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 hospi