestuarianus . The bona fide V . anguillarum serogroup O3 strains proved to be very homogeneous both phenotypically and genotypically, and the similarity of ribotypes was more than 96% . The V . anguillarum-like, serogroup O3-reactive strains from the environment were more heterogeneous in their biochemical behaviour, and showed an approximately 70% similarity in ribotypes.

J Appl Microbiol, 1997 Feb, 82(2), 157 - 67
High affinity iron-uptake systems in Vibrio damsela: role in the acquisition of iron from transferrin; Fouz B et al.; In this work, the high affinity iron-acquisition systems displayed by virulent and avirulent strains of Vibrio damsela have been investigated . This species is an autochthonous member of marine ecosystems that can behave as an opportunistic pathogen for fish and mammals . All strains tested (i) were able to grow under the restricted conditions imposed by the iron chelators transferrin (Tf) and EDDHA, (ii) secreted siderophores of hydroxamic type, other than aerobactin and desferal, that were able to stimulate the growth of the auxotroph mutant Arthrobacter flavescens JG9, and (iii) expressed common iron-regulated outer membrane proteins (IROMPs) . No change in LPS patterns was observed in response to iron restriction . Results from the assays with transferrin suggest that these siderophores could be utilized to sequester iron from Tf, a protein for which no surface receptor was detected in any strain . In summary, the overall data demonstrate that V . damsela expresses siderophore-mediated iron-uptake systems . These systems are probably involved in the survival of the species in the different environments that it can colonize, i.e . water and several vertebrate hosts.

Indian J Med Res, 2000 Nov, 112, 178 - 82
Severe cholera outbreak following floods in a northern district of West Bengal; Sur D et al.; BACKGROUND & OBJECTIVES: An explosive epidemic of cholera in the district of Malda in the state of West Bengal, was induced by devastating floods resulting from overflowing of the two main rivers of the district, at the end of July 1998, affecting 15 blocks and 2 municipalities . Diarrhoeal outbreak occurred around the middle of August after receding of the flood waters . Within two weeks of its onset, the outbreak spread throughout the district . An investigation was conducted to understand the epidemiological characteristics, identify the etiological agent, rationalise clinical management and suggest control measures . METHODS: The team visited the Block Primary Health Centres, surrounding the affected villages and also the district hospital . Morbidity and mortality data were collected and 88 patients were interviewed and examined clinically . Epidemiological and clinical observations were recorded . Rectal swabs were collected from both hospitalised and domiciliary cases . RESULTS: During the period between August and October 1998, 16,590 cases were reported with 276 deaths (case fatality rate of 1.7%) . Twenty one of 29 (72%) rectal swabs were positive for Vibrio cholerae O1, biotype ElTor, serotype Ogawa . All the strains were sensitive to tetracycline, norfloxacilin, ciprofloxacilin, gentamycin, chloramphenicol but resistant to furazolidine, co-trimoxazole, nalidixic acid, streptomycin and ampicilin . INTERPRETATION & CONCLUSIONS: Observations of the present study identified the epidemiological and clinical deficiencies in the management of the outbreak and recommendations were elaborated for its effective control.

Appl Environ Microbiol, 2002 Dec, 68(12), 6310 - 20
Conspicuous veils formed by vibrioid bacteria on sulfidic marine sediment; Thar R et al.; We describe the morphology and behavior of a hitherto unknown bacterial species that forms conspicuous veils (typical dimensions, 30 by 30 mm) on sulfidic marine sediment . The new bacteria were enriched on complex sulfidic medium within a benthic gradient chamber in oxygen-sulfide countergradients, but the bacteria have so far not been isolated in pure culture, and a detailed characterization of their metabolism is still lacking . The bacteria are colorless, gram-negative, and vibrioid-shaped (1.3- to 2.5- by 4- to 10- micro m) cells that multiply by binary division and contain several spherical inclusions of poly-beta-hydroxybutyric acid . The cells have bipolar polytrichous flagella and exhibit a unique swimming pattern, rotating and translating along their short axis . Free-swimming cells showed aerotaxis and aggregated at ca . 2 micro M oxygen within opposing oxygen-sulfide gradients, where they were able to attach via a mucous stalk, forming a cohesive whitish veil at the oxic-anoxic interface . Bacteria attached to the veil kept rotating and adapted their stalk lengths dynamically to changing oxygen concentrations . The joint action of rotating bacteria on the veil induced a homogeneous water flow from the oxic water region toward the veil, whereby the oxygen uptake rate could be enhanced up to six times, as shown by model calculations . The veils showed a pronounced succession pattern . New veils were generated de novo within 24 h and had a homogeneous whitish translucent appearance . Bacterial competitors or eukaryotic predators were apparently kept away by the low oxygen concentration prevailing at the veil surface . Frequently, within 2 days the veil developed a honeycomb pattern of regularly spaced holes . After 4 days, most veils were colonized by grazing ciliates, leading to the fast disappearance of the new bacteria . Several-week-old veils finally developed into microbial mats consisting of green, purple, and colorless sulfur bacteria.

Appl Environ Microbiol, 2002 Dec, 68(12), 6077 - 86
Sequencing-independent method to generate oligonucleotide probes targeting a variable region in bacterial 16S rRNA by PCR with detachable primers; Bertilsson S et al.; Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments . We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence . Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region . The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers . First, a double-stranded product is generated, which then serves as template in a linear amplification . After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3' end of the primers . This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target . As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of VIBRIO: The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization . An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.

Indian J Pediatr, 2002 Oct, 69(10), 909 - 10
Non-01 Vibrio cholerae septicemia and meningitis in a neonate; Kerketta JA et al.; Non-O1 Vibrio cholerae is known to cause diarrhoea as well as extra-intestinal infections in adults and children . However meningitis in children is a rare occurrence . We report a neonate who developed septicemia and meningitis due to Non-O1 Vibrio cholerae.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Jul-Aug, (4), 25 - 9
{Evaluation of polysaccharide antigens of Vibrio cholerae O139 of different origin by monoclonal antibodies}; Alekseeva LP et al.; The epitope composition of O-polysaccharides in the lipopolysaccharide (LPS) of V . cholerae, serogroup O139, isolated from clinical material and water of surface reservoirs was analyzed with the use of monoclonal antibodies . The analysis demonstrated that these O-polysaccharides were similar in their structure and chemical composition . In LPS of V . cholerae O139 clinical strains O-polysaccharide determinants occurred more often . Among V . cholerae isolated from water strains on whose surface individual epitopes of O-polysaccharide occurred less frequently or were absent appeared to be more numerous . A decrease in the concentration of microbial cells in the process of their testing by immunological methods led to increased percent of negative reactions with specific antibodies . Some V . cholerae O139 strains isolated from water were similar in the epitope composition of their O-polysaccharide and binding activity to cultures isolated from humans . As indicated by the results of these studies, cholera vibrios Bengal and vibrios isolated from river water on the territory of Russia had quantitative differences due to a higher level of the production of O-polysaccharide determinants and their occurrence in V . cholerae of serogroup O139.

J Infect Dis, 2002 Dec 1, 186(11), 1615 - 20 Epub 2002 Nov 11.
Clinical, epidemiological, and socioeconomic analysis of an outbreak of Vibrio parahaemolyticus in Khanh Hoa Province, Vietnam; Tuyet DT et al.; From 1996 onward, a pandemic spread of Vibrio parahaemolyticus infections due to one clone has been reported in several Asian countries . During a population-based study that relied on passive surveillance, 548 cases of V . parahaemolyticus infection were detected between 1997 and 1999 in the Khanh Hoa province of Vietnam . Detection of cases of V . parahaemolyticus infection abruptly stopped in November 1999, although Vibrio species other than V . parahaemolyticus continued to be isolated throughout 2000 . Of the infections, 90% occurred in individuals >5 years old; 53% of the patients presented with watery stools, and 6% reported blood in their stools . All patients had recovered by the time of discharge . A surprising risk factor for V . parahaemolyticus infections was high socioeconomic status . Like the interruption of the transmission of V . cholerae infections that had been observed earlier, the transmission of V . parahaemolyticus came to a halt without meteorological changes or changes in water supply and sanitation.

Biochim Biophys Acta, 2002 Dec 16, 1601(2), 208 - 14
Identification of a key residue in the conformational stability of acyl carrier protein; Keating MM et al.; Conformational flexibility of acyl carrier protein (ACP) is important for its ability to interact with multiple enzymes in bacterial fatty acid metabolism . We have recently shown that, unlike the prototypical ACP from Escherichia coli, the more acidic Vibrio harveyi ACP is largely unfolded at physiological pH . Mutations D18K, A75H and A75H/D18K were made in recombinant V . harveyi ACP (rACP) to determine the importance of basic residues Lys-18 and His-75 in maintaining the native conformation of E . coli ACP . Both D18K and A75H ACPs were fatty acylated by acyl-ACP synthetase, showing that neither mutation grossly alters tertiary structure . Circular dichroism (CD) indicated that rACP refolded upon addition of MgCl(2) at 100-fold lower concentrations (<1 mM) than KCl, suggesting that divalent cations stabilize rACP by interaction at specific sites . Surprisingly, mutants A75H and A75H/D18K exhibited native-like conformation in the absence of MgCl(2), while the D18K mutant was comparable to rACP . Moreover, the alpha-helical content of A75H, A75H/D18K and E . coli ACPs was more sensitive than that of rACP or D18K ACP to modification by the histidine-selective reagent diethylpyrocarbonate . Together, these results suggest that the partial positive charge of His-75 may be important in maintaining the conformational stability of E . coli ACP at a neutral pH.

Vaccine, 2002 Nov 22, 21(1-2), 138 - 45
Calibrated serological techniques demonstrate significant different serum response rates to an oral killed cholera vaccine between Swedish and Nicaraguan children; Hallander HO et al.; Serum responses to oral cholera vaccines were assessed in three paediatric vaccine trials, two in Leon, Nicaragua and one in Stockholm, Sweden . A calibrated anti-cholera toxin B subunit (CTB) IgA ELISA was used together with an assay for vibriocidal antibodies . Swedish children had lower pre-vaccination levels of antibody, but serum responses were more pronounced in Swedish children than in Nicaraguan children . Post-vaccination levels of anti-toxin antibody were generally above those found after natural infections with enterotoxigenic Escherichia coli, that cross-reacts serologically with Vibrio cholerae . Adverse events seen after vaccination were generally mild and of little clinical significance.

Fish Shellfish Immunol, 2002 Oct, 13(4), 293 - 309
The roles of haemocytes and the lymphoid organ in the clearance of injected Vibrio bacteria in Penaeus monodon shrimp; van de Braak CB et al.; In order to study the reaction of Penaeus monodon haemocytes, live Vibrio anguillarum bacteria were injected and the shrimp were periodically sampled . Immuno-double staining analysis with specific antisera against the haemocyte granules and bacteria showed that large numbers of haemocytes encapsulated the bacteria at the site of injection . A rapid decrease of live circulating bacteria was detected in the haemolymph . Bacterial clearance in the haemolymph was induced by humoral factors, as observed by agglutinated bacteria, and followed by uptake in different places in the body . Bacteria mainly accumulated in the lymphoid organ (LO), where they, or their degradation products, could be detected for at least 7 days after injection . The LO consists of folded tubules with a central haemal lumen and a wall, layered with cells . The haemolymph, including the antigens, seemed to migrate from the central tubular lumen through the wall, where the bacteria are arrested and their degradation is started . Electron microscopy of the LO revealed the presence of many phagocytic cells that morphologically resemble small-granular haemocytes . It is proposed that haemocytes settle in the tubule walls before they phagocytose . Immunostaining suggests that many of the haemocytes degranulate in the LO, producing a layer of fibrous material in the outer tubule wall . These findings might contribute to the reduced haemocyte concentration in the haemolymph of diseased animals or following injection of foreign material . It is proposed that the LO is a filter for virtually all foreign material encountered in the haemolymph . Observations from the present study are similar to clearance mechanisms in the hepatic haemolymph vessel in most decapod crustaceans that do not possess a LO . The experimental shrimp appeared to contain many LO spheroids, where bacterial antigens were finally observed as well . It is proposed that the spheroids have a degradation function for both bacterial and viral material, and that their presence is primarily related to the history of the infectious burden of the shrimp.

Microbiology, 2002 Nov, 148(Pt 11), 3681 - 93
Characterization of a novel Vibrio pathogenicity island (VPI-2) encoding neuraminidase (nanH) among toxigenic Vibrio cholerae isolates; Jermyn WS et al.; Acquisition of virulence genes encoded on mobile genetic elements has played an important role in the emergence of pathogenic isolates of Vibrio cholerae, the causative agent of the diarrhoeal disease cholera . The genes encoding cholera toxin (ctxAB), the main cause of profuse secretory diarrhoea in cholera, are encoded on a filamentous bacteriophage CTXphi . The toxin coregulated pilus (TCP), an essential intestinal colonization factor, was originally designated as part of a pathogenicity island named the Vibrio pathogenicity island (VPI), but this island has more recently been proposed to be the genome of a filamentous phage, VPIphi . In this study, it is shown that nanH, which encodes neuraminidase, maps within a novel pathogenicity island designated VPI-2 . The 57.3 kb VPI-2 has all of the characteristic features of a pathogenicity island, including the presence of a bacteriophage-like integrase (int), insertion in a tRNA gene (serine) and the presence of direct repeats at the chromosomal integration sites . Additionally, the G+C content of VPI-2 (42 mol%) is considerably lower than that of the entire genome (47 mol%) . VPI-2 encodes several gene clusters, such as a restriction modification system (hsdR and hsdM) and genes required for the utilization of amino sugars (nan-nag region) as well as neuraminidase . To determine the distribution of VPI-2 among V . cholerae, 78 natural isolates were examined using PCR and Southern hybridization analysis for the presence of this region . All toxigenic V . cholerae O1 serogroup isolates examined contained VPI-2, whereas non-toxigenic isolates lacked the island . Of 14 V . cholerae O139 serogroup isolates examined, only one strain, MO2, contained the entire 57.3 kb island, whereas 13 O139 isolates contained only a 20.0 kb region with most of the 5' region of VPI-2 which included nanH deleted in these strains.

J Bacteriol, 2002 Dec, 184(23), 6592 - 601
Vibrio cholerae phage K139: complete genome sequence and comparative genomics of related phages; Kapfhammer D et al.; In this report, we characterize the complete genome sequence of the temperate phage K139, which morphologically belongs to the Myoviridae phage family (P2 and 186) . The prophage genome consists of 33,106 bp, and the overall GC content is 48.9% . Forty-four open reading frames were identified . Homology analysis and motif search were used to assign possible functions for the genes, revealing a close relationship to P2-like phages . By Southern blot screening of a Vibrio cholerae strain collection, two highly K139-related phage sequences were detected in non-O1, non-O139 strains . Combinatorial PCR analysis revealed almost identical genome organizations . One region of variable gene content was identified and sequenced . Additionally, the tail fiber genes were analyzed, leading to the identification of putative host-specific sequence variations . Furthermore, a K139-encoded Dam methyltransferase was characterized.

Mol Microbiol, 2002 Nov, 46(4), 1135 - 47
Regulation of virulence gene expression in Vibrio cholerae by quorum sensing: HapR functions at the aphA promoter; Kovacikova G et al.; Quorum sensing negatively influences virulence gene expression in certain toxigenic Vibrio cholerae strains . At high cell densities, the response regulator LuxO fails to reduce the expression of HapR, which, in turn, represses the expression of the virulence cascade . A critical regulatory step in the cascade is activation of tcpPH expression by AphA and AphB . We show here that HapR influences the virulence cascade by directly repressing aphA expression . In strain C6706, aphA expression was increased in a delta hapR mutant and decreased in a delta luxO mutant, indicating a negative and positive influence, respectively, of these gene products on the promoter . Overexpression of HapR also reduced aphA expression in both C6706 and Escherichia coli . DNase I footprinting showed that purified HapR binds to the aphA promoter between -85 and -58 . Although it appears that quorum sensing does not influence virulence gene expression in strain O395 solely because of a frameshift in hapR, overproduced HapR did not repress expression from the O395 aphA promoter in either Vibrio or E . coli, nor did the protein bind to the promoter . Two basepair differences from C6706 are present in the O395 HapR binding site at -85 and -77 . Introducing the -77 change into C6706 prevented HapR binding and repression of aphA expression . This mutation also eliminated the repression of toxin-co-regulated pilus (TCP) and cholera toxin (CT) that occurs in a delta luxO mutant, indicating that HapR function at aphA is critical for density-dependent regulation of virulence genes.

Syst Appl Microbiol, 2002 Oct, 25(3), 403 - 15
New methods for the analysis of binarized BIOLOG GN data of Vibrio species: minimization of stochastic complexity and cumulative classification; Gyllenberg M et al.; We apply minimization of stochastic complexity and the closely related method of cumulative classification to analyse the extensively studied BIOLOG GN data of Vibrio spp . Minimization of stochastic complexity provides an objective tool of bacterial taxonomy as it produces classifications that are optimal from the point of view of information theory . We compare the outcome of our results with previously published classifications of the same data set . Our results both confirm earlier detected relationships between species and discover new ones.

Water Res, 2002 Oct, 36(17), 4255 - 62
Toxicity assays: a way for evaluating AOPs efficiency; Fernandez-Alba AR et al.; The technical feasibility and performance of photocatalytic degradation of aqueous methomyl (50 mg/L) have been studied at pilot scale in two well-defined systems of special interest because natural-solar UV light can be used: heterogeneous photocatalysis with titanium dioxide and homogeneous photocatalysis by photo-Fenton . The pilot plant is made up of compound parabolic collectors specially designed for solar photocatalytic applications . Experimental conditions allowed pesticide disappearance, degree of mineralisation and toxicity achieved in the two photocatalytic systems to be compared . Total disappearance of methomyl is attained by photo-Fenton in 60 min and by TiO2 in 100 min . Hundred percent of nitrogen and sulphur are recovered as ammonium and sulphate . By contrast, complete mineralisation of total organic carbon (TOC) is not achieved even after quite a long time (more than 300 min) . Three different bioassays (Vibrio fischeri, Daphnia magna and a Microalga) have been used for testing the progress of toxicity during treatment . All remained toxic down to very low-pesticide concentrations and in some bioassays were still toxic after total disappearance of the pesticide . Only if treatment is maintained throughout enough mineralisation (i.e . TOC disappearance), the toxicity is reduced to below the threshold (EC50%).

Immunol Lett, 2002 Dec 3, 84(3), 185 - 90
Efficacy of oral administration and oral intake of edible vaccines; Lauterslager TG et al.; To evaluate whether vaccine administration via intragastric gavage is indicative for the outcome of edible vaccines, mice were orally immunised with ovalbumin (OVA) mixed with or without Vibrio cholerae toxin (CT) in various compositions via various routes: (1) OVA dissolved in saline and intragastrically (IG) administered ('IG'); (2) OVA mixed with food extract and administered IG ('food IG'); (3) food chow absorbed with OVA dissolved in saline and fed to the animals ('food'); and (4) OVA dissolved in saline and administered via drinking bottles ('drinking') . When given to naive mice, 'IG' and 'food IG' but not 'food' or 'drinking' induced anti-OVA IgG1 responses in serum, but oral boost immunisations were necessary . Serum IgA was not induced . Oral boosting of subcutaneously (SC) primed mice enhanced the IgG1 and IgA response in serum regardless of the route of immunisation or the vaccine composition . CT did not dramatically enhance the immune response . All immunisation routes except 'drinking' induced antigen-specific IgA antibody secreting cells (ASC) in the lamina propria of naive mice . But antigen-specific antibody responses in faeces were not observed . We concluded that oral (i.e . IG) administration is distinct from oral intake . The composition of the vaccine (food or saline) did not influence oral administration . We thus suggested that the route of administration greatly influenced the outcome of oral immunisation . Although oral administration is a well-accepted route to test the potentials of oral vaccines, our study demonstrated that it is merely indicative for the effectiveness of edible vaccines . Studies on the feasibility of edible vaccines should thus be performed by eating the vaccine.

Zhonghua Liu Xing Bing Xue Za Zhi, 2002 Jun, 23(3), 203 - 5
A study on genetic polymorphism of rRNA gene pattern of Vibrio cholerae O139 in China; Qu M et al.; OBJECTIVE: To investigated the genetic polymorphism of the isolated strains using ribotyping method . METHODS: One hundred twenty-two strains of V . cholerae O139 isolated from different areas of China from 1993 to 1999 were selected and characterized with ribotyping, including 16s rDNA and 23s rDNA probes . RESULTS: One hundred twenty-two strains were differentiated into 10 different ribotypes (RT1-RT10) on the basis of rRNA gene probes hybridization (with Bgl I digestion), which consisted of 7 - 9 bands between 12 and 1.5 kb in size . RT1 and RT3, as two predominant ribotypes, comprised most number of the strains which spread to the extensive range . Nine strains, which are negative to ctxAB, zot and RS individually, belong to 4 special ribotypes . The dendrogram revealing genetic relationship among different clones of V . cholerae O139 showed that the clones belonging to RT1 and RT2 had genetic similarity on high degree, although they were isolated from different regions . The two predominant ribotypes (RT1 and RT3) were distant in genetic relationship . CONCLUSION: Results showed the clonal diversity and the wide area distribution of V . cholerae O139 strains in China, suggesting the multiple origins of O139 epidemics.

J Clin Microbiol, 2002 Nov, 40(11), 4321 - 4
Development of a hexaplex PCR assay for rapid detection of virulence and regulatory genes in Vibrio cholerae and Vibrio mimicus; Singh DV et al.; We have developed a hexaplex PCR assay for rapid detection of the virulence and regulatory genes for cholera toxin enzymatic subunit A (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and central regulatory protein ToxR (toxR) in Vibrio cholerae and Vibrio mimicus . This hexaplex PCR proved successful in screening pathogenic-toxigenic and nonpathogenic-nontoxigenic V . cholerae and V . mimicus strains from both clinical and environmental sources.

Appl Environ Microbiol, 2002 Nov, 68(11), 5641 - 6
Detection of cytotoxin-hemolysin mRNA in nonculturable populations of environmental and clinical Vibrio vulnificus strains in artificial seawater; Fischer-Le Saux M et al.; The objective of this study was to develop a molecular detection method that better estimates the potential risk associated with the presence of Vibrio vulnificus . For that purpose, we applied seminested reverse transcription-PCR (RT-PCR) to viable but nonculturable (VBNC) populations of V . vulnificus and targeted the cytotoxin-hemolysin virulence gene vvhA . Three strains, two environmental, IF Vv10 and IF Vv18, and one clinical, C7184, were used in this study . Artificial seawater, inoculated with mid-log-phase cells, was maintained at 4 degrees C . VBNC cells resulted after 3, 6, and 14 days for C7184, IF Vv18, and IF Vv10, respectively . Our data indicate that seminested RT-PCR is sensitive for the detection of vvhA mRNA in artificial seawater when exclusively nonculturable bacteria are present . This is the first report of the expression of a toxin gene in VBNC V . vulnificus . Moreover, vvhA transcripts were shown to persist in nonculturable populations over a 4.5-month period, with a progressive decline of the signal over time . This result indicates that special attention should be given to the presence of potentially pathogenic VBNC cells in environmental samples when assessing public health risk.

Appl Environ Microbiol, 2002 Nov, 68(11), 5554 - 62
Widespread N-acetyl-D-glucosamine uptake among pelagic marine bacteria and its ecological implications; Riemann L et al.; Dissolved free and combined N-acetyl-D-glucosamine (NAG) is among the largest pools of amino sugars in the ocean . NAG is a main structural component in chitin and a substantial constituent of bacterial peptidoglycan and lipopolysaccharides . We studied the distribution and kinetics of NAG uptake by the phosphoenolpyruvate:NAG phosphotransferase systems (PTS) in marine bacterial isolates and natural bacterial assemblages in near-shore waters . Of 78 bacterial isolates examined, 60 took up 3H-NAG, while 18 showed no uptake . No systematic pattern in NAG uptake capability relative to phylogenetic affiliation was found, except that all isolates within Vibrionaceae took up NAG . Among 12 isolates, some showed large differences in the relationship between polymer hydrolysis (measured as chitobiase activity) and uptake of the NAG, the hydrolysis product . Pool turnover time and estimated maximum ambient concentration of dissolved NAG in samples off Scripps Pier (La Jolla, Calif.) were 5.9 +/- 3.0 days (n = 10) and 5.2 +/- 0.9 nM (n = 3), respectively . Carbohydrate competition experiments indicated that glucose, glucosamine, mannose, and fructose were taken up by the same system as NAG . Sensitivity to the antibiotic and NAG structural analog streptozotocin (STZ) was developed into a culture-independent approach, which demonstrated that approximately one-third of bacteria in natural marine assemblages that were synthesizing DNA took up NAG . Isolates possessing a NAG PTS system were found to be predominantly facultative anaerobes . These results suggest the hypothesis that a substantial fraction of bacteria in natural pelagic assemblages are facultative anaerobes . The adaptive value of fermentative metabolism in the pelagic environment is potentially significant, e.g., to bacteria colonizing microenvironments such as marine snow that may experience periodic O2-limitation.

J Struct Biol, 2002 Aug, 139(2), 122 - 35
Interaction of the Vibrio cholerae cytolysin (VCC) with cholesterol, some cholesterol esters, and cholesterol derivatives: a TEM study; Harris JR et al.; The Vibrio cholerae cytolysin (VCC) 63-kDa monomer has been shown to interact in aqueous suspension with cholesterol microcystals to produce a ring/pore-like heptameric oligomer approximately 8 nm in outer diameter . Transmission electron microscopy data were produced from cholesterol samples adsorbed to carbon support films, spread across the holes of holey carbon films, and negatively stained with ammonium molybdate . The VCC oligomers initially attach to the edge of the stacked cholesterol bilayers and with increasing time cover the two planar surfaces . VCC oligomers are also released into solution, with some tendency to cluster, possibly via the hydrophobic membrane-spanning domain . At the air/water interface, the VCC oligomers are likely to be selectively oriented with the hydrophobic domain facing the air . Despite some molecular disorder/plasticity within the oligomers, multivariate statistical analysis and rotational self-correlation using IMAGIC-5 strongly suggest the presence of sevenfold rotational symmetry . To correlate the electron microscopy data with on-going biochemical and permeability studies using liposomes of varying lipid composition, the direct interaction of VCC with several cholesterol derivatives and other steroids has been examined . 19-Hydroxycholesterol and 7 beta-hydroxycholesterol both induce VCC oligomerization . beta-Estradiol, which does not possess an aliphatic side chain, also efficiently induces VCC oligomer formation, as does cholesteryl acetate . Cholesteryl stearate and oleate and the C22 (2-trifluoroacetyl)naphthyloxy analogue of cholesterol fail to induce VCC oligomerization, but binding of the monomer to the surface of these steroids does occur . Stigmasterol has little tendency to induce oligomer formation, and oligomers are largely confined to the edge of the bilayers; ergosterol has even less oligomerization ability . Attempts to solubilize and stabilize the VCC oligomers from cholesterol suspensions have been pursued using the neutral surfactant octylglucoside . Although individual solubilized oligomers have been defined which exhibit a characteristic cytolysin channel conformation in the side-on orientation, a tendency remains for the oligomers to cluster via their hydrophobic domains.

Photochem Photobiol, 2002 Sep, 76(3), 268 - 73
Estimation of biologically damaging UV levels in marine surface waters with DNA and viral dosimeters; Wilhelm SW et al.; We have surveyed the biologically harmful radiation penetrating the water column along a transect in the western Gulf of Mexico using dosimeters consisting of intact viruses or naked calf-thymus DNA (ctDNA) . The indigenous marine bacteriophage PWH3a-P1, which lytically infects the heterotrophic bacterium Vibrio natriegens (strain PWH3a), displayed decay rates for infectivity approaching 1.0 h(-1) in surface waters when deployed in a seawater-based dosimeter . The accumulation of pyrimidine dimers in ctDNA dosimeters provided a strong correlation to these results, with pyrimidine dimers representing more than 0.3% (up to ca 3800 dimers Mb(-1) DNA) of the total DNA in dosimeters exposed to sea surface levels of solar radiation . The results demonstrate a strong correlation between the dimer formation in the DNA dosimeters, the decay rates of viral infectivity and the penetration of UVB radiation into the water column . The decay of viral infectivity attenuated with depth in a manner similar to the decay of solar radiation and was still significant at 10 m in offshore oligotrophic water and at dimer frequencies less than 0.1% (ca 200-300 dimers Mb(-1) DNA).

Epidemiol Infect, 2002 Oct, 129(2), 245 - 51
Preferential association of the heat-stable enterotoxin gene (stn) with environmental strains of Vibrio cholerae belonging to the O14 serogroup; Sarkar B et al.; Toxigenic Vibrio cholerae O1 and O139 serogroups have the capacity of causing epidemic and pandemic cholera but are infrequently found in the environment . The other serogroups are abundant in aquatic environments but do not possess the virulence genes necessary for causing the disease . Of the 559 environmental strains of V . cholerae, collected during different periods from environmental samples in Calcutta, 9 (1.6%) harboured the heat-stable enterotoxin gene (stn) . Six of the 9 strains belonged to the O14 serogroup . Thus, V . cholerae strains carrying the stn gene revealed preferential association with the O14 serogroup . Three of the six strains harboured the tcpA gene of the E1 Tor type, which is an unusual feature among environmental V . cholerae strains . A strain that possessed the E1 Tor type tcpA also had the CTX prophage . Pulsed field gel electrophoresis (PFGE) revealed that the stn gene positive O14 strains of V . cholerae were not clonal.

J Microbiol Methods, 2003 Jan, 52(1), 101 - 14
Assessment of fluorescent-labeled bacteria for evaluation of in vivo uptake of bacteria (Vibrio spp.) by crustacean larvae; Soto-Rodriguez SA et al.; Available methods to study crustacean digestive tract colonization by bacteria are laborious, time-consuming, and do not permit in vivo assays and observation . This paper reports on a rapid and consistent technique to apply a fluorescent label to bacteria, which can then be presented to filter-feeding crustacea such as Artemia and penaeid larvae for later in situ bacterial distribution observation . Three luminescent Vibrio spp . were stained and observed inside Artemia nauplii, shrimp zoea and mysis stages, Vibrio harveyi type strain ATCC 14126, M(1) (pathogenic) and Ea (non-pathogenic) . Factors such as dye (DTAF) concentration, exposure time/temperature and sonication time were evaluated . Viability of the dye and stained bacteria were tested at 4, -20 and -70 degrees C storage temperatures for up to 81 days . Results show that 4 and -20 degrees C storage temperatures are not recommended . At -70 degrees C, both bacteria and dye are optimally preserved . Monodispersed fluorescent-labeled bacterial cells can be observed inside the digestive tract of crustacean larvae at a density of inoculation as high as 5.2 x 10(6) CFU ml(-1) . After 2 to 4 h, some leaching occurs, increasing difficulty in observation, although after 24 h, it is still possible to observe monodispersed FLB inside the digestive tract of crustacean larvae . Autofluorescence may complicate observation when filter-feeding crustacean larvae are co-fed with microalgae.

Fish Shellfish Immunol, 2002 Aug, 13(2), 159 - 70
Antigen uptake and immunoglobulin production in Atlantic cod (Gadus morhua L.) after intraperitoneal injection of Vibrio anguillarum; Arnesen SM et al.; Atlantic cod (Gadus morhua L.) were injected intraperitoneally with formalin-killed Vibrio anguillarum bacteria . Immunostaining revealed uptake of V . anguillarum antigens especially in the spleen after intraperitoneal (i.p.) administration . The uptake was time dependent in the interval 1-24 h . Most of the antigen uptake in the spleen was concentrated in areas around small blood vessels, while immunoglobulin producing cells were localised to some thick walled arteries . There was apparently little or no co-localisation of antigens and antibody producing cells . In the heart, some of the high endocardial endothelial cells of the atrium contained bacterial antigens and in head kidney some macrophage-like cells were stained . Very little antigen was found in the pigmented loose connective tissues of the peritoneum . In contrast, endothelial cells of the underlying blood vessels contained substantial amounts . In the heart, peritoneum and anterior kidney the number of antigen positive cells did not seem to change in the time interval 1-24 h . After i.p . immunisation with a mixture of V . anguillarum and Freunds complete adjuvant, the humoral immune response in Atlantic cod was low when tested 21, 42 and 105 days later . There was apparently no enhanced number of immunoglobulin synthesising cells caused by the antigen stimulation.

Fish Shellfish Immunol, 2002 Aug, 13(2), 111 - 23
Susceptibility of three different strains of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at two different temperatures to Vibrio anguillarum and temperature effect on antibody response; Hoare R et al.; Three geographically distinct-reared strains (Canadian, Icelandic, Norwegian) of juvenile Atlantic halibut (Hippoglossus hippoglossus L.) cultured at optimal and super-optimal growth temperatures (12 and 18 degrees C respectively), were challenged with a virulent isolate of Vibrio anguillarum by injection . The halibut were injected intraperitoneally with 100 microl of the bacterial suspension (1 x 10(6) cells per fish) . After challenge, temperature and strain-related differences in survival were observed . Canadian and Icelandic halibut cultured at the super-optimal temperature of 18 degrees C were significantly more susceptible to infection than those strains cultured at 12 degrees C . Total mortality at 18 degrees C for the Canadian and Icelandic strains was 56.4 and 61.85% respectively, compared to 32 and 26.6% respectively at 12 degrees C . Norwegian halibut were significantly more resistant to infection with V . anguillarum at 18 degrees C compared to the other strains, with total mortality of 13.3% . There was no significant difference in total mortality of Norwegian halibut at 18 or 12 degrees C (13.3, 25% respectively) . The specificity of the antibodies in sera from challenged halibut cultured at 18 degrees C was primarily to LPS . Immunoblots showed the presence of antibodies against O-side chain antigens . This reaction was strongest in sera from the Norwegian halibut strain compared with the Canadian and Icelandic halibut, which suggests that the difference in resistance to challenge may be ascribable to the presence of antibodies to LPS . Specific antibody levels, as measured by ELISA, increased with increasing temperature and strain differences were apparent, however these did not relate to disease resistance.

Rev Panam Salud Publica, 2002 Sep, 12(3), 157 - 64
{Characteristics of the cholera epidemic of 1998 in Ecuador during El Niño}; Gabastou JM et al.; OBJECTIVE: To describe the outbreak of cholera that occurred in Ecuador in 1998 during the El Nino weather phenomenon, to present data on the resistance of the circulating strains of Vibrio cholerae to antimicrobial drugs, and to describe the preventive measures taken by health authorities in order to reduce the impact of the disease . METHODS: The epidemiological data came from three sources: 1) the registry of the National Bureau of Epidemiology of the Ministry of Public Health of Ecuador, 2) the registry of the National Institute of Hygiene and Tropical Medicine, and 3) the final report of the Training Program for the Fight against Cholera and Diarrheal Diseases . Isolation, identification, and serotyping was done of V . cholerae in the feces samples from 10% of the suspected cholera cases that were identified between 1 January and 31 December 1998 . The suspected cases were defined by the sudden appearance of watery diarrhea, with or without dehydration, in epidemic areas . The strains that were isolated were submitted to a standard antibiogram by the diffusion method, in which the following antibiotics were tested: amoxicillin, tetracycline, trimethoprimsulfamethoxazole, vibriostatic compound O/129, nalidixic acid, erythromycin, norfloxacin, ciprofloxacin, gentamycin, chloramphenicol, and colistin . RESULTS: In 1998 there were 3 755 cases reported in 17 of the 21 provinces of the country . This corresponds to an incidence rate of 53.96 per 100 000 population . Thirty seven patients died, for a case fatality rate of 0.97% . A total of 301 strains of V . cholerae were isolated in the 637 suspected-cholera samples that were processed; all corresponded to V . cholerae O1, El Tor, subtype Ogawa . All of the strains were sensitive to tetracycline and to quinolones; 5.6% of the strains were resistant to erythromycin . The only strain resistant to amoxicillin was multiresistant . Officials in Ecuador implemented a series of preventive measures, and the surveillance system was strengthened in order to reduce the impact of the disease . CONCLUSIONS: The preventive measures helped to reduce the impact of the 1998 cholera epidemic in Ecuador, in terms of both incidence and the case fatality rate . Given the overall sensitivity of the strains to the antimicrobial drugs, there is no reason to change the current treatment regimens in the country . Taking into account the frequency of natural disasters in Ecuador and the relation that they have to the reappearance of cholera, interventions should be designed that make it possible to prevent and control the reappearance of the disease and its spread to the most vulnerable provinces of the central Sierra mountainous region and the eastern part of the country.

East Afr Med J, 2002 Mar, 79(3), 146 - 9
Carriage of Vibrio species by shrimps harvested from the coastal waters of South West Cameroon; Ndip RN et al.; OBJECTIVES: To determine the prevalence of Vibrio spp in unprocessed shrimps and their susceptibility to antibiotics . DESIGN: A prospective study of Vibrio spp associated with shrimps harvested from the coastal waters of South West Cameroon . SETTING: A laboratory based study at the Department of Life Sciences, University of Buea . Two hundred and thirty six shrimps harvested from the coastal towns of Limbe and Tiko, Cameroon, were examined for the prevalence of Vibrio spp using standard microbiologic procedures . The antibiotic sensitivity of isolates was determined using the Kirby-Bauer disc diffusion technique . RESULTS: Of the 236 shrimps examined, 73 (30.9%) were contaminated with Vibrio spp . Further, a total of 125 Vibrio strains were isolated from the contaminated shrimps . Of this number, 33 (26.4%) were V . cholerae, 55 (44%) V . parahaemolyticus, 34 (27.2%) V . alginolyticus and three (2.4%) V . vulnificus . Antibiotic susceptibility generally ranged from 68.8% for polymyxin B to 99.2% for gentamycin . Multiple resistant strains were noted, especially with V . parahaemolyticus and V . alginolyticus CONCLUSION: Shrimps maintain a reservoir of potential Vibrio spp in the coastal area of South West Cameroon . This finding is of epidemiologic and clinical significance.

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 15 - 21
Prevalence of virulence-associated genes in clinical and environmental Vibrio cholerae strains isolated in Brazil between 1991 and 1999; Vital Brazil JM et al.; Genes located on the CTX element and the Vibrio cholerae pathogenicity island (VPI) were investigated in 297 clinical V . cholerae O1 and 76 environmental O1 and non-O1 isolates from Brazil between 1991 and 1999 . RAPD analysis suggested that serogroup O1 strains regardless of clinical or environmental source were clonal while non-O1 strains showed greater diversity . PCR analysis showed that 71% of O1 clinical isolates had a complete set of CTX element target genes (ctxA, ctxB, zot and ace) and 68% a complete set of the VPI genes studied (orf1, aldA, tagA, tcpA, toxT and int genes) . The results also showed that 72.4% of environmental O1 isolates possessed ctxA, ctxB, zot and ace genes while environmental non-O1 strains rarely possessed virulence genes . Our data are consistent with the hypothesis that the CTX element and the VPI can have a mosaic structure in some V . cholerae strains, genotype diversity is due to the circulation of virulence genes which are more commonly found in O1 strains in Brazil . This study also shows that the aquatic environment is a potential source for virulence genes and toxigenic V . cholerae during epidemic periods.

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 1 - 6
Usefulness of R72H PCR assay for differentiation between Vibrio parahaemolyticus and Vibrio alginolyticus species: validation by DNA-DNA hybridization; Robert-Pillot A et al.; We compared the efficiencies of biochemical methods and polymerase chain reaction (PCR) for the identification of Vibrio parahaemolyticus strains . The 122 isolates studied, identified by biochemical tests as V . parahaemolyticus or Vibrio alginolyticus, were tested by R72H PCR assay . The results obtained with the two methods were consistent for 90% of the strains studied . PCR amplification of the R72H fragment generated two unique amplicons, 387 bp and 320 bp in length . For 11% of the strains from seawater, the results of biochemical identification did not correlate with PCR results . DNA-DNA hybridization experiments provided evidence that some strains identified as V . alginolyticus in biochemical tests should be considered members of the V . parahaemolyticus species . We therefore suggest that biochemical tests are not accurate enough for the identification of V . parahaemolyticus isolates and we demonstrate that amplification of the R72H fragment, whether the amplicon is 320 bp or 387 bp long, is a powerful tool for the reliable identification of V . parahaemolyticus.

An Esp Pediatr, 2002 Oct, 57(4), 361 - 3
{Vibrio cholerae sepsis in the neonate}; Santamaria Munoz R et al.; Vibrio cholerae sepsis is infrequent, especially in neonates although sporadic cases have been reported in older patients . We report the case of a neonate who was admitted to the intensive care unit for hypovolemic shock secondary to diarrhea caused by V . cholerae that developed into bacteremia . The predisposing factors were low socioeconomic status, home delivery, delayed presentation at the health center, and active maternal gastrointestinal infection with V . cholerae . The organism identified in blood and feces culture was identified as V . cholerae 0 -1, biotype Thor, serotype Ogawa, which correlated with the clinical presentation.

Biochem Biophys Res Commun, 2002 Oct 25, 298(2), 269 - 76
Characterization of Vibrio mimicus phospholipase A (PhlA) and cytotoxicity on fish cell; Lee JH et al.; Vibrio mimicus is a typical strain of Vibrio cholerae and produces a phospholipase (PhlA) which shares a highly conserved amino acid sequence with the lecithinase (Lec) of V . cholerae . The recombinant protein (rPhlA) produced from the phlA gene of V . mimicus was expressed in Escherichia coli as His-tag fused protein . The rPhlA was purified by gel filtration and Ni-metal affinity chromatographies . When the action mode was investigated by TLC and GC-MS, the purified rPhlA protein showed a phospholipase A activity, which cleaved the fatty acids at the sn-1 and sn-2 positions of phosphatidylcholine . However, it did not show lysophospholipase, sphingomyelinase, and phospholipase C activities . The rPhlA showed maximum activity at temperature of about 40 degrees C and pH around 8-9 . Some divalent cations could affect the activity of PhlA . The addition of Co(2+) increased the activity, whereas Mg(2+) and Zn(2+) did not enhance the enzyme activity . The rPhlA could lyse the erythrocytes obtained from the fish such as rainbow trout and tilapia . A significant cytotoxic activity on a fish cell line, CHSE-214, was observed after 24h exposure to 40 microg rPhlA protein.

Antimicrob Agents Chemother, 2002 Nov, 46(11), 3580 - 4
In vitro and in vivo activities of newer fluoroquinolones against Vibrio vulnificus; Tang HJ et al.; The MICs of six fluoroquinolones as well as minocycline and cefotaxime for 46 clinical isolates of Vibrio vulnificus were determined by the agar dilution method . All the drugs tested had good activities against all isolates, with the MICs at which 90% of the isolates tested were inhibited (MIC(90)s) by five of the fluoroquinolones ranging between 0.03 and 0.06 micro g/ml . The MIC(90) of lomefloxacin, on the other hand, was 0.12 micro g/ml . Time-kill studies were conducted with these agents and a clinical strain of V . vulnificus, VV5823 . When approximately 5 x 10(5) CFU of V . vulnificus/ml was incubated with any one of the above-mentioned six fluoroquinolones at concentrations of two times the MIC, there was an inhibitory effect on V . vulnificus that persisted for more than 48 h with no noted regrowth . The efficacies of the fluoroquinolones were further evaluated in vivo in the mouse model of experimental V . vulnificus infection and compared to the efficacy of a combination therapy using cefotaxime plus minocycline . With an inoculum of 1.5 x 10(7) CFU, 28 (87.5%) of 32 mice in the cefotaxime-minocycline-treated group survived and 29 (91%) of the 32 mice in the moxifloxacin-treated group survived while none of the 32 mice in the control group did . With an inoculum of 3.5 x 10(7) CFU, the difference in survival rates among groups of 15 mice treated with levofloxacin (13 of 15), moxifloxacin (10 of 15), gatifloxacin (10 of 15), sparfloxacin (11 of 15), ciprofloxacin (12 of 15), or lomefloxacin (10 of 15) was not statistically significant while none of the 15 mice treated with saline survived . We concluded that the newer fluoroquinolones as single agents are as effective as the cefotaxime-minocycline combination in inhibiting V . vulnificus both in vitro and in vivo.

Gene, 2002 Aug 21, 296(1-2), 213 - 20
Cloning, characterisation and phylogenetic analysis of the fur gene in Vibrio salmonicida and Vibrio logei; Colquhoun DJ et al.; The gene encoding the ferric uptake regulator protein (fur gene) of Vibrio salmonicida 87/09/1193 was located following hybridisation of an EcoRI digest of chromosomal V . salmonicida DNA with a 316 base pairs (bp) probe internal to the fur gene of Vibrio anguillarum . A 2088 bp fragment including an open reading frame of 441 bp, encoding a protein of 147 amino acids, and homologous with fur, was identified, cloned and sequenced . A plasmid bound V . salmonicida fur gene was found capable of complementing the fur mutation of Escherichia coli H1681 . Although no 'iron-box' was identified upstream of the start-codon, beta-galactosidase activity in E . coli H1681 was regulated by iron availability in the media, indicating that in V . salmonicida fur, as in other fur genes, iron functions as a co-repressor . Southern blot hybridizations demonstrated that fur is conserved amongst V . salmonicida strains and several other closely related Vibrio strains in which fur remains as yet, uncharacterized . The fur gene of Vibrio logei NCIMB 2252 was subsequently amplified using polymerase chain reaction primers external to the V . salmonicida fur gene . Comparison of phylogenetic analyses using fur and 16S DNA coding for rRNA sequences, confirmed the usefulness of fur as an evolutionary marker within the genus Vibrio.

Ann N Y Acad Sci, 2002 Oct, 969, 60 - 5
PCR and molecular detection for differentiating Vibrio species; Sparagano OA et al.; Vibriosis is an economically important disease of fish, marine invertebrates (particularly penaeid shrimps), and large marine mammals and is responsible for high mortality rates in aquaculture worldwide . Some Vibrio species are also responsible for zoonoses, whereas others are relatively nonpathogenic . Using 16S- and 23S-based PCR reactions, we obtained species-specific patterns and a 470-bp band, respectively . DNA sequences obtained on the 23S rRNA gene allowed us to identify species-specific probes for Vibrio parahaemolyticus, V . alginolyticus, V . anguillarum and for a cluster of taxonomically related species: V . carchariae/harveyi/campbelii . A phylogenetic tree based on the 23S sequences confirmed previous results obtained by Western blotting.

Infect Immun, 2002 Nov, 70(11), 6251 - 62
Phage therapy of local and systemic disease caused by Vibrio vulnificus in iron-dextran-treated mice; Cerveny KE et al.; Vibrio vulnificus is a gram-negative bacterium that contaminates filter-feeding shellfish such as oysters . After ingestion of contaminated oysters, predisposed people may experience highly lethal septicemia . Contamination of wounds with the bacteria can result in devastating necrotizing fasciitis, which can progress to septicemia . The extremely rapid progression of these diseases can render antibiotic treatment ineffective, and death is a frequent outcome . In this study, we examined the potential use of bacteriophages as therapeutic agents against V . vulnificus in an iron-dextran-treated mouse model of V . vulnificus infection . Mice were injected subcutaneously with 10 times the lethal dose of V . vulnificus and injected intravenously, either simultaneously or at various times after infection, with phages . Treatment of mice with phages could prevent death; systemic disease, as measured by CFU per gram of liver and body temperature; and local disease, as measured by CFU per gram of lesion material and histopathologic analysis . Two different phages were effective against three different V . vulnificus strains with various degrees of virulence, while a third phage that required the presence of seawater to lyse bacteria in vitro was ineffective at treating mice . Optimum protection required that the phages be administered within 3 h of bacterial inoculation at doses as high as 10(8) PFU . One of the protective phages had a half-life in blood of over 2 h . These results demonstrate that bacteriophages have therapeutic potential for both localized and systemic infections caused by V . vulnificus in animals . This model should be useful in answering basic questions regarding phage therapy.

Infect Immun, 2002 Nov, 70(11), 5990 - 6
Role of Vibrio cholerae O139 surface polysaccharides in intestinal colonization; Nesper J et al.; Since the first occurrence of O139 Vibrio cholerae as a cause of cholera epidemics, this serogroup has been investigated intensively, and it has been found that its pathogenicity is comparable to that of O1 El Tor strains . O139 isolates express a thin capsule, composed of a polymer of repeating units structurally identical to the lipopolysaccharide (LPS) O side chain . In this study, we investigated the role of LPS O side chain and capsular polysaccharide (CPS) in intestinal colonization by with genetically engineered mutants . We constructed CPS-negative, CPS/LPS O side chain-negative, and CPS-positive/LPS O side chain-negative mutants . Furthermore, we constructed two mutants with defects in LPS core oligosaccharide (OS) assembly . Loss of LPS O side chain or CPS resulted in a approximately 30-fold reduction in colonization of the infant mouse small intestine, indicating that the presence of both LPS O side chain and CPS is important during the colonization process . The strain lacking both CPS and LPS O side chain and a CPS-positive, LPS O side chain-negative core OS mutant were both essentially unable to colonize . To characterize the role of surface polysaccharides in survival in the host intestine, resistance to several antimicrobial substances was investigated in vitro . These investigations revealed that the presence of CPS protects the cell against attack of the complement system and that an intact core OS is necessary for survival in the presence of bile.

Chemosphere, 2002 Oct, 49(2), 193 - 203
Semiconductor-mediated photocatalysed degradation of two selected priority organic pollutants, benzidine and 1,2-diphenylhydrazine, in aqueous suspension; Muneer M et al.; The photocatalysed degradation of two selected priority organic pollutants, namely benzidine (1) and 1,2-diphenylhydrazine (DPH, 2) has been investigated in aqueous suspensions of titanium dioxide (TiO2) under a variety of conditions employing a pH-stat technique . The degradation was studied by monitoring the change in substrate concentration of the model compound employing HPLC analysis and the decrease in total organic carbon content, respectively, as a function of irradiation time . The degradation kinetics were studied under different conditions such as reaction pH, substrate and photocatalyst concentration, type of TiO2 photocatalyst and the presence of alternative additives such as H2O2, KBrO3 and (NH4)2S2O8 besides molecular oxygen . The degradation rates and the photonic efficiencies were found to be strongly influenced by the above parameters . Toxicity tests for the irradiated samples of benzidine measuring the luminescence of bacteria Vibrio fischeri after 30 min of incubation were also performed . 4-amino-biphenyl (7) and hydroquinone (13) were identified as intermediate products by GC/MS technique and probable pathways for the formation of the products are proposed.

J Bacteriol, 2002 Nov, 184(21), 5946 - 54
Vibrio parahaemolyticus scrABC, a novel operon affecting swarming and capsular polysaccharide regulation; Boles BR et al.; Swarming is an adaptation of many bacteria to growth on surfaces . A search for genes controlling swarmer cell differentiation of Vibrio parahaemolyticus identified a novel three-gene operon that potentially encodes a pyridoxal-phosphate-dependent enzyme, an extracellular solute-binding protein, and a membrane-bound GGDEF- and EAL-motif sensory protein . The functions of these motifs, which are named after conserved amino acid sequences, are unknown, although the domains are found singly and in combination in a variety of bacterial signaling proteins . Studies with translational fusions supported the predicted localization of the gene products . When the operon was overexpressed, swarmer cell gene transcription was induced in liquid culture . Mutants with defects in any of the three genes exhibited decreased swarming and lateral flagellar (laf) gene expression . Complementation studies confirmed an operon organization and suggested that all three genes participated in laf regulation . The lesions that decreased swarming increased capsular polysaccharide (CPS) production, and overexpression of the operon inhibited transcription of the CPS gene cpsA . Thus, the scrABC locus appears to inversely regulate two gene systems that are pertinent to colonization of surface swarming and CPS.

Eur J Clin Microbiol Infect Dis, 2002 Sep, 21(9), 676 - 8 Epub 2002 Sep 03.
Vibrio cholerae bacteremia in a neutropenic patient with non-small-cell lung carcinoma; Berghmans T et al.; Vibrio cholerae was isolated from the blood cultures of a neutropenic patient treated with chemotherapy for non-small-cell lung cancer . Attempts to isolate Vibrio spp . from a rectal swab and stool were unsuccessful . Piperacillin/tazobactam treatment resulted in eradication of the microorganism from the patient's blood . Although Vibrio spp . have occasionally been the source of infection in immunocompromised patients, this report describes the first case of non-0:1 Vibrio cholerae bacteremia in a neutropenic patient with a solid tumour.

Environ Toxicol Chem, 2002 Oct, 21(10), 2242 - 51
Oil effect in freshly spiked marine sediment on Vibrio fischeri, Corophium volutator, and Echinocardium cordatum; Brils JM et al.; The purpose of this study was to provide data to be used in The Netherlands for development of ecotoxicologically based quality criteria for oil-contaminated sediments and dredged material . In addition, the relation of toxicity to specific oil boiling-point fraction ranges was explored . Natural marine sediment, with a moisture, organic carbon, and silt content of approximately 80, 1.8, and 33% of the dry weight, respectively, was artificially spiked using a spiking method developed in this project . Aliquots of one part of the sediment were spiked to several concentrations of Gulf distillate marine grade A (DMA) gasoil (containing 64% C10-19) and aliquots of the other part to several concentrations of Gulf high viscosity grade 46 (HV46) hydraulic oil (containing 99.2% C19-40) . Thus, for each individual oil type, a concentration series was created . Vibrio fischeri (endpoint: bioluminescence inhibition), Corophium volutator (endpoint:mortality), and Echinocardium cordatum (endpoint:mortality) were exposed to these spiked sediments for 10 min, 10 d and 14 d, respectively . Based on the test results, the effective concentration on 50% of the test animals was statistically estimated . For DMA gasoil and HV46 hydraulic oil, respectively, the effective concentrations were 43.7 and 2,682 mg/kg dry weight for V . fischeri, 100 and 9,138 mg/kg dry weight for C . volutator, 190, and 1064 mg/kg dry weight for E . cordatum . This study shows that the toxicity is strongly correlated with the lower boiling-point fractions and especially to those within the C10-C19 range.

Environ Toxicol Chem, 2002 Oct, 21(10), 2225 - 32
Quantitative structure-activity relationship for the photoinduced toxicity of polycyclic aromatic hydrocarbons to the luminescent bacteria Vibrio fischeri; El-Alawi YS et al.; Sunlight can greatly enhance the toxicity of polycyclic aromatic hydrocarbons (PAHs) . Photosensitization reactions (e.g., generation of singlet-state oxygen) and photomodification reactions (e.g., photooxidation of PAHs to more toxic species) are both pathways of photoinduced toxicity of PAHs . Previously, a quantitative structure-activity relationship (QSAR) was developed for PAHs showing that a photosensitization factor (PSF) and photomodification factor (PMF) can be additively combined to describe photoinduced toxicity . That QSAR model was developed for the photoinduced toxicity of 16 PAHs to the higher plant Lemna gibba . The objective of this study was to apply the QSAR model developed for L . gibba to another organism . The organism chosen was the luminescent marine bacteria Vibriofischeri . Toxicity data used for the QSAR model were inhibition of luminescence and inhibition of growth of V . fischeri . Both short-term (15 min) and long-term (18 h) assays of toxicity were used . Light did not impact on PAH toxicity in the short-term assay, and thus the QSAR model did not correlate well with these data . Conversely, light greatly enhanced toxicity when the long-term assay was employed . The PMFs for the PAHs from the L . gibba QSAR showed a moderate correlation to bacterial toxicity in the long-term assay, whereas the PSFs showed only a weak correlation to toxicity . As was the case for L gibba, summing the PMF and the PSF resulted in a strong correlation to toxicity that had predictive value . Thus, a QSAR model derived for plants accurately described the toxicity of PAHs to a bacterial species . This indicates that the bipartite mechanism of PAH-photoinduced toxicity may be applicable to other organisms.

Mol Microbiol, 2002 Oct, 46(1), 125 - 34
MotX and MotY, specific components of the sodium-driven flagellar motor, colocalize to the outer membrane in Vibrio alginolyticus; Okabe M et al.; Rotation of the sodium-driven polar flagella of Vibrio alginolyticus requires four motor proteins: PomA, PomB, MotX and MotY . MotX and MotY, which are unique components of the sodium-driven motor of Vibrio, have been believed to be localized in the inner (cytoplasmic) membrane via their N-terminal hydrophobic segments . Here we show that MotX and MotY colocalize to the outer membrane . Both proteins, when expressed together, were detected in the outer membrane fraction separated by sucrose density gradient centrifugation . As mature MotX and MotY proteins do not have N-terminal hydrophobic segments, the N-termini of the primary translation products must have signal sequences that are removed upon translocation across the inner membrane . Moreover, MotX and MotY require each other for efficient localization to the outer membrane . Based on these lines of evidence, we propose that MotX and MotY form a complex in the outer membrane . This is the first case that describes motor proteins function in the outer membrane for flagellar rotation.

Mol Microbiol, 2002 Oct, 46(1), 101 - 11
MetR and CRP bind to the Vibrio harveyi lux promoters and regulate luminescence; Chatterjee J et al.; The induction of luminescence in Vibrio harveyi at the later stages of growth is controlled by a quorum-sensing mechanism in addition to nutritional signals . However, the mechanism of transmission of these signals directly to the lux promoters is unknown and only one regulatory protein, LuxR, has been shown to bind directly to lux promoter DNA . In this report, we have cloned and sequenced two genes, crp and metR, coding for the nutritional regulators, CRP (cAMP receptor protein) and MetR (a LysR homologue), involved in catabolite repression and methionine biosynthesis respectively . The metR gene was cloned based on a general strategy to detect lux DNA-binding proteins expressed from a genomic library, whereas the crp gene was cloned based on its complementation of an Escherichia coli crp mutant . Both CRP and MetR were shown to bind to lux promoter DNA, with CRP being dependent on the presence of cAMP . Expression studies indicated that the two regulators had opposite effects on luminescence: CRP was an activator and MetR a repressor . Disruption of crp decreased luminescence by about 1,000-fold showing that CRP is a major activator of luminescence the same as LuxR, whereas disruption of MetR resulted in activation of luminescence over 10-fold, confirming its function as a repressor . Comparison of the levels of the autoinducers involved in quorum sensing excreted by V . harveyi, and the crp and metR mutants, showed that autoinducer production was not significantly different, thus indicating that the nutritional signals do not affect luminescence by changing the levels of the signals required for quorum sensing . Indeed, the large effects of these nutritional sensors show that luminescence is controlled by multiple signals related to the environment and the cell density which must be integrated at the molecular level to control expression at the lux promoters.

Clin Microbiol Rev, 2002 Oct, 15(4), 757 - 70
Effects of global climate on infectious disease: the cholera model; Lipp EK et al.; Recently, the role of the environment and climate in disease dynamics has become a subject of increasing interest to microbiologists, clinicians, epidemiologists, and ecologists . Much of the interest has been stimulated by the growing problems of antibiotic resistance among pathogens, emergence and/or reemergence of infectious diseases worldwide, the potential of bioterrorism, and the debate concerning climate change . Cholera, caused by Vibrio cholerae, lends itself to analyses of the role of climate in infectious disease, coupled to population dynamics of pathogenic microorganisms, for several reasons . First, the disease has a historical context linking it to specific seasons and biogeographical zones . In addition, the population dynamics of V . cholerae in the environment are strongly controlled by environmental factors, such as water temperature, salinity, and the presence of copepods, which are, in turn, controlled by larger-scale climate variability . In this review, the association between plankton and V . cholerae that has been documented over the last 20 years is discussed in support of the hypothesis that cholera shares properties of a vector-borne disease . In addition, a model for environmental transmission of cholera to humans in the context of climate variability is presented . The cholera model provides a template for future research on climate-sensitive diseases, allowing definition of critical parameters and offering a means of developing more sophisticated methods for prediction of disease outbreaks.

Microbiol Immunol, 2002, 46(8), 565 - 9
Isolation and genetic characterization of a novel filamentous bacteriophage, a deleted form of phage f237, from a pandemic Vibrio parahaemolyticus O4:K68 strain; Chan B et al.; We isolated a filamentous bacteriophage, VfO4K68, from the pandemic Vibrio parahaemolyticus strain belonging to 04:K68 serovar . The VfO4K68 DNA lacked a 1,893-bp fragment present in that of the distinctive region of f237, a filamentous phage isolated from a pandemic 03:K6 strain (Nasu, H . et al., J . Clin . Microbiol., 38, 2156-2161, 2000) . The deletion resulted in the formation of a novel open reading frame (ORF) that possesses homology to the ORF 27 of ETA phage and staphylococcal enterotoxin E (SEE) of Staphylococcus aureus . VfO4K68 was able to infect the recipient 03:K6 serovar strains . These results suggest that VfO4K68 might act as a genetic transmitter and play some roles in the pandemic V . parahaemolyticus infection.

J Biol Chem, 2002 Dec 6, 277(49), 47292 - 9 Epub 2002 Sep 27.
Promoter analysis and regulatory characteristics of vvhBA encoding cytolytic hemolysin of Vibrio vulnificus; Choi HK et al.; Cytolytic hemolysin, a gene product of vvhA, is a putative virulence factor of the pathogenic bacterium Vibrio vulnificus . We have previously shown that hemolysin production is repressed by adding glucose to culture media and that production can be restored by adding cAMP . In this study, hemolysin activity and the level of vvh transcript were determined to reach a maximum in late exponential phase and were repressed when cells entered stationary phase . Northern blot and primer extension analyses revealed that vvhA is cotranscribed with a second gene, vvhB, located upstream of vvhA . Transcription of the vvhBA operon begins at a single site and is under the direction of a single promoter, P(vvh) . A crp null mutation decreased hemolysin production and the level of vvhBA transcript by reducing the activity of P(vvh), indicating that the P(vvh) activity is under the positive control of cAMP receptor protein (CRP) . A direct interaction between CRP and the regulatory region of the vvhBA operon was demonstrated by gel-mobility shift assays . The CRP binding site, centered at 59.5 bp upstream of the transcription start site, was mapped by deletion analysis of the vvhBA promoter region and confirmed by DNase I protection assays . These results demonstrate that the vvhBA expression is activated by CRP in a growth-dependent manner and that CRP exerts its effects by directly binding to DNA upstream of P(vvh).

Clin Infect Dis, 2002 Oct 15, 35(8), 921 - 8 Epub 2002 Sep 25.
Infectious outbreaks associated with bivalve shellfish consumption: a worldwide perspective; Potasman I et al.; Outbreaks of shellfish-associated infection have been reported for more than a century . Since the early 1970s, the global consumption of shellfish has increased considerably--and with it, the reports of outbreaks of infection . Most of these reports have originated from the United States, but Europe and, to a lesser extent, Asia and Australia have also been represented . The majority of outbreaks have been linked to oysters, followed by clams and mussels . Hepatitis A virus caused the largest ever shellfish-associated outbreak, but caliciviruses have caused the highest number of outbreaks; Vibrio species lead the list of bacterial pathogens . The prognosis of shellfish-associated infections is generally good, except for outbreaks of Vibrio vulnificus infection, which have a mortality rate of up to 50% in vulnerable people . Conventional and molecular techniques should be applied to better identify the causative agents, thereby enabling more-targeted control measures in growing, harvesting, and shipping bivalves.

FEMS Microbiol Lett, 2002 Sep 10, 214(2), 153 - 7
Mobilization of the Vibrio pathogenicity island between Vibrio cholerae isolates mediated by CP-T1 generalized transduction; O'Shea YA et al.; Pathogenicity islands are large chromosomal regions encoding virulence genes that were acquired by horizontal gene transfer and are found in a wide range of pathogenic bacteria . In toxigenic Vibrio cholerae isolates the receptor for the cholera toxin encoding filamentous phage CTXphi, the toxin-coregulated pilus, is part of the Vibrio pathogenicity island (VPI) . In this paper, we show that the VPI can be transferred between O1 serogroup strains, the predominant cause of epidemic cholera, via a generalized transducing phage CP-T1.

World Watch, 1991 Jul-Aug, 4(4), 37 - 8
Sanitation in the time of cholera; Misch A; PIP: Cholera, identified by violent diarrhea, cramps, vomiting, and dehydration, is spreading through Peru into Colombia, Ecuador, Child, and Brazil . Water contaminated with Vibrio cholerae is used for washing food and/or drinking thereby transmitting the disease . PAHO estimates 6 million people in South America may get cholera within the next 3 years . This cholera epidemic is the result of unsanitary conditions in which the urban poor in South America live . In fact, in Lima, Peru, 40% of the people do not have potable, piped water available . These individuals fetch their water from far away taps and private vendors both of which are not necessarily safe . In addition, 40% do not have access to a sewage system . Further, 80% of sick people in developing countries have a water related illness, be it transmitted by contaminated water or by insects and snails that reproduce in the water . Diarrhea is the most deadly of these conditions . Indeed every year 10-20 million children die from the effects of diarrhea which include malnutrition, dehydration, and shock . Yet 940 million people in developing countries have no access to safe water and 1.7 billion do not have a sanitary means of disposing of human wastes, despite the fact that the UN decreed the 1980s the International Drinking Water Supply and Sanitation Decade . Nevertheless UNICEF efforts did bring communal taps, odorless latrines, and/or pour flush toilets to 1.2 billion people . These types of sanitation costs $20-25/person whereas conventional sewers cost $350/person . Low technology supplied water averages $30/person compared to $200/person for piped water . Peru has spent $43 million on emergency medical care for cholera victims which could have provided low cost clean water and sanitation for almost 800,000 poor .

WHO Feature, 1991 Apr, (154), 1 - 3
Cholera: ancient scourge on the rise . WHO announces global plan for cholera control . (25 April 1991); World Health Organization WHO . Office of Information; PIP: Vibrio cholerae spreads quickly via contaminated water and food, especially in areas with a poor health and sanitation infrastructure . Its enterotoxin induces vomiting and huge amounts of watery diarrhea leading to severe dehydration . 80-90% of cholera victims during an epidemic can use oral rehydration salts . A cholera epidemic is now spreading through Latin America threatening 90-120 million people (started in January 1991), particularly those in urban slums and rural/mountainous areas . As of mid April 1991, there were more than 177,000 new reported cases in 12 countries and 78% of these cases and more than 1200 deaths were limited to 5 countries: Brazil, Chile, Colombia, Ecuador, and Peru, WHO's Global Cholera Control Task Force coordinates global cholera control efforts to prevent deaths in the short term and to support infrastructure development in the long term . Its members are specialists in disease surveillance, case management, water and sanitation, food safety, emergency intervention, and information and education . WHO's Director General is asking for the support of the international community in cholera control activities . These activities' costs are considerable . For example, Peru needs about US$ 60 million in 1992 to fulfill only the most immediate demands of rehabilitation and reconstruction of the infrastructure . Costs of infrastructure capital throughout Latin America is almost US$ 5 thousand million/year over the next 10 years . It is indeed an effective infrastructure which ultimately prevents cholera . Cholera is evidence of inadequate development, so to fight it, we must also fight underdevelopment and poverty .

Di Yi Jun Yi Da Xue Xue Bao, 2002 Jun, 22(6), 515 - 7
Cloning and sequence analysis of thermostable direct hemolysin-related hemolysin gene of Vibrio parahaemolyticus; Su JX et al.; OBJECTIVE: To establish a method for cloning thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus (Vp) . METHODS: After enrichment by PCR, the trh gene, along with the plasmid DNA of pGEX-3X, was digested with two enzymes followed by linkage of the gene and the plasmid, the product of which was transferred into E.coli DH5alpha . Identification of the recombinant plasmid was performed by means of PCR, digestion and sequence analysis . RESULTS: A fragment about 600 bp was identified in the PCR product of Vp14-91, which was also seen in the product of PCR and enzyme digestion of the recombinant fragment . Sequence analysis demonstrated a homology of 99.1% between the trh gene and the reference gene available in the GenBank . CONCLUSION: trh gene has been successfully cloned with its sequence analyzed, which prepares the ground for developing gene probe and protective vaccine against Vp.

Ecotoxicol Environ Saf, 2002 Jul, 52(3), 227 - 31
Hormesis responses of free and immobilized light-emitting bacteria; Christofi N et al.; The stimulatory effect of sublethal or low concentrations of toxic chemicals on organismal metabolism, referred to as hormesis, has been found to be common in the widely used Vibrio fischeri luminescence bioassay . In addition to the "normal" type alpha, we have demonstrated type beta and, possibly, type gamma, dose-response curves in free and immobilized V . fischeri bioassays developed . Understanding and utilizing data from hormesis responses are necessary in determining the toxicity of chemicals, singly or in complex mixtures, to natural biota without imposing excessive penalties to dischargers . At the same time, care must be taken not to relax environmental standards . This can only arise by fully investigating and understanding the role of hormesis in toxicity data used for risk assessment.

J Bacteriol, 2002 Oct, 184(20), 5533 - 44
Mechanism of ToxT-dependent transcriptional activation at the Vibrio cholerae tcpA promoter; Hulbert RR et al.; The AraC homolog ToxT coordinately regulates virulence gene expression in Vibrio cholerae . ToxT is required for transcriptional activation of the genes encoding cholera toxin and the toxin coregulated pilus, among others . In this work we focused on the interaction of ToxT with the tcpA promoter and investigated the mechanism of ToxT-dependent transcriptional activation at tcpA . Deletion analysis showed that a region from -95 to +2 was sufficient for ToxT binding and activation, both of which were simultaneously lost when the deletion was extended to -63 . A collection of point mutations generated by error-prone PCR revealed two small regions required for ToxT-dependent transactivation . Binding studies performed with representative mutations showed that the two regions define sites at which ToxT binds to the tcpA promoter region, most likely as a dimer . Results obtained by using a rpoA truncation mutation showed that ToxT-dependent activation at tcpA involves the C-terminal domain of the RNA polymerase alpha subunit . A model of ToxT-dependent transcriptional activation at tcpA is proposed, in which ToxT interacts with two A-rich regions of tcpA centered at -72 and -51 and requires the alpha C-terminal domain of RNA polymerase.

J Indian Assoc Commun Dis, 1982 Sep-Dec, 5(3-4), 83 - 7
Pattern of cholera in Raipur: a twelve year appraisal; Darbari BS et al.; Outbreaks of cholera were recorded in Raipur in the years 1970, 1975, 1977 and 1979-1981 . Change from V . cholerae classical inaba in 1970 to V . el tor ogawa in 1974 was also observed . The el tor strain is persisting in this area since then . In 1977, antigenic shift to V . el tor inaba was recorded but it again reverted to el tor ogawa which was responsible for outbreaks in 1979-1981 . V . cholerae was isolated in most of the months in the years 1975, 1977 and 1980 . Almost equal prevalence rates were seen in both sexes as well as in paediatric and adult groups . NAG vibrio were also inoculated . These data suggest endemicity of choloera in Raipur.

Cad Saude Publica, 2002 Sep-Oct, 18(5), 1339 - 45
{Viability of Vibrio cholerae O1 in different types of water under experimental conditions}; Nogueira JM et al.; The endemic and seasonal nature of cholera depends upon the survival of Vibrio cholerae O1 in a viable but not necessarily culturable state in ecological niches in aquatic environments during inter-epidemic periods, and investigation on the survival of this microorganism in such sites is therefore of the utmost importance . Weekly water aliquots were thus taken from 2 ponds and 2 rivers in the State of Rio de Janeiro . The samples were divided into two identical portions, one of which was autoclaved . A standardized dilution of V . cholerae O1 Inaba and of V . cholerae O1 Ogawa was inoculated in three aliquots of 100ml of these different water samples and maintained at different temperatures . Survival of the microorganisms in the aquatic environment under these different conditions was then analyzed . Regardless of the serotype, V . cholerae serogroup O1 survived in water with salinity below 0.5 per mil and at different temperatures for sufficient periods to spread through "bodies of water", demonstrating the need to constantly monitor areas of possible contamination, especially where the water is used for drinking, thus avoiding spread of the disease to surrounding populations.

Vet Microbiol, 2002 Oct 22, 89(2-3), 181 - 94
Purification and partial characterisation of a fish lethal extracellular protease from Vibrio pelagius; Farto R et al.; The purification, characterisation and lethal effect of an extracellular protease present in the extracellular products (ECPs) of a pathogenic Vibrio pelagius (7P) strain are described . The extracellular protease was purified by size-exclusion high-performance liquid chromatography and characterised by enzymatic assays . The lethal effect was evaluated by injection into fish . The native protease had a molecular mass of 39 kDa, was active on casein and L-leucyl-beta-naphthylamide (LNA) and its metalloprotease nature was shown by the LNA inhibition profile . Kinetic studies on the hydrolysis of casein and LNA confirmed a competitive inhibition of one substrate with respect to the other . The temperature assays showed that both aminopeptidolytic and caseinolytic activities were labile at 70 degrees C for 3 min . The N-terminal amino acid sequence of 7P protease revealed a high degree of homology with other metalloproteases of Vibrio species that are implicated in virulence . The purified 7P protease showed an LD(50) of 1.77 microgprotein/g fish for turbot . The quick lethal effect (<24h) and the macroscopic damage (external haemorrhagic areas, principally on fins and mouth, petechial haemorrhages in internal organs, but with no external or internal apparent necrotic areas) detected in the host were similar to those obtained by injection of total ECP and live cells of 7P strain.An extracellular protease with endopeptidolytic and exopeptidolytic activities, responsible for the lethal effect of ECP and clinical signs of vibriosis in turbot was purified from a pathogenic V . pelagius (7P) strain.

Mol Gen Mikrobiol Virusol, 2002, (3), 23 - 33
{Cholera pathogens from the new serogroup O139: molecular-genetic features and origin}; Smirnova NI; The analysis of modern data concerning new genes of Vibrio cholerae of O139 serogroup and genetic control of their biosynthesis is represented . The special attention is paid to description of molecular genetic events induced the derivation the cholera causative agent of new serogroup . The structure of the moderate phages of V . cholerae O139 and their role in virulence of the cholera causative agent of the new serogroup and variability of its genome are discussed.

Dis Aquat Organ, 2002 Aug 15, 51(1), 13 - 25
An Aeromonas salmonicida type IV pilin is required for virulence in rainbow trout Oncorhynchus mykiss; Masada CL et al.; Aeromonas salmonicida expresses a large number of proven and suspected virulence factors including bacterial surface proteins, extracellular degradative enzymes, and toxins . We report the isolation and characterization of a 4-gene cluster, tapABCD, from virulent A . salmonicida A450 that encodes proteins homologous to components required for type IV pilus biogenesis . One gene, tapA, encodes a protein with high homology to type IV pilus subunit proteins from many gram-negative bacterial pathogens, including Aeromonas hydrophila, Pseudomonas aeruginosa, and Vibrio vulnificus . A survey of A . salmonicida isolates from a variety of sources shows that the tapA gene is as ubiquitous in this species as it is in other members of the Aeromonads . Immunoblotting experiments demonstrate that it is expressed in vitro and is antigenically conserved among the A . salmonicida strains tested . A mutant A . salmonicida strain defective in expression of TapA was constructed by allelic exchange and found to be slightly less pathogenic for juvenile Oncorhynchus mykiss (rainbow trout) than wild type when delivered by intraperitoneal injection . In addition, fish initially challenged with a high dose of wild type were slightly more resistant to rechallenge with wild type than those initially challenged with the tapA mutant strain, suggesting that presence of TapA contributes to immunity . Two of the other three genes identified, tapB and tapC, encode proteins with homology to factors known to be required for type IV pilus assembly in P . aeruginosa, but in an as yet unidentified manner . TapB is a member of the ABC-transporter family of proteins that contain characteristic nucleotide-binding regions, and which may provide energy for type IV pilus assembly through the hydrolysis of ATP . TapC homologs are integral cytoplasmic membrane proteins that may play a role in pilus anchoring or initiation of assembly . The fourth gene, tapD, encodes a product that shares homology with a family of proteins with a known biochemical function, namely, the type IV prepilin leader peptidases . These bifunctional enzymes proteolytically cleave the leader peptide from the pilin precursor (prepilin) and then N-methylate the newly exposed N-terminal amino acid prior to assembly of the subunits into the pilus structure . We demonstrate that A . salmonicida TapD is able to restore type IV pilus assembly and type II secretion in a P . aeruginosa strain carrying a mutation in its type IV peptidase gene, suggesting that it plays the same role in A . salmonicida.

Rev Med Chil, 2002 Jul, 130(7), 787 - 91
{Vibrio vulnificus: an infrequent cause of septic shock}; Poblete R et al.; Vibrio vulnificus is a lactose positive Gram negative rod that lives in warm seas and can infect wounds and produce sepsis . Its infection is acquired after eating oysters or other filtering marine organisms . We report a 53 years old diabetic male who started with fever after a voyage to Central America . He was admitted febrile, hypotense, dehydrated and polypneic . Painful erythematous lesions and lumps were observed in his upper and lower limbs . After 72 hours of evolution, the lesions became violaceous, with crepitating vesicles full of hemorrhagic exudate . He developed a renal failure and a disseminated intravascular coagulation . Blood cultures demonstrated the presence of Vibrio vulnificus and the patient died 68 hours after admission.

Nucleic Acids Res, 2002 Sep 15, 30(18), 4009 - 21
Sequence analysis of bacteriophage T4 DNA packaging/terminase genes 16 and 17 reveals a common ATPase center in the large subunit of viral terminases; Mitchell MS et al.; Phage DNA packaging is believed to be driven by a rotary device coupled to an ATPase 'motor' . Recent evidence suggests that the phage DNA packaging motor is one of the strongest force-generating molecular motors reported to date . However, the ATPase center that is responsible for generating this force is unknown . In order to identify the DNA translocating ATPase, the sequences of the packaging/terminase genes of coliphages T4 and RB49 and vibriophages KVP40 and KVP20 have been analyzed . Alignment of the terminase polypeptide sequences revealed a number of functional signatures in the terminase genes 16 and 17 . Most importantly, the data provide compelling evidence for an ATPase catalytic center in the N-terminal half of the large terminase subunit gp17 . An analogous ATPase domain consisting of conserved functional signatures is also identified in the large terminase subunit of other bacteriophages and herpesviruses . Interestingly, the putative terminase ATPase domain exhibits some of the common features found in the ATPase domain of DEAD box helicases . Residues that would be critical for ATPase catalysis and its coupling to DNA packaging are identified . Com binatorial mutagenesis shows that the predicted threonine residues in the putative ATPase coupling motif are indeed critical for function.

Infect Immun, 2002 Oct, 70(10), 5355 - 62
The alternative sigma factor sigma(E) plays an important role in intestinal survival and virulence in Vibrio cholerae; Kovacikova G et al.; The alternative sigma factor sigma(E) (RpoE) is involved in the response to extracytoplasmic stress and plays a role in the virulence of a variety of different bacteria . To assess the role of sigma(E) in Vibrio cholerae pathogenesis, a DeltarpoE mutant was constructed and analyzed using the infant mouse model . The results here show that sigma(E) contributes significantly to the virulence of V . cholerae . The DeltarpoE mutant was highly attenuated with a 50% lethal dose more than 3 logs higher than that for the parental strain, and its ability to colonize the intestine was reduced approximately 30-fold . A time course of infection revealed that the number of CFU of the DeltarpoE mutant was approximately 1 log lower than that of the parental strain by 12 h postinoculation and decreased further by 24 h . The defect in virulence in the DeltarpoE mutant thus appears to be a diminished ability to survive within the intestinal environment . The results here also show that sigma(E) is not required for growth and survival of V . cholerae in vitro at high temperatures but is required under other stressful conditions, such as in the presence of 3% ethanol . As in Escherichia coli, the expression of rpoE in V . cholerae is dependent upon two promoters located upstream of the gene, P1 and P2 . P1 appears to be sigma(70) dependent, whereas the downstream promoter, P2, is positively autoregulated by sigma(E).

Microb Pathog, 2002 Sep, 33(3), 127 - 34
An exocellular thermolysin-like metalloprotease produced by Vibrio fluvialis: purification, characterization, and gene cloning; Miyoshi S et al.; An exocellular metalloprotease produced by Vibrio fluvialis, an enteropathogenic vibrio, was purified and characterized . The metalloprotease (V . fluvialis protease {VFP}) was found to have very similar characteristics to V . vulnificus protease, including a molecular mass of 45kDa, sensitivity to chelating agents or competitive inhibitors for thermolysin-like metalloproteases, and the substrate specificity . The structural gene for VFP was also cloned, and its nucleotide sequence was determined . The deduced amino acid sequence confirmed that VFP was a member of the thermolysin family . VFP, like V . vulnificus protease, showed the haemagglutinating, permeability-enhancing and haemorrhagic activities in addition to the proteolytic activity toward oligopeptide, casein or elastin.

Hua Xi Yi Ke Da Xue Xue Bao, 1999 Mar, 30(1), 28 - 30
{Purification and identification of pili of Vibrio cholera O139}; Du Y et al.; To provide the base of manufacturing V . cholera O139 subunit vaccine, purification and identification methods of V . cholera O139 Toxin-coregulated pili (TCP) were studied . The results showed the optimal expressing condition of TCP was in AKI or CFA medium, at 30 C for 24-36 h with stationary cultivation . The molecular weight of the TCP subunit protein measured by sodium dodecyl sulfate-poly acrylamide gel electrophoresis was about 20.5 kd . By using mouse anti-TCP monoclone antibody and sheep anti-mouse IgG-HRP antibody, the TCP was confirmed by dot blot immunoassay . Rabbit immune serum of TCP purified from O139 strain agglutinated both O139 biotype and El Tor biotype Vibrio cholera strains . These indicate that TCP may be an effective and protective antigen to conduct the preparation of V . cholera vaccine.

Ying Yong Sheng Tai Xue Bao, 2002 Jun, 13(6), 731 - 4
{Variation of bacteria numbers in fish-shrimp mix-culturing ecosystem}; Li Q et al.; The study on variation of bacteria numbers in Penaeus chinensis-fish mix-culturing ecosystem in 1997 . Indicated that at the beginning of culturing season, total number of heterotrophic bacteria and that of nitrate-reducing bacteria in mix-culturing ponds was low, but it was higher than that in mono-culturing shrimp pond . With time going on, the number of bacteria in mono-culturing pond increased rapidly and remained at a high level in August and September, an that in mix-culturing ponds also increased . But the latter increased slowly, and it was never over 10(4) cells.ml-1 and dropped in September . Number of bacteria in bottom of the ponds varied with the similar regulation, but the numbers was 10-100 times higher . The numbers of vibrio in mix-culturing ponds was always lower than that in contrastive pond at the same time . So, in fish-shrimp mix-culturing ponds, the contents of organic matter were lower and the total amount and variability of phytoplankton were higher than corresponding items in mono-culturing pond . It was concluded that mix-culture could stimulate and control the growth of heterotrophic bacteria, accelerate the degradation of organic pollutants, consequently fasten and stabilize the circulation of mater in ecosystem of ponds in culturing season.

Exp Mol Med, 2002 Jul 31, 34(3), 239 - 42
Cholesterol induce oligomerization of Vibrio vulnificus cytolysin specifically; Kim BS et al.; Vibrio vulnificus cytolysin (VVC) has been implicated as one of the important virulence determinants of V . vulnificus that causes serious septicemia and wound infection . An attempt was made to investigate that VVC could act as a ligand which stimulates intracellular signaling systems . Cholesterol dose-dependently blocked VVC hemolytic activity through oli-gomerization of cytolysin . Among cholesterol derivatives including 7-dehydrocholesterol, cholesteryl esters, deoxycholate, and cholestane tested, only 7-dehydrocholesterol induced oligomerization as well as inactivation of VVC . These results show that oligomerization of VVC is completely dependent on three-dimensional structure of cholesterol where specific interaction of cholesterol at oligomerization sites of VVC is very selective . These findings support the idea that cholesterol which constitute many of cellular plasma membrane could be a receptor of VVC on plasma membrane of target cells.

Bioorg Med Chem, 2002 Nov, 10(11), 3517 - 22
QSAR study on narcotic mechanism of action and toxicity: a molecular connectivity approach to Vibrio fischeri toxicity testing; Agrawal VK et al.; Quantitative structure-activity relationships (QSARs) have been established based on narcotic mechanism of action and toxicity data to Vibrio fischeri using molecular connectivity indices . The results obtained suggest that both, the degree of branching and electronic characteristic of the compounds have dominant role in the exhibition of toxicity . The information obtained in the present study will be useful in designing more potent compounds.

Kansenshogaku Zasshi, 2002 Jul, 76(7), 528 - 35
{Basic studies on Vibrio vulnificus infection: isolation of V . vulnificus from sea water, sea mud, and oysters}; Oonaka K et al.; To clarify the environmental distribution of Vibrio vulnificus, sea water, sea mud, and oysters were examined at 13 sites, i.e . 4 sites in the Tokyo Bay (eastern Japan) and 9 sites (5 sites for oysters) in Tokushima Prefecture (western Japan) . 1 . V . vulnificus was isolated from 80 (54.8%) of the 146 samples of sea water examined . It was isolated from 19 (41.3%) of the 46 samples from western Japan and 61 (61.0%) of the 100 samples from eastern Japan . 2 . It was isolated from 40 (40.8%) of the 98 samples of sea mud obtained in eastern Japan . 3 . It was isolated from 655 (30.3%) of the 2,165 samples of oysters . They were 30 (9.7%) of 309 samples from western Japan and 625 (33.7%) of 1,856 samples from eastern Japan . 4 . The density of V . vulnificus was 0.3-1.1 x 10(6) MPN/L in seawater, 0.3-1.1 x 10(5) MPN/100 g in sea mud, and 0.3-1.1 x 10(7) MPN/100 g in oysters . 5 . Seasonally, V . vulnificus was isolated from 44 (6.2%) of the 713 samples in spring, 450 (72.6%) of the 620 samples in summer, 264 (51.8%) of the 510 samples in fall, and 17 (3.0%) of the 56 samples in winter . Thus, the isolation rates of V . vulnificus from sea water and oysters tended to be higher in eastern Japan than in western Japan and to be highest in summer, then, in fall.

Environ Technol, 2002 Aug, 23(8), 911 - 8
A degradation and toxicity study of three textile reactive dyes by ozone; Kunz A et al.; Textile reactive dyes are very recalcitrant to biodegradation, causing aesthetic, acute and chronic toxicity problems in receiving waters . In this study, we present the degradation of three different reactive dyes (Reactive Black 5, Reactive Blue 19 and Reactive Blue 21) by ozone, at two different pH conditions . The ozonation process achieved a very fast decoloration rate for the studied dyes . The influence of pH in the ozonation of these dyes was only significant for Reactive Black 5 . The toxicity of dyes, after pre-treatment, was evaluated using Vibrio fisheri (microtox) . There was little change in toxicity for Reactive Black 5, but there was a reduction for Reactive Blue 19 . A significant increase in toxicity for Reactive Blue 21 was verified, caused by the increase in the bioavailability of th e cooper ion that was in the complexed form before the treatment with ozone.

Biochem Biophys Res Commun, 2002 Sep 6, 296(5), 1072 - 6
Generation of thermostable monomeric luciferases from Photorhabdus luminescens; Westerlund-Karlsson A et al.; Bacterial luciferases and the genes encoding these light-emitting enzymes have an increasing number of applications in biological sciences . Temperature lability and the heterodimeric nature of these luciferases have been the major obstacles for their widespread use, for instance, as genetic reporters . Escherichia coli expressing wild-type Photorhabdus luminescens luciferase was found to produce eight times more light than the corresponding Vibrio harveyi luciferase clone in vivo at 37 degrees C . Three monomeric luciferases were created by translationally fusing the two genes encoding luxA and luxB proteins of P . luminescens . These clones were equally active in producing light in vivo when cultivated at 37 degrees C compared to cultivation at 30 degrees C . The fusion containing the longest linker showed the highest activity . In vitro, the monomeric luciferases were less active having at best 20% of activity of the wild-type enzyme due to the partial formation of insoluble aggregates . The results suggest that P . luminescens luciferase and monomeric derivatives thereof should be more suitable than the corresponding V . harveyi enzyme to be used as reporters in cell types which need cultivation at elevated temperatures.

J Clin Microbiol, 2002 Sep, 40(9), 3296 - 9
New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh; Nair GB et al.; The sixth pandemic of cholera and, presumably, the earlier pandemics were caused by the classical biotype of Vibrio cholerae O1, which was progressively replaced by the El Tor biotype representing the seventh cholera pandemic . Although the classical biotype of V . cholerae O1 is extinct, even in southern Bangladesh, the last of the niches where this biotype prevailed, we have identified new varieties of V . cholerae O1, of the El Tor biotype with attributes of the classical biotype, from hospitalized patients with acute diarrhea in Bangladesh . Twenty-four strains of V . cholerae O1 isolated between 1991 and 1994 from hospitalized patients with acute diarrhea in Matlab, a rural area of Bangladesh, were examined for the phenotypic and genotypic traits that distinguish the two biotypes of V . cholerae O1 . Standard reference strains of V . cholerae O1 belonging to the classical and El Tor biotypes were used as controls in all of the tests . The phenotypic traits commonly used to distinguish between the El Tor and classical biotypes, including polymyxin B sensitivity, chicken cell agglutination, type of tcpA and rstR genes, and restriction patterns of conserved rRNA genes (ribotypes), differentiated the 24 strains of toxigenic V . cholerae O1 into three types designated the Matlab types . Although all of the strains belonged to ribotypes that have been previously found among El Tor vibrios, type I strains had more traits of the classical biotype while type II and III strains appeared to be more like the El Tor biotype but had some classical biotype properties . These results suggest that, although the classical and El Tor biotypes have different lineages, there are possible naturally occurring genetic hybrids between the classical and El Tor biotypes that can cause cholera and thus provide new insight into the epidemiology of cholera in Bangladesh . Furthermore, the existence of such novel strains may have implications for the development of a cholera vaccine.

Eur J Biochem, 2002 Sep, 269(17), 4351 - 8
Vibrio cholerae hemolysin . Implication of amphiphilicity and lipid-induced conformational change for its pore-forming activity; Chattopadhyay K et al.; Vibrio cholerae hemolysin (HlyA), a water-soluble protein with a native monomeric relative molecular mass of 65 000, forms transmembrane pentameric channels in target biomembranes . The HlyA binds to lipid vesicles nonspecifically and without saturation; however, self-assembly is triggered specifically by cholesterol . Here we show that the HlyA partitioned quantitatively to amphiphilic media irrespective of their compositions, indicating that the toxin had an amphiphilic surface . Asialofetuin, a beta1-galactosyl-terminated glycoprotein, which binds specifically to the HlyA in a lectin-glycoprotein type of interaction and inhibits carbohydrate-independent interaction of the toxin with lipid, reduced effective amphiphilicity of the toxin significantly . Resistance of the HlyA to proteases together with the tryptophan fluorescence emission spectrum suggested a compact structure for the toxin . Fluorescence energy transfer from the HlyA to dansyl-phosphatidylethanolamine required the presence of cholesterol in the lipid bilayer and was synchronous with oligomerization . Phospholipid bilayer without cholesterol caused a partial unfolding of the HlyA monomer as indicated by the transfer of tryptophan residues from the nonpolar core of the protein to a more polar region . These observations suggested: (a) partitioning of the HlyA to lipid vesicles is driven by the tendency of the amphiphilic toxin to reduce energetically unfavorable contacts with water and is not affected significantly by the composition of the vesicles; and (b) partial unfolding of the HlyA at the lipid-water interface precedes and promotes cholesterol-induced oligomerization to an insertion-competent configuration.

J Bacteriol, 2002 Sep, 184(18), 5121 - 9
Role for phosphoglucomutase in Vibrio fischeri-Euprymna scolopes symbiosis; DeLoney CR et al.; Vibrio fischeri, a luminescent marine bacterium, specifically colonizes the light organ of its symbiotic partner, the Hawaiian squid Euprymna scolopes . In a screen for V . fischeri colonization mutants, we identified a strain that exhibited on average a 10-fold decrease in colonization levels relative to that achieved by wild-type V . fischeri . Further characterization revealed that this defect did not result from reduced luminescence or motility, two processes required for normal colonization . We determined that the transposon in this mutant disrupted a gene with high sequence identity to the pgm (phosphoglucomutase) gene of Escherichia coli, which encodes an enzyme that functions in both galactose metabolism and the synthesis of UDP-glucose . The V . fischeri mutant grew poorly with galactose as a sole carbon source and was defective for phosphoglucomutase activity, suggesting functional identity between E . coli Pgm and the product of the V . fischeri gene, which was therefore designated pgm . In addition, lipopolysaccharide profiles of the mutant were distinct from that of the parent strain and the mutant exhibited increased sensitivity to various cationic agents and detergents . Chromosomal complementation with the wild-type pgm allele restored the colonization ability to the mutant and also complemented the other noted defects . Unlike the pgm mutant, a galactose-utilization mutant (galK) of V . fischeri colonized juvenile squid to wild-type levels, indicating that the symbiotic defect of the pgm mutant is not due to an inability to catabolize galactose . Thus, pgm represents a new gene required for promoting colonization of E . scolopes by V . fischeri.

J Bacteriol, 2002 Sep, 184(18), 4981 - 7
Conserved amplification of chemotactic responses through chemoreceptor interactions; Lamanna AC et al.; Many bacteria concentrate their chemoreceptors at the cell poles . Chemoreceptor location is important in Escherichia coli, since chemosensory responses are sensitive to receptor proximity . It is not known, however, whether chemotaxis in other bacteria is similarly regulated . To investigate the importance of receptor-receptor interactions in other bacterial species, we synthesized saccharide-bearing multivalent ligands that are designed to cluster relevant chemoreceptors . As has been shown with E . coli, we demonstrate that the behaviors of Bacillus subtilis, Spirochaete aurantia, and Vibrio furnissii are sensitive to the valence of the chemoattractant . Moreover, in B . subtilis, chemotactic responses to serine were increased by pretreatment with saccharide-bearing multivalent ligands . This result indicates that, as in E . coli, signaling information is transferred among chemoreceptors in B . subtilis . These results suggest that interreceptor communication may be a general mechanism for modulating chemotactic responses in bacteria.

Rev Biol Trop, 2001 Sep-Dec, 49(3-4), 1213 - 22
{In vitro evaluation of antibacterial substances produced by bacteria isolated from different marine organisms}; Castillo I et al.; Bacteria from several groups of marine organisms were isolated and, using direct antibiograms, identified those that produce antibacterial substances, using a human pathogenic strain of Staphylococcus aureus ATCC6538 as revealing microorganism . Bacteria which produce substances that inhibited S . aureus growth were identified through morphological, physiological and biochemical tests . Out of 290 bacteria, 54 (18.6%) inhibited the growth of S . aureus, but only 27 survived for identification . Bivalves, sponges and corals were the most represented from which 41.2, 33.3 and 29.7%, respectively, produced antibacterial substances of the isolated bacteria in each group . The marine species with highest proportions of these bacteria were the hard coral Madracis decactis (62.5%), the sponges Cliona sp . (57.1%) and the octocoral Plexaura flexuosa (50.0%) . Out of the 27 strains that produced antibacterial substances, 51.8% were Aeromonas spp . and 14.8% Vibrio spp . Marine bacteria that produce antibacterial substances are abundant, most belong in the Vibrionacea group and were isolated mainly from corals and bivalve mollusks.

Antimicrob Agents Chemother, 2002 Sep, 46(9), 3050 - 3
Functional characterization of Brucella melitensis NorMI, an efflux pump belonging to the multidrug and toxic compound extrusion family; Braibant M et al.; Two putative proteins (NorMI and NorMII) similar to the multidrug efflux protein NorM of Vibrio parahaemolyticus are encoded by the Brucella melitensis 16 M genome . We show that a drug-hypersusceptible Escherichia coli strain overexpressing NorMI displays increased resistance to norfloxacin, ciprofloxacin, gentamicin, tetraphenylphosphonium ion, acriflavine, and berberine . This elevated resistance was proven to be mediated by an energy-dependent efflux mechanism . NorMI belongs to the multidrug and toxic compound extrusion family and is the first multidrug efflux protein identified in Brucella spp.

Antimicrob Agents Chemother, 2002 Sep, 46(9), 2948 - 55
Occurrence of antibiotic resistance gene cassettes aac(6')-Ib, dfrA5, dfrA12, and ereA2 in class I integrons in non-O1, non-O139 Vibrio cholerae strains in India; Thungapathra M et al.; Molecular mechanisms of multidrug resistance in Vibrio cholerae belonging to non-O1, non-O139 serogroups isolated during 1997 to 1998 in Calcutta, India, were investigated . Out of the 94 strains examined, 22 strains were found to have class I integrons . The gene cassettes identified were dfrA1, dfrA15, dfrA5, and dfrA12 for trimethoprim; aac(6')-Ib for amikacin and tobramycin; aadA1 and aadA2 for streptomycin and spectinomycin; and ereA2 for erythromycin resistance . To our knowledge, this is the first report of the presence of dfrA5, dfrA12, aac(6')-Ib, and ereA2 cassettes in class I integrons of V . cholerae . Forty-three of 94 strains also had plasmids, and out of these, 14 contained both class I integrons and plasmids . Pulsed-field gel electrophoresis followed by Southern hybridization revealed that in the 14 plasmid-bearing strains, class I integrons resided either on chromosomes, on plasmids, or on both . Our results indicated that besides class I integrons and plasmids, a conjugative transposon element, SXT, possibly contributed to the multiple antibiotic resistance.

Mol Gen Mikrobiol Virusol, 2002, (2), 9 - 14
{Alteration of cholera toxin biosynthesis in Vibrio cholerae 01 as a result of temperate phage 139 integration into bacterial chromosome}; Eroshenko GA et al.; Infection of V . cholerae 01 (classical and eltor biovars) cells with the temperate cholera phage 139 derived from V . cholerae serogroup 0139 followed by integration of the phage genome into the bacterial chromosome significantly increased the production of cholera toxin, the main virulence factor . The level of toxin biosynthesis in the lysogenic V . cholerae classical strain increased 3-fold and that in V . eltor thirty times in comparison with the parental strains . Increased production of cholera toxin was not associated with an increase in the number of copies of genes involved in its biosynthesis but seemed to be due to changes in toxinogenesis regulation.

Curr Opin Plant Biol, 2002 Aug, 5(4), 285 - 90
Quorum sensing in plant-associated bacteria; Loh J et al.; N-acyl homoserine lactone (AHL)-mediated quorum sensing by bacteria regulates traits that are involved in symbiotic, pathogenic and surface-associated relationships between microbial populations and their plant hosts . Recent advances demonstrate deviations from the classic LuxR/LuxI paradigm, which was first developed in Vibrio . For example, LuxR homologs can repress as well as activate gene expression, and non-AHL signals and signal mimics can affect the expression of genes that are controlled by quorum sensing . Many bacteria utilize multiple quorum-sensing systems, and these may be modulated via post-transcriptional and other global regulatory mechanisms . Microbes inhabiting plant surfaces also produce and respond to a diverse mixture of AHL signals . The production of AHL mimics by plants and the identification of AHL degradative pathways suggest that bacteria and plants utilize this method of bacterial communication as a key control point for influencing the outcome of their interactions.

Cell, 2002 Aug 9, 110(3), 303 - 14
Parallel quorum sensing systems converge to regulate virulence in Vibrio cholerae; Miller MB et al.; The marine bacterium Vibrio harveyi possesses two quorum sensing systems (System 1 and System 2) that regulate bioluminescence . Although the Vibrio cholerae genome sequence reveals that a V . harveyi-like System 2 exists, it does not predict the existence of a V . harveyi-like System 1 or any obvious quorum sensing-controlled target genes . In this report we identify and characterize the genes encoding an additional V . cholerae autoinducer synthase and its cognate sensor . Analysis of double mutants indicates that a third as yet unidentified sensory circuit exists in V . cholerae . This quorum sensing apparatus is unusually complex, as it is composed of at least three parallel signaling channels . We show that in V . cholerae these communication systems converge to control virulence.

J Control Release, 2002 Aug 21, 82(2-3), 237 - 47
Oral immunogenicity of the inactivated Vibrio cholerae whole-cell vaccine encapsulated in biodegradable microparticles; Yeh MK et al.; Vibrio cholerae (VC)-loaded microparticles as an oral vaccine delivery system were prepared with 6% w/v poly(DL-lactide-co-glycolide)(PLG) in the oil phase as well as 10% w/v PVP and 5% w/v NaCl in the aqueous phase, by an water-in-oil-in-water emulsion/solvent extraction technique . VC was successfully entrapped in the microparticles with trapping efficiencies up to 97.8% and a loading level of 55.4+/-6.9 microg/mg . The microparticle delivery system with a particle size of 3.8 microm had different distribution of VC content in the core region (25.7+/-1.9 microg/mg) and surface (6.2+/-0.9 microg/mg) . The immunogenic potential of VC-loaded microparticles in comparison with PLG microparticles or VC solution was evaluated in adult mice by oral immunization, in which mice received one dose of 20 mg VC-loaded microparticles or 20 mg VC-loaded microparticles physical mixed with amphotericin B . The control group received 20 mg PLG microparticle or VC solution . Serum samples were collected from all tested mice on the day of immunization and at 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 weeks postimmunization . Sera were examined for vibriocidal antibodies by microtitration and Vibrio-specific serum IgG and IgM antibodies were assessed by the ELISA method . IgG and IgM antibodies to intact VC were detected in sera from all animals immunized with VC . The response was specific and of high magnitude . Significantly higher antibody responses were obtained when sera from both VC-loaded microparticles and VC-loaded microparticles physical mixed with amphotericin B immunized mice were titrated against VC . The immunogenicity of VC-loaded microparticles mixed with amphotericin B in evoking serum IgG and IgM responses was higher than that of VC-loaded microparticles only . These results demonstrate that VC-loaded microparticles physical mixed with amphotericin B and VC-loaded microparticles orally administered evoke Vibrio-specific serum IgG and IgM responses as well as vibriocidal antibody activity in mice . The VC incorporation, physicochemical characterization data, and the animal results obtained in this study may be relevant in optimizing the vaccine incorporation and delivery properties of these potential vaccine targeting carriers.

Eur J Clin Microbiol Infect Dis, 2002 Jul, 21(7), 537 - 8 Epub 2002 Jun 28.
Wound Infection due to Vibrio vulnificus in Spain; Torres L et al.; Vibrio vulnificus is a gram-negative rod that can cause septicaemia and skin lesions, usually in patients with underlying illnesses such as chronic liver disease or diabetes mellitus . Infections caused by this bacterium are unusual in Spain . A case of skin infection due to Vibrio vulnificus is reported in a patient whose abraded skin on his left leg came into contact with seawater . The patient died suddenly, probably due to septicaemia or bacteraemia caused by this organism . Vibrio vulnificus infection must be considered in the differential diagnosis of septicaemia, skin lesions and wound infections, particularly when a patient reports a history of contact with seawater.

EMBO J, 2002 Aug 15, 21(16), 4240 - 9
A satellite phage-encoded antirepressor induces repressor aggregation and cholera toxin gene transfer; Davis BM et al.; CTXphi is a filamentous bacteriophage whose genome encodes cholera toxin, the principal virulence factor of Vibrio cholerae . We have found that the CTXphi-related element RS1 is a satellite phage whose transmission depends upon proteins produced from a CTX prophage (its helper phage) . However, unlike other satellite phages and satellite animal viruses, RS1 can aid the CTX prophage as well as exploit it, due to the RS1-encoded protein RstC . RstC, whose function previously was unknown, is an antirepressor that counteracts the activity of the phage repressor RstR . RstC promotes transcription of genes required for phage production and thereby promotes transmission of both RS1 and CTXphi . Antirepression by RstC also induces expression of the cholera toxin genes, ctxAB, and thus may contribute to the virulence of V.cholerae . In vitro, RstC binds directly to RstR, producing unusual, insoluble aggregates containing both proteins . In vivo, RstC and RstR are both found at the cell pole, where they again appear to form stable complexes . The sequestration/inactivation process induced by RstC resembles those induced by mutant polyglutamine-containing proteins implicated in human neurodegenerative disorders.

J Bacteriol, 2002 Sep, 184(17), 4933 - 5
Filamentous bacteriophages of vibrios are integrated into the dif-like site of the host chromosome; Iida T et al.; The dif site is located in the replication terminus region of bacterial chromosomes, having a function of resolving dimeric chromosomes formed during replication . We demonstrate that filamentous bacteriophages of vibrios, such as f237 (Vibrio parahaemolyticus) and CTXphi (V . cholerae), are integrated into the dif-like site of host chromosome.

Eur J Med Chem, 2002 Aug, 37(8), 681 - 7
Preparation of a subcellular conjugate with the lipopolysaccharide from Vibrio cholerae 01 using beta-D-glucan as matrix; Machova E et al.; A conjugate consisting of detoxified lipopolysaccharide of Vibrio cholerae, a carrier polysaccharide matrix and an immunogenic protein has been synthesised and the reaction conditions have been optimised for obtaining a high degree of conjugation . The obtained construct showed reactivity with the antibodies against V . cholerae and can serve as a prospective candidate for preparation of subcellular anti-cholera vaccine.

Dis Aquat Organ, 2002 Jun 21, 50(1), 35 - 43
Vibrio carchariae, a pathogen of the abalone Haliotis tuberculata; Nicolas JL et al.; Since 1997, mass mortality of the abalone Haliotis tuberculata L . has occurred in the natural environment along the French coast . The outbreak of disease started on the south coast of Brittany near Concarneau in 1997, then spread to the north of Brittany (in 1998) and the west coast of Normandy (Golfe de St . Malo in 1999) . Between 60 and 80% of the abalone died . In 1999, mortality also affected a land-based abalone farm in Normandy during the summer . At this farm, a Vibrio sp . was isolated in abundance from abalone that had just died . The disease was experimentally reproduced by inoculation or by introducing the pathogen into the surrounding water . This vibrio, identified by genotypic and phenotypic characters, is related to V carchariae . It is similar to the V carchariae, responsible for mortality in the Japanese abalone Sulculus diversicolor supratexta, but some phenotypic characters differentiate both strains . In 2000, healthy abalone placed in 2 sites on the north and south coasts of Brittany died, and the pathogen V carchariae could be isolated from dead individuals, demonstrating that the pathogen was probably the cause of the abalone disease that has been occurring since 1997 in Brittany.

Environ Pollut, 2002, 119(2), 195 - 202
Comparative toxic and genotoxic effects of chloroacetanilides, formamidines and their degradation products on Vibrio fischeri and Chironomus riparius; Osano O et al.; Toxic and genotoxic effects of alachlor, metolachlor, amitraz, chlordimeform, their respective environmentally stable degradation products 2,6-diethylaniline, 2-ethyl-4-methylaniline, 2,4-dimethylaniline, and two other related compounds, 3,4-dichloroaniline and aniline were compared . Acute toxicity tests with Chironomus riparius (96 h) and Vibrio fischeri (Microtox) and genotoxicity tests with a dark mutant of V . fischeri (Mutato) were carried out . Our results demonstrate that toxicity and genotoxicity of the pesticides are retained upon degradation to their alkyl-aniline metabolites . In the case of the herbicides alachlor and metolachlor, the toxicity to V . fischeri was enhanced upon degradation . Narcosis alone explains toxicity of the compounds to the midge, but not so for the bacteria suggesting a disparity in the selectivity of the test systems . All compounds showed direct genotoxicity in the Vibrio test . but amitraz and its metabolite were genotoxic at concentrations 10(3)-10(5) lower than all the other compounds . The observations indicate that stable aniline degradation products of the pesticides may contribute considerably to environmental risks of pesticides application and that genotoxic effects may arise upon degradation of pesticides.

Diagn Microbiol Infect Dis, 2002 Aug, 43(4), 311 - 3
Splenic abscess with Vibrio cholerae masking pancreatic cancer; Cavuoti D et al.; A 77-year-old man presented to our hospital with a clinical scenario suspicious for endocarditis with septic emboli to the lungs and splenic abscess . Vibrio cholerae was isolated from purulent material aspirated from the abscess . Medical therapy and percutaneous drainage of the abscess were unsuccessful . The patient underwent splenectomy and distal pancreatectomy revealing a pancreatic tail carcinoma involving the spleen and colon . The patient later expired secondary to metastatic disease . This case represents the first isolation of V . cholerae from a splenic abscess but also illustrates that although newer imaging technologies have made the diagnosis of splenic abscess easier, the true etiology of the abscess may remain elusive.

Mar Environ Res, 2002, 54(1), 1 - 19
Heterotrophic bacteria in the northern Adriatic Sea: seasonal changes and ectoenzyme profile; Zaccone R et al.; A seasonal study of the quantitative and qualitative distribution of heterotrophic bacterial community was carried out in the Adriatic Sea between April 1995 and January 1996, in order to evaluate its spatial and temporal variability and metabolic potential in the degradation processes of organic matter . The culturable bacteria (CFU) ranged between 0.1 and 22% of total bacterioplankton with a maximum percentage in surface samples of coastal zones . Their distribution was generally affected by the prevailing hydrological conditions . At the coastal stations about 44-75% of CFU variance could be explained by river runoff . The changes in the composition of heterotrophic bacterial community showed a seasonal succession of main bacterial groups, with a prevalence of Gram negative, non fermenting bacteria in the cold period (April-January) and an increase of Vibrionaccae and pigmented bacteria in summer . The seasonal variations were more important at the stations influenced by rivers than offshore . The bacterial community showed a greater versatility for organic polymers hydrolysis in the offshore station than in the coastal areas . Over 60% of all isolated heterotrophic bacteria expressed peptidase, lipase and phosphatase ectoenzymes activities, in all seasons and showed an increasing trend in warm period (in July October) . The alpha- and beta-glucosidase potentials of bacteria were lower (20% on average) and showed different pattern during the year . These results suggest different role of the bacterial community in the decomposition of organic matter in the Adriatic Sea . Since only 20% of bacterial strains expressed glucosidase activity, carbohydrate-rich polymers such as mucilage might accumulate.

Appl Environ Microbiol, 2002 Aug, 68(8), 4140 - 4
Fluorescent amplified fragment length polymorphism and repetitive extragenic palindrome-PCR fingerprinting reveal host-specific genetic diversity of Vibrio halioticoli-like strains isolated from the gut of Japanese abalone; Sawabe T et al.; When analyzed by fluorescent amplified fragment length polymorphism and repetitive extragenic palindrome-PCR fingerprinting, a total of 47 Vibrio halioticoli strains isolated from four Japanese abalone species and one turban shell species formed three clusters that roughly reflect the different species of host abalone from which they were isolated . The V . halioticoli isolates from turban shells were distributed evenly among the clusters . Representative isolates from two clusters were deemed separate species or subspecies by DNA-DNA hybridization.

J Appl Microbiol, 2002, 93(2), 261 - 8
Detection and identification of Vibrio scophthalmi in the intestinal microbiota of fish and evaluation of host specificity; Cerda-Cuellar M et al.; AIMS: To develop a species-specific probe (VSV3) for the detection of Vibrio scophthalmi in fish intestine and to apply this probe to study the host specificity of V . scophthalmi . METHODS AND RESULTS: A specific probe (VSV3) based on the variable region V3 of the 16S rRNA gene (rDNA) was designed . Its specificity was tested by DNA-DNA hybridization and by colony hybridization . No cross-hybridization was found . The sensitivity of the probe was tested both by DNA-DNA hybridization and by colony hybridization . The detection limit of V . scophthalmi 16S rDNA was 150 pg or 10 cfu . Vibrio scophthalmi cells were detected in experimental samples constituted by mixed cultures when present in proportions of 1 : 10 and 1 : 100 . The VSV3 probe also proved to be reliable for the detection of V . scophthalmi in samples of fish intestine . CONCLUSIONS: The VSV3 probe can be used for the detection of V . scophthalmi in colony hybridization or DNA-DNA hybridization of amplified 16S rDNA . Preliminary results indicate that V . scophthalmi may present certain host specificity for turbot . SIGNIFICANCE AND IMPACT OF THE STUDY: The VSV3 probe provides a useful tool for ecological studies.

Analyst, 2002 Jun, 127(6), 724 - 9
Systematic search for surrogate chromatographic models of biopartitioning processes; Cimpean DM et al.; A systematic approach for identifing surrogate chromatographic models for biopartitioning processes is described . The method is based on a comparison of the system constant ratios of the solvation parameter model for biopartitioning processes and a database of system constant ratios for reversed-phase liquid chromatographic and micellar electrokinetic chromatographic systems compiled from literature sources . An acceptance filter of < or = 0.2 is applied for each difference in system constant ratio for the compared systems to provide a reasonable probability of success without outputting too many systems with limited predictive properties . Surrogate chromatographic models identified for the non-specific toxicity of neutral organic compounds to the fathead minnow and the soil-water distribution constant are tested by construction of a correlation model for the characteristic property of the biological process and the chromatographic retention factors for a structurally varied group of compounds . Although these models are not the best that could be obtained based on ranking of the differences in system constant ratios the predictive ability of the correlation models is suitable for typical applications and similar to the accepted uncertainty in the measurements of the biological property . Retention factors on the immobilized artificial membrane column (IAM PC DD 2) with 10% (v/v) methanol-water as mobile phase are able to estimate non-specific toxicity to the fathead minnow with a standard error (SE) of 0.22 log units and coefficient of determination (r2) of 0.97 for 31 compounds . Retention factors on a Bakerbond DIOL column with 20% (v/v) acetonitrile-water as mobile phase are able to estimate the soil-water distribution constant with an SE of 0.38 log units and r2 = 0.88 for 59 compounds . Other potential surrogate chromatographic models are identified for non-specific toxicity to the guppy, tadpole, Vibrio fischeri and Terahymena pyriformis as well as the plant cuticle matrix-water distribution constant . On the other hand reversed-phase chromatographic systems seem poorly suited for estimating intestinal absorption and the blood-brain distribution constant.

Mar Pollut Bull, 2002 May, 44(5), 412 - 20
Analysis of the 16S-23S rDNA intergenic spacers (IGSs) of marine vibrios for species-specific signature DNA sequences; Lee SK et al.; Vibrios are widespread in the marine environment and a few pathogenic species are known to be commonly associated with outbreaks of diarrheal diseases in humans due to the consumption of raw or improperly cooked seafood . However, there are also many Vibrio species which are potentially pathogenic to vertebrate and invertebrate aquatic animals, and of which little is known . In an attempt to develop rapid PCR detection methods for these latter class of vibrios, we have examined the 16S-23S intergenic spacers (IGSs) of 10 lesser-known Vibrio species and successfully developed species-specific primers for eight of them--Vibrio costicola, V . diazotrophicus, V . fluvialis, V . nigripulchritudo, V . proteolyticus, V . salmonicida, V . splendidus and V . tubiashii . The IGS amplicons were amplified using primers complementary to conserved regions of the 16S and 23S rRNA genes, and cloned into plasmid vectors and sequenced . Analysis of the IGS sequences showed that 37 ribosomal RNA (rrn) operons representing seven different IGS types have been cloned from the 10 vibrios . The three IGS types--IGS(0), IGS(IA) and IGS(Glu)--were the most prevalent forms detected . Multiple alignment of representative sequences of these three IGS types from different Vibrio species revealed several domains of high sequence variability, which were used to design species-specific primers for PCR . The specificity of the primers were evaluated using total DNA prepared from different Vibrio species and bacterial genera . The results showed that the PCR method can be used to reliably detect eight of the 10 Vibrio species in marine waters in this study.

Pest Manag Sci, 2002 Jul, 58(7), 707 - 12
Iron(III) photo-induced degradation of isoproturon: correlation between degradation and toxicity; Galichet F et al.; The degradation of isoproturon photoinduced by Fe(III) was investigated under both artificial and solar light . The monomeric species Fe(OH)2+ present under the experimental conditions ({Fe(III)} = 3 x 10(-4) M) is the main Fe(III) species responsible for the degradation of isoproturon . The process involves the attack on the pollutant by OH radicals generated by irradiation of Fe(OH)2+ . The major primary photoproducts were identified; they accumulate in the solution medium before being degraded . The toxicity of the solution to marine bacterium Vibrio fisheri (Beijerinck) Lehmann & Neumann was monitored during the degradation process . It increased in the early stages of the reaction and, among the photoproducts, the N-formyl derivative appeared to be the major product responsible for the increase in toxicity.

J Bacteriol, 2002 Aug, 184(16), 4520 - 8
Role of the C-terminal domain of the alpha subunit of RNA polymerase in LuxR-dependent transcriptional activation of the lux operon during quorum sensing; Finney AH et al.; During quorum sensing in Vibrio fischeri, the luminescence, or lux, operon is regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule {N-(3-oxohexanoyl) homoserine lactone} . LuxR, which binds to the lux operon promoter at a position centered at -42.5 relative to the transcription initiation site, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP) . The specific role of the alpha-subunit C-terminal domain (alphaCTD) of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated . The effects of 70 alanine substitution variants of the alpha subunit were determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli . The mutant RNAPs from strains exhibiting at least twofold-increased or -decreased activity in comparison to the wild type were further examined by in vitro assays . Since full-length LuxR has not been purified, an autoinducer-independent N-terminally truncated form of LuxR, LuxRDeltaN, was used for in vitro studies . Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRDeltaN, and 14 alanine substitutions in the alphaCTD were identified as having negative effects on the rate of transcription from the lux operon promoter . Five of these 14 alpha variants were also involved in the mechanisms of both LuxR- and LuxRDeltaN-dependent activation in vivo . The positions of these residues lie roughly within the 265 and 287 determinants in alpha that have been identified through studies of the cyclic AMP receptor protein and its interactions with RNAP . This suggests a model where residues 262, 265, and 296 in alpha play roles in DNA recognition and residues 290 and 314 play roles in alpha-LuxR interactions at the lux operon promoter during quorum sensing.

J Bacteriol, 2002 Aug, 184(16), 4466 - 74
The nptA gene of Vibrio cholerae encodes a functional sodium-dependent phosphate cotransporter homologous to the type II cotransporters of eukaryotes; Lebens M et al.; The nptA gene of Vibrio cholerae has significant protein sequence homology with type II sodium-dependent phosphate (P(i)) cotransporters found in animals but not previously identified in prokaryotes . The phylogeny of known type II cotransporter sequences indicates that nptA may be either an ancestral gene or a gene acquired from a higher eukaryotic source . The gene was cloned into an expression vector under the control of an inducible promoter and expressed in Escherichia coli . The results demonstrate that nptA encodes a functional protein with activity similar to that of the animal enzyme, catalyzing high-affinity, sodium-dependent P(i) uptake with comparable affinities for both sodium and phosphate ions . Furthermore, the activity of NptA is influenced by pH, again in a manner similar to that of the NaPi-2a subtype of the animal enzyme, although it lacks the corresponding REK motif thought to be responsible for this phenomenon . P(i) uptake activity, a component of which appeared to be sodium dependent, was increased in V . cholerae by phosphate starvation . However, it appears from the use of a reporter gene expressed from the nptA promoter that none of this activity is attributable to the induction of expression from nptA . It is thus proposed that the physiological function of NptA protein may be the rapid uptake of P(i) in preparation for rapid growth in nutrient-rich environments and that it may therefore play a role in establishing infection.

J Bacteriol, 2002 Aug, 184(16), 4351 - 8
Comparison of genome structures of vibrios, bacteria possessing two chromosomes; Tagomori K et al.; Vibrios are gram-negative gamma-proteobacteria which are ubiquitous in marine and estuarine environments . Recently, we demonstrated that some, if not all, Vibrio species have two circular chromosomes . The whole genome sequence of Vibrio cholerae N16961 has been reported . In this study, we constructed a physical and genetic map of the genome of Kanagawa phenomenon-positive Vibrio parahaemolyticus strain KX-V237 and compared it with those of V . parahaemolyticus AQ4673 and V . cholerae N16961 . The genome of KX-V237 comprised two circular chromosomes (3.3 and 1.9 Mb), similar to the structure of the AQ4673 genome . The relative positions of the genes on the genomes were well conserved in the two strains, but a large inversion on the large chromosomes, probably symmetric around the replication origin, was suggested . Although the sizes of the large chromosomes of KX-V237 and V . cholerae N16961 were similar, the sizes of the small chromosomes were very different . Unlike N16961, the superintegron of KX-V237 was located on the large chromosome . Comparison of the genetic maps of the chromosomes of KX-V237 and V . cholerae N16961 revealed that most of the open reading frames (ORFs) present on the large chromosome of the V . cholerae strain had homologues on the large chromosome of the V . parahaemolyticus strain and that most of the ORFs on the small chromosome of N16961 were present on the small chromosome of KX-V237 . The difference in the orders of the ORFs on the chromosomes of N16961 and KX-V237 implies that numerous and frequent genetic exchanges have occurred intrachromosomally rather than interchromosomally.

Zh Mikrobiol Epidemiol Immunobiol, 2002 May-Jun, (3), 58 - 60
{Invasive properties of toxigenic and nontoxigenic Vibrio cholerae of different serogroups}; Mironova AV et al.; Experimental data on the comparative study of the invasive properties of vct+ Hly- and vct- Hly+ V . cholerae of serogroups 01 and 0139 are presented . Both vct- Hly+ and vct+ Hly- V . cholerae of serogroups 01 and 0139 have been shown to be capable of dissemination into internal organs . No differences in the dissemination of V . cholerae of different serogroups in both immunologically immature and mature experimental animals have been detected.

J Infect Dis, 2002 Jul 15, 186(2), 246 - 51 Epub 2002 Jun 17.
Epidemic and endemic cholera trends over a 33-year period in Bangladesh; Longini IM Jr et al.; Despite nearly 200 years of study, the mechanisms contributing to the maintenance of endemic cholera and the causes of periodic epidemics remain poorly understood . To investigate these patterns, cholera data collected over 33 years (1966-1998) in Matlab, Bangladesh, were analyzed . Time-lagged autocorrelations were stratified by Vibrio cholerae serogroup, serotype, and biotype . Both classical and El Tor biotypes alternated and persisted between 1966 and 1988; the classical biotype disappeared by 1988, and the O139 serogroup first appeared in 1993 . Both the Ogawa and Inaba serotypes circulated the entire time . The autocorrelations revealed that both Inaba and Ogawa epidemics were followed 12 months later by epidemics of the same serotype . Ogawa epidemics, however, were also followed by further Ogawa epidemics only 6 months later . Thus, epidemics of Inaba may selectively confer short-term population-level immunity for a longer period than those of Ogawa . These observations suggest that the Inaba antigen should be maximized in cholera vaccine designs.

Chin Med J (Engl), 2002 Apr, 115(4), 589 - 92
The inherent characteristics and DNA polymorphism of Vibrio cholerae and other vibrios; Wang J et al.; OBJECTIVE: To investigate the inherent characteristics of Vibrio cholerae (V . cholerae) and other vibrios and their relationship . METHODS: Polymerase chain reaction (PCR), DNA sequence analysis, randomly amplified polymorphic DNA (RAPD) analysis and average linkage cluster analysis were used to study 3 isolates of V . cholerae strains O139, three isolates O1 biotype El Tor, four isolates O1 biotype classical and 3 other vibrios . RESULTS: V . cholerae O139 contained the genomic sequences of ctx A2-B as well as V . cholerae O1 . V . cholerae and others vibrios were divided into 4 groups by fingerprint patterns of RAPD, that is (1) V . cholerae O139 and V . cholerae O1 El Tor; (2) V . cholerae O1 classical; (3) V . paraheamolyticus and V . vulnificus and (4) V . flluvialis . V . cholerae O139 DNA fingerprint of RAPD was consistent with the El Tor biotype: average linkage cluster distance was 0, and slightly different from the classical biotype, with a distance of 2.07 . It was much more different from vibrio paraheamolyticus and others, with a distance of 6.76 - 8.54 . CONCLUSION: V . cholerae and other vibrios are polymorphic in inherent characteristics . The inherent characteristics of V . cholerae O139 are the same as El Tor biotype . O139 may have evolved from the El Tor biotype . The inherent characteristics of vibrio paraheamolyticus are the same as vibrio vulnificus.

Biochem Biophys Res Commun, 2002 Jul 26, 295(4), 922 - 8
Involvement of in vivo induced icmF gene of Vibrio cholerae in motility, adherence to epithelial cells, and conjugation frequency; Das S et al.; Previously, using global transcription profile approach icmF gene of Vibrio cholerae was identified as an in vivo induced gene . In the present study, the icmF gene of V . cholerae O395 was cloned, sequenced, and used to construct an icmF insertion mutant . This IcmF is homologous to Legionella pneumophila IcmF, belonging to the icm cassette responsible for macrophage killing and intracellular survival of the organism . The icmF insertion mutant exhibited reduced motility and increased adherence to human intestinal epithelial cells . The presence of ATP-GTP-binding site suggests further a possible role of IcmF as a signaling molecule . Triparental-mating assay, with the mutant as a recipient, showed higher conjugation frequency than wild type . We propose that the increased adherence to epithelial cell line and increased conjugation frequency of the mutant result from some sort of cell surface reorganization.

FEMS Microbiol Lett, 2002 Jul 16, 213(1), 7 - 12
Plasmid profiling and antibiotic resistance of Vibrio strains isolated from cultured penaeid shrimp; Molina-Aja A et al.; Resistance to different antibiotics was found in 26 of the 30 strains analyzed, more than 70% of the strains analyzed were resistant to carbenicillin and ampicillin and a significant correlation was found between the resistance to both antibiotics . Plasmids were found in 80% of the strains analyzed, and 11 different plasmid profiles were observed . The most common profile obtained had only a 21.2-kbp plasmid, a significant correlation was found between the presence of this plasmid and resistance to carbenicillin, although some exceptions could be detected . Plasmids were cured from a cephalothin resistant strain and reintroduced into the plasmid-free cell and into Escherichia coli DH5alpha, both strains gained resistance to this antibiotic.

Proc Natl Acad Sci U S A, 2002 Aug 6, 99(16), 10322 - 4 Epub 2002 Jul 16.
Riboflavin is a component of the Na+-pumping NADH-quinone oxidoreductase from Vibrio cholerae; Barquera B et al.; Flavins are cofactors in many electron-transfer enzymes . Typically, two types of flavins perform this role: 5'-phosphoriboflavin (FMN) and flavin-adenine dinucleotide (FAD) . Both of these are riboflavin derivatives, but riboflavin itself has never been reported to be an enzyme-bound component . We now report that tightly bound riboflavin is a component of the NADH-driven sodium pump from Vibrio cholerae.

Antimicrob Agents Chemother, 2002 Aug, 46(8), 2676 - 8
Role of active efflux in association with target gene mutations in fluoroquinolone resistance in clinical isolates of Vibrio cholerae; Baranwal S et al.; Quinolones are among the drugs of choice in the management of cholera caused by Vibrio cholerae . In this study, we demonstrate that, in addition to mutations detected in the target genes gyrA and parC, proton motive force-dependent efflux is involved in quinolone resistance in clinical isolates of V . cholerae.

Antimicrob Agents Chemother, 2002 Aug, 46(8), 2668 - 70
Chemiosmotic mechanism of antimicrobial activity of Ag(+) in Vibrio cholerae; Dibrov P et al.; Although the antimicrobial effects of silver salts were noticed long ago, the molecular mechanism of the bactericidal action of Ag(+) in low concentrations has not been elucidated . Here, we show that low concentrations of Ag(+) induce a massive proton leakage through the Vibrio cholerae membrane, which results in complete deenergization and, with a high degree of probability, cell death.

Mem Inst Oswaldo Cruz, 2002 Jun, 97(4), 511 - 6
The Zymovars of Vibrio cholerae: multilocus enzyme electrophoresis of Vibrio cholerae; Freitas FS et al.; Zymovars analysis also known as multilocus enzyme electrophoresis is applied here to investigate the genetic variation of Vibrio cholerae strains and characterise strains or group of strains of medical and epidemiological interest . Fourteen loci were analyzed in 171 strains of non-O1 non-O139, 32 classical and 61 El Tor from America, Africa, Europe and Asia . The mean genetic diversity was 0.339 . It is shown that the same O antigen (both O1 and non-O1) may be present in several genetically diverse (different zymovars) strains . Conversely the same zymovar may contain more than one serogroup . It is confirmed that the South American epidemic strain differs from the 7th pandemic El Tor strain in locus LAP (leucyl leucyl aminopeptidase) . Here it is shown that this rare allele is present in 1 V . mimicus and 4 non-O1 V . cholerae . Non toxigenic O1 strains from South India epidemic share zymovar 14A with the epidemic El Tor from the 7th pandemic, while another group have diverse zymovars . The sucrose negative epidemic strains isolated in French Guiana and Brazil have the same zymovar of the current American epidemic V . cholerae.

Infect Immun, 2002 Aug, 70(8), 4735 - 42
Vibrio pathogenicity island and cholera toxin genetic element-associated virulence genes and their expression in non-O1 non-O139 strains of Vibrio cholerae; Sarkar A et al.; A non-O1 non-O139 Vibrio cholerae strain, 10259, belonging to the serogroup O53 was shown to harbor genes related to the vibrio pathogenicity island (VPI) and a cholera toxin (CT) genetic element called CTX . While the nucleotide sequence of the strain 10259 tcpA gene differed significantly (26 and 28%) from those of O1 classical and El Tor biotype strains, respectively, partial sequence analysis data of certain other VPI-associated genes (aldA, tagA, tcpP/H, toxT, acfB/C, and int) and intergenic regions (tcpF to toxT and tcpH to tcpA) of the strain showed only minor variations (0.4 to 4.8%) from corresponding sequences in O1 strains . Strain 10259 also contained CTX element-associated toxin genes with sequences almost identical to those of O1 strains . Growth of the organism in Luria broth (LB) under ToxR inducing conditions (30 degrees C and pH 6.5) led to transcriptional activation of tcpP/H, toxR, toxT, and tcpA genes, but not of ctxA, as determined by reverse transcription-PCR (RT-PCR) . Subsequent analysis revealed that strain 10259 possessed only two copies (instead of three or more copies found in epidemic-causing O1 or O139 strains) of the heptanucleotide (TTTTGAT) repeats in the intergenic region upstream of ctxAB . Therefore, a strain 10259 mutant was generated by replacement of this region with a homologous region (1.4 kb) derived from a V . cholerae O1 classical biotype strain (O395) that contained seven such repeats . The resultant recombinant strain (10259R) was found to be capable of coordinately regulated expression of toxT, ctxA, and tcpA when grown under the ToxR inducing conditions . Serological studies also demonstrated that the recombinant strain produced TcpA and a significantly ( approximately 1,000-fold) higher level of CT in vitro compared to that of the parent strain . Virulence gene expression in two other non-O1 non-O139 strains (serogroup O37) containing VPI and the CTX element was studied by RT-PCR and serological assay . One strain (S7, which was involved in an epidemic in Sudan in 1968) showed coordinately regulated expression of virulence genes leading to the production of both CT and TcpA in LB medium . However, the other strain, V2, produced RT-PCR-detectable transcripts of toxT, ctxA, or tcpA genes in the early phase (6 h), but not in the late phase (16 h) of growth in LB medium . These results are consistent with the low levels of production of CT and TcpA by the strain that were serologically detectable . The significance of these results is discussed in relation to the role of virulence genes and their expression to the pathogenic potential of V . cholerae strains belonging to non-O1 serogroups.

N Z Med J, 2002 May 24, 115(1154), 234 - 6
Acute gastroenteritis associated with seafood privately imported from the Pacific Islands; Thornton V et al.; AIMS: To investigate a potential link between consumption of food privately imported from the Pacific Islands and presentation with acute gastroenteritis to Middlemore Hospital Emergency Department . METHODS: This was a three month prospective observational case study that included patients aged greater than fifteen years presenting with acute gastroenteritis and a history of food privately imported from the Pacific Islands . Data included case demographics, symptoms, island of food origin and food type . Stool and blood samples were collected and analysed . RESULTS: Of 358 patients who presented to Middlemore Emergency Department during the study period with gastroenteritis, 34 (9.4%) had a history of consumption of food privately imported from the Pacific Islands . The seafood came from Tonga (23 cases), Samoa (10 cases) or Niue (1 case) . The implicated seafood was shellfish (28 cases), jellyfish (2 cases), fish intestine (2 cases), seaweed or seaslug (1 case each) . Fourteen patients (41%) provided stool samples; all were culture positive for Vibrio parahaemolyticus (VPH) . CONCLUSIONS: This case series confirms a link between acute VPH gastroenteritis and consumption of seafood privately imported from the Pacific Islands . A number of public health initiatives to reduce the burden of VPH gastroenteritis among Auckland's Pacific Islanders have commenced . The Ministries of Health, Agriculture and Forestry are considering tighter controls or banning food privately imported from the Pacific Islands.

Dis Aquat Organ, 2002 Jun 3, 49(3), 221 - 6
Comparative susceptibility of veliger larvae of four bivalve mollusks to a Vibrio alginolyticus strain; Luna-Gonzalez A et al.; The susceptibility of 7 d old veliger larvae of the scallops Argopecten ventricosus and Nodipecten subnodosus, the penshell Atrina maura, and the Pacific oyster Crassostrea gigas to a pathogenic strain of Vibrio alginolyticus was investigated by challenging the larvae with different bacterial concentrations in a semi-static assay . The results indicate that the larvae of the 2 scallop species are more susceptible to the V . alginolyticus strain than those of the oyster and the penshell . Signs of the disease were similar to bacillary necrosis described in previous work . Interspecies differences in susceptibility to pathogens are discussed.

Virchows Arch, 2002 Jul, 441(1), 88 - 92 Epub 2002 Feb 23.
Vibrio vulnificus infection in patients with liver disease: report of five autopsy cases; Chen Y et al.; Five autopsy cases of Vibrio vulnificus infection with liver disease are reported . All five patients ate raw seafood 24 h before the onset of illness . The clinical presentation was of primary septicemia, with positive cultures in both the blood and cutaneous lesions . Stool cultures were positive for the organism in one patient with gastrointestinal symptoms . Autopsy examination revealed liver cirrhosis in three cases and alcoholic liver disease in two; all showed portal hypertension . Gastrointestinal mucosal changes were seen in four patients: edema, hemorrhagic necrosis, and lymphocyte infiltration . One case was of an human immunodeficiency virus infected patient in which histology showed a rare intestinal disease, phlegmonous colitis . We believe this is the first description of a case of concomitant phlegmonous enterocolitis and V . vulnificus infection . Patients with liver disease should be warned about the possibility of life-threatening infections and complications associated with the consumption of raw seafood.

Can J Microbiol, 2002 May, 48(5), 379 - 86
Use of PCR-RFLP for genotyping 16S rRNA and characterizing bacteria cultured from halibut fry; Jensen S et al.; Small subunit ribosomal genes were explored using PCR-RFLP to facilitate the characterization of bacteria cultured from reared fry of the Atlantic halibut (Hippoglossus hippoglossus) . Concern has been expressed about pathogen invasion in larvae lacking a counteracting normal flora that may aid the immune system in producing robust noninfected individuals . In this study, pure cultured representatives of normal flora that were previously found to be antagonistic towards a pathogenic Vibrio sp . were subjected to a whole cell PCR protocol amplifying approximately 1500 bp of 16S rDNA . Amplified DNA was digested by AluI, BstUI, CfoI, and RsaI, to generate restriction profiles . Before the isolates were characterized, a survey was performed to test the discriminative efficiency of the RFLP . Efficient detection of polymorphism and the resolution of species and subspecies were achieved . Using the RFLP on 103 isolates generated as many as 22 genotypes . Based on the restriction profiles, a taxonomic tree incorporating 19 reference strains was constructed . Partial sequencing found this tree to be dominated by gamma-Proteobacteria in clusters of Vibrio-, Pseudomonas-, and Alteromonas-affiliated species . Only nine isolates fell outside these genera, including the three isolates Shewanella alga, Deleya marina, and Marinomonas protea . These species have not previously been reported as halibut flora . The most frequently isolated genotype resembled Vibrio salmonicida.

J Bacteriol, 2002 Aug, 184(15), 4259 - 69
Genomic and functional analyses of SXT, an integrating antibiotic resistance gene transfer element derived from Vibrio cholerae; Beaber JW et al.; SXT is representative of a family of conjugative-transposon-like mobile genetic elements that encode multiple antibiotic resistance genes . In recent years, SXT-related conjugative, self-transmissible integrating elements have become widespread in Asian Vibrio cholerae . We have determined the 100-kb DNA sequence of SXT . This element appears to be a chimera composed of transposon-associated antibiotic resistance genes linked to a variety of plasmid- and phage-related genes, as well as to many genes from unknown sources . We constructed a nearly comprehensive set of deletions through the use of the one-step chromosomal gene inactivation technique to identify SXT genes involved in conjugative transfer and chromosomal excision . SXT, unlike other conjugative transposons, utilizes a conjugation system related to that encoded by the F plasmid . More than half of the SXT genome, including the composite transposon-like structure that contains its antibiotic resistance genes, was not required for its mobility . Two SXT loci, designated setC and setD, whose predicted amino acid sequences were similar to those of the flagellar regulators FlhC and FlhD, were found to encode regulators that activate the transcription of genes required for SXT excision and transfer . Another locus, designated setR, whose gene product bears similarity to lambdoid phage CI repressors, also appears to regulate SXT gene expression.

J Bacteriol, 2002 Aug, 184(15), 4104 - 13
The Vibrio cholerae vieSAB locus encodes a pathway contributing to cholera toxin production; Tischler AD et al.; The genes encoding cholera toxin (CT), ctxAB, are coregulated with those for other Vibrio cholerae virulence factors by a cascade of transcriptional activators, including ToxR, TcpP, and ToxT . Additional regulators that modulate expression of ctxAB during infection were recently identified in a genetic selection . A transposon insertion in vieS, the sensor kinase of the VieSAB three-component signal transduction system, resulted in failure to induce expression of a ctxA-recombinase fusion during murine infection . To determine which components of the VieSAB system are essential for CT regulation, ctxAB transcript levels were assessed by RNase protection assay in various vieSAB in-frame deletion mutants after growth in vitro under virulence gene inducing conditions . A threefold reduction in ctxAB transcript levels was observed for the (Delta)vieSAB strain; consistent with this, the (Delta)vieSAB strain produced twofold less CT protein than the wild type, and this defect was complementable in trans . These results suggest that the VieSAB three-component system is required for full activation of the ctxAB operon during in vitro growth as well as during infection . The VieSAB system may regulate ctxAB expression indirectly by affecting production of ToxT, because decreased toxT transcript levels were observed in the (Delta)vieSAB strain.

Cell Microbiol, 2002 Jul, 4(7), 397 - 409
The Vibrio cholerae haemolysin anion channel is required for cell vacuolation and death; Moschioni M et al.; Several strains of Vibrio cholerae secrete a haemolytic toxin of 63 kDa, termed V . cholerae cytolysin (VCC) . This toxin causes extensive vacuolation and death of cells in culture and forms an anion-selective channel in planar lipid bilayers and in cells . Here, we identify inhibitors of the VCC anion channel and show that the formation of the anion channel is necessary for the development of the vacuoles and for the cell death induced by this toxin . Using markers of cell organelles, we show that vacuoles derive from different intracellular compartments and we identify the contribution of late endosomes and of the trans-Golgi network in vacuole biogenesis.

Microbiology, 2002 Jul, 148(Pt 7), 2181 - 9
Cloning and characterization of a novel haemolysin in Vibrio cholerae O1 that does not directly contribute to the virulence of the organism; Fallarino A et al.; A previously undescribed haemolysin, distinct from the major Vibrio cholerae O1 El Tor haemolysin, HlyA, was cloned from the O1 classical biotype strain Z17561 . This novel haemolysin showed 71.5% overall similarity to the delta-thermostable direct haemolysin of Vibrio parahaemolyticus, and so it has been termed V . cholerae delta-thermostable haemolysin (Vc-deltaTH, encoded by the dth gene) . An ORF found immediately downstream, which appears to be transcriptionally and translationally linked to dth, displayed strong homology to the family of acyl-CoA synthetases . When expressed from an inducible promoter in Escherichia coli, Vc-deltaTH was shown to be a 22.8 kDa protein active on sheep red blood cells . Co-expression of acs with dth had no effect on the haemolytic activity or cytoplasmic localization of Vc-deltaTH . A V . cholerae Z17561 dth::Km(R) mutant showed unaltered behaviour in the infant mouse cholera model.

Biochim Biophys Acta, 2002 Aug 19, 1564(1), 99 - 106
Asp(344) and Thr(345) are critical for cation exchange mediated by NhaD, Na(+)/H(+) antiporter of Vibrio cholerae; Ostroumov E et al.; The Vc-NhaD is an Na(+)/H(+) antiporter from Vibrio cholerae belonging to a new family of bacterial Na(+)/H(+) antiporters, the NhaD family . In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity . All mutations fall into two distinct groups: (i) four variants, Glu(100)Ala, Glu(251)Ala, Glu(342)Ala, and Asp(393)Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp(344)Ala, Asp(344)Asn, and Thr(345)Ala caused a complete loss of both Na(+)/H(+) and Li(+)/H(+) antiport activity whereas the Asp(344)Glu variant exhibited reduced Na(+)/H(+) and Li(+)/H(+) antiport activity . This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na(+)/H(+) antiport . We discuss the possible role of Asp(344) and Thr(345) in the functioning of Vc-NhaD.

Mol Microbiol, 2002 Jul, 45(1), 131 - 43
LitR, a new transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ colonization; Fidopiastis PM et al.; Vibrio fischeri is the bacterial symbiont within the light-emitting organ of the sepiolid squid Euprymna scolopes . Upon colonizing juvenile squids, bacterial symbionts grow on host-supplied nutrients, while providing a bioluminescence that the host uses during its nocturnal activities . Mutant bacterial strains that are unable to emit light have been shown to be defective in normal colonization . A 606 bp open reading frame was cloned from V . fischeri that encoded a protein, which we named LitR, that had about 60% identity to four related regulator proteins: Vibrio cholerae HapR, Vibrio harveyi LuxR, Vibrio parahaemolyticus OpaR and Vibrio vulnificus SmcR . When grown in culture, cells of V . fischeri strain PMF8, in which litR was insertionally inactivated, were delayed in the onset of luminescence induction and emitted only about 20% as much light per cell as its parent . Protein-binding studies suggested that LitR enhances quorum sensing by regulating the transcription of the luxR gene . Interestingly, when competed against its parent in mixed inocula, PMF8 became the predominant symbiont present in 83% of light organs . Thus, the litR mutation appears to represent a novel class of mutations in which the loss of a regulatory gene function enhances the bacterium's competence in initiating a benign infection.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(2), 149 - 152
Cloning of the CtxB Gene of Vibrio cholerae and Its Expression in E.coli; He ZY et al.; The CtxB gene encoding cholerae toxin subunit B was amplified from Vibrio cholerae genomic DNA by PCR . The result of sequencing indicated that CtxB gene encodes 124 amino acid residues . The sequence of CtxB gene was almost the same as that of reported except for the codon of Thr 62 . The expression plasmid pGEX-CTXB was constructed by inserting CtxB gene into plasmid pGEX-4T-2, containing gst gene, immediately downstream of the T7 promoter . The expressed plasmid was introduced into E.coli BL21(DE3) cells and expression strain CTXB/BL21 was selected . SDS-PAGE analysis revealed that the GST-CTXB fusion protein was highly expression and accumulated up to 36% of bacterial soluble proteins after the induction by IPTG . A fusion protein of 40 kD was expressed as inclusion body . The fusion protein was refolded and purified . The purified fusion protein was cut by thrombin to obtain the purified CTXB protein.

Cytokine, 2002 Apr 21, 18(2), 98 - 107
Interferon-gamma increases the expression of glycosylated CD95 in B-leukemic cells: an inducible model to study the role of glycosylation in CD95-signalling and trafficking; Dorrie J et al.; B-lineage acute leukaemia cells are generally resistant to CD95-mediated apoptosis . In this report, the CD95-resistant B-leukaemia lines SEM, RS4;11, and REH were used to investigate the mechanisms of resistance to CD95-signalling . We found that interferon-gamma (IFN-gamma) treatment increased the presence of high molecular weight forms of CD95 in these cells as judged by Western analysis, and treatment of protein extracts with Peptide: N -glycosidase F indicated that the majority of high molecular weight forms were due to N-linked glycosylation . Treatment of whole cells with neuraminidase from Vibrio cholerae substantially reduced the relative molecular mass of CD95 observed after IFN-gamma treatment and partially sensitized the three leukaemia lines to CD95-mediated death . To further characterize the different steps of oligosaccharide processing that may regulate CD95 signalling, the leukaemic cells were treated with IFN-gamma and the glycosidase inhibitors castanospermine, 1-deoxymannojirimycin (DMM), and swainsonine . Treatment with DMM, a mannosidase inhibitor, efficiently reduced the appearance of high molecular weight forms of CD95 after IFN-gamma treatment, and sensitized SEM and REH cells to CD95-mediated death . However, the IFN-gamma-induced increases of CD95 on the cell surface were not altered by treatment with any of the glycosidase inhibitors, suggesting that the generation of complex oligosaccharide structures is not required for trafficking of CD95, but may instead be used as a mechanism of partially blocking CD95 signalling in these cells . In conclusion, IFN-gamma treatment of the B-lineage leukaemia lines provides a novel, inducible system for the further characterization of post-translational modifications involved in modulating sensitivity to CD95-signalling .

Folia Microbiol (Praha), 2002, 47(3), 255 - 8
Mycoremediation of PAH-contaminated soil; Bhatt M et al.; Out of a number of white-rot fungal cultures, strains of Irpex lacteus and Pleurotus ostreatus were selected for degradation of 7 three- and four-ring unsubstituted aromatic hydrocarbons (PAH) in two contaminated industrial soils . Respective data for removal of PAH in the two industrial soils by I . lacteus were: fluorene (41 and 67%), phenanthrene (20 and 56%), anthracene (29 and 49%), fluoranthene (29 and 57%), pyrene (24 and 42%), chrysene (16 and 32%) and benzo{a}anthracene (13 and 20%) . In the same two industrial soils P . ostreatus degraded the PAH with respective removal figures of fluorene (26 and 35%), phenanthrene (0 and 20%), anthracene (19 and 53%), fluoranthene (29 and 31%), pyrene (22 and 42%), chrysene (0 and 42%) and benzo{a}anthracene (0 and 13%) . The degradation of PAH was determined against concentration of PAH in non-treated contaminated soils after 14 weeks of incubation . The fungal degradation of PAH in soil was studied simultaneously with ecotoxicity evaluation of fungal treated and non-treated contaminated soils . Compared to non-treated contaminated soil, fungus-treated soil samples indicated decrease in inhibition of bioluminescence in luminescent bacteria (Vibrio fischerii) and increase in germinated mustard (Brassica alba) seeds.

J Food Prot, 2002 Jun, 65(6), 975 - 80
Resistance of cold- and starvation-stressed Vibrio vulnificus to heat and freeze-thaw exposure; Bang W et al.; The effects of cold storage and starvation on the subsequent heat resistance and freeze-thaw resistance of Vibrio vulnificus were studied . Three strains of V . vulnificus were evaluated . Cold stress had no effect on freeze-thaw resistance (P > 0.05) . Starvation enhanced freeze-thaw resistance for one strain compared to controls (P < 0.05) . V . vulnificus was not heat resistant; control populations were inactivated within 12 min at 47 degrees C . Starvation increased heat tolerance for one strain, but differences were small from a processing perspective (P < 0.05) . Cold stress had no effect on heat resistance (P > 0.05) . Cold adaptation (holding 4 h at 15 degrees C) enhanced cold temperature (5 degrees C) tolerance . This information will be helpful in the development of methods to minimize V . vulnificus risk.

J Food Prot, 2002 Jun, 65(6), 970 - 4
Growth and survival of Vibrio parahaemolyticus in postharvest American oysters; Gooch JA et al.; Oysters at the retail stage of distribution generally contain greater densities of Vibrio parahaemolyticus than do oysters at harvest . The objective of this study was to determine the effects of postharvest storage at 26 and 3 degrees C on the growth and survival of naturally occurring V . parahaemolyticus in shellstock American oysters (Crassostrea virginica) . Oysters were collected monthly from May 1998 through April 1999 from Mobile Bay, Alabama, and their V . parahaemolyticus densities were determined after 0, 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 analyzed 14 to 17 days later . V . parahaemolyticus numbers were determined by a direct plating method involving an alkaline-phosphatase-labeled DNA probe that targets the species-specific thermolabile hemolysin gene (tlh-AP) to identify suspect isolates . From April to December, when water temperatures at harvest were >20 degrees C, the geometric mean harvest density of V . parahaemolyticus was 130 CFU/g . When water temperatures were <20 degrees C, the geometric mean harvest density was 15 CFU/g . After harvest, V . parahaemolyticus multiplied rapidly in live oysters held at 26 degrees C, showing a 50-fold increase (1.7 log CFU/g) at 10 h and a 790-fold increase (2.9 log CFU/g) at 24 h (April through December) . Average V . parahaemolyticus numbers showed a sixfold decrease (0.8 log CFU/g) after approximately 14 days of refrigeration . These results indicate that V . parahaemolyticus can grow rapidly in unrefrigerated oysters.

J Food Prot, 2002 Jun, 65(6), 1049 - 53
A rapid and improved method for the detection of Vibrio parahaemolyticus and Vibrio vulnificus strains grown on hydrophobic grid membrane filters; Banerjee SK et al.; DNA probe-based detection methods were developed and characterized as an alternative to time-consuming and less specific conventional protocols . Digoxigenin-labeled probes were prepared by polymerase chain reaction amplification of the targeted sequences in the specific amplicons generated from genomic DNA . Specific probes with high yields were generated for the detection of the tlh gene of Vibrio parahaemolyticus and the cth gene of V . vulnificus . Colony (Southern) hybridization analyses were carried out using hydrophobic grid membrane filters (HGMFs) to allow biotype-specific differentiation of the two species . Eight strains of V . vulnificus and five strains of V . parahaemolyticus, including one standard (ATCC) strain of each biotype, were examined . Colony lysis, hybridization, and nonradioactive detection parameters were optimized for identification of the target biotypes arranged on the same HGMF and also on a conventional nylon membrane, thereby confirming the specificity of the probes and the comparative usefulness of the HGMFs . The experimental procedure presented here can be completed in 1 day . The protocol was designed specifically to identify the target Vibrio spp . and could potentially be used for the enumeration and differentiation of V . parahaemolyticus and V . vulnificus in foods.

Shokuhin Eiseigaku Zasshi, 2002 Apr, 43(2), 90 - 4
{Survival of Vibrio parahaemolyticus serovar O3:K6 strains under acidic conditions}; Hasegawa J et al.; The survival of Vibrio parahaemolyticus serovar O3:K6 strains and other serovars in the presence of acetic, citric and hydrochloric acids were studied . There were no differences in resistance to these acids between serovar O3:K6 and the other serovars . At pH 5.6, citric acid was more effective in reducing the number of viable cells of V . parahaemolyticus than acetic acid . However, at pH 4.5, acetic acid was more effective than citric acid . The number of viable cells decreased quickly in the presence of rice vinegar or wine vinegar at pH 4.0.

J Clin Microbiol, 2002 Jul, 40(7), 2635 - 7
Escalating association of Vibrio cholerae O139 with cholera outbreaks in India; Sinha S et al.; Between December 1999 and December 2000, teams from the National Institute of Cholera and Enteric Diseases, Calcutta, India, examined eight outbreaks of cholera, which occurred in different parts of the country distant from each other . In two of these outbreaks each, only V . cholerae O1 biotype ElTor or V . cholerae O139 could be isolated, while in the remaining four outbreaks, both O1 and O139 were isolated . The interesting feature is the escalating association of V . cholerae O139 with outbreaks of cholera; two of the most recent outbreaks, one in Calcutta and one in Orissa, were caused exclusively by O139 . The O139 strains from the six different outbreaks were genotypically closely related . These trends indicate a shift in the outbreak propensity of V . cholerae O139.

J Biol Chem, 2002 Sep 6, 277(36), 33369 - 77 Epub 2002 Jun 27.
Detoxification of cholera toxin without removal of its immunoadjuvanticity by the addition of (STa-related) peptides to the catalytic subunit . A potential new strategy to generate immunostimulants for vaccination; Sanchez J et al.; Peptides related to the heat-stable enterotoxin STa were fused to the N terminus of the A-subunit of cholera toxin (CTA) to explore whether peptide additions could help generate detoxified cholera toxin (CT) derivatives . Proteins carrying APRPGP (6-CTA), ASRCAELCCNPACPAP (16-CTA), or ANSSNYCCELCCNPACTGCYPGP (23-CTA) were genetically constructed . Using a two-plasmid system these derivatives were co-expressed in Vibrio cholerae with cholera toxin B-subunit (CTB) to allow formation and secretion of holotoxin-like molecules (engineered CT, eCTs) . Purified eCTs maintained all normal CT properties yet they were more than 10-fold (eCT-6), 100-fold (eCT-16), or 1000-fold (eCT-23) less enterotoxic than wild-type CT . The inverse correlation between enterotoxicity and peptide length indicated sterical interference with the ADP-ribosylating active site in CTA . This interpretation agreed with greater than 1000-fold reductions in cAMP induction, with reductions, albeit not proportional, in in vitro agmatine ADP-ribosylation, and was supported by molecular simulations . Intranasal immunization of mice demonstrated that eCTs retained their inherent immunogenicity and ability to potentiate immune responses to a co-administered heterologous protein antigen, although in variable degrees . Therefore, the addition of STa-related peptides to CTA reduced the toxicity of CT while partly preserving its natural immunoadjuvanticity . These results suggest peptide extensions to CTA are a useful alternative to site-directed mutagenesis to detoxify CT . The simplicity of the procedure, combined with efficient expression and assembly of derivatives, suggests this approach could allow for large scale production of detoxified, yet immunologically active CT molecules.

Acta Trop, 2002 Aug, 83(2), 169 - 76
Molecular characterization of Vibrio cholerae O1 outbreak strains in Miri, Sarawak (Malaysia); Radu S et al.; Bacterial resistance to various antimicrobial agents is common in area with high usage of antibiotics . In this study, the data on antimicrobial susceptibility patterns of Vibrio cholerae O1 from patients during an outbreak period was found to be high but variable rates of multidrug resistance . Thirty-two of 33 V . cholerae isolates harboured the tcp, ctx, zot and ace genes, suggesting their possible roles in the outbreak cases . We analyzed the molecular diversity of a total of 33 strains of V . cholerae O1 isolated from 33 patients between November 1997 and April 1998 using random amplified polymorphic DNA (RAPD) analysis . The 30 typable isolates could be separated into four major clusters containing 5, 17, 2 and 6 isolates, respectively . However, no particular RAPD pattern was predictive of a particular pattern of antibiotic susceptibility . The findings of this study showed that multiple clones seemed to be responsible for cases in the outbreaks in the study area.

Diagn Microbiol Infect Dis, 2002 Jun, 43(2), 91 - 7
Spectrum of vibrio species associated with acute diarrhea in North Jakarta, Indonesia; Lesmana M et al.; Vibrio spp was isolated from 1024 (21.2%) of 4820 diarrhea patients admitted to a community hospital in North Jakarta from 1996 through 1998 . Vibrio cholerae O1 (49.5%) and V . parahaemolyticus (30.1%) comprised the major species isolated, followed by V . cholerae non-O1 (16.9%), and V . fluvialis (9.4%) . In 938 (19.4%) patients, Vibrio was found as single isolate . Multiple infections were detected in 86 (1.8%) patients . A small number of V . furnisii, V . metschnikovii, V . mimicus and V . hollisae were also isolated . No V . cholerae O139 was detected . The majority of patients with Vibrio spp . infections were adults between the ages of 20 to 45 years . No Vibrio spp . was isolated from infants <1 year old in this study . In vitro antibiotic susceptibility testing revealed no antibiotic resistance associated with the 507 V . cholerae O1 isolates tested, except for colistin (100%) . These data implicate Vibrio spp . as a major cause of diarrhea in this region.

Syst Appl Microbiol, 2002 Apr, 25(1), 120 - 9
Characterization of Carnobacterium divergens strain 6251 isolated from intestine of Arctic charr (Salvelinus alpinus L.); Ringo E et al.; An atypical strain of Carnobacterium divergens, strain 6251, was isolated from the small intestine of Arctic charr (Salvelinus alpinus L.), fed high dietary carbohydrate . This strain showed marked growth inhibitory effects in vitro against the fish pathogens Aeromonas salmonicida subsp . salmonicida (furunculosis), Vibrio anguillarum (vibriosis) and Vibrio viscocus (winter ulcer) . The strain is a non-motile Gram-positive psychrotrophic rod that lacks both catalase and oxidase, grows at pH 9.1 (CTAS agar), but not on acetate containing media (pH < or = 5.4), on TCBS or at < or =6% sodium chloride content . Strain 6251 is facultatively anaerobic and utilises tryptone as a sole source of nutrient . Further characterisation showed the most abundant cellular fatty acid of strain 6251 to be oleic acid (18:1) (n-9) (36.0%) . Sequencing of a 16S rDNA region of 578 nucleotides and AFLP microbial fingerprinting suggested that strain 6251 is not closely related to any carnobacteria known, however, DNA-DNA similarity determinations showed high similarity (96.2%) with the type strain of Carnobacterium divergens . The unique phenotypic attributes of this strain represent new information on the biodiversity and ecology of carnobacteria and especially of the species C . divergens.

Biochemistry, 2002 Jul 2, 41(26), 8429 - 37
Carrier protein recognition in siderophore-producing nonribosomal peptide synthetases; Marshall CG et al.; Nonribosomal peptide synthetases (NRPSs) use phosphopantetheine (pPant) bearing carrier proteins to chaperone activated aminoacyl and peptidyl intermediates to the various enzymes that effect peptide synthesis . Using components from siderophore NRPSs that synthesize vibriobactin, enterobactin, yersiniabactin, pyochelin, and anguibactin, we examined the nature of the interaction of such cofactor-carrier proteins with acyl-activating adenylation (A) domains . While VibE, EntE, and PchD were all able to utilize "carrier protein-free" pPant derivatives, the pattern of usage indicated diversity in the binding mechanism, and even the best substrates were down at least 3 log units relative to the native cofactor-carrier protein . When tested with four noncognate carrier proteins, EntE and VibE differed both in the range of substrate utilization efficiency and in the distribution of the efficiencies across this range . Correlating sequence alignments to kinetic efficiency allowed for the construction of eight point mutants of VibE's worst substrate, HMWP2 ArCP, to the corresponding residue in its native VibB . Mutants S49D and H66E combined to increase activity 6.2-fold and had similar enhancing effects on the downstream condensation domain VibH, indicating that the two NRPS enzymes share carrier protein recognition determinants . Similar mutations of HMWP2 ArCP toward EntB had little effect on EntE, suggesting that the position of recognition determinants varies across NRPS systems.

Microb Pathog, 2002 Apr, 32(4), 183 - 90
Silkworm larvae as an animal model of bacterial infection pathogenic to humans; Kaito C et al.; Silkworm larvae, Bombyx mori, were examined as an animal model of human infection with pathogenic bacteria . When 3 x 10(7) cells of Staphylococcus aureus (S . aureus), Pseudomonas aeruginosa, or Vibrio cholerae were injected into the blood of fifth instar silkworm larvae, over 90% of the larvae died within 2 days, whereas over 90% survived for 5 days after injection of the same amount of Escherichia coli . Growth of S . aureus was observed in larvae blood and tissues . Immunostaining analysis revealed that S . aureus proliferated at the surface of the midgut . Infection of silkworm larvae by methicillin-sensitive S . aureus was cured by ampicillin, oxacillin, and vancomycin, whereas infection by methicillin-resistant S . aureus was not cured by ampicillin or oxacillin, although vancomycin was effective . Disinfectants were not effective because of toxicity against the larvae . Thus, silkworm larvae are useful for evaluating antibiotics for pathogenic bacterial infection in humans .

J Mol Biol, 2002 Jul 5, 320(2), 403 - 13
The systematic substitutions around the conserved charged residues of the cytoplasmic loop of Na+-driven flagellar motor component PomA; Yorimitsu T et al.; PomA, a homolog of MotA in the H+-driven flagellar motor, is an essential component for torque generation in the Na+-driven flagellar motor . Previous studies suggested that two charged residues, R90 and E98, which are in the single cytoplasmic loop of MotA, are directly involved in this process . These residues are conserved in PomA of Vibrio alginolyticus as R88 and E96, respectively . To explore the role of these charged residues in the Na+-driven motor, we replaced them with other amino acids . However, unlike in the H+-driven motor, both of the single and the double PomA mutants were functional . Several other positively and negatively charged residues near R88 and E96, namely K89, E97 and E99, were neutralized . Motility was retained in a strain producing the R88A/K89A/E96Q/E97Q/E99Q (AAQQQ) PomA protein . The swimming speed of the AAQQQ strain was as fast as that of the wild-type PomA strain, but the direction of motor rotation was abnormally counterclockwise-biased . We could, however, isolate non-motile or poorly motile mutants when certain charged residues in PomA were reversed or neutralized . The charged residues at positions 88-99 of PomA may not be essential for torque generation in the Na+-driven motor and might play a role in motor function different from that of the equivalent residues of the H+-driven motor.

Antibiot Khimioter, 2002, 47(1), 7 - 11
{Immunotropic properties of exoglycan obtained from the marine micro-organism Vibrio alginolyticus}; Smolina TP et al.; Immune system modulating activity of exoglycane isolated from culture media of Vibrio alginolyticus (strain 945-80) was investigated . The substance demonstrated stimulating activity on the humoral and cell immune system, potentiated phagocyte activity of macrophage and neutrophils, increased survival index of the animals infected by Gram-positive and Gram-negative bacteria.

J Immunol, 2002 Jul 1, 169(1), 566 - 74
Differential induction of mucosal and systemic antibody responses in women after nasal, rectal, or vaginal immunization: influence of the menstrual cycle; Kozlowski PA et al.; A cholera vaccine containing killed vibrios and cholera toxin B subunit (CTB) was used to compare mucosal immunization routes for induction of systemic and mucosal Ab . Four groups of women were given three monthly immunizations by the rectal immunization (R(imm)) route, nasal immunization (N(imm)) route, or vaginal immunization route during either the follicular (V-FP(imm)) or luteal (V-LP(imm)) menstrual cycle phase . N(imm) was performed with 10-fold less vaccine to determine if administration of less Ag by this route can, as in rodents, produce mucosal Ab responses comparable to those induced by higher dose R(imm) or vaginal immunization . Concentrations of Ab induced in sera and secretions were measured by ELISA . None of these routes produced durable salivary Ab responses . N(imm) induced greatest levels of CTB-specific IgG in sera . R(imm) failed to generate CTB-specific IgA in genital tract secretions . N(imm), V-FP(imm), and V-LP(imm) all produced cervical CTB-specific IgA responses comparable in magnitude and frequency . However, only V-FP(imm) induced cervical IgA2-restricted Ab to the bacterial LPS vaccine component . V-FP(imm), but not V-LP(imm), also induced CTB-specific IgA in rectal secretions . N(imm) was superior to V-FP(imm) for producing rectal CTB-specific IgA, but the greatest amounts of CTB-specific IgA and LPS-specific IgA, IgG, and IgM Ab were found in rectal secretions of R(imm) women . These data suggest that in women, N(imm) alone could induce specific Ab in serum, the genital tract, and rectum . However, induction of genital tract and rectal Ab responses of the magnitude generated by local V-FP(imm) or R(imm) will likely require administration of comparably high nasal vaccine dosages.

FEMS Microbiol Lett, 2002 Jun 18, 212(1), 65 - 70
Induction of an outer membrane protein of 78 kDa in Vibrio vulnificus cultured in the presence of desferrioxamine B under iron-limiting conditions; Aso H et al.; Although Vibrio vulnificus is known to be able to utilize ferrioxamine B as an iron source, its outer membrane receptor remains to be determined . In this study, we found that V . vulnificus expressed a new outer membrane protein of 78 kDa when grown in the presence of desferrioxamine B under iron-limiting conditions . The desferrioxamine B-dependent iron uptake was only observed in bacterial cells expressing this protein . Furthermore, non-denaturing polyacrylamide gel electrophoresis followed by autoradiography of the outer membrane preparation containing the 78-kDa protein preincubated with {(55)Fe}ferrioxamine B provided a single radioactive band in which the 78-kDa outer membrane protein was present as the major component . These lines of evidence suggest that the inducible 78-kDa protein may serve as the cell-surface receptor for ferrioxamine B.

Kansenshogaku Zasshi, 2002 May, 76(5), 391 - 5
{A successful treatment of Vibrio vulnificus infection}; Sasaki E et al.; A 65-year-old male patient with a history of alcoholism visited our outpatient clinic complaining of nausea and diarrhea followed by dizziness . Erythema and swelling with partial exfoliation on the right forearm to hand and right thigh were noticed . Vibrio vulnificus was isolated from the purulent discharge of the skin . Due to urgent and intensive treatment of bacterial shock and antimicrobial drugs, the patient fully recovered three months later . We believe that the patient survived from this fatal infection because; 1) the isolates were highly sensitive to a wide variety of antibiotics, 2) the antibiotic therapy was started immediately, with an alternative usage of different antibiotics, and 3) the liver dysfunction of the patient had not been severely damaged by alcohol before the infection.

Protein Sci, 2002 Jul, 11(7), 1657 - 70
The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse; Glowacki G et al.; ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue . Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse . The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs . The human and mouse ART genes map to chromosomal regions with conserved linkage synteny . The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse . We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells . Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities . Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity . Conceivably, these ARTs may have acquired a new specificity or function . The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins . In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed) . Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation . This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.

FEMS Microbiol Rev, 2002 Jun, 26(2), 125 - 39
Vibrio cholerae and cholera: out of the water and into the host; Reidl J et al.; The facultative human pathogen Vibrio cholerae can be isolated from estuarine and aquatic environments . V . cholerae is well recognized and extensively studied as the causative agent of the human intestinal disease cholera . In former centuries cholera was a permanent threat even to the highly developed populations of Europe, North America, and the northern part of Asia . Today, cholera still remains a burden mainly for underdeveloped countries, which cannot afford to establish or to maintain necessary hygienic and medical facilities . Especially in these environments, cholera is responsible for significant mortality and economic damage . During the last three decades, intensive research has been undertaken to unravel the virulence properties and to study the epidemiology of this significant human pathogen . More recently, researchers have been elucidating the environmental lifestyle of V . cholerae . This review provides an overview of the current knowledge of both the host- and environment-specific physiological attributes of V . cholerae.

Biochemistry, 2002 Jun 25, 41(25), 8082 - 6
Ligand-induced differences in secondary structure of the Vibrio parahaemolyticus Na+/galactose cotransporter; le Coutre J et al.; A detailed structural study of the prokaryotic sodium/galactose transporter (vSGLT) from Vibrio parahaemolyticus using attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy reveals stepwise increases in alpha-helicity upon binding of sodium and D-galactose . These increases in helicity correlate with decreases in beta-structural elements . The changes are accompanied by stepwise reductions in the degree of H/D exchange (HDX), suggesting reduced accessibility of water to the protein backbone . The data demonstrate discrete conformational changes from one intermediate to the next during the catalytic cycle of the protein and are interpreted in a model of the symport reaction mechanism.

J Appl Microbiol, 2002, 93(1), 108 - 16
Entry of Vibrio harveyi and Vibrio fischeri into the viable but nonculturable state; Ramaiah N et al.; AIMS: Physiological responses of marine luminous bacteria, Vibrio harveyi (ATCC 14216) and V . fischeri (UM1373) to nutrient-limited normal strength (35 ppt iso-osmolarity) and low (10 ppt hypo-osmolarity) salinity conditions were determined . METHODS AND RESULTS: Plate counts, direct viable counts, actively respiring cell counts, nucleoid-containing cell counts, and total counts were determined . Vibrio harveyi incubated at 22 degrees C in nutrient-limited artificial seawater (ASW) became nonculturable after approximately 62 and 45 d in microcosms of 35 ppt and 10 ppt ASW, respectively . In contrast, V . fischeri became nonculturable at approximately 55 and 31 d in similar microcosms . Recovery of both culturability and luminescence of cells in the viable but nonculturable state was achieved by addition of nutrient broth or nutrient broth supplemented with a carbon source, including luminescence-stimulating compounds . Temperature upshift from 22 degrees C to 30 degrees C or 37 degrees C did not result in recovery from nonculturability . CONCLUSIONS: The study confirms entry of V . harveyi and V . fischeri into the viable but nonculturable state under low-nutrient conditions and demonstrates nutrient-dependent resuscitation from this state . SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms loss of luminescence of V . harveyi and V . fischeri on entry into the viable but nonculturable state and suggests that enumeration of luminescent cells in water samples may be a rapid method to deduce the nutrient status of a water sample.

Infect Immun, 2002 Jul, 70(7), 3419 - 26
Identification of the Vibrio cholerae enterobactin receptors VctA and IrgA: IrgA is not required for virulence; Mey AR et al.; The gram-negative enteric pathogen Vibrio cholerae requires iron for growth . V . cholerae has multiple iron acquisition systems, including utilization of heme and hemoglobin, synthesis and transport of the catechol siderophore vibriobactin, and transport of several siderophores that it does not itself make . One siderophore that V . cholerae transports, but does not make, is enterobactin . Enterobactin transport requires TonB and is independent of the vibriobactin receptor ViuA . In this study, two candidate enterobactin receptor genes, irgA (VC0475) and vctA (VCA0232), were identified by analysis of the V . cholerae genomic sequence . A single mutation in either of these genes did not significantly impair enterobactin utilization, but a strain defective in both genes did not use enterobactin . When either irgA or vctA was supplied on a plasmid, the ability of the irgA vctA double mutant to use enterobactin was restored . This indicates that both VctA and IrgA transport enterobactin . We also identify the genes vctPDGC, which are linked to vctA and encode a periplasmic binding protein-dependent ABC transport system that functions in the utilization of both enterobactin and vibriobactin (VCA0227-0230) . An irgA::TnphoA mutant strain, MBG40, was shown in a previous study to be highly attenuated and to have a strong colonization defect in an infant mouse model of V . cholerae infection (M . B . Goldberg, V . J . DiRita, and S . B . Calderwood, Infect . Immun . 58:55-60, 1990) . In this work, a new irgA mutation was constructed, and this mutant strain was not significantly impaired in its ability to compete with the parental strain in infant mice and was not attenuated for virulence in an assay of 50% lethal dose . These data indicate that the virulence defect in MBG40 is not due to the loss of irgA function and that irgA is unlikely to be an important virulence factor.

Microbiol Immunol, 2002, 46(4), 298 - 303
Purification of a serine protease of Vibrio parahaemolyticus and its characterization; Ishihara M et al.; A 50 kDa protease designated as VPP1 was purified from the culture supernatant of a clinical strain of Vibrio parahaemolyticus by ammonium sulfate fractionation, Sephacryl S-200 HR gel filtration and Fractogel EMD TMAE 650 ion-exchange chromatography . VPP1 was inhibited by EDTA, EGTA and serine protease inhibitors, suggesting that it is a calcium-dependent serine protease . N-terminal amino acid sequence of VPP1 was quite similar to that of V . metschnikovii protease and antibody against VPP1 inhibited the activity of V . metschnikovii protease, suggesting the similarity of the two proteases . It was demonstrated that VPP1 or its related protease widely distribute in not only V . parahaemolyticus but also V . alginolyticus.

Nat Struct Biol, 2002 Jul, 9(7), 522 - 6
The structure of VibH represents nonribosomal peptide synthetase condensation, cyclization and epimerization domains; Keating TA et al.; Nonribosomal peptide synthetases (NRPSs) are large, multidomain enzymes that biosynthesize medically important natural products . We report the crystal structure of the free-standing NRPS condensation (C) domain VibH, which catalyzes amide bond formation in the synthesis of vibriobactin, a Vibrio cholerae siderophore . Despite low sequence identity, NRPS condensation enzymes are structurally related to chloramphenicol acetyltransferase (CAT) and dihydrolipoamide acyltransferases . However, although the latter enzymes are homotrimers, VibH is a monomeric pseudodimer . The VibH structure is representative of both NRPS condensation and epimerization domains, as well as the condensation-variant cyclization domains, which are all expected to be monomers . Surprisingly, despite favorable positioning in the active site, a universally conserved histidine important in CAT and in other C domains is not critical for general base catalysis in VibH.

Microbiology, 2002 Jun, 148(Pt 6), 1655 - 66
Evolutionary and functional analyses of variants of the toxin-coregulated pilus protein TcpA from toxigenic Vibrio cholerae non-O1/non-O139 serogroup isolates; Boyd EF et al.; The toxin-coregulated pilus (TCP) is a critical determinant of the pathogenicity of Vibrio cholerae . This bundle-forming pilus is an essential intestinal colonization factor and also serves as a receptor for CTXphi, the filamentous phage that encodes cholera toxin (CT) . TCP is a polymer of repeating subunits of the major pilin protein TcpA and tcpA is found within the Vibrio pathogenicity island (VPI) . In this study genetic variation at the tcpA locus in toxigenic isolates of V . cholerae was investigated and three novel TcpA sequences from V . cholerae strains V46, V52 and V54, belonging to serogroups O141, O37 and O8, respectively, were identified . These novel tcpA alleles grouped into three distinct clonal lineages . The polymorphisms in TcpA were predominantly located in the carboxyl region of TcpA in surface-exposed regions of TCP fibres . Comparison of the genetic diversity among V . cholerae isolates at the tcpA locus with that of aldA, another locus within the VPI, and mdh, a chromosomal locus, revealed that tcpA sequences are far more diverse than these other loci . Most likely, this diversity is a reflection of diversifying selection in adaptation to the host immune response or to CTXphi susceptibility . An assessment of the functional properties of the variant tcpA sequences in the non-O1 V . cholerae strains was carried out by analysing whether these strains could be infected by CTXphi and colonize the suckling mouse . Similar to El Tor strains of V . cholerae O1, in vitro CTXphi infection of these strains required the exogenous expression of toxT, suggesting that in these strains ToxT regulates TCP expression and that these TcpA variants can serve as CTXphi receptors . All the V . cholerae non-O1 serogroup isolates tested were capable of colonizing the suckling mouse small intestine, suggesting that the different TcpA variants could function as colonization factors.

Biochem Biophys Res Commun, 2002 Apr 26, 293(1), 456 - 62
Cloning, sequencing, and functional studies of the rpoS gene from Vibrio harveyi; Lin YH et al.; The Vibrio harveyi rpoS gene which encodes an alternative sigma factor (sigma(s) or sigma(38)), has been cloned and characterized . The predicted protein sequence is closely related to RpoS proteins in other bacteria with up to 86% sequence identity . A rpoS null mutant of V . harveyi was constructed and the phenotype studied . Comparison of the properties of the V . harveyi wild type and rpoS deletion mutant showed that rpoS affected the ability of the cells to survive only under specific types of environmental stresses . The rpoS null mutant had a lower survival rate compared to the wild type parental strain at high concentrations of ethanol and in the stationary phase . In contrast to other bacteria, deletion of rpoS in V . harveyi did not affect the resistance of the cells to high osmolarity or hydrogen peroxide, suggesting the existence of alternative systems in V . harveyi responsible for resistance to these stresses . RpoS appears not to be involved in the control of luminescence in V . harveyi even though it is implicated in regulation of other acyl-homoserine dependent quorum sensing systems.

Arch Biochem Biophys, 2002 May 15, 401(2), 173 - 7
Korormicin insensitivity in Vibrio alginolyticus is correlated with a single point mutation of Gly-140 in the NqrB subunit of the Na(+)-translocating NADH-quinone reductase; Hayashi M et al.; Na(+)-translocating NADH-quinone reductase (NQR) from the marine Vibrio alginolyticus is strongly inhibited by a new antibiotic korormicin . Korormicin specifically inhibits the Na(+)-dependent reaction of the NQR complex and acts as a purely non-competitive inhibitor for Q-1 with the inhibitor constant of 82 pM . Korormicin-resistant mutants were isolated from V . alginolyticus and the NQR complex was purified from a mutant KR2 . Similar to 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), korormicin acted as a purely noncompetitive inhibitor to the NQR complex from the mutant KR2, but the inhibitor constant increased to 8 microM, which is 10(5)-fold higher than that of the wild-type NQR complex . The inhibitor constant of HQNO, however, was only slightly affected by the acquisition of korormicin resistance . The spontaneous mutation was caused by a single mutation of G-422 to T-422 in the nucleotide sequence of the nqrB gene, which resulted in the conversion of Gly-140 to Val-140 . Thus, Gly-140 seems to play an important role for the binding of korormicin to the NqrB subunit . The fact that korormicin is a purely noncompetitive inhibitor for Q-1 strongly supports the presence of one of Q-1 binding sites in the NqrB subunit, which also has a covalently bound FMN at Thr-235 . (c) 2002 Elsevier Science (USA).

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 973 - 6
Vibrio trachuri Iwamoto et al . 1995 is a junior synonym of Vibrio harveyi (Johnson and Shunk 1936) Baumann et al . 1981; Thompson FL et al.; The taxonomic position of Vibrio trachuri was examined through a polyphasic approach using 16S rDNA sequencing, fluorescent amplified fragment length polymorphisms (FAFLP), DNA-DNA hybridization experiments, G+C content of DNA and phenotypical tests . Phylogenetic analysis showed that Vibrio harveyi is the closest neighbour of V . trachuri, sharing about 98.8% similarity in the 16S rDNA gene . Moreover, numerical analysis of the FAFLP patterns revealed that both species have highly related genomes, sharing 55% pattern similarity . DNA-DNA hybridization experiments and G+C content measurements reinforced these results, since V . trachuri and V . harveyi had at least 74% DNA similarity and 44.5-45.2 mol % G+C . Phenotypical features of both species were also very similar, except that V . trachuri utilized itaconic acid, whereas V . harveyi did not . Therefore, it is proposed that the species V . trachuri should be reclassified as V . harveyi.

Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 913 - 20
Thioalkalimicrobium cyclicum sp . nov . and Thioalkalivibrio jannaschii sp . nov., novel species of haloalkaliphilic, obligately chemolithoautotrophic sulfur-oxidizing bacteria from hypersaline alkaline Mono Lake (California); Sorokin DY et al.; Two strains of haloalkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria were isolated from the oxygen-sulfide interface water layer of stratified alkaline and saline Mono Lake, California, USA . Strain ALM 1T was a dominant species in enrichment on moderate-saline, carbonate-buffered medium (0.6 M total Na+, pH 10) with thiosulfate as an energy source and nitrate as a nitrogen source . Cells of ALM 1T are open ring-shaped and are non-motile . It has a high growth rate and activity of thiosulfate and sulfide oxidation and very low sulfur-oxidizing activity . Genetic comparison and phylogenetic analysis suggested that ALM 1T (= DSM 14477T = JCM 11371T) represents a new species of the genus Thioalkalimicrobium in the gamma-Proteobacteria, for which the name Thioalkalimicrobium cyclicum sp . nov . is proposed . Another Mono Lake isolate, strain ALM 2T, dominated in enrichment on a medium containing 2 M total Na+ (pH 10) . It is a motile vibrio which tolerates up to 4 M Na+ and produces a membrane-bound yellow pigment . Phylogenetic analysis placed ALM 2T as a member of genus Thioalkalivibrio in the gamma-Proteobacteria, although its DNA hybridization with the representative strains of this genus was only about 30% . On the basis of genetic and phenotypic properties, strain ALM 2T (= DSM 14478T = JCM 11372T) is proposed as Thioalkalivibrio jannaschii sp . nov..

FEMS Immunol Med Microbiol, 2002 Jun 3, 33(2), 133 - 8
Proinflammatory cytokine profile in Vibrio vulnificus septicemic patients' sera; Shin SH et al.; Vibrio vulnificus causes a fulminant and frequently fatal septicemia in susceptible hosts . The present study was designed to evaluate the proinflammatory cytokine profile in V . vulnificus septicemia patients' sera and the effect of doxycycline therapy on the levels of proinflammatory cytokines . Levels of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, were measured in the sera of V . vulnificus septicemic patients and normal healthy volunteers using colorimetric sandwich ELISA . The mean values of TNF-alpha, IL-1beta and IL-6 in the sera of V . vulnificus patients (n=33) increased by 210-, 232- and 40-fold in comparison with those of normal healthy volunteers (n=5), but only the IL-6 level showed a statistically significant difference (P<0.05) between the two groups . Sera from the cases for which doxycycline treatment histories were obvious were designated 'before-treatment' (TX) . All the others were included in the after-TX group . In the before-TX group (n=5), the levels of TNF-alpha and IL-1beta significantly increased (P<0.05) in comparison with the after-TX group (n=5) . IL-6 levels in the two groups showed no difference . In conclusion, the levels of the well known proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 increased in the V . vulnificus septicemic patients' sera, and the levels of TNF-alpha and IL-1beta decreased significantly after doxycycline treatment . These data indicate that proinflammatory cytokines might play a critical role in V . vulnificus septicemia like in other endotoxemic shocks . The use of doxycycline as an effective bactericidal agent and as an effective modulator of proinflammatory cytokines is supported.

Nature, 2002 Jun 6, 417(6889), 656 - 9
Filamentous phage integration requires the host recombinases XerC and XerD; Huber KE et al.; Many bacteriophages and animal viruses integrate their genomes into the chromosomal DNA of their hosts as a method of promoting vertical transmission . Phages that integrate in a site-specific fashion encode an integrase enzyme that catalyses recombination between the phage and host genomes . CTX phi is a filamentous bacteriophage that contains the genes encoding cholera toxin, the principal virulence factor of the diarrhoea-causing Gram-negative bacterium Vibrio cholerae . CTX phi integrates into the V . cholerae genome in a site-specific manner; however, the approximately 6.9-kilobase (kb) CTX phi genome does not encode any protein with significant homology to known recombinases . Here we report that XerC and XerD, two chromosome-encoded recombinases that ordinarily function to resolve chromosome dimers at the dif recombination site, are essential for CTX phi integration into the V . cholerae genome . The CTX phi integration site was found to overlap with the dif site of the larger of the two V . cholerae chromosomes . Examination of sequences of the integration sites of other filamentous phages indicates that the XerCD recombinases also mediate the integration of these phage genomes at dif-like sites in various bacterial species.

Nature, 2002 Jun 6, 417(6889), 642 - 5
Host-induced epidemic spread of the cholera bacterium; Merrell DS et al.; The factors that enhance the transmission of pathogens during epidemic spread are ill defined . Water-borne spread of the diarrhoeal disease cholera occurs rapidly in nature, whereas infection of human volunteers with bacteria grown in vitro is difficult in the absence of stomach acid buffering . It is unclear, however, whether stomach acidity is a principal factor contributing to epidemic spread . Here we report that characterization of Vibrio cholerae from human stools supports a model whereby human colonization creates a hyperinfectious bacterial state that is maintained after dissemination and that may contribute to epidemic spread of cholera . Transcriptional profiling of V . cholerae from stool samples revealed a unique physiological and behavioural state characterized by high expression levels of genes required for nutrient acquisition and motility, and low expression levels of genes required for bacterial chemotaxis.

Biometals, 2002 Jun, 15(2), 153 - 60
Bisucaberin--a dihydroxamate siderophore isolated from Vibrio salmonicida, an important pathogen of farmed Atlantic salmon (Salmo salar); Winkelmann G et al.; A siderophore of the bacterial fish pathogen, Vibrio salmonicida, was isolated from low-iron culture supernatant and structurally characterized as bisucaberin by FTICR- and FAB-MS, NMR and GC-MS analysis of the hydrolysis products . Although the cyclic dihydroxamate bisucaberin has previously been isolated from a marine bacterium, Alteromonas haloplanktis, its involvement in cold-water vibriosis of Atlantic salmon (Salmon salar) is novel . Bisucaberin production in iron-limited media was highest at temperatures around and below 10 degrees C, correlating well with temperatures at which outbreaks of cold-water vibriosis occur . Due to the very high stability constant of K = 32.2, bisucaberin is a most efficient iron scavenger which may contribute to the virulence of V . salmonicida in Atlantic salmon.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2002, 37(4), 573 - 8
QSAR study on the joint toxicity of 2,4-dinitrotoluene with aromatic compounds to Vibrio fischeri; Yuan X et al.; The individual and joint toxicities of binary mixtures of 2,4-Dinitrotoluene (2,4-DNT) and 8 nitrobenzenes and anilines to V . fischeri were determined respectively and their mixture toxicity effects were assessed using the Additivity Index (Al) . The energy of the lowest unoccupied molecular orbital (E(LUMO)) of studied compounds was calculated and was used to develop QSARs . The prediction models for the individual and mixture toxicity data were respectively: log 1/EC50=3.213-0.716 E(LUMO), n=9, r=0.830, s=0.545 and log 1/EC50=3.672-0.882 E(LUMO), n=8, r=0.925, s=0.406 . The joint toxic effects derived from AI values show that the mixture toxicity of 2,4-DNT and the other nitrobenzenes is synergistic (AI>0), whilst the mixture toxicity of 2,4-DNT and the anilines is antagonistic (AI<0) . These results confirm that the mixture toxicity is mainly related to intracellular oxidation and reduction . -E(LUMO) values of nitrobenzenes are higher . The nitrobenzenes when mixed with 2,4-DNT make the mixture systems easily reduced; the mixture toxicity effect is synergism . On the other hand, -E(LUMO) values of anilines are lower . The anilines when mixed with 2,4-DNT can make the mixture systems less easily reduced, and the mixture toxicity effect is antagonism.

J Exp Med, 2002 Jun 3, 195(11), 1455 - 62
The contribution of accessory toxins of Vibrio cholerae O1 El Tor to the proinflammatory response in a murine pulmonary cholera model; Fullner KJ et al.; The contribution of accessory toxins to the acute inflammatory response to Vibrio cholerae was assessed in a murine pulmonary model . Intranasal administration of an El Tor O1 V . cholerae strain deleted of cholera toxin genes (ctxAB) caused diffuse pneumonia characterized by infiltration of PMNs, tissue damage, and hemorrhage . By contrast, the ctxAB mutant with an additional deletion in the actin-cross-linking repeats-in-toxin (RTX) toxin gene (rtxA) caused a less severe pathology and decreased serum levels of proinflammatory molecules interleukin (IL)-6 and murine macrophage inflammatory protein (MIP)-2 . These data suggest that the RTX toxin contributes to the severity of acute inflammatory responses . Deletions within the genes for either hemagglutinin/protease (hapA) or hemolysin (hlyA) did not significantly affect virulence in this model . Compound deletion of ctxAB, hlyA, hapA, and rtxA created strain KFV101, which colonized the lung but induced pulmonary disease with limited inflammation and significantly reduced serum titers of IL-6 and MIP-2 . 100% of mice inoculated with KFV101 survive, compared with 20% of mice inoculated with the ctxAB mutant . Thus, the reduced virulence of KFV101 makes it a prototype for multi-toxin deleted vaccine strains that could be used for protection against V . cholerae without the adverse effects of the accessory cholera toxins.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Mar-Apr, (2), 50 - 5
{Cholera toxin as Vibrio cholera superantigen}; Vasil'eva GI et al.; Experimental data confirming our earlier suggestion, that cholerae toxin (CT) possesses superantigen (SA) properties are presented . When used in very small doses, CT has been found to induce polyclonal activation of T lymphocytes, essentially exceeding that observed in classical T mitogens characteristic of SA . CT, in contrast to mitogens and similarly to other SA, is shown to display this activity only in the presence of antigen-presenting cells . Experiments with the use of monoclonal antibodies to the variable region of the beta-chain of the T-cell receptor (V beta TCR) have demonstrated that CT, similarly to other SA, are capable of inducing expression of certain types of V beta TCR and causing polyclonal activation of T lymphocytes carrying these types of V beta TCR . The presence of these properties gives grounds for regarding CT as SA . The SA activity of CT has been found to be linked with its subunit A.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Mar-Apr, (2), 3 - 6
{Characterization of Vibrio cholerae eltor in the city of Kazan in 2001}; Onishchenko GG et al.; Information on V . cholerae eltor isolated in the focus of cholera in Kazan in 2001 at different periods of the outbreak is presented . The identity of strains isolated from patients, vibriocarriers and environmental objects, including their antibioticograms (sensitivity to cyprofloxacin and resistance to trimethoprim--sulfamethoxazole, streptomycin, furazolidone and nalidixic acid, which may be regarded as markers), is shown . Variable tandem repetitions in the DNA of 30 isolates strains of different origin have been determined . The results of this determination make it possible to classify all these strains as one genotype, which confirms the suggestion on the circulation of one subclone of the infective agent of cholera in the focus . As revealed in this investigation, the isolated strains are labile with respect to diagnostic phage eltor, while ctx+ strains are resistant to phage eltor ctx+.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Mar-Apr, (2), 14 - 7
{Vibrio cholerae 01, variant eltor, in the environment}; Mikhailova AE et al.; The possibilities of the autochthonous existence of V . cholerae in open water reservoirs, depending on the combined effect of different biotic and abiotic factors are considered . The role of adaptive variability of V . cholerae O1, biovar eltor for its preservation in the environment is emphasized . The data on the duration of the V . cholerae O1 isolation from different environmental objects in some regions of Ukraine are presented.

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